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Full text of "Report of program activities : National Cancer Institute"

ANNUAL REPORT 

OF 

PROGRAM ACTIVITIES 



ISTITUTES OF HEALTH 



VOLUME U 



NATSONAi SNSTITUTES OF HEALTH 

PUBLIC HEALTH SERVICE 

U. S. DEPARTMENT OF HDALTH, EDUCATION, AND WELFARE 






AepovC tf f prcrqro*/*^ a Ct' V" "!?/(? s. 



Budget 



;liiilcel .».,,«.» 



Total.,,., », 

/ lacliides Projects No's; 

Ol'FKSi: or' THE CHIEFS 

400 

40Oa 

■■■'•-'LOGY BHCTlCSIs 
^O'. J|06 

GEISxIIUiL BIOLOGY SICTKSI; 

J-iOi 4i8 

i!07 U20a~e 

U08 420b 

hXQs. 420c 

4lOo 420d 

416 420f 
kl^B. 421 

417 433 

LSmCED'UA STUDIES SEC'TICSf; 

427 427c 

427a 427d 

42?'b 

TISSUE CUXTirriS SECTICg?s 

4l6b 42&1 

428a 42.% (Ji 



. i; .tL... .v.. ,- i.. ■•. 


■ 4 ^9, TOG 





292^100 


.o 1,131,800 



549 



Sesrlal Ho.^^ j .__ 

lo Labov • '§lxl°©ffir 

3o Bot&dsMay m. 



Isidi-^dual Project Hoporfc 
Calendar Teay 1958 



?*rt Ao< 



Pr©j©0t Titles Biological studies oa the factors iiwol^ed in 
the dsrelopneiot axtd growth of tvamra in 
experlflwatal anlmlso 

A^dLnsipal Iinrestigators Ho Bo Aiiderroat 

Other Iisv-estigatoT^s ikfsy lo Cant«r 

Cooperating Vnd-tas Vtmno 



Man Years (calendar yeaif 1958) s 
Totals 5.1 

Professionsilg lo5 
Others h 



Fp&io9t Beserlptions 



Tejiti@uMr Twmrs 



Dcsdng as earliwr stis^ @®is!@@ming the @ffeet of dia©o^inued 
estrogenic atisnlation upon the develqpaBent of tsisti@iilj^ tisa@r9 
in B&LB/@ Biioe it vm found that a&as sloe de^reloped tum^s fr^a 
1 t© 12 aggrtho after extrinsi© stiiBuMtion i^s stopped o 



Tno esqperlBOBta were undertaken to aeeertain idiether the @b^ 

ser^raticHU eoiild be sonfiiwed and extended <> Eight°ve«ies°@ld stsmin 
BA.I1B/0 mles reoeived 2G^ stilbestrol-^holesterol pellets suboutane^ 
ottsljr and, fron 8 to 2h weeks later^, the pellets were reiaored trem 
ndoe i^tose testes were of nor^l sise» The »tad|f is stJH in progress 
beeaose the aniaale mm kept until the^ are 2li oonths oM bist the 
resttlt^j a@ »hmm in table Ig haw ©oafiraaed the praf^oras flndingSo 



It is clear that prolesiged or @©ntinued horssosxal stimislatiosa 
i@ not essential for the production of these tm&p»o It is also 
of intere«t that the iMijori-^ of th® t«ai@rs grew progressively o 
This stad^ will end in Februasyj 1959, when th© last Mee will h® 2li 

Biosrbhs of agSo 



550 



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finSis^ «lth tostioQlar tvmxea and Iwcauso It «ae knoim timt 
diasages isit£at«9d la t@stes ^ ostrogaDle 8tiBQ3Ati«Q persisted 
for 1^3^ mesBHim and these elaaged ti8sts@s z-oaoted prffi^ptl;sr to a 
stimsXsi^oiio 



slsad ttr^mesg agasfe^fysa (MIS/o 2 CSH)?^, Isgrhslde were needo" 
Fii-@ri@m ©sperifflE&se uith th@@@ asijeals had slmmi iMt vescStxffoen 
SsmUB cQsMbited & mamsy tmess' iasidenoe o£ lam tbaa !$$o In 
tMs exp^fisassst the mle ho^rlde im« oastsated vham 2 sntsths eld 

sad th® f^^les «esp® kept as 'wiT^jsBo 



StiIbest?ol°ehol@st@Q.-ol pellets «wsitaiaiag 20^ stilbest^ol md 
v^ghiiag about 5 ^ %!®?<i» ^@d to stably estrogesLe stiaBslaHcasi 
'Siese isez^ Isswted i&to ths sabcQtasseoos tl.88is€i@ w&SL 
tte^dugh & sU.% ia the sktsso 

^KO alee ^@r@ divided iiato 1} g?©ii|p@ a® i&^^msBo 



E®eeived pellets uhm 2 t^ 3 aootls ©M isM^h %®i^ 
ireneved 16 «adee lAtero Hesei^fod se^M pell@ts 
26 i»eks afte? resaoval of the fispst pelletSo tb®s@ 
the e^^sdsaeital ^dseiso 



B»s9lr«d pellets n^m 2 to 3 ssmths @ld «fM.©h iis^® 
U mekB later. 



Jo Beeeif ®d psllsts wlsa ^®i^ 1 adee re@ei^^ tfesis' 
pellatSo ^©s« pellets ®»r® set g-sKwedo 



ho BeoeiTsd pellets ^l^m ^Qxsp 1 alee reoeiired theii' 
fiTst pelletso ?h®@@ pellets 



lllie results are shc^a in table 2o The ssqpeslBeatal aaia@l@ 
(gs?ot^ 1) sh0B9d ® loiras* tussF istsidesoe tteui the ^&v^ h @08tt?&l@ 
aad a higSafls* iaoidenoe tlna the grai^ 3 eoastrelSo this iiadiestes 
that exposixro to the estrogea for l£ weeks had initiated eimmm 
ia the aaaaazy glaad that persisted for 26 veeks aod re»oted to 
the seoi^id stiaolAtiono Th^is^ smmsy glaed tissaes trare si!!d2m> 
to interstitial testicQlaz> tissues is their respoBse t@ stilbestrolo 



Qroup 3 ffiie® irare of scsse interest be^^e ths^ «are 11 to 12 

©M irtsm the^ raeei-ved pellets aad afe©®fe 30^ dm®l&p^ %xmm 
2$ -^ S°i ms^fes ©f agOo 



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Group 2 Semlm m,r@ «!«© of laterest boofflus© la$ d«v«loD«d 
tuiBors aft®r .caoK?yi2jg p«ll8ta for oaay 16 tTsoko „ ma- uuggeBts 
tbat^ &s ffith tesMotOs^ tusicrts^ the estrogon initiAted ohaagee 
wM3h persisted aM, aided \^ ths «nd( 

of tha iaic@, proeesdod to naligiBiic^o 



It mist b« kspft in sdiad tiat tha twox« of this «sp«Klm8Qt 
frare induced la agent=>£reo nieoo 

ljans^ESltMB or.,ageHt fron wiM^MQeo Eaa^Mar msk wlt|i udM 
His© i^^d^KF^^c^S^aaai^af l®w aotivily ascanJiag 
t© its ability to prodts@® ttis^rs in r«^ sosooptible iobred strain 
C3H sioeo A strala M13/& famle aoqoired the agent from & wild 
foster nottor dm±as 19hl cmd the agent vas propagated tfaroi^h 20 
generatioos of her desoesdantao The results ±q these adce suggested 
that the ^Id isouse agent had inereased ia acti^^ daring serial 
passage -^uraii^ strain BA.LB/0 adoeo Tm eatperiBsnts «»re p®rf&m.@d 
to tmt the aotiviigr of this line of agent. 



The first consisted of eocposiag ag^it^free sts-sdn OJl f€ 
bgr having nesborn strain C3H sdce suckled W B&LB/o f esales that 
were earzying th© 20th generation passage of the wild nouse agent o 
fhe o®3urrenoe of breast tisaors in the fostered strain G3H ni@e and 
in the first 2 generations of theif nota foeterad deseendants tms as 
followsp 

Noo nith ATerage &g© 



Fostered 10 9 U 

let Oenerstion 11 6' 1? " 

2nd « 9 $ 18 "^ 

These findings «ere ti^n @^^ared «ith earlier findings 
agent Sr^e strain C3H nise were eu@kl@d \^ viM nioeo The ^rlie? 
results vere as follouso 

Nunber Noo %iith Aferag® ag@ 



Postered 8 3 17 saeaths 

1st Oenez^tion 6 2 19 "^ 

2nd •» 6 1 22 « 

Such finding® indicate that ths ifild mm?« agent had in@r@^@@d 
in aoti^-^ dmlng passage throuc^- strain SAXiB/® feoaleso This mmsB 
tlmt 3 straim of the agent are available for studio The firsts of 

Im aetlvi-^s, sarried in isiM sd-sei the second, of aediusa aetiid'^, 
is th© wiM agmt earried to MLB/© ssdoej the thirds of high a©ti-^%-s 
is the G3E ag®st carried als© ia BALB/e ais®o 



5?^4 



'■-■'hp E3I':400 



Thai seosasd tost fop acti^ty ©f tto® «iM to^s® ag®isfc eai^slM 
ia B&IS/e nioe eoaseisted of tl^e 2>«isi|sH5dQQtiem of MM ado® to t$t® 
agento PxwloBe wosk had shosn tliat tha agent fssssa itte^ lalca 
evekAd fo» ttnaors isi tsUA siica aisd the eM®f rmsm for tinaasfoxvlag 
tiM Hild nottss «g«ist throQgh B&I^B/o f «BaI«ffi ms to aseortalA iili«t)»ss> 
it eovM thm pKt>dao6 taosp® tsasoi's ia wild sdcso 



The first step vas to establish a line of prestisablj agent free 
viM sdoe Isgr the estsiblished prooedure of siix'gical remrs^l of the 
vdld sdoe from the uterus of their Btothe? aM hs^ag them suckle ispmi 
an ag«at fs«e foster Bother, KLse frosn this line of agent free idld 
adee were foster norsed tg- MLB/s f enstlee oarzylzig the wild laoose 
agento Of lU saoh %Lld sic® ez^ 3 developed breast tusaors f^en 28, 29 
and 30 osonths of ageo This result vm astioipated because of the well 
Jaa&tm resistaaoe of vild sice to the a@ti7e agents earried luf inbred 
sdoe and, for thi@ reason, the f olloKiag experiffiesrt v&s destined to 
test for the aoti'sdtgr of the agent after its reintr^ustion into 
wiM ndoe. 

After the agent^free f eaale wild adce had foster nis^ed upon 
the B&IiB/o foibles thagr «ere aated to agent=>frde strain HIII s^les 
t© procure P= hjrbridSo Scsa© of the F^^ %^brid feasles v^W9 bred t© 

agent^^f^se I^IX nalee to procure badc^ross Ed.ee aad s@iae resei^ed 
pellets of $% stilbestrolo All of the baekoross sd©e Here gi-rsn 
$$ stilbestrel pelletSo To supply (sontrols for these F^ and 
baokoross odoe, feaales £rsm the tdJjd mouse eoloi^ nere bred t® 



the saae RIH sales and treated in exaetly the sase ssmnerr. 'Sh® 
results are shoim in table 3° 



The findings sqpported the results obtained in fogt@r@d 03H 

id.@e nentioned abo9« but thegr eertaizOy -mr® not ©osssltssiTe @^d®nc© 
that the vild House agentp after 20 passages in MLB/c fesales, 
was nuoh store effective in ^dld ndee than Hb® agent carried h^ 
Bdoe f^m the idld eolosoTo ^jadeed, it aj^ears ths.% the a@ti^% 
of the transferred agent, when reintroduoed into idld sd@e, r&w®rtBd 
to its lev degree of asti^t^, btst farther ejept^issnts are needed t@ 
establish this painto 

The laost interesting finding of the @xperis@nt «as the M.gh 
inoidsnoe of breast tuiaors in fcybride preoured Ssem. the f@!8al©s of 
the 'wild oolonjro Two reasons are possibl®o Firsts the tm^s as-m® 
beoause the hyhrids earried. the wild isouae agento If this is tru@ it 
suggests an eacesllent procedure for toting for th@ presence of this 
agent in wild ndoe^ Second, the b^pbrids were suso^tible to 
es^ogen=>indueed breast tuaisrs in the absence of the agessto l©sdl©@@ 



555 



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556 



to aajs if atach results had been asrbieipated, reoiprooal F^ b^hrMB 
fj?oBi ag«nt=freo HIII feKalee woisld bar® b«8ea obtained c The fisjding 
tl&t the F^^ ^lisdds surri^ed after reeeiYins $% stilbestrol pellets 
vas aurprising^ because, in prervious ei^erineirts, esaogr vUA feaales 
aad Bin fenales had died after reeei^iag these pelletso When it 



found that the F^ hybrids tolerated the pellets the badcoross nice 
giTen pellets to aiapliiy the exp^iaantal pro<8ed)2reo 



To determine vfaetber the agent was involved in the prodUQtion 
of tuBwrs in the fagrbrid ndce the f ollouLng expaxlBisnt is new in 
progresao Agent°free female wlM adoe are asited with agtmt-fX'ee 
SIII Btaleso Their F, feieale hsrbrids are bred to agent»free SIXX 
aalea and the F. nail hybrids are bred to agent^^^'ee Bill females o 
These mice should rereal whether the mioe used in table 3 were 
snaoeptible to estrogen induced nanaKay tuaarso It is kSi&im that 
agent-^^free F^ Iqrbrids pro@(S>od froM stufaijw G3H and RIII are highly 
susceptible h> the dehrelopsBent of auoh tuoorso 

Disappearance of the a^mfe fepa mi gjgeo This proper^ 
lie wouae aawAzy t^iwOT* *gent h^ t>een s^ntioned 



of the BK»u8e aaanozy turner agent has been sisntioned in earlier 
reportSo There has been no efaange in the I^I nicei tumors ho^e 

n@t appeared in descendants of those freia «hidi it is presuBed 
the agent disappeared whereas tusors @c@^ in ©th@r mmb@rs of 
the @@lo^o 

In last yearns repos^ foster imrsing ettsdles v®¥® Ksntionedo 
SoBie of these ha^e giTsn findings ©f intes'esto Sle^if^a agent<=>frse 
strain G3H fcffiales were eu@kled b^ agGnt°(3ar:^lng I^X f espies » 
One dewloped a tooor at 19 months and 1 at 13 months @f ag@| 1 
died tunes* free at lU msothsj 1 is Hiring at 20 mei^bs aod T aire 
living at 9 months of agso 



ElerTen agent^f^e BUI females were suckled %gr agent^carz^^ing 
G3H fomaleso Ten hare derelopad tuaorss 3 at 13^ 1 at II5 1 at 3^ 
1 at 6f and U at ^ wmMm of ageo 

One interps>etation ®f these findings is that the RIII mice are 
wry susceptible to the G3H agent bufe thagr earsy a relati7e3y w^k 
agent liiich does not increase in activit^o To inrestigato tMs 
possibility reciprocal F^ hsrbrids are being p^oewed f^a fim 
and C3H micoo The ocsurfenee of tumors in th® ^bidds id.ll rarreal 
wheth^ the tue strains are ©arxylng agents ef different aetiiritiesc 

Part B imlsded Tee Z No 



557 



TT^Mili HiawJi WlwM ■ .Mil 






<af nail szts»«Sft in siss e«n7&^ «te nllk sgeol ftor aaeBaaer @aae«i?« <?« 




TB3«61J., 1958» 



selAtiog to ^tis 99o^ts 



558 



lo Laborato^ of Bi^«3g? 
2o Of.fi©a ©f th© Chief 
3o BethMda^ MasyXand 



PBS«HIH 
Iffldi^idual Project Haport 
C^laodar I«»ftr 1958 

Pirojeet Titles AdndLnistration^ Laboiatoxy of Biologic 

Pxiiseipsa Im-«sti^torg Ho Bo Anderranto 

Othar iBtvatigfttorss AgnM Idttle^ KfttliaHne Ttoaer^ 
Oeenft Qray 



Cooperatisig tMtss 



l%a leam (ealendar y^r 1958) s 
Tetail? 3o5 

Professionally o5 

Others 3 

Pr^jeet Deseriptieiig 

Cimduotijag adniiiietmtiire a@ti'9ities for the 

laboratoTjr of Biologfo These a(grtl'<d.tiee @@nsl@t of 
8ee?etai^lj -^tiiSj, ordering aad othes> «dMai@-^f^tiTe 
respoxisibilities o 



Part B ijKsliaied Jm N© X 



559 



S®5.-'ial l0c_ 

2o Collular Biology Seetisn 
3, Bethasda, Maryland 



PBS - NIH 
Individual Pa?ojeet Repos^ 
Calendar lear 1958 



Part A. 



Project Titles Oxidative Mstaboliam in various eellulay 
structures of norssil and tismer tissueso 



Principal Imrestigatorj Dro Ruth Ko Kiellsgr 
Other Investigators? None 

Cooperating Ifeitss None 

Ifen Tears (calendar year 1958) s 

Totals 1 

Professionals 1 
Others 

Project Descriptions 

Objectives s To detersinQ whether ch@ad.cal carcinogens ha.r@ 

an inhibi^xy' effect on specific cellular oxidations ^ Yitro 

and to determine %?h©thsr th® asagnitud© of thss® effect ■can ¥© 
related to the carcinogenic potency of th© coKpsuadSo 

Methods ^ ^^ emgleyed s The effect of various carcinogenic and 
n©n°carcin6g®nlc aso dyes on glutaiaata and suceinat® oxidations 
ims studied by measuring O2 uptakes in a phosphesylating ^st©ffi 
of rat liver ajitocbondriSo The tests were T\m en norBsl asito- 
chondria and en ribofla^ln-defieient sitochondriao Th© ago 
eoB^jounds tested wsr© DAB^ MBp AB and sietl^l derivatives of 
these parent ccc^poundSo 



560 



HCI=.40l^ 



M^jUg&lgjdigggg The principal f iiidisags aro smssirised 
in the'^^^r^SH^Sn of oxidatioa is expressed as p®rc©sit 
of the cmtrol to which ao dy© was added and tos bas©d on tha jsean 
0, wptak® of it or 5 differant jsdtochondrial pfroparationso lo 
values are given for SKCcinat© oxidatiom 1^ riboflavin-deficient 
Biitochondria sine© the data ar® ineois^let© and ahoH values eloee 
to thos® of noya^l Esitochozidriao 



561 



NQI-liOii 



Effect of Aao 2^®8 on HLtoohoi^Jrial Glwtam&t® and Succiimt© CKidatioas 



Inhibition of Oxidatlffla 



K@Iative 



control 

MB 

2 m HkB 
2° H6 MB 
3c He D&B 
U» He MB 
contool 

mB 

3 Me MB 

2 ^ mB 
2» He mB 
3" He H&B 
UB He mB 
control 
AB 

3 He AB 
2 He AB 
20 He AB 
30 Me AB 
h^ He AB 



nonsaX 
% 










10 


29 


26 


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ko 


69 


15 


33 





7 








25 


hpL 


13 


18 


22 


32 


U2 


51 


38 


57 


33 


U5 








10 


9 


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16 


17 


27 


32 


27 


16 


28 


13 


16 



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38 

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6 

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28 

1? 

7 





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13 

13 







6 



2=3 
12 



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2 

12 

1-2 



2 

>1 



-ii- NCI»liOii 



The active carcinogeius, DA.B, MBg and tbsir 3" Mo 
darivativos caused significant inhibition (29 =■ $1%) of 
glutasaata coeidation in riboflairin deficient laitochoxidriao 
These carciEogens had little or no inhibitoiry effect oa succinate 
ooddation (0 ° lIRg). In the BAB and MB series and to a leeaer 
extent in the AB series, it is seen that the 2 He and especially 
the 2« Me derivatives inhibited glul^aBsffite oxidation to a significant 
©stent (32 - 69%). Howwor, these derivatives also inhibited 
succinate oxidation appreciably o 

While there &i^m2:% to be a general qualitative relation- 
ship between aelectiv® inhibition of glutamate oxidation (with 
little cof no inhibition of succinete ozidatloa) and earcinogenlcityp 
in the ease of the &&o d^ee^ thes'e is by no means a quantitative 
relationship p 

Signifiean o® to can^^ If changes in the roetaboMc state 
of oellFS'tiiiBStely iaiiit to a mligmnt state, it i.ii soneei'Wible 
as Warburg l»s proposed that alterations in the respirato^cy 
pittsra ©f cells resulting from injury ©r stress inegr pre^de the 
izme^dlate enviroimsnt for subsequent changes leading to cancer q 
It is also conceivable that the change in the respiratesy pattern 
of cells Might have to b® in a specific direction for cell survival 
and adaptation^ Such a directional oMnge adght be broi^ht about 
by various agents = eheBdcsl carcinogens, hormones^ viruses if it 
is supposed that they can all act to bring about a sirailar metaboUc 
change in nomoil cells. The results of this study as well as thoa@ 
of previous work suggest that the laachanisass of action of ©hamieal 
earciongens in ^vo may 5n;^«Jlve specific and selective inMbition 
©f certain o^daHons, such as glutamat@o 

Future resgarchs This project has been discontinuedo 



Part B ineluded Yes N© X 



563 



2« Csllular Biology Ssction 
3o BsthQsda, Maxylawd 



PIS =■ MIH 
iBdi-^dual Pro j set Raport 
Calesidar Yaar 1958 



Part Ac 



Projsct Titles Dl^ ^tithssis in norsjal asd nsoplastic 

tissues and eellSo Synthesis of pho8ptory=> 
latad Wk prsciarsors in call nwcl©io 



Prln^pal Imrestigatopj Dr. Huth Ko Kiellay 
Other Iswestigatorag Dr„ Wo C» Sdmeider 
Coopsrating tJlaitss None 

Man Years (ealandar year 1958) s 
Totals 1 

Professionals 1 
Others 

Project Descriptions 

Ob.l9ctiYe8 g To detsrasine whether the nuclei ©f cells 
activ©3y engaged in LM. synthesis ha-^e th© property ©f g©siarating 
ATP tsy Gtsidati7e phosphozylation for the forj^tion of prirairy 
Dim precursors 

Before this ©bjestive can be eonsideredj h«^®v©rp conditicsis 
most be worked out for the preparatieea from tissn@s of cell nuclei 
which are intact and r©lative3y free of other cell cessponsnfes,. 

Methods aaplgrgdi^ Tissue heseaogenates ^isre aad© la 'various 
.■ssj'3rdse'"ai®iia ' "anaSMffled iaicros©opical3j t© d©t©K9ln© ©ptiiKsa ' 
i^'viaditions for cell breakage^ prssertatien of auslsar structur© 
and for least agglutination of Bdtodioadriao Huelsi w©r© isolated 
froim the hoaaogenate by layering orer sucrose solution of klgh©r 
density followed by differential eentrifugstiono 

^ ^ ^Pqneipal findinfrss Rat and aaouse lijer nuclei ai^eared 
to be bes^^preser^d in n(»iogenat@@ mad© with Go 25 M sucrose = 
O0OOI8 M Ca Clp* PH 60I0 Nuclei once, separated from th® feoBiogemt©s 



564 



-g<= MGI'-"hOk (a) 



O0OOI8 M ca CI2 (pH 6o3)« Addition of glacoS€o6c=.P02^ (loS S 10°%), 

Mg CI2 (? X 10°^ M) and phospMt© (2 X 10"%) to th® sucros® 
jEsdium increased tha stabili-iy of th® nuelaic Hesiaspaasion of 
nuclei in th® high apoad supernatant f racstic© of th® hofflog©nate 
hsm&w&r gav® the most protection and r®p©at®d washing of aucl@i 
could b® carried out with littla visibl® daimgeo Ua® of polyTyia^l 
pjrolidone or ssrura albuisin in the sucrose m&&ivm 0V@ntual3y caused 
nuclei to become granular, Digitcnin &]^esT&d to b@ useful in 
dispersing dm^ed ndtochondria Kith no iiamediatel^ &ppai>@nt 
visible effect ©a nucleic Conditions toa:^® yet to b® Morke>.?i out^ 
however, for a aatiafactozy preperatosy procedure which -Hill 
ellMimte contaiainsints from th® nuclear fractlonp 

Siiaaificance to cancer ? Th® r3.pid uncontrolled growth 
of canci^ cilis ii^iies thai the enggraes and s-eactions involved 
in DN& {^^rnthesis are abundantly present in these tissues and that 
the nuclei of rapldOy dividing cells differ f^m. thos© ©f 
no»=dividing cells o It is claiEied that cell nuclei active in Dm 
synthesis f oza phosphorylated nucleotides idxich ar© th® prlmsj 
DM precursors and that this rsaetion is v©xy 8®n,sitiv® to 
radiation at loi^.^velSo- It would be of great interest to ksis^ . 
how the cancer cells for© these phosphoiylated iat®?ffi®da^t8s 
for JMk synthesis and .ho» these reaotioas ar© controlled in th© 
nomal cello : Th© sit© of ATP synthesis for thes® reactions would 
be an issportant considsrationo So far^ oi£^ the mitoehossJria ar® 
definitely kaoMi to catalyse osidativ® phosphosylatieas but in tl^^Mss^ 
it has receni]^ been suggested that jmclei (Mirsljy) mj possess 
this functions 

. Future research ? Attesapts to prepare intoefc nwl®i suitsbl© 
for aty^^l^'kW liiyni&sis ^11 be contijiuef^o^ "Sha u^s -.£ •jalSiyuleo') - 
ads(^bants (SEiLS and Eateola) as a means of achi(svi!^ cleaner 
nuclear preparations is being imrestigatedo The tissues considered 
for study are regemrating livsr aM som® ascites tuaor cells' 
since the nuclei frosa these ©ells unlike thgnsais caa be readily 
distinguished from i&ole cells o The possibility of oxidative 
Fhosphoiylatlon occurring in nuclei sill be studied 1^ measuring 
O2 and P uptakes under conditions which rwl® out this reaction in 
iDitochondriSo 



Part B inclKSled les 



565 



Serial Noo m^i^06 

lo LaboratojyoFlBi^x^ 
2o Cellular Biology Seetion 
3o Bethesda, Mdo 



PHS =. NIH 
Individual Project Reporfc 
Calendar Year 19^8 



Part A ^ Project title; Electron mieroscopgro 
Principal Investigators Ao Jo Dalton 



Other Innrestigatorsj ¥„ Ray Bryan (1*20, a^c„d,©) 
John Bp Moloney (lt20,b) 



May Tears 8 (Caleiadar year 1958) s 
Total* 1| 
Profeesionals 1 
Others | 

Project Description* Dmlng the past year studies hav® 
continued in four different areas o These im'olve first^ the applieatien 
of electron raieroscojgr to the problem of morphology of viruses and as 
a @m:£trol saeasure in attempts to obtain purer sas^lss of "^Irus tram 
infected tissues? second, ©ejsparison of the fine struct'v-e ©f normal 
and ne^lastic callai third, the stuc^ of a series ©f cell types la 
tissue ctdturei and fourth, the aimlysis of fractions of homogeimtes 
obtained by -rarious centrifugatieof? t<?*chnique8o 

lo In de-Uil <= in the first area and in collaboration with 
Wo H, Bryan and John Molon^, experintents ^th the Rous sarcoisa, ha^e 
continuedo An experiment designed to determine with greater accuraqr 
lihether or not there is a direct relationship between the ntmber of 
-^ral larticles per cell and the potency of a giTen tumor on bioasgay 
has been coaipletedo Th© results indicated that at the higher concentra- 
tions there is a straight line relationship between ^?iral count and 
potency but that at log dilutions in the 10^ to 10"" range^ this re- 
lationship does not holdo In this 3range particles were found in caseis 
in which the bic^asays were completely negative » In this study a new 
standard preparati^i was usedo To determine whether or not this was 
a typical result a new experiment is in progress i^iich will coiqiare 



566 



-2= KCI-U06 



the results obtained vith this new atandard preparation vith an older 
on© at log dilutions between 10"^ and 10°^ o If the results of the 
new experiment oonfira that of the pre^ous one it nay be ssoncluded, 
aiaeng other thingSj, that in the strain of shickens used, and at the 
higher dilutions, an "infectious unit" and a virus parti@le are not 
^TnoziQnnous tersiSo 

The se&ond study in7<ol¥ing the Rous sarcoma has been eoniserned 
with attsJEpts at ccneentrating vis^l particles frois tissue by means 

of differential eentrifugatioHo Little has been aseesr^jlished in this 

area with the exception of a preliiaimuy sti^ whieh suggested that 
the eumunt of dilution of the original homogeimte is an iniportant f^et@r 
relating to the final Goncentration of tbs particles o 

The tM.rd stuc^ related to the findings of Oroiqpe that intra° 
cranial insxjulstion of Rous ^irus into young chides eventuates in a 
vezy high titer of virus in extracts of the brain tissueo Examinatdon 
of brain tissue from wMch these high poten^ extracts were obtained 
did not reveal the expected high concentration of viral particles o 
This suggests that the meninges isaay b® th® sit© of extremely high con^ 
centration of vir«^o Studies are under m^ to detenaine whether or not 
this is the caseo 

Also in this area ar@ studies on the Shope fibrt^ssa &nd rabbit 
BBrxoma with Dro KilhasBo The viruses responsible for these two n®©= 
plasms are in the same group but thsy exhibit great differences in 
growth rate and in the type of cell reaction thay invoke,, It was 
thottght therefor© that a study of their fine structure would d©Hion= 
strate whetlar or not differences in growth rate and mstabelic activity 
would be reflected in differences in mozTphologfo To date n© differences 
in fine struetm*e nor in their life cycles have been found o Both appear 
in these respects very similar to Holluscum contagiosum as descrj.bed 
recently by Bto Banfieldo 

The last itera in this ©ategoxy is a study on the lyropho^ei^rio- 
meningitis vix^a with DrSo Haas and Potter » Using the ascites l^m^&im 
P=>288 as the vectorj aliquots of control tumor cells and tumor calls 
infected with UM were exaisined with the electron Bdcroscope., Saiiples 
of the same tuiBors were tested for the presence of virus by bioassay\. 
The control tumors were ne/^tivsj the infected tumors positive for L€Ho 
Numes'ous, Bsjall C500«600 f ) particles were found in the Golgi aone ©f 
Many ©f the infected cells but net in the controls o However ^ when this 



567 



=3- NCI=U06 



expsriment tsss rapeated using an ascites tfeyrnoasa as the vector^ 
no ©vidence of particles was found in either control or infected 
cella, yet asmplas of the infected ascites were positiTe for ^irws 
on bioassaya The rtssults sire equivocal to say the leasto There is 
vh© possibili% that in the first experiment we i»er® dealing with the 
coincidental preseneo of a passenger ¥lrus or of the railk agent » 
There is also the possibility that a longer residence in a particular 
line of cells is neceseaxy before LCM becosiea recognissable as a mor- 
phologic enti-SgTo Both of these possibilities are being im-®stigatado 

XI o A survey of nonsal and neoplastic cells with the electron 
fisicroscope has continued ^^ most recently la^olring pritaasy spontaneous 
and transplanted spontaneous azxi induced hepatomas 7So nori^l hepatic 
cells, normal tl^jiroid vso thyroid tuiaor cells, noraal plasma cells tso 
cells of plassia cell tusoors and the Gloudman S^pi melanc^sac, Priinary 
snd transplanted spesitaneous hepatoaias possess^ a fine structure '^®iy 
similar to nonsal hepatic cell8$ ergastoplasm being abutKlant and the 
isitodiGsadria though sosievhat sn^ller possessing an apparently nonsal 
inten^l strtsstureo The Golgi complex, hcmenrerj, tended to be con= 
ceatrated in the nuclear "hof f " . instead of being scattered in more 
peripheral areas of the cytoplasmo In addition there was no «ividence 
of lipid droplets within the -^cuoles of the Golgi eoE^lwn as frequently 
occurs in no^^sal hepatic cells a Cells of the induced hepatoma number 129 
adapted to growth ijn the ascites form showed little evidence of hepatic 
cell origin.! Only occasional strands of ergastoplasm were pressntj, the 
majoril^ of the small ribo^nucleo^protein granules appearing free in the 
Ooplasm, and the Golgi ccmplex occupying a relatively small area in 
the nuclear "hoff"o Thyroid tumor cells growing in a host umintained 
©n a high iodine diet contained but a few spherical mitochondria and 
little ergastoplasm. Large vacuoles marginated by tMck electron dense 
membranes were present in the majority of the cells and there was no 
evidence of secretory activil^o lodias retention studies gave little 
evidence of this capacity on the part of the tumor cellSo Cells sf 
the same tumor growing in a host maintained on a low iodine diet con^^ 
tftined r«lative3y large amounts ©f ergastoplasm, filamentous^^ rait©F> 
';!^ndria, an extensive Golgi cosaplsx, and numerous secretory granules <> 
This tumor gave evidence ©f considerable capacity for iodine retentiouc 

The cells of three plasma cell tumors have been studied in 
collaboration with Drso Potter and Fahcy, The tumors were n©= 7Qh29s, 
%S63 and %6klo Tumor nOo ?Oii29 adapted to growth in the ascites £ovm 
showed evidence of dediffersntiation with but a small amount ©f 
ergastoplasm and a samll centrally located Golgi cotGS>l®Xo No chi^nge 
in serum globulins were noted in mice carrying this tumor » The cells 



568 



»U- NCI=it06 

of tmaor noo 5563 eontainsd large amounts of Mghly organisod 
©rgastoplasm similar to norssal plasnsa calls o The Golgi con^lex 
■a&a high3y derreloped and associated with it were ausiarous sroall 
granulea possessing the raorphologie characteristics of sscretoxy 
granulaao In addition, aamll bodies 50 - 70 mu in diametor possess- 
ing a double pariphgral membrans (viral parfeiclss?) were present in 
this ares, Ms® earxyisig this tuaaor showed a ©oasiderabl© increase 
in oeruM globulin particularly in the ganmsa region CFahey)o The cells 
of tumor noo '^Skl also conteined large aioounts of highly organized 
ergastopl&sm, but these smoonts varied more from cell to cell than v%s 
the case in cells of trmst n©o 5563 <. The Golgi cea^lex occupied a relatiwly 
large ctytoplasmic area but consisted almost entirely of small vesicles » 
Small bodies resei^ling secretory granules were found in the Oolgi 
son© aisi similar granules of larger sise were fouzd in the Interstices 
of the ©rgastoplasiHo Mice carrying this tumor shewed an increase in 
aenm globulins also but prissarily in the bete region and s^ae of 
which ar© not present in norsffll serum (Fahey)o Further studies on the 
Cloudman S^^pl mel&noma have shotm that t^o t^pes of melanin granules 
ar® present in the melanoK® cells o Both types were found to originate 
as amall (50 •=■ 70 lau) mQlaniJs^fr©© bedie© in the Golgi zonso Deposits 
of melanin ■sere fouad in these bodies when they were in the Od to 
0<,2 u sis® range They possessed a single peripheral maaabraae and no 
definite internal structure other than the particles of melanin and 
these were readily distinguished trata mitoehondria« No ewidenee was 
foand of the melaaisation of mitochondria o 

IIIo studies on two lines of tissue cultis'e cells originating 
from one single cell clone have continued in collaboration with Dr^ 
Sanfordo Both lines har^e been shosm to possess particles baring the 
aorphologio features of virus, but mai^ more particles were consistently 
foand in and associate tdth cells of the 1cm line tl^n ifith the 
highp Similar particles were readily visualised in association -Hlth 
tumor ©ells of the first in vivo ti^asplAnt generation of both th® high 
aad low lines o Ea^tsverg by the fourth txansplant genera tion^ ^r-fcielgs 
were very rar®o On return to tissue ©ultur®, large nisabers of particles 
were again found and again were more numerous in the low than la th© 
high lineo In this latter material^ there appeared to be a dir@@t 
relationship between the number of particles and the degree of 
cytopathogenic change^ Th® method of fom^ition of the particles within 
th© cytoplasm and of their release from the cell has be^a detsrminsdg 
but the significance of their presence is still unknots, 

I?o Sti:dies on the correlation of the ultra structure and 
biochsEiistiy of fractions obtained frcaa h(SB©genates 1^ centrifugation 



569 



=•5- MJI-U06 



techniques have continued in collaboration iwith Dr, Kuffo The more 
recent results continue to suggest that concentration of RM& in a 
fraction is not necessarily related to the number of ribonusleo=prot®in 
granules found in section of pellets of these fractions » 

Vo Ttro minor studies have been couplet ed during the jearo In 
the first, in collaboration with Jo Br<MJte Gatenby, uanor of the details 
of Lubricus spemiiogenQSis have been wojrtced out, the most interesting of 
•Hhich v&B the finding of a special method of transfer of the acrosaa® 
frcsn its region of fonffiation in the Golgi substaoice to its final legation 
at the distal end of the elongating nucleus of the spermatid o It is 
carried in a special «^oplasa!io a|^@ndage frosa its original to its 
final position^ The second stujfy, with DTo Copeland, was on the fine 
structiare of the cells of the pseudobranch gland of PunduluSo These 
cells ar® presumed to be involved in the production of carbonic anigrdrase^ 
aj*i the gland itself is thought to be concerned with the degree of dis° 
tention of the swim bladder o The cells were found to possess a very 
coH^les mud highly organized ultra=»stru©tur®o The numerous rod-like 
mitochondria were closely packed in groups in the center of two cells o 
The mitochondria in each group showed the same 03*ientat4on and inserted 
between the mitochondria were numerous tightly packed tubules having 
a diameter of approximately ^0 muo These tubules were found to be 
in direct continuity with a system of branching and anastomosing tubules 
in the <^i^plasm between the mitochondria and the plasma membrane o These 
latter tub^^es were found in mai^ instances to open directly on the c@ll 
surface o Opposite the points where these tubules opened on the cell 
surface in greatest number, numerous pores were found in the qrtoplasra 
of endothelial cells lining the blood capillaries associated wil^ these 
eellSo Thus, with tb® .^eption of an intervening basement membrane^ 
thsre appeared to be direct continuity between the lumen of the capillaxy 
and the lumens of the tubules associated with mitochondria ^ sp within 
the cello Such a situation Ms been suspected in other instances but 
has not been demonstrated with the diagrammatic clariiy as 'ms found 
in the present casoo 



Part B included Yes X No 



570 



Serial Hoo KCI°it06^^^ 



PHS <=■ HIH 
Indi^dual Project Report 
Calendar Year 1958 



Peg't. B;^ Honors J, Awards, and Publications 



Publications other than abstracts from this project: 

Hagueaauj, Fo, Dalton, Ao Jo and Molon^, Jo BoS A preliudnasy 
report of electron inicroscopic and bioass^ istudies on the Rous 
sarcoma I virus „ Jo Kato CJancer Insto 20s 633=6149, 1958 « 

Dalton, Ac JoS Organisation in benign &nd saalignant cell@o 
Labo Iswsstigafciono In press o 

GatenbgTj Jo Bo and Dalton, Ao Jo« Th© speradogenesis of Lumibristis 
h^rculeuso An electron JEsicroscope stucco J, Bo B, Co In press o 

WoodsidOy Go Lo and Dalton, Ao JoS An electron microscopic stud^ 
of embryo and newborn mouse lungo Jo Ultrastructur© Researeho 

In press o 

Qaterib^j Jo Bo and Dalten, Ao JoS Origin and conne3d.oas of th© 
central nutrient in Lumbricus speraatoganeseso Nature 182s 
886»887, 1958o 



571 



lo l&h&mttsaey of Biology 
3o CsUolaup Mologsr Sectieei 
3o BetSaesda lU, Masylaad 



PBS =■ MIH 

Iffi(Sivid«sal Project Heporfe 

Calffiadar Yeas' 1958 



?s^^e&t Titles Treaapaxent siad dif£tei«a eliaai^er 



l^tAo 



0%ls®r Issvestigst«?S8 G., Algiya^ Ko Ssaferd^ So WellMa 

Ce^siatia® itMta? MCI-905 

Ifeis Ieas« (©slsBdar jmr 1^53) a 
?«yfe®ls 2 1/2 
fj^QMsimmXi 1 
Ot&eys I 1/2 

Sato MeiwiB assd Qletm Al^x® 



Obiegfei-ya i f!»9 abj«ey»9' of tbes® stadias is, first, t© repeat 
l^^^^asats reportttd ia Hajer Fisidizigs aai, seoosad, to eoctend 

of Bdee «ad; tldrd^ to detenaiae vhethsa? the hOIc agesA or 
ether faeter or f&cftm^ esnseed the plMsm eell 
eeaaais to devel«i|»o 



mfftesisa olaabers are beiiig osed^^ th&t iffi^ 
gsaft^ oells are separated f^^a ^$ Isost 
vith pe3^9s of ktto«n aiaee. 



MaJQg fJadia gBs Isi 2 expeslmeoto, aseonsasz? ttssior tisime ^th tte 
M!lc~igenr^ pUeed iQ dlf fueicra ehasOteTS in loioe of staotSiesf 
Btx«2a» «ls^ the poTes in the BtambnoBes of the diffasim ohm 
0^5 to 0.3 ja ia eise the host tissues sem rise to mmm 
H ^m the pores vere Ool to OoOl p in sise plasm eell 



572 



mi"kik 



t£afeBO9X0S®9 oSiSi& p3r(qp«ki«B of tte silk agegcto 2iifiactioa of 
pl&ssa o«ll ttfflsosv £a <^ iatox«aib vfeotber or aote tSie leHk afpot 
plaa^B a»{r s^oi^ for little is taotm of the etioloejr of tliia t^pa 
of twor. ^fSwee es^rittsafts my aaSn it possiMe to find aom 
of i^e c9iasB&t±ve factors, f^ir^ter tie pliasan eell 
taisied iB tteesd «xperiaesiit0 are aose <«8ily 
previo^J^ existing plaewt c«ll tisoro is sdc9 an^icias stoAies 
on tMs t^^ of tiisor @eai«r. 



i^^-,** 



Proposed cotti{a|g> t Uto ccstiane aa infiieatad ia ob^ctiws. 



to 




di^@r«. 
to gsafta of tbesa tvo lines tramlS te 
placed ia Aifftisim elm^bers so t^t cssly 



MBttodfl i Difftoiflo cbBsaOsi&rs as® %®iag «s^ 



: At 7 Sa^fs after li^lBBtetioa tha saae differ^aees 
of Sxsst tissisee to 9»fts of M^ and Ixm lim 



cells are eiriS^st ^dsaf^ier ttts ^asfts as« placad ia difftasim 
or dirsetl^ on tlss Stost tiesn^s. 

studies e6S£tsrite^ to 



tas vBDnarsseasuuag os xxst int®s%c%%€(& 



^^5^; ^^«^ 




still 1s@ f€faB& et 30 

of cells ffiad the oodters of 
ma for tte cells of ^& two lisss. 
vill also %e SBde m a nsSiar of difftorsne lii^s of c@lls to me 

if a oos^relatioe eaa "b® ssaide 'tertwsea ^e i^ftslsotliffi^ of the ^si^iom 



PgajbOUMi i 3 «■■■ Stfsfiies co t^ rspci^tslati^ of the h@@topG£@^ic 
%is8»es' 'of lethsOly irrediated aice f oHotdag ts^tisent ^th 
tlood vith a M^ levffiOCTte eeswA, ms> Mood ins oU&io^ t&s^ 
aice iffloetslstdS vim th& &S^ c&soia&cmi, 

OhflectiTO t !S9 dstes@ise t3Si€t!ier tli@ hsmto^ietie tissuss of t&@ 



573 



irradiated laiee «re rep(^|i>alAted iff eells frcm the injecteid lQii:&emic»l.d 
blood er l:gr eeHa ^^r(»i the irradiated flucdia«lo 



Methodg s LsQkes&oid bleod flresa & im^l&i atxtdn <!s£ 
^cTpswSeet t£»» aieeo The HesderlButi gls33d stethod vm m&d to 
determine «^ether the rep0pD3£ting cells were ef the foreign straisso 

Ma^or gladtog;8« The Injected cells «>f the lesksatcdd blood re°> 
p^alateS''li!EE9\eBEKtopoietie tlBttctea ib& evexj aamae tested o 

reaaa rdM If Isakenoid blood had been £®w^ 



^'^^'^^ 



0rer ttatll host eells reeuperated^ it 
voold heve pro^idod An a|^ro&ch for & possible m&kw of preifesxtdisg 
the shortened sarviTal Assoelsted ^th the rep«»|mlati«m of 
paletle tLssaee l^ hoaiologoas eell@o 



l*PO^gL«a fa »» GI^>«alAil^a iia grafts of thjrrold i^lMid tisstie M ?3Mti« 
M'Ok Herein and Sq^oar UbUaiaa 



To ot«S7 1^« 'Taaeular pfitteam snd rate of flow of bl@@d 
Isstto and to detenoijie ^usther differenoes in fimoti^ 
after "vazioas trea^^sents ean be oorreXated idth -vasoaler 



of ohsa^bor oa$st3°itotl«ii hstre beaadeirel^ed 
masjr sake it possible to obtain aoviss f^sei vMeh the smte ef 
of oocpnsoles tlsrongh the Mpi3Ari«i oaa be esMimtedo 



Major fiad ingas Tbm arsangeaent of eapiUasles, arterioles md 
wSE^nsHSktiMi to thTfollioIes has be«s fmi to bo differ^ 
ti»t retried in the Htsmtisre for other aMmlSo That ie^ 
follicle does not hai^e its ohb separate arteriole^ eapillAxgr 



Signifleanoe to oaaeer z^searohs These ebservatioitti add to the 
ISSSSilg^ df A p ^ a i o l ogr o f the i^r^ glandi knowledge 
need^ In studies of seoplastie th?TOid tissoes as »ell as mneml 
tis8«ie9c 



Proposed oflwrse g 7o nake more observafiims >m oapUlaxy lengths 



after the adsdlaistratiem of thloturaieilc 
Vsseislar ehtnges f oUouiag hQrpophcrseet«agr asd sabsequasst troatasmt 
vith tS^rroid sttoOatii^ honaone tdH be atiadied. 



Ftsrt B ineltsded Tes X 



574 



SeriAl MOo N^^yij 



FHS <= MM 
Zsdi.Tldts&X Project Rspox^ 
C&loBidar Tear 195B 



^^^« Bosaors, Amrds, aad PabUo«U«s>s 



I^thie, a» Bos MamLft, Bo, ssid Woiff^ Jc, fiss PiawUcsaal 
Aetlid.tr of T&i^roid leografts isitMn dlff miion 

ta aloe. Espaxlaaist&I GeU B€«aa3<oha la 



575 



Serial Hoc MCI^U Ol 



Ic Labomto^"^ Biology 
So Genoral Biology Section 
3. BethQBda lit, Maiylaxid 



PBS <= NIH 

Individoal Project Reposrt 

Calendar Tear 19^8 



Part A„ 



Project Titles The relatioaa betereea cell oheoistxy aM 
cell etmoture 

Principle Investigatorj Eo Lp Kttff 

Other Inrestigatoraj Br. ?o E'vants, aad Dr. Ao J. Ooilton 

Cos^erating tfeitox NCI=U28(c), HCI-U06 

Man Teaz>3 (caleoodar year 1958) i 
Totali 2 
Professional} 1 
Others 1 

Project Deacriptioa? 

Objective s Izirestigation of the (grtochceaieal organisati<m of 
nozml «u»l nalignant eellfl; ^th partieelar reference to problesas 
of synthesis or redi^liestion of intraeellolar structures daring 
tuoor grotftho Although studies of biosynthetie meehanissis and 
of intracellular organisation are proceeding in nmiy laboratories 
and ha-re covered a range of noraal and nali^oant tissue^ in only 
a few instances have they been coordinated in asor systaeatic fashiono 
It would, therefore, appear useful to undertake a program aimed 
specifically at correlating the two fields of inf ormtion Ib a 
single -type (or in a lisdted nmber of %pes) of tusor cell. 
Ultimately, it hoped that siaiilar studies could be applied to the 
process of agent^^induced naligi^iat transfoanaation in defined 
^st^QS of noTBEl cells, should such ^stws beeoiae available o 



Methodg s 
prsfTious 



8 Cell fractionation nethods have been described in 
Annual Reports o The ultraviolet absorption syst« of 



the %laeo Model E ultraoentrlfuge is being used to characterize 
the sedimentation properties of cytoplasmle nuoleoproteins., 
Isotopio studies of the incorporation of protein and nucleic 
acid precursors into isell fractions tdll ei^loy available 
counting equipment o For tuiaor studies it is planned to n^ke vm® 
of the Noidkoff hepatana and of the series ©f transplantable C3H 
hepatonas now availablej, particular]y ®f th© ascites hepatcam 



576 



»2° MCI-iiOl 



129 (P)c This tujBor has alyeacfy been studied tgr electron 
laicroscopgr (Brb A, Jo Dffllton)o It grows rapidly sb &. zither 
hoEiogeneous population of large single cells (Sato, BeUcin, 
and Esoner). It is amenable to counting procedures and vill 
be ueeful in isotopic studies „ For inv'estigation of the tum» 
ever of intracellular ccmstituents (sea Future Work), appropriate 
isotopioally labeled precursors Hill be injected into ascites 
turaoz^bearing miee| the labeled celle will be rescued after & 
period of incorporation and a knoan nuaber ra^inocnlated into 
"cold" isdee for further groHtho Tumcr^-er is ftsseesed bj^ com° 
parisons of the radioaotitrity in naaterial isolated frcM the 
cells at the tliae of re-inocnl&tion and after -various periods of 
growth (sse ©og« ReFssa, et. &lo. J.N„CoI„ lit 31p 1956$ Seott 
and Taft, Biecheao et BiophySo, Act© 28 « U5, 1958 » 



Resul- 



■ta s These pertain primarily to the Pi-oject as described in 
"i^ml Report for calendar year 1957 » 



l)o studies of ribofficteleoproteins in ^oplasiaic fractions o 
ao In collaboration with Drs, Ao J.. Daltraa and To Moule (visiting 
scientist), the distzlb^tion of ISM meog cytologically defined 
mierososaal subfraetions of rat liver has been under studyo It 
i%a be@ipos8ible to define a centrifugal scheme of fractionation 
which proTides (1) s fra«s*i©!a in which RNA occurs priiaarily in 
the form of free ribonucleopratein perticle@| (2) an ergasto^ 
plaasiio fraction in which the rLbonucleoprotein particles are 
attached to mesabrsnesj (3) fractions which contain BM. but^ mk 
the basis of prdliainaxy reports from Z^o Dalton, no visible 
nucleoprotein particles » If further electron oicroscopy (nm 
in progrefl8)beara out this Ifttter point, the stu^ly will have 
provid«d «7idenGe that BM is a eoai^oneat of the manbranes 
thesBselves as well as of the auclsoprotein particles » This in° 
f ^nuatioa would be of interest in regard to the msehanism 1:^ 
which HNA participates in protsin synthesis, partioularly in vie^ 
of the fact ttet nuugy tumor cells are characterised fay a def ieienqf 
in nicrosesial sembrans oaterlal. 



bo la collaboration with Dto Eo Poters<m (Laboratoa^ of Biechemietsyj 
HCI) a. stu<^ h&s been undertakaa which is directed towards th@ 
problem of speoifieily of protein synthesis among the cytoplas^c 
ribonucleoinroteinso Efforts thus f&ff have been eacploratoiy-o 
Nucleoproteins have been isolated after dee^oholat® treatment 
of ^M?le liver nicrosomss, adsorbed on ±tm exchange columns, and 
eluted under controlled conditions of pH and salt concentrationo 
On the basis of their sedimentation properties, the eluted nucl@&^ 
proteins appear to have retained their particulate form. It is 
plamied to dsterssine whether chroia&tographically separable fraeti@sis 
of nuelecprotein can be obtained fz^oaa liver and f^^sn hepst^ssa scMg 



511 



-3= TOI-liOl 



if possible, whether these fractions are related to a^rntheaia 

of proteins witMa the rsspective celiac 

2)0 Stody of isolated Qolgi materials Qolgi Btructoros 
isolated frota epidisj^jraiia "b^ iaprofyed saethods of fractionation 
(ee© Aanual Report; j 1957) haT® been shosn by electron laicroscopy 
to retain teasfy of the norphologic&l cb&reoteristics peoollaLr to 
the is gitc Qolgi cmpleXo The isolated Bsterial is ooaientially 
d@7o^ ©jf'liHft. sBd is def ioieBt in most of the sm^es aasociAted 
with the other subeellular particles o It is '?ejy rich in phos^ 
pholipid and in &cid phosphatase aoti^ty, A psper in press 
ccwere the m>vk thxm faro 

3) o StU)^ of beta=glttettroa3idas9 acti^ty in C3H mouse 
Il7©r e@ll cultures (^tk BTo Virginia Evam) olt has been found 
that the sts'alns of liver cells developed \^ Hir. Svans exhibit 
beta^glucuronidase activities 50 to 150 tliaes greater than norasal 
G3E liver. The high aetivltias are naintained in the first and 
s@c£eid generation (in C3H mice) of tumors derived from the tissue 
culture cellSo A survegr of other rapidly groaing tissues derived 
from G3H liver indicates that the elected activities are not 
related aiisply to the rapid proliferation of the tissue culture 
celiac These findings are curious in that C3H liver is nersially 
very lew in beta=>glucuroaidaeQ activity. Lm, Morrow and OresaiBpan 
have previously ehemsi that the lo^ liver activity in this im^m& 
strain is genetically detesslnedo 

Siji^nif ieance for Cancer Research ? The centiauiag juaUf icatioa 
f^^to^Ste^rs^^^sr^^^ the concept that the malignant 
state m^ be understood in terns of the structural and biocheaasical 
ori^nisatioa of the <^ncer cell. St^»iies of the intracellular 
distribiddon of SM. and of the pt^ysioal properties (sedimentation 
and ohrcffiiatographie behavior) of the ribosmeleoprotein nay c(m° 
tribute to an ^deratanding of the protein fljj'nthetic processes 
of normal and isaalignant cells « The significance of the greatly 
increassed b«ta°°glucureQidase activity in liver cell cultures is 
n^t kn&m^ Tlie question laey be raised whether this phenonenon 
is assoeiated in seae i«ay «ith the laalignant -^rans^ossaation 
-^hieh these cells have undergone in tissue culture. 

JirWer^miB feaetioaation of liver. This stuc^r i» essentially- 
complete (alectron Mcrc^eosy is still in psivgreas) and no ex» 
t@s»i<SQ is presently planned. 

(2) Isolation aad colism ehroraatography of nueleoprotein partisl®s 

©f livsr and hs^tosaa. la view of the encouraging prellaissjy 



578 



"U- NCI=U01 



results Efforts to d®7elop the chroiaatographic separatlone 
vill be continuedo Isolation and sedimentation ftnalysis of the 
p&rticle9 will be done in our laboratozy; Dto Peterson will 
carry out the chroraatographs^o 

(3) 3et£<^glucuronidG8e actlTLi^ in tissue culture cells » The 
nature of future votk related to this observation is not yet 
detexmLnedo 

(U) Investigation of the turnover of intracellular coiq>onent in 
ascites hepatoss&o Initially, the isork «ill eoofllst of studies 
of the grovth rate of the tumor and the application of centrifugal 
fractionation saethods to the cells. It will be desirable to 
isolate nuclear, isitochondrialj, raicrosossal^ cytoplasBiic nncleo» 
protein (particulate) and "soluble" fractieaaso It is then planned 
to ai^lj to the intracellular fractions the teoimiques hitherto 
eB^l(7ed to Im-estigfiite tumorrer of Wk^ Wlk and protein in tissue 
culture and sairlieh ascites cells (see Het!»>ds)o Aiaong specif ic 
questions to be eosisidered over a period of ti»e ares (a) in 
the absence of a net change in the radioaetlTdl^y in total cellular 
mk (assuming the sitoation in Ehrllch cells is also foond in this 
tuti»r)s <ioe8 transfer (^ RS!fi.°radioaoti^-^ froa nuoleus to 
egrtoplasH oeeuri (b) is -Uiere trasasfer of radiaactivi-ty fr<aa "soluble^ 
to HH& of ribonucleoproteitt partieleai (o) is there turnover 



of protein and phospholipid in the jseaihrane (structural) poxitimis 
of Bdtoehoadria and saierosesiesi (d) is it possible to obtain 
evidence relating to the fomation of raiteehosidriaj, either from 
pre°ezlstisg Mtochondria or fron other mesebranous eleuents of 

the cgrtoplasm? 



Part B ixwluded Tes 



579 



Sarial Moo fflCI^l^ 



PES ■» MM 
Cftlondftr Year 1^58 



^^Ja &ja«es,, Awfflsds, and Fablicatieas 



Ktaff, E. L., md QsXtoB, A. J. Id«ff^l£li»ticn of aoleenlBir 
f dsvi.trlia isi hsoogeo^'tes and saetlflsis of mt 
li-WTo Jo d-teastroetars Reaeapeh, I» 62»73, 195? « 






&)9ssi3ag yswk idth isolated mtesialo Fstweedinss of the 



be pnblished in bofdc fo». 



580 



1« Ifttoratoyy of Btelogy 
2 c teiMsjfBl Mology Seeticm 
3° Betbeedft Ik^ M^tylasa^ 



P8S ■» HIH 
CalMBillar I«»y 1958 



Bist A. 



Priffieipal lavestiga.'to.rs By. Walter C. SchiaeMer 

Co6p#3raitii3!g lMit»: song 

ftot*ls 2 l/i 
Oyjers l/g 

l^^^^^iisleotM®e px<@s®i3t in some! assS. sasKse? tissues aM t© 
detessaiiae the aagtalnlie pa-yitHti^ in lAiiefe tlMgj fts« £si-««a.^»i aM 
thsir x«laUondihip t® i^fee s^bi^bU of die:^rxllsosiuisIei@ a@iS Cma.)o 



Metbedgs fls(6 ae©a?yx41tofiiaie eemperaais a3f® s^aswfeea ff«ai ti®8w 
pzoteifi aM BRA. ^ aaking eoM asid ex^xaets of the tissues o fbg 
tissuee as© ssas^wsd fSfcw the aalnale uoAe? azies'yicsia aM fE©s«s 
ianediateay . ' In liqtda nitsroesii to p-mv&ik antol^ie 



fhe lieoxTxltesiiiie eca^oiaBta ps%®»ist i& the a@i^ sol^iM.® 
er^m^tm as« isolated t^ lagaas of pa^T aM ioa escahangt @I»g<^ 
aatogx«qpt^ aoA paper eleetiophox^sis. ^^tie isolated eeaipoui^s as« 

identified h^ aeaos of ahsoi^ica a^pectss., eheai^al aoal^pseSj, 
ehxcantogan^hi® assd ele^txcphovstie p3?ope?tieep esA speeifi@ 
Goloi'laietxlis reaetiaa®. ^Bam pxogsees of th@ isolation of th« 
c5an|>ou»dg is foller^ed "by siiexeMologisal assay vith the os^^mim^ 
L» ftsidephllMS B-g6^ uhieh grpeeifioally »eqiii*«s deosysl'hosiaie 



^ 



findiiiga; (A) Assay pxoeeduse: Sisee a lax>es ainotust of 
tine e^^eoded ia this xeseaseh sust "be dei>oted to the xd^ 
e£oMologi@8i as.@aysj vhi@h ans s»0t ozily tedious hut also &am° 
-what eapsleious^ it -Mas desisaM® t© detesate® uiaetlier a eibasiical 



581 



^2= mi^hm 

Ei@thGd could Too dsveloped for the detez^d^^tioa of the ds-^ 
GX^rxhosldlo ctmipouads, A oolo7lJB@tric ro&otionj, fAssLda. iBm>l7«8 
the treatsfisnt of the oolEjpomd tilth poriod&ta f olloirad bgr its re- 
action Hith thiobarbituric a@ld, ms tested botsaose its sensltlvlly 
apprcach®d tb&t of the aderoMologleal proGedureo Despite ex° 
teztsire efforts to nodif^ the nethod so that it ootOd be used for 
our purposes, it vas finally abaadoned because it p>ored isspossible 
to devise & method in which the deco^jrKl'boee of all p^risddiK® 
deoi^^bosides couM be r@scf7@red qiuaatitativelyo 



(B) Ass^ results s In the p£>er^ous annual report, a 
modified isdorobiologisal mmj procedure, tiaa described bgr msmm 
of %hioh it m.B possible to deteradao siJaultaasoasST' oa the same 
tissue sssple its cozitent of deaomaeleosides, deossmucleotldes, 
ead deoQs^uoleotides that require a ppelialQBxy «myiatl@ digestisa 
before th^ casa be detectedo The application of this setbod to 
various tissues aiad bo(^ fluids has bee& coae^leted and the results 
published (refereace l)o The- results iodioate ttsat high cosa@@£2» 
tratios^ of deos^'smeleotides smd digestible deos^ucleotides ar@ 
ehsrsst@ri3ti@al37 i&xsM in tusors aad oth^c tissues isMeh are 
actiTsly g^c^jsg or prepariog f op ^s^s^ho 

Co) Identificatiosi of deo^n&ucleoftidess Tm digegrtible 
d®©z;^u@l@otids@ observed to be present la ffiztracte of the No^lkoff 
hepatci^ Mv® h®m purified aod ideatifledo Both caqpouads pr&ired 
to be deri'^tives of deQ3^cgrtidin®=|»°diphoe^h»te (dCDP)o ia oa®^ 

the tes^dml phosptete of the dGDP wm esterified' »ith ettenol^djas^ 

while in the other^ @h@liB© ^^s so esterlf^ C^fereme 2)o 

(B) teigia aad fate of bleed deos^^tidiaeg We have pa-e^ 
vi@Q@3j established that the zmcleoside, deossrc^tidiae, acoouoted 
for esseuitially all of the deosgrrlbosidic aaterlAl ptreseait in the 
bleodo Where this desssr^idiBe origimtes aad idsat its fate ia, 
are istrigulBg questiossso In order to test tdaether the bloed 



deo2^<^tidime case frea the food ®&mma&p rats vera either fagt@d 
or fed a fi!jrathetlc diet devoid of deo^^rslbosidio aaterialo fh© 
deosjG^idiiae lervel ia the blood of rats fasted 3 d^re vas r®» 
dueed about 50 ptsr ceiat vMl® that ia the blood of rats fasted 
6 da^s^ w^s @lsmt<sd sossei^sato la the ease of the rats oa the ^tlsstl'd 
diet, th0 blood deosgr^idiae level failed to shew significass;^ 
reduetioBS after >32 dajs oa the distc Astother possibility that 
«@B cosBsidered ms tlmt the blood deo^qytidias vm produced t^ 
the iHfceetiml florao Th® blood of a gem free rat, hoswrer, mm 
f©«S3d ts ci^tsia aboixfc 50 per cent of the aoxsal level of deoi^s^tidiasc 
The results of these ps-eUMs^try expeslmsts lead to the coaclusioa 
that the tissues @f the aaSsal itself are the source of a large 
^rt of the blood deo^^idiaso 



582 



-3-- mi-km 



A atKSK^r of th© wriiMsy exoration ©f deeoyyibesidlie 
aaterial Cpr®siB»»bay deesyeytidino) has iadioatad that l«rg© 
amoonte &f ddeaqr^T^idin® mr® exo^eted )^ the xmt <m<sh dfl^o 
Prean 200=1000 naicregrffiajs of doecsyqytidiae as-s yeoerared in tfe* 
urins @@llecst«d dmi3j fram «»oh rato TMai maaxM that tha 
de^xsrcsrtidine iJa the bl©@d is @le%red between 2 to 10 times 
eash dffljo Farthenaera, sine© the bl©®d dees^sgrtidiae apimresatly 
©ilgitmtes in the tissues;, it my be @«l@tslAted that the uriimsy 
ex^etd^i &£ this @e8f»©«nd @@s°reepa»ds t@ the death @£ 100^500 
adlllea @f th® asiljml«s ®®lls dailjo 

(E) Is®t@pi@ ecKpesdatentsg In order t® rtradty' sasr® isisBply 
the de@iS2rrib@@e €;@st%iM3sg ©^i^msd@ in the A@id s@ltible f^@tim 
vM.®h stre i»temedliit«@ in DHI. ^imthesis^ it w©iaM be desirmble 
t® sdsdMster t@ the anisas&l ©r i@ef;lftt@d ti@@ae^ a spesifi© 
labeled pr@@ts*s@,? @f !M 8@ thmt all the interKediate@ w@uM be» ^ 
@@raa Mbeled auod thiis fa^iUtate theii» date®ti@»o Decsxgr^idiBe 
w@iiald be an ideal p^e<3vs©r fer thisi pii?p@@e beosuse it i® ia^ 
nmrp^mtsA &a^ intei IM ssid besause it appears in both ^rriisidiBa 
ba@,ftis ®f the im mlm^Wo SiB@© labeled daca!3r<s^rti«Siiie i® a@t 



iilabl© ©©BanareisUyg it ^« negaesaxy t® desrise a aethod f @r 
Bakiag ito It has been possible t© prepare C^labeled d«©sy^.idia® 

wsiag aa extsmofc ©f Lo a©id@phil?s@ R=26 as the sosere® @f a tffm«»= 
deeayyibesidaseo Mlligyaai qissaKtitis® e^, the labeled (se«E|3@TOd hm^ 
b®»B prei^red ajsd psrified md ®y® issiw bslag used t@ stwi^ WA 
^TSstheM^ o 

liqigig^Si® ■tgy.91»g.Z.^§Mggig It i® saw geawalJj asesptwl • 
tSatxhe redQ^licatiora ©f celmLar JMk is an obligatoiy pr«° 
requisite for cell dltdsiono Since eosntinuous and unemitrolled 
cell di^sicsi is the outetandiiog feature ©f eancer in its 
malignant phases, the study of TM. ssrnthesie is a fundamental 
approach to the solution ©f the eaneer probloao If the meshanisra 
©f DN& iss'nthesis can be dstemined and if the faetors InrrolTed 
in the ccsitrol of DM synthesis €an ba defined^ then the control 
of cell dl^iaiion and of tumor groirth should be placed upern a 
2^ti@nal basis o The studies described abore are st^ps in this 
directieno 



jj^epoaed 
soluble ( 



(courses: The isolation and identification ©f the aoid 



deo3Qrxlbo«idi(8 ooiapounds «dll be continued^ preferably 
in tissues trhich hare been exposed to labeled deosycytidinej, s@ 

that the detection of the coupounda will be facilitated. Preliminary 
atteaipts in this direeticn hure not been vesy suceeaafulj, hmemnT^ 
due t© the failure t© obtain c©neentrati%} of the administered 
deoijtyqyfcidine in the Ussues to be studiedo It is hoped ttot it 
Mil be possible to oversoae tMs problaao 



583 



studies of the urinary excretion of deosyribosidio cco^ounds 
will be continued in t»o directions s one ^iiraad mt deteradning 
ifliether deoxycytidine is the compound that is being excreted, the 
other «t detexBdniag whether the excretion is diadnifthed when fch" 
aniiml is supporting the growth of a ttssior or o&lled upon to 
replace a large part of its livePo One of the iH^Jortant queistions 
to be decided is whether the aninuil uses the deoci^cytidine it 
alreadty has for the f omiaticm of new cells or whether soae other 
precursor is used for tnaking the ne^eseazy Wko 

It is hoped that it will alao be possible to study the 
utilisation of the two unusual deeogmucleotides that M^e been 
is©lat6Ki frora the Novlkoff hepatomaj ioOo, dCDP=9tbanolaiaiae 
and dCQP-cholineo These experimfflxts will depend vcpon the 
eynthssiss ©f labeled foi-ias of these eoi^oaadSo Woric ia this 
directic^ has already begun o The a-vaiXability of these &wi= 
pounds in labeled fos^ will also be of considerable use in deter- 
ndning whether these compounds exist in other tissues and what 
their concentration iSo 

Autoradiographie experiments are also eemtemplatedo 
These sttKiies are based on the recent fii&ding of others timt 
JMk ±B aynthesised Isy soluble enaiymes obtained from the ^^topla^s 
of the cello If this is truej, one would axpest to find labeled 
IMk in the ^rtoplasm of the intact cell before it appears in 
the nusler^, The present experfjaent ar© desig?sed to test this 
point o 



Part B included Yes X 



584 



Sorfisl Ko, NGI^1*S 



PBS -^ iflH 

ladividaal Project Heport 

Calendar Year 1959 



Part Bt Honors, Awards , ajid PabMeatiens 



Rotherham, Jo aad Schneider^ Wo Co Deo^^p^bosyl omspotmds in 

finimal tissues „ Jc Blol„ Cheuio 232 « 853='8565 1958 

Schneider J, Wo Co sad RetherMia^ Jo Phosphorus coimpounds in 
animal -tiammo ?Io Deos^^t^idlns diphosptote 

eholins aiid deo:iqrs3rtidlne diphosphate ethanoXudne 
in the No^lkoff hepatoJiAo Jo Biolo Ghamo 233« 
9U8=953, 1958 o 



585 



1. ICabom^oyjf of Mol©g^ 

2. ^■miraX M.Ql&g^ B&stlasSi 
3' 3Sffi^beffiia 1^*, Mas^iMsst 



PBS > KDB 
Cftlendaz- 7«a7 19^ 

Piarfe A. 

Pzojcet Title: Studies (M& l^^bo@ytie twor». 
Friiasipel I&v»s%iSB.tor: ^sbbi Slielton 
Other lBTW8sH^i®a.ter; .Maiy E. Mee 
Coop«xatisig l^tfis 



Maa f»^ CealezKaax' ymw 19^) s 
Fxof@8Bio«Qal; 1 

Objecting; ^Sie @«ai® ef Ija^hoeytic Imiie/gsKiss @f tbe s^^^ase sbev legai^ed 
■WBsia'bilit^ in tiMgir @spa@ity t© iavidg £ioat tisstag® lo<sat«d &% e£t@@ 
distant fsoa tuner fa&cv&Atimk. ^s@ si^svi^raO. tlaag @f the lso«% is sos^ 
xelat«& vitii ^a® @x^@srt of «Ms isviusios. fb@ %«l:avi©r of tSae @ens ia 
an infe«gz«.tieffi of Molegise^ f^toars maeb as gs^owtli 3B.t@; atstsiteii^^^ 
l@@caBOtioii «&t«, axid iammolc^sel p¥@p«fti@s. It is tb© pti^pos® ®f 
studiest to attcs^t t© gos>l @u!t aM emscdssm m^w^^lj mm @f 
fa;6to?s in 0T&IS9 to assess »&%« •pg&pmrly i^smtr i%iatios3@Mp t© 
tbi ii^'msive igapasity ®f tb@ ffiea.i|p»ysr^ l^mpho&y^^ 



Stetanaaa ; lb® basie M©l©gi©al t©©l S8«®i to tlsess® s-^aaie® lis tSs® aseite® 
t^tSBOX'. Shs^Qiue^ it£ ■mm, tbe e«lls of tb@ tueo? cas% %« (£S3m@mt«& m& 
^ta eem be «3^x«ss@d on a "p«? e@ll" tests. Ei#!t afi€lt®@ t^sKSf® aw 
ewar^ntly teiisg ettsfiiea Cl#l^ :^, I#3, ^» OSlJDj Elfe, 12 djesae, aM 
P3534 

Speeifi@ ai£!rt:hM£S a£«: 

(l) DovOsle difj^asio^ cban'&^s'; ^i« sm^^ of comtsustioaa asid 
mtieeale for tlJ® w« ©f tlse clMMibess as« descyited tis tise 195T ®Mma. 
5P^g)@$tc The eeUs a«d fl'oid ia the diffoaioa cbasatens 'wese ai^lyasad % = 

((£) B»p@r elm^ato^aphy » for l!^TO3OTS©ltoB as the uasiqw <s«m= 

@titiijii)!!£l' of ceHa^sffl^ 



586 



(3) Hi«tocbe8Bieal Meijbods - fos> coimestiv© tissiie and for 



vere Bsdssc^aiBeii in perfusion ebembers 'WbialiL 
pexnitted the flold to lie ctaasiged vHsboufe 
distur'biag tlae cells. Tiie ebaators are forsBEsd 
"betnieeB two co'vexvlips aasA a siliee)B®»xubbeir 
eaalcet Ibb. thiok. Excellent prepasaticmsj, 
8t]ita.M>e for pbaa® pbotogxapby, axe oMaix^ 
1>gr gzoving the oslla in a slieet lb@tween am agar 
lAock and the tcp ©ovsz^slipo MsitloQ pi.@tuifs 
films wenw es^osed at the rate of o»e fswee p@i° 
8«eo«ifl i& a e^ua^ of ths» sat® and ehax«@ter of 
loeoBotioa& of th® ■mrijms Ipn^hoc^rtie 



Besets t 

(1) Cell -wol^sMe^hai^s aiatog ttaaoy g acowfehs C^ae ettaia^ @©a^@t@& 
and xeg^zted this areas' CpiM-ieatiion #3^' Fast 1 of this sn^ost) sosasessM 
the shane@ in 'foliate oe@t2S7lag Iji the ascites t%»op eeUs S^sxlsig tteif 
g£Oi«th. fbis WLS a di£«@t @utgs<ovyi of a stui^ offi the gx^o^sFth 3fat@@ of 
ascites ti»@s« (pvOilicaticffl j^^ fast B). F^en a pxa/stisal a^p<s@t9 it 
Kas shoiffi that tsital @ell "folwm @an ha i^eed t@ asses® as@it<*s' tmior 
gzoiryi 'vithin eestain defined and xteeog^sable l^sits of ae&mms^^ 'M 
addition^ the atisij ps«®ented the fix«t eea^zete e-videnee that th© 
ai^ssage tiwox' cell de@xeases in 'voliae 'eritli ineseasing tvm&T a@i° 
MeaetuEiaignt &S iadividnla eeUis showsd tliat as the tssor ^^ oM^ie^ 
the msKatse? of isaall ceUji ijocsf^saaed idaile the ensBiher of l&s^ e®ll@ 
d@@3'«aseido Bsaaati@ insiz<@ases in @ell -wlmm assGs^pes^ ^m issms^M-^- 
tion of an oM tumor into a nev hoit. f&¥ mum^lms the a^imssge @@11 
@f tiasDS' 11210 lafs^mse® 262 w^ ia -^elmm diaring the f ls®t gJs- tewre 
in a i%»«r bo@t. 



C2) @a^«th ,of masal eellg ,and tmer eelle in dimiaiee ehm^m 
sM^ thei y gelatiog^hip to ^teh othey g Wrmk the cyt^o^eal poisst ©f 
"^mg tlie ascites tia^r is mires' a' "pore'^iaeos' hesaiisej, In aMitiona t© 
the tua»r @ells, hetae^n 2^ ami 10^ of the seUs in ra^peffisiosj. in t3^ 
peritoneal fluid axe noxeaal celle»»l^i^hos^te@j, sDoer^hages, pol^^- 
sDOsphonticlear l@iasocytes or seairt @eM@° When ascites ti^or eell® %?« 
plaeed in-lo diffusion ehaaher@|, they &r® @l-m.yn ss&m^eiMM. t^ th@ 
mitm^ eells aM hoth tripes of eelliB ^^mt^ Parine the c&wtsm of ©%° 
perlaiKSfes desifpsed t® test the ability ©f ttasor eella t© p@@@ tha!©M#i 
mm^t93amB vith diffes«)nt pose sixes ^ it vas oheer^ted that '^te ext^sst 
sad type of grof»th ®f the noiawl eeai« differed tsa& t&mT -to tias^f o 
Is the ease of ttoe "invasiw" tmovs um.0 aM l#g, the aaesasal ®®llg 
sad tyi»©r eellg agpe^sed t© grow iMepeMenfely f roK @ash etfeer^j, ■«M1# 
ta tte ®&m ©f tte rslatlwly "iMi^intmsiing" t»ai©M I#l assS P3^3i) t3s<© 



587 



-3'-' Wil^M'^. 



s©S3»al e®ll® iSiffeyssffifeiatM lis*© fi'teoMaffitSo With th&mm ;iLAtt««> 
tWBKsyB al.@©s -felie eoaaslatajCEsy of W& f3Md is the »sl3aM"b®r@ ww© 



b3ha^@3!> of th@ n&xax&l cells ix^ the ^Temmse of tunsDr® (e@U.s %©'- 
@aaas« it appjsasw that it asay haw eoms Iwiiriffig on the iamsi^ 
p3pG!pextieffl of the %vBsBfm> fhe q>aesticra t© 1» esmtet^ iasy =■= 
tao these tvmoT eells ha've an isfluessee v^n tte laflms €^im @f 
@6lla®£m essd sBC»K3po]L^sa<sisharS,Se<»i2st?acelltilar nat:rlx»1>y »o^gia. 
c®ll©, fo tM© «M ths fiallowis!® i«mslt® Imv® hem ©telaia®ao 



(A) @ge>wth of BOMBel c fells alogifes Vhm. ttoissml ■p^ritm^e^ 
e^UM (in 1^ foiicnilag psqpcs^i^te^-'^ljpBi^hoe^rtas^. T^Jtj aae^pSMipgi^ 
25^1 p©2^«JOsplj©nael«8dP le^Ki^jr^Ss &M sast e«21»^ lf») as^ gfew® 
M aifTtssioQ ehsoalsesSj @ell Siffesmt^latloii oceio^sio Ttmig af^se 
six i«®^^f -yss l^ffiphoey^s h&v« Hisi^ppQasfed aM the g^^s^wm e@^i^u£>@ 
aa a ®b@@t of fih^oM^aots^ i!rl«r^M§!?@®& Ik the sieshe® ®f '«M.@l£ amj 
bg imst cella aiai plasma, sells » Ifeesophagee ax« still pse@@isfe sM 
grew a® i®©Iet®a eells isi areas of the filter ss% ©ovea?e^ Igr fil«®= 
t>la®%8o 

l^xQ'xypw®liss& aaalysis iMiea-^@ that coUa^iesi i@ ps^isists^ 
lif- tha@@ eeU® in saomi^ 'oas^rizsg fsrora 0^5 ag. te 11 »go p«r a&o 
aeeosdisg to the lessg^i of tSse^ tSmt they haw h«en gz^mn ia tfas 



CB| Qa'ow^ of asMwal c«lla plw ^i«er e®lls ; WTsm th® aes"" 
phelogie pbisst of •^«w» aM with ti»®'' ait of '"ligpee^il staizsSj, it 
appeals that ^^s@ sao^isal e«$lls that aeeoa^pmigr £1210 aasa £^ isct© 
the eh^sSneins do not diff es^astiate Inst x«naia a» seattexied 
aaisx^ha®»»lilEe eell«c fibers a£« ps^asnt i& 
%isl tlfesj ao aot seem to Ise asmj6iat«d vith eellso 



HiuEiy of the aoxasal sella aseonpui^flfiig l#l and P3^3» 

do diff ex^sffiliat® Isrto sheets of e^Us iema«g&iUs!^ fi'bsohlasts 
il^2e asBbgs of these @elle thie t^saof eells eolle@t. 



B^xox^xolise is ^s^b^b^ t& eha^'bez® idtere I#l aM UtlO 
ai% gf^^«& iMt to a eosssidexsM.^ leseer exteist thasi '«lth aosaisl 
cells aloaeo 



{3) ^flsae <e<altiKFe @x& claanatosfflajply of l^TOhoeyfeie twsssig' 
g®llg; ^»s»e oslt^Bfe of the aaliggMist Ijp^lioeyte of the isouse 
is a3Q esBgqptiossally difficult tMeg t@ aehieire. O&r ■pr^rl&m, 
attgspte to saintaiii the cells ^ vltgo %e^oM a peslod of a f €^ 
bours had aset is failus^. IMs year, iKWse-s^sf^ tgr sslcisg «se ©f 
msy iata «i tb® gi?0wth of the ts^^OM ^ tI^, "WI haw 1s®m aMe 



58a 



xtwtsiJSiBly to sB&iatffldsii eultwapsa of 11210 oM I#l for periote of 
tliB© usp to 72 bovons. !!Ms -was accosspliBhed Tsj taking tbe csU® 
f ox> eultmilag fttm tSie aioinAl whlLs thay we3» in tbe log pbase 
of groivth. Tl»e»lap8« phDto^sspba »hov tbat tto laode of loeo^ 
awtloo of ths cells of U^O i» mstly aiffermxfe ficm tSmt of 
the <£®lls of l^. Bates of lotsoaetloia h&'v® ^et to be cal<sulated. 

S igiaifleaiaee .^..^^^^j^^^J^^j g^yg^ ' ^I^^ tioxk of ssRsisjr isves'tl^'tosfs 
hs&s shown that the lya^bGQ.f%i& iMmSssmSjB^ of aise "viery dlcuml^ r^^' 
@@sMe ttas l^^sg^hoeytie lenk^sifta ia aas^. ^le ^^ixther eltaciiatimi 
of the ehaxeetexlsties of tbs Sieea»@ ia Mc® sbouM th@retox^f 
ha*^ a diz'^et hsarlssg v^em this Sisaas^ in isaxiL. 



^""^ '^ ^ ^- ' SSS; « x««.M«.«« or t^ «^= 



tienshlp . h g tea w en sioxwa p«?itoneai @«lls aM asaligiaa^ l3«gp)lxt@3rt«@ 
gztsfim in aiffusiosi @ha»tMirs vill @mtim@. (2) Sata tm tbg 3pe@& 
aaS waoSm of losaas^ioa of a»l%aas(& lys^ho&yim will Is® @oll@et«^c 
(3) An «l@et2«ffi nieToeeep® isttsSy of the MlatioxssSiips 'b&t-y^sk -^ts 
aaligoeM l3niphoe3rt@ asS the @«lla of th@ tiMmam filings'^ p^i3i@i<@a@} 
that it haiB ianraJal. C^) A staSy of tSs® @l«strteal passgerti®^ @f 
Ijpai^heeyfei© e®!!® fey @te«rwfctiejs of their ffielsility iu »ja ®l®®trf,© 



Bs^ B SaslMea f®s X 



589 



serial, ms, KIJ408 



PBS ■= MM 



Bairfc B; EeaaoxV; Avara»j» «x^ P«aM.&catioEQS 



SbsltosEi; E. and Hiee^ M<B. Stuaies 00 aaouse IjfnphojBBas IIo Beh&-7ior 
of tbx<e@ IsfsaptaOBSM in diffusion cbesOKTS la relation to ttaeii^ 
ia'msiTe ©apacity in the best. J". Kat. Cteeer Init. 21: 137" 
162^ 1958. 



of 'tuBOV^anS noo°°'tuaoF calX pcpula'^ions cuxiog gjccw^ of 

tlm»® as€it<ss l^isiQbosaas. J. Hat. CaxsaeiS' X£ist...2i: l6>192^ 19^- 



m.Qm, Mo E. ana Sheltoa, S. Meaaiseaeat of tba aim%<a^ aai tSie t&lal 
e«ll "mlvam dmrijsg ths g3rawtd& of as@it«s ^mo'm^ J. liato 
Cancer Inst. SI: 9 19^< 



590 



SQ/rial MOo^,^_^Jiig Part A[ ^^ 

2o Gener-al Biolo^ Ssction 
3o Bsthesda, Maxylasad 



Individual Pi^oject Report 
Calendar Tear 1958 



teU 



Project Title? Biological and biocheiaical stisdies ©n th® 
S^JI mouse leakeaaia •^■iriaso 



Principal lareetigator? JoBo Mol©a«gr 
Other lovestigatoras None 
Cooperating IMts! Mone 



Man Yeara (calendar jear 19^8) 
Totals 1 
Pi-ofessioml? 1/3 
Otheri 2/3 



P^jeot Sessriptioas 

P^4®^.^?®gi ^ ecEiprehensiT© stut^ of ths Molo^ mm bi©= 
sh®fflislE^''*'ol^''"lihi S-31 laukeada virus, with a view to^^rd sb 
U!id®r8tandis!ig of the nature of the agent aad its relatioB to 
th@ neoplastic process o 

5^=i^^.,Q'C^^^-j.-J^% ^^® leukosda virtss ^ms @stra.et©d 
tT&& "Sarseaaa" jlf •slttcnlSiH^ been «si?Ti@d in strain A^ laies for 
195 tumor generations and implanted into BALB/s/An saiego Th® 
whole cell passage ^as carried through 3 ttsaor generations in 
this jfflouse strain befor® attea^ting virus ©xtractiesio 



591 



-2» MCI lilO Pas'tA, 



ExtgRction, aiKi Filtrata.ogfo The ps-jxiediarss found sjost 
suitable few? estraction of thiS laukemia Tirus, Srm. ©ithor the 
Sarcoisa 3? or th© lauk^jsis tissues induced tf^ inoculatioii ©f 
cell fr@® extracts of th® S 37, are similar to those fouM 
effective for extraction smd concentration of the ibus sarc«®a 
-virus (see JoN.CIo l6s 8??, 1956} o 

The virus will pass through bacteriological filter candles 
of th® Mandler, Berkefald and o02 porosity Selas types o 

_, ^^^'yi^g^^^.^f^a^- Filtered and coEieentrated viz-us 
extracts of ieuk®ga.c tissues are carried in passage in n®;€bora 
Cc 16 hrso old) BfliLB/c/An iiice„ This passage is now in its 5th 
gensrationo 

The SarcOTja Ji and the T?irus induced leakeaic tissues are 
carried routine^j as i&ole cell tracsplants in csi® saonth old 
MLB/c/An miceo The induced leukeisLc tissues ssr^e as a -^irus 
source naterial^ 

Ma^lor gJadJMS * The project described herein is in the 

early" '^eveiopiBeicxtoT - stage » Only exparimenfeal results of a pr@° 
liminasy nature are available at this tiis©. 



A brief sussoaxy of these results is as follows 

^Taao r ^ p r pdncin^ ^ activity of ce^ free,,, extracts o^ 

A) Cell fi^ee extracts prepareff %' filtration from S.areom3? 
tissues^ when inoculated into newborn Esiee, will prodac® 
leukania In 85-10051 of the aninalSo The mean latent period is 
six Bjonthso It is interesting to note that extracts ("cell fr©®")^ 
prepared tgr differaatial oLtracentrifugation 023^5 will produe© 
leukemias with a aean latent period of 16 



B2, Cell free extracts of virus induced leukeraie tissues 
prepared ty filtration and differential ultraeentrifugation &r© 
apparently' Kore MologieaUy active in that i^hen inoculated into 
newbcm mice produced leukesaias in 50^ ©^ the animls isith a 
mean latent period of ? H®akSo Within 10 ^®k® follo^dng in&= 
culAtion 100$ of the test aniimls have l©i:&@E!iao 

It is this aatarial that is ffiairataiaed as a "s®l@ctiv®'' 
passage stui^o The hamatopoietic organs of first snlarf^ oT 
a. -'tuiit groi^ to ©xhibit macroscopic si^is of laukssais ar® 



f^^x 



-3= WM hlO Pert. A. 



selected for further extraction aiaid cosicsitration as a call 
£ree (virus) passage st!J(^o Ths purpose of smch a stusfy is to 
increas® the ajnoiKit of virus ±n th® soiirc© matQyialSo To date 
this iacrsass in virus contont has lEBanif ©ated itself ^ a do- 
crease in the latent period, of reinoculated aniBsalSy frosa the 
erkrenies of 8 months to 7 



Ags and Strain S^oifioity Studieao Other iisrestigators 
have aSimt^rtt^^agi^s^eB^^mWi ags specific in 
regaurds to the host aalualo It was conaidsred of interest, 
thereff^e; to design and earzy out ®2£p®ria®nts tsMch isould 
determine the host specificities of the S Ji Leiakema Triraso 

With the cooperation of DTo Asaior^oata 1*0 so kixadlty 
supplied all the test animals for this stadyj, th@ follosdag 
inhred strains of saice were tested for fflssceptibilii^ to the 
agent: mUB/c/Axi (control), C5?B1, DM, Hin, CJH and GJH 
agent fre®c. %brid aica resiating Srasi. eross®aB of these 
strains with MLB/c fesasles were simiJjirJy t®fflt@d« f h®s® 
sxperteents are in the ^r3y weeks of the tsB©t period, there- 
for© no definite results ©an be given at tMs tissp 

The age factor in the host respoi^© %jas Ksasored in E%LB/©/Ars 
!5r|,«>6., .. The inoculated aaisials ranged in ags fp<m newboi^ ("^ 16 brs^) 
through 2 months o Ft'^iaiiary results of the ©apsriseat still 
in progress shew that iaie®, froa birth through on® sioafeh of ago 
are susceptible to levakeiaia iMuctioa 1^ ii5oeu2stion of the 
virus o However, th® latent period of the older anisials group 
is stm&ishat exteesded in tiiaeo 

^atolp^ and^Patfeolo^ 

Macroscopicaiay the diseased saic® present th© tj^isal 
leukaaia piotar®o The spleen is greatly ©nlargedi th© liver 
appears enlarged and smewhat jaottledo The peripheral 2j^h 
nodes as ^eU as the sassenteric nodes are greatlj enlarged o 
The thipsOT is usmlly infiltrated to the extent of filling 
approsiflBtelgr t«o°third8 of the thoracic cavity o To date no 
ii&@roscopic parotid tuiiaors have been obs©rv@do 

Morosoopicaiaar, as described by Dto fhelffia Dtnm^ th© 
ijsrolved tissues lav© the distinguishissg cb^ractsristics of a 
g®a®ralised lys^jhocytic leiakemao 

All tissTOs obssrvsd nhether indaeed t^ idsole cell ijig)lant 
or by inoculation of cell=f ree erfcraet e:ghibited eisailar 
features, cimracteristic of a generalised 3^iapho<grtic neoplasMo 



593 



.=U- MCI hlO Part A„ 



XoukeiBic lys^hatic tissues carried out 1:^ Dto Dalton iadicat© 
the presane© of nuEsarsus virus of -yirns^lika particles in th© 
sgrtoplasra of the ol'faeted colls o Pelletiaod EEaterialj pro- 
psrad bgr extraction, filtration and ccsiceistraticn of th® 
leukosdc organs, ehov particlos identical to those found in the 
whole tissueo The particle siae is appreaisately 70 mUo Th© 
viroplaam is bound ty a liadting raeiabraaeo There is found Cal= 
though not alssays) a more electron dense center or "nucleoid" o 

Propeaed course of the projects Contii^ent upon the availa^ 
bilitgr of the alrea^ apprWeci iab<Mra!toz7- space and additional 
personnel^, the follo»lng courses of stu^ will be undertaken. 



1) Enhancement of the Tims in the tissue source through 
continued "selective'' passageSo 

2) Concentration of the extracted virus ths^ough the 
application of issproved pl^ysical and c^ochmical t©<cbniqusSo 

3) Quantitative biological investi^tioas coneersed ^th 
dose«respoQ3e studieSo 

h) The preparation of 3ta.jmigrd_ y±srm_ lots for ua® in 
quantitative biolo^^l assd biochesnical studies., 

5) Biocheadcal investigations ^th a view to«?ard a aore 
coB^Jlete understanding of the natrac© of th© virus o 
These studies isill include the role of proteins ^ lipidss 
and nucleic acids in th® infectivi-^ and isetsbolisE ef ■ 
the virus o 

6) Bio=pl^sical stMies ^th ©laphasis on th© stability of th© 
S=3f leukemia virus under vssrious physical and ehCTdcal 
envirosaaental conditions, ice„, thermal stability, stability 
in various buffer media, ©tCo 

?) Continuation and extension of the age aad strain 
specifiei^- experiments described hereiao 

8) Antigenicity studies in an atteB^Jt to dsmonstrat© 

an immunological ralatimstep to ©th^ )mam leukemia 
agents o 

9) CoHabocrative tissue culture studies are planned isrith 
Ro Ac^ Hanaker of tj^is unit« 



Part B included T®s No X 



serial lOo, j{j.Q .,. J^arlJo^^^. 

lo LabQrato32y "of Biology 
2o Qamiral Biology Section 
3o B9tli®0da, MaxylaBd 



PHS ■= NIH 
IndiTldaal Pro;)©et Rapoi?t 
Calendar Year 1958 



Part A„ 



Project Titles Biological and (^rtooh©Mical Studies on tha 
PosBibl© RslationsMps of ¥irus®s to E\smn 
Neoplasisso 



Principal Investigator: JoB, HoloneoTo This is one phase 
of a collaborative study with RgAp j^^ot; of this section ajKi 
Ro Bo Cottch of the Surgery Branch,' "COT 

Cooperating %iit3t 



!&n Years (calendar year 1908)? 
Torfe&ls 1/3 
ProfessioaaU 1/3 
Oth®r? NoM 



Objectives 8 The cwer-all ©bj©etiT@s of th© stw^ are 
compl^e^ de-tSied in reports sutoittsd t^ ths collaborating 
im^estigators mentioned above,, Tha mors iasmediat© objectivss 
of one ^©cific ptose of the problesa are as follows? To 
study, through the application of bioehei^ml, physical am 
biologi^il techniques, methods of separating frcss host tissues 
viruses or -vlrus^like agents which through further testing, 
alight be shoim to be the etiologic agents of certain h.vsm.n 
neoplasEJSo 

Methods BBPlcsared i Bryaui et al, in reeentay published reports^ 
have slio«n tlltat ibus Sarcoaias induced by low concentratioas of 
virus, will, upon extraction oJ^ the turaors ^|M onay low coa- 
centr&tions of the -^Lruso Fraqu©nt3y no demonstrable virus can 
be recovered frmi such "low dose" tumorso The techniques ®sipley®d 



595 



,2^. HGX ViO' , Part B. 



for the extraction of these tumors Wdsrs steiyiar d laboratory 
isethods dasignsd to p£>oduc@ cell free hosnogsm^s « 

It ia reasonabla to assume therefore that the failur© 
of otl^ra to demonstrate a riral association vith obtain 
huaan laalignaneies is due toj a) the law concentration of the 

b) " ^ 



agent in the host v:ts8ues and b) failure through the application 
®^ stfM^ftrd extraction procedures j to concentrate the agent 
to a sui'f icient degree to allow for further definitive testings 



We have emplegred {^©chemisal teclmiques of cell fractionation 
combined with the Mochendcal techniques of protein precipitation 



and enzymatic hgrdrolysis for the eartaraction of possible causative 
agents o The pigrsical technique oF dif f ermti&l ultracenrbrifugation 
is used for concentration of the Miterialp These techniques have 
proven to be of value in the partial purification and concentration 
®f the Rous Sarcoma virus (sea J<,H„GoIo l6s 8T?p 1956)o Siaiilarly 
through the application of these isethods a immeBliMn leuke^a 
virus of high ttosor producing activity has been separated frosk 
Sarcon^ J? of ndce (see attached report (A))o 

Major FindH.ng B s 

!i'o date four (h) tujaors (received as surgical sp^i3B©HS 
and in sufficient quantity for cytocheaical wos-k) have been 
extz^cted and the cell lELcrosonis fractions concentrated as 
described abov®o The specimens received wsrei a) a maligmnt 
melaneatia; b) multiple bladder papilleaas| c) a earcijaasa of the 
tlyroidj and d) a carcinoma of the larynx o 

Coroentrates ©f these tissues were used in cyto^thogenicity 
studies carried out l:y Ro A„ Hanaker aM RoBp Coucho The results 
of these studies are outlined in reports sutaittsd ty these 
principal investigators o 

Op?' ®©©®sion it has been found possible to derive sufficient 
volume of @one®ntrat© for animal response studies « Thus far ©laly 
negative results can be reported for the Mological studies iiwolrissg 
both ne%9bom and adult inbred micso 

Ftoposed e gm^se ^f^^^ojegts As the human tumor specimens 

(surgical and autsp^jj beemi^svailable, experimea*-® ^ill be 

cmtinaed in an attea^j* to d@aionstrate, through the applieatiea of 
f^^jhemcalj bioche^ccalj, and pl^TSioal techniques^ a husaan^tuaor 
virus relationshipo 



596 



«3- WI iao(#as-t Be 



It is b»3i.«fV9d that coiic^Jts^tiosa of ®5d;3ract®d it®t®r5^1 
is a isa^ocp fsotor for sueh a idral doBawfflstSTStiesjo It mtuM ba 
of ix^erestj} theraf o?®^ to ®stiesMi th» prassat t«cteiqu©s ol' 
propias^tioQ to ineluda cgrtooheMcsiX ps-eewlhufGs f ouxsd favorable 
for the iaoXftticn and caaacaatration of «Aaol@ cell fmetisas^ 
spocifie oBS^rBies^ celltdar (xp^msnes^ a^d aaixsl twme' viros^So 
Sueh piroeediires voold iacXiM®, 1) ths tiso of eh(S2d®al parti^aing 
agrstenS; Oogef boffar^flus'ocarboa isediai 2) sis!S^TBa,%±Q Is^d2ro3^sis 
of aoK TisraX satsslalg; 3) prot®in etisd nuolsie acsid ositamcMoB 
and precipitation taohaiqueSf, <3cgo$ th@ osa of stresng esltSg 
ph^ol eztaraetion, ato„l mni. h) ^® vbq of m&p@ s<mgitiv@ diff@s^ 
oattial Qltameontrif ligation procedm'as, Oogo; eeotrifugatioxs in a 
donsit^r gindicsito 



Part B Isseluded las STo 2 



597 



i! 


.!.jb'..)^v.-:i- 


'->@J 


2. 


Qenera: 


--/t'ion 


3o 


B®th0Sd'j_, 


^.- ,;_,,.■,; 



PBS =. KIH 
Xndl.'Tl.du&l Ficojsoi^ Hspos^ 
C&l®t3is.r J9&T 1956 



Part A 



Fro jsct Titles firal Thorapy of Taaorso lo AdaptiT® 

ISsasures for lBe3»s&8ed QeiCQl^sis in Cases 
of Ad<vanc@d Gsroinoaa, of tha C@r?i^o 



Principal Im^stigstors Eob®rt A, Hasak«r 
Other Im-estigstorss Robert Bo Couch 



Cooperating BteLtss This project coa^lessraate the oliaical 
espscts of viral theraj^ of carcinonia of th® cesprlx as coadweted 
EBjder the directi«»i of DTo Robert Eo Sjaithj CMefj Sergeryj ICIj, 
and ®xteEds this wos^ iaitiatsd l^ Robert Jo He©ba®r and BTo 
Wallace Pc Bm®, IAIJ3„ 9=752Co 



Men Tears (Calendar year 19$B)t 
Totals 1/3 

F^fessioml! 1/3 
Oti^rs Ion© 

PS?©5®ct Beseriptions 

Ob^aetives i To datarssine the ©arfcent to whieh th© oacolytic 

prop©^S®s''"©f^eno?irMs tgrpe BertJa and Goxsackie B=3 vixvis ©©Bid 
V tia©r to tiaaor paesageo 

Tumor t© ttssor passag© ©f adfaio^irus typ© B©rtte 
bM Cossackie B"3 •^Irus isas eonduetisd in patients tdth oarcin^ 
©f th® QsrriSo Th® -^dras inosula us©d •^srs pae'ei^Lred in BmlM 
c@ll cultt2r@@ inf@et@d id.th a lats pssiidrs spseirasn obtained 
fr®ri th© precsding i^tiento In addition^ attei^ts t© obtain -^irus 
inooola direct]^ froai Edncedj, infected twm^ tissue incubated 
^& a Hsaisrfcensnc© .M.®dium for 2 to 6 days war© ssadso 



598 



mi^ki6 



M,1 or Fia dia ^sf Ho apparent incraase in the oncolytic 
potsn^Sr^oFTj.-fiE' "pssage adencrvix'us typo Bertha or of 6th 
passage Co:ssackiQ 6»3 virus ^s&e observed o 

Thrae attests to produce a viral inoculunn frow minced, 
infectsd tsssor tissue yielded a positive result in one instancco 
Howeverj the low titer of these culture fluids suggested survival 
rather than proliferation of virus o The value of in vitro tusnor 
culture for propagation of virus and its adsptatloF'to'^iuaor 
destruction restains doubtfulo 

_Si|jj;nifi8an ce? This stu^ of th® oncolytic activity of 
adenovirus ^iypo^Brtha and doK^iickie B«3 virus in careinosna of 
the cervix provides a laodel for a^^luation of adaptive techniques 
in viral therapy of human tumors o 

Sropoaod Cowse g TMs program is of neeessiiy geared to 
the availatJiiil^ of "suitable cases for trislo Xraresti^tien ©f 
ezsised tusaor for Coxsackie B°3 vtoas propagation ?dll be ®2= 
tended in the atteBspfc to inereas® analytic properties of the 
agento 



Part Bo included les Ho I 



599 



Wwriai. lie, 

3o B«thefBd®,, Ms. 



PH5 =. JHH 
iBdividual Project Esport 
Calendar Tear 1958 



Part Ac 



IVo^QCt^^Tltlag Tha Aj^licatioc ©f Tissue Ctaltwrs to the 

Stud^ of Tufflor Viruses <. I, Effect of Mease 
Tissue Caltmi'® Material frmiomlj Trsated 
wi.th Hunan Letskeade Brain Extracts on Twmr 
Incideaee in Ssdegi Mieep 



Psrinsipsl Iij^estigators Retoert A^ Mamk®? 
Other XiSTdstigatorss None 



Cooperating ttoitss Parallels ssd extsnd® the joint project 
@f Dto Sarah Eo Stetwsrt and DTc Bemic® Edcfyo Pathology \jy 
DTo Me&fle Stanteno 



Man Tears (caleadar •jq&t 19^8} § 
T©tal8 2 2/3 

frofm&A&mlt 1/3 
Otherx 2 1/3 



Project Dessriptioss 



pb^eetAyesg Isrestigationj ia Swiss mice, of the 
tuBsox^ps'odu^usg 



potential of -fluids from mouse enteyo tisgaa 
©ulturespr8ffi@ua2y inwul&ted with extsmet® @f huffian leukamis 
brftin tissue » 

Msthodss TwotedbMques were applied t© asserfeain wh®th©r ' 
the IjaHe^obeduyei! ■.■Tolled ly Dr^ Sarah Stewart and Dr^ BersAccM 
Edc^ would permit isolation and raeogaitioja of an agent fr» hw»H 
leukwid bsEUiB tissue o 

P^Hfe Ao S'Sids® Bjouee ambsy© tisssue ©ultaresSg gr®i«n and Miiia= 
tained in ffiediUHi 1^ supplemented with ealf serum were inesuMted 
with extraeta of human letjkami© brain tissue o Blind passages 



600 



=<% NGI=.la6 Ca) 



of supernatant fiaids v»Te mads at bi-weekly infc«rvalfflo Each 
passage vas obsaarrad ovsr a tow weak period, then teraduated „ 
Fluid® ramowad from eultures on the second, third, and fourth 
weeks of incubation were inoculated aubcutaneoualy into randomly 
redistributed aawbora NIH Swiss luicso lAttera with mothers were 
individuaiay housadc All adce were ear^HMMrked for identification^ 
At death J, mice ware ejmjidned grossly for tumors, and prepared for 
pathological examination » 

Part Bo Ssiiaa asouee aanbxyo tissue cultures Maintained in 
medium 199 CpH loh - ?o6) were inoculated with extracts prepared 
from leukemic and non^leuksaic hws&n brain tissue. Brain tissue 
used had been stored in a d]^ ice chest o IMnocuiated tissue 
cultures were also o^intained as controls o To reduce corum inter= 
fereace with the actiidty of the l^^othetieal ■^irus, the a^mi 
supplement t@ the Medium ms withheld until sufficient time 
C20='2U hrso) ©lapsed after inoculation t© pennit cell infectiowo 
Ir^ I'^ecogijitiesi ©f inhibitors ascribed t© calf serum rabbit s^-um 
•n&s usedo Three schedules for serial passage and test were 
initiated 8 twice=week3y, weekay, and bi-weekly,, Ifefortumteay^ 
rabbit serum medium would not support mouse cultures for tw© 
weeks, and the bi<° weekly schedule wan abandoned o 

To carsy onrer cell^contained firus, call eultwes t© hs 
passaged were ground, and the mixture of ceU material and tissue 
cultiire fluids was Inoculated into fresh cultures and subcutaji^ 
oufflly into newborn Swiss mic®o The mice were previously toe^ssarksd 
t© id©s2fcii^ litter mateSp then rardoBOy redistributed among 
raotherso Litters with mothers waa^e indiTidi»13y housed^ s»d at 
1 month all mice were ear marked for idenfeificationo At deaths 
mice were escsudned grossly for tssH©r§ and prepared fer patholegio&l 
exasBtination o 

H^^or Findinj^sg 

Part A a During a 7 month period^ mouse cultures were treated 
with brain tissue obtained from k cases of acute lyn^hoi^lc 
loukeBdao Daners war® 2^ 2, $g and 6? years ©Ido Tw© huiKired 
and eigb^-^sawen mice In 3h litters ware inoculated with t^nt 
fluids from treated tissue cultureSo Four hundred md f©rt;'--aight 
Mice in $6 litters were inoculated with control fluids fi^om 
imtreated tissue cultures o At present 20^ test mice and 315 
control mica surrlTeo Sevanty^four of 90 uninoculated mother® 
remain^ . Fraliminafy gross examimtion disclosed t© date 1 mous@ 



-Uof- 



with a Mdiastiml tmar (253 dHo) &m 1 :. :om*> {268 d&n ? 

in the test gxwspi, 1 leykerad© m©xiK« C'^-' -''■ • «■ •■)n>,;/"f,i ^'Yr>:i;- 

and 1 0»ari&n tujuor in ft jaotJiero 

Four additional br&in apecimans were tested o TJieige in®lx3d« 
tissue from & $9 yro old male vith lys)phos«rcoB«j a 3'6 yxo old 
mala with chronic l^rrophoqrtie leukaid^, & 12 yr, old nmla >dth 
retieulum c«ll sarecaia, snd a 16 yr^ ©Id inal© with acute sqye3.&=' 
garaous leukeHda,™ Spinal eord from & 5h yfo old femle vith 
isyeosls fungo:Me8 'Mas also sxajainedo In 36 tost littara 210 ef 
276 -inoculated mica surriTej and in 63 confcrol HttsM 385 of 512 
inoculated soiee sur^vso Seventy of ?8 mother® ramsino Ort\ss 
exandnatd-on rsfr®alsd 2 cwarian tumors (lOj^ 33Q dSo)j, 1 lxm% 
tumor (380 dSo), aijd 1 leukeiada C211i da„) in the test group, 1 
amxim. tumor (11*9 dfto) and J* l«uk©Mi®8 i2Sk» 258s, 3U da^wtrs 
noted in tha control groujic A lung twesr and la Isukimnia appear sd 
the metl:®r8o 



A brain tissue esntrial initiated with & speeiman ©bfesiiied 
fran a 3 »©» old fesaale isd-th a eongenltal heart di^essa i«s,© tested 
in 8 litters ©f 63 zd.®&„ Oaa huafdred assd twsK'ty-four jsi©® in 
16 litters aerred as tissue eulture ©ontrolSo F0rty=niiie tost 
snise and B9 control udeo sunrise » Eight sen cf 2.k aj©thsrs reamiKo 
Ho tuaors were H©ted in either test ©r ooreferol groups o Oa© 
turaor was observed araong the moth®?®,. 



Part Bo Control ine^ula^ ecsagsriaslisg untreated ©altur® 
waterialg and eulturss treated iAth br^in tissu® extr&ets frcm a. 
2 yVo old male and a 16 jPo @M smI® wJ,th ©ong®xiital hsmrt 
disordsrSj 'wsre in^'ected into 2? ll.tters ©f ntsrwbora S^dss sidi.©©o 
One hundred and eighty^six of 21? ndc© res^iRo Oldest sciee 
are 8 tmso ©Mo H© tumors ©n gross saxaadxm'y.on wsrs found to dst*c 

Test inoeuMj eoioprisiag eultare material® treatad with 
brain tissue extracts froai 3 eas®@ (sges 2 yrsog 2 yrs^j, and 5 
yrso) of aeute lys^^o^g^ic leukendaj were injected int© 26 Httars 
of newbons S«dss Kieso On© hundred &ad siarfey^four ©f 200 Bsie« 
reiaaino Oldest ade® are 8 tmBo ©Mo H© tumors on gross exaid.Mi.= 
tion to datSc 

No tumors have been obgearred in mothers ©f either gyoup, 

Si^dilcames This project pro^des a t®st of the imougi® 
t is® iieeuHST^ a mean® for isolation ©f rSxuses ts-mi hvsmn 



602 



-4- HGI 106 (&} 

l®iske53dc brain and th© bbousq as a tast animal for tumor ±a^ 
dQction 1:^ such isolates <> 

p«g^d^,^^ej Tissue culture aopects of this project 
hav® bi^Ti^mtedo Inoculated aic© sill be nsintained and 
observed through the following yearo 



Part B included las No X 



v3 

60S 



&«;rial. Steo mzxJk- 



Ml 
Ml 



<£o a®sts£ftl Molo^ S«@ti€a 
3° Bs%Si@sdft lk» irnxf^mi^ 



fas => BBS 
GftloBSar Tsas" 1958 



^^Ao 



Ps€ijeist titles Ii!r««@tigatiea of ^sm w^« of gsffigs «b& ^^is 

©ff 



llrissipal Ite9i»iiti^t6S-s W. 1. Bgstesii 






LS 6 



Mb& fsaafs ((@al@»aa? yms- 19^) 



0b.1@@ti'^8 g fo aesestalai tte sol^ of genes ia tlie ia^^lqpsie^ 
of t'isffior© ijn Me« aad to s«lat« tl»e g@»@tis effects ifith tJtose of @%tof 
Mologiealp eit^sieftl^ or pl^^ical Agpasts te tli® ssusatiora of eaj^@s> £i& 
■i«s«io fb iSentif^ and lcc«t« ap<i@ifie i^iie« iaflnanclng tl^ osttsss^^e® 
of t^ 'mriotts kinds of tiacm aad to loealisg tlie gsne^aetisn patSs^a^ 
'liiso%ig}& iribdSh tStel? offsets 1}@@08is nsusifssto 



itetboasg All of -^isae stuOiee as® @a3fsl®a ou^ on fld@@o f|g@ M#ii» 
'" ad M^ hepatoaa fftndn used in C3Ho An agaafeofipee mM^ 

line C3Bf has %««a de^lqped aisd is also usado file lov°4M»nzy tmmr 
stzAin used is e57H» fba M|p& lung ti»or stzain ^usad is etmi& A eM 
stzain C57X> ie tbe one usad aostly ae a lov«liing tmoT e%z%i&. Stxaig 
tm Isaa laaai uead to iistxodisBa ttas l«r^»l fttllow gens and etwedn W©h 
Isaa 1ie@n vm& fo? tba ofeasa gena it eaxxias. BeeaacO^ th® stsaiB JM 
etaaxaeteslxad t^^ its ax&svsBsly lazge Isod?' sis^ has Ibean inta^odwM isst© 



^ppsop^iata ezossas bat^^^sn tltsea etxaijas asa nsda to dbtsewv® tJa@ 
@ffa<ets of se®E«gati08& of g^oaa aad to izx&xtaliiea speeifie gg^ ii2(l€> 
th@ artudies. In etxi&yixi^ ti>a MoaMur ttnor agent tlia t®st foi° tis® a^ssst 
nov eosksldai^d aa- tbfS 'best is tlia oeeurz^tise@ of tanors ia fo@t@ir^amsBM 
saaeaptlM.® f ^6ftle® ©s» tb@ ©cetssranea of t«B©i« is suee^tilsi® pwo§^ 
thss&ii^ lat^f ^Bgx^tiosis. Extsaaaous egesxls az^ isats^oitosM is -y^ 
m&iHi@£' a^g^sigpyiate f@y the spseifie ag@s^o M'b®s«Ca,&)aasyixse(iis@ aM 
si@tligrl@}>@ls»tSix$n®' a2?@ taj€@t@d iis^Tssm^&mS^ && @qieimm &i!i|»@?sioss@. 



604 



"2» mi^hii 



Xh@ Garlam tetvaehlorid® is gi-ves& bj stouush tubs. 

Bi tbe stifles o& thfe effect of coroentmtloai of os^rigeB as© is2» 
dueed pulnostazy tuno3f« tlte QAVsiBOgsn used has bsen dilieii£Cajl:i)%£kt>»«;c@m 
ii&J^eted intxaveoously aaid esqposuxes haine l)een in aetolJOlic^cMa^s® 
deaigaed ^ Or. AzsoM W. Psatt. lAiog tvaors axe nov TOotiagly emmt^. 
va&sT ■^^ diseectiiig aiesToseorpe is& limgii that bave teen flsosd l^ iBtm« 
tsacbeal iss^tlea of fixati"^ pslor to reuo-ml fron the chest es-rltyc 

B%4oy Fln&iaeBs 



Doziag the 3F«a? aMitioml data have been oMai»^ m th® ^es^tis 
^elatioJBship hetweea soxnal g£Ovth asd -lhe oceusxessee of fflaligi!iBi^i@@o 
To date aost of thie iofo^wtloe has teen oMained in z^apeet te p«&° 

twBOfts of the muse. Prlof to this w^at^ all of th® mvim l^mm. 

of the souse that had he@n related to the oeeurmi^e of pulasmsf- 
also had aa effe@t o& gxov^ as measured Ib body vei^it. ^lose 
s«lated to a deeseass' iji pulaoaas:^ tw»3:« Seeseased body «ei^t @M th® 
oae that ixiexettsiid oeeuxrense of prajtSjoenax^r tiMors iiscx'gaaes bsdy v^i^isto 
On the othe? hand; of the seiw?al tested eia^le eoat color g^&es that 
have Qo effeet oa Isedy •wei^*^, aone elteted the ©©msHfeskee ©f pa^msamf^ 



Isstesestisg x«salts are belsig oM&iaed ia es^asading this stMj 
to the rs€essi-9e ^ene for oheoe. Ohservatioas ar» l^isag H»de Sb ths 
?g sessezatiera -^ktre the eegreeatioo ratio is 3 aoxaal (1 Ob Gb assA 
2 Ob Ob) t© 1 6b<ase (db ob). ^She twors ham beei^ SMwed vith mw^hasio 
Althouipa this ^llBsi ffpeatly izaezeased bsdy vei^^^ it r@iw®s the os@w^ 
resuse of isdused pislJBOEsry tmors. She a'verai^ suRSaer of ttssors is^ 
the obese sales vaa 11.5 tihereas that in ^le zioiwal sales nas g%.7» aM 
the airerage csoaoiber in ^^ obese fesales was 16.8 ^daeseas that iisi tte 
aon»l foailes vas 27<>5« ^Ehe average wsig^ fbr the obese ss&les at 6 
sosxths ms 63.1 graw eoapared vi^ 38.3 grass for -^w wsbsI ieal@@^ 
aai for the obese fenales ms 69.9 gna» ecoipazed vith 30.I i^asm for 
the sosml f^ODoles. liomver, despite this enosaous increase ia th@ 
b@dy ''ipsi^xt of these obese anianls^ grovth neasured bgr total 1^^^ l^ini^&j 
lei^gth of the fenur^ or weight of the gast^xsnenius susele is r@dii©®i by 
the obese gene, fhese three aeasuranents are proving to be isatisfast®^ 
for giving en index of skeletal axA soscular developaent. fkes<@ x^sijilts 
together id.th the e&rlier results indicate that the genetic xel&tios^Mp 
betv@en noxsal gro^yi osiSi oceurrenee of tmors is in sespeet to i^O'siW^ 
as reflected in the slseletol asd sssssc^lar sj^taa aM sat necessaril^r £«^ 
lat«d to adipose tissue. 



la exfe^sding these studies to other tuners prelininas^ Sate hsms- 
obtained on -^le relationship of the le^ial yeUov gens (that i&^ 
creases body size) and wessaasj tunors. In C3En^i f assies ^ half of vMch 
ar® yellow (AyA) and half ageuti (Aa), of the first 52 vlggins f«6l@® 
t© d®v@lcp aaSBftey tisBorSj 50 w^® y©!!-!^ aM. 2 ymT& egp^i. SMs is 



605 



=3- ms--h2V7 



e©*taiaay e«flmiffig Mttl®'® ®&rli®r otaseywatiem, that t&® l«til*».l 
f^llew ^sa@ ea^asM tbe ssaamaxy twmr» to eqgfteftx- at an aarliss' a^c 
"mB eftast CC& final iissidexac® of ttaooss is ]b@% 3P«r& 
or %3)e feoalea a^ still li-vlog ^^b@«fe tiaun^. 



Iffi i^ts pz«8«sfe ir^udy of bgipatcnas is CJgi, @3Bf » aM CJS® wi@@ 
aM -^b@ poesiM@ «ff@et of ux>etl)an 'on tlis seemresrse of tb®«B(» t%@^s% 
an th@ C3H and 03Bf 'isaiee hai^ l)e<m aiittopsied at lit- nensthii of a®igo 
Eig|x^y<»fiir« p®x> @@s:fe of tbe eoflrf^zol C3S aaies l»d biipfttGaae ^lb»Tms& 
93 P^s^ e@iit of tile CSH »ale« tltat z«®eiiwA one ingestion of SO mg. 
of ax^grliian ha& Sa^toaaeso ©f thosjs i!«eeiving 8 iad@etic»s of 20 i^. 
isaeb of nasm^sasLf 3o p@£^^Bt l»i& lis^patonaso S^p&tcaws oceusi^sd in 7^ 
p«? ©essi of °^^ C^f @<»Bet£ols9 in 9^ p@? etacfe of ^!m C31f sHhlii® ^tli 
cai« in>sti€8& of lax^^^aan^ a»d in 90 p®s- ®&^ of tboa® tdtb 8 iMS^i^im^o 



t3tes<s is ^^viSsats® tbat em In^^stica gS vssmmm M& siwm^ is&» 

©B 8 isu® 



^^ «ff e@t li@l0«r that of ona lnj@@tiono 'ms outstaMing s^s^islt 
in this stuisr; bQ«®wp^ is tli« M^ imsMismm ©f itv^atcnas in tbs@$ 
istsaius vitlso^ tz^a'^igsx^o S^h iaeia€is@@e Imm sOmo ^«n @%®@3fv«a i^ 
'tSM C3S€ stxsino ' fMe is tlt@ M^wit in@£d®n@e of b^patonas «'««? s<sf)@£%@(t 
f&x> angr stx«in assd is mi^ M^ta? ^ba& tbat otwarrod in C3E a mm^r of 
ysaxs ago. Cane® of '^lie ineie^s^is is lasft ISQO«n° It any las ges^ig o? 
it a^ Iw Ai(Stas3ro At an^* sate our C3i strain is now an isx@@ll@sgfe iti^sdii 
for s@s®ax<eh on h^^toofts. Xt ShoiiM 1^ aMed tliat not only is tbsi^ a 
Mf^ imU^am of btspatcoias Ijut aangr' of tlse anJaals h&m asultipl® li^atmsa®. 
man ixm±<&gimm eouM vsSonMJ^ be ervisn ij^seeais^d if -^hm malm ¥®w® p@^d,t&®i 
to H-m teymA tM.s l!^ mss^ pezloa. ' 



FoljsQasai^ t'ffisosn 0€@cQ?z<ad in albots^ 10 p®? csssfe @f tSi@ @63s^s^qi1. 
€3B aal^s^ in altovt^ ^ p«if e««(t of tlios® jc^iseiiring ons inj«eti^ oS* 

Dd i^fosdSMtely 100 pasf e«st of thoea sse^iTiJig 8. ist^timmc 
no aiff«xene@ bstwecn tba agast-fs^s C3Sf 
taining C3B odea in s«i|p«@t to ling 



A waimr of banagioaas wi3«g not^ in «bs livexg of ^mm CJgl 

an& C3if waas lieiag lloit^ a2j»st eca^lataly to ^tem that 2^@i^^S 
8 injeeti@«e of -Hia 



of ^3@ @J!f €€% of o3Qpgen tansies (H& inSi3cti@fl& of piA= 
tisBO?s in stsain A ad,®@ lias Isean csossi^iiEa^ in eoop^xatis^ 
with B?. AxnoM W. Fxatto Boidng th@ yeas' mmieal e^s^rixm^M. in 
this attiay "mw® @^|pl«t®& oM <^i@ ftasialte ha^® baan pz%sai£l<sd for 
pvMisation. Ttim original o1ss«r«ati^k that the «3qpo«us« to 8^£@xi°° 
snt@ly 100 p®s> e®sit os^^ien f o? ^ hmsfs £BSsSiatal|r f oUoiflns tli@ 
isfemiPsnoua injastiea of &£1»ans(a^h)«ssfehxae@na ine£«as<i& mis 
of IMxcaS p^iQsvsmsf tmom ■mm eeSrisasd. In eMitioat it «a 
ncaMstsated that aa^osuri of tl^ siea to -mm M0k 
for iS Tatmm Issginniag ^ hormi aS^f ^Sa& ia^«etion of ^m 
liJ&g^s® in@£@as@& th@ oseiss^ane® of tmrnrsg "test s%q ia&^:$M£^«i in 




606 



Wl^ll 



iBBsdiately &T k8 h@uvs prior to tbe iaJ«@t:J.os. of -^hs emai?Qim^_^mo '-Qm. 
eT^p&msem of stxain A »ie® to only 8 p«r essl^ oxgnsen fox- !«d b0iuT(«g f'oili.-o 
lonisg the isjeetlcn of the saxcinogm x«irol-^d in a deetmm '%s& mi$ 
sa»%9r of tiHoxv l»&(se®ii lijr "yie easeinogsn. Tbm ejqposiure of rn@r«ftei3% 
fltxaia A »i@e to eig^pxoxia&ttsl^' 100 per eaa^ osQn^n fof kB heta^m <iM 
xaot &lt@7 tlie iDsifto»e« of qtoxlaaBOUs pvOjioBaxy 



it WBOM K^seax ^b&t tlie wmhmr of tunozs ind«is@("d ii^ ^m 

«pitbelim linifig the al'weoli of tl» lisigs 1)gr tiie ai'te&zCa^))«ssyime@8:^ 
exfstals lodged fa tlie Cfi^iUail^s 1)er&«si@a tbe al-v«oli is '^fm^s^ % 
tSie ®eiM9«afexatie» of ox^sm ia -^Jse aliRsoli, the msi^lms ^ssmmim i^*& 
an iii@x««8@ in os^fs^^ Ttm eff<iset of -^ae os^mgin c<;a& b@ n@M ess^ 
'Oassi tSie caseinogm is pisessBfe Sartes ®3^©«i»®. 0)3Q^gi^ So^s m^ alt^y 
tba cpitMliol Q«ina to ms&ST thm aos« svuse^ti1ba« to a lat<&x- e@«s%«^ 
nith iaam mseijais&m, Tim M|^ osqpsen tea no lesairasraM® @P'@(st <m Warn 
of tuasosrs is^ "^s® a%s<snc® ^ '^b^ @ax^iQO®iBo 



Si^Bdfieas^f to tte igyo^fasa ^ ^^ Imtm^mi 



Tat^ vilt±mtm ai» of i^teg pm&m^ of tSae Institute sni^ ^ ms 
pz^^iitsss^ion ©f san^z'o Slis sox^st i^saS to ps^'vsxs&icso is tSiSdE^i a 
^20sous^ ysm}i^M&m ®f ^^ iXi^rs^M.^!Bd iMrhml<s an£ ®3El£ia»i@ fa@t@s® 
(saissiag (sas»s@s>. laaie aasag ^m isl;insi@ fa^tozs ax« tin® @e»s@c ls!t 
cssl^r fio tber tsnaamm® a^mr istsls®!® flRstoxs %«st tiaey aim M'^ it 
px<rarcs«stt iisfl«i@sks« %ipo» iSiss £@ses«8s«ii @^ ^m iMiviSml to tii@ ira^@«i® 
«3el7in9ie faet@ss «ff«@tiag @aB@«if<> Foz' m^tsa^lm^ tMs px«ogs«m lia@ 
pa^r^ioilarlor ®q?liASi2@<t ting z^lationsMp Is^tin^ (sen@s aM ^l?Ki®@@ aM 
tisat if ii@ a£@ going to e%v^ sasieer f^rem tls® rlmm 
also ba^^ a lrao«i@S^ ' @f tli@ esa@s ooasfesoUing tli@ psQ<= 



^^^*^?l®^ ^^S ^ ^ ^S^^s 



AnaldPsis of tise s§a^i@ z^latioeetaip l>»twe«n aoiaal ss@ffth 
of ti8»z« will be eonfelBosAo ftae xeniBSftdr of 
in3s@t®a ?g sie@ for eaaS^rxiag ^» @ft(iet of tlis obss® gene win W 
autopsies and QTitmrmO. for taaow aai tetal ■mtii^» iSeslfirya. assi ^ssei^^r 
seasusvagsts idll ls« ani^. In addition a gso«9 of o-ver 3D0 of 
?2*8 bavs 1ss@n set aside for eoogpasabls data rslatii^e to 
pnlaona^ tuaors. Data tnm tbos« anjbaals vill l3@ amlyssd @tatiieti@ftU|r 
to dgr^£Bdn@ eozrelaticaae between these neasaraBentft emd maiber of 
not only in r@i;p«et to diffex«ne@s b^«e«n obes@ and i»oiHal Imt also 
correlations vitbin tbe ob@»e s»»9 end parfeletilarly ^tbln tbe 



^@«n%ly «e obtained soaie breeders of the Ooodale l»sm etwedxk 
i@L} tw& tb@ .Jaekson laboratory. Siis strain has an ae^usiaation of 
for lainse body sis® other then ob@sity. At 2 sontb® of a|^ -^s® 
■w^t^ «^pp£03djBat@ly '»'$ fpfaes biJt ax® sot fat^ ^shes^as ^o^ ®f 



607 



^4:." 



Btxsdm mi&h m A m SIM ^?^U. w3#.4 txam 25 t@ 30 s?emm at ^m mm 
iciU 1)@ (ittiii@d in Fg'@ tm^ cm»»ixm, '^^ OcoSal® Uxm ni®® ^^- 



studies as® 1)@lBg extaaOsd to waamry glaM t)aas)X% «iM 

tlKt mm Ismism Imi&o WaM of Va^ saMs a3?i@ teistg utsiiS for @tiiS^f tte 
eas^HQS!^ t«t^aieislosiS« aM tiis o^Sneer Mlf f os> 



esa »iaffia?7 gSAM tisBsoss ssui e«% Iso^m tine a^c^esmam h^sj^^^mm msd ttes@ 
iM«e«ii vim sa«liQ» t@ts»sbljosldl@. 03E?e^2 ^c® ^^ ^^^^^ piroi^^ 
for this puspoBSo ^^© iKtlee aspe 'bsisg us^ for tiife b^^toaa @tuiis@ aM 
'^ite f@Bftl@9 fO£> '^s mmesj ttmov stisHseo me nsaaasy ^wKtT stf^ sgm 
l3@ sbS@ eeilj «it^ "Ttxgiffi Fg'it @i&si@@ m» @'b%m asSaals liin ^sit 



Xffi tls@ a^l^ss^ of biiSiatenas 1» tte C3II SHOiKij, 100 03^ ml@@ Mm 

ttt li@@» f^g and ^tiss@ 100 -»ill Ise a(sfe€^siM in Ma^sli^ to ^@ 
if tM® Mgh iJBsiassiis^ mn l!@ fa,tt7i1mfeea to tl^ SOI p@ll@t@> 



1 Sjsj@@ti@o tsi vssmnmk eassS. "^mm tliat ms^t-mSi 8 Ss&^®«rliom| idU te 
ms^s^BiM t^slog the ©enifig jma^ aM their l^pateawk Sat% eOm^, ^'^ 
^»s# fo? tSsi 631 aM caaf asa<§e ^»iU l!« ai»3^si4, Os@%i£T^3iS@ ^ pA- 
sssfiisf' tm0?@ -em also 1)« acr^eft i» tSaem C5i@ asi©@ to a^l®satSm ^^i^lter 
&Tms^ ^m m^s^'^tm® 03i@ Aiffisirg fxm -^bs 03E vitb tSte adlk a^i^ 1^. 

¥@ e>s% s^stissdjog t© stuiar '^im os&saes&tem of -^ts aenfflsi^ 0m^ 
t^mGTB iM tl2® sa^^xfe'^fss® C3Bf etsaiffio ^^fi itxain ie %@@aai2Qg £n<=- 
@s-@a3s&a^j pspnlar is& @aa^@r s@ii@as^h aaS a ataiSbsf of i2iY@»ti@&t©£@ 
outsMe -^si@ I»sti%«^s ami wmj^stixis, tlxse® a^iEt°f^<s@ t«»^s%c T^m 
«7@ siss^ leltessi a'vailaMjs aM i&i@a «@ eaa lie aesossd of pw^m^ m^s^@^ 
of t22@ aiainaX ax^ ;E^ift!ii%s& of bistologie sgetiooa of tlis tts@2f@o 



^s@ £«lli®r @xl^»if<@ p%oJ«s@t Sjieieoed to aseertala vl^^£@r tls@ 
^g33grti@ GOg^t^ o-mT ^m wsms^ ttseor ag@xsfe or. -^Isug is i.%^ to & 
siiBgl® pair of ^mm& os to s»a.tip3^ seEses ^a&t Iba® 1»@^£ ii& psq^a® 
for a misi^f of ;i?@«m -»ill tm eosiisliiSM Sufing tli@ ecssdog jgasfo tt@ 
i&ta fjfi^i. tM.s p£0^@@t im^l^iisg @&i7@3rsl tlso^saaaaad sio® «ill t^i^m te 
t&lA^,a:t^ sM aaalfma aM px!)^^«x<id fo? pi&OLi@e,tio@<. 



608 



KX=41T 




io ItiffittUaad i» til® 1« 

to 
tlie Xenial yiUew ®B!f!s» S» 
l» tHe %so«ffi site ^ laj®e%ien e£> ®eM 




fbift 3 i^i«i«i Iss I 



609 



Serial Moo ^s*^ 






I'agfc S o "BOBOSBg A%&2fdS^ QSOi, P(^iLl48et%iOD8 



Ptdallcatioms 

Beaton, W. 1..° Msaaexy tfissore in Aeei£te-?£««i Mice. Asm. H,¥. 
Acad. Sei. ?!: 931-9^, 1958« 

Hestcsffip W° E. aai Prntt, A. W.: E£f®et of ao»e«s:femticn of 
Os^ys^a OS Oseurrestee of FuSnaoaxy f'usBora in 
Stxain A Miee. Jo Sat. Caoeer I&sto In psesso 



Bo8io?si: 

Is% s^@@o0Biti6aa of a/cecs^lislwga^s as a ssei^n^ist in ssMieal 
s^s@ax%!i "mm a'«asS@S th@ Jacmstws^ Boetosat« of ZdEws a@g3?e« % MieM®as^ 
Stat® md.vwsxsi'tey la Jua®, 1958» 



610 



Serial Ho. £|^aB 

1. labojeatory of Biology 

2. Oe»eml Biology Section 

3. Betbesda !*«•, BSszylairai 

PBS - HIH 

loSividtial Project Report 

Calenaar ¥ear 195S 

Part A . 

Project Title: Host-Twaor Kelatioaships 

Prineiapl Xnvestigator: Monris K. Barrett 

Other Invest igators: Maleola B. Ifelroy, Edvard J. Breyere, 
and MsiTgaret K. Deringer 

Cooperating Units; HC2»!«S1 

mn Tears Ceetenflar year 1958): 
Ototal: 5 
Professional: 3 
Other: 2 

Project Ascription: 

Objective : Our vos&ing hypotheses coaeem a field presently called 
'iSBnfflogenetics" '»hich covers an area of overlap bet^seen -yae t^o c2s.ssieai 
disciplines "but se^as to differ significantly frcaa each. Eiis is a i?sla» 
tively new field aM sjany borderlines and concepts are hasy, hwfc airpears 
to touch }spoa sevemL obscure areas in biolo^, e.g. j adaptive eas^j^s 
for^tion, sabryonic iMtastionj, differesrfeiation, irradiation prot-ectiony 
aM the broad but ill defined field of "t^ssor iMcmity." It is beecsd.sg 
increasingly apparent that biological Smtsraetioas involve scaaie undis=> 
covered "infosssation" or "recognition" systssa of genesal application. Oa? 
hypothesis esBymaa that seme au£h sjatmi is involved in the intercellular 
reactions of Bosaal differentiation, growya, physloXoo"' s^ tte"^ t&e al^s-- 
p»i diffeci'eatiation ai3& gro^^th in cai^er represent an abenation In sush 
a system. He also entertain the notion that such a br^Mo^m ^ay isot al» 
•ways be coHiplete aisd timt therein aay lie the e3©laaatioa of spontasaes^ 
legression of cancer, tesBgKsrssy reaaissioas of cancer, aM the apparent 
control of aetastasis asasS ree^jrrence. 

Bfere specifically, w are attess^ting to discover wMt a "tr^s- 
plantaticn antigen" is. At first thought it my appear to te a salver 
aonsensical laboratory plaything with no real place in astiaral pfeeac^sna. 
Htowever, it is so videspreed in nature -^lat, spei^dag teleologieally, it 
Bsust have sesae physiological fuaetitm. We believe that dissovesy of tlie 
true nature of the "antigen" and tl^ "lEaanity" ia this fieM ^fill open 
a new approach to many problcma in caseer, bo^ as to the intrinsic nat«r© 
of neoplasia and as to its contx^l. 

Methods ; Tb® teeic proeeduz® is transplaatotion of stasdardised 
tiffiore, or skin, iacfeo hosts of laaown, bist hi^y variei. geaetic consti- 
tution and observing tl^ effects of e^erise!^^! mriatims of hosrMp or 

611 



-2- 3iCI-Ul8 



tvmiTSf or both and of lasnsaisiBg iz^jectioos^ trea-taBeiit of the emtlg&aa.. 
effects of chssaieal or pl^rsical ag/sxs^j etc. Tbe dleciplinasy teclmlgues 
are those of gesssticsj iBi^uosolog^, aeiS ea^riaieiztal Burgexy* 

Major FiBdiBgs; la relstioii to ^ftat Is variot&sly called "adaptation/' 
or the F^ ^^^^ phejKssaieHion, " or "the Barrett-Deringer pheooiaeiion" there 
has been scase progress hut the isosk has heea hax^pered "by frustrating teeh" 
nical dif f ic»n.ti®s . Our eaMMt illustrating this pbenoeenon vas shosm 

(hy request) at the l2s^2Bational Genetics Congress in Mmireal and was 
wll received. 

jn this phsiacsiieason tvo questioos are of particular iisterest at 
present. One isj vovOd tiss^s of Fi hybrid origin "adapt"? W® haw 
tested 2 such ttsBors, She first appeared to "adapt" in ^uih of 2 tsisls; 
the secoM grew poorly and "vi&a difficult to j^udge. Tixas the &ami&T on 



statistiesOly '^seak data is — probably. Since F^ hybrid tiss^^s as?© con- 
trolled heteros^otes they as^ ptesvasihly scsseiffiat loore like hiamn tisstsee 

than inbred tissties in this context. 

fhe second question is does the final result depei^ only on the 
obvious change in the txmar or is the host also changed in eose VQ.y2 
Seme tijge ago -^ laade a first test of this tiy inocvOating "adapted" and 
stock sublines of the tus^r sisniltaneously on opposite sides of the hosts. 
The results indicated that under these circuEsstances both ttSBsosis gro^ as 
though "adapted." We did not feel sure of this because at this stage "pro- 
gression" had raised the transplant&bility of the stock tiaaor and also £br 
reasons that led to our investigations on multiple inoculations of tuaorsj, 
nov pi&lished. Boss& difficxilty ^m.B&^fSs^&id .. in developing suital^ tmw 
!a»,t@rials but ve no^ have additional data with another tUBor giving cos° 
pasaMe results althougji the overall transpl^x^bility weakens the eta» 
tistieal at^ects. If further eonfirfiation can be obtained these results 
say inHisence tMsiking not only in tram^luitatioo theory but also regarding 
seta^^sis. 

TSa^Te are several Mnor suggestions in ISBaKsaogenetics; is t\a^r 
isfixuQity, and in obstetrics that point to sobs peculiarity in the £@la«» 
tionship of the fetue to the acy^her. With that in iaind vs havte studied 
in progress in raice on the iiaBMnogenetic relationship "betveva a gestating 
female and her youc@. l::q>eriaEents to date have shotnoi that the resistai^ce 
of a post"parttaB fcsEEale is sigoiflcantly altered in the direction of ®e 
increased susceptibility to growth of tmasplanted eascoiaa provided that 
the sire of the px&ricm& litter aM the ttaaor corre^»cd genetically. 
ThiB effect is s^pecific and apparently is not related to the aetheds for 
lO'B^ring host resistance ^ihich have been previously reported. Bo^ieverj, 
no isaaunologie change due to an intrauterine influence has been deteetai 
in the yatms in response to the genetic constltutlcm or iMsune state of 
the aother. As interesting feature of this is that if it is generally trtag 
it refleets en th& antigenic maturity of tS^ fetus and also suggests a sort 
of nipfflide-»doi^n Issiune reaction ui^er these conditions (induced tolemaee 
In an adult?) 



612 



"3- Hci-ina 

In cositismios efforts to cbftmcterize the antigen in trntor 
jxasunity ve h&-^ succeeded in aakins seas progress. When SoXk puV 
lisbed Mb "cytotoxic ssrusa" teat it oecunred to us that stoarage of 
t\aBDr cells va&er sisxLlar conditions of Incubation vith the esacluaion 
of ersor might deplete the viability asd the antigenicity of the cells 
at different 2»tes. Shis la true. At 48 hours the cells have low 
viability SO& hi^ antigenicity; at fS hours the cells vere antigenic but 
not vl&blft. At y> hours the cells ere viable in isologovts mice but not 
in beanlogous. Ttd.a is further evidence that a living cell is not 
re^Luiifed to stisBulate tueaor Ixnunity. 



Ai^sarejfftly the antigenicity of neither red cells nor tucaor depended 
va^n JMA-pT&teisk. In collabosBtion vlth Drs. Schneider and Bothezhaa, ^j® 

have found tJiat there* is very little OTAP in Mouse bloody practically all in 
the vhite cells aM &me deteetal^e in the z«d cells, ^here is a lar@s .s^ousst 
in tiassor but vAtea a tunor susrpension v&b divided into 2 aliquotS; one brolsen 
vqp ty sonic 'aaves asi the other intact, the IMA contents of ba*h vas identical 
bist intact cells Ind^a^ed isenmity ^^lereas bn^^en c(&Lls did zkot. 

As in red cellsj, incubation of trxsor au^p@nsione in sallise 

destroys the aatigen a'fe a different rate than that for other properties o 
TuBor loses ^lability at ateut hS^ for 30 aiautes last the antigen 

persists to about 56® for 1 hour, ffhis is a©r& stable than the 
erythK^yte antigen.) 

¥e have finally succeeded in obtaining as^otber tumor suitable for 
reversing the iisteratsain relationship «ben coffi|>sred to saost of our 
previously reported induced resistance e^^periaseHts. ^e results obtained 
■were the sesae as previous observaticms. ^iiis is further evidence that 
our results do not dspead uposa a un3,que ecsabinatioii of laatexlals. fM.B 
is stj^ported hy furthsr esg^srijasnfes in eollabsjcutiom vith Dr. Sfenrtn ' 
therein ^s© found that resistance against noxsal tissue can be IMuced is 
a third strain of mice 1^ prior isocuation of blood. 

With re®5i« to «ae ijueatioa ^^hether the antigenicity of blood is 
dependent x^on the red cells^ the -idiite cells or both, we have sotae 
evidence that this as^ vaxy v±Vu 8ts«in. One set of cospleft^ ey^eri" 
Esents gave an um:^ect@& result. Thre^ strains of ailce, all of the 
eajBte H-S histoec^p&tability gro^ were selected, 1 to serve as donor a^ 
2 to ser^ as recipients. Donor blood «as frs@d of leucocytes by 
filtration thi^^ru^ glass -uool. Whole dmior blood IMuc^ resistance 
against ^asesr tuaor in both recipleat strains but filtered bloc^ IM^ee-S 
reelstan^^e in os^ recipient strain and not in the other. 



Tests of the effectivtsness of the isstravenotm route for iig^^jni^ 
sation have shoi® that '©ith orflinaty doees Int^^vetsous injections is 

effective aaid spleesssctoa^ does laot affect this but with low doses this 
rout« laasy be less effective ttoa subcu:^aneoue infection. 

A atasber of es^erliasKts involvlag both tu^sr issmmlty vssA 
heisag^atinatien are in progress. They as^ arssnged so that a large 
caaber of saterlala can be cosipared in a sost of rouM»robin feshicn. 



613 



»CI=!H8 



fhe ideet is to flad asm or noro conbinatloofl tbftt will tbsov iwv 
ligbt on the pxoKLesM inirol-ved. Thus far vs can oay only that the 
situation ie vezy complex and that aiaiple ei^laoatiaos hased v^on 
obvioua reciprocal relationahlpe like the auceesBftsl reversal of 
aiaterials sesstiooed earlier are not sufficient for g»neralimtion. 



In. Januasy I participated aa aa in^tad speaker in -^le Third 
Bbaotranfifplaatation Conference at the Bev loxk Aeadeoiy of Sciences* 



A laxse oaouDt of tlae has bs«m s;pent on editorial &^lm for 
the J. B. C. Z. sod for Cancer Research. 



SigaiffLeane e; !I!he field vith idiieh this voxk is eoneemed is 
so difficult and obscure as to bbIm clear def initloa and outlines 
alsiofit Jji^ssible. Bs-vertheless, there is a slovrly growing %odar of 
evidence idiieh indicates that ttere are interactions berfeweea tissues 
as^ their host \M.Qh in^folve critical factors in the i^tonaoena of 

definition of cancer cannot he 8i.yfsa today except ia deseriptivis and 
biologic tessBS. Tbs texae usually ineluied thia^i related to the e3&- 
peridots that m have been describing. Tba esperioeats a»y b& 
ificaat ia sdveral wya bo^ practical and theoretical. 




Tba 3B0@t t^or^satt laplicatioa is that soee of these x«sulte 
progress to%>Brd understanding of vfaat contxols the gro^Tth 
end hem this differs fic» nozBal tissues. We have al° 
a tisBor's power to overccsie host resietaaee can be 
alteredj its rate of growth, tendency to regress, and tendency to 
ssatastasise are all stfbject to escperlflEental change. !!he host's s%« 
action can be altered esq^rlsxentally, not only between slsxLlar hosts ^ 
but also at difffereat tiass and «ltes in the saas host - fhe changes 
ia the tmds Iteive eluded aoxpholt^e, chasical, or serologic definitioac 
One of the aatisans ishieh changes the host wold be aisaed by curs^t 
chnieal aethods. Sowever, one ai|^ attain a biologic description of 
the factors that s^tlaes cause qpaotasKioas tusaors of naa and an^j^ls 
to regress and that ece^rol a^tostasis. It has already been observed 
ia this laboratory (and confinead elsevheze) that such factors astern 
islly effect the outeaeg of isaay 



Belstiesa to ethers; Usere is a coaplex theoretical iate^loakis^ 
this ^vk and -Uie vesk of I>r3. S^th, hsXf and Miss I^hoff^ 
ISersia^ and Scmford. Many of the results stteodaat t^poa the use o^ 
ailllpore filters vers foreshadoised t^ the voxk c« the nature of i^ 
aatigaB ia tiaor IraRini^. fhe vmOt en adag^tion has a strong r@l&'° 
tiOB to rBeeBt develesoeots ia xadiation liiology and in tissue 



i. At prei 



Course t Sstentioa on tl^ saia e^perisoents described 



above. At pre^nt ^ otac aa^or seed is for ttBiaom vith tdiich 

like the oteve can be rsopcated but vith eertaia variations, especially 

a reversal of ^sa iaterststtia- relationships. We are stv^ying se^ssml 

%vm&'m ia a eearoh for suitable 



614 



serial S&. iC2.-IH8 



HIS - BIB 
iDOiviauBl Wso^t Boport 



B&T^ B: Hooon, kmas^f and PuMioatioas 



Ana. 25. f. Acad. Sei. 732 767-771, 1958. 



BuTott, N. K. : A critical amly»i® of "txsaoT ±mmd.ty> " S^ 
Chxwo. Dis. 8: 136-157, 1958. 



&7ye«t. If . K. : Sooe obmrmtiosa <m %bs mtm^ of the 

ia "tvBor ianimity." Asto. Un. latesaat. Cos^re 
Casicer (in p3>e8s). 



615 



2o (kmeral Biology Soistion 
3o Bet^osd&j, Hdo 



Indi'^ldual Projoot Heport 
Calendar leay 19^8 



Part,A^^ 



F3roJ«et Xltleg Stodlee on tb» Bow eartsoaa ^j^uis {. 

Qf Projects tmder tMs Gems«l Heading) 



Prinsi|»l Xm^estigators Wo Bay BsTsn 




^ 



One to r0@«Bt iB^rtaat dofvelopBtwats ia tb@ ttssss^tir^is 
^@ld in goeeralj, as well as to <»ertai& disoorezles ia this 
SAborato^, «oz% on the nm veil de^loped Rous 
lAboratossr sedel is beiog ©isrUiled in fswor of idJixs^oriented 
isKmstigatioas &a htnaan and ©the? aaassaliAn twmrs (6@@ a@@@s^ 
ing ?epcarts)o 

Mspositlon &f projeetss 

Zo OsTslepitent of tbs '9l?ias<^(shi@kea te@t=>^@teBi for 
gptamsl ^m® JjQ basis stttdies on 



The obJeetiTe of the spe^ifio project laader this 
topio sreperted last year, ziaBe3;ir ^® de?el^p=^ 
nent of the <^iiek senbrsae pook oomit teohaiqtse for 
assess @f the Rous s&rQ&uk 'viras, «as si2@@es3fiiUy 
aoeoasplished duidag the first fev aicnths of I9SB0 
It therefore tm leeger oonatitutes a research pg^jeeto 
The fisthod is a@w being easplegrai in routine Moassa^s^ 
with the result that one entire aniaaO. r@«M$ pre- 
vioosly used for @hi@ken assa^s^ i^ hmn released for 
other porposeso 

Ho Purfieation and properties of the ■'ririBSo 

Dis@@ntiaii@do 



616 



^■^2» »I-i420 Ca)-(o) 



lllo fiHX*tli©r stiMias oia th© y®latioa of reeorsfwrable 
-virus to t^Bfiosr initiatiisg do@®a 

BtiBKiLas in prograsiS in tsollaboration ^tfa 
Dr. MltoQ ar® b®ing ca^KTlsd to ooi^letloa (stts 
accos^saaQriog report uader this k@adiag)o 

TSf, Ijx7QStigatioiiB oa host respiQimiB to the virus » 

Studios under v&j sir® boixig oontinaed in 
UBdLt^ fashicm (eeo aots^oswo^ing report mder this 

heading)^ 

¥o RolatieiszaMp of tbo "slrtts to erthsr ohioken tixaor 

Tirussa^ partioularly of ths laakosiB ooe^lexo 

(CollaboratiY® studies with Dm. Bmrd of 
D*£i» Ifeivorei^- mad Bur^gstar ®f U,SoD,A„)c 

Disoontinueda 



Bart Bo isasloiad I©® I 



617 



o>. mi'-h2o Ca)-{©) 



- ?m ■=■ m.E ■ 

laSl'vi&m.l Projesdt Report 
Calttodftr Tear 19$8 



Part^Bs^ Eesaorsg Awards^ suod Publioaticsas 



FttbUoationa other than abstracts Teem this projects 



Bs^yan, ¥» R,s A ootaproheBSiTe revlow aikl diecui^lcn of tha 
probleaui of "QuamtitsitiTts Biologi@a.l Eap®risientatiG6a iB th© 
Viroa and Ganeer ?ieIda"o (ia pr@ss)o 



loners aad Awards relatisig to this projects 

BdeaiTod the hosiorar^ degree of O&etos* of S@i®i&3® frc»B 
Garaon^NeoBBn GoUegOp Jeff^rsos^ 01%-^ Tesmesgss^c, Sis 
reoogaitioB of coatribtttic»s to oaaear roseareh, part-i©ts2^r3^ 
for aignifieant vosl!: or th® Hot2@ sarseam -vlruSo 



618 



Seytal So. EdJ^LM 
1. lAbospatosy of MoXog? 
2o <leQAzal Biology Saetion 
3. Bsthesda ll(>, HEurlaad 



IMi-^aual Project Seporfe 
Tear 195Q 



itofe A. 



Proj<set Title: MoXogical and BtoebealcBl Studies on tlae 



Psix^ipal Iiswsetigator: J. B. Molooey 

Kiaa Teasre (c^essflay ysa? 1958): 
Itofeal: 2/3 
Pasifessieml: 1/3 
Otbsr: 2/3 

fhia pso jest <^as tessdaateS i& Aos^Mst 19^ i^^ favor of psoje^t 
UlO Sarfe A. aad ^10 Parfe B. 

^oj^o'fe B@€c?ip^lo£i: 

Obj&sti'gi&s; 1) ^ sfe«ay ffefitors asas«iat«4 wi-ai the laMlity of 
the virus tdth a -vlffw tossed yeeo^e^ of JjBSJger asajaarfea of tlm rirs^ 
fvtm ai^ailalOa gesxgem tlAsisas. 

2) To study t3ae sol® of Xl^idss ( a sajor faot@? ia 
^is stability of 1^ Botes vIsim} ia th« Mologieal aeti^ty of tb® 




-vlxtat ia inaeti 
of uBsatnzeted fatty acids ag Bo e iatcd vi* 
or tlae aiesoaoBe fieetion «ith ^&ieh the Tisrua is usmUy 
idoxtifisd (am ps^vioos Tsporte aad JoBoO^Io id: ^1^^ 19^o) 

^hss^ exists ^& yj'go cus s^paf^eixt isetabolie patt@3Si i3!^ th@ 
bogt^virois s>3lati082sbip> ^stBxeiA a taloaee is aaioctalned hs'^^sem 
t!^ ^soub:^ of osddisame suibatatO; endogSBOtts lipids pexoxld^s 
8Bd tSss ^t^ixz'sUy oecosrizts asttioxidazito* 



Studies «es« tmdsstaksn in tbia laStosatoxy to eaaeins th& 
'^a^iofos pbasas of tbis aasta'bolie pattdza^ tbus eaq^finssits wsse 
desiiped %id easnfied out to desozlte: l) Tba ^ yi-ro effect of 
aMed ot eaeg^ious lipids pexoslde m estaljlisbedii^cxoseQpic ) 
t«ao3Ps; 2) ffes s^latioaship of pofeasfeial lipid pe^side eoatfeeEfe of twBsos® 
t© (a) tijs laitiatieg dose of rlToa easd (b) to the aiBoaat ©f 
virus g«eo-e«sisMe fi^ tbe i^raeed 



619 



,..g« 



HraJjaoCb) 



3) 



I® SGlatiTe com!«£s:t2x»ti(on of lipide porosEids in "M^ 
induced vitb a bi^ eoRSiJcentvatioQ of virua) fiBi3. "lo^ 



of lipjlde pexoxide fonoaticsi ^uieTO 
carried msfc l!^irci3#i the iise of -Ihos! tlkiloMzMtuirle acM (ToBoAo) 
vea£tl<m of IbM msd Id^i@3?Sfii(S®%. Blo^.ogical x«spoQ8@s os* tutimsr 
prodvscifig activities ■v&x» mssL&ax&S. % ^^h@ e^lleatioa of qv^sx^i'lativts 
statietieal teeimigvuee to the assay etS* th® Bous vixva. 

Itojor fiafi&Bgs: l) fhe semilts of ^ tjtto egploratonr ;^^® ®ap®sisKBrfe® 
~ ^ in the taMjts below. 



CSiamct»ri0^ies of MpM tx«a.-teil essnt wsi^3r«!a-i;sa (em^sol) 



CesEtpSol 



Upide 






][|»-' 



5C^ 



T.S 



9.7 



^,% 



fl l^grassiiMasi^ 



6.6 



11^.0 



^oO 






«»p«i 



8 






2&3 






8^lfe5 




Mol. 8b M@a. 15: 67*^, 1957 



2) In pr«'«dLoas repoTts BxTsa !>as poiatt^ 0^ -^bat at least six 
(6) BHtsilfeetaticins of tit® eaiteeirouii pxosees axe x<8lated to tlia iMtiatiisg 
dose of -vl^ue. 'Saei results of t3se dose ?ecoTez7 e:q^xjae9sfee &»^^l}@a 
aho-m @hfirw a M^^ ^i^oifieaisfe llaear inelatlonehip Cijo:7es«is eora^l&tioB) 
of twsetT ^atm^ij^ lipiSe pexoxiie eosst:eat id.th iaitlatSja® dOM of fbe 



3)Ba 



pot^sc^ial lipids pexoxifie cess^axt of tiwer@ 



620 



=•3" KX-%SO ((1b)) 



iMweea "bsr M^ assd lav ©oatgeBfemticaa® ©f virm ttoa« ((3)) faffito» 
Btoat "bs eoosidexed (a) ^le ec«siice&tx%ti<3a of liplSe in tJte ttraor 
9^68 (h) the ectQceistxAticxi of oxldaist (eeAoaecms lipide poxosd^kse) 
asd (c) -yje cone«B!fentio& of oftturaily occmriKiis anfeio^aao^ to 
the tissues. 



Ttss x«8ttlte of esq^rlneffii^ dssl^oedl to illuelA&t« tSjetse faetosm 
shov thftt the hi|^ eonsestxtttion of lipids peroxide Cpoteetied)in 1cm 
dose tuBon aaA the lew Goaeeafexatioii of pot«mtial pexojdde In M^ 
dose tvBDTB is dTle not to ix&vmm lipidie or lipid® perosdde; bnt 
rather to a sl^oificisat iaereftse in astioxlQi&iit eoacexstx^tioaA Ib tte 
bi^ dose ttSBorSs 

Sigaificance ; 'Sms obssrvatiogm a« si^sltsd aliove, e<m%ril3ate to a 
furiiter trndErBteuBJlizis of factors ooDefiSQud vith 1^ labilitsr of a 
tuBor virus aod the role of XipiSes In the host<>virus relatioss^ip. 
It is po«siia« that application of tbese fisSiBgs wniM "bs of mlue 
ia Msaaialiaa tueor atadiee in ^ibieh attse^ts are heijsg aeds to 
exiraet and idestif^ etiole^eal agsatSo 



B&rt 1 isMsloSed Im 



621 



lo Laboratoxy of Biolo®' 
2o Ctoiua'al Biologj S@ctioa 
3o Bothasda. Mo 



Indi-rLdu&l P?oj«ot Repo(rt 
Caleadar loar 19^8 



Partr Aq 



Frojeot Titles Studies on ths Hous sa?ooasa -rirt^o XIIo 
Furthesr sttMiss oa the FeXatieo @if r@° 
oo^erable -^irus t& tvoB&r iaitisitiag do3@o 



F£lBcipal Im-estigsitorg ¥o B^ Bi^an 

OtSier lo^estigatorss Serot^ <^l2^^^ Jolm Fo K^%da?o 

Co@peratiag IMtss DTo Albsrt IMltoa asaS aa@o@iat©3o 



Han ImsB (®alcadar year 1953)s 
Teftals 1=1/3 

Profeesicssas 1/3 
Otlaerj! 1 

Prejest Bssoripitieas 

O MecfeLyee g To deteTsain® i^ther there is a qisaatitatlT® 
oozrelfttieQ betweesTthe iaeidenoe aod/or pepulatieQ desisi-^ @f vis^l 
partioles eeen in tMa seetiea eleetpoa aloFosraphs of tu!^?@ sMt 
(&} the dose of -^rirus used to iaitiate the tmamg @r (b) the @gs@mt 
of ezt?SQtable viTos deterainsd \^ bi@ss@^o 

SeriAl dlltjtifisi® of Boas mtc^bb vi^^ss ay@ 

groqps of 3 vea^ oM Qhlokmrn sad the ttssors ±adiMi®d 
b^ the k&oim denies are taken for studio Mien eaeh txmsp reaohes 
a etandasfd aixe the host ia killed aaad sections of tusaor are taken 
iaaediate:^ for fixation for eleotrcm Bdcrosaoi^o The r^mlsdjog 
tunor tissue is sealed in glass trials aM st4^>ed in a GCTg ±m 



622 



^2^ WOT. h20 (e) 



Saoh tuBODe- i® eactractsd aud aasi^®d for rims cotsbmt in tMs 
labo3?atoxy<, %® aleotx^a Esicroip^pMo studisa are carried out bj 
D-fo Daltoa in hi® laboratosyo 

jfe^qr findiagflg TSsa roafcina assays are carried on% as e©aa- 



T@ni^C^&io9e Hit\ othar wwk of th© Itbesratexy and esUj abeofe 
2/3 of th® tisaoers «sn hand haw« b9«a aa^r®d to datSo ilaal ©ml^^©® 
of th« data caanofc ba mde until all the resclts ara iOj, bat pre- 
lisaimzT- tabiUAtioaa of conplatod rasnlts oossf iris tbs qvAntitatiT® 
ooxrelations indioaited ii% ps-eliiaiziaKT- s^todias (saa Bto OaltmOfi 
report of 195?) o 

Tha ultimata objeetiw of the px^seiatj, wsm alsbospatoj 
etis(^ is to dafino tha matheBatia&l mtora of tha quAatitatlira 
@o7relati^^o 

Sl eaifieMacag Tha results Sff9 aspaoted to oontKlbnta to 
. ©"^CTageoneamiag tha awd® of aetiaa of tcaes? ri^m@B in 
briogins about tjaligisuxt traosfonsatiestio 



Part B iHsl^sd Im M© X 



623 



2. Qenss^X Biology Seetiosi 
3c B9th®ada»m. 



ladi'TidMal Fro;J«ct H«port 
Calendar Year 1908 



Parfc A. 



Projo0fc Titles Stwii®s on Rohs saffcoaa -sdraso I?o In° 

Testigatiosis oa the hoet rsspcns® to ths rlrusc 



Otbar Iis^estLgatoreg Jkss'&tl^ QsChrnXg Joisn Fo K-mds^ 

Ooepconting Uiaitss Mozi® 

Jfea leass Ceslaaadar y®ar 1958) s 
Totals 1 

Ppcfessiemlg 1/3 
Othors 2/3 

P^j®ot B®ses?iptioB8 

0b.^«etive8 8 Te d®t«rBdj!i® th@ oltimts lisdt t© iM.&h 
th@ @Q0e»Ki^^eii of Tires ia Rons sar©^!^ tisg%is @@a b® l^^(^s@d 
t^g>©%tgh @oatisiaffid selectiT® m®rSM, ^SB&g®&o 

Methodg,? As pfsvl-oiasSy S'epoK'tsdg the ^linis QGS^^xskiT&td.&si 
@i Boas tuaop "-^iss'tte has bsea progressiTsS^ iaei>@as@d t^ @@l@<3tiBg 
as a 6ou?oe of Tiros at sa@h passag® that tuaor «hi©h sig^ms-® firsts, 
and gromt aest rapidly^ ia a group ef hO inesiilatsd @M.3k@s^o 



Majeaf fjaadiagsg . Although pr©p^s@ is ^^i? (@m publieati^ 
sited ia foU^SnglecMm), the ^sms^ results ®f 



saleotive passages of the Sous fBaro&m trims isi jmmg Ghis&s o©s^ 
tinue to follov a slii^t xxj^msd tresad trMoh is soi? bigMj sigssif isasr^ 
statisticalSyo It is plasmed to @iDntia«a@ @u@h paa3ag@@j» altt^oiagh 
at less frvqvimk isfcerv&ls than t&icmwl^g uortil a liMt is 8>@ash@d 
begrond «Siioh the eoasentratioa of tItus mimot be further in<3rea@@da 
(Tbs tusaor tissue® ar® stored ia a GOg ie® ohest bet^aa ifirtx® passages )< 

3jUtBifloaace 8 If the ooscentratioa of virus is the som's© 

tuB»r tissue eaa' 'be"'i^1irther inereasedj, mm, fegr Oo5 of a log^o 



€24 



o2° ICI {i20 ((d) 



iater^lj it ahouM b« posaibla t© obtaia praotiosa 
qoantitlas of poriflad Rous sfltreom virus l:gr aotliodas mm 
aiplioabla to other triruaes that ar« present ia high (sonoenfera^ 
Utsn in their source tissues o 



Fart B inoladed lea X Ms 



S25 



=>3- i^O Cd) 



PUS -= JilH 
Individual I¥oJost R«port 
CalesKlar I«ar 13?58 



Part B8_ HbQorSj, Awards, aad Publlcatioa® 
Piablio&tioos oth^r tbtm abstrftcts Seem, this prv^oets 



Biy^an, Wo Rot Tbm snhsnodHe&t of vinss eenxteat ef Rous 

lo Aot& Ictsmato Union Agaisiot Candor (in pr®«e)< 



Hoaos's &ad Awstis relatixsg t@ tMs pr® jests ^©a®. 



626 



3. 

■em ^ms 

CBLsaSar fteas- 1958 



Ssslal So. ^ibi^ 

1. Ia■fe£>wfe©i^/ of S2,ol0g;f 
S. @(ssi»mX Molo^' S®etlm 



&S1A- 



FaK»i«et ¥ltl«: Seftxeb tw 



Ho 7o Ca»U^} 



X 2 1/3 
2 1/3 









ltelhe€» CTj^aayBd? ^^^^^^^^^^^^^^^^^' ■ ^Lfe Sf?A®gt 







of fSaeilitif^ te<sre ^«sffi bbSs^ or aa® ^s/i? isi |i>sa« 
I, f«r a VBe7fcB(^«feiOBft of tS» vosIe: of tits lalBoxa'tox^ toisi 

ilielml «i9evlmsEtetie» «ns tes»^ esi & ISaited se&l® Smi^ ^^ 
fint wuk ia Kmiitter, «aft full scale latemtossr op«mtioiis Ssi^ol^dBg 
«MMMit4a« tntumss a^ oaBceteA to lie vaaie? ^agr %9r 1^ »£M1& of @ee@BJl^@3>j. 
1958. 

Ite initial s^efiies vill isvalfe 
ia^KSSkvith 




aetiirittir «r ^8 18»® ««Kwaaa <«dg®r isKS^Js^ ©s^jsae^fetea s®^ ps©e®S!sS^ 



=.2» ^I-%SO if) 



in the iGiboxt&toxy. 



(S) BapM «xfe3:^sictiom; diffesraatxtlal vatztt-eeotrifugation wdA 
"ii@ishicig" of @«^lisB{m'b®S fraction to resaoYa exfcxaetiad aati1»>d3r» tefore 
further psoeesaiEtg ai3& e<»teen&xmtion of tlie aiczoeoiBe fVaeticm. 

(3) C^is^sx^saticm. of the parfelslly puxlfl<Bd Bdexosaas fsaetiox& 
ia an tjkltxi^'^ceis^rifus® prior to biologieftl testing. 



(k) liahsncessaB^ of -virus potessey 1^ s«l®<stlw pa4!»ae^@ B@iag 
the ^3st rapidly devieloplB^ ttaaers &8 sotsrcee of virus (appXicaMe 
Qsoly aft;er vires bag beei deraontstratad asd pz<ef«ga,ted. ) 



la aM^tiaa. to tbe pxosessine of sastterials iMch j^rtejatiaU^ 
xeskj eosstalii' e^ tvaeor virvis j, tbe proM«a of testiag for tla^e virua 
to bs -aoxtsed oat in oaeU izastaoce. la tS»' oaa@ of bia^a la&terimXas, 
biologieel teet>ffietl»a8 as« entirely iSBikaB«ii at the preoest tissB mA 
a l&z^ pree^atage of otzr isitiial effort vill ^ in -^le @s^lo£%tioa 



Bart B imlufiea T@s lb X 



628 



Ic, .Labora.toxy ©f BioX;&gy 
2„ General Bis?:iogy Seetifc.n 
3 a Bethesda, Maryland 



Individual ProJ«et Report. 



^^J, 



Projeet Titles Oanetie sstudias in aieso Ao Tumora ,to rai'^e 

B, Miiitatiswg in laisoo 



Principal lRv®stigat©rx Margaret Ko D®ringe.r 
OtJser Investigators 2 W^ E. Haston^ M, Ko Barretfc 

Coopssrating Undtss flea© 



Mas Tears (aalendar y®ar 19$B) t 
Tetali 2 

Pr©fessis^l8 1 
Others 1 



Ao Tvsmrs in 2aie®o 

io O&cmTBnQB of tvm»TB. parfci©u2®i?2y 
tyaiors^ in ag©nt=frae s'»alE CJHtsB Easso 



Ob^ Qet iiy es a In stadiss ©f aaiaassy t«iti©rs there i® csften 
a n&ed 'for"a"W©aiR ©f ad.e@ «rhi©h dees jjot have the j^kissairj traasr 



agent but i^ii©h is genetically susceptible to ito One istaeh strarix^ 
is substrain CJHeB (hereimfter referred t© m C3Ke) wM-ssh. wa-^ 
produced liy traasfez- of fertilised m& froa straia G3H t^ strain 
OJlSLo The occurrsEce of asjaaa^ t^sissrs aisd ©f ether Igrpes ©f 
tusaor© baa been obserfsd in groups ©f Tirgiag breedSag, and {or-G&= 
bred C3He fe5Ba3^s asd in a groi^ of C3H© Kales, tr^tad with 
stllbestrolo 

Ife^thpds ^^ EMplefjjIjed % A groi^ of C3B© f emles ms kept as 
•7irging°'^d°'^^?ffir^^i^ was f ©re®=>bredo The feree-bred fea^lss 



629 



were iselated when pregnant „ After birth of the litter ^ the 
young w«Br© cii.-e^iustd„aod the mothers were retumed to the breed^^ 
ing cages at aau^^ This prosodure was ©ontinued throughout the 
breeding period of the females « Choleatrol pellets ^ weighing 
6 to 8 mgo SKd containing IGjg diethylstilbestrolj were implanted 
Biibcutaneousay in the right axilla of C3He males lAiish were ©he t© 
two months of agOo The breeding females were those whieh were used 
to maintain the colory of C3He micso The farioua groups ©f miee 
were observed at regular intermls for the de^lopmeat of amnaEisry 
tumor So 

Ma.1or Findingas Kfemmy tujaors developed in h W of 99 
■rlrgln fSti^aTsnaverage age ©f 20ol imtAha, in 8^ ($hM) of 
1^5 females of the breeding soloi^ at an STerag© age of 19o2 moErfeJas,, 
in 61 (IhM) oS 82 forc@<=br®d. females at an average age of llc9 
months, and in 22 (22 M) of 98 males, treated with stilbestrol, 
at an STsrage age ©f 15 months o Data for this substrain ©orrespomiod 
quite elosolj with data obtained pr«^eus3y for the CM substraia^ 
a strain depriwd of the masmiay tumor agent tgr fost@r=nur@±ngo 
Though the presence ©f the mammasj tumor agent is .j: "mportaat 
faetor in the dwelopmesxt ©f mm&ry turners, it is wll !m©«a tlmt 
mammae tumors will develop in miee in the absenee ©f th® agaato 

MenfciiOT should be mde of th«& ^^ecretosy Actiwlt^ ©bs®wed in 
histologic sections ©f seme of the maaasary tumors wbieh ©eem'TQd In 
C3Hs ai0©o It 1^3 observed in 1 of U tumors in virgin femles^ 
in 11 of 96 tumors ocsurring in br®«ding females, in 8 of Ik tiw@r« 
oceurs'iBg in f©ree<=bred females, and in 1$ ©f 22 tumors ©c^Hsrriag 
in mles treated with stilbestrolo 

On© of the outstanding observations in this stuc]^ was th© 
high frequent of hepatoMs that occurred in C3He mlceo They 
developed in 58 (58=6?;) of ^9 virgin females at an average ag@ ©f 
2Uol monthsj in kl (30.%) ©f 155 breeding f amies at » awrsg© 
age of 21 months, in 31 (3?o8^) of 82 for©s-bred fmalma at an 
average age of 20.5 months, in $h C55o3^} of 98 males, treated 
with stilbestrol, at an average age of 23o3 nonths^ and In 19 
(90o5$) of 21 breeding males at an average age of 21oli monthSo This 
is the highest occurrence of spontaneous hepfttcxnas @mr reportsd f ©r 
aiay str^n of miceo 

Various other tumors have been observed in G3H© udcso Tha 
Mgh incidences of ovarian tmors were most stHMngo O^ai-ian t»ors 
occurred in hi ihlo$%) of 99 virgin foaales at an average age of 
2l4o3 months, in 58 C3?oh^) of 155 breeding females at an average ag® 



630 



= > JMUX=^.r:x 



of 21o5>' iBontbfj, and in 2JI4 C29o2?S) oJT 82 foroe^bred f&mlm at 
an average age of 21o6 montteo Those results record the highest 
iaeidencsB vmr ©bsorvad in jaiceo The tumors were frequently 
fflsaall and ©©©wrred Qft&n in cormeetion with (tgrstSo O^^arian egrats 
were often se®n in f ©aisles in which ovarian tumors did not ©ceuTo 

^m^teS?,> ,^^g,P^^lF^ of the Immates This experi=^ 
mont has au-^M(M inrormaiion "cenfflsxtiing th© de¥"ei.©pffient of 
mauHBaxy and other typea of tumors in a jstrain of Bdce deprived of 
the wBxfomrj tusaor agent by th© proeedur® of transferring ©va from. 
wi inbred, strain of adee carrying the agent to another strain without 
th© agento Th© results reflect on the interpr©tati<wi of th© 
relative effects ©f th® g®net5.c cesastitution, the Baammiy tumor agent- j, 
and the hormoml influence on the developaient ©f roamrrmy turners c. 



^^g^.g^TO?.,,®^ ^^^,„E?gi1^f^V This expffi'iment ha® been 
©os^leted aM the results through six generations Mt© been presented 
in a paper »iiieh has been eubodtted for publisaticsn in th© Jownal 
©f tfe® Ifetional CansaK- Institutso The strain of mi©e will b© c©»= 
tinuad and the development ©f mn;---^:sj t«Hnors will bo studied in 
subsequent generations p 

2o Detexiaination of presence or absence ®f th® mmmrj 
tuasor agent Isgr foster-nursingo 

^Objectives? To determina whether a mamHSiry tumor agent is 
p!r©sent"TS""8lKlS™BL and in strain SWR (in cooperation with I3p„ 
H®st^a)o 

An inoidencs ©^ i^ismmrj tumors of ij$ in virgin fesmles asad 
of 21% in breeding f ensiles ©f strain BL and of 19^ in breedirjg 
fefiffliles of strain SMi has been reported o In this labeacmtoxy m&mmrj 
tumors have developed in 25 (iSM) of 15© breeding femles of 

strain BLo Only ©no smmaasy tumesr Ms appeared in one ©f a group 
of 60 breeding females ©f strain SWRo 

itotheds ^ CB^torad g Groups ©f newbora female mice of strains 
C3Hf or~l!!3Be'^r9 fosfer^nursed on strain BL and on strain SWE feiaalesc 
The f oster=nursed f csmales were maintained as virgins and were 
obss3?v©d at regular interwil® for the developraent of 
tumors,, 



631 



h'- NCI-U21 



!fe^|or fJjrKJin^sg ManBnaxy tumora haTe developed ia k3 
(hBo9% foTWl^iMCW or C3H9 femlos, foater=.nursed h^ 
strain BL fcBm»l8S, at an averago age of l8o3 aioafchSo Forty-five 
@f 68 f&ml@tt have died irlthout taAaaaasy tuinors at an aT«rage «g» 
©f 20o9 fflonthso Maiamajcy tumors haw developed in 3 C3o6^} of 82 
females, fosteivnwsed tgr etrain SWE, at an average ago &f 23 montte^ 
Serventy^nine of 62 f enalee have died' without jnaomasy tumors at &n 
average age of 2li.o? months „ Thee© data seem to indieate the 
presence of a raamraaiy tumor" agent in strain BL but not in stxain 
SWRo 



oontriDttte f tsyther kn©«ledge concerning the mod 



Thi.s experiment 

will oontri^te ftsyther knowledge concerning the mode of aetdon 



of one of the factors^ tho tmarnxj Imvsa" agentj whleh is responsibla 
for the davel'»ss^®nt of maiufflary tianort! in ©erfcaia strains of misso 



Proposed courae of ^^^the^ ]?^.9A®if-^, i "^^ resnaltsfer th® strain 

SWR 8isgg€^"M!^C^F''^c^°°t©''lHae^'rimento Perlmps ati^aia SWR^ 
though not carrying the agent, may like strain BALS/e be swcsptibla 
to ito To stiu«^ this, strain SMI neiJbora mice idil be fcMter^mirsffid 
on strain C3H famales and will later be brado Th© oecmrene© of 
BSUfflEajy ttssffirs in subsequent generations of sueh lines •wi.ll b-s 
studied. 

3o Ocearrence of mamaary tvmoTs ia agent=fr®e DB«^/2 ffiis©o 



Ob^^ectiveas Strain I®A/2e, a subst^in of strain TBk/2 m.@ 
produeed°1^'^'ISe"&insfer of fertilised &m from strain riBA./2 to 
©train C57BLo The derelopmeat of wammsj tumors will b® »tiadi®d 



in group® of virgin, breeding, and fowse^bred f©nsile®s and in 

jmlas treated with stilbestrolo 

^^^^^^1^^ A breeding eolongr of DB4/2a mie@ hm hmn 
egtabliiSir°°TgTOup©F virgin f smiles has been set aoid© and a 
groiip of males has been treated with stilbestrelo JfetingSj in wMch 
th® srdjsmls will be f©r©9=.br®ds, are now being madSo 

teJ^^^JJHdtoggig Moat of tho animals in this expertoeot ar© 

still iro®r*°^elm72e male, treated with -stilbestrolj, has dw^loped 
a swssaaxy tumor at 12 months of agSo 



Si^^rriff.^mnoe to^^the prom^ r a jm of the I natittttes The prodaetion 
of anotlieffMilm'2y''''rS®r 

mother substrain i&ish will be useful in variotss ^pms of ©xperimsKts 
in yiieh studies of smsemty tumors are rmd^o 



632 



^-5- WCI=U21 



^^^^J^^i^ 



AdequAte nuaberei of Tijrgin 
stilbeatrolj b&ve been obtained » 
Sufficient breeding and forc8=brod femalos will bo obtained and 
all groupa Td.ll be observed continuously for the development of 
tuRorSo 

Uo Pfcduetioa of tumDrs of the «k±no 

OMectivesg To atteaspt to produce tunors of the skin in 
strsin mmcs^ty psdnting with ur®thano Since it has been observed 
that urethan is "noa=«5arcinogQnie for skia ©pithelium in the mouse" 
it seerasd wortfewMl© to treat mis® of strain M^ %M.ch are highly 
suscsptibl® to the d®ir©l©pia©S3t of tumors of the akias with this 
oojspotsndo 

jtethodsjCTgsleyed g A 1^ solution of urethan in ethgrlerie glycol 
is beiJ^palnieS on"^^ skin of haired and hairless aniBsals of boffeh 
esxes of the HR strains „ Other groups of isales and fenales are 
painted with stJgrlen© g3ycol alone o Twice ws6k3y paintings are 
started whan tha aaiiRala are 2 1/2 to 3 1/^ months of age ^nd are 
continued throughout the lifespan ©f the animals » 

^^J^L S^^^SML ^s* o-^ *^i® aniaials in this experiment ar@ 
still aliTOo To date n© tumors of the skia ha^e ©ceurred in aigr of 
the anis^ila. 

^^l^ggJg^?i^Q,,Pf.«^|yn^ te . Ma |Ste This project 
•Mill addlv^^^oiTOaMon conc^nim% ti^ of the genstoc 

constitution on the dQvelopHQnfc of tuswrso The effect of a 
carcinogen on the dsrelopment of a pirtieular %pe of tisior^, to 
which tha skia of animals is suse®ptiblej will be det@r3dji®do 

^.gPggM,. gg^L.'g0 ^^f!g^,!P.^^.^ Groups of strain HR males and 
f amaleslCTnerinSI' HeiessT^ll cenfeinae t© be painted 
with ureidaan in etlgrlene glycol twice a weekj sM with ethylene 
glycol aloneo The anisaals will be observed continuously for th© 
development of timers of the skin or of other Isaionso 

5o Iffiaauaogenetic laspejdsiontsg 

pbleotiYess To produce special lypes of aniraals for 
Qollabora^KS"^«-isE©ats with DTo MoKc Barrett (sa® DTo Barrstfs 
report for details) » 



633 



-6 NCI«U21 



60 Influence of tho lathal-yellow (Ay'a) gene on the 
devalopsaent of leukemia in Ddce„ 

ObJa^WM? To determine the relationship of the 
lethal<=>7ellov gene (knovn to increisise body sise) ftnd the 
oecurrenos of letikefmia (in cooperation vlth Oto Heston)o 

MeiAodB eTO >3 ^Cids^ Crosses hsT© baen jsade between 
strain 'W!/!^£'eetra.in " auaceptible to the deTslopnent of 
leukaida, and «tr«in IBRj a strain <arxying the lethal-yellcw 
geneo The Fi mio® will be painted with ^mettgrleholanthrene aiad 
observed for the occurrence of induced leukesiia and t^ntreated 
Fi ndce will be obsorved for spontaneous leukessdao 

^&M^Ml ^^® ©xperimsafe has Just been started 
nmd P^io tms results nsvB been obtained o 

j^j§SJJ^£^.S.^.. .1^^ !^l^^.._ £^9l^y^ Q^ the Inst itutes This ea^peri^ 
ment tdll den^trate iK "role of the "leiliai^yenow gene in th® 
derelopsaent of leuketraia in niceo 

-?^ gggf.^- ,PilR^^!!iii.i^f ■'¥^?.iPI!''9'i1®®i^^. ^* present oaly a portion 
of the a^ffi^"ae^^SyT^°'lffii^^rimeat hawe been p^'oduced^ 
Additional aaisaals will be produced o One-half of the total 
will be painted with 3==®ethrleholantthrene and all will be 
obserrod for the der^elopment of leukesiiao 

Bo Mutations in Rsice, 

lo Mutant affecting locomoti^io 

^j<>g^j-J g£i "^^ *®^*^ * mutaatg phano^pieaUy-like 
waltaerT^idSclTarose in strain A nsiee (in cooperation with DtTo ffeston) = 



jrg°giagi 



Crosses of affected strain A f etualas 



with Biaies of the nutant stock 7 (which carries the claractey 

waltser) were siadSo 

}fe; |e r ^ ^ flndinggg Data, obtained prwslouslyg fy«a crosses with 
strain Slpii, indl'Ste "that tho character is inherited as a simple 
recessiTec The gene exhibits aoss© lethal affect^ as indicated 
try the failure to obtain the expected Isl ratio in the segregatiem 
of th® baekoross offsprings 

The F^ offsprdjig of a cross between affected strain A females 



634 



aad w&itsey stock ? isalea wer« all ruxsnmlo Therefore th® 
star&cter in the strain A iid.^e ia not lAEiltKer aixi la a reoBSBiyire 
SIS pre^ously thought o 

, _, S|^S^.S^V^, .^^.JFm*^..."^ )?!ff.^^^«^« 'TJ* at«^ 
of this Bmtamt aa;f be of signif Icftnee iii providing another kn&m 

charaoter iMoh will be of nse in linkage studies of factors 

isfluenoiag susceptibility to various t^es of tumors o 

Proposed coarse ^_of pro, - )eqti Crosses between affected 
strain A laice end nice of" other' nitttant stocks are planned <> 

2o Mutant affecting clMiracter of the eoato 

O b, .- |ectivea g To test a smtant, phenotgrpieally-like Carae?xly 
yM.ch ar©se'''JLn''"»&aia C3Hf mieeo 



tfethods emplcyadg Mutant aniualfl of the C3Hf storain hare 
K)8's(aS''^^n axSiis of the OA stock (which carry Caracul s 
which hs:ye the geao^pe Ca/*)o The F^^ aadjaals from this cress 



ware aated to j^oduce Fg anlaalSo Data oa the oecurrence of 

the Gsotation in these ajsdmals were obtained « 

}Ssi ;\&t findini^s ? The results obtained indicate that ih© 
diaract^ ija qu^timi in strain C3Hf May possibay be Caxmejilo 
Of 10k (GW X CA)F, ffliee^ 83 (19oB%) were "Caracul" md 21 

C20o2^) were aouwalt Of 21$ F, wdcej 230 C83o6$) were "GaraeuF 
and Ii5 Cl6oli5S) 



Siffid f i oajQce to ^ the p r oj^gy. „e f ,. the ^ la st ltuteg The stedy of 
this iwjSat;'''Bsgr' proricie""a cimSctl^IiriS'la'"'C3Mf'' which mn be 
used in linkage studies of factors iisfluencing susceptibility to ' 
'various tuiaorsa 

I mposed course of project s Cresses between autant G3Hf 

Bice wi!ill"'be""3a!^e wiiii a strainhbaroagrgoag for Oaraculp If one is 
s^^ilabloo 



Part B ineloded Yes X No 



635 



i^Wi-XKlX «Wc. «vx°u<:x ^ 



PES =• NIH 
X«d±rldml Projsct Rsport 
Calendar Year 1958 



Part Bs Honors J Awaipds, and Publissatiojas 

Publlestio»ai other than abstr&cte froe this projects 

7)9Tix3g&i?g K^ Kot J/^m&ry txtmesta azsd hepatoana in agents -free 
strain cSseB 3sde©o Ae1»i Itaio Inten»to Contra Cancrtan (iK press), 



Honors and Awards relating to thia prefects; Heme 



636 



Serial Noo NCI"lt33 

lo Laboratory of Biology 
2o General Biology Soetion 
3o Betheada lli, Marylend 



PHS = NIH 
Individual Project Report 
Calendar Year 1958 



Project Titles Studies on the viral etiology of 
malignant tumors in 



Part A< 



Principal Investigators DTo Sarah Eo Stewart 

Other Investigators Dro Bemioe Eddy 

Cooperating Ifeitss none 

Man Tears (calendar year 1958) t 
Totals 6 
Professional; 2 
Other; k 

^;oject Desc ription? 

In our earlier work we raported the release of a tumor inducing 
substance in the fluids of monkey kidney or mouse embEyo tissue 
cultures idiich had been inoculated with preparation from certain 
mouse tumors o It was shown that the oncogenic agent could be 
passed serially in tissue cultureo Such fluids produced multiple 
types of neoplaams in randomly bred Swiss mice and in mice of 
inbred lines if inoculated shortly after birth „ Hamsters, both 
newly bom and animals through 2k days of age were also sus- 
ceptible to tumor induction hy this agent o 

The tumor inducing substance in the tissue culture fluids has 
now been characterised as a virus j and we have shown that we 
are dealing with only one oncogenic virus hy the following 
experiments % 

&o Rectilture of ultimate dilutions of the virus produced 
the same spectrum of neoplasms in mice as were observed with un» 
diluted virus cultures o 

bo Virus recultured in tissue cultures after storage at 
-20®G for 5 weeks produced the same spectrtan of tumor as freshly 
har-vestsd virus „ 

Co Mouse protection tests showed reciprocal cross 

637 



=2=- NCI°U33 



neutralization with aati°serum prepared from, the different virus 
sublines « 

do Filtratdon through gradocol membranes did not result 
in separation of more than one oncogenic virus <, 

The virus has been referred to as SE polyoma o 

SK polyoma was found to produce «^opathic changes in re=tiypsinised 
mouse embryo cultures o In our earlier work the cytopathic changes 
were variable o This was found to be due to an inhibitory substances 
in the calf serum which was used in the maintenance mediumo The 
cla.Lf serum has been substituted with rabbit or horse serum and 
consistant cytopathic changes have been observed o Gytopathogenic 
activity was shown to be correlated with tumor induction in hamsters » 

The virus was also found to produce hemagglutinins for several 
types of esythrocytes o The hemagglutination activity was also 
found to be correlated with the tumor induction^ 

Newly bom rabbits and rats were also susceptible to the oncogenic 
effects of the virus <> The rabbits the neoplasms were benign sub^ 
cutaneous nodules resembling fibroamso These regressed after 30 
to 120 d&ySo Sate developed renal angiosarcwnas o 

Virus neutralising antibodies were demonstrated in the following? 

&o Animals bearing tumors » 

bo Control mice without tumors but housed in the sase 
©nvircpaent as mice with tumors » 

Co IMinoculated mice in virus environment o 

do Two of the persoxmel exposed to the virus o 
Inhibition of virus was produced with certain human gamna globulins o 

A vaccine was prepared from live SE polyoma virus mixed with rabbit jtj^wffla© 

sezTBHo Hamsters given 2 injections, one when 2 days old and the 

other 6 days later^ were protected (97 per cent) when challssnged 

with live virus 2 weeks latere Sixty=seven per cent of control 

litter KMiteSs kept in a virus free enrironment and inoculated 

with the same virus challenge ^ died with tumors within $ months » 

The recovery of polyoma virus from human neoplasms ^^-^o 

The tissue culture technique described for demonstrating a virus 



638 



-=3° mi-h33 



in certain mouse neoplasms was applied to the stuc^ of human 
inaterifils obtained from cancer patienta and from autopsies o 
The following have been tested § 

(1) The buffy coat from bloods of ? adult patients with 
acute iryelogenous or acute lymphocytic leukeniiaSo Tissue cultures 
inoculated with these were carzded through 2 to 8 passages » No 
tumor inducing -^rus was demonstrated in any of these cultures o 

(2) Brain tissue obtained at autopsies from 6 adults 
who died from ngrelogenous or lymph®Qytic leukeinias» Tissue cul= 
tures were inoculated with a mince of the brain tissue in one 
series and with a trypsin digest concentrate (Ed<^ et al) pre= 
pared fr^n the brain in another asides <, fkase were carried 
through k to 10 passages o None of the mice inoculated with 
fluids from the cultures have developed parotid gland tumors o 

(3) Brain and tumor obtained at autopsy from 3 adults 
with carcinomas and one with a malignant melanoma « Cultures 
were inoculated with a mince of the tissues or with a trypsin 
digest concentrate o Fluids from 1st to 8th tissue culture 
passages failed to induce tumors in either mice or hamster o 

(li) Brain obtained at autopsy from 12 children that 
had died of acute lymphatic leukemias, and brain and tumor 
from 2 with the same disease and 1 with a Wilms tumoro Tissue 
cultures were inoculated with tissue minces prepared from these „ 
Trypsin digast concentrates prepared from 6 were also cultured = 

The age of the children autopsied ranged frtm 10 months to Ih 

yearSa 

Polyoma virus was recovered frraa 5 of these. In 2 both byain 
aK0' tumor were cultured a In both, the cultures cariying the braixs 
preparations became positive after the first passage* while the 
virus was not demonstrated in the tumor cultures until after 5 
or more passages ,. 

Of the 5 positive results, 3 were from the concentrates „ 

Since sera fron cancer patients have been found free of «nti<= 
bodies specific for polyoma virus as shown by ccsnplQraent^fisstion 
tests (Rowe et al) , one must consider the possibility of lab- 
oratoiy cantamination in the positive culture frtan the hmmn 
material,, 

The followingj hewerrerj indicate that the source of the virus 



639 



NCI-li33 



vas from the human materials tested: 



(1) All the huinsm materials were handled on a separate 
day from the mouse virus cultures o 

(2) Ncoie of the material from adults with neoplasms have 
been positive for polyoma virus, although handled in the same 
wa^ as the positive oneSo 

(3) In those Instances where both brain and tumor material 
were tested (from children) ^ the brain cultures were the first to 
become positive „ 

It is possible that the inability to demoiistrate polyoma virus 
antibodies in human sera by the c«nplement=fixation tests, nay 
be due to the antigen emplcyedo The antigen used was virus 
cultured in mouse embryo cells „ It is proposed to cai^ out 
similar tests using a virus grown in human cell cultures » 



Part B included Yes X No 



640 



Serj^l Noo MCI-If3^ 



PES = NIH 

Individual Project Report 

Calendar Year 1958 



Part Bs Honors. J, Awards, and Publications 



Stewart, S. Eo, Edctjr, Bo Eo, and Borgese, N„ G,: Neoplasms 

in mice inoculated ^th tumor agent carried in tissue 
culture o Jo Nato (fencer Inato 20 x 1223-12l43s 1958 o 

Eddyj Bo E,, Stewart, S„ Eo, Young, Ho, and Midsr Go BcS 

Neoplasms in hamsters induced hj mouse tumor agents 
passed in tissue culture o Jo Nato Cancer Insto 20? 
7^7=761, 1958 o 

Eddy, Bo Eo, Stewart, So Eo, Stanton, Mo Fo, and Marcotte, Mo JoS 
Induction of tumors in rats ty SE polycsaa virus eaibiyo 
tissue culture preparations o Jo Nato Cancer Insto 
Jano, 1959 

Stewart, So E„, and Edc^, Bo E« Properties of a tumor Inducing 
■ylrus recovered from mouse neoplasms o Psrspectives in 
Viroloar, Rutgers University Press » 1958 o (in press) o 

Stsi#art, So Eo, Ed<^, Bo Eo, and Stanton, Mo F,j Induction of 
neoplasms in mice and other msmmals Ic^ a tumor agent 
carried in tissue culture. Proceedings 3rd Canadian 
Cancer Confer®, 1958 o (in press) » 

Eddy, Bo Eo, Howe, Wo Do, Hartley, Jo ¥„, Stewart, S, Eo, and 
Huebner, Ho Jo x Heaagglutination with the SE po3yoina 
virus o Virology 6s 290°291, 1958 » 

Eddy, Bo Eo, Stenaarfc, So E„, and Berkel^, W.J C^-bop&thogenicity 
in tissue culture bgr & tumor virus from ndceo ProCo 
Soco Expero Blolo and Medo 98s 8U8=85l, 1958 o 

Stewart, S„ Eo, and Eddy, Bo EoS Tumor induction by SE po3^oiffia 
viiTis and the inhibition of tumors by specific neu= 
tralising antibodies » Amero Jo Pubo Health o (in press) c 



641 



Part A< 



PHS-NIK Serial Noo Woi 42V 

Indi^iduai Project Report lo (Laboratory of Biology 

Calendar Year 1950 2o (Leukemia^Studies Sectioji 

3o CBethesda 14„ Mdo) 



Studies on the etiology and chemotherapy of experimental 
lymphoma s 

Lo <4o Law 

Man years (calendar year 1956) 
Totals 4 2/3 

Professional; Lo Jlo Lawg 2/3 
Other ; 4 

Ao Role of thymic tissue and boae Mirrow in X^ray^iaduced and 
spontane ous leukoses ~ 

Certain aspects of this work have been continued and others 
are planned for the future o 

lo Thymic grafts (C57BL) introduced into millipore chambers 
failed to transform into lymphomasg although in many cases soots 
round cells and epithelial tissue were found 120 days after intra^ 
peritoneal introduction into thyraectoraisedo irradiated BAFje and 
also G57BL aiiceo Thymic tissue grafted under the kidney capsule, 
howe^ero transformed to lymphocytic neoplasms in nearly 3(^ 
of the animalso 

2o Following the results reported last year by Ophoff & 
Law of the leukemic potentialities of homologous radiation chiiseraSft 
experiments have been set up in which, by the Medawar technique 
(acquired tolerance) AKE bone marrow is introduced into early 
neonatal C3Hf/U« and C3Hf/Bi mice and the reciprocal o In addi- 
tion ^ thymic tissue of Alffi mice has been introduced into the 
C3H sublines so that„ in effecto C3H mice are chinreras (theoretically) 
so far as the hematopoietic and lymphoid tissues are concernedo 
Fifty percent of these mice took skin grafts (reciprocally) o None 
of the C3Hf/Lw mice have developed leukemia up to 12 months of agso 
The incidence and mean age at death of AKR chimeras closely 
approaches that of the intact AKR but there is some evidence tliat 
the n^an age at death is lengthenedo In contrasts C3Hf/Bi mice 
receiving 1=2 month AKH bone marroWo at birtho develop parotid 
gland neoplasms (25% to date)o This part of the program is 
being extended to include hemagglutinating-^antibody determisia» 
tions on AKR donors and of C3Hf/Bi mothers of the recipient raiceo 



642 



Part Ac 



Serial Ko_;^J^J|X=- 

lo ■ (nrijoratory oFlioiogy) 
2o Cl£ukemia=Studies Section) 
3o CBethesds 14;, Mdo) 



3o In coUaboration with Miss Ophoffo experiments are 
in progress to gain some insight into the abscopal effects 
of X^radiation in thymic induced lymphoiaas in C57B5^ and BAFj 
miceo The persistence for at least 28 days in thyfflectomized„ 
irradiated mice of the potentiality to transform thymic grafts 
into lymphoinas has been observed in this laboratoryo Foliow= 
ing fractiomnedo whole-body irradiation^ isologous bone marrow 
has been given intravenously to MFj mice within 24 hourso 
Periodically thereafter from 3 to 21 days^ thymic grafts ha^e 
been made to MFj miceo To date^ 12 monthSo no lymphomas 
have appeared in the bone marrow group o C57BL mice are presently 
being set upo 

4o It is the plan for the coming year to stsady the com- 
parali^e effects of isologous and homologous embryonic heraa=. 
topoietic Cli^r) tissue with isologous and homologous adult 
bone marrowo C57BL mice using several H-2^ donors and DM/2 
mice using H=2^o as weil as donors having other H=2 aliels^ 
will be usedo There is some preliminary information that 
X-rayed mice of certain genotypes die earlier of thymic lym- 
phomas than those uninocuiated controls if they receiw erabryor.ic 
hematopoietic tissues shortly after X-raying^ 

^° X^gadiation aad lymphomas o^ mice 

lo Several experiments are in progresso or are beissg 
piannedo to investigate the following aspects of X^rsy iuducsd 
neoplasms of reticular tissues; 

ad The influence of fractionated whole-body 
X-radiation (4xl6G r) is being investigated in thya^etoniised 
C57BL strain mice,, There is preliminary evidence of 40=5(^ 
neoplasms of Type A and B reticuhca cell sarcocaas in this strsia 
following X-radiation if the potentiality to develop thymic 
lymphomas is removed by thymectoi^o The role of spleen shielding 
as it relatss to the induction of these neoplasms is also being 
investigated 

bo Since our preliminary oi3servations (l^v^ & Ophoff) 
show evidence of a response of intact C57BL mice to dosages as 
low as 25 r whole-body irradiation „ if irradiated at births the 
response of C57BL/Ka mice to different dosage rates will he 

investigatedo A wide range of dosage rates will be used,, 3 r to 
CO r/hoaro in which the same total dose is given over the sam 
period of timso This will bB investigated also for RFM ffiiGe„ 
thyssectoraizeds concerning the role of dosage rates as they 
influeace the industion of graauloeytiG neopiasmso 



643 



Part Ao 



(Attachment I) 
Serial Noc NCI 427 

1 o {Liaborat'ory of Biology ) 
2<, (Leukemia-Studies Section} 
3o (Bethesda 14 » Mo} 



Co The observations of Upton and Furth have been cosi» 
firmed of a unique responsiveness of thymectomized RFU mice 
to single 350 r or whole-body X->radiationo The effects of 
fractionation and protractiosio of spleen shielding and intro^ 
duction of isologous marrow and of thyineetoiny will be studied 
in RFM miceo Intact mice will be used to study these effects 
on iyraphocytie neoplasms and thymectomized mice to study the 
influence on the X^ray induction of granulocytic neoplasmso 

do A study of the comparative responses of 3 subliaes 
of C3H mice to whole-body X->radiation is in progress o Nearly 
40% C3Hf/Bi (Bittner) mice have developed lymphocytic neoplasms 
(chiefly thymic) to date, following 4x150 r X«rayso The 
response of C3Hf/Lw C5=l(^ leukoses spontaneously) and C3ef/Fg 
(00% leukoses spontaneously) are currently under study^ CF aad 
FIA antibody was determined at 1 month of age, later at 3 months 
of ageo and at appearance of leukemiao Nearly 50% of these 
mice convert between 3<»6 months of agCo following exposure to 
the parotid tumor agents There has been seen no relationship 
between the development of lymphocytic neoplasms in this group 
and their conversiono Simiiariyo in a group of AKR/LssN raice 
obtained from Oro Jayo Animal Production^, no conversion was 
seen in this group derived originally from our colony; yet 
the incidence of thymic leukemias and mean age at death is 
strikingly similar to a group maintained in this laboratory 
in which CF and HA antibodies are found after 3 months in 
30»40% of the individualso 

Cell^^free agents and tissu e culture agent s in the induction of 
lymphomas and other neoplasms ' ' 

Collaborative efforts in this phase of work have b@en 
with Drso Dawe and Dunn, Laboratory of Pathology « M^I, Dro Kilhamg 
DBSo Dro Rowe^ LID and Dro Roosdo this sectiono 

lo The study with DTo Dunn, using C3Rf/Bi mice as recip^ 
ients of certain materials from leukemic mice has been 
The results are as follows j (See l^ble-^Page 4^o 



644 



(Attachctent I) 
^« Serial No, NCI 427 

lo CEboratory' of Biology) 
2» ((Leukemia-Studies Section: 
Part Ao 3o CBethesda 14„ Maryland) 



Average Age 
Noc of Leukemia at Death Parotid Gland Neoplasms 
Strain Mice No, % (months) Noo % 

C3Hf/Bi EXPTL 149 22® 15o0 iOol 6 4oO 

Controls 64 14 21 o9 ISoC 



13 leukemic deaths at 3-^10 months before the 1st leukemic death in controls 

at 11 months^ 

5CP^ lymphocytic neoplasms in both groups o 

Only 4 of 16 filtrates of AKR spontaneous leukeraias gave evidence of 

earlier appearance of leukemia or of parotid gland neoplasnso 

No neoplasms were observed in C3Hf/Lw mice given identical cell=free 

materialso These „ however^ were sacrificed at 14 months of agco 



2o A collaborative study with Dro Clyde Da»i'eo LFath^ 
NCIo will be reported in more detail by hiffio This project 
entails a study of the characteristics of agents obtained 
from several sources s I) from a DM lymphocytic neoplasm <P36Cj 
grown continuously in culture g 2) from a C3Hf/Bi lymphocytis 
neoplasm (Passage A of Gross) and 3) from 1^2 month old AKH 
bone Eiarroi^o C3Hf/Bi and (CSHxAKR) recipientSo 1-14 days of 
age^ develop nearly 10(% parotid glsnd tumors and epitheliomas 
of the thymus and other associated neoplasms and lesions o 
from source 2) above which is carried in P308 lymphoblasts 
in vitroo Virulence has increased thru 10^ titrations in 
tissue culture so that thymic epitheliomas now appear at 21 
days of age or earlier o All strains of mice so far tested 
have shown some response to the cell-^free material tvom 
culture but somoo notably SM and C37BLo appear quite resist@nt< 
Strain differences is response are notedo A litter effect „ 
noted earlier is still seen using our highly virulent material, 
but in some cases this is expressed in the latent period rather 
than in complete absence of neoplasms o There appears to be 
no striking relationship of the antibody status iCF & HA) of 
the mother nor of parturition age to this litter effecto 

No leukemias have been observed in C3Rf./Bi mice reeei^isg 
tissue culture materiaisp nor has there been acceleration in 
frequency or age at appearance of leukemia in AKH or CC3HkAKE) 
mice, both having potentialities to develop these neoplasms 
spontaneous lyo 



645 



Part Ao 



(Attachment I) 

Serial Noo NCI 427 

lo CI^Doraiory oiBxoIoc 

2o CIieukemia=Studies Section] 

3o (Bethesda 14„ Mdo) 



Short-term cultures of 10 to 15 transplantable leukemia s 
of spleens of several strains (including CF and liA negative 
C50 and AKR) and of several breast adenocarcinomas have been 
negative o 

Thymectomy at 3 weeks of age and irradiation at 5 days 
(400 r) do not influence the frequency or appearance of parotid 
gland neoplasms o 

The following lines appear profitable to pursue in the 
near futures 

ao Parailelistn of infectivity„ hemagglutination 
titreso and (3*Eo correlated with electron microscopy studies 
(Dro Kahiero NCI) of our continuous passage agento 

bo Attempts to transform in vitro thymic epithelium 
and parotid gland tissues with virulent parotid gland agento 

Co Factors concerned with disappearance of ?TA in 
certain transplantable leukemiaso 

do Further study of antibody status of mother in 
reUtion to infectivity of youngo 

Bo Efforts to define the specific morphologic CPE 
effects in a population predominately of a single morpholcgic 
cell type and the influence of nutritional changes in tissue 
culture fluids on CPEo A medium containing 5% calf seruns 
appears to allow CPE changes in 3 days compared to 15=20 days 
in a 'IC^ horse serum mediumo 

fo Further characterization of an "Adapted** tissue 
culture line of P°3C0 in which the agent persists and multiplies 
without sp<?cific CPiio 

3o In collaboration with Dro .ifaliace Rowe^ LID,, an 
epidemiolog.lc survey has been completedo in part„ of the 
CF and HA antibody status of many of our inbred strains of 
mice in this section,, those in the saise animal rooms with 
recipient mice (receiving tissue culture materials) and others 
in rooms isolated from theseo Ail strains of mice examined 
to date, including SIOLIo DEA/2o NH„ C3Hf/l«„ C3fif/Bio C3Hf/Fg, 
C57Klo 8^Mo and AKS have shown 30=60% CF or HI antibodies „ of 
fairly high titreSo at from 3 to 6 months of ageo In other 
animal roomS;, AKR^ C58„ C57BL„ STOEET and other strains hat'-e 



CAWachiaant I) 

»6^. Serial No p_NCL_427_ 

lo CLaboratory of Biology) 
2o CLewkemia-Studies Section] 
_A_o 3o CBethesda 14 » Mdo) 

reraaified relatiiyeiy freeo The relationship of antibody status 
to leukemia was mentioned previously and there has been ob<» 
served in C3Hf/Fg and EFM mice alsoo a lack of correlation 
between spontaneous leukemias and antibody statuSo A large 
population of these mice and also of mothers whose young 
received inoculations of cell«.free materials (C3llf/Bi and 
C3Hf/Lw> has been observed to 14=16 months of age without 
development of neoplasms other than those ordinarily seen 
among control miceo 

4o The work with Dro Lsusrence Kilhasio DlKo relating 
to the Schwartz C3H and Smss leukemic agents has been dis-- 
contlnuedo No leukemias were observed in large numbers of 
recipientSo although a parotid«>gland inducing material was 
derived from tissue culture preparations of the Schwartz C3II 
transplantable leukemia o 

A continuing collaborative study i^ith Dro Kiiham coji= 
cerns attempts to isolate a possible "leukemic agent" from 
Passage A materials (Gross) o Antibody to the PTA from s 
normal tissue sources is introduced into mouse embryo cui-- 
tures infected with virulent PTA under varying conditions and 
tissue culture fluids are then used to infect C3Hf/Bi and 
CCSHxAKR) micBo Results to date have been negative o 

Co Studies of me chan isms involved in inhibition of leukemic 

cei l'grosatF " " " ' '" ' ' 

lo The Mork with 5-fl«orinated pyrimidines has been c©h= 
tinued with receipt of the deoxyribosidc (FUDR)<. This com^ 
poundp although less toxiCo is also less effective in most 
neoplasms than the 5<-fluorouracil CFUK Dose°response and 
chemotherapeutic index studies have been done on the most 
responsive reticular neoplasms o 

Three sublines of the neoplasm P-815 (mast cell) have 
been developed thru selection; two are resistant to FUo 
exhibiting different patterns,, however o and one is resistaKt 
to FDR (5=fiuorouracil}o Cross-resistance is seen to ail 
the compounds availableo FU,; FDRo FUDH and FO {orotic seid)o 
In the early stages of development or resistance iii all 3 
sublines FODH was inhibitory ^ bat later shovfed nearly ©©saplete 
cross-resistance o 

The availability of 5~fluorocytodine (FCB) and the 
ribotide FCDRo from Sloan<4Cettering Institutes provides for 
further testing of cross^resistance with related cosspeandso 



Part Ac 



(Attachnsent 1} 
Serial Noc NCI 427 

lo (Laboratory ©jTBTologj^) 
2.5 (Leukemia^Studies Section] 
3., CBethesda 14 „ Mdc) 



2o A study of collateral sensitivities of these pyrimidine- 
resistant and of the azaserine-' and DON-resistant sublines 
developed by Mo Potter of P-C15„ is to be initiated with 
Dro Michael Potter„ as well as studies of synergistic activity 
in PGIS of fluorinated pyrimidine and azaserineo 

3o Throughout the year, all L-1210 sublines have been 
retestedfl after periods in the tumor banko Resistance to 
antl-purines and antifoiics characteristic of 5 separate 
transplantable lines„ are shown to persist after many years 
of transplantation in the absence of the respective drugs used 
for selectiono All sublines have been shown to be free of LCM 
(tested by Dro Victor Haas) and a characteristic partial 
dependence of L-12iO/AM=D and L-1210/8AG«D persists 

Do Tissae culture st udies of malignan t lymphoma 

Dro Roosa^ recipient of an NIH Postdoctoral fellowshipr, 
is continuing his tissue culture studieso 

lo In planning experiieents aimed at obtaining resistant 
populations of P388 cells in vitro we sought a n^dia which would 
be inexpensive and readily available o These cells had been 
maintained on a medium containing 4(^ human serum; we chose 
Eagle's Basal medium ^ 5% calf serumo 

In studying the nutritional requirements of this liBe„ 
it was found that Eaglets medium * 5% "dialyzed" calf serum 
would not support growtho Subsequently we found that by 
adding 1=2 mMo concentrations of either pyruvate„ serisiefl 
glyciaeo alpha=ketobutyrate„ alpfaa-keto n=caproate or 
glyoxaiate to this offidiura that growth could be completely 
re stored o 

These compounds are now being used by us in an attempt 
to propagate these cells in a defined mediumo 

Amino acids which did not restore growth include alanine „ 
aspartic acid,, asparagine and prolineo Alpha Keto acids which 
did not restore growth include glutarateo valerate „ iso'S'aler = 
ate„ isocaproate,} and adipateo Other compounds include phenyls 
glyoxalateo alpha^aminobutyrateo sodium propionate, sodium 
lactatSo suecinateo malateo citrateo iso-citratCn cis»aconit8te, 
furaarateo butyratCo cytidine„ uridine n thymidine <> hypoxas5tFdne„ 
leueovorin ((CFlo Vitamin Bi2» cholesterol^ yeast extracto 
.talker peptoneso serum ultrafiltratea and mixtures of nanj others< 



6^8 



"6- Serial Noo NCI 427 



Part A. 



■U (laboratorj? of Biology) 
3o CBethesda U^ Mdo) 



.ife are studying the relationship of these compounds by 
feeding P388 cells these growth stimulators labeled with C*^c> 
Preliminary experiments are now in progresso 

2o In another aspect of our worko we are attempting to 
select clones resistant to certain growth iiihibitorSo These 
clones when available will be used as genetic markers in trans^ 
duction type experiments concerned with the mechanisms of 
resistance in neoplastic populations^ 

Osing the same in vitro system previously diseussedo we 
have obtained a line of ceTTs ujhich will grow ire a medium eon^ 
taining a I0=fold increase in ^liiaethopterinp We x-ecently 
tested these cells after growtii in the absence of Amethopterin 
for one month and found that %k^ level of resistance was heri^ 
tableo de have also found that this line is cross-resistant 
to Aminopterino 

Cells resistant to both 5 -fiuorouracil and S^flworowridine 
are being selected in experiments now in progresso To date„ 
the level of resistance obtained is still of a low order^ 

A C02-incubatorp which has been ordered^ will gj-eat.ly 
facilitate the isolation of these lineso 



3c.. Studies have eoramsnced vising a parotid tumor agest 
CPTA) obtained from a normal tissue source CMS bone marrow) o 
Apparent rapid replication in P380 lyraphoblasts in a n^dium 
containing only 5% calf serum has been achievedo Cytopatiio- 
genie effect <CPE> observed after 24 days in th© first Dassags„ 
has appeared at 5 and 3 days respectively in the second' and 
third passageso Attempts will be isade to plaque this highly 
virulent agent on P38C or possibly on strain L or other tissue 
culture iineso Biological testing of Infectivity in raice is 
being correlated with tissue culture studieso 

4o In vivo experiments compuring the susceptibility 
of several chemotherapeutic compounds to four variants of F388o 
a lymphoid neoplasmo have been accompli shedo The parental 
population of P388'has been comparsad with„ 1} Bawe"s' derived 
line after 45 generations in mice, =«itho 2) a line in both 
its 2nd and 6th mouse transfers after having been maisitained 
continuously in tissue culture an-S with, 3) a line resistasit 
to Amethopterino 



649 



Part Ao 



CAttachiBsent 1} 
Serial Noo NCI 427 

io (iilabbratory oiTMology) 
2o (Leukemia-Studies Sectionj 
3o (Bethesda 14 » Mdo) 



Treatment with azaserine,, 6=diazo-5=oxo-t^nor leucine, 
S^fluorouracil and Aniethopterin did not differ unexpectedlyo 
Hot«everg a line in its 2nd transfer in micen after continuous 
cultivation in yitro p showed a marked difference in its 
response to IS^mercaptopurine as compared with the same line 
after 6 transfer generationso This change from an increase 
in survival after treatment of 60o7% in the 2nd passage to an 
increase in survival of only 3o2% in the 6th passage coincided 
with a change in chromosome ploidyo At the 2nd transfer^, a 
chromosome count showed 93% of the cells to be polyploidy 
while in the 6th generation „ only 36% remained with a polyploid 
compliment o 

An attempt shall be madeo when suitable material is 
available o to repeat this experiment o 



650 



Part B; 



CAttachroerit I) 

'^^' Serial. Noo NCI 427 

i o (Laboratory of^fol ogy ) 
2« (Leukemia-^Studies Section) 
3o (Bethesda 14, Mdo) 



Honprsp Awards,, and Publications 

LaWo Lo Hot Resistance des Ne^piasmes aux Agents Chimiques 
Comptes-Bendusp 74s 301«317o 1950 

Lai^o ^o 'iot Some aspects of drug resistance in neoplasms„ 
Anno No Yo Acado Sciences„ 71s 976»993o 1950 

Lawo Lo rfoo and Potter^ HoS Further evidence of indirect 
induction by X-^radiation of lymphocytic neoplasms in raice,, 
JNCIo 20s 489=493o 1958 

Uphoffo Do Eoo and Law„ Lo .lot Genetic factors influencing 
irradiation protection by bone marrow IIo The histo= 
compatibility =2 CH-2> locuso JNCI„ 20s 617=624, i958o 

Elected Affiliate Royal Society of Medicine o l958o 



(Attachment 11 
PHS"Nin Serial Noo 427 NCI Ca) 

Individual Project Report lo (Laboratory o:^ fflfologyl 
Calendar Year 195G 2o CLeukemia-Studies Section] 

3o CBethesda U„ Mdo) 



Part Ao 



Studies of the etiologyn pathogenesis and chemotherapy of reticular 
neoplasms 

Mo Potter 

Man Years (calendar year 1958} s 
Totals 4 

Professionals Potter, I 
Other s 3 

Ac Sfctiidies on drug resistance 

The origin of resistant cells is the main interesto The 
questions asked about this point ares I) do resistant cells 
antedate any experience with the specific drug or do they arise 
afterwards? 2) is resistance as a characters a somatic mutation? 

lo Employing the DON sensitive ascitic mast ceil neoplasK 
P815(, a number of studies have been initiated and now are near 
corapietiono which provide information on these questionso 

ao If the populationo to begin withj, is mixed and 
contains a high ratio of sensitive to resistant formso there 
should be observed a fundamental difference between the siarvival 
time effects when large and small initial populations arc inoca- 
latedo Survivors arc observed which are not found when 105 or 
more cells are used as initiating doseso It has been fotsnd that 
vihen surall populations of 10^ or 10^ stemline cells are inocu- 
lated into micco and DON treatment is initiated immediate lyn 
one of four different events may occur in any specific indi- 
vidual, mouses 1) indefinite survival^ 2} development of tumor 
growth whicho if removed from the mouse and retested for sensi* 
tivity or resistance „ iss aa) resistant (resistance is stable,, 
heritablfio irreversible) „ bb} resistant (resistance is unstable., 
reverts to sensitivity in one to ten transfers in the absence 
of DONIoCc) sensitive o These findings indicate DON„ though 
capable of actually eliminating some cellsc does not eiimisiate 
alio ando in fact„ does not kill all sensitive cellso Findi^sg 
that resistant populations may be developed from snsall pop«= 
lations indicates either that pre==existing resistant forms are 
actually present in samples of lO^ cellSo or that resistance 
nay develop from sensitive cells after exposure to the selecting 
agento 



652 



((Attachment II 
-2-. Serial NOo__427_NCI C a|_^ 

lo (labor^t ory^^?~Bi i ogy ) 
2o (Leukeraia=^Studies Section) 
Part A o 3c (Bethesda l-l,, Mdo) 

bo Reconstruction experiments have been used to 
elaborate on this pointo Populations consisting of known 
numbers of sensitive fstemline) and resistant cells have been 
inoculated into groups of mice (divided into treated and 
untreated groups)o The presence of 10=20 resistant ceils in 
total populations of 10^ or 10^ can readily be detected by 
survival time curveso These findings indicate the resistant 
cells arise cotnmonly after exposure to the selecting agento 

2o The studies of cortisone sensitivity and resistance 
in lymphocytic neoplasms is being extendedo Cortisone induces 
in established subcutaneous growths of tvso transplantable,, new 
lymphocytic neoplasms CP2450„ P2402)ft complete regression in 
3 to 5 dayso The infiltrative processes are less influencedo 
The regression resembles the lyraphocytolytic effects of cortisone 
on normal lymphatic tissueo Two basic efforts are being pursued; 

1) in collaboration with Dro Jack Bryant of NXA£^o cortisone 
sensitive and resistant pairs of the two lymphocytic neoplasms 
are being supplied for purposes of biochemical investigation „ 

2) because cortisone induces apparently destruction of lae^ge 
numbers of ceils„ a means for quantitatively studying the origin 
of resistance is available o These neoplasms should add more 
information regarding whether pre-existing resistant forms are 
founds or whether resistance Cas a mutation) oiay arise after 
exposure to cortisonco Cortisoneo in contrast to antimetaboliteso 
does destroy large numbers of cellso 

3a All of the studies on biologic mechanisms of drug 
resistance are aimed at ultimately devising means to control 
the development of resistanceo One experiment planned this 
year in collaboration with Dro Lavt is to investigate combination 
treatment of PC15 with DON and fluorouracilo This neoplasm is 
sensitive to both agents and the DOH/R and Fu/B lines are sensi= 
tive to the other agento The use of the two agents with this 
model system,! may indicate a theoretical means of preventing 
the development of resistanceo 

4o Further studies on amethopterin resistance in the plasma 
cell neoplasm 70429o 

Serial transfer of the amethopterin sensitive plasma ceil 
neoplasm 70429 through amethopterin treated mice selects for two 
changes? 1) amethopterin resistanccn 2) an unusual alteration 
in growth of the neoplasrao The latter is characterized by early 



653 



CAttachraent I)) 
Serial Noo 427 NCI C?) 

lo (Laboratory of Biology) 
2o (Leukemla=Studies Sections 
Pgi.LA° 3o CBethesda 14, McJ 

outgrowth in untreated hosts„ followed by regression (between 
10th and 20th days) and later recurrence (40 days and after) o 
Growth is progressive in amethopterin treated oiiceo Though 
preliminarilyo this was believed to be amethopterin dependence r, 
this has not proved to be truCo An alteration in the antigenic 
status of the amethopterin resistant 90429 cells is suspsctedo 
Total»body X-irradiation will prevent this secondary regression, 
tJork is underway and planned to further characterize this 
unusual change o 

So The collaborative work with Dro Vo HaaSo NIAIDr, on 
controlling lymphocytic choromeningitis virus contamination 
of various transplantable neoplasms has been completed in part 
and a publication has been submitted on this worko It has 
been found that LCM plays no part in the development of resistance 
for amethopterin resistance in P28e was developed completely in 
its absencco Transfer of an artificially infected P288 ansethopterin 
resistant subline (P28G AH/rII) to LCM immune mice has revealed 
a means of freeing a contaminated neoplasm from LCMo It was 
further found that amethopterin treatment of LCM immune mice 
bearing LCM infected P288 AM/rII„ did not result in the removal 
of the virus from the neoplasmo This interesting result suggests 
that amethopterin interferes with the immune mechanismo Specific 
experiments are planned to explore this problerac. WU infected 
neoplasms will be implanted in diffusion chambers into immune 
and non-icmune mice? after a period of time the chambers will 
be opened to determine if LCM persists in the neoplasmo The 
immune process may involve actual contact of immune cells with 
infected cells for humoral antibody against LCM is difficult to 
demonstrate and is probably not found in micso 

6o One type of reticular neoplasm commonly found in mice 
is the reticular cell neoplasm Type Bo or'Hodgkins like lesioRo'* 
bistablished transplantable tumors have not been de scribed » nor 
are they availableo Three tumors of this type„ Pl95n P344 and 
P353ft have been transplanted for over 2 yearso A pathological 
and biological study of these neoplasms^ in cooperation with 
Dro Dunno is nearing completiono It has been found that one of 
these neoplasms,, Pl95fl is highly sensitive to chlorambucil o A 
means of facilly treating mice with chlorambucil has been devised 
by adding o0l% chlorambucil to the die to Treatment can be 
maintained for many weekSo Attempts to develop a chlorambucil 
resistant Pl95 are underwayg initial efforts have been unsuccessful 
because chlorambucil is extremely effective against this neoplasm 
and regularly produces survivorso The pathology of this study is 
being done in collaboration with Dro Thelma Bo Dunso 



654 



((Attachment 1 1 
Serial. Noc 427 NCI Ca) 



lo (Laboratory of Bioiiogyji 
2o (Leukemia-Studies SectioR: 
Payt Ao 3^ (Bethesda 14, Mc) 

Bo The pathophysiology and [)athogenesis of plasma cell neoplasia 

"''In^^'ce'o 

There are two fundamental problems regarding plasma cell 
neoplasia which are of interest to this laboratory s I) how can 
plasma cell neoplasms be regularly inducedo 2} what relationship 
do the myeloma globulins have to antibody? The basic hypothesis 
underlying the experiments designed is that plasma cells are 
formed for the purpose of elaborating humoral antibody? neoplasia 
secondarily arises in one of these populations and catches certain 
plasma cell populations in the act of forming antibodyo The 
necessary step is to identify the offending antigen and prow 
the myeloma globulin is in fact an antibody against ito 

lo In strain C3H miceg plasma cell neoplasms appear orig» 
inally in the ileocecal regiono 70C3Hf/lw mice have been injected 
with methylcholanthrene., horse serum and Freunds adju^'ants in the 
wall of the ileocecumo Thereafter^ they will be treated with 
subcutaneous inoculations of horse serum^ If plasma cell neo» 
plasms arise and can be successfully transplanted^ immunologic 
tests of myeloma globulins against horse serum antigen will be 
exploredo Other means also will be tried with such prolonged 
stimulation with foreign antigenSo 

2o Dro Ruth Merwin has kindly donated two plasma cell 
neoplasms of Balb/c origin which were developed by her from 
diffusion chamber experimentSo Diffusion chambers containing 
C3H manynary tumors were implanted into Balb/o miceo Both tumors 
have been found to produce serum protein changes^ MPC^I elevates 
the total serum protein and produces a large quantity of globulin 
which is found in electrophoresis in the alpha globulin region^ 
The second neoplssmg Sl.:PC»2o produces a beta globulino The 
question arises„ are these globulins anti^3l!o Various expeti-^ 
ments are underway to determine this point„ which includesg 1) the 
adsorbtion by C3H cells of the specific globulin substance „ 2} half 
life studies of injected myeloma globulin from the Balb/c iseoplassn 
into Balb/c and C3H hosts„ 3) the effect of transplantable growths 
of these Balb/c plasma cell neoplasms in CBaib/c x C3H)Fji hybrids,, 
bearing skin grafts of C3Ho (In collaboration with Dfo John Fahey 
and Dro Morris Ko Barretto parts of this work are being done 5 o 

Co Leukemogenesis 

lo Two sublines of the agent-free DaAf/2 have been estab= 
lished from two familieso The natural incidence of reticular 
neoplasms in this strain will be determinedo Various litters 



655 



Till ^fLvJf ?/"'"" '"^ 2 families and have been painted 

t I "»J^>*'^h?la''threnev U has been reported that mthyl^ 
choianthrene induces about &0^ lymphocjUc neoplasos in ihu 
siralno The experiments have been designed to determine if 
this incidence Is the same in one family or whether there are 
differences between the family and the strain as a whole. Th, 
eiieci of Utter seriation and foster nursing on old STOU 
aothers is being investigated to deteroiae if biological f- 
play a ro e in the ultimate potentiality of MCA to induce 
lymphocytic neoplasms. Old STOII mothers transmit a matetnaJ 
resistance factor which inhibits spontaneous lymphocytic neo. 
plasia in i.train C5C„ It will be of interest to determine if 
this lactor also influences induced ieukemaa:, 

nRA4^/o^\ '^^^ ^f^^"" 9®"® ^^ *»«« "'*'* ^^» introduced into the 
DBAfA strain through a series of 12 backcrosses. This process 
TtlLf ?nT'!-^ extended to 20 backcrosses. arJ a new inbred 
aln^iLTin f '^^-^oP^d mm. 8j^ employing the i2ih backcu... 

genera ion population^ however,, an initial experiaeni has b^en 
begun iR which reciprocal YD3C12 mice are being painted with 
methvicho^anihrene to determine if the yellow gene influences 
the process of methylchoianthrene induced leukemogenesis! 

of tu,n^J ^ employing a single inbred strain D8Af/2, a compsr.sm, 
lUZtilZ.T?\'^ "Tl' ^' being studied^ methyleholanthrene 
Jt^ir\^°/^' ^"irradiationo DaAf/2 n.ice h^m been irradiated 

two week!,,. This dose scheduAe is fairly well tolerated by thn; 
straino 

m^.n J%,?® ^^^^^ ®*''?^" ^^ °^ particular interest because 
mice of this strain de^relop not oaly iymphoeytic neoplasms h^'i 
also granulocytic, hlstocytic and reticulum cell (Type 8> 
neoplasms. All of the studies are aliaed at deteraining wha. 
factors may fa^or the desreiopiaent of Buy specific type P 
complete pathological stady of the various neoplasms w^ 
cescfibedo 



656 



(Attachment I) 

Serial No -lJ-?.T ,-^PI-XQ J 

lo (Laboratory of"Bi ology t' 
2o (Lfiukeraia Studies Section 
3o (Bethesda U„ Mo) 



Part Be 



Publications 

Lampkiiift Jo Mo and Potter„ MoS Response to cortisone and 
development of cortisone resistance in a cortisone- 
sensitive lymphosarcoma of the mouse „ JNCIn V20; 1091=1 100 „ 
195tU 

Law„ Lo .Vo and Pottero MoS Further evidence of indirect 
induction bjr X^radiation of lymphocytic neoplasms in 
miceo JNCIc V20s 409=493., 1958o 

Nathans Do„ Faheyo Jo Lon and Potter, Mos The formation of 
inyelonaa protein by a mouse plasma cell tumor., Jouro Expero 
Medo, VlOOs 12l==130„ l958o 

DawCo Co Joo Pottero Ho^ and J^ightonn JqS Progressions of a 
reticulum cell sarcoma of the mouse in vivo and in vitrop 
JNCIo V21s 753»7C0„ 1958 » 

Pottero MoS Variation in resistance patterns in different 
neoplasmSo Anno No to Acado Sciences^ 1938 „ Cin presso) 

Fahey,, Jo Loo Potterc Moo and NathanSo DoS Myeloma proteins 
and macroglobulins associated with plasma cell tumors 
in experimental animals^ Acta Dnioo Internationalis Contra 
Cancrum„ 1958 <in press Jo 



657 



Part A» 



(iAttachme." 
PHS«N1H Serial NOo NCI 42V . 

Individual Project Report lo (Laboratory of Biology? 

CaJendar Vt^ai J 95R 2o (Leukemia -"Studies Sectio 

3„ CBefchesda 14„ Mdd 



Studies on bioctiemical mechanisms of sensitivity and resistance 
to antimetabolites 

Elizabeth Po Anderson 

Man years Ccalendar year i95G) 
Totals 2 

Professionals Elizabeth Po Anderson,, I 
Others i 

Ao St » jl^.g s _gJLJg^^£gjyL 

As reported previously,, this problem involved the invesii- 
gation of biochemical effects of 6=Bzauracilo particularly 
effects on galactose metabolismo The enzymatic metabolism of 
galactose=l-phosphate was measured in liver or blood of mice 
(normal and tumor-bearing);, dogs„ and humans before and after 
exposure to azauracilo No evidence for inhibition of galac= 
tose metabolism was observedo although such inhibition had 
been indicated by studies of Handschunjacher and .Velch on 
Lactobacillus biilgaricus„ and might !)e postulated to be mediated 
through azsuridTneanaTogs of uridine nucleotide coenzyaaeSo 
These results were correlated with galactose tolerance tests,. 
carried out through collaboration with DrSo Allan tevy,i Richard 
FritZfl and Eternard Landau of Dr^ Charles Zabrod^s Section of 
NCIo The effect of possible azauridinc nucleotide materials 
on crude and purified enzymes of galactose metabolism was also 
studieds again no inhibitions could be detectedo 

^'^ -'t-udies on azaserine and diazo^oxo-^nor leucine 

This program continues to be the major research problem 
of this laboratory o The objectites are to ocamine possible 
qualitative an^/or quantitative biochemical differences in 
the metabolism of strains of leukemia sensitive and resistant 
to the antioetaboiiteSo So far the work has been confined 
to ascitic lines of plasma ceil neoplasm 70429 sensitive and 
resistant to azaserine (and cross^resistant to 6-diaso-5=oxo= 
nor leucine J, DON),, but it is currently planned to extend it 
to other tumor lineso 



Part Ac 



(Attachment I) 
o2» Serial Noo NCI 427 (b) 

lo (Laboratory of Moiogy) 
2o CLeukemia=Studies Section: 
3o (Bethesda 14„ Mdo) 



lo Azaserine and DON have been found to be effective 
inhibitorSg at the enzyniatic level „ of various glutamine^ 
requiring reactionso In the systems so far studied^ most 
potent inhibition has been found in the reaction involving 
the amidination of formyl-^glycinamide ribotide to formyl« 
glycinamidine ribotidCp one of the steps in the de novo 
biosynthesis of purine nucleotides^ and Buchanan'"? group has 
postulated inhibition of this reaction to be the physiological 
site of action of aza serine o Our own work has indicated that 
both the sensitive and resistant tumor lines are constitu^ 
tively capable of carrying out this reaction „ ioeon the con-> 
version of forrayl=glycinaraide ribotide through formyl« 
glycinamidine ribotide to aminoimidazole ribotide „ as well 
as the further transformation of this compound to aminoimidazole 
carboxamide ribotide o AlsOn in both the sensitive and resistant 
linesn these reactions are inhibited by azaserine and by DON^ 
much more so^ in fact^ than are the same reactions in pigeon 
livero (Exploration of other mammalian tissues is planned 
for comparative purposes) □ The tumor lines are qualitatively 
alike in this inhibition^ but there is a small but consistent 
quantitative difference between them in the extent of inhibi= 
tioHo ^ork has been carried out to explore further this 
quantitative differencCo A major complication has been the 
variation of specific enzyire activity and of extent of inhi=. 
bition between different harvests of the sane cell iineo 
Various other estimates of biochemical behaviorg such as 
respiration and anerobic glycolysis have been studied^ and 
found not to vary in this fashion from one preparation to 
another o However g it has been found that the amidination 
conversion is inhibited by higher concentrations of the crude 
extract, apparently because of disappearance of either sub- 
strate or produce in side reactionSo and the enzyme concenc. 
tration for optimal conversion differs for different prepara- 
tionso Purification of the enzymes is needed to eliminate 
such side reactions and to study the quantitative enzymatic 
inhibition more precisely, and some progress has been made 
on this purificatioHo There may also be variations among 
harvests due to differences in the growth phase of the 
inoeulum„ although this has been standardized as far as pos° 
sibleo or even in the fundsraental make<°up of what may always 
be a mixed cell popalatioiio Such possibilities have not 
been investigatedg but attempts have been made to minimize 
or eliminate such variation by the pooling of a number of 
small harvests,, and toward this end„ methods of storing the 
material during accumulation of larger quantities have been 
exploredo Quantitative aspects of the biological resis» 
tance have also been studied and it has been found that the 
resistant line is less resist«Rt to higher doses but ao more 
ar(\ resistant to lower doses; this characteristic has seesised 

\)t}j only indirectly relevant to the quantitative biochemical 

pictnfeo A large portion of time in recent months has also 



HAttachtnent Ij 
Serial NOo NC^427 dv 

lo <i3Boratory" oFMologyii 
2o CLeukemia^Studies Section}) 
3o (Bethesda U, Uclo) 



Part Ao 



been given to the enzymatic synthesis,, on a preparative 
scaXe„ of the substrate forniyl-glycinamide ribotide, and 
to the modification of published procedures for this to 
a more satisfactory methodo 

2o Our original work also showed a quantitative 
difference between the sensitive and resistant tumor lines 
in the extent of azaserine inhibition of glycine incorpora= 
tion into purines; this difference was more marked with 
intact cells than with cell»free extracts o Some further 
exploration of these findings has also been made„ although 
again a complication has been variabilityo which needs 
defining through quantitation „ and this system does not 
lend itself as readily to quantitationo Separate studies on 
similar intact cell incubations have indicated that 90-lO(^ 
of the cells remain intact and viable under these conditions 
of incubation and that the cells maintain respiration and 
anerobic glycolysis well throughout the incubation? alsoo 
as indicated above, these criteria do not show appreciable 
variability from one preparation to anothero although 
freshness of the cell preparation is importanto A Qqo o^ 
5 to 6 has been found for both lines o Certain other aspects 
of methodology have been studied^ such as the completeness 
of extraction of purine products from intact cells by the 
procedure used and by other techniqueso 

Further studies on the "intact cell effect" have not 
yet been made„ but are plannedo .ife have been careful not 
to call this factor permeability since clearly the data 
could be as adequately explained by a difference in drug 
binding at the cell surface » or an enzyme in the resistant 
cello which destroys or alters the drug and which is lost 
In the preparation of the extractSo It is plannedg in the 
near future , to explore the cell entry or uptake phenomsnon 
in collaboration with Dr., John Jscquez^^ who has been 
interested in azaserine uptake in certain other cell lineso 
A related study was also undertaken by Dro Ellen Pineo 
through private arrangement with Dr^ Potter in this section,, 
and communication with Dr<, Pine on the problem has also now 
been establishedo Meanwhile „ we have continued the attesapts 
to obtain C*^ ■= labeled azaserine for this type of studyo 

Co Studies on the metabolism of ri bonucleic acid 

As mentioned in last year's reportp cell=free extracts 
of ascitic leukemia L4946 were found to have appreciable 



660 



Part A. 



ik I) 

Serial N5 <i.hp 

lo {LaboiQ'».OA> of BioXogyl 
2o (Leukemia-Studies Section} 
3o (Bethesda l4o Mdo) 



activity for the enzymatic splitting of certain polynu-= 
cieotides such as the synthetic polyadenyiic acid produced 
^y Azotobacter polynucleotide phosphorylaseo Studies on 
this reaction have been continued o The mechanism of the 
reaction is a non»phosphoroiytic splits ioCo,, a ribonuclease 
type of cieavage„ but the reaction is of particular interest 
because It possesses a type of substrate and product speci- 
ficity unusual In a nucleaseo Only 5*=phosphate^nded polymers 
and small oligonucleotides are cleavedo while a variety of 
isolated and purified 3»=phosphate-ended ribonucleic acids 
of yeast or viral origin are not attacked or are only very 
slowly split? the products are mononucleotide=5''pbosphateso 
Plans are to extend the investigation to the effect of the 
enzyme of ribonucleic aoids„ particularly soluble ribonucleic 
acid CsRNA)o from the leukemic cell source and other materiaisc 
The enzyme has been purified to a considerable degree and 
this work will shortly be written up for publicationo The 
work has been carried out in collaboration with Dro Uon 
Heptei, NIAUDo 

Do Studies on a parotid tumor agent 

A few preliminary experiments have been done toward 
characterization of a cell=free agent inducing parotid tumors 
after injection into newborn micco Some data have been 
accumulated on the behavior of this material to heat and to 
aging, and experiments are currently in progress on centric 
fugation of the materia !<, by known procedures,, and on other 
isoiative techniques aimed at the concentration of the material 
for biochemical examinationo These experiments are being done 
in collaboration with Dro Lloyd Law and Dro Clyde Dawco whose 
studies on this agent are given elsewhere in this annual reporto 

Ko As in previous years,, some time was spent in the preparation 
of manuscripts on the results of earlier research projects o 
Some of these have been published in this and preceding years? 
others are still to be publishedo 



661 



Part Be 



lAttachment I) 
-5- Serial NOoICI j^27j,b.» 

lo (laboratory oT 
2o CLeukemia-Studies Sectionj 
3o <Bethesda 14„ m„} 



Honors^ Awards and Publications 

^^a/° ^"o/r^^'^n 8° Doc Anderson, Ko Poo and Landau, Bo Ros 
tffects of 6»azauracil on galactose metabolism in mice„ 
dogso and mano JNCIo 20s 53„ 1958„ 

Kurahashio Kiyoshio and Anderson^ Elizabeth P„„ Biochimo BiopJjys< 
Acta, Volo 29„ 1930 o 

Honors 

Llection to membership in the American Society of Bioloqical 
Chemists o 



661 



ilAttaclunent IJ 
PHS-NIH Serial NOo NCI 427 {c) 

Individual Project Report U (Laboratory of Biology i^ 

Calendar Year 1950 2o (Leukemia-Studies Section) 

3o (Bethesda 14a Mdo) 



Part Ao 

Genetic factors influencing irradiation protection by bone marrow 

Delta Eo Uphoff 

Uan Years (calendar year 1956); 
Totals 1 
Professional; ^i 
Others !i 

The surveys initiated in an attempt to ascertain the role 
of certain genetically determined histocompatibility genes in the case 
of the marrow graft in the irradiated host, was continuedo The broad 
scope of this survey has made possible the postulation of theories 
which have application in the practical use of bone marrow as a 
therapeutic agent o 

Ac Graft-^versus-host reaction 

lo Inbred strains and their Fj hybrids were exposed to 
lethal total^body X irradiation and given intravenous inoculation 
of bone marrowo In many hybrids in which the parental strains 
differ at the histocompatibility=2 <H=2| locusg the hybrid marrow 
affords better protection than marrow of either parental straino 
There are some Fi hybrids in which parental strain marrow does 
not elicit a severe and lethal reaction^ although these same two 
parental strains are incompatible when they are used for recipro^ 
cal marrow transplantSo A gene dosage effect was proposed as 
a working hypothesis to explain the variability in response 
observed in the F^ hybrids treated with parental strain marrow 
in contrast to the response found among different inbred strain 
combinationso This hypothesis is not at variance with the 
graft»versus»host reaction as an explanation for the secondary 
phase of the irradiation syndrome o 

2o Further evidence for the graft=versus-host reaction 
is found in the results obtained from a "synthetic d-k" systemo 
If mice of the H=2^ and H»2 phenotypes are crossed „ the genotype 
is H=2^/H-2'''d This hybrid carries all of the antigenic components 
of strain A which is H=28 (formerly H-2^*^)o The hybrid has 
several antigenic components not present in strain Ao .^ith 
strain A as the irradiated recipient^ the H-2d/H=2'' hybrid marrow 



663 



(Attachment I) 
Serial Noo ffCI j27_ Cc) 

1 (IlBoratcrry"oT~SioTogy 
p -. „ 2o (Leukemia ^Studies Section 

i-S£iJL° 3o (Bethesda 14 „ Mdol 

gives good long-term protectiono Strain A marrow„ however„ 
reacts against the antigenic differences presented by the 
H^2a/H»2K hybrido resulting in a greatly reduced long-term 
survival o This generalization is based on results obtained 
from testing several different d^ hybrid systemso 

3o The successful use of embryonic hematopoietic tissue 
as a protective agent is also evidence for the graft-versus=host 
reaction^ since the immunologically immature fetal tissue is 
not capable of reacting against the hosto In some genetic 
combinations the use of embryonic hematopoietic tissue results 
In good long-^term protectiono 



lo The observation that a single gene difference between 
the marrow donor and the marrow recipient is sufficient to 
produce a survival pattern similar to that produced by the inoc= 
ulation of homologous marrow, was confirmed by the use of the 
isogenic resistant lines of the Street CST) straino The 
coisogenic lines which differ at the H=3 locus showed no gross 
evidence of a reaction „ while the H-1 locus gave a variable 
responseo This variability may be explained on the basis of a 
threshold effect in which the reaction is apparent in some mice 
but not m otherso This threshold hypothesis will be tested by 
the pre^immunization of the marrow donor against the host strain^ 
It may be concluded that the H=2 locus is an important factor 
in the protection of lethally irradiated mice by marrow trans= 
plantationo Although data is available indicating the existence 
of effective histocompatibility genes other than the H=2 locus„ 
these effects may result from multiple gene differences since 
a single gene difference at the H=l or H=3 locus is not suffi= 
cient to produce a uniform responseo 

2o It was found that the strain barriers can be crossed 
within the II==2b or H=2d groups, ioSo^ as long as the strains 
share the same H-2 phenotype CH»2b or H=2d) they were found to 
be compatible as marrow donors and irradiated recipientSo The 
H=2k group shows a variability of response „ depending upon the 
strain combination testedo Experiments are in progress attempting 
to ascertain the cause of this variabilityo 

3o In testing 27 inbred strain combinations, no combination 
was found in which good long=terra survival was observed when the 
strains were of different H=2 phenotypeso 



664 



•Attachme 
"3- Serial No° NCI J 

lo (Laboratory ol Biology i 
2o (Leukemia-Studies Sectio 
3o (Bethesda 14„ Mdo) 



Part Ac 



Co Preclusion of the reaction 

lo Following a successful attempt at precluding the 
reaction in an Fj hybrid»pa rental strain combination with 
embryonic hematopoietic tissue^ other strain combinations 
were testedo It was found that it was not difficult to produce 
an increased survival but in most combinations a reaction occurs 
which is less severe and more animals survive the reaction^ 
The similarity between this situation and that of "acquired 
tolerance" is more evidento Attempts at producing "acquired 
tolerance" in the marrow donor by intravenous inoculation 
of marrow in new born mice have not been successfulo Further 
work along these lines is plannedo 

2o It was apparent that a genetically non-specific 
method of precluding the reaction is necessary o ^ath the 
evidence that the antimetabolites had an inhibitory effect 
on immune mechanisms^ A^^methopterin was used in a combination 
of irradiated Fi hybrids treated with parental strain marrowo 
which produced an earlyo severe and lethal reactiono Using 
different treatment schedules^ varying degrees of effectiveness 
from increased survivals following a typical reaction„ to no 
detectable reaction were obtainedo Other antimetabolites are 
being tested to determine whether this inhibitory effect on 
the immune response is common to antin^tabolites or whether 
it is limited to the folic acid antagonistSo Tests are in 
progress^ using drugs,, such as cortisone o in an attempt to 
find a drug which is effective in precluding the reaction 
and causes minimum toxicity in the heavily irradiated animal o 

Do Chimeras 

lo i*hen mice are exposed to a lethal total-^body dose of 
X irradiation,, followed by homologous marrow Inoculationo some 
animals will recover from the graft=versus=host reactiono These 
survivors are^ in all probability, chimeras „ being composed of 
recipient plus donor --strain hecatopoietic tissue o These sur= 
vivors have been studied for tumor incidencco Of the malignant 
lymphomas that have developed a sample have been tested by 
transplantation to determine by their histocompatibility patterns „ 
if they are of donor or recipient=tissue origino Of the IC 
positive transplants,, 15 were of donor ==strain origin„ and two 
were unquestionably of host origin and one is probably of host 
origin (the confirmation is still incomplete) o Dro Do Bernard Amos 
of the Hoswell Park Memorial Institute tested several of these 
tumors for their antigenic composition and found them to Ije of 
donor strain composition o 



665 



(Attachtsent I) 

Serial N£^<>^J^S»dH-- ^'^iL^ 
I o C La bor 8 1 ory oT Bi orogyl 
2o (Leukeraia-Studies Sectijc 
3o (Beihesda U„ Mo) 



Part Ao 

Eo Serolog ical analy sis of the graft^versus-^host reaction 

In a collaborative projecto Dro AmoSo using heroaglutina« 
tion techniqueso detected antibodies that could only be produced 
by the reaction of the graft against the host in these miceo 
Further attempts at an unequivocal serological demonstration 
of the antibodies involved in these reactions have failedo 

Fo Multiple polypos is of the coloH p a genetic tract 

Lethally irradiated mice protected with bone marrow are 
studied for their tumor incidences These animals have an 
increased incidence of neoplasms of the gastro^intestinal 
systemo The control incidence for these neoplasms is deter- 
mined from data on the breeding colony o One family of the 
C57Bi-/10 H-2^ strain was observed to have an apparently 
genetically controlled multiple polyposis of the colono This 
lesion resembles the polyposis found in humans which is also 
familialo This is the first observed occurrence of this type 
of lesion in an experimental animal and may be a useful tool 
in the study of this lesiono Available stock animals from 
this family have been irradiated in an attenpt to produce a 
nslignancy from this benign lesioQo Since sufficient animal 
space and technical assistance are not available to taake a 
thorough genetic analysis of this characteristiCo no further 
work is planned at this timeo 



6l 



-5-.. (Attachinent I) 

Serial No o NCI 427 (c ) 

lo (LaSoratory o?~BioIogiy) 
2o (Leukemia -Studies Section' 
3o (Bethesda .14„ Mdo) 

Part Do 

HonorSo Awards and Publications 

Uplioffn Do E,„ Lawo i-"> lios Genetic factors influencing irradiation 
protection by bone niarrowo II the histocompatibility=2 (H-2) 
locus„ Jo Nato Cancer Instoo 20s617=624o 195r.o 

Uphoffo Do EoS Preclusion of secondary phase of irradiation syndrome 
by inoculation of fetal hematopoietic tissue following lethal 
total-body X irradiatioHo Jo Nato Cancer Insto,, 20s625=632„ 1958c 



667 



Individaai Project Report io (Laboratcry oi Biology) 
Csieadsr Year 1956 2o (Leakemia-SttadJ.es 3ecti< 

3o (Bethesda 14 „ Mdo) 

Part Ao 

k study of the etiology and chemotherapy of experimental lymphomas 

Delta Eo Uphoff and Lloyd ;ic Law 

Man Years (ealeadar year 1958); 
Total; 11/3 
Professional; Dphoffo ^zi I^Wo i/S 

Other; '>i 

This report deals priiiiarily vvith tSie tsse of X irradiatioji 
and bone aiarrow or fetal hematopoietic tissue as experinieiistal 
variables in the study of leukemia So The project may be classified 
Milder the following three major headis^gs; 

Aq Etiology and pathogenesis of experimental lymphOKnas 

Bo Chemotherapy of transplantable lyraphoiaas 

Ca RadiatioH therapy of tratisplaatable lymphosna 

Ao Etioloq^r and pa thoc^eae s_i s^ ^^of experiiseMta_l^ l|;jgpjio MS^ 

ia The stjidy of the irifiwieiiGe of thyciectomy os the 
spoEitarjeosss imcidence of leukemia in CC3Hf -x AKR)F_| hybrids asad 
on the ineidence of induced lesikesaia in CC57BL x Dm/2)Fi hybrids 
exposed to 225 r X irradiation for fowr exposures at se¥en day- 
intervals was contintjedo ThyEKCtoray increased the meas^ age by 
decreasing the ij^cidence of lymphocytic raeoplasraso but does Eot 
appear to influence the incidesjce of taaiigjiasit lyssphoms s o 

2a The extension of the study of the iaflitence of CSHf 
marrow on the high leukemic AKR strain was continiuedo In 
additions reciprocal transplants of C3Hf and AKR fetal hesnatopoi-, 
tissue were niade in lethally irradiated AKR and C3Hf miceo 
Lethally irradiated AKR mice inociilated with C3Hf fetal tissjse 
survive a mild graft-versus-host reaction and later develop thjm.c 
lewkemiao Transplantation tests were run to determine the strairi; 
of origijn of the Heoplasmo To date, they have been exoiissiveiy 
of donor strain origino With the low leukemic straisij C3Hf as 
irradiated ^recipient for AM fetal tissue ^ the resinits appear 
to be quite different o PreliEssinary re salts indicate no thyaic 
lymphomas but a lesion of the mesenteric lymph nodeo Tests are 
in progress to determine the strain of origin of this lesiorso 
The effect of thyasectomy on this genetic cosibination is toeing 
tested but it is too early to evaluate these data at this timeo 



Part A< 



,^2^ (A'tt-achment I) 

S'erial Noo 427 NCI (clU^ _ ^ 
1 c- <T3Bor 8 f of y ■ oT^i 0X0®= 
2o 0-eu(cemia Studies Section 
3o (Befchesda 14 „ Hdo) 



3c The study of the effect of exposure to 400 r„ 100 r 
and 25 r X irradiation within 24 hours of birtho on the incidence 
of leukemia in C37BL/Ka mice was continuedo A group of mice 
was also exposed to 400 r at 30 days of ageo Lymphomas of 
thymic origin appeared at the 400 r dose level while there were 
late neoplasms of nonthymic origin at the 100 r and 25 r dose 
levelso Intraperitoneal inoculation of C57BL bone marrovj 
following 400 r at birth appeared to have little effect on the 
incidence of lymphomaso Intravenous inoculation of marrow 
under the same experimental conditions decreased the incidence 
of early neoplasms although it is too early to determine the 
effect upon the late neoplasms o 

4o A study of the effect of bone marrow and fetal 
hematopoietic tissue on the incidence of malignant lymphomas 
in intact and thymectomized C57BL and CC57BLxA)Fj hybrid mice 
receiving 4 exposures of 168 r at 7 day intervals is in progresso 
Preliminary results indicate thato while adult isoiogous marrow 
decreases the incidence of thymic neoplasms,, it appears that 
fetal tissue has littleg if anyo protective effect against 
these lesionSo Of the neoplasms arising in the (C5TBLxA)Fj^ 
hybridSn a sample is being tested by transplantation to 
determine whether they are of donor or host strain origino 
Preliminary results indicate that regardless of the protective 
agent,, the majority of these neoplasms are of host^strain 
origino These results might indicate a lack of protection 
by the inoculated tissue,, In contrast to these results are 
those in the lethally irradiated mice where most reticular 
neoplasms are found to be of donor-strain origin when tested 
by transplantations 

5o The histocompatibility genotypes and their effect on 
radiation=>induced lymphoma So 

A study of the influence of complete and incomplete 
matching of the histocompatibility genotypes on the prevention 
of X irradiation^induced lymphoma is piannedo The C5TBL/10 
strain and its isogenic resistant lines will be usedo The 
C57BI/10 ScBs linCo which is 11=2^0 will be exposed to 160 r in 
4 exposures at 7 day intervalso After the 4th exposure,, one 
of three types of marrow will be given by intravenous inoculationc 
The three types of marrow areg isoiogous (H=2l*) coisologous 
CH=2d>o which differs at the H-2 locus and B/lOiPo which differs 
at the H-2 locus s the former is H=3b„ the latter a»3ao Dro George 
Snell of Roscoe Bo Jackson Uemorial Laboratory n has recently 
made available to us a new coisogenic line to add to this trio,, 
which is of a different genotype at the H-l locus,, namely H-i'^o 



669 



Part Ao 



(Attacho)enl; I> 

V.3- Serial No °_i2J. JCJ[^C dJ_ „ 

.lo ('Qboratofy o?"TsroIo(]tf i 
2n CLieMkemiawStiad.les Sectii*? 
3o CBethesda U<, Mdo) 

This line may also be used in this experiraento Preliminary 
studies indicate that malignant lymphomas may be induced in 
the C57MyiO strain by fractionated Irradiationo It is 
hoped that with the termination of other experiraentSo animal 
space and personnel will be available for this study during 
the coming yearo 

Bo Chemotherapy of transplantable leukeraias 

An attempt has been made to treat drug toxicity in tumor 
bearing as well as normal mice by bone marrow inoculatioHo 
Only a slightly increased mean survival time in the animals 
receiving drug plus marrowg as compared with drug treatment 
onlyo has been observed. Attempts are being made to determine 
some of the variables which may influence the success or 
failure of these experimentSo Animals which survive the 
drug and marrow treatn^nt will be tested by skin grafting to 
determine if they will be tolerant to a homologous grafto 
This is an important adjunct to the attempts at precluding 
the graft^host reaction in lethally irradiated mice treated 
with homologous marrow by the use of antimetaboliteso 

Co Radiation therapy of transplantable lymphomas 

There was a continuation of the attempt to treat 
transplantable malignant lymphomas by the use of high doses 
of X irradiation and isologous or homologous marroMo NtsiMrotis 
C21) attempts have been made using different morphologic forms 
of transplantable neoplasms and various marrow treatrasntSo In 
the case of one Type 8 reticular neoplasm CHodgkins=like lesioal 
the second generation transplant responded to therapy but the 
third generation transplant did noto In the former case^ the 
tumor inoculum was given by the intravenous route; in the 
latter case it was given intraperitoneal lyo It has not been 
determined whether the route of transplantation or the genera= 
tion of transplantation was the important factoro Several 
cases where the therapy appeared to be successful by gross 
observation,, histological examination revealed residual islands 
of tumor cells primarily in the mesenteric lymph nodec In these 
caseSj) the animals did not die as a result of the neoplasm but 
as a result of the irreversible irradiation damages It may te 
concluded that although the tumor growth may be controlledo the 
life expectancy of the animal is greatly reduced by the irre= 
versible irradiation injury at this high dosage levelo Further 
work is in progresso 

Part B » Not included » 



670 



Serial No, MGI^1^16 (b) 



1» Laboratoiy of Biolo^ 

2o Tissue Culture Section 

3o Betheada Ih^ Iteiyland 



PHS»NIH 

Individual Project Reports 

Calendar Year 19^8 



Part Ac 



Project Title: The Application of Tissue Culture to the 
Study of Tumor Viruses o II » Viruses 
Associated isith Huroan Tujaorst, 

Principal Investigators i Dr^ Robert A„ Manalcer 

DTo Robert Bo Couch 

Other Investigators s John Bo Moloney 

Cooperating IM.tsj Surgezy, NCIj Dr^ Robearb Ra Smithy Chief o 

Man Years (calendar year 1958): 
Total! 1 1/3 

Professionals 1/3 
Others 1 

Project Description? 

Ob^ e g tiggs g Deterfflination by application of tissue culture 
tecSSqS^ of the extent to which "sdruses imay be demonstrated 
to be associated isith a -variety of human tumors and to in^- 
VQstigate the nature of such agents » 

Methods t Some viruses associated with htsman disease are kno!*n 
to" piroSuce long term latent infections during the course of which 
the presence of the agent is not apparent » Presumably viruses 
also esist which, under usual conditions, nmy parasitiae living 
cells id-thout recognisable effects « Since the presence of a 
virus is obsdous only through some visible aspressicm of activity^ 
the extent to which viruses are actually distributed in a pep=> 
ulation is uneertaino Changes in the relationship between host 
cell aH.d virus may be reflected fcgr a visible, or cgrt ©pathogenic ^ 
response,, An alteration in the host=virus equilibrium laay re= 
suit from a change in the environment or the nutrition of the 
infected cello This raay be aceoraplished by establishaent of 
cultures of infected cells » 

Disruption of the infected cell may yield visruso Liberation 
of cell^contained viinas in this fashion is coias>3ic®t©d 1:^ the 
presence of inhibitors in the tissue horaogenate and by the lability 



671 



NCI=itl6 (b) 



of free viznaSo Stabilising buffers find dil«rtion=>sedijaentation 
techniques should be used to preisare infective concentrates of 
tissue extracts o 

Various t^es of cultured cells exhibit different degrees 
of susceptibility to infection by free virus and differ in their 
response foUosfing infection by a given agento Susceptibility 
and rsspox^e of a cultured cell is not indicative of siiailar po~ 
tential in vivo a The ctiltured cell line offers aa indicator 
system for the presence of virus insofar as it is capable of 
infection and either responds with morphological changes in 
infected cells^ or persiits recognition of infected cells ^ 
special means such as adsorption of i«ashed red blood calls c 

This imrsstigation was begun ^th two basic techniques? 
(1) Culture and observation of tumor tissue for visible evidence 
of virus activity o (2) Inoculation of a varielgr of laboratory 
cell lines ^th ainced viable tumor j, concentrates of tumor 
extracts s and ground cells and fluids from etjltures of tuEior 
tissasj followed by long term Bialntensnce and observationo Four 
blind i^ssages of cultures were considered to penait reeognitiea 
of slow!!^ grosdng agents o The first approach^ culture of tumor 
tissue^, WES conducted by the Surgery brancM, NCIo, the latter 
was carried out by the Leboratoiy of Biology, KCI, and is herein 
reported o 

A normal hunsan skin line and a bone line which originated 
frcaa human epipl^seal tissue were obtained fr<Ha Dto Wilton Esrl^o 
Huroan intestine and huaaan eaibzyo skin-^musc le cultures are routinely 
si:^plied by Microbiological Associates o 

Origiiml cultures were made in media containing horse (Ho) 
or human (Hu) seruso Skin and bone cultures (Earle) are presently 
maintained in Eagles lasdium c^itaining horse serum. Human embryo 
skin-^auscl® cultures and huraan intestine cultures (Mcrobiolc 
Assoc.) are presently maintained in Seherer^'s maintenance meditam 
containing horse serunio 

Major FindiRCTi The appended chart indicates the tusnor materia;^ 
principaSly^rrom head and neck, obtained to initiate this pro j set o 
Since the progsram %mB in its dsvelopffientel stages, ao choice of 
tuzsors V&3 inadei tissue v&s utilized as it became available if 
the necessary tissue cultures were on hand. 

No progressive <^opathogenic changes vhich could be 
initiated in fresh cuittiros by passage of cell=free filtrates 
were noted « 

Sigaificaace; Recent association of viruses ■with tumors make 
"limdaio:^ (^svelopaent of methods for detersdjaation of the extent 
to which viruses tss^ bs found in huiaan tts^^ tissue. 

sosed Cotn'ses This program Td.ll be furthm* developed &nd 
1^^^ "^^Q next yeeTo 






KOo'scM-iaX passage in owlturc-is oi. 



Hiiraan 




ICI"lil6(b) 




Part B i3ici«aded Iss 



Ho X 



674 



(Attachment I; 
Serial No» U28a 

1. Laboratory of Biology 

2. Tissue Culture Section 

3. Bethesda, llaryland 



PHS=NIH 
Individual Project Report 
Calendar Year 1958 



Part A 



Project Titles Studies in tissue culture of ttie raal3.gnant cell 
and the nornal cell from which it arises » 



Principal Investigators s ¥oR. Earle, ¥oT, McQuilkino 
Other Investigators! V. J. JjJsrans, 



Cooperating Units s Cancer Chemotherapy National Ser^^ice 

Center. 



Man years (calendar year 19S8)* Patient Days (calendar 

Totals year 

Professionals 
Others? 



Project description! 
ao Mcrocinematographic studies of tissue cells in vitro . 1) Usissg 
cultures of selected cell strains, simultaneous time lapse motion 
pictures are taken of cultures that are sirtiilar except for the selected 
■variable under study. With the aid ©f enlargeasats of selected fraisss 
and with tracings made on the projection screen qualitative guid quaiiti= 
tative descriptive studies of cell behavior are carried out. ¥ith the 
assistance of Biostatistical personnel of CCNSC procedures have beeh 
and are being further developed for quantitatively analyzing the fl'iiDS 
in order to determine such meaningful quantitative parameters as cultTO;^ 
growth ratesj rates of mitosis, intermitotic intervals, frequency of 
cell necrosis, etc. 

The first study made iiwolved a detailed analysis of a film of strain 
NCTC mouse fibroblasts #2071 j^aintaiaed ia protein^free ssdium 109. This 
study shoifjed that in this protein free chsmieally defined aiediujri the 
average timed interniitotic interval was 37 hours » Of equal significance 
T'jas the complete absence of ai^ observable cell necrosis in the fil.r. -}r, 
■J-s defined culture iT^diumo Iflfhea compared with cells of the ss.: 

" "■■'.ch l7».d been continusc". ia a prd'oeinaceoiis iiiedium ailowiag r&pxc. 

proliferation, numerous major differences in cell morphology and behavior 



Serial NOo l|28a Page 2 



wer« obviOTSo These must yat be amayaed in greater detail in order to 
oboain a bei-ter concept of tow a cell strain may dewlop a radically 
diTergent character as a result of such a chassge in its n»diao 

on acrain 20/1 in Htadixm 109<, 

c. The abw8 etwiies are being temporarily made secoadaiT" in order t& 
obtain and stiidy at once nims which might concei^bly clarify aM adt^ance 
certain other w©rk in progress in om- section on the aetioa of certain 
types ©f me-ohylcellwlose on cells of the strain 2071 iji this OT®teli5i==f!me 
medim 109 (See i^28b2) Pr^essing @f these films is m^T^ZTem 

Proposed future cotsrse of studies s To be continued as outliaed. 



Fart B iaelwied les x N© 

676 



Serial No, lt28a Page 3 



Part B Honors, Awardsj, aad Publications. 
Ptibllcatloas other thas aba tracts from this projects 
None 

Honei2*3 acnd Awards relating to this project g 

Oae film e& far c©B?5leted was used by th® Director, NCI, ©a 
a geaeral telerisioa br@adeastj a secoad has just bees released to 
another network for a similar broadcasts 



677 



Sei-i&l NOo 1428(b) 

1. Laboratory of BtologS' 
to Tissue Culture Section 
3o Bethesda, mrylmd 



PHS=NIH 
Individual Project Report 
Calendar Year 19^8 



Part Ac 



Project 1, Title!! Growth of cells of variotja establishsd strains 
in rapidly agitated fluid atuspemion cultures o 

Principal Iwrestigators? J.Co Bryant^ SoL« Schilling^ WoRoEarleo 

Other Investigators « Bo Bo Westfallj EoV. Peppers o 

Geoperatiag IMitss None. 



Man years (caleadar year 1958)8 Patient Days (calendar 

Totals y®fi^ 1958)? 

Professioitmls 

Others 

Project Description? 

Ctejecti'ves? To loore clear Hy define and to Hore closely control 
t&8 factors iaflueacing grmirih of Muamlian cells in agitated fluid 
swpenslon ctslttareso Eaphasis is placed on 1) is^ereased and laore 
efficient yield of cells for research piarpose in rapid han^stiag 
cycles ©wr extended periods ©f tim$ 2) adaptation of new straisiai 
to this type of cislturei 3) attainment of better imdarstanding of 
basic principles ^jich will allow maxlBial accwacy of control and 
reproduclbiUty^ md rosiy uvUiiaately allow greater stability of cell 
type characteristics. 

Methods ©niJloyeds These haw been d®scrib®d previoaslys special li 
liter sfaaker flasks containing a moxiraum of UOO mlo fltiid centiniisiisly 
aerated, agitated at 86UO or 12,500 rphp nuclei counts and reducing 
sugar detenainations, on small aliquot sanples, were mde regularly^ 

Major findings 8 Four large cultures of clone WGTC clone lU69 liwr 
cells were carried for hi days Itoough 9 subculturings at U-day to 
?=day interiralsi this demonstrated satisfactorily that high yields 
©f cells may be secured at frequent intenralSc While this result e©n° 
firms that repm>ted last yesr for this cell strain and for clone 929 
©ellsj the rang® cowered^, i.e. cell coEjcentration per xxoit volisros and 
678 population at si^s^cultTO'ej, was 8^t>sta?itial!ly ■ higher tfesn couM be 



•od last, yea* 
: 25 X 10^ per 
•ji't- efficiento MAxifluuii ceil pspuiatiuus ianged from 3 ^^ ( "^ blliic'f 
.'lis from the higher inoculum, and ranged from 1,75 to 5 billion eel is 
•0m the lower inoculmtij niaximuw cell concentrations per m3.. ranged 
.OH! ? t© 19 million and UJa to 13 million, respectively. These values 
;ive been exceeded only by the maxima, reported last year for a linrer 
alture carried on a 2l4=hour fluid change schedule with n© hanrestimg.. 

Harvested cells were regularly made available t© Westfall (Project Ii2&d} 

for enzymatic and nutritional studies. 

One at©ck culture of NCTC clone IU69 liver cells was maintained on 
a ib t© 5 day harvesting cycle for 126 days., Initial cell iaoeulure at 
the lo2S X 10° level was taken from the preceding eaqseriinetttj and the 
inoculum at each s1A>cult^lre was set at that level » Rates of cell pTO'^ 
liferation aad glucose utilization wer« comparable to those in the 
preceding expertraento 

A strain of raonlcey kidney cells is being adapted to groirfch in agitated 
fluid suspension culture So It has been majjitained in healthy condition 
on medium NCTC 109 si^plesagsated with 10 percent horse senm and 0^06 
percent low viscosity grade Methocelo Short periods of time in a static 
incubator appear t© be necessary as a supplementary step in the con- 
ditioning process o 

Significance to program of NCIs Our agitated fluid suspension cultures 
have given larger yields ©f cells for other studies th^ have comparable 
static CTiltureso Such cells may more nearly approach in vivo conditions 
by having proliferated under sosaditions of continuous aeration <, Insofar 
as the steady production in vitro of large amounts of cells at short in- 
tervals is of value t© the prograxa of NCI, this project is considered to 
make a definite contribution t© that programo 

Adaptation of new straias of cells to this aethod of ctiltivating has 
significance insofar as it then becomes possible to produce large amotmtg 
of previously scarce strains for various investigations o 

Proposed course of projects Numerous improvements in the procedure for 
cultivating cells in agitated fluid suspesasion cultiares are being made 
in this Section, A sinplified gassing apparatus for carefully controll- 
ing the rate of aerating a culture over a much wider range of gas flow 
rates is now r«ady t® be placed in routine use. Effective uBans of 
eliminating the time^consumiag steps of ceatrift2gati©n and pipetting at 
each fluid change are already under plaimittg. The rapid harvesting cycle 
procedure will b@ extended to other strains of cells, as nmy be desiredo 
One strain of the monkey kidiasy cell cultures is being maintained in a 
healthy condition and should be traasfornsgd to rapid proliferation with 
repeated harvesting cycles is the near fatiire (Evans project li28(c)<, 



Part B included 679 Yes x No 



Part__B_s^ Honors, Awards, and Publica&iors. 

Publications other than abstracts H-orn this project* 

1) Bryant, J.C, Schilling, E„L«, and Earle, WcR.s Massive fluid 
suspension cultures of certain maiamalian tissue cells. I« General 
characteristics of growth and trends of population. J. Nat. Cancer 
Inst. 21f 331"3U8, 1958. 

2) BiTsnt, J.C, Schilling, E.L., and Earle, W.R.: Massive fluid 
suspension cultures of certain aamraalian tissue cells. II » Glucose 
utilization and cell proliferation. J, Nat. Cancer Insto 21? 3S4.9=> 
36U, 1958. 

Notes Als© see two papers by Westfall et al (Project !428d)o 

Honors and Awards relating to this projects 

None. 



PHS»NIH 
Individual Project Report 
Calendar Year 1958 



Part A 



Project 2, Titles The growth.=potentiatiT5g effect oa cheiaically 
defined lEediim NCTC 109 of low-viscositj 
Methocel with cei'tain ceil strains in agitated 
fluid suspeasion cultures.. 

Principal Iwestigatorg JoC. Bryant, ?„J„ Svansj ¥oR„ Ear5^ and 

E„ L„ Schilling „ 



Other Investigators s 



None 



Cooperating Tfaitss 



None 



Man Tears (calendar year 1958)? 
Totals 

Professional? 
Others 



Patient Days(calenda:i: 
year 1958) s 



Project descriptions 

Objecti-ress To grow maMaaliaia cells in agitated fluid aiispensioEi 
cultures in cheaiically defined nedium NCTC 109 without serusi supple- 
Bientj by substituting a low-fiseosity non-protein, Methocel„ This 
is a continuing project o 



Methods eisployads The type of culture aad the procedures of sub° 
cultisringg fliiid change, and nuclei count have been described previously «. 
In eaqploratory experimentsj roller tube cultures were used^ rotated at 
speeds xsp to 2160 rpho In later experlissentSj, shaker flasks of the tso 
sizes were useds 250 ilo flasks containing 30 to lj.0 lalo fluid, at an 
oscillation rate of 86UO rph„ Aqueous solutions (2oU percent) of Methocel 
65 HG (27-29 percent methoijqrl, 5-$'=' 7o5 percent hf^dros^prspoxyl) of 
se-^eral low viscosity grades xjere cosibined with a^diuia ICTC 109 to final 
concentrations of 0.12 or 0<.o6 percent. The Imr -^scosity grades and 
final concentrations of Methocel were chosen to replace serum becaitse 
1) Methocel is inert and n©n=toxiCj and 2) these gx°ades and concentra^ 
tions, with NGTC 109, gi'^e approximate 3y the sai^ relative Yiscosity 
and surface tension values as dees 20 percent horse serum with NCTC 
109 « DofW'4301'ning DC^gOO silicone (5 or 10 percent solution in bensene) 
was flushed over the inner walls of flasks, which ^ere then baked at 
250*C for 3 hours. These msthod deta5.1s, especially with respect to 
silicone, are significant sine© "they have effectively reduced the loss 

681 



Serial Noo U28Cb) Page 2 
•' Project 2 
of mils by adhesion to the glasa juat abow the level of nutrient 
f3.iaid s ioSoj at wav0=«Gr«st level. 

Major KLndiKgss Medixur. NCTC 109 in agitated fluid suspension cultureSj, 
when si;5>pleisBnted with horse serums had st^ported hi^ly satisfactory 
proliferation of several strains of cells through repeated hsorvesting 
cycles o But neither cell strain 2071 nor asny other woxjild live in Medium 
NCTC 109 alone in agitated culttureso Sarlier e^.teiqpts to etA^stitute 
Methocel of high viscosity grade (liOOO cps) for serum had proven in- 
effective o 

In exploratory experiments of the present series low viscosity grades 
of Methoeel favored the formation of rings or sheets of viable cells 
on the shoulders and conical bases of special roller tubes and, in @ub° 
sequent shaker experiaents with flat^bettom boiling flasks, on the floor 
and on the wills at wara^cresto In all such cases the cells in suspen- 
sion appeared to be fewer than those adhering to flask walls o Density 
of cell aecumsilation in the wave^crest rim was often so great as to 
cause necrosis from cell creudingc 

Silic©ne=C0atifflg the flask walls was then introduced, giving a hydr©» 
phobic surface;, in order to keep all of the cells in suspension<> In 
such coated flasks strain 2071 cells on chemically defined Esedium con° 
talning low=visc©sity ffetho^-l proliferated in a coetrentioaal logarithm 
role phase wltai tm lag psriodi in 250 ml, flasks cells increased tcom 
1" million in 30 mlo ($70 thousand per mlo ) to 170 million in 1^0 ml, 
ih million per ml<.) during 9 days, with an average generation time of 
about 2o? dayso 

In a later experiment, six eultm^s in 1^ liter flasks planted with 
a much heavier inwulum, proliferated at a much slower ratej «ieir 
average increase was from 200 million cells in 100 ml. (2 million per 
mlo) to 9kS million in iiOO ml.(2o7 million per ml.) during 21 days, 
with no lag period o The maxlmam was nearly twice ^ae 500 million mxism, 
prervlously reaehed^l^ this cell strain and another strain ft'om clone 
929 cells on MCTC/sti^lemsnted with 20 percent horse serum. Average 
generation time was about 10 days, an extr@a@ly slow rate of pr@lifer° 
ationg but the rate was constant and the cxO-tures were all hsalthy. S\23?° 
culturing at 21 days to an average population of 370 million cells in 
UOO mlo gave daughter cultures idilch again slowly increased to about tbs 
same maximnm. Despite the relatively slow rate of proliferation, tl»se 
cells 8h@H«d satisfactory growth and had the noris&l morphology of the 
cell strainc No significant behavior differences appeared between three 
lew viscosity grades and two low cencentra tioas of Metrocele Glucose 
is utilised at a comparatively low rate^ 



In sumnmy, we hav9 found that grewth of a strain of rouiBialian cells 
usder these conditions, fluid suspension in a rapidly agitated protein- 
&«e culti^re medium^ p3?eviously regarded as i^ossible, can be success^ 

fudly aeconpllshedo 



682 



Serial No, U28(b) <= Page 
Project 2 



Sigrificance s The ability to grow even one strain of raammalian 
cells in chemically defined lasdium in massive agitated fluid suspen- 
sion cultures unquestionably represents a most significant break- 
through in tissue culture advance. Cell nutrition studies in the Tissue 
Culture Section (Evans, project U28c, Westfall, project U28d) with 
chemically defined medium in static cultures can now be extended to 
agitated fluid suspension cultures.. Comparative studies of nornal 
and malignant cells can also be accelerated if it proves possible to 
grow other cell strains under these conditions <. 

Proposed course of projects This advance has opened up several 
avenues of approach which should be explored. 1) Can other strains 
of cells b© grown in chemically defined mediTim in agitated fluid 
suspension cultiares idien lethocel si^pleiosnt and silicone'<^oated flasks 
are used ? 2) What is the nature of the protective effect which l©w 
viscosity Metbocel exerts ^on cells suspended in rapidly agitated 
chemically defined medium ? It appears that Methocel does not have a 
nutritional effect in this casei Medium NCTC 109 has proven to be fully 
adequate for this cell strain in static culture for four years » 

A working hypothesis is proposed for the observed behavior of 
these cells in agitated fluid suspension cultures, 1) Methocel iiiiole>= 
cules attach firmly to the cell wall ^id so strengthen it mechanically 
so that these rather fragile cells are protected from the rapid break«= 
down ^ich they invariably suffer in the absence of Methocel from, the 
chemically defined a^diumo The fact i^at low molecular weight Methocel 
molecules (circa 20 j 000 to 1*0,000) accoa^flish this, Aile high mole» 
cular weight Methocel molecules (circa lij.0,000) have not done sso may 
reflect relatively high availability of sssthyl and propyl side '-groups 
in the shorter chain molecules for attachment to the cell walls. 2) 
Methocel molecules may alter the cell wall so that intracellular mater ° 
ial cannot leak out into the medium. 3) The hard hydrophobic silicone 
coating on the flask walls may not only serve to prevent adhesion of 
cells to the flask wall, but may act in sojas manner with the Methocel 
to strengthen the cell men^sranes. h) The low rate of glucose utiliza<» 
tion presents an anomaly. It appears that Methocel in the medium has 
no nutritional value for the cells o Exploratory analyses of the siigjer-^ 
natsnt spent fluids (Westfall project ii28d, unreported) suggest that 
certain amino acids are coscpletely used i^, others only partly, durixig 
the i^S-hour to 72-hour fluid change interval. Thus the physical adapta- 
tions may be accoB^anied by drastic chgjiges in the nutritional requli"^- 
mentso 

It is certainly most significant to atterapt to expand these findings 
to other cell types o It is also most significant to explore fully the 
raachanism of "Sxis protective action, to see if it can be better tmder'= 
stood and more efficiently util5,aedo These present advances now appear 
to open Tq> potentialities of cell growth in vitro ishich will require 
extended research. 

Part Bs (Omitted) ' Yes lo x 



Serial No. U28(c) 

lo Laboratory of Biologj^- 
So Ti38i80 Colture Seotioa 
3o Bethesda, Maryland 



PHS-NIH 
Indi¥id\sal Project Report 
Calendar Tear 1958 



Part A 



Project 1, Titles Studies of cell nutrition to yjtro o 

a ■= Standardization of inoculum poptd-ation density of NCTC 
strain 2071 cells for 100^ proliferation of all culttares 
in mediuja NCTC 109. 

b =■ Studies of tl» -v-alue of coi^onent groups of NCTC 109 <. 

G = Cultivation of Ifonkey Kidney I and Monkey Kidney II in pr©teia= 
£ree insdium, NCTC 109o 

d = FurUser studies ©n developing a shesaically defiiied rasdiraa 
for C3H mouse li'^r cellso 

Principal Infestigators ?oJ<, Evans 

Other Iiwpestigatorsg W.R. EarlSj MoCoPioraisKsntij, W.To 

McQuilkia, H,Ac Kerr» 

Cooperating units s Ec Kuff (Cellular Biology, HCI hOl) 



Man years s Patisnt DaysCcalej:K?,ar 

Totals year 1958 u 

Professionals 
Others 



Project desoriptios&g a) Staadardiaation ©f inoculum sizes ©f NCTC 
strain 2071 for 100$ preliferation of all cultures in lasdium 109° 

Objesti^^eg To determine the role of inoculum size and the effect 
attributable t@ the inoculum sise p®r se in asy studies using this 
strain of cells o 



684 



. Sollal No, U28 ((c) Pag© 2 
Project JI. 

Methods? HCTC strain 20fl, «ie .si^line of clem 929 ©f st^.iia L 
cells, cultured ia cheaiisally defined medium with no added protein 
(NCTC 109) since January 1955 was abudiedo RepUoate cultures K»re 
prepared with inocula sixes raiaging ft-om 15,500 to 2,001, 900 
per 3 nl. of NCTC 109 In T=15 flasks* 

Results 8 Some proliferatioa was obtained with each inoculum size 
usedo One hundred percent proliferation was obtained with inoculum 
sizes ranging from 36,600 to 2,001,900 per 3 ml, of NCTC 109o The 
optimum range of inoculum sise as measured by the rate of prolifer'* 
ation appeared to be between 160,000 and UlO,000 aod the rate of 
proliferation of cultures from this range of inocula at ? days. 

Significances To es^^loy statistically reliable inocula of NCTC 
strain 20?1 to test the nutritional requireasnt of NCTC strain 
20fl cells f©r specific coapoaents of NCTC 109. 

Proposed course of project? The study ims been conpleted and 
pt&lished. The findings will be applied where applicable to all 
nutrition and assay studies c 

Prefect description? b) Studies of t^ value of coB?>o»ent groups 

of chemically defined NCTC 109c (1) A study of the amino acids aeedsd 

by NCTC strain 20?1 cells for rapid proliferation » 

Objectives To determins the value of the "non^esseatiaP amino 
acids ©f chemically defined NCTC 109 to NCTC strain 20-1 cells as 
coflfjared with the par«nt strain 92 9 o 

Methods « Replicate culture of NCTC strain 20?1 cells and subse- 
quent enumeration of the nuclei per flask in each treatB©at were 
used to determine the effect on proliferation of various coid3ina° 
tiona of coaponents of the amino acid mix of NCTC 109, 

Results? Unlike findings when clone 929 strain L cells were used, 
(in which no marked differences in grswth resp^ise were seen within 
21 days, the ejqierimental interval for the replicate series study, 
and were seen only after long term cultivation) witJain ? days it was 
clearly and rapidly obvious that the "non-essentials" were r^eaired 
by NCTC 20?1. 

Significance J Of use in improving aod secondarily siB?>li:^ng 
medium NCTC 109 >*ese possible » 

Proposed course of prajectt To put into practical xase the nnd<= 
ings as related t© c-Tri? findings en effect of ooB?>©a9nt groups of 
the rasdiuso 



685 



il No. k 



2) Dct«rmiEAtion of the nsquireafint of tha coezszyzns coi^onent 
groi^ of medium NCTC 10? f©r rapid growth of KCTC strain 2071 
eollBo 

Obj«ctiT«8 To ddtersaine if the six coensynias of NCTC 109 con» 
tribute essentially to the proliferation of NCTC strain 2071 
cells o 

Methods s Replicate cultures of NCTC strain 2071 cells and enuiin@r» 
ation of nuclei of each ctiltus^ per set were usedo 

Restaltss The not unescpeoted result was found that thege six co» 
ensTiaes irare not essential to the growth of tjlese cells in the 
othenriLse unaltered NCTC 109 » In view of ihe vitamin inixture in«=> 
corporated in the laedium it ims not expected that the coenzymes 
would he found to be required ^ 

Sl^nifisaaceg T@ iaapro^ and if possible t© sinplify NCTC 109.. 

Proposed courses To put iato practical use the findings, in 
results^ as related to other findings on effect of eon3>onent 
groins of the lasdiumo Paper in preparationo 

3) Requireoents of certain nuclei© acid deriTati-^yes for rapid growth 
®f NCTC strain 2071, 

C3bjeoti^«8 T© deteraiae the requirenent ©f NCTC straia 2071 eells 
for any @r all of the nucleic asid derifati-^ea incorporated is 
NCTC 109= 

Methods s Replicate cultures followed by enuiosratiem of nuclei 
of each culture in each set to dstemdne ^w effect om prolifar° 

ation ef individual or confoinatlons of the nucleic acid deri'^ati'^o 

Results g There was found to b® significant inhibition ^fb&n de@@}:y=^ 
guanosine and 5 tnsthyl cytoslne were present and inhibition to a 
leaser degree y/ihrnn deoxyedenosins was present. Only thyiaidine was 
found to be slgnifieantly stiniilatory and de^qrcytidlne was found 
to be stisnaljitory to a slightly lesser degree. 

Significances T© iapro^ and sinpliiy if possible NCTC 109 > 

Proposed courses To put into practical use the findings, in results, 
ag rtlated t© other findings on effect of coapoaeat gre^s of the 
3)^diuffi, Paper in preparation^ 



686 



Seriass Ka» U28 (c) Psige lb 
Project 1 



k) To determlBe tiss inp^rtsmce and requiremiKt of tin gl%&isr©Bic 
acid inixtare ®f medium NCTC 109 t© NCTC strain 2071 c«llSo 

Objectiw©? T© ascsertaiB the need ©f any ©r all @f th® coBapoBssata 

of the glacm>onic; acid mixtiire t© cells &t NCTC strain 2071 in miedi'iM 

NCTC 109 for rapid proliferation » 

Methods s Replicate cialttires of strain 2071 cells were prep'v'sd assd 
the nuclei of each culture @f each set were esiumsrated t@ detemdJBiei 
the influence oei proliferation of the conponentso 

Results s Th® results indicate that m^ tw© ©f th® three e&i^iQfmntB 
laay be used equally as well as the total of three „ N© siagle ca^@n° 
ent was capable of substituting for the entire mixture o 

Significances T® ia^prewe and simplify NCTC 109 <> 

Proposed ceurses T© put into practical use the findingSj in results, 
as related to ©ttisr findings ©a effect of c©n^nent grot^® of t^ 
mediumo 

$) Culti^atioa of NCTC strain 20|1 cells in medium NCTC 111, a sinpli^ 
fication ©f NCTC 109 that has s«sulted from studies on individual 
groups of cofii^^ne&tso 

C&>jecti'^res Long tens cultl'^ation of NCTC strain 20?1 cells in medium 
NCTC 11? o 

Methods? Medium NCTC 11? was prepared according to procedures for 
preparing NCTC 109 o T<=15 flasks of NCTC strain 2071 cells were plaated 
a!%l maintained on the media with required st&cultuz°Jig» 

Results s T© be obtained firom study in progress.. 

Significances To siji^lif^ medium without sacrificing quality or 
quantity of cells o 

Proposed courses To conqjlet® the study underway by 1®^ term assay. 

Projection descriptions s) Cultivation ©f Ifeakey Kidaey I and M©ak@y 
Kidney II in NCTC 109 pr@tein=fr«e ehealcally defined sasdiumo 

Ctojectl^s To determine if it is possible t® cultivate moakey kidney- 
cells of two series in a chemically defined protein°fr®e medium s@ 
that such strains of cells can be >t«ed as laboratory tools and migdsl 
cell types for '^irus studies and possibly metabolism studies o 



687 



S^ial No» ii28 (c) Page 
Project 1 



Methods snployedg Tw© cell strains (Monkey Kidney I aad II) re- 
cei^^d in tbe laboratory in a defined iredltim axKi sertun nutrient 
were adj^ted to growth in Hjedivun NCTC 109 * a very small percseiEt- 
age of seruBio This serum percentage vas so lov that it voxiLd not 
interfere with ^^irxm assays (NCI U28bl), This success suggested 
that iiMaediate efforts should be made to get the strains on the 
p3?otein=fr©e nedium. Two procedures were useds (1) Step wise 
gradual elimination of serumj (2) Imiaediate transfer of culture 
strains to coiiplete defined medium without senuno These laethods 
were both success fulo 

Major findings s For se-^ral generations cells ha7® been carried 
■ep £TQm T-'l$ flasks to T^SOs, in aaedium with a small percentage 
of seruffio Since HK°>1 could not be coatinued in successful growth 
in protein-^free sasdium it was discontinued o Since studies indicated 
Monkey Kidmsy II can be grown in defined media in aaftoimts sfttisfac° 
tory for assay work^ shaker culture studies are In progress with 
MK»II on cbealQally defined medium NCTC 109o 

Significances As postulated in objecti'^ieo 

Future course of works Limited in this laboratory o Strains ha'w© 

been lemde a'^allable to Drg Manakero 



Project descriptions d) Further sttidies on de^e^loplng a ehemlcally 
defined msdium for C3H mouse li'ror cells <. 

CSsjectlTes To determine the cos^oaition of a chemically defined 
medium timt will culture satisfactorily strains of mo\3se li'^r 
epithelial cells s To acquire basic knowledge in pl^Blcal constants 
and metabolic studies to be used as a tool for designing chemically 
defined media for oti^r Intractable cell strains in ^trOo 

Methods s Tests of the effect on cell growth and appearance of a 
nuidMr of ▼arlations in oonpositlon of chemically defined media 
were made^ as well as variations in manner of technical treati^nt 
of cultures o Appearance vpon microscopic obaer^ationj, duratiosi of 
am^valf and ability to si&oulture were used as standards for 
e^alu&tii^ significance of the eapsrimental results. 



688 



Major findings s The variables in NCTC 109 and NCTC 116^'' tes-.ed ai-.d 
results obtelned are shown in the table below. 







Treatment 

of 
CitLtures 



Basal 'defined 
raedium #116* 
defined' niedima 
#116 + inositol, 
choline, 2nd, 
folic acid 
Defined' n^dium 
#116 * nucleic 
acid derivatives 
of NCTC 109 
Uef iiied nJediuBi 
#116 ■*■ nucleic acid 
derivatives and 
inositol, choline and 
folic acid 
defined medium 
* phosphorocholi ne 



Number of 
Subcultures 



Maximum 
length of 
survival 
in days 



11 



11 



- NCTC 116 contains onlys 

- 13 amino acids, 5 vitamins and Earle"s balanced saline. 



Since the table shows some "serum effect" is still required by the 
liver cells other ideas were considered. Assuming that at least part 
of the function, i.e. the physical effect, served by serum might be 
carried out by Methocel which is non^proteinaceous, inert and non»toxic 
NCTC 109 plus 0,06^ 15 CPS Methocel medium was prepared. This concen» 
tration grade was used because with NCTC 109 the relative viscosity and 
surface tension was approxiaately that of the serum and NCTC 109 mixture 
on which liver cells have been demonstrated to grow. In one study, the 
original cultures (no subculturing) were maintained 23 days. In another 
study with sttoculturing to the tiiird generation, the cultures were main- 
tained 23 days. 



689 



Serial No. U28(c) Page ? 
Project 1 



In order to ascertain whether reduction of eleotrelytes in awidiTM 
NCTC 109 woisld impro^ tim msdium, the salt concentration was reduced „ 
Calls cultured in NCTC 109 * 10^ Ho So ■were planted in tJfe fii?e test 
percentages of saline chosen after 2 saline Tjo&sheso At 6 days after 
planting, those cultures receiirfjtg either 30^ or 1^056 saline were coja= 
paratirely better than on the other mixtures o Returning saiEple cultures 
from each treatnent to stock horae serum laedivua demonstrated tteat -©labia 
cells were still present in alio 

Rather than the electrolytic balance being vgpaet the actual c6n° 
centration of nutrients in NCTC 109 might be overMhelming the cells s© 
a series of sttKlies coiiparing the effect of percent^es of NCTC 109 in 
the final medium wex^ conducted „ The pertinent details are gi-«ren in 
tabular form belowo Daily fluid change without centrlfugation resulted 
in the continuous loss of ft^e floating cells ii. 





i 






% 

ConCo 
NCTC 
109 


liluimber of 

Subcultured 

Generation 


kaximum 

Lengths of 
Sur^^ial 
in Days 


'Demons iration of 

Rec0wrability 
t? NoMal by 
Return to Stock 
Medium 


U 


1 


12 


Hb'rmai 


" 


i 


lu 


'MOTB^ 


■■"■■' u 


1 ' 


Hi 


Poor 


3S 


2 


i6" " 


?0or 



Significance of the Projects Because of the significance of these 
strains of liirer cells, useful as tools for the study of metabelisia (See 
Westfall, NCI U28(d) and Kuff, NCI=l;01) and for tus^r irirus studies (See 
Maaaker, KCI k3B)> a» well as the fact that the strain is a prims exai!|)le 
of an intractable strain for growth on chemically defined ii©diua, the 
study assumes great signiflcanoe o Knowledge gleaned from successful haad-- 
lizg of this strain should yield insight on how to approach the problem 
for other intractable strains such as human skin epitheli m and flresh 
mouse tisstie cells o Further work with these li^^er cells assumes added 
significance because of the fact that their transformation ^ iritro to 
produce a highly differentiated epitfcelial cell tumor (probably an ana= 
plastic hepatoflra") is possibly the first tumor transformation in ^tro 



690 



Serial No, U26Cc) Page 8 
Pr@ject I 



of a normal epithelial cell. Pursuit of nutrition and metabolism 
stiidies on these cells is of primary in^sortance if one wishes t® 
resolve differences betireen the malignant cell @f this type and the 
normal li^rer cell of origin^ 

Proposed course of study: The imnediate findings suggest that sij^ee 
the cells sk,@(»@d s^ae inproveaent in dmS^tion of survival by the 
use of metl^lcellulose, ftirther derivatives and c@»@entrati@n of nethy^ 
Icelluloses shsuld be examined for possible jp^sical effects ifl|>roviBg 
the cell iBesi>raae<> The appearance of the liver cells in NCTC 109 al®s@ 
at normal concentrati<^i st^gest sme crenation and p^^cnosis taking 
place hence the nunber of efforts described in this report to allev<= 
iate this conditi@no Other msn^metabolites having an effect in tmin-^ 
t&ini!% the integrity of the cell neoferane will be investigated o 

Since basal Hssditm NCTC ll6 also sh@»ed some promise persistent 
efforts will be made t@ evolve a cbsmically deflsied medium for ti^ss 
cells tiurough newest kncfwledge on nutrition cosfoined with information 
gained from studies on the metebolism of this cell strain. The effect 
on survival tSjsae of the influence of the other coap^aent grot^js in 
medium NCTC 109 against a background of NCTC 109 with the conpoaeat 
omitted as Hell as against a background of the basal medium NCTC 116 
described in this report should be determined. Analytical studies 
(see West fall NCI U28d) suggest that owm of the pertinent grot^s for 
imisdiats axasdnati©£i is the glutathiocs and/ or cysteine grouping. 
The specific effects of the iiAibitory (for NCTC strain 20T1 of strain 
L cells) nucl@ic acid derivativ^es on the liver strain must also be 
deterainedo 



Fart B inclisded __ les x No 

691 



Serial No. ii28Cc) 

1. Laboratory of Biology 
2o Tissue Culture Seetion 
3o Bethesda, Harylaiado 



PHB=NIH 
Indl'^idu&l Project Report 
naleadar Year 1^8 



Part Bo Honors, Awards, and PiflJlieations » 

pDfi3licatiis»s other than abstracts fr&a. this projects 

Byaas, ?. J», Fioraasssiti, MoCo, Sanford, K<,K<,, Earle, WoRo, aad 
Westfall, B» Bo: Studies of Nutrieat Media for Tissue Cells in 
Vitro c IV o The Effect of Amino Acid Mixtures on the Pr©lifera» 
tion of the Long-Term Strain 20|1 of NCTC Clone 929 of Strain L 
CellSo A:©ro Jo of Hygiene, 66s 66=73, 1958, 

Floransonti, MoCo, Ehrans, Vo J,, and Earle, WoRo s The Effect of 
Inoculum Size on Proliferation of NCTC Strain 20?1, the CheadLcaUy 
Defined Medium Strain ©f NCTC Clone 929 (Strain L). Jo Nato Cancer 
Insto, 21s 79^3, 1958 o 

Evaas, VoJc, Hawkinsj NoMo, Westfall, BoB„, and Earle, WoRoS Studies 
on Cwlture Lines Derived ft'om Mouse Li'««r Parenchyaateus Cellg Grown 
in Loi^-Term Tisstaa Culture « Caaeer Research, IBs 261=266, 1958 o 



Honors and Awards relating to this projects 

Inrltation to be the Guest Speaker at the Annual Cheisistify DepartiKsnt 
Dinner^ Oouchar College, Neweafcer 19 j, 1958 » 

Iniritation to deli^sr Guest Lecture before Navy Nuclear Nursing Course, 
National N«7al Medical Center, Decei^r 2, 1958 o 

Appointed BS8iS>er General myi Pl^sical Biol^^ Review Panel, 

loff'itation for lecttzr® and demonstration of work. Course on Tissue 
Culture, University of Colorado Medical School, Tissue Culture Ass«>» 
elation swaaser, 1958 1 unable t© accept due to pressure of work in 
pr^ress » 

Continued as Consultant to Navy Tissue Bank, UoSo Naval Medical School, 
National Naval Medical Center; letter of appreciation f^om Chief, Bureais 
of Medicine and Sus^ery, Department of tfce Navy, t© Surgeon General, Uo 
So Public Health Service, 



692 



Serial Ho<, ii28 (c) 

lo LsboTAtoTj of Biology 
2 c, Tissue Culture Section 
3o Bethesda, Mazylazid 



PHS-NIH 
Individual Project Report 
Calendar Year 1956 



Part A 

Project 2, Titles Cell trftusfon^tions Ijq Titr o. 

a °° CarciBogeisiesis potenti&l of tisai^s culture mediae 

b => Ebqploratorj studies on ts®ah C3H imvao tissue in chaissicallj 
defis^ed msdia for carcinogesasis studies, 

c «=■ A stiudj of the effecst ©f ttee Stewart^Eddy polyene Tims oh 
lo^ term tissue cultured straias of cells. 

Principal Iisrestigators f c Jo E^ans 

Other Imestig&t©rs« W„ Ro Earl®s GoA« Parker 

Cooperating Unites None 



Man Tears s Patient D@ys(cal@»dm' 

Totals year 1958)3 

Professionals 
Others 

Project descriptions a) Carcinogenesis potential of tissue cultur© 



C%>jectiTess To in^stigate the possibility of tusasr production trmi 
injection of 1) the standard chick esibryo extract » horse serum s^dium.c 



2) single asj^ers of this oixture^ 3) unfiltered chick enbryo extract^, 
k) cheaioally defined medium NOTC 109. 

Methods s All the abo^e mdt& as f^sh nmtised media injected into 
se'^ral hundred ne^om NIH S»iss sdce, C3H x AKR oice md CjE ndcsc 
It is proposed to keep all sooisials 1 year or until positi'?e result, 
wfeichs^«r is shorter = 



693 



Serial NOc hZB (c) Page 
Project 2 



Results s T® datd no sisgle tumor has been ebser^d in axsy aniioalao 
Most of the Swiss mice haire been injected for at least li to 8 m&nt&@ 
and tba other strains a lesser time to d&te because of delays in 
breeding and obtaining sufficient nusber of animals for adequate in- 
jection series c 

Significai^se in cancer research s It is hoped that these data will 
answer the qnestions of idiethsr or not carefully prepared well 
standardised sradia could be responsible for the pr©ducti©n of tumors 
in vitro o 

Proposed course of studys To bring to logical tiim conclusion as 
indicated in objective and method » To s-raluate data for subsequent 
consideration of approach to carcinogenesis in "ritro on fresh tissua 
in defined media under controlled physiologic conditions o It is 
recognised that there is the possibility l^t some of the conpomnts 
of the defined medium could be infected with viruseSo It is, there^ 
fore^ necessary to g±fB consideration to sterilisation of the media 
for viruses as a step of one choice but at the ^ery least to deter° 
mine through sosts arranged collaboration the possibility of viruses 
being present in the media^ With such a project tliis is necessary 
before validation of ai^ results on carcinogenesis with f^esh tissue 
cultures in cheadcally defined media may be coapletely 



Project description? b) Eaqploratory studies «a f^sh C3H mouse tissue 
in chendcally defined media for carcinogenesis studies « 



Objecting To cultivate flresh CjH lasuse tissue in chemically defined 
media by the best technology currently kpown end to improve this 
technology wbea possible j in order to s©«i/(l) "spontaneous" triansforsa^ 
ations occur in such cells or (2) = To induo© transformations in ^sdtro, 
in such cells o 



Methods? Several series of C3H of pooled eaforyonic mouse cells haw 
been cultm^d in vitro in completely defined media for periods of tlia@ 
varying troM 2 we^TT© several months <. Aliquot of ©ells used t© plaat 
each of these series have been injected into C3H n®Ti&©ra mice &M int© 
all purpose Swisis ife^omo 

Results? To date a© tumors mar eistoryomas have arisen at the sita ®f 
injection f^om fl^sh tissiMo No cultured cells have been injected t© 
date because the technology of cultivation of fresh tissue is not y@t 
advanrsd enough to grow large masses of tissue for infections and still 
maintaiii cells for study of techniques to grsw fresh tissue in 
amounts in chemisally defined saediao 



694 



Serial No. U28Cc) Page 



SignifiGanc®8 Beyond ttoe fact ih&t frosh tissue unculturod to 
date has gl^n no tumors on Injjectlon the major sigslflcance still 
jremaias in att«n9>tin6 to carry out the objectiireo 

Propessd courses To continue to ijopratB the eheadcally defined assd» 
ium assd technique so that it is possible to propagate adequate an^unta 
of tissue for continuous injections to test possibility of spontaneous 
transformation of cells s® they will produce tuners on injection^ To 
concoodtantly atteapt to produce deliberately a traasfomation in ^Jtro 
u!»ier mare controlled conditions than previously a^railableo To at^i^" 
to advance teehnological conditions so that this can be acoos^lished. 
To Biedi:^ the proposed irothedology of attacking the problem if the 
technology is not satisfactory o Soras factors to be coasidersd in raatho- 
dology are control of gas ©xehange, aad control of filtration methods 
to achieve ^perfect" sterilization ©f solutions with respect to viruses » 

Project deaeriptioni c) A study of the effect of the Stewart-Eddy 
p®lyoHS virus on long term tissue cultured strains of cells » 

Objective? To deteraine if the polyoma virus could be propagated in 
1®^ term straisss of sells cultured in vitr®^ 



Method? Tw© strains of long term cultured ussus© liver straiSj, osae 
strain of human skin epithelium NCTC clone 2SWi and a laonkey kidney 
strate MK°I were infected in vitro by culturing a piece of parotid 
tufflor Itiea the SE polyoma v1tu®» Subsequent injections (after 1 snsath 
and 2| osnths Sa vitrQ) of flidd a&d cells were made into newborn Swiss 
mice and ntewbom C3Ho 

Results s Parotid and submandibular tumors were obtained in Swiss B!i(^® 
injected with material fromlong term liver cultures NCTC clone lli69 
and NCTC 721 injected after being subjected oise month to virus. At this 
tima no tumors were obtained from injections of husan skin epithelium 
or msekey kidney epithsliuso Swiss animals injeeted with ^iterial from 
leng term C^ msisse liver cultures, and monkey kidney cultures carried 
2^ mimths in ^tre showed parotid, kidney and lachrymal glsad tuners. 
No n9wbomir3!rSce injected with ai^ of this material at anytiEsg devel° 
oped tumors o C5H mice were included in the injected mice &fh^ias hB^m^e 
some of the infected arterial was trmi strains of C3H mouse livers 



Significances 1) Two long terra stirains of C3H mouse liver cells imTB 
capable of pi^pagting the virus. The C3H mouse liver cells in »&sg 
could be a source for large amounts of the virus <> 2) Human skin 
epithelial cells did not st^port the polyo»i virus a@ evidenced by the 
failure to produce tunsors at either of the two injection periods into 
newborn Swiss or newborn C3H miceo 3) Alteratiesw in the appearance of 
the monkey kidney cells suggested either a) <^t©l©gieal transformation 
in vitro of the a^iakey kidney cell as a result of ^ vitro infection 
^ ^se°p®lyoma virus or b) the polyoma tissae cells overgretw and 



695 



Serial No. li28Cc) Page h.. 
Project Z 



out the BMiskey kidBe>y cellsc Ths 8ignincau!ioe of the appearance of tmmra 
from infections of cultures originally containing monkey kidney cdlls and 
polyoma tximor tisstze is therefore controirersial in this izr7estigator''@ 
opiniono 

Proposed courses Sobks consideration should, if facilities are avail- 
able to laaintain such -7iru8 infected cultuz^ and aninals^ be git^en to 
conflrsdng escpsriissnts cm points 3a and 5b tsnder '^ significance" (See 
NCI U28e) with conaideration of the inplications of restaltso 



Part B inclMed Yes 



Serial No„ i428(c) 

lo Laboratory of Biology 
2, Tiaa-m CiLlture Soctioa 

PHS-NIH '• *•"»'**' "^^ 
Individual Project Report 
Calesidar Year 1958 



Part A 



Project 3ji Titles Collab®ratiosia ia Tisstae Culture with other 
organizations. 

a» Propagation of PoPcL organism isolated f^om h^ma^ sases 
of urethritis a 

b'^ DenK>nstration of B gluronidase acti'^ity in C3H ntouse li'^er 

ci£Lt^srQSo 

c<= Sttidies on Carbans^l phosphate s^thetaseo 

d- Collaborati'^ studies with Tissue Bank^ National Na'^al 
Medical Scho®!, National Naval Medical Center o 



Principal Investigators = Vo J, Evansy B<, Bo Weatfallj WoRo Earle^ 

Other Iswestigstors ~ MoCo Fioraaonti, Q«Ao Parker, HoA. Kerr 

Cooperating units <= a = Dr, MoCoShepard, Na^al Medical Research 

Laboratory, Can?) LejeunOj, Nortfe Car®liBac 
(Introduced by Dr<, Byron Olson) 

b » Dto Kuff - Cellular Bi©l@gy =NClC!i01) 

c - Dro PoP» Cohen, Dro Qeorge Brown, Ifei^o 
of Wisconsin o 

d ■= TiBSUfi Bank, Naval Medical Sch©®!^ Natioaal 
Na'^al Medical Centero 



Man Years? Patient DaysCcalea- 

Total? dar year 1958) s 

Professionals 
Others 



697 



Serial NOc hZBic) Page 2 
Pro;}act 3 

Project descriptloa s A ° Propagation of P=Pc Lo organism isolated 

from hximan cases of urethritis c 

Objectives To determine the stasceptibility of NCTC clone ZBhh skin 
epithelial cells for a PoPoL. os^ajsism (producing urethritis from tw@ 
fi-esh isolates ©f the orgaiaisn* t© determine the virulence of the 
organism in tiie cells o 

Methods 8 2li hour ©Id cultures of skin in NCTC 109 * 5% serum w©3f« 
iBis;.'^yji^ecl with injection material fr@m 2 sources (clinical cases 1*89R 
and k93) initially carried in chick endodermal cell eulture according 
to the method of Emilio Weiss „ Observations vere made of cells at aE, 
k& and 72 hours <> 

Major findings g ^ Intracytoplasmic multiplieatioa was noted ia &v<eT 
75% of the cells o Cytopathogenlcity was^ noted with (in the terminal 
stages of the es^erimsnt) an estimated 9$% t destruction of the cellso 

Lcance of th e research g Dro Maurice Shepard informs ras that the 

Ltures st^pllied subsequent to our planning tbe w@rk as well as the 
medium ha^re been of definite usefulness in clinical oM experimental 
work in the study of this P»PoL» orgaaismo 

Future course of work g No further work on the project is plaimsd with 
Dr. Shepard since he now has Idle ^^S^^eJ^^hP^^'S^ been trained and in» 
structed in handling the cells > A/^lam%is been found by Dr^ Shepard 
so he can c@ntinue work alone « 

b <= Studies on B glucuronidase activity in C3H msuse li-^r cell culttsres 

Ob j act ires To test in isolated cells 1=,®.. long tena C3H mouse livsr 
cells whether a particulate material such as B glucuronidase which has 
been found in these strains of li^rer cells; is related to the knowB 
pinocytosis activity of the cell. 

Methods 8 Hoffiogenates of cell cultures of all three lines ©f mouse llwmr 
in 2 media °^ a) stock medium^ of chick e^Tj0 extract and horse serum 
saline and lii b) a chemically defined n^dium with IC^ horse serum w@r@ 
analyzed from enjsyros acti^ityo Similar analyses for the activity were 
made on transplantable tmi®rs deri-^^ed f^om the cells (See Dr. Kuffs 
report KCI i^Ol for greater detail on methodology) » 

Ma^or find ijgg8S___ Ifeusually large amounts (for the C3H m®uae li'sisr to 
▼ivo) of ¥gluronidass were foiaad in the tissue culture cells. The 
transplantable tumors from the cells also shewed large amounts of th© 
aoti^ty (See Dr. Kuff's, NCl4i01) 



698 



;^«rial Noo Iij28 (c) Page 1 

Project 3 



Sigmncance of findings g la wlm ©f the fact that the C3H __ 
liwr has thie eneyme in only low levela the restilte assuoe aigsui^ 
ficance as orae of the few denenatratlons ©f tbe. Increased actiTifcy 
of aa amyras by raammaliaa cells in tiasm culture, rattier thaaa th® 
mere fr^qiasntly obserrod loss of an enayrw activity in snajmoaliaa 



cells ^ 2to<. The acquired ens3nn& activity is also n®t Imt ft&m 
^ ;®1J87V'"^P^***^®''° ■^*»^'»® findings als@ raise the qm^Umv, 
^ ?*^!/*®!®^'' ^" ***® **®»^ cultiffe en^iroMBsnt ha^ led U tte 
acquisition in excessive aasatmts of particular onzymBj 2) caa it 
toe used in aisorway as a practical t^l 4® deterndnSag the chaaa® ®f 
a B®rmal to a fljalignant cell ? 'c »« -^i 

PrapQsed course of pro„-)ect. At tbs lasraent a definite decisis qk 
tMFi:i net made^ -i-he priority of the urgency of seeking answers t@ 
some ©f the queetieaa bswi^ght t^ is uodecided as yet until ftxpttos- 
emigrations aad th©\(ght8 are cxyBtallizsdo 

c = Studies on Carban^l syntfeetase, 

^^ectl^ Te determine if Carfaaisgrl synthetase activity is present 
SfTlfr^^r"^® liver cells NCTC mme liver cells imiO 
cl^ie iko9) l£ vitro o 

Methods? Hoa^enata ©f various tissue culture cells were m&hr^^d 
by Dro Brown for Garba^l synthetase activity. 

lfe,1or Finding s No activity was found. 

Sigaificaace? The conclusion is drawn by Dr„ Brown that this enssjae 

ll Tll2Tl,^^^''.u^^^^ ^"^ ^^ " ^^ ^^ ^ ^^«1 ^^ detection,, 
AS a rei^h estl^^^, the enzyro could not therefore be praseat at a 
le^l of more than $% of that for the liver of a C3H iB©uae„ Since 
Ca^aagrl phosphate is considered t© be a precursor of pyriiaidias, 1) 
eit^r pyriMdtoes in tham tissue cultured cells are SS ayntlieksai 
by the cen^lete route for these liver cells or 2) Carbajurl pbaaphat® 
is pp^uoed by BmB manner other than the MBchanisa iarolving the en- 
zym hereo The deletion of this enzyoi must have occurred dt^lsg the 

^^?? "fv^*^*** ^ ^^^^ ^^'^ ^®1^^ ^^ *»ea under culture. 

Informtien on t*e metabelic patterns of sells must ultimtely ah®d 
light en the pr^le« of growth ef cells te vitr® without whiS no c®a= 
trol of cell growth «ay be obtained and STtS^T-which no cell can S 
a useful tool in raetsbolic research and in obtaining a clarified uM@r- 
standiag of the differences between the mligaaatlell and the LrZi 
cell fi-offl which it has aris^ in tissue eStSe? 

d » Collaborative studies with Tissue Bank, National Naval Madical 
School, National Naval Medical' Cmter„ WBdtcs.1 

g|M|1LgsOTipt^ dl =. studies in evaluation of the uae of ««^t 
^^^cIprar^Eile ble^ as currently finished f^om bl«d colSct^ 
ioa ageaciea as a seiErce of serum for tissue culture studies 



699 



S«ri©« No 1^28 (c) P« 
Project 3 



Qb;|ecti;y9s To deterissla® •isaefVslin^ss ©f oatdated citrated blc«d i% 
tissius cultusraiso 

Methods 8 Citratad whol® blood was twatod with bo»1jne thrcwbto„ 
fSaawma obtaisiffld taas cropped and c©ijS»ia®d ■sdLth NCTC 10? mediim and 
this nutri®rat ^as tested on hmmi skin ®pitte®lial cells NCTC 25IA^ 

been 
Rosultaj Htmsm skixi cells httvo/ctdtur^d coRtiziaoual7 in this midium 
for cf«®r 8 Konthso 



SipiifioaBce s For practical pm°po@es %^r«s 86£HBwhffl.t leas critical 
a^^SHa^^s isaixiitaisisd this "'^outdat&d^ Im^m blood is a SAtisfactoiy 
source of ®®rtm for caltiTatioa of c®11.®d 

Propossd^ omfmt T®chxjioal us® of this typ® of sexTMa ^dsar® lascesffl-aryo 

P roject d e acriptl ons d2^ Stady of potential as® of KCTC 109 a® aa 

^ectiTOs To deterMj)® by finiiiial asqwzdsBnt&tion the potential of 
!S5TC5""'i09"as am iatra^noiss fluid for possible immn «s«o 

Methods 8 J«CTC 109 hm baea ii3i,1®ct®d into jBice, rabbit® md graiima 

°pi^8 "in singl® iHj®cti©as aad multiple large aad sai&ll rolmm is- 
Sections o 

Rasuslt® &x® still in th© praliaiaary stage, Ho¥®v®Pj ih® 
'«ltJ9r®d hematopoietic activityi it h^ not been foimd, to 
J it dei®s M©t appear to be dspogited In any of t&© ©rgmsis 
teB|}OZ<&tur® ele'9%tions h&fo been ascribed to it» 

Significance 8 It is the opinion of isKrostigators at the clinical l(^-tf©lj, 
MoNoMTCo""iMt the md±m NCTC 109 jway toII proRre to be a most 'b&mtipisl, 
economical and practical adjunct to the present day intra'^no^is fl'risi 




P roposed futuys oouree 8 Contismsd aniiaal sjcpariBssntatio^ja followed 
SyjGsEMW^5SMBs©°lS°SiQiEaas after appropriate arrangessats ©od aath^s?- 
laations for clinical testis^. 

Pro;j|ect . d@@crlptipas d3a Th@ etudy ©f th® us® of ^ cloa® ©f bujnaxi 
iOn^I^SlBsffi for transplantatioao 

Methods and ^^Jeotiveas ?arioBs orgaioic aad inorganic inatarlals siach 
as'~^»3iHcaSfi^"'onffi.lipor® filter saaterial are being c©n@f.d®red as 
stipporting network for th© epltfe@'.liffil cells as wsll as for their pr0<= 
tecti'^ ability as an int©guii®at to prsy^d© a f&vorafel® ^frnixoinmnt In 
^s^iich the c@ll@ can gro^c. 



700 



Serial No. U28Cc) Pag© > 
Project 3 

Res ults g No i*e suits on the proposal abcrvBo 

Siaiiflcanoeg Successful skin epittoeli&l cell tranoplantation f@r 
either a ieaporary dresaiiig or a longer terra one has clinical b©ar= 
ing on rsparati^Q surgery in general and bum prdslema ia particular 
in lailitary asdic inco 

Future course? To pursue proposal. 



Part B Included Yes No x 



701 



Serial Wo. U28 (d) 

lo IjBboratory of Biology 

2, TiaatsB Ctaltara 

3o Betheada, mxyUad 



PHS=.NIH 
Indt»'idual Project Report 
Calendar Year 1958 



Part A 



Project Title? Studies of Cell Metabolism in vitra » 
Principal ImiBstigators Bo B» WestfaU, 



Other Ins^stigators 8 E.V, Peppers, V»J, E^ans, K,K, Saaford^ 

JoC. Bryasst, W.R. Earle aisd E„L„ Schilling = 



Cooperatij3g tJiaitss 



Man fears (caleisdar year 1958)* Patieat DaysCealeadar 

Totals year 1958)8 

Profsssioaal? 
Others 



Prolect Daacgiptioa ^ Tl^o broad ge^ral headiag abo^re c^^rers -^e 
vork of tSis ttiait as part of the problem of the cellular origin of 
cancer o A) As part of tMs a study is l^'eiiog saade of the nucleic 
acid content of cells gretm under various cultural conditions. These 
conditiona include different supporting i^dia and agitated and still 
cultiareso Different cell strains are also under study. The total 
dry iraights, protein, DNA^ BNA aisd cell populations based on count' 
of the nuclei are being oospared. B) Another study is concerned 
with the total lipid and ch@lester©l content of the cells under wsrj^ 
isg oosidltionsa G) Studies @n ttae glycogen content ©f several cell 
strains ai?e coBtinuing <, D) A continuii^ study relates to the Kateriala 
used from the nsdii:ua asitd retttri^d to the sodium by the cells tmder 
various culture conditions aad with different cell strains. E) la g© 
far as the nutritional effiscts of -rarious lastabolites are s^diated 
by enzymes sma study is being giwea t© certain en^yi^ asti^ties. 
In continuation of ttos work assoeiatod with tfee findiag tJmt two 
strains ©f Bsuse fibroblasts had greater arginaae activity than tfee 
parent Hkm so-eallsd ^highs' tm!sr<=fFr@ducii^ line of the ttmsrs 
arisljsg Srmi •&© Injsctioffi into, the 3©us® lis^ of origin ba.'v© b«©a 
assayed for aygiaas© aeti'^fity, Tfee r®s©lts ajre beii^ coiqjileG,, SQma 

702 



Serial No. Ii28(d) Page 2 



work is under way on the alpha=pho8ph©ryla8e and aayl®-l^=«lue@- 
sidasQ of the tbr&Q mouse li^r strains. It n©w appears that the 
1795 lias can no IsBger lay dmn glycogen in ttie cellj so that it 
is deemed desirable t© see which of these enzyroes haw been loeto 

Proposed course of wcwk is coatiniiation of lines of research already 
in progress as abo^e outlined. 

Significance of coag>leted works The four publications listed wder 
Part B for the year contain certain findings of s©m& ©f the studies. 
Paper nui*>er 1 suBasarizes some stmiies on the arginas© actiTities &£ 
certain of the call strains currently under study in the lafooratorr 
Sine© arginim has 's>Q&n included in the insdium fdr nutritim @f the' 
cells and has found t@ be a necessary growth factor for all cell 
strains so far tested any information relating t@ it is d® v ed of 
Talus o The eaaysie arginase is that one concerned with cos^rtiBg it 
to ureso Its acti^ty (arginase) in the cells has been ©xaniinedo 
Variations within a clone were found, so that this informtion would 
appear to point ^sp the fact that as aasorphologic change may take 
place during cultivation of cells for prolonged intenrals, emjm 
patterns may also change « Paper 3 reports the finding that in the 
absence of added pr@tein in tl^ Kadium glutaaine is aeeded by the 
sT&line of aeuse fibroblasts 2071 to renala aliire. Papers 2 and h 
record the fast that in agitated fluid suspension with continuous 
gassing betfe the nisuse li-^er and laouse fibroblast strains use large 
aiasuats of glusose and eowert part of it to lactic aeid under aerobic 
conditions. The li^er strain produces and returns to the laedium asieh 
nor© in the amount of pyrtwic asd ket^lutaric acid man does the 
nbr^last strains The study reported under h also shewed that sisaple 
fluid addition to the laedium in which the cells were gretrLng was a®t 
feasUJle as a tectelque for long term culttK^ ©f cells o 



Part B included __^ Yes x No 

703 



B&f" W Page 3 



PHS-NIH 

Indi^dual Project Rep©rt 
Calendar Tear 19158 



Pwtjio Honersj Awards., aiad Publication 

The foll@ivi»g pyblicatiosw h&r® resulted iVoni the work outlined ^&^^ 
during the yesrs 

lo Westfallj BoBo, Peppers, EoVo, Mms^ VcJoj Sasifordj, K«Kos H»'«»ktaSs 
Ho Mo, Fi@raro@Bti, M. Co, Kerr, Ho Ao, Udbhs, O.Lo aral Earl«, WoRoS 
The arginas® asad x^@da»®8e aetifltles @f cert^dji cell strains after 
long culti'^atisR 1m vitro o Jo Biophysieal and Biochefflicsl Cjrtolegy, 
hi 567=570, 1958 o"~ " 

2o Westfallj Bo Boj, Ewaxm^ VoJoj, Peppers, E„¥,j HawkisiSj N„ M.,, Bryaatj 
Jo Co, S<*iillis!^s, EoLej aad Earl®, WcRoS Observations ®e tte 
sBstatoolio beha'^er @f a cl@^ ©f saeisae liimr eells gr@Mn in agitated 
HMA stigpensioQSo 

3o Peppersj So V.^ M.©r«aimitij MoC, Westfall, BoBo^ B^aM, ¥o J..s 
aad EarlBs WoRo g Effect ©f laek ©f glutandae on Sublin© 20?1 
motsse cells Jo Nato Cancer iKsto 21j 611=620, 1958 » 

l*o Westfall, Bo Bo, Peppers^ Eo¥o, Bi^ysmt, Jo Co, Schilling, E. L. , ■ 
a^d Earl®j WoRo? Effect ®n the BBsditun of grwtto ®f cl®m ICTC ??9 
cells @M avmpstmt&m ia agitated flasks with sisqple fluid tddir:' 
Jo Nate Cancer Issst, 21g h29-k3B, 1958. 



704 



Serial No. U28(e) 

1. Laboratory of Biology 

2. Tisstas Culture Section, 

3. Bethesda, Maryland 



PKS=NIH 
IndiTidual Pro^ject Report 
Calendar Tear 19|8 



Part A 



Project 1, Titles Vitamin requtreiaaats of strain 2071 C@rigia» 
ally ttoxa. straisi L) cells as t.ested in. a 
chautically defined iradiniao 

Principal Iiwestigatorg Ko Ko Saaford, 

Other Inrsstigatorss LoT» Dr^ree, AoN, Co^'alesky^ WoRoEarldc 

Codperating Uhitss Nobs 



Mass Tsars (calendar y®ar 1958)? Patient Days(oa2jsadar 

Tot&lg year 1958 )x 
Professionals 
Others 



Project daacriptions Our objecti-ffes aare 1) to sii^lify pr©t9in= 
tm& cbeiBicaiay defined madium NCTC 109 by elimination of all 
^itatoins not required for laaxljsal proliferation of this cell strain 
and 2) to defix^ precisely the vitamin requirements of these cells , 

Methods enpl^reds Quantitative replicate culture procedures are 
usedo The cells are gemm on a modification of msdium NCTC 109 
lacking coansynsso Cells aire grovn for se^^eral veeks cm media 
lacking each of the 18 vitaminao After this period of depletion, 
quantitati'^^ procedures measure the grotfUi reaspense to additions 
of each of these /rltafldnsc, The reduced nunber of requisite -^ta^ 
Biins will then be tested as a gr@up and varied JjodiTidually Ib 
affloimt to dstain optisima balance <> 

Major findings s In prelisdnary sttsiies, these cells ^<ere tested 
in mediuiB NCTC 109 with and without ^tos addition ©f ultrafilter«d 
washed serum proteins at concentrations ©f l»2j, 0.8j and o08 per= 
cent. The reas@n for this study is e:!qplai2Md in the project on 
high and low saa?c0iaa=produeing ©ell lines. Cells were caarried for 
several transplant generations on tbes® Xeirsls with and without 
the individual vitaiaias, psntothfsnat^ , bio tin, folic aeid, i-iB.ositol, 
p=affiino beaaoio acid, and vitaaiin a.. Although there was tm 
detectable influence of the vitaain deletions ©a the cells grewB with 



Serial No. U28(e) Page 2 
Project 1 



asrjy le-a-el of serrm proteins, cells grown in control media showed 
requiremsnts for pantothenate ^ folic acid, and possibly p«amino 
benzoic acido These preliainsry experiments repealed the Hiiltility 
of detectisjg vitamin requiremsnts of cells grown in msdia contain^ 
ing e^^n these small amsunts of sertsm proteins <, The results shoi««d 
conclusively that i=inositol, required by all other strains of C3H 
■=ai©uae cells tested^ is not required by strain 2071 when tested with<= 
out serum protein addition c 

With the deiQ^nstration by S^ans and assm:iates; CUsSc) that the 
coenaynes were not required by this cell strain, present tests are 
in progress on all 18 vitamins in the coenzyme <^£res n^diunio 

Significances Although studies have been reported by Eagle and 
associates c^ vitaadn requirements of strain L cells, all of these 
studies were carried out in a sodium that was not chemically de» 
fined but contained dialy^ed serum proteins, ii^ich could contribute 
B^isy undefined supplestsntSo For this reason, a determination of 
vitamin requirements in chemically defined medium appears desiraJsl® 
not only to define the requirements of this cell strain but also to 
aid in simplifying Bffidium NCTC 109 for farther coaparatiw studies 
of nutritional requirements of cell strains. 

Proposed course of projects This project will be continued in an 
effort to fulfill the *j©ctives specifiedo 



Part B insluded les x No 



706 



1,; laboratory ©f B\ 
2. Tiaaiiae Ctatur© S^-.- 
3„ Bethwada, Maryisad 



PHS^NIH 

ladi^idml Project Rep®rt 
Calendar Tear 1958 



Part B s Honors J, kmiTdag aad Pi&lisatloaso 

Piifeli©ati@as? A siaiilar stmiy ©a aisdjas acid reqiais«!ra8fflts g K^Ko 
Smf&rds ¥o T. McQvdlklaj, M„Co Pl®raH»satij V„ Jo E^am, aad ?.Jo 
EwaaSg and W,, Ro EarlSc Study ©f aBda® aeid reqisireaaat® f©r 
iacraase is cell popuiafci®!® @f NCTC close 929 (atraia L). Jo Wat. 
Caacer lasto 20s T75-785, 1958 o 



707 



Serial Noo 1428(e) 

lo Laboratory of fiissl^gy 
2o Tissna Culture Section 
3. Betbesda, Maryland 



PHS-NIH 

Iadi'«ddual Project Report 
Caleudar Year 1958 



Part As 



Project 2g Titles In '^itr® stiaiiea ob spontamotis jnaiainary ©ar= 
cinoieas &t the strain C3H 



Principal Iwmstigators K<, Ko Sanford, 

Other Ixw©stigators« LcTo D^re©, AoB„ Co^yalesky and WoRoEarl®r 

Ce^rating Units 8 H„ B^ Anderfentj To B„ Dmmc 



Man Tears (calendar year 1958)? Patient Days (calaadar 

Totals y®ar 1958)? 

Professioi^ds 
Others 



Project descriptions 

Objectives s The original ^j©ctit« of this study (.^eh, 19S1) was t© 
deteradne whether the milk agent persists in long<=>term cultures of 
strain C3H mouse Qtsumnary carcinonas, T^ results of this early study 
re^realed activity of the agent for as long as 7 m^ths (9 tramiplaat 
generations) of culture in -gitrOo After this period, all tasts for 
the agent were negatif^, and '^e c@lls produced sarcos^s rather than 
carcinooas on injection into adoe of the strain of origin^ Sareon^s 
were produced by long-term cell strains cultured ftr-oa 13 differsat 
spontamoua earcinoEaSc The ^jecti^ of tte present study was t© 
determine the origin of the sarcetna cells. The follswlng three pd3si» 
bilities «©re censidereds 1) The original spontaneous tuii»rs a^iy 
ha^e been laixed 2) the str^ial cells of tbs tumor aisy h&-m becoms 



70^ 



Serial NOo U28(e) Pag© 
Pro^ject 2 



neoplastic in ctaltureo 3) The carcinoma cells may ha'>i?e trans- 
foriEsd to typical sarconsa cells in culture. 

Me thods ea^ lpyeds By passing the spontaneetss carcinoim for 
several gesserations through susceptible hjrbrid mice CG3H x BAXB/c) 
Pi the ©riginal C3E streuaa would be replaced by stromal cells fr«a 
the hybrid heat. Such tuif®ra ylhsn ©3q>laated to culture would cmt-^ 
taJA the tuBi©r cells and h^rid stromal cells o By suitable trans'^ 
plantation e:xp3riiQsntS; the identity of these cell types could be 
established. The culture fluid used was chemically defined medium 
NCTG 109 8i;^p3.efflented with 10 percent serumo 

Major fiiidiJiga^ Ic One strain of cells aral a deri'^d clone both 
gaTe rise to sarcoBtas, and ths cells were identified as cell® from 
the Fi i^rid host. Thus it was shown that str^nal cells of tfes 
host cultured in iritro witti the tun»r cells in a s©rma=s\^pl0j^at®d 
cheadcally deflneci' 'mBcTium can becoiae neoplastic, 2, One strain of 
cells piroduced sarcomaaj and the cells ^eere identified as strain 
C3H cells o Howe-^rj examination of the original tissue sections gh©w©d 
that on the second transfer in the hybrid host, this particular tum@r 
trasasplaat had grown as a saroMia,. Therefore, the original tumor 
msd for, culture could ha^re been mixed. 3, One strain of cell@s for 
the first four months in culture (U transplant geosrationa) on aasay 
in mice pr^uced well=differentiat©d Tmmmsj carcinomas indistisguish-® 
able from the original tumor, but after that period either failed to 
gr©w on injection or produced sarcomas. The cells that now produee 
sercosms ha^e been identified as C^ cells » This strain of cella 
arose f^^a the C3H Ba majmsazy carcinoiaat that Dr, Barrett had passed 
for II46 gemrations In (BAIB/c x C3H) F^ mice. This tumor 'oid.cb. 
has been stitsdied widely has tm-^er been known to undergo 8areoJ!iiat©us 
change u For this reason^ it appears e^i^remely unlikely that sarooim 
cells were carried as oontaminants of the carcinoma for IU6 geic»ra» 
tions of transfer in the Jbiybrid host. There fore ^ the present flisd^ 
inge seem to indicate that the carcinoma cells in l@ng°term culture 
can assume the diagnostic attributes of typical sarcoma cells wlnea 
injected into mice. 

Two clones of this strain as well as another cell strain ftmi 
a different tuaK>r still lo«ik in culture like carcinoma cells but 
fail to grow on trsuasplantationo These are being grown in diffusion 
chaiisers in 6AIB/c mice to test for tbs milk agent a&i if possible 
to step up their ■v'iruleno© for Idie mouse. 



709 



Serial Noo U28(9) Ps«e 3 
Project 2 



Significajioej One of the problems of practical as well as ttoaeret^ 
leal interest, in studies of the aUk agent is whether the agent 
continues to propagate in l®ng=terift cultna^a of the tumior^ Tfee re° 
suits ®f this project are significant in denuMsstrating activity ©f 
the agent for 7 assaths of cultwroo Of equal inportance is the ap- 
parent loss &f biological actiTity of the agent thereafter as well 
as the reguler change in the culture population t® mrc&s&B. cells a 
It has been recognized for years that sposataneous vmsmrj aad pvH'- 
monary carcinomas ft«q«antly transform t© sarcoaae ©n tr®n3planta'= 
tion in vi-roo However, there has been G@n@id®rable contrwerfsy as 
to wiie^ir°^e tumor cells haw taMiei-goiss the change or whether th@ 
sarc.@sBas arose fr^a stroam which has becoBie neoplastic » The res^lta 
of this etiady ha^e deGii@nstrated that boHi types of change can occur 
in ^tr^Bo Most significant is the present eridence that typical oar= 
cinonia cells appeajr to transform in vitro aad acqidre diagnostic 
moxphal^ic feat^ares of sarccna cSTls, 

Proposed course of projects Assays for the milk agent and attempts 
te readapt the ©ells to growth in raice by means of diffusion chasfcer 
techniqiaea are still in progress » This sttsdy will be prepared for 
publication o 



Part B included les N© x 



710 



Serlsl Koo U28Ce) 

lo Lfflboifatory of Biology, 

2o Tifflsae C^iltm^a SactioE, 
3„ Betfeeada, Ma:;^land 



PHS=NIH 
Indi'^dua^. Project Report 
Calesadar Year 1958 



Part As 



Project 3s Titles 



Studies on l@w and high sarcoma^producing liass 
©f calls deri-^d in vitro troa one cell of a 
straiji C3H w©ttsao 



Principal IxwBstigat©r8 Ko Ko Sanford, 



Other Iwestigatsrss 



LoTo Tivipr®®, A, Bo CoWlsskyi B<, B„ W«st= 
fall, E„ Vo Peppers, ¥oRo Earle= 



Cooperatii^ tJsaitss 



MoM. Woods, Laboratory of Biochsiaiatry, 

RoMo Msrwin, «> " Bioleiy 

AoJo Daltoa, u n n 

Lo Kilham, DiT„ of Biologies StaadardSj 

Eo ChUj Depto of Botas^, lal© Daifersity 

New "Ha'^eas Cosmscticut., 
DoMoScottj Depto of Chsadstryj tJai-^ersity 

of Pemisylvaniaj Philadelphiaj Pa' 



Man Tears ( calendar year 1958) 
Totals 

Pr©fee@ioimls 
Others 



Patieat DaysCcal^nday 

yew 1958) 



Project dascriptioa? This project consists of a nuoi>er of separate 
studies the objecti'^s esid results of which will he considered under 
t^ above general i%eadlng. 

Objectl-v^is Otit object!^® is t© interpret and rniderstaad the physiole^ic 
differences between these lioes of cells and to determine fee types of 
change these cells have iindergose dwriag lox^-tes^m culture » 

Study lo Objecti'ws To deteraaine the causae of 131© ns@plastic cfaassge 
in o'oltiireso The neoplastic change in these cells vaa^ possibly reatjlt 
f^oia tim chrwoososal alterations that have ocexxrred to D^itre, but until 
it is possibl® to pi^iS'^mnt adtotiss aesideats is cult-iir@,~Srobtai3a a 
control sell strains free of such abnorsaal chreasseB© atusfosrs oy typesj, 



SeriAl No, U28Ce) Pag® 2 
Project 3o 



thia hypothesis caimr-t b« readijiy validated. Another possibility 
is that the extract of entoryoziic tissue may have introduced -wkncimi 
carcinogesnis @r carcinogenic agezits that induced the change o This 
possibility h&s been tested by grewing cells in chemically defiixed 
medium NCTC 109 siQ^plemented with 10 percent horse seruBi. Thi'ee 
strains ha^a baen tested for malignaBit properties? 1) 2661 {C3H mouse 
fibrsblasts) 9 months is cultur©, 2) 292U (Chinese hamster fibro- 
blasts assayed in cheek pouches of chixiese hamsters of tiw partially 
inbred strain of origin and in corti8one'=treated syrian hamsters) 12 
roenths in culture. 3) 2881 CC3H ei*ryonic tissue minced) 12 months 
in culture, Larg® miajuers of mouse cells have beea iaject^d iato 
irradiated adults as well ae into newborn mice,, All results have 
been negative with the exception of the last iiijectlons of E661o A 
tua^r is developii^ ^ieh will be transplanted and diagnosed „ 

Study 2. Objectives To identif^r the virus^like particles cfcser^d 
by the electron naicroscope (Dr. Dalton) in the high aad low lines 
aisd in all dsrivad clones. Cells or st^raatant fluids have been 
assayed in ne^^ora mice of strains C3Hj BAIS/c, and Swiss for e-^ideas^ 
of lesionso Results so far (10 months) are negative ^ Similar efforts 
have been xasiiB by Dr, Kilha»o who has also tested possibility of 
these particles beiag the K virus. His efforts t© demonstrate heia^ 
gglutinatiag properties with different red cell systems have like- 
wise been negative. At present, we are testing by diffusion ch^nber 
tediniquss wtether these particles could be the milk ageat, since 
the original cell was from a C3H=-straia mouse , 

Study 3. Ctojectives To interpret th© difference betweaa tlwise two 
liass in ssrcoB^>=»producing capacityj more specifically^ to det©r= 
mine whether the two populations of cells are homogeneous or h8ter° 
oges^ous, aed, if heterogeneous, i^ie-ttoer the low line differs from 
ths high quantitatively in containing a smaller proportion of malig- 
nant transplantable cells or whether cells of the tw© lines are 
qualitati'^Bly different o By sieans of clonal analysis smd also 
through the chromosome studies by Dr. Chu, this project has been 
c«J5>letedo Cells of the two populations were fouisd to be hetaro= 
geneous with rsspect to chrosaosiDa© nti^bers and l^pes^ Individual 
cells from each population could reproduce in t^ derived clones 
somfB of the wide range i» chr^a^some nunbers aisd types as ws'H as 
many of the distinctive properties of tJie parent liaes^ Morpholeglc 
asad neoplastic properties of 1^ clones have beea studied by usj 
their vascuLarisatlon in host tissues by Dr» Merwin, and their 
glycolytic capacity ^ ^l^ro by Dr« Woods, These steadies havis showa 
that the two popul&ticns°oFc@lls are heterogeneous and that cells 
of the two lines are qtsalitatif«ly different? 

In establishing clones from tl^se lines, it was found liscessary 
to chasage tt» culture asdiian used, slintse single cells gessrally failed 
to divide in ths horse seru®=^ Jjryo extract culture fluid. By 
dianging the aseditm t® cfeemica-ly deftesid medium !iOTC 109 si^ple= 
with 10 percent horse serum, cloaes frms. these lisaes, as well as .frs^ 



712 



Serial No. U28 (e) Page 3, 
Project 3 



se's^ral other strains in this laboratory, were readily obtained c 
Thus the type of rnedium significantly inpro^d the cloning proeed= 
lire deifsloped In this laboratorj ^published in 19U8). 

For coiaparati'^ purposes, the parent lines as wall as ii» 
clcasoe wear© sultxsred on mnm sxipplejaeatod aedltsa NCTC 109» Signi« 
flcant chassges in t-aa^r^productug sjapaeity r©8\ilted. The chang® 
occurred slowlyj, ^ms not s^adlly re^sersed on retisra to iim original 
medium^ and issas not associated wltJi alteration in other tasoiMn proper- 
ties of thess? cell strains, with the exception of s©a® shifts in the 
chroffios««ffl XTOJ^r distribution pattern as studied by Dro Chu. The 
chasjge appears to be an eabaased cell survival ea transplantation 
ratijer -UjEa an increase in the proliferative capaeity ©f the c^lls 
in BiDTise tiss'i^ 

•Two sjaauscripts are ready for subsittai 1) ThfS properties of 
slngle=>sell defies f^poa low and high sapc©Ha=pr©ducing lines of raeuse 
cells and 2) Glycolytic properties ©f the high and low 8arcoim=> 
prodttcij^ linos and their deri"red clones by Dro Woods in collsbsra^ 
tion with m. 

Study ho Objectives T© determine whether three clones from the 
low line and ©ne £ro& the hi^ differ wit>* respect t© the acti^^ity 
of ensyo&s of the glt^ose nonophosphate sbsnt. The ensynes consid®i?®d 
haire "been glucose «6=ph«sphat« dehydrogenase, phoapheglucoaate d©hyr©= 
gen^se, asd hexskisiase^ Br. Scott has carried out this sttsdy in 
collaboration vith uso The results ha^e sho^n consistent and clearcut 
differences beti?een iim clones o With respect t© all ens^^s studied, 
tte activity is mny tiaes higter in the clo«ie deri^d fS'oM tte high 
line th^a in aj^ of the lew line clones. Low line el©a© 255$ ^ich 
arose from a cell that besarae biiiucleated and ultiasately produced & 
l^^jertriploidj !c©nonuclear cell population h&s a higher asti'^ity t?f 
G=6=PD tisan any of the other low line clones. This study has been 
coE^leted and is being prepared for publication. 

Study 5» Objsctiims To define the -yitaadn requireineate of cell clonss 
dsri'fisd from the high and low lines. Ntsasrous efforts ha-re been made 
to adapt these closies t@ cheioically defli^d aosdiim NCTC 109 lacki!^ 
any serum s«5Jpl©E»9nto Although cell proliferation aa^ continue for 
as 1©!^ as 6 weeksj, the eells in repeated efforts failed t© adapt. 
For this reason attei^ts to define tikd vitaain requireaents were carried 
out in Indium NCTC 109 containing reduced a^ratints of serum proteins » 
With the exception of inositol requireiaants, noiae of the other require- 
BHsnts could be detected, (Se® itaSel) This study was tea^rarily closed 
early in ^e y&ar tjntil a a©re satisfactory test medium could be de-r®l=« 
opsdo 



713 



Serial No. U28 (e) Page k 
Project 3 



Study 60 CIb;3©cti^jr© $ To coispare glucose utilization by cells of 
the four clones. The intake ©f glucose hj cells &f the foxir closes 
has been ssasured in repeated escperiissnts o The relation of glucose 
utiliaation to population sis® and to stages in the growth cycle 
haiTB reTealed coaaiderable unexplained. variability. Howewsr, no 
consistent difference between the clones growa in stationary cultures 
has been detected » 

Significances An®3jrsis of the diffes*©nc®8 between these two lines of 
noniaal tissue origin should prtjvide sosae insight into the types of 
physiologic chmsge that occur in culture that Ksay be associated witte 
the sasoplastic transformation of t.? jsue cells o 

Proposed course of project? Assays for the presence of the »ailk agent 

will be coB5>l®ted„ la "^iew ©f the difference between these clones in 
rates of axsaerobic glycolysis and in activity of the enzysi&a of the 
glucose^^shunt siechanlsm, im^stigatioz^s of the glucose metsbolism will 
be pursi^d ftart^er, Two loanuacript^ are alnoat conpletes 1) on two 
deri'^ati'^e linss ©f ^® high sarcoiaa"p3^oducing line which shew sig=- 
nif leant differences in argiaase actiirity, 2) on the infliaenee of 
mouse pa@iage on properties of the low sarcoioa^roducing lineo 



Part B ijscluded? Yes x I© 

714 



Serial Noo U28 (e) Page $ 

Project 3 

lo Laboratory of Biology 
2« Tlsstae Ciaturts Sectioa 
3, Betii«8d«, Marylsad 



PHS"NIH 
Indi^dual Project Report 
Cftlend«r Tear 1958 



Part B8 HsnorSj Awards, and Pviblications 

Piiblioaticias otisier tj&an abstracts from tMe projects 

Sanford^ Kc Kog Hsrwln, Ro M„» HobbSj, O.Lo, Fioranoati, HcCo and 
EarlSj, WoH, t Sttadlea «m the jdifferenoe in sarcoma^rod'aclng capacity 
of tuo llsmss of mause cells derived ija ^tro fr<m oae cello Jo Nate 
Cancer las to, 208 121=lit5, 1958 a 

Sanford, KoK.s Closml studies on imrml cells ajad &n their aeoplastl® 
trasaefergatloa in yitroo Cancer Research IB? 7!i7'=75l, 1958 « 

Chtts E^EcTo, Sanford, K»Kos and Earle, WoRo s Coaparative chroMJSiMal 
stiidies on s^mnalian cells in oulttire, II o Motsse 8arcona°°produeing mil 
strains aad tfceir derivatiims. Jo Nato Cancer lasto 21x 729°75l, 1956, 

Honors a»d Awards relating t& t&is projects 

lavltatiea to present a paper at tJie Joint Synpositim on ^sReseat Con- 
tributions of TlssxsB Cislture to Ceacer Research," Philadelphia, Pso 
April 11, 1958 » 

iMffitatloa to de»Mistrat9 cloning procedures aad to speak at Jaeksoa 
Memorial Labomtoriea, Bar Harbor, Maine, NeveriBer 25-26, 1958. 



715 



SerUl Noo ii28(©) 

lo Laboratory of Biology 
2„ Tissue Cidtxir* Section 
3„ Bethiasda, Maryland 



PHS-NIH 
ladividuBl Project Report 
Cal«nd&r lear 1958 



Part Ao 



Project hs Titles Atteiapta t® pr©pagat« tSsa polyoma vims ia 

le©g=t6rm tiss^ eultm« cell «tr*iBs. 



Principal Im^atigator? KoKc Saaferd, 

Other Ix^estigaterss LoT„ Diapi?ee, AoB„ CcwraliaiQ^-, W„RoEarie 



Ceopsrattag Units* 



Man Years (calendar year 1958)2 Patieat Daya(cal®E= 

dar year 1958) s 

Total? 

Pr®f©ssiosasli 
Others 



Project descriptiea? 

Objecti-^sg d^"^ecti^es were to detersdae 1) whether tfee pel^wm 
Tiriag could be prspagated ia estabUsfesd leag-tesna straies ©f a©use 
CU28c) or haiaster tisstie sells j 2) whether the Tirus persists asid pr®« 
pagatsa ia cell straijia ctattired jCroa Ttftss»iaduced tmsorss 3) pmnlhlB 
effecta of the virtts && cells cultursd from asraal par@tid ©r thysiaa 
tissues ef susceptible atouae sti^djaso 



MihodBt The cell strains were iafeoted by t-»© roetfesda? 1) Additi®^ 
of & small eaplaat ©f paretid t-mnr ttmm t@ tise eultups^ The toiasr 
was ebtained trm Drc Sc Stewart. 2) Additioa ©f filtered svpermtm.U 
fr©® ^irus=iBf©st®d cultures » The unfiltered material was obtaix»d 
frem Dr, Bo Eddy^ With the sxception of strsim 2071, cultures were 
gtmm m chesdoall^r defiii^d 8®diBia NGTC 109 supplsnented with 10 p@r== 
cent horse serum, Straisi 2071 cells were gr&m m tfee chesaieally 
defined nsdiiiHa al©n®c At oertain tramsplsuat geaeratieas, c@lls aad 
st^ejwatants tmre assayed for ^irs.1 asti'^^itF in mwbsra S»si3§s ©r G3H. 
Bsioe ©r ths feassster cells were isa^ected iat© mi^jom Syriaa hm;st®'rs . 



Serial No. 1*28 (s) Page 
Project k 



Results s The cell strains t«st«d and 

suia^jyized bslows 



ths results ar® briefly 




^j,.^ , of Act^^ty C*or-) Duration 

cell '^nfectioffl for iadicated eaqperias 

nuaSjer, of days ia days 
af t@? %s*sat!i!@nt 



typs 



roouse "tmrnf 

2881 e^ryo mixed tiesus 
call typ©s 



haunter Tunor 

fibrtfelasts tisstsa 



51* 



lli* 77= 



2071 C3H fibrsH= Tysmv 



Straim L 

C3H fibro^ Filt»sred U5* 71= 

fluid 




?7 





Serial No. U28Ce) Page 3 
Project h 



Activity was deinonetrated by productioa of parotid, kldsisy, or tfaymus 
tumorao Conspicuous cytopathogenic effects were not dbserred on these 
strains, but recent obser^mtions on one of tl^ie nuuamacry carcl^aomei strains 
suggests ^&t it nay be useful for in vitro assays. These rather pre° 
liminary studies which had to be teminafecT before conpletion, suggest 
that fibred l&sts laay proTide a less satisfactory s\;^8trate for virus 
replication than the carcinoma cells or 1^ mixed stAryanlc tissue cells < 

The following cell strains axe being established for studies r^^^ 
l&ting to the last two objectives abodes 

Parotid tuamr strains from SwisS;, C3H , and NIH lediite strains of 
alee, and nors^l thynms end parotid gland tissue from AEH and C3H sdcsr 

Significances A unifena source of polyoma virus as well as a suscept- 

ble strain for tests in vitro are both iiapertaat for atiadies ©f the 
inflraenee of this virus en cells in vitro o Although this projeet may 
have considerable potential signiHcaace in cancer research, tbs work 
is still in a pi°e liminary stags ©f devslopisseat. 

Proposed plans This project is now b®ing ptarsued in an effort to ofctai^i 
tha inaaediate objectives listed,. 



Part B included las No x 



718 



Serial Noo li28 (f) 

1. Laboratory of Biology 
2^ TissTiSB Cisltur® Section 
3. Bethesda, Maryland 



PHS^NIH 
IndividuRl Project Report 
Calendar Year 1958 



Part Ac 



Project Titles Studies in tissiie culttxre of tl]e nali^n&ssit cell and 
of tS&e normal c@ll ftom which it arosoo (This i@ & 
general title for all work in the section^) 

Principal Imwstigators W^Ro Earle^ 

Other Im'astigatcsrs 8 Se© 1^28 (a) thr©iigh J428(e) preceding o 

Cooperatii^ Units i Listed under parts (a) throiigh (e) 



Man y^ars (calsmiar ^ar 1958) x Patient Days(caleKdar 

Totals 1^ year 1956) 

Professionals 9 
Q-hh&Ti 10 



Project descriptions The above project title laay be considered as 
one general title for aH work in ths Section, Under the preseat 
8t;S^ead 1428(f) will be outlined a nua&er of acti^itie® not inelitded 
within U28Ca) through U28Ce)o 

lo Comitant temperature rooa 32U has recently been tun^d m^®T to m^ 
and is iKiw being calibrated ssed adjusted to leiml of necessary accurasj 
and reliability a@ rapidly as possible. As soon as practical it will 
be placed in useo This will result in ffi£>8tantial rearrangeisgat and 
sio^lifioation of equipmsnt^ and this will allow 8i3S>8tantial increas© 
of efficiency of work within the section. 



719 



Serial No. 128 (f) Faga 2 



2o Work and plans for work with the eos^jarativ© Bi^tloQ pict'iire 
ceuBsra bas been doscribed tioder {426 (a) « FtAcllltles are xumt on 'kmM 
for aaore precise and rapid d©f«lopB»nt of film. The full acti^atioa 
of tMs dl«)-v«lopnent eqtdpnent will be ptished aa rapidly as p«$@sible 
as other equipment is relocated and rearranged to make fiall use @f 
constant teiqwrature rooms 32ij^ and 322 <> 

3„ New and tmr^ accurate methods of gas tension control within cultures 
are being w&rksd out to attempt or insure better aerobic conditions 
within cultures, partiotilarly large cultures. 

Uo More precise and reliable lasthode of msdla sterilisation ar@ sew 
imder consideration end study » 



5. A mistoar of methsd^ and concepts originated or de^roloped in this 
Secti<»i have undergone nf^difleation or alteration with the passage 
of tlmeo Detailed presentation of tiasse revisions has nev«r been 
adequately fflfide» An atteai^t 1« now bs^ing made to bring such des-=' 
criptions of these T^<»to^4&teo 

60 Check Ust ©f ptSjlications fflrom tfeis Section will shortly be brought 
^=t&=date as of (probably) January Is 1959s asad will b© mad© avail- 
able as a substitute for the old list now being sent out. 

7o Patent has recently been issiaed (and assigsted to the tJaSoQcvemen^Bt) 
for design and msthod of fabrication of the design of tlsst^ culture 
flask known as the «<T» design. This is patent No. 2,658,036. 

6, Aisotber patent, basic in nature, and covering prin<siples m^ ssetheds 
for growth of fixed animal tissise cells in deep agitated fluid ©us^' 
pension has been applied for mid is at present in process (also 
assigned to U.So6oimrmient)o 

9o As a result of consultancy of W. R„ Ear2« and V„ Jo E'^'as^ with. 
the Tissue Bank, National Ha^al Medical Center, this latter orga»i» 
satio® ha@ recently coi^leted construction on and hag aetiirated use 
of a tisst^ cultwis laboratory within their oun organisation. 



10. During the last year ws h&r® had a large nuaijer of visitors tcp 
this Tissue Culture Section, ^bjs^ of these ^sitors ba^ve been tr&^ 
foreign laboratories^ or haire cone tharough in exjjeetation of ©wilua- 
ting practicability of setting 19 tissue culture work In foreign 
countries. Most of those 1^0 ha^^ come through hm9 been directly 
interested in tissue culture and its appllcsbllity in "r&rloua liaes 
of workc Maioy ha?r« had speeifie todmical Inquiries ©k whieh we 
could advise tham with respect to difficulties enco^mter®d. others 
hate wanted to see the gei^ral design and operation of the precednre® 
and equipment of the Section. A sistostantial number hai^ asked author^ 
imtion to remain and work hsr® for an extended tiass ire order t© ©btaia 
training <= WM1« «« h&v^ tried to assist worksrs ceadng tfereii^h, dw 



720 



Serial No. U28Cf) Ps«« 3 



to lack of space and equipment facilities we have been dbliged to 
linlt time alloiied to lAat a worker could otoserye io a limited auiriber 
of hours o Ifunwrous medical workers who cone through to get a geaeral 
idea of «what tiasw culture is like™ can be allowed only mittiwal 
tiBB. In each instance an atterapt is made t® evaluate the aeeds &f 
each individual ▼isitor or groupo There is no doabt, however, tiiat, 
this handling of visiting professional personnei ie extremely tiup 
consumii«c Further, the time taken yxp Is that of the professional 
staff since very few wiaitors t*io cose in are of a type whose d®sire 
for iaformtioa can be oat by untrained or subprofessloaal personi^si-. 

lie Insofar as appears to us practical we try to fjrnlah par«5St 
cultures t© allow other workers and laboratories to 'start cell strmsr^ 
in their om ©rganisatien. No osll strains are fUmishsd which &m 
coameroially airailable ir. satisfactory condition, and in general celi 
strains are furnished under coiwditiona prerioualy apprwed hj tSi© 
Director of NCI „ 



Psart B included les x No 



721 



Serial No, 1428(f) 

lo Laboratory of Bi(»l®g2?- 
2o Tt88«9 Culture SecttcM. 
3o Bcthesda, Maryland 



PHS-NIH 
Indi'^ldual Project Report 
Caleisdar Year 19S8 



Part Bo Hoaorsj Awards, and Pwtolicatioas 
Publlcatidcs @th®r thiEm abstracts frea this projsets 



See reparta Ua8(&) thru iilSCe) 



Honors aad Awards rslati^ te this projects 



Coatiataed as GQastsitant to Na-^ Tisaua Basak, UoS„ Naval Medie&l S!Bfe©&l3 
National Nafal Medical Ce£3iterj! letter of appreciation from Chisf^ 
Bureaw of Mediciias asid Surgery^ Departneat of tfea Navy, t® Si2rgi9@B 
General, Uo So PiisUc Health Service ■. 

R©qa®3t for preaaiitatioa ©f -work, Coiarse of the Tissue Cultiir© Asso- 
ciatisMBj Daawsr, Coloradoo Ifeabl® t® accept due to pressixre ©f work Ib 
progress.. 

Consultation requested by Eli Lilly Research Institute, @a cert^B 
technical points in tisatie culttirso 

Two lectisrsss by invitation ©f lastitute of Cell Physiolegyj B'jaii'srsitj 
of Nebraskao ^ 

Rsqtie^^ed to serve an Cosaalttee <m Mechaaism of Caroinogeoeslsi ?II 
Intenmtiemial Ca&cer Cmigresa. IKoable to attetid due to fact if©rk 1b 
progress made foreign trip unwise at that tiaiie. 

Presentation by Invitation at Sysposium on cultivation and use of 
animal cellsi Intens^siety SynqposiuiB, Federation of American Socistiea 
for Experiasntal Biology, Philadelphia, April 10$ aa^>llfied presenta^ 
tion now in press for Deeeafeer Federatiom Proceedings « 

Imfitatios t© serve a® Moderator of Goafereacs® e® c^ll cixltttwi t© m 
held by National Acaderay ©f Scieneess JaKaary 19S9$ Men^r ©f C&m±U&m 
on Cell CisltigrSs, Institute, ©f I^©r&t©ry ©-^ Anii^l Resowo®® of tiM 
Natiosml Acadengr ©f Scienoesi. assisted is organizli^ above S^W©@±vm. 



722 



I^aboratoty of CJhemical Haarmacoiogy 
Budget Data 

Estimat«d Obligations »..<,«»o.«,o.ooo^o.oo<.o..,»^o., 

Direct o ooaooaoaaiooaaaoas.soa^oooaoaos.aooxioaa' 

Reimburstsments 

C3binlcal o.o.a^.ooo.oo.o.,^ .o...o,.<>.o.o, 

Othero . o o » « o > o . , , . o . . . . » . o , . . . o . » o , , . . 

Total, o ,.<■.» o ,,.» o »,, , 



17 



.ooFlscal Year 1959 
,0$ 513,800 




1 / Ineludea Projects Ko'sg 

OFFICE OF THE CHIEPs 

250 
251 



CELLULAR FHABMAOOLOGF^ SEOTZCKs 

252 
BieCSBMICAL FEABMACOLCGY SBCTICWs 

253 
^QLYSACOMSXSE, SECTICMs 

261 
MACRCMOLEeOLAB CHSMISTRY SECTICMs 

262 



723 



Serial Ho. NCI 250 

1. Lab. Chemical Pharmacology 

2. Office of the Chief 

3. Bethesda l4, Maryland 



PHS-NIH 

Individual Project Report 

Calendar Year 1958 



Part A. 



Project Title ; Planning, Management and 
Coordination of Investlgationa, Organized in an 
Interdisciplinary Team Approach, on Basic Science 
Aspects of Cancer Chemotherapy. 

Principal Investigator; Murray J. Shear 

Other Investigators ; 

Cooperating Units ; 

Man Years (calendar year 1958); Patient Days 

Total; 8-1/2 None 

Professional : l^l ''2 
Other; 7 

Project Description ; 

A. General 
1 , Plan 

Tlie overall strategy of the research 
program consists of two major approaches: a) inves- 
tigation of factors in the host which may influence 
cancer J and b) contributions to principles of methoiology, 
and mode of action, in the use of drugs which affect 
tumors. At present these approaches ore followed mainly 
as separate endeavors. However, as progress is made it 
is anticipated that the findings may bo applied, in ccm- 
blnation, to the control of tiimors. This concept is 
different from what is usually denoted by 'combination 
therapy', i.e., the concurrent or sequential adralnls= 
tration of two or more eiitrlnslc drugs. 



724 



Serial No, NCI 250 
Calendar year 195S 
Part A •» continued 
Page 2 



Interesting as the polysaccharides 
are, they are viewed not as ends in themselves but 
rather as tools for the detection of important, but 
hitherto unknown, body components and of their reac- 
tions. A major objective is the acquisition of better 
understanding of the nature of the refractoriness of 
tiomors, both in animals and in patients, which rapidly 
develops after the first dose of endotoxic polysac-=- 
charldes. This induced 'tolerance" is non°speclflc 
and bears resemblances to the non-specific refractori«= 
ness induced by endotoxins in such host reactions as 
the Shwartzman reaction, fever, lethality, etc, Con= 
sequently, host responses such as these are being 
studied as a flanking attack, with the expectation that 
the findings may well be applicable to the prevention, 
or abolition, of the refractoriness induced in tumors. 

Although polysaccharide research 
is important in Itself, our main interest in these 
polysaccharides lies in the dramatic effects they pro- 
duce in the mammalian host in consequence of their in= 
teraction with components in the body fluids and tissues. 
Little is known about these postulated body components 
and the way in which they participate in many reactions. 
As more is learned about these phenomena and the sub- 
stances involved, the basic knowledge is found to be 
of value in understanding of shock of various types, 
allergies, hematological disorders, bacterial and virus 
infections, etCe 

As far as cancer is concerned, the 
possibilities transcend therapy, important as that la. 
There are reasons for suspecting that such host factors 
may have implications for: the genesis of tumors, or 
the failure to generate, under conditions of marginal 
carcinogenic influences; the rate of growth of tumors j 
the establishment and flowering of metastases; and 
early diagnosis. 



725 



Serial No. NCI-250 
Calendar year 1953 
Part A - continued 
Pase 3 

Liaison with clinical branches Is 
close and continuous In the case of the oro^ram on 

Sn^i^ ?! o t"^^^- .^" *^^ ^°^^ factors program. Joint 
work is set up and conducted In collaboration with 

ll^tr.ll'^ ^^^t ^f:o™,*^nie fco time as meritorious projeclls 
emerge from the basic laboratory work, ^ -J '^ 

2. Organization 

«<.,,^4>.-i *a., ^.- - Host factor phenomena are belns 
nJ^raJ!f/^J^ J^t ^^^ ^^ ^^^ methods of macromolecular 
physical chemistry, micro biology. Immunology, etc. The 
planning and conduct of the work in the Polysaccharide 
and the Macromolecular Chemistry Sections are lnter» 
aigitated so that they complement and catalyze each other. 

n,=,/i^ 4« *u 4 ?^f^^S this year further progress was 
made in the re-^orientatlon of the program of the Chem» 
istry Section, and in the re-assignment of, chemists, 
^rSi^S^'L^^^S^^.f?'- ^' ^- Hartwell, Mrs. D. Fitzgerald 
fnf J M^!r^ ^f^i^^ "^^^^ transferred to the Cancer Chemo= 
therapy National Service Center. Dr. A. Schrecker has 

?S"«°r}®?*®*^.?^® ^?r^ "'°^® towards biological objectives, 
in collaboration with Dr. Goldino He and his assistant 
qJoMoS'^^jr^^"?^^' assigned to the Biochemical Pharmacology 
tole^J^l'SLf oT?eSu?ts!'''^ """^'^' """ ^'"^^^^ ^^°^^^- 

Hir,o.-n«^ ^ ..u In recognition of the shift in the 
direction of the work from organic chemistry to physical 
chemistry and biophysics, the name of the Section was 
changed from Organic Chemistry to Macromolecular Chemlstryj 
^\l t^ Mora was designated the Section Head. The value 
oi the tools of biophysics to this work was emphasized by 
the recruitment of Dr, John Preiss, a Blophysicist from Yale. 

Drs. Belkln and Vivian have agreed 
to the discontinuance in this Laboratory of the projects 
on Plant Screening and Phenazine Synthesis i these are 
being brought to a close. Dr, Vivian is available for 

o^2?2f°^ ?^ ^°^ ^°^® °^^^^ *yi3e of arrangement. l#iatever 
additional phenazine derivatives Dr. Belkln may need, to 
wind up the work on Vital Staining, he can orocure from 
other sources; e.g.: from other phenazine chemists in U S . 
Japan, or elsewhere, if they desire to synthesize themj 
or, he can have them made under contract. 



726 



Serial Wo. NCI"250 
Calendar year 1958 
Part P " continued 
Page 4 

In the Biochemical Pharmacology 
Section, D;'. Goldln said Mr. Venditti accepted the 
responsibilities of Project Officer and fia&t. Project 
Officer, respectively, for the scientific aspects of 
a Drug Development Program carried out by Microbiological 
Associates under a contract from CCNSC, The work has 
been goln^ satisfactorily, and the contract is being 
renewed on a somewhat larger scale. Dr, Goldln is sub- 
mitting a separate Annual Report on this operation to 
the Clinical Director of NCI. 

3.. Major Personnel Problems 

The widely-recognized shortage of 
available young scientists, of the requisite training 
and caliber, continues to hamper the progress of our 
work. Par too much of our time and energies is spent 
on recruitment, often with discouraging results. ThiSj 
of course. Is a nation-wide problem and not peculiar 
to this Laboratory. Steps are being taken, on a national 
scale, to remedy the shortage and I have forwarded, 
separately, recommendations made upon the invitation 
of the Secretary HEW and the Surgeon General PHS trans= 
mitted through the President of the Amer^ Assn, Cancer 
Research, 

Another problem consists In the 
difficulty of moving people who no longer are contribute 
Ing effectively to the mission of a laboratory. ISiis 
situation may arise from various causes : the services 
of a particular type of specialist may no longer be re- 
quired because of a change in the previous research 
program; a worker may be InfleKlble, and umfillins to 
re-orient the direction of his woric despite approved 
changes in the situation j or, he mry h2ve orogresslv.: y 
vjorscning handicaps; otc„ 

In view of the shortage of scientific 
workers, would it be possible to imagine a new adminis- 
trative arrangement which vrould avoid drastic actions, 
end also avoid the impeding of new programs? Could such 
personnel bo transferred to an Institute's "Manpower Pool' 
(under some more^ appropriate designation) until their 
services are needed for some other program elsev/here in 
the NIH or in outside institutions? 



727 



Serial No. NCI»250 
Calendar year 1958 
Part A «• continued 
Page 5 

Neither of these two major per- 
sonnel problems will be solved quickly. Bat In view 
of the larse plans being Bet in motion to remedy the 
first one, perhaps imaginative thou^^lit ought now be 
given to a more satisfactory handling of the other. 



B. Specific 

Tlie activities of this Laboratory are, 
for the most part, described in the accompanying re° 
ports by the Heads of the four Sections, and in the 
separate report by Dr, Vivian on the Phenaaine Syn-» 
thesis project which is being brought to a close. In 
addition, the following specific items are reported. 

1. In order to meet the numer"- 
ous requests, from Investigators in other institutions, 
for supplies of our bacterial polysaccharide prepara- 
tions,' the arrangements made with Difco Laboratories 
was mentioned in the Report for last year. In addi- 
tion, by a cooperative arrangement with CIBA in 
Switzerland and the US, we prepared a large new batch 
of active polysaccharide from S. marcescens . In the 
laboratories of CIBA in Suinmlt, N. J., our strain of 
organism was cultured in 5,000 liters of our synthetic 
medium. The first steps in the concentration of the 
culture liquid and in the treatment of the collected 
bacterial colln were performed at Summit, and the 
materials then shipped hereo During the year these 
materials were proeejacd, and the active polysaccharide 
fractions isolated from this big batch. That obtained 
from the culture fluid was designated as Lot No, ?hO, 
and the one from the bacterial cells as Lot No. P45. 

We had arranged for the company 
to do this big batch xirithout charge to us. Their 
recom?>enso v/as in the form of being given somewhat more 
than half of the final product obtained. After satis-= 
fying ourselves of the activity of the polysaccharide 
in our usual bioassays, and of the quality of the product 
by chemical analyses, wc sent CIBA 100 grams of the 
polysaccharide in Septembor„ 



728 



Serial Noo NCI-25O 
Calendar year 1958 
Part A •= continued 
Page 6 

Mr, Perrault did a magnificent 
job of processing this big batch, fitting it in be- 
tween the original e:r.perimonts in new researches. 

2. In addition to the regular and 
close relationship maintained between Dr. Goldln's 
group and the Clinical Chemotherapy group at the 
NCI, collaboration in the host factor prof^am is 
arranged with clinical colleagues whenever feasible. 
One of these studies has been completed and is being 
prepared for publication together with Dr. Clyde 
Brlndleyo This deals with scrum properdin levels, 
in patients with various forms of cancer and in 
normal persons, as measured by the newer phage" 
Inhibition assay. In contradistinction with the 
report from SKI, no difference was found in the dis- 
tribution of properdin levels in cancer patients and 
in normal Individuals. 

Work on EDC levels in patients 
with various diseases has also been in progress. Other 
typos of study with clinical material are under con- 
sideration. 

3. Tolerance, Induced in the tumor- 
bearing host by the first injection of active poly- 
saccharide, is the problem of greatest interest to 

me personally. It is for this reason that I organised 
the basic science groups to develop new knowledge on 
polysaccharides and on the biologically^occurring 
macromolecules with which the;; Interact in the ellcita= 
tion of the phenomena outlined above. ("l.Plan' ). This 
provides fundamental information which is provocative 
of ideas vjhlch can then be applied in experiments de- 
signed to control tumors more effectively. 

Such application of new ideas is 
being carried out with the aid of B. Achlnsteln and 
other staff members. It has already been found, in 
exploratory experiments, that it is possible to break 
down, at least partially, the induced tolerance and 
that it is possible to Influence the rate of growth 
of several types of implanted tiamors. 



729 



Serial Mo. NCI-250 
Calendar year 1958 
Part P' - continued 
Page 7 

4. Further progress has been 
raade In the difficult methodology of measurement of 
arterial biood pressure In the mouse. The achieve- 
ments In earlier work with Pradhan (see "Publications' ), 
although gratifying as first approximations, left much 
to bo desired: the major surgery and deep anesthesia, 
required for closure of the carotid artery >. imposed 

a heavy burden on the animal and affected the blood 
pressure level. Much more satisfactory values are now 
being obtained, in collaboration with B« Achinstein, 
in both normal and tumor-bearing mice under light 
anesthesia and without surgery. 

5. Requests for consultation have 
continued to be received in a steady stream. This 
situation has been much the same as described in my 
Annual Report for last year under ''B. Specific: 5,", 
and is therefore not re-capitulated here. 

6. International research activities 
claimed an increasing share of the time. These included 
current operations, which have been Increasing in extent, 
and U.S. Government planning for major enterprises in 
medical research on a world-wide scale. 

Future Course: 

The overall strategy outlined in the 
foro-joing section is a long-range plan, vjhieh is expected 
to justify prosecution for several years at least. While 
the general objectives are clear, the tactl@s employed 
from time to time will vary, depending upon the specific 
loads that open up. 

Further progress has been made during the 
year in discontinuing in this Laboratory several lines 
of work (Survey of Compounds Tested for Carcinogenic 
Potency^ Review* of World's Literature on Folk Lore and 
Treatment of Cancer; etc.) which are now being carried 
on elsewhere. It is expected that projects on the Plant 
Screening and on the Synthesis of Phenazines will be 
closed out completely during 1959. 



730 



Serial No. KCI-250 
Calendar year 1958 
Part I^ - continued 
PasG 8 

Coordination and interdigltatlon among 
the major programs will be encouraged. For example, 
in the Macromolecular Chemistry Section success has 
been achieved in the inco3?poration of a sulfa drug in 
a polyglucose structure. i>ttempts will be made similarly 
to Incorporate drugs like araethopterln, 6-MP, etc. The 
Biochemical Pharmacology Section will be interested in 
studying such preparations as they become available to 
determine whether their incorporation in a synthetic 
macromolecule alters drug potency, tojcicity, rate of 
release into the circulation, etc. 

As another example, we have discussed 
the possibility that immunity against L»1210 may have 
been Induced in the mice which survived for a long 
time, after successful drug treatment, and which then 
rejected re-implants of this tumor. Additional mice 
have been set up in Dr. Goldin's Section to e-cplore 
this finding further. If the phenomenon is indeed 
found to be Immunologic in nature. Dr. Landy's group 
vjill participate in a Joint study for which tentative 
plans have already been sketched. 

Study of material from patients, and 
collaborative projects with clinicians, will be contin- 
ued and will be eXi^randed whenever feasible and whenever 
suitable i^ersonnel are available. 

Fundejnental studies of host factors in 
response to various types of Injury, and of the mocro° 
molecular components Involved, will be carried on with 
the o'<:pectation that leads will be obtained for appli- 
cation of attempts to control tolerance and other 
tumor phenomena. 



5?c-rlal Uo. NCI-250 
Pase 9 



PHS - IJIH 
Individual Project Report 
Calendar Year 1958 



Pai't B; Publications (no abstracta included): 

1. Shear, Murray J., /^clilnateln, Bettj, and 
Pradhan, Sachindra N. : Effect cf a bacteriai pcii,-= 
saccharide and of sevrrai other tumor-necrotizin'i 

a ;:&nt:; en carotid blcod .: res jure In mice. J. Nat. 
Cancer Inat. 21: 535-594 (1953). 

2. Pradhan, S. N, , Achinstein, B. , and Shear, M.J, 
Re3;>on3e of carotid blood pressure in mice to drugs. 
Prchlv. Internat. Pharmacodynam. Therap. i.1^ C-i.958). 

In ?reJ3. 

3. Shear, M. J. : Contributions to "Poly^ac- 
charide3 in Biology. Tranaactiona of the Fourth Con- 
for^-nce' . Tlie Jcsiah Macy, Jr. Foundation. 0. F. 
S.rin^ier, Editor, N. Y. (In ?re33). 

4. 5 and 6. Shear, M. J. : cc=author in the 
three rublicatioiia listed in Dr. Landy'3 pre ject 
fv.crt. 

I! 

7. Landy, M; , and Shear, M. J.: Polysaccharides 
cf marnvnaj-lan dvrlvativn. Their biolosicai activities, 
Includin.-^ interaction with -ro^^erdino ' Traaaactions of 
the 6th Ccn3r£3S of the Euro.:;ean Society cf Haematclcg^, 
CO'Cnhr-scn x957. S. Xar^er, Editor, Base^. : c;^. 916- 
92: (195:'). 



732 



Seria?. No. NCI -250 
Part B - Paqe 10 

PHS <- NIH 

Individual Project Report 

Calendar Year 1958 

Honors and av;ards relating to this projects 

1. Elected a Member (195Q-61) of the Board of 
Directors, Amer, Assn. Cancer Research. Chairman of 
its Nominating Committee ( 1958-59 ). 

2o Appointed by the International Union Aaainst 
Cancer as: 

a) Chairman, Committee on Chemotherapy 

(reappointment) 

b) Chairman, Finance Committee 

c) Member, T'HO«»Lialson Commltteeo 

3o Orientation Lecturer at International Symposium 
on Cancer Chemotherapy, University of Cambridqe, Enqlsnd 
CJuly). 

i|.. Elected an Affiliate of the Royal Society of 
Medicine, London. 

5. As the 5"y«ar member for oncology of the h'hcy 
Foundation's annual conferences on "Polysaccharides in 
Biology", participated in the Fourth Conference at 
Princeton (May). 

6o Declined Invitations frran the Pergamon Press 
{London) to serve as the American Editor (for cancer 
research) for a nev/ international Journal'=-="Blochcraicai 
Pharmacol ogy""«or, alternatively, as a Member of its 
Honorary Editorial Advisory Board, 

To Invited by the editors of "Perspectives in 
Biology and Medicine" to contribute an essay-type 
article on whatever aspect of nisy v/ork I v/ish to selects 

8o Served as Chairman of the Session on "Special 
SystCTis" in the conference on Screening Procedures for 
Experimental Cancer Chemotherapy, conducted by the New 
York Academy of Sciences (March). 



Serial No. NC!»250 
Calendar Year 1958 
Part B -= Pace U 



9. Appointed by the University of Bombay, India, 
to be one of the two evaiuators of a thesis submitted 
by a Ph.D. candidate in fulfillment of the requirements 
for a doctor's degree. (The usual honorarium paid by 
the University for this function is being contributed 
by me to a students^ aid fund there.) 

10. Received an invitation from Budapest, on be- 
half of cancer research workers in Hungary, to come 
there in May 1959 to give lectures and seminars. 

11. Received an invitation from the President of 
the Peruvian Cancer Society to participate in a Research 
Symposium of its first National Cancer Conaress to be 
held in June 1959 in Peru. 

12. Prepared, by invitation, several planning 
documents, on a proposed \^rorld<=wldc program of medical 
research, for an ad hoc proup of men from the NIH, the 
SGOs office, and the Department of State. This ^^s in 
preparation for the action taken at the meeting of the 
'"/orld Health Assembly in Minneapolis in Fky, 

13o By invitation of a committee of the NRC, pre- 
pared (together v/ith Dr. J. R. Heller) several memoranda 
on the financial problems of the UICC and hov/ they might 
be met. At the 7th Cancer Congress in London in July, 
served the UICC as Chairman of its Committee on Finan- 
cial Recommendations (consisting of men from South 
Africa, Sv/itzerland and the USSR); the recommendations 
of this Committee were accepted in their entirety. 

ll|.o Interviewed on cancer research over the Trans» 
Pacific telephone by Dr. Yuzo Tazakl, Director of the 
Cancer Hospital in Tolcyo. The conversation \;®s filmed, 
vrlth sound* track, at both ends for shovring on "split" 
TV screen, broadcast by the NHK television network in 
Japan. The invitation came fr<»i the Ja{»nese through 
channels. "Dr. Shear .... is the first American 
scientist to be interviewed in this series of inter» 
national goodv/111 programs arranged monthly by USIA." 
(October) 



734 



SerifiT No„ NCI- 25 1 

1 . ^abo of Chemical Pharmacology 

Bethesda 14, Maryland 



PHS-NIH 

Individual Project Report 

Calendar Year 1958 



Part A. 



Project Title; Synthesis of Phenazines 
Pr incipal Investigator; Dr, D. L. Vivian 
O^he r Investigators; 
Cooperating Units; 

MaK Years (calendar year 1958); Patient Days 

Total; 2 None 

Professional; 1 
Other; 1 

P roject Description; 
Objectives: 

L To develop new vital stains with definite advantages 
over those now knownc 

2. To prepare compounds directly related to ones known 
to have action against cancer. 

Methods Employed; Organic chemical methods. 



735 



Serial Noo NCI - 25 
Calendar Year 1958 
Part A — Page 2 



Major Findings? 

Work during the past year has been devoted to developing 
new vital stains related to neutral rede It was found that 2^ 8=bis° 
(alkylamino)phenazines were« in general, vital stains, and much of 
the experimental work has been devoted to devising practical methods 
for their synthesis^ 

During the course of this work^ preliminary investigation 
has been made of methods for synthesizing the following compoundss 

2°amino°'8°chlorophenazine 

2, 8°diaminophenazine 

2° chloro°8°methylaminophenazin@ 

2p 8°bis(methylamino}phenazine 

2=ethylamino=8-methylaminophenazine 

2= chloro-=8°®thylaminophenazine 

2- chloro-=8=n=propylaminophenazine 

2= chloro°8»isopropylaminophcnazine 

2- dhloro-^3°>n-butylaminophenazine 

2'=methylamino°8-n"propylaminoph©nazine 

2<=n=butylamino=-8-=methylaminoph©8saiziBie 

2= chloro-8=cyclohexylan?inophenazine 

2°amlino»8»chlorophenazine 

2<=anisidino°8°chlorophenazine 

2-ethylamino=8'=n=propylaminoph€nazine 

2» chloro=8=n»heptylaminophenazine 

2<=benzylamino='8=chlorophenazine 

2=n=amylamino°8=»methylaminophenaaine 

2=n=heptylamino=8-roethylaminophenazine 

Significance to the Program of the Cancer Institutes 

It is hoped that worthwhile contributions to vital staining 
will ultimately results together with possible application to the Gh®mo= 
therapy of Cancer» 



736 



Serial No„ NCI °= 251 
Calendar Year 1958 
Part A =^- Page 3 



Proposed Course of Projects 

The work is in various stages of progress for each of the 
individual compounds, but a systematized procedure is in process of 
being worked out» and it is confidently expected that successful 
syntheses of nearly the whole series will ultimately be achieved,, 



Part B included ===> No 



737 



Serial Noo NCI » 252 

1„ Lab, Chemical Pharmacology 
2„ Cellular Pharmacology Section 
3o Bethesda 14« Maryland 



PHS-NIH 

Individual Project Report 

Calendar Year 1958 



Part Ao 

Project litleg Pharmacologic Action of Tmnor^Damaging and 
Other Substances on Animal and Human Malignant and Normal Cells.: 



Principal Investigatorg Dr., Mo Belkin 

Other Investigators ; Dro D= L„ Vivian„ A., Perrault 

Cooperating Unitsg Dro D, Lavie,, Weismann Institute,, IsraeL 

Man Years (calendar year 1958)? Patient Daysg 

Totals 6 Non® 

Professionals 1 
Others 5 

Project Descriptions 
Objectives! 

{&} Finding novel drugSo primarily from plant sources^ 
and a study of these^ and of a variety of other tumor necrotizing 
compounds, to determine how they bring about cell damage and ceil 
death, (b) The morphologic and biochemical responses of noE°mal 



738 



Serial No, NCI » 252 
Calendar Year 1958 
Fart A = Page 2 



and malignant cells to various drugs with particular regard to certain 
basic functions of the cell, i,, e, » permeability, water metabolism^ etCo 

i\,?ethods Employed^ Classical techniques of pharmacology, 
histo° and cytochemistry, and tissue culture are used. New techniques 
are developed as necessary. 

Major Findings? For each of the several facets of this research 
project; the principal findings and general progress during the past year 
are as follows; 

I« Tumor Damaging Capacity of Plant Materials 

For the past several years, a systematic study has been 
made of plant materials for tumor necrotizing capacity; some 400 plant 
materials were tested. These embraced a large number of the families 
of the plant kingdom^ including many used daily as foodSo The investi= 
gation yielded several dozen plants that exhibited tumor^damaging capacity > 
In two instances, active principles were obtained upon fractionation; 
(1) podophyllotojcin from Juniperus Sabina, a diurdtic i and (2) an indole 
alkaloid, of a new structural type^ was isolated and identified from 
Alangium villosom, a poisonous plant from A ustraliao In addition^, a 
considerable number of these plants have been es^mined from the stand= 
point of their non^dialjrtic macromolecular components, mainly aqueous ° 
soluble polysaccharides,, Many of these fractions produced extensive 
hemorrhage and necrosis in solid tumors, and especially noteworthy is 
their capacity to evoke marked cytoplasmic vacuolization and increase in 
cell volume in ascites tumor cellSo (see below) 

The continued testing of crude plant materials for tumor 
damaging ability has been brought to a closer and the results are being 
embodied for publication. The only phase of the program to be continued, 
at least in part, will be closer scrutiny of a few of the active materials so 
far obtained from certain plants. Examples are certain fractions from 
Ecballium elaterium and Citrullus colocynthus o These compounds were 
derived from a collaborative investigation with Dr., David Lavie of the 
Weizmann Institute^, 



739 



Serial No. NCI ^ 252 
Calendar Year 1958 
Part A — Page S 



It Is to be much regretted that this plant program can- 
not be actively continued^ I have always felt that, if properly and 
adequately prosecuted, stach a stwdy would yield rich dividends, both 
basic and practical in their applies tiono However » the shift of emphasis 
in this Laboratory away from screenings and the development of numerous 
facilities elsewhere for this general purpose, have resulted in this 
decisiono 

II, Studies in Cellular Pharmacology 

When mice inoculated with sarcoma 37 ascites tumor 
ceils are given a single intraperitoneal injection of one of many polysac= 
charides derived from higher plants* these phenomena follows (1) in 
24 hours, there appear a large and increasing number of granules which 
stain as neutral fat| (2) beginning at 48 hours, and usually continuing 
through 96 hours, the cytoplasm becomes filled with large cytoplasm 
vacuoles, and the cell undergoes marked increase in size:. 

There are several factors apparent in what appears to 
be a basic derangement in the cellular economyo These can be enumerated 
as foUowss 

(a) These polysaccharides appear to be primarily 
cytoplasmic°damaging agents« 

(b) Not all polysaccharides that damage solid tumors 
produced this effect, 

(c) Not all ascites tumors respond allke° of B such 
tumors, only two, sarcoma 37 and the Yoshida sarcoma, eshibited these 
phenomena consistentlyc 

(d) The host is an indispensable factor in eliciting this 
vacuolar response. Several types of animal and human malignant ceils, 
grown in tissue culture and exposed for several days to high concentrations 
of several polysaccharides, did not produce either cell vacuoles or 
enlargemento 



740 



Serial No„ NCI - 252 
Calendar Year - 1858 
Part A — Page 4 



(e) The end result is a serious disturbance of the 
morphological integrity of the celL which in turn leads to a serious 
derangement of the water (and salt) balance of the cello 

(f) This is the first demonstration of an effect directly 
on these tumor cells by such polysaccharides, 

IIL Studies on Vital Staining with Neutral Red 

The work in this field for the past year was directed to 
testing a number of new vital stains« closely related to neutral red. for 
staining ability towards ascites tumor cellSo Of these, two compoonds^ 
2=ethylamino°8'=methylaminophenazine and 2='n=ppopylamino°8-= 
methylaminophenazine, both highly purified^ showed activity^ Both 
compounds gave vital staining very similar to that of control cells stained 
with neutral red of the same concentrationso In each case, the intensity 
and character of the staining reaction was fully equal to the control 

A third compound, 2=cyclohexylamino°8<=methylamino= 
phenazine» has also been tested with ascites tumor cells, and also 
demonstrated vital staining typical of neutral reds although in this instance 
the intensity was somewhat less, and the color was an orange^red rather 
than the purplish<=red of the controL 

The significance of this compound is that it indicates 
that up to the cycloheayl group^ at least, substitution by a larger group 
does not preclude stainingo The scope of possible substituents is there^ 
fore considerably enlarged by this finding that cell permeability is not 
losti and points to the possibility that still larger and more comple^s 
substitution can be made without loss of vital staining properties^ 

IV^ Evaluation of Vital Stains as Indicators of Cell Injury 
and Death 

Despite the enormous amount of work done with vital 
stains during the past half century and more, there is still no substance 
used for this purpose without some reservation as to its precise meaning 



741 



Serial Noo NCI = 252 
Calendar Year 1958 
Part A =- Page 5 



and validity as a vital indicatoro Their behavior is predicated for 
the most part on assumptions regarding loss of cell permeability^ 
changes in enzyme activity^, or in other metabolic functions to ©Kplain 
the cause of dye penetration and to indicate whethes? a cell is injured 
or deado 

This situation has been always more difficult when 
dealing with cells which do not move, such as somatic cells of 
mammals. When such cells are exposed to drugs or environmental 
conditions which damage or kill, and are observed under conditions 
which do not measure some metabolic funetion„ there are relatively 
few changes of a morphologic imture which can be observed micro= 
scopically to identify damage or death, especially in the early stages 
of such a procesSo It is in such situations, particularly vshen fresh 
material is employed, that substances like the vital dyes could be most 
useful, provided their behavior is correlated with criteria that are 
reliable and meaningful^ 

To obtain more definitive evidence for th© validity of 
substances used as vital stains, e:£periments are being conducted with 
various types of cells which have the property of locomotion, the criterion 

sssed to indicate cell v'mbilityo Such cells include several species of 
protozoa (paramecium, colpidium, amoeba, etc) and mammalian ieulco- 
cyteso Ultimately the data accumulated with these cells will be applied 
to mammalian somatic c^lls, both normal and malignant^ 

There are many vital stains to choose from, io e, g 
(fieatral red, eosin, Jansss green B« methylene blue, toluidine blue O, 
trypan blue, nigrosin, etCr, Injury or death is induced by eseposing the 
cells to a variety of lethal agents, which act by presumably different 
cytotoxic mechanismss L e„ t quinine, podophyllotoxin, formalin, 
Nitromin, acids, bases» etCo Observations are made under conditions 
which afford direct visual correlation of the behavior of th® cell and th® 
vital stain at the time of eseposure to the drugo 

Esqseriments to date indicate that the rate of dye 
penetration and type of staining response vary according to the lethal 
agent and vital stain used. For example, when cells (paramecia) are 



742 



Serial No, NCI ° 252 
Calendar Year 1958 
Part A ~ Page 6 



exposed to Nitromin, they almost at once lose the property of locomotion 
and become diffusely stained with eosin or neutral red in a matter of 
1 =2 minutes. With methylene blue however, it takes about a half=hour 
for the cells to turn diffusely blueo When formalin is used, the cells may 
never become so diffusely stained or only after a number of hours, long 
after it is presumably deado 

Vo Factors Regulating the Control of Intracellular Water 
Metabolism 

From the standpoint of volume properties and function, 
water occupies an unique position in the cellular economyo And yet, 
the mechanism of its transport across the cell boundary, and its dispo» 
sition within the cell is still largely an enigma. For many years, several 

sources of indirect evidence have pointed to a metabolic control of waters 
but direct evidence has been largely lackingo 

Reasoning from the pharmacological action of mercurial 
diuretics that the depression of water reabsorption by the kidney tubules 
was probably due to an effect on the succinic dehydrogenase enzymes 
of the tubules, investigation was begun employing mercurial and other 
drugs on water control in ascites tumor cellSo It was found that both 
organic and inorganic compounds of mercury and of other heavy metals 
caused "blebs" of these cells, indicating that more water was passing 
into, and was retained by, the cell than it could excreteo 

It was further found that 3 types of these blebs could be 
produced, depending on the metal in the compound used° 

(a) Large acentric blebs, half the volume, or more, 
of the cell in size, were produced by compounds of mercury vizs 
Neohydrin, Mercuhydrin, Mersalyl, Merthiolate, HgCL 

(b) Small "scalloped" blebs, which usually bordered 
the cell completely were produced by compounds containing arsenic vis? 
Mapharsen, potass* ium arsenite, 

(c) Large "symmetrical" blebs, which entirely 
surrounded the cell, were caused by compounds containing silver or gold 
vizs Protargol, silver nitrate, gold chloride. 



743 



Serial Mo. NCI - 252 
Cal®ssdar Year 1958 
Part A = Page 7 

Non°metai containing diuretics were without effect, vis; 
Mictine, Di&mox, theophylline, Pitressin, 

The active compounds listed above have on© characteristic 
in commons they all inhibit the enzyme succinic dehydrogenases In 
addition, when ascites cells are escposed to substances considered to be 
Li]p@cific inhibitors of the enzyme, i„ Co , sodium malonate and p^cMoro- 
Kxercuribenzoic acid, large blebs are producedc Also, when blebbing 
G(.<lls are immersed in solutions containing a tetrazolium derivative, no 
reduction to the corresponding formazan takes place, although control 
cells are very active in this respect. 

At present, the data suggest that this enzyme, succinic 
dehydrogenase, is involved in the regulation of water utilization by th® 
c©ll> Whether this is by control of water transport (outward), or by some 
other phase of water metabolism in which the enzyme acts as a metabolic 
"pu:np" to provide the necessary energy for moving water against a 
gradient, remains to be elucidated, 

VIo Studies with Human Malignant Cells in Tissue Culture 

Experiments have been continued on the effects of Nitromin 
and Me^hotresnte on two lines of human malignant cells, strain Di89 and 
Hela, it I tissue culture., Observations were made daily on fresh material « 
and on ilxed preparations stained with hematcxsylin and eosin, Oil°Red<=°0, 

and peri7dic°acid Sehiff reagent. 

The morphologic alterations and cytochemical changes 
may be summarized as follows: 

(a) Nitromin on Strain D139 cells 

Toadcity experiments indicated that continued 
eaqposure to % gamma per ml. was an optimal concentration to produce 
sufficiently gradual cellular changes. During the course of 10°°14 days^ 
the following effects were evident: 

(1) progressive cell enlargement 

(2) increased cytoplasmic granularity. These 
granules stained as neutral fat with Oil~Red°0. 

(3) a slight increase in vacuolationi the number of 
vacuoles was not Inpressive, but they assumed bizarre angular and rec° 
tangular shapes 



744 



Serial NOc r: 
Calendar yea.... 
Part A =- P&ga S 



(4) pspogsressiv© eelluiar cytolysis aad fragsr.^"---^^ ■' 

(5) beading of cell membranes 

(8) incyease in amount of PAS stainable mater.;,;.-;: 
over controls 

(b| Nitromin on strain HeLa cells 

In some preliminary experiments^, these cells ^@j>@ 
exposed for on® week to varying concentrations asp to 4 gamma per irsi. 

During this p-sriod^ a characteristic response ¥/©s 
the formation of maltiple* cyfved "sickle-shaped" cytoplasmic processes. 
Many ceils were fall of «iny« etippled-like va.cuoles» and some fi&d very 
larg® vacisoleso There was a general increase in graaiiiarityo 



Concentrations over 100 gamma per ml^ wer® too 

toxic« but cultures exposed to this dose continuously for 12 days dis^^ 
played the following changes^ 

(1) many S^rge^ misltinweleate giant cells app®£3''ed 

(2) many cells asstamed bisarre shapes 

(3| many cells were atypicaily shaped«> and oft&n 
were with huge vacuoles 

(4| ther@ was a definite increase in n^sstral fat, as 
shown by Oil-Red ~0 staining^, 

(5| PAS positive material appeared to be conemAr^- ^ ':-'■' 
more heavily abos;^t the nsacleus, as compared with contro^c 

Significance to the Fifogram of the Cancer Institutes 

These investigations are designed to obtain information conesT-rd.-: 
{&) possible differences between normal and malignant cells (b) the 
mechanisms by which necrotizing compounds produce cellular damage ^ ..-..-- 
(c) to correlate at the cellular level the pharmacologic action of drugs 



745 



Serial Nc NCI - 252 
Calendar Year 1958 
Part A - Page 9 



with the structural and functional responses of cells in relation to 
treatmento Such information is urgently needed to afford a better 
understanding of drug action, and to aid in the development of newer 
and better drugs for the use of investigators working in clinical 

chemotherapy^ 

Proposed course of Project: 

Most phases of this project will be continued, with emphasis 

on (1) investigation of the factors regulating water metabolism in the 

cell and (2} th® nature of cell damage due to chemical agents^ 

Loss of personnel has resulted in a slower rate of progress 
in the tissue culture programo 

Recruitment of a suitable replacement is being actively 

carried on, but has proven to be a slow^ frustrating and discouraging 
task,, 



Part B included — » Yes 



746 



Serial No„ NCI - 252 

Page 10 



PHS<^NIH 

Individual Project Report 

Calendar Year 1958 



Part Bo HoROPSa Awards^ and Publications 

Publications other tten abstracts from this project s 

(1) Leighton. J= . Kalla, R= L„, Kline, L and Belkin, M.s 
Pathogenesis of Tumor Invasion L Interaction between Nos°m®l Tissues 
and "Transformed" Cells in Sponge=Matrix Tissue Cultareo Cancer 

Research (In pressjo 



Honors and Awards relating to this project; 

Invited to become a member of the Pharmacology and 
Endocrinology Fellowship Review Panel of the Research Fellowship 
Branch, DoRoG« 



747 



Seri?^ Noc NCI«253 

lo Lab. of Chemical Phormaeology 
2. Biochemical Pharmacology Section. 
3o Bethesda l4, Maryland 



PHS»NIH 

Individual Project Report 

Calendar Year 1953 



Part Ao 

Pi^Dject Title; Biochemieal and Pharmasological 
Studl^aTo"!^ Ho3t=fu!Tior Relationship in TUnior Chemo- 
therapy^ 

Principal Investiigator ; Dro Ao Qoldin 

Other Inycatlgators i Dr,, Ro Darrow^ 
Dro Ac Schrecker, Dr, Lo Shuster {now at Department 
of Pharraacolosy, Tufts University Medieal School, 
Boston), S. Humphreys, J, Venditti, W, Mantel ((Biometry 
Branch } , 

Cooperating Units : 

fa) General Medicine Branch, NCI: Dr, EoFrel, 
Dr. Mo Lieblins, Dr, Po Condit (now at Oklahoma 
Medical Research Foundation, Oklahoma City) and Dr„ 
So Charache (now at Department of Medlsine, Hospital 
of the University of Pennsylvania, Philadelphia, 
Peiinaylvania).^ Df. d, R®ai;, S<gria!I Mo MCI-LuiJfe 

(b) Laboratory of Bioehemistry, NCI: Or* J. 

Lasslo. 

fc) Graduate School, Bioehem. Depto, Brandeis 
Univ., Waltham, Massachusetts; Professor Nathan Go 
Kaplan and staff. 

((d) Department of Pharmacology, Washington Unlv, 
Sto Louis, Missouri: DTo R. Burton af^o 2Dr„ RoSalvador. 



748 



Con tor. 



NCI-253 

Gaxenfiar year .1958 
Part A -»• continued 
Page 2 



(e) Cancer Chemotherapy National Service 



(f) Mlcrobiologicai Associates, liiC, 
Bethesda, Maryland.. 



Ian Years (calendar year 1950) ; Patient Days 

Total : 12-1/3 None 
Professional s 6 

Other: 6-1/3 
Project Description: 



Objectives 

1. Study of the mechanism of action of anti- 
turner agents, including metabolic antagonists, radicmi- 
me tic poisons J antibiotics, etc. 

2. Synthesis of new agents. 

3. Study of host-tumor interrelationships with 
ro3pect to drug action. 

4. Establishment of new therapeutic principles 
and procedures in the management of tumor growth in animals. 

Methods Employed 

Macrobloiogical assay procedures involving methods 
of Inhibition analysis for animals with and without tumors. 
In vivo and jLn vitro biochemical procedures. Organic 
chemistry/ procedures. 

Major Findings 

1. New antiloukoralc agents (with Venditti, 
Humphreys, Mantel, Darrcw and Shuster): Investigation of 
the haiogenated derivatives of structural analogues cf 
fcj-ic acid has been continued. In this connection, the 
American Cyanamid Company, Pearl River, N. Y. continued to 
make new compounds avaliabj.e tc this Laboratory. 

Pour of these compounds have now been inves- 
tigated in some detail employing systemic leui.emia (L-12iO) 



749 



N0I»253 

Calendar year 1958 
Part A - continued 
Page 3 

in mice, using amethopterin as a standard. 3'»l>romo° 
amethopterin was soraeiu'hat more effective than amethop- 
terin in increasing the survival time of mice with 
advanced leukemia, but was not as effective as 3'-= 
chloroaraethopterin. "Hie latter was up to 3^ percent 
more effective than amethopterin in increasing survival 
time . 

The dihalogenated derivatives, 3 '5'-° 
dichloroamethopterin and 3'«bromo 5'"Chloroamethopterin 
were highly effective in increasing survival time. Both 
drugs were capable of increasing the median survival time 
for the full period of tjTeatment (90 days) and beyond, 
even when treatment was initiated only 2 to 4 days prior 
to death of control mice. Some of them were still alive 
at 6 months following inoculation of the leukemia, and 
were presumably cured of the disease. Since the disease 
vjas systemic when treatment was started, the current 
study supports the concept that successful therapy of 
clinical leukemia may indeed be possible. 

Some of the mice apparently cured of 
systemic leukemia did not accept reinoculation of a massive 
dose of leukemic (L'»1210) cells, suggesting that they may 
have become immune as a result of treatment „ If this 
phenomenon is found to be a true imnsine response, it would 
provide a possible means for interdigitation of the anti= 
metabolite approach and the immunological approach to 
antitumor chemotherapy. 

As with amethopterin, the antileukemic 
action of the halogenated derivatives of amethopterin could 
be reversed by concomitant administration of citrovorum 
factor. 

2. Studies of tumors recovered from treated 
mice (with Humphreys): The growth behavior and responsive- 
ness of tumors recovered from mice with leukemia L-1210 
during, or subsequent to, extensive therapy with the dl-^ 
halogenated derivatives of amethopterin were studied. 
These tumors appeared in various sites other than that of 
initial implantation, or they were recovered from spleen 
or blood. The studies have shown that such tumors may 



750 



NCI°253 

Calendar year 1958 
Part ^ " eontiJTiued 
Page 4 

have various growth rates. Some of the variant tumors, 
unlike the sensitive Ii»1210, achieved large sizes 
(20=30 nnn diameter) prior to death. The tumors which vf ere 
recovered after extensive treatment showed a wide range 
of sensitivity to araethopterin. Some of them were al-=» 
most wholly resistant. 

Resistant leulcemie variants v/ere more 
susceptible to treatment with 3*5'-'dichloroamethopterin 
than with araethopterin. This result was obtained whether 
the mice were made resistant by treatment with 3'5'" 
dichloroamethopterin or with amethoyterin. 

3. Advanced leukemia, other studies (with 
Humphreys): The ability to obtain extensive survival 
time of mice with advanced leukemia permits o detailed 
study of individual mice in preclinical fashion. At 
the time when treatment ia initiated against advanced 
leukemia, peripheral blood readily transmits the disease,, 
Evidence has now been obtained indicating that when there 
is a demonstrable regression in size of the local tumor 
at the site of leukemic Inoculation, and a reduction in 
peripheral blood leukocyte count due to treatment with 
halogenated derivatives of amethopterin, the disease is 
usually not tranaralssible by the injection of peripheral 
blood into normal mice. Where treatment eventually fails, 
following such remission, the blood again may transmit 
the disease. This type of observation, plus the ability 
to start new sublines of tumor from tall blood at various 
stages of therapy, may bring woric on the leukemic mouse 
closer to that in the patient in teiros of parallel inves- 
tigations . 

Advanced leukemia in mice may serve as a 
useful tool for screening and for eherao therapeutic studies. 
This subject was reviewed (with Vendittl, Humphreys, Shuster, 
Darrow, Mantel), Tlie system provides good diserimlnation 
between active and inactive compounds. In addition, it 
provides various economies, since treatment is istarted 
only several days prior to death. It may be employed for 
the determination of optimal schedules of treatment, studies 
of synergism and antagonism, etc. 



'51 



NCI-253 

Calendar year 1958 
Part A ■" continued 
Page 5 

4« Studies with 5-fluorouracll (with 
Liebling and Ifcunphreys); Clinical reports of marked 
toxicity on the one hand and significant antineo- 
plastic activity on the other, prompted additional ln-= 
vestlgatlon with leukemia L-1210 in mice. Definite 
activity was demonstrated against both early and advanced 
L»1210. The activity was approximately equal to that 
described for some purine antagonists, but not as exten- 
slve as that observed for amethopterln. Daily and inter= 
mittent treatment were approximately equally effective. 
The useful dose ranges were sharply limited by host 
to xi city. 

Attempts to alter toxicity and preserve 
antitumor effect of larger doses by the use of inter- 
mittent therapy were not successful. 

5. Synergism (with Humphreys, Vendltti, and 
Mantel ) s Several investigators have reported antineo- 
plastic synergism with the combination of 6-=mercapto- 
purine and azaserine. Studies in this Laboratory illus- 
trated how the successful demonstration of synergism maj 
be dependent upon the schedule of treatment employed, 
Tlierapeutie synergism for leukemia L-1210 with the 6- 
raercaptopurlne-azaserine combination was most evident on 
an alternate day schedule. Synergism was less evident on 
a daily schedule, and was no longer evident on a twice- 
daily schedule. Drug toxicity for the host limited thera- 
peutic usefulness for the drug combination as well as for 
the individual drugs. The most effective levels of drug, 
alone or in combination, were invariably toxic for normal 
mice. 

It was observed (with Rirton and Salvador) 
that nicotinamide can potentiate the duration of anes«» 
thesia produced by a combination of reserpine and pento- 
barbital, 

6, Slucose analogs (with Lasslo and Humphreys): 
The effects of 2='deo2:y-D«=glucose and 2-»deo2cy«D"galactose 
were investigated on transplantable tumors in mice, in- 
cluding leukemia L»1210, variants of L-1210, and the Bashford 
carcinoma. Inhibition of tumor growth was observed with 
most of the tumors employed, and prolongation of survival 
time was generally evident. The effects could not be 



i %} u 



NCI-253 

Calendar year 1958 
Part P - continued 
Page 6 

duplicated by caloric restriction. Tlie findings are 
consistent with the conce-pt that interference with 
Slycolv-sis (with glucose phosphorylation via he."o- 
kinase; may selectively injure the cancer cell. 

7. Duration of the effects of folic acid 
antagonists in the mouse (with Levy, Charache, Humphreys, 
and Condit): Normal mice given folic acid, and sacri- 
ficed one hour later, were found to have hepatic citro- 
vorum factor concentrations more than twice as high as 
controls. The relative magnitude of this increase was 
used as a measure of enzyme activity at varying times 
after antimetabolite administration. When mice were 
given amethopterin as a single dose (360 mgra. per kgm. 
subcutaneously; an LDcq)* profound inhibition of en« 
jiyme activity occurred promptly and persisted for a 
prolonged period. Nine days after administration of 

the antimetabolite, enzyme activity was still less than 
30^ of the control values, 

8, Biochemical studies with antifolics 
(with Darrow, Schrecker, Shuster, and Prof, Kaplan's 
group): 

(a) tot i tumo r ac tion . Some progress 
is being made in attempts~^o correlate basic biochemical 
observations with the quantitative phairaacologlcal studies 
on host-tiimor-drug interrelationships. For example, with 
leukemic growth, incorporation of labeled formate is in- 
creased in infiltrated organs such as the liver and spleen, 
and is high in the local tumor at the site of leukemic in- 
oculation. This increase was correlated with the amount 
of tumor growth and the weight of the infiltrated organs. 
It was demonstrated that treatment with doses of amethop- 
terin, 3' =chloroamethopterln, or S'S'-dichloroamethopterin, 
effective in increasing the life span of mice with ad~ 
vanced leukemia, also diminished the er'tent of formate 
incor:oratlon into acid soluble adenine, nucleic acid 
adenine and tissue i:)rotein. The extent of formate in- 
corporation dropped to as little as ten percent of the 
untreated values from e single therapeutic dose, 3pproxi-» 
matlng the level in normal mice. 



753 



NCI-253 

Calendar year 195S 
Part A - continued 
Page 7 



(b) Biochemical Mechanisms . Because 
of the limitation to effective therapy Imposed by the 
toxicity for the host with the antlf olios, detailed 
studies were undertaken on underlying mechanisms of 
actloHo 

Non-=tumorou3 mice 3howv"d no 
inhibition of liver formate fixation over a wide 
range of doses of amethopterin, with different routines 
of dru2 udrainistratlon and with varying timea cf isotc., e 
incubation. No reduction waa observed even when the 
nomal rcite of formate flxuticki intc adenine had been 
increased to the level found in tumc r-bearing animals 
by such agents as carbon tetrachloride or nicotinamide. 

Although no inhibition of 
formate fixation in normal liver by amethopterin could 
be demonstrated, conditions of drug toxicity broui^iht 
about dramatic stimulation . This phenomenon was men- 
tioned in last year's report, and has now been e :ten=- 
Bively characterized. When a toxic daily dose of 
amethopterin was given, formate fixation into liver 
adenine was observed to increase as much as twenty 
fold. The increase showed general correlation with 
sjmiptoms of toxicity. It decreased again when the 
death of the animal was imininent. With single toxic 
do.5e3 there was a stimulation of formate fixation which 
was proportional to the dose of drug, and which reached 
a maximum in four days, after which it dropped back to 
normal level. Of all the organs tested, including 
brain, heart, lungs, kidney, spleen, intestine, testis, 
and blood, only liver e-chibited stimulation. 

In vitro measurements, using 
minces of the livers of toxic mice, failed to show 
stimulation, and in fact gave lower values than normal 
tissue. Such factors as lowering of the adenine pool, 
or interference with formate oxidation or excretion, 
have b- en ruled out as e planationa for the stimulation. 



754 



NCI-253 

Calendar year 1958 
Part A - continued 
Pase 8 



The work is being extended 
to a study of other factors which might influence the 
rate of formate fixation, in vivo. (Darrcw) Such 
studies are considered desirable in ordor tc hei; 
ciiirify basic mechanismj. 

9.. i^Nicotinamide and in vivo incorporation 
of fcrmate-C-^^ (with Shuater) : Injection of nicotlna- 
jnide resulted in a 5 to iOrf<~j~d increase in the in vivc 
Incorocration of fcr.Tiate C^^ and glycine-2-C-^ into 
the acid aoxubie adenine cf liver. However, there was 
ac effect on incorporatlcn cf formate into liver pre- 
teina. High do3C3 of nicctinamidc would ap.jear to 
atimulate the net ayntheaia of the adenine moiety re- 
quired for th'ij formation of new DPN. 

10. Thiadiaaoles (with Shujter): The nlco= 
tinamide antagonist 2-ethylamino~l,3.^-thiadia2ole 
(E.flT) did not appear tc act aa a competitive inhibitor 
of nicotinamide for the induced in vivc syntheals of 
mou3G liver DPN. E/^T caused a 30 percent Inhibition 
of DPR synthcoia from a high dose of nicotinamide, but 
increased by as much as 70 percent the synthesis of DPN 
from a low dose of nicotinamide. EAT also increased 
the effectiveness of low doses of nicotinamide in stlm- 
ul a tin/^ incorporation of formate C-^"* and slycine-2-=C-^^ 
into ocid soluble liver adenine. In' addition tf^T, by 
itself, WP3 found to stimulate the incorporation of 
formate-C-''^ and, to a lesser e.;tent, 3lyclhe»2»cl^ into 
acid soluble liver adenine, 

llo Organic Chemistry (Schrecker) : 

(a) As part of a project dealing with 
the preparation of amino acid antagonists to bo tested 
for potential effects against leukemia L-1210 and other 
tumors, synthesis of certain aminoketones vms investi- 
gated. Since 1-amlnoeyclopentanecarboxylic acid and 
2-methylene-3-ltetocyclopentane carboxyiic acid (sarkomycin^ 
both are active against experimental tumors, attempts 



755 



NGI-253 

Calendar year 1958 
Part A » continued 
Page 9 



were mada to prepare cyclooentanone derivatives which 
incorporate the structural features of both compounds 
(e.g, l-anilno-3-ketocyclopentanecarboxyllc acld;» 
These attempts have so far not been successful because 
of the Inherent Instability of S^aniinocyclopentanone 
derivatives, none of which has so far been reported 
in the literature. Two new routes for the synthesis 
of analogous a-aminoketones were investigated. The 
acylation and subsequent cleavage of the new compound, 
di-t^»butyl acetamldomalonate, has yielded a series of 
acyclic amlnoketones, some of which are difficult to 
prepare by previously known methods. Such aniinoke= 
tones are of value as intermediates in the preparation 
of certain antibiotics and of scintillation compounds 
used in counting tritium-labelled compounds. In the 
cyclic series, the unknown 2-=aminocyclopentanone was 
prepared by the Neber rearrangement, and some of its 
reactions were studied. This method promises to be 
generally applicable for the synthesis of ■•v=amino= 
cycloalkanones and is being further investigated. 

(b) In collaboration with Dr. Montague 
Lane, a project dealing with the synthesis of new rlbo° 
flavin antagonists was initiated. 6,7-'dlmethyl"9~C2''> 
hydroxyethyl )-=isoalloxa2lne phosphate was prepared. This 
compound has been shown to be of Interest in the study 
of metabolite-antimetabollte relationships. The synthesis 
of other new riboflavin antagonists is in progress. 

Significance to the Program of the Cancer Institute: 

These studies are designed to provide basic 
information pertaining to the host=-turaor relationship 
in chemotherapy. Prom such studies, principles for 
treatment in animals may bo established which may be 
of use in application to clinical chemotherapy. 

Proposed Course of Project: 

During the ne.ct year the various phases of 
this work will be continued and broadened. 

Part B Included: yes 



/: 



Serial No„ NCI 253 
Calendar Year 1958 
Page 10 



PHS=NIH 

Individual Project Report 

Calendar Year 1958 



Part Be Honors^ Awards^ and Publications 

Publications other than abstracts from this project? 

(1) Humphreys. ScRc. Goldin» A^ , Venditti, JoM<,» and 
Mantel, N„s Ihe Influence of Amethopterin on the Smrvival Tim© of 
Leukemic .^"ice with respect to Food Intake, Body Weight Changes^, 
and Tumor Growth, J» Nate Cancer Insto _2Q, 211=218„ 1958o 

(2) Goldin, A.^ Venditti, J^Mo* Humphreys^ S„R<,<, and 
^''antel, N^ s Quantitative Evaluation of Chemotherapeutic Agents 
Against Advanced Leukemia in MicCc Jo Nato Cancer Inst„ 21^^ 
495==511, 1958o 

(3) Burton^, RoM<,« Kaplan^ NoOo, Goldin^ A^, Leitenberg^ M, ^ 
Humphreys, So Res and Sodd„ MoAoS Effect of Reserpine and Promazine 
on Diphosphopyridine Nucleotide Synthesis in Liver,, Science 127., 
30-32, 1958„ 

(4) Shuster, Lo* and Goldin, A^ s The Incorporation of C^^ 
Glucose and C^^ Ribose into Mouse Liver Diphosphopyridine Nwcieotid©, 
J, Biol. Chemo 230 , 873-881, 1958„ 

(5) Shuster, Lo , and Goldin, Ao % The Conversion of Glucose 
to Pentose in the Bipsynthesis of Mouse I^iver Diphosphopyridine 
Nucleotide, Jo BioL Chemo 230, 883=838, 1958, 

(6) Shuster. L„« Langan, To Ac^ Kaplan, N<,Oo. and Coldin, A. 
Significance of Induced In Vivo Synthesis of Diphosphopyridine Nucleotide, 
Nature 182, 512=515, 1958„ 



757 



Serial Noo NCI 253 
Calendar Year 1958 
Part B (continued) 
Page 11 



(7) Goldin, Ac? Potentiation of Drug Ac tiono In '"Consciousness 
and the Chemical Environment of the Brain" o Report of the 25th Ross 
Pediatric Research Conference, ppo 23=30, published by Ross Laboratories, 
Columbasa Ohio„ 1958„ 

(8) GoldiHa Ao, Venditti, J<,Mc, Hamphreys, S,, Ro« Shuster« L.^, 
Darrow Ro A, « and Mantel No % Advanced Leukemia as a Tool for 
Chemotherapeutic Studies.; Acta Unio Interna tionalis Contra Canerumo 

In press. 

(9) Lieblingo Mo Eos Humphreys^ SoR»s and Goldin, AoS 
Studies of 5°Fluoro«racil in the Treatment of L=1210 Leukemia in Ti-ice, 
Cancer Research o In press, 

(10) Goldin, Ao^ Humphreys^ SoRo^ Venditti» J, Mc„ and 
Mantel, N, s Factors Influencing A nti=Tumor Synergisms Relation to 
Screening ly/'ethodologyo Ann^ N„Y, Acad„ Scio In press o 

(11) Shusters L,,, and Goldin„ ^o% The Effect of Nicotinamide 
on In Vivo Incorporation of Formate^C^o Jo BioL Chemo In press, 

(12) Shuster, Lo^ Khorana„ H,Goa and HeppeL LoAoS The 
TvTode of Action of Ryegrass Ribonucleaseo Biochim, Biophysc, Acta« 
In presso 

(13) Schrecker, AoWoj, and Trail, MoM.g Methyl Desoxypodophyl- 
late and its Methyl Ether, J„ Org„ Chemo 23, 767=768,1958, 

(14) Hartwell, J, T.,, , and Schrecker, AoV/oS The Chemist2=y of 
Podophyllum (A Review)^ Org, Naturstoffe, ^5, 83=166„ 1958. 

(15) Schrecker, A„Wco and Trail, MoM« AminoketoneSc L The 
Preparation of =Aminoketones from Di-t=butyl Acetamidomalonateo 
Jo Am, Chemo Soco 80, 1958o 



75i 



SeK-ial Noo NCI 253 
Calendar Year 1958 
Part B (continued) 
Page 12 



Honors and Awards relating to this projects 

Ao Goldiag (a) Received reappointment as Visiting Professor 
of Biochemistry at Brandeis University, Waltham, Massachusetts? 

(b) Panel Member, Board of U«So Civil Service Examiners at NIHg 

(c) Foreign Corresponding Member Societa Italiana Di Cancerologiaj 

(d) Elected to Affiliate memberships Royal Society of Medicines 

(e) Participated, by invitation, in International Symposium on the 
Chemotherapy of Cancers Cambridge, England^ July 13=15, 1958? 

(fj Invited to present work of th® laboratory atg Eastern Cooperative 
Group in Solid Tumor Chemotherapy^ January 9, 1958s American Cancer 
Society Press Conference, Febrasary 14, 1958? New York Academy of 
Science Screening Conferencep New York, March 15, 1958s Combined 
Clinical Staff Conference on New Information on Anti°Folic Drugs in Cancer, 
April 10, 1958s American Chemical Society meeting, San Francisco, 
April 16, 195Ss Johns Hopkins Medical School . May 20, 1958s Waltsr 
Reed Hospital, June 14, 1958s Gordon Research Conferences on Cancer, 
New London, New Hampshire, August 26, 1958s George Washington 
University, Pharmacology Departments November 6, 1958s Conference of 
AcMte Leukemia Cooperative Group B^, Medical College of Virginia, 
Richmond, November 21, 1958o (g) Invited to esshibit work on leukemia 
at the Trail Blazer Section of the 10th National Chemical Expositi*3n, 
Chicago, September 9-12, 1958, in cooperation with Drs« Zubrod, Rail, 
and Freio 

J„M, Vendittis (a) Elected to th® Gradmte Council, Th® 
George Washington Universitys (b) Elected to Associate membership 
in the Society of the Sigma Xij (c) Invited to present laboratory work at 
Combined Clinical Staff Conference on New Information on Anti^-Folic 
Drugs in Cancer, April 10, 1958, 



759 



Serial No. NCI-261 

1« Lab. Chemical Pharmacology 

2. Polysaccharide Section 

3. Bethcsda lij., Maryland 



PHS^NIH 

Individual Project Report 

Calendar Y-ear 1958 



Part A. 



Project Title ; The role of polysaccharide 
canplcxes in resistance of the host to injurious 
influences. 



Principal Investigator ; M«, Landy, 



Othe r Inve st i gat ors ; M» J. Shear, F, So Rosen 
and Ro Co Skarnes. 



Cooperating Units ; Macromolecular Chemistry 
Section^ LCP, Serial No. NCI-262. Biochemistry 
Section at the Armed Forces Institute of Pathology, 
V/alter Reed Army Medical Center. 



Man Years (calendar year 1958): Patient Dayss 
Total; 8 None 

Professional: I4.-I/2 
Others: 3='l/2 



Project Description ; 

Objectives: To obtain infonr^tion on raechan=> 
isms by which polysaccharide complexes of exogenous 
or endogenous origin produce effects in the mammal => 
ian host v/hich are manifested in altered resistance 
to experimentally induced or naturally occurring 
pathologic states. 



.. \ 



Serial No. NCI =261 
Calendar Year 1958 
Part A =• Paae 2 



Methods Employed: Immunological, microbiological, 
physiological, and biochemical, modified as required to 
meet the specific needs of particular problems. 

Major Findings: 

1, Last year v/e reported that fresh human serum 
inactivated endotoxin derived from bacterial, mammalian 
and plant sources. This activity was shm-m to be heat 
labile and vms clearly distinguishable from complement, 
properdin and specific antibody. During the past year 
further study has been made of this agent which, for con» 
venience has been designated EDC (endotoxin detoxifying 
component). As before, CAF| mice bearing ^^day^old lra° 
plants of Sarcoma 37 were used for the assay of endotoxin 
and Its inaetivation by EDC. Thus the standard dose of 
10 \!ig of Serr. marcescens endotoxin regularly produces 
marked hemorrhagic necrosis in the ttunors. This tumor- 
damaging action of the endotoxin Is eliminated by prior 
incubation of endotoxin and appropriate serum or plasma. 
The EDC potency of the latter may be expressed as the 
minimal amount vrhich inactivates the standard dose of 
endotoxin. 

Animal species could be grouped in three cate« 
gories vrlth regard to the EDC activity of their serai 
rat serum \i&s highly potent; chimpanzee, dog, horse and 
guinea pig sera were much less active; mouse, rabbit, 
and sheep sera exhibited no activity. The EDC potency 
of human sera varied i>;ldcly, ranging from high to barely 
discernible activity. In contrast to these variations 
of EDC potency In serum, cltrated plasma from all species 
manifested high potency of about the same magnitude. 

The Influence of time, temperature and pH on the 
inaetivation of endotoxin by EDC v/as examined. Some 
inaetivation v/as discernible as early as 10 minutes and 
vas complete at 20 minutes* This reaction did not pro- 
ceed at temperatures of 30° C. or lov/er, nor at pH belov/ 1 



Serial No. NCI-26.1 
Calendar Year 19^3 
Part A - Paae 3 



In attempting to explain the marked difference 
in EDC activity of serum and citratcd plasma j the iat" 
ter v/as clotted by recalcif Scat ion. This recalcified 
plasma had no EDC activity. For some time it was 
thounht that the clottlnn mechanism vms implicated in 
the observed difference in EDC activity bctvfeen senim 
and plasma. Hov/ever, St eventually became evident 
that the loss of EDC activity \7hich follov/ed recalci- 
flcation of citrated plasma v/as independent of clot 
formation and v^as due solely to suppression of EDC 
activity by calcium, Thrombin^treated citrated plasma 
fr«n v/hich the clot had been removed retained undimln^ 
ished EDC activityo Addition of analogous concentra= 
tions of Ca, F.c, Mn or Ba resulted In total suppresssion 
of EDC activityo Divalent cations of Co, Cn and Fe v/ere 
not inhibitory. Thus the hiph IcveJ of EDC activity 
exhibited by citrated plasma appeared to have been 
achieved by virtue of the calcltim-bindino property of 
the anticoagulant. Similar potency ^ms obtained ^-^hen 
plasma was derived from blood in v/hich clot formation 
had been prevented by other calclum=>b£nding anticoagu- 
lants, such as oxalate, fluoride, and EDTA, or by the 
collection of blood throw ah a cation exchanoe res in ^ 
On the basis of these find inns it vras anticipated that 
the addition of these calcium-binding agents to scram 
would disclose EDC activity vhere little or none v/as 
previously evident. This ^'ss indeed found to be the 
casej the addition of these agents to serum rendered 
it as active as citrated plasma. 

The Inhibition of EDC activity at first appeared 
to be a consequence only of calcium concentration? hovf- 
ever, later observations shox-jed that other factors also 
Influenced this activity. For example, sera from hiiraan 
donors exhibited considerabU differences in EDC potency 
despite the constancy of calcium levels. In at least 
one species Crat)^ serum and citrated plasma were of 
comparable potency^ 



-7<^^' 



Serial No. NCI=>261 
Calendar Year 195^ 
Part A = Page [|. 



Dialysis of plasma against distilled water re= 
moved most of the calcium. Contrary to expectation, 
the EDC potency of the dialyzed material vras found to 
be reduced rather than elevated. Stepwise restoration 
of calcium, by the addition of graded increments, failed 
to restore the activity of the dialyzed plasma, exclude 
ing the possibility that I&K'I levels of calcium were re- 
quired for EDC activity. On the other hand, addition 
of "calcium-binding" anticoagulants fulSy restored ac» 
tivity to dialyzed plasma, inasmuch as the latter effect 
vas obtained vrith plasma in which the calcium content 
had been reduced below the level of detection, it Is 
clear that neither the Inhibition of EDC potency with 
calcium, nor the reversal of this inhibition v/ith cal- 
clum=bindlng anticoagulants, v/as explicable solely on 
the basis of calcium ion. Obviously, some mechanism 
other than the binding of calcium v;as involved. 

Since such anticoagulants exerted an important 
effect even in the absence of detectable calcium, it 
was considered desirable to obtain plasma v/ithout the 
use of anticoagulant. Accordingly, human blood v/as 
passed through a cat ionic exchange resin to remove most 
of the calcium; plasma thus obtained proved to be as 
potent as citrated plasma. Dialysis, as in the case of 
citrated plasma, greatly reduced the potency of the 
res In-collected plasma. However, when this dialysate 
was recombined i-'ith the dialyzed plasma, EDC activity 
i-zas fully restored. Moreover, even after the dialyzate 
was dried and ashed, the aqueous extract of the ash 
fully restored the potency of the dialyzed plasma. 
Subsequently physiologic levels of phosphate plus bi- 
carbonate were found to restore EDC activity to the 
dialyzed plasna. Restoration was also obtained with 
caleium^bindlng anticoagulants. These findings indi- 
cate that the suppression of EDC activity by calcium 
is not direct but is probably mediated through its ef« 
fects on an anionic component of plasma which Is re- 
quired for Inactlvation of endotoxin by EDC. 



763 



Serial No. NCI'=26l 
Calendar Year 195S 
Part A ■= Pacje 5 



2, The level of EDC incltrated or oxalated 
plasma of more than 50 normal hiiraan subjects v^s deter- 
mined. The activity in these plasma samples vas of a 
uniformly hiqh order. Plasmas from some 30 patients 
with various forms of mallpnant disease v;ere also exam« 
Ined and v/ere found not to deviate significantly frcm 
the normal. Hov/ever, children v/ith cystic fibrosis of 
the pancreas i-iho had severe pulmonary infection due to 
Pseud omonas aeruciinosa vrere lackinci in this normal com«- 
ponent of blood. Other isolated cases v/ere encountered 
in v/hich EDC v/as markedly diminished or v;as absent? these 
included patients with Salmonella empyemia, Pseudomonss 
cellulitis and Salmonella sepsis in an anammaqlobulinemic 
child. 

3o Of the various naturally occurring materials 
which can be shovm to Influence resistance non-specific 
cally, endotoxins are the most potent. Accordinply, the 
very presence in blood of a normal component (EDC) v/hieh 
can inactivate endotoxins is, in itself, evidence for the 
biolooical significance of this material. Hov/ever, it 
v/as considered important to determine v/hether any evidence 
could be obtained for the participation of this agent In 
physiological disturbances in v/hich endotoxin is considered 
to play a major role. Tov/ards this end the follov/ing 
situations, leading to a fatal outcome, vrere examined; 
a) infection of mice vrith Gram negative, endotoxin-bear« 
ing orasnismsi b) radiation injury In mlcej c) hcmorrhaoie 
shock in rabbits; d) Intestinal occlusion shock in rabbits 
and dogsi and e) thermal burn shock in mice. Samples of 
cltrated plasma v/ere taken at various Intervals in the 
development of these situations. Including specimens from 
animals in a moribund state. The levels of EDC in these 
serial bleed Inos proved no different from those of the 
normal controls. If EDC does, in fact, participate in 
the defense of the host against endotoxin, indirect evi- 
dence for such participation vas not obtained In the 
form of altered levels of EDC in the circulating blood. 
V?ork has been started on the levels of EDC activity in 
tissues, the presumptive source of blood EDC, 



764 



Serial No„ NCI-261 
Calendar Year 195^ 
Part A ° Paoe 6 



k- Studies on EDC were initially made on whole 
scpuro or plasma. Despite the presence in serum of other 
factors i;.;hich are demonstrably capable of reactinci vrith 
endotoxin, the unique properties of EDC are such as to 
qive assurance that In this x/ork EDC v/as in fact respon- 
sible for the effects reported. It i/as nonetheless de- 
sirable to attempt to concentrate and isolate this activity 
from serum. At first the various Cohn procedures for 
fractionation of serum proteins, usinp ethanol as a pre- 
cipitating agent, were attempted but it was found that 
the activity v.ras lost In the ethanol precipitates. 
Commercially prepared Cohn fractions of human serum dis- 
played no EDC activity. Procedures in v/hich methanol vras 
used as the precipitating agent resulted in similar loss 
of activity. Subsequently the standard ammonium sulfate 
fractionation procedures x/ere used. With this method it 
vjas found that EDC activity v;as closely associated x-dth 
the serum albumin fraction, but definitive separation of 
EDC from the albumin fraction, or from the alpha alobulln 
fraction, x-as not effected. Consequently, curtain elec«. 
trophoresis x-ms employed in order to achieve a finer 
resolution of the serum proteins. Again no clear cut 
separation ijas achieved, but the behavior of EDC in this 
method v/as found similar to that in the ammonium sulfate 
procedure in that EDC appeared to be closely allied to 
the albumin fraction. 

Since these conventional approaches to serum pro- 
tein fractionation resulted in no significant concentra- 
tion of EDC, basically different methodology is being 
explored. In collaboration x/ith the Section on Macro- 
molecular Chemistry, LCP, the precipitation of EDC l^.'ith 
synthetic polyglucose derlxmtivcs is yielding encouraging 
pronress in the separation. 

5. Endotoxins, in addition to their toxic attrl^- 
butes, also possess individually characteristic antigenic 
properties. It therefore vras desirable to determine 
whether interaction x/Ith i£DC significantly affected their 
immunological activity. Typhoid endotoxin has been v/ell 
characterized as regards its immunological properties. 



'65 



Serial Woo rJCI<=.26l 
Calendar Year 195^ 
Part A «= Pacre 7 



and vms therefore selected for study. Rabbits given a 
sinole intravenous injection of 10 microcirams of this 
endotoxin developed pood levels of typhoid anglutininsB 
It i-jas found that this quantity of typhoid endotoxin, 
after appropriate interaction ^ vitro with plasnm or 
serum from mice, rabbits or humans, no longer vras capable 
of evoking slpniflcant levels of antibody. The conditions 
under v/hich antigenicity for rabbits of typhoid endotoxin 
vms altered by EDC i^rcre distinctive, and the same as those 
previously shovm to be required for elimination of the 
tumor damaging, Shvmrtzman and lethal effects of endo- 
toxins. 

The same conditions which brought about elimination 
of antigenicity In vivo of typhoid endotoxin were found 
to alter Its performance In quantitative precipitin tests 
v/ith typhoid antibody. Typhoid endotoxin, after interac- 
tion v/ith a potent source of EDC, vms much more efficient 
in the precipitation of specific antibody than was the 
untreated endotoxin. The course of the precipitin reaction 
of this £DC-altcred endotoxin v;as virtually Identical Tifith 
that of the polysaccharide haptene derived fron the endo» 
toxin by acid hydrolysis. The haptens had previously been 
characterized as a pure polysaccharide of relatively low 
molecular v/eight (approximately 30,000 as contrasted t© 
greater than 1 million for the parent endotoxin). These 
findings are taken to indicate that endotoxin, on intcr= 
action\/Ith EDC, is converted to a substance which is 
immunologically analogous to haptenic (degraded) poly- 
saccharide. 

6, In collaboration vrith Dr. ^faravdekar. Head of 
the Biochemistry Section at the Armed Forces Institute 

of Pathology, EDC Is being sought In animal tissues freed 
frcHB blood by perfusion. Preliminary experiments indicate 
that this line of vrork "ill be fruitful. 

7. Typhoid endotoxin is an effective ccmplement 
fixing agent, i,e., the union of this antigen v/ith Its 
antibody effectively binds complement. In contrast, the 



766 



Serial No. NCI =-261 
Calendar Year 1958 
Part A » Paoe 8 



haptenic polysaccharide derived from this endotoxin, 
even thouah It combines with antibody, fails to fix 
corapleraento Thus, in a quantitatively standardised 
system, less than 1 micropfam of this endotoxin and 
0»001 ml (0.25 ^tg of antibody N) of immune serum suf* 
f ice to bind five ^0% units of hemolytic ciuinea pip 
complement, whereas the haptene exerts no complement 
fixlnci activity, even in amounts of 100 micrograms, 
''hile determining! immunological chanoes in the typhoid 
endotoxin brought about by EDC, it v/as observed that 
after Interaction In vitro i-;ith fresh hunan serum, this 
endotoxin no longer fixed ccanplement. At first, it 
appeared that this alteration v/as due to EDC, However, 
subsequent study showed that the factor in serum respon- 
sible for this effect differed from EDC in a number of 
important respects. Moreover, partially purified prepa- 
rations of EDC from human sera did not produce this 
change. Thus it appears that the factor in fresh human 
sertim v;hich eliminates the complement binding activity 
of endotoxin is a separate ccHuponent or system, clearly 
distinguishable from EDC. 

8, By the application of various techniques, it 
has become evident that there exists in normal blood 
several substances capable of producing physicochemical 
changes in endotoxin. Elsev/here in this report are de= 
scribed certain distinguishing features of tv;o of these 
factors «, Other changes in endotoxin have been observed 
X'^hich may conceivably lead to uncovering still other 
blood components* The admixture of concentrated suspen- 
sions of endotoxin and fresh human serum results In 
greatly increased dispersion of the polysaccharide; the 
endotoxin is no longer sedimcnted at low speed and. In- 
deed, cannot be centrifuged down even at high speed. 
This redistribution of endotoxin betv/een sediment and 
supernatant is confirmed by immunochemical assay. More 
recently. It v«s found that turbid suspensions of endo- 
toxin v/ere cleared following brief incubation with fresh 
human serum at 37° Co These observations Indicate the 



767 



Serial No. NCI-26i 
Calendar Year S958 
Part A =■ Pacie 9 



presence in serum of additional substances which produce 
a variety of changes in endotoxin as reflected in iimnuno- 
loaic, toxic, and physicochemical properties. It nov/ 
seems probable that the exposure of endotoxin to serum, 
under appropriate conditions, results in multiple changes 
which may proceed either concomitantly or In sequence. 

9. a) Normally occurrincj natural hemolysins and 
hemapciltitinins of mammalian blood have been studied only 
sporadically during the past 65 years. The question of 
the immunolociic specificity of reaction betv^een these 
acients and foreipn erythrocytes Is not settled. The pres^ 
ence of specific heterophile hemagglutinins In the blood 
of most animal species has complicated the search for non- 
specific factors v/hlch, v/Ith complement, may participate 
in the lysis of red blood cells. To date, there is little 
definitive evidence that such factors exist. 

In a survey of many different animal sera for 
natural hemolysins and /or hemaoglutinins, a few Instances 
were found \»/here a non-specific hemolysin appeared to 
function in the lysis of foreign red cells in the absence 
of demonstrable hemangltitlnins. It v?as first thought 
that such a hemolytic system might involve the non-specific 
serum component, properdin, thus providing a convenient 
assay for Its presence, Ho^/ever, properdin subsequently 
vfas found to exert no measurable influence In this lytic 
system. 

The lysis of normal sheep erythrocytes by guinea 
pig serum v/as found to be dependent upon the components 
of complement and a non-hemagglutinating hemolysin. Both 
complement and the nonial hemolysin v/ere shov/n to be labile 
to heating at 56° C. for l/2 hour? the latter could be ad^ 
sorbed out of auinea pia serum v/Ith i^hole sheep cells or 
with sheep red cell stroma at 20° C. The hemolysin could 
also be e luted from sheep red cells by treatment v/lth 2% 
NaCl in the cold; the eluted substance vras then found to 
be heat stable. Furthermore, the eluted substance v/as 
found to agglutinate certain normal erythrocytes, indicating 



Serial No. NCI-261 
Calendar Year 195B 
Part A = Pane 10 



its non«>spcc£f icity. The pulnea pig hemolysin appears 
to be distinct from most other normal hemolysins tested 
because of its heat lability and its inability to clump 
the sheep red cells v.'hich, nevertheless, adsorb it. 
This substance may be similar or related to certain non- 
specific normal antibodies v/hlch function In the kilUnji 
of bacteria. 

b) There are a number of reports in the llter^ 
ature '-rhich describe the lysis of red cells foliowlncj 
surface alteration by treatment v/ith such unrelated ma° 
terials as metallic cations, salicylic acid and tannic 
acid. The lysis of these altered erythrocytes requires 
all the knovm components of complement. It has been 
established that red cells can be coated v/ith certain 
endotoxins, as demonstrated by their subsequent apgluti" 
nation in the presence of specific antiserum to the endo= 
toxin« It v/as of interest to determine if red cells 
coated v/ith endotoxins were rendered more susceptible 
to lysis as a result of the altered surface property. 

Human or sheep red cells vrere incubated i-'ith endo<= 
toxins and their adsorption of endotoxin ascertained by 
heraagolutinatlon in the presence of the appropriate spe^ 
clfic antiserum. The red cells v/ere then added to di!u<= 
tions of normal guinea pig serum {free of antibody to 
the endotoxins In question) and tested for hemolysis o 
There v/as a definite enhancement of lysis over that which 
occurred with normal erythrocytes. The four components 
of canplement v/ere required for this effect but neither 
properdin nor the endotoxin=>detoxlfylng component (EDC) 
v/ere essential. In order to determine v/hether this lytic 
activity of serum required a normal hemolysin (described 
above), the following experiment V;^s performed. Guinea 
pig serum v/as adsorbed v/lth normal sheep erythrocytes to 
remove the hemolysin. Although this adsorbed serum no 
longer lysed normal sheep red cells, the lysis of endo- 
toxin=coated cells vrais unaltered. Thus, in this system 
it appears that red cells sensitized vrlth endotoxin 
require only complement components for lysis, a system 
v.'hich is analogous to that of the lysis (by complement) 
of antibody-sensitized red cells. 



769 



Serial No. NCI --261 
Calendar Year 1958 
Part A = Paqe 11 



c) Erythrocytes of patients with the patho- 
loaic condition paroxysmal nocturnal hemoplobinuria (PNH) 
are lysed hy the subjects ^ own serum. It vi^js reasoned 
that the surface of red blood cells exposecf to trypsin 
might be modified in such a \-my as to make them susceptible 
to lysis in Isologous serum, thus simulating the lyt.lc 
phenomenon of PNH. 

The exposure of human erythrocytes to trypsin for 
periods of II4. to 20 hours at 37*' C. caused them to become 
very sensitive to lysis in Isologous serum. This lytic 
activity of the serum v/as found to be heat labile, requir- 
ing complement but not properdin. 

The lysis of PNH red cells vf&s first reported to 
require complement and properdin, although this finding 
has not been confirmed with respect to properdin. The 
fact that "purified" properdin has been shovm to contain 
significant amounts of serum Eysozyme suggested that 
lysozyme itself vj&s in part responsible for the lysis of 
trypsin! zed red cells. The addition of very small quan- 
tities of eggwhite lysozyme to human serum greatly auq=- 
mented the lysis of isologous trypsin-treated erythrocj^tes. 
These findings have been taken to Indicate that in the PNH 
condition, enzymic reactions may occur In vivo which uamssk 
or make available, sites on the red cells normally not 
accessible for interaction with senim componentSo It is 
also possible that some such mechanism is operative In the 
normal disposal, by the host, of aged red blood cells. 



Significance to the Pronram of the Cancer Insti- 
tute: Basic defense mechanisms of the host against var- 
ious types of injury ranging from shock to microbial and 
viral infection and, most recently, to tumor implantatiortj, 
are altered by the administration of endotoxin polysaccha^- 
rides. The finding and characterization of humoral factors 
which interact v/ith these polysaccharides and significantly 
alter their immunologic, toxic and physlcochemical proper- 
ties may provide a better basis for the ultimate understand" 
ing of the host response in cancer and in other pathologic 
states. 



770 



Serial Wo. NCI ==26 1 
Calendar Year 19^6 
Part A - Paae 12 



Proposed Course of Project: Recent findings in 
this Laboratory have sho^m that factors are present in 
blood "hlch can alter endotoxins in various 'Mys, Fup= 
ther study of the host factors involved in these complex 
interactions v/ill be made v/!th the follov/inq objectives: 

1) The separation of EDC from hunan plasma will 
be continued in close collaboration i/lth the Section of 
I^cromolccular Chemistry, LCP, 

2) As more of the extraneous serum proteins are 
eliminated in the course of fractionation, it will be^ 
come increasingly feasible to carry out experiments 
designed to ascertain the changes v/rought in the endo- 
toxin and, possibly, to attempt the isolation of the 
product of this interaction of endotoxin and EDC, 

3) In collaboration v/ith Dr. l.^ravdekar. Armed 
Forces Institute of Pathology, studies on EDC in mam- 
malian organs v/111 be continued. Initially, this v;ork 
v/ill be concerned v;ith attempts to obtain active tissue 
extracts free of particulate matter and to determine 
whether this factor in tissue has properties simOar to 
or different from that of blood. Inasmuch as liver and 
spleen are the organs largely responsible for clearance 
and sequestration of injected endotoxin. It is proposed 
to determine v/hether these organs constitute the princl» 
pal or sole tissue source of this activity. 

l|.) Further information will be sought on the 
properties of the serum ccxnponent involved in eliminating 
the Complement Fixing activity of endotoxin, and in the 
physicochemical alterations involved in the "clearing" 
and redistribution of endotoxin. It v/ill be especially 
Important to determine whether these reactions are se« 
quentlal, i„Co, '-hether one interaction serves to prepare 
the substrate (endotoxin) for further change by another 
serum component* 



771 



Serial NOo MCI»26l 
Calendar Year 19'^B 
Part A ■=- Pace 13 



5) It has been reported that v/ithin one or tv/o 
hours after the intravenous injection of bacterial endo^' 
toxin there appears in the serum a transferable pyronen 
v/ith properties distinciuishable frcan that of the sub- 
stance origirmlly injected. This substance is considered 
to be of endopenous origin, v/Ith the use of Immunol ocsical 
procedures it is proposed to examine the possibility that 
this "endogenous" pyrogen actueliy represents the origi° 
nally injected endotoxin ^rhich has been altered by virtue 
of Interaction v/ith various serum components v/hich v/e novr 
recognize as having such capabilities. 

6) Preliminary experiments indicate that serum of 
animals rendered "tolerant" to endotoxin has the unique 
property of markedly diminishing the pyrogenicity of 
endotoxin i-*ile leaving relatively unaffected its other 
host reactive attributes. In contrast, EDC has only 
limited effects on the pyrogenlc properties of endotoxins 
whereas it markedly eliminates the other host effects^ 

It is proposed to determine the properties of this agent 
and the conditions required for its alteration of endo- 
toxin in vitro. 



Part B included: Yes 



Serial Wo. NCI "26 1 
Pane Ik 

PHS " NIH 

Individual Project Report 

Calendar Year 19^3 

Part B; Honors, Ai-mrdSp and Publications 

Ptsblications other than abstracts frcsn this project i 

CD Skamess R. C, Rosen, F. S,, Shear, M, J., and 
Landy, M, ; Inactivation of endotoxin by a huraorai compo- 
nent, II. Interaction of endotoxin uith semm and plasma, 
Jo Exper. Med. 108, 68^^699 (1958). 

C2) Rosen, F. S., Skarnes, R, C, Landy, M« , and 
Shear, M. J»! Inactivation of endotoxin by a humoral coiti° 
ponent. III, Role of divalent cation and a diaiyzable 
ccsnponent, J. Exper. Med. 108, 70l»71l (1958). 

(3) Landy, M,, Trapani, R-J., and Shear, M. J,s 
Interference by endotoxic polysaccharides with inactiva- 
tion of coliphaoe by human serum. Brit. J. Exper. Patho 
39, (Dec. 1958) » In press« 



Honors and aiimrds relatinn to this projects 

The Head of the Polysaccharide Section vms invitedi 

1) By the International Association of Microbiolog- 
ical Societies to serve as a vice president in the Section 
on Immunology of the VII International Cdngress of Micro- 
biology held in Stockholm, Svreden, k'^9 August, 1958, In 
this capacity he organized ti;o sessions devoted to Natural 
Res i stance o 

2) By the International Society of Blood Transfusion 
to serve as a vice president in the Section on Iismunohema- 
tology of the VII Conpress of the Society held in Rome 
3«=»6 September, 1958. He also presented a paper (by imrl'^ 
tation), "Bioloqical activities of tv/o systems in normal 
blood: endotoxic polysaccharides in erythrocytes and an 
endotoxin<»inactivatinq component in sexnin", at a plenary 
session (Immunology), 



773 



Serial No« NCI'"262 

1. Labo Chemical 'Phnnmcology 

2„ Macromoiecitlar Chemistry Section 
3. Bethesda llj-s, Mao^land. 



PHS •» NIH 
Individual Project Report 
Calendar Year 1958 



Part A. 



Project Title ; Study of synthetic and natural 
raacromolecuies of bioloqieai interest, and of their 
interact lonse 



Principal Investioator? P, T. Mora. 



Other Investigators ? n. Landy, E, MerlsFs 
J. V/, Preiss, F. S, Rosen, J. W. Vood, B. G, Younq 
and M, J, Shear. 



Cooperating Units ; 

NCI Laboratory of Chemical Pharn^coiopys 
Polysaccharide Section. Serial No. 261. 

Dr, i-f, J. Df eyer J NHI, Laboratory of Celiu-' 
lar Physiology and Metabolism. 

Drs. So Farber» I, DJerassI and E. KSein, 
Children^® Cancer Research Foundation;, Bostonj Mass, 

Dr. H„ Cohen, Princeton Laboratories Inc^, 
Princeton, N. J, 



r^an Years (calendar year l9^S)t Patient Days; 

Totals 7-1/3 None 

Professional; if-'-l/S 
Other; 3 



Project Description ; 

Objectives; Study of synthetic and natural 
macromolecuies and their interaction, and the 



774 



Serial No» NCI»262 
Calendar Year 1958 
Part A - Pacfe 2 



alteration of certain biological activities such as 
ensyme activity, tumor^necrotisina and fever<»producinq 
activity, etc., throuah these m&cromolecnl&r interac- 
tions;. 

In inid<=year the Chemistry Section vrais reorpanized 
and the name channed from Orcanic to r%cromoiecular Chem'= 
Istry Section. The current experimental v/ork is In 
inacromoleeular chemistry, usina physicochemlcal methods 
developed in polymer chemistry in recent yearsj to study 
the structure of macromoiecules of biological interestp 
and of their interaction. 

Organization of matter in the macrcmolectaiar reqlon 
and the Interaction of nmcromoleculaf* components are of 
preat importance in fundamental bioionical phenomena s, 
such as in enzyme«-substrate and in ant iaen«±©nti body in- 
teractions in the mode of action of viruses, in the blo«= 
synthesis of macromoiecules throunh templates, etc. In 
ail of these phenomena, long ranoe electrostatic forces 
betv/een oppositely charaed macromoiecules appear to bring 
together the substances and initiate the blolociicai process, 
Our v;ork v/as directed tov^ard study Inp the nature of such 
macromolecular interaction between both synthetic chsroied 
polymers and natural substances, and then tov/ard the ap'^' 
plication of these studies to the biological objectives 
of this Laboratory. 



Methods Employed! Physicochemlcal and macr^iolecu- 
lar characterization methods (viscometry., osmometryj 
electrophoresis, chromatooraphy, sedimentation, etc.), 
polyinerlgatlon and organic chemical synthetic methods, 
enzyme assays, bacteriophage assay, assay for induced 
tumor necrosis and hemorrhage. 



215. 



1 ows : 



Serial No. NCI=262 
Calendar Year i95B 
Part A ■=■ Pacie 3 



or FIndlnps: These may be summarized as f( 



1) Synthetic polysaccharide derivatives v/ere em« 
ployed to study the effect of molecular parameters on 
the interaction of macromolecules. This work led to 
reversible inhibition of ntimeroiis enzymes, to detoxifi<° 
cation of certain macromolectiiar ccaapounds, to enhance-^ 
ment of certain hormonal activities in vjArOj, and to 
blockinq of the first stace of attack of T<=.2 bacterio- 
phage, 

2) The Imov/ledge qaSned frcm such macromolecular 
interaction studies v^as further extended to the bacter- 
ial polysaccharides v/hich had been investigated for a 
long time in this Laboratory. This led to the blockinc? 
of its fever-producing activity i/hile maintaining Its 
tumor<»necrotizln<5 potency, l/ork vfas started on attempts 
to understand and to control the tolerance which develops 
in tumors after one dose of such bacterial endotoxins. 
This I'trork v/as carried out in close collaboration i^ith 
Dr« Mc Jo Shear, 

3) ^^'e also collaborated with Dr« M, Landy and 

his associates In the Polysaccharide Section on the frsC'» 
tlonation and characterization of macroraolecular princl-^ 
pies from tissues and from blood* These principles 
appear to be important In host defense mechanisms. 

Details? 

1) Charrjcd derivatives of chemically synthesized 
polysaccharides (method reported in Publication #1 and 
^2) v/ere used to study the influence of molecular par&iii=> 
eters (such as molecular ^-'eiaht, dearee of branchsnOj 
nature and amount of charoed nroups introduced by sub«» 
stitution) on the chanqes in biological properties which 
occur during interaction yrith an oppositely charqed bio- 
logically active macromolecule« For example, a ncoatively» 
charged polyglucose derivative, such as polyglucose sulfate 
or polyglucose carboxyl, inhibits the action of a basic 



776 



Serial No, NCI-262 
Calendar Year 1953 
Part A <= Pacie k 



enzyme, such as lysozyme or ribonuclease, on its vreaker 
acid substrate, by selectively complexing ^'ith the en- 
zjnnc i/ith stronger electrostatic forces, '."hen another 
positlvely-charcied macrcayiolecule is added which is more 
basic than the enzyme (for example, protamine), this 
inhibition is reversed, since novr the protamine v/ill 
form the complex v/ith the acid polycflucose derivative, 
and the enzyme is liberated and resumes activity. 

The synthetic v/ork consisted of preparation of a 
series of polyglucose sulfates (J. V, l/ood) \>rith differ- 
ent molecular veir^hts, deorees of branchinn and denrees 
of substitution (reported in paper #3). Since the poly- 
glucose sulfates are stronrily anticoagulant (activity 
is in the order of clinical heparin), and this activity 
miaht render them too toxic for certain bioiogicaJ appli° 
cations, analogous series of v/eaker acid derivatives x-zere 
prepared, by introducing different amounts of carboxyl 
groups Into the polyglucose molecule (E, Merler and 
Po Maury) » 

The enzyme assays v/ere carried out v/ith B, G. 
Younpo The acid polyglucose derivatives inhibited Syso- 
zyne, rlbonuclease (both the depolymerizing activity, 
and also the action on a loi^ molecular i/eight substr®tej, 
nucleoside cyclic phosphate), hyaluronidase, cytochrome^c 
oxidase, etc. Protamine reversed the inhibition. Salt 
(NaCl) reversed the inhibition of iysozyme, but did not 
reverse the Inhibition of ribonuclease or of hyaluronic 
dase. 

In cooperative work, v/c provided several invest i«» 
gators v/ith the different acid polysaccharide deri^mtive3( 
and the follov/ing is the summary of the findings^ 

a) DTo Io Djerassi, Chiidren«s Cancer Pvesearch 
Foundation, Boston, r^ss., found inhibition of muscle 
phosphoiylase, but an enhancement of liver phosphorylase; 
also an increase in insulin activity in vivo v/ith one of 
the polyglucose sulfate derivatives* ~T!e found that the 



777 



Serial No. NCI -262 
Calendar Year 1958 
Part A == Paae 5 



effect of histamine in increasing the perraeabiUty of 
the capillaiy xi&ll of the peripheral circulatory system 
can be reversed by polyolucose sulfate. Dr. DJerassi 
is continuing to v;ork along these Hnesj, and is novj us=- 
ing poiyglucose carboxyl derivatives. Since it is pos- 
tulated that a similar increase in vascular pefmeabiiity 
may occur In the bleeding of acute leukeraiaj this line 
of work Is of mutual interest, 

b) Dr. H» Cohen, Princeton Laboratories Inc, 
Princeton, N. J., studied the Inhibition of trypsin and 
chemotrypsin by poiyglucose sulfates. He also studied 
ACTH activity Iti vivo , and demonstrated a four-^fold in- 
crease on simultaneous subcutaneous administration vrith 
poiyglucose sulfate. The increase in ACTH activity ''•^s 
even better (six-fold) with the poiyglucose carboxyl 
derivative, 

c) "Jr. R. D, Higglnbotham, University of Texas, 
Medical Branch, Galveston, Texas, found that polygUicose 
sulfate provides effective protection against the potent 
histarain releaser lt.8/80. His work brought to our atten° 
tion that toxicity of basic polypeptides, such as poly-^ 
rayxin-B, can be greatly reduced or eliminated by these 
acid polysaccharides. In our ovm experiments, lethal 
doses of polymyxin-B were given Intraperitoneal ly to 
mice, V'hen the animals v/ere in fin®,l stages of comml° 
sion. Just sufficient poiyglucose sulfate \-ms injected 
to neutralize the polymyxin's, and the animals rapidly 
recovered, VHtien poiyglucose sulfate v/as injected sub=> 
cutaneously at a site distant from the peritoneal cavity; 
and subsequently a lethal dose of polymyxin-B was admin- 
istered intraperitoneally, the lethal effect v/as also 
prevented , 

d) Dr, K, Klein Is studying, at the r4assachu= 
setts General Hospital, the absorption and the iipemJa 
clearing action of lov/ molecular v/eight poiyglucose 
sulfate, administered orally. 



SerJal No» NCI-262 
Calendar Year S958 
Part A «■ Pape 6 



In all these invest Stations v/e are interested in 
developing leads on hoi^ changes In the different macro»- 
mo2eaii®r parameters of the synthetic add polysaccha- 
rides affect various biological setivitlesp so that we 
may synthesize new products with increased potency and 
reduced toxicity. 

For the inhibition of natural ly^occurring acid 
macromoiecules a basic synthetic polysaccharide, poly- 
glucose ethylamine, t^s prepared (J. i-'. ^^ood), Scrae 
synthetic work v;as also carried out on a pilot basis to 
prepare condensation products of polygiacose v/ith knov/n 
drugs such as sulfanilamide, mercaptopurine, etc, for 
study of the effect of raacromolecuiar dimension on the 
action of the diMic?. The sulfanilamide derivative vjas 
submitted to Dr, D, P, Rail, NCI^ General Medicine 
Branchj for investigation of the permeability of the 
biood-=brain barrier. The condensation products v;ith 
drugs used in cancer chcmotheraiyy tmy lead to reduced 
toxicity or to a more gradual release over a longer 
time interval. 

Numerous nev; synthetic polysaccharides were pre- 
pared by our general method of polyeondensation of carbo« 
hydi^tesj polymers of ga lactose , ribosc, 2«deoxy ribose, 
2«=>deoxy glucose (submitted to Dr* J» LasziOj, NCI, Labo= 
ratory of Biochemistry^ for studies on the metabolism of 
human leukemic cells), etc « 

Another application of macromolecular interaction 
vras the blocking of the first, reversible stage of attach^ 
ment of T-2 bacteriophage to the cell v?an surface of 
Eo coli . This attachment is brought about by electrostatic 
"Forces betv;een the positively=charged proteins on the tail 
end of the phage and negative areas on the cell surface* 
Vith the help of Dr. ^''. Y. Dreyer,, I^IHI, L,C«P.?-i., who Is 
studying these basic proteins of bacteriophages, we 
(B. G, Young) set up a phage assay, and began to investi- 
gate the effect of these acid synthetic polysaccharides. 
Preliminary data indicate reversible blocking v/ith both 
polyglucose sulfate and carboxyl. This study on phage 



779 



4 



Serial Wo„ NC 1=^-262 
Calendar Year 1958 
Part A "» Page 7 



baocklng !s carried on in anticipation of developments 
in virus research on the etiology of cancer, and v/hen 
infonnation is available on the net charcie of sorae of 
these viruses, or v/hen they become more characterized 
and better isolated, collaboration v;lSl be arranged 
with interested investigators for study of possible 
blocking of these viruses on the same principle » 

A biophysicist. Dr. J. l.'« Preiss, Joined the 
^'!ac^omo2ecula^ Chemistry Section, to study the electro- 
static interaction forces betv/een raacranolecules. These 
are the only important operative long=range forces, and 
are instrumental in bringing together macromolecuSes, 
probably also orienting them in the interacting process 
so that they meet at complementajy sites* In these 
studies, uitracentrifugai sedimentation and light scat- 
tering methods are the major techniques to be employed « 

2) The interaction studies were extended to the 
complex endotoxic bacterial polysaccharide preparations 
from So marcescens « These polysaccharides have a vrhole 
array of biological properties, such as tumor necrosis j, 
f eve reproducing ability and other toxic properties s 
One major problem is that, after one application of the 
bacterial polysaccharide, a portion of the tumor sur- 
vives and becomes resistant to further applications of 
the endotoxic polysaccharide. 

There is no a priori reason why the same physico- 
chemical entity should be responsible for the different 
biological activities. Previous attempts to fractionate 
these activities t-rere unsuccessful, and \^hen hydrolytic 
or other drastic methods v/ere used to break down the 
endotoxic molecule and separate the activities by that 
fashion, usually the turaor-necrotislng activity disap- 
peared together v/ith the undesirable properties. It is 
possible, hov/ever, that the postulated different physi» 
cochemical entities responsible for the different bio= 
logical activities may interact to different degrees 



780 



Serial No. NCI -262 
Calendar Year 195S 
Part A - Page 8 



v/lth different nacromolecuies in the host. In that 
case, the changes in biological activities might not 
necessarily parallel each other. 

It vms knoT^ from the electrophoresis v/ork of 
others (Kahler, Malragren) that a polysaccharide prep- 
aration from S. marcescens {P'=-2^) had an overall nega«=» 
tive charge In ivater at pH 7. v/c Interacted this 
preparation in vjater v^ith different positively-charged 
(basic) macrranolecules and studied the changes in dif<» 
ferent biological activities, such as in ttjinor necrotic 
activity in mice bearing sarcoma 37 (B. G, Young) j, in 
fever-prodticing activity in rabbit and in development 
of tolerance tov/ard f eve reproducing activity (F, S, Rosen) 
The basic polypeptides, polynryxin=-B and ribonucleasej 
reduced the tumor necrotic effect of P°25, This inhi- 
bition v.fas reversed vith polyglucose sulfate and the 
effect returned to that of the original P-=-2S, The fever 
effect of P-25 vrns not influenced by these macromoleeular 
bases, 'Then, hov;ever, P'^ZS was incubated with lysosyme^ 
another basic protein, the tumor necrotic effect did not 
decrease, but slightly and consistently increased. At 
the same time, the fever effect rras reduced to one^tenth 
of the original. Also, the rabbits developed tolerance 
against the f eve reproducing effects of P-25 from Just 
one Injection of this mixture, v/hilc otherwise six or 
seven injections of P»'25 alone are needed to establish 
the same degree of tolerance to production of fevero 

Preliminary ultracentrifugal vork (with Dr„ H« 
Kahler, NCI, Laboratory of Physiology), and precipita^ 
tion studies, indicate that about 20^ of the P-25 prep- 
aration is a high molecular v/eight fraction, v/hich 
interacts preferentially with lysosyiae, forming an 
insoluble precipitate, and that this fraction may be 
responsible for the fever-producing activity^ Most of 
the material In P"»25 consists of a much lower moleculsr 
weight preparation (about a hundred thousand) which tmy 
be responsible for the turaor-necrotizing potency | this 



781 



Serial No. NCI«262 
Calendar Year 195B 
Part A •=> Page 9 



fraction apparently forms soluble c(^plexes \^ith lyso« 
gyrac without the loss of tiinior-»neGrot i 2 inq activity. 
The preliminary experiments are being extended so as to 
obtain definitive flndlnps. 

Such interaction experiments might lead to further 
fractionation of the biological effects of P-S^, and In 
conjunction v/ith the biological research in the Polysac- 
charide Section, might throv/ nevj light on the problem of 
tumor tolerance tov^rd repeated applications of ?-25» 

3) -fe collaborated x^'ith the Polysaccharide Sec=» 
tion (H. Landy, F. Rosen) in the fractionation of an 
endotoxin detoxifying component (EDC) from normal human 
serum, '/e have used a new fractionation technique by 
precipitating EDC with a polyglucose derivative^ v;ith 
encouraging results. Further fractionation experiments 
are in progress o 

Dr, E, Merler has studied the fractionation pro- 
cedures developed empirically in this Laboratory by 
A, Perrault for the isolation of endotoxic polysaccha- 
ride containing products from human and other mammalian 
tissueso As a consequence of this study, careful control 
experiments are in process (M. Jo Shear, K. Landy^ A, 
Perrault, n<=Je Trapanl) to exclude the possibility of 
incidental bacterial contamination during the preparation, 

Dr, Merler is also fractionating the stroma of 
human red cells, to study the occurrence and nature of 
the different macromolecular ccaaponents (proteins, poly- 
saccharides, lipids and their complexes) in an undeqraded 
state, and to find out what chemical or enzyiratic sepa» 
ration steps and v/hst changes in the macromolecular 
states are necessary to obtain preparations v/ith endo- 
toxic activity^ 



782 



Serial No. NCI-.262 
Calendar Year 
Part A =■ Page iO 



Significance to the program of the Cancer Instil 
ttite: Study of raacromolecuies and the forces in their 
interaction is designed to ansv/er, at the basic molecular 
levels, the basis of organization and function of livinc? 
matter* These studies of macromolecular interactions 
arc especially useful to a better understandinn and con^ 
trol of certain biological processes Important in cancer 
research, such as the elucidation of general principles 
in enzyme action, in non-specific immunological inter<= 
actions, in prevention of tolerance, and may also lead 
to the control of certain viruses. 

Proposed course of project? The macromolecular 
aspects of this work will be continued and broadened 
next years with emphasis on closer collaboration vrith 
the biological program of this Laboratory. 



Part B included: Yes 



783 



Serial No. NCI-262 
Paflc 11 

PHS » NIH 

Individual Project Report 

Calendar Year 1958 

Part Bs Honors, Av/ards, and Publications 

Publications other than abstracts from this projects 

CD Mora, P. T, and i-'ood, J. '-/.s Synthetic Polysac- 
charides. I, Poly condensation of glucose, J. Am. Chem, 
Soc, 80, 685-692 (1958). 

(2) Mora, P. T., T-'ood, J. H,, Mfeury, P., and Younci, 
B. G, s Synthetic Polysaccharides. II. Fractionation of 
Poiygiucose, J. Am, Chem. Soc. 80, 693°699 (1958), 

(3) Hood, J. w. and Mora, P. T.i Synthetic Polysac° 
charldes. III. Polyalucose Sulfates. J. Am. Chem. Soc- 
80, 3700»3702 (1958)1 

(l^.) Mora, P. T. and Young, B„ G. s Interaction between 
Synthetic PoSyglucose Sulphate and Biologically Active 
Basic Polypeptides and Proteins. Mature, l8S, li|.02-lij.03 
(1958). ~~ 

C5) Mora, P. T. and Young, B. G.s Reversible Inhi- 
bition of Ensymes by Interaction v/ith Synthetic Polysac- 
charide Macro-anions. Archives of Biochem. Biephys. 
In press. 



Honors and a^rards relating to this project? 
P. T. Mora v;as invltedj 

a) by Ueu York State University College of Forestry 
and Syracuse University to present a Graduate School Lec<° 
ture on chemical synthesis of polysaccharides. 

b) by the Naval Medical Research Institute to de- 
liver a Physical Biochemistry Seminar on enzyme inhibition, 

c) by the National Research Council Committee, 
Advisory to the Office of Ordnance Research, Department of 
the Array, to serve as a referee for grant applications. 



784 



National CaaR«r Snstitut* 

hJ 
BadoerJnolosr Branch 



Estimated Obligatioaso. 

Director >«,8o o8o«o^ 

R^linbursisfneiits 

Cliniealo » , » « « o o . « < 
Othero o a o o . o » . * ^ 



, o o , o o o , o o . o o o o o o o , » oPiseal Year 1959 

3ooo»03oo»».«.oa»c.oooB»$ 35T^700 

, o , . o » . , , o . , . . o c » o 48e,JK)0 

.0000000, .ac,,...«.ooo, 123,900 

TOtalooOOOOOOOOOOO.Oa. 96^,000 



ij Inesluies Pyojeets No'ss 



EHDOCRIHCI.O(H SERVICE? 



ai3 



8D3q 
d08e 



785 



Serial NOo NCI-SOOCC) 
lo Endocrinology Braneh 
2o Endocrinology Serviee 
PHS-NIH 3a Bethesdaj, M^ 
Individual Project Report 
Calendar Year 1958 



PgSlAo 



Project Title; Endocrine Aspects of the Progression and Therapy 
of Cancer of the Breast in Women and Men^ 

Priaeipal Imrestigators; Da Mo Bsrgenstala R^ Hertz and Mo Bo 
Lipsett 

Other Investigators: So Jacksonj To Lo Goodfriend^ Do A» Eelloggg 

Ro Ha Moyg S. Mo Kahn^ A. D. Goodmanj, Co Fo Nadler 

Cooperating Units NIHDB (Dts. J, Van Buren) 

Man Years (calendar year 1958) s Patient Days (calendar Year 1958)3 
Total! 15 patients 

Professionals 10 days 

Others 5 

Pro j est Descriptions 

Objective,?; 1» To extend and intensify detailed clinical observation of 
the natural com'se of sanser of the breast in men and wosaan in order 
to bettor ^praise the effectiveness of various Methods of therapy 
and learn more coneerning the etiology and pathogenesis of the disease o 

2 a To jjtprove i^on existing forES of honaonal therapy for palliation 
of advanced breast cancer and to elucidate the mechanlasas isvolvedo 

Mgthqds .Emloyeds 1. The acceptance of ©0B5>lete clinical rQsponsibi3.ity 
for the further management of referred patients vith proven diagnosis 
of cancer of th© breast and the application to their problsms of all 
available modes of tharapya 

2o The e3q)loration of new modes of hormonal therapy for paU.iationo 

3o The detailed analysis of endocrine and aietabolic factors operating 
in relation to the genesis and course of the disease in these patients 
\sp to the time of their deoiseo 

Ao Careful appraisal of patliological specimens obtained at biopsy 
or autopsy in collaboration with Pathology staffs 



786 



Serial NOo NCI-SOOCC) 
- 2 - Endocrinology Branch 

Maj^sJl^iSSas Metabolic Effect of tba Pituitary Horjaone„ Pgola@tin, 
These e^eriiaents wer® to deterasin© whether the pitsaitary honaon©j, 
prolactinj was capable of producing alterations in the growth of 
normal and malignant tissueo Sin©® this hormone plays an 3j5)ortant 
role in lactation^ it is of iinsportance to deteriain© whether it asight 
have some effsot t^on breast caneero Also this ia one of the horMonss 
that is removed wh©n a patient with breast cancer undergoes hypo- 
physectoisyo During th®s© studies ^ there was observed a protein 
anabolic effect of sh®®p prolactin in mano 

The preparation of hormone that was used was derived from sheep 
pituitary and x^as provided by the Endocrinology Study Section^ 
PHSp and had a potency of 20 international units per ragm^p as 
detensdned by the pigeon crop-sac assayo There was only slight 
contaadnation with ACTHj TSH^ FSH^ and LH« The reported conta- 
mination of shsffip growth hormone was between l/lO sM 1%^ This 
preparation was given 25 js^Eo; tid.o® daily^ subeutaneouslyg to 
patients on metabolic balance studies o 

The first groi^) of patients studied was made i:^) of 5 eases who have 
had previously hypophysectoray for metastatic carcinoma of the breast « 
All patients were either surgically castrated or postmenopausal a The 
horaaone was adadnistered for a 6«day period^ as described aboveo 
The urinary nitrogen excretion decreased from 1 to 4.-1/2 gssSo daily^ 
without significant change in f®cal nitrogen^ This effect took 1 
to 3 days to develop and remained 2 to 3 days after discontinuing 
the prolactin injections o The urinary aM.no acid nitrogen was 
elevatsd during the periods of psrolactin adminietrationo Potassitaa 
and phosphorus balsi'^sss were generally positive during pim'iods of 
positive nitrogen balance » In those cases with osteolytic lesions 
of th® bone^ there was no evidence of increased urinary calcium 
excretion nor was' '^here ax^ evidence of activation of the carcinoKao 
There was no evidence of lactation or increase in breast tiasue as 
measured by physical asffiuination., In order to rule out the possible 
effect of small asaounts of sheep growth hormone contaiainating the 
prolactin, a purified sheep growth horsaoi^ was given to one of the 
patients who had responded to prolaotino At a dose of 10 tijses 
maximuia contaasinating asaount^ nitrogen balance and urinary amino 
acid nitrogen were unchanged © 

In the second group of patients receiving prolactin^ there was on® 
case of a zaale with choriocarcinoiaa who did not respond to the honaoae 
and a female patient with chorioeaycintaeag who had had a previous 
pituitary stalk section and was laetatingj, and she also did not 

respond to th© drugs, A lO-year-old achondroplastic dwarf who had 
failed to show any positive nitrogen balance^ with the administration 
of human growth horirsonej, showed a positive nitrogen balance whcsn given 
sheep prolaotino A 'femal© patient with Cushiiig's Syrdromej ssaondary 
to bilateral byperplasiag showed an anabolic effect of approximately 
l-"l/2 gmso of nitrogen pw day when given this hormon® pr©paratlon„ 

787 



Serial Ho« NCI-SOOCC) 
- 3 - Endocrinology Branch 



MSS-IisdiagsJcoaM^ 



Thus it would appear that the patients with an intact pituitarj csn 
respond to shsep prolactin with a positive anabolic effeeto Lastatioa 
did not occur in any of these patient So There was no change in the 
urinary excretions of 17-l<etost9roids or 17-hydrosyeortieoids through" 
out the studies a 

These ©^©rissssnts demonstrate that a fraction of sheep pitid-tary which 
is active in stimulating the pigeon crop-sac^ can cause nitrogen re- 
tention in man^ Thusj, the species specificity in relation to a 
protein anabolic response that is noted in the action of growth hornnone 
in man has not been observed for this pituitary fraetione 

Reeant work has indicated tlsit this pituitary preparation wslj have 
3 distinct coiaponentso At the present timej, our work is being 
directed toward the separation of these cosgsonents to determin© 

whether ti© protein anabolic effect aay be separated from the prolactin 
effecto The nature of this anabolic effect is as yet unelearo From 
our animal studies in which we have coapared th© effects of prolactin 
with beef growth hormone and husaan growth hormone in stimulating the 
vgjtake of radioactive sulphur in the cartilage of hypopt-^sectiimissd 
ratSj the results to date would indicate that it requires approxiffiately 
10 times as much prolactin to stissnd.at® th© uptake of radioactive 
sulphur into cartilage in ooE^arison to the growth hormone preparations o 
A preliisdnary study in hypopbysoctomiaed rats has been done in which 
there are four grosrosj 1) controli 2) growth hormone? 3) prolactini 
4) sheep growth bormoneo Each groi^) has been injected with its rss- 
pective horswjne for a period of 1 months during \rfiich tiase body weight 
and increases in sise of tailg skullp and liiabs were deteradnsdo The 
growth honaoxm has the characteristic growth prcmoting effect that is 
well knowno The prolactin at a dose level of 100 mica'ograsas a day,, 
wMch is cojsparable to per kilo dose used in maxig had no ©ffeet t^on 
the weight gain or body growth of the I^ophysectondzed ratso Thei^© 
was no evidenoa of any stiisulation of tJ^oidj adrenal,, or ovaries o 
Thusg it Hiay be that the rat does not respond or is relatively resistant 
to the protein anabolic effect of this pituitary prepajretions 

Growth Hor,ffiQn^.« We are continuing our work on the metabolic effects 
of purified growth horsaone preparations In mane We have cossipared 
the metabolic effects of a purified growth horxaone preparation pro'- 
duced from monkey pituitaries to that prepared from human pituitasdes 
and find that the anabolic effect is essentially the sameo Growth 
honaone has been prepared from human pituitary glands by th® Bsthod 
described by C^ Ho Lig which involves an alkaline extraction and th© 
Eaterials injected in an alkaline pH of 8o5 and the ajethod dascribed 
by Mo Raben in which the groirth horaione is extracted at an acid pH 
and injected at an acid pHo A eoHgsarison of th© qssbb amounts of e&Qh. 



788 



Serial No^ NCI^-SOOCC) 
- 4 - Endocrinology Branch 



^iogjlndingsjcont^dj 



of thes© horsaones in the sams individual showed that the anabolic 

effect of these preparations was essentially tbs sai!Je» W© have 
had the opportunity of testing purified aheep groirth hormone and 
purified whale growth hormone. Neither of these substanoss was 
effective in producing an anabolic effect in mang yet both of th^se 
preparations were as active as beef growth hormone in stimulating 
th© Intake of radioactive sulphur in the cartilage of the Ig^pophyses- 
toHdsed rat. Since th© molecular weight of beef growth hormone is 
approximately 4B5<XI0 and that of human growth hormone is 27g000g it 
has been postulated that if one could degrade the beef growth honaoa© 
to a lower molecular weightj the material might be effective in maoo 
Beef growth hormone cao be treated with chymotrypsin and the molecular 
weight substantially decreased^ The isolated material that is 
obtained has been «!'all®d by Dr,, Co Ho Li the «»alphE coreo'^ This 
material has approsmaately 75^ of th© activity of the original beef 
growth hox^oae when tested in lie rate We have tested purified 
preparations of the "alpha core" in patients at dose levels from 
10 to 20 m^s a day, with an injection period of 4 to 6 dayso 
We have studied 5 separate metabolic balance cases ;, and in no 
instance have we noted any anabolic effect as measm*©d by n5.trogea 
or phosphorus retentiono 

Thus 5 in our hands j, this material does not seem to be effective 
in 3Eanc, All of the patients who have responded to human growth 
hormone have shown an increase in calcium excretion in the uria© 
during the period of hormone administrations This was not obs^ved 
during the adadnistration of ths chipiiotrypslnized beef growth horaonso 
The childrenj, under study who showed a very slight response to the 
human growth hormone^ showed the characteristic increase in urinary 
calcium seen in the adults receiving the hormone o 

A patient with pituitary adenoma^ who had moderately sever© h5?po» 
pituitarismp was given growth hormone without replacesaent and showmi 
the characteristic protein anabolic responseo The patient was then 
replaced with 30 mgrn, of hydrocortisone g 75 mgmo of triiodotl^onineg 
and after returning to her baseline j, ah® iras again given the same 
amount of human growth hormone from the same lot and again ahmmtd a 
protein anabolic response j, which was essentially the same as befor© 
replacement therapy was instituted,, ThuSg it would seem that this 
effect of purified growth hormone can be observed in patients who 
are relatively severely l^othyroid and hypoadrenalp and adsquate 
replacaaaent therapy did not increase its effectiveness aa an anabolic 
agent o 

The case of Caahing^a Syndrome and the patient with panhypopituitarism 
receiving human growth horHsone were given aa ACTH test before and 
during the last day of the administration of tfeg growth horEon©o 
There were no significant differences in response to ACTH in thes© 
2 patients before aM after tte admlniatratioa of gro^fth horasoneo 

789 



• *e 



Serial Noo NCI-SOOfC) 
- 5 - Endoerinology Branch 



Magoi: Fiqctiqgs (cofltodj 



Human growth honaon© has been adadnistered to a groi^ of patients 
\trith neoplastio diseases in an attesspt to determine whether the 
disease waa in any way altered by this growth prosaoting stibstaraeso 
Three eases of careinoma of th® breast war© given growth honaone for 
a period of 6 days. In no instance was there any isarksd increase in 
urinary calciiaa excretion? and in only one patient was there any 
siibjectiv© increase in bone paino Three patients with metastatic 
cancer of the adrenal cortex have been given human growth horEK>n© 
for a period of 6 daysa None showed an increase in the excretion 
of urinary steroid horsaoneSo One patient was on a metabolic balance 
study and showed the characteristic anabolic response to growth 
hormones, A patient with Gushing "s Syndrcmej secondary to bilateral 
h^erplasia of tb© adrenals^ showed a smrked anabolic nitrogen res» 
ponse to 5 rngfn^ of growth hormone a day for a 6«-day period a A 
patient with n^tastatic carcinoaa of the prostate with a signifieant 
elevation of aeid phosphatase was given growth horsaonej, 5 lagaio ®, 
dayg for a period of 12 dayso During this period of timeg there 
was no increase in bone pain^ and the acid phosphatase dropped 
slowlyo There was a laarked increase in aeid asainopolysaccharide 
eaKsretion in th© urinso These initial studies of the effect of 
growth honaone on patients with neoplasti© diseases have been^of 
neeessityg limited because of th© ssiaall simply of growth hormone o 
However J, as larger si^jplies become available^ patients will b© 
given larger doses for longer periods of time in order to detenaiae 
whether signifieant alteration of the disease can be produced „ 

DTo Ho Sngel has aaade a study of the effect of intravenous injection 
of growth hormone to normal and hypophysectoaiaed patients and 
determined the unesterified fatty acid concentration in tl® blood 
at intervals \3p to 6 hours following the injection of the drug^ 
Changes in a-amino acid nitrogen have also been followed ^ When 
cospared to the control e^^eriment in whieh saline is injected^ there 
is a markedly significant elevation of unesterified fatty acids by 
6 hours after th© injection of growth hpriEonao The average incraa©© 
over the control for the unesterified fatty acids is approximately 
300%a Amino acids show a small but consistent decrease during tho 
6 hour periodo Thus^ these studies indicate that growth honaone 
can act rather rapidly upon the fat of the body and mobilise the 
unesterified fatty acids as an iasaedlate energy source^. This test 
could be used to determine the activity of various human growt.h 
hormone preparations a 

Five children below the age of 14,^ with intact pituitary function 
as measured by th^oid and adrenal responaeSj, have been given human 
growth hormone o Only 3 have shoira a very slight response to high 
doses of human growth hormone^ that is 10 mgaio a day for a period of 
4 to 16 daySo This is in contrast to the high degree of s©nsitivj.ty 
shown by th© adult to much saaller doses of th© drugo 



790 



Serial NOo NCI--800(C) 
- 6 - Endocrinology Braaeh 



^gjogj^qd^ngs (oontMj 



Pituitary Gonadotr ppig, ¥e have extended our studios on th® adminis" 
tration of huaian PSH to presaenopausal wosnen with metastatie carcinoma 
of the breast who are to undergo castration aa therapy for their 
diseasea In 2 recent cases^ we have observed multiple follicular 
<53rst developisent folloving th© administration of 6 aiga» of husuan 
FSH a day for 6 dayso Both of these patients were on ifiatabolic 
balance studies; and during the administration of the drug, there 
was observed no alteration in nitrogen, phosphorus j, calcium, or 
electrolytes o There was a rise in pregnaaediol during the adminis- 
tration of the horffion©8 At th© present time, we have a sssall supply 
of a highly purified ICSH preparationj and our future work will b® 
directed toward an atteuspt to stimulate the ovaries with FSH and 
then to give IGSH intravenously and to detensine if we can produce 
multiple ovulations o Our future work is also directed toward a 
study of the blood half life and the excretion pattern in the urine 
of our purified hmaan PSH and our purified ICSH preparations when 
administered to patients who have undergone hypophysectoEy and hsva 
no circulating gonadotropins 

^i£gB3LoOafl?fy. 9f the ^raaat in Men and Womenp Ablative EMocrino 
Sur^jery^ 

Castratioa a W® have continued to perfora bilateral oophorectosay 
in premenopausal woiKsn with metastatic carcinoma cf the breast o 
We have observed an objective regression in approsdiaately IS% of 
the patients so treatedo Our shortest resaission has been 3 moathSp 
and the longest resdssion has now been over 2 years » This represents 
an evaluate of 15 patients « A nuiiber of these patients have served 
as subjects for studies of purified huaan gonadotropic honaone adminis- 
tration. These results are described under the section of metabolic. 
effects of pituitary hormones o 

Hypophysectamva We have continued our program of hypophysectos^ 
in collaboration with DTo Ja Van Buren of the NeurologJ.ij3al S©rvic®o 
In the present groiqp of 22 cases of carcinoma of the breast that w© 
are evaluating, 3 are too early to determine whether an obj®©tivs 
regression will be obtained, thus leaving 19 cases o Twelve of thaaa 
oases have shoTm significant objective regression of their disease, 
7 have failed^ The duration of resaission has varied frcsn as short 
as 2 months to as long as 12 months ^ 

At the present time, in collaboration with DTo Van Buren, we are 
evaluating 13 of our early l:OTophyseet«By cases who have come to 
autopsyo The sella turcica is removed, and by subsequent serial 
sectioning and making reconstructive models of the sella taircica, 
Dro Van Buren 1ms been able to determine the percentage of pituitary 
tissue r<OTaining in the sella turcicao In none of the eases was tlir© 



791 



Serial No^ NCI-800(C) 
- 7 ~ EndocT'inology Branch 



Ma,^or Findings (cont'd.) 



a coB5)l©t® hypopbysectoHiy, The residual pituitary volumes remaining 
in the jnajority of the cases was very small, approxiaaately 1 to 10$a 
There was no evidence of regeneration of this pituitary tissu©<> On 
stainingg the cells appeared to be essentially normals Differential 
coll counts showed tMs to be sisiilar to that found in norsaal pituitary 
glands 6 In 7 of the cases^ cancer cells were found in the sella 
turcicao la only 1 case in which there was approsdmately half of the 
pituitary remaining was there any evidence of returning adrenalg 
thyroid 9 or gonadotropin function follovd.ng the h^ophysectoH^ even 
at the tiise of relapse of th© diseases The pharyngeal pituitary 
gland was found in 6 of 9 cases in which it was sought « All but one 
specimen contained alpha cells as well as chromophobe cells | beta 
cells were distinctly unusual and iijere seen in only one patient o All 
of the pharyngeal pituitary glands were of ssaall size and fell xdthin 
the lower liiaits of normal j as established by other investigators o 
The total of all 3 laeasuri^tents of the pharyngeal pituitary fell 
bet^^en Oa5 and 8,1 miUiBieterSo. Due to its small voljiae and 
apparent absence of secondary hypertrophy^ it is difficult to 
ascribej, at least in the present laaterialj naich functional significance 
to the pharyngeal pituitary glands o 

At the present time^ we plan to do 20 cases of pituitary stalk section 
with the insertion of a small metal plate betx^een the h^othalMBUS 
end dura sellas In this mannerg we are able to interrt^t the hypo- 
thalasnic pituitary port^ system and to determine whether this will 
be sufficient to produce pituitary insufficiency and objective re- 
gression of metastatic breast cancer o At the present time^ we have 
done ^ such cases and have seen 2 objective regressionso W© have 
evidence that the thyroid^ adrenal, and gonadotropin functions are 
reduced to the point that the patient vdll develop evidences of 
deficiency if the maintenance hormone is removed o Of interest has 
been the fact that the rathsr severe diabetes insipidus that was 
observed within 12 to 48 hours after total hypophysectoEj has not 
been apparent in these patients and that frequently it is soma days 
before the patient will undergo a diuresis » This is probably 
dependent j^on the fact timt a certain ansount of posterior pituitary 
hormone is still available in the posterior lobe^ We have observed 
no cases of lactation following pituitary stalk sectiono 

Total l^ophysectOBjy has also been perfonaed in 2 cases of female 
choriocarcinoma without any significant alteration in the levels 
of urinary gonadotropin a One male with choriocarcinoma also had a 
total hypophysectofflyj and following this there was no significant 
alteration in the levels of urinary gonadotropin nor in the growth of 
the tumor o Further attespta will be made to do hypophysectwasy in an 
earlier period in th© course of choriocaj'ciaoBia once it has becose 
resistant to methotrexate o 



792 



Serial No„ NCI-aooCc) 
- 8 - Endocrinology Brsmeh 



Ma^-jor Findings Cc?oat°dJ 



Teatololactonea Preliminary studies have been made with testololactonej, 
a steroid which in one metabolic balance study when given at a dos© 
of 50 mgmo a day,, produced no evidence of nitrogen retention^ The 
cois5>ouad has been given to 2 patients with metastatic carcinoma of 
th© breast having hypercalciuriao In both cases^ there was a de- 
craas© in the urinary calcium toward normal with relief of bone pain^ 
One patient has received the coi^ound at a dose level of 50 asgSo 
intramuscularly 3 times a week for a perioti of 2 months o There haa 
b@6n no evidence of androgen aide effects in this individual » This 
cOTpound will be of particular interest if there is observed a 
significant anti-tumor effect without androgen side effects and 
without other metabolic effects that are observed with testosteroneg 
such as nitrogen retention and sodium and water retentionj, Our 
future work will be directed toward studying suitable cases who do 
not fit into our l^pophysectoay program to detenain© whether the 
compound can produce significant objective regressions for a prolonged 
period of timeo 

Pl^v35,ologv of some Cfaj^-deoxysterdida. The metabolic effects of a 
potent C2i~deoxysteroid have been described « The effects of various 
functional grot^s in increasing the glucocorticoid potency of ttese 
steroids has been stt^iedo 

Derivatives of progesterone were found to increase the Port©r-Silb©r 
chromogeas in tte urinso This urinary steroid has been studied and 
partially characterisedo Metabolic studies with thsse steroids 
have been performeda 

S%gf^^±ea^c® to th^ Program of the Institute; The direct pertinence of 
the foregoing studies to the grave problem of advanced or persistent 
breast cancer is too obvious to require discussiona 

Proposed Course of Pro.iect s The further course of these studies ar® to b® 
essentially along the lines outlined above » En^hasis is to be placed 
on balance studies and the eva3.uation of hypophysectomy in breast 
cancers Further evaluation of new steroids and related coi^ounds is 
to be madeo 

During calendar year 1958, 28 new cases of female careiaoBsa of tl^ 
bffasist WM"© admitted to our service as in~ and outpatients Three 
male cases of carcinoma of the breast were also studiedo 



Part B indudedc 



793 



Serial No„ NGI-800(C) 
PHS-NIH Endocrinology Branch 

Individual Project Report 
Calendar Year 1958 

Part B; Honors^ Awards, and Publications » 

Is Engelj Ho R.j Hallman^ Lo F,^ S^elp S. and Bergenstalg Do MoS 
The Effect of Growth Hormone on Plasma Unesterified Fatty Acid 
Levels of I^ophysectomized RatSa Proc« Soco Exper^ Biol„ & 
Med« 98s 753-755^ 1958o 

2, Lipsettg Mo B„ and Bergenstal^ Do MoS The Metabolic and AGTH 
St^pressing Activity of Certain C2i~deoxysteroid8c Acta Endo- 
crinologioa (in press) e 

3, Lipsetts Ms B^ and Bergenstaly Do MoS A C21-d®035ysteroid with 
Corticoid Activityo Jo Clino Endocrinol« & Metabo ISj 790^ 1958o 

Sfenors an d Awards selc^tti^ to this proiect s 

^K% D» M„ B^r^^ostal - 

Presented lecture on the Effect of Ejypophysectoiny on the Course 

of Metastatic Breast Caneer at Walter Reed Hospital^ Washingtonj, 

Do Ce 

Lecture on Chaiotherapy of Cancer,, presented to the Dietetic 

Association of the Tri-State Hospital Association, Washing-ton^ 

De, C, 

Participated as panel sneaber on Therapy of Breast Cancer at annual 
meetings of the American College of Physician^, 

Appointed meraber of CoiimBittee on Cancer of the American College of 
Physicians a 

Dr« ,M„ B. Lip^ett - 

Diplomate Amos? lean Board of Internal Medicine o 



794 



Serial No, NCI-803CC) 
lo Endocriaology Braiich 
2o Endocrinology Service 
PHS-NIH 3» Bethesdaj Mdo 



Individual Psroject Report 
Calendnr Year 1958 



Part A, 



Project Title; The Nature of Hormone-Producing Tumors of Pituitarjp 
Adrenal, Ovarj^ Testis, Pancreas, Parathyroid and Chorionj 
Abnormalities of Somatic Development and Grotrtho 

Principal Investigators? Rs Hertz, D„ Mo Bergenstal, Me B, Lipsett 

Other Investigators: Ao D* Goodman, Co Fo Nadler, R» Ho Moyp 
So Mo Kahn, T, Lo Goodfriend, Do A, Kellogg, F, Go Dhyss^ 
So Siegel, Ho L, Erwin 

Cooperating Units: NIAMD (DTo Robert Bates and Dro H, Wilson) 

Man Years (calendar year 1958): Patient Days (calendar year 
Totals 14. patimts 

Professional: 10 days 

Other: 4 

Project Description: 

Ob;|lectivQ3; lo To learn more about the basic metabolic phencanena 
involved in the process whereby neoplasms in hormone-produeiag 
organs produce highly excessive amounts of hormone, resulting in 
such syndromes as Gushing ^s Disease, Acroi&egalyj, I^erparathyroidismj, 
Ifyperadrenalism, and similar endocrinopathieso 

2a To maintain in tissue culture aM in animals honaone-producing 
tissues and to ascertain the factors involved in quantitative honson© 
production in vitro » 

3o To assess the capacity of new drugs to alter the course Bund 
hormonal activity of hormone-producing tumorso 

Methods Emaloyed: Ifcrmone producing tumors frcra patients with one of the 
above-named syndromes are obtained at surgery and carried in tissue 
culture in irradiated, cortisonized rats and hamsters o The supernatant 
fluid frtm the cultures are tested for hormonal activity by appropriate 
bioassay methods and the quantity recovered coapared with the content 
of a piece of tissue exactly equivalent to that originally plaeed in 
Cloture o 

Patients presenting such syndromes are cos^>letely characterized endo- 
crinologically and metabolically before and after therapy in order to 
aaeortain the relative effects of various forms of therapyo 



795 



« 2 - Serial No., NCI-803(C) 

Endocrinology Branch 

Ma.^or Findings ; At the present timeg we have under study the metabolic 
and steroid hormone stimulating properties of chorionic gonadotropin 
obtained from the urine of patients with choriocarcinoma and patients 
during pregnancyo Our studies are directed to determine whether there 
is any difference in ths ability of these gonadotropins to stimulate 
the testis of normal males as well as the testis of patients with 
metastatic chorioearcinomao The present 3 patients under study 
represent 3 different endocrine situations? a patient with metastatic 
choriocarcinoma J a patient with hypogonadism secondary to pituitary 
tiBEorj and a normal 35-year-old male with metastatic breast cancer o 
Each of these patients will reseive 4D»000 units a day for a period 
of 5 days of both tumor gonadotropin and pregnancy gonadotropino 
Evidence of stimulation will be a significant rise in the 17-keto- 
stsroid level; and in Case No a 3p who is on a metabolic balance 
study, a significant nitrogen anabolic effect should result „ 

Adrenal Cortical Cancer r, 

Our recent studies have been directed to the investigation of an isomer 
of DDDj that is 2,2-bi8-Cortho-ehloro, parachlorOy diphenyl)-(lj,l) 
diehloro-»ethane (oj,p" DDD)o This ooapound has been shown by Jo Ho Uo 
Brown to be the substance in tbs crude mixture of DDD which produces 
adrenal atropl^ in the dogo He has also reported that this will 
produce adrenal atropl^ in man when given at dose levels of 10 gmso 
a dayo This compound is obtained by recrystallizing it from a crude 
mixture of DDD and has a melting point of 77°Go It is exceedingly 
fat soluble and our preliminary investigation would indicate that the 
majority of the coa^jound is retained in the bodyo Sine© this eoa^jound 
seams to be effective in producing adrenal atrophy in the dog and to 
decrease ths adrenal response to ACTH in normal man,, it appeared 
logical to try this coa^jound in high doses in patients sufforiag from 
inoperable adrenal cancero The compound is made t^ in Go 5 ig?&o capsules j, 
administered by mouth, taken as near at meal-time as possible^ since 
there is some indication that the coi^onnd is better absorbed wh@n 
there is fatty material associated with ito Our present method of 
therapy is to start the patient on 2 gmSo a day, and, as rapidly as 
possible, to increase the dose to 10 gms. a dayo This dose is 
continued until the patient has shown a toxic effect of tha drug 
or until the steroid hormones have reached levels of nonaal or 
below normal end the patient seems to be suffering from adrenal 
insufficiency^ 

In the earlier phases of our studies, the availability of the drug 
was such that it was in^ossible to give patients an adequate dose 
levelo Our first 3 cases did not show a satisfactory responseo Cases 
1 and 2 showed no changes in their adrenal steroids on low dose thsrapyj 
while Case 3 was treated with a somewhat higher dose of the drug and 
did show a significant drop in her steroid hormones j, however p they 
did not reach normal levels and tiie metastatic disease of the lungs 
continued until tl^ patient died of respiratory embarrassMsnt „ 



796 



- 3 - Serial No^ NCI-.803CC) 

Endocrinology Braneh 

At ths present tim©, w® have been able to secure adequate asaouats 
of the drug aM ha^e sinder therapy 6 cases of adrenal carQlnon&a 
On© case J who has received the drug for a period of approxiiaately 
3 XBonthSj 10 gmso a day^ had urinary 17-ketosteroids of 175 mgjfloj, 
which have dropped now to less than 5 mgnio Her 17-hydroxycorticoids 
in the urine have dropped from levels of 25 sagao to less than 3 asgaio 
The patient has shown a significant objective regression of her 
metastatic lesions ia the chest® She is now menstruating after 
having had amenorrhsa for one yearo After 3 jEonths of therapy, 
the patient developed signs and 35B5)toais of drug tosieityj which 
was re^Qcted in increased somnolence, nauseag voDsitingg trssorg 
and the drug was discontinued « Despite the fact that the drug has 
now been discontinued for xBore than 6 weeks p the patient has continued 
to show i^roveaent in her chest lesions and her steroids er® ressaining 
at very low levels o The remaining cases are still in ths early phases 
of therapyi however. Gas® AoS, has shown a drop of urinary 17-keto-. 
steroids frfflo 175 ngaio to 15 apio and a decrease of the Mass in his 
abdomen,, After receiving approximately 250 gmsa of drugg he de- 
veloped a skin rash and the drug was discontinuedo The patient 
has been off the drug for approximately 3 weeks j the steroids atHl 
remain at the same level as during therapyo Case Go Ho is a 26-«ionth- 
old child who was started on tbsrapy in May 1957^ Since that tiEiSp 
the child has been on doses of between 1 aad 1-1/2 gmSe of drugo 
On numerous occasions, it has been necessary to discontinue the drug 
because of laild skin rasho Despite thisj, the urinary 17-ketosteroids 
have dropped from levels of 200 mgmo to 15 m^iio aM below. The patient 
has continued to grow^ appears to be in good haalthj, and the ssass in 
the right i^per quadrant does not seem to be increasing in siseo 

Case. So was a female with far-advanced Gushing "s Syndrome^ secondary 
to metastatic adrenal cancer who had diabetes and hypertension as 
^rell as the other classical findings of Gushing 's SyndrraaQo She 
was treated with Ogp" DDD and during the first 3 weeks of therapy^ 
her insulin requirement went to zero and her diabetes significantly 
iaprovedo The patient developed a severe hOToglysamic episode and 
began to becojae more lethargic^ aoa-responsivej, andj, finally^ lapsed 
into a coma and died of what appeared to be a respiratory failure o 
Her 17-katosteroids and 17-l^droxycorticoidSj, which had been markedly 
©levatedj, had dropped significantly during this period of admiaistra™ 
tion of the drugs At autopsy j> an obvious cause of the death could 
not be found; however j, a heart blood culture showed Eo colio The 
possibility exists that this patient showed evidence of central 
nex^vous system toxicity to the drugo The other patients are still 
under therapy and all have shown significant decrease in their elevated 
steroids during adaiaistration of the drugo However j, objectlT® ©videnes 
for regression of metastatic lesions has as yet not been observedo 

Our future work will be a continuation of our att^^ts at therapy 
of adrenal carcinoma and the development of methods for the deterBd.- 
nation of the drug in bloody tarine^ feces j, and body tissues o The 
preseat plans are to giv® patients Oj,p" DDD for a period of on® to 

2 w©©3£3 before bilateral adrsaaleetosy for metastatic earciaoEa of 
the breast in an att^spt to detesMne ths effect of this ecs^ound on 

797 



Sarial NOo NCI-803CC) 
- 4 - Endocrinology Branch 



IMSL^msdinEsi^StMJ 



normal adrenal glandSo Ther® also exj.sts the possibility that a 
more affective total adreaalQctoMy might be produced by this ©Oiiipound 
sinc®j if there is a significant amotmt of accessory adrenal tissue 
present in the body and this is not removed at the time of bilateral 
adrenalectoa^y^ it slight senr® as a source of continued estrogen or 
steroid homaon® formationo 

Tgstica|a|-. Tupprs. 

The beneficial effects of methotrexate therapy that have been seen 
in female choriocarcinoma have not been observed in cases of testieular 
tumors in mano W© have thus attsi^jted to study the effect of othsr 
coH^jounds which potentially might alter the growth of this timer o 
On® such QOB^ound is the dipotassium endosuccinate salt of methyl- 
eholanthrena. This ©i^n^sound has been given to patients with testieular 
ttsfBors who have elevated chorionic gonadotropin in their urine « The 
doses at a level of 200 jagma a day intravenously for a S-day period 
have thus far shown no alteration in the course of the production, of 
gonadotropin or grovrth of the lesiono We have also used a suspension 
of HKSthylcholanthrene in doses ranging from 100 to 800 mpSo a day 
intraMUseularly for a period of 5 to 7 days a In a series of 6 such 
coursesg w© ha.v® not observed any significant effect cqpoa the urinary 
gonadotropin nor in the growth of the tuseoro No evidence of toxicity 
has been noted to ©itJsr drug^ Our future plans are to study other 
chesaotherapeutic agents^ such as 2-Deo3^gluQOs© in eoaabioatioa vdth 
various chemotherapeutic agents « 

Gi^S^^^inalogues^ 

At the present time^ the investigation of the metabolic and anti- 
tumor effects of 2-Deoxyglueo3® is being made with particular re- 
., f ©rence to the effect vspon breast caneor and methotrexate-resistant 
ehoriocarcinomao This coapound has been shown to inhibit the growth 
of escperiasental animal tuasoi's and to inhibit glycolysis in leukoBjie 
cells 9 The c£«iipound has a metabolic blocking action on the utilisation 
of glucose J however g the exact sit® at \iM,ch this blocking action 
takes place isg as yetg unknown^ 

Landau et alo have given sin^e large doses of 2-Deo3syglucos® • to 
humans and obsearved aa initial h^erglye^aiag hypothermia^ and In 
2 cases of leukesmia a decrease in the white county Our investigations 
have been directed toward the continuous intravenous administratioa of 
2~0eox7glucose at levels which will produce clinical sys^jtoms and the 
administration continued for a period of 3 to 5 days a We have observed 
th© initial hypotberaiaj, soamolencej, increase in j^petit© alternating 
with occasional nauseag and hypeyglycesaia that does not s^pear to b® 
persistent despite the continuation of the drug at levels which ^d.11 
produce clinical toxic effect So It is hoped that some preferential 
interference with the carbohydratfa utilization by tumor sells will 
occur during the adisinistration of this ccfflpoundo This cojEpound 
has been administered as described above to one female with metastatic 
car.t;is,os2a of th3 ba'east vfith b^jpsT'ealcsHia and bypsrealsnria, Ot?. 2 

798 



Serial NOo NCI"803(C) 
- 5 - Endocrinology Branch 



Ma„|oy Findings IsoatMJ 



occasions p the elevated urinary caleium of levels of 500 to 700 
nigma were returned to near normal levels and the blood calcium to 
norsaal levels, When the drug was discontinued^ the urinary calcium 
again returned to h^ercaleiuric levelso It was again si^jpressed 
by anothsr additional course of the drugc Wlien this was discontinusdj 
the urinary calciusa again returned to elevated levels o Two females 
with far-advaneed methotrexate-resistant choriocarcinoffla have been 
given the coa^oimdj, and -^er© s^pears to have been a decrease in the 
urinary gonadotropin in both of these patients o The patients were 
not well enough to receive further prolonged courses of the drugo 
Two male patients with testicular tumor containing choriocarcinosaa 
were treated^ and there was no alteration in the urinary gonadotropin 
observed© One patient with metastatic functioning adrenal carcinoiEa 
was treated on metabolic balance study for a 5-day period^, and there 
was no change noted in the secretion of adrenal cortical steroids « 
However J the drug did produce a rather markedp negative nitrogen 
balance during the course of adainistrationo Our future studies will 
be directed toward the clinical evaluation of this coapound in a 
larger series of patients with choriocarcinoma and carcinoma of the 
breasts Also studies will be made in an attea^t to detersdne snore 
specifically where the sit© of the metabolic block is and to detersaine 
whether therapy with 2-D©oayglucose plus other types of eh®EOth©rapy 
may b® more effective than either agent alone <> 

StMlmJ^a-SSMQi d MQtaboliam o 

lo Characterization of steroid excretion patterns in adrenal 
carcinomao. 

The uniformly high excretion of tetrabydro-S in those patients with 
elevated Porter-Silber chrosaogens has been describedo Column and 
paper chromatograpl^ are now being used to delineate the excretion 
patterns of deoxy- and oay-letostsroids as well as tha laos"® polar 
fractions o 

2a Ketosteroids in HirsutiSMo 

A study of the excretion of "adrenal androgens" in patients with 
hirsutism as well as a coa|)arable control grovip is nearing cos^letione 
Contrary to recent papers^ it appears that even hirsutism of a severe 
degree can be found in the presence of nonaal adrenal steroids o 

3o Adrenal Function in Ovarian Agenesis «, 

It was suspected that th® coamonly reported low ketosteroid values 
in ovarian agenesis were due to an abnormality of production of 

"adrenal androgens o" These steroids are being saeasm-ed and. the 
effect of ACTH is being evaluatedo 



799 



Serial No„ MCI»803(C) 
- 6 - Endocrinology Branch 



Ma|o^J[iadings„ ( cent «d, ) 



4g The initial stages of stimulation of the adrenal cortex with 
ACTHg in collaboration with DTo H^ Wilson^ NIAMDo 

The purpose of this study was two-fold: (a) to exaadne the urinary 
steroids of the Bj^saphyseetosdsed patient who was not receiving r©- 
plaesEient therapyj' and (b) to sequentially analyze the products of 
2 pathways of steroid biosynthesis during the initial stages of 
stiaiulation with ACTHo 

5s Precursors of dehydroepiandrosteronej, in collaboration with 
DTo Ho Wilson^ NIAMDo 

Cholesterol 4G2-4. has been gi^en to patients with adrenal carcinomao 
The possible precursors of DHIA are being isolated in an attsss^t 
to demonstrate a precursor-product relationship » 

Ifetabolic Studies. 

lo A-aaethopterine 

Balance studies with patients with chorioeareinoma have been obtained 
during A-^^thopterin therapy„ Over-all changes were aiiniEal and these 
results probably represent the first report of the effects of a folie 
acid antagonist on normal tissue. 

Glucagon has been shown to exert a significant catabolic effect in 
a variety of settingSo This effect was not due to activation of 
the adrenal cortex and saay not have been secondary to hepatic glyco- 
genolysiSo 

Studies on the chesaotherapy of choriocarcinoma and related tropho- 
blastic tumors have now included observation on 34. cases o Ths 
findings are essentially as reported in Jo Ao Mo A^^ October iSj, 1958a 

Significance to the Program of the Institateg Further understanding of the 
conditions permitting the excessive hormonal production of tumors of 
endocrine origin is basic to our understanding of the Metabolism of 
neoplastic tissue generally «, In addition^ further analysis of abnonaaiitie 
in somatic growth and development should provide data pertiasnt to the 
problems of tissue growth generallyo 

Proposed , Course of Prp.ject? Further elaboration of these studies ^th 

major aaphasis on biochesdcal and pharmacological control of hormone- 
producing tumors and anomalous tissue growth is projectedo 

Part B included o 



800 



Serial No, NCI-£ 
PHS-NIH Endocrinology Branch 
Individual Project Report 
Calendar Year 1958 

Part B i. Honors 5 Awards, and Publications » 

1« Landaup Bo R.j, LaszlOj, J^^ Stengle, Jo and Burk, DoS Certain 
Metabolic and Phanaacologic Effects in Cancer Patients Given 
Infusions of 2-"D0oxy-I>-Glucose« Jo Nat. Cancer Insto 21; 485- 
494, 1958„ 

2a LandaUp Bo R.j, Levine^ Ho J^, and HertZp R,x Prolonged Glucagon 
Administration in a Case of ^yperinaulinism due to Disseadnatod 
Islet Cell Gaa-cinomao No Engo J. M0d„ 259: 286-288^ 1958o 

3.. Lipisettj, M, B« and Daiaastp B»s The Excretion of Pregn&n-3a, 17aj, 
21-triol'»20-one (Tetrahydro S) by Patients with Adrenocortical 
Careinomao ProCo Soc, Exper^ Biolo & Med^ (Dec,, 1958) „ 

4o Lipsettp Mo Bo and Bergenstalj, D, M,: Metabolic Effects of A- 
methopterin in Man, Canoor ReSo (in press) o 

5o LandaUp B» Ro and LubSj, Ho AoS Animal Responses to 2»DBOxy- 
D-Glucos® Administrationo Proco SoCo Expero Biolo & Mode, 99s 
124-127, 19580 

60 HertZj, R«, Bergonstal, Do M»p Lipsett^, M, Bo 5 Pricey E<, Bo and 
Hilbish, To Foi Chiamotherapy of Choriocareinoma and Related 
Trophoblastic Tumors in Womeno J,A,M„Ao 168! 846-854;, 1958o 

7o LubSp H„ Ao, VilaTj 0. and Bergenstalj Do MoS Familial Male 
Pseudohermaphroditism vdth Labial Testes and Feminizations 
Endocrine Studies and Genetic Aspects „ Je, Clino Endocrinol „ 
& Metabo (in press) » 

So Li, M, C, Nixon, Wo Eo and Freeman, Mo V,; Increase of Urinary 
Ci trover urn Factor Activity in Patients Receiving Methotrexate 
(A-ffliethopterin)„ Proc. Soc. Exper^ Biolo & Medo 97; 29-32, 1958o 

9o Levine, Ho J* and Glenner, G„ GoS Observations on Tryptophan 
Staining of the Pancreatic A3.plia GellSo Jo Nato Cancer Insto 
20: 63-68, 1958, 

lOo Li, Mo Co, Hertz, Ro and Bergenstal, Do MoX Therapy of Chorio- 
carcinoma and Related Trophoblastic Tumors with Folic Acid and 
Purine Antagonists» No Eng„ Jo Med„ 259: 66-74, 1958o 

llo Schatton, Wo Eo, Bergenstal, D, Moj, Kramer, Wo Mo, Swarm, Ro Lo 
and Siegel, So: Biological Survival and Growth of Cartilage 
GraftSo Jo Plastic & Reconstructive Surgo 22s 11-28, 3.958o 



801 



Serial Noo KCI-803(C) 
- 2 - Endocrinology Branch 



Pay,ij,,S^,„(eoflt«d^), 



12. Schattonp W. E,^ Bergenstalg D» Mo, Kramer^ ¥«> Mo and Wexlerj, 
HoS SurrLval of Skin HMjografts in K^ophysectoadaed and 
I^othyroid Rats^ Jo Plastic & Reconstructive Surg* 21s 
20-23, 1953, 

13a _. ,.,...„■,. ? Tumor Transplantation and Skin Hoisografting 

in ifypophysQctomized and hypothyroid RatSo Trans^ Bull„ 5s 47-»4.9« 
- 1958o 

Hoi^or^ ^^latfin^ to this„pyq.ie^ts 

Invit®d to participate in Cancer Synposium^ Indiana University 
Msdical Gentsrj, IndianapoliSj, Indianaa 

Invited to Lecture at th© Ualversity of lUiaois College of 
Medisineg Chicago^ Illinois o 

Participated in meetings of the iteerican College of Surgeons ^ 
Chicago p Illinois a 

Presented seminar at Worcester Foundation for E^eriiaental Biolo^j, 
Shrewsbury^ Mass,, 

Participated in conference on Trophoblasts and its Tumors j, lew 
York Acaden<y of ScienceSj, New York, 

The Endocrinology Branch presented the results of its research 
prograia to the following: 

1, The American Association of Obstetricians and Gynecologists o 
2e Members of the North American Society of Gynecologist 3 ^ 



802 



Serial No., NCI~S08(C) 
lo Endocrinology Branch 
2o Endocrinology Service 
PHS-NIH 3o Bethesdaj Mdo 

Individual Project Report 
Calendar Year 1958 



Psgt A., 



Project Titles Endocrine Factors Gov^erning Hormono-Indueed Tissue 
Growth aM Tuaor Formation in Animals? Bioassay of Blood and 
Urine from Cancer Patients 

Principal Investigator? Dr^ Roy Hertz 

Other Investigators: Dr^ Wa Wo TuUnerj DeTs Ao Do Goodisang 

Dffo Ra Reisfeldj MTo Ho L^ Srwin 

Man Years (calendar year 1958)? Patient Days (calendar y®ar 195S)s 
Totals 9 . patients 

Professionals 5 days 

Others A 

Project Descriptions 

GhJ^g%±y&8t lo To gain basic infonaatlon concerning endocrinological 
and nutritional factors governing hormonally conditioned growth and 
involution in organs such as the uterus^ breast aM prostat© which 
are the frequent seat of hormone-sensitive neoplasms o 

2o To develop and ia^rov© bioassay methods for the quantitation 
of hormones in the blood and urine of patients with cancer t^ho 
ar® either receiving various forms of hormonal therapy or who present 
unusual hormonal patterns « 

3o To detenaine the biological properties of steroid and related 
confounds in terms of their potential usefulness in cancer therapy o 

Methods, . Baployed s Standard methods of biological study are ssgiloyedo 
These includes 

lo The laaintenance of animals of various species on specified 
horaional or nutritional regimens o 

2o Daily observations of physical aM behavioral changes in such 
anijmalSo 

3o Gosiplete autopsy of test animals with detailed weighing and 
chetEdcal analysis and bistochemical study of specific organs « 

4.0 Surgi<3al alteration of the endocrine status of aniasals by 
gojfflad0cto]^p adrenalectosigrp Iroophysectorayj, thyroidectosnjj, pasa- 

biosiSj, lTyst©r©ctowy and other operative procedures o 



ao3 



Serial Noo NCI-808(C) 
- 2 - Endocrinology Branch 

Ma.lo£_J'indli] ig8? (a) A major effort has been made to ©atablish a 
variety of human hormone-producing tumors in the cortisonized- 
irradiated hamster or rate 

We now have 3 strains of female human choriocarcinoma ©stablisheKi 
in "the hamster and 2 in the ratp Their growth is still not quit© 
sufficiently predictable to permit standardization for quantitative 
study. However J it is slear that these tumor strains are productive 
of abundant chorionic gonadotropic hormone and in one instance of 
prolactin as veil* These tumor strains idLll prove of major ia^jortanee 
for ch@!2otherapeutic studies as well as for studies on the biosynthesis 
of chorionic goaadotropino 

Additional attsai^ts with adrenal carcinoma and with hydatid mole 
to propagate a heterologously transplanted strain have met with 
coiii^leta failures 

(b) A ctsgjrehensive survey of the biological actions- of a wide 
variety of steroids has led to the finding that certain 17-8piro- 
lactones have marked progestational activity (&«=tz and Tullnerj, 
Proca Soco Espera Biol« & Medog Nov« 1958) „ This finiing defies 
all prior presussiptions as to the structural requirements for pro- 
gestational activitjo In the monkey^ these confounds produce a 
sustained progestational effect of extreme degree for as long as 
3 aonthSg whereas such prolonged administration of progsstsroae 
itself leads to ultimate endometrial atrophyo Furiiier studies are 
in progress to determine the ultimate carcinogenic potential of 
these new steroids for ihe primate endometriuma 

(c) Further observations on the effect of prolonged but intermittent 
administration of 19-norethisteron© on the monkey endometrium and 
ovary indicate that such intermittent treatment by allowing for 
periodic menstrual sloughing of the stimiilated endometrium sustains 
the endometrium in an essentially normal state o This is in marked 
contrast with the previously described metaplastic changes resulting 
frcsn continuous administration. Hence the proposed use of such 
agents on an intermittent basis for the si^jpression of ovulation 

in women may prove cos^atible with normal endometrial functiono 

(d) Further study of the nature of the congenital abnormality of 
the urogenital syst^n in the male AXC rat has revealed the occurence 
of an interstitial cell ttmor of the testis in an abnormally de» 
veloped testis g thus simulating the clinical finding of tumor in 
©ct«>pic testes of meno Attes^ts to set off the tomorigenic process hj 
removal of the good testis and by placing the abnormal sale in para- 
biosis with a castrate male have not yielded any tumors thus faro 



804 



Serial NOo NCI-808(C) 
- 3 -> Endocrinology Branch 



mM^i^MMMJss^^^) 



(©) studies on the hypothalaiaie dependency of the anterior 
pituitary have been pursued with the aid of pituitary grafts to 
the kidney in hypopl^ectomiaed ratSe Somatic growth continue® 
in such grafted aniiaals indicating quite clearly that growth 
hormone production is not hypothalaiaic dependent o In additiong 
such grafts may b® shown to b© producing small amounts of gonado- 
tropin^ thyrotropinj, and adrenotropin by the positive response of 
such rats to chorionic gonadotropin and to aniphenoneo ThuSj, pituitary- 
d^ondence vpon hypothalamic stimuli is clearly seen to be a quanti- 
tative rather than an absolute phenomenon as is commonly thoughts, 
These observations have direct bearing on the proposed control of 
breast cancer by pituitary stalk section as outlined in project 
800(C) o 

(f) A detailed study of all non-fertile animals sent over by the 
Animal Production Section has revealed 2 very interesting abnorsaalitiesc 
In th® Panda hamster both males and females show the same type of 
abnor^salities seen in the AXG rata These includes (1) atrophy of 

one or both uterine horns g (2) atrophy of one testicle associated 
with absent vas deferens and kidney on the involved sideo Studies 
paralleling those carried out in the AXG rat are now in progress o 

In the BRSUOT mousej sterility is associated %fith huge cystic 
ovarieso Moreover j, these animala are frequently extremely obeseo 
The syndrome simulates the Stein-Leventhal picture presented hj 
obese j, sterile g, hirsute women with polycystic ovaries o Detailed 
endocrinological study of these interesting animals are in progress c 

(g) Tte currently available methods for bioassay of gonadotropin 
in human blood and urine are laborious and quantitatively uasatis- 
factoryo The development of statistically reliable a©thods of 
assay and recovery is progressing slowly and only as th© heavy burden 
of routine clinical analyses permits However ;, some progress in 
eliminating procedural and biological pit-falls has been madeo 

pj.9]po?g^ Qq^^T^® Q^i P,y,P^?.°]^' ^"^ ^^ ejected to continue and ©sctead the 
foregoing studies along the lines already indicatedo 



Part B included o 



805 



Serial Noo NCI"808(C) 
PHS-NIH Endocrinology Branch 

Individual Project Report 
Calendar Year 1958 

Part Be Honorsg Awards^, and Publications 

la YilaTj, Oo and Hertz, R«: The Postnatal Histogenesis and 

Endocrine Function of Abnoraial Testes Associated with Urinaary 
Tract Anomaly in the AXG Rato Froco Soca Exper„ Biole & Med^ 
98s 553-557, 1958» 

2a Hertz J R« and TullneTj, Wo WoS Progestational Activity of Certain 
St@roid-17-Spirolaeton©So Prooo Soce ExpoTo Biolo & Med^ 99s 
(Nov.) 1958« 

3» Landaug B^ R«e Shipj Ao Go and LevinSj Ho JoS Action of 
Insulin &n Distribution of Glucose Analogs in Eviscerated 
Kephreetoaiiaed Dogs, Am, Jo Physiolo 193j 46lg 1958^ 

4.0 Hertz J R»: Some Biological and Clinical Effects of Steroidal 
Proca of Tf Biochemo Congress^ PergaEon PresSj 1958o 

Honors and Awards relating to this projects 

1« Invited to lecture at Jefferson Medical Gollegej Philadelphiaj, Pa,, 

2 9 Lectured at Tuskegee Institute (Jo Aa Andrews Clinical Society) j, 
Tuskegee^ Alabama^ 

30 Participated in panel of the Amsrican College of Obstetrics and 
Gynecology J, Los Angeles j Calif or niao 

Ao Panel member j, annual meetings of Aaerican College of PhysicianSj, 
Atlantic City, N» Je 

5, Participated in 4th International SyB^osium on Biocheaistry of 
SteroidSp Vienna, AustriSo 

6o Participated in joint meetings of the Endocrinology and Gynecological 
Societies of Stockholmp Swedeno 



806 



Serial No, NCI-810 
lo Endocrinology Branch 
PHS-NIH 2o Endocrinology Service 

Individual Project Report 3o Bethesda,, Mdo 
Calendar Year 1958 



^dLl. 



Project Titles Alteration of Endocrine Function of Animals by 
Pharjoacological and Physiological Means » 

Principal Investigator: Dr, William W, Tullnsr 

Other Investigators s Dr^ Roy Hertz^ tea Sa Jackson 

Man Years (calendar year 1958): 
Total; 3 
Professional; 1 
Other: 2 

Project Description: 

Ob;|ei3tive3; 1, To evaluate selected synthetic cos^jounds for the ability 
to alter endocrine secretions in order to e:^lor© the potential 
usefulness of such coa^ounds in the control of endocrine tumors o 
To iaprovQ biological and bioch®ssical methods of steroid detenainatioflo 

2 a To investigate the Efflchanism of action of otMs^ounds whieh inhibit 
steroidogenesis es^loying quantitative aM qualitative Hsthode for 
detanaination of alterations in the intermediat® and final products 
of biosynthesis^ To determine the requirements of structural specificity 
and biological response in the mediation of progestational action of 
selected diphet^l con^Gunds by the adrenal glands « 

3o To induce adrenal cortical tianors in laboratory animals by surgical 
and/or chemical means for the purpose of studying steroid secretions 
of such tumors and methods for regulating tumor growtho 

Mg^hodi3_^3[losgiit Standard chaaical methods were used for deteriaination of 
steroid hormones » 

Honeal haaltl^ animals are used in both chronic and acute esqjeriments 
vith diet determined by the nature of the esperiaento Test compounds 
vere adiainistered either orally or parenterallyo 

Surgical procedures included adrenal vein cannulationj hypophysectaB^^ 
ovariectomy and edrenal©ctomy« 

Standard techniques were enployed for histology and histochesiical 
preparations o 



807 



- 2 - Serial No^ HCI-810 

Endocrinology Branch 

Ma^jo p Find ings; 1« Th© following findings are baasd on studies with 
Ogp" DDD or 2y2»bis (2-Ghlorophenyl-4-cM.orophenyl)-ljl-diQbloro« 
ethane, an isomer of technical DDDo 

(A) In dogs receiving oral treatment with o^p" DDD for periods 
of Ij, 2 9 4 and 6 days there was a marked reduction in the output of 
17-hydroxycorticosteroid3o After 2 days treatment with the drugj, 17- 
hydrosyeorticosteroid output was reduced to the low level found in ^e 
acutely hypopl^seotcssiised dogo Twenty four hours after a single dose 
of drugj tl^ rate of steroid secretion was reduced to 25 per cent of 
aosimLe Histological observations on the adrenal cortex showed that 
focal areas of degeneration developed first in the zona reticularis s, 
later in the zona fasciculatas No changes were seen in the sona 
glojaerulosae After 6 days treatsaenty 3-P hydroxy sterols were almost 
absent from the inner zones of the cortex although the glomerulos& was 
aoswala Distribution of sudanophilic lipids in the retioalaris aM 
fascieid^ta r^aained unaltered during the period in which the digitonides 
were ssarkedly decreased in these zoneso 

In these short-term ejjperimentSp the digitonin reaction was a sensitive 
indicator of altered adrenal function and paralleled reduced 17-i^droxy- 
eorticosteroid secretion* However g steroid secretion was markedly 
diminisl^d after treatment which appeared to have minimal effects on 
cortical morphology. (0» Vilar and We W^ Tullners Effects of o^p" 
DDD on Histology and 17-Ifydro:55ycortieosteroid Output of the Dog 
Adrenal Cortex^ submitted to NIH Editorial Board) o 

(B) In dogs receiving chronic oral treatment with Opp" DDD for 
periods t^ to 6 weeks thar® was a marked atrophy of the fasciculata 

and glom^ulosao These zones were s'educed to a iiarrow band of condensed 
collagenous tissue between the medulla and the glomerulosa (which re-° 
tained its structure) » Sudanophilic lipids had almost coapletely 
disappeared ftom the inner cor 'ileal zones » Secretion of 17-hydroxy'- 
corticoids was in the range found after hj^ophysectoay of normal dogSo 
There was no evidence of disturbed ©l^trolyt© balancop alteration 
of blood pressure or other a^mptcms of adrenal insufficiency^ These 
findings confirm our previous studies with technical DDDo 

(C) Following the identidfication of o,pB DDD as the active isomer 
of technical DDD, by Cueto and Brown, further investigations j in which 
the commercial product v&s used, were termlnatedo Studies on regeneration 
of adrenal cortical tissue after chronic treatment with DDD are being 
continued idth the o,p9 derivativso Regeneration was shown to occur 
from the glomerulosa inward toward the medullao R®su!!5>tion of steroid 
synthesis followed cortical regeneratiouo (Wo Wo Tullner^ Alteration 

of Adrenal Corticosteroid Secretion in the DDD-treated Dogp in preparation) , 



808 



- 3 - Serial KOo KGI-810 

Endocrinology Branch 



Ma.1or Findings (contMj 



id) In rats receiving Qhronie treatment with Ojp« DDD and 
OjP" DDD plus an^henonsj, it was found that anphenone exerted its 
typical effects of adrenal hypertrophy with lipoid accumulation 
even in the presence of o^p" DDDo The latter ooapound when given 
alone, had no effect on the size or histology of the rat adrenal 
cortex. This further essphasizes the species specificity of Oyp" DDDo 

2o A series of diaaiinodiarylalkanes was studied for toxicity | 
effects on the output of IT-tgrdroxycortieoMteroids in dog adrenal 
vein blood and progestational effects in the rabbito Those ceai^ounds 
which caused a decrease in secretion of glucocorticoids were effective 
only at toxic levels of the drugs except for 292-bi8 (p-aiainophenyl) 
propanSo 

In the rabbitg those eospounda which bad the diphenyl group attached 
to the sasae carbon atoa were progestational as has been shown for 
aBphenonsq How^ver^ none of these conpounda possessed the ketone 
group as does aa^jhenoaeo ThuSj, from these preliminary findingSj, the 
ketone groining is not necessary for the altered output of adrenal 
progestogens o 

In addition^ it was found that a pyridyl analog of aaphenone exhibited 
progestational action in the rabbito In studies reported by other 
laboratories it has been shown that this eoapound is a specific inhibitor 
of the enaymatic system concerned with ll-oxygenation in the adrenals 
of dog and asano In the rabbity such an inhibitory effect would prevent 
the formation of lip-hydroxy progesterone from progesterone or the lip 
hydrosylation of ll-deo^csorti©osterone to form corticosteroneo This 
could lead to accumulation of progesterone in amounts sufficient to 
produce the characteristic endometrial changes » 

In :'*urtber studies on mediation of the progestational action of an^henone 
by the rabbit adrenal, a method of collecting rabbit adrenal vein blood 
was devalopsda It was found that chronic aa^shenone treat^nt markedly 
decreases the secretion rate of eorticosterone by the rabbit adrenal o 
This indicates a metabolic block involving either Up or 21 hydrozylationp 
the reactions involved in the alternate pathways for the synthesis of 
corticosterone frcan progesteronoo 

In view of the demonstrated role of the rabbit adreAal in the progesta^ 
tional response to aB^henonej> the probleia of the uterotrophie response 
to amphenone in the rat was reinvestigatado SM'ies of castrate female 
rats were divided into adrenalectomized and non-adrenalectoaized grot^Sa 
Half of each groisp was treated with emphenonea The uterotrophie res- 
ponse to aaphenone occurred even in the absence of the adrenals o Th©re 
was no significant difference in the uterine weight increase nor, in 
uterine bistolo^ either in the presence or absence of the adrenalSo 
The direct action of asaphenoae on the uterus of the hypophyseetomised 
rat was si so confirmed „ 



809 



"A - Serial Io„ NGI~810 

EMocrinolo^ Branch 

!feJog Findia^s ^(confcU) 

3o! Erucie acidp a fatty add found in rapeseed oilp has been reported 
to produce a marked elevation of adrenal cholesterol in the rata la a 
series of rats receiving erucie acid chronically as a dietary suppl@@ent 
it was found that the secretion of corticosterone by the rat adrenal did 
not differ from the normal » Moreover, dogs on a similar erucie acid 
regimen showed no alteration in output of 17-hydrosycorticosteroids 
although there was naarksd elevation of digitonin-precipitable sterols 
in adrenal cortexo 

Significai; tci ^ to Cancer Research ; Control of endocrine cancer by coufiiouiids 
with a high degree of specificity for endocrine tissue and ens^e 

systoMS is of Eiajor ia^ortancQo An understanding of the site end 
Eschaniss of action of these coa?)ounds is basic to further application 
of a cheaaical rationale to cancer thBrspjo 

Proposed Course qf P2;p^1®cit;S Continuation of these studies is expected in 
accordance ^th the outline which follows: 

As Acute and chronic studies vith o^p" DDD and isoaserso 

lo One of the most noteworthy aspects of th® action of o^p" DDD on 
the dog adrenal is th© resistance of the sona glomerulosa to dassage 
in presence of almost casplet® destruction of th© sona reticularis 
aM fasciculatao For this reason^ w© propose to investigate the foUow- 
ings 

(a) The effect of this drug on the secretion rate of aldosterone 
in dog adrenal vein blood (a series of plaaea sai^les from treated 
and control aniaals is being collected currently) » W© have 
extensive indirect evidence {nosmsl electrolyte balancseg heayato" 
critp ©tCo) that there is no significant change in mineralocorti-" 
coid secretion but direct proof SEUst be obtainedo C<S!^letion 

of this study depends on facilities for aldosterone assay which 
are expected to be available shortly^ 

(b) First J the effects of simultaneously adsainistered deoxycorti*^ 
costeron© and o^p" DDD to detensiiine whether the gloai©rulosa can 
be daaaaged during disease atrophy^ 

We hev© previously detersnined that regeneration of the fascieulata 
and reticularis proceeds ceatripetally froa the glomerulosa« There 
is a possibility that sufficient depression of th® secretosy capacity 
of the latter sons can also prevent regeneration of the inner cortical 
zones from the glomerulosa when drug treatment has ceased » Such a 
possibility has potential value since j, currently 5 drug administration 
must bs continued to ©sart inhibitory effect So This second aspect 
would b® studied by intensive treatment with o^p" DDD followed hj DOG 
treatment o At the end of treaifflsent with o^p" DDDj, one adrenal would 
b® r®30V©dj, and th© second adrenal would b© taken at autop^ 2 to 4. 



810 



- 5 -» Serial No<, HCI-810 

EMocrinology Branch 

Proposed Coiarse of Project (eont"dJ 

weeks after the beginning of treatmeat with IX3C (ther© is sluiost 
complete regeneration of the f ascieulata in <4 weeks on DDD with 
return to normal secretory function) » 

(c) Th© effects of alterations in dietary potassium on the 
gloi^ralosa of th© o^p" DDD treated dog (proposed by Dr^ Berts) » 
Brown has reported lesions of the glomeralosa and early death 

in dogs receiving DDD as little as 3 weeks » We cannot confirs, tbiSo 

A high dietary eontent of potassimn jaight alter th© adrenal response 
to the drug resulting in damage to th© glomerulosao This study 

will require preparation of synthetic diets of known potassium 
contenta 

(d) The adrenal of the pi^ (dog) does not possess the clearly 
defined zones seen in the adult o, Th© developsjental stag© of 
rapid growth and organization of the adrenal cortex laay be a 
factor in th® response to Ojp" DDD so that drug damage to the 
glomerulosa laay occur at an early age^ This probleai is currently 
underway with pi^s and adult dogs being given equivalent amounts 
of the drug on a body weight basis » 

Sines© certain adrenal tsmors ar® of the minarelocortieoid'^produQing 
t^09 it would be an advantage to inhibit this type of endocrine growth 
funetiono 

In addition to chemical steroid detersainationsg careful obsarvation 

of escporimental aniiaals and preparation of synthetic dietSj, histological 

assistance is necessary for these studies » 

2o The mechanism of action of o^p" DDD on th© cells of the adrenal 
cortex is obscure o Esperiments from this laboratory and otters 
indicate that secretion or release of ACTH by the anterior pituita^ 
is not a factor in the degeneration of the fasciculata and reticularis 
after drug treatment „ 

We ar® currently working on a method for intravenous adiainistration of 
Ogp" DDD in an ®@ulsion to be used in acute esqperiraents on the dog,. 
The drug will be introduced into the art®rial circulation ©lose to th© 
adrenal glando Chesiaical measuresaents of steroid content of tte adrenal 
venous blood will be Made at intervals to evaluate effects in relation 
to tisaao Such an acuta method will permit rapid screening of other 
analogs of DDD (we have several available iK»w)a, 

M acute test method also offers a aseans to test for reversibility 
of the effects of Opp" DDDo Among the metabolites which we expect 
to examine for reversal of steroid inhibition are; (a) Cholest^-'ol 
because ofs its prim© position as a prscursor of adrenal steroids j 



811 



- 6 - Serial Noo NGI-»aiO 

Endocrinol©^ Branch 

Pyoposed Course of Pgo.ieet (eont"dJ 

evidence frosn this laboratory showing a marked depletion of adreno- 
cortical cholesterol (3P-bydroaysterols) shortly after the initiation 
of drug treatment? the inhibitory effect of the drug on 17-keto- 
steroid output (in fesoale dogs) indicating an effect in the early 
stages of biosynthesis; and the lack of effect of ACTH on biogenesis 
of adrenal steroids even after short periods of drug treatment (sine® 
ACTH is believed to exert its catalytic actions by reducing the C-27 
ccmpound to a C-21 type) ; (b) Pantothenic acid - because of (1) the 
deaionstrated effect of pantothenic acid deficiency on steroid biogenesis 
by th© rat adrenal ^ (2) the role of this vitasdn as a part of ooenzym® 
A which is a key aietabolite in lipid Bsetabolissie 

Finaily^ it is proposed to continue to evaluate selected syntheti© 
compounds for their potential usefulness as adrenocortical inhibitors 
as these coiqpounds becesee availableo 

Ba The investigation of diaodnodiarylalkanes is ejected to 
continue^ The pharmacological actions of these coopounds are being 
studied in rabbits and dogSa Several menibers of the series of reagent 
quality are available and are to be screened for adrenal inhibitory 
and progestational activity after preliminary toxicity studies o 

The probl^a of the adrenal progestogen responsible for the progestational, 
action of certain diphenyl cosapounds is being investigated o Urines 
collected from juvenile rabbits before and after treatEsent with ais^ihenone 
are being chroisatographed and extracted for pregnanediol determinations o 
As^hsQons presents a problem inasmuch as it aeco^anies the pregnanediol 
fractions Acetylation is being carried out in an attenpt to overcome 
this difficulty^ If this approach is unsuccessful^ it will be necessary 
to collect large sais^les of rabbit adrenal venous blood for progesterone 
analysis o Now that we know the marked inhibitory effect of ai^henone 
on the secretion rate of corticosterone (the end product of adr@nal 
steroid biogenesis in the rabbit) evidence in favor of excess progesterone 
secretion would indicate a block in the 21-bydroxylating enzyme syst^a 
since this step has been demonstrated as a prerequisite to lip-hydroxy^ 
lationo 

Co Development of esqserimental adrenal tumorso 

Three methods for development of adrenocortical tumors are being 
proposed? 

la Early castrations The studies of Wooleyj, with inbred mice 
and Spiegel J, with guinea pigSj, destonstrated that early castra'<- 
tion can lead to endocrine imbalanos and development of adreno- 
cortical tumor So In this laboratory^ a benign nodular h^&r-" 
plesia developed in OM rats castrated oarly in lifeo About 
20 per cent of the latter were estrogen-produciago We have 



812 



- 7 - Serial Noo NCI-810 

Endocrinology Braneh 



selected the female dog for iisitial castration e:!qperlments 
because of ths siadlaritj of tbe adrenal steroid secretion 
to that of laano 

Hot^verj, heterogeneity in available anixials presents problems 
with respect to reproducible results and transplantation bo that 
other animals J, such as the guinea pig^, will be usede Develop-^ 
ment of animal tuisors with xoeasurable endocrine secretions 
(corticoidj, androgenic^ estrogenic) would provide a valuable 
tool for cancer research and could help answer fundamental 
questions regarding adrenal->gonadal interrelationships o 

2o Over stiEHilat ion during regenerations Regeneration of 
adrenal cortical tissue upon csssation of chronic DDD treatment 
was described ia part Ao We propose to use this period of rapid 
tissue regeneration in an attempt to produce adrenocortical 
tuiaorsc, Following intensive chronic treatment of castrate 
fcsaale dogs with DDDj large daily doses of AGTH in gel would 
be administered as an e3K>gsnou3 stimulus to adrenocortieal 
tissue growth and secretion^ Clinical ei^erienoe indicates 
that in man acute damage to the adrenal cortex which produces 
cell destruction followed by regeneration can form foci of 
nodular hyperplasiao 

3o A third approach which we eacpect to ejjplore for the develop- 
ment of adrenal tusBors is the use of carcinogenic cospoundSo 
Initial eaqperiments will be made on the dogo Two methods are 
to be used: in the oztBg the carcinogen would be introduced 
directly into the arterial st^ply to the adrenal j, in the second j, 
the carcinogen would be injected directly beneath the :>apsul@ 
of the glands 



Part B included c 



813 



Serial No^ NCI-810 
PHS~NIH Endocrinology Branch 

IMividual Project Report 
Calendar Year 1958 

2igt^s HonorSy AwardSg and Publications 

Tullner^ William W.: Mediation of Progest^:tional Action of 

Aaphenone by the Rabbit Adr©nal„ FroGo Soc, Exoere Biol= 
& Med. 98j 157-160j, 1958, 



814 



Serial NOo NCX-813 
PHS-NIH 1„ Endocrinolo^ Branch 

Individual Project Report 2, Endoerinology Serviee 
Calendar Year 1958 3o Bethesda, Md„ 



£mLAc 



Project Titles The Preparation of Crude Natural Extracts of 

Hormonal Aeti^ity from Urine and Serum and their Purifisatioa 
and Phyaieal-Cheadeal Charaeterizationa 

PrincipELl lijwestigatorss DTo Ralph A, Reiafeld and Dro Ro Herts 

Cooperating Units: Laboratory of Bioehenistryj NCI (BTo So Ao 
Peterson) 

Man Years (calendar year 195S)s 

■Totals 3 
Professionals 2 
Others 1 

Project Deseriptions 

Ob.'l^ti'yess To obtain crude preparations of chorionic gonadotropie 

horjaon© from the urine of pregnant women and of women with tropho- 
blastic tsMorSo Purification and concentration of these hormones 
followed by characterization of their physical-chesiical propm'tieSo 
CoB^arlson of these properties of the gonadotropic homrones to do- 
tennine whether or not there is a ch®sieal difference between hormonss 
xdth similar biological properties but obtained from normal and cancer 
sources p respectively a Furthermore j, it would be of interest to de-» 
terasine as many physical^-cheiBical characteristics of this hormone as 
possible since this could ultimately lead to a chemical assay laethod 
which has be@n so far unavailable « 

Methods Emplo;yeds Chromatography on dlethylaminoethylcellulose and on 
hydrossyl apatite to purify chorionic gonadotropins o Application of 
bioassay techniques to HCG preparations to deteraiine their endocrine 
effectiveness o Quantitative deterasination of the ajaino acids at the 
purified product by ehrojaatogr^hy on ion exchange resins as i^^l as 
qualitative determinations by two-dimensional p^er chrtaaatograpbyc, 

Deterasiaations of N-terminal aiaino acidSj by the DKP method and of 
C-tenninal aialno acids by the carboxy peptidase Bsethod,, 

DetesTSiinations of the sedimentation and diffusion constants and 
calculation of Htolecular weight from these constants o Calculation 
of the axial ratios^ oblate and prolate^ to determine the shape of 
the gonadotropin molecule o Us© of oxidizing and reducing agents to 
detsiBiine the relation! s) bstireen the manifestation of biologieal 
effectiveness of the hormone and its structural eon^onentso 



815 



- 2 - Serial Moo NCI-^13 

Endoerinolo^ Branch 

Major Findiaga.s The honaon© extracts from the uyin© of pregnant womeng 
wh©a chromatographed on anion exchange celluloa© showed at least 
a 5-fold inci'ease in biological activity over the crude aomona 
extraetSo The purified horsaone extracts were found to contain at 
least 100 J 000 Xfj/mgo This biological activity is a|)pro3d3Hat©ly 10 
- times as M.gh as reported in tl:^ literature thus faro Hormone 

esctraets from the urine of patients with trophoblastic tutors ©inhibited 
a 10-fold increase in biological activity when ehroaiatographed on anion 
exchange cellulosso These extracts were found to contain 100^000 
lU/xago 

Slirp^tjcan^^ to tljfe FvpsTf.m of the Institute; The availability of noa- 

to3dc conesntrates of naturally occurring hormonally active substane®3 
or of ifflbibitory anti-sera capable of neutralising such endogenous 
biological effects la th© cancer patient would penait tbsrapsutie 
investigations along lines now neglected as a result of the ©a^jhasis 
on synthetic coa^ioundSo 

Proposed Course of Project ; Ertension of these studies along the liaes 
outlined above are projected and will be executed as appropriate 
personnel becojass availablso 



Part B ineludedo 



816 



Serial No, 1301-^13 
PHS-NIH EMocrinology Breneh 

Indi-vidual Project Report 
Calender Year 1958 

P§gt^; Honors s, Awards ^ and Publications 

Reisfeldj, R» A^^ Bergenstalg D» Mog and HertZj, R,: Distribution 
of Gonadotrophic Hormone Activity in the Serum Proteins of 
Norjsal Pregnant Women and Patients with Trophoblastic Timor So 
Arche Bioch^Bo & BiophySj, 1958 (in press) o 



817 



Serial No„ NGI-814. 
la Endocrinology Branch 
2, Endocrinology Setrvio© 
PHS-NIH 3o Betheadaj Mdo 

Individual Project Raport 
Qalendaff Year 1958 



PaslJ^ 



Project Title; The Preparation and Evaluation of Biotin Antagonists 
in the Study of Biotin Metabolism aa an Approach to Canc®r 

Principal Investigator; Mr« Fo Go Dhys© 

Other Investigator: Dro Roy Herts 

Man Years (calendar year 1958) s 
Totals 2 
Professionals 1 

Others 1 

Project Descriptions 

Ob^ectiv^s The principal objectives of this project are to develop and 
prepare biotin antagonists that are particularly effective in pro- 
ducing deficiencies of this vitamin in laboratory animals as well 
as in adcrobial syst^ssB A possible rout® of application of such 
deficiencies to cancer studies is described under "Significance to 
the PrograM of the Instituteo" Furthenooreg appropriate antagonists 
are to b© used to ^lock" certain bioch^d.cal reactions involved in 
the istudy of biotin metaboliam in bacteriaa 

Methods E atpJrOyQds Biotin antagonists are prepared by organic cheaiieal 
synthesis or isolated from natural sources 5 and evaluated by micros 
biological assay and biological testing in rats and miceo 

tfe.lqr Findings s. Biotin sulfone and Y C3s,4-Ureylenecyclohe3iyl) butyric 
acid have been prepared by chemical synthesis in quantity sufficfient 
for testing in rats. Both cosnpouadSp as preparedj, irere found to 
inhibit biotin supported growth in adcrobiological systems (Lo Gasei) 
€ind such inhibition to b© reversible by excess biotino Initial 
results of testing the biotin sulfone on rat leukssaia weg-e negative,, 
Th© second coapound is just now ready for testing in laboratory 
aniiaalso Other biotin antagonist of the coffiibiaative type (as 
contrasted %fxth th® coB^etitive analog type such as biotin sulfone) 
were prepared and found to be inactive ia microbiologieal syst^aso 
The ccispounds In question \mse Diacetamid® and Dibenzasddeo 



818 



Serial NOo NGI-814 
- 2 - Endocrinology Branch 

Si^niJ^ieaqGe ^o the Program of the Iisatituitet EirLdenee from ths 
literatiire indi.eat©s biotin maj- be required for the tissue 
synthesis of folic acide In view of the relative success of 
folic antagonists in the treatment of eaneerp and because of 
the drug resistance later developed to such treatment, an 
appropriate inhibitor of biotin*s role in folic acid synthesis 
adght make present treatment vdth anti-folics more effective « 

P;^,opo,ged Gffliiys^ , of „.P.yo4,^f^'fc,s lo Gci!5)letion of evaluation of coapounds 
now on handft 

.2e Setting vsp a microbial systsmg where drug resistance has 
b©en developed to anti-folics^ for study of possible effects of 
induced biotin deficiency oft-.such drug resistance 9 

3b Continue the ssarch for a more usable and effective biotin 
antagonist J, by preparation of a niMber of biotin analogs with 
deuteriujE substituted for li^drogen at specific locations in the 
molecule o 

^o Separation and isolation of the biotin vitamers described in 
the publieationo 



Fart B included c 



819 



Serial Noo NCI-814. 
Endocrinolo^ Branch 
PHS-NIH 
Individual Project Report 
Calendar Year 1958 

Part B t HonorSg Awards^ and PublicationSo 

Dl^yse, P« Go and HertZj R,j The Effects of Actithiaaic Acid 
on Egg ¥hite-Induoed Biotin Deficiency and %on the 
Microbial Foraaation of Biotin Vitamars in th© Rate Archa 
Bioeh^o & Biophysa 74.! 7-16^, 1958„ 



820 



Wagio.; 



rcitute 



General Medicine Branch 
Budget Data 






Sstlnated Obligatlonseooo. 

Director oooooo,.oooo a 0.0 = 

Relmbursemsnts 

CliMcaXo oocoo.o.oo.oc 



Total 



,o Fiscal year 1959 
4 775*000 

, 1,070,500 
' 270^000 
. 2,115^500 



Ia<Audee Projects No's? 

CHEMOTHERAPy SER\nGE; 

4-709 (c) 
4»714 (c) 
4»715 Cc) 
U«719 (c) 
h-73h (e) 



4"73| c 
4-736 (c) 

5»712 (c) 
5-737 (c) 



CUNICAL EHARMACOLOGFJr & EXSERIMEHTAL THEEASEUTICS s 



1=223 (c) 

225 (c) 

226 (c) 
227 

229 (c) 

230 (e) 

DEBMATCODOGY SERVICES 

e-T°708 (e) 
c-7»728 
c-7»730 
e»7«731 

METABC5LISM SERVICES 

6»702 (c) 

. 6-704 (e) 

6-720 (c) 

6"7aL (c)) 

M 



231 (e) 
232 
233 

234 (c) 
235 



6-7^ (c) 
6-726 (c) 
6»727 (c) 

6-729 (c) 
6=732 (c) 



821 



:.■ - ■ . « airech 

2,, Chtgffiotlierapi^ Service 
3o Bsthesda 
PHS-NIH 
Xxiidivldiiial Project Report 
Calendar Year 1958 



Part AoS 



Project ti£l(s; tixe Chemotherapy of iteute Iniukemia 

Prinicpal lavestigators? Emil J Frftirelchs aad Esnil Frei I%X 

Other Xtavestigatora: Lo Schroeder, So Bogga, Jo Shobl 

Cooperatiag Units s Clinical Pharmacology and Experineatal 
Therapeutics Service 
Duke Bnivsrsity School of M@dieine 
Jefferson Medical College 
Walter Seed Anay Medical Institute 
Jackson Memorial Hospital 
Medical College of Virginia 
Boswsll Park Memorial Hospital 
Emory l^'nivcrsity 
Bowman^Gray School of M«idiclQ@ 
Mount Sinai Hospital, New York 
Philadelphia Children's Hospital 
Mayo Clinic 
Dartmouth IJnivareity Hospital 

Maf» Yeara (calexadar year 1958) Pati@ut Days {calendar y@ar 1958) 
Totals 7 
Prof«ss tonal; 5 

Others 2 

Project Description; 

Objectives sL To study quantitatively the optimum usefulnes»i 
of drugs that are known to have activity against acute leukemiSo 

2o '^o dXt>£over a»d t@st in humans nmn chemical compounds 
vhich can alter the course of acute leukemiao 

3. ^o sttady the effects of these cosopounds on the hosto 

4o ITo test the usefulness of animals and other screening 
techniques by the collection of quantitative clinical data in man^ 

Methoda Bmployed s 

1, Crse Material: All patients referred te the Clinical 
Center with the diagnosis of &emt& leukemia are admitted to the 
study^ 3n addition there are 12. hospital units other than the 
MCX participctittg in most of the below mentioned therapeutic trials c 

2o Crit«riag Obj^ective criteria for $:h« evaluation of 
disease activity have been developed as^d appliedo th^i^ applicabi 
lity asid meaiaiag:fMHn€«s is woder continuing sntntixkf:. 



822 



3o Pharmacoiogj^ in cooperaticm with fche LahotatoTj of 
Clinical Pfeaxioacoloigys phasiaacologie disciplines are applied to 
fehe drwgs tesfcedo fhese studies ar© divided iato three major cate^ 
g©ries5 

a) foxisity s£iiidi@s of eotcpound e£ potential antitumor 
activiey given to humaas for the first tlaseo 

b) Absorption,, distribution and excretion studies ©f kaoxja 
astd pottmtial antitumor eoH$>ouads<, 

e) Analjfsis of thts influence of var^^ing dosage schedules 
and srotetea of administration ©f antitwBor activity aad toxi» 
cityo 

4o lEp@riTnental Design: Xn cooperation with biostatlsticiasis 
and eh@ Clinieal Pharmacology Section therapeutic trials were designsdc 
These inel«sde; 

Ao Cooperative comparative therapesjitic trial deslgn@d to ob= 
tain among other thing® j qtaantitative information of the relative 
effects of antim@tabolite@ of proved antitumor activity in mano 

1, Sequential Analysis ~ New drisgs are tried in patients 
with acute leukemia vHto have already received conventional aatil@i^@mie 
therapy,. Xf £h® pilot studies suggest possible human antll@i^emic> acti' 

vlty further studies are designedo Isitlally these have beea performed 
by admiMstratioa of the drug to be tested to all patients qtiialifyingo 

Major Finding®; 

Ic. Protocol II - fhis is the second definitive cemparativsi 
study undertaken by the group ^ and in it comparison is made betweeis 
3 treatment programs utilising drugs that are known to have antileu° 
kemic activity o I) Methotrexate (KSX) followed by 6-m®rcapfeopurf..ae 
C6=MP)s 2) 6»MP followed by KHK; and 3) 5®t and 6-MP given in combitta= 
tioQo to dat@ 224 patients have been entered on this study for the 
entire eooperativg group, 75 of theses patients being studied at ^lo 
fhls study is still in progress;, however a preliminary evaluation has 
been madeo l« Xfee highest remission rat« occurs with combination 
therapyo 2o 6°MP alone gives a higher remission rate than £^ alone 
i^ether given as initial therapy, or after a previous antimetabolite^ 
3,. Ihe data suggest that treatment with 6-MP do€S result ia condition- 
ing and a slight increase in response to subsequent MX therapyo 4o 
1%@ overall remission rate for all programs ia highest in childreKH 
(50%) intermediate in the intermediate age group (30%) and low in the 
adults (15%), 5o Toxicity requiring cessation of therapy occurred 
between 9 asid 25 days with an average of 14 days and was comparable 
for all treatment program® <> 6o Qualitatively JSSX. gave more islefira'o 
tion and 6^MP more GI symptoms » 7o Jaundice occurred in 15% of pS'^ 
tients on 6-MPs but in none of the patients while receivittg ISS^ 8„ 
Rsnlssion rate was <eoi^parabl<£ for the first and second courses of 
therapy o 



823 



2n 6-a2aufacil - B«eaui3e a sigaif leant ntanber of pattitnfcs 
showed i3]]|>ar©v@meat in pilot studies @£ this drssg, a double bllad, 
eoinparativ^ study of its efficaejr in a group o£ leakgmia pafeleuCs 
zmiiaetoty So all known aatll@ukemi€ therap^i^ was undeireaken^ 'TimntT' 
eight patii^ea w@re st^udied;, 15 reeelving 6»azawraieil and 13 reeeiv^ 
iiag plae«bOo Users wa« ^o difSeseinee iis the a«auber of pati@nl:8 show^ 
iag remission or signifieaat trnprovenent is. fehsse two groups, fhese 
findlGigs cn^hasls@ eh^ faet that teeaiim»i®n& oad inprovetnent caa oe€ur 
spoffitaneously 1% this group of patients « fhes@ dat^ are being pre- 
pared for p^blieationo 

3o 5°fluoroura@il • Preliininarf trials were conducted in 12 
patients witi& advanced leukemia^ (9 at mt). foxieit^ was primarily 

GX s^faptoiBSo Ho bsnefieial effect was observedo thntm data are 
b@ing prepared for publication^ 

4o Diehioroamsthopterin = foxtcltf studies aad preliminary 
elinical trials i*ere perfetmedo Twelve petienes received intransusettlas 
therapy and 36 patients rgseived oral tfeerspy^ Toxicity ©eeurred 
fairly r-ggularlf at loO lagm/kg given loMo dally^ however with oral 
administ:£'eti®n this doae pr@v@d to }m too high.. A d©5@ ©£ Oo5 iagm/kg= 
dailj? wais also to© high, while 0<,25 mgm/kgo proved to be to© loWo Thm 
patients studied had r@e©ived previous M^ (85%) and w^re ad>?a®eed leu- 
kemia pai^tents^ Only oae partial remission has occurred on therapy to 
dateo 

5o A Scady of the relationship betweea dietary felie aetd con- 
tent and antitumor effect of folic acid antagonists^ Wfeil@ massive 
d&ses of folic acid and citrovorum factor can prevent the antititsmor 
effect of aatifolic aeid ^s^^impoirnds „ no study of the effect of dietary 
folic acid cm tisv>w growth or astifcuaor effect of antlfolics has been 
yndertak^So Jm coilaboratioa with Dr, Sriggs ei WLMB^ a stiudy ha» 
be^n uadisrtak^n in mic©<, 'JSie first experlosent in progress is a compa^ 
rative study of the effect of f©lie acid fr^e diet^ folic acid free 
diet plus glycines folic acid free diet and sulfasuxidine» f^lic aeid 
free diet end folic acid on the hematopoietic tissues pf the nnusso 
This study was undertaken in an effort to define the most effective 
program for prod^acing chronic folic acid deficiency in the mouses 

Pr©po(iied Courses 

lo After ecnspl'Stion of Protocol IS a comparative study ©C 
steroid therapy is planned^ 

2o Diehloroamethopterin will, be studied in patients that 
have E©t received prior MEt therapy at a dose of 0o375 mgm/kgo per 
day orally, 

3 a Oth@}f new chemo therapeutic agents will be tea ted o 

4o liae affect of folic acid deficiency on the growth ®f 
t«f!mor@ and on the effectiveness @f asitifclic compounds in aiiimals: 
will be undertakeno 



824 



Serial Moo NCI=.4=709 (c) 
PHS.--NIH 
Individual "=l:*oject Report 
Calendar "^ear 195^ 

t ■^o! "onors. Awards, and '^blications 

)lications other than abstracts from this project: 

Frei, S», Holland, Jo'^'o, ''^chneidG^man, ''oAo, Pinkel, D., Selkirk, Go^ 
Freireich, T.,J., Silver, ^..Tc, Odd, G.Lo, and 2abrod, CoGo; A comparative 
study of two regimens of combination chemotherapy in acute leukemia. Blood: 
In press o 195So 

Frei, ^.. The Treatment of leukemia. Jo Nat. "^sdc Assoc. 50:35S-=360j, 1958. 

Frei, ^., The treatment of leukemia. G„?. 18:9^100, 1958, 



nors 'ind awards relating to this project: 

jstinguished Service Av/ai?d - av;<irded to ^r, Emil I'Yei Til b" the Rethesda Junior 
(amber of Commerce, 'September 1958 



825 



Serial No. NC; 
I.. General MedietM« 
J. Chsmo therapy S«sri. 
),. Bsthesda 



Xndlvtdsial Project Rfeprr! 



fart A? 



Profject I'itles Stsadieis -»f Metabolism of Bleed Cells an4 
Blood Formiftg Organs, 

PfiKseipal Investigators:' Jaiaga M^ Steagle^ M=,Do 

John Uszlo, M„D, (Parts F.G.H. !» 
MartiQ Liebllng, M.D. (Parit A.,) 

Otlhie-r Investigators s Dang Boggs^ M„Dc. (Parts 8^ J. J 

GooperaKtag Units;; 

2„ Or. ;j»eaa Siirk , L.ibotatory o£ Bii»«heint.*icry, NcCJ., 

3., 2;tTjest McBaiiiel^ Laboratory of Nutritiya aad gitdocrJiiiapi. , r- ,. 

Mo X.. Ac M^ ©,. 

fltr. Norm i-a Sells, Laboratory of Clinical I»v@stigatioo» ., 

Man tears t (caieadar fear 1958) t'atlent Daye (Calends 

i'rofv.B»ton»lt 3 1/4 
Othej's 1 

Project Description; 

Objectives^ To study varlou<> aspects o£ the metabolism ot the 
blood cells and blood forming organs. Speeiflcally we are interfested in 

A» Iron Metabolism - to study the pathways of fron mstabollera u-~ 
the nonnal and the patient \/ith maiignmit neoplastic disease (partJc'ijila? « * 
leukemic) as an approach to >.n understonrlnp of the anemia of malignancy 

B» Temperature and Hydrogen Ion Concentration of Bone Marcc w 
To accun(ulate data on temperatures on pH prevailing in bone marrow in all 
phases cf acute leukemia; to observe the effect^, if any„ of the chemoth«i:- 
rapeutic agents on these parameters. 

C. Nicotinic Acid ; To study the role of nicotinic acid In iron 
metabolism of normal and leukemic individuals; to observe the effectia of 
nicotinic acid on the iron metabolism of SKperimental animals and in 
vitro on retleulo-endothelial tissue. 

Dc Bone Mar row Heterograf ta ; Using the Algire graft technique 
to study the growth of human normal and leukemic bene marrow in anima] 
hosts ^ 



Part 8 iaciuded Yes 



826 



Serial EOc mi-A-lli^ 



fo produce auKtssta to human leukesaic cells 
and fractions thert^of and note tUa &ttecz on metabolism of swch cells 
in vitro, 

f o Oxidative aad Glycolytic Metabolism of Leukocytess To 



accumulate data on the metabolic behavior of normal leukocytes amd 
thosd derived from patients with the various types of l@«skemiaa 

Go Effect of Chemot hera|)e«tic Agents on th@ Metabolism: of 
Letikemic Cells : To study the effect of common chemotheraps^.Eic ageats 
on the metabolism of leukemic cells a 

Ho Stt^ar Analogs ; To study the effect of certain glucose 
analogs on the metabolism of leukemic cells in vitro; to observe the 
effects of Z-Desoxy^glucose on aaimal tumors; to observe the (sffects 
of 2"Dea©xy=-glucose on human patients o 

I,, Hematopoietic Tissue Tra ns plantation : To explore the 
poseibillties of using tissue (bone marrow or embryonic hematopoietic 
tissue) transplantation in the therapy of leukemia, certain solid 
tuHMsrs and lethal radiation in the human patient; to establish a tissue 
bank. 

J„ "Skin Window" (Rebuck Technique) in Leukemic Patients ; fo 
study the response of leukemic patient to the application of bacterial 
toxiin to a previously scraped area of the skin and to compare with the 
normal o 

Methods; Patient Material; Major Findings 

Ao Irom Metabolism ; A considerable body of data has accumsi" 
lated on serum iron and iron-binding capacity In the leukemic patlento 
This material is not yet suitable for publication. A part of this 
project has been a study of the iron metabolism of approximately 23 
cases of Sarcoidosis in collaboration with Dr„ Norman Bell of NoI.A„X.iI>„ 
This Invalved radioactive iron and chromium studies and serum iron and 
Iron blading capacity determinations. The objective has been to explain 
the anemia which complicates some of these cases. This material is ready 
for publication and will be presented at a meeting of the Southietrn divi<» 
sion of the Society for Clinical Investigation. 

B. Temperature and Hydrogen loa Concentration of Bone Marrows 
Our studies tend to confirm the one previously published report on the 
temperature of bone marrow in leukemia. An Instrument which it was hoped 
could be used to meastire pH of the bone marrotrr in situ has proved mtreli'^ 
able. We are dropping this project for the present. 

C. Nicotinic Acid ; Nicotinic Acid is Icno^ra to play some role in 
Iron metabolism. It is the only substance known to give a prompt rise 
in senmi iron. Experiments using phenylhydrazine treated rabbits to 
study iron metabolism per se and the Influence of nicotinic acid forme vl 
the basis of a report delivered to the 24th Tagusig of the Deufcseheia 
Pharmakologisches Gesellschaftj, Serlinj September 24, 1958 and the mo Se- 
rial is now in preparation for ptafe^llcation, 

827 



-Ssrlal NOo KC1-/.-714 (c) 



Data gathered on serum iron and iron<°binding capacity levels 
in dogs cm sicotlaic acid deficient diets Is li^£s^ prepfired for p3sb~ 
llcatioti. 

D. Bone Marrow Heterograf ta ; A&&ss»ta to grow hsBsan boae 
marrow in aillipore filter chambers were not consistently successful 
and therefore, it was concluded that the method was not adaptable to 
the study of the effect of chemotherapeutic agents o This part of the 
project has been abandoned. 

A side line using this technique in cooperation with Dr., 
Robert Cro^mse of the Dermatology Service to the study of hiim;^i hair 
growth has proved more successful. A paper entitled "Influence of 
the Deimal Papilla && Survival of Isolated Human Scalp Hair Roots in 
an Heterologous Host" has been submitted for publication, 

B, Igsmmolo gy: The antisera developed in rabbits using human 
leukemic cells and Freund's adjuvant proved to contain a variety of 
anti°> human antibodies rather than a specific "antimalignant" antibody 
when tested against normal and leukemic cells in vitro. This project 
has been abandoned o 

Fo Osid atlve and Glycolytic Metabolism of Letikocytssg Data 
on the metabolism (In vitro) of laukocytes from approximately 40 leu° 
kmalc patients and approximately 20 normal subjects have been obtained o 
This material is in preparation for publicationo 

60 Effftgt of Chemo therapeutic Agents oa the Metabolism of 
Leulcemle Cella ; This part of the study has resulted in a publication 
in the Proceedings of the Society for Experimental Biology and Medicine 
in 1958„ 

Ho Sugar Analogs ; In vitro studies of the effect of 2»>De80xy" 
glucose on the metabolism of human leukemic leukocytes have resulted in 
a publication of the Journal of the National Cancer Instltuteo 2°deoxy° 
D»gluco8e and 2 deoxy-D galactose were shown to be potent glycolytic 
Inhibitors of human leukemic cells and a variety of animal tumor cells c. 

A publication entitled "Effects of Glucose Analogs (2°>deoxy-D» 

glucose and 2«deoxy<-D«galactose) on Experimental Cancers", now in press 

summarizes the results of the use of these glucose analogs in the treat-^ 
ment of certain animal tumors. 

I. Hematopoietic Tissue Transplantation ; No results have yet 
been obtained In this new project. It is Intended that during the cotn° 
ing year there will be attempts at Qreatment of cases of acute leukemia 
by the transplantation of normal hee^topoietic tissue. This will entail 
developing sources of such tissue. Cadaver bone marrow, embryonic liver 
and spleen and volunteer donors are possible sources for consides'afciono 
Techniques for preserving the tissue will have to be developee as will 
techniques for determining vlsblitty of the tissues and the stsri'ival in 
the hosto If and when sources ef tissue become available we Intend to 
create a viable tissue "bank"o 



828 



■» 4 => Serial No„ KI- 4=714 (c) 



Jo "Skia Window", Rebticif Teehaigue In Leukem ia Fatieatag 

Refouck has demonstrated that the letikocyte migration normally 
goss through a certain regular cycle smder the stiBSiiafcioa of g bacte- 
rial tosin applied to the skin, Ifith the knowledge that the le«ske&ic 
patisnt does not in many oases respond i^ith a normal inflammatory re° 
action to infeetioQ we thought it would be interesting to apply the 
techniqtse to th@ leukemic patients availablso Approximately twenty 
cases have been so studied and the project is continuingo 



829 



Serial No,. J?CI»4-714 (c) 
lo G«nearal Medicine Braneh 
2o Chemotherapy Sea*vlce 
3o Bethesda 
PHS-NSB 
Xadividiial Projece Sepore 
Calendar Yeas? 1958 



Pare B; Honors, Awards g and Publicaftioas 

Publications other than abstracts from this project; 

1„ Laszlo, J, 8 Stengle, J„ , Wight, K«, Burk, D„ Effects of 
Chemotherapeutic Agents on Metabolism of Human Acute 
Leukemia Cells ia Vitroo Proc<, Soc. Exp, Biol, l^ Medo 
97s 127-131, 1958„ 

, B», Laeslo, J,, SteaglCs Jo, &»rk» D, Cerfeaia 

Metabolic and Pharmacologic Effects in Cancer Patients 
Given Infusions of 2"Deoxy-I>-eiucoseo J,N„ Colo, 211485° 
494 9 1958 o 

LaszlOs J. , Laodau, Be, Wight, K. , Burk, Do The Effect of 
Glucose itoalogues on the Metabolism of Htjman Leukemic 
Cellso JoMoCoIc 2lg475"483, 1958o 



Honors and Awards relating to this projecti 

James Mo Stengle elected fellow of the International 
Society of Hematology, September 1958 o 



830 



Serial No„ NCI-4-715 (c) 
1» General Medicise Branch 
2o Chemotherapy Service 
3o Bethesda 



PHS-NIH 
Individual Project Report 

Calendar Year 1958 



Part A, 



Pr43ject Title: The Mechanism and Management of the Hemorrhagic 
Disorder Associated with Acute Leukemiao 

Principal Investigator: Emil J Freireich, MoD. 

Other Investigators: Emil Frei 111, M,Da, and G. Richard Lee, MoDo 

Cooperating ©nits: Pathological Anatoimy, N.I,H. , Dr. L, Thomas; 

I oratory of Physical Biochemistry, N<,I,A«M„D», 
Dr„ K, Lakio 

Man Years (calendar year 1958) Patient Days (calendar year 1958) 
Total: 2 
Professional: 1 
Other: 1 

Project Description: 

Objectives: a) To identify factors responsible for precipitating 
gross hemorrhage in thrombopenic acttte leukemia patients. 

b) To study the role of the platelet in blood coagu<= 
lation, and hemostasis,, 

c) To improve the therapy of thrombopenic hemorrhage^ 

Methods Employed: 

a) In vitro and in vivo blood coaguiatiea studies o 

b) Colisan chromatography using ration exchange resins. 

c) Analysis of clinical, laboratory and pathological 
data on patients with fatal intracranial hemorrhage o 

Major Findings: 

lo Ihe association of fatal intracranial hemorrhage 
and "blastic crisis" in patients with acute leukemia. 



Part B included Yes ^/ No 



831 



Serial No„ NCl-4-715 Cc] 



In a previo' -1 report we had described the high rate of fatal 
intracranial heiHorrij.age (59%) in patients with a "blastic crisis" 
of acute iesskemiSg in contrast to a rate of 13% in other patients 
with acute leislcemia. In order to find the cause of this greatly 
increased incidence of fatal intracranial hemorrhage, two approaches 
were used; a) examination of autopsy material, b) in vitro coagula^ 
tion steadies o 

a) Examination of the autopsy material revealed that the 
patients v^ieh blastic crisis had a distinctive intracerebral lesion, 
cot .sting of hemorrhage located primarily in the white matter of 
the brain and within the areas of hemorrhage are nodules of le^emic 
cells. 1!he findings indicate that stasis of leulcemic cells occurs 
in small istraeerebral vessels, proliferation of the leukemic cells 
results in destruction of the vessel wall and formation of a leukgrnic 
nodule; hemorrhage then occurs about the nodule and coalescence of 
many such lections results in the large lesion seen grossly, fhis 
leukemic lesion is responsible for the greatly increased rat@ of 
fatal intracranial hemorrhage in the patients with blastic crisis o 

A manuscript reporting these findings has been completed and submit^ 
ted to the American Journal of Medicine for publication, 

b) In vitro coagulation studies were performed on 4 patients 
with leuksaia having greatly elevated peripheral leukocyte counts in 
order to determine whether these white blood cells interfere with 
blood coagulation. In «11 instances the leukocytes were found to 
aeee^e^ats ail a^altties of blood eoagssIat&OQ C^«®o 2;too!!sib®p]La@e£@ 
lika as&ivJLey) and mo evidissc^ of jLats^fataocs ^slth bl&od eoagialasiogi 
wss £aimj&<^ £3ow@va?, mils pesrfosmiog &hses a£i!s<El®s in oisse paC£,@!B£, a 
c?;^fibiriEog3£& was accid@Q£ally dlecovss'ada 

2, Cryofibrinogen&mia. The patient under considera'- 
tion had acute leiskemia with a peripheral white count greater than 
300,000 per cu. sxn. She had a history of several months of thrambO" 
phlebitis migrans. At the time of study she dissconstrated two areas 
of superficial venous throaibophelbitis. When hsr plasma was chilled 
to 4 C, a precipitate was noted, which disappeared when re'-warmed to 
37 Co This did not occur in serim. If the precipitate was collected 
and washed, and redissolved, addition of thrombin resulted in formation 
of a cloto This identified the cold precipitable protein as fibrincgeno 
Hov^ever only 50% of the cold precipitate would clot on addition of 
thrombin. The non clottable protein alone, however did not precipitate 
in the cold. Repeated reprecipitation and re^dissolving gave stellar 
results, that is, that both the nan^clottable and the clottable pro° 
teins were necessary for cold precipitation, Ultracentrifugal studies 
of these cryoproteins revealed two components, the first, S»-=10o20, 
was removed by clotting with thrombin, while the second, S-_ =12,20 was 
not clottable^ Th® high sedimentation of the S2q=10o20 protein and its 
low ssolubiLity at decreased ionic strength identified it as polymerized 



832 



Serial NOo MGI-4-715 (c) 



fibrinogeno These data were published in abstract form, (CliUo Res„, 
6; 197s 1958) B aad a manuscript is i«i preparatioito A total of 8 other 

patients have been tested for cryofibrinogenemla and none has bsen 
foimd to date. 

3, Studies of Antithrombin- Thrombin interaction o 
Thrombin is very unstable in plasmao This antithrombic 

activity of plasma is poorly understoodo Otsr interest in this area 
stems from the possibility that variations in antithrombic activity 
will be a precipitating factor in the production of hemorrhage, par- 
ticularly in already thrombopenic patients with leukemia. As a first 
step in this study, methods were developed for purification of Thrombin 
and anti thrombin,, 

Ao F^irlfication of Thrombin - This has been accomplished using 
GRC-50, ration exchange resin. A total of 15 separations have been per<^ 
formed to define cond|,tions for maximum yield and optimum separation and 
purification. A purification of son^ 3000 times has beea achieved by a 
column starting at ph 7, O.OSM, with a gradient increasing to ph 8, 0,5M. 

B. Purification of antithrombin - Using a MAE cellulose col^san 
separation of plasma proteins, antithronbin activity was found confined 
to a single area on the chromatogram indicating that further purification 
of this material is feasible. 

4, Platelet lipoprotein - A total of 4 studies in 2 
patients have been performed. Initial dosage was 10% of the computed 
effective dose, going up stepwise to 30% of the effective dose. The 
latter dosage gave in vitro increase in prothrombin consumption and 
stypven accelerated coagulation time. However, there was no demonstr- 
able effect on clinical hemorrhage. It should be emphasized that infu> 
sions lasted only 15 minutes. Further studies in this area had to be 
discontinued when the source of supply of fresh human platelets was 
lost. 

Proposed Course of Study: 

Further studies of purified thrombin including ultracentrifugal 
and amino acid analyses will be performed. Then efforts to further 
purify antithroml>in will be made. We hope to be able to study the re- 
action of thrombin and antithrombin, in a purified in vitro system. 
Understanding of this interaction may permit the development of aecu* 
rate in vitro assay systems, and measurement of activity in leukemia 
patients . 

Comprehensive review of clinical and laboratory data on a group 
of patients with acute leukonia is planned in an effort to define the 
relationship between platelet level, leukocyte level, fever, infection, 
and otheitr cliaical events to occurrence of hemorrhage in patients with 



Studies of platelet lipoprotein in vivo will be undertaken if 

a source of fresh hmaan platelet can be fo«md. 



833 



Serial KOo m:I-4-715 {&] 



PHS-KIH 
:idividual Project Report 
Calendar Year 1958 



Part B. s Honors, Awards, and Publications 

Publications other than abstracts from this projec'Ei: 

1, Freireich, B,J, Schmidt, P.J. , Schneiderman, M.A. , and 
Freij S, III„ A comparative study of the effect of fresh 
asid preserved whole blood transfusion on bleeding in patients 
with acute leukania. No Sngo J, of Med., in press. 



Honors and Awards relating to this projects None 



834 



lo GeHferaJi Mediciae Braaeh 
2„ Chemotherapy Service 
3o BeChesda 
PHS-HIH 
Individmil Project Report 
Cal@&da? Year 1958 



Part A. ; 

Project Tide; A Study of the Cllnieai KanifestaClon of 
Acute LeukCTiiao 

Prioelpsl Investigators: Emil J Freireieh, M^Do and Emil Fr@i £££, M„Do 

Other Investigators: Richard Shawg, MaD. , Edward Moore, Mo Do, Daae BoggtBg 
MoDc, 6„ Richard Lee, M.Dc , Arthuf Teplitskys M„I}» 
and Leoaard Carres g M„D, 

Cooperating Units: I^ose 

Han ¥e^ (Calendar Year 1958): Patient Days (Calendar Tear 1958); 
Totals 2 
Professional: 1 
Other: 

Project Description: 

Objectives ; To delineate etiologic and pathogenetic faet©?<9 
0€ the various manifestations of acute leukemiao 

Methods Employed ; The comprehensive application and correlation 
of clinical 9 hematological, radiologic, chemical, microbiological, and 
histologic methods to the above manifestation in patients with acute 

le^emia» 

Major findings : 

lo R !nal Lesions; The findings described in the 1957 Fr@j@cr. 
report have been este^dedo Major work in this area during the past yeer 
relates to the pathology of the renal lesions., The most slgoifiean^ 
finding pathologically has been the observation that approximately 50% 
of those patients with greater than 2 times enlargement of £h€ kidneyis 
have no evidence of leukemic infiltration. The correlation of this and 
other pathology findings with the clinical observations is currently 
underwayo 

2c . . Bone Lesions; During the past year Dr, Louis Thomas of 
the Lstboraeory of Pathology has con^rehensively studied the femurs of 
20 patients with acute leukemia, '^is is part of a correlative study 
which includeil clinical and radiologic observations on 85 patients 
inclusive of the above 20o 

Part B included Yes Q No j^] 



M5. 



serial Noo NCl-4-719 )Cc) 

3o Nucleic acid metabolisms 1!hi« study is still ps-lmasily 
conceraed with ss^thodology, %e details of the. development of this 
methodology sxe reported in the Projsct Report submitted bj the 
Laboratory o£ physiology. It is as&ticipated that the methods will 
be ready for application to patleats ira the next few months., tkne 
it should b@ possible to determine quantitatively most of the fre@ 
bases nucleosides and nucleotides^ A protocol has becsn devslopgd^, 
(and specimens have already b@en collected) concerning the types 
of disease and clistical situations that will bs studied, this is 
of critical ioiportanee since a coispiete analysis of a single spft'^ 
cifflsn tskms three, weeks » Priority for studies is as felkwss 
lo Normal controls „ 

2<, Patients with untreatsd acute leukemiao 
3o Sam@ patients later lAen and if remission is achievedc 
4o Patients with chronic myelogenous leukemia, 
5o Fr® ss^d post M@thotr@sat@ and 6»ffi«?icaptcipurine 3&aisiis° 
tratisa in solid tiic^r pati@ate. 



Ao Masingaal md Cmtw&l ms^mm ^mt&si mmlvmss^s A 
clinical pathological study of meningeal and central nervous systaa 
leukemic involvement in patients with acute leukemia has ^een under- 
taken. Clinical signs and symptoms of central nervous system i^avolve^ 
ment occurs in approximately 15% of the 150 consecutive patient® stu- 
died; it occurs primarily in association with acute lya^hoblastie 
leukemia, fhes@ data correlate well with findings of meningeal infil° 
tration with leukaaic cells in autopsy material. 

5o latural History of Disease: M analysis of survival; 
s^optoms, age, causa of deaths incidence of fvsmission; type of leu°° 
kemia and other clinical features in the first 150 patients admitted 
has been initiated. Record keeping has been developed to permit coa°^ 
tinuous aceustulation of these data^. fhia study will permit & current 
overall assessment of the natural history of disease under etudy^ 

Significance to Cancer Research ; Ihe continuing scrutiny etoA defiai« 
tion of clinical manifestations of acute leukemia is of significance 
to cancer research from many points ef view. This is one of the 
major areas wherein leads to pathogenetic mechanisms may be uncovered. 
In addition,, precise observations are essential to defining the 
natural history of the disease and this latter is of critical imper^ 
tance to drug evaluation, the major effort of our section,. 

Proposed Coarse of this Project:, After eogq>letion of the clinical 
study of meningeal and CHS leukemia, a prospective study of the effect 
of therapy on this syndrome will be undertaken. 

We plan to study other clinical syndromes such as pulmonary 
and hepatic leukemic involvement. 

A study of th3rroid function and metabolic rate in patients 
with leukamiag, and their relation to type and course of dis@as<^ will 
be undertaken. 



836 



2. Chemotherapy Sen 

3. Argoime Cancer Isssarcr 
Hospital, University of 
Chicago 



PHS-KIH 
Individual Project 

Calendar Year 1958 



Part A. 



Project Title: Assay of Erythropoietin in bons 
marrow suspensions 

Principal Investigators: L»R„ Schroederj, M,D, 

C,W, Gurney, M.D, 

Cooperating toits: Argonne Cancer Research Hospital 
and Department of Medicine, The 
Bniveraity of Chicago Clinics, 
Chicago^ Illinois, 

Man Years (Calendar Year 1958) Patient Days (Caleadair Year 195; 
Total: 1 2/3 
Professional: 1 
Other: 2/3 

Project Description: 

Objective: to devise a simple assay method for the 
hormone governing red blood cell production by the bone 
marrow o 

Methods: a technique was developed in which living 
bone marrow suspensions from suitable donor animals were 
incubated with radioactive iron. 

Patient Material: plasma samples collected from over 
300 patients with a variety of diseases were assayed by 
the new technique,. 

Major Findings: rat bone marrow suspensions will ac- 
tively incorporate radioiron under the influence of hisnan 
and animal plasmas. The rate of radioiron incorporation 
varies with the "cold" iron content of the test plasmao 
The cold iron content of human and aaimal plasmas varies 
with the rate of erythropoiesis in most cases and hence 
parallels bone marrow response to erythropoietisso 



Part B included Yes X I **** 



2 - 



Significance to the Program of the Institutes 

Ihe hjHQoral regulation of bone marrow activity is a 
basic physiologic mechanism as yet poorly understood. 
Knowledge of the factors influencing bone marrow response 
to stress and disease is fundamental to an understanding 
of the response to chaaotherapeatlc agents. 

Proposed Course of Projects 

A systematic study of the assay system to find means 
of negating the effect of cold iron concentration on re- 
sults, ^pon suitable modification of the assay proeeduroj 
studies of the metabolic effect of erythropoietin will be 
initiat©d» 



838 



Serial No. NCI-4-734 (c] 



PHS-NIH 
Individual Project Report 
Calendar Year 1958 



Part Bs Honors, Awards, and Publications 

Publications other than abstracts from this project; 

"Assay of Erythropoietin in Bone Marrow 
Suspensions", Nature 18151537, 1958. 



Honors and Awards relating to this project; 



839 



Serial No„ HCl"4'-735 (c> 
lo General Medicine Branch 
2„ Chemothes'apy Service 
3 o Bethesda 
PHS«=i!«IH 
Individual Project Report 
Calendar Year 1958 



Part Ao 



Project fide: The Effect of mierapy on the Synthesis of 

Desoxyribonucleic Acid by Leukemic Cells (Proposed) 

Principal Investigators: Dro George Brecher 

Dr« Etsgene Cronkite 
Dr. L«Eo Sehroeder 

Other Xmrestigatore: None 

Cooperating Units: Hematology Service, Department of Clinical 
Pathology, Clinical Center 

Medical Division, Brookhaven National Laboratory 
Vpton, Long Island, New York 

Man Years (Calendar Year 1958) Patient Days (Calendar Yeair 
Total: 
Professional: 
Other: 

Project Description: 

Objectives: to study the rate of DNA synthesis utilizing the 
in vitro incorporation of triated thymidine and radioautography in 
cells of the bone marrow and peripheral blood of patients with le«£kemia, 
and to assay the effect of antimetabolite therapy on this measure of 
proliferative capacity. 

Patient Material: patients under treatment by the Chei»therapy 
Service will be used as a source of the abnormal cells. 

Proposed Course of Project: to carry out the studies listed 
above. 



Part B included Yes 



n "^ H 



840 



PHS-N2H 
Individual Project Report 
Caleadar Year 1958 



Serial Noc KCI-4-736 (c) 
1„ Geaeral Mediciae Branch 
2. Chamotherapy Service 
3o Bethesda 



Part A, 



Project Title; the Demonstration of a Hormone Governing the 
Production of Platelets (Proposed) 

Principal Investigator; L,Ro Schroeder, McDo 

Other Investigators: None 

Cooperating ^nits: Hone 

Man Yeara (calendar year 1958) Patient days (Calendar year i958^ 

Total: j^ - . 

Professional; 
Others 

Project Description; 

Objectives; to demonstrate the existence in man and animals 
of a hormonal substance which governs the production of platelets by 
the bone marrow. 

Significance to the program of the Institute: Knowledge of 
the existence and activity of the hypothesized hormone will further 
understanding of homeostatic processes in the blood-forming organs 
and the factors governing bone marrow response to stress and disaaseo 
Variations in level of the hormone may play a significant role ia the 
thrombocytosis which accompanies scsne types of chronic leukemia and 
the thrombopenia which regularly accompanies acute leukemia. 

Proposed course of the project: Investigations in animals to 
devise suitable assay procedures and circumstances under which maximum 
amounts of the aubotances are produced. This phase will be followed by 
investigations in saan and animals into the results of admin±8tratioa of 
the substance as well as tissue culture and biochemical studies cf its 
effect on bone marrow metabolism and cellular maturation and differenta- 
tion. 



Part B Included Yes 8 I No 



841 



Serial No. NC105-7J2 (C) 
K General Itedicine Branch 
2. Solid Ttflfflor Service, 



Bethesda 



PHS - N8H 
individual Project Report 
Calendar Year 1958 



Part A. 

Project Ti tie: 

A, Coaparative Trials of Chemotherapeutic Agents in Patients with 

Sol id Tufflors. 

B. initial Therapeutic and Pharmacologics I Studies of Che«m)therspeutic 
Agents in Patients with Solid Ttroors. 

Principal Investigators: Clyde 0. Brindley, M. D., EmiLFrei 8 81, M. 0. 



Other investigators: Richard Shaw, M. 0., Harold Silberwan, M. 0. 
Jai!«s Marsh, M. 0..?nd Momtague Lufsej Mo D„ 

Cooperating Units: The Clinical Phariitacology and Experinriental Therapeutics 
Service of the General Medicine Branch enters in Part B, 
of Projects (above). Hensbers of the Eastern Solid Tumor 
Cooperative Group take part in part A & B. 

Man Years (Calendar year 1958) Patient Days (Calendar year 1957} 

Total: 3 years 

Professional: \j (C.O.B. I year; E.F. i year) 
Other: \i (Technicians) 

Project Ti tie: 



A. Comparative Trials of Chemotherapeutic Agents in Patients with 
Sol id Turaors. 

Project Description? 

Objectives: To cooperate with five other institutions in the Eastern 
Solid Tumor Cooperative Group in the application of the principles of the 
cotaparative trial in the evaluation of agents in the treatment of husRiam 
cancer. In particular the following is sought: 

1) To increase our experience so that tii>e niethodology of conducting 
such trials may be improved. 

2) To obtain information on the relative efficacy of certain selected 
agents, so that the correlation betwesffs their anti turstor acti vi ty in aninsHti 
tumors, other screening systems and in hutti#ns may be evaluat^do 

3) To establish the re@\ value of certain agent-s fn the (Sresent day 
therapy of h»man cancer. 



Part 8 included 



/Yes/ 



No 



- 2 



Seria? No. NCI -5-71 2 



lis group meets at regular 
rative trials and to 
the basic principles 

in advance by the ssjefflbers 



Methods |gpJ..Qyedl 

Our institution is a raerober of the Eastern Solid ?«?©r Coopere^fve 
Group which is composed of investigators fr<» six hospitals which 
cooperate in cosroparative clinical trials, 

intervals to plan the protocols for the ccraparative trials and to 
evaluate the results of studies in progress, 
of the cojsjparati V® trial is as follows: 

The details of the study are set down 
of the group acting with the aid of a statistician. 

2) The assignsssent of the various agents (yinder coznparison to the 
patients is done by raendosiiii seSecfeiort. 

3} Patients acceptable for the study are only those in vMom the 
size of tuiaor tsiigsses can be assayed by direct measurement or by iise@sur©= 
tinent of the masses as shown on roentgen films. 

k) The effect of the agents are deterttained by the use of only 
objective criteria including prieerily the changes in iwasured ttmor 
miisses and patient survival. For certain teasior types (i.e., royeloffia) 
mors indirect niiieasures of tiBBor activity af© applicable. The evaluation 
of the degree of response is uroade by the cooperative group mejsbers 
independently on reviewing the data without hsvsstig information as to 
the agent the patient received. To this extent the tumor response 
evaluation is '"blind". 

5) The data are analyzed in cooperation with the &l®£isticJanSo 



Patient Material 

Patients are selected only when their type of neoplasm, general 
condition and previous treatment status satisfies the conditions of 
the cotir^arative study protocols. Patients must have directly Bsseasy"- 
sble tyroor fsj^sses or other findings which correlate well with tisMor 
activity. The tumor types in the various studies are as follows: 
Study Ttwiiior Type 



Thiotepa-Ni trogen Mustard 



MelanoKBa 
Hodgkin's Disease 



Urethafoe - Plac®bo 
X-ray - Nitrogen Mustard 



Multiple Myeloma 
Bronchogenic Carcinoma 



ic Acid Antagonist *>■ Nitrogen Mustard Hodgkin's Disease 

Lymphosarconia 
ReticuluiSii Cell Sarcoma 



The first Thiotepa - Nitrogen Mustard Study has been cojmpleted, 
A total of 260 patients were studied by the Cooperative Gro«jp, 53 
caroe from the N.C.I. The study pointed up several of the problems 
and suggested solutions to many of the difficulties in performing 
comparative clinical trials. 



843 



Serisjli No. iCil-5»7S2 



Particularly it showed that %uch tri.§Js ar« feasible. Significiint 
differences in the two drugs w@s foynd in riiiessured tymor regression 



only in Hodgk 



n's disease; here Nitrogen Mustard (MechlorethiSii! 



cr 

tr 



was superior to Thiotepa (Triethylenethiophosphorsraide) = The resKiissions 
produced by Nitrogen Mustard ware rsore frequent and of longer dyration. 
No statistically significant differences in the effect of the two drygs 
was foynd in breast csrcino«iS, bronchogenic csrci norms and melsnotas 
either in reieasured tumor mass change or in pstlent survival. A l@rger 
proportion of the patients receiving Nitrogen Mustard h^d herisopoietlc 
depression warranting drug dose alteration than occurred with Thiotepa. 
For this reason the further exploration of the two drugs at different 
dose levels seems warranted. The nuraber of total cases of raelanosaa was 
small (30), so by merely including more cases significant differences 
may be found between the two drygs. The low response rate in naitfiiwary 
and bronchogenic carcinoma C^bout 25% with Nitrogen Mystaird and 10% 
with Thiotepa) raises the question of whether these tsamors are sufficiently 
sensitive to alkylating agents to be syitable for comparative clinical 

als. This poor response rste may however, be dfise in part to the 
arge proportion of very advanced cases included in the trial. Selection 

teria might be needed to exclude alm)st terminal cases. 

The sayltiple n^yeloriija Azaserine stydy has been cosiipleted. In this 

al Azaserine was cotipared to optimum ctedica-I care (placebo). Tv^nty 
two cases were included by the Cooperative Group; nine came frous the 
N.C.3. Azaserine causes severe oral mucoral ulceration and other g@stfo-= 
intestinal disturbances in most patients; this limits markedly the agvot^nt 
of dryg which can lie tolerated. No significant sntiti^iior responses wsre 
seen in either the drug treated on the pilacebo treated ptitients. 

Si cjni f icanee to Cancer Research; 

By the cofjiparati v® trial rauch more quantitative data is b©ing 
obtained on the effect of ch«M?iiotherapeeitic agents in hystssn solid tm;ior% 
than has been available previously. This information not only fiors 
clearly defines the place of a given agent in the srmmient&r'nm of the 
physician treating cancer patients but also is M~^ful in estg.bl ishing 
correlations between the antitumor activity of agents in screening syf>t@fflg 
and in hymsns. The nraethodology for such studies is being improved and 
should lead to a wide application of such quantitative studies in clinics;^ 
cancer research. 

Pro posed Course of the Project; 

The following studies are now in progress; 
l| Thiotepa-Ni trogen Mustard in Melanonaa and Hodgkin^s Disease. 

2) Urethane and optimal twedical care (placebo) in Multiple Myelcw®. 

3) Nitrogen Mustard and Jrradiation compared to irradiation tlone in 
Bronchogenic Carcinoma. 

k) Methotrexate, Dichlormethotrexate and Nitrogen Mystard in LyrsaphoRiias . 

Study 1) is a conHinuation of the first Thiotepa-Ni trogen Mustard Study 
The modifications are that Hodgkin's disease patients ar® being treated at 
various dose levels of both drugs and siBelanofws patients Fecel's^e gr®dus.lly 
increasing doses to tolerance. A dose response relation is being inv©s-= 
ti gated. 



844 



- ^ Serial) f^o.Nefj-5-712 (C) 

Br Siiydy 2) Us^^fehgne l§ bming cemp^rmd tto opEirosjH reedSciill c©r«o 
The r®a1. ¥®l(ii)# of Urethsn^ lira the lre©tei«n£ of My®low@ is iming t@s«©do 

8n Study 3)) the question of the improvement of resyUs of irradiation 
by the concurrent use of cheRotheriipy is being g^piored in lomg cancer. ' 

in Study k^ the antitw)or effects of two folic acid unt^gonists is 
being compared between thenselves and with the standard, Nitrogen Mustard. 
The corapsrisors between the folic acid antagonists is being done becsut® 
of the correlation it wwuld establish between the results in an aniwiil 
screening systew (Mouse Leukemia L12J0) and hims^n cancer. The comparJ&©ii) 
with Nitrogen Mustard wi 1 8 help to show what pliic© these folic acid 
antagonists haive in the present therapy of hmimn lywphcwas. 

Project Titlei 

B. initial Therapeutic and Pharmacological Studies of CheiwotherapsMtic 
Agents in Patients with Solid Tufwors. 

Ob!ectiv@si(A)To perform initial studies on new potential antityreor agents 
in mmn including qualitative and quantitative toxicity studies, other 
pharmacological studies and observation for «nti tysssor activity. 

Methods; The following iroethods and discipline »r& applied: 

Ei«peri Mental design: AiPeer qusl i tati ve oobses-vagjoa for toxicity 
®re made quantitative studies mre in order to Jetertsjine the optimal 
fiethod of using » given new agent. AccwwUting experience hm helped 
us conS^mb'iy in this regard. 

2) Clinical experience: The manifestation of drug toxicity ^re 
highly variable which, complicated by the dyngsatic primary disease 
process, demand careful :obsmrmtlm by experienced and well trained 
physicians. 

3) Pharwacolog'c studies of absorption, ««Gr#tion, distribution 
and tifBetabol i sra are performed in collaboration with the Pharreacologic 
«nd Experimental Radiation Service. 

Observations for antitwsnor activity are wade pari pussa with the ©bove 
after which the decision is made as to whether definitive therapeutic 
trials sre in order. 

Studies on seriwi and spinal fluid enzyme levels are done by 
following changes in SfcjbsSfDS® concentration several techniques, 
i.e., spectrophotosviietry. 
Patient Material; 

CeVonly patients with metast.stic cancer or leukemia 
refractory to or beyond conventions! therapy «re acceptable. 

Major Findings; Studies which h®ve been completed are: 

1) Toxicity and Preliiwinisry Antitumor Trial of 5-Fluoroyr®ci 1 . 

2) Toxicity and Praliwin^ry AntitUfisor Tri^l of A-139 2,5-bis 

(l-A2iridinyO-3,6»bis C2=w®thoxy@thoxy}-B-b®nEoquinone. 

3) Toxicity and Preliminary Antitisaor Trial of Dichloriwethotrexat®. 
k) Tojsicity Study on ^-AiiinopyrDzalopyrididine. 

One hundred and tv®nty nine cases on S^FIuoroisr^ci 1 were studied by ths 
Cooperative Group, 19 conaing frow M.C.8. 



845 



° 5 ° SeriaB No.NC8-5»712 (£)) 

5-F1uorouracll caused hejtopoiefeic depression and gastrointestinal 
disturbances necessitating stopping therapy after a few days ^t 
intravenous doses of 6 rog/kg/day and 15 ffig/kg/week and above. 
The drug was fairly well tolerated orally at doses of 8 mg/kg/day. 
Suggestive antitumor activity was seen in epidermoid and undiffer- 
entiated carcinojisa (grouped together) (5 out of 50 cases). 

A<>139 causes heitopoietic depression and phlebitis at the site 

of intravenous injection. The drug caused «m)od®p®£« but reversible 
toxicity when given in doses of 0.6 mg/kg/week intravenously. 
Abrupt and severe hesropoietic depression occurred at all doses 
used (0.1 to 0,3 ™g/kg,| when the drug was given intravenously daily. 
Moderate blood eount depression occurred at all doses (0.1 to 0.6 wg/kg.) 
when the drug was given orally daily. Eighty four cases were studied 
by the Cooperative Group, II from the H,CJ, Sixty four cases h^d 
tumor i!9sss®s whose size could be followed. Suggestive antittanor 
activity was seen in bronchogenic carcinoma, raaawBary carcinoisii, 
ovarian carcinoraa and lymphonfia. 

OichlormethotreKate was found to cause hesmopoietic and gastro- 
intestinal toxicity; doses of 0.5 to 1.0 pg/kg/day could be tolerated 
for several wseks by wost patients. Antitumor effect was seen in 
l^phosBsas and in adenocarc i nora^ of the gastrointestinal tract. 

4-As»inopyraEalo!>yriiKidine is tolerated equally well when given 
orally or intravenously. The drug produces nausea and voroiting in sossie 
patients and prolongation of the prothrosibin tirae due priraarily to 
factor VI 8 depression. The reversibility of this specific effect 
(though general hepatic dssts^ge occurs in anitaals as the dose is increasedl 
is being studied. 

The study of serum enzyme levels in patients receiving therapy is 
not consipleted. There are marked decreases in abnorrasally elevated seruro 
levels of lactic dehydrogenase when tumor isjasses decrease in size. Zinc 
ion will in certain concentrations i£9er®@^ this enzywiye activity of hteisian 
sera, especially when patients have disseminated n»alignant neoplasni). 

It should be ensphasized that all of the above studies are integrated 
and collated with other phases in the ascent of an antitumor agent, 
froro cheswical synthesis to definitive trial in roan. Thus the results of 
quantitative cooperative trials may be of priraary value to investigators 
concerned with snSroal tumor or other screens or to the chemist, 
More generally, the practical value of all of the prelitiinary phases of the 
iimense cheimotherapy program rwst necessarily be determined in quantitative 
studies swch as the above. 

Signific ance to Cancer Research; When an agent has shown activity in aniraal 

tjfissors or other screening systeiws, if this nwaterial is to be tested in huroans 
for antituewor activity, its pharmacology including the type of toxicity it 
!5iiay cause and its ability to cause somte deflfsonstrable antitiraor affect in 
huisians at tolerated dose levels is required. On the basis of such studies 
tmch about the value of an agent can b® found. Only when © drug has been 
found prowising in these studies is it switsble for a definitive eotsparative 
clinical trial to quan^batc its antitwor activity in a specific type of 
huwan neoplasnii. Coniiparative clinical trials are deroandis^g in resources, 
so laethods are of value which will limit the nwber of agents for which 
such trials are iimdicated. 

indirect methods of following tuRsor growth would make it possible to 
study cheifisotherapy in those several tJusior types who t»or »s§s@s ar?? 
seSdod directly d«»onstrable. This includes usore gastrointestinal ^nd 
KBiany gen i to^uri nary twnors. 

846 



II 



6 " Serial Mo = NCI~5-7l2 ',£} 



Proposed Course of Project; The studies now in progress arei 

Toxicity and PreMminary Antitumor tpJ©! of Uracil M&s^t^^rd. 

Toxicity and Preliminary Antitiroor tr5©ll of Monochloriuietho- 

trexate. 
3) Study of incidence of infections and Antibody Response in 

Chronic Lyiwphocytic Leuke»iniif) with «.nd wittwjut Adrenal Steroid 

Hormone Therapy » 
k) Preliminary antitiwor trial of if-gainopyrazf'.lopyriisiiidirse, 
5} Serwi Engyiroe .ftwdiei. iet *;©llid iasmf patients with roeasejiriDblle 

tumor masses during therapy, 

1) This toxicity and antitwwsr trial is being done by the 
niejmbers of the Eastern Solid Tumor Cooperative Groyp and is 
just begun, 

2) This study is primarily a toxicity study. The drsuig se®?,s 
to be quite toxic and perhaps further studies will be Sim^ited, 

3) This study i s wel 1 under way and shotsld be cofsiipleted 
in a few weeks, 

k) A trial to bring about sojie response in patients with 
chronic leukesaia will be isnade, 

5)) !n addition to seruw lactic dehydrogenase, it is in- 
tended thst nucleotidase and l€(ucine»tiinopeptidase be studied 
in patients dasring therapy. 

In addition to cmspleting the above studies and selecting future 
promising new agents for steady there will be a continuing reexaswi nation 
and exploration of ssi^thodology, particularly that related to exper° 
imental design. Problems in this ar&s receiving issajor attention 
at the present are; 

The extent to which toxicity studies should be contsirolledi 
Criteria for response to experiesssntal therapy, 

3) The interrelationships of various modalities of response; survival, 
decreased ttsroor size, s^gs&raatic iroproveraent etc. 

k) The problem of controls in essentially non^responding tyisMJrs, 



Jl 



847 



Serial Ko. KCI»>712 (C) 



Individual Protest Report 
Calendar Tsar 1958 



Bart 38 Hb&orBB Avard«« aod Pablications 

Ptiblicatio&BS "Dirsot Cbserration of Lesion Size asd 
I^CBQber as a Method of Folloving the a»>vth of Euman fcBnorB"» 
iQr Clyde 0. Brindleyt ]iilliott rtarkoff aiid Marvin Ao Schneiderman« 

This paper has 1)eeni accepted for publication V the Jo-umal CANCER 
and la to be in the Jamtax^oFehruaxsr 1959 issue. 



848 



PHS-NIH 
Individual Project Report 
Calmdar Ysar 1958 



Serial No„NCI=- 5-737 <c) 

Ic General Migdleine' Branch 
2„ Chemotherapy Service 
3p Bathes da 



Part Ao ', 



Project Title: Infection, Fever and Hoot Resistance in 
Malignant Diseasec 

Principal Investigator: Emil Frei III, M„Do 

Other Investigators, Dane Boggs, MoD. , Richard Shaw, M„ Do, Maurice Land3F»Ph<0„ 
John UfcZj M<,D„ and John Fahey, M.Do 

Cooperating Inits; 1„ Laboratory of Clinical Investigation, NIAID 
2o Clinical Pathology 
3o Metabolism Service, NCI 
4o Laboratory of Pathological Anatomy 

Man Years (Calendar Year 1958): Patient Days (Calendar Year 1958)° 
Total: 4 
Professional: 2 
Other: 2 

Project Description: 

Objectives ; 1„ To study the effect of malignant disease en 
the hosfc as regards resistance to infection., 

2o To explore the effects of malignant disease oa cellular 
and humoral factors of resistance , 

Methods; Case Material - All patients oo the Chemotherapy 
Service are potential candidates for these studies. 

Laboratory - fhe pathological, serological and bac<= 
teriologlcal, biochemical methods are standard except as specifically 
qualified belowo 

Major Findings: 

lo A study ©f quantitative and qualitative observations of 
fever aad infection in patients with acute leukemia has been reportp-d 
(see project report NCI-4-717 1956)^. fhe' 

mature neutrophil in patients with acute leukemia has been fo«nd to 
have normal ability to phagocytiee bruella organisms. Infections in 
patients with acute leukemia are usually preceeded by an abrupt fall 
in the absolute neutrophil counto Those observations have been pub- 
lished. 



Part B included Yfis 






849 



" -'^- Serial Ko. NCI»5-737 (e) 

2o Aatibody response in aeufcc leukeiaia„ 

fen patients with aewte leskemla along with 10 confcr&l 
smbjeces were given 5 (3 bacterial and 2 viral) antigeas aad their 
antibody response compared^ All of Ch® patieats with acute Istskemia 
had a 4 fold risd in titer with ae least nfm of £h@ antigens aad on 
the whole the response of patient® with leukemia approaehed \mT, was 
somewhat short of the normal response. There was considerable vart~ 
ability among the patients with acwfee leukemia in terms of aistibody 
response. This variability could Boe be correlated with feh® fre- 
quency o£ infections or other elinical factors o fhis data is b@ing 
prepared for p^blieationo 

3a Fever and infection in maXignast disease „ 

The frequiency aad type of infection in patients with 
chrtonic leukemia, Hodgkins disease, lymphosarcoma «, carcinoma of the 
lung as well as acute leukemia was studiedc Fever of uade£erm£sed 
«tiology was most frequent in aeiate letakemia but occurred eignifl° 
cantly in all of the above disease save chronic lymphocytic leukemia,, 
It was found aot to increase as tli« disease progressed in duration^ 
to correlate poorly with periods of rapidly accellerating disease^ 
and sjot to be associated with specific organ involvements fliue quali°- 
tative factors related to the primary gype of malighaat disease woisld 
appear to be the important factors. In contrast, infections is»ereased 
in frequency as the diseass progressed in duration and aetivitfo 

Ilse characteristics of the fevers of undetermined etiology were 
studied and found to be quite similar to those associated with infec° 
tion except fehat low grade fever was more frequent in the former cate- 
gory » 

The etiology and location of the infections was found to be 
greatly Influenced by the primary diseaseo The effect of aafeibioticsg 
corticosteroids, antimetabolites and alkylating agents with respec£ to 
the onset sdA cessation of fever aad infection were asalyzado 

4, Clostridium infections. 

Four episodes of Clostridium perfringens septiesraiap 3 
in patients with acute leukemia and one in chronic leukemia have beeis 
carefully analyzed clinically and pathologically, l^e pathogenesis has 
been inferred from these studies and definition of the 8yaq>tom eoo^lsx 
should make antemortem diagnosis possible, 

5, Pseudomonas septicemiao 

Twenty-three cases of pseudomonaa septicemia^ the majo= 
rity in patients with acute leukemia have b«en studied clinically aad 
pathologically and some of the Interrelationships with the primas'y 
disease process defined, 

6, Astibody production la chronic lymphocytic leuk@miao 

The frequency of infections and the observation that 
some patients with this disorder have hypoganaaaglebullnemia prempt«d 
a more extensive sL^udy of host resistastee in this disorder, Twaaty^ 
three such patients have been studied as regards antibody response to 
5 asitigens. Diminished response was observed in most of &he patients 
aad response was completely lacking ia soma. Response to antigsns 

850 



= 3 » Serial No. NCI- 5-737 (c) 

producirag delajred skiQ ?eac£losis baa not bean evaluated eaj^efull^ 
l«it would appear to be normals Oa© half of these patients will re=- 
ceive corticosteroid therapy aad all will fee followed to de lersaiae : 

a) fhe toterrelatioaships of ganraa globulia Isvel, aatifeody 
productioa aad frequeocy ef issf^etioiio 

b) f!&@ effect of corticost@?oid therapy on th@ frequ^aey of 
iafsctiose, (thi* is a major problem in practical cliaicat medicineo 
(tely a cositrolied study sueh as the above eaa adequately aaewer the 
question.) 

c) fhe subsequent response to a booster dose of certain o€ 
the antigens initially @mploy@de 

d) th^ relationship of the gaiona globulin level asd antibody 
production to hsmatologie evidence of disease activity;, survivslg etCo 

Finally, it is planned to extend these observations to include 
study of the homograft reaction in patients with a gamma globulinesBia 
and depressed antibody response secondary to malignant disease, fhese 
will initially include skin grafts and may be @ztended to lyzoph giode 
homografts VTith appropriate isanuniologie studies, tf hoiBOgraft accept 
tance is demonstrated, even for a relatively brief period, such pa» 
tients may be selected for total body S'' irradiation and bone marrow 
transplantation. Patients with chronic lymphocytic leukemia are in 
some ways ideal £o? these studies becaisse the disease is very sensi» 
tive to x°ray, and death xi^en it occurs results from bone marrow 
failure. 

80 Fever of Undetermined Etiologyo 

This occurs frequently, presents a problem in maasgeaent, 
and is completely undefined pathogenically, A study is currently in 
progress to defitui some of these probleos. All episodes of fever occur° 
ing on the Chemotherapy Service are comprehensively studied diagfflpst£cally„ 
Tl&ose v^ich are not obviously bacterial have viraE cultures on three tis« 
sue culture cell li/ies of the pharynx^ sersan and stool. In addition 
quantitative bacterial studies of the pharynx and stool are performed. 
A random half of such patients are placed on tetracycline for a period 
of 14 days. Repeat bacterial studies are performed at 10 and 18 days. 
1%© objectives of this study are to determine: 

a) «laether clinically unrecognised viral infections play some 
role in such fevers. 

b) the influence of antibiotics on such fevers and therefore 
the importance of obscure bacterial infection in the production of su@h 
fevers. 

c) the influence of short terra antibiotic therapy in "normal" 
bacterial flora. 

d) the importance of antibiotic administration in the production 
of superinfection with resistant organismso (fhis is a. major problem in 
the management of cancer patients as outlined above.) 



In a further attempt to define the nature of this fever efforts 
will be made to demonstrate circulating pyrogens. Circulating pyrogen 
has recently been demonstrated in experimental infection la animals 
(King et al„ J. Exp. Med. 1958). Xt is planned to take plasma from 
patient© with fever of undetermined origin, preserve it, aad inftsse it 



851 



' ^ - Serial Hbo KCS»5-?37 fe) 

iato the same patient when he has become afebrile o If in sisch 
asjtologous trasisfess circulating pyregen caa be demons Srafceds, 
attempts will be made to develop anlmsl assay systems.' X£ such 
sen be developed fractionation studies to further identify the 
pyrogen will be undertaken o 

9, Fung«2s infections. 

Oral monllieeis is a frequent problem in clinical 
cancero In eollaboratian with the NIDR s comparative study of 
saline and mystatin in the treatment of oral moniliasis is %adar° 
wayo 

Significance to Cancer Research: 

All of the above has direct or indirect applicatioft in 
the clinical managensant of patients with lealignant disease. 

Proposed Course of Project: 

to the present time our studies of host defense have basiE 
largely limited to clinical observations a These will be coatimsed 
aad extej2ded as indicated. As pointed out above more extensive 
investigations are being considered ia the following areas: 

lo Homograft reaction ia selected patients with markedly 
altered ineoune response.. 

2o Deoonstration of circulating pyrogen in autologotas 
systsanso If present and demonstrable esteasive sttidies will be its 
order, 

3o Though marked increase ia susceptibility to bacterial 
infections has been demonstrated for acute leukemia our studies 
4a4icafce that viral infectitms are infrequent, IhiSs plus the 
observatioas eoaceming lymphocytic choriomeningitis virus and 
mouse leuk^nia, suggest that work with other viruses in animal 
leukemia are in order o 

4o fhe observations of invgstigators in the Laboratory 
of Oiesaieal Pharmacology concerning natural defense mechanisms 
will be extended to patients. 



852 



.■:C:l-5-737 (c) 



Indlvidiaal Psoject Report 
Caletadar Year 1958 

Partes Boix>;s, Awards^ aad Publications 

Pabllcatioa® other than abstracts from this projects 

lo Silver, So To, Uts, J.P,, Frei^ Eo and MeCulloisgh, N.b/ 

Fever s Infection aad Host Eesistaace in Acute leukamift, 
Am« X of Medog l!25»39e Jamsuay 1958c 



Beaors asd Awards relating to this project? None 



853 



Xadi vidua! Project Report 
Calendar Ie»r 1958 



aerial !!SOoi!SiCjL=i-k!iei(^c, 
i. SMS '~ 
2, CP & m 
3o lethesda 



Part A, 



Project l^itlei Pgaetratioa of Aati-tuaor Drugs into the 

Central lervous System 



Principal Investigator: David Po Rail, C 
Other Investigators: 



Zubrod 



Ifea leers (calendar year 1958): 
fo-fcal: 2 1/2 
Frofessioaal: 1 
Other; 1 i/2 

Project Description; 



Patient Days (calendar y^ai- I958): 




Objactiveeg ^e objectives of this project are to liuantify 

the penetration of certain anti-tumor drvsgs iato 

the central nervous systm (CIS) and to atteismt 

. to elucidate 80S® of the factors influencing the relationship 

between dose of drug, plasm level, and concentration of drus: 

in the CiSo ^ 

^tho(te: rase pharasacologic technics developed and validated in 
previous years wer® tased in tbese studies » 

liajor Findings; Evidence was obtainad which strongly supports ths- 
4 ^ ^, ^^ iiypothesis, developed last year, that pi gradients 
taiportaatly affect the distribution of certain weakly, ionised drws. 
Cy means of inducing either a&etaboUc or respiratory acidosis or 
alkalosis it was possible to tset the effects of acidosis sssd 
aiisaiosis (with and vit&out concaeaitant blood-CSF pH differences), 
on drug distribution between plasjaa and CSF. When CSF p® wms lowar 
than blood, weak acids were excluded from, aad weak bss«s eoncen- 
trat€d in CSF, and vice versa, irrespective of the absolute blood 
pMo 

Lipid solubility, as ajeasured by the chloroform / a^osous buffer 
drug distribution ratio was shown to correlate, in a general way, 
with the rat® of entry of drugs into CSF. 

flaase results parmitted the forsauXation of a tentative hypothesis 
as to the fwctional characteristics of the blood CSF barrier « 
?^®«^^** *^® coiBpietely ionized at body pH will be ®xcl«ided 
from SSFo Drug® which ar« 0,2^ or raore unionized at pBl 7,% %riii' 



Part 1 included; fSS 



854 



Geriai go..MGjC~i,°223l.c) 



enter CS?.. 1!lJ.e greatei- tfee degree of vmionization, the greater 
the extsnt of drug entry irito CSf. Kie CSF/plasma ratio at 
equiiibriim will uader norajai circvoEstances vary from 0.? to i»6 
dep^adiag ■ upon the ®xiEtiiig pi ga*adiesat. Driags which are completely 
uaioaizsd at body pi will achieve a CSF/plasma ratio of unity » The 
rate at which the ®q.uilibriviiiB ratio is reached will depend, in a 
general way, upon lipid solubility. This w&j be convenieatiy sujb- 
oarized by suggesting that tise blood CSF barrier has the fraiictional 
charac'fe«ri8tics of the ceil wall, aad that GSF is phanaacoiogicai ly 
aEalogo^J8 to intrac@ll\xlar space « 

Studies incideatai to the laajior project on the distribution of 
infused emsonia between blood, aad GSF brain and muscle showed 
that this weak base conforms to th© above hypothesise fhis ob- 
servation should allow assesssifijat of the role of aismonia toxicity 
in hepatic coma. Patients in hepatic coaiaa exhibit abnonaal acid 
base balance, and blood ajBasonia valwes can b© used to predict brain 
auamonia coacentrations only if the appropriate pH values can b^ 
ffisasured or estiasated. In addition, th©rap®mtic measures which 
incidentally resitit &Sk a«id acbalnistration^ BsSt^ f glutamic acid 
infusion, leay act onl^ by virtue of the pi alterations producedo 
Studies were conducted on the distribution of siuinine, a lipid 
soLubi®, weakly ionized has®, in the dogfish (S. acanthias). An 
unnisual phenosaenon was noted. If the blood of a dogfish which had 
been given <iuinine was hemdled with utsaost care the liuinia® was 
found to be concentrated 50x in th® red blood cells, and lOOx isi 
the Isuffy coat, as cosap&red with piasam^ If care was not exercised 
in drawisig blood, these ratios fell to 5-iOx, a^d the plasssa. con- 
ceatratioa iiacraased five to tenfoido In a few preliminary «.<.- _ 
perisaeats is which blood was drawn carefully, (iiiinine was shown 
to be concentrated in the acid pericardial fluid, as compared with 
plasma o 

In prsiiffilioary experiissents ia which the t«chnic developed i^ the 
dog was applied to msnti it was shown that the e4,uilibrium ratios 
of a number of drugs ware essentially the e&&@ in isan and dog, 
but the entrj rate was much slower in Ean. 

Slgpjficanc® to Cancer Research ; fianors, both prtaajy end 

Kstastatic, fre^uisntly devolop 
within the central nervous 
systea. Little iioforisation is stvailable in the published literature 
concerning the penetration of anti-tvaeor drugs into, the OfSo Basic 
research on this aspect of the pharmacology of anti-tuss^r dr^ig® 
is necessary for the rational use of these agents in the treatoeat 
of paties^ts with t«ss&ors locatM in and aroimd the brain o 

Proposed Courses ^© following lines of investigation ar® pl^mied; 

io f\crtSser attei^te to elucidate scam of tlie basic phartmcologic 
factors which determls^e both hov much, and how fast drvi^s get 
into tt<B CSM and C3W frost plasasao Ihes®. iacltsd® syst^isic artJisriai 
press<are5 cerebral blood flow, stat® of ttoyroid function, @tCo 



855 



- 3 - 

2a Continued investigation of th© comparative ptoairoacology of CIS 
and CSF. ffce technical ease with which the ventricvilar fluid 
and brain are obtained in dogfish make this species particularly 
valuafciSo 

3" Studies in patients with suitable neoplasms will hm comtinised, 
and exteaded to include the us© of aati- tumor drugs. 



856 



Serial. 10o?iSI=i-223Cc' 



Individual Project Report 
Oalendar fear 1958 



Part D: 



Rail, D. Po, Stabenau, J. R. and Zubrod, Co 6= 
Distribution of Drugs Between ISlood and Cerebrospinal 

fluid: General Methodology e^A Effect of pi Gradient c 
J, Ffearssacoio in press 

Zubrod, Co Go, and Rallj, D. Po Distribution of Drugs 
Sstveen Blood and Cerebrospinal Fluid in tlie Various 

Vertebrate Classes o Jo Pharaaacol. in press 

Stabeimu, J, Ro^ Warren, Ko S. and Rail, B.Po 

Effect of pi Gradients on the Distribution of Aiamonia 

Istween Blood and Muscle Irain imd Cerebrospinal Fluido 
J, Clin. Investigation. in press 



857 



jlndividiiai froject Report 2o cjp- * iS 

Calendar fear i95Q 3,, ]^ 



Part Ao 



Project fities ffee Ph&nsacoiogy of Antituiaor Agents Related 
to Pur in© 

Principal Investigator; fi Li Loo 

Qfcher lEvestigatorss Mone 

Cooperating iluits: Laboratory of Pliysioiogy, ISI 

Wssk Xaars (cEisndar year 1958); 
Total; 12/3 

Professioaai: 2/3 

1 



Project Descriptions 

Obje ctives ; fo study the fate end ffietaboiissa of 'certain anti<= 
"^ traaor agents of the purime group curreatiy in us®» 

Istliiods: Standard chemical and biochemical methods vers easploy@dc 

Major ^IndiiRgE; 

lo Oa® of the asajor routes of metabolic transformation of 6= 

asrcaptopviriffi® C<S®P) is hunsaaiB m&b shown to be through its 
oxidation to 6=thiouric acid, th«s confirming the prelimiEiarj 
report of Eiiorio 

2o 6>=f%iiouric acid was successfully synthesized for the first 
' tlffi® istad its structure was established by comparison vlth 
its two iscaaers, naaeiy, 2= asad 8-thiouric acids which w©rg 
syntj&esizftd by ua<s«4Uivocal lesthodSo 

3c fhe synthetic 6*thiouric acid was identical Ik all rasp<tcts 

with on© of the metabolites isolated from the urin® of patiests 
treated wifcife ©1^. 

i^o Siiffiilarly, it was observed tisat 6-ttoiouric acid was oia© of the 
metabolites in mice injected with ^IP» 

5 c Enayaic oxidation of SMP by -iCiathijae oxidase was also shown to 
yi«sid 6-thiouric acid as one of its products » 

60 6=>^£feiojumthia®, alt&ough conceivably an iaterKedlat® iro the 
biological oxidatioKi of 6ISP to 6«'thiouric acid, was not de<= 
tectable: in the urime of mi.Va^v "himmiB- or mic^o 



Fart ® included: 



858 



Serial lo^^^^^^^ggSCc) 



7. Ey the us© of ion-exchasage ctoroaiatography, it was demoas- 
trated that there was another metabolite of 6MP which could 
be ®luted from th® chroB^tographic coluum concoiaittaiatly 
with uric acid» Its occurrence was confiru^d after the uric 
acid had beeHi destroyed bj uricase.. 

Signifi cance to Cancer Research; fhe studies of the metabolic 

fate of antitvaaor agents ar© 
important because of the light 
such studies could throw upon the mode of action of these agents » 
Further, the laechanisiB of the deYelopsasnt of drug resistaace could 
also be partly elucidated through the metabolic stwdies. 

Proposed Course ; 

1, Contijiu® the isolation and identification of all metabolitss 
of ^ in both responsive and resistant patients » 

2. Extesid such sti2dies to €MP riboside o 

3o Extend such work to other purine aatagonists <> 



859 



Individual Project Report 
Cftlejadar I©ar 1958 



Publications g 

lo Loo, foLo, and Micljael, MoE», A coiorimetric method 
for the determination of certain purine aatagonists, 
Jc Diol. Chemc, 232, 99 



too, fotc, Michael, MoEo, Garceau, AoJo, and Reid, 
6=thiouric acid, a metabolite of 6-mercaptopurineo 
Jo Am. Oh<ano Soc, in press <> 



Serial lo« 101=1-226 ((c) 

lo mm —^-^ 

ladividual Project Beport 3^ Bethesda 
Calendar Xear 1958 

Pa rt_A^ 

Project fit!®; Hmnmcoiogy of the Folic Acid Aiatagonists 

Principal Investigators David Fo Rail 

Other Investigators; Emii Frei^ III^ Montage Lane, fi Li Loo 

Cooperating Unit: KI =■ »ei =■ Choaotherapy Service 

Man fears (calandar year 1958): 
fotai: 3 
Professional; 1 
Others 2 

Project Descriptions 

Objectives : 'fo elucidate the mechanisms of action of the 
folic acid antagonists and relate than to 
clinical chemotherapy » 

Methods ; f&e general msthods have been previously described c 
Otherwise standard pharBsacologic methods were 
eisployedo 

Major Findings; 

1. (With Dto Loo) fhe fluororaetric iss'tJiod develop@d for amat&opterin 
(M^sthotrsxate) in this Service in the past years was adapted to 

■ the ffisasuresB^nt of isionochlora!iaathotr@xate (MEM) and dichloro- 
i^thotrexate {©«) ia biological fluids » 

2o Tb.e pharmacology of EGM has been coaKpared with tJjat of Eiethotrexate 
(M?X) in dogs and man. , TKM has a greater voliiime of distribution 

than MfX, and disappears from the plasiaa at a slower rate. Plasma 
concentrations of EOS are higher after paraateral as compared 

with oral drug administration. 

3. (Dr. Ian®) tto® oraJ. toxicity of DGM was determined in 10 patients 
with solid tiamors at doses of Oo5 to 3.0 JBg/kg/day, In general Oo5 
ffi5g/kg/day was tol®rat®d for periods of k2 day®, fhe evidences of 
toxicity were referable to the gastro intestinal tract and hasaa to 
poetic eystim, la 2 patients with reticuliaa cell sarccsaa tSi®r® 
occurred a significant decrease in th® siz© of issaswraMe tiaaor 
5!^S8@s following D€M adiministrationo 

PARI D ineludeds 10 



861 



a®ri®l ioo !iai-l-=226((@; 



Bis^iftQ^Mcm to C&ne^r ^e^oe^ishi A d(s-fe8i,il<gd toowlmdge of thm 

total pharmacoiosf of the folic 
acid antagonists is important 
becaBSe of iattrest in these drugs theaiselves ajsd because ttoey ©r© 
representatives of the larg© class of antimetabolite dmgSc fhe 
general priBCipi@s of the cliiaicai lise of the folic acid anisagonistSj 
which should result froai this study, imy well be applicable to other 
antimetabolites <> 

Prooosed Course? 

lo A stua/ of the total pharmacology of MIX, DCM and MEM in Eice, 
dogs and mano 

2o fhe lastem Cooperative Sroup in Solid f^aaor Chemotherapy is 
studjiBg the ccsnparative effect of MSX and lOS in iy!iphoi8as <, 



862 



Serial lo. ISl-1-227 
i. GMj 
PHS-IH 2. CP & EI" 

Individual Project Report .3« 
Calendar Tear I958 



Part A. 



Project Title: Mechanism of Antitumor Action of Purine Analogs 

Principal Investigator: Jack D. Davidson 

Other Investigators: lone 

Cooperating Units: None 

Man Tears (calendar year 1958): Patient Days (calendar year 1958): 
Total: 2 
Professional: 1 
Other: 1 

Project Description: 

Objectives : The objective of this project is to detemaine as 

specifically as possible which biochemical reactions 
are inhibited by certain purine analogs in cancer 
cells that are sensitive to these coiapo;jnds» A 
corollary objective is to elucidate the mechanisms 
responsible for inherent and acquired resistance 
to these agents. 

Methods : L-1210 ascites leukemia cells are incubated in vitro 
with and without the purine analogs and with various 
isotoplcally labeled nucleic acid precursors. The re- 
sultant distribution of the isotope in the Wk, MA 
and acid-soluble moieties is determined for the treated 
(purine analog) and xintreated cells of driig sensitive 
and drug resistant lines. 

Major Fin dings: The principal positive findings relate to 6- 
roercaptopurine (fiiJP). This compoimd inhibits 
the incorporation of hypoxanthine into nucleic 
acids in L-1210 leukemia in two different ways. 
In the strain of cells sensitive to 6MP (L1210S) the dominant block 
appears to be in the conversion of inosinic acid to adenylic acid, 
a pathway also essential in the de novo synthesis of adenine moieties 
and apparently inhibited by a nucleotide metabolite of S^SP. |n the 
leukemia subline that resists the antitumor effects of ^CP (Li2iOR), 
hypoxanthine is normally utilized only 10-20 percent as well as in 
L1210S but this utilization for both adenine smd j^uanine synthesis 
is fully inhibited by 6MP. !l!his implies that ^SP is competing with 
hypoxanthine at the free purine level for the limited enzymic pathway 

Part B included; YES 



863 



Serial MOo liCt'l'22J 
- 2 - 

to their nucleotides. High concentrations of 6MP produce this 
same block in Li210S. 'Ihe "resistasice" of L12L0R appears to be 
attributable to this low capacity to convert a significant amount 
of 6MF to the ribotide derivative. 

fhe foregoing hypothesis as to the mechanism of action of 61^ and 
L1210R resistance is supported by other experimental data. Glycine 
enters nucleic acid purines by the de novo pathway. Its incorpo- 
ration into L1210S nucleic acid adenine moieties is inhibited by 
6MP but in L1210R its use is unaffected. 

At Southern Research Institute it was found that L1210S produced 
nucleoside and nucleotide metabolites of 6^^ while L1210H did not. 
At the same tiise experiments in our laboratory showed that Sw 
penetrates into L1210R cells just as well as it enters L1210S and 
it is therefore possible to attribute the failure to foria ribosyl 
derivatives to a deficiency in active enzymes for this conversion, 

A few studies were made of the metabolism and excretion of another 
purine analog, 4-aminopyrazolo(3,^-d) pyrimidine (ifAPP) following 
intravenous administration in man. lo free k-AFP could be found 
in the urine later than 6 hours after adrainistration but a con- 
siderable fraction of the dose was excreted as the 6-hydroxy 
metabolite and was detectable for up to 2k- hours. 

Significance to Cancer Research ; Some of the laost effective antitijaor 

sigents thiis far discovered are 
pxirine analogs. A knowledge of 
their mechanisms of action and the 
reasons for resistance to their action should permit more rational 
use of combinations of agents and new derivatives to secure se^ 
q,uential or alternate metabolic pathway blocking and improved 
antitumor action. 

Proposed Course ; 

1= Further studies on 6MB to confirm and define the effective biocheajical 
locus of action, e.g. its relation to adenylosuccinic acid as an 
intermediate between inosinic acid and adenylic acid. 

2. Studies on human leukemic cells to determine whether the mechanisajs 
of action emd resistance are the same as in L1210 leukemia. 

3. If 6MP resistance in human leukemia is due to a failure to convert 
the drug to its ribotide, attempts should be made to make diester 

or other derivatives of Q4P-nuclaotide . ^Ttiese might serve to provide 
the active drug in such a form as will enter cells that appear im- 
permeable to the nucleotide per se . 

he Further studies of the phanaacology of pyrazolopyrimidines in 
humans . 



864 



Indiviiijai Project Report 

GaLendar Tear 1958 



Fart B; Honors, Awards and Publications 

Publications other tfean abstracts In 195ii attributable to this 
project: 

Jack Do Davidson j Homogeneous Counting Systems, 
ppo 88-95, in "Liqxiid SciffiitilLation Counting", 
Pergamon Press, London, 19580 



D060; I^yideoK, JoD=; Wsiggoner, J = ; and 
Dsrlin, l.Ioj ffce Counting of Dariusa Carbonate in 
a LisLuid Scintillation Spectromster e 

J a Labo ^ CliUo Med. In press » 



865 



Serial lo. lCI-l-229(c" 
1. GM3 
PIS-IIS 2. CP & Iff 

Individual Project Report 3- Bethesda 
Calendar Year 1958 



Part A 



Project Title: Phanaacology of Riboflavin Analogues 

Principal Investigator: Montague Lane 

Other Investigator: Clyde 0= Brindley 

Man Years (calendar year 1958): Patient Days (calendar year 195 
Total: 1 
Professional: 1/2 
Other: 1/2 

Project Description: 

Objectives ; 
A. Clinical (Drs. Lane and Brindley) 

To study in patients with neoplastic 
diseases the pharmacology of riboflavin 
analogues and to attempt to induce 
tumor regression and/or clinical 
riboflavin deficiency. 

Bo laboratory (Dr. Lane) 

To study the mechanism of action of 
riboflavin emalogues at the enzymic 
and cellular levels and the transport 
of flavin nucleotides. 



Ac Clinical Photofluorometry, chromatography and 
bio-assay techniques are used. 

D. Laboratory Continuous spectrophotcMHstry, bio- 
assay. Isotope, enzyme techni*iues and 
animal chemotherapy are used. 

Major Findings ; 

A, Clinical 

Galactoflavin was given in doses of 1 to 3 g-/^y 
for six weeks to three patients. Severe glossitis 
and papillary atrophy of the tongue was noted in 
one i)atient. Two patients showed depression of 



Part D included: tES 



- 2 - 

•hematopoiesis and fragsientatloia and lobulation 
of nuclei of red cell precursors in the bone 
marrow as is noted in therapy with folic 
acid antagonists. 50 to 150 mg. of galacto- 
flavin are absorbed after single daily doses 
of 1 or more gm. by mouth. 

B. Laboratory 

Chemotherapy of txmor bearing animals with 
U-6538 and 6-MP did not demonstrate synergism 
between these agents. 

The growth of L. easel was inhibited to a 
considerably greater extent by a number of 
riboflavin antagonists when flavin adenine 
dinucleotide rather than riboflavin was the 
growth limiting flavin metabolite. 6,7-"<iinie'biiyl- 
9-(2' acetoxyethyl)-isoalloxozine, U-2112, is the 
only antagonist studied which did not behave in 
this fashion. It is hoped that the explanation of 
this phenomena may shed light on the mechanism of 
flavin antagonism. Preliminary studies in an 
isolated enzyme system (D-amino acid oxidase) were 
inconclusive. Studies to determine whether FAD can 
enter living cells intact have only begun recently. 

Significance to Cancer Research ; 

A. Clinical 

Riboflavin analogues have been shown to 
inhibit rat tumor growth. Ihese studies axe 
designed to determine desirable dosage and 
routes of administration, and signs of drug 
toxicity for humans. The absorption, distribution 
and metabolic fate of these drugs are of impor- 
tance in evaluating their clinical effects. 
Pharmacologic data derived from a few patients 
shoxild permit prediction of the potential activity 
of m®nbers of this grottp of compounds. If 
significant riboflavin deficiency is produced, 
it will be possible to test the effects of such 
deficiency upon neoplasms in wea. 

B. Laboratory 

An vmderstanding of the mechanism of action 
of riboflavin antagonists may provide a clue 
for the design of more potent chemotherapeutic 
agents. 



Serial lo. , . 
Kei-l-229(c; 



867 



Serial Ho4fC2;-i»229Cc) 



Proposed Ooxirse ; 

A. Clinical 

Continued clinical trials with galactoflavin 
in combination with antibiotics in order to 
minimize intestinal bacterial synthesis of 
riboflavin. 

B. Laboratory 

1. Continued studies with isolated enzyme 
systems . 

2. Study of nucleotide entry into bacterial 
and mammallEin cells o , 

3. Study of protein binding and attempts 

to isolate the protein or proteins involved 
in the binding of B^, mm, FAD, and 
riboflavin antagonists. 



serial HboK;i=l-229Cs} 
PES - HI 
Individual Project Report 
Calendar fear I958 



Part Bg 

PuMicatiosig 



IfiE®, Mo, Petering, 1.6 o, and Brindley, COo, 
Systhesis, Pharmacology and Cliraical trial of 
the Ribofiavia Analogue, Sodium=6,7-Diinetliyi- 

9- (2 « -Ismisuccinoxyetliyl ) - isoaiioxazine U-65 38s 
Joijrnai of tk© lational Cancer Institute (in 
Press) o 



869 



serial IfcioSCI 1-230 (c) 

ras - Mm 

Individual Project Report 



lo (SMBi 
2, CP & 



Calendar fear I958 3^ Bethesda 

Part A» 

Project fitle: Chemotherapy of lfe.lignant Carcinoid 

Principal Investigator: Paul f » Comdit 

Other Investigators: S3one 

Cooperating Units: Mone 

Mass Tears (caiesidar year 195^) s Patient Days (cslendar year 1958) 
fotai: 1 
Professiossal: l/2 
Other: i/2 , 

Project Description: 

Objectives; To deterttine the response of patients with 

malignant carcinoid to chesuDtherapeutic aget&tSo 

Methods ; Drugs with Icnown antineoplastic effects vera given 



to patients with isalignaat carcinoid « fhe daily 
larinary excretion of ^-hydroxyindoles was measuredo 

|8:.lo consistaffit ch^iges in J-^dxoxyindote e.icretion 
ver® noted after autitimor therapy » 

Significaaee to Cejacer Research; Pati^snts with SBaliguant carcinoid 

present the unusual situation of 
a tumor which produces wa easily ffieaeiu-able metabolic piroducto Chaaog^s 
in the excretion of the ^•'hydroxyisidoles nsi^t provide a m>Tm subtle 
index of drug activity tbaa cbassges in tussor size 

Proposed Course; Thxs project is being discontinued because the 
"■ "^ principal investigator has left. 



Fart D included: 



870 



Serial IIOci5i.i=,23i(cl 

Individual Project Report "j] -^^^i,^^^^ 
Calendar fear I958 



Parfc Ac 



Project fitie: Fluorochrom® Metabolism 

Principal' Investigators; fi Li ioo 

Other Investigators: Margaret ©„ IGslly and Bsrnice Eddj 

Cooperating Units: DBS, Laboratory of firal Products 

Man Tears (calender year 195 8) s 
totals 1 1/2 

Professionals 1/2 
Other: 1 

Project Description: 

Objective; Elucidation of the biological, pharaiacological, 

Bxid biocheaiical factors involved in the phenomenon 
of persistence of fluorescent material in aaimal 
asid htmaa tumors aad bone after csssation of the adaainistration 
of aieabers of the tetracycline group of f luorochromes o 

gfethods ; Im addltioja to standard phanaacological and Tbiochtiaicai 
methodology, the lowaan spectrophotofluorometer, the 
Sary recording spectrophotoiaeter and the farra^d 
flt5oran©ter were used» !» additioii to the U8«al trans- 
pjjsuatsd aniieal tuaors, S-E polyoaa virus induced tvanors 
vere xisedo 



lo A siiopl®, sensitive, and specific method for the estlsiation 

of tetracycline in biological aaterial vas developed, utilizing 
a novel complexometric extraction procediareo fetracyclia© was 

extracted into ethyl acetate in the presence of barbital buJTfer 
azid calcium ions, fhe fluorescence in the ethyl ac<etat@ vas 
B&8>as\iired» A aensitivity of 0=2 ugo of tetracycline per ml. of 
plASoaa vas achieved. FroK tissue hQ(&og@nates , 9C^ of add^d 
tetracycline vas recovered emd 2 ugo per go of wet tisswe was 
BseaaurablCo An acid e^iiraction of turoor tissue of mice treated 
vitto tetracycline vas shown to contain a Auch larger asiouest of 
tetracycline in the fluorescent part of the tumor tissue tha^i 
in tlus nonfltiorescent parte 

PAR^ D' iaeludeds tm 



871 



- 2 - 

2o SpectrophotOEsetric studies of ethyienediaiiBinetetraacetic acid 

(ED32A) extracts derived from the fluorescent mouse tumor suggested 
the invoLvament of a metal-tetracycline bond in the process of 
localization. Only those cosspounds whose fluorescence was 
azahanced in the presence of calciias ion cai^ed the developiEent 
of localized fluorescence in tumors suad hones <> Other fluorescent 
and chelating compounds wer© ^aployed in similar experiisants hut 
were found to be ineffective « 

3o Issffipsters with S-E polyoma virus induced tiaaors were given 
tetracycline,. Scattered fluorescence in the tumor tissu® was 
ohserved in most cases but never in grossly houorrhagic and 
necrotic areas. In scan® cases the presence of localized flu- 
orescence eaabled the dei^ection of sites of abnoxBBl growth 
otherwise vismoticedc 

Significance to Cancer Hes®arch; Any bioehasaical or pharsEscological 

difference between norssal and 
neoplastic tissue, such as 
appears to occur with these fiuorochromes, might lead to profitable 
exploitation c Such a difference might reveal the existence of 
potentially selective toxicity upon which future development 
of specific chesBotherapeutic agents would resto The incorpora- 
tion of toxic groups in the fluorochromes that show a selective 
localization in neoplastic tisfsue ffii^t lead to the preparation 
of cftrcinolytic compounds o In. addition, the diagnostic utiliza- 
tion of this fluorescence localization islght prove fruitfiilo 

Proposed Cours®; 

lo Continued investigation of the mechanism of localization^ 
with aasphasls on the specific structural req.uiraffi@^tSo 

2° Foesibi® incorporation of these structural reqviir^gesLts 
ii^to known antitumor agents c 

3o Extend the applicatios& of the assay method t& ^h@ 
pStarsE^cologica 1 studies of tetracycline in humans o 

ko OoatitQtttd investigation of the physical chemistry of th@ 
coKBplax foriEatlo^o 

3^ Studj the possible relation betveisn viral titer of virus 
induced ttSQor and the persietesit fluorescence o 



872 



^:c-i=23iCc; 



fediTxdual Project Report 
Calendar fear I958 



Part B. 

Publication: 



Miich^ RoA«, Rail, D» Po amd foMe, <3"oE« 
Flisorescence of ^fetracjcline aatibiotics 
in ISoascc Jo Won 
Uo-A s 897-910, i958 



873 



Serial loojiglC-i-232 

1. (acs 

FIS-IIH 2. CP & Ef 

Individual Project Report 3. Bethesda 

Calendar Year 1958 



Part Ac 



Project Titlss Pharmacology of Drugs Influencing the Response 
to Radiomimetic Brugs and Radiation 

Principal Investigator: Margaret go Kelly 

Other Investigators:. Roger Wo O'Oara and Joanss© lolcroft 

Cooperating IMits: IGI Radiation Branch 

KI Laboratory of Pathology 

Maa fears (calendar year 1958)2 Patient Days (calendar year 1958): 
fotal: 2 
Professional? 1 
Other: 1 

Project Bascription: 

Objectives ; fo study effect of pretreatment with mercaptoalkylviuaax- 
dines (AEiJ, APf, APJ®?) on toxicity and tumor- damaging 
activity of radiomimetic drugs in aonnal and t«iEor<= 
bearing miceo 

fo study the mechsuiisa of protective action of AEf 
on toxicity of nitrogen Ksastard (W2) in ffionssal asid 
tisaor-bsaring miceo 

Methods; Usual pharmacologic, hesatologic, chemical^ biologic^ 
hlstophathologic, and radi(^ctive tracer tschni^ueso 



1. Coasparative toxicity of AEf, APTf and AP» was det®rmis?.®d in 
mice and doge by Intravenous, intraperitoneal, subcutaneous, 
and oral routes o Ik>gs tolerated much less of these co^pouifKls 
than did iBice> cmd developed s^ptoms of toxicity such a& 
vcaiting at dose levels of approxif^ktely i/3 th^a na^cimun 
tolerated dose, tfee toxicity of AEf ajad APMr varied witfc 
change in pH of the solution, i^il« tfeat of APf showed littls 
chaste fron pi ^ to p@ do 

2o PretreatKcnt with APf or AB® did sot give sigttificauat pro- 
tection agaiiBst toxicity of M2 iiK mice^ 



Fart 3 inclvid@d: US 



874 



seriai MOo 101-1-232 



3= Pretreatment with AEf protected mice against the acute 
toxicity of BM2^ uracil mustard, and triethyienethio^ 
phospfeoreiaiide ('ilfhiotapa), Tbut not against the acut® 
toxicity of l,4=.dimethan«sulfonjioxybutane (Myiiaran), 
colchicine, or triiaethyicoLchiciffiic acid methyl ether-d= 
tartrate o 

kc Pretreatment with AEH? decraased. the severity of SJ2 

dassage to hone marrow and small intestine, and resulted 
in more rapid recovery of these tissues o AS had no 
effect <m th® tumor- dauiagin^ action of H12, as evaluated 
by microscopic study of fixed sections of tumor tissue 
or hy analysis of siurviv&l datao 

5o Distribution studies with S- ^-labelled AW£ showed low 
concentration of radioactivity in muscle ^ skin, brain, 
and tumor tissue as cosepared. with tbat found in liver, 
spleen, kidney, and siaall iatestineo Autoradiograass 
indicate a low order of radioactivity in tyssor tissue 
(subcutaffieoua iaiplauts, meUiStatic foci in liver aad 
kidney), and a high order of radioactivity in liver, 
red pulp of spleen, mucosa of isxtestine, renal papillae^ 
end. bone asarrow. 

Si ^iflc ance to Cancer Research; Cluesaical protection against 

~ systcffiic toxicity of aLkyiatirag 

agemts without comparable protect icn of tiasor against carcino^ 
clastic action would result in tharcipeutic advantage » 

Proposed Couras of Project; 

lo fo evaluate protective effect ekgainat alkylating age».ts 
of o.ther radiation-protective ch'S^icala, as available o 

2o fo isolat® ajjd characterize the S ^^ moiety in tissuess 
of suaimals administered S^^^-labelled AK, To determine locali- 
sation of this igat«rial within cells by application of auto« 
radiograsBA' 

Ik 

3o So do parallel study with C ^labelled AEf to Etudy 

ffietabolisa of drug. 

^o fo coe^iare (by use of autoradiograia) tissue distribvitiosi 
of other sulfhydry compounds such as cystaiffl®, glutathione, and 
cysteasain^ with tjiat of AWi^ 



875 



3»yiol H9,SC2' 1-232 



art 



isot 



Kelatioia of Cfe^^jsica „ 

fteor mm&g0 end Dod^; vt-^^^-cs, La a i^^me, ox 

Ssffiiti^ aad sfosepfe loiter-. 



876 



1. &m 



iBidividual Project Report ^' ^^ ^ ^ 

Galemdar fear 1958 3- Bethesda 



Part A, 



Project ^fitie: 0b«gniicai carcinog^meBis 

Principal Bavestigators: ^r^aret Qo Kelly and Rog«r Wo O'Sara 

Cooperating Units: i^b. pathcsio^ 

Man fears (caiisadar year 1958): 
^**otal: 1/2 
Professioiials l/k 
Othsr: l/k 

Project Baser ipt ion: 

Objectives; fo determine affect of carcinogenic hydrocarbons 
in newborn 3iice<> 

Methods: Oswal hiologic and hisptopathologic techni^iueso 

Major FindiBSgas 



lo Intradernsai injection of new^m Swiss aiice witfc Ool 
ffll of 0«1^ methylcholanthrene in olive oil induced? 
(a) Multiple lung tvBsors in 11 of 12 mice within 14 

to 20 weeks 
(h) Subciataneous tumors (fibrosarcona) in 6 of 12 laic*' - 
(c) Invasion by subcutaneous fibrosarcoma of adjactnt 

tissue, including auscXe, auditory canal, eyelids r, 

salivary glands, thymus, etco 

2o Satrad'ts:;sal isijection of newborn Swiss mice witfe Ooi 
cco of 0.0^ 2,U,5,6-ditoensMaithracene is olive oil 
induceds 

(a) Multiple lims tumors within ik to 20 weeks in 12 
of 13 mice 

(b) Smbcwtaneous fibrosarcoma in 7 of 13 aM.ce in 12 to 
20 w®<ak8o 

(c) iKvasion of swrrounding tissue by fibrosarccasa with 
metastasis to liver and kidney in 2 snis^iso 

Si gnificance to C ancer Research; Potentiality of cheadcai carcinogens 

i® newborn niice is vpideterMnfd, 
atad laay Imv® some relation to huimn carcinogenesis o 

ffe® indufitio© tto© in newborn appears to be considerably short®s!i®d^ 
and tfee ffiictt air« atiii youag adwits vhmL lung twsaors develop o ffeia 
technivA«© provides a asathod of inducing a high incidence of luag 
tvigors in ®ic« for us® in evaluating asti^tvaaor activity of ds'UgS 
of interest in casacer ch©!aoth€jE;ap^„ 

Part B included; KO _ 

877 



<= 2 - 

Proposed Cours© of Projects 

fo repeat above experimeat in other strains of mice (C3ij, C57Bi,Gaf_^) 
fo investigate activity of chemical carcinogejas administered 

iatraperitoa®alij to newljom mice, 
5fo determine lowest intrademsai dos® which will produce lung 

tvnaors in Swiss mice 
f o determine earliest age of develojxaeat of lung tuBiors aft®r 

intraders©.! injection of newborn » 



87B 



Serial Mo. lCI-1-23^ (c) 
1. •'MS: 
PHS-mi 2. CP & m 

Individual Project Report 3<. Bethesda 
Calendar Year 1958 



Part A. 



Project Titles Phanaacology of Alkylating Agents 

Principal Investigators: Montague Lane 

Margaret G. Kelly 

Other Investigators: Hone 

Man tears (calendar year 1958): Patient Days (calendar year 1958); 
Total: 3,0 
Professional: 1.5 
Othsi-: 1.5 

Project ^ascription: 

Objectives ; 

A. Laboratory To study the pharmacological effects 
and anti-tumor activity of several 
recently described alkylating agents 
in mice axid rats bearing various 
advanced tumors. 

Bo Clinical To submit these agents to preliminary 
pharmacological trial in patients 
with advanced neoplastic disease. 

Methods ; Usual pharmacologic, hematologic, biologic and 
histopathologic techniviues. 

Major Findings : 

A. Laboratory (Drs. Kelly and Lane) 

lo Acute and chronic toxicity of Uracil Mustard, 
jr'5-bis(2'-chloroethyl)o«aino uracil^, has 
been determined in mice, rats, and dogs. 

2. The toxic effects of Uracil Mustard are similar 
to those of Nitrogen Mustard (anorexia, loss 
of body weight, hematologic depression, etc.). 
The irritation to the gastrointestinal tract 
appears to be considerably less, however, 
and at supra- lethal dose levels, convulsions 
have not been obtained and death has not 
occurred in less than 2k hours after injection. 

Part D included: 10 



Serial Io.lCI-i-23^ (c) 
- 2 » 



3. The advanced timors used in these studies were: 
(a) Carcinoma-Walker 256, Maumary 2iH-6 
((b) Lsanphosarcoma- -Dunning S-2778, L-2, K-i^. 
((c) Sarcoma— S- 37 and S-i80 

(d) Reticulum ceil sarcoma- -H-6867 

(e) Leukemia — Dunning IRC-74l,L-i210 
Methods of transplantation were modified to 
obtain sharp, reproducible survival curves for 
tumors listed in b,d, and e. 

k. Uracil Mustard significantly inhibited growth 
of carcinoma and sarcoma, and prolonged the 
median survival of aniimls bearing all the 
tumors listed in b,d, and e. A single weekly 
dose schedule appeared to be most effective 
and best tolerated. 

5<. Uracil Mustard was effective by oral as well 
as intraperitoneal route. 

6. Uracil Mustard was considerably more effective 
than litrogen Mustard in these studies. 

7. Studies with Indoxaa /"H^l-Dis-Cp-chloi-oethyl)- 
1' ,0-propylenephosphoric acid ester diamide 7 
and Dopan f 6 methyl 5~Dis (2'-chloroethylJ 
amino uraciij/ are in preiijainary phases « 

Clinical (Dr. Lane) 

1. Single, oral doses of up to O.U5 mg/Kg. were 
generally non- toxic to i)atients. 

2o Hausea and vomitting were rarely observed at 
doses from 0,45 to O.80 mg/Kg. and appeared 
to be prevented by pre-medication with 
Compazine. 

3. Doses above 0.^1^5 mg/Kg. occasionally resulted 
in bone marrow depression characterized by 
leucopenia, thrombocytopenia and reticulocyt- 
openia beginning about ten days after drug 
administration. Such effects persisted from 
several days to one month » 

h. 0.80 mg/Kg. resxilted in severe bone marrow 
depression in each of three patients. 



A favorable objective response (WBC from 
200,000 to 60,000, marked decrease in spleen 
size) was obtained in a patient with chronic 
lymphocytic leukemia -i^ho received 0.20 mg/lCg. 

Office a waek for four doses. 



Serial lo. iCI=l-23^ (c; 
- 3 - 

Significance to Cancer Research ; 

A. Laboratory 

L. Aniaaal toxicity (^.uaiitative and <iviantit«.tive ) 
is essential prior to clinical trial. 

2<. Information regarding dosage schedule and route 
of administration in animals may be of value 
to the clinician. 

3« GcBuparative study of agents in a variety of 
advajiced tuaaors ©ay predict their relative 
activity in h\aisan cancer more reliably than 
experimental technisLues in which treatment 
is begun the day after tumor implantation. 
Additional q^uantitative data of this nature 
as well as clinical quantitative drug comparison 
will be needed before the prognostic value of 
this techni>iue caja be ascertained. 

B. Clinical 

The liioalitative and quantitative aspects of 
the toxicity of a chemotherapeutic agent must 
be determined (in relatively few patients) 
before extensive clinical trials are instituted. 

Proposed Course; 

Ao Laboratoi*y 

Studies on Endoxan and Doi)an will be extended. 

D. Clinical 

Acute and chronic toxicity studies employing 
several schedules of administration of Uracil 
Mustsird will be conducted through the Eastern 
Solid TiBBor Chemotherapy Group. 



881 



EIS-lII! 2. CP & W 

Individual Project Report 3« Dethesda 
Calendar fear 1958 



Part A. 



Project fitle: Preclinicai ^oxicologj of Drugs Potentially 
Os©f\il in Cancer Chemotherapy 

Principal Investigator: Bavid Po Rail 

Other Investigators; M. ©o Kelly 

Goopgrating IMits: OGMSQ ■=> ISI 

Hazelton Laboratories , Falls Ghurch, ¥ac 

Ififfi fears (calendar year 19?8): 
total i/3 
Professional 1/3 

Oth^r 

Project Descriptions 

Pbj@ctivesg ffee objectives of this project are to study, in detail, 
the (iuantitative and ^iualitative aspects of th@ toxicity 
of drugs potentially useful in cancer ch^sjotherapy in 

order that new drugs may rapidly but safely be brovight up to the 

point of clinical trial » 

Methods; Standard toxicological n^thods are asployedo 

M&joT findings ; fen drugs hav© be@n studied so far: ffietfeotrgxate((Mf'X|, 
dichloro83@thotrexate (mm), raonochloroasethotrexate (BO!), Pfiz©r 1-73 
acetat®, Efeyer A-139, eEiEoethylisottoisioniuia (Agf }, aaainopropyliso^ ' 
thixironluffi, aaaiKOffisethyipropylisothiuronium, U-aminopyraaoiopyrisadiae^ 
aad 5-?iissoro«rac4i. 

fbe loajor findings can b®st be presented in light of the probl^ES 
faced in the iaitial trial of a toxic drug ia iiMia, rather tfeaja in 
t€3Ms of listijag 1J>cq"s, «B's, etco for the 10 drugs ffitwU©d siac® 
this protest began in May 19580 leWo derivatives of asfflthotremte^ 
a standard aaiti^ieutomic driij®, iB(»aochloroiaiethotrexat.@, asjd dichioro- 
methotrexate showed great activity egaiast Isukaaia in aic© and w®re 
considered for clinical trial in Baaa» 'ffee first problosj then, con^ 
certeed the qualitative aspects of their toxicity. It was isport&nt 
to know what the laature' of their toxlsity vas, smd what asigHxt b® 
expected as signs amd syniptosas of ©arly drug toxicitjo Observations 
in rats amd dogs indicated that the sjature of the toxicity and gross 
patholo^ following DSl and ICM was ©imiiar. to that s^esi after »?X» 
I» the cliaic, then, one might ©xpect to see oral ulsieratioa, h^ss- 
topo@tic depress ion 3, and gastroiEtestinal disturbaaceB at the osset 
of toxicity. 



Part D inciudeds 10 

882 



Serial Ho. lCI".i-235 
2 "= 



The ne^ct problem concerned the quantitative aspect of toxicity? 
the dose of dr\Jg to be given the first time to isano If too high, 
serious toxicity might occuTi if too low, the time retiuired to 
raise th© dos©, cautiously, to toxic levels mi^t be inordinately 
long, fh® toxicity of MIX in dogs, after repeated intraiaiascular 
administration, was ccMpared to i&at of DGM and lOMo fix© initial 
dos® for intramuscular administration in jesh was picked by toisparing 
the toxicity ratios obtained in dogs with the laaown toxic dose of 
WSK in saan, ajad incorporating a dose=reducing safety factor <, fhe 
dose picked for lOI has been shown to produce toxicity only after 
daily administration for thre@ weeks, and higher doses have be@n 
tolerated for shorter periods of timso Siniilar results are being 
obtained with liSMo 

Other q.uestions arose concerning differences in susceptibility to 
drug as related to age, sex, and route of administration. Hie 
results with BOM and WM showed no striking or important differ©aces 
in regard to these variables, biat other drugs tested in this project 
have shown such differences. 

file toxicity of a benzoquinon® alkylating agent, Eayer A-139> li^^ 
been det@rsd.ned for intravenous administration in laan by the Eastern 
Cooperative (droup in Solid fvMor Ohaaotherapy. flbiis clinical Group 
vaulted to use oral drug administration o llie con^^any had supplied 
capsules contaiaing an oily solution of the drug for oral use, but 
had presented no toxicological data. Comparison of th© toxicity of 
par eat ©rally stud orally administered A- 139 in rata showed that the 
pareaiteral route of administration was 6-10 tieses as toxic as the 
oralo Oral administration of effective aaaounts of A~i39 would have 
cntail;sd placing a lethal dose of the drug within the body, and re- 
lying upon the integrity of the gastrointestinal tract for absorption 
of only a therapeutic dose. 

The toxicity of Pfizer E-T3 acetate, an a,ctidione=-like antibiotic, 
was shown to be related to both age and sex in rats. Adult feaml@s 
were the most susceptible, tolerating only one tenth the dos® tolerated 
by adult laaleso loung f Stales tolerated .20 tiz&es as much as adult 
f«al(gs, on a i^g/kg basis, but only four tenths as nuch as yo\w^ 
males o 

SlgaiflcajRce to Cane@r R esearch; 'SSie drugs currently useful in the 
treatnent of hussaai^ neoplasms i^ust tm given at near toxic doses to 
achieve therapet^tic iresults. fhersfore differeasces in toxicity, 
even sa&all differences^ related to sex, age, route of ateinistration 
and dose schedule, can be of considerable isaportance in clinical. 
chemotlierapyo Information for pre°-ciinical toxicology is siuickly 
laade available to the Cooperative Sroxsps of the GdJEC 

Proposed Coiarse; It is planned that this project will b* coKtinusd 
aiosjg the asms lines as above o Drugs showing prossis© in Dto @oldiffi''8 
quasstitativ© aai®^ ttaor studies, or drtsgs obtained from CC^S wiS,i 
.b« put tato this toxicological appraisal before deciding on the dose, 
.route affiid B>ch®dwle of adsinistration in jaaa. 



883 



Serial K@o C°7"7_08 (e)_ 



lo General Mediciae Branclk 
2« Demnatolegy Service 
3. Betliesda 



PHS-N2H 
Individual Projeet Report 
Calendar Year 1958 



Past A: 

Project Title: Clinical Investigations @f Malignant Skin 

Diseases and of Benign Conditions Related Tlieret©. 

Principal Investigator: E„ J. Van Scott, M„Do 

Other Investigaters: Rebert Creunse, M„D. , Jo R» Andrews, MoDc 

Cooperating Units: Chemotlierapy Service, NGl 

and Radiati®n Brancit, NCS. (G-2-651(e) 

Man Years (calendar year 1958): Patient Days (calendar year 1958! 
Total: 2 
Fro£essi@nal: 2 
Other: 

Project Description: Continuation of C7'='708(c) ©£ 1957, 

Objectives; To study malignant processes in tSse skin and to carry 
out investigative tberapy of cutaneous malignant 
conditions; to study benign hyperplasia ©f the epider= 
mis in relation to malignant hyperplasia ®£ the skin 
and other organs. 

M e t hods %ployed : 

j^qsi 8 jun j^oides ; This disease is treated with chemotherape«= 
tic drugs given systemically and with whole body irradiation 
with electrons emitted from a van de Graaff generator. 

Basal cell carcin^na: Severe cases are treated with chemo- 
therapeutic drugs given systemically, with x-rays, and witli 
high energy eleefironso 

Psoriasis; Severe cases are treated experimentally with 
chemotherapeutic drugs either applied locally to the skin 
or given systemically. 

Patient^ Ma te rial ; Patients of the Dermatology Service, NC1„ 
Part B include ' i32 ^^"^ C^l 

884 



Serial Noo C°T-I08(€) 

Ma j ojc Findlia^s ; 

Mveosts Funjtoldes; In addition to the I? patients witli 
mycosis fung@ides @n whissn studies tiave been previously 
initiated, 5 additional patients have been included in 

the studies ©* tlhe Densiatoiogy Service; several m@re 
patients ©f the Radiati©n Branelt liave been studied in a 
cooperative pr®gieam<, Four patients have received Actinoo^cin 
D intravenously; one patient lias received uracil mustard 
orally. Electron beam tlheragy lias been administered wihen 
deemed appropriate » 

Therapy with Actineatyein D has had a detectable effect on 
the esythematous plaque lesions af the disease. Within 
one week following the administration @i the deug this type 
of lesiisn, present in three patients, has become markedly 
erythematcjus and has bled profuselyo This reaction has sub" 
sided within an additional two weeks with fading or ceaaplete 
disappearance ©f the lesi@in. No affects have been noted to 
^cur in the non^erythematous ttsoor type lesions ^ present in 
two patients. 

All type of lesidins in all patients have markedly improved 

f^llawiag tSierapy with high energy electrons^ The disease 
has recurred in all patents except one, who has been free 
@f the disease for 15 months at present » 

Basal cell carcinoma; Six patients with multiple basal cell 
carcinoma have been given Methotrexate ©rally, intraiBuseularly 
and/or intravenously. Antitumor affects have been observed. 
Tumors have became hemorrhagic fallowing theraf^ and have 
decreased in size. A few tumors have clinically and histo^lisg^ 
ically disappeared. Regr^wth of tusKdrs has been noted to igccur 
within several weeks t& several months following the last 
course of therapyo 

P soriasis : Many chemical agents used in cancer chemotherapy 
haire been applied locally to lesions of psoriasiso Complete 
clearing of lesions, observed grisissly, has occurred within 
one week follo»jing 24 hour exposure t@ nitrogen mustard and 
podophyllino Cleared areas tend to relapse within <one tsi 
several weeks. No local response to Methotrexate has bean 
observed, although this drug markedly clears the skin of 
lesions when given systemicaliyo 

Methotrexate, Actinomyein D and colchicine have been admin£s° 
tered intravenously to patients with psoriasiSo Within one 
week of a single intravenous dt^se there has been detectable 
fading of lesions, which has been m@st pronounced in response 
t© Methc8trexate„ 

Histological studies of fading lesions have been performed.-, 
Clearing of pssriatlc skin is accompanied by a greatly 
thickened granular layer in the epidermis, 

SjSrgg4lJ^^'^^^-&^~^-- ^ gesea gigh; Project directly concerned with 
study &t and therapeutic iafluenees on cancer and reiatee 
c©nditi«!in8„ 885 



Serial No« C^-7-708(c) 



PHS^KXH 

Individual Project Report 

Calendar Year 1958 



Part B: Honors, Awards, and Publications 

Publications other than abstracts from this project: 

lo Rabsons Ao S, , Van Scott, E, J. and Smithg Ro R. : Carcineasa 
of the anorectal junetion with extramasnmary Paget 's Disease, 

k. M. A, Arch. Patfe, 65: 432-=437, 1958. 



886 



Serial No. C-7-128 

lo General Medicine Braaelh 
2o Desmattalogy Service 
3. BetSiesdIa 



PHS-NIH 

Individual Prcjeefe Reps-rt 

Calendar Year 1958 



Part A: 



Project Title: Ser«etural aadl PSiyai®l®gical Cikaraeteristics of 
Nosraai and Patlh®logiesl Epidermia and Hair R©ot3„ 

Principal Investigster: E, Jo Vaa Se®)£t, M„D, 

Otiher Investigators: Robert G. Creunse, M.D. 

Cooperating Units: All clinical units of NCI, t© seme degreeo 

Man Years: (calendar year 1958) Patient Days (ealeadar year 3958): 
Tistal: 3 
Professional: 1 
Other: 2 

Project Description: Continuation of C7^1Z8(.c} of 195? 

pb j[ectlv e8: To investigate structural and plhysiolagieal 

citaracteristica in epiSenaal and supporting tissues 

in pursuit of understanding wide ranges ®f grawtlb. 
activity and regulatory factors £'&ere©fo 

Metltc j,8_£m ployed : Tissues are studied Silstologically and from 

reconstructions &f specific structureso Ti&tal 
number &S cells and mitotic figures tsountee 
microscopically; volume of gpeeific tissue 
structures calculated from microscippic measure'^^- 
ments; ge<^netric relatios&sltips detei-rainedo 

Further eoinpariaons are made &a the basis of 
structural elanges, particularly cihanges in Hnair 
roots, following ionizing radiation ©r drugs. 

Normal scalp l»air rts©tB are cultured in Algire 
millipore filter dkaaabers implanted in tke abdissasE 
of mice to study growtlh regulating fact®rs„ 

Pat-tent, Material : Most tissues studied or used are obtained frem 
Sidspitalised patients and out<=°patient8 &i N.C^I^. 



Part B included: Yes /X 7 N© £^^^. 

887 



» 2 '- 

Seffial N©o C=7/-?28 

n&^ov Fia^in^s; Growfeili &£ feaif appeaffs £a iatinta£eiy ifepemii upon 
an ineaet dermal papilla, a eonmeetlve tissue 
struetureo Hair r«^&£8 survive in tissue culture 
osily if thiB dermal papilla is psregent snd ine^^tc, 
In alopecia areae@ disturbed !iia.ir g£'is3)wt!i is as«soaia<» 
ted wielh Ihise.S'losieal ani ge^snetria tsS&anges in the 
Siair papilla, b^tHi ®f wfeiclh may be aeosmdarf to 
deniiciinstrable degenerative d!tanges in the n;atrient 
vessels @f tihe Ihsir papilla^ In znale tjpe "baldness" 
the lisir papilla is small; in tlhis eoniitisa n© 
Smir l&BB appeaffs t® ©eeur but tfee h&ix ptefduQei. 
is merely sfeert and narrow in calibre, botik of w&icSi 
are decreases pri&piartii&nal tiy t&e clecrease ia tike 
size &f tihe papilla^ 

Cemplefie abseaee &f mit®tie eells in feair nsots 
o<g<gurs wit&in 24 Ihsura f®ll©wing a single d®se ©f 
metfeotrexate given intravemoualy but normal ^siaubers 
of mitistie figures subsequently re=appegr,, dr^nie 
administration ®f tihe 4ryig is'' ;Ge'gempamle-i by s'atJher 
severe degenerative type c&anges in the 's&air r®ia)t 
wiliicl» tead t© persist. 

WitlSi a variety @f slhemistljerapeutic agents distlnet 
m®rj^(d)l®gieal cBianges in r®®>£8 of majsauaHy epilatci 
Ihairs are mieroweapically visibleo Specifie effects 
feave been seen in respsoise t© t%e a^iminisfiration ®f 
Metlkatreasate- Dielilor(smetti©trexate5, Aetiacssaycin D^ 
t&iis) t.eopoa,,, c®lcimide, end ,5=flv-or(@uraeil= T>e 
(seeurrence and severity ©f elhaages in Ssair rst^ts 
is preiperticmal to ©tiher general toxie signs as 
measured in counts of wihite bleod eells, platelets ^ 
ami reticuli&cytes and in ^aral ulcerationso Tii&e 
clhanges in &air nsiaits i&eeurring viken tlkese >£rugs 
are given t® patients may serve as a reliable intdess 
t® toxicity and efficacy of tSie drugs „ 

Furtiher attempts to establisti a reliable, constant 
relationsSiip between morpltolagic cSsanges in Lhtair 
roots witli d(Q)se of radiation itave been made. 
Structural defects occurring in scalp ihair ncwits 
following irradiation of the scalp witih st" ray a Shave 
been studied in twelve additional people during ti^e 
past year, T!he percentage of Jhairs wit!h tlhese de- 
fects is ia direct proportion t© tihe dose of radia^^ 
ti®n$ confirming tike previous observatiomso 

In a total of sixteen patients studied, the percent- 
tage ®f defective Isair rsots found 4=12 days 
following exposure t@ radiation laas been correlated 
witik tl»e dose ®f radiation received by tihe roots. 
ICn tSiis group ®f patients tihe percentage ©f defective 
ihairs could be used to establialh wihether ttee eijpasur^ 
doae Ihad been 100 .r ot less, ©r 200 r t<a-> '^•OQ- x. 
"She reliability of tihiis correlation &as 'oeen approxi 



•" ^ "■ Seifial N®„ C=?-?28 

Significance to Canceg Research: The studies are directly 

eisncerned vyitlk cellular reproduction, destrueti^K or 
quiescense, wfeiclj ptienomena are primary considers^ ©ns 
in a study or cancer » 

Pre>po8ed ewrse ^f ^project: Continuation along lines generally 

as indicated in the faregsingo 



Serial No. C«.|<=I28 



PHS°NIH 
Individual Project Rep©re 
Calendar Year 1958 



Part B; Honors, Awards, and Publications 

Publieationa ©tSier tlhan abstracts frsai this project: 

1. Van Scott, E„ J. and Ekel, To M<, , Geesaetric rela£ie-.n8Mps 
between tlis matrisE of tine lieir bulb and its deraial papilla im normal 
and alopecia scalp. J. Invest. DersEato 31:28l°288, 1958,. 

2o Van Scott, E„ Jo, M@rptiolc»gic changes in pil®8ebace@us units and 
anagen liairs in alopecia areata. Jo Investo Desfmato 31:35'--44j 1958o 

3o Van Sc0tt, E„ J,: Significance of changes in pilosebaceous units 
in acne and other diseases, (in press as proceedings ©f syroposimn @n 
the human integument, AAAS meetings, Indianapolis, 1957), 

4. Van Scott, E, Jo, Response of hair repots t© chemical and physical 
influences, Ciapter in BIOLOSY OF HAIR GROWTH, Academic Press, 
N. Yo, N.Y„ , 1958o 

5o Montagna, Wo and Van Scott, Eo J,, The anatomy of the hair fallicle, 
Chapter in BIOLOGY OF HAIR GROWTH, Academic Press, N„Yo,NoYo, 1958.. 



890 



Serial Meo^jg- 

2o BersimtailQgy Service 
3,. BetSsesda 



Individual Pir©jeet Rep®r£ 
Caleodaie Y&at 1958 



Part A: 

Pr©jeet Title: Tlie Biochemistsy @f Kenaal asid Patfe®l©gieal 'Epidermic 
Principal Investigate?: Simon RotSiberg, PlioD„ , EoJ. Van Seotfc, M„D. 
Otber Investigators: None 

Csaperating Units: N@ne 

Man Years (calendar year 1958): Patient Days (ealender year 1958} r 
T©tal: 2 
Profe88i©nal: 1 
Other: 1 

Project Description: Continwation @f C7"728(c) ©f 1957 

Objecti^-as; T@ study tlie bi@xyntfcesis ®f tSie majsr protein keratin " 

@f tSie epidermis and hairo Ka©wledge of the preeess will 
permit examination and cesaparisons as t@ the nature @f 
tUie protein synttiesized in nenaal and disease states. 

Evaluation and significance ©f arginase activity in nosn^ 
and patliolegical epidermis » 

MetBiods; Varying techniques ©f isolation and characterization af 
keratin are present in the literature; reproducible tech= 
niques @f isolating and characterizing keratin have been 
developed in this lab©rat@ryo 

The isolated protein is enzymatleally cleaved into peptide 
fragments, which are then separated by combined paper 
chromatography and high voltage electropfeoresiso The 
amino acid ceinp«J8ition of the peptides is determined by 
ninhydritt determination ©f the amin® acids separated by 
paper ehromategraphyo This technique permits the detecticxi 
of small differences ®f amims acids ©r peptides in normal 
and pathological epidermis ^ hair, fingernails s, and callus p 

Peptides present in isolated epidermis and pathological 
tissue (psoriasis) are separated and identified by a cesnbi: 
tion of column chromatography and high voltage electrophor: 
siSo Amino acids present in Isolated nerraal and patholo- 
gical epidermis are separated from peptides, proteins and 
other cellular materials by continuous dialysis » c®lumn 
chrssnatographya and paper chroffliat.«graphy» 

Fart B included: fes /^CJ ^®C^ ^^-^ 



^ ^ ^ Serial H@„ C-?-?30 



TSie sulflhydtl grewps i&tmed isp®n cleavage ©f elhe 
disulfide bond are measured by a PQ® (para eM@rin>= 
inere«ribema©ate) £i£ra£iQ»n„ Additional disulfide b^nds 
are t&en ©leaved wi£lh tM@glye©llic aeid aiid the sulfliydrl 
detemimedo T® keep tike reduced disulfide b-andsfrcm 
reforming tlsey are reacted wit& iodaaeetlc acid, T^en 
reaction witSi C^^ i®d@aeet©te is carried (@u£ prior t@ 
easymaeie cleavage ®f tise protein labelled peptides are 
pr-sidueed w&icSi can easily be spotted by radi®«ut@graiB @r 
eonventisnal counting te^liniqueSo 

Tlie Warburg tedbnique is used t& assay for arginas&o 
A mieri@»arginase teeSinique was developed fisr measuring 
arglnase la tl»e human liair r«a@to TMs consisted ©£ separa- 
tion 0f t&e (gimltlilne fr«ia the substrate (argiaiae in 
large excess) by liigh voltage electropfjoresis and staialag 
specifically for @mi£hineo 

ttejor^^ Findings:. Arginase activity was feuad t® be signif ieantly 

liig^er in psoriasis scales tben in normal stratum eorneura 
and normal epidermiso Serae specimens ©f normal epidermis 
possess an idiibitor @f arginase aetivityo Arglnase was 
found in feair r@®ts, tSsls a&e/wing agreement witlh previous 
observatii&ns t&at keratinising structures liave arginase 
aetivity„ 

Tfee p®sslbllity t&at ©mitiinss resulting Sxma !»ig|i 
arginase activity^ may replace lysiae in tlje keratin 
molecule obtained Sxm& p8®riasis scales was tested, 
Ortiithiass f@und as an artifact in liydroly sates ®f . kera= 
tine (as contrasted to its absence in five @t!her pr©tein 
ll^drdilysatesjj is however t&miglht noit to be present in kerati/ 

Keratin apparently may be solubilized fr-ssa normal epiiermig 
without cleavage &i disulfide b®nds<, It lias been asstsoed 
that the g©lubili2ing ©f keratin involved the cleavage of 
hydrogen bonds in additii&n to disulfide bosidso 

A n©rmal eonstituentancy of peptides in different nosmal 
epidermis is indicated in preliminary studleso 

Sigaifieaaee to Cancer Research; Basic ifflf@rmation in the process @f 
pr«iteia biosynthesis in n@naal epidermis is preliasimaify &nd 
will thus aM in evaluation of the pr®blera of growth in 
malignant epidermis o 

Proposed Course of Projeett When the amla© acid^ peptides, and mjor 
protein constituents @if the epidermis are analytically 
arrived at, biosyathetie studies with radioactive aad 
stable isf&topcs, .iajyitro aad in hiBBans,may be eoatemplated 
and carried out„ 



892 



Serial No„ C-'7'-'730 



PHS»MIH 

Individual Prsjeet Report 

Calendar Year 1958 



Fart B: Eon@rs, Awards, and Fublicaeions 

Publications other than abstracts frcaa tliis project: 

lo Rothberg, S, and Van Scott, E„ J„ , Evaluation of argiaase activity 
in normal epidenaal tissue and patiliis>l©gical strattss c&meuoio Jo Invest, 
Dermato 31:263=268, 1958„ 

2, Rot&berg, S„ , Aaaals N, Yo Acad, Sci» , 15: 1004«1012, 1958„ Possible 
significance of elevated argioase activity in ps©riasis scales^ 



893 



Serial No, G-=7«»731 

1. General Medicine Brand'x 

2. De?EsatCi)l©gy Service 

3. University @f Clkieag© 



PHS^NIH 
Individual Project Report 
Calendar Year 1958 



Part A: 

Frs-ject Title: Ghemieal Cesnpesition of 



Epidermal Lipids.. 



Principal Investigater: lic&ard P„ Reinerts©n, M„D„Coue of service 

trainee, MCX) , Vietia-r R, *estle?', PtioDc 
(Staff, University ®f Cliieage„) 

Other Investigators: K©ne 

Cooperating Units: Seetl@n ©f Dematelogyg University <&f GBiieag©, 

Man Years: (calendar year 1958) Patient Days (ealeadar year 1958': : 

Total: 1 

Professional: 1 
O'dher: 

Project Description: 

SfelgSSiaSS: "^^ identify and quaatify tike lipisS cmsstituents @f 

tlae epidersais for eosaparis@n t© tihe lipids ®f- sebaeertyt. 
glatMils, 

Metjhodg: Lipids were extracted fr«Sia specimens of i8elis,£e^i epi^emir: 
by means @f lipid solvent So Tlie lipids were separated 
int® various fraetiens by standard fraetienating teel^nique.- 
and tlhe n quant itated. 

Mi2I.J!IMtSM: I'^delhydr©ch©le8ter©l (pr©°vitamin D J, was found ijn 
tike MalpigMan layer of ftlhe epideraals, eetablis^hing 
tg»e ©rlgin of pre-'Vitamin in t&is tissue In man 
ratlher tlhan in tlhe sebaceous gland o 

Tike total pihosplholipid concentrstica in tihe MalpigA, 
layer is many times tlhat ©f the surfaee strattim c^r- 
neum ani contaiaas greater amtmnfes of unsaturated 

A particular lipid soluble substance, n©£ detectable 
in epidermis of normal indivliualo, was found isi 
umiavolvei epidermis ®f patients witiin psoriasis.. 



Part B included 



894 



Serial No„ C-l°7731 
» 2 -• 



tojg aaeej Reaearelbt; infastnafcion ©a lipM metabisilism 

in nomal and Ihyperplastic epMenals may aid in evaluetiag 
mel^absilie patatniomena in malignant epfiezmal growt&o 

ggiop^-se^ ^ CQuy8e _gf ^g.gj_eet : Fartlhe? itavestigati^ns @f ep±i.ena&l 
lipid metab®4,issa E© be earrie^ <siut in laberat^ries ©f 
Dermat®l®gy Service, NC2, as aue'^ investigatlens may be 
considered appropriate t® ®iver<^-all pr©gfam cjf £lie DerroaSoliP'g3r 
Service „ 



895 



Serial Ne.„C-?-731 



PHS-NIH 

Iiwlivi^ual Pr®ject Rep®rt 
Caies&dar Year 1958 



Part B: Hon&rs, Awards, and Publications 

Publications ©elher tlhan abstracts fri^a tSiis project: 

1» Wlieatley, Vo R asisi Relsjerts<s>n, R, P.. , TS»e presence sf vitainin=D 
precursars ia kiHcan epideemis, J, Snvesto Dermatr, 31: 51=54, 1958o 

2o Rei»erts@n, Ro Po and WSheatley, Vo R<. , Stuiies mi tlie clhemlcai 
e^smpositi©:© &i Siuman epidermal lipids, Jo Investo Dermatr (in press'j, 



896 



Serial Koo HCI-6~702-Cc) 
lo Gfflneral Stedicia® Branch 
2» Metaljolisa Ssrvice 

PHS-MIH 
lodivldtial Project Report 
Calendar Year 1953 

Part A: 

Project Title: Metabolic Study of Senas Proteins 

Principal Investigator: John L. Fehey, H. D. 

Other Investigators: Hone 

Cooperating Uiaits: For the study of neoplaa&ss in aice,- 
Laboratory of Biology (Dr. Mo Potter). 

m& Years (Calendar year 1938) Patient Days (Calendar year 1933) 
Total: 3 
Professional: 1 
Other: 2 

Project Description: 
L Qosntitative Studies of the Serura Proteins in l?eoplastic Msease States.. 

Objectives : The objective of this project is to characterise by 
quantitative cheialcal and physicocheaical technics, the ssxma pro- 
tein changes occurring in neoplastic states, and to correlate these 
changes with clinical saanifes tat ions « 

Methods Biaployed ; Chemical oethods for sertast protein^ glycoprotein 
and lipoprotein quantification were utilised. The serum proteins 
were further characterized by qtsantltative zone electrophoretic pro- 
cedures for oeasurlng protein, glycoprotein and lipoprotein fractions. 
Analytic ultracentrifugatlon was also (^ployed. A clinical appraisal 
of the patient status and disease activity was also obtained. 

Patient Material : During the calendar year, 700 patient serisa sasples 
were studied electrophoretically, and 100 sera® samples were esaained 
in the analytic ultracentrifuge. These sera were obtained frcas patieiats 
at the Clinical Center and elsewhere. 

Major Findings : 1. The diagnosis of ffi»acroglobulin©aia was ®ade or 
confirmed for 8 patients on the basis of sensa ultracentrifugal ena» 
lysess and was ruled out for 20 patients in whoa the diagnosis was 
considered. 



2„) Two additional cases of homogeneous senss gaasffla 
globulin increases not associated with sMltiple ayeloaw or smcroglobu- 
linraBla were uncovered. The leBplication of this finding in a patient 



Part B included Yes 



897 



Serial No. HCI-6-702^(c) 



3.) Hany patients ^ith recicalar neoplasas were found to 
have laarked changes in geasaa globulin levels. Decreased levels of 
the normal gasaa globulins are particularly evident in chronic lymph- 
ocytic leukoala, multiple aByelooa, asacroglobulincaiia and soiae lyaphotsso 

4. ) SeriEB 18S gassaa (eacroglobulins are often aarkedly de- 
creased in patients vith troltiple oyeloraa having high levels of serus 
6.6S ayelciga protein. 

5.) Gonverselys the serva 6.6S gaas^ globulins Esay be siarkedly 
decreased in patients with Bacroglobulinesia having high levels of 
18S ga^a^ isacroglobulinso 

6. } Cryoglobulins wsre identif led^ isolated, and character- 
ized in 4 cases of syiaptosiatic cryoglobulinsiiaa Cryoglobulin was 
found to be related to the noraal gaisE^ globulins. 

7.) The serue protein findings associated with three trans- 
sBiesible raouse plasma cell neoplasms were delineated. Two of these 
neoplassBS have serua protein changes res^bling those seen with 
plasaa cell neoplasas in sisn. 

Signlfieaace to Cancer Research ; Cheaical and physicocheaical 
tectmics such as electrophoresis sad ultracentrifugation provide 
means for evaluating sercm protein changes prior to nore detailsd 
study of individual ccanponenta. In cancer, certain individual sens 
proteins are known to increase while others decrease or are unchanged. 
Delineation of the sequence of plasma protein changes in neoplastic 
disease is needed. This aspect of the tumor-host relationships lends 
itself to quantitation. 

Proposed Course of Project : Appraisal of s«ruBB protein changes in 
specific neoplastic diseases in man and experiioental anlaalss and 
during specific clinical situations, such as with experimental cheeto- 
therapy, will be continued. 

B. Chroeoatography of SeruB Proteins. 

Objectives ; The objectives of this phase of the project are to 
define the individual serun protein changes produced by aalignant 
and other diseases, and to puri^ and characterize representative 
protein c<»aponent8. 

Methods Baployed ; Anion- eschange (9SAS-) cellulose colusm chrosBa- 
tography, preparative and analytical electrophoresis and ultracentri- 
fugation, spectrophotosaetric, chenical and inaunochaeical procedures, 
as well as tests for biologic activity are utilized. 



898 



Serial KOo ira-6"702"(e) 
-3- 

Major Findin gs; 1„) ChzOTsatograpfeic procedures have been Bsodtfied 
so tbat 3 sal. andg in BOtm iascances, 1 mlo o£ seruB aay be chrcmato- 
graphed for imalytic purposes. By cae^ining electrophorctic proc«dures 
with cbrosssstography, the levels o£ at least 14 seruB protein ccsBpouents 
can be ascertained. 

2.) Bssaainstion of semm frcKB patients with Hodgkia's 
dissase suggeiats that the asarked increase of sertSB protein-bound hexose 
observed in ectlv® phases of the disease, is attributable to increases 
of three alpha globulin cotspoaents (tmo alpha- 1 components and one 
alpha»2 component). It ^as also BOted that the copper- containing pro- 
teiQ» ceruloplasfain, \s&a increased In the f&o cases .of Hodgkin's dis- 
ease studied, 

3o) Preliielnary findings indicate that the chrmsa- 
tographic distribution of lipids is swrkedly different in serum ob« 
tained frcSi patients with biliary cirrhosis in contrast to normal 
&&Ttm or to aettm obtained in the nephrotic syndroase where elevated 
lipid levels are also found. 

4.) Monaal gaaassta globulin preparations were as^jginsd 
in det&ll, and were found to be ccsaposed of a large nusber of proteins. 
The gai^aa globulins could be divided chrcssatographically into 5 groupSo 
These groups were characterized and found to be distinctive in teres 
of ultracentrifugal and elsctrophoretic behavior, hexose content, 
i^sssunoch^Blcal properties, and physiologic activities. 

5.) Sera frora patients with mempeg histoplaaaosls^ 
and salcaonelia infections and chronic thyroiditis were exaaiaed 
chrosmtographically, and the respective antibodies were found in gaa^ 
globulin fraction 1. Although no physiologic activities were found 
in fractions 2, S, or 4, in fraction 5 were found the isoh«aaggluti- 
nins A and B, anti-D and anti-C Rh factors, the rh^aiatoid arthritis 
aev^m factor, and a part of the anti-nucleoprotein activity found in 
the aerusa of patients with lupus erythe^itosua. 

6.) Sera (Stained in disease states containing 
asarked increases of 18S gasraa aacroglobulins could be clearly distin- 
guished chronatographically from those with markedly increased 6.6S 
gasam globulins. This distinction can not be made by electrophoresis 
and has diagnostic significance, particularly in the differentiation 
of macroglobulinemia frora ansltiple ssyelc 



Significance to Cancer Research; Alteration of body protein saetsljolisra 
in neoplastic states is reflected in serua protein changes, for the 
laost part these changes have been only grossly defined. la^roved s^eans 

for Isolation^ identification and characterisation of the individual 
aernm proteisss are necessary for pursuit of the causes of abnormal 
protein saetabolisB in cancer. Substituted cellulose chrooiatography 
prosiises to be a very use&il toolj, and developssent of the asethodology 
and exploration of its applications should prove fruitful. 

899 



Serial No. SSCI-6-702-(e) 



Proposed Gotjgse of fcfee Project ; KesBar and improved procedares will 
be imrestiga£edo Oiramatogrspkie ®xs&iii!ia<:ion8 of sera obt&iaed in 
a variety of disteass sCates will con^imse in order to dsfirae better 
the senan protein changes during disease, and to esplore the possibil- 
ities for i£seful application of protein chromatographic procedures. 
Preparative protein chroasatography uill be utilized in conjtmction 
with other at«dieso 

Co Protein Metabolic Studies Utilising Isotopic Tracers o 

(fojectivea ; The objective of this phase of the project is to deter- 
mine the sites 9 ^echaniiSBs and factors influencing protein bioeynthe- 
sis^ and determining the quantity of individual proteins present 
norssally and in disease. 

Methods Baplf^ed ; Isotopically labeled asiiao acids were a^inistered 

to suitable subjectSs ^a<3 subsequently serial staples were obtained 
for protein fractionation and isotope counting. Radioiodine- labeled 
proteins are also (st^loyed. 

^.lor Findings ; A plas!!^ cell neoplasaa was shown to be the site of 
synthesis of the serisa myeloma protein. 

Significance to Cancer Research ; Specific protein synthesis by 
malignant tissue has been dearanstrated. 

Proposed Coarse of Project ; Investigations are imderway to dei'.®s»ia® 
the sites of specific protein synthesis^ and also the turnover rates 
of a nuBiber of individual proteins in patients with sialignant diseases c 



900 



Serial Ho. MCI-6»702-(c) 



PSS-MIH 
ladividsaal Project Report 
Calffindar Yeer 1958 



Part B: Koaorffl» Awards^ and PttblicatiottSo 

Fwblicatloas other thaa abstracts froas this project: 



1. Faheys, John L.g Potter, Michaels and BatbasaSj 
0aaiel. 

E^eloeea Proteins aad l^croglobalins Assoelatad 
with Plasaa Cell Tusboxq in EKperiiaesital Aaiffials. 
Acta Daio Internatiooalis Coatra Caacnai» 

in j^ceeso 



2o Msmdelsoha, Robert S«, Watkin, Donald M. , 
Horbstt, AnHi P. ^ aod Fahey, John L. 
Identification of the Vitamin B.^-bindlng 
Protein in the Sensa of Hossaals aad of Patifsmts 
with Chronic l^elocytic Leukessia. 
Blood 13: 740-747, 1958. 

3, Httthans, Daniel » Fahey» John L. , and Potter ^ 
mcbaalo 

The Fori^tion of !<^elosaa Protein by a ^use 
Plasraa Cell TusoTo 

Jouxnal of Sstperiaental Medicine, 108: 121°130s 
1958o "~~ 



901 



Serial i?o„ HCI-6-7<Kv-<c) 

lo General i^5iciiie Z.:is.z 
2<, Keeaboliasa Service 
3o Betkesda 



Ifidividual Projece Report 
Calisadar 7ssr 1958 



Part A: 



Project Title: Studies o£ Porphyrin Hstaboliss in the Timor 
Bearing Host. 

Principal Investigator: Donald P, Tschudy 

Other Investigators: Annie Colline 

Cooperating %its: None 

Man Years (Calendar year 1958) Patient Days (Calendar year 1958) 

Total: 2 
Professional: 1 
Other: j. 

Project Description: 

C^jeetives: To develop coe^ounds which interfere with 
porphyrin hiosynthesia and sttkly their effects on grosrtho 

To study the effects of vitamins, dnigs, sad horatones 
on porphyrin biosyathesiSo 

To study taxoT host interrelationships frtm tke sts^xid« 
point of porphyrin synthesis in liver and red cells. 

Methods <Mployed s 

A) Quantitative ensyiae detenainations 

B) Assay o£ nrine for porphyrins and porphyrin 
precursors o 

C) In vitro and in vivo techniques for studying 
enssyiae inhibition. 

Patient Katerial; AbnorGBelities of porphyrin aetsholi^s are 
now being studied in patients with porphyria. 

Major Findings: A synthetic analogue of ^-aaiinolevulinic acid 
has been developed which is a. potent inhibitor of the ensycse AM 
dehydrase in vitro . It has not yet been studied in vivo o 



Part B included Yea X K o QOO 



Serial Noo !ICI-6-704»(c) 
-2- 



B:^^ Infection of steroids ami studies in fidrenalectocaized 
aaiiaalSe it has b@®n shown that adreoal coirtical homoncs disaiaish 
the activity of MA dskydrsse in the liver. Previous work susgest- 
ing that the tiasor beariisg aniieal G^y be secreting Increased aatoisate 
of certsiQ steroids is in keeping with the possibility that the 
decreasQ in liver ALA dehydr&se in the tuaior bearing anlsal my be 
msdx&t&d through the adrenals. 

SigfflifieafflGe to Cancer Rosearch ; An abnoresality involving porphyria 
raet^oliss in the liver of the tissor bearing host say enable us to 
tmderstand specific chemical sechsnisffis whereby a saalignant tmtor 
affects the host. 



ggoposed Csmrae o£ the Project ; 



To continue ®£t<BBpts at Isolation of a htmoral factor 
produced by tiraors which directly or indirectly affects 
porpiqrrin synthesizing 



To exeraine further the role of endocrine organs in 
detexstining tissise levels of ensyiaes involved in 
porphyrin synthesis. 

To study laterffelationshlps of the Krebs cycle smj& 
porphyria synthesis in cancer and porphyria. 



903 



Serial Bo. KCI-6-704-<c) 



PHS-HIH 
IndlvidtsAl Project Report 
Cfileisdar Year 195S 



Part a; Hsoorss Awards j, sad Publicatioas 

Pctblieatioos other thaa abstracts froia this project: 

lo TscSmdy, Doimld Po^ and Collins^ Aimies SIsloaic 
Ester Synthesis of Delta ABi&olevuli&lc Acid; the 
Coadesssatlon of N-3 Broeioacetoiqrlphthaliaide with 

Maloaic Ester <> Journal ot Orgo 



2o Shicks ^rtia^ Laodau^ Bernards Tsekxtdy^ Hormld ?. 
The Effect of Hesose Aaalogues on the Groeth of 
Escherichia Coli. J. Baet, 75i *1^» 1958. 



Booors and ef^srds relating to this project: 



904 



Stsrial Ho, HCI~6-72i.J-(c) 

1. General Medicine Branch 

2. Metabolism Service 

3. Bsthesda 



PHS-HIH 

Individual Project Report 

Calendar Year 1938 



Part A: 

Project Title: Amino Acid Mstabolisa in Man. 

Priacipal lavestigator: Jo L« Fah«ys H. D. 

Other Investigators: ISbne 

Cooperating Units: Hone 

M&n Y@ar& (Calendar year 1953) Patient Says (Calendar year 195! 
Total : 

Professional: o 
Other : 

Project Baacription ; 

The principal objectives of the initial phase 
of this project have been achievedg i. Cc (1) to determine the role 
of L-arginine in prevention of blood asBonia elevation follo^^ing 
amino acid adstinistration in asan, and (2) to evaluate the thera- 
peutic value of L-arginine adsiinistration in clinical states sfsch 
as hepatic cooBa which are acc(^panied by blood aassonia elevationo 

The results have been published as noted in the 
individual project report for 1937. Further studies on antino acid 
fiietabollsm in man have been deferred in order to devote laore effort 
to related work on protein metabolism. 



Part B included Yss 



905 



PHS-HIH 
Individual Project Report 
Calendar Year 1938 



Sex 


rial Ho„ KGI-6-721-(c) 


1. 


General Medicine Branch 


2, 


MetabollsiB Service 


3. 


Bethesda 



Part A: 



Project Title: Mst^olisn of Hutrienta and Calories in Subjects 
vith Beoplastic Disease. 

Principal Investigator: Donald M. Watkin^ Mo Bo 

Other Investigators: Donald Po Tschudy, H. So , Ho lo Berlin, tL D. « 
Donald So Fredricksosj, M. Do » and Robert So 
Gordon^ Jr, » M. D. 

CiLinical Associates: Peter So Haeller^ Mo Dob 
lo Bernard Weinstein, M. D. , Sheraan Wei8fflBan» M0D9 
Habeeb Bacchus, Mo D. , Hham&a Waldsans, Mo Do, 
Robert Weigert, M, D. ^ and David Rathan, M<,Do 

Cooperating Units: Roneo 

Man Years: (Calendar year 1958) Patient Days (Calendar year 1958) 
Total: 8|- 
Profeesional: 3 
Other: 5^ 

Project Description: 

(fejectivae; Investigation of the over-all and interwedlary isetabo- 
lissB of fats carbohydrate, protein, saineralSj, electrolytes, vltaainsj, 
calories and water in patients during the natural course of neoplastic 
disease^ and during their responses to nutritional, physiological^ 
phansacological, endocrine, surgical or radiologic stress o 

Methods ffiaployed : 1)^ The laetabolic balance technique to quantify 
changes in the ccsBposition of tusor and host and for taeasuring the 
over-all aetabollsai of nutrients and calorleso 

2)o Calculation of insensible water loss froa 
insensible weight loss and sietabolic balance data. 

3)o Bomb caloristetry for the direct detenstlnation 
of calories in dietary Intake, and in urinary and fecal excretlonso 

4)o Measurosaents of the respiratory quotient (RoQo)^ 
and basal isetabollc rate <B.M.R.) by opea circuit indirect caloriaetryo 



^'art M Included Yes £ 



Serial No. UCI"6-72i-Cc) 
-2- 

5)o E3tiaa£es of body fat and body demsity by 
of skinfold thicknaases hj akin- fold thickness cslipers^ 
and by detenainationa of inulin and antipyrine spaces. 



6)0 Heasurement of uric acid in blood, urine, and 
body tissues and fluids by an enzynatic degradation techniqtse. 



7)o Meaaurcssent of intakOg excretion and levels 
in body fluid of B-coasplex vitjoains by aicrobiologic assays. 

8). Discrciae renal function studies. 

9). Msasur^sent of antibody production by standard 
israasnologic tedmiques. 



10). Hsssursment of plasma unesterified fatty acids^ 

(UF A),. 
11). Iteedle biopsies of liver and kidney. 

12). Clinical^, biochesical and dietary field sur- 
veys in technically underdeveloped countries. 

13). Quantitative estraction of lipids froas ti8SiB«a 
food and feces. 

14). Electrophoretic analyses of serum proteins 
(perfor^sd by Or. John L. fahey). 

15). Protein pool turnover studies using K labeled 
amino acids (performed by Dr. £kmald P. Tschudy). 



.. il^)o Msasureseent of lipid turnover rates using 

C labelled and I ■^- labeled triglycerides in «nulsified fora. (Studies 
to date perfoirsied with the aid of the Section on Metabolisa of th@ 
Laboratory of Cellular Physiology and Metabolisn of the Rational Heart 
Institute). 

17). Perchloric acid precipitation of glycoproteins. 

18). Isolation of C^^ labelled uric acid frcse urine. 



proteins. 



19). Coluffin chroemtography of proteins and glyeo-- 
20). Aniraal and tissue culture studies. 



907 



Serial No. ICI-6-721 (o) 



Patia at Material ; 

la-paticnta, ll«=l6<-57 8 

In«patients admitted 
llc=l6»57 through n«15»58 

a) Initial 18 

b) Rffi-admission 10 
3)o In-patient 3 other than above 

studied 11<=16=.57 through 

ll»15-58 43 

4), Nosioal Controls 31 



Nutrient and eaergjr aetabolism during hgrperalimentation with 
fat emulsion administered intimvenously in patients ohrtmioally ill 
vith end vithoat neoplaatio diseasoo 

Ob.1eoti-yBat Qtsftntifieation of the effects of exsess oalories 3up« 
plied bgr fat emulsion administered intravenously on host and tumorf 
evaluation of oalorio alimentation and hyperalimentation ^ fat 
MBulsions administered intravenously and by other means as ther^eu^^ 
tlo measures} exploration of the relationships among energy metabo«' 
lism, fatp protein and oarbohj^rate metabolism and tu»or growth« 

P&tlast M ater;lals Three patients with oaroinana: 1, stcaitachi 2j, pan« 
ora&sj 3» reotum«, 

ifajlor Find;i^gB; 

l)c Mministration of lipomul at slow rates did not oiroumvwit the 
"overl^uiing ^ndrome**. 

2)o In ^tients with rapidly growing tumors on Qomplete par@n<°° 
teral alimentation^ Slow infusion of lipomul resulted in development 
of lipemia after 15 days o 

3)o Increased energy expenditure with hjrperalimentation ooeura 
at slow as veil as fast rates of llpomol InfusioUo 

Sign ifiemq e to Cancer Research: Further evidence is presented ©f a 
gttiezalized metabolic abnormality associated with, but nc& necessarily 
caused by, the presence of neoplastic tissue o A complication of the 
Intravoious administration of fat emulsions at rapid rates for 25o36 
consecutive days in patients chronically ill with non^neoplastie dis» 
ease has been discovered^ investigated, and a plan established to lead 
to its eradioationo Hence, another step toward a practical mean@ of 
providing parenteral calories in concentrated foim to chrenieally 
ill patients has been taken, 

?aed Course ; 

l)o Investigation of the cause of "overloading e^drosae" and 
of the lipGmia noted above o 

2)o Study of fat utilisation rates in nonaals and in cancer 

pati(Mts ^iith a v±mi to quantitating the maximum utilisable asaomst of 



Serial Noo NCI-6-72X (o) 



Effeota of Bc^omplex vitamin defioienoies on the olinioalj, 
ffistabolio and iuBBimologio courses of subjects with and vithout 
neoplastic disease. 

Is ma BtMdlsB in 1957-58 . 



1) Study of ^2 deficiency using B];2<=lciw diet and analogs 
or entag«Maist9c 

2) Role of intestinal bioi^mthesis of B»ooiBples 'Titaaiinso 

3) Role of tissue breakdoun as a source of B^^ooiaplex vita'= 
Binso 



Hatrisnt and ener^ loetabolisai in patients vi-^ nec^lastic 
disease i«hen untreated, and during and after specific therapy. 

. Objectives; To quantify the over«all Metabolian of nitrogen 
potassiuB, phosphorus, calcium, sodium, chloride, uric acid and 
calories in patients vith neoplastic disease, and to examine ohaagee 
induoad by specific therapy directs at the neoplassoio 

Patient tfetarjL&l ? 17 patients with various neoplasmso 

tfc^or Ftodinps^ 

1) Host patients had a ayndrooie characterized l^ a negati*?® 
calorie balance, excessive fat loss, low R. Qo and high UFA con^ 
eentrations when their diseases were clinically activoo 

2) l^ecifio drug therapy in patients with neopXasmSp whose 
activity was altered by the drug, reveled a reversal toward nors^ 
of th® ayndrome, and in some instances, metabolie data suggesting 
lysis of tumoro 

3) In 4 patients who perforaaied a standard course of exercise, 
the main features were shrinkage of tuaor mass in one patient, and 
reversal of the metabolic features of the syndrome nenticmed above p in 

<»e patisito 
Significance to Oanoer Rasearch t These findings confirm and ext^d 
previously reported observations indicating a generalised metabolic 
abnozBAli-fy associated with, but not necessarily caused fay, the pre» 
e«ioe of nec^laatic tissue » They &agg©3t the abnoBig&lity may be best 
correlated with the activity of the n«^plastic process rather than 
its anat^iiQ dim^isionso An abnos^^lity such as this provides a 
ooffiSKSi denoainator for all ^pes of cancer, and hence must be considered 
a desirable object of investigation in seeking s general solution to 
tho cancer problssso ~~ 

909 



Serial No. NCI-6-721-(o) 

PypDOsod Cours|9t Additi<«al studies on G^ lab©ll«i fatty acid 
turnover, on changes in lipid metabolism, in body composition 
changes, and on the effects of exesroise. 



The RQ and BMR in patients with neoplastic disease, in 
patients chronically ill with nonneoplastic disease and in nonaal 
control So 

Ob^ieotiye^s Demonstration of an atoojanal metabolian of foodstuffs 
in patients with neoplastic disease. Investigation of possible 
factors respoisible for the abnormality. Correlation of data with 
that obtained in other studies of energy metabolism » 

Patient Material ; 41 studies in 7 noMol controls; 152 studies in 
8 patients with neoplastic. disease; 40 studies in ill patients with 
nonneoplastic disease, 

tfejor Findings : Basal metabolic rates in cancer patients who are 
trained and whose disease is quiescent are low. In most, with 
rapidly advancing neoplasia, 61^ *s are highe RQ's are low in sub«> 
jeots with active neoplasms. 

Significance to Cancer Research ; The low RQ's observed in active 
neoplastic disease reflect a generalized disturbance of normal 
metabolism in the tumor's host. 

Proposed Course; Collection of additional data on specific effect 
of feeding and of exercise o 



Nutrient and energy metabolism in patients with acute leukemia o 
Only one patient admitted. This man died the day after admission. 



910 



Serial Ho. NCI<.-6-721 (o) 



Changes In body ooiapositioQ ascompanylng the progression of 
nfioplastio disease » 

OltieetiYQSs Measure changes in fat, solid and vatsr in course of 
patients vith disease. 

Patient Ifaterial i All in A end C plus five patieats with noj^ro' 
plastic disease for control purposes » 

IfetJor Findj Lnpe? Patioats with neoplaams lose fat and beooae rela- 
tively more denser They tend to do this more rapidly than pers<^3 
who are merely aging « Fat individuals who are losing weight on 
reduction diets also beo^ae more dense. 

Proposed Course t Investigation of more patients who are also under 
study as part o f G in an att^ipt to define quantitative aspects of 
sytidTcmo mentioned underjC, aboveo 



Lipid aetabolian in neoplastic Diseasoo 

QbJectives i Idantifioatlon and qt^ntifieation of abnormalities in 
lipid metabolism in neoplastic disease suggested fcr^ nutrient and 
energy metabolism, R, Q. and B. U, R. and body ocstposltion studies^ 

H^thojis t In addition to those summarized above, isolation of lipid 
mobilizing substance from serum and urine. Fractionation of serum 
lipids o 

Patient Material : 25 norsisil oontrols and 5S patients with n®o<° 
plastic diseaseo 

llh.1or Findingst Plaas» tr F A is elevated in active neoplastic dis<° 
ease. The elevation is not due to starvation alonee 

Significance to Cancer Research ; A direct approach to quantify an abnors^ 
Metabolic process In the host aocompcuiying progression of a neoplastic 
disease is being m^^l^o 

ProDoead Coursty t Study of more patients using isotoplcally labelled 
lipids to establish rates of lipid matabolisao Correlation of data with 
other indirect aeaswrsBents of lipid metabolism and with ^n vitro studies 

vith isolated timers and neoplastio cells o Aaioal studies and isolation 

of lipid mobiliseTo 

911 



Serial ^o. KCIc»721-(q) 



PHSo^JIH 
Individual Pro j set Eaport 
Galeadar Year 1958 



Pfgt Bt Honoysg Awards, and Publioations 

Publio&tiotis other than abstracts fsron this projects 



1). Watkin, D. M. 

Sle««4»te Conseoutlve Daily Lipomul I.Vo Infuaions in Fonr 
Patients with Canoero 

Prooo Surgo Gen. Task Force Meeting cm IntraTonoas Fat 
Alimentation; Al^Bsd&j, California^ May, 1953. pp. l°4o 

2)o Watkin, D. Mo 

Ntttrieat and Ihergy Metabolism Studies in Pati«its with 
Caroincesa Receiving Slow°=rate Infusions of Lipoaul I.Vo 
ProOo Surge Gen. Task Force Meeting on Intravenous Fat 
Aliments tioni Nashville, Tenn., Nov., 195B pp« l<-i4o 

3)o lark, H. M., Uatkin, D. M« Kartess, Z. I., Kern, R. A., 
Huehroke, R. C, Sc]x°<rffenberg, J, A., Nasset, E. S., 
Peders@3, S., Aokeman, C. J., Ferguson, T. M,, EirsohP^&mo 
Jo C,, and Schaoffor, A. E. 

lAbff& t Hutrition Survey of the ArsBed Forces and Civilians x 
Interdepartmental Comsittee on Nutrition for Naticnal 
Defense} Bethesda, 1957, pp. Io76o 

4)o Watkin, D. Mo 

Increased Fat ntili2ation in the I^permetaboliai of Aoti^« 

Neoplastic Disease. 

A3TA tfoio Internatiomlis Contra Cancrom (In. press). 

5). Tschudy, D., Bacchus, Ho, Weissnon, S., Watkin, D., 
White, J., and Eubanks, Mo 

Studies of the Effect of Dietary Protein and Caloris Levels 
on the Eiaetics of Nitrogen Metaboliaa Studied with l^^l^so 
partio aoide 
J.CftIo (In press) 

6). Watkin, D. Mo 

Revision of Steiglits^"Nutrition in the Aged^in Wohl and Goodhart^, 
Modern Nutrition in Health and Disease. Second Bditi^, Lea & 
Febiger, Philadelphia, 1959 ( in press). 



912 



, Serial ^o„ MCI-6-723-(c) 

lo General Kiedicine Br&ach 
2. MetabolissB Service 
3o Bethesda 
FHS-RIH 
Individual Project Report 
Calsadar Year 1938 

131 
Project Title: Studies of Huaan Sena Albtsain Labelled with to 

Priacipal Investigator: Hetbaniel !» Berlia, M. D. 



Otker Is^vestigators: Tho^fis Waldaaxm, IS. H.^ Robert Gordoa^ M. 0c, 
a&d Jalius ^ite, Ph.O. 

Goopsratiag ISaits: Laboratory of Pbysiology, National Canc®r 
iQStituteo 

Hsm Y^ra (Calatsdar year 1958) Patieat Bays (Galeadar year 1933) 
Total: I 

Professional: 1 
Other: 

This project was divided into Project I a&d Project 11, S@e attached. 



913 



Serial Ifo„ KCI-6-723 <c) 

Fgojoct Xo 

Project Tltls: Mstributicm aad^Etes^adation of Eman Ser%^ Albttsseiia 
Labelled with I^"*^, 

Principal Investigator: Nathanlsl I. BerliQ, K. D. 

Oth4er lavestigators: Thosms Wal^oann, M. D., Julias HhitSg PhoBo 

Cooper&tifflg (^its: Laboratory of Physiology, Rational Cancer 

Instittsteo 

Project Descriptiozi: 

Ofajectiv©a ; To detarmiaa th® controlling s^chanisas of elbmaia 
synthesis aad degradation in norsals and patients vith cancer „ 

131 
Methods E iaployed; I albtfflain is given intravenously to indivldsmls 

receiving Ltsgol's Soliation, Blood and 24 hr. urine and stool col- 
lections are assayed for radioactivity for 28 days to determine th@ 
alb^aaia degradation kinetics o H Glycine is given intravenously, 

and its uptake into serus albtmln and globulin is deterslnsd by 
mass spectrcs&stry. 

131 
Patient laaterial ; I albimin kinetics of 2 norsalSg 3 patients 

vith cancers and 2 patients with idiopathic hyperalbusainenia wer@ 

studied in a control period and during albumin infusions » 

Results ; The percent of the total body albuain degraded per day 
vas increased approsisiately tvo-fold vihea the albunin pool sise 
^as elevated about 60% by albuain infusions. Preliminary results 
indicate that hyperiafusion of albuain partially suppresses albuiaiin 
synthesis o 

ignificance to Cancer Research ; This study is directed toward 
temlnlng the factors involved in the control of the production 
and destruction of oerus albunitts as a part of a study to dstenaino 
the cattse of the hypoalbu^ineoia in cancer patients c 

Proposed Coorae of Study : To continue the studies to deterssiae 
the rate of albtsain synthesis on alteration of the albusin pool 
size by albiaain infusions in patients and plassaapheresis In the 
dogo 



Pare B included: Yes g o I 

914 



Serial Kbo HCl-6~723-Cc) 

Project II o 

131 
Projeet Title: The Distribution asd Bsgradation of I AII»tS)iQ and 

I Polyvinylpyrollidone in Honaal Subjects and 

Patients with Regional Enteritis or Ulcerative Colitieo 

Principal Investigator: Bathaaiel lo Berlin, tL 0. 

Other Investigators: Tfaooas WalAsanns M. D. g Robert Gordon^ M Dc 

Cooperating Units: National Heart Institute, LCFMo 

Project Description: 

Objeetivea ; To study the kinetics of albtasin destructioss in 
patients with idiopathic hypoalbvaineaia sad to develop a diag- 
nostic test for pati^ts snspeeted of having ulcerative gestro-^ 
intestinal disease, 

131 
Methods ; I labelled albtosin is given intravenously to an 

individual receiving Lugol'e solution. Blood and 24.hour urine 

and stool 8aBq>les are assayed for radioactivity. I Polyvinyl- 

pyrollidone (FVP) is given intravenous ly, and the stools collected 

in the subsequent 4 days are assayed for radioactivity 6 

131 
Patient Material; 20 I albuain studies have been perforated 

in 13 patients vith idiopathic hypoalbtrainemia. 

Major Findings; Patients with idiopathic hypoalbtnin^Bia fall 
into tTiTO groups^ hypoanabolic and hypercatabolic. The hypoana* 
bolic has an isolated deficit in alboain synthesis vith compensa- 
tory hyperglobulinesila and hypercholesterolenia. The hypsrcata- 
bolic has normal albuatln synthesis, but excessive protein loss 
into the gastrointestinal tract. Three of these patients have 
associated chylous effusions. Three have had a llpofucsin pig&ssst 
in the siiooth nascle of the ssaall intestine. All patients vith 
hypercatabolic hypoproteinenila and those with ulcerative gastro- 
intestinal disease have an elevated excretion of I PVP in the 
stool, compared to noraals or patients with diseases not affect- 
ing the gastrointestinal tract. 

Relationship to Cancer Research : Patients with gastrointestiaal 
carcinosia frequently show hypoalbusineala. Loss of albunln fross 
these lesions into the gastrointestinal tract say be a cause of 
this hypoalbuBlnemia. 

Proposed Course of Study ; Continue to collect data on patients 
with idiopathic hypoprotelneBila to characterize further these 
syndrossea. Study the I albmln and I PVP kinetics in 

patients vsith gastrointestinal neoplaass. 



Part B included Ye a H o X 915 



Serial Mo. KCI-6-725"(c) 
lo General liedicine Branch 

2. Si^ueritlon and Metaboliem 

Service 

3. Bethesda 



ladlvldual Project Report 
Caleadar Tear 1958 



Fart A: 



Project Title: Vltssaia B^2 Bi^tabollsa 1& nonsal subjects and patients 
with neoptastic and other diseases. 

Principal lavestigator: Bonald Si. Watking M. S. 

Other Investigators: I. Bernard Weinstein, M. D. 

Cooperating Units: Mone 

H&n Years iCBlend&v year 1958) Patient Days (Calendar year 1958) 
Total: 1 3/4 
Professional: 3/4 
Other: 1 

Fzoject Description: 

Objectives ; Quantitative observations on the aetabolissB o£ vita^n 
B.^ in aan including its absorption trcm the gastrointestinal tracts 
its distribution in the organs of the body, its level in plassia, its 
binding to plassia proteins,, end its excretion by the kidney is norsal 
subjects and those with neoplastic disease. 

Methods Employed ; Microbiological assay (L. lelcfaaanniil £or vita- 
sin B.^ in plasaa, urine and tissue. Measurrateat o£ Go -labelled 
vitaistn B.^ i° blood, plasaa^ plasaa protein fractions^ uriirieg feces 
and tissues in vitro , and in organs in situ by in vivo scanning. 
Paper and block electrophoresis and coluon chrooatography of plassia 
proteins. Dialysis and ultrafiltration of plasMa proteinso Reaal 
clearance techniques. Perchlorste precipitation procedure > Studies 
of B.J antagonists and analogs in bacterial, tissue culture^ plassa 
and aamBalica systeais. 

Patient Materia l; Senm B.. levels In 97 patients and 140 noxsial 
subjects. Co B.n absorption studies in 14 patients. 

Major yindinga : Elevated B.^ l^e^^^l-" appear to be due to an abeorsml 
amount of a glycoprotein with strong B.^ binding properties. B.. 
absorption in chronic ayelocytic leukeata is ^parently norsnlj, But 
plasisa clearance is delayed. Turnover of 3-2 scab's excessive. B&rshall 
Island natives have seruiB B,^ concentrations higher than U.So nonsals. 



Part 3 Included Yea £ 



916 



Serial No« NCI-6-725-Cc) 
-2- 



Sigaiflosacc to Cancer Research ; Elm&ted plasma 3.„ levels ia 
chronic and acuCe layslocytic leukemia and Esyeloid sietaplasia &rm 
halli»rks of these diseases. The dssaonatrfition thet these im'sls 
are aot do® to increased B,^ fibsorptloag but la all probability 
to a quantitative or qualitative change in tha saetaboliss of an 
alpha- 1 globulins has farther clarified the nature of a Ketabolic 
process associated wlt^ neoplastic diseases. The desBoostration 
that sporadic elevations in plassaa Bj^„ levels are asoociatad with 
hepatic sietastases helps to clarify a hitherto tmesplained phenome- 
non and adds substantially to the evidence that elevated plas;^ B,„ 
levels ar© specific hallmarks of an tmusnal saetabollc process la 
these diseases » 

ProEoaed Cwira®; 

l)o fh@ assay for B., activity in the semaa of patients with 
a variety of malignant diseaies will be continued, 

2). Absorption^ distribution and plasma disappearance 
studies ^111 be carried out in patients without leukeaia and in 
norsaal controls. The distribution of Co -labelled 3^. in the or- 
gans of animals sacrificed at stated intervals after administra- 
tion of the vitai^ln zaay be necessary to conf ina data acquired by 
in vivo scanaingo 

3), A method for assay of B^ binding in whole plasam 
and pl&ssssa fractions will be developet to identify the plasoa and 
plasffia fractions aost able to bind B,, and dateraine their charactes- 
istlcs in normals and in patients wltn elevated plassaa B^„ levels. 
thi& identification and characterization of the escact coesiponent of 
alpha- 1 globulin responsible for B.^ binding will be attempted. 

4). Developsisnt of techniques for the quantitative isola- 
tion of vitamin E^^^ ^^^^ blood, urine, feeds and tissue will be 
undertaken to provide a^aeans of studying the incorporation of 
orally adrainistered Co Clg into B., by the bacterial flora of the 
gastrointestinal tract and by the patient hissself. 

5), Distribution of B^^^ ^'^ ^^® ^^^^ ccKBpartusents will be 
studied by constant infusion and renal clearance tschnlques. 

6)o Sistributlon of Co^- labelled B,, in norml and sBallg- 
nant tissue will be deteraainsd in surgical and autopsy spec 



?)<> Isolation and purification of B.^ binding protein, 

8)c Studlea of B.2 antagonists and asalogSc 

. Fssrther studies on the plassui clearance of B,^ ^^ ^^^° 
(chronic siyelocytic les^emia) 

10). Eelstioa of the B.^-blnding protein in leuk^sla to XoF„ 
^'' (iatrinsic factor) 



917 



S«riai KOo lSCI-6-725-Cc) 



PUS-SIIH 
Individual Project Report 
Calendar Year 1958 



Part 3: Honors, Asfards, and Publications. 

Publications other than abstracts ixaa this project: 

1). Ifendelsoha^ R. So, and Watkin, D. H, 
Sertm E,2 Concentrations Determined by 
L. leicfetannii assay in patients »ith 
Meopiastic Disease. 

J. Lab. & Clin. Med. 51: 860b366, 195S. 

2). Mendelsohn, E. S. , Watkin, D. £3., Horbett^ 
A. P., and Fahey, J. L. 
Identification of the B.^ binding Protein 
in the Serua of Sossials and of Patients 
with Chronic I^elocytic I,eukesQia. 

Blood 13: 740»747» 1938 



Honors and awards relating to this project: 



Serial MOo KS-6"726-Cc) 
I. G&mtT&l Mi&d±cin& '&v&nch 
Z, Stetrltion and Wetahollim 

Sen? Ice 
3. Bethesda 



Individiml Project Report 
Calendar Year 1938 



Part, A: 



Project Title: R®n&l clesarance studies in patients with 
neoplastic disaase. 

Principal lavsstigator: Ikmald M. f^atkin^ H. S. 

Other lnv@:&tis&tOT^i 1, BeoMrd ^einsteia, M. B> 

Cooperatliag IMits: Hooe 



Ifem Years (calea^r year 1958) Patient days (calendar yasr 
ToSal; ' 
Professional : 

OthiEr: 



1/3 



Project Oescription: 

C^jectivas ; To estlssiatfK tM glaaerular filtration rate, effective 
renal plamissa £low and tabular mass, and to investigate the ssecb^ai^^ 
isms by %ihich the kidneys htindle metajiolites such as tsric acid and 
calci^jm in patients with neoplastic disease. To ascertain the 
kidseys' role in such disorders as byperuriceiaaia and hypercal-cemia 
when these accoispany neoplastic disease. 

Methods Beiployed ; Renal clearance technique. Snzyaatic assay for 
uric acid. Ultrafiltratioc procedure for estioating filterable 

calcitB»e 

Patient Material ; 14 Renal function studies were perfonsed in 7 

patientSo 

Major FindJBjjja: Urate ireabsorpeioEi is a linear function of the 
filtered urate load. lu individtsals at the extr@stes of filtered 
load* reabsorption deviates froaa the linear fuonction. Correctioiu 
of hypercalcemia results in a amrked increase in GFR, RPF and 
TIslPAM, HyponriceiBia due to a diisinished reabsorption of urate m&f 
be a characteristic of patients with aminoaciduria and cryogletelitn- 



Significance to Cancer Research ; Pla^aa uric acid level is deteraiiaed 
in part by renal tubular reabsorption of arateg in part: by uric acid 

synthesis » and in part by the release of uric acid and its precursors; 



Part B included Ye s M o X 919 



Sariffil Mo» SCI-6"726-Cc) 



frcas tisssses umdergoing destnscsioiao The renal cont:rS.feutioE& to 
tfee sBaintenancs of plasaaiffi level ssaast b® knotaia bafore hyperarlcassia 
may b« escribed to increased syn&hesie or to tussor destruction. 
Hypercalcemia is a dangerous co^Ilcation of nceoplastic d±s&&ee. 
As In the case of hypenaricssd.a» intveetigatione of the reti&l mRch- 
anisass for clearing calcium in hyper calceala are basic to an ismdsr- 
standicg of the syndrcssis. 

Fropoaiffid Coarse ; 

1,) Additional clssraace studies ia patients with hyper- 
uricessiej esp®cialiy those with plassas levels sufficiently liigb 
to result in filtered urate loads esceeding fia-uratH!. 

2). Collect clinical laboratory data on such procedmt@& 
as coaceatration teste, blood area taitrogen levels, creatiain® 
levels, microscopic eKaaiaation of esriae sedisaent* esraMiaations of 
nstue for protosintsriaj PSP ascsretioasj creatiniae clearance® and 
infcravffissoas pyelograass for comparisoa islth discreet renal fimstion 
stutSles so that the reliability a£ clinical indices of renal func°° 
tio£t in neoplastic disease may b@ evialaated. 

3)« Correlate data obtained by cl^garance studies with 
histolo;^ of the kidney as seen in renal biopsies and autopsj Kil- 
ter ialo 

4)o develop a reliable techniq«se for dstersaiining filter- 
able calcisn in plaiHM and test its reliability and reproducibility. 

5)o Clearance studies in patients with hypercffllcesaie to ■ 
detersiiae the factors influencing tubular reabsorption of calciim 
and its relatioEi to tubsslar handling of urate and phosphorus. 

6). Further studies of protein fraction clearances in 
selected patients ^Ith proteiauriso 

7). Study additional patients with aainoaciduriao 



920 



Serial Mo» MCI"6-727Cc) 
vus WTH! -^ -ayrt-iriil Msdiciae BraBch 

ras-SflE 2. Metabolissa Servic® 

lEsdivMml Project Repoirt 3 R«tUag<|a 

Calendar y«ar 1958 j. »<s » 



Part A » 

Project Titl,®: Gross Body Ckaoposition 
Principal lav@stigstor: IDr,, Mo, I, 3®rlla 

Other lavestigators: To A. Waidsaaap M. B. ^ and Bo Ho Waekia^ g^ So 

Coopera&iQg Units: lone 

m.n Ysars (Galeadar yeas: 1958) Psfcisat Bays (ealeadas- ysar 1958) 
Tof.®l: 5 
Profess loaai: § 
Ot&i«r: 

Pjojec£ Usscriptioa; 

Objectives: Tfeis project is dsslgaed to study the changes la 
gross body ccffispositioa in neoplastic diseases, particwlarly dt^risig 
th® cemraa of the disssse, asid to correlate these changes t«ith tha 
clinical course o£ the patient and other laboratory data, &n& to 
study the a&^sia of cancer la the sarae gro«3p of patients,, 

Msthods BB>ployed ; 6ross body coaposition -Mill be determined frs» 
the body density and total body %ratero Body density ^111 be d@t®r- 
®iaed tvosB body ^ass and body voltsstej, as m^aa^ured by the Siri I&^cdy 
vol«asse apparatwg; body water will be detersained with tritiated ■ 
water, loaatological status will be deteraained by saeasureise^t o£ 
total red cell vol»ae..with Cr ^ iron twrnovei- with Pe £, &n<d red 
cell life span with C „ 

Patient Material : 3 patients with exogenous obesity; 7 patients 
with acute leukemia; 8 patients with soyeloid eetaplasiao 

Major giadings ; lihe findings can be divided into 2 areas: 

1) Gross body composition aiiid 

2) anffiaia of caiacer 
1) Grose Body Ccssipofflition: 

The Siri body volssaae apparatus has been installed &Md 
several Ejodificatioas made. At preseatj electronic caea^arfflsent 
of vapor pressiars in the ch^Hsiber is beiog ezploredo Satisfsctoxj 
replicffibility In deterasiaatioa of inert reference staEdards has 
been achieved. For sobjactSs the replicability is in general 
satisfacto:ry, but can be improved. 



Part B iaciwded ^ Yea Ho 1 



921 



-2- Serial No, NCI»6=727 (c) 

Six aoraal subjects (fvom HIAMS) have beea nms together witfe 
detenainatloa of &hs body ^^atero The principal finding is a higher 
than generally reported hydration o£ lean tiasueo Three obese sub- 
jects have been studied while on a ^tabolic balance regiioeo Satis- 
factory weight reduction has been obtainedo Preliraisary analysis 
indicates that reduction in body fat content has been achieved; 
that the calculated hydration of lean tissue changes early during 
the course of weight redaction and that the changes in total lean 
tissue are not large. The astabolic balance data has not been 
cosBpletedo 

5 individual subjects frasa other services have been studied ^ 
vhere the infonaation taight be of value in the care of these patlentSo 

2} Anemia of Cancer: 

Since the isethods utilized in this aspect of the study vere 
available, store progress has been aade in this area. 

In acute leukesia (in adults) erythropoiesis vas nostiial 
or increased in 2» deficient in 4, and absent in 1 patient. The 
red cell life-span was finite, bug shortened in 4« showed rasdora 
destruction in 1, and was noziaal in 1 patient. 

In 5 subjects with BQreloid setaplasia^ the production of 
red cells was norssal or increased in 4^ and aaarkedly decreased in 1, 
As a group j, these patients were aneaic by blood voluste standards, 
except that 1 patient was polycytheaic. The red cell life-span 
was shortened, except in the polycytheaic patient. 

Both these studies demonstrate the variability of the 
pathogenesis of anemia in these disease states, and indicate that 
each patient SBist be studied before any conclusion be reached for 
that particular individual. 

In the experimental anisal (dog), ^dies designed to explore 
the nechanisa of the control of erythropoiesis show that although 
sethyl cellulose will produce a splenomegaly, this is accompanied 
by a severe uresia due to infiltration of the kidney by awthyl 
cellulose; that splenectooy results in a significant decrease in 
the total circulating red cell volimte due to a decrease in the rate 
of production of red cellsj, and that the red cell life span is 
noroal, and that hyperthyroidisa results in acme shortening of the 
life span of the red cell. The studies designed to detersine if 
local anoxia of the kidney or a liad> influences erythropoiesisg 
requires the perfusion of partially desaturated heanoglobin; such 
preparations are dependent upon a series of operative procedures 
^ich are currently under way. 



922 



.3- Serial Noo NCI-6=?,r? (c) 

Slgalficaace to Can.e@r Research ; This project is part of the 
studiffis carried out on the area of the tusior-host relationishlpo 
I^riag the course of a aeoplastic diseases large changes in body 
weight occwre particeslarly weight loss. These Bmst represeat soaae 
chaaiges brought ahosst in the sE^etaboIism o£ the host. As a prelj^ai- 
nary and a aecessary sdjisnct to the study of these changes la the 
host's eaetaboliffliig it is necessary to ]ua.<m the (Ganges occurring 
in gross bo4y cc^positlon. 

Amassia occm:^ in cancer to a variable degree depending %pon 
the criterion o£ an^sla selected. Presently available teethodg permit 
the qnantitatioa of erythropoiesiSo Correlation of the level of 
srythropoiesia t^ith changed in body casposition vill afford an 
opport^mity to compare the lactabolisa of a single system with that 
of the whole body. 

Proposed CoMrse i The more rapid developemit of the stiedies oa 
anemia and the results obtained to date are such that this project 
will be divided into <!) gross body ccHiposition^ and (2) anas^ia of 

cancero Both will be studied in the saiae patientj, but other addltioE^al 
stitdles of aneaia will be undertaken as part of a separate projcscto 
Aside from this division of the project^ the studies outliaed above 
will be coatimuedo 



923 



Serial Sto« KI"6-729"(c) 

2. Metabolic Service 

3. Becheada 



PHS-IIH 
ladividaal Project Report 



Project Title: Studies of Mtrogea Metaboliara usiag H^^" 

Priacipal Xffltrestigator: Sossald P. Tscfe©dy» M. D. 



Oth®r Itwestigato?s: P. U. ^SLtkln^ M. 0. « S. Wdssnen, £io B. » and 
He Bscckus, Mt 0. 

Cooperstisg ^its: Laboratory of Physiology ^ HCI, 

Man Years: (Caleadar year 1958) Patieiat Days (Calendar year 1958) 

total: 3^ 
Professional: 1^ 
Other: ^ 

Froj«sct Bescriptlon: 

A) Thrmsgh the w^ of s&athesaatleal models of nitrogen 
EBStabolisBB to stisdy the effects of such variables as diet^ 
v&ttI&w hovmmesg and tvaov grotsTth on the klnetica of body 
aitrogeoo 

B) To study, the rates ®f synthesis of sfirt^ proteiim 
in vivo through the tsse of precursor product relatioaships 
and related approaches c To exteod this approach to allo^ the 
detercaicatiOQ of rates of transaaioatioa Iq vivo. 

C) To study Bsechanisna of protein synthesis and degradationo 
mthods BBployed ; 

A) Synthesis of B^^ labelled anino acids. 

B) Xsolati«m of ureas aansonias certain proteins,, smd 
casino acids frosi protein hydrolysatea. This involves ch^isic^il 
fractionation^ colusm electrophoresis and ion esschange chro^stto- 

gs-aphy, 

C) Preparation of materials for oass spectroetetry,. 
I>) Metabolic baleace studies. 



fta-fc B iaeluded ¥e a Z m 

924 



gOTial Koo lCI"6"729-'(c) 



Fa£iea£_Mg^ffi£isl; Studies of fclae. lesisSibemft&icj&l m©del of e^trogea 
ssffifiabolisEs have feoaa coisdwcted la a pffiCiest •wiKh lyssptosas'csMi.o 
S£«£dies of races of pi?o£&i& sys&hesls smd trans<Malsmcim% 2m^«e 



MBjQg FladtstjRa ! Th@ rates (, T^t& osmstmitaB «ad pool sisses coa» 
Pttted f?asi 3:liS oat 



puted frost 3:lis Batli«G9a£ical laodsl of Sim fi&tTQ asd Rltt^&siheTg 

si-si^ffi c&ndttimm« AS ion le^@l@ of proseia l»£ck«s, efeasa^es i.n 
eelo7i$ i£&i£k«9 do not af feee tha sise or r®&e of cuimover c£ tbe 
QltrogemMS f^&«^oli<s poolg m&x tlm cmlail£S:®d e%rtQ<W'Sr of body 
proceia. Wl&h l!&c7«sa@@ of psroteiQ Izitaka for a giv^B e^tiorie 
ineeke^ there is escsGea incrsatse in sis® of £^ istlr.70geQ0t£i!i m^tA- 
boiie poolo f^liiaa histh ealo^ic aM nis^ogea ii&tskis &x-® l-ac^^aaed^j 
ehare is is^ t&&s®&&&d >ra£e of eajn»over of both eke sl&soj^eacws 
axstabolic pool aad body profceia wMcfe p?«G®des posieiv® Klerogea 

A ae^ th^Qretic^l app70^.cb to £k® coesptststtioa of s@n»!i 
^■SQ'c&lu £tsr33£»ver« iS:l]iroug2a th& uzm of p:?®cuxso7 pro«l%ie£ sspocif ie 
activity casrsresj has beea deveiopad «md tssted fflsperiaaeatsiij.. 
l%e ealaula^ed e«tx«ai slbtmla tuimover is ira ehe ras^e observed 
^i£b Qtms @e&ho48. 



SlRaifieanGe £o €ane@g RaBca-gcfe; fhsse aatkods will be applied 
to the atisdy of tiaeor host imtesreletionships end the @££eet of 
£ naffiibsr of variables thereoao Th«s« pisxrait th® qusntitstive 
otwdy of the t«^3y host iaterrel&tiOEfflhip as it applies eo pRX>£eia 
sj&theais smd satsbolls@o 

Pgopcsed Coagae of the Project : T© cosaplete the theoretical 
approach to laethoiie of coaiputlag trffi&sasBioatioa rates ig vivoo 

To apply is conml subjects end pcitleate with cs:ac@r 
these esthods for the atu^ of the affect of the prese@«e '^£ & 
tmtsix oa the protein sistsbolisiB of the hoet« 



925 



Ssrial 8Jo. KCX-6-729-Cc) 



PHS-KIH 
Indlvidaal Project Report 
Calendar Year 1958 



Par£_B: EonorSg Awards, and Piablicationso 

Fublicaeiona other tiian abstracts from this project: 

lo TschwSy, DoufflM P. , Marshall. Margaret, Graff, Ada, 
and Graff, Saaoiel. 

Studies of nitrogen Ketabolisai in Bman Cancer Using M^^ 
L-Aspartlc Acid. Cancer 11: 984-995, 1958o 



Honors and atdrards relating to this project: 



926 



Serial No. 



o « (3<- \o; 



1,- General Jft»diciae Brsrach 
2 a MetaboIissB 
3. Bethesda 



Part A: 



Project Title: 



PSS~»IH 

Iiadivid^al Project Report 
Calendar Y@ar 1953 



lavestigatioQs of Hallgnent Reticular Diseases 
with Particular Attention to Those with Associated 
Serum Protein AbnorsalitieSo 



Principal Investigators: John Lo Faheyg Ho 3° 

Other Investigators: Hone 

Onits: 
Specific C&jective 2: Institute for Allergy and Infectious 

Maea@®s (Dtc Jo Uts) 
Specific Objective 3: Pathologic Anatcasy Branchj HCI 

(Bro To Dutcher) 
Laboratory of Biology s, MCI 
(IhTo J„ Dalton) 



Ifisn Years (Calendar year 1958): 
Total: Ij. 
Professional: 1 

Oeher: i 



Patient Days (Calendar yea? 19 5§) 



Project Description: 



iectj-ves: 



general objective is to define the clinical mani- 



festations of malignant reticular diseases with particular osphasis 
upon those vith jaacroglobulinemia^ cryoglobulinasia^ or other signi- 
ficant protein abnorraalitiesg to correlate the clinical tuanifegtations 
vith laboratory and histologic observations, and to explore and 
evaluate therapy o 



The specific objectives are: 
1) to record the incidence, 
clinical nsnifes tat ions. 



severity, and course of the 



2) to evaluate susceptibility to infection by clinical 
history, observations, and sseasureasent of antibody production capacityo 

3) to obtain suitable tisisue for histocheraical and (Electron 

Qsicroacopic characterisiation of the cells principally involved in tbe/^e 



Part B Incited Yes 



-m 



927 



4) to evaltiate erytbropoleses In these disease statesc 

5) to evaltjtate cisrrently available therapies and to explore: 
potentially ys«ful new therapy o 

Methods Employed ; Quantitative^ standardized^ appropriate procedtiires 
are eaiployed for each obJectivSc 

Patient Material ; This is a new project <> Previo«gs work in this ares 
was condactcd tinder Serial Hoo ifCX-6-702-(c)o 

Major Findings ; Prelisiocry res«2lts suggest that aany patients with 
asalignsnt reticular diseases have ia^aired capacities for antibody 
productioso Characteristic histologic feat^ires have been found in 
lystphoid tissue^ the bone aiarrow and, occasionally {, in other tissues 
of patients with EBacroglobulinesiac Therapy with corticosteroids 
produced an objective remission in one patient with Waldenstrom's 
nacroglobulinemia;, and symptomatic laprove^nt in a second patle&to 

Significance to Cancer Research ; Certain malignant reticular 
diseases produce specific changes in the host not found with other 
malignant diseaseso Changes such as aneaiag inability to produce 
antibodies, and hemorrhagic diathesis often jeopardise health and 
life more than do the direct effects of malignant cell proliferation^ 
How the malignant cells produce these changes is largely unknownc 
Currently available treatment for many of these diseases is unsatis-* 
factory o A basis for selection among possible therapeutic procedures 
is not available and is needed^ 

Proposed Course of Project ; DevelopsHsnt of observations as indicated 
under Objectives will be continued. 



928 



Katiojaol Cancer Institute 

u 

Pathologic Anatany BraneJi 
Bj.dyt Data 



Estimated Obligatioaaso a e » » o o . o o o o » « , o <, „ o , « o , . , « c .Fiseal Year 1959 

DireCtooooa»a,ooo«,o»a.»»,oo,.,c„.. 000.00.0 00$ 278,000 

Betmbiirsements 

CliaiCalo O O » O O O « O O O O O . O O O , , O O , o o . o O o 

Othero o o o o , o o o o o o o o o o o o . o o o o o , o o o o 96,100 

TOtal„o.,oaooooo,oooo, STVT-W 

1 / lnelud«s Projects Ko'as 

C3fT0DIAGN^TIC SERVICE; 

^2 
SURGICAL PATHOLOGY AMD PCST MCETEMs 

^3 



929 



Serial No. NCI 852 
PHS<^NIH I. Pathologic Anafeooy Branch 

Individual Proj«ce Report 2, CySodiagnoatic Service 
Calead«r Year 1958 3, Beeheada 14, Md. 



JBARLAo 

P7oj8ct Titles ExfoliaCiv® CySologj Appliod to Human Diagnostic 
Probl@ne and Kesearch Probleos 

Principal Xnves£i«aeors Dr^ Richard A. Malo^ren 

Other Snveetiga tores Dr, ElizAbeSh W. Chu 

Cooperating ISaites Not to se^ knowledge 

Man Years {calendar year 1958) s 
Totals 8 
Professionals 3 
Other g 5 

Project Descriptions 

Objectives g 

The primary responsibility of the Cytodiagnostie Service is to 
provide the staff of the Clinical Center vith an accurate and ccssplete 
exfoliative cytology service. In addition^ the Cytodiagnostic Servic@ 
collaborates in various clinical research projects evaluating cancer 
therapy, the hormonal status of the cancer patient, the course and 
natural history of the cancer lesion, and the anatomical and physio° 
logical changes in the husoan body associated ulth various patholic^ic 
conditions. The accoaplishsoent of these objectives necessitates th@ 
continual iraprovement in techniques and the origination of new 
cytologic siiethods in the laboratory. 

Methods Esiployed g 

The standard Papanicolaou staining procedure is applied to 
preparations of various huizsan specimsnSo These specimens &r^: then 
screened and evaluated for morphologic changes in the cellular elements.. 
Special procedures involving the use of fluorescent microscopy, EHlllipo.te 
filtration, and various special staining techniques rite required in w&nf 
instances c 



Q30 



PAGE II. Serial NOo NCI 8.5 2 

Patient M«tt«rtal g 

tha figures corapiled beXow are for thm laat ewalva nonChe Cl'2])o 

T ype of ffifflterial examined Accessions f ^ S^-^- do*. 

Wotmd washiogs 126 487 

Bronchial vsshings aud spu^a 183 528 

Vaginal and cervical 823 2123 

Misc«llaneou8 fluids 376 1902 
(gas&ric, prostatic^ breasC, pleural, eSc,]) 

Siaprovei^nfe technique* _479_ 2463 

TOTAL 1987 7505 

In»£ieu£a Accescioos # Slides 

NCI 1251 5629 

mi 79 200 

NIAMD 85 23? 

NXAID 142 496 

NZNDB 45 226 

mm 4 II 

NIDR 7 23 

Ref«ry«d 324 809 

Major Findtnagg 

1„ Ttie major conferibueioa of She Cyfeodlagooseie Service 6© 6h® . 
diagnostic facilities of She clinical staff has been the giarlf diagnosia 
of neoplasias of the cervix and lung and of other sites not pv&vimmly 
diagnosedo In several instances, exfoliative cytology provided the <snl^ 
tissue confiraation of neoplastic disease before deash of the p£ti®i%S. 

2o In the vound vashing project, carried on in collaboratioi^ with 
the Surgery Branch, NCZ, neoplastic cells have been deasonstraSed in n^sa&rous 
washings of cancer surgery rounds » The use of quantises ive re<gov«ry 
procedures and an expression of qualitative nature of the eells has be@ffi 
institutedc 

3o A "star" pattern observed in the Aegyrophil prep«ratlc®s ©f 
vaginal smears froia woosen with breast caxscer is being evaluated for its 
significance and an attempt is being xaede to detenaine the fai^tors 
responsible for its developsoent . This pattern «?as observed in 42X of 
women with breast cancer and in IIX of nonbreast cancer patients, llie 
nonbreast cancer cases with a "star" pattern were composed of patients 
with endocrine disorders , 

4o Sex chroBzetin coimts ar@ being done oa patients with endocrine 
disorders as a diagnostic and therapeutic aido to addlgioa, a study of 
the sex chrosaatin pattera in norssal and neoplastic tlssna® culture cells 
hs8 been doae^ 

931 



M ajor Findtnge CConfclnuad)) g 

5. £b collaboration vith eho Surgery Braoeh, « mlllipors fllCraKloK). 
e«chnique h«« baen davalopsd for identifying tumor calls in bloo<i„ Bj? 
this procedure, relatively large volumas o£ blood oay ba processed vlth 
nearly cooaplete recovery of any tiH^ir cells present., ApproKimately 700 
blood apeeiisens from patients in the Clinical Center vill have been 
processed this year and about ISO specissens from patients in the Public 
Health Service Hospital in Baltisaoreo 

6. An evaluation of use of millipore filter techniques in the 
proeessing of routing cytologic apecimsna has been sMde end its advantages 
detersgdnedo Its applicability to urine^ spinal fluid, gastriCj, pleural, 
ascitic, synovial, bronchial and breast fluids has been deisons Crated., 

Significance of the Froggam s 

lo The diagnostic value of exfoliative cytology in the clinical 
evaluation of cancer and its early detfiction is readily apparent from 
the activities of the Cytodiagnostlc Service in the Clinical Center and 
throttghottt the country » 

2o The wound wsshiog program contributes to a better zanders tandiaig 
of the factors involved in tuaior gro««th, progression and e^tastasisa and 
the reaction of the individual to his nsoplasso %n addition, the 
application of this infonaation to cancer chemotherapy may be of val«@o 

3o Althotigh the "star'^ pattern observed in the vaginal sssears of 
breast cancer patients requires further evaluation, its possible 
application to diagnosis, prognosis and therapy is under c^nsideratioa^ 

4o By the use of sex chromatin counts and techniques for hortsonal 
evaluation the endocrinologist is provided uith a valuable tool for 
guidance in diagnosis and therapy. 

5. Th@ study of tumor cells in the blood presents the potential 
of gaining a better understanding of the factors involved In Bstastasie, 
It ajay be of value in studying certain types of therspy of the es@opIa@tic 
diseases and diagnosis, 

6o The development of new cytologic techniques end the acquisition 
of specisgns fron different sites in various diseases continues to 
enhance the value ®f the ssfoliative cytology procedure as a diagnostic 
aido The value of the caillipore filter techniqua is readily apparent and it- 
is anticipated that this procedure will replace the smear preparations in 
many cytologic laboratories. 

Proposed Course of the Fgojeet g 

lo The continuation of a complete and adequate cytologic service 
for the 8&9f£ of the Clinical Center is our primary purpose „ Continued 
collaboration with the Clinlcsl Center staff in various research projects 
and the develo;»%nt of new &md Improved techniques within the laboratory 

are an important part of tk@ futur«& program c. 



Qr^? 



PAGa SV. Serial No, mi 852 

Proposed Course of the Project 



2„ A continuation of the wound ««gh£ng and blood studjf by 
quentltative techniques will be continued In collaboration with the 
Surgery Branch, . 

3o Intensive efforts will be nade to ioiprove the quentitetive 
procedures for study of blood and wound washing specimens with the 
development of a. procedure suitable for autoiaatlon as the ultiasate goalc 
In addition, it is hoped that quantitative c^tochesiical procedures X4ill 
lend thesaselves to an evaluation of this laaterialo 



Part B included Yes (•&} Nd C ) 

933 



?AGE Vo Serial Noo NCI 852 

PublicA&ions oehAr than abagracts from ehis pvojacfeg 

Malmgran, Richard A«, Px\ilt.e, John C, Del Vecchio, Pasco Ro aud 

Poteer, John Fo A Meehod for feh® Cyfeologlc Deeaegion of T.\3iEor 
Cella la Whoia Blood, J.N„C.I„ 20g 1203-1213, 1953o 

DaWUe, Sara Ho, Eabsoa, Alao S., Lfflgallals, Frances Vo, Dal Vacchio, 
Paseo Ko sokd m-hagxen^ Richard A^ Sex Chro!iBa£ln In Huaan Tissue 
CulEurs Gall Liaas„ In FrssSo 

Del Vecchio, Pasco Jk., DeWifeS, Sara Ho, Borelli^ J, So, Ward, JoAo, 

Wood, ToAo, Jr.. aiid Mal^rea, Mcbard A„ Application of VAlliT^tm 
Fiieraeloa Techniqiae to Cytologic Material, J,K,CoSo Sn Press o 



934 



Serial No. NCI 853 
PHS»NIH 1, Pathologic Anatomy Branch 

Individual Project Report 2, Surgical Pathology and 
Calendar Y«ar 1958 Postmortem Service 

3., Bethesda 14, Maryland 



Part A. 



Project Title; The Department of Pathologic Anatooiy (Pathologic Anafecwny 
Branch, NCI]!) has no specific research projects. Its 
diagnostic services are designed to serve the investiga- 
tive efforts of the N.X.H. staff responsible for patient 
care. In isaay instances , investigative study of 
pathological material is of great importance to clinical 
research . 

Staffs 

* Dr. Harold L. Stewart, Chief, Pathologic Anatoc^ Branch 

* Dr, Louis B. Thonas, Head, Surgical Pathology and PostEortera Sfirvlciiii 

* Dr. Richard A. Malmgren, Head, Cytodlagnosis Service 

* Dr. John H. Edgcotcob, Pathologic 'natots^^ Branch 

* Dr. Alan S. Slabs on. Pathologic MatCHay Branch 

* Dr, Edward Price, Laboratory of Pathology, NCI 

* Dr. Elizabeth W. Chu, Pathologic Anatoe^ Branch 

•e- Dr. Thonuis Fo Dutcher, Resident, Pathologic Anatomy Branch 

•^ Dr. Katherioe H. Herrold, Resident, Pathologic Anatos^ Branch 

4> Dr. Rowan C. Boy Ian, Resident, Pathologic Anatoagf Branch 

+ Dr. Robert Peters, Resident, Pathologic Anatos^ Branch 

-«■ Dr. Ra Gerald Suskind, Resident, Pathologic ka&ttm^ Branch 

■i- Dr. Martin Hlcklin, Resident, Pathologic Anatoa^ Branch 

° Dr. Leon Sokoloff, Laboratory of Pathology and Histocheiaistry 

" Dr. Harold R. Stanley, Oral Pathologist, Clinical Investigatioas, 

" Dr. Igor Klatzo, Head, Clinical Neuropathology Section, NI2IDB 

" Dr. Herschel Sidransky, Laboratory of Pathology, NCJ 



» These Pathologists constitute the staff of the Depart35)®ae of 
Pathologic AnatOBi^r, Clinical Center (Pathologic Anatotay Branch, 
NCI) and spend a major portion of their time in the ecrvic® 
and diagnostic work of the Department. 

- These physicians arc full time Residents in the Department of 

Pathologic Anatoo^. 

- These associate pathologists spend part time in the activities 

of the Department of Pathologic Anatcrany. In addition, several 
pathologists on the staff of the Laboratory of Pathology, KCS^ 
i.e. Drs. Swarm, Dawe, Banfield, O'Gara, Stanton and Love, ar*. 
available for consultation with the staff of the Department 
of Pathologic Anatoa^. 



935 



Page II o Serial No. NCX 853 

Projffict Descrtption g 

No specific research projects. The service functioDs of tlm 
DepartmenC during the twelve osoach period, Decomber 1957 Chrough 
November 1958 included; 

a^ 291 autopsy examinational Oa. page 4 ehere is a table which 
shows the number of autopsies performed for each Institute, The residents 
pcrfojTia nearly all of the poetraorteia dissectioas under supervision of the 
staff. A gross review is held by the staff and residents each morning 
at 8s 30. The residents review the microscopic slides of each autopsjr 
with one of the staff before completing the autopsy protocol. 

The steff assists the residents in preparing for the nuaserous 
clinical conferences in which the Departmant participates. 

b) 2273 specimens were accessioned in the Surgical Pathology Seeigion., 
A complete listing of these speeissAns, by Institute^ is given in tba 

accoapanylng table. These specii^ns are initially eacarained and deseribeid 
by a resident and their raparts are checked by tha staff. Associate 
pathologists from other laboratories or sections are frequently consuleed 
about problems in diagnosis In surgical pathology and/or assuiae respansibillSHp 
for handling certain tissues removed for specific research purposes . 

c) Cytodiagnosis Services The report for this servlcit Is subaltted 



The objectives of the Departoent of Pathologic Anatooay in th& 
Clinical Center are twofoldg first;, to furnish a diagnostic servic«». 
and second, to aid from a saorphologlcal standpoint the various clinical 
research probletes which are under study in the Clinical Center. In the 
first 2 or 3 years after the Departaient was established, taost of the 
of the staff was taken up by the diagnostic responsibilities. During the 
past year, especially, the staff has been able to spend move, tlnse in 
collaborative research on Clinical Center patients with various sieo^ers 
of the Clinical staff. 

The Bs^hods used Include those standard siethods used In describing, 
fixing and sectioning various human organs and tissues. A wld<» vaTi.&ty 
of special staining procedures are used. 

Distribution of hucian organs and tissues ; 

At present there are approxisiately 50 scientists in the various 
laboratories at N.I.H. who have requested particular tissues froia 
surgical and posti&ortem services. On many occasions it is possible to 

furnish these Investigators with fresh huxoan material. A list of th@s@ 
investigators is kept by the secretarial staff and telephone calls ara 
mad® to eh® various lave® tiga tors whenever surgical or postssortesa 
tissues are available. 



Page xn« Serial No. UCl 85 3 

Research on the dead human subject ; 

This iQSitter vas thoroughly discussed during ehis year and i£ was 
decided Shat research projects on human bodies could be done ufeer 
obtaining spacial permission from the proper relatives. In sojoe insf^ances. 
th(£ contract embalroer's services wera employed. Thus during the year. 
Dr. Van Buren has been able to calibrate a steriotaxic devtc® on 7 or 8 
occasions. We hav« also withdrawn all of the blood from the body of one 
patient for endoc Kino logic study by Dr, Hertz, Xt is anticipated Shaft 
these types of activities will increase. 

Conferences ? 

Tha staff and residents tak« an active part in numerous Clinical 
Center and Institute conferences. In addition, there are ssvsral con^* 
ferences regularly held in the Depar&aent including a surgical pathology 
conferencft, brain cutting conference and joint patholog4)Sts conference. 



Fart B Aacluded Yes (x) Na C ) 

937 



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ox 



Page V. Serial No. NC|_853 

favt B. 

List of Publications and Collaborativ Investigative grojects g 

A» Pro Louis B. Thomas g 

lo Zubrod, Gordonj Levy, Allan; Williams, George^ Thossas, Louis B.; 
^{cCullough, Norman and Lynch, Jobno Hepatitis Acaon^ 
Hospitalized Patients - Clinicopatho logic Confersoc®. 
Hospitals, Nov. I, 1957. 

2o Smith, Robert K. ; ThomAs, Louis B., and Hilberg, Albert W. 

Cancer Cell Contaioinaeion of Operative Wounds „ Cancer IjL; 
53, 1958. 

3., Schatten, Williaai E.,, Krasaer, William M., and ThoiBas, Louis B- 
Cavernous Httmangiomas Involving Veins of an %p«r Ext^esitl^y. 
Annals of Surg, 148 g 104, 1958. 

4. Boggs, David R,, Frel, Enll XXI, and Thomas, Louis 3. 

Clostridial Gas Gangrene and Septicemia in Four Fati<saf:s «lth 
Leukemia, New England J, of Med. - Accepted f®r ptsbllcatlon, 

5, MiECullough, Noraan B,; Lousia, Donald B,; Hilbish, ToF.j Thomas, 

Louis B. and Erassons, Cheater. Cryptococc©sisg Clinical StSaff 
Conferenca at JI,S.H. Annals of InteriMl Msdleine 49s 642^ 19*58. 

6o Forkner, C.E., Fritz, Richard, Freir®ich, Emil Jo, aod Ih<mas, 
Louis Bo Intracranial Bleeding Associated with Fulminating 
Leukocytosis in Patients with Acute Leukemia. Submitted r.o 
Amerlean J. of Ifedlcine for publication. 

?= Exhibit on Changes in Bones in Acut® Leukemia « Prepared wish 

Besse, B.E., Lusted, Le« B., Forknar, C.E., and Hllbish, T.F^ 
Exhibit shown at national aisefiings of the two roentgenol^lcGl 
societies, American College of Pediatrics and AMA, Brief 
exhibit publication will appear in a supple^eut of JAMA, 
MedleoraBsa, Fab, 1959. 

8» Field Studies of Cancer of I5fe«rin® Cervix » paper presenfead st 
7th International Cancer Congress, London, July, 1958 by 
Dunham, Lucia J. in collaboration with Dom, H.F., Stewart^, 
Harold i,., Edgctaab, John H., and iSioaas, Louis B. Paper on 
this subject to be published. 

9. Fox, Fo, Davidson, John, and thomas, Louis B. Maturagloa of 

Sympathlceblastoma into GangllooeuroBias Report of two Patients 
with 20 and 46 year Survivals Respectivaly, To b« published 
In Cancer, Jan,=>Feb. 1959. 



939 



Page VI. Serial Ho. WCI 853 

10 o Currant Projectes which will Lead to Publications g 

1, Bone: ChAnges in Acute Leukemia with Forkner, C.E.; 

Frel^ Emll; Fritz ^ Richard^ Bess«, BoS, and Lusted, L.B. 

2. Slntultaneously Occuxrrlng Cutaneous and Ocular Melancsxtas 

with Dr. PaCon and Dr. Von Sallman. 

11. Lectures g 

la Chang ins Patterns in Pathology - Symposiua, Washington 
Society of Pathologists;, Dec. 3, 1958. 

2. Aspiration Blopsj. Clinic Day Program of Provldance 
Hospital, Detroit, Oct. 1958. 

3^ Aspiration Biopsy. Lecture • demonstration with Dr, lUbson 
as part of short course in surgical patholc^y, In&sr^ 
national Academy of Pathology, Cleveland, Ohio, April 
1958. 

8. Dr. John H. Edgctaab ; 

1. Edgcocsb, John H. Oa tha Development of Peptic tllcer in Patients 

Treated with Prednisone or Prednisolone c Schwsizerlsch© 
Zeitschrlfe fUr Allgeiaeine Pathologic uad Backt@rlologle lis 
363, 1958. 

2. Merchant, R„F«, Lourla, D.B., Gelsler, P.H., Edgeoab, JoEo, and 

XStz, J.Po Fungal Endocarditis; Review of Literature and 
Report of Three Cases. Ann. Int. Med, 48s 242, 1958 « 

So Forkner, C.E,, Jro, Edgcon*, J.H., et al„ Pseudoinonas SepeicfeHiiac 
Antsr. J. of Medicine ~ accepted for publication. 

4o Edgeoab, John H., Van Scott, E.J. and Andrews, J.R. Morphologic 
Studies of the Skin of Patients with I^cosls Fungoides Treated 
with High-energy Electrons. Acta Dermato="venerologlca 
Scandinavlca . In Press. 

5. Toble, JoEo, EdgcOTib, J.H. and Freiraieh, E.J. Tongue Wosis 

(Linguatula Senata) Infestation in a Patient with Acute 
Leukemia, Aiaar, J, of Clin. Path. 28s 628, Dec. 1957. 

6. Current Projects which will lead to publications g 

lo Study of Patients with Mycosis Fungoides. 
2 c Study of nevi and melanoinas. 



Page VII. Serial NOo NCI 853 

C. Dr. Richayd A. Malmgren (see separate report: for Cytodiagnosic 

Service) . 

Do Dr. Alan S. Rabson g 

1, Herrold, Katherine Mo, Rabson, Alan S.^ and Smith, Robert R. 

Involvement of the Liver in Generalized Hypersensitivity 
Reaction. A.MoA. Archives of Pathology 66s 306»310, 
Sept. 1958. 

2. Rabson, Alan S., Legallais, F.Y. und Baron, S„ Adaptation 

to SeruE»"free Mediusa by a Phagocytic Cell Suraln derived 
from a Murine Lyraphoma. Nature 181s 1343, May 195^ 

3o Kirschsteinj R.K., Rabscn^ Alan S., and Kilham, L. Pulmooiry 
Lesions Produced by Fibrowa Viruses in Squirrels and Eabbitf 
Cancer Research, 1958 (in press). 

Projects which will lead to publications g 

1. Study of the hlstopathology of cutaneous lesions in 

cryoglobulinemia with Dr, John Fahey and Dr. P. Schwabs 

2. Study of hlstopathology of animals treated ulth Fluothane 

with Dr. P. Gelsler and oiembers of anesthesiology depars 
ment. 

3. Study of "malignolipin" in hunan tumors v«lth Mrr. Joseph 

Woodard . 

4o Participation in bladder cancer study with Dr. L. Dunham, 
Dr. H.L. Stewart and Dr. H. Dorn. 

5„ Participation in "Conference on Research in Leprosy", 
Carville, La„, March 1958. Presented papers Tissus* 
Culture Studies of M. lepraemurium in Serura<=free I>tediurac 

Eo Dr. EoB. Price, Jr. g 

1. Hertz, R., Bergerastal, D., Lipsett, M.B., and Price, S,B., Jrc 

Chemotherapy of Choriocarciuoma and related Trophoblastic 
Tumors in Women. J.A.M.A. 168 ? 845, 1958. 

2. Presentation of "Tustors of Soft Tissue ° Diagnostic Problems" 

with Dr. Richard Shxsnan, A.F.X.P. at International Academy 
of Pathology, Cleveland, Ohio, April 1958. 

3o Currently working on clinical and pathological stwdy of human 
kidney in acute leukemia with Dr. Frei and Dr= Fritz. 



941 



Page Vlll, Serial Noo MCI 8 53 

F . 01% T,F. Dutcher !; 

1„ Histopatliology of Macroglobullneiaia of Waldenstrom "■ pajxsr 
presented at Amsirlcan Assoc iatloa of Pathologists aad 
Bacteriologists meeting, Cleveland, April 1958 „ 

2, His topatho logy of ]MUcroglobulinemia of Ualdenstrom -^ to be 

published in A9»sr„ J. of Path, Joint author, Dr, Fahey^ 

3. Zollinger-'Elllson Syndrome - Clinicopathologic confajcence « 

to be published in Ann. of Xnt, Hedicineo 

4o Macroglobulinemia » Clinicopathologic conference " to be 
published in Aunu of Int. Medicine. 

S. Dr. Katherine M. Herrold s 

1. Herrold, Katherioe Mo, Rabsost; Alan S.j, and Sssith.^, Robert R. 

InvolvcKent of the Liver in Generalized HypetsessitiviSj 
Reaction. A«M.A„ Archives ©f Pathology 66 § 306"310, 
Sept. 1958, 

2. Working in collaboration vith Dr. Dunham on mliSiiiisal concentra^- 

tioa of hydrocarbon which will produce cancer in th® h^MSt<tr 
cheek pouchy 

Ho Dr. Itertla Hleklin g 

1. Workshop on Bacteriophage TypiiSig of Staphylococci. Meeii^S 

of Maryland Society of Pathologists and Medical Techno Xogists, 
Bethesda, Md., Oc€. 1958. 

2. Current atvdy of incidence of nazltiple cancers in autop@3^ 

ataterial, Depar^stent of Pathologic Aaatosi^, with DVo Zithtod, 
Dr. Thomss and MTc Love land. 

3. Current study &f response of huiaan ovary to FoS.H. %;ith Dr. 

Bergenstal. 

I. Dr. Robert Peters s 

1. Study of petieate with nelanos^ vh® have inela@uria &ud renal 
- changes in collaboration with Dr. John Edgcoi^. 

2. Current study in a patient with acquired egaoBisglobuliaeraia 

of lyi^h node transplanted frcra sister with different A^O type., 
Busan anti«A and anti°>B sertaa has been labeled with fluoroec' ini 
ieothiocyanate in an atteiopt to establish the presenile of A or 
B subetatice in transplaisted lyisph node. 

SlHsilsr techniques are being used in study of ABO types of 
choriocarcinoGsas in patients wieh Shase twaors c 



942 



Page XX, Serial No „ NCI 853 

'^'- Dr» Robert Peters (Continued)? 

3o A rabbit an£:i-candlda serum bus been £Iuorsc«na£ed and ulll 
be used to study lesion in patients viith candidlas is . 

4o A slide technique for determining incomplete leukocyte aafii" 
bodies has been described using fluorescenated Coombs sera 
and in collaboration uieh Dro Fordtran o£ th« blood banii: 
this technique will be used to study human ssra, 

J. Dr. R. Co Boy Ian g 

l„ Jtoport o£ vascitlar lesions in Cuo pa.tlentc with denaatomjositis 
in collaboration with Dr, Sokoloff a 

2o Assisting Dr. Thoiaas in pathological study of hamsters Infested 
with schlstosoiaiasis. 

K. Dr. R. Gerald Suskiad g 

1„ Toxoplasmosis •^ case presentation at Washington Society o£ 
Pathologists, Nov, 1958. 

2„ Study of autopeied patient vith essential pubacKiury heiaoslderosl 

3, 'In collaboration with Dr„ Love » th« effect of species varialtioi: 
on the oncolytic behavior of Co3tsacki« B3 virus „ 

Lo Dr. Eli^abeth Chu g 

See separate report from Cytodiagtujsls Service, 



Honors y Awards and MeBibershig| Sg 

1, Dr, John H, Sdgcosab ^^ elected to ntecabership of American 

Association of Pathologists and Bacteriologists and to 
American Assciciation for Cancer Research. 

2, Dr. Louis Bo Tlioinas » elected president of the Haryland 

Society of Pathologists for the year 1959= 



943 



??ationai. 
Laboratory of Pathology 
Budget Data 

E»timated Obligations <. o ......... o » <, .Fiscal Ifear 1959 

Direct. ,,0.0.0,.. 0.00 ,oo,,.,c,..$ $82,000 

Reimbursements 

Clinical . . . . . o „. o »....,......,..., „ 

Other, , , 0.0 201, g<X? 

Total 783,500 



kJ 



Includes Projects No' si 






OFraCE OF TB& CHirRTi 


'g 




500 


528 




502 


529 




525 


531 




CAKER WDmn.Qm mo PAfflo©EHESis sect: 


503 


510 


521 


50lj- 


511 


521a 


50Ua 


512 


522 


505 


513 


523 


506 


5IU 


526 


507 


515 


527 


508 


516 


533 


509 


518 


53i* 


509a 


519 


535 


509b 


520 




cmsm mmoum ssraiois 




501 


521* 




501a 


530 




501b 






PASraOLOSIGAl, TmmOUm SECTIONS 





517 



Se'fial Ho. MC,I,5Q0_ 

1. haboxatovy o£ Patteology 
2 c Office of tJm Clltl«c£ 

individual Project Repore 

CAlendar Ifear 1958 



?S£1^ 



Projee£ Title: lisCdsenesis and Faelholosy of Induced and SponemneOTiis 

twaots ol IsAboratory AQlisffila 

Ps'lneipal InTestlgator: Harold L. Stewart 

OtSier Inv®atigaeors Kattoriuvs C, Snell 

Cooperating Unites Ds-. Flojd R, Skalton^ Irban 1^6$ Research Foundatiosij, 

Louisiaim State Stedleal Setool^ Mew Orl^an^^ LauislauMi 

Man Years (calsndar year 1S>58): Patient »«y« (calendar ymt 1958) s 

Total t 5 1/2 

Profeesiozial^ 2 
Otters 3 1/2 

Projact DesertpSloas 

Objectives; to gain knowledge of tS»e etiologle and patlsffllog;!© feee^K-© 
in the production o£ n@<oplasts3 in eisperimental. anlii^is 
and to w&]m this infonaatl@« avaliabla to othar inveat^igators. 

itetli>odfflg 1. Adrenal Cortical Carclno»s 494^ a transplantables 

kon!sonally°ae£lve tumor th&t originated 1«!) tte adr^s^al 
cortajs of an Osborne '^Mandel rat tos baen studied during tiha eo^&rs® ®£ e«veii?. 
ganarati®n® of transplantation in intact^ oatura host rata. A pap»? d&scribi^g 
the pat&ftologic and physiologic. «£fects of this tumor is baing prajMrsd ^'or 
publication. Furthar Investlgatloej is h®ing a»dc of th« transfo»«tio0 
both in s^rpbolosy and In functional activity that this tumor undsr^^t^e ^imn 
transplantisd t® hypophy®ect0iatg«d rats and to n«ewbo^n rats. Wieh tfes 
cooperation of Dr, Floyd Skelton of the Louisiana Stat® inivarsiSy Sfedical 
School; blood steroid and @l«'Strolyta determinations on ths various groups^ 
of rats ar@ baisg stada. Zt is hop@d that a corralffition of patholojiic @nd 
bloclieaiieal findings will glv@ us an understanding of the behavior of ehi^ 
adrenal cortical tuii»r. In collaboration with Dr. R. C. HacCardle^ cy£alpgi@ 
essaadnation o€ the kidneys of control rats and rats bearing tmrnr 4^ 1:^ 
being SQSde to study differencaa in the mitochondria In tha e@ll@ of thu 
distal convoluted tubules. 

2c A study of th« spontaneous neoplaeras occurring in the 
JtostoBoys^ a South African rodent, is balng continued. During 1958^ 59 of 
these rodants bred in our laboratory have b«@n neeropsisd^ and tissusiS 
from 26 hava b@an ejeanlnsd histologically. 



945 



PHS-NJH 
Individual Project Report 

C«lendar Year 1958 



Part A Csonclnwed) 



3, l,2p5^6"dlbeiia«ntlhir«cene in an olive oil emulision has 
been fed Eo DBA miee to deteinaine ehe incidence of adenonaeoais in tba luns* 
of these miceo Histologic esaaination has been completed on the experitnenfial 
anioals. Additional control animals are scheduled for necropsy durina 
rkAMAmh^v 1958 



Sadueed in 

Qg 2Q^a^th. 



4. A long-tena ®£«dy of the effect of age on unEreatsd 
rats of various ages is being continued. Spontaneous tunwrs occurring in 
these rat® are being enunerated and classifiedc Because of poaaible eeeroaen 
contamination of food during 1955, part of this ea^riiuent is baing repeated 
During 1958^ 17 rats have been cxaained^ and additional rats have been s«5t 
aside for necropsy at appropriate intervals. 

5. A aenuscript entitled Nistopathogcnesls of Careinoaa 

?Q°a^thyleholaothrene has been caaplfttad. ~— -*■ -™~ 

6. Monkeys that received injections of carciaogeni* 
substances into the wall of the glandular stomach several years previously 
are being raecropsicd and studied to ascertain whether these substances 
Hsay have produced gastric carcinona. 

7. Collaborative work with Dr., H. P. Iferris; Appro:s4" 
mtely 3500 histologic sections from control rats and rats fed 2 diiUsmnt 
casclffiogesjic coaapounds, N.N^-a.T-f luoraiiyl acetamide and di^acetylamin®- 
fluorene^ have been examined. 

Major Findings; I. Study ©f Adrenal Coreical Careiaoaa 494 in intact 

mture rata indicates that this transplanted tuaor secretes 
an esscess of certain hormone® normally ejscre^ed by tSta cortex ©f eh« adrenal 
gland, and that these secretions, fefe-ough their action on tSie adeaohypophysis 
of hose rats, induce atrophy of the adrenal cortess^ 83% organs, and lymphoid 
tissue „ By inducing degenerative changes in the distal convoluted tubules 
of the kidj^y, tumor secretions impede th@ normal reabsorption of water, 
thus producing polyuria. When Adrenal Cortical Carcinoma 494 wss grown 
for one generation in hypophysectomised rat® and then returned to intact 
mature rats, its hormonal effect was like that of progesterone, although^ 
it still retained the capacity to induce polyuria, polydipsia, and renll 
tubular degeneration. This transformation in funetion was accoospanied by 
the appearance of an anaplastic type of cell not hitherto observed in any 
transplanted tumor of Un© 494. Transplantation of Adrenal Cortical 
Carcinooa 494 to one litter of newborn rats produced changes in aorpholosv 
and function similar to those observed when the tumor that had been grown 
in hypophysectoraiged rat® was transplanted to intact, mature rats. 

2. Of the 26 Sfestomys examined histologically, one 
showed an adenocarcinoisB of the glandular stomach with metastases to lymph 
nodes c Other tumors observed include papillomas of the forestoaach 
granulosa cell turaars of the ovsry, a mediastinal teratoma, two lung tuisors 
adenomas of the adrenal ©rtejs, and an adenoma of the pancreas, Hon® of eh® 
attempts to transplant tumors to these non»inbred Mastoaye has been 
successful. 



946 



Sarlal No. MCI 500 
PMS-NIH 
IndlviduAl Project Report 
C«lend«r Ifeitr 1958 



Part A (comtlnued) 



3. One hundred p«reent of the DBA mice fed 
l^2j,5;,6»diben@«nthrtteene have shown adenomatosis o£ the lungs. Mo adeno^. 
aatosls has been found In the control mice thus far exaalnedo 

4o No significant findings. 

5o Manuscript published in the Journal of the National 
Cancer Institute, November^ 1958. 

6. No signif leant findings. 

7. Rats fed the carcinogen^, N;N^;2-7 fluoraoyl acetanide 
by Dr. H. F. Morris and eammined histologically in this laboratory have shown 
an unusual nusSisr and variety of neoplasias. Ansong these lesions are: 
histislogically malignant carcinonta ^i the glandular stomachy, adenocarelnosia 
of the scsall intestine^ severe atrophy and sareoraaa of tBui salivary glassds^ 
nsurogenic esrcoffis originating in the rsgion of th@ trigesiiraal nerves «ind 
pituitary g]|,sindp adenoearciooau of the uterus^ and ae^ Anitschkow cell 
sareoae of th@ heart. Carein@eas of the liver^ ear^, and manaary gland 

are also present in a high percentage of rats fed this confound. 

Significance g Ihm kn@»lsdge gained froa a study of neoplasms in 

«. laboratory animals should provide a basis for further 
work on the sieehanism of carcinogenesis in human beings. 

Proposed Course ; It is estpeeted that a paper describing the histo° 
pathology and effects on host rats of Adrenal Cortical 
Carcinosjsa 494 will be submitted for publication in Deceifi)®r 1958^, or January 
1959. Result® of the study of th@ effects of feeding 1^2s5^6-dibengaiaehrac®a@ 
to DEA alee will be assessed and probably prepared for publication during 
1959c The collaborative work with Dr. MacGardle and with Dr. Nbrris will 
be continued; it i® possible ttet results of these eKperisents will be 
ready for publication in 1959° The essperisiental procedures sad exasain&^ion 
of histologic i^terial BSntioaed in other itesis enunscrated above will be 
continued. 



Part B isjcludeds Yes 



947 



SarUl No, SCI 300 
PHS-NIH 
Individual Projeet Report 
Calendar Year 1958 

Part B; ^onorSj, Awards^ and Publieatlons 

Publications other tStan abstracts frenn t&ois projects 

Stewart; M. L.; Snell; K. C.j, and Hare^ W. V. 

Histopatliogenesis of careinonui induced in the glandular stonach o£ 
C57BL mice by the inCramurel injection of 20'°nethylc^lanthrene, 
J. »ato Cancer Inst, 21s 999<='1035j 1958. 

Stowartp H. l>.; Snell^, K. C.p Dunham^, L. J. ^ and Schlyen^ S. M. 
Transplantable and Transaisslble Tunors of Animals. 
Fascicle 40 of ttie Atlas of Tumor Pathology^ AFXP; Was&ftingtonj, D.C 
Dccet^er 1958. 



943 



Serial No„ NCI 502 
PHS-NIH l„ Laboratory of Pathology 

Individual Project Report 2. Office of Che Chief 
Calendar Year 1958 3. Bethesda^ Md„ 



Part A. 



Project Titles Histogenesis and his topatho logy of cutaneous neoplasms. 

Principal Investigators Dr^ Jolm H. Edgccmb 

Other Investigators; None 

Man Years (calendar year 1958 }s Patient Days (calendar year 
Totals I 
Professionals I 
Others " 

Project Description s 

Ao Study of diseases of man (collaborative research) 

lo l^cosls fungoides 

Six patients have expired and have been autopsled. This 
material Is being studied with a view to publicatlono 

11 o Melanomas 

The study of naevl and melanomas continues o Dro Van Scott 
and I are enlarging the series . We have become convinced that th@ usual 
criteria for the diagnosis of primary melanom^i may apply to certain 
metastatic melanomas o 

ill. A small study of the reaction of the skin in xerodensa 
pigmentosum to light Irradiation is being studie^d., 

B. Study of diseases in animals 

lo The description of Dr. R. Bryan's transplantable squamous 
carcinoma in BALB/c strain mice remains to be published <, 

ii. Four melanomas have been produced in guinea pigs by skin 
painting with dime thy Ibenzanthraceneo The experisaent continues,; 



Part B included Yes (x) No 



9^9 



Page II, PHS-NIH Serial NOo NCI 502 

Individual Project Report 
Calendar Year 1958 

Part B - 

Hoaors ; 



Eleceioa to iteterlcaa Association o£ Pathologists and Bacteriologists 
American Association for Cancer Research 

Declined an appointiaent to the faculty of Indiana University^ 
School of Medicine. 

Publications g 

Edgcomb, JcH., Van Scott^ S.J., and AndreuS; J.R„ Morphologic Studies 
of the skin of patients ulth oncosis fungoldes treated vtth high» 
energy electrons o Acta Dermato^veoerologica Scandlnavlca (In press]). 

Bdgcoodj^ J.H. GAstroduodenal ulcers in patients treated with prednlsoc'e 
and prednisolone. Schueiz. Zeltschr, f. Pathologie 2Is 363°372; 
1958 o 

Merchant, R.Fo, Louria, D,Bo, Geisler, PoH., Edgcomb, J„Ho, and Uts^ 
J. P. Fungal Endocarditis s Review of the literature and report 
of three cases, Ann. Into Med. 48s 242-266; l95So 

Tobie, J.E>, Edgeonyj, J.H., and Freirelch, E.J. Tongue Wona (Linguatula 
Serrata) infestation in a patient ulth acute leukemia. An^Xo Jo 
Clln„ Path, 28s 628-633, Dec. 1957. 

Forkner, CoE., Jr., Edgcoob, J,H., et al. Pseudononas septicemia ^ 
Anier. J. Medicine (in press}. 



950 



Serial No, NCI 525 
PHS«NIH lo Laboratory of PaShology 

Jndividual Projecfc BeporC 2. Oiflce of Che Chief 
Calendar Year 1958 3. Bethesda 14, Maryland 



Part A. 



Project Titles Attempg to induct carcinomu of 6he bladder in haateters 
by infection with Schistosoma haffiaaCobiumo 

Principal Investigator; Dr. Sloisc Cram. 

Other Investigators: Dr, Louis B. Thosoas, Dr, Elmer Berry and 
Dro Louis Olivier o 

Man Years {calendar year 1958) s Patient Days {calendar y«ar 1958})^ 
Totals I 
Professional: I 
Other: <■ 

Project Description ; 

This research project is in collaboration with Doctors EIois«i Craia, 
Eltaer Berry and Louis Olivier^ N.I.A.Z.D. and consists of studying 
chronic infection with So haeaiatobiua (Gold Coast strain and Egyptian 
strain]) in hamsters ^ particularly with reference to She induction of 
bladder carcinoma. 

a.) 217 haiBSters were exposed to approximately 300 cercarlae 
(Gold Coast strain S. haeroatobiun) each early in 1955. Practically all 
of these hamsters have now died and have been autopsiedo Histological 
sections have been prepared o 

b.) Late in 1956 and during the first six months of 1957 j, S<, haagaato -- 
biua from Egypt became available through the efforts of DrSo Berry and 
Olivier. Approximately 150 hamsters were exposed to cercariae and 
approximately 80 of these animals are still living. 

Thus when this experiment is completed it will be possible to 
compare chronic S. haemagobivm infestation in the two groups of aniteals 
with Egyptian and Gold Coast strains of S. haematobium . 

Significance to Cancer Research ; 

S. haematobium infection has been found associated with bladder 
carcinoma in several parts of the world. This study is designed to 
produce bladder cancer in the hamster by chronic infection with S. haemato - 
bium . The incidence of bladder cancer associated with Schistosomiasis 
is high in Egypt and relatively low in the Gold Coast. The study is 
also designed to determine whether there are pathological differences 
between infestation due to S. haematobium from these two parts of Africa. 



951 



Page IX « Serial No. ;;?'-" 525 

Part A (continued) 

Proposed Course of the Ptojeet s 

Xt is anKieipaeed thaft Shese animals vill be allowed to live 
vith this chronic infection for as long as two or Shree years to see 
whether chronic bladder disease due to infestation with this organism 
will result in bladder cancer in the hamster , 



Part B included Yes C ) No (x) 

952 



Serial Mo. SCI 528 

1. Labor aKory of Pathology 

2. Office of eti« Ghi«f 

3. BethesdA; ttaryland 
P»ffi"NXH 

Individual Project Report 
Calendar ¥ear 1958 

^rt_A 

Project Tltl«: Cytochemistry of Ribosueleoproteiias 

Principal Xavestlgstor: Robert Love 

Other Investigator: 

Coop«ratiG@ «Saitas 

Man Years (calendar year 1958) s Patient Eteya ((calendar y^ar 1958) s 

Total ; 1 
Professionals 1/3 
Others 2/3 

Project Deseriptton: 

Obieealves; To study the ribonucleoproteins of noneal and neoplastic 
cells. 

Methods g Developaussit and application of a new staining isetliod for 
RM. 

Major Findings g a) The cS^mical basis of a new msthod for differeeeiatiHig 
vaxLsms forsas of RM-protein in th& cell has been &&t&x-iaiu&d 
and this study is now ready for pvsblication. b) Seven different forais of 
RKA.-proteia have been deiaoastrated In the cell, c) Two different types of MA 
have been shomi in the nucleolus; one form appears to correspond to th@ 
"nucleolinus^ " %rhlch has beesi described by the older cytologiets^^ biat which 
has not been defined eytocheraically. 

Sig;nificaHceg Rlboaucleic acid is intisately concerned with cell grawfch 
and differentiation ^rith protein synthesis and ^s^ith vi»l 
reproduetlosi. An tsuderstanding of these processes is essential to the study 
of neoplasia. 

Proposed Coarse i a} Evaluation of tltie structure and function of tte 
nucleolinus. b) Applicgtion of the new staining i^thod 
in the study of virus-^infected eells. (See project on !j|ef»castle Disease 
Virus infection. ) c) Study of RM Betabolism in rat liver during fasting 

and after feeding the starved animals <'«?li:h M. f. B. Bharadi^aj). 4) S&udy 
of tu!Bor cells treated vilth chessotherapeutlc agents ttet interfere ^Ith EMA 
laetabollsKa (with Sr. T. P. Bharadwaj). 

Part B ineladeds Yes 

953 



Serial Wo. SCI 528 
PHS-HIH 
Individual Project ReporC 
Calendar fear 19S8 

Part Bs. Honors^ Awarda^ and Publications 

Publications other than abstracts from thie project: 

Love R. 

The distribution of ribonucleic acid in tuiaor cells during laXtoalis. 

Sature 180 s 1338-1339^ 1957. 

lovc^ R« 

Interchange of ribonucleic acid between the nucleus and cytoplasm 

during laitosis of tussor cells. 

Acta I'nio Internat^ Contre Cancer. In press. 



95- 



Serial Wo. WCI 529 

lo LAboratory of Pathology 
2. 0££ice of th« Chief 
3o Bethcsda^ Maryland 
PHS-NIH 
Individual Projecr Report 
Calendar 7car 1958 

SSEJLA 

Project Tide; Viral Therapy of Tumors 

Principal Investigator; Robert Love 

Other Investigator; 

Cooperating l^its: 

Man Years C^^SQ^as' 7^^ 19S8): Patient Days (calendar year 1938); 

Total s 1 
Professional; 1/3 
Other; 2/3 

Project Description; 

Objectives : To adapt viruses to becoasae oncolytic for tumors. 

Methods ; a) Passage of Neveastle Disease Virus (NDV) through th@ 

Ehrllch ascites ^ vivo ., b) Analysis of the cytologlcal 
and cytocheMcal changes in tuisior cells infected «ith NDV. 

Ma jor Find lags ; a) KW has been adapted to infect and to destroy the 
Ehrlich aseltes tuesor without killing the host, b) k 
study of Ehrlich cells during oncolysis produced by NSV infection stsggests 
that viral HM-'protein is synthesized in the sucleus and is extruded Into 
the cytoplasm <»here it acquires a coating o£ phospholipid and forms 
prominent "ine lus ions . " 

Significance ; The study of oncolytic viruses^ and in particular of 

viruses that can be adapted to becoee oncylytic for tim&TBi. 
helps to understand the viru8°cell relationship and oay prove to be of 
therapeutic value. 

Proposed Course ; a) CosipletloQ of the study of the adaptation o£ HDf 
to the Ehrlich tumor, b) Evaluation of the oncolytic 
effect of this virus oa other transplantable tusors of tsice. c) Adaptation 
of HDV to becoiaa oncolytic for spontaneous tuoors of sice, d) Continuation 
of work on the oncolytic effect of Cossackle B3 virus on He la twstors; 
comparison of the cytopathogenlcity of rat Eela°adapted virus and naturally-^ 
occurring virus in Hcls tusaors in rats and la chick esbryos (witte Dr. R. S. 
Suskind). 



F^-t B laelud®d; ¥es 

955 



Serial Ro. NCI 529 
PHS^HIH 
Individual Project Report 

Cal«Bdsr Year 1958 

P art B ; Honors^ Awcrda^ and Publieatlona 

Publieaciozis other than abstracts from this project: 

love^ R, 

latroduction to the Conference on "The Cytopathology of Virus Infections." 

Annals N. f . Acad Sciences. In press. 

The Cytopetkology of Virus ^Infected TtuBor Cells. 
Anaals M. ¥• Acad Sciences. In press 



Honors^ Awards relating to this fro ject; 

Organised the Conference on "th& Cytopathology of Virus Infections" 
for the Net? fork Acad«sqr of Sciences^ Noveatber 7-°8^ 19S8. 



956 



Serial Mo. KCI 531 

1, LAboratory of Pat&hology 

2. Offlcs of the Chief 
3o Be£h«ada; iteryland 

PHS-NIH 

Individual Project Report 

Calendar 7e«r 19S8 

Par^A 

Projact Title; Study of Filtrable Agents from Stouse Tumors 

Priaeipal Investigators Robert love 

Other Investigators; Dr. H. Koprowski {k only) 
Dr. John Edgeoob ((C only) 

Cooperating Onits: 

Many Years Ceales^ar ye&r 1958): Patient fiays Cc«l«£sdar ye&r 1958 }s 

Totals 1 

Profeasioaals 1/3 
Others 2/3 

Projset Deserlptioas 

OfajeetAves s A. To study ths effects of inoculation ©f Be^ora and 
foetal Ql@e with cell-free filtrates of EhrXich aacitss 

tumor. 

S. To study the effects of inoculation of highly 

polyoerised ribonucleic acid and deso^ribonucleic acid into foetal «ic@o 
C. To propagate a filtrable agent from teouse l@uk@Ma 

in tissue culture. 

Methods g A. C3Hf/EeH » AKR and AL/M mice were iaoeulated m 
Indicated above. 

B. BAm/e and AL/H foetuses were Inoculated with Wk aad 

0. Extracts frora a lymphocytic neoplasm were passed serially 
in Eunkey kidney tissue cultures and inoculated into C3H/He^ x AKR and 
C3Hf/HeN x AKR newborn sice. Control mice were inoculated with esctrsets 
of noroal tissues treated in the a&e& isaaner. 

Major Findings ; A. The sjajorlty of the salee have n@w been autopsied. 

Flfty°ewo (93%) of a total of 56 C3H s AKE miee that 
had been inoculated ^ith cell-free filtrates developed tumors, the ccEseonest 
tua»rs were lyf^hocytic end other neoplasms of the reticuloendothelial 
systesi^ hepatosms^ osteosaas^ mmmry tuaors^ and lung tuiBors. Eleven {^51%} 
of @ total of 13 uninoeulated mice of the sa^ie strain developed ttaaors* 
during ths. sas^ period. All of the control tia^or -bearing siice had leukemia; 
two osteomas^ osia hepatoiE^^ aad one cortical adenoissa of the adrenal gl&M 
were also fcmnd. 

HiaetesR {^m,} of a total of 28 AL/n mice^ inoculated 
with filtrates^ developed t;aa©rs. Three, osteogenic sarcomas ifere ohBB-m^^ 
tss this group. Eievea {WQi of 22 uainocul&ted eeatrol ^lee of the ss-isa 
strain developed tusajrs durisg the saajs period.— - 

957 



Serial Mo. 
PHS-BIH 
Individual Project Report 
Calendar Year 1958 



NCI 531 



Part A (c«otinued> 



B. Prellainary observations suggest that inoculation 
these nucleic acids had no effect on tha alee. 
Preliminary results suggest that an agent capable 
of producing tusiors of the parotid gland was present in the tissue cultures 
inoculated with leukesoic tissue ,. 



Major Findings ; 
of 

C. 



Significance ; The characterisation of filtrable agents and their 
role in the production of neoplasms is of fundaesantal 
itsportance in the study of careiaogenesiSc 

Proposed Course s Completion of the study of mice under test. 



Part B included: Ho 



950 



Serial Mo. WCI 503 

1. LaborsCory of Pathology 

2. Cancer Indue. & PaChogenesl* 

3. BeCltesda^ Maryland 
PMS-HXH 

Individual Projccc Report 
Calendar Year 1958 

Part A 

Project Titles Collaborative Receareh Kequiring Pathologic Anatioa^ 

Principal Snvestigator; Thelaot B. Duxm 

Other Investigators 

Cooperatiag Utoitg; 

Han fears Ccalendar year 1958): Patient Days (calendar year 1958) s 

Total: I 1/3 
Professional: 1/3 
Others 1 

Project Description: 

Objectives t fo collaborate vith and advise other investigators at th» 
Slatlonal Cans^r Institute in phases of researels problesas 

requiring eKperisnee in anltsal patttology. 

Methods 8 This naay consist of (1) Full responsibility and separate 
publication as senior author of material reviewed in 
connection with work of another investigator^ (Z) Review and description 
of anifltals e®alng to autopsy^ and publications of the findings as a separate 
section of a paper describing the entire eK^riisent^ (3) Review and diagnosis 
of ttsBors found by investigator in course of e:^erltaents^ the Informatioa to 
be used In a tabulation of results ^ (4) Oeeaaional consultation and advice 
on pathologic aspects or aaterlal in an experis^nt. 

Major Find lags ; 1. All laaterlal has been accuaulated for description 
of three distinctive neoplasms carried by Dr. MIcImcI 
Potter. These have been studied as solid and as eeeltes ttmors. 

2. Work with Dr. John Fahey and Dr. Hlchael Potter on 
plasfss cell neoplasss. Description of tutors supplied for publications. 
Work on description of naissiary tuusors in ^le mice produced by estrogenic 
hors»neS; in collaboration with Dr. Andervoat^ has been published. 

3. Tumors produced by D7o Ruth Herwin have been described. 

4. Work with Dr. Lloyd law end Dr. Clyde Dawe^ with special 
reference to maisnary tumors in mice treated at birth with cell^free filtrates. 
HanBoary tuoffirs have a different histogenesis and szorphology from those 
comaaonly encountered. 



959 



Serial No. VC t 503 
PHS-MIH 
Individual Proj«ct Report 
Calendar Year 1958 



Pare A (continued) 



Slenlficaoce g Pathologic review la Indispensable to many types of 

cancer research. This Is best carried out by a specialist 
in patholoslc anatooy; since with the present complexitA.es of research a 
single investigator cannot know this field and his own apeelalty,. 

Proposed Course s This work will continue as in ttte pest. Vhstif as 
a result of nany observations, enough Inforaation is 
accumulated regarding any subject, publications will bo loade. 



Part B included; Ifo 



960 



SerUl Wo. MCI 504 

1. Laboratory of Pathology 

2c Cancer Xndue. & Pathogenesis 

3. Betheedaj Maryland 



PMS-NIH 

Individual Project Report 

Calendar Tear 1958 



Part A. 



Project Title; An Electron Microscopic loveatigatlon of Normal and 
Kcoplffistle flsauea. 

Principal Inveetigatori William €. Banfiald 

Other Investigator: 

Cooperating Iktlts; 

Man Years (calendar year 1938): Patient Days i^cale^dar year 1958) s 

Total! 2 
Professional: 1/2 
Others I 1/2 

Project Description: 

^jeetives: l. To deecrifea and interpret the ultra structure of 

selected neoplesos and to compare results with suitable 
control tissues. 

2. To look for and study the inciting agents or accompanying 
virus la neoplastic lesions and compare vlth slnllar agents or viruses if 
knovn., 

Major Findings s I. A study of th» epidermal lesion of aaollusc;^ 

contagiosum has led to a tantatlve working hypothesis 
of the sequence and tsethod of devclopiaant of the tasture elementary body 
from the granular ground substanes of the inclusion. 

2. A dense granule measuring 80 s llO A" has been 
described as a new structure in th& alessantary body of nolluscuss contaglosuBio 
Xt also Is present in the Shop* fibroma elementary body. It should b« 
pointed out that the developsaiatal forms of the Sliope fibrosoa virus and the 
rabbit zs^xoma virus arc almost identical. 



Sisnlfig^Rg^ s From such work comes an understanding of hmi a virus 
exists within a cell end better ic^lght for studying 

the action of viruses upon cells and possible methods of controlling ttils 

action. It is loportant in elucidating the chemical and physloehemical 

data obtained from viruses and virus infections. 

The small granule in tS^ elementary body is^ I bellevej, 

the smallest organelle yet described » 



961 



Serial No. WCIC 504 

Zadlvldual Project Report 
Calendar If ear 1958 



Part A (continued) 

Propoaed Courge ; lo Find the composition and function of the sttall 
dense granule in the elementary body. 

2. Atteiapt to fill in the gaps for the development 

of the eleaaentary body and attenpt to find the origin of the ground aubstanee 
of the inolluseun body, 

3. Nortaal stratified squaiaous epltSieliuB from various 
sites will eventually be investigated and correlated with hyperplasias and 
equasnoufi and basal cell careinofBas of various organs. 

4. Posaible limited collaboration with Dr. Dawe 
and Dr. Killham is under consideration. 



Part B included:. No 



9f. 



aZ 



Serial No. NCI 504< 

1. Laboratory of Pathology 

2. Cancer Indue. & Pathogenesl* 

3. Bethesda, Maryland 
PHS-NIH 

Individual Project Report 
Calendar Year 1958 



msjL 



Project Title: An Investigation of the Collagenous Connective Tissues 
and Slsstin. 

Principal Investigator: Willian @. Banfield 

Other Investigator; 

Cooperating 13nits: 

Men tears Cealendar yeex 1958} j Patient Days Cc*lend«r year 1958'^, 

Total: 2 
Professional: 1/2 
Otl«r: I m 

Project Oescriptlont 

Objecfcivea : 1. To elucidate age elisnges and patliiologle alterations 
in eollagsn end elastin. 

2. To uncover tneehanisin affecting the state of collagen 
in the body. 

3. To study the forastion and change of collagen and 
elastin in scare. 

Major Findings ; 1. It has already bsen demonstrated that there is 

a sharp drop in the eor.eentration of acetic aeid<>8olubl@ 
collagen in hunssn skin at one year of age. Analysis of ths results froa t^ 
skin of individuffils greater than one year of age show that the acetic 
acid-soluble eoaaponent of the skin collagen gradually disappears in 28 
percent of the individuals by 20 years of age after which there is a sharp 
decrease in the percentage of positive skins from individuals between 20 @nd 
30 years of age continuing until 40. Th^re is a rise between 4® and SO 
years and & continual rise to a high percentage of individuals ^ving 
acetic aeid°$oluble collagen In their abdominal skin^ 80%^, between 60 and 
80 ^ars of age. There is a slight decline after 80. The shape of th® 
curve is sitailar for sales and fesales but the changes are sharper and 
greater in the females for the age group above 60. 

Z. In sears tested for concentration of acetic 
aeld»soluble collagen there is no difference from the surrounding skin 
for 13 daySj, at 16 days a 2+ difference between scar and skin appears. 
At 60 days there ie a 3+ difference in the soajority of sears tested. At 
12 1/2 laonths there is a sharp fall°off in the aoiount of acetic acld°e3gtrae<» 
table collagen in the scar. Ikmmv&Xg as & group^ the concentration in 
tim scars rei^ins greater than that of the surrounding normal skin. 



963 



Serial No. WSl 504« 

ms-vm 

IndividiMil Project Report 
C«lendAr Year 1958 



Part A (continued) 



3. Hans tera have been aacrlfiecd up to 644 daya of 
age. The acetic acld-aoluble collagen of the back skin of all hamstera 
has fallen from 4+ to 3+ by 211 days of age., There is a variable but 
gradual fall^off until 440 days and by 477 days all skins haVe becoae 2+ 
or less. For abdominal skin there is a definite fall<°6ff in concentration 
by 440 days using 0.1% acetic acid for extraction and by 322 days using 
0.3311. By 561 days using thes latter concentration o£ add for extraction 
all skins have fallen from a 44- to m 2+ extractabillty or less. The data 
for correlation of these findings with pathology and age changes In organs 
has not yet been ecnpiled. 

4. Acute scurvy In guinea pigs does not alter '■ 
acetic aeld^^solublllty of the skin collagen. In chronic scurvy of 
approxlEately 1 1/2 months duration^ tl^re is a tendency for a decrease 
in the acetic aeid<»soluble eollagen of the abdoosinal skin. 

Chronic starvation la hamsters does not alter 
the concentration of acetic add extraetable collagen in the skin. 

5. George Schwars^ tsedlcal student at Duks diversity; 
has worked out the a»thod of Mlyada and Tappel for determining hydro3gy pro line 
on the crude acetic acid extracts of collagen. Eis deteminatlons as well 
as those o£ Dro Irreverre of HIAMD have conf Inxed 'the validity of the vlsu&l 
estlaate of the gross NoCl precipitate in plus readings as a caeasure of 
soluble collagen. 

Signiflcanea ; A very siiaple taethod for detecting a change In collagen 
aetabollsra has been established. A definite and orderly 
variation in the eompositlon of the skin collagen as it is related to add 
solubility takes place in taan t%iro«iighout life. This means that the collagen 
BsetaboliSiB in men is not static and can be Influenced signlfleatitly by as 
yet undetermined faetors. Whether this variation Is significant in relation 
to neoplasia remains to be determined. Whether th& continued increase in 
the concentration of acetic add°8oluble collagen la scars over that of the 
normal skin is related to the high incidence of epidermoid earcinocia in 
scars should be considered. 

A base line for hamsters has been established so that 
factors which may effect collagen metabolism can be ascertained. 

Proposed Course ; I. C««Bplete the age group from 1 to 6 months and 
continue following the normal aging process in 

bemsterSj using significant nuaiberSj, until the coneentratloo of acetic 

aeld°soluble collagen in tl^ skin remains 0. 

Correlate the pathology and microscopic age ehsmgcs 

with the change in the skin collagen. 

2. Correlate hydro^proline determinations and plus 
readings of the MaCl precipitate of acetic add extracts of skin until 
parameters can be outlined, 

3. With the electron microscope^ follow collagen 
fibril changes in maturing seerS; exomlne the collagen of the growth-ohormooe^ 
treated haisster s^terial which is now at hand;, end study elastic tissue in 
thin seetipsss if a suitable s^tfeed c«n be davsleped. 

Part B included s fes QC/ 



Serial No. MCI S04« 
FHS-NXH 
Individu«l Project Seport 
C«lendar Year 1958 



Part 6; 



Honors^ Awards, aod Publications; 

Publications othar than abstracts fron this projaet: 

Banfisldf Wllllaa 6, 

Effect of growth horsena on acatie aeid°«xtraetable collagea of 

haaset^r skin., 

ProCoSoc. Expo Biol. & Med.^ 97 i 309-312, 1958. 

Banfield, WllllaiB S. 

Collagen and Seticulia 

In Frontisre in Cytology j, 1st edition. S. Palsy editor^ Ne«7 Mavan^ Cona<, 

¥al« University Frees j, 1958 o 



965 



Serial M©. WCI 505 
P8SS-MIH 1. L«bor«tory of PAehology 

!ndlvlducl Project Report 2. Cancer Indue. & Pttthogenesie 
CAlcndar Year 1938 3. Sethasda^ Maryland 



P££LAi 



Project Title; The Induction o£ Squanous Carelnoma of the lungs in 
Laboratory Aninala 

Principal Ixsvcstigator: Mearl F. Stanton 

OtlMr Investigator: Kobert Blaekvcllj, Joseph Albreeht and staff 

Cooperating l^its: 

Man Years (calendar year 195fi)s Patient Days {calendar year 1958) s 

Totgals I 
Professioml o 1/3 
Otbers a/3 

Project Dss<eriptions 

Objeetives s To induee lung cancer in laboratory anlBsals tferough 

deposition of known and suspected carcinogens in areas 

of regenerating lung tissue o 

Metteods ; Certain aliphatic haloearbons are capable of causing isl^esaie 
necrosis when trapped in capillary vessels. I5? incorporating 
e«rei«i9gens such as netbyleholsnthrens into the inosuluai it 1$ possible t@ 
deposit the careiaogee in particular organs of Interest to the oncologist. 
The detection of oethyleholanthrene deposition by H.V. Bsieroseopy is bslng 
aeeos^lishedo 

mior Findings; Intravenou® sdninistratioa of the Smiecarbon ©loss© 
has not induced tuaors in rabbits^ rets^ or miae^ many 
under observation for one to two years o In mice and rabbits neieBier the 
haloearbon nor haloearbon ^ oethyleholanthrene cause an increase in 
spontaneous alveologcnle tumors nor do they induce careinoaias in the 
areas of infarction. In rats a suspension of ststhylcholanthrene in trieapoylits 
carried to the lungs in the haloearbon consistently induced raetastasising 
squanous careiftosias of the lungs which kill the aniiaals in six to seven 
tBonths. The histogenesis of the tunors na» under study indicates they 
arose in areas of bronchial nsstaplasia which result from haloearbon 
produced infarction. We have traced the deposition of tasthyleholanthri&ne 
with ultraviolet light saicroscopy. 

Sintniflcanee ; C^r studies xeay contribute to several o^jor problssis 

in the genesis of lung cancer In csan such as the role of 
pulnonary sfierring and its relationship to exogenous inhaled carcinogens 
which in thsetsslves wrj act by injuring bronchial ceils and subsequently 
altering their neoplastic potential. Such studies tsay also offer a new 
saethsd for studying the mechanism of nethylcholanthren® carcinogenesis. 



966 



Serial No. VCl 505 
PS8S-NIM 
Individual Project Report 
Caleadar Year 1958 



Pare A (eonCinued) 



Pro posed Co«r«e ; Study of tSte hlstogcneaia of the tuoorSc Quantitaeiva 
meaaurarasnts of dos« to reaponae. Further testing on 
ability of eearrlQg alona to ioduee carcinoBta. Testa currently under study 
inelude the lntrevexu»u6 administration of infarct inducing haloearbons 
several days prior to and aevsral days after intravenous aMthylcholanthreae 
in an effort to demonstrate ttie initiating and/or proaotlng effect* of 
these substaneeSo Further studies as outlined In Individual Project Report 
HCX 505; 1957; ar« also continuing. 



Fare B included s No 



967 



Serial Ho. VKl 506 

1. Laboratory of Pathology 

2. Cancer Indue. & Pathogatiasiiii 

3. Bachcada^ Maryland 
PHS-NIH 

Individual Project Report 
Calendar Year 1958 



Part A 



Project Title; Study of Transplanted Tusors In Hlee 

Principal Investigator: Thalma B. Dunn 

Other Investigator; 

Cooperating Units: 

Man Ye&rs {calendar year 1958): Patient Days Cealaadar year 1958): 

Total; 2/3 
Professionals 1/3 
Otiies; 1/3 

Project Description: 

Objeetives g 'S'o study various unusual tuaors which tasy throw light on 
tbe nors^l eell counterpart or cell function, and assise 
in understanding of neoplasia. 

Methods ; A nuaber of tumor lines of unusual interest are now b^iag 
carried. 

Major Findings ; Ths n^st cell tuoor which has a high content of 

haparine^ histamine^, and serotonin has been supplied to 
a nua^r of outside laboratories and a large astount of tuioor tissue has 
been given to workers in the Natloaal Heart Institute vho are studying 
serotonin and heparin netabslisa. The plasioa cell neoplasms have been 
supplied to osher laboratories^ since they have siany slBllarities to 
gultlple B^elonia in ta&n. A new source has been found from Dr, Suth Merwln°@ 
work. 

Significance ; Keopiasti@ alterations in cells which have easily 

recognised t&orphologlc or ontsbolie characters vmj lead 
to a elesrer understandiag of naopiasiec 

Proposed Course s Ko work is now b«ing done in this laboratory on 

the Bsst and plasias cell nsoplassas^ @%gept for carrying 
them in transplant j, and observing them for any alterations . 



Pars S iseludeds Ho 



963 




Se*Ul Mo. wei 507 

1. Laboratory of Patfitology 

2. Cancer Indue. & Pathogenesis 

3. Bethesda; Marylattd 
PHS-NIH 

Individual Project Report 
Calendar Ifear 1958 

Part A. 

Project Title; Effect of SNA on Newborn Rats and Mice 

Principal tnvestisator: Ross C. MaeCardle 

Other Investigator; 

Cooperating S$nits: 

Ifaa fears (calendar year 1958]): Patient Days (calendar year 1958); 

Totals I 2/3 
Professional; 1/3 
Othars I 1/3 

Project Description: 

Objectives ; To deternine whether Ribonucleic Acid will produce tusor®, 

tfethods s BM. extracted frora noncai liver of normal rats was injected 
subeutaneously ia newborn rats and »ieec 

itejor Findings ; Ho Cunors observed as yet. 

Significance s This is related to the nature of the isachanisiB of %li& 
psoduetion of turaors induced by so»ealled viruses. 

Proposed Coursft g The work is Co be continued » 



fmt B. included; N© 

969 



Serial No. NCI 508 



lo laboratory of Pachology 

2. Cancer Indue. & Fachogeuesj 

3. BeCheadaj, Maryland 
RB°NIM 

Individual Project Report 
Calendar ITear 1958 



Part A 



Project Titles Fate of hemoglobin Iron in anenlc tunor^bearlng rata 

Principal Investigator: Vlnc^ent Price 

Other Investigator: R„ E. Greenfield; Ross C. MacGardle 

Cooperating inits: 

Man Years (ealendar year 19S8>: Patient Days (calendar year 19S8): 

Totals I 
Professional; 1/3 
Others 2/3 

Project Description: 

CbjeeSivas s To deterainc fate of heooglobin iron in anenlc tumor ^bearlsg 
ratSo 

8; and 



jftjor Findings ; We found by nleroineiaeration that the iron of sueh 
rats is deposited in the strosaa of the tvmoXg °*° not in 
the viable tt^or tissue ^ 

Signlfieanee s l^is seans that this tumor tissue Itself does not 
utill^A the iron vhieh is lost fron ths» blood streaa. 

Proposed Course ; A paper is ready for publication <> 



Part B included g Jfo 



97n 



u 



ScrUL Ho>. NCI 509 



1. l«borAtory of Pathology 

2. Cancer Indue. & Pathogenesici 

3. BethMd«, Hnsirlcnd 
PHS-NIH 

ZndlviduAl Project Report 
Calender Yeer 1958 



Part A. 



Project Title: Cyroehenldtry and cytopathogeneals of cutaneous Isclone 
of psoriasis^ senile keratosis^ and Bowen*'s disease ^ 
tsicluding a description of Golgi apparatus^ minerals^ 
aitoehondria;, and rlbonucleoproCelns of norasal huaan skin. 

Principal Investigator: Ross C. ^cCardle 

Other Investigators Richard Reinertson (of Dr, Van Scott °8 IMit) 

Cooperating ^its: 

Man Years Ceal«°da3^ ye«r 1958}): Patient Days ^calender year 1958): 

Total: 1 
Profess ioisa Is 1/3 
Otiters 2/3 

Project Dsssription: 

Obieetives g the chief objective of this project was to ascertain 

possible factors concerned in tlte Initiation of proliferation 
of epldenaal cells aeanthotic grotrth; as «ell as to defect the actual 
iBorpholcgieal and histocheffiieal e^tcnges involved in the early and late . 
stages of the lesion of psoriasis and senile keratosis. 

Methods £ One of the s^tStods was microincineration to detect topographic 
distributioa of free and bound iron as well as Mg^ Ca^ e^ 
Si. Biopsy sp^elisens of early and late lesions^ and of uninvolved ekitt «e;@ 
obtained from approxiiaately 30 patients^, and ten speeineas of normal skin. 

Major Findinga s thB evolution of the lesion of psoriasis Involves 

alternating waves of hyperkeratinisatlon and parakeretinisa^- 
tlon^ each process being distinguishable by characteristic alterations of the 
€olgi apparatus ; RM and state of iron. In nonsal skln^ the outer eq\sai9ou3 
cells and ths keratinizing layers are relatively free of iron. In psoriasiSp 
on the other hand^ iron persists throughout the entire epidermis including 
the keratinous outer layer. WA persists in parakeratinlsatlon but aot in 
hyperkeratini^ation. Hortaal keratohyaline granules leave no ash residue 
of minerals, whereas the abnonael keratohyallne granules of psoriasis ere 
rich in ash cf Ce and Mg° 

We believe that the prliaary lesion of psoriasis involves 
failure in tbs epldenaal Eietabolism of iron and failure to retain adequate 
quantities of cutaneous iron^ with consequent alteration of RN4 and the 
fonnstion of keratin. The disease is not necessarily characterised by 
parakeratosis alone. 

971 



S«rUl No. HCI 509 
PHS-KIH 
Indivldtuil Project Report 
Cal«nd«r Year 1958 



Pfirt A (continued) 



Significance ? Relation to Cancer: 'i^his disease is a problem of 

growth; and we are now relating these findings to the 
genesis of Bowen^s disease and cutaneous basal and squamous °ce 11 carinoma., 

Proposed Course ; A paper is in preparation for publication^ and we 
intend to carry this work further. 



Part 6 included; No 



972 




Serial Mo. MCg 509a 

lo Laboratory of Pafchology 

2. Cancer Indue. & Pathogenesis 

3. BethesdSp Maryland 
PHS-NIH 

individual Project Report 
Calendar Year 1958 



MSIA 



Project Title; Mitochondrial lesions of the distal tubules of the 

kidney in polyuric rats bearing adrenal cortical tumors^ 
and the ontogenetic developmsnt of batonnets in relation 
to the first se@retion of urine 

Principal Investigator: Ross C. MaeCardle 

Other Investigator; Katharine €. Snell 

Cooperating ^its; 

Man Years (calendar year 1958) s Patient Days- (calendar year 1958); 

Total; 
Professional; 

Otbers 

Project Description; 

Objectives, Methods ^ and Major Findings ; Dr. Snell foussd marked lesions 
of the kidney in rats bearing adrenal eortieal tutsorSo I 
suggested that shs measure the urine output. She found excessive polyuria. 
We then examined the thalamic nuclei^ and attempted to analyse serum 
potassium and sodium. We have studied the mitochondrial batonnets of the 
kidney. Ihe lesion is in t!^ distal tubule^ consisting of globulation of 
the mitochondrial batonnsts. Thes@ bodies are difficult to stain and lose 
their stain easily^ «- indicating tkat their fatty acids beeosne saturated 
in the developB»st of the lesion. 

In order to describe this lesion^, we vere forced to study 
the ontogenetic developmsnt of mitochondrial batonnets of the rat froe 
es^ryo through the stage when urine first appears in the allantoic sac 
until maturity. We fouiad that prior te the developa^nt of t!;« secretory 
function C^'S* prior to the appearance of urine); there are no batonnsts 
but the globular mitochondria contain unsaturated fatty acids (judged by 
staining capacity). 

Signifleanee ; This represents one of the mecbanigms of action of 

a tumor on its host and it probably indicates that the 
tumor produces some substance which is injurious to the phospholipids and 
the protein of the distal tubule of the kidney thus Interfering ^ith the 
reabsorption of IS% of the water released through glomerular capsule thus 
inducing polyuria. 



973 



S«ri«l No. WC£ 509n 
PMS-HIH 
Xndlvldu«l Project Report 
CaUndar '^ear 1958 



Part A (continued } 

Propoaed Course ; We propose to dctemlne serum potasaiun and aodium 
and urine potasalum and sodium to ascertain whether the 
mlneralocortlcolde of the adrenal cortical tissue is adequate for reabsorption 
of minerals into the blood stream. We also plan to study the effects of 
various extracts of the tumor on the kidneys of normal rats. 



Part B included I No 



974 



Serial No. KCI 509b 

lo Laboratory of Pathology 

2o Cancer Indue. & Fatbogenesls 

3. Betheeda^ Maryland 

H1S-N2H 

Individual Project Report 

Calendar Year 1958 



Part A 



Project Tiele; "The Tuiaor Cell: Characteristics of Italigaant Prosoplasia" 
as part of a series of volumes of an International Treatise 
on Physiology of Protoplasm "Handbueh der Protoplasma- 
forsehung" being edited by Heilbrunn. 

Principal Investigators Ross C. !<IacGardl® 

Other Investigator: 

Cooperating %!!alts; 

]^!en Years C'»al-en<!a7 year 1958); Patient Daye-Cealsndar year 1958); 

Total: 

Professionals 
Other: 

Project Description; 

Obleetlves s The obJe€& of this monograph is to sunm^rise and correlate 
our knowledge of £he properties and possible ldentiflca<° 
tlon of a eeallgnent cell of a tus»r. 



M 



tthods ; By reviewing the literature and relating soaee new data found by 
tjor Finding: the author f, it Is clear that altl^ugh & single whit® 

blood eell of tl^ circulating blood of a leukemic mouse 
can upon injection Into a norasal host produce leukemia (notHlthstandlng th« 
serua injected with the cell); such a cell possesses no anatomical features 
by which it can be distinguished fron its normal cell of origin. Ths most 
malignant tumor constitutes several types of eells^ some of which may have 
noehlng to do with malignancy and none of which can be detected ae a 
mallgiMnt cell. The tramsplantabillty of the nalignant tumor cells is 
dlseussedj, and the cytology of several tumors is described for the fir$t 
£is»o 

Slgnlflcanee s This paper will bring together old and new data in an 
effort to evaluate the status of our knowledge of the 
saechanlsa of the tranefort^tion of non^il protoplasm to nalignant 

protoplasm., 

Proposed Course : This monograph is nearly completed and will be ready 
to submit to printers. 



W&st % included; Kfo _ 

975 



Serial Mo. MCI 510 

1. lAboTAtory of Pattwlogy 

2. Cancer Xnduc. & P«tbog«ii«sl$ 

3. Bethesda, Maryland 
PSB-NIH 

individual Project Report 
Calendar Year 1958 



Part A 



Project Titles Study of Endocrine and Reporduetlve Organs In the Mouse 

Principal investigator; Tbslraa B. Dumi 

Other Znvestlgasor; Rosa C. MaeCardle la doing soise phases of this studyo 
MTo Joseph Albreeht and Miss Betty Sanders are n&klng 
special preparations of the pituitary gland. 

Goo^rating ^Itss 

Man Years {celendar year 1958)3 Patient Days C<:«l«n<l«r year 1958); 

totals I 1/3 
Professional: 1/3 

Otfiwrs I 

Project Dejieriptions 

Objeetives ; this is a continuation of work begun several years ago. 
It is expected that it '»ill continue for a considerable 
period of tiwsi §ince tinse is s-equired for accutaulstion of animalSo to 
eecuEulete reliable data by observations on untreated micep planned proce<= 
dureSj, and reviews of tte literature regarding tiim reproductive mnd 
endocrine organs of inbred aaiee. 

Methods ? Personal observatioRS on assterial submitted frosa different 
sources^ planned essperisiental procedures in inbred mice^ 
histologie study of untreated mise at v&rious ag<se^ systeiaatie review; of 
the literature o 

Ma jog gindingsj this study is proceeding slowly because of pressure 
of other work. Gonaiderable laorpholosi® difference is 
found in tfee adrenal glands of inbred strains of mice. This seterial is 
already proving useful to other ases^^ers of the staff since it represents 
a norsifil standard of some endocrine organs in the eouse. An iisportant 
advance is a study of the pituitary gland being carried out in col labors t ion 
with Miss Betty Sanders and Mr. Joseph Albrecht. A good routine laethods 
for preparation of the oouse pituitary has been achieved. 

Significance g Since honaonal studies are an iisportant part of cancer 
research; it is desirable that reliable information be 
j»>ailabl@ regarding the endosrine and reproductive organs of iabred streine 
of ^iee. 

Proposed Course s It i® esgpeceed that this study will proceed slowly^ 
and re^uts^a several years for eoapletion. 

Part B included f Vc» ~" 

976 



ScrlAl Mo. VCl 510 

PHS^WIH 

XndlvlduAl Project Report 

C«IeQd<r ¥e«r 1958 

Part B ; Honors p Award*; «nd Publleatlons 

Honors and Awards rslatlng to this projaet; 

1, Trip tp the Soviet t^loa extending £roa May 15>June 13, as a nenbar 
o£ a delegation of six Aaerie&n woasn physicians. 

2. Citation by the Anerieaa Itedieal Wotaen^s Association as '^dieal Wosaan 

of the Year" froa Branch One^ the District of Columbia Branch.. 

3o Meabar of "faculty" for Short Course in Pathology^ Cleveland^ Ohio^, 
April 21 ° on subject: lesions in experimental anivals inportant to 
pathologists. lasions in the mouse. 

4. Scrainar at Roswell Park; Buffalo^ N.Y.^ ilovenlber 19 <> on Lesions in 
the mouse confusing the pathologist. 



Publications other than ebstraets fron this project; 

1. DusBip T. B.; and Acdervontp H. 3. 

Susceptibility of agentofree inbred nice and their hybrids to 

estrogen induced aaaaaary tuseors. 

J. fiat. Cancer Xn«£o 21? 783»8li^ 1958 » 

2. Duasi; fo Bo 

Morphology end Pathology of House Leukeffiifi " Relationship to Slan" 
Presented at New tork Aesdeny of Science Conference on "Screeaiag 
Procedures for Bxperinental Cancer Chemotherapy" Hftreh 13°15. 
To be published in Aanals of M«» York AeadeBy of Sciences c 



^71 



S«riftl No. ISCl 511 

1. Lfiboratory of Pathology 

2. Cancer Indue. & Pathogenesis 
3o Bethaada^ Maryland 

FHS-NIH 
Individual Project Report 
Calendar Year 1958 

Part A 

Project Title; Collaborative Research 

Principal Investigators Richard L. Swarm 

Other Investigators 

Cooperating ^its; 

Kan Tears Ccalsnt^ac y«ar 1958); Patient Days (ealeedar year 1936) s 

Total; 1 
Profeaaional: 1/3 

Others 2/3 

Project Description; 

Objectives g To collaborate with end advise other investigators 

particularly in phases of research requiring esEperienee 
in pathologic anatcay or the use of radioactive isotopes o 

Methods; Hethods vary widely and are dependent upon the problem aad 
the nature of the collaboration. 

Major Findiaaa ; Collaboratione which have developed include the 
fol losings 

a) Collaboration with Ore William Schatten^, Dr. H. Herbsean; 
and Dr. L. Cras^r oa & problem Involving the plastic repair of the extra 
hepatic bile ducts in dogs: this study was essentially completed in 
calendar year 1937. A few additional histologic studies have been made. A 
publication of this study not included in last year°s report is listed in 
Part B. 

b) Collaboration with Br. E. Morris on the effect of 
etidoerins glande on the occurrence of hepatic oeoplasas induced by 
2~diocetyIa!slnoflue»rlne; some progress has been a^de in the continuation 
of the hlstoiogleal study of hepatic tumc^rs which were found to have 
developed more slowly in hypothyroid animals. 

c) Continuatloa of studies on the distribution of tetra-° 
cyclins fluorescence suggested by the fonosr collaboration with Dr. Lane^. 
Dr. Rail J, Dr. Loo and others in the Clinical Phaneaeology and Sxperimental 
Therapeutics Service of !ICX. Although this collaboration has not reisained 
active^ certain ssorphologic observations reegaln of sufficient importance 

to aalntain an interest in their eicplaaation and study when circumstances 
permit. 

d) Collaboration with Dr. R. Schmid on the study of 
rodeats which have a metabolic defect in the asetabolism of bilirubin sissilsr 
to that preeeQt is certain children, liasphologlc alterations 1^ 'ch&me 

978 



Serial No. mx 511 
PSB-WIH 
Individual Project; Report 
Calendar Tear 1958 



Part A (continued) 



anifsials isave been further studied and correlated with snigyiaBtif; activity 
of the liver. I>!ro Pollsy; Chief^ Aniisal Production Vnit^, taaa been 
interested in breeding animals with this defect and a nuid>er of anlEsals^ 
the by-product of his breeding colony^ have been studied. A sterile 9 
anioal thought to carry this trait was found to have large cystic ov<arl&8. 

Significance g Collaboration is indispensable to nany types of 
cancer research. 

Proposed Course s Collaboration with Dr. Morris will continue. So!e>.a 
further work with tetrocycllne and the rats with congenital Jaundice will 
be done. 



Part B included; Yes 

979 



Serial Ko. MC I 511 

IndlviduAl Project Raport 
Calendftr Tear 1958 

Part B ; Honors^ Awards^ aiad Publications 

Publications other than abstracts from this project: 

Schatten^ tfc 0.^ Graiaer^ L. M.^ Herbeaan; H.^ and Swara^ R. L. 
EKperi!($en£al Reconstruction of the Cois»n Bile Ihtct with Split 
Thickness Skin Grafts 
Surgery^ aynecology and Obstetrics; 105s 747"753, Dec. 1957» 

Schmidj, R.J, Axslrod^ J. ^ HaGssaker^ h.^ and Swans^ R. L. 

Congenital Jaundice in Rata^p Due to a Befact in Glucuronide FovmatioKi 

J„ Clinical Investigation 37; 1123-1130^ Aug. 1958. 



Serial No. HCI 512 

1. Laboratory of Pathology 

2. Cancar Indue & Po?ho«enc 

3. Bachesda^ Maryland 
PIS^RIH 

Individual Project Kaport 
Calendar Year 1958 



Part A 



Project Title; Itorphologic alterations of cartilagic tleaue and of 
endochondral bone fomntion 

Principal Invest igators Rlchdrd h. Swarm 

Other Isivestigatora: Kona. Iliis project developed out of the collaboration 
previously done with others in mi 

Cooperating daitss 

Man Years Ccal«atiar year 1958); Patient Days Cc«ie«dar . year 1952 

Totals 1 1/3 
Professionals 1/3 
Others 1 

Project Descriptions 

Objectives s To accuisulate Infonsation about the morphologic alteration 
of normal proliferating and adult cartilage tissue and 
of neoplastic cartilage following alterations in the cartilage &M Ite 
environment « 



Methods ; Changes in the environment of cartilage tissue are effected 

by I) auto^ Iso^ homo^ or heterotransplantation; 2) altera ^ 
tion of the endocrine status of the host animal; 3) administrstlon of 
nmterlals with selective retention in cartilage as (S*^ ); and 4) by £lter<.ii» 
tlon of the diet (lathyrogenic compounds). This study is limited to 
g^erimental animals. 

Interest in transplantation e:q»erlment8 in general Is 
order to better understeod transplantation of cartilage is facilitated by 
eollaboratioQ as opportunity develops o 

latjor Findings ; Studies previously reported of a mised tumor contaislng 
cartilage have been continued, lomotransplantaitlon of 
this tutsor at times results in a tumor take and at other times r@®ult» is 
the selective preservation of the cartilage component of the tt«sor which do@« 
not proliferate^ gro^^ and have the other features of a neoplasin. 

Selective proliferation or matrix formation of the c^rtil&gc 
la tte hoisografted costochondral junction has been obtained with intr&« 
peritoneal grafts as %rell as «?ith the subcutaneous ly homografted costocho^^idrivl 
junctions. 

The pattern^ development^ and calcifisation of the sone 
of hypertrophied cartilage cells has been studied. Calcificati-^^ ;-. ;>-. 
hypertrep&ied carfcilaga bes bfien ds^nstrated with, ts^racysli; 



981 



SerlAl Ro. HCI 512 
PKS-MIB 
ZndlsrlduAl ProJ«ct Report 
Calesdar ?«ar 19S8 



Part A {coa£:lnued] 



Animals auCograft«d IntrAvenously with thyroid gland 
tissue by Dr. Richard J. Sanders were studied with Dr. John Edgeoai>o 

Slgnlflfcance i This project developed as a result of collaboration 
with other Investigators In the National Cancer 
Institute. It Is basic In nature and of Interest In understanding hoes 
tissue responses. 

Proposed Course s The project Is to be continued. Auto^jj, hoiso^j, 
and heterotransplantation of the costoehondral Junction will be atade In 
different sites end in anlraale with further alterations., Particular 
attention will be given to the alteration of transplanted cartilage in 
hosts ^Ith lathyrlstB. The hoEaotraaeplantabllity of different neoplasias 
with cartllagenous components will be further studied. 



Part B Included t Yes 



982 



S«rl«l KOo MCI 512 
PSiS-NIH 
Individual Pro>ct: Report 
Calendas: Year 1958 

PTt B ; Honors^ Aw&rds^ and PubllcAtions 

Publications otber than abstracts from this project; 

SchatteOj, W. S.^ Bergenstal^ 0. M. ; Kramer^ V. H.; Swara^ Re Lo ; 

sod Slegelj, S. 

Biological Survival aad Grovth of Cartilage Crafts 

Plastic ttttd Reconstructive Surgery 22: 11°28^ July 1958. 

S«mrai$ R. L. ; Setottesij, W. B.; Saitders^ R. J.; and Bergenstalj^ 0. H. 

The Autoradiograpliic Study of Transplanted Tissue. 

Laboratory Investigation 8: Mo. 1 CJan->Feb 1959). 

Text of preseatatloa made by Br. Stfara at a conference on autoradiography 

in Rew York in Septesa&er 1958. 



983 



Serial No, MCI 513 

1. LftboraCory of Pathology 

2. Caaeer Indue. & PaChogestesls 

3o BetSiccda^, Maryland 
PHS=HIH 
Individual Project Report 
Calendar Year 1938 



iSEJLAi 



Project Titles A Conparative Study of the Shope Fibroma in Rabbits in 
Relation to Transsaissibility by Mocquitoa. 

Principal Inveatigaeor: Mearl F. Stanton 

Other Investigators Herbert Da last ^ HIAXO 

Cooperating iPnitss Laboratory of Tropical Diseases^ NIAIO 

Man Years fe^lendar year 1958): Patient Days (calendar year 1958} s 

Total: I 
Professional? 1/3 
Others a/3 

Project Deseriptions 

Objeetiveas We have essentially eosspleted a study of tSse sorphologic 
nature of the Shope Fibronas of the rabbit which are 
transnissibls by aosquitos. 

Methods ? Cytologic and histocheaieal studies of the lesions induced 
in the rabbit. 

Major Findings s Shope rabbit fibroaaatosis is a neoplastic disease 

of viral etiology with morphologic alterations vhich early 
in its course sboi^s changes characteristic of a reactive proliferative 
response to Injury and later appears as true neoplasia. We laave stisdl«d 
the relation of the various phs8« @f developiBcnt in suckling and adult 
doaaestle rabbits and in cottontail rabbits. The rata of development and 
intensity of the latter fora of the disease is related to th@ strain and 
age of the h.9@t as well as character of the inducing virus. Only the lesions 
which progress) to show neoplastic characteristics are infectious for aosquitos 
although virus content does not change. Specific cytoplasmic inelusioffis 
in the fibrosa cells are necessary for Insect transaissibility. 

Significance ; This study contributes to our knawledge of host faetors 
responsible for the nature of lesions induced by oncogenic 
viruses. It is of particular interest that suckling rabbits and adult 
rabbits exposed to either carcinogens or ss'^lnradiation responded to a 
standard dose of the virus in the sane stanner. In e&eh of the three groups 
lethal tuBors formed (soKStiaes with 'haetastatic" fosi In other organs). 
which were characterised by the rapid and exaggerated development of "ssature" 
neoplastic cells possessing abundant inclusion bodies^, lesions were unique 
in that t^y were ssre sssily feransodssible by insects than lesions whish 
did not £.g£2isi, sush sfess'aste^isSiss.o. 

984 



Serial No, 
PHS-NIK 
lodividual Project Report 
Calendar Year 1958 



HCI 513 



Fart A {continued) 

Propoaed Course s Evidence of the presence of Shope flbromatosia virus 
is easily detected by the cytoplasnic inclusion. Attempts 
will be n&de to detereine the ultiaate fate and neoplastic charceter of 
lesions induced in pretreated hosts in sites other than the skii% using the 
halocarbon technique as a wsthod of transport. 



Part 3 included; Yes 



985 



SerUl No. BCZ 513 
PHS-HIH 
Individual ProJecC Report 
Calendar Year 1958 

Part B ; Honors, Awards^, and PublleaClonc 

PublleaClons o^her Chan sbaeracKc £ro« this projaet; 

Stanton^ Kearl F. 

A ConparcCive Study of the Shope Fibrosa In Kabblta In Relation to 

Transalsclblllty by MosqulSoac 

J. Rat. Cfinear Inst., 1958 (In preis). 



986 



Serial No. MCI 514 

1. Laboratory of Pathology 

2. Cancer Indue. & P«fchogena»i» 

3. Bethetda; Maryland 
PBS-NIH 

Individual Project Report 
Calendar Year 1958 

Part A 

Project Titles Induction of Keoplaema In laboratory Animals. Influence 
of Sex on Induction 

Principal Investigators Anibadaa S. Mulay 

Other Investigator; 

Cooperating Ignites 

mn Years (calendar year 1958); Patient Days (calendar year 1958) s 

Totals 1 1/3 
Professional: 1/3 
Others 1 

Project Descriptions 

Object Ives ; Study of the eareinogenie potential of and tfee lesions 
induced by awo dyes in nale and fesale rats. 

Metlwds ; Male and feaale Osbome°Mendel rats were fed on 8eiai«>«yntbe£ic 
'' diet containing p-=diia«ehylanjiBobengene='l»a@o»l»n«phebelesi®^ 

p-dini8tfeylamlnobenaene-l»aso=2"naphtfe8l©ne^ or p-=dlnjethylamin®«^beir6^ene„ 
Ifficidenee of tussors and other lesions was studied. 

Major Findings ; Fessale rets showed higher rcsistanee tlan nales to 
induction of tuniors by each of the cotopounds. fustor 
incidence was low in fenelas with two confounds, f hough tumor iacide«ce 
in 1:^168 and females was the same wish the third compound; it took longe? 
tiott for the f socles to develop the tuisors. Paper based on the ss findings 
has been submitted for publication. 

Si^nigieanee ; These studies ^Ip in the search for factors responsibl® 
for the resistanee or susceptibility to the inductioi:?. of 
neoplasia. 

Pro|>o8ed Course ; Other studies are in progress where homonal factors 
are being manipulated to see tl^ir influence on the 
incidence of ttisisors. 



Part B ineludedt So -- 

987 



Serial No. MCI 515 

1. Laboratory of Pathology 

2. Canear Indue o 6 Pathoganceis 

3. Bethesda^ Maryland 
PHS-HHi 

Individual Projaet Report 
Calendar fear 1958 



ParlA 



Project Titles Effect of the growth of adrenocortical adenocArelmtsm on 
this chessistry and ptsysiology of the Itoat rat 

Principal Investigator; Asabedas S. Mulay 

Othsr Inveatigator; 

Cooperating Umlts: 

Kan "Sear* Ce»l®a^fflf y««^ 1958 )s Peftient Daya C«sale»d«r year 1958 > 

Totals I 1/3 
Professional; 1/3 
Ot&er: I 

Project Deseriptlon; 

Ob jeetivas ; To characterise the steroids secreted by tSils turao;^ 
using host rat as a biological test aninal. 

Mettoda ^ fransplanting t^ adenocareinoiu in sals ssud f«B»le rats^ 

intact and adreaaleeto@i@ed . Running potasslun tonicity j, 
glycogen depdsitlon^ end sodiusi^potassiua sscrctlun tests on these enin^ISo 

Major gindin&s ? gonads in rats with adenoGarclno«Bft weigh less tfesn 
those in siailar rats wltStout the tumor. Weight of 
9varise was reduced by about 40%^ duets by 50%, seminal vesicles by 6&%^ 
epididysais by 451, and testis by 251,. These findings indicate that steroids 
^ieh influence gonads are not secreted by this tvnsor. 

Adrenalectoaised rats with adrenocortical tuator txsm»-^ 
plant can tolerate 5 al. per gn. body freight 0.16H potass iunehler ids. After 
revival o£ the tumor the saaae rats cannot tolerate this potassima load. 
Soae tusaor-bearing rats can tolerate 0.2H potaesluiaehloride; whleh is lethal 
to noroal rats. This indicates secretion of raineraloeorticosteroids 
(Deso^eortieoeterone end Aldosterone) by the tua»r and in sosie easas this 
secretion exceeds the 8«ecetion by adrenals of normal rat. 

^ine studies skew that rats with this, tutnor &b a general 
rule have polyuria. X«F@nty°four hour urine volume is increased 5 to 8 f@ldp 
it has low Spo€r. 1.01 to 1.02 compared with about I. OS for non-^tuaorous 
ratS; there is no albutainuria and no increase in urine M.F.SI. When 
compared with the perfennanee of intact cantrol ratS; adrenaleetoa^ almost 
doubles urinary sodiusa extretion and reduces poteasiura e^@retion by about 
15%; adrenocortical tusasr transplants in these rate returned the sodiisa 



988 



S«ri«l Mo. MCK 515 
PHS-HIH 
Individual Project: Report 
C«lttndar ^ear 1958 



Part A Ccontinued] 



excretion baek to control level c In Intaet rats adrenocortical tunor 
transplant reduced sodiuia excretion by about 5(71 and increased potasalua 
excretion by 10%. This suggests secretion of miners locortleoids by the 
tuBor. 

Sls;atfieance g These findings define In part ttte steroidal charaeteristle 
of t^ls tuaor. High secretion of nine ralocortlco Ids end lo*;; or no seeretlon 
of steroids which Influence gonads. 

Proposed Cougge s glycogen deposition tests to study the glueocortieold 
sacretion by tlbe tuaor have Just started o A method for 
estlsatlon of alerogram quantities of Aldosterone (pevi^etrnd at DIS) Is 
being established to study tihe sesretlon of this steroid by the tuffior end 
its ejEcretlon by the host. 



Part B included; Ho. 



SesrUl No. HCI 516 

lo Laboratory of Patliiology 
2o CaoGftr Indue. & Pathogenesis 
3. BetheadAj, Maryland 
PiiS-NIH 
Individual Project Report 
Calendar Year 1958 



P*E*-A 



Project Title: Studies on ZM laduetion of foltlple TuKors In Certain 
HbobsIs with Seevare^Eddy Polyoas vlrus-^tisaue culture 
Preparations o 

Principal Investigators ISearl F. Stanton 

Otiser Investigators Robert Blaekwellj, Sarah Steward; Bemice Kddy (DBS)^ 
Joseph Albreeht and staff 

Cooperating IMlts; mSg laboratory of Bi'Slogy^ RCI 

Project Description: 

ObjeeS:iveag This is a eontinuatlon of projects outlined in Iradividjifii 
Project Report mi 516^ 1957. 

Major FJBdiHgs ; Sueraarles of laajor findings in cooperation ^ith oSher 
investigators above will be foui^ in their reports. In 

addition^ separate investigations of host factors itsvolved in iadustion 

by this laboratory ha^;^ established the following: 

1. Varying the time of iooeulatlon^ i.e.^ in nice and 

hoaasters^ before birth or older than 24 hoisrSj, the variety of tusrars d@es 

not change but th@ incidenee and rate of developaent is ascslerated by 

prenatal ittosulation. 

to Morphologic evidence of viral paras it igatiosi of the 

target organs^ loe.^ iotranuelear inclusions and other alterations^ havs 

besn deserlbsd and studied hlstoek@rai@allyc 

3. A direct effect of the inoculum on the cells responsibi® 
for tuoor fomatlon or on their Imsdiate envlrosment is indicated by the 
Induction of typical tuseors in eBbryonic tissues estposed to the inoculum 

and subsequently iapl&nted in histoeoapetible hosts. 

4. The n&tural history and character of the tutors iiiducsd 
in haiBSters snd nice have been studied by transplantation studies. 1[he 
tuaors retain their aserphologie character after transplantation and occasional 
aQitnals bearing transplanted tu(K>re develop distant tuaor foci. However^ 
soos tumors show evidence of regression^, they do not recur after surgical 
exeisionj, show no clear pattern of imtaatatie or Invasive growth^ and in 
general retain aorphologlcal characteristics of Halted grewth potential. 

Significance g It is essential to determine the nature of the induced 

lesions and relate their character to huoan neoplasia. 'Shs. 
host factors involved in the progression of this unique variety of mssiti® 
neoplasia assy act in hussan. neoplasia of sisdlar nature. 



990 



SeffUl Wo. WCI 516 

Xxuiivlclual Project Zteport 
Calendar Year 1958 



Pare A (continued) 



Propoaad Co«gae s The devalopnesit of a slaple basic oystcai for 

tissue culture preparation enabling this laboratory to 
earry out experisents on host factors with a singla standard line of virus 
culture preparations., 



Part B included: lee 



Serial Kffl. KM 516 
PHS-NIH 
individual ProJeeC Report 
Ca lender Year 1956 



Part B; HonorSo Awards, and Publications 



Publications othar than abstracts fron this project: 

Stewart; S. E.^, Eddf, B. E.^ and Stantois^ M. F. 

Ncoplasoa in certain aMunals induced by a tumsr agent carried in 

tlssua culture. 

Proceedings of the VI£ International Cancer Congress j, London; England^, 1958 < 

Stewartp So E.^ Eddy^ So S.^ and StantoO; Mo F. 

Induction of neoplasias in mice and otlsar naamals by a tuvor agent 

carried in tissue culture o 

Proceedings of the Canadian Cancer Conference; Vol. 3; 1958 o 

Eddy; B. Eo; Stewart; So B.; and Stantofl; H. Fo 

Induction o£ tusrars in rats by So E. Polyofoe virus^anuse ta^tjo 

tissue culture preparations o 

Jo Sato Geneer Inst. 1958 o 



992 



SarUl Ho. VCl 518 

1. Labpr««;ory of Pathology 

2. Canc«r Indue. & Pathosenasis 
3 c Betltiesds, Iteryland 

PHS-NIH 

Individual Project Report 

Calendar Year 1956 



P^tt A 



Project Title; Exp^rimsntal induction of adeaocarcinona of the glandular 
seooiach in the ciouse. 

Principal Investigator; ISdward B. Prlce^ Jr. 

Other Investigate?: 

Cooperating Uhltss 

Iten ¥«ara ,<calaadsr year 1958); Patieat Daya (calaodar year 1958) s 

Totals 1 1/3 
Professional; 1 
Others 1/3 

Project Descriptioas 

Objectives ; The esperiioentsl induction of adenocarelnoam of the 

glacidular s£miach of C57BL lalce by intraaasral inject lots 
of eBethylch6la£&threne hss been reported by Stewart and Loreas^ 1942, and 
by Eare^ et al^ 1952. However^ the over-all yield of tumors produced Has 
sesall. It vas th.grefore decided to attes^t to increase the nus^er of tusa^rs 
produced by utilising the technique in cortiaonlsed ralcdo 

Methods; Routine 

Major Fiadlega s Project not yet coraplefeed. 



Sl^alfieance ; If Che taethod proves an effective oeans of laducisag 

careinoisa of Che glandular stoieach then for the first 

tiiae it will be possible to study in detail the genesis of that lesion. 

Proposed Course ; Cotopletlon of the project which Involves study of 
alcroscopie sections of the glandular stoaaach. Future 
course of the project will depend upon results obtained. 



Other Projects in collaboretloa with others; 

1. Transplantable fhio°uraeil induced Thyroid fuiaors in the Hat 
with DTo Seymour Wollman. 

2. Th's Effect of Msthotresate on Trophoblastic Disease in mn 
wltlsi Drs. Roy Eerfcs^ D. Bergenstahl^ M. Llpseet and A. Rilberg 

3. Transplantable Adresial Cortical Careiaossa in the Rat 
«itfe Dr. A. S. Muley. 

4o The Humn Xidaey In Acute Leukeraia ■= Clinical and Pathologic Study 
T.7ieh Drs. E. Frei &ud 1. Frits. 

■p'art B included; Yes 993 



Serial Mo. mi S18 
P!!S"NIH1 
Individual Projact ReporC 
Cal»ndAr Year 1958 

Part B ; Honors; Awards^ and Publleatlons 

PublleaSloae other titae abstracts from this project; 

Herfcis, R,, Bergeastahl^ D. M. , Upsatt, M. B.^ Prlce^ E. B.^ and 

Hllbisfe, T. 

ChsBothierapy of Chorioearclnooa a&d Related Trophoblastic tuasors itst 



Jtoum&l of tb» Assrlcan Medical Association 168: 845-854^ 1958. 



Other activitys 

Internationsl Acadeeiiy of Pathology S^sTgical Pathology Courg@ saitltledg 
"fusaors of Soft f Issues -- Diagnostic ProblesMi." Given at Cleveland, Ohi©^ 
April 1953^ by Drs. Richard Shim^n^ Armd Forces Institute of Pathology^ 
Washington^ D. C.^ and Dr. E. B. Price; Jr.j. national Cancer Institut@^ 
Bethesda^ Msryl&sd. 



994 



S«rl«l No. HCI 519 

1. Lftboracory of Pathology 

2. Canecr Indue. & P«chos«tt«*^<' 

3. BettMfdft; mrylaod 
FHS-tlZH 

Individual Projaet Rapotrt 
Calendar Year 1958 



msjt 



Project i:itle: Corticosteroid netabollsm of an adrenocortical adeoo* 
earelnona of rat. 

Principal Iiive9tis«eor; Aabadat S. Mulay 

Otber Investigator; 

Cooperating inltss 

^n f*T9 Ceslendar year 19S8}: Patient Days Ccalender year 1953 )s 

Totals 1 1/3 
Professional: 1/3 
OtSser; I 

Project Dessriptions 

Objgetlvas ; To estiaate 8«cr«tlon of core ieostero ids by th& tuaor 
and tk«lr eiseretion in Z^ urine of tueo;°bearlng ratSc 
PuriflCAtlonp isolation^ and clisrectsrisation of tbe sass. 

i tetlwde ? Transplanting tfea tiaaor ead eslleetlng tuaaer tissue. Collecting 
urines; extracting steroids froa tuiaor tissue and uriaee; 
retsoving fats from th@g@ extracts; vacuus concentration; second extraction; 
retBovsl of solvent at lo» teBp^rature and preesurd. Crude extract t&u@ 
prepared^ was used for prellniaary cl^onatography ^nd for soa» general 
tests for corticosteroids. 

Btejor Findings g Paper eteroaaeograa of crude twtor attract sko^s 

absorption in ultraviolet^- indicating presence of stes'olds, 
Preliffiinszy rasults in zaierogress p«r kllograa of »et tuaor tissue are 
steroids giving Blue tetrasollusi reaction 900, forsalds^ydogeaie steroids 
600^ l7"kefcQgeni« steroids 80Oj, and sfcssoids reacting witfei 2j4='diialtropfe®uyl 
hydrasana 300. Only traces of 17-OM steroids CPo'fe««r^«ilb«K' tesg) iiar® 
detected in cnsde extract. Hydrolysis of the tumor tissue with 
beta^glucuroeildase had lettle effect on these values. 

Sigaifiesnee ; Dafinltion of t^ steroid oetabollsm of tills neoplsea 
will enable us to s@e its slnilarltles and differences 
to fidrenal gland and other adrenocortical aeoplsssa. 



995 



S«ri«l So. MClt 519 
FHS-HXH 
Individual Project Report 
Calendar Year 1958 



Part A (continued}) 



Propoeed Courag s Esstraetlon o£ cortieoida froa more tutsor tissue 
and its fraetionatlon by papar chromatograplliy ars In 
progress <> 



Pert B Included J Mp 



996 



Serial No. MCI 520 

I. laboratory of Pathology 
2<, Cancer Indue. & Paeliogenttals 
3. Beehecda^ Marurland 
PKS-KISi 
Individual Proj«ce Rsport 
Calendar Year 19S8 



P&rf: A 



Project Tieles Morpliiologic study of tbe effects of radiaclon protective 
coapounda 

Principal Inv«»tig«tosrj Roger W. 0^€&t& 

Other Soveatigators Margaret Q. Kelly 

Cooperating Units: gentsral Medicine Irasuelsi; ICI 

mn Year* (calendar year 1958); Fatieat Days (eolaadar year 1958) § 

Total: 1/4 

Professional: 1/4 

Oeliers 

Project Description: 

Objeetlvess To deterffiinSj, if possible^ the siod& of action of radiation 
protective substances on £h@ tosic changes produced by 
W2 and other rsdlomliaetlc clt®«»leals. 

iifethod®; tissues of anitsals treated ^i^ith H^^; ^'^s ^^ cos^inatios^e 
of both drugs have be@n studied lelcroscopieally and- 
compared for saodlflcatloa in to%ic effects. Tlse seqtiefsce studies have 
been nade of thtg effects of th& drugs ott the pria^ry tussor^ upon the e^tetat 

of loetastaseS; and on <!hn|ysse changes in tlae tissues. Autoradiographs of 
tissue fr©s9 iBlee give S'^^~lafeeled ACT ©ff® feeing used to estlaiatg tisau® 
distribution of AET. 

Major Findings ; I. AEf protects boae «irrow and gut against fclsa 

toKle effects of IM2; without appreciably affectlisg tM 
action of £^2 on the prli&sry tuaeor or its isstaatfisas. 

2. In the tusaor systesas studied M2 appears to l»hll»it 
the spread of statasta&^s rath@r than to smrt any direct earclnoclastic 
action on the prissary tuaor. 

3. Autoradiography show greater localisation of S "^ 
in liver gut^ spleen^, kidney than in ssascls^ skln^ or tuntor. 

Sigalflcance ? I. It is of fundsiseatsl importance in the deeermisiatlon 
of ths mods of action of radiation-^protsctive and radlo"- 

xaiasetic- protective substances. 

2c Xt Is of practical i^ortance in deterBiinl^S ^he 

ejEtent of eh® protection afforded by ths particular drug being tested. 



99.7 



Serial Ho. ICI 520 
PHS-MIH 
Individual Projcce: Report 
CaUndar fear 19S8 



Fare A {con£inu«d) 



Proposed Couraa g 1. To study tbe moirphologie action of otlser 

radiation protective substances as they becoiae available c 
2. To study ia&ra«ellular diserlbu«:ion of AST ia tueiore 
and other tissues by the use of autoradiogr^iphs . 



Part B included; Ho. 



SMl«l Wo. MCI 521 
I. Laboratory of Pathology 
2„ Cancer Indue o & Fatllosanesi 
3. Bethasda^ Maryland 
P18S°NIH 
Individual Project Uaport 
Calendar Year 1958 



Part A 



Project Title: Morp1»>logic and Biologic Studies of Leukemlae and 
Lynphonaa of tha Mouse in Tissue Culture, 

Principal Investigator; Clyde 3. Dawe 

Other Investigator; }l£chsel Potter^ Williara Banfield^ Alan Rabson 

Cooperating thnits: 

Hsn Years {calendar year 1958) s Patient Days {ealend.r year 1958 }o^ 

Total: 2 
Professional: 1/2 
Others 1 1/2 

Project Descriptions 

Objectives: To observe the isorpho logic changes of lei^esila cells of 
the Qouse in tissue culture ^ and to correlate these 
changes vlth alterations in neoplastic characteristics. 

t^thoda ; This work has beess ixt progress for over three years ^ aad 
ne^ is concerned %?ith three reticular neoplasms of the 
C3A/2 mouse carried continuously in tissue culture o The methods have been 
described in previous reports.. These a&epl&sm&g and sooe of their 
characteristics are as follows. 

ttejQg FindJBgs g A. P329 is a histiocytic type of retlculu® cell 
earcoea and has tusne been cultivated for nearly three 
years. During this tis^ it has altsost eoEipletely lost its ability to grow 
into tuaors vshma iotroduced Into adult raic« of susceptible strain. Horpho° 
logically^ this cell strain has not changes greatly in yitrja^ except for the 
appearance of ssall niss&ers of polyploid giant cells. Ho«»@ver^ the ascites 
tuiaors resulting froas inoculation of @» and ll'=>sK»nth cultures into nice 
showed nearly 100% of the twsot cells to be in the hypotetraplold rasge^ 
with a high degree of heteroploldy. Also^ the tuisors derived fro@ the 
8° and ll^raonth cultures ^ere isore slos^ly growing and required tssze tltse 
to kill the hosts than did equal Inocula of the continuously anis&al^passed 
lines. These changes of "virulence'^ are of Interest inassnsch as tl^ 
saeehanlsiss involved tmy be basically related to the genesis of neoplasia. 
A tentative hypothesis is that the "attenuation" of the cells raay be to 
soass eistent analagous to loss of virulence by sicro-organiSEas «7hen cultivated 
in artificial siadia. Another possibility (not necessarily exclusive of 
the first) is that the cells have acquired nev antigen in vitro , and that 
host defenses eonssiquently prevent traissplantation. 

999 



SerUl Mo. mt ill 

lodlvidual Projeet Report 
Calendar Year 19S8 



Part A (continued]) 



To test these poatsibilltieSj, tha followisjg approaches 
are being used; 1) Alteration of raedium. After it wes found by Dr> Alan 
Rahson that P32$ would grow in a sndiuia containing defatted milk as cSm 
only protein supply^ large dosee of P329 cells grisvissg in this sodium 
vere inocul&ted into adult imk/2 mice. Characteristic P329 ast^ites tussors 
developed; and so a larger group of aniiwals T«ere inoculated with sBallsr^ 
graded call doses of cells fron tfoe saaGi msdlusa. Mons of these anlstffils 
have developed tucaore^ and it now appears that although the n«w raediuoi 
has sosae influence on 'Virulence^" this influence is not of a high order 
and will require aiore critical (quantitation. 

2) Conditioning of hosts by n^^ns of total body radiation. 
Hice treated with 42Sr prior to inoculaeion with P329 culture cells have 
developed tuiaors with higher frequency than non-s«rayed seicso This supports 
the concept tb&t the loss of 'Virulence" is in part esrely a loss of 
transplsntability caused by acquisition of new aatigeii(s). However ^ even 

in those irradiated aniraals that develop tunsors^ the tumor growth is 
extreosely slow^ requiring up to 9 or jsore ninths to kill the hoists. It 
appears that a priasary alteration in growth rate haa also occurred. 

3) Xsduced tolsrance es^erisients. A small n«snber of 



newborn mice were inoculated intravenously on th@ day of birth ^i|h 
4 X 10® P329 culture cslls^ sod again 45 days later with l.O x 10® cells 
of the saise line intrsperitoaeally. This esperiissnt is still in progress^ 
but 6-^10 i^nths after the first inoculation^ the majority of the eni&eals 
have developed imiltiple tusiors in widely scattered parts of the body^ 
indicative of blood-°bom dissemination and establisteent of cells of the 
initial i£^>culuffi. In some animals local tumors also were found at the 
site of intraperitoneal inoculationo It thus appears that intravenous 
inoculation of cultures into aewbom mice is a @or@ favorable igetltod of 
grafting the altered cells than intraperitoneal injection into adult micSc 
Whether induced tolerance plays a part in this result is still aet clear^ 
but it is again apparent that here as in the ease of 3s<°rayed hosts ^ the 
timors grG^ esetresely slowly in spite of the ix^reased ease of trans°> 
plantation. 

4) Long°tenB observations to find effect of alternate 
culture and aali^l passage on 'Virulence." fu^^rs derived from @» a@d 
ll<-Qonth P329 cultures have been re<»isolated in culture^ and cells are 
periodically returned to susceptible hosts to tsst for changes in ability 
to grow into tu^orso iesults so far indicate that the cell linss that 
have proven capable of growing in mice after lot^ sojourn ia vitro ^ retain 
their tum>r°produciisg ability ssach longer i&t least 18 months) than culture 
cells that have been passed through only the first half of the zaouse »°- 
culture °-= mouse cycle o 

B. P388. This cell strain^ derived from a lymphoid 
neoplasm of a !^SA/2 mtnB&g has been carried in continuous culture for over 
t:!»:» years. Its chief characteristics were described in the 1957 annual 
report^ and a report for publication is ia preparation. Ia contrast to 
P329|, this cell line went thr^iigh a marked i^rphologic transfon^tion in 
the course of- "adaptation" to tissue culture. Like P329^ P388 cells also 
lost mach of their "virulssee" (Killiag ti^ is proloagsd) for ssiee fey the 
iaSraparitoaeal to^jt^^ biat bave ratalKsd ths ea^aeity to kilx 100% &x 
susceptible hosts wheo injected ia desas of 10^ cells ©r e^re.^ Es^srlsssafes 

1000 



Serial ttto. MCt 521 
PHS-HIH 
Individual Project Rstport 
CAlondar Yaar 1958 

Part A vt continued 5 

Isave been in progress to test ths possibility of using the attenuated culture 
strain to lasnuni^e mice against ths more virulent iBouse°trans£erred strain., 
The results have so far not been pronisingc When living tissue culture 
cells were inoculated subcutaneous ly in Preund°8 adjuvant^ some cells 
survived snd proliferated to produee tuiaors tlbat eventually proved fatal 
to the aniesals^ though survival extended up to five months » These tutoors 
reaalned very closely locali^sed through laoet of their growth period,, in 
contrast to tuKoors derived froon the anioal^passed cell strain o An atteiapt 
to transplant etslture cells to the tails of susceptible isii^e failed. ?hig 
was done with the plan to amputate the tail and tuiaor after a period of 
tuiBor growthp and to challenge the anistals subsequently vith cells of the 
"virulent" strain. It is planned to attes^t this ej^eritaent again^ usisig 
the ear as the site for the priosry inoculation. 

fhe F388 eell strain has also been ^%tsnsively used 
udring the past year for the propagation of the tumor^indueing agent 
origistally described by Lud^ik Gross and by Sarah Stewart. Its application 
to this problem is described under a separate project. 

Co PS 15. This is a mast cell neoplasm that originated 
in a SM/2 laouse and hh&b converted to ascites form. It has been n^intalned 
in tissue culture for 10 tap>ssth®^ but with the atedlum used^ appears to be 
able to proliferate only in the presence o£ f ibrob last ° like cells isolated 
simultaiaeouely in the original e^lanatiost. Furttertsore^ the cells that 
are believed to be derived from the neoplastic i&ast cells no longer contain 
desQonstrable laetachrotaatic granules. Injection of the cultures into sdce 
of susceptible strain has failed to result in tuisors of any sort, fhe 
latter two f^satures are in contrast to th@ findings of Schindler,, who u^ed 
the sai^ cell strain^ but a aediuja eoataining citrovoriusa factor. These 
differences suggest the possible importasice of nutritional factors in tht 
preservation of morphology^ fussetioO;, and neoplastic potential of cells 
ia vitro . Work with this cell line is still only in the early stage* in 
this laboratory. 

St gnlficaace ; The isolation of thes@ cell strains from ourlne 

leukemias has provided other investigators with a systas 
for attacking certain specific problesss related to leukemia research in 
particular^ as well as to cancer research in general. In other laboratories 
the nutritional requireasents of P38S are being studied^ usitsg the platiag 
methods of Fuck. Also^ dr\2g<'resistant cell lines are being produced for 
investigations of drug resistance. Several investigators are uslsag ov^ o^ 
ffiore of these cell lines for propagation of ''polyoasa vlrus^^' and at least 
one investigator is attemptii^ to propagate the Friend agent in ?3S@ cultisras. 

For the program of the reporting investigator^ the 
project has significance in providing systems wherein it is possible to 
correlate morphologic changes occurring ^ vitro ^Ith biologic changes in 
the saxa& cell strains returxied to anl^ls. 



1001 



Serial Mo, MCI 521 
PHS-MXH 
Individual Project Repor<t 
Cslend&i: Year 1938 



P«?t A Icontiimed] 



Proposed Cougse ; 1. The ptiimty object of study regas'ding cell 

strain P329 vill be to detenoine as precisely as posaible- 
the factors respomslble for loss of "virulence." It is hopad during tha nesct 
year to be able to explore tbs possibility of transfer of "Virulence" by 
iceans of sisb-cellular fractions. If this can be aeconplished^ then it should 
be possible to identify the responsible factorCs) vithin the active fraction^ 

2. Tfee studies on induced tolerance ^ x^ray conditioning 
of hostSj, and infliaence of media on virulence will be continued as outlined 
above . 

3o fhe atten^ts to induce issramity witl* use of attenuated 
cell strains ^111 be continued o 

4o Study of the aorphologie and biologic alteratioHt of 
the recently isolated @ast cell tusaor (PSIS) will be continued. 



Part B incltaded; Ho 



1002 



Serial Mo, MCI 52U 

1. Ubormtory of Fethology 

2. Cancer Indue & Pachbgei 

3. Betheada^ Mary land 

PHB°i!SIH 

Individual Project Report 

Calendar ¥ear 19S8 



ISiLA 



Project Titlas Parotid Tumor Agent in Relation to Short- and Long»term 
Cultures of Leukeealc Tisaues of Mice 

Principal Investigator; Clyde Jo Da«fe 

Other Investigators Uoyd W. la^^ Thelam S. Dunn 

Cooperating ^its; 

Man fears (ealendar year 1958): Patient Days (calendar y®ar 1958"!' 

Totals 2 
Professionals 1/2 
Others 1 1/2 

Project Bescriptions 

(fejeefcives s At the initiation of fehis project^ th^ objective was 

to apply tissue culture of leukealc tissues to the isolatlora 
of presussptive filtrable agents capable of inducing leukeeia. Mo^ever^ w& 
succeeded only in obtaining evidence of the presence of the parotid tusor 

sgeot ("polyoiaa virus*') in sorae of the leukestic tissues eKasaioedo A report 
for publication on this project is ve^ near c^apletion^ aad the results 
are 8u^asrii;ed briefly beloe. 

Methods s Several transplanted lines of leukemia in C3H/BI ralce^ 

froB the "Passage A" series of ludwik Gross j, induced by 
cell-free filtrates of leukealc tissue^ ^ere used as well as P33S^ a 
aethyl-cholanthrene induced lymphoid leukssiia of imA/2 orlgi&o Short^^tesm 
plasias-elot cultures of these leukeiaie tissues were set up^ and filtrates 
end centrifugates of these cultures «ere injected into newborn >aic© of 
several strains Filtrates of long-tena P388 cultures Cdeserib^sd in 
]^I 521) vere also injected into newborn alee. Following the observa^ioa 
by Esbson that the polyoaia virus of Ste^rart and Eddy induced a cytopathogsnic 
effect on P33S in rallk lasedium^ a filtrate of one of our induced parotid 
tttiaors was used to iooculate P388 cultures in our routine high°>seruni 
aedluB^ and the agent has been propagated in serial passage through this 
culture systea. 

l^jor Ficdiags s Ho leukeala« indue lag or accelerating agent has been 
demonstrable in all of this work^ which includes observations on @or® than 
1500 inoculated sice. 

fwo of three leukesila lines obtained frost Gross have ehc^m 
the presence of a filtrable agent that induced parotid tusiors similar to thosa 
previously described by Gross^ Stewart^ Law^ and raany others, ^itfe one 
cell strain^ aaseh sore activity was obtained fro® fc&e short-tes-® tissue 



1003 



Serial Ho. mi 521a 

Individual Project Report 
CaleQd«r Year 1958 



Pert A i(continued) 



culture laatAriAl than from direct filtrate of the leukemic tissue (65% vs. 
0%). With the secMid cell liae„ too few oice receiving th» direct leukemic 
tissue filtrate survived to provide adequate cooparison^ although title 
short^tera culture ssaterial iaduced tunors in about 70%. of the surviviing 
recipients. With tl» third line^ neither direct filtrates nor the culture 
material showed significant activity. 

Short-term cultures of DBA/2 leukemia P388 yielded parotid 
tusors in 14% of ths surviving recipients. This vas a somewhat tanexpected 
result^ and is of interest because it suggests the possibility that this 
leukemia is & "carrier" of the parotid tusaor agent land that this agent may 
be related to the morphologic and biologic transformation of P388 in 
adapting to continuous grc^th in culture^ as previously des^ribsd. Filtrates 
of the continuous culture strain of P388 were also injected into newborn 
ralce^ with a resulting parotid tusaor incidence of only Z%. Hie sodium 
alone^ used as control material^ resulted in nsarly 41, parotid ttmor 
incidence. 

When filtrate of induced parotid tumor wss introduced 
into P388 cultures in growth medium, a definite C^ appeared in 13 days. 
Filtrates of these cultures induced parotid tussore in more than 70% of the 
surviving recipients^ and there was a striking increase in the nuiaiber of 
othsx types of twmsta appesrissgj, as well as a decrease in the latseat period 
as compared with previous active sssiterial. Approximately 90% of the anlmials 
with parotid ttaaors also had thymic ttsmors of epithelial origin. Qth&t 
c^axBtily observed tumors wares thyroid^ masmary^ tumors of renal pslvis^, 
tusors of tbB hair follicles^ osteoma® of epia®^ skull^ ribs^ and stemum^^ 
tumors of glands of t!^ nasal soicosa; oropharynx^ tongue^ and trael^a^ 
adrenal medullary tu&wrs (uncommon); and tt^ors of urethral glands 

The parotid tussor agent has been serially passed through 
10 P383 culture passages^ and tusKtr-lEiducing activity appears to have feeea 
retained. f»c othsr cell llaee^ F329 and im^ltl (of DeBruyn) have also besn 
sho^m to be susceptible o f^e have succeeded in obtaining & strain of P3@8 
that recovered from an inoculation with the agent; and which appears to be 
chronically infected with th@ agent. The physical arrst^ement of cells in 
cultures of this "recovered** strain differs from that in unexposed cultures^, 
and filtrates of tls^se cultures induce parotid and thymic tumors in a high 
percentage of recipient newborn mice. 

Using the highly potest tissue culture passage saterial^ 
it has been shown that all inbred strains of mice so far t@@ted are susceptible 
to the agent. These strains include DM/2; CoK/Bi, CoE/lJw^ e3H/Fg^ AKR^ 
C57Br, G57Blp C53^ SWR^ and StoLi. 

Significance g Although the results have been disappointing i^lth 

respect to demonstration of a filtrable leukemia ^inducing 
ageat^ this work has provided an Improved method of demonstrating the 
presence of parotid twmx agent in leukemic tissues^ and provides ms^ 
eultures systems that should prove valuable in investigations of lnterrela°' 
tiosashtps between cells and agent^ as well as in prodtsction of agent its large 
quantities for physical sad ehejaieal aiaalysis. 

1004 



Serial Wo. ml 52 Xa 

ms-nm 

Individual Project Report 
Calendar fear 1958 



Fart A (coaeinued) 



Proposed Courae g Because of liiaitaelons of tintt and technical asaiatance^; 
the reporting inveatigator and collaborators are aware 
that it will Qot be possible to follow up ssore than a snail portion of the 
interestisg leads provided by this investigation . The niore interesting 
problems, hc^fever^ are Ii«ted belowc A ciecision as to which of these will 
actually be undertaken has not been stada except whssre indicated. 

1. titration of tumor -inducing potency of tissue culture-^ 
propagated agentp and direct correlation of tumor » inducing activity with 
tissue-^culture infectivity. In collaboration with Drs. Law^ Bsryan^ and 
Rabson^ this project has been outlined^ but is not underway. 

2. Quantitative study of effects of serial dilution of 
parotid tussor agent on the number and types of tumore induced j, and the latent 
period c 

3o Effects of propagation of agent on different cell 
lines^ and in different raedl&o Will the resulting induced tuiaors be the 
saste regardless of whether agent is grown on esi&ryo ssouse cultures j, P338^ 
P329s, or 1®=IXI? 

4. Deterfflin&tion of wtesre the agent resides In trans-^ 
planted leukemias. Is it in the leukemia cells or in so£»s normal host 
cell; such as ti^ macrophage? We have inoculated nortaal macrophages with 
agent^ and observed a decline of population and decreased oetsbolic activity^ 
without clear-°cut CPE. 

So Detenaination of whether agent acts directly on 
susceptible tissues^ or indirectly. 1%is is under study ^ by aaeaiis of in 
vitro irtductlon attessptS;^ esgposing ezcplanted parotid glands of n@wb®ris talce 
to agent in culture. By iiiorphologle criteria^ Clie effect is direct. 

6. Investigation of the possibility that agent m&y <ss;iet 
in lysogenlc state in leukeioia cells. The P388 culture "carrier" liae 
supplies an approach. What would be the effect of inoculating raic@. 
(iissiunised end non-ie^aanised) with cells carrying the agent? 

7. Investigation of the psrt^ if any^, played by the parotid 
agent in the transfoneation of P3@8 cells in culture. This can be done by 
eliainating agent froia P38@ by passage through isesuns animals and then testing 
for trans for^oat ion capability. Another approach le to infect a cell strain^, 
such as L1210^ which does not carry agent and doe® not transforsa^ and then 
test again for transforming ability. 

6. Identification of agent within P333 eultnre g&IU by 
electron microscopy. Some work has been done along this line by fsto Ban£i@ld 
but the observations are still preliminary. 



Fatt. B inetedsd; f&s 

1005 



Serial No, WCI jn» 
PHS-NIH 
Individual Project Report 
Calendar Tea; 1958 

Part B ; HonorSp AwardA^ and Publications 

Publications othar than abstracts from this project; 

Dawo; C. J.j. Potter^ M. ^ and Leigl^toQ^ J. 

Progressions of & Betieuluns°call Sarcona of the House Jn Vitro 

and In Vivo. 

J. Kat. Csncsr Inst. 21: 7S3»781o 1958. 



1006 



Scriel No. NCll 522 

1. Laboretory of Pathology 

2. Cancer Indue. & Pathogenesis 

3. Bethesda^ Maryland 
PBS^HIH 

Individual Project Report 
Calendar Year 1958 



g*rt A, 



Project Title? Studlea on the Induction end Natural Hlatory of Conjwactlvai 
Garcinona In Hereford Cattle. 

Principle Snveetlgator: Heerl F. Stanton 

Other Inveattgators W, H. Byestone, Robert Stonakar^ Ray Bryan^, W. I. d^y 

Cooperating Units; Laboratory Aide Branchy Division Research Services; 
laboratory of Biology^ NCI 

Man ^ears C«Alendar year 1958): Patient Day* (calendar year 1958 )s 

Total: I 
Profe8«ional« 1/3 
Others a/3 

Project Description: 

Objeetlves g to detenainc the fate of certain proliferative epitS(<«!lial 
lesions on the eyes of cattle and to attceipt by artificial 
Bteans to induee careinonic in cattle with the products of ths tumors ^hieh 
occur naturally. 

Methods ; liable tumor tissue and cell free extracts of conjunctival 
earcinoraes of hereford cattle^ prepared by concentration 
techniques successfully established for Rous sareosia virus^ have been 
inoculated into the conjunctivas and udders of hereford cattle. Both 
young unaffected anioals and anitsals with conjunctival carcinoma have be@Q 
used as regipients of the inoeula. 

Major Findings 8 A study of a herd of 16 hereford cattle in southern 

Virginia suggests that proliferative conjunctival lesions 
are endemic in this herd. All stages of growth from sssall wart°lltee verruca 
to metastasizing squanous earcinoeas were observed. Four of th@3e &aivmls 
and their calves have been acquired and the progress of their lesions is 
now under study. Tissue and cell free esstraets of the cancer in one of 
the four cows has shown no evidence of growth after two months in the 
inoculated hosts. 

Significance g This is en unquestionably ealignant epithelial tustor 

which has its counterpart in s^n^, and follows the natural 
history of hueaan epidermoid carcinoasao The high incidence of such a l£@ion 
in a herd of cattle suggests a coeason etiological factor not as yet &ppsr@nt. 
A most likely possibility is that the initial verrucous lesion is induced 



1007 



S«ri«l «o. WC:iI 522 
FHS-NIH 
XndlvlduAl Project Report 
CalendAr t«ar 1958 



Part A (eontinued) 



b^ Insect )bo7n viruses. Subsequent progression to malignancy nay be a 
part of or separate from the initiating process. The cow serves as an 
excellent animal in whleh to study this problem^ of added significance 
is the possibility of uncovering nteans of preventing tlte disease in cattle^ 
a serious economic haeard in present day animal husbandry. 

Proposed Course s It seens likely that the precursor to tjhie aaalignant 
lesion is the hyper keratotic verrucous wart noted in 
many of the Virginia eattle. We shall attempt to eolle@t^ recover^ and 
induce typical lesions with such oatterial as well as with concentrated 
products of the raelignaat lesion. 



Part B iiKluded: No 



Serial No. MCI 523 

lo Laboratory of Pat:lioiogy 
2o Cancer IndttiSo & Pathogencisis 
3o BatSieada^ Maryland 
MiS-'KIK 
Individual Project Report 
Calendar Year 1958 

part A 

Project Title; Effects of iooislog radiation vlth es^haals on morphologic 
alterations and neoplaseia 

Principal Investigator; Richard L. Swam 

Other Investis^tors ; 

Cooperating Unites 

Man Years (caleadar year 1958 )g Patient Days {calendar year 1958), 

Totals 1 
Professional; 1/3 
Others 2/3 

Project Description; 

Objectives ; The collection of inforaation regarding the changes effected 
by ionising radiation of different types in ssan and 
e%perii^n£al anlneala. 

Methods s I) Participation in the activities of the Pathologic Anatcay 

Departieent; special eiaphasia is oade in the etudy of 
huiaan specia^ns desioastratlng change due to radiation or containing radlc- 
active isotopes. At intervale inforsoal conferences vith soise of the 
clinical staff of the ladiation Branchy KCI, have bees held. . At tSsese 
conferences^ morphological findings of clinical case ejaterial have been 
reviewed . 

2} Detection and study of neoplasms and other s!orph0l€glc 
changes in eKperiiseatal saiaals «ho have received sKtemal radiation or 
internally sdministered radioactive isotopes. 

3} A study of the distribution^ stetabolism and effects 
of radioactive isotopes of several types in man and anlte^lB„ Here the 
eraphasis Is on the utilisation of gross and histologic auto<°radiograpMc 
techniques coE&ined with ssore conventional gross and histologic eeeliaiq«es 
in the study of fhorofcrast^ S^^^ and I^^l uheU4 ®aterlal®« 

4) To facilitate the above and to assist othes- investi- 
gators^ collaboration on probletas in this field is fostered. At preeeag 
a study on the distribution and effects of S^ in laan is underway ^itfe Sr. 
J. Robert Andrews and others in the Radiation Branchy mi. 

I fejor Fi ndings; Qontlnued histologic study of hussan cases containing 
colloidal thoriuis dioside seeias to indicate the 
variability with which this fsaterial is distributed. Lack of unifors 
distribution of this colloid E^aterlal in the liver is particularly striking 
in SQsse cases. 

1009 



S«rial »o. mil 523 
PHS-«IH 
Individual ProJscS Report* 
Calendar "^ear 1958 



Part A ^continued) 



Major Fiodinga (contitujied) 

Hletologlc study of neoplaama produced In «spe rini&atal 
anisials following the adainistratlon of colloidal thorium dioxide has 
eontlDuado la the study of pritaary hepatic neoplaatnta^ particular attention 
la being paid to the prosence or absence of focal scarriog. Soam have 
presented data that would suggest that post^lrradiatlon neoplasias arise in 
post*' Irradiation scars or at least in histologically altered sites. Traas° 
plantation of soraa of the colloidal thorium dioside induced tumors has been 
perforaed and & histologic study of the trensplante is in progress <, A 
presentation of the slsailarities between come of the colloidal thorium 
dioxide tumors in mas and mouse was made at the Ap?ll 1958 annual meeting 
of the American Association for Cancer Research. 

In collaboration with Dr. J. Robert Andrews and others 
in the Radiation Branchy BCI^ the study of radioactive Sulfur iS^^} 
distribution; metabolism^ and selective damage to cartilage begun in 
rodents^ has been extended to sffia. A presentation of the autoradiographs 
showing localisation ©f S^ in tumor CchondroearcoBa), rib cartilage and 
megakaryocytes \ma made at the annual msetlsg of the Association of 
Pathologists and Bacteriologists in April 1958. The full report of the 
administration of one curie of S to a human patient has been prepared for 
publication. An eKhibit and oral presentation of these findings was 
presented at the annual meeting of the American Roentgen Rey Society. 

In collaboration with Dr. P. Rubin and others^ aniiBSls 
receiving localised rsdletion to the bones of the hind legs have been 
followed for subsequent alteration of the g»rphologie development of the 
long bones. Freaentatioa of a preliminary report on this enperii^nt wa@ 
made by Dr. Rubin at the annual meeting of the Ameriean Roentgen Ray Society ^ 
September 1938. 

Sisnlfieaaeeg With increased es^osure of mas to radiation^ it h&s 

become i^re sad more important to establish the relationship 
between ionising radiation and t^ effects of radiation^ particularly the 
late effects including neoplasia. Heed to utilise ionising radiation in 
new and unique ways for ths palliation of cancer patients a^ for tbB. 
destruction of neoplasms is recognised in our study with S^. 

Proposed Course g I. The activities outlined above under Kethoda 

are to be continued. 

2. Ea^hasis upon the biologic distribution and effects 
of internally deposited isotopes will continue. Particular interest ^111 
continue in the study of colloidal thorium dioxide in mas and anisials . 
Opportunity to study huimn patients or epeeimgns containing colloidal 
thr.rium dioxide will be utili^^^ed In addition^ current animal atudies will 
b@ brought to conclusion. Ilwo new anlisal studies will be undertaken. 
Animals with internally deposited thorium dioxide will undergo either 
splenectomy or partial h^pateetomy to see if either of these procedures 
will alter the incidei^e or type of neoplasms i<?hich develop following 
thorotr&st administration. 

Splenectomy in individuals eoataiaing internally 
deposited colloidal thorium dloxids is of Interest since a) the spleen 

1010 



SeJTial No. HCK S23 
PKlSoMlH 
Individual Project Report 
Calendar ¥««? 19S8 

Part A Cconfeiauad) 

Proposed Course {(continued) 

often cont&lQS tlse hlghese eonsentratlon of thorotrast^ b} tim sipleen 
may be the site of priismry neoplasms^ c) the spleen may be easily ressoved 
surgically In laaa^ d) certain ©^rnian workers bavc evidence which suggests 
hsraatologic iraproveinent in patlstets with colloidal thorluin dlojgide who have 
slight hematologic abnormalities, and e) transfer of some of ths colloidal 
thorium dloKlde frosi the isipleen to the liver cannot be eiecluded. 

Partial hepateetoa^ la aelBiale containing intsmally 
deposited colloidal thorium dioxide is of Interest since th@ proliferating 
hepatic pareochysm of these anltssle night lead to an increased incidence 
of prlsaary hepatic neopiassss , In a way these animals would be coii^arable 
to sosffi individuals ^ho have focal regeneration of the liver which proceeds 
in certain areas of the liver due to disease other than tM Internally 
deposited colloidal thorium dloKide» The fact that a proliferating tissue 
is aaore susceptable to a carcinogen is recognised and is tS%e basis for 
this study. 

3. Continued study of tfe® distribution of S"*'^ in tlie 
cartilagenous tissues is planned., Particular attention will be paid to the 
e«^parison of the isetaboliss of large and assail doses of S^^ to see if th^: 
large dose@ are siataboliged in en abnonsial fashion o 

4. Continued collaboration with Br. J. Robert Andrews 

,35 



and others in the Kadiation Branch on the effects of S ^ in humaa choissdro-^ 
sarcoata will be contit 



Pars B included s Yes 



1011 



Serial Ho, MCI 523 
FHS-NZH 
XndlvidiMl Project Keporfc 
Calendar ¥aar 19S8 

Part B s Honors^ Awards, and Publlcatlona 

FublicaCions oeher thm abstracts from this project: 

The follovlag paper vas sot reported last year: 

RubiOj. P. .0 BracCj, R. C.^ @u0p^ H.^ Swaraj R. h.p and Andre^fSj, J. R., 
The RadlotoKle Effects of S^^ in proving Cartilage 
Radiology 69s 7ll-=719, Hov, 1957, 

The followlfig paper is to be published In the very near futuire; 

Andrews J, J. R.^ S^ara^ R. L. ^ ScMa«shter^ L,^ Brace^ K, Cj, Rubinj, P. 

Sergeastal^ D. M. ^ ©usap^ H.^ Siegel. S.^ and Swain^ R. W. 

The Effects of One Curie of Sulfur^^ Administered Intravenotas ly as 

Sulfate to a Han vith Advanced Chondrosarcoffia. 

To be published la Radiology. 



1012 



S«rUl llo„ WC; 526 

lo Laboratory of Pathology 

2. Cancar Indue. & Pethogenesie 

3. Bctbaada^ Maryland 
PHS-HIH 

Individual Projaet Raport 
Calendar Yaar 1958 

Part_A 

Project Title; Gheaical Carcinogeiaesis 

Principal Investigators; Marie L. Hasselbach; Roger W. O'Gara 

Other Investigator: 

Cooperating Sinits: HIAID 

Man Years (calendar year 1958); Patient Daya {calendar year 1958) j 

Totals l(% 
Professionals 1/4 
Other: 

Project Description: 

Objectivea g !<. To study the ^rphology of induced tuanrs and 

detemine i^hether there is any correl&tion betiieea the 

histopathologic appearance of these tumors and their glycolytic and 

respiratory activity. 

2. To determin©j, if possible^ any relationship betveen 

dye^induced changes in Gsltoehondria^ intracellular glycolytic and 

respiratory ensyme systesa^ and the oorphologic characteristics of dye- 

induced neoplasms . 

Methods % Rats were injected with tripheny lone thane dyes C^^S^^ %r&@.n 
and fast green}. The resulting tumors were studied by the usual biologic^ 
histopathologic;, and biochealsal techniques. 

Major Findings : !<. Subcutaneous fibrosarcoii^s were induced by both 
dyes in over 90% of the rats. The tumors spread readily 

by oetastases; implantation^ and direct invasion and grew consistently on 

transplantation. 

2. The adrenal glands and gonads showed marked changes 

in anlnals bearing transplants of one subline of tuisors but not tn anisials 

bearing implants of the other sublines. 

Signiflcancc i Should supply fundanentsl knowledge on the relationship 
between altoehondrla and neoplasia. 

Proposed Course s Further studies on the respiration and glycolytic 
rates of sublines of these tue»rs are in progress. 



Fart B Iiicludeds Ho 

1013 



Serial Ho. NCX 526a 

lo LAborAtory of Pathology 
2o Cancer llinduc & PatStogenesis 
3 c Bechasdaj, Maryland 
PSSS-KIH 
Individual Project Raport 
Calendar Tear 1958 



Part A 



Project Title: Cheaalcal Carclnogsneais 

Principal lovestigators Roger H. O^Gara 

Other Investigator; 

Cooperating I^lts: 

Man Years (calendar year 19SS>: Patient Days Ccsl«ndar year 1958} : 

Total: 3/4 
Profassiooal; 1/4 
Other: i/2 

Project Descriptioa: 

Objectives : To study the effect of several popular cathartic eoatpouodg 
on the gastrointestinal tract and other tissue in mlcec. 

Methods ; ©sing 3 different strains of raiee^ six different cathartics 
were adninlstered either in the diet or by oral tube iot 
8 to 16 isonthSc 

Major Find lags : Experitsents are still la progress^ and no evaluation 
is possible at this tiaie. 

Slgnlfleanee s It i«ill provide information on possible factors in 
the etiology of huoan tuoors of the gastrointestinal 
tract. 

P roposed Course ; Ho further work is planned until the current study 
is c^Bpleted and tl» results evaluated. 



Fart B included: SIo 



1014 



Serial »o« WCI 57.7 

lo lAborAtory of Pathology 

2. Cancer Indue. & Pathogenesis 

3. Bethesda^ Maryland 
PHS»MIH 

Individual Project Report 
Calendar Year 1958 



Pare A 



Project Titles A Study of t^ Ptsyeiopatho logic Changes in the Tuaor°bearing 
Host and Tunor Lesion 

Principal Investigator; Richard A. Haloigren 

Other Investigator: Dr. Giancarlo Rabottij, Visiting Scientist £r<a} 
Milan^ Italy (Harfch 1 • Decegi>er 1^ 1958) 

Cooperating Units; 

Man Years (calendar year 1958); Patient Days (calendar year 1958) i 

Totals 3 
Professional; 2 
Other; 1 

Project Description; 

Objectives ; I. To ascertain tise biological properties of th& live? 
ffiitotic stinulant from tussor tissue when given to norsail 
aniraals at various tiraes. 

2. To study the anti^carclnogenic effect of several 
biological agents. 

3. To develop a technique for identification of the organ 
or origin of ntetastatlc cancer lesions. 

4. To study the ploidy of various neoplasns under different 
envlronmeatal and etiologic situations by use of mlerospectroii^toiBetrle 
techniques. 

Methods ; I. The heat stable^ nucleoproteln-rlch fraction of tnouse 

tuasor tissue which has been shown to produce an increase 
in tte liver aitotie laddK of @lce has been tested for Its possible otlser 
biologic effects. This aster ial ie prepared by heating a 25% honogenate 
of C3ilBA iBOuse nasBiary tuaior to 80 ^C for 20 Blnutes. The coagulated 
eaterial is removed by eentrlfugatlon and the resultant clear fluid 
Injected in I cc. doses into adult and newborn C3H mice. These anissals are 
then observed for tuiaor incidence and life duration. 

2. Hosoogenates of various nsoplasos induced by several 
agents and of different histologic type were injected into eilee and the 
growth excised. These aniieals were then challenged with carcinogens and 
tim resultant tueors Sioted. In addition fractions of soise tutaors vere 
studied in this way. 

3. Antlsera were prepared in rabbits against various 
oorsal SLt^ neoplastic tissues of the mouse and huaians. The globulin 
fraction of this asitlaera was then tagged with fluorine leotblocyanste. 
The tagged sera was then tested on seetlcoes of the appropriate tisstses by 
the Coons technique for organ specificity. 

1015 



Serial iHo„ WCI 527 
PHS-MIH 
Individual Project Report 
C«l«adAr ¥eaT 1958 



Pare A [continued) 



4p Vising the alcrospetropbotoaeter^ aeatureaMunt* Imiva 
been mads of the OBIA content of Cite nuclei of varioua virus induced tume^ra 
as well as non»virus tunors. 

Major Findings g 1. Early dat^a indicates that tli« nuclear fraction of 
G3U nouse ncaasoary tumors inhibit the development of 
aataosary tumors when injected into mice along with milk agent preparations., 
Similarly^ the Injection of the liver mitotic stimulant from mammary tustors 
seems Co inhibit the spontasteous development of mteamxy tumors. This is 
not true if the liver mitotic stimulant is administered to newborn mice in 
one injection. These findings are compatible with the observations of Gotten 
and of Rogers on the inhibition of carelnogensis by material rich in mk. 

2. Mo evidence of resistance to the carcinogenic effect 
of isethylcholanthrene was evident In the mice pre-'treated with « variety 
of neoplasms including W^ Iziduced tumors. This is In contrast to previous 
studies of this type. 

3. Organ specif lelty would appear to persist in the neoplasEsts 
as studied by the Coons technique. That isj, hepatoma and liver sections 
fluoresce when treated ^s^ith tagged anti»live:r s&t& while other normal @nd 
neoplastic tissues do sot. The same is true of lung and lung turaors &tid 

ant 1- lung sera. 

Significance g The studies on certain biologic su&terlals and theis- 

effect on neoplasia suggest th£t such material may h^ve 

a pr&nouneed prophylactic effect on carcinogenesis. 

Ths ability to distinguish the organ of origin of a 

neoplasm would offer the opportunity to tt» pattologlst to direct the 

clinician to the primary site of a metastatic neoplasm which would others 

wise be unknown. 

Proposed Course s The anti'^carclnogenic effect of a variety of biologic 
agents will continue to be studied. In addition^ the future development of 
the Goons technique as & possible test for organ specificity and l£e &ppllca» 
tlon to other systeos will be ptsrsued. The use of mleroepectrophotometrlc 
techniques In the study of various biologic material «?111 be continued. 



W&tt B ittciudedi %a 



1016 



Sisylal Mo« NCI 527 

Xndividu«l Project Report 
Calendar Tettr 1958 

Part B ; HonorS; Awarda^, and FubllcAtlona 

Publications other than abetracts from this projects 

Malngreaf R A, 

Influeace o£ Antigenic Factors in the Production of Anti'^tuiaor 

CytotoKic Sara. 

J. Mat. Cancer Intst. 20. 417 "■429c, 1958 o 



1017 



Serial 80. NCIJ33 

1. Laboratory of Pathology 

2c Cancer Indue. & Pathogenotals 

3c Betfoasda; Maryland 

Individual Project Report 

Calendar Year 1958 



Part A 



Project Title: DaveIops£B£:al pathology and function of the thymus gland 
end thyeiie tuissors 

Prineipal Investigators Eoger W, O^Gara 

Other iQvestlga toffs 

Cooperating fpnltss 

Hsn Year* Ccalendar yeas 1958} s Patient Days CcalendaE year 1958>s 

Totals 1/2 
Professional g IM 
Others 1/4 

Project Dsseriptions 

Objeefcives g - To obtain infortaation on (a) ths function of th@ thysots 
glandj, (h) ttm factors involved in the developsssat of 
epithelial thyB^soas and (c) iaflusnce of the thyajus gland on the induction 
of lyaaphoid tuisicrsc 

Ijsthodsj Surgical procedures such as thy@ectoes^^ gonadecto^^ traiis° 
plantation of neo-natal tisa^^ to adult organs^ sisd usual 
biologic; histopatbologie^ and radiation techniques. 

Major Flndlagsg l. Sieo^natal thyslc tissue transplanted into th& 

spleen of adult alee continues to grow for at least 3»6 
«?eeks and can still b® identified in the apleen 2 years after Isapiantatioss.. 

2. Intrasplenie transplsntatlon of neo'°aatal thymus 
tissue in thysaectotaised^ irradiated elce did not Increase the isicidenca 
of lymphoid tuaors. This is in contrast to th@ increase obtained t^heis 
neo»natal tissue is transplanted to the subcutaneous tissue. 

3. Thssre appears to be a eorpho logic differense in the 
s^ucleus of t!^ lymphocyte and that of the thyatocyte. 

4c A few gastrointestieal tumors and several eplthelisti 
cysts in tb@ spleen have been fotsnd in a group of mice carrying intraeplenlc 
transplants of neo^natal thymic tissue < The significasiee of these fisidlsga 
has not yet been detersined. 

Slgaifieaaee s This is a ^w eicperiasntal satbod of studying the growth 

capabilities sad reactions of the th^aus gland under 
altered eaviroasaesstal conditions. It is expected to supply iaforasatiosi 
oa factors which regulate and control the growth of the thysais gland^ aad 
oa the develops^nt of thyiiioaias and lya^boid tue^rs. 



1018 



SeyiAl No. mi 533 
PHS-niH 
Individual Project ReporC 
Calendar Year 1958 



Part A Ccontioued) 

Proposed Cour»e ; Ic The growth of Intraeplenle trcnspiaats of 
aeo^Qstal thymic tissue vill be compared in normal^, 

thyaectc^ii^ed^ gonadectomized^ £nd adreaalectonised anioals. 

2o Hae grov^eli poteatlala of eabryonic and adult th^ic 

tissue will be es^lored in neo°natal and adult hoats. 

3o Serial transplantation of thysnas iatplante will 

bd investigated. 



Part B included: Yes 



1019 



Serial Ko. iSCI 53 3 
PHS-WIH 
Individual Project Kcport 
CAlendAi: Year 1956 

Part B g Honorsp Auards^ and Publications 

Publications other than abstracts fron this project: 

O'Cara, Roger \S.g Horsi^ Robert C.^ asd ]Snfcerline^ H. T. 
Tuaors of the Aaterior Mediastiaum 
Cancer 11: 562»590^ t«ay°Juae 1958 



1020 



SerUl Ro. SCI 534 

1. Lftboratory of P«tJiiology 

2. Cancer Indue. & Paehogenesiis 
3o Bethcsda^ SferyUnd 

Individual Projact; Report 

Cales»dar Ifaar 1958 



g«gg Ao 



Projaet Title? Ineldetusa of Induced and Spontaneous Tumor* in RIH Swt&» 
Mice uaed in Sereftnlng Experiments for Potantial ViJral 
Induced Hicoan Neoplasios 

Frineipal ItiivestigatoT; S^arl F. Stanton 

OtSnar Investigator: Robert Manak«r 

Cooperating ^it: laboratory of Biology^ KCI 

H&n ¥ears Cealendar year 1938): Patient Days (ealendar year 1958 |; 

Total: 

Professional; 
Others 

Project Descriptions 

ObjectlveS s Tlte determination of the incidence and cfearaeter o£ tua^rs 
wfeleh o@eiur in tl%e variety of raiea u@sd in screening 
esperioents and the deteetion of un&fue tumors vltieh nay aris@ in the 
test miss. 

Met^d£s Dr. Man&ker cultures and inoculates newborn alee %?ith 

control and hutaan tusior inoculated tissue eultt^res. Br. 
Stanton studies the anieals for tutoors. 

ifelor Findings s As indicated above, 

Signtficaoee s Such studies are necessary to separate induced turasrs 
from those occurring sponti&neou&ly. 

Proposed Courge ; Because the mice ere not inbred it «ill b@ neee^ss^^ 
to study large numbers of laice at various age@ to hsvs a 
elear imprsssion of the non&al tumor incidence and variety of lesions which 
test anlnsals may show. This is a long-terra project. 



Part ^. inclvsded? So 



1021 



S«rl«l No. NCI 535 

1. laboratory of PjaCtioIogy 

2. Cancer Indue c & Pathog«ae8i» 

3. Betbesda, mrfland 



PBS-NXH 

lisdlvldual Project Report 

CAleodsr ¥car 1958 



Part A 



Project Titles Sssiteriaaental studies on tha effect of nutritional 

deficiencies on tb& pathogenesis of hepatomas in rats 

Principal Issvestigator s Herschel Sidransfcy 



other Investigatoirs BoESGanuel Farber^ DepesTtiaent of Pathology^ TiilJiiKe 

feiveraity^ Hew Orleans, Louisiana (Seetion A^ B^ C) 

Cooperating Onits; 

Man Years Cealendas year 195«}; Patient Days" (calendar yaar 1958); 

Totals 2 1/3 
Professionals 1 
Others 1 1/3 

Project Deserlptions 

Objectives ; To study the pathologic and cheaical changes tfeat occur 
in certain nutritional deficiencies which may be related 
to tht subsequent dsvelopssent of neoplasia In th& liver of the rate 

Methods ; A. !S»asliiorkor; a widespread protein deficiency disease i» 
infancy and childhood^ has been thought possibly to b@ 
related to the high incidence of cirrhosis and hepatoma of i^dults in kitics. 
Experimentally^ ^e have found that certain acute aialno acid deficiesK:i@s 
can produce lesions in yomtg rats «hich are siailer to those found in 
children with Kwashiorkor. Inder such e^speriaental conditions it would be 
of interest to detensine whether these young rats continued on a low gtsde 
dietary aeiino aeid deficiency would subsequently develop cirrhosis and/or 



B, BtMoaia©^ an ethyl amlog of s^tfeionine^ has been 
itrated to produce hepatomas ia long-ters rat experiraeats. Ethiosdin© 
is believed to act as a cosopetitive inhibitor of mthionine and thereby 
produces a relative taathionine deficiency state. Me have deooastreted in 
essperisiente with adult rats on acute atethionine deficient diets ghat a fatty 
liver with a similar mx differsnce develops as that found with the acute 
adaslnistratiea of etfelonine. To leara aor® about the tssechanisa by whiela 
iatcrferance with aathionine aetabolisra can induce hepatomas in rats^ 
current e^sp^rimtnta have been started in which anioials have been placed 
on a Bsetbioaine deficient^ but adequate choline^ diet. Previously it Ms 
been shown t&at rats develop hepatomas when placed ©a a choliise deficient; 
low lasthioQisie diet. However^ 8i©ilar studies with a low ssethionlnep 
adequate choline^ diet have not been described. 



1022 



Serial 2!o. ml 53' 

PISS -mi? 

Individual Project. Report 
Calendar Year 1958 



F«7t A (contlnuisd) 



Co Choline dsficiency in aeuce eisperiiaettt^s produces & 
f&tcy liver in Ch« rat while in chronic experinsenCs lliepa^onias develop in 
the rac. The possible relationship between ttie developo!ient of the acute 
fetty liver and the subsequent developsient of hepetossa le not clear. In 
acute experiiQsnts it has been found that the susceptibility to develop 
fatty liver varies with different species. A study has been undertaken 
to dftaraine whether the level of the liver estsyiaa^ choline oxidase^ taay 
be related to this altered species susceptibility. 

D. Stbionisis and 2»ac«tylat!iinof luorene have each been 
sh<;>wn to induce hepatcc^s in rata. Male rats are e»re susceptible than 
feiKiales to the carcinogenic action of 2-'aceCylarainofluorene while no ses 
d.fference has been reported «?ith ethionine. The early histopathologic 
(changes produced by each agent are similar. For this reason expariioente 
liave been started to deterraine whether these agents fed together in the 
jiet ioay act synergist lea lly in the development of hepatomas and to observe 
whether a sex difference will be found. 

Major yindinss ; A. Two publications describe the cheaieal pathology 
of acute asaino acid deficiencies. Chronii^ experissents 
have recently been started. 

S. One publication describes the siisilar sess difference 
in thm digvelop^nt of fatty liver in adult rats treated with ethionine and 
with adult rats on a seethionina deficient diet. Chronic aupsriiaents in 
which rats have been placed on i^thionin« deficient diets ere in progress. 
Rats will be sacrificed at intervals. 

C. Esperimsnts to date indicate that anlnsals eueh as 
the rat^ isouse;, and chick which are resdlly susceptible to acute choline 
deficiency fatty livers have high levels of li"»/er choline ossidsse. On 
the other handp aniisals such as the guinea pig and hamster which are not 
readily susceptible to fatty liver changes with choline defieient diets 
have low levels of this en^ysie. Studies on huisan autopsy s£at@rial and on 
a few surgical liver biopsies seeta to indicate tisat the huraan has en 
extremely low level of this en^yese. 

D. Esperiasents in which rats are fed sootrol^ ethionine^ 
2»acetylasd.nofluorene; and ethionine plus 2<-acetylamlnof luorene diets are 
now in progress for one sgonth with no findings to date. 

SigiBific&nce s Bp to the present tis^ the only nutritional def IcleBcy 
which has been found to produce liver cancer la e:q>eri{^n- 
tal aniiaals is that of choline de£lcl@ncy. It would be of much Interest 
and is^ortance to detertaisie whether other deficiencies sueh as chronic 
amino acid or protein deficienei^s ssight under certain conditions produce 
hepatoioas in eseperii^ntal anisaals. 

Proposed Course ; Rats plaeed on different diets will be sacrificed 
at varying intervals. Detailed pathologic studies will 
be eondueted and the pathologic findings will direct the course of the 
cheolcal investigation. 

{ISOfEs la addition to work described abovc^ collaborative work related to 
liv@r careiaogeaesis is beliag c&rrled out with Dr. Harold Morris. Details 
will be given ia his report.) 

Part B iav;ludsd; fes 1023 



• Serial So. W CI 53 S 

Xndlvidu&i Project Report 
Calendar Year 1958 

Part B s Honors ; Award s^, and Publlfflatlotts 

Publiea£ioa>.s other than abstrasts from this project: 

Sidraosky^ Herschei;: and Farber;, EBtmanuel 

Chemical Pathology of Acute Aaiino Acid Deficiencies: I. IMorpkologlc 
Cha&ges la Imiaature Rats Fed Threoains-^ MBthlooiae--^ or Hletldlne^- 
Devold Dleta 

A.M.A. Arch. Pa«&. 66 g 119-134^ 1958. 



Sldransky^ Kerschel^ and Farbsr^ JSasaanuel 

Chsalcal Pathology of Acute AmlQo Acid Deficiencies: II. Biochemical 

Changes In Rats Fed Threonine" or j^thloelctaodevoid Diets 

AcM.Ao Arch. Path. 66? 135-149^ 1958. 



Sldranskyj, Kerschel^ and Farber^ Easi^mjiel 

S3% Difference in Induction of Periportal Fatty Liver by Methionine 

Deficiency in the Rat 

Proc. Soc. Esp. Biol, and Mad. 98s 293»297„ 1956. 



Honors and Awards relating to this project; 

Diplomats^ Ait^rlcan Board of Pathology^ ISov^ts^mT 1956 
Hsmber^ Washington Society of Pathologists^ Koveiaber 1936. 



1024 



Serial No. NCI 301 

1. Laboratory of Pathology 

2. Cancer Pathology Section 

3. Bethecda, Maryland, and 
New Orleans, Loulalana 



PBS-NIH 

Individual Project Report 

Calendar Year 1958 



Part A. , 

Project Title: Geographic pathology field studlaa on urinary bladder 
cancer In New Orleans^ Loulalana. 

Principal Invaatigator : Lucia J. Dunhais, Harold P. Dom, and Harold L. 

Stewart 

Other Investigator: Alan S. Babson, Louis B. TboioAS 

Cooperating Units: 

Man Years (calendar year 1958); Patient Iteys (calendar year 1958): 

Total: I 5/6 
Professional: 1/3 
Others 11/2 

Project Description: Field studies on Urinary Bladder Cancer^ New Orleans, 
Louisiana. 

Objectives ; The objective of this project is to complete an esetensive 

questionnaire study of patients with urinary bladder caaeer 
in a souttem U.S.A. city with a high incidence of bladder cancer cosspared 
to two other southam cities. (Dom and Cutler, Morbidity from Cancer in tha 
United StateS; Public Health Monograph No. 29, Parts I and II, in press.) 
The data will be snalysad with a view to identifying factors associated with 
cases of urinary bladder cancer. Basic data on th$ incidence and pathologic 
(microscopic) diagnosis of the cancers are being obtained. 

tfetlaods ! For the area under study a;^ the period of study all patients 
possible with the diagnosis of urinary bladder cancer are 
interviewed, and an equal ntsober of control patients as well. The dAt& 
rscorded include residence, occupation, nodical and surgie&l history, a.tsd 
InforBHUtlon about diet and about the use of alcoholic and non-alcoholic 
beverages and of tobacco. Records are kept of tha snmll nuaiber of patients 
not interviewed, their diagnoses, and the reasons for failure to intervlet^. 
Pathologic s^tsrial is studied in all cases. 

A coding statistician and one medical social worker interviewer 
are currently etsployed in the project. Interviewing was started in New 
Orleans on 15 F<^^ary 1938. 



1025 



Serial No. NCI 501 



PHS-NIH 

IttdlvidusI Project Report 

Caleodar Year 1958 



Part A (contlnuad) 



Major FindlBga ; Data from about 100 cancer patlenta and an equal 
tausiber of control patlenta have been reviewed and 
coded. It is astiinatad that pathologic material will have been examined 
In about 80 percent of the cases at tha date on which thla report la 
suboiittad. 

SlBnlfieance ; The significance of the study lies in the hope of 

supplying a sound scientific basis for the understanding 
of factors in habit or enviromuant as they omy be related to a high Incidastce 
of urinary bladder cancer in a spaeific geographic area. 

Proposed Coarss ; It is planned to continue the study for a total of 
three or more years. Data will be tabulatetd mnA 
analysed and a report will be prepared for publication. 



Part B included; No 



1026 



Serial No. NCI 501a 

1. Laboicatory of Pathology 

2. Cancer Pathology Section 

3. Bethcada, I&ryland 
PHS-MIH 

Individual Project Report 
Calendar Year 1958 



Part A. 



Project Title: Geographic pathology field studies on uterine cancer in 
New York City, Israel, and Washington, D. C. 

Principal Invastigator: Lucia J. Dunham, Harold F. Dorn, and Harold L. 

Stewart 



Other Investigator: 
Cooparating Units: 



John H. Edgcoiab, Louis 6. Thomas. 



Man Years (calendar year 1958): 
Total: 3 
Professional: 1/3 
Other: 2 1/2 



Patient Days (calendar yasr 1958): 



Project Inscription: 



Field studies of uterine cancer (cervix and corpus) is 
New York City, Israel, and Washington, D. G. 



Ob lectives ; The objective of this project is to complete an extensive 
questionnaire study of wosien with uterine cancers in 
different geographic areas and racial groups, and to analyse the date 
obtained with a view to identifying factors suspected of predisposing to or 
causing feisffile genital cancers. Also, basic data on the incidence and 
pathologic (microscopic) diagnoses of the cancers are obtained. 

Methods : For the area under study and the period of study ©11 patients 
possible with the diagnosis of uterine cancer are interviewed, 
and an equal number of control patients as well. The confidential data 
recorded include social, medical, and surgical history, and menstrual, 
tcarital, and pregnancy data. Records are kept of the small number of 
patients not interviewed, their diagnoses, and the reasons for failure to 
interview. Pathologic e^terial is studied in all eases. 

A coding statistician and one medical social worker intervi^^sir 
are currently en|>loyed in the project. 

Major Findings ; I. Bata from 3,514 cancer and control pationts, 
Jewish and non-'Jewish white, in New York City 
and Israel have been reviewed, coded, tabulated and analysed. Pathologic 
iiMterial l^s been sitamiaed in the cancer cases. A report is being prepared 
entitled "Cancer of the uterus in Jewish woman" and will be published as a 
U. S. Public Health Service Monograph. The first complete draft of th@ 
report, including tables, is nine-tenths completed. 



1027 



Serial Ko. HCl 501a 



PHS-NIH 

Individual Project Report 

Calendar Year 1958 



Part A (continued) 



Major Findinga (continuad): 2. A preliminary report, mainly on the 
circumcision statue of loale partoora of cervix cancer patients vas prei^ented 
at the Seventh International Cancer Congress in London on 8 July 1958. It 
is to be published in Acta Unio Contra Cancrum under the title <"£>0!iie 
environmental factors and Zht development of uterine cancers in Israel and 
Ke« York City," 

3. Data frosi about 500 cases of cancer of the uterus 

in non-«hit« woaten in Hey York City have been nsvlewed and codedj, together 
vith an eqiml nutc^r of control cases. Pathologic material has been esomained 
in all cancer eases. 

4. Mta from 239 cases of cancer of the uteruss in aon«^&ite 
vosan in Washington^ D.C., and from 303 control cases have been recorded and 
some data have been reviewed. Pathologic oteterial has eo far been esasiinsd 

in about 80 percent of the cancer cases. Interview of at l®ast lOOaddltional 
patients with cancer of ths uterus azsH an equal nuo^r of control patients 
is under way. 

5. The study of 'SJancar of the uterus in non-white uosKsn" 
described under 3 and 4 will be reported separately. 

Signifieaace i The significance, of the study lies in the hop* of 

supplying a sound scientific basis for the understanding 
of factors of custom^ habit , or emviromssnt as tl^y loay be related to 
relatively high frequeocies of uterine cancer® in some groups of woHsen 
as contrastad to intermediate or relatively low frequencies in othsT group®. 

Propg yd Courae; It is estissgted thst the report oa Cancer of the 

Uterus in Jewish women will be prepared £or pubiica<° 
tion with t£s» year 1959. 

The study of non-^shite woioen in Ifashlngton will be 
coaspleted in about oste year*'s tisBS^ and tl^n data from this study and from 
the Hew York City study of non-white woasen will be analysed and a x%po«t 
will b« prepared for publication. 



Fart B lncl(ed©ds No 



1028 



Serial No. NCI 501b 

1. Laboratory of Bathology 

2. Cancer Pathology Sectloa 

3. Bethasda, Maryland 



Fsrt A 



Project Titl®; 



HB-NIH 

Individual Project Report 

Calendar Year 1958 



Geographic Pathology. Experlc^stal atudias relating 
to ths geographic pathology of oiouth cancer. 



Pvinslp&l Imi^estigator: Lucia J. Dunhaia 

Other l!Sv<s8tigatox-: 

iQoops^atiBg Units; 

Man Yeara Ccalegsdsr js&t 1958): 
Total t 12/3 
Frofessioml: 1/3 
OtI?«rj 11/3 



Patient Days (calendar year 1958): 



Project description: Eisperisaental studies on mouth canear. 

Objectives; Th® objactive of this -projact i® to essaoine tha nsueotja 
n^fsbrane of the hatastar cheek pouch for proliferative 
changes that nmy dsvalop when test substancaa are chronically oalntained 
In contact with these surfaeae. Substancea used are envirocsasntal @at@riii.l!3 
suspected of being aildly carcinogenic for nan. 

Methods s The pellets which have been prepared for insertion in th@ 
right cheek pouch of ths hsfflster include the tobacco, lio^^ 
BmshiT, arscm nut, and pepper leaf of tha Far Eastern 'l^tel quid," tb® 
plaetie oiaterlal used in preparing "false" teeth, and tobacco tars and 
control joeterials. A technical assistant is retainfsd part'time for help 
in th« sstudy. 

Major FJKdiags: The operstioa for inserting pellets in the pouch 

has been perfonmd on soore than 400 aniosals. kbout 
75 anioals survive at tha present tieae. Grose and microscopic lesion» 
of ths pouch wall have been observed in about a dosen of the several kindred 
expariiosntal aniaaals essmained. There have been no loaligfiBnt neopla^BS 
aimong thsss. It has bean demonstrated that neoplatms can be initiated its 
tb@ pouch wall by ueix% pallets coataining a known carcinogen. When tm 
rejoining animals have been essamined all findings in t£^ pouches and oth@r 
pathologic changes will be reviewed and a report will be prepared. (Stsss 
current studlesp Serial No. NCI 536, Dr. Katherine Herrold.) 



1029 



Serial No. SOIb 



PHS-NIH 

Individual Project Eaport 

Calendar Year 1958 



Pare A {continued) 



Significagjce s The signlf icai»ce is to roach an issproved undfer8tandi»s 

of chronic irritant factors as thay may relate to caneer 
of tha sitouth in nan. In addition it will be aseartained whether it U 
desirable to do further studies using isodifications of the tachnique 
describad. 

Pro^sed Course s Tha cheek pouch with the test pellet and th® oppoeit® 



or dies. Tl&s findings during the next calendar year will dictate the future 
conduct of the it3v®stigation. 



Part B included: No 



1030 



Serial Ho« HCX 524 
1. lAborfttory of fatkology 
2o Camter Paebologjr Section 
3. Betlaesda^ MaryUnd 
PHS-HIH 
Individual Project: Report 
Calendar Year 1958 



F&xt A 



Project Titles Studies of fuaer Viruses ia Ser«sa«free Tissue Culture 
Sysr— - 



Principal Im'estigator g Alan S. Eabsoa 
©tfeer Iiwestigaterg Frsaces 'f. Isgallais 

Coof^eretiag Units s 

mn Years (caleudar year 1958} s Patieat Days {calendar year .1958} s 

Total s I 
Professional; 1/4 

Other; 3/4 

Project DescriptioBs 

Objectives; Sttadies of the polyoiaa virus in cultures of mllk^a^apted 
P38S Di cells have been undertaken to attej^fc to learn 
about the action of this virus on proliferating cells in a groijtfe sridijsa 
free ©f serua antibodies and non-specific seruaj viral inhibitor®. Me have 
falso investigated the effect of ejgtraeta of selected husian tutors oa the 
sBilk°adapted P388 H^ cell system and on huoan ceil lines in the saruEa-free 
sailk laediuBi in attet^ts to isolate agents from huaan ttsaors. 

Methods g Polyeiaa virus has been studied in cultures of milk^sdapted 

P388 ©2^ cells using coaveBtional tissue culture viro logical 
aasthodso Oaeoganic activity of tiasu® culture materials has f/sen te«t«d 
in Qffiwbom haraaters according to the sjethods degcribed fey Eddy and har 
associates. Extracts of human tuaors have been handled in essentially the 
saisse Banner^ but husan cell lines as well as P388 D^ have b«®n used. 

I^jor Findings; Folyom virus has been foutid to prodvee a mrhsd 

cytopathogeaic effect ia cultures of aailk^adapted F388 D, 
cells. Undiluted inocula produce CPE in two to four days, and virus pools 

titer as high as 10' -^ 'scW^q in 0,25 ral. Detailed studies of tlm cyto- 
pathology of the process are nawia progress. 

Only a very smll nunibsr of hmmn ttaaors have been 
tavestigatad thus far and no results are available. 

Sigaifieaaceg An ^mderstanding of tha asechaniem of action of th& 

polyosaa virus in vitro will probably be of greet isB^ortaecs 
ia our understanding of viral neoplasia, fhs systeras for the study aad 
isolation of polyos^ virus way be eieeelleat e^terisaental niodels for the 
isolation of comparable agents frosi fesifiian tusaors. 



1031 



Serial lOo m i S24 
PES«>MIH 
Individual Project Re^rC 
Calendar TeAr 1958 



Part A {eon£inued> 



Proposed Coursa g The studies of the cytopfttiaology of tte> polyotm virus 
ia witro will be conKiaufid and further studies designed 

to learn fsore about the polyoaa virus and P388 H-, cell •ystaoi are planned. 

These ffill include actesapt to dsffionstrate a proliferative rather than 

destructive effect of the virus i^ ths culture ayetenso 

The work with human tuasors will be esspanded with nore 

at tempts to isolate agents froa human tuaors in huoaan €®ll lines grown 

in serum^free media. 



Part 3 included; Mo 



1032 



Serial So. SGI 530 
l< I^bo^atory of Faithology 
2c Cancer Pathology Section 
3c Beehesda^ Maryland 
PHS»NIH 
Individual Ps-ojece Report 
Calendar ?ear 19S8 



Part A 



Project titles Tissue Culture Studies of Malignant Tuaaors in Serusi-Frise 
Mediuia 

Principal Investigator: Alan S. Rabson 

Other Savestigators Frances f o Legal lais 

Cooperating ©nltss 

Maa Years (calendar year 195a): Pattest Day® (ealaiBcSar y©.ar 1958}? 

Total: 1 
Professionals 1/4 
Ot&er; 3/4 

Project Deseriptioa; 

Objectives s Adaptation of maasislian cells to 8erua<°free media has been 
undertakss to provide systems free of serun antibodies 
asid seru@ viral inhibitors. Cells gro«^ ia s@ru@°fre@ aedia should be of 
value for l£ vitro studies of anitml tumor viruses and sight be of epseial 
value in attesapts to isolate agents from hucan tuisors. 

Methods g Autoclaved non°fat isjilk has been substituted for serum in 

the growth media used for several lines of ssslignant csllsc 
The P338 D^ and P329 c@ll lines isoUted by Bave in a msdiusi containing 20% 
autoclaved non«fat milk and 80% ^diuta #199. The details of the preparation 
of the siediiia and the adaptation of P38S ^i have been given in a recent 
publication. (See Part B) 

Major Findings ; As noted above^ P383 9-. and P329 cells derived from 

lyiaphoms have 



tturlse gnalignaat lyiaphoiaas have been adapted to growth 
in the serum^free smdluia. A continuous line of cells derived frora a huasas 
epidermoid careinoaia of th@ skin of the face has been isolated in the sailk 
fi^diuzi and has been gro^m in this is@dius for alssosg one year. This cell 
strain has retained the features of an epidermoid careinona ifith intercellular 
bridges and keratin pearl formation. 

Significance; The availability of cell lines in seruts^free siedia for 

studies of tuaior viruses t&ay be of i^ortance in the search 
for viruses in husan iKgoplasBss. It is possible that even ssiall asHsunte of 
human or anlssal serua ssight contain inhibitors of a huaaan tumor virus aisd 
the use of & sarua=free culture system ©ay be necessary for successful isolatior 
of such agents. The £s>ct that polyoma virus can be grova in ailk»adaptsd 
P33@ Hi cells witli saar'k^d cytopathogenic effect suggests that a similar 
cell ciulture systea saight be useful in stfces^Jts to iaolata a eo^as'able 
virus fro9 I'siisiaan tuiserg. 

1033 



Serial Wo, mi S3Q 
PiB°WIH 
XndlviduAl Project R<spo?t 
CAlandar Year 1953 

?ayt A Cco'stiaaed} 

Propoaed Cougae s An at;teapt is beias made to isolate and propagate 
addltioaal human cell strains in tbs ssruaB-'free raediuia 
for U9« ifi the saareh £or s^ytopathogenie agents isi himan loalignant tuisors, 



?mt 3 iaeludsd; Yes 



1034 



Serial MOc MCI 530 

Individual ProJccC Report 

,CAlead&r fear 1958 

^£ft_Bs HoBora^ Awards,, aad Publications 

Fublicafcions other than abstracts front this project; 

Rabsoa^ Ao S.^ Legallais^ F. t.g ©ad Baron^ S. 
Adaptation to Seru9°°Fre£ Mediuia by & Fhagocytie Cell Straisa 
Derived from a !lu7ice I^fssphosa 
Hatssre 181; 134, 1958. 



1035 



Serial Sto. KI 5.17 

2. Pathologlc&l t&chmlGMf 

3o BetfeeadA; dryland 

Indlviduffil Project Ebeport 

C&leffidar fear I9S8 

ESSIA 

F?oj<sa£ Tieie; FaSlmlogical Technology S@ig£ioa 

FriB«sipal Imeatt^&teri Mr, J. M. AlbrecM 

O&Sier XsKvestigsSos'&s 

CoofMsrseixig 1l@i£®: 

ma fears (caUadar year 195© )s Patient Days (caleodar year 1958); 

Totals 28 
Profsssiooal: 3 ' 

Other g 25 

Project .Degeriptloai 

Stalled tissue sections are tba fundarastntal. basis of all cliaisal 

aad essperii^ntal studies of cascsr. the Section prepares histological 
sectioBS for all the iavestigators of the Ms^tiooal Cancer Iitstit»teo 
It mk@s m'&il&hla all the established rotstiae aad special stal:Qs aad 
la addltiosi develops and provides the current e^mrlis^ntai methods of 
tissua preparation such as en^yi^ stains &td specific histological 
stains c 

Movet^er 1. 1957 > October 31, 1958 

of Issveetigators ....,o ...... o ».<. . 6S 

r of Pieces of tissue ..... . . » b . 153^260 

of Bottles of iisffiue ............... 23^408 

of Blocks cut. ................. o 93^948 

of Blocks c«£ serially .............. 1,420 

of Frosea blocks cut ............... @S§ 

r of Autopsies ................... 12^717 

r of Eecuts .................... 1^44S 

r of Slides staiaed H&E ............. 111,852 

r of Slides staimd special ............ 14^627 

of Gnstalaed slides ............... 9^S03 



Photographic Service® rendered to the Laboratory of Pathology 
l^ 1957, to October 31^ 1958 



Blaek sad White .................... 401 

Color ,„ o ........... o ........ . Ill 



1036 



Serial ISO. mi 517 
PHS-IJIH 
Ijtdividu&l Project Mpott 
C&Uad&z Year 1958 



f&^t A CooaeiEusd) 



mm. 

Black 



Colo? ..,...„ o . c ........... c . . SOO 

Blsek atsd white ....„..,«...« .o ... . 289 

Color ...... o . o o ........... o . . 545 



am 



s 5 ..... o . o .....«......«., o 3,3U 

8 X 10 o ... c ............. o ... . 165 

Moisaead ........................ 165 



Fart B iaelisQeds Ho 



1037 




1 / 

Laboratory of ffayaiolojE/ 
Biadget Data 

Estimated Obligaticffls..,o,<,»„„,<,o..<..»o<,o.»»o,o, Fiscal Year 1959 
Directc « » o o o , , » o » , o o » o , , , , o , o , o « <, p . o « o o o o »$ 299^300 
Reimbursements 

Cllnlcalc o , o o <. o ,o ...,, o o ..,,».,,..., o .,,, „ 

TOtaloo.OOSOOO.O.Q 

1 / Includas Projects Ko'ss 

OFFICE OF THE CHIEP; 

90U 
918 

CMCER PffirSIOLOGY SECTION.' 

900 910 

901 919 

902 920 
905 925 
906 

ENERGY METABOLISM SEGTXCSSs 

911 

PHYSICAL BIOLOGfY SECTIONS 

914 916 

915 926 



Serial Noo NCI -901*- 

lo Laboratory of Physiology* 
PHS-NIH 2. Office of the Chief 

Individual Project Report 3o Department of Biology, 

Calendar Year 1958 Princeton University, 

Princeton, N. Jo 



Part A. 



Project Title: Ultraviolet Light and Carcinogenesis 

Principal Investigator: Harold F» Blum 

Cooperating Units: Department of Biology 

Prineeton Universi% 
Princeton, New Jersey 

Man Years (calendar year 



Total: 


1/3 


Professional : 


1/5 


Other: 






Project Description: 

During the first two-thirds of the present year I held a 
Public Eealth Service Special Research Fellowship, and was absent 
on leave from the National Csuieer Institute c Since, however, my 
activities during the last third of the year have besn intimately 
connected with my activities while on the Fellowship 2 can hardly 
report the one without the other « 

I" Objectives : Quantitative saialysis of data on experimental 
carcinogenesis by ultraviolet light. 

Methods employed : Mathematical and statistical treatment of experi- 
mental datao 

Major findings : The year of my Fellowship was devoted almost entirely 
to a theoretical stx«iy of carcinogenesis by ultraviolet light, 
continuing a project that has engaged me for several years o Sine© 
the publication of an approximate model for the process in 1950 
(Jc Kato Cancer Insto n: hS^-kS^i, 1950}, which described certain 
aspects of the data but not others, I have been concerned with 
developing a coaiplete model <> A nxanber of successful steps were 
laade during the intervening years, but only in January 1958^ was the 
step accomplished which made it possible to piill all the threads 



1039 



- 2 ■• Serial No<, NCl-904 

together to provide a complete quantitative description of this 
careinoganiG process o It has taken considerable time to put this 
material in form for publication. Soane of the final calculations 
could not be made until 1 returned to this laboratory where the 
original data were stored; and these calciilations and revision of 
the papers for publication have occupied most of my time since I 
arrived back= 

The resiiLts of this analysis cannot be expressed in brief 
space; but may be Bunsaarized by the following equation 

mv^ - m v^ ^Pl^fTij^V^ C2t^t,-t!) - ^ t,t, (i) 

where V, is the tumor volume and V the initial voltmie^ In indicate 
ing the natural logaritlm; c is a Soefficient relating to the 
accumulation of clones of replicating units, and k an acceleration 
coefficient, p is the probability and S the standard deviation^ e 
being th® base of natural logarithms; D is the dose and i the 
int^jrval between doses; t is the time elapsed between the first 
dose and the arrival at volume V, , when dosage is continuous 
throughout; tx is the duration of the dosage when this is inter- 
rupted^ and tg the subsequent time required to reach the volume V-. 
in this ease a When dosage is continued until V, is reached, t^ = t /, 
and Ife • 0, the equation besoming 

inv^ - mv^ =(r:o357i/ — ^r^ ^^^ 

The function Cl-0<.55/i} may be replaced by a function representing 
the distribution of the proportion of tumor types with interval be- 
tween doses (sarcomas /sarcomas -e- cansinoma8)o 

This model gives an adequate description rf all the available 
data; constituting, I believe, the first complete quantitative 
description of any carcinogenic process that has yet been formulated o 
Regarding the equations as purely eapirical certain conclusions may 
be drawn concerning carcinogenesis by liltraviolet light » The process 
described is continuous and cumulative; essentially irreversible esd 
non-threshold o No comparison can be drawn at the present time with 
other types of carcinogenesis because there are no data eampar8ib3.e 
to those for ultraviolet carcinogenesis o But until such data are 
available the possibility that these are general characteristics 
of all cancer processes shoiild "be entertained. 



1040 



- 5 - Serial Noo NCI-90i^ 

Although treated above as eiBgpirieal, the quantitative descrip- 
tion is built upon the concept of acceleration of proliferation of 
fundamental replicated unite, each dose of radiation accelerating the 
proliferation rate by an amount proportional to the magnitude of the 
dose a Such an accelerated pncess was recognized in the approximate 
model, but the final solution recognizes in addition that clones of 
replicated units are add@d during the develojment of the tumors, and 
that some of the cells are destroyed » These factors complicate -ttie 
pictiirsj, and while their probable role was envisioned early, the 
mathsmatitsal ea^ression has proven a rather knotty problem^ A brief 
expression of th© overall pr®e«8®ffl, compatible with (l) and (2) is 



inv xnv^ 2i * 2i |]ro317i / i ^^^ 

where 4^(t) is a function of times which describes the addition of 
clones, and f (t) is a function of ttiae, which describes tlie destruc" 
tion of cells. The first term on the right hand side describes 
the accelerated growth of the initial clones at the -time of the 
first dose; the second term describes the accelerated growth of 
additional clones as tb^se are added; and the last term describes 
the removal of cells by the destructive action of ultraviolet 
lighto 

In the above equation (5) the functions ^(t) and f(t) airs 
not directly determinable from the data, but the later has negligible 
effeet because the term which contains it is of a lower order than 
the others o Failing th© description of function ^(t), the factual 
growth curve for the tumor egamot be described, and it does not saem 
probable at the Homent that this function can be determined fross, the 
existing data, although the possibility will be explored. In any 
case th® lack of description of this function does not vitiate ths 
overall concept of accelerated growth, which seesms clearly 
established. 

It is possible, even laekijog the description of the function 
^(t), to extrapolate back to the initial volume V^ by introducing 
into (2) a value for the acceleration coefficient k, derived from 
direct measurement of the growth of these terms at the end of to 
The initial voliaue V is estimated to be lO"^ nm®, which is vSy 
much smaller than thS volume of the cancer cell 10"" laa^o One ar- 
rives at the conclusion that the replicated unit which controls 
the growth of these cancers is very much smaller than a ceJJ., al- 
though the exact volume of the former cannot be determined from 
these data. Reasoning in teUDS of modem bioch«ni8tiy and genetics, 
one arrives at the idea that the basic replicated particle is a 
nucleic eicid template » The behavior of this particle distinguishes 
it fixjm the viruses as oi^inarily understood, although the analpg^f 
is closes 



- k " Serial No« HCI-90J* 

These findings seem to provide a new point of view for re- 
garding the earcinogenic process o Needless "to say the argument 
cannot be adequately presented in brief form^ and reference mist be 
made to three papers on the subject, the writing of which has just 
been completed.. These are "On the mechanism of caacer induction 
by ultraviolet radiation; II, A quantitative description and its 
consequences; IIIo The growth curve; and IV, The size of the 
replicated particle o" These will be submitted to the Journal of 
the National Cancer Institute in the very near future.. A very 
brief report hau already been published, "Sur 1' aspect quantitatif 
de 1' action sancerogene des radiations ultraviolettes o " Bxillo 
Cancero h^: 165-166, 19580 

A book entitled "Carcinogenesis by Ultraviolet Light: An 
Essay in Quantitative Biology", was brought to coaipletion during 
the term of the Fellowship » This includes not only the general 
content of the above papers, but a more eeople discussion of basic 
aspects of the problaa together with a synthesis of ideas whj.ch it 
is not possible to inclvcLe in the shorter publications -> 

II o Objectives ; Studies of effects of ultraviolet light on mouse 
skino 

Methods eatployed ; Exposure of moi:^e ears to various concentrations 
of ultraviolet light and studying histologicsQ. changes » 

Major findings ; Many repeated doses of ultraviolet light are neces- 
sary to produce cancer, yet a single dose may produce extensive 
changes in the skino Studies have been undertaken to examine the 
latter type of reapor^e and its possible relationships to the 
carcinogenic process » A satisfactory method has now been devel- 
oped for reproducible irradiation of the mouse earo Evaluation 
of the response in quantitative terms has proven more difficult, 
but it has been possible to follow certain of the early events o 
Following a single dose of radiation from an intermediate pressure 
arc there occurs an extensive hyperplasia of the epidermis which 
has been followed by means of the incidence of mitosis o The 
maximum of hyperplasia is greater, occurs earlier, and is sus- 
tain^ longer with higher dose^ The response has the general 
character of an inflefflmatory reaction, but with special characteris- 
tics which may result from the restriction of the underlying 
photoch^aical reaction to a very thin layer » 

The aresponse to the intermediate pressure mercury are in- 
volves the dennis to a marked extents It has been our hope, that 
by using the low pressure mercury are, the radiation from which 
penetrates very little below the epidermis, to be able to study 
the latter with minimum involvaaent of the denoiso Mto Gerald Ao 
Soffen is continuing this study under xay su^rvision, as the sub- 
ject for his doctoral thesis.. 



1042 



- 5 " Serial Koo mi'90h 

A pa.p®r descri'bing the method and susnnarizlDjg the findings 
to date, lias been submitted to over ^ouinel for publication o 
"Irx'adlation of moiise skin with single doses of ultraviolet light" » 
by Ho P. BliOT., E. Go Butler, To Ho Dailey, J=, Ro D®ubs^ Ro C» Mawe, 
aiad Go Ao Soffeuo 

IISo Ob Jeetivea : Studies of ultraviolet aM photodynaanlc hemolyaiSo 

Methods employai : EKposxire of mouse ear® to various concentrations 
of ultraviolet light and studying histological changes o 

Major findings ; Research on haatolysis by ultraviolet light and hy 
photodjrassBiG e/jtion, whieh has been carried on in co3J.8&oration 
with Dro Jo So Cook wess ccaapleted said prepared for publication o The 
paper entitled "Dose relationships and oxygen dependence in ii3.tra- 
violet Kid photodynajffllc hsuolysis" by Jo So Cook and Ho Fc BluBp 
will appear in sn early issue of Cellular and Coarparativ® Pl^siologyc 

This work has shown that the kinetic relationships are the 
sam® for both foxias of h^lysis^ although the photoeheaiical reac- 
tions ar® different. The effect of dose-rat® was exasalned for 
hmolysis by ultraviolet light and found to follow the relationsliip 
ejqpected from eex'lier studies by Cook on the effect of short doses ^ 
at high dose rateo The relationship for the latter is 

t D^ = & constant 

where D is the dose, and t is the time to haaolysiso For continued 
dose at different dose rates, given throughout the time t, the re- 
lationship is 

t"' "^I = a constant 

wh®re I is the dose rateo 

It was shown that haraolysis by ultraviolet light shows con- 
siderable Oa dependence in som^ parts of the spectrum, very little 
in others o This is explained as a combined effect of direct action 
of the radiation on cellular ecsEponents and photodynamic action in- 
volving a different chromophore, possibly a porphyrin present in 
minute maouuto 

Significance to the pro,^raai of the Institute ; To throw light on 
mechEsiism of action of ultraviolet and crnicer* 



1043 



- 6 - Serial Noo NCI-904 



Proposed «5ourse of the project ; Soffle further work in this direction 
is now going on in this laboratory o Since scame aspect© of the 
description of carcinogenesis by ixltraviolet light suggest a role 
of pereaeability change, it may be fruitful to enlarge this general 
program of research, and plana for doing so are being considered., 



Part B included Yes ^ No /37 



1044 



- 7 - Serial No a KCI-90i* 

PHS-NIH 

Individual Project Report 

Calendar Year 1958 

Part B; Honors, Awards, and Publications 

Publications other than abstracts from this project: 

(l| Butler J B» G,, BlUE, Ho Fo, and Schsnidt, S= Eo : The localized 
"" character of ultraviolet effects on the urodele forelimbo 

Jo Cello CoHjpo Physiolo |0: 58I-588, 1957 (actually appeared 
in 1958} « 

(2} Blum, Ho Fo: Sur 1 'aspect quantatif de 1' action cancerogene 
"* des radiations ultravioletteso Bull. Cancer o 1^: 165-166, I9580 



Honors and Awards relating to this project: 
Seminars euid Symposia 

Sssdnars presented (all on the subject of ultraviolet 

carcinogenesis) : 

lo Laboratoire Pasteur^ lastitut du Radiusno June 1958" 

2o Departsinent of Biology, Princeton Universityo October 19580 

5. National Cancer Institute. November 1958. 

Presided as co-chaixsaan of a sjfiaposiian on carcinogenesis at 
the International Cancer Congress in London, July 1958. 



1045 



PHS-WIH 

Individual Project Report 

Ceilendar Year I958 



Serial Noo NCI-918 

1. Laboratory of Pbysiology 
2o Office of the Chief 
5« Bethesda^ Mdo 



Pfirt A o 

Project Title: Research Developnent Projects in the Field of 
Electronics 

Principal Investigator: Nathan Coffey 

Other Investigators: None 

Cooperating Units: None 

Man Years (calendar year 1958) : 
Total : 1 

Professional : 1 
Other: 

Project Description: 

Objectives ; To design new electronic equipnent eus the needs of the 
Laboratory arise; to modify existing electronic eq.uipment to meet 
the changes required in an experimentj and to supervise the re- 
searsh development projects in the field of electronics. 

Major findings ; 

(1) Further refinements in colorimetry instrumentation to provide 
more precise read -out, (Dr=. Fo Smith) 

(2) Developaient of background count elimination device to provide 
greater ease of interpretation of data collection on patients sub- 
jected to isotop® diagnosis techniques o (Mro Ro Swain) 

(3) Develojasent of circuitry and devices for continuous monitoring 
and recording of relative humidity in connection with metabolism 
studies 5 capable of being integrated with existing electronic data 
co3J.eeting instrumentation currently being used in other phases of 
same study, (Dr, A, Wo Pratt) 



104t 



- 2 - Serial No. NCI-918 

(k) Consulted with investigator and maniifacturers in determining 
specificatioiiB to be forasulated for building and/or modifying com- 
mercisd equipment for providing eommon language link between data 
reduction equipaent used in metabolism studies and available elec- 
tronic computer, to pix)vide investigator with means to by-peiss 
laborious and time-consuming manipulation of leurge amount of data 
automatically accumulated by electronic means in metabolism stiidieso 
(Dro Ao Wo Pratt) 

(5) Consulted with investigator and Instrument Maker in determin- 
ing physical configuration of gas analysis cells to be used in 
metabolism studies. Preliminatry determinations are under way in 
search for final design which will provide greater sensitivity and 
stability combined with ruggedness and portability required for 
us© by relatively untrained personnel » (Dr^ Ao W<. Pratt) 

(6) Evaluation of proposed circuitry for monitoring and recording 
movements of experimental animals while being radiated. After dis- 
cussion of problan with technician to whom problem had been assigned , 
suggest-^ circuitry which was adopted in preference to other cir- 
cuitry which had proved impractical = (Dr, Ho Andrews) 

(7) Presently being developed is device for continuous monitoring 
of germicidal ultraviolet radiation to the exclusion of other radiant 
energy simultaneously preaento Long-term stability and sensitivity 
to a wide range of intensities are other primary requirements » 
Flexible control and monitoring of input power to 0-V generators 

are to be provided also. (Dro M. Elkind) 

(8) Consulted with investigator who foresaw possible need for misro- 
scopic examination of millipores in quantities which made man-power 
requirements prohibitive « Suggested various methods for performing 
exeaninations electronically at high speedy and reecsssnended flying- 
spot techniques o Further consultations were held with investigator 
and other NIH pejrsonnel, and with representatives of eommercial 
interests, with result that recomneodation was agreed to be best 
solution and that firm commiisnents could be made when investigator 
finalized precise requirements » (Dr.. Malingren} 

(9) Design and construction of electronic circuits and automatic 
controls to convert cage for animal used in metabolism studies into 
adiabatic cage and to present graphically the input power required 
to maintain equilibrium ^ (Dr.. Ao Wo Pratt) 






5 - Serial No= NCI-918 



Signiflcanee to the program of the Institute ; The ability to have 
these refinements to eleetronic devices makes it possible to 
make metabolie analyses on noruKuL eind. tumor -bearing animals which 
heretofore were made indirectly » 

Proposed course of projeet ; To develop new and finer adaptations for 
the measurement of metabolic changes; for exangjle^ development of 
automatic computation of the large volume of available datao This 
v±H reqtiire the construction of new gas -sensing elements having 
proper electrical characteristics conr|«tible with the operating 
requirements of existing analog computers =. 



Part B included Yes £J No /x/ 



104^ 



Serial No» NCI -900 

lo Laboratory of Physiology 
2<. Cancer Physiology Section 
PHS-NIH 5o Bethesda, Mdo 

Individual Project Report 
Calendar Year 1958 

Part A . 

Project Title: Growth Stimulating FactorCs) Present in Tumor Tissue 

Principal Investigator: Florence Kate Millar, Jane Nicolet Toal^ and 

Julius White 

Other Investigator: None 

Cooperating Units: None 



Man Years (calendar year 
Total : 5 

Professional : 1-2/5 
Other: 5-1/5 

Pixjjeet Description: 

Objectives: To cbntinue the study of the growth stimulating properties 
of tumor tissue by (A) examination of a variety of tissues for these 
effects; (B) concentration and identification of the active 
factor (s) present in tumor tissue » 

Methods employed ; 

(a) Three mouse tumors and 5 human tumors were lyophilized 
and respectively incorporated as a total source of nitrogen into 
semi -synthetic diets « Control diets contained lyophilized normal 
rat liver or imiscle in place of tumor = These diets were fed to 
Walker 2^6 tumor-bearing rats which had previously reached a point 
beyond which little or no weight gain occurred and food intake 
had decreased o The effects of these diets upon weight gain and 
food intake was observed > 

(B) Walker 256 timor tissue has been extracted with a variety 
of coranon solvents to yield fractions which were tested for growth 
stimulating properties o These fractions were tested in feeding ex- 
periments involving both normal and tumor-bearing ratso Fractions 
were either incorporated into semi -synthetic diets at levels 
comparable to their concentrations in the whole tumor diet or else 
were fed as separate supplements to a casein diet„ In either case 



1047 



- 2 - Serial NOo NCI -900 

the fractions vere usually "tasted for growth effect© on tumor-bearing 
rats whiah had been previously maintained, on a ceisein diet and which 
had reached a weight plateau. Where a large enough amount of a 
fraetion was available^ the test diet was instead fed throughout the 
period of tumor growth. The effects of these tumor fractions upon 
weight change were compared with the effects of whole tumor diet, 

A few fractions were directly compared with casein as a 
source of nitrogen for the growth of young normal ratSo 

Promising fraction® are being further concentrated by pro- 
cedures such as coliHBn chromatography, dialysis and electrolytic 

desalting « 

Major findings : 

(a) Effects of diets containing mouse or human t\imor tissue 
or normal rat tissue o 

All the tumor diets examined were capable of producing in- 
creased weight gain in feanor-bearing rats. In a few cases these 
increases were comparable to those observed when similar rats ate 
the Walker tvonor diet. Where the response was negative^ this was 
usually because the animal did not eat the dieto Animals ingesting 
either of the normal tissue diets showed slight increases in weight 
which were considerably less than the increases produced by the 
Walker tismor diet. 

(B) Fractionation studies » 

1» Trichloroacetic acid insoluble fraetion <,--Althoiigh this 
fraction was carefully washed and was neutral, the rats refused to 

a) Addition of sodium bicarbonate and dialysis gavs 
a product which was acceptable, but which showed no 
growth stimulation o 

b) Following further separation by mild alkaline 
hydrolysis and precipitation of the protein at pH 5,0 
there was no growth stimtdation product by either 
the protein fraction or the ribo- and deoxyribo- 
nucleotides c 

2o Water soluble fractjon o— -Dry^ defatted Walker 2^6 
tissue was extracted with boiling water and the extractions lyophilizedo 
When this fraction was fed to tumor -bearing rats whieh had previously 
reached a weight plateau on the casein diet, the aniimls respoMsd 
with weight gains not quite comparable to those observed when the 
whole tumor diet was fed. 






•s V, 



- 5 - Serial Woc MCI-9OO 

a) The aetivity of the wa'fcer soluble fraction was 
concentrated lay adding ethanol to a concentration 
of 75 percent, which precipitated about 2/5 of the 

frastiouc The precipitate was inactive o 

b) The 75 percent ethanol soluble fraction waa 
separated on a charcoal column » The water eluate 
was very active and was found to contain 30-55 
percent NaClo It was then found that the feeding 
of NaCl itself to tiaaor-bearing rats could produce 
weight gains which were grossly similar to the 
changes produced hy the feeding of tumor diet„ 
This was observed both when salt was fed to aniaxsals 
which had reached a weight plateau on the casein 
diet and when salt was fed continuously over the 
period of tumor growths The sanount of Na in tumor 
tissue was such that, if this wers all present as 
NaCl, then the tumor tissue diet would contain 

Oo9 perisent Ns€l in addition to the amount addM in 
the inorganic salt mixture to all experimental, diets-. 
It was found that tumor-bearing rats ingesting a 
casein diet containing this much additional salt 
did eventually reach laaximum weighty although this 
oceurrsKi much later than it did for rats on the 
casein diet without added salt, 

c) The water soluble fraction is being desalted. 
electroljrkically to see if removal of sodium 
chloride (about 1© percent of the fraction) results 
in an inactive fraction, 

5» Water insoluble frag tjon ^ --The material which was 
insoluble in boiling water contained only about 10 percent of the 
total NaiSl of dry tuaior tissue o It vas fed in a diet to tuiaor- 
bearing rats after they had reached a weight plateau <= Under such 
eireumstances, the water insoluble fraction was ineffective in 
promoting weight gaino However # in further experiments this 
fraction was fed to tumor -bearing rats over the period of ixmor 
growth and compared with both the whole tumor diet and the casein 
control dieto Although animale on the water insoluble fraction 
grew poorly at first, they later showed weight gains comparable 
to that of rats on the whole tumor diet. These gains occurred 
during the later stages of tumor growth at a time when rats on the 
casein diet had ceased to gaia« Furthermore the water-insoluble 
fraction waa found to be equal to the casein diet in supporting 
growth of young normal ratso 



^7 

10^ 



~ h - Serial Noo KCI-9OQ 

These findings suggest that the effects of feeding tumor 
tissue to tumor-bearing rats can be grossly duplicated by the 
feedixig of NaCl. The residual delayed effect of the salt-free 
water insoluble fra«5tion points to the existence of some addi- 
tioneJL unidentified eonrponent which has growth-stimulating 
activity « In the earlier tests of salt-free fractions, such 
as the trichloracetis acid precipitate, salt-deficiency of the 
test anijaals may have obscured possible growth-stimulating 
effects . 

Significance to the program of the Institute ; The study and identi- 
fication of a growth stimulating soBrponent(s} in tumor tissue 
will yield infonnation about the nutritional requirements of 
the growing tumor and the competition between a tiaaor and its 
host for growth materials » 

If this growth stimulation caused by tumor tissue in the 
diet is due to the additional NaCl it supplies^ then it becomes 
neeessary to deteraiine whether the NaCl is required for growth 
of the tumor or has a sparing action. Studies of the salt and 
water balance of tumor-bearing rats will indicate the roJ.e of 
the tumor in upsetting the normal bal.anceSo 

Since this afciiBulation of growth in tumor-bearing animals 
oesurs after feeding tissue from tvanors of several species the 
above information will be of general interest to the cancer 
program ■, 

Proposed course of project ; 

1= Studies with these fractions derived from tumor tissue will 
be continued-, As there is additional evidence of growth-stimulat- 
ing activity in the fractions, further concentration of activity 
and chemical identification will be attempted o 

2o Nitrogen and salt balance studies are in progress with tumor - 
bearing rats fed either the tumor tissue diet or diets containing 
casein and salt. These GQn5>ari8on8 should make it possible to 
distinguish between the ttestabolic effect of added NaCl and that 
of timor tissue, if iaadeed the effects are actually different » 



Part B included Yes ^ No £J 



SSL 

10^ 



- 5 - Serial Wo. NCI-90O 



PHS-NIH 

Individiml Project Report 

Calendar Year 1958 



Part B : Honors j Awards, and Publications 
Publications other than abetraets from this project: 



Millar, Fo K., Brooks, Ro Ho, and White, Jc: 
Stisaulation of appetite and growth in the 
tumor-hearing rat by a variety of tissues. 
Jo Nato Cancer Insto (in press). 



Honors and awards relating to this project: 
None a 



^3 



Serial No, M!il-901 

lo Laiboratojry of Physiology 
PES"NIH 2o Cancer Physiology Section 

Individiial Project Report 5, Bethesda^ MdL=> 

Calendar Year 1958 



Part A o 

Project Title: Probable Sources of Available Nitrogen 

for Tumor Growth 

Principal Investigator: Julius White 

Other Investigator: None 

Cooperating Units: None 

Man Years (calendar year 1958) 
Total: 5 

Professional : l/5 

Other 2-2/3 

Project Description: 

Objectives ; To determine the availabilily and utilization of nitrogen 
for the rapidly growing transplant®! Walker carcinosarcoma 2560 

Methods esaployed; Comparative studies were made on rats bearing the 
Walker 256 ingesting either a 20 percent casein diet of known cos- 
position or a aJjailar diet in which the casein was replaced by 
desiceated Walker 256 tusoor tissue as a sole source of protein = 
Experiments lasing Walker 2p6 -toHDr tissue as a source of nitrogen 
in the diet had already been shown to grow larger tujaors with less 
body weight loss than coBjparoble animals ingesting a 20 percent 
casein diet. 

Paired feeding experiments: 

Rats ingesting the 20 percent casein diet showed a 
gradual increase in the allantoin excretion (end product of nuc3.eic 
acid metabolism) which increase with increased tioaor gro^rfch and 
reached a value of twice (100 percent increase) that observed 
before tisaior growth was initiated o 

Aniaals ingesting the diet containing the Walker 256 as 
& source of protein had an initial allantoin excretion approxiiaately 
twice that of animals on the casein diet„ However^ with inci^ased 
tujaor growth there was only a slight increase in allantoin excretion 
{10-20 percent). 



1054 



- 2 - Serial Noo NCI -901 

A group of animals ingesting the casein diet and showing 
elevated excretion of aUantoin had tunsors ronov©! eurgicallyo There 
was an iSBrasdiate reduction of allantoin excretion. In many cases, 
this value fell to 50 percent below the normal allantoin excretion 
and remained subnormal for a© long as 2 weeks » {E^jperiiisents were 
not continued beyond this time,) Parallel to this rapid drop in 
allantoin, was an increase in urea excretion which rsaained elevated 
as long as the allantoin rajiained suppressed » 

The exact interpretation of the®®; observations is not 
eleair at the presents It is possible that the growth of the tumor 
leads to an increased guanas© and xaathia© oxidase activity wMch 
leads to increased breaMown of guanine and xanthine to aJJLantoino 
Sinee protein synthesis (incorporation of amino acids into proteins) 
is dependent on the presence of nucleic acid, the rapid growth 
(protein synthesis) of the tusaor studied aaay be reflected by ia- 
ereas©i excretion of end products of nucleic acid metsibolisnij, 
nasiely allantoin <, 

Significance to the program of the Institute ; To obtain a better under" 
standing of tuaaor-host relationship with the islti®at@ goal of sep- 
arating and identifying the factors responsible for tmaor growths, 

Proposed course of project ; Continue this study to determine the 
real significance of these luetiabolic relationships « 



Part B included Yes £J ^o /x/ 



1055 



Serial Noo NCI -902 

lo Laboratory of Physiology 
PHS-NIH 2o Cancer Pl^siology Section 

Individual Project Report 5o Bethesda^ Mi» 

Calendar Year 1958 

Part A o 

Project Title: Changes in Nucleic Acid and Protein Synthesis in 
the Rat Small -Bowel Epithelium Following Total - 
Body X-Radiation 

Principal Investigators: Jane Nieolet Toal and Julius White 

Other Investigators: Ro Bland Williams, Jro , and James C 

Cooperating Units: Department of Pathology ^ Naval Medical 

Research Institute^ Bethesda, Mdo 



Man Years (calendar year 
Total: 2 

Professional: 1-1/5 
Other 2/5 

Project isescription: 

Objectives: Nucleic acid and protein synthesis in the rat small -bowel 
epithelium following x-radiation have been atudi®i in conjunction 
with Dro Ro Bland Williams' studies of changes in morphology and 
rate of cell division in this tissue in relation to time and dose 
after threshold to tissue-lethal doses of x-ray o 

To further study and separate the cell populations involved 
in these ch^iges, the relationship of nucleic acid and protein 
synthesis to cell division has also been studied following 
(1) repetition of the x-ray dose at selected times, and (2] 
fractionation of the dosSo 

Methods earployed ; Rats were sacrificed after several time intervals 
for each dose of x-ray « The index employed to follow "ttie synthesis 
of DNA, PNA, and protein was the concentration of isotgpic nitrogen 
found in them 5 hours after a single tracer dose of K^^ glycine. 
The behavior of DNA, PNA, and protein following x-radiation is ex- 
pressed as the percent of the N^5 concentration of aa unirradiated 
control o The PNA was examined as individual nucleotides o For the 
MA, only the purines (adenine and gu^uoine) were examined. 



- lO^C- 



- 2 - Serial Woo NCI -902 

Major findings ; This project ia complete aiid is being reported as 
papers II and III of a three -paper series » Papers I and II have 
been published (see Part B)), Paper III is being written with the 
following title: 

Effect of Total -Body X-Radiation from Near-Threshold to 
Tissue-Lethal Doses on the Snail -Bowel Epithelium of the 
Rato IIIo Dose Fractionation Studies Designed to Separate 
Biological Effects J, Cell Populations, and Time Factors 
Involved in Recovery <> 

R« Blond Williams J, Jr», Jane Nicolet Toal, and Julius 
White o 

SISMWRY OF MAJOR FINDINGS: 

1, The initial blockage of mitoses produced by x-radiation is 
not accompanied by a proportionate reduction in the rate of DNA 
synthesis.. The abortive recovery of mitosis in the proxin^ 
postirradiation period occurs while DNA synthesis is falling to 
a low levels DNA synthesis resxanes before final mitotic recovery 
begins o Final recovery is acsoaipanied by an overshoot in the 
synthetic rate of DNA^ PNAj, ana protein^ as well as in the mitotic 
index « 

2» Administration of a second dose of x-rays during each of the 
events which follow an initial dose, shows the following correla- 
tion of events and cell populations: 

a) Acute destruction and abortive recovery are related 
events and occur together o They involve cells in late 
interphase at the time of irradiation o 

b) The initial block is a distinct biological event and can 
be increased without increasing the ninnber of cells involved 
in the acute destruction or abortive recovery o 

c) The cells involved in the prolongation of interphase are 
not the same as those involved in the acute destruction emd 
abortive recovery. 

d) The decrease in DNA synthesis during the first five hours 
following irradiation can be correlated with the cells involved 
in the acute destmetiono The profound decrease in DNA 
synthesis occmrring between 12 and 1? hours post irradiation 
can be correlated with cells involved in the prolongation of 
interphase o 



1057 



ssrlfll MOo NCI»902 



e| Ce3J.8 involved in the final recovery were in an ear 
stage of Interphase at the time of Irradiation than t}u 
volved In the Initial delay, acute destruction j, and abc 
recovexy , 

f) The degree of delay In recovery or permanent sterilixa- 
tion produced by a aesond dose of x-ray®, depends upon the 
extent to which recovery has taken place from the initial 
eacpoeiire » 

5 The relative iu^portanee of dose size asad the tSaie between 
doses was studied c No eooaibinstion of doses or tiroes la as ef- 
fective as the BfiBue total roentgens adsdnistsred as a single 
dos@c 

Significance to the program o f the Institute ; This is the first 
detailed correlation of ehanges in cell division with alterations 
in nueleic acid aietabolisai in a manBBaliaji tissue following x- 
radiation, 

. These studies were liodertaken to obtain a better uraier- 
standing of the mechaaisBi® by which x~radiation interfei'es with 
cellular proliferation o When ionizing radiation is used ia the 
trealaaent of cert^n types of cancer, such knowledge of the 
tissues involved is necessary to select the most effective dose 
sikL time sequence.- 

Proposed course of project; Hone^ 



Part B included. Yes /x/ Wo £J 



1058 



k - Serial Noo NCI -902 



PHS-NIH 
IMividual Project Report 
Calendar Year 1958 



Part B ; Honora, Awards, and Publicsations 

Publlsations other than abstracts from this project: 

Williams, Ro Bo, Jr,, Toal, Jo No, White, Jo, and Garpanter, 
Ho Mo : Effect of Total -Body X Radiation from Near-Threshold 
to Tissue-Lethal Doses on Small -Bowel Epithelium of the Rat,, 
lo Changes in Morphology and Rate of Cell Division in Rela- 
tion to Time and Doseo Jo Nat, Cancer Insto 21: 17-61, 1958. 

Toal, Jo No, Reid, Jo Co, Williams, Ro Bo, Jr^, and White, J<.: 
Effect of Total -Body X Radiation from Near-Threshold to 
Tissue-Lethal Doses on Scsall -Bowel Epithelium of the Rato 
II o Changes in Nucleic Aeid emd Protein Synthesis in Rela- 
tion to Cell Divisiotto Jo Nato Cancer Insto 21: 65-76, 
19580 



Honors and Awards relating to this project: 
Noneo 



1059 



Serial No, NCI -905 

1. Laboratory of Physiology 
PHS-NIH 2c Cancer Physiology Section 

Individual Project Report 5» Bethesda, Mdo 

Galendeir Year 1958 

Part A . 

Project Title: Iodide Concentrating Mechanism of the Thyroid Gland 

Principal Investigator: So He Wollman 

Other Investigator: Jo Co Reid 

Cooperating Units : Dr^ Ruth Merwin of the Laboratory of 

Biology, NCI (Serial Noo HCI-lUlt) 

This project might coinplement research 
done in the Clinical Endocrinology Branch 
of NIAMD (Serial No, A2201). 



Man Yeeurs (calendar year 

Total: 1-1/5 

Professional : 2/5 
Other: 2/5 

Project Description: 

Objectives : To learn about (a) the mechanism by which the tigroid 
glaM maintains a concentration of iodide elevated above that in 
the blood;, and (b) the mechanism by which the thyroid gland ac- 
cumvilateB protein-bound iodine <. 

Methods employed ; Kinetic studies were made of the concentration 
of radioiodide by the thyroid, the rate of formation of protein- 
bound iodine, and the Influence of inhibitors and various physiolog- 
ical states of the animal on these processes o Some histologic 
stiidies have been made especially of the blood vessels in the 
thyroids 

Major f indites : Thiocyanate was found to increase the fraction of 
the thyroid iodide transported to the blood per minute but to 
have no effect on the transport of iodide from blood to thyroid 
gland. 

Two models describing the tremsport of radioiodide between 
the thyroid gland and blood have been developed » Predictions of 
the models fit empirical data within experimental errors Systematic 
differences between the ability of rats and mice to concentrate 
iodide seem to be largely due to higher rates of transport of 
iodide from the thyroid to the blood in rats than in miceo 



Serial Noo NCI -905 



We have also examined the arrangement of capillaries in 
the thyroid o It had been reported in the older literature that 
each follicle had its own arteriole and venule and that the 
capillaries joining than were aasociated with just a single 
follicle o In observing blood vessels in transplants of thyroids 
in Algire's windows in skin flaps, it was noticed that the above 
observation was not obeyed: that althovigh a few large follicles 
had their own blood supply, most capillaries passed several 
follicles » Dro Merwin has systematically examined the capillary 
pattern in transplants in the mouse, and we have examined the 
capillary pattern in fixed preparations o It was found that, in 
general, altho;igh the capillaries are not associated with any 
particular follicle they are very short. The short length is an 
important factor in connection with the kinetics of iodine ac- 
cumiilation by the thyroid. 

Signifieanee to the progran of the Institute ; This work is leading 
to an understanding of the relative importance of various factors 
in controlling the accumulation of radioisotopes by tissues o 

Proposed course of project : This project will be continued with 
somewhat more eaphasis on investigation of the role of blood 
flow to the thyroid in the aecumulation of radioiodine by the 
thyroid gland o 



Part B included Yes ^ No [J 



1061 



- 5 - Serial No, NCI-905 

PHS-KIH 

Irjdividual Pz-oject Report 
Calendar Year 1958 



Part B: Honors, Awards, and Publications 

Publications other than abstraets from this project: 

Wollfflan^ S. H.. ^ K®^Ja J» So, and Reed, Po Eo-, Noneoneentration 
of thiocyanate-G by the thyroid gland of the mouse,, M. J. 
Physiol « 122 ; 85-85, 1958c 

Wollman^ So Ho., and Re©d, Fo EoS Acute effect of organic binding 
of iodine on the iodid© concentrating mechanism of the thyroid 

glando Am, Jo Physiolo l^-, 28-52, 1958. 

Wollman, So Ho, and Reed, Po Eo: Transport of radioiodide between 
the thyroid gland and blood in mice and ratso Anio Jo Physiolo 
19^; In press o 



Honors and Awards i*elating to this project: 

NonSo 



1062 



Serial No» NCI -906 
lo Laboratory of Phyaiology 
2o Cancer Physiology Section 
PHS"NIH 5, Bethesda, Mio 



Individtial Project Report 
Calendar Year I958 



Part A . 

Project Title: Properties of Transplantable Thyroid Tumors 

Prineipal Investigator; S. Ho Wollman 

Other Investigator: Dr.. Edward Bo Price 

Dro John Bo Stsnbury 

Cooperating Units : The Clinical Endocrinology Branch of HIAMD 
is studying scaae of the trcmsplantable thyroid tumors (Serial 
Koo A-2a)l)o 

Dr» John B« Stanbviry of Massachusetts General Hospital 
is collaborating in a stu^ of deiodinase activity of various 
lines of tuEiors. 

Man Years (calendar year 19^) : 
Total: 1-1/5 

Professional: I/5 

Other: 1 

Project Description: 

Objectives : To study properties of transplantable thyroid ttoaors 
in the rat: Varie'ty of histologic types and their progression^ 
correlation of histology with biocheniistry and physiology of 
the tujsors. 

Methods anployed ; Classical oethods of histology; iodine metaboliam 
was studied using radioactive Iodide. 

Major findings : A survey of histologic sections of I5 lines of 
tra33splantable lines of thyroid tumors has been madeo The 
varlely of histologic patterns is very large and includes, in 
addition to the frequently reported papillary adenocarcincaaa, 
a variety of cellxilar tumors with different types of cells and a 
variel^ of vascular patterns = One tumor line produces mucins in 
quantity o Three lines of tianors contain what appear to be host 
leucocytes digesting colloid in the luaaens of the follicles o 



1063 



- 2 - Serial Nc. NCI-906 



In one of the lines the cells are polyBsorphonuclear leukocytes 
whereas in the others macrophages sore involved o This phencaoenon 
stsggests that in these tumors the leukocytes reeognize the 
co3J.oid to "be a foreign body. In one of the lines the colloid 
no longer stains with eosin sa^ is undoubted3.y abnormal, Base- 
jaent Essmbrenes are not observed aroxund the follicles in any of 
the lines. 

The growth of all tuanors^ even the ajost anaplsistie^ ie 
decreased significantly by hypophy8eeto!ay,5 but only a f®w func- 
tional lines respond to tMouracil feeding o 

A detailed study of the nature of the iodine -containing 
compounds in one line of functional tujoors has been £aad.e by 
Bobbins, Rail said Wolff o AsBong other things they find axx 
apparent breakdovm in the specificity of the iodination process 
wil*. a significant fraction of the iodine accumulating in particles 
instead of in soluble tiiQrroglobulin. 

The abilit?y of these tumors to deiodinate aonoiodotyrosine 
in vitro has been studied with Dr., Stanburyo Tuesore which had 
lost ability to icdinate could not deiodinate o Although xsost 
tisnors which iodinatad could deiodinate, there was one functional 
line which did not deiodinate.. 

Significance to the program of the Inatitute ; These tvsmjrs are 
valuable because the variety of easily identified histologic 
aM biochaaieal characteristics <. Physiologic studies will 
give insights into the correlation of structure and function.. 

Proposed course of project ; Studies of the physiology of these 
tumors will be correlated with their histologic characteristics o 



Part B included Yes £J Ko Jx/ 



1064 



Serial Noo NC2-910 

lo Laboratory of Physiology 
PHS-NIH 2. Cancer Physiology Section 

IndividTual Project Report 5, Bethesda, Mdo 
Calendar Year 1958 

Part A , 

Project Title: Tumor Autoradiography 

Prineipal Investigator: James Co Reid 

Other Investigator: Jiiliiis White smd G, B. Mider 

Cooperating Units: None 



Man Years (ealendar year 
Total: 1/5 

Professional : I/5 
Other: 

Project Description: 

Objectives : It is desir^ to obtain inforsaation on the circulatory 
pattern in tvunors and on the stability of tuBor proteins <. 

Methods a aployed; Anixsjals bearing tuasors are dosed ^th j°adioactive 
aadno acids and after various periods of time the tuasors are re- 
laov^ mid. sliced o The slices are fixed and placed in eontact 
with x-rey film, thereby registeriiig an iioase of the distribution 
of isotope across the slice <> 

Major findings : Preparation of final autoradiograms and assmbly of 
the data have not modified the conclusions contained in the last 
report, A aaanuscript has been prepared for publication which son- 
clv^es this projecto To recapitulate the findings: 1} Uptake 
of aadno acids by a tumor occurs essentially uniformly across the 
viable tissue; 2) necrotic tissue does not take up azaino acids; 
5) a viable area which has taken up an amino acid and has then 
become necrotic holds the amino acid; k) a small tuBior which has 
fixed amino acid and then becomes larger without becc®ins necrotic 
exhibits redistribution of the sacino acid; 5) a tumor esnbedded in 
muscle fixes more amino acid than does the contiguous snuscle but 
does not produce any visible peculiarities in the distribution of 
the amino acid in the muscle a 



in- 



- 2 - Serial Moo WOI-93.0 



The overall conclission of the work is that the tuisors 
sttKiied participate to a eonsiderable extent in Schoenheimer ' s 
"dynaadc equilibrium" and henee the well-known autonojj^' of tumor 
tissue must "be due to factors other than nonHiynsmic character « 

SiiSnifie^iQe to the program of the Institute ; The question of the 
relationship between the nutrition of a tumor and that of its 
host is an iE^r'tant on® in understanding the uncontrolled 
growth of tinaorso These experiments represent one approach to 
this question o The information gained is also of interest in 
connection id th the question of how necrosis affects the over-all 
metabolisa of a tumor.. This problem is a troublesome one in the 
investigation of tusaor Gh«iistryo 

Proposed course of project ; This project has been coarpletedo 



Part B included Yes £J No ^ 



1066 



Serial Noo NCI -919 

1. Laboratory of Physiology 
2o Caneer Physiology Section 
PHS-NIH 5o Bethesda, Mdo 

Individueil Project Report 
Calendar Year I958 



Part A. 



Projeet Title: Investigation of Urinary Excretion of 

Nucleotides and Their Congeners in Leukemia 

Principal Investigator: James Co Reid 

Other Investigator: Roger Lester 

Cooperating Units: General Medicine Branch, 

NCI-4- 



Man Years (calendar year 
Total : 1 
Professional : I/5 
Other: 2/5 

Project Description: 

Objectives : The purpose of this project is to determine whether 
there is a detectable virinairy excretion of nucleotides or 
nucleosides and, if so, to develop an analytical method for 
their identification and quantitative estimation » The method 
is also to be applicable to free purines and pyrimidineso 
The technique will be used to investigate the nietabolism of 
these substances in leukemic patients » 

Methods employed : The methods used are primarily chrcanatographic , 

Major findings : Following the development of a procedure and the 
examination of a number of leiikemic and normal urines as 
recorded in the last report, the leukemic series was estendsd 
to get one or two cases covering various variables such &b 
type of therapy, effect of diet, etco The data so far provide 
some orientation as to the sort of patterns observed but are 
not yet of a sufficiently systematic character to substantiate 
any general remarks. During the course of these experiments 
it became increasingly clesir that the procedure, which was 
designed to separate nucleic acid components, did not serve 



1067 



- 2 - Serial Io» WCI-919 



to separate satis faetorily a number of additional compounds 

present in urine which appear to he of interesto In addition^ 
the borate buffer which was used for the analysis of the 
nucleoside frastion interferes with the isolation of individual 
components since the borate can be removed only by methods 
whish cause considerable cleavage of nueleosides^ It was 
therefore deemed neeessary to do some fiarther work on methodology 
and this has absorbed considerable efforts The revised procedure 
is in the final stages of cheeking and appears to be satisfactory .- 

Significaaee to the prograoi of the Institute ; The ultimate aim of 
this projeet is the aeeumulation of information eoneeming 
disturbancses of nucleic acid metabolism in leukemia and the 
mechanism of the thera,peutis aetion of pxirine and nueleotide 
analogues as well as the mechanism of their toxic side effects » 

Proposed course of pro.iect ; It is proposed to apply the perfected 
procedure to a systematic extension of the clinical datao 



Part B included Yes /x/ No ^ 



1068 



Serial No» NCI -919 



PHS~WIH 

Individual Project Report 

CELlendar Year I958 



Part B: Honors, Awards, and Publications 
Publisations other than abstracts from this projeet: 



Loo J, Ti Li, Michael, M. Eo, Garceau, Ao, and Reid, James 
6-Thiouric a©id, a metabolite of S-mercaptopurineo 
Accepted for publication in the Jo Anio Chemo Soc 



Honors and awards relating to this projeet: 
Noneo 



1069 



SeriaO. Noo NCI -920 

Ic Laboratory of Physiology 
PEB-NIH 2. Cancer Physiology Section 

Individual Project Report 3„ Bethesda, Md, 
Calendar Year I958 

Part k o 

Project Title: Identification of Compound "A-20" 

Prineipal Investigator: James C» Reid 

Other Investigators: None 

Cooperating Units: None 

Man Years (calendar year 1958): 
Total: 1-1/3 

Professional : 1/3 

Other 1 

Parojeet description: 

Ob jeetives ; Identification of what is believed to be a new amino 
acid found in rat liver = 

Methods employed : Chroxnatographic methods are used for isolation 
of the substance.. The methods of physical and organie chaniatry 
are lised to accusmilate iixfoiusation which will permit an identifi- 
eation to be m£de= 

Major findings ; After overcoming difficulties which had been causing 
failure of the isolation procedure ^ considerable time has been de- 
voted to aceumalatiDg a sufficient supply of the cou^xsund to work 
withe About one=quarter gran of pure material is on hand, and 
isolation runs are still being done to increase this supply » The 
other major activity on this project has been directed to the 
development of a procedure to synthesize the presumed structure 
by an unequivocal method for demonstration of identity « Several 
schanes have been tried but have proved unsuccessful o Alternative 
approaches have been devised, however and are being systematically 
tried o 

Significance to the program of the Institute : The substance being 
st^jdied is presumably related to protein metabolism which has 
basie importance in tumor growth. 

Proposed course of the project : It appears probable that one or 
another of the sjoithetie schemes will prove suceassful fairly 



1070 



Serial Noo NCI -920 



soon, which shotild settle the identity question » The prepara- 
tion of derivatives and determination of the optical configura- 
tion of the natural product will then conclude this project. 



Part B included yes /x7 no /~7 



iQi: 



- 5 ~ Serial NOo NCI -920 

PES-MH 
Iisdividual Project Report 
Calendar Year 1958 

P art B : Honors, Awards^ and Publications 

Publications other than abstrassts from this project: 

Toalj Jeae Nicolet^j Reid^ James = , WilliaKS, Ro Bland, and 
White, JxiLius: Effect of total -body X rsd.iation from near- 
threshold to tissue -lethal doses on smalJ. -bowel epitheliiiis 
of the rato II » Changes in nueleic eicid and protein ssra- 
thesis in relation to eelJ. divisiono Jo Hat. Cancer Inst^ 
21; 65-76, 1958» 

WollHianj, Seymour Hoj, Reid^ Jaanes C ., and Reed, Franklin E»: 

Noaeoneeatratioa of th4o©ysQaat@=€"'°^ by the thyroid gisM. 
of th@ immmo -Mo J. Pbsrsiolo Igg? 85-8^^ 19|8o 



Honors and Awards relating to this pro jest: 
Kone 



1072 



Serial Noo NCI -925 

1» Laboratoiy of Physiology 
PHS-WIH ^° Cancer Physiology Section 

Individxial Project Report ^o Bethesda, Md, 
Calendar Year 1958 



Part A. 



Project Title: Protein Synthesis and its Antagonism in 
Normal and Cancer Cells. 

Principal Investigator: Mareo Rabinovitz 

Other Investigator: None 

Cooperating Units: None 

Man Years (calendar year 



Total: 


2-1/5 


Professiona: 


1 


Other: 


1-1/5 



Project Description: 



a. A conrparison of the jaeshaaisms for the formation of protein 
in normal and csuicer cells <> 

bo An evalxxation of the structural requironents for anti- 
metabolite activity of amino acid analogues on the protein 
synthetic mechanism of mama&Lian cells, and an elucidation 
of the biochemical lesions produced = 

Co Extension of the above two objectives to the tumor-bearing 
?nouse and rat with the view of selective inhibition of protein 
synthesis in cancer cells c 

Methods employed ; 

ac Ozsanic synthetic techniques are developed for preparation 
of potential amino acid ant^oniste. 

b. Incorporation of carbon -ll<- labeled amino acids into protein 
of surviving cells is measured o Such studies involve radioactive 
eoimting techniques, chromatography, and colorimetryo 



1G73 



- 2 - Serial NOo NCI -925 

Co Subcellular fractions (nuclei, mitochondria, microsomes, 
soluble proteins) are isolated by differential centrifijgation 
from cancer and normal cells which had been treated either in 
vitro or in vivo with amino acid antagonists and C-l^f labeled 
eunino acids. The kinetics of the block in amino acid utiliza- 
tion "by these fractions are evaluated, 

do Tumor-bearing animals are placed on various dietary regi- 
mens and the effect of amino acid antagonism on tumor growth 
azid protein synthesis are determined » 

Major findings ; Thi,s program was initiated at the National 

Cancer Institute on February 17, 1958, The principal activity 
for several months thereafter was in equipping the laboratory » 

Selenitsm analogues of the sulfur amino acids were 
studied as potential antagonists of amino acid incorporation 
and protein synthesis in Ehrlich ascites tumor cells » Seleno- 
methionine was found to be a very potent methionine antagonist, 
far exceeding in activity the more consaon methionine analogue^ 
ethionine, Selenocystine blocked protein synthesis in the 
tumor cells. The nature of this inhibition is not presently 
known and will be the subject for ftirther investigation o 

Several lysine analogues were ehecked for anti -lysine 
activity in both bone marrow and Ehrlich ascites tiamor cells o 
A veiy potent lysine antagonist for both tissues , S-Cp-amino- 
-L-cysteine, was found.. 



An analogue of glutamine, S-carbamyl cysteine, was 
found to block both glutamine -C^^and leucine-C^^ incorpora- 
tion into Ehrlieh ascites tumor protein. A unique characterise 
tic of the antagonist vas that it was more effective against 
higher concentrations of glutamine -C-'-^o The cause of this 
phenomenon is being investigated. 



1074 



Serial No= KCI-925 



Significance to the progrean of the Institute ; These steadies 

utilize the action of amino aicid stntiaietabolites aa a means of 
evaluating the conrparative protein anabolism of normal and 
eancer cells » In addition, structure -activity relationship 
for producing antagonism and studies on mechanism of inhibi- 
tion, both in vitro and in vivo , should furnish basic data of 
potentiEil use for cancer ehemo therapy. 

Proposed course of project ; All aspects of this project will be 
continued o 



Part B included Yes [J No ^ 



1075 



FHS-N2H 

Individual Project Report 

Calendar Year I958 



Serial No. NCI-91I 

lo Laboratory of Physiology 

2. Energy Metabolism Section 

3. Bethesda, Md<. 



Petrt Ac 



Project Title: Total Metabolism 

Principal Investigator: A.. W« Pratt 

Other Investigators: Wo Co White, F. Ko Putney, and Bo So Burr 



National Naval Research Institute {in that 
portion of work i>ert£dning to Indirect 
C alorimetry-- Human ) 



Cooperating Units; 



Man Years (calendar year 
Total: 5-2/3 

Professional : 3-1/3 
Other: 2-1/3 

Project Description: 

Objectives ; The study of the total metabolism and particularly the 
total energy metabolism of normal and tumor-bearing animals » 

Methods ^nployed ; The sttidies utilize, in addition to routine chsaa- 
ical and physical detexaiinations , the techniques of analytical 
respircsnetry as developed in this laboratory (Serial No<. NCI -911. 

1957) c 

Major findings ; 

Ao Enei^y Metabolism and Body Composition 

Wide variations in gross carcass composition have been noted 
among rats e5g)eriencing a similar (suaount of) growth of tumor of 
almost constant ccmpositiono We have observed significajit cbajiges 
in the total energy economy of the tumor-bearing smimal during the 
tumor growth period « 

Energy metabolism studies meeisure growth in terms of accretions 
of water, nitrogen, calories and change in an animal's total masso 



1076 



Seriaa Noo KCI-911 



It has not been possible to intearpret the energy metabolism 
data as changes in body comjxjsition because of the inability to 
partition the calorie and nitrogen values in terms of specific 
tissue ehangesu Therefore, a study of body composition in terms 
of water > nitrogen and calorie content in normal animals was 
undertaken. 

The initial investigation was done on a series of Sprague- 
Dswley rats fed a semi -synthetic 20 percent casein diet^ sacri- 
fieed in groups of four at 150 gms.j 200 gmso, 250 gmso, and 
500 gmso of live body weight. In addition to the determination 
of water, nitrogen and ceLLories, the carcasses were quantitatively 
separated by fat solvents into dry fat and non-fat fractions 
which were also analyzed for nitrogen eaid cailorieso 

Water content of the total carcasses varied widely ^noag the 
animals; expressed as percent of fat-free weight, the vetLues were 
much more consistent within the groups but showing a tendency to 
a lesser hydration of the fat-free mass with greater total body 
weight o 

The nitrogen and calorie content of the dry, total carcasses 
varied widely among adl the animals reflecting^ of course, the 
variations in the proportions of fat and non-fat in the dry, total 
GEarcasseSo The non-fat fractions from all the animals were highly 
\miform in nitrogen and calorie content; the v^ues were 13° 7 ±0,2 
percent and k,>6^ + 0,04 Kcal/gmo respectively. The fat fractions 
were similarly uniform containing 9^28 + OoOU^ Kcal/gm, and less 
than 0.,15 percent nitrogen, 

A number of nitrogen-calorie relationships have been assumed 
and examined. The awst satisfactory relationship assumes that the 
total dry carceisses of the animal can be considered as a simple 
mixture of fat and non-fat. An assumed straight line function can 
be established by connecting, on a plot of m@n, N/gm, vso Kcsl/g.^ 
the point at 137 rngm, N, lt65 Kcal {non-fat} with the point 1,5 saga N, 
9,28 Kcal (fat) 

It follows from our assumption that anedytical values for nitro- 
gen and calorie content per gram of dry, totsil carceiss should give 
points which fall along the asaxsaed line of average body eonqpositionc 
In addition, the position! of any point on the line should direct3.y 
reflect the proportions of f«t and non-fat in th« individual carcass c 
Both of the predictions vers verified by the data obtained from this 
series of uiimals. 



1077 



- 5 - Serial Noo NCI-9II 

In order to examine further the validity of this theoretical 
line of hody composition, the dry ©aroasses of rats fed a variety 
of diets such as chow or aemi -synthetic diets with dried tumor sub- 
stituted for casein, were analyzed for nitrogen and calorie content a 
The show fed animals were generally high in nitrogen and low in 
calories while the tumor diet animals were extremely varied but the 
points for all of the animals fell on or close to the line^, indicating 
that the nitrogen-calorie function as index of gross "body conqposition 
may be applied to any normal rat receiving an adequate ration o 

Since the line applies to rats weighing from I5O-500 gmso, the 
incraaents of dry weight gained in this range should have the same 
type of composition as that of the total diy carcasses « From the 
energy metabolism data, it is possible to compute the nitrogen- 
calorie content per gram of dry weight gained by the living animal 
during a given es^erimental period. Limited studies coarpleted show 
that calculation of the nitrogen-calorie content per gram of carcass 
weight gained by the living animal over four-day study periods gives 
points which fall in excellent relationship to the theoretical line 
of average body coHipositiono 

Bo Instrumentation-Research Bevelopment 

a) Small animal direct calorimetry; 

Our efforts to complete the small -animal direst calorimeter 
capable of measuring total heat production have been plagued with 
numerous, small problems » The sensitivity of the heat-meaeuring 
thermistor bridge, necessary for the accurate measure of total heat 
production^ has required that considerable time be spent in an effort 
to buffer the system against the small but rapid fluctuations in 
rooan t«nperat«re= Slow changes in room tenrperature can be sensed 
and adequately compensated for, thus maintaining the adiabatic 
operating conditiono Svidden changes of 2° F cannot be compensated 
for and due to the differing heat capacities of the cos^ionents of 
the systsa, shift the zero balance thus destroying the previously 
established adiabatic operating conditions o On some occasions these 
variations have been severe enough to vary the measured heat recovery 
from 97-105 percent of the measured heat input ■> 

An accuracy of 1 percent is the desired objective aM while 
a number of such measiirements have been made, this accuracy is not 
obtainable routinely over the necessarily prolonged periods which 
will be required for the animal study,, The slow but definite 
progress made to date iMicates that a satisfactory instriLment 
capable of accurately measuring the absolute heat pzxjduction of the 
animal over continuous 48-hour experimental periods can be obtained « 
In short term studies (less than 8 hours), the desired accuracy can 
be achieved routinely » 



1078 



- h - Serial No, ura-911 

It has been nesesssuiy to develop a eooipatible trEsisducer to 
measttre relative hmaidity in order that greater sensitivity and ae- 
e^iracy of the sneasurement of ealorie loss via heat off vaporization 
he achieved « This will allov at any timej a simultaneous comparison 
of heat piroduction measured directly and indirectlyo (O2 eonsuEip- 
tion) c 

Several preliminary nms showed that reduction of the i^w data 
obtained during one day's stwdy would r^tuire 5-6 man days and eould 
seriously limit the use of the calorixaetero We have spent eonsider- 
able tiiae making the total system compatible with direst aceess to 
the laB«M<, 650 ©omputero A first run using manually eoded data in- 
dicates that we have eonstrueted a satisfaetory scssnputer programo 
Delivery of a solenoid -aetivated add-p«neh maehine will give us 
direet eoo^xater aceess via paper tape and r®iuce data handling 
time frcm 6 man d^s to about ^ hoxirse 

b) Human Analytical RespirCTieter : 

The eollaborative work with the Bioenergetics Group at HJoKoR.Io 
hEis been de3.ay©d beeause of breakdown in one of the plate meters of 
the hvanan direct calorimeter. We have used this time to determine 
the absolute sensitivity of our analytical respirometer, as it has 
been modified for human worko 

We have noted previously that the aiaalytieal respirt^eter ap- 
peaired to be ssi order of magnitude more sensitive than the aneilytieal 
mass speetrcmeter in the measure of small differences of oxygen and 
carbon dioxide in respired air samples » A new procedure has been 
worked out whereby each fraction (i.e.. O2, SOa* Ng) added in the 
preparation of gas samples is weighed to an accuracy of ± OoOOOl gms<, 
Since the weight of each fraction of the final gas sample is known 
accurately the cosposition of the gas can be known accurately,, The 
results show that differences of 0=02 percent oxygen and sarbon 
dioxide can be accurately determined o 

Significance to the program of the Institute ; S-tesdies of the total 
energar metabolism offers the single precise means of determining an 
animal's general astabolic response to disease o Interpretation of the 
toted energy metabolism data as a change in the physiologic state of 
the animal will add iiaiieasurably to the definition of the disease state. 

Proposed course of project ; To continue the evaluation of nitrogen- 

calorie relationships in relation to body coo^osltion and total energy 
metebolisa in the norm^^ ai^ 'kooor-bearing animal o 

Part B ineluded Yes /x/ Ko /j' 



1079 



5 - Serial Noo NGI-9II 



PHS-NIH 
[ndividual Project Report 
Calendar Year I958 



Part B ; Honors, Awards, and Publications 

Publications other than abstracts from this project: 

Pratt ^ Ao Wo: A stoaH-anixael analytical respirometero Jo Kat, 
Cancer Insto 20: I6I-I72, I9580 

Pratt ^ Ac V7«, and Putney, Fo Ko : Observations on the energy 
metabolism of rats receiving Walker tiBiKjr 256 transplants o 
Jo Nat. Cancer Insto ^: 175-187, I9580 



Honors and awards relating to this project: 
Noneo 



1080 



Serial Noo NCI -91'* 

lo Laboratory of Physiology 
2o Physical Biology Section 
PHS-KH 5" Bethesda, Md. 



Individual Project Report 
Calendar Year 1958 



Part A . 

Projeet Title: In Vivo Measvirement of Ttanor pH with 

the Capillary Glass Electrode 

Principal Investigator: Ho Kahler 

Other Investigator: None 

Cooperating Units: None 

Man Years (calendar year 1958) : 
Total: 1-2/5 

Professional: l/3 
Other: l»l/5 

Project Description: 

Objectives ; The object of this project is to determine the in vivo 
pH of tmnors lander different experimental conditions c 

Methods employed ; Fine pointed glass electrodes were inserted into 
tumors in the leg mussl© of rats and the pH recorded for several 
hours At the end of the first hour, glucose was injected 
intraperitoneallyo At autopsy, the position of each electrode 
was determined with especial attention given to proximity to 
blood vessels, dry or necrotic tissue and distance to the 
boundary of the tujnoro 

Major findings : Capillary electrodes inserted into different sites 
in a tumor show large variations in readings « This variation 
seems to be associated with the position of the electrode with 
respect to the blood vessels of the twmor, determined by dis- 
section of the tumor at the end of an ejqperimento 

Glucose concentrations in blood and tumors have been 
measured o In noimal states where blood glucose is about Od 
percent the glucose in tuioors is very low as first pointed 
out by Warburg o In hyperglycemia, following intraperitoneal 
injection of large amounts of glucose, the glucose in tumors 
is greater than can be accounted for by residual blood in the 



1081 



2 - Serial Wo» NCI -91^ 



The relation between amounts of glucose injected and pH drop 
in the tumor as measured by the capillary electrode has been in- 
vestigated o Increasing amounts of glucose produce an increased pH 
drop until a saturation state is reached o 

Fluid injected into the peritoneum and containing or lacking 
small ions and molecules such as Na , glucose eind lactic acid 
reaches an osmotic eq,uilibrium with the blood values for these 
same materials » On the other hand the larger molecule albmain after 
5 hours diffuses from the blood into a solution in the peritoneum 
(originally albumin free) only to the extent of 10 percent of the 
blood value => 

Signifieance to the program of the Institute ; The pH of tisnor tissue 
is essentieJ. to an imders tending of chemical processes in the tumor ^ 

Proposed course of pro jest : The basic difficvilty in this work is the 
variation in electrode readings which negates correlating pH read- 
ings with other tmnor paremeters. We proiKsse to select those elec" 
trodes which pass rigorous tests for ability to follow pH changes 
of poorly bxiffered materials in an effort to get laore coasistant 
readinigSo 



Part B ineludgd no fZf yes /~7 



1082 



Serial No. NCI -915 

1. Laboratory of Physiology 
2o Physical Biology Section 
PHS^HIH 5o Bethesda, Md» 



Individual Project Report 
Calendar Year I958 

Part A . 

Project Title: Cellular Particulates 

Principal Investigator: Ho Kahler 

Other Investigator: Bolivar Jo Lloyd, Jr.. 

Cooperating Units: Laboratory of Infectious Diseases, 

National Institute of Allergy & 
Infectious Diseases - Dr. Wc Rowe 
and Janet W, Hartley, (T'rojeet serial 
NOc is not available) 
Man Years (calendar year I958) : 
Total: 1-2/5 

Professional : 2/5 
Other: 1 

Project Description: 

Ob.leetives : Electron microscopy of mouse polyoma virus » 

Methods employed : The material from tissue eultwre was partially 

purified by various methods. The partially -purified eamplss were 
examined in the electron microscope and after identification of a 
characteristic particle, these particles in aliqtuots were sedi- 
mented onto speciasen grids mounted in an ultracentrifuge cell^ 
The grids were then removed and the particles counted on electron 
micrographs o Hemagglutination titers were made of the same 
materials for which particle counts had been determined. 

Ma,ior findings ; The size of the desiccated virus particle when in the 
spherical shape is about kh mn. Virus from two tissue culture 
strains, one cultured from a parotid gland tumor and the other 
from a mesenteric lymph node tumor, appeared identical » Sedimen- 
tation experiments indicate that the particles are somewhat larger 
in solution. Particle counts from the electron micrographs were 
parallel to the hemagglutination titer. The number of particles 
was 10* times the hemagglutination titer. The end point of 
hemagglutination corresponded to the virus dilution in which the 
ratio of virus particles to red blood cells was unity. The 
uniformity of virus size is consistent with the concept of a 
single virus but morphology alone is an insufficient basis for 
such a conclusion o 



1083 



- 2 - Serieil No.. NC2-915 



Slgaificanee to the program of the Institute ; Provides additional 
evidence for the virus concept of soiaB cancers o 

Propoaed course of etudy ; Continuation of the aame type of work on 
other virus strains is indicated as well as further detailed 
stiady of the same virus o 



Part B incliKied Yes [J No (^ 



1084 



Serial Noo NCI -916 

1. Laboratory of Piaysiology 

2. Physical Biology Section 
PiS-WIH 5, Bethesda, Mdo 



Individual Project Report 
Calendar Year 1958 



Part A . 

Project Title: Characterization and Comparison of Nucleic Acids ^ 
Nucleoproteins , nucleases^ Nuclease Inhibitors of 
Koraial and Malignant Tissues and of Body Fluids 

Principal Investigator: Joseph Shack 

Other Investigator: None 

Cooperating Ifoits: None 

Man Years (calendar year 1958) : 
Total: 1-1/2 

Professional : l/2 
Other: 1 

Project Description: 

Objectives ; 

a) To determine whether the nucleic acids and nucleoproteins 
of normal and malignant tissues differ in physical^ chemical 
or biological properties » 

b) To determine the nature and amoxmt of nucleases and nuclease 
inhibitors present in tissues and body fluids. 

Mettods employed : 

a) Procedures for isolation of nucleic acids and nucleoproteins; 
controlled modification by heat, acid, alkali, enzymes, etSoj 
study of these by various chemical and physico-chemical ^roce&areB. 

b) Various procedures for preparation of extracts of tissues and 
body fluids suitable for assay of enzyme or enzyme inhibitor c 



1085 



2 - Seriea Noo mi-9l6 



Ma.lor findings ; 

Fart a e Besause of what we considered to be an erroneous report 
that reeently appeared^ a further Investigation was made of "the 
binding of Mg by DM» Earlier studies in this project were re- 
ported in a series of papers in 1951-1952» The results given 
below confirmed and extended this earlier work: 

a) In salt-free solutions^ added B«g is tightly bound by 
either native or deziatured DNA» 

b) The binding by native DNA is tigh-fcer than by denatured 

c) High eoneentrations of sodivna effectively coErpete 
with low concentrations of !fg for the binding sites » 
The binding of both univalent and bivalent ions by 
either native or denatured DM is electrostatic and 
does not involve chelation » 

Part b . The principal DHase inhibitor present in human blood cells 
is stable at too whereas th© soluble senm inhibitor is destroyed 
at that taaperatvireo It was found that this lieat lability is un- 
doubtedly an artifact which results from presence in serum of a 
labilizing factor, possibly an ensyu^o The stable inhibitor couid 
be rendered labile by the presence of serussc 

At certain pH values, both above Kid below neutrality^ 
the DNase inhibitor is more labile than DKaseo This pi'ovides the 
. basis for some methods which have been developed for differentiaHjr 
destroying the inhibitor component of enzyme-inliibitor mixtures and 
thus obtaining measure of the amoimt of each present. 

Significance to the progrsaa of the Institute ; . Interest in nucleic 
acids and nucleoproteins in connection with the cancer problem 
stems frcan key role of these substances in genetic processes, 
bacterial and viral transformations^ virus reproduction, protein 
synthesis o Interest in nuclease and nuclease inhibitors relates 
to the necessity of controlling DKase action during isolation of 
MA, particularly if taiey are to be tested for biological activity. 
In addition 9 it is possible that the amount and nature of the eazymss 
and enzyme inhibitors may be in Lome way related to the growth of 
tumors present in the hosto Knowledge of tfe binding of ions by 
nucleic acids is essential to understanding of the relationships 



1086 



>n BrancV) 
Budget Data 



Estimated Obligations « 

Direct <.0000..0<,0.80<! 

Reimbursements 

Cliaicalc.o,..,. 
Other. . o . » , <■ o e , , . c 



Total 



1_/ Includes Projects No's: 

OFFICE OF THE CHIEF: 
688 

RADIATION BIOLOGY SECTION; 



641 


675 


642 


676 


643 


660. 


644 


682 


645 


683 


659 


684 


671 


685 


672 


686 


673 


687 


674 




RADIATION HIXSICS SECTION 


624 


677 


628 


678 


629 


680 


637 




RADIATION THERAPY SERVICE 


^0"C 


663-c 


651-c 


668»c 


654-c 


669-c 


656»c 


670-c 


66o=-c 


679-c 


661-c 


689 


662=c 





.Fiscal Year 1959 
^ 423,300 




1092 



Serial No, Igl;688 
lo Radiation Branoh 
raS.=NIH 2o Office of the Chief 

Indi^dual Projeet Report 3o Bethesda, Mdo 

Calendar Teas' 19^ 



Frojeet Titles 

Developsnentg Fatirication and Maintenance of Specialized 
Equipment for Radiation Research 

Rrineipal Investigators 

Calvin R» Menclcaa 
Olher Investigators 

Edward Jo LaBrun and Ralph Co Whittakar 
Cooperating Units s 



Man Years (calendar year 1958) 
Totals 3 
Professionals 2 
Others i 

Pl-oject Descriptions 

Ob,-^eeti7Bas To design and fabricate laboratory iaistruinBnts 
and devices that are not available on tiie open market., and/ 
or incorporate refinements to said devices to make them 
applicable for the unusual services that are required in 
radiation research. To service or assist the investigators 
in servicing sudi shipment as the %n de Graaff generators 
and fabricate special parts for same, as requested by said 
iaveatigatarso To service less si^iifieant equipiasat as 
rdquBstedo The aeccxiT^mying list of projeets is coadsnsed 
ea&d does not give a et»iin:lete or wholly accurate description 
of e'^ry task invdvedo The inore significant oises will be 
designated "bgr an. asterisk («)« tf more eoioplete and aeeurats 
inform tion is desired^ it is SQggssted Ihat the investigator 
^ose name aeeompanys Ihe item 'be eoaemltedo 

It will be noted ihat a pasrt of the liat is la^solvlag 
Glass Bloxjingo This vd.ll be listed separately and ^lill be 
dosigaated as sudi« 



1093 



Serial Hbo NCI=488 



Fabrieate icasiaation eSuaiabers for 3 MeVo 

R^lae© bcagdags in 3 MeVo 

Fabricate eoimlinE^ f eg* power supply generators in 3 

Males TOO voltage gpaeers fos? 3 IfeVo 

Glass Blowiags 

Fateicato elecbrode aasembly fa* radiation es^oerisents 

associated with 3 Ife'^o 

Fabricate ^asa traps for 3 PfeVo 

Dr. Herman Stilts 

4i Fabrieate radiation seed gim, also reiaote loading devise 
and related aeeessorioSa 

Fabrieate hollow plastic spheres ef varicua sixes ;^ also 
dosage ooatainers of other assearted ^apeso 

Develope and fabricate IIK I and M H radiation thera^ needles^ 
Fabricate radiatLon iTedges of various materials and designso 
Glass Blmdngs 

^-Fabricate glsss sph^es and holders for ssuna fGS° 
radiation dosage estperincntso 

Fabrieate spseisl cylinder, with burette aadlng and 
ground glass s topper « 

X^o La^jrenec Stiila^ters 

Pevelope and build ptmch to aalce various shape holes for 
identification purposes o 

!fro Robert Swains 

IMake aid modify probe type ionization diambersu 
Refineoiaats to lateral feed of iSO scanner aleo service 

to saroso _jB 

Replace elevation screw in # 1 =» 2 IfeVo 

Fabslcate parts ss requested for low voltaj^e X-ray attadussent 

for 3 MsVa 

Build pin hole caiaera and associated parts for 3 5fe"?« 

Ibtf Robert Bases s 

Adapt incubator wito special controls, temperature indicator 
and 00a «^ flushing system* 

Pro ItortiieBr EUdnds 

Hu2nldi-ty chanbers for micro nsmlpuiationa 

Motor driven tumtsHe for unif oniiljr esqposlng cellular 

suspeasiom to ultra violet lighto 

Precision fixture for adjusting the microipso for the ortholuxd 

Fiactures for maintalniag optiinuin en"^dr<»incsital conditions for 

mammilian cells <Mring U»Vo and £°rsy exFxjsurSo 

Baveiope tools to imke speeial r?la3@ petsd pXateSo 

' . 1094 



Serial No, NGI=688 



Projset Deseription? (Continued) 

Glass Blowings 

^De'ffelops and fabricate speeial petri plates so 

designed to ndnind.ze shadow C3f fects in ultra violet 

experiEientSo 
■ss-Fabricate sjeclal tissue ctilture tubes wiiii 

optically flat sides o 

Dyo Ovaries Maxwells 

Fabricats accessories for chromatonraidiic column^ 
Glass Blowings 

Rebuild double water stillo 

Fala^icate e'&or extraction apparatuSo 

Dto Falconer Smiths 
(a.ass Blowings 

^■Fabricato glass canulae oOlO" OoDo 

Joanne Holl crofts 
Glass Blovdngs 

PabcicatQ gas collecting systemo 



Dto Jo ^i)hiteg , . ^^ 

De-rolope snd construct reuote control adjustable platform l 

dissecting microscope o 

DPo Pratts 

Construct metabolism cages o 

Fabricate plastic dehuiaidifierSo 

Develop and fabricate theraial conductivity cell block with 

double li^-passo - 

Glass Blessings 

Fabricate distillation assemblies o 
Fabricate glass carbon train assemblies <. 

DTo Jaass Reids 

Build lares capacity lyophiliaero 

Mr« Bernard Burrs 
Glass Blowings 

Fabricate gas collecting apparatus. 

Modify and repair glass traps for mass spactroirstero 

Ifro Alvin Go McNiahs 
Glass BloxdJigs 

Fateieate gas conversimi apparatus o 

Fabricate gas sair/pling tubes for use wit^ mass 

spectroKSBtero 

Part Bo ineludeds les ZZ7 Ho ,^/ 

1035 



PHS^NIH 

Individml Project Report 

CalencJar Year 19^8 



Serial No, NCI^h'l , 
lo Radiation BrancIT 
2o Rado Biolo Secto 
3<, Bethesda^ Mdc 



Jfert A, 



Rpoject Title: 

Th0 Effect of Irradiation on Susceptibility of Mica to ¥tra.l 
Infections and Changes in Vi2;nilsnce of Viral Strains After 
Ffeissage ITirough Irradiated Hosts 

I^ineipal Investigators j 

Willie Wo Smiths PhoDo and Bsmice Eddy (LoBoCo) 
Other Investigators J 

IIo Mo Aldersnan and Ruth Ec Gillespie 
Goop85:^ting Units? 

Hone 

Man Years (calendar year 19^8) s 
Totals 2/3 
Professionals 1/3 
Other; 1/3 

Project Descriptions 

Objectives ; To determins whether or not exposure t@ x=radiation 
alters susceptibility to various viral infections and whether or 
not the virulence of viral strains is alt^ed by jnssag© throiigh 
irradiated hosts o 

Methods Bnplqyed; The response to challenge with poliQajgrelitis 
or influenaa virus in Biice given just subletlml irradiation is 
cosspared with that of controls and the virulence of relatively 
resistant strains of tnfluensa virus is tested aft©r passage 
through sublethally irradiated jnice as ccanpared to control nd.oe.. 



1096 



Serial NOo WCI=61il 



Project Deseription: (Gontinuad) 

Kajgg Findings ! The susceptibility of nsiee to challenge with polio 
virus was not significantly increased by just s\xblethal irradiationo 
FcLSsage of a non«adapfced mouse strain of influenza virus through sub= 
lethaUy irradiated mice increased in virulence o In general^ the 
differences in susceptibility of irradiated and control raice to the 
viruses tested were not pronouncedo 

Significance to Cancer Research ? Sine© generalized radiation damag© 
aiay be encountered as a side effect of cancer therapy it is obviously 
important to understand alterations in susceptibility to infectionj, 
both bacterial and viral » Results of these escperiments ar© also of 
interest in connection with the use of viruses as therapeutic agent s.- 

This project has been completed .^^ 



Part B included Yes ^J No ^J 



109T 



Serial Noo 101^61 

lo Radiation Branch 
2o Rado Biolo Sect. 
3o Bethesda, Mdo 
HIS=W3H 
Indi vidua! Ps-ojeet Report 
Calendar Year 1958 



Part Ae 

Projeet Titles 

Antibodty Induction in Irradiated Mces Effect of CJbronieg 
and Intermittent Exposure to Ionizing Radiation and Fractional 
Body Shielding cei Hemolysin Rroduction 

I¥incipal In"^3tigators 

Falconer Smith, RioDo 
Other Investigatoras 

Marie Mo Grenan and Kristin Lunde 
Cooperating Units? 

None 

Man Tears (calendar year 1958)? 
Totals 5/6 
Professionals J/% 
Otl^rs 1/2 

Project Descriptions 

O bieetiY eas To determine ihe nature of the spacifie action of 
ionizing radiation on antilxx^ forming tissues in nice and ratso 

:)loyeds In eurrent e^qjeriiisnts mice are a) exposed 



to ionizing radiation in divided total doses, immunised and 
the sertrni hemolysin titer estimatedi b) in another series of 
espsriraents the contributicn of the non~splenic tissue toward 
antibody formation is observed in irradiated, splenectomiaed 
mice and rats© 

Ma.lor Findings s a) Rate of recoirery of ihe immune response 
after paired exposures to X-rays is directly correlated vjith 
the time elapsing betv/een Ihe IntEvidual exposures and 12ie 
amount of l^e individual expo sure o 



1098 



NCI»-61i2 



Project Descriptions (Continued) 



b) ^a amount of hemolysin formed by adult mice is 
directly correlated -with the amount of splesn remaining after 
surgical removal in infancy o Studies of irramme response in 
iiradiated splenectomizsd mice indicates that they majr have 
a sli^tly lower threshold for radiation injury than intact 
mice« fferaascripts describing ihe major finding for Ihe spleen 
study and the di-^ded dose stuqy are in p3r©parationo 

Sigiificance to Cancer Researdis Studies clarifying liis nature 
of injury to protein synthesis are needed to deal intelligenlily 
with the use of radiation as a therapeutic toolo 

I^g'oposed CoiSTse of Pro.ieets Biese stadies are now in the process 
of being reported on in manuscript form. Additional studies 
involving cte'onic and low level radiation are being undertakeno 



Part B included Yes ^^ Ko f^ 



1099 



(Attachnssnt I) 
Serial No„ NCI^6h2 



IfcicHvidual Project Report 
Calendar Tear 19^8 



Pa^t_Bt_ Honors J Awards, and PublicationB 



A preliminary reT5ort of %he spleen studies was made in an 
invited paper at t!^ International Congress for Mcrobiologgr 
held at Stockholm August ks, 19^8, 



1100 



Serial No„ NCMa__ 
lo Radiation Branch 
H!S"KIH 2o Rad= Biol» Secto 

Indiid-dual Project Heport 3o Belihesdaj Mdo 

CalQiidar Tear 1958 



Projeet Titles 

Hodificatic33i of Long Term Suridval and Ttnnos' Incidence 
Following Chronic Radiation Ijy Bone Marrow Injection 

PrineipLe IhvQstigatoys 

Joanne Hollerof t and Charles G.o Congdon 
Omar Sii^esti gators s 

Bnjos W®ad and Marion Matthews 
Coopsrating Units? 

Oak Ridge Natiaaal Laboratosy 

mn Years s (eaLeadas? yoas? 19^8) 
Totals 2/3 
Professionals 1/3 
OlSiers 1/3 

Project Descriptions 

^h0e|j^ss To studly alterations in Img term effects 
of chronic irradiation by hone marrow injection o 

Jfe^ods ^adoyed* Strain 2 guinea pips were exposed to 8o8r 
^^ITo^ dS^mtil their hematocrits fell to 25» Upon 
removal from the radiation field half the pigs were givea 
iatra-wanovss injection®*^ isologsms bone mazrowo 

l&JorF^^Sii.' ®one luarrow injections increased the 
Sber^SFaitols survi^rtng raore iha^n 60 days after resae^sal 
ftoin the fields and significaniay increased m© sur^TOl 
tiise of Hial© guinea pigSo 

ice to Cancer Re sjasg:^ ? Factors influencing 
BHiopenesiSo 

poposQd CoxtrsQ of Projectt Completion of pathology on 
lese gsainea pigSo 



Part B ineludads les Z^ ^'^® ^^ 1101 



Serial Ko„ mi^6hh 
lo Radiation Brandi 
FHS=.NIH 2o Rado Biolo Secto 

Individiial Project Report 3o Bathesda, Mdo 

Calendar Tear 1958 



■P art A q 

Project Titles 

Sensitization of Tumors to Radiation 
Rpineiple In-restigators 

Joanne Holl croft 
Other In'^stigatorss 

Marion Matthews and Bruce VJeed 
Cooperating Units s 



Kan Years (calendar year 1958)? 
Itotalx 2/3 
Professionals 1/3 
0-&@rg 1/3 

Project Description? 

Ob.leeti'geBg To increase sensitivity to radiation 

Ifethods Eiiiployedi 1) Mice bearing transplanted tuinors >iere 
treated with cortisone prior to or following radiation of 
the tumor or whole anmaL, 2) Transplanted tamors were 
ezposed to radiation while mice -mrQ in different atmospiieres 
of oy^f^Tia TuBjor regression, survival and bioassay studies 
were farformedo 

Major Findings s Cortiaone produced a markBd regression of 
L .^^ 1 lymphosarcoma and 70I429 plasma cell sareomao felisn 
cosiibined TJith radiation the regression was additiifBo Althou^ 
a Hbffosarcoma regressed aLl^^y following administration of 
cortisone ihis regression coriLd be accounted for in ger^ral 
vjelg^t loss of the raiceo Ho additi-^ effect on the tumor was 
seen lihen combined therapy x*as emplcyed. Cortisone did not 
extend survival tirce of ndce bearing P388 or L<#2 lyjnpho sarcoma 
treated with viiole bod:^^ radiation o Bioassay of the 7Qh29 tmoor 
indicated a direct effect of cortisone on the ceUso 



1102 



Serial Noo NCL26ij^ 



Inject Description: (Continued) 



Regression of L^l folloxdnn local radiation increased ^Ath 
increaslxkg pressiire of oatyp^ono ^%en very lar{»e L^l tumors 
were used the 03^f?en effect was not seen, A radioresistant stitlinQ 
of L ^TL did not show an oxygen effect but these were irradiated 
ti^en largSc Plasma cell sarajma 70U29 did not show increased 
regression ivlth increased oxygeno However., an Qjq^gen sensitization 
of the ascitic forms of both L =#1 and 70li29 was seen v*ien a 
bioassay of treated cells was donoo 

Significance to Cancer Re search ? Radiation Iherapy of ttaisorSo 

-St:gj ?^gd „ Course of Pro.lects Mare systematic investigation of 
ttatasr size and response to ojygeno 



Part B included lea [^ Ho /jj 



1103 



Serial Noo NGI..6if^ 
lo Radiation Branch 
H©~NDI 2„ Rad„ Biolo Sact,> 

Individual Projeot Report 3o Bethesda, Mdo 
Calendar Tear 1958 



Psgt A o 

Project Tita.es 

Effeet of Pretreatinent with AET on Radiation»Induced Tusnop 
Regression 

Prineipie in'VBsti gator: 

Joanne Holleroft 
Other Investi gators s 

Maricffi Matthews sacid Braes VJeed 

Cooperating ItaitsJ 

None 

Man Tears (©alendar year 1908) s 
Totals 2/3 
Professional? 1/3 
Others 1/3 

Project Descfripticns 

Objectives s To compare the proteetivo effect of AET on 
several tumors with ihe protective effect of AET in the 
host >*ien given prior to irradiation.. 

j fethods Emplgireds 1) Mice bearing transplanted tisHors vsere 
irracftated with or without pretreatment with AETo Effeet of 
AET treatiaent en tujBor regression or on survival tiBie was 
compared with effect of AET on decreasing radiation lathali-tyo 

2) Tmcors were treated locally v;ith radiation following 
injection of AET or saline and tua©r regression was followed* 

3) Mice bearing ascitis tumors were irradiated with or 
without preta:eatii©nt vilih AET said cells were assayed for 
viability ty subsequent transfer into untreated hostso 



1104 



Sespial Nou NCI=6iiS 

Project Dsscriptiosis (Continued) 

tfei; ^or Findings^ As xaeasurod try the redietion in tuaor MoltMoo 
folloxidnf: liiole bO(fy irradiation^ protection of L #1 lympho* 
sareoaa "ti^ kT.'^ was ne^igible^ but protection of 70^29 plaama 
cell sarcoma was equivalent to protection of "Hiis cheinical 
against 30 day lethalitjTo The amount of protection afforded 
Kij lyiaphosareoma was intannsdiatoo No protection was seen folloiJiag 
local irradiataoa of these ttimea^Sa Equivalent killing doses of 
mice bearing retiajlaiiQ cell aareraaa, H6867o Mice bearing P388 
lyKphesareoma protected 1y boio nsTroTj f oUox-dng x-^ole bod^ 
radiation sur'si'ced Ion per ihssa mice protected by AET vdiea 
treatment as giteri late in ihe course of ihe diseasoo (3) 
Bioassay of L.^ 1 asedtic cells and P388 ascitic cells shox;Gd that 
these cells were protected by AETo 

Signi ficanee to Can^r Research t Applieability of protecti-^ 
dietnicals to whole body radiation theraETo 

Proposed . CpTjrsQ , of Pro.ieet* (batimaation of bioassay of these tuBiors 
and tracer studies of uptake of AET "h^ tannorso 



Part B inslttdeds Yes ^CZ ^^ J^^ 



1105 



PHS=NIH 

Individual Krojeet Raporfc 

Calendar Year 1958 



Serial NOo NCI=659 
lo RadiatISti"BraniK 
2o Rado Biolo Sect, 
3o Bethesda^ M„ 



Part A< 



I^oject Titles 

Aetion of Eiidotoxin in Irradiatad Aniualsj Reduced Morfcalityi 
Increased Resistance to Infectioni Advanced Hematopoietie 
Recovery 

ft'inciial Investigator; 

Willie Wo Smithj PhoDo 
Other Investigators: 

Ho Mo Aldertnan and Ruth Eo Gillespie 
Cooperating Units: 

Non© 

Man Years (calendar year 1958): 
Total: 2/3 
i^ofesslonal: 
Others 2/3 

Project Description: 

^jectivea ; To dotermne the effects of bacterial endotesacins 
on the irradiated animal and to discover the physiological 
changes responsible for such effects o 

Methods BmplcBred ; Ekidotoxins are injected before or after 
irxadiation and effects noted on: (a) mortality j (b) resistance 
to experiinantal infectioni (c) hsHjatopoietic recoveryo 

Maj or Findings : The stuc^ of hemopoietic recovery Has 
extended to include a ccaparison between "recovery times" 
for lymj^iocytes^ granulocytes ^ heaioglobin concentration and 
platelets in mice recovering spontaneously^ after the injection 
®f isologous or ?ieterologous bone jnarrow and after pretreatment 
with colchicine^ carbon cr endofeoscinr. Significantly different 
patterns of recovery ware related to the different types of 
treatment o 



1106 



Serial NOo NGI°6$9 



Project Deseriptions (Continued) 

SlgnificancQ to Cancer Research ; The advance in recovery of 
bone m^rrcw cellularity following endotoxin treatment is of 
basic isnposrtanc® in cancer reaearcho Induction of recovery in 
the isTadiated marrow has hitherto been shown only f ollowijig 
the injection of living ceils j, as nearly as one can judge from 
the literatiE'Qc, The importance of this observation is not 
limited to possible applicability of treatmentg but extends to 
the general area of cell differentiationo 

Proposed Course of Project ; Efforts are being made to discover 
what processes are involved in the initiation of early recovery 
in the hemopoietic system of the irradiated animals o 



Part B included Yes fxj N© ^J 



1107 



Serial No, NCI=659 



PHS=NIH 

Individual Project Report 

Calendar Year 19^8 



Pkrt BoS Honorsg Awards^ and Publications 

Publications other than abstracts from this projects 

Smiths Wo Woj Marstonj Ro 1= and Cornfield^ Jc, Patterns of 
Hesicpoietic Reeovesy in Irradiated Mice, Accepted for publicaticm 
in Blood o 

Invited to present some of these findings at the VII International 
Congress for Microbiologsr held in Stockholm, Sweden in August of 
19580 

Honors and Awards relating to this project j 

None 



1108 



PHS^NIH 

Individual Project Report 

Calendar Year 1958 



Serial NOc KCI-671 _^ 
It, RadiaiT'SriSnich 
2o Rado Biolo Ssct^ 
3o Bethesdaj, Md„ 



I^rt Ac 



Project Titles 

Protect iv© Effect of a Colchicine and Related Comjcnands 
in Aninals E}q)Osed to X=Radiation 

Rcincipal Investigator: 

Willie ¥r, Sadths, RiJ),, 
Other Investigators J 

no Ho Aldexman and Ruth So Qillesple 
Coop^:«ting Units: 

None 
Man Years (calendar year 1958) : 



Professional; 
Other: 1/3 

Project Descriptions 

Objectives : To detsrjnine the effects of colchicine and sm^ 
of its derivatives on survival and hemopoiesis in irradiated 
ani^i^s-, 

Methods Baplqired ; The material to b® tested is injacted at 
the desired times in relation to irradiation and studies aara 
made on bona marrow, circulating cells and survival«=with 
particular concern for any evidence of alteratirai of marrow 
sensitivity to radiatieno 



111 



Serial No„ NCI=^71 



Project Descripftions (Continuad) 

Major Findings g Mortality is significantly reduced when these 
Gcanpounds are administered before (but not after) irradiation;, 
Major differences in response are related to age^ species and 
time of administraticwic. Bone marr(xs recovers its celliilarity 
and peripheral lyiipho<gfte3a granulocytes ^ platelets and hemo^ 
globin concentration racof¥er earlier under the conditions which 
produce an increase in survival. 

Significance t& Cancer Research; Schedules of tr€«tment to 

incrSseTithaJLity are belSg'^ught tiith the object of appli= 
cation to coinbinaticsrs z-adiatiOTi and shamothejpai^ in experteental 
cancer 

Proposed Course of Project : This and related prcbleras dealing 
with recovery of hemo^ieisis will be studied with the view of 
elucidating mechanisms of action „ Techniques will include the 
uptake of tritiated thymidine and special cytological preparation 



l^arfc B included Xes ^J No /^ 



1110 



Serial No, NCI-671 



PHS-NIH 
Individual Ftojact Rftperfc 
Calendar laar 



rtot Bo_£ Honoraa Awards s and Publications 

Publications other than abstracts from this project: 

Smith J Wo Wo 9 Protective Effect of a Colchicine Derivative 
in Mice Exposed to X.-=radiations Science^ 127^ 3ii0^3ia, 1958^ 



Ssjithj Wo Wo and Comfieldj, Jog Extending the Range of Dose=- 
Effect Gvirves for Irmdiated Micej Science, 128, h734i7h» 1958. 

Smiths Wo Wo, Induced Recovery of Bl©cd=.Fon!iing Tissue in 
Irradiated AnlaialSo Presented at the Second International 
Conference on th© Peaceful Uses of Atcnsic Energy ^ Geneva ^ 
Swltserlandj 19$So 

Honors and Awards relating to this projects 



1111 



HB=-Nr^? 2o Rad„ Biol., Sect. 

Ihdivichial Project l^ix)rt "- . '•-■''•■:-■•;-=■■ Hd„ 

Calandacr Tear 1958" 



Project TiUes 

Combined EndotcstKln and Vtiole Botfy Irradiation 'Treatment of 
McQ Bearing Transplarstcd ^uwsrs 

Principal Investigatofss 

Joanne Holl croft and liilXie Wo SmitJi 

Other InTOsti gators s 

Bruce Lo Weed^ Ilo I'L Alderman ^ Mar5.on M„ Matthc.JiSij and 
Ruth E„ Gilleapi© 

Cooperating Units 8 

None 

Man Toaras (ealendar year 1958) 
Totals 1/3 
Profs^aionalg 
Others 1/3 

Projeet r&scriptlong 

2MS£liSE° "^^ inp(ro\re ihe therapeutic results of X^-irradiation 
in mice bearing transpiJ. anted tumors l3y pretreating them wilii 
bacterial endbtosdn vMeh rni^ts (a) differentially protect 
the anixnal from the lethal aetion of radiation or (b) differs ntiall:; 
increase the sensitivity of -tfc® tamor to the daiaaginp action of 
the radiatioHo 

Mb thod^._ Era^oyed s, Mce bearing a transrlanted tujwor were treated 
by coi?i5inationa of endotoxin and radiation.. At hipjier leifels of 
whole bodv radiatiai bone marrov Injections ware used to prewent 
radiation leUialitgro Data on txsmor size and surTlval were used 
to evaluate results <, 



1112 



Seri^*l Nn„ NCI»672 



Project Dsscripticsn: (Continued) 

Major Fi ndings g S,. tyjiiosa endotoxin had a small inhibitory effect 
on the local growth of a lyrarhosaroomao When en<to toxin was rriven 
2U hours before vjhole bo<3y or local tumor irradiation an additit!© 
effect on tiwior recreasioi was seeno 

'-'Jhdle-boct^f irradiation of mice haarinr a disseminated 
reticulum eell sarcoma increased their survival time bat increasiri?; 
•ttie dose from 600 r to 1500 r did not yiold a steadily increasing 
survi'val timeo Endotoxin -^van 2h hours before irradia+ion increased 
tolerance to radiation and pro<fciced an inftreaao in survival time of 
the irradiated tuimor^boaring animals ^ieh vjas greater than •Qi® 
sum of the increase attributaULe to endotoxin cp radiation aloneo 

Signific ance to G anegg Research ; linp'ovGrent of radiation therapy, 

Proposed Cov trso of Fro.iect g Noneo 



Part B included 



Tes !T7 ^IZ7 



1113 



Serial No= NCI.6'Z2 



PHS^KIH 
todiTidual ft=ojsct Report 
Calendar Year 195^ 



PartJBs Honors, Awards, and Publications 

Publications other than abstractft from this projects 

Hollcroft, Jo Wo :5nd Smith, W„ W„, Endotoxin Treatmenit and 
X-Irradiaticn in Mice Boaring Transplanted Tsitnors^ Jo Nat, 
Cancor Insto ^ 3U"329s 19?8o 

Honors and Awards relating to this projects 

Nbne 



IndlviduaCL Projeet Rapcrt 
Calendar T©ar 1958 



Serial Noo N£Is6X2__ 
lo Radiation Rrand-i 
2o Rado Biolo Sect^ 
3c Bethcsda^ Mdo 



Part_Ao 

Project T^-tles i 

Service Radiation Therapy (Latoratory) 
Principle In'eBStip:ators 

Joanne Holleroft 
Other In-ssstigatorss 

Henry Meyer ' 

Cooperating Units s 

This projeet does not resemble olhsrs in ihe Piablie KeaLth Senrice 

Man Tsare (calendar year 1958)3 
Totals 1 
Professionals 
Others 1 

Rpoject Descriptions 

Qb.leoMi'^ s ° T-o madntain and operate radiation facilities and 
to adtiinister radiation to vialie and nonviable biological 
entities and laaterials in accordarjce with the needs of in- 
'westip;ators in NIK research areas other than the Radiatiori 
Brandio 

Ifethpde Eagloyeds A variolar of low energr, high energy^ low 
dose rat© J hi^ dose rate^ and rxjlsed radiation sources 
inel'ucfi.ng a high intensity electron beam are available fcr 
this projecto 

Proposed Course o f Pro;1ects To ccntinueo 



Part B included 



^es £_/ No ££/ 



1115 



Serial Noo NGI^7U 
1„ Radiation Branch 
2o Hado Biol. Sect.. 
3o Betheada^ Mdc 
FHS=NIH 
Individual Project Report 
Calendar Tear 19^8 



Project Titles . 

^mthesis of Antibody in G^H Mic© Bearing the Transplantable 
Plasma Cell Tumor X=$563 

Erineipal Investigators 

Falconer Smithy PhoDo 
Other Investigators s 

Jferie Mo Grenan 
Coopejpating Units; 



Jfen Tears (calendar year 19^8) s 
Total? l»l/3 
Professionals I/3 
Others 1 

Project Deseriptionj 

Ctojeetivess To determine why specific antibody is not 
formed at all or only in reduced quantity in mice bearing 
the plasma cell tumoro 

Methods Egfiplgyed s Estimation of antibocfy (sheep erythrocyfe 
hemolyslni} content of Btsrvm of iramuniaed mice bearing the 
tnHnorj determination of tumor sisse and distribution of 
metastases are basic to the stu^o In addition techniques 
are being developed for studies of the avidity of seraas 
(rates of specific iiranxme reactions) and for methods of 
detecting ijaccanplete antiboc^ in mouse serums <> 



1116 



Serial NOo KL;i.=CiVa 



P^ojQCt Descriptions (Continued) 

!fajor Findings; a) As tramor sia© increases fanmation of antibody 
is depressed following immunization with a particulate antlgsn^ 
b) Studies on these mice with soluble antigen (egg albundn) has 
so far failed to reveal aritibodjr of either a precipitating or 
agglutinating character,, Hosje^rerj, ths presence of an incceplete 
antibo^ (haptsne) Is STispected^, A inanuseript describing the 
major findings is in the pr-ocess of preparationo 

Significance to Cancer Research ; Elucidation of the problem of 
antibo(%r production is germane to the problem of abnanoal px'otein 
^nthesis in animals bearing this tumoro 

ft'oposed Course of Project ; Studies directed toward iiranuno-- 
chemical characterization of the serum of mice bearing th® plasnm 
cell tumor and given soluble antigens will he continusdo 



Vajrt B included Yes // No /x 



1117 



IJxcJividual Projeet Report 
Calendar Tear 19$8 



'erial Ho 
lo Radia'Cii.on brar.r;';. 
2o Rad» Biolo Sect., 
3, Bethesda, Mdo 



Project Titles 

Irranunologic Studies xvith Heterologous Marrovj 'Zranafissions 
Rrincipal 3Ji-vestigators 

Faleonor Siaith, Ph«Do 
Other Iji'sestigatorss 

Vax'le Mo Orenan and Kristin Limde 
Cooperating Units s ' 

Oak Ridge National Laboratory 

Han Yaars (calendar year 1958) 
Total? 5/6 
Professionals 1/3 
Others ' Ij^ 

Project Descriptions 

Ob.ieeti-TOss St«<3y of the immunologic factors associated with 
homologous and heterologous bone marrow transplants^ 

Ifelj^ods Emplpyeds C^ mice, selected after testing for the 
inability of their serums to agglutinate sheep ery&roqytes^ 
are exposed to lethal amounts of X rayso Shortly thereafter 
the irradiated mice are given bono marrow from Cg.^Bl mice 
selected for the ability of their serum to agglutinate sheep 
eiythrocgrteso 

Ma.lor F inding^ ss Serum from irradiated GgH mice recei-^Lng 
marrow from C^oj-Bl mice fails to agfdutinate sheep eiytbro^tes 
after recovery of the host animals o 

Signif icance to Cancer Research ; The contribution of the btaie 
marrow to the irairanological milieu of animals is poorly undeir- 
stoodo Since ihe fate of transplanted tissue depends upon iti> 
aeceptaiKe by the host these studies help \jy .idding to an 
understanding of the s'ole of the marrow in antibody formatioiso 



1118 



Serial Ko„ ISb^ZS 



i^ojest Inscription: (Contiaued) 

Jfe-O Mged, Course of Rc-o.jeets Esperiments aimed at determinirig 
■the extent to whicsh the bone mazroH contributes to antibody 
fonaation will be donso 



Part B included les ^ No [^ 



111!) 



(Attachment I) 
Serial NOo NCl»67g 



Pffi-NIH 

Individual Ftoject Report 

Calendar Year 19^8 



Part Bt Honors 5 Awards , and Publications 

Publicaticna other than abstracts from Ihis projects 

Sitd-lii, Fo, Failure of Homologous Bone Mairow to IDiduee 
a Speoific JCmanme Reaction in Lethally Irradiated CsH 
Mieeo To be published in Transplantation Bulletin aftas' 
editorial modificationo 

Honors and Awarcte relating to Hiiis projects 

None 



1120 



Serial Noo ^Jd^JL,,^^ 
lo Radiation Branch 
2o Radc Biolo Secto 
PHS^KIH 3„ Bethesda, Mdo 



lhdivd.dual Project Report 
Calendar Year 1958 



Ffoject Titles 

An Investigation cf the Relationship Between Cell Division 
aiid Irradiation Response to X<=rays and Ultraviolet in Yeast 

Principal 'Xnv&stlga.toT? 

Mortim^ M„ Elkind, Ph,D<, 

Other Ih'TOstigator'ss 



CSooperatiag Units s 

Nona 

Man Tears (calendar year 1958)3 
Totals 1/3 
Profeasionals 1/3 
Other: 

Firojeet DascriptiOTis 

O^ec^lBSi H&diation injury ^ as evidaiced by the loss of 
self reprodu ctirity or the productixjn of viable joutants, is 
conceptually associated xiiith discrete sitsso In yeasty 
conflicting reports exist with regard to the aaatoHdeai 
location of these siteSo I'i'hile roost e-vidonce favors the 
nucleus as the region of prinsary x~ray damage ^ RNA containing 
cytoplasmic particulates have been considered to be the 
primary UV sites » The latter view, in particular 5 had been 
strengthened ty ths apparent absence of a U? insensitive 
"tail" on previously reported U¥ survival curves <, Bie 3&^ray 
survival curves of haploid -through tetraploid yeasts have a 
radioinsensitivB tail which hag been shown to be due to the 
dividing moiety in the population o 

The object of this research was to undertake a study 
of the U¥ response of a misced population of yeast cells 
(dividing and non-dividing) in order to essjnine in detail 
the question of Ihe presence or absence of a UV insensitive 
Dioiettf-o 



1121 



Serial Noo NC^Z^ 
Project Inscription!? (Continued) 

Malhods Employeds The methods employed consisted cf Hie 
production and detailed analysis of UV and x=ray survival 
curves, and the effects on the preceding of various 
p^rnrutations of x^raysj U¥, and visible li^t (photoreaetivation, 
The can lines used ^^ere a pair of respiring strains (haploid 
and diploid) plus a non^respiring haploid subline „ Both 
haploid lines can be lethaUy effected by exposure to visible 
light (see report No, NCI~636)<, 

Ma4o r Findings ? a) In contrast to UV survival aarvss 
published by others 5 we found that cai'afully performed 
eaqjerimsnts revealed that the UV curve^ as well as the X-r^ 
Qi^vBf was eompound in nature o Although in iSie case of UVg 
me inflection in the survival curve is hardly as pronounced 
as in the case of X-rays, nevertheless we shoned that the 
inflection is real and results from the fact that cells in 
division are less sensitive to UV as well as to X«r£^So 
b) Throu^ the derivation of a jnatheraatical theoraSj we 
showed that the reduced UV sensitivity of dividing cells mst 
result from a basis changeCs) in the UV sensitive site 
structure of the eello This agrees with sonclusions reaehed 
by others in ecnnection id.th the site structure sensitive 
to X=ray3o c) The existence of differential sensitivities 
to UV easpcstire in a misBd population ^s also demonstrated 
in comeetion vdth mutation production and photoreaetivation 
of UV lethalityc And d) fiiile quantitati-re differences wer® 
in elldenee^ the results obtained were general and independent, 
of tX9 ploidy, respiration coupe tence, and visible li^t 
sensitivity of the cell lines usedo 

Slenifican ce to Cancer Research s A knoxdedne of ih& sites 
and mode of action of radiation at the cellular level is 
necessary for a detailed understanding of radiation effects-. 
These latter effects ^ whan understoodj may expedite the use 
of radiation as a tool in biological research in general o 
More specifically, their understanding will play a role ixi 
e3?)laining and contxolline the carcinogenic and cardnoeldal 
action of radiations 

Proposed Course of RrQ.ieetj It is iroposed to extend these 
findings to sn examination of the extent of overlap of sites 
sensitiTO to Uf and X-rays through the use of additivilgr 
e^qpsrimsntSo 



Part B included los /ZJ ' 



1122 



HIS=^NIH 

Indi<!Piclual Project Report 

Calendar Year 1958 



Serial No« MCI.^681 

1„ Radiation Braneh 
2o Rado Biolo Seeto 
3o Bethesda, Mdc 



I^ojeet Titaes 

Applieations of Soise Tissue Culture Tediniques to Hadiatioa 
Prlneipal In-vestigators 

Robert Eo Bases, Mo Do 
Othffl? Invasti-gatorss 

None 
Cooperating TJnitss 

BK--ne 

!&a Xeara (calendar year 1958)! 
Totsav 1 

Professionals 1 
Others 

Project Da script: on s 

Ob.leotiwss 1) Developnsent of tissue culture method for 
detecting materials capable of altering the radiation response 
of mammalian cej.ls» 

2) Investigation of radiation response and growth rates 
of previously iiraclated mairenalian cells in tissue Gultur9„ 

^thods Employed; Si\i^e csell techniques after Puckg et al, 
were employed o 

Major Findings; !» A quantitative tissue culture method for 
detecting materials capable of altering the radiation response 
has been achievedo 

2o The method has been applied to two compounds s amino 
ethyl isothiouronium bromide hydrobromide and 6=mercaptopui'lneo 



1123 



1 Wo., NCI-681 



frojec:fe Desoripibiotti (Contimied) 

3o Ability of indi^dual cells of a population to 
font! clones is. a more auuhentic indea: of drug or radiation 
effect on gurm^al ihan is isere entiniei'ation of the 
population at va:?ious tiiiss after treatmento 

ko In vitro J progery of a cell which has escaped sterilization 
in an iiiitial iiradiation are normally susceptable to subsequent 
irradiations,, The grotrtis rate is nonnalo 

SlgQjlgicance ,.to„ Can<^r, Rcisearifdit The method developed may be 
helpful in detecting dru ^ which can potentiate the effect of 
ionising radiation in toinor -Sieraipyo Protective ds'ugs may be 
detected as wello 

^.ig?«t8M,,Cgi;|rsa of I^o.iegt.t Sspeening of drugs is in progresso 



ThM: T-i -^ri. -T firt«ri Jen r^t Ho >% A 1 1 or 



Serial Noo NCI.^62,^^ 

lo Radiation Branch 
H©c=KIH 2c- Rado Biolo Secto 

Individual Project Report 3o Bethesdaj Mdo 

Calendar Year 1958 



Project Titles 

Sites of Action of Leiiial Irr&diation in Diiriding Ygast Cells 
Kcinsipal Si'eeatigators 

JbrUasr Mo Elkind, FhoBo 
Other Invasti gators J 

None 
Coopcsratiiig Units? 

Nons 

Man Tears (calendar year 1958) » 
Totals 1/3 
IVofessionals 1/3 
Others 

Piroject DeseriptionJ 

^^gcttvess Having founds as described an report No, iCI-676j 
that fee general characteristics of both the UV and l^^vay 
sMTvlml cwT'TOS of yeast are consistent with the preseace of 
overlap between t2ie sites sensiti-*^ to both types of irradiatioiij 
an examination of the csdatence and degree of tiiis possible 
overlap was undertaken o This examination was liKitsd to the 
dividing cell laoiaty of a uiisted popelation primarily becaus© 
of the insensitive respcs:ise of such cells^ jmrticularly to 
X==rays, is anomalous ^ "Riis anosnaly hag been imatoly rssponsi'dle 
for the view that UV and X==ray sites are anotomically or at 
least functionally separated in dividing cellSo Of eoneernj 
ax's questions of first order in cellular radlobiologyi namely 
^ere and \dxat are the primary sites of lethal irradiation 
damage in living cells « 

Methods Employed s l^e basic method involved consisted of the 
use of coupling or additivilgr experiments in which different 
<»mbinations and peXTimtations of UV^ X-ray„ and visible light 
eaqsosures (for p*iotoreacti'^ation ana photolethali-^) were 
employedo The underlying idea steisned from the large number 
of instances in ^lich it has been found ihat U? (25It mjA Hg line} 
effects ^hieh can be photoreversed ai*© nuclear ssid have nuclei© 
acid (M) action spectra „ Hence U? and X=-ray effeets which 

1125 



Serial No„ NCI^682 
ft'oject DescriptionJ (Ccmtinuedl 

are coupled and ^hhieh can be uncoirpLed Igr visible li^t 
in all lildihood involve chromosomal INA an the sites 
caaaon to both irradiations o To help inaare that the 
oonelusions readied were gesiaralg three cell linos wer® 
uatado These ware genetically related diploid and haploid 
lines J both respiring^ and a nan~re spiring haploid siiblim© 
of the precedinga Both haploid lines were shown to be aWs 
to be lethally effected Igr eaposure to visible light (see 
report Noo NCI«636)<, 

Ma.ier, Findings g a) In acMition to ttie mathematical proof 
described in report Noo HCI»676, it was shown e^pariatentally 
■bhat the reduc®d UV sensitivi% of dividing cells was not 
artif actual in natiareo That is^ it did not result j, for 
Instance^ from a larger aimmnt of speeifie b«f ncei^aetiTO 
absorptiCHi of the 2$k ap line dw® to "Sie possible presence 
of more KA in diidding cellso b) It was ^own that ther® ig 
a largg measure of overlap in sites sensitiv® ' t© both U? 
and X=r£^s and that th® ©oupling produced by pre=U? e^esures 
eould be •mdeae coneoinitant the photorsversal of lethalii^ 
produced by the U¥» @) It was shovn that it was quite imlifcely 
that there esdst two (or mere) setis of sensitive sites in a 
dividing yeast cellg one set responsible for lethali'^^ arid 
■She other responsible for eouplinj? ^th X.=ray sites o d) 
Photolethal effects, which were showi to be due to the 
deveBipmsnt <£ a heinoproteln-=like ehroroopliore (report Wo:, 
HGI-608))g do not eouple «i"& U¥ or X«rays<, Hence j, in all 
likelihood these offeets are cytoplasndc^ e) While quant ita tit- .^ 
differences ■^a&re obsesvQd^ ihe (gonelusi'^ns tdiieh f <^owecl 
from these resiilts were indspsnd®?t of the ploic^^ respiratiosj 
competenesj and visible light sensitivity of the cell lix>&s 
usedo And f) the most craisistent interpretation of these 
results is that in "dividing yeast cells the sitea sensitiii© 
to U? and X«=.rays are in appreciable mpasijr© nuclear and 
probably consist of chromosomal DKAo 



and mod 



and moda of action of radiation at the cellular level is 
nscassaxy f<a:» a detailed understanding of radiation ef rectso 
These latter effects^ when understoodj, may expedite '^m us© 
of radiation as a tool in biologiGal research in generalo 
More specifically^ their understanding will play a role in 
Q3q>laining and controlling the car(3iB0g«nic and csrciiio<d.dal 
action of radiationo 

:g^po,aed Cpuyi^e of Pro,'^e,qt; Depending on the availability of 
eqiaipaent and personnel j it would be desirable to perforan 
action spectra on the specific coupling effects obserwdo 

Part B inaudeds Yea ,£^7 K«%^7 

1126 



Serial Woo NCI .-&6ji , 
lo Radiation Brands 
■fHS-NIH 2„ Rad„ Biolo Sect,, 

Individual Project Report 3o Bethesda, Md; 
Calendar Year 1958 



Part A, 



Project Titles 

The Mitigating Effect of Ultrairiolet Exposure on X~ray 
LQ-thalitgr in Ei viewing Yeast Cells 

Brincipal In^ngstigators 

ffortmer M„ Elkind, Ph„Do 
Other Investigators? 

None 
Cboperating Ifeiitss 

Hone 

Man Years (calendar year 1958) » 
Totals 1/3 
Professionals 1/3 
Others 

Project Descriptions 

Ob^jectivess During the course of the iKork described in 
report Noo NCI=6825 ^'^ was fomd that ssnajl doses of U¥ 
could mitigate the lethal effect of %=>Tay exposure in 
di-viding yeast cells o Since this obseri?ation was new 
and bore directly on the question of oiiferlap between 
UV and X=-ray sensitive sites^ it was exaarS-ned in detail o 

Msthods E mplqgedg The ns-Uiods emplc^ed (asd cell lines) 
were essentiaTLy those described in report Noo NCI«^682<, 

^.jor Findings 2 a) It was found that small doses of UV 
(doses fflnialler than those titiich tiroducsd i.iereased X<=ray 
sensitiidty) gi-vsn before, after, or during X«ray exposut^s 
decreased the lethal effect cf X=>rays on3y for the ditddisig 
cells in ihe popiAationo 'b) It was fomd tiiat this 
protective action eould be liiotore versed by e:!qposure to 
visible light eoneomitant with the reverssl of iiie small 
ajBownt of lethality produced hj ttie prs-^UV^ And g) it 
waa found that this mitigating effect was general but 
varied quantitatiisaly in the 'three cell lines use (two 
haploid and one diploid) c 



1127 



Serial No„ NC^:683 

Project Description: (Continued) 

Ihese results are remiaiscent of DV mitigatdon of 
X<=ray dircanoaomal aberrations obsenrad by others in 
cytogaietic studies wil^ ,^:gj3e §ca ntia pollen tubes and 
ICte'osophila sperm and support tlie general conclusion 
reached in report Noo 682 l^at the sites of UV and 
X-rey lethality in dividing yeast cells are largoly 
chroinosomal DMo 

Si ^lficance .to ^ Caaeer Researehs A loiowledge of the sites 
and mode of action of radiation at the cellular leiHsl is 
necessary for a detailed understanding of radiation effects c 
These latter effects, when landerstoodg m^ expedite the 
use of radiation as a too], in biological research in general 
>bre speeifieallyj their understanding will play a role in 
©a^lainlBB and ec«itrolling the eareinogenie and earcinocddal 
action of radia-felono 

FroTX)8e d CoTtrse of ,Frp.iectg Depending on the airailabili^- ©f 
equipment and personnel j this work should be extendad to 
includes 1) a stuc^ of the action spectra of the isdtiKatiag 
action of UV| 2) Uie relationship between protein and nuclei© 
acid sjmthesis to tiiis effeet| laid 3) an in-eestigation of the 
ability of small UV doses to adtigate the X=>ray production 
of genie mutations in dividing yeast eellso 



Part B included Yes ^^ No £^ 



1128 



Serial Koo NCI«683 



Individual Pro;5ast Report 
Calendar Tear 1958 



Part Bs Honors^ Awards, and Publieationo 
PubHeations other thasi absta'acts from thia pro;Jects 

Elkind, M^ Mc and Sutton, H, A^, Ultraviolet MtigaUon of X--r^ 
Lethality in Dividing least Cells » Seienee, 128^ 1082-38 1958o 

H«jcsP8 and Awards relating to this projects 
Nona 



1129 



Serial No„ Wl^Sh 
1„ Radiation Branch 
2<, Radc Biolo Sectc 
PHS==WIH 3, Bethesda, Mdo 

IndiAridual Project Report 
Calendar Year 1958 



Fart Ao 

Project Tiiles 

The Relatiai ships Between QiroinosoHial Const! feition and 
Irradiation Response In MaBsaalian Cells 

Brlncipal Inwstigators 

JfortiiBsr Mo Elkindj HioDo 
O^&er Inwsti gators s 

None 
Cooperating Units 3 

None 

Man Years (calendar year 1958)2 
Totals 1/3 

Professiomls 1/3 
Otters 

Pro^eet Deseriptioas 

Ob.iec ti,tgS ' ^RiQ object cf this research is to perform 
cellular radiobiolo^eal studies with mananalian cell lines 
in miich (Sjuantitatiiie cytogsnetic observations are amenable o 

Methods Eis^jgggds The sin'rle cell plating techniques viiidi 
ha^T© beeji developedj notably by To To Puck and his assodatas, 
are employed in irradiation experiments in which axi order of 
statistical accuracy is sou^t approaching that forissrly 
obtainable cnly with foni^g phaga, and bacteriao TheBQ 
63^ri»ients' are being ccatdueted with fibroblast=lite dimal 
lines of the CMnese hamster Aose cells have the unique 
properties of lox* diploid chromosome niamber (22) plus almost 
complete dlstinguishabilily between tho eleven homologous 
pairs o Methods and prof&edures are under developraent or being 
sou^t to pei^ts reprodaeible X-ray and UV irradiation 
experiments I cl<aiing, chromosomal diaraeterizationj and 
storing of subclones| cytogenetic chsracterization of 
chromosomal coastitutionsi and relati^iely rapid, routine 
characterization of ehroirosomal abnarraalitieso In addition,. 



1130 



Serial No. NCI~.68U 
Project Descriptions (Continwd) 



considerable effort is eontemplated with regard to the 
improvexnant of plating efficiencies and the routine propogation 
of healtl^ cultures sinee fibroblast cell lines in general are 
Hifficult to maintain and are thought to be very sensiti-ro to 
toxic agents of ten associated with the serum source^ Ultimately 
a satisfactory solution of the preceding proH^ra may require an 
im?estigation of the nutritional requirements of these cells as 
has bean done in th© case of Hela and other cell lineSo 

MaMr^ Finfaingas a) We have fouad that ^e survival curv© of Chines® 
hamster cells in tissue culture is multihit in general character 
with a mean lethal dose about twice that of Hela S% b) w® have 
found ttat iT)eta]*agcs cf cells attached to glass can be pr&parod 
•with facility and that the cell lines under study have displa^'ed 
some karyotypic variability and instability of chroiuosome number 
as has been reported by others o e) At times ^ considerable difficulty 
has been experienced in maintaining healthy cultures with accoiapar^ring 
poor platlnr efficiencies under sraneral laboratory conditiona and 
techniques which had consistently yielded no eulturing difficulties 
and lOOf^ plating efficiencies with Hela S3o Similar results have 
been reported ly T„ To Puck who has attributed such results 
primarily to deficient serumo 

Sifsiifieance to Cancer Researdis Cell lines of the Chinese hamster 
permit the unique advantage of performing a variety of biological 
eaperlji^ntaj including radiation esperimentSj in which correlatiire 
effects on the chroirosoBjal constl-tostion of the cell can be soughto 
Such s^erijMnts are fundamental to an understanding of cellular 
radiobiologyo In addition to the importance of sudi studies to 
biological research in general, they are specifically permane to 
the carcinogenic and carcinocidal action of radiationo 

Propose d Course of Pro.ject j Because of the slow prowth rate of 
mamitslian cells in tissue cult^ire^ which limits the nuniber of 
experinsents on a given problem to about two per nonthj it has 
been found necessary to proceed simultaioously along several lineSo 
As tim- permits, and depending on the facility xd'Ui ^ieh ceH 
lines of the Chinese hamster can be handled in culture^ it is 
p-oposed to exajnin® some of t*ie follotsinc questionss 1) the 
relatSjsnship between chromosomal hoterogenilgr and ploi<^ on radio- 
responsei 2) the basis for the dependence of radiation induced 
inviability on dironwsomal integrity! 3) the nature of d-jromosomal. 
constitution in clones which devslop frcsn sirvivors to lethal 
irradlationi and h) the production and selection of nsjtsnts produced 
■fcy UV and X-rays and Vhe description of the chromosomal constitution 
of such mutants « 



Part B included? Yes £y No ^ 

1131 



Serial No. NGI-685 



lo Radiation Branch 
2o Rado Biol, Sect, 
3o Bethasda,, Kdo 



PHS»NIH 

Individual Project Report 

Calendar Year 1958 



Part Ac 

Project Title; 

Thgrmidine Uptake in Hsiaopoietlc Tissue Induced to Recover 
Following X<=>lr3radiation 

Principal Investigators s 

Willie VJo Smithy PhoDo and Victcr Bond^ FhoDc 
Other Investigators? 

Ho Mo Alderman and Ruth Eo Gillaspi'S 
Cooi^rating Units: 

Brookhaven National laboratoiy 

Ifem Tears (calendar year 1958) § 
Totals 2/3 
I^ofessional! 1/3 
Other: 1/5 

Project Description: 

Osjectives: To discover processes concerned with post= 
irradiation recovery of blood=forming tissues <> 

Methods Ehplgyed ? Autotradiography of hemopoietic tissue 
after treatensnt "with thymidine to determine the presence of 
cells cajable of mitosis « 

Significance to Cancar Research ; Qensral significance in 
field of cell pafoliferationn Specific interest in connection 
with damage to blood=>fomiing tissue following exposure to 
radiation o 

Proposed Course of Project s See Methods Bnplpyed,, 
Pfeirt B included Yes Fl No ^7 



1132 



PHS=KIH 

Individual Project Report 

Calendar Tear 15>58 



Serial No, NGI^-636 
lo RadiatlonTBranch 
2c Had, Biolo Sect, 
3o Bethesda. Mdo 



Ffeirt Ao 



Project Titles 

Recovesy of Ability to Form Antibodies in Irradiatsd Mice 
Fretreated with Endotoxin or Colchicine 

Princifal Investigators s 

Willie Wo Smithy PhoDo and Joanne Hollcroft 
Otiher Investigators J 

Ho Mo Aldentan and R«th Ec Gillespie 
Cooperating Units; 

None 

Man Years (calendar year 1958) s 
Totals 2/3 
Professiosials 1/3 

Other: 1/3 

Project Descriptions 

Cfojectivesg In stn^y of rasovezy ppocsessas it is important 
to see how various hsnopoietic functions are related o Tha 
question to be answered here is Esther or not abi}d.ty to 
react against foreign tissue is associated with the advance 
in recovery time of cellular elements as seen following 
isologous at heterologous bone marrow in;3ection or treatment 
with colchicine or endotoxin e 

Methods E^l^ed s Transplantation of hcsaologous ascites at 
intervals during recovsry frosn irradiation in tr^ted and 
untreated aniisals and observing response,, 

Significance to Cancer Research ; This problem is of increasing 
imporbance as interest wJ.dens"Tn whole=body irradiation^ both 
as a hazard and as a therapeutic toolo 

Proposed Course of Project; See Methods Esnplciyedo 



Part B included les. / /' No ^J 

113.1 



Serial No, NCI^67 

n il - t - n III I I i H a I III un a 

lo Radiation Branch 

FHS^KIH 2o Rado Biolo Sect... 

Sadividual I¥oject Report 3o Bethesda, Md, 

Calendar Year 1958 



Pf«jeet Titles 

Induction of Radiorasistane© 
Prtoeipl© Si-rostigators 

Joanne Holleroft 
Othsc Ih'^osti gators 8 

Mari«i Matthews and Bruce Weed 
Cooperating Iftxitss 

None 

Man Years (calendar year 1958) s 
Totals 2/3 
Professionals 
Others 2/3 

fttjjeet Descripftions 

0b;1ecj4^3s To imrestigate spontanea! s and radiation indueed 
radio re si stance in lyansplaated tuasorso 

Methods SsBployeds Transplant irradiated and non^irradiatad 
tumors serially and stuc^ changes in sensittviiy to radiation 
by laaasurint^ tojaor regression or ty bioassay of ascites tTixs»r 
cells o 

Ma;1cr Fji KHjigg^s L ^1 l^nnphoaareoffla beeasie more resistant 
to radiation after 18 pasnagas^ each passage having been 
irradiated with 1000 ro %is resistanee is manifest in 
regression of the ajlid turaor aft«r radiation as vrell as by 
bioassJQT of irradiated ascitic tuasoro ISie resistant line Ero>?a 
slif^htLy niore rapidly than the non=irradiated line and grows 
in {CyDBk/zJFx^mk and C3H as vjell as A ale^ SareoBia 29668 
did not change in its response to radiation from the Uth to 
the l6th generation^ and irradiating each generation with 2000 r 
did not alter the response or growth rate of this tumoro 



1134 



Serial No. NCI...687 



Project Descriptions (Contijiitiad) 

Pic^OEpscjd CottriB_e of Pyo fleets To continue atudtjrang the differences 

in the 2 linesofL^l^d to stnd^ new tumors « 

S^^dficaRCQ J^...9ancer_Re3ear.dig Radiation theraj^ of tujUGTSo 



Part B incawdad Yes £^ No ^J 



1135 



PHS-NIH 

Individual Project Report 

Calendar Year 1958 



Serial No„ KGI=62li 



lo Radiatdon Branch 
2o Rado Fnysc Sect, 
3o Bethesda. Mdo 



Part Ac 

Project Titles 

Radiation Injtuy and Repair 
Principal Investigators 

Howard Lo Andrews j, HioD„ 
Obher Investigators: 

Louis Mo Caiaeron 
Cooperating Units; 

Walter Reed Aray Medical Center 

Vlan Tears (calendar year 1958) s 
Totals 2/3 
Professionals 1/3 
Others 1/3 

Project Descriptions 

Objectives t To gain insight into mechanisms by which ionissing 
radiations affect living tissues o 

Methods Bgaplqysd; Work on the fractionated dosage schedules j, 
started in 1957 ^ has been completed but data analysis remains 
to be donso 

A second phase of the project involves studies on swine 
at high doses and in the mid<=lethal range. Both of these 
projects are of prime importance to the Department of Defense 
and serve to ccEDiplete data obtained at NIH on smallar animals o 



Fkrt B included 



Yes ^ No ^ 



1136 



Serial No„ NGI=62ii 



PfiS^NIH 

Individual Project Report 

Calendar Tear 1958 



Part Bg Honors 5 AwaardSj and Pctolications 
Publications other than abstracts from this projects 



AndrafwSp He Lo^ Species Differences in Response to High Radiation 
Doses, Rado Res., 9, U694i77, 1958o 



Honors and Awards relating to this projects 
None 



1137 



Serial No, aCI^628., 
lo Radiation Branch 
2c, Rado Physo Secto 
3„ Bettiesda, Mdo 
PHS-NIH 
Individual Project Report 
Calendar Tear 1958 



Payt A« 

Project Titles 

Dosiroetiy of Hi^ Faergy Radiations 
Rpincipal Investigators 

Hoxijard Lo Andrews, PheDo 
Other Investigators? 

Louis H» Cameron 
Cooperating Units « 

None 

Man Years (calendar year 1958)8 
Totals 2/3 
Prof e ssional s 1/3 
Others I/3 

Project Descriptions 

Objeoti-ceas To develop inethods aid instruments for 
measuring radiation delivered at depth by high intensity 
pulsed beams o 

Methods EmEXoyed s Extensions of previous work with chemical 
dosimeters was planned but practically no work has been done 
during 1958 because of lack of personnel o Ihe need of this 
xflork continues and perhaps it can be resuised in the future,, 

Part B included Tes ^^7 ^o I^ 



1138 



Serial No. FJCZ.629 
1« Radiation Brandi 
TWS^NIH ; 2o Tlnd. Physics Seetc 

Infuvidaal Project Report 3o Bethesda, Mdc 

Calendar Tear 19^ 



Payt ^Ap 

Project Titles 

The Kffeet of Ionizing Radiation on Amino A^ds 
ftdncipal Invastigator: 

Chasfles Ro Maxwell, PhoDo 
Other In-ve sti gators s 

None 
Cooperating Unitas 

None 

Man Tearss (calendar y^r 1958) 
Totals S/6 
Professional: i/^ 
Others 1/^ 

PJrojeet Descriptions 

Obi eeti ires s The objective of this project is to determine 
the medianisa of the chestieal reactions induced "ig ionisijig 
radiation in aqceous solutions of amino acids. This is pas^ 
of a long range proprain tc aecuraulate inf ontstion on siaple 
^stens of biological interest so that general principles 
may be ascertained and applied to complex ^sterns which 
are not amenable to thorou.^3 direct in-wsstigationo 

fejBio^ E inpi^Qyet^i Solutions of amino acids are irradiated 
with ionizing radiation and ttion analyzed f cr the products 
fOCTiedo Since the products are sensiti-ve to radiation it is 
necessary to determine the yield of each product as a funetioR 
of dosfi to as lov7 a dose as possible and to extrapolate theso 
•values to obtain Uie initial yield of the ppodact at very 
low doses before secondary reactions distort tha pietas'Qo 



1139 



Serial No„ NCL=6g£ 

Projeet Description: (Continued) 

Once the products of the simple ivater-amino acid-radiation 
system arie found the effect of the introduction of other chsaicala 
into -the system are studjedo Substances are selected^ in accordance 
with the rapidly exjanding coneeiJts of radiation cheiaistrys liiieh 
as-Q thought to accentuate or repress one or more of the various 
intermedLate processes by vdiich radiation is belieired to produce 
its effects o 

Since the final chemical (and ultimate biologieal) effect is 
liie result of the f ormaticsi of a complex micros copically-hetero=> 
renous mixture of excited molecules j HgOg, H and OH free radicals 
in discrete "tracks", ths influence of tha physical distribution 
of ttese acti-ve intermediates as well as possible differences 
in relative abundance and/or chemical nature is investigated by 
using different sources of radiationo lb date 50 KV »=rays5 
150 O electrons, and $ Kev alpha psrtieles have been us@do 

One of the more iasportant, if not the most important, active 
intennediates is the OH f3*ea radical <, The role of tJiis radieal 
in the absence of the H free radical and the host of exeitsd 
maleeules in the ionisation track is s ladled ty diemieally 
produeing this radical in the system being studies o To data this 
has been accomplished ly the action of Fe^+ and HgO^o 

Maj or_Fi nd±nep s A redetermination of the initial yields of the 
previously identified produeta from irradiated glycine by the mora 
nehsitiVB analytical procedures -srfiich we have devolopad has shoT^m 
that ws do not have a material balance^ There is another prodfuct 
of the reaction which should be identif iedo 

It was early observed that the concentration of HCOCOOH in 
the irradiated solution appax) aches a steac^ state with increasing 
dose t^ile the yield of the other products continued to increase « 
It lias now been establishsd that this is the die to competition 
for Ihe OH free radical and that glycine is relatively rlnsensiti-ve 
to oxidation by ihis radical,. 

Irradiations in the presence of Fe®* and in the presence of 
both Fe®* and Br" have shown that the mechanism for the formation 
of HCOCX)CH is toe disproportionation of two radicals formed by 
the action of the OH free radical on glycine » These expssdments 
also indicate that the as yet unidentified compound is forriKsd 
from the same radical o 

It is becoming more and no re evident that HCHO is not formed 
by any of tha "usual" reactions e^)seted of fres radicalSo It is 
not formed by oxidations with Fenton's reagent which is 'rrriot-ai t© 



Serial No< 



Project Deseriptions (Continued) 

proceed throu^ a free radical mechanism involving the OH radiealo 
Fe^* ion which is knoT-n to oxidize moat organic free radicals 
and tiiieh morQ than tripl©s the yield of HCOCOOH hag no effect 
upon the yield HCHO„ The presonce of specific competitors for 
the OH free radical whidi drastically reduce the yield of 
HCOCODH also have no effect upon the yield of HCHOo Initial 
specific yields as a function of glycine concentration show a 
linear dependence characteristic of formation by direct 
absorption of energy and incompatible with an indirect nsjchanismo 
Yot the finite yield indicated \iim this data is extranolated 
to aero concentration is incompatible with a direet meehaiiismo 

S igpif i oggice , to Caacer Research s Although not directly connected 
with Cancer rasearch^ it is of great interest because it seeks 
an understanding of the mechanism for ihe effects of radiation 
which is Msed empirically as a tool in the clinical treatment 
and laboratojy study of eaneero 

.P£o^S. 6.^-^gigL8g^ o ^, i^Pj'^^,!^^,* ^ork vd.ll continue in an effort to 
fully elucidate the msehanlsE of the effect of radiation on glycine 

SpeeificalLy the -Kork vdll be directed towards 

a) Identifying and determining the unknown product indieatsd 
by an ijicoraplete material balances, 

b) Determining *tiie mods of H atom attack by irradiations in 
D20 and measuring the yields of D^, HD, E^, CHaOOQi^ 

e) An in-vestigation of the 0^ present system as well as ^e 
Og free s^rstem upon whidi oar efforts in the past has been 
priMariiy concentrated o 

c) An investigation of the effect of pH upon boththie 0^, 
free and 0^ present ggrstemo 



Part B IndLudsd Yes ^ No^ 



1141 



Serial Noo NC:^:637 

lo Radiation Branch 

2» Radc Physo Seeto 

3o Bethesdaj Mdo 
PHS-NIH 
liidividaal Pro;jest Report 
Calendar Tear 19^8 



Part A,,, 

Project Titles 

Development of Hi^=.Intenslty X-ray Source 
Principal In"vesti gators 

Howard Lo Andrews, PhoDo 
Other In-vestigatorss 

None 
Cooperating tJnilas 

None 

?!an Years (calendar year 1958) § 
Totals 

FS'ofessionals 
Olhers 

Project Descriptions 

Objective{°is To obtain hi^ intensity beams of X-rays ^ bo-Si 
stea^ and pulsed, from the 3 I'fev generatoro 

I'Ipthods Ejmplpy^(js The previotis attack on this proMem 
included the use of untried target raaterialso The use of 
liquid targets coupled Tdth either high speed target scanning 
and defocussing seems indicatedo No progress was made on 
this project during 19^8 due to lack of personnel o 



Part B included Tes ^^ No 



114 



9 



Serial Noo NCI^77 
-^ 1, Kado Pi^sies Seeto 

2o Radiation Branch 
HB=MIH 3„ Bethesda, Mdo 



Individual Project Report 
Calendar Year 1958 



Part Ao 

Ftojeet Titles 

Properties and Mechanisra of the Benzoic Acid =■ Ferrous 
Sulfate Radiation Dosimetero 

Principal Jii-^stigators 

Charles Ro Mascwailj PhoBo 
Other Invest! gators 2 

None ' 

Cooperating Units s 



Man Yearss (calendar year 1958) 
Totals 5/6 
Brofeasionals 1/3 

Others 1^ 

Rrojeet Deseriptions 

Ob.^egtiVBS' The initial objective of this project was to 
detexTnine the mechanism of fee reaction vdien an air saturated 
solution of benzoic acid and ferrous sulfate in dilute sulfiffi® 
aeid is used as a radiation dosimetero ^is solution wag 
proposed by Dr, Gail /^dams as a sensitive dosimeter for the 
chemical evaluation of a do3e of ionising radiatlon„ Ih 
view of liie widespread interest eshibited by ra(fi.ologists in 
this dosia^ter we felt that an investigation of the meshariisjm 
of the dosimeter vjould be of value » When the preliwiaary 
observations x^ere examined in light of the result of project 
Noo NCI-6783 this objective was extended to inclmfe a stisciy 
of the role of other organic solutes ^^n added to •Qie ferrous 
sulfate dosimeter o 



n /. Q 



Serial Noo NCI.^77 

Project Descriptions (Continued) 

Methods EmpIcyM ' "RiQ yields of ferric ion as a function of dos© 
and ejcperiiaental ccnditions such as acid concentration j ferrous 
, and solute concentrations and ferrous/solute ratio were measurod 
and compared with the yields of ferrie ion when small amounts 
of HgO^ were added to the dosimeter under the same conditions o 
■Rie rate of ferric production in a post irradiation phase of the 
irradiatton induced oxidation was Beasured and compared with the 
rate of production in the HgO^ induced oxidationo 

Standard chromatographic techniques were used to separate 
and identify soma of the organic products in the irradiated 
bensoie acid dosinetero 

Jfe3oy-gte^ =ng8L The role of all the organi© solutes studied^ 
bensoic - acids ethyl alcohol, etl^lene glycol and isopropgrl aleohol— 
wsPB found to be the saiaso "She meehanisra of the reaction teas found 
to consist of tw> partSo The first part in-vsjl-was the radiation 
induced oxidation of a small amount of ferrous ion and the fonnation 
of a small amount of HgOg^ During this pfoase and at large solute/Pe'^ 
ratios the action of all the orpanie solutes are the samec The HgOg 
yieldg which is in the abseme of an organic solute is equal to the 
sum of the yields of the H free radicals and HsOa produced b-r the 
radiation decompositirai of water , is increased by an amount equal 
to the yield of OH free radicals from this dccosiipositiono 

There radiation induced reactions are then followed by the 
oxidation of a much larger amount of ferrous ion by this H3O2 and 
the dissolved osygsno It is in this phase tiiat the final yield 
of f ejric icn and the sensitivity and stability of the dosimeter 
is determinedo It has been shovm that -aie oxidation during this 
phase is simply the reaction of Fenton's reagent studied in project 
Noo K[CI~6T8 and is independent of radiation o 

Proposed Course of Pro.jecti This project will be terminated after 
the puli!Lication of the a,bove results o 

•Siere seems little point in continuing the effort to separate 
and determine t^e organic products frcan irradiated benzoic acid 
solutiono Most of the pisjduets are fcrmsd during the post irradiation 
phase of the reaction and the n^chanisra of this rhase can be more 
easily investigated by the methods employed in project NCI=^78o 

Part Bo included Tes £^ No 



1144 



PFS=NIH 
Individual Project Report 
Calendar Year 1958 



Serial Noo B^^b28^____ 
lo Radiation Braneh 
2o Rado Physios Secto 
3o Bethesda, Kdo 



Project Titles 

The Action of OH PVee Radicals upon Organic Compounds 
in Aqueous Solutions 

Rrineipal In^stigator: 

Ghaples Ro MaxiJSlLj, Ph^Do 
Other Investigators? 

None 
Cooperating Units s 

None 

Man Tears s (calendar year 1958) 
Total J 1/3 
Professional t 1/3 
Others 

Project Descriptions 

Ob.leetJTOSi The objectives of -tiiis project are to determine 
the jaechanism of ^e eheinical action of OH free radiealSc, 
produced from Fe^ and H^Og, upon aqueous solutions of 
cheajical conrooundso The action of ionizing radiation is 
generally agreed to proceed principally throupji H and CM free 
radicals and HgOgo "Eiis project was xmdertaken to determine 
the role of OH radicals and H^Og from another source >n.tliout 
the presence of "the H free radical,, Hie original objecti'^ 
to stuc(y the effect itinon glycine was extended to include oiSiej' 
eojnpounds vjhen the findings f rem the glycine investigation in 
aerobic sdlutiDns were in disagreement with Ihe currently held 
general medianisms based upon vjork on these other compounds <> 
The new analytical tediniques which xife had developed were 
readily adaptable to the stu<fy of almost any «)mpound and 
since th^ --^ould r>rovide data whiCh should clearly and concisely 
differentiate between two mechanisms proposed in 19^9 for the 
reaction in the anaerobic condition the work was extended to 
cover thiso In addition to ita ob'vlous application to radiation 



ll4f 



Sariai Noo NCI^76 



Project Descriptions (ContinBed^ 



chemistry we felt that the finding would be a distinct contribution 
to free radical oxidations since the mechanisms being tested ha^re 
been in vopue since 19h9o 

Ifejfegds. Employed a OH free radicals were produced tiy mixing tT*o 
solutions of the organic compoirndsj one containing a ferrous 
salt and the other hydrogen peroxide ^ Samples trere remo'^ed at 
measured times and analysed for Fe^* and HgO^ and in some cases 
for the oxidation producto This method was possible beeause of 
an analytical method davaloped tgr us -rfiieh made it possible to 
stop the reaction at any time and analysis for both reactants 
and producto Pre-sious Ijnvcstigators had waited until one or the 
otter of the reactants was exhausted and had based their mechanism 
upon only the stoichiometry of the canjieted reactiono The actual 
kinetics of the reaction as v;e obser'!?ed it pro-^dded essential 
additional dnta f ca:' differentiating between basically different 
mechanisms each completely consistent with the previous stoichiometric 
datao With plycii^j which is ofprimary interest to us because 
of project NCI<=629j vje determined the kinetics of the appearance of 
products as well as disappearance of reactants o In general vre did 
not do this vdth other compoundso In some cases the effect of pHc, 
temperature and the presence of other ions are inirestigatedo 

J^lojL-|linJJJlg8' A medianism for ^e action of Fe^ and H 

^pona& saterate d solutions of glycine has been de'veloped and 

was pTiblisiBd in 1957 <• During the same year extensive data on t^e 

kinetics of the action of Fe^* and HsOg in both air satui'ated 

and air ftee solutions of several otl-ier organic compounds were oolleets 

The effort tdiis year V7as directed toward the analysis, 
evBluationj and preparation for publication of the above datao 

A, The kinetic data for the rates of HgOs consumption and 
Fe** oxidation of several organic comTX>unds in air free aqueous 
solution clearly proves that the reaction proceeds by the oxidation 
of an organic free radical by the Fe^* ion formed in the first 
step of the reaction mechanism and not by the initiation of a 
chain oxidation by HgOgo Thus for tha first time it is possible 
to clearly choose between the two mechanisms proposed for this 
reaction in 19h9 (Jo Chem, Soco, Sl5, 2li27, 19h9} (Jo Amo Chemo SoCc, 
JS^ 37771378U, 19lt9)o 

B» An analysis in 19^7 of preliminary data on the rates of 
Fe** oxidaticsi and HgOg oansuKption in the oxidation of se'wral 
organic eoripounds xn air saturated solution indicated that the 



1146 



Serial No» NCI^76 



Project Descriptions (Contintjed) 



reaction and medianism for all theae compounds was similar 
to that which we proposed that year for "Uie oxidation of flycino 
and different from the peneral mechanism proposed in 19h9 by 
Kolthoff and f'edialliao (J. Ani„ Chem. Soc„ JJl, 3777, 7?8J4, 19h9.) 
At that time it appeared that only a minor ef^oort would be required 
to differentiate between the two mechanisms and additional 
observations were madOc 

A analysis ^is year of all the data, howeverj reveals that 
neitheif metSianism is entirely correct. At large soltrteA'e^'^ ratios 
the extent of oxidation or chain length is determined by some 
reaction other than the coiTp5tition of solute and Fe** for an OH 
free radical a? observed by us for glycine or competition of 
solute and Fe^ fas* an HRO free ra«iieal as proposed by Kolthoff 
and medialliao At lew solutsAe®* ratios the chain length is 
contarolled l?y one or the other of these eon^jstitionso ^a bu3.k 
of the evidenee indicatesj, but does not prove, that the reaction 
paroceeds throupjh tfae repieneration of Hj^O^ and not ^ the forma ti©a 
of and chain oscidation by an organic I^droperoaldee TJie enddenee 
suggests but a^in cbes not prove that the undllineatod termination 
step is a radical-radical disproportionattono 

Propos ed Co^isrse of Proieets The project will be terminated as so<m 
aa one or both of tw manuscripts novi on hand are publishedo 

The first manuscript on the medianism of the air free 
oxidation will be submitted soon,, 

The second nanuscript on the mechanian of the air saturated 
oxidation is not of as high a standard as I wewld lilee and may not 
be submitted for pablicationo The securing of new data t« more 
firnsly establish the mechanism would require much more work aad 
the use of more elaborate teehniqaes thiiiM pareviously usedo I ara 
not sure that this effort is warrented at this timeo 



Part B inoLudeds Tes £^ No 



1147 



PHS-NIH 

Individual Project Report 

Calendar Year 1958 



Serial iiOo l;;L;j.^;^.L'C 

ic Radiation ''Branch 
2, Rado Rjyso Secto 
3o Bethesda„ Mdo 



Fart Ao 

Tro^ect Titles 

Radioisotope Administration by Iontophoresis 
Erincijal Investigators 

Howard Lo Andrews , HioDc 
^her Investigators s 

Dorotl^ C, Peterson and Louis Mo Cameron 
Cooperating Ifeits: 

None 
'Man Years (calendar year 1958) % 



Professional s 1/3 
Other ; 2/3 

Project Descriptions 

Methods Bnplqy^eds A variety of radioactive isotopes are 
adndnistered to guinea pigs by jB.ss3.5ig an electric current 
through an electrolytic solution and into the skino Animals 
are sacrificed at intervals and the isotope distribution 
determined o 



Major Findings? Metallic ions vai:y treniendously in their 
^otsin fcindteg ability so that some can be moved only a 
millimeter or so through the skin while others enter prcsnptly 
into the general circulationo 

Significance to Cancer Research; If scam penetration with 
localiaation can be achieved this jsay prove to be a t?sei"ul 
method for treating skin lesions by radiation , In particuiae 
electrolytic applications may supplement beam therapy in 
mycosis fungoideso 

Proposed Course of Projects It is hoped to continue this 
»ro:ffc and searoh" for more satisfactory isotopas than havr- '■ 
studied to date. 



art r> jUifej-uusu 



wo /x 



Serial No„ NGI=2=6;^_.^ 
1 „ Had iatfOTTBranch ' 
2o i^do Thai% Serv^ 
3o Bethesda, Md, 



PHS=NIK 

Individ val ^Project Report 

Calendar Year 1958 



Project Title I 

SeTwice Radiation (Clinical Therapy) 
Pj-incipal Investigator: 

Stanley E„ Sneider^, M„ Do 

Othor Investigators: 

J„ Rcfoerb Andrews g M., Dcj, H^maan Suit^ Mo Do and Robert W, 
Swain 

Cooparating Units? 

In other Public H^^lth Service Hospitals where ra'iiation 
therapy is practicado 



J&tient Days (calemiar y®ar 1958): 



?5an Years {calendar year 1958): 
Total? 1/3 
Professionals 1/3 
Others 

Project Deseriptions 



Objectives t To provide radiation theragy for those patients 
reQuirin'i~8uch in the course of the disease for which they 
are being investigated by WIH research groups other than the 
Sadiation Branch, 

Patie nt tfaterial s A total of 119 patients were tr^ted with 
IheTollowing distribution asecrdir^ to disease categories^ 



1149 



Serial Nc„ NCi^^=>:^,:>..vc) 



Project Description I (Continued) 
Malignant Diseasa; 

Prtmaxy Treatment 1 
Metastasis or recmTent Ij. 

Iftearus 
Recurren"t X 

Chariocarcincsiia 
Uterus 1 

Metastasis 2 

Tostieular 
Metasta.ais 3 

Vagina. 1, 

Renal 

Metastasis 1 

WiJjns 

Metastasis 2 

Prostate 

Metastasis 1 

Breast 

Metastasis 10 

Head and fJesk 

Disease after surgery 6 
Ifesopharynx 1 
Tljyroid 1 

Adrenal 

Hi®ochX"OBioc!3rtc«na 1 
Cortical 1 

Pancreas 1 

PituitaiT 

Cwaihii^s 2 
ChrcDiophcfo© 2 

GUcsmta 6 



EpendyraoBna-apinal 1 

Lung 2 

Eosinophilic 

Granulcjna 1 

Hodgkins 11 

I^nnphoaarccana h 

Rsticiiltm? Cell. Sarcoma 3 

Chronic I^jrmphatis Letikaraia "I 

Chronic Ifyolofjenous Ijeukossda 3 

Acute lymphatic Leukemia 7 

Acute Jfy'elogenous Leukemrta 5> 

Nelantsna 

Mata stasis 3 

Soft Tissue Sarcoma h 

K[ycosis Fungoides 8 

Basal Cell 3 

Benign Disease j 

Ti^TOUS (syasthenia ) 3 

Acute fkrotitis l 

Unknown QraniiLation 1 

Inf laiamatoiy Miscellaneous 1 

Keloid 1 

Radiation effect on lialr grcwtb J 



1150 



Serial No. MCI-? -650(c) 



Project Descriptions (Continued) 



Methods Employed ; A wide range of photon energies were used and 
varioxiS methods of application were asnplqyedo The following are 
the number of courses of treatment using the noted energy? 

103 cases given 180 courses of 2 mev irradiatiomo 

1 case given 1 course of 200 kv irradiation o 

12 cases given 20 courses of 60 to liiO kv irradiation^ 

9 eases given 9 courses of electron beam therapy <, 

h cases given k courses using radium or iridium^ 

1 case given 1 course using intraperitoneal radiogoldo 

Major Findings £ Responses to radiation therapy have been consistent 
with the response reported by others o 

Rroposed Course of Rpoject; To continue,, 



Part B included Yss fy No ^ 



1151 



Serial No„ NCI^-^--j5glCcj_ 
lo Radiation Branch 
2„ Rado Ther„ Sanr, 
3, B&thosda^ Md„ 
PHS^NIH 
Individual Project Repcxrt 
Calendar Year 1958 

Bart Ao 

Project Titles 

Eisetron Boans Radiation Therapy 

Principal Investigator; 

Jo Robert Andrews^ M,, Do 

Other Investigators? 

Eugene Jo Van Scottj Mo Bos, Howard U AndrewSj Ph„Do, 

Robert W„ Swain, Hennan D„ Suit^ M„ D^ and John H^ MgctsaS:., M. @ 

Cooperating Units? 

NCI^General Medicine Branch, C7=708(c} 

Man Years (calendar year 1958); Patient Days (calandar year 1958): 

Totals 1/3 
I^ofessionals 1/3 
Others 

Piroject Descriptions 

Cbjectivesi To stucfy the syraptomatiCj, histologic and other 
oG^ectiTvi^responses of patients with agrcosir fungoides to 
radiation of the entlr© skin with high energy electrons,. 

Methods ^nploryed s Sectrons with an initial energy of 2,1 
M&v are appIi©T\iy a Van de Oraaff electron accelerator as a 
fan=shapsd beam^ F^tients are moved under this beam on a 
incfoile table and by rotating the patient into several different 
positions the entire skin surface can be irradiated with th© 
electron beain« Weekly radiati<xis of approodLnsately hOQ x'ads 
are given to the skin of the entire bocfy at a single sitting 
with total doses being of the order of 1000 to, occasionallj,, 
3000 radSo The jrajcimwa depth of penetration of this beaa is 
tissue is 1 ciso and the doses cited ar© average doses thromgh- 
out this depth. Continuous clinical observations are snad© and 
photographic and biop:^ material has bean obtained where 
appropriate , 



1152 



Serial No. NCi-^-^^.,-/ 



Project Description: (Continued) 

Batlant Material ; In the program were 9 patients for a total of 
5I treatments,, 

Kajor Findings ; The project has been continued very much as 
pa^viously recorded and the major findings are the same„ In other 
words^ it has been found that all cases of mycosis fungoides are 
responsive initially to electron beam therapy and in some the 
initial response is very great and relief of sympt<ans is extremely 
effectivso Electron beam therapy^ where applicable^ has c«ae to 
be considered the method of choice in the definitive management 
of this disease-, On the other hand^ all responses are teinporaiy 
but the minimum time for the persistence of response seems to be 
a matter of months whereas maximian time haSg in a few in stances ^ 
been a yearc It has not been demonstrated that either initial 
syraptoBiatic response to the therapy or the persistence of the 
response are any better at the highest dose levels » 

Rroposed Course of Project s Instrumentation is being developed 
?ar ilie determination of mean tissue oxygen tension^ Whan this 
is working satisfactorilly alternate surfaces of patients will be 
irradiated under conditions of air breathing and oxygen breathing 
and mean ojcygen partial pressures will be determined under the 
COTiditions of irradiation and differences, If anyj, in radiation 
response will be investigated « It is thought that nycosis fungoides 
la an excellent tumor for contrcsUed^ experimental clinical studies 
because the patient can be his own control f&c alternate surfaces 
can b© treated under different conditions. In addition^, the 
lesions are relatively radiosensitive j resp<asses are fairly rapid | 
mtd,tiple lesions are available for studyi and the lesions present 
where th^ can be readily inspected or biopsied » 



Part B included les /%7 No 



1153 



Serial NOo NCI=2-65l(c) 



FHS-NIH 

Individual Rroject Report 

CSalendar Year 1958 



I^rt B; Honorsj Awards^,: and Publications 

Poblications othor tl&n abstracts froiR this projects 

Van Scott J, So Jog AndrmsBg J, Ro and Mgcosab^ J* Hoj 
Mycosis Fimgoides with High Energy BleetronSc Accepted. foE" 
p^±>licatioiJ in Acta D^rsKitologica^Venerealegioa , 

EdgcQjfoj, Jo Hos Van Scottj Eo Jo and AndrawSj Jo R^j Hi3to= 
pathologic Changes in the Skin of Patients with f?Sfcosis Fungoid^ 
Following 'Rierapjr with High Hkiergy Ea^estronso Accepted foar 
pttolication in Acta Dennatologica-=V®n@realogicao 

Honors and Awards relating to this projeets 

Non@ 



1154 



Serial NOc NCI=£-65Wc2 
1» Radiation Branch 
2, Rad, Thero Serv^ 
3« BGthesda^ Md, 
PHS=NIH 
Individual Rfoject Report 
Calendar Year 19^8 



I^ii; A ■ 



!¥ojQCt Titles 

Tijna as a Modifier of Clinical Radiation Response 
Principal Investigator; 

Jo Robert AndrewSj> M<, Do 

Other Investigators? 

Hennan D^ Sultg Mo Do^ Stanley E, Sneider^ Mo Do^ lawsnee 
Schlachtsrj, Ec B^j Theodore Rckiisiaoii^ M, Dc^ James ko Ros®, 
Mo Do and Jack Levinj Mo Do 

Gc<»p@:mting Units; 

Non@ 

Man roars (calendax" year 1958) s Patient Days (calendar year 1958): 
Total; 1 
Professionals 1 
Others 

Project Description: 

Objectives and Methods Bnployed ; This project is an extension 
cF"proJ€>ci G«3=6^5Tc7^' "f'or"cai9ndar year 195? and it conttaaes 
studies pr®viotzsly undertaken in much the same fashion as has 
previously b©en reported a The objective is to study the dose= 
time relation^iip for the control of sqwamous cell earcinosoa 
with specific reference to possibilities for modifying th® 
radiation response insofar as improved normal tissue toltsranc® 
is concerned by extending the irradiation period over a pro=:> 
longed tisaQo Criteria for the attairanent of the dsjectiva ar® 
control of the presenting lesionj, dmation of life after 
treatment g complications (ulc^E'ation and necrosis) in, th® 
jresQRting lesion^ and radiatiffin ccxnplications in normal 
tissusSo 



1155 



Serial NOc 



Project Dascription: (Continued) 

This current annual report of this jwoject is given the form 
©f a report on this stu<^ for the period beginning in June of 1955>' 
and ending June 30^ 1958= Inasmtash as more than three months nay 
be required fca* the administration of treatment patients admitted 
into the study since July 1, 19$Qg are not included in this rapm't^ 

Patient Material and I-kj or Findi ngs; Svamaxy report of project 
fSTth'e per'iod^une l^'SS to JunojSg 19$B„ 

Noc of patients s 30 

NOo of patients living: 10 

Mean duration of life; 

8o5 months J, range h to 19 months following initiation of 

theraiy in 20 patients dead cr lost to follarop 
lOoli months J, sr-a^® U t© 39 months in ©ntla:'© Series of 

30 patients 

If®o of patients living without clinical evidence of diseassj 8 

Mean field sis@; 73 css^ 
Doses 7000 t/7S days to lOgOOO r/lOO days 
. Mean dose; 8870 r 
Mean duration; 960 3 days 

Radiati«i complications; (necrosis of soft tissue j, brain (1) or 

bone J, trismus j, fibrosis 5 extensive edesB) 10 patients 

Control of local disease; lU j^tients^ 9 of whom are living 

Cause of death; 20 patients 

Vris&xy or its local extensions 13 
Merfcastatic disease; 2 
Radiation cemplication; 3 
Intercurrent disease; 1 
Lost to followups 1 



1156 



t I 



u <P 



hi 



8 8 

O vO 



I. 



o 

8 



vO 



(^ 





i 


1 




8 


n 




< 

u 


^ 


g 


43 


e 


CD 


Is 




OS 


E^ 


S 





e- o 



\A 







§ 


"^ 








o 


U 








°H 


■2 


fei> 






-93 


_ C 


a 




n 


^n 


,fi o 


T« 




43 


+3 o 


> 




V) c 


?5 -H 


•H 


•H 




« ® 


SSfH 


3! t-e 


(H 


s 


o 4* ■ 


Oi 


oi . 


o 


o 


gg. 


o S 


;§^ 


!§ 



1157 



Serial No» NCI-^^giiCcj 

Project Description! (Continued) 

A previous survey* had sxiggested that an exposure dose of 
lOpOOO r delivered in 100 days was at the limit of tolerance 
under the conditi<»is of radiation employedo Present practice is 
to aim at a tumor exposure dose of 9000 r in 100 daySo 

Eight patients have been admitted to the program since 
July 1^ 1958g busfe of these eight the therapy program has been 
coBipleted on3y recently in but threso 

Proposed Coto'se of Project ? It will b® proposed to one or more 
©f the joint it^^ groups in radiation problems that a larger 
scalsg controlledg and randomised experiment be done to determine 
whether or not there is argr real superiority in the high dose= 
extended treatment time method as compared with the mere 
conventionala lower dose=>8harter treatment time methods <, 



4frSe9 ^aft Bs Publications 



Part B included Yes /TJ No ^ 



1158 



Serial No, mi^^6Sh(c) 



PHS-NIH 

Individual Project Report 

Calendar Tear 1958 



Rart Bg Hon^rSj, Awards^ and Publications 

Publications other than abstracts from this projects 

Andrews^ Jo Ho^ Ruibinc, Po and Swain^ Ro W^^ The Dos®=*iine 
Relationship in Radiation Therajy.. High Dose^ ProlcKiged Tline^ 
Large Volume Eadiation T'harajys The Limits of Tolerance g Aiqo Jc 
Risentgenolog Radc TheraRr and Kuelear Medo^ 7£s 61i=:-73j 1958<, 

Andremsg Jo Eo and Snaider^ So £<,§ The Modification of th© 
Radiation Response,, Accepted for publication in the Amerisan 
Journal of Roentgenology ^ Radium Riarapj and Nuclear Medieins:, 

Honors and Awards relating t© this project s 

Nona 



1159 



PHS=NIH 

Individual Project Report 

Calendar Year 1958 



Serial NOo NCI^=656(c) 
lo Radiation Branch 
2o Radc. Thero Serr^ 
3o Bethesdaj Md, 



Part A, 

Project Titles 

The Distribution of Sulfur=»30 in Htsrein Tissues and Tumcars 
Princiial Investigator: 

Jo Robert Andrews, Mo Do 

Other Investigators 8 

Lawrence Schlachterj Mc Do^ James A<, Rose^ M, D„, Jack Levin^, 
Mo D^j, Richard SKarmj M, Bog and Eaael GtHap^ Ao Be 

Cooperating Units s 

llCIs^Pa-Uiologic Anatcoy Branchy WCI-523 

Man Years (calendar year 19$Q)i Patient Days (calendar year 1958} s 

Total: 2 
Professionals 1 
Obher: 1 

Pro;3ect Descripticais 

Objectives : The objectives of the project are to determine 
the distrj&ution of Sulfur =35 in hunanss ^^ determine the 
biological effects of S-=35 on both normal human tissues and 
human neoplasms and to determine the possible usefulness of 
S=35 in differentiating certain human neoplasms o 

Methods Baplqyed ; The general method eag)loyed in studying 
distribution^ dSsimetryj, and possible diagnostic differentiation 
of certain human neoplasms is the intravenous injection of a 
10 mco amount of S-35 follcawing which periodic radioactiviigr 
assay studies of the urinary excretion^ the blood level, the 
bone marrow content ^ and selected normal tissue and neoplasm 
content of S=35 are made. There is involved the collection of 
numerous specimens by both venapuncture and biop^o Radio= 
activity assay problems are concerned with the preparation of 
biological material for direct counting and with the cheraical 
extraction of S=35 from tissues for coxmting as sxilfateo 



1160 



Serial Noo NC.I°2°4$6(c) 



l^oject Dascriptionj (Continued) 



Patient Material t The patient inaterial has consisted of three 
patients with chondrosarcoma ^ tsie with chordoma, one with ostscsgeni© 
sarcOBiaj and one with mixed salivscry gland ttanor,, The last tw© 
were included in the study because the initial diagnosis of these 
patients was chondrosarcoma , These studies have shown a rather 
jH*edictable pattern of urinary excretion of S-3$3, eighty per cent 
of the administered dose being excreted within hB hours o Infcar=? 
nation on the relative distribiition of S=3^ in skin and ncffsial 
cartilage has been obtained and the differential concentration of 
S»35 i^ the latter has been found to be greater by a factor of 3" 
The concentration of S=35 in the three cases of chondrosarcoma 
studied has been found to be higher than the concentration in skin 
by a factor of anywhere from 7 to 30c This concentration ratio did 
not obtain in the case of the patients with osteogenic sarcceaa and 
mixed salivary gland turaor and^ in this respect, the S-3.? uptake 
study assisted in the proper identification of these tumors o Th® 
ehordana tissue did show a differential uptake of about 10 compared 
with skino 

The human dosimetry of S-^^^ has been developed in respect to 
the blood levels attained and in respect to the rate of removal 
frcsn tha bloodo The general pattern of the rate of removal from 
blood is similar in all the patients studied but th© dose levels 
attained vary sanevfhato An attempt has been made to measure the 
radiation doss to bone marrow by S=35 but^ to date, this has been 
unsuccessful because there has been no consistency in the results 
bbtainedo Tliere has been developed^ however j, a method for 
determining the proportionats amount of bone marrow and blood in an 
aspirated specimen of bcaie marrow by preliminaiy labeling of red 
blood cells with Chrouiiuin<=^lo 

Two patients were given large doses of S-^S^ the total dose 
in one being 1000 mCo in three equal increments given in 22 days 
and the other being 666 mco given in two increments 10 days apart ^ 
Both these patients had chondrosarcoma and both nonnal and neoplastie 
tissue effects w©r® studiedo The principle norsnal tissue effect is 
that of severe depression of hematopoiasisg the platelet counts 
being reduced to levels of about 10^000 and the white counts to 
levels of 1000 in both patients « Jfeximum depression occurred at 
about the fifth week after the initial increment of dose and per- 
sisted for about 10 dayso . Sp<^tanaous recovery of hematopoiesis 
occvjrred in both patients o No other normal tissue effects were 
observedc In the case of the patient receiving the hi^er dose 
cessation of tumor growth vras cfo served for a period of five months,. 
Auxiliary studies have included numeroo-s radioautographic observations 
of nonnal and neoplastic tissues. 



1161 



Serial NOo NCI^^g6(c) 



Project Description: (Continued) 



Propo sed Cours e of Pr ojects To continue the study along the lines 
devei'^e3~ibov"e o 



Bart B included Yes /xj No 



1162 



Serial No. NCI»2=656(c; 



HIS=NIH 

Individual Project Report 

Calendar Year 19^8 



I^rt Bi Honors Awards ^ and Publications 

Publications other than abstracts from this project t 

'Sane 

HonoK'B and Awards relating to this project s 

Exhibit entitled "Some Effects of a One Curie Dose of S=35 
in a Case of Chondrosarcona'' «as awarded a Cartificat© of 
Merit at the Annual Meeting of th© Ajnerican Roentgen Bay 
Society held in Washington^ Do Go^ Ctetober 19^8 o 



1163 



Sex-ial Noo ^J^^^OiH 
1, Radiation Br^^i 
2o Rad„ There SearT^ 
3o Bothesdaj, Mdo 
PHSoNIH 
Individual Project Repcsrt 
Calendar Year 1958 



?art A, 

Project Titles 

Clinical Radiation Dosimatry 
Rpincipal Investigator: 

Robert Wo Swain 
Other Investigators: 

None 
Goopez%ting Units: 

Nona 

Man Years (calendar year 1958); f^tient Days (calendar year 1958] 
Totals lc=l/3 
Professionals 1/3 
Other: 1 

Project Description: 

Objectives : To develop methods for and to detenain® radiation 
dosage distributions for various types of radiation therapy 
used in the treatment of patients » 

Methods Bnployed ; Chemical and thin«=walled ionis^tian ehaajbars 
for electron beam therajgro Ionization chambers and photo- 
graphic methods for Supervoltage X=ray Therapy „ 

Major Findings ; Ionization ohaiEbors and electronic circvdts 
ics" plotting Tsodos© curves have been constructed,, The 
circuits require some minor refinsasents before being usedo 

Small sis® ionisiation chambeirs for determining the dose 
in cavities have been constructed vax'ying in design and 
materials usedo None of these has resulted in a chamber 
giving satisfactory results,, 



1164 



Serial Noo NCI-2^6o{c) 



Project Dascripfcions (Continiied) 

The photographic dosimetM' scanner has been modified to Riv© 
increased resolutionc 

Calibratioxis for electron beam therapy have been simplified 
by calibrating ionization chambers with the chemical dosimeters 
as a standards 

Scsne data have been obtained in the determination of absorbed 
dose using chemical dosimeters far larger field simes than 
previously reported but the work has not been concliidedo 

Proposed Course of Project s Work on the automatic plotter will be 
contihueclo ""^ 

As new ideas are conceived or new naterials become availabl»s 
attempts will be made to construct suitable small sis® ionissatiori 
chambers o 

Pinal data ^ould be obtained for the determination of 
absorbed dose using chemical dosimeters in the coming yesTo 



F&rt B included Yes /°°7 No 



1165 



HIS=NIH 

Individual Project Report 

Calendar Year 1958 



Serial NOo NCr-^--.^l(e) 
lo Radiation Branch 
2o Radc, Thexo Serv,-, 
3= Bethesda, Mdo 



I^rt Ac 



Project Titles 

Continuous Visual Monitoring of 2 Mev X<=s*ay Therapy 
iVincipal Investigators 

Jo Rctoert Andrews^ Ho D., 
Other Investigators! 

Hiilip Rubins Mo Do and Robert W^ Swain 
Coopesating Unitss 

Nona 

Man Years (calendar year 19^8): Batient Days (ealendar y«sar 1958); 
Total; 
Rrofessionals 
Others 

Project Descriptions 

Ob j actives 8 This project was completed and reported in 
detaiFHirthe Individual Projeet Report for Calendar Year 
1957 » This report is being submitted for the purpose of 
bringing Part B up to datSc. 



Part B included Yes j^ No /^ 



1168 



Serial No, NCI^2^66lCc; 



PHS^NIH 

Individual Project Report 

Calendar Year 1958 



Part Bs Honors^, AwardSj^ and Ptiblications 

Publications other than abstracts fron this projects 

AndrewSg Jo Ro., Si^ain^ R» Wo and Ruibing Po^ Continuous Visual 
Monitoring of 2 Mqt Roentgen Therapgr^ Anio Jo Roentgenolo^ Had. 
ThQX^Rf and Naelear Modo^ 7£, Ik^lQ, 19$8o 

Honoirs and Awards relating to this projects 



1167 



Serial No« NC.I=2=--662(c), 
lo Eadiati'^rm^ncl-ii 
2„ Rado Thero S&rv'o 
3o Bethesdaj, Md« 



PHS"NIH 
Individual Project Reporfc 

GaLendar Year 1958 



Ifert A 



£,■ 



Project Title; 

Radiation Therapy foa* Advanced Carciaosia of the Bladder 
Following a Siiirgical Diversion of th® Urinary Stream 

J^rincipal Bwestigatcrs 

Herman Do Sultg M, Do 
Other Investigators J 

J, Robert ArKirewSj, Ho D^ and Stanley Eo Sneidarg Mo D, 
Cooperating Itaitsj 

None 

Man fears' (ealendar year iP58)s F&tisnt Days (calendar year 1958); 

Total; 2/3 
Professionals 2/3 
Other; 

Project Descriptions 

Ctojeetives; Estimation of probability of resolution of locally 
aSvaiiced. Flsdder carcincma (extension into perivesical tissii® 
and/or invasion of pelvic organs) by means of high dos@ 
(7=^000 r/50-56 days) 2 Mev x«ray therapy » 

Method.8 Bm|>lqyeds (a) A review of the earlier experienc® wj.th 
carcinoma of the bladder treated in this branch showed that 
of the tcyfcal of 8 cases treatsd^ t^so had died short3y aftisr 
cospletion of their x^ray thex'apy as a result of ureteral 
«A5struction| in both of these instances post morteia study 
did not demonstrate pelvic tusaosPo In two other instances an 
ileal bladder was constructed a few months after therajy to 
relieve ureteral ©bstructiono Because of these findings th© 
protocol was altered so that all patients considered for 
high doss radiation therapsf to bladder earcinotsBS liave had 
an ileal bladder constructed prior to isiitiation of therapy o 
This procedure has been psrfonaed by the S\irgical Branch of 
the Cancer Institute « 



1168 



Serial No„ NCI^>-^662(e ) 



Project Description E (Continuod) 



(b) X?=ray tectaiqueo The planned dose has been 7-8000 r/S0^$6 days. 
In all cases a 2 Mev Van de Graaff has been used to produce the 
x«ray beam (12 mu Cu HVL^ 100«125 cm<, TSD)o The entirety of th@ 
pelvic contents ha.re been treated to a dose of 5000 r/35 daye by 
means of an opposed field technique o If the jatient has tolerated 
the therapy sati3factapiJ.y and no metastases have been detected^ 
the urinary bladder and the residual tumor mass is jxradiated to 
an additional 2«33000 r/m=21 days by means of anterior cbliqu® 
wedg® fields or an arc rotation technique <, 

Pa t ient M ater ial t I^tients with advanced carcinoma of the bladder 
IS^'ii'Sr^l^ivesical tissue cr other pelvic tissue) have 
farmed this group « During the calendar year 1958 ^ w® haYs sesn 
10 jatients. Of thaseg one was an early bladder tumor and was 
not treat@d| two had metas-feases demonstrated after diagnostic 
work^up her© (periaortic nodes in one^ and metastasis t© humerus 
in the other) and were treated only with palliation as intent, 
IVo patients (80 years old^ ?9 years old) showed general 
deterioration ejhile in th© hospital and were given only palliative 
type of therapgTo Thtis 5 of the 10 were treated according to the 
protocol of this projects 

lk^[OT;__FindiBg^sj (a) In ®v9ry instance there has been erfcensivs 
resolution of "the tinsior mass as assessed six weeks after the 
ccaapletion of treatment o In two instances at six weeks aftex- 
therajy^ cystoscopgr and deep wall biopsy revealed no tuaiorj bcAh 
of these cases have been examined at post mortem and no pelT3.e 
tumor »^s foundo In th© other 3s tumcar persisted and these are 
being cxsrrently evaluated for additional therajy by means of an 
ijnterstitial radiation technique (see project NCI«2=^8C)o 

(b) These $ patients tolerated th© surgeiy and irradiation 
without majca* complicationc There wsre no instances of bladder 
disecsnf ort during or following the tJberaj^o Gystoscopsjr during 
aiid after treatment has shown only mild bladder wall reactions 
and no ccaplications secondary to telangiectasia have developed., 

(s) The clinical status of all bladder carcinoma fully 
trea-tad here is indicated in the table r, 



1169 





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1170 




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Sor-lal No. NC 1^^662 (c) 



Project Descriptions (Continued) 



Rpoposed Course of Project s That this project be continued until 
nearly 2o cases have teen treated to full dose levels following 
urinary stream diversion to assess these points; (a) effectiveness 
of 7=8000 r in resoivinR the tumor raassj (b) acute and chronie 
reaction of the bladder wall to this dose levels (c) in conjunction 
with project NCI=2=668(c)a detennine if supplemental high dose 
localised radiation can effect local cur©^ (d) incidence in this 
group of patients dying with metastatic tumor but with control of 
the disease in the region treated^ iceoj the pelviSo 



I^rt B included les [^ 



1172 



Serial No. NCI-2^663(c) 

1, Pad iaFi en Branch ' 
2„ Rado TheTo Serr^ 
3» Eetheadao Md^ 



PTIS-NUi 

Individial Project Report 

Calendar Year 1958 



Part Ac 



Project Title; 

CarcinoBia of the Eaop^iagus Treated by 2 Mev Rotational Therapgr 
Principal Imrestigatra': 

Jo Robert Andrews » Mc Do 

Other Investigators; 

Hersan D^ Siiitj M^ Do* Stanley Eo Sneiderj Mo Doj Lawrenc© 
Schlachter, Mo D„, Theodore Rc^inson, M„ Do and Robert W, 
Suain 

Cooperating Units? 

None 

Mati Yesirs (calendar year 19^8) g Patient Days (calendar year 19S8) 
Total; 1 
Professional: 1 
Others 

Project Description: 

Cbjectives s The determination of both the limitations and 
potentialities of radiation therapy in the management of 
patients with advanced carcincana of the esophagus and^ as an 
important by='product of the study^ to deterMina the relation- 
ship between the development of radiation pneumonitis and its 
severity and the dose of radiation to the lungs , Possibilities 
for protection of the Itmgs by the introduction of phanna= 
cological agents by aerosols was also to be studiedo 

]^tien t Material s This project was inactive dialing calendar 
year 1958 because appropriate patients did not become available. 
Active efforts to procure patient material were not mad© 
because adequate means and personnel for the definitive study 
of pulmonary physiology are not presently available c. 



\ 
Pfeirt B included Yes ^^ No ^J 



1173 



FHS=NIH 

Individual Project Report 

Calendar Year 1958 



Serial No„ NGI=2^668(c} 
1, Radiation Branch" 
2„ Fiad, T>jero Sorv- 
3n Bethesda. Md„ 



Eart Ac 



Project Title: 

Radioactive Isotopes in Interstitial Radiation Therapy 
Principal Investigator: 

Heman D„ Suit^, M„. Do 
Other Investigators; 

Robert W, Swain and C5alvin R^ Mencken 
Cooperating Units s 

Nona 

Man lears (calendar year 191^8).' Patient Days (calendar year JS'^B] 
Totals 1/3 

Professional; 1/3 
Others 

Project Description J 

CtojectivQs s (1) To develop a technique and equipment for 
placing small radioactive sources^ as a permanent iroplantg in 
inaccessiJDl© anatasnlcal regions ^ so as to deliver a high total 
dose locally but a low dose to th© operator and other medical 
and nursing staff. (2) To develop technique and equipment 
for achieving dose distributions ccmparable to that obtained 
with radim needle therapgr but with greater flexibility as to 
dos© distribution and with reduced radiation aTposur® to 
operator and staff o 



1174 



Project Descriptions (Continued) 

Methods Employed s A„ For the radioactlye material iridium {7T'--lS"i 
was selected for these reasons s (1) Half-life «• Ih days. Therefor < 
in the caae of permanent IjnplantSj a tot^l of 3=5=7 cc, radium 
equivalent woiild be the total amount required even for extensive. 
peliric implants. Length of half'=lifa Is not important for non-- 
permanent i;mplants as the mcc radiinn equivalent would b® approxi- 
mately the sane for a.iy t;sable half=-'lif» (2 days to n&ny years], 
(2) Its mean energy of gamma ray emiasioam Is 1*00 kvj this is nr. 
energy photon that is absorbed equally by bone^ soft tissue or fat.. 
The beta rays are nearly all screened by the stainless steel 
sheatho (J) The Isotope is available as variable length wires 

©r of seeds of 3 smo lengthy In b6t.h -inptar.r.fliK ths- ,-^;iisid*« (■ii,.fT.f>ft; 

is 0«5 raur, 

B„ Equipment developed for the persrianant implant technique! „ 

(1) Needl9s=-iRiese are 12 o$ cm^ in length and hav^ a. break in thelT 
contlJiuity^ near point of attachment to the pistol^ to effect a 
sharp drop in the eapilSary preB3V5r& as the plisnger is witlidra>?n., 

(2) The gun ha. 9 a spring operated jnagassine that has a capacity of 
2^ seeds o A plunger deposits one seed at a time j'ost beyond th^ 
end of the needle | this plur^ger is activated by a trigger which 
causes the thin drsasig to which the pltmger is flxed^ to rotate 
Ther© is a guage fixed to the gun which indicates the depth iv. 
tissue that each seed is being deposited,, (3) A separate d«ri <> 
has been constructed for loading the magazines by remote control , 

C, Needles constructed for the temporaiy implant techrdque- - 
These are simple stainless steel tubes (same six© as that used for 
20 guage needles) with one end closed and pointed and the other 
end open and containing a hole for suturing o These have been 
built to hold radioactive wires of 30 and 50 mm, ler^th„ 

Patient Material s A, For the permanent Implantg h cases have been 
triatea'and these have had residual tw3.ac following high dose sx- 
temal beam therapy (2 neck masseS;^ 1 carctncana of cervix^; 1 
carcinoma of bladder) o 

Bo The iridium wires in needles have been used as pruriar}' 
therapy in 3 head and neck cases (1 tongue, 1 floor of mouthy 1 
parotid carcinejRa)e 



1175 



Serial No„ NCIc=2-668(c] 



Project Description 5 (Continued) 



Vbl^ot Findings t The cases treated thiia far have been explcratoi'y 
in nature but have shown that the eqiiipnent functions as planned ., 
In the permanent Implant cases a dose of the order of i2gOOO« 
1^8 000 r has been ci'^'Q" to the central area of the implant 
(calculatsd for total decay) and these doses have been tolerated, 
Ifajor implants can be performed with a dose of 20»$0 niTo to th€> 
operatorj this is appx eciabljr less than dose for cofuparable 
implants performed with either radon^, gold, or radiumo 

Proposed Course of Project s (1) Implant residual masses after 
coepletion ol planned therajy for car cinema of bladder ^ to deterroine 
if very high dose local therapy can produce local control of the 
lesion. This will provide a good location to determine practicality 
of the aquipwent as designed at mcnsent and nay perhaps suggest 
improvements, (2) Make measureiaenta of dose to operator and staff 
in a variety of cases c Establish dose to the tissue for these 
implants by phantcm techniques and direct measiirements where 
possible o (3) Consider other isotopes that have different physical 
properties, but usabl® in the same equipnento 



Bart B included Yea £y No ^ 



1176 



PHS^NIH 

Individ>jai Project Report 

Calendar Year 1958 



lo RadiatToiT'Brancir^ 
2c Rad. Thero SerVo 
3o BethQsda,, Md„ 



F^rt A, 



Project Titles 

Rjarmacolegical Modification of the Radiation Respoyise 
ft*incipal Investigators 

J^ Robert An^ewS;^ Mo Do 
Other Investigatara: 

Ri^ Ccndit/ M„ Doj Alan Le'vy^ Mo Do and Eugene J„ Van Scott^, M„ 
Cooperating Units s 

None 

Man Years (calendar year 1958)-' i%itient Days (calendar year 1958): 

Totals 1/3 
l^ofessionals 1/3 

Other; o 

Project Descriptions 

Ob jectives ; This project had originally been conceived as 
a testing project for the hxanan application of so-<salled 
radiation protective chemical agent So This was planned in 
anticipation of having conpounds available in addition to 
AETji the human phancacology of which had previously been 
studied on an informal basiSo This study has been rspcrted 
and it was published in 1958c 

l^tient Material s Five patients ware studied in a dose-> 
response stu^y for bofth orally and intravenously administered 
AST and on© patient was studied en intravenous AETo Excluding 
this lasts in whom the study vas vary limited ^ all patients 
exporieneed reactions characterized by nausea | scrae had 
vcssaitingi and sooie experienced mild degrees of vasoaotor 
collapse with one instance of severe t^ypotensiono These 



Serial No, NCI-2-65yvcj 



Project Description ! (Continiied) 

responses occurred to doses of the drug which on a molecular ^ 
scavenger basis and assuming homogenous distribution would have 
been by no laeans adequate to "capture" a significant proportion 
of the calculated yield of radiation induced reactive deconipo=- 
sition products of watero The study was inactive during 19^8 
because other drugs did not become available for testing <, 

R»oposed Course of Project s It is proposed to resume and to 
continue' jAiarmacoliogical testing as drugs become available a 



Bart B included Yes 1%1 No 



1178 



PHS-NIH 

Individual Project Report 

Calendar Year 19?8 



Rart B; Honors^ Awards^ and Publications 

Publications other than abstracts from this project; 

Condltj Pod Levyg A^g Van Scottj Eo Jo and Andrews^ Jo Rr,^ ov 
Ef facts of beta-aiBinoeffcliylisothiuroniUBi br«anid© (AET) in mane 
Jo PhanRo and Exp. Thar^, ISS, llkl, 1958. 

Honors and Awards Relating to this Frojoats 

None 



1179 



Serial Noo NCX32^670(c1 
lo Radiation Branch 
2« Rado Thero Serir„ 
3„ Bethosda, Mdo 

Ihdiiddual Projeet Retxjrt 
Calendar Year 1958 



Ptyt,Ao 

Project Titles 

Studies on Thyroid Caaicer 
Principal IhVBstigatorg 

J» Robsrt Andrews J, Mo Do 
Other In-vestigators s > 

Theodore Robinson j, M„ Do 
Cooperating Units s 

None 

Man Years (calendar year 19$8)« Patient Days (calendar year 1958) s 
Total t 1 
Professionals 1 
Others 

Project Descriptions 

Ob.lectiTea and Methods Employed s To study the factors influencing 
the response of oarcinoraa of the thyroid gland to radioactive 
iodine in as^ciation with thyroid hormone treatsnentc To 
study the tumoirer rates of primary carcinoma of the thyroid 
and metastases and their response to hormonal and other factors. 
This study inwlves a rarie-ty of considerations including 
teehnicolopical^ biological, endacrinolopical, and pharma- 
cological o Technical studies are concerned with problems 
associated vjith the identification and evaluation of radio-- 
activity in specific organs or anatomical sites. Biological 
studies are concewied with the relationship of the histopatho= 
logical characteristics of thyroid cancer to radioactivity uptake 
and responses Hormonal considerations are concerned with the 
relationship of tumor control to the hormonal status of the 



1180 



Serial Noo NCX^g^?Q(e') 



PSwJect rsesofiptions (Conttotssd) 



indi'vid^sal, and in ©safijlnation with radioawti-we iodSjaoc Alao 
being sljudiod is th® relationship of 'bsmovsr rates (of TlAI^lja.) 
of thjrroid tximor in relaldonship to the ingestion of thyroid 
piwrjucts snd other subatanee® to see if radioactive substances 
nay bo held in twor tissue for a laiger period, thus increasing 
the amottnt of racttation to i^e tissue o Pharmacological studies 
ai^e sinilarly concerned -aiih these relationships o 

PaU eat ,liSa.teria^» Proved cases of carcinoma of the thyroidj either 
priinar:^.' or secondary or botho 

Major Findings 8 Findings from this stucfc;- seem to show that the use 
of radioactive iodin© along vilth thyroid products seeroa to have a 
tumaricidal effect beyond that of laere racH.oactivity a lone o IDue 
to the fairly recent origin of ihis combined technique equivalent 
studies to iifhich our resets a>uld be oojnpared are laot readily 
available c. Results also seem to show tiiat tumo-ror rates of tuiaor 
may bs visibly altered fcrjr IJie use of endocrinological agpnts^ 
thus lacireasing the amo^mt of radiation to thyroid tuaor if these 
agients are given in reLationship witii radioactive iodine «, 

PrqPff..gf <? Go^^ , pi; ftro„1es|i,t To oant^msBo 



Part B inal^<fed Yes /^ Wo ^^ 

1181 



Serial .No„ liCU2^19( c) 
lo Radiation Rrancfi 
2„ Rado Thero 3orv, 
3= Bethesdaj Md„ 
PRS-NIH 
Individual Project Report 
Calendar Year 1958 



I^rfc A< 



Project Titles 

Feasibility Project 
Rrincipai investigator : 

J^ Robert Andrews g M.s D, 
Other Investigators: 

Herman Do Suit^ M„ Do and Stanley E„ Sneidar^ Mo Do 
Cooperating Units t 

None 

Man Years (calendar year 19$B)t Patient Days (calendar year 19S&) 

Totals 2/3 
Professionals 2/3 
Others 

Project Description! 

(fcjeetives s This project overlaps with NCI-I5=000 and will 
Ee conibined wnder one number in the future , Essentially this 
projects have to do with the admission and study of patients 
presenting with unusual (from the radiation point of view) 
technological problems or with lesions of unusual or special 
interest to a clinical cancer 3@rvlc®„ Included have been 
patients who subsequently have been transferred to one of 
the other named projectsj generally a radioisotope study „ 
patients with "bcs*d©rline*' cancer such as the histis^ytos©®^ 
patients with Bgrelonja or suspected i^jrelonaj, and children or 
young adults with undifferentiated cancer of the head and necko 

Fbecept in the case of patients transferred to ether 
projects this case material is not yet appropriate for fonGal 
reporting.. 

Proposed Coi't'SQ of Project; To continue as number NCI-15==000, 



F^rt B included I'ss J^7 No ^/x^ 

1182 



1 o Radi^tlon'^^an'rh 
-"c Rado ThQTc Serv,- 
3. Eethesda^ Md,, 
PHS=NIH 
Individual Project Report 
Calendar laar 1958 



I¥o.^ect Titles 

Medical Radiation Hazards 
Principal Invest igatosrj 

Jc Rdberfc Andraws^ IHc D„ 
Other InvestigatcfflTS : 

Non« 
Cooperating Units? 



Man Xeara (calendar year 19^8); 
Totals 1/3 
Professionals 1/3 
Others 

Project Descriptions 

ObjeetJyes : The entire subject of raedieal radiation tia^ar-is 
i^s reviewed in detail with r©feren(?@ to the diY@r&i. &»rm^tu 
of samatie and gonadal injuayj, genetie defects ^ &xs& radiation 
oarcinogeneaiSo Reccanmendations for reducing medical 
radiation hazards were evolved „ This stui^ was publiahsd tn 
calendar year 1958 » 



Part B included Tes /x7 No 



1183 



Serial No, NO 1=68? 



FHS-NIH 

Individual Project Report 

Calendar Year 1958 



Part Bi Honor Sg Awards » and Publieations 

Publications other than abstracts from this project: 

Andrews J Jo Ro, Medical Radiation Hazards and the Responsibilities 
of the Medical Profession, Medo Anno District of Columbia ^ 27, 111- 
119, 19^8, ^ 

Honca-s and Awards relating to this project: 

None 



1184 



Surg??.";; Branch 
Estimated Obligations,., .,,,...,,,„,...,,,,,,., ,piaoal Year 1959 



Director, oaao.o, 
Reimbursoaents 
Cliaicelo , , , » , 
Other, o , o a , . . , 



Total,,., 



Ij' Includes Projects No'a: 

OFFICE OF 2HE CHIEF; 



10»750 (c) 9»756 (c) 

10^751 (c) 10-759 

9-752 (e) 10-762 (c) 

8-753 (c) 1©»765 . 

8«75^ lo»768 

&-755 Ce) 10^773 



1185 



.4 296,^00 




PHS-NIH 

Individual Project Report 

Calendar Year 1958 



Serial No„ NGI»10"750(c] 
lo Surgery Branch 

3o Bethesda, Md, 



Part Ao 



Project Title: Removal of Cancer and Other Tissues for Histologic, 
Biochemical and Other Studies as Required by 
Scientists and Other Investigators for Correlated 
Studies o 

Principal Investigator: Dr, Robert R, Smith 

Other Investigators: Dr. Alfred So Ketcham and Dro John F. Potter 

Cooperating Units: None 

Man Years (calendar year 1958): Patient Days (calendar 

Totals 1/2 year 1958): 

Professional: 1/2 

Others 

Project Description: 

Objectives : 

The object o£ this project is to supply human tissue for 
histologic, histochemical, biochemical and biophysical studies to 
scientists working in various branches of the National Cancer 
Institute, who require tissue for studies o In addition, it ia^Iies 
furnishing general surgical coverage of patients admitted to other 
branches of the N.Colo and, or other institutes, and assisting with 
their study projects « As a rule, such studies are done as coopera° 
tive projects with various investigators but on occasions, specific 
types of tissue are required for specific studies „ For ensanpls^ 
one investigator requested that specimens of skin and its associated 
neoplasm be made available for keratinization studies, and various 
muscle biopsies and, or biopsies of liver tissue under specific 
conditions have been requested. These materials are obtained 
during operations for standard therapeutic reasons „ 

Methods Eagiloyed ; 

Methods es^loyed are standard surgical procedurei>c 

Patient Material ; 

Material for this project is obtained mostly from patients 
hospitalised for study in other projects in other branches or 
institutes. Occasionally, it is necessary to admit patients f©r 



11; 



Page 2 

KCI°10-750Cc) 
Serial No. 

specific studies in which a specific type of tissue is obtained„ 
There have been several exanqples of this, mostly at the request 
of the dermatologist who wanted large volumes of basal cell 
carcincHO&s for a specific study. Even in these studies, data 
for other projects were obtained; for exaiiq>le, the wound seeding 
study and another exanqile is a beryllium granulosoao On several 
occasions patients were admitted by other Institutes for material 
for special in vitro studies (mostly thyroid adenomas) and 
patients with obstructive Jaundice in order to obtain bile for 
biochemical studies. It is difficult £T<m the data available to 
determine just which cases were admitted for specific studies. 
There were 172 minor operations done to obtain tissue for biopsy^ 
and, or biochemical, biophysical, and histocheralcal studies. 
In addition, there were 26 major operative procedures performed 
for other investigators. This surgery work load was derived frosi 
answering 582 requests for consultation. 



Presented in reports of other projects by other investi= 



Significance to Cancer Research ; 

This project Is more or less a service function for 
other branches of the National Cancer Institute and the National 
Institutes of Health. 



The number of such cases will continue at about the 
present level in proportion to the patient load. 



Part B included Yes / / No /KX / 



1187 



PHS-NIH 
Individual Project Report 
Calendar Year 1958 



Serial No. NCI-lQ-751(c] 
1 . Surj>ery Branch 
2. 
3. Bethe.da, Md. 



Part A. 



Project Title: 



Evaluation of Radical Surgery and the 
Development of New Surgical Technique as 
a Therapeutic Means of Palliative or Definitive 
Therapy of Cancer, 



Principal Invertigator 
Other Investigators 



Dr„ Robert R. Smith 

Dr., Alfred S. Ketcham, Dr„ John F, Potter, 
Dr„ John Dillon, rr„ Robert Hoye and 
Dr. Marvin Romsdahl. 



Patient Days (calendar 

year 1958): 



Cooperating Units: None 

Man Year, (calendar year 1958): 
Totals 1 
Profesr ional: 1 
Other: 

Project Description: 

Objectives : 

The objectives of this project are to develop and improve 
methods of obtaining increased palliation and/or definitive cure 
of cancer. 

Methods Employed ; 

This project is made up of a number of separate parts, with 
clinical and laboratory components. The clinical part consists of 
evaluation of radical surgery, mostly head and neck and pelvic cancer 
patients. The. e patients have proven cancer with no clinical evidence 
of pread beyond the operative area. The citudy involves review of 
specific type, of case recordti which now are beginning to accumulate 
In numbers which allow some analysis of method and re.ults. 



1188 



Page 2 

NCI-10-75I(.e) 
Serial No. 

Cervls Cancer =■ Patients with more advanced carcinoma of the 
cerviK uteri without evidence of distant spread are admitted for 
study. These patients receive as complete a work-up as possible 
and are followed in a continuing study to detsrmine the biologic 
behavior of this type of cancer influenced by various treatment 
regimens o The cases that are operable receive excisional surgery <- 
A total pelvic exenteration consists of removal of the contents of 
the pelvis, including the rectum, uterus and adnexa vagina, urinary 
bladder and all pelvic lymph nodes <, An anterior exenteration is 
the same except the rectum remains intact. The important change 
in operative procedures is the use of an ileal conduit in place 
of a wet colostcsny, in cases in which it is necessary to remove 
the urinary bladder. 

The problem of urinary diversion has in itself become 
a special project. As experience with the method develops, it 
becomes apparent that an ileal bladder has eliminated many of the 
complications experienced in early exenterative surgery methods „ 
The patients with ileal bladders are followed very earefully„ 
A method of retrograde urographic studies has been developed 
Blood electrolyte and kidney function studies are done on these 
patients ;, and studies on absorption of electrolytes from the 
isolated ileum are under way, 

Dr, John Dillon has started an analysis of the cervix 
cancer patients admitted to the Clinical Genteti to study the 
"unnatural history" of cervix cancer, and to evaluate the t^everal 
methods of treatment. 

Head & Keek Cancer - Patients with primary cancer of the head 
and neck area are admitted for coo^lete work-up . Of special interest 
to this project is the group of paranasal sinus cancers. These 
cases are subjected to radical excision by a combined neuro- 
surgical face approach. This procedure has been described previously. 

Dr, Robert Hoye is analysing the group of head and neck 
cancer patients who have had an autopsy performed, and Dr, Marvin 
Romsdahl is reviewing cases of carcinoma of the larynx and pharynx^ 
These cases are reviewed with the intent of determining the results 
of different therapy, changes in the biology of the cancer produced^ 
or associated with therapy, and the ooclal and economic rehabilita= 
tion of this group of patients. 

Patient Material s 

Cervix Cancer Program - Since £he opening of the Clinical 
Center nearly 130 patients with proven epidermoid carcinoma of the 



1189 



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NCI- 10- 75 1(c) 

Serial No» 

cervix have been admitted for situdy. Some of these were 
admitted primarily for the virus study program but a majority 
were amenable to definitive surgical therapy o About 12% of 
85 cervix cancer patients seen here before May, 1958, were 
found to be inoperable on admission because of supraclavicular 
lymph node or lung metastases. Only two patients refused 
treatment. Seventy- two patients (857.) were explored, and of these, 
51 (71%) had a definitive operative procedure carried out. Thirty 
of the 51 (58%) had a total pelvic exenteration, 8 (15%) had an 
anterior exenteration (leaving the rectum intact), and 13 (25,47.) 
a radical hysterectomy and lynq>h node dissection. 

As a rule, the cervix cancer patients seen at the Clinical 
Center had advanced disease, about one-half of them represent 
radiation failures and only a few can be classified as Stage I or 
early Stage II. Survival data of this group of patients are still 
incomplete. Our experience with radical pelvic surgery at this 
center is a little over five years. The longest follow-up of a 
living patient following total pelvic exenteration is 54 months, 
and two other patients are alive over 44 months. Seventy^eight 
per cent of the deaths (18 of 23) were due to advancement of 
local recurrent and, or metastatic cancer. There were tw© opera- 
tive deaths (2.8% of patients explored, or 3.9% of patients who 
had a definitive operative procedure) . Three deaths occurred from 
unrelated causes in patients viithout evidence of cancer six months 
to 34 months after surgery. Sixty-six per cent of patients x^jh© 
died had distant metastases on autopsy, (7 of the 12 had generalised 
metastases, while 5 had only isolated metastases). Seventy-eight 
per cent of the group who died had local recurrent cancer in the 
pelvis. This study did demonstrate that the wet colostomy method 
of urinary diversion is not satisfact