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



NATIONAL 



1959 



NA'nONAL CANC. 



: ; ;MAT10NAi:«r(WTl'ES m HEAWH : ^: 



.:^^u 



Indi-?ddvsal Project Report 
Caleindar Tear \%9 

Fart A» 

Project Title J Stodies on the tIteI etiology of ne^ls^s 

Principal Jiwestigators Sarah Eo Steiart 

Other Birestigatoraij Bo Eo Eddy, BS^LYPjHIH 

Cooperating Units: Sloan«£ettering Institute, No lo 

Man Tsars (calendar year 1?^9)» 
Total: S 

I^fessionali 1 
Others it 

Project Descilptiont 

Ao Work on Po2y<M® vimsi 

lo Developaent of resistance to S E polyoma vires o 

la the early work «ith leukemia cell^free extracts it isas 
obSQ2rved that of general litters of nsrsbem niice iaocwljated with 
certain parejarations, all jsdce in a given Utter naght dea^slop 
tusaors while the rest of the litters rei^Sned negative o S"aeh 
results hare been interpreted as a litter effect fcgr Law ©t el 
(Jo Nato Cancer lasto 16 s U95, 1955) », Wo haTo now shoim°°&^ 
this all»ar-nonQ re^onse was probably not due t© a pecularitj 
of the litter of isice inoculated but m.s dae to the mol^er ef 
th© littero In mai^ laboratories Kher© the tumor^indtscing agent 
was not coafflidered as an inf aetlQ»i^ ':"druss the f^aale 2d.ee tmt 
had norsed an inocslated litter ^©re reased for breeding o In 
oar experience mice tiiat had nursed infected litters «@r® ne^ar 
reused (Jo Nato Cancer Insto 15s 1391^ 1955) and idLth tissue 
celture propagated polycBsa -elr^s SO^IDO percsnt of h^bxdd 
(C3Hf/Hen X AKE)Pt, "^c® injected deireloped multipls types 
of neoplasias (Jo Mato CaiKJsr last,- 20? 1223, 1958 )o 



541 



7 



In more recent studies (C3Hf X AKR)fJj^ l^brid jnlce wars 
found resistant to the oncogeexic effects of polyoma virtas after 
it had been carried for 3 to 13 serial passages in tissue 
aultures Bi&de frcsn S^lss mouse embryos^ -whereas SuisB mice 
retained full susceptibility to the virus. It thus appeared 
that vMle the virus had become adapted to the Sviss i&ouse b^ 
passage in Sviss raouse mbgyo cultures, it had lost the &bilit^ 
to infect the hybrid miceo Esperiiaente were perfoxmed to 
explore this possibililyc It ^s fcsind that ^e resistance 
to the oncogenic effects of polyoena viras after it had besn 
casr^ed for 3 to 13 serial pas^ges in tissue cultures roads 
from SHias siouse einbryos, idiereas Siriss mice retained full 
susceptibility to the vlruso It thus appeared tteat ?djile 
the virus had becoioe adapted to the S«is3 mouse t^ passage 
in Swiss mouse embryo cultures, it had lost the abiHtgr 
to infect the hybrid ndce, Ijixpcrijnenta were performed to 
explore this possibility. It vas found il^t the resistance 
of the hybrid nice vaa due to maternal antibodies tzansssitted 
to th@a in the milk of their C3Kf/Hen scthsrsc Although 
Hie st£%in G3H/^en had not prsfviottaly nursed an inoculated 
Utter, they Hsre housed in an en^lroxsnent contaadnated ^th 
polyoiia virus while the Swiss nice were not. The Esilk of 
lalce ^lich had been inoculated with polyoEa virus had as 
high hemagglutination inhibit!^ titers &@ were foimd in their 



In a colony of mice with latent poly?a^ virus infection 
transfer of maternal antibodies te the offspring fcgr «ay of atdlk 
may account for aiy irregul&ril^ of results following inoculation 
of this -siruso Maternal antibodies Eiay also explain the lack of 
spontaneous parotid gland taasB's in an infected colosny, as it is 
cosauion for newborn Bdce to nurse almost i^aedlatsly after birtho 
Maternal antibodies transferred to the of^^ring rmj alio ea^jlain 
the absence of these nsGplassis in the offspring of tumor bearing 



2o Tissue culture plaques of S E polyossft virus. 

The cytopathogenic changes produced i^ S E polyopia viri;^ 
in mouse esibsyo cultures make it possible to ^p3y the tissue 
eulttire plaque method for deteTsoining if only en@ virv^ produces 
the spectrum of n»)plaass obsezved in inoculated mica, Dr,B 
has shewn that In hamsters plaqued virus produced tha same 
i^es of neoplasms as that not plaqued. Since in hamsters the 
virus produces oily sarcomas end vascular lesicsis the res^dts 
are not as <»nelu^ve as they would be in mice. 



542 



Pol^KBa. ■?irus used in this study «as subline #6069 
derived from a Sid.8S moss® that had bsea iajected shorfe3y 
sf tsr birth with ^^irus sublina #3ii65> which had beea passed 
through a 220 muo gradacol filter p During a 2 month pes'iod 
the moasa dsTeloped primary neqplaaBss^ nssjigr of ishieh war© 
multiple, Thasa consisted of lavLltipls laassajy edsnocsretooKas, 
hair follicle i>pidernioid earcinoaasg edpidermoid carcinasia ©f the 
gingiva, ple^iorphic tumors of the parotid, exorbitai lacninal asd 
tz^cheal submueosal glands, tfcgrroid casxineaa epithelial thgnaesiap 
EiesothslioBJSs of ths pleax«, renal aarcosaas, renal tubular 
lesi<%!3, aad osteogenic sarccssss of the skoll aixl spine. The 
iQOQse t^s killed idien 66 dsys old aod a culture t^s scMe in 
mouse embxyo cells frcsn a since of one of the parotid tuacors 
and one of the Tnsm&rj adenocarcinoesas. fha supernatant fluid 
froB the 8th passage in mouse erabXTO cultures vas used in tli@ 
plaqse studies, The technique for plaqssing has been described 
by Dr, Mdy (ProCo III Canadian Cancer Cenf, 1959). 

Fluids from cultures made frran 6 different plaques 
inoculated into neii^bom S»lss mice iodoced tumors in 86 percent. 
Little "variation ^sa noted in the tgrpes of tumors observed in miee 
inoculated ^th the plaqued and non plaqued virus . Htce that 
received virus froa 3 of the plaques appeared to have a greater 
varie'^ of tisiors than those that received th© othersi howevsr, 
the difference hetusen these and the other 3 plaques tfas prG^ibly 
qmntitative rather than qualitative as the aiajorlty of the 
triors vhicfa have been ohssived appeared in sosie of the sdce. 

3. The histogenesis of epithelial tli^measms induced 
in jaic© inoculated with polyaaa virus. 

This tuisor baa been observed in about 30 percsat of 
inoculated mice. It occsrs as a circBEseribed tli^yisic bbss 
which in sasalltaasors is seen to arise in tt^ medulla, fh© 
lesions ^aiy in sise frj^i siicroscoplc foci to rnasses which almost 
ccsaplatsly displace the lungs. Mlcroscopie szaisinatioa of serial 
sections isade frca early tt^ors indicate that their origin ^&j 
be in Bassall^s corpuscles. An increase and a l^rperplasis of 
these cells %as observed in scsaae early tt^jors. 

li. CSieadcal nature of S E poly«sjm virus. 

In collaboration ^^i-th cheialsta froia th® Slean^Kettering 
Institute and Dr. B. Sddy^ HIH, th© following results fe'sr® 
obtained on the chsadstry of polyoma virsej 



543 



An jjifecticus nucleic acid rsaistsHt to tha action of 
ribonucleese (Mass) but which was ijmcti-B^ted bgr deo^xibonucleasa 
(DiasQ) was isolatad frcaa fflouse ejnbiyo caltures infected ^th 
p6iy&s& virus. Two inethods of preparing ns^leie acids £rtm the 
tissue cultores »8r« esspltir^eds th&t of Gierer and Schrfinm 
(Natisre 177 s 129?, 1956) «ind the one described by Kirbgr (Bischeia<, 
Jo 66i h9$s 195?) » Both g&vs •ttie saia© rssultSo Control nijcleic 
acids were prepered fswa non infected ciaLtureSo Mouse einbr/o 
cultures inoculated with the nucleic acid preparations frcsa the 
infected cultures yielded intact polyoma -^Irus with the HHaee 
treated preparations ajid not with the nueleic acid treated with 
DNase,. This M&a demonstrated tgr <jytopathogenic changes in tissue 
cultures, ty hemagglutiMi'^on of guinea pig erythrocytes ©ad by 
tuBor inducfelcn in haiaatera and raiceo Neither enigrme was foimd 
effective on the intact virus o 

Bo Work with humian neopl£3B»<, 

le Oellular proliferation in tissue cultvsre induced 
with a substance dorired from cell-free concentrates from h&mxi. 
neoplastic naterialc 

Focal areas of proiif erating cells or pocks were produced 
when c^l«f?ee c<siceatrates derived trtm 2 different hmsm nec^MmB 
and from the urine of 3 children with neoplastic disease war® 
inoculated into sionolayered cultures prepared from priasffisy 
tzypainised cells from hmmn smtlai. sM frcsa h^^san eiabsyo ndiica« 
!Sis cells i^re snaintained in Earleoa ©ediwa 109 (HCTC) contsising 
$ percent horse serum and antibiotics o The increase Jii." tii>ll 
aultiplication caused them t© pile up f<midag discrete foci 
sessetiBies measuring Od5 cnio in diameter, i^ieh were scattered 
throughout the aonolsyer of nox^ial cellSo The first evidesace of 
cell tsonsfomatlon in both types of cultures ^s a lm& of 
Biigratics! of the cells resulting in a whirl; this is shzmi in 
the first figure o The continued grc^h of -Uiess foci in aianicQ 
cells is illisstratsd in figures 2=ito 

^opathogendc changes were usually observed in the first 
few tissue culture passages o They were also seen in control 
cultures idiich received similar concentrates prepared frosi hmmn 
amniotic tissue but were found only- in cultures witli the original 
inoculum and after the first passage <> 

3 
The tiaaor-=c©ll=free concentirates were prepared bjf a 

3 ""¥^©16 cells" were noCobVeiwed'''''om"''e3Siii^ 
after Spinco centrifu^tioQo 



5^14 



modification of th© lasthcd described i^ Moloaey (Jo Sfato Canear Iijsto 
16 J 877, 19$6)f for purification of the Ho«s earcoaa Yixnso The 
uslne eoncenfesates wera prepared as follcpsrss A morning oriixs specimen 
■sdth the pH ad^justed iio 6,6 was caiitrifuged in an IntematieaRl 
refriges^atsd centrifuge at 3000 rpm (lUOO x g) fee 30 sBlnutes at 
0° Co to resBov© all cells and gross precipitates o The cleared urine 
tsas than centrifuged in & Spinco centrifuge at i}.0,000 rpm (101,000 Xg) 
for one hour and the pellets nhich imre asually clear and gelatinous 
were suspended in a •yelume of OoO^ M sodium citrate, in ^shich the 
pH had been adjusted to 6„6 vith citric aeid, so aa to give a 20=fold 
coi^ents^tion of the naterialo The suspended pellets -mTQ then 
h^ogenized in a Potter^Elvehjem hesaogKniser and the hcaiogenats sms 
centrifuged at 3,000 rpjn (IhOO S g) for 30 minotes at 0*^ C. Th© 
si^raatant fluid >*ich was generally clear or lad «i3y ^i tCLi^M 
opaleseenee %ss inoculated in 1-2 lal. amounts into 2 &%, pfescr^tion 
bottles ^iSx the inonolssrered anznioai or human embryo s©lls and were 
Incubated «t 37® C» Tumor ccneentrates ware inoculated in the same 
roannero Serial passages to other Bonolayered ouLtures were sad® 
by transfer of 1=2 mlo of culture fluido The fluids were ^langsd 
at 'Bsakly intervals^ 

Proliferative changes Tmx@ producsd \^ fi^e specisisnsg 

(1) A surgical specimen Snm a papillomatous gro^h of the 
tcsjgue in a 73 yeax old malei the patient also had multiple 
epidersQoid earcinceias of the buccal mucosae and a rectal po3^<> 

(2) An autopsy specimen from an esibryonal tuisor of the liTor 
in a 22 month old childo (3) Dilne from a 6 year old bey 
with r^n^ l^ngjhoqytic leukeEdao (h) Itelna froa a 6 year old 
girl ^Hi a renal neuroblastoiaao (5) Urine from an 8 year old 
girl id.th H^^cins disease. 

The cellular proUf eratiTe changes produced by the papillras® 
extract were observsd after the second passage in aimioa cell 
cultures and on ths initial culture of t-h© hmm,n embryo aft©j? 
2 ^raeks" iaculaticaio Tha call transfoiaatiKi produced by the liver 
tumor concentrate ^^s not observed until after the $rd passage 
and vas not reproducible on all traisf ers until after it bad 
been carried for h weeks in the husasn embjy© eultiareSo 

The cultures inoculated vith the urine coz^entrates were 
slew in sho^dng a cell trs^isformaticai @M the initial changes were 
observed only an the huasan ©abryo cultures. The ©ri^sal cultures 
"Kere reta^siirlsed 3 to U 'feLiaes ®ada duzlag a ii=5 Konth period o 
Focal arfJBs laLth abnortnal siigyation of the cells ifere observed in ths 
inoculstsd cultures in 2 to 3 isoathSo Transfer of the culture fluid 
to ani8t*^i cultures produced the saa® f^al Issions as obseired 
uith -^e tvmxxr concentrates o 



545 



Although sffi'ial passage in tisstte c«iLtia''e of the proliferative 
affect is readily acccaapliahsd l^ use of th3 culture fluids it appears 
that cellular p&rticles or cells are ascessary to produce the effect 
(sfraa though iMtially the i^tsrial i^s cell^'frQe) o Acti^rity ia lost 
by iiltrati<m and fcgr contrifagation ot t^a tissue cultor© fluid, 
Othsr ixmtsmaes of aimllsr behavior Are knoMn to occur. It is ksuBnm 
that in order to transfsr TariesllA idxus in tissue culture it is 
aecessaxy to carxy over gbH fr&^entSo Also influenswi Tires las 
been shown to exist in an incomplete fojsn (noa trsnsferab3^) when 
groHsa in certain cells » At present na do not kno» -yae aatasrs of 
the gr«fth accelerating agent bijt studies ar© in progrees which 
indicate ttot the cells transf osmad by the papillcsm agent ha^s a 
specific antigen which is deiaonatrabla by isse of the fluorescent 
antibo<^ mathoio 

2o Uterine and gastroiatsstinfil lesions observed in 
hamsters inoculated ^th fluids frsaa tissue cultcras t^ich had 
raeaived preparations from huaaa autop^ usterial fnsa. patients 
that had died of c&ncer. 

From a total of 263 inoculated haiasters of both sexes that 
hav© been autopsied 171 vere fenslese Of the 1?1 femles 18 
percent derreloped a hemorrhagic lesion of the uterus o In nany 
of these there isere ssacerfited fetuses, ssveral id-th extrusion 
of one or more fetus into the peritoneal cavity » Hemorrhagic 
lesions of the Gdo tract hsfe also been obserred in xbs^ of 
the bainaters of l^th sexes » At first it sjas belie^ired to be of 
the sams ethology as the uterine lesiim^ but it has also b®@n 
found in numerous control animals , The uterins lesicai has been 
seen in oa3y k of 90 control f eEaale haasters T^iich Mve bQ®a 
autoj^iedo 

A histologic study of these heiccrrhagie lesions is in 
progress o 



Part B included Tes 



548 



serial 1^. W^l-^3% 



Gslessdar Year X959 
Itert S. Honors, Amrds, acd Publicati<ms: 
FuMlcatloms otlser thea etetmcts fr^a this project: 

Bsriodiesls; 

Stsi^rt;, 3. E., EMy, B.So, Ii^Jia, M., aoS Lse, S.: Hie develcEMsfc 
of s^sis^sse la laiee to ttaaor Induction by S S polyom virus « 
Sattsre Cia press )« 

Ste^rt, S. 1., Eddy, B. S., waS. Staatffia, M» F : Seoplasaas la roteits 
iiadtieed by S E poly^ viFUS. Acta Uao lat^raato co»tr& caiiesr 
15: 8^-^, 1959<. 

mmyorca, G.A., Mdy, B. E., Stevayt, So Sa, Huntsr, W. S., Friesad^ Co, 
aea Beaflicfe, Ac : Isolation of is3fectio\ss deoa^ribon«clelc acid 
froa S E polyma lasfect^ tissw sultar»8» !<, A pi^liaisaaay 
r^port» Proco fetioaal Acad. Sc,, Decsster 1959 (coMmsiieatsa 
tjy Qilbert Dslldorf ). 

Ste-s^rt, So S. asad Mdy, B. S. : TvmoT iMtxtioa by S S polyisM virus 
^sd tl© iidiifeitlcja of ttraopB by specific i^ufex^izissg aatibo^ieso 

^ M. So Pisb» ^sath h9i :£m^}^K^ 1959o 

Stsmrfe, So So, Mdy, B, So ai^ Stassfjoa, Mo F.: laductioa of rao^ls^s 
in laice ©ad otSier saamls l:y a t^Esor ageat carried in tiss\'^ 
eultura. Parac. ^ird Canadian Causer Conference. 287-305 j. 1959 « 

Eddy, B. B., Stewart, S= Eo, and Stantoa, M. F.: Induction of t«s»s^ 
in rats by tissvss culture prc^rations of S B polyo^ viw^o 
J. Bat. Cancer Inet* 22: I6I-I7I, 1959o 

Eddy, B- So, and St®^rt^ 8. E.: Physical properfeiea and bsm^wfeimtis, 
6S1& eytos^ttoaggaic effects of the S E polyoaa vlru3« Ps«^o Third 
Caaadiaa Cancer Coafersnce. 301-3^^, 1959^ 

Mdy, B. 2., St®«art, So &., liiS^scbstein, B. L., assd Ifoiasg, K. S»; 

IBfl^M5tioa of subcutsaj©o«s nodules in rabbits ^th th@ S B polyosa 
virus- latu^: 183; l^-l&T, 1959. 

Eddy, B. E. and Stemrt, S, So, Chametaristics of tfeS B ^l^m^ viraso 
Mo Jo P«bo Ifflaltli Si^: ikBS-liSS^ 1$^$. 

mdkat 

Stewart, So go : Polyt^ Virus, Section A* 2a Advances in 'nnm 
Baseareho Aeade»ie Ps?©ss, ^v ¥o2k (iE press )o 



gte^rt. So lo esA m&j, Bo go: A ^vl@^ m. th© m©l©g^.Qal Fx-opgrt:!©© 
(la press. 



|)ress7) 



c:)i7 



3o - : ia, Sd« 



GaleiGiar Xsay 1959 

F^jeet fi^e: DM ©ja-fehssis is s^aaai aoS ae^lastde tl8S«® 
^ cells. .Sjmtbesi® ©f pb&sphoigrla^ BM 

Frimipal lawsti^torr K«^ K^ l[i«lley 

Ot&ss® la^sti^tor; ¥« C. Scbneider 

G«5ep«:ss.tiag Usd1^: Icssi® 

Maa YsasB (calej^ar year 19^}: 
Sot^: 1 

Frof@s!Si(^ial: 1 
(^^©r: 

Protest fisscrlptissi: 

Oltjcctivies; SJ^ loog j?aa^ o^^etstiv® of ^is study is to ^Ib 
soias \isi@3r3tai2dlBg of ens^oatie eozs&rr}! ssectaanisass in TMk S3m.ttesis 
^ vMeh cell gsftjath sod latiltiplieaticm are r«gula^&a. Bseeat findings 
ea tfee j^tm® of MA, pi^eursors iMicat^ tba-fe a stMy of -fell© fosa^ticaa 
of deo:g^ueleotids tslpt^spfeatss ia ,a©ga^ gj^ tuasr cells Bj:lght 'te 
feasible aM wytSMiile at this timu 

The iissdiat© objetstii^/iss v»;re coneeETOiid witSs (l) Sis-rolofpffisat ©f 
&a asssiy s^tlK^ to detect sjod a^asuse &£03^z»acleiotid@ kisms® activities 
ia -mslcms kii^s of i^ss^ 3zsd tv^or cellsK aM (2) dewilopsssssti ©f 
ij^iTo^^ E^tiiOds fox' isolating an^iei in '^M.ch -msloaas ©Gfei-rXtier-- 
leading to DE^ ©jpatl^sis Eight te 8t«ai©t, 

Ms-yaotoi ^2@ a@4^y ss&tbcS useS t^ lorabas^ for @aasuti£ig 
d@o}!^^£el@oti§@ kismse activity ia extss/gsts of S<. coli •was @@l@etefi for 
this st«ay« Ttm astlsad g^^ads ©a tl3.Q -as© of p32„3_afe@lM teosgfsaasliiotiaas 
^tddeh ar@ ccmverteS t@ tripho^batss '^ Maass setitm ia 1:^ p^g®i^^« of 
AS*. 2te sstlKJds use^ for pjfspai'at^.ea of p32.3iafe@ied ^;@«^-:^iiicle©tid©© 
ves® follotssd tte©ti^. Il^s® ijaclvjieia ap^s-eial Effit&o^ fes* culttsslBg 
So' soli ia p32, leola-iiGo aofi hyd?olyais of Isbelod DM to 5'"^eo3^» 
SEml^otides, ^pas-atiora of tiss ixi£.ividual iausleotiS@s 1^ ioB ©xcteBg© 
ohs^^tDgi^phy sM id@sct;ifieatiQ» ^ spsctx^ ebas®£t@slsti@s« 

I^^SsMs for ieolatias asu^l^i iavolwd suspa^iag th@ sells i^ 
^aspiotas Isisds of sjodified 8«ex©f3# ©oliitioBS;, x^txsxlj^ tfee cells fey 
■wa^imss ssssteBic&l swaas ®^ s«p©gutJ^^ t&^ isijsslsas' fssctioa 1^ 



5'-l8 



» 2 - Seifial too ffiS-^Ofe 

intact e@ll ¥@s@ ei^asldarsd desixable. 

FriEcipal flMSg^^;. ©as tecteaiceO. pro'fel^as ii£volim& ia tS« 



ps-epamtie^ of S^ox^stocXsertMes S'soffi tia^ffit;^ asid of 

of P^-la^l@d Bt^l®otig@3 fstxa 3. eoll ba^ Isesa satisfastorlXy 

low p&ospbat® Mdit;® for F^ lBcosep©3?ati<», vigoroes &®gatie® stjela as 
is po@@iM» isi & ^s "meiM^ eyllM®? is zj^seessasy. !:^i@ glasswit<@ 
aeed ssot bs alteolutely stssH.®, iHst aa laitially ©tesdle a^aium aM 
Ix^ofiul^^ of a pure c%a.ti£Fe of 1. eoli Stsuin 3 are t^cessas?^ to avoM 
l&Tm aisGusts of acid prod\^tion in t^ sssdiusi b^ exfexnoe^nss organiiszos 
^ihich can cause g^onth to stop "b&foxm tb® phospimt^ Me ^en ®:^ai;mt@!i 
fi^tt ths ssediwa. At a pbosphate eozaeesstsaticm of 33 ;a affllss/lOO eiIo 
iastead of 80 ;i a»les as gejsesally vis^ for p32 i^efposatitm, tSi@ 
txtilizatioa of phasphate iato MA is sare ©diciest , test this is ©ffs@t 
1^ th@ la?^7 voljam of etalture s^ui3?ed to produee -^i® ass^ axsmmt of 
WA, Wo special difficiilties -mm «J3ematss®a in. ps®pas?ing WA- 
mESl@ops^t9isi IbQT al&alii^ Si^etiosi. a^ ps^cipitatioa vWa pescbloric 
aeia, Sa tbi hydiolyBis ©f DM afte? B^as« aigsstioa, l?y di®at©s®s@, 
it ie ffiee@smiy t!sst tfc© diestsrase \^ fre® of 5'-s8ttcleotiaas«. las^ 
@si@l@<3tida@@ aetiviti@s -imm feus^ is tb@ diestsxase f xaetioiss dS^taima 
%gf acet<23© ps^ipit&ticsa of sBak& vejSKu after the l^cpMlis^ vwmm imd 
1a8«a stos^ tmzmn for soa® tiass. Si® aueleotifies® aetivity isas 
fiestsfisysia by bsatlag tS^ diesteras© soivstioa for 20 mixast®® &% 60® 
at pS 6.5 ±a i^tm precise® of s@snra all5*gata<. IHje pTOcipitat® ^ra® 
e«ntriffu®ed off mS. tte el@ay sispermtaat eolistioa m& dialyz®* ov^sfiii^ 
at 5® ffi^iiMt O.oe M fxla^lCl. buffer, pH 3.86 ®!© ps^amticaa has Imea 
tept froz«a for a asmth *5d.«ioj«t loss of activity, i^arly quaatitatiw 
Isytosiysis of 1. eoli SHA, to 5 ' -deojq^mi^leotides ^aas obtaima by -^jg 
coabia^a aeti€»a of ^s© aM dlestes^sa. fl>e issdi-^ldwal m^l^^id®© 
^s« s^amtM \^ eolvsm ehasaatogssphy oa Dowsx 1 <-2x iB tb§ ®i-ffil 
scslv^t systsffio Fs<® a di^st coataiiii^ II.6/1 isolgs of ss«eleoti^«s, 
1,8 ^ saoles of d-CMP, 4-®IP aM lesP wre ©Mslaed. d-AUP yarn isst 
s"ec©-!}e2^ wster e^sditions ps^vailtng at j^cva 



®2S f ©IKwiag j^thod has tm& Agvel^psd for isolatlsg atselei 
fr«i aseitss lJsrg>at@B^ sells.. 

Solufcioa 1 « 0,25 M sisero©®, 0=003 M CaClg^ 5 x 10°^ M IfeClp^ 
0«0I M Mstidim, pH 7«^. 

Sol^ioa 2 - 0.25 M smtom, 1 x lO"**" M CaClp, 2o5 x 10°^ M 
Mgeig, O0O25 M bistidirag, pH T.^. 

Qss& ml. of 6^i!^i£^€d ceUs fr^a 3 day t:^m£iplaQts as© ^sa^bsS 
twiee -^tlj 5 ^©l^ssgs of g©luti<» Ij -yisa r©stjsp®aded to 10 sale ■^^th 
©ol'orfeiaa 1. To -ais s^i^piai^iaa, 0»CS g., of ^.gitsaSa sots a/^^ aefi tiw 

549 



" 3 - ferlsl Bso mi-k(A 

A3J, o^sralJisas si® ©arriet otsfe ia t&8 eoM (O® «■ |?®}o fte B^m^^&n&lmi 
is psistsesi th^ou^ a So o 22 ijafpoSssBic s^^l® imtil :®o®t of %te c«lls 
as® bSfs^MS ®^ ■^5® a;B8l®i f©s* 1^ ^sst part aw fs"@® ©f adteri-sss 
e^rtopla^e stes^* fbs ai:^1®r of passes tSisou^i tlie lasoCL® d^pes^a 
<m f^ parfcioalaT e@ll pfepasstic® asd tbig fore® wsed to puisfe tOs® 
st2sg«igi«sia tteeu^ tte Be®ai@4 C®11 p2^^|>a2«.tiois® froa ts®]£ffi:^tsmtB 
tt^i^ oiaer tbaffi 5 daj?® aye sbs^Sj asos?® s^sistasat -feo feseatege.. 0®ss©5£>allyy 
fas* 5 ta^ -teassplaatsj; 5 to 10 passes ar@ s^^«i@at to bs^aSs: ^ist of 
^i@ G^USo 'Bm lm^@3& cell siagpaisasiosi is c@^t£lfs^s^ at 1^)0 s^^ for 
7 sisutsSSj tte si^p@®aataiit oolufciOQ is plpstted ©ff sssS ^iSm s^&M&r 
©g^fesist is s^sssigeatea t® 10 ml. ^-^ soltzfclmi 2. fl8« smLs^xisicm is 
a^lB pss^sfi m-^s^ tfcis^s tlsE^aag^ tb@ a@€«LL« to f^my® «syt<^lEsM© 
t@^3 ts^<m m^h&t @^ to bs'sak mi&t Qf tls@ r^aJU&iisg ^it&l® e@lls. &t^T 

^iMsh is reMtivsly el@&a. a© aiscl®! ia sel^sfeiem 2 ai?© raas^aMy 
st@M® id-thatga^iBg 3figp®atsga ^^asMxsg ai^ «st©s?agi® ia tte eoM f©r hehj 
4ay®. AEaljf^is for cytoeh2«^s ©xiias© aM ASas® astiirlti^s slics^^fi 
2 to h pes-eeat of tJiie total bo^togsaste setivity ps<@s@at iB tM tmsle&T 
f'mc%l(m. la ®vM€bc@ t&st ttese auclei caa sxipporfe osidati-ve 
pliosptos-ylatioa isa^ fe'iffla» It s'^aias t© te s@©a ^d^^ter ^^m^' pois®8©© 
setiTiti@s ia m^l^ie ©sM a^sBt3te®i©» 

&2odificati®is of ^^ s'teov'® proc®dure Mv® team ^ag«^ to i»ola.t® 
msslsi f»» 2©ZB^1 &M s®@®m>xis'feii^ s?at li-s®? aM fisssa »Yiteff 
bep-a'feGssa eells« 



S3.fflifica^@ tp^eajMigig; k sti^ of t2s@ ^nz^s^B ismsll-^3^ isa M& 
s^TH^tesis is Esssiffil, aiaa ttsaor e@lls say la@lp t© ps^vi^® & tesis for 
isaSssrstaMisg tM Batusf® ef tieecat rolled gjrewtls i® caaa^ff.. Ato-ro all, 
"Us© lEstatsolle aetii?iti@8 a£ tfe© csiscer c@ll apg^af t® te ^a.i?@d to 
BM @|atl3De@i@ as ^3@ of its ps-isiei^al activiti@»o 

g^«CT_ ^@@as@h; a© Siii€i3;ymKjl®®ti^ kiiKas© aettviti^s of 
•mxicms t2fp®s of Bos^al ass^ tisssr ceils 'aiH 'tei foiir@stl^te«!. ^th tte 
assay ^tbc^ sKm d©wl^?©i asd attes^ts ^11 fe® ssad© to d@t©ssd45» 
©f tl:^ eoaditi^QS ^liicfe isfl^ss^:® thsse aeti'^ti@s ia eeUs. 



f^T%. 1 iael^^eS; Yes ' m X 



550 



Serial NQq mi'-kpf, 

lo l^b©^3E^""o?""BT©l'oiF 
2o Ollulsr Biology S@®tJ.oa 
3o Bstiiesda, M» 



lndi"sldual Project Haport 
calends Tear 19^9 



iS^^J^ 



Project Titlag Electron mierofleppijr of noraial and raaligaant 
cellSj including sttsiies «ax ^nsBeso 



Bdacipal Inreetigatopg Ao Jo Dalt«a 
OtSi®r Ismsstigatorsg 



Co©s>amtiag UJaitag 

Bsyaa (MCI=420) 
Potter (NCI4427) 
Moloney (MCl4il0) 
Maaak®? (MCl4a6) 
BelMn 

Sanf©i?d (NCl4i28) 
St9i®JPt (HCX-Ii33) 
MssTdn (NCI«4ait) 



Man Tears (C&l -jndar year 19$9) i (A«go 1- Dec. 1) 
^ofcal« o83 

Professlomlg o5 
Others o33 



Project Descriptiong 

(1) !me stffldy P* plasaa cell tumwa In collaboratioa 
with Drso M©l©nagr and Sfersdn, began MXh Drc Potter in lf$7» 
has continued witSiowfc interf^iption daring thiai interwalo Matarfjal 
so far anaJyaed with the elecxtron mics'oseope iacledee two Pilgria 
tcffiorsj two turners f^m Dro Dmnj, five ©btaiasd l^r Dr^ Marwin and 
one derolopad 1^ Dr, Pottero Among these j, foar ^®r@ »spOffiUaeoMffi''s 



551 



= 2 - NCI-ii06 



S8Sd six ars considered to ba^e bean iBducedo Eight tumors arose 
in Esice nhich prestoaably ca3:Tied th« fflasBsaaxy taaor sgento The 
oth^" tsfo arose ia aaiamla fyee of this ^igsnto 'WMIq vas'iaM.ons in 
Gltrastructaral morphologsr were noted sjaong thes® tissaorSj th© 
differences coiald not b® eorrelated lailii tha differences in serum 
globalin patt^HS® induced in mice ca&nying the tmwrs^ A fefctar® 
e$s!2Q0Q to all t\smvts so far exaniiaed has b@sa the presence of 
■^Irus^like particles in the <^ftoplasm of the tumor cells « The 
particles vea^ f omsd to be formed on the fflembrsaes of the e&do» 
p3asmie rstiettltsa, the membrgme forming the outer shell of the 
particlac It ^as thought &% first ti^t the preeeace of thse® 
psrtieles eoisld be ccrrelAted nith the f&et tl^t all of the tumors 
arose in sales carrying the immmry t«mor agent^ but ^eir presence 
in eell3 of toilers arising in sdce free of the agent slimin&te 
this possibility. Forthermore^ bioasss^s m. cell f^e exts^icts 
of se^ral of these tumors can now be em)sider@d as imving 
derooiastrated the absence of the ts/aams^r tmsw agent in the tiaffiors 
tested. The sigrdfic&s^e of tJiese particles is taaknwn %t present, 
but ^sir regular oceiKrance in plasma cell timore and their 
absence fTtm vmv^l plasma cells suggests that thej are related 
in scene v^ to the neoplastio state of the plaszoa esUSc, In 
eonnecti<m tdth this atudr & search is ecostinuing for a sois'ce of 
huB^n sicreloBaa cells plentiful enough to allow analysis %dth tha 
eleetrsn mlcroscopeo la addition, with Dro Potter a staiy of ths 
cytogenesis of plASsa eell® -ssith the ©leetron Kicroseope is ui^er 



(2) ^th Drso ISDloa^ and Manaker a ®tts3y i® being ©airied 
out on the awMphology of the Molonigy leukeada agaato This agent 
has alyeajfy been claract^zlsed tentativelyj but further work is 
necessary before a definitiv© doscription can bo gi^«Eo In the 
saterial e^sucsined so far the particles cci^idered to be the 
infeetiT® agent Mv® not been j®rticalar2y noDSBrous but they 
have b®ea identified in spleen, l^rssph node and thyraus of infested 
aaimS^o It is hoped that with the us® of tissue ealture and high 
pot©iQCsy tanaas's seai^quantitativQ studies tmj be ^awisd out ^lish 
should answer ths question as to whether or not -Ui® psrticles 

are the infective uraits of the Moloney le^emia viruSc 

(3) IiJith DTo Sarah Ec Stewart j tissue cultuz^ preparaticBS 
.of hmmn sm^esi aad j&ouse esibryo cells %hich shoiir s^esAthogesdo 

effects foliating e:!q>ossr8 to ceH-fSr©© extracts of hu»»n taaas'© 
jBre being ©^adned for the j©ssible pa^sen®© of ^dnxS'^lSJc© 

farticleso To date three sai^les of zoaterial have been exajsined 

with sss®ntial3y negati'?'© results o 

ik) A stady ■sdth Dr= Sasfosd Mb jast been initiated 
^hieh iacludss sn aEi%s5^ of a stamisi, ©f tissue owltur© 
eells grcssK from sialifasy glsai ssplsatSc fhss® esHs ^iU b® 



552 



3 = NCI=J406 



studiecl following infdction with tho po2ycs!s& vims and ^th the 

twsor agent. No reeults have been obtained to datOo 



(5) D?o Belkin had previously foand that S°3? ascites 
cells developed n^siosroiss qftoplafiniic vatsuoles eei e^osure to 
certain plant polysaccharides » It %as considered of interest 
to detensine the site of origin of these vacuoles ^hidi in turn 
sdght be a lead indicating the cell eoraponents acted \jp<m ly the 
polysaccharide e An electron inicroseopie asmlysis has been 
initiated with the aim of settling this point o Preliraiiazy 
result® indicate that the vacaoles which becesae visible with 
^e light sdcros^pe following trea-bnent id.th the polysaccharide 
actually are derived at least in part from pre <=exis ting sviixal^^ 
scopie vaaiolea present in untreated S°3t cells o Marg^ of these 
pre-=^8xisting vsewoles contain vlrus=like particles in various 
stages of development and seme of the enlarged vacuoles of the 
treated cells were found to contain virus^like particles shoeing 
laorphologic evidei®e of degax^rative change p It is hoped that 
future studies involving varia'^ons in length of exposure to and 
concentration of polysaccharide will deterssdlne whe-^ier oa- not 
these prelisdz^ry observations are consistent and significant 
and ^'hether the aeticKi of the pinlysaccharides is specifically 

at the site of vinss forBia-y.ono 

(6) Dro Love has d«vel^>ed a technique involving 
staining with a methyl^ie blu©=iittolybdat® solufeic^ i^icsh 
visualises different eoap<^ents of the nucleus and cytoplasm 
depending upcei the length of time cells are escposad to the 
solution. Since molybdenum is a relatively heavy metal, it 
vas considered worths^hile to test Aether these differences 

in staining reaction observable with the light micr^^cope could 
be observed in greater de-^il and fiith more accura«^ Mth the 
electron microscepeo Matezlal has been harvested for this study » 
Ho»7ever, no observations have been n&d® to dateo 

(7) A program has been propped whichj in eoilBb<s®tiea 
•^th DTo Ray Bsyan, Mould involve the screening of pellets of 
extracts of hun^ua tumors ^th ^e election sdcroscope to detenaine 
the presence or absence of virus^like particles in these tumors o 
The program envisages the use of the elytron ideroscope as a 
priroaxy screening tool in this studyo Th© work involved wcnld 

be well beyond t!^ capabilities of the unit as it exists at pr®s©ntc 
The only way in %Mch this program could be activated i)d.thout. 
adversely affecting other research would b© to recruit additional 
personnel and to purctose mere equipment ^ Further detailed 
discussion followed hj administrative coasnilanents regarding 
recruitment itf technical help and purctese of additional 
equipaent ^il be neeesssry before this prog^m can be initiated o 



Psrfe B inslBded Jm X m 

553 



- h - ICl4i06 

FHS'=9IIH 
ijadiTidual Project Sepoirt 
Calsadar Year 1959 

J^yt^ o Honors^ kmrdSf and Publications 
Publications other than abs'te&cts fpoa this pa?oJeetj 



Qatanty, J<. Bo and Dalton, A<. Jog SpsfEiogenesia 
^ Lj^ ^ic^g. jierctdQuS o An electron Edcroscops study o 
Jo E^^T" H^S^T'qytolo 6s ii5°52, 19S9o 

Ck^elaM, Dp Eo and Delton, A, J>3 An association 
betifs^j Biiteeh«K3ria and ihe sndoplasaaic raticuluia in 
cells of the psetsdobranch glAnd of a Telsoato 
Jo Biepaars. Biocheia, Qjrfeolo Bt 395"$^^ 1959 o 

¥©odsid®5 Go Lo and Dalton, Ao JoS The ultrastructar© 
of long tisau® frm. newborn and smbsjo zalcso Jo 
Ultmstract«e« Ms. 2t 28=51^* 1958 o 



Honer® ajid A^E^rds rel&tizsg t@ tias project « 

Prssideatg H©h York Sosisty of ElsctiHsa JE-croseopistSj 
1959=^o 



55^ 



1. X&lsossitoxy of BioliOgsr 

2. CeUiaXar MolGgy S®eti@i£ 
3o 



IMividwal Project mpon, 

Calsaaar Yea? 1959 



^gt A. 

Principal Im-ssti^tog-: Waltsr C. Sclm®M©r 

Coc^pssBtisg Units: toMi 

ISia Yi^rs Ccaler^ss' yeas' 1959): 
fbtal: 3 

Ps^fessicssl: 2 
Sbtal: 1 

Psfojeet ^scglptlcm: 

Objecti^s; fb isol&t@ aad iSimtify tise Seessyriboauclsosides 
a£^ deo:!3r7il»o»uel@otiS®3 ps^ssat ia nossial aM eens@7 tiss\sse aM to 
dsts^^ii^ tl3@ isstabolie patls^ays in ^Ich tbsy as® involved ^A ^t^lr 
£<slatiGn@hip to -^Si® s^'U^esis of Seox^pTilKmucleic acid Cs^)<> 

MetlM^s ; ^^ d@o:^2fltosidic c^^kkjMs e&r@ @@p&£^t^ frsM ti@@«@ 
psoteiios aod M& ^ ^M&g eold aeiS @2t^mct8 of t^ tisst£ss. 'Sbe 
%i@3^@ &r@ !£'@@9ve& UQd@? ai&@stl28si& m^ fsossa isiiii&ifttsly ii^ li(|,^d 



^1^ ilmos^ri'b&Bl&lc co^spoisz^ p?@s@is& in tte acid solutil® ex^s'aet 
ase isoMt@d tssing p^s? as^L coltam elis-c^atogTapby aM pap®? el@{rli<o° 
pbos^sis. ^^ isola.t@d cos^otipds aife idsntified \ii' wsass^ cf a1>30iption 

spects^; cb:^c^t^s^Me aM. el^otrq^lzoT^tic 'behavior^ and eolOTisastsl® 
and @ics>9M@logieal ssactiox^. 

Boir^is^ assays of S@o^rri^si&i@ ccsi^us^s t:€S% msMs vith tSis 
os-^£ii^a; L. aeiSopMlus B.''26f ^shloh @p@clfieally £@quis?@8 tl^e® cosi«- 
pousude for grcM^th. Uriz^ eesstlss ^^£@ collected ^ily in asodlf i@d 
plastic cB^&j mMcli v®^ atstoclaved each ^y« His uriss sasi^les ^@£i§ 
filt€S«d aad t^eat^ vitSa peztsbloric acid pslor to ami m^us^^nt of 
dso^?il39@idic ccstipow^Sa @i9 c^spo^S® ps^s^nt in th@ uTin^ ^@^ 
purifi^ cMt^sto^apMeally. 



555 



- 2 - Serial ^= miAo-J 

BiM. ^G^^i3^B±m \3Bis^ a traMeos^Til^siSase pzQseat ia sKtraeta of 
L. aciSopMliss E-26. !£lse eeo3S^^Mias>S<<;^'^ fozsssd was pmlflsS 

easa to coaeist entirely of deosqreTtMis® oa ths ^asis of Edcj?oMological 

aetivlty. 

A« E^getiffl @tMi®a. Our i^da ob^t ia stuSyiag ^to oslsiax^ 
e3oc3^1f^~a^°l^^^^sidic ecs^toimSs -u&e to detapaixae vbetl&ar tls®@@ 
co^oi:^© GG^ ft-cm "^^ &i®t or iss^stlsal flos& sM ^d-isthsr tto 
aiii^il &mL istilisis ^^B in situatiosas ^Ssers £^id DM n^tbsesia i® 
sis^@0saxy (ps^sezsc© of a tvasor o? s^^sssatisg li-«@7). M^^tou^i %hesQ 
sti^iss bav% ^»@n ec^lieat^ Bot c^sl^ Isgr t«clmieal af.-ff iculMes as 
wen &@ l3gr ^>®2latloEi© ia Infiividval excxetlffii pattesffis, l^i® follmdag 
tmss^tive ea^slisalosiB h&yfs be^a reacts. 



1. fl^ dso^rrlbosidle ccgqsomsds in tfas uriss) aire sot S@^v@S 
sBtirsly fs'O^ ili@tasy coostitusatfi. ^Siis me sbo^m ^ studies 
oa fasted rats msd oai ztats fed a ofeeaieally (l@fi!Sf@d, solublg 
asaiiK) acid diet (Mffidly furoisbsd fey Dr. MiltcHi Winitz). 



iatsstijml tract, siac® ttey -w^r® ©xsrated Ija tbs urioe of 
ge^a frse aal^ilso 

3. AStsr partial hsj^ts&ies^, the esoereti^i of deoa^ribosidie 
ea^>ots^s appears to d@clixs» aisd rascb^e a MMsnaa le^l 2-3 
days after nm q^raticm. Since this did iiot oeeur ia sate 
^Mch wr@ iaparotosaiaed and iis^laated vit^i a li<9®r 1@^ 
from eaotS^r sai^L, it insula appsar tiat disriag the period 
ia -»Meb DM is 'bsiag r^idly e^tbesizod ia the liver i^ 
deo:<3rsdl3osidie e^potissis 33o^sial24r @»cr9t®d ia tb@ urlm &r^ 
diverged isito s^thetic pa^Rssys. Va£ovivm.t@l^, this ©j/js®- 
tica pattssa isas oot ©Ijserved is 100 p®re«at of ^am hepsfcsctOTised 
rats @tt:di^ sad' thus additimml @2@erl'£eats ^dll ba mm®mt^ 
to detessala® -Kiiy this ^ms j^t tnse. 



^. la ths eas® of rats feisariEg tsaa^laate of tbe ^;Yiloff 
fespatJ^, tl® THiaasy esse^tioa also ap]^a^ to h& decreased 
at a time v^n ^m ti«>r is grofc-iag ssost mpidly. '^^ agsiBy 
S»3!se-^r; tl^rs -ms® ©sse^tiosis to tMis coaelu3i<m. 

5= St^i@s Qsi tls@ ideatity ©f tlibs isriaary ccsapouMs, el'^KS®^^ 
iaeoiplst®, iMieat® -y^t t^ tmS&s cct^pouM prstisat i@ 

dso3^eytidi^ aad tliat ^iasr ssra?iiite of d«0s;rttrMiae @M aa 
^mid©atifi«d swlsotide jsateri^ sr® also @acrst^. 



556 



- 3 - S®3?ial ^, mi'kOl 

B. leo-^ie sti3fli®s . It %-&& hsspefi tisat tS» i^satlf icatioa of the 

a ^eeific ps@cux€@? of SH^^ deos^ytiSliae, lalieled vlth Cr^, Such 

933»@ri^mts "imxm also iBgicatetl by on^s- ps^vloue fia&ings timt tM@ 
pyrliaiSi&e imcleosids is fouod in Bszisr tissues , iacltafliag tissosps, as 

tSse sHJor deos^n^ltdsidic eoi^posiant. !3is t»e of this ps^gcm'so? couIS 
t^n ts Q:^@cted to @lisciSat8 ^^ -gs^-m^ iavolvi^ in th@ sjQthagsi@ 

of ■pjrMi'^d^s J®A asKleotiSes la a stsai^tforwasd ^lasss*. 



Att^^ts to ym@ tMe pz^eurso? ia vi-wa h&va 
tss^ora^ily ^bscatss® the inj@et@d ffiit@rial \iB8 f&aa& to b@ aIs»Bt 
vsaifo^ssly £ists>itat@S l^is-ou^ioijt tba "bo&y of tbs animl. imsis the 
£sm9U2it of s^icsaetivlty lo@alis@£ in & singSA tissias vas too 1<m to 
isolation i»sth(AdJLs. 2n osSer to cis«tBiv«nt ^sis prol>2i^, the 
of 4303(yc^lgiBe vith & w3Sih hig^r specific &ctivi^ voksM "bs 
For this pu£p@g®; a@o:s^ytiaiii» lahel^ -^-^ txltitsi ^las 
oisS®m& fs&^ aa outsiSs c<mts^y3tor« Mtsr a six raos^th SeXay v% va:^ 
isfosassd that the ^miifmcttsr®? I^S l^ea tssahle to px@pas« tl:^ osatoslal. 
^Sf^-^Tj ths s@nufaett2s%7 i^as aot ^b® ^ -m originally sp@oifi@& aM ' 
tim s^tsrial is l»ing reosSsred. 



In. the issantiassj, eev<ex®l in vitgo e^q^riiassKts ha^ 
using latieleS dsoi^s^ytidine. Suspensions of a C^ ascites h^&isom. 
^T® ineiil^t@d ^th tlm laheled smte^ial and the pr»ii«Bt8 isolated aM 
identified. !ghs DM of -»i@ cells Taesese la1»l@d pstsg^esivel^ durins 
tl^se e^psrisses^. In tS® fi^t e:^@£^^at8, the eells ^mv® ii^ut^t®d 
in the ascitic fluid l^cause this ^s thou^xt to be s>ox« plbQraioloiiealo 
lo^^-^r; tl^ suspensions mpiSly Itecas!^ asid asd the sadicsbctiw 
pri^tsets f02S3^ iz^icatM that degxsi^tion vas occm^ilng. 

It vas foua^ that a mn^ svl%&\ils incubation ^ivra consis^d 
of a b^sffe?^ isotonic salts soltsfeion.. I^ this zaediian, tise wMn p^^^^t 
fos@s£ vas th@ nucleoside; d@oa^ui^dii3e. fhs^sidins easd usaeil vex® 
also fo3ai@d but at a leirel osee-flfth as great, "^m specific activity 
of the coi^KSunds isolated stmmi. that the deoxycytidise added sM the 
deo^yuridin® foxi^d beea^ p?ogs«e@si-«^ly mos® dilu^ duslng the 
insulation, mis indicated -^aat thei^ is mi ex^o^as&us soui^e @f 
deo3!ycyti£ine in these cells. 

Mtboui^ only ^©11 ^isunfes of raait^cti-elty -mt® f&sA in ths 
nuc2sotide fraction of these cells, it vas si^icient to isola^ aM 
piBElfy these ccs^to^sQds. Tkms® studies, altbou^ incc^lete, iMica-^ that 
thes^ tmy be 1^ or mxs^ deois^onscleotidss present, ^he ^peets^ of thei^ 
c^^oisMs indicate l^^t 6 contain deo^yeytidine, 1 deoxytssddiii^ @M. 
1 ^nsidine. !iase 2®st Mv® not l^en siaffieieatiy puslfigd to oMaSa 




roaa -a© 
lovi^ff h^jatesa, it is i^cessa^ t© hav® fMi^wsti-^ pr^j^tsatiosis 

of ^®s© csg^o®^©. Such p2?^amti<ms w^iM also te -m^^ u®®f?sl in 
detasMsaing th© ps?@©gi^e s^ ec^seatmti^ of ttese cc^^ouM© ia o^w 

557 



^ h " ^rial lo, ]^I-^T 



of plu^pbo@tl2srao2^ai!te-l,2->C^^ (SSA) has %e@n CGsaplated aM tbs 
s^o^hssie of tl@ pbosphocholios (PC) aaalog is ia progrsssc 'Sis 
B^s^Ubs&lB of iaoxsrcytiSisis tslptao^hat® CaC!EP) (unlalaXed) has also 
'been cc^letad. 8iao3 t£^s@ coaE^pouiifis e^ sew evsdJL&'^e, the 
ensSfssatic s^nr^^sis <3f &CDP-C aM dSD?«S Isy vasmL aM tiSBor tiS8%@s can 



^& obs^cal syat^sis of the lattes* cossipounSa has saver 
vssy sa%lsf)s«to2y dtje to lo^ yl^Ms sstd side s^sctiOKis. In 
&S&SV to SBks f^sll 11^ Of ^i@ £sdi<^stiv« imteslals; ecss of tbs 
i^^fer £^t$K^i> of Waossw&f "^ aasass of vhich tissse c^^<»iDd8 could 

1^ 1^^®, e^ l^isjg imie@ti^ts4e ^B^ s^s^ts to fete &s^ i2a:oacli2ffiiv®« 

SigaifieasM?® to eaaeeg geseaafch : !Sie f&ct that ths sspathssis 
of MA i@ osis of t!^ ohll^toxy reacticsss is. ths prodc^tioQ of 
!^^ cellS; IK)*^ x£Os^a wsd t^dlgassntf 1^« finmsed i^ae atteatioQ of 
msse assS. i^:s@ iavssti^tors oa ^hs» stuSjr of this uoique c^Hular 

s^t@slal. !&@ eltsslSatitm of the stesctieos hy isMeh IHA. is foassfi is 

^MB c<s^id«2^ to lJ8 «s^ of -fee SK>st isEgsorfeasfe proM.^® in csss&^v 



Sfe® ®2g>®yi!^2ts a©seffi'^a s^v® ax© hi^iLy s®Mveat to 
stti^es. ^l2@ uslnas^ ©^ses^icm stisdiss surest that tisaozs @M. 
othe? ^o^rlag tissues sasy call ^^ptm the dsoxycytidizae p?»s«xst in tte 
tissi^s i^ circulating ia. the M.00S &s a huildiag hiloc& fos> Mk 
e^tl^sis. She in vitaro @^^sii^nts @ho^ td>at at least oaae tuEKjr 
c«ll is ca^'bls of imMag its tmx teoj^ytidixss. Whs^ier ^si& 
eapahili'^F is sha2®d ^ othar eells or whether this ability fulfills 

tbs S€I^UiS^S@^S of ti^ t\^Dlf OSll is SlOt I3I0^&> ThSB® 8X9 g bCI%%'9@S'^ 

isspog^aist qtsisticms \9hich w3b% ¥e rssolwS ^for@ th@ control of 
Wk s$mt^@iS; esiS. heneSj of cell miXtiplicatioQ; csua l» satisfaetoslly 
appsoasb^. !&d idsstity of ^s@ &go:i^H!%leotide6 px^ssent ia the 
aeid solT&il@ @^3^e-^ of tissuss is also isaportaat fsrasi this^«a2&ap@isit. 
It shouM "bs i^iStios^S that of the pygiroMI-na &eox^i»l«otideB mm 
Ti3^^si., 3 c<m%aiu d@o:QfcytiMae/ 3; d@osyuxldiQe asS 3» th^raMlseo 
fM.@ sug^s^ that tl^s® may bd s<sds is^r^ deo^sraueleotl&es ia tSsa 
l^pat«Ma si2e@ the isotcfiic &^^7£±mnts is&ieated tSie pses^ice of 
Ik 



558 



Serial Wo, mi-kOJ 



Pgi^osed eougs®; Th® mls^3^ @:^s^ion studies vill Its coa- 

ti&is@&. It is bQ!P@& that glaring tte s^^cl y®&v it vill tse po@s£l3le 



to resolve @am of tl^s iasoosistei^ies tlmt bav» l^$a o%s@snp^ and 
to 4s8id8 ^l^tba? tS» aaLsi&L eaa> la reality, ue® eztfiei^Qous 
deo»3r7ibo@i£ie co^»otmds fo? MA. s^ttesis in ttmasa aod x<egessd£iatii^ 

•m^ wsi'M 031 t^ purlficati^i of Ite ZHdi^ctiv® i33iel®®%id@s 
will cositisssie. ^siSss tise Sstexsoiosfitim of the Msatity of tl2@@@ 

ccsi>oi2Ms, it ^0ill 1^ l^>d7tes£t» to M^^ tba specific &ctivi% of 

m@m c^^om^ Im^msm tl^ latter -^11 enable s Sscislo&t -lo Ise Bsa^ 

a® to -^^ utilisatioa of tli@ et^gtafsssa© la -^3® s^ttssis of MA, 

mth tte ehmie&l m& eazysmtle syiathssis of dCDi'-O aM 
dCD?-E vill ^ stuSiet. Xhs sozj^itic foxsaatioa of these coai^ouMg 
is especially li^ostajst ls$ca%^® tbei? ps<9seQce in ths hspatcsaa 
^sSicat@s tl^t the tts^? has a hotter aiselsaiiiffia for theiv fosmtioa 
thaa &o®s no^^l liv^r^ f sera vMeh tb®y appear to ^ a'tees^ or 
p^es^s^ i& @usb l©^?;®? coi^®atratlosi« Sis patSnisys ^ vbich thess 
ss® ^ilized vill also b@ studied to deedd® vh»tbes? -^2® 
of ^tmm eo^ounSs in th@ tisaor is 4ts@ to ov®xpredisctimi 
OS* liisfficieiQit istilisatioaio 



Pan B ii^li^tds ¥es X 



559 



« 6 - Serial lo= JSCl-^kOi 



Cal^i^&r Ysex* 19^9 



Part B.^ le^3S®> ^"^2511®., a^ Piifelic©ti®as. 

SchasiSsr, ¥Ea.t@r C: Mitoehfoadrial mta^alitra. ^ 

Mvsaices ia ^azymlo^ 21: 1-72, F. F. Sbsd^ ed. 
Iate^ci«ac® Pu&lisbsrs, Hesw Yosffe, 1959» 



560 



Sgrial lo. KIl-ij08 

2. C@Xlulas> Biology S@etic^ 
3. 



CaleaSar Year 1959 



BsrtA» 

Principal Imrestigator: T^^ma Smltaa 

O^^r £a^/ast.lgat076: ^rgr Rice 

Cooipesa^lBg U&lts: Koss 

tea feasre fcalsiiaar yeas? 1959)- 
Itotel: 2.5 

Ps^eesioc&I 2 
Ol^r: .5 

Project D®s«2?iptio®; 

Objecting: S'b eti^ \be pJ^aiolo^ asA imapboicgy ©^ l^^a^SuS" 
ci^ic txm>T c@ll@ astS t£«ir £<@latloasMp to 1^ sosraal oelle of the 
bost ei&l^^o ^ te^vlor of th« tva^r cells is an iatspution of 
Mological factors stsoh as, groHtb X9.t9» loc6i3»%i<Ei s^t®; sstabsslis^, 
jj^mesiologieal prQ£s@?ti@S; aM ths ii3cfeegs^ti<m of thiss® factors mmt Ite 
eo!^iS@z<e& @|^ij^t ^2@ tejekgsrousid of boat iiifl^si^c®. It is the pxirpos® 
of ^ssse 8ti^i®s to att^t to 8©st ouife aM esaai^ s®pasat@ly ses® of 
the®® faetoys ia ss^y to assess fflOi%? ps^«3?ly the yslatiosMhip 
lb@t«^ei txmsw csUs &M tS^ £2.oi@sul eeSJ-s of 'gb® host. 

M&Ube^;. Bss feasie Mologic&l tuol U£@d ia tbss© stuMss is ^bm 
asclts© tiffloy. f^ frse calls of tte p®sltoaeal fltrid of tis® am^® 
ii@%^ stf^®£ &B s@p7%E@£stiisg -^ sio^^al tissue that is in ecss^imst, ^th 
tte asei^s ®@llg. 

Sp®eifie i^rJjki^s: (l) Boi^fel® diffusion eljgaabsr: F&r a®tails 

of cmsst2iistioa @si. tjss, &®e 1957 asEmml x^eport. S^ cells aM fluid', 
is tte aiffusioa ebssfeer "j^s^ saalfsed ^-"{2} Hietoeteaieal ®a^seSs»-» 
fo? G^msssti'^ ti@s%!a aM elas^ie^ls ^&lag ^ the izitraselltila? ^t^x, 
CS) fepsr ctos^atc3®ea.pS^«»f©S' l^stso^fpipslii^, ^»e laaiqia® cfflMtiti2©!St 
of colla^sa, (k) Heetsfeffi ssic20sc^^°-of eollagsa filths aM cell 
vO-tsSiStsi^ttass o 



561 



= 2 « S®sial m. KI-lK)8 

1. GjffliiT^ a sid i^alatitm of &swsl cells ia di^f^i^ ebajategs ; 

In ee@kiag t@ obl^aia cQ@c£@t@ ixsfom&tloa a'toufe the isebaviosr of ss>ssaal 
cells in the ps'sseae® of tmor c®lls, the b^mvior of vamal eeULs 
alo^@ isas stt^Ll®&. In so doing -^ fotasd oursolves dra^an izsto a 6o» 
^«^tr<K»ld pol^Bico'>s@lt2c-^u9rtdy at fisvt sz^ then vlVa. ®ss^Bamlwsmc ¥@ 
lwli®v!g that ^ havis tl!@ mms at haM to solv® consuslmlv&l^ tte 
pvoMffiB of ^s fate waB. ftmction &S tb@ f ?e@ cells of ^i@ ?e^lculoo 
eniotSslial sjst^a. 

f^ aiffusioQ chasiter -^eelmiqiss baa pxovlded ^^ @vMm£c@ ia 
BvsipQfSt of th3 l$si»boc^ts«>to~fil3^l3ila@t traztsfoxBiatiosi. %» tseliniq.ise 
has' p@sssitt®S us (a) to gs^Of^ cslls of ^sstmm mmSa^r @xsBl typ^ fop loaig 
periods of tism in an isologous or hete^ologoi^ ennxasBssaoib, (b) to 
esE^nizis eell selaticmsbips in stained, tSaoSjs-wamxt prepaxati^os, ais& 
(c) to m^ljss® cbiMeallj tb@ ecmtsats of ttm cbs@bs?s. Wbsa tSis tT»® 
cells of tbs p@^tosssal ^taid of aice are gzoua in di^tasion ebaaibes^p 
tsansfos^atimss Occti? t^ich lead to tbs fossatloo of an orgasai^id 
tlss^ss. 'Wm l^^^tocjte laod^Latss into an indiffes'^at cell ^Jhicb, in 
twWf has the ei^aisity to bect^as a fibroblast or plasaa e«ll. ^t» 
laaeroplm^ also app^irs to hs,v9 the ssse capacitsfo Qzanolocyt^^iesis 
OOCYSTS in ths chasil^rs and mast cells ar@ still ps@@esst after l4o Sa^. 
Qolla^n <»a® idsntifi^ in iitm cboEBbera after the csUs bed grown for 
t«o -g^sks aM the eolls^fn eststsnt of th@ ehasol^rs c^atini^d to ii^r^oas 

^t&. tl3^o 

tEiere is v^osm to assv@e tbat in ^b» aaiml the fx€$ eelXs of tb@ 

3«ticuloo«sdothelial ^srst@a are re^ponsivs to tSae e^mrochaaging environ^ 
j^nt ^sat they esssomster Is^ virfeia of thsir ssobilitj, aM that they 
^«mLd respond to ti:^ a^s^sml emrirrasssnt created It^ a vound, a tissor, 
infection^ or aastabolic distux^bsnce in a a^sner not tsililce that ol@@rved 

tn tbi§ diffusiosi cbmber. 

2. Furthsr stgadies ea t^ pgastratioa^ of l3B^i^?ytie ttsgao r eglls 
thgqn^ cellttl^e g^aibrana fnters ; 3a previously r^porti^ ^sfe, it 
•ms stated that t'mor cells ^@re not able to penetrate cellulose 
sfflssibr^se filters (l^pe M) laving an average pore sia© of 0.8 Uo 
Mere s^e^it stxsdies have caused vm to revise this stat^ssnt. 

'^e eeHs of th@ hi^ily invasivis X^IO tusor penetrated 1^ 
AA filter 100 percent of the tizBs after 30 dasrs'gmuth in the chaaiber 

(kO ci^ssib^rs) and established a visibl© emms^ of gsmth on ^3e 
"Boa«e®ll side" ^ th® ehssSser. ^ s^lati-yely asM-inirBBiw tts:^^^ 
L#L> ^as not able to @@taMish visible ^mfi^ on i^ no<n<=>eell sid@ 
of the chasabers t^ 120 days (SiO cbs^tes?®) but in on® chaadsar I#l 
cells ■«©r® microse^ically visible ©n tSie SEKsa-cell ©iie csf tto© staiaei 
cesKlsar filter. Issaal cells infereaiK*^ into the eba^bsm ni^ tAs 
I#l tt^or cells •^» aMe to psnstmt© -^e center. Jlll1i@r a^ \?®j?e fots^ 
o® the s^S6«-©®ll Sid© of -^s eS^^rs. Ss a^lti^si, tJ^s^ -^r© cli^s 
of ywssgsd*'^ call ^sssts f©^ss£ eosisl@t®at3^, these ^^r@ protebly 
tMor c@lls^ 

562 



» 3 » BsriBl Sto. mi-koB 



@qi2^ chaste ; ssay o? ^^ soot "^ aM;@ t9 p^ss'tsa'to oat a^ifieiel la&gsleT 
aod est^Misli grsvfth at a Sistaat sita. IMs dij^ersziee in the eepscii^ 
of c^ll pc^iOatiozss to s^kr^vs asiy o^sute la alfliag or hSMsv±ag the 
estaMlslsasat of s^w Blt@3 of grovtb 1;^ tl^as taaK>s@ vMle tbay s^e 



3, febaviog of ttj^sr cells ytam. gi^eyaa on eaqplaafea of ccBSiactiviB 
%i.ssvis in Sifftisiosi obssi'tes^: We faav® x^eportad pTOvlously "&&% aowssS. 
pexltosisal cells %@bave4 aiff9£*sat2^ isi ^ss diffissica cba^ilis? aceosam 



to tl^ tssso? tS^t mB s^ecfm ^th ^i@s. In tbe ease of tho "in^^asivs' 

tuisors I^ S3i£ £1210, lOze siosml c^Hs a^ tiBior cells a,pp®aired to 
gro^ is^spenSasUy, %fut in ^xs ea@@ of -^^ '^umoi&'va@i-99'' tus»?8 I#l 
aM F333, t£^s% ^s@3 a gsfinite association %ett;e«a iSis t«o csll tsp#s 
in -^Mch tls@ tisior csHs- gre^ in S@finits cliasgpe Wis>% v®^ s&rsmm&^ 
and ®neloeed "by tl^ tiQxmJ. cells. 

W@ imv9 followeS v^ this ol^ermtiwi 1^ gsor^ing U210 aM X#l t^s^stS^f 
v^tSi @:9lants of sosi^l eossseetive in the saae cimi^s„ Tba filssoMasts 
of isbs stSsststaneoos fat paiSs of t2ie sssKise groi? luxuriantly in the 
difft^ion cb^ibsr. % intsodxjsing ascites cellfl into tbe msss ebaml^r 
\d.th tbe filKromastS; %tm ^lationships Itetueen the tw cell t^pea could 
be studied. Tb® s^sults of -^lis esiperjbssent 'n&ce stsl^ing. Ixs^n^tion 
of the stained filters am viAeh tb@ cells grew showed that tiievs vm 
absoliitely no associatiem ^tizean the invasive L1210 cells asS tise aon<- 
nectivs tissij@ fl'bSQ'ikLsjstBo 'Soq tumor cells grev as frse fl<^ting; 
independent entitles. 'Bm cells of tise Q«:m>invasiv!@ L#l grs^ in 
int^^te sissociation with the coasseetive tissiie fUm^blastSo Whssi tte 
^@s of tiier fibroBlastie gs^^th sts^p^, the tta»r cell grc3»«f^ also 
@t(^p@d. In aany places tts ad-mneing ed^ of the gro'tTing c@m:^cti've 
tisstse sheet vaa alieaS of tl^ ttams? cell gs^f^th. We hope to i}::<^?>or@ 
furtl^r -ys® reasons fos* -ais esfe5?aordiBa^ Mhavior. 



Si^ iifieaaee to cancer researeh : 'Bts l^j^iioeytic leiifesaBias in 
alee very. closely £%s@^l® the l^i^b&cytic letikasdas in ssan. &e furtter 
elucidation of ^m chas^c^ristles of t^ disease in laice should Imvs 
a direct ^^aring ^oa the disease in mastp 

Propos ed eoig se of tte pro jects f^) Xnvesti^tion of t^ 
r@ls.tios!ship het«e@n nosiml cells and nsXi^t^xA l^^i^eytss gfo^n in 
diffteioQ chaisibsrs ^11 c^timis. Specifically xse vouM li&@ t@ isolate 
aM gro^ as pvss® lit^s the soxs^ lys^hcK^yte, imcrqphage, polyssospl^ouclear 
leucocyte, and ^se smst cells. We \Kmld XiJSe to isolate and g^su ^isor 
cells that as® tmcoaitamiimted 1^ nsr£gal cells. ISte cbasi^ters as@ c^i^ 
stnast<^ in such a %my t£mt it is possihl© to isolate tte eell@ fr^i 
so^ of its p:^s2ets. We ho^ to leasa s^isthing of tise cells' 
^eta^lis^ 'by stui^dng its pn^^ssts. 



563 



•^ k ' Sgyial mio mi-kcB 






(3) HsctEWi saics©seog?e st^^ of s©latio©sMps '^t^^@® thi 
mll^a^t l3^!^cyte aad cellB of the iavated tlsssss. 



(U) A stu^ of tS2@ surface dias^ of aoxsal si^ saligsaoat 
l^s^hocy^s Is^r o'bseTvatifm of tbsir ssoMlity in an slectxlc fieM« 

(5) Blsctron ^czoecopy of cells^n pst^ijctioaa anfi tSi® 
effect tlssgon of the prgsenee of t^^or e«lls. 



Sayt B ifflclMgd: Yss X 



5Bi| 



» 5 » Sss-lsl fe« KI»J*f5B 



GaleMasf Year 1959 



^rfc B» IfeMrs, A^B2^, aM Pablleeticms 



Pjalslieatloas oldsar •feaa abstsscts fs^osa -^lis project: 

S&glt^i, 1. 82^ Me®, Mo !«.: Bslatiea of iavasiv® e®pacity to 
passage of l^^bocjtic cells tlxmi^ c®llulos@ wm^tosm 
filters. lBtis2@ l8k: 1^79-^0, 1959o 

l^iseltoaj, ®. asS ^iX-toi, A. J. : Eleetsssa ssie^aecopy of ©ffl^ripolesl©. 
JouTo Biopbys. ai^ Biochsm. Cytol<^ 6: I^esibe? 1959. 



565 



Serial |fo„ HCI-lj-Og 

2o Cellular Biology S®ction 
3» Bethesda, Mdo 



Individusl Project Report 
calendar Year 1^59 



Partju 

Prc.ject Titles Electron microseope studies on -SFarious 
norm&l^ -rirus Bssociated &nd ^iligasmt 
cells o 

Principal Iixvastigston Robert F» Z©ig©l 

Other Ii'rs-estigatorss 

Mo F. Stantoa 

Ho A„ Mamker (MCI=it.l6) 

¥<, Re Br^n (ICI=li20) 

Ao lo Weiabsrg 

H. ¥o H©rtdl©r 

Coopsratisjg Um.tss 

HGI| Latoratosy of Biology, Labor® toxyof Bioshesd-stsyj 
Laboi^tory of Clinical Pharfflacology^ HTlM)s 
aiaieal EMaoriHOiogr Branchj NHIg Laboratory 
of Csllular P-jgrsiolo^ and Metabolism. 

M^ I@as*s (calendar josr 1959) s 
fotali 1 1/3 

ProfgssioKsls 2/3 
Others 1/2 

Project BesesiptioEig 

Psrt oa©o A smjor portiem ©f ojsr tim© aad ©ffort ■ssas 
speat ia prepailag amd @x@ialniag tissa® frwR the Bovine 
Ocular Sqaajaoas eareiaemao This stydy b©gan id.th the 

e©ll©8tiea of the tisse© fesa Hersfos'd cotis at Bay Maao? 
Farm is Lsi^es, Del, and Srem the Walter Heed expezdaieatal 
£@.rm. ia dryland „ The cooperation of th® raach foreissn 
a^id Ms staff ®as gr©stly appreciated » ?h® tissjj® was 
fixedj sabodded aai ©xaaiasdo Fortioas ©f the 



566 



•= 2 - NGI=U09 



tissue were taken for tissue culture work by Dr, Manaker 
and for tl^ concentration of the microsomal and possible 
virus fraction hgr Br. ¥» Ro Bsyanc Parts of the tissue 
*ich presumably represented the early plaques in the highly 
keritinised epithelium were stored in glyeerine for a period 
of time befcre fixation. This treafenent is reported to aid 
in the preparation of this difficult tissue for sectioning 
uhile preser^ng any viable virus partieles which might be 
present o After a craasiderable amount of micrcaooj^ i^e 
perforaied it ^B.e decided that in the particular tissue as^indned 
partieles ^th the morj^ol^gj of viruses were not present arsi 
thai further efforts te visisalisa them Here not warranted » 

Part two involved the study of virus production in pan- 
creatic acinar cells of a young chick,, Previous observations 
on the virus particles coiKserned with lyiuphosnatoeis and the 
Rous virus indicate a vezy different pattern of formation. 
The observations inade in this study indicate the possibility 
tl^t reai^r diverse tissues may be concerned with the patterns 
of virus behavior and reproductive qrcle.. Much more work h&a 
yet to be done to elj^rldat® this problenio However^ a brief 
paper is in the process of being written to document this 
finding o 

Part Idirsej, mm under way^ is an attempt to reworkj w-th 
more up to date teshniquaSj a study begun by Dr. Dalton aM 
Dr, R» Handler on the albuaen glands of the chicken oviduct o 
Staining techniques should enhance arri aid in the localisation 
and identification of active regions of the cells o Hopefully, 
this study will contribute to a better understanding of the 
secretory cycle of this copiously produced proteinaceous 
substance = 

Part four is a preliraiimry stidy concerning the effect 
produced by the incubation in various concentration of insulin 
on fatty tissue frosi a diabetic patient. It appears that 

increments in the titers of insulin produce an increase 
in the nuraber and sise e>f prins^totic^like vesicles in the 



567 



° 3 " KGI=li09 



HdssibrsBes sM ^topl^sm of the fat cells » It is hoped that 
furrier stislies m&j eo-afirm the Sgrpothesis that, in part at 
least, iissulin plaja some role iss alleging blood sagar to 
enter th© r&rious cells of the bocfyj presamab]^ &b a. membrane 
effect on th© end or^sso This study ^ia@ begtm «ith Dr, Ao 
Weinberg o 

Part five coBC©s=ns sase prellsdaazy observatioas on 
ffiillipore=f liter ehajabsred calls of ssttsdias organized by 
Dro E= SheltOHo This aiproach shoold b® a fertile one from 
the point of view of elucidating the details of qyto^diff ©r- 
©ntiation of sem@ of the cells of the heiaopoietic bqtIqBo 
This work is b@isg held in abeyance at presento 

Part six a In a comprehessive stw3y of jsicrososaal fractioas 
of lOF^ikoff EepatcBia cells, a co°^uthorship in a paper M.th 
Dr^ Eo Ktaff has resulted o A large Hismbsr of -OTiiriously prepared 
spaGimens tmr® examis^d and photographed « It is believed that 
sera© insight into the basic building blocks of egrbology sasj be 
gained through this and siaiilar studies o This type ©f stiKJyj 
pr€f«r"iously parsaed by Dr« Kaff and Dro Daltoa and moabers of 
the Seetionj is basic to th© oltiaiate cca^capt of cellalar 
morphol©^ &nd phgrsiologTc It is at this level of endeavor 
that these two tsnas becoms virtually ^naonomouso 

Part s@Tsn involved th© testing and standardisation of 
a new lead l^droxide staining techniqee suggested by Dr» Daltono 
A short not© for priblication ressiltsd. 

Part eight is coneeraed tfith the dsvelopaent of a doctor&'b© 
thesiSs parts of vhieh are being prepared for publication* 

Part nine« During Dro Daltonfs absence considerable tiei® 
and energy was spent in supervising and atteiapting to coordl^te 
the instal'^Ation of a ne'^ electron microscope and other 

laboratory ©quipaent wMch sboald facilitate and aid th© 
tforMag ci^ditions of th® latoj'atos^-o 



Part B included Im No X 



5B8 



1. laboratory of Molo^ 

2. Cellular Blolo^ Sectioii 
3' Betfeesda, Md. 

PIS-IIH 
■JMividml Project Bspcs-t 
CaleMar Year 1959 

Part A. 

Project title: DiffusioB cbam"ber aM tr&aspaj?sat cimmfeer research 

P^Bcipal lawsti^tors: i5uth M. Merwin 

Other iBY-estigatox^: ftel^m Buosa 

Copgersting Uaits: loaie 

M^ Y®a2« (caleMar year 1959): 
Tbtal: h 

Professioml: 2 
Others: 2 

P^ojeet (Seseslptioa: 

Pgoblea 1 — IMisc tioa of plasjaa-cell timoTB i Buth Msywia as& Sheika Item 

Objectives; The object of tlssse stiadies -sms to ^tessdae y^ 
plsffli^-cell ti^ss axes® ia MLB/c sice c&ssyiag latraperit^seal dif- 
fusion chsmbers ^Meh co&talssd C^ ^^sss^-tvs^r tisgias. S3^€riBs@£itt@ 
■^STQ set 1^ to tsy to f lisd owfc vhlch of t^ ss^]^ factors p^sesat ^^E'© 
essential for the IMtictioa of the t^sorso S^ach factoss as tl^ 
folloidjQg ^«sr® coasidered: the elites* itself, the bcB©lc@3us asad 
tisaQrous smti^s of the tis@tie 'cd.thiB the chsmher, the preeeae® of the 
ws^mrj tus»7 ageBt in the tis8%@ in the chasabere (Dr. Mltmi foiasd 
virusoli&e particles Ib tte ii^^aced plasssa^cell t^^ors sugg@@tisig that 
a virBs Bight fee iavol'^d ia the ttjsor iM^iCtioa)o 

ilethods; Bifftasion cha^'ters i£®t® ±^lasib^ as ^l&xil:s or coq-> 
taiBisjg -wariots© typ&B of tissues. Methods that would extsmct aM eoa- 
ceatrat® aey ulnsB particles present isa the tmior cells fs&r® es^lq^d 
to olsteija preparatioias which ^sere asss;^ for the preeesce of virus 
by iffljeetiag th^ iato 33e^K>2ii sice. 

Ifejor fiadiags; Tim ®ost isportent f IMiag so far is ttet a 
blaak eh^ibsr eaa IM^c® a plas^ cell ttmsor, '&i& fiMieg ffisSss it 
sees less likelj that a Tirus plays aa teportastt role ia iMueisg 
ths t\mo»8. Cs®e reports bj Drs. FaSsey aad Potter oa stiaSies Had© 
id.th these iMi^ed pla^a cell tumors )» 

Propp^jsed coaa:^; To detesaiae -feljy the blaak ehasiters iBfi-uee 

the t^iiaors. Tbs possiMlitl@s cos^idered are that the chamber or ecsss 
part of it s^j act as aa a-S4u.-vaat or as aa asatigea or "^th or that the 
ehasiter ^y act as aa irri'teat ©r that the t3?p© of careiaogesic stissuliss 
eatasisg i±s sarec^&s, ^^sieh also arise ia these ej^ertoeats^ say aff©et 
■the plasis, cells o 

569 



" 2 •» Serial So. HCI-J^l^t- 

Si^aificasse to cashes? s^eseareh: ^@3@ tusaors &Te similar to 



t^^KO^ ia 1^^. Qaae of tbe tiaaors is of pasticul^r 
intes-est 'bsQa.-m& it catmes s^ s,lmox^al pro-^ia to appear in th@ vsiss 
just as certain hw^si s^tsloaa tissiosfS do. 



Profel^^ g«°Mtlgenicity^ of plass^ cell ti^r tl60t;^ la isologous tosts . 

Objective i fbe object of tliss® es^erfaeats ■aas to flafl o«t 
wb®tlaey plessa cells ax«i aatigeMc is aice of tlae strain of origia of 
the tm^r. Shis aseased a possiMlity sisc© ia G3H saice ths tics^s® do sot 
tsJs,® ia 100 p®re@at of '&© tosts aM siaee id^us-liSEe pasrtieles bav© 
teea o"bBer^®d ia th@ cells aad aJ.so siae® tfe® e©lls foim an sTbaDSEsl 
pxoteis. 

gjgtlKsds; Difftoioa ebssiters eoa-fcaiaiag plass^. cells i^se placed 
ia hjpgri^aiae liosta. 

Ma jog fisMa^; Bie ose liae of sa^ligaasat plssEa ee3J.s stijdieS 
(that ^7&^Wr msMatig@aie ia mie® of tb® strain of ©sdgiBo 

Sifflificaae® t® e^iesg g^seageSa; ©lis aatigeaie differease 
■fegt-^eea Snost aM ttMog* Mght te «^loit®d ia tsylag to f IM a sstfeO'd for 
coat^olliag tte gs^cmth of tM® tgp® of t'-Mos*. 

Fgpposea coBg^®: f© i?@p®at tte t©st wsiag plas^i cell ti^rs 
that have &T±^m. ia tte ®^^Tims&ts reported ia tls© fis«t probl^a. 
PreliMaa^ e25>®ri^ats iafiisatsi ttot a good ascites forai of tSj© 

timors is a^eSeS sM tSaess &e© ■feeiag dsv@lQS>@^o 

Protae^ ^I'-Ylrm ®tMi®8» 

Objeetiw; It ea^s®a possiMe that tusaor viaruses sight "be sjoi^ 
iafecti's© ^ea tisstss coatsiaisiis the® ^s plBCsd ia fiJ.ffKSioa chm£t@T3^ 
Tba Yl^m ia the ch^b@T ©iglat ^ less affected % host seaetims ( j«st 
as feetesologot® tississ ^thia th® etembess ©m^-roe mmh Icager thaa 
wtea plaaed dix^ctly ia coatsct ^th feos■^ tissues) @M tisst tSa© etaaber 
Bsi^t tht^ s@r^ as a Isog eosttoi^S se'ojfa^ of ri.-m3o 

Msthpas; DiffMsioa cbsB^r© -aere aseS, 

Ifefag filings; fte mwm.-sj ^amor agtat does aot seesa to te ®s^ 
mors iafectl-^ 'jjfeea ia ;-ite the Sifftssloa chamber tbaa wl^a aa egt^l asaoxsat 
of iafecteS tiss^is is plaeed ia ©a opea ehassbero 

Si^fie aae® t^ e^^ii^i^T gsgeaa-cM; If viaf«s@s isere saass^ ^M'ecti-^ 
^dsea piaeed ia cfesiabes^ it isi^t psoiriae a t®tt@r assl^K^ thaa those 
available for tisB)OS'"Vir?ss assajo 

PgQgosed eoagse; To see ^aether ot^r txmar iMtseiag vl^ases 

aet life© tfes ^^^xy ts^or ®^at. 



570 



Rroblem 4"-^To sttidy- the effect of local s-irradiaticai on aj-s?s^ 
resistaat tmiorso 

Objjectivess Tae isiraediato olJ^ectivs is to test the sarcomas 
arising in. the e^erJments cLeseribed In problera ,1 to see If one caa 
"be found that wiii'ljs radio-res is test t-fiiea it is still small eEcaxgh. 
to be observed in tra^isparsnt chasibers^ Saxccmas tested previously 
have been foviad to be x-adiosensttive tiien they are small althougli 
resist aat when larger.. 

Methods; S'tsaors in traaspas'snt cb.aEfeers Tifere obssrvsdo 

Major findJBgs; Sc5Ja®a 

Si^gaif ic^ce^ to cmicer resgg^^; To help in uadsrstasdlng 
the interaction ■fcst^feiHf'raSxo-'resistaat tumxjr -cells and relatlvelj 
radio-seasitive host "olood vessels in 'she x-esponss of certain 
tumors to treatment with s~-irradiatioa» 



part B iacludsds Yes X 



571 



- 4 » Serial m. WGl'-hlk 



FEB-WM 
IMividml Project ItepOTt 
Gals^ar Imx 1959 



Bs^B. loBos^, A-Kss^, asid PuSjlieatlesjs. 

^jrelB, 1» M, aM Algis^, @, 1= : S^i^tiMi of gl®^»e@ll seopla®^ 
P5?oe. Sos. la^ff. Mol. Med. 101: '^3T''^39, 1959- 



572 



'2. ©©jsesral Blol^^ Secst-ioa 
3° letlussaa 1^, !&}.^:yiaa 



a:^viaual Project import 
Cal®£^ar Imr 1959 



Bart J.. 

Project Titl®: iBvesti^tion of ite sole of gsaes asid tbsir 

s^latiss^Mp to iKsa«genetic factors in tS^ 

PriBclpal Znvesti^atos': ¥. So Eestoa 

aM AraoM ¥. Pmtt 

C<^p8rBtia3g ISsaits: ®CI»5i21 

l^^a lears (ealeritor ysar 1959) J 
Sotsl: 6 

F2€>fes8iGS3^: 2 

OtSser: k 

Project Bsscriptloa: 

S^SSMUS* ^ asc®rfeain tte otI® of genes t& ths de'if©l<®smnfe ®f 
^sso(i>3 In mics suod to x'slst® ^m gnostic effects -^th thos« of \si3i&r 
Mological, clsi^cal or pbgfsical a^n'^ isi th@ cau@ati(^ of canc@r in Mce-c 
To idssatif^' ai^ loeats Sipse&fie ^is@s is^ueziieii^ tl^ occuxfeoc® of 

tlt@ -vsslc^sB MnSa of tiH&o£@ essS to €i@t@£S2ias the gmis-sifitiois path\<ays 



^:ffiods; W@ coas&imjs to v^s Bic@ «xelu0i-v«ly as ou^ e:^@x>lasi@nt^ 

EB^Balg. ¥@ a^^ ea^ir^dj^ 8 st^^ias i^cl\s3iitg C3E-*& hl^ m^s^:^'=tws&r 
aad M^ fespatraa stmin, CSif-'like C3S bat vltSusut ^i® mmsiT^^t'miQT 

s^iHt, A-«^M^ limg t?;^r sM lii^ s^^S2?y tt^or stsjaln, ¥M«»Miat 
carries tfe® l^^^ml ^Hofa" gea®, C571Ii aM C571>»1©^ a^i^^isy-'to^r aad 
l®^j liiEg-ttissor straiss, is«»tl^ sts^to vi-^i -rossr lag@e 1k^ Bis@j, aM 
a^»--^®8 stsain tl^t Dr» Ssisbi^ia "bseot^t fs<OB Japan and tlaat has a 
rslativsl^ high issiSssse© of tmsmay ttssssB. In aMitiesi -m harm tte 
asutaat stock Vob ^lat csrzl@s tte rBsessiv® gen® otese anS also CBTrl&& 
pisteld, 

l2?g@fil2sg -teslssiqsses ays ?^ed to pa^d'Obce desisted hybrid g®i2®s?a» 
tl^as ia isMeh segragation of ^® mtS®r iat®s®stiag gex^s «M®r st«.ay 
cas bs olssesijsd, and tbe occ\53Ps^ie@ of tm©ss Ji'- ol^eg'v^g. isi 12^ 
■v-sxlous s®gs?8^at3o Ml ani^djs are eMagsiM^o In 1^ E^ffisss^/ 
ttsso? iso'& ti2® snisKils as?« Mli^ aftar tS:^© s^Bsmsy t\»er te^ &yp^&r&S.f 
Is^ ia tl^ ptslsMsasy twasr ^i©i^ sM 8ct® of tbs stu^iss ©a hgp^it^aa 
tSje aai^^ls ar© Mlled at srp©eific &^s. All isnas^aal tiffios®^ aM all 
■U^s?s ©glsiag la tfe@ atefsffis® of tiss s®^^^ tHsass* sgeat ss-a 

573 



roiitiasly eoanfesd ts^.«p tte di©s®8tisg aieroscc^ a^ter tSia luisgs 
baipg Issea isxflated -^tlj fi^.ti'i?s« Fos-bey-atsysing is eaqslog^ to add 
ei.: ®lisis^te tl» ^aamsy tsaaor ag^aife, aad carcSxsogeais exie injects ©y 
fe^ in appstspslste disp^^ioos o? solistions. Sa tSi« os^iasa 8t<^i®6 

@3g>O0J2s©3 laa'v^ bisesa cs^ried «mt in iMt^'bolic ehassljsrs esppeeiaLly 
a@£»iga®«l l3y Dr* AsaoM W. P:mtt«. Bate ar® saalyzea id.-Ui acceptable 

tasts for si^iificaae® aud cosrslatiea pyoc®aux«eSo 

^jor FiMia^; 1Sa@ ra-^feer es^zssiv® project to stMy t^ssttmr 
tSas gigsetie coaatsnsl of tbe aaaaiasy tJ^or a^at or vi^js ia csoss«s 
tet^^'ssa C3H aM C^TBL 1ms ls@@n c^isleteS sM the results px^asatsd for 
puMic&tiosa. Q®!m c^stirol oimr -^:^ prc^as^tion of tl^ agaist vas coa» 
fisjssd;, "but tb® s®gr®^ticm data did aot sn^spost aa inte:i^retation of 
sisgl® gs2jg ccsitrol, "biife s^ggastsa tlmt t^j® ageat can Is© pyqpagaMd 
in tlie pr®se2^© of os® or sore of sevBTol ger^Se Si:® (^gseg^-feiOQ ©f 
tJ^B« ges^s as s^vsalgd is the oceiarreace of Eaasaaaxy ttssoss la 
appropriat® fost^r-'aus^sed tsst C3Sf sM C^Sla f^ailea ^as ^Jt ap^reat 
5ja t^ f ig^t teslscross vii^s^ it jai#it Imv® bs@a expected tafe lastesA 
ia 'Uis s®s^^ Mcksrsss. Srsss, a f<^^@ lackiag aa^ of 1^ gei^s for 
pst^js^tlssg tbe agefflt appa:matly d^ss aot ©liadaat® I^Bi a@es& Imfe 
ttes^ is s© maltipllcati^i of islMg ageafe aoS. 'thsmigh dil^ioa it is 
lost ia s\ate©«n^acfe ©sKes-atiesss. ®^r@ ^las evideaee "^lat f«aBales that 
do props^t® sad tsaasmit tib® agest caa v&ny ia tfe© quali^ o^ qi^stity 
of tte stgsat proStaeefi px^smmMy depsMiag \:®feaa tS^ aiMter tijey Im-v® 
of tiss gs^s for prepagatissg ■^i® ag^fe. ' fasKwra cyHier tfeaa i^ai^ffy 
t\sm'm -mr^ also talralat©^.. Of tSws®, tepat^^s-wsre -^^ issst fE®» 
qusatly occn^dag t^aos® ia ^se CSM" asai C31e tsst gKSi^s, aM 
rsstioalis e®ll ty^jys 's^r© ^m sa^ f^eqt^afely occiigring tmors of tte 
h^2?id g^o\i>s* Bgticulraa cell ttjis^rs ©eeusrefi ia as hi^ as 26 Tji.ie©at 
of tbs s®ca^ lmekcr©ss g^m^o 

B3S @ti^Qa 1^ ®ff@ct ®f twittjaa caa fegiga-tas^ ia sslss ©f 
straias G3H, C;^;, aad C31® sale® isss eca^let^ diislas "^^ y^gar, ®5sa 
■H^ s®3Qlts '«s©2^ accspted fo^ ptiM,ieati«m. A sas^risi^l^ Mgh 
itKJldsBs® of S^atfisms TSBs fo^s^ ia i^B® -^kxm® stvaSxis, aM tfe® 
Gee^sr:s^Be@ of tlie tt^^s^ ^ms si^iifieaatS^ i3^£@as@S t?ith tt£^tli^%» 
oas®^ tisiKJra 03? lesi^ss 3MxKj@d with tte tsrsthsa ^ss® puSsm^x^ ttJE^r© 
s^itei b^ass^iiagije l€3ioas s<^ of •kMcIi a^s© ©viSsatl^ ls®s^agi©» 
sMo^elicassis ia tSie liv®r. Wi®b® Iteas^sfeagic l@si«sia s^asg'^lisg© d©^ 
h^M-mtTs oectsr -witlssjsfe tig^ttea. ®^ 4ae«®as@ ia "siwrntaasoas" 
32spat<mas tSjiat v@ 'aa-^^ otesri?^ ia ©is." C3H liB®s has xastosMefilj 
■feasa d-oe in part to a ^astie ebsa^ ia tb® stsaiao Part of th© 
ins2^®g#, Iso^evar^ M& besa di^ to a eiiaa^e ia ai«t= C3Hf sale© fed 
Pusdaa JstoaBtosj Cto-u bad a sigaifieaatiy l<m@T ©ceinreac® of 
'hspB.%m&® th^ aid ttos® fed tfe© M?I pelX©ts, tl^ ai@t m mif 
soutiaely f@@a to our eolo^y. Ia reJ-atiosi t© this \'® hBjm also 
i^corSM p:isat©r gro"^?^ ^ritla ■&© KI p«ll@ts thaa vitis tlj© Farlaa 
■iii-:tJl. 'Ife® prsses^og© 03? sfesgae^ ot -fete s^^msy tisa'&s' sgtJat haS a© 
effect 'jfioa tt» ©Ge\KE®ae® ©f tl® l^pa-fecsm®^ 

574 



- 3 » Sterlal m. mi-klj 

Furtbar s?ssti3.ts ha^© Isega okr^iasd'cHa tbe effects ©f spesific 



All of tbe C31¥ F^ fej;^aiEg ©eiS vlygla fmsslmB for ffittaSyiiig th® 
©ffeet of -y:!© letlial j'sHm? gsaj® cm ^sa^asry tx®os® have b®w tffi@a 
a^^;cpsi®a« f^s^ ^es -v^tsy little d±ff^3E«i^e Issti^sea tbs yellow aM 
tbs agmtl ^rs@diS3fs. Of tbs IC^ ^llow tetedlasg f^mles ^ pe3?8sat 
d@v«l®p®d siiBs^^- ttsisr© at sa avers®s a^© ©f 7.9 afflaths, aM <^ the 
106 @|pt£fei tesedis3ig fa^slass 97 pes^®^ fevslspi^ iwmmsy tiassors at 
SB swysg® a^ of 8.5^ sraaths. In "^s visigla f®sl®s tljsse ^ms very 
lit^e differgjM'© ia iacM©ae@ of ttmox^^ Imt tlss t^^oss e@sm ^^ at 
a ©ash lates" age ias tte agouti f espies. Of -Si© 88 ^llow vlygla 
f^aales 100 p^s^eat deiralcped asaffiasasy ti^oi^ at an avexage ags of 
80 1 ^sattis, ajffid ©f tte 111 agouti visgia f<Bsmlm& 96 p^^reesit developed 
BSHiasy tisssss at an avesmg® ag© ©f 15 • 2 mmi^^, TixaSf it appears 
tisat the diSfersK28 eaiised t^ tM® ges® is . "baeicsdly a bosaooal 
differeace ^ffiich ee^ ba seen in tb® virgin fes^des, but this hosmoBal 
diff@r®s2c® is ol>literat©d li^ bx'eediBg tSse fessales. It ehouia fcs 
aotsd tJsat tl^s© tota coaafis® little's ©asiiey obssrvation that tb@ 
^llo-ef f®;^es had tl^ir t^isors earlier, latst do n©t coxjfi^i his 
©■feser-'yation ttet the final ix^sideac® im ths ^sllows vas Imar thaa 
that ia tJi® Etsi-ysaUo^;© ^ich hs& aJ-^sys 8«€is3& issprol^Me to \is. 
©IS fact tteat appsT>xiBat8ly 100 p^se^sat ©f tlis f^jales of all grot^e 
ia oiir e^p&rism^^ M& -fetors iMiea"fc«s «3® gGoS b®alth i^is^iiaM ijs 
ous" ssis^l®, aad tMs ^y 3^ pa?% aceo^mt for th® diffe^ssac® Ijst^esa 

^ta coll©et®d SiariBg t^ ysar ix^leate a positiv® effect of 
■tJje letlml f^ll^^ ^ssm also tss •S^ oceur3Pence of sspontaiaso^® feepatcsffiaso 
fM® bas "besffi sho^ai in f^^ hybsld jmles finsa erossas 'betweea stsaiiss 
C3S asd i®. Freliiai'mz'sr calealatl<ms ^^r® im^ ©sily aa tte teta fsBSi 
®al©s th&t li'^^ to -toa 16 moatljs of s^;s s«t for aut^s^. Of "Sie 63 
yell®?? sales 100 peg-e^at IsM tepatoiaas, ^hs'm^s of the 9^ e®Mti ^l@s 
88 psrceat bM !i^atc^a®o Aa ©v®a gi^ater diffss^sKj® ^as sbo-sta is 
tte av©rag© aiabsr ©f tepateiaas p«sr aaiaal. Bt» s^sHow iEal@a avsssgsS 
3 Si^»at06sj; p«r asaissalj aM tbe agouti ^Iss aveiag®^ caa^ 2.02. 
St laas tern tos-^ ttet tS^ letfeal y^Ujcm gea® iBC2@as@s boOy i^eight 
aM it did isi tJiis esmerS^at. ]&i aMiti<m total tejdy lessg^ aM 
l®23i^ Of tb® f<8^aar ts^s® i^asi^®! ajsfi -y^ ®sstrocn^Bi'>s® ^ogel® -w-ais 
^@i^i@d. la aMa^s of this a^ tte ssuscl® ^si^ts -mv® mat 
@atiafaetos^ fo? eos-x^latio^ ssbI^^sb but tSs® ^@1@^^1 msasiias€^S!!E2r&® 
•m-mc Sjs tli<s eo^Mmd gsofi^s '&®r® a^s a positive eosi^latic® tet^j®sa 
©ee^jx-s^ssc® of b^atJ^as aM each of ^js tlisse ffisas\srssieBt®, "bo&y 
«ssi^t, toa^ Isag-feli, ®M leaglfe of fsms^c 



iaeffsas® t&© <xei?2'£'^5see ©f ptslsosmsy t^^rs, ^^m^ tu^ss®^ ©si, 
Isspat^ss. ass« tota @x^ad -^s® sug^stit^ that laaay of tS^s® 
specific genss ®sff®sti]^ ©eem'S^BG® of tija varim^ ti^soi® do so 
ttocmgli ps^s®sg©s also rela-feeS to JKJxmal gs^^itli as ^as«s®i In 
stele^al B^. mmevQ^T de\«f&loptesat Ijut i^t as sstasis^d ia toiy T-Jsight 

575 



- h '■> ferial fc. KJI-^17 

'SMi stt^T of •®!® ©ffsiTGt of e.^:^'®®. t®Essi«m cat iBdustiOffi of 
p-alisssary ■•i5ssa©»s la atraisa A tA&& ifissi® Ib e©(^s^^a%Xmx ifitSx 
By- IrsisM W= Fts..%% baa 1j®sa ss^i^^a^st retafitefi ©sdjag to Slsoy^^ ef 

liBS relsticaasbip tetw^^n aiasfer of iMtaed p^ljMjms^j txaer® aM 
ciKssestsmtion of os2?@®ja to -ttiieh t&© assSa^ls %"®2^ ®5spos@d for iSm 
foxiT coasQBtj&tioss^ 100 p®H'£;@at, kO pesv.€at, 23. pexcesffe (sir), se^-. 
3 pareeat^ 



is ccsatViMtis^ t©'&mr4 a mas^ tlaorc«i^ iajsg,©2®tas^l2ig of tSs® etisifcsgy 
of caster, fli* psfoggaia e^tmsiss® %to l^sEtsse® ®f gi^ass la ©Mel®^ 
«^s^ii. to ths fex:S@at of .Dsg^'Stirii^ ®ff®8'^s ©f ©p^cille (g©aes ■«§?«» a 
Eis&ss" of diffes®^ 'typ<ls ©f ear^®^.. *&© psogsE® also ^s^basia^s tiie 
selatiossMp of ®®Eies to otJier toso^sii faetore ijs tte ®tiolc^f cif eazfflsro 

of e.ma3®r tlie getass couts^lliKg tSa® ps«^a^tiea of tb@ vlssjisesi stouM 
also te soi^idsy^d. ®s® parogras is BiBlpteg t© -^te tto® eff®?5tii ef 
S2!igci>?le ^ffl©s od sp@cifi«5 types of casisi&s' t© Isog^ssal ®ff®ot£t a»i to 
aaxaial gs'o^li gS stel^rlal sM ssuse^lax' ®l^E8®fes => or tesic:fi&M.y t® 

■fete €£-ae®r pieoeestJ t© lassteMi^r.o All of tfe^sa ec^trl^iu:!® torsms^ & 
teoafier «M®.t®taErli3ig o^ tte ee^assaMcs of cas»®r »■ -feh® sppy^ash to 
the pseYaaticffl of aaasse? - aM 'teMs ^xm± fe® -fefes ult3jgsa^ slm of thm 



fog^ this lis© of w©2^i t© ■&© eo3Sfeii^w<a. Mfiiti.offlai mR'k »%- ia, 
p-^ogx^S3 iael\ffil@s sfeaai©s ©f t^ «fff®ct ®f tltgi letSsal y^llmf gm& 

■tests for tte mftmit aS ^hm i^j^ ©*R€s.@ «m sp^aataa-so'os p^aasssmry 

tii!a&g^ la Fi aaai F2 ^j;^s143 tet^K^g© BtmAn A bM stjpaia m tijst te.s 
fe®®a B%l®Qted for Issg® tetly sis®,, t© ©«® if ttes® is a positive 

l32fl^jg2®gi ^' tbss® HuAtiplts @M. pagsiM;/ i3ea«i^@«ifiG ^s^s for 

dvjsjef gss^ ®v^ "tj@ plaa to tsst -feis gsa® i^^a a Eoagbsr e>f -tsase-^s.. 

B»® v*:«^p®slami? for stMyi^ tl^ p^sslbl® Esstoai^ %■ 'ilhlsh 
tlss lethal ysHcrw geae iasr^asss poilKoaa^'^ tisms'^ raa ist© jjcsscbIIce." 

that til® g@22@ i2iss^.'as®s aoBual gro^t^ ^sM.eb ^m& thou^it to te v 



576 



- 5 " Seriaa Mo mn-hrf 



KI diet tl® fiiffes-gise® la teSy v®i^t ©f tte ^llow sssd tb@ tew® 
ees.ts«Xa is ssot great s® %-© as^ tovlE-g to r©p@at tte stMy lasieg 
Fm^iim latex^tosy elK2^ ttot doe® x&svlt in a x^eas^'bl® vejl^a^ 

H© mjiff Isav® stssia M aic© tlmt Ik-o fsuteum left with i^ -atea bs 
Tstiss'issi t© Japsm. IMs stmia cs^ fssDsa Ji^aa ®M l8 wajmlstsd -fee 
as^- of -felse straisss is this cotssfesy. lofwgv«i', tls® f®i^l®s of thfe 

is r^iP- -Isi ^B FiQ gs^^iutiosi ©f bs-etJ^r X eist«y iafespe^iag, ms& ^m 
plasj to cost-iiass to iate-^e^ t&© strain, t© totesMss esos® aee^rately 
it® isaifisaca of s^Ej^ify tmioses, to me if it has -^ asj^a^ t«©a' 
ag^ist, &ad if so Jkjw tte sgeat ©i?i^ar®s ^th tlmt is our ottes" strains ■. 

In Mie st?£ii@s cm tl3@ @ff<s«te of ti^^^a a& ptsQjscmasy ti'^os^s 
^33 ^l&n t@ ccHStlm® ths sitMy of &ob@ v&Bj^sm® asd to lo^ fm?tte? 
into tb® qwstioa of ^slistJ^r or sot -^igy® is a fiaiasgii^ effsct of 
tba M^ cffisceatraticm of ©:?ygeB oa t&s pul^onas^ tisatsg. 



Part B lacliMffifit les 



577 



- 6 « Serial 1®. MJI-^l? 



Cal^aiaa:^ Tear 1959 



?^|_^. losos's, Aws2^, aM BiM.ica,tioss 

Jc H®2^fi. JuLy<»At3g. 1959' 

of ©po2it:aisso«s bepstamas sM. tli« iiac^isas® of tMs iasM^sc® 
wi^ uretlsaa to C3H, C3^, ai^ C3fe '^tl® ©ice, J» &t, 

®^®8t of gsEstis g^gs^^tioaa ®i •^b® %ssas^ssi<^ of W.m 
^assasy tiMor agsat la isles* J, &kt. C@^er 2ast« 2^; 

(m^sh i960). ■ 

^o fes-boa, ¥* 1.: Sits ©f fexss Actloji aM Ca2«lEeg®2®siB» 

MiKKjgs'spji oa Sesseties aj^ Cs^e®r pj&liste'd % Si.. Do ABsSersea 
&®i>.1:tal aM ©sasr lEstituts. 1959 « 

5o Hsstosij VI. E..-; Sfe© ing^ct of ggaeties upoa caacsr Fsseayslio 
mil. EosTOll P&I& l^sarial Institute » 1959, 

60 H8stosi;s W. 1. : fevl@v of tbs bodte Caaadiaa (Jaacsr Ooaf@s?©ftc@ III, 
AoIoBoS. Balletia, 1959. 



578 



lo Laboratory of Biology 
So Qeaeml Biology Section 
3° Bsthesda. M. 



Ijadividiaal Project Beport 
CaleMar Year 1959 

Protest fitle: Host»>T\^^r Belationships 

Principal IsTestigator: Msrris K. Mrrett 

Othisr Investi^.tors : ^Icolm B« ^Iroy, M'^ard Jo Ireygre, an)S 
^r^ret Ko Bex'inger (isi. collatso3ra,ti©a) 

Coops^^tirig UMts: B^I-421 



Yeays (ealei^ia.a' year 1959) ^ 



Others 2 

Project Bescjdpv.ioa: 

Qb-jective; Our vo2fei3Sg li^otMs ^.^ coi-cei as a field pr^sestly 
called "^isssussegeaetics" vliieh covers sm as^ga ©f o-^rlap "bet^esn tJse 
tt-sD classical discipliiigs feut se*^^ to differ si^ificsoitly fr<sa escfec 
Shis is a rfiJ.ati-wely aew f ieM aM. saE^ ^EdeiiiEes sM eoasepts are 
basjj, Mt appears to to^seb i&poja seves^O. olssc^xre areas ia biology, eogo^ 
adapti-x^B easyssis fossatios, tmbr^roMc iad'aetioa, diff©r@atiatioa, 
irradiatioa preteetion, aad tlis fes-oad Irat ill defiasd field' of "t^^or 
ilSEBaitjo" It is Ijee^lag ijiev^esingly appaseat tliat biological iBter- 
aetioaos iiavolve s^ae tssdiscoirered "lafossgitioa" or "s^ecc^Hitioa" system 
of ges^ral applicatiou. Ovs* bjpotlassis ass^^es tS»at soae suab system is 
iavol-'i'ed ia tb® intescellislar 3?eactions of sor^al diffe3?eiatiatica^ 
gro^h, physiology Msd tbat tSie el^ssosiial differeatiatioa aad gro^^tSi te 
caaeey rapreseat ®a afcgs'satioa ia siseb. a systeso We also eEfcertaiia 
tbe iKJtion that mxih a. fcz®aMosa msy Bot sl'^ays l38 ccs^lete sM tM.t 
thei'eia say lie tfee e^^lsyotatiosi of spoatamsotsss s®gs®®sicm of e®ae©r,. 
t&m§OTB.Ty r«BsissicBs of e^jc@r, aad tise apparent eoatsol of sietastasis 

fibre specific^ly: vm as@ att^ptiiig to discover -s^at "tissue 
IsLsiiaaity" aM "tauasplaatatiosa aatigeae" ar©. At first tbo\2@it tliey ssay 
appaar to le s&thar BfisHseasical labos^toi'y piaytMs?^ Mth no r©ai plae© 
ia natural pljssoaesa. Ictfevgr, tbey as® so ^^Mespread ia lostiaj® tMt^ 
speakiBg teleologieally, t^ey msst ba-^ scsse physiological fyjaetioso 
¥e tselieTs tiiat discovery of the txifs iiattsrs of tte "anti®aa" sM -fehe 
"i^imity" ia tMs fieM ^d.il ,oj>eB a BS^f appreacli to m^iy psolaless ia 
cEGcef, 'totli as to tbe ijatslssic ^tm2<e of usqplasia aM as t-C' its 
control. 

579 



™ 2 - Serial lo. KJI-J^-lS 

Sfethods: Tk^ Ijasic proesd^a^® is trsasplaatatioo of B%asi&s.j^z&& 
tiroos's, or skint, in.-^ hosts of toao-j^jbist hi^ily varied gsasetie ccm» 
s-^itstion aM ol^srvlsg tfes effeets of ea^esdEsental vsrlatioa of hosts, 
or tuBOTs, or teth assd of i^-oaiziiig iujectioas, trealaasnt of -feJse aatl- 
g&aSf efftets of eb^iilcal or plsysieal s^ata, etc. ^le dlsciplizsasy 
tecbniQ,i;ses ax^ 1^k3S@ of ge^stics^ issms^lio^f essA ex^rSjotrxbaX euTg^syo 

!fe,1oy_Fi^i£^: In relatiosssbip to ^hat ia -mriously eaUed 
"aa&ptatlOT^ of a -feajsEor, or i^m "^i h^rid phssssaeaon, " or tb® "Sasneett- 
IJsx-jiger pi^aomsmsa, " iksi^ of i^aich is a good t«s9i, ^iera hava 1>een x^ 
sa.joy positive fisdiags to cbaags vie-^i^iffits previously e^^ressssd. Aaotter 
ti;^©? "adap-fced" ^Hsfe oaiy poorly aad -^as drofpped liecausa of teclmicai dif- 
ficulty. AiaotlsQr atti^t to leassi -gfeether siaMltaaaeoasly le^len^sd "©-fcock" 
and "aiapt©d" tisEors i®t®2^et jieMsd xssctmviaciBg data "^feat, t£^E«si ^-ai 
psi^vious data, lea-v®s tSse qt^stion opea. AnotSjer attss5>t to see if byteld 
ttssor adapts ^-m a ms^tlvm s@eult oei poor data; -^v^ ^^ak data Mve 
points Ixstii direetiomo (HMs has a l^ariag tm "dosage" ejects )« With 
T&sj^ct to a I^l^sld fet;2S -^^ ba-^ ol)@«r?@d no «td.d@:i&e of a rsactioa to 
tSi® pure stsssla Esther » ■&•(!* tbs sotteg' is chaa^ed, as ^iH l>e 
latsTa 



Ovsr a period of 3f®ars tter© Mv® Ijsen several iMicatiems that 
the Histocc^patiMlity-a (l-S) c<ss>le3c of entigeias Eiighfe have SK^e 
special featm^s vith suspect to tS^s® plssaoss^na. Our oiEfecrc®s@s 
in-mkv^ diffsrsi^sss at this sts^csng loe^ss Imt th@ x^sults gsssisraXl^ 
suggssted -fctet ^shatever tsasasplsatability changes occusrred did not 
iuvol-v^ cb^2^s Ib ^d@ antics ef^^lmx, la sgs@ 't^s^ tMs @@^m (^do 
W® as^ ia the early sta^s of ^sosfe with i^^ tts^rs, la -^Aa c&b® •^tes 
twiors aM ^ds^Ls ars all alii® at this loc\2s» She thought is that 
if ehaagss oceiar ttey isust "i^ 'based oa o-Sser locio (^m of tli© t^^r© 
is na^ grs&'liig ia abotsfe 50^ of ti® fos^i^a straia ■*?«& th® results s^g^st 
that wl^t ^ are gettisg is "iCTUjaoselsctioo" essd not "adaptatioa. " If 
this is so -^ vlU ha%r© eri iJEScfcesestiag {^portmsity to try the sffect of 
otiisr outsrossss on the Bsms ttraors. Siis ssi^t throw soass light t^jon 
tJae s^latim of "the ^teid phsiK^mum" to "sosatie sax^tion, " "ts-ass- 
dtictioa/' and msB like. 

Ws have had a losg ©taadiag Ss-Sserest ia th® "isadividtgaXitsr" of 
tssaors. Tim varisd Isehavior i^HiticB^ afeo'v® is ce® agpsct of ^lis, 
Aaot^er is our tsatati^i?® ofeseri?aticm that ia msm ecsaibiiaatioas ti^jor© 
jsay aot, as is ^aesslly feliswd, grmf as veil in th@ Fj^ byteid a® 
ia the parssEfe stmia« We tM^ that -m hav® seen -^ais 3 ti^s« If it 
is tsnis it sKj r®^iJiire sczs^ rsvisi<Hi of tis® sispl® eoascspt of "osm 

'j^ ^itigea," -Kith "cMplete dcaalMace." Aloag "SJith this x^ Imnr® 
careftil, frmqvis^ ssssijr^sests of a ssai^r of spsist^^sotas ssasssas^ 
ttsaors is jaic@= Ss® e^sc^sd @x&2«i^ ^mriaticm ia growth -^bs ote®r5r©d 
asos^ jidce lut^ ^os« -mriatioss wsr» n® greater thaa -yKss® te&55@ea 
ssaltiple t^ssors as a sissgl® laoiis®, ISi® latter eaa hardly tie eai^®4 
1^ the hossKs^l or ^tetelie ©tat^ss of tSs® host, 

WiMsi respeet to -Ife® q,mstioa of tjl^-y^r "adaptstioa" iffivol-ros 
th© iats-odiaetiegi iato tte e@ll ©f sostthi^ liks 1214 prot©is, a -tyiriis^ 
a pla^a^K®, or tte li^j ^5® hav© lfe@®B caqg«ratiBg ^th Sr. IMSmy cm 
a fia%ll pKs,1e2t«, le Isas s t@c!i».iqtBg for a&sds'MKg ®ueh ^terial 
cffl TOS^ fism ss^mmL glass pa^tlei@s sM tl3®s ps-qpelilisg it tlas'TCgla 



a cell SS.SS ia ''cbB esats'iftise. 'Sh.& idsa is to esctract possible tgaiiS" 
ducers frcsa oija straiEj, iaject theai into ttaaos- cells of another stsjaia, 
and see if tha ceils are eh^s^d ia tbeir latei^ction with tSie Tirst 
strain. So far tbsre iJas fceea mueh t©eMieal difficulty aad few 
resiilts l^ut those obtaii^d enco-uz^gg pe2^istencg = 

All the &iKne iavol-ves stuiSy of t>!S inte^ctioa of a hoeiogssft. 
aad its host. We bavB les® "been iat@s«st@d ia the fact that ia ^udo® 
teeedlBg populatioas a fet^'® is a hi^gsaft feut its istesiactioa witfe the 

tost seeas to 'bs ej^sptioaal. (fhis is also sisggested in ebozio- 
©pit^lio!^. ) To stmj -S^Ms bqvbtsI. et^Maatioas ©f ia-strsia aM ©ateross 
s^tiBgs of laice vese set i-^# as reported last year; tte ^otheifs aod 
offspriag i-ser-s o'feser^d for possiMe. effects. The vbtj iaterestiag 
©utcose laay "be epitcsiised as follows. Smmg fm^les of a gi^en stxaisi 
those paroj2S (aot pregEssit) 1^ a ^ile of a diff©2«at straia e^iMt 
"toles^sjce" tosas-d a tisasr of tte seeoad stxiaiEo The tolerasace is 
spec if ie for -l&s strain of t&s ^1@ sM is ssot s@@a ia fei^les pasisji® 
by a third sts^in^ Tolexmae© caa ba ©'bserved either as ©a iaersased 
Bisaber of tates of ti^jor ia xaaiaasRaaised fi^iales or as veak resis^ase 
in li^ssjniEed females ^ flae effact appears to iacs®ase ■altli multiparityo 
A vssx&iBQvtgt reporting tMs is ia p3?£SSo Further @3^eT_-_25^ats ia pregsess 
ba-ve iMicated tJaat t^ toleraae© ean also b@ sees &s an iacseased 
sttrvival of skiia h<asogi?afts. ,fi!;;e\,v' "s^^i a tiaor of tis© tbird st^ds. is 
used tfcs ^ecificitj shifts to tlmt stsaia. Eq the case of the latter 
tvsssoT thsxe sa^ fee a Msarre bost-ttseor sjelaticashipo A few oljserm- 
tiOBS s'o^est timt ttere is s<^e sort of aigs^tios of th© tissusc- ffe® 
is^l^at starts out as e localised imssi after attaiaiag a isaodesate size 
it teccssBs craterifosra (aot ule&s^-feed) aM ws.j grow as a lar^ cix^le or 
crescent with xkj grossly appaseat tias©? ia the ceat^ro Siis has aot 
feeea seen iB t&e strain of origia of "fehe tuEEoy« 

O-fehex" incidental aad testativ® ofesermtiois oa tl^ effect ©f parity 
ara as follows., ae toleraace pgfsists for at least I50 fiayso It ao®s 
siofc result fr«sa ^ere escposTsre to tb© ^O.s ia partitioned cages ^^itho-at 
B^tiag. .^pay^atly a previm?j3ly existijag isssme &t&te ws,y Ise abrogated 
tsy ma3.tiparity« Finally, •sliea f^salss are i^samized agaiast tiss^ses of 
a givea sts^isa, satisgs to ssales of that stiBia aaay sfesult ia fesar 
fsrfcile imtiags» 

W© coBtiisue *mriea at-is®2^ts to leasa SG^-aisag of the s^t^sre of the 
aatigea ia tuEOS" terosaity aM ha» it say differ frtm tb® associate M.eod 
grorap aatigeasc Briefly , oia? observatioasi coEtiat;® to sho^ that tte txfo 
antigeas differ is their lability to hsatisgj to foi^Lia, aafi to soaic 
•^Iteaticffi, ^hei&er t^ aati^iaic saterial is derived tram. M-ood or frc® 
tuffior- tisstftg. The tisaor iKEoaity aati^a is p3?®s©at ia feta,! tiss^ss 
IsQfoTS the Sao^sa Mood g^oi^ aatigess appsar. She latter is trs^ «v«n 
^ea by"fexdd effibryoa (i.So^ coataiaiag oaly a half^dose) a.m usei as 
azr&i^a. Wtea a MIM/c traiajr is isoewlated iato C5TM»/S-2^ (a ceiS(^eBie 
liHs) s^eipieats the latter as?© sesistasit to a DM/2 ttassr. Ml th©se 
ssatsrlals ase tto© saae at tlse H-2 lecias aad skje© of the 3 possifel@ cc®» 
biEatiesas yisM hsffiaggl^feiaias ia ^gggii^-aiaised smimls alth©i?gh tiMor- 
i^KiKlty is stseego 



581 



- k » Soi-lal So. K:I«4i8 

¥niB proMss se^ms at i^reseat to 'e© gettissg sssre c«»f>iicat'Sdo 
•Slsare are ssveraO. iMicatioas tlmt sse^s ©f o^je= ideas nay te too slg^ls 
am direct « It appears to -us iih&t skia gs^ts aay peiait aa aggTCaeli 
to proMeffis for Hhieh timos- grafts aiss tisssuitalsleo But i^ as® »5t 
s-stisfied -«rltli p^seat tectoifoe® fos' sMa gxmffciiag. Aceordiaigiy -^g 
are tr^ring to dsvelop a tecbsigisg for oWaiaissg depex^aMe ses^slts 
^ea Easy gs^sfts are plsced slKJSltes^oissly oa a siaigle 2^elpi©at» 

reached. If -feliis casa te Soa© it sesss possiM.® to sst i^ @2^eslii®ats, 
is'^olTljag "crisscross" aad "rois^ robim" tyg®s of trmasferj tlmt -will 
pes5sit obser-s^tion aM satfeesatical asal>^is of sioltifaetor effects ia 
a i3ay sot heretofore possible. !Siis far^ fair s^sscess bas 'bsea atvlmiEefi 
witls as saay as 20 ^^£t& am a ©iagls s-s©«s@o As aa iBcMeatal tiadiag, 
•?3e bave eo^isBsad tS^ ssportefi s^slativg iaccss^atibility of a sale- gsaft 
OB a female 3?eeipieat witMa stsaia C57EL* 

As teekgro-oaS for bcsk of tbe afeovB it feas teas aecessa^y to do 
a la$"gs ssoisat of serolo^. ¥s liav^i ea^iet@€ detesssiimtiosas of Mood 
factor 8istig©Hicitj i^siag tSie 7 stsaiias wadsi? sttsSy ia this laloratoryo 
The 12SS of saefe strala as "both sseipieiat as^ fio^r bas ^eMed a k^ 
plae@ taM© tMt shouM ■grave & i2s©fal jpefes^uc® la f^sfeiase ■»@r&o 

B?o Molossey tes rspor-feed a, vlrs:^ aerivwi fs^ our Sarecs^ 37= 
His ^^3± ■m.B dop^ with MLB/c ®lee. ¥® fea-e^e tSi®TOfoE<© ©ufc<^si@d a 
large xi'asates' cf strais. A saice (ia vsliieh oijr ti^os? i^ caxried) foy sigas 
of l©-s^oses sad Im-^-e fo^s^ bks©. Mso.f Df« Br@^re is ftsmishiog 
"tol^s'aat" fesales of a resis-^fe stimis t© fe. ^losse^ to see if tMs 
sort o? tol®-£a.mie affee-fes s^sistsne® to Tlras. 

¥e texsa atit^spsied abotrfs 50 old wd.ee of stmia C57BL/H»2^ to s&e 
if, as siagges"bea fey Miss TJpboff , ^p©stasi©o^ag pol»8 of tSie aiioaeis^m 
®M eolosa occisi"» Essy cce^r to abottt 1^ ©f ssdss aM aboKt 50^ 
of femles at afeo'itrfe l8 ®mtlis of a^. 'Bm tiso lesion -teM to oeexsr 
togatte^o 

A l^'ge as^imt of tlsi® has te@a ©paat osa i^itorial dw:'^©s -^-sith 
the Jot^aal of tte fetiosal Caaeer Instittsfce asS Caaeer P^sc-arcli^s. sssS 
oesasioi^liy for otlner jotssaalss 

I parteieipated, "by isivit^tion^ la the foUosriag meetiagSc 

Fraacis D@lafieM Sbsgital^ leir Xos&,. ga\'e a general seMearo 

toericjm Assoeistioa of leM aad leek S^rgeoas, Washington, 
iB^idtea diseussoro 

^ellclK>las Seia:l^.5fS; Palm Bsaeh, a weel; of s@^Eaa=s Gsa gessetic© 



582 



difficult m^ dbBcntm as 



fieM w:§.tfe ^?Mcli this %-ork is concess^d is 
s© difficult bM o'bacuvs as to sste clear defiMtlon sracl outlisses 
almost 3^ipossifel®o le^mrfctelessj the?® is a slowlj gx'mfiHg Isodj @f 
evifeac© ^hich IMicst®® that tlsere s^: iate^actioas Itetw^ea tissis®® 
asad tteir host ■sMch i®-rolv© esdtieal faetosB ia the pheaosjeaa of 
cancer, ^abs^ral©^ ^ad tfaasplamtatioa of aosasal tissues » A defesssiM.® 
defiaitioa of causer casmot ba gi-v©a tcdaj escept ia descriptive aM 
Mologic tej^So Bte tesa® tssiMlly IxjclMed tMogs x^lated to tte 
@3q»erlffie£t@ tMt ^^s Im-m te@a d^sscribi^. ^0^ ®sp@rl^eats asy 13@ sig« 
nif leant in several -asys toth psmctlcal ®sad theoretical = 

fh® sjost is^os'toit fe^lieatioa is timt sore of ttese sssislts 
surest psogi^s® to^iW iiad®rsts3aaiag of nh&t cosit3POls the g;gcm&i of 
a caaeer aM hem •&!& differs fr«j oidsmI tiss^^So W@ have als^a/S^ 
ss®n tJmt & tismr'B poser to ©uesee®® hont T&eistmsc® eaa tse alt@iM; 
its xate of gsswthj, tesstescy to rggjress^ @M teMejaey -feo sietastasis® 
®3?g all subject to e3^«rlB8©Btal clms@e= ©s® host's reaction caa te 
altsi^a ®speri3s^B.taily, aot c^slj tet'^ea sisaiMr hosts, tafe also at 
differeat tij^s aM sit®s iss the ssa© "aost. fte cbaages iB ISie t^aaor 
have elided sospbolc^e, cbeaiieal, or serologic defiffiitic^o Cutg of tbe 
aatig®as isMeh sMss^s tte tost "wouM Ik® aisseS ^ ciasreat eh^aical 
B^thodSo M ii^^rfessat a,sp©ct of tb© 'Ijjbrid pheiasMeaoa" is Its pogsiM© 
3?©latioMi t® "ts^uiisductioBo " ]fe^3Ptoiyfe possibilities with »®a£d to tSse 
effects of parity can te im-giaed ija E©lstioffi to s®TOEal cc^^lieatioas 
©f ps^gisancsf iBslT^aiog Ki disease ®M etoriocasciaoi^o Iixfeexps^tatioas 
B-Ts tJsisiss®o Mb'^sver, os© isight attala a biologic d©@eripti©a of tte 
fsct©s« that s«»stlj®ss essase sposataEeotus ttssss?© of sma aad ©aiBsals to 
ysgs^ss asd that coats^l i^tastaeis., It ims edsesM^ feg@a o'bser^ed isx 
tMs latoratoa^ (^ (eesafi^^ else-^|j©r@) tfeat st^h factors imtesially 
affect tbs ©jstec^ of ^mss^ cli@Bot^:mpsutic ©s^esl^rts., 

^^ti^i}0___ot^M: K!®r@ is ®, co^l®^ ttecr^^tiisaX iate?l©cfeiag 
^t^iigsgw tbii' TO^^aM tl® -aosk cf IteSc '-Mtli, Is.'w, wen'ian^_ano^Sanfora,, 
;and Mi ss U phoff . Ifejy of iiha z'esislts atteadaoat ijgoffi tlis use ©f 

Mlllip©2@ fiitir® ^m^e® fes'eslisdtwed hj -^i® ¥©xk on tte smtm^ ef t&e 
aatigea ±n. t^m&r i^mmltj. Tkm ysork ®i adaptatioss baa a sts.'m^ 
rslatioa *© 2?«§eeat d«v«l©ps®ats ia j^^iatioa Molcgy aM is tissxas 
t3fffiB0plaatstioffio 

Proposed cotigses E3st@asi<m oa the M,lia @:^©2li^ats daserlted 
atjove. At ps^iseat, ow ^tjor s@«4 i© for tis^rs vtth ^M.e& e:sp®ri®s!ats 
lite tfe@ alsove eaa te 2?ep®at«d fcut with certaia ■mriatf.ojs®, @8p©eielly 
a i^verssl of the iarfceretsaia mlatiOMMpso We as^ st«dyi^ m-mTal 
tm^Ts la a sssre'h foip sisita'bl® ©ii©s<. 



Part B. i^lMeds fes 1 



583 



•» 6 - Berm.1 &. Kl-^i-lS 



GaleMar fear 1959 

^r-|^e loaorSj A'^j'arfis, m>d Rsblicatla^as 
Foblicatioasj 

9l5-0-9^2, 1959 » 

Bsejet&f S. J. : Effect of iaoetilatiaii of paelsed es-ythyocytes oa 
suTA'l'^ral of skia 'hi:m>gssi£ts la 2^ts« Proc, Soco Isp, 
Biolo Ifea, 101: 7^4-7^7^ 1959» 



©ice isdiie'efi "to^ stsmia-'Sp^cific latlsHgSc Jo^to Casieer lastt 
(in press. ) 



;8^ 



2=, G®aQ?ffll Bicjlogy Secti©a 
3c Bathesdaj Mdo 



KIS°SIH 
&.l&i^5&T Year 195? 



Project Titles Geastie stadias in saieso Ao f\sBics*8 ia sdc® 
PriBcipal liwastigatcs's Margaret Kc Deriager' 
Othss,' Investigatoa'Sg ¥» Ec Hestcsi, Mo Ko Bas'ratt 

Cooperating IMltsg Hons 

Man YeEs« . (calendar year 1959) i 
Totali 2 

Bfofessionalg 1 
Othsrs 1 

Project Dssczlptions 

Ao TOTiors in siieso 

le OcsmrsasQ of Ksas^ary tissors in agent^f^© DM/2sB iBi©©c 

Obd^^^|8 Sts'sin mk/2@Bs & substrain of stg-sin HBk/Sis 

■n&s 5^^^©3^^ the transfer of fertilised o^ freau stsaia IiEk/2 
to sti^ia c57BLo Th@ de-^elo|^.eat ctf ma^issy tsiEBSS'a and of ©feher 
ttsKiii's iM bsisg stisdisd in group© of virgiaj breeding^ aad 
feres^br^d f©imlg@j and in males treated td.th stilbestrolo 

MgtSigds^^^^^^^^ A gro^ ©f DPA/2e f eraales i^s kept as 
r irgiS^'^^^'^^Ser^oup is b®is^ forcs-^brsd,, The force°>br©d 
fessalss ar© isolated t^ea pregnimto After ths bir-Ui of th® 
littor^ th® young ar® discarded, sad ths mtsthsrs ar@ r®tu2^ed 
to th® br®©d3^ cages s.t (sacBa TMs ppocedi^e i@ eontinued. 
•throaghoat the breading p^ied of the fesmlaSo Cholesterol 
psllstSj ^"sighiag 6 to 8 !Hg« sad ccffrfcaialng 10^ di®tl^l<= 
stilbsstrolj s®r® implanted subetstasas^'asly in ths right ajdlla 



585 



NCl4i21 



of DM/2q sales tMt is-srs mae to two meaths of agSo Tha 
hreedsig fsrrjales wqk-© tissue tSjat ^®a?8 us^ to asaiataisx the 
colK^'- ©f dm/2® mxeeo Th© vsxl€i^s gros^ ©f sdce ©s-a 6b- 

tvsamBa 

^te_F^^^^s A coi^iderabl© aymber of th© strain DBA./2@B 
snSjsals of i^^^ea-lBsat ara still aiiT© bset a sufficient atDabsr 
haT© beea awtopsied to ii^eats the prebabls tr©ad of the rasultSo 
Ths incldsRe© of KMBnasy tuiaors ai5)sar® to be considerably losr@r 
in ag©ist«='f2r8e DM/2eB animsls tl^t developed frosa tsansferred g^& 
than in strain DBl/2„ In the coIoh^ of sts^ain DBA/2 (^th the 
agent) J 6$ of 81 breeding failles (80o2^) hav® developed maiiBaaiy 
tusiors at an avarags age of 1h<,$ inosiths, MaisBaaxy tuniors have 
devslopsd in oas strain ]B/L/2e breeding feajsle (at 20 months of 
age) aral in ©ns DM/2© mala (ta^atsd Kith stilbestrol) at 12 
months of ag®. Ho smMmrj tumors have been obse2v®d in SHy of 
tJae BBk/Ze im^l@st kept as virgins (approsdjstely one°fo5n^- 
are still H-slag but ®i^ over 19 moiiths of ag©) os" in arsy of th© 
fores-bred teimlea (msiBt of the anisBls of this groap ar® still 
ali.ve and oa3y essQ°fo^«^ ©r® more thira a ysar of age)? These 
restalts sesj be con'fe'astad iHiti-i these obtained previously fca? 
strain agent °fsp@® C3HeBo mm^&rj tsam's ©s®uyr«i in ij^ ©f virgin 
fenaleSj in SUoBjg of breeding fsmleSj in Iho^ of forcs^brsd 
feKSles, and in 22. ij^ of males treatsd with stilbastrolo 

meat -d^ff'@i^^'''^^^Sflon" cisic^'s^^^iks "d!e^l©piaieHfe of 
aaiaoaaiy and of ether -^©s of tisnors in a stx'aia of adca deprived 
of i^e sissjaasy t«3i®r agent l^ the pr^edur© of tx^ossf eiring o^a 
f2?osa an inbred sts^in of Mice cai'sying the agent to another stgeia 
•■^.It'vTist th® ag®atc The results ^ill refleot esi the inteypretstlc® 
of the TislatiVQ effscts ©f the gQJsstic eoastitttti<mg the msM^sj 
ts^es? agentj as^ ■&© hormoiml influene® ask the defvelopaent of 
laarsiasy t?ffl^'S» 

^^^g^^ed^^^^^of^tho^^^gjeotg Flirfcher data on the 
cte velo'^Si^QF'l^^fs"'isr^e "fi^e^red females «ill be 
eolleetsdo WJ^n autopsies have been perfonaed on the rauaining 
virgin fsmales and on the force "bred feitsles, the infonoatien 
eb-Jiainsd 1^.11 be etsabined ^th that ft^n the feimles of the 
breeding coloisy and th® umtex^l wiH be prepared for publioatdoao 
Th® straisj ©f laiee 18111 be ccsjtinued and th© develepaent of 
m^n^Mrj tumors ^d.!! be studied in subs^uent generations o 



586 



JIGI=J421 



2o Prodiietion of tumors of ths skin 



Ob^ectd.'rest f© atteimpt t© p^edise© twiors of the skin 
±n stJiSto^M"'^^ t^ palnfcing «!& isr©-^^^sio Sii^e it has b«9a 
statsd that isrstfeaa is "a^a«a®rciss>g®sie for ^cia epithelicni 
$M the jaosse" it s©is®®d ^orti'^hils t© treat sjise ef strain HS^ 
Kliich iSir® higlily si^c©|?feibls to ^e d®T@l©^®Rt of t^saors ffiC the 
ski^j i^ith this ctEspeando 

glyeolTF^^^^"^"'^!® a^cin of halr^ ®k3 hairlsss animals 
of bc^ B8>:as of th© HS straino Other groi^ of imlee and fei^Ies 
are psinted td.th etligrlene glycol alone o T^ee weekly paintiags 
are started when the aaiiaals are 2^ to 3| months of age and 
sre continued thsijughotst the lifespan of the aniiaals^ 

^J^^^^^^gg Data obtained indicate that uretban is 
prob®b'^nSC"l^^^g@nic for -ys® ^cin of strain HR Hdese Hoh» 
everg ttsaors of the skin have been observed in 8 of 18 hairless 
f eri^lea a^d in 3 of 8 hairless males that M^e been painted with 
urethan in e'^jlene glycol. Tiso of h hairless males , painted 
M.th ©tiiyleae glycol aloa®, detal^jed ttmora of the skinc None 
of ths tim^srB arose in th© painted ar®£o 

Fainting the akin ^th iuretl^n does induce pulsions^ tusaors 
aisd hesiorrl^gic leBlmm of the liTer in haired and heirless anis&ls 
of strain W^ Seais of th® lesiosas in ths liver can be classified 

©s heiBsngloendothslici^a ^hile others appear to be bleod^filled 
sp&ceSo 

^Si^^jfj^se^^ to^ %he^ ;^o^ffm of _g|QZg^itat® g This stady 
ifill alSaS'OTm^'m''cmc^Si^'°^^^'°^^!^QncQ"''o?°genetic consti^ 
tuition ©n the developaenfc of tsaaorso The effect of a carcinogen 
^n the dsvelopaent of & particular l^e of tuiaor^ to which the 
skin ©f aniiaals is gtsseeptibles sill be dstersdnedo 

^^i^,,„^^^,,,^^^^„^B^S^^ Groups of etrsin HR aales 
and feiaales (both haired snd hairlaes) sdll be painted with 
isretban in ©tl^rlene glycol t^ce s weekg and with etl^lene 
glycol alone. The animals id.ll be observed centiatsoasly for 
the deveiopsient of tumors of the skin ©M of other lesioas., 

3o Influence of the lethal'^'jellow (A^&) gene on tha 
dsvelopssnt of leukemia in adeao 

^^^^^ss To deteTOsin® the relatiosaship of ths 
lethsl^^^^^gene (knoim to insress© body sise) &M th® 
oeetirroncs ©f lejikW-s in treated and un1a*©ated ©niiBEls {in. 
eooperaHos. ^th DTo ^ston) ^ 

• 587 



NCI=J|21 



l^lsth^s^^^^^geds Cr©ss@s have been trnde between s-^rain 
DM/25 a'straia siise©ptibl® to th® de-^lojsaent of leukeEda, and 
s'traiB YBR5 a straisi easTsring tha lettel-yellofw gena., Th© Fi 
laie© havs beea j^tebed 3 times a week fee 7 ■^eelM (7 diffeyaat 
palntiBg sitss feav© been used) ^th Oo2^ 3=saQtigrlcholanfehr®ne in 
ethero A sse©M groap ©f F» snissls has be@a mslataiaed ^thout 
treatmsnte Tim t^o groups sr© ©bserred at reg«lay inteir^als 
for th© ds^eloi^iSiat of lej^esdao 

Ma.j^;Hndigg8g The large aaajoH-ty of the treated and of 
the ■i^T^^^°'^^mla of tMs esparisoeat ®r® still alive » No 
reticular si®opla^as of angr type have been obsessed in any of 
the anisjals that fea^e been autopsiedo 

MsEErasy tusers hs'SB de'^eloped in 13 of the untreated 
females and in 1 of th© treated feraaleSo All of these fensles 
were th® yellow segregantSo 

Si^-g^egngetojtojgo^gmoft^^ This 9xperi= 

ment ^ur'l^^^^^^"^M^^'*'^'°°^eT©ISS^IIosf gen® in the 
dsvslop^snt of leukeiBia in ssiceo 



^^posed cg^ge^jifthe^groj^t The g^oup of untreated 
S°^S°'^e°^^^^^SS^''M^r'3=Bietl:^lcholanthrQne will be 



anissa: 

observed at regular intef-^^ls for •Uie de-rolopment of ttjaisrs 

aad of ©thes" lesions o 

1^0 Hecs'otising arteritis in stsaia BL/Ds sdcea 

^Jeet^^f Dsslng roafeine autopsies of breeding fessale 
Edes orsirain"BL/D8 j the ©varies ses^ noted to be enlarged 
and pal® o This suggested cresrian twKors and therafoiPe histolog^.c- 
sections of these ovaries iser® asd© and exsainedo The ssiero*' 
seople ©ssKis^ticsQ explaiasd the gross appearance fog* it vas 
foaM ■yjat th® corpora lutsa ^er© h@a-?l!ly infilts?at©d rdMi a^loid 
and enlarged e This fiisding fc^s of payticislsr iatas^stj beoasase 
this same straia ©f Bsise ^as s'aps^d h^ Sokoloff aad Habessssaim 
to Mrs an idiojathle necrosis of boaso At the ?© aest of DTo 
Lsoa Sc&oloff of HIAMD & stx^ of the availsbl® histologle siatei-lsl 
f?03a i:'o^d.n© autopsies of a gst^op of breeding females v&s s&d® 
and the ^tssdal ua® presented, !y isrritetionj at th® Cosfersac© 
on the G^f^safative Pathol©^ of As^thritis and Bhsiasati^i held 
at IIH effli Fsbymsy 5 «^nd 6g l^$9o 

^^^a ^^^^^ i A st&Sj isas mad® of the histolegiQ 
s©cti''S"''^'°^S©?^Hes fr<sa a grossp @f bs'eediag females of 
sts^ir* BLo In additiosj aestiosss of kidneys ajjd of t^Eaorgy- 
ishea presents '^^^e, also a'^ailable fos* stva^o 

588 



=■ 5 " rrU-L'^-!'^.'- 



^J^^^Jto^Rgss Ok gross exandsKitl^i th© orariss of stsmir. BL 
wsre ^'^n^'^II^iS to bs enlarged and pal®, on rnierc^cc^e 
(sxamination ths sections ic«^sal®d infiltssatlcaa of an^l'^id into the 
e«^^papa lufeeac Tha extent; of iafilta^ti^a v^^®d £t(M smll foai 
to 3jirge Fomided areas enti£>33y arspXaoing %im corpora lut^« So^^b^ 
tiiiss i^@S9 tsas c&leS^eatloii in ths eents^l ptsiaon €^ the angrloid 
jaaesc, Asgrl«id ^ib observed In one or both ss^aries ei:f 59 s^ the 73 
bfssdiijg femles of stsaia BL? In additioa to sjsQrloidj striking 
■oscular lesioas ^er@ observed ia (m& os* li^sth 0?a^i©s of 32 brsediag 
f einalssa The vascular lesion 1ms beesi described as a thick@ising 
of the lislls of the vesssls dae to the depssiti^s of ©osinopfeilie 
laatesdal ^thin the latissa ai^ the aodiao ^snosie aad frag^ejstatimi 
of th© nuelei oeeuered in ei!«h areas* The prssenca of arteritis 
was stot detected oat gross esmdn&t^mi eith@7 in -yis ovaries or in 
other organs a Thss^efore, no data on ths distribution of ths lesion 
are available sine© caiipletQ laioroscopic smr^B were act dcmep 
HOW0T9P, the vascular lesica 'sjas noted in jsics-oseopie ssetions 
fr<»i the parirenal adipose tisaua, isi vessels of the ads^sml corteXjj 
in vessels ©f the utonaa, asoJ in vessels of the messntex^c, In 
addition to the vascular lesion, Fsnal disease was obssrved in 65 of 
73 breeding females o Mi8roseopi@ally,the reml disease was the 
t^e that has been described 1:^ Donn and that cseui'S pssiiicular^ 
in strain A mi©©. ManaEasy t«mars ocemred in 12 of ths 73 breeding 
fenales between ths ages of 11 and 22 months and pulmoi^sy tugi©i-*s 
in 16 of 73 feraales bet-^^en 15 asd 23 months. 

provicfed" mope" 'SlSaSme^e®ra^^'i;S'°^SHe&isti€S ©f a highlj 
iabred strain cf ffiiee., This '^ll iaesrsss® their usefateess in ^s 
stu^ of vasdoss diseases « 

KT>pos^6d^^_oowgse ef^^^^ Tl^ Eatarial has besa ^apar@d 

for p'uSji'ea^'Jaa "ia 'iiie°^^^S^s^f -yie €@if®rsmj® oa the 
Coagparative Pathology of Arthritis aM lh©^^.tiOT which ^11 bs 
poblished in ^e EawmbBr issue of Lsiborat^^ Invsstigatlonp 

5o AH^loid la the orgwss of s-fesmia BL mieeo 

jg<]f>^ectivesg The observation that -^s eospsra l«t©a of t&e 
oveTlea oi^'" s^femin BL bs>®©dis3g fej^lss i^sra ©ft©a h©am3y iu- 
filtrated with ajuflQid ^^s^tsd th® pr©s®afe study ©f tha distribiMoa 
of auyloid in th® organs ©f strsin BL esI©©,, 

t fetho ds E tajgilo^ed t Groops of isale axid female aaie© of strajja BL 

' '"^ve 'iiSsn Is®^ aal&"'and sill be a?2top®ied at 15 months ©f ag©o Ssetisns 

of all organs sill b® mad® assd dala on the distributLois of ss^loid 
^11 be secured froa thasio ■ 



589 



- 6 - HCI-421 



l£4SLli^li£* Ifo aijiaals ba^s bean aMt©psi®d and tbss'sf or® 

Sig^jilcaace to thajBTOgj^__ofjfeh£Bist^^ TMs stsdy ^11 

p!rQ^dl°''SM''^^3^^^ISr^^^^^°°^^°°^S^^^^sties of os® of 
'fch® highly inbpsd stmiiss aaisatiaiissd at the Iiisiitat®. 

P^^^ed^a£S8^tts^^Je£feji Data oa tfes distribation ©f 

a2srlt€!a°'tSir'BI"°elc^H"^a°°^^m^rial prepared for publicatioSo 

6, iHBiiunegeaetie experimants, 

Objectiy eB s To prodaee ^©ci^l types of aadEals f sff 

soliaKoraiSve ' experinisxjfes with rs-o M* Ke Barrett (s®e D^c Bsrrattfs 
y®port for details) « 



Part B iasluded les X Ho 



590 



101-421 



lHdiTid»ml Project Ifepert 
CalsJ^ar Tear 1959 



Furfc Bt Hmi<^8, Awards, and PublicatioES 



Peblieatiens other ih&n abstracts frcasa this projects 

Casdisgerj, H<. XoS ^fe^!sasy tuaops asd im'p&tams in 
agent ■^fre© strain G3BsB ntice. Acta IMiOo inteysjato 
eont?^ cancro I5s I50=l5lj 1959* 

Dsrimger, M, Koj Occurrane© of tiasersj parfcleularly 
iBBiisaasj tiwiorsg In agent^free staraist G3WB mIcso 
Jo Mato Cancer last, 22; PS^S^IOOS^ 1959 o 

mriiigeVg Mo KoS Maerotiajjig arteritis in ©tr&in 
HL/©3 saice. Labs»tozy IwrsstigstAon (Ns^eaber 1959) o 

Hestoa, Wo Eo, ¥lafeakis, Goj sM Deslagers Mo KoS 
High incddene© of spcataneoxss hepstes®© and the iaersas® 
of this ineidence isildi sarettesa in C3Hp CSHTj susd G3Hs 
mle sdcQo Jo Nato Cancer last, 21*! (F@b, 1960)o 

Essteug ¥o So, Vlahakis^ Go, ssid Deilnger, Mo Kog 
Delayed effect of g©ri©ti© segi^gstl^ on the ts^asslssioa 
of the EJSBBs^yy t?saor agasit in »de®o Jo !?ato Gsae©r lasto 
2i*g. (Maa?o 1960)o 



591 



PHS-WIH 
Individiial Project Report 
Calendar Year 1959 



Serial No. HCI 427 

1. (Laboratory of Biology) 

2. (Leukemia -Studies Section) 

3. (Bethesde 14, Md,) 



Part A, 



Studies on the etiology and -therapy of experimental 
lymphoma s 

L» W, Law 

Man years (calendar year 1959) 
Total: 4 2/3 

Professional; L. W, Law, 2/3 
Other s 4 

A , Role of thyroic tissue and bone nia rrow_^in _ Xz.^T" 
induced and spontaneous leukoses 

lo The experiments are now nearly completed 
relating to -the influence of thymic grafts CC57BL) 
introduced into millipore chambers, intraperitoneally, 
in thymectomized, fractionally irradiated C57BL and 
B/iFi hybrids- Despite the persistence of tissue for 
long periods of time within the chairfcer, no evidence 
of transformation to neoplasia has been seen.o 
Approximately 20-30% reticular neoplasms, type A and, 
B of Dunn have been observed but -these are unrelated 
to thymic tissue. Interestingly, no plasma cell 
neoplasms have been observed in mice of these geno~ 
types although B/vLB/c and C3H mice appear tc be 
sensitive to the plastic ma-terial„ 

2, Bone'-marrow chimeras, developed in the 
C3Hf/jj^ and CBHf/^^ strains of mice by introduction, 
of I^KR bone marrow into <12-hour old mice have 
developed lymphocytic neoplasms at a frsq-uency and 



Part B incliided Yes 



i92 



Serial No. NCI 427 
1» (Laboratory of Biology) 

2, (Leidcemia-Studies Section) 

3. (Bethesda 14, Md.) 



Part A 



latent period of non-chlmeric controls, nor has the 
introduction of thymic AKR grafts into such chimeras 
resulted in an influence of the leukemic picture = 
Approximately 10% of the Bittner subline recipients 
have presented unilateral parotid tiamors when re- 
ceiving bone marrow from AKR donors ]cnown to be HI 
positive o 

3, Isologous bone marrow given immediately 
following the last of 4-weekly irradiations (acute 
X-ray) completely prevented the appearance of 
thymomas in thimiic grafts introduced at 3, 7, 14 and 
21 days after irradiation on C57BL and BAP^ miceo 
Thus, the inhibitory effects of isologous bone marrow 
is a permanent long-lasting influence =. As discussed 
above the presence or absence of thymic tissue, and 
as shown here, the introduction of isologous bone 
marrow are factors which do not influence other 
neoplasms of reticular tissue. Such neoplasms^ 
chiefly involving monocytes, appear after 12-=15 months 
of age in these mice. 

B o X^^ ra^j ^/tion and lymphomas of mice 

1. A study is in progress of the effect of 
total body X-radiation on the induction of lymphomas 
in C57BL/Ka and RFM mice, irradiated in utero at 
10,5 days and at later periods just prior to parturitio? 
at 17 to 19 days. The 10=5 day schedule was selected 
as a result of the findings of Russell and Major of 
the induction of somatic cell gene mutations in. coat 



593 



Serial No. NCI 427 

1. (Laboratory of Biology) 

2. (Leiikeraia-Studies Section) 

3. (Bethesda 14, Md.) 



Part A. 



characters in mice irradiated at a presumably very 
sensitive age, 10.5 days. To date many of the groups 
have passed sis?; mon-ths of age and no evidence has 
been observed of an increased frequency, nor of 
earlier appearance. A negative o'atcome in these 
studies could conceivably rule out any striking 
sensitivity of fetal tissues but could not, because 
of nuinbers of animals alone, rule out a probable 
influence comparable to what may occur in human 
beings to the possible response of the fetus to radia- 
tion received during roentgenographic examination of 
tlie mother. 

2 , Work is continuing on the comparative 
sensitivities to fractionated whole~body irradiation 
of 3 sublines of C3H mice, C3Hf/x,w/ CBHf/gi and 
C3H/Fg, tlie latter a high-leukemic line studied in 
this laboratory over the past several years. Several 
interesting obser^^ations are worth considering; 

a. C3Hf/Lw mice are relatively resistam;:,. v.-o 
date only 20% lymphocytic neoplasms have been recorded < 

bo Samples of C3Hf/Bi slice differ in their 
responses. Our stibline CBHf/g^jVj^^, maintained for 
several years here develop 50»60% tiiymic lymphomas, 
A sample of these obtained directly from Bittnar 
were not more sensitive, however,, than the C3Hf/j^,^.. 
subline, 

c. An early appearance of tliymic lymphomas is 
seen in C3Hf/pg mice. This type does not ordinarily 
appear in this subline and thymectomy does not 
influence the spontaneous occurrence of the reticular 
neoplasms. In •i±iis connection a collaborative study 



59^ 



Serial Ho. NCI 427 

1, (Laboratory of Biology) 

2, (Leukemia-St-udies Section) 

3, (Bethesda 1,.., Md.) 



Part A. 



has been undertaken with Dr, John Maloney in an 
attempt to recover a cell~free material from CSHf/^g 
spontaneous neoplasms using the Maloney techniques. 
Heretofore, extracts and filtrates and tissue 
culture materials have been ineffective in <12 hour 
old test mice of various strains. 

3 . A study in collaboration with Drs . Rowe 
and Hartley, NIAID, has been concluded concerning 
the relation of infection of parotid tumor agent 
(polyoma) to lymphocytic neoplasms in J^KR, CSS arid 
C3Hf/gj^ (X-ray induced) aiice and the observations 
submitted for publication. There was found to be 

no correlation of HI antibody with leukemie appearing 
at various times throughout life, nor was there 
obtained any evidence to indicate a greater risl?; of 
developing leukemia in mi-ce infected with polyoma virus < 

4, As soon as facilities are available to ais 
section, it is planned to initiate a study using 
chronic gamma irradiation; of the influence of dose 
rate on the induction of reticular neoplasms in 
C57BL/Ka an<3 DBA/2 mice. Dosage intensities from 
approximately 5. r to 80 r/hour will be used in %i7hiGh 
the same total dose is given over the same period of 
time. This will be accojuplished in one strain for 
intact and for thyme ctoml zed mice in order to differ'^" 
entiate the influence on thymic and non=thymic neoplasms 



595 



■■rial Ko. NCI 427 



Pari 



1» (Laboratory of Biology) 
2o (Leukemia=-Studies Section 
3, (Bethesda 14, Md.) 



C. Cell^rge _and. tissue culture agents in the 
induction of lymphomas and other neoplasms 

Various phases of this work are done on a 
collaborative basis with Drs, Rowe and Hartley, NIJL'D 
Dr, Dawe, L, Path», and Drs, Kahler and Lloyd, L, 
Physiol,, NCI, 

1, Preliminary results have been reported 
of the relationship of antibody status (HI) of the 
mother of several strains and sensitivity of the 
litters to -Izhe carcinogenic stiumlus of a P~388 
passaged highly virulent parotid timior virus, -All 
mothers showing HI antibody titers >200 delivered 
young which resisted (ttm-sor induction) virus. 

Spontaneous convertors' , tumorous mothers or those 
infected by suc3?ling their inoculated litters all 
behaved similarly, Asa additional resistance factor, 
i-iot related to antibody, needs further study, Th.:ls 
is reflected in absence of tumors in nearly 30% of 
mice, randomly spread throughout litters, A:aother 
interesting observation concerns the appearance of 
a significantly greater number of s: tumors, 
over controls in (C3H x AKRlj'^ mice 5 8 months 
of age; these mice completely resisted the early 
carcinogenic effects of virus. The possible re^ 
latlonship of this phenomenon to antibody titers 
needs inves tigation , 

Foste^ :■■£ litters bor- :i posit 
mothers or ^ -; mothers and ^.,...„i,_ _„t£.§.§. ^^'^ 

HI antr: :;-o be transferred to litters both 

transpl- .y and through the mother's milk. 

Transfer: her route appears equally efficacl 



596 



Serial No. NCI 427 
lo (Laboratory of Biology) 
2o (Leukemia^Studies Section) 
3„ (Bethesda 14, Md.) 



Part A, 



2, I\ study of the comparative effect on CPE, 
hemagglutination titers (HA) and infectivity (tTimor 
induction in mice) in relation to number of virus 
particles is underway o Several interesting 
(apparent variants) are being investigated? a) 

a thymotropic substrain, b) a strain giving rapid 
CPE but with an unusual induction of t\amor tyjpes 
in mice, c) a strain with high HA titer, giving 
rapid CPE which induces neoplasms in hamsters but 
apparently not in mice, and d) a hamster 
kidney^passaged strain which is non'-carcinogenlc 
for mice. Titrations down to 1 x 10"°*^ (particle 
t;;&vtnt <30) are being studied, 

3, Other projects being continued ares 

Sensitivity of Hl-negative adult CSHf/j^-j 

and (C3H2cAKR)F3_ mice to parotid tumor agent 
the role of thymectojj^ in response to thym.o ■::•:':> 
pic virus. 

Transformation in in vitro organ cultures 
of salivary glands to parotid tumor agent = .?:■ 
preliminary report is in press , This v/ork 
to be continued by Dr, C3.yde Dawe^ Lo Path. 

Study of the host cell~virus relationship 
in a 'carrier line'' of P-388 in which complete 
CPE does not occur. 



597 



Serial Mo. NCI 427 
Ic, (Laboratory of Biology) 
2o CLeuiceniia~Studies Section) 
3o (Bethesda 14, Md.) 



Part A. 



D . Studies of mechanisms involved in inhibition 
of lexikemic cell growth 

]\ collaborative study has been initiated. wiUi 
Dr, Wallace Brocknian, Soiathern Research Institute 
investigating the biochemical mechanisms of re^- 
sistanceto 5-°fluoropyrixrd.dines. The sublines avail= 
able for study developed in this laboratory are: 
PS15/pu, P815/puj^, and L1210/po» '^-S biological 
characterization and standardization of these 
lines have been completed o Rt present the bio- 
chemical work involv©?j8 1) incorporation studies 
in sensitive and resistant sublines of orotic vs» 
fluorootic and uracil vsa fluorouracil 2] capacity 
of S and R sublines to metabolise ribonucleotide 
and derivatives o It is planned to study the enzyra.e 
changes, particularly kinase and phosphorylase, in 
all variant lines since Reichart et al,. (Nature 183; 
939 J 1959) , have shown differences for both enzymes 
but involving different neoplasms^ during the 
stepwise development of resistance. Uracil instead 
of 5~fluorouracil, however „ was used, 

A study has been initiated testing for 
probable collateral sensitivities to Amethopterin., 
6-mercaptopurine, cytoiran and DON of several variant 
lines of the mast=cell neoplasm P815o This has 
he'sn prompted by the observation that certain L121G 
variant lines, particularly L1210/th and L1210/6mp 
show a strikingly increased sensitivity to the 
5=fluorinated pyrimidines « 



598 



Serial No, HCI 427 

1, (Laboratory of Biology) 

2, (Leukemia-Studies Section) 

3, (Bethesda 14, Md,) 



Part A, 



So studies in mangaalian somat ic genetics and 
carcinogenesis 

Within the next year it is proposed to establish 
a new experimental method to study certain basic 
problesBS concerning carcinogenesis and inaiamalian 
somatic cell genetics in collaboration with Drs= Rose 
and Bradley, University of Melbourne^ Guest Investi'^ 
gators at NIH, The method a contin^tous injection 
technique" provides that tissues either in situ 
or grafted can be continuously, directly and locally 
influenced by the infusion of materials. 

Attempts at genetic transformation of cells 
wis. concerns 

1) Alteration of histocompatibility 
characters of t-umor tissue using coisogenic 
strains of miceo 

2) Alteration of drug sensitivity to drug 
resistance using established variant lines , 

One experimental situation pertaining to 
mechanisms of carcinogenesis that will be studied 
concerns the possibility of influencing thyniic 
lymphoma induction in C57BL mice^ Here thymic 
grafts will be made into the renal capsule of 
fractionally irradiated mice and attempts made by 
the use of different substrates (NA) to influence 
Ixfrcphoma production and regeneration of thymic grafts 

3) Attempts to transform normal cells (salivary) 
glands (for example), to neoplasms through con" 
tinuoutf infusions of DNA-extracted malignancies. 



599 



Serial No. NCI 427 



1, CLal>oratoa:y of Biology) 
2„ (Leulcemia-'Studies Section) 
3. (Bethesda 14, Md.) 



Part A, 



Dr, Roosa, recipient of cin NIH Postdoctoral 
fellowship, is continuing his tissue culture, studies 

1» Nutritio nal re quirements of P"388 in cell 
culture 

As noted in last year's report an established 
cell culture was found to grow optimally in 
Eagle ' s Basal Media supplemented with only 5% whole 
calf sertim. However, when the serxim was dialyzed 
growth did not continue.. For sustained growth 
in mass culture using dialyzed calf serum it was 
found that the addition of pyruvate or L-serlne 
restored the growth promoting activities of the 
v/hole sesrum. Subsequent work showed that for 
isolated (single) cells both pyruvate and L~serine 
(or glycine) were necessary for growth o 

By the use of radioactive tracer e;eperiments 
V7e attempted to determine the metabolic relation^ . 
ships be-tween these compounds and P~388 lymphc- 
blasts o The cells were allowed to grow in the 
dialyzed serum medium in the presence of either 
radioactive glucose, pyruvate or serine and after 
a 10-fold increase in protein had occurred the 
specific activities of the protein^serine^ glycine 
and alanine and the pyruvate in the medixim were 
determined o These experiments showed that all 
of these compounds were synthesized from git 
It was also established that L^serine^ glycii..e 
and alanine in the cellular protein do not arise 
from exogenous pyruvate, and that the effective 
supplements therefore act in some way other than 
as simple precursors for these amino acids. 



600 



Serial Bo, HCI 427 
1. (Laboratory of Bi&logy) 
2o (Leukemia-Studies Section) 
3, (Bethesda 14, Md.) 



Part A. 



We also were able to establish that pyruvate 
can be replaced by ot^keto^-n-butyrate both for grcvrth 
in mass culture and from isolated cells « •'? 

late and <^ -keto-n-caproate serve as growth 

in mass culture only.> A series of other keto 
acids, fatty acids, Kreb's Cycle intermediates, 
purines, pyriiaidines, vitamins and amino acids 
-.s/ere found to be ineffective, 

2 . pevelppment of . -Methods, of analyzing the 

genetic char" -•"•"•' ^ tics o_f.. these cell--- i-;-;^^ 
in vitro and o techniques , 

With the development of an adequately defined 
medium it became possible to grow populations of 
P=38S cells both in mass culture and from single 
eells, studies with large populations %\fere 
usually observed by the use of protein dete:ard3:ra« 
tions and these studies were often supplemented 
with nuclei counts » By allov/ing a few cells to 
develop in a petri dish maintained in an inc-nn? ' : - 
having a constant CO2 atm,osphere xire are ablr 
isolate clonal stocks. We are then able to sruc.v 
tliese clones quite readily as they develop into 
colonies under the pressures of various grovbh 
iiihib iters . 

The intr5.paritoneal injection of these 0--; 

into histogenitically compatible mice leads to 
an ascitic t'OKior, One can then test the ge- 
cf th?^- resultant tumor ei-ch by in vivo trea 

ting small n'oiabers of cells in tissue 
..-V.......C...,,..;. c.'-T ■"^'•^■'■".ting the resultant colonies in 

Uae press various concentrations of inliil 



60; 



.11 



Serial No. HCI 427 

1, (L3l>oratory of Biology) 

2, (Leukemia-Studies Section) 

3, (Bethesda 14, Md.) 

P art A ,_ 

3o Selection of drug resistant variants 

Variant lines of P-388 cells were obtained by 
selection of resistant sublines growiri'.; in the 
presence of gradually increasing concentrations of 
various growth inhibitors. Several distinct 
lines have been obtained and are carried continu- 
ously in different concentrations of these drugs. 
To date distinct resistant steps to 2\methopterin 
(representing a final lOO-foid increase over the 
minimxim inhibitory level) have been selected,. 
The isolation of these variant lines has followed 
a stepwise pattern and each step has paralleled a 
similar step in cross "-resistance to Aminopterin , 
Other resistant lines include two steps to four 
other inhibitors; 5'=-Fluorouracil, 5 -Fluor our id ine, 
S'-Fluorodeosjyuridine, and 8-Azaquanine , In addi- 
tion to these lines several variants containing 
double markers have been isolated. Due to the 
recent isolation of these lines only one has been 
testedo Under cloning conditions this line retains 
a well marked resistance to both S^Fluorouridine 
and .f!>methopterin » The other double marked lines 
are resistance to S^Fluorouracil and i^methopter inl- 
and 8=Azaguanine-5~Pluorouridine resistance, and 
an 8=Azaguanine=-Amethopterin resistance » 

The resistance obtained in each case is 
heritable as it is retained during growth in the 
absence of any inhibitor for periods of greater 
than 100 generations » 



602 



Serial Mo._„_lCI... 427 

1. (Laboratory of Biology) 

2. (Letikemia-Studies Section) 

3. (Bethesda 14, Md.) 



Part ^. . 



It is possible to obtain ascitic tumors after 
tlie intraperitoneal injections of from 1-3 million 
cultured cells in histogenitically compatible 
mice. We are interested in deterraining if the re- 
sistance obtained in culture . is retained after 
animal passage » Results here are somewhat slow due 
to a lengthy latent period, but initial results 
show a retention of resistance in raice although 
it is still of a low level. 

E2(:periments are in progress at the present time 
to obtain resistant populations of these cells by 
,4E vivo selection. These lines v,;hen obtained shall 
also be tested in cloning type experiments to 
determine the level of resistance and the percentage 
of isolated cells capable of in vitro growth in the 
presence of various concentrations of inhibitors ., 

4 , Preservation of ^ variant cell lines 

It has become £ e to preserve the many 
variant cell lines being established, Tb.us they 
would be available if a culture line became 
accidentally contaiainated or if further ^vorls: with 
a particular resistant step becaiae warranted at 
some later dateo 

We chose to explore the possibilities of 
freezing and have established a relatively simple 
technique using apparatus already available = To 
date all but one of the resistant steps have heen 
successfully preserved. The longej;,t test period 
has been a period of six months » 



60 



Q 



>erial Mo_„_ HCI 427 

1, (Laboratory of Biology) 

2. (LeiLlcemia^'Studies Section) 
3o (Bethesda 14, Md.) 



f'art T\. 



Plasraa cells in cell culture 



Recent studies witlrt Dr. Michael Potter of 
this labor at03ry, include attempts at growing- 
plasma ceil tumors in cell culture. These 
studies are aimed at developing a relatively 
sample systeKi for establishing clones of these 
cells so that the protein patterns of tumors 
arising from single cells may be studied (clones 
would be used to establish tumor grov7th in laice] . 
It is anticipated that through these methods it 
will be possible to determine if the Bence Jones 
protein and the f>'erxam protein are produced by the 
same or different cells, 

^ » „. Agent which T>'£odvr''--^ ^. rr^j-trro^ ■>>■■ y c ■^'. ■•'^_fecj^_in 
g."g.8§.,„,ael-lg- 

Studies are in progress using a parotid turoc: 
agent (FTA) obtained f3-:om a normal tissue sourc^ 
iM<R bone marrow). Ti^is agent apparently repli--: 
in P="388 lyznphoblasts in a media containing onX; 
calf sermtto To the present time fourteen succe- 
passage generations of this agent have been goev,, 
Ifhen the 'wash fluids at the time of culture IysIb 
are Injected into newborn mice a variety of tuirios: 
(predominately of the parotid gland) appear aftar 
six v^eelcs. 

Animal strains which have already .:^..-.^.. - v--... 
this agent include G3Hf/Bi and CC3H 2; AKRlF^'s. 
There does not appear to be any xi^ealcening of the 
oncogenic properties of this agent after contin".:,- 
ous tissue culture passage^ nor is there a sign.!.;' 
cant change in the type of tiaaor arising after 
continuous passage in cell cultures <, 



60i 



Serial No. NC I 427 

1, (Laboratory of Biology) 

2, (Leukeraia-'Studies Section) 

3, (Betliesda 14, Md.) 



Part h. 



Experiments are already in progress to 
determine if the activity of -the sample is 
altered by alternating passages in cell culture 
and animals. The reisolation of active material 
has l>B&n successfxally accomplished from a paro» 
tid tmtior in the first attempt and unsuccessful 
V7hen attempted from a mammary tumor. 

Collaborative esrperiments already in progress 
with Doctors Law , Dawe, Rowe, and Kahler are 
concerned with studies of several variants of 
this agent. Included are examinations for cyto- 
pathogenicity of tissue culture lines, mouse 
sensitivity and hemagglutination, 

7o Organ culture studies 

Studies are currently being planned with 
Dr. Law to study -the effects of the Maloney 
Agent (and possibly otltier viral'-like ager 
thymic and other tissues in in Z4i^. orga ., 
cultures . 



605 



Serial NOo NCI 427 



lo (l^oratory of Biology) 
2o (Leukemia^Studies Section) 
3o (Bethesda 14 „ %do) 

Part Bo 

HoKorSo Awards^ and Publications 

DawBo Co Job Law„ Lo Woo and DuhHo To BoS Studies of 
parotid tumor agent in cultures of leukemic tissues of 

raicso JPCIo 232 71 7^797 „ i959o 

t^Wo Lo Wo, Some aspects of the etiology of leukemia o 
Proco of the Third Canadian Cancer Conferenceo ppo I45--l69o 
Academic Press o NoYo l959o 

Roweo Wo Poo hartley Jo Woo Law„ Lo Woo and Huebnero RoJoo 
Studies of mouse polyoma virus injection IIIo Distribution of 

antibodies in labo mouse eolonieso Jo Expo Medo 109? 449o462o 
1959o 

Bowe„ Wo Poo Hartley, Jo Wo, Brodskyo loo Huebnero Ro Jo,, 
and L^Wo Lo Wo Observations on the spread of mouse polyotna 
virus infectioHo NaturCo ie2o 1617„ 1959a 

Uphoff„ Do £oo and Lawo Lo Wo An evaluation of some 
genetic factors influencing irradiation protection by bone 
marrowo JNCI„ 22; 229-241, l959o 

DawSo Co Joo and LaWo Lo Woo Morphologic changes in 
salivary -gland tissue of the newborn mouse exposed to 

parotid-tumor agent in vitro o JNCio 23; 1152-1178„ 1959c 

LaWo Lo Woo DawBo Co Joo Ro!sje„ Wo- Po, and Hartley^ Jo Wo 
Antibody status of mice and response of their litters to 
parotid tumor agent (polyoma virus) Naturco 184 i po 14200 i959o 

Chosen a delegate (USA) to the WHO Expert Advisory Committee 
on Cancer o 



606 



?HS -NIH 
Individual Project Report 
Calendar Year 1959 



Serial Ho. NCI 427 (a) 

1. (Laboratory of Biology) 

2. (Leukemia-Studies Section) 

3. (Bethesda 14, Md„) 



Part h. 



Studies of the etiology, pathogenesis and che?nO"» 
therapy of reticular neoplasms in mice 

Michael Potter 

Man years (calendar year 1959) 
Totals 4 

Professionals Potter, 1 
Other s 3 

A . S tudies on drug resistance 

Two questions on drug resistance have been 
asked s 

1) When do resistant cells develop? 

2) Miat component of the cell changes when 
a sensitive cell toecomes resistant? A gene? 
Employing one model system the DON-sensitive ascitic 
mast cell neoplasm P815, evidence was obtained that ' 
resistant cells could develop in the presence of the 
antagonist. However, the demonstration of persisting 
sensitive P815 cells in the face of long courses of 
DON was a disappointing development, A drug sensi~ 
tive tumor which would be destroyed by a drug is 
essential to the purpose of demonstrating xdien and 
how resistance develops. 

Three model systems are being explored for their 
value in contributing more inf oarmation o 



Part E included Yes 



607 



Serial Imp., issci 427 (^) 

1. ( Laboratory of Biolog:^'-) 

2. (Leiikemia-Studies Section) 

3. (Bethesda 14, Md.) 



Part A. 



1 a. An jMaetho pterin sensitive , ascitic, tetraploid, 
lymphocytic neoplasm; P288_ 

Like other DBA/2 lymphocytic neoplasms, greatly 
prolonged survival times are obtained with Amethop- 
terin treatment. Full resistance to amethopterin 
does not develop in a single transfer generation? 
yet JNraethopterin fails to cure mice inoculated with 
doses of 10^ or more P288 cells. A technique has 
been developed to er.plore the question. The inocula-- 
tion of 10 ceils will initiate a successful transplant 
in about 80% of mice„ Five treatments of Amethopterin 
3 mgm/kg given on days 0,2,5,7,9, to mice inoculated 
with 10 sensitive cells results in the development 
of a transplant in less than 5%; with 10 cells of a 
full resistant line about 80%. Cells were isolated 
from mice inoculated with sensitive cells after 
Amethopterin treatment of 25-30 days duration. 
When doses of 10 of tiiese cells were inoculated into 
mice, the above-m.entioned Ame-'chopterin treatment 
resulted in successful growtli in roughly 30-60% of 
the cases depending on the isolate. Mice following 
Amethopterin trea-tment ^.rhich developed ascites did 
so at a much later time than similar mice inoculated 
with a full resistant line, suggesting the cells ware 
not growing during a treatment but were persisting. 
Evidence has been obtained that this characteristic 
is heritable. The finding explains that sensitive 
cells can change to a resistance though this is not 
3 complete resistance. 



608 



Serial No, NCI 427 (a) 

1. (Laboratory of Biology) 

2. CLeiikeinia~Studies Section) 
3« (Bethesda 14, Md.) 



Part A. 



1 b. With Dr, George Briggs, a folic acid deficient 
diet is currently being studied for its effect on 
P288 and P288 AioR2 . Surprisingly the results now 
nearing completion, consistently demonstrate the 
folic acid deficient diet produces a better survival 
time effect in mice bearing the Amethopterin re- 
sistant subline than the i\methopterin sensitive subline, 

2- The search for a supply of cortisone sensitive 
lyrophocytic neoplasms 

Cortisone sensitivity in lymphocytic neoplasms 
of mice mimics in many \vays the response of hviman 
acute leukemia to cortisone, i.e., the regression 
of large established transplants, followed by re^ 
currence, is similar to the profound short~lived 
remissions in man. One cortisone sensitive neoplasm 
(P1798) failed to maintain its sensitivity over an 
extensive period . A search for new neoplasms is in 
progress. Several methylcholanthrene induced 
lymphocytic neoplasms of DBA f/2 origin have failed 
to respond. X-radiation induced -fchymic leukemias of 
strain C57BL are being tested. A good working model 
system of this type should throw light on the time 
of origin of cortisone resistance in relation to the 
initiation of steroid treatment. 

3. 5-Fluoro uracil sens itive plasma-cell neoplasms 
of strain BALB/G origin. MFC 1.2.4 

Established transplants of 2 cm. in diameter 
can be completely regressed x-zith 5"=f luorouracil . A 
fourth MPC plasma-cell ttanor, MPC<-3 fails to respond 
to 5-fluorouracil. This model system has great 
potentiality; but is in need of intensive exploration. 



603 



serial No. NCI 427 (a) 

1. (Laboratory of Biology) 

2. (Leukemia~Studies Section) 

3. (Bethesda 14, Md.) 



Part i\ . 



Doses of S'^'flujiTOuracil (20 mgm. kg.) given daily 
(6 times weekly) are the most effective. Toxicity 
results after 2-3 weeks and the dose must be changed 
to 10 mgm. kg. This maintenance dose can be given 
over a 2 -month period but the tumors recur in the 
presence of this treatment o The development of 
5-fluorouracil resistance is in progress. MPC 1,2, 
are currently in ascitic form. The ascites are 
useful for initiating the tumor with controlled 
doses of cells. The ascitic tumor cell population, 
however, is sparse. Mice with fully regressed MPC™4' 
of several months duration accepted a new graft of 
MPC-4 and died. The opportunity to explore immunity 
is presented. The development of this system in a 
quantitative way should provide an e^rcellent means 
for learning more about when and how resistance in 
cells arises. 

B. __ 

1. Induction of plasma-cell neoplasm s in mice 

A controlled means for producing plasme -cell 
neoplasms in mice would provide a methodology to 
learn certain facts regarding multiple myeloma. 

A new means for producing plasma-cell 
neoplasms is under investigation. Follotiring the 
observation of Dr. Ruth Merwin that millipore 
diffusion chambers implanted intraperitoneally in 
mice cause the appearance of plasma-cell neoplasms 
in BALB/C mice, other means have been sought. 
Sixty-ft'ur mice were given incomplete Freunds 



610 



Serial Mo. MCI 427 ^^^ 

1. (Laboratory of Biology) 

2. (Leukemia~Studies Section) 
3« (Bethesda 14, Md.) 



Part A. 



adjuvants and a filtrate of a culture of staphylo- 
coccus intraperitoneally . One-half were painted 
with methylcholanthrene? the other were controls. 
Two plasma-cell neoplasms have appeared in the MCA 
group and two in the controls . These reseaible 
grossly and microscopically the tumors described 
by Merwin. 

Mice immunized intraperitoneally with 
horse serum before the adjuvant injection are being 
similarly observed , It is hoped that by stimulating 
specific antibody formation before or during carcino- 
genesis, that a n^eloma globulin producing trans- 
plantable plasma-cell neoplasm can be obtained which 
will permit a study of the antibody activity of the 
myeloma globulin. 

2. Investigation into the nature pf mveloraa_„ 
globulins 

Currently 10 transplantable plasma-cell 
neoplasms are being maintained in this laboratory. 
Employing the technique of Immunoelectrophoresis, 
the myeloma globulins are being characterized. 
The myeloma globulins produced in mice bearing MPC 2 
and 3, of Beta mobility on paper, have been found 
by iramunoelectrophoretic analysis to be antigenic- 
ally unrelated to a gamma 2 globulin or any other 
globulin in normal serum. 

These two identical myeloma globulins can 
be clearly and strongly precipitated in agar by 
rabbit anti MPC~3 serum, or by rabbit anti MPC-3 
serum, or by rabbit anti MPC<-3 serum absorbed with 
whole normal BiVLB/C serum, but are not developed 
by rabbit anti-normal BALB/C serum. The list of 
tumors in Bi^LB/C is being extended, 6 are now 
available to determine whether there is an infinite 
or grouped antigenic variation. 

611 



Serial No. NCI 427 ^^^ 
lo (Laboratory of Biology) 

2. (Leukemia-^Studies Section) 

3. (Bethesda 14, Md.) 



Part A. 



C Studies in methylcholanthrene (MCA) induced 
in strain DBa f/2 Lw 

1. Mice of strain DBA f/2 Lw develop generalized 
leukemia very rapidly following MCA painting. Mice 

of one month of age are begun on a course of 20 
paintings over a period of 7 weeks. Four weeks 
follovjing the last painting the first wave of leukemias 
appear. Mine cell free filtrates were prepared from 
leukemia tissue of individual cases arising early and 
from pools of several donors and inoculated into 
newborn mice of strain DBA f/2. Leukemia tissues were 
ground in sand and 10-20% extracts were centrifuged 
lightly and passed through Selas filters . To date 
in 159 mice under observation, for a minimal period 
of 5 months and a maicimum of 11 months no cases of 
leukemia have been observed, 

2 . A control pedigreed colony of DBA f/2 
mice is now 1 year of age (minimum) , The incidence 
of leukemia and allied disorders is being determined. 
This is a tedious task for old mice of the strain 
often die unexpectedly without forewarning. 



Mice of strain BALB/C have not developed leukemia, 
following MCA skin painting. A study has been initiated 
to determine if the difference between BALB/C and 
DBA f/2 in response to MCA is controlled by a single, 
two or more genes . Mice from a pedigreed colony of 
BALB/C/An maintained in the laboratory have been mated 
with DBA; the Fx hybrids, first generation and second 
generation backcross to DBA f/2 are being painted 
with MCA to determine the incidence of leukemia. Thus 
far no cases of leukemia have appeared in F^ hybrids ,, 



612 



Serial No. NCI 427 (^) 

1. (Laboratory of Biology) 

2. (Leukemia-Studies Section) 

3. (Bethesda 14, Md.) 

Part B. 

Honors, Awards, and Publications 

Potter, M. Biologic studies on the develop°- 
ment of DON resistance in a mast-cell neoplasm of 
the mouse. JNCI, V23, 163-181, 1959. 

Potter, M., and Haas, V= H, Relationships 
between lymphocytic choriomeningitis virus amethop~ 
terin and an amethopter in -resistant lymphocytic 
neoplasm in mice. JKCI, V22, 301-809, 1959 o 

Fahey, J. L., and Potter, M, Bence<- Jones 
viroteinuria associated with a transplantable 
mouse plasma-cell neoplasm. Nature, 184j 654'=655j 
1959. 



613 



[ndividual Project Report 
Calendar Year 1959 



Serial MOo 427 NCI (b^ 

1. (Laboratory of Biology) 

2. (Leukemia-Studies Section) 
3.. (Bethesda 14, Md.) 



Part A, 



Studies on Biochemical mechanisms of sensitivity 
and resistance to antimetabolites 

Elizabeth P. Anderson 

Man years (calendar year 1959) 
Totals 2 
Professionals 1 
Others 1 

A, Studies on azaserine and dia2o-02?: 0"norleucine 

This is a continuation of work reported in 
previous years on biochemical differences in the 
metabolisEi of asaserine-sensitive and^^resistant 
lines of plasma cell neoplasm 70429. 

1. Two new azaserine-resistant lines of 70429 
have been developed by procedures previously used 
in these laboratories for the selection of 
drug-resistant variants. These lines are novi7 being 
studied biologically, and will also be studied bio- 
chemically, in comparison v/ith the AzR-la line 
previously isolated by Dr. Michael Potter of this 
section and already studied in some detail by us. 



Part B included Yes 



614 



Serial I\io. I'jci 4^2/ (b^ 
1. (Laboratory of Biology) 

2. (Leukemia»Studies Section 

3. (Bethesda 14, Md,) 



Part R. 



2. Enzymatic studies on the azaserine-inhibited 
conversion of fonnyl-glycinamide ribotide to 
fomorl-glycinamidine ribotide in 70429S and 70429 
AzR-la have been continued in the attempt to 

define further the quantitative differences observed 
foetv/een the two lines in this conversion. Toward 
this end some further work has been done on purifica- 
tion of the ens3fme. Considerable work has also been 
carried out on the development of methods for 
enzymatic synthesis and for isolation of certain 
substrates for this work and on the accumulation of 
a store of these substrates, 

3. Arrangements have been made for further 
studies on possible differences between these sensi" 
tive and resistant lines in concentrative cell 
transport and/or breakdown of asaserine and DON; 
these follow up earlier observations on differences 
in inhibition observed with intact cell suspensions 
but not with cell-free extracts. These studies will 
be carried out in collaboration with Dr, John Jacquez, 
at Sloan-Kettering Institute, in the months of 
February and March. 



1, Work has been completed on the partial 
purification and the study of properties of a 
phosphodiesterase from ascitic leukemia L 4946, 
This enzyme is capable of hydrolyzing certain 
5 ' -phosphate-ended ribopolynucleotides to mono- 
nucleoside-5 '-phosphates; the mechanism of the 
reaction has been e^^lored and found to involve a 



615 



Serial Ho. ECl 427 (fa) 

1, (Laboratory of Biology) 

2, (Leukemia-Studies Section) 

3, (Bethesda 14, Md,) 



Part P.. 



stepwise cleavage of one nucleotide unit at a time 
from the end of the polymer chain. Siabstrate 
specificity has been investigated? the ensyme also 
shows activity against both ribo- and deo.'ayribo-oligo- 
nucleotides, but it is unable to hydrolyse simple 
esters of nucleoside-5 ' - phosphates because of a 
requirement for a nucleoside moiety on each side of 
the susceptible phosphodiester bond. This is the 
first enzyme to be reported with such a substrate 
specificity and represents, therefore, a new type 
of phosphodiesterase. Similar hydrolytic activity 
against ribopolynucleotides has been found in all 
of a number of other types of tumor tissue examined. 
This work has been carried out in collaboration with 
Dr. Leon Heppel, NliMM). h report of the work was 
presented at the Federation Meetings in Aprils a 
paper on the complete work has been written and will 
be submitted shortly for publication in Biochim. eto 
Biophys . Acta . 

C, 

1, Much time in the last few months has been 
devoted to compilation of the literature and prepara- 
tion of the te::t for the chapter on Biochemistry of 
Cancer for the 1960 j\nnual Reviews of Biochemistry, 
which should be completed shortly. This chapter 
will be written in collaboration with Dr. Lloyd Law 
of this section. 

D, 

1, j!\s In previous years, some time was spent 
in the preparation of manuscripts on the results 
of earlier research projects. 



616 



Serial No» NCI 427 J^) 

1. (Laboratory of Biolocy) 

2. (Leul^emia-Studies Sec-tion) 
3o (Bethesda lA, Md„) 

Part B . 

Honors, ^fiwards and Ptiblications 

Anderson, E. P., Majrwell, E, S., and Burton, 
R, M., Enzymatic syntheses of C -labeled uridine 
diphosphoglucose, galactose-l-phospliate, and 
uridine diphosphogalactose. J, Am, Chem. Soc, 
81s 0000 (1959) 

Yen, C. Yo, Anderson, E. Pc, and Smith, P. K», 
Effect of X"»irradiation on incorporation of 
ureidosuccinic acid-C;""!'^ into nucleic acid pyrimidines . 
Radiation Res . , 

Honors 

Election to membership in the Society for 
Essperimental Biology and Medicine. 



617 



PHS-NTE 
Individual Project Report 
Calendar Year 1959 



Serial Mo. WCI-427 (<^^ 

1. (Laboratory of Biology) 

2. (Leiikeinia-Studies Section) 

3. (Bethesda 14, Md.) 



Part A. 



Factors effecting transplantation of he matopoietic 
tissue in the irradiated mouse 

Delta E. Uphoff 

Man Years (calendar year 1959) 
Total : 1 

Professionals 1/2 
Other: 1/2 

In past reports, emphasis was placed upon the 
genetic factors influencing irradiation protection 
by bone marrow. From the survey which was initiated 
in an attempt to ascertain the role of certain 
genetically determined histocompatibility genes in 
the case of the marrow graft in the irradiated hosts 
several generalizations can be drax-m. There is 
presumptive evidence indicating that if genetically 
incompatible marrow is used as a protective agent, 
a graft versus host reaction ('secondary disease') 
will occur resulting in the death of many of the 
treated mice„ The severity of the reaction and 
degree of mortality varies with the strain corabina-" 
tions and is probably a function of the antigenic 
incompatibility of the strains employed. Foreign 
marrow has not been demonstrated to produce ' secondary 
disease' if marrow donor and irradiated host share the 
same histocompatibility="2 (H»2) phenotype. The H~l 
and H-S loci have little or no effect upon this 
reaction. 



Part E included 



618 



serial Ho. MCI 427 (c) 
1» (Laboratory of Biology) 

2. (Leukemia-Studies Section) 

3. (Bethesda 14, Md,) 



Part A. 



As a result of this study on the genetics of 
marrow transplantation, it became apparent that 
there are other factors influencing the long term 
survival of these lethally irradiated and protected 
mice. The premature clinical application of marrow 
transplantation as a therapeutic measure in terminal 
cases and the resulting failure to obtain evidence 
that the marrow transplantation was successful, 
emphasize^ the urgent need for a more complete study 
of factors influencing the acceptance and survival 
of the marrow graft in the irradiated host. The 
absence of technical assistance has made progress 
very slow and has necessitated the abandonment 
or indefinite postponement of several aspects of 
this study. 

A. Adult marrow transplantati on 

1, The survey of the genetic factors inxlu- 
encing irradiation protection by bone marrow was 
continued in an attempt to establish an all inclu-= 
sive working hypothesis. All strain cornbinations 
tested- within the H-2^ or H-2^ groups have been 
found to be compatible as either marrow donor or 
irradiated host. This lack of strain specificity 
but dependence upon only H-2 specificity, sets 
marrow transplantation apart from transplantation 
of p-c,her tissues. No combination was found in which 
good long-»term survival was obtained when strains 
were of different H-2 phenotypes. The H-2^^ group 
of which five inbred strains are available is more 
complect. The reports of hemagglutination studies 
on these strains indicate an incomplete matching 
of the antigenic components associated with the 
H-2 locus. In transplanting marrow in irradiated 
host cortfiDination of H-2^^, this incomplete matching 



619 



Serial No. MCI 427 (c) 
1. (Laboratory of Biology |i 
2„ (Leukeittia^Studies Section) 
3 , ( Betliesda 14 , Md . ) 



Part A. 



of antigenic components manifests itself in a 
moderately severe reaction from v/hich an appreciable 
nxmbex of animals survive. The presence of this 
' secondary disease coupled with the serological 
evidence of an antigenic difference between strains 
of the H~2^ group would indicate that these data on 
the H~2^ coiribinations are not at variance with that 
of the H»2b and H-2^ groups, 

2. The new coisogenic line made available by 
Dr, George Snell of Roscoe B. Jackson Memorial 
Laboratory made possible the confirmation of the 
previous report that a single gene difference at the 
H-1 locus does not produce a visible reaction = This 
new coisogenic line completed a coisogenic trio and 
made possible the testing of the additive effect of 
a gene difference at the H="l and Ii"-3 loci simultane= 
ously. The double gene difference xfas not a suffi- 
cient stimulus to produce a visible reaction. 

So In treated mice of some genetic combinations^ 
there appears to develop a transieint anemia following 
initial protection « In some cases this anemia 
appears to be related to a tendency for hemmorrhage . 
It was not considered an urgent problem for study 
since it only infrequently results in death of the 
host. iVlthough it may prove profitable and enlighten- 
ing, the study of this phenomena was indefinitely 
postponed. 7memia of a more severe nature vill be 
discussed in a following paragraph (B 2) » 



620 



Serial Mo. HCZ 427 (c> 
1. (Laboratoary of Biology) 
2= (Lexokemia-Studies Section] 
3, (Bethesda 14, Md.) 



Part A, 



4, Homologous marrov/ inoculation following 
irradiation at the mid-lethal ' dosage level has 
been observed to be lethal within 30 days post 
irradiation in several laboratories. Although the 
genetic combinations were few (2) and the groups 
v/ere small, the confirmation of these results by 
three different investigators led to the immediate 
acceptance of this phenomenon. In an attempt to 
determine the role of the H-2 locus at this dosage 
level (500r-550r), one strain of the H-2^ phenotype 
was selected as irradiated host, two different H--2 
marrow donors were selected as well as one each of 
the H-2*^ and H~2^ phenotypes. At least 90% of "the 
animals in all four groups at the 500r and 5,;0r 
dosage levels survived thirty days or longer. These 
results would indicate that the mid^lethal zone 
effect may not be a problem for all genetic coarSDina- 
tions. A limited number of strain combinations will 
be tested in an attempt to determine what factors 
are involved in this lethal effect. The mid-lethal 
zone effect has been considered a major problem in 
clinical therapy where the lethal range has not heen 
established. The full significance of the present 
observation is not immediately apparent and specula- 
tion at this time would be premature. 



1. In an attempt to preclude the graft-»versus"'hos 
reaction fetal hematopoietic tissue was used as a pro"- 
tective agent. It has been reported previously that 
in one genetic combination no visible reaction 
occurred when young (14 day gestation) fetal tissue 
was used rather than adult tissue. Comparative data 
are now available on four irradiated Fj_ hybrids 
receiving parental-strain fetal hematopoietic tissue 



621 



Serial Ho, NC I 427 (c) 
1. (Laboratory of Biology) 
2„ (Leukemia-Studies Section) 
3. (Bethesda 14, Md„). 



Part A 



in sever*, different combinations. In several, but 
not all, hybrid-parental-strain combinations, no 
reaction was observed if early fetal tissue was used. 
In the other combination and with late neonatal 
tissue, a syndrome occurs which is less severe and 
more animals survive the critical period than 
following treatment with adult marrow of the same 
genetic combinations. Similarly, good survival was 
observed in strain cornbinations of the H~2^ group 
following protection v/ith fetal tissue. In combina" 
tions in which there was a gene difference at the 
H~2 locus, the long-term survival was usually bettei: 
following treatment with fetal tissue than with adult 
marrow but never reached as high as 50 percent. 
Since this mortality represents a major obstacle in 
the use of fetal hematopoietic tissue as a therapeutic 
agent, a study is underway, in an attempt to deter- 
mine whether this mortality results from an immune 
response and its relation, if any, to secondary diseas; 

2 . In certain strain combinations an anemia 
was observed following initial recovery from lethal 
radiation. Unlike the transient anemia mentioned 
above, this anemic condition is quite lethal. In 
collaboration with Commr. VJilliam McFarland, National 
Naval Medical Center, an attempt was made to deterraine 
the cause of this anemia. In testing animals of 
different genetic combinations as they became available; 
it became apparent that this was not a simple problemo 
The types of anemia detected were hypochromic anemia, 
hemorrhagic anemia resulting from slow blood leakage 
into the small intestines and kidneys, and a possible 
hemolytic anemia; this latter diagnosis has not yet 
been confirmed. The possibility e^rists that this 
late type of anemia may result from an immune response 



622 



Serial Ho.o.,_. ..jjjj^X_ 

1. (Laboratory of Biology) 

2. (Leukemia-Studies Section) 

3. (Bethesda 14, Md.) 



Part I.., 



initiated by a red cell sutigen a^t governed by 
the H~2 locus which has been described for some 
inbred strains of mice. The genetic tests have not 
yet been undertaken to check this hypothesis. The 
complexity of this problem of anemia and the lack 
of technical assistance made necessary an indefinite 
postponement of any further hematological determina- 
tion. The genetic studies will continue in an 
attempt to have available more definite information 
of a genetic nature at such time as this study may 
be resumed. 

Co Preclusion of the homolc 

1. The preclusion of the reac tion follov;ing 
treatment with ^-methopterin gave hope that this 
might be a genetically non-specific method which 
might have practical clinical application. Other 
drugs tested were found to be too toxic in the 
irradiated and protected mice to be able to deter- 
mine their efficiency in blocking the immxane response 
under the es^perimental conditions used. Lmong the 
drugs tested were thioguanine, 5-fluorouracil and 
6-azauracil. Cortisone and aureomycin were found 

to be ineffective in a limited number of trials. 
Since time'-dose relationships have been shown to be 
very important, the negative results thus far obtained 
from drugs other than Amethopterin should not be con- 
sidered all inclusive since very minor alterations 
in the treatment schedule have proved to be the 
difference between success and failure of the treatment. 
Further work is planned, 

2, In collaboration with Dr. Raymond Popp, 
Biology Division, Oak Ridge National Laboratory, tests 
were made on irradiated animals treated with homolo- 
gous marrow and one of the various drugs listed above 
to determine by their electrophoretic hemoglobin 
pattern if the graft was destroyed as a result of -the 
drug treatment. In a sample of twelve mice ranging 

623 



Serial Mo. i>iv:.^ -^z'l (c) 

1. (Laboratory of Biology) 

2. CLeiiI<eniia-=Studies Section) 

3. (Bethesda 14, Md.) 



Part A. 



in age from three to twelve months post irradiation 
all hemoglobins were of donor-strain origin. This 
would indicate that the drug action is not one of 
slow destruction of the graft and recovery of the 
hosts hematopoietic system but probably one of a 
direct effect upon the immune response of the graft 
against the host. 

3. Since the irradiated and protected mouse 
tolerated the drug treatment less readily than the 
non-irradiated animal and these treated animals 
were demonstrated by their hemoglobin patterns and 
tumor transplantation tests to be chimeras a 
somewhat different approach at drug treatment was 
attempted. It appeared that 7^.methopterin in the , 
presence of an antigen permanently blocked the 
immxHie response against this specific antigen. 
hxi attempt was made to produce 'tolerant ma£row 
donors by means of treated and non-irradiated mice 
with Aiaethopterin and injections of foreign spleen 
cells in various coiribinations , l^.en these pretreated 
animals are used as marrow donors in the same strain 
as that of the spleen cells used in the pretreatment, 
the results v/ere not easily explained. Under some 
pretreatment schedules, the marrow donors appear to 
have been sensitized against antigens of the host 
strain causing a more severe reaction than was 
e^ipected from that particular combination although 
the early deaths described by others in presensitiza- 
tion vxperiments did not occur. Under other conditions, 
there appeared to be prolonged survival although as 
yet an actual preclusion of the reaction has not 
been observed. In lieu of more promising methods of 
altering the homograft reaction, further attempts 
will be made using a slightly altered treatinent 
schedule. 



624 



Serial No<. i><c X 427 jg) 



1, (Laboratory of Biology) 

2, (Lexikemia-Studies Section) 

3, (Bethesda 14, Md.) 

Part A^ 

D. Chimeras 

1, The previously reported study of the 
histocompatibility patterns of the malignant lymphomas 
arising in the irradiation induced chimeras iiras con™ 
tinued. The more recent transplantation tests in- 
volved the high leukemic AKR strain as either marrow 
donor or irradiated host to the exclusion of those 
of other strain combinations » The es'periments 
involving the AKR strain will be discussed elsewhere ■ 
in this report; only the transplant data will be 
given here. Of the positive transplantation tests 
for homologous marrow chimeras, the neoplasms 
proved to be of donor-tissue origin in 49 cases and 
of host-tissue origin in 11 cases. In the chimeras 
produced by homologous fetal tissue inoculation, 
15 neoplasms were of donor tissue origin and 2 of 
host tissue origin. A smaller sample of tests were 
conducted on animals receiving drug treatment (CI). 
Six of these neoplasms were of donor tissue origin 
and one of host tissue origin, thus corroborating 
the finding of Dr Popp (C 2) ^ that these mice are 
true chimeras in a second strain combination = The 
neoplasms involved in these tests v/ere primarily 
lymphocytic and frequently involved the thymus » 
In appro::imately one-fourth of the tests the trans-- 
planted tissue failed to grow. These negative tests 
included several plasma-=-cell neoplasms and four 
adenocarcinomas of the small intestine which were 
transplanted as possible plasma-cell neoplasms. 
Adenocarcinomas of the intestines occur in untreated 
mice but are rare. The nxamber found in this sample, 
which may be a ve2:y small percentage of those 
actually occurring in these treated animals, repre- 
sents an increased incidence of this neoplasm. 
Several other neoplasms of unusual types v-7ere observed 



623 



Serial Ho„ NCI 427 Cc) 
1. (Laboratory of Bio3.ogy) 
2» (Leukemia-Studies Section) 
3o (Bethesda 14, Md.) 



Part Ao 



in this small sample of treated animals. Diagnosis 
has not been completed, hox-^ever; they are considered 
by Dr. Thelma Dunn to be similar to those observed 
to occur in mice treated at birth with the Polyoma 
virus . No routine attempt has been made to dei-on- 
strate the presence or absence of a viral etiology 
for these neoplasms c 

2. Reversal of the functioning donor type 
hematopoietic system to that of the host type is 
known to occur with vaiiform regularity in animals 
pj^otected with heterologous marrow. There has been 
little evidence that reversal occurred in animals 
receiving homologous marrow therapy. The occurrence 
of a single case of reversal of homologous chimeras 
led to the postulation that the greater the genetic 
difference between the host and donor the greater 
the probability of reversal' occurring. Of the 
14 host type txamors observed in these transplanta~ 
tion esrperiments all represented marrow donors and 
irradiated recipients of -the same H-2 phenotype, 
i.e., either both H-2", o- F-2^. These data would 
indicate that the prerequisite for 'reversal' need 
not be a 'great ^ genetic difference between host 
and donor. 

E, Transfer of im munity 'b^ 

In collaboration with Dr, Victor Haas of lll?/S~j.. 
ejcperiments are in progress attempting to transfer 
immunity to the LGM virus from one mouse to another 
by means of lethal total body X-irradi£ tion and 
isologous marrow inoculation. Although the e:;rper.lments 
are still incomplete, it would appear that under these 
e^'perimental conditions it is not possible to transfer 
immunity to the living antigen. 



628 



la 



Serial Ho, NCI 427 



1, (Laboratory of Biology) 
2 c (Letf emia^-Studies Section) 
3. (Bethesda 14, Md.) 



Part h. 



F. Multiple polyposis o£ the colon, 8 gen etic trait 

The development of a line of C57BL/10 H-2 
carrying the genetic trait for multiple polyposis 
of the colon was continued. Attempts at producing 
a malignancy from this benign lesion by irradiation 
were not particularly successful due to the high 
incidence of malignant lymphomas induced in this 
strain by the irradiation. 



627 



11 



Serial No. NCX 427 J^) 

1. (Laboratory of Biology) 

2. (Letikemia^Studies Section) 

3. (Bethesda 14, Md.) 

P art B^ 

Honors, Awards and Publications 

Uphoff, D. E. Alteration of homograph reaction 
by A-raethopterin in lethally irradiated mice treated 
with homologous marroxiz. Proc. Soc. Erp. Biol. & Med,, 
99; 651=653, 1958. 

Uphoff, D, E., Law, Lo Wo An evaluation of 
some genetic factors influencing irradiation protec- 
tion by bone marrow. J. Hat. Cancer Inst. 22s 
229'=24i, 1959 o 

Fellow of the American Association for the 
Advancement of Science. 



623 



PHS~NIH 
Individual Project Report 
Calendar Year 1959 



Serial Ho. NCI 427 id ) 

1. (Laboratory of Biology) 

2, (Leukemia -Studies Section) 
3= (Bethesda 14, Md.) 



Part A. 



Pi study of the etiology and chemotherapy of experi- 
mental lymphomas 

Delta E. Uphoff and Lloyd W. Law 

Man years (calendar year 1959) : 
Total: 1 1/3 

Professionals Uphoff, 1/2 s Law, 1/3 
Other ! 1/2 

^' Etioloc 

1. Attempts at altering the incidence of 
leukemia in the high leukemic AKR strain vjere e:-:tended 
to include the use of marrow of the four available 
H~2^ strains and treatment at ages ranging from three 
to eight months . Although secondary disease '' occurs 
between 30 and 90 days following lethal irradiation 
and homologous marrow inoculation, an appreciable 
number of animals survive making possible a study 
of the incidence of leukemia in these chimeras ^ as 
well as the effect of varying degrees of reaction 
upon this incidence. The latter may prove to be an 
important factor since one of the results of a severe 
reaction is the depletion of the lymphoid elements . 
Although this study is still incomplete and no 
general statements may be made on the oirer-all 
incidence of leukemia in these animals, at this tirae, 
some interesting observations can be made. Animals 
which are three to five months of age at ttie time 
of treatment may die as a result of the treatment 
e.g. from secondary disease , or may recover and 
eventually die of leukemia or from some other cause. 



629 



Serial No. HCI 427 (d) 

1. (Laboratory of Biology) 

2. (Leukemia-Studies Section) 
3= (Bethesda 14, Md=) 



Part P.-, 



from 
Of those developing leukemia/ transplcintation testSo the 

indications are that these neoplasms are primarily 
of donor tissue origin even though the donor marrow 
may be of a low leukemic strain e.g. C3Hf . /i few 
neoplasms proved to be of host tissue origin and 
are considered "reversals . These are discussed 
elsewhere in this report. By six months of age 
some untreated AKR mice, particularly the females, 
m.ay be e.irpected to succxunfo to leukemia. Mice 
si;: to eight months of age with advanced lymphocytic 
leukemia of thymic origin at the time of irradiation 
may die shortly after irradiation apparently as a 
result of the tcbxic effects of the destruction of 
large masses of tumor tissue, A percentage of the 
animals which survive the acute irradiation damage 
will die of a "typical secondary disease' . Other 
animals which succuiiib e^diibit an unusual type of 
liver damage, the cause of which has not been 
determin'ed. During the second and third months 
post-irradiation a few mice die of leukemia o Trans- 
plantation tests show these to be of host tissue 
origin. Unlike those previously mentioned, these 
are considered to be animals which were preleukemic 
or in the early stages of the development of the 
disease at the time of irradiation. The malignant 

cells were not completely destroyed by the irradia™" 
tion and went on to develop a neoplasm which eventu-= 
ally killed the animal. This might be e^i-pected 
since a dose of 700r has not teen demonstrated to be 
sufficient to destroy most neoplasms.. Some mice 
which survive these episodes may live long enough to 
develop leukemia with a transplantation pattern 
similar to that described for the animals which are 
younger at the time of irradiation. Of these mice 



630 



Serial Ho. HGI 4?.7Jd.) 

1, (Laboratory of Biology) 
2„ (Leukemia-Studies Sectioxi) 
3, (Bethesda 14, Md„) 



Part A. 



that develop leTilcemia^ the age at death appears 
to be the same regardless of the age at treatment 
ioG., the older the animal before trea-fcment, the 
shorter the latent period. In the reciprocal, 
transplant i.e., AKR marrow in one of the four 
irradiated hosts, the incidence of le-akemia is 
relatively low. A few of these neoplasms hsve been 
transplanted and were shown to be of donor tissue 
origin as might be expected. The high incidence of 
leukemia of donor tissue origin in the irradiated AKR 
mice and the '"low incidence of leulcenaia in the re™ 
ciprocal combinations might indicate that rather than 
an indirect inductive effect of the irradiation as ' a 
causative agent, there may be fundamental differences 
between the AKR strain and the other four strains of 
the H~2^^ group. To test the role of the thymus in 
the induction of these neoplasms, thymectomized AKR 
mice will be treated with marrow of the Street, strain 
in a manner similar to that of the intact mice. 

2, The study of the effect on -fcha incidence 
of lymphomas of esqposure to 400 r, 100 r, and 25 r, 
at birth with and wi-thout bone marrow inoculation 
at the 400 r level and 400 r at 30 days of age was 
completed. Intraperitoneal inoculation of G57BL 
marrow following 400 r at birth appsar-ed to have 
little effect on the incidence of IjiTsphomas , while 
an intravenous inoculation appeared to decrease the 
early neoplasms. Pathological analysis has not been 
completed making further generalisations impossible 
at this time. 

3 , The study of the effect of bone marrov/ 
and fetal hematopoietic tissue on the incidence of 
malignant lym.phomas in intact and thymectomized 

C57BL and (C57E.lJ5cA)Fj_ hybrid mice receiving 4 exposures 
of 1S8 r at 7 day intervals and -treated wi-th C57BL 



631 



Serial No. NCI 427 (cl) 
1, (Laboratory of Biology) 
2» (Leiikemia^Studies Section] 
3o (Bethesda 14, Md,) 



Part A. 



marrow or fetal hematopoietic tissue was continued. 
In the Fi hybrids at 13 months of age, the incidence 
of reticular neoplasms in the controls was 64%; in 
the fetal tissue inoculated group 34%; and following 
marrow inoculation, 22%, Similar results were ob- 
served in the C57BL strain (85%, 40% and 30%, respec- 
tively) » ^Transplantation tests of the neoplasms 
arising in tlie hybrid mice indicated that the major- 
ity of the neoplasms were of host tissue origin. 
These results might indicate a lack of protection by 
the inoculated tissue although the occurrence of an 
occasional neoplasm of donor tissue origin and the 
time of onset would indicate the role of other 
factors. The differences in response of the 
X-rayed mice receiving marrow and fetal tissue may 
result from differences in the population of cells 
introduced. The earlier appearance and higher in- 
cidence of neoplasms in the mice receiving fetal 
tissue may reflect a potential inductive capacity 
of the enferyonic hematopoietic tissue. 

4. The previously proposed project to study 
the influence of complete and inccmplete matching 
of the histocompatibility phenotypes on tlie prcven- 
tion of X-irradiation-induced lymphoma has not yet 
been attempted because of a lack of animal space 
and technical assistance, h somewhat less extensive 
project was temporarily siibstituted for the proposed 
study in an attempt to determine the influence of 
the H~2 locus on the prevention of the induction of 
lomas in C57BL/Ka mice. 



A single gene difference at the H-2 locus 
(H-2^ versus H-2<^) does not prevent lymphoma indue- 
tion in thymectomised C57BL/Ka mice, whereas H»2^ 
bone marrow from C57BL/10 (differing at loci other 
than H=-2) is effective in preventing the induction « 



632. 



Serial No. NCI 427. (d) 

1, (Laboratory of Biology) 
2= (Leukemia-Studies Section) 
3. (Bethesda 14, Md.) 



Par t A. 



Einbryonic hematopoietic tissue (C57BL/Ka) , though 
strikingly effective in producing ijmiediate thymic 
regeneration, is not (unlike adult bone marrow) 
very effective in reversing the carcinogenic process. 
These studies are continuing in the following 
aspects s 

a) Study of the dose-response effects of 
embryonic hematopoietic tissue 

b) Influence of other H~2 allels such as 
other H-»2^, H'=-2«3, H-2^, ^1-2^, using both 
embryonic and adult hematopoietic tissues. 

Bo Chemotherapy of transplantable leulcemias 

Attempts at treating drug toxicity in tumor 
bearing as well as normal mice by bone marrox'? inocula- 
tion was continued. A series of AKR mice were treated 
with therapeutic dose level of thioguanine and sn 
intravenous inoculation of C3Hf bone marrow. The 
leukemias arising in these treated mice were tested 
by transplantation and were found to be of AKR 
tissue origin. This would indicate that transplanta- 
tion of marrow under these conditions was either not 
successful or that 'reversal' had occurred in 
virtually 100% of the animals treated. With the 
possibility existing that in these animals receiving 
antimetabolites and homologous marrow, a state of 
immunological non-reactivity (or ^tolerance-) toward 
the strain of the marrow donor may e^ist, a second 
group of mice will be treated and tested by skin 
grafting. 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 antimetabolites. 



63 



o 



Serial No. NCI 427 fd) . _..__„ 
1„ (Laboratory of Biology) 

2. (Le-ttlceniia~Studies Section) 

3. (Bethesda 14, Md,) 



Part ho 



Following the report of Loutit and colleagues 
at Harwell of sensitivity of one malignant lymphoma 
to high level X-radiation, protracted over 24 hours, 
we undertook a study of the response of all available 
morphologic forms of reticular neoplasms (a total 
of 10) , Striking responses were obtained with a 
type B neoplasm (P195) and with a type A neoplasm 
(L11371) . Work is continuing v^ith the latter, a 
chronic neoplasm in C57L mice to study the influence 
of dose rate and protraction of irradiation, of tlie 
effect of isologous versus homologous bone marrow 
following irradiation and the permanence of response 
in later transfer generations. 

Part Bj 

Not included. 



634 



mi 

Serial MOo .'42b=»A 

1. Laborif^y of Biology 

2. Tissus CixLtisr-e Section 
3o Bethesdaj Jferyland 



IndividTjal Project Report 
Calendar Year 1959 



Part A 



Project Title? Merocirffimatographic studies of tissiae cells 
in vitro. 



Prjjicipal Investigators I WoT, McQuilki% WoR. Ess-le< 
Other Irwestigator « ¥, J, Evaaso 



Cooperating Units s Honec 



Mali Ye^=s ( calendar year 1959)' Pat3.ent Days (calendar 

Totals year 1959) s 



Others 

Project Descriptions 

lo A study of the alteration of I!K^rphologic and ■'oeha;riQral character" 
istics of strain NCTC-929 (strain L) cells resultirig fi-onx grcsvth 
In cheraically defined, protein-feee msdiufflj IC'rc-109<. 

Objective? To dstsradne q^jantitative asd qualitatiire differeness be- 
t^3een the isiorphologic and behavioral characteristics of cells of thi; 
t«ell kaoim strain ^len it is Eiaiataiiied is its accustosied protein 
meditus and after it has been adapted to the nutritional environiiient 
of the chesaically defined pro-&3ia<=fre8 iti^ditBi HCTG"109= 

Ifethods Ei!f)lc^ds Detailed study and analysis of many tiiije-'lapse 
laotion piettjres of both the parent and the adapted cell strains^ 
Quantitative tisis data is obtained by correlating the frame fre«» 
q^isney wiW. the obser^s-ed ©ceirerenee of ©vents in the filsi. 



Part B included Yes x Mo 

635 



NGI 
Serial Ho=l!.28ajPage 2 



fe^or fiadiBgS! Some qmte definite alterations ha^e occm^ed in the 
cell straisi as a resiilt of adaptation to and establishment in the 
chemically defined proteia-f^ee mediian. Quantitativelyp the average 
generation tisa is about I/3 longer | the rate of lai^ation of cells 
on tiie glass siibstrate is reduced by at least 90 per cent ^ and the 
d\2ration of the cytokinetic period has bean estimated tentatively to 
be about l/5 as long, Qiaalitati'O'elys the cells show a less contact, 
less reft'actile aad less granular aiOE'ph©logy| cyclosis is altered to 
a more peristaltic type motioai the cells maintain a more independent 
existence with a decreased affinity for each other and conseqttently 
appear to have less msAv&l infltienoes cells do not intermittently 
round t^ and spread oirt as farmer ly^ but almost exclusively round up 
only for the prophase stage of ni3.tosis| cjrbokinesis is not only of 
shorter durationj but occis's in a much less •violent maimer, Uo statis" 
tically significant findings of cell death ha'Te been obssr-^ed in either 
cell-jnsdixffii ^stem« Hows-^er, a fei? spectacular cell deaths by coiEplete 
and sudden disintegration have heen observed in filjos of one subline 
©f the d'i-rived cell straino 

2 . Analysis of film of a strain of monkey kidney cells maintained on 
cheraically defined^ protein'^i^ee nBdiusao 

Objectives To determine the proliferation rate in this cell=>H!sdiujn 



Jfejor findings? The average generation time ©f this epithelial cell 
strain is approxiisately 3? hours, ■tshich is con^sirable to the previously 
deteriidjied time of fibroblast cells in this sarae niediuni„ This rate of 
proliferation indicates a popul-ation increase of approximately 2i^»fold 
in a 7 day periods 

.Significances These studies indicate the methods that are being 
en^loysd and the efficacy of the cinematographic tedanique as an in^ 
stroment of research in population dynamics of living cell cultures 1^ 
yitro o 

The first study^ above, characterises some of the factors of cell msr° 
phology and cell behavior of an established in vitro cell strain of 

mouse fibroblasts that cannot ba deteriiiii^d in any othei'" ■a'ay. It further 
elucidates soms alterations that have occurred in this cell strain as a 
restslt of a radical change in the nroritional environmsat , 

The second study ^ above 5 confirms the rapid proliferation rate potential 
of the iffiport.ant monlcey kidney strain sdaen greisn in vitro in cheEiieally 
defined^ protein^free Hsdiuni HCTC lQ9o It also dsraoSs^ated that success- 
ive generations of particular individual cells casi be studied eineraicro" 
graphically (in this case up to six generations).. 



63i 



NCI 
Ssrial lo. ii28aj Page 3 



Proposed course of project j To coatiniie the present line of iir^estiga- 
tion of the arteratisa in prolif ©ration rats 5 mfispholegy and cellular 
behs'/ior in cell strains^ as corralatsd Mth oncological trans forraationss 
viral activity, and nutritional changes. It is proposed that refinements 
and extensions of observations will be made by the addition of the tech'^ 
niqiiss of higher magnifications, phase microscopy, and perfusioao 



Part Bs Honors, Awards, and Publications. 

Films have been profvidedgby request, to the British Broadcasting Co., 
for 72S8 in a forthcoming science series Tf broadcast entitled "The 
Last ScoTjrge". 

Data flcom 2, aboire reported in sorfeicle "Srowth i^ vitro of a long teria 
strain of laonkey Icidney cells in uiediuia lilGTC 109 fl'ee of any added pro= 
teinj " pisblished in Aiuericaa Jotirnal of Hygiene, n<3<¥^ 1959, (see section 
Ii28-C). 



637 



KCI 
Serial No, i!.28(b|__" Project 1 
lo Labcffalory of Biolo^'- 
2o Tissue Culture Section 
3= Bethesdaj Iferyland 



Indi'^ridual Project Report 

19S9 



Part A 
-^--^ 1, 

Project/ -2-^3.6 5 Rapidly agitated fluid suspension ciilt'<2res far the 
grosTtli of CBlis of various established cell strains < 

Principal Inirestigatorss JoC, Bryaats E. L. SchilliMgj ¥oR«Earieo 
Other Ixrirestigatorgs Moseo 
■ Cooperating Units? 



ffea Years (calendar year 1959)* Patient Days (calendar 
Totals Year 19B9)' 

Professionals 
Other s 



Project Description? 

Objectives! To continue attempts, described in last yearns re= 
portj to define ai'id control more closely the factors influenciiig 
growth of raarinsalian cells in rapidly agitated fluid suspension 
cultures. Es^lxasis this year has been placed on 1) adaptation of 
new cell ' strajjis to this tjpe of culttsre^ and 2) detJ-eloping and 
testing of a nei-i gas^netering device for controlliEg tke flow of 
gas tiirougii the cultures above the fluid, 

I'&thods Ec^jloyeds These have been described in earlier reports s 
special l|- liter sl^aker flasks containijag a laaximisa of liOO^itSO mlo 
fluidj continuously aerated through the gas phase, sad agitated 
on a shaker platf or® at 861tO revolutions per hour (rph) * ' Huclei 
counts and reducing sugar analyses x^ere made regularly on small 
aliquot saiuples. 



Part B included les x JJo 



o o o 



Ssriai 3}Jo. Ii28(b)j Page 2 
Project 1 

The old gas«=F^tering de-e-ice (Si^lej et al,j Annals N,Io Acad, Sci = 
63(5) s 6665 1956) ^'a.s replaced earlj dm-ing the year by a similar 
asid more acctsrate electronically controlled gas metering ^stesoCSee 
ii28(f)o The gas phase of each agitated cultwe receives one short 
■bisrst of gas diaring each iS-second iater^al« This caa be "S^aried at 
will. Kiree different gas ndxtures haire been -osedi a) $ percent 
C02s 25 percent 02, and ?0 percent N21 b) $ percent CO2 ia sir| 
c) 10 percent CO2 in aire 

IfejoQ? Findings s The kind of gas mixtiare used, of those listed above, 
had no effect on the proliferation rate of the cell strains studied; 
either a monkey kidnej cell strain SGTC 3196 (LICI-iS) or a mouse 
fibroblast strain 20?1, The mediisra ^ss pro-fceia»&'ee chemically de= 
fined mediim NCTC 109 ST^pleifJsnted with Bfethocel. 

The sTiccsssfiil demonstration dwiiig the two pre^ioiss years that 
established cell strains sush as HGTC clone 929 Srosi strain L and 
KCTC liTer clone IU69 cosld be repeatedly carried throiigh short 
hanfesting^siibciiltisring cycles £&£■ high net yields at high popiala^' 
tion Is'^els laade it advisable this ^ar to estend the procedizres to 
other cell strainsj siich as strain ICTC 20?1 firom clone 929 mouse 
fibroblasts^ a menkey kidney cell strain, and a hiaman skin strain» 
The elimination of seriM and the si&stitistion of Bjsthylcelltalose as 
a si^plej^nt to eheisiieally defined mediiMi ICTG 109 has been given 
major ei^hasis. Project 2 of Serial Ko, U28(b) repca-ts the sttsdies 
of the factors isc^olired. 

Significances Use of these methods for ctalti'^ating mammalian cells 
in agitated fliaid sv^snsion cultiires srnkes possible the production 
in iritre of larg© amoiants ©f cells of various strainSj both nofrsial 
aad'^Srr^iant^ xmder much more aeeijrately controlled experimental 
conditions. Consistent airailability of large qiaantities of cells of 
a given strain at ^eqiaent internals atid -isnder rigidly contrelled 
esqjeria^atal conditions f er long term bicscheiaical stisdies is noia of 
major iir^ortance in vm^ lines of researeho 

Proposed CoTSPse of Project? The precsdm^e may be extended te other- 
selected strains of cells ^^ich s^e ispo^'tant for caaeer rssssrcho 
A nsjor long-term ©bjsctiTe is t© deteriaine the conditions necessary 
for maintaining a rapid aerobic hffi'¥8stiBg='S^1bcultl3ririg cycle fas' 
•grarioTSs cell strains. lafsroveiaent of the procedures is a coatia't^ing 
objective o The proced'^sre will be extended to incorporate a e©ntiauoi?.3 
flow apparatus for continuous growth of the cells imder steady state 
conditions. 



639 



Serial No. ItaSCb), Page 3 
Project lo 



Pgrt^B^^ EonoTSs Awai'dSj and Publications s 

(Piislications otlaer than abstracts t^om this project i) 
(Honors and a^Tardss ) 

A paper- presented Aprils 19S8 ^J i'iWitatioa at the "Syii^joss-um 
on Culti-vation and Use of Anisiai and Plant Ceils"" j an Intersociety 
^uposiiim of the Federation of American Societies for Experimental 
Biologjj Philadalphias Pa., was based in large part oa this project 
and presented current resiilts fi-oia this project., The paper was 
P'oblished in aaiplifiad foria as follows? Earlej \hRo % LoBg==term 
colti^ation of siiinial tissue cells in large cultiires. Feda. Proe. 
llihjt 967''9'lhs 1958 « PuMication vsas tos late to be included is. 
om" 1958 annital report. See Earle project ii28(f) fixr listing. 



6H0 



NCI 
Serial Mo. hZQ Co) - Iroject 2 
lo Laboratory of Biology 
2«. Tisstie Culttire Section 
3» Bethesdaj Fiarjl^sid 



PHS-N3H 

Individ'ual Project Report 

Calendar Year 19S9 



Part A 



Project 2, Titles The grow&h^petentiatiDg effeet on chemically 
defined medi-jsss; ICTC 109 of l-fethocel irith cer= 
taia cell straias in a) rapidly agitated fltaid 
suspension eiiLtm-es and b) small statio cultm^eso 

Principal Investigators s J. G, Bryant^ ¥.Jo Bsaas, Ec. L. Schillings 
and ¥. R. Earls <> 



Other Investigators ! B> 3, ¥estfall, E,¥, Peppers « 



Cooperating Unitss (Project 2b) Ha"^ Tissue Baxikj, Naval Ifedical 
Center, (P.G. Bakken)o 



Project descriptions 

Objectives? To gro?'? manuaalian cells in agita'bed fluid snspensisB 
cultizres in clieriiically defined Esdluia KCTC 105 ■sd.tho\£t serum si2ppla'= 
nsnt by siastitutiiig a lios'^proteia cheaiically defined Ifethocel,, 
En^jhasis is placed on a raonlcey kidney cell strain o 

I-fethods Emplc^ed? The type of etiltiirs aad the basic procedtarss are 
considered in Project 1, Serial 1^28 (b) , 

Major Findings? Esqjloratory investigations repea-ted last jeaTf lao-ose 
fibroblasts of sti'ain M3TC 2071 in different t^pes of eijltm-'e flasks,* 
Trariotss grades and concenteations of Methocelj and tilth a silicoaa 
coating on the walls of the flasks j ^lere cs^firffied Idiis 3?^ar» .4s an 
e^iample, in two eult'ores of one expsriirient, strain 2071 sells prcf=^ 
liferated fcsr 1? days at a moderate logarithiJiic rate eroresssd as 
i}»86 days and l5,*63 days respectively average generation times « Popu^ 
latioa increased from 8l and 63 sdllioii cells in 100 silo to 911 and 
78S million cells in IjOO sale Repeated har-'^esting eyclesj hoKBvar,^ 
have not yst been attained fcs- this strain w fs^ any other cell strain 



Part B incteded Yes x Ko 



HCI 
, Serial Mo» 1^28 (b)^ Page 2» 

Projsct 2 

in this tssdlvM in agitated cultiiTeso 

ifexiy experajaents hava bsea earried out in agitated jQ.i2id 
suspension cultures isrith the rasakej kidaey cell strain ECTC 3196 ( 
(LLOWS. from Lill^ Research Laboratories) » This strain has been 
proliferating rapidlj in static ciilt-ores ia protein-&ee chemically 
defjxed mediura KCTC 109 for llj. months (See Evans project l!.28(c). 
Generally tha agitated ■ fluid s^speasioa eijlttsres of these eells 
have prssentsd aii anomaljs the cells resiain healthy in appasranee as 
either single cells dr small irr-egislar loosely packed cliaaps of eellss 
but show little 'if axgr in.cjr'eass in numbers cf^er periods of tiirsa isp 
to t'rjo months <i 

In a few cases these moKlcey kidney call cultTares have sho^^ 
sloH prolifes'atioii. For exaa^ilej oae c-altisre oa ?^T(~- 109 lEeditM 
STippleiiisntsd t-jith a Im-? ifiscosity Tfethocel (25 cps) at 0=12 per-*- 
cent gave a fovr-^foid increase la 21 days. In another ej^eriment 
where Methceel sijpplemant vjs.s at lauch higher concentrations (25 
cps at ls65 percent and iiOOO cps at O.Ii.5 percent) two cultisres iia? 
creased in population froia abotit 28 o 5 millioa to 12? millioa., in 
75 to 80 ml, froidj, a Ito5-"fold increase ±n 3U days. This similarity 
in proliferation rates betxaseH these txfo e3q5erii!ients occurred des'^- 
pite a x-ride dzlfferesce in actsjal "viscosities of the complete mediimi 
the relative viscosities x^ere 1,>3 in the first case and aboiit 10^0 
in the second e:cperi?&ent. This appears to eliminate Tiscesity of 
the liiediiua as a significant factor » 

Other factors studied in maryr experiments with monliey kidrsey 
cells in agitated fToiid susp-ension ciiltxTres iacltide the follo-s5^g? 

1) Doi&ling the concentration of 13 aBiiao acids of NCTC 109, argp 
cystines glu-Mg, histg iso, leu, lys^ methj phs^ thrj trypj tpf^ and 
•g-al, had no disceraable effects 

2) Jlsthocel •vriscosity grades tested j 10, l5s 25? aad iiOOO epsj sko^isd. 
no consistent discemable effect. The fsv? cases where sIoet prolifer^ 
ation did occm' ^sere x-jith 25 cps aad is.000 cps J'fethocel, 

3) The eoncantratlons of Bfethocel t-ested Xi'ere priscipally 0,06 and 

Co 12 percent of the lou viscosity grades, b-ot included in sosne ei^er-- 
ijssnts higher concsEtrations ^ to 1,6 pereeat of 10 cps., C-©i5.ee2itra'=> 
tioB of I&thocel had no apparent correlatioa with cell popislationo 
h) Cell oeacentratioas in the inoculyia aad ia fresh fluid at each 
fitiid change ^ere tested at SOOjOOO/mlj 1 milliorj/iiiL^, and 2 adllion/iHlo 
These t-?as no diseernable effect, 

5) The effect of tx'jo different glucose conceatrations in the fresh 
liiediiiiiig 100 sdlligrais percent (normal f^' Earlei's saline aad for 
mediuffi ICTC 109) and 26 iag« pet. 5, was ez-caiBiriSdo The hi^er cos2centra-= 
tion of gl-ucQse in the fresh ESdiiEa -mas accoiapaaied by an iacrsased 
rate of glucose ^ilisation h't/o not tj ass^^ increase la cell prolifsr^ 
at ion ratSo 

6) Horse serisii as supplesEeirt (20 pereesat) t«a,s less effective thaa 
t-jere Bfethecal st^pleiasntss the c^titis'es disiatep*ated withia a short 
interval of timsg tiaether in silic©ae°esated flasks or in uneoated 

flaSiiSa 

642 



^risl I©, li28 ih), Psa® 3 



t) Dst^i'saiEstiea Jmst b©fcap® each flisid «&©ag® ©f total prssteia 

©ad t©tal ffiuelslc acids «ia aliquot® g^j^lss ©f e®ll saspsssii^ 

tiss bei-«©€ia tfe® '?al'^s ifctaiasd @sid feis sselfii eoeat-So This 
GESTi'slatii^ si^t as ©spscted :^e^ ths static E©n"=pE'elif@2'ativ© , 
gatts^ ©f t^ e'sltiir®So 

c©lls ia i^plies-t© roller t^® flask® sss c^^ried ©at to dstii^te 

the ©ffsot 9f csi»taia ©-«;&!©? ^^^?iafel©go A 3°li ^sy Htdd dsesig® 
istsr?al ga'?® sj^h highSF pa>©llf®ratiras rat@s t&aa did th® 7=dagr 
iistervalo Ifetlioc©! of 50 op© "^se©§ity gp®^ «bs sli^tly esg?@ 
©ffectivs th£S5 \?&B t5 eps lfeth©e®l, Mstfes©©! at O.U perssat 
csmceatratijm ^«as sll^jtl^ ^i^ ®£i®ettvm thaa it ^ss® at O.O6 pgr^ 
cent ecseestffatiissa. ^^ hi^est ds'^a gpe^d^ 20C^ rph^ fsas ^&r« 
©ffsetitre t^a ^'j?© ©l&lses' 1(XK) r^ ®r 500 2^ draa sp©«dSo ^® 
isdieatisa tkst J&itliscisl ©f hi^©? -^isessitj gs?ads aad at hi^ep 
e&acsatjfaticms ^s sass^ ©ffsdti'w© ^®s cos^i^red t© ^sdt fte't&sg* 

A e©ES3q?^Ht ®:^l«^ates^ ©^©fi^aat yith ®^tie T=15 replicsat© 
csltv^® flasks i2rr©lf®d tbs ^ass ©f six differaat isdse@®itj p-sdss 
©f ^1^©e©l at s@v©ral diffi©p©2it 2?®lati^?@ly bi^ ©®ac®istrati«SEig 
fer ghastlier rfcrala ©f csllss h^©a ©pii^®lial strain mTQ 2$)A 
i^^ el^^ ^KJ gJillj. Eses® cells fead pr®rl©?^l^ b®@a gg'Sfe-a ia 
sli@aieally defined radi^ ^fC 109 is ste.tie flasks t&t s&m^Bl 
iKSB'^as csaly Ki^ coasider^Xe diffleulty. sj^ hse^ raceufely. be®a 
f©^ t© p?©lif€i?®ts at a ssdgrats rate ia th© sa^ asdiua st^spl©<= 
^istsd ^•ai l&'yasssl ia a^tatsd fltaid siss^sasioa cultures (E^ssjs 
ps'ejset Is28 (e) )<, ^all© tlis ©alls proliferated ©aly @l®5ljs all 
ts'eatssnts did ^fs^ ss^g isepeass ia ^11 aB^>©rs ^©r th® iaoctsliss 
psi^alati^ Istfeseel coac^aferatieas .tead kjo efffeet ^a prelifera^ 
tiea.. Of %h& siz 'Viscosity grades o£ Mstiic«®l tastsd^ 10^ 50^ 100^, 
UOOj ISOO aaS l|(XK) sps, the 100 eps grade gssm tha hi^st pE'olif®s'= 
ati®a ra.t9Sg bj a sigaifleant mss'gin. 

Sigaificsacs? Siscs some strains of smE^aliga calls csa L^e® b« 
©itiisr g£*3?sa ES'" at least- gssiattaissd In msMstwB . agitated fltiid stss- 
pssslea ©islt^QSs ©ell asSs-itim sttidies ia the Tiss^ GEltw© 
Ssetiss iEvm^ pr©J®at k2B (e)j Ifestl&ll Ii28 (d) ) isitls efe^sieally 
dis^ssd ^sdi\'ia ss?© saosr beisg ©rbsssded feess etatio etO.tts'^s e©ist®laiag 
3 t@ 15 ®lo fltiid ®aeh t© ®gita-b®d fliaid sTisp®ESioa ©xaltta'ss coatalis= 
100 si. t@ 400 slo n^d ©ash. C6^®i»ati's« sttsdles ©f aeraal @E^d 
s^li^a^it e©lls esa fc« aec©l@rat©d ®s it bseosssa i^ssibl© t© gr©^ 
ass^ ©six sts'alms ia cJasisicall^ defied g^di® ia agitated ealttspeso 



6^3 



NCI 
Serial Mo. 1^28 (b), Page k 
Project 2 



Proposed Com'se of Projects The use of methylcellulose as a 
protective agent is bsiag stiadied more in'beasivel^ with the 
selected cell strains used so far^ ia order to attain rapid 
proliferation and frequent harvesti2ig«'S*ci2lt\3risg eyeless this 
study mil also be erbendsd to other selected isgjortaat strains 
of siaiijEialian cells. Similar Ij potentially protect ire chesaically 
defined non^protein agents such as dextrsns and PfP (polyirinl° 
p^rolidone) Biay be investigated for their effect on the groxcfch 
of cells ia chesiically defined media ia agitated ei0.tures. 

The sBchaaism of the protective effect of Ifethocel s's^ple-' 
raent -onder these conditions is being explored. The extent to 
tSiich Ifethocel has a ntrbritive effect^ if aigry in addition to 
or coftplersentary with its protective effect^ needs elucidation. 

P-iirther plaanf-sg for futtar^e estensions and applications of 
this study is based on the following facts. 1) Present deep*- 
freesing methods for preserirlng cells include the ^ise of glycerol 
as a protectix^e agentj glyeerolj while it is a kao'sn chemical 
entityj affoa'ds only liEiitsd protection and is at best inadeqiiatei 
also^ seriM is present in caltiare flirld. To our knowledge, no 
long«=>t3rm tissue cultiires groim on chemically defined media haire 
been siiajected to fr-aese -drying sttsdiss. 2) Preservation of cells 
hj frees8«drying ^'ill certainly be tased to a greater degree in 
the near fatrn'-e bs cause it may reduce the possibility of cell 
Changs d-oring extended internals of tiaejand because it may save 
tisa and labor. 3) J'fethocel has been shcmsi in oisr laboratezy to 
protect cells of certain strains in agitated and in static caltiires, 
in the absence of ser^Sj allcfi-ring the ^ise of protsin=»freej cheEdcally 
defined insdiae Therefore it is recosaaended that the lise of Ifethscel 
instead of glycerol as a protectix^e agent d^j^^^ and _aft^_ ^'eese-^ 
drjdngs for calt'ures of cells which have been growi in protein^fres 
chssdcally defined msdiaj shoiiLd be studied by authorized iisyesti-" 
gators » 



6i|4 



NCI 
Serial Woo h2& (b). Page 5s 
Erojeet 2» 



Part B Eonorsj Ax-jardsj aad Publicatioass 

Publications other than abstracts froai this projects 

1) Bryantj J.C^j Schilling, EoL.j Earle^ ¥oRos aiid E^anSj ¥oJ»i 
I&.ssiv3 fltsid suspension c'olt'ures of certain tisstie cells. Ille 
E:!q>lorator3'" supplem.entatioii e^qseriments with cheniisally defined 
BiediuiB IGTG 109. J. lato Caxicer Inst. 23t 000°000j 1959^ (In 
Press). 



Paper I? of the sei-ies "The gro"Kth«poteK.tiating effect of chemicsllj 
defined mediura WySC 109 witli Ifethocel on strain 20?1 cells cultures 
in silicone ^coated flasks'^ is in preparation. 



snors and aBarda relating to this project i 



645 



mi 

Serial NOp h28 (c)- Project 1 

1. Laboratory of Biology 

2. Tissue Culture Section 

3. Bethesdaj tJaryland 



Individual Project Report 
Caleadar Year 1959 



Part A 

Project Ij Title; Studies of Ceil Nutrition in vitroo 

ao Cultivation of ffenkey ICidney Cells in protein-^ 
free medium NGTC 109 and their use in mrus 
research. 

bo Cultivation ©f feesh C3H mouse fibroblasts in 
eheniieally defined isiediiMi, 

c. Cultivation of a long term strain of human 
sicin epithelial cells in icediura HCTC 109j a 
chemically defined medium free of added pro=> 
tein= 

do Studies on si-ng>lifica.tion of chemically defined 
protein°f^ee medium ?JCTC 109. 

Principal Investigators VoJ. Evans » 

Other Imrestigatorss J. Co Bryant j W.R.Earle, W.T.McQuilkinj 

HoA. Kerr J G. A o Parker » 

Cooperating Units s Lilly Research Laboratories! Tissue Bank^ 

Naval Sfedical Schooli Dr„ Eo Sheltonj 
Cellular Biology Section, 

Man Ye^?s? (calendar yesr 1959) Patient Days (calendar year 

Totals 1959) 

Professionals 
Other s 

Project description? a) GultivatijHi ©f monkey kidney II in msdiina 
NCTC 109 a protein^free chemically defined 
medium. 



Part B included Yes x No 



646 



NCI 
Serial l?o, U28 (c)s Page 2 
1 



Objectives To determis^ if It is possible to grow monkey kidney 
cells in long term cultivation in protein^&ee chemically defined 
rnediunij, 30 that such strains of cells <san be used as laberatdry 
tools and model cell types for Tims stiadies and icetabolism studies o 

I-fethod eiqjloyed? Strala W^ = Lilly Research Laboratory = was im- 
mediately trans fesrred (after recei-vlng thsm in this laboratory) 
from a raeditJin containing IfediiM (Morgan) 199 and 1% servsm to Ifedima 
IJCTC 109, Fluid change schedtxles were varied to obtain desired popu=' 
lations and cult-ure beha'^ietr, 

fejor findings? Tlie cells have ultimately been satisfactorilj'' 
cult-i3red in all sises and types of ctilture flasks used in the labor ° 
atoryo Fluid resicrral schedules were xforked out for optimum use of 
cultures for ¥irus assay studies particularly £ar polio virus., Rates 
of gro^ith were determined on the micr ocinemat ©graph «, A report in 
Moveriber issue of Ax^rican Journal of Hygiene gi-^es details of tittij^" 
of polio virus grown in the cells as i^jell as details of cultivation 
and behavior o A clone strain on this medium has also been obtained 
and is ^asll established in T=>60 cultures » 




i!®(Siia7'''°ASea^°^rEii™vaiue' ilia Virus' studies' since poU© viri^ 
Type III groHs in it at as least as high titer as this virus will 
grow in fresh monkey cells. It is expected^ as the inforn^tion 
becomes mcsre general by way of the published papers the strain 
mil assume great significance for those investigatcs's desiring a 
strain of cells free of "wild" viruses.. The fact that serum is not 
required may make it additionally valuable for studies xifeere serum 
presence may interfere in any way with virus cultivation as it irjeil 
might in measles and mraigjs virus cultivation for vaccines » 

Proposed courses Continuous maintenance of s'train is proposed with 
the ejqjectation of making it available to other laboratories for 
virus stud5.es o It is in use for studies in the methodology of both 
cultivation ©f large agitated fluid suspension cultures and (in 
collaborative studies) in possible cisltivation of viruses from human 
tumors o 



Project description? b) Cultivation ©f fresh C^ mouse fibroblasts 
in medium KTC 109s a proteis-'f^ee diemic&lly defined mediiau. 

Objectives 1) To establi^ new strains of CJH mouse fibroblasts directly 
in chemically defined aKdiusn s© that an atterr^t can be made to deter-° 
mine blether cs" not cell transforations to tmiiffi-^producing cells in 
vivo are influenced by the cos^josition of the mediumo 2) To induce 
^rSs formations in vitro in such calls at the appropriate timSo 



647 



MCI 
Serial Ko, ii.28 (c) Page 3 
Project 1 



I-lsthods en^lojfBd; Several series C;^I mouse fibroblasts ha^-e been 
planted in iritro by one or the other of two riiethodSo 1) Pooled 
minced eSryomc meuse cells ©f ij^ying types have been usedo 2) 
Explsnts of small pieces of tissise from the epigastric fat pad ha^e 
been retrieved Broai dotsble millipore filter chanibers and planted 
in vitro imder finely perforated cellophane in JKJTC 109 mediim free 
oT serim., Control ciilt-ures haire been established in meditaa KCTC 109 
with the addition ©f 10$ horse serian by Tolinne. 

Results s One "strain" of cells i>om the pooled enfcryos has been 
in vitro 9 moaths in the coit^letely defined medi-umo The cell type 
appears to be predominately fibroblasts.. Grotsth is extremely 
slot'sr Qonparsd x-rlth the control strain td-th the serum. To date no 
daiightsr generations can be established ii'om ito Nevertheless ^ 
it is alive and groidng at least in amoisnts satisfactory for explor°« 
atory micro injection Herk» Each new series has been maintained 
longer than its ' predecessors indicating definite progress toward the 
desired objectii^eo The escploratory studies td.th tissixe adapted in 
vivo in the do'oble miliJ-pore filters prior to retrieval and implants^ 
?IS in v itro in the chemically defined medium saay have mere promise 
of eoS'inuot's rapid cm.tivation than that from pooled eai>ryoaic tissue, 
These cultm'es morphologically appear like all fibroblasts grm-m in 
v^ro^ asd sesm^ at least after a shosrt period, to be ''holding their 
oSS^in the chsraieally defined mediTHa^ 

Significances Gontinuad success appears to be far mm-e likely now 
in c-altivation ©f &'esh tissue in chemically defined mediratti attain- 
ment of the ultimate objective for the etiltivatioa is definitely hope- 
ful » 

Proposed Cotirse of Projects 1) To contin-oe with the strain avails 
able I 2) To develop others for continuation of and to iii^rofe mediia-a 
^id methodolo^ of cultivations to pursue as rapidly as possible xd.th 
thera the cbvioizs objectives of the cultivation of ftesh tissue in 
chemically defined mediutua (See section ii.28 (c), project 2) for essplan^ 
s:tatj and preliminsry restO-ts on the general objective «) 

Project descriptions c) Cultivation of a long term strain of humaji 
skin epithelial cell^ in medium KTG 109» 

Objectives To determine if it is possible to p'ow human skin epithel- 
ial cells in the proteia-^free chemically defined medium M5TC 109 in 
static cultm'es. 



S. ei^loyeds Cells of the clone strain KCTC 2Bhh of htmrnn skin 
carried in KCTG 109 ^Ith 1(^ human serum ^ere ^adually depleted ©f 
serum. The first reduction ims to $% serum and then to 0<,J2$%c 
Partial fluid rsnetsals mave also used initially in this adaptation to 
retain population. Since the cells beoama &ee floating on" this 
regime centrifugation pries* to flitid ress^jal i^s required ^ Periodic 
return to the small ■s-oluiae of serum i-jas alternated x-rf.th cultivation 
on the coHiplete ICTC 109 mth no seriaa until finally cultiratioa en 

648 



SCI 
Serial No. 1^28 (c) Page it 
pyoiject 1 



sei'um free nsdium with no periodic returns to serum containing 
medium was possible ^ 

Restalts! A long-term strain of hraan skin epithelial cells has 
be6n adapted to routine CTslti'S'ation in a chesically defined laediiMi 
free of added proteina The groTsth rate is relatively good. 

Significances This is the first long-term strain of hiaiaan cells 
ever to be groi^n roistinely and continuously in a proteia^f^ee 
eheraicallj defined medi^M. The potential of these cells for manj 
aspects of virological and cancer research, is considerable » 

Successful epithelial cell transplantation for either a teit^ioras^ 
dresslngj or a longer tsria one, or as a stimulant to i^e recipients 
owi cells has a clinical be^'iag on reparative swcgerj in gsx^ral 
and da biirn problems in particular ia military jusdiciaeo 

Putas'0 CoisrsQS To pm'sue at a minor expleratory level the potential 
gr0i?th of these skin cells in rnass arasunts in fluid suspension 
cultures in eheiaicallj defined iBsdiumj (See Series li28(c), project 
III, Cheaieally defined sodium) to laake the breadth of our know^ 
ledge on the cultivation of this strain of cells available t© those 
^o fflight propose to use the cells as an integument or integument 
substitute and also to th© virologists for their use particularly 
■d-th respect to possible cultivation of possible human tunor viruses o 

Project description? d) Studies on siH?)lificaiiion of medium. KCTG 
109 o (See Dr.. Sanford^s report also.) 

Objectix''es To sisiplify coB?)ositi©n of medium to the maxiimam with- 
out sacrifice of quality or quantity of cells, 

Jfetheds ei^leyeds Replicate cultures, ©f strain K3TC 20?1 (strain L) 
mouse fibrobiasts fello^d by eauiueratlen of nuclei ©f each culture 
in each set to detersains Idae effect of proliferation of individual 
or combinations of cos^jsnents. Long^tersa cultivation of cells ©n 
media that have bees designed as results of findings i'rom e^Eplora- 
tory studies by replicate culture procedures. 

Results s m>d±mi ^TG 11? has been formulatGd and put to prolonged 
tssto 15iis B^diusi W^TG 11? has been derived from raediiM 109 by 
oaittiag coQasjmss, sodium glucuronate, deojfygua/iiosine, d%,02y° 
adsnosins, and 5=s©thylcgrbosiae, and "essential'^ unsstixrated fattv 
acids. 

Significances Bfedium M3TC 11? is sis^jler and s^Jsstaiitially chea^ss^ 
^o prepare tliaa msdium KGTC 109. Further it has bean demonstrated" 
that apparently for both excellent quantity and quality of cells 
the ffnly nucleic acid derivatives required for strain L are th-«isii" 
dxne and dee:icycytidiseo It has alse been revealed that adenosine 
•criphospha-bs xs not rsqtdred by these cells? this suggests that" they 
m^st xmv3 .SS2B rascIiaaisHi as yst midiscwered £m- pL-oduoiiig it. 

649 



NCI 
Serial NOo hZ8 (c) Page S 
Project 1 



Futiire, course of projects To cois^jlete exploiratory studied on 
the necessity fcs' the so=?,called eoii>iKation of rediacing st5b=> 
stances, (glutathione, cysteine and ascorbic acid) in inediiiia 
KGTC 109 o To collaborate mth Br. Sanford (as the result of her 
findings on the Titamn studies) on redesigning a conpletely 
siB?)lified chemically defined nsdium for strain 2071 (strain L ) 
cells « To use findings on, strain 2071 moisse fibroblasts (as proto- 
type cell) to e'^olvs better rasdia for fresh tissue and for sell 
strains so far not grsr^^n in ehexd-cally defined laedium. To use 
siniplifiad i^ditM In the first interssdiary metabolism studies 
done xd.th cell strains grown in -Fitro in ehevaically defined ri^idiraa 
free of the interfsrsnoa from and possible influence of added prop, 
teise FixTthermoTSs for esaaiplej eaperiinents are now being planned 
to st'tsiy the possible adaptation to protein^free chemically defined 
rnsdium K€TC 109 of certain cell strains which ha'^e been preirioiisly 
intractable to such rusdimuo Such a cell strain is liver clone 
iCI^ 1^69<, The siipplements to be used in an atten^it to effect 
adaptatioxi include these groups s 1) ffethocel and spermaej 2) 
pyriwa'be, oxalacetate snd alpha«»keto glutarate, and 3) ser-um as a 
controlo SperiBine has been found by Ifeger (IJatiare 103? 182? j 1959) 
sjid others to influence cell perriBability of certain bacteria and 
laight pro¥8 to ixifluence similarly maismalian cells » Special nu-^- 
tritional needs of the a.ntractable strains might possibly be m.et 
by the three key is3tab©lic intermediates. 



650 



NCI 

Serial Ho. k28 (c)^ Page 6 
1 



P^tB HonoTSs Afe'ordSs sad Publications. 
pTiblicatioas other thsa abstracts f^om. this project? 

1) Evans^ ?.Jo^ Fioramontis M.C.j and Earle, W.R.s 
Studies of nistrient media foa:' tisstse cells. V. 
Tb.e effect ©f the coenzyrsses of B^diuni ICTG 109 on 
the prolifsi-ation of SJGTG strain 2071 snd JiGTC 
strain 2937. Aia. J. Hjngos ?0s 28°33s 19S9o 

2) B^ranss Y.J., Kerr^ H.A.j McQuilkiiij W.T.j Hull, H«.A.s 
and Earle J ¥.R,§ GrsKth ^ yitro of a long-tersa strain 
of msnkej kidney cells in rae'diism W^TC 109 ;tree of aagr 
added protsia. Am, J. Hjg.j 71s 19$9. 

■s 

/ 

Honors and a^ssrds relating to this projects 

Centiaued as 'w^mber of General and Celltalar Biology Review 



Continusd as Coas-o0.t®nt to Ns^ Tisstje Bsnk, U.S. Hs^al 
Dl^dical Schools Mational Ka'^al Ifedical Center, (Ewans) 

V 

By invitation presented paper at Atlantic City Aprils 19^9 

in tlie Tissue Ctilttare Association ^^B^osiisia on the &s?olm«» 
tion of Cell Population, Title ©f papers Large scale long 
term cultivation of cells in chesically defined FiSditJis, (Etssis) 

Presented paper by isredtatioa before C©FM.ttee on Cell Culture 
in a conference sponsored by the Institute of Laboratory Animal 
Resotsrces and the Maticaal Researda Council held at Ifee Ifetioaal 
Acadea^ of Sciences Jaaiaary, 1959, Sisbject of paper? Hu.tritional 
Standards f&k^ Cells in Tissue G^tisj^. (E^aas) 

Participated hj iarltation in seetisg at Wilson Halls ^7 "^9^9 

on Lihsracteriaations Preservation and S^ply of Tisstae Cialture 
Cell Liness sponsored by Viruses and Cancer Study Section 
Panel Research Grants Branch, latienal Cancer Institute. (Srans) 



651 



HCI 
Serial No. Ii28 (e) - project 2 
lo Laboratory of Biologj 
2o Tissue Cixlture Section 
3o Bethesda^ Iferylasid 



Individual Project Repeat 
Caleadar Year 195*9 



Pas^t A 

Project 2 « Titles Cell transforiGations in vl^o. 

a) Garciaegeassis potential of tissue cultm'8 rasdia^ 

b) S"feudiGs on fresh C3H mouse tissxie ia chemically 
defined ii-sdia both ^th &nd mthout the addition 
of sojT^ horse seranio 

e) StTidies ©a fresh C^ mouse tissue ia chendcallj de- 
fined medima for carcinogenesis <> 

Friacipal Swestigatorss ¥o J<. Evans aiid 6»A. Parker, 

Otter Iis?sstigatorss ¥. R. Earle^ and Ann Palko. 



Cecperating IMtss lone 



Ifen years « Patient dajs (Caleisd^" 

Total? yeas? 1959) 



Others 

Project descrlptisa? a) C^?einogesjesis potential of tissi^ cijltm^ 



Objectives? To iswestigate the possibility of ttM©r productioa ttom 
isjactlon of 1) the standard chick eKbs:=yo extract ^lorse sermi ffisdiiamo 
2) Siagls Kjssiisers o£ this ndxtiirej 3) IFafiltered chick esferyo esferacto 
k) Chesdeally defined jngditaa ICTC 109$ 5) Used filtered sT^rssatast 
fltaid froja sioiase liirsr cells is -^ritroo 



Part B inclnjded 

652 



S@cti<m EfOo iiS6 (c}^ Fs^s 2 
Project 2 



I^theds oa|)lQ:F®ds All %^ aibw® isa@di& i^®cted iato se^ral 

siaissal® ar® beiag k®|>i ess® j@ar md©@s i^ihym&ktj rap@?v®B@s» 

Resets s A sajcritj ©r ssdimls hs^ r«®ch®d ths t«s-Msal p®ri@d 
of ®2^«riis9ss^l plsfflo dross eassdsffitica re^&ls sio class* c^ 
i^iet^sst of 33^ p^rtio^al^ ^azlablAo Histolosicsl preparation 
haiT® b®@ia s>®t^^s3ed ®ssi a largo i»Bi)®r of ania^So ObservatliKBa 
ha.'?8 b@ea to dats o£i tha Ssiss sstd i^i^rld etrainso F!r«qtesQt an^ialiss 
hsve b®6a rs'^aled isj t&« l^-^jSasitle tlssvsSf kid^^js ll-mv md ®ali<° 
^8Tf gX&sdSo S®^®3'al ea^s of «mlers@d spldon aod l^i^ ssodss hs^M 
bs©ss ^@no H®®^ iafHts^tioa of l^i^o^ic colls h&3 &QciscT®d of 
saliwsy gl^jl®^ lEidaoy aad li^rar o Th.® flxidiiegs eo f ar lase® i^t 
to ba flml]^ eorrolated to flsid o^ if aa^ meiplsstic tiias^e i@ 
&3B0G±&t®& f9i^ aa^ epaclfle tfp® of fluid li^®otis@s isito as:^ of 
1^® @ts>ai!is of aai^,lao 

SigEifiomc@s It is lK^®d timt tlio datsi obtslmd S^oia i»^®ctii!g 
the 3 sitraina of ^2%iml3 ^tja ti&® aferosiBatiosud laadia aad thoir 
ooa^oaoss&s will mppl^ basd^lino knowlsdge to tho phoB«Bsao» of 
e@ll ts>a^fQri6atioa in yitro aad/ or i,miispi0)mB±a in Im^ t®r^ 
tiss^ evltisroo Eeeotiil^^of tbis preiblea «ill aid ia asss^gss'isii 
1^9 q>s@sti@s of liiy e@lls tr8&fifein& aftar e(»K&i^mo@@ c^tiv£ti®st isi 
yitro . It Hill &lso @sto lerag tora ties^a o^ti^s straisss of add^l©s<^ 
al sigaiflcance in indasferial a|jplieati®n as r@lat@d to cb@^t^@ra°^ 
po^lic tastissg aM oti&er fioMs. 



Prcpossd eojsrss of psfojsets ?o cmtianfii tltis atodj^ as tarigijially 
plaaaad. Sao^ld it b« ascortainsd tliat th® is»dia or ai^ of tii@ir 
eoa^oioonts eosstaia agesste timt ar@ ospablo of prod^iag pa^olosi<=> 

eal aiid/ or s^cplastie ehas^s ^i^n i^®et@d iato m^o^n tsslmlSs 
tfeoffi a fortile i^®a of ftet«p® gttsdgr is ©p«a Jsr ia« idaatificstien 
fflBd isolation of ^® ®gsssts asad fa? ftetbor studios on toe cosfcrol 
and oliMnatlQSi of sissh t^iangos. 

Project d@scripti@@3 b) S&ndios on fls'osh Cjg lao^so tUem «alti2r@d 
in ohs^cally dsilmd ^dicaa ai^pls^afead vith 



Ote4®etif®« T© dffltorsdae tSa® tia® th® trasssfoTBatioB® occw ±si 
flpo«&3y ieolatod OKijryoaic CJS bovss cells tdian tfeoy ar« cttlt«r®d 
^ S^i o» ^iisaically defined sedi^ sisppL&aoated ^th horso 



l99^od @si;loy®d8 ?iss^ &>oa 18«1^ day old C J! eeinpyos v&s mincod 
and onltwod in seadim mfC 109 -^ IC^ horso sar«iao Rotstino establish^ 
iseat ©f tm ®©riaa of e^twos (3^% jkO^) by t*i® usml l^m^&tmj 

pg'ocedw®® ^ag carried osto 



653 



ucx 

Ss2>lal SJ©o 1|28 Cc)i, T-3^Q 



fetraeesXaf aad is^ya^s©^!^' iajeeticas ®f "i^s tissra® 'm^ sad® 
accs^dissg to ■ys® getedjale g&®sa i^ tli@ tafel® ia t&® results @®etic^c 

Rssss^tsi Tm £®llmi^ %abl& tm. pag© 6 giif®8 tli® results t® <tet© 

& t&i:r<5 gsriosa still %& h® csrs'isd ©^^^ t© d®fias t© ® sharpss* 
d©gre© t&® ©splGTS-ts^y data so fte elyssiissd in tfe® fls'st t'^© @^l^?a= 

p5id.©Lis©s ties^ of p:'iss2y ig^^-tasceo Xa additi^a t® tfe© data ia tise 
%^1© ss-2^ si^ls i^s®2^ati^ial data ^i t!s© fegfes^lcs" ©f l^laatad 
tlssi^ la Mie ©J® k&B fe^sa eollgctsdo At tla® sssssatj^ ^®y fes?@ iittl® 
eritical sigBlfleESG© feiit t&sj ^^j b® @sp@ct®d t@ ©ss^ is^jm^^ass® 
s-§ subseqtsesfe eoj'.ilg'M^ data sr© ©@ll®st©di8 I^lsists ©f tiess® 3? 
^id 51 ^ays ia ^ts?o reissued ^ ist© ©ffiall dier©^ ^ss®@s a^ tfe© 

@sd ^S days ^ ''^.^^'E ■®P<sb i^®Qt>lm i^i^^i. th© ii?i@ sM s^s©q®s^&ly 
ites £'®s«^b®do STss^ 121 daji ia g.tff® ^^a Issjsctsd @pf©sd ia tfe@ 
©y® and afisoted ife® ptspilo Tiss^i^'lW dagrs ia yitgQ b«®a®s lai^l^ 

csr^^® S't^t^s'Qd, f igs^ l4i days is ^itre ^5^1 i^ecti^i pf®d^€©d 

cells ia ^© t^sai^s ©r© ai^s^afes fmsifeg-a cells asi ©t?©id ^th ®ssg»- 
jsitssss aM dsfiaits iaillts-atieei Iji ■^@ a®r^i tissii© ^c^So 



654 



NCI 
Serial HOo U28 (c), F@s@ 
project 2 



cm GILLS IS inmo 





s».iss n 


SERIES II 


Days 
ia vitar© 








Ttsore 


1?G 


fmissrs 


fC 






QroiT^ 




6?©-t5t& 






AcoeXersti^i 




Aeceleratica 


3? 










51 






=. 




?3 






o 




93 










103 








** 


10? 






= 




121 










135 




S'-g- 






m 






^ 




1U8 






♦ 




166 






4- 




m 


♦ 






- 


198 






4- 




1P9 


4- 








23^ 


* 








262 


* j 






1 



^•Tfeis dat^a Is based on 
accsleratedo 



jtjdgessat of ^ea eultisp© greijfe ^©ppsa^ss 



655 



NCI 
S^ial ij28 (c)j Psg® 5 



SigsiflGsiie®? &©6@ ©splsg-atery data ^®^ t&at @®lls e^lfcwed 
is' 2^g£ Ms?® tfe© .. eap^ility (^ the ijsjsctisa procedijr© ussd) 

affesr ©splaatatiaa m& mTl®@ 22 MiSiia k'^ m^ike^ In esries 

Istas^al sad iss sss>i@s II wi.tS&ia © 26 ds^ p®s"i©dc ' ^ie is 
■ resasr'cabl^ closs esrobsrati^ ©f tli® t^© a^ s>sgg@gr&s tisat pes— 

th& Sx^d to 6tfe ssnya of 1^@ir US® ia gitge ia ea-fisr t© d8tay<= 

islai i^ til© 28 ©ad 26 daj iat®nral S8i®ri«"ftetli®? sisffressd. 
It Ko€ld g©©a l^at ■ ttls isk£(^m&tl®n is s^cess^y ia erdsr t© 
knoM 1&© pss-iod of tii^ dtis'lag ^ieh isfestigatlssjs ^y bs sad@ 

^©s© data suggest sasgr l©adSo First it.istll b@ ^ssfwsd t^t 
acssleE's.tsd grcKrfek of tfe@ eislttis?®® eeeisrs prim* t© tfe® tlmB 
ia t^ Hi"® |f til© cvltw^ ^®m t&© e®ll8 id-ll ©a iB^®«ti^ 

kaoid^ afes©list©3^ ^-^etlisr tMs ha® eaasal sigalficifflss « is 
2a©2^2j ps^allsl @eeisT®M®o I£ t&sr® is tlj®' o^ept ^at tli@ 
tr^fflsfes'.^.ti^ ■ is asssxsiat®d ^-^ eestaaiastiesi fey "^a ps^ic^lat® 
£g®£^t« at a lo^j- titer tla^js 1&® istejwal ©f tis® ©f tfe® c@lls |£ 
vitgg^ si^t fe@ s^iierded as t&® p®ri^ rsq^Sred t© rsis© mk@ 
ti^SSr. 6uX"fiei@JBt2^/to ^©Id ®ffis^ @4^^ to p6?od®s® tSa® t^^arso 
If til® ts'aasfei'ffiatie® is essasider^d as resulting fee® hss-isag 
grot® fe© calls ^ad©2> aaasrcfeic coaditi^ie «ith coBfiotsitssr^ alte?"^ 
atlsjas ia ©©11 ^tfbslis® l^a t^s p^^ied ®i grotstSs ia gltro is 
sig25i:d.cssfe as t^® ^ ia mleli suoh ® trassitioa laasTa^Fplac® o 
If it is p®ssibl@ tliat t&© c^aeitj @f th® sts-aiss of ©alls t© 
pr^Tsee tmim^s ssi isj®otisa is 3?©le,t®d t© a sslsetiea ©f esllis 
is the pepislati^ tk®s co2asid®r©ti@ss ^@uLd h@ gl-mn %& '^^Tja, . 
esrlj closing stwIS^s sad to oesiSj's^tiea ®f th@ data slr©®dj ' 

t±m ©f tr^isf^sati^a is q^t© ms^S'&Wg as s'ewsaXed ia thsg® 
stsiissj. sosss cessidsratisa jd-^t Is® s±v®n to tli© ^yit ©f a 
liiPlt®d 6ti3^ ©f a f®-3? ssaaths oa tijs <^?Biffld.os ot diS'^ssi^ 
pat-tss'iis ia ^@ e©llss pas^icBlayly if elsaaia^ studies ®re eas?yi®d 
oBt ^id csiilirs^tion &S lai^sad straia. data is aibtai^ted gs cle@@d 
gtrsias» 

^op^sad cs^»p© of gt^i^s ?@ 8K»fli?^ reatalt© to date asal pm-s^ 
sms bijp0t!i®g>>.@ ©^sidss^ad ^ad®r si^fiesRcso" 

Pr©^®et dsseriptisas e) Studies ^i ^®^ G^ ^ms© tisstjs ia 
efeeadcall^ d®fia®d ^dii's fas' ea!Peis®f®at®is. 

C^jectit'esj 'f© is«^®stiga-fe® 1) Mli@tSi®? ^ ast C^ ssm®© fife@« 
blasts gr©ra 1® da®^©ally dsflasd radii® 1GS3 109 'sitii s© bla^fes 
^&m. 'te cfc^^-sallj d®fi?^.d ^^^b WTG 109 laitk si© added ®©sn^ 
will pr©d^@ t«isrs ^©n ijsjecti^ is&© 1^© sts'aia ©f ^igla as^ 
t) if so affesr fe©^ loag a etsltitrati©!! tiss ia ^tr©<, 



656 



W7. 
Ssrial Mo. hZB Ce)^ P®g® 6 
PP©5©ct 2 



^t&ods @^loj®d§ fissee llfsss lg»l6 day ©Id G^ ©^s^os isas 



of tij^. 

djs^eally dBfimd ^sdi^So Each mw ^^ ha® b@®a isereasir^lj 
batter groidsjg ttess its pg>®d©c®ss@s'= a® e«s=r®at ^s® Mas bs@a 
isa ^it?© e^sr 9^^ths» Th©=e^ Siardlj t@.fe@ co2©id©r®d as a strsia 
of ^Hs it is possible ^&&t it i^ill ©^.tlaij® t© g^m Bsid -Bltisatsgly 
bse©g32 €®©o %@ is^lsats f^s® cells of tfeos® i^^ffal sss'i®© Mf® 
b®aa mafe ©r^sr t-se calls Ma^Q fe®©ia ^ ^its?© 26^ 37s Si, 1^1 sEd 

tlis e@ll^ iB all mssss ee^iss k©s l3@®i slesj, 

Srifaifisgmess ^®^© is ps-oaiss l^^.tfe® last ssFies'^d fe^® f^ 
f^«2*s m^l®® t&at a st^*^ (siiail^ %& tJsat ^s® described is psrt 
b)^ saf feseag^dsd oisfe TEd.tfe cells ©sps^d s§alj t® aisfei®2£& fe^i^ 
a e@^2^%®3^- ^^aissll^ ds^^d o©^®@iti©a. It ^s is possifei® 
^'©a'trsr s^t^©l ®aa fee ®btaiE@d ©f s^t^@ c^Kiiti®as as ^ssj ^y 

r®^©al g^ iafgs'isa.ti^a sa the r®l© ®f i^die ia ta?a2ssf^'^fei^» 



FT^^eesd eei^'gas' F\^t;li©s' ©^ i^lsts&s fip®a g^s?© e®?!®© p*©®!^ 
fas' a X^^r p^.'^lod ©f ^©m tlssBs is cSa^Meally d©as®<i if^di^t 

F9ll^?i^ ©^ g^ ©f t^ e^iceptg adraiae@d la part- b ®f ■feMs 



657 



lo Laborates'j of Biolegy 
2= Tiss\^ Ciiltwre ^feetion 
3c Betlissda, Maryl^ad 



Indi^dual Prejsct Report 
Calead^p Tear 1959 



Part A 



Project 3j Title g Collabce'atioas ia Tlss^ Gultigp© ^tli 
otiisT org^oisatic^So 

a) Collabor&ti^i s^vdy ea steroid eaeji^ 
activity of stabllissd taottsa, husssi 
li^«r and othsr e®ll etraias ^ ^itg©o 

b) Project description b=> Collsbofrativ® 
sttxdiss OS) deterraisatiea &S ?@ latins 
antiganicitj of Ic^ag t®ria strairas of 
hu3iaa skis cells sj^ Se®^ skisi csllso 

c) QroK^ of his^ai skia cells' ia classically 
defiasd issdi^a im rspidly a§itatad fluid 

SQ9p6S2SiC^ C^ttirfiSo 

d) Studies ea th@ possible iss& of KCK 1C9 
AS ffiffl ist£>s9'@is@ie3 f®edij3g solisfeleso 



Prisseipal Izs^sstigatcs's ?» Jo E^raaso 

Other Lofrestigatori? Jc Co Byjaat^ E<, Lo S«^ailliEgp ¥oHc 

Sas"l©„ 

Ceoperatiag Ifcitss a) 0, Toskiasj imi©,, 

Laber&ts^i®® of li®cla©El.istrj ®ad 

^tabolis®, 

b) Mclo Fisk^ 

c) Ks.-^ Tisst^ Basika 
Paul Bakksso 

d) Bx-o Jo ¥©lst@Ea©ls®, 
Veteran© lospit&l^ 



Part B iaclttded X®s Wo X 

658 



Serial No. h2Q (c). Page 2 

Project 3 



¥s.ii jQBTS (caleridar je-^v 1959) Patient Daj=-s (calendar 

Totals jeax 1959) 

Pro re sslonal ; 
Other J 



Project description? a) Steroid en^insB activity of stabilised 
of siouss fibroblasts, mouse liver cellsj, humaii Irvrer cellss 
human skin epitheliumj Hionlssy kidney cells = 

Methods employedt Hsai?y birb prists T-60 cnltiires of stabilased 
cell strains of rasixse fibroblasts ;,■ mouse liver cellsj hiauan 
livar cellSj h-usaa skin epithelium, have been prepared bj om" 
methods of eultifating T"=60 ciiltiires for BTo Toirikins for his 
analysis o (Sae his report for techm.qi?.es txsed)^ 

Resiiltsi The rssuj-ts are tabulated beloHo (See page 3) 



659 







* 'S- O -*■ * * 8 



o o o o o o 



o o o o o o -^ 



O O O O O O ■ 4- 



O O O O O O 8 



o o o O O O 



o o o o o o o 



I 






if|r|iip 



« 



^ ^'^ sfr fc* o 

lllliti 



u 



g 



f 






II 

r 



660 



NCI 

3©?i&l Noo U28 Cc)s Pag® 



Sigsiflcaaces Thes® esples'ator^ data cellseted a?® of d©finit® 
val^ ia S5^9 acG^?atsly clsssii^iag eell straiaso In additiesig 
sffida.st^siiss as th®s® da£±m ^^® pr©eis@ly ^iich e®lls ia th« 
mi^sd c©ll pep^latioa in as «£rgaa |£ viT© ^ s^:?;poa©ible feaf 
smllar sctlTltj la tfe© ^ol© ©rgea (^"^sai» slice.. lafts'aa* 
tioB ±n tills §f @s is OS® of th.® aost isrgoiit ftstSaiaeatal ssssds in 
•th@ adTsaesseat ©f tJs© practical applicability of t&a loBg^term 
tisss© ctsltura ta c^^cey s-sssg^ch «ad ia other fieldso 

Ps'crposed CQt&rss ©f projscti CoBtiauatiosi of thia geasrsl typ® 
of cdllafe&rsitica -^i@n apppaprist® colls^os'iitiv® effcrts can ba 

srrsmgsd» 

Project d©sc7iptioiis b) Collsbor&ti'^ studies est deteroiBatioi^ 
of relati-fe asiti§€nicit^ of l<H2g %®r& sttsdsm of huaaa akisi s^ 
fr®sh skia c@lls» 

Objseti'?®? To ds'tersii^ if speoies aatigenieitsr is alt«r«d affeer 
proloaaged s^tivatioa ^ -fl^o at a fapid rate of prolifsratloa 
in ehssdc&ll^ deflmd ^dlmSo^ wit& added serim a2»i ijs chsid.- 
eallj defied s^dim isitfeo^ ®e^ ssriSBo If it is altersd fey 
smch cultisr@ condition^ husmi viruses prope^ated ^thin '^;e cells 
aigSit b® ®sp®et to prodtsca Tir&l tesors in fflice cs* affect e3ip®ri° 
matal aai^lB m^@ readily tSsaa virus Srom origieal tumor tissue 



^•^©ds ©ifsloysdg Ssf^eral grass of calls ^e cult«s«d acced- 
ing to lesg ter® Isrgs seals tise^ cultws pyocsdures of the 
laborstesy, TImj mis^ tfesn injected isit© Mce aj^ various ias^©l= 
ical test© wsir® r-m by D?. Fiako (Sse Ssrial Noo l4l5)o 

Be stats 8 itoleg-atj^y s^sults sJas^d tliat antigeaicity of tSses® 
cells £pp-.srs to h®v® beea redisced fey -^e mt^ds of etsltivati^ 
in g'i;^o ° 

Sipiifieaaoe 8 To dat© n© h^ssa -^insses have beeia lanaqtiiToc^ly 
isolated &om hums t-ao^So lafcE-aatioa ik>oa ^lis straSy ^y 
aid ia ^klHg tijsse 1«^ taria altered cell straias eigaiflGaat 
tools fe^ h->3mn tnms^ r±s\^s ressarcfeo By tfeeir use t^mjrs of 
hussa ©eigir. si^t ths© be sttisdisd iM e^peri^atal aaisalso 

Proposed cows® 8 Det&ils indicated ia D2°o Flak 8 s mp^t (Ssrial 
lOo 1^15) o Altss'atioas ia both the cells <^ad ^eeipieats will be 
attested to ac&ieve tfa® ©b^ectiWo 



661 



Project descr-iptioag c) Gro-w-th of human skiB cells in daeiaieally 
defined SReditari in rapidly"- agitated fltaid staspension c^ti^eso 

Objectives To define and control factors inflisencing growth of 
hraian skin epithelial cells in agitated fl-oid si^psasion cxiLt«res<, 
SsEphasis has been placed on adaptation of this strain ©f cells to 
ciilti-^ation in chendcally defined a^dium ICTC 109 -sd-th n© added 
serrai for the potential in Tirological researchj htamaa timor research 
and reparative surgery, 

Methods en^loysd? These bascially ha-^e been previously described 
(See Project 2^ li28 (b). Modifications sre thought, ho^'ever, with 
this strain, to be responsible for soi?^ of the success of greatho 
Small 250 inle shaker flasks are lined with Dow silicone iB% con<= 
centration in bensene and baked at 250°C for 3 hours) snd sre 
inoculated with 25,000^000 cells in 30 nil, of NCTC 109 containing 
liOOO GPS nsethyleellulose at Ooh5% concentration. The cultures xfere 
continuously gassed with $% CO2 in air. Cultures were eentrifuged 
in the culture flask prior to fluid renewals and 80^ renewals were 
madeo Populations were determined fttm Mclei counts of aliquotso 
As soon as a culture reached maximum capacity of fluid allowable 
in the shaker flask culture they are transferred to increasingly 
l&rger sise shaker flasks that can still be centrifuged, the largest 
being a 500 mlo flask. 

Results? Cxiltures gron as single cells and snsall cell aggregates « 
In one culture an inoculum population of 25 irdllion cells, under 
every other day fluid vqub^&Is, has increased to Sh2 million cells 
within a reasonable interval of 31 days. Cultures £p©ia the original 
culttere so established have been maintained fcs* 3? aad U3 days and 
are still under cultivation., Harvesting far dau^.ter cultures is 
feasible, Ta@ results of using the 0,li5^ liOOO cpg concentration of 
Jfethocel have been encouraging t^ to nowj although there fears been 
indications in the appearance of the nsedluta that this viscosity 
may be to© heavy to permit raaxiiiom proliferation. Future i3K©stig£" 
tions using l®is8r concentrations ©f irsthocel are being planned, 

Si^ficancss This is the first strain ©f hugaa cells that has been 
gre«n in coE^sletely chesiically defined issidium and also the first that 
has been ^ma in such a nssdim in agitated fluid suspension cultm^eso 
The pofe®ntial fer virus assay and virtss production as well as possibly 
reparative surgery should be far raachis^ sad should be investigated 
by those isjifsdiately concerned in these prdslems. 

Proposed course? Effective means of t^ing this cell strain for its 
full potential significance is open for consultation and collabora- 
tiono (See Series No, i}l5). Continued iE?)reveinsat in the technology 
of cultivation of this strain of cells M,ll be made, including the 
point n^ntioaed earlier with respect te modification ©f coa©entra<= 
tion and type of asethocel far iupro^ing tiie aieount and qmlity of 
growth, 

662 



2« Tissue Cyltiire Seetioa 
3„ Bethesday fferylaad 



Indiiridual Pa?o^ect Report 
Galend^" Year 19S9 



Part A^^^^ 

Project Titles St'odies of Cell Efetabolism ig. vitro » 

Principal Invrestigators B. Bo Westfall, 



Other larestigators? Eo V. Pepper Sj, ?oJo E'f^isj KcK= Sanfordy 

Jo G, Bryasitj WoRo Earle, Eo Lo Schillins 



Cooperating Units 8 Mobs 



Man Years (Calendar year 19^9) Patient Days (calendar: 

Totals year 



Project Beseriptions 

As befers in reports covering ths w>Tk of this ^o^ these 1b° 
Testigaticsas sre part of the problem stijdy of tlss cellular ©rigin 
of canc^'o In act^jslityj sevsral aspects of cell rEetabolism are 
under considsrstioa, Thes® are 1) Studies on nucleic acid ia loug-' 
term strains of tissii® eulti^esi 2) Studies on glycogen conteEti 
3) Studies on the lipid and cholssterol cententf h) Stitdies on mater-= 
ials stich as andno acids reiuo^ed from ths msdiiM as ■^bII as those p'ato 
into the imdxwB. bj the cdlls dTsrijag ^&wbh. in the lasdirarii 5) St^alles 
on the kato acid content of used issdiissii 6) Steadies oh the eaz-yms 
acti-^lties^ particiilarly srginas© and catalase aetiflt-jj of long=° 
term cells and of trauors derived Stom. injections of these cells o 



Part B included 

663 



Object5:"res Tae f first objectiTS is to collect iaforiaatioE on these 
long=>terra strains » The itLtimate objective is to learn if signifi^ 
cant differences exist between 1) cells of different origiRj 2) 
cells grown under different culture conditions^ and 3) cells of 
different tumes* producing abilitjo 

Ifethods Ej^loyeda Cells aad raedi-um .-erom l©ng"=tenji cialtures in the 
Tissue Cultio'e Sactioa are used in all these stales o Adaptation 
and tin© consiiming modification and validation of c3i.ssic analyti- 
cal niethods are reqiiired for practically all procedis?es aiaployedo 

Ifejar Findings; Findings in the nucleic aeid studj as t-jell as 
those in the glycogeiij the lipid, and the cholesterol stisdies 
are being assembled in manuscripts » Strain differences occ«r in 
respect to the content of all ©f these materials » Seven straajis 
of cellSa ^or exsitple, of mouse fibroblast origin had no detect- 
able glycogen. Interestingly enough^ hoxse"^erj after several years 
in yitrog skin epithelium still stores glycogen, and t%m of the 
^Mfee iJyer epithelial cell strains studied also store it. Strain 
1195 ^ich -w&s derived £com. a hepatojHa arising on injection of tissue 
culture cells of liver, has lost its capacity to store glycogen! 
neither doss the tumor tissue ^-eni mouse infections of these cells 
any longer ha^e the capacity to store glycogen o 

The cholesterol content of the cells in all cell strains studied 
in chemically defined medium "sriLthotit ai^ serum is decidedly less than 
x^en serum is present in the siediumo 

Nucleic acid stud.ies to date "ha\fe been ' confined to mouse cell 
strains » DM is higher per nucleus in strain L fibroblasts (KGTG 
929 and !CTC 2071) than in cells of fresh mou^e tissue^ and is also 
constant, i,e»j 17 raierogrsins per million nuclei 72 hours after 
fluid change o RM content per cell is 2 to 3 times as great as the 
DM content per nucleus. 

IsTe^pecti^e of the origin of the cell strains , alj&a keto acids 
are fonaed by all of the cell strains tested? these acids are 
liberated into the meditsm at considerable concentrations,, 

The ensyms activity of asny of the cell strains has been e:£a!i'ained<, 
The findings on the similarity of keto acid production for all strains 
are puzslingo The excess may bs discharged into the medium because 
of a ratio use of them at a gi'^'en tisiSo Sirnil^°ity aaiong all stra5.as 
studied in respect to alpha keto acids production points to the 
considerable similarity in metabolic behavior from strain to strain 
of cells in at least this on© respect,. 

From the ensyme studies there is the demonstration that cells in 
vitro can keep some of their characteristic easyme actiijltyc, and in 



66^1 



NCI 
Serial Noo U28 (d)^ Page 3 



fact show increases in activity! soioetimes they lose all meastirable 
activityo For example the arginase activity of some cells was 
markedly increased <> This indicates that the cells are relatively 
unstable o In the light of oxjt present state of ignorance of the 
causa of cancer^ such changes in enzyme acti'^ity of cells might have 
significant bearing on the cancer process. Staafield Roger's has re= 
ported ETicleic acid froBi Shope Papilloma viri;is induced high arginase 
activity in rabbit skin cells so therefore tumor agents iiiay alter 
enzyme activity df cells to the degree that a specific alteration 
is associated with tumor production » It therefore might be possible 
that the bodies seen in electron microscope sections and Judged by 
Dr» Dalton to be virus-^lik© in the two tumor lines of Sanford id.th 
high arginase actiifitjr could be associated with this type ©f phe^^ 
nonienono 

The s"feudies in cell sjstabolisffl are considered to have particular 
significance because the cells studied are from carefully controlled 
long established cell strairis -sjhere characteristics and behavior in 
tissue culture have been ©bseru'ed for long periods of time. Uniferndts'" 
of treatment of the culture and the availability of much inforniation 
of the effects on reinfection of cells malces these cell strains of 
unique valus for call n^tabolism studies o Considerable ejnphasis on the 
arginase activity and catala?38 activity -studies shews that strain 
differences do occur « In particxtlar all of the liver epithelia are 
high in arginase. The catalase activity j, though Ism cen^ared with 
fi^esh livers is ©sfer-ssisl^ high cornpared with fibroblasts. Catalase 
activity is low in all lines of fibroblast origin o High and low 
tumor produciag lines of fibroblasts (see Sanford 14.28 (e) are both 
higher in arginase thait is their parent line,; 

Significances Cells can synthesize cholester©! nhen they are carried 
in chemcally defined medium lifaich does not contain cholesterol or 
fatty acids. 

The finding of low g].^'c©gen in the liver strain is xmique in that 
it in^lies that cells of this tumor strain have lost certain ensyrae 
activities or a sirigle key enzyme concerned with the deposition and 
mobilisation of glyosgen. 

Cells obviously raaise protein since they grow. HoT<ieverj in the 
rapidly growing non^^isintegrating culture, they do not throw asjgr 
of this protsin a«ay into the raediim. This point is a unique and first 
finding which has come out of the use of the corr^letely chexnieallj 
defined issdium, Tliis point has been demonstrated x\rith both the strain 
L cells and the moakej'' kid^ney cells each of xdiich have been groim. on 
protsin^fpse njsdiuiR. 



665 



KCI 
Serial Ho, 1-28 (d). Page h 



Proposed eoTsrse ef the xfork; Great emphasis will be put on the 
eontimaatian of the atisdies concerned id.th materials removed from 
the roeditffl-i amd those ptst into the raediiam by the cells, Signifi^ 
cant results ^ould be attained from the tmique cell strains v^ich 
haTe been csarried f&c long periods of time in jnediim to viiich n© 
protein has been added. These should be particularly significant 
in aiding the progracj on sinplication of the completely defined 
raediisrft as well as suggesting leads in designing defined media for 
cell strains which so far haTe not been groim in any such defined 
media o Such studies will entail the use of tagged materials using 
radioacti'5'e preparations o This t^ork will be unique because metab=> 
olisju studies ^ ^itro to date ha^e been confused by not having been 
done on long term strains cultured in the absence of added protein. 
Glycine and serine for exarnple increase growth rate of cells j they 
might therefore be incorporated into the nucleic acids of the cells, 
IsJhsther or not this is so needs to be determined , ^3any investi° 
gators using serum in the medium h&-^e said glycine snd serine are 
not essential in the ifjediijmi so this controtrersial point on the 
function of these amino acids assumss significance. This and simi- 
lar studies id.ll be approached through the use of radioactive iso~ 
tope tracers. Also since cjrfcidins and thymidine are used in the 
chendcall^!" defined msdiuaij studies are now being planned to in=" 
Testigate the possible role of Bl2 and folic acid in their ufcilisa- 
tion of these two nucleic acid deri-rati-res by means of tritium 
tagging. 

Xv''ith an increased number ©f clone strains of cells now able to 
grox'f in chemically defined medium free of added isroteia^ and with 
increased and increasing simplification of thisj.t sdiuiiij plans are 
noxs beisag made to continue study along the indicated lines. 



666 



Ssrial Noo U28 (ej =. pi-oject 1 
lo Laib«mtes7 of Bi©l©gr» 
2o Tiastss Cxiltar® Sectiea, 
3o Settoadap Msapylessdc. 



CslejiiiiM' yeer 19B9 



Part A 



Project Title I Vitasija rsq>2i7@s3nte of a st?aia of aoissa 



Othes" XsE?estigat$^s8 Lofo D^^m?®©^ Ao Bo Coival»8ky, WoRoEa^l©. 



Coc^Tatisng ^^t:83 Noas 



Hs£& Isars (ealssda? ^raa? 1959) $ P&tlest Da^ (calsodsr 

Tcftals sraag" 1959)? 

Professiossalg 

Frojsct B@ss2>iptioa8 Ovse objectives ^^ i) te si^lifly pr©fe®iffi= 
i^@ ohsEically ^fiaed ^ditm KCfC 113 1?3r eliiaiaatiea ©f all 
vitaat^ Eot rsquijj'ed fej •^s© cells fea? rspid ppolif ©ratio® o t) 
t© defiias prscissly tha vitamin req'ssirsasBts of tlies® cdlle Tsndsj^^tlie 
eapsrissatsl coaditicms ossdo 3} la particxElarj to ©val^st-e Ut^ 
iat®yr®lati«a^sips &i folic acidg Bi2s azicl ta^sidias for oell 
p?olif®r&tiQsio 

M©^©ds s^lo^ds Cslla of a strsla of ©012^ fl^dJlssts^ SCfG 
30TI wsr® grssm f^ 2 mGU^,a ooa ^dia laekiag eaoh of tJi® 18 
vitaaijiSo Stralffi 30TI is sts'sis 20?! adapted t© gre^th in ^@sd.mllj 
d®fljsgd cosffi^rg® £ffs® B^di^mo After tMs period, ^iicfe -m foisai 
jsse®ss^y fas? ■^teaia dspletiosij 1^ gFosrth ^®spmim of t&® csXis- 
to th® added 'Stasia -ssas q^satitati'f^ly ssasiss^do A a@w Ksditea 
ccetaiaiEg the requisit® -^tasdns is beiag ®^almt®d sad ees^gsps-d 
f©3? ^s^ffeli stims].ati©R mth th© ©^igisaal ssdiwEp . 

667 - ,. .. 

?a3?t B iiieluid&d ies ko /-^ 



Seg-ial Ho^Ji^J©)^ Pago 
Project 1 



AjDgKg {^idismlj, pyridosdffiSa nlacia^ iai®cis^uaid«j rlbeflaviaj Bi2s acid 
paalio-IJ»isaats, €fe©lis^ ehlerideg folio aeidj, tMaains-S, p-=>a!aia© feeiiBoiq*^ 
ass^^ic scidj aad ieeffiitolo Ezp^yissats iira still in process ©a bietia 
Fsrt&@r s^altrntioa of B12 s^qisis^^atsiss )h® abseae® aad p3f®s©ne© ©f 
f©M.e aeijd ©ad tlj^mdi^ is also ia pg'«P^'P®'> VilwaialisSs ^lieh la this 
sgedi^asi, ©s*® r^t!^®d f^a* cell stas-^'s®! Iis«^ o®®ia id®afeifisd ®® |®rf.o«' 
•ys^aata, chslia® chl«^idSs aiaelEMidSs ai^i pibollatino Vltesiins swfc 
appaysatly rsquix^'d fss> e@ll sm'^'^al bat •dsi^ stlHalsfee iiacy®®asd 
Q©11 p'slifss'sti^i 2?&t®s ar® fsllSj, p^ldo^l «a? ppfidiari^j, «md 
pessibly bj-otijia 0^ stSjsteaia ©ppss?® ss0i t© r®qMr® pp-idj-sal at all, 
b^ C6afii"2^tcry teste eo^js^iisg this ^ariasfc «i"& th© origiaal atraifa 
^® la prcfg=ssSo All ©f t^s® Titssdas h^® beess SjSSffi^^d la t-h© 
^^ae® sf e®sis^i®®o Tk® r«s?tfe©r iawasffeigatiea of th® p©a3S}>l® sjip 
poa.'tsse® of Big ^11 b® c^^ed ©52I. ^& di©M.eal3^ dsftsssssd |ffi'®t®ia=' 
fr®® ^diiia M3TG 11? eoateis^jig ealj' tss of th® f©s&' ssacleic aeld 
desdfati'wsj, d®©:g|aejildia® asad tlajffiidii*®j ?«8q^±j?@d bj •feeits e®13^ is 
tM.s s^dit® ss &©ss2 bj S^aaso With ^s mif medi^aaj. tSss intas^la^ 
ti©a^.ip ©f tls©^ eo^oj^at© iw eell p'e^tli aa^ b® ®^alt^t®do 

Sigaificsacs? Stedi®© by Chsj^ i^d ©timers hat® diffi^s ttefc dtsl^-aod 
s®rss p2'®t®iffis M^ b® esffit^dssfesd ^-^ rass^ dlffte?®sEfe witagaissso W&t 
this r®asogi ®. sjstss s^ish es th© ©s® dsv®lqp©d ia tbis laborat^yg 
fee® ©f added proteias ®isd stlso satibietlcs ^lai«^ ©®eM fsfes" iatg^« 
eellialss' bastsrlal gspt^tlis is ^asiiq^ tt^ &je&w&t® sfetadi®® of ^tassia 
ip©qi2irs»2its ©2^ iK&®rs*©latiesa8hipSo ^»s® s-iisdi®® @s?® si^flsa^fe 
as a st©p in sig^lif^iag sadStia ICf^ II3 im^ ftet&®r ess^a2ii.tii?© 
atisdiss «m ssssfeltio^al r®qM3?®M!Ht@ ®f ®©11 gteaigs. With B&Si, s. 
css^letslg?- defied radi^^ z-adaesd to ©a2y t&es® coffi^a®5ats 1^8 etlls 
acttsslly jrequir®, tlxs rols of ea^ ©f le^ empmm^^ is :s£fe®rfe^d3.iai?j 
^8t&bclisis cass tla@a b© ©asMisBd ^aoder rigidly coatyollsd @esiitie.-ag/ 



668 



MCI 
Serial Ho. IjgBJe)^ - Pro j©st 2 

2o Tiseus Cultior® S@eti(%a 

3o Bethasidaj Ifes^lmi 






Psr>. A 



Frajeet 2 5 Titles Ssyc^s^atmis ty^JSJeoffsatiOB md pereietaac® of 



l^^jaeipal Iweetigatcgrg loKo Sanft^dc 



sad WcHo Esrls. 



To Dmm^ I^aboratcsry of Pathologjo 



Han I©®rs (aal®adar j&w 19$9)i P®ti«nfc Days (ealssKSar 

Totals ysasr 19$?) s 

Fr©ibgsi«3ss®l« 
Others 

ObjsotiiP©! 'Hii® p(r©>et i® di'^ded into thre« parts with t4jr@© 
diffsifeat abj©©tl^?@s as follows s Pa^^ 1.. T© dsterMns ishetfe®®- 
tix® ss^^sj tvm^ ageafe p^g-sists is l©Bg°t®3!^ cell _ats'aiBs 
cslt-ersd S?^ apoataasoiss ^i^aaay em'eiaeaa® of strais C;^ Ed.c©o 
Psi*t g» To 9ss^jm th^ iaflT^sie® ©f tw© tjpss ©f eialt^s^ i^di«a 
&B. i®,iHt®agsie® ®f €©X1 diffeifsiatiBti^i la losjg^teym ctilt^gf@o Fart 
3o f© ©splsia tiss sarG©^.t€ras fesssafspssstioa #d.^ occtsrs f?®- 
qtseatl^ la ttos© e©ll strsiago 



Psrfe B imjl^^d I®s So sc 



669 



NCI 
Serial Me. 1*28 (e). Page t 
Project 2 



Metiieds @^loj@di Losg'^^ra o«ll strains vsir® establi^ed ±& 
c^tw3 ^3^ 9 prisssy tvmspa tokea frea strain C3@ fee^la mi^ 
^th the a^rat aiid f^ess 9 traasplented tiiatere earrlsd £qt %\s& o£> 
B^QtTQ gsmrsti^s in S^^id adco to rsplAcs 1^ C^ stream Hlth 
@ts>6^ 1^^ 1^® ibj^id h«st. SmSi t^smpXaistod tosors ^lAn ex» 
plastdd to «mlt^!^^ ^?&uld esatadn tii® t^;^@r c®ll8 azsd h;^rid 
stros^l csllSo By s^tablo trsssplaotrnticfs ex|»8rii»&tSj, sM b^r 
f3s» of @iagl@ call cl^^s, ^e Idsirtity of thoes c«ll t^pee could 
fee estabUshedo 



Major fiisdiisgss %e sm^^xy t\se«r egeat sppsasrs to pss>@ist in 
cert&ia of tlse@@ e«ll arts>eii!s fer p@?iod8 of &mt & ^sTc Eq^° 
ev®T, tys scti^tj of tl&s egent conld b® d@!»»zi8:^&t9d in cultures 
that h£d ^2£ide?g@£S3 a s^fcc^g&tous <^&sisg@o DifferoBtiAtiei of 
^y^ary carei^i^ calls was ssaintaisisd in e®rtai& cell strains 
c^tisred in mrvm^s^phsi^ntsd ^@^&&13^ dsflmd aediixB bat not 
in fmdtim cesataining mnm. &i^ chick ®mbsyo extr&cto Sbcp®?iB@:it&X 
®^d@i3C® indicat®@ ^^&t iib.® serco^atoiis che^ige in th® c®ll p@pu° 
l&ti@3:!0 ^3^ s^stalt ^€su ©ithsr of t^o oatlT®!^ diff«?ont phen^^xms 

1, Malignant t3:'ansf(^^^tion of th® str^i^l cells in cultts's ssd. 

2, Mcg^ologic &lt@r&ti633 of the saaligns^t cells of epithslial 
origin so as to rsss^ls cells of s®e«nchjssal origin . 

CQltTS!>®@ of t^^o of th®m st?iidn@ hm^ hs®n &xMmi^& hj l&vwa. and 
Hsu ^o h£9@ psf@par@d mai-mcfftpt ^& %h9 dxe&sos&BQS of th® csll&o 



SigBificastc@s A prei>lea of practic&l as W'll as thiecs^tical in° 
terest in @tiadi®@ of the naMsary turaep a^^nt is ^0*^6? th® ag@Bt 
ccsstintsss to ps>opag3,t® in long°t®ra cultisres of ^o t^tsssro His 
ros^ts of this etud^ ss® stgaifieaist in deaiKastrating aeti'^tj 
in certain of ''^i® c@ll strains f@3> p^iods of o^rar a ^^, 3h@ 
role of cisltur® is^dia in ssdst&ining coll dif f@i^i£tiatiea in 
rapidly growing popaLstions of eslls h&s c^sialder^l® bearing on 
call tran8fcs>@ation st^siisso 



The fSs^tssnt repesrta of sarecsoatons transf«?QSitio& in 
carcinomas tr^iisplantod ^ ylyo &r in vitro ha^o r@s«lt@d in 

eonsidsrable s®atre?^risf aa" ^o «h9t^r~^l3 1® a e®ffiOTPsl«a of t*e® 
carcin<^a c®ll ^r th® rosult of a i^ligaaist ehango in the gtsr^ia,. 
^® restalts of these ©asperiasaats ^o«sd that both processss ocewred 
in ctalt^sr©o 

Fropossd eo^s® of pr©J®s*3 A is^nuscript ia pr«parati«^ will b© 
ooi^l®t®do . Certain ©f thmm atraias will bo emttnmd fes* ©leetg-ses 

Eiicrescep® studies by DTo Daltms., 



670 



Id 
SssriEl IOo^l<28 (e) ^ Project 3 
1„ l^^^Sj of Bi©l©gy 
2o Tiseiss Cislti^^ S@ebi^Q 
3o B®tfeesdas Marylaad 



Isdi-sld^l Pr@J®ct ,R©psrfe 
CalsBfte" leer 1^09 



Psrt A 



Prefect. 3j, !fitl©§ Cell ts'aasfiisrjsati^* la fite® o, 
J^iacipsl Isi^stifatsjs'g I.I. S®aff5rd., 






Ce€^2*atSi3f tM.t®s 



Totals f®sr l^F)s 

I¥©f®ssl®j2als 

C^jectl^©? This p'ejeet ss«ssists &t a si^^^ss' ®f gusperat® studiss 
^ieh Mil ba c^siiered m^ ^s ab^ te^teg. 

St^j l* fr&c@&m-m i&s- tis© g^©Kt5j @f siagl® isolated felssa© e®!!©,, 
Sffffi'ts h&v@ fes®a nad® t® sii^lif^ i^l l^g'm^ tis© ©l^jsg tst^^ssigs® 
smd to ps-gpsF® si!asp©ssi©ia® ©f singl® eslls ^o® Sf®Al;f l^tosed 
tisstseso Aa i^g'S'if®d ©l^aiai; ssf&cs^iiaxHs hsM b©®s d@^iP®l©p®cl« , fhi® 
stisdgr is ei^aificast. teea^ a fKPoeedm-^ fer ^e^tii of siogl© is€!l«.t®d 
cells is rtsqiaisit® f©s" stisdi®s ©b ®s11 traa® ^r^ttesis m '^Itg*©,. 



Sfeta*^ go Alt©rati©Hs ^ithis a clesss® @f msmm t^^ss- ©9ria.> A el©®al 
analysis of a 1©^ ss-gljass® He© ©f a el^® ©f tmvjm tmsm' ©®lla Ms 
she»i li©r®ditaiT dif^s^®ffie®s te ©rgiim®® asiiislty ^tMM -^Ms Hbso 
Dif lter®iie@s ia sasj^ aetivity te®fe^®sa es®lls @f ® els^ rsf?®al ©il- ' 



671 



Ses^ial lOo U28 (e) Pag® t 
Project 3o 



Ueatieno 



St^dy 3o A clooal stiad^ of aas^QS® palaooaiy ceroiitssHftc A si£gls» 
cell elesis ef pnlmimiej oareinosaa la bsiag studied fear «r^dsacd 
of i3££S>ee@ait@u8 change o 



Prep@s3d coii£>se of projeets Thsss stvedies ^11 bs eoB^let«da 
F^filisr etisdi^s mi tSia isaligoastt transf«»'aati8» of soarmal tissiis 
odlls ia yitafo gill b® pisrs^d. An «:^viffi8zst is beiag plaa^d 
to t9sF"tha yol® of fistaer^^iosis &s a faetop i& this 



672 



/ NCI 

Serial HOc 1*28 (a}^ Page 3 
Project 3o 



P^b^ Hea©?®^ Awards aad Pi&llcatiens?- 

P^licatiojas ©^®s? tfesa ^sstrmctss 

Saaferd-s S,>Ioe r&r^iSj, RoH.,, Hctobs, GoL.g Jomgg JoMo aad 
EM'lSa W,H,o t Gl©aal sa&ljsis ©f ^exlsjat call ii^s tr«^s= 
fer^sd t© saligaaat c^lls la ti@g%ss c^slttsrso Jo Mate Caaesr 
last. 238 1^9. 

lEllt^iisj§ of aai^l passags on a lina ©f tissi^ culttar® <^llsc 
Jo Mato Caae©? lesto 23* 1959o 

W^dSij Mo ¥,, Ssaf^dj Sol.j, Bwk©j D.^ asd EaylSj WoR»8 
Glyeelytio js'spsr'sies of hi^ 82^ le^ sareo^i°:prodQei3^ HjasB 
asd el^sss ©f i^sss ti®®ia=Gultt?re cells o Jo Nate Cesieer Isast, 
238 195^0 

SasiT^ I.Ioj ¥s®t&Us BoBo, WoadSj MoWoj and Barls^ l^oRoS 
l^tsbolie TsristicmB ■^ithiss a el^ ©f gsoiase flbr^lasts 
tes3Sisf®£'ffisd t© i^^lEsti© eellg ^ ^tgo, Aeia Ifeio laterSo 
Casts'® CaaertSo In ps^ss.o 



Messrs s^lstiag t© tM^ prejsets Swltatiea t© preseisfi< a p^>s? 
t© ^s Washiiigt!^ Patb©lsgiits Associatitm ©atitXsd ^Prodvitisaa 

©f ^.ligsaj^cj ia ties^s© eiiltii^©s &f ®iafl©=^^ll ^'igia'»o loFoIoPo 



673 



SSi'iai v^e„ 4HO ^©; « frojeet 4 
1» Laberatozy of Biologi^ 
to TXssrm C\attxre Ssetiea 
3. Bstfessdsj Jferjlaad 



FHS°WIH 
ladividual Project R®p©rt 



Psrt A 



Project h$ Titles Se«» afl&ct^of pel^tsaa inf®eti©Q ra ^lls 
in loag'=^sra tisst^ ct3ltia?©o 



^isicipgil Isi9«stigat«rs SoSo Ssu^^^ 



Q&^^jt&tisig ^aitas A.Jo Dslt^ae Xaboratopj of Blolefy^ 

R„Co JfeeCsrdl©j, Labcg'aterj of ?atliolog^-o 



Maa Years {calesdar j^ssc 1959) « Pstiest Dafs (calsEdar 

Totals I®sr 1959) 

Professi^mls 
OthsyB 



Pjfoject Sescrlpti^ig 

Objeoti^ess OtH* c&^©oti*^s are t© dstss'sias 1) cs^m^t±m 
affects of poljros^ inf®cti^ ^ 6 esll straias ^ -ritr©^ 2) 
e^dsae® fes- aad peysistaEC© of "^iriss i& 3 csH fftr^S 
ctslttsrsd ^^ j^retid txs^rs^ 3) s«®© ©ffscts ©f pol^ism ^ a 
ol^is of cells f^esB E©«se ^li^sry glaad tississo 

l&^ods ®j^l^®d« Cells &®to b@ea iafeetsd fej filtered ssfes-aets 
of parotid tussaps.. Assays f^ ^litis activitj haw fesss ssad® iat© 
Saiss Mca l®ss tbgn th hot^s ©f 6g©« a#^©grsjM.c e^ldesos as 
liall as q^i^titatl^e grmUt ?8sp«®ses ©f eslls after S^f©ctism 
basre be®a ^de <m rsplicat® ealtm^s. Both li'^iBg c®lls snd ■i'imd 
and staia pK^^'atisaas hav® l^©a e^^,j^d„ Issa^ for saliiasat- 
p©t#ati®litl®s ef iKf®ot©d eslls h^m fe#©a Bs.d© iat® sdca» 



P^t B imluded leg I© :s 



Ssplai NOo U28 (e), Page 
Project lio 



Frepaoratieae h&vs b««n givva t© DTo Balton for eleetrcm islcro^ 
soope studies and to Dr. MacCardls for exaoiaatioa of golgi asfd 
other esll detail«» 

Major £lsidias9s Slsc I«&g«>t@i'ffl straisss h«re 1>e«a c«i;)ar«d ^th 
respeot t« iBaistdQance of -^l^ftl actl^ty sad ^opsthogecie 
effect of polyoaa iaffectioao Of tfe®88, ts© straias of adult 
C3S fibre^lssts aad (@@ ertrain of cSsissses hasster tiss^ eella 
sh0«r ao e^@plci20t29 ef facts of iafeotlen. Tkuna etv&lms & @lss» 
of oBjmssj ceroi&^p a cloise of •aliTaxy gland(bot& psrotid aod 
s^sEaxLlla?^} cells firoa ss;^^! tissoe, fistd a straia of C3^ enS>r^oa» 
ic cells i^e susceptible. Effects of izifectloa of the clis® f^oa 
TBOtml parotid «sd siibsssillery tissise &9 beiag iivrastigated, ^raiss 
cQltm«d £r€» parotid trausra haevs beea studied ^th respect to aetiT° 
it^ of ^in^f cell ssqphologsr, tp^sspla&taibillty, especially f«r 
ceai»^i9<m with the treated noraal cells, l&mor assasrs ha?e been 
but esperi^sats are still In progress, so results eanoot be 
issrised at mis tisss. 



Sipai^canoas ^is prejeet asjr bss^ sigaiflca&ce froa t«o poixxts 
of ^l3i?o A class of eells has baea established that stay prme 
&m of ths ^^t satisfactory fee plaque teetaoiques. Cells are 
susceptibl® sM f€SE>ffl a ccsttiasious epithelial aextole^r oa the flask 
flosTc EmQeaSHjfs, possibls rasoplastie effects of tha ^Irus @a these 
cells is yitro ^ if {O^seist, i?ould pr&fide a useful tool tor controlled 
study 0? oarei^geaesia M yitro . 

!^r^»03ed plsas ^sis project ^11 be etatisued izi an effort to e^taia 

the ismdUU $^j®cti^s listedo 



675 



serial ^&^,MtiiL. 

to Tissn^ CiLLt-nr® Ssctitm 
3o B#ti3ssd£, HasTlAsd 



iBdtfithial Pt-oject Report 
Cals&dar Tear 19B9 



Part A 



Project Titles Sttsdies ia tissue culture of tla® ssaligaaBt o@ll 
md of th» acrml cell £rosi «hidi it a?es3o (This 
is a fsnsral title for all vos'k in tliis S«eti»a)o 



Priacipal Iznrsstigators WoRo Saris <> 

Ottear Iiwestigaterss Se« U28 (a) threts^ li28 (e) precadiisgo 



Co@p«7atiBg Uhitss Usted ^sodsr parts (a) threogh (f) 



Han Tears (calsudar ^ar 19$^} s Patlaat Da^ (c&l@!2idar 

Totals 19 I»ar 195^) e 

Prof«seio»al3 Bh 
Othars lo| 



Projoct Desori|>ti<ai2 Tho abev® prejsct ti®^ be 
general titls for all is&rk in the Seotisa, ^ad«r t&® prs^st s«b= 
head Ii28 (f) vill be ootliasd a tmi>®¥ of aeti<?ities swt iiseltsded 
tfitbia li28 (a) tliroii^ 2^8 (@). 



lo Cosistasit ts8iierat«re lo^ 3Sh h&s hma ealiisrated te&t3ti<^l7 
to a eatisfactorj dsgs^e ®f aeon^ae^ ssd reliability o The rsoa has 
already been plaeed ia @se (Sapfes^jer 1^9), bat ©ely for a 8^bet®a= 
tial part of tiae ctsltures^ isitil sufficient t±m ha@ pmmd sM 
eaoue^ extresB c@ld %®atiier seassssal ehas^es bat® oee^sred to thor« 
oughly establish the Img tem rellsbilitj of the tes|)@ratisre coa^ 
trol. 



Part B isKluded I@s x 



676 



Sasfial I0o|^to6 {£1^ Pag® 2 



asd ibie ^11 be s^ffUatially i^arthsr iaer«A8»d «lim "slot" panels 
foar 3«acl&iag eolt^Fss &>oa ostsids tii« room b«v» b««a coiqplet«do fm 
ine«3>&t«s>8 bsro %«®a diasardsd and others ffiXl follsfir st a latsr d&too 



2o ¥«^k emd pls^ for w«E>k with ^« eetfiaporAtl'Vd aotim pl@tur9 
be99 Iseca deeoribsd wdsr U28 (a)o SqiatpasBdi is do haad asd £&oiUti®s 
8X« being inatallsd for tmee is^elM afid rapid da^l^piBSufe of fllso 
Fvll activation ef this dsvelopaasit equipaeat ie beine aecos^ilia^od m 
othsr aqislpa»at is s^lecatad assd r^s^zTaRgad to Bai» fall ^a @f c®ia° 
staat teapaz>&ti!g>9 Eee» 3^3 EMHk 3^29 s^ ^^Ic r^osm 331» cootsislEsg 
ttes ££99 iilai d«Mr«lopl£ig ssaeHias, 

3c Bss^ aeoisrat® ssstlstM® Sm' G&^^&HLsig gaa taiaai<»i tdtMa cultm^e 
ar« baiog waerkaa (&^ M S3& attaint to iaaora leettar aercbic coisditiQ^ 
isltSiin ciiXt«ir««c Z& collaboration vi^ tha laatansmett Daaisa Ssctios^; 
H^saarch Saevi.e«^p a& i^psratisM for alsctvoialoally eositrelliBg Um 
gas flo« "^iro^^ tii@ gas pi&aao of agitatad flmld amp»naijm &^%v^@s 
bas baen dsirised and put ia asa (Saa 1^8 (b)o Social atteatio^ is 
also baii^ gi'msk t« alteratiosas in cvltmm deei^ so as to insura m»^ 
conti&Qoiss asid oess&rollad aarobiosis as^ sr aaaar^ioeie ea ai@y b@ 
neadad to stisdr ^i^ isiflwsBCfi of thasa two mjer fbetors ea eall p>f«ri^c 

ho Msra praoisa assd raliable satbods of asdia stariliaatien arf» nm 
coQsidaratiea sM itnd;f . 



$0 Tlaa eb^ek list ®f p^licati^aa £ros tba Tisavw Cultis?® Sastim 
has baoa bro^t i^«to<=dat8e February 1^9= aad is ntfw wailstels ia 
liadtad mi^&r for dietrJLbmfel<»o ' ' 



60 Battar aqisSpisdat for ^a^iner pipattas (usa is larga 
iaov on harai aad is baiBg tasUd 



?« Oldsa eollaberatioB is being aaiscb&iswd aith dasisasrs af Comlssg 
Glass Wss'ks ia davalopisBafe of a battar daeiga of all glase w&tar dis- 
tillatioa aq^sipjaant. Oaa trial raolt ia new osi baad for tes^lngo 

80 A patanfe emmeijo^ priaeiples Kud. ns^ods of gre^irlh of fl^d aMaal 
calls ia daap s^itatad flwid suspeasioa eultupas is atill ia ^m®§& 
(assigaad to IMtad SUtaa Qmervmnb)o A pat«n& iat9rf«s«®ae® by 
oaaa of tJsa pfearisace^ical oospaaias leas rasolwd ia issw^ ©f E®rl@ ®t 
al, except iasof^ m tiie leetbods m^ be applied t® tdrel^ical 
res larek. IS&ls exeaptiea is uosettled to data^ 

Pataat ( assl^aed to VoS.QcnrarBBSBt) oa the design of tha ^f" 
sirias ©f c«ltiare naaks baa baaa isaasdi pateat coterisii rsdlcaX 
iaad^atieaa ia M»ricati«Q is ia process pe^^.Uag dispesiti^ of ^ 

iBtsrf^raae® &@tim by m^ Csraia^ Sla®8 Werkso 



677 



Serial m. h2B {f), Pag® 3 



9, The coBstsltaaey of ¥.Ro Ess^ls aad ?o Jo S^aas «ith the Tiss^ 
B&isk» N&tioaial Sl&^&l iisdie&l Ceafc«x>^ hfts be«a costiiinied. They a?8 
in oo&tinisid ioft«?aittent consultation and eollabcnr'atieaj, incltiding 
1} asthodology of &>«esing asaA proeenratlon of calls, pKrtieQla3r2y 
boBS sasremi eolls^ 2) -us« of tlis alcroeiaeBiatogra^p aad 3) adffipts^ 
tioa of httBOB akla apitlisUttBo All of tboso etibjeeta Actually fit 
ia •VBTj clos®lj ^th liass of rseesrch in the Tissue Culture Section^ 

SoCoIo 

lOo During tto last jresar «8 eoatinosd to hs^o ssi oxtr^iasly lix^ 
xsa&gr of vlsit^rss ssscT o^ ^ob hsvo boon fSran fesraiga laboratoxdes 
or i«ho bAiro cosiB tbs'ou# to evaluato tbs prsctieability of sotting 
vip tissuo cult^ro mipk in f eloign couotrioso Still aasftber grovqp 
has booss prii^srLly i&torostod in ous- mthods idiioh h»ra a&istiaimd 
eulturo 8ts>aias or^or sangr jsars ft«o £p&a. dstBoaatrable plsurspneigiajnia^ 
lik@ iiiilsotioQs. Praetic&Uy all ^siters hiS99 boon prioarily istoro^ted 
i2i tissu® culture arad its as^lieobility ia '9a?ions linss of worko 
ha;^o hsd 6|>9cliic t«<Smical inquirlss en «hi(^ «s could advis® thsst 
i9ith ;«ep9ct to difficulties oBooustcrsdo Olhors haire insat«d to @®9 
^^ gensiral dssi^ and op«£>«:ti«& of the prooedvres aad equip^at of 
th® sectlesa^ Soss have aslced aulhorLeatioa t@ reaain ssA wsork hsr@ 
fOK> an oxtoaded tim ia order to obtaia trsd.aingo ^Atlle wb limm tried 
to assist such workers., lack of spase aad equip9i@Bt fbeiUtias hm® 
S&tmd m to Hsit m» tiaa allosad to uhat a v@rkar could c^ses^ 
la & liMted tmi^m' of hrarao la ea<^ iaatanee «e ttj to ©raluate the 
needs of each iaSi^idTOl ^sitor or p'os^o Iheye is a® do^t^ htm/mrg 
that this h&adling of -slsitiag professimsial pos-smml is ezls«B@1^ 
%lm eoasuaiago Further, the tiae taken rsp is that of the professio»I 
staff because yvgj few Waiters are of a typo whose desire for iaf ®'m= 
t±&& can be smt bj ua^raiaed or ssfoprofesaioittl ptHPsoisaslo 



llo JxmoSm^ &8 spears to ua pce^aatlo^ k@ trj to 
cultiBfos to alios? other twrkers and laboepateries to start cell steaiaa 
in thel? OHB orgaaisatiea. lo eell sts-aias are fbraiehed ^&i«^ mte 
coneroially oTsdllsblef or available £eim e^er 80uroes» ia s&tiS'^ 
Ikctovy Goaditioao Cells strains l^araished bjr Tissoe Cultiis^ Se^iesg^ 
KCI, tmdsir c^sditicas prevloaii^ apprc»«d bgr the Direetor^ of m@ 
latioaal Caaeer Institute^ 



12. fhe Zeiss imerted (Plaj&ktoit) isioross^pe is at pre8®»!& eesi^dered 
loj SaoF ^e best alsrosecpe for tissue culture sim® it vm fl£<st. ±b^w&^ 
duced iato this field b|r %@rk aad pi&Uoatlcas of the Tiest^ Crnltw^ 

Sectienj Labor&tcfy of Biol®gy, HX.So. R^mmr^ this instr^ssi ms 
»&t origS^Oly deaigaed for tissue culture «Qrko Js, receisl e@af^r®£!i@®@ 
ia this Section^ held i0i1& the Chief HLorosospe (^ical asid m^mH^l 
B®sig:»rs of ^Sm Zeiss Gtsi^&^, esteasi'^ disesssiQas ^»r@ ^mriM m 
to redesign this sd.orosc<^ particularly fes* tisane culture matk^ tmr- 
ia@^©ratiag iato it ^vs^lmjs Ganges i^oh h;^® hmn msSst iM ^is 
imtrt2s©sKfe ia ^e Tissue Cisltw® Seoti^s Lsteoaratasy ©f Blelsfy.^ 1>,G=I, 
suggested bj its psrsssasslo, 

678 



Serial HOo hZB (£), Page h 



Part B EmeBPSs kmatda^ @a& Tvtlle&ttimao 
P«i»Ueatl@as Other than Absbraots Sroa this project g 

1) see 7«p<^3 hZB i&) thrcrogb li28 Ca)« 

2) S^rXd, ¥.RoS LQS««t«ra culti<9mtiaa of asdml tissue coUe 

in lArg® culiw®8c F®dno Pr«>Co 17(U) « ^?-??U» l^o Preisen&ed 
1^ is^t&tion April 1908, buk pi&tlished too late to be iiieludad 
in 1906 fismoal rsparto Sas 1(28 (e). 

Ummts laad Awards relating to this projeets 

Contiawd «s coestsltaBt to Tiaem Basis, V.S. N®^«a ISadieal 
Schools KfttioBal STenral lisdieal Canter, 

C^tiznj»d o«easuLtation requsatad by Eli Lilly Hesaareh In3ti<» 
tiate on variotis techaieal points in tissna oultixra. This 
eonsultati'sa arrangasssn!^ is coasiderad of si^batantial ^ral«a ia 
advaBcefflesKt of tha verk of tha Tiss^ia Gtat^^ra Sa«niott« Ifational 
Canear Ihstitutao 

Served vi^&n ii3H.tatiim aa Modarator of Confarasoa on Call Cvdti^^p 
January 13» 19^9 at tha Hatienal Acadaiey of Scionoas toidar the 
Auspices of nta Insrtituta of Laboiratery Animal Resocsrcasj ITatleml 
Reaaaroh C@\s3@iXo 



Sar«rad vpmi invitatim in three eoafaraneae doring 19$9 && ^Chir^ 
aeteriaatioQ^ Fresariration and S«^p2jr of Tissna Culture Cell Lime^^ 
sponsored by Tirus and CanfBsr Stu^ Section Panels Research Qrmts 
Rational Cancer Institute. 



I2»^tad to giw a series of laetures at the Instit^sta of Call 
Boreielogy; Qhi^rareity of Nebraska, ^ring 19$9o This inritati^ 
tsaa held owx- at aqt request untU Spring 1^ because of 
of trark on the constant tessparatura rooa and other equipisKato 



Bresanbad a paper m invitation, "Loag^tern L^ge Scale Tissua 
Culture^^ befcsra tha Gosi&atics Chaasiats Society, Hsff Irnkg m^ 
1, 19B9o 



Addressed vpm infltation the E±0i S&tcol Teachers of Eoanoks^ f ao 
on «LoBg»tera Imes^ Soale Tissue Culture in C^ccr E®sesr@h°«;v 
April 190^0 

Addressed vspm imltatioa the Inatrumeat Design Section, Di^giesi 
of Eeseards Senrices Saainars HIHg on Sissus Cialture, April 1^59o 

Zstrited to address the Isistrumnt Design Section^ Di^riaion of 
R®^ar«fe Sarvias ^simr, NSH, ea the %sc®y ©f Tiss^ Ctatisp® 



^^^8 



Series Ho. 1*28 (f), Page 5 



«9isioa Btslaioa sm sc®na?io aai tUsm for program^ Oetdi^? 19B9c 



Jxsftted to gtr* p®p@r asd partieipat* la s-eoud tsble dlsctisgi^i «m 

a^eia^9Eissls» Bdrliffij, D®3«Gbe? 19$9o W&« 'mshla to accept 6i3@« 



^s^-'-ed to pr«p87® outlists s>«^ei? articla for tsew Sele&tllle 
Aiadrlcsn j^tgaaina ppesestiog irork of tho Tissus Cnltiire Ssctifm^ 
Labe7ato£7 of Blolo^, N.CoIc Teatatiirelr accepted iaid.tatio!a 
peaddng auyserisatloiia 



68Q 



Serial Ko. ICI-^HO 

1« Laboj^toiy of Biology 

2. Virus Oxicology Sssctioa 

3. Betiiss4a, IfesylaMl 



iB^vidiml Pro;J®ct Bsport 
Caler^r Year I959 

P?*>5@ct !Eitle: Molegieal esi& feiochKsslcal studies on a Mirir*e 
letsk^aia viMS i-scoTRBssd fsc^ Ssxcma 37 

Principal Izi\'estl®atoi?: J. B. ^lamy 

Otter Isvestigg-toys: Soise 

Ccop®3?a-feisis tfeiits: Ica^ 

l^aa Yesrs (caleMar y^ear 1959); 
fetal; 2-2/3 

Tsof@BS±aml: 2/3 
^t,al: 2 

^jscti^s: A cMp2^beiisiv« sttiSy of the biolgffl and bi'Scb^jiist:^-- 
of l^m Isi^mia virus, witSi a vl®w t@i^rd an uadars-taading of ths nattjr© 
c^ ^e agjsnt aacL its relatiem to 10% neoplastic pxt>c@sac 

fethodB ^ployed; 

Origin, Source as-d Ssfessctioas ©f the VItqb 

SarcGsa 37 's&ich liad heen carsled tgr M. E.. ^rsett, of tMs 
Sastitrste^ for 195 txmor geaesstioas in A/ra Mce ^as traasplaated to 
SALB/c m.Q@ aM carried ia this stssia fog' 3 tiai©? gensratioES tesfos^ 
att^^tiag to ext:eact -^ss leisfesBia vir^^ It is of intes^st to uot® 
tJj&t aftsr 50 passsgas ia saics (MlLS/c) th.® sascosa stlJJ yi©Ms the 
Ig'^^a virus. SlMllsrly let^eaia os-igisially i8teg@d Is^ cell-fi?©e 
®:^2s.et® of Sare^^ 37 is i^eaflily tgs^splaatable ^ItMa ©otise sts®,is 
E2sd tSiss® -Kftol® cell iMuced lei^^d^ts Cdevelopiiss in 100 pesceat ©f 
tls£ s®cipi®32t@ la 10-lij fiays follOKlBg I. P. iiK)aiJlation) serv© as aa 
©xcellsat sourc® of ^&® ■i?l:rus. For adfiitiosial virs^ soui^es s®® 
bslo^ -m&Qv tig^ v^lggts. 

Ssfej^etioa of tl3© ¥iyus fi'Sga Soosce flsstsss 

f®o ps'ece&ssTes M-i?^ ^g®B ^®v^leped for tte recoi^esy of t&® 
le-oksiaia TiriJS ft^ssj seux^e tigsxxes. 2fe©s© as^ (1) a stai^la2?iisea 
isetlKsa ©f diffes^ntlal etmtrif^^tiisa Celtesiaatlsg Mgh as^ law ^©®ds) 
assd (2) & stajsiargiaed t@ehaiq\^ a^l^-lsg "tec^riological ^Itimtieaa 
i^-fesds in ei^feiaatioffi ^tJi fii^@g^Hfei6il iiit^ceistslf«gBti<»^ & 

681 



° 2 « SsTlsO. m. ^1=S*10 

■^Mcb cesstaia t3^ biologicailly aetl-ve vlsms aaterialc Swsh fractl«m 
Ssri'^^sS fr^ Saxcam. 37 @M tl2s virus istS^issS leuk^ESie tissues ^vere 
^1^^^ iB biol^ical, pb^ical sM bloabeMcal stales ^ Uks resets 
of -^Mch ere Sescriljed tel©w. 

liolc^ical 

1» Tk!& reeo^'esy of rlT'M fstsa Imjkssie uaterlal is priBXirily 

d@p€xid3nt cm c^icsmtratioia of t^ ag^st is tlte eouirse tissuse. To 
^te33is» fsKM -HMoh of tbe tlar®« lasoet gspossly affect®* tissuts (li'v^r, 
spl®®a, Ij^Epla a^®s) th® gs'eatest aaeo^aat of vli?ua could te a«c©'v®SE®fi, 
pools 6f ijSieTO tismsss -^^e frasti^mted s^spasately accorfiiJig to 
pr©e@tur® (l) a1^v@. As fie-fes^aisssd bg' latssst periods (tis^ to death 
^'ith leukosis follO'iTiKtg vlrvB iiaoet&Iatimi) aad pereentaget iacideosce -v^iiss 
It 'Kas fouM tliat l®t^®sic li-vsr is a s«elatl"WBly poor'sou^s of the vis^aso 
L@yk®^c sfpl@@sis asii lys^h ssf^Uss asr^ fisse&tl&lly cd^g^retMa, bo*^ 
yleMls^ M^ coaeeat3fatl«m® of t^ virus. Cell-fr®® concentrates 
(9.^ ^o squsimlsBt p«sr ssl.) of l@^enlc l^as^h xaofies vh@a dllts^a 10°*'^ 
prM^icM l®T^;@siia is. 100 percent &£ tte test. aMjmle '^th & mson la'^^sist 
period of l«-<,5 BsoatSaSo 

g. fte l^st specificity of tte l®«&easia ^r«s, tJith re@aM to 
"totli &@@ aM stsmisi of Wb recipieiat; ^ms iav®sti@a.1»sfi<> ^^'■'■MIM/q 
Bic© s®S!giag ia age frcm 1@§© than 1 tS&r@B@jh 67 Says were iBOCul&t«€ 
^tb vinjs g^co-wred fsrai @i-^sr SEsecssa 37 or th» -ubole c®3Ll isd^Md 
lexiteiiis^ C@^ a'^ve). A 100 |^rc@nt I@i]^@ssS£ iiaciS@i^@ -m^ olsteis^ 
ia all a^ grs^s^ ^w-jigver, tis® latest, period -ma soi^what extesaaist is 
tiffi@ 1^ the olS@r ^c@» Bssialts of mx ®3^@rlaent so«f ia prc^rsss 
IMlsats tJmt M.e@ as c^. as 5>>5 Ecaatiss at tis® of viras iiacsulatioaa 
■Sis T^iltli le^jtesda -^th latest periods a® short as 3-0 m^^hs. atjmia <->«> 
©3t de&sssiiae tte d^gs^ee of eteaia specificity of tli@ le«k^9ia vinm 
tfee follGwlBg straisas of sdce, klMly fts^ai®b©d fs«^ tSj® iiaiby^ eolcmiss 
of E. B. teaer^cat, "^si^ ixioetiLatsa: MZ^/c (spefer^aee); G31^ G31f, 
A/II!, Swiss Ci®^ml ptapose)j I, RIIlp DM/2, aaS C57BI« ®t« restsltieg 
®:!©®slsffisistsl data isadieate tMt tJs« l@\^iL®iai® virus is s»t stsaia 
specific, fbe iaeiSeiac® of Ij-^hoejftic let&i^ia (esssstlally lOO pes^sat) 
ana the iissa latest periods wm^ tto igibrsd straia® t^t®a ©hosM n® 
sigaificaat deviatiaa tssm tho^ of tte s^fsreiEas© stsaiB» Mie® of -IS^ 
G5T1L straia sbsf^efi & relatival^ low Imi^feiada incideac® (§8,6 p®wea.t)« 
©lis f iMlBg Hl^t iMicate a d@^^e of rssls'feaac® of this st5:^ta to 
tfes leiisala -virus. This ia^s^stisg poiat is being iav®stigat®fij 
witaa @3^@riji®a'ts ia ps^ogrsss i&si^m& to detsraiine tfea effects of "totli 
^2s eegiceatratioa of virus ia -^s iffioewl-iaa asad tte ag® of tl^ bost 
at tfe© tiss© 



atioa ©X varus is •»© iffioewx-sffla sma tijs ag® < 
©f vinss ixses^a^mti^i Cs®« also lCI°»J{-10((a))o 



682 



la ass sfforfe to ie-te^ais© tbe susceptilsility of tb® prog^ay 
derived f rcsa ©tsaias sbowis^ & l©w epc^ls^csouffl is8eid®ac« of the 

spgcificity tsMc^ liight ^ e^p&mn% by <^is^ of str&i£& sasatiu^, F^ 



l3ji%^lS M(^ ^@3<e lja&e%3l&t@d idth the virus. 'Btmees pi<oeaziy 
fr<^ crosses ^t^®si tte smiles of tii@ isilSFSd stsulss listed abovs s^ 
the fsmlss of tl^ BAIS/c stmia. AU of tlie siMmI@ of th@ ljybri& 
^s^^ 45.eS of l©%^®saia aM tJs® av®2^^ tis® t© deat^ dM a©fe w.Ey 
si^aifiQsatly fssM tte ^laa survl'tfal tiss® of tise iateed MLB/c ffsfersjjs®' 
ctrais. ®s?^, as SMiea-lsfi, sao dilisfeloa effect of sfesaia specificity 
-^ms apparent ^ tlis o^ of straiB imtiis^. 



3o Sslecti-v^ vls-;^ passage: Ibi po-^aocs" of the virus hss 
issrs&ssS ths'o^j^ a series of sxi^Tlsm&%& tessaad "sel«ctlv« visus 
passa^." ^^33 p^osedim^ ^^s to B@3.@«t 1^ first asaiaals of an s::^ri» 
iseor^ tl^t pf^ssBteS so^^rosc^lc @vlS^ac@ of liri^aais. 'Sha Moatopoietle 
os^asa® ^$@s^ @}ct^£@«ti^p flltes^, wsS. ^^ visvs ooQCdof^vated ^ dif- 
f@rais.tial e€3Strif^agati(m. Ba^s^toiss isie« -w&x® tbsa issoculated s^i^tsta^ousl^r 
id.tli -ya® e©ll-"f:ff@e vir(i3 eoasezitrats. Usi^ siaeh a psctc^dims aM 
aft^r 8 passa^s tiss psteaey of tte vis^^ Ims ^®®b iiic2«afi@S as sbofaa 1^ 
a SmivmBB In ^m latsn^ psrio&s (tis t® Misk«sMa ize&uctiozi) fsraa tte 
esifese^ ©f 6a % ^c^stbs t® 10°12 ^mks toT 100 .p©s«®at of tlie aaisralBo 



pr@s@a>ts til® i^imX piet«£i% &f l^^boc^ic l<gvj^@3iia, sissilar to that 
-fAilQh ocQix^B 0p{mtgi££OU3l7 la sati&v®. MI tbs dieeassS sic€ sidiiMt 
gr©s'U.y eiilas^gd splefsis (lO^SO tisss iKJSSBal tmi^t) susd llves'Sc 
^^ psripbssfal sosS g%s@!£^ss>ie o&Ses s!^m isassi-?@ ij&vol-viis^at> aiid tli@ 
tSj^sus is iafiltssitsS to the ®xfe@at ©f co^fsriag las® esbsTtOT poytic© 
of 'lbs &wrt. 

Bo !s®^3^®s!s otli@y ttea ^feos® of tte l^s^bocytlc type bav® tesaa 
otesyvM g^'^ssly ©r ©ic^sscopically la as^ of tlse t@st eaiEmlSo 



TOVi®Wd tSl® tiSS13® 



S^a^^ DOTS& of tte IsboOTit®sy of PatbolfE^- 



p$s;@S€i!S@ a s^^Hsi o? @li^tlj i2^.<ss3t^ Bi2sl@us £n2s?st:ms!i@d ^ a sis^^ro^ 
2lsi of tese^Mlic ey&oplas^' Mi'totie figusRss isgs^ fs^qpssasfe. '^© 
ajpebitectm^ of 1^^ Ij^^li S3as>a®s -ms -usi^lly fiestroiyea sM tSs® 3a®9^3Uisti« 
c®lle @xfe®?^f^ "bejos^ Ito c&psul©-. Isfilts^tieii Is^ inecplastie s&IIb 
ia tfeg li'rer "sas eos:MJ®a.ts:®it©d ss^fuM .tte bigs>ati« vessels » I;' _ 
aSvaaeed dissas® sJiote-sS iaf iltrati^ of ^^piastie sells ia v - „ . 
glaEfls, tfcs! ki^Ei^fS, l«iiag@# asi, ia -K^ a^isiage0« 

A sJMlasr histolegie pietus^ 'fc-as »®eogais@S ia all tSa^ asie® 

cell®, lo vsriatiosa \ms 4@ts«t©S ia miom of iiffer@at straias sr 

■Bs© earii®© felesS of 3^i3&«ffiic sdes ps®s©at®fi tte f^Himtag 
pict^asej IwreasM. WHS C^-^9,1^ ^^)^ asc^eas^t HC (7^000,000 m^),, 
fisiOT''@as@fi fe^aoglo'feia (10, 3S (^ffiS,/lOO sale M.©^)j aesl tees^isasisS 'M^fesi- 

683 



5. Sb© prod^icticsi of hete^olc^otss aatisiginaa: A psreliaaiimzy 
Qi2aly@is of tlss data fztm an expesdssesit bow in pTogress Isdicatas 
th&t it its poaalbls to pxoduce leu&^iia virus aatisenaa ia zsli^its. 
S!K:h a l^t@£<olo@oi@ azxtisss^ia cesitaios aawfexaliaiBg aatitodJcs agaiskst 



the virus. Altboisg^ it h&s "bmn fousd tSsat Boaaal rabbit senna doss 
i»3t ixsaetivate ths leukaatia a^ent tii® specificity of the antisasTaa 
aaa its effsctiv® tit®r ssast 1® attJdiscL. 



6. Bout@s of is^oculatioa: '&& virus '^ias fotmd to b@ eff@ctiv@ 
-i^sa iz»>ci2lat@d 1^ ajs^ ooe of tbs follcraiaig routes: suteu&aass^us, 
iatmv©s0us, iat^-acerebml, aafi intsaperitonesd. Ssperliraiats coa^ari^ 
t^@ @ff@€tiv^£^ss of these roulss are gtill in prosress "^lerefoxQ 
at^solutd valtEss as^ not available at tMs tiB»« 

Of iBterest is t&e finding that @&thers vhich bad cambalised 
t3a@i? vlrus«iz^9culated you&g, later disd of leukesila. ^Qa» poseibilit^ 
of th@ oral i^vsction of leulsssaia 1^ virus coneentrat^s le qq^ \s^t 
invsstigaticm. ^ifi possibilitj sbouM ^ coosidersd In the bousiz^ 
of sBl^als ia qt^atitativ® bioassay ®>^ari»ttnt©<. 

7» 1Ss8 sffe«t of tbygsec^a^' or a npleaaet^^ on tlis viral iadue- 
tion of l^^aks-iaia Ib a&ult sdce ie being studied, li^t veeic old Mc® 
■uer® inoe^ilatsd witla virus h& hours following th» operation, '^m 
rssolts to date ar« as follows: 

So. Positive Incidence 

lb. laocxslated percent^ 

Coatrol (isntreated) 2k/21 88.9 

Spl!g33ect<sdzed 12/15 80.O 

a3J3Ssetcsais®d 3M 21 .Jj 

^Pereent of inoculated saice dsad or dying vith leUkiemia. 



%^s @:^@risssnt is in progress, th@refox«, no averse latsnt 
period fig«22©3 ar@ available. 

^Shis strllsiisg effect of ^^issctts;^ on ^le viral izsduction of 
letateaia in laice is being stisiied ftot^r: (a) effect of splessctoa^ 
ax^ thyasectomy (saiss iMivid^ml), (b) effect ©f iS^psctcssy at varioiss 
tisse intervals follo^riap virus inosuLatioa bxssS. (c) effect of reJ^^laa^- 
tion of tSs^sn^ at various tis^ intervals fol3.o^ng tl^ns@cti^^ asd rin^ 

8. "^e prefiactioa of tiaor variants; Atfe^ts to develofp 
localis®d leia&saias (lyi^biogareca^a » IcKjalized) t^ieh V012M yieM ■^i© 
Isi^^fflla vi^ws bav© r®sult®d in tls® develqpaest of th© follcj^fing t'-sass- 
variBats: (a) a lyi^osarecsa which a^ss® ia the thg^ic asd iztguliml 
sod® es-ea of «m ad.^Q.t sous® wMch hsA s^ceived len^jsla virus st Msf-th;, 

68^ 



» 5 ° Serial lo. mi^klO 

Cl>) aa ascitic fojca ©f Usm'S' vaslssit (a) elsove, origiaially _ 
h^ the ints^pesdtoise&l ixioetili^tiQii of the lyttq^hsa&Tcam., (c] 
localized l^pi^boeaTcos^ gs<cnm subeutoseousl^, o^igijEsilly iaduced 1^ 
tlie i@ocT2latiOEa of rlrxiB laSuced leukssiie splftssi sM C^) & localized 
Ics^^hoearco!^ gSGwn s^23»:ut&99eoQ8ly oslglasally ix^uced ^ tiie iaeculation 
of a vir^ iz^iteed pool of ler^msLc lys^h mf&SBs spleens estd ll'Tisrso 
All of ^sese tim>rs ase« s^ataissed la routioe vbale cell tissor txsBSo 
plant @@s^e@; with passage evexy 1^ da^. Xn all eases the adtalt mice 
beerixsg t!a© localised gro^^th also develop a gss^saliged lyfflahocsrfeic 
letEkssda. 

It is hi^ly sigiiif leant t£^t all of t2%e tisnor variants rs@as^'° 
less of tbsir fossa (aolid 03? ascitic) yield the leuks^da virus* VIstjis 
ps^paraticsis, deri*ytsd froai such tisaaors, ^®a iEsoculated-iato trn'^botn 
mice issues l^s^&ocytic leiskemla vith latesrt periods as short &b T -y^eelss. 
Msan response values &m not availaMe at this tl^ie. 

IPhysical 

1. ^IS&e B^sptolcgy of the leu^^a virus as seen vith t^ 
election Mcrascc^^e is e^^letely descrihed in SCI-^loCa). 

2, Stability: ®se leiakeBda visus retains its biological activity 
Ct\3S23r producing activity) vbsa prepared in citrate buffer and stor^ at 
t«^©s^t^sr®3 b@low <-50O C. Citsate-vinss saterials prepaxed in a^ll 
aliquotfl (0.3 ail«} are stable asd n^rodijclble (biological activil^) 
^hen sealed xsmdiBT -memm aod stored at tl:^ toaperature of frozen COgo 
Such "stas^ard" j^terials say be used in quantitative biologiceO. esi& 
MochesBical studies. 

Siailsrly the Sarcosm 37 » the %%s^ss- variants and the virus iMuced 
letikesslc tissises smintain their capacity as ^diole cell issplants and 
vin» source materials vkk&n stor^ at -SO© C. 

Of comiderable techoieal i^ortance is the finding that ^bs 
letske^a virus pxeparsd tn citrate buffer vill vithstasd lyophilisatio2& 
without appareist loss of activity as coas^ared to a f^rozen aliqvot of 
the se®e virus preparaticas. Such frosen°4rled ps^pazations have l^ea 
sex^ to selected cancer iaveetigatoi^ in this couatsy and abrt^do 

Bxperi^gats tucsu in pit^gress iMicate that th® citrate-viros 
siispeBsicms are stable at 37® C for at least 90 jaiatttes but are 
ametimted at 56*> C. withia a period of 30 sjiautes. 



685 



» 6 - s®r5Ai mc mi^kio 

Pixgposea epuara® of p roject; la aMitioaa to the studies ps«»po8®a 
above t^ foUo'^^lBg cooxb^b of inv^sstl^tioa viU te taMerts^ea: 

1. MocbaBical inv^stii^tioos cci^c@XKi@d ^th a ssore cosi^l®-^ 

i23»ie7i?tQsdi£g gS tb@ imture of the vlsiss. lEiee® st\£Sies vilX include 
12^ yol® ef psotsiffis, lipid©, aM mseleic acifig ia t5te iafactlTi-^ 
aaS i^tatiolisa of tia© virus. 

2. Bio-pl5;^-Bical stMies vi^ es^Sjasis oaa tSse stability of tli@ 
vinss tma©r -varioiss pl^sieal ^sS clsaaical @a-?i£0^@»&ttal cOTsSitioES, 
i.e,^ t^sml stabili^, stelsility ia m^ous salt aaS lasffer isedia, 

3. Sh© effect of total bodjf x^irKafliaticm assd/or beat <ja tl:^ 
Is-teut p®riod of tbe virtm todisced l«mk^da ia' edult ®ie@ will be 

■ sttaaied. 



k» Att^l>ts will 1^ imS® to €2os8 tbe gpceies 'MrrimY with 
^^basis em tli@ viral iMuctic9i of Istio^ala ia vats ased ' 



^o Furific&tioa eM. cma&^i^r&tiosi. of the virus -^jstrngh th@ 

application of Ssjprovsd pi^rsical aisd cytosbsaical tecboiquss. 

6. 'Bm dgi/^cpraafe of q\3Eatit&tiv® procedos^s for tli® bicassay 
of tJ^ Tirus e^loi^iag ^sponse criteria other -aiaa "tJse to d@a^ ■^th, 

l€1ik@Ql@>« 

7. QtiaEtitativ® tims-^oe€°2@cov@^ studies of virus fecm 
levsk^-aic tiss\s©s vlth particular ^s^lmaiB oa tlie racovegy of virus 

fTOg_lst^c^ie ^ teyala. 

8» Mtigeaieity sttjdies to deaosastrate an Isaaunological 
relatic^ship (if a»y) to oti^r Isaowi teuk^da agents = 



Part B iseltsS®d. les X So 



686 



T " Serial »o. mi'hlO 



Sadivldiial Projeet Beport 
CaleMar Ye&v 1959 



FuKLieatioiss ©t&ar tl^a abstmcts frcsa this project: 



'^lom:yf J. Bo : Biological stales on a l^i^hoid l«ul£i9isla virus 
ejctssctsfi fs'aa Sasecssa 37. I. Ozlgin and iatsoductory 
iavsstigations. J. Sat. Caacer last. Sh-. {April i960). 



687 



Sarial ^o^mi^iODM 

lo Labera^osy"" ofm^B^ 

3c B9thesda, Mdc 



FBS=>HIH 
IisU-ridii&l Project Report 
CSalsndar Ysar 1?59 



Part A. 



Pro j est Title? Collaborative biolegieal, biochemical and 
pigrsical studies on a mouse leukeada viras 
recovered trcm Sareoi^ 37 <. 

Priseii^l Isr^GStigates'j Jo Be Moloney in ojllaboratien «dth 
those listed belowo 

Other Is3?^sstigators3 To Bo Duim:, Laboratory of Patholo^rj^lGI 

AoGoldiHj Labor&tcsy of CSieHi^il ?lmstmL&olo^sMGI 
Mo Bechciglj LabOTat<a?y of BiochssdetsypKCI 
Ao JoDaltoHj LsboKitory of Bi©logysNCI=?li06 
Eo Jo Breyerej Laboratory of Biolo^j!ICl4;l8 
Ro Ap Hasakex", Laborats^y of Biolpgy,NCI^l6 
Mo FiRkj Laboratosy of Biolosr^MCI=4il5 
No Ida, Present Address? Mo Do Ar^e^rson 

Hospital Si.M Tumor Institute, Eoustsfe^ TeXo 

mn Years « (Galsadar year 19^9) 
Totals 2; 3 

Professional g 1/3 
Other* 1/3 



Projetet Dsgeriptions 

^je^^^^s To studjjr, through th© collaboration of other 
In^Q^^^^^gthQ biologieals biissheiBical and pl^sieal natsar® of 
the le^mdM vlnss and its relation to the neoplastic process ■> 

^fe^^^^l^;^^_^^a^J|yj^;^^J^^lngS8 Mai^ of the studies 
dasc2^^°'^1^'^S°la"'llir3^y"^^^Sa8ntal stagey thersfore^, 
only espsslstsi^al reseslts of a preliEiiiasy Jimture are a'^milsbls at 
this tiisso 

lo Ths pathology of the viras induead disease has bs©a 
described ia HCI-iilOo In csllaboratioH «ith Dr. Dtarm of th© 

Labm^tosy 'j£ f&thxilog- a ecsaplet® Korphogaaie stisdy ef th© 
induced Isiikesda te® b©@ra m^artaksno fhiss ass attest is being 



683 



HGI-iilO(a) 



siada to nor© ecaplstslj urjderstand tm disease process through 
vhe «iriy deteotlon of mieroseopie lesions » At p(redetarmned 
iiitervals of time, folleKijjg virus iaoculatdon, a complete 
hiateloglcal esasaination is m&d& of tha organs snd tissues of 
infected iai23 to detarsolne the pattern (if aagr) of infection 
airi/o?. infiltration of the gsaaralised dlssag0„ DefiaitiTe 
results fycm this study &re not a-snailabla at tMa time due 
to the x^hort pariod ©f obsss'f^tiona 

2o rhe use of this rixns for the production of indicator 
ttsKOTs in studies of th© chssnothex^gy of leakeiDia l&s tifo distinct 
advantages g (a) Ths iadeced nsoplasBi is a h|^t tujno? and (b) the 
5.^uc3d dis&^se is elBdla? t-o l^at which cmeurs spontaneous^ir in 
aat'ire. In cllaboration ^th Ao Ctoldin and So Htaaphriea of the 
Lab^^at'Dry '^ Ch^aieal PbsCTsasologgr sttzdies ha^s b€«n undertaken 
on ths ©heissethex-aj^r o^ the virus induced iQUkKuia ia, yivog These 
studies have hem. made possible t^ the produeticzi oF s^'B.eient 
quantities of staiviardiaed (knami biological activity) virus 
EiateslaXs and throu^^h an accumulated knsjfledge of the virus^host 
rslationship. It is possible to produce in one working d^ 
sufficient irix^us to iiioculate two million aice all of which 
M.11 die id.th IsukeKia tji-Uiin a known period of tiai8<. Further 
it is possible to deterssine grossly when a test aniEal first 
develops leukeinia aiKi to estiaiate the time to death of the animal 
(average of 19 days) f olleiising first indication of palpable spleen 
and/or lysiph nod©o Engjlcying such a biological ^stsm, selected 
anti-'leukeniia drugs are being tested to detexaine their effQctivs== 
ness as (a) anti=l©ukssBia vir».s coapounds, (b) anti='rirus=induced 
lQuk®d^ coiapouadSj, and (c) any.=«hol3 call induced leukemia 
e<sap©uads„ The latter system ^s origix^Hy a virus induced 
leukemia and oarsded as a vhole cell passage series » 

To date no sign5.fic&nb increase in survival tiias of the test 
animals in relation %o the e<xitrols hae been notsdo However, there 
is an indieati^a that th© spiaen weight of -fche treated aninatls is 
considerably redusod in relation to tl®t of tlie untreated aniESls 
(sae also repsrt hj AoGoldinj et ^,>) 

3o Prsliisinsry biochesdcal studies cm mice with advamssd 
virus induced 3yniphs^^ie le^aMa showed a severe depression of 
liver eatalase aetivi^o This level ^ssls belcu tl®t of ar^j^ls 
b^ofing solid tiMorso 

Gollsborativ® studies id.th Mo Hechcigl of the Laboratcsy of 
Biocheiaiatsy are new ia progress to datsrsdne ttie l^jae (f ollc*fing 
virus inocaal^tion) at i?hich livsr catalsse activity is defH'essed 
and to relate thig finding with the onset (itEasrossopic) of leukemia 
and further to eorrs3at© this ^ei^nim tie depression id.th the 
TQQarexj ©f virus fre^ ^q affected liv©ro 



683 



-3= 13Cl4ilO(a) 

ii. The morpholosr of the leeksiaia virus Is bsiag studied in 
coilaboratioa wi'bh Ac Jo Dalton of the LaboratoKy of Biology.. 
Eleetron mictoBCGp® stKdiss ef 'rir^ae induced Isukeada Igvsph nodaSj 
spleen f liver and tl^mus glsads have b®«n c&rrled out,, SlMlarlj 
studies te'g's beea ss&d® <m ertsd© aad partially purified Tirus 
©orieent^ates prspsrsd from ths abora tissaes. ?irea particles 
(r®seKsbli2ig the A tjp© particle described by Bemhard) lave b®@n 
foi'M in all materials exsmiaed., 

Witliin the tissues ths particles ars foend ia tha int9r=> 
ceHtalsr spaees » occasioaaliy idthia vaceoles in the ©ytoplasm 
and infrequently aboat the iifmer stsrfaoa of the plasaa membrane <> 
Ths Tirus, bouM Igr a double lindting m^abrane, averages 8? mu 
isi diasistsr sijd it possesses a somswhat electron dens© nusleoid 
U8 sju 3ii diameter o 

Addltioi^l electron ndcroscops studies aro isn progress 
to more clearly define the particle, its relationship to the 
disease process, its nature or morpfeslsgy in tha induced 
localised lymphosarcoinas (ss8 HCI^iilD) and to quantitatively 
relate particle nvsmber in tissues sM pre|:«ired vinas ceaicentratss 
to ttsnor producing activilgro Such etudies ^11 be of gr©at 
assistaiscs ia virus purlfisation investigations o 

5o la report NCI^tlO it w&b noted that the C57BL strain 
of ioice inightj to a degr©©^ b® resistant to infection bgr the 
lesikeud® vlnaSo Applying tMs fixiding aad in eoUaboration with 

Eo Jo Briars and Mo Barrett (S®© NCI=iil8) who Mve sbosn tMt 
j^^^ influences the host suscgptibiH-^ to solid tumor is^laats, 
aS^^ ^jas us3d©rtak®a to detsraine the effeet of repe&tsd cross 
Eatings of C5?BL mic® and a susesptibls strain of laiee on tha 
viral iMsction of leukendac These ii»?in©logioal experiaeats ar© 
still prelirf^iasy and no fissdings are available at this timeo 

6 c Th@ le^SsQiBla virs® has bssa successfully siaiataiaed in vitr© 
for extended peslods of tlma in thes® id.Bsue culture ^steias ly «=^=«===» 
Eo Ao Ifenaker of the Laboratory of Blolo^o Tbss© ^steias ar® 
as follcmss (a) ¥irus indaced le^eiaia spleen cells on Earls os 
mome liver csllSj (b) ?irus induced leukeisia spl^a cells on 
normal mous© spleen cell®, aM (c) Infection of normal mouse ®pl©®a 
cells ^th cojKjentratsd virus ©xtS'^ets^ For cca^lste details Bee 
H0I'=lil6 l^ Dro Hanakere CollaboratlTe efforts have been lisdt®d 
to salsiag a'^ail^ble virus pr©j®r®tioa® ®M. leukeaie aniimlSo 
In additdcm there Her® h^s available sdce basing a lo^lised 
leukesda (lysiiphoeareMa) deseslbsd ixi NCl4tlOo TMs tum^ has 
also been successfully cultured in v itro 1^ Re Maj^kero 



690 



° U » NCI"iilO (a) 



7o In collaboration ^th M<. Fink of the Lsab^story of 
Biole^ the iimimologic&l properties of the leukeiaia virus are 
being inyestigatedo 

To dat® tha antigsaic aatare of tJte agent rejEains obacure for 
it has Slot been possible to demonstrate any heiaagglutination os" 
csojaplemsat fixing antlbt^es associated with the virus » Fluorescent 
antibody studies with Ro Malmgreaj Labcwttosy of PatholoQTi, '^s ^ 
progress ©raJ thus far no positive resalts are available » 

It is apparent timt aside froa the possible prodi2Ctl<m of 
aeetraliBljsg antibodies in a hetcrologoas agrstea the ^iros is 
indeed weakly antigenic » 

80 In the lltsratur© there -m-s reported the ©as® hiarfeosy of 
a obild ^ich deYelojKsd leukomia, beisag bom to a Isiakeiaic laothero 
In soreral of our expsrlmeirtB it has been noted that the "vLms ia 
appar©Jitly timsMitt ed ;g|ortl^ca^l3y to iiie offspring of letakeaic 
psireatSo iffiF'sFtHe^e^eT'oFthQ W-^ generation a high iaeldesice 
of lei^^enda is fomid in these siice bora of par^sts ^ich i^d 
x®c®iT©d Tirtis vhaa newbomo T© d©t@2«sisse the route of traassaissioa 
of the -cdr^ss to the y«2ng a sesles of eixpexlicsnts ^&s ds^ised in 
a>llaboration Kith !» Ida, Mice kaomi to be pregismt were 
inoctO£ted ^th id^tis at varioiss stages of gestatlono Thus cm® 
z&n det@2?mine ^rhether the virus is t^S'SJossiitted via the crrma or the 
plaeontEo These esp^isients are in progress and no definitiT® 
results are a'railable at this tiaeo 



Part B iKelsdod Tea Mo X 



691 



Serial MOo KCI=1a15 

2,, V'±EV.8 oncology Sestion 
3o Bathesda, Md.o 



Individml Project Report 
Cffilsadar le&r 1959 



Part A. 



Project Titles Application of iisimBnologiesl tsolmiques to 
the problem of deaoasti^ating virusee in 
hraaaa asd othsr tismors, either direct3y or 
Ijif refvsaling th© preseaca of specific antibo<^o 



Priaeipal lOT-estigators Maiy Ao Piiik 

Other la^QStigatorss 

Cooperating Oaitss 

Wo Ho Brjan and associates (mi^iZO) 
Jo B, Moloney (HCI-ltlO) 
R« Ao Maimgren (NGI°S27) 
YirgiHia E^ffins (MCI=i^28) 

JSaa laars (caleadar year 1959) s 
Totals 2 
ProfassioBals 1 
Others 1 



Project Descriptions 

lo ^^J^^^s Th© Hsajor ©bjeeti-^e is to reveal^ h^ 
the use of i^a^SloglcS teehniques^ -rii^ses vblch ar© aesoeiatsd 
id-th huaaa tumors either as passenger -virases or as etiologieal 

agents of th© msligasncyo Tbs ti^o modes of attack ="= demoastrstioa 
of the "elros ^th specific antibody j, or dsmoastrsitdoK of specific 
ai3tibo<^ with a knoan i?lrus='=caa be considered together-o 

Preii^iaa^ to the anticipated work ?d.th hmmn. materials, 
knoBS asiaal viruses ^ieh produce maligsmncieis have be©n osed 
as models for the testing aM ds^elopaieat of methods ^hich 

sdght b® S|splicable to th© hwmn problenio 



69 



o 



HCI=U15 



2o |fetood£_e ^gWedg UsiHg the Rous sarcoroa -virus of 
haY© bsea ssad© to apply ths follo^lMg techniqsaass 

lo Prodwction of aHr&ibodies. Formlin treated and haat 
treated fraetlsms of iaf ©eted tiasse have been zisised 
Hith FreuiJdcs adju^Jit aiid iajected into the 

homologous species o 

Batreatsd infected tissue fractions mixed ^th Fr®uM"s 
adju'^nt hav© be©a injected into rabbits o 

Bo Tests for presence of antibody § 

a) Ccfflipleiaent fisatioao 

b) HeroBgglutination-=inhibitiono 

e) Precipitatioa^^in fluid mediai ia gslSj biIcfo 
and smcro methodSy single and donble diffasiono 

d) Flaor©ec©nt antibo<^ techniqeea^^on tissue sections 
to re-ysal presence of '^irusi in gel diffnsion t© 
rsTssl an ©thar^ia© imlsible precipitate o 

e) ^persensiti-^ty taohniqueso 

lo Imaodiate-type reactions "-in yitre Schalta«mis 
t-e'sbMqy;®! ia ^jo anap^laSi'so" 

2o Del^ed-type r&s£B!mB^■'Jk±l^ tests of tubercGlia 
igrp®o 

Ao Production of antibodies = Graaps of chickens were 

successfully isaaisjized against a challenge injection ©f 
Rous sarcoma "idras by the pre^ous ad333iaist3^tion of 
forsalin°treatsd or heated ^rus mixed laii^ Fremsdos 
adjiwaato 

Antisera were prepared in rabbits by injecting th® 
extract of letakeaic tissue Eiixed id.th Fr©nnd«s adjwant<= 
The reselts from ths neutralisation test are not complete 
because of the long incabation pexlodj and until tM.s 
time ws '^11 not know -^tether or not the antiserma contains 
©MdYiral antibodies o 

MLB/c mice are being injected with fonnslin<=treat©d 
or heat treated leukenda virus « 



693 



- 3 « mi-hi5 



Bo Attemptis to demonstrate -dral antibodies or antigen® g 

lo Rocg sarcoiaa jirm o Unsiuccessful attengjts were 
mde ^o^&mimt^^B antibody to Rous sarcoma Tlnis 
in the sertan of chickens Aich had regressed several 
tumors iaduGed by the ^ij^aSj aeai whose serum contained 

a high titr® ®f naatralising sntabcdyp Tha teehffliques 
of complsment fixation ®M precipitation were triad 
first p HeB!agglQtiimtion°iniiibi"y.oa studies could 
not be m&d® because the virua did not agglutinate tha 
©syttooi^tes of aagr specsies tested smder varioas 
©::^©2'imental conditions „ 

The most hopeful lead was vjith studies emplaning 
a flE5oresc©in<=>tagged antibody in gel^Kliffasion stadiesi 
it is believed that a specific antigen=«ntiboc^ 
aggregate not d^aonstsabls by other teshniquQe jsiigfet 
be rsTealsd by this ussthcsdo 

In cooperation with Dr. RoAo MalragreiSj the 
fluorescent-antibody technique isas successfully 
applied to show th® presence of ep©eifieal2y reacting 
Rous sarcoma -^Lrus in chicken taaorSo 

^^ ^^^^ 3-© ^fcggia_jr3ys aSo COTrplesaent-fixation tests 
^^aleS'TSe^resencQ^of antibody against mouse 
antigens^ but not against viral antigens = Mcrosome 
fraetiGfns from either normal or leukemia tissue 
reacted equally well M.th rabbit antisera against 
nosroal or leukesnic tissueSo These findings persisted 
after treatment of the antigenic praparations idth 
Genetrono 

Attespts Hers isads to demonstrate a viral 
hsraagglutinin^ as a preliininiary step to tii© deiaon- 
stMtion of antibody throagh its role of inhibiting 
heBsagglutinaticsio Esythrocgrfces of man, chicken^ 
rabbity gainea pig^ sheep and mouse tave been tried o 
Mieroscae preparations of infected and aon^infeetod 
tissues have been used fresh and frosan^ after 
treatment M.th heat^ formalia or tsypsin^ Several 
times it -ma observed ttet heaaagglutinstic^ did 
indeed ocsm's bat iii?srisb3y Hith th© norsaal tissas 
to the B&V3B extent as tdtij the leukeHdc tisssa©o 



B3h 



k - NCi=Ui5 



A lytic phancsnenon has basn observed repeatedly 
when tl^ lauksjjsic tissKag biit not the norBsal, t«s 
added to guinea pig erythrocjies » We were taimble to 
repeat this with a laore highly refiaad riraa 
preparation th® one tixB® this preparation was 
ai?®i3^bl©<, The 3ytie effect tos inhibited by 
noTB^l rabbit serum &a well as the szxtisora againet 
©ithsr the norraal or leuk^aic tissaso 

Tests for !^irperseaaitivi% to i^e -rlrus are 
beissg done in mice iiaauidzed with the killed Tiros 
Bdsed ^th Freiindta adjuwsnt and sabsequently boosted 

ifith live viraso Thes© teats iaclad® th® highly 
sensitive anapfe^laGtic tests, and a skiu test for 
del&yed°%p© fe^srsensitivitjo Teehniqees for th© 
demonistration ©f d©layed-%p© l^ers^isiti^ty hav® 
been saeeessf ally a©t lap using a solcbl© protein 
satig®Qo 

In collffiberation isith BVo Eo Ao Ifcljngren fluorescent 
aatibody studies are bsing doBe<, 

ko Sigaif ioanee ;to ^io°^f <J;iggJ:_X^-^?:SH!L£'^^S!i?.-^Si^£? 

One of th© asajor objecti'^fes of eaneer researeh at th© 
present time is ttet of deterEsining whether viruses ar® 
iswolved in th© causa tioa of htaraan cancer o Sine© laost 
of the knoKn tismor virosss are species specific, sork 
with haiaan tumor viruses Hill iwjdoubtedly reqair© sior® 
knowledg© iiwolving iHsasanological factors as wall as th® 
develo^ent of ©xparisasntal methods ^bich tak® sach factors 
into eonsidaratioao The work in progress j ssid that 
projected are dir©ct«d toward both of th®sa endSo 

5 o ?r opos^^om's© _ , o£..! ^Bilgg^ s 

Continuation of stcdies tJith Moloney leukesoia vires 
until an adequate iisaunologiGal test syetesa Ms be®a 
derisedy or roled out as a praeticsl po@sibili%ro 

Apjplieatioa of aay fiiKi,';iJg8 to the h\JKaa txsmor tItub 
problem && ths Bsa-feerials hQcmi® available^ 



695 



5 - KGl4i.l5 



Atteimpts to cross speciss barrier of infectXTity in 
viruses as applied to the establishment of human tuinor 
viruses in SBice^ This will constitute the major part of 

the research project dicing the coming year,, 

Plan of att©B;^ts to cress species bandLer of 
infectiTity In -rtrKSss as applied to the establislsaent 
of hisaan tmsor -viruses in mieeo 

le Rationales (1) ^Irna ©oatained in hraaan esll doss 
not infect jsiee because of immsne reaction against foreign 
species J whieh destroys medium for propagation of the ^irusj 
aKd/or (2) outer coat of viral partiele bears hwmxi antigens 
aM an iKBimie response against these prevents viral maltipliss- 
ticaio In either ease, a broakdcfim of the hosfs response to 
foreign spesies should pro^de an environment eondueiTa to 
viral xEUltiplieationo 

If one oeuld break doram the isBSEHna reaction ag&izst th® 
hwrnn antigens so that human tissue would sarvive, tirus 
eottid propagiat® and perhaps become adapted to the new hosto 

Bo General esperiraental apprcaohs Break down imBiisnologioal 
reaction betiseeii th© host and the injected virus ^containing 
tissue fcgr? 

lo Alteration of th© hoetg X=ray 

Cortisone 

Cre£ti'?a of J^potbyroid stats 

CS'eation ©f deficieney ©f 
vltaiains knosn to isfleeae© 
antibody productionj e^gcj 
l^atothenis acid and 
j^ridozine 

Develop tolerance by injection 
©f huBjaa (and in. addition^ 
viral<=>infe©ted husaa) tissue 
in otsro ©r at birth o 

DssensI^Se by feeqaent in^ 
jeetifflas of ti2^ aiaouats 
of foreign tissue o 

2o Alteraticm ©f th© iaoouluaig 

Sid the inosulisa of as laaeh of 
the host material as pesaible. 

Groi? eells in tissue caltvirsj 
both in sertjsi^eontaiaing 
saedioBiji and in chsasleally 
defined mediomo If the 
696 r-elativs antigsrAcitj of -a 

spesies is l@ssen.-£:d -/Iff. 



6 - NCI=itl^ 



growth in tissas Gulttsre, ■ 
this approach might bs 
expected to reYeal pressnce 
of -^rus more certaialy than 
iBoculation of fresh tisss3o ■ 

Attempt to reduce species anti<=' 
genscity fc(y ja:dor freesiug of 
inoculuaij and by lyophilisation, 
so long as these procedures do 
not advers83y affect the 
infectivitgr of the 'girusp, (There 
is evidence from huraan homO" 
grafting -Uiat these physical 
procedures do reduce the anti^ 
g@!EK5.city) o 

Co Spseifis e3cp®ria®ntal approach g 

1, It is proposed that all of the methods outlined ia»i©r 
A IWaii on of the _^ hcet b© attempted first with th® Roos 
iiFcoS''°9lTOs'"1Eo^<'Se gystsnio This choice is rasde 
tesause of the large body of backgroand iaforjsatioR on 
tMs tussor virus o Aetasl ©3tp©rijaeRcfcal work is afe^aitisg 
the cosipletion of an aaisssl roosjo 

2o In collaboratlT© studies Bith DTo Virginia 'S^&m'<s, 

TissEe Caltare Sections results from a s^ill lamabe? of 
saisaals iaosalated with hmsan cells ^ich have been 
in tissue cal-ttira give a steong indication that tl^ 

antigeiixicity of these cells has been reduced for the 
2aoas®o These ®xp®riffl®ats will be contlBQed trntil a 
Talid eosielusioa can be made regarding the effica«^ of 
the procedures in r®duoing the aatig©nicl'^ assoeiatsd 
^th sp©ci©s specificity o 



Part B insisted T®® I I© 



697 



Serial m. HCI=Ul5 



Individual Project Report 
CalsEdar Tear 19$9 



^ Bo RcaorSj Am&TdSg and Publications,, 

Maliagrea^ Ec A,>y Fiuk, Mo A.., and MillSj ¥»? Damonstration 
of the iiitrac©31alar location of Rous sarccsaa -rlrGS antigen 
by fluorescsia labeled aatisarac Jo Mato Cancer Instcj) 
accepted for pablicationo 

Bs" iH^tation froa Tissae Bsakj latioESl Jisval Medical Gents^j 
deli"r©r@d a saries of six ©v®aiRg Isctt^^s on Basic Imamolog^ 
to the staff of th® Matiocal Haval M®dieal Center o 



69 



o 



1. Laljoratoxy of Biolc^ 

2. Tirus Omsolog^ Seetioa 



Cali^^aar Yeas* 1959 



Project S.itle: ¥lml SSaerapy of Sts^rs. I. Maptive ^astsres 
for iKSseased CSasolysis ia Csses of Ad-v^aeeS 
GareiiissES ©f the Cervix 

Principal lavesti^tor: Bolssrt- A« |.&iEa&©r 

Gt^s* la'viesti^tos's: Ho'berG 1. Go\ieh, M^sarS D. Mela«^lia 

Cospesjstisag Uaits: This project coz^leseafes tlae eliaie®! 

aspects of •rixal therapy of eareia^® of ths 
eervix as eosiSueteS t^der the direetioa of 
Br. Sotert S. ^dth. Chief > Sxsy^sy, ICI, aM 
esteMs tlae ^vj©2% iaitisted 1^ Drs. Bo'tes^ J« 
Itietee? aM Wa3Jace P. Bo-^p lafeoratoiy of 
lafeeticiEs Diseases, MAJS^ 9--T52-C. 

Mi® lesra (ealeafer js&t 1959): 
fi0fcs3.: 1/3 

Ps^fessioaal: 
O'&ligr: 1/3 

F^J'set Bsscription: 

pb,j@etiwai T© ecMtiai'se tlse iB-vTgsti^tioBs ia ps«gs«ss to 
detsrMss tM @3£teat to ■wMeh tl^ ©neolsrfeic properties of Co2^®cki@ 
B»3 vii-es coisld te ©ahamesd 1^ ti^sor to ttsor passage 

^ ^qgg : Ssjsor to tiamr passa^ of Coaiaekie B-3 ^is^ss v^b 
cojsdv^iS ia patieats -with e&s'ciiae^ of tSse cervix. Aa srMtrary goal 
of 10 serial passages "esas est. Sas virus iaoc^a V2s©d ■^eye ps«psrefi 
ia Hela cell etslttires isfeeted vd.tSi a lats positive specissa oMaixsM 
fycHJ t^ prscefiiBg patiaat. E^ prepas^tioa of iEoewla iu ^itlaafi 
t^'gtS eultiss^s of '^sus iafeetsd tts^or tissiis ^»3 tsisatisfactoiy. 

^jog_fiB|la^; Fifteea patients ^th csi^vical caseiacmas -^jsye 
ts^ated vita Co^ssaekie B-3 "vls^us. Ia th© eoij^ss of this t^eatagsrfe, foiss" 
aSaitiosal i^ssages ■?-^r© s^de. A total of tea tvm.or to tissor passages 
of -sdrus have aoif baea jsaSe . 2be tssth passage lias 'fesea iaocialstefi into 
t-E*o patisats. lo ses^ts isSicati"^ of iaereaseS oneolytie aetiiri%" 
solelj diss to ps,ssage ia cerrieal t',^ors ^^i-s oMaiaefi. TM® cliaiesl 



693 



Serial Io« 1I3I»U6 



Sigaif icggce : ®a±s st^.j of the oaeolytie activity of 
Co2SgaeMe B»3 'rf.riis is cB.T&lm^. of tl^ ee^vix pisovides a ^»lsl for 
eveslmttioii of adapti^^© -fegeMiqiJss la visul tlMgrapy of l»3®aa tiaos^o 

P^^sed^eOT.^rse: This progissi is of s^cesslty gearsd to tte 
aijailaMXitj of stni'fesble eases for tyial. For tMs ssgasoaj, a Itait 
of ten serial vIttsb passages te eer^ieal tisro^s was set to pso^de 
data foi" evsilttatioa of the acapti'^s^ usasxires used. Tlast limit Ms 
as^i Isgea s^acfesd. Final deeisiaa of tbs eoias^e of tMs project ia 
esBsctsd Ib the asar futiire. 



Fa,rt B iael-aded 



TOO 



Sssrial lb. IfCI- 

1. Isboratosy of Biol©^ 

2. Tiros Cacology Sectioa 
3- BgtbssiSa, M. 



CaleaSs^ Year 1959 



ITt A. 



Project 'Iitl«: fhe Applicatioa of TIbsvb Qnltux& to the Sti^ 
of SiKsr ¥i2i^es, I. Effect of Motjse fisEua 
CBlfes^ I'fe.tsslal Previoxisly Treated ^tb Emmra 

P^iacipal Iniresti^tor: Eolierb A. Ifeaaker 

Ccsc^ratiag Iteita: PaafsHelg aM ©steads tlse ^iat project of 
I3r. Samh 1. S"&s^?srfc (lCI''ii-33) assd Dr. ^zaie© 
Eidjy BSj L¥P| Pathology %■ m\ Mearl Staatoa^ 



Maa fs®s^ (ealeaSas' year 1959): 
Tbtal: 1-2/3 

Prof ©ssiogal : 
OtSisrs *, 1-2/3 



Ob;jeeti^s_: lax'^stigatioaj, ia B^-rias sice, of tte tTMsr-psofitifiiai 
po'teatisl of fltsids f xcisi sons© ssa'es^ra tissiae ciil'tasies pse-^d-oasly iiciseu^ 
la'teS ^th esti'aets of liissasa IsiES&esaic brais tissae. 

feb ^Ss; I^BO -feeclmisrass ^jere applied, (l) Follo^slEg t&e 'feasle 
psocedBre tss@d bj- Drs. SasBh E. BteHart asfi ifessaie© MSj, cuittas'ss of 
wlaol® S%slss siotsse emb^jos tser® 5saista.ia®S ia ^flitm 199 s^3@p3.©Esat©fi 

le^kesde teaia tissue, ferial bliafl j^assag® of fluids tms isade at 
'bi'^vB^'Mly ijatervmls., Bac^ passa,g© -^jas t^s^siiiia'bad after ^4- f^iete of 
iasiilatimis &^?feosa Siriss sice %jere ii»c^jlateS witli c'altB^e jEats^ial 
sf^S^y tJae seeos^^ tM.Kl, asS foiii'tb %^ek of lactabatios. Coatrc*! Mc@ 
received %iati"aat-©c«. tissue etal-tes'a isaterial, (£) Ml fe'tijsaa, braia 
tisssse spesi2®as -^-re're retested is, StJiss soisse e®b2^«o e'dltus©© SiaiiTtela©^ 
iia ^eMxm. 199 s^Wplei^ateirS td.tla raMjit s®s=«ss. Serial "bllM passage of 

e©lis aM st^e3?imtsat fl^lifis \^Te ®5©S as is©e«la fos* passage. 

Ml mice 'a?®se iS©ati^€sd IMlTldimlly aM ©jtssiBsd periedieallyo 
At death., jffiiee tjes© 8sasa3.2s@d grossly for tisasysj ^ad speeisaess %^i°® 
yesoired for pa.tl30l<^ical escasisatioa. 

701 



- 2 - Seslal fe» mi-hlSis,} 

^J22LS^SI^£* ^^^^ ^©si^sis of braia tissue fxt^ cases ©f 
Isiskssila @M 5 speeimeES ©f braia ' tis^ts® frc^ zson-le^il^esaic sow««s 
•i^re tsste4» E:^ gsssults olstaiaefi ^itb 131^ inocixlated contstJl miee, 

287 imiffiocT2lat-e^ siot&er siiea, aM 971 test laice sboBed ao 0i©aifis&at 
aiffes@Hce ia tfee t:^®s of tiaiors ■sMch oeeurred ia the thr^ gr©ii>8 
1,5 to 2 ^ars after iaoeuLatioa. Ibr ¥@re sigaificaat fiiffereaces 
obs®rvBt ia tte si»ljsr of -tjiaoys foi^ la tla@ test gsoi^ wliea e^aret 
■^tjia tl3S aimter fc^Jid ia tli® coatrol groi;^. I^st tta80?s v&re feasM 

's©re t^ aali^assies sost fs^^-?2satly eneotratesed, 

S^^fic^^: fflais project pso-^Sed a test of tfee s«it©,Mlity 
of ao-ass tiss^js etJlttir® as a jssans for isoiatissg -vis-UBes fT<m le^esiie 
lix-aia ©Bd of ''cte ssouse as a tast anissl for tisiaar iMtaetioa ^ SBch 
isolates « 

P^^^d^jo^se: S-he project tes "besa texiaimited. 

Part B iBGlMefij Y®s So X 



702 



Serial lo. imi-kl6ih} 

1. Laljorsto^ of Bioioggr 
2e ?lrus Oncology Section 
3. Bstfessda, m. 



ras-ias 

IMlTid^sl Project Bsport 
CaleEter Year 1959 

F2t>j®ct Title: ^^ Applieatioa of Tisst^ Cultm© to tb® 
Sttsdy of Ts^y Viruses. II , Yinsses 
Associated ^tb Itssaa Ti^Drs 

Pxlacipal laTCstigatorss Bofees^ A. Ifeaaker^ fetert; B. Coiieli 

Otlier Investigators: ES.vsa.r^ B. McLsugbiia 

Cooperating tTalts; Dr. Eobert B. Saith, CMef, Siargesyj, SCS 

fen Yssrs (calesasr year 1959): 
®3tsl: 1-1/3 

frofe&sleml: I/3 
Ot&ey: 1 

Project tescriptios: 

Ob^eeti^^: DetSKMsmtioa, 1^ applicatioa of tlssia® cislt^ffi® 
tQsM^qm&f of tlse eisfceat to -^icM ^rosas ^y fee ae2soast^,t®d la 
association witls a variety of Si^^^i timors sasd to iave©ti^ts tM© 

fetho^s: T^o t@cfe1siq.12es were eEgslojfed to diselose tte presence 
of viruses ia staY^iesOlj-pesoved ttsaors: (l) Ctsltm-e aud obssrvatioa 
of t-tsor tissue for -visible ©videse® of virus activity. (2) laoeislatiegs 
of a I'ariety of e©ll lises ^th asineed irlable trasor tissta®^ ofeser^ratioa 
for ® ©oatb or aorej, aad biiad passage to frsah cultures for aa 
©Etsimleat peflod of ofessrvatioa, TbB first tectoiqias -^^s cashed oa 
l?y tie S^ar^ay Bsofflch, -^CJ... Fluid© aM cell ssatsrial tvtm this so«2«e 
-«^es^ fisffther ia^irssti^.tea by tbe sesoM tsstoique. Ese latter %'as 
carxled 02a "fey tSie latos-a-tosy of Biology, ¥iras Cfeeolo^ Sectioa, ICIj 
asd is l^s^ia reported, 

Tb& av&llabl® sell liass lacl^idea a aossal hsmsm "atM" lias, s, 
"Ijos®" liss wM.cSi ©rigi^ted fro® hmsasi epip^seal ttssvBf aad a ^DE&sy 
^iiissy liBe. Stese were oMaiasd frosi Br. Miltoa Sarl@ as stock 
G'ialtta'ss. I'j^aa embryo slsia-^osele siafi h^assan iateetiae calttsres "5s©s% 
oMimised f2^^ llierobiological Associates j, ^tfeesda, M» 



70: 



Serisl So. lGI"J*l6Cl3) 



1^ jgr_f i^jB-gs : A tot-sl of 15 suygie®liy-e25sised t".Mors 
■^^j'& iatrcdiacsd iato tbs a-^ailaMe cell liass as fieseribefi. Iei 
aaditiOB;, 27 i^eslseas of tissue e^ttsis^d tvs^r laat^slal xsgceived 
from Dr. 1. Cotash of tlss Sus^esy Br^^ach, SCI, 'tjes^ sisilarly 
processed, lo eviaence of -^firus activity bas ^®a observed to date. 

SijBifieagce: "Eae asgoeiatioa of vinasss -with t^jsors is 
ajS±waX&' makes saasdatoiy tM d©v®l<^^eBt of sistliods for i.et®s^ffis.ti©a 
of the extent to tfrMsli visnises imy I?® foxsoS in Imaaa ttssor tiss^is. 

durlHg t«e foUovyiffig year* 



^,st B iaeluaea: Yss lb X 



70;^ 



Serial lo. KI«-Ja6(c) 

i. la.'boT&tQT^ of Biologj 
S. Virus Oneology Section 
3» Betbesda, m. 



PSS"1IS 
Iisfiividml Project tep-ort 
CaleM^-v Tear 1959 



Project ^ffitle:; fhe spplicatlos of fciss^se c^2lt"ar© to tJse stt^y 
of ti2K>r -v-lr^ssso III. Ifeiateaaace of a 
le-yk^aia ¥irus of alee in vitro 

Prineipal Ia\^stigator: lolsert A. ^mak®? 

QthsT iB-TOstigators: loj^ 

Coopea'atiag 0nits: loag 

^m Tears (ealssia? ^ear 1959): 
Sotal: 2»2/3 

Frofessioaal; 2/3 
Others s 2 

Project Bfese^ptloa: 

0b^ec;sl^£: To detsHsiE.® tte possibility of ro\x&ixii& ^ jgltso 
ssaiBteaaae© cf tl^ s^iase lets&aaia iTisiss isolated Ijgr Dr. J. B. Ifoleaeyy 

iCI, 

^;^gs; Sss e^^erl^ntal pattern ccmi^riB&&'. (l) Tests for tfe® 
psrsisteace of t>i® le^eMa agent in eisltiss'es of ssi^d tisjanis, spleea, 
aad Iji^la aofies o1?fcaiaed fro® lei2ka®ic aai^ls. (2) Attesgsts tO" ®M&pt 
tise letfeeMa ageat to sa es'batolishffid ^oii©® liver cell (M,) lime (Earl®) 
wit^ iaoeiila of lei&.€mic tlsst^s» (3) Betesaisatioa of tte possifeili-fej 
of ia vitgo lEifeetioa of nosajsl mouse thjmns or spleea tissue, (h) 
Investi^tioa of t-lie suitaMlity of ma'ose spleen emlt^res for the 
sBa^;fceaaaeg of the leTi2k<»ia ageat in serial passage. (5) COmi^aeterige- 
ties of passisager viK5s®s i-aiish sight ccofoas tba iatesps^tatioia of 
th@ 2®s^t-s o'btaisea in eHltws'es coatainiag tli® le'AeMs ageat. 

la the abseaee of d©f iaitivss eytopathogeaic activity tgr the 
le^issia agsat isi^er tlie cos&Lticsais e^loyed, all tests for -fch® 
pressjiQ® of -virus ^er® sm^s la iaice. Eesults ^sss© a-^ailalsle ^^ to 8 

S^jQg fis^iags; 1= Thsi pei^isteaee of tte le*^iia ag^at la 
tbs fl^ds of pri^jry cuLtwrss of tissises f re® letJk^aic aal^als f ©2? 
e^teM®d psricjis -^ms dssosstratea= Altes^'fee ia ;^w - 'M vttm_ 
passa^s ^-re:f!© also safle. Is oae @2^eri®sB.t; a^oefcoid eells, \^\eh 
'hsM. taeir orl-gia ia tbs li^ssr or la sisfcis^^s of tlj^iaiSj, @pl@sa 
sn& Ism^ astes of le^&smic Mee^ "^isiNg calt^zssd for a psriod ©f 33 iSajs« 



705 



- 2 - Serial lo M01~kl6{c) 

SsiEples of flijlds takes of tbs lOt^ bM. the 33rd daj of cT2ltm« 
■were di'\rlded 5jsto 2 parts. Qa® part was filtered throxxgh a 'feetsrls* 
tight filter to remove cell material, ^th filtex^d asd laa^ilte^ed 
sables vsTQ iajected lato litters of ssi?bom aiee. A to^l of 35 
ales ^'©r® thi23 iEoci5latedo WitMa 3 to 6 sostlis, all tiat ose died of 
leiskeiaia. lo evideace of the p:^ssae© of a^LLigsmat cells ia tbe 
cialtares ^^s not®d. lo difference 3.a latest pesrlofi ^?as saoted foT 
those aaisjsls tMt received oesIj fltaids •tsliea c<^ared to those asisBls 
tlist vrnre iaoe*«ilated irith fluids eoataiaiag viaMe cell ataterlalo 

To ia'vestigate tbe alsility of a Ij^boid cell to ^intaisi the 
virus, a l^fg^feoss-rccsm wMeb had its origia is aa aais:^! previously 
iBoe-«iLated vitla active ©stract of l@i&saic tisstESS, isas obtaisied fr^ 
Dr. Jolm B* Moloasy. Cultisres of tl^ saligaant cell -^gre asiat-aiaefi 
for 7 ^"ee^s. Fltaids eoateiaiBg cell s^tssial pxoduced tiaaors ia 2.5 
t?ecks wlsen iscsnls/md into snice sutosutaasoasly. Fliiids saa^led a^feer 
41- acd 7 "^^eks of c^tu^e ^ere filtesed thsoti^ Selas filters of 03 
porosity t-o resoYe e®3J.s. Lsuk^da rss'ul-feed in 8o perceat of aaiisal© 
inoeiil-a*l>sd s^oaatally isdth fl^iids frcm k ^'eek c^alturesj aiid in 50 
percsat ef asi^als iEostslated aeoxsatally %ri-aa flisids fs*esi 7 vesk 
euitvxes. Bea'tbs f?^ let^^aia oectari'ed tetweea 6 aM 9 aoatliB after 
iBoauLatioa» IPso BOBtla old wd.Q@ also s^ceived ttes® fluid iaoe^a. 
Deaths frc© leisfeesia eecuys^-d la 7»5 to 9 snaths ia 2 of 6 asx^^Ls 
iKseulatsd -sith tl3s ^ 'esesk HiiidS;, SEd ia 2 of 6 aaaissals isocaLa.ted 
■tjith thB 7 'o'eel: fluids. ^Ose restslts siiggsst tbst tlse Ijs^bosase^^ 
sell s^^pOi^s t&e "^r^ss poorly if at all. 53ie ps^senee of tJ^ a^snt 
say have 'sees d^ie to a stall popvd^tioa of asaotber cell tji^. 

2. Aa attss^t 'to establish tlje ietskemis virus ia Sarle's 
cloned aK>use liver' cell lis® ■^as sad©. Over a pes^lod of 2.5 lEoaths, 

h additions of splees and Ips^h ac^es fro® leiskeMc siee -^ere ^ide 
•feo j^^ase liver cult'ures is flasks. Slax'se days after tSse last ®ddi» 

tioB of tisstas, fliaids vers filtered throxs^ a Millipore M filter 
Bssd issed to isoetalate fresh flasks of the i^tjse livsr liae. At this 
tl^ t&e incKJtd.'ma ^sas tested for activity "by tbe iaoeial&tioa of 2^^» 
Isosia jaice. All aalssls iooGislatsd died id-tb Is^akemia 3 to 5 i^^tbs 
later. 'Bssts laiei'e Eade of Q.t2ids fn^ tte cultures isocijiated 'aiith 
tl^ filtsHts ^giasiag vitli the s&soM day post-iafectioa. Miae tests 
'Hsre iffide duriag tb© aext 2 MSS'febs. loss of tbe aniggls ia^ectsd 'aitfe 
tbsse 3^."fes2lals bss 'feeecsse leia&sjaic d'<jri2g 5 to 7 laoaths' o'bses'vatioao 
Evidently, no adaptetioa occtarrsd. 

Hie original caltiires appas^iatly as® swpportiag cell ^terial 
frosj the tiss\:^ laocals. le^^assia •sas produced ia aaisala 2.5 to 5 
ssoatlis af-fcsr ssoaatal iEjsetioa of etjlt^a'sg fl'oids takes 8 weeks i, 
9»5 '^?&®kef @s& 1^ ''Jesfes after t^se ctalti^es 3?©eeiv©d th® last 
iaoc'olation of Isvfessaic tissisg. Fluids is t52@se ctalttires "t^i^ rotsrfei^J 
elsasged e<^let®ly thrice t^s 



706 



- 3 " Ses-S^-al So. WrOl-hlSic) 

3. To detesaais©' th® possiMllt-j of J^ vltTO iafectioa of 
spleen asd tbjfJHus t-issi^ ei2ltu:^s prepared fxask saosaal BMS/e sice, 
a pc'fesiiti cell-free esfcraet of le^^^iaic ^u®8 tissi^s ■^as ©IrlJEijEed 
tfero'ogb the courtasj of Dr. Jolm B. I&loaey. This ®stss.et vas wsed 
to iafeet caltisrss "riaieSi h3.6. teeea ^iat&iuid for i? fiays isa vitro. 
■She lei'lsesda agent ^ms preseat in both cHiturss 1^^ dsys thgreaftsr. 



l*-. Serial passage im© atte^t«d ia ao^se ®pl@«R cuItiis^So 
Tlssee e^merisejats '«5srs ear^iea otzt. Caly tbe first of these bg,s 
tees, leader %^y si^fficieatly losg to yi@M sigsaificaat 5?est0.ts. Sli© 
e^^erfjseat coasistsS of 3 s©ries3 of colt'or'es. A scbedisle for passage 
of omi^teuyh. of tfee fl'oids frcsa tisfces of each series to fresh -feiates 
each veek was adlssred to as missh as possible. This first series qo£i» 
sistad of Bniafected control ciilt^ires, tbg secoM consisted of eialtus©s 
infected -tsltM ceil^'f rse erferaet of lei^essie ^siase tiss'j^s pr©pas©d 
hs Br. JoliSi B. l-loloaeyj aMiSie tMsfi eoz^isted of c^slttmss iafeetefi 
■^jith 0.01 al. of vi-able^ sai-aeed le^^esic spleea, l^spla ssoiasp ®^ 
tb^'Tsus of a soass bsarissg tfee l^tJa traasplaat passage of leis&^Eic esils. 
Fluids in eulttsres tfere cbaaged ems^letely 3 ti^s each 'Keefe, le-^sos-a 
sice frere iEOCvilstecl vritli fliiids asd c-ell ^t@s?ial f3?i^s each psssags 
i?it!i tli© esjseptios of tfee 9t&, 12fej, ai^ 13th. 'B^ last passa^ ^ifl® 
vas the iHli, 

lo l(St2s©sda eecmrred to 'iiie iiac^tilated control Mc©. So 
lei2]£esjia liss l^eea observed 'b©yoM th© fotas^ch passage of fluids frtm 
erfcract-iafeetsd ctd-tBres. Osse of 7 laiee that -aes^ isjoswlsted -Kitla 
Esatarial fyaa -fcMs fourth passage etsltm?@ died of letsbs^ia ia 
3.25 KOB-Sisj t4is r^saiader are still 'esiBg o'feser'^ed. ©ag l©iak<g®ia 
ageat ia t^ss culti'xss iafseted ^th tlssiss l^s teea KaiataiE!®fi fes" 
11 serial passages. Oes of T ale® Sjioculated 'td.tb 11th passage eraltm'^ 
■material is leiskeiaic 2.5 siffi^a-fclis aft«r Ji^aaatal isjectioa of teat 
culture S2a*&srial» Lei&tssias M'to oeeisTaS ia aM^als ^ased to t®©t tte 
prior passages for px^seace of i^ss e^at. The ageat c©a lis ^liataii^i 
ia "^troj bwt its Issfeavior tis^er tl^se eoiiditiOBS is asot ^t eleas-^ 

5o 'Swo virases ^iicla ps^ixce ^patitis is laice ^se igolated 
dinrisg tbsse sttsdies. Ths first isolated "^as wssb exfe@asiv®ly is- 
irestigatefi than tl:^ secoM. A paper reportiag the results obtJiisssfi 
for pi 



TtiB hepatitis ^flriis saost exfeeasi-s^ly studied te,s Iseea 
desigaa.t€d M?»A59- 2t is sta'ole to ©tber^ a cferaetesl^tic 
diffareatiatiag it frss tli@ seeeg^ egest isolstad. Eeatiag at 
560 Co for 30 minutes Sesti-o^rea iiif activity. Iiafeetioa ia -felie 
Blil®/e ^o-ose isi2.uces iisjEaBlty. Sert® f x-oa IjgBRsa© ©aisssls ha.B a 
i-slatiTely Mgh as-iitralisatioa tit«r. Ta^.B® p:raperties Siffereatiate 
tMs agent fsim o^ers tliat hB.-^B 'baea psev3.o\aslj reported ia tli® 
lita^^atUi-is. feaa-jiS mottes^ traasmit s-esistasc® to infeetlea to tlaelr 
Tiroge^^ •witMn bs'isrs of Mrtfe» Ite x-lros has bgss pattsagsS ia tissts® 
ciilttis^s of larls's sotsse li-rer line asfi iu movse sple«a ctal-fetires. 
Gytc^atbogeaic eliaEge® s.tb ■pTQ&vs®^. ia teiai systeHss. 



- k ' Serial So. K2»^i6Cc) 

gig^iflcsjige; IMiat@Baace ®f tJs® le\2teaic ageat la vitro 
ss&es possibi® eositi'olled stt^ea ^nd pesisits isi'vestl^ticsis not 
possiMe Itt -gl-i^o. fh@ Meatificatioa of a passen^r viras 
(feepatitiij s'^sS det-elc^s^at of i^^ssfis for its prepagatioa will 
pesait its elisiimticsa tbro«^ i^sjsMsatioa, aaad thias a ®©s® 
a&quate c<^trol of th© Mologieal stMies oa le^-sM.a. 

S^^^.SS^®' '^^® program iriH "b® ftot&ss? €e-relo!pga 
sad S2^&aa&t fitiiiag th© ^©seS y©as"a 



■P&rfc B inclMed: Tes lo X 



70 



p 



Serial lo. W.ClA-20ic) 



2. ¥isTis tocology Section 
3' 



IjaaiYidml Project Bsport 
Calesfiar le&r 1959 



Protest Title: Stt'Mes oa the Boas sayeos^ vir^s. 

IIIo l^H'ther stiMies cm tSie relation of 
reco'wera'ble visms to tiB^sr iaitiatiBg doss. 

Principal lavestigstor: W. Bay Bsg^aa 

Other Investigators: BoToi^j Celaanj, Joha P. IC-vefiar 

Cooperatiag Uslts: Mfeert J. DaJ-toaa aad associates (lCI-^06) 



^M Years (eaiess^r jsar 1959) s 
Total: l"l/3 

Profsssioaal: 1/3 



Project D©sc2''iptioa: 

ObjaetJ-ges; ,fo dstes-Ma® wietaer tbere is a tt^Btitsti.Y® 
correlatioa bet-^'sea th® iacideac© asd/or population density of mral 
particles sssa ia tisia seetion eleetxoa mieKsgraphs of t^asoys aai: 
(a) '&be doss of -vir^s •used to initiats tfes t'saaors, or (b) t^e a^o-smt 
of sstractaMe V3,rffis dstesMaed Ijy Mossssy. 

gg gggal eo^ gat; ®se ®3g3sri®eatal ^oyk as8ociat<ed witli tliis 
protest ■®B.s e^jlet®d la Qatdb&T 1959; sM tlie ^tMods eaog>l©y®i. are 
therefore saot reitasated (see 195^ import). 

g^jor fi^lags: Assalyses ©f tlae Moassay data sfeORiF tfeg aas© 
correlatioa 'bet'jsea t^e saouat of ^-irsis estsaetsM® f^oa t\^©rs aisa 
the iaitistisg dose of ■vi^iss as -^as ps^vioiasly fo^^S;, sM reportsd<, 
It is tbe cesrelatioH of these flMisgs irit^ virus-partiel® camrbs 
imder the eleetroa ^es-oseope t-Mt coastittstss the chief pisgpos© of 
this pro^eet. The electron sicrograpMe sttidies ag^ ia progress is 
Dr. Baltoa-s MboTatory, "^St hav^ aafe ^t "beea eo^letsd. Fisal 
©valiiatioKS s-sait tbese results. 

SigBJf ieaaee ; Tk^ resmlts ar® ejected to eoatrilmt© to fcasie 
kao'ifle^ge coaeeraiag the ssode of aetioa of ttsmr ¥lr!^©s ia feriHglBg 
afeout Bs,ligsBat traiasfoi^atioa. 

Proposed GO^JTS® of project: Ccm 



aM t^iritiBg of TepoTt toT pts'olieatioa. 
_^rt_B iBcl^taed: Yes lo ] 

703 



Serial lo. Kl-teoCd) 

1= laborato^ of Biolo^ 
2= YIt^b Oaeology Sectica 
3. Istbssd®, M» 



CaleMar fear 1959 



Piojest Title: StMies oa Bo\ss sarsosa -^rirus. I¥. lavestiga- 
ti^tioas 6a Isost s^spoas® to the Tlrus (a) ia 
eMc&sas smS (b) in Japasese qmil 

Priseipsl fev-iestig&tor: ¥. ^y &^?®s 

Other la-TOsti^tors: Fo J. lauscher, BorotSgr Caloaa, 

Coqgersting Waits: J. A^Bb^js^-Q aad associates of the 

G«s®f5?ea Life ^seareli C©ja.t@r, Tassp®, Flao 

Ifea fears (ealesfiar jq&t 1959): 
!fetaL: 2-1/3 

PTOfessiosial: 1/3 
Ot&er; 2 

Fso jeet Beseriptios; 

Ob^ecti-TOs: (a) fo dsteEsai^ tJie uLtiaate llait to ^aieb tbe 
eoaceatsatioa of Tirus la Sous sarccsi^ tisstse caa "k® increased tteotigh 
coBtim^d selesti^^ serl^ passages ia cMckeasi ("b) To detesaias tfes 
su5.tal>ilitj of Japs-jssse g.'jsail (Coti'^^x japoaaica) as a host for the 
sti^.y of Boiss sas'c^sa virss- 

^^^s: (a) Fast e^sr-leaee Ms stosa timt tlxe virus c(m.^ 
c&ntmXi&n of lo^as tusor tisst^ .eowlS te progs^ssively iaes^ased l^y 
seleetiBg as a sowsce of vistis at eaeli passage that t?®o? ^bicb 
appease first;, aM g3?eif ssost smpifily. Is a grotsp of aljout kO ebiekeas. 
Siscli genial passages &Te teiag ecmtiataea soutinely;, sad ^11 'm saia- 
tsiaed tmtil sueh tisss as aa t^per lisit of visas CQEceatratiosi is 
raaelaedj aM ao f«rtiis2* isaerease -Hitla passa^ is detectable « 

C'b) E2@loESt-o23r sameriiBgats la eollalsosstioa with Br. E&yMX;£s 
hsL-^rst slio^m that Wb Japaisess t'^aall is Mghlj s^iseeptibl© -tc tbe Hoiss 
sa2«ecsm v±T%mo Wut'^.qt studies are teisg eon&vcte&p iaclMlfflg 
passages ia q-«mil siiallar to t&ose is cMeSeas (see Ca))y to detes^ise 
■ai^ther tbs qmil (a ves?y sssail f©-^?!) can repJ^ce cMcfeeas in 
lafeorato:^ st-odies ©f t^e ^d.ras. 

^M.JM^ME' C®) ^t "^^ t3«© of tte last ^port (1958) a 
t«tal of o-mr hQ passages ted ^©©a seSs sad tbe overall poteacy of 
ttBSKJr tis0»3 esfesacts had te@a ias^sassd by 1=5 log wiits, tteOBgb 
seleeti-^?© p.assag©s. TI2© 3tn^ is aow ia the &2s& passage aM. pofceaey 
tes teea te"&ter iaGreased Isy O06 log tssits (('-5- fold) d^riag t!ie last 



710 



Serial Mo, W£l-k20(i 



year. This represeats a total iaci<®ass of 2.1 log imlts (ovsr 100 
fold) ia pot-easy o-ver tbe sstsrials ©"(ja.ila'bie "i^fosm this slr,^ ve.s 

(b) Pa^lisiaargr sti^ies bav^e slao^m that tbe loijs sare^sa ylrtiB 
titrates Out as fay in qftmil as la tlis ssost s\asceptibl# cMeksas yet 
iavsstigatsd. Also, tbe yields of virus fsc® ttMors of qtmil iMueed 
i-ritli stx'oiig fioses ar© ©tual to t&e Mgtest yieMs ofetsiagd froa tSie 
jsast susesptiMe cMckesa. 

Signlfiesaea; (a) !Ehe pas^os© of attssg^ts to ij^ssase •vis'as 
contsat of i^moT tissis© is to pexmit piarifieation of tlse virus ia ' 
qtamtities sfieq^mts for ptoysicalj QhsmiQ@l aM l^smjologieal st\^i©s. 
Tiie poteaeies alreasSj acMsi^ed iroisld js^le it ps^etical at tiais tl^ 
to i23aa.er&ake pisrificatioa stMies. Bsne-vBr, -^se task will he easier 
tbs M^^r tl^e \^TVs eontsat of tiasoK* tiss?^, ^sd at-fcss^ts to^aM tMs 
esd -Kiil lie ^stpotrnd mitil tlie t^psr Mological lisit of vis^i^ eon- 
ceatratioa h&& "bass dsfiaitely estaMisfesd. 

C"b) If the sesults with qi^il ara readily ^eprodtsciMe tlse 
qpail ■trill ccsastitute a vstj v&L'm.'ble Isost for sttsflies oh tise Sotss 
ssareffi^ -rlros. The cMef advastagg -sill Ise in tli© i^alles* slse ©f 
tla(g t-est asii^l, \-3Mela is ao lar^r tlMsa a 3 ^J ©M cblek t?l2SB ftill 
gsfomii. !13ais ^^uld pes2iiit 5 to iO ti^s as mssh ia-vesti^ti-i^s 'SFOife 
to te Sose ^thla tSie sase space as is acm fe-foteS to cMekejas. 

Future cQUT^e of projaet; (a) Selective passages -«dH te 
eoatiBiSd ^aitil as upper li^t of ^t^b cose©Bts®tioa ia t^s^a? tis©^3© 
lias teea i'eaefeed. ("b) Itjse exfcensi-^a e3^erissats ^11 be carsded out 
to tsst the Eepsxjdttsi'eility of tb/& pg^lisiaasy fiadisg -^th q.imil @m& 
to detsssdse their s^taMliti" bs test aais^ls for tMs visias^ 



T^Tt B isolt^ed: 



" 3 - Serial Is, ICI-teoCd) 



CaleMar fear 1959 



^gt_B: losorsj A'tsardSj asi FuMicstio^ 



Publicatioas atl:^©? tlma absstracts fTsm tJsis psojeet: 

Bsysa, ¥. K. : labaae^ssat of vItxib yieM of Koiss sasecssaSo 
Acta tJaio. IstsEmt. cost?© le caiaeer 13% 761'-T6Tj, 1959 « 

Bgyaa, H- 1. : Q,iaaati'fcati^;is Mologleal espeslsseatatloa ia th© 

¥iras asd caiicer fields. J. fet, Caaser last, 22: 1S9-159, 1959<, 



EoEOi'is aEd A'^mrds s^iatlEig -bo this project: kob© 



712 



Serial !3o. WCl~h 

1. labosatosy of Molo^ 

2. Vi^u® ^ticoloi^ Sectiosj 



CaleMar Year 1959 

Project Titl©: Searela for t^^asr isdtseiag viri^es asosig 

S20W kaoija to lia^ie s viral etiolo^ 

PriBcipsl iB^sti^tor: ¥, lay B?y®a (Dis-ec-^r of Seetlssal 

Loa ia ^2t 



Otiser In-isest-i^tors: M. A. Fi^j B. A. m.mk&Tg P. J. Patischsr, 
Boro-fehy Calnsa, Jobs. P. S^ns^r 

Cooperating ffixlts: H. B. Mdervcmt aad assoeiatss « ICI"^00 
A. J. Daltoa ssS. associates - KI-^-06 

Maa Years (ealeaasr jear 1959): 
TotBlz T.5 

Prof essioaal : 2.5 

Projeet Description: 

Qbjeeti'^es: To fietersjizse t-jiie-fel^r vario^^ s^^aaliaH t^®ors 

sssoeiateS ^Mth viml etiological ageats, 

gsB&gml ^•fcatsiaaat ; TMs projec^t is part of a las^r eol" 
lalKsratlT© stijdy aM lEvoi-^s four Indspeiadeat ia-vssti^tors @M ti«o 
research sssistsists of professioaal gimfi« mlAia the ¥irBS CMsologj 
Beetioa» 

fisse activities of -feliis Sectioa coast-itiate a psls^s^ part of tise 
larger colla'boj.'atiTe stufly wMeh iavolv^s otlser S®ctioas of t-be Lalsoratorj 
of Mologj, ®M ©t!xer lafeoxatories of the latioaal Caster lestit-utej 
®s tsell ss teo Py,Mie Health Service lioj^itals. Coorfiiastioa tsetnissa 
seetioBS aad Is-toi-atories is pro-rldefi "^ -fete eMsf of tbe iMt&v&toTj 
of Biology. Ovsrall direction ®sd eoorf.iB.stioa of tl^ joist st-isaies 
ia-^ol'vi^ P^ Ssospitals ia proifideS Is^J' the Associate Bi^getor ia Clarge 
of l©ssare^, ICI, 

Mstlaoas e^lc^M; Slece ti^or visijses of latosatory aaisale 
®.T& species speeifiCj Immsi. tmoT ^rsses;, if tfes^ e^dst^, ^j also 
fee @j?^€©tsd to te speeies sxjeeific» This coastitu'ljes t&e ssajor 
obstacle to as a3J.»otit attack ob t-fe h'si^m proM^a '^tii existdsg 
teoi^ledge aad tscMi^iaes^ siace Mological tests for vd.s'xasas eaimot te 
csffrled out *ritMa tbe saas species. 

713 



- 2 » Sea-ial lb, ^lAsoff ) 

5Ss0 iK>st pr€E2isiBg epprocicli to the hmmzi p^oMeia at '^jfes pseseat 
t3ija is tlmt of t'.sl2g IxyBaa cejJLs gjoi.'a ia -fcissi^ cultu^ as Biological 
ia.dieatos's of viarus acti-nrity.' Tiaia fipproaeh -sFas thsTsfq^s s'i. 
fis-st. am bas eow fceea la prog2=Sias for 
aM associates - see Ms report;, KI°^-l6)-„, 



A second approaen lavei-^s %sb vse or r^jen-o as'vieiopsga-cs in 
the field of imajsology assd tb@ ia^eetioa of la^ratory asaimals la tfes 
l^te foetail or Besss'feal sta-ts for tbe iBfii^tioa of iassass^logical 
toleraace tonaM forslga (ht/^aa) ^ifiral aatigeas ia 'Mbo^itOTj aislsals. 
E:^?lorato?2r studies, ©sly, ba-ve beea easi*ied out tiros fer, pesidiag 
the availaMlity of aai^al 2«oes eo^ ^©Ser coastruetioa (sae s^pojt 
of Br. M. A. Pink - KJI-U5). 

TcB third app^jaeh sou 'feeing p'os^^^d involves tbe isasc^slation 
of both filtrates of ss2S^Lii^i t^aae? tiss^se aM ti^sr tiss\2e f^apisats 
ca-bo the etorioallai^toic sembraEes of Isota eklckea aad Japsssese qi.uBil 
e^s* Tschaieal ps-osefitases are teiag Se'^^loged -^t^ t^ssors of 
laljoratosj" aai^^ls set sio'!? ksoTsa to hB.-ws s vimL etiology. St^^ies 
iam>lvtog liii^ai csB.ee2s ^fill "bg iastig^tsS as so«m as ^bs teebaiesl 
ps^esdi.i^@s ba'^^ bsea st?i^"Ici®a'fely a®"wels^©d to instjre a fair eMae© 
of success, Uatil resea.-fely t^s@ stifles -^qt® coMxaets^ "fey tl^ besS 
of tl^ ¥2^us C^eologj- Seetioa, bat fea'^ aay^ "besn tussiB& over to 
Di^ F= Jo ^.tiselisr tsho s^cently jDiasS ti^ staff. 

Ia gSaitioa to tiss stoi?© biological appsoacbgs, stt^ies ia 
coila'Dos^tioa witli Bi:». Baltoa eM Ms associates of the Gq11^23^^ 
Biology Ssctioa iavol^s ej^^iaatios of ti^r tissi^s 1^ electsoa 
:®2.croseqpj for e"rissBe«: of' 'cb& presescs of islrtss parfcieles. Toi^x^ 
this BtiAf sssaiSstg t'jsors ars ©stra^tsd aM processed fe^ setliods tS^t 
mvs pyoi^d svx:eessfi3l ia ■'jjork "vrl-^ fe^osjn ^li^al -bis^r irirusesj sM 
ceaceatrated f sactisos fjihleh ^oiJlfl be es^eetsd to eoataia particles of 
tte sisse of -v-irases an^ Serl-ved % sltraseatrifj^tioa. fisg pelletised 
isatarials are f issd x^iile ia ■^is ceatilftige tubes aa.d tl^s ps^sssssd 
fos" this sactioa elestrossaies'oseopy* l^smisstioris of all strata ©f 
tSie isellets ia loagit\3fiisasl seeti^^s pso^ida© a basis for the istestioa 
of ¥irus er vS-E^J^-like partieles, if tlsey as® psessat (Se© sepo^ of 
Drs. Balt-oa si^. Eei@©l)o 

Fisally. a s^jor part of the o'^erall s^ssarcli ps^Ject is^'el-s^s 
tbe sixgilsr t-estiag ©M st^iSy of ssEimliaa tilers otijar t&sa tljose of 
hmmas. "but -^laicli ase Eot asw ks^o^KS to ba^sB a visml ©tiology. Siaee 
Buoh. i^terialg caa 1% piit bseS: iato iaflieator laosts of tbs sai^ 
species (vario-as lalxjsatoffy aa:l^^s}j a iseaais is tbiss provided for 
pro'^isg tfe® ©ffieaey of tl^e %"asi.ous pros^fitssss sM appr^ickes QHtliaefi 
atoiise- for tl^ detsctio® of t^^r vir^isas. 

^jQg flMia^; ^js piast ^®r tes Mea speat in the fiei?el®^ast 
of ^thofiSj 2^er5iitisg of aew p©ra©is©lj bM. the s^^sisllsg of lafeaga- 
t&ri®s ®ad aaissal ^'©©aso ^-feSjoi^li aetsai isa^ssstigatioas a»s siE^eadj 
iSMBTimjg tiss psogs^-m is aot yet ia ftaH ^eratioa. ^ f isml r^sislts 
&V& a'^?a,±la.¥Le f©r ^©poytiss at tMs tis®. 

71-1 



3 - 



^2J®S^§_22^S2.* K'tsslsg Ishe ealsMar s'say i960 Htm foUcmiag 
jTiSa years mil "be ^ voted to tMs pKJjsst: 



fotal: 


13.5 


F^ofesslQjaal: 


3o5 


O-titees*: 


10 



Part B isclisdecl: 



Seflal No« HCI«250 

1. Lab, ChemJcal Pharsffieology 

2. Office of the Chief 

3. Bethesda Itj., ^1afyiand 



PHS-NIH 

Individual Project Report 

Calendar Year 1959 



Part A. 



Project Title : Pianninq, Manaaemcnt and Coordina- 
tion of Investicrations, Ortianlzcd In an Interdisciplinary 
Team Approach, on Basic Science Aspects of Phamacology 
in Cancer, 

Principal Invest icj&tor g Fmrray J, Shear, 

Other Investigators ; Adrian Perratilt. 

Cooperating Units ; 

f%n Years (calendar year 1959) s Pat lent Days 

Totals 11 Mone 

Professional? 2 
Others 9 

Project Degcriptions 

A. General 

• I. Plan 

The strategy has tv/o main facets s a) inves« 
tigatlon of factors in the raaranmlian host i^hich are c®pa«» 
ble of influencing msjor phenomeim in msHgnsncy •«• vls.^ 
genesis, arov/th, invEsiveneess and metattasisi and b) 
contributions to principles of methodoio^g and raode of 
action^ in drug treatment of cancer. Although these ti^'o 
main lines of work v/ould appear to be unrelated ^ some of 
the findings will require interdig Jtstloa of the imnrnRolog* 
ical and the chemotherapeutic approaches for their exploi-* 
tat ion. 

The points made in this part of the report 
last year continue to have relevance. 



716 



Serial no. HCI-2$0 
Calei^ar Year 1959 
Part A - Pape 2 



2. Organization 

Rc-orlentation and rsorganigstien of the 
r-fec?«aiiolecular Chenisti^ and of the Biochemiemi Phan?®- 
eology Sections, described In the previous annual reports 
have now been accomplished. 

Dr. A. W. Schrecker is perfox^inc? ably, with 
high morale, together v^lth Dr. J, A, R. Mead ho pisns to 
stay v/ith us and become a citizen of the United States i 
they v/ork well and fniltfully together. Dr, M. A, Chlriaoa 
Joined Dr. Goldin«s grotjp dtiring the year and is taking 
hold in a quite satisfactory fashion. Mr. John Vendltti 
is functioning, to our eorapiete satisfactlonj as Assistant 
Project Officer in the scientific operations usider cofi" 
tract arrangement at Hlcrobiological Associates. The work 
of Dr. Qoldin snd his ©tsff h^s Increasincsly been receiv» 
ing favorable recognition. So rasny rcqisests sre beinp mB.de 
of Dr. Qoldin to provide his experience mnd judgment to 
affairs of chemotherapy on the br«»der scene that, in view 
of his ii^hole -hearted co-operative spirit^, there is nov/ 
the danger of his bec^^ing ©verie®ded. 

Dr. P. Te Mora is eontinsiing to fsature^ ©s 
a biological scientist ®nd as a section leader^ in a 
gratifying fashion. The work he and his coilmborstofs 
hsve been doing has attracted favorable esss^ent from out« 
standing research men in his field both her® and abroad. 
The following personnel changes are being madei Dr* D, 
Vivian'? s retirement for disability becf^aes effective at 
the ena of 1959, '^nd his technician is being transferred | 
Dr. J. Preiss is also being transferredi &n^ Dr. E. Merler 
is leaving for ® university appointment in January I960. 
Recruitment for replacements is in progress. 

In the Polysacchsride Section a Iso* there 
is a continual ferment of exciting Ideas and findings, 
which go on despite personnel turnover. In the spring. 
Dr. R. Sksrnes left to work in Prof« GrabarSs laborstory 



717 



SerimI Ho. UCl^2^Q 
Calendar Year 1959 
Part A » Pa^e 3 



in Paris for a year under an NIH Fellowship and, at the 
end of June, Dr. F« Rosen returned to Boston tinder an 
appointment at the ChiXdren^s Medical Center of Harvard, 
The repiaceiaents are two younp raenj Dr. G« Michael vho 
recently received his Ph.D. !n Microbiology at Rutgers 
under Prof, Werner Brsuni and Dr. \l, R. Keene, \sho had 
Just completed his internship at the Johns Hopkins Hos- 
pital. Both men shov/ signs of promise. 

Toi-^rds the close of the year, realignment 
of personnel amonji the Sections v/as carried out to depict 
more realistically the actual supervisory relationships. 
The najor change was the shifting of Mr, Perr®ult and his 
subordinates to the Office of the Chief, 

B, Specific 

The activities of the Labormtoi'y are described 
in detail in the aeeosnpmnyi^o reports by the four Section 
Heads. At this point, a few specific itesss are selected 
for mention. 

1. Further integration of the labormtoiy pro- 
gram v/lth the clinic is in progress. Dr. Qoldln contlmscs 
to maintain & close and regulsr liaison v/ith the CHnicsl 
Director end the staff @f the Medicine Branch, Stepa are 
being taken to extend to clinical R^terial from ths Siir«» 
gery Branch the findings made, i/ith aninial mmterlsl, in 
the sections dealino with niacra'noleciiiar blolo^ ©ad 
chemistry, 

2. Dividends are beginnina to be obtained from 
studies of host factors in the marr-mai v/hieh affect bsc^- 
terial polysaccharidet and bacterial eellt. These fir4d*»- 
Ings are valuable in themselves b«tp in addition^ the 
bacterial cell has been employed as a "raodel" for the 
masOTmllan cell. Extension to tumor ceils of enr nev-er 
concepts and imterlais Is yieldino positive progrcssa 



718 



Serial No. I4CI-250 
Calendar Year 19^9 
Part A - Page k 



3. V/e arc still v/restling with th@ d iff lets It 
probieai of unambiguous isolation of polysaccharides with 
endotoxic properties from marcaaallen material. This de» 
veloped from the experimsnts of Dr» E. Merier x^hog® ©t- 
tempts to confirm and to systesjiatige the empirical pro- 
cedures of Mr. Perrault hinhlighted the necessity to rule 
Qntf rigorously^ the role of contarainatlna bacteria by 
definitive culturlng of samples of the nmteriai taken 
throughout the processing procedures, Mr. Perrault hss 
now succeeded in governing the protracted imniptilationsp 
over i^r^ weeks. In the open laboratofy so ih^t bacterial 
contamination (as checked, independent iy, at every stage 
by Mr. Trapanl) is not a factor. The final products ob« 
tained in this v®y, however, have b^en unejspectedly large 
in amount, snd hicjh In nitrogen content, as veil as de^ 
void of endotoxic characteristics, v/©rk Is in progress 
to split off the polysaccharide isolety frojn the proteins 
with which It is presufsably bounds 

I^» Requests continue to be received by the 
Chief for consultation, advice, and help in mzny differ- 
ent kinds of scientific matters. These appeals com® fram 
various parts of the NIH, other Federal Goverrment Dspart«» 
ments, universities, scientific organ £ sat ioas, indtastry^ 
and Embassies of fereipn countries. The requests come 
from, this country snd abroad ^ and are made dowsi the line 
frora Directors of Research to recent graduate® and stu«» 
dents. These demands are laet v/ithin the limits of fe^s^* 
ibiilty. 

5. A good deal of time has had to be given by 
the Chief to the affairs of the American Agiocl^tlon for 

Csncer Research in discharge of his official responsibil- 
ities la that organisation as Presid®at«»EI®ct ^ as Giairsmn 
of its new Policy Casaitte®, and as Its representativs In 
the V/a§hington area* 

6, Item 6 In the report for 1958 stated that 
'^International research activities claimed gn incrssslRg 



719 



Serial Mo. NCI -250 
Cslendap Year 1959 
x%rt A « F©ge 5 



share of the time" of the Chief. This "was even more the 
case In 1959. He continued to contribute, v/ith others. 
In the drav/lng up of proposals for v;orid-.\^ide cancer re» 
search through \^fD^ He re-organ! sed, as Chairman of the 
Finance Committee of the Intematior^l Union Against 
Cancers the obtainSnci of ne\-f financial support for the 
UICC, and continued to serve as Chaiman of the Chemo- 
therapy Conanittee of the Union. In his capacity as an 
officer of AACR and of UICC, he devoted (Ijy Invitation) 
considerable time to staff work for legislative circles. 

Future Course: 

The General philosophy of the pregrata of the 
Laboratory is smch the same as described in the report 
for 1958. Coordination and interdiqltatioa among the 
various projects in the different sections will eontiniss 
to be encouraged. 

Directing research is relatively easys a) v/hen 
Instruct ions are issued by the Boss r-fen^ and foilov/ed by 
a docile staff (lee,, Regimentation)| or b) v/ben in a 
group of creative original men eachj, separately, follov/s 
his o\m bents with the Chief merely being their admlnls-> 
trative servant (l.e.j Lalsseg-Faire) . Giving leadership 
to taientedg strong-^v/illed ^ temperaiiiental scientists is 
difficult enough when each v/orks on a completely independ- 
ent project, but the difficulties are rnultiplisd sjsny^foid 
when the objective is to rralntain their enthusiasrn for 
working toif^rd a cOTimon target and vlth raateriel in common.. 
But it is v/orth the efforts 

Discussions have hean held by the Chief with 
several of the staff of the Surgery Branch to the end that 
this Laboratory might explore postibie differer^ces betvesn 
the blood sera of eancsr and non^saneer patient® with re- 
gard to the newiy-uneovered cytocidai factor for tumor 
cellsi also, to deterrain® ''-tiether hursian cells (nonsml and 
malignant) behave as do those of mlcQ with respect to 
capacity to absorb out thi§ factor frc^. norstml hurnan blood 
serum. In the liaht of the work of the BjBrkiunds In Sweden^ 



720 



Serial M©^ NCI -250 
Calendsr Year 1959 
Part A " Page 6 



and of Fcdoroff in Western Canada j this is on the docket. 
It only rermlns to persuade the leadincj men in th« Labo- 
ratory to give these a sufficiently high priority. 

At first sight the mechanism of carcinogenesis 
by chemicals might seem to have no obvl<ms relationship 
to the other lines of v/ork In this Laborstory, However^ 
there are now reasons for suspecting that the host factos 
presumed to be involved in other neoplastic phenomena 
under study by us jmy also play e part In the generation 
of tumors by carcinogens. Hence \-ie propose to explore 
this hunch during the coraing year. 



Part B included! Yes 



'21 



Serial No* !^'CX»250 
Page 7 



PHS-NIH 

Individti®! Project Report 

Calendar Year 1959 

Part B . Honors, Av^rds, and Pubiieations 
Publications (no abstracts Included): 

(1) Landy, M. » Trapaal, R-J., and Shear, M» J<,s 
Inset ivat ion of endotoxin by a htsmoral component, IV. 
Alteration In the iimauno log leal properties of typhoid 
endotoxin. J. Exper. Med. llO t 731-750 (1959). 

(2) MorSp P. T., Yottngp B. G., and Shear» M„ Joi 
Reduction of Toxicity of Cationie MacroRsolecules by Com^ 
plexinq with Anionic Derivatives of Synthetic Polyglycoses. 
Nature, l&^t !.!.3i«-U3 (1959). 

(3) Mora, P« T,, and Shear p H, J. 2 Reversible Inhi- 
bition of Macrooiolectiles (Ensymess etc.) hy Eieetroststic 
Interaction with Derivatives of Synthetic Polysaccharides « 
Internationale Union fSr Relne and Annexvandte Chemie, Kom" 
mission f^r I%croc5olec^le, Symposium liber Makrooiolek€le in 
■•/iesbaden, P^croffnoleculare Nattirstoffe tind Modelle, Vor« 
trage V C3^ Verlag Cheml®, GsnbH. v^einheim (1959). 

Honor i and at^mrdg pertaining to this projects 

1. Elected Vice-President ( President °E 2 ect) of 
American Association for Cancer Research at the Annual 
Business Meeting^ Atlantic City,, April 1959. 

2, Appointed Ch©in?an of the newly-cf«ated CoM-altte® 
on Policy^ of the Amer. A®@n« Cancer Research^ 

3« l%ny people have coinniented on the ineiuslon in 
the lead article in the "Saturday Evening Pest'' for Oct« 
31, 1959 of an interview (and photograph) with the Chief 
on cancer cheraotherapy- It is interesting that they coa« 
sSder this to be "recognition and sn henor^f 



722 



Serial No. NCI-250 
Calendar Year 1959 
Part B •» Paae 8 



k-, CBS has made a filn, \/ith sound trackj, of the 
Interview of the Chief by Dr. Tom Doolcy (of Laos) for 
broadcast In January or February some evening in the 
program "CBS Reports ..•..". 



723 



Serial m>, HCI-g53 

1. L^os>atos^ of Chaaleal Fhanaacol©^ 

2. Bioeh@mieal Fharmaeolo^ Section 

3. B@th©sda Ihg Maryland 



fiIS«NZH 
Individual Project Export 
Calendar Year 1959 



Part A, 



ProJeet Tltle i Bioeheoiical and Hiarmaeologieal Stisdies 
Of HOBt-Tumor HelationBhipe in Tumor Cheaotherapy* 

Principal Investigator 8 Dr* A. Soldln 

Other Inyestisatora » Dr. A. Schr@elc@?j» Dr. M. Chirigos, 
Dr. J.A,H. Mead, S. iSimphreySa J. Vend! t tig H. Mantel 
(Biometry Branch), Dr. R. A. Darrour (now at national 
Institute for Medical Research, Mill Hill, London, ^igland. 

Cooperating Units i 

(a) General Medieine Branch, NSIs Dr. 1. Frei and 
staff. Serial Mo. mi 226, 

(b) Laboratory of Biology, NCIs Dr* J. Moloney. 
Serial No. HCI 420. 

(c) Laboratory of Neuro anatomical Sciences, MMDBi 

Dr. J. Cammeiraeyer. Serial No. HIMDB EF-1. 

(d) Graduate School, Biochemistry Department, Brendeis 
University, Waltham, Massachusetts s Dr. Hathan 0. laplan 
and staff? Chemistry Department, Dr. Orri© Frief3iaaiii« 

(e) Department of Pharmacology, Washington UnlverBltj 
School of Medicine, St. Louis, Missouri j Dr. R. Birtcn and 
Dr. R. Salvador. 

(f ) Department of Pharaacology, Tufts University M@dieal 
School, Boston, Massachusetts 8 Dr. M. Friedkin, t^, L. Shiseter, 

(g) Oklahosaa Medical Research Foundation, ©klahoaa City, 
©klahoxaas Dr. P. Condit. 



724 



Calendar yeas? 1959 
Part A « ecmtismea 
Pag® 2 

(h) Iftiiveysity of Miaial Medical School, mmX^ 
FloriSat Dr. J. Colsiqr* 

(l) Caneer Ch^ootherapy National S®rviae 0@i^t@F3 
MiC2>obiolosiosX Assoeistes^ Ine.« Beth^sda^ Mefyland 
(Contract Mo. SA.43»ph»2371). S©3?ial Ho, HGI 2l6o 

Man Yeaps (oalenaar y@ar 1959) t Petient Dasrs 

Totals 13 l?on@ 

Professional i 6 
Others 7 

Proj©et Descriptions 

Objectives 

1, Stu^ of the msehanlBia of action of antituasor 
agents, including metabolic antagonists, all^lating agents, 
antibiotics, etc. 

2, Synthesis of new agents. 

3. Study of host-tuEKjr Interrelationships with r^speet 
to drug action. 

4. Establishment of new therapeutic principles and 
i^rocedures in the management of tumor growth in animals » 

Bfethods Etoployed 

Macrobiologieal ass^ procedures involving methods of 
inhibition loialysis for animals with and ifithout tumors. 
In vivo and in vitro biochemical procedures. Radioactive 
tracer teehni<mes . Organic chemistry proeeduras. 

^Jor Findings 

lo Survivors of Syst©iie Leukaaia (with lii^hreys, 
Venditti, Chirigos, and Chapman) s St had been reported last 
year that the dihalogenated derivative of asethopterin, 
3S5'-diehloroamethopterin (DCM), was capable of eliciting 
extensive increases in survival time of CDBA inice with 
advanced leukemia, and that some of the mice actually survived 
for an indefinite period of tim@. 



725 



CB.l®n6w year 1959 

Page 3 

A Bt\&&Y ^&s eonduoteS to Set@rmine the relationship 
between duration of therapy and antileukaBie effeet with 
sen. Survivors of eyet^mie disease v^ere obtained only 
when eideouate doses were administered over an extended 
period of time. The length of tiiae necessary for daily 
treatment to be continued before any significant smssiber 
of survivors could be obtained proved to be 30 to 60 days. 

Vlhen daily treatment with SCM was discontinued prior 
to 30 days^ very few of the animals survived. As had been 
observed earlier with amethopterin« most of the animals 
succumbed within several days following discontinuance of 
therapy. After about 2 weeks of treatment the animals 
usually showed no gross evidence of the disease^ and 
inoculation of blood or transplantation of tissues into 
other mice frequently failed to transmit the disease. 
Microscopic examination of the tissues of such animals 
might also show no evidence of the disease. That such 
apparently cured mice still harbor the disease was &mmn^ 
strated by the regrowth of the leukemia when treatment 
was discontinued too soon. 

2, Studies on liaimmlty (with Itesphrey®, ChirigoSj 
Chapman^ and Vendltti)} It had also been reported th&t 
mice surviving systemic leukemia were refractory to 
reinoculation of a massive dose of leukemic cells. Thes© 
studies have heen extended, nice surviving systemic 
leukemia L1210 show transplantation iaamnity not only to 
reinoculation of the sensitive line of leukemia LlSlOj, but 
also show a high degree of transplantation iiosmmity to 
inoculation of an amethopterin x^sistant subline^ M46r^ 
(which is also markedly resistant to DCM). The survivors 
of systemic disease were also iramne to a BCM resistant 
subline (^6»3R) of leukemia L1210. Following inoculation 
of either on© of these resistant tumor® (or th© se^aitiv© 
tumor )« many of the mice showed no evidence of leukemic 
growths while in others the tumor grew slowly and attained 
a large sise at the site of leukcmie inoculation^ prior to 
death. The letter observation suggests that the disease 
does not readily become systemic in the mic@ that have 
acquired inrn&mity. 



726 



Calenaar yeas' 1^9 
f aft A - eontiimiia 

F®g© 4 

Similarly, 'out of strain" BM^cto miee iBiamisea 
against leukemia L1S10« without the benefit of drug 
therapy (mei^ly toy implanting th© tumor and l©tting it 
reg3?e@s} w®r© also demonstrated to b© laaain© to subseq^snt 
inoculation of th@s@ resistant variants. Thus, in "in 
strain ■ mioe the therapy is required to elicit the imsmn© 
T®BpoaB9, In out of strain" mice the initial is^lant 
regresses spontaneously mi^ the miee become imaasn@« 

The above results provide a separation between ttMior 
cell resistance to therapy as opposed to tumor cell 
resistance to host imimnlty. The ttsro variants of leukeiaia 
L1210 (B^R end F^-3R) ez*© resistant to antifolic therapy 
but are inhibited by the induced immunity of the host. 
Thus, although the immune mechanism may contribute to 
therapy in the in strain' miee, resistance to therapy 
does not necessarily ia«>ly abrogation of the effectiveness 
of the isMune response. This is in agreement with the 
previously reported finding that failure of therapy is n©t 
necessarily associated with the development of tumor cell 
resistance. 

It has been observed that the homograf t response to 
the amethopterin resistant subline L12lQ^Wi6R may be over- 
come by treatment of the "out of strain'' mice with amethopterin 
or DCH. On treatment with these drugs the tumor grows 
progressively and the animals succumb. However, if the 
"out of strain" mice were inmamised by preinoculation of 
normal spleen of EBA mice or with leukemic tissue, antifolie 
treatment did not, suppress the established Ismmlty. 

The above observations raise questions conceKilns the 
role that host imaainity may play in influencing the host- 
tumor relationship in chemotherapy. To i?hat extent is the 
antileukemic action of the antagonist attributable t© imMine 
responses influencing host tumor coB^atability rslatlonshipsf 
That the successfully treated leukemic mie© ^hm evidence 
of ii^aanity would suggest that at least part of the curative 
action of the halogenated derivatives results from therapy 
so influ^icing the host=tum©r relationship that tte mice 
develop imrsamity to the disease. The imaam® state in turn 
may help to eradicate the disease. If true, what are the 
detailed mechanisms and can advantage be taken of this in 
therapy? 



727 



Calendar year 1959 
Fart A ° eontimaed 
Page 5 

3, S^isltisatlon of a Resist ©at Tu^r to Therapy 
(with HuQ^hreys, ChLrlgo&s VsncSitti^ and Chapman) s A 
model experiment has been condiieted showing that it is 
possible to sensitise an antifolie resiistant subline of 
leukemia LiSlO to therapy. This vsm aeeoa5>li8hed by prior 
inoculation $si6 ti^atm^tit of the sensitive line with !)GM. 
The tiaaor ®aployed was the J^6r s»esistant variant of 
leukemia LISIO, whieh is highly resistant to methotrexate^ 
end markedly erossoresietant to ^M. WiSn eontrols live 
for about 10 days« Treatment with ECU, even when treataent 
ie initiated on the day of implantj extends the survival 
time only to approximately 20 days. The survival tiiaa of 
the mice with this resistant tumor may be increased to a@ 
mueh as 60 d^s if the animals are preinoeulated with the 
sensitive leukemia and treatment with DCM is initiated 
after the disease has beec^ae systemie. 

Presumably the trestment of the mice with the sensitive 
systemic leuk^oia elicits an immine response in the host 
which in turn, contributes to the therapy of the resistant 
leukemia. This presus^tion would appear to be warranted^ 
since mice that had survived systemic leukemia LISIO f6l<» 
lowing extensive therapy (day 7 to 90) were demonstrated 
to be wholly or partially immune to reinoculation of either 
the sensitive or antifolie resistant variant. Th® possibility 
is not excluded^ however, that the sensitive leukasic cells 
may have sensitised the resistant cells directly or following 
therapy. Bie study indicates that the ehaiiotherapy of 
cozi&inations of sensitive and resistant variants -may be 
eE?)loyed to study immunological and other factors pertaining 
to transplantation, and to investigate means for the ia®r©ir®« 
ment of therapy of resistant variant®. Resistant sublines 
may serve as convenient markers for studying these inter» 
relationships, 

4. K0ute of Admiiaistr©ti©n of tetifolies 

a. ^arvival time (with ¥enditti)g Th© ezter^ive 
survival tim® observed ©n treatment ©f sale© with ad'^sne©d 
leukemia was obtained with subcutaneous afeinistrstion ©f 
DCM. A study has now been conducted shCMing that en oral 
administration of SCM, there is a marked loss in antileuk^s 
effectiveness, teethopterin has also shown reduced effecti%'©ness 
^1 oral administration. 



728 



CaleMaf ^©ss? 3.959 
Paa?t A •=• c©ntimi©a 
Page 6 

b. Effect of route of antifolie ©dminietratioa 
on fonaate lacojfporatlon (with SeiM?eckep> Meaa^ and Igrnch)? 
Since the inhibition of fosumte ineoi^oi^ation Into the 
aci€- soluble adenine of I@uk@mie spleens can ses^e as a 



measuffe of effective levels ©f antifolic drugs. In vivo, 
it was eaiplosred to eesspaye the effectiveness of Wff^^t 
routes of administration « In leukesiie mice» the @\ibc^tane©ii@ 
and intraperitoneal routes produced similsr levels of 
inhibition «rith 1X3M* Maziosum inhibition t?@3 observed when 
formate was injected 20 oiinutes after the adodLMstration 
of the drug« Even after 7 minutes^ near^mssiiaal inhibition 
was obtained. However, with oral administration of ^M, 
masisBun inhibition was not reached before mi hour, ^r@ov©f>, 
imoh larger doses were required to produce inhibition levels 
eoQ^arable to the ones found on parenteral ad!sinistration» 
It would appear, therefore, that DCM 1b onlsr ©lowly absorbed 
froa the intestinal tract, ssi^ that wiQh larger dose© ai^ 
needed to produce sa aati tumor effect thsn hj th© parenteral 
route. Since one of the main tossie effects of BCM is that 
produced in the intestinal tract, our studies womld eaggest 
that the oral rout® of a<teiinistration is inferior. This Is 
of iB^ortance in its iii^^lications for clinical therap^r* 

5. Slew Antileukeiaic Agents (with Venditti and Htoiphs?®5rg)is 
Cytoxan, a cyclic phosphaaide ester of nitrogen imstard, mm 
investigated in some detail against l^kemia L1210 and 
several of its resistant sublines. It was found to be ®©re 
effective than a series of alkylating agesits including 
uracil mustard, nitrogen sDsustard, triethylen© melaniae, 
degranol, ehloraosbucil, hydroiijainon® imetardj, ssreolysin^ 
or nitromin, in increasing the survivgl ti©© of rale© ^f^ith 
advanced leukemia. It was at least a® effective as aE@tiiopterisi« 
In agreement with data of Dr. M. Lane, its sntileukraie 
effectiveness against advanced leuk^da was d^onstrable 
over a wide rang© of schedules of therapy, Intei^ttent 
administration of the drug appeared t© provide more ©ff ©stive 
therapy. 

The antitumor effectiveness of Cytem^i was not diMnlsh©a 
in the treatment of sublines of leuk^i® U210 which were 
resistant to the entifoiies or the aatipurines. The anti« 
leukemic action of Cytoxan could not be blocked fey nicotirismide, 
as had ti%®n reported elsewhere. 



729 



eaiendas? 3r©®3? 1959 

Fag© 7 

Sttiaies with Cytoxan against early transplantable 
Imkaida L1210 suggest that th® aetion of this alkylating 
agsnt results in the eetual destz^etion of entileukemie 
@ell©. A single dose of CytOKm. was so effective against 
©a^ly leukSEsia that mm^ ©f the miee survived. 

By comparison with the treatment of normal eaiee^ it 
was demonstrated that the therapeutic effectiveness of 
Cytossan was limited by the toxicity of the drug for the 
host. This toxicity was less limiting than had been 
previously observed ^ith S^f luorouraell . 

Cytoxan was also demonstrated to he effective against 
adenocercinoma 755 in mice, retarding the growth of th© 
local tuasor, and increasing th© survival time of the sdee. 

Cytoxan derivatives « A collaborative research 
progrm is in ps?ogFees"%?ith E>r. Orrie l^ietoan at Brandeis 
Uhiversity, who is synthesising congeners of Cytoxan, 
Structure^ activity relationships are under investigation 
with these coi^ounds in this laboratory. 

6. Fyrasolo fsrrimidiaes (with Venditti and Frei)i 
A detailed study was conducted on the effectiveness of 
the pyraaolo(3,J*»d) pyrimidines against transplsntabl© 
mouse tumors. 

A series of pyrasolo(3,it*d) pyrimidines, substituted 
in the 4 position, was coiapared with 6-iaere apt ©purine and 
amethopterin for ability to increase the survival time of 
mice with advanced leukegda L1210. I^iere was a wide rang© 
of antileukemic activity among the various pyraiolo 
pyrimidines employed, but none of th^a was as @ff ective 
as 6-mereaptopurine ©r amethopterin. 4-tein©pyras©l© 
(3,4-.d) pyrimidine (4APP)f 4-n«pr©pyla!!dn©pyrasol©C35^-a} 
pyrimidinej 4-beasylaiaisK>pyrasoloC3s^-d) pyriiaidinei asid 
4»amino-l«m@thyl pyrasolo(3,4«d) pyrimidine were the mest 
effective of the pyrasolo pyrimidines tested. Th&^ w©re 
approximately 30 pew cent as effective as g^©th©pt©rin in 
increasing survival time of mice with advanced leuk@iiit« 

4a?P failed to produce appreciable increases in the 
survival time of mice with an 8«asagaanine resistant subline 
of L1210, also resistant to 6-mercaptopurine. On th© other 
hand, 4APP did produce marked inhibition of gro^ffth of the 
local tumor in mice bearing th© antipurin© resistant leulcaaia., 
indicating that cross resistance was not cesislete. 

730 



Fag® 8 

4APP and th® 4«b€insyl derivativ® lishiblted the ^mth 
of relatively early a^enoearcinoma 755* but failed to 
increase the life span of the miee. In contrast, 6»iB)Speapto« 
purine psvdueed a marked increase in the survive! time of 
the mice bearing edenoeareinoBm 755 ®nd also resulted in a 
high incidence of tumor free survivor®* 

4AP? was ineffective against sarcosia 379 It failed 
to inhibit local twaor grojjth and failed to Incraas© the 
survival time of the mice. 

7. Evaluation of ^tileukemic Agents Srsploying 
Advanced Leuk^oia L-ISIO in mee (with Venditti, Kline^ and 
Mantel) J The assay procedure es^loying advanced leukemia 
in mlces developed in this laboratory, had originally been 
employed to evaluate the antileukemic efficacy of 38 eoij^oaridSa 
including 23 that had he&n @i^loyed in the Ggllhora-Hirsehbei'g 
report « on investigation of diverse sy@t®as for C sneer 
Chemotherapy Screening. This assay system is now in full« 
scale use in the Drug Development and Evaluation Program 
(Contract Ko, SA-43»ph»2371). A total of 125 eoi^ounds has 
been evaluated in this system with respect to their relative 
capacities to extend survival time of mice with l©uls©aia 
LiaiO* Xneluded in this list are alkylating agents, anti« 
bloties, purine and pyrimldine antagonist®, nicotinamide 
antagonists, eolchieine derivativea, etc. Two solid tmsor 
assay system® have also been developed (Adenocarcinoma 755 

and Sarcoma 37), and are currently being aaployed in th© 
©rug Bevelopment end Evaluation frograa (detail® are listed 
in report on Contract SA-43«ph«237lI» 

8, Virus- induced Leukemia (with Moloney and liimphr@ys)8 
A collaborative program has h®®n instituted with ©r. J. 

Moloney t© study the effect of ehemotherapeuti© agents 
against the Moloney vis^s leuicemia, l^lcmey obtained this 
virus from sarccsaa 37. It induce® lyiashoeytic leukenila in 
mice, and is not strain . specif ie in tMs regard. The indueed 
leukemia is transplantable as th© whole sell, but is strain 
specific* Sufficient studies have been convicted to dat© 
in this program t© d^n©nstrat® that the virus induesd laakemia 
provides a useful tool in chemotherapeutic studies. Onee th© 
disease has developed it appears to b@ hlgSsly refractory to 



731 



Cal®adaz» year 1959 
?as?t A » ©entitled 
Page 9 

therapy a Little or no increase in survival time wss 
obtained with am@thopte£>in« Cyto£an» or 6-meFeaptopu?ine. 
On the other hand, definite reduction wes observed in the 
sise of the sple@n» liver, thymus , end nodes following 
therapy with these drugs. This work is to be extended 
to include study of the astion of drugs on (a) the vii^e 
itself, (b) the induced leukemia, and (c) the transplanted 
leukemia. Treated animals are being esaisined for micro- 
eaopie pathology hy Sr. Thelma Sunn. 

9« Spontaneous Tumors (Mth Hus^hreys, Chirigos^ and 
Chapman) s Chemotherapeutlc studies have been undertaken 
with spontaneous maimnary tumors in C3H mice. Although the 
initial study has not been completed, it is already evident 
that the spontaneous tumor is highly refractory to amethopterin, 
6«mercaptopurln©, and Sytosan. 

10. Combination of Ch^aoth©rapy and Surgery (with ColBlq^^ 
Chirigos, and Bimphreys)8 Some evidence ha® been obtained 
that the combination ©f sxargery plus treatiaent with S-aereapt©- 
purine is more effective than treatment alone, in inhibiting 
tumor growth and possibly in increasing the survival time 

of mice with adenocarcinoma 735* 

11, Biochemical Studies with Antifolies. 

a. Inhibition of purine synthesis (with Sehreelcsp^ 
Mead, Daz>row and Lynch) t Studies were continued on the 
inhibition of purine synthesis de novo in normal and l^akemi© 
mice by amethopterln and 3 • , 5 ' -licM^jamethopterin (PCM)« 
as measured by the inhibition of fox^ate-C-*-** incorporation 
into aeid«80luble adenine of spleen and other organs, A 
small parenteral dose of either drug produced extensive 
inhibition in spleen or leuksaie tis^ae® whai it was adi^n^ 
istered less than one hour before the injection of labeled 
formate. For instance, a single dose ©f 1,5 sg/kg of SCM 
produced over 90 per cent Izshibition in leuk^d© splssns 
when it was given 20 miiautes before administration ef f©rsiat@« 
With this 20-ffiinttte interval, the dose ratio ef BSM to 
amethopterln for equal inhibition, as detesmined in a©e@« 
response studies, was about 2, However, the is^Uilbitory 
effect of the drugs w®s transitory, and the effect of BSM 
was xmch less persistent then that of amethopterln. By 
extending the Interval between the administration of drug 
and of formate, recovery curves were obtained, Thsj showed 



732 



Calendai? year 1959 
Parfc A « eontimied 
Page 10 

thati, with an Interval of 6 houses or vadv®, the dose ratio 
of DCM to amethopterin for eispel inhibit ion^ be© am© about 
35 to 1. It should be noted that, to obtain Inhibition 
lasting fos» 24 hoiirsg about 75 sjg/kg of DSM wes i?equlred. 
This is the dose that produces maxlMim siarvivel times in 
leukemle mice on daily treatment. The finding indicates 
thet» in therapy j, it is necessaz^ to maintain an effeative 
dose-level between consecutive injections. 

B^ measuring dose^responee x>elation@hip8 with 
aii^thopterin and SCM in both normal and leukemic spleens^ 
it was found that DCM was relatively more effective than 
amethopterin in the latter. This ma^r account for the 
more favorable effect of DCM in prolonging life in leukanie 
mice. 

b. Mechanise of reversal by citrovopiaa factor 
(with Schreeker^ ^ad* and X^mch)* IJreatH^nt with very 
large aaounts of citrovoru© factor {QW) esaised partial' 
reversal of the inhibitory effect of asiethopterin ^kd PSM 
upon foz^ate incorporation into the acid»soluble adenine 
of leuk^Diic and normal spleens in vivo « Increased doses 
of CP were needed with increaseSl doses of the dnigs, 
Reversal was more pronounced when CF was administered 
before, rather than after* injection of BCHo ^e current 
theoiy of the action of folic acid antagonists does not 
appear to e^lain these results. Since amethopterin blocl^ 
the fOMiatioa of tetrehydrofolic acid from folic aeid^ and 
since OF can yield tetrahydrofolic acid by a different 
pathway, supposedly unaffected by amethopterin, formats 
incorporation should not be inhibited at all hj amethopterin 
if sufficient CF is supplied, ©ur results show th®t this 

is not the case. Therefore, amethopterin (and BOl) imst 
inhibit the transforsaiylation reactions in purine nucleotide 
biosynthesis or inhibit the transport of CF into e^XlB. 
The latter has been d^iongtrated fey Wood and Hitchings In 
bacteria. &sr study suggests that this may also b@ tru© 
for leukemic cells and for nosmal spleen c^lls^ 

c. Correlation of biochemical indices ifith therapy 
(with Schreeker, Mead, and liamphreys)8 Morsial liv#r wes 
found to be rather insensitive to ^itifolio drugs. fM@, 

a dose of 75 mg/kg of DCM failed to produce inhibition of 
formate incorporation in that organ. Sine® format® incorporation 
in leukemle cells is Mich higher than in liver, and since it 



733 



Csl®naai? year 1959 
Part A •=> eontiiaied 
?ag@ 11 

Is greatly inhibited by &CIS» it e@n s@i>v@ as a meaius^© 
of syetemie infiltration during th@ l^uk^aie proe®s@ as 
affected by treatment. Foraiat© insorporetion ^®s meaeiired 
in leukemic mic® that were treated daily with 75 wg/kg of 
BCM from day 7 to 17 after inoculation. In untreated 
control ndce, formate incorporation rose sharply as the 
livere becasoe Infiltrated. Treatment reduced inco^oration 
to the levels observed in normal livers. With cessation 
of treatment, incorporation rose sharply again after a 
delay of about 3 days, indlcsting relsifiltratioa of the 
livers with leukemic cells, tealogous results were observed 
in spleen. 

d. Reeietaaee studies (with Sehreeker and Meaa)i 
Studies have been instituted on gmethopterin^indueed 
inhibition of format® incorporation in an amethopterin* 
resistant subline of L1210 leulcsmia (I^R) . Large dose® 
produced extensive inhibition of formate laeorporatioa in 
leukemic spleen and local tumor with the sensitive md 
resistant subline. With small doses, inhibition ^m leia 
pronounced in the resistant subline. The differential 
effect was more pronounced in the local tumor th^ in the 
infiltrated spleen. The data suggest that the d©§@-r©spon@e 
curve for inhibition is shifted toward the larger dosep in 
the resistant subline, in agreemi^t with Michol's dencription 
of resistfint cell populations. The difference between the 
sensitive and the resistant leuk^a would appear to be of 

a quantitative rather than qualitative nature. 

e. Dlhydrofolic reductase (^ith Br, FriedMsi, 
Humphreys, and Schreck@r)8 Studies are in progress on th© 
inhibition of dihydrofolic reductase by ^tifolics in 
sensitive and resistant tumors. Evidence has been obtained 
that diliydrofolic reductase activity is higher tn the 
resistant subline studied than in the sensitive leukeiaia. 

12. Mine ^id Metabolism 

a. Tyrosine and Tryptophan (with Ohirig©s)8 
Preliminary studies have shoim that tryptophan and tyrosine 
are present in significant amount® in tumor as well as in 
normal tissues. A survey is being made of endogenous levels 
of tyrosine and tryptophan in tumor tissue as ec^!i>ar@d t© 
normal tissue. 



734 



Calendar year 1959 
Part A -» eontliEiQca 
Page 12 

b. Transemlnasd (with ChirigO0)s Transaminase 
©etivitjr is being surveyed in a series of transplant sble 
Bn6. spontaneous tumors. Several amino acids are being 

studied to determine whether individual traaeaMinases 
esist for eaeh essential saino aeid. 

c. Transport (%rith Chlrigos)8 Transport of 
aromatic amino acids in solid tumors is being investigated 
in relation to inhibition by alkylating agents @ueh as 
the phenylalanine saistards. 

A system of growing leukemia in mouse brain has been 
developed in order to ascertain whether di^ugs effective 
against this tumor depend on a transport meeh@niai to 
exert their antileuk^oie effect. 

13* Pyridine nucleotide Synthesis (with Harrod, Langan^, 
and Kaplan)} The injection of eBaserine w@s demonstrates 
to inhibit markedly the synthesis of diphosphopyridine 
nucleotide (BM) resulting frcsa nicotinamide iajeation. 

The amount of DPN in mouse liver is increased markedly 
on injection of a nua&er of pyridine derivatives, of t^hlch 
nicotinamide has been demonstrated to be most effective, 
raising the level by 8-10 fold. 6«!?ereapt©purine, thio« 
guanine, and nicotinic acid inhibit the synthesis ©f E>FM* 
Azasertne, unlike the other inhibitors of this system, 
actually esoAsed a marked reduction in the nos<mal levels 
of the pyridine nucleotide. Th^r® ua® an ^proximately 
9-fold deoreafee of pyridine nucleotide level, as eos^ai^ed 
with the level in normal animals. The mtElMM decrease 
occurred in 1«2 hours and recovery occurred in appr^:imat-©ly 
24 hours.. S-DiasO'-S-oso-^norl^ucine {WM) behaved ©Imiiar-Iy 
to asaserlne in lowering mouse liver DFSI. Sine© ©laees^ne 
and DOH are known to inhibit nucleotide synthesi© ©f pr©eurseFs 
of CPM, this may account for the inhibition of BFl synthesis « 
Additional investigations ere in progr-ess and it Ib hop©d 
that these studies i?ill be of value in elucidating thm 
pathway of DFH synthesis ^d it® re^^atioa in tlm intaet 
animal. 

14. Formation of Hiootinic Acid Analogue of DiphoB«> 
phopyridine S^eleotide Following Hicotinaiside Adndnistrt^tion 

(with Langan^ Kaplan, and Shu8ter)s A pssridlne imcleotid© 



735 



Calenaap year 1959 
?sa?t A - eontismed 
Page 13 

isolated fT<m the livers of miee injected with nieotinamid® 
has been identified as the nieotinic aoid analogue of £)FM, 
Evidence has been obtained that it is an Intei^iediate in 
the synthesis of J>m f£^m both nieotinszoide and nieotinic 
acid» as well as from other pyridine oosapounds. Inhibition 
by asaserine of DFH synthesis fr^Q nicotinamide allowed 
accmmlatlon of nieotinio aoid analogue. 



15 • Interaction of Hicotinesiide with Reserpine and 
Chlorpromaeine (with Birton, Salvador* and Biii!phreys)g 
Nicotinamide «dll set in conjunction t^ith reserpine and 
ehlorpranasine to reduce the sponteneous activity of mice 
end to increase the duration of anesthesia. 



16 ♦ 3« acetyl pyridine tojticlty (with Cammesraeyer and 
iiutaphrey@)s Studies ere in progress to investigate the 
patholo^ of the ataseia associated with administration of 
3- acetyl pyridine. 

Significance to the Progr^a of the Cancer 2nstitute t 

Th%@e studies are designed to provide basic infosmation 
pertaining to the host-tuiaor relationship in ehesaotherapy^ 
from both a pharmacological and biochesdesl point of vies?. 
Proa such studies, principles for treata^it in enimals are 
being established which may be of use in application to 
clinical ehesiotherapy. 

Proposed Course of Project t 

Daring the nest year the various phases of this wos'k 
will be continued and broadened, ferticulas' ei^hasis 
will be placed on phasing into the newly i^dertaken areas 
of reseercht indicated above. 



Part B included! Yes 



736 



iwial Ho, 1CI»253 
Calendar year 1959 

Fag© U 

Individual Projeet Report 
Calendar Yesr 1959 

Fart B . Honors, Awards, and Publieatlons 

l^ibllcetions other than abstraets from this pipojeets 

(1) Sister, L., Khorana, H. Q,, and H@pp@l, L. A. t 
The Mode of Action of Bydgraes Ribonueleese, Biochim. 
Biophaica Acta ^, 452«46l, 1959. 

(2) Ooldin^ A,, ¥®nditti, J. M,, HamphreySa S« K., 
Shuster, L,, Deyrow, R, A.j and Mantel, N. s Advanced 
Xieukemia as a Tool for Chemotherap@utie Studies, In 

press, ACTA UNIO ZNTEHNATIONALIS CONTRA CAHCHUM, (Fs^e@@ding© 
of 7th International Cancer Congress, London, England, 
July 6«12, 1958). (Reported in lest years' report) 

(3) Liebling, M*, Biiaphreys, S. R., end Ooldin, A, 1 
Studies of 5-Fluorouracil in the Treatment of L1210 Leukemia 
in Mice. Cancer Research !£, 116^121, 1959 « 

(4) Shuster, L,, and Goldln, A. 1 Soae Biochaaieal 
Effects of 2-Sthylasaino»l,3*^thiadiasol@« Bleehemieal 
Pharmacology 2, 17-24, 1959. 

(5) Shuiter, L., end Qoldin, A,s Th© Effect ©f 
Nicotinamide on In Vivo Incorporation of Forssat@»C2.^. 
J. Biolo Ch«Ha. ,^7^^133^ 1959. 

(6) Ooldin, A., Humphreys, S, R., Venditti, J. M,, 
and Mantel, H.s Prolongation of the Lifespan of Mice 

with Advanced Leukemia (L1210) fey Treatisent with Halogenated 
Derivatives of ^sethopterin^ J, Hat, Cancer Inst, 22, 
811-823* 1959. "^ 

(7) Huu^phreys, S. R,, and Goldin, A«i Investigation ©f 
Tumor Variants Recovered from Mice with Sysitesic Lsuk^^a 
(L1210) Follonring Estensive Therapy with 3%5'-DiehI©r0as!©th©p= 



terin and SVBJPoaJO-S'-Chloroamethopterin, J. Mat. Cancer 
last. 2^ 633-653» 1959. 

(8) Lasslo, J., Humphreys, S. H«, snd Ooldin, A, 3 
Effects of Glucose Analogs (2«DeojKy-l>«gi%&®©se* 2-I)@0sy»B- 
galactose) on Isperimental Tumors. J. Hat. Cancer Inst., 
in press. 

. 737 



Cslendap year IS59 
Fart B « contlmed 
Fsge 15 

(9) Qoldin» A.« and Kujqphr*eys9 S. H«s Studies of 
IiB^inity in Mice Surviving Systemle Lm2k.maS.& LISIQ, 
J. Nat. Caneer Xnst.^ in press. 

(10) Venditti, J. M.y Bxm^h^Qy&, S. R., and GolSin^^ k»% 
The Effectiveness of Cytoxan Against Mouse Laxkemis L1210 
end Resistant Sublines. Canee? Chemotherapy Reports ^s 
6-8, May, 1959. 

(11) Venditti, J. M.0 li(2iiiphrey@» S. R., and Ooldln^ h,t 
Investigetion of the Activity of Cytoxan jUgainst Lm&k^aia 
U210 in Mice. Cancer Research J^, 986«995* 1959. 

(12) Narrod, S. k., L@ngan» T. A.^ Jr.« Kaplan^ H* 0«, 
and Coldin, A.t Sffect of Aaaserine (o«diasoesetyl»L«serln@) 

on the Pyridine Nucleotide Levels of Mcnase Liver. Nature 183 j 
1674»1675* 1959. ""^ 

(13) Charache, S., Condit» P. T., and m^i^hr^Sa S, H.s 
Studies of the Folic Acid Vitaiains. X7. The Persistence 
of Araethopterin in Matmoalian Tissues. In press j, CAI^^. 

(14) Charache, S,, Coadit, P. T., Levy, A. H.j 
Hi2^hrey8» 3. R.« end Goldin, A.s Studies on the folic 
Acid Vitaalns. V. Sffect of Folic Acid on the Citrovonsi 
Faster Activity of House Liver. In press^? CAHSM. 

(15) Langan, T. A., Jr., Kaplan, K. 0,, ^id SMsterj, L,i 
Formation of the Nicotinic Acid Analog® of Diph©sph©pyridi.ne 
l^ncleotide Follo^^ing Hicotinemlde AdMnistrationa J« Biol« 
Chmk. 2^, 2161-2168, 1959. 

(16) Goldin, A., Venditti, J. M., Klin®, I.s mid Mant®!^ Mo ^ 
Evaluation of Antil€uk^d.e Agents ^pl^ing Advanced L^Ril^irda 
L1210 In Mce. Caneer Research 19, Caneer Chi^otha^apy 
Screening Bat® t?, ^29^U66^ Nov^er, 1^9. 

(17) Venditti, J, M., Frei, 1., ma doldin^ A.8 lffeeti%-s« 
nese of Pyra£Olo(3»4-d) fyrlmldines Against firss^pl ant able 
Mouse Tumors. In press, CAHO^. 



738 



Fart B •» e©ntisiu©d 
Page 16 



Hono3<s and Pm&p&B relating to this projeets 



A» Qoldin s (a) Received a?©appointai©at a@ Yisitissg 

Fffof S8@6i? '6i"'Woeh^sl&%w7 at Brandeis tJhiversitFa Walth^ 
9la@saGhusettei (b) Panel Msinb®?, Board of U. S. Civil 



Servioe Es^alnep® at H2H5 (e) Foreign Corresponding 
m&^Qr Societa Italiana ©i eanceyologiaj (d) Affiliat© 
!!iei&bex>ship» Royal Soeict^r of M@di6in@i (@; Appointed 
to Caneep Ch^ooth@rapy national Service Center Ps^g 
Evaluation Pan®l$ and to its Ooomiittee on E^@risi@ntaX 
Th@rapeutiesi (f ) Appointed to Cane@3:> Ch^ootherapy Study 
Seetion« DiTieion of Research Grants^ KIHi (g) % 
invitation^ served on Sgantining Conoaittee for consid@s°ation 
of Saehindra Hath ?radham as candidate for l1i.D« degr@ej, 
January 28, Oeorge Washington University School of ^dieln©^ 
(h) Invited to present work of the l^bi?^ato2^ at Inter- 
departmental Research S®ain©r* BM.ver®ity ©f ^fylaad 
School of Medicine, March 4| (i) lisvited to present work 
of the laboratory at tMiversity of Pittsburgh Medical 
School, May 4 and 5 1 (j) Korainated to att^d Public 
Health Service Orientation Conference for Cosmissioned 
and Civil Service Personnel, May ll»12i (k) By invitation^ 
presented data to, and participated in, lasr ilgentS'€oimiitt@© 
Clinical Study Panel, June l6| (1) Slogether ^"ith General 
Hedicine Branch, SKJI, presented work of the laboratory ©n 
closed circuit television broadcast, July 31| M Imlt^ 
to participate in syraposium on "Cossparison ©f Prestnt St©-teui 
of Ch^aotherspy of Transplanted Rodent Tumors and the 
Chemotherapy of Human Tuiaors, Mt» S)esert Island BiolegiesX 
Laboratory, Bar Harbor, Maine, Mgust lO^llj (n) By 
invitation, participated in CCISC Conference on E^oerissantal 
Clinical Cancer Ch^iotherapy, Mo¥@sib©r 11-12, 

J. Venditti ; (a) By Invitation, participated in 
dordon' itesearch Conferences on Cancer, les? L^idon^ H^w He^sMre^ 
to©ist Sl^-Septesiiber 4. 

A« Sehrecker t (a) Invited to spend a month as ©seat 
worker "in 'tHe Graduate Department of Bioehasisti^g Brand©!© 
l&iiversity, Waltham, Haasachsisetts. 



739 



Serial Ho. MCI-252 

1* Lab. Chemieal Phari^aoology 

2. Cslluiar Pharmacology Section 

3. Bethesda l4, Marylan<3 



PH3«MIH 
Individual Project Report 
Calendar Year 1959 



Part A. 

Project Title: Phaisaacologlc Action of TuraoF-Daiaaging 
and other bhesslcal Agents on Anlaal and Itoaan Malignant 
and Nox^ial Cells « 



P rincipal Invest itgator? Dr. M. Belkin 

Other Investigators: Dr. J. W, Prelss, Dr« D. L. Vivian, 
*A7Terra\iltV 

Cooperating Units: Dr. D, Laviej Weisiaann Institutes 

Israel, tir, A„ *ii'.' Dalton, Cellular Biology Sections, 
NCI-406. 

Man Years (Calendar Year 1959); Patient Dajs: 
*"" Total 6 °° ' Hone""^ 

Professional 1 

Other 5 

Project De scriptions 

Objectives? 

(a) To study tumor neerotislng coispouni^s^ froja 
plant and other sources, to detersaine how tliey bring about 
cell damage and cell death, (fe) ®ie oasrphologic and bio-' 
chemical responses of nonaal and msllgnant cells to various 
drugs and chemical agents with particular regard to certain 
basic functions of the cell, i,e., permeability, water 
metabolism, enzyme behavior, etc. 



iho 



Serial m, HCI-252 
Calendar Year 1959 
Part A " Pag© 2 

Methods Employed t 

Classical techniques of pharsmeology^ cyto« Bn6 
hi®toch®^©try, ®nd tissue culture are used. H®w t®ch« 
nique® are developed as necessary. 

Major Finding s 8 

For each orlhe several facets of this project, 
the principal finding® and general progress during the 
ps3t year are as follows: 

Ao Tuiaog Daaaag inj^apacity of Plant MatogislSg^ 

The systematic testing of crude plant aaterisl© 
for tumor damaging capacity ha@ now h^^n discontinued. 

Tti® results of the final test® have been era« 
bodied in t^o BianuscriptSj as follows s 

1) 27^ of 105 plant materials from 82 species 
of Australian medicinal and poisonous plants induced damage 
in transplanted sarcoma 37* Four plants, arbitrarily 
selected, were also screen€«§ against seven additional 
transplanted mouse tutors. Each of the four plant® in^ 
flicted dsiaase upon a number of tusnors^ fe^t; not always 

the ssifje tumors, A certain degree of selectivity appear® 
indicated,, 

2) Of a series of 65 plant utaterials fro® 58 
plant species cojmsonly used a® foods, 3^^ produced dssage 
in sarcoma 37 s 

Mien the polysaccharides, dialysed from a 
number of the^e food plant®. Mere tested ageinst eolid 
sarcoma 37* pronounced tusmjr hesi^rrhage and necrosis was 
produced 9 Alsog these polysaccharides could induce typical 
enlargement and vacuolisation of sarsosa 37 arid ©tlier 
ascites tui^r cells (see belovr). 

These raarked effects produced toy these poly- 
saccharides uere obtained only after parenteral adiiiini0trs» 
tion (loP, or !*¥»), Mon- absorption or inactivation in 
the .2ut appears to be c^ite coifspletej since no effect was 
obtained when polysaccharide was given orsllyj even ©t 



7^11 



Serial liOo ICX»g52 
Caiend®i» Y@®? 1959 
Part A « Fag© 3 

doses ten tim@s the M.T«D* 

So Studies in Cellular Phamaeolo^, 

Z. Factors Regulating the Control of Xntra» 
cellular Water Hetabolism, 

In the previous Annual Report, th@re i?as 
described the production of 3 type® of blebs in ascites 
tumor cells t^en the^ ^ere esqposed to certain chei^cal 
agents. The fox® of these blebs wer® (1) acentric (2) 
s:^ssaetrical and (3) scallop fnumerous small blebs -i^ich 
border the oell)« 

This study has been extended to include 
six additional mouse and one rat ascites tui^rs. The c@lls 
of all these tuisors responded with the foz^aation of one ©r 
the other of the 3 type® of blebs, depending on the agent 
used. 

The chemical© capable of pro^eing cell 
bleb@ all have the property of reacting ^ith sulhydryl 
groupso Such reaction is of 3 possible kinds s by alkyla=» 

tion^ by oxidation or by ^ercaptide formation o 

It is of interest that the majority ©f 

chemical® producing blebs are those ^ich forsa ^rcaptide- 
linkages, with a few exceptions: iodoacetaraide, an alkylatlBg 
agent produced scallop blebs i hydrogen peroxide «, an oxiilising 
agents produced acentric blebs i and KMnOj^a CuClg and por^- 
phyrindine^s also oxidising agents, proTOced syisistrieal 
blebs, 

fteie capacity to produce blebs -mh^n aete^ 
on by certain chemical agents appear® to be a g@n®ral prop« 
erty of raalignant ascites cells. To date, cell^ of 5 n©3:^al 
tissues, exposed to appropriate agents® have failed to reset 
in this manner* 

Tm) iaiplicatione may be drawn at the present 
times 

1, blebs are foKsed as a result of inhibi- 
tion of suldryl^ containing enzymes (one of ^ich is sucsiBic^ 

dehydrogenase) which .is associated with a disturbance in 



742 



Serial .Mo, MCI-a52 
Calendar Y©ar 3.959 
Fart A « F©g@ 4 

th® ifater regulating capacity of the c©ll« 

2. there appear to be structural an^ ftmc» 
tional differences between the cell membrane of malistisnt 
cello 6n6 (several) norsaal cells, which are responsible for 
their different behavior when exposed to blebbing agents.. 

II, Studies on Vital Staining with Meutral 
Red, 

•Riia prosraia, concerned isjlth the a^th©=» 
sis and testing of new congeners of neutral redj, has been 
tenainated. 

A lasnuscrlpt* describing the preparation 
and properties of some 2^8»dlsubstituted phenazines used as 
intermediates plus 2 new vital stains related to neutral 
redj, has been accepted for publication in th® Journal ef 
Orggaiic Chemistry C®®® below )« 

IIIo Evaluation of Vital Stains ©s Indicators 
of Cell Injury and l#ath. 

Investigation a® to the validity of 
vital stains as indicators of viability has continued. 

The behavior of several vital dyes^ and 
other dyes not generally considered as such^ vmB ohBerwe^ 
with motile protossoan cells as test objects (paramaQliaE^ 
colpidium)* Eb^eriisent® f^er© designed so that optimal c©n« 
centrations of stain and toxic agent allowed death with Buf- 
f icient fiiownees so that the behavior of the dye could 
readily be followed » 

Observations to date peiTsiit a nuiaber of 
tentative conclusions s 

lo Vital stains may vary considerably 
with regard to speed in indicating cellular damage^ i,e,5 
eosin penetrates faster than nigi^sini* etc^ 

2. "me behavior of vital stain® (like 
neutral red and crystal violet) Hhich concentrate in the 
cell interior in localised areas appears to 6epeM iKJre 



7i|3 



Calendar Year 1959 
Fas»t A « Page 5 

upon the condition of the localised stained ar^a than 
upon the total physiological state of the c#ll« Thla 
typ® of stain is coi^aratively aluggish in Indicating 
sell damage 9 

3, Loss of the property of selective 
pei^eability to a dye appears to be a more sensitive cri^ 
terion and takes place rapidly to indicate cell dsasage 
(©osin, trypan blue, briilisnt ^ital vM an^ fast grsen 
stain faster than neutral red), 

4e Soiae toxic agsnts allow penetration 
of certain stains feosin, f®st grsea) even though th© c®ll@ 
are still iKJtile and appear relatively undamaged, 

2?o Studies with Baman J^Jign&nt Cell® in 
Tissue Culture, 

As part of the progpaia eoncenied ^ith ths 
natujpe of cell dassage hj chemical agents, th@ effect of 
Suramin Sodium (Haphuride Sodium) was studied on culture© 
of HeLa, m89s Dl64 and Da27o 

After exposure of these cultures to 
Suramin^ 1 lag/al, for 24=>48 hours, the folloifing ch®«ges 
were observed j 

1) Disruption of the early reticular 
pattern of growth, 

2) Pr®@3sentation or "beading" of the 
cell membrane and cytoplasia« 

3) Retraction of cytoplasmic pi^cesseSo 

4) A populatios of prsdosiinsntly i^imded 
cells. 

Although all the cell lines e^iMt@d th® 
above qualitative changes^ the time required to evoke these 
changes an^ang them varied eonsiderablys with D«22T shoifing 
the greatest sensitivity. ThQ various rates of response to 
Suramin and other chemical agents like hydi^ cortisone are 
teeing exploited to determine whether by this iiieans it will 



1^'4 



serial Mo« !fGI"252 
GalenSar Y©ar 1959 
Part A « Pag© 6 

b© possible to distinguish th©s© ^ c©ll lines ©n@ tvom. the 
oth©r«, a Esost difficult thing to 60 on purely M>si?hologic 
appesrasieea and a desirable obj©cti¥e« 

¥, Effect of Highep Plant Polysaeehsrldes on 
Ascites TuiiQF Cells, 

As described previously, isany plant pol3r» 
saccharides will, ^en given loP* to saiee with ascites 
tusjsrs, esuse spelling w:i6 vacuolization of the csllSe 
The initial findings have been published (s©© below). 

Further experia^nts have indicated the 
indispensable role of the host in eliciting this ph®noEi« 
@non. 

lo Sarcoma 37 ascites cells were exposed 
in vitro t© golden rod polysaccharide for one hour, then 
washed 3 tiims in balsnced salt solution and injected 2e?8 
into nozroal mtee. Marlced cell vacuolization was evident 
in less than 20 hours « 

2e Nbi^aal mice were injected lePo ^th 
golden rod polysaccharide* After intervals of 30 aimitea 
to 72 hours after such aiMinistration^, the mic© were in= 
jected loPo with aareo!fiE 37 ascites cells^ and beginning 
4 hours later, cell samples ^ere exasained* ¥acuoliEation 
was evident when the cell® ^^re inoculated 1 hour after 
polysaccharide injectionj increased up to intervals of 
36 hours after polysacehsride injection (depending on the 
dose) and then fell sharply after this periods 

3» Mice carx^ring sarcoisa 37 ascites 
tujQor ifsre injected !«?«, with golden rod polysaccharide o 
At varying tijse interval®, ioe,, from 1 t© 7 hours after 
polysaccharide injection, the cells wer® r®H©¥@d, w&^h®6- 
3 time® with balanced salt solution^ and then transferred 
to new, nonaal hosts » These cells were then eiiasained for 
the next 96 hours « 

This ©3q)®riB^nt disclosed that cells ©x-' 
posed in vivo to polysaccharide for as brief a periods as 
90 ssinu^es, and then transferred to a new^ non°tr-sated host 



w 745 



Serial Ho, IICX«252 
Calendar Year 1959 
Part A « Page 7 

would go on to produce a good vacuollsation effect %n 
24-48 hours « 

A number of implications follow from 
these experiments t 

1» The polysaccharide is adsorbed on 
the cell surface , when the cell is in contact with it, 

2e The cells need not cosse into prior 
contact with the polysaccharide in order to pspoduce 
vacuoliaation« 

3. ^©n noi^al host© are injected with 
polysaccharide J, they are "stiisulated " in some fashion snd 
can induce vacuolization in cells transplanted I»P» as 
long a® 36 hours later, ^hen presumably little or no poly- 
saccharide remains as such. 

4 6 The participation of the host Is in~ 
dispensable to the induction of cell vacuolisation and 

swelling o 

EseperiBssnts are in progress to detenaine 
whether the host produces antibodies in responae to poly^ 
saccharide administration in a manner similar to that 
established for the polysaccharide® of Qrans-negative 
bacteria s and whether such a substance react® with the 
polysac©haride at the cell surface (gee !» above )« Also 
to determine more closely which organ system of the host 
participate® in this phenoiaenone 

It may be conjectured that these vacuoles 
arise from vacuoles already present in the eell^ or de ^v© 
from cellular stinActuree which might give rise to them^ 
such as the ©ndoplesmlc retieulunj^ 

Prelisiinary studies with the electron 
mici^scope (with Dr, Dal ton) indicate that these vacuoles 
arise from pre<^exi@tins vacuoles^ some of vihich surround 
the virus particles knowi to be present in this tui^oro 



746 



SeMal No. MCI«252 
Galena as* Year 1959 
Part h " Page 8 

VI. Electron Liactivation of Plant Polysaccharide. 

Polysaccharide, obtained frora golden xx)^ 
f lower, was exposed In vacuo to 7 K.¥. electrons, eialtted 
by a hot tungsten fiT^isrit and eccelereted by a gr|jd arrange- 
sent. Exposures ranged from 9 x lO-*-^ el©ctrons/cm^ to 
50 •»• lCr-2 electrons/ca^. Control samples of polysaccharide 
were similarly handled but not radiated, (!mes© pro©©Su2?es 
were carried out by Dr. Preiss). Both treated and untreated 
polysaecharide was injected I. P. into silce with 3««^ay 
sarcoma 37 ascites j and cells were examined every day for 
4 days« 

The polysaccharide controls always ex» 
hibited typical cell vacuolization c The treated saiples^ 
however* displayed progressive Inactivation. with increasing 
dose, from H6^ inhibition of the v©©uolisation phenosienon 
at the lovfest electron exposure, to 9B% inhibition at the 
highest. 

It has "been calculated « that the degree 
of Inhibition of golden rod polysaccharide by the electron 
source employed approximates the esjpression from the target 
theory of single hit inactivation^ the fonraila for which is 

A ^ Ao e -^ 

where A ® residual biological activity after 
irradiation 

Ao ^ biological activity of controls 

D == dose expressed as ionlsations/cc 

¥ ® is the apparent target volu^ae^ 

It was found that it takes about l6 times 
as many electrons to attenuate the ' activity of the golden 
rod poller hy a given percent as it does to attenuate the 
sucrose hydrolysing activity of invertas© to the s^i^ de»» 
gree» Since the material densities are about the a^sss the 
molecular weight of the active isoiety of golden rod poly- 
saccharide laust be of the order of 7500, 



747 



Serial Ho. HCI"252 
Calendar ¥eai» 1959 
Part A » Page 9 

Goiaparison molecular '^eightg obtained 
by this electron b©am technique for the ©nayiaes invertase 
and ribonuclease are 110^000 and 15,000j whereas values 
for the seme substances obtained by conventional methods 
are 120,000 and 13»500;, respectively. 

Significa nce to the Prograia of the Cancer Institute? 

13iese studies are designed to obtain basic infor« 
matlon concerning (a) possible difference® between normal 
and malignant cells, (b) the mechanisms by which necrotis» 
ing corapounds produce cellular damage and (c) to correlate 
at the cellular level the phanaacologic action of drugs 
with the stmctural and functional responses of cells in 
relation to treati^nt. Such information is sorely nesded 
to afford a better understanding of dnxg action, and to 
aid in the development of newer ^nd better drugs for the 
us© of investigators working in clinical chej^therapy, 

Pro poaed Course of Project: 

Most phases of this project will be continueSj 
^th emphasis on (l) investigation of the factors regul®t« 
ing water metabolism in the cell, (2) the nature of cell 
damage due to chenjical agents and (3; the d<svel©p«^nt of 
ii^roved tools with which to recognise such damage » 



Part B included «==» Yes 



748 



serial Ho. NCI-'252 
Fag© 10 



PHS-KIH 
Individual Project Report 
Calendar Year 1959 



T.B^^mM.z l^nors, Awards, and Publications 
Publicationg other then abstracts fyo ra thia p rojects 

(1) Leighton, J., Kalla, R. L», Kline, I,, sn^ 
Belkin, M» j Pathogenesis of Tunrar Invasion I. Inter« 
action between Noz^al Tissues and 'Tranaf ox^sd " Cells in 
Sponge-Matrix Tissue Culture, Cancer Research 19.' 23-27 
(1959). 

(2) Lavie, D. , Willner, D,, Belkin, M, , an^ Hardy, 

W. Q» ; New Compounds frosa Plant® with Mti-fumor Activity. 
Acta X? biss 177-181 (1959). 

(3) Belkin, M, « Hardy^ Wo Q,» Perrault^ ^«5 and Sato, 
H« 5 Swelling and ¥acuolisation Induced in Ascites Tumor 
Ceils by Polysaccharides from Higher Plants. Cancer He" 
search l£s IO5O-IO62 Cl959)» 

(4) Tivian, X>* L», Hogart, R, E^s, and Belkin^ M^ i 
Phenasin® S^theses, X. 2j,8"Di®ubstituted Phsnaslne© fede 
as Intensediates for Hew ¥ital Stains, together with Tsjo 
New Vital Stains Related to Meuti'al Re<2. J, of Ox'ganic 
Chemistry j» In ?re®Bo 



^Honors ®nd^^ Aifarda, _ relating , to ^ this ^ project i Kons 



749 



serial M®a MCI»2S2 

lo hBbc Chemical Phs.rm&eolQgy 

3„ Bgtiiesda 14^ Maryfesd 



PHS = NIH 



Part A 

the inliibltiofB o^ ©ssaymes aad othsr biolegieaEy^aetive macr®m@i@e®l®s„ 

PFJgielpal %^©sligstgr; Po To Mora 

Oth®g tovestigatog-sg P., Maiafj, E„ M®rl®F^ Jo W„ Fr@lsa;^ 

Jo Wo \V©od» Bo G, Yo«sgs a^d M„ Jo Shear, 

Coopgyatl ag Uaitsg NCIs Laboratory ©f Ch#mi®al PtermaestegyB 
Polysaselsarid® S@®ti®a. Serial M©, 2S!o 

Bl©eli®miijtry. L®lberat©rj of Pfeysicsl Blelogj^ MIAMDo Serial MOo 
NIAMD=10So 

Pg>®f@ss©if Eo Wolff, Faesiitf @f Mediels©» D^^-trsltj ©f Ksiisys 
Maa Year® CCaleisdag ymr lB5B}t ^B^^JdMSL 



T«^S:; 


12 


Ps'«!l"©sa;k®a1? 


®-a/2 


Otl^eai';? 


5 1/3 



750 



S@s^i M©o NCI » 3i2 
€^l®-mdm^ jmr 196S 
Part A - Pag© 2 



Pro|eet D@§eglptl@^; 



Obj@eUves; Tfe® €^J®c4lve of th© program of fe® Maer©» 
moleeulsr Chsmistry S©€«ii£^ was tw©f©lds 

1) Th® feadameatai stsidj ©f Sat©raeti©a ©f, ^pssitelj- 

2^: Biophysical appllesti^i ©f fesowiedge tliiss gaiaed 
is @s£zym® i^Mbltiosi, bloekiag of toz§e% of oertalsa maGi^mol@@®l^s, 
Irah^itlng phage mnltipMeatios, @te« 

Methods EmpSoyedg 

Thg m®t!i©d3 were s®l@et®d ss s@©d©d, fs^oas «Si@ fi©ld® 
ol p®iym€i» assd sjEtfe@tie ©rgaale ehemislfyj poljm®rlaati@si m^tfeeds,, 
pf ®|sarati©© s^ safestitaSed d©ffivativts» ®ISo i frem ph^rsteal e^»©mislfyl 
viseomstiy, polyeleelroljl® titrallos, speetr^feolom^ts^, etSo » aisd 
from maeromolseular e^emtaSrys 9iltrae®slrifisiatiQsi^ elsetrophertsls^ 
chromatography, et€. ; a©d the data w©r@ carrslated ^Ith F@stslta fs'om 
various ©Bzyiae assays, plmg® assays^ tosielty dsttrmteatlsae tumor 
ss^gfosis asd f@ves* pr©d®stlea la agsfejaSSs ete« 

Major Flsid^igas 

^^ ^S2Sf jgMbitiTOs Th® @i®©tr®stetie f©f e@s Is 
«li® lBt@ractl®i^ of aismerosi© ®uzfm®B asid their ©isbstrst©® ^®i^® 
stiadi©do Th© activities of eiisymes ^©s*® feloeked wim. Bjrdh^tiQ 
cteas-ged macrom©l®eiiiar isMfoitoi's wMsli liad sIeeiIIs? but Mgh©r 
eharg® thaa th® st^slrateo Uade? th®ss eosditieris tli® ©n^jsaes w©fs 
dfe-ected to isal®yact with th© IMilbltors a^d foi'msd asa laaetiv® s&raplms. 
However s th® ©azymie aetSvity was completely reso^^fed wh®a tli® 
eompl©s ^as dlssoelat®d bj addiag a s®c©sid maeromo^eislig- w^4sh had 
a stroidg and opposite charge to the IrMfeltor maerosnoi©eisl.0o The ls= 
hibltor was tSasss removed from th© c©mples and tis© eszysa® was £!©■!&? 
free to approaeh th@ sisbstrat®. 



751 



serial H©, HCI = 2i2 
CaltEidar j®aw iS59 
Fart A - Fag® S 

l& fhmB® st^di@s it ^as o^r plan to l®&r^ alKHit th@ 
forces which bring th® s^^jme &nd %h® B&hstT&t® togetli®?, by 
compstlssg ^ith and overcomliig th@s® forces n^ith ths Sutrodisetiosi 
of a third, s^thstic eompoiasat. Th@ @S!sym® Inhibitors w®r® pre^ 
parsd by addisng charged groups &mh as sulfate or earbo^l fifosps 
to th® syathetic polysaeeharidsso Th@ @y2%th@tic polysaecliarids 
derivatives can be prepared ^ith great v^H®%y in slz@» ©Irnct&ir®, 
charge, etCo V^'ith th@ir help we inlssided to Isarsi som^tkk^g about 
the satisr© gud lh@ str@^gth of forces ^Mch e^st b@t^@©£i ©iszijmes 
mad stsbstrates iii th® early stages of isateraetioi^. Th®s® forces 
appear to be general, and isi the neimsrous mm&yme systems we hav® 
studied -we alivaiys cossld achieve IMilbitioii of the ©ssasyme activity if 
vas chose for bioGking a synthetic substance '^hich had &n opposite 
charge to the net charge of the @sizym®c This m@cha^sm is most 
obvious ^here the substrate is also a snacromolecul® or ^hen the 
substrate l^s a definite charge. A large nmmb&r of ssisyme s^° 
strate Issteractions fall mid^r thes© d@IIMtio3is. 

The sy^t^m ^© st«adi@d most in detail ^a® rlbcaajieleas® 
(RNas©) asd riboaueleic acid (RNA). '^e chose this system feseaiis® 
th© stractttr® of the ENas© Is completely knowno With small aisioiiMs 
of the synthetic aMonic (negatively --charged) polysaccharide deriva» 
tives, SQch as polyglecose sulfate, ^e coisid completely IsMbit list 
RNase, ^hieh is a cationSc or positlvely°charged prot@i@ at th© pE 
t?her® it has the highest activity oa the HNA, ^hich is a n®gatlv@Iy° 
charged macr©motec82i@o W© studied ^hich type of polyglucos© sislfat® 
is the most effective in isshibiting the ENas® and wq immd thst the most 
important factor is the degree of sulfatioa^ that is t!i@ €hai*g@ desisity. 
If th® degree of sulfatioia ©n th® polyglncos® sulfate -©as S p@r ai3hjdiro<= 
gl^gcose isnit, «rhich is the maidmsam possible, this preparation IsiMb^^ 
ited RNas® more than a thousaad times more powerf^^ thaja the 
hitherto known best Inhibitor (h©parin|, assd several tisr.es better ^lap. 
|©@s si^stiteted poiygMess© s^slfst^s with 1 or 0. S sulfates p&r f ^hy=> 
drogliacose mslts., 

Similar qisantitative studio® ^@re dose comparing liiMb<=' 
itory potessey with charge dfsasSty, asing several polyglaeos® sarbosyl 
derivativesa In this case to© th© charge dsaslty was foissd to b@ the 



752 



serial No, WCI ° 282 
Calmdar jmr 1@59 
Part A » Pag® 4 

most irApertaist mser®mol@eisl®? param©t®r. Similar g'ts^ts i^@re 
obtalasd -^^ith othsr tsisyisi® systsmSo Th©rtfor@ It appears tisst 
th® most impm-taal force la the ®arly stages of luteraetieis fe®tw@®ia 
th©ss enzymes asjd the aiibstratss is prs^bably electrostatic. It is^ 
kno-«rn that ossly sleetrostatie forces can S3d:©sid bsyoad sbosit 5 A 
b@twe®Q i?Et®ractii3g maeromol®es3l®s, tfeerefcsff© it is ssot safprislag 
that th®s® forces ay© ©pes-ativ© ia brlag!ag tog©th@r th® esisym© a©,d 
lh@ siabstrat®. 

Th® molecular weight of Hi® IrMfeiling sisbstasees ^as 
fotssid to fos of less importance* ®xe©pt that It was always si®e©s®arj 
to liave a maeromoiecular sebstaace '^Stfe a molsc^ster ^slglst ef at 

i©ast 2, 000 to obtain Sishibitioa. Th® strsscts?® was ©f ®t©ii less 
importeac®. la all of tla®g@ e^®rim©iits the iisMMtisig ajsstlseti® poly- 
saccharidss w®re compact, spherics! macromol®c®l®So Tbis sllou^ed 
a study of the macyomoi®C6sIar iffiteractiois im a thr©s dlraeusioisal 
basts which caaaot b© d&sk® ^ith IMtar masF©m®l@ctsl®s; aisd thin 
approach Is opesisjg up th® posslMMty ©f d®t@rmfeiBg th& sis© of th® 
aetiv© sites of ^nzymeBo 

Th® RNas© activity isas tuhSbltsd also on th© small 
substrate snolscules cycMc oridylia aad cjtidyltc 3=5 phosphat©So 

Aiaother eissym® system which "^as stsidi®d M some 
detail was th© astiois ©f lysosym® «^ tfee c©ll waE of Micyoeoc^ag 
iysodeikticBSo W® det®rmia®d th® amount of polyglaeos® salfat® 
oeeessary to obtain q5sa^titati^'© IsihiMtioa, aad iound it to b© appros:i<^ 
mately eqsial to th® amo^at of th© lyisosyme» which has about th© 
sam# znoiectslar ^sight as th@ iiMblto^. 

Other catioMc essymss -^hicfe w©r© isihiblted by th® 
synthetic auiQuic polysaccharid® d®rivatlv®@ ^©r@ hyaliss'ojsidas©^ 
DNase, trypsisis chymolrypsisi, cytochrome"© oxidastp ©tc. Th© 
work on trypsiss asid ehymotrypslis IssMMtlon was does® Jointly with 
DTo Ho Coheu of PrMcstoa Lafeoratories, 



753 



Serial Ns. MCI - 282 
Cal®iidar jear 195S 
Part A « Pag© S 

To study tfe@s® ph@Eiom©iiae and to l®ar^ moi*@ aboat 
th@ fimdam©atal natssr® of ensym® sisfestfat© Isteffactios, phjsiasl 
chsmieai studies w©r@ carried out ©a the Interaction of two ©pposit®!^" 
charged macromol©C8sl@s. Tilratiou of polyeatiojis wi^ polyaaions 
was foMSxd to follow stoieMomttric ©qsslvalsac®. The Isit^Faelioa of 
such macpomol®cisl®s -^as dJUfertRt h(m®v®rs from the ialeraction of 
low moleeialas' ■weight icmn, Bime® a firm ©I©etr©static complex ^as 
formed ^Mch und@? ces'taia conditlceis sissh as st Mgksr coaeestra" 
tioss led to precipitation of th® macromol©cialsr eompsag^tSo TMs 
phsaomesioa (precipitation) caa b® ased to fraetlomt® laasr^molgcsilar 
compoasats; and this prlseipl® was applied in the fFsetionstion of a 
blood compcm^nt, d®3orlfe®d later* 

la th@ study of ®uzjm@ ssfestrat® i%t®raetl©si , th® 
Ught^scattering method '^as also €mploy®d in th© ©arly part Gi tti© 
year by Dr. J. W, Pr®iss, Dr. Pr®iss, ho'^@v®r, b@eame mor© 
interested in appl^g his @l@ctroa«fo©mbardmts&t m@"lla©d to MologiesI 
problems, and after cosastriscstisjg a lo^ ©ssergy electron gaiSj la® stsditd 
th® dtstrssctioES oi tumor s^erotisfeg activity of th© eadotoxlc polysae^ 
charld® from S. marceseaas. H® also applied the ®lsetroga'=fe©mbard«=' 
m©M teshsilqiai'to ®azym®s. Dr. Pr®is3 plaiae to costlssti® his espgFl» 
m®®ts and liatgrest in ®l®etroiit«bombardm@ist testeiqiies la a^otlisr 
Isstitut®. 

II, Blockig g of tonicity ®f catioMe macromolgffialgs. 

PoljmjKte, sisomjcls, streptomycin, of protamif^©, 
ia dos®s «»hich caiase the death ©f mice fs'lthfe 25 to 40 snmut^Ba esM 

b® coaateracted if a n^^rB.Mzing eq'^vaterJ ©f asiioisis sygith@tis poly 
saceharld© derivativ© is administered hj th© sam© iatrapsritoasal 
rossi® 10 misates after th© Ipjeetion of tli@ to:!£ie dos® c^ tls@ eatioisls 
astibiotlcss th© moriba^d miee r©cov®i;*©d i^pidlj and is a Is-?/ mls-eles 
appeared to be aormaL TMs ^as a rapid and drastic revisrsioa oi 
toxic symptoms. Oar fisd^gs wer© reported ia a lead as^isle In 
"Halsre", If two mliaistes prior to th© appMsatioa of tli©s=© eatiosiiic 
macroEiolecales polygliaeose sulfsts was applied at a sit® far from 
the perltosi®ai cavity, (md@r the sklia ©f tte© n®ck)> there was eo 
toxic effect t^om th® isthal dose of th§ ssstibiotiese SIseg siasfe rapid 



75^4 



S@s-tel ^^©, MCI " 2@2 
Cal^dar y©ar 1959 
Pai^t A «• Pag® 6 

disseKilnatios ©f the p©lygloc©s@ sulfat© iat© tli@ peritoneal eavity is 
uallk®ly ^mdes* thss® conditions, iS is possible that th® poljgkicos© 
stalfate stimulated th® relsaa® of asaicmSc pelysaccharldes from ®sa=> 
dogeaosss soisrces. \l' ® st&idi^d lh@ ©Mcieiie j of th® dMer®3st poly» 
glusos® siilfats d©pivat4ves la detosdfieatioa, aad w© fosissd agaisi 
that ths most hi^ly charged derivatives ^®rs by far th® most aetiv©, 
Th®r® ^as a direct correlatioa bstw®©a the aMlitj to ee^st^rsct tfe© 
toxicity of cationic maei*omoi@ciil©s and tfe® ehsrg® dSEsitj of th® 
anionic macromol©calss. Simiiaf fsstilts '^®r© obtaisstd with th@ 
polyglucos® cafboxyl dsrivatives. 

Elo AppUcatiosi of maeromol®cafcir fcttt®gaetion to 
the bacterial poljs acch a rjd® fgom S. mage@3c©s^ . 

T'h® tumor^^scpotisiag activity of the is©gativ@ly«' 
charged polysaccharide compies preparation (Lot Mo^ P'-SS) was 
redaced by RNase, hy prolamise or by oth@r maeroeatioaso Howe-t^®!', 
whea lysoaym® was applied, w@ foimd 4m©^€ct@dly that th@r@ '^as a 
significant iEcrease in th© tiamor necrotizing propsrty ©f the F='2S. 
while at the sam® lime it appeared that th® f®v®r prodiseisig activity 
^as red«seed» C^rrsat research Is imder -way to study this iist®?ac= 
tion between P-25 and lysoayme and to s®parat@8 if possibi®, fee 
f©v@r pro'iaciag unit of th© P<='25 from thg timior nscrotlsfeg portioa® 
Uitracsntrifagatioa esperim©sts ^@f © ear^lsd oiat Jotetly ^^ith Dr, 
^. Carroll of NIAMD, asiisg a a®"^ fractissatioa msthod, @mpl©yiag 
asi aaaiytical c®E ^Ith a moving platformo 

IV, laMMtioa of phag® T2« 

Rseeutly it m-as fotaad fey other Isavsstigstors tliat 
th@ tail of th@ T2 baeteriophag®. ^Msh sttaehss to the E, c©li eeE 
waE, contaisss a positive protein similar to iysosyms, aad that th® 
attachment happens as thca^ ®lectrostatle forces act bet"^'®ea ths 
positive tail of th® phage and SBSgative ai'sas oa the cell dus'lag ths 
early part of the isitgractioa, ?v@ tried to s®@ if this electrostatle 
interaction eoiiid b@ pr©v®sst®d fey bloekissg tha positive tail protefc 
with 3S®gativ© synthetic polysaeebarid® d®rivatlv®So Vv® fousid tfeat^ 
Bpou short iacsbatiojs at the pH of 4. 6» polygliaeos® sislfaric acid 



755 



S©^i®l H©o MCI => SS2 

Fas*! A - Pag© T 

Cor poljgiss®©®® ©aifbQsyl deHv^ti^® at @©m®^Mt Mgl?®i* d©seg| com- 
pletely inhlMted ths T2 plrng®, ^Ml@ sniffle ae£d at the same pE' 
did a©ft redaee th® viability of the plsag®. After these laei^batloii 
steps, the misttars eoQtaisiiffig the aeiioQle mmcromolseisl® assd the pMge 
was dii^ed a thos^aisd fold aad platad ^id^r isormal co»diti©isao At 
th© lower pH of 4o S, sialfmic acid pas'liaiiy redaeed the vSaMlily of 
the phage, bat th© ®ff©et was s^versibl© by addltlosiai Ssssiilsatlea at 
a hSf he? pH before plattegc Th® inMbStory s®ti®si of p®lygSssc@s® 
sttlfsii'i© acid isas act ff®v®i'sibl@o These ®t88di®s& shcm that elsarg®d 
maeromolscoles have a special rol® in phage IsMbltion asid d®mon° 
strate th® inhibition of viruses by the iiiti>od«icti«Hi of a 3yss$h@tie 
material «rhlch has a Mgh@r charge d@£sslty thasi th@ recsplos>« aiad 
thos either denatures the virus oe* ©a®s©® th® vir^ t@ p2'®f®r®istl.©lly 
attach to th© iaMbStoro 

Diarisig the course ©f our et«dl@s other poiyglucose d^rlvatiT®® 
were also employed, and w© te&dverteMly f®®®d on® ©th®r tjp® ©f 
effective iuMbitor to T2 phag®o At pH i„ S, m©l@C48l®s ^Sth tw© al= 
dehyd© groups were imnd t© b® effective teMbitors, aad both large 
aad small mol®c«l®s sais^e-d ©qisaliy good.iahlbitisra^ tfe®!*©!©?© itils 
«as not a macromolecelar Inhibition, Th® ald®hyd©§ employed ^®rm 
gleeose dialdshyde or polygli§©os® dlaidshyd@o 

Th® T2 phage was s@l®«t®d f«» laMMtioa sisee IMs phag© Ss 
©aa© ©f the most stadled virsises, assd sfee@ It Ss @asj t© do qmsitltaiiv© 
^ork with this syst®m„ These ©spsrimests ^'©g*® doae la preparatioa 
for ©steasion of s«ch lisMbitlea ®tsidi®s to aoimal vifsssds. 

The above espef tmeutal ^orK rsproted Isi §®etlogss l=IV was 
carried o@t lu e®llaborati©ii with B. Go Yo^sigo 

Vo Sy^theti® Polysaccharides. 

fo the e©wse ©f preparalioEi c^ sii©gativ@ly surged 
polygl^os® derivatives a s©s^®3 «^ poiygliseos© ©arbo^l derivatives 
^as prepared ^ilh dl^eriiiit amoseals ©f earfeo^l groups p@r raonome^ie 
isisit (P, Maary)o A® already meMiossed, these products ^bt® m^d to 
study the charge dep^adease of EWas® and T2 phags lisMbltle«« Also, 
these preparatioas w®r® io^nd to esihaisee ACTH aetivltjo TMs latter 



756 



Serial M©o MCI - 2iS 
C&l®^d^w j®^r 1SS9 
Pas't A = Fag® 8 

work ^as dea® i® eollsboratioii with DFo M„ CshSfS!, Prlasslos 

polygiucos® sttlfat@$ whl€h &r® strosig a»tl°>€oagalants, Th® 
carbosyl deHvatl^ss ai*© son^tosle i, po at 10 mgo p@r moiss®, Prefo 
E. Wolff of the U£ilv@s>s% of N&ncy fomd that th® Ilp@mla»si®ar£!tg 
activity of th@ f«3lly stsbstitnted p@iyglsso®e sullat® £@ several times 
Istghtr thasa that ef heparin. W® &r® em°?sMly @^sg@d with Pr€d> 
Wolff ^ a Gol^borativ® attesmpt to syssth^sl^© a p^lygMcos® dsrlva^ 
tiv@ sabstitutsd with nsgativs grotips ^Msh ^lE liav® Mgb llp@mla° 
cl@arisig activity B,nd no ai3tt°c«^g@!asst or oth^r t©^s side a«tivi^. 

The differeat aaioaie polygleeos® d®i"ivatiir@s 
w@r@ missh te dessiaiad; moE>@ thass two do3@@ ^i^ir^rslt^ Siiv@slig®tof !3 
requested samplsso several of tli©m fos" f^sgareli appll©®tloiss in 
immisnology and iu sssymology. 

Th© rtst of th© syiitlj©tie progs^m, ^hieh Is eas'S'l^d 
©m ^ith Jo ¥^„ Wood, also iaeisidsd prep^rati©!! ©f positively cliarg©^ 
synthetic polysacehaiids derivati^sso Th@s® cstionle polygl&seQS© 
derivatives af ® golag to be used t@ stisdy Hi© isMMtiojj of M©i®gieally 
active aoioais macromoleeMi©©, Preliminary ^©rk showed th^t tliej 
are aimiferly efftctiv® Sn IsiMfeltlag ©azymes with s®l K,,gatl^® siasFg©^ 
aad th@y can also s®rv@ as p?®e^itatjiig and fi^actlossatlsig agents @f 
anSosai© masfomoleculeso 

The sjatlistie m®lliod ©f pE-epagfeg polysaecfea^d®® 
from mofiosaeehafide by ehemical p@lyccmd©usatlmi prce^din'© "^as 
®Hlejid®d to sium@£>ous other ssgars. High molecular ^©Iglil p©ajm®rs 
of 2=d®osy=>gliseos©» ribos®, rlsamssos®, galaetc®©^ maltog©, massaess 
arabinos®, €te. were c^taisi®d in good yieldo 

Th@ sysst!s©tie program oa ph©gisgia® elssmlstrj was 
terminated spoa lis® disalsiMtr f ®tlr®m®M ©f Ds'o D» L, VIvissio 

VIo Chtmisal stia-iles c^ the pol^sagehagidf g'Sgipi©:a^ fi^om 
So marc©sctas. ~ ' ~ 

Th® ^rg® seals s©^ poiysaeetisrld© preparations 
of Mfo u P@rra^il designated Lot MOo P^iS was stsdisd by 



757 



Serial NOo MCI » 2$2 

P&ri A - Pag® 

D^, Eo M©rl@ro H© foiaad tliat th© preparation is hem©g®iso»^s etee^ 
tFoph©s"@tlsaliy but coiataiiss two comp©si®ssts whieh s@dim©iit at 
di^epeist rates ia th© jsltraeeatf if«g@o Th® h©avS®F eomp®si@zst appears 
to b® an aggregate of a ligM@r c®mpeK®st ^ith a f®^ feissdred thosssaad 
molecelai* ^eighto The ¥°4i& is a strongly segativ® maes'om©l©c«sis» 
similar to P«25 « Titration of th@ nsgativ® groyipg, £@ eoHaboratioB 
with Dr. Ho Saroff of NIAJ^ID, sho«^®d that th@ pK^ ©f m® r.®gativ©ly 
eharged groups is abo?st 2„ 7, asid it is most likely to 8©m@ from a 
carboxyi of th® maaramic ssid lypSo Th@r@ was oisiy a v®s^ small 
amount of amino acid associated tirilh tb@ P°45 preparation. Th@ 
gr@at bulk of th® polysacclmrid® is a polym®r of gltseos®, with Oa 3% 
phosphorus attached to it in mom® organieally^bonsid form. 

VIL Ssareh for endotoaie polysaccharld® compleseg 
from mammaljaa^sgurcesa 

Dr. H, k.artiK of th# UMversity ©f FrassMart^ 
reported obtainiag pyrogenic preparatiosis from hianaa felood eom- 
ponents saach as from r©d e®il stroma usid^r st©ril® eonditionsj 
using a strong phenol esstractioa. Hoi^evssre Dr. M@rl©r did not 
obtain pyrogsnic material by this mstbod. Dr. Mora^ while ia Eisrcp® 
to attesid aia Saternatioisal maeromolecisiaf sjrmpcsstem, %^it®d Dr. 
Martiix'' s laboratory, and together viUh Dr. Martin prspar-sd a 
prodaet from stroma, and erarreHtly w® ar© attempting to reprodac© 
the 5/iartia method iss o«r laboratory. 

VHI. Fraetioaa tioa of blood for aeompcmeat ^Mels 
B@@traUs@s ©nd^ai e aet^ty"('EDC')o "' =--—-™~--— ■--—===— 



After the findings in the Polysageharld® S@eti«» siiadsr 
Dr. Lasdy, that hsman seraim eosstalsa® a compoaesat 't^hish isaetii?£t©s 
th© ©Bdoloxie prod^st from basteria, w© (Mora» Yo^iisg) attempted t© 
fractiosist® farther this factor in collaboratissi ^^Itii Dr. Landy* s grot'.po 
In oar fractlossatloss scheme a s@lectl%'e preelpitatios ^Wi polyglnees® 
sulfate was ©sed. Uposa preeipltatioa isa a Karr©^ pH rssige asd spoa 
re-=solisliois of the preeipltate, most of th© aetivity was reeayered m 
a small fraction of the s^rum, Siieh fractloisatlon @Kp©riin©Ets ■w®r@ 
temporarily s«sspesid®d bist it I® plasmed to st&rt this ■^ork agala Im 
eoUaboraticm trith th@ Polysaceharid® S@etiois. 



758 



Part A » Pag® 10 

to Bi©m©dl@al E®@gar@ls_aad_lgJhg_Fr^^ 
of the ~ 



Th® ©sasjme blc^kSag sft«sdl@s ar® designed So isliieiclat© 
tfe© g®aeg*ai forces ^hiefe SsitSat® the eiazyms^siibstrat© lateraeticsa 
assd which probably oH®M th® ®Mym@s" approach t© t&@ s^l^sts-st©® 
so that complemesitaFy sit©s ms^te '^Ith a vsry lilgli per€®Maf@ of 
e^sctive coHisioaSo Siasli glisdies a^© b.Iso fefos'mativ© with irffigafd 
to Ih® natBT© ©f th^ acti^© e®®t©rs ©f t&@ ©asymsso Th®s© f5issda=> 
mental st«sdi@s ca® Isav® practical ®oas©q53©2ie®s to s®l®etiv®ly te° 
hibit&sg certain eiaaymle aetivitieso 

by asmlogous ®!®etr®§talie lisl©raeticm wMh ©pp@sit®ly efeirg®d 
maeromol®e®l@s may h® ®s@fial la ^arm®eol©gj» 

Thess studies ©f ma®i*om©l®eislag' l0t®3'actl©giis «Msli felosk 
bioiogieai activities hav® lmp©s^^s@ fea rslalio^ to tk® imtisr® sisd 
m®chaaism of aon-speelfi© r®®istaae©e a sssbject sa th@ progi-am ©f 
this Laboratory and espeelaliy ©f tli® Polyaaeelias'ld® S@eti©ii« The 
fiandamesatal work ©a mae^omolecalas* iiiteractSost mm^ iacr®a@© otsr 
kEowl©dg@ to th@ abov® fi®ids a®d stimislat® f^tture '^^ork ©ss siqii° 
specific p®sislas!s© factors, aM g@ii®raEy ©la Immnaologieal fastarrs 
1^ casscep r@s®a7eho 

Tfe® @s4e«isiois ©f masr©m©l©cailar fet@racliosi to pr®slplta<=> 
ti«H» and thias fpactioaatiosi of bioiogfeal ®Btitl®s reps'gseEts a m@vj 
method ©I separating d®M®at® Mologleai macFom©l®ei®l©s iisd^r 
cosiditiosss wSi©F@ t&e activity is ©asilj loslo 

Th® pteg© studies giif© «ss lisfsrmaMoa en the satm'® ©I 
th© forces isaitlatsiag attechm©ist, Sisch forces migM b© similar in 
ffiMmal vSraseSo These stsidies &r@ das® la preparati©!! to apply 
sseh SfflformatioES t© the possible bloekiiig of t^mor vir^aSo 



759 



Serial Koo MCI = 2S2 
Caleada? year 1§§© 
P&n A - Page 1,^ 

Pro|?osed coay s® of projeet 

The ftmdamsnta! studies 4^ macromoi«eular M«> 
toractfon asd the Hophysieal applieatiosss wili b« ecntisi»®do 'Virm 
and enzyme isMbition wUl b® fgarth®? st«idi@d, Appiicatioss of virtas 
bloeklag will b© attempted oa som@ t®m©ir virsaso The macix!« 
molseular i33leraeti<MS related to th® blosSclag of bioiogieai factors 
associated with QOii«§p@cific rssisSaae® ^iii be coati&usd also and 
exteaded la eoilaboratiosi with the Polysaecharlde Sectioii and 
tmder th@ direotion of the Chief of this Laboratory. Also frao^ 
tiosmtion by macromoiecylar c<»npl€si£ig &nd by other methods 
will be carried out for separatism of blood factors participatiag 
in noB^speellls r@sistai£e@c 



Part B SsBclodeds Yes 



760 



S©?M1 M©o MCI = 2il 
C&l^adag' year 1@59 
Pags B =■ Pag® 1 3 



PHS=MS5 
IsadlvidiMl Projset Report 

Calsndar Y^air- 1959 



Part B 



Henors, Awards, &nd Fiab Meatioaa 

PisbMeatJQas &th@r tb^is abstracts l^om this projset; 

(1) Mora, P. To aisdYoasig. B, G,? R«verslbl® Mii&itiosi 
of Eaz^rmes 1^ Znteraetio^ with S^t!i€tie Polysaeehairid® M^croaMoas, 
AFsfeives Bloeham. Bioph;fSo 8|» S«20 C18§8)o 

(2) Mora, P, T<, „ Yoissag:, B. G. asad Shear, Mo Jo z 
Redactioa ©f Toxielty of Catiossle Maeromol®si8l8s by Complsxisg ^'ith 
Assloaic Derivatives of Sjxithgtie FelygMcoses. Nstur©, JM^^ 43i°4SS» 
(1958)o 

(3) Moras Po T„ Merlsr, E^ and Ma^ry, P^s Sj^thstle 
Polysaccharideso IV^ Prepsratioa of Carfeosyl DsriimtiY®® ^ P©ly= 
glacos®, Jo Am, Chem. SoSo. §!_« 544g=5451 {105S)o 

(4) Mora, P, To, Sli@ar, M. JoS R#versiMs Milbltiea ef 
MacE-omolsenits (Enzymes, - etSc ) by Electrostatic Iiit®raetio2i -^ith. 
D@ix vativss of SyntfeetSc Poiysaccbaridss. Iiit#r!^tl®aai® UMcs fif 
Reiss® as&d Asigewasdt® Ch@mi@« Kosnmi@si@^ fir Maeromol^eil©, 
Sympositsm ^b®r Makromolek^le in ¥<i@@bad@sio Macr©mol€Ci^r@ 
Natarstolf© yad M©d®Us« Vortrag© V CS, V@rlag Ctemi®, GmbHo 

(5) Mora, Po To, Wood, J, W, aiad McFarlaisd, Vo Wo s 
Synthetic Polysaccliarideso Vo Polymerlaatioa of Varlsis® Aldoses, 
J. im„ Ch®mo SoCo %^^2£» (198IS)o 



76: 



Serial No, HCI -=262 
Calendar year 1859 
Part B -= Pag® IS 

Hoaors and Awards g elatteg to this pirojects 

Po _To_ Mora; a) nominated to tb® NCI Spsciaity 
Fe31ow»h^ Boardi bj lavited by the Commissiosi «m Maepo° 
mol«cttts3, l!it«raatiOBial UMon of Pare and Applied Chemistry to 
papticSpate in a Symposimsi at Wiesbad®]^« G@3*ma<i^» and to 
ppeseat oar work on sasym© Isahibitioa; c) Issvit@d to giv© semis^^s 
at the Medical School, Yale Universfityi lastltet® foi* Mae?©mol@c«slar 
Chemistry, Univ®i>sity of Freiburg, Gss-mai^j C. N, Ro So C®rjt®r fosr 
Maeromoleeular Research, University of Strashoarg, F?aac®s asad 
DepartmesaS of Biochemistry, Ualversity of Oxford, Eaglaiad^ d) 
Isivited to orgaMz© a symposittm as Cfeairssaaa oo PoiysscchaFid® 
Synthesis for the ISSth National Meetisag of the AmeHeasi chsmlcal 
Society, N®^ York. 1900. 



762 



Serial Mo., MCI-261 
le Lab, Chasiesi Pharaaacology 
So Folysaceharide Section 
3. Bethesae 14^ Marsrland 



PHS«NXH 
Individual PiTOject Report 
Calendar Year 1959 



iMLI^ 



Project Titl e? Kie r^le of polysacchari*!© complexes in 
resistance of the host to injurious influences « 

Principal Inveeti^atori M„ Landy 

Other Ihveatig&tors : W« Eeene, J« Go S^chael, Fo S^ Rossn^ 

R«Jo Trapani and Mo Jo Shear* 

Units? Cytochsiaistrj Section* Labe Biochemis- 



r^r gg if i e 



;ry* NCI, Serial No, NCI=33^o Biochealstry Branch, Amed 
Forces Institute of Pathoiogyj WRAiCo Rocky Moimtain 
Laboratory, NIAID* laiminologlcal Research Laboratory ;> 
State Mcteriological Institute, StocJkijolma Sweien* 

Man Years (Calendar Year 1959 )« Patient ,D®.yf •■ 
Ibtal 6 1/2 

Professional 3 1/2 ^o"® 

Other 3 



Project D escription? 

Ob Jecfeiv^e® s 

To obtain information on huisoral and cellular' 
factors in the host ishich contribute to natural resist- 
ance to infection and to neoplasia, Bn6 the mechanisms 
by which these factora are iRfX\5enced fey polyssscharide 
co3^1ex@@ o 



7G3 



Calendar Y^ar 1959 
Part A - Page 2 

"°'°^ocSemica3!7''Siunoiosical , microbiologicsa^ and 
physiological, EKMSified as required to tmet th© specific 
n@©«3s of particular p2^bl< 



^ij There has been continued interest in the extent 
to which the lipid and the polysacchai^d® moi©tl®® of bac«- 
terial endotoxins account for the characteristic host r@= 
3pon®®3 elicited by these cen^lexeea A nimfeer of report® 
have appearsd in ®uppox»t of th® vi©i? thst the endotoscic 
properties of these substaiices is attributable ©:sclusiv©ly 
to their content of fiamly bound lipid C^iPi^ ^)» '^^® 
issue has been investigated in collaboration with Dr» 
Edgar Ribi of the Rocky ^untain laboratory* NIA2D, Dr* 
Ribi has developed methods for obtaining endoto2d.n® of 
lovf lipid content from Salmonella enteritidja by @xtrac« 
tion with water saturatiS^iSfF^hyl e€H©r7"°'Still worm of 
the remaining bound lipid could b® relieved by non-hydrolytlc 
procedures o Endotoxin thus prepared contained only lo2^ of 
bound lipid in contrast to a lipid content of 19° 2^ ixi a 
product prepared by th® traditional ]^ivan procedure^^ lo©o9 
extraction of bacilli ^th trichlozssacetlc acid and phenol. 
Both products were extremely potent endotoxins* There was 
no significant difference in their potency a© regards the 
following! in rabbits «==• capacity to prepare for the der^ 
mal Shwartsman reaction j pyrogenicityi and letha3.ityi in 
mice «=^ induction of hemorrhagic necrosis in sarcomas i 
and lethality. It is thus evident that a large proportion 
of bound lipid can be reuioved from bacterial endotoxin 
without significant reduction in it® biologic activities* 

S, The effect of ©ndotoxic polysaccharides on 
tutior glycolysis was investigated in collaboration ^th 
Dro Mo Wo Woods, Laboratory of Biochemistry, HCI^ Endo- 
toxins in concentrations of 0,003 to 0*3 parts per million 

effected a @ev®ral»fold ©tiimlation of aerobic acid fojrasa- 
tion from glucose by the S91 uK^use iselanoHm, Anaerotois 
glycolysis was increased to a lesser extents Sie degree of 
stimulation obtained with a series of endotoxin prepara^ 
tions derived from different sources by various isolation 
procedures paralleled their potency in eliciting typical 
endotoxic phenomena ^ vivo» The findings indicated that 
the properties required""^^ elicitation of reaction® in 



76-1 



S@rlal lfo« HCX«26l 
Cia®n^a3? Yeas* 1959 
Part A -=• Page 3 

th© host by the®e isaerosiolecular materials may b© the Bmm 
B3 those Involved in af lectins glycolysis in vitro, 

3o Studies on an ®n^ot©xin»detoxifyins agsnt 
(EDG) in anlmsl tissues were continued in collaboratic^n 
with Dr, Waravdeka-fft BeEd of the Biocheodstry Branch at 
the Arm®^ Forces Institute of Pathology. Incubation 
in vi^ro of thin slices of rabbit liver, kidney or ®pl@en 
wTtlT"^^, ma roescen® endotoxin pro<Sueed no significant 
ehanglTn the tty^jr-cTi^naging potency of this polysaccharidt* 
However^s 40^ h«»QOgenates of these organ® altei*e«S' this @ndo« 
toxin so that it no longer eli sited he^^rrhagic^necrosis 
in Sarcoiaa 3To E^erisiental isoxlc tias th©i?@®f t©r restricted 
to rabbit liver ^ioh was freed of bloo<3 by thorough p®^- 
fusiono Liver homogsnates were fractionated li;^ standartS 
cytoeheiaical and eentrifisgal pi!©e@dur®s and it w®® found 
that neither nuclei nor mitochondri® possessed en(iloto3d.n« 
detoxifying activity^ The supernatant j^ich r©ssain©d ^m^ 
activ®j but this activity was lost ^©n the ®iC2^©om®s 
were rM®ov@do Ifowevsr^ combination of microssom©® ®n^ 
supernatant r@©tor©d encSotoxin^inactivating poteneyo Fr©-- 
limlnary experiments on th© experimental condition© of 
test idiich influ©nc© th® inactivation of endotoxin by this 
tissue preparation indicate that it imj be different fr©m 
the ESX; in blood, 

4„ An inveatigstlon if®8 begun of th® effects of 
selected carcinogen© on natural iiraimity of the irouse. It 
hss been demonstrated by other investigators that large 
doses of selected carcinogens {methyleholanthrene^ ben£o« 
pyrene, and others) produce marfsed depletion of host 
l3?TOphoid ti®isue„ Xn addition^ there is suppression of 
bone marrow activity and hei^rrhagic necrosis of the Wimll 
intestine in Jiiice receiving large do©e® of carcinogens. 
Because the tissue damage produced by carcinogens re^ 
sembles the tissue destruction that results from total 
body irradiation* it seems reasonable to assmrae that th© 
tissue dsffisage produced by cErcinogens likewise is 
associated with profound alterations in host resistance « 
It is also ISicely that these alterations in host resist^ 
ance would play a significant part in the pathogenesie 
of tumors resulting froia these carcinogens « 



765 



Serial No, MCI-26I 
Calendar Year- 1959 
Part A - Page 4 

Our viork thus far has been primarily concerned wltJi 
determining the toxicity of msthyleholanthrene for the 
CAFt mouse « The observations of other's have been confina- 
ed and it has been determined that 0.1 ®ia/ks« of methyl- 
cholanthrene is sufficient to produce maximira tissue dssEage 
without lethality. In addition, it has been found that 
the hematocrit and the clearance by the R,EeS„ of a stand- 
ard dose of colloidal cartoon injected i»v, are th© most 
sensitive indicators of lymphoid and bone marrow depletion^ 

5o In last yearns annual report we described the 
characteristics of a subatance in serua designated S>C 
(endotoxin detoxifying eois^onent) and the experimental 
conditions under which it altered isolated endotoxins 
in yi tr o_ so that they no longer were capable of eiicit«= 
ing endlo toxic or antigenic responses in the isansaaliaii 
host, These studies have been extended to ascertain 
whether endotoxin in its native state on the bacterial 
surface was likewise susceptible to inactivation by an 
agent with the distinctive features of ESC» Tsdo charac" 



teristic attributes of the endotoxin of intact S^^nella 
tyghosa, the induction of hemorrhagic necrosis TrT^irc^^ 
"^ and the stisulation of specific antibody forjsation in 



rabbits were the test situations eiaployed to measure 
changes in the endotoxin of ty^ihoid bacilli following 
interaction in vitro Mth seruao The endotoxic and anti» 
genie activitresoF°t3»phoia bacilli killed by heat or by 
chemical treatment were abolished by EDCj the same agent 
previously shovm to inactivate isolated endotoxino In 
contrast;^ the endotoxic and antigenic activities of viable 
bacilli were unaffected h^ EBC; they were, hot^evers 
eliminated by a different system in fresh serum. This 
inactivatiOHa of the endotoxic properties of viable ty<=> 
phoid baoillij hy the sera of various animal species was 
found to parallel their bactericidal activity for ^« 
ty phosa o It was ©hofin that the sasie components in aerura 
were required for both effects^ vis^s noraal antibody, 
all four of the recognized components of cos^lergent and 
divalent cations, SGrura rendered deficient in any one 
of these six constituents of the system no longer was 
capable of eliminating endotoxic potency or exerting the 
bactericidal effect. The quantitis^ of serum requiredj 
and the kinetics of the interaction in vitro and in 



7B6 



Serial No. MCI- 261 
Calendar Year 1959 
Part A «• Page 5 

certain in vivo situations « were strikingly similar for 
both effects, attesting to the intimate association of 
these change® effected in the bacilli by sennas These 
changes appear to be similtansous events; it has not 
been possible to effect a separation of the killing of 
the bacilli by serum from the loss of their toxic and 
antigenic properties, Bswever, the essentiality or in» 
evitability of these changes for the bactericidal process 
itself remains to be d©teiiained» Miich of the two huiSDr=> 
al systems in noraal senasa inactivates the endotoxin of 
typhoid bacilli depends on whether the organism© are 
viable or not. The concomitant elimination of endotoxie 
attributes of typlioid bacilli in the bactericidal process 
of normal Berom has obvious important Implications for 
the host-parasite relatlensMp*. 

6, The plssaa of febrile rabbits that have been 
treated with bacterial lipopolysaccharides has been re*» 
ported to contain "endogenous pyrogen", a substance ^th 
fever producing capabillti®s quite different from thos® 
of the originally injected polysaccharide. This led to 
the hypothesis that endogenous pyrogen is a product of 
tissue injury produced by the injected bacterial ©ndo>* 
toxin, Kowever^, an equally plausible hypothesis which 
we are exploring is that endogenous pyrogen is th© in« 
Jected endotoxin which has been altered by the host cells 
and plasma « Reduction of the pyrosenicity of endotoxin 
following Incubation in vit ^^ with normal hussan serum was 
first reported severaT°Vears agOe Studies in this 
Laboratory have sine® denionetrated that detoxification 
of endotoxin by human plasma is not liasited to pyro«» 
genicity and that alterations in other biologic proper'^ 
ties of endotoxin are equally impressive. Tliese obser- 
vations lend strong support to the contention that en« 
do toxin can be profoundly al termed hy the host^s plasma 
and that endogenous pyrogen laay, indeed » be altered 
endotoxin o 

Experiaients were designed to study in detail 
the pyrogenic properties of endotoxin that had been al*» 
tered in yitro^ by Incubation with hus^an plasms» The 
following' preliiainary data are available! 1) Altered 
endotoxin produces fever in endo toxin" tolerant animals; 



7B7 



Serial Mo. MCI-26I 
Calendar Year 1959 
Part A - Page 6 

2) There is no leukopenia associated with the pyrogenlc 
response! 3) Animals do not become tolerant to daily in^ 
Jections of altered endotoxin^ 4) There is no acute re«» 
fractorinests to altered endotoxin ^en multiple injec- 
tions are given over a short period of time. The above 
properties of eniSotoxin altered by plasma have been the 
very ones used to distinguish endogenous pyrogen from 
bacterial endotoxin. 'Hiese data ar© therefore interpreted 
as providing support for the hypothesis that endogenous 
pyrogen is endotoxin which has been altered toy the host's 
plasma, 

7, Adrenalectomized Mce are known to be unusually 
susceptible to the lethal properties of bacterial lipo^ 
polysaccharides. Treatiaent with 400-500 micrograias of 
endotoxin are required to kill normal !iiicei on the other 
hand, only 2-5 micrograms are necessary to kill adrenal* 
ectoraissed mice. It was, therefore, visualised that this 
specially prepared aninial might provide a sensitive and 
generally available test animal for the assay of endotoxin 
and for the endotoxin detoxifying component in plEsmao 

It waa found that the detoxification of endo^ 
toxin by EDO could indeed be shoxm in the adrenalectomized 
mouse. However, the quantity of this factor required Vfas 
greater when tested in the adrenalectomized laouse than wlien 
the reaction mixture was tested for other biologic proper* 
ties of endotoxin^ Consequently, the adrenalectoinised mouse 
has but linilted usefulness for assaying EDO since only 
plasmas containing large amounts of the factor, could be 
readily assayed by this procedure „ 

80 The bactericidal eystem of normal and iaamine 
sera, in which antibody and complement are the principal 
participants 9 causes the death or lysis of . Sram^negatlve 
bacteria. The susceptibility of individual strains of 
these organisms to the lethal effect of seruia varies 
widelyo The means by which fre^h serum evokes this cidstl 
effect is not known, nor ia infonsation available to 
account for differences in sensitivity to serua an^ng 
various species and strains of Gram°negative bacteria^, It 
has been reported that the endotoxins of these bacteria 



768 



Serial Ho. iCI-SSi 
Cslendar Year 1959 
Part h » Page 7 

C®ojaatic antigens, iipopolysacGharides) interfes?© with the 
bactericidal effect of ©et^oia in vitro „ We have sought to 
determine whether ®usc@ptibilI¥3nco~s@rum of ^tiganlcslly 
identical or closely related strains was correlated with 
differences in their content of endotoxin sndl/or soimtia 
sntigen, Tito siich closely relatsci strains of Esc heric hia 
coli were isolated from a sias3^ clone and vfeic^'TCDim ' to " 
(II?Fer imrkedly in their sensitivity to seinsmo The mxp®T^ 
nmtanfe fluids of boiled ©uspension® of these organism® 
served as antigen ps^parations «fhich were readily adsorbed 
on erythrocytes o Such antigen«coat®d erythrocytes ^^r® 
agglutinated by specific antibody. Ihe extracts from the 
seniza resistant strain contained laach sore of the cell 
sensitising antigen thmi did the preparation from the sen- 
sitive strain o It saas fo\ind that aeetonfe-killed and dried 
cells of the resistant ©train-, as wsll as extracts of this 
cultures were considerably more potent antigens for rabbits 
than their counterparts from the sei^ara susceptible strain, 
Furthermore th@ cells and the extracts of the resistant 
©train exhibited sarkedly greater endotoxic potency 
(induction of heiaorrhagic necrosia in Sarcoma 3T) than 
analogous products from the sensitive strain. These find- 
ings indicate that the quantity of somatic polysaccharide 
coi^lex on these bacteria vmj detenaine their susceptibility 
to the bactericidal system of fresh serum. 

The occurrence in norjisal semam of antibodies 
specific for bacteria of several genera of Enterobacteriacese 
{25 strains of Escherichia^, Salssonella and Shigella were 
examined) waa investigated » It ifas found that antibodies 
reactive ^ith each of these strains were present in norf^alo 
human and rabbit sera, The&e natural antibodies are pres= 
ent in low concentration^ taat the (jiantitiss are sufficient 
to pennit nsaximal expression of the bactericidal action of 
these Bev&o The bactericidal action of nox^^l serum against 
small inocula of E^ coli was not increased by the addition 
of specific ijssmne" antibbdy, 

9o The findings in studies on the effects of nor« 
mal serum on the endotoxic polysaccharides of Grasn^negative 
bacilli and on their ifiability^ presented in another see=> 
tion of this reportj, were utilised in a search for humoral 
systeias ^ich might affect tuimsr cells. Exploratory ex«» 
periments revealed that seru® exerted an impressive lethal 



7B9 



Serial Ho, MGI=.26l 
Calendar Year 1959 
Fart k » Page 8 

effect on tussor eells^. Incubation of Sarooaa 37 aseifee© 
cells ^th nonsal human serusa abolished their capacity to 
proliferate in micep ISiis lethal effect of semai on tm^r 
aells ims investigated systeiaatic&lly and it was found that 
this activity was ©xlilbited by a system distinctive by 
virtue of its sp®ci@© distribution, th© saany coi^onents 
required for it® actions and th® rang© ©f sious© tusiors it 
affected., Biost of th© ®xp@ris^ntal ifork ifss carried out 
with cells of Sarcoma 37 in the ascitic form and with 
fresh normal human seruSo Cells ^id seru® were Interacted 
vitr^ for 1 hour at 37*C«, in a ratio of liao^ c®lls per 



"oT~seruBi, and th© effect® on th® cell© w©r© ascertained 
in four ways, a) Failure of the cells to proliferate in 
vivoo b) Loss of metabolic activity, vrith glucose as "^b^ 



rate, as measured in the Warburg respirometer (in collab-^- 
oration with Dr, M, W„ Wood®), or by the reduction of neo^ 
tetraswliua, c) Uptak® of eosin as evidence of c®ll death « 
d) Mbrphologic changes» notably the extensive blabbing of 
the cell j^mbraneo The®® four parasseters yielded consistent 
result® in the assessment of cell viabilitja !Eie cytotoxic 
activity of serum was ellnslnated by heating at 52® C« for 1/8 
hour, or h"^ absoi^ption with imahed sarcoma cells at 4* Co 
The original cytotoxic activity was restored on combining 
the heated seruai with th© absorbed one^ an indication that 
at least two factors were required o Inasi^ch as ®u©h h®Et 
treatment Inactivates CS, and 'Since the absorbed s®n^ 
was found to retain its hesaslytie activity, it appeared 
that coii^lemtnt loight be involved » '^gts with the standard 
reagents RIb R2e R3 and r4 prepared fro® husian ©eris® showed 
that absence of any one of the four recognised coaponents of 
coieplement resulted in loss of cytotoxic activity. Appro- 
priate combination of these reagents restored activity^ 
Addition of EDTA to serum resulted in loss of cytotoxicityi 
restoration of activity ifas accomplished by the addition of 
Ca*^ or f^'^j thus, divalent cation was also essential for 
the reaetiono The reaction between human serum and the 
tumor ceils was explored as regards serum concentration^ 
number of oellSj, tea^eraturej and incubation time^ Sertm 
diluted 1j8 showed undisainished activity vfhile a ItlS dilu- 
tion produced a partial effects On® jbIo of sex'^tis destroyed 
the proliferative capacity of an inoculin ms Iw^rge as 3^0^ 
cells and exerted a considerables but ineoiBplete effect on 
7x10' cells o The reaction proceeded optitsally at 37® Cog 



770 



Serial MQo mi-'26l 
Calendar Yeajp 1959 
Payt A = Page 9 

with a lethal effect exerted within 2 minutes. One-hslf 
hour was re<^ir©d for cytotoxic acfclon at 20® C, Uat at 

4*C» c®ll Yisbility remained unaff©ct©^o 

Th® cytotoxic effect obtained with human @@nm 
was also esshibited by sera fro^ ohiasssansesa rhesu® s»nk®y, 
cow# pig# <3ogj> cat J, rabbit, chicken ®nd goose o Slon-eyto- 
toxic sere were thos® from horse, ®h®©p, goat, g^in©a 
pig, hamster, mt and e^'US©« Th€ factor absorbed fi^ra 
human seru® by Sarcoiaa 37 cells could also b® v®mD-w®6 by 
other m>uBe ascites tumors including th© Ehrlich ana Ks^hB 
carcinoraas^ hepatomas 129 suid 13^ and Sarcoma 180 a® i^®ll 
a® by cell suspensions pr©par@d froia solid tiMors of Sai?=» 
cosaa 37 and Melanoma S«91« Susp©n®iona of no3:'S!ial mouB@ 
cells of liver* lung^ kidney^ heart, imscles ffin^ erythrO'=» 
cytes effected little or no absorption of this factor, but 
spleen absorbed it coapletely. Preliminary eKperis^nt® on 
the eunditions for adsorption of th© factor on tmmv qbIIBs 
its eiution fro® the®® cells, its heat lability and ^her 
properties indicate that it b^gha^e® like a nonRml, antibo^Iy 
reactive with receptors on these mouse tosK>r- cells, SBnm 
injected into micei.po iisaediately prior to or following 
ioPo challenge with Sarcoma 37 ©©cites failed to exert a 
cytotoxic effect ^ vivoo 

^©re is thus present in the normal ©®raji of 
a ntaiBber of species a factor resembling antibody ^tuich/, 
together with complement exerts a lethal effect on nouse 



tusaor cells ^ yitgOa The action of nonaal serum on these 
cells bears a s'Crlic'i'ng resemblance to the effects produced 
by hyperinsmme seruiOp 

lOo Human serum has been shoim by Dr* Bo BJoAIimi 
of the State Bacteriological Laboratory^ Stoclchola, Sweden^, 
to contain a cytolytic factor coupled to an inhibitor^ • 
The factor was dissociated froa^ its inhibitor by dialyiis 
and obtained in a relatively pure state follo^sfins electro- 
phoresis « In the isolated istate it displayed a lytic 
effect on four established cell strains of human origin « 
In collaboration with m\ BJorkluna, exploi'^atory esq^sarl- 
ments '^ere carried out to exaisiine th® effset on raoaee 
tiMor cells exerted by thi® factor (CW) and other components 
separated from noraial huisan serum bj paper curtain 



771 



Serial No* HCI-261 
Calendar Year 1959 
Part A - Page 10 

electrophoresiBa CF produced -marked laorphologic change® 
in Sarcoma 37* Krebs, Ehrlieh and other ascites tuiaor cell© 
manifested by multiple lesions in the esll laeiabrane. 
Following this in vitro interaction the S«37 cells no longer 
proliferated in mice. The cytotoxic effect produced by 
this heat stable factor was manifestly different from that 
of the antibody-complement iQrateia described in another 
section of this report. 

Significanc e to the Prograa of the Cancer featltut©? 

."""'TKere' is ' present"' in""'^he riorraal' s'einia "oT'^ny^sp^ies 

a systeia of normal antibody and cossplement i?hich IB lethal 
for various mouse tumor cells in vitro. The effects exerted 
on tumor cells duplicate those r@porfi"d for hyperisssmne 
serum* Purthenssore h@at stable fractions have been isolated 
from noimal senm ^Ich are also cytotoxic for tu®or cells , 
•Eiese findings offer tangible evidence that the host may 
possess norasal components which influence the growth and 
behavior of at^ical cells and thereby contribute to 
natural resistance to neoplasia. 

Proposed Course of Project i 

"""l* Th© following parameters of natural infinity 
nfill be measured in mice that have been treated with toKic^ 
carcinogenic doses of ffiethylcholanthrene and bensopyrenes 
ao Survival of an heterologous skin graft, 
bo Resistance to a standardized skin infection, 
c. Antibody response to a well-defined antigenic 

stimilu@« 
do Reaction to the toxic and lethal properties 
of bacterial polysaccharides. 

2o Investigation of the endotoxin detoyJ-fying coi3° 
ponent in plasma will be continued ^th the objective of 
elucidating the relationship toetX'feen endogenous pjrrogen 
and plasma-reacted endotoxin, 1?o obtain such inforr/iation 
attempts will be made to pr^ pare animals whose plasaia is 
deficient in SDC, Medical and surgical extirpation experi« 
raents Mill be carried out, including the following s ll) 
treatment with nitrogen imstardi (2) hepateotoii^j (3) pan« 
createctorayj (4) splenectomy! (5) end nephrectoiaye Sueh 
animsls will be tested, in addition^ for their ability to 
produce endogenous pyrogen. 



772 



Serial Ho, NCI«26i 
Calendar Year 1959 
Fart A « Page 11 

3b Woric on the purification of the endotoxin i3e"- 
toxifying syatem in raaismalian tissues will be continued in 
collaboration with Dr. Vlaravdekar, Arroed Forces Institute 
of Pathology,. 2^e relationship of this system to known 
engyraatlc activities of these tissue preparations will be 
examined. The effects of various experimentally induced 
pathologic states on this tissue activity in rabbit liver 
urill be studied, 

4o A number of additional ©trains and species of 
Qrais-negatlve bacterial genera will be examined to deter« 
mine whether the correlation between susceptibility to the 
bactericidal action of normal seruia and content of soiaatic 
antigen and/or endotoxin, as found in E, coli, represents 
a general phenoDsenon. "" 

5, Studies will be initiated to cosspare the par« 
ticlpation of natural v®e iMaine antibody in the bacterid 
cidal process as regards the following: 

a. Their effectiveness for sensitizing bac" 

teria to the l®thal action of coaplesaent^ 
b« ThelT relative specificities as revealed 
by quantitative absorption tests with 
hofflologous and heterologous sicroorganism® . 

6» Study of the coisplement«dep©ndent system of nor^ 
mal een«8 lethal to mouse tumor cells will be continued witi 
the following objectives: 

9 The component of the s;^tem which res@m°« 
bles natural antibody will be concentrated 
and its properties exanilned in greater de«» 
tailc Infoi'mation will be sought to ac» 
count for its bisarre distribution in 
animal species » 

b. ISie range of tumor cells affected by this 

system will be ascertained, 
c« Experiments iflll be designed to account 
for the lack of serum cytotoxicity 
in vivo o 
de In collaboration with Dr. Mo ¥« Woods, Lab« 
of Biochemistry, NCI5 the raechaniam of the 
injury inflicted on the tuiaor cells Mil b© 
investigated with special ^tention to the 
striking effects on glycolysis. 



773 



Ser-ial flOo MCI«26l 
Caiendar Year' 1959 
Part A =» Page 12 

,7o ^e findings with serum fractions (prepared by 
Dr„ Bjorklimd) have made it evident that in normal human 
©©main at least tiio separate components are present vyhichj 
in the isolated state ^ are capable of exerting strong 
cytotoxic effect on sssuse tias^p cells. It is proposed to 
examine the conditions under which these effects are exerted 
and to detessaine the extent to which indi¥l<3ually or 
collectively they contribute to the cytotoxic effect of 
normsil senimo 



Part B included -=»— YeSo 



774 



Se3?ial HOo MCI-261 
Page 13 



PHS-NIH 
Xndivic^ual Ps?oJect Report 
Calendar Year 1959 



P art B o Honors, Awarda, and Fublieations 
Publications oth@r than abstracts from t his project? 

(1) Landy, M, , Trapani, R^J,, and Shear^ Mo 3<,z 
Inactlvation of endotoxin by a hui^ral component. X?c 
Alteration in the iagminological properties of typhoid 
endotoxin. J. Exper, FSsdo UOs 731=750 (1959 )c 

(2) Rosen, F, Ro, and Landy, M, : Inactlvation of 
endotoxin by a hiimoral component. V« EDO activity dipping 
Oram-n®gative infection and shock. Zeito f. Xes^inltat®° 
forsch, nB: October (1959 )» 

(3) Landy, M, , Trapani, K-'J*, and Rosen, F. S, s 
Inactlvation of endotoxin by a huiaoral component. ¥1* 
Two separate syateias recjulred for viable and killed 

)nella ty phoss a J, Clin. Xnve^tigc In pr«ss J^j 



HSifli^ 



Honors end awards relating to this project? 

The Head of the Polysaccharide Section was in¥ited 
to participate in Bn International Conference on Biolosicai 
Effects of Endotoxins in Relation to Isssunity held in 
Freiburg, Geraany, JUly ST-Sl* 1959» 



775 



Serial MOo ICI-800(C) 
la Eadocriaology Branch 
2» Eadocriaology Servlee 
FIS-UIH 3« BethesdEj Mo 

Individual Project Report 
Calendar Year 1959 



l^kA" 



PE-oJect fitles Endoeriae Aspects of ths Progressioa aud fhers^y 
of Cancer of the Breast ia Womqb and Meao 

Principal lavestigetors: Dro Do Mo Bergsastalp Dr^ Re Herts and 
Dro M= Bo Lips©tt 

Othe? Ic^estigatorsi Dr^ So Mo Kahn, I^o T<, L, Goodfx-ioadj, Dr^ 

D» A^ Selloggj, Dr« R, E» Moy, Dr„ H« Schwartz, Dro So Gsnuthy 
DTo Jo Le^Sj Mr^ So Jaoksoao 

Goop®ratiag Uait: (Br^ J^ ¥aa Bureis) 

Man Tears (calendar year 1959) s Patiect Days (calssdar year 1959); 
Totals 16 Patients 

Professiomis II Days 

Other: 5 

Project Deseription; 

gbjeetlvess lo To extend and intensify detailed clinical observation 
of th@ natural eours® of fsaaeer of the breast in men acsd X'foaen in 
order to bettar appraise the effestiveaess of various s^thods of 
therapy and learn more concsrnijQg th® etiology and pathogenesis 
of the diseases 

2 a To ii^rove won. existiag foras of horaoaal therapy for palliatios 
of advaneed breast cancer aad to elucidate the Eseshanisffls ijwolv&lc, 

Methods Employed; 1^ The aceeptane© of eoaplete cliaieal responsibility 
for the further iaanage^ent of referred patients with pro-^en diagnosis 
of eancer of the breast and th© application to their problesss of all 
available modes of therapy, 

2o The ei^loration of new laodes of hormonal therapy for pallia tiono 

3o The detailed analysis of ©ndoeriae and Estabolic factors operating 
in relation to the genesis and eoijrss of th® disease ia these patiant: 
up to the tlBi®. of their dsaiseo 

A-i, Careftsl appraisal of pathological apeelMens obtainsd at biopsy 
or autopsy ia eollaboration with Pathology staffs 



776 



Serial No^ ICI-800(0) 
- 2 - Eadoerinology Branch 

Hajoy Findiaess MataboMc effects of gro->^h^omoae: 

1, Extensitr© metabolic balaac© studies with chymotrypsinised 
beef growth hoj?3Eoa©j, whale growth lionEone, and sheep growth 
horffiono have revealed these products to be insetiv© ia maao 
Huffiaa gffcwth horiEoas was showi to ba an ®ffeeti^/e aaabolie 
agent in a v-ariety of clinical stiadies, A significant ©ffeet 
of huffiaa growth hormone isas an increase in urinary calciiaa ©s- 
cretion frcm 20 to 150 ingo daily, 

la an atteE?>t to demons trat© the possible role of gro^rfch honaoae 
ia tiimo? gro%jth, ws have adisiiiistired human gro^^th honaon© to ten 
patients vdth metastatic breast eaacerp three patients ^th laetas- 
tatie adrenal cancer, and t^o patients with Metastatic careinoma 
of the prostate. In no instance Has there clinical exacerbation 
of the disease. The measurable parssEetera of tusEor activity ia 
the last two grom>a were unchanged^ Froaa ti^ previous studies^ 
it is apparent that an Inersas© is jH'iisary ealoitsa following the 
adsiaistration of grou'th hormoae is an inadequate criterfei of tumor 
growtho la th® patients undergoing a rsaiission following hypo- 
pbysectoBsgrj the urinary eal-sitaa was tmehanged following gro%fth 
horaon©o Only in those patients ia whoa there was s'^idenee of 
tuffior progression did growth hormoR© produce soe© rise in the 
urinary ealciusEe, Thus, for these short periods of adBJinistrationj 
we could find no e^^idence to suggest tliat hisman grc^h horson® 
facilitates or stimulates ttaaor aeti'^'ity^ 

T^^^Lof eaacor of •^juM^iklS.Jl^aao 

EgD,cyplffl"seal stalk section « In col3.aboration with Dr^ Jo 'van Bur en 
of MINDBj we have perfos^aed hypopli^seal stalk section in II patients 
with metastatic breast eanaero This was dons with the esqsectation 
that the procedure would be slroler^ -thas decreasing th© morbidity. 
However g for reasons whieh are not apparent at this tS:asf j thB 
morbidity in this groixp was considerably higher than ia ow h^-po- 
physectoBsy group,, .Furthermore^ ths results of treateent ha'^e not 
been as good as those following hypopJ^ysectoEgr, It haSg therefor© ^ 
been deelded to tenainate this study at this tisi®, and to int@nsivsly 
follow those patients who had been sjfljjsctsd to stalk ssctioa^ 

The data wl.th respect to endocrine function following stalk section 
revealed pituitary-endocrine relationships ■ not heretofore described. 
Adrenal function alaost unif^^y decreased to hypopl^sectoi^ IstsISo 
Gonadotropin excretion was variable for m&nj months after siirgsry. 
Four patients have demonstrated normal thyroid function that eould be 
sc^pressed by the ad3ainistratioa of triiodotl^roninSe from thes® 
dataj several tentative conclusions may be drawns 



■777 



Serial ilo^ IGI-aOO(G) 
" 3 - E-ndocrinologjr B3?aaoh 

1„ Folloid-ng hypophyseal stalk seotionj there is trariabl© 
destruction of th® anterior pitaitas'y glands 

2o Contras-y to studies in th© dog^ ACTH piroduetioia appeared 

to be tb0 Kost sensitive iMicator of pittdtary glaad destruetiojSa 

3e la the abssnc© of hypothalisffiic-pituitesy vasettlar eonaeetions, 
the thyroid-pi tuitaspy esds ■vms Eorml in s©v®ral patients and 
gocadotropin production coatiauede 

Though antes'iojp hjjpopS^seetcsay has been coatiaued io favorable 
pati©ats for the perpos© of ha-ying ttes® patieets a'S'ailable fo^" 
study-, the th©3?ap@atio results do aot differ from those pff®¥iously 
r©port®d« 

2~D^oay^luco3Q,o 

Th® sts2di®s oa th© effects of 2-»d@o:^gl«GOs© oa breast cancer 
imv® h@®n brought "to a clos®« Incoaclnsiv© data suggest that 
8?^pr©asioa of ti® metaboliQ acti-^ity of brisast caiiee? as :sanif©stsd, 
by reduced ealciisEi oscrotion saa b© obser^ad in properly sel@et©d 
cases o Thsse observations as?® sutmitted for pt±>lication by Dro 
So Kaha aad Dr« Do M<, Bergsastalo 

SienifieaacQ to tha Pro^qa of th© lastitate; Ths direct psrtinenee 
of the foregoing studies to th© grav© problesa of advacsed or per- 
sistant breast caaesr is too obvious to require dJ^eussioBo 

ProQOg ed Corn's® of Project; Th© farther cours® of th@s<8 studies are 
to b@ ©ss®atially aloag th© lines outliiaed abov®o Emphasis is to 
b© placed oa balance studies and tte ©valuatioa of h^ophyseetasy 
in breast cenesro Further ©'valuation of &&u stsroids aad related 
coa^ouods is to b® saad®o 



Part B iaelud©d„ 



778 



Serial Io« HGI-'SOOCG) 
PBS=J?IH Endocrinology Braacb 

Iadi¥idual Project Pt^ort 
Calendar leap 1959 



Part Bo S^norSp A^^ards, and Piiblicatioas 
Hoaors and Awards relating to this projects 

Co-chai3?fflan, Morieaa College of Physiciaa 6th Postgrada&ta 
eourse oa Sndoeriaologyo 

Coiasaltaat to Coopeyativ® Groi:Q5 Study on Ltmg CanCs?o 

Presaated a paper at tl® sieetings of Th© Association of 
Ameriean PlsysiciaaSj Atlantic City, H, J„, J&y 1959* 

^g^oy Barta 

Invited to leeture at Acadec^ of Medieia© of Glevelaud and 
Cujahoga County Msdical Soeietyj Cleveland, OhiOj Novsjabej? 
19, 1959« 

Participated ia sssd-aianual laeetiag of Th© Medical and Chiyargisal 
Faculty of ths State of I&ryland, Ocaaa City, Mdo, SeBteabe? 18, 
1959. 

Moderator on panel of the 4-5th Annual Clinical Congress of tbs 
American Colleg© of Suffgeoas^ Atlantic City, Mo Jo, Septeafoer 28, 
1959o 

Participated in meeting of the Brooklyn Acadesay of Pediatrics 
held at Bsthesda, Mdo, May 20,, 1959» 

Gfesiissan of the Endocrinology Section of th® Asisricaa Association 
for Gaae©r Research at thsir 1959 meeting, Atlantic City, Ifo Jo, 

April 11, 1959« 

In'^it©d speaker of The Eastern Conference of Radiologists, 
¥ashij3gton, D, G», March 1959= 

Dr^„ B, Lips^tt 

LsctiLred before the Rouse Staff of Georgatows Univs^sity Msdical 
School J Hovard University Medical School j Washington Hospita3. Ganttsr-jj 
and Washington Sanitariimo 

Invited speaker at Piedmont Postgraduate M©dieal School, C1(^ssoq, So C^ 

MsEsber, Faculty of Postgraduate cossrs©, M®:°iGaa College of PhysieianSj, 
Bethesda, Md„, Pebo 1959o 

V 779 . 



PHS-UIH 

Individual Project Report 

Calendar Year 1959 



Serial !lo^ BCi~803C0) 
lo Endoeriaology Braach 
2o Endocffiaology Serris® 
3, Bethesda, Md, 



Pagt A. 



Project Titles The Nature of Horiaon©-=ProduQing Tumors of Pitidterjs 
Adrenal, Oayy, Testis, Pancreas, Paratt^oid and ChorioEi 
Abnomialities of Somatic Developmsnt and Growth* 



Principal Investigators: 
Dr, Mo B, Lipsett 



Dr, Roy Herts ^ Dr^ D, M, BergoBstal and 



Other Investigators s Dr» Ro H, Mojj Dr„ John Lavds, DTo Sa Genuthj 
Dro D, Ao Kellogg J, Dto T» L« Goodfriendj Dr^ H» Sehwsrtz, Mr» 
Ho L., Erwin, Mr, S* Jackson and MTo Po Rayford 

Cooperating Units: Dr, Ro Bates and DTo Ho Wilsoaj NiAMDi 
Dro Lo Garren, Pharmecologj Branehs KCI, 



Man Years (calendar year 1959): 
Totals 16 

Professionals 11 
Others 5 

Project Description: 



Patient Days (calendar year 195- 
Patients 
Days 



Qb j^Q cti'gess lo To learn more about the basic metabolic phencmsna in- 
volved in the process whereby neoplasms in horwoae-^'oducing orgaas 
produce highly excessive aasounts of honisone, restdting in such sya- 
dromes as Gushing "s disease^ acromegaly^ hypepparathyroidi^a, liyper- 

adreaalismj and sisiilar ©ndocriaopathi®s« 

2 a To maintain in tissa® culture and in animals hormone-prodaciag 

tissues and to ase®rta3.a tha factors involved in quantitative hormoae 
production in vitro =, 

3o To assess the edacity of new drugs to altar the course an.d 
honaoaal activity of honaone-prodscing tssmorso 

Methods Employed: Hor»oae prodtaeing tusors fro® patieafcs td.th one of 
the above-named syodrc^es are obtained at siargery and carried in 
tissue culture in irradiatedj, cortisosised rats and hamstsrsa Tha 
supernatant fluid fro® the cultures are tested for horaonal activity 
by appropriate bioassay saethods aM the quantity recovered coiapared 
with the content of a piece of tissue exactly equivalent to that 
originally placed in culture^ 

Patients presenting such syndromes are coi^letely characterised 

endoerinologieally and aetabolically before and aft®r tbsrapy in 
order to ascertain the relative effaets of variotss foras of tbsrapjo 



780 



Serial lo^ HGI«803CC) 
- 2 - EadocriaolQ^ Bs-asach 

Ma,1or Findiags; 

1» StMi^e on Ghoriocarciaaffia^ 

(a) Fifty patieats with Qhorlocas^einosaa or relatsd metastatic 
trophoblastic diseas© h&v® nou been studied frcssa both a thera- 
peutic and more basic aspect, Tw©nty-=-on© of 44. patients who 
vare admitted more than 3 months ago show no reaidaal evidence 
of dis©as© for periods rasaging from 5 to 4? aonthSo Eight 
patients are now resistant to metbotrssat© with parsistent 
diseas© as e-^ideaced by coatinuiag hosmoae titer. Fifteen 
patieats have died followiag iaitial respoas© with si!bs®qu©at 
de^elopsBsnt of drug resistaaeeo These data provide a very adeqimt® 
statistical basis for th® ©■valuation of ssethotrexatsi therapy ia 
advanced lastastatie-trophoblastic diseas©^ indieating that almost 
half such patients can b© eff©©ti-^©ly fr^^sd of evideaee of residual 



(b) Th® problsffi of the methotrexat© resistant patient resaias 
our most pressing cone©rno Repeated therapsutie attests with 
nitrogen mustard j eytoxaSj and actiaes^cia ha-^e yielded no signi-" 
fieaat responses a 

Sine© Mayj 1959 w© have treated 8 methotroxate rssistaat chorio- 
carincma patients with Tincaleukobiastia®, aa alkaloid dsiri'^ed 
from th© psriwiakl® plant (¥iaoa rosea) (Kobl©5, Gutts gjRd Bs©r, 
1957) o (Se® also project SOS for o«ir aaisial studies indicating 
th® sensitivity of heterologously traasplaat©d choriocarcinoiaa to 
this ag®at)o 

Thr©® of these pati©jits now hav© nonaal gonadotropin titsrsj stiggestlag 
th©ir eoi^lste rsidssionj th® longest being 5 Months in dm?ation« 
Two additional patients Bhow@d defiaite but rapidly reversible 
evidence of t^mor r©gr©ssioao. Ths nmsaining thres patisnts showed 
no apparent respons® to th© drugo Thus m© appear to have at least 
some r®co«rs© against methotrssat© rssistance and possibly a highly 
pot©pt agent for primary therapy, Lisiitations in available supply 
of th© drug has retarded tbs progress of osar stMieSa 

Reversible toxie sff^ets of ¥ineal8ukoblastiae hav© included; (a) 
bona ,»arrow depression, (b) alopeaia, (c) aeurotoadoityy (d) 
stomatitis, (®) malaise and depression, and (f) local phlebitis „ 

Th® Host significant aspect of these findiags is the fact that 
Viacaleukoblastine is ch^deally unrelatsd to any previously used 
oncolytic agent^ thus opening a larg® area for investigation ia 
the alkaloid fields Thes© stsadies are sohmitted to Cancer Research 
for publication,. 



781 



Serial lto„ KCI=-803(G) 
» 3 - Sndocriaolo^ Branoh 



MaigL^mM^I (con«t«) 



(c) The problem of the isiune relationship b©tvs©0a chorio- 
careiacroa aod the host subject has been frequently raissd siace 
this tasor is deri^sd from th© sygot© which eoatains both pateraal 
aad Estes-nal gensso W© have therefore ander taken a study of the 
blood groins of women vdth ehoriocarcinosaa in relation to their 
husbands" blood groups » fhea© studies iMieat© no widssae® of 
cross-iffisatmisatioa between husband aM yXf® even in those patisats 
vhose tissues are extensively ia'^aded by tismor,, It is inferred 
that this sssbryonic tissue lacks sigaificant aatigeaicity fos? the 
host at least as far as circulating sinti-body formation ie con- 
cernedo Further studies regarding fissed tissue antigens are in 
progress o These observations ar© being subsaitt®d for pdalieation 
in the Aassriean Journal of Obstetrics and Gynecology in collabors.- 
tion with Dr<. Paul Schmidt of our Blood Bank, 

(d) Honaal placenta has been shown to contain two types of steroid 
responsive ©nsysass - an estrogen activated del^drogenass and a 
steroid transhydrog©nas©c Thsse ©nzymes have b®«a deaonstrated 

in very small asounts in ehorioeareiaoKa tissue and fkm further 
eharaaterisation of th©s© onzjsms as ^i©ll as thsir higtoche^eel 
localisation in tumor as well as placental tissue will be of great 
interest (r^-o Donald A» Kellogg)^ 

(q) M®thotr®sat® has been found to eo^letely inhibit t!^ trass- 
fonaation of phenylalanine to tyrosine^ This interference is 
consistently observed in tb© methotrexate treated patient sM say 
play a vital role in the saechanissi of action of th® folic anta- 
gonist (Q?o Theodor© Goodfriend) :, 

During the past year^ w® have treated 14 patients with laatastatie 
adrenal cancer with o^p' DDD« Five patients ha.r® shown objecti'^© 
regression of cancer « There hav© been 7 deaths in the group o All 
patients with functioning adrenal csnser have shown sos® suppressios 
of steroid output with th© dr^„ 

y© are starting to correlate pla^^a levels of o^p» DDD '«dth dosage 
eM responsso Prelisdaary observations suggest that some of tb.® 
poor responses to o^p" DDD occurred is spit© of adequate absorptioa 
of the drugc 

In collaboration with Dr, William Tullaer, w© have i.nitiated a 
study regarding th® disappearance rate of Ojp" DDD froja pl.&^s&^ 
This vill b© exasuiaed by both standard sh^eal teebniqu®s and the 
use of tritiated o^p" DDD« 

In 2 of the patients whose ketosteroids had reached norissl levels^ 
p^er chroBsatography revealed that a large proportion of tS^s® was 
dehydroepiaadrosteroaso This suggests continuing lou-grad® activity 

.. 782 



Serial lo. MCI-»aD3(G) 
-> 4 - Siidocriaology Braaeb 

of the cancer, ©vea though a significant remission was obtained o 

Sttidiea in Steroid Metabolism^ 

1« In collaboration with Dro l^ilsoa of NIMD, w© have initiated 
a study of the urinary E©taboIit©s of ij^ortaat precursors of 
hydroectt'tiaone and d©bydro^iaadrost©ron©9 Fr<aa this sti^^ it 
should be possible to delineate ths areas of easysBatiG blocks in 
a variety of types of adrejaal caneer« Due to the ^ery large ex- 
cretion of all types of steroids in these patients j, conaiderabl® 
purification has to b@ psrforEed in order to aceuratsly msasur© 
any single metabolite, 

2o Xa last year's report, we outliaed tbe project to study the 
initial stages of stimulation of ths adrenal cortex ^ath AGTH, 
in collaboration with Dto Wilson« This has been ct^leted and 
we have eho^ai that ti>© biosyatheais of adrenal androgens is less 
de|>®nd6at on AGTH than is the synthesis of Cortisols, These re- 
sults ar,e being reported in detail In the Journal of Clinical 
Endocrinology and MetabolisBJa 

3o The study of the precursors of debydroepiandrosterone in 
collaboration id.th Dre Wilson has proved difficult becafsse of 

the probl«@is encountered in separating on® of tl^ metsboliteso 
We ar© still actively pursuing this^ however a 

4., A study has been initiated to det@rgdn@ the proportion of 
^19^2 ®©'^^o3.ites in thB uria© arising from dehydroepiandrosteroneo 
Tritiated dehydroepiandrosteron® has been given to ovariestoiaissd 
patients, as well as patients with adrsnal cancer. SMd •&© specific 
activities of ths urinary C19O2 metabolites seasuredo This will 
yield information about thg role of debydroepiandrosterone as the 
precursor of tiB androgenic. metabolites excreted in tbs urine c 

5o The previously described study of vozsen with hirsutissa has 
been eoE^jleted and has been accepted for publication in the 
Journal of Clinical Metabolisao We were unable to correlate 
any of the CX9O2 or Cj^gO^ jsetabolites or pregnanetriol mth eitbsr 
tl® extent or as'^erity of tis hirsutisiBa 

60 The availability of orally effective progestational cou^ounds 
free of estrogen activity psx^aitted an es^dnatioa of the effects 
of high doses of progestino In contrast to progesterone^ Provera 
caused a negative nitrogen balance only once in 7 attsssptSe It 
did not have any anti-aldosteroa® aetivityo Provera failed to 
suppress gonadotropin excretion in post-asnopausal patients and in 



78; 



Serial Mb^ NCI-a03(C) 
- 5 - Endocrinology Branch 



^^ogj-indiflgs (eoa'to) 



3 girls with preeocious psaberty. Partial chayaeterisetioa of 
urinary metabolites of Provera was uodertaken becatsse of tl^ 
findiag that th© ac&iiaistratiea of this Gcsapouad resulted ia 
large iner©as©s in th® oxcretioa of Portor-Silber cbroaogenso 
Although the struetua?© of the functional group was not deter- 
Hdnedj, kaovm fuactional groins wsr© ruled out as a soure© of 
this reaetioQo This study will not b® pursued^ although ther© 
iSp undoubtedly, a previously uoresogaised phecylhydraain®- 
reacting groi:^ in iBetabolitss of Provera, Thes® studies are 
"being r®port®d in th® tosrieas Josarnal of Obst©tries aad 
Gyaacologr, 

7o An asspheaon® derivative which has the capacity to block 
ll^bydrosylation has b®©n studied^, aad a test for pituitary 
ACTH reserve standardized o This will prove usefiO. cliaically 
in evaluating patisats with pitiaitary tumors* 

So In eollaboration with Dr» Leoisard Garran of th© Phanaacology 
Branchj, NGIy we hav© exaEtdned th® influence of noa-thyroidal 
hyper eat abolie states xipon the metabolism of Cortisol o The 

disappearaaee rats of ^-C-^^-Cortisol aud th® sseretion rate of 
eortisol have bean Bsasured in patients with leukeiaia before 
and after ©orreetion of the hypesssatabolie states No differeness 
have besa obsarvedo This study will b© e^qsanded to iaelud© the 
effect of fever iMused by pyrogens on th® sjetabolisan of oortisolo 

tetabolijm^ 

1» In th© patients with paahypc^itoitarisBij, low doses of Cortisol 
and trliodotbyroniae were shows to have significant eatabolic 

ef feet So Th© influane® of th® endoerin© status of tb® patient 
with respect to the eatabolie effects of eortisol had not pre- 
viously been examined 9 Frosa our studies ^ it is apparent that 
physiological ©ffeeta of these, honaoaes cannot be G«pared unless 
the endocrine s'tatus of the subject is wellMiefined^ 

These studies have been sabsaitted to Metabolism^ 

2c, Glucagon was shown to produce a rise in plasma anesterified 
fatty acids 4S hours following administratioffio Its eatabolie, 
effect has been studied and demonstrated to be independent of 
hepatic glycogenolysiSo These studies have been subsitted to 
the Journal of Laboratory and Clinical Medicine o 

3<s A congener of t®stosteron®j, testololactone^ was shown to be 
metabolically inert in 2. balance studies^ It didj, however , 
aarlradly reduce the hyp@rcal€i«5?ia aceoe^anyiag osteolytic bone 
disease due to carcinoma of the breasto There hav© been no ob- 
jective regressions of diseasso 



78.'i 



" 6 - Ep^osrinology Branch 

The effects of growth honaone have b®en exasdnsd in 3 patients 
with astivQ aoromegalyo No nitrogen retention was observed^ 
although the urinery saleium excretion increased = These same 
patients deiaonstrated an ability to retain nitrogen whan tes- 
tosterone was giveso This phenoiEenon has not previously been 
noted J and iaplies either different sites of deposits of protein 
or different aaeohanisms for nitrogen anabolisms In eollabcration 
with Dro Julius White g of the Laboratory of Physiology ^ we have 
begun an investigation into certain parameters of protain asetaboli^ig 
using N3-5-iab@led glycine« W© intend to study patients with acti¥© 
acromegaly before and after treatments N^5-giy©inQ will also b® 
given to patients on metabolic balane© di^ring periods of adminis- 
tration of human growth hormone and testostaronog in aa effort 
to delineate tl^ possible different meehardsass of nitrogen retentiosc, 

SienifiQanee to tSa Program of the Institute; Further understanding 
of the conditions psrsaitting the exeessive hormonal production 
of tumors of ©ndoeriae origin is basic to our understanding of 
the Esetabolism of neoplastic tissue generally^ In addition^ further 
analysis of abnonaalities in somatic growth and development should 
provide data pertinent to the problems of tissue growth geserallyc 

Proposed Course of Project; Further elaboration of these studies with 
major eaiphasis on biochasaical and pharma©ologiea3, control of horsnone-- 
producing tumors and anosalous tissue growth is projectedo 



Fart B included-. 



785 



Attaelment I 

Serial Ho, SGI«80,3(C) 
PiB"KIH Sndocriaoiogy Branch 

Individual Projaet Report 
Calendar Year 1959 

Part Bo Honors J Awards ^ and Ptiblicatioas 

Publications other than abstracts from this pyojeets 

lo Lipsettj Mo B» and Bergenstalj Do M»,; The Met-abolic end 
Gorticotrophin-Suppressing Astivity of Certain G2i-D©c.3cy- 
steroidSo Acta Endocrinol, 31: 33-398. 1959c. 

2a Lipsett, Mo Be and Bergemtals Do Mo 5 Mstabolic Effects of 
Amethopterln in Man, Caneer Resoareh 19; 89"-925 1959= 

3o Engel, Ho Ro, Borgenstal, Do Mo^ Hixonj W. So and Patten^ 
Jo AoJ Effeet of Htanaa Growth Rormone on UFA and Plasiaa 
Amino Acid Nitrogen in Man, ProCo SoCo Espsro Biolo & Medo 
100s 699-701, 1959o 

A„ LubSj Ho A«g VilaTj Oo and Bergenstal^ D„ Mo J Fsffldlial Msile 

Pseudohenaaphrodism With Labial Testes and Partial Feffiiaisation; 
Endocrine Studies and G®a©tie AspactSo Jo Cliajo Endocrinolo & 
Metabo 19*. 1110-1120, 1959« 

5o Bergenstalj, Do Mop Lipsett, Mo Bo^ Moj^ Ro Ho and HertiSg Rot 

Regression of Adrenal Cancer and Suppression of Adrsnel Faustiofl 
in Man by o^p" DDDo, TranSo AssoCo Asarc. PbySo 72; 341"- sl959o 

6o Lips©ttj, Mo Bo and Bsrgenstalg Do Mo? The Catabolic Effects of 
Low Doses of Cortisol and Triiodotbj^ronin© in tba Fanh^opituitary 
Sub j sot o Metabolism (in pr©ss)„ 

7o Lipsatty Mo B09 Engel^ Ho R^ and Bergenstaly Do MeS Effect of 
Glucagon on Plasma Unesterified Fatty Asids and in Nitrogen 
Metaboliasao Jo Lab» & Clino Medo (in press), 

89 Lipsettj, Mo Bo and Riterj, Bc= D^s Urinary Ketosteroids and 
Pregnanetriol in Hirsutism^ Jo Gliao Endoerinolo & Ifetab„ (in 
press) o 

90 HertZj RoS Gonadotropin and Adranocortieotropin frosa Rat 
Pituitary Homografts as Manifested by Host Response to Chorionic 
Gonadotropin and Amphenonea Endocrinology (in press )«, 



786 



- 2 - Endocrinology Braneh 

Part Ba (cofif'to) 
Honors and Avrerds Rslating to this Projsct; 

Dr^ Roy Kartg 

Rs~appoint«d to serve on th© Postgraduat® GoBEBitte© of ths 
Endocrine SociQty during 1959~60o 

^^sabsTg Awards CoBBiitte© of tb© Endocrin© Socletyc 

Participated as gusst loetijres', Sqtaibb Centennial Leetwl^s 
PrograEip February 1959t> 

lij^ited to lecture at Mayo Clinic^ Rochester, Minnesota, April 
27, 1959o 

Guest speaker, Minnesota State Gyneeological and Obstetrieal 
Society J, Duluth^ Kiinn.j, April 25^ 1959o 

Lectured at the University of Southern California Medical Sehooly 
April 23s 1959<. 

Partiaipated in meeting of the New York Cancer Society » April 
14, 1959o 

Chairman^, sseetiag of the Kick Carter Clubs BethesdSp Mdoj lovmber 
6, 1959o 

Participated in seaainET of Armed Forces Institute of Pathology 3 
Deceasber 1, 1959o 

Drp, Dp Mo Be 



Invited to participate in confereae© on Th© Biological Aeti^ities 
of Steroids in Relation to Cancer j, GCHSCg VergenneSg ¥t=3 Septo 1959= 

Dr. M. B^ Lipsett 

Participated in meeting of the Hick Garter Club, Betbesdaj Moj, 
November 6^ 1959o 

Participated in Brook Lodge Conference on progestational agents „ 

Invited speaker at Postgraduate Course^ Freedisen's Hospital, 
WashingtoSp Da Co 



787 



S©risl No, KCI-aoSCO 
ic, Sndoeriaology Branch 
2» ladoerinology Service 
P!S~MIH 3o 
ladividuel Project Rsport 
Calendar Year 1959 



Part Ao 



Project Title: Eadocrlne Factors Govarniog Hcrmone-Induced Tissue 
Growth and Tumor Formation in AcdjEals; Bioassay of Blood and 
TJrlae trcm Cancor Patients 

Principal Investigators DVa Roy Hertz 

Other Investigators J DTo Wo W, Tullriar^ Dro Js Lewis^ Dr, Ho 
Sehwartz, Dr, S« Genuth and Mto H,, Emis. 

Man Years (ealendar year 1959)? Patient Days (calendar year 1959) s 
Totals 9 Patients 

Professional: 6 Days 

Others 3 

Project Descriptionj 

Objectives? 1,, To gain basic inforssation csoneerning endoarinologisal 
and nutritional f aetors governiag horaonally conditioned growth 
and imrolution in organs such as th© uterus j, breast and prostate 
vhich are th© frequent seat of horEone-sensiti^e neoplasms o 

2o To develop and ii^rov© bioassay methods for the quantitation 
of hos^ones in the blood and urine of patients td.th eariser who are 
either receiving various fones of horaoaal therapy or who present 
unusual honsoaal patterns a 

3o To detenaine the biologieal properties of steroid and rel8.ted 
compounds ia terms of their potential usefulness in eancer ther apy^ 

Method 3 _Ewml oye^t Standard lasthods of biologieal study ars eisploysdo 
These include; 

lo The maintenance of anixaals of various species on specified 
horaocal or nutritional regimens o 

2a Daily observations of physieal and behavioral ehanges in such 
animals o 

3 3 CoB^jlete autopsy of test animals with detailed weighing and 
sh«aioal an^ysis and hlstoehesaieal study of specific organs o 

Ao Surgical alteration of the endocrine status of animals by 
gonadeetojay, adr®nalactoi?yg hypophyseetojay^ thyroidectoBiy, para- 
biosis, hysterectoHiy and other operatiire proeedureso 

788 



Serial mo mi^OBiC) 
~ 2 •" Endocrinolo^ Braaeh 

^Ia,■^or Findiagat lo (a) Four stfains of hufflRa choriocarcinoma 'm.v@ 
now besn established as hetsffologous traasplaiats ia tte hamster 
chesk-poucho Two of these straiiES no long©? require ps-ioi? eor- 
tisone adiaiaistration to fee haasstero Saeh of these tm&ora grows 
rapidly for 2 to 3 weeks and then undergoes necrosis^ liquefaction, 
and total resorption during the ensuing week or twoo Continued 
cortisone injection does not protect the tmaor from the host 
reactiono Histologically the tumor tissue eontains both syneytio- 
trophoblast and eytotrophoblaato The tumor cells are highly pleo- 
morphic « The tumor tissue is riehly vasetdariaed and one may 
estimate that at least half of the tissue mass is laade up of newly 
proliferated endothelial channels filled vdth blood cells which 
er® clearly of host origin,, There is abundant mitotic activity 
but in no instance have metastases or even invasion through the 
cheek-poueh been obaervedo 

(b) The endocrine properties of the transplantable ehoriocsst'eiaoffia 
are very strikiago The tismor is productive of large amounts of 
chorionie gonadotrophinso This is readily detestable in the tujnor 
tissue^ in the plaasaa and in the urine of the hamstero Bswevers 
it is undetectable in the animal "s peripheral tissues such as the 
liver J, kidney J aplsen or gonads o It would appear that the chorionic 
hormone has sufficient molecular sise to be excluded from entry 
into peripheral tissue cells but can still be filtered throt^h the 
glomeruli of the kidney^ This suggests some selective handling by 
the kidneyc Moreover^ the absenee of d^aonstrable horjuon© from the 
gonads p even though the latter shov marked trophic response to th© 
hormone, presents a paredosa Moreover j, neither slices nor hQiEO-° 
genates of the tissues mentioned iimotivate •Uie hormone u^jon incu- 
bation at physiological pH for 4 hours at 37o5°Go This syatea 
Bsay be e^qjected to provide mich additional material for study of 
the biosynthesis^ distribution^ fate and mechanism of action of 
this vital hormone of normal and abnorssal pregnancyo 

(g) ^prcpriate studies have indicated that^ unlike norBsal 
placentsj, choriocarcinoma is not productive of detectable amounts 
of estrogen^ progesterone^ or sortiooid<, 

(d) A hamster in wliich a ehoriocarcinoiaa tumor has becoia© re- 
sorbed completely resists reinoculation with the ssiae tumor straino 
The serum of such a resistant hamster will inhibit the tuiaor frcm 
taking in a previously uninoculated haMster^ thus desaoastrating 
the passive transfer of imminity to a ii^terologously transplanted 
tumor o That this is not the effect of non-specifis anti-hufaan 
serum is shoim by the failur© of potent anti -human-globulin rabbit 
seru® to trensBsit such resistance to tumor inoculationo Hox-jever^ 
much needs to be done to clarify th® true nature of this interesting 
effecto 



789 



;:cs.!. .'-^^- i:--'; 



- 3 - Endocrinolo^ Branch 

l^ogji^-diags (con«t.) 

In collaboration with DTo Bozicevich of mMD we have failed to 
demonstrate any signifieant htsmaa antigenicity of ehoriocaroinosa 
tissue when adaainistered in Freund«s adjuvant to th© rabbit or 
by tuffior iiEplantation in the haastero These tests for anti^human 
siabstanee ware carried out by eosr^lasjent fisationj Oa©lit©g'lony 
plat® and by Bentonite flcaiiLatioao It would sesm that this 
jjriHjitiv© esabryonal tissue laeks tlie capacity to indue© anti- 
human antibodies in the rabbit and hamster and yet ean ijEpsfft 
tumor resistance in the hamster q The data are suggestive of a 
tumor-speeifit resistance factor not related to orthodox anti- 
bodies <> Further studies are in progress to identify this factor o 

(e) The availability of one of thss® tumor strains on a weekly 
turnover basis has peradtted ths devQlopment of a ehemotberapeutie 
screening procedure against this straino A uide variety of hor- 
monal agents of estrogenic, androgsisiCg progestational and corticoid 
type have been found not to affset the normal growth rate of the 
tifflsoro This strain was_,deriv©d from a methotrsxate-resistant 
patient but the transplanted twaor exhibits a moderate sensitivity 
to Bethotrexat® in sob-lethal dosss^ 

Ths periirijjkle plant, Vinea rosea^ has been found to contain 
several ingredients of alkaloid type which are highly inhibitory 
to the tuaorg the most potent of these being now identified as 
Vinealeukoblastine and Leurosiaeo Related alkaloids derived 
froH this plant such as LochnerinQs perivine, reserpine^ catharan- 
thins 3 and viadoliae are totally inactive at to:sie dos® levels o 
These observations have been extended to the therapy of human 
chorioearcinoaa (cfo 303) o 

In addition an extended series of unrelated alkaloids h&v® b@@n 
tested^ all with negative effectSo * 

(f) A tissue culture laboratory has been developed and nm^roos 
methodological probl€Bas worked outo Choriocarcinoisa from th® 
hamster has 'b®®^ successfully planted in roller tubes and T~flaskSc 
Hormonally active siqjernatant fluid has been repeatedly recovered o 
Quantitative studies have been "ondertaken to deterssine ^^ates of 
hoiraone-produstioa in vitroc These in vitro systems vd.ll provide 
esoelleat material for analyzii^ th® nutrition^ and emriroiaasntal 
requirements .to hormone formation and th© relation of horsione 
formation to cellular proliferation and differentiatiofio 

2o Chorionic gonadotropin has b®©n identified only in the hora©j; 
monkey and chimpanzeeo la the Maoaeus rhesus iwsnkey vm have found 
detectable levels in the serum and urine during pregnancy only 
between day 20 and day 35 follo'^ing timed matingo The hormone then 
abrE5>tly becomes uadetectabla even in extensively concentrated 
plasaaa or orinso It is also absent fross peripheral tissues such 

790 



Endocrinology Byaaeh 



(con'te*) 



es l±v®Sg kidaey ^nd spl§e!i throughout pregnancy. Although it 
is readily deteetabl® in th© fetal chorion as long as the pas-i- 
pheraL plasma resaaias positive, it is no longer detectable in 
this tissue which ia usually regarded as its site of formatioa 
after day 35 of pre^ancy^ It is possible that it is present 
in a form which beeoiaee unavailable to the test rat or mouse o 
It is also possible thair the -Isvela may Xall below that d©~ 
teistable hj our current assay methods o Farther atteaspts at 
concentration of large pools of plasma and chorionic tissue 
late in prsgnan*^ will be undertakenj since th© period of de- 
tectability in the monkey corresponds to the peak of quantitative 
output in human pregnancye In tJ® human » however , easily detectable 
levels persist throughout pregnancy^ 

The pfesencsa of hormone from day 20 to day 35 of pregnancy in 
monkey plasaa and chorion has provided a tool for tl^ evaluation 
of survival capacity of ©ctopieally transplanted chorionic tissue 
in this species o Since these saatings are not brother to sister 
it was desmsd desirable to attarapt to depress tl^ maternal "re-- 
slstancgf' to ectopically placed chorionic tissue by prior irradia- 
tion of and cortisone adudnistration to the mother which was to 
receive her own chorionic villig Various routes of dispersion 
of villi Imve h&sn tried in a total of 12 such pre~treated pregnant- 
lapnJssySp but all have been directed toward distributing the villi 
throughout the pulffionary bed via the right hearto In no instance 
has there been hormonal or histological evidence of a takSo In 
factg. no villi have ever been recovered at subsequent autopsy 
after periods ranging from 30 to 150 dayso Thus our attests 
to essperiiaentally reproduce hormone-producing ectopic villi with 
potential malignancy have met with eoE^lete failure -thits far.- 

3o Further studies on the biologisal properties of husfian chorioM,c 
gonaiiotropin have been directed toward tm development of mors 
sensitive and less laborious aethods for bioassayo One of the 
initial effects of the hoarmoa© is to produce an ovas'ian bjperesiia 
which is grossly visible but not quantitatively assessable by 
the naked eyeo Radioiodinated husan serus alfcuiasa has been found 
to increase in the hyper eadc ovary at a rate which is proper tioaal 
to the aaaoant of chorionic hormone adainisteredo This provides 
increased sensitivity over the mouse assay and with proper auto~ 
matization niay provide a very practicable method for the ntsierous 
assays which our clinical and biological work requireSo The 
initial data are encouraging and fartbar efforts tot^ard the da- 
velopment of this assay are being pursued^ 

4q Previous studies have indicated that th© ovary of the rat and 
rabbit undergo a substantial degree of di^ereatiation and grcvith 
prepubertallyo This process is pituitary-dependent since it will 
progress noraially in ovarian grafts in l^opbyseetdsised :ffiale or 

791 



Serial Mo o iK;i-»a03{C; 
Sadocriaology Breaeh 



[S (coa^t,) 



fomala hosts « We imr/Q att^ig)tM to detss-aine the rcl® of tho 
hypothalaaus in this process o Extensiw© h;^othaiaiaic dastruetion 
ia the rat is without effect upoa pre-pubertal gonadal de^elopiasat. 
Further studiss of possible neural factors Inwolved in this process 
are ia progress » 

5o Furth®? studies of th© functionsGi. potential of pituitary 
grafts in hypopiQrseotomiaed rats show that these ectopic glands 
produe© AGTHp growth hormoaQ-, and gonadotropin i^thout h3?pothalaEu. ; 
regulatiooo 

6» Th© Dunning rat leukessaia which baa prsTiously been carried 
in Fischer rats has been suoeessfully adapted to the Spraga®- 
Dawley rat and is now in th© 70th transfer gsaoration in this 
straioo It has developed increased %irulence" with thia 
adaptation sine© it kills in 7 to 9 days in th© Spragi^-^Dawley 
strain and in 14- to 17 days in th© Fisohar stra5jio 

This tumor is sensitive to laethotresat© and to bydrocortiaons: 
Cosabiaatioa studies have clearly shown that half -ssasdJEally 
effeetiva dosss of each of these drrjgs ar® not at ell sdditiv®,, 
- Ifenee it is clear that each of thsss ag®ats produces its inhJ.bito?j 
effeet by a distinct ffieehanisEo 

It has been shovn Bsicrobiologically that biotin deficiency iBpis^ars 
the capacity of numerous ffiicroorganisma to synthesis© folic aeid» 
AttsE^ts have been mad© to detsrssine nhether biotin deficiency in 
th© leukiaaic rat will reduce the r®q«ir®aeat for m®thotr®aate for 
ma:KiBial inhibition of tussor growth. No such relationship coui.d 
be d^ionstratsd nor did th© partial biotin deficiency produesd 
by egg-white diets li^air th® growth of the tumor signifieantljc 

7o We have found that if an immature f^aals rabbit is first, 
treated with one-tenth of th® effective dose of follicle- 
stijaulatiag hos-jsoae for 10 daySg then subsequent adsdnistratioii 
of even excessive doses of the sajse horaone proves to-tally 
ineffective o This ws have eallad a desessitisation phsnoffisaoiio 
Further studies ha^e indicated that mrtisone bloska this da- 
sensitization phenosusnon whereas aatihistarainiss do act do so,; 
This suggests a possible iaasuna laeehanissa and furthar ©Jtpsrisents 
will help to elucidate this problemo 

Proposed Coarse of Protest; It is expected to coatinus and ©steM 
the foregoing studies along the lines already indieat^o 



Part B incladedo 



79 



^ 



Serial lo, MGI»808(C)- 
Sadocriaology Branch 
PHS-HIH 
Individual Project Report 
Calendar i'ear 1959 

Part ,B^ HojaorSj Awards ^ and Publications 

Publications othe? than abstraets from this projects 

io Goodsoaanj, A, DoS Effects of Pantotheaata. Deficiency and of 
OffiQga-Metii^rlpaatotheaate on Rat Gortieoaterone SQeretion„ 
Federation Proc, 18; HOo 1, Mareh 1959, 

2a, Herts, R,i Ckoriocarcinoffia of Woiaan Maintainsd in Serial 
Passage in Hamster aad Rato Proco Soqc E35>@ro Biol,, & Med^ 
102: 77-^15 1959o 

3. Hertz, R»? Growth in the ^opl^-sectoiBisQd Rat Sastdaed by 
Pituitary Grafts » Endocrinology (in press) „ 



Honors and Awards relating to this projects 

Drr, Roy ,%rta 

Elected snsgabor of the Research Advisory Council of the Aasrican 
Cancer Society for a term of 5 ye^rSo 

Mssnberp Endocrinology Panel of tbs Cancer Ghsssotherapy National 
Service Center » 

Participated in conf©ren«30 on The Biological Activities of Steroids 
in Relation to Cancer ^ CCKSC, Yergennes^ ¥t„, Septoiebsr 1959o 

Guest speaker. Southwest Section of AEsriean Association for Cancer 
Researehj Inc» (Noble Foundation) j Ardmos^e^ Okiahosiaj October 16, 1959= 

Chairman, Panel on Endocrine Aspects of Cancer Palliation^ Anmisl 
Postgraduate Assembly of the Endocrine Society, San Francisco, Ca- 
lifornia, November 1959t> 



793 



Serial I'lo* IGI-SIO 
lo Sadoeyiiiology Braaeh 
PHS-KIH 2o Endocrinology Ssrvie© 

Iftdi'^idual Project Report 3o Bethesda^ ¥Ao 

Galeadar Year 1959 * 



Part A., 



Project Title: Phasnaacological and Physiological Studies on 
Alteration of Endocrine Funetion in Animals « 

Principal Investigato?; DTo Williaia Wo Tullner v 

Other Invastigators: Dr*. Roy Herts 

Man Years (calendar year 1959) s 
Total: 3 1/4 
ProfessiOBal: 1 
Other: 2 1/4 

Project Deaeriptioa? 

Ob.iectiveaii lo To evaluate selected synthetic eospounds for tbe 
ability to altar endocrine sscretions ia order to ssplore the 
potential usefulness of such ccaspounds in the control of endo- 
crine tumor So To develop and is^rov© biological snd blochemieal 
methoda of steroid det8rBsination„ 

2o To investigate the jueehanisia of aetion of cojspounds which 
inhibit steroidogenesis employing quantitative aM qaalitatiire 
methods for determination of alterations in the i-atenaediat© and 
final products of steroid biosynthesis o 

3o To investigate the interrelationship beti^en th® anterior 
pituitary, adrenal eortex and th© reproduotv/e system pai'ticularly 
with respect to the secretion of androgens and estrogens ay tiss 
adrenal cortexo 

4.0 To indue© adrenal cortical tumors in laboratory amiaals for- 
the purpose of studying steroid produetion of sueh tumors aad 
means of inhibiting tumor growth o 

Methods EiapltQ^ed; Normal healthy aniiaals were used ia these e2cpari" 
mentso Stock diets ■were used except for special post "Operative 
reasons or ©Kperisients related to nutritional effects on horsone 
soeretiono 

Both standard surgical teefcsiques ar^i those which have he&n de- 
veloped for these studies ha^r® been en^loyedo These inslude 
adrenal vein sanauiatioEg h^ophysect<sayp adrenaleetoiays o'^arlscGox 
emd endocrine organ transplants o 

Standard chesieal Eiethods ^^ere used for the qusJititati'ra assay ox' 
steroid hormones o 



■ ■• Z " Endoericology Braaeh 

^4or_^^^s^^j 1^ The foXlowiag fiMiags ar© based on pbasfjaasolo- 
gical studies with o,p« DDD (2j2-»bis(2-»ciiloroph©Byl-4.-ebloropheErl)- 
Ijl-diGbloffoethaa®) o 

A a In oar previous e3?)s?iffi©ntal studies j, th® problem of insoKplsts 
absorption was fx-equeatly associated ^th ths oj?el adaainlstratioa 
of fat^soluble coKpomids such as o,p" DDDo This has led to ds^"©- 
lopmsnt of a soitabl® vahicle for the intravenous adraiaistpatios 
of sueh ocm^ousads (Poblisation HOo 3)o 

Using this iatravsnous method for th© invsstigatioa of the effecta 
of Ojp* DDD In ssverel series of dogs, it '^as found that after 
48 hoKisB of treatmeatj th© rate of seeretion of adrenal coi-tical 
17~hydrox7 steroids decreassd ssae-ksdlyo Prelisdnary obssrvations 
ii^icate that theys is also a profound reduction in secretion rates 
of cortioosteroa® aad aldoeteroae so that with respect to th© major 
sad products of the dog adrenocos'tieal biosyathetic pathway^ the 
effects of thia drug reseabls those follow-s::^ the disappearaace? 
of adrsnocorticotropia after bypophj^sectossjo B^we^er, in no 
isistaaoe could tbis iaMhitory sff®ot on adreaocortical steroid 
output b© reversed by adrsEOoorticotropic hormoa© indiceting that 
the drag aets directly on the adreaai cortsxo Intra^siioua strtdi©s 
with OjP" BDD are being conducted em-rently to datsrsaines effects 
of ti^ - dose alterations a.s related to inhibition of adrenoco.'i'tieal 
fuoetiofflj methods for iacraasing coResatratioa of the coiapound per 
unit ¥oltBS5s of vehicle I and its distribution in th© body tissues of 
the doge 

The dsTslopment of aa intrairenous ssatalsioa contalniag Ojp"BrtD 
makes possible aeat© ©^>®riJE®nts i^yhich aiay elucidate ths Ejechaaisin 
by which this drug @s®rta its action^ Several adrenocortieal ' 
metabolites are to b© tested for their ability to bloek th© iahi- 
bitory actloa or revsrs© tba pharsaa-cological ©ffoots, W© propose 
to investigate tha ©ffeets of sio^aloaic acid^ squalenej cholesterol 
and pantothenic acid as relat-ed to th© adreaocortical InMbitorj 
action of o^p" DDDo 

Be Sine© a siethod has bsen devissd for the ietraveaous adaiiiis^- 
tration of fat-soluble co:^oiMidgg it has bsen possible to begia 
ths evalimtion of other aaalogs of DDD partiotilarly vith rsspeot 
to th® aetioa of thsa© analogs on the adreaal cortsxo In aeut© 
stadies on the dog^ it was found that difluorcdiaitrodiphenyl- 
dichloroethane inhibits 17~l^dro3yeortieosteroid secretion,, Potoiac;? 
eos^arisons with o^p" DDD ar@ und^rvay and otbsr -halogenated anslcgs 
are to be tested in an effort to find compounds of greator pots&sj.„ 

Co Previous investigations of th© acute phanEacclogie action ef 
orally-adadnistered o,p« DDD in the dog h^¥® been a-stended (Publica- 
tion Ho„ 1) o Within 24. hours after a singl© oraJ- dqs© of ths drug. 
there was a coE^jlete si^pression of 17~l^drosycortieost@roid out- 
put although there were no apparent histological changes in the 



j'b.) 



Serial HOo MGI-8I0 
- 3 - Endocrinology Branch 

Ma.|or Fiadings (conHe) 

adrenal cortical tissues ^ Exogenous adrenocortico trophic 
hoicmjne did not reverse this acute inhibition of adrenocortical 
sesretiono 

D«, A series of dogs received ehronic oral treatment with 
OjP" DDD for periods up to 90 dayso Aftsr 90 daySj, the ©©11 
architecture of the adrenal cortex was cossplstely destroyedc 
In animals tlat died in apparent adrenal insitffieisacsy aa long 
as 3 months after cessation of treatsentj, there was ao evidence 
of adrenocortieal regeneration such as vf© had previously observed 
at intervals following 3 weeks treaisaent at a soi^arable daily dose 
level o 

Eo Chronic, oral administration of o^p" DDD to the rhesus aonk©y 
(Macacos rhesus) had no obaarvable effect on adrenocortieal funotioHc 
Adrenal venous blood levels of hydrocortisone of treated anisuals 
were within the normal rang®« There t^'es no evidence of histological 
alteration of the adrenal ccrteso 

Although Nelson and Woodard had reported that treatissnt ^th 
technical DDD produced no histological change la the rat adrecal 
cortexg early functionel effects, without eoncoTd-tant histologicEU. 

change as observed in the dog^, indicated that the problem warrsaited 
reinvestigation especially with th© potent isos»r o^p' DDDo Fo11g--v-- 
ing chronic oral treatment with o^p" DDD^ the adreiial secretion of 
eorticosterone of tb© treated anisals '^jas not significantly differeai 
than in untreated animals o 

The above findings in the monkey end rat fortter ©n^shasis© the 
Harked species specificity of this cosapomido 

2o In a continued investigation of the endocrine effects of 
diaadnodiphenyl ccHspounds, studies were liiaited to diaminodiphenjl'- 
iBethane (Methylenedianilina) (Publication No« 2)o In the castrate 
feanale rat, chronic treatment vdth this coa^jound produced a'irenal 
hypertrophy, uterine enlargeBsat and u'as goitrogenic « Histological!.) 
tlM adrenal cortex showed marked lipid acc^ssulatiofio In the dog., 
acute intravenoi^ administration of diasd.nodiph©nylss©thaji© produoed 
a 7 to 8«»fold decrease in 17«bydro3^corticoid output 5 laijititss after 
adndnistrationo This decrease was accoi^anied by a concoKiitarit 
transient increase in blood pressurSe In the issaturs fexEal® 
rabbit,, this eoa^ound elicited a progestational proliferatica of 
the endometrium which was shown to be adrenal-dependent <, In this 
respect J the oon^jound is three times as active as eiaphenon©, 
AdrenalectonB(y as well as either cortisone or corticosterone-treat- 
ment blocked the progestational effect of dieEsinodiphenylseths 



798 



i4/fi8J2 



ns. 



- 4 •" EBdoeriaoIogy Braneh 

Majog^FiMin^ (con'to) 

However J there was a lack of progsstatioaal respoas© to tbs 
ctKBpouad vrhsn the rabbits were not prised with estrogeao yith 
the exception of the pressor qualities and a lack of anesthetic 
action, the effects noted above are similar to those of aas^honoae 
(3, 3~bis _(p-»aiainophei?jrl)-2-butanone)o These findings dssaoHstrate 
that the pethyl ketone structur® of ajsphenone is not essential for 
the endocrine responses observed,, Beea^SQ of tosdcity, no further 
studies affe planned with this coi^ouad although tte possibility of 
obtaining less toxic structurally-related coM^ounds is not beicg 
oivarlooked Inasiiauch as the sjiMttetricEl diphenyl st^actur© is com&on 
to several potent inhibitors of ad^enocortiGa^, ensyma syst^as iia 
laboratory animals and laano 

3o Studies on the interrelationships of the anterior pituitary ^ 
adrenal cortex and reproductive system have been continued* 

Ao The problem of sez hormone secretion by normal adrenocortical 
tissue and th© relationship of adrenocortical secretion to the 
state of -the reproductive system requires clarifications Pre- 
liminary experiments g in which rat adrenal glands vera transplanted 
under the kidney capsule of castrate imles contiguous to hoaiologoiis 
transplants of seaainal vesicles frosa other castrate EaleSg indicate 
a degree of androgenic stimulation of the serainal vesicle secretory 
epithelium^ It is proposed to determine the role of the anterior 
pituitary trophic honaones in this syst<^ffip beginaing lAth injeetioa 
of whole rat pituitary extracts followed by ccaabinations of adi'eno- 
corticotropin and the gonadotropins « 

Bo Although the uterine ©ndometriuHi requires pretreats^nt with 
estrogen to obtain the optisauaa response to progesterone j, it has 
been observed (KLsaw et alo) that high doses of progesterone alone 
can induce progestational proliferation of the rabbit endoaetriuiE., 
in order to detenaine whether adrenal estrogen secretion might b© 
sufficient to induce the response to progesterone alone,; adrenal- 
ectcKiized rabbits were treated with progesterone* Progestational 
proliferation of the endometrium occurred in th® absence of the 
adrenals indicating that estrogenic hormone secretion by th® adrenal 
is not a factor in the direct action of p?ogestsron©o 

ChCTiical characterization of the adrenal cqiEpounds responsible 
for the progestational actions of assphenon© and diasdnodiphenjl- 
methane is also a part of the proposed course of -fehes© studies o 

^o Development of a direct method for the assay of adreaocortico« 
tropic hormone (ACTH) was undertaken. Using th© acutely hypopb^r- 
sectofflized rat, it was found that tbs adrenal secretion of cortico- 
sterone was a sensitive indicator of injected ACTH at low dose levels 
A similar method was described at the laeetings of th© Federation of 
American Societies for Essperimental Biology (Lipscomb and Nelson) = 
These studies have not been pursued furtl^ro 



Serial !lo« UGI-SIO 
Endocrinology Brancsh 



Major , Findings (conHo) 



5o Because the current expsrisieists on ths iadastion of adrenal 
tuaors in the dog as?© of a long-term nature thsre is no evidenee 
at present aa to the suceesa of espei'lEeats initiated within the 
past yeaTo Postnatal ovariectosjy of s©v©ral litters of poppies 
has provided a series of animals Mhlch. will be exasdned for 
adrenal pathology in the coaiing y©as?6 Likevds©,, a aeries of dogs 
received intensive chronic dosea of Ojip" BDD which ij®?© adeauate 
for destruction of adrenocortical tissuse Treatnient was coscludsd 
and after several months, blbpsies TdJ.1 b© taken to det-erain© 
whether the rapid regeneration of adrenocortical tissue folloid.iig 
drug treatment has resulted in cellular alterations » 

Significance to Blo-ffled3,€^ Hesoareh and the Program of th e linatittitQa 
Basic understanding of the functioned interdependence of the adrenal 

cortex and the reproductive systea and their respective steroid bio- 
synthetic patterns add not oaly to our knowledge of norisal foactioa 
but also provide information need^ for the pharmacologic control 
of abnormal growth and secretiono Cosi^ounds such as those reported 
her®, which are highly selective in altering the function of endo- 
crine tissues, offer a possible means for control of endocrine 
cancero 

Proposed Course of Projeot a It is e^^ectod that the above studies 
will be elaborated and continued as already indicated under 
Mantqy Findin^s^ 



Part B included 



793 



o 



Attac!ffii©?it I 

Seriai Ho, HCI-SlO 
PKB-HIH ladocrinologj Braaeh 

Individual Project Report 
Calendar Year 1959 

Part, B» Honors, Awards ^ and Publications 

Publieatioas other than aba tracts froa this projects 

1„ Vilar, Co and Milliaa W. TuUner, Effects of o^p" DQD on 
Histology and 17-^5rdro3C3rcortieoid Output of the Dog Adrenal 
Cortex, Endocrinology 65s 80, 1959o 

2o Tullnar, Hilliam VJo Endocrine Effects of Methylenedianiline 
in the Eat^ Rabbit, and Dog* Eadocriaology (in press) o 

3a Tallner, "Hillisa Wo and Roy Hertz o Effest of Intra-yenous 
Ojp" DDD on Plasma 17-i^drosycortisoid Seeretion in the Dogo 
Endocrinology (in press) « 



Honors and Awards relating to this projects 

Elected ^feEiber of ^ericas Association for Canc©r Research. 



Results obtained from this research project were presented to 
the following professional grot^s: 

1, The Endocrine Society,, Atlantic City, N, J» Jtiae 1959=. 

2c The SXI International Congress of Pl^sielogical Scieaeeg 
Buenos Aires, Argentina August 1959 <» 



'99 



Serial So^ MCI-"S13 
i, SMocrinology Bs*aa©h 
2o Endocrinolo^ Ssruic® 
PIS-MR 3o 



Individual Project Report 
Calendar fear 1959 



Part Ac 



Project Title: The Ps^aration of Grade Natural Estraots of 

HormoRal Activity from Uxdae aad Serum aM their Purifieetion 
and Pbysical-Chcaaiesl CharacterizationSo 

Priacipal Inveatigatofss DTo Ralph A^ Reisfeld, DTo Roy Bsrtz 
and Dtc diaries F, Uadler 

Cooperating Uaits: Laboratory of Biochesaistrjj NCI (DTo Eo Ac 
Peteraoa) 

Man Tears (calendar year 1959) s 
Total: 3 
Professional; 2 
Other; 1 

Project Description: 

Obj^egtiyss: To obtain crude preparatioas of ehorioais goaadotropic 
boraoae froas the urine of pregnant women and of woesq with tropho- 
blastic tjjsiorso Purifieation aad eonceatratiojs of these borsoaes 
followed by ehax-aetsrisation of their physical-shaisical propsrtiss, 
Comparison of these properties of the gonadotropic horssoneis to de- 
tarsia® whether or not there is a ehajaieal diff ersnc© b®tv;sen 
hormones with aiailar biological properties but obtsiaed fross 
noKsal and cancer sources j r®BpectivQlj^ Furtherisoraj, it.wuld 
be of interest to determine as maay physical-shssiGal ' charaete-- 
ristics of thi-s hormone as possible sinee this could ultlaately 
lead to a eh^sioaJ. assay saethod which has "bean so far unaHfailable- 

Mfithods EaoloTadi la Tuaaor tissue frosB patients with chorioear©incffi£ 
was ©xtraeted and frasstlonated aceordiag to ■&© tseliiiiqaes of 
Sober and Petersoaa 

2o Cellular, fractionation of freshly prepared eheriooareiricsa 
tissue reeo'^ered as heterologous transplants from hamsters ^las 
carried out by the techniques of Hogeboo® and Schneider,. 

3o Urinary concentrates vere prepared followii'jg apprr^riate ad- 
sorption and elution procedures as preseribed toy Drs-> Sober ar-d 
Petersons, 

4o Bioessay of these fractions and eoneentrates were carried out 
by the aouse uterine weight laethodo 



800 



Eadocricol.ogy Branch 
^ 2 ^ 

Ma.ior Flndla^s; !» Th© fractions frosi tumor tissue hair© been 
eheaaically and biologieally charaeteris®d (Publicatioa No« l)e 

2» Preliminary data iadieat© that most of tbs hormoaal activity 
of th© traaor call is in the small grandar fraction with little 
in the S5:?)ernataat fraetioao No activity is detectabl© ia the 
roaaioing cellular fraetioaSe 

3o Furthar ehsiaical charaetsrization of the ahorionie horaone 
B.3 £'eeov®red fi?<Ma the urine of tiaaor patients soggeats some 
eheaaical differances from that obtained from normal pregnancy 
urine (Concentration of Gonadotropic Hormone from the Urine of 
Patients with Trophoblastic Timors, R,, Reisfeld and R» KartSy 
abstract presented at aestings of the Msrican Ghesdeal Societ.fa 
Septeaber 1959) » 

SignifiQanee ^o the Program „of the It^etituta; The availability of 
aon-to3d.c concentrates of natiarally occurring horraonally active 
substances or of inhibitory anti-sera capable of neutralising 
sueh endogenous biologieal effeets ia the caaeer patient woiald 
permit therapeutic investigations along lines sow neglected as 
a result of the es^hasis on synthetic eoi^oundso 

Proposed Course of Project; Extension of these studies a3.oag t^e lines 
outlined abcw© are projected and vill be esecated as appropriate 
personnel becomes available^ 



Part B included. 



801 



SMocrinology BssEfi.ch 

Indi"Fidual Project Report 
Calendar Ysar 1959 

Part Be Honorsj Awards, and Fublicatioas 

Ptsblieations other thaa abstracts from this projects 

lo Kadler, Charles P, and Reisfsldj Ralph A, 3 Goaadotropic 
Honaoae Distribution in Proteia Sxts-asts of Trophoblastio 
in Maxio J» Lab* & CliMo Med, (in press) « 



802 



1» Sssdoeriaoiogy Bi'aaeb. 

PHS-KIH 2, EndoeriEology Ssjcvice 

Indi'^idual Project Report 3*. Bethesda^ Md^ 

Calendar Ysar 1959 



gartA. 



Projeet Titla: Th® Preparation aad Svaluatioia of Biotin Actagoaists 
in the Study of Biotin Metabolism as an Approach to Cancers 

Principal Invsstigaton Mr* Frederick G„ Dhjrae 

Other Investigatorss Dto Roy E&rtz 

Man Tears (calendar year 1959)? 
Totals 3/4 
Professional J 1/2 
Others 1/4 

Project Description: 

Objectives? Th© prisaeipal objectives of this project ars to da^elop 
and prepare biotin antagonists that are particularly ©ffestive 
in produsing d©fiGieaoies of this vitamn in laboratory anisials 
as well as in sderobial systeaSa A possibl© rout© of application 
of aueh deficiencies to eancar sttsdies is described under ''Sig- 
nificance to tbs Program of the Institute," Furthermor©^ appro- 
priate antagonists ar© to be used to "block" certain bioetesmcel 
reactions involved in the study of Biotin Metabolism in bacteria. 

Methods EjHtDloyed; Biotin antagonists are prepared by organic chesieal 
synthesis or isolated from natural sotarcesj, and evaluated by sicr<5" 
biological assay and biological testing in rats and mce« 

Ha,|qr Finding?? Further testing of Biotin Sulfone and Y(3»4-i'reyl8rie- 
cychlohe^l) butyric acid on rat letsk^aia did not result in sig- 
nificant inhibition of such leulceaia. 

Excess biotin, (abo^e ttet required for mndmvsssi growth) ^ addsd to 
cultures of J^^ ara biaosus was found to gi'^e rise to a f i¥e-~fold 
increase in secretion of folic acid hj this orgaais® into th® 
surrounding iaediuffi« Th© folic acid was ^asured after 3 days 
incubation of J<, a^abinosua and at constant levels of ssasiatiEa 
growths It was expressly noted that excess biotin did^.S9i i^" 
crease the growth response of the folic acid assay organisja 
Cko caaei) « This effect seems to be specific for biotia-folic 
insofar as tested sinee an excess of pantothenic acid or ribo- 
flavin did not cause a significant increase in the secretion of 
folic aeld by Lo arabinqsusa 



803 



:^ Bsi'&acn 



This folic increase was found to take plac© oaly ia tJae freetion 
elaborated into the lasditsm and not in th© eellej, vhsr® the folic 
acid contant vas found Jbo. .b,e-<soEstaiat.o. Fortheraor®,. this folic 
asid aee^etlonwas found only wh©E Ui& ©Kcess biotin ims added 
at the start of iaeubation or i«dthin 24 hours thereafter-'- Biotin 
added after Lo agabinosus had attained maxim^jm groi^th did not re- 
sult in any Increased folie seeffetion,, 

These findings are of particular interest because of a pre'vious 
report in th© literaturs suggesting that biotin isay be required 
for the tissue synthesis of folic acid in the reto 

c A deuterium deri-yatiye oiT. YC3s4.-W©yleG®'eyclohe3syl) butyric acid^ 
— -{G^) prspased by substituting deuterium for hydrogan gas in the 
catalytic hydrogenation process by which UCB is usually pr@par®d|, 
did not show a significant increase of anti-biotin activity over 
that of th® regular WBo 

SignifiQan^;|io.,tl3j8;Progjt;aB.,of th© Institute,; Evldena© from, the lits- 
ratur® iadieates biotia may b© required for tha tissue synthesis 
of folie acid, and findings presented ia this report sho's? a 
positive effect of biotin on the synthesis of folic acid in sdero- 
organisffio In view of tha relative aueeess of folic antagonists 
in th© treat^nt of cancer, and because of the drug r-esistanee 
later developed to sueh treataaentj an appropriate inliibitor of 
biotin's role in folie acid synthesis saight make present treat- 
ment with antir-folics more effective ^ 

Proposed Course of Pro.leet; !» Further study of th® relationship 
of biotin to the biosynthesis of folie aeid in mcroorgaaissase 
Developsaent of antagonists to biotia and to int02=asediates in this 
partieular biotin»folie functiono Extension of sueh studie-s to 
laboratory animals for investigations of tha possible tissue 
synthesis of folie a@id, 

2o Evaluation of biotin antagonists j, and also aatliastabolites 
of certain biotin vitamers ia sicrobiologieal systssis where dri;g 
resistanee has been developed to anti^folicso Extension of saeb 
studies to i'ats and hamsters id.th tumors resistant to ssethotresata . 

3o Separation and isolation of the biotin vitamers described in 
a pi&lieation of previous findings in eonaeetion with tMs project. 



sm 



i - ..-, HCI-4-709 (c) 

1. General Medic iae Bratsich 

3« Bethesda 
PES- KM 
Individual Projece Report 
Calendar Year 1959 



Part A.% 



Project Ti£let The Chesotfearapy of Acwte LeukeMa 

Psrincipal. I«vesfeigafeors; Ensil J Freireieh and Sail Frei III 

Other lEVcstigators s L, Sehroeder 

Cooparatisig Uaits; GliBical Fhasnaacology and Sxperiaeatal 
Therapeutics Service 
-Jefferson Medical College 
Walter Reed Asuf Is&dicai Institute 
Jackson M@myTi.al Hospital 
Medical College of Virginia 
Eo£vell Park ^^rial Hospital 
Msaavj Ifeiversity 
■Bow!aaa«Gyay School of Medicine 
Ifeaat Siaai Hospital, Ifew York 
PhlladelpMa Obildren's Hospital 
Mayo Ciisiic 
Dartmouth ISniversity Hospital 

Man Years {caleada? year 1959) Patisat Says (calendar year 1959) 
Total: 7 
Profassioiaai: 5 
Other; 2 

Project Baseriptioas 

Objectlvea: 1, To stady q»saa£itatlvely the opt^iian usefulaesa 
of drags that are kaowa t© have acti'^rity against acute lesdceaia, 

2. To discover and test is huasaas new dtaaiical compowids 
%7hich caa alt@r the co«2ree of .^».wwt.B ^au!;a.«a4.^c 

3, To study the effects of tliese ccmipouads tm the ho8t» 
4» To test the tjsefulness of aaiatals and other screeaiag 

teelmiqisas by the collection. of qwaatitatlva clinical data, ia sano 

Methods gffiployed; 

1, Gas© Material: All patients referred to the Clinical 
Center with the diagnosis of acute lesi&eisaia are a&aitted to the 
sti:idy. In addition there are 12 hospital tmits other than the 

SiCI participating in most of the below as^tioned therapewtic trials. 

2. Criteria; O&jectiv© es-itaria for the evaluation of 
disease activity have beaa developed and applieda Hjsir applicabi- 
lity and aeaEiagfnllness is taader eontisBlag scrutiny. 

Part B iaslKdad ^S 

805 



Serial No. MCI 4-709 (c) 



3 J PharsEacologj; la cooperation witli £he Laboratory of 
CliEieal Plisssacology, pharBsacoiogic disciplines are applied to 
the drugs tested= Tliase studies are divided into three major cate- 
gories: 

a) Toxicity studies of coss^ownd of potential antittsaor 

activity givesa to hmmus for th® first tijae» 

h) Absorptioa, distribution and exeretion studies of kaowa 

and potsBtiai antitisBor corapounds, 

c) Analysis of the iafiueace of varying dosage schedules aad 

routes of admiaistratioa of astiStaaor activity and toxicity. 

4. Ejcparisseatal Design: In cooparatioia with biostatisticiaus 

asid the filiaical PSiarEiscology S^ctioa therapeutic trials ware desigasdo 

f&asQ iaslads: 

A» Cooperative comparative therapeutic trial designed to ob- 
tain asjong other thiags, quantitative iafonEStioM of the relative 
effects of antimetabolites of proved aatitssffisr activity ia man. 

Bo Sequsatial ^isalyais - Hew drugs are tried in patients 
with acute leiskessia who hsve alrsady received conventional anti- 
letakeffiic therapy.. If the pilot studies sssggest possible huaaa aati- 
leukasElc activity further studies are desigaedo Initially these have 
bsQR parforsasd fey adosiaistratioa of the dmtg to be tested to all 
patients qualifying. 

Major Findings; 

1, Protocol II - Tais is the second definitive cOTjparative 

study tsndertakea by the groups and in it cosaparison ia aade between 
three treatsaat prograaas latiXising drugs that are known to have aeti 
leukeiBic activity. I) >IethotreKate (MSX) followed by 6»Biercaptop«riae 
(6-SSP)j 2> 6-MP followed by MTX; and 3) MIX asad 6-HP given in coa- 
binatioa. To date 32S patients have besa entered on this stady for 
the entire cooperative groap, 92 of these patients being stiadied at 
WCl. fhis stBdy has just been cosapletedj and although final analysis 
has not been eoaapleted. Preliminary evaluation of the data on 215 
patients has revealed the following: l. tfee highest remission rate 
occurs with ctmibinatioia therapy althotsgh this is not higher than the 
rate x.7ith 6-MP plsas that with MTS. 2. ^e raasission rate with either 
6'W or MXX is not diflareat when givea as initial therapy or as 
second phase therapy after treatisent with the other agent. 3. 6-»MP 
alone gives a higher ressissioa rate than WiK. alone whether given as 
initial therapy s or after a previous aatiraatabolite. 4, the overall 
remission rate for all programs is hig&est in children (50%) iater- 
mediate ia the intermediate ag® group <30%) aad low in the adwlts 
(15%). 5, HK was essentially inactive ia adults «5%). S. SosieiSy 
reqwiriffig cessatioa of therapy occBrr©^ batween 9 and 25 days with as 
average of 14 days and was coroparable f®r all trsatssgat prograjas. 7. 
QisaiitativeXy MTX gave more oral ulceration aad 6«-MP Ejore Gl sympfcosjs. 

806 



serial KOo Mil 4-709 (c) 
- 3 - 

2. ?TGtOQ.ol III. - this is fcfee third definitive comparative 
s£udy tmdertakes by the groisp, fhe objective of this stwdy is £© 

develop a ssethod for the evaluatioa of sew ageats iu patients with 
acufce lleukesaie, essrly in the course of 'cheie illaeas, before the 
use of 6-MP or M!K. Th^ proposad methe^ 'Koisld ssseasssre the dmrafcioa 
of a reasissioa iadaced hj adreaal sseroidse fcoi deeemsiffie wSsether elie 
anfcilet!J;9iaic actioa of a drug c&n b@ d©£eee:Qd by its ability to pro- 
long asch a resaissloB. For this sttsdy an agesafc of provea activity 
C6-MP) was selected for stnidy. All paSisats ia remission froja sSeroid 
therapy recaive either 6-MP or placebo aisaia£eaaace therapy and th& 
durafeion of r^sissioa is aseasusred. to date 59 patients have eia£ered 
the study (11 at; Kl). 'Bt®. 4&t& have not beea aaalyzedo 

3. Dichloroasiafchopteria - Foilowissg initial toxicity sfciffidies, 
a coafcrolled eosaperstive study of dichloroasssthopterin and amethopt- 
eria vjas trader takes, ia patisats less t&aa 20 years of age who had 
received ao prior affiS;ifolic tfeerapy„ To date, 9 patients have eatered 
this study, (3 at ICl), Ihese data have aot been analysed o 

4. Gytoxaa - this compowsEtd has feeea stssdied by the groap with 
too sjajor objectives: 1=, to deteraiisie whether the coraposaad is active 
egaixist hiffisaa leiikeaiias and 2= to detenaiEe whether dosage schedule 
effects the activity of this cospo^mdo In the Li210 laoMse letskeasia 
aiad other lao'sse and rat transplaated timore cytosara is hi^ly active 
aad activity is changed by alteratioa of dose scheduleo To date 96 

patiesatis have entered the study (30 at Ml}' 30% of patieats have ahcnm. 
objective improvsseat , bat ia oaly 13% was this sufficient to qaalify 
for partial resaission^ Bosage schedola (daily or weekly) did not show 
sigaificsat differeaces although soataishat laore ijaprovesssent was noted 
with the weakly sehesfcisc Toxicity was aot significaatiy different 
for She £v?o sshedwles. 

5,-. Methyl- glyoxal-bis-gaanylhydrasoae. fhis co^omid has &eea 
stwdied saqaeatially ia patients with acssta le^skeaia. 36 patieats 
have entered the sttidy (13 at 1^1). to date, oo favorable responses 
have beea ssoted. 

6„ Misreofcisie - t&ia coaaposmd is highly cytotoxic ia tissue csjltwre 
but iaaetiv® agaisst aairaal ttastors, 9 patieats (5 a£ BCI) have received 
this cossposKd, No beneficial effects have h&esx acted. 

7, 6-agatsridiae - fhis coiapoiimd is the riboside of G-azauracil. 
It has beea studied ia 4 patieats (2 at mi). It has been abie to 
produce a fsll ia Isaskesaic le^skocytes isa the peripheral blood, bat this 
effect has fesaa traasieat and ifeversible despite coatiaised therapy. No 
ae»rotoKlcity hes beea aoted at doses of 20 tistes the maxiisal dose 
tolerated for 6 aaaisracil. 

Proposed Gotsrse; 1. Protocol 111 may provide the cfeesjotherapist 'tutth a 
powerfai tool for the st?sdj of aew cfeasKStherapefstio ageats ia patieats 
x^ith acste leakaaia. If so, it will be istiliaed as a design for atisdyiag 
sa^- ageats, 2, Stadias of 6 Aaasirldias will proceed, 3» Steadies of 
the effect of route of- adsaiais£ra£i®a ©a tlie actioa of aatifolic cs^possds 
Bvch Es dichloroaHetfeoptsria \<iS.ll fee ^sa4®f£akea„ 

807 



Serial Ko. ^X-4-709(c) 



PHS-KIH 
ladividaal Project lapojrt 
Calaadar Ifear 1959 



Part__B»_£ Hsaors , 

Fttbllxstlsas othar thsssi abstracts fsrom £&is project: 

lo SfeiMer, B, I,, Frei, Bo XXS, foufeyj I. H. , Saraaa, J., 

Frsiraich, E. J, Brisdleys S» O's ssd Clesaats, I. Observatlosis 
©« tfee feiisaaa tosisiSy aad phaessaco logic activity of 6-A2au?sell = 
lar Eesearck, ia press . 



SoKosrs and awards relatiag to tfeis project: 



asrial MOo ^5GI-4■••715 (c> 

1. Gmesral MadiclEs Sraacfe 

2, Ch®sto£h&rapy Serviee 

PHS-NIH 
Iffidividual Pro j eel: Espo^t 
Cslessdar Year 19 5S 



VaxZ A. 



Pr©1ec£ Title: 'B^e Mse&aaisiB aad Maaagesssnt of £he HssJOE-rfeagie 
Disorder Associated with Acss^s Lasskesia 

Priscipal Iaves£igator: Essil J Freireica^ M-D., 

O'caev Iav©stiga£oris : L„ R. Schroedas, M.B. aad B©il Frei IIIj H.B- 

C<50parat:iag Halts: Pstkologiesl AaaSc^y, H.S.I. , Br. L. a«s!gas. 

Blood Baiik, U.S.S., Dr. Klimaa, Dx„ F, Sclmidfc 

B^n Years (caleB<?.ar year 1959) PaSioaS Ssys (caleadar year 1959) 
Total; 2 

Prof aseiosal ; I 
Otfisir: 1 

Project SeseripSioa: 

Objectives; a) To idsnSify factors rsspsasible for precipiSafciag 
grcss hetaorrfeage Ir ^Isrombopeaic acw'ca le^kasaia patients, 

&) To stndy the srola of the platelet in blood c©sgu- 
latioii, and hssossasls. 

c) To -i-gipeov® th® thsrapy o£ ^hrosabopeaic hasEorrfeags . 

Methods Ei^loyscl: 

a) In vitr© aE«l is vivo blood eoagulatioa e^sdieso 

b) Colsssa chrossstograpby esiag cation <ssefaange reslas. 

c) Analysis of cliaical, labosratory a&d pathological 
data on patieafes with isessorrhage. 

HvSjosr fisdiags: 

1. Pisrlficatioa of Thro^in - Pa?ified thrcaabin is poorlj 

soluble. A£ low ioaic sferaagtfea and at pfe values below 6.5, the 
proteia is irreversibly precipitated. This efearacteristie has issada 
tfee isolatioa aad ecaceaS:ra8:ioa of suffieieat proteia for further 
chsssieai smdy vsry difficult:. Progress has beea tsadQc By appropriate 
selection of ph aad ienic straagfcfe val^jes^ batch preparaeioa procedures 
hava besa improved, fhsse saay lead to isolafcioa of snSiici^nt material 
frs® hQth bovias sad hissaa sources £0 allow for fursi&er chaaical s£«dy„ 

2. Aatil&rosabisi thrcss&ia iatsraefcioa - l5ffi,affl £hro^ia is 
less stable than Bovine throsbia in both hsaaa and feoviss- plassja. 
This is ferae wb®a Slarosbin ac£iviey is @s£:i®e£®d by eif;:h®r clottiag 
hiisssaas or bovia® fiferissogea or by its es^erass activity oa fosyl 
argissiae m®E&jl ®BteXo For bo^fe eajsass th© sraSe of loss of acSiviSy 



809 



Serial M©„ mi- A- 715 (c) 
- 2 " 

is a fisnctioii of cossceaSrstioa of eesytsa added, Whsa djrc3®bia 
is added to plasma fisrthsr coaversioa of psfothroabin to throssfeia 
is daereased ia proportion to tha quaatttj of t&rdasbia addedc 

3. Ee-triew of cl laical aajl laboratory data oa a groiap 
of pa£ieaEs with acuta lei&eaia has fesea ?jadesf£aksa to define ths 
relaSionsljip of cliaical fessaorsriiags So plstelefe levels, fevar, ia- 
fecSioa, osrgGa sisSj e£e„ Pr@lisair»sry saalysis iadicates fchat: 
platelet level corralatss besC wi£fe ellsiesi homoerfeageo Eeiserrhage 
raralj occurs- wkea piaSelst levels as^a a&ove 20^000 per cu»sssi. sEtd 
ocevirE store regularly &t levels bslow 5,000 per euoi^ao 

Prcpasecl Course of Study: 

io Studies of purified thjrssabia and £h® throBi&in aEfci» 
tfiroi^in iiitarraefcioa will be coatiauad, ISsa biological activity 
of the produces of thts iaSerractioa will ba studied. 

2. la colla&oraSioKi with the Cliaical Ceater Blood Baak, 
©oB.S„5 WQ have iKitiatad sSudl^s of fresh pla&elet trans fwsioas ia 
patients with aeute leukaiaiao Tha platelaSs are obtsiaed by platrelet- 
pfeoresis frost sslecSed donors. This techaiqsis may allow for greatly 
iiser©as©d availability of fresh piatslees whiefe has hit&erto limited 
oar studies in £ljis araa„ If fefeis soarcs is aa effective oae we plaa 
£o stts4y the effect of prophylactic plafceleS; traaefasions on feemorr~ 
feage and survival of patients with actste lasakemia. 



810 



Serial IfOo 1CI=U"715| 



PHS-IIH 

Individiml Project Keport 

Calendar Year 1959 



P&rt_jjo lonorSj Awards j, and PublicatiOES 

1, l^itz^ go D»j Forkn®r, C E. , Jr.^ Frelreich, E. J,, Frei, E. Ill, 
SliOMaSj, Lo Th& association of fatal intracranial haroorrhage end 
"blastic crisis" in patients with acute leiikemia. Mew Engo J, of 
M@d. 261:59-64, 1959- 

2, Freireiefe^ g„ J., *2homas, L. B. , Frei, E, III, Fritz, R. B,, and 
Forkaier, Co E», Jr» A distinctive tjpe of intracerebral hemorrhage 
associated with "blastic crisis" in patients with leukemia, Cancar, 
la press o 



Serial Eo. NCI-4-719 (c) 

1, General Hedlci&e Branch 

2, Gh2®o£herapy Service 

3, Bethesda 
PHS-NIH 

Individual Project Report 
Calsadar Year 1959 



Pare A. 



Projees Titia: A Study of the Clinical Masifestaticm of 
Acute 



Principal IssvesSigatore: Eiail J Freireich, M.D. and Essil Frei HI, MoD. 

0£hsr lavsseigatore: Lo R. Schroeder, M<,S. , Richard Shax^, M.9o a 
Edvard Moore, M.D. , Dane Boggs, H.D. and 
Arthur Teplitsky, M.D. 

Coopers tlag Baits: Hoaa 

Mae Year (Caleadsr Yesr 1959): PatlSEfc Bays (Calendar Year 1959): 
Total: 2 
Profess iosal : 2 
Other: 

Project BsscripSioa: 

ObjacSivss: To dell-aeate etlologic aad pathogeaeSic factors 
of £ha various saaifsstatioas of acafca iaitkessia« 

Methods^ Employed; The coiapreheasive application aad correlatioii 
of cliaical, heisatoiogical , radiologic, cfeemical, microbiological, aad 
histologic siethods to the abovs manifestation ia patients with acute 
lei^ke^ia . 

Ma|_^^^_Had£^s : 

lo MeaiEgaai aad CsEtral Kervoas System Involvemeat « A 
retrospective clisical~pathological stwdy of meaingeal leakemia tn 
150 patiessts ^ith acute le^eiaia has been cosapletedo "She clinical 
syadroaie of oeaiageal le^emia which results prisaarily froaj iacreased 
intracraEial pressure was recogaized ia 25 (1?%) of the 150 patients 
studied. It occurs prisaarily ia children with acute lymphocytic 
leuketsiao Eight of the 25 patieats developed the first evidence of 
mesiageal leakeaiia uhile in rsaissioa« Review of the literature ia- 
dieat@s a sharp increase ia the frequency of this syndrojES since the 
introduction of chemotherapy, Theae data suggest that the lacreasiKg 
iisportaace of this cliaical syndrcme results froia the use of cheso- 
therapeatie agents effective against the systesaic disease bat rsiativsly 
ineffectiv© against the laeningeal involvsraent. Study of the central 
nervous system in these patieats revealed that the eyadroEs of sieaiageal 
le«keiaia was assjsciatsd with a high frequeacy of intaraal ccpsssiKi- 
cating hydroeaphalus and with leuk^aie iafiltratioa of the srachaaid^ 

Part B included NO 

812 



,/:ao MCI 4~7i9 (c) 
- 2 ~ 

Laakejaic Isvolveajaat of the dura, choroid plexus or arachtioidal 

grasuilatloas ts-as s©t related to tfee occurreace of msaingeal iesjkeaiao 

2„ Hat^ral History of Disease . A review of tbe clinical 
ccusrs-a of 1?8 patients has he&n vsadextakeu. Aciate mjelogsnous lesfee^ia 

was found in all aga gEo«ps 'K&ile aesste lyapliocytic lewkesia was fo®a-d 
prisKirily ia cIslMrsE. (IMder 20 years of age). Oaly 20% of the 
paSieats with aeute ajalogeaoas lewka^ia s&owed any iasprovejaeat with 
c&sasoSkerapj, IS'is response rate for pa£isats with lymphocytic le^ejaia, 
Qsxv-evsr, was tsach be^tsSo More £tisa 90% of th& e&ildrea witfe ac^et:© 
Ijaphocytis letsltssia iraspoadsd £o cheao therapy „ In contrast onlj 40% 
of elis adal£s (graatsr £haa 20 years of ags) wi£M ij^liocy£ic Isssksmia 
r@sposdsslo Tiua freq^saey of rsspoffise £o efeetasthes-apy correlated with 
sjsrvivsl of th© patisa£„ Mediaa survival for chiidrea with lyaaplioeytic 
l®33kemia being 11 ®os,, assd for adults with l^phocytic lesakeiaia, 6 saos. 
Survival also related £o £he extesst of reaissioss. Median stsrvival for 
child4>3a feavieg a coapleta remission beiag 13 ^js.j sad median survival 
for children showing so improvement: being 2 isosiths. 

SigRificaaee to Gather £aaearch; fha coatiaaissg scrutiny aad defiai- 
tioa of cliaical siaaifestafciosis of aca£s la^zkesaia is of sigaificaece 
to caacsr research from stsEy points of view. %is is one of the 
ffiajor areas wljeraia leads to pathogssistie saselianiems may be aacoveredo 
In addition, prscis© observations are essesttial £o defiaing the 
Eateral history of tlia disaas® aad Skis latliar ia of critical ijEpor* 
taace to drug evaluationj the saajor effort of osjr ^ectiosio 

Propossd Goarse af tfais^^^^Fgojecg; 1= Studies of saeniageal lauksEsia 
will ba ssstaaded hj steady of bTo©d braia barrier, ability of cfe^sso- 
tharepeutic ageaSs asd other materials ts eater th& C.N. So la addition 
a prospective stisdj £© assess t^ freqaeuiey aad jaaEageaiaat of tfeis 
sjiidTosis will bs ?sadertakeno 

2« Malysis of data cofficeraiag aataral history will b® 

c€S!2^iistffid, aad fsEsrtlaar data acctssaslatedo 

3. Ifoeleic acid asetatolissa, fte detarsaiaatioa ia the 5iria® 
of p-ariss and pyriaaidine bases as well as laucieosides aad asjeleotidss 
feas rscesatly becssae possible after 3 years of extensive jsethodological 
iavestigatioES carried oat by tisa Laboratory of P&ysiology (See' their 
Project Report). Stu4ies ia laaa are noxa ajsdarii/ay coaparSaig aerials 
witfe patisffits with tastreated acsste leakeaiao 

4. Boss lesioEs. A coia|>reheK@ive retrospective study ©f 
tko. clinical aad s-ray saaifestatioaB of bous lesioas ia acssta lesskesia 
was coadiscted ia 85 patiejstSo la 20 of th©se patients tlia satire fesser 
was reiaoved at astopsy arid the pathologic fiadiags correlated with the 
elinical aad radiological f ladings, Fivs distinct types of Iasi©as 
have been eaeojaatssred aad thair patfeogeaesis has bsea, ia pas-tj iliaei- 
datedo 



813 



Serial lo, lCI=ii-=7l9(c} 



Individual Project Report 
Calendar Tear 1959 

Part B. Sonors, Awards^ and Publications 

1. Moore, E, Wo, fJioisas, L. B», Shaw, R„ K, , and Freireich, S, J. 
Thm central nervous system in acute leukemia; A postmortem study 
with particular reference to hasorrliages, leukemic infiltrations, 
and the syndrois® of meningeal leukemia, A»M.Ao Archo of Int, Medo 

In press. 

2o Hilbish, To Fc, Besse, Bo E.,, Lusted, L. 3=, Baves, M. L<,, ^hoHias, 
L, Do, and Forkner, Co A, Acute leukemia. A, Mo A„ Arch, of Into 
Medo l0!^;7i^l"=T47, i959o 



814 



Serial m. MCI- 4- 734 (e) 
lo Gaasrfil Medicisse Braac-fe 
2o Clsaaio£h®rapy Sarvice 
3„ Beekssda 



Fas- HE 
ladividiaal Project Report 

Gaieadar Yeax 1959 



Pare A. : 



Project: Sisls: Assay of Eryi:hropoie£ia ia bosas 

FrlECipal lavas £igat©srs: L. E. Sehroedesr., M.S., 

C, W. GBsraay, M.S. 

CoopeK-atiag Ifeits: Argonse Caaeer g.esearch Hospital 

feive^sitj of Gfeicago Clinics , 
GaicsgQj Illinois 

Ifea Years {Calea&r Year 1959) Fatisnt Bays (Galesdar Year 1959) 
tot&U 1/2 
Prof ess ioaal ; 1 A 
Qthet: 1/4 

FroJaeS Ssscsiptioa: 

Objectives: 'So perfacS a sisals in vi£ro assay asetfessd for esrySlsropoletin. 

Methods; SBspaasioas of li-^iEg hemaeopoiefcic caiis froa £h@ feosse aa^s-ow 
QT spleaa of SKltable doao? aaisals were isiCKbated wifcfe ra«iioaetive iroa la 
the preseacs of test sad coeSrol plasmas » Cell-bound radioaetive iSGU was 
measB^ed in a scit^tillatiois co«m&er, 

FaSioEt: Ma£erial: Plasnjs gasaples foom patients wifch a variety ©£ hesa- 
£ologie disorders were assayed ^ 

Major FiEdiags: Hsssaa ery£feropoietia could be assayed by tfeis teehsiq^e 
aad 5:ha desHOQStraeed activity was iadepesadeat of the iron coi*£sat of tae 
plaataa. Ilodificaeloa of tfea ©rigi^ally- described assay and rigid control ©f 
it<m eoaeaatratioa ia £fee test system viaa aeeessas-y to achieve this resBlts 
hut tfce additieaal steps involved did ssot vitiate the utility of the assay, 
fh® seasitivity of tsia assay appeared to be greatly increased by £fe® iss© ©f 
tseiaatopoiatic cells fresa aiiiraals wliose QR<iogasso«g erytferopoissis was gasppress;; 

Sigisificaisce to the Frograsi of ths iBstitute: 

fhe h^Eorel rsgalatioia of boa® Esarrow activity is a basic pfeyaioiogic 
sjechaiaisHi as yat poorly ssiderstoodo Isaowledge of the factors iaflasississg 
bos® ©arrow respoase to sts-ees aB«! disease is fmidasasatal to aji uaierstaEsdimg 
of the respoasa to eliesotherapasieie agents. 



Fart S Sot iacludad 

815 



Serial l!o„ ECl 4 734 (c) 

- 2 - 

Whssi the assay iaeShod is ready for appllcatlcm i£s ecoaossy of ffiaterial^ 

asd sisaplici^y siissld psrait detailed investigatioss of erythropoiesis ia 
health aad disease tha.t have hitherto cot besn feasible. 

Proposed Comrse of Projsst: 

As tlia saajor fixsdiags are the restalts of aspeE-isgaats considered pse- 
liasiaary arid sj:plora£orjf t:&ey req«ir© ceafissaeloiSo OptsQ coafizwafcis-aj 
a detailed stndy of tise kiaatics of tfea assay is proposed, followed hj a 
ccxQparieos of this tsekaiq^se ^^ifch esiefiiag assay saethods to defiae its 
ace^raey ©ad applicabiXifcy;, 



816 



Serial i?Oo £«;i-4-735 (c) 

lo eenesal Mediciae Brasssh 

2. Che^jfcherapy Sers^ice 

3. Befchesda 



PHS-NIH 
SsdivMsai Project Repos-i 
Caieadar Year 1959 



Part A. 



Projset flscle: Tha Effsst of ISiarapy an the Syafchssis of 
Bssoxyriboauclelc Acid by Le^saic Cells 

Frirtclpal laves £i§a tors; Dr. Gaorge Brecher 

■Other Ia,vas£iga£or® : Sr. Eijgsae Croaklte 

Cooperating Sai'£s: Hessatology Service, Sspto of Giiaical Pathology, 
Ciiaical Geate? 

^a Yaars (GalsEdar Year 1959) 
fcfiai; 1 

Profsssioisal: 3/4 
Othesr: i/4 

Project Bescription: 

Objectives: To stady the rate of mk syutlsesis of ieakeMc cells 
of the peslphsral blood aad bon® laars-ow la aa ia vitro system 

Methods: laeabation of le.ukmlc cells with tritiasn- labelled 

tfeysidias and Ideatifileatioa 0f la'eslled cells by radioautography ^a.s 
ssssd as an indicator of DMA sya£h©3is» 

Patient Material: Multiple paripheral blood aad boas aarrow 
spaclsaeas were obtaiaed from patiests witfe acute leukeaia being car©d 
for by the Ch^EO therapy Service of m;I. 

llajor FiEdiags: tke syathesis o£ WA by lesskeaie calls eoatitases 
ia vitro for at least four lioiss'© with little or ao dieiastioa ia rate. 
S?o aew cells ©Efcsr t&e ayathetic phase of the gensrative cycle dKX'iag 
is_viero isacisbatioa, aad aoaa divide o 

a® addition of large ajsoBsts of labelled tlsysaidioe to tfee syst^as 
has ao apparaat deXeterioias affect aad persaits the praparatioa of 
satisfactory aiitograpfes ia less thasa a wesk., 

fhers is ao correlatioa apparaat between Wk synthetic activity 
asd the ss^al criteria of cellislar itra^taritya 



Pas-t g MOT m^WMB 



817 



serial Ho. J^2-4-735 (c) 
- 2 - 

theze is a ^i&e rassga in rates of DHA sysfehesis by leukessic cells„ 
la KCjae of the patisBts ' cells esamiaed did the rate approach wfea£ 
siighs • hs-^e fesan espsctedg and in no case of le?2l?.e3aia did the rat:© is 
tliQ cells of the peripheral blood escged that of calls frosa paeisnts 
with irifeetiosis moiaosucleosis. 

Sisaificaac© to Gaaee 



X£ ±s sow fsasibis to issplore Shs effee£s if assy, of aaeil®«ke®ic 
therapy o© BMA syatfeesis as me&snK@d by this system, 

Aatisipstiag sssccess or failure is taa trsatiaeat of aeuSe leukemia 
wifcli a spsclfic agaat is sow possible oaly la a statistical fsshioa, 
siaaniagiess for the Isdivideal patient. 

If tha efSeet of tlssrapy oa the rate of BHA syathesis by le^cimie 
eells is fotjad to predict evestsal resposssivaaess of the iadividaai's 
disease, it coiald E'esult ia a trasaeadsus savisg ia tia^ and patieat 

Proposed eoasrse of tlia project: 

Wsvi' titat BGme kaav^ledga of t&e ira vifcg@ kiaetics has beea gaiaad, 
tfes pTOce&ra ^?iil be Bsed to mzannvB tfee effect of aati- leukesie 
tiaerapy on caliular MA syathesis aed attempt to correlate this with 
rss^ssion of the disease. 



818 



Serial No, NCI- 4 736 (c) 

1„ General Medicine Branch 
2„ Chemotherapy Service 
3. Bethesda 



PHS-NIH 
Individssal Project geporfc 
Calendar Year 1959 



Part A« 



Project Title: The Deaonstration of a Hormone Governing the 
Production of Platelets 

Principal Investigators: L.R, Scteceder, M.D. 

Glenn £. Lee, M.D. 
Other lavsstigators: Mone 

Cooperating Units: None 

Man Years (calendar year 1959) Patient days (Calendar year 1959) 
Total: 11/2 
Professional: 1 
Other: 1/2 

Project Bescripfeion: 

Objectives: to demonstrate the existence in man and animals of a 
hormoaal s^stanee vjhich governs the production of platelets by the 
bone marrowo 

Methods: Bsscaabopeaia was prod»sced in esperimsntal animals by 
injections of saponin, by the passage of blood over columns of Dowex-50 
resin beads, by prolonged thrombopheresis throtsgh an indwelling vascular 
catheter, and by the administration of specific platelet antibodies. 

Major Findings: 

Severe throsabopsnia cotsld be regislarly produced by thrombopheresis 
or the administration of platelet antisert^ with negligible side effects. 

Plasma harvested at intervals from throi^openic animals, pooled, 
and administered in large amount to norssal animals resulted in oae case 
in marked megakaryocyfcic hyperplasia of the bone marrow of the recipiaat. 
Control plasmas, saline, and plassaa from throsabopenic animals harvested 
at different tiaie intervals did not have a similar effect. 

Significance to the Institute: 

Proof of the existence of a hormone governing platelet prodt^tlon 
will add substantially to an tmder standing of the homeostatic mechanisms 
that control bone marrow function la the no^aal. Derangement of these 
mechanisms may have a rol® in the throa&openia that is a part of the 
aciste leiskaaias and th® thrombocytosis that accompanies chronic myelocytic 
lesskemia. 

Proposed cotirse: 

Confirmatory eKperimsnts are ia progress « If they support tfee 
reported observations, two lines of investigation are proposed. 

Part B included MO PI Q 



Serial Noo HCX-4-736 (c) 
- 2 - 

(1) repetition aad expansion of the iaitial type of experiment 
to establish the statistical validity of the hormone's existence and 
to define the tlme-cossrse of its appearaace aad utilization, as well 
as the threshold aad laaximum stimuli for its prod'action. 

(2) investigations in smaller experimental animals to develop 

aa econofflical assay that is not species specific. Various laaneuvers will 
be employed to enhance the responsiveness of the recipient animals in 
attempting to increase sensitivity and specificity of the assay, 

^en the assay system is developed, a survey of human plasmas for 
activity will be made. The collection of such plasmas has been begyn. 



82 J 



lo Seasffal Madiciae Branch 

2o Solid Tiffisor ServicSj Chemothet&'^-i 

3, Betlsesdaj Marylaad 

FHS"HIH 
Individual ProjscS Repots 
Galeadar Year 1959 

Frojact: Title: 

A, Coffipasrsti-ys Trials of Cfeesiotberapsistie Ageats ia Patien£s 
t*ii;a Solid Tj^sorso 

B. laitial Therapeutic asd Plsansacological Studies of Cfeemo" 
Sherapaafeie Ageats is Fasisnts i^ith Solid Tuaorso 

Friaeipal lavsseigafcors: Clyde 0» Brisidley, M. B, aad Esil Frsij, III, 

M, Do 

Other Issvesfiigstors; ifcafcague Lsae, M« B., Earold Silbersaant M» Doj, 
Jas2©s Mars&j Mo !>« j Mehasd ^cfeanic. Ho Bo and 
Harold Faileaj M. Do 

Cooperating Oni'css I) Clinical PharsEaeol©gy and Esperifseatal Tfeara- 
paweics Service o 2) The Eastera Solid TtssKsr 

■ Mass Years (Calesdsr yesr 1959) ■ FatieEt Days (Calsndar y@sr 1959) s 

Total: 3 years 

Frofsssioaal: 1 1/2 (E-oFI 1/2 year.. , Co0„3o 1 ysTo) 
Other: Teclniiciaas 1 1/2 years 

Project Title: 

Ao Coaparativa Trials of Ctseaotherapaufcie Ag©n£s ia FafcisaEs 
wi£& Solid Ttss^orSo 

Project Descripeioa: 

Objectives; To £ske parfc in the Eastern Solid Tusjor Ches50£herapy 
Groisp ia £lse applieafeioa of £ha principles of fefea C<^pars.£ive 
Clinical Trial ia £he evaluatios of eheisieal ageatg ia the 
trefitoeafe of htJEsaa caaeer. The preseat priiaary objecfcives 
of the group ar@ the isaprovsiseae in the ssethodology of £h@ 
Comparseive Clinical Trial in husan carjcer patieats aad ths 
^ase of £hs Coaspaifafciva Trial £o establish the relafeiva effeee- 
Siveaass of selacged chswsthaTs.p&utic. agsrats ia smao 



Far£ B iaeluded 



82. 



¥®s^ 



^©O 



Sarial Soo KCI-5-712CC} 



These data caa he used to (1) evaluaSe £he correlafeioa betwesn tha 
sctiviSy of afease agsnCs la aaimal tussor sereeaisag systeass and in 
paJ:ieats ivi£h eaacer sud (2) £o eseablish their relative usefulness 
ia the current Sksrapy of feumaa cancer o 

Thsas ars uEdargoing coafcinuisg ^sdificaeioa wi£h experience o 
Tha caacssr ebiesaotberapist » pbai^iacologist and biosBietriciaa operate 
as a uni£ la these sfewdies, 

1) FsaisEt Selac'cioao The effect of prior fefearapy and axesn£ of 
disease oa respoasa so tharapy is beiag iacreasiagly defined as data 
frosa qusa£iative studies sccuaiulates. For st«<Sies involving direct 
eoapsrisoas i£ is desisrsble to use sensieive tuaiors 0^yisplioma} o For 
studies of iHsessitive gussors reeaiat Mstorical controls m&j be 
sstisfsctoryo 

2) laadoKaisationo This will becosas more effective as factors ia- 
flasacisg response to therapy are dsfiaedo Tbess factors can be 
quastitatively assayed by rsgressioa aaaiysis. 

3) Bsterssinatioa of Effect and Analysis of Sata, Staiadard criteria 
for respcsEse to therapy have been developed <> Objective ehaages in 
tsjsor sise froa the basis of these criteria though syraptoasatie 
slteratioas will be recorded and analysed ia parallel » 

Case evaluation witla esiphasis on quantal as well as quali- 
tative responses has been iisprovedc Evaluated cases will be coded 
on IBM cards „ This should allov? for rapid and complex interiia 
ssalyses of studies j a procedure which in the past has been cusabersosie < 
In additions a cosiplete, «p»to»date . analysis of all studies will 
be laade at each taeetiag. This will be facilitated by the use of 
Status Reports for all patients Etill on study « 

Major Fiadiags; 

1) Thiotspa CTSFA) - nitrogen asustard (HH,) study - The first 
portion of this study indicated that in Eodglin's disease Hli« produced 
a sosaewhat greater rasaission rate and the remissions produces were 
significantly loagero However j as there was soma evidence that the 
dose of HHg ia this study was closer to the laaximally tolerated 
dose, it was decided to obtain dose-response inforssation for both 
TSPA and HH^" it bseasa^ quickly apparent that the dose-response 
carve was steepj i«eo that staaller doses were relatively ineffective „ 
In additions it woald appear that at mssiinally tolerated doses the 
therapeutic effect of p^ sad TSFA are similar « 

,r 

2) %eloBa Stiadyo fha first ssyeioEja study involving the coaparisoa 
of asaserine ssid a placeb© has been completed » Asaserine is inactive „ 
In the eursrettt study urethaae and a placebo are beisg eompsredc It 

is anticipated that the Watural history of syeloas smd the place of 
urethana in its trastmeat (a highly controversial area) will be 
^quantitatively defiaedo/ 



822 



Serial Mo. 1JCI-5-712CG) 



Wi^h tMs baskgrouad i£ is plaaaed to esaiaiae dsjsgs that are active 
agaiQSi: ssous® jsyalosaa (s disease wMc& Is aorpkologically and 
bioclsessicaliy very siEiilar feo hiEsaa rayeloaa). 

3) X-irradiat?.oa aad HH« - The place of these tlierapeutie 
Eoduli-£iss is the treatseag of Itsag eaacer is iasdequately defined, 
'fhe relati-js affect of tlaese agents ^ aloae and ia cosablnstloaj on 
Utsor slss aad SKr^ivsl is baisg sSudisdo 

4) Lyaphosaa Study « DisM.oriiS®tho£iresa£e, meeho£re:sa«:e (bofcls at 
£wo dose le¥al8) and M^ are beiag cosaparad in lyiapfeosaecoisaj 
reticuiua eell aarsoaia sad Isdgkia's disesse, PreliiEinasy analysis 
indiaasea that t&s folic acid sa£agonis£s feave aatituiBor properties 
comparable to ES^ agaiase lympiicjsarcosa and refciculasa cell sarcosaso 
As with 11„ tlae aose response .carve is steep o 

Frojese Title: Bo Initial TMerapsueic aad Pfearascological Sfcudisa 
of Chsmotherspeutie Ageaes iu'f&tients with Solid Tuiaorso 

ObJecSivgsj^ 1) To obtain ijiforsaafcioa oti £te ©pfcimua route and ssiode 
of admnlstratioss eosicity sad spectrtas ef asstitusor activity of 
nsw cliasistlierspesatic ageata in cancer patients c 2) fo esplore saw 
msShods for tfee detareiEstioa of growth rata of tvmoxs ia ssano 

|fetb.od3s For tl'as first ob-j.@c|:lv« the procedures are as follows; 
First t&e writiag a protocol for the' initial trial of the drug vistag 

tisa ds-ts availsble froas aaimal antit«ssor and toxicity trials as 
guides o Then dose routes asd dose levels 4re essplorad while obssr-" 
vations on patisat reacfcioa, organ fsasctioa and tisssor growth rata 
are observado Drag or t?3iGor KStabolic products may b® followedo 
IJfeaa tfea bast roate of adizjialstration^ the primary liaaitiag toxicity 
sad isa?;is3ya tolersted dosa of aa agent have besa detersaiaed, tfee 
drug is given ia two or tSirae dose levels to patieats with a broad 
spsc£r?im of tusaor types while observations for aatittssor activity 
ar® Esade, 

For tfee seecnd objective ssrtam easysss levels dsid tiassor 
sstabolic products are assayed end tusaor rassses are measured directly 
as patisets raceiva efeerapy. A correlation befcweesi c&asages ia sar^ss 
easyase levels or fjssor sastsbolic product eKcretioia aad clsatiges ia 
tmsor grois?£h rate ana so?2g&e. The tiasor metabolite sad sasysaa 
studies iEvolve the use of the speetropfeotosaaters radioisotopes 
a^d the asiial biochemical teclmiqueso 

gatieat ^ ^ lfe.t ® riglj_ For the esploratory dr«g toxicity asd aatitmsor 
effects oaly patieaits yitth caaeer or leiskeaia for ,^ko^ ot^ar a^r© 
coaveatioasl therapy offers little prospect of palliafcioia ©re 
acceptable „ For the steadies oa tssaor growth rates p patieats with 
saaay types of eajseer and oa various therapeutic regimeas are iacladedo 



823 



Serial Ho. HCI-5-712CC] 



ffgJog^igdigg3_^ SsriEdias ^^Mels have baea eompleted ia th& last 
year are as follows: 

1) fojEieity ssd Aafci^iaisor Txial of Urscil Mussasd. 

2) Tonicity and AatiStasor Trial of Cytoxan. 

3) Tossiciey asd M£l£tiz^r ?ri&l of IfemoelilorsaafchoSs'esateo 

1} The optiaya oral doss of Bracll S&sstas'd was fouad to be 0.2 sg. per 
kilogram once weekly. lEtravaasus use of this agenE was no£ explored. 
It had smaifcasor activity ia Ecdgkia's diseases lysspliosarcoiaa sad 
t&s chroaic leaksadas. le caused severe he£icpoi@£ic depressios of 
both lei&ocy£ss aad throabocySes aad occasionally vaiy saskad gas£ro" 
inteseinal disturbances .. 

2) Qytoitsxi (CyclopaospbaEiiis) was tolerated by sost patients at oral 
doses of 4 ago per kilogrsa daily for several weeks. Iratrsveaoiisly ' 
tha sassiffiue tolerated dose for saveersl weeks vjas 20 ssg. per kilograis 
once weekly. Aatittssor activity was seen ia lymphosarcoma &nd ovarian 
careiaGsso Tke drag depressed the leukocytes relatively acre than 
tfes thrombocytes. Alopecia was troublesosaso 

3) Mcnochlonsetliotresste ca^ised pro&ouzioed folic aeid antsgoBist 
toxicity SEd isapairsisst of fespatic fuaction at doses of 0.05 ssig. par 
kilogrsEj daily witMs oaa to two weeks. 

Ia those patieats vjith metastatic eaacer in whoia tka levels 
of the serusa easyssas lactic dehydrogeaase or riboauelease were 
elevated; these ensf^^ levels decreased significantly when t^saor 
regressisa was breaglifc sboat by effective therapy. Liaiitatitms of 
tiaese tests as iadisators ' of tiiEor growth rates are that ia soise 
terssiaal patients these sensa levels also fall wfees therapy was aot 
eff active. Twssor growth was being curtailed by sautritional changes 
ia these patiesta. Also, !saay patieats with metastatic cancer have 
no elevatioffis prior to treatEent and subsequently no cfeaages ia 
these ssayises durisag therapy. 

Mi^.MitSggS®-, ^g-5MCQ ,g ,^^s<eage1i ;_ A aeceasa^ prslisiaary to a 
clinical trial of a proisising agent is a dstersniaatioa of its rebate 
of adj^inistration, primary side effects and probable optiisus dose 
raage . 

Isdireet aaethods of foliowiag tuisor gro^h rates would 
fssilitsts the trial of new agents in patients ia whois the neoplasas 
are sot dirsetly ssaasurable; this is the case particularly for ssaay 
tisBors arisiag from tlie gastrointestinal tract. 

Fgogg8g d_C'gers e of ^groject Stisdias now in, p ggggggfl. 

1) Tonicity sttidy on parenterally administered dieMonaethotrasate 



2) Aatit^^o? trials in patients with malignant carcinoid. 

3) Ssr«s3 easyme st^^dies in solid tssasor patients with laeasarable 
tasEor aasses daring therapy.. 

4) Cellular .easyas stedies on patients receiving pyriaidine aatag= 
ouistSo 

82^ 



Ssrial Ho. KCI-5»?12CC> 



la I) above the cwo drugs sre bsiag givsn jsareafcerally for 
several weeks j in prsparatioa for a ccMspsrafcive aneitmsor trial 
using 'che paraateral roufce for drug sdminis£ra£iono I» ' 2) the 
urinary escretion of 5 -hydroxy iadole acetic acid is being followed 
as an isdes of t«a«5r activity wMla patients are given new ^iti- 
titiEor ageata. In 3) these studies ars being estended to include 
cthar eassnaaa, glutatMoaa reductase aad riboaucSaass , in s.n'. 
effort to find one wMefe would h& sensitive in gastroiatestiaal and 
gssitourinary carciaossaso In 4) above the cellular enzyiaes cott" 
csraed i^ritli ■pyximldlne EStabolis® are being studied. The cite of 
sctioa of cartsia pyriiaidiae aatagoaists caa thea b® sought. 

As with the comparative eliaical trials coatinuing efforts 
-are being sads to improve our saathods of conducting these prelimiaary 
studies o 



825 



Serial lOo ICI-.5--T12(C) 



PHS-IIH 
Individual Project Report 
Calendar Tear 1959 



Part B . Honors, Awards, and Publications 

1. Lame, L, Petering, H. d., and Brindley, C» 0. Synthesis, 
PharMacology and Clinical Trial of the Riboflavin® Analogue, 
Soditaa-6,7-Siffiethyl-9- C 2 ' -Hemsuccinoxyetfeyl) "•Isoalloxaainia, 
U-65380 J, lat. Cancer Inst. 22, lo. 2 3^9-361, 1959- 

2o Brindley, C. 0., Markoff, E<,, and Schneideniisan, M. A. Direct 
Observation of Lssion Size and Kumber as a M«?thod of Following 
the Growth of Htaasan Tmaorso Cancer Vol. 12, 139-l^!-6, 1959» 

3. Zubrod, Co So, Scbeideriaan, M., Frei III^ E., Srindley, C. 0. 
dold, 6. Lo, Shffiider, B«, Oviedo, P.^ Gorman, J= , Jonas, R, Jr.; 
Jonsson, Uo, Colsky, J., Chalmars, f», Dederick, M., lollaxid, J.; 
Selawy, 0., Hegelson, W.,, Lasagna, L« , Owsrb, A, Appraisal of 
Methods for the Stiidy of Chemotherapy of Cancer in Man; CosparatiYe 
Therapeutic 1?rial of Nitrogen Mustard and Thiotepa« J. Chrono Dis. 



Gold, a. L., Hall, T. Co, Shnider, B. I., SelamT, 0.^ Colsky, J, 
Ac H., Jr, Dederick, M. M«, Holland, J, F., Brindlsy, Co 0., Jons 
Cancer Besearch 19 (9); 935-939^ 1959 



826 



i>HS-NlH 

Individual Project Report. 

Calendar Year 1959 



Serial No. NCI 5-737 (c) 

1„ General Medicine Branci: 
2„ Chemotherapy Service 
, 3o Bethesda 



Part A, 



Project Title: Infection, Fever and Host Resistance in 
Malignant Neoplastic Disease 

Principal Investigator; Bmll Frei III, M.D» 

Other Investigators, Harold Silberman, John P, Utz and John Fahey 

Cooperating Units: 1, Laboratory of Clinical Investigation, NIAID 

2, Clinical Pathology 

3, Metabolism Service, BCI 

4, Laboratory of Pathological Anatomy 

Man Years (Calendar Year 1959): Patient Days (Calendar Year 1959): 
Totals 4 
Professional: 2 
Other: 2 

Project Description; 

Objectives ; 1„ To study the effect of malignant disease on 
the host as regards resistance to infection„ 

2a To explore the effects of malignant disease on cellular 
and humoral factors of resistance. 

Methods g Case Material - All patients on the Chemotherapy 
Service are potential candidates for these studies « 

Laboratory - The pathological, serological and bac- 
teriological, biochemical methods are standard except as specifically 
qualified below. 

Major Findings: 

lo Acute Leukemia 

Ow: previous s£«adies have indicated that (a) antibody response 
is normal or at best, only slightly subnormal. In patients with acute 
leukemia, (b) the phagocytic ability of polymorphonuclear leukocytes 
is normal in such patients and (c) a marked fall in the absolute poly- 
morphonuclear leukocyte usually precedes infection. Current studies 
using the Rebuck skin-wiadow technique indicate that at sites of ex» 
perimental inflammation patients with acute leukemia (a) frequently 
do not show polymorphonuclear leukocyte mobilization (b) that this 
ability to mobilize leukocytes correlates highly with susceptibility 
to bacterial infection and (c) that ability to mobilize lesakocytes 
correlates moderately v;ith the circulating polymorphonuclear cell 
count. 



Part B included Yes 



827 



Serial No. NCI 5»737 (c) 



2o Chronic lymphocytic leukemia 

■The study reported in part in the £958 Project Report has been 
completed. The results are as follows: (a) lypoganBnaglobulinemia is 
frequent and tends to be progressive, (b) Response to antigenic stimu- 
lation is depressedo The degree of depression correlates well with 
susceptibility to infection and moderately v;ell with the ganma globulin 
levels (c) Isohemagglutinin levels are nsarkedly depressed, (d) Delayed 
skin sensitivity is normals 

"Siough a retrospective study tended to implicate prednisone in 
infections ia patients V7ith CLL, a prospective controlled study of 
prednisone has indicated that; (a) the improvement in well-being and 
rapid and often complete decrease in organ and lymph node size occars 
with the use of prednisone, the WEC usually increases. This seemingly 
discordant effect is unexplained „ (b) pradnison® did not increase sus- 
ceptibility to infection in this group of patients. 

3. Fever of Undetermined Origin in Neoplastic Disease 

A retrospective and prospective study of such fevers has revealed 
the following: (a) Viral, bacterial and fungus infection is not presents 
(b) Tetracycline, as compared to a placebo, did not influence the course 
of the fever, (c) Tetracycline adversely affected bacterial flora, par- 
ticularly as regards resistant staphylococci and monilia. (d) Bacterial 
infections occurring during therapy were somewhat more frequent and more 
severe in patients receiving tetracycline. 

Attempts were made to demonstrate circulating endogeno%iis pyrogen 
in man, 300 c.c, of plasxaa were taken from patients with fever of 39.5° 
C and returned to them later during afebrile periods. Three such patients 
were studied, two with fever of malignant disease and one with a fever 
produced by exogenous pyrogen. We were unable to demonstrate a circulating 
endogenous pyrogen. 

4. Specific Infection 

Pseudomonas and clostridial infections have been described (See 
previous progrsss reports) 
(a) Moniliasis. A prospective study has indicated that 

(1) Thrush occurred in 157. of the patients 

(2) 31% of the episodes of thrush begin during the last 8 weeks 
of life 

(3) Antibioties and corticosteroids contribute to susceptibility to 
monillal infection but are relatively less important than the 
severity of the disease. 

(4) Nystatin mouth wash (as compared £o a placebo) significantly de- 
creased the duration of the thrush. 

(b) Cryptococcosis. Disseminated cryptococcus infection with extensive 
hepatic involvement (not previoasly described) in the almost complete 
absence of host cellsslar response has occurred in 3 patients vjith 
lymphoma. Biese cases are under study. 



82B 



- 3 - 

5o Host response in patients with Hodgkins disease 

Early and late patients with Hodgkins disease are being sfcudied 
as regards 

(a) Circulating antibody response 

(b) Delayed skin sensitivity 

(c) Skin homograft response , . , ^ ,. , ^ ^ *■ 

The interrelatioaships of these tferee factors of host defense as 

well as their correlation with stage, extent and duration of diseases sus- 
ceptibility to infection and prognosis are under atudyo 

Lcance to Cancer Research 



Aside from importance in the clinical management of patients with 
cancer, the effect of cancer on the host has broad implications as regards 
the nature of the neoplastic process. 

Proposed Course of Project 

Most of the above work is clinical with application of standardised 
laboratory techaiqsieso Ihe clinical work will continue = With th& rapid 
expansion of laboratory techniques and approaches to psoblesas relaeing 
to feost defense (as represented at KCI by Fahey, Landy and others) it 
becomes imperative that they be applied to patients. 



829 



Serial Mo. 101-5-737(0 



Individxial Project Report 
Calendar Year 1959 



Part B. Honors, A'ita.rds, and Publications 



lo Forlmer, C. I., Frei III, E,, Edgcomb, Jo H., and Utz, J, P« 
Pseudomonas septicemia; Observations on twenty= three casaso 
Am. J. M@d, 25(6) 677-889, 1958, 

2. Boggs, D» Bo, Frei III, Ec, Ehoffias, L, D. Clostridial gas 
gangrene and septicemia in four patients with leukemia o 
lo lo J, Msd, 259; 1255=1258, 19580 

3o Aronson, So Bo, and Shaw, K. Corneal Crystals in 2i.fultiple 
syelomao AcMoA. Arch Ophth. 6I? 5in-5l!.6, 1959. 



830 



SmrUl Noo KGI- 1-223 (e) 
lo GMB 
PHS-MIH 2o CP & ST 

Xndividtial Prsjecfe Report 3, Bethesda 

Calendar Year .1959 



Pare A. 



Projeeg Tielas Peaetrseioa ©f Antl-'feiaor Dctags.lnte® th& 
■ Csafcsal Nervous Sysfeess 

Prisieipal lavesglgefeors J3m^id Po Sallg C&srles Go Z«sbr@d 

Gooper@£te§ Ufeltsg Nose 

Msa ^ears (ealeEdar yssr 1959)? Pafciea£ Days (ealendar year 1959)? 
Totals 2 1/2 

Professloaals 1 
0£fe®rs 11/2. 

Frojscte l>eseripfeicfE8g 

^Jl£l^2£° *® ®bjec£i%'es of eisis project are t© qwanfetfy 

fefe® pesseeraeioa of eerfeaia aaei^-tiaaor dmgs iat® 
She cea£rsl aer^CRsa syse«si (GSS) SBd fea aefeiMpt 

to ©lueidate sc@e of tSie factors inf lf£@s&ei^ '£!j@ reslatioaskip 

bataesa 4&&& of dnsg^ plasma le^wale, askd coaegatratisa of dr^g 

in tke CNSo 

Bfetjfegagg t^ phsa^acalogie feee&aiee d®=?el<^®d assd walidateed ia 
'~''"°''*"~" prsviotas ysars w©?e used la e&ese BtMdi©6» 

Ma^jor, Ftodimss ffe® stodies Chis y®ar progressed slaag £lir@® 
Iifi@8S (l).ae£@^ts t& iMd e©ffidlgi#Qs ehat 
a leer efea bisod CSF barrier j> (2) a££®^es eo d«lineacei t&e 

charast^risstea.ef dmgs wfelefe fail t® eaCsr CSF (3> a££^^es 
eo @,xteskd £hs sttsdy of dste blood CSF barrier tn vzaxnc 

(1) Arfearisl kypoteuatmi and aeeta c©reiEoa@ _a&biais£raeies failed 
fea ffllfesr dr^ aaery iiat© CSFo ^a blaod CSP b'arriarj ©fisagssnad 

by ©5sr e@efeEiq©@Ss, is iiataet ta S^-ze-hmsr old dogs« 

(2) Smdiss Is €ha dogfish dmoiaserseed east salfsailife aeidj men 
afeer 96 fesrarsa falls, to eaear CSF la sjore efefsa ersee quantities ^ 
Paraslaekipp^srle aeldj so eSi® oefeer feaadg if gi^raa £3®©a will 
aehsl'sr® diffaslesi eqisllibritaB b&ewe^a blo®d aad CSFo Ifes half«tl^@ 
for fcfels aqailibriM la 50 femsrso Ufe&e«ig& sulfassllie seid is 

of tens glireia a dissQssiatioa c^staat of 3a2 f®r the firse hydr®g@a9 
i€ iSs isffi faeEg a seroag aeid asid gfee pK of 3,2 refsrs e@ ^e 
dissocisfeloa ©f e&ffl secsad feydrogaa, Parssteolaj^ptariss aeid is a 
Snse w@sfe orgassie ele€£roly.£es aad ate«2£ «sk© parfe Ira 6€C>0 1& 
%m£iss&&lsitm€ at besdy pHo 



Psr'g B l^lud©i? tlS 



83. 



=» 2 » 



l^e ei^Bp£ri@on b@t«3'esai these Cera eesi^GiBids indieates eha£ 
tmdis@oei&£ed dnss »i1^^ -peaeei:a€a in£9 die splsial f iuid| 

ha^e.-wrnxg ia ^l£e of edsaqtsate elmsg disseeiaEed ecaBpouRds^ 

sulfsBilic scid far issssasiceis t?ill £i&e eiiig@r spinal fluid isa 

more t&ass trace qusistltiasu thus the ao&ioa th&t £he bl@od 

CS? barrier is lipoldal isa naetsreg aad eossequently is persseabl® 

to usidi£sociag@dg bug is^@x@&able eo disse&isCed drug is sersBgf±iehedc 

(3) S&i;idies sa the blood CSF barrligr in 16 pafeieats bx-^m beem 

ccffiple£sd' eissis year. ^Sipyrimes s^lfadi®aisaeg aad parassiao 

hipptsrat@ have beess £l&e test eostpmrndso CSF «ras sera^lsd frs@ £he 

1%^'bar areas ias^ead of the eistaraal area as in the dogo '^^eae 

studies h&ve shessffi that dse barrier is esseatially the saaie ia 

@aa &e ia tt^e dog^ the slBgle diffsreose noted has been that 

tl^e rate of approach to eqnilibrita is miseh slower in eian„ 

Tke half'<giss!:s@ f@r both ag&tipyrine end stslfadiazine are aboistt 

5 fold larger in man as e^^ared »l€h the dog, 

Sif^ifieaag? fc® Cr^Bear Resaarehg tmioxa, both prfiaary and 

metastasie, frequs&tly de^®!^ 
. «riehin ths central ner^otss 
syst(^. Little isfos^atloa is a^^ailable in the published literamr@ 
eon€@miug the pene&ratiem of an^i^tt^^r dnsigs Int© th@ CNS» 
Basle research «ai tbtis aspect ©f the phanaaeolc^y of aatl-=t«^or 
drugs is @eeess@ry far th(t ratioiiral use of these agents im ^@ 
treateaae ®§ ■^attesita with tiMors losaeed in and &zmzkd fete 
braisio 

Pros^Qssd Cegrgeg fh© followlag lines of io^estigaglon are plaaaedg 

lo Farther &tte^^e £o elsjieidatis sfsm of gh@ basis phasma^i^logie 
factors t$hieh da^ssmiae both hs^ mm^^ and h&v fast dnsgs geg 
into the 6HS and GS? f£'<aa pla^sa. Biese lastede eerebral blisod 

flo^a state- ©£ thyrald fisEetiesj ete. 

2o G&&zimx&d inr^estigatioa of the eea^ara^l'^e phassasieolosy «sf CHS 
and CSFo ffea te«lasieal ease with wfeieh the ^entrieular f Itsid 

and brai® ar® obtained ia dogfish sake this species parties isrly 
¥altsable» 

3c Studies iK pafeiesaEffl with saitebl© n®C|>lafflis will b@ etaafein^sais, 
and extended m isselude the us© of aBti<=t^or dnsgSo 



4. Studies with c<ss^mmd9 ststgh ae gltacose tiliieh Bnter bralss and 
CSF by etereo«>8peeifis transport eeehani^So 



832 



Serial NOo K^2-l-223<e) 



PHS^NIH 
Individual Pr@jee£ Report 
Calendar Year 1959 



pare _Bc 



Ralls 0° Pos SfeabaoaiSs Jc Ro aad Ztsbrsd^ C„ G, 
Ltstrlbnttmi oi Dn^gs Between Blood as&d Csrebrospinal 
Fluids General Methodology and Effeee of pH Gradien&o 
J, Ssamacolo 125? 185»1©3, 1959« 

Zssferads Co G»6 aad Rallg D» P. Disgrib«£io» of Drtsgs 
Betwe®!^ Bl3>s»d aad Cerebrospinal Fltsid in ttLfk Varloi^s 
Vsrfeebraee ClasseSo J. Pfesrmeol. I25s 193»1998 lf59o 

StabanaUs ^^ ^^a VJarr^Qg Ko S» arad Rail;, So Pa 
Ef£ee£ of pH Gradisiste (m £he Dis^rlbfiglosi of -taiGnla 
Bee«e@n Blood smd tesele Brsia and €er@broi^inal Flt£ldo 
Jc Clio. lawseigatioQo 38s 373»383o 1959 » 



833 



CalsadajfYear 1959 



Psrfe Ac 



ProJ<a££ Titles f^^s Pkansaeelagy of tetittSE©? Ageags R^lafe^d 

C#gp€!raeing Sales g Nsn® 

Ifea Y®ars (eaieadar year lf59)s 
Totals 1/3 

C5tli8rs 

Objeefeiyass fo stu4^ tfee fata snd ia;@£ab«ii^ ©f cercain an£i° 
etffii0f agrafes ®£ ehe pmrlsjie gy©!^ ei£rff@n€ly ia tsee., 

^efeodss Sfiaada^d e&esalesl ®red bi®e&^5leal tmthoda were e®pl9y®do 

'if&?@ f@%s:ui t0 @^p@?£ eh@ pre'Hotss reported findings. 

Zo ffeffi psagtrsgio® of §HP t&rmjjgh f=h® blood«GSF barrier in thm 

dog after sa l<,Vc iaj®efei©n ®£ efe® dr^g was d^soaseratod 
spseferopboe^M-strieally by aa«^iaiEg tfee. yltra'violfee 
absorpeloa sp^tra of the GSF ss^lss obtaiesed by eisteriial 
p«me-£'ar®o tfe® id^seiey ©f tfee dnsg ia da© CSF was e@nflm€d 
by paiJar eliri^agograpfeya 

3„ 5^'aiourle ©eld was pr^ared f®r sasysae iahibigi®® sfcudiesc. 



SJiMJllj|gl^t^,go..CgafeQ.g,,,Rf8®ag,fcfeg fea jsesidies of the ®@tab9lle 

fae^ ®f azieigts£!j3r agents ar@ 

sueh seuill@3 e^^ld elsrsm ^on eh@ m@d@ o£ &£ei@n i»£ £h@s@ agSB^e, 
l^rfe&erg ^lis tasdissi^s of, gfe® dg^el^^safe @f drag reslsgaoe^ ess Id 
els® b^ pareiy elmgldaisd tferasagh efes ®atab®lie e&Bdi«9» 

ESmSM^-^MSsm g Ceagism® efe@ isalstim sssd id@Btififeaeim ©£ all 
jseEsb^llees ®f^lF arsd €® riboside is baeh jr#ss>@ssi';?« ead r<&sietaBe 

Pare B inel«£d@dg fSS 83 -j 



titer Iffil Nc 
FHS-MIH 

G&lmtd&r Ye£.r 1959 



Tmt Bs 



S®fio3 8Xs 3039 (I95f)„ 






I. Gm 

pas-NiH 2„ CP & Sf 

Csi^da^ Y®ar 1959 



Pas-e Ao 



Pr^jeet fifeles f&a MseaboliM of Folle Aeid Aneagmisieso 

Frincipal Isweseigaeorss Ti Li Lea aad Visseaatt To Oll'^erio 

Otkar lBvas£lga£orss Koae 

Cooperating 'laies 2 Ce&motfeerapy Ser^leSg GMBg NGI 

Man Years (celesdar year 1959) s 
totals 4 2/3 

Profgssi^als 1 2/3 
Ot&ars 3 

Pr®Jaee Deacsriptii^i? 

MflfeMss Stassdard eh^lgal fiad bi^k^sieal Bee^ddsg ineltsding 

la fhm sxeree£ioa ®£ ffi«thotr@sag® (MKt) .and dlehlarraaefeko^ 
6rejiaste (BG^) at va?ie«E® pl'^s by ^arl^ss Bol??«B!fea was 
smdi^do tt was fois&d tliag b@th dcugs ecasld be @zt:fa6t@d 

qssaat^toti^gly tat© »'^b*jesaoi fra® a pH 3 btsffer a®lmtim^ 

2. fl&e £|s^lie®£i0a of i<93i'=>®^.!!iaiag@ elasraraaeagragihy t© fe&a 

aepsyseioa aad isol@fel@a af ^arieiss fsilie aeld derl<tfa£i^@s 
was iasaas4i?€ly la-s'^seigseedo Ife was dise®<-^&red t&ae tk%sm 
&i^®%mds^isnld ba et^tr^e^ii&t&ely fraetisssated and ptsrifisd 
by t&@ ts8@ of a eelluloss loa^o^s^haage syattma Hixeu?@s 
©f el©s®ly >r©ls£@d f®lie 'seid deri'sratl'resj, 8V®s eh®ae 
dlff©?ias ofsly by oae fimecloaal groy^g w@r@ effi®i®aely 
reaal^®d tetd eket? separaee ee^o-a^tso Sl&is prseedur® 
a£f @r4s f ©li@ seld aaesgoalsts @f a hig&@7 pt^iey thassi ; 
liiEk@?es> aelsieveda Several pa|>er ekrsEBatagraphle sysfeosa 
V&T& also d^v@l@p^ £@r £hs eepaffatlisn @f etese f(3lie agid 
•affi£ag@ai8ts» 

Psse B te©leds^g HO 



836 



Serial Ho. WCI-l-226(c) 



tn 0«« «xg€r^«nfe; the ab©w; ffi,f.fes5>i>lic*. wa® idfwfeiil^id s« 

•- llt%'--apm^ph^tmek^xii:&liy diiimmit fxm. 3% 5^-dlgfe't9r@= 
falie aeid wag i8@iag«,<i= Simils.rl3?a fsllowisg ferasas^t 
wiefe DeMs M99Kgfeang®d drug (lOZ @f das®) ikmI a iE©«eafe®lle« 

eaidase failed fe© fiai^ro'yfe tfe® r®€s?f«ry sif oaefesnged dTtsg 
effid sg€«b®lle€c. H© reds^eftd K-M's«sl®g(3^s to fsli^te ffi€ld 
was is3let«d ffsiB feh® eirln® pf ©ie® i«je€e@d wiife SCMc Chily 
tfeg un£liafsg«d dnsg (10% ©f d»g#) was r«#®^erabl.@ frsaa t&@ 

4c. In ©OT!nee€i@n wifefe fefee smdy s»f fefee assfeod for Am d@fe«mto~ 
a^lsB ®f €i*® f®Ue «eld ae.>g«g<SBi®6g„ eh® ©xldstis^. ©f »:^ 
by p@£a8SiiaB. pewEaaganata vss is6i?®8£ig£fe«do At |>H 8 6k@ 
p*®d«£@t was 284^di«K^®°6<=f g@rtd&a@ ©eufbssEyli® ©eidp efes 
8gr^eetgr# ©f wfei@li wag @®tabllgited by 0g^wi§« fefdrs'lytie 
deaffiinaeie^ 6® ih® 2a4'^dSliydra(sy ©^.^oitead, and elg® b^p ss. 

Si^iftcmfeg m Cam&^KmmAT^% ffe® §t^t®s sf fefe® !aE.ffieab@li55 fa's® 

b«.€a««® @f the lighfe g^^eh seisdi^s 
€(9»gld throw «^sa £&e m^m gf aeel,#a @f tfetse agmatSa FtErtfeft^Tr tfe© 
rggffllts @f s^fe s^diss ®ay §!«r^^ as mt tetellig@a€ gssid® t® s;fc« 

Proposed egiajse? 

is@lafel®n and ideratlf leafel^si @f ti*« ^tfeaboliets @£ KCH. 
CssseiBtsm sisllar w®^k im MZo 

fe@ 8fc«sdy ehe tmtstl drag balasie^c 

3o Ap^ly ®ie?®bi@l©gieal t®€tei«si K^s 6^®l«afe<g cte atti^ifey 

@f"eaeh iE«tab©lie^. wieh r#spsee t® eh« aefeiou @f fslte ss£d. 



837 



lo cm 

FHS"KIH 2o CP & ET 

Isidtvldw^l Prajeee Report 3o BetbesdA 

Gfflleadar Y«fe©r 1^59 



?att A<, 



Project Ti&las ^tchaaiss of Mel^issor A££i®a of Pnsia£ Ma logs 



Kan Years (eaiesdair ymv 1959) § 
Toeals 2 
Pr©f esaloaal s I 
OtSsesrs I . 

Projeee Bsaeripfciwis 

pb|eefciyegs fhe objseti's'e of £&is projeet is £® defeetEsiaa aa 

sp&eifieally as possibl© %iiiigh bloehemle&l reaetimis 
@r@ inklb&eed by certaia purine axi&logs ia eanesir 

cells £has are sstisi^lve' t& th&s^ ee^cmade. A eosrollary obJ®e£i'^e 

is t& eltseldat® the eteehaais^s responsible f@r ii^er^ig and aeqtsired 

regisea^ee £@ eiiaee ageaCa. 

Bfe^thods s Radioactive pnslnes, phosphorus aad pisriae asialogs wstv® 
e^loysd with b&th isseact ©alls aad eatsysis pr^aragiona 
t@ st«3dy tha effaces &£ t&a drtsgs i^mi imslsie aeid petaboliss in 
seii3iti'«'@ and rasistanc si^use aad himim letskfisiaea 
A raathod® logical advanes was esade is £he dsf^alopseat of a ratio 
raeordi^ talgra ^isXet ^<}nitor for tsse in cdbatst ekri^Eographyo 
This permit £@d detest ioa of &im:^ms&&a slisCed £r«^ ion eselaaiigtg 
coltssQ&s by gradi^i£ techniques despite ths prssessse 9i changiog 
ultra vi@l@t absorpei<s» isatriasie £0 tha foraie aeid elisaago 

Ms4®y .f^^?° ^® ^m:k initiated laafe y®ar ©a 4"sssla©pyras©l©« 
(3;4<^d> pyrtesidina emsabolifia ia @ssl was €^ii'" 

eluded asd is bsiios published (2>. 

S&t£dies eS tha ^echaai^ ®f aetisn @£ 6«^ereapt^tsri»ei (^^) 1^ 
LI2I0 l@jsk(miffi wsr® ©st^ad^ t® eoafi^s th& fissdtogs reporeed 
last yaaro this ££;beMded dsing does rseponee studies with b^th 
saiiaitiv® aQd rsslstaat L1210 ee;lls ^s m®&sm:& th& @£f@eSs &f 
graded ^mmttB ei 6MP v^m, the ^eilissti^ of hyp®KSBthii$@ for 
ad^iylie aad guaaylie agid syafefegsis^ All ssfeilisafeiisia ©f &yp®« 
saathia® was pr©f«sadly islnibitsd hfv^sf l<m e^aessaeratioas ©f 
<5» ia elm "resiseaat" e®ilB ^^11® ealy ntiliz&tim for ad^ylie 



Part B isEledeis 'yES 

833 



Serial NOo S8SI-1-22? 
w 2 " 



acid moieties was affeceed in ehe"8e2isi£iv®" eells rnitil high 

ccneeaerasi^&s of 6MP w@r© s^icjysdc This indicates a block in tdi® 

e@avsFSiesa of ££(@sinie &mtd to &d@&yX±e acido tt^ bass ®ol@ey 

o£ the int^^i^e^dise® adsnylestsesiraie a© id was synthseixsd \mt 

daspiCe eh@ use @f ^i@ as « igafrisr ie was bo6 posslbla £d dmtesmixss. 

£hs sl£e ®£ -li^Sie blo-ek @©r@ pr®eis@Iyo A fijial r^osfe af all ®f 

these &MP s»d£@s is beiag published (3) a 

Studies hs^e b®€Si iBi£iated to d@g@miia@ ^egher ehe aeehaaisiss 

of 6M? aetlissi aad rssisganea deteimisiigd iM £h® aoiss^ are appllsabl® 

£0 feasaa leisk®ffii®o Pok this va>rk $FS' was sysjcliagisffid wieh rsdic-^ 

setiva sisifssro Use ©f this saterial wi£h i€s iahgrene high 

aaslytieal seusiei'^ity has neeessieafced eli® develop®<mt of new 

teehaiqoes for Isolaeloa of mesabsliees ©f ghe dnsge ffeis w©sk 

is is pifagrssso 

S^lggjJie^g® ^?:j>^teag@g_ R^ g SfflB® ©£ the Eose effect!'?® aa£l» 

ttmor ageaSs £h«s far diseo^eyed 
are pniina analogs a A knowledge 

o£ ehe stschaniss-s o£ thslr aetioa a^d ^s sr@asoas £07 resistance t& 

their actisr^ slhcmld e<3Btribu£e to tha rational d&^elopmtmt of m&XB 

effective aatitimies' chmotfeerapy„ 

pgeposad Gotsrs®? 

lo Coiitisuatioa of effoi'ts to deterssisa the ^echaaisias of aetioa 
and resistas^e® to 6M? in htssas letiki^ia eellsc 



2.. Sti£dies of efea aaetabolim of 5MP in 

So Fiirthsr efforts to dafia^ precisely the si^ifieant bi@sh®5^i@ai 

a©tl©a of 65IP re^ssssibie f@r iea ^tl£<®^y eetl^ity ia 1,1210 



833 



Serial NOo iSI-l'»227 



Calendar ?sar 1959 



Part B , 



Fublieael@as other than abstrases in 1959 aecrlbutftble t® 
ehls projeets 

Naehaa, Do G»g Ds^idseag Jo D= g Waggeaers, Jo Go | esd Berlin^) 

N. Z. fhe Cisnmsieg 3f Ba?i«M Gfirbotsats is. & Liquid Seistillagl^i 

Sp«€€rose£ero Jc Lab. & CliOc md. 52s 915=917, 195Sa 

(Ifeia V7a8 rep®re«d as "In Press" las£ year) 

Shawj, R„ Ko^ Sh>£M@S£, Ra N, | Davids^ig J, D. | Rail, D, P^ | a&d 
FS-eig Eo Stales with ^a ^sperimantal Mtietasor A^&^tg 4«teln&« 
pyrazol0(394"d)pyrlBiidiaeo Gaaeers ia press 



SaTideoa^ J. S„ Sadies oa £he l&cltaraiai of Mti@a @£ 6»Hsr@8pt<»» 
p@r£a@ £b Ssasi^ive aad ResietanS L1210 Le^emla in VlerOo Casaeer 
Research, ia press "'""" 



8H0 



Ic GMB 

FHS-NIH 2<, CP & EE 

lodl'y'idtsal l^ojeee Repmt 3o Be£li@sda 

CsleRdsr Year 1959 

Part A. 

Project Tl£l®g Phat»a£@logy of Ribofls'^ln Analogue 

Principal laaveeeigatsrs ftoieagu® tsm& 

Othsr iB.'Vssfcigators g Koa© . 

C.oop®ra£iBg Uaifess N<?3S,e 

Maa Years (ca leader year 1S59) g 
Totals 1.0 
Professioaals 0»5 

Cthari 0„5 

Projeee Descripeioas 

_^jee£^5£2 k. - Cliaiealo ^o iadtie© esaior regressiem and /or' 
slinleal riboflavia def teleaey ia patients wieh 
aeoplds^im dissasss shrsugh the tsee of vtboiX&vtn 

So La boragory o to sfeisdy eha meehaniffia of aetioa of 
riboflairin fioalogues at she ecll^lar le^el aad eh@ 

traaeporig of flavla eiuelsotldes iisto eells, 

jfet^odsg A„ Clialeal, Ph^e^aglteorffiBatryj eferaas^grsphyp 

bio-aesay aad Isoee^e (Fe^.®) techniques are ?as®d. 

B«» LabQratpryo Spaeg^r^pfeofejaastryo blc^as^ayg' 
is0£ape aad exts^e eeslmiqtsss are uiasd. 



miMjMl^s? 



^° gl^ggl* J£ has b©ea possible for e!i^ firsfe etea 
e© isiduc© ribof lavia defieieaey wieh a rlboflavia 
a!a£&s«Kslse in 2 pstleats wlgh tEe^^lasels dissaees es^d 
the fi«disigs ha^« b^a de^^as&sated phote^raphiealSy ead 
bis€hCTieaIly. H@ dafiaite m&M&tlan of sff^es @n 
gt@or gx&'^th C(f3«sld be @ad©o' 

®° iLabera!i;@r^, A nisaber of riboflavin asal©gj2©ffl fea^® 
bste ees£ed i&r Elieir iEblbi£©ry effeee «m grcswda &£ 
L. easel in ehs ^ressaca ^£ riboflavin aad ies a«a@l©®'? 
eids8< Various ae£i«3a pa&£ems were observed toe 

definite i!Bfor@ael@si e<m@@rQist3 £h0 si€es of a^gag^ism 
was a©t da-^®li^ed. Cell ®a£ry afeMdless, aeill Isi ps^gresse 
feav© 182^ leaded £!sae f lavia ad^aia® dimjelffioeida probably 
©a£ers Lo eas®l as aa iataet e©1®«sI©o 



8^11 



serial NOo M2I° 1-229 (e) 

-:. 2 = 



;igni£ieaace to^^Caneer Reeearehg 

Ao Cllni&ala Riboflavin analogues have been shiswQ to 
isakiblt rae CtEsor grts^elio Tiee developaenc &£ a regtee^ 
whieh will result in eliniesl riboflavin deficiency may 
pemi£ the e<«)'alua£lQsi oi ghe effects of this deficiency 
oa the growth of tuaors Iss aaoo Gosbisiation chenotherapy 
studies say also be feasible a Ira additioa^ a system is 
presided for the further elueidatimi of the physiological 
role of rlbo£la<^in in msoAc 



Bo toboratoryp Aa «mdersea&disig of the Beehaaisai of 
action of riboflavin antageaists i^ay aid in the d@siga 
of more potent ehoBotherap^gtlc agents <> 



^^B3^J^£it° 



Ao Clinical C<^feiaaisd cliaical erials with gal&^t&Slmta. 
and detailed sttsdies of amy antitumor eff^ts and bio» 
chemical changes will be m^d^rtfiken., 

^^ l^bpratpry. Further sttidies &£ cell @ntry and &i 
isolated ensysie syetess will be otade. 



842 



FHS^NIH 
iKai^idsaai Projeeft Ripest 
Gal®adar Year 1959 



PffiTE^o 



yeMa.rm&&&lc»^y mnd Cliaieal Trial of th^ Rthmil&vin<& M&1^^'.&.^ 
f-6538„ Jo Hs£o Canesr las£, 22? 346-361b 1959o 



8i|3 



ladi^ld^l Project Report 3 = 

Cal^dar Year 1959 



partjio 

Frajsct Titles Stsdies on fetrssyeliaesg 

So Ch^iesI DetezsiaatioB of fetrscyeliaes ia 
?issu@3 aad fisstse Fluids 

ISo Tisssie Distribstiim ©£ Tetraeye lilies ia 

Asissthi&tisad Dogs. 

ISXo lataractiotts ©f Tetrseyellaes witfe garbitBirisseSp 

(Hiis is & ea&tiem&tijs^ oi.wosk previo«isly r@p@rt<s^ 
3££ds? 'ih% ^itlag "^luGroeltrostt Bfetabolim"o) 

Frfsicipal Isi^sstigseor? K«r£ W= Kt&a 

m& Years {eeleailar year 1959)? 
Totals 2 2/3 
Frofeasimsls I 1/3 

Others I 1/3 

Frejeet ©escripslmig 

!o fG d@vei«^ a sietliod for the 4et^@iaatlc^ of tetrasyelinss 
£b tisi^ses, ^icb ;d>o»ld b® isdsp^deat of tl^e problems ls» 
hssTfiat ia bloaasayso 

XI» To determine fcfee volisiss of distribation, plasma proteiss 
blading, and axteat ©f eatry lafc© spiasi flwidj red blood 
calls, sad skelstal a^scle of tetreeycliae &nd d<mst&yl^l&T'- 
t&tr&&j^lisi® i& anesthettsed doge. . 

HI. Is s£sgi% ths biadiag of tetraeyeliaes to ^11 ^©IscuIqSs 
niseleie aeids» amd prstaiso 

1= Sol^^t ®str«stioag flsjor^istry 

II o ffes tetraeyeiiaes ware deter@iK®d by £l«sorc§s®£ry ©f 

their et^leses with csleitaa and bsrbitalj, afggr ssgraegioa 
isitQ ©thyl ssetatas as desss-lbad :la Fart lo Fla^ss protsia 
bisdi^ -KSs detemiaad by a isedifi©ffl£i®a ®f tk® tssssal 
eq?3ilibrl5s^ dialysis ©©tliodso A small voisms @£ lestmle 
sslise in s ^11 bgg is dialysed agsia©t spprosfeately 10 

Fsrt B iaeli2&ds MO ^^ ^ 



serial NOo !Cl"l»23i(e) 
» 2 ° 

sld@ aad Oistsld^ £k« bag is ae&ieved vlth a Elleing shek®r^ 
and ^isllibsiea is @stabll@h^ wiehin 4 lumrso Itte plasna is, 
el^asg l&eld at isore aearly physiologisel sondlgions than in the 

<ss«sal met^odao 

Giiateasxy phaxssaeol^ie t@£hsilqi2«s vers oehert^is^ %3s@do 

IISo Di8ferib«s£ioa b®€we®s few©- liquid ph«8«So 

Flssoresesraee and ffll^ra^lolee absorption spe^trosc^yo 
Eq«siiibriisa dialysis o 

Ic (Wie& DTo Loo) 

fetresyelln® ^^s foimd £9 be extra€e8bl« logo sthyl aeetate 
in d&@ presence of calci^^ ions and barbital b^fsr^ (^ar 
6&S optim&l pK reage of 8.5='9»08 a distribueioa ratio of 
apprcfxismtmlj 2Ssl in tawos of t&a orgfi&ie phasa »ss obs^r^ed. 
ffee ®tErseei<m did not take pla^e if either Csi^ or barbital 
war® OTsitted fr^ the systessi aor ewild Ca"*"^ b@ r^plaeed by 
^-^-^, or barbital by other bisfferao "Sse extraetlod wag 
attribtsted to the fonaatiost of aa imeharged aol«e!islar e<a^l<gK 
€6asisti2^ of tetraeyeliaeg Ga-'^*, aad barbital„ fh« ecsBplex 
is strozigly ihmre&^sa&t aod ean b® leeeasisred ia eh« «thyl 
aeetate phaa@ in eo^eeatrations as loa as lO^^M. ttstheds 
;?«re readily daveloped for detesminations over the ue^sal 
therapeutic raags of e<meentv&ttcae ±n plaaaa^ red eellSs 
spioal f itsid, aariaes aad bile. A.tta^t8 to apply tho aethod 
to tissues were at first tmsueeesefislo Ihis v/aa belie9-«i to 
bfi diae t& the pr^seaea of phosphates f^iehg tmd@r the alkalin® 
eORditioas of tha «xtra€t£os» font elaei«aa pho^hate pree£pi» 
tafeess which adsorb tetrseyeliaeo th& difficulty was finally 
o9«r€@ie by the tsse of Pb4^ to precipitate phosphatss at pHSc 
Ixeess ?&M- v£s thos praeipitatod vlth XO^^g th@ latter did 
aot interfere with the estraetioa step., Hiis modified raethod 
v&a s«eeese£«illy applied to deterrs^agioas in skeletal amsel^^ 
li^^ kidney, ead 'Cmor tisa^iesc fkm dr^ was^ ia ea@h e&a& 
extrae^d frca the tiss^ h<»oge&ates with dilute EC I vith 
reeo<9eri«8 of 80<»ieo%c l&e blank readings were g@an@rally 
^ery loi?^ and no signif least false posit I've reaetions h&v& 
h&m icmde tkm aethod appears to be highly speeifie for 
certain tetraeys lines. S<iseh asall changes in ^e @ol@s^l# .: 
o€€i8r ia 4=^i<-s desdlseehylaaiao^e sad anhydra«t®eracj^lisi- i... 
preheat or grestly redssee, the fonsatioa of estraetable 
fluorese®kfe €<sipl«ses imder the eonditiesas ^l@y@io lhe> 
method has also b®®a siseeessffelly ^s®d to des®mia® d€i\ 
ehlor^-tetraeyeliae Sm elssssas md bed^ ilisidso Little ..^t., 
'^as d«3s« rjitk ehlor-aad oky-='t&trm.:^linm bees^s© eg' tasir 

845 



serial Hoo i^Iol«231(©) 

.= 3 c 

Il„ (With Dfo asll) 

Plasss €«»Ke«»£rafeiona of teerae^lia® <W) aad d^g«feiiy%° ; 
§hlesmttmy^liM% (I^T)^ after slngl® lo-^. do8«Sg folleweia 
fir8fe-=>order disa^peeraaee eisr««8 aftes 2. holers following^ -driia 
s«l»liniEt?fitioac f^ plassa feslf^^eiaees were 3c 1-4.4 hoisrs for 
IC aad 6»14 h«sas« for KiST (ranges for 3 dogs oa eaefe dusg). 
Eserapolfttloa eo s«ro &isa^ of £h« lin«££ s@ei°lGgAri€limiis 
ploes» pe£@i££®d iket €ai€«£latio^ o£ voliaaea of disferll»stlon| 
£faesft «ssfe 0,5&-'O.64 1/fcg for TE and lol«lo4 1/kg (llfeisrs 
p®r kg body weiglafe) for I^St (rasxges for 3 dogs tm eaeb dr*sg)c 
PUma'prstffiia biadiKg smdies^ by eq«illbri*8ffi dialysis at 
370b • isadiesSftd 50£ bisjdiag of KCT and approxisaately 35% btsd- 
ing of TCc la Cezfiifi of d£® sereia eone@n£r&fcloas of fr@e dnsg^ 
th« ^ol«a6«s of dlstribtstioa w«r«» therefor^s, approsifflaeely 
0„94 aad 2„5 1/kg for TC aad i^STa respcefeivelyo lti«»€ 
rssffilss iiidieat® tlm« DICf and, to a leeeer @K£ei!&e» IS are 
■ e®a€«aerae«d ia sorae ^sfera-srass^lar ee^artaettt of Eh« bodyc 



'^ia piefcure was 8«ippor£ed by seaa«gres@nes of ecmeentratlone 

in skeletal sascl*, ahiefeg affeer 6 krsog were lo2-l,5 and 
2o3»2<,5 tisms the (total) pl&m& eoafieaeratieas for fG aad 
IM^ X9.s^mntw&ly„ In sesb® of £hese ms^&rissmit&s- ^onegaaS 
ia^ra^^iKBeaes iaf^ssioas of dr«sg wer® gl^'ca^ ia addleioiai to a 
iosdias dos®s ^ order to saiae&ia relatively emistant plasesa 
levels o 

D^lB^ tSss ec^rse of thea& e}{p«rlsiesi6s, b@£h drugs ^tered ehe 
spinal flteid ia eoae^ttraeiotts eqval Co approxlsae@Iy 20% of 
the etm&esitz&tioaa it®e ia plasffifto Sq^ilibration foeeee^ 
plssSM a»d ^iasl fl«sid was probably so& achieved^ 0ad 
e3s^srlsa«ats are ia progress to dofeftsatiae the e^nesi&grasicm' 
ratios afe«r allotting several days for eqtaillbration. 

eoasidarable momsits of these dnags vere fouad Co b® 
aaeoeiaeed vi£h the red blood eella. Eq«sllibraeioa b®ew««o 
red eolls md plaaaa took pla«« within IS rainutes in vitro^ 
and tha r&ee of dnsg disappearanes frora red sella elosely 
followed the plasasa diaappearasee eisrves in vlw>o Becaase 
of variability in the red eell«to«>plasisa eoneentra^iffia ratios 
ia different esq»«rlBea£88 the effaeta of (Ca-H-> aad pH «r@re 
st^i^ds ttsiag waaheS hissan red sella s^ap^ided ia iaotmie 
aaliiM. tt v&s io&A that a 2«fold and 4»fold &me&R.tT&ti.tgn 
In red eella took place in the ease of TC and IMlf^ respestiTc' 
at pH7o At pH 6o5°7 = 5s Ca-H- (ta eoaeeatratisne €«^parable tc- 
those fonad physlologieally) ssark^ly reduced the amount e£ 
either drug associated with eella, wfeilSe at pH 7«8 and abm^.!, 
a second affect of Cffi4-»- bee^e apparent ^ieh resKlfead ia 
increasing the ascmnts of drug botsnd to the sells o Altkoisgb. 
the interpretation of these results is E®t yet ©lesr^ tb-- 
p®iat e^t ^k& i^sreanee of eonslderii^ di^'g-aleat isetal i: 
ia trying t® ^der stand ^he di8£rib*gtioa ©f these drsggs. 



846 



Serial Ko. !ICS«l<»231(c) 

a, 4 - 



IIIo ^« forsta£i«m of eas^l&ssaB of £e£rseyelia@ (SC) wi£& Ca^- 

aa<d otties di'^alssjl: metals ia well teowio C(^»laKaticn ^it& 
barbitisr&Sss £:ad ^tth p^dlk@£«me8 has sot been previcmsly 

described &o o^ kaof^Ii^ge. Distrlb%iCiot& b@£«?@«a!i etkyl 

fo? s^dyls^ ehsss ec^^le^gs. IC^ soiEssesitraeieas vers &&%&£<=■ 
sisisd ia ©a«& fikas®, af£®r eqisilibrseioa, ff«s efes romslta 
of tfeese s£s34i®s^ it «m® iraferrad ehae '^ fosais aa iisloaiaed 
em^lss \iith CsrH- &M barbitals Similar e^^leses ife?6 
ebser'^d ^l^ Zs-H- ssde £o a mssk lesssff estraitg wi£fe E%-H-» 
tet l&^H- fail©4 e® fom extrsc table c<^iexsSo Ktmobarbital 
asdi f>eiis@barbli:ail ^&t& saord potos^S IJusa bsrbie&l ia cs^sisg 
©jsts-aeaisa. Mefefeyl ssrabssifeasiea ©£ «ae of £&@ niferegeas i® 
&h« barbitrsrsse ssel^sa did la&t eteag® its poeaa^y iss ekis 
regards b®£ siabsfeiesSiea of both Blerog®ffis rsadered £lia 
moi@eale iu@.Tto DiphssylliySsiatoia as<l oxasolidiaedioaes bad 
ao e«^l.©s^f©2»isg a€£ivl£y» aor did a ^arlaty of othe? 
X£ @£is later foC;.S t;tot s«:@3 p^&ik&to&ms also fo£^ @stj?s£t&ble 
cfi^lesss wiafe 1€ assd Sa++o Cylle p^diketoness iJKmevsrs w@pe 
eff@@S;iv#s indisatiag tbat a ge<»striciiil £om able £& ehelag® 
is ret^ired^ esiJ @@ggeati!^ tkat &^6 sdttare of t^ biadiag is 
diff®sr«!at taaa ^ith barbieasssaso 

Att€@p£s were @sd@ to d&test d&^^es ii£ ^Itsoreseasies sss.^ 
tsl£r&<^-^i@l@g &b@ofp€l(His tshiek sigSst a&ees^smy coag>iex&tlo&o 
An iaersjsee ia fl^^oKsse^iC® iat^sity ^«%s soted ie eqia«@t3s 
sol^i:ioas cgferitetsble to th» fosssfeiea of a barbital emi^le^„ 
Ho slsBiil®Bs.t cbdages in &bosspti<m sp^t^a or s^ti^fil dmislty 
«€:?e obssr^^s &ow®^eif, sugg@sti^ thst th® b&rbigssFatie st&j 
ao£ bisd go tks dsrcsofjbosfie par£ of ^« tetraeyeiia© soleeKle, 

Eqssilibris^ dialysis ss^ies ifsr® earried o^t to d@c©ssise 
shstka? te£7&£yeliii&8 biad to m;ia«£l®ic e&M& sjsd proteins o ^se 
sio&t strike ressalts ^ere obtsisad %;it:h BM„ Biisdisig ^as 
obsesrred ia t&© preseaes of Ca-H-j, %i+£, or Za++o Thesre isfss a© 
biadiag is the ab@@&€& of div&lesat @@tal iosis, H@st d©m£ts33rati@^. 
of mk isssrsaasd its ability to bisd tetracycliaeso There was 
iB^sli less bisdiEg t© SHA= Bsese s®n^ albiaain bo«md t@tra== 
eyeiiSiS t® a ^all eat^t. 'Sae degre® of bisdiag ^aa greatly 
tosreased by Z8H-f-» ijsst a©t by &H-?- ©sr l%++o 

SigBif i^ffee^ £©^^ pss£&s^^ i@@aarefeg Istrseyeliaes eahibit tte ability 

t© semeostrafc® sel^ti^mly ia 
esrtais tissjaes,, iaclsdiag saB& 
tim&s cells o '^is biaidiag lasy be & to&l for 5s^r«wiag ^ tet©w«» 
lodg® of tl^e es^o^itioi or stnse^srs of ttsi^r e@lls «mde^ ^^rio^s 
coadltioaso 

Ae imd@£-staadiss.g ©f tfea fsetoxrs ©lsle.k go^csna the €®ll?sl3r dis-* 
tributioa ©f ^ass dregs ®sy ba of gems&l vsl®s ia eh^sogfeairapmtie 
probl^iSc fk© tstjfasyelisaas ar® al@o'cf iHterest b^sissm of tk^iz 
aatibiotie py^ss-eieso ll^sidsti^a ©£ e&® s^^ssi^ is t&ls iastssee 
of seltsetive esl'lslax- essielty wsEsld b@ ©f vital int®x®Bto 
•ffiie frssest sS^iss ©a tke e^^leasiag ^r^a^ti®© of tets'aeyeilass sxs 
|jrellaisa?i&s ©M®d st ^i®§@ g^alsu 

8i|7 



serial H©c NCI- 1-231 (e) 

» 5 » 

frt^o&M €^xm g fssreher ^joirk ea th& biadSag of fceferfieyellaes is 

cells is plsasi©*!, iav©lt?ii^ 

(@) ia ^1^0 stodiss of dresg Sis^rilMtioss is eis8!s<ss £b dogs md 

(b) @mdi@@ of bl^clistg to red eallSg ssd eismi;© h(Mogeaag«s tmdsr 
va^f isig eeaiiSltioiis of pH snd di^^l^it sstal iem eaoeesaerati^m. 
(e) qmseitustiv® 6tMi9s @f bisdins t® ptsrlf led tUm& etm^mmts^ 

(d) cytesk^ieal ®mdi®@ «m e&e e^^osieiraa of £fe® fhmsme^t 
Baeesrial ^ieh psrsises In ei^ors &St&s g@£r@eyelin@ 



8i|8 



S^isl Ho, iCI- 1-232 

la (^B 

PHS-NIH 2„ CP & ET 

Xadi^idisei Project RaporC 3o Betlaesda 

Caleadar Tear 1959 



Par£ A. 



Projsee Tielas Phasaseology of Dnags Influsaeing ehe Respmiae 

e@ Radii^li3@tie Drugs and Radlatiem 

Principal la-g'estigater s ^rgar®e Go Kelly 

Other Iav®s£iga£orss Rsger Wo O^GarSa Tl Li IsOO 

Coapex'selng Bsiess EKI Laboratory of Pathology 

Man fears (calendar y«ar 1959) s 
Totals 2 
Profasslosals 1 
oeliers I 

Project DeaerlpeiQQS 

,^J©g^«s s f © se^dy she mechaatsa of proteefel're ef f eefe ©£ ■ 

AST oa t^i&ity of aierogera emseard (HK2) ia 
^osn^al aad £«^sr^b@ariag alee and raSSa 
to detessiisa l^alisag&on of S»35 asd C^14 lebelUd AST 
in tissues aad viehia eells by a!»plleetioa of eoeoradiisgras 
seetmiqufiSs msA tQ es^ar® this with distrlbuCioB ®f o^er 
s^lflaydry (g^i^cssdso 

T© evaluate proe^^iv* eff^t agaiasg radlaffiteeeie drugs aad 
radiasloa of oth@r el^eses of €^ss99^mds(in addition So mareepts^ 
alkylgtsaaldiaes) aad to as^Ior@ possible advaafiage of ec^bioaei^ms 
of pr@£ffisCi^e ffigeafeso 

^ehods s Ustsal ^fearsseo logic ^ h^aeologie, eheasiea]^ biologies, 
histe^agholc^ic; sad radioaetlTft Craeer te£liaiq«s@So 

lo Preereaeasae with AlSt hsd no affeee tm tha desroasa is 
pari|»heral blood e^si^C produeed by mil in aersal siee^ ra&e^ aad 
dogao Sa rats b&ariss subeataaaoua Is^laaes of Duaning le^aksaiSj, 
eka assoelatsd alsar^ iiigreasa i® per£|^eral leukoeyee ewmfe was 
pravanted or ^icMlsed e@ aboue tlaa esse degree in raes pratreae^ 
with ASf as is T&ta gi^'ea HH2 ealyi 

2» Sfea Esisaiag loQk^ia la rats is K©re saaaiei'^e to aitrsg^i 
E^seard therapy tfeaa assy of ehe 0®©rs ia saiiea ^Uh wa ha-^a usad» 
Aa M&zQ&Q® isk ^s^lv&l of rafes beariag e&is e^^nr ean ba obeaisi^ 
ag relafel^ely aKi">eosl.e d«se Is^ala ®f HHZg ^ss pefflslesiag a 
sere prsels® ^almei^a af'assy dose redaeeim effeet ©f prseraafemt 

P£r6 B .ia^lsdedg fSS 

849 



Serial N©. 1-^1-1-232 ■ 

» 2 - 

with P^o So i&x ss Q.SXI be j'i^ged ff<a@ asaainatioa ^f swt9l'u&l 
dasa (ata£is£ieai evalmitioB has aofe beaa sade as yefe)^ AST 
dass not si^laise £he tissor^daaagins s££@e£ of io^ dosss o£ UK29 
asd at higber dose l«vsl9 vf RN2s, p;@£rea^eag with AST raeialte in 
a tk£r^@u£ie advaag&ge by pr^eecelsi^ die host £r@ss &hs e^£ie 
@f£ee£8 of siierogen Tss&st&x6o 

3. 3o Tolersace go tbft letiiallgy o£ A3gT eotsld aee b@ d@a£@ns£?a£ed 

ia ^ig@ after rs^eaged dailyp ©^(sry 2 dsySg ®y ^^@kly a&sinisesaeis® 
. o£ ^(^^letiial d@ses of AEfo 

4o Saa O'Gsrs (I,ab<, Paefeo) for resaits @f eistoradioarapMe sfeSdy 
^ifih S-35 sad c«i^«lab@led ^ff ead g&imsorees md S-35„tabelM 

5, (With Dr, Lpo) smdles ha'^e bssa smarted eo d^ter^ioe die 
eff&ce iSQ £dKi&lty sttd ttmot d^^ging aeei^a e£ ?adic^ia@£ie dr^s 
£Qd TsAt&tto&f, oi es^esmds ^iefe geaieraba fre^ radicsiso fd 
dssss £^-b«£yl perb^jseatSj dt«£^b«fcyi perosid&g b«is©y.l p®r®»id@8 
s@d a bydrss^Ia&iiig resgeat eo^osed of forsofsa sulfaee^ hydrog^r^ 
psrasidsg aseorble ecidg and SOT A ha-?;© be@i tssesd agaicst HN2»> 
Tfee hydrosylatiag sreageae did oofc decrease eh® tOKleity of HN2 
asd say ha^e saereassd i£o ffee ©fees' 3 eeapoisidsa however » gaire 
effaeeive pro^eceicsi against aa LD30 ot HN2 vih&a glvaa 30 to 60 
aiit^ses bsfor® ths sltrogeQ su8S&?do 

Slgnlfig aKce to Ca a s ay Researdt g. C&^ileal prote££l3n against 
sys£e^ie £osleiSy of alkylaeis^ agents @r ?adia£iaa vl£hotag 

cffi^arable -psotmittsxi of ttmor against eareiaoelastls a@£iora 
wmsld r@ai£l£ ia a £l&®7^®u£le ad'9an£ag®o 

pg^<^08ad^£ei£gs@^of Sfead^g Co^pasaetva sssedy of asiaary eaerefeism 

prodt£c£s of 6^14^ labeled ASf £a dogs sod ia sise ead ra€a Isa ^se 

h<^@ of gs££lsig @sm@ iafossatl^a or tsdaerBtassdisg of £li@ fmd@rlyi@g 

basis 3>f the sps^i^s differsoee ia toslel^y^ 

To sss^less £h# study &£ eeilolar dis€rib6Si@a ®i l&b^lsd ASf iia 

@ie® by u&d ®f s£igroaTs£®?£di^?^Sc 

fo e??al!2aee e&® prseeeit^e effeefe oq tssiaity asid ^Bs6r<°d^ssigisg 

a@£l@a 0£ mU aad radia^loa of £r@e radieal geataratiag a 

©iEgly &n4 ia ceasbiaaeioa &-iieh MT, 



850 



Serial N©o NGI<^1°232 



PES-NIH 
Indi<^id?2al Project Reporfe 
Calendar Year 1959 



■\c£ioas of Sj, 2e>a2iRoee;hylispehl!ur(5alt«gi Br^ HBR (ABr}o 
Tosieity aad P?o£ee£i'^e Sffecfe ©gainst Nitregea SSasfcard 
Tojcieiejs by KargaraC Go Kellyg Dsvid Po Rallg Glsns E. 
TxttsiTs, Roger W„ O^Gsraj aad Char las Gp Zubrodg 
Jo Phanso & Ess^ ISier. la press » 



85: 



1. GHB 

PHS'^'NIH 2„ CP & If 

Individisai Project Report 3o Betfeesd® 

Cal<mdar Year 1959 



Part: Ao 



Projeee TiSlea Cliasic®! Car«siiaog<saesis 

Piriacipal SEvesEigaeos-ss Margaret Go K©liy aad Roger Wo O'Gara 

Coopez-afciag toifees Lsb„ Patfeolegy^ ISl 

m.n Years (ealeadar year 1959) s 
Toesis 1.0 
Professionals 1/2 
GChers 1/2 

Projeefe Dascripfeioas 

O bl^gi^fg sg To deeessiiss fefee effse£ ©f careinogejaie hydx:oearb(sms 

Mefetodas ©ssisl biolegie aad felse©paefeoi®gie tffiebaiq4i@a» 

lo We h&'^e &miiitfmd the results r^oresd last: year em. the 
indisfitioa of hmg. ttmavs in Gensral Piarpos® (Swiss) ale® follouiag 
sisbes£s58je©U8 inJ^eeiSKi of R@wb©ms wish eiefeer OaOS ajg ®f l92j,50 
6«dibsasaia£hrseen« or Ool ®g ©f 20«raa£feylcliolaa£br€ne in ©llvs oilo 
E^aa eotssideyably l«3^°sr dose ls^<S:le ehan efeosd r@port«d &h<mm 
prbdtss© s high iaeld^e© of snaleipls lisag ttmor® in fehis sfersia, 
\f£ar s«bea£aaeoas iajaceisa of aitber Oo02 ©g of dlbesjsaafeferaeaa® 
or 0o03 @ag of me^ylcholsaehrene fes» aswbsrsi Gasseral P*irpos@ mieag 
EUielplQ Imig adaa«Mas were foaad in 50-807„ of th® sateals killed 
in 8 wesksg and in 90^1007. ©f €fe® Miis:als. killed ia 16 we^So Two 
lower doss levels of dibaaz®n€hrae^i® (0.006? asid OoQ022 ®g) in-^. 
dac®d Itmg e^oro is 25% of £fea aafeala killed ia 8 weaksa End la 
55aad 33% r®s|i®6S:li?aly, of those kilUd U Ih weeks, K lo»®r 
dose of a&thyleholaaehren® (0,01 mg) iadueed lisssg t^ato£-s is 25% 
of fe&e ®ie© killed In 8 we^s, 67% of ehosa killsd £a 16 we@ks^ 
and 100% ®f ehos® klll^d^in 24 waekso la gsnsrsls ehe iaeideac® 
of @ies with Imig ad^smias snS th@ aasber of £ismors per affeeissd 
ECUS® ifssreesad direetly as fefe© dos® was iaereased and el^ Eima 
bgfor® sserifiee h®@ l@isgfch®a®sl„ 

la additie® £o fibyosara^ssa (eoeal iaeld^ea c®.. 10% ag 16 *je^«>a 
's^ia oEh@r paefeelogie lesisas hava bissa ©bsej^jrsgdo Four ®le®, gi'^®a 
©ef&yI(eholaffi£l3a'@a® sabsweaa^msly as js^sfb^ras dwels^ed i©t^®sla 
(is 9^129 14s ffisd 21 *?a@kss r&si5©eelv€ly) j, I d«^®lop@d a ehygsia 



Pare B iaeltideds 130 g52 



serial N©c 1131° I- 233 
o 2 - 

l>'s^fef^ias i had a lesisn of the paaCKeas„ (Me of the alee ^tvmi 
dibez^san'chracsne devdiopad an intradttggal papill«saa of £hs m^sasr; 
gli^nd. HiseoliosiG se^ei'S^s of mlee killed Afcer 21 we^s £r@ aet 
yee available' f@? s&tsdyg ead ^11 ba rsporSed lager, 

2. Aa K?sltaa&i®a has been reads of £&© eusc^sptibiliey of aewbi&m 
C3H and €<»57ii aiea t£» stsb«£!Uta@o@U8 a<^iiiistra£i9a @£ earelsegsas 
bssi£us@ adtsls iaii^s of tbese seraisss ars rela£i^9«ly r@sl8gaa£ e@ 

Issjg fefflior l^dias£l«ra by isferaTeis®Bs &®'sr©ll ag subesat 
a&iaistragiQa sf cayeiasgenle feydyeea^beas. 



50 aa«»bora C3H ®i@® gl'^^a 0^06 lag dibsaEsaeferae^oe sisbeiataaemsslys 
hmg tumors &sr@ i^stmd is 12% 'df lOi® @iea killed in 16 ^saks^ in , 
44% ae 18-23 v&dks^ ffisd iia ?4% of Cfee asies killad ia 24«27 w©efc8o • 
Th& iaeidetted of subeueaaeo^ss gusores in this strain was 24?ec, ^^^ 
eariieat £tsior beiag &@t©d in 8 ^@akSo Os&e mea&& had eaearrsets 
o£ both eyssa 

Of 30 ssewbom G3H ate® gi'^aa Ool @g ssthyleholaathrsne subet^taaam^sly^ 
hsns tamot were fous&d ia 33% of aiee killed ia 16 wesks^ aed S3% . 
of thase kill0d iij 24^28 weeks. The iasldsaee of -subeutanec^s 
£miors was 40%. Fo^ir ssiee had pepillc^tas of th® skiits &b^ 2 had 



f^ 



>57BL Meeg 

(le limg t^isor nodule was fo^d ia 2 of 22 @iee killed in 12 
to 24 wa^s afger 0a06 og dib^axasudarae^sae and ia ncme ai 17 miea 
killed Iza 12'»27 wesks after a@£hyleh<>laB€h7eaCo the iaeid^ea of 
s@b<gts£aQe«m8 txm&sa has been 10% is the dibeasaaChragene gT9&p tmd 
35% in Che siachylehdlaaChrana grotsp^o \ddiei(mal pathologic lesions 
ha^e d<£val<qpad ia ehs ssice gives EadChyleholanthreaes one mouse had 
a thyiEeaaa^ and aaoeher had lauk^ic is^laaCs in limgSs thysius li^«r^ 
splesB l^^h aedes» aad kidney. In 5 of 10 aiee raaiaiaiag 27 vsdes 
afieer iajsesicm of s^ehycholaathraaa^ a dsforaity &£ the ehsat has 
raeeatly beas^ no£ed« the xiphoid proeeas is protuberaaeg and th^ 
s£erasM is depressed aad e^nea'sre. 

3« A c^aparisoa has b@e^ made of ehe rala&i'^e st£s@epeibiliey eo 

Ifsng t&&ox iadtsffitioa of sai^.e injected as aewboms^ and ©f sales in<° 

Jeeted &t Ig^a end 6 ^^^s of agSo Gsaeral P«erp®s@ @iee wer@ issed 

in this study g and were gi^ten eithsr 0c06 isg dib^nzanthraeena of 

@.l mg i&ethyleholaathrsne subeutanemstsly. Eight weeks after in<^ 

jeetloss 6f elghsf £ar@isa@seng die insidenee of Itsag tssaors was 

abo^t 50% la th® @@«fb«mss and 10»20% ia Che anj^als iM$m.t&d &t 

ls3s or 6 weeks of agSo By U ws^Ss the insid^ea ia the 1 w®^ 

old grm&|» was 6@^arabl@ to thas in newborns (86% aad 83%f, r@«psggi^@ly| 

while the ln(sid«n@e ia ^e older age grasps was still low.. By 

24 weekSg all of the miee injeeted at 1 week of age had l^sng ^mats^ 

while ia those iajeeted at 3 waeka of age^ 17% of the dibeaseat^aeeae 

groi^g and a^® of t^'se gi'^en sss£hyl@h0aaghrea@ had dssfslep^ 

l@as ^SBorso £@ additles^a ^@ aessbsr of tisstore p@r aff@eted aosse 

d®er@S8ad as the ag® &t 5sfeieh the earelaogen was gl^^a was ia» 

&ress@d(awrag@ of l<°3 psr affseted isotsse in the 3 and $ weeks of 



a. 3 ^ 

4;, ££fes£ o£ Intraperltc&eal adbiaistraclen of eh&se cereiaagmi^ 
lias bees sSudied, Mewbora General Pisfpose jsiee were injegted 
incrsp@rit£mseil7 wieh either 0,015 sg of dibens^aehraesn® ®f 
0„02S and 0„03 sag of meshylekolsaehrsne eoaeaised in Oo05 aal o£ 
olive oilo Oaa ©£ 3 isiee killed 24 weeks^ and Jl of 34 killad 
be£f^m 35 asd 42 w&eka had i^leipl® hm^ eimorso In addlfeioag 
3 of ths miee killed in 41 t»s«ik8 hsd h^atessas^, 1 had a heraanlgi^raaa 
of Che liters 2 had hspatle eyses^ 2 h»ad remal'e^aes,, ons had a. 
lasion @f thm pero£ld gla^d^ another @ papllleaas of the skiig:, and 
ehs ovariaa eissu® of 3 miea was largely destroyed and e&@ o=yrery 
filled with large ehaRneis dilaesd \^ik blood o 

Sigaif ieataes ^to^^Caaeer^^^ Ressarehg *Sse grieaeer sssseepCibilifey of 

aewborra asd very youag suekliag 
Esiee Co chsaical earei&ogeQs saay be of is^or£aii€@ in seteisg safe 
standards for €h@ ^i^sae papislatioa.. Furehertsorss eh@ ase of 
QSt^borQS offers tk& possibility of a aor@ sensitive sys£«^ with 
iihich to -deeersisa ehe careiaogeaieifey of food additi-^ssg efeCo 
Careisogea imduced Ctaeors ^ould be isseful isi eh^ioCherapy s^udieso 

.^c^osed Coarse of the Proj^ees 

To es-slisaeffi she susesptibility of sewborsa mi€.@. tm a few ©th@r 
eareia©geaSo 

To d@t@ssai£i® «^t@£her sulraisissal do3@ l^vsls of 2 or mors eareSsiogene 

h£-^s aa addieive eff ese aad wo<£ld r@@«sl£ ist lung 6i£ffior iadi£€£i@i% 
ia as'^bom ©ieoo 



fo es^i^se ehe possibility of usiiig; Ci^ors iradueed i^ ne^borsi 

as a todl. in evalua^iag the relael'^s an^i'^^eussor aeei^ity of dnsgsi 
of iaearess in eaassr eheaogherapy. 

To d«t@s@ins ehe ®££eet of eareindgeas ac^ixiisesrsd to pregimssg 
Eile@ by iaersveit^ss or ia£rfi^erie®»eal rcsste m& £h£ ineidsnes of 
£tira3irs in th@ir pr&geay« 



854 



serial N©o JC2" 1^234 (©) 
lo am 

Isdiyldu:al Ps-oject Report 3o B^thesda 

Cal^dasr Y«ar 1959 



Par£ Ao 



Project Tiele? Slasraeeology o£ Alkylaeing Agsnts 

Pjfiacipal Xc^estigaSsrs Moaeagaa Lane 
Otfeer Isi^ssgigaE'©rss None 

Ifen Years (ealssdar yesr 1959); 
Tofealg 2„0 
Profeseisssals Oo5 
oeherg 1„5 

Pr®Jeet Deseripeioas 

^° jLajbpgategyo To study die phaxsjaeologieal eff®e£s ^id 
&ni.t"timoT activity of various alkylating ageaCs in sies 
and rats bearisg as&ablishsd timers so as t<s develop ie^^ 
Sormsttloa afeich mighe faellitae® the elisieal ferial of 
new agaatso 

B= Gli?iie@la A^ Tq sKteis eteas® as@at8 to prftlissioary 
pharisaeologisal £ri£l is paeieats wltb ad'iiressead a@^la@tl@ 
diseases. 

Bp ?o eorrelats tks offsets ®£ alkylating 
ags^gs cm £err^in@ti<es xsi^ '^arloss h^cag^logie parasE@gssr^<, 

^^^^ ' ' 

Aa Laba>ragary« Usual pharmaeolegles heaaafealogiej bielogie ead 
liieei§pa4hol«gie e®ehniqu«9o 

B» Cl^tc&l. P^3di9fie€i'^@ iron traeer smdies (i&@i®di£Eig 
astarasl body eeimeiag) j, e&^ieai degfirmissa&i^aas ©f irosSe 
and usiial elijsleal eeelmiqu@3c 



}^SE. 






CQsieity of eyel^haspfesBid® has bis@n defined is 
ai€e assd ratSo Calika HN2, tfea dnag had little sffess @a 
gassr&^istt^stissal epithelii^.p It inv^^ed a ieark@d @@ga'^ 
karycs^yte hyperplasia Sa- e&€ splsea aad b<ma& sarroa 6® 10 
days eftsr adteialatraeioa. fl&a IJ>SO f «>r ssi@e ^se ebm£.t 
425 @g/kg and f®r rata was 150 tsg/kg. 

2. Cyel^li9^&ao£id@ has b&m. s^im& to ba ^ore @f£ee£ive 
thaa other alkyla^isg agssgs aad ssthotrssate Ik^ prol^^isg 
^9 gssrvi<&al of ®iee wiefe ad^?siaeed L-1210 les^mlBo It ®aa 



Fart B iselsda^s 'tlS 

855 



serial no„ s?ci-I-«Z34(e) 
<. 2 - 

lim of 1^^1210 l«ka®ia (afeieh Is slso 6»KP rssiatanfc).- 
3, The ag^jt has been sbowa £o bs esasidfirably more effeeSi^a 
on s ©see wsekiy tvB&tm&&t scfeeduls t&an on other reglmai^So 
4., VJhan given ok such & sehedal®, eyelophosph^ide has been 
3h&sm.t:a ^sresdMC® masked laersasss ia s^r^^lval of animals bea7c:> 
isag i?@ll ss!:3.blf.s!b@d s:mo7s(l3^i^h«^a 2^2» earelacsBa 24i^§e 
Ssmsaiiag i®i^^.^eis ^d ^uanisg lymhossse^m} , and £o b® far 
©©re Mi&ettw® tks& slerogea sasfcardo 

lo 4 study ef sSae p&a^seolegy ©f saracil saastard. ijas e^^iaeed 
is C90|>@?aei^ ^itli Brs. Hall ai^d Coleky of the Eastern Solid 
Te^or GM^s©f:&israpy Sro?!^. ffee design of ihta &txsdy was based 
^<m is£e»raai:i@fs prs^ioosly obt:aiQ«d ia aaimal aad cllai£sl 
studies a^ MSIo fS£9 aystffiale eoxieity of oral dosas of lessscil 
sisstard ia aiaa was foaad to bs stellar eo tfeat of intre^eacms 
&SSS of ai£r®g«gs ^sa&arde i.Q,„ aa^ses^ -ipjffixi&iags assorssla 
and bsas sasrr®» degraression, f&e follo^iag doses ware found £© 
ba tolerate f ©r § w®s&s witli eaial^al eoaieifcys 0,015 a^/kg 
dsilye ®.2 sg/kg esiee we«Bkly| 0.45 ®g/kg as a aiagle doses 
la this stmt^s objeeti^a tssker regr®ssi®i was setm in s®§® 
ps£leHfes' '^th Hodgkia*s dis<aas©3 l:^sj|5laossrceffiSa «ad &hz<Ml& 

2o Fsrselsiaeeie ssadies ware eesi^Ietsd ia or@ pattm^t assd 
ar© ia progress ia a s^s^d patiaaSo IfpS^k® of F©S§ by r®d 
cells was afeewa £o b« Is^airsd 4 hoars af Eer £ dose of nitvegmt 

B iijslf iegiaga to. , Sgg§er_lg .s^reh s 

^' IS^SES^Z" ^•' ^® 3C?2diea ^i£t| Israeli mastsrd «sad i?ig;ls 

eyffil«3^l£os|!)fe8mide &SV® ^>hasig©d tfe® ^ortasc® of s^aliaati^ 
£fee psrfss^ssea of drugs ia aaissl tanor syst^ss c® tli© basis 
of cpti^£!is se&ed^ales of tr@at^@8it. 

2, Sie resislts of. the sfesdies with c^lop&osph«®ids 
i® soasa lei;&^is gav« €ii€ourag®ssa£ fc© dia evalKstlen ©f t&i® 
slkylstiiai sgeat is adslt aad chlldh^ h%®as leiskiSaias, is ^iefe 
€<^ditlo(sa It h&s prodiseed s^s« "e.^^l©t© r<2sissi^is"o 

3o l^e coffisistsffltly grisaeer &nti'^ f,mQis ©ff^fe© 
©bteiasd 0i£& iESss^icteat trefitessat is aaisjsls has provided a 
patterts for tfee desiga ©£ cliaieai progr^s ^lc& c^^sr© £Sis 
affects of differeat se&sdsslee,. 

S" SU^Eli- •^° ^® q^salitael's?® sad qsiaatitaSi^s aspects ©£ &Ue 
toKicity- of a ch^ss-tkerapeatic egeafe must be dsses^teed pries- 
to estsEsi^© elislesl £rial„ 

2o a® f©rr®kismeic stsidles ssy prs^id® issii^&t: isto 
elae avola£loia of &a®s£©logie elaaa§®s wfeiek are iadseed by 
alkfiaeisg ag®at,s» 



856 



- 3 - 



sb^^mm£S^sm^' 



^' toboraaogyo e®^ietiosi of se««diss of ®ffa«fcs ©f 

B» Siigtiealc Cfi®£iam£loa ©f £sxir®kiR«t:lc sisudies 
c^urr'Sffifely^ progress ^£h nitr<og@£i snsstard sad oSIsiSe: 



857 



ssrisi woo miL'='i'^^ji^{&} 



€sl®sdS3f Year 1959 



<cyelopfe©spl3s®ide). Cancer Ckmo^stas^j Reports^ No., 3^ 1-^6^ 
1959 u 

ales &k! ale® -^& h-^llia Im^mU. J, Ha£„ €m&®t I®s£o 23^ 

Lssi®, M.s ■&© sati!;?ffi©^ activity ®f 5«bis»(2»-eliloiro®£lsyl)» 



858 



lo (5M8 

Xndivldtsal Projeet Report 3., -i 

Calendar t@ar 1959 



^ggrt^A, 



Projeee Titles Preelinieal Texieelogy of Dr^s Poeeatially 
I73«£«sl la Caneer Chiemothdrapy 

Priseipal lR-«^eseigae«rs David Po Rail 

Oeher lE'^esfeigstorss Msrgeret C Kally 

CooperatiEg Ussitss CCNSC » NGI 

Hazleton Laborateries^ Falls Ghurehj, Va^ 

Maa Years (csleadar year 1959) 
Total 1/3 

Oeher 

Project DeseripSions 

Obieetiveag Tfee objecti'^'es of ehis project ar« t© study, in detail 
ehd quant leati's^e end qtsali^aei^^® aspseCs of Che 
tiosisl^y 'of drugs potentially useftsl in caaeer Cheso° 

therapy in arder that nev drugs assay r^idly b^e safely be brmsgfet 

!£p to dia p&iat of elinieal trialo 

Metfeedsg Staiidard Sexlgologleal methods are oaaplsyedo 

I^jor FindlagBg fhs studies this year Tofi^^ be«n eone^raad ul^ 
gySoiUiRe a a®» alkylating agcnt^ de^asr^e^topurlaeo 4 esis&pyrasol® 

pyriraidines eitrogsn oasstard^ a ecmblnagicMi of various d@ses of ^ 
ra@reaptop^rine and asetk^gerinj and aetinflayelng as well as furtixer 
sttsdies s» deri-^^aeiT^s of agse^hopterin and its hal@gsaag®d dari^eti'^?®^ 
and £»?3. S€«idi4i8 osi the effeet of age and s^soa the tOKl@ity @f 
S'^TS ware axfceadsd, Gas^ratioct ©f obele rats ineraas@d their 
stsss^tibility t® E»73 5^10 fold and brms^t it iat<» the rasigs @f 
noxtsal fs^alsa fhere was no dlffer^siee in i»73 toxicity b&&(;»e!t 
(Bale aad fisssale dogs, the pt2!a@nary lesissk obesr^ed iQ patimt@ 
who ree^i'^md E-^IS was r^rodtsc«d ia th@ d.sgso Sttsdies @£ ssigrasgosj 
misstard and e£, 6>^MP e^saf irt^ed in g«as?al previously r®e<3rd@d 
t92cisologi€aI datao Of partieislar if^terest is th@ fiadiag that 
6<^a7eapt<^uriise in the d@g ixieraases senaa transasiijc^s® aegi'^lty 
at sisb-ls^ial levels „ 

the d^ssaistratiiea of the eff@fetlvea«ss of €ffe©saa against r@4©ne 
eiasors whea this alkylatisag ageat was giv^ea at widely epaeed 
internals by Dr. Lane ©f this Serviea Isd t® a study of t^ 
toxieol^gy of oj^w^m. using wldlsy spae@d dsse §€had@la:s in d€>g'0 
and rats, Ifee sd^attEage^ in tersas ©f adaiiaistrati^s ©f ®Qir® drag^^ 
®f tl^ less freqisaat dr^g a^^inistratioa sehadulss was elsas-ly 
showoo Of partieislsr iatarests also, ia th« poselbiiiey tllat' ia 
th® rat l©-.i4 d®ys follawisag a. stgb-|®gfeal dose ©f eyt®sffla (aat^ alas 
^^Sfe©pmi?i«) ^k@g-® Is a p@irt©d of d@«rpag®a m^m^tihillxj £© ife© 

Pffirfe B iaelisded? "^S 



- 2 o 

£fe® le&hal aetioa of th& driBg in (jisesfeioBo Such aa ob®®s^seisa 
amild serife as ^& basis for an expIaK&tioa of th%. iiserea@@d 
£&srs|sei2eie bigaefie @f cerfcaia dose sehedulea« 

One poeenti&ily Istporesnt smdy wi£h ds& ^sssthop£erin deri'^a£i'v-@s 
has b@@sj £b3 cosparietm o£ £be toxlei^y of ehtts® sgtmts «h<m gi^te 
by & caast&nt ira£u@itm m'er « 48 hour period sgainsg r@p<iae(Sd 
dally &(teini@sratloao In dogs £he ce^stAiae 48»h^£r infusion is 
more £9£l@ ^mn is daily a«ipinls£rael<^ for 14 daye« 

Si,galf iggGge.'fee „C^,si^,sr Reseagebg %© dr?ags cjsrreistly «ss@fttl ia the 
treafeaaae of htsaffla tseoplassia tsjisfe be gi'«^®i at nm&v tojsle dase© ea 
aehie-sr® fcters^assie srasial£s« flierefore ev^» eaall dif£®rffiaa«s ia 
tosici&y rslsged eo sesj, sge^ rot££e ®f adQiiai8tra£losi £i?.d doss 
gchaduia^ saja be of eozislderabl® £ffi|>@rtaQee in eliaiesi igheaot^erapy 
@iBce tl3i@ ^sy lead to larger doses safely gi^en to mmi. Isti&ssmtimi 
fox presllaieal eoacicology is quickly aade available t& the 
Cooperaeii;® Giroups of fcis© OTNSCo 

Proposed^^ Coarss g It Is plamsed thafe ghis pr©j®e£ will bs GsaSiaaed 
aloag the sajse lia@3 as ab©^®., Drugs skcwlssg prosise Ik BTo 
Goldias qissEfeiCatlve aalmal tmtoT sEadiesj or drugs obfeaimed fri« 
0€HS€ will be pu£ into tht& toxieologieal appraisal before d®^ 
elding can th& dosSg rcmte asid sehedule of adsiais&raei^m ia aaisto 



860 



Serial No. KCI-l---:i35 



ladividttssl Ps-ojeefc Rep©rE 
C«l(mdsr Yaar 1959 



Pare Bo 



Sha^s Ro Ko| SfealmsHj Ro N»'g ©a^idsoa^ Jo Do| Ralls Do Peg aad 



861 



Part ko 



Serial fe, KI- 1*236 
PHS-NIH £^ @!B' ■ 

Ual^adar^f^ar 1959 3, Bethesda 






Ms© \'@.srs (eslasidaig- yaar IS 59)8 
Oe%«r g 

^J^tl^gss T© jsbs^rys iff tsfee fej!»p.®£s:fr" "'■^^^'' '"•'■ ''='=2® felgfid is 

.131 ^«.. - i »!«.*' ^ 



'ih% saallgoasfey is fefeaged sad tfefe ^sfcl^tgs sr^ «aM%tabslie tfe@ 

Ms|0ff ^,giadiag8 g tfes st^i^s are ia piregjrsgs and gisffieiesne 
dafea are a®e y«£ a^^ailablgo 



sf an-allt- ' - - lease ss£|mr' 

ia fe,yg(£EfelsyT#idl«^g ass ls^s!s£igaci©6Ti tet© it;© affeefe s«Js Si^ ' 
pa,TSBm.t@SB wfelefe hyper thys'si Id las is teowa e© ir ''^,-; 3-1.5 v :*, 
pssffitfe a beee^r ^sadeTsCaadiag of this t^tsb^ii; 

a©©t^elafc®d_ 



hfrj%2ta:r la ard«»3f £® estsblisli f?hfife ^fe<s ife§tegiq®*£ ££€••' 



862 



Serial No. KGI- !-23? 
lo GMB 
PHS-NIH 2„ a? & Ef 

1i^ii7id«8al Proje€£ Report 3o . Bethesda 

Calsaidar Year 1959 



ia££l" 



Pi-oJecS ?i£l@S Bio'i!a@ai€sl and Pfeffis^Eacol^^ieal Smdias of 

0£feer IaR?®ffi£ig®£orss Hoae 

Sfea fsars (ealesdar year 1959) g Pati&ae Dftya (calendar year If 59) a 
loeals 1 

Pr©fessi;3®b®ls 1/2 
?^&®^i 1/2 

Pr0J®ee Deserl^SiQ® 

1) T@ d^^eiop practicable aad reliable €eehaiques £&z gvmA 

in vitro of ealls frcM htaaa bi<^@y tsaesrlslo 



2) To ®^l@y e&fflse maehode 

a) f@r sgtsdyiag ehe tsaehsalsiE o£ aceicm of ^erap@t£ei€ ag^mtec 

b) iE& ds^elopittg a saat f^r eh® sessslei^l^ of a patsisae^s 
t^&s £(S» seleeted eherape^Sie .e^poisads. 

g) as a's&ffiaiss of 8t«2dy£ag bielogigal 8std bioeS&ml^al 
v&'sl&tl'sm. of h«Biaa nsoplae^Sc 



Magliodss €alle ar® handled as simgle £@11 mispesesiio&s aad grs^ja 
as BQissolayer sh®®€8 or €l@a@@i, ^n. glaaso ^ defisKed 
ffiadla, ms^plemsntud v&ea raee&ssary wi£h ssreaa or otk@r 
biol@gi@&l8a 

Maigr.nRd^g§g Work dairiag eh« flrse balf year of ekis prejset 
has isvol^ed mly set^iag .9^ aethods for gzmtk 
&n<& me&mxsm&t of aeaadard dell lisi@e< 

M^^S^m^S^mS^lB^^S^' ^^ ®sas«$y^®!^s of 8?««refe «d g&© 

©eeabolisK of sssmal asd ti@^lasgi€ 
sells In yltz& presides s ®@a®® ®f 

esMisiag 1^ eoadleioRs reqisired fer saasl3Kffil gr^efe rae®s te ai&s^al. 

€©lls Mj b® s®li®sse4wly killed @ir ialaibitedo 



fare B M&hi4.&dt H@ 

863 



i>237 



S@"ealled plat lag e®«haiqu«s pra'jfide a as©"- - •'-* 

kin^ties of dir«8g aetisa m* p«r« e®ll str®, I. 

feyfgSo S«sels sfeisdies fshoiald aff@rd iaslgb . .ate--.^«,*.,fr.j>«.i 

®f aetiesi of thesapeistle drtigs and may al,. f@r 

ffiSfdifieafei^ of £y©®£E4<is£a8€h«dwle» ia ^.s/^ ■ ---^ t-*-'- 

hiseeelsgj&ieal m: tmtti.tlm&l e&filmiq^^ee %' 
aaaku peseibl^ the ssct&lyatB ef e«ll»l&r po^: 
dssring ^® natxsrsl hist®3fy of atMt^l&m.B &r x 



Ic Stedf @f £l&e'«&ati@n &S sel@€t@d drags $»& s@is@ ^stsbiisted e.@ll 
s^rairae to order to ds^elisp tsi^ek^ds axid gai» a !atmrl64g# ®f tfe« 



tiam^s^ wish sf e€i«l atg.ggatioat m ss^abllshing s^ehods i@T 
dbeaining a high rsesrvery of 'liable eslls frsn biopsy @3e@rial 
(iaelssding bia®d esil6igx"e)o 

3 , Stwdy hi8t®eiii®si«al ®©«&©«2s ■ fox id«eifyisig spaeifte ©®ll fe;^®®- 
4o Ate^pt "eliaieal eeets" ©f fe&@ sansiel^ity of biopsies ®£ 



864 



General Met 
3c, Bathesds 



PHS-HIH 
Individual Project Report 

Calendar Year 1959 



Part A: 



Project Title: Clinical Investigaticsas of Maiignaat Skis 
Digeasas and of Benign Conditions Related 
Thereto. 

Principal Investigator: E. J. Van Scott, M, B*o 

Other Investigators: Eisfeard P. Reinertsonj M« D. , Arthair 
Zc Eisea, M. Do 

Cooperating Units? Chemotherapy Service, KCI 

Man Years (calendar Year 1959} : Patient Days (calendar yaar 195: 

Total: 2 

Professional : 2 
Other: 

Project Description: C©ntimaation of G-l«708(c) of 1953 

Objective @g To ©tudy msligasmt processes Ib the skin s»d to 
carry ©uE investigative therapy of catsaeouE snsligQsat 
conditions; to study benign hyperplasia ©f the epidersis 
in relation to malignant hyperplasia of the sliia aad o-disr 
organs o 

Methods Saployed; 

Mycosis fun^oides; This disesse is treated \s±th cfeaiBo- 
therapeutic drugs given systCTiically sad with electron at 
X- radiation when indicated, and is further studied aad 
treated by other means derived frsss observations on the 
insaunologic behavior of the disease o 

Basal cell carc in^a; SeiTere cssss are treated with cfeass" 
therapeutic drugs given er^r ■atemtcallj, with s-rays^ and with 
excision^ The nature of the tumor is investigated in the 
patient by studying changes in the tismor occurring in 
response to locally administered agents and to alterations 
in the stromal envimment of the tuniaro 



Part B included: fes f^J Ifo /__/ 

865 



- 2 « Serial lo. C»7-708(c) 

Psoriasis: Severe cases are treated experimentally with 
chsmotherapeutic drugs either spplied topically to the 
skin or given systemically. The nature of the disease sEd 
the cutaneous lesion is studied in the petient. 

Pati^t_Mate£i^: Pa£ien£s of the Dermatology Serviee, ICI 

Mvcgsi8_ FungyOides; Beneficial but temporary response of the 
disease" has been observed following cbesaotherapeutic drugs 5 
3E~rays and electron radiation; no new data has been collected 
relative to these forms of therapy. 

A single patient with a previous diagnosis of lEycoBis 
fungoides has been observed to be totally free of this 
disease for over two years, the original disappearance 
of cutaneous lesions having been associated with a 

rather marked drug eruption, l&ole blood from the patient 
has been transfused on two occasions into another patient 
with laycoDis fungoidesi no detectable effects on the 
recepient's dise4,8e have occurred frsHu this procedure. 
Attempts have been made to sensitise patients with M, Fo 
to & number of drugSo In one patient this has been 
accfflEplished with resultant skin eruption, marked eosinophilia, 
fever end pulmonary infiltration suggestive of Loeffler's 
syndromeo Of five cutaneous tumors observed in this patient j 
three disappeared clinically, one underwent marked reduction 
in. siae, and one remained unchanged » (Remaining cutaneous 
tumors treated with s«ray. 

Basal Cell Carcin^Kua; Patients with sufficiently severe basal 
cell cancers have not been available for systemic tl^erspy with 
chaasical agents during the past year. 

Various lines of study on epidermal and tumor growth have 
been pursued in patients with this disease, as well as 
with other diseases » Aut©» transplantation of epiderrosl 
tissues free ®f donor site str^aal connective tissue 
indicates that & tissue may be either incapable of survival 
in a new envirffioment , or that it assumes the characteristics 
of epidenaal tissue aormally resident in the reseipieat sitSo 
Thus, for essmple, oral mucous miSHferane upon £rimsplaste6i©n 
into the superficial coriusa of skin' uadergoes EKidulstion iat® 
epidermis J but upon implantation into the deep dermis its 
incapable of surviviage Similar manipulation of bmsal cell 
tvmsT tissue has gives preliminary indication that this tuir&sr 
cannot survive in the deeper dermis unless sccoiiipsaied by 
the strasasl conaective tissue of the donor site« 

886 



» 3 « Serial So. C»7-70S(c) 



j'sogjaais; Ms?^ cheaical agents ^sed ia eancer ds^ao-^ 
therapy have beea applied tcpic&lly to lesions of 
psorlasiso Complete clesriag of legiossg clinical m.4. 
histologic, has occurred within one week following £wen£y° 
fottr hour applications of nitrogen Bnsstardg podo|^ylliag 
colehisinej mercury , assd taro Cleared areas tend to 
relapse within one to several weekso No local response 
to Methotressate has beea observed, although this drug 
marksdiy clear© the skin of lesions when given systemically» 

Mstfeotresse@j Acfcincs^cisi D, 5 FSaorouricil sad colchicine 

have beea administered systemicelly to patients with psoriasis., 
Cleariag @£ the skin has occurred following all these chemical 
agent So 

Serial histological studies ©f lesions have indicated that the 
lesion of psoriasis may be characterised && hyperplasis of the 
epidsrmiSj, that hyperplasia is ©lowed with cytotoxic sgeatSs, 
allowing norzaal kerstiniaatioa to tske place » 

Siigaificance to Cancer Eesejsrch ; Project directly concerned 
with study of, and therapeutic influences on^ csneer aod 
related cois^itioBSo 



867 



Serial No. G-7°708Ce) 



Individual Projeefe Ref^rt 
Calendar Year 1958 



Fart B: Honors 9 Awards g sad PublicstioQS 

Publications ^tfeer than fibstracts fresn this projects 

1„ Sr^er, L„ Mo 3 Waitej, J, H. ^ Edgcoaab^ Jo Ho ^ Powell, C„ €09 
Toj^hya Jo Ho 8 Van Scott » E. Jo, aad Smith, Ro Ro Buffn 
Following Accidental Esposure T© High Eaergy Radiations Asm, 
Surgo 149; 286°2939 1959. 

2= ¥an Scott, E. J. snd Eeinertsoa, Richard Fo Reiaertson; 
Morphologic and Physiologic Effects of Chemotherapeutic 
Agents in FsoriasiSj Jo Investo Dermat„ (in press) o 



lo Mesaber of Bersaatolog^r Service (Eo Jo Van Scott) elected £0 
Board of Directors, Soco for Investigative Dermatology, 

1959-1964 o 

2c Member ( Eo Jo Van Scott } elected to membership » Amero 
Dermatological Assoc « 



868 



Serial KOo G-7°?28 

lo General Medicine Branch 
2o Dermatology Service 
3, Betkesda 



PHS^KXH 

Individual Project Repore 
Galendar Year 1959 



^ElA- 



Project Title: Structural and Hiysiological Charaeteristics 
of Hormal snd Pathological Epidermis and 
Hair Roots, 

Priaeipal Investigator; E, J. Vaa Scott, Mo Do 

Other Investigators: Richard P. Reinertson, M. D. , Robert 
Go Crouase, Mo Do (Research Fellow) 

CooperatiBg Units: All cliaical units of I3CI, to some degsreeo 

Man Years: (calendar year 1959) Patient Days (calendar year 1955 

Total: 3 

Professional: 1 
Other: 2 

Project Description: Continuation of C?°728Cc) of 1958 

Objectives; To investigate structural and physiological 
characteristics in epidermal and supporting 
tissues in pursuit of understanding wide ranges 
of growth activity of epidermal tissues and 
regulatory factors thereof. 

Methods Employed; Tissues are studied histologically aad froaa 
reconstructions of specific structure So Total number of 
cells and mitotic figures counted micro seopicallyj volujae ef 
specific tissue structures calculated from microscopic 
measurements, geossetric relationships c 

Further comparisons ©re made on the basis of structural 
changesj particularly changes in hair rootSj following 
ionising radiation or drugs. 

Hormal scalp hair roots are cultured in Algire millipor-e 
filter chambers implanted in the abdosaen of mice to study 

growth regulating factors. 



Para B iacludsdi ¥&s / S / mc / "7 



Serial Ko„ G-?=?28 

- I -- 

gatieaS Material ; Tissues studied or used are from 
hoapitalised patients and ©ut=>patients of Dermatologgr 
Service and other clinical services of NoCdo 
Differentiation of epidesmsl cells and regulatory 
influences on keratlnlsation are studied in patients 
by manipulative auto^implsntation of epidermal tissuec 

Malor gindJEgs; The essential dependence of hair growth 
on the connective tissue dermal papilla has been 
further elucidated in humans from histological studies 
of geometric relationships within the hair root., Mitotic 
activity of the germinative matrix is directly pro° 
portionate to the number of cells in the papilla, 
1 mitosis normally and uniformly present per 8 papilla 
cellso Pemianent baldness in alopecia areata has been 
found to be associated with destruction of the dermal 
papilla. 

Mitotic activity, decreasing in successively hi^er 
levels of the hair bulb, is found to further be a 
function of the area of the dermal papilla to which the 

matris cells are exposed and to the transverse thickness 

of the laatrix at each level,, 

Reversible damage to the growing hair roots of the human 
scalp has been found following a number of chemotfeerapeutlc 
agents s including methotrexate, di<=chloro°methotre5iate, 
5-fluorouraeil, ectinoaaycin D, thio°tepe, colcemid, cytosen 
and vinca leukoblastin^ Dssage to hair roots has been 
found to be & sensitive indicator of systemic effects ef 
these drugs, occurring with equal or greater frequency 
than changes in WBG, platelets, or reticulorytsso 

The process of keratinisation ©f epidermis has been 
studied in feuiaans by imbedding -isolated epidermis intra° 
derraally. Differentiation of cells and keratinisation 
occurs in the central aress of masses of implanted calis, 
suggesting several mechanisass by which keratiaisation axid 
differentiation may occur, 

Sl^tjle ssice to Cancer Research: The studies are directly coa^ 
concerned with cellular reproduction, destruction or 
quiescamee, phenomena primary in studies of cancer o 

Proposed teurse of Prelect ; Continuation along liaeg ge 
as indicated in tl5.e foregoing o 



870 



Serial Ho. C-»7-728 



PHS«1«IH 
Individual Project Report 
Calendar Year 1958 



Part B: Honors, Awards j snd Publications 
Publications other than abstracts from this project: 

1. Van Scott, E. J., Evaluation of Msturbed Hair Growth 

in Alopecia Areata and Other Alopecias. Ann. B.Y, Acad. Sci. 
(in press). 

2. Crounse, R. G. and Stengle, J^ M. , Influence of the Destoal 
Papilla on Survival of Isolated Hvnaan Scalp Hair Roots in An 
Heterologous Host. J, Invest. Dermato 32: 477-479 ^ 1959. 



87; 



Serial Ko. C-7-730 

1. General Mediciae Braicich 

2. Dermatology Service 

3. Bethesda 



PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part A; 



Project Titl«: The Biochemistry of Kormal and Pathological 

Epidermis. 

Principal Investigator: Simon Rothbergs Ph. D. 

Other Investigators; Eugene J. Van Scott, M. D. , Robert 
G. Crounse, M. Do (Research Fellow) 

Cooperating Units: Hone 

Man Years (calendar year 1959) : Patient Days (calendar year 
Total: 3 1959) 

Professional: 2 
Other: 1 

Project Description: Continuation of CI-^JSO of 1958 

Objectives; To study the biosynthesis of the major protein ^ 
keratin. In normal and abnormal epidermis. Knowledge of 
this process will permit examination and comparisons as to 
the nature of the protein synthe sized in normal and 
diseased states. 

Evaluation of the differences between the major keratinous 
protein (or proteins) of normal and pathological huiaan 
epidermis in order to determine whether an abnormal keratin 
does or does not eigist. 

Estimation of the arginase content of the dissected coEpoas: 
of the human hair root. 

Description of the Krebs-Henseleit urea cycle in human 
epidermis,. 



Fart B included Yes / S / No /___/ 



872 



Serial ': ' 20 

" 2 - 

Mf^jh-OSSA Solubil3.zstion, isolation and purification of the 
major protein fractions present in normal and pathological 
epidermis are made. Comparisons are made of the fingerprint 
patterns produced by the individual proteins in order to 
detect differences in these proteins which may otherwise be 
undetectable. 

The peptides obtained by trypsin aad chymotrypein hydrolysis 
of the purified protein fractions are separated utilising, first, 
one dimensional descending chromatography and then high voltage 
electrophoresis. The location of the peptides is visualized by the 
ninhydrin reaction with the peptides. "Fingerprint' patterns of 
the protein fractions are compared and the peptide differences 
between different pH fractions of the s®ne tissue (epidermis, 
psoriasis scale, or ichthyosis seals) or between similar pE 
fractions of different tissues revealed. 

The possibility that cleavage of disulfide bridge occurs when 
the protein derivatives are solubilised is tested by titrating 
sulfhydrl groups, using jg^daloroosereurlbensoate according to 
the method of BoyeXo 

Hiaaan hairs obtained by epilation ©re separated into their 
various ccsaponents by microdissection. Arginine assays are 
performed by a microadaption of previous incubation procedures 
plus the microdiffusion techniques of Conway for daSenainatioa 
of the ammonia (resulting from urease hydrolysis of urea). 

Evaluation of ornithine transcarbanylase, and arginine synthe° 
tase in epidermig is achieved by two methods. Tlie first 
method utilises pre-esisting techniques, assaying with an 
excess of substrate in each case, tstiile the second employs 
radioactive C»14 arginine, aad labelled ornithine and 
citrulline derived thereof, is tracer amounts. Products of 
the tracer reactions are separated fey high voltage electro^ 
phoresis, eluted, planchatted sod counted in windot'?less gas 
flow system. 

Major Findings ; The peptide patterns obtained for each of the 
nine different protein fractions reveal major sljiailarltiss 
between the proteim fractions of esch tissue. Although 
differences are found in the peptide patterns of normal 
epidenais, psoriasis scale and ichthyosis scale, it is not 
certain whether these peptide differences ©re due to ^baoraal 
keratin. 



873 



Serial Ko. G-7-730 



Soluliilisation of protein did not require cleavage of 
disulfide bonds; however s, s difference in hydrogen bond- 
ing of the proteins of £h& epidermal tissue Is indicated 
from the different peptide groups present in the fractions 
examined, 

Prelivainary findings indicate that glycine^C'"'^ incorporation 
into the proteins of the epidermis is maximally reflected 
in the stratum corneum 25-30 days after intravenous adniin^^ 
istration. 

The inicrodissected components of epilated human scalp hair 
roots assayed for arginine activity showed activity in the 
sheaths but not in the bulbar portion of the epilated roots. 
This finding discounts any local relationship of arginase 
activity with the protein synthesis of the keratinous 
material of the hair shaft. 

Utilizing the two methods described above, the ensjnneB of 
the Krebs^'Henseleit urea cycle other than arginase, are 
found to be absent in fresh surgieaXly obtained human epi- 
dermis . 

Significance to Cancer Research : 'Although the diseases 
discussed in the work are benign, comparison of the protein's 
synthesized by the call in normal and hyperplastic epidermis 
would seem to be important. Evaluation of the differences 
may also help to indicate the nature of the alteration of 
normal protein to form abnormai protein. 

Other basic information in the process of protein htosynthesii 
in normal epidermis should aid irs evaluation of protein bio^ 
synthesis in malignant epidermis,. 

Proposed Course; Continued evaluation of the ''fingarprinf 
peptide patterns produced when the protein fraction is digest - 
by only one ensyme (trypsin or chymotrypsin) ^ will lead t© a 
clearer peptide pattern and should thus enable a more critical 
appraisal of th® peptide (o? issisEO acids in peptides) differ^- 
ences between the protein fractions. 

Studies of the biosynthesis of protein in epidermal tisB-aas 
in vitro and in humans are now in progress and contiauation 
of these studies is intended. 



874 



Serial Bo. G=>7°73£ 



Individual ProjecK Reposx 
Calendar Year 1959 



Part B: Honors, Awards, and Publications 

Publications other than abstracts tr:csa this project: 

lo Rothberg, S, The Evaluation of Keratin Fractions in Normal 
and Abnormal Epidermis. J. Invest. Dermat. (in press) 



875 



2. Mstabffiliasa S«snrie® 



ladlvidssal Projece S@pr©£ 
Sai«adsr Yesr IfSf 



Frojese Tifclet S@rim Pro£«iais Studies 

Prlssipal SKvestigaeor: Jolsa X.o Fafesy^ MgSc 

Sthar lavsstigstsrt For S:he sSisdies with ya4io iodissa6®d gas^a glefe^iisa, 
fosio To Wfil^ism assd Patsl Sclm&h 

CJooperaSlag Ifaita: far th& Bt^j ©f asopla^sa in asics - 

L®b®raeory of Bieiogy, SSS CBj^o E. Potter) o 
For the st@iy o£ "asto irasisae" diseases iss s^a •» 
Laborffi&o^y of Metabolim^ MSI (DTo So Goo^smn.),. 
For tha setsdy of red blood cell affi£ibodies - 
Blood Bank, Clinical e®nter {®r„ ?« Schaidt)^ 

Ska Years Ceal«adar jb&v 1959) PatisaS: ©ays Ceal«m4as: ymx 1959) 

Total 5 3 

Profsssiossl: 1 
©£her: 2 

Project ^ecEiptioffl; 

Ofejgctlvess %s ®l>Jec£ of ebls proj«cfc ia to detes;®ia® p&ysicocfeKmlcal, 
E^tabolic £3Bd o£her sharae&esiseies of eha sertsB proc^iias foimd noiei^lly 
simS ia associatioa %d£l& sisligiaaat disease^ th& rol® of selected protsias 
lat £b3 !aodi£ic£tioa or @a&if@3&a£ioi£s of disease, ssS tS&@ us^ftsls^ss of 
ssr^ prot^ias &s ®s@£ly aceessibid indices of disease Actlvitiy or 
t&«rspffiMtic eff®G£:iv« 



^thods EBploy®d ; Qaaneitative aad q«alil:as:iv« chfiBieal, physical^ md 
imsmadieBlcsil proee^ses^ as veil as eests for biologic activity &r« 
@s@d so iasasm'S and eharacte^lsd, ^^K^ poosiblo!^ iadivid«aal prot@ln@<, 
Radiolsotopically labelled (S^^^o? C^ ) protein* ia tracer arotsats ars 
®eillaa4 for laefcaboXic stssdiaso ?raseionatlon of the ct^lex ®ist«sr« of 
aosaal serss® protstas ®r« doa® by ©lactrophoresie, ehrtJ^stograpfeie^ 
solffi&ility and oeter proce^reso 

Pasiaat ^terial; Sera; wejre; ofetaiasd frtaa patistata adaitted to t&© 

gfetafeolisffl Service, mi, fr^s coopsratiBg enits t&roughoat thm fetiosi&i 

esase? Sns£it«s£s and eh® Satiosml Snsfcitistes of lealtfe aisd also fr« 
pfeysieiaas is tfe® ssetropolitaa Waglsiagtoa area and throisghoist tfe@ 
cmmtxjo Clinical infos^atioB was available on all of these patlssts sM 
ssl®et®d patients were afeitted to the eiinieal Center for detailed stssdy.. 

Fart B iaelsded Yes.._l.. lo 

876 



Mt^fg FiadJBgsg The. gimm. glebalias ©f ncntsal @«imb «f@r@ @l@arlf ®bmm 
£® b@ s larg@ fasaily ©f r®la£«d bsst noe' id®nfeieal py©e®ia isi©l®.eui®s^ 
Sicbgre'ssp® wifelsin ehls pg©fe@ini fsasily w®y® eh@a iiiv@s£igae@«i im detail 
rafebiy tfeaa fer®®eiag £!i® gsooia gX®b«lin as a heis^genemi® maito l^e Bia,J©r 
gr^i^ps ©f g^ssoa gX^bicllns^ wish uler&e^ntrifssgal s@di!<Mntagi©a ce^ffigignes 
©£ 18s aad 60 6S^ ^an b@ s@pA3?at<id by aal6Xi<°<§s€hang@ @@ll%il©s@ ghrcaaafiogsfspt^'-. 
and £fe® lafe£@r growp can b« fiajffehig? ssbdivid^d into 4 @r aidre 6«6S fracfcicns. 
Detailed characterlzatlGn @£ £hes@ fractions revealed that £he 6,6S g^tr^a 
globulins were J, indeed^ a family of saolecales sharing similar t;sl£racentz ■ 
fagal and iaamsaoGheniiGal properties but differing in electric change, 
Gontsnt and antibody sctivityc 



A similar family of aolec^les was found when myglraaa proteins. £xcm 
tk« s@rs of 38 patients with smltipla my^lcma^. ver@ SKSiainM in d^itail 
These asyglesaa preteins, which were either hemogeaeous or cciabinattotis of 
& £@w closely related proteins^ appeared to b« representative of protsias 
norsoally present in 8@r^'^ in sntall asiount^ Similar observations were made 
on the sermi SKjrelosna proteins associated with 8 transplantable sso%*se plesmr 
cell necplafisaso 

The individ^saltty of the sayeloasa protfinSj coupled with the previor.;-: 
daasonstratlon that mf&lcm.& protein is fosised in 6h(g plaasBS-call ttmot , 
indicated that eaeh pl«ysjfia,"e®ll stalignancy was . fsmctionally distiBcfc m><': 
that each tmseiT cmld be identified by its soluble sensa predt^ct,, the 
a^elcssa protein,! 

It follows fr^s t-h« above c©asid@rati©n feha£, as th@ variety of r^elosu 
protaias represent & v&tl&ty of pi^«A-!- ^ -iV. vj®epls3ins^ th@ spigetnifflj of 
aorsal 60 6S gasma global tea ves>- a#nt a spectrsga of liBdi%r^ 

ncrosal plass^ocellsc 

Individtsality of gsas?a»gl©bwlin. pKR^tselrLi?' ''35i« -.,,-t, r/,i.«_--, 
st?idy ©f s«sisa antibedi«-0. Many antibRdl^s • 
global iffiS^ b^t a n^sa^^r of antibodies u&r.^ £< 
gl^b^zlinso ^y an antlg«» iadtsegs a part.ic^iv, 
big dstersainsdo '^^"* -""''- ■""■ -^ - ^"--■'^■■■- '-■ ■- 
different iadiv:_ 

aHtig®a and thsfe. si-./ss^ ,■ ^ ..:-i£ifaAs^tt«,ii"_^iaj:ii,ii.. ■ <,•:;-■ 
fo^snd to a aingl® aafeigsis... CsBbination ef p' 
t«chnicSy hewswery have been deve' ' ' * ' .:.iL. trecogs.LSL. ; 

Investlgatiem of th@s@ sppar@Qt <: 

Significance te Gaar.-- -jessnted her* 

coasistsnt with , '" .^iiasf ctI^ab q- __ 'usjac AriSi.-:_ 

single csll £h® :, - apparently ©pr®.:-, jasis to £h«, • 

sites so fr®q58€atlj teiiC©UB^.@r®d in cliaii'sa.l ■;.i:z.Q.'c'la&c 



^11 



'.:- - Serial' HOc HCI° 6--- 702- C c) 

The a^eicaaa protein feas proirsd to bs an iasportant mgeabolic sssrkar 
for pla^as'cell seoplassiSo It is accessible^ rsadily identified sad easily 
quantifi^do With the desaoastration in ntou8@ tumors of a direct correlation 
bet^@ea tba esuanssit of ti^sor and the saioimt of serum Gcyelossa protein^ it 
vGuld sean that serial quantitative measuresent of the serum SD^eloiEia protein 
say be laore valuable in assessing th@ clinical course and response to therapy 
of any individual patient than serial bons laarrow aspirations or skeletal 
roentgenographic examinations particularly in view of the uneven distribution 
of tsyelcmia cells and the difficulties in evaluating quantitatively the « 
roentgenographic bone changeso 

Proposed Course of the Project; It is proposed to Investigate the effect 
of malignant plai^aa-'cells on the n^saining noztoal plass»-c@llS; the synthesis 
of E^elosia proteins and related proteins by the malignant plasma- eel Is ^ th@ 
laetabolic alterations induced in the gamsa globulins by plasn£»cell 
malignancy^ and the significance^ in tersis of normal proteins^ of the finding 
of isyel«na proteins of the beta globulin eleetrophoretic mobility and s^ltiple 
coisponentSj, 6a 6S > 15S^ in the ultracentrifuge^ and other properties 
suggesting a relationship to proteins normally formed in the plasma* cells o 



878 



Serial No> HCI>6>702(<s) 



PHS»NIH 
Individual Project Report 
Calendar Year 1959 



Part B ; Honors, Awards^ and Publieations 

Publications other than abstracts: 

lo Fahey, J,Lo and Horbett, A,P«, Fractionation of Gassna Globulin: 
by Anion- Exchange Cellislose Qiroaaatography, Jo Blologicsl Che- 
234 ; 2645-2651, 1959, 

2o Fahey, J,Lo and Morrison, S»Go 

Separation of 6, 6S and 18S GaHaua Globulins with IsohsEaggiistirj: 
Activityo J« Laboratory and Clinical Medicine, in press , 

3, Goodman, H<,Go, Fahey, J,L, , Malsagren, R.A, and Brechar, G„ 
Separation uf Factors in Lupus Erytb.es5stos«s Ssrtmj Reacting 
with CcsEponents of Cell Nuclei, Lancet, 2: 382-383, 1959. 

4, Fahey, J<,L, and Potter, M, 

Bence Jones Proteinuria Associated with a Transplantable Motjs'^ 
PlassBa-Csll Neoplasm, Nature, 184: 654-655, 1959, 

5, Fahey, J„L, , Potter, Mo, Gutter, F^J, and IHmn, T,Bo 
Distinctive Hyeloaaa Globulins Associated with a New Piass?a-G?.i: 
Neoplasm of Strain C,H MicSo Blood, in press , 

6o Potter^ M„ and Fahey, J^L, 

Studies on Eight Transplantable Plasisa Cell Neoplssas of Kiee 
Jo Nat'lo Cancer„ Inst^ , in press . 

7, Dunn, T„B„, Potter, li. , Fahey, J^Lc- and Merwin, R„Lc 

Horphology and Sensn Protein Changes in Plasaia-Gell Keopiassi.; 
in Mice, Archivio "del Vecchi" per I'Anatoaia Patoiogica, 
in press o 



879 



■Serial Ifo.^Cl- 6" 704C.e) 

Ic General tediciae Breach 
2o ^tabolis® Service 
3o Sethssda 



PHS-ISIH 
Iffidivldwal Proj®et R«port 
Calendar Year 1959 



Part A; 



Projset Ti£l®: S£8sdi©s of Porphyria Metabolic ia the Taaor Bsarisg 
Most and Porpfeyrias 

Principal lEvestigator: Bonald P„ Tschudy 

Other Investigators: Annie Collins, ^i^anisel Hells^n 

Cooperating Units: Son© 

SSaa Years (calender year 1959) Pati«nt Bays (calendar year 1959) 
Total: 13/4 
Professional: 3/4 
Other: 1 

Project Bsscriptimi: 

dsjeetivas; To stu&j tssaor host iaterrelationships froas the standpoint 

of porphyrin synthesis in iiver and red c«.Lls„ 

To study the effects of vitaad.ns, drugs and horaones en 
porphyria biosynthesiSc 

To s£«dy bioch«aical abnorEalities of acute intermittent 

porphyria^ 

Methods egployed; 

Ao Q«3aRtitatl e e&zyi@e detes^inations 

Bo Assay of urine for porphyrins, porphyrin preosrsora, 

uric aci# aed a@ino acids 
Co Use of C s^^'S<>s^; acetate asd pyru /ate in ho@ogenates in 

aeasoring their rate of converaioas to fatty acids, 

cholesterol and CO^^ 

Patient Material; Red sell eazyase studies tsill be perforased ia patisnts 
with aallgsancies asd patients with acute prephyria are being st^disdo 

tejor gjadln^s : The synthetic analogue of S-asinolevuliaic acid vhieh 
is a potent inhibitor of ALA. dehydras« in vitro has not been active 
ia vivo by methods used thus far^ 



Attempts to deaonstrate a hs@K>ral factor produced by 
tusors ^hich causes the decrease ia hepatic ALA dehydrase have be<ga 
incoaclasiv®^ Adrenal cortical horsoaes diaaiaish the activity of 

Part B. included Yes Mo .1 

880 



Serial lo. aei-6-7G4<c) 

hepatic ALA dekyiras®^ b»t tks d«ereas® a€ this &asjs& ^cc^rs 
ia the adrsasleeti^izsd tiasor bearing hosto The ftnzyms decrease la 
therefore aot a^scoadary to adresoal eertical activity^ Xajeetloss 
o£ "toKohexsose" prspsratione fail to eatsse any decrease in ALA 

dehydrasa^ 

gigalfieaaea to gsae@g Rsseareh ; The decrease in hepatic ALA dshydrss® 
foand is all tvesr bearing hosts exaniaed thus far (vhea the 
is visibly enlarged). This suggests a biochemical abnomality 
to a^ligasnt t«ESK>re„ Sttidies of this abaoraallty say @nab @ 
IS3 to assderstand s.^@eific @@chasisas whereby a @a ignaat- t«3@ov affscis 
the host. 

Proposed Coar se of '.-ha Pvojaet ; 

A. T® s'ady rslatlonehips of carbohydrate setabollaa asd Krebs cycle 
activity to porphyria syatheais and porphyria. 

1, To stady farther chesaiesl abaoraalities ia acate porpl^ria. 




881 



lerial Ko .NCI-o-TZl-fc) 



Ir, General Medicine Branch 
2o Metabolism Service 
3, Bethesda 



PHS-NIH 
Individual Project Report 
Calendar Year 1959 



Part A; 



Projegt Title s Metabolism of Nutrients and Calories in 

Neoplastic M*;'>ease„ 

Principal Invest igatQi-; Donald M„ WatkiUj, Mr, Do 

Other Xnvestigatorss Donsld Pc. Taehudy^ MoD„, SJi, 1, Berlin^ M„D„ 

Clinieal Assoeiates; Peter So Muelleff^ MoD,. ^ 

Robert Couelis, M.,D„ 
and Leon Rosenberg^ Mo Do 

Cooperating Unitss Surgery Braneh, NCIj Biochemistry Branehj, NCI 

Man Years; (calendar yesr 1959) Patient Days {calendar year 1959) 
Totals 4„3 
Professional: l^S 
Others 2.8 

Project Description: 

Object Ives s Investigation ©f the over-all and intermediary metsbolisat 
©f fatp carbohydrate J protein^ minerals ;, eleetro^e©®^ vitamins^ 
celeries and water in patients during the natural course of ngopiastie 
disease, and during their responses to nutritional, physiological^ 
pharmacological; endocrin«; surgical or radiologic stress^ 

Methods Baployed ; l)o The snetabolic balance technique to quantify 
changes in the ccKoposition of ttMooz and host and for measuring the 
cwer^-all metabolism of nutrients and calorieso 

2)o Calculation of insensible vater loss frosa 
Insensible weight loss and metabolic balance data^ 

3)o Bomb calorimetry for the direct determination 
of calories in dietary intake, and in urinary and fecal excretionSo 

4)c Measurem®nt3 of th® respiratory quotient (E-QO 
and basal saetabolie rate (BcMoRO by open circuit indirect calorimetry ^ 

Part B included Yes X No 



882 



— 2- Ssrial HOp HCI -6"721--(c ) 

5). Sstisssateg of body fat aad body density by 
of skinfold thicknesses by skia-fold thickness 

e&iipere and by ateans of tritiated vater^ and helltn dilution* 

techaiqiMS (^ with BTo ^. I„ Berlin)., 

6)0 l^astsrffinsnt of uric acid in blood, uriae, aad 
body tissues and fluids by an eazy^atic degradation technique^ 

7)o Measureaeat of plasm unesterifiad fatty acids ^ 

(0 ? A)o 

8) a Quantitative extraction of lipids from tissue, 
food and feces » 

9)0 Protein pool turnover studies using 1 ^ labeled 
amino acids i'^ith Qr^ Sonald Po Tschudy)^ 

Patient Material; 

1) In-pati@ats, 11-17-58 1 

2) In-patients admitted, 

11-17-58 through 11-16-59 

a) Initial 14 

b) Read£sission 5 

3) SSorssal Controls 22 

Major Findings ; laterpretatioas of data froa investigations conducted 
during the past 12 oonths has advanced appreciably our miders£andii^ 
of a syndrose often observed in patients t;?ith neoplastic diseasSo 
This syndroaie which for purposes of identification will be called the 
"host-weight loss" 8yndri:fflEej, consists of host weight loss, negative 
calorie balance, a change in body coeiposition characterized by a 
decrease in th@ per cent fat and an increase in the per cent lean 
body ffiass, a low basai respiratory quotient CK»Q, ) and an increased 
basal unesterifisd fatty acid (WA) concentratiORo 

A„ An 11 year old boy with renal asaiuoaciduiria, a unique 
condition which is analogous to neoplasia in that it robs the body 
nitrogen pool of a^ino acids required for synthesis of protein in 
noraeal organs and tissues, has de^>astrated the key features of the 
syndrome nteationed abovec Supplesentation of his dally diet with 
ffisounts of threonine (IcO g@o ) aad lysine (0^5 gra^ double the 
quantity being lost in his urine was associated with inerea«4 growth^ 
positive K balanee and an iserefise in total daily energy cjEpendlt^srSo 
IS^^- labelled aspartis asid tunusv®? studies were perfonasd in this 
patient £0 saeaaure nitrogen kinetics at two different levels of 
protein intake, 4 g^ per kilo of body weight per dey aad 2 gSo per 
kilo of body \ji!»lght per deyo Mass spectrographic deterainstioas 
essential for the interpretation of these stisdies have not jet 



883 



~3- Serial aa. HCI-6-721~Ce) 

Sl ^lficance to Saacer R^eaarch ; Tfeeae findings stsggcst by analogy 
that ffisopl&stle ^isssas say indtice the above sssttta^sd syndroaa by 
6st?sctissg s^lffio £€ids tT<m th.9 body altrogtta pool &sA that ideatifieation 
of aaS stsppls^atatioQ of ths host dietary with the estraeted saiao aslds 
say of £er a aseaas o£ rsvsrelBg the aanifeatationa of the syadro@e ±n the 
host. 

Propossd coarae of project ; Carffaat stt3^i®s ia this patleat are tea £ lag 
the hypothesis that tbs growth observed dssrisg threooise and lysine 
suppl^sststioa vae the result of the pati@at'3 rsseiviag the speelfie 
aaiso acids j, not merely s&onspeelfic nltrogeiio Projected studies lael^d® 
the forsi^latlost Sr(^ tsrisary ssaiao acid eseretioa data qoaatified by eoli^a 
chromatography of a ch^sically defined dietary suppl^^nt which will 
provide this patissit %?ith a total dietary of the highest biological val@e.o 
This @@pplssest ^lil then be tested for its ability to reverse the 
senifestatiosis of the host-weight- loss ayadroae ^ich this patient dlsplaysc 
luring thsse isvestigatioos the relatiofaship of total calorie espeaditisre 
to proteia gyatls^sls ^111 be quantified by the aaastar^aent of insensible 
water loss^ by indirect calorimtry and by changes in body ca^ositlon 
bet^jsea tws points ia tisse^ K^^-labelled aspartic acid ttarnover studies 
will be isioorporated into the eaperiaental design to provide infonaatioa 
on sitrogea kinetiss for cosparisos with the two previosss stisdies perfonied 
at two levels of proteia Intake which were stationed abovec 

Bo Previous investigations of this project have d^aosutrated that 
ingestion of nos^elly adeqtsate dietary protein and caloric hyperaliseatatlos^ 
^ith iatravensttsly ai^inistered fat ^^Islons stiamlate t^iK^r protein 
synthesis aaore thaa host protein synthesiSo ^her studies before, daring 
cjid after eh®sotherapy of syeloproliferative diseases have sho%m that; only 
after redaction of U^or protein synthetic capacities is the host able to 
respond to the protein anabolic effects of dlete These findings suggested 
t^hat a potest physiologic anabolic stiseltss for the host vhlch wsuld not &t 
the sa^ tis^ either stissslate or- poison the tesaor «o«3ld persit asasurs^sBt 
Qsder tinsly physiologic conditions of host-ttaaor cogBpetitionj particularly 
for e^ao acids and calories, tssder ciretssstaaeee etsch that the host ^omM 
have saore of a co^etitive advantage than is @ore conventional escperi^antal 
designso Isercise was selected as the @03t physiologic anabolic stisal^s 



five yovssg patients with neoplastic disease have been observed before, 
dfsring and after stany consecutive days of ez^relse on a sotor-driven 
treadaillo Jaixs of these ^sfhose studies have beea coeplsted <one is still Ib 
progress) shotted the follotylng in association with exercise: 1) loss of P 
possibly d^se to loss of tissise high in P and low in M and reutilizatlon of H; 
2) a decreased excretion of ssric aeidi 3) a loitered basal respiratory 
quotient; 4) & lowering of increased €FA coac^itratlons dee to active 
neoplastic disease btst an increase in WA concentrations already in the 
norsal range; and 5} a change in body ec^positloa characterized by a loss 
of fat and ^atero 



884 



-4- Serial ^OoKe£>6-721-{e) 

Sigal£leagc@ to Cancer Easearda ; Thes® stsdies sisggeat that organ 
asd tls@s3S az&abollsa caa eccar unSsr tka lafl^esice of a ooxsal physiologic 
stimslos isi th@ p?es@aee o£ naoplastic 4isaas«c The eh«ag«s in ths 
psrs^sters saea8«s?@4 say b<s inte7p?at«d aa shoving that «s«rcis@ do«s 
stls^late «f£eGtiv« host cosspstition with ttmor fo7 the ingredients of 
protein eyntheslSo 

Pgop@s@4 coBggg of project ; Gtaipletion of present «tig^ of a patiant 
with chronic ssyelocytic lemk^aia, an iiSeal disease for st@% h®ea«ase of i^s 
long msd benign ecmrs® and the ssssltipl®, easily laeasarable parameters it 
affords for studyo Reerssit^mt esA stisdy of suitable nora^l controls in 
tha ssss age grosspo Setereination of the quantitative rslationships hetwesn 
total body caloris esp(»idit«re and the synthesis 9f protein dssrlng s^erclse 
in t@s^r-h@sring &ni. fiontrasor-hea^ing hoatSo 



So Previoess invsstigations tander this project have demonstrated that 
the^basal respiratory qisotient C^oQo) is lov, the basal plassa ssaesterified 
fatty acid (CM) concentration high and the basal aetabolis rate (i^) 
variable frim very low to very high in patients with active neoplastic 
disease vho are eons^aing an adequate dietaryo 



To e3:ssaine the ^tabolic rate C^); the R«Qs and the plasma WA 
concentration throughout the day as well as wider basal conditions^ IS 
"fesding- fasting" stisiies were perforsed in 13 patients. Syring th@ 
first day, th@ patient ate his vassal constant dieto froia the evening 
of the first day to the coming of the next following day^, he fasted,, 
Msasssr^gssnts of the ssstabollc rate, the RoQo, and the plassaa UFA concentrate- 
ions were siade periodically throughout the st^y« 

Patients with active neoplastic disease either showed a low RoQo and 
high WA at the beginning of their fast period or d^^nstrated a change to 
low RpQ« and high UfA within 12 howrs of th@ beginning of the fasto Patients 
with inactive disease aad normal stsbjects required frc^ 16 to 24 hotsrs and 
g(^3@ti@es 36 ho33rs to reach the ssse low RoQo 's and high UfA concentrations 
observed in the patients with active neoplastic diseasCo The oetabolic 
rate r^saiaed elevated in patients with extrcs^ly active disease despite 
fasting, whereas in patients with less active disease sod in norsal sisbjeets 
the ssstebolic rate v&b fo%md to be lower at any parti^alar point in ti^e 
relative to, the "fesdiag,'-' day^ ■ 



Sianificaace to Cancer Research ; These studies deaonstrate that patients 
with active neoplastic disease respond to fasting by a shift isem a 
^tabolic SBixtu?e with an R„Qo of 0o8 f.>j 0o85 to a i^tabolic nizt^e with 
an RoQc of 0o6S to 0c7 and by increasing their plas^ UFA concentrations a@ 
a ^aans of saobilizing fat in far shorter tiaes than do patients with 
inactive disease or storssal s^sbjectSo There is no direct relation between 
fltetabolic rate and changes in H^Qo and l^A concentrations, since several 
patients with active ^■^-S<r^-? ■: hs^ noraai or eves low ggetabolic rateso Tkase 



885 



"S» Ses-ial Mo^ lie£°6'72I-Cc) 

fisidiBgs snggest tks prsses&ce of aa abnorsaality 1& lipid eaobllizatioa 
aad «3tllisati©B in tlie patieat vith aetive eeoplastie dl8«aes not aot«d 
in aoiTffiel subjorets or i@ ps&i@nts 111 ^ith oehoir diseases., 

g«Eag® Ci^gee of pgoject ; Additional stales ia patiests with very 
ac&iv« &®opIasl:ic disease b.ts& is other aooneoplastic conditions 

associated with hypexssstsboIifflB ®©eh as pregnancy, feyperthyroidi^, fever, 
acute aoKiety aod chroaie exercise. 

Wa A3 a stisp In dsvelopiag further the role o£ the asino asids in thm 
geoesis of the ho@t<-%?eisht-Io8s syndrome, the effieat^ of water solisbls 
chemically defimd diets (stspplied by the Siach€@istry Braach) in host 

fisainteoasice &M repletion were studied in two patients transferred to the 
Mstaboli^ Service fro® tk® S^as'gery Ix-sRch with iaaetive laeoplastic 

dis®as?®„ 

^ta frc® these studies indicate that diets eontainiitg L-aasino 

acids are suitable for both s^inteoance end repletion bat are EK»re efficaeiosss 
when given at the 50 calorie per kilo of body weight level than at the 35 

caiori® par kil® of h®S.y wsight: levelo They also sssggest that D-L asims 
acid ssixmres @sy also be efficasioiisly sjsed b/ the host after &n 

eqtiilibratioa or aijustseat period of about 6 dsyso 

Siaaifiea aee to Caaeer Eesearch; These diets say be varied la their 
cmip9Siitt&& exactly to co^fos:^ to the optiawsi biological veltae for & 
given patientc Hesse th<sy provide an iavalaable tool with vhich to 

approach the dif f ictslt probl^t of the role of the g^iao acids in the 

gesmsis o£ the host-%?@ight<-loss syndrome 

gjiittige eosrse of project ; laves tig® tioa into the reason behind the 
observation that purified i^ino aeid diets req«3ire sore ealories for 
a givea degree of nitrogen retention than do isonitrogenoiss diets 
GJtmp(sa@4 of ^ole prcteiao As greater supplies of mttao acids are 
evailable^ stisdles to develop specific ss^^t^res of amim» acids vith 
highest biological v^ltses for oosi^l subjects end for patients ^ith oje^ 
specific Bsoplastis process ^ill be tssdertakeno 



886 



-6- Serial No. NCI- o"721-(c) 

PHS-NIH 
Individual Project Report 
Calendar Year 1959 

Part Ba Honors^ Atjards , and Publications 
Publications other than abstracts frojn this project: 

1) Levenson^ S,M„ , and Watkin, D.IL , Protein requirements in injury 
and certain acute and chronic diseases o Federation Proceedings ^ 
December^ 1959 (In Press), pp. 31-68, 

2) Gyorgy, P„ , Chairman; Wallace, W,M. ; Hegsted, D„M, ; Harper, A.E, ; 
Waterlow, J.C,; Watkin, D^M,; Ross, M.H. ; and Scrimshaw. N, S, 
Sytaposium on protein re4uirement and its assessment in Gian, 
Federation Proceedings, December, 1959 (In Press)^ 

3) Shaw, R,K„, Moore, E„W, , Mueller, ?, S„ , Frei, E, , III, and Watkin, D, M, 
The effects of actincKnycin D on childhood neoplasms,. A>Mc. A, J, Dis, 
Child, (In Press), 

4) Watkin, D„M„ Protein nutrition in neoplastic disease, Food and 
Nutrition News, 30: issue #9, pages 1 & 4, June 1939^ 

The following laanuscript has been stibaitted for approval: teller, P=S, . 
and Watkin^ D.M, Plassja unesterifled fatty acii (UFA) concentrations in 
neoplastic disease, (For publication in Jo Clin» Invest,) 

As of Novesaber 15, the following manuscript is in the process of 
final typing prior to sisbssission for approval: Watkin, B^Mo^ and 
Stainfeld, J.L, Sietabolic relationships bgtwaen tujsor and host 
during large-dosage prednisone therapy in hmaaa neoplastic diseas®, 
{For publication inJoNoCoU) 



887 



Serial Ko. g€I-6-723(e) 
lo General Msdicine Branch 
2« Metabollsia Servlcs 
3, Betkesda 

PHS"M2H 
Xadlvi<hiRl Project Report 
CaleodAr Year 1959 

,131 



Fr©j©et Title: Studies of lagan Sensa Alfeasia Labelled '^itSi X 

©£her Savestigatoffs: Issbert Gordosi, McBo (Ml) aiu£ J«sli«s White, PhoSo 

Csoperasiag Ifeits: Laboratory of Physiology Hatioiml Casscar lestit^st® 
l&tioaal Heart Zsiistituts 

mu Years (calendar year 1959) Patieat Bays (calendar year 1959) 

Total; 1 

Prof@ssioE!al; 1 

Othsr: 

•^his projsct was divided iato Projects X and Ho See attachedp 



888 



1, Gsaex'&I Kadicine ^ximch 
2» Matabolism Service 
3, BethfisdA 

PHS-HIE 
Sa41vid®al Project Report 
CslesSar Tear 1959 

Pmject I 

?&xt A; 

?E-©jes£ Title: Mstributioa araS Begradation of Hiaiisa Seztm Alfe^a 
Labeliea wltfe I^^^c 

Ps*iHCipal Iavfaseig®t©]f: flu«Mas A„ Walfeasa^ Mo So 

Otter lavessigafeors: Julias ^ite, ?KU^ 

Coc»p3ra£i£ig IMlts: Laboratory oi I^yeiology, Kstiosa&I Cancer 

Prejeet ©aserlptioas 

^jectivas: T© Set&tmiss th& eoatrolliog Bec&aais^s of alfe^mia 
syasfeesis &n4. dsgradatioa ia nos^als asd patieats with eaacero 



w&(&®lvt®^ Is&g&i'B Solmtismo ' Blood assi 24 hmir uriss sad stool 



mthodn ^^lm®&% 1 &lhmln is givaa iat^aveaoasly to issdivi^sals 



radioactivi&y for 2S days to det«raiB« the 
3o H^5 eiyciaa is give® isttravsnoffisly^ 



eoII@st:10!ns &x® ass&yad for 

jalb^iss &gr®da£i©© kin©ticsc 

and its uptake Isitc ser^^ alb^ia asad glolmlitt is deterssiioed by 

@ias9 spsstrt^^try^ 



Pa£i@a£ Material ; The studies were performed oa & pstisat '&?ls:h a 
c«?sb@llar h^sangioblastossio 

Ifejor gjadisga ; Ths percent of the body allsisEBla degraded psr day 
was iacrsas^ 60 p@rc@rat aad tka rate of albmin synthesis desr@as@d 
approsigstely 40 perceat £ollo«dsg daily infusioa of 30 grass of 

alb^ain a day for 10 dayso 

Sigaificasicg to €an€©r Research ; This stsdy is directed toward 

detsxeising the factors iisvolved in the control o£ the prod^ctiosi 

sM destruction of semsa albtsain, as a part of a stisdy to deterssi£s@ the 

Gm^® of the hypoalbsi^is^sia ii^ cancer patientSo 

Proposed Coisgs© of St^y ; To c«mtiEsae the studies to detersiRe thm 
effect of alteratioas of the alb^^ia and gasoa glob^lia pool sizes 

©a the rat® of syatfeesis ©f tfess© proteleSo 

Part B inclmi^d: Yes lo S . 

889 



i@rial ao. KCI-6~723Cc) 

lo General ^sdicisi« Branch 
2, ^tabolisa Service 

3o l£tb@sde 



-ma 

Xsdivi^al Frojact Rspore 
Caleadar Y«ar 1959 
Project IX 



Pars A ; 



131 

Project nfcle: -a©, Mstrilmtion asid Begradatioa of l"""" Albvain aM 

I Polyvialpyrollidoa® ia Patients with Idiopathic 
%p®prot@iQ^^a and Gastrointeetioal BeopIaffiK 



Friiacipal Investigator: Thoena Ao Waldfaama, M^Bc 

Othsr laweetigators: Rater e So Qoxdon, MpBo 

Q}op$ratiag Iteits: Matioaal Hftart Iastit«t«, LCFM^ 

Project Description: 

Objeetivga s To stssdy the pathogenesis^ clinical pattern and the 
kinetics of albia^n destruction in p&tlents with idiopathic hypo- 
albsssinesiap 

Sfethode ^ployed ; I^^^ lfiS>eled alhessia is given intravenoesly to 

an individual receiving I^ols aol^ti&no Blood.and 24 ho»r isrine and 
stool saaples are assayed for radioactivityo I polyvii^lpyrollldone 
(PVP) ia given intravenously and stools collected in the subsequent 4 days 
are assayed for radioactivityc 

Pati@at Material : 30 2^^^ albwain stedies have been perfos^d ia IB 
patients vith idiopathic hypoalbi^in^iac 

131 
iiajor findings ; On the basis o£ the I alte^in stisdies and clinical 

data patients with idiopathic hypoalb^in^sia fall into two ssajor gr<mps 

the hypoanabolic and hypercataboliCo the hypoanabolic. patients have an 

isolated deficit in albsasaia synthesis with prolonged I albwain 

siarvival and secondary hyperglobislin^ia and hypercholesterol@@!i&o 

All of these abnormilitiea are corrected by albt^in infssiono The 

hypercatabolic patients have normal or slightly increased alb«BBin 

synthesis but rapid degradation or loss of albts^Sc All of th@ patients 

vith idiopathic hypercatabolic hypoproteincssia and with ulcerative. _, 

gastrointestinal diseases show an elevation of the :sacros^l@c^le I 

PVP in the 4 day stool collection following intraveaotis injection 

Gcsapared to nor^ls and patients with diseases not affecting the 

Part B included Yes I Ho 



890 



-2- Serial Mo,HCI-6-723Cc) 

Project II 

g&strolatestiaai tracto 1!his SKggasta tfeat tfee gastroiatestlBal fcract 

4s tfee @it;a of protein loss in pstisnte wit'a iiSiopsthic hypercafea&olic 
i^ypQpro£sxiss^&&o 

Approsiffiae.sly 25 pezrcsat of tteis patients with this syndsroasa reported 
±1 th@ litsr£^t)sra !:mv« had idiopathic chylous cff^sionsc At l&p&xatms^ 
or Crosby capsisle bloptgy S e£ 6 patients sttsdied h&ve shcnm markedly 

dilatad lymphatic eha^sels in the jsjm»al 



Relationship to Csne®!? Rosaarch t Isss of protein into the gastroiateseiisal 
tract ^aj h® fsetor in ths hypoalbisiinssia assoeiat@d with gastrointsatinai 



C@@gs@; To iSontii3^@ ths sttsdies directed tor^ard «)nderst£i!^ding tte 



pathogaaesis of feyp©alfea^a®aie both idiopathic and that associsfcsid v?ithi 
ncoplastie dis®sss®o. 



891 



-3- Serial ^ o„ liei-6-723(e) 

PHS-SSH 
££iSivid@al Pfojsct Report 

CalsEidar Year 1959 
Project II 

Part. S; Meaors, Awards^ aad Pablieatissns 

F^ilcatl'^ss oeh®r than abstracts: 

lo Gordon, R„So Jro, Bsrttar, foQcj ^alSmsm, ToA^ 

Idiopatl&ic %pealb«aiifi®£ia: Clisiic&l St&if Confcr«sie@ 
of the lagloiial Xnstltiates o£ Maaltk^ Annals of Into 
1^0 51; 553-576, 1959„ 



892 



Serial Ho« HCl-6-725 Cc) 

lo 6«ne?al S^dlciss Branch 

3. S«thcsda 



PHS"i"£H 
Indi'^idual Pr^jsce Saport 
Cal«Bdar \mx 195? 



^£Li" 



Feojsct Titl«; ^itaaiu B,, setabollaa ia nosraal s(abj®c«:s end 
l^tisnts yfth ascplascic end other di&caseso 

Frifficipsl laves tl,?ators ®<msld St., Watkia, E,So 

Otfe©r laves £lgatori; Xo laifssard U@ln8t@ia, Mo Bo 

€oop@ratlsg Units: 



M®a Ysara (ealftadar y«ar IS 59) Pati®a£ Bays (calendar year 1959) 

Tofcal: 2 1/3 

Prafsssioaal; 1 1/3 
Oehsr: 1 

Projeet Baseripeion: 

Object lyes; Qaamtiti'tlv® obsfirvaSioes oa ths eateboliara of vi£aaia 
S„ is ^SE iacluding Us absorptioa fr«a the gastroiatestinal tract, 
ill 4istrib?3ti©a in tha orgat.s of th« body, its level ia plassa, its 
biadiag to plaSEia protains and its estcretion by the kidney in nor^l 
stsbjssts sad those with neoplastic dlsaasso 



^tho4s fepIoy@4i Micro^.iological assay C U leichaaan nii) 
B, „ in plasaa, arine and visseSc ^asureiBSat of Co^^-lab« 



for vitasin 

-„ ^^^..^s, »«^ ..-^>,^-„ ,, -labailed vitasiin ' 

a\1 in blood, plagaa, piasm protein frsctious, uriae, feces and 
tisssies ia vitro, and in ozj&na in sita by in vivo scanaiag„ Pap®r 
and block alactrophoresis aai coliffln chroeatography of plasaa proteinso 
aiffiiysis and laltrafiltratioa ?f plaoBa protetnso Renal clearance 
tecbalqiseso Fsrchlorate preclTjitation proce&srCo St^ies of Bx2 
antagonists and analogs in bacterial, tissue c«l£yire, plasm and 
Lien syst 



Patient tfaterial '. Fourteen adaiaaions on 11 patientSo Of these 6 
had chronic ^elocytic leukssia, ?. had Addiaoaian peraiciotss an^sia, 
and 1 each had a^eloid aataplasia^ scute myelocytic laakeaia, careinc^a 
of £he pancreae, esxA c&rcinoffia of the l«sngo 

SaaJQg Fladinge; The B, .-binding pretein ®f the plasssa of noraal s®bj®ets 
and of stsbjects with Mronic myelocytic leukemia has been fmM is fm 

saroiaacoid frsetioB of plas^ sad displays the characteristics of a 

glycoprotein^ farther pjgrlflcatioa of this seroESBCoid fraetioa has b^eai 



Part B isicl^ed ¥es X l3o 



893 



"2~ Sariai feoKI-6°?25°Cct. .. 

&cls;l«vad by ^ai@a°©Kcl5S3Sis@ o®Il©l@s® csis^ia ctero^atogffaplayo T^e 
sar«sHsscoid frsceiea of ffi03f®al plassa appears £o have tSarae cs^paaeass 
to ^hieh S. „ is betss^o In th.® sesc^^eoid fraetion of plaa®a frcsa 

a B, „-fetadiag c^^saeat wfeoeo elsstioa chssracSceristics a^e tfes ss^s as 
OE&e if t&ffi coapaaeae;® i© aorsaal platss^o At£ffisp£s t& ©seablisk ttot 

A sesl«» of s£»lie© ^i£b Kbs ^e&ylaaids an4 seal Me saalogs of a 
^2aoesrbo3^1ic fosB of c^saocobalsfflin Cob£aia®4 fr^ BsTo So L®s£®r Ss^ltls 
of £sie Slaso Lelnoraeogies) ^iS®?® c&TxMi ®«at ia vitro with 'L^ Isieteaimii 
aad is '^-Ivo jssiag aj^®"lafeell@4 vil:^l© Sl2 ^^'feie^^ IndUatsd that tfe©s@ 
CfiiEpom^s eumld c«a3p«t« eff<Be£iv®lj ^iek eyaaaoeobalaBaiB for blEdiag 
sites oa plasms aad in gaatr©ia£©8£iEsi abusorptiaa on iatriasic f&ctor„ 
FiG5^?®vsr, eliaiesl su?di«s la 2 pati©ats %?ith ckrwaic ej^elocyfeie 
l&^^iim. sEd high plsaaa B.^ coaeeatrafiiona coatrolled by parallel 
steiles la 2 paf:i®at« with «.d®s©esrclEo@a aad nottml plsasaa vieaais 
112 GOiacsQ-es-atioffiB faiiisd to zm^&l snj tharapwatic affect oa cferoaie 
siy«loey£le 1« 



■figaigiemacQ fco Caaecg B<S8<5ageh; "^hts r®sial£s of tfeais® 8t«sdli©s st2gg@st 
£lte£ £h® ®bB®E^®lIy high plasma §.„ e®HC@atratioaffl in syeloprolifsrativ® 
^isessrfers} sr® csswse^ by tha pre^jsaea ©f ass lae^esssd qssaaeiey of a 
asrasally oeearsiag eosspoaeat of tfea s@r<sss0col«l fffsetiois of plagssr 
Tbas® resalt® thetSQioxQ as® cl^ar indicetioas foy additioaal work on th® 
k3.a©tis8 of this co®p®aent aa«l on tb« precise roi© of tb® iacreased 
q^saatity of B1.2 iis plassaa" la- tfee Eyslcprolif^retive procegSo Stssdiea wi5-_ 
£b® S3_2 ©afeagSSiats indieata that tfeesc ec^poisads have eertsia properties 
as 1^2 aaalogs tat tbat these aris aot c3,laiaally isapofftsato Thay do 
howevlr provi«i@ ®ac©5srag®ieiit for ^rtMr ®f forts oa the part of tfee 
vltsjaisi «la<gE3i3ts to pr®dac© sisailar analogs ia th® top® tliat sosse m.j 
prova eliaicsliy ®£f©ct;iv©o 

Prgg^g|_£0g£E^t Studiffis now ia progress ^©sigaffii to astiaat® tfe® ^g 
plssssa elsaraacs a.nd tfe© tarinsry, f«cal aad biliary excr«tioifl of €o ' ■=• 
labelled vitasis ij^^.ffi&iBlst©r«d iatravanoasly te^sad to tfe® pati@a£s 
own plasma protsia illl Is® carried o«st ia patl©nts with c^iroal© 

and ■ferithoi^t aaoplastie diseaseo 

€maesat5rsti®s ®f t&e active ej3^ea®a£ of tl2« aer«MMCoid fri9.e£;i0a oS 
plasBa fsro^ pstieats witk chronic syelecs'tie l®»k^ia aad its fartfess 
sjgbfsaetioffistioa asd pssrifieatioa '^ill be attsseptsi. AMittoual stisslies 
will also bs peK'forsM to dsEosstraSo coaclesivsly whether or n^t tlita 
frsetida will sebstituts fer iatrlasla f?iet©r in the patisiat witM 
Addis®siaa p©rBiei®as aa^iiae 

Rse©a£ly as (atfeylsaide ^erivativ® ®f tha soBosarbosjlic fosrsa of 

89^4 



-3- BmUi ^OeMSI-6-725-Ce) 

Gy©a0s©feal®alB has b®®a obtslsM fr<^ ®r„ E^ L®s£®r ^Ith of tto Glamj 
LaboratorlsSc This derivative skc^ld be invest iga£sd iu the sai^s ^araaei 
us^. Ib @:!^:a£d.sl££s th@ ^sthylasaids and the analiS® deriva£:iv@9o Smdi«® 

cc^!p@'£S! ®f f@et:lv3ly for bisidlag sicss o«& plass^ ssd imlirifflsiG t&et&r 
sbS y@t appear to bs biologieally ioaeciv^ in oaissalian sjrstess will Is® 



895 



=4- S©?isi Ho, NGI°6-?25Ce) 

PHS-HIH 
Indlvidsaal Prejsct Report 
Calendar Y®ar 1959 



Pars .Be. Honors j, Awards^ and Ptsblicatissna 

Piablleafeions oehgr than abstraets from this projects 

1) Wainstsin, loBo, Wisiasauan, S.M» ^ and Waeklaj, B„M„ Th@ 
plasssa vi£smiii Bj^2 binding substance: Xo Its dstsetion 
in the sercsMfioid fraetion of plassaa frosa norssal s?sbj©s£s 
and patienEs wieh chronic snyelocytie ImMmxla^ J,-, Clin= lnv@st„ 
38; 1904«1914^ 1959, 

The following SBan«sG?ipt8 frc^ this proj©gt Imv® be«n ssibssisfesd 
for approval: 

58 
Weinstein, ioBo, and ^'Jatkin^, DoM, Co B- absorptisHi;, plaesaa 

transport 5 organ distribution end ©xerataon in patisats wi£h 

iny@loprolif®r«£iv9 disorders^ solid tHEaors and nsaneoplasEie 

diaeas®,, (To be aubaiitted for publication t© Jo Clla, Invastc. ) 

W@in8t@in^ to B, ^ Weiasama, S.Bij, and Watkin^ D.Mo Tb@ 
plasao* vltfflain B,, binding sijbstans®: Il„ E^rtter pKrifiGcSico, 
from nomal and enronic sayslocytie Imsk^is plasaa by 
fractionation ©f sercroseoid on anion- eKefeang® ©@ll«loa© eolmpxis, 
(To b® 8fubaniet®d for publication t® Jo Clin, Isvasto) 



896 



3o letkead® 



FSS-IXH 
lyidividiasl Frajsct Report 
' eal©©dsr Year 1950 



SMMJl' 



Project Titl*: Mvml el@ar®ias® sfesii®® isa'patisefes with 
Friseipal Isvesgigator: Soaald Mo Watkia, M,B« 

dospsrseiag IMits: Msa© 

Msa Years Ccsl®adsr year 1959) Fatissafe &ys Cseleffi^as y^ar 1951^ 

T@tel; 1/24 

©thers l/^§ 

Pxsject Sascriptio©: ♦ 

r@5^l plsssss flow aM tisbtsiss ®&^s a®4 feo iav@©tig®£(2 tte ^^shaa- 
is®*, by ^%iclii thm kidasys femail© oatafeolieas s«3S& as imIc seid aai 
calcic ia pa£lsa£8 •?#it:fe aeoplastie ^iseasao To sasesrtsisa £lia 
kida@y§' r©l® ia sssch ilsarders a® kyperssrie^ls md tsyperealcaais 

K-sS:^@jia feploy®^; Isaal clearasMse «@chaiqt!a<, KKsyasstie assay fosr 
:^-lc acii» StttrafiltratiOB psrocedar© fos estteatiag, fil£@s®bl@ csL . _ 

gatiaafc mt&sMli Two rsEi©l fsgactioa stisslies were pasrfos^Ka^;, ckss -:" / 
®ac& of £^0 p®fci®ats 'fe^ieh eryoglssfealiaeaiao 

^jor glBsSiags; Urat® rsafesarptioa is a lisae&r fisKCtios ©f £fe® 
fliterffid srat® Isado X© isidividoals aS: tfea «st:r^s ©f filtered 

of feypesfsalee^is s-QsaslS® te a ©a^ksd iaesrease la GfS, IPF asad 
^^M, lypo^arie^Bia das fto a ^islBished reabsorpfcioa of us&tB asey 
b@ a c&asae^eristic ©f p®«:i#a4a witk ®airo©acidis2"ia sad ery ©global istst 
ftse p£6i^a£ %?i£h cry©globis1.ia^ia reabsorbed only 60% q£ £ilt@resl 
^srate lo^do Eowaver;, tfes other patieat reabsorbed fch® ttorKsl 901 of 
i±lt®rmd los4„ 

Bi.i;aifi€aaes to Saaeag Raaeagefe; Flss^ ssric acid levSl is dsteralsis; 
ia part by rssal t^abalar r©absarpfci©© of UT&tB, ia psr£ by ©rie scid 
syattesis^ asjfil ia part by t&© rei©as® csf asris acid sBd its pres^rsoxs 

fast B iaela4®d Ysg^ ^3_?._„ 

■ 897 



-z- Beti&i ^^.^Mk.&zMkiEL 



£?a^ tlssses sss^sffgsiag dastimstioao The r®aal soatribisEtioa to 
ek@ esi®£S!saa«s of pl&^sa level wa^t be ksacr^a before feyp«r«3sie:®siia 

lyps^sslsasia is a dsag®?«sus cosBplieation ®f sasopiasele diseasst. 
As in th& case of feypsnaxicesiaj iavestigaticsaa of eSaa rsaal ®ec&- 
aalss® for cl«ari®g calcisga ia bypercalcesaia ars basic to aia «m4er- 
staaiiag of th® BjwdtimBo 

1) Additiassal slsarsae® s£isdl®s la pstieats with hypar- 
srisCTia, espsclslly those witfe plasaa levels sniilclentlj Mgh 
to resslt ia filfcefsd ssrsta l©sds sseseSijs® t^^^a£©o 

2) €0ii@et sliaieai laborst^ary 4&t& on sikjIs ps-ocedssrc® 

ss esasfiffitratioa t®s£®, blood isrea aierog# Im&la, creaEiaiffi® 
levslsj, asicr08c®pie saiaEsiaatiea of «sriR@ s^gilj^sBC^ esfisiaatioa of 
isria® for proteia^rimy FSP ssc^ffltiou®, crsa^aiaa elearsaces aad 
iEtravsffioss py@l®gra®s £®r csEpfisri-sea with diser@«£ raaal ftmetioa 
stsiiss so tfefit t&® rsliaMiity of clinical iiji^ictfia of r®aal itmetion 
in ©soplaselc dis^ss© aay te fiv®l®a£®d„ \. 

3) Cterr®I®£s dats ©beaiasd &y clearance sttsdi©^ ^tk histology ©f 
eb.® ki&ey g,® sasa la sreaal biepsies aad siatopay^ esaterialo 

4) Bsv@lop a reliable tsclmiqa® fer detemiaing Wltersble calsi?as 
i§s piaass® ®ad test its rellsfeility aad reprod«cib!^lity« 

5) SI®®raaca s£ig4i@s in pstieBts ^ith feypsErcalc^ala to detag^ae 

tb® fasEoffs ifflfi®®seiag ta&ula? yesbsorptioa of ealclim and its relatloa 
to tsi&alsr Issn^liag of rasaSe and pboapfeoruso 

6) Fasthar s£«ad[|.ss ©f psroaeia fs-sctioa elaarsaces ia aeloeted p&tisats 
^Ith pifoasiasssiao 

7) St:®dy aUiti&n&l patlsaes ^isls e^aoaci«fe>srta.> 



89S 



Serial No.. mi-b-'lZljc) 
i„ General Medicine Branch 
2„ Metabolism Service 
3o Bethesds 
PHS-NIH 
Individual Project Report 
Calendar Year 1959 

Part A; 

Projeet Title: Gross Body Composition 

Principal Investigators No lo Berlin, MoDo 

Other Investigatorsj Do Mo Wafckin, MoD, 

Cooperating Units: None 

Man Years (calendar year 1959) Patient Da^a {ealender year 1959) 
Total: 4 
Professional: 4 
Othes; 

Proj@8t Description: 

(^jeetivec; This projeet is designed to study the changes in 
gross body cooipositien during the course of neoplastic diaeases^ snd 
to correlate these changes with the ellni@al eourse of the patient 
and oth@r laborstorf dAta^ and to study the aa^oia of e&neer in ths 
Sfime group of patients „ 

Methgdg^ ^^sjpl.oygd; Gross body ceanposltion will be determined fr«3 
the body density and total body water^ Body density will be dat«r'= 
mined from body ma^s and body volums^ as ese&iiured by the Si?! bcdy 
voluDt@ apparatus; body water will ba 4at«rnilned with trltlated 
water., HamaCoIegl«al status will be df^terait&ed by maasu^essaint of 
total red sail voliss^ with Cr^^^ iron twrncwer with Fa^^, and red 
sell life span with C^*.-, 

Patient fete rlsl ;- 6 patients with variaua neoplaarjs; 4 subjects 
(with DrSo Miadon end Buskirk, NIA}©)| 6 patients (U with neoplasms^ 
not studied on a sEetabollc balancs regiiaa) 

!^iog Fiadin.gs : 

1-:, Gross Body Cosaposition: 

Ifcdification €»f the ssathsd cf analysis of hellisa and electronic 
aeasjjr^ent and recording of the tKi^jeraturs in the ehasib@r have 
been satisfactorily craapleted^ In general j repllcatd runs on a 
patient do not differ SBore than 100 cCo 

Analysis of th® changes in body ccsaposition in 3 ob«9SQ swbjscts 

has b@®n eossplstado Excellent agre^sasgnt between th® calculatsd 
fat loss as aasas^red ia this project and froa saatabollt balance data 
was obtained^ Th@ eosabitrntiosn '©f thess 2 procedisrss frarnishes an 
adaquat© and satisfactory approach t© th© steady of both short end ler.j;; 

tejrsa chsaggs in gross body GOmposition,, 



Serial No. NCI°>6>727Cc) 

It is estimated that tbe standard deviation in tha difference between 
2 estijastes of total body fat, froea densitoasetry and body water 
measurements is approxisuately 0<,5 kgo 

Analyses of the other patients studied awaits the' completion of 
isetabolic balance datao 

2o Ani^Jia of cancer: 

Two patients with ciyeloid aetaplasla have been treated with 
large doses of testosterone^ Qae patient died of oiassive bleeding 
before a therapeutic effect could be aehievedo The second patient 
shows objective evidence of a hessatological response; as raeasured by 
body density and water this patient showed an increase in body fat whil® 
under therapy; the metabolic balance data are not yet available. 

Significance to Cancer Research ; This project is part of the studies 
carried out on the area of the tiaaor-host relationship. During the 
course of a neoplastic disease, large changes in body weight occur, parti= 
cularly weight losso These Eust represent sosae changes brought about in the 
Ketabolism of the host. As a preliminary and a necessary adjunct to the 
study of these changes in the host's metabolissa, it is necessary to know the 
changes occurring in gross body cisaposition, 

Anemia, occurs ia cancer to a variable degree depending upon the 
criterion of an®aia salectedo Presently available methods perasit the 
quantitation of erythropoiesiso Correlation ©f the level of @rythropoi©sis 
with changes in body craaposition xvriil afford an opportunity to co©par© the 
SQatabolisa of a single systsa with that of the whole bodyo 

Proposed Course ; The initial phase of installation and modification of the 
equipsaent has been coepleted^ The data fri^a the 3 obese subjects indicate 
that the goals originally set can be sceto At present, recruitntsnt and long 
tevm follow>up of selected patients is underway. 



son 



Serial No„ HCI-6-727Cc; 



PHS-NIH 
Individjjal Project Report 
Calendar Year 1959 



Part B ; Honors, Awards^ and Publications 

Publications otfeer than abstracts: 

la Hathan, D060, Davidson, J„Dej, Waggoner^ J<,G„ , 

and Berlin, Nolo The Counting of Barium Carbonate in 
a Liquid Scintillation Spectroiaetero Jo Lab„ and Clino 
Med„ 52: KOo 6, 915-917, 1958, 

2o Hathan, DoGo, Berlin, Nel., Studies of the Production and 
Life Spaa of Erythrocytes in Myeloid Ifetaplasiao 
Blood, 14:Noc 6^ 668-682^ 1959^ 

3o. Kathan, D„G„ end Berlin, N, X, Studies of the Rate of 
Prodtjction and Life Span of Erythrocytes in Acist® 
Letik«aiao Blood 14: Ho» 8 935-949, 1959o 



Serial ^. ..mir^-imic}. 

So 



lailvi^mi Pr©J©st Repose 
fel®ai®r Yeas- 1©59 

Pifiaeipsl IssvestigsEsr: ^asld Po Tssh®^ 

CoQ^ermtlmg Ifeits: L£ls®?aSos:j of Fhysiolsgy 

ISia Y@®rs (calsadar yess 1959 Pstisat Bays Cefil^edsr yesr IfSS) 

t®tal% 2 1/4 

Fr©f®88ioaals 1 1/4 
@£h®rs I 

?r©j©e£ ^seriptisa: 

Cfej@eeivQs: 

Ac Thrmsgh gisa us® of @as:h^£ical ^sdols of £iit^3g@si 
E^tf^olis® to stv^f ih® ef facts of sueh vsriablas as 
4£€ig; bx>TSs^ts&&, tmsiSTC growth aad rjs4i£ti(m oa £he kinatles 
of body aisrogeso 

Ac Syaebasis of S^^ labelia^ aaiao acids 
Bo Isolfttiost of nz&s ss^ amnasiiA f resi vzlas 
Co Pr«p«sr«ti©is ©f materials for 



Pasieafc Mat^glal: A pseiwat wifch lyasplwaasTGOTa has beoa «e^l®4 



-£hro^ssh maaz »£ she eous'se of her dlse&aeo P&tiaaes itl£h ^iilgis<mel@s 



Majsg gJBdiaga; The grsatfe ©f a loeal t»®or asss Clysphesayesm) te 
tha leg easm@A & slighe isssreass la body nitroges9»@ as&abolle poel 
3is@ ^Ith us ehaag® in ?st@ eonseaat for asetabolis pool eisrsse^e^-c 
Tasr® ?5®3 a siighfe lacreasa is. 4aily bo4^ nltrogsa iaco^poffstisao 
IvsMi&tifm ©f £fee leg ssass <1000 r - I dose) e«3se4 s»rk«d eaisrges^at 
©f 'e©dy sitsegeaosss ^stabollc peol -wieh great dserease ita yaea s©ass£aat 
f©s: Bsta&alic poai £3sa©v®i% Bo^ eisesgea isworposratioa sr^aisssd 

Pass B iael?i4ed ^@®,_I_„ 1® 



302 



<-2~ SsrisI lb. M€£-6°?2fCe) 

Sisaigicaac@ to Caacer Researck ; Tfe®s© aasch^s p@s®ie tte 
qsantitative stvMj of th® tjj^er beariag hsst ^itk regard t© 
nit:r©g®Q ^ta&olis® asd th(& effssts ©£ saay variables s^ds 
as diet, eherapy, eCCo tbereoa, 

Pggiposad CoBgse of tha Projact i 

Ac T@ etttdy tke affects of ra<i2.a:ti@B, ster&id'a ani t^^eir 
grsvth «»r« exeeQaively., 



SO; 



-LiimCsL^,... 



PHS-BIH ' 
ladivid^al Project Report 
Caisjadas: Year 1959 



F%sS»Iie&£ioQS othss tham abstracts ix(m this pr@j@e&: 



lo Studies of tbe S£f@et of 9i@t&^ Prot®lB araS Qflo^iQ Levels 

Soaald P, Tsek«aS^, Habeeb S&cch^, S!^^?msJ Welss^n^ 
BoaaW U. tetkia, ^rjoria Sabaaks sad Jaliss 'ihlee^ ■ 
J„ Glioc iBvfSstigatloa 38, $92-901, <l§5f) 



304- 



FHS'-ilH 
SndiVidtsal F£©j«et E®posr£ 
Calei!<3flr Y®as 1959 



ISSLAs 



Projset Ti€le: ItEvestigatioa of l^ligaaat Setie«ler Diseas® sad Sm^m 
Protein AbnoisaalitieSo 

Principsl Investigators: Joim L„ f&h.&y^ M„Do 

Other Investigators: Heme 

Cc»op@?atisg Halts: In specific aspects of the vjorko 
Pathologic Afisti^sEy Branch^ NCI 
<Djre^ To I>iitcSi@r aad So ISscCardile}^ 
Laboratory of Biology ^ NCI 
(Ihfa J, Daleoti) 
Institute for Allergy aad Infectious 3i3©ases 

(©To Jo UtS^ 

Man Years Ce®len^s^ y®«r 1959) Patient Bays (calgEdsr y©a? 1953) 
Totals 1 1/2 
Professional: 1 

Other: 1/2 

Projeet Bascriptioa: 

Objectlves s Tfee clinieal feati blocteiieal charsetaristies of sssiigiiaiai:. 
r^eieular sod relstsd . dis:#ss@s sr« r.© bfi defia©d esd @splored with 
particular atfeeneion to 'suacroglotelin^aia^ cryeglobElla^sia or cthev 
sigaif leant serum proaiMa abaoH^liti©s:„ It is fes^@d thas t© gsis -i^, 
. insight into the sssch^lsas raspoasibl® for th© .jsajor disebliag imatur: 
th@§«g llln^samsg to «i4ri^ diagnosis mM progsosis'j, ©sd to Improve 



Bfethod® g^Xs>^ed ; Chpaicai and physieedagaieal tesfeaies^ iscl^sdiES 
electrophoresis 5 wltraeesatrifiagstioa aad ehrOTiatosraphy ar@ @Eploy@4 r"LV-: 
quantitative aad cpjslitative ass@s^j®at of sen® protsia Ghai5g®§„ 
Qsiaatitative is^amblogic techniques ®r@ mployed for assgsssseat of sen^n 
antibody levels and of r«spons© to antigemic atikalstioao Histologic 
evaluation is coadtscted by histoch^ssical iKSi3aohi@toIogiCj, eleetroa 
iaieroseopic and standard procedssreso Rigorous elinicsl ^vsl^atica 
according to a specific protocol bassd on prsvio^s ©spexieasso 



Part B included Yes X W3___ 



905 



-2- Ssrial M6oggfcMl;i°Ce) 

MajQg ^ladiage; Gssssa iffiscr@glob®liis®aia awiy occssr ia pati@aes with a 
vsri@tj df (slls^e^l sad la^o^fiSGiry laaaifaseatioaso M»7phologie st«sdl«s 

plasss-eelis «?it£a ln£?assiclsar i&cltssioos in pati«aes with ssallgBsat 4is€A8e 
a^d @aa70gl(^uliii^iao %@ iatraaisclda? (aaS iatracytoplssmic) laclsselomt 
^©re sho%m Co bo ir^iarkably sisailfir to th@ clrcelatlag gaasHi sacroglolmliQ 
sssggestiiag a eos^osi ld@atl&y asd lad«e4 that tlie lyaqpbocytold plaas^-mlla az» 

ehs siee of ssadroglobulin fossatloa,. 

SleaS vigsos5i£y ^ay b« s»rkediy @l0vat®4 by la^ga e^o^s^es of ssnsa 
mcsroglob^IiEseo @is@$t 4(^{mse?atloa chat sac?oglo¥«alla i@d^«iS elavat@S 
hl&&d viscQBitj was tba l^sediate caeee of cardiac falltsre la o&e patisBt 
^itli IMltsd Inat oehertfis® &4&qvAt6 cardiae resarve paxaitted Bat&hllBkmsa.t 

of m th@r3p^£ie ir@g&^ss dsisiga^d to keep the s«st@ saeroslob^aliiis h«l«m ^h@ 
erisiesl lsiv@lo 

Sigiaiflesae® to Saacag R)sa®ayeli r Tha fisding of a characteristic c@ll in 
giacroglob^Iisa^d.c lysiphcsia should ia^rovc tha rscog&ltioa of this dis®as@o 
It i^ill bs of ps^tieular lQt®r@st to kno^ if related aoraal cells ar@ 
responsible for s;»^a ^acroglo&Blia symthssis^ for the gaassa Kseroglob^lias 
snoi^lly soBtalm a sp<scial grotsp of antibodies as noted in the ^£'>6-°702Cc)o 
?h@ c@ll3 &i fO£Ssati@n for t1a<ss@ antibodies are jjEiportant to iass@aIoglsts and 
6@col@gl@f:@ €one@n£©4 ^ith tlsssss trassplantatlos^ and i^s^mityc 



Rscogaitioa of thm Bjmptcssa of esccssslve blood viscosity vill allo@ 

pr«^p£ tfe®rapy to avert ^?laat seay ©tfesrwis® he a progressively deteriorating 
clinical eosraSb 

Proposed Cojarse of the Project ; Therapettstic ohaervationa in BMcroglotelinesBis 
^Umms ^n c©asism@ wisfe sSMles o§»8@?^aei0a8 la eryoglefeullE^ie €is@ss@So 
^@ affeg^a ®f ffisiigB^fe m£. MaaF-asiagsaiSfc disease ^t& dysgJSE^globwlineads 
on lionaal g^ma global ia coaponsats and i^ssne saechaalssss will be exploredo Aa 
effort will be saad© to define the 'criteria by which noa malignant diseases with 
associated eryoglo&salins or increased gai^s sacrogiobulias i^y be distinguished 
fro^ malignant dlsssses with sfjsdlar changes, -. 



306 



Soxisl ^OoMSI-6~732<'Cc) 



Xsaiividsial P?oj®ct Report 
fel®s4ss Year 1959 



„ ?gg £__l; Isaosrsj A^iaMs^ aa^ P^licaglotts 
FsjbiieaaieiEai ©£her Shss a&sSrasts: 

fhs H.^-s tops etiology of tfe® B^CE-oglobisiiiassia of l^a Ideas trcsi 
J. lafcc Saac^r lasto 22: S87-917, 1959o 

2o Ziibrod,, Q,Q^ , Hilbieh, Tofo, fahey, J<.Lo, Satoher, Tofc , Fssc, F,,A„ 
sad ©Bser^aHj, EcFo 

WaldeastrJ^'s ^croglofesallaeaila, Aimals of Istt, fedo 50; 
1010-1027, 1959„ 



907 



Serial No, HCI°6-738{c ) 

lo General Medicine Bra.i 
2„ Metabolism Service 
3o Bethesda 



PHS»NIH 
Individtsal Project Report 
Calendar Year 1959 



Patt A; 



Project Title: A Study of the Factors Controlling Red Blood Cell 
Synthesis and Destructiono 

Principal Investigator; Thoasas A« WaldsHarm^ MeD„ 

Other Investigators: Hathaniel I„ Berlin^ MoDo^ Shennan Mo WeisssEsn, MoD„ 
and Edgar Levia^ M<,Dc, 

Cooperating Units; None 

Man Years (calendar year 1959) Patient Days (calendar year 1959) 
Total: 1 
Professional: 1 
Other: 

Project Description: 

(a>j@ctiv@3 ; To stwdy the factors controlling red blood cell synthesis 
^l^d destruction in nonsal and in pathological stateso 

51 

Methods Bgploy@d ; Quantitation of erythropoissis by th© use of Cr 

labeled red calls to deteraiiae the total circulating red cell volynse, 
Fe^^ labeled siderophilin to detenaine the rate of red cell pred-dctio-n 
and C^^ labeled glycine to determine the red cell life 8psn<, Assay of 
erythropoiesis stisimlating activity in the fasted rat by tfes red cgll 
radio-iron uptake and ngticalocytosiSo 

Patient ^terial ; A patient with e cystic cerebellar hesangioblastosEa 
and polycythesaiao 

Major Findings ; The steadies of spleaectosay and isethylcellalose infusion^ 
in the dog^ in part previoasly reported, have been cosjpletsdo Follo^?ing 
splenectojsy in the dog^ there was a 31 percent desliss in th@ total 
circulating r©d cell vol^ssa persisting for at least the reaiaiaisg 5 sso., -- 
of the st«dyo Ho change ^as noted in the red cell life span bat there ^.^ 
was a 36 percent decrease in red cell synthesis as estiseated hj th@ Fs"'"' 
red cell tumovero The reticulocyte percentage increased 138 percent cs 
a result of an increased intravascular reticaiocyte Bjstiiration tlsse 
following splenectosayo Ko significant hestatological changes were noted i 
sham operated controls a 

Part B incltsded Yes x Ho 



SOS 



:I-6'738Cc) 



We h&ve utilized methylceilwlose infesions in the dog in an attsapt 
to produce an esperisneatal snodel of hyperspleniaajo Although moderate 
splencHaegaly and anesia with a shortened red cell survival was achieved 
the animals developsd ssarked renal dasEag© and uretnia,, Following 
at^inistration of 125 grains of dessicatsd thyroid daily to doga there 
was a 25 percent increase in the total red cell volume. There was a 
comparable increase in the rate of red cell synthesis but no change in the 
red cell life span. 

Arterial shunt studies directed toward determining the sits of 
erythropoietine production have been continued but no new conclusions are 
available at this |iiGeo 

A non-dialyzable factor with ffiarked erythropoiesis stisiulating 
activity was isolated fr<Hn a cystic cerebellar heaangioblastoiaa in a 
patient with the association of this ttmor and polycythemiac This is 
the first d^minstration of earythropoietiae like activity in any ssaterial 
other than urine or plassiao It swpports the concept that tfea 
polycythesoia associated with scm& eerebellar h^saangioblastosas is catssed 
by the production of a red cell stiEulatiag factor by the timoro 

Sifjiaificanca to Cancer Research : Aneaaia is a ssajor ccssplication of the 
neoplastic diseaseso The pathogeneses of the an^aia is only partically 
«aaderstood„ A knowledge of the factors involved in erythropoisais both 
in nonoal and decreased states is reqtsiredo 

The studies performed on the patient with cerebellar hessangioblastossa 
and polycyth^aia have aided our understanding of the pathogenesis of this 
assoeiationo 

Proposed coiirs® of Stedy : To continaa the stady of the factors controlling 
erythropoiesis in the experissental sniiaal aad mano To conduct studies 
directed toward detasrsaining the ch^sical nature and jBode of action of the 
cereb@llar erythropoissis stiictslating faetor^ 



909 



Serial NOo MCI- 6°- 738(c) 



PH3-NIH 
Individual Project Report 
Calendar Year 1959 



Part B ; Honors^ Awards ^ and Publications 

Ptiblications other than abstracts: 

lo Berlin^, Melo^ Waldmaniij T„Ae^ Waissiaan, S^Mo 
Life Spaa of Red Blood Cell„ Physiol^ ReVo 39; 
577-616^ 1959o 

2o WeisKZsatiy So M« ^ Wal&sann, T^Aa, Berlin, Nolo 

Qssaatitative Measureaient of Erythropoiesis in the 
Bogo ATOo Jo Phyaiolo in press, 

3o Waldsann^ ToAo, Weisssoan^ Sail, , B^rlinj, Nolo 
The Effect of Splenectosig?' on Erythropoiesis in 
the Dogo Blood j, in pressc 



910 



Serial Hg„ ^I_852 
PHS-SIIH 1^ Fs£ho logic AastoJH^ Braacla 

Individual Project Report 2^ Gft©dlagB©stis Sesrviee 
Calea«iar Year 1959 3„ Be£hesda 14, Md. 



Project Titles Esfoliative Cytology Applied to Wi^&n Biagiaostic 
Problems aad Rssesrcli Problem 

FriEcipal Xi&vestigators Dr. Richard A. MalsBgrsa 

Other Ssvsstigaterss Dr.. Blisabetli M. Cfeis 

Co©peraei-flg Uaiesi Nobs 

Maa Years (Caleodar year 1959)2 
Totals 8 
Professionals 4 

Others 4 

Project Descgjptioa ; 

Tfee priisary responsibility of the C^Eodiagaostic SesnTica is t© prs-rlde 
the stsff of the Clialcal Center with aa accusate aad cogrplea© ©s^fcsllative 
cytology service o la additioi^;, the Cytediaga©stic Sei?vlc© collafeorat'ss is 
varioiss clisical ressss-ch projects ©"^eluaSlsg eaassr thasapyj, tbs fesrssissasl 
states of the casacer pstieat, the cous'ss sad sat'^ral histe^ of tha cisscsr 
le.sioa, astd the saat«^Bieal aad physiological ebosges isa the feisas body 
associated with vsriosas pathologic coffiditl®as» Ths sees^s^lishisast of these 
objecti'^'es aecessitstee the coa£in«£l iispToves^at la tschaiquss and the 
origiaatioffl of ae« cytologic laethods is the laboratory,, 

Methods Eiaployed g 

The stiaadard Papaaicolaou staiaing psecedurs Is applied to praparstisss 
of various hisman speeinjeaSo These spsclsssas a'^e thsa screened -ssd evsluaSed 
for Esorphologlc chajages ±n the call«lar elessents* Speelal procedures 
involving the use of fluoreseeat isieroseopy, millipore filtration, and y&tlciss 
special staiBing techniques are required In oany instsjiceso 



91: 



Fafcieafc Material ; 

the figures cosapll&d hmlcm are for the last tuelv© asciiths C12) . 

fype of mterial eatamlned Ac^ssloas HUdgs 

Wound wasblBgs 126 685 

Broacbiel -washings and sputa 116 324 

Vagiaal and cervical 1,079 2^961 

Misc®llaaeou8 Fluids 531 1^388 

(gastric, prostatic, breast^ pleuralj, etCo) 

Zaiprovaaent tQchmlquas 186 439 
Bloods 



1,509 5J^1 

3,547 11,758 



2,450 


9,076 


140 


414 


182 


658 


188 


417 


X22 


369 


3 


8 


10 


50 


451 


761 



Institute AceesBlons # Slides 

KCI 

mi 

M£AW 
NIAXO 

mmB 
mm 

HIDE 
Referred 

Ma^or Findings; 

la The laajor contribueioo of thm Cytodia^ostic Service to tte 
diagnostic facilities of the clinical staff hss b®@n ths early diagnosis 
of nsoplasffls of thm c®rvix and lung and of othmw sites not pre^^iousiy 
diagnosed o In several instances, exfoliativa cytology provided tte© only 
tissue confinaation of neoplastic disease before death of the patient <> 

2o The quantitative ©valuation of wound washings j subajitt©d by the 
Surgery Branchy, liCI has been continued with the d^ionstration .of aeoplastie 

cells in njany of the washings o 

3,,. In collaboration with the S.urgery. Branch, mi and the Po So- Fufelie 
Health Service Hospitalj, Baltiiaore, approsteately 1500' blood specimens will 
have been collected this year frcaa cancer patients for essmination for 
cancer cells o Cytologic esaminatloa of this material by the streptolysia 
Eillipore technique indicates that cancer cells are jsore often found in 
cases of non»resectable cancer than in resectable cases o The cells are 
snore frequently found in local blood fr<sa the tisaor site than froa peripheral 
blood. In a number of patients in which cancer cells have been fouad in £h® 
local blood there has been no recurrence of the disease to date follo%7lng 
surgical treatioento 

4o The use of the sex chrcssatia detescsination on patieats with various 
endocrine disorders has contributed to the ultimate clinical macagessent of 
these patients o 

912 



PAGE in. Ssriai |fo„ lg.I_852 

Ha jQ g FJfadliags (Co ntinued}: 

5„ The application of the laillipor® iiltsatiaa £eebaiqu@ to va^ioiss 
B^tva vagiaai cytology spseimsns has increased tli@ efflclsncy and dmcvm&sed 
th@ tisae required to process th@ speeimsHo It. has also pesmittsd a 
diagnosis to be mad® which ^ould have been l@ss likely hy the older 
processing usathods, 

60 The fluorsscent antibody t@chiiiqu@ for the dsiaonstration of anti-^ 
nuclear substances in the seriaa has besQ applied to the diaguostic study 
of systemic Lupus eryth^ssatosis and related diseases « The davelopggat 
of this procedure was done in eollafeoretion %?ith Dr. Howard Goc^an of 
the National Heart Institute and the s^thod is now employed in the routine 
study of patients with Lupus, cheusaatoid arthritis^ Sjogren's disease &nd 
other collagen diseases » 

7o Exsminatlon of approximately 70 urine speciatens using the mllllpang 
filter technique in collaboration with Bro George Gwen of NXAXD has revealed 
the presence of intracytoplasnsic bodies Ira macrophage like cells o Xhese 
bodies were observed in the cells of virtually all the speciesens escasinsd 
regardless of the age, sex or medical status of the patient o This obser» 
vation is in disagreement with the report of others who found intracytO'^ 
plastic bodies of the san^ sort in association with viral diseases , 
particularly aeasleso 

Si^gnlfic ance of the Program; 

lo The diagnostic value of exfoliative cytology in the clinieal 
evaluation of cancer and its early detection is readily apparent fri^ 
the activities of the Gytodlagnostic Service in the Cliaical Cerater assd 
throughout the country o 

2o The wound washing program contributes to a better uaderstandiag 
of the factors Involved in tuaor growth^ progression sad Esetsstas^s aad 
the reaction of the Individual to his neoplasia., la addition, the 

application of this Information to cancer chessotherapy laay be of v®laa„ 

3o The fluorescent antibody technique applied to serum of patients 
with arthritis, systesilc Lupus erytheasatos is and collagen diseases provides 
one of the most sensitive tests available for the demonstration of anti- 
nuclear bodies in the serum o Its use contributes to the ecraaplete 
evaluation of patients with these diseases and ©ay be of help in gaining 
a better understanding of the etiology of these diseases, 

4„ By the use of sex chromatin counts and techniques for hormonal 
evaluation the endocrinologist is provided with a valuable tool for 
guidance in diagnosis and therapy. 

5o The study of tiaaor cells in the blood presents the potential 
of gaining a batter understanding of the factors involved in metastasis o 
It may be of value in studying certain types of therapy of the neoplastic 
diseases and diagnosis. 

913 



?AGS I¥o Seri£-1 Bo. HG1__852 

Signific ance^ of the Program CCon£inaf,d); 

6. The developsent of n«w cytologic tecljniques and the ecquisitioa 
of spscimass from different sites in various diseases continues to 
enhance the value of the exfoliative cytology procedure as a diagnostic 
aido The value of the millipore filter technique is readily apparent and 
it is anticipated that this procedure will replace the ssaear prepaeatioas 
in many cytologic laboratories o 

7o Excretiim of active virus in the urine is a well docuaseated 
phencsienon in soase human viral diseases. Whether the virus is intracellular 
or extracellular at this tiiae is not clear however « The devslo^ssnt of 
methods for the d©Bons£ratioa of virus ia the urine is therefons of 
potential value in the anaamentariusi of virus study and in the SKpansion 
of our knowliiidge of viral diseases. 

Proposed Course of the Project; 

I. The continuation of a compleS;© and adequate cytologic service 
for the staff of the Clinical Center is our primary purpose. Continued 
collaboration with the Clinical Center staff in various research projects 
and the development of new and improved techniques within the laboratory 
are an lasportant part of the future program , 

2» A continuation of the wound washing and blood study by 
quantitative techniques will be continued in collaboration with the 
Surgery Branch » 



Part B included Yes (:g) No < ) 

914 



PAGE ?. Serial No. MCI 852 

Part B o 

Publications othar than abstracts from this project; 

Del Vecchio, Pasco a., DeWitt, So Ho, Sorglll^ Jo I,, Wasd^ Jo S., 
Wood, To A., Jr.,, and Malmgren, R. A. Application of Millipore 
Filtration Technique to Cytologic Matarial. J.KX.X. 22: 427-431, 1959, 

DeWitt, Sara Ho, Rsbson, Alan S., Legallais, Frances V „ ^ Del Vecchio, 
Faseo Ro and Malssgren, Richard A. Chrcsaocenters Reseasbling S£k 
Chresnatin in Human Tissae Culture Lines. J.HoC.I. 23: l039-=95, l959o 

Malmgren, Richard A. and Potter, John F«, Cancer Cells in the Circulating 
Blood, Southern Medical Journal. 52: 1359-62, 1559, 

Honor Awards a nd Mesaberships; 

1. itosrican Medical Association A^-ard of Merit for Exhibit, "Tumor 
Ceils in the Blood" by Drs . Potter, Kaiser, Malmgren, Hilberg and Fruitt. 

2. Incentive award to Mr. Pasco R. Dal Vecchio and Mr. Philip R. 
Joraa of the Scientific Reports Branch, Division of Research Services for 
development of Millipore Filter Holder, October, 1959. 



315 



Serial NOo NCI 853 
PHS-NIH lo Pathologic Anatomy Branch 

Individual Project Report 2, Surgical Pathology and 
Calendar Year 1959 Posts^rtem Service 
3o Bethesda 14, Maryland 



Part Ao 



Project Titles The Departzoent of Pathologle Anatomy (Pathologic Anatomy 
Branch, NCI) has no specific research projects <> Its 
diagnostic services are designed to serve the iavestiga^ 
tive efforts of the NoI.H, staff responsible for patient 
care„ In many instances^ investigative study of 
pathological material is of great importance to clinical 
researeho 

Staffs 

* Dro Harold L» Stewart, Chief, Pathologic Anatomy Branch 

* Dro Louis Bo Thomas, Head, Surgical Pathology and Postaortem Service 

* DTo Richard Ao Malmgren, Head, Cytodiagnosis Service 

* Dro John Ho Edgcomb, Pathologic Anatomy Branch 

* Dro Alan So Rabson, Pathologic Anatomy Branch 

* DTo Edward Price, Laboratory of Pathology^, NCI 

* Dro Elizabeth Wo Chu, Pathologic Anatomy Branch 

+ Dro Thomas Fo Dutcher, Resident, Pathologic Anatomy Branch 
+ DTo Ro Gerald Suskind, Resident, Pathologic Anatomy Branch 
+ Dro Martin Hicklin, Resident^ Pathologic Anatomy Branch 
+ Dro Jack To Lasersohn, Resident, Pathologic Anatomy Braaeh 
+ Dro William Co Roberts, Resident, Pathologic Anatomy Branch 
+ DTo SeyoKJur Mo Sabesin, Resident, Pathologic Anatomy Branch 

* Dro Leon Sokoloff , Laboratory of Pathology and Histochemistry, NIAMD 

® Dro Harold Ro Stanley^ Oral Pathologist^ Clinical Investigations, NIDH 
" Dro Igor Klatzo, Head, Clinical Neuropathology Section, KIKDB 
® Dro Herschel Sidransky^ Laboratory of Pathology, NCI 



* - These Pathologists constitute the staff of the Department of 

Pathologic Anatomy, Clinical Center (Pathologic Anatomy Branchy 
NCI) and spend a major portion of their time in the service 
and diagnostic work of the Department o 
■¥ ° These physicians are full time Residents in the Departsseat ©f 
Pathologic Anatomy o 

* ~ These associate pathologists spend part time in the activities 

of the Department of Pathologic Anatomyo In additioa, several 
pathologists on the staff of the Laboratory of Pathology, NCI; 
ioe= Drso Swarm, Dawe, Banfield, O'Gara, Stanton and Love, are 
available for consultation with the staff of the DepartEient 
of Pathologic Anatomyo 



916 



Page rio Serial Eo. mi 853 

Project Description ; 

No specific researeh projects,, The sasvice fimctioas of the 
Departssent during the twelve ajonth period, Uovember 1958 through 
October 1959 includsds 

a) 265 autopsy eieaminations . On page 4 there is a table which 
shows the number of atitopsies performed for each InstitutSo The residents 
perform nearly all of the postmortem dissections xmder stspervision of the 
staffo A gross review is held by the staff and residents each morning 

at 8; 30c The residents review the microscopic slides of each autopsy 
with one of the staff before completing the autopsy protocol. 

The staff assists the residents in preparing for the ntuserous 
clinical conferences in which the Department participateso 

b) 2601 specimens were accessioned in the Surgical Pathology SectioUo 
A complete listing of these specimens, by Institute, is given in the 
accompanying table . These specimens are initially esamin@d and described 
by a resident and their reports are checked by the staff. Associate 
pathologists from other laboratories or sections are frequently consulted 
aboot problems in diagnosis in surgical pathology end/or asstsse responsibility 
for handling certain tissues removed for specific research purposes o 

c) Cytodlagnosis Service; The report for this service is subiaitted 
separately o 

The objectives of the Department of Pathologic Anatomy in the 
Clinical Center are twofold? first, to furnish a diagnostic service 
and second, to aid fr<«n a morphological standpoint the various clinical 
research problems which are xmder study In the Clinical Center <> In the 
first 2 or 3 years after the Department was established, saost ©f the titsje 
of the staff was taken up by the diagnostic responsibilities. During the 
past year, especially, the staff has been able to spend more time in 
collaborative research on Clinical Center patients with various maabers 
of the Clinical staff. 

The methods used include those standard methods used in describing, 
fixing and sectioning various human organs and tissues » A wide variety 
of special staining procedures are usedo 

Distrib ution of human organs and tissues ; 

At present there are approximately 30 scientists in the various 
laboratories at NdoHt. who have requested particular tissues from 
surgical and postmortem services o On many occasions it is passible to 
furnish these investigators with fresh human material o A list of these 
investigators is kept by the secretarial staff and telephone calls are 
made to the various investigators whenever surgical or postaiorfcesa 
tissues are available,. 



917 



Page IIIo Serial lOo KSI_S53 

Coaf erences g 

Th@ staff sad resldants take aa active part is s&uo§rous Clinical 
Canter and Institute conferences o In addition, there are several con- 
ferences regularly hald in the Department including a surgical pathology 
conference, brain cutting conference and joint pathologists conference o 



Part B included Yes (s) Ko ( > 

918 





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Ul 



PAGE Vo Serial Bo. NCI 853 

Part B --. 

List o£ Publlcatioas and Collabogative lEvestigatlve Projects g 

Ao Dr. Louis B» Thoiaas ; 

lo Boggs, David R., Frei, Emll HI, and Thosaas, Louis Bo 

Clostridial Gas Gffiagrene and Septieeisia in Four Patieats 
with Leukeasia. New England J. of ifed, 259; 1255-1259, 
Dec, 25, 1958 » 

2. Fox, Frederick, Davidson, John and Thceias, Louis B. Maturation 

of Sympathicoblastoiaa into Ganglioneuressa: Report of Two 
Patients with 20 and 46 Year Survivals Respectively,, 
Cancer 12 g 108, Jano-Feb, 1959 » 

3, Fritz, Richard Do, Forkner, Claude Eo, Jr., Freireich, Eail J., 

Frei, Efiiil IIX, and Thomas, Louis B. The Associatloa of Fatal 
Intracranial Hemorrhage and "Elastic Crisis" in Patients tsith 
Acute Leukemia, New England J, of !fed, 261 ; 59, July 9, 1959. 

4o Paton, David and Thosjas, Louis B. Simultaneous Occurrence of 
Primary Malignant MelanonKis of the Eye and the Skiao A<.M,A„ 
Archives of Ophthalnwlogy . 52; 645-652, Ctetober 1959 o 

5o McFarland, Jaujas; Thoasas, Louis B., Gilbert, Joseph and Morrow, 
Andrew, tfyoeardial Necrosis Following Elective Cardiac Arrest 
Induced with Potassium Citrate, To be published in the Jc of 
Thoracic Surgery, 

6c Freireich, Esail J,, Thomas.. Louis B,, Frei, Bail III, Frits, R.D., 
Forkner, C.E. A Distinctive fyp® of Intrscersbral Haiaorrhaga 
Associated with "Elastic Crisis" in Patients with L®uksiBia„ 
Cancer, la press, Januairy 1960 o 

7o Moore, Edward Wo, Thosaas, Louis B,, Shaw, Richard K„ aad Freissich, 
Emil J, Central Nervous System in Acute Leiiksssia; A postisortem 
study of 117 consecutive esses with particular refaranne to 
heasorrhage, leukemic infiltration, and the syndroms of sisai-iageal 
leukesiia, AoM,A„ Arch, of Int. Med, In press, 

8, Clinical Center Staff Conference at National Institutes of Eealtho 
Moderator; Von Sali?aann, Lwdijig, Discussants; Kaufman, EerbsrS 
E,, Haase, Gunter; Bartter, Frederick C, and Thoiaas, Louis 3o 
To be published in Annals of Internal Medicine, 

9o KaufEtan, Herbert E, and Thosaas, Louis B, The Vitreous Opacities 
Diagnostic of Familial Primary Aissyloidosis , To be published la 
Annals of New England J, of Mgd„ Dec, 17, 1959, 



920 



AGE ¥!<, Serial No, HCl 853 

List of Publications and Collaborative Inv@s£iga£lva Projects (ConSlaued) ; 

10 „ Hiibish, Theodore F., Besse, Byron Ec, Jr., Lusted, Les B., 

Daves, Marvin L., Thomas , Louis Bo, and Forkaar, Claude E,, Jr. 
Acute Leukemia; Skeletal Manifestations in Children and Adults, 
A.MoA. Arch, of Internal Medicine 104; 741, Nov, 1958, 

11 o Current Projects which will lead to Publications ; 

1, Cancer of the Uterus, Dunham, Lucia J., Casper, Julian, 

Dom, Harold F., Thomas, Louis B., Edgcoaib, John Ho, 
Syaseonidis, Alexander and Steisart, Harold L, 

2, Study of Clinical Center Patients with CarcincKsa of Cervix 

Treated by Pelvic Exenteration with Dr. Robert Smith, Dr„ 
John Dillon and Dr, Jack T. Lasersohn, 

12, Lectures i 

1, Aspiration Biopsy, Lecture - desKjnstration with Dr, Alan S. 

Rabson as part of short course in surgical pathology. 
International Aeadeiay of Pathology, Boston, Mass,, April 
20, 1959, 

2, Moderator - Clinicopatho logic Conference, Annual Scieatlfie 

Assesably, lledical Society of the District of Colusbia, 
Novesaber 25, 1959, 

B. Dr. John H» Edgcomb g 

See separately submitted Annual Report, 

C. Dr, Richard A, Mali^ren s 

See separate report for Cytodiegnosis Service, 

D, Dr, E.B. Price, Jr. ? 

1, Kinsey, D,L,, Price, EoB,, Jr., and Smith, R,a. Effectiveness 

of ChesEotharapy in Relation to Extravascular Invasion hj Ee^oUc 
Tumor Cells o Accepted for publication in Cancer, 

E, Dr. Thotaas F. Dutcher s 

1, Dutcher, T,F, and Fahey, J,L, The Histopathology of the Ifeero- 

globulineiaia of Waldenstroa, J.N.G.I, 22s 887-917, 1959o 

2o Zubrod, Charles G,, Hilbish, T,Fo, Fahey, J,L,, Duteher, T,F,, 
Fox, Frederick A,, Ossensan, Elliott F, WaldenstrHm's Macro- 
globulinemia; CPC at the National Institutes of Health, 
Ann„ Into Med, 50s (4), 1010-1027, April 1959, 



PAGE VXX. Sesiai KOo NCI 853 

3„ Current ggojects ^-hlch will lead to Fublieatioas g 

lo Edgar, Ro aad Duteher, ToF» The His £opatho logy of the Blssg- 
Neel SyndroHKo 

2o The Syndrona of occult gastrointestinal loss of plasma proteins « 
Waldisann, Thoraas A., Steisfeld, Jesse Lo, Duteher ^ Th^sas F., 
Davidson, Joha Do and Gordon, Robert S,, Jr., will be presented 
at the Aiasrican Gastroenterological Association H^e£ings in 
the Spring of 1960 o 

3o The InsDaunocytocheaiical Demonstrations of Intranuclear Localisa- 
tion of 18S GasE^a Mscroglobulin in the Macroglobulineisia of 
Waldenstrom. 

Fc Dr- Martin P. Hicklin s 

lo Marsh, H„Hc and Hicklin, Martin D. Survey of Staphylococcal 

Types in Hospitals in the District of Columbia, Maryland and 
Virginia, In press. Medical Amials of the District of 
Columbia o 

2o Deisoastratien of Bacteriophage Typing of Staphylococci., 

Annual Meeting of Maryland Society of Pathologists, Baltiisog'g, 
Mdo October 24, 1959 o 

3o Projects % 

lo Incidence of Multiple Cancers in HXH Autopsies, 1953=1957, 
with Mr. Don Loveland, Statistician, NCI. 

2o The Ovarian Response to Human FSH with Dro Louis Bo Thcaas 
and DEo Mortimer Lipsett, NCIo 

Go Dr. Ro Gerald Suskind g 

lo CPC on toKoplassESsis with Dr« Jacobs and Dr, Kayoe will fee pub- 
lished in Annals of Into Medicine o 

Ho Dr, Seyiaour Mo Sabesin g 

1„ Current Projects ^hieh will lead to Publications g 

lo Electron Mieroscspy — =■ Learning techniques ©£ Eleetroa 

BJicroscopy is laboratory ©f Dr=, Williasa Banfieldo Will scon 
begin project under Dr„ Banfield on Electron Microscopy ef 
collagen in various diseases, ioSo, rheumatoid arthritis o 



S22 



PAGE VIII. Serial Noo HCI ^53 

2» Case Repsffts to be writteas 

lo Cdsgenital heart witli smaleiple defects^ sitna tsi x^arsas 

and agf&oeais of spleen o 
2o Dissesinatsd Gryptocsccosis la a pstieat ^ith. Hodgkin's 

Disease o 

t. Dr. R„ Co Boylan g 

lo SeporE of vascular lesions in £^o patisats with derma tos^ositis 
ia collaboration with Dr„ Lsoa Sokoloff ^ 

2o Aasistiag Dro Louis B. Thotaas in psehologicai study of haiasters 
infested with schistosoasiasis, 

Jo Pro Ellaabeth Chu g 

See «eparete report froEs Cytodiagnosis Searvice, 
Ko Pro Alan So gabsoa g 

I9 Rabsonj, Alan So and Legallais, F.Yo Cytopathogeaic Effect Produced 
by Poly«Ksa Virus in Cultures ©f Mllk°Adap£®d Jferine Ly^hossa 
Cells (Strain P388D1}» PrsKio SoCo Espo Biol, and Msdo 100 g 
229-233, 1959 

2„ Ro Love and Rabson, AoSo Cytochenjical Studies of Milk -Adapted 
Murine LymphoKia Cells (Strain P388D1} Infected with Polysaia 
Virus o J.N. CI. In press o 

3» Malmgren, R.A., Rabotti, G., and Rsbson, A. So Intracellular 
Localization of Polyoma Virus Antigen Demonstrated with 
Fluorescein Labeled Antisera. 

4., Rabson, A. So and Kirschstein, R.L. Intracranial Sarco-sas Produced 
by Polyoma Virus in Syrian Hamsters. Archives of Pathology, 
In press » 

5. Rabson, A. So and Legallais, F.Y. Gro^jth of Hisiaan Epiderraoid 

Carcinoma in Tissue Culture Usiag Nonfat Milk Msdluao Archives 
of Pathology 68 ; 342, 1959, 

60 DeWitt, Sara H., Rabson, Alan S., Legallais, Frances Yo, 

Del Vecchio, Pasco Roj, and Malnsgran, Richard A, Ss3S Chrsjsiatin ia 
HuBian Tissue Culture Cell LiaeSo JoNoCoIo la presSc 

* o Current Projects which will lead to Publications ; 

lo Morphological studies of patients with cryoglobulinemia in 
collaboration with Dr, John Fahey and Dr, Paul Sch'sjabo 



323 



PAGE S3C Serial HOo NGI 853 

2c Mospliologieal studies of patients wifeh hyperparathyroidism 

in collaboration with Dr, Fred Bartter and Dr. Robert Smithy 

Lo Dr, J»To X^sersoha g 

lo Projects ; 

lo The function of the eosinophil °- Experissantal procedures 

Eosinophil response in blood of guinea pigs is being ccispared 
during active and passive anaphylaxis „ In collaboration 
with Dr. Richard Mal^reno 

M„ Pro William C. Roberts g 

lo Projects ; 

lo Report of case; ^nnps ^ocarditis with Dr. Sam Foko 

2o Idiopathic siedial cystic necrosis - report of case. 

No Dg» Katharine M. Herrold g 

See separately subsaitted Annisal Report. 

Oo Pro Robert Peters g 

Morrow, Andrew j Peters, Robert j Waldhausea, JoAo, Bloodwell^ RoD. aad 
Bra«n-aald, E. Supravalvular aortic stenosis g Clinical hsHSQdjnassie 
and pathologic observations. Circalatioa. In press. 



824 



Ssriel MOo mi 500 
1. iMhox&toTy of Pathology" 
2= Office of the Chief 

3„ Bethesda^ ^syl&nd 



ledividual Project Report 
Calendar tear 1959 



Fart A,, 



Firoject Titles Sistogesaesls aad Bs£hoIogy of Induced and Sponfcaaeous 
Tumors of Laboratory Aainals 

Pslacipal Iavas4:ig&£or s Bsroid L. Stewart 

Other Investigators Ksthariass C. Snell 

Cooperatisag Ualtss 1. Br=, David Johason^ MIM© 
2, Dr. John 3. Cailioua;. WIM. 

Ifea Years (calendar year 1959) s 

Total! 6 1/3 

Professiojmls 2 

Others 4 1/3- 

Project Dascriptioa; 

Objectives g fo gsia knotrledge of the etiologie aad pathologic 
factors in the production of neoplasBS io eiq>erisss3a'tal anisials and to 
laake this inforjnstion available to other lawestigetors. 

Ifefchods g 1. Adrenal cortical carcinosns. 494, e transplantable « 
honconslly "active tusior that originated in the adrenal cortex of an 
Osboms-i&sdel rat^ has been studied during the course of 12 generations 
of tr&ssplantatioa in intact^ nature hosts. CSiesHical estraction and paper 
chrojsatograpfey studies of the ttsmor tissue are being nade by Ttx. David 
JohnsoQo k s^ibliae of this tumor, designated 494H, that developed when 
ttffisjr 454 ?ms grown for one generation in hypophysectoaised rata aad then 
returned to intact satisre rats, has been studied for 6 generations o Trans° 
plsatation o£ tumor 494 to bypophysectomised rats is being repeated to 
detarsaina whether trass fonsse ion to a tumor similar to 494H will occur 
agalOo In collaboration with Dr. E. C. KacCardle a paper dealing with fche 
cytologic ehsages in the kidsseys of rats bearitsg tuaior 494 is being prepared. 

2, A study o£ the spontaneous neoplasiss occurring in the 
tlastoffiys^ a South African rodent^ is being continued. To date 119 of these 
rodents have been necropsied^ and tissues from about half of thejn, have been 
esamined histologically. Several tuiuors have been transplanted to jastomys 
pre-treated with various doses of irradiation^ since transplantation of 
tumors to untreated Mastomys has been entirely unsuccessful . 

3„ A long°"terni study of the effect of age upon untreated rats 
of various ages is being continued. Spontaneous tumors occurring ia these 
rats ere being emsaerated and classified. 



925 



Serial No. 
EMS -HE 
lEdivi,duaI Project* Report 
Calendsy Year 1959 
Part A (coBSinasd) 

4<. Monkeys tfesfe received injections of carcinogenic substances 
into the wall of tfee glandular stomach approsiaately 10 years ago are beiog 
necropsled and studied to ascertain whether these substances may have 
prodticsd gsstric carcimynSo 

5= Collaborative work with Dr. :^rold P. Morris g ApproxiiGataly 
4000 histologic sections from rats fad either di^acatylffinsinof luorsne or 
2,7-fluorenylenebissceeamide have been essmiaed. A ronwacript is being 
prepared on the effects of these two cotapounds upon various organs of the 
treated rats, sad sspsrsee nsnijscripts are being written on unusual lesions 
of the salivary glands^ cranial nerves^, and glandular stotaach. 

6, Collaborative work with Dr. John B. Calhoun; Esperiments 
of Dr. Galhemi suggest thst rats living tmder a certain amount of stress 
in a free^-racging eonEauaity develop more tmaors at an earlier age tSma do 
rata living under the usual laboratory conditions » We have necropsled a 
nuit3>er of £hsse rats and are studying the tissues histologically » 

Ife^^iqr Findings g lo Dtiring 12 generations of transplantation 
into intactj, asatmrs^ jsale and female Osborne -j^ndel rats Adrenal cortical 
carcinoffis 494 has saintainad a consistently siiailar histologic pattern of 
cords of uniform polygonal ceils* All tumor^bearing rats have developed 
polyuria^ poiydlpslaj and degenerative cimnges in the distal convoluted 
tubules of the kidney and have shown atrophy of the adrenal glands^, Be^t 
organs,, lymphatic tissue^ and pituitary gland. Preliminary analyses of tumor 
tissue' by Br, Johnson indicate that this tmaor secretes an excess of steroid 
horsEKsnes at least sisailar to those present in the nornsal adrenal cortexo 
A sublinaj tumor 494H| developed after feransplsntation of tumor 494 into 
hypophysectosjised ratSj> differed in histologic pattern from tumor 494 in 
that it contained Isrgs^ bisarre cells in addition to the uaifornij, polygonal 
cells of tuEHjr 454, i^en tumor 494H was returned to intact^ adult male 
and ferasle rats, it retained its ox-m distinctive histologic pattern^ yet 
it also induced polyuria, polydipsia^ and renal tubular degeneration. Bsts 
of both seisss inoculated with tumor 494H developed hyperplastic vimsss&xy 
glands filled with milky secretion. In the feaales^ the uterine horns were 
enlarged and the vaginal epithelium mucified; in the taales, the testes aad 
accessory sea organs were esstressely atrophied. A few inoculated with 
tumor 494II have developed asaasaary carcinoiaa in addition to hyperplasia of 
the jDSnanary tissue, fhis tusaor induces developnent of the secondary ses 
organs in goaadectomised and in hypoplsysectoiaiaed rats but not of the 
testes or ovaries <, 

2. Txaiaors found in Mastonr^s have included a feu carcinomas of 
the glandular stoiaach as well as papillonas of the forestousach, ivmg tujaorSp 
adenoiaas of the adrenal cortex^ granulosa cell tumors and luteoaas of the 
ovary ^ and lytaphomss. The incidence of hepatonas has been unusually' high. 



826 



Serial Uo. ICI 500 



Individiual Projecfc Report 
Calandar Year 1959 
Psrt A (contiaued) 

3, Mo significaat fiadiagSo 

4„ Mo sigaificaat findings = 

5, Eats fed tte carcinogeBj> 2»7 fluoresylsnebiascetaaide by 
Dr, H. F. Morri0 assd e^^ffiiaed hisEologically in this laboratory have shomi 

an unusual ntasiljar and variety o£ naopIasiBS„ Ajoong £hsse lesioas are; 
histologically !^llg3ssa£ carcinojm of the glsadular stosESch^ sdenocsarelKossa 
of the sjGsall latestlne^ seyere atrophy and sarcoss of £he salivary glands^ 
neurogsaic ssrcoi^ origirtstisag in the region of tka trigeminal aerves and 
pituitary gland^ adsEocareinosja of £lss u£eras. Sarciaocas of £lie liver ^o 
ear^ sod ajasESiasy glaad are also preseat in a high percentage of rats fed 
tais compousdo 

6, Histologic study of rats from the eKperia^nt of Dr« Calhoun 
Isas rsot bsec cotapleted, PreliiEiaary data isdicates that probably the txffisor 
incideace its his sfcrassad rats is little if at all higher than ia rats 
living under nonaal laboratory conditions. 

Sigaificanceg 'Sh& knowledge gained from a study of neoplasras 
in laboratory asisals should provide a basis for further work on the 
mechanism of carcinogenesis ia hmssa beings, 

grop; osed Gotsrses Stitdy of Adreaal cortical earcinoaa 494 sad of 
its siibiiae;, 494H will be continued,, aad it is expected that ia collaboratioa 
with Dro Johnson detesraiastlons of the hormones produced by these tmaors 
will ba completed. Collaborative vjrork with Sr„ ^sCardle oa the cy to pathology' 
of mitochondrial batoasets ia the renal tubules of polyurie rats bearing 
adreaal cortieal tumors vjili be contiauedj and a nanuscrint on the subject 
should be ready for publication during 19&0» Collaborative work with Dr^ 
Morris fe expected to be ready for publitsation during 1960o the. esipariicsatal 
procedures and exaiaiaatioa of histologic cmterial laentioned in other items 
eaumarated above will be coatiatied. 



Part B included? Yes 

927 



Serial ^o. _EC1 500, 

Individual Project Report 
Calendar Year 1959 

^£tJB. HoEOTs^ Awards, and Publicatioas 

PublicatloBs oth.&r ebea abstracts from tMs projeess 

1, Stewart^ H. L., aad Ssellji K= C. 

The MsSopafelao logic of esperisnentsl fcmEors of tbe liver of £lie 
ree. Ctepfcer 17 in lbs ffeysiopatliology of Gaacer <F. Hoinburger^. 
ed.,) mui B. loeber, Inc^ Hew York, 1959. 

2., St&wsxtg Eo Lo 

Kis Gsacer Xnvastigator, 
Cancer Ses. 19s 804-818, 1959 

3« Snellji Ko G.;, sad Stewart^ E. L. 

Variations ia Histologic Patfeera sad FuEtctional Effects of a 
TraasplaKKabls Adsssml Cortical Carcisjoma in Intact l^pophyaec 
toaisad, aad Mewbojfa Sats^ 
J, Nato Cancer last. 22: 1119-1155, 1959. 



928 



Serial No. NC£ 502 
PHS-NIH Ic Laboratory of Pathology 

Individual Project Report 2o Office of the Chief 
Calendar Year 1959 3o Bethesda, Md„ 

Pare A o 

Project Titles Histogenesis and histopathology of cutaiaeous laeoplasiaso 

Principal Investigators Dr, John H<, Ec%c«sib 

Other Investigators s None 

Man Years (calendar year 1959)2 Patieat Days (calendar year 1959}? 
Totals 1 
i^ofessionals 1 
Others - 

Project Description s 

Ao Study of diseases In csan (collaborative research} o 

io Mycosis fuagoides 

Ten patients have expired and have been autopsied„ Two 
articles have finally appeared in print (see pisblicatlons and eaclosed 
reprtae8)o A larger pathological article is conteaplated to iaclude £ha 
clinical and pathological observations isade in the ten i:^cropsl€d cases o 

ii, Melanoosas 

The study of naevi and s^lanoseiis contisLu^ss^ Ue now hav@ 
autopsied fifty patients wifeh advanced Biielano©a<. Dr» Rothbesrg and I have 
Isolated a ssoall quantity of black caaterial fr^a the urine of a a^lanuric 

patient o This material will be studied by physical and chemical s^thodso 

One uoman vith a oalanoma excreted large smoisatB of gonado-^ 
trophic honsKjne in her urine. The slgaificaisce of this finding is baiag 
stud led c 

ill a Because of insufficient amterial the seroderasa pi^issatostim 
study has been diseontixs^'^d for the present c 

Be Study of diseases in aniiaals 

io Four s^lancHaas have been produced in 51 guinea pigs by skin 
painting with dimethylbensanthraceQe o This espsrimisnt has been eospleted,, 

iio The hairs of chinchillas are beis^ exanised for the p^er iodic ity 
o£ melanin production in these animals „ Chinchillas have an agouti coat, 
and the pattern of melanin production in hair appears to be very regular 
but ^ery c<«aplez<, ■ 

823 



PAGE II ^ Serial Nei. NCI 502 

Co Miscellaneous collaborations and efforts 

1. With m. R. Lov«, HCIo 

I am seadiag slides on C3K mice injected at birth with the SE polycssa 
virus ^ The sniatals are over one year old^ and this sxperiiaent contiauas 

as an effort to detect aay late effects of the infectlosio 

2„ With Dr» R. Hertz, NCI, 

Dro Hert? and 1 have agreed to prepare a descriptive paper correlatiag 
the pathological and clinical observations on those patients «;ith adrenal 
cortical carclaogia who have been autopsledo 

3„ I am studying necropsies of sis adults and one child uho disd 
with cytoEsegalic inclusion disease „ 

4<, I have helped Dro Lo Dunheiu in drafting ssaall parts of her 
aaonograph on the cervical caacer study „ 



Part B included res (x) Ko ( )) 

930 



Serial Mo. NC:£ 502 



Parfc 



Honors s 



Lectures ia pathology, Howard University 

Appoineiaaat as mttSi&T of a sissioa of go^^rsmenc pathologists to 
RusslSo 

Publle&tions g 

Forloaar, CoE„, Jro, Frei, Easil III, Edgco:^, John Ho, aad Uez, John. 
PseadcsEoaas aepticesaiao Aa^ro Jo Medo 25s 87? -890, 1958 o 

EdgcoiJi^, JoHo, Vaa Scott, EoJo, and Andrews, JoRo Histopathologic chaages 
in the skin of patients with oncosis fungoides foilouiag th&rapy t;)itb 
high eaergy clectiroaso Acta dezBsato-venereoio, Proc. llth lafeeraafcc 
CoEigro Derasat, 1957, Volo II, Po 457-462., 

Van Scott, EoJ., Aadrsvjs, JoRo, aad Edgc€«d>, J.H, Therapy of isycoais 

fuRgoides with high energy electrons „ Acta denaatc>vene7eol„ Froco 
llth I»temat„ Coagr, Deraato 1957, Vol„ II, p„ 451-456 = 

Craa^r, LoM., Waits, J„Bo, Edgcssab, JoHc, Powell, C.Co, Tuohcy, J„Ho, 

Van 'icott, EoJo aad Smith, R.Ro Bum followisag accidental exposure 
to high energy radiation„ Ann. Surgo 149 g 286°293, 1959, 



S31 



Serial No. NCX 525 
PHS-NXH Ic Laboratory of Pathology 

Individual Project Report 2. Office of the Chief 
Calendar Year 1959 3^, Bethesda 14, Mary lead 



Part A. 



Project Title: Attempt to induct carcinoma of the bladder ia hamsters 
by infection with Schistos oma haematobium. 

Principal Investigator; Dr. Eloise Cram. 

Other Investigators; Dr. Louis B. Thomas, Dr. Elmar Berry and 
Dro Louis Olivier. 

Man Years (calendar year 1959) ; Patient Bays (calendar ye-ar 1959) : 
Total; 1 

Professional; 1 
Other ; - 



This research project is in collaboration with Doctors gloise Cram^ 
Elmer Berry and Louis Olivier, N.I.A.I.D. and consists of studying 
chronic infection with S. haematobium (Gold Coast, strain and Egyptian 
strain) ia hamsters; particulerly with reference to the induction of 
bladder carcinoma., 

a.) 217 hamsters were exposed to approkimately 300 cercariae 
(Gold Coast strain S. haematobium ) each early in 1955. Practically all 
of these hamsters have now died and have been autopsiedo Hlstologicel 
sections have been prepared. 

b.) Late in 1956 and during the first sis months of 1957, So haemato- 
bitsm from Egj^t became available through the efforts of Drso Berry and 
Olivier. Approximately 150 hsaisters were exposed to eereariae and 
approximately 80 of thase animals are still living. 



when this eisperimsnt is completed it will be possible to 
compare chronic S . haematobium infestation in the two groups of anisaals 
with Egyptian and Gold Coast strains of S. haematobium . 

Sig nificance to Cancer Research ; 

S. haematobium infection has been found associated with bladder 
carcinoma ia several parts of the world. This study is designed to 
produce bladder cancer in the hamster by chronic infection with S. 
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 thase two parts of Africa. 



S32 



rags lie Serial NOo HCX 525 

Fart A (coatinued) 

Propo ssd Course of th a Project; 

All but approximately 30 of the aalnsals have now expired after 
living froE two to aeariy three years following infestation with 
So haematobiim. Grossly, no bladder tumors have been observado Buring 
tha nest year histologic sections of the autopsied anisaals will be per- 
formed so that this esperisssat cen be completed. 



Part B includad Yes ( | Ho (k] 

933 



>jeTz.&.i uo. bA,L s^^<^_ ^ 

I. Laboratory of Fjatfeology 

■2. Office of £lis €Msf 
3. Bethesda^ Maryland 

ladfvldual Project Report 
CalsEdsr tesr 1959 



Fart &.. 



F!;.'ojsG£ Title; Cytoclsesaistry of aibestscleoprsteins 

Pirlseipsl iGvesSigsSici-s Robert Lova 

OShar IsvestlgaSors 5 Br. ■?. P. Bhasad^^aj (BjC,D,E,L) 

Br. E. S. SttSkiad <A) Friiacipai Iiaveatigstcr (S) 

STo Mo p. Salssan <F) (smiD) 

Dr. A. J= Daltoa (A) 

Brc K. 0. Ellea (Wistar Issseltute) (K) 

Dro H. Kobsyashi (S^I) 

D^o L. Kilfeaja (J) <EBS) 

GoopsratiEg Qtiitss 

mm Years (calendar year 1959 )s 
Totals 11/3 
Professionals 2/3 
Qfkett 2/3 

Frojaet Bescriptioss 

Object Ives I To sfcu4y the riboeuclsic acid tuetabolism of sorsaic 
aeopXastie sad viras«»infected cells, 

Hethodg I A. Fuxth&t develofuasot of new atainiztg procedures for 
r Ibosuc IsQprotelss , 

B. Study of the cytochemistry of riboaucleoprotaisis i» rat liver 
calls duriisig starvation find reȣeedistg. 

Co B^vestigation of thB structure at^d fts^ction of the liuclsoliausi. 

P» Devslopa^at of s siagsle stsin for laltochoadriao 

E. Study of tha riboniiclsoproteia E^taboiisia of Ehrlich sssi£e» 
calls siecoa^aiayiag the mtotie abaonaslities prodxjced by colcMciaec 

Fo Cytoefes3aieal &nd bloeheaical stains of BsLfi cells infected 
with a DM virus (vscciaia), 

G. Cytochemical studies of Saia cells itsfeeted with &. ae?^ 
(probably IM.) virus j, B^-i. 



93if 



ladivldtial Project Eeport 
S&le&d&T Yesr 1959 

Part A (coatlnued) 

H. Gytodb&mic&l studies of eacolysis pro(Sueed by Nswcas£ie 
Disease ?lrus la the Ebrlieh ascites tin^r. 

I, Afctes^ts to infexbit mitosis by treatnsesst of Sbrli&h ascites 
tusKsr cells ox P388BI tissite culture ealls with riboauc lease, 

J» Sttsdy of tbs eytoebfimisSry of squirrel sad rabbit fihvasm. 
virus lafeetiorso 

K. Iiaves'cigatlos of the nstuse «nd properties of a riboauslease 
coafcasiasst of bo^iae testicular sad Btr&ptococc&l hyaluroiaidssa. 

L. Aetessptis to isodify EM. asetabolism by treaSsasaS; of Ebrlieft 
ffisoites cells with osasariae and 2«deo3^glueose. 

Bfi jog _ Fiadi^a^ ; A. 1. "The clieiaical basis of th& £©luidine blue= 
nsolybdsss (fBM) Bsstbod of stsiaii^ nusleoprotei«0 has been dstesasiaed, 
2. la addition to dia seven forsas of RM-proteia originally deliEeated by 
the fBM is©thod<, tifo otfear forms of lM»»proteia have baea recognised o 3o It 
has beea slioxm that alsctroa°deiise izsslybdate is deposited at sitas of 
metachrosBStic staiaicg hf tha 13M teethod. 

B. file chsxigee in the riboaueleoproteins of the x&t liver were 
studied by the fBM method. Souse fo«M of RMP were usaff acted by starvstios j 
others disappeared and reappeared after re-feediBg« The results suggested 
that soa^ of the eytoplastaic EKP was fosned ia the uucleuSo 

Q. The nucleoliaus has been observed in a nnsSt&x of noranl and 
neoplastie eells. The nucleoliaus wss usually large in neoplastic cells and 
was definitely increased in siae ia cells iafscted with Hewcastle atsd M."^! 
virus, ia liver eells during re»feediag after starvatioa, in cells prevented 
from entering propase by colchlciae asd in regeaerating rat liver eellss fed 
ethioaiae (by courtesy of Sr. H. Sidransky). 

D. A simplified irpa-heiBatoatylln staia suitable for use oa blocks 
fi^ad ia forssol-subliasate has been developed. 

E. Depend Isag on the dose^ colehicioe produced either jEsfcsphssa 
arrest or eos^lete iahibition of mitosis of Ehrlich cells.. In the rortcar 
iastsnce^ auclear paracbroBsatiE failed to diffuse iato the spiadle soae at 
the end of prophase. Ia the latter case, ahere thQ oaset of mitosis was 
inhibited^ there was an iacxeaae its the sise of the nucleolus asd aucleoliaxsB 
and as increase in the parachrosatin of the interphase cells „ The results 
suggest that the diffuse naelear parac&rosMtin sasy play a role in the 
genesis of the spindle fibers. 



935 



Xsdlviduai Ffoject Report 
Calendar Year 1959 



Part A (coatiaued) 



F. The deveiopEssat of isotile vaccinia virus psrtleles has bsea 
observed under the phase adcroscope. Grssiular BHPj as well &s SM was 
3ssccla.t:ed -g^ith the foroatioa of the cytoplasmic inclusions produced by 
vaccinia viruSo The eytoclieiaissl studies bsve been correlatad with virus 
titration sxid biosbaaiicsl analyses of the sucleis acids of tae cell. 

60 Preliasinsry studies indicated thst there are marked differeaess 
between the eytop«t!iogeaie effect of ^-1 virus oa isonkey kidney and oe 
RsLa sells « A aismber of cytopathogeaic effects thst have not bees dascrlbsd 
befcrs have baen observed <, See, Dr. Suskiadfe report. 

H. A study of the cytopstlsology of Elirlich ascites ceils isaf eeted 
with He-^castle Disease Vlnss has been published. 

I. Riboauclease, even in doses of 40 mg^n/mX., had ao significSKt 
effect oa the growth or SM? cetabolissi of any of the cells tested. 

J. Igiterifil froiB squirrel and r&bbit fibromES has b^en collected 
but feas not yet been studied e 

K. All preparstioES of byaluronidase tested contained ribonueleaseo 
The properties of the contaroinant riboauclease have been elucidated aad ecaspares 
with those of crystalline pancreatic riboaucie&se. 

L. Asaserine aad 2»deosyglucose treatasent produced ao detectable 
effect on the growth or on the nucleic acids of Ehrlich ascites tus^r cells-, 

Sigaificanee i The role of RM toetaboliem of the cell is of 
fundaasental issportsnce in the understanding of genetics^ protein synthesis^, 
cell grox'?th and differentiation^ neoplasia and virus isfectioso 

grogqsad Course s 1) Continued investigation of the cytochemistry/ 
of RKP. Devalopiaent of the lEBM staining procedure for electron microscopy « 
2) Further studies of the nucieoliaua and of ESF xKtsboliso in various 
systems? (a) cells infected v?ith Wk and BM. viruses using newly -developed 
techniques and autoradiograpl^. (b) Cells in which protein synthesis or 
cell division is inhibited by various procedures = 



Part S included; Yes 



93S 



Indtvldu&l Project Report 
Caleadar Tear 1959 

gayt B. Hoaors, Aw'erds^ and Publications 

Pubiisatioas other than abstracts ftom tbis project ? 

lo lj>VQp E,^ sad Bfearsd^jay<, T. ?, 

Two types of riboaucleoprofcein ia the aucleolus of isassialisa calls „ 
mtntB 183, 1453-1454^ 1959, 

2, Love. R.p and I,ileS;, R. H. 

Siffsrestiatioa of Buelsop?oteias by ioactivstlQjs of protela^-^osad 
aaiia© groups sad stainiag with toluidiae bl«e ssd SEm^siuas icolybdat:? 
Jc Hlstochea. aad Cytochas. ?, 164»18lj, 1959. 

3„ Lovs^ E. 

the cytopathology of virus°'infected Cussor cells* 
Asrac H. Yo Acado Sci» 81, 101-117, 1959o 

4o Blsaradx'jsj^ T. Po^ asd Love, S. 

Cytology of rat liver eelis dtsring stsrvatioa aad re£eediEg„ 
Jo Mat. Csacer iBSt, 23^ 6S5-715, 19S9. 

5o Bltsradwaj^ 1. T.^ sod liove^ R. 

Saaasaetrstioa of isdtochoadria in tissues fixed for cyfeochssalcsl 
and gensral staining procedures. 
StsiB fecfeaol. la press. 



S37 



Sariai kc*= ._„_____™__„ 

1. laboratory of Faefeology 

2. Office of the Ghiaf 

Individual project Report 
Celeadsr Year 1959 

B srfc_A . 

Project Titles ¥iral Taerapy of Tuisors 

PriEcipal IsveaSig^ter; Robert Love 

Othsr Inves£iga£or8 5 Br. E. G. Si^kiad (C) 

Soapsrating Units; 

Ifea Tssrs (csleisd^r year 1959 )s 
Totals 1 1/3 
Prof essloasl ; 2/3 
Other t 2/3 

FsajeeS Descriptloias 

Object lyes; To adapt viruses to beesHne oneolytie for tx3iaoES« 

jaetbodes A. Psssage of Heajcsstle Disease ¥irus (SD¥) through 

various tvuaors in vxvo» 

B. Aaaiysis of the oncolytic effect of tba virus. 

C. Coxaparisoa of ti» oaeoXytic effect of r&t<^HeIja adapted and 
uusd@pt:@d Cox@aekie virus on Bsid cells in rats end chick eso^ryos^ 

Major giadia^ s; 1) NDV ^as adapted to infect scd to destroy the 
Eferiich ascites tsis3or« 2) Tha Ekrlicli adapted virus grew poorly in the Krebs 
2 tisaor sad produced no oncolysis » After 18 passages in vivo through the 
Rreba 2 tuteor the Ebrlich»adapted NDV becaine oncolytic. It has aot beea 
possible to adapt the original chick eajbaryo HDV to tbe KTebs tusaor by 
in vivo passage, 3) Cliick er^ryo WV prodwsed sotoe oncolysis of the F388 
mouse ascites ttissor wMle the Ehrlich-edapted virus had ao effect o After 
& f ex? passages in vivo tlirougli the P388 tunsor tbs chick ea&ryo HDV hecsws 
more definitely oncolytic while the Ehrlich-adapted virus tjas still without 
effect on tuaor growth. 4) Stela ceils could not be gro^m with sufficient 
regularity to permit a study of the effect of Coxsackie virus oss Hals eell®^. 

Sigaificance; The etdsptatioB of a virus to a given tusaor dose not 
cotjfer ojicolytic properties for otl^r tiaoorSo In eosae instances & virus 
is adapted to one tensor fEsy be easier to adapt to another. On the other 
hand adaptation to one tuinor sn&y hsve the opposite effect and render the 
virus Bs>re difficult to adept to another tusasr. 



838 



'fex-t k (ccstisued) 



Sii;l.::. :X!I 529 

Individual FrojeeE RepsrS 
Cslsadar Year 1959 



Feosoaed Coarse; a) Coajpletiois of slse project of WV sad 
Ehrlieh, Ksrebs 2 aod F388 cells; b) aa sfctaa^t will be Bade fco adapt cfeick 
SE^ryo IS5¥ to £he Sferlieli t'jifio? wlehoufi tha use of tissue culeure; e) fursbei 
studias of vlrus-^isd-acsd snsolysle will be ujade ^^ith spoataneoiss or 
^•ims-issdaced tusasrs. 



Paart B included s Yes 



S39 



Celsadsr Yeas 1959 
Bar£_E. Soaors^ Awards^ sad Fisbllcatioas 
PubiicatioBS oSlier thas abstrasts from eMs p^srojeets 

I^ LovSj, Re 

InC^cductioa £0 £lsie Coeferaaee oa "fhe Cy£op&thology of ?irus 

Ann. H» ?. Aesdo Bei. 81 j 4-5, 1959. 

2. LovSp R» 

Editor of above ^ 

Asa. H. 1= Aesd. Sci= SI: 1-213^ 1959. 

The cy tops tho logy of vinis-iafectsd tuaior cells. 
Asao K. ¥0 Acad, Sci. 81s 101-117, 1959, 



o *l 'J 



lo Laboratory of Paeliology 

2. Office of £be Chief 

3. Betbasda, ¥^xyl&-ad 

ladlviduel Project Report 
Calendar Year 19S9 

Projact Title; Study of Fil£rabJ.iS Ageafcs froiss Ifeuse TtaiHors. 

l:»rlaci|jsi lavestigfitor ; Sobert: Lova 

O'cher Ixvyestigators; JoM H. Edgcojsb (A oaly) 
Alsn S. Eabson (B osly) 

Cooperating Salts ? 

^&a Yesrs (c®leadar year 1959) 
Totals 1 2/3 
Profssssiossis 2/3 



Fsrojeet Bescs"iptio?ss 

ObJa£t|^gg£? Ac To propagate a filtssble agent from usous® leukams, 
ia tissue culture. 

E. To gfemly She cytopatfeoiogy of polyonsa virus infection in tissue 

MsShods; A« Esstracts from a lytaplsocytic aeoplasm were passed 
serially ia lisjnkey feidasy tissue culttjsres and inoculated, ioto €3H/laH ^ k¥S. 
mx6 C3Sf/ Isl s AKS aewborA snice. Coatrol asiee were iBoc«latsd r^lth. esfiffacts 
of acra^I tissues tre&ted In the smm msnn&T, 

B. P388D1 BiuriRS lyssphojcs calls grown in & ailk laediuES w.2re 
itjiscted with po Ijoias virus atwS studied by vas^ious cytocheaiicsl tsc1:^atques» 

l^jQg Findings g A. A variety of fcusvors were produced ia sdce fey 
iaocul^&tioa of the first passage sjoakey kidaey nsterial ceataiiaing the 
lewkemis SKArect, 

B. Ia P388Si calls infected vilth polyois^ virus, tbs ribontxeleo^ 
pro tela of the nuelear parachroastin and elis nusleolirsus could not be 
deraoBetratedo The cueleolus was eaiarged and vacuoles coataitiiag a diffuse 
end a ooadeased forts ef sbnonaal ribonacleoproteia appa&red ia the stsclsuSc 
The ja'jclei were &t first eiilarged but Xates: shrgtils^ bec^ae pykaotic and 
disintegrated^ Sssclesr fragmsnts frequeatly «ader?^ettt phagocytosis by other 
less dssasged tissue cialture eel Is « Deoasyriboausleic acid cotsld aot be 
deffioastrated x^ifh eertalsfcy ia associatioa with the B«?ly-foriBed sbsaonsaX 
riboaucleoproteiss in the K«clsias» 



941 



Isdtvidua.! *r©jeGt 3.epor£ 
Caleadar Ye&r 1359 
Fart A (coati-asiad) 

gij|a^lesa5g,s The nature of fclse iateractioa between visfue sad 
host cell sad £lae factors devermiaing wheSher She outera?ie will be aeerosis 
or psfoiiferstioa is of fundaaaati^al lE^Kjrtance is the study of viruS'^indueed 
neoolasi®. 



^S£s A. AIX the survivirig mice will be &eld oa test 
fcr 18 wQuthB &t th& end of which tisss the results will be giKS.lysad» 

B» la view of re£-ortg that the infectious nucleic acid of polyosa 
vinia! is dsoj:yriboa«;leic acid sew teclsaiques will be applied £o defeect 

any sewly fortsed BSSl in She nucleus <. 



Bsrt E tnzlud&^i fes 



942 



Serial no. _ l^Ql :: 

iBdividusl Project Report 
CalsBdar Tea^ 19S9 

?art_B. Honors^ Avaxda^ and Fablleatious 

Fabliesfcloas otfesr thSB abstracts from this pyojeetg 

lo I^\=-ej S.^ sad Ssbsoa^ A. S. 

CytocbesElca.! studies of lailk'Sdapeed sasrise lyropbosaa cells 
(strsia P388B1) iafecfced ^sith polyo^ vi:e-tJSo 
J. Hat* Caacer Inst» Hovasabsr 1959. 



9i|3 



i = Liiborator/ -of "Pafhoiog 
2. Offiee of the Gh.tef ' 
3o Befcliesda^ Maryland 

Ittdividu®! Project Report 
Calendar Year 1959 



Part A. 



Project Titles A review of the surgical and autopsy sections frcsn 
Veterans of World.Wsr I wbose cause of death has 
beea listed as lung cancer « 

Principal Investigators Kstherlne McD, Herrold 

Other Investigators? Harold L« Stewart, Irfiborstory of l^thology^ HSI 
Harold F, Bora, Biotaetry Branch, BSS, HIH 

Cooperating Bnitse 

Man Years? (calendar year 1959) 
Totals 

Professionals 
Others 

Project Bescripttcss 

Objectives s The objective of the study is to review ail the 
cases from veterans of World War I vhich have been dlagtto&ed as liing 
cancer and to determine whether the findings aieet the criteria for prinary 
lung cancer => The secoad objective is to classify the tumor according to 
the histological typcc 

Ifethods g This review is being done in conjunction with the 
epideialo logical atudy of the national Cancer Institute on the relationship 
between sinking and death from carcinooa of the lungo 

Pilot studies using Syrian hamsters are In progress to detertalne 
an espsrifl^ntal approach for studying the pathogenesis of lung cancer. 
Intratracheal instillation and transplantation of newborn tissue, includirtg 
bronchi and lung tissue into weanling hamsters have been tried o Mtesapts 
to produce trachisal fistulas in hamsters so far have been unsuccessful o 

Major Findings s fhis study has net been in progress a eufficiasfe 
length of time for evaluation of the significant findings., 

Significance s tbs knowledge gained from this review of limg eaa«ei- 
cases s^y elucidate the isiportance of proposed etiological factors. 

Proposed Course s fo continue revieiJing the surgical aad autopsy 
material sections from the cases of veterans of World War 1 and to perform 

espariasental research relating to lung cancer <, 

Part B included? No 



Serial Ho. ligX.,136°a 
PH3-NIH 1. Laboratory of Pathology 

Individual Project Report 2, Office of the Cbiaf 
Calendar Year 1959 3. Bethesdaj Md, 

Far t A o 

Project Title: B^tpsriatsatai studies' relatisig to the mininjal coaceU" 
tration of hydrocarbons xmicb. will produce cancer 
in hamster cheek pouches « 

Principal Investigators Dr. Katherine McB. Herrold 

Other Investigators: Hone 

Man Ysars (calendar year 1959); Patient Days (calendar year 195S) ; 
Total: 1 
Professional: 1 
Other: - 



Project Description; 




The objective of the study is to determine the miniiaal conceatratif; 
of ksoxm carcinogens which will produce cancer in hamster cheek poijehss 
and to study the histolytic changes during the early initiations and 
proESJtion stages. 

Methods t 



Test pallets using C.P. beeswax as a vehicle and containing various 
concentratiOiis were prepared. The hydroea,rbons , \dbich are being used in 
the experiisjent are 9, 10, dimethyl - Ij 2 - bensanthraceae and 20- 
methyicholanthrana. The pellets have been inserted in the right cheek 
pouch of 83 hamsters and will be left, in situ for indefinite periods. 
Pellets consisting only of beeswax have been inserted into the cheek 
pouch of ffiale and female hamsters and will serve as controls. The tech- 
nique being used is the one developed by Dr. Lucia Dunham (see Serial 
Mo. HCI 50lb). 

Major Findings : 

A marked difference has been observed between the action of 20 
methylchoiaathreae and 9, 10 dimethyl 1, 2 benzaathracene on the ss-acoiis 
membranes of the hamster cheek pouches. Concentrations of 2-5 ssgrns and 
above of 9^, 10 ditaethyl 1^ 2 benzanthraeane produce acute inflaaaoatioB^ 
ulceration and necrosis of the hanster cheek pouch and within a period 
of 10 Ssys to two weeks the pellet sloughs out. 



Page. lie Serial No„ KCI 536-Ca! 

Fart A (Contiaued)s 

The end stage ia many of these pouches is atrophy and in a few of 
the eases asoplastic changes have occurred. With lower concentratioEs 
of the carcinogens^ epidermoid carcinoma has developed in many of the 
haiastsr cheek pouches so far eKamined, The livers of the tussor bearing 
animals reveal prominent hsaatopoiesiSo 

High concentrations of 20 methylcholanthreae have produced only 
negligible changes in the hamster cheek pouches and no tumors so far 
have developed. The control animals which have been examined reveal 
no significant pathological changes in the pouches. 



Complete necropsy examinations are being performed on all hamsters 
and the pathological changes v/ill be correlated and evaluated. The 
findings will dictate the future course of the experimental study a 



Part B Included Yes (x) No ( ) 



Page III. Serial Ko. HCI_53§=(a) 

Part B, Piiblications g 

A case report has been prepared sad She paper is ready to be 
submitted for publicatioQo 
TITLE? Aberrant Parathyroid Gland in Phsrsmgeal SubmucosBo 



847 



Serial S3= HSZ 303 
X. Labora&oKy of Pathology 

2. Casseer Indtse. & Bashogosesis . 

3. Bet&esda, mtjl&ad 

ladividual Frojecfc Report 

Calendar Year 1959 

Projsat Titles Collaborative Ressareli leqtalrlag Mfebclogie ^atos?^? 
Friasipai Iisv©stig&£©r s Tlielisa B. I^uzsa 

Sc-spessslBg laiitss 

¥s.n Years <ssl£ad£r year 1959): 
Totals 2/3 
Professiosals 1/3 
0£l5srs 1/3 

Obje^lvgss fo aollsbormt® wiek aad advise otlasr iavastigatoes 
ae tfee Bstiszial Caaeer laseitute ia plsases of research problems x&qnixitug 
sKpsrisjsc® is sai^l pathology. 

MB|fe6de; Tkis seay eoaslst of {1} Fall ssapoESibility aad separate 
piibli^sasion ss saaler aatfeor ©f saatariel reviessed ia cotmseeioa witii work 
of assatlger iavesei^toffj, (2) Review sad dascriptioa of saisasls cesaisig to 
autopsy^ aad ^^iieatios of ehe fiadiags as s separate seceioa of a paper 
daeerifeisg the ea£ire e^^eriusBtjp <3) Seviesi? and diagnosis of tyasors 
fotisd fey lE^j^sfcigsSor iE co'jrse of ejES»eriE^K£s, the iaforssatioa to be 
sssed is a temsistio^ of resulfeSj <4) Occasioaal consult&eion aad advice 
on patfec logic aspects or arterial in ae esperissisae. 

fe^JQg FlTadlagg? 1» With Dr. luth Jferwia. Mlee which fead bees 
imp lasted ia£s:ape?l&(^3aslly ??it!i 'tsadwiche^' coatalaiTag s varieey of 
differsat Sissies have bees sutopsied, 'Ihe results have aot beaa tebulsted 
but a sigaificaat maEbar of plasca cell ESoplasB^, difftissly seeded on 
gssrltoseal surfaces, have beess feuasd. Spontaneous txsasors of this typs 
aave aevs? beaa fotiad. Flssss cell aeoplasBS of this type Mve nefi-? 
been fosmd la s MLS/c Hale HK>use ^ish received i«j estrogen^ in a G3H 
ssouse^ sad ia a taousa with a blaak "aaads?ish»" fhis eliasisstes several 
factors --se^ eetrogea stissulation^ lAIS/s genetic eoastitatien^ &ni 
"foreign" tissue its easdwich— »as necessary factors for the induction of 
the plsssES cell aseplasaES, Other oeoplissins^ particsilarly fibrosarcosss, 
have also bseia feiied but these msuslly appear in close association with 
the saa<^j'ich. Sr. Michael Potter and Dr. Joha Fahsy are stiidyisg Sbe 
absoEEsal proteias ©f ttess tisssors^ Dr. Jack Mlton possible virus 
particles J aad 'M^ Eose MaeGardle the Solgi s.|>paraeus, 

3^B 



PSS»1IH 
Calendar Year 1959 



Fart A {coatisued) 



2o With I5r» Katlaariae Sanford. Tbe aKjrphology of fctaaors gro^m 
ia tfea ssouss frcsia tissue culfeurs ceils . 

3, Cooeiaued work wish Br» AsdervoEt ob EssEssry taseors, 

4= With. DSc John 'Disney a& laaksasia produced by a celX'^frea 
filtrate.. BMB/c ssise ware lajecSsd with spleen eatraets frois aorsal 
23ic©5 and fsosj "virus lea^jsia" mlce» Autopsies have beea perf erased at 
freqaeaS iiieervsls oa iai?@ fros 2 groups . A£ present tii^ (12 ^K^sks after 
iajectioa) ao diffsresee Iss beea detsctsd. 

SijIglfl^Sees Bafefeo logic review is liadispaasabla to i^tiy tfpes 
of csacar s'esesrct*. This is baat sasxsried otjt by e specialist ia pstbslogic 
sssto^y sisea "sjitb t&a present ess^lesities of resesrels a siagle 
ia\?estigj?.£os caissot fcse^? tMs field snd hie owa speeislty. 

^^oposed ^ Go^yge s This work i^ill eostisst® as 1» tise past. 
ss a result of ciasiy o&sarvatioees, eniougl^ Isf&sn^atios is ssctMulated 
rsgardi^ag auy 6abj®ct-> publiestiotis ^ill bs i^de. 



Part B iaclisdedg Ho 



949 



3. Bethesda^ Maryland 



PHS-NIK 

Individual Project Report 

Calendar Year 1S59 



Pare ACl) 



jest. Titles An Electr©n Microscopic Investigation of WQnnal and Heeplastic 
Tissues. 

Farincipal Investigator: Hiliiam G. Banfield 

Other Investigators: Dr. Dewe^ Dr. Rabsen, Dr. Ramirez, Dr. Lemgley^ 
Darlene C. Brindley 

Cooperating Units: 

Man Years (calendar year 1959) : Patient Days (calendsr year 1959} 

Total: 1 1/2 

Professional: 1 
Other; 1/2 

Project Descriptien: 

Objectives ; 1. To describe and interpret the ultra structure of selects^ 

neoplasms and to compare results x^ith suitable csntrol tissues . 

2. T© look for and study the inciting agent ©r accompsnying vires 
in neoplastic lesiems and compare with similar agents or 
viruses if known. 

Major Findings: 1. The polyoma virus was described f@r the first tiiae in thixi 

section obtained from cultured cells supplied by Dr. Dciws 
(see publication). A larger viral form probably different frsa the polyssxa was 
ale® present in this material. The same cells gr©T;i7n by Dr. Sabson in s milk i 
and infected with polyoma contained only the 300 A® polyojsa particla and rs 
the larger particles. The 300 A° pslyoma particles grew in cultures afte.^ 
the inoculum t© 60" for 30 minutes. 

2. Skin lesions vjere examined from raallgnent and benxga 
acanthosis nigricans and txHB©r from the malignant lesien. Ck?ntrsl^ sections fry:;. 
normal and jaundiced skin were also examined. Saall particles 50-lOOA^ in 
diameter were found in great abundance in the basal cells ©f the malignant 
acanthosis nigricans j, less frequently in the benign snd least in the contral. 
Their exact identity has not been established but they are prebably ferritin whicb 
3© ' not been described in skin. Dr. Helen Gurth end her husband and 
Dr.. -lade it possible for us to obtain these lesions. Dr. Eabson could 
grow nothing from these lesions in tissue culture - 

■3. Two lesions of m©lluscum csntagiosuja were ebtainsd fre-21 
Dr. Marc Weiaer. The virus on gr@ss visual- examination baa not grm-m in tissue 
culture of keratinizing epidermal cells (Dr. labson) ©r on rabbit slcin or in 
rabbit testis. . 

4. In collaberstiein with Br. I®ngley vm have submitted a 
.paper ©n the structure ©f the rete mirable ia the kidney ©f the sat . 

950 



:irial m. mi 504 



Individual Fffeject aep€srt 
Calead&r Year 1959 



Part A (1) (cojatinued) 



Si^nificaace; The viral nstura of the polyoma agent was established bayend 
any reasonable doubt . The ultra sts'uceus'e of a gate mjgable 
in the masraalisn kidney^ similar to that in the swim bladder of the fish has 
been described for ths first time. A previously undescribed 50"100 A* dense 
granule in the basal layers of the skin has been found, 

^oposed Course;^ 1. With Dr. Sabson and Dro Dswe work out a life cycle e-f 
polyoma snd sttenpt to find the significance of the largs 
particles in the serum cultures and their absence in the milk cultures. 

2. Attempt t© identify the 50=100 A® particles in the ekin 
and the sr^all dense granule in the molluscusa el^entary body. 

3. Continue in the direction noted under project descgipelv 

4. In collaboration with Dr. Ramirez investigate the ultra 
striseture of a lyspbesa in the hamster and mycosis fungoides. 



951 



mis-wm 

Iridividual Project Eeport 
Calendar sear 1959 

ject Titles An Investigatien ®f the Collagenous Connective Tissues and Elastia. 

ncipal Investigator; William G. Banfield 

Other Investigators: Darlene C. Brindley 

Co«.perating Units: 

Yesrs (calendar year 1959) ; Patient Days (calender year 1959) ; 

Totals 1 3/4 

Professional ; 1 
Other; 3/4 

Ps-Qjsct Descriptiens 

Oblectives; l.To elucidate age changes and pathologic alterations in collager 
snd elastin. 
2.T© uncover iDechanisras affecting the state ©f collagen in the 



^: 1. In haasters there is an increase in the abdeminal skin 

collagen soluble In 1:3000 acetic acid between 28 and 42 days 
ing the time of development of spennatogenesis and the appearance cf niany clear 
cells in the pituitary. Additional age changes have been noted but no definite 
correlations have yet been made. 

2. As an outgrowth ®f the studies ®n ageing hamsters we have 
9 transplantable tunscrs ant&ng v;hich are two melanomas^ a reticulum cell sarceoja,, 
a lymphoma,, 2 possible adrenal cortical tumors and an epidermoid carcinsHna. 

Significa nce ; The data being collected on the hamster is yet t@ be evaluated, 
A fairly good base line for the normal developsent throughout the life span ©f th: 
ssale hamster should be established. 

P roposed Ceurse; 1. Complete the histological portion of the ageing hasster 
program and analyze the data. Fill in the data with ssre 
sld hamsters where needed. 

2. In collaboration with Dr. Sabesin study the collagen 7 
the f®@t of a patient with rheisjatoid arthritis. This c@llagen shows w&ny 
pathologic changes. 

3. With the electron microscope and in collaboration with 
Drs. Sskoloff and Obrien^ stjudy the collagen in ochronosis. In addition follow 
collagen fibril clianges in maturing scars^ examine the collagen of growth h®i*JEene 
treated hamsters and study elastic tissue in thin sections if a suitable method 
can be developed. 

4. In collaboration with Drso White^ Reid and Jackson ccl 
the c@llagen at various sites in the rat for rate of decay of Cl^ labelled glycine 
when it is stepped after feeding fre® conception to about. 4 months after bis'tb, 

352 



PHS-IIH 

Individual Project Eapo-fS 

Calendar Year 1959 

Part Bs 

Benorsj Awards p sad Pub lic&t ions s 

Publicstisns other than abstsasta tvem this ps©jee£s 

1. Banfisid^ Wo G,^ and Bo Brindley 

Asi electron microscopic study ©f tiia epidertssl lesien ©f 

BssSiscus c©at®gi©s?imo 

Am. M, Y, Acad. Sci., 81s 145=163, 1959 

2o Bsnfield^. U. G, 

BsKse granule in the elementery body of xsolluscum cantsgiosia;; . 
Jo BiophyslCo and Biocliem. Cyt©l,^5; 513-514^ 1959 

3, Bsnfieid, W. G.^ and Do Brindley. 

Aeetic ficid«=soIuble collagen in bui^n scars « 
Sus'go Gyneco ©nd Obst.^ 109? 367-=369^ 1959 

4o Mafieldj W. Go 

Age chsages in the acetic acid-seiuble collagen ia htsman skin. 
-tech. ©f Fatb. In press o 

5o Banfield^ W. G.^ Bawe^ Go J.^ and Brindley^ Do Co 

IntraeellulSF snd extracellular particles in tissue cultures 
inoculated with paifotid tuaier agent ifoly&tB& virus) ■, 
J. Bat. Cancer Inst. In press. 



953 



Serial Ko. _M9lA9.h 
lo Lsbors£ory of Fatfeology 
2o Cancer Indue , & FafehogeEesls 
3 c Betbssdaj, Maryland 



Individual Project Report 
Csleadar Year 1939 



Fart A. 



Project Titles "Sib induction of Squaraeus Carcinon® of tba laisgs is 
iMhat&toTj Asiffials 

Prineipal Invas£igetor s Msari F, Stan&eQ 

Oth&t: Investigators; Robert Blscfeeil^ Joseph Albrechfc and staff 

Cooperaeiag Uniess 

imn Years (cslendsr year 1959) s 
Total: 5/6 
Frof assiossal g 1/4 
Otiiar; 7/12 

Project Description? 

Obj|ee£ives; To iaduee lussg cancer la laboratory animals tferoxigli 
depoeitioa of kaowa Si3td siispected carciaogsns in areas of regenerating Ititig 
tissus. 

Methods s Certain aliphatic haloesrboas are capable of csausing 
ishetaic necrosis wbsa trapped ia capillsry vessels. By incorporating 
carcinogens such as ESthylcholaEthraae into the inoculum it is possible to 
deposit the carcinogen in particular organs of iatereet to the oncologist , 
The detection of methylcholanthrsac deposition by U.V. microscopy is beisg 
accos^lishedo 

|fe4or Fiadisgs; Satravenous adiaiiaisfcr&tion of the Isalocsrbon alotse 
has not induced tuaors in rabbits^ rats^ or siice^ Tsmny binder observation 
for one to two years « In mice and rabbits neither the teiioearbon nor 
halocsrbon 4- Esstbylcholsnthrene catjse an increase in spontansgus alveoiogeiiic 
tumors nor do they induce carcinoajas in the areas of infarctiono In rats 
a suspension of atsthylcholsnthrene in tricapoylin carried to the lungs in 
the feslocarbon consistently induced matastasising squaxnous carcinoiiais of 
the lung which kill the aaioels ia six to seven months. The hietogcnesis 
of the ttsEors now under study indicates they arose in areas of bronehisl 
metaplasia which result from fealocarbon produced infarction, ¥e liavs 
traced the deposition of n^thylcholaathrene with ultraviolet light microscopy,. 

Sig^nificance g 0-ar studies vmy contribute to several asajor probleass 
in the genesis of lung cancer in nan such as the role of pulmonary sc&rriog 
and its relationship to eKogenoue inhaled carcinogens which in thesiselves 
fijay act by injuring bronchial cells and eubssquently altering their neoplastic 
potential o Such studies tsay also offer a new method for study itsg the 
asechanisia of ©ethylcholaathrene carcinogenesis, 

95^1 



Saletsdar Year I9S9 
Fert A (eoistiijuad) 

Ffoposad Courses Study of the Iiistegenesis of £l^ tas!ors,> 
QusstiSativs Eje&suressiSBts of dose to respoase. Furfclier testing oa abilifey 
of ssarrlsg aloae to isiduce carciaoiaac Teste eurrsatly under study isclude 
the iatravesoiss adjsiaistratioa of infarct induclsg ^locarboas sevesal 
days prior to and several days affcer Istraveao'os sa&ti^IchQlsaElsreae ia as 
effort So damossSrste £lie Initiatisg aad/or proiuoting effeets of ttese 
substaacss. Farther sSudies as outlined in Indl-vidual Project Report 
NSI 503^ 1957, are also contiauiBgo 



Fert B included s Mo 



955 



Serial m, _Ml 5Qi 
I. UihoTatQsy of Bs&laology' 
2o Cascer XxiduCo & PaSliogeaesls 
3, Befehasda^ l^rylaad 



Individual FrojeeS Report 
Csieadar Yea? 1959 

Fart A. 

Project Titles Study of frsasplaaeed Tssek)^^ ia l£tee 

Priseipai Xawestigseor s ^eisa B. Dusm 

Othsi" X»%?a3tigat:or s 



laa Yssrs {calendar yesr 1959) t 
feSsl: 5/6 
Professieaals 1/3 



Ob.'|ag£iws t To sfiisdy various ususisal ^^isors chiefs E^y t&r©?? 
ligfet oa fc^ saonssl call coaa^srpsrS or cell faactica^ sad assist la 
usdessfcsadiag of aeoplasia, 

l^ttods? & aus&er of Ciisaor liass of uousual iafierese a^e iSsS? 
bsiEg car^lsd. Asoag tube® are (1) B^se call ESoplesm^ (2) FlaSES ssll 
asoplssBSf (3) WxmctiovBl sdxeaisl coreisal aesplasia, (4) Orgaaoid fcximors 
of uterus ia B4IS/c sales ^ (5) OsSee^esie s&rcoiaas. 

ISajor gltidlagSj (I) Mice feasrlEg tbe ^ast cell tuasor h£ve hBon 
seat to 3 saad>er of isbersSsries is £l^ Ifeited States aad also to Swedes. 
"EiM^t tlsstsa feas siso beea supplied to © aissber of iaveatigiators st efes 
Hati&mt^l Issseita-fcas of ^sith. Bi«s £;«^>r is the aseites form is carried 
by Br» Hieliael Fot£ar^ aad tn the solid form in this labcrstory. 
Batsc'hz'o&iatlc granules are s£lll abu&dant xa the n^llgosnt cells afser 
5 years ia Srsaeplaat. Tmncr tissue supplied frcsa this IsboyaSory bas 
seceatly been issad by Br. Ed^sard Kora of She Ifatioasl Seert Institute for 



(2) Se-j^'-aral plaessa sell lines have produced aleeratioES ia fcfcg 
kidssy of ttHsor-^beasriisg sice. These tuisjr iiaas Imve bees supplied £o 
IS^. Eaipla Colesiaa^ & siadiesl ssudsnt workisig uades Dr. J. F. A.. I&jI^eiis 
at tba ^lEiverBity of Alabssa Medical School. 

(3) The sdesaal cortical asoplasm iacoastaatly groduees 
ebssgas ia female sice^ sad oSher s»rp!» logic altsratioBS < 



viriiisiBg ebssg 



956 



Serial 'Ho.._lgX 506 

Individjial Froject Eeposrfe 
ealesdar Year 1939 



Fart A Ceoatiaised) 



' (4) Several tussss of Vsm uterus are ao^ carried in MIB/e 
ssiee, Shsss are ren^rl^bly orgSEoid^ and oae tasscrj after sevea iioafelas 
gtaasth, produe-ss a cystic ssvieyj lined by &w&fsiss.ttii&l Hsaeosa aad serosa^ 
®ad i^ijst: appears £o be a sffiootih Euascie oater layer. 

(5) Os£eogsalc esrecaas. these eaaaars often nsfeastasisa to 
the Itsags asid are beitjg used la oS&sr lab&rstorias for studies on tmaax 
spreads k l^rge ssalfelBxscleated "osteoclastic" cell is regularly found 
ia oaa of eha «:i:5S2orSe In £issas ctaltare (by Br. Clyde Ba^) the smslti" 
nucleated cell disappears te£ i£ resppesrs %fea traasplsated beck to aiasc 

SigEillga^es Maoplestis alteratrioas ia cells ^icfe Issve 
easily raeognissd i^rpholsgie ©r asatabolie elis,rac£er3 ©ay lofid t® a 
clearer imdarstandisg o£ asoplasia. 

Proposed Cesrses "Sie trajssplssst befesvior ©f ttese umssasl 
tiisaors is bsisg a&sarved. Studies m:Q plssaasd to deterasiae wliesber 
the uteriae £i:^5rs raspoEd to endserias alfcerstioas. 



Part B iscladedi lo 



957 



■Serial Ho. ,.,151.507. ^ 

l» iaboraeos-y of Fatholegy 

2. Canear lEduc. & BstSiogeeesis 

3. Besfeesda, m^laad 

Individual Project.lepore 
Calendar Year 1959 



P^gt A. 



Project Tielss Factors Sawolved la AborSiva Mitosis in Ensma Isir 
Follicles iu the Catsgaa (restisg) Pi^ss of ^@ 
er©®£ls Cyele 

Frlssipal Xavessls®^©^' Soss C. ^cCssdle 

Otfess- lawsstlgators ? E. J. Vaa Seotfe 

ScopsEsati^ Ufeiess 

l&a less-® (csleBdgy year 1959) •/ 

To&alj i/2 
f:esfe@sioBal| i/4 
Otmrj 1/4 

F£-©jae£ Sssss-iptioffis 

^j£et|^g92 We dlseovased ehsi: the epithelial cells of hmsBn 

h&lx follicles is the restiag (eatagea) pisaee of tha JMiir g7c»7th eycle 
reveal svidescs of arrested M.eo£ic f igus^Si, wSteyees in tte ae&ively 
growis^ pisasa £ew if say such abortive aitofeie figures could be foimd. 

^H^ss A taisr©seop£f. stisiiy of biopsy speciajaas. 

gi^saificaaea; We sre ii^terested is^ ds&enai&iicts x^l^t^er basal 
&nd sq'jasssua cell tiss^rs possess sueh figai^es.. We are also iaSereeted 5.a 
tryiag t& detajnalEs ^bsah©? sosaa ssafiMssslly oeciasriisg miSotic lahibieor 
stay he pre@@sst dtsris^ elie esssges pl^se aiid nsceralised la t"^ mi^en 
plms@j— a situstioa sisiilss: to tlse pyodactioa of «ce£ylcbolitj« sad its 
b^efifeio^ns by sla^lisseaserasa, 

BroiK>S|ed Ck>«gae; f isishi&s it for fl«ai proofs sad we pilsB 



^ri: B issludeds lOo 

958 



Serial Ho. mi 508 
1« Laboratosy of Baehology 

2. Caac®^ Bsdue. & Fatiiogeaesis 

3. Bafchesda, Maryland 

Individual Project Eeport 
calendar Year 1959 



^^^• 



Reject fltla; Cytology of Antibody»produci^ Plasaffi-cell Tis^srs 
of Miea and Wsmn Ifoltiple JS^lome 

friaclpal luveseigator: Ross C. HscCar^l® 

0£ber' laves tigs tors?, HisSxasl Fot£er awd Joba L. Fal^y 

Msa Years (eelsailsr year 1959 )s 
Tosali 1/2 
Prof essionfel g 1/4 
Others 1/4 

Pr©-jest Eteseriptlcas 






wess Te detessaisie fcfee ia£racell»lsE site of produetlea 



of ^stibcsdies @nd to aseestsisa e&a eellulsr orlgta aad fate of plass^ ©ellSi, 

^thoda s By eytologlcal tectesiques for Golgi apparatus aitoslsoadria 
e.nd Wkf t^e sSudy thz tmeox tissue and also plssa® cells iaduced to foMi ia 
miee iaje^ted witfe rabbit serum or Freuad^s adjuvaaC 

mjoT Figdiags ; Bse Juastmauclear cleer eytoplasnaie "hof by 
vrbich tbe plasi!^ eell ie elsaracteriaed cosslBts of tbe Golgi apparatus sad 
ti&e ce&£ro@osis. ^e pl&sa^^eell ttsasrs of vd&e possess three differest 
types of Golgi &p^t&tnn eoaslstit^ of: t. & i&xn sissall strands; II. s 
diffuse set:i?orl(i and IXX. ® Isrge t&iek l^pertrophied ball siet^ork. Two 
of tbe tutors prodtsfiing ba£s-globulia can be distlaguisl^d frojsj the otSer® 
by tlieir (steraeterisfcic l^rpartropliled Solgi apparatus ityp& III). However;, 
the typs of Golgi appsrst?;^ does ssot appear to be related to felse electro- 
phoretle Bsobility of tlie globulla, but ratl^r to the speed of productiea 
of the proteitt. IJhe cytoplasm of plasoa cells is rich in B^, but tha 
Golgi sres is free of it, 

Flsssa cells of plss^°«ell ttsHKire appear to derive fr«ws lympho^sB-^ 
like cells t^hich in turja probably detive froai tsadifferesstiated ly^feo-^ 
cytogeaic cells of the genaisisl centers of lyeiph nodes. 



S59 



serial llo, _^1_508_ 

Isdi'v-idBSl Project Beport 
Calendar Tesr 1959 



I'&xt A (cosSlaued) 



Slgaificssce ; Suek isfssrssaeioa amy lead to batter Kssderstaadiag 
of SSia jseclisniss of produetissa of antibodies aad £o & mstlsod ©f coiatrolllag 
the differeatlaeioE of plss^ sells in pisscsa-cell tiaaosrs asd sessltiple 

ggpposed Soisrsa; We istesd to purstie this probissn ia aa afifeesspe 
£o prove the celtelar origis of pl&saa eelXa. 



Part B iasliadeds Ho 



' 360 



Ho. _._gii,gO£'. 
-. i^boratory of Fatbology 
2c Csaeer laduc. & Patfeogsaesis 
3o lafcassda. Ms^ylsad 



Indlvidi^l Project Report 
Calendar Yeas 1959 



Fart M.. 



FrejeeS TiClej Tlse frotopiasa ef f^jsior Calls: CSisracterlstics of 
iBaligaSiit aeoplffistic psrptopXasm 

fripcipal Investigator: Eoss C. I^cCardla 

CoopssraEisg Units; 

Man fssrs (calendar yssr 1959); 
Total: 5/6 
Frofessieaai; 1/4 
Othsr: 7/12 

Project BascripSloBs 

Objgctlveas This sasuscrip^ coasises of about 300 typewrieses 
pages beiag written la asoaograph for© as part of aa loeematiosal Baadbucfe 
4er ?ro£opl&ssaf©S3chuag edited by ^iibrunn sad Weber » Xt: describes 
Che eytological end sotee physiological cbsractsristics of tssnor cells„ 
It sostaias scatffi aew i^terlal sueh as plasi^-cell tumors ssid pituitary 
tasaors thas lias ao£ been pablisked elsewhere, 

Vmp D ssd^ ^ Course ; To be seat fco the. printers oa J&mMxy 10 « 
Tfeis ^111 finish the work. 



Fare B included; Ho. 

861 



Serial Mo. -__iei_51Q 
1, Lsboraeory of Ps£bol©gy 

lEdividual Project Report 

Caleadar Year 1959 



mtt A. 



Project fit:lss Study of EsidocriEe sad Reproductive OrgSKS ia Sfes 
MBJ2Se 

Friasipal XtiveSfcigator s ^Haelssi 3. D?ss£i 

Other Saves tigstoirs s Rcss C« IfecCa^dla is doisg seia® ptesss of £bis 

study, ^, .^sepfe Alferee&t ®ad S^, Bass Bsrgssass 
er® ffi^ls^ speeisl pxepsrs£ioBS of the pi£uit;asy 
gisad. 

Uss Years (s^^^Jsdas: year 1959); 
foe^ls 3/6. 
Frofessloaals 1/3 
Ofefe^s 1/2 

Project Beserif^Slsai 

Objectives g fljis is s coatisuatioii of msrfc begua ssversl y^airs 
ggo. It is espscted Sfest it will coatisajsa fo? © coEsiderable psriod of 
tiass siaca tiss is required for aessssHjlstioE of sasisaal©. To sscuaaslate 
reliable dafea by obsssvatioas oa uiatreaead miee^ plaimsd psoeedu^es^ 
aad sevies^s of tlsa lit^raSssrs regarding tbe reprodisettve assd eadoerir^s 
©rgass of iabsed siee. 

Metbods ; PsrsoKil obsservstioas as ^terial sabraieted fros 

different so^^fsasj, plsssssd 33£peria@Qt:sl |»?oeedta?es i® isbred ssiee^ 
feissologic study of usSirssted mise at varloas sgaS; syssei^ele review 
of £bs lifeeratwre. 

!^.'}or glag^iflgSi iSiis study is proceeding slowly beca^sss of 
pressure of ofees ^ork, -Coaslderable Korphologie difference is fcasad 
ia tlse sdreisal glands of iabred straias sf sice. SM© tmtetl&l is 
already g»rovias useful to otfear Biaoa&sss of tfee staff siace ie ?ep?£S©s£s 
a noEi^l st&ad&rd of sc^jb eadoerlne orgasis 1x2 tbs e^tuse., A bagismiag' 
has beea ssde on writiag up raetales oa fe&a edreaal glasd. fhs litesatssre^ 
wlsicli proved ^ssss^eSedly -^oltsalnous, hs« beea ravistjed. Ansjyphous 
particles seea ia tfee aedulla in Senksu^foj^aol f issd adseaals are beiag 
studied by Dr, Sam Spieer, He feas found shsll&s particles in the sdroi^l 
medulls of othar species » Ifercurie chloride is required to preeipiesfie 
them sad ne previous dessri|ssioB hss been fousd of tlasss bodies. 
I^jcatioa of tbs pieeal glasd of tbs wotise bss also been determiae-d^ asd 
sojse observations ®r© being asade, 

962 



Individ«£l froject Sepore 
Caleisdsr Yeas: 195§ 



Part A (eoHtiaued) 



|^^;^^BC©2 SiKse liostaoaal studies ase an iH^Kjrtant part of 
caacsr s^sssarch, it is desirable that reliable iaforantioa be available 
ragasdlng tl^ eadocriue B.M repsodtsctive orgaas of iabred strsias of ssise, 

Froposed Courses Xe is eKpected £lss£ fcfeis 8£udy will proceed 
slc*?lyg sad ireqsilsre ss-ye^al ye^rs for eoE^letioa, 



Fart B included t Tes 



c 



^3 



Isidivid«al Project Report 
Gsleadar Year 1959 

Part 1. Honors^ Awards^ aad Ptajlicatioas 

FubliGseioas othBT £hsn absferacts from this projects 

Bisim^ Tbeleas B. 

Cs«eer Eesesreli ia efea Soviet UoioB 

J. Aaso l-^d, Hotaea's Assoc« 14i 314-316, 1959. 

Mt^, C J., I^i9^ L. W., and Buan, T. B. 

Staidies of ?.^'6«id Tus^r Agent ia Cultures of l,e«keffiie Tissues of Mica 

J. mt. CmcBx txist. 23 1 717»797, 1959, 



l«jsors esd Awards relstiag t© £his projects 

lo Frssidesst, Wasfeicgtoa Soeiety of Bethologists 

2o Biits^er^ BoBsd of Bisectors ^ im&sic&n. Assoclatloa for Caracer Sesesreho 



96i| 



Seriiil MOo HGI 

Ic I^iboratory of Fa&ology 

2. Csacer lEdae„ & Pathogenesis 

3. Bethesda, Marylsad 

Xadivlduai Project Report 
Caleadar "fee? 1959 

frojact Titles Collaborative Resssreh 

Frls^lpal Xnvastlg&tdr s Siclssrd L. Sx^arsa 

OShsr lavestlgstorsj 

Coopsratisg '{Jaitss 

Mas Years (calendar yeas 1959): 
f ©tsi ; 1 
Professicaals 1/3 
Oi'liers 2/3 

Projsct Beacxiptioa: 

Ob^ieetlyes g To collsbsrate with and advise otber irivestigaSors 
particularly ia phases of research requlsfiag esqserleace in patlielogle 
asistoaiy m: the use of rsdiosctive Isotopes. 

^thodg; MeCfeods vary widely &nd are dependent upon ths problea 
aad tbe esSeat cf the collsborstion. 

I^jor Findiags; Collaborations ijMch have developed Include fcfee 
following? a) Collaboration witfe Dr. H. Morris on the effect of eadosriRe 
giaads oa the ocesjrreaee of hapatic neoplasms induced by 2-dificatylsiHino" 
f luorlKa. Sam& progress Ms beea ESde ia the coatiauatioa of the 
histological study of hepstie tumors which were found to have developed 
wore sloi^Iy in hypothyroid ssiEals. 

b) Collaborasioa with Dr* S. S. Spicer of the Laboratory cf 

Pathology and Histochemstry (KI&M)) on a study of naacopolysaeclssrideso 
Thsougiii the study of tissues labeled with S^ it has been possible to 
cotif Irsi the fiadiags of others that esuch of the ©cid nsicopolysaccharides 
particularly of smciiss are sulfated. In additioa, ho^ever^ soEsa asid 
Baacopolysaecharidss were fousd to coatsin no S^^„ Dr. Spicer has gone on 
to ideistify these aoa-sulfated acid naieina ss tliose which have a high 
contest of sialic acid. Presentation of this t?ork was s^de at tfea Histo-^ 
chemic&l Society laeetiagji April 1959. 

Ixi edditioa as a result of past collaborations interest eoQtistses 
in; a) the distribution of tetracyclines aad their fluorescent patters Ib 

necrotic tissue and certain neoplasas; b) the sstabolisirs of bilin^bia a^d 
study of tissues froia aaiasals with a congenital eaayEsatic defect ia the 
metsbolista of bilirubin. This coageaital defect is &n inability to coEJi^gate 
bilirubin ssd certain other chejssicals sad horiEoaee with glucurosic asid. 

965 



Serial So„ HGX S13 

lEdividisal Project Report 
GaXesdsr Tear 1959 
Part A (coaSicusd) 

ISurisg th& past year £be pyiaeipsl iuves tigs tor i^s coasts Ited 
with a Euaisisr of scientists about Sheir research work sad feas iectured 
in the tbsree N.I.H. Isotope Traisi^ Courses given dmring the past yesr = 

gi^ifieSBcs; Cellsborstion ia iEdispensabie te s^ay types 
of caaeer research. 

groposed Coarse s CollsborstioES listed ■sill be contiausd ss 
desired » 



Fart S iaeludsds Ho 



966 



Saslal ISo M£I_5I2 __ 

1 IsbQX&tOTj of F&ttelogy "" 

Xadlvidtial Frojset Ssport 



miA 



F:-roJec£ Titles Traasplsa£atioa Seiidias with PsrCie-alss Esspfeasis oa 
tbs Morplielogical Altssatieas o£ Cartilage Tisstse a^d 

friaeipsl lavsetigators Elsteird L. S^aicsi 
Qth&T Ssvestigators s 

Maa I'ears (cslssdar ysa? 1959); 
Totals 1 
Fyof sssiosial s 1/3 
Other; 2/3 

db^gcS.ivQS g To sscsaaalaee infoirastitm ebotit: tsreasplaatad eiss^s^s 
Hitb as essfbasis oa s^rpSsslegie alterations of nonml proliferstlsrsg aad adult; 
cartilsge tissue sad sseoplssn® floowiag transpl©ntstion« 

Mstboda^g Cbanges in the eKviroEffisnt of cartilage tissue &tB 
effected by I) sueo^ iso, horns or baterotraKsplarsfeatioB; 2) sltarstion of 
the eadosE-iEe ststtas of the host aniaal; 3) administration of material 
salactively retsiaed ia eastilsge {S^^}; end 4) by slfcaration of the diet 
C lathyrogesic coiapoimds). This study is limited to aisperiaffiintfil anissalSo 

Interest ia trsasplaatation e^erinients in general exists sad In 
order to better uaderstaad tbe traasplantatioE of cartilage, study aad 
collaboration in the traasplaatatioc of other tissues is jssde ais opportuBity 
develops. 

j^Jor ^ittdiags ; Studies previously reported of iso«» &nd hssK^ 
traiisplasfcation of a mixed tijisor coatalaiBg sosaa cartilage tissue have bees 
continuad. Adaptetioa of febis twnor to esrtaio hybrids asd homogrsfts has 
been ascompllsked. Attempts to taodify the amount of cartilage present ia 
the tuffior have aot been very successful » 

Salactive.prolifsrstioa or matrix forsEatioa of the cartilage ia 
the hoasografted costochoBdral junction fess been further studied using fetal 
and newborn cartilage as xfall as adult cartilage. These ejjpariments 'v^ere 
set up as chronic es^srlEjents and the only preliminary iaforsatioa available 
is that these grafts behave in a fashion sissdlsr to those previously studied^ 



967 



Serial lOo _.J:C1.512 _ 

ladividisal Frojece Report 
CslSBds^- lesr 1959 
f&Tt k (eosfeiaued) 

k pilot &xps.Timsnt with oaly vesy f&a miiiaals has beesa s£areed 

is whlcii cartilage fros a aewbora anijm.1 wss plscsd in tissue eultare 
fluid contaiaing pslyotsa virus sad tbBn fesasogrsftad to adult aalasals. 
fhase saisals are being observed for ths developnsaat of ceoplasmSo 

Bogs autografts^ istrsveaously wifels thyroid giasd tissijs by 
I5T» Rieh&rd J, Saadssrs w^ve studied '^tth Br. Jolm Edgcossb. Survival of 
th& grsfted thyroid tisstts ia sed sdjaceat to puliaoaa,py blood vessels kas 
bsea d.s3oast2'a£sdo Obsarvatica of surviving aBlusals for ehronic cbaages at 
thB slt«:E of the grafead' thyroid tissue is being issde. 

Sferlag the past js&t sfeadies were pesforsned with a trsE-splsrstafels 
cartilsgeaoas ssaoplssta is rabbits obtaiaed from Dr<, B. Levy o£ Houstoa.f 
Texas ., "Hiis tussor i^as tratssplastab le by itstraoccuiar transplaatstion in 
Br« Levy's rabbits^ Isj our Isands, sewboma aaiaals were transplanted wita 
the tisiEor ia tbe hopes tb&t iu the newbora deeresssd iEaeauBologic sensitivity 
sud iBsreased asjoaats of grcs^tb honaose laight asks posisible grestsr growth, 
ftesa subcutsaaoijjs tsrsssplasit© did incsesse in sise initially hat regressed j, 
presiffiiably due to a hosmg'^^ft reaction. InterestiEgly eaougfe^ all of the 
iBt££oceuler trenspiSEts we have tried bave also been ustsuccessfui. Oae 
possible esplaaation of these results laight be that the saissals with wliiefe 
Dro Levy has had success trsnsplsntiag the tumor have all been closely 
related to eseh other « We ere not an^d-ous to have a tumor whose gro??th 
eSD be isslHtaiiied only in the iatraoecular site. In an effort to effect 
subcutaneous or intrataiscular traEsplaatatioE of this ttaaor we have 
successfully trassplanted tissue to fetal rabbits in atero. Furttser 
attesspts at ia utero traasplsstation are planned for this tissaor." 

Sigaific&aces "Shis projact developed as a result of collaboration 
with other investigators in the Hetional Cancer Institute . It is basis ia 
nature SBd of interest ia understanding host-tissue responses. 

Proposed Gour'se i Ihis project is to be coatinued. 



Fart B incliadeds Yes 



oq3 



IrsdiTidual Project: Report 
Calendar 'tsjar 1959 

Part B . KoaorSj,- Awards^ aad Fablica£ioas 

Fublissvtions otiier tssa abstracts from tMs project; 

Ic Swsr% R« L.^ Scfeat£sa, W. E.^ Saaders^ S. J.^ aad Bsrgeastalj-, B, 
Tlis AuSoradiograpMc Study of TraBsplasted Tissue. 
Lafeorstosy JirvrssSigafciea 8 s 149-161 CJss«Feb„ 195S) 



S69 



I. Isboratsry of Fatbology 
3. 



Individual Projast leport 
■ Caleadsr Year 1955 

Fart A. 

Projaci; Title: Ostogasietic. Bsvelopmsat of Plsssm Cells In Mice 

Principal Iirires&tgator; goes C. l^cCardls 

OSber lavestlgstorst 

€oopera£ios Units? 

&& Years (csiesdar ysar 1959) 
Total I 1/2 
Ps.'ofessiotssls 1/4 
OSkass 1/4 

Project Baser i|Jtioas 

Ob ^active® ; Tbs lya^ph nodes, splean^ &nd Ileocecal region of 
0sice from fstus eo old ags are bsissg &ssxsd.iSi&d to deternsisse the age at 
t?hieli pMsjsa. cells first appear asd to daterKlae the rate of productioa 
at later stages, fhe cytologieal c&sraeteristlcs of plasaa cells sre 
beiag atudisd to &BCBTtB.lti the 8t&t& ef the Solgi eppssattiS;, Kitoehoiidria 
and cytoplesaic EH& at diffarest st&ges of saturation of tfea plassa eellc 
As atfcSEEpt is baisg sasde to detsrsaisas tha cellular osigia of plasasg cells,, 
end to detanaise ^isether plesna cells cast ba fosnaed xmder artifielsl 
stiissulaticfQ prior to t&eir sorsal date of ©ppaa^ance. It is aot yet 
kco^s whether each typa of protein satibody is produced by a single ssparate 
pl&sjss. cell, or ^Jjetlser all ara pxo4iiced ia the ssase cell at different 
stages of saturation ^Mch imy er n&y act be reversible. 

Mstfeods ; Fretsnd'^s adjuvsnt vill be utilised as a stltstulsnt to 
fornstioa of plsssa cells. This csa be iajected into ths fetus as well 
&B in tbe ne«rbora. Tissues vill be t^tksa st various ages azid fined and 
staiued cytoiogicslly, 

l^JQg Fisdlags; Id the aaeuse, plasma cells first appear la taa 
lyiaplietic tissue of tfee, iatestinsl ssseatery &t about ten dsys after btvtho 
MoEe were fouad before birth and none lisaedistely sfter birthD Plsssa 
cells at tea days of sge (called tentatively puberty plBSj^ cells) csa 
be distinguisliad from tfejse of tfee adult si^asa by tha sisa of thsir 
Golgl apparatus wMch is distissctly saiallar ttoa that of tlie edult pleS'Sis 
cells. It is not known riib&thss such age&ts as Freimd's adjuvant eould 
induce the ten«^ay old nause to form adult piastre cells, or "uhethsr a 
puberty plasssa cell assrely differsatistee into an adult form. 



970 



ladividtml Project: Report 
CsleBdar Year 1959 



(eoaSiaued) 



gianlfieaaga; fitsss firsdicgs and the stiidies plaaaed ars 
bslie\?2d to fee of f-oadsssaaesl ii^ortanc:© t© sa isaderstaadisg of She 
gaaesis of & plassa-ceil tassor la the isouse, s.nd possibly also of aiuleiple 

Fgopogsd Soarso; It is ifitatsded to pursue this study^ particulsfly 
with £ viav? to daterEjinisg the relative susceptibility of aewbora sad 
adult; ad.ee to the forr^.-tioa of plssn^ cell fcusK>rs, 



Fart B iacl«ded; No 



3, Beg^®4s^ m^Msd 



Isiivi&Ml Project Eegort 



^^4*. 



Feojsee Titl&i Bsdsesi a^ Spoataaeoas Mespl^ssa© i© tefe©^®£©^ 

f®f:ais 1 1/3 

Fr©f©ssi«aii 1/3 
Qtti&st I 

Qi Mp®,t^ i^^<s&d\y ss© dfss i® sale sad fessle safes. B. lafl^sea 
0f p^i^s^ find t^isssptea^d twsss© oa liissu© and s^sbbs steaissssr ©f rsss* 

^^|si A, llal® sad fasaX® 08borse«l^isdel mtM ^re fed ob 

S. A smsQ^m issdueed ia o@bes'ffis«^adel safes ia e&i@ Isfeorasosy 
was tr^ssplssi^d is ^il@ ^is^ls of £h@ aa;^ et^sio. Asotl&e? groisp &£ 
m^le aad fea^le ©8&oEae»l^Qdei gats tms fed ©« seratss^fcliefeic diat 

pal^ibla sad reae^d a siis ©f ab©u£ 4 ess, is difil^tarj ti&^s m%4 mnm 

fe&ass assies, A p&p&r besed ca thsee fisidtege Siss basa publi©&gd. St?adies 
osj goaadeetosl^dj adrssmleefeosjisad, aad Itosstoae ereaeed sat© sre seill 
isi p?03?@s§ asid no £l£idis^3 as'S yet^ available oa tf^ia. 



872 



Serial 'Siu^m^^A^ 






Psrfe & i&&s.tisk%s&4) 



B. Seisms @Mol@s® vslt^s &i B&ssi&^ &td i^^tms^ h^simg t&Z^ 
^se^e g>e3|^.©ei'^sly 3 ssd i £iss®8 £J^ nalass tog no^s&l g'ae® t?iSlm'5s6 
AM©Mss lavels fos" liver fetssuiS ©f £B®es-»b©effiiig saes ^ere alxswfe 3S% 
Im^ti feMsi €139^ fog- g&g ll¥iar tissue ©f smiesel sses, A g®|s©r te-a®a 









m^is^l, ^feltah ssiglfc fee ^©e^oasiljls fo:^ diff arrises is tsassos' g^m^® feate^e^ 



Fast B iaelisdeds Ifea 



973 



aJ®?i®l &. .MUB^ 






MgS^j.' ^asss-s, tessds^ ^[4 ^alblie®tioffi@ 

" as© 4y©@, 

S^oe, S®e* 1^. Bi©l. & Med. l@Os 32S«3a2^ l§5f , 

P^©e. S©s. S^, Biol. & mS, IQlt 763»?66, 19S9, 



974 



Dw;aa^i& iSi., t ^vRjJ^^_£A^^ 



3 c l0gte8d®, Sferylfissd 






teLA» 



Frsj«?.et Title J lffe«fe «3f fete ©s-ss^jefe ef Msmmiz&'Stisml Sarsim»ii osa 

mis J^BSB iml&^&s y&ss 1959)% 
fosalj I 

Otter? 2/3 

usiag &0@£ rat s® a fei®l©s4ael £©©«: saisil,. 

fesmls 3^t@; latest ^4 sdrsss&Isc&oMi^d. E^imisig po^ssiisa tolsirs^-e 
ssgss asKl glys©ggs d®|«©iei®a tssEe oa sdsr^aleeSrasised saes wltk sad 
^l^&G«t iTSBo»s. Sodiu® a^ poSa9gi«® @$udis@ ©a s®£8 wtfch «»d ^ifelio^t 

m.1ag Fte^ia^s^ fot^sslum t»le»mse tssfcs sfew fe&sfc tMs tm^t 
seeretas saiassalossortieoM®, A paper h&^d <m sfesse fis^is^s Ijm hem. 

st^y «sse s?e|>or£ed Sssfc ygsr. Ssw3£taa eat! poSassAiaB study laadiests 
!3iiii©y©i©eosrfclaoid ©eeretiaa by fete tttsay. A pspsg- based oa l&sge fiadisigs 
is ia psspaystlcsa. PKsliiaimsry reaul&s oa glyCog®© d&positiee tssss 
Indicate zMt this £:»a^!? a@3?@t@s little^ if sss^^ gXuecsos'&ieoid©, 



Si^ifie^esg f&ese fiadiisg^ ia psst, defisse e&e sseroidial 
«l^yaefeeristiss of £lsi@ Sue©?. Failusa to syae&s®is@ ee:rteia stet'oldg by 
eliis emssB lavites @a issquisy ©beast fscSers gesponsible f©r £lsls feilm^a^ 

ggo ^&ed ^CouE@©g Test© for glweoeoyticoid eeeretioa by tliis 
txsmz as® ©till uisd^y @t«sdy. I^tsbollsm es^nriissiiits sr@ beisig plaimed to 
is'^stigat© fseters ^feiels felosk syssSlae^ls ©f e©ygaia steroids by tte t«eor 



ijfE ® iaeludsds So* 

375 



2o Citiicei: leduEo & ji?a.t,. 

Irsdividuel Project Report 
CalSBdasr fear 1959 



Fart Ao 



project Title? Studies on ehs taduc£ioa of Hulttple Ttmors in Cert&ia 
l&ffia^ls with StewaxC'-'Sddy FolyoEsa Virus -=6 is sue Cultux-e 
Prspsratioas 

Priaeipsl Investigators MesrI F. Sfcsstoa 

Otaer Investigators s Sobert Blackrasll^ Ssrafe S£ewsr£^ Bemiea Eddy (BBS),, 
Joseph Albrecht sad staff 

CoopsratiEg Uteltss DBS^ laboratory of Biology^ I3SI 

flais Years (caleodar year 1959 )s 
Totals" 1/2 
Profess ionale l/^ 
Otfears 1 "4 

Project Descriptions 

Ob^leiati-tfess 'Shta is a coBtiauatioa of projects outlined is 
ladivisluffll Project leport MCI 516, 1957. 

l^jor Fiadiagag Svmmrtes of aiajor f ladiags ia eoopersfcion ©Itb. 
oeher investigators %?ill be foimd in tbeir reports o In addition^ separate 
invastigatioas of host faetors iavolved in induction by this leboratcry 
have established ths folloxo^ijsgj (1) In collabcraKlon wi&h Br8„ Kafeler and 
Stei*£r£ &n analysis of tus^v incidence has been nsde in aice inoculated by 
liffiitlssg dilutions of the vinss culture preparations that were subjected go 
differential high speed cents: ifugat ion » All fractions induced eusior© 
sltiioiigb sKsst of the activity could be sadinsnted at a particle sise 
eons is tent vith that of the virus. The variety of tvanors did not vary 
significfiBtly in the different fractions snd no significant incidence of 
leuketa!^ was observed ^ The iimtisg dilution and fractionation ello!t?ad 
an analysis of tumors that follow ©inisaal doses after ioag*term study. 
t4&-txy non-specific iesiooa particularly in the kidneys and salivary glmads 
of older sssice indicated either abortive etteiapts at cell proliferation or 
regression of miniiml lesions,, Older aiice also had 45 significant Eurober 
of etaaors particularly bone tusaers that showed clear evidence of acquiring 
characteristics of invasion and siet&staseSo 

(2) fbs transplantable haiaster tuajors refeirred to is MCI 51£(i^sii) 
have eoEtinued in transplant , Serological atteiapte to detect antibody 
in grafted hisaastere have been made on a limited basis. Thus fsr significssfc 
MI antibody has not been detected in the grafted aaiaisls„ Prese-at studies 
are being carried out on grafted 'newborn haiasters. >liieh of the work with 
grafted ttnnors is directed toward detertainissg whsfc influence host factors 
may have en the behavior of grafted tusjors and Induced tis^rSo T5?o systems 
are used 2 (a) varying host factors (sge, sexg csj-rcinogen esposurs^. ffiutogeti 
ea^osura^ etc.) Isi aniiajais grafted with tussor lines of knoism behavior? (>) 
itidtisis^ tmsDTB of varying type and behavior frcsis single tissues exposed 

976 



Serial Ho. _..l:g_gl_516 

ladividtsal FrojecS Expert 
Csieadar Year 1959 
Fart A (coiitinaed). 

£o ■virtis-eislt'ares aad is^lsBtsd tato £l&e ebaek pouch of weanliiag Issastsrs, 

(3) A criticsl analysis of the tvmors in saiee feas beea publisaed 
(Referesiea 1). Original observafcioas ia this study iaclsjde conGlus5.ve 
support fo?: theory that the active agect ia a visrus and relatien of twaor 
developsans So availability of passively trSBSferred aatibody. 

Sigaificaacag The polyoa® viEws-ttsaors serve as e^jeptiotml 
essgexlsasEital tools. ■ Tfee if?ide raage of epscies fcfeafe respond find convea lessee 
'in working x^itk small redests is &n advesstsge over all other virus tumors, 
Secoadly tliere le ZhB advantage over chesaisally induced tuaiors hath, ia fcbe 
short l&tsnt period aed opportuaity of following £hs progress and behavior 
of £liS eu?aor© ia raiation to virus as detected by sj©ej virologicsl sad 
ffiorphGlogleal tacliaiq^ses includissg fluoreseaace antibody teeiaaiques. ifcek 
of our ssora receat activities ceatey on perfecting the fluorescesce teelmique. 
FiHffilly there is good evidence to indicate that inductiou mad prcgressioE 
proceed ia a slow step-wise fashion ishicb xs&y allow separate sttsdy of the 
ti»'o TBoat isaportant sttri&utes of tramors nasaely cell proiiferatioa aad 
the asq^jisifeioa of agressive behavior patterns by tise proliferetiag calls. 

Proposed Gotarseg The davelopiaeEt of a sisaple basic system for 
tissue culture prepsratiea eimblisg this laboratory to csrry out ssspsrir^iats 
oa host factors with s siagle staadard line of virus culture preparations^ 
Develoi^^at of fluoreseeat antibody techaietiss for tfea deteetioa of poiyoam 
vijrus &utigen. Freparatioss of 'a report oa tlia bistogeassis of tujsors wMcb 
develop in tfee hasjstero 



Part B. i-Bsiisdad^ Yes 

977 



X&dividujil Project Seport 
Calendar 'fear 1959 

Fart Bt Honors^ As-jards^ aad Pufclicafcloas 

Publics tioss other fcfesa abstracts from this projects 

1. SeastoQ, H« F.p Stewart;, S. E., Eddy^, B. E,, sad Blaelasfell^ 1„ 
flSQ oaeoganis effect of tissue culture preparstioas on poIyoEE 
virus oa feSal ssisso 

J. Mafc- Cancer Institute. DaceH&er 1959. 

2. StsatoHo M. F= 

"Ete local oHCogenie resposse of liaii^tsr tissues ts polyosia virus = 
CsKCsr Eesesrcfec la press. 

3;> Stews?t^„ Sc E...^ Eddy, B. B.^ aad Stanton^ M. F. 

Heoplasi^ ia rodeat:s induced by S. E. Polyoast virus » 

AiJTA Uaio Interaatioaala CoaSra le Cancer }C^- 842-851=, 1959. 



978 



SarlAii 5!o. ^gl 518 

i. labosBtory of i^fcbology 

3 c Betliesda^ l^rylasjd 

Xndlvidusi Project Report 
Cales^dsr "fear 1959 



Part Ao 



Project Title I Sspsrlaeatal Induction of AdenocarciEOEa of the 
GXasdular Stos^ch ia tl^ Mouse 

Prlaciisai laves.eigaters Ed^^yard B. Price;, Jr. 

Otlies Investigators s 

Gooperaeissg Uaitss 

Maa Years (calsadsr year 1959) s 
Totals 1/2 
Professionals 1/2 
Others 

Project DascrlptioB s 

Objectives t The eKperiraantal indiistion of adenocarcinojas of the 

glandular -stosmch of C57BL laice by istrssaursl injection of sstfe^lcfeolacEfeeae 
hse been reported by Stevrsrt and Lorens ia 1942 aad by Esre et &l is 1952 » 
HoKsves-j the overall yield of tuisors produced by this sietlKsd was saeall. 
It was decided to attea^t the ssEa procedure ia cortisonlaed mce to 
determine if the yield of tumors obtained '5?o«ld be greater tbaa tbat '^ith 
ajethyleliolaathrene aloaeo 

Msthode g Soutiije 

B^jor Figdia;ge g The project is not as yet coBapletsd^ but the 
findings to date suggest that cortisone has aot enhanced the yield of 

saetliylcbolssthrene induced tui^rs of ttie gl&aduler stosasch ia mice, 

Significaaee ; Studies of the getm&ta of earciaoma of tfee stonmoh 
would be greatly enhanced if a asstbod could be devised for the e:speris^ot;«^l 
industioa of tlmt lesJ.oa, 

Proposed pourse ; This study Isas been delayed by the coffipletloa 
of the collaborative projects listed belov^?. It is proposed to coffiplete 

the project o 



879 



Isclividisal Firoject laport 
Galeadsr Ysar 1959 

Part A (coa£iaued) 

Qthet projects in collaboratioa with others s 

1, Tgj^splaa tabls tbi oagaclI--li5d«eed thyroid Smaors la the 
rat ^isfe Bro Seyassur Wollasasi., Report msbnttt&d to Br." 
WollESis oa October 15^ 1959. 

2. Trsas|?l^table_ adrem^ Q&Tcljtm^ in the rat with. 
Sr„ A. S. Mulav. Reporl; submitted to Sr, Hulay ojs July iS^. 
19S9„ 

3 o gathologic fltsdlagg la tha huxaaa kldaey ia patieKts witfa 
gciEte lea^gla vilth. Brs..E. Frei and S, F^its. Report 
subffilttad to STo Frel oa June 15, 1959 « 

■ ^^ ^h@ fate of iatraveaously iBjecfced w&lS£Sims& cells la ths t 

laEg and tbe gelatioKshlp to t1ia_e f£ecfclveaesg o£ _ clseaot'tea'aa/s 
Ais a^^risBSistffil study with Srs. D. L. Kinsey sad E. Eo Smit!i„ ' 
Report eubsaitted to Sr, Kinsey my 30^ 1959. 



Pert B. iacludeds Ye® 



980 



Icdividual Frojecfe Rapor£ 
Calendar Year 1959 

£^t^. Honors. A^-fsrdS;, sad Fublic@£iosis 

pablicafcions other tisaa absfcrssts fecHn this project i 

Kinsay^ B. L.^ Prise^^ Em-jard S., Jr',, and Ssithj Robert S„ 
Effectiveness of Cheuiotaerapy in Ralstion to Ex£ravascular Ssvasioa 
by EaJbolic T^simx Sells. 
Accepted for publication by SA^EE. 



98: 



wm-^nm 2. Csaeer ladue. & mthogm^Bis 

Cslsssdmr fear 195S 



^ElA' 



Brajest titles SsirtieosSesoid Ifetsfesllssa of ss Ads@aos®g£4eal 
Ad^@€STCisiss£i£ of i^t. 



Otteffs 2/3 

^^^^^Si T® ^sSsfsiefe, ^ssify^ i®olst@^ @^ Mesa&iff j: 



sad pg@.®@svi^ tfeg Si»©r £igs«®, eoilseSiag arisie® fsro® ewaQr-feesriEsg 

wife @6tea©i s®d ssfSfStiag th/B sstessg fsosa solid yesidsss* Ss^a^feion 
^i llfids fs-sra efea e^raei: bj fseasiag ii®d salfeiag sue £®afelgi^®. 

aqi^oM 2r©sid«© vkth ©Shyi fssetete, pasrif ieafci^i ©£ £fee extei^e aid 
s®j^/»?issg selyl as®es^© i» vssus, sesiroid sesii^s tkss pssgsssd^ ©©Issst 
els^oa^&®gf®pfed itssio 5 f£(®©Si«^i8 o^ ^3 ftlisoiiis^^ @i.l.l@isi£9 c&l%^3i» T^s@ 

sohm^t ^ystssB^g p®.^^ &tstp& developed to lo@a&@ s&@?oid sp&ts^ tM 

aad %?lt& varieii® @teais©X ®g®s£s« 

^j^„ F ladit^s I Prsliadaary results sbe^ iadicseioas of 
steroids elegfit© eelQSteissHslosi®^ ssjdffos6©a@dio®s, pyojestes^s^ %-dro2^* 
tasSos^rof^j and @ lagge spofc of hlgfely ©olsy ssegoid^ Eosre pslsi? fefess?^ 



382 



::=3raili ^',^.„iHr^.„;%y'. 






Fsre A Ce«eiwe^) 



Sigaifi^^ae@,r Id®©fei£i@®ai®a ©£ £&s steroids ®ass@&®d fejf thig 
frs^ tM.t mi the aes^l ©is-saal gla®^, 1^1® is teffs ^y leaS to face©r® 






ef t^^ BZmmMiBi, t® eellsst @ii@5Bg& s^ter^l 1® ©se& fsaeti^^ &@ 



f&zt 1 iffieiwdedj ^o 



983 



Sazial K«„ _J^_I_52Q_ 

1. Isb&satQXj of Fstliology 

2. Saasar iBdue., ^ ^thagersesia 

3. Bethesdaj, Msxylaad 

Isdi\''idual Project Segorl; 
Saleadar Yesr 1959 



Part A» 



Frsjaet Title; Morolaologie Study ©f tasEf fasts of RadiaUoa 
Frotsetive Sora^ojssids 

frlacipal Ssvesti^sSors ; Bs^er W. O^'CSsss sad l&rgsret G. Kelly 

¥sm lesre (ealsa^a^ ^®sff 1959) j 

Fs-sfassiessXs 1/4 
Qth&st 1/2 

gbjegtiggas Tq deSeEsissej if possible, £fee moda of sc&loa ©f 
radiation prose^tive sabstssses oa £^ fcosdLc clai^es produced by ^^ &nd 

^Jteds? teteradisgsaphs of tiasiss fress islse givaa S^S sad €"* 
labeled AET ^ese ^aS to esSii^ee eissue distvibutxim of ABT. 

^jQT^F^^^s 1) AuSoradiogra^ of tissue blocks s&ow 

lossligpstios oFl^^'^^^ia li-ysr, g«t, bsae sssTow, rsd pul? of £lss ©plaerip 
sad isi the proscisal ecswol-atsd tsjfeslss asd papillae ®f the kidsey. 
Coacan&^stioa of S^S ^^ |_g l^-jj ;^ lyaplaQld tissxie (lymislj aadss^ tkyssi® 
gJvSad sad follislss ©f ehe spleas). It. is ©lao lo^ ia sk&lBml aassele^ 

e, fetv' sstisfaetosy taieroautoradiographs Mve been ©btslee^ to dnte^ but 
eliey appssr to substaa£iats efes ofeservsaisss listed ab©v® sad ss^ggeel; 
£fes£ t!is isferecellulss distsibatioii is cMsf ly g© fes' cy$epl^ssj„ 2s ^aa 
red palp of tfes spleesj, tl^ labeled MT appear® £0 be ©sSsseslluXas' saslies- 
£Ma assoelased t?il:b say parsicalsr eell. Ths *os®la esaSstes very lis&Ie 
rsdlosstivity, fe«£ ^faat is t^&v®. sppessrs eo b© ©oaesatrsted about afee 
isrges aeu^ous. 3) Autoradiogrspfes of titssue blocks fsross laiee givea 
C^*=Xsbeled .4ET s®\?e aa ideKClcal picture So th&t obgsised %'ith S-'S^lalsaled 
AET. Sn eoseras^^ assSossdiegrspfes of stos^l aisd £«s33Bor='bsari^ iaice killed 
30 Hjlaut®s af geg adisiraistsatioa of S^5 md G^^ Ifibslsd shiaiira® ase S^S 
labeled aodlt^ sislfeee sljew a lo^ but jaalfoir® distsibaticsi of rsdi^astivity 
tl3KO!^S?sa£ efes body tiesuss. 



984 



Serial Hoc _j mn^52Q__ 

ladivMusl ProjeeS leporfc 

Cslei^dar Year 1959 



fart ii (eoatiaued) 



Slgaifiesace; XS assy help ej^lsia tbs %^ariatloa is £ha effsctive- 

aess of AST ia diffeseat species ef ssixESigj, e.g.;, between the dog sad ehe 

SSOUSSo 

ggoiosed Seggse; 1) To Btndy th& niospltQ logic actious ©f ot&er 
rsdiaei-aa proteetiv© siibstaaees as ttey bacos^ available. 2) To study 
the eomparaeive tisssse di8£sibu£ioa is dogs arad godeatSo 



Pas'e B issludsds Yes 



885 



Xadivi«3issl, Projae£ Report 
€&l@.rdax 'fear If 59 



^E£=1.* Heaea-s, #,?jards., siad Fablie&tioiss 
Fabliieatioss otter £&aa sbstrsstrs fr^a t&is project; 



S., Sail, Bavid P-, Trivers, Gleaa E.^, O^GsrSj loger W. 

Aatloas ©f Sj,2-aiaia©eSfeylesot&l«2roEl?sa 3r, ®1 (MT). Tosd-eity sad 

Frstsesivs Effect Agaisist Hitsogea Mastard To^cifcy. 

J. TaB.xsm.cologj aisd EKperiE^aasl Sierspsuties. la press. 



986 



FIIS-1'IIH 
livifeal frojset Report: 
CalsBdar Tsax: 1939 



Pro jest TlSi®? Morphslogie asd Biologie Stiadies of Laukeasias aad 
LysfhGsas .©f the l&isse in Tissua Galture 

Friaeipal ItivestlgaSor ; Clyde J. Sasse 

Gnhst Ii%v&ettg&toTBx Micbasl Potter 

Cooperatiag Visits s 

ll^a Years (calsadar yesr 195f): 
fo£alt I 5/6 

Otliart 5/6 

rrojeeE Beaesiptioss 

^jgct^ess To observe the lEorplaologic chsjagss of leukemia 
cells of the sjoase its tisaae eulfcure^ s.nd to correlate these G&asges ysiti: 
alterstioss Ib aaoplastlc c&araeteristlcs <. 

Mst^dss CoafelHsso^s oulturss of Isukemias or lyaipbosssB are 
:atsblisfead bj- s^ehods dessri'bacl la prsvioos reports (19S7p 1958). 'Ifes 
^;5to logic slisracSeristies la cultiires aed ia cosapa&ible hosts are eooiparsd. 
as -fe'ell as the biolsgic differesses bea-jeso t^ cnltnxe IIkss and 
raoa^cultBsre (eostisiuo«s ssjase^pasgaga) iiass» Those 's*ec-slEsa*s tlsat l«wg 
been studied are described below o 

^^£«SM4SSS.' ^^' Ee£lcui?jai sell B@.xcon& F329« Little 
:^dvsnes ia terras of po^-itive findings bag besR sssda tflth tliis IIess bayo^iL 
£feos£ desc^ifeed la tfee 1958 report. Several e^sbiiues tims fead bess csrr -; 
^or as loag as :-j-jo yea.r§ 'tvase! lost tferoug!;; ascldeat> isterruptisg ttj-e 
_ £udy on -affaet of altsrrtasa psssege Stirougl^ cultures ssd saice^ I'fee 
rigieal culSuxe lins feSB been isatoeained^ howaveiTj aad- sevexsi lii^as 
asived frosa tte host eulfc^sr© liess a^a beiag carried in msuBe passage. 
^. l3c^ liaes froaa tiss0S"3 tliat .grew in mice iBoeulatsd as sewborss,, or 
afSer l^irradlaSios^ witfe let'j«virulense ct'ltasrs call©, ^ire.bsissg 
■-^-aintaiaad iii soese passage. 



Atteaspts to trssisfer 'Vitulesce" . £c the svlrulent ?.ise kave 
sea aasitceassfal^ fwo sp^roaehas ^^jete ueedg as follamBt (i) FiltrESes 
lid exude esifersats f?o3s s&ort^^'tes'ffi sulttirss of the viruleiifc, taos3sa:'»ps.-s?;S(; 
'529 iiae were added te cultisres of. the avirulaat iiaa. AfSer 24 hours 



987 



Serisl Mo. _._.MGI^5^ 

ladividisffil Projacfc leport 
€aleadsr Yssr 1959 



?ar£ 4 (coatisusd) 



these cultures were isajecSed IP iato cosEpstible ©ice. Ho t«Eiors appeared,, 
C2) A spoaSaaaotss his&loeytic aeopiasm very sisalXar to the ©rigiiisl P329 
aaaplssiEB %ia© eaeoaatered la sa old MXS/c s; BBA/2 H!o?zse. Sitscs F329 ted 
arises Ik a !Eii\/2 s^use £Ms s,i£iisl:loa provided us with & viruletstj, cj&©lo« 
gicslly ideatic^l tuiusr^ dlfferiag fresas P329 in its F, genetic eosseitetioa 
(sad sssociated ht&to-'COT^^tthilttj ehasactasrs) as wall as ia virwlesce^ To 
test: tb,s possibility tfeat s 'Vlruleaee fseSor" adgfet bs traasferrad direcSlj 
froHi sail to sell by coatact^ Eiised populst^ion oaltnT&s isare prepared fey 
addiag cells of tfes vir«l@si£ ?|_ £sjaser to saltures of the avlnalexit P329, 
After &■■ W3sk of csatacS ia thes£ lalsffisd calt.jjres, the lalssed populstioas 
^jsre iajecfc JF ii^So EM/2 and _/BAIB/c s 1BA/27F| hybrids » Tusaors grew in 
sad killed tlta F|_ hosts^ but no tumors appeared in ths BM/2 hosts, 
iadieaSlEg tliat e ^Virulence factor" eitSier had sot basn traasfersed off th&t.. 



also fes concluded tiist If &n "ayirttieace factor" fead beea transferred, it 
bad ao£ be©a fcrsasf erred to all of ths. virulent cells. 

B. F388, Cells froia this lyispljold seoplsssn have been earsigd 
in eulture for over three years » Three separate isolations &te being 
carried aad each of t&ese lines 8ffio^;?s a cimrseteristie cell sorpfeology^, 
eltIso«.gh tlie sliailaritiss withia the groap asfs greatsi' thaa the differeseas. 
All of these llsies shew sasked laorpho logic sad biologic alteratiotis th&t 
wars described is Isst je&r^s report. la addition^ studies by ti8s=>la.pss 
cinetnatograpliy ars bsieg amde. These show that site masmsr of locoiaotion of 
tlie altayed tissue cultar© cells is aot sisailar to that of lymphoid cslls^ 
butf like t&eir eytologic featmres ia fixed preparatioas, is more siailsr 
to EsovajBSKt of rs^QSoph^es ia culture. 

I>r« Eob«£rt Roosa of the leukemia Study Section has used F3o8B| 
ceils to study developsant of drug resistance ^ vitro. He bas subsiiefeed 
tuaKjrs., derived frosa cultures reslstaat to several aati" leukemic drugs^, 
for bistologic study* At preseat it appears fclsst tuaoES derived from eelia 
resistant to S^fltsorouridiae eati be distiaguished from tumors derived £tom 
other ?38SDi eislture sublines, t&is poiat needs further study to 
sstsbiisb- Essre objectively 'Ai&t the special features of the S^iluoTouTidina 
resistant cells are isi t&tmB of physical or Mstocheinicsl q«all£iaSc 

Co F81S. 4etiv3 studies of this culttsre cell line^ derived fross 
jssst eeii tajaor FSIS^ h&v& bssa discoatinaed, although & £m} ealtarss are 
still Hsaiataiasd c TM eulttsres sio longer coatain sny cells ideatif table as 
derivstiva® of mast esllS;, and cultures inoeulsted into asica fail to 
produce tiiisKjrB, fhe cells that continue in culture resesible fibroblasts^ 



S88 



Serial S?o. mi 521 

i;tsdivlGtmI Project Eaport 
Caieadar Yssr 1959 



Part k (costist5a*i) 



S l^alf JGime® ? fhe chaage ia taorphology and the eomplate or 
partial loss of virulaace of the above neoplasms during propagation is vitro 
sasms likaly £0 bs of £midS3mnt&l iiaportaiace^ perhaps comparable to £he 
loss of viraiejsce of faS&ogeaie bacteria and viruses t-?hea cultivated in 
vitro . Sfes pheaomeiaoja smy tasll be similar to tbat originally descsibed 
by Saaford for Mgfe aad Im^ emrasr-prodttcissg liaes developing frcisi aorsssl 
ffio'jse fibroblasts ta ^tro. It per&aps feas somawbat different aiasaisg 
tli^n tits pteaosssoa observed in Sanford^s systasj^ insassuch as is our 
e^erisssaes the Beoplsetic cells had a.lt&&4y possessed ttsairor^prodBalag 
qizalitlas before beis^ placed in culture. If tfee secsssnisia by wMch loss 
of vxTul&nce. c-ecusrs could ba dstensined, it is eosceivable (reajotely) 
timt thB cssage could be isdueed is vivo, thuB iucreasiBg survival of tsisior- 
bear lag snissls. Oa tfes othsr fesad, ia tlis aaalogous bacterial systesis,,, 
Biethodg have sasver bses fousd foi* deersasiai;, viruleaee ^ 2^£> thot3g,ls 
trassfsr of ^1% tos bsea shown to coafer i scresse d viruleace on previous Ij 
sviruleat strains ia vitro* 

Pronged goug 8g _; Tl^s aegativQ results in all sttes^ts £0 restore 
vixuleace to ?32§ cslls ^vb fraakly bsea discouraglagj aittowgb all the 
avesroes of approach liave aot beea closed. For ssaE^le; (1) transfer of 
purified DM. e2strssts from virulsKt calls to avirulent ones isas not baea 
atte?npted« (2) Treasfsr cjf aviruleat cells withia laillipore cM^erSj 
f oiloE'sd by release of the calls into the sase feost anisaal might provide 
sdvaatages for ©uscessful traasplesEtatioa. (3) Tracaduction of a 
theoretieel viruleaee f motor by tj^sas of a virus could be attempted^ 
particularly &b tte F32f liae hsts been showa to be susceptible to iafeetloia 
by poiycffis virus. Ho^^ever. ej^>sriH®ats along these liaes have been aad 
still ara beiag deferred ia order to pursue iateasively other projects 
coneeraing iatereellulsr relationships in tissues infected ^ith polyotaa 
virus ia vitro* 



f&Tt B iacludeds Ho 



189 



Serisl MOo MS_|.^521a^^^^^^^^ 

1. LsboraSory of Pathology 

2. Caticas: Istdue. & FafeliogeEesis 

3. Beth-ssca, Marylaad 

Bsdivxd-a&I Project Eepore 
Calaadar I'ear 1959 



Fart A 



Fro jest Titles Tisssse Culture Studies Invsseigating £te l^cbsaisa 
of Ttisor • ItsdasSion by Parotid Tuajor Agent (Polyoss 
Virus) 

Prlasipal Imrastigators Siyda J< DaH® 

Otfeer lavessigstors 2 L. W. Lssr 

Gaoperatitjg ifnits; 

Man Years (calendsr yasr 1959): 
fotai? 11/2 
FrofassioEtsls i 
0£lser; 1/2 

^jec^^^s This project is aa esSeneloa of Fsrojact 52is deseribec 
in t^ 1958 report. Ths lavestigatioa of parotid ttsasor ageafc ia leukeisic 
tissues of ssiee tes been publissssd, sad tbB obsesvaKioas oa ebaagas iadused 
by the sgent oa Bswbosts issuse ealivs^ glS3sds ia vitro are ia press. The 
objeetj.ves of this project a^a to del:eisaiae She iaf lueace of several host 
factors oa £b.e developsssnt of the fejo baeic typsg of respoase to tlie virus 
JB vitro; nsaaly, the eytolytie sad tlis pre lif erst i%»s rospoasee. 'She 
liGSt faetors pi^ssatly being iavestigated ares cferoEologie age, speeias 
differeacea, iasgmologic sS:ate with respect to polyonja virus isfectios^ 
stEge of cellular different iatioa at tisse of isfection, sad specific 
inf l^ieKce of issseae^sal eleseats on the developsieat of th& dlffereot types 
of response by epitheliam^ 

Matlaiodg ; fhe investigation employs both tfee gelatia sponge mMtTts. 
xmthod of tissue culture described is publication (2) below^ aad the 
ssiivsry gland xudissnt culture tecSmiques first described by Bargfeere aud 
later sKsdif iad by Grobsteim* Sjibaasillary gland tissue or subisssillary 
rudiEseats at various stages of developaent are cultivated on platss^ clot 
edjaceat to galstia sponge. Xa the case of adult subsuasillsry glaad^ it 
is necessary to repl&ce tfee aposges after 7«>10 days of cult«:re because 
duritsg elsst period adult glaads secrete & ^■sotB&s& that digests tbs orlgiaaX 
gelstia sponge. 



990 



Ssris.1 lloo J^lJ2lA^^ 
CS.UM&X Yeas- I9l§ 



Fart A (soseiaued) 



fo study iaflueaees of state of sellislsr dlffarsEtiseloa asid of 
Eorphogeastic evocators oa e&e £ype of resposise £o vinss^ sufesaasillary glasd 
radiEsaats are sulfeared with £ad v^ifclsout virus^ at vsrious eesges of develop-^ 
laetst: rsEgiag fs'OEa the earliest: sissgle-bud aal&ge to stages of sdeaoasere forasation 
and fuTiOtloas-l seioa^ dsvelopsEeut. To study the effects of virus on the 
epitlisiial ot B^seacbysisl rwdi^afc© alonS;, these cosspoaeats are first 
separated fro® oaa msotlier by Sfee trypississtioE teckaiq-ae of erobstsia» 
Coupisd with racoHsbitsatioB e^arls^ats, this approach smkss posaibla tha 
saalysis of lstarcell«las- relstioasfeips sssd their influeaea on th® 
rasalt of viral lafectloa. 

Major FlEdlBgs; Iffiereasiag chroao logic age beyosd the rsS5?boi*ti 
period does fsot diisii^ish the proliferative response of subamsillary glaod 
to parotid ttsEior sgeat in vitro. Precise quaatit<atioa ia this system is 
difficulty but sstitmtiosiS are that the proliferative response is asor@ 
pronouKcad is adult glsnds thaa in glands of aewborae, ia vitro, Csrtais 
qusliSstlve differeaees hsva been Eoted also^ 'Aich will aot be described 
here. Ho signlficimt differeacs h&s been soted bet&?sesk adult otsle and 
adult fansale res^ase. Glands froiH sales &t 4, 6^ sjsd 20 weeks ©Ed at 15 
jaoaths of age give sppsoxLasately slHsilar respoass. 

Is the opposite diractioE our observstloas are iacois^Iatei. but 
indicate thus far that there is little oir qo prolifes'Stive sespoase by the 
epithelial elea^ats in glaads ia the eesrliest stages of ssrphogeisesls. Cells 
of the eplthelisl as well as of the jneseaahysjsl portions of the jaidiesfcas 
are infected^ however^ as indicated by typical auelear lesions and seerosiSo 
The cytolytic pheaons^aoa appears to be about as narked ia the rudliaental 
sasseBGhyae ss in the adult mesanshyiae^ but the epithelial response is 
predoTBiaaatly, if not eatirely, cytolytic ia the ensbryonic rudia^Kts, i?hersse 
it is chief ly proliferative ia epithelium of adult glands in vitro . Eesults 
with sepasrated epithaliai and oeseEchyiaal eoHsposients of rudlaKKts are sot 
yet at hasd. 

A siagle eKpe^itBEst teafcisg the respoBse of subEsaKlllary glaad 
from a 15"Hioath»bld ssoisse, "Eaturally" infected by parotid tuajor egsKst aad 
haviag a liigh HI BBTvm titsr^ h&s been carried owt. Ths proliferative 

srespoase at 30 days vas just as a-arked as in adtilt gland from noB-isiauaised 
saice. Further, the uninfected coatrol cultures showed ao resposse to virus 
at ail, itsdicatissg that latest virus ?v'ss saot present or saot effective In the 
maual Eaaaer^ even though added virus was able to act. 

Studies of differeaees of species response have not bees eoapletedo 
It has been fouod^ hoK-ever^ that parotid ^ subsaasillary, thyroid, sad thysEus 
glaad tissue frosj tt-Jo hissssj fetuses did act respond ia either a cytolytic 
or proliferative mmmet\ Studies with rat aad haiBSter salivary glsads are 
tmder way. 

991 



Serial Mo. _lCI.521a, 
CaleHdar Yaas 1939 



Fart A <ecB£is.ued) 



It is i^Qvtsat to £he Iceerpretstloa of tisa above fissdiags £© 
sscertaiQ £lse biologic s^saiag pf £&e issr^aologis changes observed ia 

cultures i-isfacted with tlie vinss, Isitisl sttesspts to trmisplanS fcbe 
altered suIShtss to siies resulted to devslopseiit of virus^isdused tumos's 
is the 2-irrsdiated adalt secifiiests^ fe«t jsot ia tlie growth of tussors 
from ffee trsasplsafead cells ,. 

lenet'fsd aStetspts So gr&ft tfee cultures are uader wsy^ Slassds 
froa adult ssics Itave besa ressved surgically, iafectsd aad cult^s^ed for 
30 days, end £liea raSurasd to the originsi donors, as well ss to isoiogous 
hosts. Ia sosse asses they h&ve. been placed under £ke capsule of the 
rerosiEing subaSKillsry giaad. CuXtares Isave beea traosplaated to issaiualged 
&nd Koss-iissjRJislEed ed«lfcSj as wsll as to ae??lsora asice. Growth of Etjraera a£ 
the sites of txaaspiase tes not ys£ beea observed af£er periods r&eigiag 
upward froja sijs ^aeks, 

§i,SMi^SS£° Bscsuse of fche failure of altered cultures t© 
grow iEte fciisaors ^hea Srsasferred to mice, ths cfesages is vitro catmot 
at preseat be accepted ss fully equivalent ' to ejsEor genesis ira vivo; . Hows\»er,s 
it sMSt also he reeogsised tSmt Che traasplantsbility chsraeteristics of 
eoa^irgbl;^ simXl aad early tumors induced ^ vivo is sot known , It Is 
kasowH tfeat avea large salivary glsad trasore succeed in bseomiag est^blislsed 
in tse'a hosts ia njueh less than 100% of the attempts, Virus "induced thytaie 
tensors grow only rarely on traasplaatatioa. It is otir tentative assujaptioa 
that the proliferstlve and lEorpbologic cbaages ia cultures are iss fact 
equivalsat to tfes early efesBges ia vivo at a stage wbere coatinued growfi!^ 
is still highly depecdeat ob host laflaesces., 

If this assissjj^tioa saa be verified then it ia apparent that t&e 
f iadiags described sbove offer a ehalleKge to the coaeept that either 
early chrosologie sga (n^fisiisag ?) or relatively uadifferentiated state 
favor ttssBor isduetio:^ by this sgeat. On the contrary, it appears that the. 
aewbora mouse is peculiarly sissceptibla to the ttanar-'induciEsg effects of 
the ageat because it is precisely at this age that two aecessary sonditioas 
overlap ia tisse; ssisely, (I) susceptibility to iafectioa by virtue ©£ 
iiicoE^leteiy developed Itmrnts^ respoase, and (2> capability for proiifereti^'e 
respoEse by virtue of relstivaly^ though aot cos^letely advassced stats of 
Esorphogeassis snd/or differentiation. iHbiis coaelusioa is supported by our 
previow-s observations thst the apparent refractoriness of sdult eaiisal© 
could be brekea do-im by (1) iatroductioa of a massive dose of virus hj Tv 
route; and (2) by is^airisg the iaKcuae response hy irrsdiatioa prior to 
virus iaoculatioa. 



992 



Serial Eo. ^MLSSla^ 



Fsril: A (soiitiassd) 



"ghess is s gT®e£i«al vslus te fchase observatioES witls regard to 
sesreb. fsr viral S^ssjor 4ges.tg in hsm&ns. If aaslegoas sgeots exist la 
huiBsms^ is should sppear alms saltarss ©f adisle tisswe wa^ld provide ajora 
fsvsrsble test syse©ES tfea cuXetsres of tt©'?Jbora or fstal tissues. Also^ 
£l»£ state of isssjHsity of tbB daaor of £ba tissue £0 be Isfected ia sultisre 
could be disregarded;, as tkis ??ottld n<st afface t!^ response (asst^lag ubq. 
of Eoa-isasuaa sera frssj Isstsro logons sourses ia fefee etslt«rs asedi^Bs). 
These tuo co^sidesatisiss &h.&BM. faeilitats the isse of tfels syatam as s 
jsodel £01* detecstie-s of t«ac5l?«'iBduciBg vinises. 

T?fo feypothesas ®s £© tfe inttereallular sssd cell»visa@ ireisSio^hip 
ceasexBad is tiaaor iasducticsfss by this vinss ®re uBder eongidara£lea as 
follows : 1) Three^fsctor^ or double dependence thB&xy, The proliferative 
raspoasa of sslivssy glaad epit&elium £0 infectioa &y the sgsat: ia dependent 
ca tfes prior asSablistosst of a eereain le'^/el of diffesrestiation ia Ste 
itsfeated eell,» fbAs lavel of different istiois ia tuns is depeadaut (as 
"shoim by aiabryologic studies) upota spaeific iaterrelsSioaships bet^jeea tha 
spitlislisl sells sad etj® supportfesg laeseachysse. If ehls iaterrelatioxmMF 
h&s not bssB asblevsd (is essbryogeeasis) or Ms been iaaterrupted (separation 
of epitheliiaa fro® st3?®Ea ia prepsrasioa of tnoaolayer cultures), £hs pro® 
lifereSive reeponsa is sufplsatad fey ® cyt©lyt:ic oneo The epeeifieity of 
auseeptibility ssd of regposse ©f cexSai© ssrgsns asd cells to the &g&at rsBy 
thus be based oa a coss^ssitive relaSiossMp beSaeea cell sad virus for the 
coasplSK 53®£eri©ls (Mf ?) by wMc!j,£&e mesesQl^ia&l supportiBg cells aorssslly 
coatrol diffssrenSiation and funstioE. 

Aceordisig to this concept^ a third eleasat^ else sasssnehyssal 
si3ppor£isg sell; Batst be takea iato coESiderstion'ia tbs study of viras--=-sell 
reXatloaship© la tumor gsaesis. Tamos' developaasat would be dependea'S cot 
only on tfej preseaca sad actios of visus in tlia teinior cells^ hut. also oa 
tfee issiatenaacsj &t least: for a period dusriag £lse evolution of the tsjesor^ 
of the dependent rel^tioasltip of tbs iafec£ed cell with a properly 
fuBctioaitig asaseashyiEal supportiag cell, fals concept if double dapeadeace 
would give the BsasendjgFiaal sell the rola of maiat&iffiing the "fealaiiae" 
betT^ees virus sad host call \4iich xsaiay virologists ls&v& believed essasitial 
for davelo|S2sant of vims tmjoss, Wmn tiie supportive cell is reaaovsd froEs 
ths picture^ th& iafeetad call is no longer able to ooisspete sad the viras 
saalEiplies aad d&Bttojs ths cello 

"Ehis eoaeept is consisteBt wish the observatioa of ussuacessful 
fcraasplaatatioa of tissue cultures asd toawrs. ye have aotsd that the 
raaseachynml cells ia our tissue culttisgs ted usadargone very aearly eossipiet® 
aesrosis at the tist^ trsasplaatstioa was atten^ted* Seace, it woKld follot? 



993 



Serial Ms» _mtS2'm_ 
SaXeadar ¥sar 1959 



Fsrt A (casitiaiiad) 



that £&£ epitbslial cells, tteugh si;sread aloag a prollfera&ive pattoay 
would Qvastiisliy saccoisb feo visratl ef feefes because tha systesa had beea 
deprived of fc&s tliiicd iaf liisaee. Tlils ^oaM also ejEplaia the failure 
of groT^Sls of £fes showers of essholts. pjlEnmary E^tsstases £fea£ occur froia 
the sallvasj tusors. 

. Tke sbo\^a eeaespt co-ssld &QcmmZ for a favorable isafl^sesae of 
viral aatibodj on tismoT gta^th^ oaee Issitiated. As tb& saesesshyiosl cells 
h&ve been, BhcMsi, to h^ letfeslly respoasive £o viras isfestloE, tisair 
psrotactioa by sosse mesas wo^ld be secessary to steintsin thsa la tfeeir 
suppsrtivQ fuaetioia^ Hera agaiU; tlie time of developaseEt of satibody 
respoQse la £'qs ssouga Imppsas to be ideal for provisiou of this p3i:otes£:i©n 

2) lwo"fsc£or^ dose-rsspoxise tbs^apy. A reaoBstructiosi of th^ 
cisais sf events secording to £Ms eoasept z%ms as follows s The first step 
ia viral isfestioa involves tlss sesenshyKal cells isa contact with epitlislls3®c 
In tbe absesce of satibody,* virtss sailtiplies ia these cells ajsd evestuslly 
produces eyto lysis ^ ^■?ith loesl release of inaseasely increased^ coaceatrstioss 
of viifus. la tbs sdult gland ^ this high vtr«s conceatratloa infests tha 
differeatiated epitlaigliTSffi^, resultisg iu a proliferative rsastion, wfeers&s 
is poorly differeatisted embsyoaie eplt^aliajn it produces saly eytolysis,, 
because the asdiffareBtiated epitfeeli-aa is more "seasitive." If this 
concept t<?are eorrsct^ it should b© posaible to induce proliferative affec-ts 
ia sssbiryoaie epithslitsn by uslsg a lesser virus doss. "Siis is diffie«lt to 
sefeieve its a eossplete orgSBj siace virus boosts its concantratioa by 
rsplicatiag in the j^senefeyi^l cells, Vitna dose sowld be coatrolled 
caly is a pure epitfealial systesi. 

It will bs lusted that evea this coaeept ic^uires sense es^lsxatioa 

for the difference ia reaction of esJbryonie epitheliuss as coiapared ^jith 
adult epitfeslium, assiHaiug that the dose level to which, they are exposed 
its ovg&si eulttssras is the serss. this tiseory is sa&ithstie to the first in 
that its pressise is that less well«differenf:isted epithellsm la w^t& 
susceptible to either the cytolytic effect or the proliferative effect sf 
virus, tha type of respoass depeedi^ oaly on tte initial doss. th&. first 
theory holds that ei^ryoaic and adult eplthsliasa reSporad differeBtlj to 
tfe© smis dose of virus becsuss of iafluexsces of a third factor^ the. 
coatrolliag aaseaehj^ise . 

Proposed Courae; fha projects helow have beea saleated for 
iutvx& iswastigatlons chiefly on the basis that ttey will be of vsiise ia 

deteraislag tha validity ©f one or ti® other of the sbo\'e eoacspta of tisssr 
genesis. 



89/4 



Serial 1©. WZl S2Ia 
Year 1959 



1. The sSudles of dxffereaces of species responee ^ vitro 
^7ill be CGStisued a£ Isase uatll tfee fissestas aad rat fesve bees insltided. 
Estsasion to qtjisr saisials will depsnd oes results wieh Sliese feii?o« We are 
espscis-llj? interested is sbtaiElsg iaforstatloa as So wheSlier g proliferasiva 
response ±n a given species correlates rsith the ability of tfee salivary 
glsad vmsex^chys^ of £hat species £o proaote lEorphogeassis asd differaaSiaeion 
of salivary glaad epitbaiiusn of the asouse. 

2. lavestigseion will be costiaued on the effect is jLl^SH «5^ 
tixe virus c© £lia sepsrsted Sisbisisillsry rudiaaats o£ the aiouse. 2£ she 
first eoacspt is correct, tisa virss sfeould prod^see eiehesr necrosis or ao 

effect at sll on eit^r isolated capsular ^seael^^s^ or isolated epiSl^Iisl 
rudiEsat, If ps'oXifsratioa occars in isolatied epiclialial rudiasBt, the 
f lrs£ coacept ^?ill h&vB lss£ much of its support. 

3« It is hoped to ba able to study tkB effect of viral arsSibody 
on tlis prollfsrative pheaojsenon 1^ yl£go. fbis caa be doae by repestiiog 
the espssissEt es origiaslly carried out, but addiag aatibody to the saedliEss 
at various tljEg isi-ervsls after iafectioa. 'This espsriE^nt will require 
S&W3V&1 of snti-'ijsouse aatibody from tbe antibody preparation. 

4. Cotjtisued attes^ts t?ill bs tmde to fisd a way to scMevs 
trsasplsstatioa of tko. converted cultures to Esice« Possibly the sdditios 
of aatibody to cultures after the iuitiatloB of tbe nsospfeo logic cfeaags but 
prior to traeisplasstatioa woal4 be helpful. Aaotfefir possible tsetic vjould 
be to gros? tSie cultures la ssilllpore chssSi&'ss in the nsouse before retKsviBg 
the^ ^3xd- plaeing is eoatact with host tissue. A successful result could 
bs interpreted to iadieata sa itif iueace of isclogoue serum eEvlrossaent 
durlag the soaversios process. 

5. As a double cheek ca the presently-held vtssn that a relatively 
advanced state of dif fsreistiation of the epitaelissa actuslly favors the 
proliferative type of respoase, cultures not eji^osed to virus will be 
esrried for 30 days or ajo^e^ after which tla^ virus will be added end fchs 
results observed. Although the epitheli^a survives well for 30 dsys^ by this 
tii^ it has lost aasch of its differentiated ch&rscter^ and theoretically 
should give a lees prosounsed proliferative response th&n fully differetitisted 
e.dul£ glands. 



:t B iaclisdedj Ye® 

395 



Serial So«.^IJ2Ia_ 

ladiviclvml Prcjecfc leport 
Saiendsr Yesr 1959 

Bart^B.. Honors, iteards, asd Publics£ioas * 

Pisblicatioas other £bsa abstracts frojs this projeee? 

1. Bawe, G. J.f l&^, L. W.^ sad Dmsn, T. 8. 

S'cudies of parotid-tiiraor ®geat to cultures of leoksasic tississs 

of alee. 

-J. mt. Casces last. 23; 711-191 , 1959. 

2. Bawe^ G. J., and law^ L. W. 

Morphologic cimjsges ia salivary-gland tissue of ehe newbora 
mouse esqjosed to pasotid»t«ax>r ageat in vitro . 
J. Siac. Caacer Inst. 23: 1157-1177, 1959. 

3. Banfield, M. e., Bawe, C. J., sad Briadlay, Barleae G. 
latracellsalar stjd a^tracellulsr particles iu tissue cult«rs 
inoculated with psrotid^fcimor agesst (polyoma virus). 

J. mt. CmMiox last. 23; 1123-1135^ 1959. 

4. La'^^ L. W.^ Dawe^, G. J., Rot^e, W. P., sad Hartley, J. W. 
Antibody statics of taice and response of tfesir litters to 
psrQtid»t!sa5r viriss (po lyona visnsa). 

Mature, 184: 1420, 1959. 



S96 



^,^., ..=.... „.y. _jiei_52_2_ ^ 

lo I^borstosy of FaSholc-gj 
2o Cssees Indue « & Pstliefgenasis 
3, Be 
PSS-HH 
Isdlvidxsal Project leport 
eeleadsr Year 1959 



Project Tltlas Studies on the Indueeion sad Jlaturai History of 
Cotijtsaetival Csreiaoa®. in Hereford C®t£l© 

Friseipsl Iis^/estlgstoff ; Msarl F. Staatoa 

QthBT X?sves£igffitosss Robert. Solgel, Sofeest Mssssker, Say Bryas, 
W. I. Gay, 

Cooperatisg, Units s I^aborffitory Aids Branch, Divisioa of Research 
Services^ Laboratory of Biology j, KCI 

W-n Years (Galeadar year 1959)2 
Totals 1/2 

Prof essisnsJ ^ 3./4 
Otlisrs 1/4 

Froject BescriptioB J 

Objeetlvg^s; To deteradee the fate of certsln proliferative 
e^ife&elial lasioas oa th& eyes of cattle aad to aSt^spt by srtifisial 33e®as 
to iadaee esrciasjss in es^ttle with the products of the tuiaors which aecar 



Methods g Viable tisEor tissue aad cell free estrscts of coajuefeival 
eareiTS0iass &£ hereford e^jetle^ prepared by coaceatratioQ teslmiques 
suceesafuXly established for aous sarcosaa virus, Imve beeo iuoculated iato 
tfes coBjuEstiva® fiad udder® of liereford cattle. Both youeg ucaffeeted 
SHiisals aad aslnals ^ith eonjuactival carcino^i have h&en used as reei|siea£s 
of the iacculs. Dr. 1. Eeigel does electroa microscopy on biopsied tua^re^ 

^jor FiadJEga; A herd of 500 cattle ia Eastern !ferylaad h&s 
besQ studied. Ten to 2^ of the herd has eye tisaors. Systecatic folio's? up 
of progressing lesioas with photogrjspha Is in progress. Biopsied ststsrtal 
has been used for electron microscopy sud tissue ctsltase study ss «?ell as 
inoculation o The cosjuBCtiva of 4 cattle have beasj iaoculated with tiijsor 
estrscts or culture aisterisi. Thus far we his^e. uncovered ao evidence othas' 
th.©a epidemiological evideaee that this ttsasor is etiologically related to 
aicro»osgaaisjias, Bsvjever, further work is ia progress through Eiorp&o log leal 
studies by Br^ Seigel and tissiie culture studies by Br., V^rmk&r, 

Significaace; Ihis is an m^uestiosiably iMllgaaat epithelial 
tuEKsr X'jhich has its counterpart in «5®a„ and follox^s the natural history of 
htaasa spiderBioid carcisiOHss. Ihe high incid'sace of such a iesioB iti a herd 
of cattle suggests a cossraon etiological factor net as yet apparent o k most 

397 



isrial i5o„ Wi'&..S?^^,.^.... 

Cslsadar Yasr 1959 
Fart i%. (cosfciaiisd) 

likely possibility is tM.t £be isitisi versTKsous lesioa Is isaducsd by 
iBseet-borKS viruses c Sisbsesueat prog^"essioa to maligsiaasy isay bs a 
part of or separate fsos tha initlatisg process. The cow serves as ss 
eKSsllaat aslTsal isa wMeli to study this probissa^ of added sigsif isaase 
is ths p©S£-ibili£y of UECovsriag snssas of preveaEing ths disss©© io eatile, 
a BBTious ecososaie hasard la praseni: day aniimi husbandry. 

■ Frop-osad QonxBst It seeiss likely tMt the presarsor to the 
aaligaaat lesiGH is tb& hyperksratotic verrticous wari: aoted in smny of 
She VirgiEis sasels. We sfell afetea^S to collect, rescwer, asad induce 
typisEl lesioas r^tth mieh. sstsrial as well as with conceatrated produets 
of tfes Ejaltgsant lesioa« 



898 



Serial lo. HGI 523 



io laboratory of Fashology 

2. Career Xadtjc, & Fstbogsnssis 

Individual Project Report 
Galeadar Yesr 1959 



■g^:t_A' 



Project Title; Effects of Xoaising Esdistioa with Emphasis oa 
Morpljologic Alfceratioss aad Heoplasius 

Frisieipal Saves tigstor; Eielmrd h. Swarm 

Qth&T Ia-5,?es£isato3rs: 

CooparatiEg Uaits; 

^E Years (caleadav- year 1959 )s 
Tesal; 1 1/3 
P^ef ssB ioaal t 1/3 
Others 1 

Frojeefe Beseriptioas 

SkJ^^iSSf.- ^® GollesKlos of isfom^tion regsrdiag the efcsages 
effected by ioaisissg' radistie-a of different typss ia nsn asd experiiE^rital 
ssiccals. 

I^tliods ; I) Detection asid study of seoplssnss ssd ofehsr EsoE'phQlogic 
cfesnges ie e3^ss:is3®atal aaiissals who have received ex£erEal radiaeion or 
intertsslly admiKistered riidiosctive isotopes. 

2} A Study of the distributiou^ astabolism sad effects of radio«> 
active isotopes of sevssaX types is oan ar«d animls. Here the emphasis is 
on the titillsetioa of gffosss s,nd histologic autoradiographic tecbslques ia 
the gtady of thoriiEH dioside^ S^^ aad 1^31 labeled naterials, 

3) Partieipatioa io the activities of the Bathologic AEatosay 
Departs^at. Special eBSpfeasis is lasde in the study of hmm.n spacls^as 
dsmoEStrstiag chsage due to irradiation or cosstsiaiag radioactive isotopes =, 

4) Xo facilitate the above sad to assist other itsvestigstorSj 
collaboratiQn on probleias ia this field is fostered. Studies oa the 
distrib-atloa of S"^^ in hasan tissues have beeo is collaboration v?ish clisleal 
studies of Dr» J. lobert .Andrews asd others in the Radiation Braiseh^ HCI. 

^4g£^^^^^S' From the coatlauisag stisdy of the reported effesfce 
of colloidal thorium dioxide administered iatrsveeously in snaa, the picture 
of focal sesrsing of the liver emerges as a lesioa x^hlch at tis^s is 
described ia association isith ncoplasjus, but is E»re frequently tas sale 
patholsgic lesion in ama. Sfest of tfee anin^ls asd certain of the his^a 

999 



Serial Hoc _1CI 
FES -MIS 
232dividual Prsjeet Eeport 



Tart A (ceatirj.53ad) ^ 

psfeieBts fe'ith sssllgsiist vascular tisssors £oll©5?lBg iatrsveaous colloldsl 
diosids fea-vQ litsls or so foeal scarriisg ia the liver or spleea sltfeough 
searriEg is asea ia segiossi aodes sad s^bca'ceneous tississ coatalaiag 
thoK-iiisi dioxide. Is e^srissatal asstisaals scarring of thB sabcsstanaous 
tissues al?eut estrat'ssatsd Sbsriaia depesits deaa not appear to be ss 
esSsaslirs s® tas ssarrlsg ia smt^ Ob th& other ^^aRd, tmaors &t the site 
of sube'ataKSous esferavassSioa of tSioTo£ras£ are more cosssos ia redestis 
than feassa ps-tissts. Bs viex? of elsese fiadiags furSter seudy of thB 
corsrelatioa s£ fibrosis aisd ssoplsaia in She liver sad sple©a ©f hunaa pstieata 
Tsith. ttmxofs&ut dsposies is p Issued. 

Wosk witli tfes traseplaatsbls vascular tussors of the liver &nd 
splean Ib mice Ms coatiaued. Serial traasplaatseioa has resulted is soiise 
iaereses ia tfee growth rs£s aad ssorphoiogis cljanges-p FraliEsiaary storphologie 
studies iaslieats tli^e ths slower growth raee is sssocisSed wieh i*eli 
differesSisted Sus^rs fe"hich Itave ssaall vasculsr strucSissss, Faster growiog 
ttimors liave cells prsseafi in laore of aa ©ggragafee with less well defined 
vasculsr sssuctisres, A eareful stody oS' ebe lassato logic system ie ia progrsss 
in these aaiaals with vssculsr ttsaaors. 

Study of rsbblts xstth ia£ravenou8iy isajecSed colloidal thoritsm 
dioxide has c«mtiniied. Iksriug £he past year ?:wo asore aniiiaais feave died with 
Xivar sad apleeu has^^sgioendoehQliosas, These saiE^ls stewed no fibrosis. 
The avo^ser of rabbits on e^eriiaeae bsve baes sj^li but the following data 
shows K daficl£a £raK4« In 12 snizrals is.jeeted ??itli thoTottast (lot 220) 
3 ®aiss.Is reeeived 2 isjacfcioas of 3 ec. and 9 eaiszais received a single 
Injection of 3 ce. Of the 3 aniasals wbieh received a total of 6 ec, oae 
died wisboxst txjiBor after 19 jnoBtl^. The otiier t%7o both developed tisaor &t 
36-37 ssoatfee. Hoes of the aaisssls given a single injectioa of 3 ce. developed 
£utK>r; however, ealy 4 of tfes 9 ia tbis group lived loijger tfeajs the 38 
ffiOEths. (Six in the group died without tuinor withia 35 iiK)a£hs after 
inject ioss e ) Of the 4 aais^ls wMch lived 38 saontha or loiager only ojse 
which is yat ©live h&s lived 43 saost^. 

Tliorotr©s£ gives intravenously apparstafcly ftenda to izscraaee the 
Qissiber of deaths frosi infection^ etc. While this hse not been &n object of 

direct studyy it has pisgised otir esperirasats oa c&reinogsnesis, pisrtieuiarlj 
those ia rats. An e^qserisKat started in OM/H rats with Dr. Bab® to study 
tlie effects of partial hepstsctoiay ®nd spleneetotsy on the incidence of 
neoplasia has bae*a virtually radueed to a pilot ea^serissnt by auaserous 
deaths pris^rily froja puteooary infection. Siiailar but less eastessive 
difficulties wer© easier ieaead t'a'ith e:^}sri23en£s with mice and rabbits = 

One patient with thorotrsst (02-14-58) was studied in tte Slisieal 
Canter during the past year. 



lOOQ 



Sas-ial Noo IICI S23 



Issdividaal Project aepost 
Gslesdar Year 19S9 

Part A (eos£iEued) * 

Si^ificEBcas With increased exposure of iBan So radiation^ it 
isas bscoss tssstb sad mors issportSHS to establish the psthogesesis &nd 
clmraeter of irradiation injury aad irradietioa induced aeoplasia so that 
injury iesdiag to ceoplssia ess be preveaSed if possible atid so tbB.t sadiaafe 
ecargy esn bs used la the jaost satisfactory a®CEar to detroy aeoplasiaSo 

Proposed Course; I) The activities outlined imdaz Jfetbods are 

to be eoatlRued- 

2) In an effort to reduce the latest period for aeoplasia to 
develop^ efe® edmissistratlsa of thorotrast to aeiffeora rabbits aad tke use 
of litter ajste coatrois reeeivisg tfeorotrast at a later age is piaasad= 

3) Close stten£io33 iJill be paid to the reports of others studying 
htsBsa patients with iatraveaously adTsiei©tered thorotrast. 



Fart 1 iseiudads Mo 

10: 



Serial lb. ^^ ^'- 

1. Laboratory of Pathology 

2. Caneer Iad'uc.& Pathogenes 
3- Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

Calendar Year 1959 



Part A. 



Project Title; Pathologic Findings in Hvman trophoblastic Disease. 

Principal Investigator: Edward D. Price, Jr. 

Other Investigators: 

Cooperating Units: 

Man Years (calendar year 1959)! 
Tbtal: 1/2 
Professional: l/2 
Other: 

Project Description: 

Objectives ; Despite the voluminous literature on the morphologic 
aspects of choriocarcinoma and related conditions, reports of well-studied 
series of large numbers of cases are rare. It is felt that the availability 
of a relatively large number of such cases, particularly an ample series of 
necropsies of choriocarcinoma, at the Clinical Center provides suitable 
material for a careful morphologic study of this group of conditions. 

Methods : Routine 

Major Findings ; She project is still in the planning stage and 
there are no significant findings as yet. 

Significange ; She study may help to clarify aome of the confusion 
which still exists about the morphologic asi>ects of this group of conditio»ji. 

Proposed Course ; Study of the clinical records, autopsy report S; 
and pathologic material available. 



Part D included: No. 



10 L' 



Serial Mo. mi 526 

Ic laboratory of ethology 

Isdividuai Project lepoyt 
Calendar Year 1959 



eare^ 



F^rojset Titles e&saisal Carci&ogefiesls 

Friacipai Swestigstor s Msjfle L. Basse Ibaels 

Otkmz Suvastigafiors Soger W, 0*Gars 

Coopsrstitig BtaitS3 WlkW 

^n fears CcalsBdjsr year 1959 >s 
Totals ■ 1/2 
frafessloiaal; 1/4 
0£lisrs 1/4 



frojees SeseriptiGss 



I) To st^Sy t!ie asorpfeology of llgh£ gresa ead 



fs,s£ greea ia^ased &«^>r@c 2) to stxidj the cl^ages isa ssi£oclioadrla asd 
ia the laSs-sselltilaar glycolytic &&d respirstosy sa^^js^ systrems iadueed 
by these dyes^ 

witfe fss£ s^sn asid ligliS grass dyas (3% a^d 6% gquso^ss solutions). 

^j[OT_Fte4i^©s I) Sabcufesaeous fibresareossas *?ere induced in 
ovsr 90% ©f mjiis^Is by both dy@s v!iea iajeeSed as a 3% or 61 solution ia 
distilled ^&t&z. Sie first tmjor arose 7 Hssnlibs after tl^ sesrt of 
iajectioas ia a fa©£ greest trsated snis®!, ffee tujaors spresd readily by 
a!at:gstsses^ i^lsatetiea^ sad direct iavseioa snd grew eoasistesfcly ob 
traBSplfmestioa. 2) "^se adresa.! gleaads @sd goaeds showed osarked feype&smis 
and byper£replsy ia eniimls beartag traasplmies of one subliae of tussors 
but sM>t iM siaisials bssriiig isfsl^its of ths other subline, these lesiosis 
®re eimllsr to tfeos® ttet fesva been reposted in aai^ls vith hypsr'^'olesis., 

Sigaifiesace; Should sui^ply f«sjdai^a£al ksm^ledge oa tbe 
relatioaship batweea mitocbondria end neoplasia. 

Fropased go;ir@eg Mo further Hos'k is plaimed at the preeeat feisss. 



Part B itaeludeds Iss 



IOl 



ladividual frojeet Sepore 
Calessdsr Year 1959 

_^££_B. H0EOSS5 k^axdBg ssd FubXicsEions 

Publiegticms otlssr thaa abserseta from this projects 

Hesselbacli, M. t., «ssd O^Gara, E. W. ' 

Fast: Sre«a« sad Llg&t Or^ea-iadueed Tisj^rss laduction, Morgfeology^, 

asid Effect oa Bast. 

4» ^£0 Caseer 2a@fe« Is prsss. 



10 u,. 



1„ Lafcors-toey of F&thology ^ 
2 c Sascsr Indue = ■& Pstbogeaesii 
3. Betfeesda^ Msrylaad 

ladi-^idual Projset: Report 
G£j.sadar Year 1959 

Fagt A. 

Frojeet Title;' C&ejsical Csrcinogesesls 

Frlasipal Ixs^^astlgator ; Eoger W. O^Gara 

Other IsvestigatGrSi 

Cooperating Salts s 

Msa Years (saleadar year 1959)? 
Total? 1/2 
Profess iosal: 1/4 
Otbarj 1/4 

^ojec£ Inscription; 

Objectivee; To stsdy several popular cathartic compounds for 
possible csrciaogenic properties. 

MaSbodsg Usiag tiiree differeat: ssraias of aice^ six different 
cat' Issrtics tjsss adsdaiaterad either ia the diet or by stomach tube for 
15=20 m:>t 



-^jgy,_.^^^j.^j^g.° S^ BAlS/c nale nsiae livisg to oae year or older? 
S of 25 control mice ted lang tunrars, one bad a sqtiamous cell carciKoina 
of the subtsaKillsry gland, one had an interstitial cell tusaor of the testis 
erad one showed reticular call hyperplasia of abdoaiinal lymph nodes. Mice 
recaivixjg cascara, rheut-arb, castor oil, or pbaaophtha.leia respectively 
in their food (poiMered Derwood pallets) had essentially the saioe ineideaee 
of tumors as the eoatrol aniisals^, although there ware adnor variations <. 
Ihree of the 12 anissals re-^eiving podopl^lltm in their diet developed ^ 
hepsfcoBSS^ eighS shewed either lyaiphoid tumors or marked reticular sell 
hyperplasia of iiver^ spleen sad lyiisph aodes, tt-7o had benign polyps of the 
cecam or coloa^ and two had Itmg tiasors» Oaly 7 out of 14 auiaaiis receit/iag 
caloajsl survived beyosg one year of age. Three of these had lung tuiBorSj, 
one had carciBoaffi of the bladder sad imrked reticular cell hyperplasle of 
liver J spleen, and Ijnaph nodes, and one had a hetnangioiDa of the liver » 

C57BL BSls mice fed cathartics by stousch tube for 15 months i 
26 control mice showed 2 hepatonas, 2 lymphoid neoplasias and no gastrointesfcix 
nsoplassas. Hiae mice fed calocssl plus nsagaesiiaa sulfate developed 1 feepsSosm^ 
1 lyn^hoid neoplasm, and 3 gastrointestinal neoplasms. Four sdditioasl 
gastrointestinal neoplassss were found in inice receiving the other cathartics 
==!!Sking a total of 7 gastrointestinal neoplasias in 74 aniimls (4 were sasali 
papilloEjas of the forestcsEach, 1 was a s^ushioijs cell carcinonsa, and 2 iv-ere 
adenoHiSs of jejunum and cecusa respectively. 

100: 



Ssjtlal ISO, ^X 526£ 

jHdividtESl Freject Report 
Saleiidsr ta&s 1959 



sices Msst o£ &^se died ©f ssi^fleidosxa at a relasivel^? 
early age C^-iS isoat;!^) sad sot saseh ^^sefui iafosa^eion could be ofetalnec, 

glgaifiej^eqg f^ reealSs ofessis^d aspecially with MIS laics 
si^gest £fest; po<3spkyXl»a3 aad perhaps also ealos^l tsssy lisve isa^ carcisjogeaic 
pr&psreieSe Tke liver S,s4 £fee reticuioessdctfeelial system saera to ba the 

tisBi^s 2sog£ affected « 

grogggsd Cagysa^; Ho fiirtljar work is plSEsaed ia the isasiediate 
futtxse but t^ wsrk wita eal©«5el aad podsp^yllusa s&suld be repeated t^lag 
larger grotips of aaisisIS;? asd parbsps differsat stsraiiss sad species „ U 
ef G3H ffiiss laight bring out say latest tsadeBcy to'^axd p-eoduction of 
hepatcK&Sj, aad use of less ki^lily inbred (general purpose.) strsise might 
sccesfcuate ssy carcis«^eaic effect ob otlier ©rgans. 



«sc 



Part B iaeludeds No 



ioi 



*G- 



1« LabossScry ef SfeSliQiogy 

2c Cancer Indac. & Bsskogeaasis 

Isdividiaai Frajact Rapost 



Project Titles A Sttsdy ©f tdas fhysiop&tlaole^ic C&aages in tae fisso?:- 

Frisci^l iBvestigafcors lldsard A. J^la^tea 

OSltes Investigators 5 

Goopsxseisg ISiits? 

1-^ia Years (calaadasr year 1959): 
totals 2 1/3 
Frof sssloaal s 1 
Gt^sars 11/3 

Fifojsct Besci'iptieas 

Objec^/esi I. So Btudj further the properties of the liver 
EiiSoSie stisirulaQt is tissorSo 2. To apply the fluorescent antibody tselmiq-ae 
to the study of £ha tumor viras^ to autoiasautje pbsaojneaosi^ and the detection 
of M&,. 3o To evElaate host snd sell fs,ctors ia the metastasis of various 
mouse SiaTsors. 4o To study the possible interrelatioaship of earciaogea 
aad virus iaduced aeoplasiss. 

^fc|jMs° i» 'El® swpensstaat frsetion of various tissues heated 
St 80°C for 20 saiautas were iajected subcutaneous ly into C31 raice^ ThsBe 
stJiijmla were thea ehalletsged with eeveifal carcinogens and the tismor iueidsace 
and latent period moted. In addition sob© mice bearing S3®4 issaasary ttmoxs 
were treated with the heated supertjstaat from C3EEA. laanssary ttsmor. 

2, Antisera prepared ia rabbits agaiast P388 tissue culture eel Is 

Infected with the poiyosa virus was tested by i^ans of the frao:eeseenS aiitlfeody 
technique on P388 tissue culture cells infested -with the polyoES virus for 
antiviral aatJ,body ia &n attetj^t to detaonstrate the intraaellulsr loeatioa 
of the virus SBtigen. Saetioas of salivatry gland tumors were also estamined 
after asposuxe to the assssB fluoreseeia labeled atjtisera. 

Chickeai aud turkey aati-Roase virus sera was labeled with f luorasceia 
isothiocyasate and tested oa seetioas of Rous sarcons in an effort to 
damooatrate the intracellular location o£ the viral antigen. 

Sertaa from patiSBts wife Lupus Eytheti^toais and other diseases 
were used itj the iadirect fluoreseeat antibody techalque oa seetioas^ btbs&ts 
and tissue culture cells of various noraml and seoplastic tissues of aalmal 
aad hujaaa origiSo Blocking tests ware also conducted using serum frots 
tuBos-bearing aniEsals and hust^as, 

1007 



Serial Ifo. _^1.52' 

Isdlvidual froject Report 
Calea4ar Year 1959 
Fa.rt A {contims&d) 

3, A assEbsr of spostsaaous and Srsssplanted casuse ttsEosrs h&ve 
been trsusp lasted iseo various sites. Xlia tse£s.s£a£ic pattexn aad cell 
characteristics ere ths-n beisg observed., fbe tujsors baiag studied include 
spGsteaecras saaasiary tisacrs^ MJ4 induced ssrcoiaas, trsasplaafcabls Essusssary 
tuisors^ eax'ccisas^ ostsogaalc sas-coass aad S9X ffielanoaao (Ib collaboration 
laith Bs. Elisalsetli Shu, Patholo'gie Anatoi^^ M5I). 

4. Miss injected wish ths polyoss^ virus and treated wifili 
ETtethylehoIffiathrens stsd tirethaa are baiag observed for ttiisor developisenfi:, 
(In collsbora£ion xsith. DTo Clyde B&sa and Dr, Lloyd l£.w„) 

sa.jog Fiadiagss 1) (&) Injection of th& liver mitotic stii^latiisg 
fsctor ia fc'hs nwcleeproteia rich heated superaafcaat of C3HSA. mmss^ry ^SMaox 
into mice bearing the Eaaisaary tissior bas resulted ta increesed growfeh of tbe 
Sraasplsat: over th&t observsd ia cotJtrol C3HB& tsasmaxy tusnor^beariisg mice^ 
(b) Injeetion of eha livsr mitotic stimilatisjg factor in the heated 
supssrastSGC from Eorisal souse liver had ao effect oa tb& incidence or latent 
period of s^thyichoisnthrese iaduced tuasars. (c) Injactiosa of th& liver 
mitotic stiisulatiag factor ia the heated supersataat from a trsasplaated 
ssasiEary tisiKsr had no effset oa the iKcidence or latent period of 
jaethylcholaathrese iaduced ttsaors, (d) Xajectioa of the liver mitotic 
stisailsting factor ia the heated superastsnt from & tr«®splsnted EJethyl'» 
cholsathme induised sareoss ssjarkedly deersased the iaeideace sad ineressad 
the latent period of WA It^ixcmd sarcoisas, (e) lajectioa of the liver 
mitotic stias^latiKg- factor in the heated supensatsat from nonml liver resulted 
ia a decrease in the ntsmber of virethaa treated mice hsvisjg luag tunsjrs sad 
a decrease in the Euiaber of t^jiaors per ssoussc (f) A similar decrease is 
the aisBsbar of mice ■^ith uretten iadisced tiassors sad a decrease ia the niaj^jer 
of tuESjrs was fo^ad in xalce treated with the liver mitotic stlmulatiag faetor 
iB the heated supemataat of a traasplsnted tx^asEsry txsanor. 

2) (a) Is a collaborative study with Br. Alan Rabsoa aad Dr, 
Giasearlo Rabotti of the laboratory of Jfethology, -SCI, the iatramjelesr 
location of the polyosa virtts in P3S8 tissue culture cells was demonstrated 
by the fluorescent antibody techalque. The viral antigen could ssot be saaa 
in sections of polyosm indaced salivary glsad tujuors^ however, (b) Irs 
collaboration with Dr. ifery Fink of the Laboratory of Biology^ HCI, the 
Intraauclear and iBtrscytoplasmic location of Sous sarcosa virus astlgen 
was dsEoastrated ia seetioEs of Rous sarcoss tusaora using the fluoresseafc 
antibody technique, (c) Bs collaboration with Dr. Ho«?ard Goodisaa of the 
National ^.art Instit^te^ a techniqtse was worksd out for th& dessoEStr&tioa 
of aatinuslear aatibodies ia the serum of patient© with Systemic I^pus Erythesatos 
and other related diseases, 'fhe aatinuclssr laaterial will react t-iith the aaclel 
of ell cells tested includiag norssal and neoplastic and fro© htssan^ rabbits^ 
guinea pigs, mice and rats. Ho aatinuclear raaterial was dssKjnstrable ira 
the serma of caaeer patients, A bloekitsg effect on the asstiauelear ssterial 
in L4ipa3 patients sera by sara from sancer patients was siot dssjonstrable. 



10' 



'I'adlvidvjs.1 Frojeet Report 
Calsadsr Year 1959 



Part. A (saatiiiuad) 



(s-1) fe aoi labors tiim witfe Br. Howard Goods^a of Slie MstiOEal 
Eesr£ lastltueej, ^c Jobs. Fahay of fcha Gsseral ^diciae Srsucli, HCI, sad 
Br» George Brecl-^r of th& Clinical Pathology Bsmrfis^nt of ttke Cliaisal 
Ceater. whan serosa of LtiiJus paCieats was fractioaated aad the fractions tested 
by tlie fiuoresasTiE; antibody ■ CeelmiquQ £fee setivity was fotmd fco reside is 
both £li3 7S £sd I8S glofeulia fractioss,. 

gigsifie^^es 1} The sfcisdies of £lse liver mitotic stinsalating 
fsstor in etsaors indtc&te tlja£ £bis factor tesds to pvovsote the growth o£ 
an eseablisbed tutsor i?feile it has an inhibitory effect oa tbs «arc5.aogenie 
effect of isethylcbolaathreae sud urefcfeaa. Tke rela£ie?asbip of thsse obssrva-- 
tions to t5imor §rm?tli acd csrcitjogeaesis awaits fisrtljer study. 2) f!ie 
iEtracelluIar locfitioa of tha txmm: viral antigsES is of basic isiportaBce 
to the s&udy asd understanding of virtia neoplass^« 3) the study of serosa 
factors T^lth aatiauciear activity issy wall contribute to an uaderstatsdirsg 
of the etiology and trestsseat of the eoilagSB diseases o It is also of 
iaterest ia te^as of the eosipositioG of tka career ceil nucleus ttet it is 
not disfci»g«lsljBble frota ths normal by these seryis fsetors. Of eqissal icterest 
is the abseiace of a dassoastrable difference in tfee sema nucleoprotsias of 
cascar aad aonml iadividuala by this system, 

Froypsed Course t 1) k stialy of tfes growth factors associated 
with the tiassor legloa will be continued with emphasis oa isn^^roved tecfeaiques 
for detection aad cterscterisstion, 2) Sba siae of the fluorescejat antibody 
teehaiqae for the study of virus ttaaors will be eastersded. 3) Farther 
studies of the host cell relationsMp iis tlse aietsstatic procsss will he. 
uader taken. 



Part B included s Yes 



1009 



ladividtsal Project Report 
C^lesaisr Tesx 19S9 

^y£_B. S>aors^ Awards,^ aad Fubllcseiosss 

Pu'&licatioBS other tlaau abstracts frosa tkis i^rojeets 

1. GoodEan^ E« C.^ Fshay, J, L,> Jfelasgrea^ R. A., and Beechar^ G. 
Separafcioa of Factors In Iwpas Ei^tfeeEiatosis Serum Breasticg 
with Qsmgon&^ts of Cell Muclaic 

LSRcet 2 s. 7099% SSa-SSS, 1959, 

2. IfelE^rejjj R. A,o Fiak^ M* A«^ sad Mills^ W. 

Besasastratioa of the iBtrscellulas Localisation of Rous Sarsoasa 
?iras Aastigea by Fluoresceia Labeled Antissra. 

J, Sato Cancer Insto la press. 

So HalEgEeH^ E. k.g BaboSfc^ G.^ and Sabsos.,, A. S. 

Intrscelluisr Locallas£ioa of Polyonaa Vims Aafeigea Beasoastrsfcee 
with Fluoireseeia labeled AaSisera. 
J, Kiat„ Canesy Inst. In oresa. 



101 



Serial gOo liQ1333 

1. laboratory ©f 'F&thalo-^ 

2o Caacer lEsduc» & Bsfeliogenssis 

3. Bethesda^ msylsxtd 

iBdividissl Project Heport 

Caleadsr Year 1959 



gs.rfc__A. 



Fsrojeet Title: Ssvelopssntal BslSsolo^ sad Fimction of fhe iSsyasis 
Glssd aed Thymic ^jHsors 

Friaclpal Im^ssf igairor t Soger W. O^Gara 

Qth&T Xssv"©stiga£ors I 

Msa fesE-s (calsndsr year 1959 )s 
Tstals i/2 

Profssaieaels 1/4 
Ofefeer; 1/4 

Project Usscripeiss s 

pbje.a£lv<e3g fq ©bteto iaformfcioa oa <a) t^ fusctioa of tbs 
fc&ysass gland^ (b) th& fastors iavolved ia the growth of the thyiaus glssad 
asd ia tbe developmaag of epithelial th^csoE^Sp (c) the influsace of t'ns 
tfejnass glasd oa She iadssetioa of Xysspheld tt^ao^s, 

Mettsadsg Sargicsl procedures sad the usual biol@gle^ feisto-= 
pstholaglc^ a&d rsdiatioia t&alsiiqxiee , 

Major Figdiag:s; I) Serial studies of aao»na$«l thytaie glands 
trassplantsd into the splees of adult isice ebss& tk&t the th^aic tissue 
rsgeserates fro® its csm cells snd aot frois isafiltreted host lyv3phocyte& o 

2) Adreaaleeto^ plus ore^iecto^ enhanced ^deretel; the grofwth of isera" 
eplsMs t^:^sst3 tresisplssts btat ovArectosE^ did aot. fhyiseetosay did not 

appreciably ea^ace tis& growth of ifitMispiesjic thymte- transplassts. 

3) 0!ie iatrssplsnlc gl^f^e tiraospleat b&a resulted in a ttanspl&nt&hle 
squemjus cell eas£iQ«aaa. tmw ia its 3rd passsgeO ^) Additional studies 
confiESB psrevious reports that leukeasis does aot dexrelop is irradiated 
tfegmssetosi^d CSTBl naice wbsa given iatrasplenic trasasplasts of neo-astsl 
thysHss glands. 

SipiKificaaees iSais is s aaw espariasental ssethod of studying tks 
growth eepabilities sad r^ctis^s of the thyjjjus glaad under altered 
eEviroaa»Btsl conditioas, St is e:qpected to supply isfo3nmtio» on factors 

which regulate aad control the groi?th of the thymus gland, sad cm the 

developiKEt of thjwsonas sad lysaphoid tumors. 



101: 



serial m. ^_ML^L 

Xsdividi^l Project Report 

Csleadar Year IfSt 



'F&vt A (eos£iia'Jsd) 



froposed Cearss; 1) Additional studies are beis^ laade of the 
comp&x&tiv& effeets of neo'^eata;!. t^rmlc tissue x^hen tra&sp lasted into 
tha liver^ spleen^ and subcutsaeous tissue of tfejrEoectoraised irradiated aisSc 
2) The asture of t&e epicheiial cysts found ia tlie spleea after traasplaa-» 
tatioa ox sso-aatal t^usie tissue is beicg furtlier iavestigated by 
coispafflsea -jfith tsras^plaats o£ htonchi^ skia^ aad stosach frois neo-natsl 
laiceo 3) Greups of Irrsdiatsd ssd.ce will be fed aatiimtilbolites sad 
ali^latitsg sgestss (a) duriag tlie feriod of Irradi&tion sad (b) duriag 
the period follc^lsg radistioai, in sk attest to iiaflueaee the ioeidasce 
of radlatioa-iaduced laakesiia dus'iBg the prelewfegals period. 4) Cholssterol 
pellets coataiaiag horsicaes will be is^lasted subcutaaeously iato 

laics to study the loag^terss effect on the tfesfsst^. 



Fart B iaelijdeds Ho 



101: 



Ssria.i u^^. i.,%,^ i^i'^k _ ^ "^ _^_^ ^ 

lo Ijaboracory of Pattelogy 

2, Caaeer laduCo & Pa£b,ogenssis 

Individual Project lepor£ 
Csleadar Year 1959 



i^lAi 



Project Title; Incidence of ladaeed amd SpoataasoiJS Tmaoxs in HIH 

Swiss Mice Used ia SorseuiBg Ssi^isariEisats for PoS:sBtia,i 
¥lra.l ladused SuBJSa neoplasias 

Prisci»al lavsstlgator s Mearl F» Staatea 

Other laves eigstors Eob©r£ I^Eaksr 

Cooperssiag Efelfca: I^bc?££ory of Bio logy j, ^I ' 

&laa Years (c«ileEdsx jesr 1S59); 
fotels 1/2 
Prof assioasl s 1/4 
Others 1/4 

Prajeet S^sseriptloKs 

Qbjeetiyes g Sbs dstermiaatioa of She iacldsace aad chsraeter of 
tissors '«?hiefe oeeur isa the variety of mice used in scrsenitsg esperitaents 
sad tbe detectioo of uEiqtsa fc^jaors wfeicli soay arise ia ehe test xaieso 

l^ttodss Dr. I^ESker cultiires ead inoculates newborn laiae v?itli 
eoQtroi assd human tiKsor iaoeulated tissue cultures^ Dr, Stanton studies 
the ©aiaals for tumors. 

Major Fiadita^si Issults are in very preliasiimry stages of analysis.. 
Of 106 tuffior-bessing animils (all over 6 months of age) the followieg 
percas£.ages have ba^a aoted: breast t"osa>rs in 20%^ pulraonary alveologenis 
tutffiors 3ltj ratieulo-endotlsalial £usx©rs 11%, uterine leicnsyoHSS 6%^ 
subcutsaeous fibr©ssreoa®s 5%. Ia additioa tlia followissg rare tusssors Isave 
been fo-asd; 4 EdeaocarciaoBKS of the kideey (2 froia a group iaoculssed ^ifch 
tke sai^ fflsterial and 3 from the sas® source of material), 2 thyroid sdeBcsms^ 
1 salivary gland sdeKosss, 2 lasst cell ttHBors^ 1 osteogenic ssrccass, 1 ovariaa 
earcinoiaa, X endoasatrial csrcimotsaj 1 pituitary adenon^, 1 eutsioaous hydradenossa.^ 
aad I liver bsmsagioimo Tbe incidanee of multiple ttsaors is remarkably high 
is this group of sisiESiSc 

ieance ; Sash studies are cecesssry to separate induced 



tuffiors from thoge occurring spontaneous iy. 

Proposed .gourse; Bei^ause the mice are not inbred it will ba Eacesssrj? 
to stady large Mssfeers of miee at various sges to have s clear imprsssioa of 
the noraial ttaaor laeideiscs aisd variety of lesions tjhish test aaiissls laay 
shQ^'« This is a loi3g»tsna pro jest. 

i.:t E isstededj lo 101 ^'^ 



1. Laboratory of Patlioiegy 

2<, Caacer iBdise^ .Is Bet3sogeneais 

3= Bethssda, l^rylsad 



ladivldual Project Eeport 
Csleiidar Year 195S 



Part A« 



Picoject Titles EsparlBjaatal Studies on th& Effect of Hutritioaal 

I^fiaiaacies on tbs. PssSiogeaasis of SepaSosas iia l&ts 

Fpiacipal lavestigstors Heractel Sidrnasky 

Otfear Savastigaeers 2 1) Tsuaeo Baba, laboratory of Pathologv^ MCI 

(Section A (i)) 

2) Eaisanuel Farber, Bept, af Pathology assd 
Biocfeaasistryj Tuiaise Uaiversity, Hew Orlaeiss 
(Seceioa B <1)) 

3) ¥.8. Mlth&ndex^ Laboratory of FioefsemistrTc 
mi (Section B (2)) 

4) larold P. Morris^ Laboratory of Bioebeasistryp 
mi (Section C (4)) 

Coo|seretlag tmitss 

Maa fears (calendar year 1959) s 
fotal; 2 1/3 
Profess loaals 1 
Others 11/3 

Project Bescripttoss 

Objeeti%'eSi Te study the patliologic and chemical changes tltat 
oecur ±n certaia sutritional dsfieiesclea which imy bs related to the 
subseqtieat develops^sst of neoplasia is the liver of the rat. 

Hathod s g (A) ^ashiorkor, a widespread protein deficiency disease 
in iafsasy assd childhood^ baa been considered possibly to be related to the 
high incidence of cirrhosis atsd hepatoE© oi adults in Africa. E:^eria^atally^ 
we fesve previou0ly found tbst yeassg rfits force«fed purified diets devoid ia 
certain single esseatiai amino @.cidS;> threonine, tseehioaiaa, or histidine 
develop saay lesions which sre siiailsr to those found in teashiorkor, la 
order to le®m additional itsfonation under our experimental conditions^ 
deficiascies in valine or lysine have recently been studied. Also studies 
are is progress to determine the influence of level of esrbohydrats iut&ke 
on the ebanges produced by the amino scid deficiencies. In order to slistilate 
more closely the natural diets in areas where kKrashiorkor exists, esperiseanfes 
have basa carried out in which yonng rsts have been force»f ed diets 
contaiaing certain vegetable proteins as the sole source of protein. 



lOiq- 



ladividusl Project Report 
Calendar Yesr 1959 
Bast A (co'titisuec) 

1, Choline deficisacy in acute esperitaeats ■ produces & fatty 
lives in£&s rat ^hiie is eferonic esjjserlEiasits feepatosa dsvelops in £he 
ret» The possible relaSiotssMp "aet^aesx tim develcpasat of tlie acate fafcey 
li'ver sad Slsa subsesjuaat develo^BSsat of IsepatoBja is not slear. Xa scu£e 
esperirjsats tlis susceptibility t© davelop fafcty livers tes been found £o 
vary ia cerSaia speeisSc A study has bsea conducted to learss whether the 
level of the liver essjiae^ etelitie oKidase^ in 8 species of SQiimls sad 
in mmx^ msrf he tel&te4 So this altered susceptiibility. 

Ib additioa^ s stady lias been uxsdertakes is ordei° to obtais a 
Htosra clear auderstandlag of fe&e relatioas&ip of u^tliloniae to choliise la 
scuta eholiae deflsiaacy.. 

fxevions-ly it lies haen sfeowa that rats develop tepatojsas ^feas 
plssad OS. & choline deficient, lew usethioaine diete More receafe studies 
reveal t!&as: euMosise, aa eSfeyl imalog of nsstMorsiae,, produces hepatoses . Ie 
£he rat. Ethioaise is believed to &ct as a eoBspatitive inhibitor of jBe'eliioaiae 
aad thereby prod^sces a relative ssafchioaiEa defieiency state- Msfefeioaise 
but not sholins coa^Ieesly isiMbits this action of afchioiiine. To leara 
Esors about tlie snee&snisa by tshicli iaterfereace with metbioniae laetabolism 
c&n iaiiice hepatoasss in rafcs^ ctsrreaS ej^erisESBts have beeia started in ti?hish 
snisials have haen plseed oa a inetbloittiise deficient, but adequate choline , 
diet. 

€. Ethioaiae fess receatly been reported to produce bepstosms in 
tte rafco Altbojsgh rmcb. ssqserisseatal work Ms been performed oa the scute 
actioBS of athioEiae, little Is lasowfi about its actios in chromic 
esiperitaeats lesdisig to feepatoisao Several studies have been started to study 
certsia aspects in tke davelopEssot of hepatoses. The influence of partial 
hapstectoEjy before placing r&ts on etbiomiae is uader investigatioG, 
P-HydroKypropiopheaoQe (^F) has been described to inhibit the developmest 
of liver tussore caused by butter yellow. The affect of FBP on kepato?m 
forjastioa dus to ethiosiine is praseBtly be&g studied. 

EthioHina sad S-2»fluorenylsceta!aide (!I='2'«>PM) have each been 
shovm to icduee hepatou^s is» ra£s= l^ie rats are more susceptible than 
feimles to the careiaogeaic aetioa of N«2'»FM while no sex difference has 
beea reported with ethioaise/ T&s early his t ops tho logical shaages producad 
by sacli agsat have been reported to be siailaro For this reason^ esperis-iatits 
have been started to determine xjfeather these agents fed together is the diet 
assy act synergistisally in the dsvelopa^nt of hepatomas and to observe 
whether s sex differesce tsill be foxiado 

Studies have bean started to leara ssore about the ses difference 
iR liver tijaaorogesesis due to H"2-'FM. Mts of both sesses of the Osboras" 

Meadel strain and Buffalo strain have beea sacrificed at iaterv^als in order 
to study the early- histologic ehssges in the liver. 



1015 



Serial Ho. _^gC£535 _ 

IXidtvi4-as.l X-rojeefc Eepox't. 
Galeadsr Isas 19S9 
Fart A (coatiKtaed) 

Ms j or ^ ^ FlBdisgs I Ac X) Oaa publieseicm describes £ha chesalcal 
pathology of sosSe vsliae asd lysiae deflcisncas. 2) Preliisiaary studies 
sppssr £0 iedieate £iss£ tb,a level of earbobydrste isstake is iaiportoat ±n 
pro.duslsg tfea psthologie elsaagea described ia £lie above assd psrevious gab» 
lications. If £!ie carfeobydseate iatake is lot^red while all otfeer nutriests 
are kept eons taat, few patlto logic cMages result. 3) Eats force-fed eertaia 
vegetable proteins (cosaj casssvs^. ailo, wlbeat^ sad rice) for three dsys 
develop many pathologie cfeanges eimllsr to those described with previous 
asiffio as id defieisacieSo 

B. 1) Oss pitblicatioa describes the various levels of liver clsoliae 
oxidase in hsuk assd in 8 species of anisjals. 2) Preliaiiaary studies reveal 
£|2SS girowth and psthologie cfcsjages due to choline deficiency are highly 
dependent on the level of diet:ary nsthionine iafislce. 3) Chronic esperis^nts 
ill wliicli rats Issve baea placed on a nethionine deficient diet have sow beea 
is progj^ass for 11 niosSfes. "B&ts will be followed for aa additioiaal 12 
moa£b,s „ 

C. 1) Espei'iseats ia which rsfcs were partially fespatectosaised 
aad then placed on aa ethioaine diet:, have now been followed for six months 
with.no fisdiQgs to date. 2) Esperiicents in which rets were fed ethioaise 
with and without ra? have beea followed for aine laoaths. Preliciiaary 
findings indieste that "SW does not alter the early a^rphologic changes 

due to ethioaiae, 3) E^eriiaests in which rats were fed control, ethioaiaej 
K-2-FAA;, and ethionine plus 'E-'Z-FMA diets were observed, l&le ^sad feimle 
rats fed the ethionine plus M-2-FAA. diet died with hes>atomaB at &n 
earlier tisae thaa ia all other groups. 4) Experiments in which rats xijsre 
fed K-2«FM and sacrificed at racnthly inteirvals reveal that the hiatopsthologi 
changes are different in the xoale than in the feisale. The laale rat develops 
changes earlier and of a different type than in the fetnale. 

Signifieaace ; Up to the present time the only nutritional 
deficiency xi^hich has been found to produce liver saacer in e^terisaental 
saiisssle is that of choline deficiency. It t^rould be of imsch interest and 
importance to determine whseher o^'her deficiencies such &b chronic 
amino scid or protein deficiencies asight «nder certain conditions produce 
hepatonias ia experiaiantal anisaals, 

.gouyeas Eats placed on different diets will be sacrificed 



at varying intervals. Betsilad pathologic studies will be conducted and 
the pathologic f indijsgs ??ill direct the course of the chemical investigation. 

(note J la addition to work described above^ collaborative work related fco 
liver carcinogenesis is being carried out with Dr. H. P. Morris, Details 
will be given in his report.) 



Fart B iacludeds ^ee 



PES -If 

ladividual- Project lapor?: 
Calendar Year 1959 

£g£L.lz, Hosors^, Awards^ §.na Publications 

Publications other Sbaa abstracts from this project 

1, SldransB^, ^rschel, and Baba^ Tsuaeo 

Chesaical Pathology of Acute Assino Acid Befisiarsele'ss III. 
Sferphologic and Biochemical CIssages is Yotxag Eats Fed Valine'- 
and Lysiae-Davoid Diets, 
Jo Nutrition. la press, 

2o Sidrsnsky^ Herschel, acd Parber^ Ewsmnu&l 

Liver Choliae Oisidaee Activity ia ¥sixi aisd in Several Speeles 

of Anliosls. 

Arch, Bioeheni. Biopbtya. In press, 

3, Sternberg, William H., Sidrsaskyj, Herschel^ sad Ochsner^ Seys-our 
Prissry Malignant Lymphotxias of tbe Limg, 

Cancer I2i 806»819^ 1959. 

4, Sidransky, Eerscbsl, and Friedaaan, Lorrsins 

The Effect of Cortisone and Antibiotic Agente on ExperiiEsat^l 

Pulsjonary Aspergillosis. 

Am, J, path. 352 169-183,, 1959. 

5, Pearly Haurios A,^ and Sidransky, ^rseliel 

Fungus Infections of tlie Lsj^ in Patients Treated with 
Corticosteroids and Antibiotics 
Bullc Tulane M. Fac. 19 g 35-42^ 1959, 

6« Sternberg. Willisia H. j, and Sldransky, Herschel 

Pathologic Observations on Pritmry Haligmat Lympfeosnas 

of tbe Lang, 

Bull. Tulane M. Fac. 19 s 1-10^ 1959, 



Honors, Awards relating to tbis project: 

Mesfijer^ Azserican Society for EKpsrinsntsl Pathology, April 1959 
Meiaber, Aiserican Association of Path. & Bsct, April 1959 



I0l7 



Serial lo, ,^I..gOL. 

1. Lsfec33'st®E'y of Fa£|jol©gy 

2. Gaacex ?s£h©l©gy Seetios 



FHS-MIH 
ladlvidual l^oject; lep©r£ 

Calendar Year 1959 



lasLA. 



Project Title: Geographic pathology field studies ©a ux±iR&ty blaMssr caacea' 
in New OrleanSj, Louisiana 

Friacipal Investigator: Lucia J. Dunham; EareM F. Bonaj aad Harold L. Stewart 

Other .Investigators: Alan S. labsosj, lo^is B. Thos^s 

Ceopersting Units; 

msa Years (calendar year 1959); Fatisnt Days <caleadar ye^s: 1959); 

fetal: 1 S/& 
Professieaal; 1 i/3 
Otbsr: 1/2 

Project Bescription; Field studies ©a Ifeinary llasldsr Csnesr^ ls5>? @rlee.as. 
loaieieaa. 

gfejegtiws: Tis© obj®eti¥«e @f this pr©jec£ is to eesssplsSa aa sssSessi^a 

quest ioas^ir© study of pasisats with m"i£?ary bladder essses^ 
ia s ®ms£her» S,S.A,. city with a high iacideEsa ©f bladder cmtQBt ecE^asfsd to 
tw© other southern cities. CBara aia^ Cwtler^ I'larbidity frc^ Casicer ia eh® 
Ifelted StateSj, Fufelic Healtfe Mosaograph fo. 36) . The 4s£a t^iil be sasl jfs®a. with 
® •^iew to identifying factors asaeciated with eases ©f urinsry bl®«ld©£ esseer, 
Bssi© data on the ineidsne® e,n4 pathel®§ic Ci^ic.s'©se©pic) diagaosis of tbs 
cancers are being obtait%e4. 

M@tho48;_ For the area midsr &tndj ®sd the period ©f Btudj ®ll pstiests 
psssibl® with the iiagsosis ©f sarissry bladder caecar sre 
interviewed j, and an ®q\s&l niMser of control p&ttentB a® wall, fhe ©sse 
recorded include residence^ oecispatiosj sasdleal sa^ surgisstl history j, sad 
iafoKB^tioffi ab®ut diet aad about eh® «»© ef alc©b®lie aad ?.oa-slcoh©lic 
hs-??®rages sad of £©b®ee©. I®c©r4s are kept ©£ the ^aall sisber ©f pstiea'is 
B®t iatsrviswed^ their diagaosesj, and th© r®as®ns for failtsre to iateririew. 

A coding statistician sad on© atsdical serial worker iater-^iewsr 
are currently ©asployed in th® project. laterviewiag was started ia lew Srieaa© 
©a 15 Fs&rusry 1958. 



1018 



Serial l0., mi 5QI 



Calendar Year 1959 



f&Kt A Ce©a£in«ed) 

Major Findings; ©aea fre® ab®is£ 200 caaser p@tiea£s sad asi ©iiual 
nuiabe? of e®al:?ol patients have been zevi&^&d asd 
e©ded. St is eatisuated efeat patholsgie isss£©rial will hmw® beea ex^aiaed 
ia &h@ut 90 pe:^cen£ of the oss®@ m£ ab® dee® on w&lcb £lais ^e^©r£ is 
subsaieted. 

Significance: The significane® of £&® study lies in £hs hop® of 

supplying & sound scisnfific basis toz th®\mde'sstmi4tng 
cf fsceors in h&bit &v envi?onasent as they fs&y he ^slated t& & higb 
incidence of urinmry bladder csncer in a specific geeg?aphic «res. 

ggeposed Course ; It is planned £o eoatlaae £bs s£«dy foi: a tetal of 
three ©r s»ye years. Saea will be £afeulsted sjssS - 
analysed and & report will bg prepared for publication. 



Mrfc B included; lo 



1019 



3. Bathe sd^j, Hsyylsjid 



PHS-HIB 

Individual Project EepjE-t 

Calaadar Year 1959 



^£LJ. 



F£roj@e£ Ti£l®; Gsogrsphic pathology field studies oa uterine csBcer in 
Hew York €l£yp Israel^ aad Washisgtea, D.C. 

Frisiieipsl Investigator: Lueia J. Dunhsm, Hffiir@M F. Dorii^ s&4. Harold L. Stm^aTt 

Othe? Zsivestigatorsi Jolm H. Idgccab^ Iiouis B. Thcsr^s 

Cooperating ttsits: 

Maa fears (ealenday year 195f ) : fatl<&nt Bay© (caleHdsr yesr 1959) % 

Total; 5/6 
Prof essieoAl s 1/3 
Other? 1/2 

Project: DescriptioRs field studies of uterin© cancer (eervis asd eorp?3s) ia 
Hew York eity^ Xsrael^ sjsd WasbiagtoHs S.C. 

©bjsctiyes;^ The ©bjective of this peojee£ is to cojEplete mx estsasi'.?© 

questioaaair® study of woaea with aesrin© ssaeers ia diffsrsst 
geographic areas sad racial groups^ aad to saslys® tke data ©btaiaea with a 
view t® ideatifying factors saspeeted of predispesimg to or ©aijsirag femsla 
geaital cancers. Also,, basic date on the iasidsses aad pathologic (saicroscepie) 
diagnoses of the cancers are obt&ined. 

^thods ; For the area uader study mid the period ©f stmy sll pseieats 
possible with the diagaosis ©£ aterine csacsr are istsrsfiswed., 
sffid ©a ©qiisl iaiaaher of eoatrol patieats as 'sj®X]|., TIs© coafidsatiel dsts sesoiirdad 
iselisde Eocial^ isedical^ and surgical history^ and sseastrualy isarisalj, and 
pregsaacy data, lecords are kept of the ssail a«saber ©f patients aot isees- 
viewed^ their diagsoses^ and the reasoa® for faiisjre to iutoivvlei'? . Fsth©l©gic 
ssatsrial is studied ia all eases , 

A codiBg statistieisa &nd oae s^dicsl social worker iHter^^iewer 
©re eurrsatly s^loyed in. the project, 

^^9¥... f ig^jgtsss 1. Data froia 3j,514 eaaeer sad eoatrol pa&ients,, Jgwish sad 
noQ^Jswish ^hite^ in Mew York City aad Israel bavs baea 
■£a¥i@wed^, coded, tabwlatad amd saalysed. Pathologic ©atsrial has bessj exssaijied 
ia the easacer cases. A report has besa prepared sstitied Csaces- of the Stesrys^ 
Fare I^ Jewish sad aon-Jewish whit® woaea la Msw York City sad Jex^ish wos-sb ia 
Israel. She B®v&n authors^ iEsludiag Br. J. Cssperj, Fetah Tlq-y^i, £srsei aad 
Sr, A. Syaeosiidigj Thsssaloni&a^ Greece^ srs currsstly editiag the tasaussript 
preli^nsry to prablicatioa. Twelve additiOKsl critics srs also reviswing tlie 
msEiaseript whiclTwill h® published i» the near fafciar© s® a W. S« Fublie Hsalas 
Service Monograph. 

102G 



Sesriai llo. _^IJgla_ 



Individual Projscfc Eepsrt 
Calendar Year 1959 



^ELA Ceoatinued) 



^JOT Fiadia^s; 2. P^yt SI. fSegyo w«Ksea las few ¥©s-k City. 

Bata £r{sa About 500 c&aeu o£ cases^ ®f the utei^s ia 
a©n«wfeite tjsmea la Sew fork City bave bseu reviewsd m&d codedj, togeehss: witfe 
aa ©qisal assafeer of c&atsol cases, Bstbologic ia©t:®rial has been sKSiEinsd ia 
all ©asssesr eases. Sata are reedy eo b© traasf erred t© puneh c&tda £©t 
aachiae ©aalysis. 

3. F&Tt 11. HcEgro woiaea is WasMngtoa, B.C. 
Bat® ixom afeoat 400 cases of cancer of the utesna© in Heg^o wosaea sad fr^s 
about 400 control cases will be asseasblad by Feb^isary I ©r Mssrsh I, 19&0. 
Bats fresa the entire Part IS study of Hegso w^^o will b® coded and SESlys-fid 
d-ariag 19S0. 

MggMiSgggSl ^^ signlficfiuaee of the study lies ia tbe hope ©f 

supplying a sousd scieatiflc basis for the uEderstsrsdiHg 

«f factors ®f cust^Sj bsbit, or eaiciroisiaent as tbey-saay be related t© 
relatively btgh fresqsseacies of ttterine cancers ia scss® groups of wcsssa as 
contrasted to interasediste or relatively low freq?seaci®s in other groups. 
In ®dditi®n^ & critical ©-srsliistiosi of the atudy^ prepared at tb® sss® tisas 
@£ tfee report^ should provide a useful coimaent&ry ©s teebaiques ia ebe 
epidemiology of career. 

freposed Coar^ses It ia plasmed to ptabllsts sSie reg^srt ®f Fart I ®f tfj© 

study^ to prepsre the report ©f Fart SI for fviblic^^ttom, 
&tid t© explore other teehaiqiaes thsa were «s©d ia these retrospective studies, 
The ©ther t@chaiq«es to be coasidered iaclude: C^) ® prospeeti-ve study of 
©saeer of the uteriss ia a large group of hesltlay persoas^ such as eErollees of 
the health Issarasice Flaa of Greater ^sw ¥©rk^ aad {1} a comparative geseti© 
study of rates of careers of the uterus in white w^aeSj iabred and ojatbred 
trirasial isolate groups of woB^a aad Kegro fe'cssea. 



^art 1 included; Wq 



1021 



Sesrisl Mo. ^iJ2|^ 

2. C®ac®s' Fetlaology Section 

3. BethesdSj Maryland 



FHS-MH 

Xndividual Fsroject Report 

Calendar Yesff 1959 



^IS^ 



Frsject Title J Geographic Fethology. Sxpes'issssatal studies ysla^issg to £be 
geographic pathology of ssouth cancer. 

Friacipal Savestigstor: Lucia J. ©usshass 

Other lavaaeigatoirs: 

toops^asing l^il:«: 

Maa Yeairs (caleadar yeas 1S59) : Paeisat Dsys (caleadsr yesr 195S) s 

Total; 2/3 
Profaesional • 1/3 
©the?; 1/3 

Fg-ojeefe Description? Ssp©ylm@El:al s£B4ies ©a soufife easeer. 

Ob.jec£iv@g.; The objective of this project: is t& esajsia© tha sj^eoas ssssfesaEe 
of th® hsasstfiff cheek poach fog" ptoliter&tive cImEges £tot ssey 
d©^@i®p wfeea eest substaaces s^e eteoaieally sssiatsiaed in c©a£acs: with Sfease 
SMS'faees. Sisfestaaces used ay® eavirojmentffll ast«gri®ls s«sp®c£eii ol b@£s^ 
mildly ca^einogeBic for msn. 

^thgds; Ths pellets which bsve been prepared for iasertloa ia feh@ rigbt 
cheek pouch of the hsasstss: iaclMs the to&sce®^ llim^ gsmhl^^ 
srec® tmt^ aad pepper leaf ®f the Fm- Es^tesm '%©&©! qaifi'% sh^ plsstie 
ssaterial us«<l ia prepasring "false" teeth^, ana toteec© tss-s aad coats-ol 
saaterisis= A t@ehsjic«l assistaat is ^©taiasd part-time for iselp is tlie study, 

Majig g^^Figdiggs ; The operation for iasgrtiag pellet© ia tm poush liss been 
perfosmsd oa sKsr® thsB 400 asisjals. Mo aa:lsals air.E-^i\?s 
at tha psr®©@at time. Gross aad aieroseopic le®i©ag of the sauclj -v^ssli l?^¥e feeera 
observed Isa about 15 ©f the several huadred esperisssatal emimBle eseskinsd. 
There have b@©n sso asaligiaasit aeoplasss ^aoag thess. It fess fesaa 4a»as£ra£sd 
that Beoplasiss cam be initiated in the powch wsll by uaiag pellets corstsiBirsg 
a kaowE earcisogea, Whea tissues from all the sraimals &©■?© b©ea ®3jsained 
ail fiodiEg® in the pouches sad othe^ pstfeolcgie chsages will be ssvie^fsd 
and a report will fee prepared. C^© curremt stiidiss. Serial 1©, SSI 53§,, 
B? . Eatherisae lerrold) . 



1022 



Serrlal Mo. JCIJQlb 

Calendar Year 1959 

Fagfc^ A <c©a£in«3ed) 

Sigalfic&aee; The sigaificancs is t© sreash a® ia^roved «E,(des-8£sading 

©f efescaic irs-itane factors ®s they may relate £© cancer 
©f the iffiouth in w&n. la addition it will be aseartained whether it is 
desirable t@ d© further studies usiag tasdificstions of the teefenique desesised. 

Pro gosed Cow gse; Tbe cheek pouch wich the test pellet^ sad the opposite 
"-^ ' ' pouch, are both exafflined when the hajaster is sacrificed 
or dies. The findiags will dictate the future eeaduct ©f the invest igatioa. 
A repojrt will be psepaBed for pisblieatioss. ©ae or asesre esgjesiHieBtal approseses 
of possible use for the hio-ms&y oS weak csrciKogecs will be expl©"sd. 



f&tt B iacluded; So 



1023 



Ia^i-^i4«a3l fEojseS Sages'* 
S©isaJ<^®s Yeas ISS9 

I^ojec^ tiUa. Sfe^iss ^i Fc^lyc^ nsiss is SemB^FTSe YUB^se GM^ 
Systems 









SejTto eolMbosaeloa wtti« Sr. Htlltso isaafLW. 

IIZI ^^it^m^ nBB BI cells ii^"s0Sed ^i£b polyem vis^^<. 



Serial m. mi 524 






Jasfe & (eoatlBugd) 



polyoes vig-u® ia vifes© ^11 psfofeSlsly be of g?eafe is^ytaEee to owf imde^'^ 
st^idiss of viral seo^i^i®, Uss systes^ fes- £ls© Isolseioa of pelyi^m vi'ms 



la F388 Bl €s©ll® in ssessa»fsree ^iiiiss ®ill fee e^a^isuad. Sysees^ i&s g&s 
gsm-^t^ ef eks ceils ^sd visr.is la a cssaf>lseeiy d^aieall^ defiised w^hm @se 
fes5,as iSe^'elegei ^sd s^s&U fsellieaee bioeh^ical seudieg of efee vissas^&sss 
sail ej'g£sia« Mtas^ts to fii^ a pmoliiisss&ive vlrus^lKJSt c©ll sysesa i© 
eul^?£; Mve b@@@ ;i<s@ueeassf^i but id.ll bs oos&ii£u@d. 



f&ist i iagludig^; 1f0S 



1025 



S'^Ti&i i&. mi sm 






fe^_l. &ssr@j Ais^^is^ mid Bsbiicatiom 
ft&lieat;l®as @tl®s^ etea sterlets fe-om £M® fTOj@gt:s 

lfiik«'®8feftgs! rnssi^ I^ss^hstm Sells (S^^i® F3S8 Bl) 
F«©s. S©g. Isp, ^i®l. & m4. im% Ilf«>133^ BSf . 

e5?t©e&smig®l gtadies of mi&«®&pt©il l&criTO I^psaplsOTa Sells 

J. »e« e^e@r liist«^ ia ^®m^ 



1026 



3, Batte®4®^ I'^gylaai 



Cslsadar t&^ 19B9 



mkA^ 



Fr©J@et Titles ktt^ts to Isolate Oaeqgsffiie 1?i3fus©s fso® 

6MSS f&sffs Ce&i^?i&s' y-gss- l0Sf ) 
Ofe^s? 2/3 



mthsSBi t^m&tB of £?@@^ &i;s^@ eis^s'S a£% prep@!^@d s^d 
issei3l®g®d isifeo Slssis® esileM^cs of Is^se eells^ iaSo ssewfeesa Syrtes teasfeers 
^^ into £^ml %gs@t@r@ in u^^o. Bliad ^@ss@g@d o£ tke feis@$s@ eulg-oire 
fluid© a£@ t:s^e ©t i^®r^l@ to f@tsl @s.S s«£^?a ^^i^teS'S. ^sa £is@t^ 
cssl£yi£®3 %m&d Mv« bssa gs^^ i^ s^swa<»£r@@ Esedltsit to fivold possible 
pt'QSetss® of een^ ®iitib«^l@!i o? ©ensa viml iahlbitsnrs . 

lias b@aa isolft&ed s^d gso^n fes- sc^ro jrem.'s Is a eoivm^Sted t^dima coB^i^l^^ 
su&oei^v©^ ssQofal: tsilk. Culiasses) of ftlils eeXl atsaln as:^ Imjeulated 
'?i£^ sa£«asts ©£ Irg^E Ike^js fcrnsrars sied sbser^/od fe» peesible syeog@gfei« 
sffeet®. "Ssfg sSuidg' Ms bees ia psogscss fos less ftl^ cmss yegg ead sso- 
©ytopsitMs «geats tev© been isolated yefe. ISie feasasegr isecsalatiea studies 
lisve becB i© progges© ijs 8 soaths e«d ©oly oac imteuMted assts^I Ms 
defvelfisssed s fet^or. 

Essrsce© ©£ 33 fass^a feiasoE-s Isave bsea isocula&ed fefem far* fhass 
IscltMa Will® em»r of £te kidaeyj jaasotteliowi of £^ pesieasdiisa^ fedgkiss's 
dl@©@@©; sqasasoi^ cell csreifisosaa of she arsl csvicy, ei^syoaal slibdj>E§?o'=' 
aas-cos^^ gsjagllo^uxo&lastoiaa, sysaoviai ss^eos®^ UEjdiffe^eati^ted sss:sia^.«i, 

sfeoffioeaiesiisoi^jj ^sd v®rio«® feypss of .leakssaias 



1027 






fast A Cessssia^si^) 



\?iir'4S will fes ©f' gress iims^taace ia els© stiMy of ^sma e^ne^srs 
s@i®«£ad fegi^s f£m»rs t^l^ th&- feigs?ae c?*lt5sre SfSt®© &sA sstesl s|ss£©k-; 



FasfS B lael^ssSi Yes 

1028 



EiiiWt-iil 43© » 









1029 



B&tiMl llo. ,^,i£, 532 

3. BeSlmsda^ m^ylssid 



Sadi%''idual ^ojeet Report 
• Ssle^ar ¥e«s- 19S9 



^1.^ 



Fj?ojes& fit!©? S&adis® ef Smia fwisss pjf©4M@d isa tos®£@r® &y 

frteeipj^l l^&atis0^t&-s% Alsfi S. Sables 

@tl@r Ss??®§tig®£©£ss toUls L, S2.ifsstet«in, Bivisto® of Blologieal 

1^3 ¥©S£'® Ceele^^g' sfsss- IfSS)? S^eieat Says <eales4g? yes-g' lf5f>s 

fsssf©©si«^©is 2/3 
Otfesrs 1/3 

^^l^^s f^ sespcsass ©f Systes fesa@feess fe© iaasasesateal 

vira® S'M te la&ss g^ae of tlieir felol©sieal propsrfcies, 

lg.^ggi.t Fs@ls of polfOE® vlnj® sro?!?B ia tailk«=®dape@d F388 ©1 
calls feaw feeaa prepared aad tiSasfeed by gsrial t€a«fold dilafciosg ia t!i« 
@a^ e@il s^t@is. faiyi^ @i^JS£!t^ of vlrua bsve besa isumulated i%%&s@» 
e@?@bsally is&o yotsog Sp^iaa li^g^te^s. tha sais^ls have beesi $a?@fully 
observed f©r ewlds^se of iasxaessaiai disease sad ecs^let:@ aaSopsias Mwe 
beea perfossad oss i^is®is fo^siul 4esd &s affixibuad, S^offfeioas of bs®iia tmssr© 

^.ll^^^J^^^g Srae of glie fiEsdiisg© fesva boea praseated Is 
© ps^sr felmfc is a©w is p^®@. "^ s«sai®sy of £M§ ^pes* follotisj 



as-lg® f?fosi <i©asse«tiv© tisB^B in ete p£a asteg oss sfee marfsee of Sfea bsaia 
and i£% £te sulelj, as^ f^osa £te ^vesiti&ig of @smll oaaiBgesl and eerebrsl 
v@@@3ig«, ^s& &m^'m Mvm. dsveloped ia'as£lssal@ lmseula£@d on %1^ day of 
hitth i^sd at 3, 9^ m^ 12 dsys of sge, isisd they have beeo fotsad 1^ m&isml& 
which Mve died as @o®ts s@ 31 dsy@ afg©r ^.s^oeulseioe, Hisfeoiogiesily^ 



103G 






gars A (e'Sittlmiad) 



iae?@c@r®te^lly isisml®£®4 Sou® ©sressm visiss, , f^ taster i;',s»if0 ®l-s© 
^esesJsia S:^ rar® s^ssBs^aasl E?KS«ifs of else brsla is s^a *ie& M^s fesm 

he saeeasgful," 

giaallj^g^i "Uss dffi^astratiea of ^isgsffil s^«oess i^oduscad 
by pel.H/-o^ virus is ^^ge^© suggesS® £^ possi&ili^ £&s£ siiailss' SyEsssss 

ts^or SzmB tes^a la^o^i^eal s@£Coiass fo?r virus ieolafeioa stedie©.- 



vivtsB In yfflJi^ Sj'Tisa tesss&ss'S g^y &© a ug©&l s»s £&t stodie® «sf fete 
Kseste^iiK^ ®f viral gmi^s fcs^atio®. X£ is assiessble fos hisSog«£®si© 

sias© d©atlj slisisld h® oere fspld ss^ eertsiis witfe a sp®€®«©ecupfi^ fe&sa- 

iats^essalsl l^sisses s§s n^a? In progress. 1^ vivo iafaeeivitySifes^eio©® 
of vir«3 go^l© i6i?i6fe tess^sB tissws ewlesj^e isfeettvifey ssd ksacn-m temgglttSiai© 
eonteat asra siso is pg^^s^S. ths &i0Sol@gieai typo© of txm>^^ i^g-edssm-a 
tJith SEisll isseisl® ove^ loag pssiod© o£ ei^ will fee of coastdss^la 
iaserest stese S^ psesilsillty ®f gllsis® predssstl^s lay fe^ vlS53@ tes m>t 
teen eiiasiMted. irises^^s as-© h&isQ presew©^ asepeleally £s<^ bsaiii t^smm 
pvodiseed by v®syi®g d®88S oi vlsise ®Ka3 ©fetsa^feg ^111 h& ®gii@ So feiteato 
feli0 ©swat @£ vistss is ttes© ttmiVB to datesffiiss ^fea^es? e&are is m^ 



Fasrt B i®sl«d@di Ye© 

1031 



Sa^toi Mo, KI S32 



1959 



1. Xae^er«aial Ssireo^s Predassd fey Boly®saa ^ir«© is Syrisss 

A. -S. SaSeoa ted E, i. Kisrscfesteia 
ilfelsives ©f BaSlasIeg^* Ik ps©®s 



1032 



3. Bethesda^ Maryland 



PHS-KIH 
■,2M:-.aaal Project Report 
Calendar Year 



Sffsct. of species variation on the oncolytic properties of 

Goxsackle B3 virus on heterotransplants of HeLa cells 



isipal Investigator: E. G^erald Suskind 

3r Investigator: Robert Love 

Years (calendar year 1859) : 
Total : 1/6 
Professional: 1/6 
Other : 



;iect Bascription: An attaiapt xvas niade to compare 2 clonal strains of Cossacfc 
B3 virus, one oncolytic and the other non-oncolytie for 

srotransplants of HeLa in rats with their behavior in heterotransplants of 
in the chick en).bryo. The project has been temporarily abandoned 
the difficulty of producing reproducible HeLa tiimors in the chick 



r'O. 



1033 



PES-NIH 
;-duai Project Report 
Calendar Year 1S59 



■.ject Title; The cytopatbological and cytocbemical changes of parainf. 
vii'tis infection in different cell systems. 

/ftvestigatOi": R. Gerald Suskind 

..„ „...,. -.viiaator: Robert I>ove 



Years (ealsxidar yaar 1959) : 
Total; 1/6 
Professional: 1/6 



3ject Desc.ri|>t3.03; 

Qbjscti\[es; The eytopathologicsl changes prsduced by infection with £. 
recently described niyx'ovirusj, particularly with regard '■ 
iinges in nuclsic acids and nucleoproteins are under study in different. 
steEJs. Mates of virus syatbesis ss measured by the hemadsorption tecbr,.iu. 
- --. correlated with cbaiiges in nucleic acids and nucleoproteins as observed 
:--t:icular'ly by toluidine blue-BJOlybdate staining. Differences have beer 
jsrved in infected HeLa cells, where nuclear degeneration and syncytia;. 
.:;a£ cell formation is produced^ and in Infected monkey kidney cells I'j'hs:, ^- 
5 virus proliferates without causing cellular degeneration. The nuclec- 
Dtein and nucleic acid alterations are compared in these cell systems. 
:or'adiographic and fluorescent antibody studies to correlate the nucla*: 
-nges with sites of viral synthesis are planned. An opportunity is ps. 
study the cytological aspects of a virus host cell relationship in s : 
...t.. wbicb the saiae RHA containing virus can live in sjnnb.iosis with one ca,: 
destroy another^ a i*eIationship which is of basic interest in the pathog - 
'-■^ cancer. 



is in preparation for the April Federation meetisg.-; 



103i( 



i . Laborato.. 

3= Bethesda^ tiasylai 



PHS-HIIi 

Individual Project leport 

Calendar Year 1959 



Part A 



- ject Title: Further observations on nucleoprotein staining with toluidine- 
blue molybda^a 

.'■.cipal Investigator: Robert hove, M.D. 

'■:'- Invest5.gator ; R. Gerald Suskind^ M.D. 

.- v'ling Units; 

Man Years (calendar year 1959) : Patient Bays (caleridar jjear ■ 

Tonal: 
Professional ; 



iact BescriptiOii: (See report of Br. Love^i HGI-528). In additioxi to the 
3 stages already described a 4th stage has been observ- 
Hala eells and ascites tumor cells. Hitherto uridescribad S.KA gfaaules "- 
;n observed in the Golsi aoae of Hela cells. 



rt_B» Manuscript in preparation. 



1035 



^-land 



idual 'fi:cj£c£ Report 
.calendar Year 1959 



Fart A 

-jjact Title: Comparison of the oncolytic properties of Nexi7castle Disease. 
for iisouse lymphoma cells (F388) in vivo and ia_vi tro 

/acipai Investigators R. Gerald Suskind 

■^investigator: Robert Love 

-^,:s (calendar year 1959) : 
Tutai: 1/2 _ 
Professional; 1/6 
Othars 1/3 

"iriptions Previous studies of Goxsaekie B3 virus in heterotrs- 
of EeLa cells have shown that serial passage in^ ]"'^'^'- 
/ means of adaptation for oncolytic properties or this virus^ a process -miiy 
[ be reversed by passage in vitro. An attempt was made to reproduce this 
' phenomenon with Newcastle Disease ¥irus and mouse lymphoma cells CP388) in _v.^v: 
' ^^"^ jLn v itro. It vjas found that an unadapted (egg adapted) strain Newcastle 
I Disease Virus was oncolytic for P388 tumors without further adaptation^ t-^ 
' a st-T£iii oncolytic for Shrlich ascites cells failed to destroy P388 tumor 
^Vuber details see report of Dr. Love). 

;u____B Presentation of a case of essential pulmoaary hemosiderosis ■' 
pathologic Conference^. Clinical Center. 

C&.3e reports (In preparation) Carcinoma of the liver associe 
hemihyper trophy in a 22 month old infant. 

Case report: (In preparation) Congenital Toxoplasmosis. 



1036 



1. l&hox&tQxy of FatliGiQ^" 
2c Fat&oiogieal fecbaology 

Isdividusl Project Eeport 
Caletsdar Year 1959 



Pars A. 



Fro jest Titles BaSholsgicsl Teslmology Seetioa 

frinelpal Xsvestigators Joseph K. Albrecht 

QthaT lavastigators s 

Cooperstiag Ualfcst 

MaE Years (calesdar year 1959); 
Tofcsl; 30 
Frofassloaals 4 
Others 26 

Project Baseriptios; 

Stalaed eissue seetioss ars the fuEclassa£al basis of all cliBlcal 
and eisperlHseatsl studies of caacer. T&e Seetioa prepares histological 
sectioss for all fete isvestigatQrs of the Hatioaal Caacer Institute^ Ifc 
ffiskes a-«'ailable all the established routine and special siiaias and in additioa 
dsvalops aad previdsa tlie currexst e^sperimental mstlaods of tisaua preparation 
such, as etisyase staiBS aad specific histological staisso 

Movssabe r 1„ 1958 <» O cfeober 31. 1959 

^ NuE&er of iavssfcigators . . » . . c . = » « . , . » 73 

MtH!&er of pieces of tissue ........ o .. . 215,993 

HuE^ser of beetles of tissue ........... . 28,757 

Hoisifeer of blocks cut ... ... ........ . 112^404 

Hiaaber of blocks cu£ serially ........,, o 1,602 

Kusber of fresea blocks cut ......... o . . 1,259 

Kuffiber of suSopsies recorded . .... 21,430 

Ht'E-^er of recces ................. 1^167 

Euiifcer o£ slides stsiased H & E 133,933 

lussber of slides stsiaed special .......... 20,020 

Biatjer of uastsiised slides 11,155 



1037 



Serial Moo _^^;£Jj,7 

ladlviduai Project Report 
Csleradsr Year 155% 

^&stogT&phtc Sesviea xesid&xed to the Laboratory of Pathology 

l§ovfiEl^2S' 1., 1938^ to October 31^ 1959. 

Blsck & Waite „ ...=... o <=. o c , . » 223 

Color ^ »,.,» c .„....»...<,....«.. o 85 

Bl&ek & IJhi&e . c . ^ . . . .».,. = . 104-3 

Color .,....., 700 

Black & lihlta , 200 

Color .,..,...,......, . . o 450 



4 K 5 ......... 3943 

8 K 10 ... o ......... c ......... . 101 

l-^'anted 365 



Part B included g No 



1033 



Serial No« KCI~90^J^ 
1= Laboratory of Physiology 

2. Office of the Chief 

3. Department of Biologj'- 
Princeton University 
Princeton, K. Jo 

PHS-NIH 

Individual Project Report 

Calendar Year 1959 

Part Ao 

Project Title: Ultraviolet Light and Carcinogenesis 

Principal. Investigator: Harold F. Bltm 

Cooperating Units: Department of Biology^ Princeton University 
Princeton, Kew Jersey 

Man Years (calendar year 1959) '• 
Total : 1 
Professional : 1 
ether : 

Project Description: 

I„ Quantitative Studies of Carcinogenesis 

Ao Carcinogenesis by iiltraviolet light. 

Last year a quantitative model was formulated which de- 
scribed experimental data on the induction of cancer in albino mics 
by ultraviolet light. The process was shotm to be continuous, 
cuEiulative and essentially irreversible and non-threshold. Equa- 
tions were derived describing this induction in terras of the con- 
cept that the tussor consists of an incx-easing number of clones 
whose rate of prolifex-ation is aceelerated by repeated doses of 
ultraviolet light. E2q»eriaiental data support this concept. By 
this model the initial volume of these tumors could be calculated. 
This volume calculated to be smaller than a tumor cell, which in- 
dicated that the basic replicated unit governing the growth of 
these tumors is of subcellular dimensions. (See attached bJ.biio- 
graphy references h~^)o 

B. Carcinogenesis by other agents » 

Since cancer may be considered as a growing part of its 
host, it should be influenced by factors that affect the overall 



1039 



- 2 - Serisa NOo mi-92h 

grov/th of the host. Hence ^ this lends a degree of uncertainty 
to the interpretation of results of caacer studies in which time 
of appearance, the incidence in a population, therapeutic action 
on cancer, the action of endocrines, etc. are concerned (5)o 

A paper entitled "Quantitative Aspects of Cancer Induction 
and Growth: As Illustrated in Carcinogenesis "by Ultraviolet Light' 
(8) was called to the attention of Congresswoman Leonor Ko 
Sullivan of Missouri as having a bearing on the problem of toler- 
ance limits for suspected carcinogenic dyes in food. Upon the 
request of Congresswoman Sullivan, I replied explaining the in- 
feasibility of setting tolerance limits for carcinogens. This 
letter appears in the Congressional Record of August 27, 1959» 

Congresswoiaan Sullivan's letter stimulated me to return to 
quantitative analysis of data on other carcinogens, which I had 
attempted earlier without much success. The particular point in 
question is whether the action of chemical carcinogens, like that 
of ultraviolet light, may be cumulative. The latter appears true, 
but I have met again with little success in a quantitative analysis 
because most of the data have not been taken in a way that will 
permit accurate analysis of this aspect. The attempt at analysis 
will continue, but it seems clear that to obtain really adequate 
data would require extensive, carefully planned experiments, in- 
volving the participation of considerable personnel on a long 
teita basis. 

The possible relationship of the findings with ultraviolet 
light to the "fail-out" problem has been discussed in a brief 
paper to appear soon in Science (12) entitled "Environmental 
Radiation and Cancer" o 

EIo Studies of the Acute Effects of Ultraviolet Light on Skin 

A single dose of ultraviolet light evokes a strongly hyper- 
plastic response from the epidermis. The proliferation of the 
epidermal cells for a time reaches rates that are higher than 
those found for the cancers that develop after repeated doses. The 
possible relationship between these two things has been of interest 
to me for a long time, and it has seemed possible that some infor- 
mation about the carcinogenic process might be contained therein. 
Five years ago I began studies on the effect of single doses of 
ultraviolet light on the ears of strain A mice, which have pro- 
ceeded slowly because of technical difficiilties and limited assist- 
^ance. However, it was possible to make a qualitative and semi- 



lO'lO 



- 5 - Serial NOo KOI -90^ 

quantitative study which has been briefly described in an 
article appearing in the Journal of the National Cancer 

Institute (5)o 

The quantitative aspect of the study has been recently 
pushed forward with considerable success by MTo Gerald Soffen^ 
as the basis of his thesis for the doctorate, imder h^ super- 
vision, MTo Soffen has held, since September 1958? a Public 
Health Service pre -doctoral Fellowship. 

This study has provided consistent quantitative results , 
parts of which may be described by relatively sisiple equations. 
Mr. Soffen and I, and latterly Dr. Roger Pickham, have attempted 
to find a meaningful general equation to describe all the re- 
suits^ but with little success up to now. The attempt will 
continue^ however, and it seems likely that success will 
iiltimately be achieved. But in the meantime, partial analysis 
shows some interesting relationships which seem to have basic 
significance . 

Attention foeusses on the proliferation of the cells 
of the epideimis, which may be measured accurately by couiiting 
mitotic figures at different times after the exposure to xiltra- 
violet light. The accompanying figure shovjs an example of the 
relationship found. The curves represent the incidence of 
mitosis, properly corrected and plotted on a logarithmic scale 
against the time subsequent to ejcposure. A m-miber of curves 
are included which represent different dosages of radiation 
from an intermediate pressure mercury arc. A similar series 
of experiments with exposures to a low pressure merctuy arc 
yielded the saai® kind of curves; but with a somewhat different 
dosage relationship., which seems explainable on the bases of 
difference in penetration of the radiation from the two sources. 



10141 



Serial KOo HCI-90i'. 




,re lo CoiirBe of epideraial proliferatiori. following 
exposure to uLtraviolet radiation as measured by the 
incidence of laitosis. The Mtotic counts have been 
corrected in terms of the nuEiber per unit area of 
skin; this takes account of the increase in totel 
population due to increase in number of cells ^ since 
growth only occurs in one dimension. 



The effects of five different doses of radiation are 
represented by the five curves, the effect increasing wi-.h 
dose. Early in their course the cvirves are straight lines, 
indicating that the proliferation rate of the cells is constant. 
Later the rate falls off, passes through a maximma, and t,hen 
the curves again become straight lines, but with negative slope. 



10i|2 



- 5 - Serial NOo mi-^k 

It has generally been, assianedj due largely to scattered 
studies on himan skin that exposure to ultraviolet light is 
followed "by a latent period before hyperplasia or other 
changes begin. It is to be noted that the curves all reach 
the base line for mitosis in nonnal skin at about I.5 days 
after the exposure, and this might on first exsunination be 
thought to represent a latent period, particularly if only 
one curve were examined. However, the crossing of the base 
line at the same point, and the finding that the mitotic 
counts prior to that time (I.5 days) are lower than those 
for the normal tissue, indicate that the ultraviolet light 
has rendered a portion of the epidermal cells incapable of 
dividing o Pending a more complete mathematical analysis, 
these curves seem best explained as the result of influence 
of injured or moribund cells on the surviving proliferating 
cells. Thus the higher the dose of ultraviolet radiation, 
the greater the initial destruction of cells (resulting in a 
lower starting point on the ordinate), and the greater the 
initial rate of cell proliferation. 

The swelling of the cells and their nuclei after the 
dosage with ultraviolet light, indicates increased permeability 
and suggests the possibility that the increased rate of pro- 
liferation depends upon the release of material from the 
moribvmd cells which cem be used by the survivors in their 
replication. Such an explanation is in accord with tents,tive 
suggestions regarding hyperplasia which appear in ray recent 
book {l)g and also with a working hypothesis of carcinogenesis 
that is presented therein. The latter assumes that it is the 
acctunulation of templates, called for convenience "tems," in 
some of the tissue cells, which confers on them the inherited, 
rapid rate of proliferation that characterizes the clones of 
cells making up a cancer. With this possibility in mind, 
experiments are now being undertaken to study the effect of a 
second dose of radiation following the first. The analysis of 
the findings already obtained on single doses will, of course, 
be continued, since this must form a necessary basis, for the 
exploration of successive doses. 

Studies of the Effect of Non-ioniaing Radiation on Permeabili ty 

Studies of permeability changes in red blood cells, 
following irradiation with ultraviolet light and with visible 
light after photosensitization, have been going on in this 



1043 



~ 6 - Serial NOo KCI-90Ji 

laboratory for some time. A few years ago, Dro John Cook, in 
studyiiig hemolysis by ultraviolet light, discovered here an 
unejqjected relationship, in that the hemolysis time varies as 
the square of the dose. Later while Dro Cook held a Public 
Health post-doctoral Fellowship we collaborated in studies, 
the results of which have appeared this year in an article in 
the Journal of Cellular and Comparative Physiology {$) ^ The 
dose-squared relationship has not been satisfactorily ex- 
plained as yet, and the question of mechanism has become more 
important with our finding that the same relationship holds 
for photodynamic hemolysis, where an entii-ely different photo- 
chemical reaction is at base. 

Becaxise of the possible relationship of permeability 
changes to carcinogenesis, I have atteaipted to find a 
theoretical explanation of the dose-squared relationship. 
The basic idea is that both membrane permeability and 
colloidal osmotic pressure are increased propox-tionately by 
the radiation. The analysis is not yet in shape to present 
for criticism, but I hops to write it up in the near future. 

An account of earlier studies on permeability cha^iges 
with photodynamic action, in collaboration with Professor J. A. 
Green of Rutgers University, and Professor Arthur K. Parpart 
of Princeton, will appear in an early issue of the Journal of 
Cellular and Comparative Physiology (ll). Prehemolytic changes 
in photosensitized rabbit red cells were studied. Na-K exchange 
and glucose disappearance were studied while the cells were 
being exposed to brief periods of visible light in air. Brief 
periods of photiosensitization markedly accelerated Ha uptake 
and K loss while only slightly increasing the rate of glucose 
disappearance. It appears likely that the acceleration of 
cation exchange X'fith photosensitization is the resizlt of ceil 
surface alteration rather than of an effect on cell metabolism. 

Significance to the program of the Cancer Institute : To obtain a 
better understanding of the mechanism of carcinogenesis by 
ultraviolet light and of acute effects of ultraviolet light 
on the skin. 

Proposed course of project ; To continue this program. 



Part B inclui3.ed 



IQ^k 



- 7 - Serial Woc KGI-9QU 

PHB-MIH 

Individual Project Report 

Calendar Year 1959 

Part B; Honors, Awards, and Publications 

Publications other than abstracts from this project: 

lo Bltm, Ho F»: Gsreinogenesis by Ultraviolet Light: An Essay 
in Quantitative Biology . Prfnceton University Press, 
1959, PPo 5^ 

2<. Biuia, E» Fo: Time's Arrow aad Evolution , (ed. 2) Princeton 
University~Press , 1959 » (Paper hound edition. } (First 
edition 1955) « 

3. Blxsa, H. Fo, Butler, S. G., Dailey, T. H., Daube, J. B., 
Ma,we, Ro Co, and Soffen, Go A.: Irradiation of mouse 
skin with single doses of tiltraviolet light. J, Hat„ 
Gsjicer Insto 22: 979-995* 1959<. 

k. Blrnkg, Ho Fo: Goordinated growth of body parts and cencer. 
Jo Nat, Cancer Inst. 22: 229-259, 1959. 

5 . Blum, H o F . : On the mechanism of cancer induction by ultra- 
violet radiation. II o A quantitative description and 
its consequences. Jo Nat. Cancer Inst. 2^: 519-355, 1959o 

6o Blum, Ho Fo : On the mechanism of cancer induction by ultra- 
violet radiation. III. The growth curve. Jo Sat. Canc&r' 
Inst. 25: 557-5^2, I959. 

7. Blum, Ho F.: On the mechanism of cancer ind.uction by ultra- 
violet radiation. IV. The size of the replicated unit, 
Jo Hato Cancer Inst. 2^; 5V3-350, I959. 

80 Bltm, H. F. : Quantitative aspects of cancer induction and 

growth: As illustrated in carcinogenesis by ultraviolet 
light. Aaierican Scientist V7: 25O-260, 1959. 

9. Cook, Jo So, aad Blum, Ho F.: Dose relationships and oxygen 
dependence in ultraviolet and photcdynajaic heffiolysiel 
Jo Cell. Gos^o Physioio ^: 4l-60, I959. 



1045 



Serial Ko< 



n-SQ^ 



lOo Blum.) Ho F«, Butler^ E= Oo^ and Schmidt,. S. E^: Regeneration 
of liEib abnormeJ-ities after ultraviolet Irradiation. 
Jo Ceil, Compo Physiol. ^2: I77-I86, I958. (While dated 
195B, this paper appeared in 1959) « 

llo Green, Jo Ao, Blum, Ho Fo, and Parpart, Ao Ko: Prehemolytic 
stiadies of photosensitized erythx-oeytes . Jo Cello CoHip,, 
Physiol.; 1959' (This paper has been scheduled for the 
Atigust issue of this journal, which has not yet appeared.] 

12 o Bltaa, H. F.: Environmental radiation and cancer. Science <, 
(This paper should appear before the end of this year.) 



Honors and Awaitis relating to this project: 

SeiEinars and Syzaposia 

Apill 7 Seiainar "Ultraviolet Light and- Cancer,.," Departsient 
of Biology^ Rutgers University. 

April 15 Lecture "Quantitative Aspects of Growth and Origin 
of Cancer Induced by Ultraviolet Light," at the 
axmual meeting of the American Society for 
Pharmacology aiai Esqserimental Therapeutics. 
Atlantic City. 

April 16 Sesainar "Back of Natural Selection," Swartfcmore 
University. 

loveaaber I7 Sigmimd Pollitzer Lecture "Carcinogenesis by 

Ultraviolet Light; Eacperiment and Interpretation^" 
le^y York University Medical School. 

December 2 Paper "ExperiiEeatal Photobiology, with Special 
Reference to Studies on Skin," at Symposivaii on 
Sunlight and the Skin under auspices of the 
CoEsaittee on Cosmetics of the American Medical 
Association, Clinical Meeting, Dallas, Texas. 
I also appeared in a television broadcast in 
connection x^th this aieeting. 



1046 



serial Noo 1^1-918 

Laboratory of Physiology 
Office of the Chief 
Bethesda^ Mio 



IMividual Project Report 

Caleislar Year 1959 



Part Ao 



Project Title: Research Developaent Projects in th© Field of 
Electronics 

Principal Investigator: Hathan Oof fey 

Man Year® (celeMar year 1959) : 
Total: i 
Professional: 1 
Other: 

Project Description: 

Ob^iectiveg ; To design new electronic equipment as the needs of the 

LaJsoratory arise; to laodify existing electronic equisaaent to asset 
the changes reqviired in an esqoeriment; and to supervise the re- 
search development projects in the field of electronics. 

Major Findings : 

(1) Modification of recording spectrophotometer to permit fluoro- 
scopic deterainations to be sisdeo (Dr. Juries C. Reid) 

(2) Consulted with investigator end maaufactwrer of transducers 
«sed in gas analysis cells in metsholism studies in effort to 
further ia^iTsve operating characteristics . Mditional tr^3,sdueers 
producing an opposite reaction are being checked in an effort to 
increase available sensitivity through an additive effect » (Drc 
Ao Wo Pratt) 

(3) Adiabatic cage previously constructed further refined to in- 
sure long periods of unattended operation with incx-eased safety 

to equijaaent and escperiBJental anisials. (Dr.. A.. W* Pratt) 



10147 



- 2 = Serial NOo KOI-918 

(h) Farth®r saodifications to cosmercial analog-to -digital 
convgrter-scsaner-printer device to provide compatibility with 
ad-planch device of another sa^iufacturer to provide coMEon- 
langiiage link to available electronic computer., (Dr. Ao Wo Pratt) 

(5) Provided tsagjorary ffil®ctronic circuitry to ©ak® possible a 
detaraiination that greatgr magnification and resolution was pos- 
sible with thg electron saicroscope than was provided by the 
asanufacturer, and that such peraaanent rsodifications were desirable. 

(Bto H, Kahler) 

(6) Coaglat®d d@velopB§nt and constructs electx-onic circuitry 
to insur® flexible control, long-tcisi stability aiid aaonitoring of 
input power to ultx-a-violet generators in P V gexmieidal device^ 
(Dro Mo sakiisd) 

(7) Consulted with investigator concemiiTg feasibility of 
el®ctix>nic GO\mting of colonies on glass dishes and exaEJined 
eq,uipiaent perforssing similar functions at other govemssent in^ 
stellation. Recosssgnded certain electro -sBschsnical aids to in- 
crease speed and reduce operator fatigue using existing optical 
lasthod. These recosaiendations now being iE^lessnted. (Dro Mo 
Elkind) 

(8) Presently consulting with investigator and instrusaent maker 
on equipment to be used in studying behavior patterns and condi- 
tioning of experiEsatal anijaals when exposed to aiidible and shock 
stiMali* Ho GODstruction under way at this tijae^ (Dro Fo Smith) 

(§) Presently conferring with investigator concerning electronic 
circuitry for electrophoresis apparatiis. Certain circuit arrange- 
raent appear to be desirable to reduce cost and increase versatility 
of the equipasnt. No construction is yet under way. (Dro Fo Smith) 

Signifjgenee to the progr^ of the Cancer Institute ; The ability to 
have these refineissats to electronic devices Bakes it ^ssible to 
saske asetabolic analyses on norssel said tuinor-bearing anisals 
which heretofore were made indirectly. 

Fro: p3sed sourse of project ; To develop new aM finer adaptations for 
the messuresaent of laetabolic changes; for exasjple^ devgloptasnt of 
autoa^tic coiaputation of the large voluise of available data. This 
will i-equire the construction of new gas -sensing elements having 
propar electrical characteristics compatible with the opsx-ating 
requiresaients of existing analog computers. 

Part B included Ho 



1048 



Serial Ho. I'IGI-900 

Laboratory of Physiologj'- 
Cancer Phji-siology Section 
Bethesda, Mdo 



PHS-NIH 

Individual Project Report 

Calendar Year 1959 



Part Ao 



Project Title: Studies in Txaaor-Host Relationships 

Principal Investigator: Julius White 

Other Investigators: Florence Kate Millar and Jane Hicolet Toai 

Man Years 

Total : 8 

Professional : 5 

Other : 5 

Project Description: < 

Objectives : To continue the study of the growth-stimulating factor (s) 
present in tumor tissue: A) To define the role of sodium as a 
dietary factor responsible for weight changes in tumor-bearing 
rats. B) To observe the influence of tumor tissue dietary facto, s 
on the changes in nitrogen utilization and the changes in electrolyte 
metabolism which occur during tumor growth. 

Methods Siaployed : Sprague-Dawley rats bearing either the Walker 256 
tumor subcutaneously or the ISirphy-Sttirm lynrphosarcoma intra- 
muscularly were maintained in metabolism cages on any one of the 
following diets: (a) a 20 percent casein control diet (C-20) 
adequate for optimal growth of normal rats; (b) a diet with 
lyophilized Walker tvmor tissue replacing casein as a nitrogen 
source (T-2U) (the sodium level of this diet was about 5 times 
that of the C-20 diet); (e) a 20 percent casein diet which con- 
tained either the soditca equivalence of the T-24 diet (C-20 ^ 0<.9fj 
NaCl) or twice the sodium equivalence (C-20 + I08 



Food and water intake were measured and urine and feces 
collected daily. Urinary excretion of Na, K, CI, phosphorus^ total 
nitrogen, urea, NH3, allaatoin, and creatinine were measured. A 
procedure for analysis of Na and K in solid materials (diets ^ fecez 



10^49 



- 2 - Serial Noo NCI-9OO 

dried carcasses, and tumors) was developed involving a wet diges- 
tion with HaSOi^ and HKO3 and subsequent measixrement of the Na and 
K in the digestion solution. For CI content of diets, carcasses 
and tiamors, the Schoniger catalytic coaibustion technique was used,, 
followed by Gi measurement with a Cotlove titrator. Daily nitrogen; 
sodium^, potassium^ chloride and water balances were calculated, the 
water balance being based solely on the difference between water 
intake and urinary output. In a few es^eriments tumors were re- 
moved surgically during the course of the balance study. At the 
end of the balaace study period (usually after 25-55 days of txMor 
growth), animals were sacrificed and carcasses and tumors lyophilized 
for analysis. In some cases the necrotic center of the Walker tumor 
was separated from the viable outer shell and the two portions 
analyzed separately. 

Major Findings ; The daily Na excretion of normal rats was dependent 
on the Ka content of the diet ingested. Rats bearing the Walker 
tvimor showed a drop in urinary Ufa beginning 15-20 days after 
tumor implantation even though the rats were in positive nitrogen 
balance. This drop in Na excretion occurred in rats ingesting any 
of the esqperimental diets. For example, the daily urinaiy Na of 
rats on the C-20 diet eventually fell to about 10 percent of the 
normal daily output; on diets containing more salt, urinary Ua fell 
to 50 percent of normeJ. . The drop in Ka excretion "represented an 
increased Na retention which was maintained throughout the ensuing 
period of ttmior growth. Chloride and water followed the Na pattern 
while K remaiaed relatively unaffected. 

Periodic single supplements of KaCl were given to normal 
rats on the C-20 diet and to the same rats after Walker 256 tumor 
implantation. During the normal period and the eaarly stages of 
tumor growth (prior to 15 days), 80 to 100 percent of the dose was 
excreted. Twenty-five days or more after tumor implantation, salt 
"supplementation" resulted in almost complete retention (80=97^ of 
the adiainis"bered Na was retained) <. 

The adrenals of rats bearing the Walker tumor and ingesting 
the G-20 diet showed marked hypertrophy when compared with adrenals 
from normal rats. TMs hypertrophy wsis characterized by enlarge- 
ment of the zona glomerulosa . Adrenal hypertrophy was considerably 
lessened and the zona glomerulosa reduced in tumor-bearing rats in- 
^ gesting either the T-2i8- or the C-20 + KaCl diet, presianably as a 
result of the increased level of Na in the diet. 



1050 



- 5 " Serial Koc HGI-9OO 

Preliminary balance studies with rats 'bearing the Marphy- 
Stuna lymphosarcoisia intramuscularly have been carried out to 
ascertain the roles of. necrosis and tuiaor size in relation to Ha 
retention. In rats bearing this fast-growing^ relatively non- 
necrotic tumor and ingesting the C-20 diet, the daily urinary Ha 
output fell to a minimal level after only 9 days of tujiior growth^ 
reiaained low for 5~7 dayS;, and then rose to normal levels. The 
return to normal after 7 days of this Ha e^ccretion contrasted markedly 
v/ith that of Walker tuiuor-bearing rats on the same diet. The weights 
of the lymphosarcoiaas at sacrifice were 51 "to k2 grams. Ho hyper- 
trophy of the adrenals was observed in animals bearing the Ijm^ho- 
sarcoBEa. 

The 1958 Annual Report described the gradual increase in 
allantoin excretion during tmaor growth;, the effect of diet on the 
allantoin excretion pattern of tujaor-bearing rats, and the imme- 
diate reduction in allantoin excretion which followed sur-gical 
removal of the tumor. This study was extended to include the 
effect of timior extirpation on Na excretion and allantoin excre- 
tion simultaneously. A rat bearing a Walker 2^6 tumor was fed 
the 'T-2k diet for 20 days until daily analysis showed Ha retention 
and elevated allantoin excretion. Upon removal of a ^'^-^rsxa tuiaor j 
the Ha and allantoin levels returned to noriaal within 72 hours. 
Six days after tuiaor extirpation the Na excretion began to decrease 
and the allantoin exex-etion 'to increase; a small laass was palpable 
at the origineuL site. On the ninth day an l8-graiffi tiamor was re- 
moved and again the Ha and allantoin returned to normal. 

Significance to the program of the Cancer Institute ; The study of 

electrolyte changes in both tumor and host and the correlation of 
electrolyte metabolism with nitrogen utilization should yield in- 
formation on the competition of the tusBor and the host for avail- 
able metabolites. 

Proposed Course of Project ; Studies of the growth-stimulating proper- 
ties of tumor tissue will be extended to factor (s) other than the 
already identified Ha^ with special interest in their effects on 
the changes in nitrogen utilization and changes in electrolyte 
metabolism which oceur during tximor growth. 

Contiauence of present studies will: (a) provide infoi'ms- 
tion on the role of tumor size in evoking and sustaining Ha reten- 
tion and adrenal hypertrophy, (b) enable us to ascertain whether 
the coincident Ha and allantoin excretion effects dui-ing tumor 
growth may be related, and (c) show the carcass -timor distribution 
of the Ha retained by the tumor-bearing rat. 

Part B included Yes 



1051 



Serial Noo NCI- 



PHB-KIH 

Individual Project Report 

Calendar Year 1959 



Part B . Honors, Awards, and Publications 

Publications other than abstracts from this project: 

!*E.ilar, F. K.., Brooks, R. H= , and White, J..: StSffiulation. of 

appetite and growth in the tusnor-bearing rat by a variety 
of tissues. J, Kat. Cancer Inst, ^: 685-696, 1959. 

White, Jo, Toal, J<, 1«, Mllar, W. K», and Brooks, Ro E=: 

Walker carcinosarcoiaa 256 tissue as a dietary constituent « 
IIIo Sodium as a factor in stimulation of water and food 
intake and growth in tujaor-bearing rats. (Accepted for 
publication; to appear in Jo Hat. Cancer Inst., Jan. 



1052 



Serial Ho. mi-^3 

labovaXory of Pbsrsiology 

Caaeer Physiology SeetioB 
• Bethgsda, Mo 



PiB=fOH 

Individual Project Report 

Calendar Year 1959 



Fart Ac 



Projeet Title: Iodide Coneentrating SfeehanisiB of the fhyroid Slend 

PrineipaQ. Investiga-tor s Sa2f2»«r Ho Wollmsn 

Man lears (calendar year 1959) : 
SotaL: 2/5 
Professional : 1/3 
Other: 1/5 

Frojeet Description: 

Objectives ; To learn si>out (a) the saechanism by which the thyroid glend 
asaintains a concentration of iodide eleifated above that in the bloody 
and (b) the seehanisiE by which the thyroid gland aceimulates protein =• 
bound iodine o 

Major Findings ; This year sjost of our ^ork on this project has bean 
theoretical. We have set up siSE^e models of the thyroid gland 
and obtained from thea a q\iantitative understanding of rates of 
binding of radioiodine, rates of trsmsport of radioiodide into end 
out of the thyroid 5 rates of equilibration of radioiodide between 
the thyroid gland and bloody and of the clearance curves. These 
Hsodels are an extension of earlier published models, where oi-ganie 
binding is blocked, to the case where binding is peiraittedo Agree- 
aseat between the simple siodels and escperiaental data is ramrkably 
good except in the case of data from hypophysectoHsized slice » This 
preparation requires a Eiore coiaplex model with internal heterogeneity « 

Sijgpiifisanc® to the progr^i of the Cancer Institute : This work is leading 
to an understanding of the relative iisportsnce of various fsctoi-s 
in controlling the ace\anulation of radioisotopes by tissues. 

Proposed Course of Project ; This project will be continued with soise- 
what BKJre ejBcphasis on investigation of the role of blood flow to 
the tl^i'oid in the aceuBsulation of radioiodine by the tli^x-oid gland. 

Part B included les 



1053 



- 2 - ' Serial Koo M3I-^5 



PHS-KH 

Individual Project Report 

Calendar Year 1959 



Part B o Hoffiors, Awards, and Fisblieations 

Publisations other thaa abstracts froa this project; 

Ifollffian^ So Ho, aM R®ed^ Fo So: Transport of radioiodide between 

the tiayroid gland aad blood in mice and rats. Mo Jo Pbysiol? 
1^: 113-120, 1959= 

Wolliamij, So Ho*. Coasentration mjd organic binding of radioiodid® 
by th© thyroid gland. Anno H« Yo Aeado.Seio In press. 



Honors end Awards relating to this project; 

Iisvited leetiire: "Concentration and Organi® Binding of Radioiodide 

by the Thyroid Gland o" Presented at the Sys^osiiisi 
on Modem Concepts of Thyroid Physiology hsld by 
the Kew York Acadeay of Sciences on Mov^s^er 5-7^ 
1959. 



1054 



Seriea Noo mi-^G 

Latooratoiy of Physiology 

Caneer Physiology Section 
Bethesda^ Mdo 



Individual Project Report 
(Calendar Year 1959 



Part Ao 



Project Title: Properties of Transplantable Thyroid Ttaaors 

PrineipaL laves tiga"tor: Ssyasour Ho WollBsan 

Other Investigators : Sro Edward Bo Price, IP NCI 

Dro Leonard Warren, 1ST KIAMD 
Dr» Saasuel So Spieer, IPH NIAIffi 

Cooperating Units: NCI, Laboratory of Pathology - 

Dro Bkiward Bo Priee (Serial Uoo mi-3lQ) 

HXAJS3ij Latooratoiy of Pharmasology and fosisologj'- - 

Dro Leonard Warr«n (Project Serial l?Oo not 

availeble) 
HIAMJ, Laboratory of Pathology arad Eistochsaistry 
Dro Sanuel So Spieer (Project Serial Hoo not 

available) 

Related Project: The Clinieal Endocrinology Braneh of NlAli3 is 

8t\slying sosi© of the transplantable thyroid tiaaorso 

Man fears: (ealendar year 1959) 
Total: 1 
Professional : 1/5 
Other: 2/5 

Project Description: 

0"b jestives ; To study properties of treaisplantable thyroid txjuors in the 
rat: Variety of histologic types aod their progression, correlation 
of histolo^ with biochsaistry and physiology of the tiaaors. 

Major Findings ; Some of the lines of transplantable triors of the thyroid 
gland which we have "been studying contain a good deal of intra" 
cellular periodic acid - Sehiff positive naterialo Hraaogenat@s of 



1055 



- 2 - Serial lOo Kei-^6 

these tajfflorB are very viscous and this BUggested th© presence of 
imicinso Other lines contained eosin negative colloids An ettej^t 
was therefore asade to characterize the mucins in almoBt all our lines 
of tusBors incliiding those with eosin negative colloid « Itoiy of the 
lines contained acid Mucins in the colloid as indicated by staining 
by basic dyes. Tbg aeid group was shown to be sialic acid and not 
sulfate by a variety of histochesaical procedures including the 
dependence of stainability on pH end by removal of stainability by 
a specific sialidase preparation. 




CCK)E)2-C%0H 



In rats carrying on® of the tusEors with intracellular acid mucins^ 
the sialic acid level was elevated in serum and iirine. 

Soiae of the laore funetionally active thyroid ttasors contain 
neutral aaucins as does the thyroid. In an autorsdiographie stiady it 
was obsex-ved that in a tuaor with neutral imcins all follicles 
forced organic iodine, whereas in a tumor with acid isucins in the 
colloid some follicles were non-functional- Hovever^ the relation 
of acid mucins to tigroid function is unclear as yeto 

Significance to the program of the Cancer Institute ; These tumors are 

valisable because of the variety of their easily identified histologic 
and biochemical characteristics » Physiologic studies will give in° 
sights into the correlation of structure and function. 

Proposed Oougse of Project ; Studies of the physiology of these tumors 
will be continued. 



Part B inslisded Yes 



1056 



- Serial Hoc IiCI-906 



FSS-KIH 

Individual Project Report 

Calendar Year 1959 



Part B o Honors^ Awards, aad Publications 

Honors enA A^^ards relating to this project: 

Invited Lecture: "Progression in Transplantable Thyroid Tumors 
Arising in Rats Fed Thiouracilo" Presented 
at the S^a^^siuffi on Conditioning Factors in 
Neoplasia held at the annual iseeting of the 
Asserican Association of Pathologists and 
Bacteriologists in Boston^ April 23=25, 1959. 



1057 



Serial No. NCI-9IO 

lo. Laboratory of Physiolo^ 
2o Canc(gr Physiology Section 
5o Betbgsda^ Mdo 



Individual Project Report 

Crigndar Year I959 



Part Ac 



Projeet Titls; TuBK>r Autoradiography 

Prinsipal Investigator: Jaases Co Raid 

Other Investigstor: Jiilius White 

Man fear© (calendar year 1959) ' 

Total: Hone - Cosatpleted project 

Professional : 
Other: 

Proj©et Dsscription: As stated in the last annisal report this project 
was soa^leted in 1958= It is listed again this year for the piir- 
p3ss of reporting a publication resulting from the project, Se© 

Part Bo 



Part B ineliaaled Yes 



1058 



Serial Koc 91O 

PES-KH 

Individual Projeet Report 

Cslendar Year 1959 



Fart B o Honors, AwajxLs, aM Fablicationa 

Publications other thaa abstracts frosa this protest : 

R©id, Jamgs So^ and >^M,te, Jwlius: An aatorsuiiographie study of 
tus&or dynamics o Jo ll&t. Caneer Inst, 22: 8^^5-867^ I959. 



1059 



Serial Ho. NCI -919 

Larooratory of Physiology 
Cancer Pbyeiology Seetion 
Bethesda, Md. 



PBS=fflH 

InsLividual Project Report 

Calendar Year 1959 



Part A» 



Projset Title: Investigation of Urinary Exer@tion of Nucleotides 
aM toeir Congeners in Leuk^Eia 

Prineipal Investigator: Jaiags So Reid 

Cooperating Ufeiit©: General Ifsdieine Bransh, NCI -4-719 (c) 

Man Years (ealsndar year 1959): 
Total: 2 
Professional : 2/5 
Otbsr: l-l/j 

Project Description: 

Objectives: Tbe pisrpose of this project is to detaiiaine whether therg 

is a detectabls urinary excretion of nucleotides or nucleosides, aad^ 
if Eo, to develop an analytical method for their identification and 
^xmntitative estimation. The saethod is also to be applicable to 
free lajirines and pyrisaidines. The technique will be used to inves- 
tigate the metabolism of these substances in leukemic patients ■> 

Methods Employed ; The jsethods used are prisarily chromatographic. 

^jor Findings ; As was statai in the last report^ technical difficultigs 
cropped iip when the analytical procedure developed earlier was 
applied to urine. The correction of these difficvslties has provel 
more troublesome than was ejipeeted, new probl^as tending to arise as 
old ones are dis]^sed of » Tl^se range in character from practical 
ones^ such as lyophilization losses, to chemical ones, such as the 
effect of variation of loading and washing volumes upon the sharp- 
ness of group separations in the preliminary stripping columns. 

These difficulties have gradually been resolvsi although 
xanthosine recovery is still son^what low and there remains a 
tendency to non-reproducibility in the performance of the aamonium 
acetate gradient syst^a used for the analysis of the major nucleo- 
side fraction a Both of these effects are probably related to 



lObO 



- 2 - Serial NOo IICI-919 

instability of the pH in certain regions of the gradients' It i© 
antieipated that the cause of this can be discovert and controlled, 
but so far it has stood in the way of our publishing the procedure. 
Insofar as the analysis itself is concerned, however, these items 
are not critical ones and urinary analysis has therefore been 
resjs^d with the running of a nox'mal control^ which is in progress 
at the saosent and appears to be going saoothiy. Th® eollection of 
l@«£]£@siic urines has been going on steadily and a nusber of sped- 
isens are on hand. 

With ehroaatogrsphie problesas apparently under control, 
serious consideration has b@@n devoted to the resaaining njajor 
problem to be dealt with, naiosly quantitative estimation of the 
individual ccsiponents of the mixed fi*actions obtained in the 
urinaiy chroraatogrfiuas . Although it was always reasonably certain 
that this could be aceontplished, in crude fashion at least, by paper 
chroismtography, the con^lexity encountered in the urinary patteniis 
made it apparent that this s^thod would gravely cosiplicate an 
already unwieldy procedure » 

As an alternative, the possibility of aaploying diffei-ential 
spectrophotcsastry has been investigated. It was soon found that a 
liinitgd treatasent based on the selection of n points from the ultra- 
violet spectrum of a mixture of n eoarponents does not give accept- 
able accuracy even for simple mixtures.. Application of Bsore 
powerful Bjathenatical curve-fitting techniques, however, gives such 
excellent results in preiiajinary trials that a thoroiigh test of 
the possibilities of this approach is underway. This rsqiaires the 
aid of an electronic computer. A program for the MM-'650 has been 
written by Dr. Arnold Pratt, Laboratory of Physiology, and is under 
test. 

Signifieaaee to the prograsa of the Caaser Institute ; The ultimate aim 
of thi© project is the accvmailation of information concerning dis- 
turbances of nucleic acid metabolisaa in leukemia and the aechanism 
of the therapeutic action of nueleotide-related analogues as well 
as the mechanism of their toxic side effects. 

Proposed Course of Project ; 

1} The developaent of a standardized analytical procedure based 
on diffex-ential spectroplKjtometry will be a jaajor activity for the 
iEsnediate futtu-e. 



1061 



- 5 = Serial Ho, KGI-919 



2) 1?h@ sssss of nuaerieal data being genei-ated reqiiires sosb© 
instrtsaentation for data reduction not only to save time but to 
avoid erporso ThXe, will involve digitalization and autoaatiG 
piinching of data from the Caxy spestrophotossaeter atKl autoiaatic 
saa^le handling eGuijffiento Mditionel lyophilization equipisent 
moBt also be built o 

5} Ghixsaatography of urine specimens c^i proceed while l) and 2) 
are going on end it is es^pected that a survey of tiie acute leukeiic 
group vill be achieved this year. 



Part B ineli^ed Yes 



1062 



h - Serial NOo NCI -919 



PHS-KIH 
Individual Frojeet Report 

Calendar ¥@ar 1959 



Part B ° Honors, Awards, and Bisblications 

Publications other than aSJstracts from tiiis project: 

Loo, To Lo, Michael, Mo Eo, ©arseaa, Ao^ and Reid^ Jo Co: 
6-Thiouric acid, a lagtabolite of 6"asersaptopurine< 
Jo AJHo Ghemo Soe, &; 5059-501*1, 1959» 



10G3 



Serial NOc HGI°920 

lo "" Laboratory of Pbysiologj'- 
2o Cancer Pi^ysiology Seetion 
5o Bethesda, Mo 

Individual Project Report 
Calerjdar Year 1959 



Part A= 



Projeet Title: Identifieation of Cos^und A-20 

Principal Investigator: Jeanes Co Reid 

.Man Years (ealendar year 1959) 
Total: 1-1/5 
Professional : I/5 

Other: 1 

Protest DeEsription: 

Objectives : Identification of what is believed to "be a new aiaino aeid 
fomid in r&t liver* 

Methods Employed ; Chrossatogrsphie ssethods are lised for isolation of the 
substance. The saetbods of physical aasd organic ch^nistry ar® iised 
to aecvesslate infoiaation which will penait an identification a 

Major Findings : Activities on this project have been devoted to orgenie 
ehesiistry in an attea^t to develop both a synthetic procedure for 
the ccKi^ound and degradative procedures for converting it to known 
substances o These have so far been imsuecessful but work is con- ' 
tinuingo 

Significance to the progrssa of the Cancer Institute ; The substance 
being studied is presusaably related to protein mstabolism which 
has basic iagportance in tuaor growth « 

Proposed Course of Project ; Evaluation of synthetic and degradative 
seheEfis in progress will continue » 



Part B included No 



IOG'4 



Laboratory of Physiology 
Cancer Fbyslology Section 
Bethesda, m. 



PHS-HIH 

IMividual Project Report 

Calgndar Year 1959 



Part Ao 



Protest Title: Prot©in Syntheeis and its Antagonigm in Normal and 

Oancer Ceils 

Prineipal Investigator: Marso Rabinovitz 

Mara Years (ealendar year 1959): 
To'tel: 2 
Professional : i 
Other: 1 

Proj©et Description: 

Objectives ; 

ao A eomparison of the meehanisms for the formation of protein 
in norsaal and cancer cells « 

bo An evaluation of the structural requir^jsnts for antimetabolite 
activity of amino acid analogues on the protein synthetic mechanism 
of asam^ilian cells, and an elucidation of the biochemical lesions 
produced. 

c. Extisnsion of the above two objectives to the tusaor-bearing 
mouse and rat with the view of selective inhibition of protein 
synthesis in cancer cells » 

MettKtds SEployed ; 

&« Organic synthetic techniques are disveloped for preparation 
of potential amino acid antagonists » 

bo Ineorporation of carbon-l4 labeled amino acids into protein 
of surviving cells is measured <. Such studies involve radioastive 
countiKsg techniques^ ehrossatographyj and colorissetryo 



10G5 



~ 2 - Serial NOo Nai-925 

(g^ SabseiluLar fractions (nii©l©i, BJitoehondria^ Mcrosomes^ 
soliiblg proteins} ar@ isolated by differential esatrifugation 
f rQ«n cancer and noraml cells which had b€©n treated either in 
vitro or in vivo with asaino aeid antagonists and C-1^ labslgd 
©Eiino asidso Tim kin©tiss of th© block in assino acid utilization 
by these fi-actions are evaluated.. 

do ftaaor-bearing aaiEials ar® placed on various dietary regiaens 
and the effec%of amino acid antagonism on tusaor ggoirfch and pro-= 
tsin synthesis are detejissinedo 

!-lajor FiadingfjB ; Several asaino asid anaLogues which contain a sulfur 
atoEi in place of a asethylene group of the side chain have been 
synthesized » These ineli^® ifr-thia-L-glutajaine, U-thia-D-glutamin® 
and %-th±a-L"hoiBoglutaaineo Two aew coffipotinds, 4-thiaaiethionine 
and ij-thiaisoleucine# have be@n prepared, and an iaagroved synthesis 
for l*-thialysine was developed. Several other analogues, such as 
a-amino-p-shlorobutyrie acid (valine antagonist) and OHm@tl:^lthr@onins 
(isoleueine antagonist) were synthesisedo 

W@ have previously shovn that s»thionine sulfoximine reduces 
by half the protein synthetic rate of washed Ehrlich ascites cells 
because it prevents the biosynthesis of the essential amino acld^ 
glutasainso This inhibition has now been shown to be shaz-ed by the 
nuclear and cytoplasmic protein-foraing syst^sSo 

The iBix@& disulfide of ^eth^l mercaptan and cysteine, which 
may be called 4-thiffiaethionine, inhibits protein synthesis by 
Ehrli@h ascites cells at low concentrations. This iahibition 1@ 
not prevented by isethionin® but is by glutathione c The analogue 
is non- toxic to Eiceo 

Certain aMno s^id analogues which replace their metebolite 
in the early stages of protein synthesis will later block the 
protein-synthetic mechanism. This biocheEiie^ lesion can be 
assgnified by the use of several amino acid antegonistse The 
Inhibition is only partially prevented in the presence of the 
aorresponding natural amino acids » fhe cause of this phenomenon 
is being investigated o 

A potent lysine ant^onlst in the Ehrlich as©it®s cell^ 
i|-thialysine, is well tolerated by mice on a lysine-deficient diet. 
Is Zi^ ejf^riaents iMieate that th© antagonist will interfere 
with lysine-€^^ incorporation in tuasor and liver ^ but not in 8pie©n= 



1066 



Serial No» MI =925 



Signlgjgance to the progrcan of the Cancer Instjtut® ; These 8t4aii©8 
utilize the action of asiino acid antimetabolitea as a lae^is of 
coB^paring protein anabolism in noiiaal and caneer cells » In addi- 
tion j Bt3ructure-activi% relationship for producing antagonism 
and studies on Bsgchsniam of inhibition, both in vitro and in yivo^ 
shoiaid furnish 'bajsic data of potential vm& for selective inhibition 
of cell types aM specific protein-fonaing systeais^ 

Proposed CovarBe of Project ; All aspects of this project will be con- 
tinued o 



Part B included Ye® 



1067 



- h - Serial Noo MSI =925 

PES=fflH 

Individual Project Report 

Salendar fear 1959 

Part B ° Honors, Awards, aad Publieatione 

Publications other than abstracts frcaa this project: 

Ba^iaovitz, M,s ©^ Tuve^ Ro Ko : Mtiisetsbolit® activity of 
lysine misdoguss on lysine incorporation into rat bone 
imrarow protein in vitro. Proco Sos» S3cp= Biolo Ifedo 
100: 222-224^ 1959, 



106S 



serial Noc KOI -^7 

Laberatory of Physiology 
Cancer Pbysiology Section 

Bethesda^ Mdo 

PHS-HIH 

Iijdividual. Project Report 

Calendar Year 1959 

Fart A » 

Projiset 1?itle: Sialic Aeid in tbe Siiyroid gland 

Frineipel Investigators: Seysour H. Wollman 

Dro Leonard Warren, HIAMD 

CooFsrating Unit: SIAMD^ Laboratory of Pharmaeology and toxicology • 
Bro Leonard Warren (Projeet Serial Hoo not 

available) 

Related Projeet: Dro Robert Wo Bates, Section of Sndosrinology, 

Lgfljoratory of Hutritioa md EDdocrinology^ MMS 

Man Years (ealendax year 1959) 
Total: 1 
Profession^ : l/5 
Other: 2/5 

Projeet Description: 

Ob j1@stivea ; To stuiy how the concentration of sialic acid in the tbjiroid 
gland varies with physiologic state of the gland » 

Major Findings: fhe finding that sialic acid, a nine-carbon amino sugar 
""^ derivative is a constituent of the colloid in thyroid tiasaors led us 
to exaaine 



ClEfe OTAc 



Sialic ^ ^< ciH 

ACM —^ /%-A 

WX3Q CSK)H)a-SE2pH 



1069 



" 2 ° Serial NOo KI«927 
» 
noimal tbyroid glands for Bialie acido W@ obtained a pure prepara- 
tion of tbyroglobvilin frosa Bro Edelhoch and found that it contained 
on© p®r cent sialic acido The eialis aeid concentration in the 
thyroid gland was highest in glands from hypophyseetoiisized rats^ 
less for non^ls, and least for rats fed thiouracilo The 8ia2.ie 
aeid eonceatration in the thyroid began to decrease after feeding 
thiouraeil for laare tissn 24 hours and i'eaehed a minimusa by about 
9 dayso It is not surprising that the sialie acid concentration 
was decreased since thyroglobulin is resorbed in the thyroid glands 
of tMouracil fed rats. Sine© it is believed that thyroglobulin 
is proteolysed before thyroxine is liberated^, we investigated whstjaer 
a product of proteolysis i:BSiely free sialic acid could be found in 
thg thyroid glKid. fh@ eoncentrstion of free sialic acid in the 
thyroid was lower in hypophysectoaiized rats than in normals » In 
rat® fed thiouraeil, the concentration of free sialic acid in the 
thyrcdds was highest when the concentration of total sialic acid 
was decreasing asost- rapidly (after apppoxiiaately 2 days of thiouras-^il 
feeding )o 

Sl^piifiganse to th© progr am of th@ Ganeer Institute ; Sialie acid is a 
pr^MHinent ccaiponent of ease transplantable thyroid tuasoi-So Studies 
on sialie acid in the aorsral gland were desirable to get insights 
into reasons for the presence of the sialic acid in tuaorso 

Proposed Course o f Projeet; This project will be completed after a fei; 
additional studies » 



Part 3 inclined lo 



loyn 



Serial Soo NCI -911 

Laboratory of Plays ioiogy 
Sae2%y Metabolism Section 
Bethesda, McL, 



PUS-HIH 

Individual Project Report 

Calendar Year 1959 



Part A= 



Project Title: Total Metabolism 

Principal Investigator: Ao Wo Pratt 

Other Investigators: W- Co White and Bo Eo B\?.rr 

Man Years (calendar year 1959) - 
Total: 5-2/5 
Professional : 5 
Other: 2-2/5 

Project Description: 

Objectives : The study of the total metabolism andj, particularly^ the 
total energy metabolism of normal and tumor -bearing animals.. 

Methods Eiap loyed ; The studies utilize, in addition to routine chemical 
and physical determinations, the techniques of aaalyticai respii^o- 
Hietry as developed in this laboratory (Serial No.. KGI-911, 1957 
and 1958)0 

Major Findings: Energy laet^bolissa studies measure growth of aa animal 
as aceretions of nitrogen, calories, water and change in the . 
animal's total Eass. It is necessary, therefore, to ejcpress an 
ojaisal's growth and change in body composition in terms of these 
variables. 

A systematic study of several nitrogen-calorie relations was 
initiated during the previous reporting period and has been con- 
tinued during this reporting period. VJe have detenained that the 
diy total carcass of a sacrificed animal can be described ±i\ chemxcsl 
terms by its fat and non-fat content. It vras found, too, tlmt the 
nitrogen and calorie content of these two body compartments re- 
mained constant relative to Keal and nitrogen content among individual 
animaiLs over I50 to 500 gram body weight range. It was assumed 
that a. dry, total carcass could be considered to be a mixture of 



1071 



- 2 - Serial Noo ECI-91I 

these two entities^ and tha,t the proportions of fat and non-fat in 
an individual carcass could he described by a straight-line func- 
tion on a plot of ssgffi N/gm and Kcal/gm of dry, total carcass 
=911, 1953)0 

The equation of the theoretical line was 

= -29 A Kcal/gta + 27k (l) 



As a t«st of the theoretical line, the dry total carcasses from pO 
animals having body weights ranging from 150-^DO gi-ams and also 
having been maintained on various diets were examined for nitrogen 
and calorie content. Using the principle of least squares, a 
straight line was fitted to the data points. The equation of the 
fitted line was 

mgm N/gm = -29,2 Kcal/gm + 275 (2) 

The good agreement between equations 1 and 2 is apparent." Variance 
of an animal about the theoretical line was 3.6149 (mgm u/gm}^ and 
the standard deviation was I.9 asgm N/gm. The 99^ confidence limit 
was approximately two standard deviations. Further analysis re- 
veals that the variation about the theoretical line is primarily 
aniiaal variation. In fitting the experimental line, it was as- 
sumed that the value for Kcal/gm was given and the value of agm 
U/gm was varying. Both variables, however, can vary in this system; 
therefore, an extensive bivariamt analysis is being carried out in 
an effort to establish the expected normal variation within rather 
precise limits. In this way the nitrogen-calorie relation will 
give a meaBure of gross body coEtposition and also an indication of 
change in elemental costposition of the fat or non-fat coiapartment. 
This woiald be indicated, of course, by experimental points which 
fell outside the zone of normal variation. 

With the development of this Eaathematical technique, it, ap- 
pears profitable to study the change in gross body composition and^ 
if present, change in elemental composition of the carcass of the 
tumor-bearing animal. Such experiments are now in progress. 

The available data, though insufficient, indicate that the 
carcass of the tumor-bearing aniffial undergoes severe gross coiaposi= 
tional changes during tuaaor growth but that no discernible change 
in elemental coiaposition occurs. This is a point of considerable 
importance: first, in landers "tending the general me"tabolisia of the 



1072 



- 5 - Seri^ Noo' ICI-9II 

tumor -bearing aniioal and, additionally, in the construction of a 
matheiaatical model from which one can predict, solely from the 
energy metabolism data, the tumor growth and the change in car- 
cass weight and composition. To this end, the derivation of a 
satisfactory sathematical model based on the nitrogen-calorie, 
weight change relations is our most icxportant objective. 

Meas;ireEent of the animal's total energy metabolism in- 
volves both indirect and direct calorimetric techniques. It would 
appear that we have achieved a routine operation of the direct 
calorimeter with controls that compensate for wide variations in 
the tempe L-ature of the water supply and for modei-ate variations in 
room temperature. A routine study involves four to six days of 
continuous animal observation divided into two-or three-day periods. 
The equipaent has been modified to record data on punched paper 
tape, which xfhen converted to punched cards can be processed in 
the HoIcH. computer (IBM-650) « The computer program has been 
written by our group and thus allows for the necessary flexibility 
for processing the data. 

The present experiments are designed to measure heat pro- 
duction and heat loss, thus providing a measure of calorie balance 
in the tumor-bearing animal. A prime objective is to determine 
the agreement betv/een the indirect and direct calorimetry of the 
tumor-bearing animal. At present we are beset with changing 
electrical characteristics of the carbon dioxide transducer. We 
are unable to cosg>easa-fce for these' electrical changes and have been 
forced to undertake re -instrumentation of the carbon dioxide trans- 
ducer. A considerable amotmt of progress has been made. 

During this reporting period both the routine analytical 
and isotope mass spectrometry have been consolidated under Mr. Burr. 
The majority of the work in this area relates to the routine activ- 
ities of the laboratory. However, a number of pilot research pro- 
jects have been corapleted in an effort to better utilize the 
analytical instrument in our physiological investigations. One 
such project which will be pursued further is the measure of an 
oxygen balance in hopes that an independent measure of metabolic 
water can be achieved. Additionally, Mr. Burr is collaborating 
with meiabers of the Radiation Branch in a study of the abstraction 
reactions of hydrogen atoms in radiochemical reactions of organic ccs 
pounds in aqueous solutions (NCI -697). 



1073 



Serial Noo HCI-9II 



Significance to the program of the Caaeer Institute ; Studies of the 
total energy ffietabolism offers the single precise means of de- 
teiminijig an animal's general metabolic response to disease. In- 
terpretation of the totaJ. energy metabolism data as a chejige in 
the physiologic stete of the animal will add immeasurably to the 
definition of the disease state. 

Proposed C ourse of Prcieet; To continue the evaluation of nitrogen- 
calorie relationships in relation to body composition and total 
energy metabolissE in the nonaal and tumor-bearing animal. 



Part B included 



107!4 



Serial lOo SCI -911 



PHS-HIH 

Individual Project Report 

Calendar Year 1959 



Pai't B o Honors, Awards^ and Publications 
PuMications other than abstracts from this project: 



Pratt, Ao Wo, and Putney, F.. K. : Gross body composition and total 
energy metabolism in nonnal rats. Ado Jo Physiol. 107: 660- 
666, 1959„ 



1075 



Serial Hoo I®S1-91^ 

Laboratory of Physiolo^ 
Physical Biology Seetion 
Bethesda, m.o 



PHS-llH 

IiidividMal Project Re^rt 

Calendar Year 1959 



Part Ac 



Project 2itle: In Vivo ileasur^sent of T^^r gH with tfo@ Capillary 
©las® Electrode 

Principel Investigator: Ho Kshler 

Man Years (ealendar year 1959) : 
Total: 2 
Prof essiosial : i/j 
Othsr: 1-2/5 

Protest Description: 

Objectives ; The object of this project is to deteisine the in vivo pH 
of ty^ors imder different experiment®! conditions.. 

Methods Bgigloygd ; fine-pointed glass electrodes, selected after pasB= 
ing rigorous tests ^ w©r© inserted into tis^rs in the leg Emsele 
of rats and the pE recorded for several hours = At the er^ of the 
first hour, glucose was injected intravenously or intraperitoneally^ 
and the effect of this upon the pH olsserv^o At sajtopsy^ the posi- 
tion of ©sch electrode was detexiained with respect to the gross 
anattmical features of .the tuisor? 

Major Fi Mi^s ; In contrast to previous vork in which the glucose \^S3 
injected intraperitoneally, the intravenous injection produces a 
different blood condition o ©as intraperitoneal glucose causes 
fluid to flow from the blood stream into the peritoneal cavity and 
hence causes a dessication of the extra-peritoneal body. On the 
other hand, the intravenous glucose injection causes aa increased 
blood volume which is repeatedly 8e@n to enhance the bloai volt;ase 
in tisaors. On our pE diagrams the intravenoiis injection causes & 
ffiore rapid drop in pH aad a ssor® rapid return to noi-mal levels. 



1076 



- 2 " Serial Koo Hei-91^ 



The Hovikoff Hepatosja which becomes necrotic was used to 
test the pH dixjp of neerotis versus solid tumor tissue after 
intrsvenous injection of glucose- It was fovuod that the solid 
t?aaK>r tissue gives several tisses the pH drop of the necrotis 
tisstLe» In 12 necrotie and 25 non -necrotic tuamrs the pH drops 
were 0.l6 pH aM 0<,65 pH unit®^ respectively. 

Sigaifjisa nc® to the program of the Caaeer Institute ; The pH of ^mor 
tisEiiss is essential to an understanding of ch^siesl processes in 
the tusor. 

Proposed CoHr§e of Pr ojeet; It is expected that this project will bs 

conclusi^ dtiring the next year. 



Part B inelMed lo 



1077 



Serial No« liCI-915 

Laboratory of Physiology 
PhysieaL Biology S®etion 
B©th@@da; Mio 

Iiadivldual Project Report 

Calendar Tsar 1959 

Part A o 

Proj®et Title: Gellwlar Particulatgs 

Principal Investigator: H<. KaliL@r 

Other Investigator: Bolivar J» Hoyd, Jro 

Coopssrating Units: Wig laboratory of Biology, Leukeaia Sti^diss 
S©etion - Dro L« W. Law (Serial Noo NCI-te?) 
ICI, Laboratory of Pathology - Dro Co Jo Bswe 
(Serial Ho, NCI "521a) 

lilAID^ Laboratory of Infeetious Diseases - 
Dro Wo Po Rowe (Projeet Serial NOo not available) 

Man Years (calendar yaar 1959) *. 
Total: 2-1/5 
Professional : 2/5 
Other: . 1-2/5 

Project Description: 

Objectives ; Parotid Tmaor Agent or Polyoma Vir^is — The relation between 
concentration of particles as determined by electron ndcroscope 

technics end various biological effects » 

Methods Bstployed : Partially purified virus was sedimented on to 

collodion filais nounted in ultracentrifuge cells. The eoncentra- 
tion of the virus was then determined by counting electron aicro^ 
scopic images of the virus « Dilutions were then injected into 
newborn mice for txuEor induction assays. Aliq,uots wei*a also issed, 
for cytopathogenic effects^ HA aiad HI titer. 

It is not possible to report on the resiilts of thie work fox- 
another 6 months » 



1073 



° 2 - Serial Ko» mi-Sl^ 

Ma.1or FiMiogs ; Expsrimeats started l=-i/2 years ago with Drsc Stewart 
and l^jdy vera eonelusdedo fhesg preliiainary tests were to find in 
what 0i2@ class their polyosa vinas was= Tissu© cjjlt^are fluids 
wer© centrift8g@d in density gradients # and fi-actions teken from 
the ultracentrifuge tubes were injected into newborn Esice<. At 
dgath th®s® w©r© autopaied by Dr^ Stanton for t^sor oseurr@nc©o 
The eouGlusion reached frcsa these experiEjente was that the size 
of the virus particles was consistent, within large er^or limits^ 
with the values found later from electron mcroscoj^. 

Pr®liEinary tests on Ifolongy's Lguk^Bia virus obtained 
froa sous© spleens showed that there was a siaailarity of particles 
in the aouse tuaor and the normal spleen control « Methods for 
differentiating between normal and vims particles aaist be 
utilized. 

Cell "free filtrates of Murphy °Sturai I^fi^hosareoaa extraets 
were found to induce bony growths in young rats which could be 
propagated by cell-fras extracts of the growths. It turned out 
timt •yiese effects were caused by pleuro-pneusionia-lite organias^ 
(PP.LD) which see® to grow well in certain tujaorso No evidence 
was obtained to implicate a virus in the propagation of this tmK>rc 

Signifie^aice to the progress of the Oaacer Institute ; Provides evidence 
bearing on the virus concept of sosse cancers o 



Proposed Course of Project ; Continuation of the ssaie type of work on 
other virus strains ^ as well as further detailed study of the sasig 

virus. 



Part B inelisaded Yes 



1079 



" 5 - Serial Noc NCI -915 



IMividual Frojest RejKsrt 
Calendar Year 1959 



Part Bo Honors, Awards j, sad Publisations 

Publications oth©r than abstracts frcMn this project: 

KaJaler^ H., RovSg W» Po, ULoyd, Bo J,^ and Hartley j, J« Wo: 
Elgotron Esieroscopy of eous© parotid tvamr (Folycma) 
■vlrm. Jo Ifato Cancer Insto 22: 61*7-657^ 1959. 

©aviHj, M. A.,, and Lloyd ^ Bo Jo: Knives of high siliea contgnt 
glass for thin-see tioning. J. Biophys, m^ Bioeheao 

Cytology li 507 (only), 1959« 



1080 



Serial Noo mi-9l6 

Lsbosatory of Physiology 

Physieal Biology Section 
Bethesds^ Mdo 



FHS~HIH 

Individwal Project Report 

Calendar ¥@ar 1959 



Part Ao 



Pro jest Title: Sharseterization and. Cooiparison of Husleie Aeids^ 
iKGleopiroteias, Hucleaees, aud Haeleaae Inhibitors 
of Sorsial end Malignant Tissues aiid of Body Fluids 

Prineipal Investigator: Joseph Shaek 

Mbzi Years (calendar year 1959) > 
Total: 1-1/2 

Professional : 1/2 
Other; 1 

Projeet Description: 

O^jestives; 

a) To deteisdne whether the nucleic acids and nucleoproteins of 
noraal and Esalignant tissues differ in physical, ch^aical or 
biological propei'ties . 

b} To determine the na,ture axA azsount of nucleases and nuclease 
inhibitors present in tissues end body fluids. 

c) To develop new proc®iures for the study and characterization 
of nucleic acids. 

Methi^a ^aployed; 

a) Pro<gedia37es for isolation of nucleic acids and nucleoproteins j 
conti-olled ^^iification by heat, acid^ alkali, ensyaes, etc; study 
of these by various chesaieal and physico-chanical procedures. 

b) ¥arious procedures for preparation of extracts of tissues and 
body fluids suitable for assay of enzyme or enzyme inhibitor. 



1081 



° 2 - Ssrial Noo KCI-916 

Ma.ior gisidin^s ; 

a) Fwrther investigation of the interaetion of deoxyi-iijonucleate 
and ffiagnssium ions indicated that 

1) the hindlng is essentially independent of pH in the 
rmigm where no titration of the nucleate ocgurs, aM 

2) "^ae qiaantity of % toomid is strictly proportional to 
the concentration of MA at a given fx«e J^ ion eoncen- 
trationo 

b) Trypsin destroys the Elase I inhibitor derived from hisaan white 
blood cells both in the absence and in the presence of BSese 2^ 

but does not affeet th® DKase lo Shis destructive action of trypsin 
can be prevented by the presence of soybe^i trypsin inhibitor 
The destruction of B^ase I inhibitor by a factor present in human 
serum is, by contrast ^ not prevented by this soybean inhibitor <. 
These findings have provided the basis for laethods of assaying 
DHase I inhibitor in the presence of a relative excess of DlJase I 
^sd of assaying BSiase 1 when inhibitor is present initially. Use 
of these proeediires have shown that noitnal huroaa urine, which con- 
teins large asiounts of BSase I^, sontains no soluble IMase 1 inhib- 
itorsc 

Slgnifisance to the prograai of the ganger Institute ; 'She significance 
of research on nucleic acids and nvicleoproteins in connection with 
the cancer probl^ is related to the key role of these substances 
in genetic processes, bacterial and viral transf omsations , virus 
reproduction, protein synthesis » Interest in nuclease and nuclease 
inhibitors relates to the necessity of controlling DHase aetion 
during isolation of DHA^ particularly if they are to be tested for 
biological activity., In addition, it is possible that the ^sount 
and nature of the enzys^s and enzyme inhibitors may be in sc;^ way 
related to the growth of tusBors present in the host, 

PropQged Course of Ps-oject ; 

a) Further coa^parison of nucleic acids aiKL nucleoproteins derived 
fnm noz^tal acid malignant tissues. 

b) Psurther studies of the nucleases and nuclease inhibitors of 
the bcjdy fluids of norssal hujmns, cancer ^tients, and appropriate 
es^erjjj^ntal enisals. 

In view of the rather specific association of high DI?ase 1 
inhibitor content with the white cells of blood, it is contea^lated 
that considerable attention will be given to studies of this sub- 
stance, particularly in relation to various hematological diseases. 

Fart B inelMed Yes 

1082 



5 - Serial Uoo K02-916 



Individiaal Project Report 
SaleMar Year 1959 



Part B ; Honors^ Awair^s^ and Publications 

Publieations other than abstracts fsxm this project t 

Shaek, Jo, and ByntESj, Bo S..: Detersaination of th© interaction 
of deosyrifeonuclgst© and BSgnesium ions by saeans of a 
ffigtal ion indicator. Hatur© l81^: 635-656, 1959, 

Bhe^k, Jo : Th@ influgnce of sodium end magnesiim ions on the 
action of deosyribonuclease II o Jo Biolo Sheaio 22^: 

5^5-3006, 1959, 



1083 



Serial Hoo mi-9Z6 

I^or&tory of Pijysiology 
Pi^'-sical Biology Section 
Bethesda^ Wl. 



PHS-lilH 

Individiial Project Report 

Oalendar Year 1959 



Part A= 



Project Title: Purifieation end Characterization of the HyKojaa 
Virus Transforming Agent 

Principal Investigators: Joseph Shack end 

Lawrence Kilhaia, Division of Biological 
Standards (Project Serial NOo is mst 

available) 

Men Years (calendar year 1959} : 
f o-tel : 1/2 
Professional : 1/2 
Other: 

Parojeet Bescription 

Objegtiv^s; Work during this year was eoncem^ with both transforming 
agent prepared froa layxc^sa virus a) by heat ^one (TAM) and b) by 
a so2^in^ heat-urea trealanent (URM-^AM)o A major objective was 
tci piarify these to a state svii table for stisdy by methods of chesais- 
try and electron microscopy as well as in teitas of their transforsins 
potency » 

Methods Esxsloy^ t Procedares for prei^ration of transforsaing agents 
and determination of their susceptibility to varioxis enzymes » 
Ktmeroiss jsethods used in preparation of viruses and viral products^, 
e.go differential sedimentation, genetron ti-eataent, etc. 

Major Findings : 

a) Att^^ts at substantial purification of rayzoasa virus as well as 
of transfonaing agents from extracts of txssors were without success » 
Later vork involving Eyxoma virus grown in tissue culture was, 
however, very proiaising in this respect. It was foxind that active 
transforming agents could be prepared from the tissue cultures 
as well as from extx-aets of tuaorso 



IO8I4 



- 2 - Serial NOo KOI -^6 

b) Atta^ts to isolate es. active seperated (deproteiniz^) nucleic 
aeid by "Ui© phenol proeedures wei-e unsuccessful. 

e) In addition to diffeanances in susceptibility of TAM and WEA- 
TM to deoxyribonusleas© (reported last year), signif leant differ- 
ences w@rg found in the behavior in different types of tissue 
culture. TM is effective in & variety of cultures such as 
squirrel and asonkey kidney, rat sabiyo, smd rabbit kidney 
(either dcsagstic or cottontail )» UREA-^SAM was active only in the 
kidney cultures derived from rabbits, the natural host of both 
fibroaia and B^rxoasa viruses o It ^as found that both TAM sad IIIREA- 
TM can enter the cells alone (in absence of fibrosa virus); 
however, any UREA-^SAM that enters is destioyed in a relatively 
short tiffis (15 minutes to 5 hotirs in different experiments), 
whereas TM remains undestroyed for as long as 5 dayso 

Significance to the Frograa of the Oaacer Institute : The significance 
is related to the role of viruses in cancer etiology » 

PropoE@d goMTse of Project ; This project vaa ended in Sept^sfljer 1959 
fiS a result of the teraination of Drc Kllham's virology progr^. 
It i@ Isoped that the techniques ssad approaches developed in the 
course of this project will find application in other projects 
now under consideration. 



Paxt B inelwied Tes 



1085 



Serial Ho. KSI=926 



PHS-HIH 
Iiadividual Project Report 
■Calendar fear 1959 



Part B ° Eoaors, Awards ^ aM Publieations 

Biblieations other thsa abstracts froa this project: 



•Shask^ Jog and Kilhaia^ Lo: E®latioa of ^rxoisa deosQrribonueleic 
acid (dm.) to fibrcfflia-^fxoEa virus trans forisationo Froe< 
Soe= laspo Biol. ^©d» 100: 726-729, 1959. 



1086 



1^ Radiation 3rarieh 
2o Office of tlie Ghisi 
3»* Bethesda, Md., 



Individual Projeet Report 
Calendar Year 1959 

Part kg 

Rrojeet Titles 

Development J Fabrication and Maintenance of Specialised 
Equipment for Radiation Resoareh 

Principal ]jn vest iga tor : 

Calvin Ro f-'^encken 
Other Ihvestigatorss 

Edward J, LeBrun and Ralph Co IvTiit taker 
Cooperating Units s 

None 

Man Yeass (calendar year 195?) 
Total: 3 
Prof essional J 2 
Others 1 

Project Description: 

Oajectivess To desijrn and fabricate laboratorjr instruments 
ST?fevIces that are not available on the open imarket^ and/or 
incorporate refinements to said devices to make them appli-' 
cable for the unusual services that are required in radiation 
research o To service or assist the investigators in servicing 
such equipment as the Van de Graaff ne^-s5r*ators and fabricate 
special parts for sanCj as requested bv said investip;atorso 
To service less significant equipment as requestedo The 
accompanying list of projects is condensed and does not glre 
a complete or wholly accurate description of every task Involveo 
The more significant ones v?ill be designated by an aaterick ^--)c 
If more ccssplete and accurate information is desired.;, it is 
su^fested that the investigator whose name aecompanys the iti-in 
be craisultedo 

It will be noted that a part of the list is involving Glass 
Blowing o This will be listed separately and ?Jill be designated 
as sucho 



1087 



Serial F.o, NCI»688_ 

Project %sGription: (Continued) 

Major Projects; Radiation Branch 

Dr, Hotr/ard Andrews! 

Fabricate niscellaneous parts for, and assist in repairing 

and servicing 3 Mev Van Der Graaff o 
Rebuild rotating device for 3 MeVo 
Modify TV camoa support „ 

I-lrs , Peterson : 

Design and fabricate, pivoted mouse cage to detorrine 
radiation response o 

Mrc. Jfurphy: 

Fabricate ionization probes as requestado 

Kr, Robert Svaii«s 

Design and fabricate device to motivate ionization cban:ber by 

remoted control. 
Fabricate Isodose Scanning devices 
Fabricate ionization cbarbcrs for continuous monitoring of 

2 Kev x=ray beauio 
Desirm and fabricate boring device to rrachino tbe cone of 

the 2 Mev« 
Service to 2 Hc>Vo Lifting roechanisirio 

Dr, K, Blkind: 

Build T5V radiation apparatus for cellular worko 
Fabricate fixture for maintaininn control temperature of 

mamrnalian cells during radiation,o 
Convert lantern slide projector for the purpose of exaninfjig 

fixed and stained colonies on petri disJies, 
Fabricate j^as fixture for petri dishes during phase inicroseopjr- 
Microscope narking device for irairkin.^ cones on petri dishes.-, 

Dri So Sneider: 

Design and fabricate skull clanp for radiation treatrr-ent of 

the head and necko 
Fabricate treatnent wedf^es as per specifications » 

Dr, n„ Suit: 

Development of needles for interstitial radiation thsrapf^'- 
with cobalt and irididiuis wirso 

Dro Ko Brace; 

Develop adjustable tubular ^au^es for neasuriiig treatmsnt 

distances c 
Design and fabricate r®B«»t9 h[fdraulic control for Lumeon 

csamerao. 



1083 



Project Description: (Continued) 

Di% Po Ries2s 

Fabricate holder for syringe radiation, including a 
monitoring ionization charber to be used on 2^0 KVP 
x=ray" machine <> 

Dro Maxwall: 

Kodify recorder to pull paper chropiatographs across {^as 
flox' counter at same speed ;:s chart o 

Fabricate hot air heater bath for low teinjjerature evapora= 
tion of corrosive liquids^ 

BTo Falconer Smith: 

Fabricate agpjlutination test raeko 

Design an.l fabricate automatic drain for colorimetero 

Mss II o Gump: 

Adapt dissectin:^ microscopes and investigating microscope 
to be used in technique for nakinirr vrindow transplants ^ 
Al.so associated toolinr reauired for viindoxT splintSp etc. 



Glass BlcMxngs 

^ Develop and fi'abricate special petri plates so 

desipined to minimize shadoT-^ effects in ultra violet 

experiments o 
-"- Fabricate special tissue culture tubes with 

optically flat sideso 

Dr, Elkinds 

■s- Fabricate nodified type Tc=60 tissue culture flasks o 
Fabricate glass cylinders e, 
Repairs on vacuum apparatus o 

T)r„ MaxiJell: 

Fabricate ciirornato^-raphy colunmSo 
Repairs on water still., 
Fabricate condensers. 

Dr, Falconer Smith: 

* Fabricate tubes with optical bottom for photographic study.. 

Dr^ Ilm-jard Andrevjs; 

Fabricate glass cylinders for use with 3 Mev, 

■s- Fabricate electrode assenfcly for radiation purposes o 

Dr, P„ Reisz: 

* Fabricate degassing; apparatus used in radiation experiments r 

* Fabricate quarts distillation apparatuso 
Fabricate glass discs « 



1089 



Dr, Robinson; 

Fabricate at.csTii.aea'ec 

Fabricate special type drying tubes o 

DPc Roset 

Modify Erlenreyer flasks^ containing internal well, 



Departnant of Physiology 

Dro ftrattj 

Modified thermal conductivity cell block with dual 

construction o 

Fabricate lucite hujrddity cells o 

Dr, 1"^ Ko Miliar: 

Specially constructed aluminuTr support for lyophilisero 

nr„ Mo Rabinovltz: 

Stainless steel drying racks for chromatography paper o 

Dro Kahlen 

Modifications to ultracentrifuge rotor,, 

B, Eo Burr: 

Miscellaneous adaptations and ncdifications for mass 
speetrometero 

Dro Reidi 

Glass Blowing: 

* Fabricate glassware for modified lyophiiizing apparatus 
using glas? plp8c 

Fabricate chroriatograpl^ colusnnSo 

Dro Pratt: 

Glass Blowing: 

Fabricate mononeter with tungsten electrodes o 

Repair glasswareo 

Dto Rabinovita; 

Glass Blowing: 
Modify Warburg flasks o 

Dto Whites 

Glass Blowing: 

* Fabricate lyophiiizing apparatus o 
fabricate all glass separators o 

Mr„ Burr; 

«■ Fabricate gas sampling tubes for use in mass spectrometer „ 
Fabricate nitrogen condenser for saTCe, 

* Fabricate control valve for liquid nitrogen autoinatic fills 

Dr, Kahler: 

•5?- Fabricate all glass electx 



I^rt B included Ies/™7 



lo Radiation Braneh 
2o Hade Biolo S©cto 
3o Bathesda, fflo 



FHS=4n:H 

Iadi"d.dual Project Report 

Calfindar laar 19^ 

Fwjset Titl»s 

Antibody Prediction in Irradiated Mces Effect of Chronic 
mid. Int@2^ttent Exposure to Ionising Radiation « 

Principal Investigators 

Fal©on®r Sinith 
Other In'wstigatorss 

Mari® Mo Gi^nan^ John 0?«as 
<J©op®rating Ifeitss 

Non@ 

Mssn Xesrs (eslendar ysar 1S?S?) 

Totals 1 1/3 
Professionals I/3 
Oth^s 1 

Pro j set Daseriptions 

Ob,1<9j3tivB.g8 to detensdjie ths effects of chronic gpsrnia 
radiaticn @3cposure on antilx>dy f orsiatibn and to oba«r^ 
details of 'tin9«^3os9 relationshipa in split radiation 
esposuras as ^astirod by the capacilgr of Bdee to fona 
sp@©if ie antibody;., 

M:yigd.cE Kp;i,eiye4s 1) Exposure of sic® to eob«lt®° source 
is baing arrangado At present -^1© calibration of the so^a'CS 
is tjnder way„ 2) Escpostir© of adc© to a standai^ x»ray @ovrc@ 
is being carried ono 

feJoX-Zjji^&a^ss 1) Reeo'^ssT' of iuBmnii responses after two 
doses of s»rays ^^en at -varying internals of time deiaon- 
^trata a relationship apparently cozaparabl® to obserfation® 
3md9 in s-fcudies of lethality assiciated with split x«>raj 
®3ip®iST2r0s o 2) ■JfeO.tiple, radiation exposures are cuBalativ® 
in liieir injurious effect on the iramune response in. asiee 
although the ioijurious effect is not apparent in so fsr as 
clneulating Khite blood cell eotsni is conossisdo 



Serial Noo HCI-^lia 

§imM^^§M,.^LSM^^^I®^M^' studies attesnpting to 
olaariJ*;^ tte natur® of chronic and rasltipl© radiation injury 
to pro<^ss©s of prot©in synthesis is gSKBaia to standard 
radiation tbsrapy procsdures,, 

P^I?QM4-mttrsQ_ofjggo39et? Ghronic exposure studies with 
rale© of nmrioua a^Sj tssing tha nowly developsd Co®" soure® 
sr® to l^gino S»ray espostir© studies with divided desss 
ar® Hearing co?apl©tion. Chronic e2qposur®s of splenactosniz^d 
ssdes idll be undsrtakon in order to deterraine the role of tYm 
spl©®n In antibody foiination in such preparations o 



Paris B included Yes J^ Ho ^J" 



1092 



Serial No, NC^^ 



PHS^inH 
Indi-ricfeal Project Report 
Calendar Tear 19^ 

EonoTBg Awards^ end J'u'blieationss 

FablxcatioRg othey than abstracts fyosn this pro.iet^tg 

H©im1ysin fos^^tlon in inic© following partial 5 total 
sples^eteii^ of sp2e@n transplantation. P^c. SoCo J^s 

Honors ai^ Airards s^elating to this projects 

N©a@ 



1093 



Serial Noo MCI-^g?, 
lo- Radiation Branch 
2o Rado Biol. Sect, 
3o Bethesdap Maryland 



PHS-KIH 
Indi^dual Project Report 
Calendar Tsar 195? 



Project Title I 



Aeticn of Endotoxin in Irradiated Animals? Reduced Mortality 
Increased Resistance to Infection | Advanced ffesjatopoietic 



Principal Iiwestigatori 

vailie ¥o Sinitis, Ph» D, 
Other In'sestigatorss 

Ho M« Alderman and Ruth G. Sisspson 
Cooperating Units s 

lfei,n Tsars (calendar year 1959)! 

Totals h/B 
Professionals l/$ 

Others 3/5 

Project Dsscxlption! 

Objgc^^ss To determine the effects of bacterial endotoxins ok 
tiie irradiated animal and to discoTO? the physiological changes 
responsible for, such effects, 

^,ttio^3,..^elcr7M» Endotoxins are injected before or after ir- 
radiation snd effects noted om (a) Biortalityj (b) resistance 
to ©srerimental infeetion| (e) hematoyMsietie teco^mrj, 

Kajor Findingg? In addition to earlier findings we ha-^ learned 
I^Tatr^aV^Snt^tesdn, unlike colchicine, is effective in quit® 
young snisnalSg (b) endotoxin , unlike colchicine is effeeti^ns 
in guiisa pigs and (c) unlike the action of eolehieii^ in rrdeaj 
hsmstsrs and rats^ etsJotosdn is effeeti'^ when gi-^en after, teat 
r^t before, irradiation. 



Serial Ko„ mi^S9' 

^■oject Description; (Continued) 

Significance to Cancer Research ; The advance in recovery of 
bone marroxor celluiarity following endotoxin treatirent is of 
basic Sjnportance in cancer research » ]jiduction of recovery in 
the irs^adiated jnarrow has hitherto been shox^n only following 
the injection of living cellSj as nearly as one can judge frcan 
the literature o The importance of this observation is not 
limited to possible applicability of treatment, but extends to 
the general area of cell differentiation « 

Proposed Course ^ of Rs'ojeGt ; Eff csrts are. being jnade to discover 
^aT"pi^oces^es~are~lHvorTCd in the initiation of early recovery 
in the hsjhopoistie systeir of the irradiated anistalso Sources of 
ebemical fractions of the lipopoSy saccharide are becoming available 
and an effort to define the fraction responsible for tho initiation 
of h«3nopoietie recovery iJill be inadQo We shall atteiiipt to make 
both a biological and chennical definition c 



Part B included Yes /x / Mo 



1095 



Serial No« I!CI"6?9 

Individual Project Rsport 
Calendar YmT 19$9 

Part .Bo s Haaorsj, Awards 3 and Pub Ilea tio?is 

Publicatisis other than abstracts from this project; 

Sxnlth, VJ^ Waj MarstoHj, Ro lo anc3 Comfieldg Jeg F^ttes^s of 
Haaopoiettc Recovery in Irradiated MicOo Accepted for publicatirei 
in Blood o 

3hvited to present sope of these findings at the VII International 
Gonf^ress for ^'Hcrobiologjr held in Stockholittj, Sweden in August of 

Gfl Brecherg W^ \J^ Saiith and E, Po CriXTlcit®^ Strahlenschutz 
dureh Qranul02ybeu9 Chapter in " Phgrsiologie iJnd Fhysiojmthologie 
der ¥eissen Blutsellsn" Edited by Ho Braunsteiner^ Ked« Uni'S', 
IClinik in Wierio Georp Thieine Verlag^ Stuttgart o 

Honors and Awards relating to this project; 

None 



1096 



Serial No« NCI^71 

lo Radiation Branch 
2o Hade Biol, Seeto 
3o Bethesdaj Md^ 



Ihdividual Project Report 
Calendar Year 1559 

Pairt Ac 

I¥oject Title; 

Protective Effect of a Colchicine ard Related Compounds 
in Aniinals Exposed to X^Radiation 

Rrincijml Investigators 

Willie V. Sjnithp PhoDo 
Other Investigators: 

Ho Mo Aldersjan and Ruth Go Simpson 
Cooperating Units s 

Kcne 



Man Tears (calendar year 19^9)! 
To-bal; :k/§ 
Professioral: l/5 



rS-ojest description: 

Objectives; To determine the effects of colchicine and sone of 
its derivatives on survival and hemopoiesis in irradiated aniiralSo 

Methods Employed; The material to b© tested is injected at th® 
desired tiines in' relation to irradiati<m and studies are made on 
bone marroHj circulating cells and survival»td.th particular concsni 
for any evidence of alteration of marrow soisitivlty to radiation o 

Ifejoy Findiii^s; Mortality is significantly reduced V7hen these 
compounds are administered before (but not after) irifadiationo 
Ikjor difforencas in response are related to age, species and 
time of adrcinistjrationo Bcxie marrov: recovers its cellularitj 
and peripheral lymphocytes, granulocjrtes, platelets and hemo--- 
globin CGsicentration recover earlier under the conditions which 
produce an Increase in survival o The observation that very young 
anirals do not respond favorably to this treatment loay be a clu© 
to the xneehanisiR involvado 

!5if?iifieance to Cancer Reg^gghs Any findings which are, eoneemed 
9HS^ensitf^ty~t'o "rad'iatlSrand recovei-y frosi its daaiaging 
effects are of interest in cancer research and this project 
qualifies under both categorieso 1097 



Serial I'Oo !:CI^71 

T-1'Oject Descriptiwi: (Ccsitinued) 

■ apposed Co^arse of rojeet ; This and related problems dealing 
Kxth~reeOTS5ry of " hemopoiesis x-;ill be studied mth the view of 
elucidatinp mechanisms of action o Techniques viill include th© 
uptake of tritiated thyridine and special cytolo^f^ical preparations , 
Much of this material is ready to be written up for publication,, 



1093 



Ssrial Noo KGI«671 



FES-HIH 
Individual Project Report 
Calendar Year 1959 



fert go ; IlonorSg Av^arda, an:l Publicaticais 

Publications orfcher than abstracts froai this projsct; 

'Sirdthj Wo y. p i^otective Effect of a Colchicine Derivati\'e 
in Mice Exposed to X-radiation, Sciences 12? 9 310-31(1 s 1558 o 

Smithy V/o l.'o and Cornfield^ Jo 3 Extending the Range of Dose= • 
Effect Curves for Irradiated. Mic©j Science, 123, h73^7hg 1958^ 

Smith, I-Jo W,, Induced Recotrery of Blood^Fomdng Tissue in 
Irradiated AninalSo Presented at the Seccmd 3Jitematiorial 
Gonferoiee on the" Peaceful Uses of Atondc Energy ^ Geneva^ 
Switsorlandp 1958o 

Honors and A-^jards relating to- this project: 

Fon® 



1099 



Serial Noo .ES^fi^^ 
lo Radiation Branch 
2o Rado Biol.. S®eto 

- 3o Ba-thesdaj Mo 



PHS-«IH 

Indiiddual Projaet Report 

C^^xsJar Tear 19S? 

f^jset Title: 

Synthesis of Anti"body in C^H Biiea leaping the Transpl^tabl® 
FlsLsm. Cbll TssTOS-j X-5l563<. 

Priiicipal In^stigator: 

Felsoasr Ssdih 
OtJisr Ia"?ieBti^tors2 

l&sde Mo Grsnanj John Oi^ns 
Cecj^rating Units? 



¥^ci Years (calendar ysar 1959) 

Total: 1 a/3 
P^feseioRsls l/3 

Otiiars 1 

Pi?o;}®ct Descriptions 

QM^cti^a.; Characterisation of fee isEsune Btechaniem in SEies 
bearing ths transplsntad plasma cell ttitaora and to datsraln© 
eosabined ®f fectis of ladiation and specific antibody on %msiv 
g^wth and dairelopmnto 

Lo^gdj 1) Ssrums of mice iuBMniaad both with 
, sheep erythrocytes) and soluble (boirlsie eertam 
albozain) ssoasa time after tumor tyaagplant are compared tiith 
thsir non^tiaiBor bearing eonteols as to specific antibody 
content o 2) Serum titers are sstimated ty seaeitiife iflsmmo^ 
chsaiical tssts involving either aggliitination of tanned 
sensitised erytl^o^tes or their hemolytic powero 3) Passi-^ly 
tr^isferred antitedy ia studied In tojjjor bearing ini<^ to eompax-® 
Its half life with that in control laicee 

|^^^_^jid^^! 1) Capacity to form antibody both to partieulsts 
and solnbi© anti^n can bs eors®Xated with sia© {vsl^t) of tte 
tuiBoro 2) thie eosrelation does not est@nd to thg eseondoxj r®- 
spons®5 tisis a wall defined sseondary iEsmjns response g Kay b® 
sliei^d in mica bearing tusiors of 5 ^ems *®i^t or nK>reo 3) 
Failxtr-8 to detect antibody in mice bearing X-$563 tumors is not dx 



_. Serial Noo JSr^l 

^^SS-MsMMm' (Continued) 

heicodilution sinee blood -voltairas with tunrars over 5 grams 
a'rorage only about l/3 more than their controls^ U) No 
@irid©nee was found for possible sesoun componsnts which would 
interfere with antibody determination « $) Half life of pas» 
si'vsly transferred antibody is about the sams in tumor 
bearing Hiee as in their controls j, henca antibody,, if fonaed^ 
should b® detectable, 6) 2pCX3 r *to»r dos® in a single 
83tposurs caused regression of a tumor » 

Sigjiiff ieance, to cancar research? Ba^relepB^nt of a si^ificant 
specific antibody useful in treating the tuasor as an adjunct ol 
radiation therapy is predicted on a thorough under atajaJing of 
ths nature of protein eynthesis in tiie presence of this tuasor^ 

Frp^sed couyse.^,of the laro.ie^cts Ds"^lopiasnt of a specific 
antibody to the tuEior will be attempted as an adjunct to 
r^3^t io n therapy » 



Part 3 iaeluded Teo £^ 



1101 



Serial NOo NCX;^76_ 

2o Eado Hole Sect. 
* 3o BsthesdSs Mdo 

Individjaal Project Report 
Calendar? Yea? 195? 



Pagt,A.3 

Project Titles 

An inTestd^gation of th® Relationship between Cell Di-risl^ 
aM Irradiation Response to X=>rays and Ultyarlolet in least 

Principal Invest^ator s 

r-fortimsp lU ELkindg PhoDo 

Othsr Investi^atcjrs s 



Coopos'atiRg Units s 

Nona 

Ifen JeasB ( calendar year 19^9): 
Totals 
i^ofessional: 
Others Q 

JVoJect Descriptions 

Cbjectivess Radiation injury, as evidenced by the loisa (£ 
seS^reprodsicti'siity or the production of vlabla mutents<, is 
conceptually associated vdth discrete sitae » In yeasty 
conflicting imports exist with regard to th© anatomical 
location of tlise sites <> While most evidence favors ■&© 
nucleus as the region of priaiary x*ray damage , RNfi. containing 
cytoplasmic particxilates have been considered to be tl^ 
priraary W sites «, Th® latter view^ in partie^^lar^ had b&en 
s-^engthened ty the apparent absence of a W insenaitive 
"tail" on previously reported UV survival ctirveso fh® x-raj 
survival ciEcves of haploid through tetraploid yaasts have a 
radioinsensitlve tail ^ich has been shown to be do® to ths 
dividing Hioiety in th© popaLatioiio 

The objeet of this research was to landertaJse a stmt^ 
of tfes W raspoase of a mixed population of yeast cells 
(dividing and non-dividing) in order to exajnine in detail 
tb® qussidon of the presence or absence of a W ins©Rsiti\^s 

1102 



S-arial Moo mi^676 

Fz-o^GQ'b Csscriptloas ( Continued) 

!Mtod m_^.pl ^gdi The methods eraployBd cor»isted of the 
production and dstailed analysis of W and x^ray survival 
eiarves^. and the effects on the preceding of various per- 
mutations of Xu.raySj, OTy and visible light (photoraaetivatioa} i, 
Sis cell lines tased were a pair of respiring strains (ha.pl<^d 
and diploid) plus a non-respiring haploid subline o Both 
liaploid lines can be lethally effested by expostire to visibl© lights 
See repcH't No^ HCI^36o 

|^og_ Kiniany^ 8 a) In contrast to W sttrtdval ciarves publiahsd 
^ otSrss we foaai that carefully perfonasd eaperimente revealed 
that th© UV cimreg as well as the X-^Tsy cwrven is corapound In 
nature o Although in the case of UV^ the inflection in th© 
stjnsival cisrve is hardly as pronounced as in the ease of X^s^ys 
neverthalGss \j» shewed that the inflection is real and results 
fx'mi ths fact that cells in division are less sensitive to U? as 
^m:ll as to X"srayiSo b) Through the derivation of a mathematical 
tfcaorsm^ w© sheared that the reduced UV sesisitivity of dividing 
s@lls aiiast result from a basic change (s) in the D? sensitive site 
structure of the cello This ageees with conclusions reached by 
Olivers in connection with the sit® structure sensitive to X^rayso 

c) Ths ©sd-sterK© of differential sensitivities to W exposiire in 
a mixed poptO^ition was also demonstrated in connection with 
mutation production and photoreactivation of U? leiiialilyo' Aud 

d) wiil© quantitative differences were in evidence^ the results 
obtained were general and independent of the ploidyg respiration 
ccaipetsnce^ and visible li^t sensitivity of iHcie cell lines tisedo 

fica nce to Ca ncer Research s A knowledge of tii© sites and 

?F^tion of""radi£ "" 



"oF^ction of radiation at^tiis cellular level is necessary 
fc*" a detailed understanding of radiation effects o These latter 
eff estSc, when uiBierstood, may expedite th© use of radiation as a 
tool in biol(^ical research in general » More specif ically-g thslr 
understanding will play a role in explaining and controlling th© 
carcinogenic and carcinocidal action of radiatlono 

Proposed Course^ of ^Pro;)lect; It is prqjosed to extend thesa 
' Ings to an examination of ths extent of overlap of sites 



sensitive to UV and X»rays through the use of additivity exper- 
iments o 



Part B included leajg? Ho/^ 



1103 



Sarial Hoc NGI-676 



HiS-NIH 

3^K3i"^dual Project Report 

Calendar Year 1959 



Part B; Honors g As^asH^^, and PubHeationa 

Publications otbar than abstracts from this presets 

lakindj, Ho Mo and Suttonj, H^, The ItelationsMp between Di^sioa 

and X-Ray Sensitivity j, Ultraviolot Sd^sitiTilg^, and Photoreacti^atioa 

3sx leasts Badiatlon Research, 10^ 283-295 » 1959« 

Honsps and Awards relating to this project: 



1104 



PHSc^JJIH 

Sidividual Project Raport 

Calendar Year 1959 



Serial Ho. KCI-681 

lo Iladiation"^an^ 
2o Redo Blol/Secto 
,3o Betheada^ Mdc 



Project Titles 

Applications of Scsne TLssas Oultixr® TeGhrdqaes to Eadlatioa Hasaar 
Principal laves tigators 

, Robert E„ Bases ^ Mo Do 
Other Iwestigatcarss 

Hon© 
Goj^ex-atiug IMta: 

None 

fen Tears (calendar year 1959 )s 
Totals 1 
Professionals 1 
Others 

Project Inscriptions 

^ jeetiyes s Exploiting single cell techniques previoissly 
deTOiop8d°to detect materials capable of altering the 
radiation response of manBaalian cells o 

^ ^thodg anp loyed; Single cell techniques modified af t©r 
"Kiek'w^pe'appliedo HeLa cells "vjere i^ed in the aasayso 

rfajy Ein ^ ^ ss l) Actinczuycin D enhanced the killing affects 
ofXraJs^OT'^single cells exposed to the drug just before or 
after irradiation and during clone f t2?matioao 

2} Cells irradiated in medium having 85^ of iSie -water replaced 
by DgO were made more sensitive o Exposure to such meditss aloae 
sterilized normal cells upon prc^ressive exposure o Survivors 
of DgO asposare were abno[E.'raally sensitive to sulseqLieat irradiation, 

3) Ifeterials tested which failed to modify tl^ radistloa rs^poBse 
of mammalian cells ^?es ea^alase^ riboaacleasoj, spexmineo 

h) ^s mechanism of action to actdnomycin D is unknowns Thas® 
f indii^s haire been inecaporated in a paj^r si&mitted to Qma.@T 



Ps'sjeot .Deseriptions(Oontirru0d) Serial Hoo MCI^^Sl 

Signific ^^g to Cancer Research s Aetinasnjrsia D appeals to deserve 
eriSe'Sr®^l'uation°"as!' "'"aii " adjunt to radiation therapy in htsaan 
eaacer tl^rapjc 

Pg0;3OSQd ,f^;agS9 ^of^^ Projects 1} It is suggested that actincjnyein D 
^ ©vaEateTin 'ahimai or7wid human csneer chanotherapy as an 
adjunct to radiation ■Ktsrapgr., 

2) Tim effect of actinomycin D and oiiiBr Bsaterials on the post= 
.Irradiation status of cells deserves fm-ther studSTo This may lead 
to an tjncterstanding of repair mechanissjs in the p<^t irradiation 
stat'os of calls o 



Part B inelu^&d les ^ Ko £^ 



1106 



Serial Koo KCI^8l 



IiKiividual Project Eeporfc 
Calendar Year 1959 



Part Bg Honors 5 Awards, aad Publications 

Publications otter than abstracts from this project g 

Bases J Ro E^j Some Applieatiotss of Tissu© Cvslture 
Jfethods to Radiation Hesearchj. Canoes' Raseas'chp 
.^ 311«3l5p 1959. 

Honors and Awards relating to this project? 

Ion® 



1107 



PHS-MIH 

IndiTidaal Project Report 

CalerKiar loar 19^9 



Serial Mo, ^Z;;68g___ 
lo Hadlatioii Braiish 
2o Bado Biolo Secto 
3o fethssdaj fMo 



'•% Ae 



-reject Titles 

Sit^s of Action of Lethal Irs-adiation in BLviding least Cells 



Mjrtijnsr Mo Elldndj Ph,Do 
Other InTOstigatos^s J 



Cooperatiiig Units s 

ifen® 

Ban Years (calendar yeas* 19^9)? 
Totals 
Professionals 
Otfesrs 

Frojeot Descriptions 

Qb jectJ^@3 1 Ikving foiandj as described in r®p<s't Koo HCI«^76<, 
that ths gersral characteristics of both tis W and X^-ray 
siir^ival cm^es of yeast are eorsistent with tl® presenc© ef' 
overlap bst^esn the sites sensifci-?® to both t^ss of irradi^-feioaj, 
^1 exaiaination of the existence and degree of this possibiB 
cTOBrlap "Efas tmdertakano This exaadnatioa was lindtsd to t he 
dividing esll laoiet^ of a laised popi^ation primarily bseaiisa 
of the insensitive response of such cells^. parfeicislarly to 
X=.rayBg is ancsaalo'uso This anosnaly has been niainly responsible 
for tte vie*j that W and X^ray sites are anotoraically isr at 
least fimetionally separated in dividing cells » Of concerns 
are qtaestions of first order in cellular radiobiolcgyi nasaslj 
where and what ere tls priiaary sites of le-feal irradiation 
damage in living cells o 

fethg^s B np lgged s The basic method in-^olTed corsisted of %hB 
'Sie"^ eoSpEng^or additiirity esperimeats in ^Mch different 
Gcanbinations and psiiantatimis of U?^ X-ray^ and visible light 
@3q)osi3res (for photoreactivation and photolethaHty) vere 
8ii5)lojedo Th@ under Ijdng idea si&mned from tiB larg® nsafcer 
of instances in which it has been found that U7 i2$h w.i Hg lis®} 
effects wM.ch can be photcs'etrersed are nuclear and harm mjcleie 
acid (M) action spBctrs. .. Hsnse OT and I-ray effeots xsfeioh 

1108 



Ssrial Noo NGI^82 

Ps'oject Inscriptions (Gohtinusd) 

are eoapled and wliich can he uncoupled b^ x-isible light 
in all liklihood imralva ssueJsar Ni as the sites 
ecsnraon to both irradiations « To help insure that the 
ctmclusions reached T3©re ganeral^ three cell lines w^e 
lasedo Thsse were fjenstieally related diploid and haploid 
ISxiBSg both respiring p and a n(m»r®splring haploid sublte 
of tbs preceding o Both haploid lines were shown to be 
able to" be lethally effected by expostsre to visible lights 
(Sss report No« NCI=636) 

a) In addition to -fch© natheiaatical proof 
in repeat Noe NCI«6765 it was shown experimsntally 
tl^t the reduced W sensiti-rity of dividing cells was not 
artif actual in nature ^ That is, it did not result i, feg? 
irstancej from a 3*irg6r amouEt of specific but non<-active 
absorption c£ tte 251i m. line di® to the possible presence 
of la^-e M in dividtog cells o b) It was shcssfn that tlsrs is 
a large neaaire of overlap in sites sensitive to both W 
and X«i"a^ and that the coupling prodiiced by pr©=»UV espcsus^s 
could be tindone concomitant tJ® photoreversal of lethalily 
produced by ti® OTo c) It was dicmi tlmt it was quite imllks] 
that there eslst two (csr mare) sets of sensitive sites in a 
dividing yeast c®ll| one set responsible for lethalityj and 
tlm other responsible for coupling id.th X=3>ay sites <> d) 
Photolethal effects ^ which were showi to be due to the 
de'?'al©|3Renfc of a hemqprotein^like chraaophor© (report- Noc 
IIGI'^58)g do noffc coaple with W or X-rays » Ifene©a i^ ^13. 
likelihood these effects are cytoplasmic o e) WoUb quanti- 
tative differences were observed, tl^s conclusions which 
follosfsd from these results were independsnt of the ploi^^ 
respiration congietencej and visible light sensitivity of the 
cell lines ijsed* And f ) the most consistent interpretation 
of these results is that in dividing yeast cells the sites 
s^isitive to D? and X«.rays are in appreciable aseasure nuclear 
and probably consist of chroeiosocml DM« 

Si^i^c imc® to Cancer Ee aearchs A knowledge of the sites 
am"mocb d? action of radiation at the cellular level is 
necessary for a detailed understanding of radiation effects o 
Those latter effects, when understoodi, may expedite tha us© 
of radiation as a tool in biological research in general o 
riara specifically, their understanding will play a role in 
explaiMng and controlling th© carcin<^enic and carcinocidal 
action of radiaticsvo 

gfoposed Cour se of Project s Depending c«i the availabi3Li%' 
ofe^Sptienb' and psrsonSla it w«jld be desirable to perf^a 
aeti^ spscte® on th© specific coupling effects observedc 



1109 



Serial NOo NCI»682 



Ihdi^daal Pro ject . RepOTt 
Calendar Tear 1959 



F^t B£_ Honors J, Awards j and Publications 

Publicatdons other than abstracts ftcxn this projects 

Elkinds lU lU and Suttonj, Hoj Sites of Action of Lo'toal 
Iriadiations Oe-srlap in Sites for X^Hay^ Ultraviolet^, 
Photoreactlvationj and Ultraid.olet Erotectiwi and 
Reacti\''atioa in Dividing least CslLsg Radiation Besearchj 
lO, 296»312s 195?o 

Honors and A^mrds relating to this protests 



1110 



PHS-NIH 
Indi-g-idiial Project Report 
Calendar Year 19$9 



Serial KOo NCI.^3$ 
lo Radiation Branch 
2o Rado Biolo Secto 
3o Bathssda, Md, 



Project Title; 

ThgTEidine Uptake in Hsnopoietie Tissue Induced to Recover 
Following Xi^irradiation 

rTincipal Investipators j 

milie '-0 SMtbg PhoD<, and Victor Bond , PhoD^ 
Other ihTsstif^ators 5 

Ilo Ho Aldgnifjan and Ruth G„ Simpson 

Gooperatihg Units? 

' Brookhaven Naticmal Laboratory 

?fen Years (calendar year 19^9) i 
Totals ''h/$ 
Prof Qssional : l/$ 
Othar; $/$ 

Project Description J 

CbjeetiTesj To discover processes concerned with post-« 
T^S3iat£Si recovery of blpod^^f arming tissues o 

Methods Employed; Autoradiography of hemopoietic tissue after 
^'Sea tiPonTljr^^thjnnidine to. determine the presence of cells 
capable of mitosis , 

Signific anca to Cancer Research ; General significance in field 
o^" cell proliferation. Specific interest in connect icn Trf.th 
dajnaf^e to blood-^fonnlns tissue following exposirce to radiation o 

Broposed Gourse of Project i Progress in this project has been 
"How due to pressure of other committments and technical diffi^ 
culties =^ a specific course cannot be outlined until thorough ■ 
studies of the preparaticKis on hand have be®i nade but we plan 
to proceed along the lines already indicatedo 

Part B included Yes ^ No ^ 

1111 



Ssrial Mo^ 

lo Radiation Branch 
2c Rado Biol, Sscto 
3o Bethesda. ■■do 



ras^NiH 

Individual Rrojoct Report 
Calendar Tear 19^9 

fart Ao 

Project Titles 

F-Qcoveiy of Ability to Form Antibodies in Ixzadiatad y.lce 
Pretreated with i&idotoxin or Colchiein'^ 

Principal Inrestigators : 

Willie VTo Sinith, PhoDoj Joanne Ilollcroft and Jerome Cornfield 
Other IJivostiriators: 

Ilo yo Alderman and Ruth G, Simpscm 

Cooperating Units: 

School of Public Health and Hygiene, The Johns Hopkins Univa-^tj-j 
Baltiracras Jfferyland 

Kar Years (calendar year 19^9) s 
Totals ii/$ 
Professicsial; :i/5 
Others 3/5 

Project Descriptions 

CSajeetives; In study of recovery processes it is ijnportant to 
see how various hemopoietic functions are related^ Tne 
question to be answered here is whether or not ability to 
react a^ainst^ foreipn tissue is associated with the ad'^anc© 
in recovery time of cellular eleinents as seai following 
•isologous or heterologous bone Emrrow injection or treatjKsnt 
with colchicine or endotoxin ^ 

Me thods Eteplcyed; Transplantation of homologous ascites at 
'iniiervals during recovery from irradiation in treated and 
untreated animals and ohservinc response » 

r %jor bindings ; (a) Recovery of transplantation i^aaunity to 
ascites cell's of TiBk orijrin in IAF]_ mica following just sube 
lethal i2*radiation was evicJent at a time vhieh corresponded 
fairly closely to the time of recovery of other hemic elements: 
granulocytes, erythrocytes, lymphocytes and plateletSo (b) An 
injection of is!i4ogous bon® marrow did not hasten raooYsry but 



1112 



Fs-oject Description; (Continued) ;- 

an injection of young spleen cells did hasten recovery of this 
LTiflunQ mechanismo (c) A pre^irradiation injection of bacterial 
(SYidotoxin did not significantly alter the time of recovery but 
an injection of colchicine caused a significant delay in VBCOvesy 
(d) A sionificant gain in statistical inforr-ation is achieved by 
utilizing' survival times as well as survival percentages in 
analysis of the -^atac This is achieved by a procedure which is 
b^inf? T.'orlced out by Sjnith and Cornfieldo 

Significance to Cancer Research ; Recovery processes after 
daioa'^ing radiation are ot f^oneral interest because of the 
vd-despread use of radiation in cancer thera^ro Hecovery of 
this imnuno irechanian is of particular interest for if i-r. can 
be delayed vjhile other recovery processes advance, successful 
homologous transplan<:ations mipjit be more readily achieved 
than in the tmtreatad irradiated caseo 

Rroposed Course of Project ; Additional experiments viith the 
object of delaying recovery of transplantation iai3nunity are 
coiitemplatedo 



ftirt B included Tes // ^o ZE 



1113 



2a Sad. Biol, Sact, 
3o Bethesda^ Md^ 
RIS«NIH ■' 

Individual Pro j set Repoii; 
Calendar Tear 19S9 



Fart Ao 



Project Title; 

ReccTfsry Processes and Effects of QidotcMdJi and ColchieiB© 
!nreatBisnts <m ReeoTfsry in V^y Y<mng Ar^isals 

P2=incipal Jiivestigatoars 

Willis W, Ssiith, Hi<,D. 
Oth^* In-vestigators: 

Ho Mo Aldannan and Ruth 0, Simpson 
Cooperating Units s 

Ntme 

Man Tears (calsndas* year 1909 )s Patient Days (calendar year 19^9) s 

Totals k/§ 
ProfessiGnal; 1/$ 
Others 3/$ 

Project Description; 

^jeetives? To brcaden the studios ws are inking of T®<sorery 
Itrm. radiation daamgeo 

Method? amplcyed z See related projeetas NCI==65'9s ^71 » 

Hajor Find ings s (a) We have rae®itiy found that Biabhj if not all,, 
of tile diff srehca in survivorship of weanling as ecstjared to oldc-^r 
mica caa be attributsd to a latar h^opoietic r^Qormj in th®' 
young than in the. older anisnals,.. (b) It spp^rs Uw.t jomig sie© 
(5 «ko old) are rsspsasive to endotoxin b^ not to eolchiein® 
ptretroatsRento 

Significane® t o Cancer Re sear<dit A better und®rstaiiding of %h® 
factors'l&ifluSicing dssnage 'aad ' r®soY®zy Is -of ofe^ous significasse 
in an ®r®a >*©re radiation is used as ext^sivsly as la eancef 
therapy e "Ihase probl®?.s are also of general -biological iap<5Ptancec 

Proposed Course of Project; This involves prSmrilj*-^ soaparati^® 
studies oTTiOT^bietie recc^ry under different e<mditions as 
regards, to (a) age^ (b) species and (c) typ® of tr@®ta®nto 

l^rt B included Tes ^ No/T? 

IIIH 



2o Rado BioXo Sect, 
3-. Bsthesdaj, Mo 
FHS=WIH 
Individiial J¥ojaet Report 
Calendar lear 1959 



Part Ac 



Project Titles 

The Study <^ Basic Vinjs='C@ll Interactions Using Q^iantitEt-we 
Tisstjs Culture Techniques 

Principal 3iwestigatoir8 

Royce So Ikjckart, Jro^; PheDo 

Other Imreatigatcsrs J 



Cooperating Units s 

Laboratoiy of Cell Biology^ 
NIA.ID 

Ifen Yeax^ (caleniar year 19^9 )s 
Totel? I 
Professional ? f 



Project Description: 

Objectives t To cariy out a qssantitative stu<fy of the viri-ss- 
celT^iiSionships in the phsnomenon of -virus interf erens© ., 
The study is to gain insight on thg effects of -rtrti^as oS' 
ceHSj, the role of interference on tte fonaation of Tildas 
carrier cultures ;» and to obtain the paraaieters of the int8i'= 
ferance system neeessary to carry out studies on tlie iiifera.-= 
cellular nature of the interference a 

^tfipds^ flnployed : Methods enployed hare been thos® necessaffy 
?iOT 'the^qua^^tiT© assay of ¥irus8s (publishsd| "Sons Factoi^r 
Influencing the Interaction of Western Ek^uiae Sncephalccrijelitia 
Virus and Selected Eos% Cells^ Jo. Inf , Ujs. 103s 163^ 1958) anx 
for the qualitatiire manner of handling td-saue cultxo?® cells ace 
determining their ability to form clones o 

Jfajor F inditi gss Previous work showed that Western EqsiTiB 
incephaiS^elitd^ Virtis inactivated at 37® can pro-beet cells 
from "the cytopathic effects of fully virulent haaologons rixus . 
Data obtainod suggest that cells so protected are -oaable te 
produce virus until they undergo several generations of growth 
after w'nich they become once again fully st^eeptiblso About 
50 per cent of cultures consisting of a protected populatiosi 
©f cells became virus==carrier cultures when active tItus was 

1 1 1 cr 



Project Description? (Oontinusd) 

added prior to their inversion to the sisscep'bible steteo 
Gayrior cultxjres consisted of about one per cent iafec-fced 
cells irs the presonee d protected cells= Tm supernatant 
fluid from a carrier culture contained an excess of pro- 
tective materialo These resvilts ara consistent icith Vbb 
hypothesis that the interference phencsnsnon is responsible 
fc^ ths induction of tiie virus-cairier sta-fae and its pr©^ 
longed maintenance is the result of a d^mamic eqailibriram 
betifean interference and virus jsrodaeticGo 

r icaace t o Cance r Research ; The inTsstigation is 

jd to attest" to understand th® nature of th© ehangss 

teoyght about by inactive -virtisss -which are capabl® of 
protecting cells against tte cytopathic actions of other 
viruses D An understanding of the raetcbolic altes'atiojss 
effected in cells by an infective virus and a Esjaner in 
which these can be changed to ps^ect the cells mil all«f 
a rational approach for the de-vslopnent of subst-ancsa 
effective in conbatting virus infections,, perhaps tumor 
viruses as well as cytopathic virus eso 

Proposed Course of Projects To caa'ry out espsriiaents in- 
vestaiiSing the'biochsS!^ changes caused hy infectioa 
and interfcs'encs to attempt to find if iriterfererKe is an 
intracellular biochemical inhibition of virus multlplloatioa.: 



i^art B inoludsd ^®s,^7 ^^^J 



1116 



Individual Frojaot Rsport 
. Calendar lear 19^9 

Paij^^g Honors » Awards j, and Publicationa 

Publications other than abstracts £i?om this project: 

Ro Z« Lockartj Jroj, 

The Prediction and Maintenanoe of a Virus Carrier States Thc- 

Possibls BoIq of Homologous Interference., Virolo^g in press o 

Honors and Awards relating to this project! 

Guest lactiire fca* the Tissue Cialtm'e Cc«rss sponsored hy th® 

Long Island Biological Association at Cold Spring Harbor j, !few lork.-. 



1117 



HiS-NIH 
]Didi'vldual Project Repca-t 
Calendar Year 19S9 



c'p Hado Biol. Secto 
Jo ]fetii0sdaj, Maryland 



Part Aq 

Project Titles 

Rspais' of X=ray Damaga in Cell Lines of ths Ghinss© Hajast®^ 
^wn in Cul'te'e 

Principal Investigator s 

IfertS^isr Mo SaidLnd^ Phc Do 
Other Ini^stigatoffs t 

Mone 
Cooperating l&iitss 

lifons 

Ifan Years (calendar yea? 19^9) 
Totals 2 

Ps-ofessionals 1/3 
Obhsr? 1=2/5 

Project Inscriptions 

Ob;^ectives s To determine idisthei= or txcrt sublethal d^aage 
accisiaulat^d by maraaialian cells surriTing X=.ir2=adiati«i i& 
heritable o 

|fethod^ finployedi ^ii^jriwed single cell tissue aslbm® 
tec lioiques were used fw pis-poses of s l) raaldng dense 
cell snspensiansi 2) irradiating under eonts'ollsd 
conditions! and 3) assaying for stirviring cells ^ SUfta?- 

fiT8 Kv X»rays were usedo 

Ifed^ KLn dlnasg 1) Sublethal dsauage is not transmitted to 
tte progeny ^ surviving cells « 2) After an acute ssposure 
to X^raysj surviving eells rapidly repair their da^iag® bsfor© 
their Urst division post-irradiationo 3) The kinetios of 
reeosrery displays at least one maalmura foUowed by a isinim'jSi. 
k) Repair of daatag© is sspressed by the reactiiratio;'. of 
sensitive sites inactivated by ths X=.2=ay exposn^'e, 5) Rspair 
of daraag© does not involve a tame delayo. 6} ^e syBtsmC's) 
responsible for recovery in surviving cells is not attenuated 
by repeated exposureso That is, sxE'viving cells can undergo 



1118 



Projeet inscription g (Oontinued) 

^< 
repeated cycles of dsiaage and repair o ?} '.the magiiituds 
of recfwesy as a, function of time is ttepaadent upon tUs 
reeovery raediiaao 8) Iii spit© of the disproportionate 
symhetic capcities of lag phase ceils eoa^rsd to log 
phsse ceULSf, r©eovery in both types of cells is ssseiirfcially 
tbe ssaaso 

Significance ^ to Career Jteseaychs She repair ©f stsblethal 
%=T&j dsiriage " is " a " new ' ' fi!ndSg^f considoj'able IraporfcaD^c® 
vdth regard to tte natixre and location of ths sites in a 
call ssnsitive to lethal ii'radiations Sis application of 
this findijqg to biomedical and particularly canssr reseas'ch 
ar® many and incl^ide questions r®lating to? X) radiatioa 
therapy I 2) mutogsnisisi 3) d^® rate dependence of radiatioa 
effects I k) ganeraX mafflraalian radiobiolo^j $) the cyto^- 
genstics of radiation daaage and repair | and 6) the bio« 
ch®nil3la?y of radiation dasiags arai 5?®paii?o 

Proiposed^^ Cpiirse of th®^ Pro;1eets Tha documentation osf tte 
m^if^^^Mion hM b^mcoB^lotedo A nmsb«s' of addititsml 
protests alxmd at a Bios-e general laiaiey standing of l®tbal' 
aisd recovery edufeet-s in maimsalian colls ar® planned or in 
progrssso (Sae Project Numbers mi^M^}i,6sXih)^fi$U®}s<&9lid\b^, 

In ad<K.tion5 it is of partieialar interest to ptjrsiis tb? 
aspects of these JtincS.ngs that relate to radiation thsrapyo 
This should iiwol^-© tjork oa a cellular and an an^xa.! lefelo 



Part B included les ^^ ^^7 



1119 



Serial Ho. mi-=6 91 



PHS=NIH 
Bsdii^dsiai Project Report 
Calendar leas? 1959 



Part BoS Honors i> Awards « arsi Publieations 



Publieatione other than abstracts tran ihis projects 

IQLklnde Mo Mo and Sutton, Hog X=.Ray Damage and RscOT-ery in, 
llosmlXsn Cells In Culture^ Ifetm-e, iBk^ 1293^1295 Octo 2k, 195'S 



Honors and Avards Belating to this Prpjeot : 

Invited lecttiress 

Titles "X^-Ray Danjaga and Repair in tlajBnalian Cells Gs^ami ia 

Cult'uai'e" 
lOaees Biology Divisions Oak Ridge National LaboS «^«3y 26 ^ 19^3 

Titles "XujRay Experiments 'with ^fe!^pllalian Cella" 
Place? Uayal Radiological J^f&me Labj San JVansiseo^ Calif „? 
Septo 3s 19^9 

Titl@5 "Cellular Reecwery Related to Radiation Therapy" 
Plaess Radiology Cepto^ University of Calif orrda Jfedieal Cs.nt«i 
San Si'aRciscOj, Calif o| Sapto 3* 19S9 

TitlsJ "Cellular Aspects of Radiation 'Ei^rapy'* 
PLaees li5th Annual Mastii^ of ths Radiological 3ooi©ty of 
Wcsrbh Americai Chioago, Illo« Ner^c 17^ 19$9 

Title? "Radiation Genstiss" 

Places National Reaeareh Council Cojimittes on Radiobiologyg 
itwitGd confersnsa w. radio« and laoleoislar biol^ji 
Gould House J, Ardsley^ No Yo^ Deco 3i 19$9o 



112n 



PHS^NUi 

Individual Project Hsport 

Galendes' Year 1959 



Serial NOo l^I-69lCa^^ 

2c Rado aiolo Secto 
3o Jfethesda« Md<, 



Part^ Aq 

ft'oject Titl©s 

The Dependence of LethaXity on X^ray Dos© Rat® in Chines® 
HaiTgstar Cells Grovm in Culture 

Principal Ixivestigator; 

Ife'tiKsr Mc Elkind^ Pho Do- 

Other Im^stigatcs's s 



Cooperating Units ; 



Man Tears (ealeeidar y^ar 19B9)t 
Total g 2/3 
Professional J 1/3 



Iroject Dascsiptions 

Objectives t The obser^i'U.oa tliat sis^Tlng sells t© 
^irradiation rapidly repair thsir daisage without a tic® 1,'^ 
(See Ps-ojeet Kfoo NCI-691) suggests that iBHiallty should be 
dose rate <^pendsnto SpsdLficallyj) this observation stiggests 
tlmt at reduced doss rates the killing effeet (suppression 
of -onliinited division) should 'be rsducedo 

?fethod3^ ^ lihployods Saproved single cell tissias cult lire 
^SHS^SswereTiased for pm-pc^es o£s l) makii^ dense 
cell suspensions I 2) irradiating under s<xi trolled 
caiditionsj and 3) assaying for sts^rLving cells ^ Hfty- 
five Kv X=»ray3 were usedo 

jss Siis work is in prc^esso Thus far wo ha^^ 



found that redi^e dc^s rates have a greater rather tlian 
l«s^^ killing effect on Chinese haaster cells grosm in 
cSture as indicated by the liKsan lethal doseo The dose rate 
rai^e frcm 720 rad/rain to 9 rad/min is under investigation,^ 
Frelimnary data indicats that the latter dose rats deci'ess^es 
tb@ mean lethal dose by about a factor <^ 2 compared to tbs 
fontjsro Ho^ever^ although 1ii© raeaa lethal dc^e decreasea -s-ritli 
decreasing dose rate^ the hxtness nuraber increases © 

1121 



r^oject Description! (Gontimied) 

^ gnif icance to G^CQr ^Reseagchg Bie ancsaolous natijr© cf 
'^^S0~f indi^s"^®' of significaiKje primarily as th©7 relate 
to ths infc3CTiation deriTed from Project #NCI=691o 

Proper ed Co urse of Projects This wosrk will be pia'sued in 
Grd@^ to seek an explanation of these seemingly anomolous 
results o Dae to ps-actical limitations (equijanentj persoreialg 
and to seme extent techniques) the range of dose rates which 
can be exsiiiined ijith good experimental control is liraitedc. 
!nie impcs'tance of uncbr standing these results justifies 
cceisiderabls addltioiml effarto 



Part B included "^es^y ^,^7 



1122 



Lidi-^dusl Project leport 
Calendar Year \9S9 



Xq •f-o>tiici'liXOii 'ii'f.'cXViiMi. 

2.0 Rado Biol, Sect* 
3o Bethssdas Mcaxylar 



Project Title 1 

Division Delay, Growth Rate, and Radiossnsiti"d.ty of CMnes® 
Hamster Cslle Which Stirvi*^ X-Irradiationo 

Principal Inirastigators 

Mortijnsr Ho Elkind, PhoD« 
Otiier In'^stigatorss 

Nono 
CJooperating Units s 

None 

Ifeai Years (calendar year 19 59)' 

Totals a/3 
Professional: 

Others 2/3 

Pi-oject Description; 

Obieetiveat Sxj"blethally irradiated cqIIs repair their dMsage 
rapidly (aes NCI»»691)o The purpose of this study is to detsrEiin® 
•father or not« 1) reco'^ssy is associatad ^th division in s\;'.r»- 
■yiYlng c©lls| 2) post irradiation growth rate of aur^^jig sells 
is dose depend©nt| and 3) radiation sansiti'vity of sur'wlTLng csHs 
is doss dspendantg 

^tjiods^ Eia^^py@ds In addition to thosa listed for Project NCI'.-5Fly 
th® following specific Kssthods ^r© ©saployedr 1) ^ S^^M^ ©sserTa- 
tion of th® oRsat of di^Lsion in siarrl^ng cells lasing -pfAn^ udero-^ 
scopyi 2) detemdnation of tha termination of delay and siabseqissnt 
growth rate using a. colony spreading techniqae (ratrypslnigation)§ 
and 3) Jnsasisrensnt of dslay^, growth rate, and radiation sensitivity 
post-irradiation using a no-^l 3G-ray teehnicp© arssi %t^' ■<ZTV^T±OTi of 
^sibl® colony formation^ 

,Ma.|oy. Fifidiji^a; 1) Lethally irradiated cells grow as rapidly ©s the 
controls for at least a foi* divisions depending on ths dossw Iri 
contrast to this^, sm*^viRg cells sniffer a- delay which 5-ncr®ae0s 
with tifi® does- they reeaiTGdo 2) Aftar poat-.irradiation delay ,« 
surTlving oslls grov aa rapidly as the controls.- And 3) ths 
radiation aensitivity of sur?i-s«rs after the onsst of division is 
constant and about ths sams as that of lag phase eellSo 

1123 



.Si.gni,,f igane©, tgLi?,^c.^„ , R® a@ar.qh 8 Mtotdc fedsx iix asaMnaliasi 
radiobiology has often besen iss@d as an index of sur?imIo To 
th@ extent that these observations are applicabl© to ^ ^SQ. 
situations, aiitotic irdex as a measure of ^alsili^- can bs 
Ysry misleading., Sindlarlyj the observations that gro^h 
jrat© and the radiation sensiti^t? of survi'djig cells &t® 
essentially independent of the x-ray doss to which they were 
exposed is of fitndajBsntal interest in tte basic and applied 
areas of radiobiolo|y« 

Proposed, Course of Prelects ^e quantitation of these observations 
as a function of dose in bsst perforrned with log phase cellgo In 
addition^ the division delay dependence on dose is required f02' 
(MCI<=691 (d))o For these reasons and othersj the tsefarjolo©?- of 
^o^dng Chines© hauister cells in suspension (log phase) warrants 
deirelopa^nt and will t« -^gorously pursuedo 



Part B included I@s /"^ Mo 



1121+ 



PHS-^>JIH " 

Indi-sidual Project Report 
Calendar Tear 195? 

Project Titles 

Th& xt-®nt of the Dependence of X-^ay Lethality on Chromosoj-na] 
Baitiage in Chinese HsiRster Cells Grown in Culture o 

PElneipal In'?Bstigators: 

Mortimer M. Elkind^ Pho Do 
Other In-^sti^tors: 

Charles Oreen'bila.ttp Mo D, 



Hiotoblology Section 5 Physical Biolos^ Laboratory ^ NIAl© 



Totals 2/3 
Profaasiomls I/3 
Others I/3 

Project Deaeriptions 

^JjseM^fs A8 a result of a number of radiation stydiiss ^^ith 
OTltured roamnalian cell® 5 Puck has eoneludsd that x-*yay lethality 
rsEfults from ths production of observable ehro!ao@OBial damagSc 
This conclusion^ if eorrect, is of basie iniportance and has man^- 
imnlieationSo Th@ objeeti"TO of this study is to se® to what extent 
Pudc's conclusion is justified in Ghinesa hamster cells of eloss 
to normal karyotype 

^^thods Emrloyedg Quantity ti've eytogsnstis obesr^ations of 
control and irradiated cells are made in ths context of ths 
lethality and r@co^@sy effects* d«serib©d in Projset Noso 
NCI-6915 NCI-691 (a)g MCI«691 (b)o 

tk.ip; ^ Findings s Biis work is in progrssso On® of ths nsain 
reasons for starting this study ^as ths t^covbtj observations 
described in NCI~691, The rational is ths foUo^ings 1) •sai?'- 
•vising t^lla irrsnadiately after exposure are damagsd cells c. If 
the a,€Cuimalation of chromosomal damage kills a eell, sur-^-^Xig 
cells iimediately after e3cr>osure should possess sublethal 
chroiTiosomal damage o 2) Stxr"^ving eells rapidly repair their 
sublethal daipaga before division (ss® NCI 691 ^d NCI 69I (b)» 
^erefor® unless irradiation induces a state of lability with 

1125 



r^^-;gard to ih& display of QhroKosomal dajsage^ surviving csil8 after 
reeo*s®ry and particularly their pro^r^ should not display chro5®-> 
somal dsmiag^e 3) Sur^mng eells suffer a di-vrisicn dslay dtaring 
'fM.ch (in interphasa) they repair their dajtiagSe In contrast to 
tliis^ leljhally irradiated ©ells divid® as rapidly as th® conts'olg 
for at least a few di^ilsions (s©e NGI".^l(b))o ffencsj eytc^notic 
obssrvations of a^ eells irradiated sni^t display vaxjing degjrsss 
of daina§8 depending upon %?hetl:er surviving or rion-sur'vi'd.ng eells 
happen to be observed ^jhich^ in turn, depends on ^©ther or not 
reco-very and post-irradiation dslay ha-va tsnninatedo Fksws-^rj, if 
these observations are made bsfors division dslay is oifsr., liiich 
is ssqserieneed only by survi-^ing cell©., then all aitoges obssr?©d 
will be In lathally irradiated cells <> If unlimited division fc 
thea® ceils is suppraased because of ob^rvable cjytogenstis daisg.gSj 
then all jnitoses should display chross) somal abbsrations ^nd indeed 
the frequency of these should be directly correslabXe with survifaO. 
as a function of dos®<, 

Thus far our studies p conducted with lag phase eells, have shD^^i 
that only a fraction of the mito^s observed during division de).f^^-' 
aavB ary observable chroKO somal aberrations o Others have I5, 2,, ■ 
aberrations but with frequencies too Iob to explain shap-8 of tb;. 
©xirvival «mrv® in detail,, 

Sigrdfieance to Cancer Reseayehs If furthes" work bears out our 
initial observations that lethally iiradiatsd sell® do net display- 
on a quantitati"^ basis # chroiaogoiiml damage corralatabl® Kith 
lethality, then the chromosomal hypothesis of Puek as a whole 
is in doubt o Bris w>uld not mean that x-t&js eannotps'odx'.ca at:.- 
rations or that laany such aberrations do not constitivbe lethal 
urationso Rather, this ^«-ald rsan that observable ehroswssossal 
dajBiage can be at test only on© isechanism responsible for csaXl 
deatho Clearly, subtle, optica3J.y ui^stectable chromosoi^al 
dama^ or in the limit, genie daisiaga eannot b® imled oiit on th^ 
basis of cytogenetic observations <, By the saia8 tokens b2>-5{®Y64'^ 
non-genie damage related to viability becoMes a significant 
possibility "by iKplicationo 

FroTx?3ed Course of Prg.leet; The quantitation of ttes© obs-2E'^m= 
tion 4s exfcen^ tiould be test perforsied -with single log phas® 
eells for technical- reaaonso Henss^j the teehniqaea required for 
th© production of such cells require devisl©p7®nt before th® 
cytogenetic aspects are pursued furthero 



B; included Im ^^ S© ^^ 



1126 



Seals,. , -_^;^.,_,..,,^ 

lo Radia'^on Branch 
2. Rado Biolo Secto 
3 c Bethgsdas Eai-ylaad 

Individxjsl Project Report 
Calendar Xoar 19S9 



?QXt Ac 



Pro j est Ti-aes 

Tl® Itepsac^nc® or Indej^ndsnce of Recovery in Oulturads 
Haamalian Calls SwikI^Ie® X«IrradiaM.on and Thsir Radios- 
s(Kisiti¥iiy as litigated by Ifeoig Action 

Prlaeipal Ijivssldsatoa's 

Ifortiffisr M. Elkindj Fho Do 
Ofchsr Iiw®sMgators 2 

William MbfeleTg Mo Do. 

iJoopea-'ating Units s 

Clinical Pharraacolc^y and E:q)e£lnental Therapeutics 
Sections General Ifedicim Branchy MCI 

Ifaa Tears (calendar year 1959) 
To-lieJ.s 1/3 

feofesaioaalJ (lIolLEo) 
Ofctert a,/3 

Pi'oject Dsscriptiong 

In Project No. liCl^91g rscorery ia eslXs 

ition was described^! and It was fts'thes- 
repos.'ted that reccvery waa fcmnd to ba independent of the 
physiological stat© of the cellso In generals radioseii- 
sitiirity is known to be modifiable -fela the action of <^ug&>o 
For instaiK!@s asdnoetiiylisothiuronium bronide (ABT) Is 
kamm to decrease radiosansitxTity by about 2s o This stuc^- 
is ons aimed at assajaining ths degree to whieh recor^ry ±b 
gmrmTsl ia manmalian cells o Specifically the question asfced 
is§ Do surviving calls recov'sry qualitatively and quanti- 
tatiTely to the same extent and irr©spective of tteir. prd)a-> 
bility of survival foa^ a ^"ren dos©? In th© satie cell lin@g 
thsrsforss vi® will inquire whethar or not the mitigating 
aetion of drxsgs on the probability of surviiral is reflected 
in the repair of sublethal damage in isia-'^vir^ oellSo SinsB 
the dspsndence of radl<^ensit±^ity on dnsg action is in its® If 
of comiderable interest^ a sseond objectlTe in tMs i302'k 
is to &csminQ the inflisencs on sitr^val of different drugs o 

1127 



Project iS:sc5.ap'':.3.oas (Cpntintad.,!,' 

ffethods , Sraplpyeds Inprc^ed single call .tisstss cult'sar'© 
•teSSHass were usad for purposes of s 1} making dense 
cell suspensions! 2) iFra&ating under controlled 
ccajditionsj and 3) assaying for siunriving cells o Fifty- 
five Kv X-rays were ijsedo 

fe j ^_|la diag3 s This wcjrk is in progress o Toxicity studies 
have hsBn perf orssed with AET^ S^broreouracil^ and ^^f lu©?o- 
ds<sysiridin©o Thtjs far^ only AET has been scaaiined and onXj'" 
with regard to its irxO-uence on radios ®2Siti^1ye As f&md 
in other systesis^ AET decreases tlm radios srasitivity by 
about 2Xo 

Signifieaiice to ^Cancer R esearch s !His mitigating action of 
'^^5"'^^ds°cf Ijaportance t^JiSTregard to the idsnt ideation of 
tte sites of action of 1® thai irradiation and the radiation 
response of malignant tissue « Squally jjispcirtant fs^csa. k basi® 
sxid spplied point of ¥iew is a Imcs^ledge of the p<^sibl© 
infl^jsaee of tl^ action of drugs on recovesyo For instanseg 
tf a giTen drisg iTiitigates (perhaps evai decreases) the radio-' 
sensitivity of irealignant cells it cdghfe still ha^e iaportaat 
vsQs in therapy if it also ropressss recovery o 



l^SBgjQ^JSggg.^® ,9C,,„^Q 4^S^ ' ^^ ^ planned to exaniiags tte 
'^StSn^^°°ofsiv5ralarags "KrMeh have been implicated as 
relation itdtigatir^ agents o 



Part B ineludsd Ies£^ Ifo^^gJ 



1123 



3. Hado BioLo Sect, 
3 c Seth3sda.i> M., 



PHS=.WIH 
3isdividyal Project Report 
Calendar Isar 1959 



Part Ac 



Project Titles 

A Stisdy of the Jfesponse of Hunan Cells (Hela) to X»RayB 
Principle Inre^stS^atc^s 

Rojre® Ze Lockartj JTob Pha D^ 
Other Imrestigators § 

rlOEliijner rio EUdndg Ph^ D^ 
Cobpai'ating Ifoitss 

Norn 

I&n Years (calaidar year 19$9)i 
Totals I 
Professionals § 

Others 

Project Description? 

Cb^ectivisss Eto Mo M» Elkind usir^ Chinese hamster cells has laade 
'detaiiedfanalyses of survi.val eiarg^s resulting ft-oa single and - 
fractionated doses of X^-rsySo He has established that thes© c^l?3 
have a threshold amounting to a hitases nxsssber bettfean 8 and 10 
and that ster^vors were able to repair tl^ir sufc-lethal darsgac 
The amoimt of damage siiffered or the repair >rMch oceis's is di 
related to the hitness mambero Ife'o Robert Bases^^ dwiBg vb&.&.. 
studies on Hel© c^ls noticed that one of his cell lines cbang.= f 
Sw&si a hdtness nismber of 2 to about 12 « The pm'pos® of tJiis 
inyestigation was to make a ccmparatiTe stu«^ of two lir^s of 
Hela cells from tb® same parental stock Hith hitnssg nisbars of 
two and fi'TOo Experiments were done to se© if reco^eiy ffcsi 
radiation damage was a general phenc^ssnon rather than tiniqu© for 
Chinese hamster cells and to see -what r^latioriship esdst^d befefi:; 
hitness nuajber and recoveryo 

Ifethods Qiiployedg The methods xssed were those far estebll;": 
sWi7ivaI'""b'ymeEn3 of counting clones of cells res'olting feoa th& 
grwrfeh of single cells and for irradiating cells aftsr attaclssi- ;; 
to glass petri dishes o These raethods are bofeh Bnplj dsscsribsd 
by a?o Mo Mo Slkind= 



1129 



":foJeet Bese^lirfiiojas (Gontimiad.) 

,^Gr K. ^^3|g8 g Cfeis HeLg strain" had a hitaess msS>@T vbloh 

vSisTSraJo3 to 8>ith a rae^i letimi'd^e varying batif®®^ 
21^ 2"o- aiHi 95 ffo respactivelyo la reBpfmm to a first ds^e 
of ,X«s?8ys cerresponding. to about 10 percent surd.val^ ■ o®lls 
wore capable of undergoing an amount of reeorery about eq?jal 
to th^x idtness ntssbsro- This cisans th« cells hairs regained theis? 
threshold and have the same sensitivitjo Th® othes" strain of 
Hsia had a bitsiess niMbsr of t'^o for eerveml mtsiths wiiii , a saan 
Is-^jal dose of about 230 r^ yet shaded about 5 fold racotraiyo 
Sis reason for tM^ has n^b yet b©en dstesained as the cell atS'ain 
was not stabl© idth ragard to its xadiatioa response ^id gradually 
sMftsd so that it ^as indiistinguiishabl® froa the other atraiao 

Th®s® studies tmve confimed Eir^ ELkiad'ls studies ^th tb@ 
QiiBss© hsastss* cells, and hasre ehc^n that t^ba yecopasy phencmenon 
app@a.?@ to be a general onso Indlfaticsis are that both liitness 
ms^er and sensitivity to 3Ursys ar& scasswhat ■siariable fsjcaa 
espafiissat to ^eptrim^it and at least in one c®ll line iHBtsbleo 

Si^figTO^ to_^^^Csaioeg Hese^ohg TasBQ atadtes are part of a 
p" G^aa"to'^d«t^2iie t&TelSes^and kin^ oS danage i^sh ©ells 
smff ®^ as a result of S^aray iMixlto The imderstanding of tb.e 
nisisrous pera^stsirs conta?olling tba ri^qtions of e@lls t® 
ionisiisg radiation is most in^jorfcant bo1& t^mi the fiiRdai!©ntal 
aiidi^stas<3ing of its actions irmi a biologieal point of vie^ 
aM fsm the practical applications 3ji ta?eatiaant of tumors q 

^^^o^aed^ ,!^H!f?, .P^«i^^^9^^ "^^ attempt to understand wh^ 
^^selTsfe'SrrSeiS^^fc'ed in its respoas® t© X«ra^So 



Paa?t B i^Iu&d ^©^^7 %^ 



1130 



X. Hadl„ Branch 

2. Hado Biol, Seci 

3, Bethesda, M. 




PKSc»fnH 

Individual Project Report 

Calendar Year 1959 



Studies of effects oX an antibody in relation to dciralopif^nt 
of an aseites (P«388) plasma call tumoro 

Principal Ih^CBstif^tor: 

Fal<»ner Sjtiith 

Other In"TCsti gators s 

Jofe Owens 
Cc!op®rating Units s 

None 

ifen Teare 

Totals 1/2 
Professionals 1/2 
OiJiSr? 

Project Descriptions 

Objecti3gg.s ^0 e3Jpand preliminary obsertfations on tuis^ricidal 
affects of antibody prepared against an aseites t-airior= 

_lfethqds„ emg^p^ed? Antibody prepared in rabbits against wasted 
cells frealiy laken from ''die aseites fluid of the ttssor ?^338, 
7 days after transplant is intrapsritoneally or intravenously 
injected at intervals of tins after the tm^r is intraperitonsally 
inoculated o 

Ma ,1 or, findings % Preliniinary obser-^atiors indicate a sasll effect 
of the antibody on the growth of the neoplasia^ via»o mean body 
vieight in ^^ups of ntice receiving Oo2 ml of rabbit antiseriAsi 
intraperitoneally e^ry other day from the day of inoculation. 



1131 



fcOlews a l®ss eisdldng inerease and esrsi'ml tiiss is 
ppolong®d fey atout 2 days as sajinparsd to saline treated 
contiola inoeulated with this rapidly lethal lasssoro 

Sig nificant to eaaeey, .y;^,?®^:^^.^ ■^ spseif ie antitasnof 
preparation is possible outeoBis of this sfejdy and it In 
©aEectod that ^at is learned from the properties of tfe© 
preparation under stu<^ my lead to soin© mdsrstanding ®f 
naturally produced qrtoly^i® agents » 



Psrt B inelisd^ 



I®s O' ^® ^ 



1132 



Ssrlal No. .JS^gk 



lo Radiation Branch 

2o Hadiation Physics Sec-t. 

3<. 



PHS-NIH 
ladi-^dual Project Report / 
Calendar Tear 19^9 

Part. Ap. 

Pss2j®et Title J 

Radiation Injtisy and Rspaix" 
Prineipal In^ffestlgators 

Ho L^ Andrews 
Othsr Xn'^stigaters: 

Major JaSies Shi'^ly 
Cooperating Units s 

"feltsr Heed Artay I^dical Center 

Kan Tears 2 

Totals 2/3 
Profesaionals l/3 
Othert 1/3 

?K>5ect Deserlptioas 

Ob^^^^wes Te gain insist into laaehanisms l^r •Hhich ionising 
radiations affect liring tissues <, 

,?fethod8 Ein^loy^sd; Repair processes at hi^ x»ray doses vers studied 
by doss fractionation* A second phase involves studies on swiiie^ 
animals of interest because their size apparoximals that of ftsanc 

Repair processes appear to be operative e^n at 



Part B included Yes / / No 






Serial too .lC|^i6__ 
lo Radiation Braneh 
2^ Radiation Physics Secto 
3c BethesdSj Maryland 

PHS-NIK 

Indi'^diaal P2?©ject Report 

Calendar less- 1959 

Project Titles 

Bosisietzy of Exga Energy Radiation 
Pr3.neipal liTfesti^ton 

H^. Lo Andrsx?3 
O'&ier In-eigstigators! 

Lo'uis Cs^S^TOTt 
Cooperating Units: 

Nons 
Ifen Tears I 

Hon© 

Project Deseriptions ♦ 

ObJegiAross To de-^elop insthods and instnsEJsnta for K^aaiiriDg 
'hi#s intensity pulsed s^ray teum, 

M|tthgds„En?plgx,egl£ No progress was made dxiring 19^9 dn© to laek 
of personnel B 



Part B indludsd les £^ Mo /^J 



11314 



Serial NOo NCI-^9 



io Radiation Branch 
2o Rado Physics S©ct< 
mS«N2H 3 c Bethesdap Mdo 



Sidividtjsl P!p©j8Gt Report 
Calandar Year 19^9 



Fartr Ac 



Project Titles 

Tli3 Effect of laaising Radiation on Amijio Acids 
l^incijal iRvestigaton 

Charles R„ F^:stf©llg PhoDo 
Other Investigators s 

Coop@ratii^ Units: 

Hone 

•'!lan Years s (ealendar year 19^9) 
Totals 2 
Professional s 1 
Oth©rs 1 

l¥oJ8Ct Descriptiwij 

(5)jBCti7ess The dbjeetiv© of this project is to determine 
the mechanism of the chemical rsactisxis indueed hy ionising 
radiation in aqueous solutions of axtiino acids o This is part 
of a long range program to accumulate inf ormaticai on simpl© 
gS'-stsffls of biological interest so that general principles 
Hsy be ascertained and applied to complex systems which are 
not amenable to thorough^ direct investigationo 

Methods Bnplqy[®d^i Solutic^s of amino acids are irradiated 
^ItH'T^SEIng radiation and then analysed for the products 
formsdo Since the products are ssnsitiTQ to radiation it is 
necessary to dstersdne the yield cf each product as a function 
of dose to as low a dose as possible 'and to extrapolate these 
values to obtain the initial yield of the ps'oduct at ^©ry Im 
doses before secondary reactions distert the picture o 

Onee the products of the simple watsr=®Biino acid=?adiation 
system are found the effect of the introduction of other chemicals 
into the system are studiedo Substances are sal®eted, in aceordanc© 
with the rapidly expanding concepts of radiation chessistryj ishieh 
lire thought to accsntus,t© or repress csie os* asore of the Tarieas 
inteMiediate processes by #iich radiation is beli@T®d to produe© 
its effect So 

1135 



Serial Noo ^^^9. 



■3Ct D®seription« (Goatinusd) 



Since the final ^chemical (and taltiirsate biological) affect is 
th© result of the f orjtiation of a ccasplex Kiicroscopical3y'=het9ro=' 
genous sdjcturs of excited molecules, H2C^s H and OH fre© radicala 
in discrete " tracks'' s the influence of the physical distribution 
of these active intsnnsdiates as well as possible differences in 
rslativ© abundance &nd/^ choaiieal natiare is investigated by using 
different soiK'ces of radiation » To date $0 K? x=>raySj 1^0 KV 
QlsetrcmSs and ^ Mev alpha partieles have been ussd^ 

Caie of thQ more impcsrtantj if not ths most iasportants, ^eti"r© 
intsnaadiates is the (M free radical o The role of this radical 
in the absence of th© H free radical and the ho^ of excited 
molacules in the ioiiisaticsi track is studied by chemically jS'Oo 
dijc5.ng this radical in the aQTstem being studied o To date this 
ims been acecsmplished hy the actiosi of Fer •«• and HgO^o 

fejor Findings; Th® developaent of n gas chroinat ©graphic technique 
T^^WeTSoStloxig concentration and determination of ffiicroaolar 
&moarAs of acetic acid produced frora glycine by sjnall dos@s of 
dxradistlon has mad© possible seme detailed observations on the 
msebaaisE for the production of this compound^ Initial yields of 
acetic acid as a function of glycine concentration have showi that 
its like NHj and ECHO but unlike HCOCOCH^ doss not become independent 
of concentration as doas the yield, of psroducts friss most organic 
solutes <, 

Of more significance have been the jaas? spectrograpMc ®sss?i« 
ination of th© acetic acid produced when glycine was irradiated in 
D2O0 Acccrding to the mechanism proposed hy Weeks and Garrison 

Bard Mp Weeks and I'^arron Mc Garrison^ Rad„ Research £, 2^1 (1958) b 

acetic acid is fcoaed by the reactions: 

1} H + MgGI^COOH =^ Mj + ">CI^COCH 

2) ^GHgCOO! * KHgCHgCO® «#» GH^GOOH ♦' NK^CES0(H 

In D«0 t&® corresponding roacticKis wotsld be: 

1) D -s- KD2CH2CO® =«^ MDj + oGILGOCD 

2) «>CH2C0aD -s- M)2CH2C0CD ==#» CKX0O3 ♦ !©2CHG0CD 

and the acetic acid formed (after the removal of the exchangeable D 
CHI the carboy group should be.GH^COdie Mass spectrographie analysis 
of th® acid isolated ho^jsirer has sho%m a mixture of 1/3 CHgDGOCS and 
2/3 CH-jCOOHo, Thus there maui^ at the. least^ be a second asthed for 
the fcaaticsa of acetic acid^ 

1138 



SsriaX No. mif629 

jsct Dascs'iption? (C^sitlBtsad) 

With ic/Q exohang© colissm chrOTiatog3?apty, C^^ labeled glycin©5 
and liquid seintillati<xi counting we have qualitatively ccaifinsed 
the work of Wecsks and Qazrison; as to substantial yields of aspartic- 
and diarainosueciniG acids in air free irradiaticsiSo We cfoserr© on© 
but not the SQCcsad isajor xmknrasa product reported by themo Sine® 
W8 have observed the yields of these products to be a function of 
dossj, they ^dll have to be investigated at low doses before their 
part in the entire msehanisis can be ©valBatadp And of course the 
unlcno^sm product will n@ad to be identified » 

These results show that the mechanism proposed by Weeks and 
Garrison which seemed to account so beautifully for Kost of the 
data assailable to them at that time is^ at best^ only partly 
correct sxid that ccaisiderable raore ?fork is neededo 

A fffejor effort this year has b©®i the design and procurement 
of a cobalt 60 iiTadiation facility which isiU b® put ia operation 

early next yearo 

Significance to Cancer Research i Although not directly connected 
id!tK^S'ReS°7ei3areh7"TrTs°'''^S"great interest because it seeks an 
understanding of the inschanism for th® effects of radiation which 
is used ffiTipirically as a tool in th© clinical traa'teent and 
laboratory'' study of cancero 

Proposed Course of Project: Work will eontinue in an effort to 
K31y eT^^ate"liie meeiiaKism of the effect of radiaticsai on glycine ^ 

Specifically the work will be directed towards 

a) Identifying and determining the unknown product indicated 
by an insonipletQ siaterial balance o 

b) An investigation of the C^ present system as well as th© 
Oj free syst^n upon which our efforts in the past has been 
p^ijfnarily eoncentratado 

e) An investigation of the effect of pH upcai both th© 0^ 

frea and 0« jE'ssait systa^o 

d) Study the role of disproportioeiation reactions by irradiating 
a ptsra d or 1 aiaino acid and assaying ensyiaatically for the optical 

isc^arc 



Part B teeluded Yes ^ No ^ 

1137 



Ssrlal Noo NCI=629 

PHS=.NIH 

Individual I^oject Report 

Calendar Year 19^9 

Part BoS Honors 3 Awards and Pyibllcations 

Publications other tlian abstracts fSrosj this projects 

I'kxwellj Charles Re and Peterson, E„ Co, The Effect of 
a-radiaticsx oa Aqusous Glycine, J. pb^So chemo 633 935 (l9S^9)o 

Razors and aiv-ards relating to project? 

Dec, 29s 3.958 •= AMS Meetings Washington^ Do Coj presented 
iijvited paper at symposia on "Radiation 
Ci>®mists:y" 

C@G= 29^ 1959 =■ AMS Meeting, GhicagOg Illinois, presented 
invited paper at symposia on "Radiaticai 
Cheajistry of ftroteins and Amino Acids" 



1133 



Serial No. HCI^37 
1« Radiation Brsneh 
2. Radiation Physics S©et. 
3"„ ^thesda, Marylard 



PHS-NIH 
Individiaal Pnxsjeet Report 
Calenaar Year 19^9 



Projeet Titles 

DsfelopB^nt of Hi^^Intensity X-Say Sotarea 
Pjfincipal Int®3tigators 

fe^ard Lo Andrews 
Othsr In-^ssti^torsj 

Ecbsrfe W= S?^in 
Coopsrating Unitss 

None 
¥sB.n Years s 

NORS 

Projeet Descriptions 

Obje^y^^g,? Tb obtain high intensity t^ams of x^ra^ra , pulsed 
or st-®ady f^si the 3 Mst ^nerator. 



Methods Eaii^loyedt No progress 
1959 dn® to lack of personi^le 



was jnads in this problem d\jring 



Part B included Tes .£j^ No ^^ 



1139 



Sarial Noo J?CI."680 

1« Radiation Braneh 

2e Radiation Physics Sect. 

3« Bsthesdag Mazyland 



PHS-UIH 
Sidividual Project Report 
Calendar Tear 19^9 

Project Titles 

Radioactis"© Isotope Administration by lontophorsses 
Principal Investigators 

Howard L» Andre eJS 
Other Investigators s 

Lo^s C&^tB3?on and HoTO'&ij C^ Peterson 
Cooperating Units s 

Hon® 

Man Tesrfs? 

Totals 1 
Professional 2/3 

Ottisrs 1/3 

Ps^ojeet Descriptions 

PAlQcti^s and._Mej^pd^,.aaployM» A '^lety of isotopes wsre 
administsred throu^ the skin of guinea pigs 1^ the aT:plication 
of elsetrie currente Isotone distributions were dstei^ned by assaying 
skin sections and if«rtinent orpms. The distribution of P^®' seewsd. of 
siiffiei©nt interest to wasTsant an attempt at traior treats^nt, X%lker 
sareoras in rats w®re treated by the local application of P^® "by elaetrie 

Jfe.1og-?S^ ^^lll^-^ fmaor concertt-rationg of P®^ several tisies hi^er 
than in norasal tissue w©rs readily obtained. Calenlatlons is>3icatsd 
that doses of a fs^ thousand rad wsre delivered to a ©3od f^astion 
©f th© tmror tissue. In sorse casas there was a t€inporary slowing 
of tuEsr gi?©wth ard in ona case a complet© regression. In ^nera^ 
ho^s-^rj the results were unsatisfaetoiye 

5ignii;,5,can^ to Can^z;- Researeh.s . Thsre is probably little pracstlml 
application to eanear trealsnant ^-yiou^ other tiJBaDrs itd^t respond 
satisfactorily » 



11 'in 



Smial Noc NCX»680 



ao^^4^yra©.,g£p£o3get: Itere seems lit.tle chanes of 
ijEpro^iKlng the coneentration ratio of the adsainistered isotope, 
Although some jeoj?© anisnal trials isould be of interest they 
should i&v® a low priority » 



Psrt. B included . les £^ No ^^ 



im 



2o Rado Fhysies Segeto 

3o B©thssd,a.j Marjlaiid 

Project Title? 

fh® Abstraction Rsaetions of Ifydrogen Atoms in the Hadiolyais 
of A<^sous Organic Solutions o Part !« AsnineSj Andno AeidSj, 
Amides j, Peptides and llaiolSo 

Principal T4i^®stigators 

Dr„ Peter Rissa 

Other Investi^-torSt 

Dr„ Charles Ro IfexHsll 
MTo Bss^najpd Bust 

Cooperating OhitsJ 

Eaergr MstaboliSBi Section^ Laboratesy ef Rspsi^logrs Ife-tiosml 
CaBcar Institxita 

Ifen I«ars (calandar year 1959) 

Totals 1 
Profsssionals 1 
Others 

Project Descriptions 

O^.ieetivee? The setion of ionising radiation on aqu®0'as sol-tations 
produces H atosss, OH radieala, hydrogen and hydrogen p-sro^ddSo In 
■Sie absanee of dissol-^d oxygen, S atoms may react with satnarat-sd 
organic soltites "fcy abstracting H atoKs from sarbcn^ nit'^ogen^ 
sulfur and o^gen to produce i^dro^n mid an organic ite® radiealo 

For solutes which have moare than one sits at -which H atom abstx^asticn 
n^y occur^ there is at present insuffielsnt informstion toT predicting 
the site, or sites of reaction. The objectiws of this project are to 
study the meqhanisia of" the abstraction reaetions for such solu'bes 
and thus to deterad-ne the" nsturecf the organic free radicals prod-aced, 

^;^odg^ -SmBS^oyed « When aqasous organic solutions sx-a exposed to 
ionizing radiation, hydrogen is produced by t%?s distinct proc®ss'SS-- 
The primary sftolecular hydrogen yield .arises from "chs raeoabin&tion 
of H atoms in a series of sasall regions of high radical ecnesntra-* 
tion ( known as "bpursS) and is ind@pendsnt of add®d solute "urA^T 
certain conditions^ Additional hydrogen is fonssd fros the redactions 
of H atoms uhich diffuse out of the sptira and reset -with th© organic 
solute^ 

Analo^us processes cecur ^en organic solutes dissolved in B<?jO 
are irradiated and the primary uralecular D2 yield is kno^j fros 
previous studieso 

^ F, S. Baintoht gad^.ggg^.gjjPPlg.J?3l2^^g,^^^^^^^ ^..-■■Al^.? 



serial lOo IS^^l^ 
m^odp_ EjmkyjMt (Continued) 

e(D^)s the n-mher of D« mol®<mles forasd psr 100 ev absorbed j 
Is iqSal to 0.^ for 220 k^ 3c-rays» Additlonja D„ or HD is 
fowasd from the reactions of D atoms with the solute. In tha 
present Btndy solutes were dsuterium labeled on nitrogsn or 
mltw by axehangs in D«0 ( 99.5 atom %)s> leading hydrogen 
stablv bo^snd to carbon under the corditions of the ©3cperiiiients« 
'Sirae for the case of acetamide dissol^d in D 0^ hydrogen in 
ssssll of the primary inoleeular yield slight bl fomsed by reactioaa 

1 or 25 

kn 

1) B •!• CH3CO NDg «--«^ Dg * CSi^CO m 

2) D -^ CSjCO KHg -21«^ HD + CHgQO HD^ 

T^^ hydTo^n (Dg ^ HD) prodijced during radiolysis of a deg&ssed 
solution of acelainide (or of other solutes) tjas ©xtracted on a 
¥an Sljks apparatus and its isotopic composition determined by 
iW^s«.sp9ctrois8try. From th© observed yields of Dg and HD 
/I^CD^) said Q{^)Jp the relatiw contributions of reactions 1 
mid 2 ean be m@.mT®d. If both contribute appreciably $ ths 
ratio of the rats ©onstantSs kn/k^g can t^ calculated sine© 



ft 



« G(}32) - gCDg) 



Maior Findings: The observed yields j eorrscted for the primary 
i^ft^TlT^ yield are sumarised in Table lo The results show 
«iat D atoms react with glycine « aeetainid© and glycylglycins at 
neutral pD by abstracting from carbon and not from nitro^n. In 
strongly basic solutions, idiare the dipolar ion of glycine is 
coa^rbsd to KDg CHgCOOPj, an apprseiable fraction of D atoa® 
gbstraet from the -M)g ^^up of glycine » A similar result is 
found for n-butylasdne „ fhe relative rate constants for ab» 
straction from nitro^n vs^ carbon are shown in Table lo 

for substaneea containing tfca =SD ^oup, n«batylthioi and 
eyst®in®p the fonaation of RSo radicals predooinates in 
abs-feaetion rsacticns, Bius for cysteins the rate of abstraction 
®f dOTts^iua from sulfur is appro xiasately lit times ©^ater than 
tfea rats of abstraction of hydrogen from eas'bono 

An intgg^sting aspect ©f ths resulte of Table 1 is the fact that 
that fat® of mora than half of tfc© D atoms produced by radiation 
in neutral solution is unaccounted for by- abstraction reactions o 
In the cas® of glycine indirect -evident^ suggeete strongly that 
H stoffig esn react with the dipolar ion to give aasjjonia and 
'?GHgCSX)'° radicals o® 



® So S. l-fes^ll. Annual Report, NCI=629, (1958* 1959) 



A siirdla? .reaction appears to take plaes isith asddeso In the cas® 

of the tMol2» tliS rl'ssing D stoins probably reset to produco D^S'^ 
•Aich is known to ba a mjor product in the radiolysis of feysteins. 

1143 



Saslal Mo. NCJ^X 



'J.® I« ^- H®l®.ti-?@ Rate Constant for Abstraction Reactions at 2^0;, 


■ /.■at© 1 


GOg) - g(S2> 


G(ffl}) 


ke 


^Cb) 


GH^GOHDg 1 


0O3 ^ ^02 


06k 1 


<o08 


<ol2 


2^ ND^CHgCOO 


cOl 4 .02 


o5? 


<oO^ 


<o03 


3o ©^CHgCCi^DCHgCOO 


eCa ± 0O2 


ciOi 


<o07 


<oO? 


h. JIBgCHgGC^ 


0I2 


0^ 


.31 


.31 


1. CH3(CHg)^g 


1 oa 

j 


.90 


. o23 


.6? ^«> 


6o m^iGii^)^ 


.h$ 


ogll 


lo9 


12..I* '^«) 




\ ,0 


0037 


llio3 


ll4o3 ^^^^ 



{&} k«s refers to the rate of abstraction from carbon, kx to th© rate of 

absts'astion from alenmits other than capboj\o Fton nite^gen for solntss 
X «■ 5| From gialf\sr for solutss 6 and 7o 

(b) k^ is tli@ rate omstant psr C»H or Hc,H ^1^0 

(e) ealeislat@d sss\2ssing 6 equally rsactiTs seecndasy H atoms « 

(d) ealffulated assrjisdng jpeaction of "Sie "^ytiary H atom only« 



ll^il 



Serisl Hoo NCI«^? 
mSMJM§^^^ Gontirmsd) 

Ttm results for glyoyl^yeine are of significance for. th© 
aqueous radiation eheiaistay of proteins ^ sineo they indicate 
that tte reaetions of H atoias with the psptide bond will not 
produs® f£^© radieals vit^ the odd @leetarori on th© nitrogen 
of the p©ptid© bondc 

§^^M^SS.^.SSS^E^^^^^' ^is project eontributes to 
th® knowledgs of the initial fr@© radical reactions which as® 
a S5£jor part of the initial radiation dasiage in biological 

sjstssnsc 

!Voij03©d TO,'ar^, of pro.leet? The rats of abstracting a deutsri-o® 
atom from a =43-D or =4J»D bond laay be 5=^10 times smeller than th© 
rat® of abstracting an H atom fi^m a C^K or N«H bondo In ord@f 
to ®^sl-aat© the insportsnes of this deuteri-om isotope effect^ 
^x> radiolysis of sosi^ repr^ssentati'BB compounds (CD-CQJH^ imd 

mfE^QcS) id.ll bs studied in light water.. 

Th® extension of th® prQS®nt approach 1» other functional 
groups (hydssssid© and guanidinlura) is bsing consideredo 



Part B inel\jd®d I@s £17 No ^^ 



1145 



?HS=Mn! 2c. Kado Ther, 3erv, 

Individual Rcoject R®pog=t 3o Bethasda^, ¥Ai, 



Galsndar Year 19^9 

J^^oject Title: 

Sepvic© Radiation (Cliiiical Therapy) 
l¥incipal Sivsstlgator j 

Kirkland Barace^ Mo Do 
Other In^estigatorss 

Jo Robert Andrews, Mo ;Qo 
Cooperating Units: 

None 

¥s.n Years; 

Ffofassicn&li 2/3 
Others « 1=4^ 
Tot^l? 2=.l/3 

* Project Descriptions 

Oijeeti'^ess To pro^id© r^diaticgi therapy fcr those patiaits 
^^irin'g'sueh treatsaent ia th® course of the disease for Haich 
they ar®^ being investigated by NH! research groups other tMn 
the Radiation Branch,, 

Patient Sfaterials A total of 189 e(Kisultations i^ere sesn ia 
IK'e Tarious dspartxnents of this Institutisa during th© past 
12 months, A total of I8li eoarses of radiation thm^&w w©^'® 
adsdni stared o 

Methods Ekaplogred: A ^d@ range of photcsi energies were «s©^ 
and various m^tinods of application, ^©r© «nploy®d<, Folloidng 
are the na^er of eourses of treatjaents at the Tario-ag ®n@rgi©s 

noted? 

l60 courses of 2 M@v irradiatioa ; 
7 courses of 2$0 kiloTolt radiation 
6 courses of llo kiloTOlt radiation 
k eourses of 60 kilovolt radiatira 
^ cdursQS of elactrcsi beas therapgr 
1 e^Tical rgditm applioatii^ 

Ifejocr Findings ; Respcsiss to radiati<«j therapy has been @on» 
sistent with responses reports by others „ 

Pp oposed Course of Project ; To continue » 

Pasft B insludfld ^efiC7 

11 lift 



lo Radiation Branch 
2<, Rado Ther„ SerVo 
3= Bethesdaj, I-!do 

Individual Project Report 
Calendar Year 19^9 



Part 



i¥oject Title: 

Ghildhocxi Cancer 
Principal Investigators 

Kirkland Braces Mc Do 
Other IhV9!gtigatorsj 

Jo Ro Andrews, Mo Bo and Louis V« Avioli, JTo D« 
Cooperating Units j 

None 

Man Years (calendar year 19^9 )s Patient Days (calendar year 1959): 
Total: l/k approx, 200 

ftrofessionals l/k 
Others 

Project Description; 

Ctoj active ; The objective of this is to collect data on the 
treatment of malignant diseases of ehildhoodo 

Patient Tlaterial ; All children under t.he age of 18 who have 
TSiignan't~"tOTrorso 

I'fajor Findings ; Malignant tumors of cliildren are extremely rare 
and we have collected only 19 cases o We can only say at this 
point that histological diagnosis is particularly difficult and a 
very poor guide to the rapidity with which tumors will spread^ and 
their radiation sensitivity. This prograsa is significant to bic-- 
logical research only in that we hope to gather enough date fras 
these extrnmsly rare cases to classify thsss tumors and d©tei\minQ 
what place radiatiai therajy has in their treatsaento 

Proposed Course of Pro.ject; He propose to carry on with this 
projeci until we have patfiered sufficient case rater ial cm which 
we can draw some typ® of conclusion » 



Fart B Included Yes ^ No /t, 



2o Hado ThBSo SqsTo 

PES«.N1S ■ . . 

BifSi-^l&Eal ProJ©€t. R^«^t 
Gaa-aadas? Tear 1.959 

I^o;*©et Titlss 

Sim® as a ffodtO.©®- of SLinisal Ifediation Bospsmse 

Jo Hsbssi; to&QWSg Mo Do 

Ofeer Irarestigatorss 

Hemaa Do Suit^ Mo Dog Stanly So SnsicteTs K Dog Sirklsnd 

Go i&'aeee lio Doj Loais ¥o Avioli^ Mo D* sad Gordesi Sbas-p^ Mo D^ 

CsopsratiJjg Units s 

lh.n leases (ealesscto ys^ 1959)? 
. Totals 

Project Deseriptioa? 

Ob j ^tg^amxdjfe^^^^^^^glgg^ ; fhiss p^-ojeet is a» e:gtsnsi©a 
^^^^^(c)T'"S"^®5^teal dcsiim©txy^ and gsn^al cUalcaa 
sspeeta of this iES-ojeet ware snorted in detail In 19SB mid tm 
vBsdatioas hav® be&n introduced os- dbsarrod in 1959 o 

Pstisnt I'kteslal and Ma>1cg Jlndli^at the patients included in 

12 or moE^ moaths agOo Th® asediaa sar^^al time of this ^oop 
of 40 is 10 months o The patients ineludsd in -fche atu*^ s^^j ±n 
gsnarsl^ thesa wi-^ rathsK" Tar ad^arased caneer of the hesd. sad 
sjacko It is siofc likely that tha isadiaa surtiTal tis^ of 10 
Biorsfchs is isforsa than would bscm bsaa attained had thss® patients 
h&en treated in a mare ©(Msviantioml macner tdth a siaaller dcse 
gi-smi or®* a shoff-ter payiod of tisaSo Lcwal cutan®3«s and ©tlies' 
ROTsml tissu® reactions haT® tean mildo la respect to th© @!dB 
r®®pom® ths^ mild reactions s^s probably Ia2»ge3y related to 
abses^tioji eharactsrisfelcs of a 2 Msv X^s-ay bsasi ratfegir than to 
the orbendsd ttee of is^adiaticuo In T&sp@Qt to other nasml 
tissue reaotisK'Js th® opposite may be the caseo. this case raatei'fel 
is pr@s©jitly being critically re"elgw®d and trog-ksd t^ for publieatlo 



PrcJ^ts Pkti©at adtdssioRg to the projset ha'^-^S! 




_ sueh tiffi© as ths ease mat^ial ha® 'os@n 
and •6ro2»fegd tap sso t&st th@ pTOJect csn hk 



Part 3 inelufed "i&a^J i^o/I7 H^lS 



Ss'ial Noo MCIgg^6gf^j 



Gaiejsiar leax 19^9 



Part^Bi Honors^ Awer^, and Publicatdorss 

Publieaticss® other than abstracts fr<M this ps-o^sets 

AsdreiJS^ J, Ro sad Sneidsr, So Eo, Th@ Modifies'U.on of th® 
Hadiation Response, Mo Jo of Roentgenolo and Raditim Tbm&pjs 
81: ]i85s 1959o 

Honors sxid Awards relatirg to this projeots 

None 



1149 



Sei'ial Mo» M^^sti 
lo Radiation Bi.>.„...... 

_3„ Betbasda^ Marylmid 



PHS-MIK 

Indi^daal Project Report 

Galersiar Tear 19$9 

Project Title? 

Distribtition of S-J^ in Exainaii Tissiias arri Ti»s>rs and -^.e Clinieal 
Effects of Large Doses of S-.3S. 

F^ineipal In'^stigators 

Jo Rolsss^ AndrswSj *^p Do 

Otl^S" IJivestigatorss 

Laiarenca Schlactes*, M,, Dc, Jsi^s A, Ross, Me Hop Jaek Le^ln^ Mo Do.. 
m,cha2*d Swaawij Mo D<.^ and B&^sl. Qompj A© Bo 

Coopsjpating' Units s 

NCI«.Pa-&ologie Anatoasgr B^andi, NCI»523 

M^. Tears (calendar year 19$9)t Patient Days (eslendar ysar 1959)" 

%)tals 

Profsssionalj 

Others 

Project Dsg@riptions 

Ob^lectiyes, arai Mathod_s _E|jiRlogMs '^i^e eonesptnai aspects of th® pro-. 
ject r®aain tha sanss as reported in detail as KCI-2-6^(c) foi» the 
ca2,®isdar year 5-9^ ■> ^® study has bean ^-^relj handicapped 'bssa.riss 
apsropriate patients ha^§ not beooffi© s^milatij®. 

PatlEiit %j^.gi^ 8 One otJisr eass has b@en studied, 'ftis is tte 
ca©© of an elderly jaale presenting -sJith massive tliorasic srallj, 
intrathoraoiej snd extrathoraeie chondresareoitia<p Sttidiss ssf Ihe 
S-.35 uptake in tussr and skin shoi^d e>ne®ntration of the S^3$ 
in the tuinor 1» be ^atar than' "Siat in fee skin 1:^ f asters fross 
5 to 5> depending upon the time orj pssiis^^ only tha horsnal 
variation g of the biopsies « ^ preliMnasy distribution study -ssas 
rej^atsd following the administeation of gro?sth horsnon© aM ri@itter 
the rQlati-WB nor the' absolute concentration of S»'35 in twSK>r or skis 
diangedo FoHo^djig the preliminary studies 38S asie of S-Jj i^r^ 
gi-^en and two rnonths later anoliier doss of 335 2S0o ia.s given o Ti'^32'® 



1150 



fetie^..^^^^? (Contin-aed) 

was on^ slight depresslcsi of the white and pdatelst ©ounts in the 
jsrioi li^.tween the t«o doses but Ij weeks folloi-dng the sseoM dos© 
thsre was marked depression of the platelets and moderate dgpr©s«° 
sion of th® peripharal white cells o "Eies© recovered aftar t^M 
Bfflnths bat 6 iK>nths after the sseond dose the patient fe'ss fomd to 
hai?Q a severe anemia and thromboeytoi^nis idth peteehiaSo This is 
premseably. related to persistessce of heraspoietie depression by the 
3-3^ bat clinical studies for the abaolute identification of th® 
e&G.^ of the peripheral blood findings have rstt been cojnpletedo 
On the other handj a patient treated similarly in th© pre-sioua 
calendar year remains ^mli insofar as norjisal henjopoietie fEnetion 
is concerned and without ayssptoras insofar as the local mass of 
■fesBor of the i^ItIs is coneerned. Both patients rssei-^ed leeal^ 
intensive external beam x^^ray therapy to the lesions folloTdng 
ihe sulfur studies » In th® case of the asale ^th the th^^eic 
wall neoplasm ^is treatise nt becanre j^cessary baeauss of reeur^ 
renee of uncontrolled, ulcerati'ro tuisjor of the ©xtertasl slsst 
iiall,. 

fS&Sio.SSd^5ss SS§L^ ^ f,^M^^J ^ese studies vill be ptarsued verj 
imch as has been outlined al»i^ and in preidous rspcrts if eas® 
saterial becomes available » It has beeojt® apparent feat careful 
attsntirat has to be giv©n to the matter of h©iBopoi®tie rsasrvB 
and •He ar© prepared to do "Sie studies necessary to an undarstsMing 
of HiQ tffects on this resas"^. 



F^ B- insluded I®® ^^J ^^ ^7 



115i 



SasM-Tidml E?ojsat Report 
GalssaSas- Year 1959 



Part Bt Honors j> AHards, aM ftjblicatioiss 

Publteations ©tl»? -Sian ai>ateaets ftom tliis pjojoett 

Asij3sM&3a ^v ^e» SisaFsij Re Lag Scblacht®?; Le-^ Brac©9 K^ 0^^ 

Swain, Re W„, Ths ISffeets of Qm C3arl« of Sulfw^^ Mminists^d 
lJxt>ra:ireno)3sl7 as Sulf&t© to a ^«n with Adwsnesd ChosjdrosarecEsiBo 
This pap^ was accepted for pisfellcation in t^e Aib» J«, of 
RoentgQiiole end Radiiaa Therepy in 3^58 and Wjs galley proof 
has been received but f er rsaaons unknonn to me it has not h^isrs. 
published^, 

HoacK's and Asrarda rela,ting to this is-ojeets 



1152 



1„- Radiation Bx-andi 

3o B©-fchssdas Mdo 

Ihdt-rldml Projeet Reporfc 
Cai^idar Tear 1959 



Barfc Ac 



Rfoject Title t 

GliJiieal Radiation DosirostEy 
Pi'iBeipal Jiivestigateps 

Refcerfe W» Swalu 

Other Investigators: 

.Cooperating Units $ 

l&n r«ars (calendar year 1959)? Patisnt Days (calendar y®H= 1959) § 

Total; 11/3 
Rrof@ssi€sials 1/3 
Others 1 

Project Descriptions 

Cbj@etivQsg To develop siethods for arid to determji® sf©.dia.tioa 
doisg©~cfi"stributioas for i^arious types of radiation thei^-p-f used 
in th® tr^tment of f^tisnts and to ds^is© E©thods f csr isapE'Cffssi^fit 
of i^diaticsi distributicsi -within the prntisito 

Methods ^pl^e^s Ch^dcal" and icmisatissi chssibers for ©Iscfe'oa 
"^sem CSS'apyr^csiisaticHi ehasabsrs and phot^i^.0iia gisthods for 
Supsr^oltsge X'='ray Th®ra^o 

l^j eg Findings g Work s^ ^® isedas® plotter bkd bs^ s topped du® 
io" a ~i^©:rlag@ of parsc^snslo At th® |^©s@nt tissa^ all ©Igstrical 
and ine^ianieal eoispsasats ha^s b@sn asseaibled for ss^-^mato^tie 
plotting of isodos® distribu-tions in «at®r using a mmll Issaiaa,':^ 
ti^ chamber. ■ As tims persiits^ t3?ial ?uas will b® ^ds t© dst'SS'- 
SjiB® desirable s<^if ications » 

To ijnpr«wa de^,g@dists?ib-dti^is for trsats^it of ^tl-satg^ *?®dg@ 
filters hav® b®®i ds-yelopad fosr use on the 2 M©v Vaa ds Qffmaff 
gensratos's ■. I^elissinary dosag® distributicms w®r® Esde usisg 
filja dosiiaeta^- M®thGd.s„ Ths studiss ss^d® indies t® a psssibl© 
iimecHraey in th© f ilsi method ^ieh isiU bs investigated ^li®n 
th© plotter is c«®pl8tedo 

1153 



Serial lo ,. ^«2=660( c r 
H*oJ©et Dsseriptioa: (Gsitinssd) 

The dosimeters in the 2 Me'^ Van ds Graaff Generators ars being 
replaced hy t^ansmiBslaci ionisation (^.sj&ssk-s cosmeeted to 
evsrrant integrators which can b@ pi3?@5©t to turn th© X'^'ays off 
at ths mxd of a prescribed dose« This should lead to great©? 
asouraey of treatsaent and s?educ® possibilitj of ov©r©?£po.su?© 
since at th® present timsj the teshnieiasi is required to tusB 
off the generator whsn a laeter indicates the pE'sscribsd dose* 

I^oposod Cgarse of F^ojact; Work on ths isodose plotter w5.11 bs 
ctmtlnusd Wiih the "oSJ^^ve of laalcing it as accurate as possible 
and as ecsapletely aut<saatic as practical » 

¥ith ths a^ilability of the 256=Ghann@l analys©?, a projeet -^411 
b® undertaken to datsr^tdne x-ray spisctrao 

Search will be eontinued for small ionisation ehaisbs^oj' oth®s' 
types of dosi^stsrs to facilitata asssssatsit of dosag® durfeig 
treatrs®it of patients ^ 



?s.Tt B includad Yes £J ^© ^ 



115k 



lo Radiatdon Braseh 
go Rado Thsrapy Ser^iae 
3o Bsthesdaj, mrjl&rd 



HiS-NIH 
Indi-^dual Project Seport 
Calendar Tear 19^ 

Frojset Titles 

Aa Appsrsisal of Rgnal Function aiid Infection in Ad-?sKe©d 
Bladder Malignancy Treated wife Ileal Bladder Oonstru&tlaa 
and Z«-Irradiationo 

Principal Investigators 

Jo Robert AndrewSj M, D„ 
Other 3iif®stigatorss 

Jack Ls-^in, M, D. ^ Heraan D» Sisit ^ M^ Dc 

Coopesating Units? 

Nons 

Man Tears (calendar year 1959) Patient Days (eal®ndar jB&r 19$9) 

Total? 
PK>f®ssionals 

Pf^5®e"& Descriptions 

Objesti^^g Ewluatlon of the results of tirlnary str^ais 
■di^srsion and radiation l^erap^ for ad"fanc«d ear©inoins, of 
the "cOLadder^ and an apprsisal of renal function and infection 
in th@@@ easssc 

Jj^^^^-g^plQgM^ Patients with Stage G arsd D careinorja of 
the bladder J ^ada 3 and isy had iirinary streajB difersios % 
conatr^etion of an il®al bladder- (sisrgery p^rfonrad fcy tl'ia 
S^rgieal ^aneh of the National Cancer Institute) ^ and g-ab». 
sequently had high doses (7000. to 8a>3 r in ? -sss-ks) of 2 
Msv s:»2*ay tharaj^ d@li*5®s^d to the bladder aM the su2Tound3.ng 

pdl'9ie @tS*U@tT2T@S9 

^^^a^^.^^^^s Nine patienta have been so treated ^ These 
cases and 2 patients who hair® had di-^srsion of -UmlT nrinarj 
etreajn following x-ray therapy ha,'«B eonstituted th® source for 
th@ stucco 



1155 



Ao The eosnbination of di^raion of the tarinasy gts^sm and s.=. 
irradiation to the pel^s has restsltsd in marked sympto- 
matic impro"^i?®nt as manifested by control of dystiriaj, 
freQaency^ urinary tract obstruction^ and hematuria., 
Ihere has bsen at least tempor&iy control of tuiiSDr fol- 
lowingirradiation. Howe-^er, 9 of the 11 patients srs 
now dead J 7 of the 9 with distant metastases » Tlie tm 
living patients ha"^ survivsd 2$ and Jli ironths reepsetiTOly* 

Bo Renal function and bacteriological studieso 

Renal ftaietion has been studied in these patients tjy means 
of the blood urea nitrogen j, ssinsin creatinim, creatinine 
elearancea and fractional PSP excretion. Infsction of th® 
urinary tract has been assessed by the use of qaantita.ti"^ 
urine cultures, FoUovdng dlTsrsion of the urinary streasa 
and treatnisnt with irradiation j renal function as Yneasured 
by the blood urea nitrogen^ saruia creatininSj &rd OTP ®sam» 
ination indicated a prosr^Jt and striking return to. normal 
le-velSa However, the endogenous creatinine clearance snd 
fractional PSP excretion did not show any such isipro'ssinente 
On the contrast renal function as measured by thes® tests 
m,B markedly depressed b This surprising diTOrgsnse of .data 
is as"y©t unexplained. 

Thsr© was no significant change in the percentage of positi^^ 
quantitati-i® urine cultures post-operativelyft PresiiBsabljg 
therefor®, infsQtlon of the upper urinary tract is not 
eradicated ty urinary stream di'vsraiono In these patients 
the absence of clinically acute pyelonephritis does not rule 
out the pi^sense of a silent infectious process occurring ixi 
ths kidneys o 

Al^ou^ none of our patients demonstrated e-OT.d®nce of 
tirinary tract obstruction foSlowing urin.a2y stream di^^rsiors 
our data suggests to us that daaags to the kidneys froffi prO'^ 
longed urinasy tract infection and ureteral obstruction ±3 
not necessarily corrected by relief of urs-nary traet obst2n;ie- 
tion^ and the extent of bacterluria reinains unaltered » '^b 
haira concluded that our patients remain in a aiarginal strnttsa 
with respect to renal function despite the apparent benefit 
indicated hj the ssrura creatinine ^ BMj and I¥Po Tha pros®- 
dur® ®ass however, of real symptosaatie benefit to ti^ patient 
and greatly facilitated radiation therapyo 

It is suggested that renal function is ss>re cornpromi^d in 
these patients than appreciated previously ^ ' and that fur-bha? 
gtttdy is n®@ded to .©"raltaate Ihe long t«2® @f fset® and benefit® 
ft-om urinary stream di^srsion and irradiation in eane©r of tlm 
bladdsr. 

Part B included les .HJ" ^ ^^ 

1156 . 



ladiTidssal f^Jeet Esp^t 



3o B9-feh®sd£s M. 



ifedioactlT© Isot^es ia Iatea?stitlal Radiation Th©rapj 
Fxlnclpal l^xrsstigat^i's 
Haman D, Stsit^ H^ Do 

Eob83?t We Ssain and Galvin Ro Jfeaekm 

Cooperating Units $ 

ffen lea^s (calendar j®ss? 1959)' Patient Bsys (calsK<!ap ysa? l$'59)s 
Tetalt 

Ofcheri 

I^ojeet Bascriptions 

_^^ec^^ss (1) fo da-relop a t@ehnique! and eq^psasat fss? 
pSflinig smll radieactiva scns^ees^ as a peimisn^nt iapSjmfcs:. 
in iimccessibla aaatcaaical rsgieasg so as to dsllmf a high 
total d^e locally but a Icm dess to th@.op®2=st»«£' and «K&a2= 
madieal and nrn'sifig s'teffa (2) To de-s^lop taeliriiqs^' ead 
sqidpjiient far achisvijig dos® dist^ibutlKis ec^parabl® t3 
that obtained «lth rscEyja seedl® therapy b®t ^th grea.-'^g' 
flssd.biliti' as te des® distilb'^tidn asal idth redhead 
yadlation es^osx^e to (^arator and staff « 

JHethods S^Lg-ed^^^aM . j.fel^L,^^ln^ J 'Shis ^as d^aa^ltsM isi 
^'tg3X"IiriE© 3SSH^l^"^®BTrepsspt of calsisd&r yesi> 1^58 o 

si^l^^^Hi5"d^5SiJ£s b©@a loasgd t© I^o Gilbert Klatefearg 
Mo Do Aj^iarson Hospitals, to vm® in a eeatrolled Btn^f ^ ts&Gsm? 
ef the urir^ry bladdsra Gassa will fee ts'®atsd ca a 2=out5j® 
basis using ext^'Ml b®ain radiatiiai tbasapj' in bsth gs^d^iS aad 



1157 



Serial KOo MCI-2.=^8Ce} 

Fx'ojsst B®sesipt.i©Ea2 (eosittoi@d) 

si^plmrni^xi^ radioactl^'^ Jjspl^tation tberapy in, ons 
g^m^o Ths M« D. Aades'son Hospital has tl® patient 

s^Assrial and tb® ©ths? rssou^es to cond^jst an @st®ssiv@ 
teial of th@ ii^'tejsasKfeo' 



Bas-t B iislndsd 



^^^ ^^O 



1153 



Indl'^dKal P^^eei Report 
Cal®Ma^ I®as' 1959 

ll£i=S* S«>ss)S^s A'H'aj?dSs a^ PubHeaticms 

PofelicatioE® ofchar than abstracts trm. tMs protest i 

Koa^g aad Air^ds relating to this ps^Jeets 

An ^tdbit on this s^tgrial was pg©pa3*ad sM shmu si tls© 
Amasaal Msstings of ths iimesican Jfemtgan Raj Soeisty is 
Oinciis^ti and &t th@ Amnsal Ussting of tb® Badiological 
S<ffiietj of North te®?ica in Chieago whare it refeei^td a 
citetlosio 



1159 



Serial r-Joc l]CI ^^JQ (p} 
1- Radiatiai Branch 
2p Rado Thevo SbtTo 
% Bethesdaj, :M^ 
PKS4IIH 
Individual Project Report 
Calendar Year 19^9 



I^rt A, 



i^oject Title: 

Studies on "Hiyroid Cancer 
Principal Divestigators 

Jo Robert AndreirSj Ho Do 
Othea: Investigators: 

Theodore Robinson, J?„ D^, Jasses Rossj M. D^, 
Cooperating Units: 

N<xie 

Man Tears (calKidar yeas? 1959): Patient Days (oalsndar year 
Totals 

Professiosial: 
Others 

Project Description: 

Qbjeeti'yes and Methods Bnployeds To study the factors inflwancdaig 
^'e''r'©sp(ai'se' oi^' 'carcincS' 'of "the thyroid gland to radioactive 
iodine in associaticHi with thyroid hormons tr^trasnto To stns^' 

. the turnover rates of primary careinoiaa of the thyroid and 
metastases and their respcsiss to honaonal and other faetor-So 
To study the hormone effect ^ and hyparthyroidisia on thyroid 
tumorso This study involves a variety of considerations iBel"u= 
ding technieologicalj biological^ endocrinological and pmrrsra- 
cblopical aspects o Technical studies are ccncerned with problems 
associated with the identification and.'©valuatic« of radicsiGtiTitj 
in specific organs or anatoiBical sites p Biolo-ical studies are 
ccKicemed with the relationship of the histopathological char^" 

- acteristics of thyroid cancer to radioactivity uptal<e and response; 
tlormonal consideraticsis are eoncemed with the relaticmship of ' 
tumor control to the h<j®monal status of the individual alons 



IIBO 



Serial 

Project Descriptionj (Goitinued) 

and in ccmibination with radioactive iodineo Also beinn studied 
is the relationship . of turnover rates (of RA.I«131) of thyroid 
tumor in relationship to the ingestion of thyroid products and 
other substances to see if radioactive substances ray be held 
in tumor tissue for a lonf^er period^ thus increasing the amount 
of radiation to the tissues, 

Batient tfeterial ; Proved cases of carcincsa of the thyroid^ 
©i^Ker primary "or secondary or botho 

Major Findings; Findin^^s frran this stut^ seem to sho:? that the 
use of radioactive iodine kloig with thyroid homone seems to 
have a tumoricidal effect beyond that of _m©rn radioactivity 
alonoo T)ue to the fairly recent orijrin of tliis combined technique 
equivalent studies to which our results could be compared are not 
readily available* Results also se^ia to shot-? that mere thyroid 
hormone alone nay have sowe effect on causing regression on 
tlxyroid tumoro V/cack vdth sriall animals seems to shox? that admin== 
istration of thjrroid hormone causini^ the animal to retain the 
hyperthyroid state increases the radiation respcnseo It also 
seems to Increase the radiation response to tumor withiji the 
animal and possibly hgrpertl^rroidism alone in one form of tumor 
may have a s<Mnevihat slowing effect on the p:ciffch of the tumoro 
Results are questionable as to whether or not turnover rates of 
functioning thyroid tumor siay be demonstrably altered by the use 
of endocrinological agents o 

Proposed Course of Project ; To continue^ In addition to ccntiaiiiag 
those observatdoias which ar@ described abo^e a sttidy of heaaggl^itis 
ating atitoii^imaae anl^bodios in rssposas® t© thy^oglobulis and ttasss' 
fractions is a3^o beiag imdsrtateao 



Part B included Yes /y Ko ^ 



1161 



3„B©th8sdas Maryland 



Indi-vid-ual Project Reporii 
Calendar Tear 19^9 



Project Title? 

Hadiation Induced Dysplasias of Bona 
Principal Is;wBstigat03?» 

Philip Rubin, M, Do 
O-ttiar Investigators: 

Haa©l QoBip, A, B. , Riehard Swarsig Mo D«g aM J, Rotert Ar^s^w®^ H, 3^ 

Cooperating Urdts: 

Strong Ifemorial Hospital, D©partinent of Radiologgrg Uni'^rsit^i- of 
Rochester 5 Rochester , New York 

Man Tears! 
Totals 

Professior^lf 
Others 

Project Itescriptions 

OMecti'VBS and Msthoda Bnployedg This pro^eet coneamsd the indiactien 
of dysplasias of bone by irradiation ^th either internal eisitt-ers or 
extariml x«irradiation. The iatenial esd.tt@rs employed were S^-J^ aM 
Sr»»90o S»35 affects largely epiphysial gro^h and E^tsptiysial ehaii^s 
while Srto.90 affect® largely diaphysial groirKi« Estemal iiradiatisn 
was ^Tsn 89leeti"^ly to either an entire' bonsg the epipli^sis^ tte 
Bsetapltysis or the diaphysis. Novel Bseans for precise local irradiatAsa 
of the eaperii^ntal aaiii^ were de^ralopsdo 

^IPi!^. ■illi^^^-'^r' ^* ^^s fourd that local irradiation of porfeione of 
growing bone in appropriate doses caused charaeteristis deforsdties 
of the tone a Thes^ deformities were relalsd to certain t>^Qr@tieal 
aspects of Ixjn© HK>deling ard to the clinical ©stebchondrodys^diiiisSv 
On theoretical bases good eorrelation eouM ^ shown l^t'sean th® ©a:^. 
pg?i.E©ntally induced radiation dysplasias and ^@ ehara.ct^risties of 
the clinical' osteochondrodystrophies ef unknoiim cause o fb® rss'olts 
of this study ha'^ been published » 

Pyenoead Cepyse, of JF^^ects This project jias brotsght to cessplstion at 
the Uniirarsity of Rochester studies coisaTsneed by D^^ KsiHp R^bin bxA 
others ■^lile Dr. Rubin was at the Natioral Cancer Instit-ats^ 



PaS't B included? Tes , 

116: 



Ssii^ia,! Hot. SB&^::^ 93,..-. 



Calenday Tear 19 $9 



Payt Bo Honors, A^'asiiSp and Ptsblieatlons 

Publications other than abstracts troia this projects 

Rabin, Po, tod3?ews, J, R. , Swarm, R, and Gurnpj H*, Radiation 
Induced Dysplasias of Bone^ Asnsrica Jowmal of Resatgsnolo^ 
and Radimi llierapy, 82$ 206, 29^, 

Honors and Awards Relating to this Projects 

None 



1163 



Ssrlal No. K0I;^6g|. 

2t> Had, Th^« 8&rWo 
3o Bethesdag Md^ 



PHS^HIH 
Individual F^ojset Rep<s^ 
Calsndar Year 1$B9 



fert A« 



i¥oj©et Titles 

Developient of Wedga Filters for a 2 Ma?^ X^ray Bsasn 
I¥incii»l Sliest igatosf! 

Stanly S» ^eidep^ M, Do 
Other Sivesti^t^sj 

Hermn Do Suitj^ McDej Robert ¥, S^la and Jo Robert Andrews^ MoDc 
Cooperating Units: 

Nona 

Man Years (ealendar yeas' 1959) 
Total: 

Rpofessionai: 
Others 

IVo^QCt Deseription: 

Cfoj eetiygs i Tha isodose eorvss used in coa"3'(^tional sadiatioa 
therapy lis flat across th® fieldj that is^ thaj ar© falrlj 
parallel with th@ surface bsing treated » Ke ha^e andigrfcakers 
to change the shape of the isodose curves produe@d in a fe^si 
of Ses^ys from a 2- Kev Van d® Graaff aeselsrator so that th@ 
©urres fona an acute instead of a right angla ?fith th© e^it^ral 
axis of the |)®aai<, Hi® ideslisssd eurres w©uM begin at om 
edge of the field at a point and ascend at a gi-r©n angle (h$^ 
eg' 60®) in a straight line aeross the width ©f the fieldo, 
This ■ distortion of the radiation disttibuticsa in a fi®ld ±b 
useful -f^sn two or ssore fields are coE^ined in the •to@atm@nt 
teehniquea for certain anat«^lcal sites in sharply disaiaishing 
tlse radlaticKx "given outsid® of the -mhrn® of th@ dis@as€i 
bearing tissue and in ^rtially or coi^lat^lsr a-roiding str^js^ 
tures ^^ch are sensitive to high dose irradiation,, Although 
wedge filters have beai in clinieal use in a few radiatioja 
therapy centers f<^ 20 years, we planned to study wedg© design 
and ccsnposition (effect of atosnic nuseber) in an attempt to 
produce "idealised" angling of the isodos© surf as® ? 



llGii 



serial KOo NCIo6Sli 



Epoject Description! (Ceaatinusd) 

In additirai to the desi^i of ths wedge' filters themsel-^es 
W9 hav© been concerned with (1) ths possibilitgr of using just 
two Hedged filters fos* specified angled isodess cur^© (®<.go h^) 
over a r^xge of clinically useful field sizes | (2) the ©ssplc^iBsnt 
of Incite bolus irjaterial at the inaehin© head to ccmpsnsat® f os' 
air "gaps •ahen the x=ra,j beam enters the sixrface of a patient 
obliquely I and (3) the ©valuation of this trsatment t©ehniqu@ in 
actual ease nsaterialo 

Methods Bnplcyed ; On. a s®t of standard isodoss curves lines 
were drawn at 1»5^ or 60® to the central axis of the field » IliQse 
lines started at one @dge of the field and ascended across th@ 
field » Th® starting point is taken to be at the sxirface sdg® 
pffo^acted into the phantcaa j?ather than the gscsastric edgs» Th© 
angled line is planned to have a single isodose value and this 
raluQ is taken to be th© value of th® standard isodose cur^e 
which th© newly drawn angled line intsrsects at Urn field ^dg®^ 
At various po^Jtits along this angled line differences "in pss?-^ 
centages of cdepfeh dog® between th© rslvi® of the angled line and 
i^e regular isodos® , etspves ar® dstsnainedo Th® aiaount of lead 
required to achieve this reduction in thei depth dose is then 
read f^osn an absorption eurye. for the 2 Ms^- bsara in lead^ This 
absorption