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A!S'iA!«YSlS,  OF  PROGRAM  ACTIVmES 
MAJIOKAL  mSHTUTES  OF  HEALTH 


IMATlOHAi.  CANCER  IMSTITUT'E 


NATIONAL  tWSimJTES  OF  HEAIJTH 
PUBLIC  HEMIH  SEK.^ICE 
S  DKl^ARTWENT  OF  mMJK  EDUCATION,  AND  WELFARE 


PAGE  I 


1.     National  CaHce'r  Institute  2 .  Laboratory  of  Biol»gy 

INSTITUTE  LABORATORY  OR  BRANCH 


3.     Office  of  the  Chief U. g.    ItQQ 

■SECTION  OR  SERVICE  LOCATION  (OTHER  THAN  ©ER^   NO. 

■*  BETWESDA). 

6.  Biological  Studies  on  the  Factors  Involved  in  the  Development  and 
PROJECT  TITLE     '       Growth  of  Tumors  in  Experimental  Animals.     '^ 


7.     H,  B.  Anderv«it 

PRINCIPAL   INVESTIGATOR (S) 


8. 


OTHER  INVESTIGATORS ; 
fi     PROJECT  DESCRIPTION 

Testicular  Tum»rs 


The  precsding'  report  suniinarized  an  investigation  with  strain  C 
mice  designed  to  utilize  testicular  tumors  to  study  a  dependent  tumor. 
The  study  is  now  virtually  completed  and  will  be  published  as  soon  as 
time  is  available  for  writing. 

Findings  reported  last  year  remain  unchanged  with  the  exception 
of  those  dealing  with  transplantation.  This  phase  of  the  study  is 
still  in  progress  but  the  results  to  date  are  as  follows. 

Sixty-nine  tumors  were  transplanted  to  ascertain  whether  they  • 
were  able  to  grow  in  estrogenized  hosts  only  or  whether  they  were  a* 
longer  dependent  upon  the  hormone.   Of  PO    tumors  transplanted  fr»m 
mice  that  Were  carrving  stilbestrol  pellets,  8  failed  to  grow  in  either 
estrogenized  or  nonestrogenized  hosts,  2  were  dependent  and  10  became 
independent. 


h7 

]S^h3.^        PROJECT  DESCRIPTI'^N  (Cont.) 

r  ^  Of  U9  tumors  that  continued  to  grow  after  removal  of  pellets, 

'  «^       5  failed  to  grow,  2  were  dependent  and  U3  were  independent. 

It  is  seen  that  as  transplantation  proceeds  more  tumors  be- 
come independent.  Consideralily  m^ve   work  is  necessary  to  analyse 
the.  data  but  the  results  reported  here  are  sufficient  to  establish 
cloarly' that  earlier  reports  in  the  literature  concerning  the  high 
degree  of  dependency  of  this  type  of  tumor  were  probably  the  result  »f 
transplanting  tumors  before  they  had  achieved  independence  in  their 
original  hosts.  This  finding  with  testicular  tumors  raises  two  questions. 
First,  have  other  types  of  dependent  .t\amors  been  transplanted  too  early 
or  do  they  remain  dependent  in 'their  original  hosts?  Second,  what 
factors  are  involved  in  the  relatively  slow  development  of  autonomy 
in  testicular  tumors?  Many  skin  tumors  of  mice  are  known,  to  evolve 
from  papillomas,  but  the  difference  between  papilloma  and  carcinoma 
can  be  detected  histologically.  Thus  far  no  such  difference  is 
detectable  between  dependent  and  independent  testicular  tumors.  The 
problem  is  complicated  further  by  the  observation  that  during  a 
16  weeks  sojourn  in  mice  a  stilbestrol  pellet  produces  histologic 
changes  in  testicles  which  persist  for  many  months  after  removal  of 
the  pellet.  Tumors  may  even  develop  in  a  few  of  these  altered  testicles. 

xipparently  in  some  tumors  the  development  of  malignancy  is 
a  slow  process  and  such  tumors  may  be  used  to  test  the  effi. . .  •  c  - 
of  preventive  procedures. 

The  preceding  report  mentioned  three  experiments  started 
to  study  testicular  tumors.  These  are  still ^in  progress,  but 
have. progressed  sufficiently  to  supply  results.  The  first  was'  per- 
formed to  ascertain  the  susceptibility  of  various  F-,  .hybrids  derived' 
from  strain  C  and  other  inbred  strains.   None  has  proved  to  be  as 
susceptible  as  strain  C  but  (C  X  DBA/2)F  and  (C  X  '^)F-,  hybrids  are 
developing  testicular  tumors  in  sufficient  numbers  to  indicate  their 
value  as  test  animals.   I^brids  between  strain  C  and  strains  C57BL, 
I,  or  RIII,  are  developing  few  tumors. 

The  second  experiment  censisted  of  giving  stilbestrol  pellets 
to  other  Inbred  strains  to  compare  their  susceptibilities  to  induced 
testicular  tumors..  .None  has  aporoached  strain  C  in  this  respect.  Of 
the  strains  tested  strain  C  exhibited  a  unique  susceptibility.  All 
strain  C  animals  developed  testicular  tumors  before  a  tumor  arose  in 
any  other  strnia.   A  few  tumors  have  «ccurred  in  DBA/2  males  and  one 
in  a  strain  C3F  male.  Strains  RIII,  y,  I  and  0^7  BL  are  t©o  susceptible 


PROJECT  DESCRIPTION  (Cont.  )  ^  ..  ^ 

to  the  toxic  effects  of  stilbestrol. 
-  #       • 

The  third  study  was  designed  to  ascertain  the  influence  of 
age  upon  susceptibility.  Results  between  young  (2  months  old)  and 
older  mice  are  not  available  but  mice  less  than  one  month  of  age  were 
very  svBceptible  to  toxic  effects  of  stilbestrol  and  those  that 
survi-^ed  have  not  developed  tumors.  The  chief  objsctive  of  this 
experiment,  however,  is  to  determine  the  susceptibilities  of  2,  6 
and  12  month  old  animals. 

Breast  Tumors. 

But  on§  experiment  gave  sufficient  findings  during  the  year 
to  report  at%bis  time.  This  experimeRt  consisted  of  administration 
of  stilbestrol  and  .progesterone  to  agent-free  strain  C3K  females 
to  ascertain  their  influence  upon  the  occurrence  of  breast  tiimors. 
Our  untreated  breeding  females  show  a  breast  tumor  incidence  of. less 
than  ^fc. 


Group        No.  of 
mice 

TJo.  with 
subcutane- 
ous tumors 

Average 
age  in 
months 

No. 
living 

10'^  stilbestrol 
pellet 

Ul 

10 

2li 

18 

10'?  stilbestrol 
pellet  plus 
progesterone 

39 

2U 

20 

9 

20^  stilbestrol 
pellet 

37 

10 

19 

XI 

/ 


PROJECT  DESCRIPTION  ("C^rit.) 

Living  mice  are  between  21  and  26  months  of  age.  Ttmifrs 
are  called  subcutaneous  tumors  because  they  have  not  been  diagnosed 
histologically. 

It  is  seen  that  thus  far  both  10^  and  205?  stilbestrol 
pellets  have  increased  the  incidence  of  tumors.  Also,  progesterone 
plus  IC?^  pellets  produces  more  tumors  than  do  10^  pellets  alone.  ■ 

This  experiment  was  performed  to  determine  the  influence 
of  progesterone  when  administered  to  agent- free  mice.  The  aim 
of  the  work  was  to  apply  the  findings  to  our  colony  of  wild  house 
mice  which  are  known  to  carry  the  mammary  tumor  agent  but"  develop 
few  breast  tumors  when  bred  or  given  pellets  of  stilbestr»l.  It 
is  considered  essential  to  obtain  breast  tumors  in  these  animals 
and  to  test  the  tumors  for  the  presence  of  the  agent.  Exploratory 
findings  with  agent-free  strain  G3F  mice  suggest  the  use  of  b«th 
stilbestrol  and  pr%.gesteroae  in  the  wild  mice. 


PROJECT  RE.ORT  FOR^    (C«nt.) 

SERIAL  no; 


11. 

BUDGET  ACTIVITY: 


RESEARCH       X  ADMINISTRATj ON 

REVIEW  •^.  APPROVAL  TECHIMICAL  ASSISTANCE) 

12. 


COOPERATING  UNITS,  OF  T^'E  PUBLIC  HEALTH  SERVICE,   OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,   FACILITIFS,   OR  PE-^SONNEL  FOR  THIS    PROJECT   IN  EITHER 
1956  or  1957» 


13. 

IF  T~aS  PROJECT  RESEMBLES,  COIr^LEMENTS,   OR  PAJliLLELS  RSSE/vRCH  Da-JE 


ELSEvrfHERE  IN  THE  PUBlIC  HKILTH  SERVICE   (¥..T!"OUT  INTERCHANGE  OP 
PERSONNEL,   FACILITIES  OR  FUNDS),    IDENTIFY  SUCH  RESS'RCH: 


i^OJECT  REPORT  FORM  (C»nt. ) 


SER,L\L  NO. 


15. , _ 

PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROK  THIS  PROJECT  DTJRIM} 


CALENDAR  YE/lR  1955 


Andervont,  H.  B. j  Biological  background  for  experimental  work  •n 

tumors.  In;  Canadian  Cancer  Conference,  Vol.  I;  2-2U.  Academic 
Press,  Inc.,  New  York,  N.  Y.  1955» 

\ndervont,  F,  B.  and  Dunn,  T.  B.:  Transplantation  of  hepatomas  in 
mice.  J.  Nat.  Cancer  Inst.,  l5t  l5l3-l52U,  1955. 


16.  

HONORS  AND  AWARDS  TO  PERSONAL  RELAtING  TO  Tf^IS  PR-'JECT  DURING 
CALENDAR  YE\R  1955. 


H.  B.  Andervont,  President,  American  Association  for  Cancer  Research. 


PAGE  1 

...  „  PROJECT  REPORT  FORM 

1 .  National  Cancer  Institute    2 .  Laboratory  of  Biology \ 

INSTITUTE        ~  LABORATORY  OR  BRANCH 

3/  ^  '    '     ■■"'"':■    .  .  ''  h.  ,    '         '    '  $.  hoi 

,$ECTioiJ  ok  ^mm'  ; — ' LocA'rloM(tf  'o^m  ium  feETH.) '  &tk.   Mo. 

6 .Sedimentation  behaviour  and  homngefteity  of  enzymes  and  other  compounds 
,  PROJECT  TITLE    in  crude  'solution  (continUeti  project). 


7.  Drg.  G.  F.  Hogeboom  and  E.L*  Kuff 

PRINCIPAL  INVESTIGATOR (3)  .,  '  ,  ' 

8.  Drs.  W.  C.  Schneider,  B.  R.  Hill,  J.  L»^lrvin;  Miss  V.   F.  Embrey 
OTHER  IWESTIGATORS 

9. PROJECT  .DESCRIPTION 

This. project  is  a  collaborative  effort  and  includes  several  phases,  some 
'of  •which  are  not  yet  comgleted. 

Methods  employed;  As  described  previously,  the  method  employed  involved 
centrifugation  under  convection  free  conditions  and  subsequent  sampling 
and  analysis  of  the  fluid  column  at  successive  levels  from  top  tn 
bottom.  The  techniques  were  devised  in  this  laboratory  (G.  ^.  ^ogeboom 
and  E.  L.  Kuff,  J.  Biol.  Chem.  210,  733-7^1  (19^1i)). 

Major  Findings  and  Proposed  Course  of  ^^rojectt 

(a)  In  collaboration  with  Dr.  Borroughs  R.  Hill  of  the  City  of 
Hope  Medical  Center,  an  investigation  was  made  of  the  sedimentation 
behaviour  of  the  lactic  dehydrogenase  present  in  the  serum  of  normal 
and  leukemic  patients.   (It  had  been  previously  shown  that  the  lactic 
acid  dehydrogenase  content  of  leukemic  serum  was  greatly  elevated). 
The  centrifugation  studies  conducted  in  thxs  laboratory  demonstrated 
clearly  that  the  enzyme  present  in  leukemic  serum  behaved  in  exactly 
the  same  fashion  (within  the  limits  of  experimental  error)  as  dii 
lactic  dehydrogenase  obtained  from  normal  serum.   It  is  our  under- 
standing that  Dr.  t'Ih  will  continue  the  work,  using  electrophoresis 
and  other  methods  of  fractionation,  and  that  the  results  will  be 
published  when  this  latter  phase  is  completed. 


PROJECT  DESCRIPTION  (Cont.) 

(b)  Several  years  ago,  it  was  shown  in  this  laboratory  that  over 
50  per  cent  of  the  total  nitrogen  of  isolated  liver  mitochondria  was 
■  in- the  form  of  soluble  proteins."  It  was  also  shown  that  the  major  ta.-"' 
component  of  the  mixture  (apparently  a  monodisperse  globulin)  was  ''■ 
absent  from  the  mitochondria  of  a  hepatoma.  A  number  of  attempts  to 
determine  the  significance  of  the  latter  finding,  using  standard 
techniques  of  protein  fractionation,  have  not  met  with  sufficiant  success 
to  warrant  publication.  It  is  also  true  that  liver  mitochondria  contain 
a  number  of  enzymes  that  can  be  obtained  in  the  soluble  phase.  With 
the  new  technique  of  analytical  centrifugation  mentioned  above,  initial 
studies  have  been  made  of  the  DPN-cytochrome  c  reductase  and  glutamic 
dehydrogenase  released  from  isolated  mitochondria  by  treatnBnt  with 
sonic  oscillations.  The  results  thus  far  indicate  that  DPN-cytochrome  c 
reductase  is  polydisperse  and  thus  is  probably  firmly  bound  to  the 
structural  fragments  pf   mitochondria,  .At  the  centrifugal  forces  thus 
far  employed,  glutamic  dehydrogenase" has  not  been  sedimented'far 
enough  to  warrant  any  firm  conclusions  as  to  its  characteristics. 

During  the  next  year,  it  is  planned  to  make  a  further  study  of  the 
soluble  fraction  obtainable  from  liver  mitochondria,  with  respect  both 
to  the  enzymes  present  (e.g.,  ribonuclease  ani  desoxyribonuclease)  and, 
if  possible,  to  the  protein  that  is  absent  fr«m  hepatoma  mitochondria 
but  present  in  l-^^-rge  amounts  in  liver  mitoc'-  ondria, 

(o)   It  is  also  planned  to  investigate  the  sedimentatL on  behaviour 
of  a  number,  of  enz.ynes  localized  in'  the  soluble  fraction  of  the 
cytoplasm,  including  several  of  those  which  are  involved  in  purine 
metabolism  and  have  not  been  obtained  in  a  pure  state  ("cf.  Schneider, 
W.  C,  and  Hogeboom,  G.  ";i. ,  Intracellular  distribution  of  enzymes  IX. 
Certain  purine  metabolizing  enzymes,  J.  Biol.  Chem.  19$,  I6I-I66  (1952)). 

(d)   In  collaboration  with  Dr.  J..L.  Irvin,  a  study  will  be  made 
of  the  heterogeneity  of  desoxyribose  nucleoprotein  isolated  from  the 
nuclei  of  liver  cells.  Some  work  on  the  rate  of  incorporation  of 
labelled  amino  acids  into  the  nucleoprotein  (in  collaboration  with 
Dr.  Stetten)  is  alsc  contemplated. 

Significance  to  Cancer  Research;  It  is  hoped  that  the  work 
outlined  above  will  provide  a  rational  background  to  future  similar 
studies  of  neoplastic  tissues. 


PROJECT  REPORT  FORM  (Cont'd.) 

10.  hOl 

SERIAL  NO. 

11. 


BUDGET  ACTIVITY;,. 

RESEARCH      X  AH-lINISTRATION 

REVIEW  lit.  k^?m'VkL  TECHNICAL  ASSISTANCE 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEAIjh  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FCR  THIS  PROJECT  IN  EITHER 
1956  or  1957 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLE^■IENTS ,  OR  P:\R\LLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PHBLIC  HEALTH  SERVICE  (wriHOUT  INTSRCHANCE  OF 
PERSONNEL,  FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PROJECT  RE^T'RT  FORM    (Cont'd) 

m.   J4OI 

SERI'^L  NO. 

15. : . 

PIIBLIC-TIONS  OTHER  THAN  ABSTRflCTS  FROM  T'^S   PRUSCT  DURHJG  CALEJID/iR 
YF'VR  1955 

E,   L.   Kuff,  G.   1^'.   Hogeboom,   and  M.   J.   Striebich,   the  Sedimentation 
biihaviour  of  alcohol  dehydrogenase  and  urease  in  crude  solntions, 
J.   Biol.  Cham,   212,  ii39-UU8     (1955). 


16. 

HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  TFIS  PROJECT  DURING  CALEND/vR 
YEAR  1955. 

F»r  honors  and  awards,  see  Annual  Report  of  Kuff,  hogeboom,  and  Dalton. 


PAGE  1 
PROJECT  REPORT  FORM 


1, National  Cancer  Institute 2 .   Laboratory  of  Biol»gy 

IICTITUTE  LABORATORY  OR  BRANCH 

3. Cellular  Biology  Section  k^  $•  U02 

SECTION  OR  SERVICE  LOCATION(IF  OTHm  TH^IN  BETHESDAj        SER.    NO. 

6.  The  relation  between  cell  chendstiy  and  cell  structure 

PROJECT  TITLE  ^  '    '       "  " 


7«  E«  L.  Kuff,  G»   ^»  Hoge^oom^  A.   J.   Dalttn 
PRINC IPAL  I  WEST  IGATOR  (S )  ^~" 

8.  M.  F.  Embrey 


OTHER  IWESTIGATORS 

9*   PROJECT  DESCRIPTION 

Objective;  (a)  To  determine  the  distribution  of  enzymes  and  other  com- 
pounds of  biochemical  importance  among  the  particulate  structures  re- 
leased by  mechanical  disruption  of  normal  and  malignant  cellj  (b)  to 
characterize  morphologically  the  intracellular  structures  thus  released; 
and  (c),  thus  to  arrive  at  an  interpretation  of  the  fine  structure  of  the 
cells  in  terms  of  biochemical  function.  Results  obtained  by  the  use  of 
more  precise  and  sensitive  methods  for  investigating  the  sedimentation 
properties  of  cytoplasmic  particulate  material  have  strengthened  the  con- 
clusion that  these  particles  fall  into  a  relatively  limited  number  of 
distinct  classes,  each  of  which  exhibits  a  peculiar  array  of  biochemical 
properties.  Simultaneously,  electron  microscopy  has  greatly  broadened 
our  knowledge  of  the  fundamental  structural  units  of  the  intact  cell. 
Correlation  of  these  two  fields  of  information  is  an  obvious  necessity. 

Methods  employed;  The  following  methods,  developed  in  this  laboratory, 
have  been  employed  for  the  observation  and  separation  of  groups  of 
cytoplasmic  particles  from  homogenates  of  tissue:  (1)  size  distribution 
analyses  of  the  particles  based  upon  the  rates  of  sedimentation  of  their 
specific  biochemical  characteristics  (see  the  last  annual  report  for 
description  of  method);  (2)  differential  centrifugation  according  to 
schedules  based  upon  the  size  distribution  analyses;  and  (3)  density 
gradient  fractionation.   In  the  last  method,  which  is  currently  under 
investigation,  a  small  volume  of  homogenate  is  layered  over  a  pre- 
viously prepared  continuous  sucrose  density  gradient,  and  the 
particles  centrifuged  (in  the  SW-39  rotor  of  the  Spinco  Model  E  ultra- 
centrifuge)  to  their  iso-density  levels.  Fractions  obtained  by  any 


PROJECT  DESCRIPTIOW  (Cont.) 


of  the  above  methods,  and  characterized  biochemically,  have  been 
prepared  for  election  microscopy, by  fixation  with  osmic  acid  and  em- 
bedding in  methacrylate.  Structures  observed  in  thin  sections  of  the 
fixed  fractions  were  compared  with'lihose  found  in  the  whole  horaogenates 
and  in  the  intact  cells. 

Results;  As  descrifesd  in  the  last  annual  report,  size  distribution 
analyses  of  the  cytoplasmic  particles  in  rat  liver  homogenates  had 
demonstrated  three  major  classes  of  particles.  Of  these,  one . undoubtedly 
corresponded  to  the  mitochondria.  Electron  microscopy  has  now  revealed 
that  the  mitochondria  are  well  presei^^-ed  during  the  process  of  homogeni- 
zation  in  30=^  sucrose,  a  fact  of  some  importance  in  interpreting  the  ' 
many  results  hithert^  obtained' *)y  differential  'centrifugation.  The  • 
nature  of  the  other  two  groups  of  particles  is  not  yet  understood.  One 
of  these,  rich  in  acid  phosphatase  and  uricase,  has  been  tentatively 
identified  with  elements  of  the  ergastoplasm  of  the  intact  cell.  There 
is  some  evidence  that  the  other  group,  containing  the  non-mitochondrial 
DPN-cytochrorae  c  reductase  activity  of  the  cell,  may  be  made  up  at  least 
in  part  by  structures  derived  from  the  Golgi  complex. 

Considerable  attention  has  been  devoted  to  the  question  of  the 
intracellular  distribution ' of  ribonucleic  'afcid  (RNA)  in  view  of  the 
mounting  evidence  for  the  participation  tf  this  material  in  protein 
synthesis.  RNA  has  bten  found  to  be  associated  with  all  three  of  the 
above-mentioned  particle  groups  in  liver  horaogenates:  about  10/^  with 
the  mitochondria,  30%   with  the  "aoid  phosphatase  particles",  an4  '^0% 
with  the  group  of  smaller  particles  rich  in  DPN-cytochrbme  c  reductase. 
The  remainder  is  riot  sedimentable  in  30°^  sucrosd  at  the  speeds  attain- 
able with  the  SW-39  rotor.  Ifoon  severe  starvation,  the  RNA  associated 
with  the  acid  phosphatase  particles  disappears  almost  completely,  while 
that  of  the  smaller  particles  remains  Tit  is  known  that  starvation  re- 
sults in  loss  of  the  ergastoplasmic  elements  of  the  liver  cell  as 
examined  in  situ).  In  the  very  ra-^id  growth  situation  provided  by 
Strain  L  fibroblasts  grown  in  tissue  culture  ^by  the  method  of  Dr.  Harry 
Eagle)',  preliminary  studies  have  shown  that  iTiOSt  of  the  crboDlasmic 
RNA  is  in.  the  non-sedimentable  fraction. 

The  sedimentation  characteristics  of  liver  gl^roogen  as  it  occurs  -> 
in  sucrose  homogenates  has  also  been  studied.   If  a  density  of  l*$i   gm,/cm. 
is  assumed,  the  relatively  polydisperse  glycogen  particles,  show  an 
average  "molecular  weight"  of  about  20,000,000,  a  value  from  $  to  10  times 
those  reported  for  liver  glycogen  isolated  bv  standard  chemical  methods. 


PROJECT  DESCRIPTION  (Cent. ) 


Significance  to  Cancer  Research;  It  is  felt  that  a  combination  of 
the  techniques  of  cellular  fractionation  and  electron  microscopy  will 
result  in  a  considerable  extension  of  our  knowledge  of  the  spatial  and 
functional  arrangement  of  intracellular  materials.  This  information, 
p?n*ticularly  with  regard  to  components  such  as  ribonucleic  acid,  may 
ife  fel^'ftol  ip,  u^derstand?^^  tiie.  proeesses  df  .celluLat  groi^hj-  &M   in 
■plr^tidniar,  IM  &if  I'erdTitxa^ing  tfeweori  ndlr'ffi'ai  an'S-  afcnofmal  ftgefearastfev 
of  protein  synthesis.   In  addition,  the  ability  to  correlate  appear- 
ance under  the  electron  microscope  with  known  biochemical  properties 
would  greatly  facilitate  the  investigation  of  many  situations  not  now 
accessible  to  study  -  for  example,  the  very  early  stages  of  malignancy 
where  changes  might  be  restricted  to  too  few  cells  to  analyse  by 
presently  available  methods. 

Proposed  course  of  the  project;  During  the  coming  year  it  is  proposed 
to  investigate  in  more  detail  the  sequence  of  events  which  occur  at  the 
time  of  disruption  of  cells  during  the  preparation  of  homogenates.   Pre- 
liminary observations  of  unbroken  and  partially  broken  ceils  in  homo- 
genates indi6«tes  that  this  approach  may  be  the  best  means  of  determin- 
ing the  intracellular  situs  of  origin  of  many  now  unidentified  particu- 
lates. This  aspect  of  the  project  will  be  carried  out  with  the  help  of 
a  new  microtome  for  thin  sectioning  and  in  collaboration  with  Dr.  F.  S. 
Sjostrand  who  will  be  visiting  here  from  the  Karolinska  Institute  of 
Sweden. 

With  the  more  precise  methods  of  cell  fractionation  now  available, 
it  it  proposed  to  continue  the  study  of  the  major  ribonucleic  acid  con- 
taining fractions  of  normal  cells  and  to  compare  them  with  the  correspond- 
ing fractions  derived  from  rapidly  growing  ceils,  both  normal  and  malig- 
nant. An  interesting  situation  is  provided  by  the  liver  cells  of 
animals  which  have  been  subjected  to  severe  fasting.  As  mentioned  above, 
the  ergastoplasmic  elements  are  l«st  from  such  cells,  and  there  is  a 
great  reduction  in  the  volume  of  cytoplasm.  TTpon  refeeding,  theTe  is 
a  rapid  resynthesis  of  cytoplasm  without  cell  division,  together  with 
characteristic  evolution  of  the  reforming  ergastoplasm.   It  is  prooosed 
to  compare  the  cytochemical  changes  occurring  during  this  period  with  the 
findings  in  the  more  usual  situations  where  growth  is  accompanied  by 
cell  division. 


PROJECT  RE:^ORT  FORM  '  (Cont'd) 


10.  1;02 

SERIAL  NO. 


11. 


Ri:SEa.RC;'  "'  ^  ,0'I>;iSTRATi-N 


12 

COOPER^'^TTNG  Ili^lTS  Oh'  THE,  p-TELxC  iIMLTH  SAVICS,   OH  OTHER  ORG.UIiZi-^lUld, 
PROVILING  FITOS,  FACILITIES,  OR   PFRSOMSIL  FCE  T"IS   FROJEGT  iiv  EITHER 
1956  or  ]'?57 

D^".  F.   3..  o.^o.=^*-,rand,  Karolinsk?   Institutet^  Sufd-n, 


■"'i?  T^^3   mojECT  ■Hi!;aii;t'lBfeV'GO>''PI'SS'ENTS,   or  P\R:\LLELS  RESEfJlCF  DONE 
ELSF^'-'ERE  IN  TIE  F^'BLiC  TTE'-lTt  S '^JIVICE,    (WITFOIlT  INTERCHANGE  OF 
PERSONNEL,  FACILITIES  OR  FJltoS),   IDENTIFY  SUCH.  RESE/JICH 5  ■  " 


PROJECT  RE^'^RT  FORII   (Cont'd) 


lii.     U02 


SERIAL  NO. 
1^. 


RT'ELICATIONo   OT^^R  T'^-.N  /IBSTR'lUTS  FR'M  T^-IS   FRCJZCT   DURING  C/iLE^TD/Jl 
YE'i-R  10^5 

T-'ogeboom,  G,   'W,^    ^.nd  Ki:ff,  E.   L.- ,  Relat-ion  between  nell  structure  and 
cell  chemistry.   Federation  Proceedings  lIi ;  633-638      ri955). 


16. 


HONORS  AND  WARDS  TO  PERSOM'JEL  REUTING  TO  T'-'IS  ^^ROJECT  DITRING  CALENDAR 
YE!\R  3-955. 

(a)  G.  '-.  Hogeboom:  By  irvitation,  addressed  the  Open  Scientific 
Session  of  the  Federation  of  American  Societies  for  Experimental 
Biology,  San  Francisco,  April,  1955.  (See   ref .  l5) 

(b)  E.  L.  Kuff ,  by  invitation,  presented  paper  (with  G.  '-^^   Hogeboom 
as  co-author)  at  Fourth  International  Sjnnposium  on  Enzymes:  ^"nits  of 
Biological  Structure  "-  Function,  on  "Sedimentation  and  biochemical 
characteristics  of  cytoplasmic  particulates."   (To  be  published  in 
Proceedings  of  the  Symposium) 

(c)  G.  H.  Wogeboom  acted  as  chairman  of  the  Session  on  Enzymes  and 
Cell  Structure  at  the  above-mentioned  Fourth  International  S3miposium 
held  at  the  Henry  Ford  Hospital. 


PAGE  1 


PROJECT  REPORT  FORM 


1»  National  Cancer  Institute    2 .  Lalioratory  of  Biology 

INSTITUTE  LABORATORY  OR  BRAMCH 

3.  Cellular  Biology  Section    h .__ ^ «  UoU 

SECTION  OR  SERVICE  LOCATION(IF  OTHER  THAN  BET"ESDA)  SER.  NO. 

6.0xidatiY8  metabolism  in  various  cellular  structures  of  normal  and  tumor 
PROJECT  TITLE      tissues. 


7.  Ruth  K.  Kielley 

PRINCIPAL  INVESTIGATOR(S) 


OT"ER  INVESTIGATORS 

9.   PROJECT  DESCRIPTION 

Title !  Oxidative  metabolism  in  various  cellular  structures  of  normal 
and  tumor  tissues.  Xanthine  Oxidase. 

Ksthods  employed!  Xanthine  oxidase  and  DPNH  dehydrogenase  enzjrmes  were 
isolated  from  the  supernatant  fracti'^n  of  liver  cells  bjr  protein 
fractionation  methods  described  in  a  previous  annual  report.  The  be- 
haviour of  these  enzymes  in  response  to  changes  in  environmental  con- 
ditions was  studied  by  quantitative  measurement  of  their  activities* 
Xanthine  oxidase  activity  was  measured  spectrophotometricaily  by 
following  the  rate  of  uric  acid  formation  from  the  oxidation  of  xanthine 
at  290  mu»  DPNH  dehydrogenase  activity  was  also  measured  spectro- 
photometricaily ■fey  following  the  decolorization  rate  of  dichloropheno- 
lindophenol  at  600  mu.  The  extent  to  which  these  enz,ymes  couple  with 
various  nitrogen  compounds  was  determined  by  estimation  of  either  the 
reduction  products  formed  or  the  reactants  disapoearing.  The  methods 
were  chemical,  spectrophotometric  or  enzymatic  depending  upon  the 
nature  of  the  product  analyzed.  Molybdenum  in  trace  amoimts  was  de- 
termined by  a  special  modification  of  the  colorimetric  thiocyanate 
method. 


PROJECT  DESCRIPTION  (Cont.) 


Objectives;  (1)  To  obtain  information  on  the  mechanism  of  reduction 
of  various  nitrogen  compounds  by  xanthine  oxidase  and  Ijy  other  as  yet 
unidentified  enzymes  of  liver  and  tumor  and  the  relation  of  molybdenum 
to  the  reduction  process,  (2)  to  identify  and  characterize  other 
enzymes  in  liver  and  tumor  tissues  which  catalyze  similar  reduction 
reactions  and  (3)   to  study  ways  and  means  by  which  knowledge  gained 
in  these  investigations  can  be  applied  to  cancer  problems. 

Major  Findings;  It  has  been  found  that  the  xanthine  oxidase  of  liver 
can  be  coupled  to  the  reduction  of  various  types  of  nitrogen  compounds 
representing  a  wide  range  of  oxidation-reduction  levels.  The  mechanism 
of  electron  transfer  to  these  nitrogen  compounds  appears  to  require 
molybdenum  as  a  specific  metal  activator,   ^^ophosphate  inhibited 
these  reductions  indicating  that  the  reduction  process  is  metal 
catalyzed.  It  was  shown  that  the  molybdenum  present  in  the  enzyme 
is  very  tightly  bound.  All  attempts  to  remove  it  reversibly  have  so 
far  been  unsuccessful.  Further  evidence  that  the  reduction  of  nitro, 
nitroso  and  probably  other  related  groups  including  the  azo  group 
proceeds  through  an  intermediate  step  between  the  dehydrogenase  and 
the  final  transfer  of  electrons  to  the  N  acceptor  was  obtained  b^  the 
demonstration  that  the  rate  of  reduction  was  not  directly  proportional 
to  the  total  dehydrogenase  activity  (xanthine  and  DPNH) .   In  the  re- 
action of  the  enzyme  with  ox^rgen   or  dyes  such  as  methylene  blue  or 
dichlorophenolindophenol  on  the  other  hand,  the  total  dehydrogenase 
activity  was  the  rate-limiting  reaction.  Examples  of  the  types  of 
nitrogen  compounds  reduced  by  liver  xanthine  oxidase  and  their  re- 
lative reduction  rates  are  as  follows:  Inorganic  Series;  nitrate  18, 
nitrite  2h,  hydroxy lamine  5h.  Organic  Series;  p-nitrophenol  3$, 
p-nitrosophenol  2^3,  p-dimethylaiuinoazobenzene  (DAB)  20,  2,$-dinitro- 
phenol  108,  2,It-dinitrophenol  6^,   2,6-dinatrophenol  $0,      In  general, 
it  may  be  said  that  for  the  oxidation-reduction  levels  they  represent, 
the  organic  compounds  wore  considerably, more  reactive  than  the  in- 
organic. The  azo  group  of  DAB  was  significantly  reduced  although 
at  a  relatively  slow  rate.  With  the  dinitrophenols,  the  position  of 
the  substituents  in  the  ring  greatly  influenced  the  reactivity  of  the 
compound.  2,5-dinitrophenol  containing  nitro  groups  para  with  respect 
to  each  other  was  more  active  than  its  isomers. 

Further  studies  on  nitre  and  nitroso  reductases  of  liver 
confirmed  previous  suggestions  that  a  second  enzyme  was  present  in  the 
supernatant  fraction  of  liver  cells.  The  enzyme  was  found  to  be  a 
flavoprotein  specifically  catalyzing  the  oxidation  of  DPNH  in  the 
presence  of  suitable  electron  acceptors,  but  unlike  the  great  bulk 
of  diaphorase  activity  found  in  the  liver,  this  flavoprotein  has  the 


PROJECT  DESCRIPTI'-^N  (Cont.  ) 


unique  property  of  being  able  to  couple  with  nitro  and  nitroso 
compounds  (other  N-containing  groups  have  not  yet  been  tested).  Al- 
though xanthine  was  not  oxidized  by  this  enzyme,  there  is  a  suggestion 
that  xanthine  oxidase  is  closely  associated  with  this  second  flavo- 
protein  enzyme  since  the  latter  is  released  from  crude  xanthine 
oxidase  preparations  by  heat  treatment. 

The  effect  of  TSPA  (N,  N' ,  N' '  -triethylene  thiophosphoramide) , 
an  anti -cancer  agent,  on  the  aerobic  oxidase  activitj'-  and  on 
p-nitrosophenol  reduction  catalyzed  by  liver  xanthine  oxidase  was 
tested  tQ  determine  whether  either  or  both  of  these  electron  trans- 
ferring pathways  of  the  enzyme  might  be  affected.  The  results  were 
entirely  negative  showing  neither  inhibition  nor  activation. 

Preliminary  work  on  the  determination  of  xanthine  oxidase 
levels  in  blood  showed, that  the  activity  was  limited  to  the  plasma 
component  and  that  it  could  be  conveniently  measured  spectrophoto- 
metrically  provided  the  sensitivity  of  the  instrument  were  stepped 
up  sufficiently  with  a  photomultiplier.  The  feasibility  of  determin- 
ing xanthine  oxidase  levels  in  blood  was  looked  into  because  there  is 
some  interest  in  examining  the  blood  levels  in  cancer  patients. 

Significance  to  Cancer;  Xanthine  oxidase  as  one  of  the  principal 
enzymes  of  purine  catabolism  is  of  special  interest  in  the  cancer  field 
because  of  its  relation  to  nucleic  acid  synthesis  and  breakdown.   Its 
presence  in  excess 'or  absence,  its  inhibition  or  stimulation  might 
possibly  reflect  on  the  chemistry  of  normal  or  abnormal  growth.  Haddow 
et  al.  found  for  example,  that  xanthopterin,  a  potent  inhibitor  of 
xanthine  oxidase  in  vitro,  often  induced  hypertrophy  of  the  kidney 
in  rodents.  He  also  observed  that  xanthine  oxidase  concentrates  from 
cow's  milk  when  injected  into  mice  with  spontaneous  mammary  tumors 
produce  apparently,  anti- tumor  effects. 

Recent  developments  in  the  chemotherapy  of  cancer  and  leukemia 
with  various  purine  analogs  point  to  the  importance  of  xanthine 
oxidase  as  one  of  the  cnntrolling  factors  in  the  effectiveness  of  the 
treatment.  Dietrich  and  Shapiro  found  that  flavotin,  a  riboflavin 
analog,  potentiates  the  carcinostatic  action  of  8-azaguanine  through 
inhibition  of  tumor  xanthine  oxidase  (7^5  mammary  carcinoma).  This 
inhibition  results  in  the  accumulation  of  xanthine  which  in  turn 
inhibits  guanase,  an  enzyme  destroying  8-azaguanine  by  deamination. 
The  over-all  effect  of  flavotin  then,  is  apparently  one  of  preventing 
the  destruction  of  the  carcinostatic  purine.   In  this  connection  one 


PROJECT  DESCRIPTION  (Cont. ) 


might  consider  the  possibility  of  limiting  the  synthesis  or  activity 
of  xanthine  oxidase  by  antagonism  of  the  molybdenum  component  rather 
than  the  flavin  component  of  the  enzyme.  The  toxicity  experienced 
■with  flavotin  may  very  well  be  due  to  inhibition  of  a  large  number  of 
flavoprotein  enzymes  whose  activities  are  more  directly  and  vitally 
concerned  with  the  energy  metabolism  of  cells  than  is  xanthine  oxidase. 
As  far  as  it  is  known,  the  only  flavoproteins  containing  molybdenum 
in  mammalian  tissues  are  xanthine  oxidase  and  possibly  aldehyde  oxidase. 

Another  aspect  of  the  relationship  of  xanthine  oxidase  to  cancer 
is  the  ability  of  the  enz"\Tne  to  reduce  the  azo  .group  of  carcinogens. 
After  the  results  on  the  reduction  of  DAB  were  reported,  it  was  learned 
that  similar  observations  were  being  made  at  the  Chester  Beatty  Cancer 
Institute  where  an  extensive  progr.'^.m  of.  research  on  xanthine  oxidase 
is  being  conducted.  Furthermore,  their  observations  indicate  that 
there  is  an  inverse  relationship  between  rate  of  reduction  and  carcino- 
genicity (private  communications).  Mueller  and  Miller  have  shown  that 
DAB  is  also  reduced  by  an  enzyme  in  liver  microsomes,  presumably 
TPM-cytochrome  c  reductase,  a  flavoprotein.  It  is  highly  probable 
that  other  metall«-flavoproteins  of  the  xanthine  oxidase  type  such  as 
liver  aldehyde  oxidase  and  the  DPNH  dehydrogenase  described  in  this 
report  can  also  participate  in  reductive  detoxications  of  azo  carcino- 
gens.  The  relationship  between  carcinogenesis  caused  by  DAB  and  the 
riboflavin  level  of  the  diet  becomes  clearer  when  it  is  learned  that  the 
enzymes  involved  in  the  reduction  of  DAB,  as  far  as  we  know,  are 
flavoproteins. 

Proposed  research;  On  the  hypothesis  that  tumor  growth  may  be  influenced' 
by  changes  in  the  balance  and  activities  of  critical  enzymes  in  t-hese  ■ 
tissues,  it  is  proposed  that  investigations  be  made  on  the  effect  of 
mimosine,  an  iinnatural  amino  acid,  on  animals  bearing  soontaneous  tumors. 
Studies  en  the  physiological  effects  of  t;'is  compound  are  limited  to 
observations  on  rats  in  which  the  symptoms  were  those  of  a  deficiency 
disease  including  marked  alopecia,  diminished  growth  rate  and  in  many 
cases  of  long  duration,  cataract.  The  mode  of  action  in  terms  of 
enzyme  systems  affected  is  unknown,  but  on  the  basis  of  its  known 
structure  (3-hydroxy,  U-Oxy,  (Uk),  1-pyridine  alanine),  several  possi- 
bilities can  be  considered.  The  most  likely  sites  of  antagonism  wouJ^d 
appear  to  lie  in  enzymatic  reactions  in  which  pyridoxine  is  implicated. 
There  is  already  work  by  Dietrich  and  Shapjro  on  the  carcinostatic 
properties  of  (-'.esoxypyridoxine,  a  pyridoxine  analog  and  antagonist. 
Other  possibilities  are  that  mimosine  may  interfere  with  protein 
synthesis  or  amino  acid  metabolism  involving  phenylalanine  or  tyrosine. 
At  the  present  time,  arrangements  are  being  made  with  Dr.  Evan  Horning 
to  obtain  the  compound  in  quantity  from  imported  plant  material. 


PROGRESS  DESCRIPTION  fCont.) 


Some  investigations  on  the  xanthine  oxidase  levels  in  blood 
plasma  of  mrmal,  non-cancerous  and  cancerous  subjects  are  being 
considered  in  collaboration  with  Dr.  F.  M.  Kalckar,  The  purpose  is 
to  determine  whether  xanthine  oxidase  levels  can  be  correlated  with 
the  degree  of  differentiaticn  typical  of  the  tumor. 

From  the  standpoint  of  cellular  structure  and  function,  it  is 
of  some  interest  to  know  whether  enzymes  occurring  in  one  part  of  the 
cell  can  be  coupled  to  enzymes  in  another  structure  of  the  cell.   In 
the  intact  cell  where  enzymes  are  acting  in  heterogeneous  systems, 
such  a  possibility  does  not  seem  to  be  out  of  reason.  The  xanthine 
oxidase  system  would  appear  to  be  an  appropriate  model  for  experiment- 
ally testing  out  such  a  possibility.  There  is  evidence  that  in 
mammalian  tissues,  "xanthine  ocidase"  consists  of  two  components,  a 
dehydrogenase  and  a  terminal  oxidase  system.  The  purified  xanthine 
oxidase  of  rat  liver  is  an  enzyme  no  longer  associated  with  its 
natural  terminal  oxidase  system,  but  in  the  process  of  separation  and 
purification,  has  become  an  oxidase  reacting  directly  with  atmospheric 
oxygen.  The  nature  of  the  natural  terminal  oxidase  system  of  xanthine 
oxidase  has  not  been  determined.  It  would  be  of  great  interest  and 
significance  if  it  were  localized  in  the  mitochondria  for  the  linkage 
would  then  provide  a  possible  means  for  the  utilization  of  a  large 
amount  of  energy  resulting  from  the  oxidation  of  xanthine  and  hypo- 
xanthine  for  useful  cellular  work  through  the  mechanism  of  oxidative 
phosphorylation.  With  these  ideas  in  mind,  it  is  proTD:  sed  that  ex- 
periments be  tried  to  determine  whether  enzymatic  coupling  can  be 
achieved  between  the  xanthine  dehydrogenase  of  the  supernatant  fraction 
and  the  electron  transport  mechanism  in  mitochondria. 


PROJECT  REr-ORT  FORM    (Cont'd) 


10. 


kok 


SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 


RESEARCH  X  AMINTSTRATION 

REVIEW  /k  A'-^RQVAL  TEC^-^ICAL  ASSISTANCE 


12. 


CO-OPERATING  UNITS  OF  THE  PUBLIC,  HEALTH  SERVICE,  OR  OTHER  ORGAN IZ -,T IONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  "^ERSOMNEL  FOR  T^'IS  PROJECT  IN  EITHER 
1956  or  1957 


13. 


IF  T-'IS   PROJECT  RESEMBLES,   COMPLEMENTS,   OR   "-ARALLELS  RESEARCH  BOM 
ELSEnPERE  IN  THE  PUBLIC  HEALTH  SERVICE   (WITHOUT   INTERCHiVNGE  OF 
PERSONNEL,  FACILITIES  OR  FU1©S),   IDEivTIFY  SUCH  RESEilRCH: 


PROJECT  REPORT  FORM  (Cont'd) 


lii.   liOii 


SERIAL  NO. 


15. ^ 

PUBI.ICAT IO^B  OTHER  TPAN  ABSTRACTS  FROM  r'lS  PROJECT  DURING  CALENDAR 
YEAR  1955 


Klelley,  R.  K.,  Purification  of  liver  xanthine  oxidase.  Federation 
Proc,  lU:  235  (1955). 

Kielley,  R.  K.,  Piirification  of  liver  xanthine  oxidase.   J.  Biol. 
Chem. ,  216:  Uo5  (1955). 

Kielley,  R.  K.,  Reduction  of  2,U-dinitrophenol  by  liver  xanthine 
oxidase.  Third  International  Congress  of  Biochemistry,  page  ^^ 

(1955). 


16. 


HONORS  'IND  A¥\RDS  TO  ^  ^^ONNEL  RELATING  TO  T'-^IS  PROJECT  DIRING  CALENDAR 
YKAR  1955. 


PAGE  1 
.  .     PROJECT  REPORT  FORM 
1, National  Can<j«r  Instituta 2«  Laboratory  of  Biology 

it^tJiTWE : t^wmfM  oft  mm   

3.  Cellular  Biology  Section      It*  $  U04 

3EcT!dK  &k  SffiVKg ' —     t.«)AfTt)H(l^  (^rMi  TriAU'BfiM.)  Sfcft.No. 

6. Electron  mieresawjy  and  phas^e  contrast  microacopy  of  normal  and  malignant 
PROJECT  T.fTLE         cells 

7.  A»  J.  Dalton  and  Marie  D.  Felix 

PRINCIPAL  INVESTIGATOR (S)       ' 

8»  J»  Bronte  Gatenl^,  Harry  Eagle^  Clifford  Grobstein  and  W»  Ray  Bryan 
OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 


A  comparative  study  of  the  Golgi  complex  with  the 
electron  microscope 

The  objective  of  this  project  is  to  determine  the  essential 
variations  in  the  fine  structure  of  the  Golgi  complex  in  representative 
cell  types  in  several  phyla  of  the  animal  kingdom. 

The  methods  employed  are  fairly  well  standardized  now  -  fixation 
in  chrome-osmium,  double  embedding  in  fom^l-agar  and  n-butyl  methacrylate 
followed  ¥y  thin  sectioning  for  study  with  the  electron  microscope. 

The  major  finaiings  have  l|eai  the  demonstration  that  (1)  the  double 
membranes  and  small  vesicle  components  &f  the  Golgi  comples  were 
regularly  present  Ih  all  vertebrate  cell  types  studied  but  the  large 
vacuolar  component  occinred  regularly  only  in  highly  specialized  cell 
types.  (2)  The  fine  structiire  of  the  Golgi  complex  of  vertebrate  and 
invertebrate  cells  is  basically  similar,  the  double  memTirane  systems 
and  the  small  vesicle  component  being  present  in  both.   (3)  The 
walls  of  contractile  vacuoles  of  protozoa  and  soonges  possess  double 
membrane  systems  and  small  vesicles  similar  morphologically  to  the 
Golgi  complex  of  vertebrate  and  invertebrate  cells. 


PROJECT  DESCRIPTION  (Cont.) 

The  significance  of  these  findings  is  related  to  the  over-all  ■ 
concept  which  activates  much  of  the  work  of  the  unit  -  the  concept 
that  in  order  to  understand  the  mechanisms  involved  in  the  development 
of  neoplastic  cells,  much  greater  knowledge  of  the  structure  and  function 
of  normal  cells  is  needed.  The  results  obtained  from  this  study  suggest 
first  that  the  Golgi  complex  is  universally  present  in  animal'  cells 
and  second  that  the  Golgi  complex  is  possibly  involved  in  control  of 
water  balance  in  cells  generally  since  its  structure  is  so  similar  to 
that  of  the  walls  of  the  contractile  vacuole ^of  many  protozoa. 

It  is  proposed  to  study  a  series  of  somatic  cells  ef  inverte- 
brates and  the  contractile  vacuoles  of  several  more  protozoa  to 
make  the  above  generalizations  somewhat  more  secure. 

A  comparison  of  normal  and  malignant  cells  with 
the  electron  microscope 


The  object  of  this  project  was  to  determine  whether  any 
significant  differences  in  the  fine  structure  of  normal  and  neoplastic 
cells  could  be  detected. 

The  major  findings  have  been  that  while  variations  in  the  amount 
of  various  cell  components  were  noted,  neoplastic  calls,  as  might  be 
expected  possess  all  the  essential  components  of  normal  cells.  The 
fine  structure  of  chromatin,  nucleoplasm  and  nucleolus  was  found  tc 
be  the  same  in  both.  Both  normal  and' neoplastic  cells  possess 
ergastoplasm  in  variable  amounts  but  there  was  a  tendency  in  what 
might  be  termed  anaplastic  tumor  cells  for  the  small  granule  component 
of  cytoplasm  (thought  to  be  responsible  for  cytoplasmic  basophilia) 
to  lie  free  ifi  the  cytoplasm  rather  than  to  be  associated  with  the 
membranes  of  the  eragstoplasm  (the  usual  situation  in  normal  Bells), 
The  Golgi  complex  in  tumor  cell  types  which  retain  some  degree  of 
function  possesses  all  three  c®mponents,  (double  membrane  system,  small 
vesicles  and  large  vacuoles)  but  the  Golgi  complex  of  more  anaplastic 
tumor  cell  types  possessed  only  the  double  membrane  system  and  small 
vesicles. 

•     ■   „ 
:  While  it  was  not  expected  that  a  significant  qualitative  differ- 
ence between  ■'normal  ancJ  neoplastic  cells  would  be  found,  such  a  study 
was  indicated  since  if  such  a  difference  were  found  it  would  be  of 
considerable  importance  in  diagnosis.   If  a  real  and  consistent  corre- 
lation is  found  between  variations  in  the  fine  structure  of  the 
ergastoplasm  and  Golgi  complex  and  th»  degree  of  anaplasia  of  malig- 
nant, such  a  corj-elation  could  eventually  be  of  some  value  to  the 
CTtopathologist  iii'44i^g'^osis. 

In  the  future  i^is  proposed  to  study  a  series  of  tumor  cell 
types  ef  varying  degrd,p'  of  anaplasia  to  determine  whether  the 
variations  described  above  are  consistently  associateddin  a  general 
way  with  the  degree  of  anaplasia ^ 


PR':^JECT  DESCRIPTION  (Cont.) 

Phase  contrast  and  electron  microscopy  of  tumor  cells  developing 
nutritional  deficiencies  in  tissue  culture 


The  objective  of  this  project  is  to  determine  whether  there  are 
any  specific  alterations  in  the  fine  structure  of  tumor  cells  during 
the  development  of  a  deficiency  state  resulting  from  the  absence  of  a 
specific  amino  acid  or  vitamin. 

In  addition  to  thfe  usual  methods  for  electron  microscopy  of 
tissue  sections,  a  method  of  double  embedding,  first  in  formol-agar 
and  then  in  methecrj/late,  vias  developed.-  This  double  embedding 
method  makes  possible  the  accurate  orientation  of  cells,  tissues  and 
organs  and  also  greatly  improves  the  preservation  of  cells  on  free 
surfaces  and  in  tissue  culture.  During  the  development  of  the 
deficiency,  living  cells  have  be-n  studied  with  phase  contrast  and 
comparable  cells  preserved  for  study  with  the  electron  microscope. 

The  major  findings  to  date  are;  (1)   piodification  of  the 
fine  structure  of  cells  appear  to  be  specifically  related  to  a 
deficiency  in  a  particular  amino  acid.  For  example,  changes  in 
mitochondria  and  the  ergastoplasm  occur  in  tyrosine  deficiency  which 
have  not  lieen  noted  in  dying  cells  in  control  cultures  nor  in  cells 
developing  glutamine  or  folic  acid  deficiency,  Cej.ls  developing 
tyrosine  deficiency  have  been  studied  in  detail  but  the  studies  on 
glutamine,  folic  acid  and  glocuse  deficiency  are  still  in  progress. 

The  significance  of  these  results,  which  are  meager  at  present, 
is  in  the  suggestion  that  specific  changes  may  occur  under  these  con- 
ditions and  that  these  changes  may  be  pinpointed  in  relation  to  bio- 
chemical and  radio-isotope  analyses.  While  these  changes  may  in  many 
cases  be  an  "end  result"  and  thus  not  necessarily  indicators  of  the 
site  of  utilization  or  metabolism  of  a  particular  amino  acid  within 
a  tumor  cell,  the  very  real  possibility  remains  that  the  combination 
of  electron  microscopic,  biochemical  and  radio-isotope  studies  may 
eventually  give  us  this  information. 

It  is  proposed  to  continue  this  study  with  a  more  detailed 
analysis  of  the  changes  induced  by  glutamine,  folic  acid  and  glujose 
deficiencies.  This  material  is  already  fixed  and  embedded  but  not 
sectioned  or  examined. 


PROJECT  ■DESCRIPTION  (Cont.) 

A  study  of  the  cells  native  to  the  peritoneal  fluid 

The  objective  of  this  project  is  to  develop  a  greater  under- 
standing of  the  structure,  function  and  origin  of  cells  native  to 
the  peritoneal  fluid. 

In  this  study  living  cells  ■were  examined  with  the  phase  contrast 
microscope  both  as  an  aid  to  identification  and  as  a  prerequisite  for 
study  with  the  electron  microscope. 

The  major  findings  have  been  that  the  primary  cell  components 
of  the  peritoneal  fluid  are  lymphocytes  and  macrophages.  The  intro- 
duction of  foreign  particulate  matter  to  the  peritoneal  cavity  is 
followed  by  an  early  rise  in  neutrophilic  granulocytes  which  is 
followed  in  t\irn  by  a  more  prolonged  rise  in  the  number  of  macro- 
phages. Under  these  conditions  numerous  transitions  between  lympho- 
cytes and  macrophages  were  founri.  There  was  no  evidence  that  meso- 
thelial  cells  transformed  into  macrophages.  Neither  was  these  any 
evidence  that  mesothelial  cells  contribute  significantly  to  the  cell 
population  of  the  peritoneal  fluid.. 

There  has  recently  been  a  great  increase  in  the  number  of 
papers  published  which  described  work  using  ascites  tumors  as  tools 
in  cancer  research.  It  is  surprising  that  in  none  of  these  has  any 
significant  attention  been  paid  to  the  cells  native  to  the  peritoneal 
fluid.  On  the  contrary,  the  idea  that  the  majority  of  these  cells  are 
der.ivatives  of  mesothelium  appears  to  have  been  accepted  by  many 
investigators  in  soite  of  the  lack  of  evidence  for  such  a  derivation. 
In  the  study  of  the  development  of  ascites  tumors  and  in  the  associated 
problem  of  identification  of  tumor  cells,  the  information  obtainable 
by  the  application  of  the  newer  methods  is  important.  Our  work  has 
made  a  beginning  in  this  direction. 

It  is  proposed  to  develop  this  project  further  by  determining 
the  reaction  of  normal  cells  of  the  peritoneal  fluid  to  the  intro- 
duction of  tumor  cells  and  to  study  the  early  stages  in  the  develop- 
ment of  .ascites  tumors,  with  both  the  phase  contrast  and  electron 
microscopes. 

Other  projects  involving  electron  microscopy  but  which  will  be 
covered  in  detail  by  the  investigators  responsible  for  preparing  the 
materials  ai«e : 

(a)  Electron  microscopy  of  tissue  fractions.  Drs .  Hogeboom 
and  Kuff . 

(b)  Electron  microscopy  of  the  process  of  embryonic  induction. 
Dr.  Grobstein 

(c)  Electron  microscopy  of  the  Rous  tumor.  Dr.  Ray  Bryan 


PROJECT  REPORT  FORM  (Cont'd) 

10.   Uo6 

SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH    X  ADMINISTRATION 

REVIEW  k  k??ROVkL  TECHNICAL  ASSISTANCE 


12. 


CO-^^ERATiNG  UNITS   OF  THE  PUBLIC   HEALTH  SERVICE,   OR  OTHER  ORGANIZATIONS, 
PROVIDING  RTlvIDS,   FACILITIES,   OR  PERSOMEL  FOR  THIS   PROJECT   IN  EITHER 
1956  or  19^7 

In  cooperation  with  Prof.   J.Bronte  Gatenby,   University  Zoological 
Department,  Trinity  College,  Dublin,   Ireland. 


13. _     __     _ 

IF 'T WIS   PROJECT  RESE^''BLES,  COMPLSI^IENTS,   OR  PARALLELS  RESEARCH  DONE 


ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE   (WITHOUT  I'JTSRCHANGE  OF 
PERSONNEL,   FACILITIES  OR  FUNDS),    IDENTIFY  SUCH  RESEARCH: 


PROJECT  REPORT  FORM  (Cont'd) 

ili.   Uo6 

SERIAL  NO. 

15. . 

P'^LIC^TIONS  OTHER  TH^N  ABSTRACTS  FROM  THIS  PROJECT  DllRING  CALEMDAR 
YEAR  1955 

Gatenby,  J.  Bronte,  Dalton,  A.  J.  and  Felix,  Marie  D. ;  The 
contractile  vacuole  of  parazoa  and  protozoa  and  the  Golgi 
apparatus.  Nature,  176j  301  (1955). 

Dalton,  A,  J.;  A  chrome-osmi-um  fixative  for  electron  microscopy. 
.\nat.  Rec.  121:  281  (1955).   (Note.   While  this  is  of  abstract 
length  it  is  the  definitive  article  on  this  subject). 

Felix,  Marie  D.   and  Dalton,  A.   J.:  A  phase-contrast  microscope 
study  of  free   cells  native  to  the  peritoneal  fluid  of  DBA/2  mice. 
J.    Mat.   Cancer  Inst.   16:  Iil5-hii5      (1955). 


16. 

HONORS  \m   AWARDS  TO  PERSONNEL  REUTING  TO  TWIS  PROJECT  DITRING    ' 
CALENDAR  YEAR  1955. 

Invited  to  take  part  in  a  symposium  on  "Mitochondria  and  other  cell 
inclusions"  sponsored  by  the  British  Society  of  Experimental  Biology 
at  Oxford,  England,  Sept.  19-23.   *.t  this  symposium  I  was  asked  to 
serve  as  chairman  of  a  section  on  the  Golgi  apparatus. 

Invited  to  take  part  in  Symposium  on  "Cancer  Cytology  and  Cjrbo- 
chemistry"  soonsored  jointly  by  the  N.  Y.  Academy  of  Sciences  and 
the  Damon  Runyon  Memorial  Fund  for  Cancer  Research. 

Invited  to  serve  as  chairman  of  section  on  cytology  (electron 
microscopy)  American  issociation  of  Anatomists  Annual  Meeting, 
Philadelphia,  1955. 

Invited  to  take  part  in  symposium  on  cancer  research.  Harvard  Univ. 
Medical  School,  Harwich,  Mass.,  June,  1955. 


PROJECT  REPORT 


l»National  Cancer  Instituta    2»  Laboratory  of  Biology 

3 .  Cellular  Biology  Section  h . 5 .  U07 

SECTION  OR  SERVICE  LOCATION (IF  OTHER  THAN  BETH.)  SER.  NO. 

6.  Precursors  of  Deoxyribonucleic  Acid  (DNA)  in  normal,  Regenerating  and 
PROJECT  TITLE  Tumor  Tissues. 


?•  Walter  C.  Schneider 

PRINCIPAL  INVESTIGATOR (S) 


8.  Mrs.  Leona  W,  Brownell 


9.   PROJECT  DESCRIPTION 

Objective;  To  determine  whether  precursors  of  DNA  exist  in  normal  and 
tumor  tissues  and  if  so,  to  isolato  and  identify  the  compounds 
present  and  determine  the  metabolic  pathways  in  which  they  aro  inter- 
mediates. 

Methods!  Tissues,  obtained  under  anesthesia  and  frozen  immediately  in 
liquid  nitrogen,  are  extracted  with  ice  cold  perchloric  acid,  Deoxy- 
ribosidic  compounds  are  isolated  from  such  extracts  using  both  ion 
exchange  and  paper  chromatography*  The  compounds  are  detected, 
identified  and  quantitated  with  the  organism  lactobacillus  acidophilus 
R-26  used  in  conjunction  with  spectrophotometric  and  chemical  tests. 

Fajor  findings;  Using  more  refined  methods  of  isolation  it  has  bee;:) 
possible  to  show  that  deoxycytidine  is  present  in  rat  liver  and 
accounts  for  essentially  the  entire  deoxvribosidic  activity  of 
extracts  of  this  tissue. 

In  contrast,  extracts  of  the  Novikoff  hepatoma  showed  three 
striking  differences  in  the  type  of  deoxyribose  compounds  they  con- 
tained. The  deoxyriboside  fraction  accounted  for  only  about  $0  per 
cent  of  the  microbiological  activity  of  extracts  of  this  tissue  and 
contained,  in  addition  to  deoxycvtidine  deox^oaridine  and  thymidine. 
The  remainder  of  the  microbiological  activity  has  not  yet  been  identi- 
fied but,  judging  from  its  behavior  on  ion  exchange  resins,  probably 
contains  either  nucleotides,  small  polynucleotides,  or  phosphorylated 
([derivatives  of  nucleotides. 


PROJECT  DESCRIPTION  (Cont.) 


Studies  of  rat  liver  undergoing  regeneration  have  shown' that  the 
total  amount  of  deoxvi'ibosidic  compounds  present  is  approximately 
doubled  as  early  as  2k   hours  after  partial  hepatectomy,  -Further- 
more, in  contrast  to  normal  liver.  2^-50  per  cent  of  the  total 
deoxyribosidic  eompoiinds  appear  to  be  the  unidentified  nucleotide 
type .   ■  -  ;  ' 

Significance  to  cancer  research;  The  fundamental  problem  of  cancer 
research  is  to  determine  why  cancer  cells  are  able  to  multiply 
without  restriction  whereas  the  multiplication  of  normal  cells 
occurs  at  a  controlled  rate,  Deoxyribonucleic  acid  (DNA),  more- than 
any  other  cellular  constituent  now  known,  must  be  intimately  in- 
volved in  the  solution  of  these  problems  for  several  reasons.   In 
the  first  place,  DNA  is  not  only  a  major  comoonent  of  the  chromosomes, 
but  also  occurs  in  the  cell  in  a  constant  amount  directly  proportional 
to  the  number  of  chromosomes  present.  Furthermore,  before  cell  ., 
division  can  occur,  the  entire  DNA  of  the  cell  must  be  reduplicated 
to  insure  that  each  daughter  cell  contains  the  full  complement  of 
DNA.   In  addition,  DNA  alone  or  in  combination  with  protein  has 
been  found  capable  of  transforming  cells  of  one  genotype  to  another 
.and  even  of  Inducing  cancer. 

The  fact  that  DNA  occurs  in  such  constant  amounts  and  is 
capable  of  inducing  heritable  changes  makes  it  of  the  greatest 
importance  to  determine  how  this  compound  is  formed  in  the  cell, 
in  terms  of  the  prtcursor  compounds  and  enzymes  involved.  Until 
such  inf orma-^j. on  is  obtained,  attempts  at  blocking  DNA  sjmthesis, 
and  henoe  at  controlling  cell  division,  must  be  entirely  haphazard. 
The  results  obtained  so  far  in  this  project  represent  a  considerable 
advance  toward  the  achievement  of  this  goal  since  they  demonstrate 
that  specific  nucleosides,  hitherto  unrecognized  as  precursors  of 
DNA,  are  widely  distributed  as  tissue  constituents.  Since  these 
compounds  are  qualitatively  different  in  the  normal  and  tumor 
tissues,  the  s\Tithesis  of  DNA  in  the  latter  may  proceed  along  a 
different  pathway.   If  this  proves  to  be  the  case,  the  possibilities 
of  controlling  cell  multiplication  in  tumors  would  he  greatly 
increased.  The  continued  study  of  DNA  precursors  by  means  of  their 
isolation  and  identification  as  well  as  by  the  elucidation,  through 
enzymatic  and  isotopic  methods,  of  the  metabolic  pathways  involved 
is  consequently  of  utmost  im  ortance  for  cancer  research. 

Proposed  course:  The  identification  of  the  nucleotide  like 
compounds  will  receive  the  greatest  emphasis  during  the  ensuing 
year.   4  stirvey  of  the  occurrence  of  these  compounds  in  various 
tissues  is  also  pla'-^ned  and  it  is  hoped  that  it  will  also  Ise  (; 
oossible  to  begin  work  on  the  metabolism  of  the  deoxyribose  compounds. 


PROJECT  FORM      (Cont.) 

10.       U07 

SERIAL  NO. 

11. 

BITDGET  ACTIVITY  J  ~"      '     ' 

RESEARCH         X  ADMINISTRATION 

REVIEW  ^.  APPROVAL  TEC^'NICAL  ASSISTANCE 

12  i ■ -  "  

COOPERATING  UNITS   OF  THE  PUBLIC  HEilLTH  SERVICE,   OR' OTHER  ORGAN " ZATIONS , 
PROVIDING  FUNDS,   FACILITIES,  OR   PERSONNEL  FCB  THIS    PR'JECT  IN' EITHER 
1956  or  1957  '      • 


13. 


IF  T"TS   PRVECT  RESEI'BLES,   COMPLEMENTS,   OR  PARALLELS  RESEARCH  DONE 
ELSEW'ERE  IN  THE  PnBLIC   HEILTH  SERvICE   (WITHOUT   INTERC'-IANGS  OF    PERSONNEL, 
FACILITIES  OR  FUNDS),   IDENTIFY  SUCH  RESEARCH; 


PROJECT  REPORT  FORM   (Cont'd) 


lii.    Uoy 


SERI'iL  NO. 


1$.  ■■  ■_  •__•'„ 

PITBLICA.TTONS   OTHEft  T"^N   IBSTR/ICTS  FROM  T^-^IS   PRVECT  DURING  CALENDAR 

YE.AR  1955 

1.  Schneider,  W.  CtS  Deoxyril^osldes  in  animal  tissues.  J,  Biol. 
Chem.  2J6,   287  (1955). 

2.  Hogeboom,  G.  w.  and  Schneider,  W-.C:  The  Cytoplasm.   In  The 
Nucleic  /Vcids,  Vol.  2.  Edited  by  Chargoff  and  Davidson, 

New  York,  1955. 


16. 


HONORS   ftND  WARDS  TO  PEftSONl.EL  RELATING  TO  r'lS   PROJECT  DURE>JG 
CALENDAR  YEAR  1955. 


PAGE  1 
PROJECT  REPORT  FORM 

1 .  National  Cancer  Institute   2 .  Laboratory  of  Biology , 

BIc-TITTITE-  '  LABOR 'VTORI  OR  BRIKCH 

3.CellulTi:^  B-L0log7  Section U- ^-   hOS 

SFCT;~TO:rSE'AniCL  LOCAT--TJ?Tf  OC^lR  TR.N  .TETtTIsDA)      SER.NO. 

6.   studies  on  t^-e  morpVolotj.s-  a.id  phy3iolog-.^  c."  t'fio   lyr:phrcytic  tumors 

PRajLcT  tTtTs 


7.   Emma  ohelton .■• 

?RlF«IP'iL  IIvVESTIGi^.X.!Fs7~ 

8 . 

OTPEi".'  Ir'VEiJTIGATORS 

9.   PROJECT  IESCRIPTjOK 

Objective:  To  correlate  differences  in  the  laorphology  and  physiology 
oFTwoTymphocytic  tumors  with  the  diifer-ences  in  their  behavior  in 
the  host. 

^'"ethods;  In  order  to  t^.st  the  ability  of  the  cells  of  lymphacytic 
ascites  tumors  to  pass  t'lrough  membranes  of  known  pore  size,  measured 
numbers  of  cells  are  sandwiched  betwetn  two  pieces  of  Schleicher  and 
Schuell  membrane  filter.  Th=:-  cells  are  enclosed  by  sealing  the  membrane 
filters  to  a  circle  of  Ivicite,  The  sandwicn  is  placed  in  the  periton- 
eal cavity  of  a  mouse.  By  making  biopsy  punctures  of  the  abdomen 
and  counting  differentially  the  cells  in  the  ascites  fluid  it  is  poss- 
ible to  tell  with  considerable  accuracy  the  time  at  which  the  cells  ■ 
escape  from  the  chambers ^ 

Major  Findings; 

1.  Cells  of  Lymphoma  i2    (diploid  tunor,  highly  invasive)  survived 
and  grew  in  chambers  (10  mice)  for  a  period  of  50  days.  The  cells  did 
not  escape  from  the  chambers.  When  the  contents  of  these  chambers  were 
injected  subcutaneously  into  mice,  tumors  were  produced  in  every  case. 
Similar  results  were  obtained  w?i®n  ch^ambers  were  placed  in  17  additional 
mice  for  periods  ranging  from  2  to  28  da:(,'S, 


PROJECT  DESCRIPTION  (Con't.) 


2.  Cells  of  Lymphoma  #1  (tetraploid,  relatively  non-invasive) 
escaped  from  the  chambers  and  produced  ascites  tumors  in  four  out  of 
a  group  of  10  mice.   In  five  of  the  remaining  mice  killed  after  35 
days,  a  few  viable  cells  were  present  in  the  chambers.   Inoculation 
of  the  chamber  contents  has  produced  a  subcutaneous  tumor  in  the 
host  mice  in  only  two  cases.   In  the  last  mouse,  killed  aftef  $9   days, 
no  viable  tumor  cells  were  observed  in  the  chamber  and  no  tijimor  has 
appeared  at  the  site  of  the  inoculation  of  the  chamber  contents. 

The  final  outcome  of  the  project  cannot  be  foreseen  as  yet, 
since  the  results  are  not  clear-cut.  The  question  of  leaks  in  the 
llJambers  due  to  incomplete  sealing  of  the  filter  is  raised.  The 
indications  are  that  the  cells  of  L*^'2  have  a  greater  capacity  t« 
survive  and  miiltiply  in  the  chambers  than  do  the  cells  of  L0., 

Significance  to  cancer  research;  The  nature  of  the  invasive  properties 
of  tumor  cells  is  little  understood*  These  experiments  are  being 
carried  out  in  the  hope  that  they  will  shed  some  light  on  this 
phenomenon. 

Proposed  course;  /Idditional  experiments  with  chambers  constructed  of 
membranes  of  graded  porosity  are  under  way.  It  is  planned  to  apoly 
this  technique  to  a  greater  variety  of  ascites  tumors  than  the  two 
mentioned  above. 

Cinematographic  studies  on  the  locomotion  of  these  cells  in  vitro 
are  planned. 


PROJECT  REPORT  FORM  (Cont'd) 


10,     ii08 


SE'^FiL  MO. 
11. 


BUDGET  ACTIVITY: 

RESEARCH  X  AD"'"INISTRATION 

REVIEW  &  APPROVAL  TEC^-'i\"ICAL   ASSISTANCE 

12. I 

COOFERATING  UNITS   OF  THE  PUBLIC  HEALTH  SERVICE,    OR  OTHER  ORG^NIZ.ITIONS, 
TROVIDING  FUNDS,   FACILITIES,   OR    PERSON^ffiL  FOR   T'-^IS   PROJECT   IN  EITHER 
1956  or  1957 


13. 


IF  THIS   PROJECT  RESETiBLES ,  COKPLE'ENTS,  OR   P.'^.RALLELS   RESE'IRCH  DONE 
ELSElfli'HE'-E   IN  THE  PUBLIC   HEALTH  SERVICE   (WITHOUT   INTERCHANGE  OF 
PERSONl'^'EL,   F^'CILITIES  OR  FUNDS),   IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEJ-iS   lii,   l5  "<  l6. 


PAGE  1 
PROJECT  REPORT  FORM 


1 . TTational  Cancer  Institute       2 .   Laboratonr  of  Biology 

lilSTITTntE  ~~       LABOR 'iTORT  OR  E-:\KOH 

3»Celli-'la:''  Bioiog/  Section    k. __5*Ji2^ 

'   3FCf:rrcrSE-'V^Zcl ^^       LOC'\T:.V>Tjn"^'  0':^E'H  TH.N  :iETTTE3DAj      SER.NO. 

6.   StucaeP__on_bhejno_rptolu^,r  a-id  phy3iolcg-,_'-__o/^  t-y  ryan.ohc corbie  tumors 
HICJLCf  TITIE        ~       "         " 


7 .   Emma  3he  Iton . 

PRI-NOIP-'iL  IIv\fESTIG'i':'CiT*Sl 

8. — 

Gl'FEr?   I:  VEdTIa/iTORS 

9.      PROJECT    "itSC'.IPT.iON 

Obj-ctive:  To  correlate  differences  in  the  morphologrr  and  physiology 
^T~l^oT.ymphocyt;  c  tumors  with  the  differences  in  their  behavior  in 
the  host. 

T'ethods:  In  order  to  t-st  the  ability  of  the  ceils  of  lymphscytic 
ascites  tumors  to  pass  firough  membranes  of  known  pore  size,  measured 
numbers  of  cells  are  sandiiiched  betWb>on  two  pieces  of  Schleichc-r  and 
Schuell  membrane  filter.  The  cells  are  enclosed  by  sealing  the  membrane 
filters  to  a  circle  of  lucitj.  The  sandwicn  is  placed  in  the  periton- 
eal cavity  of  a  mouse.  By  making  biopsy  punctures  of  the  abdomen 
and  counting  differentially  the  cells  in  the  ascites  fluid  it  is  poss- 
ible to  tell  -with  conside-'-able  accuracy  the  time  at  which  the  cells 
escape  from  the  chambers. 

Major  Findin^^st 

1.  Cells  of  Lymphoma  f2  (diploid  tunor,  highly  invasive)  survived 
and  grew  in  chambers  (10  nice)  for  a  period  of  50  days.  The  cells  did 
not  escape  from  the  chambers.  When  the  contents  of  these  chambers  were 
injected  subcutaneously  into  mice,  tumors  Wo-re  produced  in  every  case. 
Similar  results  were  obtained  when  chambers  were  placed  in  1?  additional 
mice  for  periods  ranging  from  2  to  28  da:s,'S. 


PROJECT  DESCRIPTION  (Con't.) 

2,  Cells  of  Lymphoma  #1  (tetraploid,  relatively  non-invasive) 
escaped  from  the  chambers  and  produced  ascites  tumors  in  four  out  of 
a_ group  of  10  mice.   In  five  of  the  remaining  mice  killed  after  35 
days,  a  few  viable  cells  were  present  in  the  chambers.   Inoculation 
of  the  chamber  contents  has  produced  a  subcutaneous  tumor  in  the 
host  mice  in  only  two  cases.   In  the  last  mouse,  killed  aftef  59  days, 
no  viable  tumor  cells  were  observed  in  the  chamber  and  no  tumor  has 
appeared  at  the  site  of  the  inoculation  of  the  chamber  contents. 

The  final  outcome  of  the  project  cannot  be  foreseen  as  yet, 
since  the  rosults  are  not  clear-cut.  The  question  of  leaks  in  the 
chambers  due  to  incomplete  sealing  of  the  filter  is  raised.  The 
indications  are  that  the  cells  of  Jj}2   have  a  greater  capacity  t« 
survive  and  raaltiply  in  the  chambers  than  do  the  cells  of  L#l. 

Significance  to  cancer  research;  The  natiire  of  the  invasive  properties 
of  tumor  cells  is  little  understood^  These  experiments  are  being 
carried  out  in  the  hope  that  they  will  shed  some  light  on  this 
phenomenon. 

Proposed  co'^rse;  /Idditional  experiments  with  chambers  constructed  of 
membrT.nes  of  graded  porosity  are  under  way.   It  is  planned  to  apoly 
.this  technique  to  a  greater  variety  of  ascites  tumors  than  the  two 
mentioned  above. 

Cinematographic  studies  on  the  locomotion  of  these  cells  in  vitro 
are  planned. 


PriOJECT  REPORT  FORM   (Cont'd) 


10.      U08 


SE^I/iL  NO. 
11. 


BUDGET  ACTIVITY: 

RESEARCH  X  /iD'-'TNISTRAT  ION 

REVIEW  &  A'-^PROVAL  TEC'-'NICAL   ASSISTANCE 

12. 

COOPERATING  UNITS   OF  THE  PUBLIC  HEALTH  SERVICE,    OR  OT  HER  ORG  AN  IZ  iTI OI© , 
PROVIDING  FUNDS,   FACILITIES,   OR    PERSON^iEL  FOR   T'-'IS   PROJECT   IN  EITHER 
1956  or  1957 


13. 


IF  THIS   PROJECT  RESEMBLES,  COKPLEi'.ENTS,  OR   P.'^/ftALLELS   RSSEflRCK  DON'E 
ELSE'aIHEwE   IN  THE  PUBLIC   HE\LTH  SERVICE   (WITHOUT   INTERCHANGE  OF 
PE^iSONNEL,   F\CILITIES  OR  FUNDS),   IDENTIFY  STICH  RESEARCH: 


NO  ENTRIES  FOR  ITEi^IS   lU,   iS  "-  l6. 


PAGE  1 

PROJECT  REPORT  FORM 

1» National  Cancer  Institute   2,  Labor attry  of  Biol»gy 

INSTITUTE  ■  ■   LABORATORY  OR  BRANCH 

3.  Cellular  Biology  Section   ib . ; ^ .  ItO? 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THA.N  BETHESDAj   SER.  NO. 

6.  studies  on  liv«r  carcinogenesis  in  the  mouse 

PROJECT  TITLE 

7.  Emma  Shelton 
PRINCIPAL  INTESTIGATOR(S) 


OTWER  liJVESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objective;  To  study  the  changes  occurring  in  the  parenchymal  cells 
of  the  liver  of  the  mouse  prior  to  the  formation  of  a  tumor,  and  to 
develop  methods  for  studying  these  changes. 

Methods: Nutritional,  biochemical,  histochemical 

Kajor  findings;  A  study  was  made  of  the  tetrazolium  method  for 
measuring  succinic  dehydrogenase  in  order  to  answer  the  questions: 

1.  Do  tetrazolium  salts  accept  electrons  directly  from 
succinic  dehydrogenase? 

2.  ^^ow  sensitive  is  the  tetrazolium  method  in  comparison  with 
oxr</"gen  uptake  as  a  measure  of  enzyme  activity? 

3.  Can  the  histochemical  tetrazolium  method  be  used  as  a 
reliable  quantitative  test  for  succinic  dehydrogenase? 

It  was  found  that: 

1.  Since  tetrazolium  reeuction  is  inhibited  by  antimycin, 

the  tetrazolium  salts  do  not  accept  electrons  directly  from 
succinic  dehydrogenase  but  require  the  mediation  of 
cytochrome  b. 

2,  The  tetrazolium  method  is  a  relatively  insensitive  method 
for  measuring  succinic  dehydrogenase,  being  in  the  neighbor- 
hood of  twenty-five  times  less  than  oxygen  uptake. 


PROJECT  DESCRIPTION  (Cont.) 


3.   In  spite  of  the  lack  of  sensitivity  of  the  method  and  the 
place  in  the  enzymatic  chain  of  elgctron  transfer  where  the 
enz3matic  activity  is  measured,  ratios  of  activity  of  liver 
to  kidney  to  brain  are  identical  using  both  tetrazolium 
^       and  oxygen  uptake.  Thus  the  tetrazolium  method  can  be  used 
as  a  valid  and  accurate- quantitative  measure  of  succinic 
dehydrogenase. 

The  very  small  tumors  appearing  in  the  livers  of  mice  fed 
o-amincazotoluene  have  a  higher  succinic  dehydrogenase  activity  than 
do  the  cells  of  the  surrounding  parenchsma  as  measured  hj   both  the 
tetrazolium  method  and  by  oxygen  uptake. 

Significance  to  cancer  research;  In  order  to  study  the  earliest 
changes  taking  place  in  livers  undergoing  the  malignant  alteration, 
histochemical  methods  of  known  accuracy  must  be  developed.   In  future 
exoeriments,  t'ne  tetrazolium  method  can  be  relied  upon  to  give 
accurate  estimates  of  the  succinic  dehydrogenase  activity  of  cells 
at  the  microscopic  level. 

Proposed  course  of  project;   Since  the  strnin  A  mouse  is  a  poor 
breeder,  and  is  thus  not  too  readily  available  for  use,  experiments 
are  under  way  to  ascertain  whether  a  high  percentage  of  tximors  may 
by  produced  in  the  more  readily  available  strain  C  mouse. 

A  quantitative  cvtological  and  histochemical  study  of  the  early 
stages  of  tum»r  formation  in  the  livers  of  these  mice  is  planned. 


PROJECT  REPORT  FORl^I    (Cont'd) 

10.     U09 

SERIAL  WO. 

11. 

BTTDGET  ACTIVITY: 

RESEARCH  X  ADMINISTR/ITION 

REVIEW  "<  APPROVAL  TECHNICAL  ASilSTAUCE 

12. 


Co-operating  units  of  t^e  public  health  service,  or  othf.r  or  can  izm  ions, 
providing  ttjnds,  facilities,  or  ^eisonijel  for  tuts  _^roject  in  either 
1956  or  1957 


13. 


IF  TWIS  PROJECT  RZSE^''BLES,   COMPLEMENTS,   OR   PARALLELS  RESEARCH  DONE 
ELSE'.ir"SRE  IN  THE  PTOLIC  HEALT^T  SERVICE   (WITHOUT   INTEPC^ANGE  OF 
PERSONIMEL,   FACILITIES   OR  FUITOS),    IDENTIFY  ST^CH  RESEARCH: 


PROJECT  RE^'iRT  FORf:  (Cont'd) 

li;.  it09 

SFR.I'rL  NO. 

15. 

PUBLIC-'.TlONS  OTWER  TW?.N  .\BSTRACTS  FROM  T"IS  PR'^JECT  DURING  CALENDAR 
YEAR  19^5 

Shelton,  E. :  i^epatomas  in  mice.  I.  Factors  affecting  the  rapid 
induction  of  a  high  incidence  of  hepatomas  by  c-amincazotoluene. 
J.  Fat.  Cancer  Inst.  l6i  107-12?,  1955. 


16. , 

HONORS  AND  AWARDS  TO  PKR30NML  RELATING  TO  T^'IS  PROJECT  DURING 
CALENDAR  YE/'iR  1955. 


PAGE  1 
PROJECT  REPORT  FORM 


1 .  National  Cancer  Institute     2 .  Laboratory  of  Biology 

INSTITUTE  LABORATORY  OR  BRANCH  '  ~^ 

3.  Cellular  Biology  Section k-  $.  UlO 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  T'-iAN  BETHESDA)SER.NO. 

6. Transparent  Chamber  and  Dif fusion  Chamber  Research 

PROJECT  TITLE     '        ^      '""    ^ 


7.  Glenn  H.  Algire ,   

PRINC IPAL  IWEST IGATOR  (3 )      ",       ~~ 

8 .Doctors  C»  C.  Congdon,  Y.  J.  Evans,  L»  W.  Lav,  R«  M.Merwin  and  J.N.  Weaver 

9.  PROJECT  DESCRIPTION 

No.'l 

Research  program  in  Transparent  Chamber  ■Project 

J^oblem:  Developments  in  Transparent  Chamber  Methods 

Objectives;  To  modify  transparent  chamber  designs  to  attain  increased 
simplicity  of  construction,  and  longer  useful  duration  of  the  chambers. 

Methods  employedj  Transparent  chambers  are  constructed  about  an 
internal  splint  of  plastic  which  provides  better  support' for  the 
skin  fold  than  earlier  designs  of  chambers* 

Major  findings:  These  new  forms  of  internal  splint  chambers  are 
being  utilized  in  current  work,  in  order  to  determine  their 
applicability  to  problems  in  which  observations  must  be  made  over 
periods  of  several  months. 

Significance;  It  is  hoped  to  extend  the  useful  duration  of 
transparent  chambers,  so  that  observations  of  cellular  and  vascular 
reactions  to  carcinogens  may  be  carried  on  for  longer  periods  of 
time  than  has  been  possible  heretofore. 

Proposed  course;  To  utilize  these  chambers  in  long  term  studies 
of  cellular  and  vascular  reactions  to  chemical  carcinogens. 


PROJECT  DESCRIPT'ON    (Cont.) 

Wo.   2 

...problem;  Factors  affecting  the  .survival  of  heterografts  in  diffusion 
chambers. 

Objectives;  From  preliminary  experiments  reported  last  year,  it  was 
found  that  himan  (HeLa)  cells  survived  for  at 'least  50  days  in  dif.fusioo 
chambers  in  mice,  but  after  two  weeks  extensive  areas  of  cellular  de- 
generation were  found.  Current  studies  are  directed  toward  a  study  of 
immune  factors  in  the  survival  and  growth  of  hum.an  cells  in  diffusion 
chambers.  Dr.  Virginia  Evans  has  furnished  the  cultures  of  HeLa  cells 
used  in  these  studies. 

• 
Methods ;  Human  cells  (HeLa  strain)  from  tissue  culture  were  placed  in 
diffusion  chambers  using  filters  of  estimated  porosity  O.ij.  microns. 
Observations  were  made  either  in  vivo  using  transparent  chamber  methods, 
or  as  fixed  and  stained  specimens  after  intraperitoneal  implantation 
and  later  removal. 

Major  findings:  HeLa  strain  of  human  cells  proliferate  rapidly  in 
diffusion  ch-ambers  in  normal  mice  for  about  2  weeks,  after  whxch  time 
areas  of  cellular  degeneration  are  noted,  but  laany  surviving  cells  are 
still  present  for  at  least  50  days.  Extensive  destruction  of  HeLa 
cells  occurs  wit'- in  7  davs  if  placed  within  diffusion  chambers  in  mice 
which  have  been  immunized  by  subcutaneous  inoculation  of  this  cell 
strain,  two  we  ks  prior  to  implantation  of  the  diffusion  chambers, 
indicating  that  circulating  antibodies  pass  through  the  filters. 

Significance  to  Cancer  Research;  This  is  a  study  of  the  reaction  of  an 
animal  to  grafts  of  human  cells,  and  the  pissibility  of  eliminating 
destructive  effects  in  order  to  obtain  growth  of  human  tumors  in  ex- 
perimental animals.  ' -ossible  application  to  diagnostic  problems  of 
cancer  will  be  considered,  as  well  as  studies  on  the;  mechanism  of 
action  of  drugs  capable  of  affecting  tumor  cells. 

Proposed  course;  Experiments  will  be  undertaken  on  the  effect  of  the 
porosity  of  membranes  on  survival  of  heterografts  in  diffusion  chambers. 
This  will  be  done  to  determine  whether  it  would  be  possible  to  find 
a  pore  size  that  would  allow  cell  growth,  but  would  not  allow  antigenic 
material  to  escape  from  the  chamber  and  immunize  the  host.  Will  it  be 
possible  to  obtain  prolonged  survival  and  growth  of  human  cells  in 
diffusion  chambers  by  blocking  the  escape  of  antigenic  material? 

The  studies  will  be  extended  to  other  cells  of  human  origin, 
both  normal  and  cancer. 


'^R'^JECT  DESCRIPTION  (Cont.) 

No.  3 

Problem:  Mechanism  of  action  of  Chemotherapeutic  Drugs  on  Tumor  Cells. 

Objectives;  To  determine  the  mechanism  of  action  of  various 
tumor-damaging  drugs  on  tumors  in  terms  of  their  circulatory  effects, 
or  direct  cvtotoxic  properties. 

Methods  emrjloved!  Studies  of  the  reaction  of  tumor  cells  to  injected 
drugs  were  made  under  three  conditions. 

(1)  Using  diffusion  chambers  in  which  implanted  cells  are 
isolated  from  contact  with  host  tissues  by  porous  membranes; 

(2)  Solid  vascularized  tumors  where  circulatory  effects  may 
be  involved J 

(3)  Solid  vascularized  tumors,  as  influenced  hy   injected 
drugs,  plus  procedures  to  alter  the  circulatory  response. 

I^ajor  findings;  ^;ouse  sarcoma  cells  growing  in  diffusion  chambers 
placed  intraperitoneally  are  not  ai'fected  by  bacterial  polysaccharide 
injected  intraperitoneally.  Subcutaneous  injection  of  podophyllo toxin 
results  in  mitotic  arrest  without  extensive  necrosis  of  cells  growing 
in  diffusion  chambers  in  the  absence  of  blood  supply.  This  result  is 
in  contrast  to  the  hemorrhage  and  necrosis  wh  .ch  occurs  in  solid 
vascularized  tumors  following  injection  of  either  of  these  drugs. 

Preliminary  evidence  has  been  obtained  that  increased  blood  : 
flow  caused  by  local  warming  of  tissue  may  orevent  the  tumor-damaging 
action  of  podophyllotoxin. 

Significance  to  Cancer  Rtjsearcht  Evaluation  of  therapeutic  agents  in 
cancer  depends  upon  understanding  their 'mode  of  action.  Transparent 
chamber  methods  for  microscopid  observations  in  living  mice, 
supoleniented  by  diffusion  chamber  procedures,  help  to  determine 
whether  an  agent  acts  directly  upon  the  living  cell  or  indirectly 
through  its  effects  on  the  circulation  of  the  host  animal. 

Proposed  Course  of  ^reject: 

1.  To  apply  the  diffusion  chamber  methods  to  studies 

of  the  effects  of  various  chemotherapeutic  drugs  on  cells  isolated 
from  host  tissues  with  particular  reference  to  leukemia  and  to 
human  tumors, 

2.  To  continue  studies  on  the  role  of  circulatory  responses 
in  the  action  of  tumor-damaging  agents. 


PROJECT  DESCRIPTION  (Cont.) 

No.  h 

Problem!  Mechanism  of  radiation'  protection  by  bone  marrow.   " 

Objectives;  Lorenz  and  coworkers  have  shown  that  injections  of 
isologous  or  homologous  bone  marrow  will  protect  mice  against  amounts 
of  X-radiation  which  are  lethal  to  uninjected  animals.  The  mechanism 
of  protection  is  not  clear.   One  hypothesis  is  that  the  injected  bone 
marrow  cells  survive  and  proliferate  to  take  over  the  function  of  the 
marrow  of  the  irradiated  mice;  another-,  that  protection  results  from 
some  as  yet  unidentified  humoral  factor.  The  object  of  these  experi- 
ments was  to  determine  whether  radiation  protection  can  be  brought 
about  by  bone  marrow  cells  enclosed  within  diffusion  chambers.  A 
positive  result  would  support  the  humoral  theory;  a  negative  result 
would  be  in  accord  with  the  cellular  rfepopulation  theory.  This  work 
was  done  in  cooperation  with  Doctor  C.  C..Congdon. 

Methods;  Determination  of  the  minimal  effective  number  of  marrow  cells 
necessary  for  protection  when  given  intraperitoneally;  inclusion  of 
this  number  of  cells  in  diffusion  chambers  (and  multiples  of  this 
dosage)  for  intraperitoneal  implantation  into  irradiated  mice  and 
determination  of  the  effect  on  survival  as  compared  with  controls. 

T^jor  findings;   It  has-been  found  that  marrow  cells  grow'within 
diffusion  chambers  which  are  placed  intraperitoneally.  The  dosage 
required  for  protection  when  marrow  cells  are  injected  directly  into 
the  peritoneal  cavity  has  been  found  by  Congdon  to  be  approximately 
5,000,009  cells.  This  number  of  cells  and  twice  this  population  en- 
closed within  diffusion  chambers,  were  placed  intraperitoneally  in 
previously  irradiated  mice.  The  results  were  negative,  that  is,  no 
protection  was  a'fcrded  by  bone  marrow  cells  enclosed  within  diffusion 
chambers  and  placed  intraperitoneally.   In  following  up  this  negative 
result,  the  volume  of  fluid  in  which  the  cells  were  suspended  proved 
to  fee  an  important  factor,  since  Congdon  found  that  protection  was  not 
obtained  after  i.p.  injection  if  the  cells  were  suspended  in  the  same 
volume  of  fluid  (.02  cc)  as  used  (necessarily)  in  diffusion  chambers. 

Significance  to  Cancer  Research;  Studies  on  radiation  protection  are 
of  importance  in  enhancing  the  value  of  X-radiation  as  a  therapeutic 
tool  in  cancer. 

Proposed  Course;  This  aporoach  has  been  discontinued. 


PRnjECT  DESCRIPTION  (Cont.) 

No.  5 

Problem;  Invasiveness  of  leukemia. 

Objeetives;  F'ouse  leiicemia  cells  differ  greatly  in  their  capacity  to  ■ 
invade  the  tissues  of  the  host.  Studies  ■were  undertaken  to  determine 
whether  this  property  is  correlated  mth  the  ability  of  these  cells 
to  pass  through  filters  of  graded  ooroSityr   Parallel  studies  of  . 
the  migrator^,^  behavior  of  leukemic  cells  are  being  carried  out  using 
time-lapse  motion  pictures.  ' 

Methods ;  Fragments  of  leukemia  1210  were  placed  in  diffusion  chambers 
formed  from  filters  ranL,'ing  from  h' millimicrons  to  O.U  microns 
aver-ige  pore  diameter.  Bi'^logieal  and  histologic  studies  were  made 
to  determine  the  fate  of  the  cells.  Siinilar  studies  are  in  progress  ■ 
using  leukemia  5l78,  a  less  invasive  neoplasm.  These  tumors  are  pro- 
vided by  Doctor  Lloyd  Law. 

Major  Findings;   In  the  systems  so  far  tested,  if  has  not  yet  been 
possible  to  consistently  prevent  the  development  of  leukemia  in  the 
hosts.  Current  studies  are  devoted  to  finding  out  whether  leukemic 
cells  pass  through  the  pores  of  the  filters  or  through  the  seal 
uniting  the  parts  of  the  chamber.  Alternative  exolanations  for  these 
results  are  poftsible  and  are  being  tested. 

Significance  for  Cancer  Research;  These  studies  are  concerned  with 
factors  which  promote  the  spread  of  cancer  cells  in  their  invasion 
of  normal  tissues.  Results  of  such  experiments,  using  a- series  of 
filters  of  graded  porosity  and  leukemlas  of  diverse  invasive  properties, 
will  contribute  to  an  understanding  of  the  mode  of  transmission  of 
leukemia  through  membranes,  and  to  a  characterization  of  membranes 
in  terms  of  their  properties  in  a  biological  system. 

Proposed  Course;  Studies  will  be  continued  as  described  above. 


PROJECT  DESCRIPTION  (Cont.) 

No.  6 

Problem;  Studies  in  carcinogenesis  using  diffusion  chamber  methods. 

Objective:  The  diffusion  chamber  method  for  "tissue  culture  in  vivo" 
provides  a  new  approach  to  certain  problems  of  chemical  carcino- 
genesis. Earle  and  associates  have  found  that  normal  adult  fibroblasts 
grown  in  tissue  culture  in  a  completely  heterologous  media  in  the 
absence  of  deliberately  added  MCA  give  rise  to  sarcomas  when  inoculated 
into  mice.  One  possible  explanation  for  these  results  has  been  "growth 
in  the  heterologous  media,"  This  explanation  would  be  ruled  out  if 
normal  or  embryonic  mouse  tissue  underwent  a  malignant  change  when 
enclosed  within  diffusion  chambers. 

Goldblatt,  following,  experiments  and  theories  of  Warburg  ''1927), 
has  presented  evidence  that  normal  fibroblasts  in  tissue  culture  undergo 
a  malignant  change  if  subjected  to  conditions  of  intermittent  anoxia, 
'■'■hrough  selecting  filters  of  increasing  thickness,  and  of  consequent 
greater  diffusion  distance,  one  could  study  the  effect  of  this  variable 
on  neoplastic  change. 

The  immediate  objective  is  to  ascertain  whether  aiult  or  embryonic 
subcutaneous  tissue  enclosed  within  diffusion  chambers  undergo  neoplastic 
transformation. 

I^ethods  employetl; 

1.  To  determine  whether  the  materials  (plexiglas  II,  and 
Millipore  filters)  are  carcinogenic  for  mice  -  (by  intraperitoneal 
and  subcutaneous  implantation). 

2.  Fragments  of  muscle  from  young  mice  (strain  C3w)  were 
enclosed  v:ithin  diffusion  chambers  and  placed  intraperitoneally 
into  adult  hosos  of  the  same  strain.  After  6  months  these  were 
reimplan^-ed  (open)  into  new  hosts  which  had  received  UOO-I4.25  r  total 
body  irradiation.  Another  series  will  be  done  after  12  months. 

In  other  experiments,  subcultures  were  made  each  month  for  6  months 
before  testing  for  neoplastic  transformation. 

Major  findings;  It  has  been  found  that  fragments  of  muscle  from 
young  mice  produce  a  vigorous  outgrowth  of  connective  tissue  cells, 
with  numerous  dividing  cells.  Adult  connective  tissue  cells  also 
migrate  out  in  diffusion  chambers. 


PROJECT  REPORT  FOM    (Cont'il) 


10.  -  Uie  • 


SERIAL  NO. 
11. 


BUDGET  ACTIVITY:     ■     ,  ■ 

RESEARCH      X  ADMINISTRATION 

REVIEW  &  APPROVAL    -     ■     TECHNICAL  ASSISTANCE 


12. 

COOPERATING  UNITS  OF  THE  PUBLIC- HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  T-'IS  PROJECT  IN  EITHER 
1956  or  15^7 


13, 

IF  TWIS  PROJECT  RESE?''BLES,   C0MPLE1«ENTS ,   OR   PAR^fiLLELS  RESEARCH  DONE 
ELSEVcHERE   IN  THE   P^IE^LIC   HK4LTF  SERVICE   (WITHOUT  I1^!TERC"-'ANGE  OF 
PERSONNEL,  FACILITIES   OR  FUNDS),    IDENTIFY  SUCH  RESEARCH: 


PROJECT  DESCRIPTION  (Cont. ) 


Normal  connective  tissue  cells  have  been  carried  in  diffusion 
chambers  in  mice  for  6  months.  Reimplantation  (open)  into  irradiatei- 
hosts  has  not  led  to  the  production  of  tumors. 

Normal  connective  tissue  cells  have  been  carried  in  diffusion 
chambers  with  subculture  at  intervals  of  1  month,  and  biological  tests 
were  made  for  the  development  of  malignancy  at  the  end  of  6  months.  The 
results  have  been  negative. 

No  mice  have  developed  tumors  as  a  result  of  subcutaneous 
implantation  of  materials  used  in  making  diffusion  chambers.  These 
experiments  are  not  yet  completed. 

Significance  to  Cancer  Research^  -Increased  understanding  of  factors 
involved  in  the  transformation  of  normal  to  neoplastic. cells  is  of 
fundamental  importance  in  cancer  research." 

Proposed  course  of  project;  This  experiment  is  still  in  progress. 
Various  chemical  carcinogens  and  related  but  inactive  compounds  will 
be  used  in  studies  of  the  effects  on  cells  and  organs  using  diffusion 
chambers. 


SERIAL  NO. 


15. 


PUBLICATI'^NS  OTHER  THAN  ABSTRACTS  FROM  THIS  pR'^JECT  DURING  CALENDAR 
YEAR  19?^ 


Weaver,  J.  M. ,  Algire,  G.  H»,  and  Prehn,  R.  T. ;  The  growth 
of  cells  in  vivo  in  diffusion  chambers,   II.  The  role  of  cells 
in  the  destruction  of  homografts  in  mice,  J.  Nat.  Cancer  Inst. 
15:  1737-1767  (1955). 

Algire,  G.  't.,  and  Merwin,  R.  M. :  Vascular  patterns  in  tissues 
and  grafts  within  transparent  chambers  in  mice.  Angiology  6; 
311-318  (1955). 


16. 

HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  T'-^IS  PROJECT  DURING 
CALEIWAR  YEAR  1955  • 


Page  1 
project  report 

National  Cancer  Institute       2 .  Laboratory  of_  Biology 

INSTITUTE  L.-.BOF.-.TORY  OE'BRt.WCH  "   " 

Cellular  Biolog.y  Section    4. 5.  414 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  EETHESDa)  .    SEFJAL  NO 

Transparent  Chamber  Research ' 

PROJECT  TITLE  """  '    '  ■"        "  ' " 

Ruth  M.  Merwin  


PTJNCIPAL  HvIvESTIGaTCRCS) 


Dr.  C«  C.  Congdon,  Dr.  K.  K.  San  ford  and  Dr.  G.  H.  .-.Igire 
OTHER  INVESTIGATORS 


PROJECT  DESCPJPTION 


Problem  #1.   Fate  cf  homologous  bone  marrov;  cells  injected  intravenously  into 
irradiated  mice.       Ruth  Merwin  and  Charles  C.  Congdon 

Objective;   To  study  the  mechanism  by  which  marrow  injected  intravenously  pro- 
tects mice  that  have  been  given  vdiole  body  irradiation.  It  has  not  yet  been 
proven  whether  the  introduced  cells  repopulate  "the  tissues  of  the  irradiated 
animals  or  whether  the  injected,  suspension  provides  soaie  substance  that  enables 
the  -irradiated  cells  to. recuperate.  If  cells  from,  an  animal  not  genetically 
like  the  irradiiited  one  were  injected  and  if  one/2Jtefttify  these  introduced 
cells  and  estimate  their  numbers  it  snould  be  possible  to  find  aut  whether  the 
injected  cells  repopulate  the  marro'w  or  whether  the  introduced  cells  only  tide 
the  animal  over  until  its  ov.ti  ceils  can  divide. 

j,eth»d:  Mice  of  the  LAF  strain  were  irradiated  with  900  r  and  each  was  pro- 
tected from  this  otherwise  lethal  dose  by  an  intravenous  injection  of  homo- 
logous juarrow  from  a  mouse  of  the  C3li   strain.  At  various  intervals  thereafter 
the  marrows  of  the  protected  LAF  mice  were  tested  to  see  if  homologous  cells 
ViTere  present.   Tue  iriarrows  were  injected  into  LaF  iiiice  each  of  which  carried 
a  nonvascularized  homograft  of  Harderian  gland  tissue  from  a  C3H  mouse.   Non- 
vascularized  homograft  do  not  initiate  imir.unity  in  the  LaF  host  but  if  the 
injected  marrow  being  tested  contained  homologous  cells  immunity  vrould  be 
initiated  in  the  h»st  and  -vvould  cause  the  graft  to  disintegrate.   The  percent- 
age of  the  grafts  disintegrating  and  the  interval  before  disintegration  pro- 
vided a  means  of  determining  v;hethsr  the  injected  cells  incre^.sed  in  number. 

jiajor  findings:  aU  of  the  marrows  taken  from  the  protected  LaF  mice  whether 
they  were  taken  within  6  hours  or  not  until  150  days  after  irradiation  and 
protection  showed  the  presence  of  homologous  cells  except  about  a  third  of 
those  taken  within  4  days. 

An  increase  in  the  number  of  these  homologous  cells  vias  indicated  by 
12  days  after  injectien,  and  no  decrease  in  number  seemied  to  take  place  before 
1$0  days,  the  longest  intej;;val  after  which  the  marrows  were  tested. 


Page  2 


A  few  tests  of  the  blood,  spleen,  lymph  nodes,  and  thymus  of  the  pro- 
tected mice  indicated  that  homologous  cells  were  present  in  them  also. 

Significance;  It  is  important  to  understand  the  mechanism  by  which  injections 
of  bone  marrow  protect  anijnals  that  have  been  giveh  an  otherwise  lethal  dose 
of  irradiation. 

Proposed  course;   The  data  obtained  will  be  prepared  for. publication. 

Problem  #2,   The  effect  of  local  roentgen  irradiation  on  the  formation  of  nevr 
capillaries  after  injury,       Ruth  Merwin  •    ' 

Objective:  In  a  previous  study  it  vj-as  found  that  the  capacity  of  vessels  to 
form  new  sprouts  v«fas  affected  ty  doses. of  irradiation  over  500  r.  The.  interval 
before  new  vessels  invaded  the  coating  of  exudate  around  wires  placed  in  the 
skin  became  longer  as  the  dose  increased.  At  present  a  study  is  being  made 
using  a  higher  dose  to  find  out  v;hat. dose,  will  completely  prevent  the  formation 
of  new  capillaries.  Also  the  growth  of  new  capillaries  around  wires  placed 
in  the  skin  a  year  after  irradiation  is  being  studied. to  find  out  whether  tne 
endothlium  will  have  regained  its  capacity  to  grow  without  delay  into  an  in- 
jured area.  ,     ■    .      ;  .... 

Methods;  The  growth  of  capillaries  into  the  coating  of  exudate  that  forms 
around  wire  stitches  placed  in  the  skin  of  mice  is  being  studied  microscopic- 
ally using  transparent  chambers. 

Major  findings:  Preliminary  findings  indicated  that  a  dose  of  2,000  r  com- 
pletely prevents  the  growth  of  new  vessels  around  some,  but  not  all,  wires. 
One  year  after  local  irradiation  the  delay  before  sprouts  form  in  an  injured 
area  is  approximately  the  ssjne  as  when  an  injury  is  made  immediately  a:fter 
irradiation. 

Significance;  An  understanding  of  the  changes  in  normal  tissue  irradiated*  ,  , 
locally  is  of  importance  because  it  is  difficult  to  irradiate  a  tumor  without 
including  the  surrounding  normal  tissues.  The  deleterious  effect  of  irradia- 
tion on  nornial  tissues  sometimes  limits  the  dose  that  can  be  given  to  a  tumor^ 

Proposed' course:   To  Continue  as  stated  above. 

Problem  #3.   Tumor  growth  and  vascularization,-  Initial  vascularization  of 
transplants  of  tumor  and  normal  tissue,  Ruth  Mei-win  and  G-lehn  Algire 

Objective:   The  object  of  this  study  is  to  evaluate  the  significance  of  the 
activity  of  the  graft  and  host  vessels  in  the  initial  vascularization  of  grafts. 

Methods  employed;  Microscopic  observations  were  made  on  grafts  placed  in 
transparent  chambers  in  living  mice,  With  the  usual  methods  of  studying 
grafts  one  must  focus  through  the  graft  in  order  to  see  the  area  in  vAich  the 
graft  and  host  vessels  establish  connections.  Therefore,  grafts  were  placed 
so  the  area  in  which  these  changes  take  place  would  be  beside  rather  than 
under  the  graft. 

Major  findings;   The  surviving  vessels  in  grafts  ff  normal  tissues  produce 
sprouts  which  may  be  active  in  establishing  corinections  between  the  vessels 
of  the  graft  and  those  of  the  host.  On  the  otiier  hand,  the  vessels  in  grafts 


Page  3 

of  tujnor  tissue  survive  only  in  some  grafts  and   do  not  produce  new  vessels. 
Grafts  of  nonnal  tissue  of  the  size  used  did  not  cause  host  vessels  to  form 
new  sprouts,  whereas  grafts  of  a  siriiilar  size  of  a  mammary  adenocarcinoma 
caused  a  rich  network  of  new  sprouts  to  form. 

Significance;   The  methods  used  here  provide  a  means  by  which  comparisons 
could  be  made  between'  the  effect  of  various  kinds  of  stimuli  and  various 
kinds  of  tamers  on  blood  vessels. 

Proposed  course:  It  is  planned  to  compare  various  typss  of  tumors  in  their 
capacity  to  stimulate  host  capillary  proliferation. 

Problem  /f4t  Effect  of  irradiation  on  the  capacity  of  mice  to  develop  immunity 
to  homografts.         Ruth  Merv/in  and  Ks-thsrine  Sanford 

Objective;   The  objective  of  this  study  was  to  find  out  how  long  after  irrad- 
iation mice  are  incapable  of  d;;veloping  Immunity   to  homografts. 

Methods ;  A  nonvascularized  homograft  will  net  initiate  immunity  but  it  will 
be  destroyed  if  the  host  becomes  immune.   The  interval  after  irradiation  and 
after  the  injection  of  an  ijni'aunizing  dose  of  homologous  cells  before  a  homo- 
graft  disintegrated  was   taken  as  the  interval  during  vjhich  mice  could  not 
develop  immunity. 

Major  findings;   Preliminary  results  indicate  that  there  is  much  variation 
between  mice  but  that  mice  can  acquire  imjnunity  as  soon  as  '11+   days  after 
900  r  and  protection  by  isologous  marrow  or  as  soon  as  11  days  after  irradia- 
tion with  450  r. 

Significance;   To  help  identify  the  natur-e  of  the  changes  taking  place  in 
cells  that  become  neoplastic  (see  the  report  of  Dr.  K.  K,  Sandford) 

Proposed  course;   To  continue  as  stated  above, 

414 

SERIAL  NO. 


BUDGET  ACTIVITY: 

EES^AECH  X  ADiZNISTR^.TION 

RSVIM  &  APPROVAL  TECHMIC^  ASSISTANCE 


12, 


COOPEIxiTIKG  UNITS  OF  THE  PUBLIC    PCi-.LTH  SERVICE,    OR  OTHER  OIiGaNIZATIONS, 
PROVIDING  FUNDS,    F..CILITIES,    OF   PERSONNEL  FOR  TliiS   PROJECT  IN  EITHER  1956 
or  1957 
Dr.   C.   C.   Congdon  is  at  Oak  Ridge   National  Laboratory,   Oak  Ridge,   Tenn, 


IF  THIS  PROJECT  RESEiiBLES,   C0.v.PLEl,ESIT3,    OR  P.iRALLELS  RiiSEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC   HEALTH  SERVICE   C.ITHOUT  INTEFJCH.\NGE  OF   PiFSONNEL,    FACILITIES 
OR  FUNDS),    IDSiMTIFY  SUCH  RE3E.-.RCH; 

414 

SERIAL  NO. 


15. 

PU^^LICaTiUNS  OTH.R  TILU\f  ABSTFuiCTS  FROM  THIS  Pi>,0J3CT  DUPING  CaLE'.NDaR  Yli^.R  1955 

Viiscular  patterns  in  tissues  and  grafts  within  transparent  chambers 

in  mice,  Glenn  H.   jilgire  and  Ruth  M.   MenA^in, 

;jigiology  6:     311-318,  1955  '  '  ' 

16  ,  . 


H0M01.S  .nlO  HVlAicDS  TO  PERSONITfiL  RELATING  TO  THIS   PROJECT  DURING  CALENDAR  YEaR 
1955. 


PAGE  1 

PROJECT  REPORT  FORM 

1 .  National  Cancer  Institute   . 2 «  Laboratory  of  Biology 

INSTITUTE  .  ,       laboratory"  OR  BRAICH 

3.  Cellular  Biology  Section 1;.  ,      ^  ^'     ^^^ 

SECTION  OR  SERVICE  '       ~     LOCATION(IF  OT"^R  THAN  BETHE3DA.)     SER.    NO, 

6»  The  mechanism  of  destruction  of  homngrafts 

PROJECT  TITLE 

7.  J»  ''''»  Weaver .   

, PRINCIPAL  INVESTIGATOR(S) 


1.   G.   ".Algire 

OTMER  ItJVESTIGATORS 


9.      PROJECT  DESCRIPT-'^'iN 

Objectives;  To  elucidate,  the  mechanism  of  homograft  destruction.  Work 
published  in  1951;  (J. N.C.I.  IS:   ii93-^07  and  509-5l7)  suggested  that 
such  grafts  are  nrt  destroyed  by  circulating  antibodies  and  further 
data  published  in  19$5  (J. N.C.I.  15:  1737-1767)  suggested  that  they  are 
destroved  by  contact  with  host  l^/mphocjrtes.   There  was  some  evidence  in 
the  latter  work  that  these  lymphocytes  release  a  diffusible  specific 
cytotoxin  f antibody?)  at  the  site  of  the  graft.  The  purpose  of  further 
work  in  1955  was  to  see  if  thi-S  was  so. 

T^iethods  employed;  Single  and  double  diffusion  chambers,  tissue  cultures, 
serologic  methods,  transparent  chambers  and  conventional  cytologic 
methods  fsee  following  section  for  further  details). 

Fajor  findings;  "'"odified  intraperitoneal  diffusion  chambers  were 
employed  with  two  adjacent  comoartnents  separated  by  cell-impenetrable 
membranes  with  a  pore  size  of  about  one  micron.  Lymphocytes  from 
C57BL  mice  previously  immunized  with  C3H  tissue  were  combined  in  one 
compartment  with  C3'~'  "target"  cells  (spleen  or  tumor). 
'*s  expected  from  previous  work:,  this  resulted  in  the  death. of  botto 
the  l^TOphocytes  and  the  target  cells.  Similar  "immunized"  lymphocytes 
alone  or  target  ceils  alone  had  been  placed  initially  in  the  second 
compartments.  Both  of  these  two  types  of  cells  died  in  these  latter 
compartments  whereas  cc-itrol  nonirrmuniaed  lymphocytes  or  control 
G57BL  target  cells  survived  here. 


PROJECT  DESCRIPTION  (Cont.) 

Furthermore,  target  cells  combined  with  dead  "immunized" 
lymphocytes  (frozen  and  thawed)  in  intraperitoneal  diffusion 
chambers  with  one  compartment  did  not  survive  whereas  such  target 
cells  did  survive  when  combined  with  control  dead  nonimmunized 
lymphocytes.  Whether,  as  might  be  expected,  dead  target  cells  will 
destroy  "immunized"  lymphocytes  in  diffusion  chambers  has  not  yet 
been  studied. 

These  results  can  be  interpreted  most  simply  as  signifying 
that  homografts  are  destro\'Bd  by  antibodies  which  are  bound  to  host 
lymphocytes  until  these  i-\Tnphocytes  contact  the  grafted  cells  and, 
in  addition,  that  this  destruction  of  the  graft  is  accompanied  by 
the  death  of  the  host  lymphocytes  which  is  caused  by  antigens  released 
from  the  dying  grafted  cells. 

Further  attempts  to  destroy  target  cells  in  vittio  by  combining 
them  with  living  or  dead  "imm.xmized"  lymphocytes  and  efforts  to 
visualize  in  vivo  by  means  of  modified  transparent  chambers  the 
interaction  of  living  graft  and  host  l-/mphoc^^es  have  been  unsuccessful 
to  date  —  quite  p<3ssibly  for  various  technical  reasons. 

Proposed  Course;  The  above  findings  will  be  extended  and  methods 
will  be  sovght  to  visualize  in  vivo  the  interaction  betwesn  living 
grafted  cells  and  host  lymphocytes  and  also  to  cause  this  to  occur 
in  vitro  where  the  mechanism  involved  can  be  more  easily  analyzed. 


PROJECT  REPORT  FORt^   (Cont'd.) 


10.l£l6 

SERL\L  NO. 


11. 

B'HXJET  i'.CTIVITY: 


RESE/IRCH"         -X  ADfaniSTRATlON 

REVIEW  k  A   PROVAL  TECH1IIC\L   \S3I3TANCE 


12. 

CO-JPERATIMG  imiTS   OF  THE   PUBtIC   I-^ALTH  SERVICE,   OR  OT'-'ER  ORGAri.'ZATION, 
"PROVIDING  FUrroS,  FACILITIES,   OR  ?£?^.SON:IEL  FOR   T'-I5    ^^'"JECT   I'!  EITHER 
19^6  or  19^7 


13._ ^_^     

IP  Tf:5  ^RoJfic^T  tt^3Et^6Lr.5,  Col^^^LKi.ErTS,  or  P.-.R-JLLEi3  ftESi'-^dp?  COilE' 

ELSEV^'JERE   II'"  THE  PUBLIC   HEMTH  SEP.7ICL    (w  THOIJT   IIJTERCH'k'GE  OF 
^EPoGlf-IEL,  F.-^CILITIES  OR  FUi-JDS),   IDENTIFY  SUCH  RES-',RCH: 


PROJECT  REPORT  FORM   (Cont'd) 

Hx.       hl6 

SER.IAL  NO. 


15 . 

PUELiCATIOtJS   OT^ER   TT-AN  ABSTR/^.CT5  FROM  T"IS   PROjSCT  tillRING  CALENDAR 


YEAR  1955- 

Weaver,  Algire,  Prehn;  J. M.C.I.  l5: .1737-1767,  1955. 


16.  

HONORS  AND  AWARDS  TO    ^IRSOKJEL,  RELATING  TO  T^^-'IS    PROJECT  DURING  CALENDAR 
YEAR  1955.  ■■■ ■    •         ■      • 


SUPPLEMENTAL  PROJECT  DESCRIPTION  OF  SERIAL  NO.  Ul6  LABORATORY  OF 

BIOLOGY  -  DR.  JAMES  M.  WEAVER 
PROJECT  DESCRIPTION  (Addition) 


Significance  of  work;  "Cancer  iinmunity"  has  long  been  a  subject 
of  considerable  interest  and  of  intensive  investigation.  Latency 
of  cancer  and  occasional  Spontaneous  regressions  of  cancer  in  man 
may  well  be  manifestations  of  an  immime  reaction  of  the  host  to 
the  tumor.  If  such  reactions  could  be  inteisified  at  will,  the 
benefit  to  the  host  is  obvious.  However,  the  mechanism  of  re- 
jection of  homologous  tumor  grafts  in  animals  and  of  homografts 
in  general  has  long  been  disputed.  This  mechanism  has  certain 
features  in  comnion  with  better  understood  phenomena  of  immunity, 
such  as  reactions  to  bacteria  or  foreign  proteins,  but  the  search 
for  antibodies  to  these  tissues  has  usually  been  to  no  avail. 
TTntil  the  nature  of  this  mechanism  is  clarified,  it  seems  unlikely 
that  attempts  to  utilize  it  to  combat  cancer  in  man  will  be  success- 
ful. It  is  hoped  that  the  research  program  described  above  will 
contribute  toward  an  imderstanding  of  this  mechanism. 


Sni.    19T:!'B'J'-' 


PROJECT  REPORT  FORM 
1.  National  Cancer  Institute  2,     Laboratory  of  Biology 


INSTITITTE                   LABORATORY  OR  BRANCH 
3.  General  Biology  Section   i;.  5.  ^^'^ 


SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THA1«I  BETHESDA  SER,# 

6,  Investigation  of  the  role  of  genes  and  their  relationship  to  -n«n-genetic 

PROJECT  TITLE 
factors  in  the  development  of  cancer* 

7.  W.  '£♦  Heston 

PRINCIPAL  II'JT?ESTIGATOR(S) 

8,  Margaret  a,  Deringer,  Thelna  E.  Dunn,  Delta  Uphoff,  George  Vlahakis 

OTHER  im'ESTIGATORS 

9.  PROJECT  DESCRIPTION, 

Objectives;  To  determine  the  effects  of  genes  on  the  occurrence  of  tumors, 
particularly  of  the  lung  and  of  the  mammary  gland,  of  the  mouse.  To  obtain 
some  indication  of  the  number  of  genes  involved  and  where  possible  to  ident- 
ify specific  genes.  To  ascertain  the  manner  in  which  the  genes  are  related 
to  non-genetic  factors  in  the  induction  of  tumors. 

Methods  employed:  This  report  covers  only  certain  faicets  of  a  long  term 
program  in  which  each  year  certain  sub-projects  are  completed  and  certain 
new  ones  are  initiated.  Methods  employed  in  various  facets  of  the  program 
may  be  much  the  same  with  new  procedures  interjected  when  needed. 

In  the  study  of  pulmonary  tumors  a  number  of  Ihbred'-  'trains  of  mice 
are  available  with  specific  incidences cof  pulmonary  tumors  ranging  from  90 
percent  in  strain  A  to  less  than  1  percent  in  strain  C57L,  Seven  specific 
genes  located  on  five  specific  chromosomes  have  been  shown  to  be  associated 
with  the  fccurrence  of  pulmonary  tumors  -  some  through  the  effect  of  the 
gene  itself  and  some  through  linkage*  The  occurrence  of  these  tumors  can 
be  increased  by  extrinsic  factors  including  the  carcinogenic  hydrocarbons, 
urethan,  nitrogen  and  sulfur  mustard  and  radiation,  and  the  nuinber  of  tumors 
occurring  offers  a  valuable  quantitative  measure  of  response.  Through  proper 
matings  specific  desired  genotypes  can  be  produced  and  these  can  be  studied 
in  relationship  t»  any  of  the  extrinsic  factors.  Through  transplantation 
of  lung  tissue  various  genotypic  combinations  between  host  and  lung  tissue 
can  be  created.  The  carcinogens  can  be  introduced  to  shorten  the  duration 
of  the  project  and  also  to  study  and  compare  their  specific  effects  in  com- 
bination with  specific  genotypes.  Gold  thioglucose  has  been  injected  t« 
increase  body  weight  comparable  with  effect  of  the  lethal  yellow  gene. 

In  the  study  of  mammary  gland  tumors  a  number  of  inbred  strains  are 
available  with  certain  incidences  of  mammary  tumors  and  differing  in  ability 
to  transmit  the  mammary  tumor  agent.  The  line  of  strain  C57BL  here  at  the 
NCI  is  outstanding  and  valuable  in  our  studies  in  that  as  shown  by  Andervont 
it  will  not  transmit  the  agent  for  more  than  one  generation.  Through  ap- 
propriate matings  and  foster-nursing  of  young  the  relationship  of  the  geno- 
type t»  the  agent  can  be  studied. 


Page  2 

Major  findings:   -  Lung  Tumogs. 

In  an  attempt  to  determine  whether  or  not  the  lethal  yellow  (A/)  gene 
of  the  mouse  increases  the  •ccurrence  of  pulmonary  through  some  general  effect 
related  to  its  increasing  body  weighty  non-yellow  a  a  sibs  have  been  injected 
with  gold  thioglucose.  Following  this  treatment  the  aa  mice  gained  weight  so  that 
'their  average  weight  was  approximately  equal  to  that  of  the  A^a  mice  without  ^'old. 
Furthermore  the  average  number  of  lung  tumors  observed  in  aa  mice  injected  with 
gold  and  later  with  dibenzanthracene  was  about  the  same  as  that  of  the  A^a  jnice 
without  gold  but  with  the  dibenzanthracene.  ■  There  are  some  unexplained  sex  differ- 
ences in  average  number  of  tumors  which  may  be  clarified  with  further  experimenta- 
tion. Before  publishing  we  want  als»  to  get  data  on  effect  of  gold  on  eccurrence 
of  lung  tumcjTS  in  Ay"a  mice  and  also  the  effect  on  occurrence  of  lung  tumors  •! 
holding  dowri  the  weight  of  Aya  mice. through  dietary  restriction.  This  study  is 
underway. 

In  the'  study  in  which  we'  were  attempting  to  determine  whether  or  not 
the  effect  of  the  A^  gene  in  increasing  occurrence  of  pulmonary  tumors  was  local- 
ized in  the  lung,  the  results  were  somewhat  irregular.  This  was  done  by  trans- 
planting AJa  lung  into  aa  hosts  as  well  as  Aya  hosts  and  transplanting  aa  lung 
into  A^a  hosts  as  well  .as  aa  hosts,'  The  greatest  number  of  t'omors  occurred  in 
the  transplanted  lung  when  the  genotype  of  the  transplant  and  that  of -the  host 
were  the  same.  The' highest  incidence  of  tumors  occurred  in  aa  transplants  in  aa 
hosts  and  the  next  highest  in  Aya  transplants  in  Aya  hosts.  The  least  incidence 
•ccurred  in  Aya  transplants  in  aa  hostg  and  next  to  the  host  occurred  in  aa  trans-' 
plants  in  Aya  hosts.  This  experiment  will  be  repeated  before  publication, 

■Reticulosis  involving-  particularly  the  axillary  and  inguinal  nodes 
has  been  found  in  a  high  percentage  of  these  AYI''y  mice.  Although  scattered  cases 
of  this  condition  have  been  observed  previously  in  a  number  of  our  stocks  this  is 
■the  first  time  we  have  noted  it  in  a  high  incidence  in  any  group.  It  has  not 
been  recorded  in  such  an  incidence  previously  in  AYF;j_  mice.  The  cause  of  the  con- 
dition* is  not  known. 

In  further  dose-response  studies  on  the  induction  of  pulmonary  tumors 
in  strain  A  mice  with  dibenzanthracene  th©  nature  of  the  response  curve  has  been 
determined  in  the  region  of  very  minu+e  doSes,  A  straight  line  response  has  been 
noted  between  dose  ,5  in^.  and  dose  .01  rag.  with  the  point  for  zero  dose  deviating 
upward  from  the  extension  of  this""  straight  line.  The  points  for  dose  .00^  mg, 
and  dose  ,0025  mg,  also  deviated  upward.  Response  was  measured  in  terms  of  aver- 
age number  of  nodules,  and  the  lower  points  that  deviated  upward  represented 
group's  in  which  not  100  percent  of  the  animals  had  one  or  more  nodules,'  It  may  be 
that  some  theoretical  res^xmse  points  can  be  established  statistically  assuming 
variation  in  the  animals  having  no  tumors  and  these  points  may  not  deviate  from 
the  straight  line  response. 

Such  dose-response  studies  have  also  been  made  on  the  induction  of 
pulmonary  tumors  in  strain  C3Hf  mice  with  urethan  in  doses  ranging  from  1.2^  mg. 
t»*hO  mg.  No  increase  in  tumors  was  noted  below  the  dose  of  10  mg,  and  at  doses 
10,  20,  and  UO  mg,  the  response  was  so  slight  owing  t»  the  resistance  of  the 
strain  that  it  -was  impossible  to  determine  the  dose-response  relafp.onship.'' 


Page  3 

The   outstanding  observation  in  this  c^roup  of  C3^'h'   mice  was  definite  evi- 
dence of  increase  in  incidence  and  average  number  of  hepatomas  with  ini;rease  in 
do>sage  of  urethan.  This  was  noted  in  the  males  only.  This  is  the  first  obser- 
vation, to  ovir  knowledge,  of  the  induction  of  hepatomas  in  mice  with  urethan. 
An  experiment  has  been  set  up  to  verify  the  observation  before  publication. 

Manjmary  uland  Tumors; 

otudy  of  mammary  g'land  tumors  in  strains  without  the  mammary  tumor  agent, 

Ln  oui-  C3Hf  strain,  mammary  tumors  are  continuing  to  arise  with  an  inci- 
doncp  of  approximately  25  percent  and  an  average  tumor  ape  of  approximately  20 
months.  -Although  our  colony  is  now  in  Ahe  20th  and  30th  generation  of  inbreeding 
since  the  original  foster-nursed  litter  from  which  this  strain  was  started,  we 
still  havi^  seen  no  evidence  of  high  tumor  lines  segregating  out  as  wfuld  be  ex- 
pected wore  the  agent  present. 

In  July  19'i3,  a  litter  of  Andervont's  subline  C3HfAn,  that  in  his  labora- 
tory had  shown  a  considerably  lower  manunary  tumor  incidence  than  had  our  C3Hf, 
was  obtained,  and  from  that  litter  a  colony  has  been  bred.  Of  23  C3Hf/An  breeding 
females  tl'.at  have  been  autopsied,  6  or  26,i  have  had  mammary  tumors  at  an  average 
at;;e  of  13i3  months,  the  remainder  died  without  mammary  tumors  at  an  average  age 
of  I'i  months.  It,  tlierefore,  appears  that  in  r>ur  laboratory  the  incidence  of 
tumors  in  his  line  is  going  to  be  comparable  with  that  in  our  line  and  significan- 
tly higher  than  that  in  his  line  in  his  laboratory.-  Explanation  for  this  differ- 
once  piay  be  one  or  more  of  the  following.  1)  In  our  laboratory'  th^  animals  are 
fed  Peerwood  diet  whereas  in  his  the  diet  is  P;?.rina  chow,  2)  in  our  laboratory 
the  females  have  been  bred  throughout  the  year  whereas  "they  are  not  bred  during 
the  winter  months  in  his  laboratory.  3)  in  our  laboratory  the  females  are  housed 
in  the  same  large  room  with  animals  ^dth  the  milk  agent,  although  on  opposite 
sides  of  the  room  and  on  different  racks,  whereas  in  his  laboratory  agent  fre« 
animals  ai'e  not  kept  in  the  same  room  with  animals  with  the  agent*  Since  breed- 
ing has  been  shown  t©  be  an  important  factor  in  producing  mammary  tumors  in  our 
C3Hf  line  (the  virgins  having  an  incidence  of   less  than  5  percent  whereas  the 
breeders  have  25  percent  or  more)  the  second  possible  explanation  is  probably  the 
most  important.  The  third  explanation  would  imply  infection  with  the  agent  and 
as  indicated  above  we  have  seen  no  avidence  of  this  in  our  own  C3Hf  line. 

The  large  mass  of  data  on   the  relationship  of  the  genotype  to  the  milk 
agent  yias  now  been  prepared  for  publication  and  sh%ul'*'  be  submitted  in  the  near 
future.  The  genetic  control  over  the  propagation  and  transmission  of  the  agent 
is  very  pronounced,  the  agent  being  eliminated  by  the  third  backcross  generaticn. 
Once  eliminated  the  agent  cannot  be  revived  by  again  introducing  a  suitable  geno- 
type. The  agent  cannot  be  caused  to  arise  de  novo-.  The  agent  can  be  transmitted 
by  the  males  and  once  introduced  into  the  line  in  this  way  is  immediately  built 
up  to  create  a  high  tumor  line, 

Tfc  test  further  for  the  presence  of  the  agent  tJhcse  data  were  tabulated 
to  show  parent-offspring  correlation*  A  positive  correlation  appeai-ed  only  where 
it  was  certain  from  other  observations  that  the  agent  was  present. ^  In  other 
lines  although  quite  a  number  of  tumors  were  present  there  was  n«  parent-off- 
spring correlation  which  was  in  line  vjlth  the  previous  conclusion  that  these 
turners  arose  in  the  absence  of  the  agent. 


1  Page  U 

The  data  also  were  tabulated  to  determine  whether  or  not  more  tumors 
occurred  in  females  of  later  litters  than  in  those  of  early  litters.  With  the 
exception  of  the  few  females  that  wer«  infected  by  the  male  and  were  infected 
after  the  first  or  second  litter  was  born  so  tumors  occurred  more  in  later 
litters,  there  was  no  difference  between  early  and  late  litters  either  in  groups 
where  the  agent  was  known  to  be  present  or  in  groups  where  it  was  absent. 

Cleft  palate  and  harelip. 

In  I'^aser's  interpretation  of  his  results  on  induction  of  cleft  palate  and 
harelip  in  strain  A  mice  with  cortisone  he  has  contended  that  the  cortisone  did 
more  than  increase  the  occurrence  of  an  anomaly  that  occurred  spontaneously  in 
the  strain.   His  arguinent  was  that  with  cortisone  he  got  some  cleft  palate  with- 
out harelip,  whereas  cleft  palate  without  harelip  never  occurred  spontaneously. 
Since  I  had  contested  this  j.nterpretation  end  s'ince  we  had  the  opportunity  to 
observe  a  large  number  of  newborn  strain  A  micfe  we  have  gathered  data  on  these 
anomalies.   Of  1313  males  born  8.3  percent  ha/a  both  harelip  and  cleft  palate,  .6 
percent  had  harelip  without  cleft  palate  and  .23  percent  had  cleft  palate  with- 
out harelip.   Of  1351  females  born  6.29  percent  had  harelip  and  cleft  palate,  .37. 
percent  had  harelip  without  cleft  palate  and  .22  percent  had  cleft  palate  without 
harelip.   Thus,  without  treatment  of  the  mother  the  animal  can  have  a  cleft  palate 
without  evidence  of  the  harelip. 

Significance  to  program  of  the  Institute:   One  of  the  approaches  to  the  control 
of  cancer  in  man  is  through  the  study  of  the  factors  in  the  development  of  cancer 
in  mice,  the  manner  in  which  these  factors  become  effective  and  the  way  in  which 
they  are  related  to  each  other.  Basic  among  these  factors  .are  the  genes,  but  the 
observable  effect  of  the  genes  is  greatly  influenced  by  nongenetic  factors.  The 
results  reported  help  to  round  out  our  knowledge  in  this  area  and  it  is  hoped  that 
such  knowledge  will  be  of  assistance  in  understanding  the  factors  causing  cancer 
in  man  and  in  directing  investigations  on  cancer  in  man. 

Proposed  course  of  project. 

The  studies  will  be  continued  much  along  the  same  lines  as  in  the  present 
report.   Linkage  studies  that  were  not  at  a  stage  suitable  for  reporting  now  will 
Ite  continued  and  possibly  concluded  in  the  coming  year.  Data  such  as  those  on 
the  transplanted  foetal  lungs  will  be  written  up  for  publication  as  soon  as  Dr. 
Steffee  has  finished  going  over  the  slides. 

The  experiment  for  verification  of  induction  of  hepatomas  in  mice  with 
urethan  will  be  carried  on  and  possibly  concluded.   This  will  be  extended  to  in- 
clude repeated  doses  as  well  as  single  graded  doses. 

The  dose-response  studies  on  the  induction  of  pulmonary  tumors  are  being 
extended  to  urethan  in  strain  A  mice,  the  strain  C3H  having  been  too  resistant 
to  give  analyzable  results  for  the  dosages  used.   It  is  necessary  to  get  results 
from  some  carcinogen  in  solution  since  it  is  realized  that  the  straight  line 
relationship  observed  with  the  dibenzanthracene  dispersion  may  have  represented 
only  distribution  of  DBA  particles. 

Studies  on  the  effect  of  the  A^  gene  in  increasing  pulmonary  tumors  in 
mice  are  being  extended  to  include  data  on  AJa  mice  treated  with  gold  and  also 
on  i^Pi.   mice  whose  weights  are  held  comparable  with  that  'of  their  aa  sibs  through 


Page  5 

The  experiment  attempting  to  localize  the  action  of  the  A^  gene  in  in- 
creasing the  occurrence  of  pulmonary  tumors  will  be  repeated  before  the  results 
are  published. 

A  new  but  related  project  on  the  effect  of  concentration  of  oxygen  on  the 
occurrence  of  pulmonary  tumors  in  strain  A  mice  is  getting  under«^  in  collabor- 
ation with  Dr.  Arnold  W,  Pratt.  This  study  is  based  upon  the  observation  ef  in- 
creased rate  of  radiation  induced  mutations  in  Drosophila  and  other  microorganisms 
with  increased  concentration  of  oxygen,  increased  melanotic  tumors  induced  with 
radiation  in  Drosophila  larvae  with  increased  oxygen,  and  increased  spontaneous 
melanotic  tumors  in  the  larvae  with  increased  oxygen  but  without  i-adiation.  The 
studies  will  include  2  month  old  strain. A  mice  injected  with  dibenzanthracene 
and  expose*  to  various  concentrations  of  ©xygen,  and  als»  newborn  strain  A  mice 
exposed  to  various  concentrations  of  •xygen  but  not  injected  with  dibenzanthrac- 
ene. 


10. 


11. 


12. 


13. 


15. 


16. 


U17 


SERIAL  NO. 


^,UDGET  ACTIVITY: 

RESEARCH      ^  Administration 


P.EVIEVif  &  APPROVAL         Technical  Assistance 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDIi-lG  RTNDS,  FACILITIES,  OR  PERSOMeL  FOR  THIS  PROJECT  IN  EITHER.  1956 
or  1957 


IF  THIS  PROJECT  RESEl-BLES,  COMPLEMENTS,  OR  PAFiALLELS  RESEARCH  DONE  ELSE>fHERE 
IN  THE  PUFiLIC  HEALTH  SERVICE  ('.-JITHOJT  INTERCHANGE  OF  PSRSONNE;.,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  RE^-LAHCH: 


lU.    U17 

SERIAL  NO. 


PL^r^-LICA.IONS  OTIiER  THAN  ABSTRACTS  YRCH   THIS  PROJECT  DURING  CALENDAR  TuAR 
1955. 

Deringer,  Margaret  A,,  and  W,  E.  Heston 
Development  of  pulmonary  tumors  in  mice  segregated  with  respect  to  the 
three  genes:  dominant  spotting,  caracul,  and  fused.  J.  Nat.  Cancer  Inst, 
16:  763-768,  1955. 


liONORS"AND~AWARDrTO  PEPSCN^^IEL  REUTING  TO  THIS  PROJECT  DURING  CALENDAR 
yf:ar  1955. 


PAGE  1 


PROJECT  REPORT  FORM 


1 . National  Cancer  Institute  2 .  Laboratory  of  Biology 

INSTITUTE  LABORATORY  OR  BR.ANCH 

3»General  Biology  Section    h- 5'  Ul8 

SECTION  OR  SERVICE        LOCATION  (IF  OTHER  THAN  BETHESDA)  SER.  NO. 

6. Host-tumor  Relationships ^ ■       

PROJECT  TITLE     .  "^ 


7«M»   '(.   Barrettj  ^I.   B.  Kelroy,  M.  K»   Deringer 


PRINCIPAL   INVESTIGATOR (S) 


OTHER   IWE3TIGAT0RS 


9.      PROJECT  DESCRIPTION  '  • 

In  multicellular  organisms  the  morphology  and  physiology 
of  each  cell  and  each  tissue  is  strongly  influenced  by  other  cells  or 
tissues  in  its  environment.  There  seems  to  be  a  cellular  ecology  which 
maintains  organization  and  balance  from  earliest  differentiation  to 
death.  Ample  experimental  evidence  for  such  intercellular  influences 
exists. 

Cancer  may  be  vievjed  as  a  disturbance  of  su^h  an  ecological 
balance  and  from  such  a  view  one  may  derive  a  definition  of  cancer.  A 
definition  in  these  terms  is  the  only  definition  of  w.hich  I  am  aware 
that  does  not  require  frequent  important  qualifications.  For  example, 
some  normal  tissues  have  enormous  proliferative  oower  and  may  even 
be  invasive  'whereas  some  malignant  cells  multiply  slowly  and  may  remain 
localized  indefinitely  but  in  the  end  the  normal  usually  responds  to 
controlling  influences  and  the  malignant  does  not.' 

Such  a  definition  is  primitive  but  its  constancy  is  a  recom-  ,. 
mendation.  The  primitive  quality  is  due  to  a  lock  of  knowledge  of  the 
nature  of  the  forces  or  influences  that  modify  the  intercellular 
balances.  At  present  we  c.?ll  these  influences  "imrr.uno  gene  tic",  for 
want  of  a  better  term.  Q-or   research  is  directed  at  learning  more 
about  the  nature  of  these  influences.  The  possibility  of  final 


PROJECT  DESCRIPTION  (Cent.) 

practical  application  of  the  knowledge  is  suggested  liy  the  occurrence 
of  spontaneous  remissions  and  even  regressions  in  cancer  of  both  man 
and  animals  -  regressions  that  appear  to  be  brought  about  by  a 
resistance  on  the  par-t  of  the  host.     

Methods 

The  basic  procedure  is  transplantation  of  standard  tumors 
into  hosts  of  known  genetic  constitution  and  studying  the  effect  of 
exoerimental  variations  on  the  host,  the  tumor.,  or  both*  -  The  dis- 
ciplinary techniques  involved  are  those  of  genetics,  immunology,  and 
■experimental  surgery r 

Major  Findings 

A  review  of  this  field,  including  our  major  findings  and  their 
place  in  the  general  perspective  was  reported  in  press  last  year.  This 
paper  entitled  "The  nature  of  tumor  immunity"  has  now  appeared  as  a 
chapter  in  a  book;  see  publications. 

We  previously  reported  that  a  host  can  be  made  immune  to  the 
implantation  of  a  tumor  by  prior  inoculation  of  washed  red  cells  or  by 
stromata  but  not  when  the  cells  or  stromata"have  been  broken  up  by 
•relatively  gentle  procedures.  We  also  reported  that  the  antigen  was 
easily  destroyed  by  heat  or  by  formalin.  Others  have  strongly  sug- 
gested that  the  antigen  concerned  is  a  hemagglutinogen.  Available 
data  indicate  that,  genotypically,  these  antigens  are  related  but 
we  believe  that  ph  ^otypical  differences  exist  and  may  be  important. 
Accordingly  we  have  accumulated  unpublished  data  that  indicate  such 
differences*  Hild  sonic  vibration  destroys  the  "resistance"  antigen 
but  not  the  hemagglutinogen  for  mice  (dextran  technique).  This  work 
has  -been  hampered  by  "histocompatibility"  limitations  which  prevented 
observation  of  the  2  types  of  phenomena  in  the  same  mouse.  We  have 
now  succeeded  in  adapting  a  tumor  so  that  future  observations  can  be 
made  in  the  same  mouse,-  The  "resistance"  antigen  (for  mice)  is 
destroyed. by  heating  for  30  minutes  at  U2-I4.U  C  whereas  the  hemagglutino- 
gen (for  rabbits)  rem.ains  potent  after  30  minutes  at  56  C.   In  one 
strain  of  mouse  a  high  degree  of  resistance  can  be  induced  by  certain 
red  cells  but  such  mice  do  not  have  hemagglutinens  demonstrable  by 
the  new  techniques.   (Mr,  Melroy  has  done  most  of  the  serologic  work). 
Another  aspect  of  the  differential  characterization  of  this  antigen 
is  found  in  a  study  of  incubation  peri-ods.  We  reported  a  freakish 
incubation  curve  that  seemed  to  characterize  the  development  of 


PROJECT  DESG^IPTI"N  (Cont.)  ^   , 

resistance  following  inaculation  of  red  cells.  This  -work  is  very 
laborious  but  we  have  made  some  additional  progress.  The  first 
notch  (decrease  in  relative  immunity  during  the  3rd  week)  in  the 
curve  has  been  see'n  again  and  additional  evidence  for  the  second 
notch  (5th  week)  has  been  obtained.  Tha^'"^' gain-loss-re  gain"  phenomenon 
has  not  been  reported  in  immunology.  We  do  not  know  what  it  means 
but  it  seems  to  have  something  to  do  with  the  particular  antigen  be- 
cause it  does  not  occur  when  tumor  is  used  as  the  antigen.  This 
might  represent  an  obscure  biologic  difference  between  normal  and 
malignant  tissue  but,  because  the  cell  type  was  different,  this  is 
uncertain,   'vll  these  points  permit  us  to  continue  with  the  hypothesis 
that  there  is  something  peculiar  about  this  type  of  immunity  snd  the 
antigens  that  generate  it, 

A  paper  reporting  the  effect  of  multiple  inoculations  of  a 
tumor  and  the  implications  of  the  result^  in  interpreting  Lewis'' 
"breaking  down  pf  resistance",  Greene's  "Constitutional  factors 
peculiar  to  a  tumor  bearing  host"  and  Casey's  "secondary  XYZ"  effects 
appeared  in  the  December  19'?h   issue  of  J. N.C.I,  which  came  out  in 
January,  1955»  Tfee  crux  of  the  matter  is  that  the  cachectic  host'  " 
cannot  muster  such  .immunologic  defenses  .as  it.  previously  possessed  and 
this  loss  is  nonspecific.  As  an  outgrowth  of  that  work  we  reir^vesti- 
gated  the  effect  of  incision  and  partial  excision  on  the  progress  of  the 
disease.  The  work  with  tumors  transplanted  within  strain  and  out.  of 
strain  has  been  concluded*  Neither  simple  cruciate  incision  nor  partial 
excision  increased  the  number  of  metastases  and  the  latter  prolonged 
the  survival  of  the  hosts.   (Details  given  in  previous  report).  We  are 
lifiw   observing  the  effect  in  spontaneous  mammary  tumors.  Accumulation  of 
a  properly  matched  series  is  very  slow.  So  far  the  results  are  in 
agreement  with  those  in  transplanted  tumors.  These  results  are  contrary 
to  several  reports  in  the  literature,  including  one  in  the  J. N.C.I,  for 
19h9   and  one  in  Krebsartz  fer  1952. 

Following  our  discovery  of  a  maternal  influence  on  growth  of 
a  tumor  and  the  specificity  of  "adaptation"  we  attempted  to  find  a 
clear  extrachromosomal  (aside  fr^m  milk  agent  (a))  or  X-Y  chromosomal 
inj;^luence  on  turner  growth.  A  special  tj'pe  of  F^'  hybrid  produced  ty.  , 
Dr.'  Deringer  furnished  a  very  elegant  substrate  for  this  investiga£ioh 
but  we  were  unsuccessful  in  demonstrating  an  effect.  The  experiments 
have  been  abandoned.  ,     . 


PROJECT  DESCRIPTION  (Cont.) 

Several  years  ag»  Cl»udman  reported  that  mice  ,ff  strain 
C3Fe,  obtained  by  transfert-ing  fertilized  C3H  ova  into 'C57BLK  mothers, 
were  tolerant  to  a  tumor  of  C57BIK  origin.   If  this  were  true  it  would 
furnish  an  interesting  and  instructive  variation  to  the  other  known 
examples  of  "pseudohybridization"  or  "acquired  tolerance."  However, 
Cloudman  reported  tolerance  in  generations  F^  to  F  .  Tolerance  in 
the  parental  generation  woul*  be  easy  to  believe  btrt  not  tolerance 
in  filial  generations.  The  istablishment  of  strain  C3He  by  Dr. 
Deringer  gave  us  an  opportunity  to  reinvestigate  this.  A  total  of 
I7U  C3He  mice  of  generations  Fg  to  R  failed  to  yield  any  takes  of  a 
tumor  of  C57BIK  origin  an^  there  also  were  none  in  1;  mice  of  the 
parental  generation.  ThuiS  no  confirmation  of  Cloudman's  report  was 
obtained.  A  manuscript  reporting  these  negative  findings  is  in 
preparation. 

I  participated  in  the  Gordon  Cancer  Conference  as  an  invited 
discusser  but  no  manuscript  for  publication  was  involved. 

A  considerable  amount  of  time  has  been  spent  in  reviewing 
manuscripts,  ap plications  for"  fellowships,  and  grant-in-aid  applications. 

Significance  "     '  , 

The  field  with  which  this  woi*k  is  concerned  is  so  difficult 
and  obscure  as  to  make  cle^.r  definitions  and  outlines  almost  impossible. 
Nev..rtheless  there  is  a  slowly  growing  body  of  fevidence  which  indicates' 
that  there  are  interactions  betw.cn  tissues  and  betwefen  tissue  and  its 
host  which  involve  critic^J.  factors  in  the  phenomena  of  cancer,  embryology, 
and  transplantation  of  normal  tissues.  A "defensible  definition  of  cancer 
cannot  be  given  today  except  in  descriptive  and  biologic  terms.  The 
terms  usually  include  things  related  to  the  experiments  that  we  have  been 
describing.  The  experiments  may  be  significant  in  several  ways  both 
practical  and  theoretical. 

The  most  important  implication  is  that  some  of  these  results 
suggest  progress  toward  understanding  ©f  what  controls  the  grewth  of  a 
cancer  and  how  this  differs  from  normal  tissue.  We  have  already  seen 
that  a  tumor's  power  to  overcome  host  resistan<?e  can  be  altered;  its  rate 
of  growth,  tendencj'-  t^  regress,  and  tendency  to  metastasize  are  all 
puljject  to  experimental  change.  The  host's  reaction  can  be  altered, 
ex^ei-imentallv,  not  only  between  similar  hosts  but  also  at  different 
times  and  sites  in  the  same  host.  The  changes  in  the  tumor  have  eluded 
morp'-ologic,  chemical,  or  serologic  definition.  One  of  the  antigens 
which  changes  the  "host  would  l>e  misgad.  by  current  chemical  methods. 
'Jowever  one  might  attain  a  biolog4<C'  description  of  the  factors  that 
sometimes  cause  snontaiieous  tumorS  of  man  and  animals  to  regress  and  th^t!, 
centrol  metastasis.  It  has  already  been  obaaj^^?^  in  this  laboratory 
Tand  confirmed  elsewhere)  that  such  factors  materially  effect  the  outcome 
of  many  chemotherapeutic  experiments. 


PROJECT  REPORT  FORM  (Cont'd) 

10.  Ul8   / 

SERI1L  NO. 

11. 

BUDGET  ACTIVITY:  ' 

RESEARCH   X        ADMINISTRATION 

REVIEW  &  APPROVAL    TECHNICAL  ASSISTANCE 


12. 

COOPERATING  UNITS  OF  THE  PUELic  HE/iLTH  SERVICE,   OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  F\CILITIES,   OR  P2RS0WJEL  FOR  TKLS  PROJECT  IN  EITHER 
1956  or  1957 


13. 

iF  TT^tS  pftojEC*  kfiSffifeLK,  toMt^LEiEN'rS,  oK  mALLELS'RTSEAftcH  bofffi 

ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE   (WITHOIT:   IKTiIflCHANGE  OF 
PE[iSONNEL,  FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH} 


PROJECT  DESCRIPTION  (Cont.) 

The  experiments  on  incomplete  excision  may  lessen  the  fear 
of  some  surgeons'in  attempting  palliation  in  certaxn  selected  cases  , 
and  thus  lead  to  amelioration  of  a  few. 

Relation  to'  others 

There  is  a  complex  theoretical  irterlocking  ^^^ween  this 
«ork  a«d  the  work  of  Drs.  Algire,  Grobstein,  Prehn,  and  Sanford. 


PROJECT  RE^^ORT  FORM   (Cont'd) 


lU.    iil8 

SERIAL  NO. 


1?. ^ 

PUBLICATIONS   OTHER  THAN  ABSTRilCTS  FROH  T"IS  PROJECT  DURING  CALEIO/m 
YEAR  1955 


Barrett,  M.  K.  and  Hansen,  W.  H.:  Resistance  of  mice  to  multiple 
inoculations  of  a  transplantable  tumor.  J,  Nat.  Cancer  Inst.  15: 
Ull-h20,  195U  (A:;Teared  in  1955). 

Barrett,  M.  K.:  The  nature  of  tumor  immunity.  In  Origins  of 

resistance  to  toxic  agents,  M.  G.  Swag,  R.  D.  Reid,  and 

0.  E.  Reynolds,  eds.  'icademic  Press,  New  York,  p.  308-333,  1955* 


16. 

HONORS  AND  AWARDS  TO  ^E^^SONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955. 


PAGE  1 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute    2.  Laboratory  of  Biology 

ITSTITUTE  '  LABORATORY  OR  BRAWCH 

3.  General  Biology   U .        5»  Ul9 

SECTION  OR  SERVICE  L0CAT10N(IF  OTHER  T^AN  SEE.    NO. 

BETHESDA) 

6.  Gastric  Cancer  Investigations _^ 

PROJECT  TITLE 

7 .  M.  K.  Barrett ^ 

PRINC IPAL  INVESTIGATOR (3 )  '      ' 


OTHER  INVESTIGATORS  " 

9.  PROJECT  DESCRIPTION 

QlBjectives 

Efforts  have  been  tiireeted  at  finding  methods  f tr  producing 
or  studying  some  of  the  supoasedly  etiologic  factors  in  gastric 
cancer.   Many  etiologic  or  predisposing  factors  have  been  suggested 
but  laboratory  investigation  of  them  has  been  held  back  by  a  lack  of 
adequate  methods  for  producing  them  in  animals.  Our  work  has  com- 
prised a  series  of  attempts  to  produce  starting  points  from  which 
gastric  cancer  studies  could  go  forward,  Tn  addition,  we  have 
attempted  to  stimulate  work  in  this  field  generally  through  th<? 
activities  of  the  Gastrointestinal  Cancer  Committee. 

T^ethods 

New  techniques  of  experimental  surgery  (some  developed  in" 
this  laboratory)  and  physiology  have  been  used  to  study  gastric 
polyps  iu  dogs  and  peptic  ulceration  in  rats. 

Findings 

It  was  reoorted  last  year  that  when  artificial  polyps  are 
constructed  in  a  dog's  stcanach  the  mucosa  of  the  polyp  undergoes  de- 
generative changes  ending  in  atrophic  gastritis.  A  paper  reporting 


PROJECT  DESCRIPTI'^N  (Cont.) 

this  appeared  in  Gastroenterology  for  March  1955. 

In  keeping  with  our  general  ideas  regarding  tissue 
specificity,  we  adopted  an  hypothesis  that  the  stomach  does  not 
digest  itself  because  some  "recognition  factor"  prevents  it.  A 
recognition  factor  or  specificity  might  exist  between  the  enzymes 
and  the  tissue,  or  between  some  element  of  the  mucus  and  the  tissue, 
or  among  all  three.  If  this  were  so  the  mucosa,  which  is  relatively 
immune  to  the  corrosive  action  of  its  own  secretions,  might  be 
readily  attached  by  foreign  secretion.  Under  properly  controlled 
conditions  gastric  juice  was  transferred  between  strains  of  rats. 
It  was  found  that,  although  each  strain  was  resistant  to  its  own,., 
juice  for  a  period  of  U  hours  (or  more),  the  glandular  mucosa  of  "■ 
each  was  vulnerable  to  the  juice  of  the  other  during  a  period  of  3 
hours  (or  less).  A  manuscript  reporting  these  data  has  been  sub- 
mitted to  Gastroenterology.  ,  .  .  _  :  "' ~  ":  ',' 

Significance 

The  experiments  on  gastric  polyps  should  influence  the 
clinical  management  of  polyps,  in  the  direction  of  radical  treatment, 
gsner^.lly.  They  also  provide  the  long  sought  method  for  producing 
atrophic  gastritis  regularly,  although  better  methods  are  still 
desirable.  ,•  •  • 

The  studies  on  peptic  digestion  may  have  far  reaching  im- 
plications, which  ai  present  are  speculative;  One  might  ask  some 
questions.   Is  the  pepsin  specific  in  an'immuno^enetic  sense?  If  so,' 
do  other  enzymes  have  a  biological  specificity  in  the  natural  state 
and  do  they  lose  this  specificity  in  vitro?   (As  pepsin  may  do). 
Is  a  different  enzyme  from  pepsin  involved  in  the  initial  attack 
upon  the  mucosa?  (If  so,  the  specificity  remains").  Does  the  mucus    'v,- 
(jontain  a  mucopolysaccharide  which  is  antibodyrlike  in  function?  :  If  so, 
is  this  where  -lysozyrae  enters  the  field  in  peotic  ulccr  r.ad  ulcera- 
tive colitis?  Does  gastric  mucosa  sometimes  contain  groups  of 
cells  whAch  are  immunogenetically  aberrant?   (Most  clones  of  cells 
do  contain  atypical  cells).   Is  the  chronic  ulcer  case  an  example 
•f  this  with  an  area  of  permanently  lowered  tolerance  and  the  acute 
ulcer  case  merely  a  case  of  temporary  imbalance  of  tolerance  for 
transieftt  causes?  If  so  the  first,  should  always  be  excised  for 
cure,  the  second  may  get  well  without  surgery.   Is  this  the  reason 
that  nearly  all  gastric  cancers  (at  the  surface)  ulcerate?  If  so, 
one  might  be  justified  in  deliberately  augmenting  ulceration  in 


PROJECT  RE^CRT  FORM  (Cont'd) 


10.  Ul9 

.  SERIA.L  NO.  " 

11. 


BUDGET  ACTIVITY: 


aESEARCH     X        ^DMINISTR;'VTION 
REVIEW  k   APPROVAL   "   TECHNICAL  ASSISTANCE 


12. . 

.  COOPERATING  UNITS  OF  TITE  PUBLIC  HEALTH  SERVICE,   OR  OTHER  '" 
ORGANIZVT   '^NS,   PROVIDING  FUI^IDS ,   F.\CILj.TIES,   OR   p■SRSONI^IEL  FOR 
THIS   PROJECT  IN  EITHER  1956  or  19$ 7 


13 . 

IF  TWIS   PROJECT  RESEMBLES,   COMPLE-'ENTS,   OR  ?  JIALLELS  RESKIRCH  DONE 
ELSEWHERE   IN  THE  PLTBuIC  HEi'iLTH  SERVICE   (WITHOUT   INTERC^'iNGE  OF 
PERSONNEL,   FACILITIES   OR  FUNDS),   IDENTIFY  SUCH  RESMRCHt 


PROJECT  DESC'.IIPTION  (Cont.) 

some  small  lesions  -  a  long  shot.  At  any  rate,  this  appears  to  be 
a  new  and  provocative  idea  in  gastric  digestion  -  and  there  has  not 
been  too  many  such  during  a  century  of  investigation. 

Gastrointestinal  Cancer  Committee  work. 

\  large  amount  of  time  has  gone  into  work  with  this 
Committee.  A  small  Si'-mposium  was  held  in  Stone  House  near  the  end 
of  last  year.  At  the  request  6f  the  editor  of  Gastroenterology  I 
prepared  a  short  summary  of  the  proceedings  and  this  appeared  in 
Gastroenterology  for  June  1955 •  There  has  been  a  demand  from 
abroad  for  this  reprint.   I  don't  know  why. 

I  carried  the  major  burden  in  organizing  the  6th  National 
Gastrointestinal  Cancer  Conference  which  was  held  in  New  York  in 
April.  Editorial  work  and  proof  reading  of  the  proceedings  have  been 
time  consuming  but  are  completed  and  the  complete  Proceedings  appeared 
in  Gastroenterology  for  October,  1955.   This  is  much  more  rapid 
publication  than  was  obtained  previously. 

Proposed  Course 

We  are  planning  tec'riniques  whireby  we  may  be  able  to  deter- 
mine whether  the  specificity  of  gastric  juice  for  gastric  mucosa 
also  extends  to  unrelated  tissues.   If  the  specificity  resides  in  the 
enzymes,  such  an  extension  could  be  very  important.  Other  attempts 
will  be  made  to  see  if  stronger  implication  of  the  enzyme  can  be 
obtained. 


PROJECT  REPORT  FORM  (Cont'd) 


lU.  Ui? 

SERilL  NO. 


15. 

PUBLICATIONS  OTHER  THAN  ABSTR.'ICTS  FROM  THIS  PROJECT  DURING 


CALENDAR  YEAR  1955 


Parrett,  M.  K.,  Lcfco,  T.,  and  Hansen,  W.  H. :  Degenerative 
changes  in  the  mucosa  of  artificial  gastric  poljrps  in  dogs. 
Gastroenterology  28:  393-Uol,  1955. 

Barrett,  M.  K.:  Summary  of  gastrointestinal  cancer  symposium, 
November  19,  195U.  Gastroenterology  28:  969-971,  1955. 


16. 

HONORS  AND  AWARDS  TO  PERSONNEL  RELATII\IG  TO  THIS   PROJECT  DURING 
CALENDAR  YEAR  1955. 


PAGE  1 
PROJECT  REPORT  FORM 


1.  National  Cancer  Inst.  2.  Laboratory  of  Biology 
IM3TITUTE     '  LABORATORY  OR  BRANCH 


3.  General  Biology  Sectioni;«  ^.1|28  (a) 

SECTION  OR  SERVICE       LOCATION( IF  OTHER . THAN . BETHESDA )       SER.  NO. 
Studies  •n  the  Rous  sarcoma  virus.  I.  Development  of  th« 
6»  virus-chicken  teat-system  for  general  use  in  basic  studies  tn 
PROJECT  TITLE  carcinogenesis  and  other' tfio logical  reactions. 


7.  W.  Ray  Bryan 


PRINCIPAL  I ^iVEST IGATOR  ( S ) 
8.  John  B.  Moloney,  Dorothy  Calnan 


OTHER  INVESTIGATORS^ 
9.  PROJECT  DESCRIPTION,  53  y^  X'-'td'Cioo 

Objectives;  To  bring  about  adequate  control  of  the  biological  and 
experimental  factors  which  cause  excessive  variations  and  interference 
with  orecise  systematic  investigations  involving  the  Rous  sarcoma 
virus-chicken  test- system  (a  valuable  system  for  crucial  studies  on 
carcinogenesis,  particularly,  from  the  yiewppint  of;  viral  etio.iagy).  . 

Methods  employedt  Sinc,e  satisfactory  methods  have  now  been  worked  , 
out  and  the  principal  objectives  noted  above  have  been  accomplished!,  , 
this  project,  is  currently  of  minor  importance,  consisting  only  in  . ,_ 
the  further  refinement  of  existing  procedtres.  The,  details  of 
methods  are  therefore  not  reiterated  here  '(See   IRSli  report  under 
this  heading). 

Major  findings;  The  major  findings  developing  from  this  study, 
which  had  been  in  progress  for  about  U  years,  have  been  detailed 
in  a  comprehensive  review  of  the  subject  published  during  the  past 
year  )see  references  1  and  2  below).  The  contributions  of  this 
laboratory  to  the  problem  are  summarized  as  follows; 

1.  Control  of  the  variations  in  initial  potency  of 

extracts  was  accomplished  by  showing  a  correlation  be- 
tween tumor  initiating  dose  of  virus,  and  the  '/leld 
of  virus  in  the  tumor  tissue  extracts.  By  using 
only  tumors  initiated  by  strong doses  of  virus,  the 
tumor  source  material  can  be  kept  highly  potent,  and 
predictable . 


PROJECT 3DESCR I PTION 

2,  Control  of  the  variable  loss  in  biological  activity 
of  different  virus  oreparations  was  accomplished  by 
the  use  of  citrate  reagents,  in  which  p-itency  remains 
stable  for  at  least  7  days  at  ordinary  refrigerator 

temperatures.,. 

Preservation  for  long  periods  of  time  (at  least 
■'   *  .   2  years)  was  accomplished,  by  freezing  in  citrate 

solutions  and  storage  in  a  COj  ice  chest.  This  find- 
ing permits  the  use  of  standard  virus  preparaticfns 
■'' .  *   for  quantitative  biological  studies  and  inyostigations . 
extending  over  long  periods  of 'time i''-*^  .^di'i 

3.  Control  of  variations  in  susceptibility  among  different 
test  lots,  of  chickens  employed  at. different  times  was  • 
accomplished  by  the  use  of  stable  standard  preparations 
of  virus  (see  2)  and  bioassay  methods  based  upon  refer- 
ence to  standard  preparations. 

li.  The  variations  in  susceptibility  among  individual   - 

chickens  of  a  common  lot  was  brought  under  statistical 

control  by  studies  on  the  distribution  of  chicken 

sensitivities  and  the  selection  of  appropriate 

statistical  methods  applicable  thereto. 
mor-^"-  ■  ■  ■   ■       .   "   , 

Future  course  of  project;  It  is  planned  to  initiate  sometime  next 
spring  or  summer,  a  study  on'  different  types  of  host  chickens  in 
their  responses  to  the  Rous  sarcoma  virus.  The  study  will  involve 
various  inbred  lines  of  c'nickens  derived  from  the  Regional  Poultry 
Laboratory,  U.S.D.A.,  at  East  Lansin'g,  Michigan,  and  will  be  in 
collaboration  with  Dr.'  B.H.-  Burmester  of'  that  laboratory.  The  ob- 
jective will _ be  to  find  lines'  of  high,  intermediate  and  low  genetic 
susceptibility  to  the  virus'^  for  further  use  in  contemplated  studies 
on  virus-host  interaction. 


■  ^ir"  y'.i 


PROJECT  REPORT  FORM  (Cont'd) 


10.   1^20  (a) 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY 


RESEARCH    X        ADMINISTRATION 
REVIEW  &  APPROVAL     TECHNICAL  ASSISTANCE 

12. — — 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS , 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957. 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUfeLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF 
PERSONNEL,  FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PROJECT  RS^nRT  FORM    (Cont'd) 

lU.    U20  (a) 

SERIAL  NO. 

15. 

PUBLICATIONS  OTHER  T'^AN  ABSTR.\GTS  FROM  T^'IS  PROJECT  D'lRING  CALEfffi-'iR 
YEAR  1955 

W.  R.  Biyan;  Biological  Studies  on  the  Rous  Sarcoma  Virus. 

I,  General  Introduction.  J.  'Jat.  Cancer  Inst.  i6:  285-286,  1955. 

W.  R.  Bryan:  Ilsidem.   II.  Rgvisw  of  Sources  of  Experimental 
Variation  and  of  Methods  for  their  Control.  J.  Nat.  Cancer  Inst. 
16:  287-315,  1955. 


16. 

FON'-^RS  AND  Wi'^RDS  TO  ^/ aSONwEL  RE_-iTING  TO  T' " IJ  PROJECT  DURING 
CALENDAR  YE\R  1955. 


PAGE  1 
PR'-iJECT  REPORT  FORM 


1.  National  Cancer  Institute    2 »  Laboratory  of  Biology 

Institute      '  ^    laboratory  or  branch 

3,  General  Biology  Section h- g.U20  (b) 

section  or  service  location  (IF  oti-ter  t^'An  BETH.;    ser.no. 

6.  studies  on  the  Rous  Sarcoma  virus.  II.   Pvirifi cation  of  the  virus. 

PR''^JECT  TITLE 


7.  John  B.  ^ioloney  and  W. -Ray  Bryan 


PRINCIPAL  INVESTIGATOR (S) 
8.  Dorothy  Calnan 


OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives; 

To  obtain  the  Rous  sarcoma  virus  in  sufficient  quantity,  and  in 
a  sufficiently  high  state  of  purity,  to  permit  its  physical  and 
chemical  characterization,  as  well  as  its  general  use  in  investigations 
on  mechanisms  of  carcinogenesis. 

Methods  employed; 

(a)  Enzymatic  degredati-n  of  impurities  by  the  use  of 
hyaluronidase  and  trypsin,  (b)  precipitation  of  the  virus  by 
protamine  sulphate,  and  (c)  differential  ultracentrifugation,  were 
combined  to  produce  a  purification  techaique  which  yields  a  product 
of  h   to  12  fold  higher  purity  Con  a  nitrogen  basis)  than  methods 
previously  developed.  A  report  of  the  procedures  has  been  published 
(see  reference,  below). 

It  is  apparent  that  these  same  procedures,  carried  through 
2  or  more  additional  cycles,  might  accomplish  final  purification 
of  the  virus  if  stronger  sonrce  materials  were  available  for  their 
application.   (See  further  discussion  under  "Proposed  course  of 
project",  below). 


PROJECT  DESCRIPTION  (Cont.) 

Significance  to  cancer  research;  The  rapidity  of  action  of  this  tumor 
virus  (microscopic  tiutiors  in  2  or  3  days,  gross  tumors  in  5  or  6  days) 
indicates  that  it  may  enter  directly  into  the  intracellular  reactions 
involved  in  malignant  transformation.  This  virus  may  therefore  be 
capable  of  guiding  the  investigator  directly  to  the  elements  or  re- 
actions involved  in  the  transformation  from  the  normal  to  the  neoplastic 
ptate^  Studies  on  the  chemical  interactions  are  dependent  upon  the 
availability  of  practical  quantities  of  essentially  pure  virus. 

Proposed  course  of  project;  (a)  Further  refinement  of  present  success- 
ful  separative  procedures  with  emphasis  on  increasing  the  final  yield 
so  that  additional  purification  cycles  can  be  undertaken,  (b)  the 
overwhelming  amounts  of  non-virus  material  ("impurities")  to  virus 
in  the  present  starting  extracts  of  tumor  tissue  cause  relatively 
great  losses  by  any  method  of  separation.  Efforts  are  therefore  being 
made  to  increase  the  virus  content  of  the  tumor  tissue  to  be  used  as 
virus  source  material  (See  under  project  III). 


PROJECT  REPORT  FORM  (Cont'd) 
U20  (b) 

10.  SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH             X  ADMINISTRATION 

REVIEW  ^  APPROVAL  TECMICAL  ASSISTANCE 

12. ______ 

co6*ffiA'ftW6  ilWiM  oP  fM  'mtit  HEAttH  SffiVtdE,'  6ft  ofH^TR  okGAiiiZAf  ^'onS, 

PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957 


13. 

IF  THIS  PROJECT  RESE-fitES,  d'6l^^KEiN'iS ,  Oft  PARALLELS  ftfi^fiAR^J^  tloM 
EISEW"ERE  IN  THE  FiBLIC  HEALTH  SERVICE  (WITHOUT  ItJTERC"ANGE  OF   PERSONNEL, 
FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEMS  lli,  15  and  16 


PAGE  1 
PROJECT  RE'^ORT  FORM 


1.  National  Cancer  Institute2.  Laboratory  of  Biology 

INSTITUTE  LABOR.^TOR^'  OR  BRANCH     ' 

3.  General  Biology  Section  h • S .   Ii20  (c) 

SECTION  OR  SERVICE        LOCATION  (IF  OTHER  T".\N  BETHESDA)   SER.  NO. 
Studies  on  the  Rous  sarcoma  virus.  III.  Ihe  quantitative  relation- 

6.  ship  between  inducing  doses  of  virus  and  the  amount  of  virus ^ 

PROJECT  TITLE    extractabl^  from  experimental  tumors. 


7.  W.   Ray  Bryan 

PRINCIPAL  INVEST IGATOR(S) 


8.  Dorcthy  Calnp.n  and  John  B.  Moloney 

0T^"ER~IWESTii';Af0R3  '  ■  , 

9.  PROJECT  DSSCRTPTiON 

Objectives;  The  original  objectives  of  this  researc>i  w.:.re  accomplished 
dm-jng  the  past  year  ind  a  report  of  the  firdings  was  given  in  the 
publication  listed  below. 

M.^jor  finding? ;  The  anioant  of  virus  extractable  from  experimental 
iu'iors  vas  found  to  >>':  related  to  the  dose  of  virus  usal  for 
initiating  the  tumor.   Above  certain  initiating  dose  l2\/-els  (2^0  or 
more  ED^O  units),  the  virus  rields  ap:)roached  an  upper  limit  asymptoti- 
cally, but  at  lower  virus  initiating  doses  the  yields  were  highly 
correlated  with  initiating  dose.  Doses  of  1  ED^O  unit,  or  less, 
procLuced  tumors  from  which  very  little  or  no  virus  could  bo  recovered 
on  ext.actj.on. 

Significance  to  cancer  research;  In  addition  to  the  practical  useful- 
ness of  the  information,  which  permitted  methods  for  pi  educing  source 
tissue  of  constant  high  potency  (see  project  I),  the  results  of  this 
study  have  imoortant  implications  with  respect  to  concepts  in  cancer 
research.  For  example,  it  has  been  generally  assumed  by  workers  in 
this  field  that  tumors  having  a  viral  etiology  should  yield  demon- 
strable virus  on  extraction,  and  that  the  failure  to  demonstrate 
virus  in  filtrates  of  turners  is  justification  for  regarding  them 
as  being  of  nonviral  etiologyt  The  above  findings  show  decisively 
that  this  is  not  a  valid  assumption,  and  that  under  certain  re- 
producilile  experimental  conditions  even  the  most  potent  and  most 


PROJECT  DESCRIPTION  (Cont.) 

rapidly  acting  tumor  virus  known  is  capalile  of  initiating  tumors 
from  which  little  or  no  virus  can  >»e  recovered  on  extraction. 

Since  the  frequencies  of  experimental  tumors  are  also 
related  to  initiating  dose,  the  probability  of  detecting  virus 
in  tumor  tissue  extracts  decreases  as  the  frequency  of  the  tumors 
decreases.   A.t  the  extremely  low  frequencies  with  which  "spontaneous" 
tumors  occur  in  nature,  therefore,  one  would  not,  on  a  basis  of  these 
findings,  expect  to  demonstrate  the  presence  of  a  viral  agent  even 
if  the  tumor  should  be  of  viral  origin, 

Proposed  course  of  project?  The  developments  in  this  project  have 
opened  up  new  areas  of  investigation.  The  most  significant  problem 
to  be  investigated  is  that  of  the  reason  for  the  observed  correlation 
between  initiating  dose  and  virus  ^/ield.  The  final  answer  to  this 
question  will,  of  course,  involve  both  the  mechanisms  of  virus  re- 
production (in  this  particular  case)  and  of  neoplastic  transformation. 
>Tany  ye=>rs  would  probably  be  required,  therefore,  for  substantial 
progress  toward  this  end. 

The  present  efforts  on  this  project  are  confined  to:   (a) 
The  development  of  biological  materials  which  will  be  necessary  for 
the  more  'hasic  studies,  such  as  a  transplantable  tumor  L'.ne  of  low 
dose  origin  which  will  provide  ample  amounts  of  tumor  tissue  yielding 
little  or  no  virus  on  extraction;  and  similar  stable  tumor  lines 
yielding  extremely  high,  as  well  as  Intermediate  potencies,  (b) 
Exp'i.oratory  experiments  on  possible  mechanisms  which  rould  explain 
the  correlation;  such  as  the  presence  of  inhibitor  substances 
(suggested  by  certain  results  of  earlier  investigators),  or  differ- 
ences in  amounts  of  virus  actually  reproduced  (electron -micrographic 
studies  with  Dr.  Balton  -  preliminary). 


PROJECT  FORM    (Cont'd.) 

10  .j£20_j£)____ 
SERIAL  NO. 

11.  

BirOGET  ACTIVITY; 

RESEARCH  X  AmiNISTR\TIO.N       , 

REVIEVJ  ^.   APPROVAL  TECKMICAL  ASSISTANCE 


12. 


COOPERATING  UNITS   OF  THE  ^UBLIC  HEALTH  SERVICE,   OR   OTHER  ORG.INIZ'TIONS, 
PROVIDING  FUNDS,   FACILITISS,   OR   ^ERSONl^EL  FOR  T'-'IS   PROJECT   IN  EITHER 
1956  or  19^7 


13. 


IF  T'-'IS   PR-^JECT  RESEIIBLES,  COK^'LEI'iENTS,   OR  ^'-R'-LLELS  REoEAxxCH  DONE 
ELSEWHERE  IN  THE   PT^BLIC   HEALTH  SERVICE   (WITHOUT   II'TERCH'INGE   OF 
PERSOi^-^L,   FACILITIES   OR  FiTNDS),   IDENTIFY  STTCH  RESE'RCH: 


PROJECT  REPORT  FORI>^   (Cont'd) 

lU.  HgQ  (c) 

SERI'vL  NO. 

Ig.       

PUBLIC/\T10NS  OTHER  THAN  ABSTRACTS  FROM  T^IS  PROJECT  DIEING  CALENDAR 
YEAR  19^5 

Bryan,  W.  R,,  Calnan,  D.  and  Moloney,  J.  B. :  Biological  studies 
on  the  Rous  sarcoma  virus.   III.  The  recovery  of  virus  from 
experimental  tumors  in  relation  to  initiating  dose.   J.  Nat. 
Cancer  Inst.  16:  317-335,  1955. 


16. ■ . 

HONORS   AND  AWARDS  TO   "fRSONlviEL  RELATING  TO  T^'TS   PROJECT  DURING 
CALENDAR  YEAR  1955. 


PAGE  I 

PROJECT  pj:port 

•  *  National  Cancer  Institute '_  2 ,   Laboratory  of  Biology 

INSTITUTE  .  ■  LABOIUTORY  OR  BRANCH 

I*  General  Biology  Section I4. 5.   I42I 

SECT  ION  "CR 'SERVICE  '        LOC.iTION'^  (IF  OTHER  TIliiN      SERIAL  NO 

EETHESDA 

) ,  Genetic  Studies  in  Mice  ' A.  Genetic  Studies  of  Tumors  B.  Ctenetic  Study  of  a 

PROJECT  TITLE   '        '  '       "'  • 

Mutant.  .Urogenital  Abnormalities  in  Strain  A  x  C  Line  993$   Rats.    

' .  Margaret  K.  Deringer 

PRINCIPAL  i:TE3Tir-AT0P(37  "  ' 

i.  Dr,  V.  E.   Heston     -  Dr.  M.  K.  Barrett ^ ] 

OTHER  INVESTiaiTORS  • ■■ 

'.  PROJECT  DESCRIPTION: 

Objectives; 

A.  Genetic  studies  of  tumors 

.  li  To  study  the  effect  of  the  injection  of  the  mammary  tumor  agent  into 
agent-free  strain  C3Hf  mice  duririg  the  .late  stages  of  pregnancy  at  which 
time  the  agent  has  been  reported  to  disappear  from  the  blood  of  mice 
which  carry  the  agentj  and  to  see  if  the  agent  can  be  transferred 
tlirough  the  uterine  wall  to  the  fetus  (in  cooperation  with  Dr.  Heston). 

2.  To  study  the  effect  of  the  subcutaneous  injection  of  2~f^™ii^oszotoluene 
on  the  prQcluction  of  hemangioendotheliomas  in  strain  Hn  mice.  It  has  been 
observed  that  hemangioendotheliopias  developed  spontaneously  in  l8.'i  of  a 
group  of  untreated  strain  HR  mics»  No  other  strain  of  mice  has  been  des- 
cribed in  which  hemangioendotheliomas  occur  frequently  but  they  have  been 
produced  by  subcutaneous  injection  of  o-aminoazotoluene  in  strains  BALB/c, 
A,  C3H,  and  C5'7BL. 

3.  To  study  the  incidence  of  mammary  gland  tumors  in  virgin,  in  breeding, 
and  in  forced-bred  females,  and  in  stilbestrol  injected  males  of  strain 
C3HeC57BL  (a  substrain  developed  in  this  laboratory  by  the  'transfer  of 
fertilize*  ova  from  a  strain  C3H  mouse  to  a  strain  C57BL  mouse). 

U.  To  determine  whether  a  mammary  tumor  agent  is  pi'esent  in  strain  BL 

(Bagg  L)  and  in  strain  S'^  (Swiss)  (in  cooperation  with  Dr,  Heston). 

$,     To  study  the.  relationship  of  certain  known  genes  (W  -  dominant  spotting, 

Ca  -  caracul,  and  Fu  -  fused)  in  mice  to  tjie  production  of  pulmonary 

tumors  induced  by  the  injection  of  a  hydrocarbon  (in  cooperation  with  Dr* 

Heston). 

6,  To  produce  special  types  of  animals  for  collaborative  experiments 

with  Dr.  I^'i,  K^  Barrett  (see  Dr,  Barrett '.s  report  for  details), 

B.  Genetic  study  of  a  mutant 

1.  To  test  a  mutant,  phenotypically-likc.waltzer  which  arose  in  strain 
A  mice  maintained  in  this  laboratory  (in  cooperation  with  Dr.  Heston). 

Urogenital  abnormalities  in  -strain  a  x  C   line  993$   rats. 


i'ACE  2 

lit  autopsy  contreiiital  absence  of  one  kidney  was  observed  in  a  rat  of 
the  strain  A  x  C  lino  993$   colony*  Subsequently,  a  number  of  rats  of 
this  strain  wore  observed  to  have  one  kidney  missing,  and  a  number  were 
observed  to  have  othor  urogenital  jibnorinalitiL-.G.  The  extent  and  inci- 
ili'iu'i'  of  those  nbnovma.l  conditions  hars  bc>'n  obnorvcd. 

Method H  omploycd : 

Al  1.  llroups  of  strain  C3Hf  mlro  have  boon  injected  intraporitoneally  with 
0»5  ^'0«  O-'C  f  S%   cell  free  extract  of  a  mJUiimary  t^iland  tumor,  from  a   strain 
C3H  mouse.  The  [.^roupn  of  animals  were  injected  1-3,  U-6,  and  7-9  days 
boforo  the  birth  of  tho  youncj  immodiately  followin^^  the  birth  of  the 
young;  and  when  thco  first  litters  wore  weaned.  Two  groups,  maintained 
as  virgins,  have  been  injected  at  h$  and  65  days  of  ape  respectively. 
Unlnjooted  female  mice  have  been  allowed  to  produce  one  litter  each  and 
then  have  been  retired  at  the  time  at  which  these  litters  wore  weaned. 
Approximately  200  female  sff sprint;;  of  mothers  injected  before  the  birth 
oj"  the  yonnf^  have  been  kept  as  virgins.  The  animals  in  this  experiment 
will  bo  maintained  as  long  as  possible  and  a  study  of  the  occurrence  of 
mammary  i:l''^nd  tumors  and  of  other  typos  of  tumors  will  be  made. 
2»  One  hundred  retrain  HR  mice  (equally  distributed  as  to  sex  and  as  to 
profjonoo  or  absence  of  hair)  wore  injected  with  100  mt^:,  of  o-aminoazo- 
toluone  dissolved  in  olive  oil.  The  injectioiis  were  startecf  when  tho 
animals  were  2  to  2  -."V  months  of  age  and  were  given  in  monthly  doses  of 
10  mj%  each.  One  hundred  uninjcctod  animals  served  as  controls,  illl  •f 
tho  animals  were  observed  for  tho  development  of  tumors  and  of  any  un- 
usual lesions. 

3.  One  hundred  C3Hi  females  have  been  set  aside  as  virgins  in  order  to 
study  the' development  of  maiumary  gland  tumors  in  them.  One  hundred  C3He 
females  are  being  forced-bred  for  the  same  purpose.  These  animals  are 
isolated  when  prcgnaat  .'iiid  nre  rc;turned  to  the  breoding  cage  within  2U 
hours  ot  less  following  the  birth  of  the  litter.  One  hundred  males  have 
been  injected  subcutaneously  in  the  right  axilla' with  6-8  mg.  pellets  of 
10^  stilbestrol  in  chclestrol  for  the  purpose  of  studyiiig  the  development 
of  nvuim*\ry  gland  tumors.  This  will  aliso  be  determined  in  the  case  •f  tho 
brooding  females  which  are  used  to  maintain  the  colotiy  of  C3He  mice. 
Ij,  Newborn  strain  C3Hf  or  C3He  femnlu  mice  are  foster-nursed  on  strain 
BL  and  on  strain  S^.'JY(   females.  The  foster-nursed  animals  are  maintained 
as  virgins  v-md  are  observed  for  the  development  of  mammary  gland  tvunors.^ 
^',  The  mice  \iBod  in  this  exvxrimont  were  olfspring  produced  by  mating 
F-,  hybrids  of  strain  IJCaT'u  and  A  with  strain  119li»  Tho  animals  were 
injected  intraperitoneally  with  O^S   mg,  of  1,2,5,6-dibcnzanthracene  at 
two  months  of  age.  They  wore  autopsled  at  10  months  of  age  and  were 
observed  for  the  development  of  pulmonary  tumors  and  of  aixy   unusual 
lesions, 

P,  1,  Stivnin  A  mados,  ,  -howing  the  waltaiiir;  chi^.racter  were  outcrossed  to 
strain  119l4>  F]_,  F2*  ^'^'^  backcross  generations  wore  produced  and  data 
were  collooted  regarding  the  presence  of  the  charaott.r  in  the  different  " 
groups  of  animals, 

A  rtuttnu  .saarch  for  atawrrmfilities  of  the  urogenital  system  was  made 
at  autopsy  in  youii^.-;  animals  (1  to  i8  days  of  age)  and  in  adult  animals 
(2  to  17  months  of  a^e)  of  the  -strain  A  x  C  lino  9935  rats. 


P.UJE  3. 

Major  findings;  ■ 

jv,     l.'Tn  the  group  injected  1-3  days  bcforo  the  birth  of  thtj  young,  11  of 
a  group'  of  57  females  have  dovclopod  mammary  !,':1-.uki  l.mnora  at  an  avorago 
ago  of  20,9  months;  the  non-tumorous  ajiynals  diofl  at  an  average  age  of 
21,1  months.  In  thu  group  injected  [|-6  days  before  th^,.  birth  of  the 
young,  13  of-  a   group,  of  $3  females  have  developed  maimnary  gland  tumors 
at  .an  average  age  of  20.8  months;  the  non-tumorous  animals  died  at  an 
average  a^  of  20,2  months.  In  the  group  injected  7-9  days  before  the 
birth  of  the  young  h   of  a  group  of  37  have  developed  majranary  gland  tumors 
at  an  average  age  of  l6.0  months;  the  non-tumorous  animals  dying  at  an 
average  age  of  19.8  months.  In  the  group  of  animals  injected  at  the  wean- 
ing of  the  first  litter  5  of  a  group  of  52  animals  developed  mammary  gland 
tumors  at  an  average  age  of  2lj..l,|.  months;  the  non-tumorous  animals  died  at 
an  average  age  of  20.1  months.  One  fumale  of  a  group  of  l6  injected 
following  the  birth  of  their  young  developed  a  m<-jnuna.ry  gland  tumor  at  25 
months;. the  non-tumorous  animals  died  at  an  average  aije  of  19»9  monthn. 
Nine  of  195  of  the  offspring  of  injected  mothers  developed  riKimmary  yland 
tumors  at  an  average  age  of  20.1  months;  the  non-tumorous  animals  died 
at  an  average  age  of  20,6  months.  Three  of  57  of  the  females,  retired 
after  weaning  of  their  first  litter,  developed  mammary  gland  tumors  at 
an  average  age  of  18,3  months,  the  non-tumorous  animals  died  at  an  aver- 
,  age  age  of  21.1  months.  In  the  groups  injected  at  kS   '^nd  65  days  of  age, 

5  of  Ul  females  in  the  former  and  2  of  k3   females  of  the  latter  group 
developed  mammary  gland  tumors  at  average  ages  of  15..0  and  19.0  months 
respectively;  the  non-tumorous  anim.'ils  died  at  average  agc_,s  of  20.0  ,and 
20,3  months, 

2,  The  incidence  of  hemanyiioendoiholi^mas  yind  of  hepatomas  has  bi,un  in- 
creased in.   the  animals  which  have  been  injected  with  o-riminoazoioluene 
as  compared  with  the  untreated  animals.  Hemangioendotheliomas  were  ob- 
served in  13  of  23  injfoctcd  haired  a.nd  ,12  of  22  injected  hairless  males 
as  compared  with  8  of  2k   untreated  haired  and  2  of  16  untre  .ted  hairless 
males.  They  were  observed  in  13  of  22  injqcted  haired  and  in  17  of  25 
injected  hairless  females  as  compared  with  6  of  23  untreated  haired  and 

6  of  25  untreated  hairless  females.  Hepatomas  were  observed  in  8  of  23 
injected  haired  and  in  6  of  22  injected  hairless  males  as  compared  with 

3  of  2[|.  untreated  haired  and  1  of  l6  untreated  hairless  .males.  They  were 
observed  in  lli  of  22  injected  haired  and  23  of  25  injected  hairless  fe- 
males as  compared  with  1  of  23  untreated  liaired  Lnd  none  of  25  untreated 
hairless  females. 

Multiple  tumors  of  both  therje  types  •ccurred  more  frequently  in  the 
injected  animals, 

3,  No  results  to  date, 

Ii.  Six  of  a  total  of  l6  strain  C3Hf  or  C3He  females,  fo;iti..r-nursed  by 
strain  3L,  have  developed  mammary  gland  tumors.  The  tumors  have  appeared 
at  an  average  age  of  13.0  months,  the  nontumorous  animals  have  died  at  an 
average  age  of  15.0  months.  All  of  tl^  C3Hf  or  C3He  females,  foster- 
nursed  on  strain  SWl,^,  are  still  alive, 

5,  In  th>;  outcross  resulting  from  crossing  (A  x  WCaFu)Fi  and  ('iCaFu  x  u)F-| 
animals  with  strain  119)4,  the  segregants  that  Wk,re  fused  were  less  sus- 
ceptible to  induced  pulmon^iry.  tumors  than  were  the  segregants  that  were 
nonfused.  No  significant  difference  in  susceptibility  was  observed  be- 
tween the  segregants  with  dominant  spotting  and  those  without  spotting 
or  betwe(;n  the  caracul  and  noncax'acul  .'segregants,  (Geventy-five  of  IlO 
animals  exhibiting  domina,nt  spotting  developed  pulmonary  tumors  following 
intraperitoneal  injection  of  dibcn'/,o.nthracone.  Sixty-nine  of  107  animals 
without  Roottlng  developed  pulmonary  tumors,,  The  difference  between  the 


PAGE  h. 

two  is  not  significant)!  X  -  .33,   P  is  botw-cn  O.^j  iind  0.7.  Thirty-one 
of  58  animals  with  caracul  coat  and  2?  of  h2  animals  with  normal  coat 
developed  pulmonary  tumors.  The  difference  between  thciTi  is  not  sig-nifi~ 
cantj  X^  -   .05,  P  is  between  0,8  and  0,9,  Twenty-seven  of  ^0   animals 
with  fused  tail  and  39   of  52  aniiuals  with  normal  tail  developed  pulmon- 
ary tumors,'  The  difference  between  the  two  is  significant!  X  =  h»9't   P 
is  between  0,02  and  0,03, )  Growth  curves  showed  that  the  average  weight 
of  mice  that  were  fused  was  less  than  that  of  mice  that  were  nonfuse^l. 
and  the  mice  that  were  caracul  weighed  less  than  those  that  were'  non- 
caracul.'  There  was  no  difference  betx^een  the  average  weights  of  the 
males  with  and  without  dominant . spotting,  but  the  females  with  spotting 
weighed  more  than  those  without, 
B,  1,  In  the  (II9I4  x  i.)F2  animals,  75  of  a  total  of  63h   animals  (11,8-0 
exhibited  the  waltzing  character  and  in  the  (119U  x  j4)-iij3C  animals,  97 
of  a  total  of  251  animals  (38. 6;^)  exhibited  it,  .These  results  indicate 
that  possibly  this  character  is  due  to  a  single  recessive  gene,.  No 
further  results  at  present  available, 

.ib normalities  of  the  urogonitaJ.  system  in  strain  A  x  C  line  993$   rats 
were  observed  in  13ii  of  a  total  of  7U8  females  and  in  137  of  a  total  of 
813  males  autopsied  at  1  to  18  days  of  age,.  In  animals  autopsicd  at  2 
to  17  months  of  age,  Ii5  of  I89  females  and  12  of  62  males  were  abnormal, 
•The  principal  manifestations  were  the  absence  of  one'  kidney,  or  the'  pres- 
ence of  one  cystic  kidney.  These  were  accompanied'  generally  by  other 
abnormal  conditions,.  In  females,  they  included  a  missing  ureter,  uterine 
horn,  oviduct,  ovarian  capsule,  or  ov;iry;  an  incomplete  ureter  or  uterine 
hornj  a  cystic  ureter,  uterine  horn,  or  ovarian  capsulej  or  hypertrophy 
of  the  remaining  kidney,  with  vaji'ia^tion  in  the  degree  of  abnormality,  ' 
In  males,  they  ijicluded  a  missing  ureter,  epididymis,  vas  deferens,  or  " 
vesicular  glandj  atrophy  of  the  te,stis,-  vesicular  gland,  or.  epididjanisj 
cystic  ureter;  undescended  testis;  and  hj'pertrophy  of  the  remaining 
kidney,  all  also  varying  in  degree. 

Significance  to  cancer  resco-rch;  .   " 

A,  It  is  already  well  ^established  that  the  genetic  constitution  influences 
the  development  of  tumors,  .ill  facets  of  this  project  will  add  more  infor- 
mation to  that. already  existing  concerning  the  mode  of  inhcritiince  of  var- 
ious types  of  tumors, 

B.  The  study  of  the  mutant  may  be  of  significance  in  providing  another 
known  character  which  will  be  of  use  in  the  linlcage  studies  of  factors  in- 
fluencing susceptibility  to  various'  types  of  tumors. 

Proposed  course  of  the  project; 

It..     1.  This  study  will  continue  through  1956, 

2,  This  study  will  be  completed  early  in  1956  (four  experimental  females 
remain  to  be  autopsied)  and  the  results  x\rill  be  prepared  for  publication, 

3.  This  project  will  continue  tlirough  1956, 

U,  It  is  anticipated  that  this  experiment  will  continue  through  1956, 
5»  The  results  of  this  experiment  will  appear  in  the  December  1955  issue 
of  the  Journal  of  the  N-ational  Cancer  Institute,  . 

6.  Special  types  of  animals  will  coatinue  to  be  produced  for  experiments 
with  Dr,  Barrett, 
B,  1,  Project  will  continue  through  1956, 

The  results  of  this  experiment  have  been  i^rritten  up  and  the  manuscript, 
has  been  submitted  to  the  Editorial  Eoar«l  for  appro\'a,l  for  publicfrtxon  in 
the  Proceedingpof  the  Society  for  Experimental  Biology  and  Medicine, 


PAGE  5. 


LO.      J42I 

-?— pj  '-J  :jr,  ^  ■ 


LI. 


BUDGET  ..CTI\r[TY: 

rese;jrch        X /JDraraSTIt.TION 

KEYim  &  .iPPROV;A  TECHNICAL  .,.SSIST;.NCE 


L2. 


-3. 


COGPEIi^TING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,   OR  OTHER  0RG;.NIZATI0NS, 
PROVIDIi''G  FUNDS,  F.XILITIES,   OR  PER30:.;NEL  FOR  THIS  PROJECT  IN  EITHER  19^6 
OR  1957, 


IF  THIS  PROJECT  RESEIMBLSS,   C'HPLEISNTS,    OR  P.JIALLELS  RESE.JiCH  DONE  ELSHaPTERE 
IN  THE  PUBLIC  ESilLTH  SERVICE   (iflTHOUT  INTERCFLJ^GE  OF   PERSOiiNEL,  FACILITCES 
OR  FUNDS),   IDENTIFY  SUCH  RESEARCH: 

.I4.   U21 


SERL:.L  NO. 

5. 


PUBLICx.TIONS  OTHER  THx.N  .JBSTR.'>.CTS  FROM  THIS  PROJECT  DURING  C.^LEND..R  YE..R  1955 

1.  Deringer,  M.  K,,  and  Lorenz,  E.:  Results  of  exposure  of  newborn  HR 
mice  to  X  radiation.  J.  Nat.  Cancer  Inst.  l5:  923-929,  1955. 

2.  Deringer,  M.  K.,  Lorenz,  E.,  and  Uphoff,  D.  E, :  Breeding  behavior  and 
tumor  development  in  (C57L  x  ^x)Fi^  hybrid  mice  receiving  X  radiation  to 
ovaries  shielded.  J.  Nat.  Cancer  Inst,  15:  931-9l4l,  1955» 

3.  Deringer,  M,  K«,  and  Heston,  vj.  E.J  Development  of  pulmonary  tumors 
in  mice  segregated  with  respect  to  the  three  genes:  dominant  spotting, 
caracul,  and  fused.  J.  Nat,  Cancer  Inst.  I6:  763-768,  1955. 

h»     Deringer,  M,  K,,  and  Heston,  W.  E.i  Abnormalities  of  the  urogenital 

system  in  strain  A  x  C  line  993$   rats,  (Submitted  to  the  Editorial  Board 
of  the  Journal  of  the  National  Cancer  Institute). 


P^GE  1 


PROJECT  RE-'ORT  FORM 


1.  national  Cancer  Institute 
Tf^fTfUTE      ^     ~~" 


2 .  Laborator;/  of  Biology 


LABORATORY  OR  BRANCH 


3«   General  Biolog;'-  Section 
SECTION  OR  SERVICE  '' 


U._ -5.     Ij23 

LOCATION) IF  OTHER  THAN  BETH.)        SER.    NO. 


6.   Developmental   Physiology- 


PR'-^JECT  TITLE 
7'.  Clifford  Grobstein 


PRINCIPAL   INVESTIGATOR(S) 


OTHER  INVESTIGA.TORS 


■9.  ■  PROJECT  DESCRIPTION 

■4.  Objectives:'  To  increase  'understanding  of  the  interaction  of  tissues 
and  the  corrfponents  of  rudiments  in  normal  development  as  these  relate  to 
the  differentiation  of  new  cell  types,  and  to'  apply  iiiformation  obtained 
to  pathological  problems,  in  particular  to  problems  of  tumorigenesis  and 
malignancy. 

B»  Methodst  Developing  rudiments  of  mouse  organs,  e.g.  kidney,  salivary 
gland,  and  axial  skeleton,  are  separated  into  their  components  and 
cultured  in  isolation  and  in  various  recombinations  to  determine  the 
degree  to  vjhich  their  development  is  interdependent  and  the  nature  of 
the  interactive  mechanisms  involved.  Development  is  observed  in  vitro 
and/or  on  reimplantation  into  the  artterior  chamber  of  the  eye. 

Major  findings;  Study  continues  of  trans-filter  induction  between 
embryonic  spLnalcord  and  me tanephro genie  mesenchyme.  Methods  have 
been  developed,  in  collaboration  with  Dr,  Dalton,  for  electron  micro- 
scopy of  the  filter  during  induction.  Cvilture  methods  have  been  further 
refined  to  permit  elimination  of  the  clot,  thus  making  possible  study 
of  the  induction  in  defined  nutrient  media  as  these  become  available. 
Preliminary  studies  on  intact  rudiments  with  the  amino -acid- vitamin 
mixture  of  Eagle  indicate  that  it  can  be  used  if  supplemented  with 
l|orse  serum  or  mouse  ascitic  fluid. 

The  nature  of  the  trans-filter  inductive  influence  cannot  yet  be 
specified.  Filters  in  contact  with  inductively  active  tissues  may  show 
three  types  of  penetration  depending  on  tissue  type,  filter  type,  and 
duration  of  contact:  1)  Cytoplasmic  processes  identifiable  with  the 
electron  microscope;  2)  Granular  or  filamentous  material  demonstrable 
by  staining  or  phase  contrast;  3)  Diffuse  material  present  after 
alcohol-formalin  fixation  and  digestible  by  tr-'/psin.  Efforts  are 


PROJECT  DESCRIPTION  (Cont.) 

continuing  to  determine  possible  correlations  between  the  several 
kinds  of  penetration  and  biological  activity,  and  to  "trap"  biological 
activity  in  the  filter  in  the  absence  of  living  tissue, 

Metanephrogenic  mesenchyme,  which  never  forms  epithelial  tubules 
by.  itself  in  vitro,  does  form  tubules  when  implanted  into  the  eye  or 
brain  of  adults  -  despite  the  fact  that  tissues  from  these  sites  are 
negative  as  inductors  in  vitro. 

Parotid  epithelial  rudiments  undergo  characteristic  parotid-type 
branching  in  sub-mandibular  mesenchyme  -  suggesting  that  the  mesenchyme 
of  the  two  salivary  rudimentis  shares  a  common  property  not  found 
previously  in  mesench-^mtie  from  non-salivary  sources,  and  that  the 
difference  in  type  of  branching  is  a  function' of  epithelial  rather 
than  mesenchymal  properties. 

Preliminary  experiments  by  Dr.  Robert  Auerbach,  N.I.H.  fellow, 
indicate  that  lens  induction  by  the  optic  vesicle  in  the  mouse  can  be 
obtained  in  vitro  and  that  the  process  may  be  able  to  be  analyzed  by 
methods  similar  to  those  used  for  kidney,  salivary  gland  and  cartilage. 

D.  Significance  to  cancer  research:  A.s  noted  in  last  year's  report 
these  studies  bear  on  the  nature  of  factors  which  sta'kilize  or  disrupt 
tissue  architecture. 

E.  Proposed  course  of  project:  Continued  study,  is  planned  of  the 
nature  of  the  mechanisms  of  induction  in  the  several  in  vitro  systems 
available.  Approaches  in  progress  include;  l)Electron  microscopy 

of  the  filter  betwe^;n  interacting  tissues;  2)  Variation  of  filter 
oorosityand  other .experimental  conditions  in  an  effort  to  separate  the 
three  t:/pes  of  filter  penetration  for  correlation  with  biological 
activity;  3)  Determination  of  the  nature  of  the  factors  responsible  for 
tubule  formation  in  implanted  kidney  mesenchyme;  ii)  Culture  of  the 
interacting. system  in  defined  media  so  as  to  simplify  identification 
of  active  materials. 


PROJECT  REPORT  FORM  (Cont»<l> 

10.   U23 

SERIAL  NO. 


11. _ 

BUDGET  ACTlVmi 


RESE?JICH  I  AD-'^'INISTRATION    .  ,, 

REVIEv^  ^  APPROV/i  TECHNICAL  ASSISTANCE 

^ '•fo6p^^ilmrmin:$,  OF  the  VnBLici  hil/.lt h~5e^vice,  or  .6ther  oRGaNr^vi-iuiNS , 

raovSiNG  FUNDS,  FACILITIES,  OR   pERSONIJEL  TOR  T"IS  PR'aJECT  IN  EITHER 
1956  or.  1957: 


'if  T-lS  PRijteCT  R-^ryBLga,  doM  "'LEMENTS,   Ok   -:^!\RJl'LLEI5  R'^SL-lkuH  1^)1^^ 
ELSK^-ERE  IN  THE  PUBLIC  HEALTH  SERVICE   (WiTHOUT  INTERCHANGE  OF 
PERSONNEL,  FACILITIES  OR  FUNDS) ,   IDENTIFY  SUCH  RESEARCHj 


PROJECT  REPORT  FORM  (Cont'd) 


111.  It23 

SERIU  NO. 


'pTIBLICATIONfe  OTHEft  TF/IN  fiBSTRICTS  FROM  T^iiJ  t'KOJECT  DURING  GALJiNUAk 
TEAR  19^5 

1,  In  vitro  studies  of  cartilage  induction  in  mouse  somite  mesoderm 
(with  ".  uoltzer)  J.  Exp.  Zool.  128:  333-358  19$$ 

2,  Tissue  disaggregation  in  relation, to  detemir.ati->n  and  stability 
of  cell  type.  tan.  i^.  Y.  Acad.  Sci.  60:  ,1095-1106  19$$ 

3,  Tissue  interaction  in  the  morphogenesis  of  mouse  embryonic 
rudiments  in  vitro.  In  "Aspects  of  synthesis  and  Order  in 
Growth",  PrincetoR  nniv.  Press  19$$ 


16 . : , : 

HONORS  AND  AVJ'iRDS  TO  -PERSONNEL  HiiLATiNG  TO  T'lB   PRr^JECT  DURBIG 
CAiENDAR  YEAR  19$$. 


PAGE  1 
PROJECT  REPORT  FORM 


1,   National  Cancer  Institute     2,   Laboratory  of  Biology 


INSTITUTE  LABORATORY  OR  BRANCH 

3.   General  Biology  Section  -     li«  ___^ ^'         U2I). 

SECTION  OR  SERVICE      -  .     LOCATION  (IF  OTHER  THAN  BETH. SERIAL  NO, 

6,  Tissue  Compatibility  Studies 

PROJECT  TITLE 

7.  Richmond  T.  Prehn 


PRI  'CIPAL  IN\^ESTIGATOR(S) 
Joan  PI.  Main 


OTHER  IIWESTI  GATORS 

9.   Project  Description 

Object;  The  long  range  objective  is^  of  coarse,  the  understanding  of  the 
physiology  of  cancer,  which  in  turn  will  some  day  lead  to  control  of  the  disease. 

It  is  axiomatic  that  the  well-being  of  the  cancer. cell  is  dependent  upon 
its  social  acceptability  to  the  normal  cells  of  its  host  -  i.e.  upon  its  recog- 
nized failure  to  arouse  a  significant ■ immune  response  or  foreign  body  reaction. 
The  cancer  cell,  in  order  to  survive,  produces  little  or  no  detectable  response 
of  an  antigenic  nature,  but  is  apparently  .accepted  by  the  host  organism  as  a 
legitimate,  if  somewhat  undisciplined  part  of  itself. 

This  fact  stimulates  the  question:  why  are  some  tissues  or  cells  antigenic 
and  others  not?  The  facile. answer  that,  "only  foreign  cells  are   antigenic,"  not 
only  begs  the  question  -  it  is  net  .correct.  The  discovery  of  a  variety  of  auto- 
antibodies demonstrates  the  cogency  of  the  question.  It  is  not  irrational  to 
believe  that  the  understanding  of  growth  m  general  and  of  cancer  in  particular 
may  await  a  better  understanding  of  why  cells  are  antigenic  in  some  cases  and 
not  in  others.  Such  knowledge  might  make  it  possible  to  produce  autoantibodies, 
i,.e»  to  induce  imjnunological  growth  inhibitors  artificially  against  cellular 
types  such  as  cancer  which  probablj''  have  little  or  no  natural  antigenicity. 

The  immediate  object  of  t.'ie  project  is  therefore  to  investigate  the  causes 
or  sources  of  cellular  antigenicity  or  its  lack  -  to  investigate  the  origin  of 
the  ability  of  the  organism  to  "recognige"  some  cells  as  endogenous  and  others 
as  foreign  and  x\Thy  it  sometimes  makes  mistakes. 

Methods  and  Results:  The  immediate  work  of  th.e.  project  can  be  conveniently 
divided  into  three  related  categories: 

I.  The  phenomenon  of  specific  aq^iired  tolerance:  —  The  original  observa- 
tion was  that  when  strain  DBA  mrlce  were  exposed  to  an  X-ray  dosage  of 
800  V  and  then  protected  from  the  lethal  effects  of  the  radiation  by 
the  intravenous  injection  of  (E>lLB/c  x  DBa),Fi  bone  marrow,  the  DBA 
mice,  throughout  their  subsequent  life  were  tolerant  to  skin  grafts 
from  BALB/c  donors.  Subsequently,  it  was  found  that  bone  marrow  from 


:^age  2 

BALB/c  rather  than  F-]  hybrid  mice  would  acconplish  the  same  result. 
The  effect  was  profound,  almost  100,1  of  the  homografts  surviving  as 
though  they  were  autografts.  The  tolerance  produced  was  apparently, 
specific  for  the  antigens  contained  in  the  bone,  marrow,  since  resist-  . 
ance  to  ot'fier  antigens  such  as  those  of  a  C57-L  skin  graft,  returned 
as  soon  as  the  mice  recovered  from  the  acute  effects  of  the  radiation. 
Further  studies  suggested  that. second  set,  htmografts  are "accepted  as 
yell- as  first  ..set  by  "tolerant"  mice*  To-date,  attempts  to  reverse 
the  tolerance  by  injections  of  im/iiune  splenic  tissue  have  failed.   -  . 
Preliminary  results  w?ugge.st  that  the  tolerance  producing' factor  ,may 
be  subcellular  and  'pcrhaps  capable  of  Sijrial  transmission  in  irrad- 
-  ■   irited  hosts.  The  phenomenon  is  not  limited  to  strain  BALB/c  grafts  . 
to  strain  DBA  recipients,  but  has  been  demonstrated  wi'th  (CxDBA)  F]_ 
grafts  to  (Lxi\).F]_  mice  and'  with  B'ALB/c  grafts  to  C3H,  It  is  apparent 
however,  that  certain  strain  peculiarities  do  exist  although  these  ^ 
have  not  yet  been  fully  explored,  A  qualitatively  similar  though 
quantitatively  much  weaker  effect  'has  been  obtained  by  substituting 
L.D.  50  dost.s  of  nitrogen  mustard  for  the  radiation^ 

II.  Antigenicity  of  methylcholenthrene  induced  sarcomas* /b  r.umbcr 

of  investigators  have  reported  the  apparent  production  of  isologous 
immunity  to  methylcholanthrone  induced  sarcomas.  Since  it  is  the 
prevailing  opinion  that  tumors  are  non-ant igenic  in  the  animal  or 
strain  of  origin,  the  results  of  these  investigators  have  been  attri- 
buted to  an "unrealized  genetic  heterogeneity  of  materials.  However, 
it  has  now  .bee"n  found  in  this  laboratory  that  such  immunity  can  be 
regu,larly  produced  by  MCA  induced  s.arcomas  but  not  by  spontaneous 
sarcomas  or  mammary  carcinomas.  It  is,  therefore,  a,pparent  that  if 
the  immunity  produced  is  duo  to  the  small  residual  genetic  heterogen- 
eity, then  MCA  indufced  sarcomas  are  for  some  reason  peculiarly  sus- 
ceptible. The  only  other  possible?  explanation  would  seem -to  be  that, 
due  to  some  peculiarity  01  the  HCA,  those  tumors  may  differ  from  the 
others  by  being  antigenic  in  th©  animal  of  origin  -  i.e.  they  may  be 
capable  of  producing'  true  autoifiraunization^  Fortunately,  experimental 
means  are  available  to  distinqaish  between  these  alternative  hypotheses, 

III,  riiscellancous:  A  number  of  miscellaneous  but  related  experiments  have  • 
been  done  during  the  past  year:  a»  The  growth  of  strain  C  skin  grafts 
in  (CxDBA)  D'A  backcross  mice  has  been  studied.  Results  indicated  that 
5  or   more  histoconipatibilitv  genes  were   segregating.  ,  b.  The  claim 
of  Hardin  and  "Jerder  that  multiple  successive  skin  homo-grafts  could 
produce  a  state  of  acquired  tolerance  was  inv^^stigated.  The  results 
did  not  substantiate  this  claim,,  c.  Red  cell  a;._glutiaation  within 
di.lfusion  chambers  was  studied.  Fed  cell  agglutinins  could  not  be 
demonstrated  in  the  chambers  in  vivo  even  in  immune  mice,  d.  The 
immunising  power  of  red  Qells  in  diffusion  chambers  was  investigated. 
It  was  foimd  that  the  diffusion  chamber  prevented  immunization  even 
though  the  red  cells  themselves  remained  potentially  antigenic,  e.  It 
was  found  in  a  preliraiu-iry  expcrimtn,t  .that,  contrary  to  the  results 
obtained  at  other  sites,  intra-diffusion  chamber  homologous  cells  were 
killed  when  diffusion  chambers  were  implanted  intra- spleMcally  in 
immunised  mice,  f.  An  attempt  with  the  aid  of  X-radia-oion  and  bone 
marrow  to  produce  an  effect  in  mice  by  the  Rous  chiaken  tumor  agent 


Page  3 

was  an  apparent  failure,  g.  An  attempt  to  "adopt"  tumor  cells  to 
a  foreign  strain  by  prolonged  growth  of  the  tumor  cells  within  diifusior, 
chambers  in  the  foreign  strain  gave  possibly  slight  but  generally  in- 
conclusive results.  This  despite  the  exposure  of  the  cells  to  the 
foreign  medium  for  a  period  of  9  months,  h.  A  preliminary  study  of 
the  efficacy  of  various  routes  of  administration  in  producing  immunity 
to  homologous  red  cells  has  suggested  that  the  subcutaneous  is  signifi- 
cantly poorer  than  the  intravenous,  intraperitoneal  or  Intracutaneous 
routes,  i.  A  statistical  study  of  the  influence  of  the  occurrence  of 
a  mammary  tumor  upon  the  probability  of  the  occurrence  of  a  second  in 
the  same  mouse  was  undertaken  in  high  tumor  strain  C3H  mice.  The  re- 
sults are  not  yet  apparent,  j,  A  study  was  made  of  the  effects  of 
trypan  blue  on  MCA  carcinogenesis.  This  was  inconclusive,  k.  Pre- 
liminary results  suggest  that  the  "immunization"  of  adult  BALB/o  mice 
to  the  mammary  tumor  agtnt  interferes  with  the  growth  of  transplantable 
BALB/c  mammary  tumors. 

Major  Findings  and  Significance:  Two  findings  are  possibly  of  major  importance: 

a.  The  production  of  specific  acquired  tolerance  by  X-radiation  and  bone 
marrow  administration.  This  is  a  significant  advance  in  our  under- 
standing of  the  mechanism  by  which  an  animal  is  capable  of  distinguish- 
ing foreign  from  endogenous  cells.  It  is  too  early  to  say  whether 
this  work  will  in  addition,  have  any  direct  practical  application 
though  it  is  apparent  that  a  useful  investigative  tool  has  been  dis- 
covered. 

b.  The  antigenicity  of  isologous  MCA  induced  sarcomas.  This  work  demon- 
strates an  app.irently  unique  characteristic  of  such  tumors.  It  is, 
however,  too  early  to  forsee  where  this  observation  will  lead. 

Proposed  Course  of  project: 

A,  With  regard  to  the  specific  acquired  tolerance  produced  by  radiation 
and  bone  marrow  administration,  it  is  planned  to  investigate  the 
follovring.  Some  of  these  studios  arc  already  under  way, 

a.  Farther  studies  of  specificity  and  possible  strain  limitations. 

b.  Effects  of  varied  dosages  of  radiation  and  of  marrow  on  the 
phenomena, 

c.  Effect  of  varying  the  period  between  radiation  and  antigen  admin- 
istration, 

d.  Can  tolerance  be  produced  in  immunized  mice? 

e.  Effect  of  different  routes  of  foreign  antigen  administration. 

f.  Influence  of  tissues  other  than  marrow  as  a  source  of  foreign 
tolerance  producing  factor. 

g.  Study  of  possible  changes  in  transplantation  specificity  of  the 
skins  of  tolerant  mice  or  of  grafts  in  tolerant  animals, 

]|.  Fnjrther  study  on  the  coll  free  nature  of  the  tolerance  factor  and 
its  serial  transmissibility. 

^*  Can  tol6rahce"'fe«;''irt'Vferscd  by  itilirrbw  andX  radiation  treatmeht. 

■  ■  ■   •  ■"  '  ■•':,;:'^;  ■■■'■■' d.^  ■  ,^  ■','■.' ; 

It  is  doubtful  if  time  will  permit  all  of  these  studies  to  be  initiated,  let 
alone  completed  during  the  follotxing  year. 

B,  With  regard  to  the  antigenicity  of  HCA  induced  sarcomas,  a  initial  ex- 
periment is  now  in  progress  to  determine  v;hether  autologous  MCA  tumors 
will  show  the  same  antigenicity  as  isologous.  The  future  of  this 


Page  U 
investigation  must  await  the  results  of  that  experiment. 
10.   U2li 


11. 


12, 


13. 


SERIAL  NO. 


BUDGET  ACTIVITY: 

RESE:  RCH  X  /iDMINISTRiiTION 


REVIEI'J  &  APPROVAL  TECHIJICAL  ASSISTANCE 


COOPERATING  UNITS  OF  THE  PUBLIC  HEAL-TK  SERVICE,   OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,   OR  PERSONIffiL  FOR  THIS  PROJECT  IN  EITHER 
1956  OR  1957, 


IF  THIS  PROJECT  RESEMBLES,   C0HPLEI-1ENTS,   OR  P.^JIALLELS  RESEilRCH  DONE  ELSE- 
^dHERE  IN  THE  PUBLIC  HEiVLTH  SERVICE   (liTITHOUT  INTERCHi^NGE  OF   PERSONNEL, 
FACILITIES  OR  FUNDS),   IDENTIFY  SUCH  RESE^iRCH: 


lii.  U2U 

15. 


SERI/iL  NO. 


16. 


PUBLICATIONS  OTHER  THAN  ABSTRi.CTS  FROM  THIS  PROJECT  DURING  CALENDiiR 
YEilR  1955. 

1.  Main,  J.  M.,  and  Prehn,  R.  T,:  Successfull  skin  homogrr.fts  after 
administration  of  high  dosage  x-ray  and  homologous  bone. marrow. 
J.N. C.I.  15:  1023  -  1028  (1955). 

2.  '/feaver,  J,  M.,  Algire,  G.  H.,  and  Prehn,  R.  T.:  Growth  of  cells  in 
diffusion  chambers  II.  J.N.C.I,  l5:  1737-176?  (1955). 

3.  Prehn,  R.  T.,  Algire  G,  H.,  and  VJeaver,  J.  M.:  Diffusion  chamber  in 
homograft  research  Trans.  Bull.  2;  Hi?  (1955). 

k»     Prehn,  R.  T.,  and  Main  J.  M. :  Lack  of  immunizing  capacity  of  homologous' 
cells  within  diffusion  chambers  J.N.C.I.  -  In  press. 


HONORS  ;JJD  AteiRDS  TO  PERSOMIEL  RELiJTNG  TO  THIS  PROJECT  DURING  CALENDiiR 
YEAR  1955. 


Pa,^e  1 
PROJECT  REPORT  FORM 


■^»  National  Can(^er\^In£tvtute  2.  ,  Laboratoj^y  of  r^.xj^lo^y 
insfitute  ~    '"      '     '  LAEOPATORY  OR  BRANCH 


3.  C-eneraT  Biology'-  Section     h, .    ^'______ 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THi-'N  EETHDSDA)  SERIAL  NO, 

6.  '^  •  "Eff'fects'of  the  a'^  and"»b  Genes  on'Mouse  Metabolism  and  Physiology" 

■  "'  PROJECT  TITLE   —     — 

7.  George  L.  vjolff ' ^| ^ '_ 

PRIl'CIPAL  I!"/ESTTGAT0RCs1  "    "'         "  "*~  . 


OTHER  IN'^ST  I  GATORS 

9,  PROJECT  DESCRIPTION; 

Objective:  The_ objective  of  this  project  is  to  determine  the^ primary  aad  plei»- 
tropic  effects  of  the  A^  (lethal  yellow)  and  ob  (obese)  genes  on  mouse  metabolism 
and  physiology,  especially  as  related  to  the  increased  lung  cancer  incidence  in  :■. 
iVfa   mice  and  to  the  hormone  balance  in  mice  carrying  the  A^  and/or  the  ob  genes. 

Methods  Employed:  An  inbred  strain  of  mice  carrying  the  -AJ  and  ob  genes  is  being 
developed  in  order  that  the  effects  of  these  genes^  both  singly  and  in  combina- 
tion, may  be  s\.udied  against  the  identical  genetic  background. 

The  anaerobic  glycolysis  of  Harding-Passey  melanom.as  (in  the  presence  or 
absence  of  exogenous  insulin  and/or  testosterone)  after  transplantation  to  normal 
and  gonadectomized,  male  and.  female,  Ob-  and  obtb,  A-^a  and  aaj  mice  is  determined 
by  means  of  a" Warburg  re'spirometer  apparatus. 

The  anaerobic  glycolysis  of  kidney  minces  (in  the  presence  or  absence  of 
added  insulin  and/or  testosterone)  of  male  and  female  Ob-  and  obob  mice  at  various 
ages,  after  growth  of  the  Karding-Passey  melanoma,  or  after  exposure  to  high 
temperature  (35°c).  has  been  studied  by  the  same  method. 

Acid  and  alkaline  phosphatase  activities  of  A^a  and  aa  mouse  lung  and  Harding- 
Passey  melanoma  homogenates  are  being  determined. 

The  possibly  differential  resistance  of  Ob-  (non-obese)  and  obob  (obese) 
mice  to  high  temperature  stress  is  being  studied  by  exposiag  the  animals  to  35°C 
for  2ii  hours. 

Major  Findings:  The  yellow  obese  mouse  phenotype  (presumably  A^^aobob)  has  been 
produced  and  found  to  be  viable. 

The  rate  of  e'stablishment  and  growth  of  the  flarding-Passey  melanoma,  six 
weeks  after  inoculation,  has  been  found  to  be  higher  in  obob  mice  than  in  Ob- 
mice. 

The  in  vitro  rate  of  anaerobic  ^glycolysis  of  the  Harding-Passey  melanoma 
grow-n  in  obob  females  was  found  to  be  higher  than  that  of  melanomas  grown  in  any 
other  sex-genotyne  group,  including  ovariectomized  obob  "females.  In  the  presence 


Page  2 

'.' .  . 
of  exogenous  testosterone  this  difference  has  a  probability  of  less  than  ,91  of 

being  duo  to  chance. 

Stimulatory  effects  of  exogenous  insulin  on  melanoma  glycolysis  were'-tb- 
served  in  the  presence  of  added  testosterone. 

In  vitro  Fridney  mince  r;lycolysis  of.  obob  mice  has,,^  in  general,  been  f»und  t» 
be  higher  than  that  of  Ob-  mice  at  30  and  63  days  of » age  and  after  2U  hours,'  ex- 
oosn.re  to  3$°C.  """eraale  kidney  glycolysis  in  all  of  these  categories  tended  t»  be 
higher  than  the  corresptndi  .g  male  kidney  glycolysis. 

Growth  of  the  Harding-Passey  melanoma  depresses  the  in  vitro  kidney  mince  ■ 
glycolysis  of  obob  mice,  but  seems  to  have  no  effect  on  that  of  Ob-  mice. 

Testosterone  inhibits  in  vitro  kidney  mince  t.lycolysis  of  Ob-  and  obob  mice, 
while  insulin  has  been  found  to  stimulate  kidney  mince  glycolysis,  especially 
after  the  mice  had  been  exposed  to  35°C. 

The  partial  contrel  »f  the  potential  glycolytic  capacity  of  mouse  kidney  tnd 
Harding-Passey  melanoma  b  -  an  insulin:  anti-insulin  sj'-stem  and  a  pleiotrspic 
effect  of  the  obob  genotype  on  this  system  is  suggested  by  the  data  (sbtained. 

Significance  to  Cancer  Research:   In  order  to  obtain  an  understanding  of  cancer, 
it  is  necessary  to  learn  how  genes  control  the  balance  of  hormones  regulating 
normal  and  tumor  cells  so  that  any  difference  between  the  latter  may  be  explftitei 
in  the  treatment  of  neoplasms. 

To  explain  the  relatively  high  incidence  of  lung  tumors  in  mice  carrying  the 
Ay  gene  as  opposed  to  a  relatively  lower  incidence  in  aa  littermates,  it  is  nec- 
essary to  know  which  specific  metabolic  reaction  is  under  the  control  sf  this 
gene.  The  solution  of  this  probl>..ra  might  ultimately  rovide  a  method  of  prevent- 
ing lung  tumors  and  of  treating  Ivng  tumors  cheinotherapcutically. 

The  use  of  the  Harding-Passey  melanoma  as  a  bio-indicator  of  the  hormonal 
balance  and  the  study  of  the  combined  and  separate  effects  of  the  Ay  and  ob  genes 
on  this  hormonal  balance  are  designed  to  provide  clues  to  possible  relationships 
between  altered  metabolism  and  lung  cancer. 

Proposed  Course  of  Project:  The  determinations  of  alkaline  and  acid  phosphatase 
activities  in  A -'a  and  aa  mouse  lung  homogenates  and  in  Harding-Passey  melanomas 
grovm  in  Aya  and  aa  mice  will  be  continued  and  extended  to  determine  specific 
phosphatase  activities. 

As  socn  as  the  new  strain  carryir.g  bcth  the  Ay  ana  ob  genes  is  sufficiently 
inbred,  determinations  of  the  in  vitro  r  tes  of  glycolysis  of  Harding-Passey 
melanomas  grown  in  normal  and  gonadectomis,ed,  male  and  female  mice  of  the  four 
iiJ f c rent  phenotypes  (AyaOb-,  A/a obob,  aaob-,  aaobob)  will  be  begun.  Similar 
determinations  will  be  made  on  non-maliv^nant  tissues,  e.g.  kidneys,  of  these 
mice .  " 

The  role  of  adrenal  steroids  in  the  insulin;.  :inti-insulin  system  in  relation 
t»  the  Ay  and-  ob  genes  xv^ill  be  investigated.    &'-• 


12. 


13. 


page  3 


10.         h26 

SERIAL  NOT 

11. 


BUDGET  ACTIVITY: 

RESEARCH  X  ADFUNISTRATION 


hSVISVJ  &  APPROViiL  TECKNICiiL  ASSISTANCE 


COOPILRATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,    OR  OTHER  ORCrANIZATIONS, 
PROVIDING  FUIMDS,  FACILIT.TES,   OR  PERSOMEL  FOR  THIS  PROJECT   IN  EITHER  19^6 


OR  1957. 


IF  THIS  PROJECT  RESEIIBLES,    CG:'IPLSr'IENTS,    OR  P^.RALLELS  RZ'^'Eii-RCH  DONE  ELSEWHERE 
IN  THE  PUBLIC   HEALTH  SERVICE   (VflTHOUT  Ii\ITERCHAtU.E  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),   IDE^'TIIFY  8UCH  RLSE/aRGK: 


NO  ENTRIES  FOP   ITEMS  lU,   l5  and  16. 


PRCJECT  UEFORT  F03f.: 

1,  National  Cancer  Institute '    .2,  Laboratory  of  Biology 

IIISTITUTE  L/i30R/.TCaY  GS  3Ri;r!CH 

3.  Leukemia  Studies  Section    4,  _____^______________  5.  427 

SECTICrJ  CR  SERVICE  LOC^iTION   (IF  CTHER  HWIl  SERIAL  f!C. 

SETI-ESDA) 

6.  Studies  on  the  etiology  and  chemotheraoy  of  experimental  lymphomas, 

7,  Lloyd  '/J.  Law 

PRINCIPAL  If IVESTIGATCR ( 3 ) 


8,  ['.lichael  Potter  in  certain  collaborative  proiects. 

OTFoER  inVESTIGATCRS 

9.  PROJECT  DESCRIPTICn 

Our  investigations  in  leukesriia  and  other  lymphomas  in  mice  come 
under  two  major  headings:  1)  Studies  of  the  etiology  and  pathogenesis  of 
the  disease,  end  2)  Studies  of  mcch::':isns  involved  in  inhibition  of 
leulcemic  cell  ■"rovjth  throu;;h  the  -.'.se  of  certain  selected  compounds, 
particularly  such  antirxtabolites  as  ontifolics,  antipurines,  and 
antipyrirnidines,  g.c;,.  methotrexate,  6-mercaptopurir;c,  8-a3sguanine, 
azaserine,  6-azaur3cil,  and  the  pyrazolo  pyrimidines. 

Heretofore  major  etiiphasis  has  been  placed  upon  lymphocytic  neo- 
plasms. As  may  be  seen  fror/i  this  report,  our  interest  is  now  extended 
to  include  etiology  and  therapy  cf  otlier  raorphologic  forms,  such  as 
Type  A  and  Type  3  neoplasms  (Dunn),  plasma  cell  neoplasms,  etc. 

1)  Studies  of  the  etiolotry  and  rjathoienesis  of  experimental  leukemias. 

A.  The  r5le  of  the  thymus  in  spontaneous  cases. 

Additional  data  are  novj  at  hand  concerning  the  role  of  thymic 
tissue  in  the  production  cf  lymphocytic  neoplasms  in  (C-SM  x  AKR)F]^ 
mice,  AKR  thymic  fragments  significantly  increase  the  incidence 
and  time  at  death  from  leukemia  and,  in  the  majority  of  cases  (57 
of  77),  the  transplanted  thymic  tissue  is  neoplastic,  C3Hf 
thymuses  have  n^:  such  influence  and  none  of  the  recovered  thymic 
frngnents  were  found  to  be  neoplastic,  AKR  spleen  likev;ise  is 
found  to  be  ineffective.  This  study  has  been  extended  (with  Or, 
f.l.  Potter)  to  include  the  role  cf  AKPi  thymic  tissue  in  thymectomized 
(AKR  X  C3H)  mice.  It  is  knov;n  (from  a  small  series)  that  the 
incidence  of  lyir.phomas  in  thynect"mi?.ed  (AKR  x  C3H)  mice  is  56% 
(conpared  to  53%  in  intrct  controls),  but  the  average  age  at  death 
follo7;ing  thymectcny  is  increased  from  14.8  to  19,0  months. 
Furthermore,  lymphocytic  neoplasms  have  net  been  found  in  the 
thynectomized  mice. 


(Project  description  continued) 


SERIAL  m,       427  .  p^j^gg  2 

3.  ?he  influence  of  ^irafted  thymic  tissue  following  exjosiire 
of  recipient  mice  to  ^c-^radiation. 

Certain  data  are  rovj  ^vriiable  concernin'j  the  influence  of  • 
C573L  thymic  tisst'.e  rr.fted  into  thynectcmiied,  irrndisted 
_    .   ^  ;:  ^25  r  (C573L  x  A)Fi  nice: 

1,  Thyme c tome ctomy  ccnr;letely  inhibits  the  indnction  of 
lymphocytic  neoylasrns  (but  not  ty-;e  A  reticulum  cell  neoilasrcis) . 

^..  5r^fted  thymic -tissue,  from  1  tC'IC" 'da'y""''-C'idC573L  mice, 
becosiiec  Iculreuiic  in  3".%  o::  the  esses  if  transfer  is  made  either  on 
day  1  or  day  7  after  ::-rcdiatioi:. 

3.  TrsTiSplcntEtion  studies  of  these  induced  neoplasms  indicate 
that  those  arising  early,  rt  5  snd  6  months  (4  cases),  transplant 
to  C573L  and  the  F},  r/lorcas  those  arising  loter,  C  to  10  months 
(9  cases),  g^ovj  progressively  only  in  the  "Ft^  mouse  (no-t  in  C57BL,- 
origin  of  thymic  tissue). 

These  results  provide  the  information  suggested  by  I'^aplari  of 
an  "indirect  mechanism  of  x-ray  induction"  dependent  on  the  post- 
irradiation  state,  but  also  provide  further  data  on  the  thymic 
"sphere  of  influence",  indicating  that  this  tissue  is  capable  of 
inducing  lymphocytic  neoplasms  fcllc-r/ing  repopulatio-n  vjith  F^- 
recipient  round  cells. 

The  follovjing  additional  e>:  ;eriments  -r.si-ng  this  model  are 
nov/  underway:  •      ' 

a)  Influence  of  strain  A  thymic  grafts  of  various  ages  1^  10, 

30  days  on  the  inductih:  of  lymphocytic  neoplasms. 

b)  Study  of  the  pcrsisteiice  of  the  post-irradiation  state. 
Thymuses  from  1-7  c\-3y   Old/SL  'onors  have-  been  grafted  at  1,  7,  14, 
and  F;o  days. 

c)  Influence  of  G673L  P.nd  A   bone  marrow  on  the"  induction  of 
leu'cemia  in  grafted  G573L  thymuses  in  thymoctcmized,  irradiated 
(C573L  X  A)  mice.'   ■■  ... 

C.  Influence  of  thymectomy  i n  CSM/Fg  subline. 

nearly  30/o  lymphomor.  have  been  observed  in  a  milh-agent  free 
subline  of  the  C3H  strain  obtained  from  Ur .  Figge.  Appro'dmately 
one-third  of  these  r,?opli, sms  are  lymphocytic  but.  very  fevj  involve 
thymic  tissue,  Cn  tiie  othar  hand,  fractionated  ;c-radiation  (4  ;: 
■90  r,  weekly),  produces  a  preponderance  of  lymphocytic  neoplasms. 
Thymectomy  has  been  accomplished  in  2  groups:  those  observed  for 
the  -  spontaneous  disease  and  a  group  recei.vir.g  x-radiaticn. 

LJ .  The  role  of  cell-free  materials  in  the^  induction  of  leu'cemia  and 
parotid  gland  neo'^lasms. 

In  a  study  conducted  jointly  with  Dr.  Thelma  Dunn,  Pathology, 
results  have  hceii   published  concerning  the  incidence  of  leukemia, 
parotid  gland  neoplasms  and  other  neoplasms  in  C3Iif/Lw, 
(C3K  y.  AKa)Fi  and'  (G3K  x  C3H/Fg)Fi  mice  following  introduction  of 
leukemic  materials  (extracts,  centrifugates,  and  filtrates)  from 
the  high-leuhemic  lines  AKR,  C56,  and  C3Hf/Fg»  The  incidence  of 

(Project  descriptiors  continued) 


SERIAL  nc.  427  PAGE  3 

'  leukemia  and  the  age  at  death  were  found  not  to  be  influenced 
by  the  introduction  into  young '(;<:.  24  hour  old)  mice. 

Certain  other  observations,  however,  v/ere  of  interest: 

1»  Pcrotid  gland  neoplasms  were  found  in  all  3  groups 
of  test  mice,  especially  in  the  (CSH  x  C3H/Fg)Fi  cross. 

2,  In  association  vdth  the  parotid  neoplasms  in  this  latter 
group  (25%)  nearly  all  the  test  mice  shovjed  hinhly  invasive 
subcutaneo^us  tumors. 

3.  Many  other  uncommon  forms  of  neoplasms  vjere  found,  etq». 
adrenal  medullary  tumors,  early  mammary  tumors,  but  these 
occurred  among  the  control  as  v;ell  as  the  experimentel  mice. 

Since  Gross  has  suggested  that  subline  differences  may  play 
a  part  in  the  response  to  a  leu':emogenic  agent,  this  v/ork  has  now 
been  extended  to  include  105  C3H/Bittner  subline  mice  inoculated 
<  24  hours  with  centrif ugates  (9500  R.Ti)  and  filtrates  (3erkfeld 
and  Selas)  from  12  different  spontaneous  AKR  leukemias.  These 
same  preparations  have  been  given  simultaneously  to  85  C3I-]f/Lw 
subline  mice. 

.7e  had  observed,  at  the  last  annual  report,  t'lct  a  transplan- 
table adrenal  medullary  neoplasm  gave  nearly  40%  parotid  gland 
neoplasms  in  C3M  and  (C3H  x  A'-R)  mice  v;hen  tested  at  the  G2,  G3, 
and  G8  transfers.  Consequently,  a  series  of  studies  v/ere  com- 
menced relating  to: 

1.  Influence  in  different  inbred  mice; 

2.  Effects  of  different  types  of  filtration; 

3.  Tests  for  activity  after  various  periods  at  -60''C. ; 

4.  Titration  of  stored  material. 

More  than  250  mice  were  set-up  and,  to  date,  after  11  months, 
no  parotid  tumors  have  been  observed  and  the  ability  of  L5665 
(the  adrenal  medullary  neoplasm)  seems  to  have  disappeared  with 
the  later  transfer  generations. 

Crosses  between  the  2  sublines  discussed  above,  C3Kf/Lv;  and 
C3H/Fg,  have  now  been  made  reciprocally,  and  inoculations  of  cell- 
free  materials  from  AKR  and  G3H/Fg  leukemics  are  being  accomplished 
into  infant  mice  to  study  the  unusual  occurrence  of  a  high  incidence 
of  parotid  gland  neoplasms  associated  vath  subcutaneous  neoplasms. 
The  subcutaneous  growths  appear  to  differ  morphologically  and  bio- 
logically from  those  seen  spontaneously;  for  example,  in  old 
C3H  strain  mice. 

E.  Study  of  certain  -/rotectivc  factors  in  induced  and  spontaneous 
tyrnghomas. 

1.  3one  marrow  repressive  factor. 

a)  Data  are  now  nearly  completed  on  the  influence  of  high- 
leukemic  (AI'R)  bone  marrow  and  low-ieukcmic  (CSIlf)  bone  marrow  on 

(Project  description  continued) 


SERIAL  UC.       427  PAGE  4..  ,  ,..v 

spontaneous  and  x-royed  induced  ieukemia  in  (CSHf  x  AKR)Fi  mice. 
It  is  clear  that  C3Hf  bone  mferrov;  represses  lymphomas  in  these 
hybrids  i36'0  whereas  AKR  bone  marrov;  influences  the  incidence 
and  mean  age  of  leukemic  death  in  o  positive  direction  (75%), 
It  is  clear  also  that  4  monthly  IV  inoculations  is  most  effective 
in'  repressing  lymphomas  in  the  F^   hybrid  (7%  at  12'. months  com- 
pared with  27%  in  controls). 

b)  This  work  is  being  extended  (by  liiss  Deita  Uphoff)  to 
study  the  influence  of  CSlIi  bone  marrov/  in  t!ic  AKR  strain. 
Periodic  inoculations  of  bone  marrow  into  AXS  mice  of  various 
ages  from  2   vreeks  to  3  months  have  been  commenced. 

c)  It  has  been  fcund  (Uphoff)  th:t  iatact  bone  morrow 
colls  from  certain  H-2  lines  will  protect  against  other  11-2 
irradiated  mice  (mortality),  e.g.,  CSS  bone  marrovj  gives  maximum 
protection  to  (C3Mf  x  AI-R)Fi  mice.  This  v;ould  appecr  to  be  a 
good  experimental  model  to  sti'.dy  the  relationship  of  protection 
against  1)  mortality;  2)  thymic  cegcneration;and  3)  induction  of 
lymphomas  by  radiation.  Such  studios  are  nov;  in  progress. 

d)  The  role  of  bone  marrov;  in  protectioh  against  x-ray 
induction  of  Type  A  rnd   B  reticulum  laeoplasms  is  being  investigated 
in  D3A/2  thymectomized,  irradiated  mice,  and  in  BAFj  thymectomi:3ed, 
irradiated  mice.  In  both  these  groups  there  appears  to  be  an 
induction  by  x-rays  of  Type  A  end   B  neopljsn'is. 

2»  Liaternal  resistance  influence  (i.iRF )  ■ 

An  attempt  to  further  characterize  an  influence  of  re- 
sistance to  leukemia  in  AKR  and  C5G  mice  by  foster-nursing  uoon 
old  (>  32  wee!:)  STOLI  mothers. 

Although  AKR  mice  in  most  instances  show  less  leukemia, 
yjhich  appears  late  in  life  (beyond  1  year),,  there  are  found 
litters  which  develop  tlie  disease  as  expected  even  though 
fostered  by   old  STCLI  mothers, 

F2  and  F3  mice  obtained  from  AKR  mothers^ fostered  on  old 
STCLI  mice, are  now  under  observation  to  determine  if  LBF  can  be 
passed  on  in  successive  generations, 

Follo'ving  reciprocrJ.  cresses  are  nov;  being  accomplished 
in  the  hope  of  studying  the  influence  of  iLRF  oti  radiation-induced 
lymphomas : 

(Old)  STCLI  X  C57X/::a 

(Young)   3TCLI   x  C57a./Ko 

Fractionated  irradiation  at  90  r  x  '',   of  7  days,   and  at 

22,5  r  X  4,.  of  7  days,   '  ,,     ..; 


(Project  description  continued) 


SERIAL  NO.   427  PAGE  5 

2)  Studies  of  mechanisms  involved  in  inhibition  of  leukemic  cell  growth, 

A.  Studies  are  now  underway  to  develop  resistant  and  dependent  variants 
of  other  lymphocytic  neoplasms  and,  in  addition,  of  type  A  and  B 
reticular  neoplasms.  Cur  interest  here  is  to  determine  if  cross- 
resistance  and  collateral  sensitivities  are  the  result  of  specific 
drugs  used,  or  are  dependentin  part  on  the  neoplastic  cell  population. 
Dr.  Li.  Potter  has  established,  in  ascitic  form.,  a  battery  of  15  such 
neoplasms  from  vjhich  selection  will  be  mode.  From  this  group  an 
Azaserine-resistant  plasma  cell  neoplasm  7C429,  which  exhibits  a  new 
pattern  of  resistance,  has  been  established.  This  resistant  popula- 
tion of  cells  will  be  of  interest  since  specific  sites  in  biochemical 
pcthvrays  at  Vijhich  Azaserine  acts  are  Itnoiw  (in  pigeon  lines  and 
E,  coli  systems).  The  response  to  various  antileukemic  agents  is 
being  studied  of  neoplasms  which  do  not  show  exponential  growth  in 
the  ascitic  form, 

3,  In  collaboration  with  Dr,  Arnold  ".'Jelch,  Yale  University,  studies  ore 
in  progress  of  several  urr.cil  and  orotic  acid  analogs  which  have  shovm 
great  promise  as  inhibitors  of  bacterial  grcvjth.  Three  of  these  com- 
pounds: 6-az3uracil  and  the  methyl  and  benzyl  derivatives  of  6-uracil 
sulfone  have  been  shown  to  give  striking  inhibition  of  the  neoplasms 
L1210,  L4946,  and  L5178  only  if  given  at  least  3  times  daily.  It 
has  been  found  also  that  if  given  in  the  drinking  H;:0  (5  mg/ml) 
optimal  inhibition  is  attained,  thus  simplifying  the  projected  work 
of  1)  developing  resistance  to  these  compounds  and  2)  studying  reversal 
mechanisms, 

C.  In  collaboratiw  work  with  Dr.  Charles  Elichol  of  Yale  University  studies 
are  being  made  on  1)  content  of  PGA;  2)  capacity  to  alter  PGA  to 
compounds  measurable  as  OF;  an:'  3)  differences  in  sensitivity  to  PGA 
antagonists  in  in  vitro  systems  employing  ascitic  forms  of  our  L1210 
resistant  and  dependent  variants. 

Certain  findings  to  date  are  of  interest: 

1,  An  extremely  high  activity  of  ascitic  cells  in  contrast 
to  lymphomatous  cells  he s  been  found,  making  possible  the  de- 
velopment of  cell-free  systems  to  study  such  changes  as  cell 

permeability  and  differences  in  enzyme  systems  as  they  relate 
to  resistance. 

2,  Antipurine  variants  have  a  far  greater  capacity  to  convert 
FGA  to  CF-like  compounds  in  comparison  with  the  sensitive  ascitic 
cells,  and  also  shov;  a  striking  sensitivity  to  inhibition  of 
this  conversion  by  A-methopterin.  These  results  fit  in  v/ell 
with  studies  on  formate  incorporation, and  suggest  a  shift  in 

the  metabolic  pattern  from  utilization  of  exogenous  metabolites 
to  one  involving  de  novo  synthesis  from  precursors  in  the  cell. 


SERIAL  []G.     427  FA3E  6 

10.  427 
SERIAL  f]C. 

11.  . 

BUDGET  ACTIVITY: 

RESE/iRCH     .       /T7  ADini-!I5TR/iTI0N  /~7 

REVIEW  &  APPROVAL  Z7       'lECHrJICAL  ASSISTAHCE  Z7 

12.  Laboratory  of  Fatholoav" 

CCOFERATIIJG  UNITS  OF  TFE  PUBLIC  HE/iLTH  SERVICE,   OR  CTEER  OSGArilZ/iTICIlS, 
FROVIDIHG  FUriDS,   FACILITIES,   OR  PERSOIIMEL  FOR  THIS  PROJECT  lU  EITIiER 

1955  or   1957 

*  Provided  personnel  for  collaborative  worl^  in  certain  projects. 

13.  [■lone, 

IF  THIS  PROJECT  RESELiXES,    C0L;PLEL;EI ITS ,   CR  PARALLELS  RESEARCH  DONE  ELSE^JIiERE 
IN  TI-IE  PUBLIC  I-IEALTH  SERVICE    CVITMCUT  INTERCII/iNGE  OF  PERSONNEL,   FACILITIES 
OR  FUNDS),   IDEr^TIFY  SUCH  RE3ERACH: 


P/.9E  7 

14,  427 
SERIAL  fJC. 

15.  

PU3LICATIGII3  CTHER  THAN  ABSTRACTS  FROU  THIS  PROJECT  DURIilG  CALEIIDAR  YEAS   i9J 


Mandel,   U.   George  and  Lloyd  7J.  Lav/;  "The  Effect  of  4-AGino-5-ir.-iicl£:scie- 
carboxamlde  oii  tl;e  Carcinostatic  Action  of  S-Azagwanine."  Cancer  Resesroh 
14 ;  COP-11,  December  1954, 

LavJ,  L.  v7,  :"Stiidies  or;  Trsnsformations  to  ResistsTiCt  and  De:jende::ce 

iii  Leid:emiG  Cells,"  Origins  of  Resistai.ce  to  Toxis  Ano-"ts,    pp.   r68-?;S6, 

1955,  published  by  Acedemic  Press,  Inc,  New  York. 

Law,  L.   :'J, ,   Thelma   3,  Dunn,   "nd  Feter  J,   Boyle:  "IJeopfesiris   in  the  C3H 
Strain  sad  in  Fj  Hybrid  ilce  ef  T\70  Cr'/sses  Fcllo^/iiing  Intrcduction   of 
Ex"^rEcts  and  Filtrates  of  Leidie-inic  Tiscaes,"  J,  list.  Cancer  Inst,   16(2) : 
495-539,  October   195:3. 


16.  Hone. 


HCHORS  ArJD  AWARDS  TO  FERSCUIIEL  RELATIi'C  TG  T^IC  FRCJECT  DjRIIIG  CALErDAR 
YEAR  1935, 


PAGE  1 

PROJECT  REPORT  FORK 

1,  National  Cancer  Institute  2.  Laboratory  of  3ioloqy 

INSTITUTE  UBORATORY  OR  BRANCH 

3.  Leukemia  Studies  Section  4.  _______________________  5.  427(a) 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN     SERIAL  NO. 

BETHESDA) 

6,  Studies  on  the  etiology  and  chemotherapy  of  experimental  iTinphomas. 

PROJECT  TITLE 

7,  Luchael  Potter 
PRINCIPAL  INVESTIGATCR(S) 

8,  _j: 

OTHER  INVESTIGATORS 

9,  PROJECT  DESCRIPTION 

1)  Leukemoqenesis :  chemical 

F/ 
DBA  12   mice  have  been  painted  with  0»2%  methycholanthrene  in 

ether  3  x  /wk  in  various  experiments,  Liany  of  the  experiments  in 

progress  here  have  revealed  that  this  mouse  develops  many  different 

morphologic  forms  of  reticular  neoplasms,  mainly  Type  A  and  3  reticulum 

cell  sarcomata.  Some  of  the  information  obtained,  it  is  hoped,  will 

reveal  a  method  for  inducing  such  types  of  reticular  disease  with 

greater  regularity, 

A.  Path  of  action  • 

/Jithin  cages,  mice  have  been  selected  and  painted,  while  others 
in  the  same  cages  h^ve  not  been  painted.  This  study  has  been  de- 
signed to  determine  the  role  of  absorption  through  the  skin  as  the 
path  of  introduction  of  the  carcinogenic  material.  If  the  non-painted 
cage  mates  develop  leukemia  to  the  same  degree  as  the  painted  mice, 
greater  emphasis  will  be  placed  on  studying  the  oral  route  of  this 
material  in  leukemogenesis.     Ten  cages  have  completed  the  painting 
schedule;  in  these  cages  there  are  32  painted  and  28  non-paintedmice, 

3,  Crude  tobacco  smoke  condensate 

Crude  tobacco  smoke  condensate  has  been  obtained  and  is  being 
painted  on  the  skin  of  D3A^/2  mice.  The  carcinogenic  activity  of 
this  crude  material,  if  based  on  the  presence  of  hydrocarbons,  may 
increase  the  incidence  of  lymphocytic  neoplasms  in  these  animals 
prior  to  one  year  of  age.  The  mode  of  action  of  known  carcinogenic 
hydrocarbons  in  this  mouse,  i.e-. .  a  cumulative  action  on  a  distant 
system,  administered  during  a  susceptible  physiologic  age,  suggests 
this  type  of  biologic  test  may  indicate  the  presence  of  materials 
leukemogenic  to  the  mouse.  Through  the  cooperation  and  kindness  of  a 
commercial  firm  a  large  supply  of  crude  tobacco  smoke  condensate 
is  being  made  available,  and  with  the  supervision  and  cooperation  of 
Dr.  J,  Hartwell  of  this  Institute,  concentration  of  hydrocarbons 
by  vacoum  distillation  will  be  carried  out  and  this  material  will 
also  be  tested.  In  experiments  thus  far,  the  presence  of  nicotine 
has  limited  the  use  of  this  material,  and  probably  obscures  the 
action  of  substances  which  may  be  present.  The  complexity  of  any 
(Project  description  continued) 


SERIAL  NO.   427(a)  PAGE  2 

biologic  test  of  this  material  is  taken  into  consideration, 

C.  The  influence  of  bone  marrow  on.  methylcholanthrene-induced 
lymphocytic  neoplasms. 

DM  ^/2  mice  painted  in  the  routine  manner  for  10  or  20 
treatments  have  received  2  intravenous  bone  marrow  injections 
follovang  the  last  treatment.  In  each  case  the  bone  marrow  is 
of  DBA  V2  origin.  The  dose  of  bone  marrow  consisted  of  that 
amount  of  bone  marrow  obtained,  from  2  femora.,  2  humeri,  and   ...  .  . 
"  tibia  from  one  mouse.  The  injections  vrere  given  on  the  2nd 
and  16th  day  following  the  last  painting.  Ninety-six  mice  are 
involved  in  the  experiment:  48,  10  painting;  48,,.  20.  painting; 
each  group  further  subdivided  in  half  and  half  treated  and  not  treated 
v;ith  bone  marrow, 

D*  The  comparative  action  of  x-radiation  and  methylcholanthrene  in  the 
induction  of  reticular  neoplasms  in  the  DBA  ^V2  mouse. 

The  D.3A  ^^   mouse  develops  lymphocytic  neoplasms  in  the  thymus 
and  in  other  organs  in  the  reticular  system.  This  mouse  also  has 
a  moderate  incidence  of  spontaneous  reticulum  cell  sarcomata,  of 
the  A  and  3  type,  Gonadectomy  in  this  mouse  has  been  reported  by 
Kirschbaum  to  have  tvjo  effects  on  raethylcholanthrene-induced  lympho- 
cytic neoplasms  in  the  D3A/2  mouse:  (1)  increase  the  incidence  of 
lymphocytic  neoplasms;  (2)  to  overcome  the  physiologic  barrier  of  age. 

In  order  to  test  in  the  same  strain  mouse  whether  the  mechanisms 
of  leukemogenesis  are  the  same,  B3A  ^/2  mice  have  been  irradiated 
tvith  125  r  every  7  days,  for  4  exposures;  subgroups  of  thymectomized 
mice,  and  gonadectomized  nice,  have  been  included.  Other  pertinent 
groups  of  mice  are  included,  luore  animals  are  being  added  to  the 
series. 

Group  At  10 


-  12 

months 

63 

13 

20.3% 

21 

5 

24  % 

42 

0 

0  % 

19 

0 

0  % 

30 

12 

40  % 

Intact  X-ray 

Gonadectomized  X-ray 

Gonadectomized  None 

Thymectomized  X-ray 

Intact  tiCA 

It  has  been  found  by  examination  of  the  autopsy  data,  and  /leukemia 
tissue  sections,  that  the  character  of  methylcholanthrene/is  quite 
different  from  that  of  x-ray/  A-fadistion  appears  to  produce  a 
monotonous  type  of  lymphocytic  neoplasm  in  the  thymus,  whereas  in 
the  methylcholanthrene  group  lymphocytic  neoplasms,  in  combination 
with  type  3  reticulum  cell  sorccmsta,  have  been  found  v;ith  great 
frequency.  Gonadectom.y  thus  far  has  not  greatly  augmented  x-radiation- 
induced  leukemia  in  the  DBA  f"/2  mouse.  Further  studies  are  in 
progress, 

(Project  description  continued) 


SERIAL  NO.  ^-27 (a) 


PAGE  3 


2)  Genetic  Studies 

In  1946  Furth  described  multiple  osteomata  in  thymectoinized 
AKa  mice.  Those  tumors  appeared  in  animals  of  12  months  of  age  or 
over.  Normally  this  type  of  growth  would  not  be  seen  in  these  mice 
because  tlie  mice  v/ouid  be  dead  of  leukemia  before  the  osteomata  could 
appear.  Eaployincj  papain  digestion  of  mouse  carcasses,  skeletons  of 
(C3H  X  AXR)F-,  mice  12  months  and  older  were  found  to  contain  those 
neoplasr^is.  They  ore  multiple;  commonly  appear  on  the  skull,  pelvis 
and  loirer  extremity;  almost  never  in  the  upper  e::tremity;  more  common 
in  females  (over  80?^  of  females  hove  one  or  more  of  these  lesions 
after  14  months).  An  interesting  finding  has  been  that  these  lesions 
have  not  been  seen  in  only  2  of  37  (AKR  x  STOLDFj  and  (3TCLI  x  AKR)Fi 
mice  of  similar  age.  This  problem  is  of  real  interest  since  the  inci* 
dence  of  leukemia  in  the  2  AKR  hybrids  is  also  different,  3TGLI  is 
known  to  contribute  a  nursing  influence  which  delays  and  inhibits 
leukemia.  Further  studies  employing  other  A!<;R  hybrids,  and  genetic 
studies  of  backcross  animals,  are  undervjay. 


Osteoma 

ta 

Strain 

Sex 

No, 

No,  of 
animals 
bearing 
tumors 

Total 

number 
tumors 

C3H  X  AI<R 

2? 

27 

21 

95 

db" 

28 

15 

32 

STOLI  x  AKR 

99 

17 

2 

3 

a& 


3)  Hormonal  Studies 


A,  Gardner  has  reported  that  estrogen  treatment  of  C3H  mice,  of 

the  Strong  subline,  developed  thymic  neoplasms  follov;ing  estrogen 
treatment,  .'Jeekly  doses  of  !^0  mgm  of  estradiol  benzoate  are 
being  administered  to  2  sublines  of  C3H,  The  G3H  F/Lw  and  the 
Z^   line  from  Bittner,  Gross  has  reported  that  subline  differences 
exist,  ivhen  he  attempts  to  induce  leukemia  with  extracts  of  AKR 
leukemic  tissue.  It  is  of  interest  to  determine  if  a  similar 
difference  exists  with  estrogenic  induction  of  thymic  tumors, 

3,  A  study  of  leukemoqenesis  in  (DBA  x  CE)Fi  and  (CE  x  D3A)Fj^  mice. 
Virgin  females  of  this  hybrid  type  develop  hyperestrogenism, 
characterized  by  a  proliferation  of  the  endometrium.  Reciprocal 
hybrids  of  these  strains  have  been  produced,  though  it  has  taken 
some  time  to  get  enough  CE  mice,  and  the  first  group  has  been 
painted  v;ith  methylcholanthrene.  Lymphocytic  neoplasms  in  the 
virgin  females  will  be  transplanted  to  determine  if  these  neo- 
plasms are  dependent  on  this  abnormal  hormonal  state.  The  role 
(Project  descri{)tion  continued) 


SERIAL  no.  _  427 (^.)  PAGE  4 

of  rjenetic  facttfrs,  goneid^t^oKij ^   etc*  Virill  be  evaluated.  To 
date,  no  lymphocytic  neoplasms  Kav«  been  found  in  the  D3ft  x  CE- 
hybrid  mice, 

4)  The  development  _o|,  transo^la^ntabl^  retl,ejAlaj^  ng^cjilnsms  of  the  mouse^ 
in  ascites  form,  for  the  study  of  chernqtherapcatie  ffl,echg.n,ismG« 

A  spectrum  of  transplantable  tuniors,principally  in  D3A/2  mice, 
has  been  developed  in  the  last  year  in  order  to: 

a.  increase  the  range  of  morphologic  forms  vvhich  can 
be  studied  vath  chemotherspeutic  agents; 

b.  to  test  differences  within  morphologic  classes  of  response 
to  the  antimetsbolites,  azaserine,  methotrexate, 

,  6-mercaptGpi5rine. 

For  this  purpose  the  follovjihg  tumors  have  been  established;  and 
sorae  of  their  ciiarecteristics  ore  listed, 

A.  Lymphocytic  Ileo'^lcsrr.s 

A  series  of  10  ascitic  lymphocytic  neoplasms,  originating  in 
the  D3A/2  mouse,  has  been  developed.  l\!ith  these  ascitic  tumors 
studies  on  drug  sensitivity  Vi/ere  carried  out;  further,  using  cell 
doses  varying  from  10  to  10"'  cells,  titrations  of  these  tumors 
were  undertaken.  In  order  to  establish  true  sensitivity  or  resistance 
to  a  given  antimetabolite,  vjith  survival  time  ys  the  end  point,  the 
cell  population  must  be  uniform.  Studies  o  f  titration  reveal  that/Semi- 
unifcrmity  is  acquired  in  most  Ccses  gradually,/  ^trrig;,t  line  re-   log- 
lationships  between  cell  dose  in  log  units  r;nd  days  of  survival    arith- 
sre  the  criteria  for  unifornity.  Si-uh  relationships  have  been        mic 
found  to  hold  for  Ll2i0,  and  for  G  of  the  turiicrs  in  this  series, 
P268,  F312,  P335,  These  s.t,rBight^, lines  have  been  found  to  ex- 
tend from  cell  doses  of  TO"  to  IC,  Some  tur.iors  have  never  shown 
this  character  or^have  developed  it  very-  slowly,  •  L517S---3rrd-F330,  ■ 
c.nd   the  plasma  cell  tumor,  70429,  are  examples. 


In  many  experiments  employing  antiraetaooiites  it  has  'oeen   diffi- 
cult to  obtain  good  results  with  early  transplant  generations, 
principally  because  the  tumors  take  too  long  to  kill,  thus  allowing 
the  animals  to  live  after. the  maximal  optim?!  dose  of  the  anti- 
metabolite can  be  given. 

From  .this  work  facts  regarding  the  adr.dnistration  of  antiinetabolites 
to  animals  bearing  tumors  of  a  logarithnic  -rr.d  a  non-iogarithmic 
character  are  being  learned. 


(Project  description  continued) 


SERIAL  no.     427(e) 


PAGE   5 


I,     Lymphocytic 


Mo. 


IndEJ.ction 

or 

oriciin 


Strain 


288 

fviCA'" 

312 

I,!CA 

335 

L.CA 

ope 

LiCA    ■ 

LICA 

433 

Z-ray 

5178 

ncA 

553 

Spent. 

421 

X-ray 

413 

LICA 

3A/7: 


RETICUL/lR  I!E0PLASI.:3 


Time  of  death  from 
doses  of  1-3  ::   10^ 
ascites  cells 
At  Days 

generations   survival 
7-11    13.6  -  n. 


II.  Reticulum  Cell  Sarcomata  (D3A/2) 


228 
329 


KCA 
hiCA 


II,  HodqIcins-li::e  Lesion 

195     MCA       " 
L7235   Filtrate  ?  C3H 

IV.  Plasma  Cell  Tumor 

70429   Spent.      G3H 

V.  Granulocytic  Leu!:emia 

I-I5530   SpoRt.      CSe^ 


7-11 


—  7  '■ 

4 


1/ 
17 

42 


-  13.1 

-  11.7 

-  16.7 


About  30 


i!ot  ascitic 


20-40  days 


Trans:?lgnt  gener? 
tior.  at  which 
converted  to 
ascitic  ti'.mor 


days 


*Fie  t  iiy  1  c  h  o  1  a  n  t  h  r  e  n  e 


(Project  description  cc;itin«ed) 


SERIAL  NC.  4r,7(a)  PAGE  6   • 

3»  Tiie  Ret  id;  I  am  Cell  Sarcomata  (Type  A  snd  3) 

Dunn  in  her  review  described  z   classiiicrtioa  of  retieular 
neoplGsms  of  the  moi-se.  An  im'jortsnt  category  of  neoplsrns  in  the 
mouse  has  not  been  evaluated  es  to  the  relationship  to  humsH  disease,. 
cell  type  involved,  and  response  to  chemotherapy.  The  relationship 
of  these  neoplasms  to  the  more  chronic  lynijihornats  of  mail  has  been 
repeatedly  cited.  Type  A  and  3  neoplasms  hEve  been  transplanted  in 
DBA/?,  mice.  Ten  neoplrisms  sre  Egsiti  available  and  have  been  trans- 
plsnted.  These  necplEsras  sre  being  converted  to  the  sscitic  form 
where  the  cells  arc  studied  i/ith  ohsse  micrescopy.  Two  Type  A  neo- 
plasms have  been  converted  to  sisGites  turners,  Ti\'o  Type  3  neoplssms 
have  been  carried  for  3  gerierstions.  These  neoplssms  ^rc  sppearing 
so&ner  and  it  is  hoped  this  most  interesting  cell  type  '.jill  be  avail- 
able in  the  ascitic  form.  Studies  thus  far  reveal  these  Type  A  tutaors 
to  bo  sensitive  to  triethylene  rx-iamine.  The  problems  of  adrainis- 
tering-this  drug  effectively  s-;;;iin  hsve  isportant  bearing  on  human ^ 
chensothejjapeutic  problems. 

TmQ   Type  B  neoplasms,  L.7':.35  in  C3H,  and  P195  in  the  D.B/i/"  mouse, 
are  imiqwe  in  their  transplant  ch&r?'cter.  These  tunors  require  many 
months  to  develop,  .'teen  they  do  appear,  it  is  first  in  the  spleen 
and  then  generalises  in  the  lyivph   nodes.  Studies  of  the  morphologic 
changes"  during  transplaiitatiosi  are  being  carried  out  with  Dr.  Clyde 
Dawe  and  Dr.  Thelina  Durn  of  the  Patholo^/  Section,  These  tumors  have 
not  yet  been  tested  v/ith  cheraGtherapeutic  agents, 

C.  Plasma  Cell  Tumor 

The  plasma  cell  turnGr  #704-19  arose  ir-  :_',3  ileocecal  region  in 
a  C3K/He  mouse,  and  originally  was  a  plasmj  c^ii  neoplasm.  Following 
20  transplant  generations  as  r  solid  tumor,  it  v;as  converted  in  one 
transplant  generation  to  an  ^iseitic  tumor.  The  ascitic  tumor  cell 
no  longer  resembles  a  differontiotcd  plasms  cell.  At  ascitic  genera- 
tions 3^  9,  snd  17  this  tumor  ?;as  titered  by  inoculating  doses  of 
cells  which  varied  from  10?  to  10",  Cell  doses  of  lO'*',  10*^  cells  gIo 
not  hill  anymore  rapidly  than  doses  of  10*.  In  5  experiments  utili-  " 
2ing  165  animals,  employing  doses  of  .SS  "  2,?.5  x  10^  cells.,  87  per- 
cent of  control  animals  were  dead  by  the  40th  day,  whereas  only  15 
percenit  animals  treated  with  ?.0  or  more  daily  doses  of  5  mgm/kg  b.w, 
azaserine  were  dead  at  the  40th  day.  It  has  been  found  by   these  and 
other  experiments  that: 

1,  Aza serine  is  a  povjerful  inhibitor  of  this  tumor, 
?:,  Aza  serine  is  relatively  non-toxic  and  osn  be  given  for 
extended  periods,  thus  providing  a  chtmothvirapeutic  model 
for  the  study  of  a)  the  developmtnt  of  resistance,  and 
b)  the  reasons  for  eventual  curative  failure;  c)  the 
mechanism  of  action  of  asaserine;  d)  some-  crude  idea  of 
the  rate  of  resistant  lines  developing  in  a  group  of 
animals  can  be  explored. 

3.  Tv7o  resistant  and  probably  dependent  lines  of  this  tumor 
have  been  isolrtcd.  Others  are  being  tested. 

4.  Azaleucinc,  anotrier  analog,  hrs  been  obtained  and  has 
been  found  to  else  be  inhibitory  to  this  tumor.  Studies 
on  dosage,  cross  resistance,  and  other  properties,  are 
undervrey, 

(Project  description  continued) 


SERIAL  no.  427 (a)  FA3E  7 


D.  Granulocytic  Leukemia 

This  turner  was  at  first  z-   ohlororna  and  hss  only  recently 
developed.  It  has  developed  virulence  rapidly.  It  is  in  ascitic 
form  and  consists  mostly  of  myeloblasts  i/ith  some  myelocytes  and 
more  differentiated  forms.  Tic  studies  have  as  yet  been  undertslcen. 

5)  Radiation  Induced  Leu.':ernia 

A.  The  pathophysiologic  mechaiiism  hj   which  thj^mic  lymphocytic  neoplasms 
are  iriduced'  in  C57X/Ka  mice  are  not  clearly  defined.  The  most 
strildng  influence  thus  far  depends  upon  fractionation  of  tlie  x-ray 
exposures.  This  suggests  that  an  inj ury-regeneration  phenomenon  may 
be  involved.  The  fractionation  procedure  has  been  carried  out  by 
Kaplan  at  4,  8,  and  16  C^y   intervals  with  little  difference  in  the 
incidence  of  leukemia.  An  experiment  is  underway  in  which  groups 
of  C57X/Ka  mice  are  receiving  '^:2Z   r  four  times  at  intervals  of 
16,  30,  and  45  days.  This  proiongstioa  of  the  interval  is  designed 
to  break  up  the' injury-regeneration  time-relationship,  if  such  a 
relationship  is  the  determinin'j  factor, 

3.  The  development  of  the  Rf  str:;in  (with  Zr .   L,  il .   Law) 

The  Rf  strain  of  mice  has  been  reported  to  have  a  moderate 
incidence  of  granulocytic  lei:;:e;.iie ,  following  the  e::posi;re  tc  single 
doses  of  128  r.  Rf  mice  hr.ve  been  bred  in  this  laboratory  for  the 
last  year,  and  good  breeding  colony  hcs  been  established.  Liice  from 
.  this  colony  have  been  used  for  prilirainary ,  confirmatory  experiments 
regarrling  the  incidence  of  granulocytic  leLncemia,  The  mice  have  hee-a 


!)laced  in  the  following  groiv^s 


Leuhemo.^enic  Treatment 


1.  Intact  L-6  r  single  dose 

?.  Intact  128  r,  4  doses,  7  day  intervals 

3,  Thymectomized  i'-.G  r  single  dose 

4,  Thymectomized  1"S  r,  4  doses,  7  day  intervals 

5,  Intact  Fainting  with  methycholanthrene 

6,  Intact  :Veekiy  injections  of  25  y  estradiol 

7,  Intact  breeding  colony. 

Leukemias  are  beginning  to  appear^  Several  chioromas  and  non- 
thymic  leukemias  have  appeared  in  various  groups.  This  mouse  may  be 
the  instrument  ijhereby  further  information  on  the  etiology  of  granulo- 
cytic leukemia  will  be  forthcoming. 


PAGE  8 


10.   427(a) 

SERIAL  m. 


11. , ^___> 

BUDGET  ACTIVITY:  

RESEARCH    /  x  /     AD1;!I1!ISTRATICI!    /  / 

REVIE.J  &  APFRCVAL  Z7     TECMTIICAL  ASSISTAFICE     /     / 

* 
j9^  Doctors  Clyde  Davje  and  Thelma  Dunn,  Lab.   of  Prithology,         .    •      • 


COOPERATiriG  UniTS  OF  TItE  PUBLIC  "EALTH  SERVICE,   OR  OThER  ORGArllZATICnS.PRO- 
VIUiriG  FU[1DS,   FACILITIES,   OR  PERSONIIEL  FOR  THIS  PROJECT  II!  EITHER  1956  or 

1957 

*  AH  aspects  regard! nrj   the  ;-r;t!iology  of  reticular  disesse. 

13.  Hone. 

IF  TlilS  PROJECT  RESEK3LE3,  CCFiPLEiJEnTS,  OR  FAR/iLLELS  RESEARCH  DONE  ELSE.VMEl 
IN  TI-E  PUBLIC  HEALTH  SERVICE  (.riTI^CUT  INTERCHAMGE  OF  PERSGNriEL,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  RESEARCH/: 

14,  15  &  16:  NO  ENTRIES  FOR  ITEMS  14,  15  &  16. 


PAGE   1 


PROJECT  REPORT  FCRf.i 


1 «  National  Cancer  Institute ^ .  Laboratory  of  3iolor]7 

iriSTirjTE  LA30PJ\TCRY  Cri  3RAMCII 

3,  Leukemia  Studies  Section ^-.__ 5,  -127(b) 

SECTICn  OR  SERVICE  LOCATICri  (IF  OTHER  THAN  ISLUll*)     SERIAL  NG. 

6«  Studies  on  the  etiology  and  chemotherapy  of  exnerimental  lyrnrhomas, 

PROJECT  7IILE 

7,  Bernard  Shacter 

PRI11CIPAL  iriVE3TIGAT0R(3) 

e.     - ^ 

CTI-ER  iriVESTI  GATORS 
9. PROJECT  DESCRIPTION 

Project ;     Biochemical  raechanisms  of  resistance  to  metabolic  antagonists  in 

chernotherr! py  of  experimental   lymphomas. 

Objectives : 

The  purpose  of  this  project  is  to  discover  the  mechanisms  in- 
volved in  the  development  of  resistance  to  the  nrowth  inhibitory  action 
of  agents  initially  active  against  experimental  lymphomas, 

methods  enroloyed; 

The  effects  of  kno'/m  c'r.emotherapGutic  agents  on  various  metabolic 
activities  of  lymphoma  cells  crc  determined,  in  order  to  establish  possible 
differences  in  the  action  of  the  agents  on  sensitive  as  opposed  to  resis- 
tant cells,  f/.etabolic  activities  studied  include  uptake  of  radioactive 
precursors  i;ito  proteins  and  nucleic  acids,  effects  on  significant  enzyme 
systems,  and  effects  on  concentration  of  important  cellular  metabolites, 

Liajor  findings; 

In  the  course  of  determir.ing  the  effect  of  amethopterin  on 
glutathione  and  ascorbic  acid  levels  of  sensitive  and  resistant  leukemic 
cells,  it  was  found  that  there  was  a  decrease  in  liver  glutathione  levels 
cf  animals  bearing  the  rapidly  groiving  lymphocytic  leukemia  L4946  in 
ascitic  form.  Administration  of  3  mg/!:g  amethopterin  to  animals  bearing 
rapidly  gro'ving  tumors  vjas  iollov;ed  by  a  cessation  of  tumor  growth,  as 
measured  by  change  in  total  volume  of  ascitic  oelis,  and  by  a  return  of 
liver  glutathione  levels  to  considerably  above  normal.  The  techniques 
of  measuring  changes  in  total  volume  of  ascites  cells  and  changes  in 
liver  glutathione  levels  are  being  applied  as  a  simple  yet  precise  means 
for  determining  action  of  other  possible  chemotherapeutic  agents  on  the 
growth  of  experimental  lymphomas. 


(Project  description  con'td.) 


SERIAL  NO.  427(b)  PAGE  2 

SiqnificaHce  to  cancer  research; 

One  of  the  major  problems  in  the  use  of  chemotherapeatic  agents 
for  the  clinical  control  of  leu'conun  is  the  eventual  development  of  re- 
sistance to  the  action  of  the  drug,  such  that  the  leukemic  process  can  no 
longer  be  controlled.  If  it  v/ere  possible  to  establish  the  mechanisms 
involved  in  development  cf  resistance,  it  might  be  possible  to  institute 
measures  to  either  avoid  this  undesirable  effect,  or  else  to  circumvent 
it,  perhaps  by  combined  therapy. 

Proposed  course  of  project; 

During  the  next  calendar  year  it  is  proposed  to  investigate  some 
of  the  mechanisms  luhich  have  been  suggested  as  leading  tov/ard  resistance. 
These  include  a  study  of  deaminction  of  S-asoguanino  by  lymphomas  sensitive 
to,  resistant  to,  and  dependent  on  the  agent,  since  increased  deamination 
of  asaguanine  has  been  proposed  as  a  mechanism  for  resistance  to  the  agent. 
The  primary  effort  will  be  directed  tor/ard  a  search  for  alternative  pathi/ays 
of  synthesis  of  esseutir.l  products  by  resistant  cells,  as  the  possible 
mechanism  for  development  of  resistance. 


3ESIAL  no.         427(b)  PAGE  3 

10.  427(b) 

3ERI/J.  no. 

11.  

BUDGET  ACTIVITY : 

RESEARCH       Rl  ADUiniSIMTICrJ 

REVIEV;  &  APPROVAL  TECI-SIICAL  ASSISTAIJCE 

12.  None 

COCPERATiriG  UIJITS  CF  TrE  FU3LIC  HEALTFI  GERVICE,   OR  CTKER  CRGArJIZATICNS, 
FRCVIDIllG  FUI-©3,  FACILITIES,  OR  PERSONNEL  FCR  THIS  PROJECT  If]  EITHER 
1956  or   1957 

13.  None 

IF  THIS  PROJECT  RESELIBLES,   CCi.iPLEl.IENTS,  OR  PARALLELS  RESEARCH  DONE  ELSE- 
WHERE IN  THE  PUBLIC  [EALTH  SERVICE   CJITKOUT  IMIERCI-IANGE  OF  PERSCFINEL, 
FACILITIES  OR  FUNDS),   IJENTIP/  SUCH  RESEARCH: 

14.  No  entries  for  Items  14  &  16, 

15.  ^ \ \ 

PUBLICATIONS  OTIER  TI-IAN  ABSTRACTS  FROU  THIS  PROJECT  DURING  CALEfSAR  YEAR 
1955. 

Shacter,  3,,  Interrelations  in  Respiratory,  Phosphorylative  and  Nitotic 
Activities  of  Ehrlich  Ascites  Tiimor  Cells:  Influence  of  Dinitrophenol, 
Arch.  Biochem.  and  Biopliys.  57,  3C7-4G0  (1955). 


PROJECT  REPORT  FCRl/i 


PAGE  1 


1,  Mational  Ccncer  Institute 

INSTITUTE 


?.  Laboratory  of  Biclocry 


UBCRATORY  CR  3RANCH 


Leukemia  Studies  Section 
SECTICri  CR  SERVICE 


5.   427(c) 


lccatigm  (if  other  ti-ian        serial  lic. 
beth;) 


6.  Studies  on  the  etiolociv  and  chenot.'ieraoy  of  experimental  lymphomas. 
PROJECT  TITLE 

7.  S.E.  Reauae 

PRIIJCIPAL  II]VESTIGATGR(S) 

8.  None. , 


OTHER  INVESTIGATORS 
9.  PROJECT  DESCRIPTION 


Project:  Studies  on  the  etiology  and  chemotherapy  of  experimental  lymphomasi 

Objectives:  Investigate  genetic  and  biochemical  mechanisms  involved  in 
the  development  of  drug-resistance  and  -dependence  in  riiouse  leukemias, 

fviethods  employed;  1)  Model  bacterial  systems  have  been  employed  for 
genetic  studies  of  the  frequency  and  mode  of  development  of  resistance  to 
purine  analogs  v;hich  are  effective  antileukemic  drugs,  2)  In  vitro  studies 
have  been  made  of  specific  biochemical  reactions  considered  likely  to  be 
involved  in  the  development  of  resistance  to  craethopterin,  3)  Patterns 
of  activity  of  a  number  of  antipi'.rine  drugs  h::ve  been  studied  with  respect 
to  their  effect  on  a  number  of  drug-sensitive,  -resistant  and  -dependent 
leukemias. 

Ka.lor  findings:  1)  No  organisms  tested  heve  proven  to  be  satisfactory  for 
the  genetic  investigations, 

2)  The  reaction  of  most  interest  in  this  phase  of  the 
work  has  been  the  cleavage  of  aminopterin  at  C-9  to  yield  pterdine  and 
p-3minobenzoylglutamate  (measured  as  diazotisp.ble  amine).  Although  such 
cleavage  has  been  reported  ic  a  number  of  microorganisms,  no  such  activity 
has  been  detectable  in  aminopterin  (or  amethopterin-)-resistant  leukemic 
lines,  Leukemias  sensitive  to  this  drug,  as  well  as  those  dependent  upon 
it,  appear  also  to  lack  cbility  to  cleave  it.  Liver,  kidney,  spleen,  and 
skeletal  muscle,  :;rom  both  normal  and  leukemic  mice ^  shovired  no  such  activity i 

3)  The  adenine  analog,  4-aminopyr3zolo-(3,4-D)-pyrimidine 
(APP),  and  guanine  analog,  4-hydroxy-6-anilncpyra2olo-(3,4__)-pyrimidine 
(riAPF),  have  been  investigated  for  their  ability  to  inhibit  various  leu- 
kemias or  to  satisfy  the  drug  requirement  of  anti-purine-dependent  lines. 
APP  inhibits  all  sensitive,  resistant  and  dependent  leukemic  lines  tested 

to  the  extent  of  40  to  60  percent,  including  one  (L-4946)  which  is  naturally 
resistant  to  other  antipurines,  AFP  does  hot  substitute  for  8-azaguEnine 
or  thioguanine  in  lines  dependent  upon  these  drugs.  Purified  KAFF  does 
not  inhibit  any  of  the  tumors  inhibited  by   APP;  an  unpurified  sample  had 
shown  some  such  activity.  An  interesting  point  is  that  HAP?  appears  to 
satisfy  the  antipurine  requirement  of  an  8-azagu3nine-dependent  leukemia, 
AFP  inhibits  the  growth  of  an  amethopterin-^dependent  line.  Other, 
(Project  description  continued) 


SERIAL  lie.  427(c)  PAGE  2 

variously  substituted,  members  of  this  series  of  compov.nds,  as  vifell  as  the 
(4, 3-d)  congener  of  APP,  liave  shown  little  or  no  antileukemic  activity. 
APF  is  quite  toxic  to  mice  (C/Daft)Fi,  producing;  detectable  weight  loss  at 
o'oses  of  7,5  mg/hc  X  3.  At  levels  of  15-20  mg/!cg  X  5,  severe  extraorbital 
hemorrhages  may  occur. 

All  data  obtained  so  far  apply  to  activity  in  terms  of  y/eight  of 
tumors  grown  as  subcutaneous  lymphomas.  Survival  times  of  nice  carrying 
the  same  leukemias  as  ascitic  tumors  are  not  prolonged  by  APP. 

Significance  to  cancer  research;   1)  If  one  or  more  bacterial  strains 
can  be  found  which  imitate  the  responses  of  our  leukemir. s  to  the  pyraaoles 
and  other  antipurincs,  it  is  felt  that  much  c?^n   be  learned  about  the 
genetic  aspects  of  resistance  and  dependence  which,  thus  for,  has  not  been 
amenable  to  a  direct  analysis  in  the  leukemic  material,  Cf  particular 
interest  in  this  respect  are  dcta  on  the  frequency  of  mutation  to  dependence 
and  resistance;  allelic  relationships  among  genes  affected;  genie  interactions 
such  as  suppression  v;hich  may  occur.  At  present,  available  technics  are 
inadequate  to  permit  a  direct  attack  on  such  problems  involving  mutation  in  ■ 
leukemic  cells. 

2)  Detailed  knowledge  of  biochemical  events  involved  in  the  expression  of 
dependence  or  resistance  offer  expanded  hope  of  effective  chemotherapeutic 
attack  against  fulminating  or  refractory  leukemia  encountered  in  the  clinic. 
Negative  information,  vjhile  rarely  conclusive,  can  help  narrov/  the  area 

in  which  fruitful  exploration  is  li!:cly  to  be  made.  It  is  tentatively 
concluded  that,  in  our  leukemic  m.aterial,  cleavage  of  aminopteria  does 
not  play  a  role  in  the  expression  of  resistance  to  this  drug, 

3)  The  activity  df  4-aminopyra3olo-(3,4-d)-pyrimidine  against  antipurine- 
resistont  mouse  leukemia  suggests  that  it  might  be  of  interest  clinically. 
Little  appears  to  be  known  concerning  its  pharmacology  or  toxicology. 
Given  such  information,  it  night  prove  satisfactory  for  trial  against 
antipurine-refractory  leukemias,  or  used  in  combination  with  antifolics 

or  other  an ti purines. 

The  fact  that  APP  inhibits  leukemias  resistant  to  other  antipurines, 
whereas  other  antipurines  show  cross-resistance  omong  one  another  against 
these  same  leukemias,  suggests  that  AP?  is  active  at  a  biochemical  locus 
different  from  that  of  other  purine  rnalogs.  The  failure  of  APF  to  substi- 
tute for  8-asaguarjine  for  the  8-a3aguanine-dependent  tumor  is  consistent 
with  this  view.  These  and  other  data  suggest  several  different  points 
of  action  of  purine  analogs  in  the  sensitive,  resistant  and  dependent 
leukemias.  Further  analysis  of  the  interactions  and  cross-resistance 
patterns  should  yield  insights  into  the  biochemical  mechanisms  underlying 
theip,  as  i>;ell,  perhaps,  as  specific  points  vjhere  rational  chemotherapeutic 
attacks  might  be  made. 

Proposed  course  of  sroject;  1)  Further  attempts  will  be  iTiade  to  Obtain 
suitable  bacterial  strains  to  serve  as  genetic  models  as  discussed  above. 
Emphasis  will  be  placed  on  obtaining  antipurine-sensitivo,  -resistant 
and  -dependent  lines  of  organisms  which  provi'r'e  opportunity  for  relatively 
extensive  genetic  analysis  (e.g.,  Escherichia  coli.  Salmonella  spp,)« 
^^  Iji  vitro  studies  will  be  concentrated  on  the  problem  of  pinpointing 
the  loci  of  action  of  APP  and  IiAPF  in  the  leukemic  lines  in  which  they  are 
active  i^ietaboT  ically.  An  attempt  will  be  made  to  follcvj,  qualitatively, 
purine  synthesis  by  tumor  homogenates  and  ascitic  cell  suspensions  and   ~ 
the  effects  produced  by  the  pyrs^soies  in  t!iese  systems.  If  these  surveys 
are  successful,  it  may  bo  possible  to  extend  and  quantitate  them  by  means- 

(Project  description  continued) 


SERIAL  I!C.  427(c)  PAGE  3 


of  radioisotope  studies  in   collaboration  vjith  someone  competerit  In   scsh 

technics.  If  such  brei  sr.d  cell  suspensions  prove  satisfactory,  tiiey 

could  be  used  in  plsce  of  the  intact  animals  now  being  used  in  studies 

of  reversal  ox  activity  of  APF  and  FIAPP. 

3)  Many  difficulties  have  been  encountered  in  attempts  to  study  the  effects  oi 

the  i:yrozoles  combined  vjith  normal  purine  bases  or  other  purine  analogst 

Those  problems  arise  as  a  result  of  the  lov^j  solubilities  of  the  compounds, 

toxicity  of  many  combinations,  etc.,  so  that  activity  measurement  in 

terms  of  tumor  vjcirht  often  become  unreliable  at  best  and  impossible  at 

ivorst.  Shacter  has  shown  that,  under  certain  conditions,  ascitic  tumor 

packed  cell  volume  can  be  correlated  vjith  antileul:emic  activity.  Host 

liver  glutathione  levels  con  also  be  used  as  a  measure  of  such  activity. 

It  is  planned  to  investigate  the  extent  to  which  these  criteria  can  be 

used  to  replace  tumor  weiqht  as  a  measure  of  intileuhemic  action. 


SERIAL  Ho.       427(c)  PA3E  4 


10.         427(c) 

SERIAL  MC, 


11. 


3UD3ET  ACTIVITY: 

RESEARCH  fT]  ADLlIIlISTR/iTia!       /~7 

REVIEW  &  APPROVAL  /~7  ■        TECIirilCAL  ASSISTAFICE     /     / 

12.  Mono. ^ 

COOPERATIMG  UIIITS  CF  T!-!E  PaSLIC  MEALTH  SERVICE,   CR  OTIIES  CRGAniZ/iTICnS, 
FROVIDIMG  FUfSS,  FACILITIES,   t'R  FERSCIIIEL  FOR  TI-IIS  FRCJECT  III  EITIE,^ 

1956  or   1957 

13,  none. 

IF  TI-IIS  PROJECT  RESEI.:BLES,   COf.^LEl.lEf .TS  ,   CR  PAR/iLLELo  RESEARCH  DOilE 
EL3EV/IIERE  III  TI-IE  PU3LIC  I-EALT:-!  SERVICE    (?JITI-!GUT  inTERCrlAIIGE  OF  FERSOIinEL, 
FACILITIES  OR  F'JIIDS) ,    I,";EI!TIFY  S'JCM  RESE/.RC:^: 


NO  ENTRIES  FOR  IlEI.iS   14,    15  &  16. 


Genoral  Project         1|28 

Serial  Nc. 


Study  cf  chemically  defined  medium  and  cell  nutrition. 


Page  1, 


PROJECT  REPORT  FORM 


1,  National  Cancer 2,  Biology 


Institute  Laboratory  or  Branch 

3.  Tissue  Culture h^     Bethesda,  Md« 5.  [i28(a) 

Section  or  Service  Lo.caticn  Serial  No, 

6.  Development  of  a  defined  medium  for  the  cultivation  of  strain  L  cells  in 
Project  Titles    vitro. 

7.  V«  J.  Eva-ns _^ ^ 

Principal  Investigator (sp 

8.  J.  C.  Bryant,  W»  R.  Earle,  K,  K.  Sanford,  B.  B.  Westfall,  M.C,  Fioramonti, 

and  W.  T.  McQuilkJJi, . 

Other  Investigators 


9.  Project  description: 

Objective;  To  develop  a  satisfactory  defined  medium  adequate  for  pro- 
ducing proliferation  of  strain  L  mouse  cells  in  order  to  study  their 
nutritional  and  metabolic  behavior* 

Methods  employed;  Replicate  cultures  are  prepared  and  enumeration  of 
nuclei  are  made  after  exposure  tc  defined  media  for  varying  intervals. 
Such  media  were  devised  from  the  collected  information  on  the  nutrition 
of  bacteria,  mammalian  and  .avian  cells  in  vitro  end   the  data  from  physi- 
cal and  chemical  fractionations  of  naturally  occurring  medium  on  which 
the  strain  L  cells  are  maintained. 

Major  findings  t   Several  media  have  been  devised  and  tested.  Media  have 
been  tested  containing  amine  acids,  amides  glutathione,  vitamins,  coen- 
zymes, unsaturated  fatty  acids,  desoxyribosides  of  nueleic  acids,  car- 
bohydrate sources  and  inorganic  salts,  No  antibiotics,  fungicides  or 
protein  sources  are  included.  A  medium  has  been  devised  which  allows 
a  continued  (11  months  to  date)  reasonably  rapid  rate  of  proliferation 
for  clone  L  cells.  More  recently  the  unsaturated  fatty  acids  have  been 
omitted  from  these  media  with  no  deleterious  effect  upon  proliferation. 
Vitamin  B  12  has  been  added  wi.th  a  resulting  small  increment  of  additional 
growth.  In  a  continuation  of  this  ^^rork  the  essential  amino  acid  require- 
ments of  strain  L  have  been  determined  as  well  as  the  essentiality  of  the 
coenzjrmes  group  and  the  nucleic  acid  derivatives  group. 


Page  2, 

Significances  A  chemically  defined  medium  is  essential  for  determing 
difference s~in  the  physiological  between  normal  and  malignajit  cells 
and  for "research  in  the  nature  and  origin  of  malignant  transformation, 
The  present  medium^  essential  in  all  respects,  serves  as  a  prototype 
medium  for  a  prototype  cell  in  tissue  culture.  Such  a  medium  also 
makes  tissue  cultured  cells  a  most  valuable  tool  for  chemotherapeutic 
and  virological  studies.  Actually  the  best  chemically  defined  medium 
described  for  strain  L  cells  so  far,  from  these  studies  has  already  been 
found  to  be  an  exceptionally  excellent  medium  for  virus  studies  with 
human  cell  strains,  when  it  is  supplemented  with  heterologous  serum  in 
,  small  volumes* 

Projected  studies;  Continued  research  will  be  done  to  determine  the    ' 
essentiality  of  each  of  the  groups  of  components  as  well  as  the  in- 
dividual members  of  the  group  and  to  fortify  the  medium  with  additional 
known  growth  promoting  material.  At  the  same  time  metabolic  studies 
are  in  progress  using  this  medium, (See  report  B,  B,  Westfall) 

10,  U28(a) 

Serial  No, 

11. 

Budget  Activity: 

Research    x         Administration 

Review  &  Approval      Technical  Assistance 

12. ; 

Cooperating  units  of  the  Public  Health  Service,   or  other  organizations,  pro- 
viding funds,  facilities,   or  personnel  for  this   project  in  either  1956  or 
1957, 

Medium  has  been  made  available  to  cooperating  units   of  the  Public 
Health  Service  such  as  the  Microbiological  Institute, 

13.        ' \ 

If  this  project  resembles,  complements,  or  parallels  research  done  else- 
■  where  in  the  Public  Health  Service  (without  interchange  of  personnel, 
facilities  or  funds),  identify  such  researchj 

The  organization  rf  Dr.  Harry  Eagle  in  the  Micrc^iolcgical  Institute  is 
also  working  on  cell  nutrition  in  tissue  culture, 

lU.  U28 (a) 

Serial  No, 


15. 


16.. 


Page  3 . 


Publications  other  than  abstracts  from  this  project  during  calendar  year 
1955. 


Studies   of  Nutrient  Media  for  Tissue  Cells   in  Vitro .     I,  A  protein -free 
chemically  defined  medium  for  cultivation  of  strain  L  cells.     Cancer 
Research  i6sOO-00  Jan  1956,     V.J.  Evens,  J.  C,  Bryant,  M.C,  Fioramcnti, 
W.  T,  McQuilkin,  K.K,  Sanfcrd,  and  ¥.  R,  Earle, 

Studies   of  Nutrient  Media  for  Tissue  Cells  in  Vitro.     II,     An  improved 
protein-froc  chemically  defined  medium  for  long  term  cultivation  of 
strain  L  929  cells,     V.   J,  Evans,   J,  C,  Bryant,  VJ.  T.  McQuilkin,  M.C, 
Fioramonti,   K.  K,  Sajiford,   B.  B.  Westfall,   and  W,  R.  Earle,  Cancer  Res, 
16 1    00-00,  Jan.  1956.t 

Honors   and  awards  to  Personnel  relating  to  this  project  during  calendar 
year  1955* 


Page  1, 


PROJECT  REPORT  FORJI 


1,     Natirnal  Cancer 2,     BiolcQT 


INSTITUTE  L/vBGRATORY  OR  BRANCH 


Tissue  Culture h*     Bothesda,  Md. $,   U28  (b) 

Siictirn  or  Service  Lrcr?-ticn  Serxal  Nc, 


6 .     A  study  rf  the  stability  cf  prctcin-frce  chemically  defined  medium. 
Frcject  Title 


7,  V.  J.  Evans, 

Principal  Investigator (s) 


!.  W,  T«  McQuilkin 

Other  investigator  (s) 


9.  Project  description: 

Objective;  To  determine  whether  the  chemically  defined  medium  is  stable 
biologically  after  being  maintained  in  solution  at  5  C  for  a  prolcpgej^ 
period  cf  time,  '  ',' 

Methods  employed;  Cultures  of  strain  L-929j  mouse  fibroblast  cells  were 
maintained  for  a  period  cf  six  weeks  in  protein-free  chemically  defined 
medium  NCTC  109  the  component  solutions  of  which  had  been  stored  at  $'^C   - 
as  long  as  3  to  ii  months.  The  cultures  were  fluid  changed  and  serially  ■ 
transferred  in  the  manner  cf  routine  cultures. 

Major  findings ;  The  morphology  and  cell  proliferation  of  the  cultures 
appeared  excellent  and  equal  to  stock  cultures  maintained  in  chemically 
defined  medium  that  wrs  made  up  fresh  every  twd  weeks. 

Signiflcsnce;  Reports  in  the  literature  of  the  development  of  chemically- 
defined  culture  media  and  accepted  data  regarding  the  stability  of  certain 
cf  the  component  solutions  indicate  rather  extreme  instability  cf  certain 
cf  the  components  of  the  medium.  According  to  this  information  such 
constituent  elements  as  glutathione,  coenzjTnes,  glutamine  and  vitamiji  B- 
vitamin  B-12  solutions  must  be  kept  fresh  or  in  a  frozen  state  until  im- 
mediately before  use.  To  know  that  the  chemically  defined  medium  is  not 
so  biologically  unstable  mptes  possible  substantial  saving  of  labor  in 
the  repetitious  preparation  of  the  medium.  It  also  makes  possible  a  wider 
range  of  use  of  the  medium  in  experimentation. 

Proposed  course  of  project;  To  study  other  aspects  of  the  stability  of 
the  medium  with  the  view  toward  further  simplification  of  its  preparation. 


10,  U28  (It) 

Serial  No, 


Page  2, 


II, 


Budget  activity: 

Research  x 


Administraticn 


Review  &  approval 


Technical  assistance 


12,  None 


Cooperating  units  rf  the  l\iblic  Health  Servicej  or  other  organizations,  pro- 
viding funds,  facilities,  cr  personnel  for  this  project  in  eithep  19SS   cr 
1957. 


13  •  None 

If  this  project  resembles,  complements,  or  parallels  research  dene  elsewhere 
in  the  Public  Health  Service  (without  interchange  of  personnel,  facilities 
cr  funds),  identify  such  researchi 


1^-     U26    (^) 

Serial  No* 


1^,  '.None 


.Publications  other  than  abstracts  from  this  project  during  calendar  year 


16 ,  Nono 

Honors  and  awards  'to  personnel  relf.ting  to  this  project  during  calendar 
year  1955. 


i'agc  1 


1,  National  Cancer ^   2.  Biolog[__ 


Instituto  Laboratory  or  Branch 

3,  Tissue  Culture h.   Bethesda,  Md. 5.  i|28(c) 

Section  cr  Service  Lccaticn  Serial  Wc. 

6 .  The  effect  of  horse  serum  residue  and  certain  chemically  defined  supplements 
Project  Titlet    on  pro  lie  ration  of  strain  L  clone  929  cells  from  the  >■ 

mouse, 

7 .  V,  J>  Evans  ■ 

Principal  Investigator (s ) 

8 .  M.  C.  Fioramonti,^  K,  K.  Sanford,  W.  R.  Earle,  J.  C ♦  Bryant,  W.  T.McQuilkin. 
Other  investigators 

9«  Project  description;  ..,..• 

Objective  To  develop  a  partially  defined  medium  incorporating  washed 
serum  residue  and  free  amino  acids  as  well  as  other  growth  factors  that 
is  capable  of  supporting  proliferati.cn  of  strain  L  cells  as  the  whole 
serum. 

Methods  employed;  The  replicate  culture  techniques  accompanied  by 
nuclei  enumeration  methods  were  used  to  obtain  data  on  the  action  of 
horse  serum  residue  obtained  by  ultrafiltration  supplemented  with  certain 
chemically  defined  media.  The  horse  serum  residue  was  obtained  by  ultra- 
filtration procedure  as  described  by  Sanford  et  al.  The  supplements  vjere 
those  of  Morgan,  Morton  and  Parker's  mixture  199» 

Major  findings;  Quantitative  experiments  were  used  to  test  the  effect 
of  supplements  to  a  basic  medium  of  the  residue  fraction  remaining  after 
ultreJiltraticn  of  horse  serum.  This  stu.dy  was  carried  out  on  washed  cell 
suspensions  of  clone  929  strain'  L  cells,  origjjially  obtained  from  a 
strain  C3H  mouse t.  These  cultures  were  planted  in  T-l5  flasks  on  glass 
substrate  and  changes  in  population  levels  were  determined  by  enumeration 
of  the  nuclei  at  seven,  ten,  thirteen,  fourteen  and  twenty-one  day  inter- 
vals. The  unsupplomented  fraction  of  the  horse  serum  was  incapable  of 
maintaining  the  inoculum  level  beyond  seven  days.  This  residue  medium, 
siipplemented  with  the  amino  acids,  amides  and  amine  of  horse  scrum  was  less 
effective  in  maintaining  population  levels.  The  further  addition  of  niacin, 
p-aminobenzcic  acid,  niacinamide,  pyridoxine  HCl,  thiamin ,HC1,  d-Ca  pento- 
thencitej  i-inositol, 'choline  chlrridej  riboflavin,  ascorbic  acid,  gluta- 
thione, cysteine ;HC1,  biotin,  folic  acid,  vitsmin  A,  vitamin  D(calciferol), 
tween  80,  menadione,  vitarain  E  and  ATP  as  contained  in  mixture  199  of 
Morgan,  Morton,  and  Parker  gave  population  levels  superior  to  those. obtained 
by  use  of  unfractionated  horse  serum.  Under  the  experimental  conditions 
used,  addition  of  the  other  components  of  mixture  199  to  this  medium  gave 
no  added  increase  in  proliferation  and,  in  fact,  indicated  a  possibly 
inhibitory  action. 


15. 


16. 


Page  2 

Significances  Development  cf  a  chemically  defined  medium  is  cf  major 
Importance  for  ultimate  comparison  of  normal  and  malignant  cells 
and  this  was  one  step  in  the  development  of  such  a  medium. 

Proposed  course 

.of  project: Continued  study  to  develop  a  chemically  defined 

medium  'oF'general  use. 


10,     U28(c) 
11. 


Serial  Mo, 


Budget  activity; 

Research   x  Administration 

Review  &  Approval        Technical  Assistance 


12 i       None 


Cooperating  units   of   the  Public  Health  Service,    or  other  organizations, 
providing  funds,   facilities,   or  personnel  for  this  project  in  either 
1956  or  1957. 


13 »   None 


If  this  project  resembles,  conplements,  or  parallels  research  done 
elsewhere  in  the  Public  Health  Service  (without  interchange  of  per- 
sonnel, facilities  or  fimds),  identifj^  such  research: 

The  organization  cf  Dr.  Harry  Eagle  in  the  Microbiological  Institute 
is  also  working  on  cell  nutrition  in  tissue  culture. 


lU.   I428  (c) 


Serial  Mc, 


Publications   other  th^'H  abstracts  from  this  project  during  calendar 
year  1955. 

The  effect  of  horse  serum  residue  and  chemically  defined  supplements 
on  proliferation   of  strain  L  clone  929  cells  from  the  mouse.     Cancer 
Research  v,   l5:   00-00,   1955.  M.C.  Fior?iiicnti,   J.C.  Biyant,  IaT.  T,  Mc 
Quilkin,  V„  J,  Evans,  K.  K,  Saufcrd,  and  ¥,R,  Earle, 


Honors   and   "wards  to  personnel  relating  to  this   project  during  calen- 
dar year  1955* 


Page  1 
PROJECT  REPORT  FORM 
1.     National  Cancer 2.  Biclcsr 


Institute  Lnboratcry  cr  Branch 


3,     Tissue  Culture  Section ii.  Bethesda,  Md, g«  1^28  (d) 

Section  or  Service  Location  Serial  Nc» 


6,  Simplification  of  a  protein-free  chemically  defined  medium  NCTC  109 
Project  Title 


7.  V.  J.  Evans 

Principal  Investigator (s ) 


8 ,   J.  C.  Bryant,  N.M.   Hawkins  end  M,C,  Fiorrjfionti,  K,  K.  Sanfcrd  sxid  lAj^RtSarle 
Other  Investigators, 

9»  Project  Descriptioni 

Object ivet     To   determine  the  essentiality  of  individual  members  of  ccrapcn- 
ent  groups  of  coenzymes  mixtures  nlucurcnic  acid  mixtures  and  fatty  acid 
new  in  the  complex  chemically  defined  prctein-frce  medium  NCTC  109  used  tc 
grew  strain  L-929  cells. 

Methods  employed:  Replicate  cultures  of  strain  L  cells  are  preparecl  and 
enumeration  of  nuclei  are  made  after  exposure  tc  the  test  medium  for  vary- 
ing intervals.     From  chojiges  in  the  number  of  nuclei  en  test  medium  in 
relation  tc  t  he  complex  but  complete  medium  containing  at  least  all  the 
essentials  and  perhaps  some  non-essentials  for  strain  L  it  is  possible 
tc  determine  the  essentiality  of  each  individual  component. 

Major  findings.  Exploratory  data  indicate  that  it  may  be  possible  to  omit, 
Glucuronclactcne,  methyl  linclenate,  methyl  arachidcnate,  diphcsphopyridine 
nucleotide,  cocarbcxylase,  flavin  adenine  dinuclectide,  uridine  triphopyri- 
dine  nucleotide. 

Significance i  The  data  indicate  that  it  is  possible  tr   prepare  for  practical 
purposes  a  simpler  medium.     Further,   it  is  learned  that  the  substances 
which  can  be  omitted  are  not  essential  nutritional  requirements  for  this 
strain  of  cells  because  their  absence  does  not  result  in  lessening  of  growth 
or  cause  death  of  the  cultures. 

Projected  studies;     Continued  siinplificaticn  of  the  medium  will  be  pursued 
and  the  data  will  serve  tc   act  as  a  basis  for  determining  the  nutritional 
essentials  for  other  normal  and  malignant  coll  strains. 


Page  2 


10«  ^28  (d) 

Serial  Nc, 


11. 


Budget  activity! 


Research     x  Administraticn 

Review  &  Approval      .  ■,  ,  Technical  Assistance 


12.     None 


Cooperating  units  of  the  Public  Health  Service,  or  other  crgrnizationS, 
providing  funds,  facilities,   cr  personnel  for  this  project  in  either 
1956  or  1957 


1 3 . 

If  this  project  resembles,  complements,  or  parallels  research  done  else- 
where in  the  Public  Health  Service  (without  interchrnge  of  personnel, 
facilities  or  funds),  identify  such  research} 

The  organization  of  Dr.  Harry  Ergle  in  the  Microbiological  Institute  is 
also  working  on  cell  nutrition  in  tissue  culture, 

li^»     1^28    (d) 

Serial  No,  ', 


15.     None 


Publications  other  than   abstracts  from  this   project  during  calendar  year 

1955. 


l6  «  None ^     

Honors  and  awards  to  personnel  relating  to  this  project  during  calendar 


Page  1 


PROJECT  REPORT  FORM 


1,  National  Crncer 


Institute 


2,  Biology 


Laboratory  or  Brajich 


3»  Tissue  Culture 


lit     Bethesda,  Md, 


Section  cr  Service 


Location 


Serial  Mo* 


6«     Supplemental  studies  of  the  effect  of  ultrafiltrate  of  whole  chicken-egg 
Project  title  J         extract  on  the  cultivation  of  cells  in  vitro. 


7 «     V«  J.   Eva-ns 

Principal  investigator (s) 


8 ,     W.TtMcQuilkin,   J.  C.  Bryant,  W*  R,  Earle,   K.  K,  Sanford,  M,C.  Fioramonti. 
Other  investigators 


9«  Project  descriptions 

Objective;  To  supplement  the  study  of  ultrafiltrate  of  whole  chicken -egg 
extract  that  was  carried  cut  and  reported  in  19^h>   by  studying  certain 
aspects  of  the  stability  of  the  ultrafiltrate. 

Methods  employed i  Replicate  cultures  of  strain  L  cells  were  cultured 
on  egg  ultrafiltrates  that  had  been  1)  heated  to  6^0  for  1  hour,  2) 
dried  and  reconstituted,  3)  frozen  for  over  two  months  and  ij.)  a  compari- 
son was  made  in  long  term  cultures  between  the  ultrafiltrate  made  from 
fertile  eggs  and  that  made  from  non-fertile  eggs. 

Major  findings;  The  proliferative  ability  of  the  ultrafiltrate  was  not 
diminished  by  heating  cr  by  drying  and  reconstitution.  The  two-^onth 
old  frozen  ultrafiltrate  had  a  proliferative  ability  comparable  with 
that  of  fresh  embryo  extract  medium.  The  fertility  or  non-fertility  of 
the  eggs  used  as  a  source  of  the  ultrafiltrate  appeared  to  make  no  dif- 
ference in  its  proliferative  ability. 


Signif icr.nce:  These  findings  emphasize  the  biological  stability  of  egg 
extract  ultrafiltrate  and  its  potential  usefulness  as  a  substitute  for 
embiyo  extract  in  tissue  culture  media. 


Page  2 


Proposed  crurse  of  projectt  Stock  and  experimental  cultures  of  various 
cell  strains  have  been  and  wi.ll  increasingly  be  carried  on  this  medium, 

10.  128  (e) 

Serial  No» 


11.  , : 

Budget  Activity 5 

Research     x  Administration 

Review  &  Approval  Technical  Assistajice 


12  ,  None 

Cooperating  units  of  the  Public  Health  Service,  or  other  organizations, 
providing  funds,  facilities,  or  personnel  for  this  project  in  either 
1956  or  1957. 


16.. 


13.   None  , , ,  _  . •  "'  • 

if  this  project  resembles,  complements,  or  parallels  research  done  else- 
where in  the  Public  Health  Service  (without  interchange  of  personnel, 
facilities  or  funds),  identify  such  research s 


Hi.       ii28   (o) 


Serial  Mo, 
15 .        In  Preparation 


Publications   other  than  abstracts  from,  this  project  during  calendar  year 

1955. 


Honors  and  awards  to  personnel  relating  to  this  project  during  calendar 
year  1955. 

¥,  T.  McQuilkin  used  this  material  as  a  basis  for  a  thesis  submitted  to 
George  Washington  University  in  partial  satisfaction  of  the  requirements 
for  the  degree  of  Master  of  Arts  in  Zoology,  which  was  awarded  in  October, 

1955. 


Pago  1 
PROJECT  REPORT  FORM 


1,  Naticnal  Cancer 2,  Biclcgy 


Institute  Labcratcry  cr  Branch 

3»  Tissue  Culture li.  Bethesda,  Md. $,     li28    (f) 

Sccticn  or  Service  Lccatirn  Serial  No, 

5,  Develrpment   cf  a  defined  medium  frr  the  cultivaticn  cf  human  skin  cells. 
Project  Title 

7,  V.  J«  Evans  and  W.  R.  Earle 

Principal  Investigator (s) 


5,  See  Itcm'l2«  

Other  Investigators 


?,  Project; description: 

Objective;  To  develop  a  satisfactory  defined  medium  adequate  for  producing 
proliferation  of  the  human  skin  strain,  §Yl6h.m     This  is  essential  for  use 
cf  this  cell  strain  in  problems  of  cajicer  research,  homcgrajCting  and  viral 
research, 

"-  Major  Findings {  Exploratorj''  data  recently  obtained  indicates  that  the  ' 
human  epidermal  cell  strain  #1769.  will  grow  in  the  chemically  defined 
medium  NCTC  109  as  already  worked  out  in  this  laboratory  provided  there  is 
human  or  horse  serum  supplementation  at  low  concentration, 

Significg.ncet  On  these  media,  NCTC  109  previous  exploratory  virological 
studies  have'  been  successful.  For  cajncer  research  and  humaji  transplanta- 
tion work  such  a  defined  medium  is  imperative.  Information  resulting  from 
studies  of  such  a  human  strain  may  also  serve  as  exploratory  data  for  pre- 
paring a  tissue  preservation  medium  for  the  living  cells.  The  implication 
cf  usefulness  in  nutritional  and  meta.bclic  studies  in  relation  to  skin 
cancer  are  ma.nifold  and  obvious. 

Proposed  ocurse  of  project;  a)  With  this  strain  as  with  all  the  others, 
The  essentiality  of  all  the  individual  components  and  component  groups  cf 
medium  NCTC  109  are  being  investigated  tr  improve  the  chemically  defined 
portion  of  the  medium}  b)  Similarly  the  action  cf  the  horse  serum  is  in- 
vestigated to  detcCTiine  its  biochemical  and  biophysical  function}  c)  The 
chemically  defined  media  will  be  used  tr  study  long  term  preservation  cf 
normal  tissue  at  refrigeration  temperatures]  d)  Tr  use  NCTC  109  as  medium 
for  studying  (the  biochemical  products  and     -     the  rejectirn- 
accoptance  phenomenon,  (See  separate  project) 

^0.   U28  (f) 

Serial  No, 


•5561  -ics^ 

3UJN     •91 


•5561 
s'uoN     •5X 


l£li_LEH£S 


jqoj-cosoj:  qons  j^Tq.uop-t:   *(spunj  ao  soTqixxoisj 

*XSuuos:t©d  JO  o2uuqoaoq.uT  q.nji{q.xtt)   ooxAjog  qq.X'eoH  ofl^li^  ^^1  ^  saoiiK 

•0SX9  oujp  qajposoj  sxoxiTSJBd  jo   'sq-ugiuoxdmoo,  'saxq^^osoj  q.09Cjjd   sxq^  JI 

ouoM     'CX 


•  saoq.^§xq.soAux  jjxuss  **I*0*W^^ 

uoxsxAjodns  aopun  xo"^-3s«^Sc!  sjucg  ongsfi  £q  ouop  jCxi^^U'i.oTi  sbw  >iajjM.  sxitq.,jo 

qoTiH     •aeq.uoo  X'^'^TP^W  T^-^^K  I^ujxcj.-aM  *looqog  x^^TP^H  X'-^-^^^M  'Jl-Ucg  onssfi 

*i.56l  -i^ 

95X  iioLtq-xo  ux  q.ooCoj:d  sxi^q.  joj  x^^^^'^saod  aj   *soxq.xxX3i2J  'spimj  SuxpxAjad 

^sujxq.pzxuBgjj  jcqq.j  gj    ^ooXA-iag  qq-X'-^sH  oxxqtij   oqq.  jj  sq.xun  guxq.-o'jgdjjo 


>     uoxq.-aaci.sxuxiirpv                    x          qojcuosoH 
^ ;.A!q.xAxq.O'e  ci.ggpna 


Page  1 


PROJECT  REPORT  FORM 
Naticnrl  Cancer ^ 2,  Biology 


Institute  Laboratory  cr  Branch 

3,  Tissue  Culture k-   Bcthesda,  Md. g.  I;28  (g) 

"Section  or  Service  Location  Serial  No, 

6,  The  effect  of  scrum  fractions  on  the  proliferation  of  strain  L  cells  in  vitro. 
Project  Title 

7.  K.K.  Sanfcrd  and  B.  B.  Westfcll. 

Principal  Investigator (s ) 

S •  W.  R,  Earle,  J,  C.  Bryant,  V,J,  Evans,  E.  V.  Peppers,  M«  C.  Ficramontij  and 
Other  Investigators     W.T,  McQuilkin, 

9»     Project  Descriptions 

Objective t  To  examine  those  coraponents  of  the  large  molecular  portion 
of  serumThat  appear  to  be  essential  for  the  rapid  proliferation  of 
strain  L  mouse  cells  in  vitro,  with  the  ultimate  objective  of  isolating 
components  that  could  be  added  to  a  chemically  defined  medium  for  the 
purpose  of  increasing  the  rate  of  cell  proliferation. 

Methods  emplcyedt  Horse  serum  was  fractionated  by  a  modification  of  Cohn's 
low -temperature  procedure.  Method  No,  10,  The  effect  of  four  serum  fractions 
on  the  rate  of  proliferation  of  a  clone  of  mouse  fibroblasts  was  tested 
in  quantitative  experiments. 

Major  findings:  All  protein  fractions  isolated  from  horse  serum  were  found 
to  promote  growth  of  strain  L  cells.  When  tested  in  a  protein-free  basal 
medium,  the  gross -globulin  fraction  could  be  substituted  for  the  large 
molecular  portion  of  horse  serum  to  yield  comparable  rates  of  increase  in 
cell  numbers.  After  removal  of  the  gamma  globulins  from  the  gross  'globulin 
fraction,  the  residual  globulins  could  also  be  substituted  for  the  proteins 
of  whole  serum  with  only  slight  decrease  in  numbers  of  cells,  A  lower 
rat'e  'of  increase  in  "cell  numbers  was  obtained  with  the  albumin  and  gamma 
globulin  fracti'ons  tested  at  a  concentration  of  1,3  percent;  when  tested 
at  a  higher  concentration,  how'ever,  (2,6^)-  the'  rate  of  increase  in  numbers 
of  cells  grown  in  gamma  globulins  was  the  same  and  in  the  albumins  was  less 
than  that  of  cells  grown  in  the  same  concentration  of  the  unfrattionated 
serum  proteins. 

Significance  to  cancer  research t  Although  a  protein-free  chanically  de- 
fined medium  has  been  developed  for  strain  L  cells,  at  least  four  other 
cell  strains  in  this  laboratory  grow  in  the  defined  mediura  only  when  a 
small  amount  (0,0^  to  1  percent)  of  serum  protein  is  added.  To  pursue 
carcinogenic, metabolic  and  nutritional  studies  on  these  strains,  defined 
serum  fractions  appear  to  be  essential  at  the  present  time. 


Pag©  s 


Prrposed  course  cf  this  project;  ,  Experiments  will  be  continued  testing 
the  relative  value  of  certain  commercial  serum  fractions  prepared. as  ■ 
nearly  as  possible  according  to  the  methods  developed  in  the  present  stu(^, 

10,  U28  (g) 
Serial  Mo, 


11 . . 

■  Budget  activity: 

Research    X  Administration 

Review  &  Approval         Technical  Assistance 

12,     None "     . \ ■  ^'      ' , 

Cooperating  Units  of  the  luijric  Health  Service,   or  other  organizations , pro- 
viding  funds,  facilities,   or  personnel  for  this  project  in  either  1956  or 
1957. 

If.  this;  proj6ot~relemblei3,  ccmplcments,  or  parallels  research  done  elsc- 
where  in  the  Public  Health  Service   (withotit  interchange  of  personnel, 
facilities  or  funds),   identify  such  research: 

The  orgajiization  of  Dr.  Harry  Eafvlc  in  the  Microbiological-  Institute 
is  also  working  on  cell  niitrition  in  tissue  culture. 


13. 


lU,     U28    (g) 

Serial  No, 


15. 


Publications  other  than  abstracts  from  this  project  during  calendar  jcdt 
.1955, 

The  effect  of  ser-um  fracticns   en  the  proliferation  cf  strfein  L  mouse 
cells   in  vitro.     K,  K.  Sriifcrd,   B.  B.  Westfall,  M.G.  F^cramonti,  W,T« 
McQuilkinj  J,  C,  Bryant,  E.V.  Peppers,  V,  J.  Evans  and  WJl,  Earle, 
J,  National  Cancer  Inst,  16j789-802,  1955. 

l6.  None ^ _.____>__^ 

Honors *and  awards  to  personnel  relating" tr  this  project  during  calendar 
year  1955. 


Page  1 
PROJECT   REPORT  F0RI4 
1,     Nationr-1  Cancer      2'i,     Biclcgy 


Institute  Laboratory  or  Branch 

3,     Tissue  Culture It,  Bethesda,  Md, ^5,  k2Q  /y^-^ 

Section  or  Service  Location  oerial  No, 


6 ,  S tudy  of  chemically  defined  mediwii  and  cell  nutrition  for  clone   92.9  cells-, 
Project  Title 

7.  K.   K.  Sanford 

Principal  Investigatcr(s } 


8 ,  M«C.  Fiorajionti,  V/.T.  McQuilkin,  J,C.  Bryant,  V.J.  Evens  and  ¥,R.Earle, 
Sther  Investigators 


9»  Project  descriptiont  Attempts  to  simplify  and  improve  a  protein-free 

chemically  defined  culture  medium,  NCTC-108  for  the  proliferation  of  strain 
L  mouse  cells. 

Objective)  To  determine  which  components  of  the  chemically  defined  mix 
are  essential  for  cell  proliferation  and  to  establish  the  optimal  con- 
centrations of  these  components. 

Methods  employed}  The  effects  of  varying  concentrations  and  depletions  of 
components  on  the  proliferation  of  strain  L  cells  have  been  determined  by 
quantitative  replicate  culture  procedures , 

Major  findings!  Of  the  26  amino  acids,  amines,  and  amides  of  the  protein- 
free  medium  NCTC  108,  those  essential  for  proliferation  of  strain  L, cells 
^  have  been  determined.  From  dose-response  curves  on  those  essentials,  new 
mixtures  have  been  devised  and  are  now  being  tested, 

•An  analysis  is  being  made  of  the  effects  of  the  desojqj^ribosides  on  cell  pro- 
liferation. 

Proposed  course  of  project;   This  project  will  be  ccntinued  until  the 
effect  of  the  dcsoxj'ribosides  and  other  nucleic  acid  derivatives  on, -cell 
proliferation  have  been  established*  The  study  of  the  amino  acid  mixtures 
will  be  completed  scon, 

•U>,  Significance  to  caneer  research;  A  simplified  chemically  defined  medium 
"Is  iof  primary  significance  in  order  to  determine  differences  that  may 
exist  in  the  nutritional  requirements  of  malignant  cells  dji  vitro  as  com- 
pared with  the  normal  cells  from  wi:ich  they  arise. 


10,  U28 


Seri 


ial  No, 


Page  2, 


11. 


Budget  activity { 


Research  x 


Adrainistraticn 


Review  &  Approval 


Technical  Assistance 


12,  Nrne 


13, 


Cccpcrating  units  of   the  Public  Health  Service,  cr  ether  organizations,  pro- 
viding funds,  facilities,  or  personnel  for  this  project  in  either  19$6  or 
19^7. 


If  this  project  resembles,  complements,  or  parallels  research  done  elsewhere 
in  the  Public  Health  Service  (without  JJiterchange  of  personnel,  facilities 
or  funds),  identify  such  researchs 


The  organization  of  Br.  Harry  .Eagle  in  the  Microbiological  Institute 
is  alsr  working  on  cell  nutrition  in  tissue  culture. 


lU.  U28(h) 


Serial  No, 


1^,  None 


Publications  other  than  abstracts  from  this  project  diiring  calendar  year 

1955. 


16,  None 


Honors  and  awards  to  personnel  relating  to  this  project  during  calendar  jqpt 

1955. 


Page  1 
PROJECT  REiX3RT  FORM 
1,     Naticnal  Cancer 2.     Biclcgy 


Institute  Labcratrry  cr  Branch 

3.     Tissue  Culture U.     Bothesda,  Hd, 5.  ii28    (j) 

Secticn  cr  Service  Location  Serial  Nc, 

6 ,  A  study  of  the  preservation  and  storage  rf  strain  L  cells. ' 

Project  Title 

7,  V.  J.  Evans 

Principal  Investigator (si 

8,  W.T.  KcQuilldji 

Other  Investigators 

9,  Project  description; 

Objective t  To  determine  how  long  strain  L  cells  could  be  preserved  at 
5^C  with  no_.  cr  infrequent,  fluid  changes. 

Methods  employed}  Flasks  of  stock  cultures  were  placed  in  the  cold  room 
for  periods  ra-nging  from  three  tr  eight  weeks  during  which  times  the 
medium  was  either  not  changed  at  all  or  was  changed  at  infrequent  inter- 
vals. When  cultures  were  withdrawn  from  the  cold  storage,  they  were  fluid 
chajiged  imraediately  and  then  maintaiiied  in  the  usual  manner  to  determine 
whether  the  cells  wruld  revive. 

Major  findings}  All  cultures  in  cold  storage  fcr  3  weeks  survived  and 
recovered  satisfactorily  -generallj'-  within  one  week  aJTter  removal  from 
cold  room  and  return  to  routine  procedures.  Cultures  could  survive  and 
recover  from  an  additional  three-week  cold  storage  period  if  returned 
to  normal  treatment  fcr  about  a  week  beforehand.  No  cultures  survived 
when  stored  at  ^'^C  fcr  5  weeks  cr  longer  even  with  fluid  changes  at  the 
end  of  the  third  cr  fourth  week. 

Significance;  The  usefulness  of  such  preservation  methods  was  under 
consideration  for  the  mcintenance  of  strains  at  minimal  expense.  This 
work  partially  corroborates  the  work  done  during  the  same  period  by 
Swim  and  Parker  and  reported  in  Prcc,  Sec.  Exp,  Biol,  and  Med,,  vol,  89j 
Aug, -Sept,  1955. 

10.  1428  (i) 

Serial  No. 


Pago  2. 


11; 


Budget  Activity t 


Research    x 
Review  &  Approval 


Administ ration 
Technical  Assistance 


12 ,  None 


Cooperating  units  of  the  Public  Health  Service,  or  other  organizations, pro- 
viding  funds,  facilities,  or  personnel  for  this  project  in  either  1956  or 
1957.  


13.     None 


If  this   project  resembles,    complements,    or  parallels  research  done  else- 
where in  the   Public  Health  Service    (without  interchange  of  personnel, 
facilities   or  ftinds),   identify  such  researcht 


1^.  128  (i) 

Serial  No, 


15. 


Iniblications  other  than  abstracts  frrm  this  project  during  calendar  year 
1955. 


l6.  None 


Honors  and  awards  to  personnel  relating  to  this  prcject  during  calendar 
year  1955. 


Page  1 


PROJECT  REPORT  FORiM 


1,   National  Ca.ncer 2,  Bi elegy 


Institute  Laberatery  or  Branch 

3,    Tissue  Culture li,  Bothcsda,  Md. g.  U28  (j) 

Section  or  Service  Location  Serial  No, 

6,  DeveloFment  of  a  defined  raediuin  for  the  cultivation  of  mouse  liver 

Project  title:    epithelial  cells. 

7 ,  V.  J.  Evans ' ' 

Principal  Investigator (s) 

8 ,  W.  R.  Earle  and  N.  M.  Hawkins ■_ 

Other  Invjstigators 

9,  Project  description: 

Objective;  To  design  and  test  a  defined  medium  which  will  support  prolifer- 
ation cjid  allow  determination  of  effect  of  individual  components  of  the 
medium  on  substrains  of  mouse  liver  cells  of  low  and  high  tumor  incidence, 
(designated  //II4.69  aJid  #1795  respectively). 

Methods  employed;  Replicate  cultures  are  prepared  and  enumeration  of  nuclei 
are  made  after  exposure  of  various  intervals  to  the  defined  media.  Such 
media  are  devised  from  collected  data  on  the  nutrition  of  bacteria  and 
mammalian  and  avian  cells  in   "vitro  and  also  from  the  data  on  physical  and 
chemical  fractionation  of  naturally  occurring  media. 

Major  findings;  The  prototype  media  NCTC  107,  IO8  ,    109  for  strain  L 
cells h'ave  been  found  unable  to  support  continued  proliferation  in  the 
normal  clone  liver  strain  lii69«  However,  the  incorporation  of  serum 
fraction  such  as  gross  globulin  as  well  as,  in  as  little  as  O.Og  gm, 
percent  of  amino  acid  free  horse  serxm  residue  produces  minor  prolifera- 
tion of  the  cells.  The  essential  amine  acids  and  amides  for  this  strain 
have  been  determined.  Exploratory  data  indicate  to  be  identical  with  those 
required  for  strain  L  cells.  VJith  this  medium,  glucose  and  amino  acid 
utilization,  keto  acid  formation,  and  formation  of  lactic  acid  on  both 
the  high  and  low  incidence  tumor  cell  have  been  determined  in  replicate 
cultures,  (See  report  of  B,  B,  Westfall) 

Exploratory  data  from  cultures  of  the  high  tumor  producing  strain  .of 
liver  cells  when  subjected  to  medium  NCTC  IO8  in  which  the  amount  of 
glutajTiine  has  been  decreased  show  almost  twice  the  proliferation  that 
'the  low  tumor  producing  strain  shows. 


Page  2 

Significance  i   A  partially  defined  medi-um  for  studying  the  behavicr  cf 
liver  epithelial  cells  has  been  devised,  A  slightly  mcdificd  medium 
will  support  prclif eraticn  alsc  cf  a  strain  cf  liver  cells  cf  high 
turner  incidence.  This  suggests  a  difference  between  a  ncrmal  and  malig- 
nant cells  in  tissue  culture.  It  must  be  learned  if  this  explcratcry 
data  can  be  confirmed  and  whether  additional  differences  can  be  demcnstratcc 
in  vitro  from  a  nutritional  point  of  view. 

Proposed  course  of  project;  Efforts  iidll  be  mrde  to  substitute  for  and 
augment  the  effect  of  the  0,0^  gm  %   of  amino  acid-free  protein  in  this 
■  medium.  It  is  proposed  tc  do  this  by  introducing  further  purified 
fractions  cf  serum  proteins  and  tc  -supplement  the  medium  w-ith  single 
materials  which  can  be  closely  attached  tc  the  protein  residue.  It  is 
further  proposed  to  continue  tc  demonstrate  nutritional  differences 
between  the  two  cell  lines,  .and  to  constantly  attempt'  tc  devise .media 
free  cf  any  protein  for  these  strains  also, 

10.  Ii28  ( j) 

■   Serial  No,  .    ■      '  ..  .■ 

11. ,^ \ , . 

Budget  activity? 

Research     x         Administration 


Review  &  Approval  ■  Teclmical  Assistance 


12. ^-   _    •  -  •  '"   "  •  '  ••  • ,, 

Cooperation  units  of  the'  Public  Health  Sgrvice,  or  other  organizations, 

providing  funds,  facilities,  or  personnel  for  this  project  in  either 
19^6  or  1957. 


Laboratory  cf  Clinical  Investigation,  National  Micrrbiclogical 
Institute , 


,13,  '  None* 


If  this  project  roserablcs,  complements j  or  parallels  research  done  else- 
where in  the  Public  Health  Service  (without  interchange  of  personnel, 
facilities  or  funds),  identify  such  research:. 


1^*  ^28  (j) 


Serial  Mo, 
l5«  None 


Publications  other  than  abstracts  from  this  project  during  calendar  year 
1955.  ■  ■     , 


16,  None 


Honors  and  awards  tc  personnel  relating  to  this  project  during  calendar 
year  1955. 


Generc-l  Project       I|.29 


Serial  Nc, 


Studios  cf  the  influence  cf  cell  pcpulp-tirn  en  prcliferaticn, 


Page  1 


Project  Report  Form 


-^*  -Ngitiicna,!, riancp.r.  ,.i. 


^ 


stitute 


2. 


BJcJ.r. 


hij  CI  r.gy r~ 

Laboratciy  cr  Branch 


3«     Tissue  Culture 


Secticn   cr  Service 


U»    Bp-h.hp.ciHa  _,    MH-, 


Location 


5*  li29fa) 

Sdrial  No, 


6,     Studies  of  the   influence  of  cell  pcpulr?.tion  on  prclif eraticn« 
Project  Title 


7.  K.  K.  Scnford 

Principal  Investigator (s) 


9. 


G,  L.  Hobbs  and  W,R,  Earle 


Other  investigators 

Project  description;  Single  cell  studies  and  the  development  of  cell 
clones. 


Objective {  To  develop  clones  of  human  skin,  mouse  riypmary  carcinoma, 
and  four  strains  of  mouse  fibroblasts.  Each  of  the^iS clones  is  desired 
for  specific  research  projects  which  will  be  considered  •under  other 
headings.  The  development  of  a  clone  of  mouse  manimary  carcinoma  is  for 
use  in  a  collaborative  study  with  Dr,  H,  B,  Andervont  on  the  relation 
of  the  milk  factor  to  the  m'-.llgnant  cell  in  tissue  culture.. 

Methods  employed;  A  sieved  cell  suspension  is  prepared  and  drawn  into 
capillary  pipettes .  Capillary  segments  are  cut,  each  segment  contain- 
ing one  isolated  cell.  The  segments  are  embedded  in  plasma  clot  in  a 
Carrel  flask,  and  the  culture  fluid  is  renewed  thrice  weekly. 


Major  findings;  Attempts  have  been  made  to  define  more  accurately  the 
conditions  allowing  the  proliferation  of  single  isolated  cells.  Some 
progress  has  been  made. 

Significance  to  Cancer  ResearchtThe  development  of  clones  of  cells  is 
of  fundamental  importance  for  future  studies  in  carcinogenic  cell 
transformations  among  populations  of  cells  in  vitro,  virus  studies,  and 
in  studies  of  the  differences  in  the  physiology  and  nutrition  of  malig- 
nant cells  and  the  normal  cells  from  which  they  arise  in  culture. 


11. 


Page  2 


Proposed  course ■  Renewed  efforts  will  be  made  to  develop  clones  cf 
of  project {      the  several  cell  strains  listed -above,  and  to  define 
more  accurately  the  conditions  that  allow  the  proliferation  of  single 
isolated  cells. 


10.  'h29    (a) 

Serial  No, 


Budget  activity; 

Research    x         Administration 
Review  &  Approval    Technical  Assistance 


12,   None 


"Cooperating  Units  of  tho  Public  Health  Service,  or  other  organizations, 
providing  funds,  facilities,  or  personnel  for  this  project  in  either 
19^6  or  1957.  •      ■ 


13.   None 


If  this  Project  Resembles,  Complements,  or  Parallels  research  dene  else- 
where in  the  Public  Health  Service  (without  interchange  of  personnel, 
facilities  or  funds)  identify  such  research. 


lU.   U29  (a) 


Serial  No, 
1$,       None 


Publications  other  than  abstracts  from  this  project  during  calendar  year 
1955. 


l6 ,   None 


Honors  and  awards  to  personnel  relating  to  this  project  during  calendar 
year  1955. 


.PROJECT  REPORT  FORM 
1,  National  Cgjicer 2.  Biolcgy 


Institute  ■  Laboratory  or  Branch 

3.    Ti.ssne  Ciilti^rfi  .Section . ^^  ^'  -    ■  ■  Rp,t,hP.,sdn.  .    Mfi . _- — ^'      li?.9    (b) 

Section  or  ScrvicG  Location  (if  other  than  B 

6.  Growth  .of  massive  fluid  suspension  cultur'es  of  animal  tissue  cells. 
Project  Title 

7.  W.  R.  Earle ■ .      '  ' 

Principle  Investigator (s)     ~  i 

8.  Jay  C.  Bryant,  E.  L.  Schilling,  B,  B.' Westf all, Ellison  Peppers  and  V, J. Evans. 
Other  Investigators 

9»  Project  Description:  Growth  of  massive  fluid  suspension  cultures  of  ; 

both  normal  and  malignant  animal,  tissue  cells.  Application  of  these 
methods  in  defining  factors  resDcnsible-  for  proliferation  of  represent- 
ative tissue  cell  types  in  such  .cultures ,' 

Objectives}  The  general  objective  of  this  project  is  to  develop  methods 
and  equipment  for  growing  massive  fluid  suspensions  cultures  of  animal 
tissue  cells.  Factors  receiving  attention' in ' the  attainment  of  this 
objective  include  particularly  various  types  of  fluid  media,  control' of  , 
air  inflow,  control  of  pH,' and  regulation  of  volume  and  compositions  of 
culture  fluid  in  relation  to  nvLmbors  of  cells  arid  rate  of  proliferation. 

Methods  Employed;  The  standard  Brxmswick  type  platform  shaker  enclosed  in  . 
an  incubator  box,  which  had  been  developed  previously,  was  used  for  con- 
tinuing studies  with  massive  cultures.  The  shaker  was  operated  at  about 
12,000  revcilutions  per  hour,  and  the  size  of  culture  flask  used  was  l|-  liter. 
Continuous  aseptic  flow  of 'a  gas  mixture  cf  S%   GO2,  20^  cxygen  and  7$t  nitro- 
gen, saturated  with  water  vapor,  was  maintained  through  each  culture  flask. 
The  rate  of  gas  flow  through  each  flask  was  held  constant  at  either  llj.0  or 
280  ml,  per  hour.  Phenol  red  at  a  concentration  of  ,002  percent  was  used  in 
all  of  the  culture  fluid,*  as  a  pH  indicator.  The  fluid  was  either  renewed 
periodically  without  appreciable  loss  of  cells  or  was  increased  by  increments 
of  fresh  fluid.  The  rate  of  proliferation  of  cells  in  suspension  was  deter- 
mined periodically  by  nuclei  counts  of  carefully  sampled  aliquots. 

Major  Findings:  Considerable  progress  has  been  made  during  the  year  in 
attaining  better  control  of  the  major  factors  governing  the  growth  of  cell 
suspension  cultiires  in  shaker  flasks.  With  all  cells  used  the  concentra- 
tion of  glucose  in  the  medium  has  been  increased  up  to  several  times  the 
coneentration  in  Earle 's  normal  saline  in  order  to  maintain  cell  growth 
during  intervals  between  fluid  changes  unlimited  ty  exhaustion  of  glucose. 


General  Project  h30 


Serial  Mc. 
Studies  en  the  cultivation  of  epithelial  cells* 


Page  1 
PROJECT  FORM  REPORT 


1.  National  Cancer ^^   2,  Biolcgy 


Institute  Laboratory  cr  Branch 

3.  Tissue  Culture U.  Bethcsda,  Md, 5.   U30(a) 

Section  or  Service  "Tccaticn  Serial  No, 

6,  Stiidy  of  a  strain  of  H-uman  Liver  Cells « 

"^  "'Prc3e5t;  Title: 

7,  V.  J.  Evans . 

Principal  Investigator (s) 

8 ,  N,  M,  Hawkins,  W.  R,  Earle,  and  B,  B.  Westfall ^ 

Other  Investigators 

9»   Project  Description: 

Objective:  To  cultivate  a  strain  of  hvu-aan  liver  cells  for  long  term 
stiidies  for  ultimate  ccmpariscn  of  normal  human  cells  and  malignant 
cells  derived  from  it. 

Methods  Minced  human  liver     prepared  by  trypsin  treatment  together 
with  stirring  and  subsequent  tissue  culture  cultivation  in  human  serum 
and  chick  embryo  extract,  has  yielded  a  strain  of  cells. 

Major  findings:  For  the  first  time  a  hximaji  liver  strain  gives  some 
promise  of  continuing  tr  grow  in  vitro »  This  is  contrary  to  data  from 
■  ^  cr  6  other  endeavors  tc  cultivate  human  liver  for  any  prolonged  inter- 
val. To  date  this  strain  of  cells  subcultures  readily  in  fluid  suspension 
and  exploratory  data  indicate  that  it  may  grew  in  shaker  cultures  thus 
facilitating  metabolic  studies. 

Significance  to  Cancer  Research;  In  addition  to  significance  tr  cancer 
research  the  strain  of  cells  should  be  an  invaluable  tccl  to  virc-logists . 
Techniques  developed  in  establishing  this  strain  are  of  usefulness  in 
establishing  human  liver  strains  for  comparative  studies  in  the  pl:ysiology 
of  the  normal  and  malignant  cell. 

Proposed  course  of  project:  Since  adequate  riiicunts  of  tissue  and  fluids 
are  obtained  from  the  large  culture  those  -urill  continue  tc  be  used  for 
glucose  and  glycogen  analysis  and  for  comparison  with  similar  studies  on 
the  ether  stx*ains  of  mcuso  and  human  epithelial  cells  in  the  laboratory. 
Attempts  tc  produce  a  malignant  transformation  of  this  cell  in  vitro  will 
be  made.  Since  the  HeLa  strain  of  cells  from  a  human  cervical  carcinoma 
has  given  rise  tc  small  identifiable  grotrths  in  the  anterior  chamber  of 
the  mouse  eye  similar  techaniques  may  be  worthy  of  consideration  for  other 
strains  of  cells  developed  and  in  particular  for  this  human  liver  epithel- 
ium,   :^   '-rain  of  cells  will  be  cloned  as  scon  as  feasible. 


Page  2, 


10«   f}30  (a) 

Serial  Nc, 


11«  ^  ■ 

Budget  Activity? 


Research     x  Administraticn 

Review  .  &  Approval  Technical  Assistance 


12.   None 


Cccperating  Units  cf  the  Public  Health  Service,  or  other  organizations, 
providing  funds,  facilities,  or  personnel  for  this  project  in  either  1956 
or  1957. 

13.  None 

If  this  project  resembles,  ccmplements,  or  parallels  research  done  else- 
where in  the  Riblic  Health  Service  {without  interchange  of  personnel,  facil- 
ities or  funds),  identify  such  research: 

la.     ^30  (a) 

Serial  No.  .   . 


15.     Hone . ^ " ^ 

Publications  other  than  abstracts  frciii  this  project  during  calendar^  year  1955 


16,    Mone ^ 

honors  and  awards  to  personnel  relating  tc   this  project  during  calendar  year 


1955. 


PROJECT  REPORT  FORM                  ^^S®  ^ 
1.  National  Cancer 2,   Biology, 


6. 


INSTITUTE  UBOR/iTORY  OR  BRANCH 


3»     Tissue  C-ulture ll,     Bethesda,  Md. 5.     1|30  (b) 

Section  or  Service.  Location  .  .  Serial  No, 


Preservation  cf  culture  cells. 


Project  Title 


7.     V^  J.  Evans  and  W.  R.  Earle, 
Principal  Investigators. 


See  Item  12 

Other  investigators. 


9»  Project  description:   Now  that  cells  can  be  cultures  in  substantial 
amounts  is  it  desirable  to  be  able  to  preserve  these  cells  in  a  latent 
state,  for  extended  periods  cf  time.  The  most  premising  method  reported 
to  date,  has  been  reported  by  Polge,  C.  (Nature  l6i|j  666;  l9i;9)  who 
used  glycerine  impregnation  for  pjreservaticn  of  bull  spermatazoa,.  The 
Tissue  Bank,  Naval  Medical  School  has  utilized  this  method  for  the  pre- 
servation cf  skin  and  cornea.  It  is  desirable  to  learn  the  length  of 
preservation;  whether  this  inexpensive  material  (dry  ice  and  glycerine) 
is  satisfactory;  and,  whether  this  material  is  less  toxic  to  cells  than 
other  materials.  Tissue  culture  viability  of  preserved  tissue  will  serve 
as  the  index  of  the  physiological  conditions  of  the  cells. 

Methods  employed;  Rabbit  comea  cultures  were  planted  in  a  thin 
plasma  clot  under  perforated  cellophane  in  Carrel  3.5  flasks,  A 
nutrient  medium  of  horse  serum  and  which  embryo  extract  lA  Earle's 
saline  was  used.  One  half  of  a  rabbit  cornea  was  used  in  each  culture 
flask.  Initially  fresh  medium  and  wore  planted  within  30  minutes  from 
the  time  of  removal  from  the  eye.  Soaked  corneas  were  placed  in  test 
tubes  containing  1^%   USP  glycerine  by  volume  ±a   Earlets  saline>  or, 
in  Ringer's  saline  for  1  hour.  Division,  explantation  and  incubation 
of  unfrozen  corneas  took  place  within  an  hour.  Frozen  comea  were 
frozen  either  with  (l)  no  soaking,  (2)  after  soaking  in  glycerine  in 
Earle's  balanced  saline  for  1  hour  and  (3)  after  soaking  in  1$%   glycer- 
ine in  Ringer's  saline  for. 1  hour.  The  excess  fluid  was  decanted  and 
■  the  tubes  of  corneas  were  first  immersed  in  a  container  of  carbon 
dioxide  alcohol  slush  at  -760C  for  3  minutes.  At  the  end  of  1  hour 
■  at  -76°C  the  tubes  containing  corneas  were  removed,  thawed  by  immers- 
ing in  a  water  bath  at  38'^C  and  corneas  were  divided,  explanted  and 
incubated , 


rage  x 


Page  2 


In  the  exploratory  skin  study  to  date,  the  prccediires  were  approxi- 
mately similar  to  that  used  to  the  cornea  study,  except  that  human 
senm  was  substituted  for  horse  serum,  and  the  skin  used,  was  of    ... 
human  origin. 

Major  findings  t  In  the  study  on  rabbit  comea.  the  fresh  cornea 
showed  excellent  migration  of  epithelial  and  fibroblastic  cells  in 
all  instances  within  U8  hours.  Corneas  which  were  soaked  in  dilute 
glycerine  or  soaked  and  then  frozen  showed  a  slight  lag  in  migration 
but  were  soon  indistinguishable  in  migration  and  cell  appearance  from 
fresli  corneas.  Corneas  frozen  without  glycerine  protection  showed  no.' 
migration  in  28^  of  the  cult\ires.  The  remainder  showed  retarded  mi- 
gration and  severe  cell  injuzy.  Corneas  preserved  by  the  1$%   gly- 
cerine, in  S^%  Earle's  saline  by  volume,  and  frozen  and  preserved 
at  -76OC.  have  been  fctmd  tc  be  viable  in  tissue  culture  up  to  6     s 
months.  Exploratory  data,  obtained  tc  date  on  skin  preserved  ty  these 
methods  does  not  appear  tc  be  viable  in  tissue  culture. 

Significance ;  Before  beginning  the  study  the  feasibility  of  using 
human  corneas  was  considered.  Since  insufficient  numbers  could  be 
obtained,  the  rabbit  cornea  was  selected  since  most  experimental 
work  to  date  has  used  this  source.  The  tissue  viability  test  was 
used  to  test  the  physiological  condition  of  corneas  preserved  by  the 
above  methods  because  it  was  considered  of  greater  critical  useful- 
ness than  either  the  clarity  of  the  graft  after  transplantation  or 
tests  of  the  respiratory  enzyme  systems.  The  results.  :0f,  the  study 
demonstrated  for  the  first  time  that  rapidly  frozen  .glycerine  im- 
pregnated corneas  were  consistently  viable,  -Fovr  -factors  rather  than 
any  independent  one  appears  to  be  responsible  for  thisj  (l)  glycer- 
ine soaking,  (2)  fast  freezing,  (3)  fast  thawing  and  {k)   the  use  of 
a  highly  buffered  balanced  physiological  saline,  such  as  Earless 
saline  .rather  than  Ringer's  Salfjie, 

.Corneal  endothelial  cells  (which  are  reqixired  in  a  viable  state  for 
successful  keratoplasty)  may  assist  in  establishing  criteria  for  the 
preservation  of  cells  cultured  in  , large  masses  and  stored  in  glycer- 
,ine.  If  it  can  also  be  demonstrated  that  corneal  endothelial  cells, 
which  are  preserved  by  glycerine  impregnation  and  stored  at  dry  ice 
temperature,  exhibit  viability  in  vitro,  possibly,  cells  of  less 
specific  function  may  be  preserved  'in  a  like  manner.  The  human  skin 
which  has  been  preserved  by  a  modified  glycerine  method  serves  well 
as  a  clinical  homograft  dressing.  In  spite  of  the  failure  to  date  to 
establish  viability  in  vitro,  it  still  appears  important  to  learn 
whether  or  not  improvements  in  methods  of  preservation  of  skin,  so 
that  viability  in  vitro  is  demonstrable,  will  yield  a  superior .dress- 
ing. 

Serial  No, 


Page  3 


11. 


12. 


Budget  Activity 

Research       x         Administration 
Review  &  Approval         Technical  Assistance 

Cccperating  Units  of  the  Public  Health  Service,  or  other  organizations, 
providing  funds,  facilities,  or  personnel  for  this  project  in  either 
19^6   or  1957. 

a)  Tissue  Bank,  b)  Dept.  of  Ophthalmology,  c)  Naval  Medical  Research 
Institute,  National  Naval  Medical  Center, 

13.  None 

If  this  Project  Resembles,  Complements,  or  Parallels  Research  Done 
Elsewhere  in  the  Public  Health  Service  (without  interchange  of  person- 
nel, facilities  or  funds),  identify  such  research. 

Jh,   U30  (b) 


Serial  Mo, . 

15. 


Publications  other  than  abstracts  from  this  project  during  calendar  year 
1955. 

The  viability  of  fresh  and  frozen  corneas  as  determined  in  tissue 
culture.  Mcpherson,  S.  D,,  Draheim,  J.  W.,  Evans,  V,  J,  and  W.  R, 
Earle,  American  J,  of  Ophthalraclcgy  -  In  press. 


16,  None 


Honors  and  Awards  to  Personnel  relating  tc  this  Project  During  Calendar 
Year  1955. 


Page  1 
PROJECT  FORI!  REPORT 


1,  National  Cancor        2,  Biclcgy 


Institute  Lc.bcratcry  cr  Branch 

3,  Tissue  Culture  il.  Bethesda.  Md, 


S.   h30  (o) 

Serial 'Nc 


Sccticn  cr  Service  Lccaticn  Serial  Nc 

6,  Studies  en  the  cultivaticn  of  human  cndccrine  secreting  tissue  in  vivo, 

SojecT~Title 

7,  V.  J.  Evans 

"Principal  Investigator  (s ) 

8,  N.  M.  HaiJkins,  W.  R.  Earle 

ether  Investigators 

9»  Project  Description: 

Objective :   To  cultivate  cells  of  endocrine  tissue  as  made  available 
by  the  EnH'ocrinclogy  Branch  of  the  National  Cancer  Institute  in  order 
to  learn  the  extent  and  duration  ci  hormone  secretion  of  isolated  cells 
in  vitro. 

Methods  employodi  Additional  islet  cell  tumor  tissue  from  the  pancreas 
of  a  clinically  diagnosed  oasc  of  carcinoma  of  the  pancreas  has  been 
obtained  from  the  Endocrinology  Branch,  of  the  National  Cancer  Institute, 
Dissociation  of  the  tissue  tc  small  but  multiple  colonics  for  explanta- 
tion  of  cultures  has  been  done.  This  method  has  been  successful  for 
cultivation  of  other  humaji  epithelial  cell  strains. 

Major  findings}  Presently  this  new  strain  of  cells  is  still  in  an  early 
stage  of  cultivation  in  vitro. 

Significance  to  cancer  research;   The  development  of  techniques  for 
growing  human  endocrine  tissues  in  vitro  so  that  they  continue  to    _  ■ 
function  should  have  considerable  practical  value  for  therapy.  From  the 
point  of  view  of  cancer  research  any  endocrine  assays  or  data  from  histo- 
chemical  assays  should  serve  as  valuable  indices  of  the  changes  in  cells 
in  vivo  and  in  "^{itro  both  in  clinical  and  experimental  conditions. 

Proposed  course  of  project:  Experiments  will  be  continued  tc  culture 
various  tissues  of  endocrine  origin  as  they  are  made  available  from 
services  in  the  Clinical  Center  and  other  sources.  Methods  must  be 
developed  for  culturing  such  highly  specialized  cells.  The  duration  and 
amount  of  hormone  secretion  and  study  of  the  morphology  of  the  tissue 
growth  V7ith  photographic  recordings  will  be  made.  Histochemistry,  bio- 
chemical analyses  and  morphology  and  mouse  chajiiber  techniques  will  be 


Page  2 


employed  and  correlated  with  function  and  therapy.  Attempts  will  be  made 
to  devise  new  methods  of  assay  of  fiinction  by  grafting  as  suggested  by 
mouse  chajnber  techniques  and  as  justified  by  the  growth  of  cells  in  vitro, 


10.  U30  (c) 


Serial  No, 


11  • 


Budget  Activity} 

Research 


Administration 


Review  and  Approval 


Technical  Assistance 


12. 


Cooperating  Units  of  the  Public  Health  Survicc,  or  other  organizations, 
providing  funds,  facilities,  or  personnel  for  this  project  in  either  1956 
or  19?7. 

Endocrinology  Branch,  National  Cancer  Institute,  and  Transparent  Chamber 
Unit,  Laboratory  of  Biology,  National  Cancer  Institute, 


13.  None 


If  this  project  resembles,  compleiiionts,  or  parallels  research  done  else- 
where in  the  Public  Health  Service  (without  interchange  of  personnel, 
facilities  or  funds),  identify  such  research: 


Hi'.  U30  (c) 

Serial  No, 


15.  None 


PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR 

1955. 


16.  N6ne ' , 

Honors  and  Awards  to  Personnel  Relating  to  this  Project  during  Calendar 
Year  1955. 


Page  1 
PROJECT  REPORT  FORI>^ 
1,   Naticnal  Cancer 2,  Biolc^ 


Institute  Laboratory  or  Branch 

3t   Tissue  Culture It,  Bethesda,  Md.  5»  I|.30(d) 

"Section  or  Service  Location  "  SeriaT'No, 

6. 


7. 


A  study  of  long  term  human  skin  strains. 

Project  Title 

V.  J.  Evans  and  W.  R.  Earle 

Principal  Investigators 

..tn  ■■>,:■;; 

See  item  12, 

Other  investigators 

9.   Project  Descripticnj   Objective:  a)  To  produce  and  maintain  long 
term  human  epthelial  cell  tissue  culture.  This  will  tend  to  an 
understanding  of  tissue  culture  cell  metabolic  needs  as  a.',  preliminary 
to  transplantation  studies,  b)  To  determine  by  continuous  in  vivo 
transplantation  of  cells,  the  possibility  of  spontaneous  in  vitro 

—  malignant,  transformation  of  human  skin  cells. 

Methods  emplc^^ed  j  a)  Human  skin  from  a  65  year  old  man  obtained  by  • 
the  Tissue  Bank,  Naval  Medical  School,  was  separated  into  dermal  and 
epidermal  elements,  A  cell  suspension  of  epidermal  elements  was  planted 
in  human  sei'um  and  has^bisen  carried  in  vitro  now  for  over  2  years,  A 
subline  ^^ras  established  in  horse  serum  for  experimental  work  where  human 
serum  is  undersirable,  b)  Shaker  studies  were  established  according 

—.to  the  procedures  of  Earle,  Biyaiit  and  Schilling, (c)  The  strain  1769, 

'^''^ "*'•■•  human  skin  has  been  implanted  in  the  anterior  chamber  of  the  G3H  mouse 
-  eye,  and  the  pouches  of  golden  hampster.  Mice  were  irradiated  and 
cortisine  treated,  as  described  by  Toolan. 

Major  findings:  Human  skin  has  been  successfully  cultivated  over  two 
years  m  human  soru.m.  The  subline  on  horse  serum  has  been  carried  now 
for  over  6  months.  The  details  for  preparation  of  the  strain,  descrip- 
tion of  the  cells  and  related  use  of  the  cells  have  been  reported  in 
a  manuscript.  In  exploratory  studies  the  strain  on  human  serum  when  in 
■the  presence  of  vi.able  homogenous  tissue  will  elicite  what  appears  to 
be  a  rejection-acceptance  phenomenon  (see  report  on  rejection-acceptance 
"  phenoraen  in  vitro),  b)  The  complete  detail  of  methods  employed  vnd 
size  of  cultures  attained  in  the  report  of  Earle,  Bryant,  and  Schilling, 
"Growth' cf '.massive  fluid  suspension  cultijres  of  animal  tissue  cells". 
Fluid  and  cells  from  these  were  made  available  to  Dr.  lAfestfall  (see 
report  of  Westfall  and  Peppers)  for  studies  on  utilization  of  glucose, 
storage  of  glycogen  and  production  of  keto  acids,  etc,  c)  No  overt 


Page  2 


turners  were  found  in  the  anterior  chamber  cf  the  mouse  eye  or  in  the 
hampster  mcuth  pcuch.  This  '^as  contrary  to  the  findings  cf  tumor 
tissue  when  HeLa  (Human  cervical. carcincma)  was  implanted. 

Significance;  For  the  first  time  a  strain  rf  human  skin  cells  has  been 
cultured  in  vitro  for  over  twr  years  and  continues  to  grow  luxuriantly, 
resembling  morphologically  epithelial  cells,  Nimiercus  types  of  studies 
have  been  made  with  it.  It  is  known  to  support  some  l5  viruses.  The 
strain  can  be  grown  in  amovints  adequate  for  metabolic  studies.  It  has 
been  possible  to  initiate  problems  related  tc  hcmotrans plantation. 
The  cells  have  possible  application  in  clinical  use,  particularly  in 
reparative  surgery.  They  may  have  additional  significance  in  investi- 
gations on  cancer  therapy,  both  because  it  supports  viruses,  and  as 
a  stable  strain  the^,-  may  be  of  value  in  screening  chemotherapeutic  agents, 
Comparative  studies  on  their  metabolism  with  that  cf  epithelial- cancers. 
might  be  significant. 

Proposed  course  of  study t  Tc  continue  studies  on  the  nutrition  and  metab- 
olism of  the  cells  for  ultimate  use  in  clinical  work.  To  confirm  studies 
on  homcgraft  response  and  to  continue  studies  on  large  shaker  flasks  tc 
facilitate  the  above. 


10,   J|30  (d) 


Serial  No, 


11. 


Budget  activity: 

Research      x 
Review  &  Approval 


Administrative     x 
Technical  Assistance 


12. 


Cooperating  units  of  the  Public  Health  Service,  or  other  organizations, 
providing  funds,  facilities,,  or  personnel  for  this  project  in  cither 
19^6  or  1957 


Much  of  the  work  on  this  problem  has  been  dcno  ly -personnel  of  the 
Tissue  Bank,  Naval  Medical  School,  National  Naval  Medical  Center, 

13  »    None '  

If  this  project  resembles,  complements,  or  parallels  research  done 
elsewhere  in  the  I-\iblic  Health  Service  (without  interchange  cf  per- 
sonnel, facilities  or  funds)  identify  such  research; 


Page  3. 


1^»     |f30  (d) 

Surial  Mc, 


15. 

FYiblicriticns   rthjr  than    abstracts   frcra  this  project  during  calendar  year 

Perry^   V.  P.,  Evans,  V.   J,,   Earle,  W.  R.,   Hyatt,  G.  ¥.   and  Bedell,  W,C,- 
Lcn,;;;  Term  Tissue  Cultxire  of  Human  Skin,     Am,   J,   cf  Hyi^iene,   Jan,        19^6. 

Bassett,  C.A.L,,  Evans,   V,J,,  CaTipbcll,   D,  and  Earle,  W.  R.   -Character- 
istics and  Potentials   rf  Lcnn-term  Culture  of  Human  Skin.     N,M,R,   I, 
Project  Report  00?   081.10. 11. 

l6.     Secretary  of  Navy  Cominendation  Medal  tc  Hospitalni?Ji  Vernon  P.   Perry  IC  for 
for  his  participation  in  this  work  as   carried  en  under  our  supervision  in 
the  Tissue  Culture  Section^   Laboratory  of  Biology, 


PROJECT  REPORT  FORM 


1,  Naticnal  Cancer 2.  Biology 


Institute  Laboratory  or  Branch 

I,  Tissue  Culture it.  Bethesda,  Md. ,    5«  U30  (e) 

Section  or  Service  Location  Serial  No, 

6 ,  To  adapt  strains  of  cells  to  media  of  heterologous  compos ition , 

Project  Title 

7,  V«  J.  Evans, 

Principal  Investigator (s ) 

8,  W.  R.  Earle  and  see  item  12 

Other  investigators 

9«  Project  Description < 

Methods  a)  Present  experimental  studies  include!  subjecting  human  malig- 
nant  cells  and  huraan  cells  from  non-malignant  skin  to  media  of  various 
combined  mixtures  of  human  and  horse  serum  until  horse  serum  alone  could 
be  used.  The  cells  were  maintained  in  Carrel  D-3.5  flasks  and  Earle  T-30 
and  T-60  flasks.  After  intervals  of  120  hours  or  more,  the  less  foreign 
and  it  is  presumed  less  toxic  serum  concentrations  permit  subculturing  of 
substrains  of  cells  for  study. 

Major  Findings:  It  has  been  found  possible  hy   the  method  described  above, 
that  cultTores  of  human  malignant  tissue  (HeLa  l88l)  initially  propagated 
in  human  serum,  can  human  scrum,  can  be  adapted  to  heterologous  horse 
serum  medium  to  produce  HeLa  strain  1985.  By   a  similar  procedure  a  strain 
of  normal  human  skin  epithelium  grown  in  human  serum  and  designated  strain 
1769  has  been  adapted  to  horse  serum.  This  adapted  strain  was  designated 
strain  2198.  Both  horse  serum  adapted  strains  have  been  found  of  usefulness 
in  homografting  problems  in  mouse  chamber  work  (see  report  Algire  et  al.), 
and  for  cultivation  of  hujiian  viruses  (see  report  Habel  and  JlcBride,  National 
Microbiological  Institute). 

Slgniflcancei  Both  these  strains  have  already  been  found  of  significance 
in  studies  of  cultivation  of  human  viruses,  (Strain  1985  has  already  been 
used  in  studies  on  mechanisms  of  homografting.  The  mechanism  of  adapta- 
tion should  be  an  interesting  situation  for  study  by  the  immunologist  in- 
terested in  the  problem  of  homografting  as  well  as  by  the  protein  chemist, 
Habel,  Gregg  ajid  McBride  of  the  Microbiological  Inst,  and  Dental  Listitute 
reported  results  on  large  numbers  of  horse  serum  adapted  cjiltures  submitted 
to  them,  from  this  laboratory,  In  one  experiment  cells  --dapted  to  horse 
serum  were  less  susceptible  than  those  grown  in  human  serum  and  less  sus- 
ceptible thaji  fresh  kidney  to  poliomyelitis  virus.  However,  a  second  ex- 
periment indicated  rn  equal  susceptibility  of  all  three  types  of  tissue' 
culture  to  the  three  types  of  policmyse litis,  HeLa  cells  grown  in  horse 


serum  were  just  as  responsive  to  pclio  virus  isolated  from  23  separate 
stock  emulsions  of  polio  virus  as  those  grown  in  human  serum. 

HeLa  cells  grown  in  horse  serum  in  suspended  cell  shaker  type  of  culture 
and  then  carried  in  a  maintenance  medium  for  2[i  hours  pnd  tested  for  pclio- 
iryeletis  as  efficiently  as  those  grown  in  human  serum.  This  indicates  that 
viruses  are  able  to  propagate  in  cells  growing  in  the  sho.kor  type  culture 
and  an  advantage  is  gained  where  hcrse  serum  is  used  both,  because  of  its 'i* 
availability  and  because  the  specific  antibiodies  of  hum&i  serum  may  be 
eliminated. 

Projected  plant  Further  study  of  problems  of  homografting  in  mouse  diffu- 
sion chambers  with  both  strains  of  cells  and  their  respective  substrains 
to  determine  likenesses  and  differences  in  behavior  will  be  made, 

10.  It30  (e) 
Sbriar"No, 

11,  ^wm^'^^  ' 


16. 


Budget  Activity: 

Research      x  Administration 

Review  &  Approval        '    Technical  Assistance 


12. 


Cooperating  Units  of  the  Public  Health  Service,  or  other  organizations, 
providing  funds,  facilities j  or  personnel  for  this  project  in  either  1956 
cr  1957 

Laboratory  of  Infectious  Diseases,  National  Microbiological  Institute; 
Tissue  Bank,  Naval  Medical  School,  National  Naval  Medical  Center;  Trans- 
.  parent  Chamber  Unit,  Laborafrry  of  Biology,  NCI 


13.,  None 


If  this  .Project  Resembles,  Ccmplomonts,  cr  Parallels  Research  done  elsGwhon 
in  the  Public  Health  Service  (without  iiitcrchans^^c  of  personnel,  facilities 
or  funds),  identify  such  research: 


Publications  other  than  abstracts  from  this  Tofoject  during  calendar  year 
1955.         ,.  ., 

Perry,  V.P.,  Evans,  V.  J,  and  Earlc,  VJ, -'R.   Cultivation  cf  large 
cultures  cf  HeLa  Cells  in  hcrse  serum.  Science  121$ 8o5,  1955. 


Honors  rnd  awards  to  personnel  relatin,-;,'  tc  this  project  during  calendar 
years  1955. 


Page  1. 


PROJECT  REPORT  FORM 
1,   National  Cancer  2,'   Biology 


Institute  Laboratory  or  Branch 

3.   Tissue  Culture ii,    Bethcsda,  Md,. 5.  J^30(f ) 

Section  or  Service  Location  Serial  No, 

6,  Studies  on  the  utilizaticn  of  a  long  term  strain  of  skin  epithelium  for  pro- 

Project  Title    pagation  of  virus, 

7,  V.  J.  Evans, '^ '2 

Principal  Investigator (s) 


¥,  R.  Earle.  -  Also  see  item  12 


Other  Investigators 


Project  description;  To  test  whether  hu.man  skin  epithelium  will  support 
cultivation  of  certain  human  viruses. 

Method  -  More  than  2000  roller  tube  cultiure  of  a  long  term  strain  of  humrji 
skin  "cells  from  a  6$   year  old  man  were  studied  for  cytopathogenic  effects 
and  production  of  complement  fixing  rntigens  when  exposed  to  numerous  sig- 
nificant viruses . 

■Major  Findings }  To  date  those  viruses  apparently  multiplying  in  this  cell 
strain  are;  adenoid?!  pharyngeal  conjunctival  viruses  types  1,  2  and  3j 
Coxsacki  virus  tjpo   B-3j  herpes  slmplexj  B  virus j  vaccinia j  mvimpsj  en- 
cephalcnsrocarditios  virus j'  lymphocytic  choriomeningetis  virus;  St,  Louis 
encephalitis  virus;  and  yellow  fever  virus. 

Significance;  This  strain  of  ^^ells  for  virus  studies  has  significance 
because  of  its  being  an  epithelial  coll  of  human  origin.  Many  viruses 
have  not  been  propagated  in  vitro  since  this  cell  strain  grows  luxuriantly 
in  large  amcunts,  and  maintains  many  viruses  in  rapid  proliferation,  it 
offers  substantial  potentialities  both  as  a  basic  research  tcol  and  as  a 
practical  basis  in  large  scale  p«^:ducticn  of  viruses.  Already,  this  cell 
strain,_  coji  be  maintained  in  a  defined  medium  supplemented  with  only  limited 
amcunts  of  serum  for  these  specific  studies  on  viral  propagation.  In  other 
cases,  where  desired,  an  entirely  heterologous  serum  may  be  used. 

This  tissue  may  be  assumed  to  be  non-malignant  until  exhaustive  studies  prove 
otherwise. 


12. 


16. 


Page  2, 


Prcposed  course  rf  project:  Exploratory  data  on  the  cultivaticn  of  this 
strain  cf  cells  in  mass  amounts  in  the  shaker  type  culture  will  be  con- 
firmed for  other  studies  but  the  obvious  value  of  this  strain  of  cells 
from  a  normr.l  tissue  source  useful  for  vaccine  production  is  suggested 
for  consideration  by  virologists,  end  will  not  be  studied  by  our  group, 
Continuaticn  of  efforts  to  demonstrate  any  potential  malignancy  must  be 
pursued.  Methods  presently  available  for  doing  so  will  be  tested  and  new 
ones  will  be  sought.  Methods  of  controlling  possible  development  of  malig 
nacy.  in  tissue  culture  must  be  tried  and  their  validity  tested. 


10.  h30    (f) 


..  Serial  No, 
11 •     None 


Budget  Activity: 

Reserxch  x  Administratirn 

Review  &  Approval  TechnJ.cal  Assistance 


Gcoperating  Units  of  the  Public  Health  Service^   or  ether  orgrnizaticns^ 
TDrcvidin?  funds,   facilities,   or  perscnnel  for  this  project  in  either  1956 
ov  10^7  / 

L.-^.bci-c  tc?.y  cf  Infectious  Diseases,  National  Hcrphr logical  Institute^ 
Clinical   Investigation,   National  Institute  cf  Denta]   Research;  Tissue 
E,.\nk  Naval  Medical  School,  National  Naval  Medical  Center.     Over  2000 
cultures  have  been  furnished  the  first  twr   of  those  for  virological  re- 
search: 


13-.       N-nc 


It   tiiis  pr.-.^GcL  resembles,   ccmpl'j.ients-    or  piv-diGis  research  done  elsc- 
wnere-in  the  "^nblic  Health  Service   (without   ii;.terchange  of  personnel, 
facilities   cr  funds),    identify  such  research. 


lit.   U30  (f) 

Serial  Nc , 


15. 


fublicaticns  other  than  abstracts  from  this  project  during  calendar  year 
1955. 

Long  Term  Tissue  Culture  of  Human  Skin,  Perry,  V.  P,,  Evans,  V,  J,, 
Errie  /J.  R,.  Hyatt,  G,  W,,  Bedell,  \i,   C.  Am.  J.  of  %giene,  (Jan) 
1956.' 


Honors  fnd  awards  tc  personnel  relating  to  this  project  during  calendar 
year  1955. 


Page  1 
PROJECT  REPORT  FORM 


1,  National  Cancer  2,     Biology 


Institute  Laboratory  or  Branch 

3 •  Tis3»e  Culture  ___;_  _ _  _  -V- - h .Bethesda,  Md. ^ .h30  (g) 

Section  or  Service     "  Location  Serial  Mo, 

6,  Rejection-Acceptance,  phenomenon  in  vitro, 

Project  Title 

7,  V.  J.  Evans  and  W.  R.  Earle 

Principal  Investigator (s) 

8,  See  item  12, 

Other  Investigators 

9,  Project  description t  Tc  determjne  if  viable  and  non -viable  tissue  frag- 
ments  in  vitro  will  elicite  an  in  vivo  homog raft -like  reaction. 

Method:  Fresh  and  freeze  dried  tissue  have  been,  procured  from  the 
Tissue  Bank,  Naval  Medical  School  and  planted  in  Carrel  D-3#5  flasks, 
_.-.:  with  plasma  and  a  fluid  medi'tn  phaie ' ccntairting  st^'ain  1769  (long  term 
human  epithelial)  and  allowed  tc  clot.  Observations  were  made  at  14.0,96 
and  120  hours  tc  determine  if  rejection  of  strain  1769  has  taken  place. 

Major  Findings:  In  an  exploratory  series,  observations  suggest  that  there 
is  a  recognizable  horaograft-like  reaction  in  vitro.  The  214-72  hour  re- 
jection phase  of  strain  1769  is  evidenced  by  the  formation  of  a  definite 
zone  of  partial  or  complete  growth  inhibition  at  the  periphery  of  the 
fresh  tissue  explant.  This  inhibition  zone  has  not  been  observed  with 
freeze  dried  tissue  explants. 

Significance  to  Cancer  Research:  If  these  proliminaiy  observations  can 
be  confirmed  they  might  well  be  most  sighificant  in  further  elucidation 
of  the  hcmograft  response  that  occurs  in  vivo. 

Projected  Plans:  To  repeat  and  expand  these  findings  with  different  types 
of  tissue jboth  viable  and  non-viable,  of  homogenous  and  heterogenous 
origins, 

10,  l|30;(g) 
Serial  No, 

11, 

Budget  Activity: 

Research      x  Administration       x 

Review  and  Approval        Technical  Assistance 


12. 


Page  2 


Cooperating  Units  of  the  Public  Health  Service,  or  other  organizaticns 
providing  funds,  facilities,  or  personnel  for  this  project  in  eithor 
1956  or  1957. 

>  f .  ., 

Tissue  Bank,  Naval  Medica:l  School,  National  Naval  Medical  Center.  Much 
of  this  work  was  actually  done  by  Tissue  Bank  Personnel  under  supervisici 
of  N.C.I.,  senior  investigators. 


13,   None 


If  this  project  resembles,  complements,  or  parallels  research  dene 
elsewhere  in  the  Public  Health  Service  (without  interchange  of  person- 
nel, facilities  or  funds),  identify  such  research; 


lli.  '  il30  (g) 

Serial  No, 


l5 ,   None 


Publications  other  than  abstracts  from  this  project  during  calendar 
year  1955.  ■ 


16.    Wone „   

Honors  and  awards  to  personnel  relating  to  this  project  during  cal- 
endar year  1955. 


Goneral  Project  _Ii31 

Serial  No. 


Studies  cf  cell  trans fcrmaticn  in  vitro. 


,  - .  .  Page  1 

•  '  ■     PROJECT  REPORT  FORM 

1,     Nat irnal  Cancer ,  2.   Biclogy 

institute  laboratory  or  Branch 

3«  Tissue  Culture lu  Eothesda,  Md. g.  U31  (a) 

Section  or  Service  "  Location  Serial  No. 


6,  Studies  of  coll  transformation  in  vitro . 


Project  Title 
7.   K.  K,  Sanfcrd 


Principal  Investigator (s) 
a,  G,  L.  Hcbbs,  M.  Fiorajncnti,   and  W.  R.  Earle 


Other  Investigators 

9.  Project  description:     Characteristics   of  two  ].inos  of  cells   originating 
from  a  single  adult  mouse  cell,  , 

Objective;     l)  To  define  quantitatively  the  sarcoma-producing  capacity 
of  cells  of  these  two  lines  as  dependent  on  the  numbers  of  cells  injected 
into  mice   end  as   influenced  by  the  period  of  cell  groirt.h  in  vitro,  2)  To 
determine  whether  the  difference  in  sarcoma-producing  capacity  of  these 
two  coll  lines  is  related  to  a  difference  in   immimolrgic  properties  affect- 
ing their  transplantability  t'"  the  mouse  strain  of  origin  and  to  define 
the  autonomy  and  immunologic  specificity  of  the  cell  lines  through  hcmolo- 
■■  -gcus  transplajitaticn  studies.   3)  To  compare  the  proliferation  rates   in 
vitro  of  the  two  cell  lines,  k)  To  compare  the  growth  in  vivo  of  these 
two  cell  linos,  g)  To  ostablish  and  analyze  the   chango  in^sarccma-producing 
capacity  induced  in  these  cell  lines  by  mouse  passage,  6)  To  determine 
differences   in  oxidative ' metabolism  of  those  two  lines  of  cells,     7)  To 
deterrainc  differences  in  the  nutritional  requirements  of  these  two  lines 
of  cells. 

Methods  employed;  Quantitative  procedures  in  vjhich  leicwn  numbers   of  cells 
were   injected  into  both  X-irradiated  and  ncn-irradiated,  immunized  and  ncn- 
iramunizcd  mice  of  the  inbred  strain  of  origin  have  been  used.  Injections 
of  cells  were  also  made  into  foreign  inbred  strains  of  mice.  Proliferation 
rates   in  a  stock  horse  serum-chick  embryo  extract  culture  medium  have  been 
determined  md  nutritional  requirements  are  being  studied  by  the  use  of 
quantitative  replicate  culture  procedures .     In  collaboration  with  Dr,  Mark 
Woods  the   oxidative  metabolism  of  the  two  cell  linos  is  being  investigated. 
In  collaboration  with  Dr.  Ruth  Morwin  an  attempt  is  being  made  to  determine 
the  la,tent  period  for  sarcoma  production  from  cells  of  these  two  lines   in- 
jected into  mice  treated  so  as  to  be  unable  temporarily  to  develop  a  homo-    ■ 
graft  reaction.  Cell  growth  in  vitro  has  also  been  studied  ty  implanting 
known  numbers  of  colls  into  diffusion  chambers,  each  plaood  in  the  perito- 
neal cavity  of  a  mouse. 


11. 


Page  2 

Major  findings?  1)  Marked  differonces  in  the  sarcom?-producing 
capacity  of  these  two  cell  lines  were  demonstrated,  2)  The  sarcoma- 
producing  capacity  of  cells  of  these  two  lines  was  found  tc  be  depen- 
dent on  the  numbers  of  cells  injected  into  mice  and  was  influenced  by 
the  period  of  cell  growth  in  vitro.  3)  Both  lines  of  cells  were  found 
to  be  antigenic  tc  the  strain  C3H  mouse.  Immunologic  cross  reactions  , 
occurred  between  these  two  linos  rnd  also  between  these  lines  and  a 
different  clone  of  cells,  clone  929  of  strain  L.  h)   The  cells  of  these 
two  lines  grew  specifically  in  the  strain.  C3H  mouse  and  not  to  any  '  . 
extent  in  other  strains  of  ■mice'  tested,  5)  In  mice  treated  so  as  to 
be  unable  temporarily  to  develop  a  hcmogrsft  reaction  (see  report  by 
Dr.  Ruth  MertTin  )  marked  differences  were  observed  in  the  latent  periods 
for  tumor  development  from  injections  of  equivalent  numbers  of  cells 
of  the  two  lines.  These  data  indicated  that  the  main  difference  in 
sarcoma-producing  capacity  of  these  two  cell  lines  was  not  a  difference 
in  their  trans plan tability  tc  the  mouse  but  was  a  difference  in  their 
ability  tc  survive  and  grow  in  a  non-resistant  host.  6)  The  malignancy 
of  cells  of  the  low  sarcoma-producing  line  could  be  markedly  increased 
by  animal  passage.  After  one  generation  in  a  mouse,  the  cells  were  - 
changed.  This  cell  transformation  lasted  for  approximately  6  months  of 
growth  in  vitro  for  the  one  tissue  cultiu'e  line  studied,  7)  The  prolifer- 
ation rate  of  cells  of  these  two  lines  when  cultured  in  a  horse  serum- 
chick  embryo  extract  mediiim  was  not  significantly  different,  8)  Cells 
of  these  two  cell  linos  could  not  proliferate  on  chemically  defined 
mixture  'ICTC  107-108  tinless  0,5  to  1  percent  serum  proteins  were  added. 
The  amine  acid  requirements  appeared  to  be  the  same  as  those  of  strain  L 
cells.  9)  Initial  studies  by  Dr.  Ifcods  indicated  a  marked  difference 
between  these  two  cell  lines  in  their  rates  of  aerobic  and  anaerobic 
glycolysis  and  in  their  rates  of  respiration. 


Significance  to  cancer  rcsearch;_   This  analysis  of  the  behavior  of 
cell  lines  that  djjifer  markedly  in  their  ability  to  produce  sarcomas 
in  the  strain  C3H  mouse  and  that  have  transformed  from  nonnal  to  tumor- 
producing  cells  should  provide  some  information  as  tc.  the  type  of 
metabolic  change  that  these  cells  have  und.:rgone  in  their  transformation, 

Proposed  course  of  project;  Tc  complete  the  studies  outlined  above  and 
■  to  pursue  particularly  the  biochemical  studies  in  an  effort  tc  detect 
the  mecho.nisras  underlying  the  changes  in  malignancy  of  these  cells. 


10.   1|31  (a) 

Serial  No. 


Budget  Activity: 

;  Research   x         Administration 

Review  and  approval   Technical  Assistance 


12'. 


Cooperating  Units  of  the  Public  Health  Service,  or  other  organizations, 
providing  funds,  facilities,  or  personnel  fcr  this  project  in  either 
19^6  or  19^7. 

Cooperation  with  Dr.  Dean  Eurk,  Dr.  Mark  Woods,  Labcr"tcry  of  Biochemistry 
and  with  Dr.  Ruth  Merwin,  Laboratory  of  Biology,  N.C.I. 


13.   None 


If  thn.s  r>rcject  resembles,  compleracnts,  or  parallels  research  done  else- 
where in  the  Public  Health  Service  (without  interchange  of  personnel,  facil- 
tics  or  funds),  identify  such  research; 


lli.  hn(a)_ 

Serial  Ho. 
l5.  None 


Publications  other  than  abstracts  from  this  project  during  calendar  year 
1955. 


l6.   None 


Honors  and  awards  to  personnel  relating  to  this  project  during  calendar 
year  1955. 


PROJECT  REPORT  FORM 
1.  Naticnal  Cancer  2,   Biology 


Institute  Laboratory  or  Branch 

3,  Tissue  Culture U.   Bothesda,  Md $.  I|.31  (b) 

Socticn  cr  Service  Location  '  Serial 


No 


6»  studios  of  cell  transformations  in  vitro 


Project  Title 
7.  K.K.  Sanford, 


Principal  Investigator (s ) 
1,  G.  L,  Hobbs  and  I'J.R.  Earlc , 


Other  investigators. 

9.     BrojGct  Description;  '  A  study  of  the  tumor-producing  capacity  of  clone 
y29  of  strain  L. 

Objective:,  To  test, the  sarocmarproducing  capacity  of  a  clone  of  cells,  clone 
929,  derived  from  a  single  cell'  6f  strain  L.  Strain  L  was  originated  in  19iiO 
from  an  explrnt  of  subcutaneous  connective  tissue  taken  from  a  strain  C3H  mouse, 
After  two  yerrs  rf  growth  iii  vitro,  cultures  of  strrin  L  gave  rise  to  sarcomas 
when  injected  into  strain  0311  mice.  The  incidence  of  mice  developing  sarcomas 
in  19ii3  from  the  injected  cells  was  68  percent.  Throe  years  later  in  19li6, 
this  incidence  hrd   dropped  to  1  percent.  The  object  of  the  present  study  was 
to  explain  this  change  in  sarcoma-producing  capacity  of  the  cells. 


Methods  emplcyed;  The  sarcoma -producing  capacity',. of  the  cc^s  Was  been  tested 
by  injecting  the  cells  intraniuscularly  intf^  X-irrJidiatpd' and  non-irradiated 
strain  C3H  mice  as  well  as  into  foreign  strains  df  mice  and  strain'  C3H  mice 
immunized  with  clone  929  cells. 

Ma j or  findings ;  VJhen  clone  929  cells  were  injected  into  X-irradinted"  strain  ' 
C3H  mice,  the  percentage  of  mice  developing  sarcomas  was  comparable  to  that 
obtained  in  19li3  when  strain  L  cells  wure  first  tested  for  sarcoma-producing 
capacity.  An  injection  of  clone  929  cells  was  found  to  induce  an  immune  re- 
action in  strain  C3H  mice  that  completely  prevented  the ■ growth-  of  clone  929 
cells  subsequently  injected.  It  was  thus  demonstrated  th-^t  the  apparent  decrease 
in  sarcoma-producing  capacity  of  these  cells  resulted  from  the  development  of 
an  incompatibility  between  the  tissue  culture  cells  and  the  strain  C3H  mouse 
rather  than  from  an,y  dembnstrablc  ch.r'nge  in  the  mclignancy  of  the  cells.  Two 
sarcomas  derived  from  clone  929  cells 'were  foiind  tr  grow  iti  all  strain  C3H 
mice  injected  after  serial  transfers  in  vivo.  A  specificity  of  the  sarcoma 
tissue  for  the  strain  C3H  mouse  was  also  demonstrated, 

Signific-'^nce  tc  cancer  research;  At  the  present  the  only  method  for  deter- 
mining  the  malignancy  of  tissue  culture  cells  is  to  inject  the  cells  into 
animals  of  the  inbred  strain  from  which  the  tissue  originated.  The  signi- 
ficance of  the  present  study  is  to  point  out  that  negative  results  of  such 
a  test  do  not  necessarily  indicate  a  lack  of  malignancy  of  the  cells  tested. 
It  has  been  demonstrated  that  an  incompatibility  may  develop  between  the 
cultured  cells  and  the  mouse  strain  of  origin  such  that  the  cells  become 
antigenic  to  the  host.  The  change  observed  earlier  in  the  sarccma-producing 


Page  2 


capacity  of  this  tissue  culture  strain  has  thus  been  explained  as  a 
change  in  the  transplantability  of  the  cells  and  not  as  a  change  in 
their  malignancy,.    It  has  also  been  demonstrated  that  cells  grown  for 
10  years  in  a  heterologous  culture  medium  have  not  lost  their  specif icty 
for  the  animal  strain  of  origin. 

Proposed  course  of  sub-project:  This  sub-project  will  not  be  continued. 


10.  ii31  (b) 


Serial  No,. 


11. 


Budget  Activity  J 

X  Research 

Review  ?Jid  approval 


Administration 
Technical  Assistance 


12 ,  None 


Cooperating  Units  of  the  Public  Health  Service,  or  other  organizations, 
providing  funds,  facilities,  or  personnel  for  this  project  in  either  1956 
or  1957 


13.  No'" . 

If  this  project  resembles,  complements,  or  parallels  research  done  else- 


where in  the  Public  Health  Service  (without  interchange  of  personnel, 
facilities  or  funds),  identify  such  research j 


^'  ^31    0>) 

Serial  No. 


15. 


Publications  other  than  abstracts  from  this  project  during  calendar  year  1955. 


The  tumor-producing  capacity  of  strain  L  mouse  cells  after  10  years  in  vitro. 
K,.  E,  Sanford,  G«L.  Hobbs,  and  W.R.  Earle,  Cancer  Research,     In  press. 


l£ • ,  None. [ ^ 

Honors  end  awards  to  personnel  relating  to  this  project  during  calendar  year 
1955. 


Page  1 

PROJECT  REPORT  FORM 


1,  National  Cancer 2,  Biology 


3,  Tissue  Culture   h»   Bethesda  Md. ^ 5»  Lgl  (c) 

Section  or  Service  Location  Serial  No. 

6,  Study  of  two  strains  of  mouse  liver  epithelium  frcm  the  ssme  single  cell. 

Project  title*  c^ 

7 •  V»  J «  Evans, 

Principal  Iftveatigatoris) 

8.  N,M»  Hawkins  and  W.R.Earle 

Other  Investigators 

9«     project  Descriptions 

Objective;  To  study  two  substrains,  arising  from  a  common  clone  of  mouse  liver 
epithelium.  One  of  these  strains  (1795)  is  of  high  tumor  producing  ability  the 
other  is  low  in  tumor  production.  The  primary  objective  is  to  have  available 
two  such  strains  for  comparing  their  metabolic  and  physiologic  characteristics. 

Methods  employed {  Cell  cultures  have  been  studied  in  vitro  and  have  been 
continuously  injected  in  both  x-irradiated  and  non-irradiated  C3H  mice.  The 
behavior  of  these  cells  in  replicate  culture  has  been  studied  for  their  re- 
sponse metabolically. 

Major  findings;  Mouse  liver  clone  (li|.69)  was  originated  from  a  single  cell 
of  strain  721  in  April  19li.8,  Metabolic  studies  will  be  dealt  with  in  detail 
by  Dr«  B,B»  VJestfall,  This  clone  cell  strain  has  shown  a  low  capacity  for 
developing  tumors  on  injection,  only  1  tumor  has  been  found  in  non-irradiated 
mice  vjhile  2  have  been  found  in  x-irradiated  mice  in  over  200  animals.  Practi- 
cally 100^  tumor  incidence  was  obtained  frcm  one  of  these  lots  on  trocar  in- 
jection into  both  non -irradiated  and  x-irradiated  mice.  Tissue  culture  from 
one  of  these  tumors  were  grown  to  produce  strain  17 95 •  This  strain  developed 
a  much  enhanced  capacity  in  a  much  shorter  interval  for  its  cells  to  give  rise 
to  tumors  on  reinjection  into  C3H  mice.  The  incidence  of  takes  vTith  this  strain 
is  81.0^  on  injection  in  x-irradiated  C3H  mice  and  69,8^  by  injection  in  non- 
irradiated  C3H  mice.  Tumor  tissue  on  subinoculation  into  irradiated  mice  was 
100^  while  those  in  non-irradiated  mice  was  only  10^, 

A  manuscript  is  in  preparation  deseribing  these  two  cell  strains. 

Significance  to  Cancer  Research;  A  comparative  study  of  the  metabolic  and 
physiologic  behavior  of  these  two  strains  in  tissue  culture  appears  to  be 
of  significance  in  understanding  the  characteristics  of  transition  from  a 
cell  strain  able  to  give  rise  to  practically  no  tumors  to  one  able  to  give 
rise  to  a  high  percentage  of  tumors. 


Page  2 

Projected  work:  Clone  cells  of  this  high  incidence  tumor  strain  will 
he   used  for  experimental  work  tc  compare  and  to  deteimine  if  tiffliors  can 
be  obtained  from  the  second  pure  cell  strain  as  well  as  the  mixed  cell, 
strain  (179^),  Studies  are  still  in  progress  to  determine  if  the  mixed 
cell  strain  (17 95i)  continues  to  give  the  same  high  incidence  of  timors  or 
whether  this  incidence  is  lessened  ty  continued  proliferation  in  vitro.« 


10.  "  U31  (c>> 
11. 


Serial  number 


Budget  activitys 


12 ,   None 
Cc 
■  '  \1 


X  Research  Administration 

Review  and  approval        Technical  Assistance 


None I . • 

Cccporating  Units   of  the   Public  Her^lth  Sorvlce ,   or  other  organizations,  pro- 
\ldtog  funds,' facilities,   or  pnrscnnel   rcr  this   project  jji  epither  19^6  or 


13.    "N^re-  „._'.■ ■         . ^ ■ 

■'If  trd;!  prefect  re^orobles,   complements,  -  or  :para'j.lel3  research  done  elsawhere 
-■in  the  l-u.jlic  Keallth  Service  (without  interchange  of  personnel,  facilitie's 
or  lunda )  „   identify  such  resear':;h ,  ,  ,  ,     . 

ll;.     1|31      'jt' )     ._.  '■ 
Serial'  iiC,      ■        ■ 


15.-  'Ncne-"     - ^ .■__ ■ ■ '        ■  ,    ^  ' 

•■Piib-lications  other  thaii  atj tracts' from  'i.his  project  .during  calendar  year  ,1955 ■ 


1.6.     None   '     ■■■ '     ■  '  _  . ^ ■  .    .  - 

HOTiors  and  awards  to  persomiei  relating  to  this  project  during  calendar  year 
1955. 


:i<iS.  C^Md: 


General  Project       l|.32 


Serial  Nc, 
Ccmpariscn  micro cinematography • 


•                                 Page  1 
PROJECT  REPORT  FORM 
1,  National  Cancer  2,  Biology 


Institute  Laboratory  or  Branch 

3 » Tissue  Gultnre k ,     Bethesda     '  5:«:'i|-32  (a) 

Location.        '"    ~  '    /Serial  No, 

6,  Studies  with  the  Comparison  Micrccinematograph. 


7. 

Projec 
.  W.  R. 

t  Title. 
Earle 

Princ 

ipal  Invest 

,igator 

■  (s) 

8. 

,  W.  T. 

McQuilkin 

(part 

time) 

Other  Investigators 

9»  Project  Descriptionj 

A  three  optical  system  time  lapse  cinematograph  has  been  developed  and  con- 
structed in  the  NIH  Instrument  Shops.  This  is  to  be  used  in  comparative 
study  of  normal  and  malignant  cultures,  and  comparison  of  cultures  under 
different  experimental  conditions.  Particular  attention  will  be  paid  to 
factors  which  influence  population  changes  in  cultiires, 

,  One  person  was  given  preliminary  training  in  use  of  this  instrument.  Final 
adjustments  were  begun  on  the  instrument  after  the  long  delay  reported  last 
year.  Several  experimental  film  strips  were  run  on  behavior  of  various  cell 
types.  Person  being  trained  recently  resigned  to  take  outside  position  at 
a  much  higher  salaiy.  No  replacement  has  been  authorized,  but  Ifr,  McQuilkin 
(Biologist  GS-7)  is  much  interested  in  the  equipment  and  is  carrying  out 
further  part  time  work  on  it.  This  equipment  needs  full  time  work  on  it  to 
make  it  of  practical  usefulness  however, 

Significajice  to  Cancer  Research}  This  camera  is  a  powerful  instrument  in 
¥tucfy  and  comparison  of  various  normal  and  cancer  cell  types  ■under  controlled 
experimental  conditions. 

Proposed  course  of  project.  As  technical  assistance  is  available  the  studies 
projected  arc  as  follows:  l)  Comparison  of  cell  strains  with  various  nutri- 
ents missing  from  medium,  2)  Transition  of  epithelial  cells  to  more  spindle 
shaped  forms,  allowing  possible  clearer  understanding  of  transition  of  car- 
cinomas to  "sarcoma"  and  transition  of  epithelial  cells  to  "fibroblasts" • 
like  forms,  3)  Influence  of  cell  population  density  on  manner  of  migration 
and  the  morphology  of  epithelial  cells,  ii)  Action  of  specific  reagents  on 
cells , 


U32  (a) 


10.  Ii32__ 

N:: 


'^-  ^  Page  2 

11. 

Budget  Activity: 

.  Research    x  Administration 

Review  and  approval        Technical  Assistfjice 

,12,  _  None 

---"-•     C  cooperating  Units  of  the  Public  He  a  It'h  Service,   cr  other  organizations,  pro- 
•*'  ■      viding  funds,   facilities,   or  personnel  for  this  project  in  either     1956  or 
1957 


13.  None '  "  '  • 

If  this  project  resembles,  complements,  or  parallels  research  done  elsewhere 
in  the  Public  Health  Service  (without  interchange  of  personnel,  facilities 
or  funds),  identify  such  researchs  , . 

11;.  Ii32  (a) 

Serial  No. 

l5.  None, '-^ '  I '    

Publications'  other  than  abstracts  frcji  this  project  during  calendar  year  1955 


l6.  None 


Honors,  and 'Awards  to  Personnel  relating  tc  this  project  during  calendar  year 

1955,     ■•-'•'?^'  •;, 


FkyE   1 
PROJECT  REFCRT  FCRk 


1.  national  Cancel"  Institute ?:,  Laboratory  of  3:.olocr/ 

inSTITUTE  L'lBCR/.TCRY  OR  3R.'".rc:-! 

3.  Leulrernia  Studies  Section 4,         . 5.  433 


SERIAL 


6.  Studies  on  t!;s  eticlocp/  of  nio;.'.se  leu!:emias  end  neoolasns  of  the  ogrotir. 

FRCJECT  TITLE  and  adrenal  gisnds. 

7.  Sargh  E.  Ste:^fsrt 

FRII  !CI?AL  If] VESTIGATGR  (3 ) 

6,  [lone 

GTrlER  If!VE3TI3ATCRS 

9.  PROJECT  DESCRIFTICn 

Froject:  Studies  on  the  etiology/  of  iriouse  leulienias  and  neoplasms  of  the 
parotid  and  adrenal  glr.nds. 

Obiectives ;  To  find  good  sources  for  the  parotid  gland  tumor  agent  and 
the  leukernis  agent  in  order  to  recover  them  and  determine  if  they  are' 
identical. 

I^iethods  emjlcyed; 

I.  rievjborn  mice  were  inoculated  v/ith  cell-free  extracts  prepared 
from  iei'henuc  mouSe  tissues,  from  parotid  gland  tumors,  and 
from  other  tumors  arising  in  mice  having  received  e;:tracts 
of  leukemic  mouse  tisst^es. 

For  controls,  similar  nice  r/ere  inocLilsted  viith   the  xollov;ing: 

a)  Extracts  prepared  from  tisst'.es  of  normcl  strain  C3M  mice. 

b)  Extracts  from  C3H  mammary  tismors  carrying  the  mil!':  agent, 

c)  Extracts  from  human  lymphomas, 

d)  An  active  mumps  virus, 

e)  Hethylchoianthrene  applied  to  the  s!:in  of  newborn  mice. 

II.  3y  using  a  method  prcvionsiy  described  (Stewart,  Proceedings  of 
American  Assoc,  Cancer  Research,  April  1955) , attempt  to  recover 
a  virus  from  transplanted  ien!:emias,  parotid  gland  tumors  and 

adrenal  gland  t-amors  and  test  these  for  carcinogenesis, 

III,  Study  the  histopathclcgy  observed  in  the  organs  of  mice  (irradi- 
ated" and  ncn-irradiated)  carrying  transplanted  mouse  tumors, 
and  of  mice  inocs^icted  with-  the  virus  recovered  in  II  to  note 
if  there  are  similarities. 


,'!5C  r  total  body  radiation. 

(Project  description  continued) 


SERIAL  r!0«  433  PAGE  2 

Ks.jor  lindings: 

I.  Two  good  sources  for  the  parotid  glsnd  tumor  ogent  have  been  found; 
one  is  AKK  leukemia  »60,  extracts  of  which,  on  repe?.ted  tests  over 
s  ?-yeaif  period,  have  yielded  an  incidence  of  50  to  80  percent 
parotid  glsnd  tumors,  some  of  the  mice  have  had  both  leukemia  snd 
parotid  tumors  "nd  others  only  ieu'^emia.  The  other,  CSH  leukemia 
*19,  has  yielded  Bbout  a  30  percent  incidence  of  parotid  gland 
tumors  and  adrenal  tumors  over  a  period  of  3  years. 

Other  sources  for  the  parotid  gland  tumor  agent  have  been  an  AKR 
leulcemia  #RIL6,  CSI-I  leul:emia  #11?'4,  a  paraganc'liomn ,  snd.   a  mamriiary 
tumor.  The  last  two  arose  in  (C3Hf  ::  AKR)  hybrid  mice  as  a  result 
of  inoculating  with  AFCR  Icukemir:  extracts. 

rione  of  the  mice  inoculated  with  extracts  prepared  from  carotid 
gland  or  adrenal  gland  neoplasms  from  normal  tissues,  from  human 
lymphomas,  or  from  mill:  agent  msniraary  tumors,  have  developed 
parotid  gland  tumors  or  lcu!:emias, 

iviice  receiving  mumps  virus,  '  nd  those  receiving  methylcholanthrene, 
have  remained  free  of  leuicemis  and  parotid  gland  tumors, 

II.  A  virus  has  been  recovered  from  the  following  leul-emias;  AKR  lou- 
kemias  »60  and  ffRIL6,  and  frca  G3H  leu'cemias  »11!^;4  and  #19.  These 
4  leukemia s  have  repeatedly  been  gocd  sources  fcr  the  p:"rotid  gland 
tumor  agent-.  It  has  not  been  possible  to  recover  a  virus  from  the 

.parotid  gland  tumors,  or  from  the  adrenal  tumors.  Extracts  prepared 
from  these  tumors  have  not  yielded  parotid  gland  tumors.  '        '  ' 

This  virus  is  highly  lethal  for  nev/b.  i  v.iicc-,  especially  for  strain 
C3H.  A  few  (C3I-!f  x  AKR)Fi  I'.ybrids  have  survived  the  inoculations, 
and  4  out  of  15  which  received  virus  after  serial  passage  developed 
parotid  gland  tumors  in  from  5  to  12  months. 

III.  Five  C3H  parotid  gland  tumors  transplanted  into  irradi"ted  mice 
grew  in  from  50  to  EC  percent  of  the  first  passage  transplants 
and  retained  their  original  histology  even  after  repeated  trans- 
planting. Cne  tumor  has  been  carried  for  3  years  r'P.i  2   for  2 
,  years,  i'.iice  carrying  these  tumors  lived  for  from  3  to  6  months  and 
•developed  marked  hepatomegaly,  cardiomegaly,  hemorrhagic  adrenal 
glands,  -enlarged  spleens, . and  edematous  peripheral  nodes.  The 
microscopic  findings  were  as  fcllov/s:  The  sinusoids  cf  the  adrenal 
cortex  and  cf  the  liver  were  so  greatly  dilated  with  blood  that  in 
many_  there  was  almost  complete  atrophy  of  the  parenchymal  cells.  I;iarke' 
myeloid  metaplasia  and  erythropoiesis  were  observed  in  the  spleen 
c.n;d  liver.  Congestion  of  the  kidney  gioneruli  and  other  organs  was 
,.  alsa  observed.  From  the  histologic  findings  a  diagnosis  of  hypervolemi; 
has,  been  made  (Dr.  Tlxima  2vSin) »     Atrophy  cf  lymphoid  tissue  w"s 
generally  found,  i.xtastasis  of  the  parotid  gland  tumor  to  the  lungs 
has  been  a  common  findin-';. 


(Project  description  continued) 


SERIAL  MC.   433  PAGE  3    , . 

These  same  tumors,  vihen   i.:ioculated  into  no::-irrsdiated  mice, 
failed  to  grow  in  a  high  i^ercent  of  transplants,  aud  vj'ien  they 
did  grow  they  were  rapidly  transforraed  to  ssrcornos,  liice 
carrying  the  sarcomas  did  not  develop  hypcrvoleirJ.a,  This  vras 
also  true  in  those  instances  v;here  the  tumor  crowth  was  slow  and 
the  mice  remained  alive  4  to  5  months  (tumors  prinoipslly  sarco- 
matous). The  myeloid  metaplasia  end  lymphoid  atrophy  observed 
in  the  irradiated  mice  was  also  found  here. 

All  mi".e  v;ith  the  transplanted  tumors,  whether  sarcomas  or 
parotid  gland  tumors,  developed  a  ::Tanulo-:ytosis;  the  white 
blood  counts  were  frequently  over  lC0,0C0/ci5bic  mm.  Also, 
all  developed  a  severe  anemia,  the  hemoglobin  frequently  droooing 
to  3  or  4  grams/lCO  cubic  ml. 

The  histologic  changes  observed  in  mice  that  developed  an  acute 
infection,  after  inoci'lrtion  vjith  t!!e  filterable  agent  (Pro- 
ceedings Am.  Assoc,  Cs::cer  [{esearch,  April  1955),  were  similar 
to  those  observed  in  mice  with  the  parotid  gland  tumor  in  that 
myeloid  metaplasia,  lyr:phcid  atrophy,  and  hyperemi?;  of  the 
different  organs  ivere  also  noted.  Severe  anemia  with  a  granu- 
locytosis u'os  also  present, 

Significance  to  cancer  rcsaarch: 

It  is  felt  that  this  study  has  contributed  to  the  study  on  the  etiology 
of  the  parotid  gland  tumor  end  leuheniias. 

Proposed  course  of  oroject; 

To  be  continued  with  more  emphasis  on  demonstrating  lec'cemo genie 
activity  of  the  agent. 


SERIAL  no.  433  FAGE  2 

fa.ior  lindincis; 

I.  Two  good  sources  for  the  parotid  glcnd  tumor  agent  luwe  been  found; 
one  is  AKR  Inukemifi  w&G,  extrccts  of  which,  or.  repe'ated  tests  over 
a  2-year  period,  l:>cive  yielded  an  incidence  of  50  to  80  percent 
parotid  glnnd  tumors,  some  of  the  mice  hcve  hod  both  leuliemiei  snd 
pnrotid  tumors  nnd  others  only  leukemia.  The  other,  CSH  leukemia 
#19,  has  yielded  about  a  30  percent  incidence  of  parotid  gland 
tumors  ond  odrcncl  tumors  over  :i  period  of  3  years. 

Other  sources  for  the  parotid  gland  tuiViOr  ogcnt  have  been  an  AKR 
leukemic  W:RIL6,  C3H  leu!:c!Tii?  #11?.4,  a  paragnnqliomn ,  and  a  mammary 
tumor.  The  last  two  arose  in  (C3Hf  y.  AKR)  hybrid  mice  os  a  result 
"of  inoculating  with  AKR  Iculcemia  extracts. 

None  of  tiie  mice  inoculated  with  extracts  prepared  frcra  parotid 
nland  or  adrenal  r;lar.d  neoplrsms  frotn  normal  tissues,  from  human 
lymphomas,  or  from  mil!:  agent  laammai-y  tumors,  have  developed 
parotid  gland  tumors  or  lcu!:cmias, 

iviice  receiving  mumps  virus,  '.nd  those  receivinc  mothylcholanthreno, 
have  remained  free  of  leukemia  and  parotid  gland  tumoi's, 

II,  A  virus  has  been  recovered  from  the  following  leulcemias;  Alui  Icu- 
kemias  #60  and  »RIL6,  and  frcn  C3H  Icukemias  »11S4,  and  ^l*?.  These 
4  leukemia s  have  repeatedly  been  good  sources  for  the  parotid  gland 
tumor  agent'.  It  has  not  been  possible  to  recover  a  virus  irom  the 
.parotid  gland  tumors,  or  from  the  adrenal  tur.iors.  Extracts  prepared 

from  these  tumors  liavc  not  yielded  parotid  gland  tumors. 

This  virus  is  highly  iothrl  for  nv,'.;b:  i  i.iicc,  especially  for  strain 
C3H.  A  few  (C3Hf  x  AKR)?";  liy.rrids  have  survived  the  inoculations, 
and  4  out  of  15  which  received  virus  after  serial  passage  developed 
parotid  gland  tumors  in  from  5  to  12  months. 

Ill,  Five  C3H  parotid  gland  tiiniors  transplanted  into  irradiated  mice 
grew  in  from  50  to  CC  percent  of  the  first  passage  transplants 
and  retained  their  original  histology  even  after  repeated  trans- 
planting. Cne  tumor  has  been  carried  for  3  years  z\it   2  for  2 
.  years.  IVdce  qarrying  these  tumars  lived  for,  from  3  to  6  months  and 
'developed  marked  hepatomegaly,  cardiomegaly,  hemorrhagic  adrenal 
glands,  enlarged  spleens,,  and  edomatous  peripheral  nodes.  The 
microscopic  findings  were  as  fcllov;s:  The  sinusoids  of  the  a'drenal 
cortex  and  of  the  liver  were  so  greatly  dilated  with  blood  that  in 
many_  there  was  almost  complete  atrophy  of  the  parenchymal  cells,  L.arke- 
myeloid  metaplasia  and  erythropoiesis  were  observed  iu   the  spleen 
Ein.d  liver.  Congestion  of  the  kidney  gloneruli  bvs\   other  organs  was 
a  1  SO;  observed.  From  the  histologic  findings  a  diagnosis  of  hypervolemia 
has.  boon  made  (Dr.  T!:clma  Dunn).  Atrophy  of  lyr.phoid  tissue  w"s 
generally  found,  littastasis  of  the  parotid  gland  tur.or  to  the  lungs 
has  been  a  common  finding, 

(Project  description  continued) 


SERIAL  NC.   433  PAGE  3 


These  same  tumors,  when  inoculated  into  no::-lrrc:!iated  mice, 
failed  to  cjrovv  in  a  high  ;^';Grcent  of  transplants,  and  ivhen  they 
did  grow  they  were  rapidly  tremsiormed  to  saroowos,  liice 
carrying  the  s"rcomas  die  not  develop  hypervolemia.  This  vjas 
also  true  in  those  instances  where  the  tumor  growth  vjas  slow  and 
the  mice  remained  alive  4  to  5  months  (tumors  principally  sarco- 
matous). The  myeloid  mctaplosia  and  lymphoid  atrophy  observed 
in  the  irradiated  mice  was  also  found  here. 

All  r;:i",e  v;ith  tl;e  transplanted  tumors,  whether  srrcomac  or 
parotid  gland  tumors,  developed  a  ^'ranulo^ytosis;  the  white 
blcod  counts  were  frequently  over  lGO,?CO/cuhic  mm.  Also, 
all  developed  a  severe  anemia,  the  hemoglobin  frequently  dropping 
to  3  or  4  grams/lCO  cubic  ml. 

The  histologic  chan{;e3  observed  in  mice  that  developed  an  acute 
infection,  after  inoculation  vjith  tlic  filterable  agent  (Pro- 
ceedings Am.  Assoc,  Ca:-.cer  ['{esearch,  April  1955),  were  similar 
to  those  observed  in  mice  v/ith  the  parotid  gland  tumor  in  that 
m.yeloid  metaplasia,  lyr.ph.oid  atrophy,  and  hyperemia  of  the 
different  or;;;ans  wore  also  :";Oted,  Severe  anemia  with  a  ;]ranu- 
locytosis  u'os  also  present, 

Significance  to  caiiocr  rcsoarch; 

It  is  felt  that  this  study  has  contributed  to  the  study  on  the  etiology 
of  the  parotid  gland  tumor  and  leuhomias. 

Proposed  course  of  'roiect; 

To  be  continued  witli  more  emphasis  en  demonstrstinr;  lci'''.emo3enic 
activity  of  the  agent. 


PAGE  4 

10.       433 

SERIAL,  NO. 


11.  

3UBGET  ACTIVITY : 


RESEAPvCn     /x~7  ADL'IIIIISTR/vTIOn       fl 

REVIEsV  &  APFRCVAL  [J_     TECI-INICAL  ASSISTAflCE     /~7 


12,         None, 


COOPER/iTiriG  UTIITS  OF  THE  PU3LIC  HEALTH  SERVICE,  OR  OTHER  CRGANIZ/^TIOIiS, 
PROVIDING  FUrSS,   FACILITIES,   OR  FERSCMNEL  FOR  THIS  PROJECT  IN  EITMER 

1956  or  1957. 


13,       None, 


IF  THIS  PROJECT  RESEMa.E3,   COI^'iPLEtiEMTSi  CR  PARi'XLELS  P^SEARCH  DOI'E  ELSE- 
WHERE IN  THE  PUH.IC  HE/i,TH  SERVICE    (/JITMOU?  INTERCH/'iNGE  OF  PE3SCNFIEL, 
FACILITIES  OR  FUNDS),    IDENTIFY  SUCI^I  RESEZ-.RCK: 


PAGE  5 
14.  433 

SERI/iL  r!C. 


15. 

PUa^ICATIOIIS  OTHER  7F.LV.  k3STR{XT3  FRGL,  THIS  PROJECT  DURIHG  CALEriDAS  YEAR 

1955 


Ste?;art,  Ssrah  E. :  "[leoplssr.i.s   in  mice  inoculated  ^ith  cell-iree 
extracts  or  filtrates  of  leu!:emic  mouse  tissues,   I.  r.'eopiesrns  of  the 
carotid  and  adrenol  glands,"  J.  Net.  Cancer  Iiist,  _15:   1391-1415, 
April   1955. 

3tew"rt,   Sarah  E. ;  "Heoplssms  in  mice  inoculated  with  cell-free  extracts 
or  filtrrtos  of  leu'cernic  mouse  tissues.   II.  Leircernia   in  hybrid  mice 
produced  b'j  cell-free  filtrates,"  J.  Hnt,  Cancer  Inst,  j^:  '1.1-53, 
August  1955. 


16. 


HCriCRS  AMD  A:a/ARDS  to  FERSCIIIiEL  iJELATIHG  TO  THIS  PROJECT  DURIFIG  CALEHDAR 
YE/iR  1955. 


General  Project  1^3l4 

Serial  No. 


Metabolic  studies  on  tissue  cells  in  vitro, 


Pr.ge  1, 
PROJECT  REPORT  FORM 
1.  Naticnal  Cancer 2,     Biology 


Institute  Laboratory  or  Branch 

3,  Tissue  Culture h*     Bathes  da,  Md. 5«  1|3L   (a) 

Section  or  Service  "Location  Serial  No* 

6,  Metabolic  Studies  on  Tissue  Cells  in  vitro 
Project  Title 

7,  B,  B.  Westfall ; ^ 

Principal  Investigator (s) 

8,  E.  V.  Peppers,     V,J«  Evajis,  K.K.  Sanford,  N.  M.  Hawkins,   J.C,  Bryant, 
E,  L.  Schilling,     M,C,  Fioramonti,  G,  L.  Hobbs,  and  W.R.  Earle, 
Other  Investigators . 


9.  Project  description;     Alpha-keto  acids  in  tissue  cultures, 

Objectj.v,-;;  No  information  was  ?vr.ilable  on  the  behai/ior  of  alpha-keto 
acids   ill  "the  medium  during  growth  of  the  cells  in  tissue  culture,  and 
since  these  are  important  substances  in  the  metabolic  cycle  it  was 
deemed  desirable  to  study  certain  key  ones  both  to  ascertajin  any  changes 
that  might  be  taking  place  d\3ring  groirth  of  the  cells  and  to  see  if  they 
might  substitute  for  the  comparable  a-minr  acids  in  culture  as  certain 
ones  have  been  found  to  dc  in  mammalian  growth. 

Methods;     Existing  methods  for  the  estimation  of  the  alpha-keto  acids 
were  modified  end  adapted  so  as  to  give  good  separation  of  the  alpha- 
ketoglutaric  acid,   oxal-acetic  acid  and  the  two  geometric  isomers  of 
pyruvic  acid  din itropehnylhydraz ones #     The  methods  wore  then  applied 
to  the  ultrafiltrates  of  the  horse  serum  and  chick  embryo  extract  and 
tc   the  protein -free  supernatant s  from  the  media  after  growth  of  the 
cells  in  the  media. 

Major  findings ^  The  medium  supplied  to  the  cells  was  f oimd  to  be  low     ' 
in  keto  acids,  but  in  those  media  showing  good  growth  of  the  cells 
the  cells  were  quickly  able  to  restore  the  level  to  a  more  ne?-rly  plasma* 
like  concentration. 

Significances  This  study  was  partly  an  orientation  work  aimed  at  getting 
methods  well  in  hand  and  determining  concentrations  of  the  keto  acids 
supplied  to  the  cells.     Further  work  is  planned  with  different  strains 
and  various  media  to  see  what  effect  these  combinations  have.     This  is 
part  of  the  program  concerned  with  the  general  metabolism  of  the  cell, 
It  is   therefore  proposed  to  extend  the  study  as  indicated. 


10.      Lt3U  (a) 
Serial  No« 


Page  2, 


11. 


Budget  Activity; 

Research      x 
Review  &  Approval 


Administration 
Technical  Assistance 


12 »  __None 

Cooperating  units  of  the  Public  Health  Service,  cr  other  organizations, 
providing  funds^  facilities,  or  personnel  for  this  project  in  either  ' 
19^6  or  19^7.  •   '   ' 


13 •  No  resemblance  or  complementation, ^     ■      ^  

If  this  project  resembles,  complements ,  or  parallels  research  done 
elsewhere  dn  the  Public  Health  Service  (without  interchange  of  person- 
nel, facilities  or  funds),  identify  such  research: 


^»     U3i;  (a) 

Serial  No, 


15. .  

Publications   other  than  abstracts  from  this  project  during  calendar  year 
1955, 

"a-Keto  acids  in  tissue  culture,,  I,  The  concentration  of  pyruvic  and 
a-ketoglutaric  acids  in  the  ultrafilt rates  from  horse  serum  and  chick 
embryo  extract.    B.B.  Wostfall,  E.V.  .Poppers,  and  JAf.R.  Earle.  J ^  National 
Cancer  Inst,  l6;l:  August  1955* 

l6.  None 

Honors  and  awards  to  personnel  relc?ting  to  this  project .dui'ing  calendar 


year  1955. 


Page  1 

PROJECT  REPORT  FORM 

1,   Natirnal  Cancer 2,   Biclogy 

Institute  Labcratrry  cr  Branch 

3,  Tissue  Culture k»   Bethcsda,  Md. 5.  k3h   (b) 

Section  cr  Service  Location  Serial  Nc» 

6,  Metabolic  study  en  tissue  cells  in  vitro, '_ 

7.  B.  B.  Westfall _^ 

"Principal  Investigator (s) 

8 ,  E,Vv  Poppers .  K.K.  Srnfcrd,  V.  J.  Evans,  N,  M.  Hawkins^  and  W.R.Earle. 
Othor  investigators 

9.  Project  description:  Fraction  of  horse  serum  proteins  for  use  in 
cultivation  of  cells  in  vito. 

Objective:  It  had  been  shown  horse  serum  freed  of  its  readily  diffusi- 
ble substpjiccs  by  ultrafiltration  when  properly  supplemented  gave  ex- 
cellent growth  of  the  tissue  cells  in  culture.  As  a  first  attempt  to 
see  if  the  growth  was  perhaps  associated  with  some  one  of  the  more  or 
less  well  recognized  discrete  fractions,  separation  by  means  of  a 
modified  Cohn  low-temperature  technique  was  carried  out  and  the  fractions 
tested  for  growth  by  Dr*  K.  K,  Sanford,  If  one  of  these  contained  all 
the  growth  stimulating  factors  it  would  have  been  a  distinct  forward 
toward  getting  a  well-characterized  medium  for  growth  studies. 


Methc^ds i'  As  noted  above,     • 

"Major  findings;  Growth  was  reasonably  good  with  all  fractions,  but 
better,  with  some  than  ethers;  the  gross  globulin  fraction  was  that, 
one  giving  as  good  proliferation  as  the  original  serum  residue.  The 
study  indicated  that  growth  was  not  necessarily  associated  with  any 
particular  moiety,  or  if  such  should  be  the  case  that  it  was  effective. 
If  time  and  suitable  facilities  are  available  further  work  on  frac- 
tionation is  contemplated.  It  is  hoped  that  outside  sources  may  be 
found  that  might  undertake  such  fractionation. 


10'    U3U  (b) 

Serial  No, 


Page  2. 


11 .  ■ 

Bndgot  activity: 


Research    x        Administraticn 
Review  &  Approval    Technical  AssistsnCG 


12.  None 

Cccperating  units  cf  the  Public  Health  Service,  cr  ether  crganizatirns, 
providing  funds,  facilities,  cr  personnel  for  this  project  in  either- 
19^6  or  1957. 


13. 


If  thl:^  project  resembles,  complements,  or  parallels  research  dene, 
elsci-huxo  in  the  Public  Health  Service  (without  interchange  of  personnel, 
facili-r"e3  or  funds),  identify  such  research i 

It  has  been  ruinored  that  Dr.  H.  Eagle's  group  and  Dr.  H.  Stoinman 
have  been  working  along  similar  lines,  but  we  have  no  direct  infor- 
mation , 

Serj.aj   i<'c^ 


15. 


Publications   other  than  abstracts  from  this  project  during  calendar 
year  1955. 

Effect  of  serum  fractions  on  the  growth  of  strain  L  cells,  K.K.Sanford, 
B.B.Westfall,  M.C,  Fiorr^nonti,  W.T,  HcQuillcin,  J.C.Bryant,  E.V.  Peppers, 
V,J»  Evajis  and  W.R.Earlc.  J.  National  Cancer  Inst.  l6:3s  Dec.   1955. 

l6 .     None . •       - 

Honors  and  awards  to  personnel  relating  to  this   projecst'  diiring  calen- 
dar year  1955 


Page  1 
PROJECT  REPORT  FORM 


!♦  National  Cancer 2,  Biology 


Institute  Laboratory  of  Branch 

3»  Tissue  Culture !;.  Bethesda,  Md. $,   ii3li  (c) 

Section  or  Service  Location  Serial  No, 

6,  Metabolic  studies  on  tissue  cells  in  vitro. 


Px-oject  Title.  ■ 

7,  B.B.  Westfall 

Principal  Investigator(s) 

8,  E.V,  Peppers,  V.J.  Evans,  K,  K.  Sanford,  M,  M,  Hax-zkins,  J.  C.  Bryant, 

■  E«  L.  Schilling,  M.  C,  Fioramonti,  G.L«  Hobbs,  and  W,R.  Earle, 

Other  investigators, 

9,  Project  description:  Chemical  studies  on  the  changes  in  the  nutrient 

■  mediwii  when  cells  are  grown  in  large  quantity  and  volume  of  the  shaker 
flask. 

Objective}  No  information  was  available  as  to  change  in  nutrient  in 
the  medium  during  grcvrth  of  cells  on  the  large  shaker  apparatus.  Also 
since  growth  was  very  good  end   it  was  known  that  the  quantity  of  certain 
of  the  amino  acids  were  supplied  in  low  a-moimt  it  x-jas  deemed  desirable 
to  see  if  any  of  these  were  limiting  in  concentration  since  Fischer  had  " 
demonstrated  in  19^3  that  the  free  amino  acids  lysine,  histidine,  arginine, 
valine,  leucine,  is oleucine,  threonine,  phenylalanine,  tryptophane,  methio- 
nine, cystine  and  the  amine  glutamino  were  needed  for  good  growth  of  cells. 

Major  Findings;  In   one  study  with  the  HeLa.  strain  of  cells  it  was  found 
that  ty  using  the  medium  made  up  of  human  placental  fluid  and  whole  egg  ■' 
extract  ultrafiltrate  with  their  attendant  low  concentration  of  rjnino 
acids,  certain  of  the  amino  acids  appeared  to  be  exhausted  from  the  medium 
in  a  3-day  interval  with  a  heavy  inoculum  and  good  growth. 

Significance {  This  study  was  primarily  part  of  a  program  tct  the  study 
of  cellular  behavior  under  different  conditions,  ca-ncer  being  thought 
of  as  mainly  a  phenomenon  in  the  growth  of  cells.  The  strain  of  cells 
under  study  were  human  malignant  cells  of  epithelial  origin. 


Page  2. 


Methods i     The  cells  were  grcwn  en  the  shaker  flasks  with  ccntinucus 
gas  exchange  and  ocntinucus  shaking.  The  mediijin  was  examined  for  free 
amine  acids  by  paper  chrcmatography;  for  alpha-keto  acids  by  extrac- 
tieri,  and  paper  chrcmatography,  f cr. lactic  acid  and  glttccse  by  adapta- 
tion of  existing  spfectrcphotometric  .methods. 

It  is  proposed  to  continue  and  extend  similar  studies  to  other  cells  and 
to  various  conditions  of  maintenance. 

10,     _i:t3U   (a) 

Serial  No", 


11. 


1^. 


Budget  activity: 


Research  x  'Administration 

Review  &  Approval  Technical  Assistance 


12,   None 


Cooperating  Units  of  the  1-ublic  Health  Service,  cr  other  crganizati'^ns, 
providing  funds,  facilities,  or  personnel  for  this  project  in  either 
1956  or  1957. 


13,  .  No  rescmblarice  or  complementation  known. ■ 

If  this  project  resembles,  complements,  or  parallels  research  done  else- 
where  in  the  Public  Health  Service  (without  interchange  of  personnel, 
facilities  cr  funds),  identify  such  researchj 

i^«       lilh  fc)     ■- 

Serial  No, 


Publicr.ticils  other  than  abstracts  from  this  project  during  calendar 
year  1955. 


"The  change  in  concentration  of  certain  constituents  of  the  medium  during 
growth  of  the   strain  HeLa  Cells."    B.  B.  Westfall_.  S.V.Peppers  and  W.R, 
Earle.r     fhe  American  J.  of  Pfygione,   l6{3{  April;  1955. 

l6.         None 

Honors  and  awards  to  personnel  relating  to  this   project  during  calendar 
year  1955* 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute  2.   laboratory  of  Pathology , 

INSTITUTE  LABORATORY  OR  BRANCH 

S'oo 

3.  Cancer  Pathology  Section  4.   5.  NCI  §^» 

SECTION  OR  SERVICE  LOCATION(IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

6.  Histogenesis  and  pathology  of  induced  and  spontaneous  tumors  of  laboratory 

animals . 

PROJECT  TITLE 


7.  Harold  L.  Stewart 


PRIHCIPAL  IN v-ES'.ClGATOR(S ) 
8.  Katharine  C.  Snell 


OTHKR  INVESTIGATORS 


PROJECT  DESC^IIPTION        .:  ,        - 

Object:  To  gain  knowledge  of . the  etiologic   and  pathogenic  factors 

involved  in  the  production  of  neoplasms  in  experimental  animals, 
and  to  make  information  obtained  available  to  other  investigators. 

Methods:   1.  A  fascicle  on  transplantable  and  transmissible  tumors  of 

animals  has  been  prepared  for  publication  in  the  Atlas  of  Tumor 
Pathology  under  the  auspices  of  the  Subcommittee  on  Oncology  of  the  Committee 
on  Pathology  of  the  National  Research  Council.  This  fascicle  Includes  the 
history,  transplantation  behavior,  detailed  pathological  description,  and 
microphotographs  of  50  of  the  most  widely  used  animal  tumors.   (Dr.  Lucia 
Dunham  has  collaborated  on  this  work.) 

2.  A  study  is  being  made  of  the  pathology  of  spontaneous  and  Induced  tumors 
of  the  liver  of  animals.  This  study  will  form  the  basis  of  papers  to 

be  presented  at  the  Symposium,  on  Liver  Cancer  at  Kampala,  Uganda,  and 
Leopoldville,  Belgian. Congo^  in  August  1956  under  the  auspices  of  the  Unio 
Internationalis  Contra  Cancrum.   It  will  also  be  published  as  a  chapter  in 
the  revised  edition  of  The.  Physiopathology  of  Cancer  (F.  Homburger  and 
W.  H.  Fishman,  editors). 

3.  A  series  of  untreated  rats  of  seven  strains,  males  and  females,  breeders 
and  non-breeders,  is  being  necropsied  at  various  age  intervals  and 

subjected  to  detailed  pathological  examination  in  order  to  enumerate  and 
classify  spontaneous  tumors,  to  study  tissue  changes  with  increasing  age, 
and  to  furnish  controls  for  present  and  future  experiments. 


Coo 

SERIAL  NO.  NCI  509*  PAGE  11 

PROJECT  REPORT  PORM  (Cont'd) 

9.  PROJECT  DESCRIPTION  (Cont'd) 

4.  An  attempt  Is  being  made  to  produce  adenomatosis  of  the  lung . by  feeding 
l,2;5^6-dib'3nzanth3:acene  to  DBA  mice. 

5.  Monkeys  that  received  injections  of  carcinogenic  svbstances  into  the 

,    wall  of  the  stomach  several  years  previously  are  being  necropsied  and 
studied  to  ascertain  whether  these  substances  may  have  produced  gastric 
neop'.a^ms,  ■■•■  '-  -■       ■  •  ■  , 

6.  A  study  is  being  made  of  the  tuiftOrs  occurring  ir.  the  !1H0  strain  of 
irice.  Th«^;se  mice  are  of  interest  because  of  the  fact  ihat  their 

ancestors  received  a  single  doee  of  methylcholanthrene  in  an  effort  to 
proiucc:  an,  hereditary  gastric  carcinoma. 

Major  ♦:Jn-iirigr,:   1.   Fascicle  on  Transplantable  and  Transmissible  Tumors 

of  Auiinals.  The  manuscripts  on  50  tumors,  together  with  300  micro- 
pbotogrpph*i,  havrt  been  completed  and  these  have  baen  approved  for  publicatiort 
by  the  M.I.H.  Editorial  Board. 

2.  The  study  of  liver  tumors  is  in  progress  and  microphotographs  and 
lantern  slides  are  being  prepared,  i 

3.  A  total  of  404  untreated  rats  have  been  necropsied,  the  series  of  ■ 
necropsies  is  complete  on  the  Marshall  520,  the  AXC,  and  the  Osborne- 

Mendel  strains.  Microscopic  evaluation  of  the  material  is  in  progress. 
Unusual  tumors  noted  have  been  carcinomas  of  the  adrenal  cortex^  a 
carcinoearcotaa  of  the  jejunum,  and  tumors  of  the  lung  and  testis. 

4.  Necropsies  and  evaluation  of  sections  of  the  DBA  mice  fed  1,2,5,6- 
dibenzanthracene  are  being  made,  but  it  is  too  early  in  the  experiment 

to  report  any  significant  findings.  ^  ..„.». 

.  .,;■   .  V  ■■'       ,  •;  "    ■      '  '■■■"■     "  '■      •■   '       ■  •■•orfuci'.Oiir: 

5.  No  monkeys  necropsied  to  date  have  shown  gastric  carcinoma  nor  any  ^' -'.v' 
lesions  that  can  be  attributed  to  the  injection  of  carcinogenic 

substances  into  the  wall  of  the  stomach.  '<<   •<•  >  "-  ./  !  . 

. 'Gin.brr.  :iw  Tnvii  ^irfrj  J 

6.  Various  tumors  of  the  ^HO  strain  of  mice  have  been  obseifWiff,  amJ' 
these  are  being  studied  histologically  and  evaluated.  There  have  been 

no  carcinomas  of  the  glandular  stomach,  but  tumors  of  the  nervous  system^   ' 
lung,  uterus,  and  reticulo-endothelial  system  have  been  found.     ■  'iv  ..  ■,. . 

Significance  to  Cancer  Research:  The  knowledge  gained  from  a  study  of 

neoplasms  in  laboratory  animals  should  provide  a  basis  for 
further  work  on  the  mechanism  of  carcinogenesis  in  the  human  being. 


erial  No.  NCI -&ef     PROJECT  REPORT  FORM  (Cont'd)  PAGE  III 

.  PROJECT  DESCRIPTION  (Cont'd) 

Proposed  Course  of  Project:   It  Is  expected  that  the  fascicle  on  Transplant- 
able and  Transmissible  Tumors  of  Animals  will  be  published  in  1956. 

The  study  of  the  pathology  of  liver  tumors  will  be  completed  and  ready  for 
publication  by  the  summer  of  1956, 

Experiments  dealing  with  carcinogenesis  (enumerated  as  parts  3-6)  will  be 
continued. 


PAGE  IV 


PROJECT  REPORT  FORM  (Cont'd) 


10,  NCI  ^e^- 


SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         0         ADMINISTRATION       /T 
REVIEW  &  APPROVAL  fj         TECHNICAL  ASSISTANCE  £1 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13 »  None  to  my  knowledge 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  V 


PROJECT  REPORT  FORM  (Cont'd) 


L4.  NCI  '%m- 
SERIAL  NO. 


L 5. PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 

Stewart^  H,  L.  and  Hare,  W.  V.   Chronic  gastritis  of  the  glandular  stomach, 
adenomatous  polyps  of  the  duodenum  and  calcareous  pericarditis  in  strain 
D.6.A.  mice.   J.  Nat.  Cancer  Inst.   In  press. 


L6.  None 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955. 


PAGE  I 


PROJECT  REPORT  FORM 

1.  National  Cancer  Institute     2.  Laboratory  of  Pathology  '•      - 
INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Pathology  Section 4.  New  York,  Israel,  Washington^D.C.5.  NCI  -54^ 

SECTION  OR  SERVICE  LOCATION  (IP  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6,  Geographic  patholdgy  field  studie's  on  uterine  cancer  in  New  York  City, 

Israel,  and  Washington,  D,  C.   '  ^  -'  '  '  '   •  "  '  "'  ■-■■■■ 

PROJECT  TITLE 

ti??;Lacia  J.   Dunham,   Harold  F.    Porn,   Harold  L.   Stewart  ' 

PRINCIPAL  INVESTIGATOR(S)   - 

8.  John  H.  Edgcomb,  Louis  B.  Thomas 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Field  studies  of  uterine  cancer  (cervix  and  corpus)  in  New  York  City,  Israel, 
and  Washington,  D.  C.  '    -rfs^'Hiv- 

Objectives;   The  objective  of  this  project  is  to  complete  an  extensive 

questionnaire  study  of  women  with  uterine  cancers  in  different 
geographic  areas  and  racial  groups,  and  to  analyze  the  data  obtained  with 
a  view  to  identifying  factors  suspected  of  predisposing  to  or  causing 
female  genital  cancers.  Also,  basic  data  on  the  incidence  and  pathologic 
(microscopic)  diagnoses  of  the  canci?.rs  are  obtained. 

Methods:   For  the  area  under  study  and  the  period  of  study  all  patients 
possible  with  the  diagnosis  of  uterine  cancer  are  intervievjed, 
and  an  equal  number  of  control  patients  as  well.  The  confidential  data 
recorded  include  social,  medical,  and  surgical  history,  and  menstrual, 
marital,  and  pregnancy  data.  Records  are  kept  of  the  small  number  of 
patients  not  interviewed,  their  diagnoses,  and  the  reasons  for  failure  to 
interview.   Pathologic  material  is  studied  in  all  cases. 

A  coding  statistician  and  two  medical  social  worker  interviewers  are 
currently  eoployed  in  the  project. 

Major  findings:   1.  Data  from  about  2,000  interviews  with  Jewish  women  in 
Israel  have  been  reviewed  and  coded. 

2.  Data  from  about  2,000  interviews  with  Jewish  and 
non- Jewish  white  women  in  New  York  City  have  been  reviewed  and  coded. 

3.  Data  from  about  400  interviews  of  non-white  women  in 
New  York  City  have  been  reviewed  and  coded. 

4.  Data  from  about  20  interviews  of  non-white  women  in 
Washington,  D,  C,  have  been  reviewed  and  coded.  This  study  was  started 
late  in  1955. 


SERIAL  NO.   NCI  h^  PAGE  II 

PROJECT  REPORT  FORM  (Cont'd) 

9.  PROJECT  DESCRIPTION  (Cont'd)  •)>«.!'  t 

5.  Pathologic  material  has  so  far  been  examined  in  about 
■j,i'   ^P;. percfnt_ of .  the  above  cases.  ..,-.■ 

'  7''r:A  ■>        .  H.;/n  '  6*  The  data  from  apqiitJZ, 500   cases  have  been  transferred 
to  punch  cards  for  IBM  analysis. 

7,  The  medical  literature  on  geographic ■  and  enytronaje|»t;al, 
factors  in  uterine  cancer  has  been  reviewed,   ,j 

Significance  to  cancer  research:  The  significance  of  the  study  lies  in  the 

hope  of  supplying  a  sound  scientific  basis  for  the  understanding 
of  factors  of  custom,  habit,  or  environment  as  they  may  be  related  to  ■ 
relatively  high  frequencies  of  uterine  cancers  in  some  groups  of  women  as 
contrasted  to  intermediate  or  relatively  low;  frequencies  in  other  groups.. B 

Proposed  course:  The  proposed  course  is  to  tabulate,  analyze,  and  appraise 

the  material  referred  to  above,  and  to  prepare.reports  of  these 

studies  for  publication.  At  the  conclusion  of  the  project  it  is  planned 

.  to  undertake  clinical  studj,es.  in  geographic  pathology  on  patients  with,/ 

cancers  of  the  esophagus,  stomach,  and  large  bowel.  .  .■;.-,v?  hr,.:; 


■.'5  J  X-i     ■■ 
-  -:f!4  h: 


i   .v.-l:.a 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI  -^J^- 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         /^   .  ADMINISTRATION   ^   £7 
REVIEW  &  APPROVAL  £7    TECHNICAL^ ASSISTANCE  £37 


12.  Office  of  Biometry.  Office  of  the  Director,  National  Institutes  of  Health 
COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


■lAi  i-J~> 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  IV 


PROJECT  REPORT  FORM  (Cont'd) 


14.__NCI_5i5u_ 
SERIAL  MO. 


15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 

Dunham^  L.  J.  and  Dorn,  Harold  F. 

Techniques  in  the  Geographic  Pathology  of  Cancer :  '5  V'i3aH 

Schw.  Zelt.  fUr  Allg.  Pathologie  und  Bakterlologie,  Vol.  18,  No.  4,  1955 

Printed  in  Swtt«erland.  •  v«iiy.'S,: 


16.  None 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 
INSTITUTE  LABORATORY  OR  BRANCH 


nnnr 


3.  Cancer  Pathology  Section      4. 5.  NCI -^a^ 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Geographic  Pathology.  Experimental  studies  relating  to  the  geographic 

pathology  of  mouth  cancer. 

PROJECT  TITLE 

7.  Lucia  J.  Dunham 

PRINCIPAL  INVESTIGATOR(S) 

8.  Marvin  S.  Burs  tone   '  '  •      '  ■  • '-     

.  -  OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Experimental  studies  on  mouth  cancer. 

Objectives:  The  objective  of  this  project  is  to  examine  the  mucous  membrane 

of  the  hamster  cheek  pouch  for  proliferative  changes  that  may 
develop  when  test  substances  are  chronically  maintained  in  contact  with  these 
surfaces.  Substances  used  are  environmental  materials  suspected  of  being 
mildly  carcinogenic  for  man. 

Methods:  The  pellets  which  have  been  prepared  for  insertion  in  the  cheek 

pouch  of  the  hamster  include  the  tobacco^  lime,  areca  nut,  and 
pepper  leaf  of  the  far  Eastern  "betel  quid,"  the  plastic  material  used  in 
preparing  "false"  teeth,  and  tobacco  tars  and  control  materials.  A  technical 
assistant  is  retained  part-time  for  help  in  the  study. 

Major  findings:   Since  the  project  is  a  long-term  one,  there  are  no  major 

findings  at  present.   It  is  hoped  to  maintain  the  experi- 
mental animals  for  at  least  two  years  before  gross  and  microscopic  study 
of  the  pouches.  The  operations  used  for  testing  substances  has  so  far  been 
performed  on  107  hamsters. 

Significance  to  cancer  research:  The  significance  is  to  reach  an  improved 

understanding  of  chronic  irritant  factors  as  they  may  relate  to 
cancer  of  the  mouth  in  man. 

Proposed  course:  The  study  will  be  extended  to  more  animals  and  to  a  wider 

variety  of  suspected  substances.   Current  experimental 
animals  will  be  studied  with  care,  when  illness  or  death  intervene.   In 
addition,  improved  techniques  of  study  will  be  attempted. 

."ilJ/Tra 


PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 
SERIAL  NO.  ' 


ill.. 


iiaai 


BUDGET  ACTIVITY: 

.,..  qi  .,->.  ,,,         j^ggg^jjcH  /x7         ADMINISTRATION  ff']^^^^:- 

REVIEW  &  APPROVAL  /~7         TECHNICAL  ASSISTANCE  7~7 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PROVIDIN 
FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  OR  1957 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS >  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  F/^ILITIES  OR 
FUNDS) ^  IDENTIFY  SUCH  RESEARCH 


ft^  o« 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Office  of  the  Chief 4. 5.  NCI  40^ 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Histogenesis  and  histopathology  of  cutaneous  neoplasms. 

PROJECT  TITLE 


7.  John  H.  Edgcomb 


PRINCIPAL  INVESTIGATOR(S) 
8.  None 


OTHER  INVESTIGATORS 
9.  PROJECT  DESCRIPTION 

During  the  past  year  the  project  has  included  the  following  activities: 

A.  Studies  of  diseases  of  man  (collaborative  research). 

i.  Mycosis  fungoides 

The  reaction  of  three  patients  with  mycosis  fungoides  to  electron 
beam  therapy  has  been  studied  pathologically.  This  type  of  therapy  causes 
remissions  of  cutaneous  tumors  in  this  condition, 

ii.  Melanomas 

Naevi  from  the  Skins  of  patients  with  malignant  melanomas  have 
been  examined.   It  appears  that  more  naevi  from  such  patients  show  junctional 
activity  than  is  the  case  of  naevi  from  control  patients. 

B.  Studies  of  diseases  in  animals 

i.  Further  studies  are  being  made  on  C  strain  mice  bearing  Dr.  R. 
Bryan's  transplantable  squamous  carcinoma.  Many  of  these  mice  develop 
severe  leukocytosis.  Attempts  to  transform  this  disease  into  granulocytic 
leukemia  have  been  unsuccessful, 

ii.  An  attempt  to  produce  melanomas  in  guinea  pigs  is  being 
continued.  As  yet  (2  years)  no  melanomas  have  developed. 


PAGE  II 


PROJECT  REPORT  FORM  (Cont ' d ) 


10.    NCI  •&£?- 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH        ■  /x7  •   AD^  :     [j 

REVIEW  &  APPROVAL  £7    TECHNICAL  ASSISTANCE  £7 


12,  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  i 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Patholoav    .  . 
INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &. Pathogenesis   4. 5.  NCI -^^ 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAt  NO. 

6.  Accumulation  and  dissemination  of  information  relating  to  the  normal  and 
pathologic  anatomy  of  laboratory  animals,  particularly  mice,  with  special 

emphasis  on  neoplasms. fj  j.-  :■  •^;i:.r  ■....:..,...,.  .  , 

PROJECT  TITLE  M/l-wbgat  . 


7.  Thelma  B,   Dunn 


iiinjs^ 


PRINCIPLE  INVESTIGATOR(S) 


8.  None 


OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 


.*i:„i(i- 


Objectives:   To  accumulate  information  relating  to  the  normal  and  pathologic 

anatomy  of  small  laboratory  animals,  and  to  publish  it  in  such 
a  form  as  to  be  readily  available  to  laboratory  workers  throughout  the  world. 

Methods:   Individual  study  of  particular  phases  of  pathologic  anatomy  of  ; ., 

laboratory  animals;  collaboration  and  consultation  with  other 
scientists  at  the  National  Cancer  Institute  in  projects  requiring  the  review 
of  histologic  material  obtained  from  experimental  animals;  review  of  iqaterial 
sent  in  for  an  opinion  by  scientists  from  outside  laboratories;  carrying  in 
transplant  of  unusual  neoplasms  in  mice,  so  they  can  be  kept  for  continued 
study;  and  accumulation  of  literature  and  references  relating  to  the  subject. 

Major  findings:  The  present  project  has  been  carried  on  through  a  number  of 

years.  During  the  past  year  a  paper  has  been  published 
with  Dr.  Law  describing  the  morphology  of  an  unusual  neoplasm  of  the  parotid 
salivary  gland  in  mice,  with  a  review  of  previous  reports  of  salivary  gland 
tumors  in  mice.  Several  transplantable  tumors  are  being  carried,  which  have 
not  been  described  before  in  the  mouse.  These  are  a  uterine  adenocarcinoma, 
a  parathyroid  neoplasm,  an  actively  secreting  adrenal  cortical  neoplasm, 
and  a  mucoid  adenocarcinoma  of  the  ovary. 

Significance  to  cancer  research:  Since  the  mouse  is  used  so  regularly  in 

cancer  research,  it  is  necessary  that  the  normal  and  pathologic 
anatomy  of  this  species  be  well  understood,  if  reliable  conclusions  are  to 
be  drawn  from  cancer  studies. 


SERIAL  NO.  NCI  568- 


PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


9.  PROJECT  DESCRIPTION  (Cont'd)  !,ii«Gi_i.??J!l' 

Proposed  course  of  project:  Work  on  this  project  will  continue  as^it  has 
in  the  past.  -  Phases  of  the  subject  now  under  special  study  are. 

a.  Systematic  review  of  the  :endocrliie:  system;  of  the  mouse rv:#,  Ross ^ 
MacCardle  is  collaborating  in  this  by  carrying  ""^^f  "^^««/^l^^^^  ■''°.  „ 

the  pituitary.  A.  study  of  the  endocrine  organs  of  different  inbred  strains 
at  different  ages  has  been  bfegun.  ■    ■  ^mpji' 

b.  Continuing  collaborative  study  with  (1)  Dr.  Lloyd  I^"  «^.'"J^^>^"=^^;;Jj|^ 
various  filtrates,  and  of  mice  developing  leukemxa^  (2)  Review^of  stmilfx 

material  from  Dr.  Sarah  Stewart;  (3)  Dr.  W.  E.  Heston  of  ^/'>^.J^^^ ^^^""^^^l  . 
strain  C3H  and  C57BL  in  which  a  variety  of  tumors  appeared;  (4)  Dr.  H.  u. 
Andervont  on  mammary  tumors  in  mice,  and  the  pathologic  changes  in  wild  :^ 
mice  and  (5)  reviev,  of  small  numbers  of  slides  from  Dr.  Margaret  perirjgar,,, 
Dr.  Elizabeth  Jones,  Dr.  Michael  Potter,  Dr.  Vincent  Price,  and  Dr.  Robert  . 
Greenfield,  and  others. 

c.  Continuing  review  of  unusual  lesions  sent  from  outside^sputG|s^  ,  Amot^^  '' 
those  recently  received  were  several  from  Dr.  W.  U.  Gardner  of  Y^le,; 

Dr.  Michael  Klein  of  Florida,  Dr.  P.  Loustalot  of  Switzerland,  and  Dr. 
H  I.  Pilgrim  of  California.  Opportunity  to  review  these  uousual  lesions 
Is  of  great  value  since  it  gives  a  chance  for  comparison  with  other 
-labotatdrieis;^.^'-.  U'-^axo;-!  a;lt  tyj-  i^niin.isrs  no'.-  .:'mit:>of}  o'"'  :    ■■•    • 

.ai  Continuing  observation  of  unusual  transplantable  tumprs.   Important  ^^  , 
information  has  often  been  gained  by  observing  the  behavior  of  transplanted 
tumors,  especially  where  they  have  a  hormonal  effect. ,  •      :  ;...,.T  •.;  use;:^:!^-^ 

e;  Systematic  review  of  literature  relating  to  enjiocripe,organp  inmlp,^,.^,, 

-       •haS'^been- started,  ....■■■  .   .;..  .  .:.;.iii   j:..^ii.x.!;j,4-ici 

'..k   y-i.':-  >r  ■■  ■        .       '  :;■■;.    .io sal fs;/>  im^  yo-f-  iii \-in -^9 

..iaaj;d*f'«  .-aill  ..'.-fu'lun  'br.  :   ;      ■■         ^.l^«ii'uml'>■o■^■^■ -hrsii -yuti  i-.! 

J-;    ■..rxjfr^o.o  ui  i-fgooTifJ  j.'-j  hyii.-i'x:x-  :    r.^  ^.lasauii?- :6>rfT     .:  aa«ibHi>.- totfiS' 

■  '■:..  ;•;  ...iioivotq  3-.    .  ..        . :  sv- ,  .... .,;;  ffi: -tossls  Y'T^vlTr.i? 

,;-  0--      •■•:'■■■'  .■     ■'••■■  ■"■;■■■>' '.3 sob  ti'jod  Jl<-ia 

hivx'idHintv;  i'. 

:,:■,■  .    •  ■      -  ".      ,  •■'    b'--«iJj'iTI  -P-    f5i"!0- 

;■  ■       ■  '    :■  ...      ■     .,.'  ,,         ,'  ;,..      ,  ;.    .;.jif£.-J,l-liH-'giS. 

'  :  ,  ■ . .      .ij'Mi-'O  •   ' '    ■■  ■■■ 

..,■     .;,..J.ji:'!-!       .!:■-    '^O    yW(03Si/Ti; 

■jfcbu3e   t:  '  '     ■■    nvCitb   .xi 


PAGE  III 


PROJECT  REPORT  FORM  (Cpnt ' d) 


So  3 

10.  NCI  -^oe- 

SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH   .      iW:  ADMINISTRATION       £7 

REVIEW  &  APPROVAL  /"7     TECHNICAL  ASSISTANCE  /~7 


12.  None 


COOPERATING  U'JITS  OF  THE  PDBLIG  HEALTH  SERVICE^  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OK  PERSOUNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13.  Not  known. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  IV 


14.  NCI 


PROJECT  REPORT  FORM  (Cont'd) 
03 


SERIAL  NO. 


15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 

Dunn,  T.  B.,  Morphology  and  a  Cure  for  Cancer,  Journal  of  the  American 
Medical  Women's  Aseaclation.  Vol.  10:  101-109.  April  1955. 

Law,  L.  W,,  Dunn,  T;  B.,  arid  Boy Ie>  P.  J.  Neoplasms  in  ttid  C3H  strain  and 
in  F,  hybrid  mice  of  two  crosses  following  introduction  of  extracts  and 
filtrates  of  leukemic  tissues.   J.N.C.I.,  16:  495-539.   1955. 

Dunn,  T,  B.,.  Mammary  Tumors  in  Laboratory  Rodents.  A  lecture  to  a  class 
in  vetefinary  pathology  in  a  course  oti  Diseases  of  Laboratory  Animals. 
Delivered  December  5,  1955.  This  lectuife  to  be  published  in  a  textbpok 
related  to  the  course, 

Andervont,  H.  B.,  and  Dunn,  T.  B.  Transplantation  of  hepatomas  in  mice. 
Published  in  J.N.C.I.,  15:  1513-1524,  from  Proceedings  of  the  Conference 
on  Experimental  Hepatomas. 


16.  HONibRS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955:  ■■•  ■  .       '  ■■■■-■.■  '^V 

Elected  Secretary-Treasurer  of  the  Washington  Society  of  Pathologists,  1955-56, 


PAGE  I 


PROJECT  REPORT  FORM 


I.  National  Cancer  Institute     2.  Laboratory  of  Pathology  __j 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue',  fy.  Pathogenesis  4. 5.  NCI -»ib^ 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  An  electron  microscopic  investigation  of  normal  and  neoplastic  tissues. 

PROJECT  TITLE 

7.  William  G.  Banfield    ^;  i  ^   

PRINCIPAL  INVESTIGATOR(S) 

8.  None 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives:   To  describe  the  ultra  structure  of  selected  neoplasms  and  to 
compare  it  with  suitable  normal  and  control  tissues. 

Methods:   The  usual  electron  microscopic  methods  will  be  used. 

Major  findings:   The  microscope  is  not  yet  in  operation. 


PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI  -5^-?- 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 


RESEARCH  /x/    ADMINISTRATION:  ,   X-A 

REVIEW  &  APPROVAL  /"7    TECHNICAL  ASSISTAnC^  /T' 


12.  None '  '  ':  .:'.:,.,..  ....  ..-^  . 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  tRO-   ' 
VIDIN!?, FUNDS,  FACILITIES,  OR  PERSONNEL,  FOR  TH,IS  PROJECT  IN  EITHER  1956  or  57 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2,  Laboratory  of  Pathology 
INSTITUTE  LABORATORY  OR  BRANCH 


3.  Of  flee  ;of  the  Chief         4. .      5.  NCI  50  6 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHBSDA)   SERIAL  NO. 

6.  Pulmonary  Tumor   ;__ .  .  -   ; 

PROJECT  TITLE 


7.  C.  Harold  Steffee   ,.  -.  ;  ' 

PRINCIPAL  IKVSSTIGATOR(S) 

8.  None ^ 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION         ..  ;        ,    . 

objectives:   (1)  To  gain, basic  information  on  biologic  aspects  of  cancer, 
using  pulmonary  tumors  as  a  tool  in  this  endeavor.   (2)  To 
gain  information  regarding  the  etiology  and  pathogenesis  of  human  lung 
cancer,  and  to  determine,  the  carcinogenicity  of  various  substances 
suspected  as  etiologic  agents  in  human  pulmonary  cancer.  This  requires 
finding  a  method  for  producing  similar  tumors  in  animals. 

Methods:   (1)  Biologic  studies  have  been  done  using  tumor  transplantation 

and  genetic  techniques.   (2)  Attempts  to  induce  experimental 
bronchogenic  neoplasms  in  animals  haye  used  embryo  transplantation  techniques 
and  also  the  intratracheal  administration  of  known  carcinogens  and  suspected 
carc^ihogens  in  adult  mice.  : 

Major  findings:  The  study  of, the  effect  of  size  of  the  original  lung  tumor 

and  the  s.ize  of  the  injected  fragment  upon  transplant- 
ability  has  yielded  -the  following  information.  The  data  suggest  that  th^re 
is  a  critical  size  of  pulmonary  tumors;  smaller  neoplasms  transplant  poorly. 
When  pieces  of  varying  size  of  large  tumors  are  transplanted  the  percentage 
of  "takes"  is  essentially  the  same  for  all  sizes  except  the  very  smallest. 
During  the  course  of  this  experiment;  appvoxiraately  20%  of  the  animals 
died  over  ^weekends  and  were  too  autolyzed  for  definitive  autopsy.   Because 
of  this  and  also  because  the  results  obtained  are  of  border-line  statistical 
significance,  it  was  decided  to  repeat  this  experiment,  checking  the 
animals  on  weekends.   Insufficient  time  has  elapsed  with  the  second  running 
of  this  experiment  to  permit  accumulation  of  definitive  data. 


SERIAL  NO.   NCI. 506  PAGE  II 

PROJECT  REPORT  FORM  (Cont'd) 

9.  PROJECT  DESCRIPTION  (Cont'd) 

The  hypothesis  that  malignant '  cells  dbisdrved  in  the  sarcomatous  trans-   - 
formation  of  the  alveologenic  adenoma  of  the  mouse  are  derived  from  host 
stroma  has  been  tested.   It  ha^  been  found  that  this  hypothesis  is  incorrect, 
(2)  Previous  studies  with  embryo  mouse  lung  transplants,  using  methylcholan- 
threne  as  a  carcinogen,  produced  7  sarc0fflas,  1  squamous-celi  carcinoma,  .  . 
and  2  adenocarcinomas  in  a  total  of  25  such  transplants.  Repetition  of 
these  studies  with  refinements  in  technique  by  which  it  is  hoped  more 
epithelial  tumors  may  be  obtained  have  not  been  completed  as  yet,  A 
similar  study  using  cigarette  tar  as  th6  "carcinogen"  has  failed  to 
produce  any  gross  tumor  in  13  such  transplants. 

An  experiuent,  in  which  several  types  of  cigarette  tars  have  been  adminis- 
tered intratracheally  to  mice  together  with  suitable  control  aobstances 
has  not  yet  bean  completed.  Mice  which  received  intratracheal  cigarette 
tars  and  died  during  the  course  of  the  experiment  to  date,  have  shown  no 
increase  .in  pulmonary  tumors. 

Collaborative  studies  on  the  carcinogenicity  of  various  chromate  fractions, 
carried  on  with  Dr.  Anne  Baetjer  of  The  Johns  Hopkins  University  have  not 
yielded  gross  tumors  to  date.  Microscopic  studies  have  been  completed 
on  approximately  half  of  the  animals  so  exposed,  and  these  sections  show 
.chiefly  a  non-specific  inflammatory  reaction  and  rarely  the  formation  of 
tiny  granuloraata.  -  ., 

...■.■'  ■  ■■  ■■<'•■■-  .  .  ;:;/i!SjJOd:t/!<.  If;;-  Xo'3-*^-"'   .  .■   ■  ■■  ^■■■-    ■'•'■ii 

Significance  to  "cancer' teseatch;-  Study  bf  the  relationship  of  tumor  slJse 

to  transplantablllty  is  a  problem  in  the  fundamental  nature 
of  malignancy.   Definition  of  the  exact  point  at  which  benignity  becomes 
malignancy  will  sharpen  the  focus  for  further  biological  and  biochemical 
r studies.  The  fact  that  the  sarcoma  cells  which  often  arise  on  serial 
transplantation  of  mouse  lung  tumors  are  not  derived  from  the  host  serves 
,  to  eliminate  this  one  possibility  in  the  search  for  the  source  of  these,  :.: 
cells.  The  importance  of  this  search  to  the  general  problem  is  one  of  .-.. ■ 
understanding  the  fundamental  cell  behavior  as  well  as  ap  understanding 
of  the  occasional  carcinosarcomas  which  are  observed  in  the  human.  The 
Other  experiments  performed  in  this  project  represent  a  more  direct  attack 
on  the  human  cancer  problem.   Bronchiogenic  carcinoma  is  now  in  many  areas 
the  commonest  malignancy  of  men,  and  seems  to  be  increasing  in  frequency. 
Many  laboratories  throughout  the  country  are  engaged  in  the  search  for   ; 
etiologic  agents  but  all  of  this  work  is  seriously  hampered  by  the  lacl^ 
of  a  suitable  ..test  ohject.  Thus,  we  have  placed  much  emphasis  on  trying 
to  produce  an  analagous  tumor  experimentally.  Simultaneously,  we  have  ;.;■ 
studied,  the  effects  on  experimental  animals,  of  two  substances  which  haveth 
been  implicated  in  human  neoplasia^  namely  cigarette  tars  and  chromateis. 
Though  these  are  but  two  pf  the  many  suspected  etiologic  agents,  they  iat 
least  represent  a  patt  of  the  attack  on  the  vast  problem  of  human.  luhjg;,ci 
cancer.  u   io 


JERIAL  NO.  NCI.  506  PAGE  III 

PROJECT  REPORT  FORM  (Cont'd) 

).  PROJECT  DESCRIPTION  (Cont'd) 

Proposed  course  of  the  project.  Since  the  principal  investigator  is 

leaving  the  National  Cancer  Institute  in  December  of  1955,  many 
of  these  studies  of  an  exploratory  nature  will  be  terminated.  The  animals 
bearing  transplanted  pieces, of  lung  tumors  in  the  study  of  the, effect  of 
transplant  size  and  tumor  size  upon  transplantability  will  be  allowed  to 
live  out  thcair  life  span  in  order  that  this  data  and  this  experiment  can 
be  completed.   In  the  study  on  sarcomatous  degeneration  of  transplanted 
lung  tumors,  no  further  transplants  will  be  done  even  though  all  of  the 
tumors  have  not  yet  undergone  the  degenerative  changes.   I  believe 
sufficient  data  is  already  at  hand  to  support  the  conclusions  given  above. 

Experiments  on  embryo  transplant  techniques  and  with  intratracheal  _^_  ,\ 

adinini is t ration  of  cigarette  tars  wLH  be  terminated  and  the  results-  '   •  •   "i 
prepared  for  publication  if  this  seemis  Warranted .   The  remaining  slides 
in  the  collaborative  study  with  Dr.  Baet jer  Will  be  studied  but  no  hew 
experiments  are  planned  in  this  area  since  the  grant  to  Dr.  Baetjer  will 
terminate  in  1956.  Another  collaborative  study  has  been  planned  with  the 
Medical  Director/  Randall"  B.  Haas,  Vf,   S.   Public  Health  Service  Hospital, 
Manhattan  Beach/  to  inveistigdte  othier  possible  medhariistns  of  pulmonary 
carcinogenesis.  These  experiments  will  include'  intratracheal  administration 
of  carcinogenic  hydrocarbons  adsorbed  on  carbon  particles,  to  tuberculous 
and  non-tuberculous  animals',  'to  iietdrmlne  the  lipoid  solvent  effect  of 
caseous  necrosis.  These  experiments  will  also  include  studies  on  certain 
enzymes  which  have  been  reported  to  cause  squamous  metaplasia  of  bronchial 
epithelium  in  humans.   The  animal  work  in  these  experiments  will  be  done 
at  Manhattan  Beach;  the  pathologic  material  will  be  examined  by  the  principal 
Investigator  in  his  tiieW'locatiari.  .-•.It'  is- expected  that  this  experiment  will 
get  underway  shortly  after' the  fir6t  Of  the  hSxt  daleridar,  yegir..  We  haye 
prepared  the  carbon  particles' for  these  exp'eirlments,  hdve  tested  their 
toxicity  on  guinea  pigs,  and  will  shortly  ship  them  to  Dr.  Haas. 


:J  yiri^'i 


PAGE  IV 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI  506 
SERIAL  NO. 


•f' 


11. 


'  ,  ^:;i:>  ■'So  ;■;*«:.-■ 


BUDGET  ACTIVITY:   'io  ■•v-ki.3'S   tsffS   wi  :s';j-.:  aJ  -aiitj; :  .:t'.j    -../OiUq    • 
«;*.:.':  -RESEARCH  /^ADMINISTRATION 


A./ 


';   /   ,    REVIEW  &  APPROVAL  £;/    TECHNICAL  ASSISTANCE?  7^/'   =  ^ 

COOPERATING  UNITS  OF -THE  PUBLIC  HEALTH  SERVICE,  OR;  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES/  OR  PERSONNEL  FOR  THIS-  PROJECT  IN  EITHER  1956  or  1957 

1)     PHS  Gratit  C'-603,   providing  funds  to  Dr.  Anne.  M.;  Baetjex,  Department    • 
of  Environmental  Medlfeine,  The  Johns  Hopkins  University,  615  North  Wolfe 
'    Street,   Baltimore,  Maryland,    f or  experlTOents  on  inhalation- and  intrat     . 
tracVieal'  injection  O'f;.chromates,i   ',    rsrsio  o'^BfjiiEjftswuiOdi  ' .rfijaaS  isi.aS:  '.   ■■'■■'' 
•  ■       '■-         ■.■•:;..,.,  V     ;.  .         ■■;.  I  w';t>?j;v;'.  :;■.■.«.■■■;  ■  s*^'-dT    ■  :,^{M.n..'noni^:\:■■■ 

2)     U.   S.   Public  Health  Service  Hospitdl,  Manhattan  Beach,   Brooklyn  35>N.Y. 


'>j3*':*fswl»«.r'oaa!--.  iiii4  sfj- 


"iip^rifi  •  »s.iaoitoan'  &|j\)::^^i:i 


13«  No  similar  research  kndwn^  to  be  in  progress  elsewhere.   -     . 

IF  THIS  PK)JECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute  2.  Laboratory  of  PatholoRy 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  eancer  loduc.  &  Pathogenesis  4. 5.  NCI -^.Qg- 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  NO. 

6.  Mitochondrial  changes  in  the  liver  of  X- Irradiated  mice. ■■  . 

.  PROJECT  TITLE 


7.  Ross  C.  MacCardle  and  Charles  C.  Congdoo 


PRINCIPAL  INVESTIGATOR(S) 


8. 


OTHER  INVESTIGATORS 


9.   PROJECT  DESCRIPTION: 

Objective:  To  determine  whether  cytoplasmic  elements  are  the  primary  focus 
of  initial  damage  in  X  radiation  injury,  and  to  study  the 
anatomical  basis  of  recovery  from  X  radiation  injury  that 
follows  injection  of  bone  marrow  into  irradiated  mice,  we 
studied  mitochondria  and  other  ;CytQlogical  structures  by  the 

Methods:    usual  methods  at  early  periods  postirradiation. 

Major  findings:   It  was  found  that  mitochondria  of  the  liver  certainly  are, 
altered  long  before  the  animal  succumbs,  and  the  mitochondria  - 
recover  their  normal  state  after  bone-marrow  treatment.  This 
may  be  a  factor  in  X  radiation  injury.  This  has  led  us  to 
study  the  state  of  the  hepatic  and  serom  phospholipids  and 
choline,  the  mitochondria  after  x-raying  the  liver  only,  and 
after  shielding  the  liver. 

Significance  to  cancer:  Having  found  a  definitive  change  in  mitochondria, 
we  now  proceed  to  study  the  mitochondria  of  the  reticulo- 
endothelial system  in  spleen,  bone-marrow  thymus,  and  lymph 
nodes.  We  are  studying  the  thymus  of  C57B1  and  LAFj^  mice  in 
which  X  radiation  produces  lymphocytoraa.  This  involves  a 
cytological  study  also  of  thyraectomized  mice  in  which  X  radiation 
fails  to  produce  tumors. 

Proposed  course:  We  wish  to  finish  the  study  of  the  liver  and  bone-marrow 
after  X  radiation,  and  study  the  other  cytological  structures 
which  we  have  already  preserved  in  this  project.  We  wish  also 
to  make  a  comparative  cytological  study  of  the  thymus  of 
various  strains  of  mouse  in  respect  to  their  susceptibility  to 
formation  of  tumors  at  different  ages. 


^7 

10.  NCI  403- 
SERIAL  NO. 


PAGE  II 


PROJECT  REPORX  FORM.  (Cont'd) 


IX. 


BITOGET,  ACUVJtY.: 


RESEARCH         [^1         ADMINISTRATION       /_/ 
REVIEW  &  APPROVAL  [J         TECHNICAL  ASSISTANCE  7j7 


12.   None     '.-.:V  •.■'•  :'^  ...■:;. ,:;/■■•  .  r.  ,  '    ■^:;\;  ■..,;::       ■ 

COOPERATING  UNITS  OlF  THE  PUBLIC  HEALTH  SERVICE,  OR  ,<)THER  ORGANIZATIONS,  .PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13.  None 


IF  THIS'  PROJECT  :RBSEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH i DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTEI^CHANGE  OF. PERSONNEL,  FACILITIES  . 
OR  FUNDS),;  IDENTIFY  SUCH  RESEARCH:     r  :         ,  .  :  ' 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


14.  NCI  »ee- 

SERIAL  NO. 


15. 


PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 

The  present  part  of  this  project  in  respect  to  tumors  will  be  done  probably 
in  conjunction  with  Dr.  Lloyd  Law  of  NCI.  The  remainder  of  it  is  being 
done  with  Dr.  Congdon  v;ho  is  now  at  Oak  Ridge  National  Laboratory  in 
Tennessee. 


16.  None 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955 


H'jjr. 


PAGE  I 


PROJECT  REPORT  FORM 


I  Mo^^nna^  rancer  Institute     2.   Laboratory  of  Pathology __ . 

•  INSTITUTE  LABORATORY  OR  BRANCH 

I     ■       ■  5  NCI  "^'0^ 

^-  SEmON  OR^kvtcE"'"-^"^^  'location  (IF  OTHER  THATlEfttESDA)  ' SeHa^ 

6.  Comparative  ^tudy  of  the  macroscopic  fortn  and  cytological  structure  of 
normal  human  epidermis  and  human  hair  follicles  to  evaluate  changes  in 
pathologic  states  such  as  in  basal-cell  tumors,  squamous-cell  tumors, 

intraepidermal  tumors,  acne,  mycosis  funeoides.  and  other  diseases _^_„ 

PROJECT  TITLE  -    H  ,   ;■        -^^  -   > .  •       ,-• 


7.  Ross  C.  Macr.Ardle  with  Dr.  E.  J.  Van  Scott  of  NIK 
PRINCIPAL  iNVESTIGATOR(S) 


8. 


OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 


Wooden  models  are  being  made  of  whole  hair  follicles  in  health  and 
disease  that  reveal  striking  differences  in  size  and  Pattern.  Minerals 
and  athercytoiogical  constituents :wiil:he  studied  at  different  levels  of 
the  follicle.  This  study  of  models  has  ;|ipt  been  attempted  previously. 


PAGE  II 
PROJECT  REP.QRT  FQRM  (Cont'd) 


11. 


;\;BWKJEX  ACTIVITY:  ., 

RESEARCH         /x7   ADMINISTRATION       £7 
REVIEW  &  A^PRdVAL' £7  .  TECHNICAL  ASSISTANCE  £7 


^^ • COOPERATING  DNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  FRO- 
SSlwf  FUNDS,  FACILITIES,  or  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


ITAOIXisiiVfeiii   iSiiilj" 


"'Tr?r:^;3fi  tDUC4>: 


^^'tp^THTS  project  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DQNE  ELSEWHERE 
iS  TOE  Pmic  HeS^^^^       (WITHOUT  iNTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 


FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology - 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &  Pathogenesis  4. ' 5,  NCI  SCt 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  NO. 

6.  Relationship  of  Np.di  oxidase  (cytochrome  oxidase)  granules  to  mitochondria 

in  tioraal  and  malisnant  tissue - 

project'  title 


Ross  C.  MacCardle 


PRINCIPAL  INVSSTIGATOR(S) 


None 


OTHER  INVESTIGATORS 


PROJECT  DESCRIPTION 

During  the  past  two  years,  it  wae  discovered  by  the  use  of  ultracentri- 
fugation  that  the  Nadi  oxidase  granules  and  mitochondria  resident  in  the 
living  cytoplasm  are  anatomically  independenu  of  each  other  in  situ  in  the 
normal  intact  cell  of  enithalrvn  of  the  eoidtdymis,  kidney  and  liver  of 
mouse.  The  form  of  the  Nadi  o;.iJase  granule  is-  distinctly  ..different  from 
that  of  the  mitochondrion.   It  ha&  now  been  found  that  in  hepatoma  of  the 
mouse  they  are  also  anatomically  separate.   However,  in  the  case  of'  the 
malignant  dependent  thyrotropliin-secreting  transplant  tumor  of  the  pituitary 
gland,  the  Nadi  oxidase  granules  can  be  dislodged  by  ultracentrifugation  to 
one  pole  of  the  cytoplasm  along  with  the  mitochondria,  whereas  the  granules 
of  the  normal  pituitary  gland  cannot  be  moved  by  the  same  centrifugal  force 
required  to  move  the  mitochondria  --  the  mitochondria  of  a  normal  gland 
thus  being  moved  to  one  pole  of  the  cell  leaving  the  Nadi  granules  unmoved. 
This  may  not  necessarily  mean  that  the  mitochondria  and  Nadi  oxidase 
granules  of  a  tumor  are  anatomically  associated,  however.   It  may  indicate 
that  the  viscosity  of  the  malignant  protoplasm  is  less  than  in  normal  cells, 
thus  permitting  the  granules  to  be  moved  with  greater  ease,  or  it  may  mean 
that  the  weight  of  the  granules  of  this  tumor  differs  from  that  of  the 
granules  of  the  normal  gland. 


PAGE  II 
PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI  BQir        ^ 
SERIAL  NO. 


BUDGET  ACTIVITY:  '■■      ■  ^  -  ■  ■.^. •■-,■;  r.r"."-^"":Tr^n""' 

RESEARCH         /x7   ADMINISTRATION       £7 
REVIEW, &  APPROVAL  £7   TECHNICAL^ ASSISTANCE  £7 


12.   None 


COOPERATING  U>[ITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEl'IENTS ,  OR  PARALLELS  RESEARCH  DONE:  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCH^^NGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS);  IDENTIFY  SUCH  RESEARCH: 


s^rortf- 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  1 


PROJECT  REPORT  FORM 
1.  National  Cancer  Institute  ..  2.  Laboratory'  of  Pathology 

institute;  .   ,   .       laboratory  or  branch 

3.  Cancer  Sd^uc.  &.  Pathoi^eaesis  4.  . S.NCI-^W- 

SECTION  OR  SERVICE   "  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO 

6.  Intracellular  cytology  of  thyrotrophln- secreting  tumors  of  the  anterior 
lobe' of  the. pituitary  gland  of  radiothyroidectotnlzed  mice,  and  of  human 
pituitary  tumors »     . 

PROJECT  TITLE, 

7.  Ross  C.  MacCardle         .  .    ■    , .  ,. 

PRINCIPAL  INVESTIGATOR(S.) 

8. 

OTHER  INVESTIGATORS 


9.  PR9JECT  DESCRIPTION: 

Object;  The  object  of  this  study  is  to  ascertain  and  define  the  earliest 

morphological  alterations  that  occur  in  cells  of  tissues  in  their 
change  from  the  normal  to  the  malignant  state  of  protoplasm  in  respect  to 
a  possible  analysis  of  the  underlying  cause  of  the  malignant  transformation. 
This  tumor  of  the  mouse  represents  one  of  the  most  valuable  experimental 
tumors  because  it  resembles  pituitary  tumors  of  man.   It  clearly  consists 
of  a  single^  hlstologically-identifiable  type  of  tissue.   One  or  another 
of  the  constituent  cells  of  the  anterior  lobe  proliferate  in  different 
states  of  hyperplasia  and  malignancy.   The  gland  is  stimulated  to  become 
hyperactive  in  producing  the  thyroid-stimulating  hormone  for  a  thyroid 
gland  that  has  been  essentially  destroyed  by  treatment  with  radlolodine. 
Finally,  the  hyperplastic  tissue  becomes  malignant  to  produce  10  times 
as  much  hormone  as  the  normal  gland.   The  amount  and  kind  of  hormone 
produced  by  a  tumor  can  be  determined,  and  the  cytologlcal  alterations  of 
the  same  tumor  can  be  identified  and  related  to  the  function. 

Methods ;   Cytologlcal  methods  can  detect  alterations  in  cellular  minerals 

(microincineration),  mitochondria,  Golgl  apparatus,  nucleoprotelns, 
cytochrome  oxidase  granules. 

Findings ;   The  major  findings  so  far  indicate  that  the  Golgl  apparatus 

first  becomes  hypertrophied  and  more  readily  metallophilic  as 
oore  cytoplasmic  iron  becomes  deposited  in  the  vicinity  of  it.   The 
staining  properties  of  the  mitochondria  of  the  tumor  are  different  so  that 


SERIAL  NO: 


NCI  ,&ee-  ^ 


PAGE  II 


PROJECT  REPORT  FORM  (cont'd) 

9.   PROJECT  DESCRIPTION  (Cont'd) 

they  cannot  be  destainediais  easily  as/thdse- of  the<  ttorttial  gland.  ;Most-of  .  ; 
the  cells  of  the  malignant  ,thyrotrc>phin'-secretlng  tumor  appear  to'cirlgihate 
from  alpha  cells^  thus  suggesting  that  TSH  may  be  formed  in  these  cells.  At 
least  in  the  malignant  state.  The  cellular  constituents  of  the  hyperplastic 
tissue  have  not  been  identified  with  production  pf-TSHi  and  there,  is  no.  .; 
!  evidence  that  alpha  ceiis  of  the  normal  gland  produce  thyrotrophinii  :  :■  ' 

Proposed  Course;  The  proposed  course  of  the  project  is  to  vtudy  the  fetal, 

:i  t  1'  adult -and  hyperplastic. glaiiid  to  compare  the  relation  of  .. 
cytologicali changes  to  prdducti6n> of  hormones  with  those  of : primary  and  . 
transplant  malignant  tumprs!.   It  is  clear  that  a  study  of  the. effect  of .  ; 
TSH  on  the  cells  of  an  already  developed  TSH-tumor  should  be  made  to  .  s 
determine  the  cellular  organoids  first  affected  when  the  activity  of  the 
tumor  begins  to  diminish  in  the  presence  of  TSH.   Comparative  cytological 
studies  of  human  and  animal  pituitary  tumors  should  be  accoinplished.;  ;  It:  .  i 
is  proposed  to  study  the  effect  of  radioiodine  on  young -and .adult  dwarf -> 
homozygous  mice  that  lack  pituitary  alpha  cells. 


HCMa:* 


\Wi^T^'mm^.  s^^i'^y.^-   , 


?.:t!«;3fcj; 


i.;;^jl[5,  3lfc!' 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


10.    Nci-»ee- 

SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         /^    ADMINISTRATION       fj 

REVIEW  &  APPROVAL  £7         TECHNICAL  ASSISTANCE  £7 

12.   None 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  OR  195/ 


13.  No 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  tV 
PROJECT  REPORT  FORM  (Cont'd) 


14.  NCI  -^ear 

SERIAL  NO. 


15. 


PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  195 

MacCardle,  R.  C,  and  C.  C,  Congdon,  1955: 

"Mitochondrial  changes. in  hepatic  cells  of  X-irradiated  mice." 

Am.  J.  Path.  31:  725-745 


MacCardle,  R.  C.   1955: 

"Characteristics  of  mitosis  in  tumor  cells." 
In  press:  Annals  of  N.  Y.  Academy  of  Sciences. 


16 .  None 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955. 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &,  PatboReneais  4. 5.  NCI  4et- 

LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  NO. 

6.  Writing  a  brief  Monograph  entitled:   "The  Tumor  Cell:   Characteristics  of 
Malignant  Protop?.p.sm"  as  part  of  a  series  of  volumes  of  An  International 
Treatise  on  Physiology  of  Protoplasm  "Handbuch  der  Protoplasmaf orschung" 

being  edited  by  Heilbrunn  and  Weber. 

PROJECT  TULE 


7.  Ross  C.  M^cCardle 

PRINCIPAL  INVESTIGATOR(S ) 


OTHER  INVESTIGATORS 


9.   PROJECT  DESCRIPTION: 

Object:  The  object  of  this  monograph  is  to  summarize  and  correlate  our 

knowledge  of  the  properties  and  possible  identification  of  a 
malignant  cell  of  a  tumor. 

Method:   By  reviewing  the  literature  and  relating  some  new  data  found  by  the 
author^  it  is  clear  that  although  a  single  white  blood  cell  of  the 

Major  findings: 

circulating  blood  of  a  leukemic  mouse  can  upon  injection  into  a 
normal  host  produce  leukemia  (notwithstanding  the  serum  injected 
with  the  cell),  such  a  cell  possesses  no  anatomical  features  by 
which  it  can  be  distinguished  from  its  normal  cell  of  origin.  The 
most  malignant  tumor  constitutes  several  types  of  cells,  some  of 
which  may  have  nothing  to  do  with  malignancy  and  none  of  which  can 
be  detected  as  a  malignant  cell.  The  transplantability  of  the 
malignant  tumor  cells  is  discussed,  and  the  cytology  of  several 
tumors  is  described  for  the  first  time. 

Significance  to  cancer:  This  paper  will  bring  together  old  and  new  data 
in  an  effort  to  evaluate  the  status  of  our  knowledge  of  the 
mechanism  of  the  transformation  of  normal  protoplasm  to  malignant 
protoplasm. 

Proposed  course:     I  propose  that  it  now  be  printed  in  book  form,  and 

it  is  nearly  ready  to  go  to  the  printers.   I  was  invited  to  write 
It,  and  I  understand  that  it  was  accepted  in  advance. 


PAGE  II 


PROJECT  REPORT  FORM  (pont'd) 


10.    NCI-404r  / 

SERIAL  NO. 

11.-  ■■  ;,..■'  ■,  .  ■ 


BUDGET  ACTIVITY: 


RESEARCH         /x/    ADM7.MISTRATI0N       /_/ 
REVIEW.  &  AFFROVAL  £7  .  TEGU.NXCAL  ASSISTANCE  /j' 


12.  None 


COOPERATiKG  CNITS  OF  THE  PUI^LIC  RRALTH  SERVICE,  OR  O'/HER  ORGANIZATIONS /PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13.   None 


IF  THIS  PROJECT  RESEME.LES,  COMPLEMENTS,  OR  PARALLELS,  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  Of'  PERSONNEL,  PACILITiES 
OR  FUNDS),  IDEHTIFV  SUCH  RESEARCH: 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  I 


PROJECT  REPORT  FORM  ■ 

1.  National  Cancer  Institute 2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &  Pathogeoesis  4. 5.  NCI  -&e4-  ^ 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  NO. 

6 .  Intracellular  cytology  of  human  and  animal  tumors. 

PROJECT  TITLE  •   .    . 


7.  Ross  C.  MacCardle 

PRINCIPAL  INVESTIGATOR(S) 

8. ' 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION: 

Object:   Little  is  known  of  the  basic  intracellular  cytology  of  the 

cytoplasm  of  human  or  animal  tumors.  Most  of  the  modern  studies 
deal  with  electron  microscopy  (a  method  limited  to  tissue  fixed 
in  one  or  two  ways)  and  certifugal   isolation  of  mitochondria 
of  a  polyglot  tumor  (hepatoma)  of  the- mouse  (the  "mltophondria" 
being  of  questionable  integrity  and  identify  after  crushing  the 
cell). 

Method:   Using  light  microscopy  and  ordinary  cytological  methods,  I  have 
been  studying  the  minerals,  Golgi  apparatus,  mitochondria  and 
mitotic  figures  of  various  tumors  in  an  attempt  to  evaluate  some 
of  the  remarkable  features  found  by  electron  microscopy  in  tumors 
that  have  not  been  studied  by  ordinary  methods.   I  have  been 
making  a  comparative  cytological  study  of  white  blood  cells  of 
circulating  blood  in  human  leukemia  in  relation  to  the  structure 
of  cells  of  the  bone  marrow.   I  have  found  that  the  Golgi  apparatus 
is  in  the  form  of  widely  dispersed  dictyosomes  in  the  argyrophobic 
cytoplasm  of  the  young  transplant  Sarcoma  37  in  mice,  whereas  it 
is  a  small  compact  network  in  richly  argyrophilic  cytoplasm  of  the 
older  vascularized  transplant  tumor.  This  seems  to  indicate  a 
detectable  difference  in  slowly-growing  and  rapidly-growing 
malignant  tissue.   I  am  also  studying  basal-cell  tumors,  squamous- 
cell  carcinomas,  superficial  intraepidermal  tumors,  Bowen's 
disease,  and  Xeroderma  pigmentosum  in  the  skin  of  man. 
Proposed  study:  I  expect  to  continue  this  study  along  the  same  lines. 


Page  II 
PROJECT  REPORT  FORM  (Cimt'd) 


10.  NCI  -see^  / 

SERIAL  NO. 


11. 


BUDGET  ACTIVITY:     ■  ' '  '!■^■ 

RESEARCH         /x7    ADMINISTRATION       fj 
REVIEW  &  APPROVAL  £7         TECHNICAL  ASSISTANCE  /[J 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  195^  or  1955 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  14,  15,  or  16. 


FAOE  I 


PROJECT  REPORT  FORM 

1.  National  Cancer  Institute     2.  Laboratory  of  Pathology  '-         ■ :' "  ■ 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Pathology  Section      4 .-  •  ■ ;.5 .  NCI  -S^aa- 

SECTION  OR  SERVICE  LOCATION  (IP  OTHER  THAN  BETHESDA)  SERIAL  NO. 

6.  Mechanisms  of  tumor  invasivenesa>  and  the  influence  of  chemical  agents  upon 

them. __«________«-.^ '  ■ 

PROJECT  TITLE  ■,  :  .-■.;:?  i  ■   >v .i^li*i '?«•;■ »» ss ;i: ii i  ^  *; 3  ;,«.,.,.■ 

7.  Joseph  Leighton  .   '  .   .  .  .,/;  V;,  •;  j  ■■^:-  ■■■  ■. \ _______ 

PRINCIPAL  INVESTIGATOR(S)  .  ^«-  t-. 

8.  Murray  Belkin  (Laboratory  of  Chemical  Pharmacology),  Ira  Kline  (Laboratory 

of  Chemical  Pharmacology) 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives:   (1)  The  further  development  of  quantitative  methods  for  eva:laiating 

a)  the  destructive  invasion  of  normal  human  tissues  by  human 
cancers  in  tissue  culture,  and  b)  the  interaction  in  vitro  of  host  cells  and 
tumor  cells. 

(2)  The  study  of  the  mechanisms  of  local  invasion  and  of 
metastases  in  tissue  culture  with  special  emphasis  on  the  metabolic  require- 
ments of  these  malignant  processes  using  as  experimental  tools  biochemical 
agents  such  as  cortisone,  hyaluronidase,  stilbesterol  and  analogues  of 
metabolites. 

(3)  The  appraisal  of  the  influence  of  chemical  agents  on  tumor 
invasiveness  in  which  the  pharmacologic  effect  is  considered  in  terms  of 

a)  altered  rate  of  invasiveness,  b)  changes  in  the  organization  and  differen- 
tiation of  the  tumor,  and  c)  the  responses  of  the  host  tissues  which  may 
tend  to  limit  the  spread  of  the  tumor. 

(4)  The  discovery  of  the  conditions  which  must  be  supplied  for 
the  tumor  in  tissue  culture  so  that  progressive  growth  may  be  obtained  from 
a  large  percentage  and  a  wide  variety  of  types  of  clinical  cancers,  making 
chemotherapeutic  studies  possible. 

(5)  Cultivate  tumors  from  the  operating  room  to  observe  the 
extent  of  differentiation  in  vitro  as  a  supplementary  aid  to  the  evaluation 
of  sections  made  of  biopsy  and  autopsy  material. 

Methods;  Sponge  matrix  methods  for  tissue  culture  are  being  used  in  this 
work. 

Major  findings:   (1)  Tissue  culture  studies  on  the  destructive  Invasion  of 

normal  tissues  by  cells  of  human  cancer  have . been  further 
extended.   Invasion  by  several  cancer  cells  has  been  observed  including 
a)  strain  HeLa  (Gey),  which  arose  from  an  epidermoid  carcinoma  of  the  , 


dV<5> 
SERIAL  NO.  IICI-$«-  |>A^  IX 

tioiia  uron  9»iii  (€«i»t*4> 

9.  PROJECT  DESCRiniOK  <ewH*0 

cervix>  b)  «tr4to  KB  |Batl«>  «%l«|  M^if  .fv«a  «  •ieclnoiiia  of  t^^ 

c)  strain  J-96  (08go«4>  vkUl  «Im«  twom  k  iiibna«jrtlc  Uwhcmia.  A  variety 

of  normal  huiRantl9s««8  4t«  t*«iitfif4  ijnf  r«fUce#  by  chtie  tumor  cells. 

(2)  further  ttvilif  oi  ir«t4>tlom  of  sponge  matrix  methods 
have  been  developed  vliicli  fCMilk  tie  ifB«ilit«#BB*  •i   Ike  speed  of  the 
invasive  process. 

p)  The  spontas«»«fl  t««Mlora»«ioit  of  normal  human  connective 
tissue  cells  into  a  histolof  Uallf  MUgMftt  lwiior>a8  been  observed.  A 
paper  describing  this  event  tf  l»  fr«M  to  2£i£S££>  Studies  are  in  progress 
on  the  biologic  malignancy  •#  tM«ji«li  i^e  in  eultures  where  it  is 
combined  with  normal  human  tts«««s  awl  6sk  Heterologous  animal  transplantation. 

(4)  A  study  OR  one  foleiitial  chemotherapeutie  agent ^  NCI  #3022, 
a  water  soluble  podophylletextn  Aevivatlve  has  been  completed  and  a 
manuscript  describing  our  experlnsMs  is  being  prepared. 

(5)  Several  **iienMl**  tell  lines  originating  in  other  labora« 
tories  have  been  ejcamined  in  s|««g«  «8t«ls«  In  some  instances  these  "normal*' 
cells:  are  morphologically  iftdlttlaguifhaliie  from  highly  malignant  cancer. 

'The;  biologic  properties  of  thes*  tells  in   terms  of  their  invasive  capacity 
is  being  studied. 

Significance:  The  productivity  of  this  ftoject  has  In  large  measure; 

developed  from  the  aalMre  of  its  organization.  It  has  been 
one  of  close  coilaboratioA  ani  iiilegfation  of  efforts  of  personnel  of  two 
laboratory  departments.  Pathology  and  Chemltal  Pharmacology. 

Methods  have  been  developed  vhieh  fermit  the  experimental  9t;udy  of  the 
invasive  powers  of  human  eaneer  so  that  the  metabolic  requirements  of 
invasion  as  well  as  potential  inhibitors  of  invasion  can  be  studied. 

Observations  have  been  made  in  combinations  of  tumor  with  normal  tissues 
that  may  contribute  directly  to  our  understanding  of  the  invasive  process. 
Cells  growing  out  of  several  normal  tissues  invade  tumor  cell  masses  to  an 
extent  comparable  to  the  invasion  by  the  tumor  of  other  normal  tissues.  , 
This  raises  the  possibility  that  in  some  instances  normal  cells  in  the  body 
may  enter  and  divide  a  tumor  noJule  giving  rise  to  several  new  centers  of 
tumor  growth.  A  second  observation  which  may  be  significant  is  a  negative 
one.   In  Invasion  experiments,  one  almost  never  sees  degenerating  forms 
among  the  normal  cells  in  the  areas  being  invaded  by  tumor.  It  is  as  if 
the  normal  cells  disappear  completely,  either  by  migration  or  very  rapid 
dissolution. 

Proposed  course:  The  principal,  investigator,  has  planned  to  accept  a  position 
at  the  University  of  Pittsburgh  in  May>  1936,  where  he  will  have 
an  opportunity  to  teach  as  well  as  to  continue  his  rftsfearch  work.  The  project 
will  be  pursued  there,  and  the  facets  of  it' that  are  of  direct  interest  to 
the  Laboratory  of  Chemical  Pharmacology  will  continue  to  be  studied  at  the 
N.I.H.   It  is  planned  and  expected  that  many  aspects  of  the  fruitful  collabora- 
tion existing  up  to  now  between  Dr.  Belkin|  Mr.  Kline,  and  the  principal 
Investigator  will  contisuf ,  regardless  of  the  distance  between  cities. 


PAGE  III 


PROJECT  REPORT  FORM  (Conf^d) 


10.  NCI  %i9r- 

SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         ZH/    ADMINISTRATION       fj 
REVIEW  &  APPROVAL  /~7    TECHNICAL  ASSISTANCE  /T 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


1 3 .  No  similar  rea earch  is  known  to  be  in  progress  elsewhere. 

IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH 


PAGE  IV 


PROJECT  REPORT  FORM  (Cont'd) 


14.  NCI-&g'g— 
SERIAL  NO. 


15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING , CALENDAR  YEAR  1955 

Major  contributor  to  An  Introduction  to  Cell  and  Tissue  Culture ,  by  the 
staff  of  The  Tissue  Culturie  Course,  Cobjperstown,  New  Vork>  1949-53, 
Burgess  Publia^jlng  Company,  ,1955,. 

r'N'saw.,..  ,  •         ■■■■■■■■■■■■■'■        \,j:  •'-^-.-sr-'-  ,/■*;;,■•-;■  :i  ;>  ^ 


16.  HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955 

Guest  Lecturer  and  Consultant  on  Tissue  Culture  to  the  Department  of 
Pathology,  New  England  Deaconess  Hospital,  July  11  &  12,  1955. 

Lectures  at  Camp  De trick,  Maryland;  Naval  Medical  Research  Center,  Bethesda; 
and  Tissue  Culture  Course  at  Cooperstown,  New  York. 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology '• 

INSTITUTE  LABORATORY  OR  BRANCH 

\  .■',■■  vT// 

3.  Cancer  Indue  &  Pathos enesis   4. .    ;  5.  NGI-4a3- 

SBCTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Effect  of  neoplastic  diseases  and  of  various  stresses  on  the  pathophysiology 

and  morphology  of  certain  endocrine  organs  in  man  and  in  animals. 

PROJECT  TITLE 

7.  E.  M.  Nadel  . '    ; 


PRINCIPAL  INVESTIGATOR (B) 
8.  None  (See   Item  #12) 


OTHER  INVESTI.GATORS 

9.  PROJECT  DESCRIPTION   w 

Quantitative -measurement  of  polar  reducing  corticosteroids  in  man  and  in 
animals  by  the  use  of  recently  developed  paper  chromatographic, fractionation 
techniques. 

Objectives:  To  quantitatively  measure  the  .polar  corticosteroids  present 
in  the  bloody  urine,,  and  tissues  of  man  and  animals,  (normal, 
stressed^  and  tumor-bearing)  and  to  qorrelate  where  possible  the  clinical, 
course  with  the  adrenal  and  other  morphologic  alterations  and  with  specific 
corticosteroid  excretion  patterns*  .      ; 

Methods:  The  term  stress,  is  used  in  its  widest  concept  for  the  purposes  of 

this  report  and. includes  dietary  deficiency  and  excesses  as  stresses, 
hormonal  excesses,  and  deficiencies  as  stresses,  x-ray  irradiation  effects, 
stress  by  physical  and  microbial  agents,  carcinogens,  and  the  effect  of 
carcinoclastic  and  potentially  carcinostatic  drugs. 

Animals:   Stressed  animals,  normal:  controls  and  tumor-bearing  animals  are 
prepared  biologically  and  the  clinical  course  followed.  Tissues,  blood, 
and  urine  are  collected  during  the  course  of  the  observation  period  and  at 
post-mortem  examination,  for  chemical,  histsOchemical,  and  histopathological 
study.   Extraction)  purification  and  analysis  are  done  by  specific  applica- 
tions of  chromatographic  and  spectrpphotonietric  methods. 

Man:   Clinical  patients  from  the  Leukemia  Service,  NCI,  Surgical  Service,  NCI, 
and  Psychosomatic  Service,  NIMH,  are  being  studied  in  addition  to  non-patient 
controls.  Analyses  for  polar  corticosteroids  are  performed  on  the  urine,  of 
patients  before,  during,  and  after  the  administration  of  ACTH. 


SERIAL  NQ.  mi  ^2^  PAGE  II 

PROJECT  REPORT  FORM  (Cont'd) 

9.  PROJECT  DESCRIPTION  (Cont'd) 

Major  findings:  The  pattern  of  exexetlon  of  the  new  tiolar  corticosteroids 

has  now  been  studied  extensively  in  guinea  pigs  with 
leukemia,  with  liposarcoma,  following  stllbesterol  treatment,  following 
-  ACTH  administration,  following  x-radiation,-  etc<  .  (1)  There  are  qu^intitative 
differences  in  the  excretion  of  6-^  hydroxycortlsol,  Steroid. lla,  and: 
Cortisol  in  neoplastic  disease.   (2)  There  is  a  decrease  in  6-p  hydroxylation 
in  scurvy  in  guinea  pigs.   (3)  Stilbesterbl  treated  scorbutic  guinea  pigs 
respond  to  ACTH: less  than  'do  conCrbi  guinea  pigs.'  (4)  There  are  strain 
differences  in  the  response  of  guinea  pigs  to  ACTH.   (5)  There  are  3  polar 
blue-tetrazolium  reducing  corticosteroid  zones  in  the  urine  of  man  in 
contrast  to  3  such  zones  in  the  urloe. of  guinea  pigs  as  determined  by. paper, 
chromatography  after  ethyl  acetate  extraction,   (6)'  Th6  response  of  clinical 
patients  to  ACTH  is  by  an  increased  extraction  of  both  free  and  of  conju- 
gated corticosteroida.  The  principieknoftn  free  eprttcoBteroids  are  6-P'^' 
hydroxycortlsol  and  tetrahydro-F  and  Cortisol  (F).     '- ' 

Significance  to  cancer  research:  The  presence  of  compouridS^iiriore  polar>  " 

than  Cortisol  in  the  urine  of  man  and  of  guinea  pigs,  indicates: 
(1)  Conclusions  based  oh  th6  excretion  of  cfarticosteroids  without  specific 
reference  to  their  identity  must  be  re-evaluated,  e.g.  the  principle^   ;  . 
corticosteroid  in  the  albino  Hartley  guinea  pig  is  Cortisol,  whilethe.  .  . 
principle  corticosteroid  in  Strain  2  and  Strain  13  guinea  pigs  is  not 
Cortisol.   (2)  When  hydro lyzed  urines  are  extracted  atid  theit  glycogen  r 
potency  compared  with  that  of  extracted  unhydcoLyzed  tiritie  there  is  little 
difference  in  the  biologic  activity  of  the  2  extracts;  Inasmuch  sts  the  : 
free  v,ncohjugated  polar  urinary  corticosteroids  are  the  biologically  active 
materials  responsible  for  nearly  all  of  the  glyoogenlt  activity  of  hydrolyzed 
or  unhydrolyzed  urine  it  now  becomes  important  to  study  these  free  corticos- 
teroids in  some  detail.   (3)  Up  to  the  present  insufficient  attention  had^: 
been  given  to  these  new  polar  corticosterdid«  for  2  reasons:  a)i  they  were  not 
khowh'  to  be  present  prior  to  their  recent  discovery  by  us  in  the  urine  of 
goinea  pigs  and  of  man;  and  b)  previous  methods  of  extraction  .actually 
destroyed  them. 

Proposed  course:  It  was  thought  prior  bo  1950  that  Vitamin  C  def icient - 
;  :   ,   (scoxbutlc)  guinea  plg«  had  poor  adrenal  function..  We, 
showed  that  adrenal  activity  was  actually  excessive  in  scurvy  by  (a)  measuring 
the  corticbid  in ' the  urine,  (b)me?asuring  It  in  the  blood,  (c)  demonstrating 
the  biological  activity  of  purified'  extracts,  (<J)  isolating  and  crystallizing 
the  major  product,  (e)  identifying  it;  as  hydrocortisone  (compoaod  ,F)  and 
(f )  by  histological  study.  These  studies  exploded  previous  speculations 
"i/and  served  to  seifc  up  hew  problems,'   •'     '     '  '^     ;  .:;;;}    ,  ' 

;  Despite  a.  high  circulating  blood  tltre  of  glucocorticosterolds,  the  glycogen 
in  the  liver  of  Vitamin  C  deficient  guinea  pigs  is  paradoxically  low.  It 
was  found  that  the  guinea  pig  cannot  deposit  glycogen  in  its  liver  in  the 
absence  of  Vitamin  C,  even  though  Cortisol  were  administered  together  with 
glucose  or  fructose.  This  is  being  further  explored. 


PAGE  III 

v^.  ISA^         PROJECT  REPORT  FORM  (Cont'd)         fj:--   _fSi«  ^^f  :;.  ^/--fU 

9.  PROJECT  DESCRIPTION  (Con,t.,'d)...»s3:)  l»®&1  ?S?^a  tE^l./.^^ 

Further  studies  on  the  guinea  pig  indicated  six,  oth^i;:  upsuf pej:ted;;Urinary 
corticosteroids  were  present.  All  have  since  been  isolated  and  all  but 
one  identifiiddv  One  of, these  is, a,  previously  unknown,  compound,  6  BOH  Cortisol, 
This  new  corticosteroid  has  now  been  found  in  human;  urine  together.  v;ifh  a 
new  series  of  C2iOg  compounds.   Isolation  of  these  new  steroids  is  in 
progress  to  provide  sufficient  amounts  for  biologic  testing.--   :_  "    .>;;;?: 

The;; quantitative  pattern-  of  the  corticosteroid  excretion  .is,  currently  being 
investigated  in  guinea  pigs  under  various  stress  combinations;,,  sych  as 
x-radiation^  dietary  deficiencies,  drug  effects,  neoplastic  diseases, 
environmental  alterations,,  .and  an  attempt;  is  being  made  tp,  corarelate  adrenal 
morphology  with  the  excre.t,ion  of  specific  cor tico^sterpid's.,  This,  typ^  of 
investigation  will  later  b^  extended,  to  other"  endocrine  organs.  ,  ^ 

A  clinical  study  developing  from  the  observations  in  guinea  pigs  and 
utilizing  Clinical  Center  material  from  the  Leukemia  Service  and  Surgical 
Service,  NCI,  and  from  the  Psychosomatic  Service  of  the  NIMH  has  been 
started.  The  purpose  is  to  study  the  alteration  in  the  excretion  of  specific 
corticosteroids  during  the  course  of  leukemia,  e.g.  in  remission,  in  relapse, 
under  therapy, while  intervening  infection,  during  active  disease,  and  to 
note  whether  the  pattern  changes  which  the  adrenal  is  challenged  with  ACTH. 
These  patterns  are  compared  with  those  of  non-neoplastic  patients  and  patients 
with  neoplastic  diseases  other  than  leukemia. 

Autopsy  material  from  the  patients  who  have  died  with  leukemia  is  being 
collected  for  a  histopathological  study  of  the  effect  of  leukemia  on 
various  endocrine  organs. 

Biosynthesis  of  steroids  in  normal  and  tumor-bearing  animals  is  being 
studied  in  vivo  with  a  Visiting  Scientist.   New  methods  of  detection  are 
being  developed  with  the  aid  of  radioisotopes.   In  vitro  studies  utilizing 
a  simple  perfusion  apparatus  are  contemplated. 

A  "library"  of  frozen  urine  aliquots  and  bloods  from  clinical  patients 
and  animals  is  being  collected  for  future  study. 

In  addition,  a  long-term  study  is  planned  on  the  corticosteroid  patterns 
occurring  following  the  use  of  methylcholanthrene,  total  body  radic^tion, 
and  other  potentially  carcinogenic  agents,  in  order  to  detect  any  specific 
quantitative  and  qualitative  changes  in  corticosteroid  excretion  pattern 
in  carcinogenesis.  A  related  study  on  humans  exposed  to  radiation  has  been 
proposed. 


4V/ 

SERIAL  NO.  NCI  %idr  PAGE  IV 

PROJECT  REPORT  FORM  (Cont'^) 

9.  PROJECT  BESGRIPTION  (Cont'd)  '-^^i    ^'  ^  n  :- 

A  continued  istvidybn  the  effect  of  vatious  stressed  (see  Methods  Jabbve)rvo 
Is  contemplated,  oh  the  guinea  pigs*   "iiS"^  wr?  anrf  bi^^  03  vfs«  eWT 

The  effect  of  various  potentially  therapeutic  agents  on  the  course  of  'i'; 
cancer  in  the  guinea  pigs  is  being  studied  with  the  hope  of  picking  up 
alterations  in  the  specific  corticosteroid  excretion  patterns  associated; 
with  c^rcinostasis.  ''^ 

■.     1  i      '•■'.■■  ■  '       .  SK.'.:  I:-  'bi-  ;  ■  .v 

A  continued  study  on  carbohydrate  n»etabolistn  in  the  guinea  pigs -is  being 
pursued  witli  the  hope  of  applying  this  fundamental  knowledge  to  the  in  vitro 
perfusion  studies  that  are  planned  when  facilities  become  available; 

:  .    .  :iai«r'iD  J,sa.i:ffi.jD  jii 

..,  '        ■  \  '  '■    i-'.r-l  i-Mi^  .  ^'V1 

oiilaijqu'  1;.    .    . 
,jS!qBiu;:,  ni  .   ,    .  : 

oJ  hmr,   1^3? ,        ■  '  ■•!•.■■.• 

.nyjr-  ^}'<>:_'h  .  :.  .  ■  ■  '■■.., 

■  \if.i:'-    .  ■  .     ■   .,   ■  .  ..    -    .       ■ 


10.  NCI -5*3- 


SERIAL  NO. 


PAGE  V 


PROJECT  REPORT  FORM  (Cont'd) 


11. 


BUDGET  ACTIVITY: 

RESEARCH         /x7 
REVIEW  &  APPROVAL  /"7 


ADMINISTRATION       h,l 
TECHNICAL  ASSISTANCE  7~7 


12 .  E.  Frei.  ClinlGal  Center.  NCI.  L.  Cramer,  Clinical  Center,  NCI. 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  None  to  my  knowledge. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL, 
FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH 


PAGE  VI 
PROJECT  REPORT  FORM  (Cont'd)  ^' 

14.  NCI-5£» 
SERIAL  NO. 

15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 

Effect  of  neoplasms  and  of  stresses  on  the  excretion  of  specific  corticos- 
teroids in  the  guinea  pjig, 
Nadel,  Burstein/  and  Doirftnan. 
Clin.  Res.  Proc.^  Feb.  1955  (Prpc.  Am,  Fed.  Clin.  Res/f^ 

Urinary  excretion  of  Cortisol,  6-p  hydroxycortisol  and  an  unidentified 
steroid  (Steroid  Ha)  by  guinea  pig  with  leukemia  or  with  liposarcoma. 
Nadel,  Burstein,  and  Dorfman. 
Proc.  Am,  Assoc.  Cancer  Research  2:  37,  1955, 

.Corticosteroids  in  the  urine  of  normal,  and.  scorbtitic  gulin^i  pigst  'isolation 

■"'^■'arid  ffuantitative  determination;,  ■■"■'  ' 'pV-^  ;■''*'-•  i  '  '  '    '^'-.^'•j'H,  ?'•'"•-■'■■  '  ''■' 

BUratein,  Dorfman/ and  Nadel.  '     '  ,  ' i  HP  .^  .mtlJIim-  .mn^^^mmy 

J,  Biol.  Cheira.  April  1955. 

Isolation  of  polar  reducing  corticosteroids  from  human  urine. 

Nadel,  Burstein,  and  Dorfman. 

Arch.  Biochera.  &  Biophysics.,  Accepted  October  7,  1955. 

Dissimilarity  in  alkaline  phosphotase  staining  of  Keratohyaline  granules. 
Nadel  and  Wodinsky. 
..„ Jj  Histochem.  &  Cytpchem.  S.ept.  1955. 

'  Tfon«pdrallel ' changes  in ' changes  in  cholesterol  and  ascorbic^acid^-in  the 
'  adrenals  of  malaria  parasitized' chicks,  ■;•'  ;;'■,,  «.Vp,'>'f' '  /■?:'•  '■:y~  l■xy^s.,^^.'. 

Taylor,  Greenberg,  Josephson,  and  Nadei. 

J.  Clin.  End.  &  Met.   June  1955.  (Proc.  Endocrine  Society) 

On  the  defect  in  glycogen  deposition  in  the  livers  of  scorbutic  guinea  pigs. 
Nadel,  Mulay  and  Saslaw. 
Endocrinology  May  1955. 

16.  None  __«______->_ 

HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955. 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology         .  ■i'y.^X.'O^"    . 

INSTITUTE  LABORATORY  OR  BRANCH  • 

■   '    ■   -y;  '  S*^ 

3.  Cancer  Indue.  &  Pathogenesis  4.     ,  5,  NCI  -S-^^^ 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BBTHESDA)   SERIAL  NO. 

6.  Effect  of  microbia'l  and  other  agents  on  the  development  and  course  of 

neoplastic  disease  In  animals.  .  ^'  ^   •   '  ■   '    "'"••■' 

PROJECT  TITLE  :  '^ 

7.  E.  M.  Nadel 


PRINCIPAL  INVESTIGATOR(S) 
8.  None  (See  Item  #12) 


OTHER  INVESTIGATORS 

PROJECT  DESCRIPTION  ,  ,  - 

The  present  project  title  includes  3  sub-.projects  gathered' under  the  project 
title  of!   (a)  Mutual  aspepts  of  the-  pathophysiology  of  leukemia  and  malaria 
in  animals.  When  it  became  lobvio^s  that  the  project  on  perfusion  studies 
could  not  begin  because  of  the  administrative  uncertainty  of  ispace;,  facilities 
and  moving  date  to  the  Clinical  Center  laboratories,  2  other  projects  related 
to  a)  were  undertaken.   .....  .-  i    •  ,.  .   .■  .  -^  ■ 

These  projects  include:  (b)  The  effect  of  certain  viruses  on  the  course  of 
cancer  in  animals  and  .  (c)  the, mode  of:  action  of  certain  living  and  chemical 
agents  on  the  developinent;  and  cou?:se  of  cancer  in  animals. 

Objective:   (1)  To  explore  the  activity  of  certain  potentially  carcinostatie 

and  carcinogenic  agents.   (2)  To. explore  certain  immunogenetic 
and  endocrine  aspects  of  resistance  to  malaria  infection  and  to  viruses 
helpful  in  a  study  of  the  carcinostatie  potentiality  of  these  microbial 
agents.   (3)  The  mode  of  action  of  certain  virucidal  and  raalarlacidal  drugs 
as  carcinostatie  agents. 

Methods:  The  overall  problem  is  studied  by  the  combined  use  of  techniques 

in  pathology,  microbiology,  hematology,  genetics,  chemical 
pharmacology,  and  immunology.   Inbred,  hybrid  and  specific  backcrosa  animals 
are  studied  for  resistance  to  micro-organisms  such  as  the  malaria  parasite, 
neurotropic  viruses,  etc.,  and  the  effect  of  these  on  the  course  of  trans- 
plantable tumors  is  studied.   Combinations  of  drugs  and  of  viruses  are 
studied. 

Major  findings:   (1)  Guinea  pigs  with  leukemia  lived  significantly  longer 

after  they  were  experimentally  infected  with  the  virus  of 
lymphocytic  choriomeningitis.  (LCMV).   (2)  Guinea  pigs  made  immune  to  the 


SERIAL  NO.  NCI  -^^Sr"  PAGE  II 

PROJECT  REPORT  FORM  (Cont'd) 

9.  PROJECT  DESCRIPTION  (Cont'd)       ■  ,  -:;-.;. 

,LGM  virus  wer.e  afforded  no  protection  when  the  virus  was  injected  after 
transplantation  of  the  tumor,  (3)  Four  drugs  (ethionine,  anjethopterin,  and 
"  a  :purine  and  pyrimidine  ati^Iogue)  were  studied  to  find  that  dose  at  which 
each  individual  drug  was  ineffectivfe.  Combinations  of  these  drugs  at  their 
individually  ineffective  dosages  successfully  proloriged  the  life  of  tumor- 
bearing  mice,  more  than  100%  in  comparison  to  controls.   (4)  Repeated 
therapy  with  the  combination  of  4  drugs  to  tumor-bearing  mice  resulted  in 
the  development  of  a  resistant  tumor.  This  resistant  tumor  is  now  being 
used  as  a  means  of  studying  the  mode  of  actioii  of  other  potentially 
carcinostatic  drugs.  .■[■■^T\bIT-'.S\>Vii 

Significance  to  cancer  research:   (1)  A  neurotropic  virus  (LCMV)  niade 

relatively  benign  by  repeated  passage  to  mice  (300  generations) 
was  able  to  prolong  the  life  of  leukemia  guinea  pigs  from  19  to  over  30  days. 
The  virus  did  not  of  itself  appear  lethal  to  guinea  pigs  though  other 
-strains  of  the  virus  apparently  are.  This  increase  in  survival  is  greater 
^  . than  that  previously  reported  frqia  this  project,  on  the  inhibitory  effect 

-  of  malarial  infection  on  the  cc)urs6  of  Leukemia  in  mice.  This  type  of 
study  has  therapeutic  implications  for  use  in  mati.   (2) , By  judicious  use 
of  a  tumor,  resistant  to  four  different  types  of-  chemotherapeutic  agents, 
the  mode  of  action  of  potentially  carcinostatic  drtjgs,  with. different 
chemical  structure  can  be  studied.  ,; 

Proposed  course:  Attempts  to  enhance  the  duration  of  the  inhibitory  effect 
of  the  microbial  agents  under  study  are  being  made. 
!   These  include  the  judicious  use  of  hormones,  xrray  irradiation,  drugs  and 

-  multiple  sequential  use  of  microbial  agents,  various  pyrogenic  materials 
(e.g.  polysaccharides,  etc.).  The  possibility  of  the  use  of  viral  therapy 
in  the  form  of  the  LCM  virus  for  leukemia  in  nian  must  be  considered. 
Continued  studies  on  the  possible  similarities  in  the  genetics  of  .resistance 
in  mice  to  malaria  and  to  leukemia,  and  the  role  of  endocrines  in , such 
resistance  are  underway. 


■a:    i-ui     .-,■:.  :■.  .^ftiPiivv..-  j.i<p:ri: 


PAGE  III 
PROJECT  REPOKT  FORM  (Cont'd)' 


10.  NCI  »£A— 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         /x7     ADMINISTRATION       fj 
REVIEW  &  APPROVAL  /"7     TECHNICAL  ASSISTANCE  /T 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 

Cooperating  personnel  assigned  to  the  staffs  of  Dr.  Greenberg,  Chemotherapy 
Section,  Laboratory  of  Tropical  Diseases,  NMI,  Dr.  Haas,  Office  of  the 
Director,  NMI,  Dr.  Jay,  Laboratory  Aids  Branch,  Office  of  the  Director,  NIH. 


13.  None  to  my  knowledge    ■   ■   ■ 

IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH 


PAGE  IV 
PROJECT  REPORT  FORM  (Cont'd) 

14.  NCI-S-^^- 
SERIAL  NO. 

15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 

Effect  of  malaria  on  leukemia  in  mice. 
Nadel,  Greenberg,  Coatney. 
J.  Infectious  Diseases. 

Differences  in  survival  of  several  inbred  strains  of  mice  and  their  hybrids 
infected  with  Plasmodium  berghei. 
Greenberg,  Nadel^  Coatney, 
J.  Infectious  Diseases. 

Synergistic  inhibitory  action  of  amethopterin  and  a  diaminopyrimidine 
Oh  leukemia  L  1210  in  mice. 
Nadel,  Greenberg. 
Cancer  Research, 

Increased  resistance  to  malaria  of  certain  inbred  strains  of  mice^  their 
,.  hybrids  and  backcrosses, 

Nadel,  Greenberg,  Coatney,  and  Jay. 
Am,  J.  Path. 

Resistance  to  quadruple  combination  therapy  in  leukemia  L  1210  in  mice. 

Nadel  and  Hilgar. 

km,.   J.  Path,,  June  1955.  (Proc.  Am.  Assoc.  Path.  &  Bact.) 

Inhibitory  effect  of  lymphocytic  ehromomeningitis  virus  in  thg  course  of 

leukemia  in  guinea  pigs, 

Nadel  and  Haas. 

Fed.  Proc.  14:  March  1955.  (Proc.  Am.  See.  Exp.  Path.) 

Backcross  studies  on  the  genetic  of  resistance  to  malaria  in  mice. 
Nadel,  Greenberg,  Jay,  and  Coatney. 
Genetics,  Sept,  1955. 


16.  None 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

6'/3 

3.  Cancer  Indue.  6e  Pathogenesis  A. 5.  NCI '525- 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Use  of  isolated  organ  perfusion  techniques  in  a  study  on  the  pathologic 
physiology  of  tumor-bearing  animals. 

PROJECT  TITLE 

7.  E.  M.  Nadel 

PRINCIPAL  INVESTIGATOR(S) 

8 .  None  \ ' 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objective:   Study  changes  in  physiology  accompanying  tumor  growth  to  learn 

how  tumors  divert  energy  sources  away  from  the  host^  as  a  means 
towards  the  control  of  tumor  growth. 

Methods:   The  perfusion  methods  used  have  been  used  successfully  in  the  hands 

of  Dr.  Leon  Miller,  v/hile  the  micro-methods  to  be  used  have  been 
described  by  Drs.  Scholander,  Kirk,  and  Natelson. 

Major  findings:  There  have  been  no  major  findings  inasmuch  as  work  on  this 
project  has  been  delayed  by  unforeseen  circumstances. 

Proposed  course:   It  is  hopefully  anticipated  that  work  can  be  started  on 

this  project  shortly  after  the  contemplated  move  from 
Building  8  to  Building  10. 


PAGE  II 
PROJECT  REPORT  FORM  (Cont'd) 


10.   NCI  •&£»• 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         /z/  ADMINISTRATION       £7 

REVIEW  &  APPROVAL  /"7     TECHNICAL  ASSISTANCE  '  l~l 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  I 


PROJECT  REPORT  FORM 


National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABOR/iTORY  OR  BRANCH 

3.  Cancer  Ifldue.  &  PathoRenesis  4. . ^ ^ 5 .  NCI  -§4:9- 

SECTION  OR  SERVICE  LOCATION  (  IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Induction  of  adrenocortical  tumor. in  the  rat  and  study  of  the  adreno-cortical 
steroids  of  the  tumors,  thus  induced.  Also,  the  effect  of  these  steroids  on  the 

steroid  metabolism  of  the  host. 

PROJECT  TITLE 


7.  A.  S':  Mulay 

PRINCIPAL  INVESTIGATOR(S) 


8.  W.  H.  Eyestone 

OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objective;  To  find  out  if  the  steroids  produced  and  the  enzyme  systems 

involved,  in  the  adrenal  cortical  tumor  differ  either  qualitatively 
or  quantitatively  from  those  in  the  adrenal  gland. 

Methods:   Transplanting  and  harvesting  of  the  adrenal  tumors.  Determining 

activities  of  enzymes  like  choline  esterase,  adenosine  triphosphate, 
phosphomonoesterase,  and  alkaline  phosphotase.   Quantitative  and  qualitative 
metabolism  of  steroids  by  tumor  slices  under  different  given  conditions,  on 
fresh  tumor  tissue.  Extracting  steroids  from  harvested  tumors  stored  in 
deep  freeze  (when  enough  material  is  collected),  purifying  the  extract, 
separation  of  various  steroid  fractions  by  chromatographic  methods,  and  then 
their  characterization  by  various  physical  and  chemical  methods. 

Major  findings:  Adreno-cortical  adenocarcinoma  has  been  induced  in  9.57o  of 

the  female  Osborne-Mendel  rats,  under  experimental  treatment. 
This  adrenocortical  tumor  has  pronounced  effect  on  the  adrenal  glands  of  the 
host.   Considerable  quantity  of  formaldehydogenic  steroids  have  been  found 
in  the  extracts  of  these  tumors. 

Significance:   Successful  conclusion  of  this  program  may  give  us  some  insight 

of  chemical  and  enzymatic  changes  which  transform  normal 
adrenal  gland  into  adrenocortical  adenocarcinoma. 


SERIAL  NO.  NCI  *i6  PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


9.  PROJECT  DESCRIPTION  (Cont'd) 

Proposed  Course:  A  considerable  portion  of  the  next  calendar  year  will  be 
devoted  to  separation  and  identification  of  the  steroids 
in  this  tumor.  This  involves  growing  and  collecting  the  tumor  material, 
extracting  the  steroids,  separating  different  steroid  fractions  chromato- 
graphically,  and  collecting  enough  of  each  fraction  for  further  separation 
and  then  physical  and  chemical  characterization.  At  the  same  time  a 
technician  is  being  trained  in  enzymologic  and  metabolic  procedures  for 
working  on  fresh  tumor  tissue. 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


0.  NCI -5^:6- 
SERIAL  NO. 


1. 


BUDGET  ACTIVITY: 

RESEARCH         /x7    ADMINISTRATION       [j 
REVIEW  &  APPROVAL  l1         TlECHNICAL  ASSISTANCE  l~l 


2.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATION,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


3,  As  far  as  I  know,  it  cjoQS  not. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  IV 
PROJECT  REPORT  FORM  (Cont'd) 

14.  NCI  Hfr^ 
SERIAL  NO. 

15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 

Mulaj,  A.  S.  and  Schlyen,  S.  M. 

Les:'.cn5  induced  in  C573R  mice  with,galliux  citrata  and  -nethylcholanthrene. 

Am.  J.  Pathol.   In  prass. 


16.  HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955 

Elected  to imerabership  in  Society  for  Experimental  Biology  and  Medicine 
(Rational)  "  ' 


PAGE  1 


PROJECT  REPORT  FORM 


National  Cancer  Institute  2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

Cancer  Indue.  &  Pathogenesis  4 . 5 .  NCI  512- 

SECTION  OR  SERVICE     •        LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 


6.  Sex  difference  in  the  incidence  of  hepatomas  in  rats,  fed  a  carcinogenic 
azo  dye,  and  study  of  hormonal  and  other  factors  responsible  ;for  this.  • 

difference. ___«__^ ________ 

PROJECT  TITLE 


7.  A.  S.  Mulav    .  :  .  '/  ^    ■  ■   ■"'.-■    ■  ■■     :        '.    ■"■'■■      :  •  :■'  '  .-   -     ■■'-  '^^ 
PRINCIPAL  INVESTIGATOR(S>     -• 

8.  None 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION  ;•::-.:■• 

Objective:   To  determine  the,  role  played  by  sex  hqrmones  on  the -Incidence  of 

hepatomas  induced  in  rats  fed  carcinogenic  azo  dyes^  and  see  what 
part,  if  any,  is  played  by  other  so-called  "residual  sex  differences." 

Methods:  Male  and  female  rats,  intact,  gonadectonized,  and  with  or  without 
estrogen  or  androgen  treatment,  are  fed  a  synthetic  diet  of  known 
composition  containing  £-dioethylaminoazobenzene.  All  groups  of  rats  thus 
treated  are  kept  under  identical  conditions,  and  a  few  animals  from  each 
group  are  sacrificed  at  definite  time  intervals.   Livers  of  these  animals 
are  examined  for  macroscopic  and  microscopic  hepatomas.   From  these  observa- 
tions, effect  of  the  treatment  on  the  relative  induction  time  and  the 
incidence  is  calculated. 

Major  findings:   Intact  male  and  female  Osborne -Mendel  rats  fed  a  carcinogenic 

azo  dye  in  multiple  deficiency  synthetic  diet  of  known 

composition,  for  10  months,  showed  a  marked  sex  difference  in  the  incidence 
of  hepatoma. 

Significance:   Evidence  thus  gathered  will  forge  a  link  in  the  chain  of 

environmental  factors  necessary  to  induce  neoplasm  in  animals. 

Proposed  course:   Experiments  on  various  treatments  to  different  groups  of 

animals  are  just  starting  and  will  run  through  the  next 
calendar  year. 


PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI-^t^- 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARGH  •        /x7  ■   AMIINISTRATION   :    ■  ■■  [j 
REVIEW  &  APPROVAL  H         TECHNICAL  ASSISTANCE  /~. 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  As  far  as  I  know,  it  does  not. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS ,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


.>y  t&hnoi:. 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


14.  NCI-§4^ 
SERIAL  NO. 


15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DDRISG  CALENDAR  YEAR  1955 


Symeonldls^  A.  and  Mulay^  A.  S. 

Histopathology  of  the  adrenal  glands  of  rats  fed  a  low  protein,  low  riboflavin 

diet  alone,  or  with  £-dimethylaminoazoben2ene. 

J.  Nat.  Cancer  Inst.   In  Press. 


16 .  None 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955 


■)  mj^k 


,0M  J. 


.ji 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Office  of  the  Chief  4.  5.  NCI  •S^'fr- 

SECTION  OR  SERVICE  -  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Induction,  Pathogienesis  and  Morphological  Alteration  of  Tumors  Arising  in  Bone 
PROJECT  TITLE 

7.  Albert  W.  Hilberg 

PRINCIPAL  INVESTIGATOR(S)       

8.  None  '■  '       ■    "      -  '  - 

OTHER  INVESTIGATORS     - 

9.  PROJECT  DESCRIPTION  •  . 

Objectives:  To  develop  a  consistent  method  of  producing  bone  tumors  in 

animals  which  are  similar,  morphologically,  to  those  in  man 
and  to  study  the  events  leading  to  tumor  formation  or  alteration  of  tumors 
as  affected  by  transplantation. 

Methods:  Animals  have  received  Injection  of  beryllium  oxide  by  various 
routes  such  as  intravenous,  intramedullary  and  intratracheal. 
At  intervals  skeletal  surveys  by  x-ray  examination  and  blood  serum  phosphatase 
levels  are  determined.  All  control  animals  and  injected  animals  are  autopsled 
and  bony  tissues  examined  grossly  and  microscopically.  Special  studies  of 
tissues  are  made  using  histochemical  and  x-ray  techniques. 

Transplantation  of  spontaneous  bone  tumors  into  various  sites  such  as  liver, 
kidney,  subcutaneous  tissue  and  intraperitoneal  spaces  is  earried  out  to 
study  effects  on  morphology  of  tumors. 

Major  findings:  The  rabbits  injected  with  beryllium  have  developed  a 

lymphoma,  2  bone  sarcomas,  and  1  bone  sarcoma  developed 
in  a  mouse  and  is  being  successfully  carried  in  serial  transplantation. 

Morphological  variations  in  spontaneous  bone  tumors  by  selective  site 
transplantation  into  kidney  have  revealed  a  differentiation  of  undifferen- 
tiated cells  into  bone  after  bone  had  failed  to  develop  in  subcutaneous 
transplants.  This  represents  a  possible  redifferentiation  of  anaplastic 
cells. 

To  date  some  40  spontaneous  tumors  primary  in  bone  in  mice  have  been  found 
and  classified  by  morphological  type.  A  detailed  description  is  being 
completed. 


SERIAL  NO.  NCI  -Si*-  PAGE  II 

PROJECT  REPORT  FORM  (Cont'd) 

9.  PROJECT  DESCRIPTION  (Cont'd) 

Significance  to  cancer  research:   Bone  tumors  are  said  to  be  among  the  most 
common  neoplasms  in  children.  These  tumors  develop  at  any  age 
and  represent  a  major  problem  since  cures  are  rare  and  treatment  usually 
mutilating.  Attempts  to  learn  oore  a^out  the  development  of  bone  tumors, 
Including  contributing  factors  in  development  and  the  biological  and 
morphological  behavior  of  these  tumors  can  give  valuable  information  toward 
the  goal  of  prevention,  treatment)  and  cure  of  bone  tumors.   Information 
concerning  specific  substances,  such  as  beryllium,  which  produce  bone. tumors, 
may  be  of  value  because  of  the  industrial  uses  of  beryllium  and  its  compounds. 

Proposed  course:   Continuation  of  the  study  of  animals  injected  with 

beryllium  oxide.   Continuation  of  selective  Bite  transplan- 
tation studies  of  spontaneous  tumors  arising  in  bone,  with  particular 
emphasis  on  transplantation  to  the  liver.  The  classification  and  description 
of  all  spontaneous  tumors  arising  in  bone  in  mice  is  a  continuing  process  as 
these  tumors  are  received  from  many  sources  within  the  various  laboratories 
of  the  National  Institutes  of  Health. 

Collaboration  in  beryllium  studies  in  intratracheal  injections  and  in 
studies  of  Rous  sarcoma  will  continue  until  completion. 


PAGE  III 


PROJECT  REPORT  FORM  (Cortt'd) 


10.  NCI  •aiO' 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         /x7    ADMINISTRATION       £7 
REVIEW  6.  APPROVAL  £7         TECHNICAL  ASSISTANCE  £7 


12.  None  to  my  knowledge 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13.  None  to  my  knowledge 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  IV 
PROJECT  REPORT  FORM  (Cont'd) 


14.  NCI-§4^ 
SERIAL  NO. 


15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  195! 

Hilberg;  Albert  W. 

Morphological  Variations  Itt  an  Osteogenic  Sarcoma  of  the  Mouse  when 

Transplanted  to  the  Kidney 

J.  Nat.  Cancer  Inst,   Inpress. 


16.  None 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology ' 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Pathological  Technolosy  Sect.  4. 5.  NCI 


SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Pathological  Technology  Section 

PROJECT  TITLE 

7.  Mr.  J.  M.  Albrecht '      ■  -  ■  ■ 

PRINCIPAL  INVESTIGATOR(S) 

8.  None ■  •  

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Stained  tissue  sections  are  the  fundamental  basis  of  all  clinical  and 
experimental  studies  of  cancer.   The  Section  prepares  histological  sections 
for  all  the  investigators  of  the  National  Cancer  Institute.   It  makes  available 
all  the  established  routine  and  special  stains  and  in  addition  develops  and 
provides  the  current  experimental  methods  of  tissue  preparation  such  as 
enzyme  stains  and  specific  histological  stains. 

January  1.  1955  -  December  31.  1955 

Number  of  Investigators  67 

Number  of  Pieces  of  tissue  105,361 

Number  of  Bottles  of  tissue  15,921 

Number  of  blocks  cut  52,356 

Blocks  cut  serially  ..................  464 

Frozen  blocks  cut 722 

Number  of  autopsies   14,635 

Number  of  recuts 920 

Number  of  slides  stained  H&E  67,562 

Number  of  slides  stained  special  .....  13,701 

Number  of  unstained  slides  .........  7,805 


SERIAL  NO.  NCI  -frtS" 


PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


9.  PROJECT  DESCRIPTION  (Cont'd) 


aJwlMafii 


Photographic  Service  rendered  to  the  Laboratory  of  Pathology,  .January  1.  1955 
to  December  31,  1955  ' 


GROSS 


•  Black  &  White  _.•,,  .  ....:/  'S'^^'-il^;::"^...^ ■  .■■•84 

Color  . , 94 


MICROS 

Black  &  White , 380 

Color 333 

-LANTERN  SLIDES  ' ""  "   ^ ,  -'^r  .  ^v. 

Black  &  White   .......,,..., 103 

Color .......,.,,,....,..    ,,  ,.    ;    ;      195 

PRINTS 


^  X  5        ,    .; .1548 

8  ^  IP .    .        32 

14,x  17..,,.    ...    .^, ..,..,>..,,,. ,;.-...   vr.    ■,...    ./v.'^.    ...■   :;'5    ■■ 
.Mounted.  ..,.:.,.,:..,.  ..,..-..,,   >,  .^.,;    .::;   ..   :.--*-v;  ..<>  .  .^^   •■28a\  ^' 


10. 


r/7 

NCI  rH:3- 
SERIAL  NO. 


PAGE  III 


PROJECT  REPORT  FORM  (ebnt-»d) 


11. 


BUDGET  ACTIVITY: 


RBSBARCH         /x/ 
REVIEW  6>  APPROVAL  [J 


ADMINISTRATION       /_/ 
TECHNICAL  ASSISTANCE  ll 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEM"ENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERK)NNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  IV 


PROJECT  REPORT  FORM  ;  (Cont ' d) 


14.  NCl-§4» 
SERIAL  NO. 


15.  None 


PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CAUNDAR  YE^  1955. 


16. 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955. 


Willie  D.  Morgan  received  cash  award  June  1955  for  a  vacuum  device  and 
siphorvacuum  cap  which  aids  ah; the  Cfl^  utility  bottles  frprt 

"large  cans  or  bottles  ot;  reagents.  ;  ■  ;  .   .  .»   ..  .  ■.   ,■'•..     .  ,^\ 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute.    2.  Laboratory  of  Pathology  '    ■  , 
INSTITUTE                     LABORATORY  OR  BRANCH      ,  , 

3.  Office  of  the  Chief 4. ■ .  .  .:"  -  '  5.  NCI-»6^ 

SECTION  OR  SERVICE             LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  NO, 


6.  Heterologous  Transplantation 


PROJECT  TITLE 


7.  C.  Harold  Steffee  and  Katharine  C.  Snell 


PRINCIPAL  INVESTIGATOR(S) 
8.  None 


OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives:  To  produce  neoplasia  of  human  tissues. 

Methods:  Human  fetal  tissues  have  been  transplanted  into  the  cheek  pouch 
of  the  hamster,  the  host  animal  being  treated  with  cortisone. 

Major  findings:  The  transplanted  fetal  tissues  generally  persist  for  a 

reasonable  time  in  the  hamster  cheek  pouch  but  no  evidence  of 
neoplasia  of  these  tissues  has  been  obtained,  A  major  obstacle  in  this 
program  has  been  the  development  of  dissecting  aneurysm  in  many  of  the 
cortisone-treated  hamsters  with  premature  death  of  the  animal  resulting  from 
hemothorax  or  hemoperitoneum.  Similar  lesions  have  been  found  in  the  control 
animals  treated  with  cortisone  but  not  given  any  fetal  transplants.  That 
this  finding  is  not  limited  to  hamsters  is  evidenced  by  the  discovery  of 
degenerative  changes  in  the  aorta  of  each  of  two  mice  bearing  functional 
adrenal  cortical  carcinomas  (Dr.  Thelma  B.  Dunn). 

Significance  to  the  program  of  the  Institute:   The  original  objective  of 

these  studies  was  to  produce  human  carcinoma  in  organs  which 
•re  difficult  to  study  in  experimental  animals  because  of  the  lack  of 
analagous  neoplasms  in  the  animal.  These  sites  include  the  lung  and  the 
gastrointestinal  tract.  Had  we  succeeded  in  this  endeavor  we  would  have 
had  an  extremely  valuable  tool  for  testing  a  number  of  suspected  carcinogens 
for  these  tissues,  and  a  tool  which  would  have  been  much  more  comparable  to 
the  human  situation  than  is  otherv/ise  possible.  Perhaps  additional  studies 
will  permit  us  to  attain  this  objective.  The  significance  of  the  aortic 
lesions  to  cancer  research  is  at  best  rather  nebulous.   It  may,  however, 


SERIAL  NO.    NCI  •5f>t- 


PAGE  II 


PROJECT  REPORT  FORM  (Cottt'd) 


9.  PROJECT  DESCRIPTION  (Cont'd) 

provide  us  with  further  insight  into  the  fundamental  effects  of  adrenal 
cortical  hormones  on  the  connective  tissues  of  the  body.  This,  then,  is 
an  area  in  which  we  are  seeking  fundamental  knowledge  with  no  immediate 
applicability  to  the  cancer  prohlem. 

Proposed  course  of  the  project:   A  number  of  hormones  related  to  cortisone 
will  be  tried  in  an  attempt  to  attain. our  original  objective.   . 
Additional  studies  will  also  be  carried  out  to  try  to  define  the  precise 
relationship  of  cortisone  to  the  development  of  the  aneurysm. 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI  -SOJ- 
SERIAL  NO. 


a. 


BUDGET  ACTIVITY: 

RESEARCH         /x7    ADMINISTRATION       [j 
REVIEW  &  APPROVAL  [1         TECHNICAL  ASSISTANCE  fj 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13 .  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  14,  15,  OR  16 


^b'  ffjoO)  ;cv;-.-'{   r>;r';rH>i 


)rTjv.TTDA  -ndirM 


'i^^'iHf.  /   viMTVa« 


■■■:  V    jKf  t^I 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute    2.   Laboratory  of  Pathology 

^   INSTITUTE  LABORATORY  OR  BRANCH 

^f 

3.  Cancer  Indue.  &.  Pathogenesis  4. 5 .  NCI  4-H- 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

6.  Chemical  and  histopathological  changes  induced  in  the  adrenal  glands  of  rats 
fed  a  deficiency  diet.   (This  diet  is  usually  used  in  conjunction  with  azo 

dye  feeding  for  the  induction  of  hepatoma  in  rat.) . 

PROJECT  TITLE 


7.  A.  S.  Mulay  and  E.  M.  Nadel 
PRINCIPAL  INVESTIGATOR(S) 


A.  Symeonidis 


OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objective:  To  find  the  factor  or  factors  absent  in  the  deficient  diet, 

which  are  responsible  for  the  induction  of  the  histopathological 
changes  observed  in  the  adrenal  glands  of  rats  kept  on  this  deficient  diet, 
and  others  deficient  only  in  one  or  two  known  factors.  To  study  the 
chemical  and  metabolic  changes  in  the  adrenal  glands  of  these  animals, 
concomitant  with  the  histopathological  changes. 

Methods:   Starting  with  a  complete  synthetic  diet  of  known  composition, 

different  diets  are  prepared  from  which  a  known  factor  or  factors 
are  withheld.   Such  diets  are  fed  to  different  groups  of  rats  and  the 
chemistry  and  histopathology  of  their  adrenal  glands  is  compared  with  that 
of  the  adrenal  glands  of  rats  on  complete  diet  of  known  composition. 

Major  findings:  Histopathology  of  the  adrenal  glands  of  rats  fed  a 

multiple  deficiency  diet  of  known  composition  (one  used 
in  connection  with  hepatoma  induction  in  rats  with  azo  dye  feeding)  is 
described  in  our  preliminary  paper  "Histopathology  of  the  adrenal  glands  of 
rats  fed  a  low  protein,  low  riboflavin  diet  alone  or  with  p-dimethylamino- 
azobenzene"  in  J.N. C.I.   In  press. 

Significance:  The  role  played  by  the  adrenal  gland  in  the  induction  of 
hepatoma  in  rats  fed  multiple  deficiency  diet  containing 
carcinogenic  azo  dye,  has  been  demonstrated  in  our  laboratory  (J.N. C.I. 
14:  805-817,  1954,  and  Endocrinol.  57:  550-558,  1955)  and  in  others 


SERIAL  NO.  NCI  54*  PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


9.  PROJECT  DESCRIPTION  (Cont'd) 

(Richardson:  Cancer;  6:  1025-1029,  1953,  and  Griffin:  Cancer  Research  13: 
77-79,  1953).   Knowledge  of  the  factor  or  factors  responsible  for  these 
changes  will  take  us  a  step  nearer  to  the  understanding  of  the  neoplastic 
process. 

Proposed  course:   Critical  experiments  to  confirm  our  first  obserVati&tts  -. 

on  the  altered  histopathology  of  the  adrenal  glands  of 
rats  on  multiple  deficiency  diet  of  known  composition  are  in  progress. 
Better  part  of  the  year  will  be  taken  up  in  confirming  the  histopathology 
picture  and  analyzing  for  concomitant  chemical  changes  in  these  altered,  v, 
adrenal  glands.  Experiments  with  complete  diets  of  known  chemical 
composition,  deficient  in  only  one  or  two  known  factors  have  just  been 
started. 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 

LO.  NCI  Jh^ 
SERIAL  NO. 


LI. 


BUDGET  ACTIVITY: 

RESEARCH         /x7    ADMINISTRATION       fj 
REVIEW  &  AP?WVhL  /~7         TECHNICAL  ASSISTANCE  /"7 


L2.   None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  As  far  as  I  know,  it  does  not. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH:       , 


PAGE  IV 
PROJECT  REPORT  FORM  (Cont'd) 


14.  NCI 


^ 


SERIAL  NO. 


15.  PUBLICATIONS  OTHER  THAN  ABSTRACTiS  FROM  THIS  PROJECT  DURING,  CALENDAR  YEAR  1955 

Nadel,  E.  M.,  Mulay,.  Av  S.>  and;;S,aslaw,.  L.  D. 

On  the  failure  of  glycogen  deposition  in  the  livers  of  scorbutic  Guinea  Pigs. 

Endocrinol,  56:  584-589,  1955.-  i';       J.   :  ,  ■.  3  • 

Symeonidis,  A.,  Mulay,  A.  S.,  and  Trams,  E.  G.  ■ 

Effect  of  prolonged  pretreatment  with  desoxycorticosterone  on  the  liver  of 

hepatectotnized  rats. 

Endocrinol.  57;  ,550t55B,  ,195^;:   v;^ 

Mulay,  A.  S.  and  Eye stone,  W.  H. 

Transplantable  adenocortical  adenocarcinoma  in  Osborne -Meade 1  rats  fed  a 

carcinogenic  diet. 

J.  Nat.  Cancer  Inst.  16:  723-739,  1955. 


16.       •^■■^"  ■■ 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955. 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  l^aboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &  Pathogenesis   4. 5.  NCI  -§49- 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

6.  Tissue  Culture  of  Mouse  Leukemia s.     '■  ^   ^. - •     

PROJECT  TITLE 


7.  C.  J.  Dawe 

PRINCIPAL  INVESTIGATOR(S) 


8.  Michael  Potter  and  Joseph  Leighton 
OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objectives:   Development  of  methods  for  isolation  and  continuous  cultivation 
in  vitro  of  mouse  leukemia  cells. 

Methods:  Methylcholanthrene-induced  leukemias  in  ascites  form  in  DBA  mice 

are  used  as  a  source  of  leukemia  cells  which  are  propagated 
alternately  in  gelfoam  matrix  in  roller  tubes  and  in  mice.   By  inoculating 
the  tissue  cultures  intraperitoneally  into  mice  at  successively  increasing 
time  intervals  after  explantation,  it  is  possible  to  determine  the  survival 
time  of  leukemia  cells  in  tissue  culture  and  to  perpetuate  the  cell  time^ 
which  otherwise  dies  out  in  tissue  culture  alone. 

Major  findings:   In  the  four  months  during  which  this  project  has  been 

underway,  it  has  been  possible  to  carry  one  line  of 
lymphatic  leukemia  through  6  tissue  culture  passages  and  an  equal  number 
of  mouse  passages.  During  this  time  the  maximal  survival  time  in  tissue 
culture  has  increased  from  7  to  13  days.  The  possibility  that  a  morphologic 
change  may  have  occurred  in  the  leukemia  cells  is  under  study. 

Significance  to  cancer  research:   For  studies  of  growth  characteristics, 
nutritional  requirements,  and  susceptibility  to  therapeutic 
agents,  it  is  desirable  to  be  able  to  cultivate  pure  populations  of  leukemia 
cells  in  relatively  controlled  environment  such  as  is  obtainable  in  tissue 
culture.  The  purpose  of  this  project  is  to  determine  whether  leukemia  cell 
lines  can  adapt  to  continuous  tissue  culture  propagation  as  a  result  of 


SERIAL  NO.  NCI  «3-  ■'■■^^  'X.iiW\!r-  ■:  PAGE  II 


PRO JECt  REPORT  FORM  (Cont'd)         >™H£ii-r£:'_ll:; 

9.   PROJECT  DESCRIPTION  (Cont'd) 

repeated  exposure  to' tlssbeeiil'ttlire' environs      If  this  prdves  to   be  so 
the  method  can  then  be  applied  more  generally  to  the  isolation  of  various 
types  of  leukemia  cells  in  tissue  culture.   At  the  present  time^  no 
satisfactory  method  of  achieving  this  end  is  available.      . 

Proposed  course:  The  method  described  will  be  continued  for  at  least  a 

year^  at  which  time  the  results  and  possibilities  of  the  approach 
will  be  evaluated.  Additional  cell  lines  will  be  added  to  the.  experiment.'.. 


'=Ji)Si,-i3,- 


■     •.  i       ■    ■  ^■^:^^Z>^Li::'  ■■■    ■    ■■:'■' 

■.■'■'  .  iu\^-:jj ..b  <:  •  ■  ■    ■  •       ■ 


■3/3   utjQuni: 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


10.  Nci-^ty 

SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         /x7     ADMINISTRATION       [j 
REVIEW  &  APPROVAL  [j  TECHNICAL  ASSISTANCE  [j 

12.  None 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  14,  15,  or  16 


■i»  ■?«■ 


■'^^T>    »'SI'T>iT-ff.H?^ 


iO  '8'Sjtti,!)S;jivv    .,     -.',.■■■>  .;/?Ai:iJ}M^i:iS.i   ^Y^iMi.: 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology ■"  ■  ■   ''• 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &  Pathogenesis  4.        ■^_^   5.  NCI  -§4-»' 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Tissue  culture  in  a  perfusion  chamber  designed  to  use  whole  blood  as  a  source 

of  nutrient.  .■■■.-.   -  ■■- '  ^"  '  "   '  '  '  ' ^__ 

PROJECT  TITLE 

7.  C.  J.  Dawe 

PRINCIPAL  INVESTIGATOR(S) 

8.  None .     _. 

OTHER  INVESTIGATORS  :   •      : .  •   T    '  '  7'. " 

9.  PROJECT  DESCRIPTION;  .:;.;;'.  w  r^  ■ 

Objective:  To  determine  the  applicability  of  a  new  type  perfusion  chamber 
to  special  problems  in  tissue  culture. 

Methods:  A  lucite  perfusion  chamber  has  been  designed  and  constructed  to 

permit  culture  of  tissue  explants  in  a  flowing  plasma  medium 
derived  by  membrane  filtration  of  whole  blood,  circulated  through  the 
chamber  under  physiological  pressures  in  a  gravity  system.  The  chamber 
permits  continuous  microscopic  observation  of  the  cultured  cells. 

Major  findings:  This  iapparatuB  has  hot  yet  been  tested  in  relattoh  to  ;it;s 

effects  on  all  viability  and  growtli'.  The  hydraulics  have  , 
been  shown  to  function  satisfactorily  as  outlined  above.' 

Significance  to  cancer  research:   Using  fresh  heparinized  whole  blood  as  the 
circulating  medium  it  becomes  possible  to  bathe  tissue  explants 
in  an  environment  presumably  very  similar  to  that  provided  by  the  plasma 
component  of  the  circulating  blood  in  vivo.  Oxygen  tensions  of  the  medium 
can  be  controlled  by  varying  the  oxygenation  of  the  circulating  red  cells. 
The  investigator's  objective  in  developing  this  chamber  is  to  study  the 
effects  of  the  medium  so  derived  on  the  survival  and  proliferation  of 
leukemia  cells  in  vitro.   The  extent  to  which  leukemia  cells  can  proliferate 
while  actually  within  the  plasma  of  the  peripheral  vascular  system  is  at 
present  unknown.  Other  applications  of  the  apparatus  are  apparent.   For 
example,  it  would  be  possible  to  observe  the  effects  of  metabolic  products  of 
one  cell  type  on  another  cell  type  by  connecting  the  chamber  in  series. 

Proposed  course:  The  growth  promoting  or  inhibiting  effects  provided  by 

this  system  will  be  studied  primarily  on  mouse  and  human  leukemia 
cells,  using  human  blood  as  the  circulatory  element  Initially, 


PAGE  II 


10.  mi  iVr- 
SERIAL  NO, 


PROJECT  REPORT  FORM  (Cont'd) 


si  ISSK 


11. 


:?{:*:*  !■ 


BUDGET  ACTIVITY!' 


RESEARCH         /x/     ADMINISTRATION       i_l 
REVIEW.  &.  APPROVAL,  /"7  .   TECHNICAL  ASSISTANCE  /"7 


12. None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES^  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS-,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


■\^«'.-liK^ 


I  ■^  A  *  .. 


.  .   ■■  <^:\    hitkl^i/ i': ' 

:.;:;■$/-    !>'*<> 3 fjg,,r 

^     •■••I'Aijt*/-  Sif:?  ■  : 

^.  .  "f'J  ■fiMs 

■    ■ aiuow    1 

:.   -n:  -fto'  ^q- 

■^^-iU  a^   ::3asu<': 

■■  !  ■   •■■■         •■I^-^«  :Sjyjli  i 

•    ■■■irimiiii'ghi.r.i.i 

vH' 

PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 

14.  NCI  -^rtir 
SERIAL  NO. 

15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 

Cytologic  studies  of  sputum,  secretion,  and  serous  fluids  of  patients  with 

malignant  lymphoma. 

C.  J.  Dawe,  T.  B.  Woolner,  E.  M.  Parkhill,  and  J.  R.  McDonald. 

Amer.  J.  Clin.  Path.  25:  480-488,  May  1955. 


16.  HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955: 

Certified  in  Pathologic  Anatomy  by  American  Board  of  Pathologists  (April  1955) 
Completed  all  requirements,  including  thesis  and  examinations  for  degree  of 
Ph.D.  in  Pathologic  Anatomy  from  University  of  Minnesota.   Degree  to  be 
conferred  March  1956.  Thesis  title:  "Hodgkin's  Disease  and  Its  Inter- 
relationships with  Other  Disorders," 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &  Pathogenesis  4..        :      .  .  .       5.  NCI  Sifn 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN,  BETHESDA)  SERIAL  NO. 

6.  An  investigation  of  the  collagenous  connective  tissues.       

PROJECT  TITLE 

7.  William  G.  Banfield 

PRINCIPAL  INVESTIGATOR(S) 

8.  None  ; ,  ■  ■ __^__ ■  .  .   ■     

OTHER  INVESTIGATORS  '   .        . 

9.  PROJECT  DESCRIPTION 

Objectives:  The  immediate  objectives, are: 

1.  To  elucidate  the  age  changes  in  collagen. 

2.  To  uncover  mechanisms  affecting  the  usual  state  of  the 
collagen  in  the  body. 

Methods:   1.   Collagen  from  human  skin^  scars  and  Achilles  tendons  are 

subjected  to  dilute  acetic  acid  in  order  to  establish  more 
definitely  the  normal  limits  for  the  age-swelling  pattern  of  Achilles  tendon 
and  to  measure  the  amount  of  soluble  collagen  present  in  theee  tissues. 
Variations  in  the  reaction  of  skin  and  Achilles  tendon  from  the  normal  are 
investigated  through  the  patient's  history  and  possible  causes  of  such 
variation  are  subject  to  animal  test.   The  maturation  of  scars  is  studied 
with  respect  to  changes  in  acid  soluble  collagen  and  histology. 

2,  Hamsters  are  tested  for  changes  in  skin  collagen  after 
hormone  treatments  or  gland  removal. 

3.  A  successful  search  has  been  made  for  a  stain  which  will 
react  differently  to  collagen  from  a  young  Achilles  tendon  than  to  collagen 
from  an  old  Achilles  tendon. 

Major  findings:   1.  A  possibility  has  been  found  that  chorionic  gonadotropin 

and  an  adrenal  cortical  hormone  each  may  increase  the 
solubility  of  human  skin  collagen. 

2.  A  method  for  differentially  staining  collagen  in 
young  and  old  tendons  and  in  young  and  old  scars  has  been  developed. 

3.  Recent  scars  contain  a  large  amount  of  acid  soluble 
collagen  whereas  old  scars  do  not. 

Significance  to  cancer  research:  A  knowledge  of  collagen  metabolism 

including  collagen  maturation,  may  help  in  the  understanding 
of  connective  tissue  tumors.  Such  knowledge  should  also  contribute  to  an 
evaluation  and  possible  control  of  the  stroma  which  accompanies  and  even 


SERIAL  NO.  NCI  -Hrfr-  PAGE  II 

PROJECT  kEPORT  FORM  (Cont'd) 

9.  PROJECT  DESCRIPTION  (Cont'd) 

seems  ne^cessary  for  the  continued  growth  of  many  tumors.   Staining  techniques, 
especially  if  placed  on  a  hi stoch^nflcal  basis,  would  be  invaluable  for 
studying  the  collagenous  components  of  tumors. 

Proposed  course:  The  measurement  of  theacid  soluble  collagei^'ln  scars  will 

be  continued  until  all  stages  in  the  maturation  of  a 
scar  can  be  represented.  The  scar  specimens  will  be  studied  using  the 
newly-developed  differential  staining  technique  for  cqllageh  and  cohv6tltlonal 
histologic  stains.   The  screening  of  skin  and  tendons  for  their  content  of 
acid  soluble  collagen  will  be  continued  and  leads  will  be  followed  by 
animal  experimentation.  Animal  experiments  to  determine,  possible  effects 
of  gland  removal  on  collagen  development  will  be  continued.  The  mechanism 
by  which  old  and  young  collagen  is  differentially  stained  will  be  investiga- 
ted and  an  attempt  will  be  made  to  improve  the  method.   The  electron 
microscope  will  be  used  to  further  the  project. 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


10. 


SERIAL  NO. 


LI. 


BUDGET  ACTIVITY: 


RESEARCH         U/ 
REVIEW  &  APPROVAL  /~7 


ADMINISTRATION       L_l 
TECHNICAL  ASSISTANCE  /T 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHi\NGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  IV 


PROJECT  REPORT  FORM  (Cont ' d) 


14.  NCI  -6^6- 
SERIAL  NO. 


15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1W5 


Banfield,  W.  G. 

Width  and  length  of  collagen,  fibrils  during  the  development  of  human  skin, 

in  granulation  tissue  and  in  the  skin  of  adult  animals. 

Journal  of  Gerontology  10:  IS-'l?,  1955,: 


16.  HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955. 

None. 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &  Pathogenesis  4. '"  "' "  '   5.  NCI  52€"- 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Effects  of  Ionizing  Irradiation  with  emphasis  on  the  morphologic  alterations 

and  neoplasms  '■  ' 

PROJECT  TITLE 

7.  Richard  L.  Swarm    .  ■  ■  "    .  ■  .  ■      '• ' 

PRINCIPAL  INVESTIGATOR(S) 

8.  None ] ^  -  '■     ■  '  ; -   -  . 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives:  The  collection  of  additipnal  information  regarding  the  develop- 
ment of  neoplasms  in  man  and  laboratory  animals  following 
exposure  to  radiation  injury  is  one  general  objective  of  this  study.  Some 
interest  in  the  type  of  irradiation  change  other  than  that  which  is  manifested 
in  the  formation  of  neoplasms  is  maintained.  The  different  effects  produced 
by  different  types  of  x-radiation.  Alpha  irradiation  and  Beta  irradiation 
are  of  particular  interest.  A  knowledge  of  the  changes  effected  by  different 
types  of  radiation  in  existing  tumors  will  be  sought. 

Methods:  Three  types  of  approach  have  been  made.   (1)  Participation  in  the 

Surgical  Pathology  and  Post-Mortem  pathology  division  of  the 
Pathologic  Anatomy  Department  of  the  Clinical  Center.  Here  a  review  of 
accessioned  specimens  is  made  possible  by  actual  participation  in  the  study 
and  diagnosis  of  specimens  as  they  are  received.  An  effort  is  made  to 
select  and  categorize  anatomic  material  showing  radiation  change.   (2)  Detec- 
tion of  neoplasms  and  other  changes  in  animals  who  have  been  given  whole 
body  irradiation  of  varying  amounts  at  significant  intervals  prior  to 
anatomical  study.   (3)  A  study  of  the  distribution  and  effects  of  some 
radioactive  isotopes  given  by  different  routes  to  animals  and  therapeutically 
or  for  diagnostic  purposes  to  man  will  be  made.  Here  the  emphasis  has  been 
on  the  effect  of  I-V  administered  colloidal  thorium  dioxide. 

Major  findings:  A  new  project.  Of  interest,  however,  is  one  human  case 

which  was  studied  during  life  by  the  General  Medicine  Branch 
of  NCI  and  which  was  later  studied  anatomically.  Study  revealed  the  presence 
of  neoplasms  in  the  liver,  spleen,  and  bone  of  a  patient  who  had  stored 
thorium  dioxide  in  the  reticulo-endothellal  system  for  many  years.  Studies 
of  this  are  as  yet  incomplete,  however,  from  the  observations  made  In  this 
case  and  other  reported  cases,  an  etiologic  relationship  between  the  storage 
of  thorium  and  the  development  of  neoplasms  in  man  seems  to  exist. 


SERIAL  NO.  NCI  -We--  PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 

9.  PROJECT  DESCRIPTION  (Cont'd) 

Proposed  course:   (1)  An  interest  in  the  morphologic  effects  of  radiation 
on  human  tissue  and  in  particular  on  previously  existent 
neoplasms  will  be  maintained  and  further  developed.   Particular  emphasis 
will  be  placed  on  the  study  of  the  effects  of  different  types,  and  energies 
of  irradiation.   Study  of  human  case  material  (speciii^ens)  from  the  Clinical 
Center  will  be  the  important  phase  of  this  activity,   (2)  The  anatomic 
study  of  animals  exposed  to  whole  body  irradiation  will  be  continued. 
(3)  The  study  of  the  distribution  and  morphologic  changes  effected  by  the 
administration  of  colloidal  thorium  dioxide  given  intravenously  to  man  for 
diagnostic  purposes  and  experimentally  to  animals  will  be, continued. 
Knowledge  of  the  pattern  of  storage  and  effects  in  man  will  be  furthered 
by  a  proposed  istudy, of  human  material  .at, .the  Clinical,  Center,  The  Armed 
Forces  Institute  of  Pathplogy,  and,  of  material  submitted  by  Dr.  William 

LOOney,-.  .;■;,  >:,.'?  ,,  ■   ,,.,..,:•.      i  =  :.-;,  ::  .■!„   ;   .i^w.  .•  ^   ■        ■,,;•.:.. 


^X'nz  -U: 


PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


10.  HCl.^2S- 
SERIAL  NO. 

11. 


BUDGET  ACTIVITY: 

RESEARCH         /x7     ADMINISTRATION       £j 
REVIEW  &  APPROVAL  /~7     TECHNICAL  ASSISTANCE  /"7 


12. None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  195fr  or  1957 


13.  IF  THIS  PROJECT  RESEMBLES, COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 

(1)  The  work  up  of  anatomic  specimens  from  patients  who  have  received 
irradiation  in  the  Clinical  Center  will  complement  the  work  of  the  clinical 
services  particularly  that  of  the  Radiation  Therapy  Branch.   (2)  The 
anatomic  study  of  laboratory  animals  given  whole  body  irradiation  has  been 
limited  to  hamsters  supplied  by  Dr.  W,  Smith,  Radiation  Branch,  NCI.  Other 
pathologists  have  assisted  Dr.  Smith  in  this  study.   (3)  Research  on  the 
morphology  of  damage  in  animals  produced  by  I-V  administered  thorium  dioxide 
has  been  made  by  many  investigators  in  this  country  and  abroad.  Only  two 
European  investigators  have  succeeded  in  producing  neoplasms  in  animals 
following  I-V  administration  of  colloidal  thorium  dioxide.   Experiments 
designed  to  confirm  or  refute  these  findings  are  contemplated.  To  my 
knowledge,  no  study  of  the  morphology  of  thorium  aggregates  in  human  tissues 
like  that  proposed  has  been  undertaken  elsewhere. 


PAGE  IV 


PROJECT  REPORT  FORM  (Cont ' d ) 


i'a.3 

14.  NCI.5^-S— 
SERIAL  NO. 


15.  None 


PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 


16.  HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955 


Elected  to  Fellowship  in  the  American  Society  of  Clinical  Pathologists  and 
to  membership  in  the  Washington  Society  of  Pathologists  and  in  the  D.  C, 
Section  of  the  Society  for  Experimental  Biology  and  Medicine. 


:>;i^3HHSi  L.   .    .,-  ;  TDSLOKf  aiHT  ^J[ 


PAGE  I 
PROJECT  REPORT  FORM 

1.  National  Cancer  Institute 2 .JLaboratory  of.2.?tholog^ 

"institute    '   '  LABORATORY  "or  branch" 

3._0ffice  of  tjbe.  Chief 4. 5  .NCI_f*9-;;i_ 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BEfflESD^  SERIAL  NO. 

6.  Morphologic  and  histochemical  study  of  a  human  carcinoma  of > the  floor  of  ■ 
the  mouth  in  material  from  the  patient,  from  long  term  tissue  culture  of 
the  tumoF  and  from  heterologous  transplantation  of  tissue  culture 
material  to  the  cortisone  treated  rat.  


PROJECT  TITLE 
Alan  S .  Rabson 


PRINCIPAL  INVESTIGATOR (S) 

8 .  Gerald  "Suskind   .       ,    .    .    ■...; ^^  _„ , ,  j_  [■■    ]■ '"•        '-■■..■■       _  ._____„_____ 

OTHER  INVESTIGATORS       ' 

9.  PROJECT  DESCRIPTION 

Objectives:  The  objective  of  this  project  is  to  determine  whether  or  not 

there  are  significant  morphologic  and  histochemical  differences 
in  tumor  cells  after  prolonged  grov/th  in  tissue  culture  and  in  heterologous 
hosts  . 

Methods  Employed:   In  1954,  a  tumor  of  the  floor  of  the  mouth  was  excised 

from  a  patient  at  the  Clinical  Center  and  cells  from  this 
tumor  have  been  grovm  in  large  quantity  in  tissue  culture  as  recently 
reported  by, Eagle;  (Strain  K-B)  .  The  original  histologic  sections  of  this 
tumor  as  well  as  subsequent  recurrences  are  available,  in  the  files'  of  the 
Pathologic  Anatomy  Branch),  for  morphologic  and,  histochetftlcal  Study i  The 
cells  in  tissue  culture  will  be  studied  on  cover-slip  preparations  and  in 
sponge  matrix  tissue  culture.  Solid  tumors  in  cortisone  treated  rats  have 
been  produced  by  injection  suspensions  of  the  cells  in  tissue  culture,  and 
are  being  studied  with  a  variety  of  fixatives  and  stains. 

Major  Findings;  The  project  has  only  been  in  progress  for  a  short  time  and 
no  significant  findings  are  available. 

Significance  to  Cancer  Research:  The  potential  value  of  cell  lines  of  human 

tumor  cells  in  biological  and  chemothera- 
peutic  studies  has  been  questioned  on  the  grounds  that  the  cells  are  variants 
of  the  original  tumor  and  have  only  a  limited  relationship  to  it.  It  would 
seem  to  be  of  considerable  interest  if  it  could  be  demonstrated  that  cells 
grown  for  many  generations  in  tissue  culture  and  subsequently  in  heterologous 
hosts  are  morphologically  and  histochemically  similar  to  the  original  tumor 
from  which  they  were  derived. 

proposed  Course  of  Project:  As  described  above  under  Methods  Employed,  the 

morphology  and  histochemistry  of  the  original 
carcinoma  of  the  floor  of  the  mouth  will  be  compared  morphologically  and 
histochemically  with  the  tumor  cells  in  tissue  culture  and  in  cortisone 
treated  rats . 


10  ■  NCI  $^^9- 
SERIAL  NO. 


PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


11, 


BUDGET  ACTIVITY: 

RESEARCH      ,   ,  /xj 
REVIEW  &  APPROVAL  /^ 


AiDMINISTRATION       /_/ 
TECHNICAL  ASSISTANCE  /~7 


12, 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE/  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957 


13 


,IF  THIS  PROJECT- RESEMBLES ,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HE-UTH  SERVICE  (WITHOUT  INTlERCH/iNGE  OF 
Personnel,  FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  III 
PROJECT  REPORT  FORM  (Cont'd) 


14.  NCI  j^^ 
SERIAL  NO, 


15. 


PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955 

Pretest  of  Forms  and  Field  Techniques  For  Use  in  the  Detroit -Windsor 
Air  Pollution  Study  by  A.F.W.  Peart,  C.P.  Anderson,  A.S.  Rabson  and 
W.L.  McEwen,  AM/i  Arch,  of  Indust.  Health  11:  47,  1955. 

The  Effect  of  Gamma  Globulin  on  Subclinical  Infection  in  Familial 
Associates  of  Poliomyelitis  Cases  II.  Serological  Studies  and  Virus 
Isolations  from  Pharyngeal  Secretions,  G.C.  Brown,  A.S.  Rabson,  and 
J.H.  Schieble,  J,  of  Immunology  74;  71,  1955. 

C-Reactive  Protein  in  Serum  of  Patients  with  Leprosy.  A.S,  Rabson, 
International  J.  of  Leprosy  23:  155,  1955. 


16. 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955. 


PAGE  I 


PROJECT • REPORT  FORM 

1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Office  of  the  Chief 4. 5. NCI  5-3^6— 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Attempt  to  induce  carcinoma  of  the  bladder  in  hamsters  by  infection  with 
Schistosoma  haematobium 

PROJECT  TITLE  ' 

7.  Dr.  Eloise  Cram  and  Dr.  Louis  B.  Thomas 

PRINCIPAL  INVESTIGATOR(S) 

8.  None - 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

This  research  project  is  in  collaboration  with  Dr.  Eloise  Cram,  NMI,  and 
consists  of  studying  chronic  infection  with  S.  haematobium  (Gold  Coast  strain) 
in  hamsters;  particularly  with  reference  to  the  induction  of  bladder 
carcinoma, 

(a)  217  hamsters  were  exposed  to  approximately  300  cercariae  each  early 
in  1955. 

(b)  Approximately  177  infected  hamsters  are  still  living  and  will  be 
allowed  to  live  as  long  as  possible, 

(c)  Forty  infected  hamsters  have  died  after  162-305  days  after  exposure 
to  cercariae.   These  animals  have  all  had  heavy  Schistosomal 
infection  of  the  intestines  and  liver,  but  only  slight  infection 

of  the  bladder.   Pathological  study  of  these  animals  is  incomplete 
at  this  time.   No  lesions  suggestive  of  neoplastic  change  in  the 
bladder  have  been  seeo. 

Significance  to  cancer  research:   S.  haematobium  infection  has  been  found 

associated  with  bladder  carcinoma  in  several  parts  of  the  world  and 
is  thought  possibly  to  be  a  cause  of  bladder  carcinoma.   This 
chronic  infection  study  is  possible  because  of  Dr.  Cram's  work 
in  getting  S.  haematobium  established  in  hamsters. 

Proposed  course  of  the  project; 

(1)  A  continuation  of  the  study  of  Gold  Goast  strain  S.  haematobium 
in  hamsters, 

(2)  Similar  study  of  chronic  infection  with  Egyptian  strain  S.  haematobium 
when  infected  snails  become  available  in  Dr.  Cram's  laboratory. 


10.  NCI  »3e— ■  ■ 
SERIAL  NO. 


11. 


PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


!(>!ii3iO 


BUDGET  ACTIVITY: 


RESEARCH         /x/     ADMINISTRATION.   .   [J 
REVIEW  &  APPROVAL  [j  TECHNICAL  ASSISTANCE  [j 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


:.;fi=, ;!: 


-t  sl^T 


13.  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),'  IDENTIFY  SUqn  RESEARCH  :  .; 


s  t-ftoni'.- 


NO  ENTRIES  FOR ,14,  15,  or  16 


PAGE  I 

PROJECT  REPORT  FORM 

1.  National  Cancer  Institute     2.  Laboratory  of  Pathology 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &  Pathogenesis  4. 5.  NCI  526 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Metabolism  of  Cortisol  into  higher  hydroxylated  derivatives  in  guinea  pigs 

with  and  without  neoplastic  disease. [ 

PROJECT  TITLE 

7.  E.  M.  Nadel  and  S.  Burstein 

PRINCIPAL  IN\nESTIGATOR(S) 

8.  None 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives:  This  title  includes  three  sub-projects t   (1)  Isolation  and 

elucidation  of  structures  of  new  Cortisol  metabolites  in  vivo 
(Dr.  S.  Burstein).  (2)  Study  of  enzymatic  transformations  involved  in  specific 
tissues.   (3)  The  incorporation  of  C''-^  acetate  into  Cortisol  and  metabolites. 

Methods:   The  methods  previously  utilized  include,  extraction,  paper  and 
column  partition  chromatography,  infra-red  spectrometry,  ultra 
violet  spectrophotometry,  incubation  techniques  using  slices  and  homogenates 
of  liver,  adrenals,  and  other  organs,  use  of  radioisotopic  techniques. 

Major  findings;   (1)  6-p  hydroxycortisol  and  Steroid  Ila  (as  yet  unidentified) 

have  been  isolated  from  the  urine  of  guinea  pigs.   (2)  6-p 
hydroxycortisol,  Steroid  2  (as  yet  unidentified)  tetrahydrocortisol  have 
been  isolated  from  the  urine  of  man.   (3)  Radioacetate  is  incorporated  in 
increased  amounts  into  Cortisol  and  corticosterone  in  the  adrenals  of 
scorbutic  guinea  pigs,  and  both  steroids  have  been  identified  for  the 
first  time  in  the  tissue  of  the  guinea  pig. 

Significance  to  cancer  research:  This  work  provides  the  background  material 

for  the  continuation  and  extrapolation  of  similar  studies  on  the 
tissues  of  tumor-bearing  guinea  pigs. 

Proposed  course:   In  this  collaborative  project  we  will  endeavor  to  complete 

such  studies  during  the  coming  year.   Such  a  study  is  now 
feasible  because  of  the  closer  association  with  Dr.  S.  Burstein  on  the 
reservation  as  a  Visiting  Scientist  from  the  Worcester  Foundation  for 
Experimental  Biology. 


PAGE  II 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI  526 


SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         M  ADMINISTRATION        [j 

REVIEW  &  APPROVAL  /"7     TECHNICAL  ASSISTANCE   /~7 


12.  None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER  ORGANIZATIONS  PRO- 
VIDING FUNDS^  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  None 


IP  THIS  PROJECT  RESEMBLES;  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH 


NO  ENTRIES  FOR  14,  15,  or  16 


PAGE  I 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute     2.  Laboratory  of  Pathology ■  '".  ■  ■■. 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Cancer  Indue.  &  Pathoaenesis  4.  5.  NCI  ^34:- 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Morphology,  pathogenesis  aihd  tra'nsplantability  of  spdntanedus  neoplasms 

within  the  canine  species.  .  ., 

PROJECT  TITLE 

7.  Louise  S.  Lombard _^ . ___ 

PRINCIPAL  INyESTIGATOR(S) 

8.  None   ,     .,     '   ■  .".••;■•'  •- '  -  .  ;  ■'   ■  '  ',  '  ^  ^  [     ■      '   .  ■    ■    ■'  ^      .•  •:- 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives:   The  objective  of  this  project  is  to  study  the  morphology  and 
pathogenesis  of  spontaneously  occurring  neoplasms  within  the 
canine  species  and  to  obtain  transplantable  canine  neoplasms  which  could 
be  utilized  in  morphologic,  chemotherapeutic,  metabolic,  endocrinologic 
and  irradiation  studies. 

Hethods:  Through-  the  use  of'  cortisone  and/or ^t^        irradiation,  a 

spontaneous  ianaplastic  thyVo'id  carcinoma  was  transplanted  by 
various  routes  in  heterologous  puppies  and  studied  motphologically. 

Major  findings:  The  transplantable  canine  thyroid  carcinoma  was  grown  in  a 

wide  variety  of  tissues  with  growth  occurring  in  almost 
1007o  of  the  irradiated  heterologous  puppies.  The  morphology  of  the  tumor 
remained  essentially  unchanged  throughout  the  35  serial  transplant  genera- 
tions. Lymph  node  and  lung  metastases  were  found  in  irradiated  animals 
receiving  either  fresh  or  frozen  tumor  tissue  (the  thyroid  carcinoma  was 
preserved  by  storage  at  a  -60  to  -70°C.  for  several  months). 

Significance  to  cancer  research:  Transplantable  malignant  neoplasms  in  the 

dog  would  offer  tumors  in  a  larger  host  for  morphologic, 
metabolic,  biochemical,  hormonal,  and  irradiation  studies,  as  well  as 
including  another  species  for  the  testing  of  chemotherapeutic  substances. 
The  transplantable  canine  thyroid  carcinoma,  intracerebrally  inoculated, 
is  now  being  used  as  a  test  tumor  for  the  efficacy  of  radioactive  boron  in 
the  treatment  of  brain  tumors  (Univ.  of  Penna.). 

Proposed  course:  The  transplantable  thyroid  carcinoma  will  be  transplanted 

to  untreated,  closely  inbred  puppies  and  in  irradiated 
puppies  receiving  cysteine  and  bone  marrow.  Morphologic  studies  and 
transplantation  experiments  will  be  performed  utilizing  other  spontaneous 
canine  tumors,  especially  leukemia. 


PAGE  II 


PROJECT  REPORT  FORM  (Cotit'd) 


10.  NCI  ^a^- 
SERIAL  NO. 

II. 


BUDGET  ACTIVITY: 


RESEARCH  .        /x/    ADMINISTRATION  r;   '  7_/ 
REVIEW  &  APPROVAL  [j  TECHNICAL  ASSISTANCE  £7 


12.  School  of  Veterinary  Medicine^  University  of  Pennsylvania  Facilities, 

-Personnel  (Dr»  Mark  Al lam,.  Dean)  1956. . . .  :—  , 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  OR  OTHER 'ORGANIZATIONS'  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  57 


13.  None  ' 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,- OR  PARALLELS  RESEARCH  DONE  ELSEWHERE  ' 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FAClLlTlEfS  OR- 
FUNDS),  IDENTIFY  SUCH  RESEARCH 


PAGE  III 
PROJECT  REPORT  FORM  (Cont'd) 


JTAy 

14.    NCI- 521"" 
SERIAL  NO. 


15.  PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 


Urbain  Leblanc 

Early  Veterinary  Pioneer  in  Cancer  Research 

J.  Amer.  Vet.  Med.  Assoc,  126:  363-365,  1955. 


16.  HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955 

Society  of  Phi  Zeta,  May,  1955. 

American  College  of  Veterinary  Pathologists,  Nov.,  1955. 

Washington  Society  of  Pathologists,  Nov.,  1955. 

Conference  of  Research  Workers  in  Animal  Diseases  in  North  America,  Dec,  1955, 


Note:   Collaborative  research  is  being  done  with  Drs.  H.  L.  Stewart  and 
H.  B,  Andervont  on  adenomatous  gastric  lesions  in  strain  I  mice; 
with  Dr.  W.  R.  Bryan  on  rapid  and  slow  growing  Rous  Sarcoma;  with 
Dr.  H.  P.  Morris  on  experimentally  produced  pituitary  adenomas  and 
hepatomas  in  rats. 


M'^ 


ii;-'-;joru 


.1  Ti>SL-o>j; 


;r  ■  r.^jj'^:  ^^'iii-v^  ^iiroAirff'ii/^  <. 


PAGE  1 
PROJECT  REPORT  FORM 


1,'    National  Cancer  Institute       2,     Radiation  Branch 

INSTITUTE  L.'.BOPJiTORY  OR  BR/UMCH 


Radiation  Biology  Section       h»  5»       62U 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THi'.N  BETHESD/J       SERI/iL 

NO. 


6,     Description  and  Treatment  of  the  High-Radiation-Doso  Syndrome 
PROJECT  TITLE 

7«     H.  L.  Andrews  and  K.   C.   Brace 
PRINCIP/iL  INVESTIGATQR(s) 


8.  H.  Gmp 


OTHER  INVESTIGATORS 

9,  PROJECT  DESCRIPTION 

Objectives;  1,  To  characterize  the  syndrome  produced  by  x-ray  doses  up 
to  about  100  times  that  lethal  in  30,  days.  Emphasis  in  this  project  is 
on  function  rather  than  on  pathology,  2,  To  study  agents  capable  of 
modifying  the  high-dose  syndrome,  3.  To  study  the  effect  of  dose  rate 
and  fractionation  on  the  high-dose  syndrome,  , 

Methods  employed;  Doses  from  1000  r  upward  are  administered  with  the  3  Mev 
x-ray  generator.  The  guinea  pig  is  the  animal  of  most  interest  because  of 
the  sharp  break  in  signs  of  radiation  injury  at  6^000  r.  Typical  studies 
made  before  and  after  radiation  are  blood  coimts,  blood  electrolytes,  pain 
threshold^  pinna  reflex,  electrical  impedance  of  body  tissues.  Survival 
time  s  are  carefully  recorded. 

Major  findings;  Using  200  KVP  x-rays  at  55  r  per  minute  we  have  found  in 
the  guinea  pig: 

1,  At  6,000  r  there  is  a  sharp  change  from  a  5  day  death  in  depres- 
sion to  a  death  in  less  than  2lt  hoiars  with  marked  signs  of  increased 
central  nervous  system  excitability, 

2,  lAJhen  barbiturates  are  given  prior  to  irradiation,  irradiation  of 
even  15,000  r  produces  only  the  depression  normally  seen  with  less  than 
5,000  r  and  the  survival  time  is  about  k   days  instead  of  the  1  day  or  less 
obtained  ^^^ithout  medication,  A  series  of  depressant  and  anti-convulsant 
drugs  are  without  effect  on  survival  time  although  some  prevent  the  appear- 
ance of  the  high-dose  syndrome. 

Significance  to  cancer  research;  Any  increase  in  knowledge  of  the  biological 
effects  of  radiation  are  of  potential  value  in  radiation  therapy.  It  is  of 
interest  that  the  large  doses  used  in  this  project  are  not  large  in  terms  of 
local  doses  delivered  for  therapeutic  purposes. 

Proposed  course  of  project;  Much  of  the  work  already  done  will  be  repeated 
using  the  3  Mev  generator  to  obtain  a  greater  relative  radiation  dose  to 
underlying  structures  in  the  central  nervous  system.  The  high  dose-rates 
obtainable  with  this  generator  i\Tiii  be  studied  for  biological  effectiveness 
and  will  permit  an  extension  of  studies  of  agents  modifying  the  high-dose 
syndrome. 


lo.Nei- 


62h 


SERL'JL  MO. 


PAGE  2 


11,  BITOGET  ACTIVITY: 

Research  -       /C7 
Review  &  Approval  /~7 


Administration       /^ 
Technical  Assistance  /~7 


12.   COOPEIL'.TING  UNITS  OF  THE  PUBLIC  HE/iTH  SERVICE,  OR  OTHER  ORGi.NI  CAT  IONS, 

•   PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PliOJECT  IN  EITHER 
1956  or  1957: 

None 


13.     IF  THIS  PROJECT  RESE!'IBLES,   COi^IPLEIlENTS,   OR  PiiR.'XLELS  RESEj^CH  DOI^ 

ELSEVJIIERE  IN  THE  PUBLIC  HEALTH  SERVICE   (WITHOUT   IKTERCaiNGE  OF  PERSOl'MilL, 
FACILITIES   OR  FTOIDS),   IDENTIFY  SUCH  RESE/JICH: 

None 


NO  ENTRIES  FOR  ITEMS  U4,  l5  &  I6, 


PAGE  1 
PROJECT  REPORT  FCRIl 

1,  National  Cancer  Institute        2.  Radiation  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

3«  Radiation  Biology  Section  h^ , 5.  NGI-627 

SECTION  OR  SERVICE         LOCATION(IF  OTHER  THAN  BETHESDA;   SERIAL  MO, 

6,  Change  in  Tissue  Constituents  by  Radiation 
PROJECT  TITLE 


7.  H,  L,  Andrews  and  E,  J.  Liljegren 
PRINCIPAL  INVESTIGATOR(S; 


None 
OTHER  II\rVESTIGATQRS 


9,  PROJECT  DESCRIPTION 

Objectives;  To  determine  changes  in  amino  acid  concentrations  produced  by 
x-irradiation  in  various  body  tissues  of  the  guinea  pig. 

Methods  employed;  Assays  of  various  tissues  were  made  by  separation 
columns  and  paper  chromatography.  Normal  ahimals  were  compared  with 
those  receiving  various  doses  of  x-rays« 

ria.ior  findings;  Experimental  work  has  been  completed  and  the  results 
are  being  analyzed.  There  are  changes  in  amino  acid  concentrations  but 
it  is  premature  to  discuss  them  in  detail  now. 

Significance  to  cancer  research:  Any  increase  in  our  knowledge  of  the 
biological  effects  of  radiation  is  of  potential  importance  in  radiation 
therapy, 

Proposed  course  of  project;  Unless  the  data  are  more  striking  than 
presently  appears  this  project  will  be  terminated  with  publication  of  the 
findings  « 


10,  NCI.627 

SERIAL  NO. 


PAGE  2 


11.  BUDGET  ACTIVITY: 

Research        /^ 
Review  &  Approval  f^ 


Administration      /V 
Technical  Assistance  £J 


12.  COOPERATING  UI^ITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONIffiL  FOR  THIS  PROJECT  IN  EITHER 

1956  OR  1957. 

None 


13..     IF  THIS  PROJECT  RESEMBLES,   CQlPLEIffiNTS,   OR  P/iRALLELS  RESEARCH  DONE 

ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL, 
FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 

None 


NO  EOTRIES  FOR  ITEMS  li;,  l5-&  l6i 


PAGE  1 


PROJECT  REPORT  FORPI 


1,     National  Cancer  Institute       2»     Radiation  Branch 

.,  INSTITUTE  *  LABOR/iTORY  OR  BRilNCH  ■ 

3.     Radiation  Biology  Section      h, ___, 5«  NCI>628 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  TH/JM  BETHESDAJ       StlRI/iL  NO, 

6,     Dosimetry  of  High  Energ;y  Radiations 
PROJECT  TITLE 

7*  H.  L,  Andrews 

PRINCIPAL  INVESTIGATOR ( S ) 

8.  R«  E,  Murphy 


OTHER  INVESTIGATORS 


9k  PROJECT  DESCRIPTION 

Objectives:  To  develop  methods  and  instruments  for  determining  the  radia- 
tion doses  delivered  to  tissues  at  energies  up  to  3»S  Mev  and  at  high  dose 
rates. 

Methods  employed:  The  usual  types  of  ionization  chambers  used  for  radiation 
dosimetry  are  not  designed  to  read  correctly  at  the  x-ray  energies  avail- 
able from  the  3*5  Mev  generator  and  are  probably  incapable  of  measuring 
the  high  intensities  anticipated  when  the  generator  is  run  at  full  output. 
Chemical  dosimeters  appear  to  have  a  response  independent  of  photon  energy, 
and  to  be  capable  of  accurately  recording  high  dose  rates.  Work  has  con- 
centrated on  two  chemical  systems,  the  production  of  HCl  from  chloral 
hydrate  and  the  oxidation  of  ferrous  iron  to  ferric, 

i^fa..1or  findings:  As  an  extension  of  the  main  problem  the  chloral  hydrate 
system  has  been  adapted  to  depth-dose  determinations  by  the  addition  of  a 
gelling  agent.  With  this  dosimeter  a  beam  of  radiant  energy  can  be  visual- 
ized and  measurements  of  local  radiation  doses  made  with  a  probing  pH 
electrode.  The  gel  absorbs  radiation  almost  exactly  as  does  water,  and 
hence  its  response  will  be  a  good  indicator  of  tissue  dose  in  complex 
structures  not  amenable  to  calculation  or  to  measurement  with  other  methods. 

Significance  to  cancer  research;  A  most  basic  requirement  for  good  radia- 
tion therapy  is  that  the  tumor  dose  be  made  as  high  as  possible  relative  t« 
the  dose  delivered  to  healthy  tissue.  Any  method  which  can  improve  the 
measurements  of  dose  delivered  to  deep  body  struct-ures  should  improve  the 
ability  of  the  therapist  to  keep  the  tumor/tissue  dose  ratio  high. 

Proposed  course  of  project;  Measurements  at  high  dose  rates  have  not  been 
made  because  of  target  failures  when  attempts  were  made  to  operate  the 
generator  at  high  power.  As  this  difficulty  is  overcome  chemical  dosimetry 
will  be  applied  to  high  dose  rates  and  to  short  pulses  of  both  x-rays  and 


PilGE  2 


MCI-628 

SERI/iL  NO, 


PROJECT  DESCRIPTION  (CONT.) 

elGctrons,  These  measurements- will  then  provide  a  basis  for  the  extension 
of  research  in  radiation  biology  into  a  dose-rate  range  never  before  reached 
in  ,the.  laboratory. 


10.     MCI-628 
SERL'iL  NO, 


PAGE  3 


11,  BUDGET  ACTIVITY: 

Research        /T/ 
Review  &  /^jproval  /V 


Administration      ^£7 
Technical  Assistance  /^ 


le,    gooperj;tihg  mits  of  the  public  he/xth  service,  or  other  orgaotzations, 

PROVIDING  FUIJDS^  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957: 

None 


13.      IF  THIS  PROJECT  RESEMBLES,    COMPLEffiNTS,    OR  PjJUXLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HEj'XTH  SERVICE  (WITHOUT  INTERCHi^NGE  OF 
PERSONNEL,   FACILITIES  OR  FUIffiS),   IDENTIFY  SUCH  RESE/JICH: 

None 


NO  ElMTRIES  FOR  ITEIIS  lij,   15  &  l6. 


PAGE  1 


PROJECT  REPORT  FORM 


1,  National  Cancor  Institute  2,  Radiation  Branch 

INSTITUTE  L'xBOPu.TORY  OR  BRilNCH 

3.  Radiation  Biology  Section  h, g.  MCI-629 

SECTION  OR  SEliVICE         LOCATION  (IF  OTHER  TIL'iN  BETHESD/J '   SERIAL  WO, 

6,  The  effect  of  ionizing  radiation  on  amino  acids 

PROJECT  TITIE 

7.  Charles  R.  Maxwell 

PRINCIPAL  INVESTIGATOR(S; 


^«  Dorothy  C,  Peterson 


OTHER  INVESTIGATORS 


9,     PROJECT  DESCRIPTION 

Objectives;  The  objective  of  this  project  is  to  determine  the  mechanism 
of  the  chemical  reactions  induced  by  ionizing  radiation  in  aqueous  solu- 
tions of  amino  acids.  This  is  part  of  a  long  range  progrrj-n  to  accmnulate 
information  on  siraple  systems  of  biological  interest  so  that  general 
principles  may  be  ascertained  and  applied  to  complex  systems  which  are 
not  amenable  to  thorough,  direct  investigation. 

Methods  employed;  Solutions  of  rmino  acids  are  irradiated  with  ionizing 
radiation  and  then  analyzed  for  the  products  formed.  Since  the  products 
are  sensitive  to  radiation  it  is  necessary  to  determine  the  yield  of 
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  product  at  very 
low  doses  before  secondary  reactions  distort  the  picture. 

Most  of  the  study  is  done  with  $0   iW  X-rays  as  the  ionizing  radiation. 
The  effect  of  dose  rate  and  ion  density  is  investigated  by  irradiating 
with  electrons  and  alpha  particles.  Protons  and  neutrons  will  probably 
be  used  in  the  , future. 

Ma j or  findings ;  Earlier  work  has  shown  that  x-rays  induce  four  reactions 
in  aqueous  solutions  of  both  glycine  and  alanine.  One  of  these  reactions 
was  shoT>rn  to  result  directly  from  the  absorption  of  energy  by  the  dissolved 
moleculej  the  other  three  were  shown  to  be  indirect,  e,g,,  the  res'ult  of 
energy  absorbed  by  the  water  which  is  generally  considered  to  produce  H  and 
OH  free  radicals  and  H2O2. 

The  work  this  year  has  been  concentrated  on  studying  the  role  of  the 
above  active  intermediates, 

1)  Irradiations  in  the  presence  of  dissolved  oxygen  have  shown  that  the 


Page  2 

NCI-629 
SERI/X  NO, 


PROJECT  iiEPORT  FOM  (Cont.) 

reductive  doamination  of  glycine  to  acetic  acid  is  the  action  of  the  H  free 
radical,  . 

2)  Experiments  using  OH  free  radicals  produced  chemically  from  Fe"*"*"  and 
H2O2  have  shown  that  the  oxidative  deamination  of  glycine  to  glyoxalic 
acid  is  the  action  of  the  OH  free  radical* 

3)  Failure  to  observe  the  formation  of  formaldehyde  as  the  result  of 
OH  radicals  distributed  uniformly  in  the  solution  by  the  reaction  of 
Fe"*"*"  and  H2O2  has  lead  to  the  conclusion  that  the  reaction  producing 
formaldehyde  is  peculiar  to  the  high  local  concentrations  of  radicals 
along  the  discrete  tracks  of  ionizing  particles, 

k)     Investigation  of  the  effect  of  alpha  particles  from  Po^-^  upon 
aqueous  solutions  of  glycine  has  shown  that  this  densely  ionizing  particle 
induces  the  same  reactions  as  docs  x-rays  and  electrons  but  in  a  different, 
ratio.  The  relatively  high  yield  of  formaldehyde  is  in  agreement  with 
finding  3). 

5)  The  kinetic  measurements  associated  with  obtaining  result  3)  have 
provided  considerable  insite  as  to  the  mechanism  of  the  "catalytic" 
action  of  Fo"*"*"  ion  on  the  action  of  H2O2  on  aiuino  acids  and  will  result 
in  a  separate  publication. 

Significance  to  cancer  research;  Although  not  directly  connected  with 
Cancer  research,  it  is  of  great  interest  because  it  seeks  an  understanding 
of  the  mechanism  for  the  effects  of  radiation  which  is  used  empirically 
as  a  tool  in  the  clinical  treatment  and  laboratory  study  of  cancer. 

Proposed  course  of  project;  Continuation  of  the  work  will  be  directed  to 
completing  thu  glycine  and  alanine  investigation  and  extending  the  work 
to  the, effect  on  more  complicated  amino  acids. 

Specifically; 

1;  The  mechanism  for  the  large  effect  of  very  small  concentrations  of 

dissolved  oxygen  upon  the  glycine  reactions  will  be  studied, 

2)  Measvirements  upon  the  influence  of  glycine  concentration  and  of 
temperature  upon  the  relative  frequency  of  the  various  reactions  will  be 
refined, 

3)  Studies  upon  the  effect  of  pH  upon  the  reactions  will  be  made, 

k)     Studies  upon  the.  effect  of  x-rays  on  phenylalanine  and  tyrosine  will 
be  initiated. 


PAGE  3 


PROJECT  ICEPOKT  FOlttl  (Cont.) 


10.    MCi-629 

SmiiiL  NO, 


11.      BUDGET  ACTIVITY: 

Research  /X/ 

iieview  &  Approval    /^ 


Administration  // 

Technical  Assistance     /~7 


12,     COOPERi'JING  UiMITS  OF  THE  PUBLIC  Haj;j.K  SEIIVICE,   OR  OTHER  ORGANIZilTIONS, 
PROVIDING  FUNDS,   FACILITIES,    OR  PEi"iSOm\JEL  FOii  THIS  PicOJECT  IN  EITHETt 
1956  OR  1957: 

None 


13,     IF  THIS  PROJECT  IcESiilMBLES,   CQ'IPLEMENTS,   Oi  P;ju.LLELS  RESEiffiCH  DOME 
SLSEVJHERE  IN  THE  PUBLIC  HEi'.LTH  SERVICE   (WITHOUT  INTERCHANGE  OF 
PETiSONHEL,  FACILITIES  OR  FU1\^DS),   IDENTIFY  SUCH  I^SEi'.RCH: 

None 


PAGE  k 
PROJECT  ilEPOItT  FQiHi  (Cont.) 


lii._NCI-629_ 
SERIAL  NO, 


15,  PUBLia.TIONS  OTHER  THAN  ABSTR/lCTS  FROM  THIS  PiiOJECT  DURING  CALE^D;il 
YEAR  1955: 

The  Effect  of  Ionizing  Radj.ation  on  /imino  Acids 

II,'     The  Effect  of  X-iiays   on  Aqueous  Solutions  of  Alanine, 

N.   E.   Sharpless,  A.   E.    Blair  and  C,  R,   Maxwell,   Radiation 
Research  2,  135-lJ-ti4   (1955) 

III,   The  Effect  of  Electron  Irradiations   on  Aqueous  Solutions   of 
Glycine,      C,   R.  Majaiell,    D,    C,  Peterson^   and  W.    C,  VJhite, 
Radiation  Itesoarch  2,  h31-k3Q>   (1955) 

16,  HONORS  AND  AWJiDS  TO  P:'iR30Hl\IEL  RELATING  TO  THIS  PROJECT  DUIiING 
C/iLEND/Ji  YEiui  1955: 

None . 


PAGE  1 


PROJECT  REPORT  FOiM 


1,  National  Cancer  Institute   2,  Radiation  Branch 

INSTITUTE  LilBORATORY  OR  BR^INCH 

3.  Radiation  Biology  Section  k, .  5»  NCI-63I1 

SECTION  OR  SERVICE         LOCATION(IF  OTHER  THAN  BETHESDAj   SERIAL  NO* 

6t  Late  effects  of  irradiation  in  anijnals  protected  from  early  death 

by  bone  marrow  or  antibiotic  treatment 

PROJECT  TITLE 

7»  Willie  W,  Smith,  pathological  studies  by  George  Brecher  (Clinical 

Center),  Katherine  Snell,  Richard  Swarm 

PRINCIPAL  INVESTIGATOR(Sj 

8,   Ilo  M.  Alderman,  Ruth  Gillespie 
OTJffiR  IWESTIGATOR ( S  ) 


9,   PROJECT  DESCRIPTION 

Objectives!  To  determine  the  extent  to  which  life  expectancy  is  reduced 
in  animals  which  have  been  exposed  to  severely  damaging  radiation  and  then 
treated  in  such  a  way  as  to  prevent  early  death  from  infection,  hemorrhage 
or  anemia  and  to  determine  where  possible,  the  cause  of  death  in  these  animals. 

Methods  employed;  Animals  exposed  to  radiation  which  causes  death  in 
the  absence  of  treatment  are  treated  with  bone  marrow  or  streptomycin 
and  are  then  observed  for  the  remainder  of  their  lives.  At  death 
autopsies  are  performed  and  histopathological  studies  made.  Various 
treatments  are  applied  with  the  object  of  prolonging  life. 

Ma j or  findings ;   (l)  In  hamsters  surviving  the  first  month  following 
exposure  to  x-radiation  the  median  survival  time  appears  to  be  linearly 
related  to  the  radiation  dose  rather  than  to  the  proportion  surviving  the 
first  month.  Treatment  with  streptomycin  or  marrow  appears  to  prevent 
early  death  but  not  to  promote  total  recovery,  (2)  Three  treatments 
applied  to  hamsters  surviving  a  month  after  irradiation  have  failed  to 
alter  significantly  the  survival  time,   (3)  Histopathological  studies 
have  shown  a  high  incidence  of  gastric  ulcer,  pneumonia,  vascular  and 
renal  changes  and  neoplasm,  depending  upon  the  initial  radiation  dose 
and  the  time  of  death. 

Significance  to  Cancer  Research;   (l)  An  evaluation  of  the  total 
efficacy  of  treatments  which  may  be  used  to  reduce  the  deleterious  effects 
of  therapeutic  treatment  with  x-ray;  (2)  an  estimate  of  the  incidence 
and  type  of  neoplasm  which  may  be  expected  after  whole-body  exposure 
to  varying  doses  of  radiation;  (3)  an  estimate  of  the  physiological  condi- 
tion of  the  animal  after  exposure  to  varying  doses  of  radiation. 


PAGE  2 

SEilKL  NO, 
PROJECT  iiEPaiT  FOrxl'I  (COW.) 


Proposed  courso  ox  project;  In  addition  to  continuing  this  project 
r.long  the  lines  indicated  above,  functional  tests  will  be  applied  to  the 
animals  surviving  various  exposures  and  treatments.  These  will  probably 
include  resistance  to  oxperiraental  infection,  ability  to  mobilize  leuco- 
cytes, ability  to  respond  with  polyerythcmia  to  repeated  exposures  to.  ' 
hypoxia,  resistance  to  toxins  and  possibly  liver  and  kidney  function 

"t/GSuS  « 


PAGE  3 


PROJECT  REPORT  FORM  (CONT.) 


10.  NCI-63I1 

SERIAL  NO. 


11,  BUDGET  ACTIVITY: 

Research  /^ 

Review  &  Approval  f~] 


Administration       // 
Technical  Assistance  /V 


12.   COOPERATING  UlilTS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOIi  THIS  PROJECT  IN  EITHER 
1956  OR  1957: 

Dr.  George  Brccher,  Clinical  Center  Pathologist, 


13,   IF  THIS  PROJECT  RESEMBLES,  GOMPLPilENTS,  OR  P.jRj'.LLELS  RESE/JiCH  DONE 

ELSEWHEiiE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL, 
FACILITIES  OR  FUITOS),  IDENTIFY  SUCH  RESK'JICH: 

None. 


PAGE  li 
PROJECT  REPORT  FOmi  (CONT.) 


lii.      NCI-63li 
SERIAL  NO, 


15,  PUBLICATIONS  OTHER  THAN  ABSTIi..CTS  FROfl  THIS  PROJECT  DUIiING  C;.LENDjdR 
YE/Jl  1955: 

X- Irradiation  in  Hamsters  r-nd  Effects  of  Stroptoinycin  end  I'larrow- 
Spleen  Horaogenr.te  Treatment,  W.  W.   Smith,   R,   I,  I'fcrston,   L,  Gonshery, 
I.  M,  Alderman  and  H,   J,   Ruth,  Im.    J.   Physiol.  l83,   98   (1955). 

16.  HONORS  AND  AWARDS  TO  PERSONNEL  RELVTING  TO  THIS  PROJECT  DURING 
CALEND/Jl  YEIJi  1955: 

None, 


PAGE  1 

PROJECT  REPORT  FOIiM 

1,  National  Cancer  Institute   2,  Radiation  Branch 

INSTITUTE  LABOjrt.TORY  OR  HRi'.NCH 

3,  Radiation  Biology  Section  h, " g,  NCI-636 

SECTION  OR  SERVICE         .LOCATION  (IF  OTHEii  THAN  BETHESDA;   SEiilAL  NO. 

6,  The  Lethal  Effects  of  Visible  Radiation  on  a  Strain  of  Haploid  Yeast 

PROJECT  TITLE 

7,  Dr.  Mortimer  M.  Elkind 

PRINC.LTi.L  INVESTIGATOR(S j 

8,  None 

OTHER  IfJVESTIGATORS 


9,  PROJECT  DESCRIPTION 

Objectives;  The  objectives  of  this  investigation  are  to  delineate  the 
parameters  which  control  the  sensitivity  of  yeast  cells  to  visible 
radiation,  and,  where  possible,  to  correlate  the  effects  observed  with 
processes  within  the  cell. 

Methods  employed;  Actively  growing  (log  phase)  yeast  cells  (haploid 
Saccharomyces  cerevisiac)  are  harvested  from  a  liquid  growth  medium 
(yeast  extract  plus  dextrose),  washed  by  centrifugation,  and  resuspended 
in  a  potassium  phosphate  buffer.  Immediately  after  resuspension,  the 
cells  are  essentially  insensitive  to  the  emission  from  a  300  watt,  incandes- 
cent lamp  slide  projector  (3^0  m/u  to  750  m/u),  VJith  time,  the  population 
becomes  increasingly  light  sensitive  to  this  emission  as  measured  by  the 
ability  of  the  cells  to  grow  into  visible  colonies  x^rhen  plated  on  agar 
containing  growth  medium.  The  cells  are  irradiated  in  the  same  buffered 
solution  and  are  kept  at  a  temperature  of  from  1,0-2, Co  C  during  irradiation. 

Jfcjor  findings;  A,  For  a  given  composition  of  the  buffer  solution  and  a 
given  temperature  of  storage,  with  time  the  colls  become  progressively  more 
light  sensitive,  B,  For  the  saiue  length  of  time  of  storage  the  sensitivity 
is  strongly  dependent  on  the  temperature  of  storage.  For  instance,  2  hrs, 
of  storage  at  30°  C  will  produce  the  same  sensitivity  as  about  28  hrs, 
storage  at  l-g-°  C,  C,  For  the  same  length  of  time  and  temperature  of 
storage,  the  sensitivity  is  strongly  dependent  upon  the  pH  of  the  buffer 
solution  and  to  a  lesser  extent  upon  the  molarity  of  the  buffer  solution, 
D,  Suppression  of  oxygen  tension  of  the  buffer  solution  at  the  time  of 
irradiation  decreases  the  sensitivity,  E,  CompoTed  to  log  phase  cells, 
resting  cells  do  not  develop  appreciable  light  sensitivity  in  the  course 
of  storage. 


PAGE  2 
NCI -6 36 
SERI/iL  NO, 

PROJECT  REPORT  FORf'f  (Cont.) 


Significr.ncG  to  cancer  research;  This  research  is  a  phonoinonological 
study' ox  a  process  present  in  a  biological  system  which,  if  at  all,  has 
not  received  rauch  attention  in  the  past.   In  addition  to  the  technical 
need  for  a  knox^rledge  of  the  scope  and  extent  of  this  effect  as  it  might 
present  itself  a,s  an  artifact  in  other  radiation  studies  with  this  organism, 
this  work  is  related  to  oancer  research  as  basic  biological  research  in 
general  is  so  related. 

Proposed  course  of  project;   0[3timumly  the  culJiiination  of  this  work  would 
probably  consist  of  an  identification  of  the  biological  processes  responsible 
for  light  se;isitivity.  With  this  in  mind  it  is  planned  to  complete  the 
exploration  for  the  apparent  pertinent  parameters  involved,  and  to  examine 
the  action  and  absorption  spectra  of  these  cells  within  the  limitations 
of  available  equipment  and  techniques. 


10,  NCI-636 
SERIAL  NO. 


PAGE  3 


11.  BUDGET  ACTIVITY: 

Research         f^ 
'  Review  &  Approval  // 


Administration       fl 
Technical  ^issistance  ri 


12,   COOPEitATING  UNITS  OF  THE  PUBLIC  1Ij^.AL^j:'H  SERVICj^],  Ou  OTHEA  ORGANIZATIONS, 
PROVIDING  FUNDS,  I'ACILITIES,  OR  PERSONNriL  FOR  THIS  PROJECT  IN  EITHER  ' 
1956  wR  1957: 

None 


13.      IF  THIS  I'ROJECT  RESEMBLES,   CQIPLEIIENTS ,    a..  PiR^ALLELS  RESEJJiCH  DONE 
ELSafflERE  IN  THE  PUBLIC  HE^'XTH  SERVICE  (WITHOUT  INTERCHANGE  OF 
PEliSONNEL,   FACILITIES  OR  FUNDS),   IDENTIFY  SUCH  RESEiARCH: 

None 


PAGE  k 


1^*       MCI-636 

SERI/J.  NO. 


1^.      PUBLICATIONS   OTHEii  THj-il\I  ABSTIiACTS  FROil  THIS  PK0J3GT  DURING  CiiEND/Ji 
.,   TEiiR  1955: 

Mortimer  M,  Elkind  and  Carl  A,  Beam,  "Variation  of  the  Biological 
Effectiveness  of  X-Rays  and  Alpha-Particles  on  Haploid  Saccharomyces 
cerevisiaej"  Radiation  Research,  3,   88-1 OU  (1955). 

16.     HONORS  AND  AW.mDS  TO  PERSONNEL  REUJING  TO  THIS  PROJECT  DURING 
C/iLEND/J{  YE/iR  1955: 

None  « 


PAGE  1 
PROJECT  REPORT  FORM 


1,     Mational  Cancer  Institute  2,     Radiation  Branch 

INSTITUTE  L/iBOFu'^TORY  OR  BR/J^CH 

3,     Radiation  Biology  Section  k»                                                                  5»  NCI-637 

SECTION  OR  SERVICE  LOCi.TION(IF  OTHER  Tm'.N  BETHESDA;       SERI/J.  NQ, 


6,     Development  of  High-Intensity  X-ray  Sdiifce 
PROJECT  TITLE 


7i     H.  L,  Andrews _«___^ 

PRINCIP/iL  I1^VESTIGAT0R(S; 


R.  E.  Murphy 


OTHER  IIWESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objectives;  To  obtain  from  the  3  Mev  generator  the  x-ray  output  which  it 
should  be  capable  of  producing. 

Methods  employed;  The  3  ^'fev  Van  de  Graaff  generator  given  to  NIH  by  the 
Liggett  and  i'lGycrs  Tobacco  Co.  is  capable  of  producing  x-rays  at  intensities 
never  before  reached  in  the  laboratory.  It  had  never  been  used  as  an  x-ray 
generator  and  when  attempts  were  made  to  utilize  its  capabilities  x-ray 
targets  failed  by  melting.  Failures  occurred  at  only  about  l/k   of  full 
power  and  hence  the  possible  generator  capability  is  seriously  restricted. 
Since  target  fadlure  allows  cooling  water  to  enter  the  accelerating  tube 
each  failure  represents  a  minor  disaster.  Pending  remedial  steps  the 
generator  has  been  operated  conservatively  to  insure  continuity  of  service. 
Improved  operation  is  to  be  expected  from:  1,  higher  voltage  operation, 
2,  high  frequency  target  scanning,  3.  use  of  targets  of  high  atomic  number, 
hi   improved  target  cooling. 

Major  findings;  A  study  of  failures  of  the  gold  targets,  and  the  general 
theories  of  x-ray  production  have  suggested  the  following: 

1,  Since  the  efficiency  of  x-ray  production  increases  with  voltage 
operation  at  the  highest  possible  voltage  will  give  increased  output  for 
equal  target  heating.  By  careful  attention  to  details  we  have  raised  the 
routine  operating  voltage  from  3»0  J'lev  to  3.5  I'fev,  For  speeial  purposes 
operation  at  3,8  Mev  appears  possible  but  can  not  be  counted  on  for  daily 
use, 

2.  All  target  failures  appear  to  be  due  to  a  burst  of  high  current 
lasting  for  perhaps  one  microsecond.  If  this  is  the  case  sweeping  the 
incident  electron  beam  over  the  face  of  the  target  at  very  high  speed 
should  reduce  lofal  heating.  Preliminary  experiments  with  300  kilocycle 
scanning  indicate  its  feasibility  and  indicate  the  direction  for  future 
equipment  design? 


FIJJE    2 


NCI-637 
SERIAL  NO. 


PROJECT  DESCRIPTION  (CONT.) 


3»  Since  x-ray  production  is  proportional  to  the  atomic  number  of 
the  target  material  steps  have  been  taken  to  replace  the  gold  (Z=79) 
targets  with  thorium  (Z=90),  Thorium  .targets  have  been  obtained  from  the 
AEC,  and  will  be  installed  when  suitable  welding  techniques  are  proven 
satisfactory, 

U.  Delivery  of  cooling  water  to  the  back  surface  of  the  target  has 
been  improved  and  steps  are  planned  to  reduce  the  trauma  caused  by  target 
failure. 

Significance  to  cancer  research;  With  the  high  outputs  potentially 
available  from  this  generator  radiation  doses  can  be  administered  at  dose 
rates  never  before  available.  It  is  possible  that  quite ' different  biological 
results  will  be  obtained  when  a  given  radiation  dose  is  given  at  say  10,000 
r/rain  rather  than  at  the  more  usual  ^0-100  r/min. 

Proposed  course  of  pro.jccti  These  were  covered  under  "major  findings," 

This  project  moves  slowly  to  avoid  disruption  of  existing  radiation  schedules 

but  within  6  months  operation  at  full  generator  power  can  be  expected. 


PAGE  3 


10.  "CI-637 

SERIiJ.  NO, 


11.  BUDGET  ACTIVITY: 

ResesTch        /TJ 
Review  &  Approval  ri 


Administration      f^ 
Technical  Assistance  f~] 


12,  CDOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVIClI,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSOMEL  FOR  THIS  PROJECT  IN  EITHER 
1956  OR  1957: 

None 


13.     IF  THIS  PROJECT  RESEl'lBLES,   COIiPLEi'ISNTS,   OR  P/HALLELS  RESE.'JICH  DONE 
ELSEVJHERE  IN  THE  PUBLIC  HE.'.LTH  SERVICE   (WITHOUT   liWERCHANGE  OF 
PERSONNEL,   FACILITIES  OR  FUNDS),   IDENTIFY  SUCH  RESE/JICH: 

None 


NO  EI\ITRIES  FOR  ITEMS  II4,   l5  &  I6, 


PAGE  1 


PROJECT  REPORT  FORM 


1,  National  Cancer  Institute   2,  Radiation  Branch 

INSTITUTE  Li^BOR/xTCEY  OR  BR/.NCH 

3»  Radiation  Biology  Section  h, __, ^ 5.  NGI-638 

SECTION  OR  SERVICE         LOCATION  (IF  OTHER  THAN  BETHESDAj   SERIilL  NO. 

6,  A  study  of  the  neuropathology  of  massive  doses  of  x-rays  in  the 
guinea  pig 


PROJECT  TITLE 


7.  Kirkland  C.  Brace 

PRINCIP/iL  INVESTIGAT OR  ( S ) 


8«  Howard  L.  Andrews 


OTHER  IIWESTIGATORS 


9,   PROJECT  DESCRIPTION 

Objectives ;  To  determine  the  effect  of  massive  doses  of  x-rays  on  the 
central  nervous  system  of  the  guinea  pig. 

Methods  employed;  Guinea  pigs  are  exposed  to  various  doses  of  x-rays 
and  the  brains  removed  at  various  intervals  after  the  exposure.  The 
tissues  are  examined  microscopically  to  determine  the  time  course  of 
and  type  of  pathology  that  could  be  observed. 

Major  findings;  The  only  readily  observable  changes  with  doses  of  less  than 
25 J 000  r  of  x-rays  are  in  the  granule  cells  of  the  cerebellura.  Doses  of 
less  than  6,000  r  do  not  show  any  changes  in  the  central  nervous  system. 
Doses  of  in  excess  of  6,000  r  produce  a  peculiar  pyknosis  of  the  granule 
cells  which  appears  about  an  hour  after  the  exposure  at  a  dose  rate  of  50  r 
per  minute.  Almost  90  per  cent  of  the  cells  are  involved  after  8  hours. 
Death  occurs  at  about  2k   hours.  The  neurological  symptoms  observed  are 
closely  associated  with  the  amount  of  pyknosis  present. 

Significance  to  Cancer  Research;  It  has  long  been  reported  that  the  non- 
dividing  cells  of  the  central  nervous  system  are  higlily  resistant  to  radia- 
tion. It  appears  from  this  data  that  the  cerebellum  is  particularly  radio- 
sensitive in  the  guinea  pig.  This  may  explain  the  particular  sensitivity  of 
the  medulloblastoma  which  is  derived  from  the  same  anlage  as  the  granule 
cells. 

Proposed  course  of  project;  With  the  availability  of  new  equipment  we 
expect  to  repeat  this  study  at  much  higher  dose  rates  to  determine  if 
there  is  any  change.  We  also  plan  to  extend  the  study  to  some  other  species 
of  animal. 


PAGE  2 


PROJECT  REPORT  FCRii  (cont.) 


10,  NCI-638 

SERIAL  NO. 


11,   BUDGET  ACTIVITY: 


Research         /6c7 
Review  &  Approval  f] 


Administration  /~]    ': 
Technical  Assistance  ^ /~7 


12.  COOPEIL\TING  TOJITS  Oi^  THE  PUBLIC  HE/iLTH  SERVICE,  OR  OTHER  ORG/J^J'IZATIONS, 
PROVIDING  FIMBS,   FACILITIES,  OR  PEi^SOWNEL  FOR  THIS  PROJECT  IN  EITHER 
19^6  OR  1957:  . 

National  Institute  of  Mental  Health 
(This  project  was  reported  last  year  by  NINDB,  lA   that  time  Dr.  Aivord 
was  principal  investigator  along  with  Dr.  Brace,  Dr.  Aivord  has  left  NIH, ) 

13,  IF  THIS  PROJECT  RESEMBLES,  CaiPLEi'IENTS,  OR  PAR,'.LLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HE/iLTH  SERVICE  (WITHOOT  INTERCHANGE  OF 
PERSONNEL,  FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEi.RCH: 

None  I 


NO  ENTRIES  FOR  ITEflS  lli,  l5  &  16, 


PAGE  1 

PROJECT  REPORT  FORM 

1,     Mationg.l  Cr.ncer   Institute       2,     Radiation  Branch 

INSTITUTE  UBOII/.TORY  OR  BR.'iNCH 

3.  Radiation  Biology  Section  h. 5.NCI-639 

SECTION  OR  SEIWICE         LOCATION  (IF  OTHER  TffixN  BETHESDiJ   SERIAL  NO. 

6 .  A  study  o.fetiiT:.-ULUiUJijja^aB3^a§y  of  life  span  of  the  nucleated  red  cell 

PROJECT  TITLE 

7.  Kirkland  C,  Brace      


PRINCIPAL  INVESTIGATOR! S) 


8,  Paul  D.  Altland,  NliJ'ID 
OTHER  INVESTIGATORS 


9,  PROJECT  DESCRIPTION 

Pro.ject;  A  study  of  the  life  span  of  nucleated  erytlirocytes  of  the  birds, 
reptiles,  and  amphibians  using  dU, 

Objectives;  A  better  understanding  of  the  remarkable  resistance  of  the 
lower  forms  and  particularly  the  poikilotherms  to  the  effects  of  ionizing 
radiation. 

Methods  employed;  The  erythrocytes  of  various  species  of  animal  are 
labeled  in  vivo  by  the  intravenous,  or  intraperitoneal  injection  of  glycine- 
2-Cli4,  The  methyl  carbon  is  incorporated  into  the  hemoglobin  of  the 
erythrocyte  and  is  carried  with  the  coll  until  it  is  destroyed.  The 
determination  of  the  life  span  is  based  upon  the  interpretation  of  the 
fall  in  the  specific  activity  of  the  hemoglobin  following  injection  of 
the  glycine, 

!lajor  findings;  The  life  span  of  the  bird  erythrocyte  has  been  determined 
by  several  other  methods.  We  have  repeated  the  determination  to  check 
the  validity  of  our  method.  The  value  of  I|.2  days  we  obtained  for  the 
duck  erythrocyte  confirms  previous  work.  The  specific  activity  of  the 
hemoglobin  of  the  box  ti:irtle  remains  almost  unchanged  after  500  days 
after  injection  and  the  specific  activity  of  the  hemoglobin  of  the  toad 
remains  unchanged  after  270  days  after  injection.  We  can  only  interpret 
this  data  as  showing  that  these  cells  are  extremely  long  lived. 

Significance  to  Cancer  Rasoarch;  An  understanding  of  the  effects  of 
ionizing  radiation  on  a  cell  depends  on  knowing  the  normal  physiology  of 
the  cell.  Those  extremely  long  lived  cells  of  the  poikilotherms  may 
partially  explain  the  resistance  of  these  cells  to  effects  of  radiation  and 
could  explain  the  general  radio  resistance  of  the  animal. 


PAGE  2 


serl;l  no, 

PliOJECT  REPORT  FORM  (corit.) 


Proposed  course  of  project:  The  work  on  the  turtle  and  toad  has  been  set 
up  to  continue  for  several  more  years,  We  hope  to  determine  if  a  finite 
life  span  of  these  cells  does  exist.  If  possible,  this  study  will  be 
extended  to  somo  other  species  especially  the  fish. 


PAGE  3 
PROJECT  REPORT  FORM  (cont.) 


10.     NCI^639 

SERIJ.L  NO. 


11,  BUDGET  ACTIVITY: 

Research         /S7  Aijministration       /  / 

Review  &  Approval  f~]  Technical  Assistance  /  / 

12,  COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SElRVICE,  OR  OTHEit  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  OR  1957: 

National  Institute  of  /arthritis  and  Metabolic  Diseases 


13,  IF  THIS  PROJECT  RESEI'IBLES,  CQiPLEi'lENTS ,  Ou  P;j"ulLLELS  RESEiu'iCH  DONE 
ELSEVniERE  IN  THE  PUBLIC  HEiXTH  SERVICE  (WITHOUT  INTERCHANGE  OF 
PERSONNEL,  FACILITIES  OR  FLWDS),  IDENTIFY  SUCH  RESJiU'iCH: 

None 


1$.     PUBLICATIONS  OTHER  TH.uN  ABSTRACTS  FRQl  THIS  P.tOJECT  DUilING  Ci.LENDiJi 
YE.'-at  1955: 

I^rkland  C.   Brace  and  Paul  D,  Altland,   7ted  Cell  Survival  in  the 
Turtle,   The  i'merican  Jom-nal  of  Physiology  183,   91   (1955 )» 

16,     HONOItS  AND  AWixRDS  TO  PERSONNEL  REL/.TING  TO  THIS  PiiOJECT  DURING  C;XJ»JD;jt. 
lE/ii  1955: 

None, 


PAGE  1 


PROJECT  REPORT  FORM 


1,  National  Cancer  Institute  2,  Radiation  Branch 

INSTITUTE  L/.BORjVrORI  OR  BR/.NCH 

3.  Radiation  Biology  Section  k, ^  5,  ^^^-^^Q 

SECTION  OR  SERVICE  LOC;iTION(IF  OTHER  TH/J  BETHESDAj   SERLIL  NO. 


6,  The  effect  of  x-radiation  on  the  anaphylactic  response  in  mice 
PROJECT  TITLE 


?♦  Falconer  Smith __^_^ 

PRINCIPAL  IIWESTIGATOR ( S  ; 

8 ,  t'larie  M.  Grenan,  (Hazel  P.  Gvunp,  not  presently  attached  to  this  project) 
OTHER  INVESTIGATORS 


9,  PROJECT  DESCPIPTION 

Objectives;  a)  Obtain  a  quantitative  expression  in  terras  of  combining 
ratios  of  antigen  and  antibody  for  the  increases  susceptibility  of  the 
irradiated  mouse  to  anaphylaxis,  b)  Laprovo  the  tissue  specificity  of 
leucocyte  and  other  tissue  antigens  and  study  the  effects  of  their 
homologous  antibodies  on  irradiLated  mice. 

Methods  employed;  The  responses  of  passively  iminunized,  irradiated  mice 
to  varying  concentrations  of  antigen  are  tested  using  hen's  egg  albumin 
and  its  homologous  antiserum  (rabbit)  as  test  prepare.tions.  In  addition, 
tests  are  carried  out  with  mice  using  intravenous  injections  of  mouse- 
leucocyto  antiserum  and  erythrocyte  preparations. 

Major  findings;  Rabbit,  mouse  leucocyte  antiserums  (prepared  from  peritoneal 
exudates)  given  intravenously  is  more  harmful  to  irradiated  mice  than  to 
their  nonirradiated  controls.  Since  a  similar  result  was  observed  with 
hen's  egg  albumin  antiserum,  additional  v'jtudies  of  a  quantitative  native, 
using  this  preparation  will  be  made. 

Significance  to  Cancer  Research;  Anaphylaxis  is  a  coinmon  response  to 
protein  by  appropriately  sensitized  mammalian  tissue  and  is  apparently 
enhanced  by  radiation.  It  is  considered  possible  that  specific  tissue 
sensitivity  can  be  obtained  which  in  turn  may  produce  an  additive  effect 
when  combined  with  x-radiation.   In  addition,  studies  of  the  effects  of  x- 
rays  on  the  anaphylactic  response  jirovide  information  on  the  biochemical 
behavior  of  the  reactive  tissue. 

Proposed  course  of  project;   Improvement  in  specificity  of  tissue  antigens 
by  the  isolation  of  specific  cell  types,  the  preparation  of  antisera  to 
these  and  testing  of  the  antisera  will  occupy  a  major  portion  of  the 
calendar  year  assigned  to  this  project. 


PAGE  2 


PROJECT  REPORT  FORM  (cont.) 


]_0,   NCI-6iiO 


SERI/J.  NO. 


11.  BUDGET  ACTIVITY: 


Research         fxj 
Review  &  Approval  f~J 


Administration  £J 
Technical  Assistance  f~] 


12,  COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUl^DS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  , 
19^6  OR  1957: 

No  other  units  cooperating, 

13.  IF  THIS  PROJECT  RESEt'lBLEG,  COMPLEMENTS,  OR  PARi'iLLELS  RESK.RCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE  (VJITHOUT  INTERCILa\lGE  OF  PERSONNEL, 
FACILITIES  OR  FU1\IDS),  IDENTIFY  SUCH  RESE/JICH: 

No  knowledge  of  parallel  studies  elsewhere. 


NO  ENTRIES  FOR  ITEMS  lit,  \$   &  l6. 


PAGE  1 
PROJECT  REPORT  FORJl 


1,     National  Cancer  Institute  2,     Radiation  Branch 

INSTITUTE  LIBORATORY  CR   BRANCH 

3,     Radiation  Biology  Section  h* g,NCI-6Ul 

SECTION  OR  SERVICE  LOCATION (IF  OTHER  THAN  BETHESDAj        SERIAL 

..  NO. 

6,  The  Effect  of  Irradiation  on  Susceptibility  to  Viral  Infections 
PROJECT  TITLE 


7.  Willie  W.  Smith,  Bernice  Eddy  (L.B.C.) 
PRINCIP/vL  INVEST IGATOR(S) 


8,  Ilo  M,  /ilderman,  Ruth  Gillespie 
OTHER  IIWESTIGATORS 


9,  PROJECT  DESCRIPTION 

Objectives;  To  deterraine  whether  or  not  exposure  to  x-radiation  alters 
susceptibility  to  various  viral  infections. 

Methods  employed;  The  response  to  challenge  with  poliomyelitis  or 
influenze  virus  in  mice  given  just  sublethal  irradiation  is  compared 
with  that  of  controls. 

Major  findings;  The  results  thus  far  indicate  that  mice  exposed  to 
just  sublethal  radiation  are  no  more  susceptible  to  the  challenging 
injection  of  polio  virus  than  are  controls.  Experiments  with  influenza 
are  in  progress. 

Significance  to  Cancer  Research;  To  promote  a  more  complete  understanding 
of  the  effects  of  irradiation  and  to  enable  one  to  anticipate  possible 
deleterious  effects  of  irradiation  used  therapeutically. 

Proposed  course  of  projects  This  project  will  be  continued  along  the 
lines  indicated.  In  addition,  we  plan  to  study  the  effects  of  sublethal 
radiation  on  response  to  several  other  noxious  agents. 


PAGE  2 
PROJECT  REPORT  FORM  (Cont.) 


^Q  NCT-6i;l 

SERIAL  NO. 

11,  BUDGET  ACTIVITY: 

Research  /x7  Administration  /V 

Review  &  Approval     /~/  Technical  Assistance     /V 

12,  C00PER.;TING  UI^ITS  of  the  public  HE.^LTH  SERVICE,    OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,   OR  PERSONl^L  FOR  THIS  PROJECT  IN  EITHER 
1956  OR  1957: 

Dr.   Bernice  Eddy,.  L,   B,   C. 

13,  IF  THIS  PROJECT  RESEMBLES,   CaffLSI'lENTS,    OR  P/Jli'iLLELS  RESE/^RCH  DOME 
ELSEl'JHERE  IN  THE  PUBLIC  HK\LTH  SERVICE   (V/ITHOUT  lOTERCHANGS  OF 
PERSONlxIEL,   FACILITIES  OR  FUNDS),   IDENTIFY  SUCH  RESE/JlCH: 

None. 


NO  ENTRIES  FOR  ITEl'lS  lU,   l5  &  l6, 


PAGE  1 

PROJriCT  REPORT  FORM 

1,     National  Cancer   Institute^       2,     Radiation  Branch 

INSTITUTE  LABOR/.TORY  OR  BRANCH 

3,     Radiation  Biology  Section       h, 5.  NCI-61;2 

SECTION  OR  SEjIVIGE  LOCATION   (IF  OTIiER  THAN  BETHESDAj       SERIAL 

NO. 

6,  Antibody  production  in  irradiated  mice:  Effect  of  fractional  body 
shielding  on  hemolysin  production  and  of  radiation  dose  rate  on 

hemolysin  production 

PROJECT  TITLE 


7*  Falconer  Smith 

PRINCIPAL  IWESflGATOR(Sj 


8,  H,  Jeanette  Ruth 

OTHER  INVESTIGATORS 


9,  PROJECT  DESCRIPTION 

Objectives;  The  objective  of  this  project  is  to  determine  the  effect  of 
x-radiation  administered  under  the  indicated  conditions  on  the  immune 
response  in  mice. 

Methods  employed;  Peak  anti  sheep  erythrocyte  hemolysin  titers  of 
individual  mice  are  determined  at  weekly  intervals  following  exposure  to 
x-rays,  radiation  dose  rate  being  varied  in  some  instances  while  in  others 
the  volume  of  dose  of  x-rays  is  varied. 

Ma j or  findings ;   It  was  recently  shown  that  bone  marrow  or  spleen  homogenate 
was  ^^^ithout  effect  on  radiation  damage  to  the  immune  response.  Shielding 
varying  fractions  (60-90^)  of  the  body  provides  varying  degrees  of  protection 
to  the  inmune  response  at  a  ;:;iv3n  total  body  dose  as  determined  by  serum 
hemolysin  titers  at  one  to  three  weeks  after  x-ray  exposure. 

Significance  to  Cancer  Research;  Antibody  production  reflects  one  aspect 
of  protein  synthesis  by  living  tissue  and  is  therefore  directly  related  to 
cell  metabolism,  a  problem  of  much  interest  in  cancer  research. 


PAGE    2 


PiiOJECT  REPORT  FOffl'I  (cont.) 


10.         NCI~6It2 
SERIAL  NO. 


11,      BUDGET  ACTIVITY: 

Research  /x7 

Reviex^r  &  Approval     [^ 


Administration  f~] 

Technical  Assistance  /] 


12,      COOPER/xTING  UI\IITS  OF  THE  PUBLIC  HIL'iLTH  SERVICE,   OR  0  HER  ORGANIZ..TE  ONS, 
PRO  IDING  FUNDS,   FACILITIES,    OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  OR  1957: 

No  other  units  cooperating. 


13,      IF  THIS  PROJECT  RESIilBLES,    COfffLaiENTS,   OR  PiiR^lLLELS  RESE^'iRCH  DONE 
ELSEWI-iERE  IN  THE  PUBLIC  HEj\LTH  SERVICE  (WITHOUT  INTERCH.;NGE  OF 
PERSONNEL,   FACILITIES   OR  FU"NDS),   IDENTIFY  SUCH  RESE/JiCH : 

No  knowledge  of  parallel  studies  elsewhere. 


PAGE  3 
PROJECT  REPORT  FCRM   (CONT.) 


ll;,    NCI-6U2 
SERI/iL  NO. 


1^,      PUBLICixTIONS  OTHER  TIL'.N  ABSTR/.CTS  FRQl  THIS  PROJECT  DURING 
Cj.LEl®/Ji  YE/Jt  1955: 

Falconer  Smith  and  H,   Jeanette  Ruth,  Hemolysin  Production  in 
Irradiated  Mice  Given  Spleen  or   Bone  Marrox\r  Homogenate, 
Proceedings   of  the  Society  of  Experimental  Biology  and  Medicine 
90,   187   (1955). 

,16,      HONORS  AND  AW/J1DS  TO  PERSONNEL  REK.TING  TO  THIS  PROJECT  DURING 
GALEND/iR  YEiJi  1955; 

None, 


PAGE  1 


PROJECT  REPORT  FORM 


1.  National  Cancer  Institute   2,  Radiation  Branch 


BISTITUTE  LABORATORY  OR  BR^^NCH 

3.  Radiation  Biology  Section  k. ^^NCI-6i|3 

SECTION  ..OR  SERVICE        .  LOG  TIONIIF  OTHER  THM  BETKESD* )   SERIAL  MO. 

,6.  Long  Term  Survival  and  Tumor  Incidence  Follomng  Acute  or  Chronic 

Irradiation 

PROJECT  TITLE 

7.  Joanne  Hollcroft 

PRINCIPAL  INVESTIGATOR! S) 

8,  Eliza  Miller,  Charles  C.  Congdon 

OTHER  INVESTK.'ATORS 


9,   PROJECT  DESCRIPTION 
Objectives: 

A,  To  study  long  term  survival  and  carcinogenesis  of: 

1)  Chronic  irradiation  of  guinea  pigs  T-jith  or  without  injections  of 
bone  marrow  and  determine  the  effect  of  bone  marrovi  treatment  in 
aleukemic  leukemia. 

2)  Acute  radiation  doses  i«dien  the  animals  are  protected  from  the 
acute  irradiation  effect, 

B,  Metho(i  employed: 

1)  Family  2  guinea  pigs  were  exposed  to  8,8  r  per  day  gamma  irradiation, 
•when  their  hematocrit  number  dropped  to  about  25  they  wers  removed  from  the 
radiation  field.  At  this  time  half  the  pigs  were  given  intravenous  injections 
of  bone  marrow.  Survival  and  tumor  incidenee  were  studied. 

2)  C^Hb  mice  were  x-iiradi;^t cd under  the  folloijing  conditions: 

(a)  liOO  r  at  birth, 

(b)  laOO  r  with  sham  spleen  shielding, 

(c)  ijOO  r  v.d.th  spleen  shielding, 

(d)  900  r  -with  spleen  shielding,  and 

(e)  900  r  iidth  chemical  protection  of  cystiene  and  anoxia 


PAGE  2 


NCl-61i3 

SERIAL  NO. 

PROJECTION  DESCRIPTION  (CONT.) 
C,  Major  findings: 

1)  Intravenous  injections  of  bone  marrow  suspensions  decreased  the 
number  of  early  deaths  following  limited  chronic  irradiation.   These 
injections  may  have  prevented  so'  e  aleukemic  leukemia  in  male  guinea  pigs 
but  seemed  to  have  no  effect  on  aleukemic  incidence  in  female  guinea  pigs. 
The  hematocrits  of  the  animals  developing  aleukemie  leukemia  appeared  to 
drop  lower  after  removal  from  the  radiation  field  than  animals  lAioh 
recovered  more  completely.  Of  the  animals  which  recovered  from  the  chronic 
irradiation  the  bone  marrow  treated  animals .seemed  to  have  a  longer 
survival  time, 

2)  Survival  time  following  acute  ejcposures  was  not  influenced  by  spleen 
protection  or  the  fact  that  the  animals  were  irradiated  at  birth.  The  , 
mice  receiving  cystienc  and  anoxia  protection,  lived  longer  than  the  spleen 
shielded  animals.  In  the  mice  irradiated  with  900  r  an  increase  in  the 
following  lesions  appeared: 

(a)  Adrenal  tumors, 

(b)  ovarian  tumors, 

(c)  glorerular sclerosis, 

(d)  rp'^clofibrosis  as  noted  in  the  sternum, 

(e)  lens  damage  to  the  eye.  An  increase  in  the  number  of 
reticular  endothelial  neoplasms  T,Tas  noted  in  the  animals  protected 
chemically  and  an  increase  in  miscellaneous  carcinomas  and  sarcomas  icls 
seen  in  female  spleen  shielded  mice.  In  both  groups  pyelonephritis  occurred 
less  frequently  than  in  the  controls, 

Significanee  to  cancer  research:   Irradiation  is  a  well  knoT«/n  carcinogen. 
The  long-term  effects  of  aci'te  doses  of  total  body  irradiation  are  of 
consequence  in  considering  the  role  of  radiotherapy  in  treatment  of  caxicer. 

Proposed  course  of  project: 

1)  Influence  of  hematocrit  number  and  white  blood  count  as  i-ell  as 
total  dose  •n  development  of  aleukemic  leukemia, 

2)  Completion  of  histologic  studies. 


PAGE  3 


10.   NCI-61;3 
SERIAL  WO, 


11,  BUD"'-5T  ACTIVITY: 

Research         /x7 
Review  4  Approval/^ 


Administration       f~~J 
Technical  Assistance  f~~J 


12,  It  is  hoped  that  Dr,  Congdon  of  the  Oak  Ridge  National  Laboratory  will 

continue  ^^d-th  the  pathologic  diagnoses  on  these  studies. 

CGOPSRATIMG  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZ lTIONS, 
PROVIDBTG  FUNDS,  FACILITIES,  OR  P5RS0WNEL  FC"'.  THIS  PROJECT  IN  EI'r^ER 
1956  or  1957. 


13.     'None, 

IF  THIS  PROJECT  RESEI-ffiLES,   CCMI'LMEi^ITo,    OR  PARALLELS  RSSE/\.RCH  DONE 
ELSK'HERE  13  THE  PUBLIC  HEALTH  SERVICE   ('-'I'fflCUT  IWTERCHtNGE  OF  PERSONNEL, 
FACILITIES  OR  FUNDS),    IDi^ITIF^^  S' CH  RESE/iRCH. 


NO  ENTRIES  FOR  ITEI"?S  lij.,   l5  &  l6. 


PAGE  1 
PROJECT  PlEPORT  ^OKl 

1.     ?Tational  Cancer  Institute       2.     Padiation  Branch 

E'^STITUTE  IJIBORATORY  OR  BRAJICH 

NCI-6UIi 


3,     Radiation  Biology  Section       h, ^* 


SSCTia^   OR  SERVICE  LOCATION    (IF  OT^'KR  Tl-Uil'-J  BETHESDA) SERIAL  NO. 


6.     Modification  of  X-irradiation  Treatment  of  Localized  Tumors. 
PROJECT  TITLE 


7,     Joanne  Ilollcroft 

PRINC IPAL  INVESTIGATOR! Sj 


8,  None ^ 

O'fflER  BIVESTI'-ATORS 


9.   PROJECT  DE'^CRIPTION 

Objectives;   To  deterinine  in  what  manner  the  tumor  reacts  to  ionizing 
radiation  cind  in  what  manner  the  radios ensitivity  may  be  inci'eased,. 

Methods  employed;  Vary  the  physiology  of  the  host  prior  to,  during  or 
follo^cLng  irradiation  of  a  transplanted  lymphosarcoma  L-1,  follow  tumor 
gro-wth , 

Major  findings; 

1,  Attempts  todiange  blood  supply  to  tumor  by  administration  of 
adrenalin,  histamine,  priscoline  or  diathermy  did  not  greatly  alter  the 
response  of  the  tumor  to  x-irradiation, 

2,  VTien  a  single  dose  of  cortisone  was  admjjiistered  either  prior  to 
or  following  irradiation  the  increase  in  response  was  found  to  be  additive, 

3»   Thiotepa  may  increase  the  response  if  given  at  the  proper  time 
prior  to  irradiation. 

hf     Fractionation  of  a  dose  of  UOOO  r  into  k   equal  doses  given  2  minutes 
apart  produced  the  same  regression  as  the  same  dose  given  daily  but  if  the 
dose  is  given  at  2  day  intox'vals  the  effect  is  less* 

Significance  to  Cancer  Research;   To  find  methods  of  increasing  usefulness 
of  radiotherapy. 

Proposed  course  of  project:  Continued  studies  on  fractionation  and 
comparison  of  fractionation  treatm-ont  of  radiosensitive  tumors  id-th  that  of 
radioresistant  tumors.  Studies  on  radiation  induced  radioresistance.  Use  of 
antimetabolite  and  cytotoxic  poisons  to  potentiate  radiation  effects. 


PAGE  2 


10.  yCI-6iii| 
SERKL  NO. 

11.  BUDGET  ACTIVITY: 

Research  /x/ 

Review  &  Approval/"^ 


Administration  /V 

Technical  Assistance  /^ 


12.  GOOPERATBIG  UfllTS  OF  TKE  RJBLIC  HKILTH  SiiRX^ICE,    OR  OTHER  ORGANIZATIONS, 
PROVIDDIG  FUI'^DS,   FACILITIES,  ,  OR.  PERSONNEL  FOR  THIS   PROJECT  IN  EITHER 
1956  OR  1957: 

None 

13.  IF  Tl-nS  PROJECT  RSSETiBLES,    COI-ffLEt'IENTS,   OR  PARilLLELS  RESKIRCK  DONE 
ELSKNEl^E  IN   THE  PUBLIC  HE.\LTH  SERVICE   ('TITKOUT  INTSTiCPLuNGE  OF 
■r-ERSOM'EL,   FACILITIES  OR  FUNDS)   IDS^TTIFy  SUCH  RESEARCH: 

None 


NO  ENTRIES  FOR  ITHfS  ik,   l5  &  l6. 


PAGE  1 


PROJECT  REPORT  FORM 


1,  National  Cancer  Institute    2,  Radiation  Branch 

BISTITUTE  MBOPATORY  CR  BRANCH 

3..  Radiation  Biology  Section    li. g,WGI-6i|g 

SECTION  OR  SERVICE  LCGATIO]\t  ([F  OTHER  TE:\N  BST^-'ESD.OSERIAL  NO, 

6,  Effect  of  X-irradiation  on  Disseminated  Lymphosarcomas,  L#2  and  L2C» 
PROJECT  TITLE 


7.  Joanne  Hollcroft 


rREICIPAL  raVESnGATORTsy 


8,  Charles  C.  Congdon 

OTi-IER  INVESTIGATORS 


9,      PROJECT  DESCRIPTION 

Objectives;  To  determine  the  LD  100  g,  L-2  tumor  and  devise  a  feasible 
treatment  schedule,  Treatmen"-.  of  L2C  guinea  pig  leukemia. 

Methods  employed;  A/HeN  males  bearing  L-2  lymphosarcoma  were  irradiated 
starting  on  the  10th  day  after  transplant.  A  bio-assay  of  the  liver  spleen 
and  tumor  from  these  mice  x^as  performed  by  innoculating  A/HeN  males '  stib- 
cutaneously  i^rith  these  tissues  at  various  times  follomng  treatment, 

A/HeN  males  bearing  L-2  tumors  were  x-iraiiated  and  treated  subsequently  with 
bone  marrow. 

Family  2  guinea  igs  bearing  L2C  lymphocytic  leukemia  were  treated  T>jith 
x-irradiation  plus  bone  maA-rowj  thiotepa,  x-irradiation  clus  bene  marrow 
or  thiotepa  alone,  (This  work  was  started  by  Dr.  Congdon,) 

Ifajor  findings;   Kith  doses  greater  than  2000  r  the  greatest  number  of  "takes" 
occurred  when  'the  tumor  fragments  were  transplanted  1,  2  or  3  days  after 
treatment.  Few  transplants  grew  from  tissues  taken  immediately  or  h  hours 
after  treatment.   Two  daily  doses  of  1000  r  or  li  daily  doses  of  500  r  were 
found  equally  effective  in  inducing  the  transplantability  of  tumor  fragments 
1  day  after  the  end  of  treatment.  No  tumor  fragments  grew  after  transplantaticn 
from  animals  receiving  I1.OOO  r,  >Jhen  tumor  fragments  were  irradiated  in  vitro 
id.th  itOOO  r  1^%   of  the  tumor  transplant  grew. 

In  treating  mice  bearing  L-2  tumor  with  irradiation  followed  by  bone  marrow 
longest  survival  time  (11  days)  occurred  when  the  animals  were   irradiated 
with  5000  r  to  the  tumor  and  900  r  to  the  body  given  in  2  equal  doses  h  hours 
apart. 


PAGE  2 

NCI-6It^ 
SKR.nL  NO, 

PROJECT  DESCRIPTION  (COWT.) 

Family  2  guinea  pigs  bearing  L2C  lymphocytic  leukemia  have  been  treated 
successfully  mth  3'  x  iiOO  r  x-irradiation  plus  bone  marrow,  thiotepa  or 
a  combination  of  thiotepa  and  x-irradiatlon. 

Signif ioaiee to  Cancer  Research;  Radiotherapy  of  leukemia. 

Proposed  course  of  project;  Use  of  the  bio-assay  technic  to  study  radio- 
sensitivity  of  other  disseminated  neoplasms.  Study  of  the  number  of  cells 
innoculated  vs  time  to  death  in  hopes  to  extrapolate  this  information  to 
the  effectiveness  of  radiation  treatment.  Continuation  of  radiation 
treatment  of  guinea  pig  leukemia. 


10,      NC I -6ii5 

SmiAL  NO. 


PAGE  3 


11. 


BUDGET  ACTIVITY": 

Research  /x/ 

Review  &  Approval  /~J 


Administration  /~7 

Technical  Assistanoe  /^ 


12.  COOPER'.TING  UNITS  OF  THE  PUBLIC  HKILTH  SERVICE,    OR  OTHER  ORGANIZ.\TIOMS, 
PROVIDING  FUNDS,    FACILITI'S,    OR  PERSONNEL  FOR  r^IS  PROJECT  IN  EITHER 
19^6  OR  1957 : 

None 

13.  IF  THIS  PROJECT  RESEMBLES,    COl^^LEI'MT TS ,   OR  PARALLELS  RESKIRCH  DONE 
ELS0«IHEB1E  IN  'THE  HIBLIC  HKJAm  SERVICE   (^^^ITH  UT  DJTmCHAJ^TGE  OF 
PERSONNEL,   FACILITIES  OR  FUNDS)    IDEf^TIFY  SUCH  RESK'lRCH: 


NO  E[\ITRIES  FOR  ITEMS  \\x,   1^  ^'.  l6. 


PAGE  1 
PROJECT  ItEPORT  FOM 


1,     National  Cancer  Institute         2.     Radiation  Branch 

INSTITUTE  L/iBOPu.TORY  OR  BPuliICH 

3.  Radiation  Therapy  Service    h, , ^  5»NCI-6'^0(C) 

SECTION  OR.  SERVICE  LOCATION  (IF  OTHETi  THAN  BETHESDRj   SERIAL 

NO. 

6,  Sorvlco  Puidiation  Therapy 

PROJECT  TITLE 

7,  J,  Robert  Andrews,  IWD,,   and  Philip  Rubin,  M«D, 

PRINCIPAL  IblVESTIGATOUSj" 

8,  Robert  W.  Swain 


OTHKi  INVESTIGATORS 


9,  PROJECT  DESCRIFTION 

Objectives;  To  provide  radiation  therapy  for  those  patients  requiring 
such  in  the  course  of  the  disease  for  which  they  are  being  investigated 
by  research  groups  other  than  the  itadiation  Branch, 

Methods  employed;  Consistent  with  contemporary  radiotherapoutic  practice 
employing  a  wide  range  of  photon  or  other  energies, ^  radium  and  radio- 
isotopes as  indicated. 

Patient  material;  Those  patients  present  with  a  wide  variety  of  neoplasms 
for  which  radiation  therapy  might  be  of  palliative  or  other  value. 

Ila. j or  findings ;  Variable  as  would  be  expected  from  the  diverse  nature 
of  the  material  studied  and  the  widespread  diseases  generally  present. 

Proposed  course  of  project;  To  continue. 


PAGE  2 


PROJECT  REPOiiT  FOitfl   (Cont.) 


10.  NCI-6gO(C) 

SEicIAL  NO. 


11.   BUDGET  ACTIVITY; 

Research         /~7 
Review  &  Approval  [^ 


Administration      /V 
Technical  Assistance  /~7 


12.   COOPEtiATING  UNITS  OF  THE  PUBLIC  HEi'.LTH  SERVICE,  Oit  OTHER  OliGANIZATIONS, 
PROVIDING  FTOIDS,  FACILITIES,  Oli  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  Ou  1957: 

None 


13,.     IF  THIS  P..OJECT  F^SSEMBLES,    COMPLEMENTS,   Ou  P/JL'iLELS  RESKJtCH  DONE 
ELSEMlEIci^.  IN  THE  PUBLIC  HEALTH  SEIi.VICE   (WITHOUT  INTERCH..NGE  OF 
PETtSONNEL,   FACILITIES   OR  FUNDS),    IDENTIFY  SUCH  RESE/JiCH: 

None 


NO  EOTRIES  FOR  ITEMS  lli,   l5  &  l6. 


PAGE  1 

PROJECT  REPORT  FORM 


1»  National  Cancer  Institute    2,  Radiation  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Radiation  Thoraov  Service  h* S,   NCI..6gl(C) 

SECTION  OR  SERVICE       rOCATION  (IF  OTHER  THAN  BETHESDA;    SERIAL  NO. 

6,  Electron  Beam  Radiation  Therapy 
PROJECT  TITLE 


7.  J.  Robert  Andrews,  M.D, 

PRINCIPAL  I1WESTIGAT0R(S} 


8.  Philip  Rubin,  M.D»,  and  Robert  W.  Swain 
OTHER  INVESTIGATORS 


9,  PROJECT  DESCRIPTION 

Objectives:  To  deliver  effective  doses  of  ionizing  radiation  to  the 
epidermis  and  corium  by  means  of  controlling  energies  and  character 
of  the  ionizing  radiation  to  limit  the  effects  to  this  zone. 

Methods  employed;  Ionizing  radiation  is  an  effective  treatment  for  a 
variety  of  multiple,  malignant,  superficial,  cutaneous  neoplasms  including 
mycosis  fungoides.  Bowing's  disease  and,  possibly,  Kaposi's  sarcoma. 
Ionizing  radiation  may  be  administered  as  x-ray  therapy  but  in  this  case 
there  is  generally  such  absorption  of  radiation  in  deeper  tissues,  including 
the  radiosensitive  bone  marrow,  as  to  limit  the  amount  of  radiation  to 
less  than  an  effective  dose.  The  absorptions  of  electrons  in  the  energy 
range  (<.  2,0  Mev)  available  is,  however,  limited  to  less  than  1  em,  of 
tissue.  This  makes  possible  the  administration  of  effective  doses  of 
radiation  to  superficial  neoplasms  without  affecting  deeper  structures. 

The  physical  factors  associated  x^ith  the  production,  dosimetry, 
and  clinical  use  of  an  electron  beam  arc  being  thoroughly  studied.  Clinical 
studies  of  skin  erythema  and  skin  blistering  doses  arB  being  performed  and 
compared  with  x-ray  effects  as  to  relative  biological  effectiveness.  The 
effects  of  various  doses  on  superficial  cutaneous  neoplasms  are  being 
studied.   In  addition  to  clinical  observations,  appropriate  biopsy  material 
is  being  obtained  and  all  observations  arc  being  carefully  documented. 

Patient  material;  Cases  of  mycosis  fungoides.  Bowing's  disease,  Kaposi's 
sarcoma  and  other  primary  or  secondary  superficial  malignant  neoplasms, 

Maj or  findings :  This  project  was  initiated  only  in  the  last  six  months. 
The  response  of  the  limited  niunber  of  patients  so  far  available  for  study 
indicates  that  this  type  of  ionizing  radiation  is  clinically  effective. 

Proposed  course  of  project;  As  indicated  in  methods  above. 


PAGE  2 
PROJECT  REPORT  FORM  (cont.) 


10.NCI-6'^1(C) 

SERIAL  NO. 


11.  BUDGET  ACTIVITY 

Research         f~J  Administration       /V 

Review  &  Approval  /~]  Technical  Assistance  £J 

12,  COOPERATING  MITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FIM3S,  FACILITIES,  OR  PERSOMEL  FOR  THIS  PROJECT  TO  EITHER' 
1956  OR  1957: 

None 


13,      IF  THIS  PROJECT  RESEi'lBLES,    CQlPLEilENTS,    OR  PARALLELS  RESEARCH  DONE 
ELSEIfffiERE  IN  THE  PUBLIC  HEALTH  SERVICE   (WITHOUT  INTERCH/ii'^GE  OF 
PERSONNEL,   FACILITIES   OR  FUITOS),    IDENTIFY  SUCH  RESE/iRCH: 

None 


NO  ENTRIES  FOR  ITEMS  II4,  l5  &  I6, 


PAGE  1 
PROJECT  liEPORT  FORM 


1,  National  Cancer  Instituto    2,  Radiation  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

3,  Radiation  Therapy  Service     it, ^,  NCI>6g2(C) 

SECTION  OR  SERVICE  LOCATION(IF  OTHER  THAN  BETHESDA;   SERIAL 

NO. 

6,  The  Relative  Biological  Effectiveness  (RBE)  Factor  for  the  Destruction 

of  Human  Carcinoma ^   ^  

"PROJECT  TITLE 


7.  J,  Robert  I>xidrews,   H«D. ,  Philip  Rubin,  M,D. ,  and  Robert  VJ.  Swajn 
PRINCIP;i  INVESTiaATOR(S} 


8,  Eugene  J,  Van  Scott,  M,D.,  a.nd  Richard  P.  Reinertson,  M«D. 
OTHER  liWESTIGATORS 


9,  PROJECT  DESCRIPTION 

Objectives ;  To  determine  the  relative  biological  effectiveness  (RBE) 
factor  for  the  destruction  of  hur,ian  carcinoma. 

Methods  employed;  Human  multiple,  superficial  cutaneous  neoplasms  are 
treated  with  a  range  of  doses  by  a  variety  of  ionizing  radiations  (including 
low  voltage  unfiltercd  x-rays,  medium  voltage  filtered  x-rays,  2  Mev  x-rays, 
the  gamma  rays  of  radium,  and  0,9  and  1,5  Mev  electrons)  and  the  cancerocidal 
dose  determined  for  each.  In  addition  to  grossly  observable  clinical  effects, 
microscopic  histological  studies  have  also  been  undertaken. 

Patient  material;  Cases  of  multiple,  superficial,  epithelial  cancers  of 
the  skin. 

Major  findings;  Major  findings  can  not  be  reported  at  this  time  because 
this  project  was  initiated  only  within  the  last  six  months. 

Proposed  course  of  project;  As  indicated  in  methods  above. 


PAGE  2 
PROJECT  REPQIT  FORM  (cont.) 


10.  NCI-6^2(C) 
SERL'X  NO, 


11.  BUDGET  ACTIVITY 

Research         fl  Administration       fl 

-Review  &  Approval  /V        Technical  Assistance  /V 

12.  C00PER.;TING  units  of  THE  PUBLIC  HEALTH  SERVICE,  OR  OTi-iER  ORGi'.NIZi.TIONS, 
PROVIDING  FUITOS,  FACILITIES,  OR  PERSOMEL  FOR  THIS  PROJECT  IN  EITHER 
1956  OR  1957: 

None 

13.  IF  THIS  PROJECT  RESEilBLES,    COflPLEl^iENTS,    OR  P.iRiULELS  RESEi'JlCH  DONE 
ELSEl'JHERE  IN  THE  PUBLIC  HE^'XTH  SERVICE   (WITHOUT  iNTERCHiUMGE  OF  PEIiSOIWEL, 
FACILITIES  OR  FUl\fDS),   IDENTIFY  SUCH  RESK'uRCH: 

None 
NO  ENTRIES  FOR  ITEMS  lit,  \$   &  16. 


PAGE  1 
PROJECT  REPORT  FORM 


1,  National  Cancer  Institute  2,  Radiation  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Radiation  Therapy  Service  U, gt  NCI-653(C) 

SECTION  OR  SERVICE         LOCATION(IF  OTHJR  THAN  BETHESDA;   SERIAL  NO. 

6,  The  Influence  of  Oxygen  in  Modifying  Radiation  Response 


7,  J.  Robert  Andrews,  M.  D. 

PRINCIPAL  investigator(s; 


8.  Philip  Rubin,  M.  D,  ,  Robert  W.  Swain 
OTHER  liWESTIGATORS 


9.  PROJECT  DESCRIPTION: 

Objectives;  To  investigate  the  possibility  of  modifying  response  of 
neoplastic  cells  to  ionizing  radiation  by  altering  the  oxygen  content  of 
blood» 

Methods  Employed;  Appropriate  patient  material  (see  below)  is 
irradiated  over  bilaterally  more  or  less  symmetrically  enlarged  Ijmiph  nodes 
with  equal  doses  of  ionizing  radiation  (2^0  KV  X-rays)  under  different 
conditions  of  oxygen  satiu-ation  of  hemoglobin  and  dissolved  blood  oxygen. 
One  bilaterally  symmetrical  area  is  irradiated  under  conditions  of 
normal  atmospheric  press\ire  and  oxygen  tension.  The  other  bilaterally 
symmetrical  area  is  irradiated  under  conditions  of  normal  atmospheric 
pressure  with  lOOJ?  inspired  oxygen  under  which  conditions  the  differential 
oxygen  tension  is  increased  by  a  factor  of  approximately  5» 

If  a  differential  effect  is  observed  it  is  proposed  to  extend  the  experi- 
ment by  irradiation  of  similar  patient  material  under  conditions  of  re- 
duced oxygen  tension  (normal  atmospheric  pressure  but  reduced  differential 
oxygen  tension)  and  under  conditions  of  increased  oxygon  tension  to  above 
norraal  atmospheric  pressure  by  radiation  in  a  pressiu'e  chamber.  It  is 
further  proposed  to  investigate  directly  the  oxygen  tension  in  the  tissues 
exposed  to  ionizing  radiations  by  the  use  of  appropriate  instrviments. 

Patient  Material;  Cases  of  Hodgkin's  disease  or  lymphosarcoma  with 
bilaterally  and  symmetrically  enlarged  lymph  nodes  in  previously  untreated 
areas. 

Ma j or  Fj.ndings ;  Major  findings  can  not  be  reported  at  this  time  because 
this  project  was  initiated  only  within  the  last  six  months. 

Proposed  Course  of  Project;  As  indicated  in  methods  above. 


PAGE  2 


10.      NCI-6g3(C) 
SERIAL  NO. 


11.  BUDGET  ACTIVITY: 

Research        ff       ■   Administration      f~J       > 
Review  &  Approval  f^  Technical  Assistance  ri 

12.  COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  0THJ3R  ORGANIZATIONS, 
PROVIDING  FU'NDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  OR  19^7:  ,  _   .  . 

None 

13.  IF  THIS  PROJECT  RESEflBLES,  CQIPLHIENTS,  OR  PARALLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF 
PERSONNEL,  FACILITIES  OR  FOTJDS),  IDENTIFY  SUCH  RESEARCH: 

None 
NO  Ei^RIES  FOR  ITEMS  lU,  l5  Sc  16,  .    : 


PAGE  1 


PROJECT  REPOPiT  FORM 


1,  National  Cancer  Institute  2,  Radiation  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3,  Radiation  Therapy  Service  h* S,   NCI-6^1i('C) 

SECTION  OR  SERVICE  LOCATION(IF  OIliER   THAN  BETHESDA)   SERIAL  NO. 

6,  Tiine  as  a  Modifier  of  Clinical  Fuadiation  Response 

7»  J.  Robert  Andrews,  M.  D. 
PRINCIPAL  INVEST IGAT OR (Sj 

®»   RTJlip  Rubin,   M.  D,  and  Robert  '-'.   Swain 
OTHER  IWESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objective;  To  evaluate  the  prolongation  of  treatment  time  as  a  modifier 
of  clinical  response  to  ionizing  radiations. 

Methods  Bnployed;  Irradiation  of  appropriate  patient  material  (see 
below)  to  tumor  doses  confirming  with  the  equation, 

D  =  2750  tO.23 
over  approximately  a  100  day  treatment  period. 

Patient  Material;  Cases  of  squamous  cell  carcinoma  of  the  head,  neck, 
larynx  and  uterine  cervix  in  Stages  II  and  III, 

Major  Findings;  Major  findings  can  not  be  reported  at  this  time  because 
this  project  was  initiated  only  vrithin  the  last  six  months. 

Proposed  Course  of  Project;  As  indicated  in  methods  above. 


PAGE  2 


10.  NCI-6gi;(C) 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

Research        [J  Administration      fl 

Review  &  Approval  [J  Technical  Assistance  f~] 

12.  COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER, 
19^6  or  19^7  s 

None 

13.  IF  THIS  PROJECT  RESKIBLES,   COIIPLEMENTS,    OR  PARALLELS  RESEARCH  DOM 
ELSEV/HERE  IN  THE  PUBLIC  HEALTH  SERVICE   (VJITKOUT   INTERCHANGE  OF 
PERSONl^L,   FACILITIES  OR  FUNDS),   IDENTIFY  SUCH  RESEARCH: 

None 


PAGE  3 
PROJECT  REPORT  FORM  (cont.) 


11;.  NCI..6g[;(C) 

SERIAL  MO. 


15.  PUBLICATIONS.  OTHER  THAN  ABSTRilCTS  K^OIi  THIS  PROJECT  DURING  CiXEND^Jl 
121  Ji  1955: 

J.  R.  /mdrews  and  Jog  M,  Moody,  The  Dose-Time  Relationship  for  the 
Cure  of  Squamous  Geil_CaiicinDma:     Presented  at  the  Inter»-/unerican 
Congress  of  Radiology,  V/ashington,   D,   C,   1955j  Accepted  for  publica- 
tion by  the  /merican  Journal  of  Roentgtinology  and  Radium  Therapy, 

16.  HONORS  ;.ND  AVL'J^DS  TO  PERSOM^IEL  RELATING  TO  THIS  PROJECT  DURING  CALEi©/Ji 
lam  1955: 

None 


PAGE  1 


PROJECT  REPORT  FORM 


1,  National  Cancer  Institute  2,  Radiation  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Radiation  Therapy  Servlco  k, g^  ;'-TCI-6 55(C) 

SECTION  OR  SERVICE         LOCATION(IF  OTHER  THi^N  BETKESDA;   SERIAL  NO, 

6,  Detormination  of  Radiation  Tolerance  to  the  Brain  by  ileans  of 
Electroencophalographic^  Studies 

PROJECT  TITLE 

7.  Philip  Rubin,  M.D.,  and  J.  Robert  Andrews,  M,D. 

PRINCIPAL  INVESTIGATQR(S; 

8«  Dr«  Cosimo  Ajmone  Marsan 
OTHER  INVESTIGATORS 


9,  PROJECT  DESCRIPTION 

Objectives:  The  use  of  electroencephalographic  studies  while  treating 
various  brain  tumors  will  be  employed  to  determine  if  any  damage  has  been 
done  to  normal  brain  tissue.  It  is  assumed  that  physiological  changes 
reflected  in  the  electroencephalogram  may  precede  the  severe  structural 
anatomical  changes.  No  attempt  has  been  made  to  correlate  electroencephalo- 
graphic changes  in  definite  areas  of  the  brain  with  isodose  curves  which 
will  give  exact  dosage  to  these  points. 

Methods  employed;  Serial  electroencephalographic  studies  will  be  done 
during  the  course  of  treatment,  beginning  with  a  base  line  study.  Aberra- 
tions in  brain  waves  in  certain  areas  will  be  correlated  with  the  dosage 
level  at  these  points.  With  the  sharp  beam  alignment  and  available  isodoee 
c\irves  this  can  be  calculated  with  sufficient  accuracy  to  have  considerable 
correlative  values. 

Patient  material;  Appropriate  case  material  which  includes  a  wide  range 
of  brain. tumors  which  have  proved  to  be  inoperable,  but  show  some  evidence 
of  radioresponsiveness  on  the  basis  of  the  literature. 

Major  findings;  Major  findings  can  not  be  reported  at  this  time  because 
this  project  was  initiated  only  within  the  last  six  months. 

Proposed  course  of  project;  It  is  proposed  that  in  the  next  year  this  study 
will  be  considerably  amplified  in  conjunction  with  an  expanded  activity  in 
brain  tumor  research  in  the  NINDB, 


PAGE  2 


PROJECT  REPCSRT  FORI'f  (cont.) 


10. 


NCl-655(c) 

SERIAL  W07 


11.  BUDGET  ACTIVITY: 

Rcsuarch         [j 
Review  &  Approvr.l  // 


Administration       [J 
Technical  Assistance  [J 


1956  OR  1957  5      ■  ■  '■  ■  - 

None 

t.        TE  THIS  PROJECT  RESEMBLES,   COiffLElENTS,    OR  P/JL'XLELS  RESEARCH  DONE 

''•    eL™^e'S1he'public  HkLTH  service  (wnwri™^^^^      OF 

PERSOOTMEL,  FACILITIES  OR  FUNDS),   IDENTIFY  SUCH  R^Sl^iJlCH: 

None 


NO  EOTRIES  FOR  ITEMS  lU,   l5  &  l6. 


PAGE  1 

.  PROJECT  REPORT  FORM 

1,  National  Ce.ncer  Institute       2.  Radiation  Branch 

INSfifUTE  "  LABORATORY  OR  BR/JJCH 

3.  Radiation  Therapy  Service   1;. g.  MCI-656(^ 

SECTION  Ce  SERVICE  LOCATION(IF  OTHER  THAN  BETIIESDAj        SERIAL  NO. 

6,     Attempt  at  Radiotherapy  vrith  s35_Sulfate 
PROJECT  TITLE  . 


7.     Philip  Rubin,   M,  D-  and  J«  Roberfei/.hdi-ews,   M,   D. 
PRINCIPAL  INVEST IGAT  OR ( S ) 


J.  A.  Holler  oft,   Marion  Matthews,   Raymond  Gottsch.^.lk,  I1.D« 
OTHER  irVESTIGATORS 


9.   PROJECT  DESCRIPTION 

Objectives ;  s35  given  as  sulfate  selectively  concentrates  in  cartilag* 
and  tissues  containing  abundant  sialfated  mucopolysaccharides.  Since  the 
beta  radiation  of  S35  is  very  weak  (in  the  neighborhood  of  pO  lev, )  it 
must  be  determined  first  vrhetiier  this  can  destroy  cartilage  cells.  The 
s35  is  laid  doi'm.   in  the  ground  substance  of  cartilage,  a  chondroitin  sulfuric 
acid  polysaccharide.  It  is  postulated  that  the  path  of  the  emitted  electrons 
will  be  adequate  to  reach  the  cells*  To  date,  no  evidence  of  cell  destruc- 
tion has  been  shown  with  the  dosage  used  in  adult  cartilages.  It  seems 
essential  before  one  embarks  on  the  use  of  large  amounts  of  this  radio- 
isotope which  will  mean  considerable  expense,  that  evidence  of  some  radio- 
toxic  effect  should  be  shown  on  cartilage. 

Methods  employed;  The  follovxing  animal  experiment  was  therefore  decided 
upon  and  is  being  carried  out  as  outlined  below: 

1,  Thirteen  litters  of  white  suckling  rats  were  utilized  weighing 
between  8  to  25  gms, 

2,  Varying  amounts  of  radioactive  sulfate  were  injected  ranging  from 
the  toxie  level  (2,0  Mc/gm, )  to  the  postulated  therapeutic  level  (0,1  Mc/gm, ) 

3*  The  animals  that  have  died  during  the  procedure  were  bottled  in  10^ 
formalin  and  are  to  be  examined  at  a  future  date  by  Dr.  Gottschalk  for  the 
pathology  and  tissue  assays  which  are  pertinent  to  the  procedure, 

1;,  Weights  of  the  animals  will  bo  taken  every  week  to  obtain  a  growth 
curve, 

5,  X-rays  of  all  the  bones  in  the  body  will  be  made  every  two  weeks  to 
determine  the  effect  of  the  administered  S35  on  epiphyseal  centers  and  bone 
growth. 

Patient  material;  Application  to  Patients  with  Chondrosarcoma;  If  the 
above  work  suggests  that  groiving  cartilage  can  be  destroyed  by  radiosulfur, 


PAGE  2 

NCI-6^6  (130 
SERL'A  NO, 
PROJECT  itEPOuT  FORM  (cont.) 

ari  ottempt  vill.be  made  to  utiliZG  this  agent  on  a  clinical  basis.   Only, 
niticnts  w;th  advanced  chondrosarcoma  who  have  been  treated  with  surgery 
and  external  radiation  previously  will  be  candidates  for  this  treatment. 

I'laior  findings:   Definite  disturbances  in  the  epiphyseal  and  metaphyseal 
i^feiT^J^^n'of  long  bones  have  been  observed.  This  remains  to  be 
correlated  with  tissue  assay  and  radiomicrographs  of  tissue  sections. 

Significance  tq3'.ncexJl^s_3archi  A  new  method  of  treating  chondrosarcoma 
ij;  the  ani'iiar~jxporiraents  prove  successful. 

Proposed  course_o^£ro,loc:y.  Continuation  as  outlined  above  until  studies 
are  completed  which  will  be  in  three  months. 


10.  Mr.T-yA  rX^ 
SETtlAL  NO. 


PAGE  3i 


11.  BUDGET  ACTIVITY J 

Research        f^ 
Review  &  Approval  /"^ 


Administration  f~] 

Technical  Assistance     f^ 


12.     CCX)PER/i.TING  Ul^IITS  OF  T  ;E  PUBLIC  HEALTH  SERVICE,   OR  OTHER  ORGMIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,   OR  PERSONI^JEL  FCR  THIS  PROJECT  IN  EITHER 
1956  OR  19571 

None 


13,     IF  THIS  PROJECT  RESEifflLES ,    COiiPLEilENTS,    OR  PiLRilLLELS  RESE/JiCH  DOIME 
ELSEWHERE  IN  THE  PUBLIC  HE.'J.TH  SilEVICE   (V/ITHOUT  INTERCHANGE  OF 
PERSONNEL,  FACILITIES  OR  FUNDS),   IDEOTIFY  SUCH  RESE^'J^CH: 


NO  ENTRIES  FOR  ITEMS  lU,  l5  &  l6. 


PAGE  I 
PROJECT  REPORT  F-^RM 
PROJECTS  WHICH  ARE  BEING  DISCONTINUED 
N.C.I.  2.  General  Medicine  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

Nutrition  and  Metabolism     h. ____«_______^  5*  NCI~70l(C) 

SECTION  OR  SERVICE  '  LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

A.  Meticorten:  Metabolic  and  clinical  effects  of  massive  doses  of 

PROJECT  TITLE    Prednisone  in  subjects  v^ith  malignant  disease 

Donald  M.  Watkin 

PRINC IPAL  INVESTIGATOR ( S ) 


OTHER  INVESTIGATORS 


i^OJECT  DESCRIPTION 

Objectives:  To  quantitate  the  effect  of  massive  doses  of  Prednisone,,  a 

synthetic  steroid  reputedly  lov;  in  midesirable  side  effects,  in 
malignant  disease  not  amenable  to  more  conventional  therapy. 

Methods  Employed:   Metabolic  balance  technique  I  -^  labelled  human  serim 
turnover . 

Renal  function  studies . 
Electrophoretic  analysis  of  serum  protein. 

Patient  Material:   Two  women^  one  with  multiple  myeloma  and  one  with 

lymphosarcoma,  and  three  men,  one  with  Hodgkins  Disease, 
one  with  multiple  myeloma,  and  one  with  prostatic 
carcinoma. 

Major  Findings:  All  subjects  demonstrated  negative  nitrogen  balance;  all 

demonstrated  retention  of  sodium  and  chloride.   Theoretical 
phosphorus  balances  could  not  be  reconciled  with  calcium 
and  nitrogen  balances.   Potassi\Jm  balances  were  negative.   Uric  acid 
excretion  was  slightly  elevated  in  three  and  dramatically  elevated  in 
two  patients.  Marked  negative  phosphorus  and  nitrogen  balances  and 
ma:rked  elevated  urinary  uric  acid  excretions  were  associated  with 
objective  diminution  in  tumor  size  in  lymphoma  and  Hodgkins  Disease. 
I  ^     albumin  turnover  rates  were  accelerated  by  Prednisone  therapy  in  one 
patient,  genal  function  studies  before  and  during  treatment  indicated 
a  marked  reduction  in  renal  plasma  flow  apparently  due  to  reduction  in 
cardiac^  output,  an  example  of  forward  failure.  Electrophoretic  studies 
of  plasma  proteins  revealed  for  the  first  time  a  change  in  the  charac- 
teristic pattern  in  a  patient  with  multiple  myeloma. 


NCI-701-(C)  PAGE  2 

SERIAL  NO. 

PROJECT  REPORT  FOEM  (conb'd) 
f  •    ■       ■• 

Numerous  side  effects  including  acute  psychosis,  congestive  heart  fail\ire, 
paroxysmal  tachycardias^  systemic  moniliasis,  pulmonary  embolism  and 
severe  acne  were  observed  during  massive  Prednisone  therapy. 

'  Significance  to  Cancer  Research;  Prednisone,  because  of  its  potency  greater 

than  Cortisone  and  freedom  from  side  effects 
when  given  in  smaller  doses,  suggested  a  means  of  giving  enormous 
corticoid  doses  to  patients  with' hopeless  malignancy.   The  -reporttjd  •:■• 
studies  indicate  that  in  massive  doses  the  hormone  has  numerous 
undesirable  side  effects,  that  it  is  effective  against  malignancies  in 
the  lymphoma  group,  and  that  it  can  induce  some  changes  hitherto  un- 
reported in  multiple  myeloma:.  Except  for  experimental  purposes,;  however. 
Prednisone  in  massive  doses  cannot  be  recommended  as  a  therapy  for 
malignant  disease. 

Proposed  Course  Oj'  Project;  The  discrepancies,  between  theoretical  and  actual. 

phosphorus  balances,  changes  in  myeloma  proteins, 
changes  in  resistance  to  disease,  and  alterations  in  intracellular 
electrolytes,  especially  in  the  myocardiim,  are  avenues  which  may  be 
followed  as  suitable  patients  present  themselves  and  As   new  synthetic 
hormones  are  developed. 

B.  Metabolism  of  nitrogen,  calcium,  phosphorus,  electrolytes  in  metastatic- 
prostatic  carcinoma. 

Objectives;  Accumulation  of  metabolic  data  in  patients  with  extensive 

metastatic  prostatic  maligna.ncy  be.fore,  during  and  after  various 
therapeutic  maneuvers. 

Methods  Employed;   Metabolic  balance  technique.  Red  cell  turnover  deter-' 

minations . 

Patient  Material;   In  195^ j  two  patients  were  studied  before  and  after 

orchiectomy.   In  1955;  one  of  these  returned  one  year 
after  operation  for  a  follow-up  metabolic  study.   In  addition^  two 
other  patients  with  extensive  metastatj.c  disease,  both  several  years 
post-orchiectomy,  were  admitted  for  study  of  the  effect  of  hormone 
administratjon. 

Major  Findings;   The  beneficial  effects  of  orchiectomy  in  reducing  calcium 
and  protoplasmic  loss  were  ma-intained  one  year  after 
operation.   Stilbestrol  administration  was  of  equivocal  value.  Massive 
Prednisone  dosage  superimposed  on  stilbestrol  administration  in  one 
patient  produced  a  temporary  clinical  improvement,  but  in  the  long  rion 
resulted  in  marked  wasting  of  bone  and  protoplasm. 

Incidental  Findings;   The  patient  treated  with  Prednisone  demonstrated  a 

unique  afibrinogenemia  which  was  followed  throughout 
his  course  and  which  seemed  to  improve  under  Prednisone  therapy.   This 
was  not  due  to  prostatic  fibrinolysin  but  to  a  lack  of  fibrinogen 
production. 


PAGE  3 
CI-701(C) 
ERIAL  NO. 

PROJECT  REPORT  FORM  (cont'd) 

Significance  to  Cancer  Research:  Quantitative  evaluation  of  the  natural 

course  of  the  disease  together  with 
quantitative  measurements  of  the  effects  of  therapy. 

Proposed  Course  of  Project;  Continued  search  for  more  patients  with 

extensive  prostatic  disease,  follow-up  on  patients 
already  under  study,  further  investigation  of  the  relationship  of 
prostatic  malignancy  to  afibrinogenemia. 

C.  Metabolism  of  fluoride  in  leukemic  subjects  with  and  without  chronic 
fluorosis 

Objectives :   Investigation  of  possible  role  of  fluoride  in  genesis  of 
leukemia . 

Methods  Employed;   Metabolic  balance  technique. 

Patient  Material;  During  195^  two  leukemic  patients  with  fluorosis,  one 

leukemic  without  fluor-os is  and  one  non-leukemic  without 
fluorosis  were  studied.   In  1955 j  three  patients  without  fluorosis, 
two  normals  and  one  with  multiple  myeloma,  were  studied. 

Major  Findings:   On  an  intake  of  distilled  water,  patients  with  fluorosis 
excrete  fluoride  in  the  urine.   Patients  with  and  without 
fluorosis  excreted  a  fixed  percentage  of  fluoride  intake.   Ljukemia  or 
multiple  myeloma  did  not  alter  the  fluoride  excretion  pattern.  Balances 
of  nitrogen,  potassiiwi,  phosphorus,  calcium,  sodium  and  chloride  were 
not  significantly  altered  by  fluoride  administration  at  levels  of 
5  mg.  per  day. 

Significance  to  Cancer  Research:  The  essentially  negative  results  of  these 

studies  should  help  allay  the  fears  of  many  ". 
laymen  that  fluoride  in  drinking  water  m.ay  prove  harmful.  No  evidence 
has  been  obtained  in  these  studies  to  indicate  any  harmful  effects 
of  fluoride  administration.   These  studies  of  course  do  not  rule  out 
fluoride  as  a  carcinogen  but  do  demonstrate  the  absence  of  any  measurable 
toxic  effect. 

Proposed  Course  of  Project;  Any  further  pursuit  of  this  subject  will  depend 

on  the  ability  of  NIDH  to  perform  fluoride 
assays.   At  the  present,  no  extension  of  the  program  has  been  planned 
as  far  as  NCI  is  concerned. 

D.  Role  of  B  complex  vitamins  in  tissue  anabolism 

Objectives;   To  observe  the  utilization  of  B  complex  vitamins  in  tissue 
anabolism  induced  by  steroids  or  hyperalimentation. 


PAGE  k 
fGI-70l(C) 

>:rial  no. 

PROJECT  REPORT  FORM  (cont'd) 

Methods  Employed;  Metabolic  balance  technique.   Bioassay  for  vitamins  in 
food  and  urine. 

Patient  Material;   One  patient  studied  initially  in  195^  was  carried  over 
into  195,^ 

Major  Findings:   No  direct  relationship  was  noted  between  vitamin  retention 
and  tissues' anabolisra.  The  small  quantities  of  vitamins 
required  and  the  inaccuracy  of  the  bioassay  technique 
may  account  for  the  negative  findings. 

Significance  to  Cancer  Research;  The  domonstratiun  of  the  essentiality  of 

certain  vit;arains  in  the  growth  of  normal 
or  tumor  tissue  could  lead  to  development  of  effective  antimetabolites. 

Proposed  Course  of  Project:  The  bioassay  procedures  originally  conducted 

by  the  Endocrinology  Branch  are  no  longer 
available,  he>nce,  no  continuation  of  this  project  is  contemplated. 

E.  Metabolism  of  human  cerum  albumin  administered  intravenously  in  large 
doses  to  subjects  with  malignant  disease.  ■ 

Objectives:   Quantitative  measurasBiipnt  of  the  metabolism  of  large  amounts  of 
human  serun  albiwiin  given  intravenously. 

Metboda  Employed:  M'-tabolic  balance  technique. 

Eluctrophoretic  analysis  of  serum. 

Turnover  of  l''-3-'-  labelled  human  serum  albumin. 

Patient  Material;  One  normal,  one  subject  with  Hodgkins  Disease^  one  patient 
with  multiple  myeloma,,  one  v;ith  lung  cancer  and  one  with 
face  cancer. 

Major  Findings;  Balance  data  indicate  that  the  albumin  was  gradually 

utilized  over  a  period  of  about-  h   weeks.   Isotope  studies 
show  an  increased  rate  of  albumin  turiiover  luring  and  after  albumin 
administration. 

Significance  to  Cancer  Research;   Albumin  is  readily  available  plasma 

~   constituent  frequently  low  in  patients 
with  advanced  cancer. 

Proposed  Course  of  Project:   Further  evaluation  of  the  data  compiled  in 

these  studii.:s.   Wo  new  studies  planned  at 
this  time. 


PAGE  5 


PROJECT  REPORT  FORM  (cont  d) 


LO.  NCI-70L(C) 


SERIAL  NO. 


LI. 


BUDGET  ACTIVITY: 

RESEARCH         [YJ         ADMINISTRATION       /  / 
REVIEW  &  APPROVAL  I      7    TECHNICAL  ASSISTANCE 


L2. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SlilRVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


L3. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HE/VLTH  SERVICE  (WITHOUT  INTERCmvNGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEMS  i'+;,  15  &  l6 


PAGE  I 

PROJECT  REPORT  FOM 

1.  N.  C,  I.     ' 2.  General  Medicine  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3    ■        __ 4.  _ „ _: - 5."  NCI..702XCJ 

SEGTIWJ  OR  SERVICE  LOCATION  (IF  oflKR  flBN  BETHESDA)   SERIAL  NO. 


6 .  Metabolic  Study  of  Se  rum  Proteins . , — . 

TrOJ^GT  TITLE 

7.  J.  L.  Fahey.  J.  L.  Steinfeld.  J.  M.  Watkin.  H.  A.  Sober  and  E,_A^_Petersgn_ 
PRINCIPAL  INVE^.TIG;iTOR(sT 


OTHER  INVESTIGATORS 

9A.  PROJECT 'DESCRIITION:  Electrophoretic  Studies  of  the  Serum  Proteins  in  Neo- 
plastic Disease  States.  (J.  L.  Fahey) 

Objectives;  The  objective  of  this  project  is  to  electrophoretically  quanti- 
tate  the  serum  protein  changes  occurring  in  neoplastic  states 
and  to  correlate  these  changes  with  specific  aspects  of  the 
clinical  status  of  the  patients. 

Methods  Employed;  Development  and  refinement  of  a  quantitative  method  of 
zone  (paper)  electrophoresis  was  completed  during  the 
year.  Systematic  follow-up  of  patients  admitted  to  the 
Metabolic,  Acute  Leukemia  and  Solid  Tumor  Services  was  instituted  so  that 
serum  protein  data  can  be  correlated  with  a  clinical  evaluation  of  the  type 
and  extent  of  disease,  disease  activity,  nutritional  state  of  the  host  and 
therapy  employed.  Sequential  observations  in  the  same  patients  are  an  im- 
portant aspiect  of  this  study. 

Patient  Material;  Patients  admitted  to  the  Clinical  Center  for  other  pro- 
jects were  utilized  in  this  study. 

Major  Findings;    Zone  (paper)  electrophoresis  can  be  utilized  for  quantita- 
tive measurement  of  serum  proteins. , 

Long  term  patient  follow-up  was  instituted.  A  study  of 
the  serum  protein  changes  during  immune  response  to  antigen  challenges  in 
leukemic  and  control  patients  (in  collaboration  with  the  Acute  Leukemia 
Group)  revealed  no  striking  correlation  of  gamma  globulin  changes  in  pre- 
liminary evaluation.  However,  the  study  is  not  yet  complete. 

Genei"al  assistance  to  all  clinical  groups  has  been  utilized  to  evaluate 
serum  proteins  of  patients  with  multiple  myeloma  and  of  patients  in  which  a 
gamma  globulinemia  was  suspected.  None  were  found,  however. 


NCI-702(C)  PAGE  2 

PROJECT  REPORT  FORM  (Cont'd) 

Significance  to  Cancer  Research;  Delineation  of  the  sequence  of  plasma  pro- 
tein changes  in  neoplastic  disease  is 
needed,  for  this  is  an  aspect  of  tumor- 
host  relationship  which  lends  itself  to  quantitation.  Also,  the  effects 
upon  the  serum  proteins  of  anti-tumor  therapeutic  regimens  is  generally  . 
unknown. 

Pr<;'posed  Course  of  Project;  Emphasis  will  continue  to  be  upon  serial  deter- 
minations throughout  the  course  of  illness  in 
the  patients  studied,  and  upon  correlation  with 
clinical  data.  The  immune  studies  will  be  completed.  Evaluation  of  the 
effects  of  cortisone,  similar  steroids  and  other  chemical  agents  will 
continue.  , 

9B..  PROJECT  DESCRIPTION:  Measurement  of  Albumin  Turnover  and  Total  Exchange- 
able Albumin  in  Patients  with  Cancer,  Control  Patients 
with  Weight  Loss  and  Volunteer  Normal  Subjects. 
(J.  L.  Steinfeld) 

Objectj-ves!  To  determine  whether  the  decreased  serum  albumin  concentration 
found  in  patients  with  cancers  is  due  to  an  increase  in  plasma 
'   '      volume  or  failure  of  albumin  production  to  keep  up  with  albumin 
degradation  or  metabolism  -  and  further  to  ascertain  if  the  metabolism  of 
albumin  in  cancer  patients  proceeds  at  a  normal,  increased  or  decreased 
rate.  ■■:-■■..-' 

Methods  employed ;  Commercially  available  (Abbott  Laboratories)  I  ■'albumin 
is  checked' for  free  radioactivity  and  satisfactory  prepa- 
rations (one  milligram  of  albumin  nitrogen  and  100  .  Iv^ 
are  injected  ihtravenoucly  into  patients  oh  relatively  constant  caloric 
,';'■  and  nitrogen  intake.  Frequent  serial  sampling  of  serum  radioactivity  as 
■  ■•  ^  well  as  daily  determination  of  total  urinary  radioactivity  permits  calcu- 
,  latibn  of  total  exchangeable  albumin  and  albumin  turnover. 

"^l   "f^atient  Material;  Adult  female        11  admissions   308  patient  days 
'■■  "'        '   ■     Adult  male  5  admissions   14.0  patient  days 

Out  patients         3  admissions 

Major  Findings;  The  major  findings  of  this  project  during  the  pt  si  year 

have  been  significantly  decreased  total  exchangeable  albu- 
,,^^....j^^_  ,  .  min  found  associated  with  relatively  normal  plasma  volumes 

"'" and  the  decreased  or  low  normal  turnover  rates  of  albumin  in  oancercus 

patients.  This  is  evidence  against  a  much  increased  albumin  turnover  with 
failure  of  albumin  production  to  keep  pace  with  the  increased  destruction 
..being'  the  factor  responsible  for  the  low  serum  albumin  concentrations 
i^een  clinically. 


'io 


Significance  |to  Cancer  Research;  In  order  to  understand  the  physiological 

relations  between  host  and  neoplasm  it  is 
necessary  to  investigate  the  possible 
Sources  of  tumor  food  supply.  Since  serum  albumin  concentration  is  low  in 
cancerous  patients,  one  might  hypothesize  trapping  of  .serum  albumin  by 
tumor  and  use  of  that  albumin  for  metabolism  or  tumor  growth.  It  is  of 


NCI-702(G) 


PAGE  3 
PROJ^T  REiORT  FORI-l  (Cont'd) 


importance  to  learn  which  nutrients  tumors  can  and  cannot  use  since  such 
understanding  may  contribute  to  the  development  of  effective  anti-tumor 
agents . 

Proposed  Course  of  Pro.iect;  In  collaboration  with  Dr^  Robert  Milch,  the 

tissue  distribution  of  I"'-^ -^albumin  in  tumors  as 
compared  with  normal  tissues  will  be  determined 
through  the  counting  of  J-^^-'-alburain  found  in  various  tissues  obtained  at 
operation  or  at  autopsy.  These  determinations  will  be  corrected  for  the 
II3I  blood  content  of  the  various  tissues. 

Also  the  distribution  and  turnover  of  serum  albumin  and  other  plasma  pro- 
teins in  cancer  patients  will  be  investigated  using  endogenously  labeled 
plasma  proteins  as  with  S^^  or  C-^  amino  acids, 

90.  PROJECT  DESCRIPTION:  Fate  of  Intravenously  Administered  Albumin. 

(J.  L,  Fahey) 

Objectives;  llie  objective  of  this  project  is  to  characterize  Che  dis- 
appearance rate  and  distribution  of  the  products  of  albumin 
loads  administered  intravenously  to  patient  in  various  neo- 
plastic and  nutritional  states. 

Methods  Employed;  Complete  metabolic  balance  techniques  have  been  utilized 
to  follow  nitrogen  and,  in  certain  instances,  calcium  and 
phosphorus  balances  in  selected  pateints.  Intravascular 

distribution  of  albumin  and  other  serum  proteins  have  been  measured  by 

electrophoresis  and  radioactive  iodinated  albumin. 

Patient  Material;  Average  Stay 

No.  Days 

Admissions:  Adult  Male     4  35  days 

Adult  Female    2  25  days 

Ma.ior  Findings;  Intravenously  administered  albumin  is  partially  available 
as  a  source  of  nitrogen  for  tissue  and  tumor  needs. 

Significance  to  Cancer  Research;  Patients  with  neoplasm  develop  low  serum 

albumin  levels.  The  cause  of  this  pheno- 
men  is  obscure.  Determination  of  the  fate 
of  intravenously  administered  albumin  loads  under  several  conditions 
should  help  to  clarify  this  problem.  Nutritional  evaluation  of  intra- 
venously administered  albumin  can  be  undertaken  at  the  same  time. 


Proposed  Course  of  the  Project;  Early  completion  of  analytic  data  is  anti- 
cipated. Correlation  and  evaluation  of 
findings  can  then  be  completed. 


NCI-702  (C)  PAGE  k 

PROJECT  REPORT  FORM  (Cont'd) 

9D.  PROJECT  DESCRIPTION:  Chromatography  of' Serum  Prbte ins.  (J.  L.  Fahey) 

Objectives;  The  objective  of  this  project  is  to  further  characterize  the 
'  ii  serum  proteias  frOiB  normal  and  tumor-bearing  patients  by  ap- 

plication of  new  methods  of  protein  separation.    '  ,'J.Li 

Methods  Et-aployed;  Drs.  Sober  and  Peterson  of  the  Laboratory  of  Biochemis- 
■;  tr-y,  NCI^  have  designed  systems  of  substituted  cellu- 

lose columns  on  which  protein  components  move  differen- 
tially under  pH  and  salt  gradients  in  controlled  temperature  conditions. 
.  .  ,  iif.'  Individual  serum  protein  components  are  identified  by  electrophoretic 
, ,  j  and  spectrophotometric -means.  '        '  '  • 

Major  Findings;  Within  the  past  year  equipmerrt  has  been  assembled'  and  the 
fj^-.tj.  chromatographic  procedure  establishei  in  the  clinical 

area.  Further  work  on  the  method  has  resulted  in  some  re- 
duction in  the  time  and  complexity  of  the  procedure  required  to  carry 
„ .,_  j  out  an  analysis. 

Significance  to  Cancer  Research;  The  alteration  of  body  protein  metabo- 
lism in  neoplastic  states  is  reflected 
by  serum  protein  'changes.  These  have  as 
yet  been  only  grossly  defined  by  other  analytical  means.  Fxorther  identi' 
f ication  and  characterization  of  the  serum  proteins  themselves  Are 
,.  necessary  in  order  that  the  causes  of  protein  abnormality  may  be  studied 

Proposed  Course  of  the  Project;   Further  effort  will  be  devoted  to  develop 

ment  of  a  modified,  more  rapid  adaptation; 
yj...  of  the  present  column  chromatographic 

'.  technic.  When  this  is  achieved  a  clinical  survey  will  be  feasible  in 
,.., which  sera  from  patients  with  a  variety  of  neoplasms  and  in  various 
.  states  of  disease  activity  will  be  tested. 

.9E.  PROJECT  DESCRIPTION:  Evaluation  of  Protein  Metabolism  by  Means  of  Isotop< 

Labelled  Amino  Acid.  (J,  L.  Fa^iey) 

ft  -...-  wol  "^^^^   study  has  been  delayed  because  of  the  basic  de- 

.v.  ;,;  cision  to  change  the  method  of  protein  fractionation 

'•  .  .  from  the  cold-alcohol  m:thocl  of  Cohn  to  the  substi- 

tuted-ceiluiose  column  technic  of  Sober  and  Peterson,  However,  this 
method  requires  further  development  before  it  can  be  efficiently  applied 
to  the  requirements  of  such  a  study.  Work  with  the  method  is  already 
underway  as  noted  in         report. 


NC 1-702 (C)  P^GE  5 

PROJECT  RErORT  F0R14  (Cont'd) 

10,     NCI-702(C) 


SERIAL  NO. 


A,   B.   C.   D  and  E 


BUDGET  ACTIVITY: 

RESEARCH  ^         AlI'iINISTI^iTION  O 

REVIEW  a  APPROVAL  O         TECHNICAL  ASSISTANCE  [J 


GOOPERi^TING  UNITS  OF  THE  i^UBLIC  HEALTH  SERVICE,   OR  OTHER  ORGANIZATIONS,   PRO- 
VIDING FUNDS,   FACILITIES,   OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or 

1957. 

A,  The  Acute  Leukemia,  Solid  Tumor  and  Surgical  Services  of  the  NCI  have 
made  available  blood  samples  from  patients  admitted  to  study  on  those 
services  and  have  collaborated  generously  in  providing  the  clinical  in- 
formation necessary  for  adequate  interpretation  of  tlrie  serum  protein 
data, 

B,  None, 

C,  None. 

D,  Drs.  Sober  and  Peterson  of  the  Physical-chemistry  Section  of  the  Labora- 
tory of  Biochemistry,  MCI,  have  been  responsible  for  the  development  of 
this  chromatographic  method  and  are  actively  collaborating  in  the  appli- 
cation of  this  technic  to  clinical  studies, 

.     E,     None. 

13.     ^ 

IF  THIS  PROJECT  RESEI-iBLES,   CO^iPLEilENTS  OR  PARALLELS  RESEARCH  DONE  ELSEVfflERE 
IN  THE  PUBLIC  HMLTH  SERVICE   (WITHOUT  INTERCIi'iNGE  OF  PEn.^ONNEL,   FACILITIES  OR 
FUNDS),   IDENHF?  SUCH  RESEARCH: 

A.  None. 

B.  None, 

C .  None , 

D.  None. 

E.  None. 

No  entires  for  items  14,  15  and  16. 


1-.   N.C.I. 
INS  TI  TOTE 


PROJECT  REPORT  FORM 
2,  General  Medicine 


PAGE  I 


LABORATORY  OR  BRANCH 


3.  Metabolism 


SECTION  OR  SERVICE 


h. ^.  NCI  703(c) 

LOCATION    (IF  OTHER  THAN  BETHESDA)  SERIAL  NO. 


6,  Estimation  of  Red  Cell  Survival  In  Patients  With  Advanced  Neoplastic 

Disease  Before  and  After   Treatment. ■• 

PROJECT  TITLE 


7.   J.  L.  Steinfeld 

PRINCIPAL  INVESTIGATMTsT 


8.  None 


OIKFil  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objectives;   To  determine  if  measurement  of  red  cell  life  would  be  a 

useful  parameter  for  ascertaining  the  value  of  a  proposed 
therapy  for  cancer  by  first  correlating  such  studies  of 
red  cell  life  span  vjith  approved  methods  for  clinical  and 
laboratory  evaluation  of  cancer  therapies. 


Methods  employed: 


100  micro-curies  of  Sodium  Ghromate^l  in  8cc  of  ACD 
solution  are  incubated  with  50cc  of  patient's  blood 
and  after  washing  throe  times  with  saline  to  remove 
free  radioactivity,  the  labelled  red  cells  are 
reinjected  into  the  patient  from  whom  serial  samples 
of  blood  are  taken,  radioactivity  measured  and  red 
cell  life  span  is  estimated. 


Patient  Material;  Admissions 


Adult  male 
Adult  Female 
OPD  Visits 


3 

1 

20 


90  pt-rdays 
30  pt-days 


Major  Findings:  Twenty  studies  in  eighteen  patients  have  shown  that  red 
cell  life  span  is  variably  decreased  in  cancer  patients. 
In  only  two  instances  was  the  patients'  status  stable 
enough  to  perroit  two  full  studies  of  one  month  each 
to  be  carried  out  before  and  after  therapy.  No 
conclusions  may  be  drawn  at  this  time  as  to  value  of 
this  technique  for  judging  efficacy  of  cancer  therapy. 

In  a  collaborative  effort  with  Drs,  H,  Chaplin  and 
P.  Schmidt  of  the  Clinical  Center  Blood  Bank,  red 
cell  survival  of  blood  stored  and  frozen  at  -1;5°C  for  vary- 
ing periods  of  tirae  xi^as  found  to  be  in  the  normal  range. 


NCI-703(C)  PAGE  2 

PROJECT  REPORT  FORM  (Cont'd) 

Significance  to  Cancer  Research;  Objective  measures  of  judging  the 

— . ^ destructive  effects  of  cancer  therapy 

on  cancers  and  beneficial  effects'  on 
the  host  are  urgently  required  at  the  present  time.  Such  techniques 
as  this  -  although  involved  and  time  consuming  -  would  be  of  value  in 
select  cases  if  correlation  were  high  between  red  cell  life  span 
and  clinical  status.. 

Proposed  Course  of  Project;   To  continue  as  outlined  above  and  to  pursue 

~  further  with  Drs,  V.  Price  and  R,  Greenfield 

studies  of  the  tissue  distribution  of 
labeled  red  cell  components  at  surgery  and  at  autopsy,  comparing 
normal  and  cancerous  tissues  after  correction  for  the  blood  content 
of  the  tissues • 


NCI-703(C) 


10,  NCI-703(C) 

SERIAL  NO, 


PAGE  3 


PROJECT  REPORT  FORM  (Cont'd) 


11. 


BUDGET  ACnVITY": 

RESEARCH  fTT 

REVIEW  &  APPROVAL  /         / 


ADMINIGIRAHON  /         / 

TECmaCAL  ASSISTAKCE/         / 


12,  None 


COOPERATING  UNITS  OF  IHE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSOmiEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957. 


13.     None 


IF  THIS  PROJECT  RESEI-fflLES,   COl-iPLEMENTS,   OR  PARALLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE    (WIIHOUT  INIERCHANGE  OF 
PERSONNEL,   FACILITIES  OR  FUNDS),   IDENTIFY  SUCH  RKSEARCH: 


No  entries  for  items  lU,  l5  and  l6. 


PAGE  I 
PROJECT  lilCPOKT  FOm 

L,  Nj^.Ij 2.  General  Medicine  Branch : .-__ 

INSTITUTE  "  LABOMTOkY  OK  BMNCH 

5. A. , 5.   N0I::204Lia 

SECTION  OK  SERVICE  LOCATION   (IF  OTHER  THAN  BETHESDa)  SiiiRIAL  WO. 

The  Influence  of  Delta  Amino  Levulinic  Acid  and  Some  of  Iti;  Analogues  on 

j.  Tumor  Growth, ^ 

PROJECT  TITLE 


D.  P.  Tschudy 


PRINCIPAL  INVESTIGAT(m(S) 


OTHER  IN^/ESTIGATORS 


PROJECT  DESCRIl-TION 


Objectives;  Since  delta  ajnino  levulinic  acid  is  an  intermediate  in  porphyrin 
synthesis  and  one  carbon  transfers  we  are  attemptinp  to  demon- 
strate a  growth  promoting  action  of  this  compound  in  tumors, 
along  with  direct  demonGtratlon  in  tumora  of  the  enzyme  which  converts  delta 
amino  levulinic  acid  to  porphobilinogen.  We  are  attemptinf^  to  inhibit  tumor 
growth  by  means  of  chemical  analogues  of  this  compound. 

Methods  employed;  Organic  synthe.'Jis,  tumor  transplantation,  tumor  growth  rate 
methods,  paper  chromatograptiy,  extraction  of  enzymes  from 
turaors,  piianuacological  methods  and  histologic  methods. 

Patient  MatqriaJ. : 

Ma.ior  Findings;   Delta  amino  levulinic  acid  has  been  synthesized  by  a  now 
method,  A  number  of  chemical  analogues  of  this  compound 
have  been  prop'. red,  some  of  which  had  never  previously  been 
synthesized.  Pharmacologic  and  enzyme  aludiojj  in  animals  are  not  completed. 

Significance  to  Cancer  Reaearch;   If  inhibition  of  "one  carbon  fragment"  utili- 

zabion  can  be  attaine(J  by  this  mo't  lod  along 
with  partial  inhibition  of  porphyrin  syn- 
thesis, antitumor  activity  may  result.   S/nergistic  increase  of  anti-tumor 
activity  of  folic  acid  antagonists  may  also  be  produced. 

Proposed  Course  of  Project;   If  we  are  successful  in  demonstrating  the  enzyme 

synthesizing  pori)hobilinogen,  we  may  attempt  to 
demonrrtrate  its  intracellular  localization. 

Growth  inhibitory  activity  of  any  of  the  compounds  will  be  studied  in  other 

systems  including  human  tumors. 


PAGE  2 
PROJECT  REPORT  FORM  (Cont'd) 


10. 

NCI-70A(C) 

SSitlAL  WO. 

11. 

BUDGET  ACTIVITY 

■    -RESEARCH 

HI 

ADMIillSTRiTION 

D 

REVIEW  &  AI^ 

r'ROVAL 

D 

TECHNICAL  AS3ISTAWCE 

D 

12. 

13. 


COOPERATING  UNITS  OF  THE  PUBLIC  HE/iLTH  SERVICE,   OR  OTHER  OHSAWIZATIONS,   PRO- 
VIDIWG  FUl'IDS,  FACILITIES,   OR  PERSOm\!EL  FOR  THIS  PROJECT  IN  EITHER  1956  or 
1957. 


IF  THIS  PROJECT  RESEMBLES,   COMPLEMENTS,   OR  PARALLELS  RESEi^RCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HE^lLTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES      , 
OR  FUNDS),    IDENTIFY  SUCH  RESEARCH: 


No  entries  for  items  14.,  15  and  16» 


PAGE  I 
PROJECT  REPORT  FORM 
N.C.I.  2.   General  Medicine  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

3.  ^. 5-  NCI-706(c) 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

6.  study  of  the  Value  of  a  Skin  Test  Using  Polysaccharide  in  Cancer  and 

PROJECT  TITLE  Non-malignant  Diseases 

7.  John  H.  Tuohy  and  Murray  Shear 

PRINCIPAL  INVESTIGATOR (S) 


OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION: 

Objectives:  To  examine  the  sensitivity  and  specificity  of  the  test  for 

human  malignancy  in  which  a  small  measured  amount  of  bacterial 
polysaccharide  is  injected  intradermally  into  the  skin  of 
humans  with  and  without  malignant  disease. 

Methods  Employed:  As  indicated  above. 

Patient  Material :  None . 

Major  Findings:   This  study  has  been  in  abeyance  during  the  reporting  year  due 
to  the  requirements  of  the  Solid  Tumor  Chemotherapy  Program. 

Significance  to  Cancer  Research:   Implicit  in  the  objectives  listed  above. 

Proposed  Co\irse  of  Project:  This  study  may  be  abandoned  in  view  of  the 

increased  activity  in  the  Solid  Tumor  Chemo- 
therapy Program. 


PAGE  2 
PROCfECT  REPORT  FORM  (cont'd) 


10.   NCl-7o6(o; 

SERIAL  NO. 


BUDGET  ACTIVITY: 


RESEARCH         JTJ  ADMINISTRATION       /~7 

REVIEW  &  APPROVAL  /  7       '^       ^L  ASSISTANCE  /  / 


12 . 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  195 6  or  195' 


13- 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEyVRCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEMS  1*4,  15  &  16 


PAGE  I 
PROJECT  REPORT  FORM  ^ 

fj^Q_j^  2.     Gctif.Tal  Modiclru.'  Branch 


INSTITUTE  '  LABORATORY  OR  BRi^NCH 

k.       ' ___^  !3.NCI-707(C) 

SECTION  OR  SERVICE  LOCATION  (IP  OTHER  THAN  BETHESDA)   SERIAL  NO. 


Evaluation  of  Blood  and  Plasma  Water  Content  in  Patients  with  Malignant 
PROJECT  TITLE  '  Neoplasms . 


John  H.  Tuohy 

PRINCIPAL  INVESTIGATOR (S: 


OTHER  INVESTIGATOR:; 


PROJECT  DESCRIPTION, 

Objectives:  To  determine  the  relative  difference,  if  any,  between  the 
moisture  content  of  the  blood  of  patients  with  malignant 
disease,  patients  with  other  forms  of  acute  and  chronlo  illness, 
and  normal  individuals. 

Methods  Employed:   The  chemical  determination  of  moisture  content  of  whole 
blood,  serum,  plasma,  and  tissue  by  the  use  of  the 
Carl  Fischer  rea^orit. 

Patient  Material:   None . 

Major  Findings:  This  project  has  been  temporarily  abandoned  and  was  not 
active  during  the  reported  year. 

Significance  to  Cancer  Research:   Carried  to  completion  with  a  positive  rftsult, 

t'hls  would  furnish  a  diagnostic  test  for 
malignant  disease . 

Proposed  Course  of  Project:   Indicated  above. 


10.  NCI-707(C) 
SERIAL  NO. 


PAGE  2 


PRtJECT  REPORT  FORM  (cont'd) 


11. 


BUDGET  ACTIVITY: 

RESEARCH 

REVIEW  &  APPROVAL 


fTJ     ADMINISTRATION       /  7 
I     7   TECHNICAL  ASSISTANCE  /  / 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  .    ITEMS  \h,    15  &  i6 


PAGE  I 
PROJECT  REPORT  FORM 

1.   N.C.I. 2.  General  Medicine  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Dermatology h. '>.  NCI-708(C) 

SECTION  OR  SERVICE    LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

6.   Dermatologic  Research 

PROJECT  TITLE 


7 .  E»  J.  Van  Scott,  Richard  Reinertson,  C.  B.  McCall;  Jack  Walte,  Ross  McCardle 
PRINCIPAL  INVESTIGATORS 


OTHER  INVESTIGATORS 


A.  PROJECT  DESCRIPTION  -  Statistical  Incidence  of  Types  of  Pigmented  Lesions 

on  the  Palms  and  Soles  -  E.  J.  Van  Scott;, 
Richard  Reinertson,  and  C.  B  McCall 

Objectives:  To  determine  the  incidence  of  pigmented  lesions  on  the  palms 

and  soles  J  and  to  determine  histological  types  of  those  occur- 
ring on  these  sites. 

Methods  Employed:   Examination  of  palms  and  soles,  of  randomly  selected  group 
of  individuals,  for  presence  of  pigmented  lesions. 
Biopsy  removal  of  random  group  of  such  lesions. 

Patient  Material:   Random  selection  from  patients  in  Clinical  Center^  em- 
ployees, juvenile  home. 

Major  Findings:  Approximately  2%   of  over  7OO  individuals  examined  have 

been  positive  for  pigmented  lesions  on  the  palms  or  soles. 
Approximately  ^5  lesions  have  been  excised  from  40 

individuals  and  have  been  exEunined  histologically.  Over  50^  of  these 

lesions  have  been  found  to  be  "Junction  nevi." 

Significance  to  Cancer  Research:  Si'ice  Pack  reported  that  the  incidence  of 

nevi  on  the  palms  and  soles  is  less  than 
l^j  and  since  k   to  12^  of  melanomas  are  sail 
to  occur  on  the  foot,  an  accepted  ciorrent  therapeutic  procedure  is  to 
prophylactically  excise  any  pigmented  lesion  from  the  palms  and  soles 
on  the  rationalization  that  such  lesions  are  unduly  prone  to  develop 
into  melanomas. 

The  data  of  this  project  questions  the  validity  of  such  reasoning 
and  opens  for  re -evaluation  the  role  of  the  ,;-inction  nevus  in  the 
development  of  melanoma. 


NCI-708(C)  ,   ,  PAGE  2 

SERIAL  NO.  ' 

PROJECT  REPORT  FORM  (CONT'd) 

Proposed  Course  of  Project:   This  project  is  completed,  pending  the  final 

compilation  of  the  data  and  its  review  by- 
statistician. 

9. '\B. PROJECT  DESCRIPTION  -  Relationship  of  Junctional  Activity  in  Pigmented 

Nevi  to  Development  of  Melanomata  -  E.  J.  Van  Scott 
and  Jack  Waite 

Objectives;  To  determine  incidence  of  activity,  junctional  or  otherwise,  of 
nevi  in  patients  with  and  without  melanoma. 

Methods  Employed:  Nevi  on  the  backs  of  three  groups  of  patients  will  be 
nxjmbered  by  a  constant  method.  The  three  groups  of 
patients  will  be: 

a)  Normals  (patients  without  melanoma). 

b)  Patients  with  metastatic  melanoma. 

c)  Patients  with  primary  operable  melanoma. 

Randomly  selected  nevi  will  be  excised  from  the  three  groups  of 
patients  for  histological  examination. 

Patient  Material:  Patients  hospitalized  in  the  Clinical  Center  or  followed 
in  Admission  and  Follow-up  Clinic. 

Major  Findings:   Nevi  from  three  individuals  have  been  excised  to  date.  No 
findings  to  report  at  this  time. 

Significance  to  Cancer  Research:  This  project  attempts  to  establish  whether 

or  not  there  is  some  generalized  bodily 
influence  which  promotes  malignant  change  in 
"benign  nevi"  found  on  the  skin  of  patients  with  melanoma. 

Proposed  Course  of  Project:   Continuation  as  outlined  above. 

9.  C.  PROJECT  DESCRIPTION  -  Histologic  and  Histochemical  Anatomy  of  Certain  Skin 

Lesions,  as  Correlated  with  Gross  Anatomy  -  E.  J. 
Van  Scott  and  Ross  McCardle 

O'ff-jectives:  To  establish  the  gross  and  microscopic  anatomical  changes  which 
occur  in  the  skin  and  its  appendages  under  normal  and  pathologi- 
cal conditions. 

Methods  Employed:   Punch  biopsy  specimens  are  serially  sectioned  and  stained 

with  different  stains .  Sections  are  examined  microscopical, 
ly.  Balsa  wood  models  of  cutaneous  structures  are  made 
from  projections  throvigh  a  microscope  of  these  structures;  resultant 
models  are  72  times  normal  size. 


PAGE  3 
fCI-7Q8(c) 

;erial  no. 

PROJECT  REPORT  FORM  (cont'd) 

Patient  Material:  Skin  specimens  obtained  from  patients  in  Clinical  Center; 
Employee  Health  Service. 

Major  Findings :  Reco  istruction  models  thus  far  made: 

Condition  No.  of  models 

Normal  hair  follicle  of  back  .  2 
Follicle' involved  with  acne  (back)  -  ,.  ■^.rh 
Follicle  involved  with  basal  cell 

carcinoma  (back)  1 

Normal  follicle  of  beard  2 

Beard  involved  with  alopecia  areata  2  , 

Follicle  of  normal  scalp  1 
Follicle  involved  with  alopecia 

areata  (scalp)  2 
Follicle  involved  with  early  male 

baldness  ...  2  ■   , 


Total  number  of  models  ,l6 

'        Corirelati'bn  of  inicro-scopic  changes  vith  gross  changes  CQncurrently 
-  being- mad^. 

Significance  to  Cancer  Research:  ■  This'  study  relates  to: 

1.  '   Elaboration  of  anatomical  changes  taking  piace  in.  the  skin  at 

the'  time  of  development  of  skin,  cajacer.   .  ; 

2.  The  study  of  controlled  growth  and  .quiescence  of  the.  germinative 
cells'  in  the  hair  bulb,i 

3.  The  effects  of  the  hormonal  milieu  on  the  growth . of  hair  and 
sebaceous  glands .'  ■  ' 

k.     Suggests  the  possibility  of  the  study  of  hair  growth  in  tissue 
cxilture  as  a  means  of  studying  certain  condition&  which  may 
affect  cellular  mitosis. 

Proposed  Course  of  Project;  Continuation  as  outlined  above. 

).     D.  PROJECT  DESCRIPTION  -  Amino  Acid  Content  and  Enzymatic  Activity  of  Skin 

and  Mucous  Membranes  -  E.  J.  Van  Scott  and 
Richard  Re inert son: 

Objectives:   Quantitative  determination  of  certain  amino  acids  and  arglnase 
activity  in  the  various  layers  of  normal  and  pachologic  skin. 

Methods  Buployed:   Tissue  to  be  analyzed  includes  epidermis  and  corium^ 

'^'      mucous  membrane  of  upper  esopha-gus  and  vag"Lna^  and  liver, 
obtained  from  autopsy  material;  also  includes  scales  from 
patients  with  exfoliating  skin  diseases,  malignant  tissues  from  skin 
of' patients  with  either  primary  or  metastatic  disease;  hair,  nails. 


PAGE  h 


NCI-708(C) 
SERIAL  NO. 

PROJECT  REPORT  FORM  (cont'd) 

Chemical  methods; 

1.  Arginase:  Method  of  Krebs  and  Henseleit,  modified. 

2.  Cystine:   Okuda  titration  of  hydrolyzed  tissue. 

3"  Arginine;   Sakaguchi  method,  as  improved  by  Sakaguchi. 

k.     Other  amino  acids:   Paper  chromatography  of  hydro- 
lyzed tissue,  elution  of  color  from  spots  on  paper  and 
quantitative  estimation  by  colorimeter. 

Patient  Material: 

Major  Findings:   Determinations  of  arginase  activity,  arginine,  cystine,  and 
free  siilfhydryl  have  been  made  on  several  specimens  of 
psoriatic  scales,  plantar  calluses,  epidermis,  and  scales 
from  exfoliative  dermatitides.  The  amount  of  arginine  in  psoriatic 
scales  has  been  found  to  be  significantly  less  than  that  found  in 
plantar  calluses. 

Method  for  analyses  by  paper  chromatography  is  being  refined. 

■  Significance  to  Cancer  Research:  The  amino;,  aeid  pattern  of  total  whole  skin 

of  animals  has  been  reporte.d.  to^^hange  when 
the  skin  undergoes  malignant  chaxige.  The 
amino  acid  content  of  the  various  layers  of  the  skin  is  not  known 
and  particularly  is  it  unkno\m  for  human  skin.  This  project  attempts 
to  partially  define  the  values  for  various  chemical  constituents  in 
discrete  layers  of  normal  and  abnormal  skin.   Changes  in  the 
chemical  composition  of  cancerous  skin  may  be  accounted  for  by  volume 
changes  of  certain  layers  of  the  skin  rather  than  elementary  changes 
brought  about  by  the  malignant,  growth  per  se.   On  the  other  hand,  the 
actual  chemical  composition  of  specific  cellular  elements,  or  layers, 
may  change  under  pathological  conditions.  This  project  attempts 
to  clarify  such  points. 

Proposed  Course  of  Project:   Continuation  as  above. 

9.  E.  PROJECT  DESCRIPTION  -  Chemotherapy  of  Skin  Cancer  and  Skin  Diseases  by 

Iontophoresis  -  E.  J.  Van  Scott 

Objectives:   Evaluation  of  local  effect  of  drugs  in  skin  tumors  and  skin 
diseases  of  substances  introduced  by  electric  current. 

Methods  Employed;   Introduction  of  substances  into  skin  by  iontophoresis 
apparatus;   observations  of  gross  changes. 

Patient  Material:   Incidental  procedure  on  patients  hospitalized  for  other 
major  purposes. 

Major  Find.ings:   No  demonstrable  effect  has  been  shown  to  occur  in  normal 
skin,  psoriatic  skin^  and  in  tumors  of  mycosis  fungoides 


PAGE  5 

JCI-706(C) 
5ERIAL  NO. 

PROJECT  REPORT  FORM  (cont'd) 

following  the  iontophoretic  introduction  of  thallium,  mangamese, 
cobalt , methotrexate . 

Significance  to  Cancer  Research:  Project  attempted  to  illicit  any  direct 

effect  of  substances  introduced  locally  into  f 
skin  tumors. 

Proposed  Course  of  Project;  Discontinuation  as  a  discrete  project. 


PAGE  6 
PROJECT  REPORT  FORM  (cont'd) 


10 .   NCI-708(C) 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY 

RESEARCH  JYJ  ADMINISTRATION        /  7 

REVIEW  &  APPROVAL  /~7         TECHNICAL  ASSISTANCE  /  J 


12.   None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  1956  or 
1957. 


13. 

IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCIiANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEMS  1^1,  15  &  l6 


PAGE  I 
PROJECT  REPORT  FORM 
N.C.I.  ■2     Genera!  Medicine  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

Acrtc  Leukemia ^  • 5  ■  NCI- 709(C) 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

The  Chemoi-herapy  of  Acute  Leukemia 

'PRO.JECT  TITLE 


Emil.  Frei  ,  III 

PRINCIPAL  INVESTIGATOR fgy 


E.  J.  Freirelch,  James  Stengle,  Richard  T-  SUver,  G-  Lennard  QoJ.d 
OTHER  INVESTIGATORS 


PRO.TECT  DESCRIPTION- 

Ob.'jectives  ■   To  study  the  effects  of  chemical  com-DOunis  on  the  course  of 
acute  'eukemia. 

Methods  employed-   I.  All  patients  i-eferred  to  the  Clinical  Center  with 

the  diagnosis  of  acute  Leukemia  are  admitted  to  the 
Gti'.dy.   (for  participating  hospital  units  see  #12). 

2.  Objective  criteria  I'or  the  e\/-aLua';ion  of  disease 
activity  have  teen  developed  and  applied 

3-  One  of  the  following  methods  xor  evaluate n-^,  drug 
effect  wl 11  be  app ; ied  depcniini  upon  the  degree 
pf  kiowa  antitumor  ac.;ivlty 

a)  Pi '.ot  studies  in  a  few  patients  with  varying 
dosages  for  dru-^s  of  potential  antitumor  activity. 

b)  Sequential  aria  lysis  i  e..  administration  of  the 
drui  to  the  number  of  patients  necessary  to 
establish  with  confideiK-e  limits  thai,  it  is_,  or 

■is  not,  influencing  the  course  oJ'  the  disease  and. 

c)  comparative  .studies  of  dru-^'s  of  proven  human 
antiiiojnor  activity  in  an  effort  to  detertiii-ie  whether 
3i,:,njf leant  dif.t'erenceg  e/cLst 

h.      Combinations  and  varyi.i/j,  dosa/^e  schedules  will  be 
stu'iie'-l  when   laboratory  evidence  or  th'ioretical 
factors   sugjesi.  possible   incrf.-ased  antitumor  activity. 


WCI-7Q9(C)  PAGE  2 

SERIAL  NO. 

PROJECT  REPORT  FORM  (cont'd) 

Major  Findings'   1   A  preliminary  review  of  our  data  indicates  that 

.  combined  Metho .rexate  and  6  Mercaptopurine  therapy  .  . 

is  not  superior  co  either  one  above. 
2.   The  evidence  as  to  whether  the  administration  of 
Methotrexate  at  Less  frequenc  intervals  alters  the 
therapeutic  toxic  ratio  is  inconclusive. 
3»  Pilot  studies  using  MetHoorexate  at  intervals  frequent 
enough  to  mainoain  constant  blood  levels  have  revealed 
enhanced  therapeutic  effect 

Significance  to  Cancer  Research-  Trial  in  humans  of  potentially  effective 

chemical  compounds  is  of  obvious  importance. 
Quantitative  comparative  studies  designed  to  detect 
s  i.ight  but  statistically  significant  differences  are 
essential  to  direction  of  drug; development  into 
profitable  areas. 

Proposed.; Course  of  Study  The  most  intense  activity  will  obviously  focus 

on  the  drugs  that  show  major  activity  in  the 
animal  screen.   Leads  that  develop  as  a  result 
of  studies  by  the  pharmaco  logy  group  such  as  dosage 
schedules  routes  of  administration,  etc.  will  be 
exploited. 

In  an  effort  '.o  increase  the  number  of  clinical  trials 
other  hospital  units  have  been  inc  Luied  in  the  study 
and  it  is  hoped  that  more  will  be  added. 


PAGE  3 

PROJECT  REPORT  FORM  (cont'd) 

0  NCI-709(C) 
SERIAL  NO. 

BUDGET  activity! 

RESEARCH  ^TJ         ADMINISTRATION       '      ' 

REVIEW  &  APPROVAL   '   ^    TECHNICAL  ASSISTANCE  f      ' 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  '-956  or  95^^ 

Department  of  Pediatrics 
University  of  Buffalo 
Buffalo^  N  Y, 

Roswell  Park  M  morial  Institute 
Buffalo  3,  New  York 


3.   None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCRANGE  OF  PERSONNEL,  FACILITIES 
OR  FWros),  IDELITIFY  SUCH  RESEARCH- 


NO  ENTRIES  FOR  ITEMS  L^l  '5   &  l6 


FAOJ.0  I 


H;ai,,CT  U^lJ'OiiT  FOluM 


1 .       NC.J 2,       Uumr.Ml  Medici luHrnncli 

1  N;'T.LTUTK  Ly\iK)o'A'ri)  iY  OK   Blu\llCV 


3.      Non 


Jone  ]|.        None  ''.Nt;  1-71.0(1;) 

-,CT.10N  o;<  Si'^mciv I'A..  .Mi'U  (.11-'  (':Tnp;i'!  Tiiy^i^:  rn';'ri)i':;">l")A)         ;.>.,;;:Ia1,  Wo. 

ltit:4)rjti.  uLlai'v    ^iota,boliSlll  I11   M.md 

"OTfFimT! — 


!■  :iN(;iPA.i.,  iNV;';;;T;i:CATo;;,(t:i) 


1.      Alton    lM.'i;;lec,    MJI.,    n(ni.n(.l    Tseluulv,    M.1I. 


OT!M,;'  .ilvv.';oTK;Atw,;;; 
rivui  i',(.;t  n  ;  c.i.MrTniM 

Project:      .Inheriiu'di  .'vry  Mel.Mluil, i  :iiii    In  l''i.'in 

Ob.juctives;      At.  v.'irieu;!    i.j  111   ;:   111,  nili'.'vr   of   Uw   ncvncl  1  iii  I'.'i  1.   In'/incii.  ;;   col.  I  .'tborato 
wi  111   IIk'  cl:lnical  {.roup  .i.n  'Ti  attempt  to  m.'ike  pronliie  biochem- 
\r.:\\    litudion  on  pationts  with  known  deJ'ecto  in  Jnturmed;l,'ir" 
motaboliriin,   or  to  uxploro  pospible  diriturbnnno;i   in   1  nt.rnn. 'di /M-y 
mi.;tai.)olism  in  pftientn  w' th  nuopl.-uitic  diwu.'uie, 

fi  tl  od;;:      Inxr]]  pati^Jitti   .'ire   ndridited  io   tJi.     (.'iMi' t.m  1    iiiiMi.lcin''   nurvlcie   ru'id 
th  )  details  of  the  prof.rnnis,  ond  thoir  J.ntopration  with  probluma 
of  patiunt  caro,   ary  worked  out  by  consultation  between  iiKiiribura 
of  the  f-enoral  medicine  staff,  and  th>.i  nonclinicfil  inventit'.ntor. 
The  apocifio  1; 'bora tor \'  luetbodfi  utiliiBed  ni-'e  r/ither  variable, 
nnd  depend  on   tJw    f.yp.>  nl'  in-nblom  nndor  atudy.     In  goMeral,   they 
ha.vo  followed    l.li.     p,'i|l,Mni   nl'  ;;imple   i.ixploratlon  o,l'  the  propunce 
or  ab.'ionci'    in   hhiMd    'Mid    nrin      o\'   |in.dii(' I.;:    id'    1  nl.oriiiiid  1  ary 
metaboli  sin. 

i'ati>  nt  Mat.irlal:      {199S  caieiKlar  year)     j\i  timuS  it  in  possible   to  under- 
take- ntudios  in  patients  nlreadv  on  tho  survico.     At  oUier  tinuig 
special  recruitment  ia  noedod.     For  oxamplo:     The  donire  of 
Dr.  Muisttir  to  study  the  antlno  pcid  pattorns  in  phan;ylpyruvic 
olipophrenia  waa  li.d  to  nucli  fjpecial  rnrrui  tiii' nl,. 

Admissionn:                                              Ne.  Averwr.^  ;;tay 
Day.? 

Childr>:n  male  1  2Q 

Child rni    I'Miiiale  1  20 


Page  2 


PROJECT  REPORT  FORM  (Cont'd) 


Major  findings;   These  are  reported. by  the  separate  laboratories  collab- 
orating in  the  studio,?. 

Patients,  knovm  to  have  phenylpyruvic  oligophrenia,  were 
admitted  on  February  28,  19$^   for  studies  to  be  carried  out  by 
members  of  this  Section  in  collaboration  with  Dr,  Samuel  Bessman 
(Associate  Professor  of  Pediatrics,  University  of  Maryland  Medical 
School,  Baltimore)  and  Dr.  Sidney  Udenfriend  (National  Heart  Institut. 

AdmJ.ni  strati  on  of  flutamine-  (7.6niMole  per  kilo)  led  to  a 
dramatic  drop  in  phenylpyruviC'  acid  excretion — to  one-third  to 
onti-fourth  of  th^j  control  level, 

a.  Oral  administration  of  glutamine  and  aspara£,ine  (7.6 
mM/kilo)  does  not  lead  to  dangerously  high  levels  of 
blood  ammonia  nor  to  very  freat  e::cretion  of  ammonia,, 

b.  Blood  levels  of  glutamine,  5  times  the  norms!  level, 
were  maintained  for  about  3  hours. 

c.  Glutamine  administration  does  not  appear  to  have  affected 
the  excretion  of  phenylacctylglutamine,  or  of  creatinine. 

It  may  tentatively  be  su' gestc^d  that  the  strikinf-  decrease  . 
in  phenylpyruvic~"acid  excretion  after  glutamine  administration  is 
due  to  transajnination  of  this  keto  acid  to  phenylalanine. 

Significance  to  CANCER  Research;   This  program  is  of  importance  to  the 

National  Cancer  Institute  program  because  it  represents  an  attempt 
to  utilize  directly  the  newest  developments  in  the  nonclinical 
branches,  in  the  study  of  the  natural  history  of  neoplastic  disease. 
In  thi?  way,  the  theory  and  the  technique  of  biochemistry,  tissue 
culture,  biology  of  groT^^bh  and  radiobiology  can  be  made  available 
to  the  clinical  studies  at  an  early  stare  of  development.  It  is 
hoped  that  from  such  active  collaboration  will  come  new  leads  as 
to  the  pathogenesis  of  neoplasia. 

Proposed  course  of  project;  At  the  present  time  no  specific  projects  are 
being  carried  on,  since  the  ones  proposed  last  joar  have  either 
been  completed  or  developed  into  full  projects  of  their  own. 


PAGi:;    3 


PROJECT  RiiPOilT  FOrn^i   (Cont'd) 


0. 


NCI-710  (C) 


1. 


BUDGi:;!  ACTIVITY: 


RESEARCH  £3^   aDMNI  :.T?UiTION        r~~7 

REVIii;;>[  &  APPROVAL  /~7   TECHNIOii  ASSISTANCiI;   /~7" 


From  time  to  time  patients  iidll  be  studied  in  cooperation  mth 
other  Institutes  at  the  National  Institutes  cf  Health,  but  i-jithout 
2,  specific  allocation  cf  other  research  funds, 

COOPERATING  UNIT3  OF  TKj.  PUBLIC  h.. ,1/111  SERVICE,  OR  OTHER  0.-;rANI2ATI0NS, 
PROVIDING  FUNDS,  FACILIT1.,S,  OR  PER30KK;:.L  FOR  THE  PROJECT  IN  EITHER  19$6  or 

1957 


3,       Does  not  apply 


IF  TEI':.   PXJ.^CT  RE3Ei'IBL..,S,    CriiPL.EEETS,    OR  PARALL.!,LS  RESEilRCL  DONE  ELSE- 
WHERE IN  TH^  PUBLIC  KEl/n;  ECl^VICi:,   (vlTEOUT  INTERCIlAKG.  OF  PERSONNEL, 
F.lC1LITIES  or  funds),   IDx^NTIFY  SUCI:  RESEi.RCH: 


NO  ENTRIES  FOR  ITEMS  ll;,    li;;  &  16 


PAGE  I 


N.C.I. 


PROJECT  REPORT  FORM 


2.  General  Medicine  Branch 


INSTITUTE 


LABORATORY  OR  BRANCH 


3.  Solid  Tmor  Chemotherapy  k. 5-  NCI-71l(C) 

SECTION  OR  SERVICE         LOCATION  (IF  OTHER  THAN  BETHESDA)  .   ^ERIAL  NO. 

S.   Assay  of  Compounds  for  Antitvimor  Effect  by  Injection  into  Skin  Metastases 
PROJECT  TITLE 


7.   T.:C.  0.  BrindLey     

PRINCIPAL  INVESTIGATOR ( S ) 


OTHER  INVESTIGATORS 


PROJECT  DESCRIPTION 


Objectives:  To  develop  a  method  of  assay  of  compounds  for  antitumor  effect  in 
humans  by  the  injection  of  these  materials  into  subcutaneous 
tumor  masses. 

Methods  employed:  Needle  and  syringe  injection  of  lesions;  determination  of 

antitimior  effect  by  direct  caliper  measurement,  comparative 
photographs  and  histological  study.  A  patient  having  a 
remission  of  her  malignant  disease  was  studied  by  endocrinological  survey 
and  a  search  for  immune  response  to  the  tiamor  was  made  by  serological 
tests  and  by  transfusion  of  this  patient's  blood  or  plasma  to  other 
patients  with  similar  malignancy. 


Patient  material- 


Admissions ' 


Adult  males 
Adult  female 


No. 

0 

7 


Average  Stay 
Days 

52 


Outpatient; 


NiJmber  of  patients 
Number  of  visits 


10 
20 


Major  findings-  The  study  so  far  has  shown  that  (l)  extensive  histological 
change  may  be  caused  by  antitumor  agents  before  changes  in 
size  can  be  demonstrated  by  caliper  measurement  or  changes 
in  appearance  occur,  and  (2)  the  quantity  of  antitumor  agent  which  must 
be  injected  locally  into  tumor  masses  in  order  to  cause  regression  in 
size  of  these  masses  is  relatively  large  and  approaches  the  systemically 
tolerated  dose  in  some  cases. 


NCI-711-(C) PAGE  2 

SERIAL  NO. 

PROJECT  REPORT  FORM  (cont'd) 


One  patient  in  the  study  had  a  complete  regression  of  all  metastatic 
lesions. 

Significance  to  Cancer  Research-  For  many  agents  the- effects  on  biological 

systems  and  the  antitimor  effects  in 
animals  are  not  closely  correlated  with  anti- 
tumor  effects  in  the  human.  The  number  of  agents  to  be  tested  is  large. 
So  a  method  whereby  agents  can  be  safely  tested  within  a  short  period 
of  time  in  humans  woyld  be  useful.   The  possibility  that  the  injection 
of  subcutaneous  metastases  could  be  developed  into  such  a  method  should 
be  explored. 

Proposed  course  of  the  project:  For  the  past  several  months  suitable  patients 

for  this  study  have  not  been  available.   The 
project  has  been  temporarily  stopped  for  this 
reason.  When  such  patients  become  available  in  quantity,  it  is  planned 
that  the  project  will  be  resumed.  The  patient  having  the  complete  re- 
mission of  her  disease  will  continue  to  be  studied  for  possible  causes 
of  this  remission. 


10.  NCI-711(C) 
SERIAL  NO. 


PAGE  3 


PROJECT  REPORT  FORM  (cont'd) 


11. 


12. 


BUDGET  ACTIVITY 

RESEARCH  JT]         ADMINISTRATION       /~7 

REVIEV7  &  APPROVAL  /  7     TECHNICAL  ASSISTANCE  /  7 


None 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATION,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  195^  or  195 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  H^LTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),  IDENTIFY  SUCH  RESEARCH; 


Project  700C  was  a  similar  study  as  carried  on  by 
Dr.  R.  D.  Sullivan. 


NO  ENTRIES  FOR  ITEMS  lU,  15  &  l6 


PAGE  I 


PROJECT  REPORT  FORM 


N.C.I. 

insttttte" 


General  Medicine  Branch 


LABORATORY  OR  BRANCH 


i.  Solid  Tumor  Chemotherapy 
SECTION  OR  SERVICE 


5-  NCI-712(C) 

LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 


A.  Controlled  Studies  in  the  Chemotherapy  of  Solid  Tumors 

B.  Preliminary  Studies  of  Chemo therapeutic  Agents  on  Human  Solid  Tumors 
PROJECT  TITLE 


A.  C.  0.  Brindley;  B.  I.  Shnider,  J.  H.  Tuohy 

B.  C.  0.  Brindley,  B.  I.  Shnider^  J.  H.  Tuohy 
PRINCIPAL  INVESTIGATOR(S} 


None 
None 


R  INVESTIGATORS 


A.  PROJECT  DESCRIPTION  ~  Controlled  Studies  in.  the  Chemotherapy  of  Solid 

Tumors 

Objectives:   These  are  two-fold:   (l)  To  test  the  validity  of  the  experimental 
design  of  a  cooperative  randomized  controlled  study  of  chemo- 
therapeutic  agents  on  a  comparative  basis,  and  (2)  to  determine 
the  relative  effectiveness  of  a  test  chenlotherapeutic  agent  against  a 
standard  drug  in  the  treatment  of  a  number  of  selected  solid  tumors  in  man. 

Methods  employed:   (l)  Determination  of  experimental  design  including  the 

format  of  the  cooperative  study. 

(2)  Selectioa  of  patients. 

(3)  Selection  of  test  and  standard  drugs  and  their  admini- 
stration according  to  an  established  protocol. 

(k)   Antitumor  effect  is  judged  only  on  the  basis  of 

objective  measurements. 
(5)  Collection  and  synthesis  of  data  from  all  cooperating 

groups. 


Patient  material:  1955  (April-November) 


Admissions: 


No. 

Adult  male   20 
Adult  female  31 

Children,  male    1 
Children,  female  0 


Average  stay 
days 

90   . 
90 


PAf  E  2 
NCI-712  (e)  '■ 

SERIAL  NO.  *:•  -   '  •  . 

PROJECT  REPORT  FORM  (cont'd) 

Outpatients: 

In  Admissions  &  Follow-up  Department 
Patients      77 
Visits      122    ,  „      ...    V  ..,;,  V 

In  home  or  outside  hospital 
Patients      20 
Visits        20 

Major  findings:  The  study  is  now  in  its  formative  stage.  At  present 
Triethylene  Thiophosphoramide  as  the  test  drug  and 
HN2  as  the  standard  are  being  compared  according  to  the 

design  indicated  above.   Twenty-nine- (29)  patients  have  been,  or  are  now 

in  the  study.  :  •  :  .  ■,.■. 

Significance  to  Cancer  Research:  By  matching  patients  and  the  drugs  being 

compared  in  a  completely  randomized  manner , 
and  by  basing  anti-tumor  effect  solely  on 
objective  criteria,  this  study  represents  an  entirely  new  departure  in  the 
field  of  cancer  chemotherapy.   It  is  the  first  major  effort  to  transfer  the 
experience  in  experimental  therapeutics  in  other  fields  to  study  of  chemo- 
therapeutic  agents  in  malignancy.  The  cooperative  endeavor  of  which  this 
project  is  a  part  has  achieved  acceptance  by  the  Clinical  Panel  of  the 
National  Cancer  Chemotherapy  Center.   It  is  being  suggested  as  a  pattern  of 
study  for  other  cooperative  groups.  There  is  hope  that  in  this  way  drugs 
Showing  promise  in  more  preliminary  studies  can  be  critically  and  rapidly 
evaluated  in  large  numbers  of  human  cases . 

Proposed  course  of  project:   In  the  course  of  the  next  calendar  year,  it  is 

anticipated  that  five  other  institutions  will  join 
in  this  study  following  the  experimental  design 
originated  by  the  Solid  Tumor  Chemotherapy  Group  of  NCI  at  the  Clinical  Center. 

Our  own  activities  here  will  consist  in  increasing  our  experience  and  parti- 
cipation in  this  study  by  expanding  our  out-patient  activities  and  bed 
capacity. 

9.  B.   PROJECT  DESCRIPTION  -  Preliminary  Studies  of  Chemotherapeutic  Agents  on 

Human  Solid  Tumors 

Objectives;  To  test  rapidly  agents  which  have  shown  promise  in  other  studies 
(animal  screening,  pharmacological,  etc.)  for  their  toxicity  and 
antitumor  effect  in  humans . 

Methods  employed:  Agents  will  be  studied  in  selected  patients  using  sequential 
analysis  and  objective  criteria. 


PAGE  3 

rci-7i2(c) 

)ERIAL  NO. 

■  PHOJECT  REPORT  FORM  (cont'd) 

Patient  Material:   (Estimated  -  calendar  year  1956) 

8  patients  per  agent  x  5  agents  =  Uo  patients..,..  _..,,-.'■... , 
Average  days/patient  «  60  days 

Patients  participating  in  "A"  may  also  be  included  in  this  study  upon 
completion  of  their  participation  in  the  Controlled  projestT-'.  ,-.-",-,. 


Major  Findings :   None  ... 

Significance  to  Cancer  Research: .  A  logical  part  of  a  drug  development  program 

and  sequel  to  animal  screening  is  a  rapid 
preliminary  method  for  the  assay  of  an 
agent's  toxicity  and  anti-tumor  activity  in  humans.   It  is  a  necessary  pre- 
liminary to  the  final  critical  evaluation  of  any  chemotherapeutic- material. 

Proposed  course  of  project:   As  agents  are  furnished  from  intramural  and  outside 

sources,  they  will  be  systematically  evaluated  by 
this  technique. 


PA(5E  k 
PROJECT  REPORT  FORM  (cont'd) 


10.   NCI-712(C) 

SERIAL  NO. 


11.  A  &  B 


BUDGET  ACTIVITY: 

RESEARCH  /xj  ADMINISTRATION  /  / 

REVIEW  &  APPROVAL  f^  TECHNICAL  ASSISTANCE      /  J 

12. 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957. 

A.  Cooperative  Groups; 

By  virtue  of  requested  grants  it  is  expected  that  the  following  5 
institutions  will  contribute  to  the  cooperative  study  of  which  this 
project  is  a  part. 

Department  of  Medicine,  University  of  Miami,  Miami,  Florida 
Roswell  Park  Memorial  Institute,  Buffalo,  N.  Y. 
Lemuel  Shattuck  Hospital,  Boston,  Mass. 
Johns  Hopkins  Hospital;  Baltimore,  Md. 

Georgetown  University  Medical  Division,  D.  C.  General  Hospital, 
Washington,  D.  C. 

B .   None 

13.   A  &  B  -  None 

IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OP 
FUNDS),  IDENTIFY  SUOH  RESEAROH: 


16. 


PROJECT  REPORT  FORM  (cont'd) 


Ik.        NCI- 712(C) 
SERL\L  NO. 


15.   A  &  B  - 


PUBLIC       OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR  YEAR 
1955 

A.  Acceptance  by  Clinical  Panel,  National  Cancer  Chemotherapy  Center  and 
its  establishment  as  model  for  other  such  projects. 

B.  None 


PAGE  I 


PROJECT  REPORT  FORM 

1.  N.C.I. ^^  2.  General  Medicine  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  h. 3.NCI-7.13(C) 

SECTION  OR  SERVICE         LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

6.  Energy  Metabolism  and  IV  Fat  Emulsion 

PROJECT  TITLE 

7.  C.  B.  McCall 

PRINCIPAL  INVESTIGATOR (S) 

8.  Pr.  Donald  Watkin 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives :  To  measure  the  effect  of  hyperalimentation  with  intravenous 
fat  emulsion  on  respiratory  quotient  and  energy  expenditure 
in  patients  with  inoperable  cancer. 

Methods  employed:  After  suitable  training  period,  by  an  open  circuit 

method  employing  high  velocity  low  resistance  values  and 
Douglas  bags,  measure  Og  and  COp  in  expired  air  and 
calculate  RQ,  oxygen  consumption,  and  calories/square  meter/hour 
during  three  periods:  ..l)  control,  2)  while  receiving  IV  fat,  and 
3)  follow  up  after  IV  fat  discontinued. 

Patient  materia  L :   Patients  on  Dr.  Watkin 's  metabolic  service  admitted 
for  IV  fat  study. 

Major  findings:   Five  individuals  have  been  studied  to  date.   There  has 
been  no  consistent  pattern  diiring  control  period, 
precluding  any  statement  as  to  the  effect  of  the  extent  of  the 
tumor. 

Two  patients  diiring  the  second  period  (i.e.  receiving  IV  fat) 
showed  an  abnormal  i.y  low  RQ  to  a  level  suggesting  ketone  body 
formation.   There  was  no  consistent  BMR  change.   In  one  case 
fasting  RQ  was  relatively  high  with  subsequent  fall  while  receiving 
IV  fat. 

One  individual  in  follow  up  showed  normal  RQ. 


NCI-7  3(C) .  PAGE  2 

SERIAL  NO.    ■"  ■  ,      , 

PROJECT  REPORT  FORM  (cont'd) 


Significance  to  Cancer  Research:  This  study  may  help  in  part  to  answer 

the  question,  whether  one  can  protect 
a  patient  from  his  cancer  by  supplying 
calories  and  how  is  IV  fat  emulsion  used  by  body. 

In  -general,  this  type  study  might  prove  useful  on  a  number 
of  metabolic  patients  under  a  variety  of  conditions,  such  as, 
type  alimentation,  extent  of  tumor,  and  nutrition. 

Proposed  course  of  project: ■  Finish  the  patients  already  started  and 
'        '      add  others  until  we  have  information  as 
outlined  complete  on  6  patients. 


PAGE  3 
PROJECT  REPORT  FORM  (cont'd) 


10.   NCI-7L3(C) 
SERIAL  WO. 


11, 


BUDGET  ACTIVITY: 


RESEARCH          .pCJ...       ADMINISTRATION       r—j 
REVIEW  &  APPROVAL  _/" J_       TECHNICAL  ASSISTANCE  /    / 


12.  NONE 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957. 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL, 
FACILITIES  OR  FUNDS),  IDENTJFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEMS  ik,    15  &  16 


"vrr 


PAGE  I 


PROJECT  RaORT  FORM 
1.  H.C.I.         2.  General  Medicine  Branch 


INSTITUTE  UBORilTORY  OR  BR-'IWCH 

3.  Leukemia  Section k. 5.NCI-71A  (C) 

SECTION  OR  SERVICE  LOCATION   (IF  OTHER  TliilN  BETIiSSm)        SERIAL  NO. 

6.  Iron  Metabolism.  


PROJECT  TITLE 


7.  James  Stenglc 


PRINCIPAL  IiIVESTIG.iTORCS) 


OTHER  IlWSSTia.TORS 


9.     PROJECT  DESCRIPTION 

Objectives:  a)   Does  the  iron  metabolism  of  the  patient  with  malignant  neo- 
plastic discasj  differ  from  that  of  the  normal? 
b)  Is  the  leukemic  cell  or  solid  tumor  competing  for  iron  at 
the  expense  of  normal  body  needs  and  may  this  be  a  partial 
explanation  for  the  anemias  characteristic  of  these 
diseases? 

Background;   Recent  work  in  the  National  Cancer  Institute 

(Price)  indicates  that  certain  mouse  tumors  have  a 
high  iron  content.  It  is  commonly  noted  in  bone 
marrow  observation  that  stainable  iron  is  lacking  during  an 
acute  exacerbation  of  leukemia  but  may  reappear  during  re- 
mission.  It  has  been  reported  (Simmons  and  Everett)  that 
normal  rat  loulvocytes  incorporate  considerable  amounts  of 
radioactive  iron  administered  orally  or  parente rally. 

Methods  Employed;   In  leukemic  patients  serial  serum  iron  o.nd  leukocyte  iron 
determinations  will  be  made  and  correlated  with  hemoglo- 
bin and  leukocyte  counts. 

In  vitro  studies  of  normal  and  leukemic  leukocyte  uptake 
of  radioactive  iron  will  be  made . 

In  solid  tumor  patients  seruin  iron  and  hemaglobin  values 
will  be  followed  and  surgical  and  autopsy  specimens  analysed  for  iron 
content. 

Some  of  the  above  work  will  be  carried  out  in  cooperation 


Page  3 

PROJECT  RETOltT  FOHM   (cont'd) 


10.   WCI  -  7U   (C) 

SERIAL  NO. 


11. 


BUDGET  ACTIVY: 

RESEARCH                      /X7         ADMINISTRiTIOW                [J 
REVIEW  &  APPROVhL  [J         TECffiTIC>.L  ASSISTANCE  fj 
12. 


G00PE.<ATING  Ui\IIT3  OF  THE  PUBLIC  HE;.LTH  SERVICE,    OR  OTHER  ORGANIZATIONS,   Fi^0- 
VIDING  FUNDS,   FACILITIES,    OR  i- EitSONNEL  FOR  THIS  PROJECT  lij  EITHER  1956  or  1957 


13, 

IF  THIS  PROJECT  RESEI'iBLES,    C0I%^L5i«iErir3,    OR  P-uULLELS  RESE^iRCH  DONE  ELSEiJHSRE 
IN  THE  PUBLIC   HFALTH  SERVICE   ('.v'lTHOUT  INTERCH.1NGE  OF  lEr.SONNEL,   FACILITIES  OR 
FUND) ,    IDENTIFY  SUCH  RE3S.11CH: 

No  entries  for  items,  14,  15,  and  16. 


PAGE  I 
rROJECT  liEt^ORT  FOhJyi 
1-   N.C.I. 2.     General  Medicine  Branch 


INSTITUTE  lABORiTOx{Z  OR  BRANCH 

3.  Leukemia   Section U. 5.  NCI-715(C) 

SECTIOil  OR  SERVICE  ~  LOCATION   (IF  OTHER  TE'iN  BETHESDA)  SERIAL  NO. 

6,  The  Hemorrhagic  Diathesis  Associated  with    -cute  Leukemia. 

PROJECT  TITLE 

7.  E.   J.   Freireich 


PRINCIPAL   liWE 3TIGAT0R( S) 


Emil.  ■Freii::JII 


OTHER  li^IVESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objectives:  a)  To  identify  factors  responsible  for  precipitating  gross 
hemorrhage  in  already  thrombocytopenic  patients, 
b)   To  study  the  effectiveness  of  fresh  blood,  platelet  trans- 
fusion, and  other  blood  products  in  controlling  gross 
hemorrhage  in  leukemic  patients. 

Methods  'Employed;   In  vivo  tests  of  bleeding  tendency,  in  vitro  tests  of 

blood  coagulation,  clinical  observation  and  measurement 
of  gross  bleeding. 

Patient  Material: 

Ma.ior  Findings:  Major  effort  thus  far  has  been  directed  toward  setting  up 
techniques  and  procedures. 

Significance  to  Cancer  Research;  Hemorrhage  ranks  with  infection  as  the  two 

major  causes  of  death  in  acute  leukemia. 
At  oresent  no  effective  prophylaxis  or 
therapy  for  thrombocytopenic  hemorrhage  in  leukemia  is  available.  Many  of 
the  now  chemotherapeutic  agents  for  leukemia  as  well  as  other  tumors  cause 
throm-boponia  and  bleeding.  Any  affective  therapy  of  thrombopenic  bleeding 
could  greatly  prolong  life  of  acute  leukemia  patients  as  well  as  improve 
the  effectiveness  of  chemotherapeutic  agents. 

Proposed  Course  of  Project :   Observations  of  coagulation  mechanism  will  be 

made  periodically  in  acute  leukemia  patients  in 
an  attempt  to  define  any  changes  associated  with 
the  onset  of  clinical  hemorrhage.  Patients  suffering  from  hemorrhage  will 
be  given  fresh  blood  transfusions  and  other  blood  products  and  the  effect  on 
the  clinical  bleiding  and  coagulatio'.:  mechanism  assayed. 


."AGS  2 
PROJECT  REI-OHT  FOWi  (Cont'd) 


10.  iIOI::a^(G}., 
SERIAL  NO. 


11. 

BUDG"'T  ACTIVITY: 


RESSilRCH  Jl^  ADl-ulMI3TlmTI0N  '  /27 

RHVia;  ;^.  ilPi  ROVhL  /S/  TSCHl^IIGAL  aSSISTaIICE     £} 

^^^   Division  of  Biologies 'Gontfol     g^^^^  ^^^^ 


COOPER.iTING  UNITS  OF  TM  PUBLIC  Hm  iLTH  SERVICE,    On  OTHER  ORGANIZATIONa,   PRO- 
VIDING FUNDS,   F-iCILITIES,    OR  PERSONNEL  l^'OR  THIS  PnOJEGT  IN  SITiIER  1956  or  1957 


13. 


None' 


IF  THIS  Pi.OJECT  RESEMBLES,   GOi-lTLi^-iJlNTS,    OR  P.iR-LLELS  ESSE  iRCH  DONE  ELSEWHERE 
IN  THS  PUBLIC  HEiLTH  SERVICE   (l/ITHOUT  lilTERCEii'JGE  OF  rSRSONNSL,   FACILITIES  OR 
FUiMDS) ,    IDENTIFY  SUCH  RESE.\RCH: 


No  entries  for  items  14,  15  and  lo. 


PAGE  I 


PROJ'^CT  REPORT  FORK 
1.   l^u.I. 2.   General  I'ledicine   Branch 


INSTITUTE  LrlBOE-.TORY  OR  BR-iNCH 

3.  Leukemia  Section ^A. 5. NC 1-716  (C' 

SECTIOii  OR  SEiWIGE  LUGiiTION   (IF  OTHER  TliAN  BETHE^M)        SERIAL  NO. 


6.  Nutritional  Aspects  of  the  Therapy  of  Acute  Leukeraia 

PROJEGT  TITLE 


7*   Emil  Frei  III  a:a4,,.Emi'l^F3reir.elch  . 
PRINCIPAL  INVESTIG..T0R(  3) 


OTHER  INVESTIGATORS 


PROJEGT  DESCRIPTION 

Objective;   To  determine  the  effects  of  nutritional  factors  contained  in 
animal  tissues  on  the  course  of  human  acute  leulcemia. 

Methods  Employed;  Various  animal  tissues  are  emulsified,  analyzed  for  chemi- 
cal composition  and  presence  of  infectious  agents,  and  if 
tolerated  by  experimental  animals,  are  administered  to 
to  acute  leukemia  .atients.  The  course  of  tiie  disease  is  followed  by  clini- 
cal observation  of  the  patient  and  various  hematological  deterroinations  on 
blood  and  bone  marrow. 

Patient  Material :  None . 

Major  Findings;  Emulsified  mat-^rial  has  been  prepared  from  hog  tissues  and 
stored  at  -50°G.-  Studies  in  animals  are  in  progress  and  no 
evidence  of  toxicity  has  been  found  to  date. 

Significance  to  Cancer  Research;  The  major  approach  to  the  therapy  of  neo- 
plastic diseases  has  been  and  continues  to 
be  the  use  of  cytotoxic  agents.  As  a  new 
approach,  based  on  the  assumption  that  the  neoplastic  cells  are  capable  of 
maturing  and  returning  to  normal,  nutritional  non-cytotoxic  factors  will  be 
used  in  the  treatment  of  acute  leukemia.  The  discovery  of  a  factor  or  fac- 
tors that  would  promote  the  maturation  of  leukemic  cells  would  have  immense 
significance . 

Proposed  Course  of  Project;   The  pig  tissues  now  available,  if  found  satis- 
factory after  screening  in  animals  will  be  ad- 
ministered to  acute  leukemia  patients  that  have 
been  proven  refractory  to  conventional  forms  of  therapy.  This  will  be 


NCI-716(C) 

PAGE  2 

PROJECT  REPORT  FORM  (Conf) 

administered  by  nasogastric  tube  and  the  course  of  the  disease  carefully 
observed.   If  any  changes  occur,  further  separation  of  component  tissues 
will  be  used  to  identify  the  responsible  factor.   If  no  changes  occur, 
other  tissues  and  other  routes  of  administration  will  be  attempted. 


PAGE  3 


PROJECT  Ri^ORT  VQRA   (Cont'd) 


10.     NCI  -716(C) 

SERIiiL  NO. 


11. 


BUiXJST  ACTIVITY: 


RESilARCH  /x/ 

REVIEW  &  ..IPi^ROV.xL  /J 


ADMINjoTRiTION  /Z/ 

TEOHiaCAL  ASSISTANCE         /^ 


12. 


U.  Z.    Department  of  'griculture  -  heats  Research  Division,  Beltsville,  Md, 

Dr.  gilis  and  Dr.  Hiner  supplied  and  prepared  hog  tissues. 

COOPER-TLfc  Ul'JITS  OF  TI€^  PUBLIC  HS.iLTH  SERVICE,    OR  OTEER'  ORGiUlIZ^iTIOi'lS,"  PRO- 
VIDING FUIIDS,   F.iCILITIES,    OR  PERSONNEL  FOR  THIS  i  ROJEoT  IN  EITH-R  1956  or 
1957 


13 .     None 


IF  THIS  PROJECT  RESE1-':3LES,    G0MPLEi£NT3,    OR  FARhLLSLS  RESEaRCH  DOjIE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE   (iJITHOUT  INTERCaUIGE  OF  PERSONNEL,    F.xCILITIES  OR 
FUi'iDS),    IDENTIFY  SUC;I  RESEiiRCH: 


No  entries  for  items  1^.,  15  and  16. 


I       PAGE  I 
PROJECT  REPORT  FORM 
K.C.I.  2 .   General  Medicine  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

Acute  Leukemia h ___^  ^  .NCI-7'-7(C) 

SECTKJN  OR  SERVICE    '  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

Infections,  Fever,  and  Host  Resistance  in  Acute  Leukemia^ 
PROJECT  TITLE 


Richard  T.  Silver 


PRINCIPAL  IlWESTIGATOR(Sj 

].      Dr.  John  Utz,  NMI  Dr.  E.:dl  Frei :  Dr.  John  Fahey;  Dr.  Grace  Beal.  NMI- 
OTHER  INVESTIGATORS  Dr.  Robert  Kolb  L.B.C. 


PROJECT  DESCRIPTION 

Objectives'   1.   To  obtain  data  relating  to  the  natural  history  of  fever  and 
infections  in  acute  leukemia. 
2.  To  define  the  mechanisms  of  impaired  host  resistance  to  . 
infection  in  acute  leukemia. 

Methods  employed-   Fevers  of  known  and  unknown  origin  have  systematically 
been  studied  according  to  a  previously  defined  protocol 
in  over  30  patients  with  acute  leukemia.   Comprehensive 
bacterial  and  viral  data  have  been  collected  and  analyzed  in  conjiinction 
with  a  group  in  the  Nationa'.  MicrotiologicaL  Institute. 

In  order  to  define  the  capability  of  '.e'okemic  granulocytes  to  phagocytize 
opsonocytophagic  determinations  have  been  made  in  11  patients.   The 
antibody  response  to  6  challenge  antigens  has  been  measured  in  11  Leukemic 
patients  and  in  7  "normal  controls."  Alterations  in  serum  proteins 
d\iring  the  immunization  procedure  have  been  measured  by  paper  electro- 
phoresis 

Major  Findings:   The  tabulation  of  the  relative  incidence  of  fever  of  known 
and  unknown  origin  in  the  leukemic  patient  has  not  been 
completed.   Of  major  importance  in  problems  of  patient  care 
is  the  finding  that  many  fevers  previously  ascribable  to  leukemia  per  se> 
have  been  demonstrated  to  be  due  to  a  complicating  and  remedial  bacterial 
infection.   The  marked  susceptibility  to  bacterial  infection  does  not 
seem  to  apply  to  viral  diseases . 

The  opsonocytophagic  tests  have  been  concluded.   No  difference  in  the 
phagocytic  activity  of  the  mature  polymorphonuclear  cell  in  leukemic 


NCI -7 1-7  (C)       -  ?AGE  2 

SERIAL  NO. 

?R©JECT  REPORT  F9M  (cont'd) 

blood  during  active  disease,  bacterial  infection  or  in  hematological 
remission  (as  compared  to  nonrKiL)  was  noted.  Hiagocytosis  by  the 
more  iicffiature  celLS  of  the  granulocytic  series  of  both  Leukemic  aftd 
normal  blood  is  minima.   This  confirms  previous  reports  in  the 
literature,   The  susC2ptibiIity  to  infection  does  not  bear  a  direct 
relatio-iship  to  the  absolute  mature  polymorphonuclear  count;  but 
■   rather  to  a  ratio  of  mature  poly^totaL  WBC   Significant  differences  .  ■ 
in  the  response  to  challenge  antigens  and  in  the  electrophoretic 
pattern  of  the  serum  proteins  have  not  been  observed.,  although 
final  results  must  await  completion  of  the  immunization  procedures  in 
the  remaining  normal  controls/ and  statistical  analysis.. 

Significance  to  Cancer  Rssearch-   In  spite  of  the  voluminous  leukemia 

literature,  there  have  been  no  comprehea- 
'"  sive  observations  relating  to  fever  and  ■ 
infection  in  these  patients.  Although  there  have  been  many  individual 
■  studies  pertaining  to  phagocytosis  antibody  response  and  electrophoretic 
patterns  in  leukemia  there  has  been  no  study  to  the  best  of  our  know- 
ledge^ which  has  attempted  to  correlate  all  these  parameters  with  serial 
clinical  observations.  Thus,  it  is  hoped  that  information  obtained  from 
this  study  will  augment  our  understanding  of  the  natural  history  of 
acute  leukemia  and  aspects  of  host  resistance  in  general,  and  aid  in  the 
further  improvement  in  the  clinical  management  of  the  leukemic  patient. 

Proposed  Course  of  Study  The  present  study  should  be  concluded  within  the 
"  next  6  months . 


PAGE  3 

PROJECT  REPORT  FORM  (cont'd) 

NCI-7I7(C) 

SERIAL  NO, 


BUDGET  ACTIVITY: 

RESEARCH  JTJ       ADMINISTRATION        I      / 

REVIEW  &  APPROVAL  I     7    TECHNICAL  ASSISTANCE  /   7 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  L956  or  L957 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDEl^ITIFY  SUCH  RESEARCH)- 


NO  ENTRIES  FOR  ITEMS  1^,  15  &  I6. 


:)  \Am 


PAGE  I 
PROJECT  REPORT  FORM 

1.   N.C.I. 2.  General  Medicine  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 


h.  ^.NCI-7'.8(C) 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  NO. 


6.  Pulmonary  Physiology 


PROJECT  TITLE 


7.  C.  B.  McCall 


PRINCIPAL  INVESTIGATOR(S) 


OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION  -  Ventilatory  studies  on  patients  with  airway,  lung, 

or  pleural  involvement  by  neoplasm. 

Objectives :   To  study  and  correlate  changes  in  tumor  size  with  pulmonary 
function  and  blood  gas  studies  and  clinical  course. 

Methods :   Obtain  baseline  determinations  of  cotaL  vital  capacity,  timed  - 
VC,  maximum  breathin^-j  capacity,,  residual  air,  total  lung  volume, 
I   nitrogen  rinse  out,  and  fluoroscopy.   These  determinations  will 
be  repeated  at  suitable  intervals  and  correlated  with  X-ray  size 
of  tumor,  sput\:iBi,  wheezes,  chest  pain  and  dyspnea. 

Patient  material:   All  patients  on  Solid  Tumor  Chemotherapy  Program  with 
suitable  pulmonary  lesions. 

Major  Findings:    A  major  portion  of  my  time  in  past  five  months  has  been 
devoted  to  first, learning  techniques  and  determinations; 
second,  learning  their  proper  interpretation  and 
evaluation  and  thirdly,  calibrating  instruments  and  developing 
more  accurate  methods.   In  addition,  during  this  time  I  have 
answered  k2   consults  from  NCI  -  these  tests  being  especially 
valuable  in  patients  before  and  after  pulmonary  surgery. 

As  to  the  above  project,  I  have  to  date  only  baseline 
determinations  from  which  no  conclusions  can  be  drawn. 


WCI-T'-8(C) PAGE  2 

SERIAL  NO. 

PROJECT  REPORT  FORM  (cont'd) 


Significance  to  cancer  research:   Such  studies  may  shed  some  Light  on 

'the  effect  of  various  tumors  and  their 
growth  on  pulmonary  physiology,  parti- 
ticularly  ventilation^ and  possibly  provide  some  clue  as  to  the 
cause  of  dyspnea. 

Proposed  course  of  project:  Perform  studies  as  indicated  on  all  suitable 

patients  admitted  to  So '.id  Timor  Chemotherapy 
Program. 


PAGE  3 
PROJECT  REPORT  FORM  (cont'd) 


10.   NCI-7l8(.C) 


SERIAL  NO, 


11. 


12. 


13. 


BUDGET  ACTIVITY: 

RESEARCH  [~T~]       "ADMINISTRATION       j -J 

REVIEW  &  APPROVAL    /    /_   TECHNICAL  ASSISTANCE  /~7 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  OR  1957. 


IF  THIS  PROJECT  RESEIffiLES,  COMPLEMENTS,  OR  PARALLELS  RESKf\RCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL, 
FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEl/B  lU,  I5  &  I6. 


PAGE  I 
PROJECT  REPORT  FORM 
N.C.I.  2.      General  I'-L.  1-1  c I. le  Branch  


INSTITUTE  L^BOR'^TORY  OR  BRANCH 

3.  Acute  L^ukeuia 3. 5-  NCI-T19(C) 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)   SERIAL  WO. 

b.  A  StuJ-y  of  the  Clinical  Ma:iiibstatiOiis  of  Acutg  Leukapia 

PROJECT  TITLE 


7.  Enil  Frsi,  III  . 

PRINC IPAL  lOTESTIG/iTOR  ( 3 ) 


8 :     Richard  T.  Silver,  Richard  Svarm,  E.  J.  Van  Scott,  Charles  Wells 
OTHER  IWESTIG/lTORS 


9.   PROJECT  DESCRIPTION 

Objectives:  To  deliaeate  ebiologlc  a-t-id  pathogei:etlc  iactcrs  of  the 

various  ma'iifestatioas  of  acute  leukenia  with  particular 
emphasis  on  the  oral,  neuroloj;ic,  hepacic,  rc^al,  and 
osseous  lesio  s  and  the  ab..orTial  uric  acid  metauollsiii. 

Methods  employed;  The  coraprehensive  application  and  correlation  of 

clinical,  heroa to logical,  radiologic,  chemical,  micro- 
biologic, and  histologic  methods  to  the  above  manifes- 
tations in  patieiits  vjlth  acute  leixlceiriia . 

Major  Findings;  Though  data  concernii  g  the  above  has  bee:i  collected, 

it  has  not  as  yet  been  analyzed  and  correlated.  It  is 
appare^it  that  multiple  factors  may  contribute  to  these 
manifestations,  e.g.  the  leukemic  process  per  se,  antimetabolite 
therapy,  steroid  therapy,  infection,  anemia,  etc. 

Significance  to  Caxicer  Research:  A  study  of  the  imtural  and  modified 

history  of  acute  leukemia  and  its 
complications  is  of  obvious  importance 
to  cancer  research. 

Proposed  Course  of  Project;   It  is  anticipated  that  some  of  the  information 

gained  from  this  study  will  have  practical 
application  in  the  clinical  management  of 
this  disease.  Therefore,  at  least  portions  of  the  study  will  continue 
indefinitely.  Moreover,  additional  diagnostic  and  e.cperimental 
procedures  will  be  applied  when  it  is  apparent  that  the  present  methods 
are  inadequate  to  explain  the  observed  manifestations. 


10.   NCI-719(C) 
SERIAL  NO. 


PAGE  2 


PROJECT  REPORT  FORM 


11. 


12. 


BUDGjiT  ACT:-VI'iT 

RESEARCH  Jxj       ATMIND.S'rPATICN  /     / 

RE\^Fv/  S:  APPROVAL  /     /       TEOffl^ICAL  ASSISTAITCE  /      7 


C0OP£;P.4T:r;v:i  UirriS  O?  the  public  HMLQH  service,   or  OTIiER  ORGANIZATIONS,   PRO- 
VIDING FlTIl^JS,    FACILITIES,   OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  I956  or 
1957. 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSE- 
WHERE IN  THE  PUBLIC  HEALTH  SERVICE  (V^THOUT  INTERCHANGE  OF  PERSONNEL, 
FACILITIES  OR  FUNDS),  IDENTIFY  SaCH  RESEARCH: 


NO  ENTRIES  FOR  ITEMS  \h,   15  &  I6 


\.bi\n  -i: 


PAGE  I 


PROJECT  REPORT  FORM 


N.C.I. 


INSTITUTE 


2.  General  Medicine  Branch 


LABORATORY  OR  BRANCH 


k. ^___  5.   NCI-720(C) 

SECTION  OR  SERVICE      LOCATION  (IF  OTHER  THAN  BETHESDA)        SERIAL  NO. 


Svalual       Aiaino  .Aci.d  Metabolism  InlHuman  'Subjects  by  Utilization  of 
PROJEC'    jE  Parenteral  Routes  of  Administration 


John  L.  Fahey 

PRINC IPAL  INVESTIGATOR ( S ) 


OTHER  INVE3TIGAT0F.S 


I.   PROJECT  DESCRIPTION 


Objectives:  The  general  objective  is  to  study  the  human  metabolism  of  intra- 
"~~~~~~~~~   venously  administered  amino  acids  in  normal  and  disease  states. 
Specific  objectives  are  (a)  to  establish  the  identity  and  amount 
of  individual  amino  acids  essential  to  achieve  positive  nitrogen  balance, 
and  (b)  to  approach  the  problem  of  determining  the  role  of  individual  amino 
acids  by  observation  of  the  effects  of  incomplete  amino  acid  mixtures. 

Methods  Employed:  Optically-pure  1-amino  acids  prepared  by  the  methods  of 

Greenstein  and  obtained  from  him  have  been  placed  in  aqueous 
solution  and  administered  intravenously  at  a  constant  rate  of 
infusion.  By  means  of  complete  collection  techniques  metabolic  balance 
data  have  been  obtained. 

Total  nitrogen,  ammonia  nitrogen,  urea  nitrogen  and  alpha  amino  nitrogen 
have  been  determined  on  appropriate  samples  of  urine,  stool  or  plasma. 

Patient  material: 


Admissions: 


No. 
2 


Total  Stay 
300 


Major  Findings:   1.  Mixtures  of  pure  1-amino  acids  containing  all  of  the 
"essential"  amino  acids  (Rose)  were  adequate  to  achieve 
nitrogen  balance  in  the  single  malnourished  adult  main- 
tained on  a  balance  study. 


NCI-720(C)  PAGE  2 

SERIAL  NO. 

PROJECT  REPORT  FOR!/I  (cont'd) 

2.   '5inission  of  .l.-Arginine  from  the  intravenous  mixtiore  was  capable. of 
producing  convulsions  and  coma  and,  in  preliminary  dog  experiments, 
has  been  lethal. 

3-  Administration  of  the  toxic  (arginine-def icient)  solutions  resi^Lted  in 
a  marked  rise  in  blood  ammonia  level  of  the  dogs  which  coincides  with  the 
development  of  convulsions  and  coma.   The  rise  in  blood  ammonia  can  be 
prevented  by  coincident  or  prior  administration  of  1-arginine. 

Significance  to  Cancer  Research:   Further  information  on  amino  acid  disposi- 
tion and  nitrogen  transfer  within  the 
intact  subject  will  provide  a  better  base 
from  which  to  investigate,  tumor  metabolism  and  the  metabolism  of. the 
tumor- containing  host.  Ciinically,  development  of.  means  of  managing 
the  toxic  effects  of  elevated  blood  ammonia  levels  would  be  important 
in  the  management  of  hepatic  damage  states  and  in  conditions  of  alkalosis 
in  which  ammonium  chloride  administration  is  indicated.   Safe,  profitable 
intravenous  administration  of  amino  acids  is  important  in  the  general 
medical  care  of  many  patients  with  neoplasm. 

Propos'id  Course  of  the  Project:  A.   Further  investigation  of  the  conditions 

determining  1-Arginine  requirement  (i.e. 
anesthesia,  rate  of  amino  acid  administra- 
tion or  ammonium  administration,  and  possible  similar  protective  action 
of  other  amino  acids)  are  to  be  continued  in  preliminary  dog  studies. 
Extension  of  these  observations  to  human  subjects  in  normal  and 
pathologic  states  is  planned  with  special  interest  in  hepatic  dys- 
function states  resulting  from  cirrhosis,  tumor  invasion  of  liver,  and 
from  the  presence  of  tumor  in  the  host. 

B.   Further  efforts  to  establish  the  requirements  for  other  amino  acids 
will  proceed  after  clarification  of  the  role  of  1-arginine. 


12, 


13. 


PAGE  3 
PROJECT  REPORT  FORM  (cont'd) 


10.   NCI-720(C) 
SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH  ]TJ  ADMINISTRATION        /  7 

REVIEW  &  APPROVAL  HI  TECHNICAL  ASSISTANCE  /  / 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  I956  or  1957 

Drs .  Jesse  Greenstein,  M.  Winitz  and  J.  Birnbaura  in  the  Laboratory  of 
Biochemistry,  NCI,  are  investigating  amino  acid  requirements  and  metabolism 
in  rats  utilizing  intra-peritoneal  administration.  Constant  consultation 
and  exchange  of  information  as  these  studies  have  progressed  have  been  of 
mutual  benefit. 

The  Laboratory  of  Biochemistry  has  also  prepared,  and  made  available,  the 
purified  1-amino  acids  utilized  in  this  study. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS,)  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEMS  lU,  I5  &  16. 


PAGE  I 
PROJECT  REPORT  FORM 
N.C.I. 2 •  General  Medicine  Branch 


INSTITUTE  LABOMTORY  OR  BR/iNCH 

Nutrition  and  Metabolism    h.  5-  NCI"72l(C) 


SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO. 

3.  Metabolism  of  Nitrogen,  Minerals  and  Vitamins  in  Subjects  with  Malignant  Disease 
PROJECT  TITLE 

^  Donald  M.  Watkin,  M.D.  and  Donald  P.  Tschudy.  M.  D. 

PRINCIPAL  INVESTIGATOR(S) 

3.   Jesse  L.  Steinfeld,  M  D  ;  John  L.  Fahey,  M.D.;  and  Donald  P.  Tschudy,  M.D.; 
OTHER  INVESTIGATORS    in  addition,  Residents  Montague  Lane  and  Charles  B. 
McCall  and  Clinical  Associates  Wroth,  Roush,  Mohler, 
Gold,  Silver,  Schroeder,  Fritz,  Landau,  Levine, 
Forkner,  Schick,  Goulian,  Flick  and  Paton 

).      PROJECT  DESCRIPTION 

Objectives :   Investigation  of  the  over-all  and  intermediary  metabolism  of 
protein,  fat,  carbohydrate,  minerals,  electrolytes,  vitamins, 
calories  and  water  in  the  natural  course  of  malignant  disease 
and  in  the  response  of  the  disease  to  nutritional,  chemothera- 
peutic,  endocrine,  surgical  or  radiologic  therapy. 

Methods  employed:   The  metabolic  balance  technique  is  used  to  identify  changes 
in  tumor  and  host  by  measuring  quantitative  differences 
between  the  intake  and  output  of  body  constituents. 
Quantitative  measurement  of  water  balance  together  with  analysis  of 
expired  air  (Dr.  McCall)  are  used  to  estimate  energy  expenditure.   Radio- 
isotope techniques  are  utilized  to  measure  albumin  turnover  and  red  cell 
survival  time  (Dr.  Steinfeld).   Electrophoretic  partition  of  plasma 
proteins  is  used  to  identify  abnormal  plasma  protein  patterns  and  observe 
changes  during  the  course  of  a  study  (Dr.  Fahey) .   Heavy  isotope  (N-'--') 
techniques  are  used  to  study  the  metabolism  of  urea  and  uric  acid 
(Dr.  Tschudy). 

Patient  Material: 

In  patients  January  1,  1955  1 

In  patients  admi-tted  January  1,  1955  -  December  31^  1955 

Initial  Clinical  Center  Admissions  l8 

"Second  Clinical  Center  Admissions  h 

Third  Clinical  Center  Admission  1 

In  patients  other  than  above  studied  during  1955  -  H 

Total  Deaths  3 

Total  Autopsies  3 


PAGE  2 

NCI-7gl(C) 

SERIAL  NO.  PROJECT  REPORT  FORM  (cont'd) 

Outpatients; 

Screening  8 

Follow-up  6 

Research  1 

Total  patient  days  attributable  to  patients  studied  on  this  project- 3692 

Major  Findings:  Because  of  the  large  number  of  different  studies  which 
were  completed  under  the  general  heading  of  NGI-70l(C), 
a  separate  report  on  each  study  is  given  herein. 

A.  Nitrogen,  albumin,  mineral,  electrolyte  and  energy  metabolism  during 

hyperalimentation  by  intravenously  administered  fat  emulsion  in  subjects 
with  malignancy  and  cachexia;  Donald  M.  Watkin 

Objectives:   Quantitation  of  effects  of  excessive  calories  supplied  by 

fat  administered  intravenously  on  host  and  tumor/  evaluation' 
of  caloric  hyperalimentation  as  a  therapeutic  measure; 
exploration  of  the  relationship  between  energy  metabolism,  pro- 
tein metabolism  and  tumor  growth. 

Methods  employed:   a)  Metabolic  balance  technique  utilizing  liquid  constant 
diets , 

b)  I  -^^  labelled  human  serum  albumin  turnover. 

c)  Open  circuit  measurement  of  respiratory  quotient 
{B.M-)   and  basal  metabolic  rate  (B.M.R.). 

Patient  Material;   Two  men  and  one  woman  with  inoperable  carcinoma,  one  man 
with  sarcoma,,  and  one  man  with  post-gastrectomy  cachexia. 

Major  Findings:  All  subjects  maintained  weight  on  liquid  diets  during  control' 
periods .   Fat  emulsion  intravt,nously  increased  caloric  intake 
by  1/3^  resulted  in  weight  gains,  retention  of  protoplasmic 
constituents,  and  an  increase  in  total  albumin  and  rate  of  albumin  turn- 
over. R.Q.'s  in  patients  with  rapidly  progressing  carcinoma  were  so 
low  as  to  suggest  ketone  formation  during  control  periods.  All  patients 
showed  low  R.Q.'s   during  fat  emulsion  infusions.   Side  effects  from 
infusion  of  emulsions  were  back  pain  with  the  initial  infusion,  BSP 
retention,  and  a  moderate  anemia  during  the  infusion.  A  striking 
finding  of  general  interest  was  the  ease  by  which  cancer  patients  can 
consume  extremely  high  caloric  intakes  when  the  combination  of  liquid 
diets  and  intravenous  fat  emulsions  are  used.   In  one  patient,  hyper-  • 
alimentation  was  associated  with  objective  increase  in  the  rate  of 
tumor  growth. 

Significance  to  Cancer  Research:   Practically,  these  studies  show  the  feasi- 
bility of  feeding  adequate  calories  to 
cachectic  patients.  From  a  theoretic  point 
of  view.,  they  suggest  the  presence  of  a  defect  in  carbohydrate  metabolism 
in  patients  with  advancing  cancer.  They  show  that  fat  supplied  calories 
can  be  utilized  to  spare  protein  in  cancerous  subjects. 


PAGE  3 

NCI-7gl(C) 

SERIAL  NO.  PROJPJCT  REPORT  FORM     (cont'd) 

Proposed  Course  of  Project:   The  abnormality  in  carbohydrate  metabolism 

suggested  by  R.Q.  studies  v/ill  be  actively 
pursued.  New  emulsions  of  fat  will  be  tried 
with  the  ultimate  goal  of  providing  adequate  nutrition  completely  by 
the  parenteral  route  and  hence  eliminating  cachexia  and  starvation 
as  a  cause  of  death  in  cancer  patients.   Abnormalities  of  energy 
metabolism  during  hyperalimentation  in  cancer  patients  will  be 
studied. 

B.   Renal  excretion  of  uric  acid  at  various  plasma  uric  acid  levels  in 
subjects  with  leukemia  and  other  malignancies:  Donald  M.  Watkln 

Objectives :   To  investigate  the  relations  among  the  renal  tubular  reabsorption 
of  uric  acid,  plasma  uric  acid  level  and  uric  acid  production. 

Methods  employed:   R  nal  clearance  technique. 

Patient  material:   Two  patients  with  leukemia,  two  with  multiple  myeloma, 
and  one  with  reticulum  cell  sarcoma.   In  two,  serial 
studies  were  performed. 

Major  findings:   One  subject  with  chronic  myelocytic  leukemia  studied 

before,  during,  and  after  treatment  with  myeleran  demonstrated 
a  depression  in  mrlc   acid  clearance  with  no  change  in  plasma 
uric  acid  level  during  therapy  followed  by  a  marked  increase  in  uric 
acid  clearance,  a  decrease  in  the  absorption  of  filtered  uric  acid,  and 
a  decline  in  plasma  uric  acid  level  after  completion  of  treatment. 
This  was  accompanied  by  an  increase  in  glomerular  filtration  rate, 
renal  plasma  flow  and  TmPAH.   The  reciprocal  relationship  between  the 
tubular  transport  of  uric  acid  and  PAH  is  an  incidental  observation 
corroborating  similar  findings  made  by  NHI  workers  utilizing  rabbit 
kidney  slices. 

Significance  to  Cancer  Research:  Uric  acid  is  in  man  the  end  product  of 

purine  metabolism.   In  leukemia  and  certain 
allied  malignancies  plasma  uric  acid  levels 
are  high.  This  level  is  determined  in  part  by  renal  tubular  reabsorption 
of  uric  acid,  in  part  by  uric  acid  synthesis,  and  in  part  by  the  release 
of  uric  acid  from  tissues  undergoing  destruction.   The  renal  contribution 
to  the  maintenance  of  a  plasma  level  must  be  known  before  any  interpre- 
tation on  uric  acid  production  or  release  from  tissue  can  be  made. 

Proposed  Course  of  Project:   Renal  function  studies  de'signed  to  measure 

glomerular  filtration  rate,  renal  plasma  flow, 
TmPAH,  loric  acid  clearance  and  renal  tubular 
transport  will  be  conducted  in  subjects  with  leukemia  or  other  malignan- 
cies associated  with  abnormalities  in  uric  acid  plasma  levels.   Every 
effort  will  be  )iiade  to  study  these  subjects  serially  before,  dui'ing  and 
after  successful  chemotherapeutic,  endocrine,  radiologic  or  sxirgical 
therapy . 


PAGE  k 
TCI-7gl(C) 
■5ERIAL  NO. 

PROJECT  REPORT  FORM  (cont'd) 

In  addition,  the  information  acquired  will  serve  as  guides  to  more 
elaborate  studies  of  the  uric  acid  pool  and  uric  acid  synthesis 
carried  out  with  the  aid  of  heavy  or  radio  isotopically  labelled 
uric  acid  and  its  precursors. 

C.   Vitamin  3^2-      Vitamin  Big  metabolism  in  normal  subjects  of  various  ages 
and  in  subjects  with  malignant  disease;  Donald  M  Watkin 

Objectives :   ^Qualitative  and  quantitative  observations  on  the  over-all 
metabolism  of  vitamin  By^^i    its  levels  in  blood  plasma,  its 
distribution  in  the  bodies  of  normal  subjects  and  those  with 
cancer,  and  its  excretion  by  the  kidney. 

Methods  employed:  Microbiological  assay  for^itamin  B-jo  i"^  blood  plasma  and 
urine .  Measurement  of  CO   labelled  radioactive  B^  p  in 
blood,  urine,  tissue  and  feces. 

Patient  Material;  To  date  all  studies  have, been  conducted  in  roughly  250 
presumably  healthy  individuals  without  known  malignant 
disease . 

Major  Findings:   Normal  values  for  blood  levels  of  B-^o  ^^   various  age  cate- 
~  gories  have  been  established.  Absorbtion  of  vitamin  'Bj_2 

from  the  G.I.  tract,  after  oral  ingestion  has  been  related  to 
dose,  age  of  subject  and  the  subject's  gastric  acidity.  Urinary  excretion 
following  various  intramuscular  doses  of  B^p  has  been  quantitated  for 
presumed  normals  aged  20  -  100  years.   Renal  clearance  of  B-,  p  in  various 
age  groups  has  been  quantitated  at  low  and  intermediate  plasma  B-ig 
levels .   Recent  studies  have  demonstrated  the  mc-chanism  of  B-i  o  clearance 
at  high  plasma  levels. 

Significance  to  Cancer  Research:  Vitamin  Bnp  pl^-ys  'in  indispensable  role  in 

the  metabolism  of  all  foodstuffs  and  in 
hematopoiesis.   It  has  been  used  with  ■  ■■ 
reported  success  in  treating  neuroblastomas.   Plasma  B,  ,-j  levels  in 
leukemia  are  extraordinarily  high.   B-]_2  antagonists  are^being  developed. 

Proposed  Course  of  Project:   The  techniques  for  the  microbiological  assay 

using  L.  Leichm  nil  and  for  the  analysis  of 
C0°'-'  labelled  radioactive  vitamin  B^o  s-re  being 
developed.  Bloods  will  be  collected  from  all  patients  with  cancer 
admitted  to  the  Clinical  Center,  assayed  for  B-j^gj  ^^^   analyzed 
electrophoretically  for  plasma  protein.   In  selected  patients,  B^o 
■distribution  studies  using  the  renal  clearance  technique  will  be 

riO  ^ 

performed.   Distribution  of  radioactive  CO"  labelled  B-,p  in  normal 
and  tumor  tissue  will  be  determined  in  surgical  and  autopsy  specimens. 


PAGE  5 


rci-7gi(c) 

JERIAL  NO.  PROJECT  REPORT  FORM  (cont'd) 


D.   Leukemia:   Donald  M.  Watkin 

Objectives:   Characterization  of  over-all  metabolism  in  chronic  and  acute 
leukemia  and  the  changes  induced  by  anti-leukemic  therapy. 

Methods  employed,:   Metabolic  balance  technique.   Discrete  renal  function  studies. 

Patient  Material:   In  195^  three  patients  with  chronic  and  one  with  acute 

leukemia.   In  1955 ^  one  patient  with  chronic  myelocytic  and 
one  with  acute  lymphocytic  leukemia  as  in  patients;  one 
patient  with  chronic  myelocytic  leukemia  as  out-patient. 

Major  Findings:   Myeleran  therapy  in  chronic  le-ukemia  results  in  a  marked 

early  increment  of  phosphorus  and  uric  acid  excretion  followed 
by  a  reduction  in  uric  acid  excretion  to  bslow-control  values 
with  continued  therapy.   Methotrexate  therapy  in  acute  leukemia  induces 
an  increase  in  phosphorus  and  uric  acid  excretion.   6-Mercaptopurine 
therapy  was  associated  with  a  temporary  diarrhea,  although  no  definite 
evidence  of  a  sprue-like  syndrome  could  be  proven.   Hydrocortisone  in 
combination  with  6--MP  induced  a  remarkable  increase  in  uric  acid 
excretion.   6-MP  over  a  prolonged  course  reduced  plasma  and  uric  acid 
levels  to  extremely  low  values . 

Significance  to  Cancer  Research:   These  studies  provide  quantitative  means  of 

evaluating  the  rate  of  progression  of  the  leuke- 
mic process  and  the  extent  to  which  this 
process  may  be  altered  by  antimetabolite  therapy.   Thuy  point  toward 
possible  mechanisms  of  action  of  antimetabolites  in  leukemia. 

Proposed  Course  of  Project:   Selected  leukemic  patients  who  are  sufficiently 

well  to  cooperate  in  balance  and  renal  studies 
will  be  studied  with  particular  emphasis  on  the 
excretion  of  uric  acid  and  on  calcium  and  phosphorus  metabolism. 

S*  Nitrogen  Utilization;  A  Study  of  Nitrogen  Utilization  in  Human  Tumor 

Patients  by  Means  of  N-^^  Labelled  Amino  Acids. 
(D.  P,  Tschudy) 

Objectives:  To  study  the  effects  of  malignant  tumors  on  the  organism  with 
respect  to  over-all  rate  of  protein  synthesis,  size  of  nitro- 
gen metabolic  pool,  rate  of  incorporation  of  nitrogen  into  the 
tumor  and  other  aspects  of  nitrogen  metabolism. 

Methods  Employed;  Synthesis  of  isotopically  labelled  amino  acids,  isolation 
of  compounds  from  blood  and  urine,  preparation  of  samples 
for  mass  spectrometer. 

Patient  Material :  Noj.         Aver,  stay 

Admissions:         3  5 

Ma,ior  Findings;  Tumors  contain  higher  levels  of  isotopic  N  than  serum  pro- 
teins examined  at  the  same  time.  Rate  of  labelling  of  urea 
and  ammonia  is  about  the  same  in  tumor  and  non  tumor 


WCI-721(C)  PAGE  6 

PROJECT  REPORT  FOR^i  (Cont'd) 

patients.  Some  cancer  patients  may  excrete  less  isotope  than  normals. 

Significance  to  Cancer  Research;  Following  the  mathematical  model  of  Ritten- 

berg  and  San  Pietro  we  will  quantitate  the 
effects  of  cancer  on  the  protein  synthesis 
rate  and  size  of  the  nitrogen  metabolic  pool  in  patients. 

Proposed  Course  of  Project;  Data  is  being  applied  to  various  mathematical 

models  for  calculations  of  both  rates  and  rate 
constants  for  the  over-all  organism  with  re- 
spect to  nitrogen  metabolism.  The  physiological  validity  of  these  models 
may  be  tested  by  direct  experimentation  and  measurement  of  certain  rates. 


Page  7 

PROJECT  REFOxlT  FOM  (Cont'd) 


10. 

NC 1-7 21(C) 
SERIAL  NO. 

1]. 

BUDGET  ACTIVITY: 

RESEARCH 

HI 

REVIEW  &  API  ROVAL 

D 

12. 


13. 


ADMINI'-^TRx.TION  [J 

TECHI\fICAL  ASSISTANCE  [J 


COOPERATING  UIMITS  OF  THE  PUBLIC  HKiLTH  SERVICE,  OR  OTHER  ORGANIZilTIONS,  PRO- 
VIDING FUi^DS,  Facilities,  or  personnel  for  this  project  in  either  1956  or 
1957. 

A,  B,  C,  D  and  E  -  none 


IF  THIS  PROJECT  KESEf'iBLES,    COFlPLEMENTS,    OR  rAR-xLLELS  RESEiiRCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  rISALTH  SERVICE   (WITHOUT  INTERCHiWGE  OF  PERSONNEL,    FACILITIES  OR 
FUI^DS),    IDENTIFY  SUCH  ftSSEARCH: 

A,   B,   C,    D  and  E  -  none. 
No  entries  for  items  14,   15  and  16, 


PAGE  I 


PROJECT  REPORT  FORM 


1,  N,  C.  I. 


INSTITUTE 


2,  General  Medicine 


LABORATORY  OR  BRANCH 


3.  Metabolism 


SECTEON  OR  SERVICE 


U. , 5,   NCI-72£(C) 

LOCATION   (IF  OTHER  .THAN  BETTIESDA         SERIAL  NO. 


6,  Measure  of  Blood  Volume,  Red  Cell  Mass  and  Total  Circulating  Albumin  in 

Patients  with  Cancer. 

PROJECT  TITLE 


7;  J.  L.  Steinfold 


PRINCI PAL  INfc tTGATORTsT 


8,  None 


OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPnON 

Objectives:  To  determine  whether  there  is  a  consistent  decrease  in 

patients  in  the  various  stages  of  carcinoma  growth  in  the 
red  cell  mass,  since  conflicting  reports  in  the  literature 
state  that  there  is  and  that  there  is  not  a  decrease  in 
the  red  cell  mass  in  patients  with  carcinomas ♦ 

To  determine  whether  the  decreased  concentration  of  serum 

albumin  seen  so  frequently  in  patients  with  cancers  is  a 

result  of  an  absolute  decrease  in  albumin  or  is  the  result 
of  an  increase  in  plasma  volume. 


Methods  employed; 


Patient  Material: 


Major  Findings; 


Blood  Volume,  Red  Cell  Mass  and  Plasma  Volvme  are 
determined  using  the  isotope  dilution  method  with 
II3I  albumin  or  red  cells  labeled  indth  sodium 
chromate^l, 

80  Patients  Admitted  for  other  NCI  clinical  research 
programs. 

Red  Cell  Mass  was  consistantly  decreased  in  80  studies 
in  60  patients  with  advanced  carcinomas.  Plasma 
Volume  -  in  the  absence  of  congestive  heart  failure  - 
was  within  norma.1  limits. 

Recalculation  of  the  conflicting  data  available  in 
the  literature  using  the  concept  of  "body  hematocrit" 
and  a  body  hematocrit:  peripheral  venous  hematocrit 
ratio  of  0,91  reconciles  the  reports  so  that  the 
available  published  data  are  consistent  with  the 
present  findings  at  the  NCI, 


NCI -722(0) 


PAGE  2 


PROJECT  PEPORT  FORM  (Cont'd) 


Significance  to  Cancer  Research;  In  order  to  adequately  characterize 

a  metabolite  it  is  essential  to  know- 
not  only  its  concentration  (previously 
available  as  hemoglobin  or  albumin  concentrations)  but  also  its 
volime  of  distribution*  To  further  characterize  any  metabolite  the 
rate  of  production  and  rate  of  metabolism  also  must  be  determined, 
however,  this  project  is  concerned  with  only  the  first  of  the 
above  two  requirements. 


Proposed  Course  of  Project;  To  extend  these  observations  to  include  more 
~  patients  with  cancer  in  various  states 

(  extent  )  of  disease  and  to  make  repeated 
observations  on  the  same  patient,  throughout  the  course  of  his  illness. 


PAGE  3 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI-7£2(C) 
SERIAL  NO. 


11, 


BUDGET  ACTIVITY: 

RESE.'^JICH        fT~J 
REVIEW  &  APPROVAL/   7 


ADMINI S  TRAT  ION        /    / 
TECHNICAL  ASSISTANCE  J  7 


12.   None 


CCOPERATIMG  UNITS   OF  THE  PUBLIC  HEALTFI  SERVICE,   CR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,    FACILITIES,    OR  PERSONN-EL  FOR   ffllS   PROJECT  IN  EITHER 
1956  or  1957. 


13.   None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEI^'IENTS,  OR  PimALLELS  RESEARCH  DONE 
ELSElfflERE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITIiOUT  INTERCH;J^IGE  OF 
PERSONNEL,  FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


No  entries  for  items  lii,  l5  and  l6. 


PAGE  1 
PEDJECT  REPORT  FORl^I 


National  Cancer  Institute 2,  Surgery  Branch 

INSTITUTE  lABORATORY  OR  BRANCH 

•     .    ,  NCI 

.    ^.  ■ -  5._JZ5QiCi_ 

SECTION  OR  SERVICE  LXATION  SERIAL  NO. 

Removal  of  Cancer  and  Other  Tissues  for  Histologic,  Biochemical  and  Other 

Studies  as  Required  by  Scientists  and  Other  Investigators  for  Correlated 

PROJECT  TITLE  Studies. 


7.  , ,  Dr,  R.R.  Smith.  Dr.  J.  H.  Waitet  Dr/ W.  E.  Schatten 
PRINCIPAL  INVESTIGATOR(S)     " 


Dr.  A,  Ship    Dr.  R.  Miller     Dr.  W.  Kramer 
8 .    Dr.  R.  Milch    Dr.  L.  Cramer     Dr..H.  Herbsman- 
"  OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION"  ■  .     - 

Ob.iectives; 

The  object  of  this  project  is  to  supply  human  tissue  for  histologic, 
histochemical,  biochemical  and  biophysical  studies  to  scientists  working 
in  various  branches  of  the  Cancer  Institute  who  require  tissue  for 
studies.  As  a  rule,  such  studies  are  done  as  cooperative  projects  with 
various  investigators  but  on  occasions  specific  types  of  tissue  are  re- 
quired for  specific  studies.  For  example,  one  investigator  requested 
that  specimens  of  skin  and  its  associated  neoplasm  be  made  available  for 
keratinization  studies,  and  various  muscle  biopsies  and /or  biopsies  of 
liver  tissue  under  specific  conditions  have  been  requested.  These 
materials  are  obtained  during  operations  for  standard  therapeutic  reasons. 

Methods  Employed; 

Methods  employed  are  standard  surgical  procedures. 

Patient  Material; 

Material  for  this  project  is  mostly  obtained  from  patients 
hospitalized  for  study  in  other  projects.  An  example  of  such  a  case  is 
a  patient  with  an  islet  cell  adenoma  of  the  pancreas.  This  patient  was 
admitted  and  studied  mostly  by  the  Endocrinology  Branch  but  material  was 
used  in  a  number  of  correlated  biochemical  and  histologic  studies. 
K   patients  with  extensive  melanomas  and/or  basal  cell  carcinomas  were 
admitted  especially  to  obtain  tissue  for  special  studies  by  investigators 
in  other  branches.  It  is  difficiilt  to  differentiate  patients  admitted 


PROJECT  REPORT  FORM  (Cont'd) 


PAGE  2 


NCI~750(C). 


SERIAL  m . 


Patlenlii,  Material;  (Continued) 

specifically  for  this  project.  However,  127  minor  procediores  were  performed 
for  other  branches  of  the  Cancer  Institute,  and  62  minor  operative  pro- 
cedures were  performed  for  other  institutes  on  patients  admitted  by  those  ; 
institutes  to  obtain  tissue  for  biopsy  and/or  biochemical,  biophysical  or 
histochemical  studies. 

Ma.ior  Findings; 

Presented  in  reports  of  other  projects  by  other  investigators. 

Significance,  to  Cancer  Research; 

This  project  is.  more  or  lesjs  a  service  function  for  other  branches 
of  N.C.I,  and  N.I.H. 

Proposed  Course  of  Project; 

The  number  of  such  cases  will  continue  at  about  the  present  level 
in  proportion  to  the  patient  load. 


11, 


BUDGET  ACTIVITY 


RESEARCH  /X: 

REVIEW  &  APPROVAL     / .  "7 


ADMINISTRATION 

TECHNICAL  ASSISTA1>ICE     /Z7 


(No  entries  for  items  12,  13,  15  &  l6) 


PAGE  1 
PROJECT  R:PORT  FORM 


1.   Matlonal  Cancer  Institute       2.   Surgery  Branch 

■  p:sTiTuTE  '  ■      ij:Bmnmrm~mKEm 

3. h.  5.  NGI-75l(C) 

SECTION  OR  SER.'Kfe  LCC/iTlOlvi  (lE  OTiiER  T^L^J?  j^^Tilii^M)        SERIAL  JJO. 

Evaluation  of  Radical  Surgery  and  the  Development  of  New  Surgical 
6.       Techniques  as  a  Therapeutic  Ifcans  of  Palliating  or  Definitive  Therapy 
PROJECT  TITLE  '  ;       of  Advanced  Cancer. 


7.       Dr.  Robert  R.    Snith 

PRIMCIPAL   IWESTl(lVrCR(S ) ■ 


Dr.    John  11.  Waite  -  Dr.  Williaj-n  E.    Schatten 

OTHER  i-westig;.t:rs  " 


9.   PROJECT  DESCRIPTION 

Objectives:  The  objectives  of  this  project  are  to  develop  methods  of 
obtaining  increased  palliation  and/or  definitive  cure  of 
advanced  crncer,   present  day  therapy  depends  u.pon  the 
complete  removal  of  cancer  or  its  local  destruction  by 
physical  agents  J  I.e.,    svrg^r^j,   radiation,  or  chemotherapy. 
Salvage  by  any  of  these  methods  is  low  (l5-30%),  and  points 
to  the  ineffectiveness  of  the  tools  that  we  now  have  avail- 
able. Examples  of  this  type  are  found  especially  in  cancers 
of  the  pelvis  and  in  the  head  and  neck  areas.  Clinical 
material  from  these  two  areas  is  readilj/  available. 

^iethods  employed:  Patients  with  advanced  cancer  of  the  pelvis  and/or  head 
and  neck  areas  are  admitted  for  study.  These  patients  do 
not.  have  dissemination  of  their  disease  beyond  the  opera- 
tive area.  This  project  is  in  realitjr  composed  of  a  number 
of  projects  of  specific  sites  but  because  of  the  relative 
infrequency  of  cancer  of  specific  areas,  studies  must  be 
arranged  to  take  advantage  of  the  clinical  material  as  it 
becomes  available.   In  the  report  of  a  v-ear   ago,  study  of 
paranasal  sinus  cancer  was  Mentioned.  Seven  patients  with 
this  tjn^e  of  cancer  were  admitted  for  surgery  and  in  only 
two  were  conditions  right  for  the  more  radical  type  of 
surgery  to  be  performed.  This  surgery  consisted  of  resection 
of  t:  e  floor  of  'the  anterior  cranial  vault  including  the 
cribriform  plate,  contents  of  the  nose,  the  orbit,  and  the 
involved  antrum  nnd/or  hard  palate.  T\irenty-eight  primary 


PROJECT  REPORT  FQffi4  (Cont'd)  PAGE  2 


HCI-75l(C) 
SERIAL  NO. 

tlethods  employed;  (Continued) 

carcinoinas  oj?  the  cervix  were  admitted,-  The  rndicsl 
surgery  consisted  of  pelvic  lyraphadenectomy  associated 
with  the  radical  hysterectomy  and/ or  pelvic  exenteration. 

Patient  material;  As, noted  in  the  previous  project  reports  there  is  .   .  '  • 
considerable  overlap  of  patient  materialj  that  is,  a 
patient  is  used  in  more  than  one  project.  There  were, 
however,  7  patients  admitted  for  the  prim?r  antrum 
study  and  28  patients  adinitted  for  study  ox  cervix  canccr»  "  • 
Tht.  total  number  of  hospital  days  for  thxs  project  was 
l666  with  102  out-patient  followup  visits. 

Major  findings;  Of  the.,  28  peryix,.gast,.s,  adiiiitted,  some  of  which  were   • 
admitted  and  treated  in  the  previous  3''ear  but  never 
reported,  17  pelvic  exenterations  were  performed.  An 
additional  8  patients  had  radical  hysterectom;/  and  node 
dissections,  and  1  patient  had.:a.  radical  resection  of  a 
cervical  stump  cancer,  plus  radical  iliac  node  dissection. 
Complete  analysis  of  this  group  of  patients  is  not  possible 
at  this  time,  mainly  because  a  sufficient  length  of  time 
has  not  elapsed  since  surger",-  to  allow  adequate  evaluation. 
It  is  possible  at  this  time  to  note  that  in  our  hands  as 
the  surgery  is  perf orrbed  at  this  hospital  the  operation  is 
relatively  safe.  There  has  been  one  postoperative  death 
in  this  group.-  This  patient  died  about  ten  days  after 
radical  pelvic  exenteration.  She  died  as  a  result  of 
intestinal  obstruction  complicated  b^r   peritonitis.  We 
feel  that  for  the  tyoe   of  clinical  material  presented  at 
this  hospital  the  radical  surgical  approach  to  advanced 
pelvic  cancer  gives  worthwhile  palliation  and  increased 
longevity.  More  complete  analysis  of  this  group  of  patients 
-  •   should  be  available  x-jithin  the  next  year. 

Of  the  7  paranasal  sinus  cancer  patients,  only  2.  of  them 
■  were  suitable  for  the  radical  resection  described  above. 
Both  of  these  patients  have  done  x^fell  and  are  still  free 
of  disease  after  a  relatively  short  postoperative  period. 
Our  experience  with  this  small  num.ber  has  shown  that  the 
operation  is  safe.   It  does  not  produce  objectionable  deformi- 
ties and  is  well  received  by  the  patients.   In  2  of  the 
patients  admitted  for  this  stud;/-  the  disease  was  found  to 
extend  into  the  middle  cranial  fossa  and  definitive  surgical 
procedure  could  not  be  performed.   In  one  of  the  7  antral 
cases,  the  patient  died  with  disseminated  cancer  throughout 
his  lungs  and  mediastinum.  This  patient  had  a  radical 
resection  of  the  maxilla  but  not  the  anterior  cranial  fossa. 
The  other  patient  had  a  modified  radical  antrum  resection 
and  is  free  of  disease  at  the  present  time.   The  7th  patient 


PROJECT  REPORT  FORM  (Cont'd)  PAGE  .3 


NCI-75l(C) 

SERi^'.L  WO. 


Major  findings;   (Continued) 

has  had  surgery  performed  only  a  short  time  and  is  not 
available  for  evaluation. 

Significanco  to  Cancer  Research:  We  believe  that  the  developraent  of 
effective  means  of  palliating  and/or  curing  advanced 
cancer  is  one  of  the  fundaraental  purposes  of  the  Cancer 
Institute's  program.  Documentation  of  the  course  of 
cancer  of  these  areas  as  the  patients  continue  through 
the  course  of  their  disease  adds  considerably  to  the 
total  knowledge  of  the  natural  behavior  of  cancer.   It 
should  also  be  pointed  out  that  this  project  provides 
considerable  material  for  both  excised  adjacent  normal 
tissues  as  well  c?.s  the  cancer  itself  for  scientists  to 
use  in  other  projects  in  the  Cancer  Institute. 

Proposed  course  of  project:  During  the  coming  year  it  is  proposed  to 

contijiue  the  studies  as  outlined  above.   It  is  anticipated 
that  in  the  future  as  in  the  past,  one  of  the  m.ain  problems 
with  pelvic  cancer  which  requires  the  excision  of  the  urinary 
bladder  is  a  means  to  control  the  urinary  stream.  Studies 
are  underwaj'^  at  the  present  time  in  which  the  ureters  are 
placed  in  ?n  isolated  loop  of  bowel.   This  looks  promising 
but  is  still  not  the  ideal  method  of  maintaining  adequate 
urinary  drainage.   It  is  hoped  that  additional  cases  of 
advanced  but  locally  operable  paranas.-'l  sinus  cancer  will  be 
obtained  in  order  to  f-urther  study  the  neuro-physiolor-ical 
changes  involved  in  this  type  of  radical  resection.  As  in 
the  past,  it  should  be  pointed  out  that  the  neurosurgical 
portion  of  paranasal  sinus  study  was  undertaken  in  conjunction 
with  neurosurgeons  from  the  Institute  of  i'eurological  Diseases 
and  Blindness. 

11. 

BUDGET  ACTI\^ITY: 

RESEARCH  /x/         ADMDJISTRiiTION  £7 

RE\?IE\'if  &  APPROWvL  [J  TECHNICAL  ASSISL'.NCE  [1 

MO  ENTRIES  FOR  ITEMS  12,  1,3,  lU,  15  &  l6. 


PAGE  1 
PROJECT  REPORT  FOM 


1.     NationaJ.  Cancer  Institute ...,,      2,     .Surgery  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

3.  4, _»«__ 5.  NCI-752(C) 

.  SECTION  OR  SERVICE    LOCATION  {IF  OTHER  THAN  BETHESDA)     SERIAL  NO. 

6.  Clinical  Investigation  in  the  use  of  Viruses  in  the  Treatment  of  Human  Cancer. 
PROJECT  TITLE 


7.  Dr,.  R.  R.  Smith  and  Dr.  R^  J«  Huebner 

PRINCIPAL  INVESTIGATOR(S) 

8.  Dr.  W.  E.  Schatten.  Dr.  L.  B.  Thomas  and  Dr.  W.  P.  Rowc 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives: 

To  develop  means  of  treating  human  cervix  cancer  using  cultin'e  fluid 
containing  live  virus,  attempting  to  reproduce  in  vivo  the  in  vitro 
observation  of  the  destructive  action  of  viruses  on  cells  in  tissue  culture. 

Methods  Employed; 

APC  (adenold-pharyngeal-conjunctlval)  viruses  grown  in  5%   chick  serum 
in  Hanks-Simms  solution  containing  HeLa  cells  was  filtered  through  a 
sintered  glass  filter  and  tested  for  sterility  and  safety.  This  fluid 
was  then  injected  by  direct  needle  injection  into  the  cervix  tumor  mass 
via  the  vagina,  and  from  above  directly  into  the  mass,  or  by  intra-arterial 
cannula  passed  up  through  the  femoral  artery  into  the  lower  abdominal 
aorta.  It  was  necessary  to  determine  the  antibody  levels  of  the  serum  of 
the  patients  Injected  to  make  sure  that  the  proper  virus  was  used.. 
Following  the  injection  it  was  necessary  to  obtain  culture  swabs  from  the 
vaginal  fluid  and/or  biopsy  tissues  or  cervical  scrapings.  Blood  cultures 
were  taken  regularly.  The  patients  were  thoroughly  studied  for  evidence 
of  systemic  manifestation  of  a  virus  disease.  At  the  start  of  this  inves- 
tigation, it  was  determined  that  using  this  agent  in  susceptible  individuals, 
a  local  necrosis  could  be  produced  without  the  production  of  any  recogniz- 
able systemic  disease.  In  a  number  of  instances,  following  the  use  of  the 
local  chemotherapeutic  agent,  more  definitive  surgical  therapy  was  insti- 
tuted. In  8  patients  radical  pelvic  exenteration  was  possible  following 
the  virus  therapy.  In  an  additional  3,  radical  hysterectomy  and  node 
dissection  was  performed.  These  were  standard  operative  procedures  which 
have  been  modified  as  described  in  Project  751(c), 


PAGE  2 
PROJECT  REPORT  FORII  (Cont'd) 


SERIAL  I-D, 


Patient  Material; 

During  the  past  twelve  months  19  patients  were  admitted  especially  for 
this  study,  for  a  total  of  224-2  patient  days.  35  out-patient  visits  were 
made  by  this  group.  As  pointed  out  before,  this  patient  material  was  avail- 
ble  for  a  number  of  correlated  biochemical  and  histological  studies  as  well 
as  the  radical  pelvic  surgery  study  as  described  in  Project  751(C). 

Ma.ior  Findings: 

To  date,  28  patients  have  received  virus  fluid  for  therapeutic  studies 
of  their  cervix  cancer.-  kn.  additional  6  patients  received  control  material 
consisting  of  the  tissue  culture  fluid  containing  everything  but  tlie  virus. 

The  findings  during  the  past  year  have  confirmed  those  given  in  the 
report  last  year;  that  is,  patients  whose  sera  does  not  contain  antibodies 
to  APC  virus  develop  an  area  of  necrosis  at  the  site  of  injectionj  when 
antibodies  are  present  such  necrosis  has  not  been  observed.  In  24-  patients 
that  have  been  more  thoroughly  evaluated,  injection  of  these  viruses  in 
amounts  up  to  ADO  cc.  of  ctilture  media  did  not  produce  appreciable  systemic 
disease.  On  three  occasions,  an  influenza-like  disease  was  produced,  con- 
sisting of  general  malaise,  photophobia,  and  fever.  These  symptoms  dis- 
appeared without  therapy  in  a  few  days ,  There  was  no  pharyngitis  or  con- 
jionctivitis  as  observed  in  upper  respiratory  infections  caused  with  APC 
viruses  occurring  spontaneously.  Local  necrosis  progressed  to  extensive 
cavity  formation  in  the  vaginal  pelvic  portion  of  the  cancer  in  20/5  of  the 
patients.  There  was  moderate  slough  of  the  lesion  in  52^  of  the  patients, 
in  20^  a  slight  to  questionable  necrosis  was  observed,  and  in  S%  no  response 
was  observed.  Injected  virvis  was  recovered  from  tissue  biopsies  and/or 
vaginal  smears  as  late  as  17  days  after  the  treatment,  A  prompt  antibody 
rise  was  observed  in  all  cases.  Extensive  cyi;o-histologic  studies  of  • 
repeated  biopsies  and  smears  failed  to  show  aiay   specific  recognizable  effect 
that  could  be  attributed  to  the  virus.  Increasing  amoiints  of  necrosis  were 
observed  on  biopsies  taken  during  the  period  of  slough  but  rtb  inclusion 
bodies  or  other  specific  viral  effects  were  demonstrated.  In  no  instance 
was  there  complete  destruction  of  the  cancer  observed,  Regrowth  of  the  tumor 
was  observed"  in  the  area  of  slough  as  early  as  10  days , 

It  soon  became  apparent  that  the  drug  did  produce  local  changes  which 
we  are  convinced  have  a  specific  effect  upon  the  cancer  tissue.  No  obser- 
vable effect  has  been  noted  oh  the  other  tissues  of  the  body.  It  is  apparent, 
However,  that  the  prompt  rise  of  antibodies  prevents  the  continued  growth  of 
the  virus  in  the  tissue,  thus  limiting  its  effectiveness  as  a  destructive 
agent.  Attempts  have  been  made  to  use  cortisone  in  increasing  amounts.  This 
did  not  prevent  the  rise  of  antibodies.  Its  effect  on  the  local  lesion  in 
conjunction  with  the  virus  used  remains  to  be  settled.  It  did  seem,  however, 
that  the  best  results  of  the  virus  were  obtained  in  patients  that  were  closer 


PAGE  3 
PROJECT  REPORT  FORM  (Cont'd) 

NCI-752(C) 

SERIAL  NO. 

Ma.ior  Findings:  (Cont'd) 

to  the  terminal  portion,  of  their  disease  j  that  is ,  that  they  were  debilitated 
and  more  serio-usly  ill.  This  suggests  that  the  nonspecific  defense  mechanism 
of  the  body  is .probably  a  factor  in  the  limitation  of  the  use  of  this  agent 
as  it  exists  today.      -    '  '    '. 

Significance  to  Cancer  Research; 

This  program  helps  to  carry  out  one  of  the  fundamental  aims  of  the 
Cancer  Institute,  that  is,  of  developing  new  and  more  effective  methods  of 
therapy  of  common  malignant  neoplasms.  It  also  helps  to  identify  and 
categorize  the  newly  discovered  APC  viruses  and  helps  to  delineate  the  re- 
action on  human  tissue  of  the  virus  n^od  in  the  experiment.  If  a  method 
can  be  developed  to  allow  the  propagation  of  this  virus  in  the  tumor  tissue, 
it  is  reasonable  to  expect  that  the  continued  propagation,  not  only  locally 
but  in  the  disseminated  disease,  might  be  developed  into  an  effective  means 
of  controlling  cancer. 

Proposed  Course  of  Pro.iect: 

During  the  coming  year  it  is  hoped  that  this  project  can  be  pursued 
more  vigorously.  Ehjring  the  past  year  and  a  half  it  was  recognized  that 
this  was  a  new  approach  and  that  caution  was  necessary  to  prevent  misi:aider- 
standing  in  using  human  material  in  this  type  of  study.  We  are  sure  now 
that  this  material  is  not  harmfiol,  tliat  it  does  not  produce  aggravation  of 
the  growth  of  the  neoplasm,  and  that  we  are  doing  these  patients  a  service 
by  studying  them  and  providing  the  care  that  is  available.  It  is  believed 
that  the  course  the  project  should  take  is  as  follows: 

(l)  As  noted  in  the  i^  vitro  study  of  the  effect  of  this  virus  on 
HeLa  cells,  the  degree  of  destruction  is  directly  proportionate  to  the 
concentration  of  the  virus  present  in  the  fluid.  It  is  hoped  that  this 
virus  can  be  concentrated  100  to  1000  times  so  that  tremendous  amounts 
of  this  material  can  be  given  to  the  patients  in  a  smaller  volume  of 
fluid.  This  is  a  problem  in  production  of  virus  which  is  being  studied. 

(2)  The  serial  use  of  APC  viruses  whose  antigenicity  is  not  exact 
and  will  allow  the  use  of  multiple  viruses  through  multiple  injections 
to  produce  the  same  type  of  effect. 

(3)  Continued  studies  to  alter  the  host  resistance  to  infections  in 
an  effort  to  produce  a  viremia  which  would  allow  contamination  of  all 
of  the  cancer  cells  and  a  more  complete  destruction. 

(4)  Repeat  the  histologic  study  in  an  effort  to  definitely  detennine 
the  mode  of  action  of  this  material. 


PAGE  i 
PROJECT  REPORT  FORM  (Cont'd) 


-JJCJr25£(£l 

SERIAL  NO. 


11. 


BUDGET  ACTIVITY 

PkESEARCH'      ■   ■    .         /Tj  ADMINISTRATION    -  /~7 

REVIEW  &  APPROVAL  /~7  TECHNICAL  ASSISTANCE  /"~7 


(No  entries  for  items  12,  13,  H  and  16) 


PACE  1 
PROJECT  REPORT  FORI! 


1.  National  Cancer  Institute'         2.    Surgery  Branch 

INSTITUTE  lABOPJlTORY  OR  BRANCH 

3. ■  4.._ .       5.  NCI=2i   _ 

SECTDN  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  10  . 

Cytologic  Determination  of  Number  of  Tumor  Cells  and/or  Recurrences 
6.  -"Recovered  or  Found_.ln  an  Operative  Vtound  Following  Removal  of  a.. Primary. 
""project  TITLE  Cancer  in  Continuity  with  its  Regional  Lymph  Drainage 

■  Area  Containing  Ibtastascs . 


7.     Dr.  R.  R.  Smith 


PRINCIPAL  DIVESTIGATOR(S) 


3.    Dr«  A.  ]/l.   Kilbcr&-.  Dr.  J. .  K.  V/aite  and. Dr.  W.  E.  Schatten 
OTHER  INVESTIGATORS 


9,  PROJECT  DESCRIPTDN 
Ob.icctives : 

This  project  is  the  clinical  part  of  PrdjectNo.  754  dealing  with 
the  seeding  of  operative  wounds  ty  cancer  cells.  Its  primary  objective 
is  to  demonstrate  the  frequency  and  the  nature  of  tumor  cells  found  in 
the  washings  obtained  from  operative  vrounds  from  which  operable  cancers 
have  been  removed. 

Methods  Employed; 

..•Patients  with  locally  advanced  priinary  cancers  of  the  head  and  neck, 
•  pelvis,  or  other  appropriate  sites,  with  regional  lymph  node  metastases j 
have  .been  admitted  for  primary  surgical  therapy.  For  , the  most;  .pair t,  ,  ,.• 
these  cases  have  been  those  with  extensive  metastases,  in -other  words, 
•  the  borderline  operable  ones.  The  surgery  performed  is  mostly  the  standard 

operative  procedures  accepted  as  treatment  for  the  specific  cancer  sites. 
.  In  part,  these  same  patients  provided  material  for  Project  751(C)  in 
which  the  limits  of  opcrability  have  been  f lurther  defined  by  a  more  exten- 
sive resection  with  and  \./ithout  primary  reconstruction. 

Following  the  removal  of  the  tumor  and  its  metastases,  the-  ctperative 
-area  is  thoroughly  washed  \;ith- a  fine  saline  spray.  The  aspirate  is 
fixed  in  an  alcohol-ether  mixture  and  in  the  Pathology  Lab  is  centrifuged 
down,  tiie  sediment  being  treated  by  laking  the  red  cells  which  are  present 
in  the  fluid.  The  remaining  sediment  is  then  smeared  and  studied  in  the 
usual  Papanicolaou  technique  and/or  clTompod  together  in  a  block  and  the 
usual  histologic  sections  prepared. 


PAGE  2 
PROJECT  REPORT  FORI!  (Cont'd) 


NCj:~753(C) 

SERIAL  NO. 


Patient  !'atcrial; 

25  patients  were  admitted  specifically  for  this  project  for  a  total 
of  1737  hospital  days,  100  outpatient  visits  were  made  by  these  patients. 
It  should  again  be  pointed  out  that  nearly  all  these  patients  were  used 
in  other  studies,  and  in  some  patients  admitted  for  other  projects,  wound 
washings  were  taken.  This  was  demonstrated  by  the  fact  that  during  the 
calendar  year  of  1955,  72  different  patients  had  wound  washings  studied 
in  the  Cytodiagnostic  Laboratory.  .   .  ._ 

Major  Findings; 

Complete  analysis  of  all  72  cases  is  not  available  at  this  time,  "The 
results  of  a  study  of  36  of  these  cases  have  been  tabulated  and  was  pub- 
lished in  the  December  1955  issue  of  the  J.N.CI, 

In  summary  of  the  36  cases,  woimd  washings  were  positive  in  9  instances 
and  in  an  additional  5  cases  the  washings  contained  suspicious  cancer  cells. 
When  the  wo\and  wasliings  were  positive,  local  recurrences  had  already 
developed  in  2  of  these  patients  arid  in  an  additional  case  in  which  the 
washings  were  suspicious.  In  2  instances  where  the  washings  were  negative, 
local  recurrences  had  already  occurred. 

The  finding  of  clumps  of  tumor  cells  in  25^  of  this  small  series 
would  certainly  make  one  suspect  that  this  figure  \Jould  represent  a  minimimi 
number  of  cases  that  could  be  expected  to  demonstrate  local  recurrence  of 
tumor.  However,  it  should  be  pointed  out  that  the  finding  of  tumor  cells 
in  a  wound  is  not  necessarily  assiorance  that  a  recurrence  will  develop. 
It  is  possible  that  washing  of  the  woond  would  destroy  the  implants,  and 
the  host  factor  which  allows  the  recurrence  to  develop  could  possibly  con- 
trol the  remainder.  On  the  other  hand,  the  finding  of  negative  wound  wash- 
ings would  be  no  guarantee  that  recurrences  would  not  develop  because  of 
the  difficulty  in  assin-ing  ourselves  that  the  washings  are  truly  negative. 

The  important  point  of  this  work  to  date  has  been  that  malignant  cells 
in  wound  washings  can  be  relatively  easily  identified.  Most  of  the  washings 
that  were  examined  contained  no  isolated  tumor  cells  but  small  fragments 
which  could  be  readily  identified.  In  one  instance  this  contained  a  piece 
of  cancer  one-tenth  of  a  millimeter  in  diameter.  Cell  structure  could 
very  easily  be  identified  in  this  case.  In  all  instances  cells  or  clumps 
of  cells  seen  in  the  wound  washings  were  comparable  when  compared  with 
the  tissue  biopsies  of  the  surgical  spcciiiicn.  In  all  cases  in  which  the 
washings  were  considered  positive,  the  tumor  cells  identified  corresponded 
in  morphological  characteristics-  with  the  tumor  cells  seen  in  the  tissue 
section. 


rJIJ'JECT  REPORT  ETCRM  (Cont'd) 

PAGE  3 


NCI-753(C) 


SERIAL  m . 


Significance  to  Cancer  Research; 

Demonstration  of  cancer  cells  in  operative  wounds  again  points  up  the 
deficiency  in  our  present  methods  of  cancer  therapy.  On  the  positive  side 
it  demonstrates  a  possible  cause  of  failure  of  this  type  of  therapy,  and 
certainly  suggests  a  means  of  approaching  the  problem  from  a  chemotherapeutic 
standpoint. 

£rgpP8e^  Cpuygg  of.Prp.Jep.t; 

It  is  proposed  to  continue  this  Study  as  outlined  above,  further  delin- 
eating the  frequency  and  the  characteristics  of  a  seeded  wound.  It  is  hoped 
that  this  series  will  be  allowed  to  increase  to  the  point  where  it  will  be  a 
statistically  significant  one.  It  will  be  necessary  to  continue  to  follow  the 
patients  that  have  been  so  treated  over  the  next  2-3  years  to  further  determine 
the  ratio  of  local  recurrence  and  failvire  of  therapy  to  the  finding  of  tumor 
cells  in  an  operative  wound. 

It  is  hoped  that  within  this  next  year  the  results  of  the  laboratory  part 
of  this  experiment  (Project  754)  will  have  progressed  to  the  point  where  chemo- 
therapy of  operative  wounds  can  be  brought  to  the  operating  room. 

Ll,  BUDCET  ACTIVITY; 


RESEARCH       Al  ADIIINISTRATION 

REVIEW  &  APPROVAL  /Zj  TECmilCAL  ASSISTANCE 


.5. 


PUBLKATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR  ZEAR  1955 


Paper  entitled  "Cancer  Cell  Seeding  of  Operative  Wounds" 
By:  Robert  R.  Smith  and  Albert  W,  Hilberg 
Published  in  J,N.C,I.,  Vol,  16,  No.  3,- December  1955 


(No  entries  for  Items  12,  13  and  l6) 


PJ.CiE  1 


PROJECT  REPCRT  FORM 


1.   National  Cancer  Institute       2.   Surgery  Branch 


3. h.  ■     5.  NCI-75U 

SECTIOfI  OR  SEli;Kii  '        LOd/.'i'lOM  (Ih'  Ol.m  mU\T  BETHSSDa)        fJi^Ria  Mu. 

Evaluation  of  the  Nature,  Cause,  and  Means  of  Preventing  Seeding 
6,  of  Operative  Wounds  Using  Transplantable  Animal  Tumors. 

PROJECT  TMLE 


7.       Dr.  Robert  R.   Smith 

PR Wc ipA L  t^iV£6Tli\T0R(  S ) 


8.  Mr.   Richard  V.  Eck  -  Dr.   John  H.  Waite  -  Dr.   Ross  Miller  -  ^ 

OTHER  IlWESTId'.TCRS  Dr.  Arthur  Ship  -  Dr.  William  Kramer 

9.  PROJECT  DESCRIPTION 

Objectives; 

The  objectives  of  this  project  have  been  broadened  during  the  past 
year  from  methods  of  producing  experimentally  in  animals  multiple 
recurrent  tumors  in  operative  wounds,  to  the  establishment  of  the 
standard  experimentally  seeded  operative  wounds  which  provides  methods 
of  testing  the  efficacy  of  various  therapies  aimed  at  preventing  the 
wound  seeding. 

Methods  employed; 

As  pointed  out  in  Project  No.  753(C)  a  frequent  finding  in  radical 
surgical  therapy  of  cancer  is  the  presence  of  tumor  implants  in  the 
operative  wound.  In  trying  to  evaluate  the  effectiveness  of  any  treat- 
ment applied  to  an  operative  wound,  and  in  order  to  demonstrate  the 
safety  as  well  as  the  effectiveness  of  any  given  chemotherapeutic  regime, 
it  is  believed  necessary  to  develop  in  the  animal  a  situation  which  is 
comparable  to  that  in  the  human.  This  would  allow  a  statistical  analysis 
of  a  given  form  of  therapy  over  a  short  period  of  time. ,  In  order  to 
develop  this  experimental  tool  the  V^  carcinoma  in  rabbits  has  been 
utilized  to  produce  in  our  hands  a  seeded  operative  wound  which  in  practi- 
cally 100>?  of  the  cases  produces  multiple  tumor  implants  in  the  operative 
wound  within  a  2  to  3  week  period.  K  ascites  tumor  injected  intraperi- 
toneally  produces  numerous  implants  in  the  peritoneal  cavity.  S-91  mouse 
melanoma  produces  multiple  implants  in  the  lungs  when  injected  intravenous!-, 
or  subcutaneously  in  thst   area,  Mr.  Richard  Eck  has  developed  a  method  of 
raising  an  air  bubble  on  the  back  of  a  mouse  by  injecting  air  in  a  subcu- 
taneous pocket.  Turaor  suspension  injected  into  this  area,  following  which 


RiGE  2 
PROJECT  RiiPCRT  FOM  (Cont'd) 


NGI-75U       ■ 

Methods  employed;   (Continued) 

2 

the  air  is  removed,  produces  a  wound  with  multiple  implants.  With  V 
carcinoma,  dissection  of  the  axilla  and  removal  of  the  pectoral  muscles 
produces  a  setting  very  similar  to  that  seen  in  the  operating  room  in 
which  a  radical  mastectomy  was  done. 

During  the  past  year  the  procedure  has  been  perfected  to  the  point 
where  the  three  types  of  tianor  described  above  have  been  standardized 
so  that  in  our  hands  they  produce  consistent  results.  The  three  trans- 
plantable tumors  provide  the  opportunity  to  test  the  cancer  study 
qualities  of  a  compound  or  a  procedure  in  vitro  as  well  as  in  vivo.  ^ 
mixing  in  the  test  tube  the  compound  to'Te  tested  with  the  inoculum,  allo-v 
ing  it  to  remain  in  contact  for  a  stated  period  of  time  and  then  applied 
in  the  air  pocket  or  intravenously  or  intraperitoneally,  would  allow  a 
fast  screening  method  to  determine  the  carcinolytic  properties  of  a  given 
compoimd.'*^   .   -   .    • 

Ano-jiher  factor  which  requires  study  is  the  safety  of  the  use  of  a 
given  compound  oij  tissue.  It  is  believed  that  any  compound  that  would 
be  of  any  value  clinically,  inust  not  damage  to  any  degree  large  blood 
vessels  or  adjacent  nemres.  Any  extensive  necrosis  of  normal  tissue 
would  contra indie ate  its  use  in  such  a  study.  To  test  the  efficacy  of 
this  a  system  has  been  developed  whereby  the  brachial  plexus  of  the  mouse 
and  rabbit  is  exposed  and  the  chemical  under  study  is  applied  directly  to 
the  brachial  plexus  and  observations  made  in  this  manner.  An  additional 
test  for  the  safety  of  the  drug  under  study  was  obtained  by  washing  the 
operative  wound  on  one  side  of  the  animal  and  leaving  the  opposite  side 
contaminated  with  tumor  tissue  to  grow  untreated.  In  most  instances 
these  animals  acted  as  their  own  control. 

Major  findings; 

1.  Using  the  methods  outlined  above  it  has  been  shown  that  the  washing 
of  the  wound  with  a  saline  spray  caused  a  decrease  in  the  number  of 
implants  that  grew,  but  in  no  instance  was  it  possible  to  prevent  the 
development  of  tumor  implants  by  the  spray  washing  or  blotting  of  the  woui: 
with  saline,  '   ■ 

2.  A  group  of  animal  tumor  implants  were  tested  with  podophyllin  resin 
drugs,  pod'ophyilin,  and  alpha  peltatin.  Toxicity  studies  showed  that 
over  10  rag.  per  kilo  of  alpha  peltatin  produced  severe  generalized  tcaic 
manifestations  and  local  toxicity  consisting  of  d^^mage  to  nerves  was 
apparent  with  as  little  as  1  milliliter  of  f  mg.^  alpha  peltatin  producing 
nerve  paralysis.  Mere  concentrated  solutions  of  th-e  drug  produced  a  co- 
agulating type  of  necrosis  involving  tte  entire  axilJLa.  In  l6  animals  ir. 


PAOE  3 
PROJECT  REPORT  FORK  (Cont'd) 


mi-ish 
SERIAL  MO. 

Major  findings;      (Continued) 

which  the  wound  was  washed  with  alpha  peltatin  solution,   allowing  the 
drug  to  remain  in  the  wound,   the  tumor  continued  to  grow  luxuriantly 
in  all  dilutions  well  into  the  range  where  toxic   symptoms   occurred.      In 
fact,   it  was  the  observer's  impression  that  in  certain  instances  the 
alpha  peltatin  seemed  to  cause  the  tumor,   if  anything,   to  grow  better 
on' the  treated  side  as  compared  with  the  controlled  side. 
3.     Additional  chemicals  -  NCI  -  1136,   3022,   lQ9U,  podophyllotoxin, 
alpha  peltatin  and  crude  podophyllin  resin,   were  all  ineffective   in 
reducing  the  number  of  lung  tumors  in  S-91  melanoma  injected  intravenously. 
In  some  experiments  these  compounds  caused  an  increase   in  the  number  of 
lung  tumors  up  to  100%.     The  possibility  of  this  being  due  to  a  stress _ 
phenomena  seemed  to  be  borne  out  when  the  same  results  Xirere  obtained  with 
heat  stress,   cortisone  or  systemic  formaldehyde   stimulation.     The  possi- 
bility of  using  systemic  podophyllin-t^npe  drugs   in  conjunction  with  local 
washing  was  tried  but  inconclusive  results  were  obtained.     Because  of  the 
extensive  local  tissue  damage  it  was  suspected  that  this  drug  in  its 
present  form  has  very  little  to  offer  at  the  clinical  level  at  this  time. 
h.     Formaldehyde  seems  to  be  the  most  effective  m.eans  yet  available  to 
prevent  the  seeding  of  operative  woionds.     Toxicity  studies  in  mice  and 
in  rabbits  as  outlined  above  show  that  in  mice  1%  formaldehyde  left  in 
contact  x^rith  the  brachial  plexus  for  five  minutes  produced  no  symptoms. 
In  rabbits,    using  a  |%  formaldehyde,   no  damage  could  be  demonstrated  to 
the  brachial  plexus  in  the  11  animals  tested.     1%  solution  left  in  contact 
with  nerves  for  20  minutes  produced  nerve  damage.     |^  formaldehyde  solu- 
tion prevented  the  growth  of  tumor  implants  in  3  of  5  animals  tested  and 
allowed  a   single   implant  to  develop  into  others.      In  saline  control  ani- 
mals,  consistent  results  were  obtained  in  growing  large  numbers  of  implants 
in  the  area. 

5.  27  different  chemicals  have  been  tested,  both  in  vivo  and  in  vitro 
studies.     The  ones  that  show  promise  of  results  and  possible  application 
at  the  clinical  level  are   (1)  citric  acid,   a  1%  solution  which  lowers  the 
pH  to  2  which  increases  to  2.5   in  contact  with  tissues.     A   1;^  solution 
was  between  90%  and  95%  effective  in  preventing  the  growth  of  K     ascites 
tunor.     At  the  other  end  of  the  spectrum,    sodium  carbonate  with  a  pH  of 
11.2  dropoing  to  10.9  when  in  contact  with  the  tissues  produced  no  appre- 
ciable nerve   injury  when  left  in  contact  with  the  plexus  for  5  minutes, 
and  was  lOO/o  effective  in  the  prevention  of  growth  of  the  K     ascites  tumor. 
(2)  Ethanol,  a  23/^  solution  was  also  100^  effective  in  the  in  vitro  test. 
It  has  not  been  tested  as  yet  in  the  actual  operative  wound  seeding, 

6.  f^ertonic  solution,   such  as  10^  sodium  chloride,   is  also  effective 
in  diminishing  the  number  of  tumor  implants. 


,^ 


PAGE  h 
PROJECT  REPORT  FORM  (Cont'd) 

NCI-75U 

SERL-.L  NO. 

PROJECT  DESCRIPTION  (Continued) 

Significance  to  Cancer  Research; 

With  the  development  of  methods  to  artificially  create  a  seeded 
■operative  wound  it  has  been  shown  that  a  standard  formaldehyde  solution 
produces  results  which  will,  if  applicable  to  humans,  greatly  increase 
the  effectiveness  of  cancer  therapy  as  it  exists  today.  The  immediate 
applicability  of  this  type  of  procedure  to  the  clinical  cancer  problem  as 
it'  exists  offers  opportunities  for  immediate  benefits  even  though  the 
ijltimate  answer  for  cancer  will  probably  not  reside  in  this  study. 

Proposed  course  of  project; 

It  is  proposed  to  continue  the  studies  of  delineating  the  effects 
of  these  chemicals  on  seeded  wounds,  seeking  to  find  the  ideal  chemical 
which  will  prevent  100^  the  seeding  of  an  operative  woiind  and  at  the 
same  time  leave  the  wound  in  a  condition  which  will  allow  primary  heal- 
ing. 

It  is  recognized  that  eveh  though  therapy  of  an  operative  wound  would 
become  100^  effective  in  preventing  thirMevelopraent  of  tumor  implants, 
therapy  of  cancer  would  still  not  be  100^  effective  because  of  the  disser "  - 
nation  of  the  tumor  implants  which  occurs  at  the  time  of  surgery  or  shortl;/ 
thereafter.  It  seems  reasonable  to  expect  that  the  ideal  therapy  would 
entail  a  drug  which  could  be  used  systemically  and  would  be  effective  in 
preventing  the  implantation  of  lung  or  liver  metastases,  and  locally  to 
prevent  the  implantation  of  tumor  cells  in.  the  tissues  of  the  operative 
wound.  Dr.  William  Kramer  is  investigating  the  use  of  colchicine -like 
drugs  and  Dr.  Roes  Miller  the  use  of  several  arsenic  compounds  to  try 
to  fulfill  these  criteria. 

11- 

BUDGET  AdTlVlTY: [ 

RESE/iRCH  /X7       ADMINISTR^iTION  £7 

■  REVIEW  k  APPR0V;.L  /~7         TECHNia.L  .ISSISTANCE  £7 

NO  ENTRIES  FOR  ITEMS  12,    1.3,   lU,    l5  &  l6. 


PAGE  1 


P.iOJjiCT  RLPOPiT  FORM 


1.        National  Cancer   Institute  2.       Surgery  Branch 

-TFHTTfTJTE "     LA.JQRATQHY  OH  bHAKCH 


3.  '• U. 5.  NCI-735(C) 

SiiCtiffl   OS  S,:ft\/lCr.  LOGATIOi:   (IF  OTHLaii  THAij   BETHESDAi       SERIAL  NO. 

The  Development  of  New  Methods  for  Treatment  of  Lung  Cancer  Using 

6.  Regional  Chemotherapy:      Clinical  Research 

PROJECT  Ti'i'L^ 

7.  Dr.    John  H,  Waite 


PRKCIpAL   liJ7ESTllrATUk(b) 
Dr.  William  Kramer 


9.    PROJECT  DESCRIPTION 

Objectives;  The  chief  p-urpose  of  this  project  is  to  improve  treatment 
of  those  ccncers  of  the  lung  which  are  found  to  be  beyond 
hope  of  surgical  cu.re  b^'  resection. 

Methods  employed;    The  plan  of  attack  is  one   of  combined  surgery  and 
chemotherap3^.     l/hen  open   thoracotomy  reveals  a  tuaor  not 
amenable  tO'  surgical  resection^   catheters  are  implanted  in 
the  vascular  channels   supplying  the  diseased  lung,   and 
exteriorized  thr'ou?;h  the  chest  wall.     After  the  patient  has 
recovered  from  his  thoracotomy;    a  potentially  chemothera- 
peutic  agent  such  as  nitrogen  mustard  is  injected  thorough 
the  catheter.     Serial  rnessurements   of  the   si7;e  of  the  x-ray 
shadoi'j  cast  by  the  tunor  are  made.      Changes^    if  observed, 
are  compared  with  the  changes  in  other  lesions  in  untreated 
portions   of  the  lung  fields   if  other  lesions  exist.     Although 
the  project  was  conceived  primarily  to  get  a  high  dosage  into 
primary  lung  carcinoma,    a  certain  percentage  of  which  have 
been  shown  to  be  slightlj"-  susceptible  to  general  chemotherapjr, 
it  was  also  planned  to  stud^^  metastatic  tumors  when  discovered 
at  open  thoracotomy  and  unsuitable  for  surgical  excision. 

Thus  far,  we  have  followed  the  policj^  that  it  would  be 
VLnfsir  to  subject  inatients  with  definitely  known  inoperable 
t'omors  to  the  risk  of  thoracotomy  when  the  possibility  of 
benefit  from  regional  chemotherapy  is  as  vet  unproven. 


■^ROJZCT  R.POilT  FORK  (Cont'd)  PA(S  2 


^imm  HO. 


Methods  Employed:  (Continued) 

Therefore,   ^^re  have  careful  .y  worked  up  every  case  before 
surgery,   rejecting  as  unsuitable   those  patients  with 
obviously  non-resectable  tumors.     Most  of  thes^  non-explor-. 
able  patients  are  placed  on  a  suitable   study  progran  in  ihe 
General  Medicine  Branch  while  receiving- palliative  x-ray 
.    ,  therapy.     Lung  tunors  found  to  be  resectable  at  open  opera- 

tion would  of  coarse  be    -dven-' the  ■  benefit.,  of ,  the  best 
excisionsl  sia^ger^'-  availaole,    tlius  viaking  this  sub-group 
also  unavailable  for  regional  chemotherspj''  studies.     The 
excisional  surgery  patients  vjould  contribute  to  lung  cancer 
studies  ^oj  stiiiiulating -our  thoughts  toward  better  surgical, 
procedures,   by  being  available  for  wound  seeding  studies 
under  Project  no.    753(C),   ard  as  subjects  for  early  diagnosis 
studies. 

One   of  the     ajor  points  to  be  worked  out  is  the  exact 
placement  of  the  catheters  with  regard  to  the  blood  supply 
to  the  neoplasm,     Tliree  sets   of  vessels,   viz,    the  pulmonary 
vein,   the  puLmonary  artery  and  the  bronchial  arteries,   are 
available.     Zxpeririients  under  Project  no.   7$6,  Dog  Research 
in  Catheter  Implantation,   are  being  used  to  develop  a  tech- 
nique for  these  different  implantations. 

Patient  i'aterial! 

m  patients  have  been  studied  during  a  total,  of  20  admissions. 
l5  of  these  admissions  were  durjng  the  current  calendar  year. 

No.  Average  Stay  Days 

'Admissions:    Adult  males  9  SO 

Adult  females  '5  60 

Outpatient:    Number  of  patients  2.0 

NuTiiber  of  visits  1)5 

!1ajor  Findings; 

Seven  patients  have  been  found  to  be   suitable  for  the  stucfy 
in  that  woncup  conf  jj^raed  the  presence  of  lung  tumor  and 
possible  curability  hy  excisional  surgery.      Of  these  seven 
patients,   two  proved  to  have  a  bronchogenic  carcinoma  suit- 
able for  pneumonectomy,   and  hence  not  available  for  catheter 
implantation.     No    urinary  bronchogenic  patients  have  yet 
been  found  in  whom  thoracotom;^/  was  indicated,   but  who  were 
found  inoperable. 

In  five  patients  metastntic   tumors  were  found.     Two  of 
these  metastases  were  ap-iarently  solitarj'-  and  it  appeared  - 
best  to.  treat  these"  patients  by  excisional  surgery  which  was 
accordingly  performed.     This  left  tturee  patients  with  metastatic 


PAGE  3 
PROJ..:CT  REPORT   FORM  (Cont'd) 


I\TCI-755(C) 


?''ajor  Findings;   (Continued) 

tumors  available  for  catheter  implantation,  the  catheter 
being;  implanted  in  the  pulmonaiy  arterjr  in  two  cases  and 
in  the  pulmonar?/-  vein  in  one  case.  Nitrogen  mustard  in- 
jected daily  has  been  the  chemotherapeutic  agent  studied 
so  far  to  establish  base  line  values.   One  patient,  with 
metastatic  adenocarcinoma  from  a  colon  primary  showed  no 
apoarent  change  in  the  size  of  her  tumor  until  the  time  of 
death  from  a  cerebral  metastasis , six  weeks  later.  The 
second  patient,  also  with  metastatic  adenocarcinoma  from  a 
colon  primary/,  has  shown  a  slight  regression  of  the  treated 
pulmonarj'-  metastasis  during  the  same  tjjne  that  a  new  meta- 
stasis has  appeared  in  the  contra-lateral  lung  and  grew 
constantly.  Perfect  access  to  the  pulmonary  artery  bed  was 
maintained  in  this  patient  for  the  nine  months  that  lie  lived 
after  catheter  implantation.  Both  of  these  patients  have 
shown  minimaL-  bone  raarroX'J  changes  compared  with  the  changes 
usually  seen  after  intravenous  administration  of  nitrogen 
mustard.  In  one  patient,  with  metastatic  cancer  from  the 
cervix  uteri,  the  catheter  was  implanted  in  a  radical  of 
the  pulmonary  vein  and  the  pulmonarj^  vein  tied  off  between 
catheter  implantation  site  and  the  heart  with  a  thought  to 
gain  access  thereby  to  the  bronchial  vascular  bed.   On 
injection  of  nitrogen  mustard  into  this  patient,  she  developed 
an  abscess  of  the  treated  portion  of  the  lung  which  appeared 
to  start  first  at  the  tuj^ior  site  but  later  spread  to  the 
entire  lobe  requiring  subsequent  lobectomy.   Seven  of  the 
lit  patients  worked  up  have  been  not  suitable,  because  of 
proven  metastases,  or  because  of  the  disease  found  was  other 
than  a  suitable  carcinoma.  Three  of  the  four  far-advanced 
patients  were  fo^ind  suitable  for  m.etabolic  studies  on  Dr. 
Watkin's  service. 

Significance  to  Cancer  Research:  Continuing  increase  in  the  incidence  and 
death  rate  due  to  lung  cancer  demands  maximal  efforts  to 
control  the  disease.  The  primary  or  basic  science  approach 
involves  efforts  to  determine  the  biolocic  nature  and  environ- 
mental relationships  of  lung  cancer.  While  of  great  hope, 
this  research  has  yet  to  produce  applicable  results,  A 
second  phase  is  seen  in  programs  aimed  at  the  discovery  and 
treatment  of  lung  cancer  in  its  earliest  stages.  This  attack 
has  succeeded  in  curing  up  to  ^%   of  all  patients  seen  by 
extirpative  surgery,  the  only  curative  treatment  now  known. 
The  third  phase  of  lun:;  cancer  research  involves  improvement 
of  treatment  of  the  disease  when  it  is  past  its  earliest 
stages,  and  the  large  nuimber  of  well-advanced  lung  cancer 
patients  no\<t   seen  provides  an  urgent  motivation  for  the  clini- 
cal investigator  to  search  for  an  approach  such  as  described 
in  this  project. 


PROJECT  REPCET  FORM  (Cont'd)  RiGE  k 


NGI-75g(C) 
SERIAL  KO. 


9.   PROJLCT  DESCRIPTION  (Continued) 

Proposed  course  of  project;  Thus  far  it  has  been  shown  that  catheter 
implantation '  into  the  pulmonary  vessels  is  a  feasible 
■  procedure  and  that  it  provides  accurate  access  to  the  vas- 
cular bed  for  a  prolonged  period  of  time.   It  is  planned 
to  continue  the  studj'-  as  outlined,  using  patients  with 
operable  metastatic  and  broncho-enic  carcinomas.   It  is 
hoped  to  get  a  number  of  bronchogenic  carcinomas  to  test 
this  method  inasmuch  as  this  is  the  type  of  turaor  which 
has  been  shovm  to  be  slightly , sensitive  even  to  intra- 
venous a  dill  in  is  trat  ions  of  mustard.  \Je   are  also  considering 
whether  it  might  not  be  justifiable  to  perform  thoracotomy 
with  catheter  implantation  even  on  patients  with  clinically 
inoperable  tumors,  inasmuch  as  so  little  of  honestly  thera- 
peutic value  is  available  to  this  group  today. 

k   new  method  of  gaining  djjrect  access  to  the  bronchial 
artery  bed  is  under  investigation  in  the  animal  laboratory. 
Project  756,  and  may  be  available  soon  for  clinical  evalu- 
ation. 


11. 

SufiGET  ACTWlT/: ' ' — — ^ 

RiiSLARGH  /x7       ADil  IN  IS  TUITION  AJ 

REVIEW  5: ,  I.??[i  mh  L  /y         TECHN ICA  L  ASS  ISTANCE  A7 

NO  EKTRIES  FOR  ITEiIS  12,    1.3,    Ik,   l5  &  16. 


PAGE  1 
PHOJECT  REPORT  FCBIi 


1.       National  Cancer  Institute  2,       Surgery  Branch  

Institute L^son^TokY  on  i^mm 

3,  h.  -  5.-  NCI-756 

gi^CTION  OH  ^^UVKJE        L'Mr.TICN  (IF  OTiGR  m.f  dilTiiEiJDA)      sekl.l  ^iu. 

Development  of  New  llethods  in  Treatment  of  Lung  Cancer  using  Regional 

6.  Chemotherapy:     Laboratory  Research 

PROJECT  TiTi^' 

7.  Dr«    John  H.  Waite 

8.  Dr.  William  Kramer  -  Dr.  I-filliam  Banfield 

OTHER  IMVSS'iia'.iOR!:; ' 

9..  PROJECT  DESCRIFnON 
Objectives; 

This  has  been  a  key  project  for  the  development  of  techniques  which 
were  later  used  in  the  human  lung  cancer  project  755(C).     Using  the  dog 
as  an  experimental  animal,  various  methods  for  the  catheterization  of 
pulmonary  vessels  at  open  operation  were  studied,   in  an  effort  to  potentiate 
the  known  effect  of  parenteral  nitrogen  mustard  on  bronchogenic   tumors  and 
to  provide  a  satisfactory-  route  icr  evaluation  of  new  possible  chemothera- 
peutic  agents  as  they  are  developed. 

Methods  employed; 

Fine  polyethylene  catheters  were   introduced  at  thoracotomy  into  a 
bronchial  artery  or   into  a  branch  of  ?  pulmonary  artery  or  pulmonary 
vein.     The  catlieters  were  brought  out  throu.^ii  the  chest  wall  and  allowed 
to  remain  in  situ  for  a  long  period  of  time.     The  continuing  patency  of 
the  catheters  was   tested  and  possible  deleterious  effects   on  the  surround- 
ing tissue  were  watched  for.     By  injectJjig  diodrast, . serial  angiograms 
were  obtained  under  various  experim.;ntal  conditions  and  studies   on  approxi- 
mately 50  dogs.      It  was  also  attempted  tc   deten.iine  x^hether  the  differential 
damage  to  normal  dog  lung  structures  could  be  accomplished  bjr  the   injection 
of  extremely  high  doses  of  nitrogen  mustard. 

Major  findings: 

Implantation  of  catheters  at  open  thoracotomy  was  perfected.  Catheters 
can  be  left  in  for  periods  of  time  as  long  as  six  months  providing  continued 
access  to  the  pulmonary'  circulation.  The  feasibility  r.nd  harmle.ssness 


PROJECT  REPORT  FOmi  (Cont'd)  R'^GE  2 


NC 1-756 

^rl;l  no. 


Major  findings:  (Continued) 

of  the  catheterization,  technique  was  demonstrated  satisfactorily, 
enabling  us  to  bring  it  to  the  human  project.   Previous  experiments  had 
shoxm  that  differential  damage  to  the  bronchial  -lucos?  could  be  obtained 
hj   injection  of  large  quantitities  of  mustard  directly  into  the  bronchial 
arteries,  which  vessels  are  knuwn  to  be  the  direct  blood  supply  to  broncho- 
genic carcinoma  in  humans.  Direct  application  of  this  technique  to  humans 
did  not  seem  possible  because  of  the  variability  of  bronchial  vascular 
supply  in  humans,  and  because  of . the  likelihood  of  finding  difficulty  in 
the  dissection  of  finer  hilar  structures  in  advanced  carcinoma  of  the 
bronchus.  We  have  continued  the  studies  with  attempts  to  gain  access  to 
the  bronchial  vascular  beds. 

Injection  into  the  pulmonary  artery  branches  failod  to  produce  specific 
burning  of  the  m.ucosa,  although  of  course  this  does  not  ni^eessarily  mean 
that  mustard  so  injected  in  huraans  would  not  cause  a  greater  effect  on 
the  bronchogenic  carcinoma  which  is  much  more  sensitivo  than  the  normal 
tissues  of  dogs.   In  another  attem.pt  to  gain  access  to  the  bronchial  vascu- 
lar bed  the  pulmonary  vein  to  the  affected  lung  was  ligated  near  its 
entra.p-cc  to  the  heart.  Diodrast  injection  of  these  preparations  seemed 
to  visualize  the  bronchial  vascular  bod.  However,  nitrogen  mustard  inject- 
ions failed  to, produce  specific  burning  of  tht  bronchial  mucosa,  and 
further  studies  on  the  collateral  vessels  which  hrvo  developed  after  pulmo- 
nary vein  ligation  have  revealed  that  these  are  actually  conn.:^ctions 
between  pulnonary  and  systemic  veins  which  sidetrack  the  bronchial  vascular 
bed.  The  most  "pronounced  local  effect  on  Imig  tissue  as  compared  with  a 
general  body  effect  mcasirred  by  bono  marrow  examinations,  occurs  immediately 
after  ligation  of  tho  pulmonar'/  veins.   Injection  of  pulmonary  v^ins  with 
nitrogen  mustard  occasionally  produced  >:,nough  daiiiage  to  cause  local  necrosis 
of  lung  tissue. 

Investigations  have  now  beguai  on  utilizjjig  th^  thoracic  aortr  itself 
as  an  access  vessel.   If  the  aorta  were  temporarily  obstructed  at  the 
level  of  tho  diapliragm  and  ^  quantity  of  chemotherapeutic  agent  intro-  . 
duced  into  the  thoracic  aorta  it  would  immediately  be  carried  to  tlie 
bronchial  circulation,  chest  wall,  mediastinal  l^niiph  nodes  and  other 
structui\,s  usually  involved  by  locally  advanced  bronchogenic  carcinoma. 
Methods  of  accomplishing  this  at  open  thoracotomy  and  h-y   aorta  catheteri- 
zation in  the  intact  anlnial  are  being  studied. 

Significance  to  Cancer  Research: 

Continuing  increase  in  the  incidence  and  death  rate  due  to  lung 
cancer  demands  maxlfial  efforts  tj  control  the  disease.  Tho  primary  or 
basic  science  approach  involves  efforts  to  determ.ine  the  biologic  nature 
and  environmental  relationships  of  lung  cancer.  Wliile  of  great  hope, 
this  research  has  yet  to  produce  applicable  results.  A  second  phase  is 
seen  in  programs  aimed  at  the  discovery  and  treatm.ent  of  lung  cancer  in 


PROJECT  REPORT  FORM  (Cont'd) 


PAGE  3 


NG 1-756 
SERia  NO. 


Significance  to  Cancer  Rcsoarch:   (Continued) 

its  earlibSt  stages.  This  attack  has  succeeded  in  curing  up  to  $%   of 
all  patients  seen  hj   extirpative  surgery,  the  only  curative  treatment 
now  known.  The  third  phase  of  lung  cancer  research  involves  improvement 
of  treatment  of  the  disease  when  it  is  past  its  earliest  stages,  and  the 
large  number  of  well-advanced  lung  cancer  pati^-nts  now  seen  provides  an 
urgent  motivation  for  the  laboratory  inve3tigat6r  to  search  for  an 
approach  such  as  described  in  this  project. 

Proposed  course  of  projoct; 

Continued  studies  will  be  made  to  determine  the  most  advantageous 
vascul-^r  route  of  drug  administration  in  bronchogenic  crrcinoma,  The 
project  will  provide  a  continuing  opportxmity  to  evaluate  changes  in 
a  catheterization  technique  to  be  employed  in  humrns.  After  deciding 
on  a  standard  technique  we  would  like  to  use  that  technique  for  toxicity 
studies  in  dogs  before  using  new  drugs  in  hum-^ns  by  the  regional  vascular 
approach. 

11. 


BU£)(JST  AcTtVlTV: 


RESKIRCH  /17       ADHINISTR'_TIOIJ  £7 

REVEilJ  &  APPROVAL  r?         TUGHI^IICAL  ..SSISTAMCE  /~7 


NO  ENTRIES  FOR  ITEIiS  12,   1.3,    lii,   15'  &  16. 


PROJECT  REPORT  F0RI4  PAGE  1 


1.       National  Cancer  Institute  2.       Surgery  Branch 

"  toJS'Tll'UTE  UiBORjlTORY  OR  BR-'iNCH 


3.  h.     5.-  NG  1-757(0 

SECTION  OR  Service         location  (1F  other  TH^M  B^THkSM)      SERIAL  m. 


6. 

Prevention  of  Operative  Site  Recurrence  by  Improvement  of  Biopsy 
Wound  Closure  Technique 

PUoJECi  'TITLE 

7. 

Dr.    John  H.  Waite 

PRlNdlpAL  'lMVESTia\TOR(S; 

8. 

Dr.   Robert  R.   Smith  -  Dr.  Albert  Hilberg  -  Dr.   Horace  Herbsman 

OTl-lEK    iWVEtJTi&iTORS  ' 

9,     PROJECT  DESCRIPTION 

Objectives; 

,The.  purpose,  of  this  project  has  been  to  gather  some  experimental 
data  concerning  the  validity  of  biopsy  wound  closure  techniques,  to 
determine  if  any  currently  used  techniques  are  satisfactory,  and  to 
develop,  if  necessary,  improved  techniques  for  management  of  biopsy 
wound  closure. 

Ifethods  employed: 

Patients  with  apparently  primary  operable  carcinoma  of  the  breast 
are  prepared  for  surgery.  A  routine  local  incision  is  made,  and  the  sus- 
pected tumor  excised,  examined  and  submitted  for  frozen  section  histodiag- 
nosis.  The  cut  surface  of  the  excised  tumor  is  carefully  washed  to  procure 
positive  c,ytologic  material  for  comparison  with  later  skin  surface  washings. 
Cases  with  positive  frozen  section  are  suitable  for  study. 

The  local  wound  is  closed  by  one  of  the  standard  methods  vinder 
study,  and  the  skin  area  completely  cleansed  and  redraped  for  radical 
mastectomy.  At  this  point,  the  closure  is  carefully  examined  for  gross 
escape  of  serous  or  bloody  fluid.  Any  of  this  serous  or  bloody  fluid 
which  does  escape  is  carefully  aspirated  for  cytologic  bxamination,  and 
in  addition,  washings  are  taki^n  from  the  margins  of  the  closed  biopsy 
wound  or  any  cover  which  may  be  fixed  over  it  for  cytologic  examination 
to  determine  whether  cancer  cell  escape  has  occurred  with  the  method 
under  evaluation. 


PROJECT  REPORT  FORII  (Cont'd)  PACE  2 


NGI-757(C) 

bKKL'iL  i\IU,  ....•• 

Methods  employed;   (ContJJiued) 

The  present' s-cope-  of  ■  the  project  is  limited  to  the  following 
closure  methods: 

1.  Closure  with  a  tight  interlocking  continuous  suture. 

2.  Closure  ^^rith  a  tight  Interlocking  continuous  suture  followed  by  ?. 
plasticized  film, 

3.  Suture  closure  with  a  sheet  of  rubber  submitted  to  the  surrounding 
skin. 


Patient  material: 


No.         Average  Stay  Days 


:\djTiissions:  Adiilt  males  2  lb 

Adult  females .  ■,        '^  ......  .        „,      ^^ 

Outpatient:  Number  of  patients       10 
Number  of  visits         IjO 

Major  findings: 

Five  breast  carcinomas  and  one  chondrosarcoma  have  been  carefiilly 
studied  according  to  the  methods  outlined.  That  the  opportunity  exists 
for  wound  seeding, to  occur  from  the  biopsy  site  appears  probable  from 
the  following  data: 

1,  Three  of  these  six   patients  yielded  cr.ncer  cells  from  the  skin  sur- 
face, using  cytologic  detection  techniques. 

2.  In  all  six  patients,  oozing  of  serous  fnd  bloody  material  through 
the  closed  biopsy  wound  occurred.  This  occurred  despite  a  tight  closure, 
and  it  was  noted  that  the  bleeding  usually  occurred  from  the  suture  needle 
puncture  site.   It  was  reasoned  that  the  blood  escaping  from  the  biopsy 
site  might  easily  contain  a  suspension  of  tumor  cells. 

Closure  of  the  biopsy  site  with  a  continuous  tightly  interlocking 
suture  was  unsuccessful  in  sealing  the  v7ou:id  as  was  Aeroplast  (an  aero- 
solized plastic  spray  which  dries  rapidly  to  form  a  film)  which  was 
applied  in  all  of  the  five  breast  ccrcinona  patients.   Oozing  from  the 
closed  biopsy  wound  occurred,  lifting  the  dried  plastic  film  from  the 
skin  surface  in  a  few  seconds'  time. 

The  four  patients  who  were  found  to  have  benign  tumors  on  biopsy 
contributed  to  the  program  by  yielding  cytologic  control  specimens.   2  cc. 
of  a  dye  placed  within  the  depths  of  one  of  these  biopsy  woiinds  appeared  ■ 
immediately  on  the  surface  of  the  skin  through  the  suture  needle  puncture 
sites,  imitating,  we  th^nk,  the  case  with  which  a  tumor  suspension  could 
reach  the  skin  surface. 


PROJECT  REPORT  FORM  (Cont'd) 


PAGE  3 


NCI-757(C) 

9.     PROJECT  DESCRIPTION  (Continued) 

Significance  to  Cancer  Research; 

Wound  recurrence  following  biopsy  and  local  surgery  is  not  uncommon. 
In  treating  one  of  the  most  common  cancers,  that  of  the  breast,  leading 
clinics  admit  to  a  12^  local  recurrence  rate.  The  two  most  likely  factors 
are  inadequate  surgical  margins  and  spillage  of  cells  at  the  time  of 
biopsy.  Exfoliated  cells  have  been  recovered  from  skin  surfaces  by  the 
principal  investigator  even  after  more  than  usually  careful  attempts  at 
biopsy  site  closure  and  sealing  off,  using  conventional  techniques.  A 
simple  method  which  would  guarantee  100%  riddance  of  skin  surface  tumor 
cells  would  be  of  immediate  value  to  all  surgeons  in  treatment  of  many 
common  malignancies. 

Proposed  course  of  project; 

Thus  far  none  of  the  techniques  in  current  practice  for  biopsy 
site  closure  is  even  theoretically  capable  of  preventing  escape  of  a 
tumor  cell  suspension  from  the  closed  biopsy  wound.  We  will  evaluate 
other  methods  in  common  use.   In  the  meantime  we  will  be  trying  to 
develop  a  foolproof  technique  under  the  animal  project  no,  758.  We 
will  continue  to  get  washings  from  the  operative  wound  site  to  correlate 
positivity  of  skin  surfrice  washings  vjith  the  radical  operative  wound 
washings,  and  we  will  continue  to  follow  these  patients  clinically  in 
order  to  correlate  tumor  recurrences  in  the  operative  site  wi.th  the 
positivity  of  skin  washing  cytolog.-. 


11. 


BUDGET  ACTr\fITY: 


RESEARCH  /x7       ADMINISTR/iTION  £7 

REVIEW  &  APPROVAL  /~~/         TECHNICAL  ASSISTANCE  /V 


NO  ENTRIES  FCR  ITEMS  12,    1.3,   lii,    iS  &  l6. 


PROJECT  REPORT  FORil  PAGE  1 


1.       National  Cancer  Institute  2.        Surgery  Branch 

BISTtt'tJfE L^SOPulTORY  OR  BRaMCH 


3. Ii-. 5.  NCI-7$8 

SECTIOM  OH  Si^RVKE  LOCATION'  (IP  OTflER  TJ14N  BETHEiifiA)        'SERI'.L  NO. 

Prevention  of  Operative  Site  Turior  Recurrence  by  Improvei-nent  of 
6.  Biopsy  Wound  Closure  Techniques:     Laboratory  Research 

PROJECT  TITLE 


7.       Dr.   John  H,  Waite 

PRINCIPAL  raVESTIQ\TOR(Sj 


Dr.  Horace  Herbsman  -  Mr.  Richard  V.  Eck 
OTHER   INVESTiaiTORS  '  "" 


9.   PROJECT  DESCRIPTION 

Objectives; 

The  purpose  of  this  project  is  to  evaluate  methods  vrhich  have  been 
proposed  for  biopsy  wound  closure  by  laboratory  techniques  on  the  experi- 
mental andjiial.     A   closiire  method  is  sought  which  would  prevent  the  escape 
of  tumor  cells  fropi  the  skin  surface  into  the  radical  surgery  wounds 
after  preliminary  biopsy.      If  currently  used  techniques  are  fo-und  to  be 
unsatisfactory,    new  techniques  will  be  developed. 

Methods  employed: 

■  For  experimental  convenience,   a  blue  dye   is  used  to  imitate  the 
turaor  cell  suspension  which  must  be  retained  within  the  biopsj'"  wound 
in  order  to  prevent  radical  surgical  wound  contamination.      Using  rabbits 
as  experimental  animals  we  have  made   standard  biopsy  wounds,   closed  them 
with  continuous  interlocking  sutures,   and  injected  dye   into  the  biopsy 
wound  cavity,    to  determine  the  quantity  and  pressure   of  fluid  retained 
by  the  wound  closure  being  tested,   before   leakage   occurs   onto  the  skin 
surface. 

Thus  far  three  methods  of  closure  have  been  evaluated; 

1.  Closure  with  a  continuous   interlocking^  suture  as  described. 

2.  Closure  with  a   continuous   interlocking  suture  overlaid  with  a  plastic 
film  sprayed  onto  the  skin  surface. 

3.  Closure  using  Raney  sur:?ical  skin  clips. 


PROJECT  REPORT  FORI-I  (Cont'd)  PAGE  2 


MCI-7g8      : 

SKdiAj   MO. 


PROJECT  DESCRrPTrON-tC-ontiritlca')-  '■--      '  ."  .  ■  __     '        '  .     •' 

Mr.  j  or .  f  indings : 

•     '  1.  :  After  clasiire'  of  •  the"  standard  biopsy,  wound  with  a  continuous 
interlocking  suture  only,   1-2  cc.    of  dye   injected  under  2  cm.    of  water 
pressure  appeared.  JJTimediately  on  the^  surface  of  the  closed  biopsy  wound. 
The  appearance. of  this  dye  was  not  at  the  incised  skin  margin,   but  always 
at  a  suture  needle  puncture  site/- the  same-., locus  from  which  bloody  oozing 
was  invariably  noted  after  the  clinical  closure   of  biopsy  wounds  in  huraans. 
Apparently  the  skin  defect  created  by  a  suture  needle  puncture  should  be 
regarded  as  continuous  with  the  incision  into  the  underlying  tun  or, ^  and 
a  likely  BO'urce  f 'or"  turror  cell  "escape.        v 

2,  Sealing  of  the  closed  biopsy  wound  with  Aeroplast  (an  aerosolized 
plastic   spray. which  dries  rapidly  to, fcrpi  a  film),  permitted  about  three 
times  as  mueh  dye  to 'be  in^edted '"under  a.  slightly  higher- pressure.     However, 
if  only  sli'-;ht  manipulation  of  the  closed  biopsy  wound  was  performed,   dye 
immediately  burst  from  beneath  this  very  fragile  seal,   easily  dissecting 
the  plastic   seal  off  of  the   surface  of  the  skin.     Thus,    this   type   of  a  seal, 
including  the   older  collodion  seal,  would  seem  to  add  little  protection 

to  the  biopsy  site. 

3.  Application   ofRaney  clips  to  corpt  skin  margins  resulted  in  an 
extremely  efficient  closure  -permitting  'Ux   injection  of  about  ^0  cc,   of 
dye   under  about  lu  cm.    of  vrater  pressuro,   before  appearance  of  the  dye  at 
the   openings  of  nipples  or   sweat  glands. 

Significance  to  Cancer  Resegrch;  - 

The  intact  skin  overlying  a   s-ft  part  cancer  must  usually  be   incised 
to  obtain  a  biopsy,   rupturing  the  envelope    ox   normal  tissue  which  theoreti- 
cally should  be  removed  intact  with  its  indwelling  m.alignancy.-    The  pre- 
vention of  escape   of  malign?nt  cells  through  this   incision  constrbutes   one 
of  the   oldeat  problems  in  Cc-^ncer  surgery..     Many  methods   of  closure   of 
biopsy  wounds  have  been  recommended  e-np^jr^icrlly.      It  is  hoped  that  utili- 
zing the   testing  methods  described,  we  will  be  ablo.  to  recommend  or 
develop  -^    standard   tiethod  for  biopsy  wound  closure  backed  by  experimental   . 
data. 

Proposed  course  of  project; 

The  most  important  untried  method  for  wound  closure  is  a  method  which 
has  been  recommended  using  tho  cementing  of  a   sheet  of  rubber  over  the 
closed  biopsy  wound.     This  technique  will  be  tested,   and  further  experi- 
ments will  be  condu:;ted  on  the  Ranejr  clip  method  of  closure. 


PROJECT  RE^PORT  FORM  (Cont'd)  '  PAGE  3 


10.  NC 1-758 

SERL'-L  NO. 


11. 

BUDO^T  ACTIVITY: 


RE,SEi;RCH  frf      ADME'JISTRATION  £7 

REVIff."!  &  APPROVAL  /"/         TECHI-Iia.L  ASSISTANCE  /~J 


NO  ENTRIES  FOR  ITEMS  12,    13,   lU,    l5  &  l6. 


PROJECT  REPORT  FORM  PAGE  1 


1.        LTational  Cancer   Institute  2,       Surgery  Branch 

INSTITUTE L.B01i;T0RY  OR  BR.'i!CH 


3.  h.  ' 5.  NCI-759 

SECTION  OR  SERVICE  LOi^TiO^J   (11?  i^lM  Tii.H  BIDTHSSD..)        SERL'.L  NO. 


6.        Ttchnic   of  Hypophysectoiiiy  in  the  Guinea  Pif 
prO^CT  TiTi£ ^ ' 


7.       Dr.   Lt,ster  M.   Cramer 

"p^^inTpXTFTESTmr^rrsT 


0.       Dr.   Eli  Nadel 

OTHER   BIVESTIG'.TORS 


9.      PROJE.CT  DESCRIPTIO'^- 

Objectives; 

To  develop  and  perfect  a  technic  for  hypoph^z-sectomizing  the   guinea  pig. 

Methods  employed; 

Guinea  pig  hypophyseal  fossae  were  appruached  trans cervic ally, 
transcranially,  and  transbuccally  and  the  pituitarj-  gland  renoved  by 
suction.  Animals  m:>intained  postoporativel3^  on  antibiotics,  clyses, 
and  steroid. 

Major  findings: 

No  complete  hypophysectomies  were  accomplished,  the  Injaiting  factor 
being  the  extension  of  anterior  pituitarj^  colls  up  around  the  stalk  to 
the  hj'pothalamus.  ^ 

It  is  felt  that  radioactive  substances,  such  as  yttrium.,  implanted 
into  tht,  pituitarj"  fossa  will  offer  a  better  chance  to  perform  a  com- 
plete hjTDophysectomjr.  Neither  investigatur  is  authorized  to  use  radio- 
active substances,  and  the  project  is  arosently  shelved  until  an  interested 
personage  with  the  necessary  radioactivity  clearance  can  be  included  in 
the  study. 


PROJECT  REPORT  FORl-I  (Cont'd)  PAGE  2 

NCI-759  •  ■ 

&mt.1  V.IO. 

PROJECT  DESCRIPTION     (Continued) 

Significance  to  Cancer  Research: 

Continued  developments   in  the  f ic^ld  of  endocrinology  demonstrate 
the   Mportance   O'f  pituitarj^  hormones- upon  cf-ncer  •■  tissue  *      If  "complete, 
removal  of  the  gland  could  be  easily  done   in  rjuinea  pigs,   a  very  useful 
tool  xsrould  be  available  to  further  modifj'-  this  control. 


Proposed  course  of  proJL:Ct;'     ■ 

Pending  the  availability  of  radioactive  j'ttrium,   no  further  work 
is  planned  on  this   project. 


11. 


rese:.rch  /T/     ;.D'ii'asTR.TioN  A7 

REVIEW  ic  IJpyRLNLL  H         TECffi'lICL  ASSISL^NCE  /"T" 


NO  ENTRIES  FOR  ITEiMS  12,   1.3,   lU,    15  "x  l6. 


PROJECT  REPORT  FGIG'         '  '         P;.GE  1 
1.   National  Cancer  Institute       2.   Surgery  Brnnch 

-TH^TTTITTE li^BCii-xTORY  OR  br;.itch 

3. Ii. .  S.  NCI  760 

SECTION  OR  Sfiftv'iCE  LOCATION   {W  OTIffiR  TH.UI  3ETIDLSDA )  SERIAL  NO. 

Th>..  Studjr  of  the  Effects   of  Beta  Nn.phthylr.mint  and  U-Aminodiphcnol  on 
Qastrointestinal  -'.nd  .Bladder  ;''ucosa  by  Direct  Contact  in  Dogs. 

a.  DevelopCTont  of  a  Surgical  Tuclinique  for  In  Jivo  Study  of 
Drugs   in  the  GastrointestLnal  and  the   Isolated  Urinarj^  Bladder. 

b.  A  Studj^  of  the  Techniques  for  Handling  the  Genitourinary  Tract 
6,  after  Cystectomy. ; ' 

PRD'JECT  TI1*Le  ~ 


7.       Dr.  Donald  Cole   -  Dr.  Willian  Hueper 
PRlNdlR.L  INViSTia-TOR(S; 


■oTl-M  INVEST  IG-.TORS ^^ 

9.   PROJECT  DESCRIPTION 

Objectives; 

A  study  of  the  carcinof^enic  potentialities   of  certain  aromatic   "nines 
and  azo  compound  yt^llow  ;b.      These  comp>^unds  along  with  yellox^  ,..B  and 
yellow  OB  are  involved  in  food  dycs  in  comnion  use.      This  iS  the  problem 
of  Dr.  William  Hueper  of  tlie  Environmuntal  Cancer  Section  of  the  National 
Cancer  Institute  that  lie  will  report  in  detail. 

Ifcthods  em-ployed: 

An  acute  experiment  was  devised  in  order  to  study  the  naetabolism 
of  the  djes  in  an  isolated  segment  of  the  gastrointestinal  tract  for  the 
length  of.  time  it  may  bq  present  duriiig  norraal  digestion.      In.,  effect, 
in  vivo  test  tubes  were  created  by  isolating  the  gastrointestinal  tract 
of  dogs  tliroughout  its  length  from  the  stomach  to  tht,  rectum  with  no 
reconstruction  of  the  ana tony.      In  another  group  of  chronic  experiments, 
in  vivo  test  tube  bladders  and  segments  of  gastrointestinal  tract,    stomach 
To  colon,  were  isolated  with  reconstruction  of  the  anatomy  for  what  was 
believed  to  be  the  best  fiuictional  results.     A   total  of  19  acute  experi- 
ments were  performed  on  the  gastrointestinal  tract.     The  dogs  were  prepared 
preoperatively  so  that  their  gastrointestinal  tract  was  clean.     The  organ 
was  satisfactorily  isolated  at  the  time  of  surgery,   bejjig  caroful  to  main- 
tain the  integrity  of  the  blood  supply.      Tlie  drug  under  study  was  then 
placed  into  the  isolated  loop  and  speciraens   obtaiiied  from  the  isolated  loop 
at  various  intervals.      In  the  chronic  'experiments  10  mg.    of  yellow  AB  were 
fed  in  meat  balls  to  the  dogs.     After   tliree  weeks  they  were   sacrificed  and 
autopsied  to  be  examJ-ned  for  metabolic  analysis  and  pathologic  changes. 


PROJECT  REPORT  FORli  (Cont'd)  Ri( 


NCI-760      ' 

Methods  employed;   (Continuod) 

Two  dops  hr-d  pouches  formed  with  implantation  of  ^  grsra   of  yellow  ;.B  and 
two-. others  had  jejunal  loops  and  colon  loops  prep.ared  and  the  drug  placed 
into  the  isolated  segments  ia  a  variety  of  circumstances.  Multiple  pro- 
cedures were  carried  out  to  ascertain  the  best  method  for  diverting  the 
urinary  stream. to  obtain  an  isolated  urinary  bladder  and  still  maintain 
life.   The  drug- was  placed  thro-ugh  the  dome  of  .the  empty  bladder  which 
was  then  closed  with  silk  sutures.  The  urethral  ope:ning/was_  then  closed 
in'a  similrr  manner;-"^ '■'""■  "       ' ""'-■■ ''~ ■""•"'  ""  "' 

Major  findings; 

"■  The  result  of  the' cxperinient -is "not  complete  as  yet.  The  dyes  were 
not  left  in  place  long  enough  to  produce  any  histologic  changes,  .'.s  for 
the  chemical  degradation  products  for;ned  by  the  compounds^  definitive 
results  are  not  as  yet  available.  ..'.s  in  the  similar  instance  of  using 
humans,  the  placement  of  ±h^   ureters  in  the  colon  or  on  the  skin  produced' 
a  hydroneplirosis  and  hydroureter  with  ascending  urinary  infection  which 
made  it  impossible  to  maintain  adequate  kidney  function.  The  placement 
of  the  carcinogenic  agent  in  the  bladder  with  the  urinary  stream  diverted 
has  not  boon  completed  as  yet. 

Significance  to  Cancer  Research; 

A  more  thorough  understanding  of  the  possible  carcinogenic  effect 
of  certain  dyes,  would  possibly  l^^nd  tu  -  useful  prophyl^actic  measure  in 
the  prevention  of  c?,ncer.'.  •   . 

Proposed  course  of  project: 

Sec  report  of  Dr.  William  Huepor  re '■.warding  future  course  of  study.   • 


11.  _____ 

BUDarT  ACTIVITY: ^ ~ 

,     rese,'-RCh  /tj      ;DniNiSTR.\Tioi-j  /y 

■  REVE^J  i  ;.PPRaV.-.L  £7         Tt.CHNIC:.L  ..S3IST;,.NCE  £7  . 
NO  ENTRIES  FOR  ITEI-IS  12,    13,    lij.,    i$  :k  16. 


PROJECT  REPORT  FORM  PAGE  1 


1.        Np.tional  Cancer  Institute  2.        Surgery  Branch 

IMSTITUTE L;Sta.'.TOnT  OR  BRAMCH ~~~~ 

Memorial  Hospital  & 
3.  li.   Sloan-Kettcring  Institute  i;     NCl-761- 

"S^CTIO-'.^  OR  SERVICE        LOa:.TiOM  (IF  G^nm  T^-N  MTHESC;.)        gERnL  l^Ol 


6,        Blood-Pressure  Sorvo-Reg-ulator 
PROJECT  "TITLE 


7.       Dr.   Jolin  H.  Waite    . 

PRINC  1P;.L  IN\7ESTli.-TCR(  t ) 


8.      John  Laughlin  -  Naorai  Sager  -  Williara  Poppall  -  William  Rowland 

othf:r  JiivEST  la'.  tors  '  ' 


9.   PROJECT  DESCRIPTION 

Objectives;  ,      _. .      . 

To  construct  a  machine  for  automatic  regulation  of  blood  pressure  in 
shock  viuc  to  vasodilation^   and  in  hypotensive  anosthtsia. 

Methods  employed; 

The  mean  arterial  blood  pressui'-e   is  measured  directly  with  a  trans-     •.■ . 
ducing  manometer.     Appropriate  vasopressor   (norepinephrine),   or  vaso- 
depressor (arphonad)    solution-  is  c7:drn;inistered  continuously  intravenously 
by  a  variable   spe^^d'  infusion  pump.     The  pump  speed  is  regulated  by  a 
signal  generated  by  a  servo-loop.     The  servo-loop  signal  measures  the 
difference  between  the  blood  pressure  measured,   and  the  blood  pressure 
clinically  desirable  for  the  patient  being  treated.     T:  e  therapist  thus 
needs  o^nly  to  set  the  control  panel  for  the  desired  blood  pressure,   and 
the  auto-regulated  machinery  administers  blood  pressure  regulating 
substance  as  needed. 

T'ajor  findings; 

Construction  of  the  blood-pressure   servo-regulator  apparatus  has  been 
completed.     The  apparatus  has  been  tested  on  a  series   of  experimental 
animals   (dogs)   in  the  Physiological  Laboratories  of  the  Sloan-Kettering 
Institute.      Necessarj'-  refinements   in  design  and  construction  have  boon 
made  r'nd  completed.      The  apparatus  has  now  been  moved  to  the  recoverjr 
room  of  the  Memorial  Hospital.      The  participation  by  the  principal  investi- 
gator has  been  on  a  consultative  basis  d^oring  the  calendar  year,   1955. 


PROJECT  REPORT  FORiM  (Cont'd)  PAGE  2 


NCI-761 
aERr.L  WO. 


PROJECT  DESCRIPTION     (Continued) 

Sit^nificance  to  Cancer  Research; 

The  raochine,    if  successful,  will  be  an  aid  in  maintaining  blood 
pressure  in  patients  with  neurogenic  shock  due  to  radical  surgery  or 
drug  reactions.      It  will  also  provide  a  method  for  controlled  lowering 
of  blood  pressure   (hypotensive  anesthesia)   in  patients  undergoing 
radical  surgerj'-  for  cancer. 

Proposed  course  of  project; 

The  apparatus  will  be  evaluated  in  controlling  the  blood  pressure 
of  the  next  six  patients  requiring  vasopressor  agent  administration  to 
maintain  blood  pressure  at  the  Menorial  Center. 


11.  

BuB'GET'ACTWir^ — ' ' — ~ 

RESEL\RCH  /^       /J^IIEIISTR-.TION  £7 

REVIEI'-!  &  AP?ROW.L  /~7         TECrffiia.L  .ASSISTANCE  A7 


12.     Sloan-Kettering  Institute   -  I''crr:orial  Hospital,   New  York  City 

COOPEUVriMG  JtilTS  OP  THii  PuBlIC  HL' LT:rS-^R\/iCh,   OR  aH'Ml  ORa.MIZATlOWS, 
.    PROVIDING  FJiiDS,    F  CILITES,    OR  PERSOlll^EL  FOR   THIS   PROJECT   IN  EITHER 
1956  or  1957. 

WO  ENTRIES  FOR  ITE.iS  1.3,    Hi,    15  &  I6. 


PROJECT  REPORT  FCMl  R^iQE  1 


National  Cancer  Institute       2.   S'urger^r  Branch 

ii.  5.  :ici-762(c) 

Blood  Volurr.e,   plasna   Volume,  Red  Cell  Mass  and  Total  Circulating  Plasna 
Protein  and  Electrolytes   in  Cancer  Patients  D"ar:ng  preoperative  Bowel 

Preparation  and  the  Period  after  Radical  S^Jirger-.^.  

"PROJECT  IITLE '' 


7.  Dr.   Rcoert  ;..  >^ilch  -  Dr.    Jesse  L.   Steirifeld 
PRi-xP/lL  llfV£STI3i^TcR(Sj 


\.   Surgical  Staff,  :'.C.: 


9.      FRO.^CT  DESCRIPTIMf 

Objectives; 

Although  considerable  data  have  been  presented  from  several  Isbora- 
toriss  concerning  the  netabolic  responses  to  surgery  and  other  forms   of 
tra^'Pia,   little  attention  has  been  directed  to  nietabolic  changes   in  patients 
undergoing  various  preparatory-  procedures  during  the  imnediat-e  preopera- 
tive period,   the  operative  and  immediate  postoperative   oeriod.      This  is 
particulsjrly  true  of  female  patients  undergoing  extensive  preparation  of 
the  bowel  for  pelvic   surgery  prior  to  contemplated  exenterative  procedures. 
The  objectives  of  the  present  study  are  to  determine  the  above  parameters 
seris.lly  in  patients  undergoing  "vigorous"  as  opposed  to  "mild"  prepara- 
tion of  the  gastrointestinal  tract  for  both  pelvic  and  extra-abdom.inal 
surgical  proced'jres. 

''ethods  employed: 

.Injection  of  known  small  amounts  (approxiniately  10  microcuries)   of 
1  ■'-    labelled   human  series  albi-r-iin  and  measurement  of  its  dilution  at 
15  and  30  minutes  after  injection  in  both  whole  blood  and  plasma.     Coi;5)led 
with  serial  determinations  of  xhe  plasma  concentration  of  red  cells   (hemato- 
crit), plasm.a  protein,   and  senum  electrolytes,   it  will  be  possible  to  cal- 
culate the  total  amounts  of  these  various  substances  v/hich  are  circulating 
in  the  plasma  and  the  effect  of  bowel  oreparation  of  thiem  from  calculated 
data  on  red  cell  mass,   plasma  volume  and  blood  volume. 

pa~ient  material; 

-ati&nts  undergoing  surgery  by  the  N.C.I.    Surger;.'-  Branch  /rill  be 
stu'Jied.     Daily  EKG's  and  if  possible,  ZSG' s  will  be  taken.     I'o  patients 
admitted  during  this  year  for  this  project. 


PROJECT  REPORT  FORM  (Cont'd)  PAGE  2 


MCI-762(C) 
SERIilL  KO. 


9.   PROJECT  DESCRIPTION  (Continiaed) 

Major  findings; 

None   to  date. 

Significance  to  Cancer  Research; 

In  order  to  improve  our  methods  of  treating  cancer j  mere   precise 
information  is  necessary  concerning  the  metabolic  status  of  cancer 
patients  coming  to  surgery.  This  project  should  help  the  surgeon  pre- 
pare patients  for  major  surgery. 

Proposed  courae  of  project; 

During  the  next  year  it  is  planned  to  study  patients  undergoing 
radical  pelvic  surgery,  both  during  the  preoperative  and  immediate  post- 
operative periods.  It  is  anticipated  that  approximately -2ii-l 8  patients 
will  receive  such  therapjr  and  be  available  for  such  studies. 

11, 

"DUDGET  ACTITTTyI 

.    RESK'iRCH  /p  ADMINISTRATION  £7 

REVIEV.  k  AP  ROVi\L/y  TECHNICAL  AbSISTASCE  £7 

NO  EFTRIEb  FOR   ITE.  S  12,    13,    ih,    l5  &  l6. 


PAGE  1 


PROJECT  REPORT  FORM 


1.        N"tional  Cancer  Institute  2.       Surgery  Branch            

-EISTTTUTE                                           ■  UBOPu-TORY  OR  3R.U-ICH 

3.  h. ^-   NGI-  76.3 

SECTION  OR  SERVICE         LOCATIOW  (i^'  OTHER  TR-.N  BMHesdA)        SERia  'No. 


6.       The  Effect  of  Prednisone  (Metacorten)    Upon  the  Healing  of  Wounds 
PROJECT  TITLE  ' 


7.       Dr,-  Lester  I'l.   Cramer 

PRINCIPAL   IHVESTI(i-.TOR( S ; 


•     "OTHER  I^]VESl'I(i;'roRS 

9.      PROJECT  DESCRIPTION  '         •     '  ■         . 

Objectives; 

Because  of  its  greater  anti-inflammator;^'-  action  and  its  lesser  effect 
upon  ion  netabolisiTi,   prednisone  is  rapidly  replacing  compounds  E  and  F  in 
a  large   segment  of  clinical  medicine,    especially  in  the  management  of 
rheumatoid  ?rthritis,   chronic  pulmonary  asthma,   and  many  ocular  and  derma- 
tologic  conditions.  ■        . 

The  inhibitor^,r  effect  upon  wound  healing  of  compouiids  L  and  F  is  well 
docuTiiented.      It  becomes  Imperative  to  know  if  this-  new -.Gompound  will  have 
greater  wound  healing  inhibitory  effect  as  night  be  expected  from  its 
greater  effect  upon  inflajumation. 

Methods  employed;  '  ■    . 

280    C57BL  nice  were  divided  into  two  groups   of  li|0  each,   and  then 
each  of  these  subdivided  into  lli,  groups  of  10  each.      One-half  of  all 
the  animals  were  given  prednisone   .25  mgm/20' gm*   daily  for  5  days  before 
wounding,   the  other  one-hnlf  served  as  pair -fed  controls. 

The  wound  consisted  of  a  2  cm.  incision  in  the  anterior  wall  of 
the  distal  stomach.  5-0  nylon  thread  was  used  to  close  the  wound  in 
linear  fashion,  used  a  continuous  horizontal  mattress  suture  for  serosal 
coaption. 


P;.GE  2 


PROJECT  REPORT  FCRM   (Cont'd) 


NCI-763 

SERni  wo. 


Methods  employed;   (Continued) 

Prednisone  was  given  daily  postoperatively,  and  all  animals  were 
weighed  daily.  A.  group  from  .each  of  the -prednisone  and  control  groups' 
iras  sacrificed  on  each  das'-  for  fourteen  days,  and  the  wounds  studied  in 
two  ways:  (1)  Strength  of  wound  as  determined  by  introduction  of  con- 
pressed  air  until  the  suture  line  bursts.  (2)  Histologic  character. 

Major  findings; 

_'.ll  of  the  technical  aspects  of  the  work  have  been  completed. 
I.  Bursting  strength  ..re  suits. 

a.  Curve  has  been  established  for  the  healing  wound  in 

thu  mouse  stomach  which  is  comparable  to  the  accepted  curve  for  a  similar 
xiTound  in  the  rat  stomach. 

b.  The  curve  increment  of  .healing  strength  in  the  mouse'  ■ 
treated  with  prednisone  is  ox  the  same  shape  as  the  normal  curve,  but 
is  very  slightly  lower.  Limited  studies  using  cortisone  showed  that 
healing  strength  was  about  the  same  at  i^iid  points  on  the  curve  as  that 
found  with  prednisone. 

II.  Histology'  results. 

Not  available,  slides  are  being  processed. 

Significance  to  Cancer  Research; 

The  use  of  new  steroids  as  a  palliative  agent  in  treatment  of  cancer 
produces  serious  problems  in  tlie  surgical  management  of  patients,  especi- 
ally, if  it  could  be  shoxm  that  these  drugs  do  inhibit  wound  healing. 

Proposed  course  of  project; 

Until  complete  analysis  of  the  material  already  at  hand  has  been 
performed,  it  remains  impossible  to  definitely  state  whether  this  drug 
has  any  effect  on  wound  healing.  It  may  be  necessarj-  to  run  a  few  more 
anLmals  to  further  determine  this  point.  


11.  •  

BuDGE'T'AOTWITt: ^ ^ . — _ 

RESK-.RCH'  /T/       ;.DMnJISTR-.TION  /7 

REVEi'j  St  :.?mm:L  fl      technical  ;.ssistance  f~f 


NO  Ei'ITRIES  FOR   ITEMS  12,    1.3,    lIi,   l5  h.  l6. 


PAGE  1 
PROJECT  REPORT  FORM 


1.        i\Tg.tiong.l  Cane  or  Institute  2.       Surger::  Branch 

INSTITUTE-  L:.BOR.iTbR^  OR  BRjiCH 


3.  k.  .  .  5.    NCI-76I. 

SSCTIOK  CR  SKRVIOE  LOdJl'lOM  (1^'  OTtli^R  m.i']  jJE'i'tiESUA )        SUSl'L  NO. 

6.       The  Transplantation  uf  "Conditioned"  Endocrine  Tissue 

PROJECT   TITLE 


7.       Dr.   Lester  K.   Cra-ier 


8.       Dr.  Williajii  Schatten  -  Dr.   Williain  rlohler  -  Dr.    Jesse  Steinfeld. 
OCiiER  IJI^SI^I'Q'.TORS 


9.      PROJECT  DESCRIPTION  

Objectives: 

It  has  been  shox>ni,  but  not  conclusivelyj    th?t  the   imraune  response 
to  hoLiografted  tissue  may  bo  abrogated  bj'  riodifying  the  antigenicity 
of  the  donor  tissue. 

Wo  plan  to  use  a  tissue  cvlt-or^  :p.ethL>d  of  modifying  neonatal  rabbit 
thyroid  glands,   ?nd  then  trans nl-^nting  then  to  hjrpothyroid  recipients. 

Methods  employed: 

Thyroidectorry-  is  periorined  on  rabbits  less  than  iiB  hours  old,   and 
explants   of  these  thjToids  grown  in  media  consisting  of  Eagle's  nutrients 
and  serum  of  the  recipient  rabbit.     Thyrotropic  hormone  is  placed  in  one- 
half  of  the  cultures,    and  all  of  the  culture  have  histologic   section  and 
I  --^     uptake  performed  on  a  representative  sample. 

After  a' suitable  lengtli  of  time  in  tissue  culture,    the  explsnts  are 
placed  subcutaneously  in  the  anterior  abdominal  x\rall  of  a  rabbit  which 
has  been  thjToidectomized  two  weeks  previously.     Thyrotrophic  hormone  is 
cdministered  two  dajrs  prior  to  and  ten  days  after  the  homografting, 

1.31 
I  ■      uptakes  will  be  determined  over  the   graft  bi-weekly  for  a  three 

month  period  at  which  time   grafts  which  are  not  functioning  will  be   sacri- 
ficed.    Functioning  grafts  will  be  left  intact  and  I-'--^-'-  uptake  determined 
monthly  for  at  least  six  more  m.ontlis  at  x-^hich  time  histologic  examination 
will  be  performed. 


Pi.GE  2 


PROJECT  REPORT  FORii   (Cont'd) 


MCI-761i 


Methods  emplo;^ed;   (Continued) 

It  is  .contenplated  using  the  transparent  chajnber  for  some  of  the 
transplants  when  function  has  been  demonstrated  '.  Controls  are  liver 
treated  in  the  same  fashion. 

Major  findings; 

Presently  we  are  working  on  technics  for  ,>ving  TSH  in  vitro,  for 
counting  I'^-^-'-  in  vitro,  and  determining  optirnui-;i  tiiTiing  for  the  condition- 
ing.   Tliree  gr'afts  have  been  in  recipients  for  two  weeks.  Two  of  these 
have  demonstrated  no  function,  the  third-  one  has  picked  "up  6;^  of  the  I  •  , 
but  the  control  transplnit  of  liver  tissue  has  also  picked  up  6/j  of  the 

Significance  to  Cancer  Research:      •  ■" 

In  the  treatment  of  cancer  of  endocrine  glands  it  is  often  necessary 
to  remove  the  entire  gland.  This  necessitates  that  the  patient  continue 
taking  substitution  drug  treatment  for  the  remainder  of  life.  If  a  method 
of  producing  a  functioning  homologous  graft  could  be  developed,  possibly 
using  tissue  cultured  grr'fts,  considerable  benefit  to  clinical  management 
would  be  obtained. 

Proposed  course  of  project; 

It  is  planned  to- continue  th^^  experiment  as  outlin>^d  above.   If 
it  can  be  shown  that  these  grafts  grow  r-nd  function,  one  m.ight  consider 
the  possibility  of  attempting  the  sam^  procedure  in  humans.  It  is 
doubtfxil  if  during  the  present  year,  progress  on  this  experiment  will 
develop  to  the  point  that  would  allow  such  a  study. 


11. 


BUDGET  ACTIVIiT: 


RESE'.RCH  (V     ;.dministr,;tion  n 

REVIEla'  2t  ..PPR0V..L  £7         TECHNICAL  ASS1ST..NCE  /7 


MO  ENTRIES  FOR  ITEMS  12,    1.3,    H-l,   l5  &  l6. 


PROJECT  REPORT  FORM 


1»  National  Cancer  Institute 2,   Surgery  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 


A. _ 5.  NC 1-765 


SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  NC  . "" 

&.     Effect  of  Carcinogenic  Agents  using  a  Bronchial.. Pouch  Preparation  in  tho  tr 
PROJECT  TITLE 


Dr.  John  H.  Waite 
PRINCIPAL  INVESTIGATOR(S) 


Dr.  W,  C.  Huepsr  and  Dr.  William  Banfield 


OTHER  INVESTIGATORS 

?.  PROJECT  DESCRIPTION 
Objectives.: 

(1)  To  introduce  and  utilize  the  blind  pouch  technique  into  broncho- 
genic carcinoma  carcinogenesis  study.  Results  may  be  added  to  current 
evidence  concerning  the  etiology  of  human  lung  cancer. 

(2)  To  provide  a  satisfactory  large  animal  tumor  for  treatment 
studies. 

Methods  Employed; 

As  performed  at  present  the  pouch  is  fashioned  from  the  left  lowe-^- 
lobar  bronchus.  A  left  lower  lobectomy  is  first  performed,  amputating- 
the  bronchus  more  distal  than  usual.  The  lower  lobe  bronchus  is  then 
re-amputated  at  a  higher  level,  severing  the  cartilagenous  ring  and  endo- 
bronchial structures,  but  leaving  the  peribronchial  structures  intact  as 
a  vascular  pedicle.  The  left  main  bronchus  is  sutured  as  are  both  ends 
of  the  pouch,  forming  in  effect  a  pseudocyst.  The  end  of  a  fine  poly- 
ethylene catheter  is  implanted  into  the  lumen  of  this  pouch,  and  the  othe 
end  of  the  catheter  is  exteriorized  through  the  chest  wall  where  it  is 
buried  beneath  the  skin,  available  for  injection  purposes. 

Suspected  carcinogenic  agents  will  be  injected  into  the  bronchial 
pouch  via  the  polyethylene  catheter  at  regular  intervals.  Suitable  coritr:'.- 
where  the  v«hicle  only  is  injected,  will  be  included.  Serial  roentgen- 
ograms will  De  taken  to  detect  and  follow  the  progress  of  any  left  hilar 
radiodensity  which  may  develop.  The  animals  will  be  followed  for  four-  t-: 
five  years,  after  which  time  they  will  be  sacrificed.  It  is  proposed  to 


PAGE  2 
SERIAL  NO. 

Methods  Employed;  (Continued) 

study  the  following  agents  initially:  3,4  benzpyrene,  tobacco  tar,  and 
internal  combustion  engine  eschaust  concentrate. 

If  sufficient  animals  and  facilities  are  available,  rates  of  uptake  isf 
radio-labelled  samples  of  these  compounds  may  be  studied.  Further  e^cperijiier  . . 
will  be  conducted  to  attempt  production  of  a  bronchial,  pouch  or  bronchostomc 
continuous  with  the  skin  surface,  for  carcinogenesis  studies. 

Ma.ior  Findings;  ■ 

In  a  pilot  group  of  3  dogs  that  were  studied  for  technique  development 
only,  all  three  dogs  survived  the  procedure  and  in  all  three  a  satisfactory 
pouch  preparation  was  found  at  sacrifice  two  weeks  later.  Pathologically, 
the  pouch  appeared  to  be  identical  to  a  pseudocyst  lined  by  healthy  bronchial 
mucosa  and  filled  with  a  non-infected  gelatinous  material. 

Significance  to  Cancer  Research;  ■•■•■.■;:-•-■.,,:, 

To  date  the  majority  of  evidence  purporting  to  bear  on  the  genesis  cf 
human  bronchogenic  carcinoma  is  adduced  from  the  ability-  of  .a  suspected 
carcinogenic  agent  to  produce  a  tumor  when  painted  on  the  skin  of '  a~.  rabbj,t  . 
or  mouse .  In  interpreting  the  results  of  these  experiments ,  not  only  must  • 
the  heterogenecity  of  the  e3q)erimental  animal  with  respect  to  humans  be  co'f^ 
sidered,  but  dissimilarity  between  epidermis  and  bronchial  epithelium  plays 
an  important  role.  Exposing  the  experimental  animal  to  an  atmosphere  ladon; 
with  the  suspected  carcinogenic  agent  has  almost  always  been  unsuccessfur: 
in  producing  tumors.  This  has  lead  to  the  conclusion  by  some  investigators? 
that  the  agents  are  not  respiratory  carcinogens  and  by  others -that  the 
animals  respiratory  defense  (the  cough,  the  dilution  of  the  carcinogenic 
agent  in  respiratory  secretions,  and  the  action  of  cilia)  are  so  f f ficier!'.. 
as  to  make  exposure  to  the  carcinogenic  agent  negligible  using  this  tecliXLlqac 
There  is  much  logic  to  support  the  second  hypothesis,  and  a  good  method  to 
provide  contact  with  a  strong  concentration  of  the  suspected  agent  over  a  prc= 
longed  period  of  time  and  against  which  contact  the  animal  may  not  have  an 
effective  defensive  mechanism  might  be  of  some  value  in  settling  this  point. 

We  feel  that  a  study  on  the  uptake  of  radioactive  benzpyrine  from  our 
pouch  preparation  alone  might  yield  sufficient  data  to  justify  the  project. 

11.  BUDGET  ACTIVITY;    ^    '.. 

RESEARCH         ^O    ADMINISTRATION:       /~~7 
REVIEW  &  APPROVAL  A7    TECHNICAL  ASSISTANCE  /^/ 


(No  Entries  for  Items  12,  13,  15  &  16.) 


PAGE  1 
PROJECT  REPORT  FORII 

1.        T\Tacional  Cancer  Institute  2.       Surgery  Branch 

IMSTlTU'i'!^ WiaOiiVl'ORY  OR  BIlVJCH 


3.  h.  5.  MC 1-7 66(C) 

SECTIO^J  OR  SERVICE  lOOATIOW  (IF  oThER  fmi]  feEthESDA)      SERL'.L  NO. 


6.  C crab ination  Therapy  of  Esophageal  Carcinoma. 
PROJECT  TITLE  " 

7.  Dr.    John  H.  Waite  -  Dr.    J.   Robert  Andrews 


pRTNCmL  i^;\/EtiTlL^iTuR(t.)' 
8.         Dr.  Andrew  G.  Morrow 


OTHER  IlI^TESTiaiTORS 

9.      PROJECT  DESCRIPTION 
Objectives; 

This  project  will  attempt  to  develop  a  satisfactor;;'-  method  for 
treatment  of  squamous  carcinom.a   '-..f  the  esophagus.      Iraprovement  of  ■ 
operability,   resectability,   and  cure  rate,  will  be  sought  by  a  pro- 
gram of  preoperative  rotational  supervoltage  radiation,     A  byproduct 
of  the  studj'-  will  be  the  availability  of  the  recently  irradiated  esopha- 
gus for  intensive  pathologic  study  to  evaluate  the  effects   of  rotational 
supervoltage  on  this  tumor,     B}/  use   of  tube  feedings  during  the  entire 
period  of  preoperative  radiation  an  attempt  will  be  made  .to  improve 
caloric   intake  and  reduce  the  incidence  of  hemorrhage  and  perforation 
during  the  course  of  therapy. 

Methods  employed; 

(1)  Complete  workup 'to  determine  the  extent  of  tumor.     Patients  with 
metastatic  carcinoma  or  with. regional  Invasion  of  a  vital  structxjre, 
such  as  heart  or. lung,  will  not  be  suitable. 

(2)  Insert  plastic  Levin  tube  at  esophagoscopy  to  provide  maintenance 
and  improvement  of  nutrition.      Consider  insertion  of  a  nuchal  esopha- 
gostomy  tube  if  patient  develops  hypopharyngeal  irritation  due  to  the 
indwelling  Levin  tube. 

(3)  Radiation  plan  of  study  bj'  Dr.  Andrex^^s  as  coinvestigator.      In 
general,   he  plans  to  give  external  two  million  volt  therapy  by  rotation 
teclinique,   beginning  with  low  doses  and  increasing  gradually  to  a  total 
of  6,000  to  7,000  "r"  axial  dose  over  a  period  of  six  to  seven  weeks. 
If  difficulty  in  achieving  a  satisfactory  vertical  width  of  port  is 
encountered,   the  possibility  of  delivering  supplemental  gamma  rays  from 
an  available   intra-luminal  source  will  be  considered.      The  presence   of 
the  indwelling  Levin  tube  would  be   ideal  for  this  technique. 


PROJECT  REPORT  FORM  (Cont'd)  P^'^^E  2 


NC 1-7. 66(C). 

SERL\L  No. 


Methods  employed;  (Continued) 

ii.  Surgery  (with  Dr.  Morrow)  about  four  weeks  after  completion  of 
radiation.   If  resectable,  total  esophagectomy  with  dissection  of  the  ^  ■ 
regional  nodes  will  be  performed  with  iiranediate  or  delayed  reconstruction 
of  gastrointestinal  continuity. 

Evaluation  criteria  will  include  (l)  changes  in  tumor  size  by  serial 
roentgenography  during  radiation,  (2)  extent  of  radiation. reaction- and 
tumor  fixation  at  the  time  of  surgery,  (3)  extent  of  twaor  and  lymph  node^ 
metastases  by  pathological  examination  of  surgical  speciiaen,  and  {h)   clini- 
cal course  oa  patient  after  treatment. 

Patient  Material;        ■       "   ' 

Patients  studied  during  the  calendar  year  1955  are  reported  by 
Dr.  R.  R,  Smith  under  Project  No.  751(C),  "Evaluation  of  Radical  Surgery 
and  Development  of  New  Surgical  Techniques  as  a  Therapeutic  Method  of 
Palliating  or  Definitive  Therapy  of  Advanced  Cancer."  This  included  the 
initial  3  patients  studied,  for  an  average  of  Ul  hospital  days  and  one 
outpatient  visit  each.   ... 

Major  findings; 

Of  the  3  patients  studied,,  1  patient  was  unsuitable  for  the  program 
because  of  extreme  senile  debility,  and  2  p?tients  were  suitable.  These 
patients  both  satisfactorily  completed  a  course  of  rotational  2-raillion 
volt  therapy,  and  came  to  surgery  one  month  after  completion  of  therapy. 
Maintenance  of  alimentation  by  tube  feedings  was  very  successful  in  both 
patients  and  in  itself  maj  prove  to  be  a  significant  contribution  to  the 
treatment  of  this  lesion.   Of  the  2  patients  brought  to  surgery,  one  proved 
incurable  by  virtue  of  abdominal  metastases  found  at  the  time  of  prelimi*- 
nary  laparotomy. 

An  esophagectomy  was  performed  in  one  patient.  Resection  of  the  lesion 
was  not  hindered  in  this  one  instance  by  preliminarjr  radiation,  on  the 
contrarjr  it  is  felt  that  the  prelirainrry  radiation  probably  improved  the 
resectr^bility  of  the  lesion  which  wps  in  the  region  of  ti:-e  r;rch  of  the 
aorta.  This  patient  expired  postoperatively  due  to  a  culmination  of  tempo- 
rary hemorrhagic  shock  during  surgery,  poor  cardiac  action,  nnd  pulmonary 
edema  postoperatively.  An  operative  mortality  of  about  l5^  is  expected 
for  this  tjTpe  of  procedure  but  will  be  acceptable  because  of  the  certain 
early  fatality  in  esophageal  carcinoma  treated  by  current  methods. 


PROJSCT  REPCRT  FORM   (Cont'd)  R'.GE  3 


MCI-766(C) 
SERIAL  rIO. 


PROJECT  DESCRIPTION  (Continued) 

Significance  to  Cancer  Research; 

Carcinoma  o^f  the  esophagus  is  the  fifth  highest  in  frequency  among 
internal  cancers  in  ndiilt  males.   This  frequency  and  the  poor  results 
available  from  present  methods  of  treatment  make  it  an  ideal  subject  for 
cancer  research.  Adequate  siirgery  and  adequate  radiation  each  fails  in 
its  own  way  to  control  more  than  2%   of  the  entire  group  of  patients 
presenting  themselves  with  esophageal  carcinom.a.  The  chief  advantage 
of  supervoltage  rotational  therapy  x-ray  is  appearing  to  be  better  immediate 
palliation,  although  the  incidence  of  eventual  recurrence  and  death  probably 
will  not  be  much  lower.  Combination  of  surgery  and  supervoltage  rotational 
therapy  is  being  tried  at  several  centers^  utilizing  surgery  as  the  pri- 
mary treatment  and  roentgen  therapjr  as  a  "mop  up"  treatmt^nt.  For  the 
reasons  already  enumerated,  the  principal  investigator  feels  that  the 
initial  use  of  radiation  may  provide  superior  results. 

Proposed  course  of  project; 

Sec  "Methods  employed"  above.   The  availability  of  material  will 
make  this  a  slowly  developing  project.. 

11.  _^ 

BUDGET  ;.CTlVriY: 

RESE..RCH  fTJ       -:.DimiISTK-.TION  [J 

REVL.V^'  k  EPPRO'vV.L  /T"         TECHMCAL  ;.SSIST..MCE  /~7 

NO  ENTRIES  FOR   ITEI'IS  12,    13,    lU,    l5  ?c  16. 


1 .  Ifc^-— =-   ^=--=~ 


".^.il  ^'---.---zi  isscoisted  with 


5.  :ri-- 


.-■C  Z^T'.    --■ 


.^agec"^  "x  i=_e  per- 


PnDJECT  REPORT  FORII  (Cont'd) 
NCI-767(C)  P^^^  2 

SERIAL  NO. 


9.  PROJECT  DESCRIPTION  (Continued) 

Methods,  employed; 

Temperatures  of  the  patient  from  various-  body  suxfaces  v;ill  be 
recorded  simultaneously  with  a  multi-channel  thermocouple  which  will 
also  I'ecord  the  prevailing  room  temperature.  Various  physiologic 
determinations  on  the  patient  will  be  obtained  using  standard  methods, 

Patient_  Ija  terj-.al ;  " 

liaterial  for  this  project  \irill  be  obtaiiied  during  operations,  on 
patients  ad-nitted  for  other  projects. 

Ha  .lor  Findings  t 

None.  Not  started  yet,  ■  r  ■ 

Siftmficance  to  Cancer  Research; 


The  information  to  be  derived,  from  this  study  will  be  used  to  help 
render  major  surgical  procedures  for  cancer  more  successful. 

Proposed  Coijrse  pf^Pro;ie_ct; 

During  the  coming  year  many  of  the  patients  undergoing  major  surgery 
for  cancer  will  be  studied  diuring  their"  surgical  proced-ures.  Constant 
body  temperatures  and  other  physiologic  data  will  be  determined  to  be 
correlated  with  the  procedure  being  done,"  the  room  temperature  and 
htmiidity,  and  the  other  contributing  factors.  ■         ■  ■ 


PROJECT  REPORT  FORM  (Cont'd) 

PAGE    3 


10.    _2Mcl 


SERIAL  NO. 
11,  _, .„ 


BUDGET  AGT.IVJTY: 

RESEARCH  ^  ADFETISTRATION  [J 

REVIEW  &  APPROVE!  L     [J  TF.CIMICAL  ASSISTANCE  O 

12.  N  0  N_E  .^ .  _-_____^ .___ 


C00PERATI:mG  units  of  riC  PIjTELIG  IjEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT. 


13, N  0  N  r 


IF  THIS  PROJECT  PEEIQIES,  COliPLEIENTS,  OR  PARALLELS  PvESEARCH  DONE  ELSEl-fflERE 
IN  THE  PUPLin  HEALTH  SERVICE   (WITHDUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR 
FUNDS),   IDENTITY  S'JGH  RESEARCH. 


15.  NONE 


PUBLICATIONS  OTHER  THAN  ABSTPulCTS  FRDII  THIS  PROJECT  DURING  CALENDAR  YEAR  1955 


16,  N  ONE 


HDNDRS  AJ^E)  AWATxDS  TO  PERSOMEL  REL/ITING  TO  THIS  PHDJECT  DURING  CALENDAR 
YEAR  1955. 


p:iOJECT  REPORT  FORM    ■                                        PAGIl  1 
1.        National  Cancer   Institute  2,       Siirgery  Branch 


fiTSTlruTfi  LlBOiIvtcry  or  branch 


3.  U.  " 5.  yci-763 


6.        Studv  of  the  Role  of  the  Pronerdin  System  in  Natural  IiTiinunit3?- 

PROJECT  'knam  ' 


7.       Dr.  TJiiiiain  E.   Schatten 
PRINCIPAL  ll<i\i^^TiCiU'ali(S) 


Dr.   Lester  I'l.   Cramer  -  Dr.   Horace  Herbsnan  -  Dr.   Robert  R.    Smith 
OTHEIR  lN\/hS'i'iaii'i'okS '  ' 


9.    PROJECT  DESCRIPTION 

Objectives;  •      ,        ■    ' 

To  evaluate  the  effect  of  altering  the  serum  properdin  level  in  an 
attempt  to  abrogate  the  immune  mechanism  that  plays  a  role  in  the  neutrali- 
zation of  viruses  and  in  the  prevention  of  succescful  homo-  and  hetero- 
transplantation of  tumor  tissue. 

Methods  employed: 

One  stud"  that  would  indicate  whether  or  not  zjrmosan,   a  carbohydrate 
that  combines  with  the  serum  globulinj   properdin,    is  effective  in  alter- 
ing host  resistance  would  be  to  ascertain  whether   or  not  z;;Tnos3n  is  effect- 
ive  in  the  heterotransplantation  of  tumor  tissue.      If  zjTdosan  were  effective, 
it  would  strongly  indicate  that  properdin  pla3''s  a  role   in  the  natural  resist- 
ance of  host  anuials,   since  zymosan  has  a  specific  action  on  properdin. 
This  study  will  be  carried  out  by  adrainistering  zymossa  intravenously  to 
rats  in  different  doses  at  different  rntervals   of  time  prior  to  and  aftar- 
inoculation  of  rats  with  tujnors.      The  S-91  melanoma  of  mice  will  be  used 
for  heterotransplantation  in  this  study  of  the  effects  of  altering  properdin 
levels   in  rats.      If  it  is  proved  that  z^nnosan  is  effective  in  successful 
heterotransplantation,   then  transplantation  of  tumors  from  patients  to  rats 
will  be   studied,     Tuinors  will  be  transplanted  ijito  groups  of  rats  tliat  have 
been  pre-treated  with  zymosan  .-'nd  with  zjrmosan  and  cortisone.     Tumors  that 
survive  transplantation  and  grov;  will  be  periodically  transplanted  to 
other  rats,    some  pre-treated,   and  others  not  treated.     This  will  be  ?n 
attempt  to  adapt  a  transplantable  tumor  strain.      Tumor  groirrth  will  be  mea- 
sured directly  following  subcutaneous   inoculation  and  b     weight  of  the 
tumor  following  ijitraperitoneal  inoculation. 


PROJECT  REPORT  FORM  (Cont't)  PAGE  2 


HC 1-768 
SERML  NO. 

Methods  employed;   (Continued) 

If  heterotransplantation  of  tumors  can  be  effected  successfully,  the 
oncolytic  effects  of  viruses  on  tumors  will  be  studied  by  injection  of 
viruses  into  the  tumors.  .Following  vjxus  injection,  siilocutaneous  tumors, 
will  be  measured  and,  biopsies. -taken  periodically  for  histologicsl  studies. 
The  effects  of  different  viruses,  on  a  tumor,  as  well  ?s  the  role  of  zymo- 
san, cortisone,  and  other  agents  in  enhancing  virus  effects  will  be  studied. 
Also,  this  would  provide  a  method  of  increasing' the  virulence  of  a  virus 
by  passing  the  virus  thru  a  patient's  tumor  to  "train"  it  against' the 
tumor  before  adrninstering  the  virus  to  a  patient. 

Major  findings;  '  ,         . 


This  project  is  in  its  preliminary  phase.  However,  it  is  believed 
that  there  was  a  definite  difference  in  the  size  of  heterotransplanted 
tumors  in  rats  that  were  treated  with  zymosan  prior  to  tumor  inocula- 
tion when  compared  to.  rats,  .that  were-  not  treated.     \ 

Significance  to  Cancer  Research; 

If  huma-n  tumors  could  be  heterotransplanted  into  other  animals 
successfully,  this  would  provide  a  method  for  studying  individual  turiiors 
of  patients  in  laboratory'-  animals.  This  would  be  a  method  of  testing 
many  chcmotherapeutic  agents  in  laboratory  animals.  As  mentioned  above, 
this  would  also  provide  a  method,  for  the  evaluation  of  the  oncolytic 
effects  of  viruses  on  tumors  snd  perhaps  this  would  be  a  method  of  con- 
ditioning a  virus  to  become  more  virulent  with  regards  to  its  effect  on 
a  particular  tumor.   If  zjmiosan  were  effective  in  altering  host  resist- 
ance so  that  a  tumor  could  be  transplanted,  it  might  also  be  used  to 
alter  the  immune  mechanism  that  prevents  continued  propagation  of  a  virus 
following  its  administration.   This  antibody  response  following  virus 
administration  is  one  of  the  major  probltns  that  now  presents  itself  in  ■ 
the- virus  treatment  of  carcinoma.. 

Proposed  course  of  project:  .     . 

Heterotransplantation  of  tumors  will  be  performed  in  a  sufficient 
number  of  rats  that  are  pre-treated  with  zymosan  and  with  zymosan  and 
cortisont  to  determine  the  effectiveness ,. of  this  treatment  in  hetero- 
transplantation.  If  •z-ymos'.'n   does  promote  successful  heterotransplanta- 
tion, then  further  studies  wilt  be  carried  out  as  outlined  above. 


PiiOJECT  REPORT  F0RI4  (Cont'd)  PAGE  3 


10.  NCI- 76 3 

SERilL  iv'O. 


11. 


Budget  acthity: 

RESEARC:i  /x7  ADMINISTR^ITION  /T* 

RE/IEiJ  1  A??R0VA.L,/7  TECHNICAL  ASSISTANCE  /7 


NO  ENTRIES  FOR  ITEJ4S  12,    13,    lli,   13'  ?:  16. 


PROJECT  REPORT   FOM  PAGE  1 


1.       N^.tional  Cancer  Institute  2.       Surgerj;-  Branch 

liSTITUTE LiillftlTORY  OR  iTUvAQU 


3.  it. 5.  MCI-770 

ggcTION  OR  5^\llC&  LOCATTO?!  (1^'  CTH^R  T'^J' br'j^HESDA )      SERI/.L  NC. 

Study  of  the  Effect  of  Growth  Hormone   on  Cprtilage  'Transplants  and 
6.         of  the  Sxxrvival  and  Growth  of  These  Transplants 

projj,ct  title ' ' 


7.       Dr.  William  2.   Schatten 


8.       Dr.   Lester  '.'.   Cramer  -  Dr.   Horace  Herbsman  -  Dr.  Delbert  I'l.   Ber^^enstal 
OTHER   ItTVESTlG.\TORS 


9,   PROJECT  DESCRIPTION 

Objectives: 

To  develop  a  method  of  stirirolation  of  growth  of  cartilage  transplants 
and  to  stud"-  the  survival  ard   rrox^h  of  homotransplants  and  heterotrans- 
plants   of  cartilage. 

Ile'thods  employed; 

Hypopbysectomized  rats  are  used  iri  this  study  because  they  are  sensi- 
tive to  small  amounts   of  growth  hormone  and  becc?use  non-treated  hypophy- 
sectomized  animals  are   oetter  controls  than  normal  rats   in  this  experiment. 
Three   groups   of  rats  are  being  studied.     Autografts  of  xiphoid  and  costal 
cartilages  are  transplanted  in  one   :;,roup,   homografts   in  another,   and 
heterografts  from  rabbits   in  the  third  group.     The  cartilage  grafts  are 
transplanted  into  the  axilla  and  abdominal  wall.     Rats   in  each  group  are 
sacrificed  at  weekly  intervals.      On  the  second  and  third  days  prior  to 
sacrifice,   S-35   is   injected  intraperitoneally.     After  the  animals  are 
sacrificed  the  cartilage  grafts  and  the  aniraal's  own  costal  and  tibial 
cartilages  are  removed  f  r  stLidj"".     The  prima r-y   method  of  studying  acti- 
vity of  the  cartilage  is  by  deternining  the  S-35  uptake  of  the'  cartilage 
as  expressed  in  counts  per  100  mg.    of  dry  cartilage  per  minute.      The 
transplants  are  com.pared  with  animal     cartilage  that  was  not  transplanted 
and  also  with  the  transplants  of  p   control  animal  that  did  not  receive 
growtli  horm.one.      The  growth  of  cartilage  transplants   is  also  evaluated 
by  means  of  comparisons   of  measurements  and  wet  weights   of  the  cartilages 
taken  at  the  time   of  transolantation  and  at  the  tiime  of  sacrifice. 


PROJECT  RjiPORT  FORM  (Cont'd)  KiGJi  2 


NCI-770 


9.   PROJECT  DESCRIPTIOW  (Continued) 
Major  findings; 

The  project  is  in   oh?  preliminary  phasa,      1%  is  believed  that  the 
data  that  has  been  accimulated  up  until  the  present  time  shows  that 
autografts  of  cartilage  can  be  stimulated  by  gr^/'th  hor'none.     Homo- 
grafts  and  hetfcr.graf-c::  of  cartilage  can  be  stitnUlat^u  to  a  lesser  extent 
by  growth  hormone.     In  addition,  het.erografts  of  cartridge  inL  anim-^ls 
treated  with  growth  honnone  do  not  take  up  as  nuch  J-3$  as  hett-rografts 
in  non-trur.ted  hyp  jphysector.iA.:ad  anmals.     This  finding  is,    of  course, 
of  great  interest  and  is  bcir^-  more  fully  investigated, 

Signif icanr'e   to  Cancer  Research; 

It  is  go'-.^rill"    csrecd  that  honologous  cartilage  grafts  surx'ive 
transplantaxj-on  ior  vaxying  periods  of  time.     The  exact  reasons  that 
cartilage  grafts  are  not  affected  oj  an  immune  response  as  are  alinost 
all  other  tissues,    is  not  known.     It  is  also  not  known,  how  well  homo- 
grafts  of  cartilage  do  survive.     It  is  difficult  to  perform  man;'-  studies 
that  might  give  this  answer  bocause  of  the  low  mutabolic  activit;^  of 
cartilage.      It  is  believed  that  the  uptake   of  S-35  by  a  cartilage  graft 
will  be  a  critical  test  of  "letabolic  survival.      It  has  been  shoi-m  that, 
after  dialysis  of  cartll-^ge  rc'  [j.8  hours  following  removal  of  a  speci- 
men from  an  anm'.l  tha-i;  h.s  received  S-35,   9^  of  the  sulfur  is  bound 
in  ■  n  organic  couipound.      Srlp>^'^.te   is  takc;n  up  by  cai-tilage  cells  where 
it  is  probably  attach.-.d  to  the  mucopolysaccharide  molocule  and  thereafter 
appears  in  the   ground  substance.      It  is  hoped  that  this  study  will  indi- 
cate some  of  the  reasons  that  honografts  of  cartilage  svirvive  transplanta- 
tion. 

It  is  also  the  purpc-se  of  this  experiment  to  determine  if  cartilage 
grafts,    including  autografts,   homografts  and  heturografts,   can  be   stimu- 
lated by  growth  hormone  following  transplantation.     If  this  could  be 
achieved,    it  would  have  clinical  application  in  various  reconstructive 
procedures  in  which  cartilage  grafts  are  used. 

Proposed  course  of  project; 

Three  groups  of  hypophysectomized  rats  will  be  used  for   studying 
autografts,   homografts,   and  hettrografts  of  cartilage.     There  will  be  iiO 
rats  in  each  group,   20  servijig  as  controls,  and  the  other  20  reciving 
growth  hormone  following  transplantation.     Following  transplantation 
of  all  ijO  ratsj   10  rats,   5  cuntrols  and  5  treated,  will  be  sacrificed  each 
week  for  k  weeks.     S-35  will  be  given  to  the  rats  prior  to  sacrifice  as 
outlined  above.     Growth  of  the  transplants  will  be  studied  as  outlined 
above.     If  autografts  can  be  stimiilated  to  grow  by  the  use  of  growth 
hormone  in  hypophysectomized  rats,   normal  rats  and  other  anixiial  species 
will  then  be  used  in  an  attempt  to  sec  if  growth  hormone  will  stimulate 
the  growth  of  cartilage  transplants  in  those  animals.     Also,   cartilage 


PROJECT  RiLPCRT  FOWi  (Cont'd)  PAGE  .3 


NCI-770 


Proposed  courso  of  project;  (Continued) 

transplants  will  be  performed  and  period  of  time  allowed  to  lapse  prior 
to  adjiiinistration  of  growth  hormone.     This  will  be  done  in  a  separate 
group  of  aniraals. 

The  fact  that  heterografts  of  cartilage  have  ben  found  to  take  up 
less  S-35   in  hyp ophyse  eternized  animals  that  receive  growth  hon-aone  than 
in  h^'-pophysectomized  animals  that  are  not  treated  indicates   that  hj'po- 
physectomized  aniraals  may  not  be  able  to  react  to  foreign  tissue  with 
an  immune  response.      If  this  finding  that  Wc?.s  present  in  the  first  few 
experiments  that  were  done   is  corroborated  by  our  further  exper jjiients 
it  would  be   indicated  to  heteroti-'ansplr'nt  tumors  to  hypophysectomized 
rats  in  an  attempt  to  sec   if  the   tumors  would  not  be  affected  hy  host 
resistance  and  would  therefore  progress  at  their  own  rate  of  growth. 
If  this  were  true,   then  such  turners  could  be  passed  one  or  more  times 
in  hypophysectomized  rats  and  t!ie  tujnors  night  become  adapted  to  the 
new  hosts.     They  might  then  be  transplanted  to  normal  rats  and  the  tumor 
developed  as  a  transplantable  tumor. 


11.  

BUDGET  :XTI'/rrY: 


RESK\RCH  /x7       ADMINISTRATION  £7 

REVIEW  k  APPROVAL  /T"         TECHNICAL  ASSISTANCE  /~y 


NO  ENTRIES  FOR  ITEIIS  12,    13,    lii,   1$  ?c  l6. 


PROJECT  REPORT  F0RI4  PAGE  1 


1.       Nrtional  Cancer  Institute  2.       Surgery  Branch 

INSTITITI'E  LVBOliLTORV   Uk   'dk'MA 


3.  h.  5.  NC 1-771 

SfeCTlor  OR  SERVICE        LOCATlOiJ  (IF  OTl-i^R  TiUW  BETH3SDA)        sERi'.L  NO. 


6.       Experimental  Reconstruction  of  the  Extra-hepatic  Biliary  S3^stera 
PROJECT  TITLE 


7.       Dr.  IJilliam  E.   Schatten 

Pki!101P.'.L  i*JVESTiti.TcR(SJ 


8.       Dr.  Lester  11.   Cramer  -  Dr.   Horace  Herbsman  -  Dr.   Robert  R.   Smith 
dTHER  IMVESTICATORS : ' [ — ' 


9.   PROJECT  DESCRIPTION  " 

Objectives; 

To  develop  a  successful  method  of  reprir  of  commoii  bile  duct  defects. 

Methods  employed; 

Adult  mongrel  dogs  are  selected  as  experimental  animals.     A  chole- 
cystectomy and  ligation  of  the  comi-aon  bile  duct  is  performed  in  ordtr. 
to  closely  simulate  conditions  encountered,  clinically.     Tiiree  daj^s 
following  this   operation,    a  second  operation  is  performed.     At  that  time, 
a  split-thickness  .graft  of  skin  is  taken  from  the   -bdominal  wall.     The 
common  duct  region  is  exposed  and  a  portion  of  comiiion  duct  proximal  and 
distal  to  the  area  of  previous  ligation  is  excised  in  order  to  create  a 
defect  that  is  at  least  2|  cm,    in  length.     The  skin  graft  is  then  used 
to  form  a  tube   over  a  rubber  T-tube,   and  the  T-tube   is   then  introduced 
into  both  ends   of  the  common  duct  and  an  accurate  anastomosis  between 
the  comm.on  duct  and  skin  graft  is  performed.     After  the  graft  is  sutured 
in  place,   a  tab  of  omentum  is  placed  in  the  region  of  the  graft.      The 
.end  of  the  T-tube   is  tied  off,   brought  out  through  a  stab  wound  in  the 
right  upper  qi:ia.drant,   and  then  placed  in  a  subcutaneous  pocket  in  the 
musculature  of  the  right  upper  quadrant  of  the  abdomen.     The  skin  graft 
is  constructed  into  a  tube  in  the  direction  of  Langer's  lines   in  one-half 
of  the  dogs  and  is  constructed  so  that  the  long  axis  runs  perpendicular 
to  Langer's  lines  in  the  remainder  of  the  dogs.     The  T-tube  will  be 
allowed  to  remain  in  plrcc  for  tliree  months   in  one-half  of  the  dogs  and 
for  six  months  in  the  remainder.     Thu  dogs  will  be  observed  for  three 
to  six:  months  following  removal  of  the  T-tubes  in  order  to  observe  them 
for  evidences   of  bile  duct  stricture  formation.     The  dogs  will  then  be 
sacrificed  and  gross  and  histological  studies   of  the  coMnon  ducts  will 
be  performed  at  the  time   of  autopsy. 


PROJECT  RiiPORT  FOffli   (Cont'd)  R/iGE  2 


NCI-771 
SER1/.L  NO. 


9.  PROJECT  DESCRIPTION  (Continued) 

Major  findings;  ,  .   ' 

At  the  present  time  the  above  procedure  h?.s  been  carried  out  success- 
fully in  ik   dogs.  The  procedure  has  been  performed  in  three  other  dogs 
and  these  do-js  have  died  at  varying  interv^.ls  postoperatively  due,  to' 
leakage  of  bile-  in  the  region  of  the'  graft.  Technical  points  have  been 
learned  from  the  loss  of  these  three  dogs  and  none. of  the  last  nine  dogs 
has  been  lost  because  of  bile  leakage. 

Significance  to  Cancer  Research; 

The  repair  of  common  bile  duct  defects  is  one  of  the  most  difficult 
problems  in  surgery  today.  This  is  i^videnced  by  the  multitude  of  experi- 
mental and  operative  procedures  that  havJ  been  employed.  There  is  general 
agreement  that  it  is  advantage. ous  to  preserve  the  sphincter  of  Oddi  in 
reconstruction  of  the  biliary  tract.  For  this  reason  it  -would  be  more 
preferable  to  repair  common  bile  ducts  by  means  of  a  successful.  tr.':'ns- 
plantation  of  tissue  to  bridge  existing  defects  than  to  effect  repair 
by  means  of  Roux-en-Y  procedures  tiiat  are  advocrted  today.  The  proposed 
method  of  reconstruction  of  the  common  bile  duct  will  alloinr  preservation 
of  the  spincter  of  Oddi  and  therefore  will  be  of  valuu  as  a  now  surgical 
technique.  If  successf  iJ.,  this  method  will  be  recommended  to  surgeons 
for  clinical  application. 

Proposed  co-urse  of  project: 

This  m.ethod  of  common  bile  duct  reconstruction  will  be  performed 
in  a  number  of  dogs  so  that  a  total  of  20  will  have  survived  the  procedure 
and  cm  then  be  observed  for  evidences  of  stricture  formation  over  the 
proposed  period  of  tine. 


11.  

BUDGET  ACTIVITY: 


RESiL.\RCH'  /T/         ADiIIi\IISlTR;iTIOM  £7 

REVIEM  &  APPROVAL  7^  TECHNICAL  ASSISTANCE  /~7 


NO  ENTRIES  FOR   ITEl"iS  12,   1.3,    lU,    l5  'i  16. 


PAGE  1 
PROJECT  REPORT  FORl^l 

1.  N.  C.  I. _^  2,  Endocrinology  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

3,     Endocrinology  Service         4-.  5.  NCI-800C 

SECTION  OR  SERVICE  LOCATION  (IF  OTIiER  THAN  BETHESDA)     SERIAL  NO. 

6,  Endocrine  Aspects  of  the  Progression  and  Therapy  of  Cancer  of  the 

PROJECT  TITLE 

Breast  in  Women  and  Men 


7.  Roy  Hertz,  Delbert  !:.  Bergenstal.  and  M.  C.  Li 
PRINCIPAL  It'TVESTIGATCR(S) 


8,  James  A.  Pittman.  Harold  Altman  (M.  G.  Sherer  and  A,  Breslow  up  to 

OTHER  INVESTIGATORS  7/1/55) 

9.  PROJECT  DESCRIPTION  -    • 

Objective s; 

1,  To  extend  and  intensify  detailed  clinical  observation  of  the 
natural  course  of  cancer  of  the  breast  in  men  and  women  in  order  to 
better  appraise  the  effectiveness  of  various  methods  of  therapy  and 
learn  more  concerning  the  etiology  and  pathogenesis  of  the  disease. 

2,  To  improve  upon  existing  forms  of  hormonal  therapy  for  pallia- 
tion of  advanced  breast  cancer  and  to  elucidate  the  mechanisms  involved. 

I'fethods  employed; 

1.  The  acceptance  of  complete  clinical  responsibility  for  the 
further  management  of  referred  patients  with  proven  diagnosis  of 
cancer  of  the  breast  and  the  application  to  their  problems  of  all 
available  modes  of  therapy, 

2.  The  exploration  of  new  modes  of  hormonal  therapy  for  palliation. 

3.  The  detailed  analysis  of  endocrine  and  metabolic  factors  operat- 
ing in  relation  to  the  genesis  and  course  of  the  disease  in  these  pa- 
tients up  to  the  tiiri.e  of  their  demise. 

A^  Careful  appraisal  of  pathological  specijnens  obtained  at  biopsy 
or  autopsy  in  collaboration  with  Poithology  staff. 

Patient  material; 

No.     Average  Stay  Days 
Admissions:   Adult  females  4-3  43 

Outpatient:   "Tumber  of  patients      LA 
^^umber  of  visits       272 


PAGE  2 
PROJECT  REPOP.T  FOmi  (Cont'd) 


NCI-.800(C) 

SmiAL  NO. 


m^oT   findings  -  12/l/5^-12/l/55; 

.1.  The  momber  of  ...cases  of  breast  cancer  which  have  come  under  our 
observation  now  eqioals  213,  These  patients  have  been  studied  with 
varying  degrees  of  intensity  but  they  all  have  contributed  to  the 
general  clinical  background  and  teaching  functions  of  the  group.  This 
activity  continues  to  provide  constant  liaison  vxith  local  physicians 
and  medical  institutions  for  additional  patient  referral  to  the 
Clinical  Center. 

2,  The  necessary  clinical  precautions  to  be  taken  in  the  adminis- 
tration of  massive  parenteral  estrogen  therapy  in  patients  with  breast 
cancer  and  now  rather  completely  determined,  thus  making  this  form  of 
therapy  an  entirely  feasible  procedure  in  this  and  other  clinics.  In 
the  face  of  the  hi^ily  variable  clinical  conditions  present  in  patients 
with  advanced  breast  cancer,  it  seems  increasingly  unfeasible  to  . 
attempt  to  determine  definitively  whether  this  form  of  therapy  presents 
any  distinct  clinical  advantages  over  more  conservative  forms  of  es- 
trogenization.  Nevertheless,  our  own  clinical  impression  at  this  time 
is  that  in  certain  selected  cases  massive  parenteral  estrogen  adminis- 
tration presents  certain  advantages  for  prompt  and  intensive  hormonal 
therapy. 

3,  Additional  data  on  estrogen  withdrawal  bleeding  in  elderly 
patients  with  advanced  breast  cancer  indicate  an  incidence  of  appro- 
ximately 15  percent.  These  obser^/ations  confirm  the  marked  difference 
in  endometrial  response  in  senile  women  from  the  uniformly  positive 
response  seen  in  women  under  4-0  years  of  age.  This  difference  in 
response  suggests  that  an  additional  factor  other  than  estrogen  is 
essential  for  the  activation  of  the  hujuan  endometrium, 

4-.  The  failure  to  encounter  a  single  case  of  endoro.etrial  carcinoma 
among  all  of  our  breast  cancer  patients  treated  with  prolonged  and 
intensive  estrogen  confirms  the  lovj  level  of  carcinogenicity  which 
estrogens  have  for  the  endometrium  in  patients  over  4-0, 

5,  In-".tial  studios  on  the  use  of  massive  intravenous  androgen 
therapj-  hf.ve  proven  discouraging.  Local  and  systemic  tolerance  is 
not  gor.d  and  lie  have  failed  to  detect  any  material  blood  level  of  i 
circulating  androgen  by  bioassay  even  after  as  much  as  750  mgm-,  tes- 
tosterone propionate  given  intravenously.  Moreover,  the  mechanical 
problem  of  rendering  such  androgen  solutions  free  of  particulate 
material  has  thus  far  proven  insurmountable.  Accordingly,  our 
clinical  efforts  in  the  development  of  massive  androgen  therapy  in 
breast  cancer  have  been  momentarily  suspended. 

6,  Techniques  for  complete  balance  studies  of  breast  cancer 
patients  have  been  developed.  These  patients  can  now  be  followed 
with  respect  to  balance  of  sodium,  potassium,  calcium,  nitrogen. 


MCI-500(C) 

SERIAL  MO. 


PAGE  3 
PROJECT  REPORT  F0RI4  (Cont'd) 


Ma.jor  findings  (cont'd.) 

phosphorous  and  creatinine. 

These  techniques  have  thus  far  permitted  a  -coaplste  characteriza- 
tion of  the  metabolic  action  of  the  newer  corticoids,  suggesting  a 
greater  field  of  usefulness  for  these  compounds  in  the  practical 
palliation  of  breast  cancer  patients.  One  of  the  more  immediately 
demonstrable  effects  is  a  rapid  correction  of  hypercalcemia,  a  fre- 
quently fats-l  complication  in  patients  with  breast  cancer  with  ex- 
tensive osseous  metastases, 

7.  The  aforementioned  balance  techniques  have  provided  more  re- 
liable quantitative  data  on  the  B-complex  balance  in  patients  under- 
going protein  catabolic  effects  of  exogenous  corticoids.  Although 
excretion  of  biotin,  riboflavin,  pyridoxin  and  folacin  is  unaltered 
during  corticoid-induced  catabolism  there  is  a  distinct  and  repro- 
duceable  loss  of  pantothenate.  Thus,  the  participation  of  this 
dietary  trace  factor  In  the  steroid  catabolic  effect  is  suggested, - 
In  addition,  these  data  extend  earlier  observations  indicating  a 
retention  of  pantothenate  during  androgen  induced  anabolic  states. 

8.  Cytological  studies  of  the  vaginal  smears  of  patients  treated 
with  estrogen,  androgen,  corticoids  and  progesterone  have  been  con- 
tinued. No  evidence  of  metaplastic  changes  have  been  noted, 

9.  A  new  steroid,  17  ethyl  19  nor  testosterone  which  is  knoi-m 
to  be  highly  anabolic  has  been  p^.rtially  evaluated  for  its  relative 

' androgenicity  in  a  series  of  six  breast  cancer  patients.  The  compound 
thus  far  seems  to  present  no  marked  advantage  over  other  androgens 
available, 

10,  A  study  of  the  effects  of  potent  pituitary  follicle-stimulating 
hormone  preparations  on  the  himian  ovary  has  been  conducted  in  six 
patients  Just  prior  to  surgical  o-variectomy  for  palliation  of  advanced 
breast  cancer.  Marked  follicular  stimulation  was  observed  in  4  cases 
and  no  response  in  2.  No  detectable  rise  in  serum  or  urinary  estrogen 
accompanied  these  effects  and  no  detectable  estrogen  was  found  in  the 
ovai'r-ian  vein  blood  obtained  at  the  time  of  ova.riectomy.  Thus  the 
tro;-ihic  response  of  the  human  ovary  is  not  accompaniad  by.  the  readily 
detectable  changes  in  blood  and  urinary  steroid  level  seen  when  the 
human  adrenal  is  activa.ted  by  ACTH. 

11,  Attempts  to  modify  adrenal  cortical  function -in 'breast  cancer 
patients  by  Amphenone  administration  ho.ve  led  to  a  more  compl:;te 
appreciation  of  the  clinical  toxicity  of  this  compound  and  its  con- 
sequent limitations  as  an  Inliibitor  of  adrenal  cortical  function  in 
man.  Nevertheless,  this  information  has  proven  usefiil  in  application 
of  this  agent  to  other  endocrinological  problems  to  be  described  under 
project  803, 


PAGE  4 
■PROJECT  REPORT  FORI'!  (Cont'd) 
NCI>>^00(C) 
SERIAL  NO. 


Slgnificange  to  cancer  research; 

The  direct  pertinence  of  the  foregoing  studies  to  the  grave 
problem  of  advanced  or  persistent  breast  cancer  is  too  obvious  to 
require  discussion. 

Proposed  course  of  pro.i'ect; 

These  comprehensive  clinical,  endocrinological  and  metabolic 
studies  in  breast  cancer  patients  are  to  be  extended  essentially 
along  the  lines  already  described  above.  A  nev7  departure  vjill  be 
the  evaluation  of  the  place  of  hypophysectomy  in  the  management  of 
breast  cancer,  a  study  to  be  carried  out  in  cooperation  with  the 
neurosiirgical  staff  of  N,I.N,D.B.  In  addition,  studies  of  the 
effect  of  radiothyroidectomy  on  the  clinical  course  of  breast  cancer 
will  be  undertaken. 


11.. 


BUDGET  ACTIVITY: 

RESEARCH  [J       ADl'IINISTRATION  [J 

REVIEVJ  &  AFPPOVAL  [J       TECIffilGAL  ASSISTAI'CS  [J 
NO  ENTRIES  FOR  ITEl^iS  12  &  13 


15.. 


PUBIJCATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955: 

1,  The  effect  of  meticorten  and  meticortelone  on  thyroid 
function,  by  M,  G,  Sherer  and  B,  N.  Siefring  (J.  Clin,  Endo- 
crinol. &  Me tab, )  In  press. 

2.  Amphenone:  Toxicity  and  effects  on  adrenal  and  thyroid 
function  in  man,  by  R.  Hertz,  J.  Pittman  and  M.  Graff  (J, 
Clin.  Endocrinol.  &  Me tab, )  In  press. 


16.. 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955: 

1,  Dr.  D,  M,  Bergenstal  was  invited  to  attend  the  International 
Rheumatology  Congress,  Rio  de  Janeiro,  Brazil,  August  1955. 

2,  Dr.  R,  Hertz  V7as  invited  to  participate  in  the  meetings  of  the 
Societe  d'Endocrinologie  d'Haiti,  Port-au-'Prince ,  Haiti,  Dec. 
1955  (unable  to  attend). 


PAGE  5 
PROJECT  REPORT  FOPJI  (Cont'd.) 
NCI-800(C) 
SERIAL  NO. 

16.  HONORS  AND  AIJARDS  (Cont'd.) 

3,  Dr.  R.  Hertz  is  Invited  to  preside  over  forthconing  session 
of  the  Endocrine  Society,  Jime  1956. 

A,  Dr.  R.  Hertz  was  reappointed  Chairman  of  Endocrinology  Panel 
and  Member  of  Executive  Coromittee  on  Growth,  National  Research 
Council. 


PAGE  1 
PROJECT  REPORT  FORM 

1.  Kf.C.I. 2.  Endocrinology  Branch 


INSTITUTE  LABOPJITORY  OR  BPiillCH 

3,  Endocrinology  Service   A.  HGI-3Q1(C) 

SECTION  OR  SERVICE       LOCATION  (IF  OTHER  THAN  BETHSSDA)  SERIAL  NO. 

6.  Endocrine  Aspects  of  Cancer  of  the  Prostato  in  M-^.n 

PROJECT  TITLE 

7.  Ro7A  Hertz,  Deroert  M.  Borgonstal.  M.  C,  Li,  and  Lois  F»  Hallnan 

PRINCIPAL  INVESTIGATOR (S) 

8.  James  A.  Pittman  and  liarold  Altman 

OTHER  BTVESTIGATORS 

9.  PROJECT  DESCRIPTION  - 

Objectives;  1.  To  improve  non-surgical  therapy  of  cancer  of  the  prostate 

2,  To  increase  understanding  of  endocrine  factors  involved  :' 
genesis  and  course  of  prostate  cancer. 

3,  To  gain  basic  knowledge  of  the  metabolic  action  of  cer- 
tain steroids  in  man, 

ITethods  employed;  Detailed  clinical  and  metabolic  observations  of 

patients  with  advanced  prostatic  carcinoma  who  are 
no  longer  amenable  to  accepted  forms  of  clinical  management. 

Patient  material; 

No,      Average  Stay  Days 
Admissions:  Adult  males  4  51 

Outpatient:  I^Iumber  of  patients       2 
Number  of  visits         4- 

Ma.jor  findings;  1.  The  administration  of  chorionic  gonadotropin  to  3 
patients  with  prostatic  carcinoma  just  prior  to  or- 
chiectomy has  shovm  that  such  gonadal  activation  even  in  the  senile 
man  will  aggravate  the  disease  as  manifested  by  exacerbation  of  pain 
and  elevation  of  acid  phosphatase.  The  expected  rise  in  urinary  ex- 
cretion of  biologically  active  estrogen  was  not  detected.  The  spermatic 
vein  blood  obtained  at  the  tim.e  of  the  orchiectomy  also  contained  no 
detectable  androgen  or  estrogen.  Thus,  although  it  is  apparent  on 
histological  and  clinical  grotmds  that  the  senile  human  testis  can  be 
activated  by  chorionic  gonadotropin,  this  trophic  action  on  the  testis 
is  not  reflected  in  detects^ble  increments  in  steroidogenesis  as  is  the 
case  in  the  activation  of  the  human  adrenal  by  ACTH, 

2,  Further  studies  of  the  mechanism  of  the  elevation  of 
serum  acid  phosphatase  in  prostatic  cancer  patients  have  yielded  no  new 
data  of  value.  Thus  we  have  failed  in  our  attempts  to  transmit  this 
activity  to  human  fibroblasts  in  tissue  culture.  This  was  attempted 


PAGE  2 
PROJECT  REPORT  FORM  (Cont'd) 
KC 1-801(0) 
SERIAL  NO. 

I-ia.ior  findings  (cont'd.)- „.",.,.• 

on  the  theory  that  this  enzymatic  activity  may  possibly  be  associated 
■  v;ith  a  virus-like  agent  v;ho^se.  titre  rises  in  advanced .  prostate  cancer 
patients.  Initial  impressions  that  this  elevation  of  enzymatic  acti- 
vity represents  the  failure  of  an  iriiibitory  agent  in  normal  serum 
have  failed  of  confirmation. 

Significance  to  cancer  research;  The  high  frequency  of  prostatic  car- 

.,-     -.  cinoma  in  man  and.  initial  inroads 
upon  it  by  hormonal  therapy  indicate  the  pertinence  of  this  study  to 
cancer  research. 

Proposed  course  of  pro.i'ect:  Additional  studies  of  the  clinical  and 

endocrinological  factors  involved  in  the 
extension  and  growth  of  advanced  prostatic  cancer  will  be  undertaken. 
I'lajor  emphs.sis  will  be  placed  upon  the  evaluation  of  several  atypical 
estrogenic  compounds  and  corticoidal  steroids  in  the  therapy  of  pros- 
tatic carcinoma. 

11 . : 

BLTDGET  ACTIVITY: 

RESEARCH  .  U       ADi-IEICESTEiATION  £J 

REVIEI7  &  APPROVAL  /^       1201211011.  ASSISTANCE     /7 


NO  ENTRIES  FOR  ITE^S  12,  13,  15  &  16. 


PAGE  1 
PROJECT  REPORT  FORM 

1.  N.  C.  I. 2.  Endocrinology  Branch - 


INSTITUTE  LABOFt/iTORY  OR  BRANCH 

3.  Endocrinology  Service     K. , 5.NCI-.8Q2(C 

SECTION  OR  SERVICE  LOCATION  (IF  OTHJl  THAN  BETHESDA)  SERI/iL  NO. 

6 .  Preoperative  Hormonal  Therapy  of  Cancer  of  Cervix 

PROJECT  TITLE 

7.  Roy  Hertz,  John  H.  Walte,  and  Robert  Smith 

PRINCIPAL  IWESTIGATOR(S) 

8.  James  A,  Plttman  and  Harold  Altman 

OTHER  INVESTIG/\TORS 

9.  PROJECT  DESCRIPTION  ' 

Ob.jectlves! 

To  determine  the  value  of  steroid  therapy  as  a  palliative  measure 
in  advanced  cancer  of  the  cervix. 

Methods  employed; 

Patients  who  are  considered  amenable  to  definitive  surgical  therapy 
are  pre-treated  for  4-  to  6  weeks  with  massive  doses  of  various  steroid 
hormones  singly  and  in  combination  and  then  are  operated  upon.  Their 
progress  is  evaluated  by  frequent  pelvic  exaralnatlon,  serial  biopsies 
and  smears  and  by  histopathological  study  of  the  surgical  specimens 
removed.   Pyelograms  ajid  barium  enema ta  are  also  employed  to  eva.luate 
changes  in  the  pelvic  viscera.  The  amount  of  vo.ginal  bleeding  is  ■ 
checked  from  day  to  day.  The  patient's  general  clinical  status  v/ith 
respect  to  v;elght,  appetite,  blood  status,  and  pain  are  also  closely 
observed. 

Patient  material; 

No.    Average  Stay  Days 
Admissions:  Adult  females  3  10 

Outpatient;  Number  of  patients        3 
Number  of  visits         12 

Ma.jor  findings; 

No  extension  of  our  findings  beyond  last  year's  report  has  been 
made  due  to  the  justified  preocupation  of  our  stirgical  staff  with 
the  virus  approach. 


NCI-g02(C) 

SERi;.L  NO. 


PAGE  2 
PROJECT  REPORT  FORM  (Cont'd.) 


Significanco  to  cancer  resoaroh; 

Palliation  of  advanced  corvical  carcinomas  urgently  needed  in 
view  of  the  low  over-all  5  year  survival  rate  of  patients  with  this 
disease  {UO-^Ofo),  ... 

Proposed  course  of  pro.ject; 

The  further  course  of  these  studies  will  depend  upon  the  interests 
of  the  surgical  staff  Xirhich  is  at  the  moment  justifiably  concentrated 
on  the  virus  approach  to  cervical  cancer,  •        • 


11. 


BUDGET  ACTIVITY: 

RESEARCH  ^J      ADMNISTR/.TION  ■      /^ 

REVIEt'J  &  APPROVAL     /7       T3CMICAL  ASSISTANCE  .  U 


12. 


COOPJ?a"-Tr:G  TIIIITS  OF  TH3  PUBLIC  HE/.LTH  SERVICE,   OR  OTHER  ORGANIZ/.TIONS , 
P^,OVIDirG  FU^IDS,  FACILITIES,.  OR  PERSOMEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957: 

Surgery  Branch,  NCI  -  Dr.  R.   Sraith  and  staff. 


NO  ENTRIES  FOR  ITEFS  13,  15  &  16. 


PAGE  1 

PROJECT  REPORT  FOPuM 

1.  N.  C.  I. 2. Endocrinology  Branch 

INSTITUTE  L/iBOPJ.TORY  OR  BRA-NCH 

3.Endocrinolo^  Service     /^. 5.NCI-803(C) 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)  SERIAL  NO. 

6. The  Nature  of  Hormone -Producing  Tumors  of  Pltultar:;-,  Adrenal,  Ovary. 

PROJECT  TITLE 

Testis,  Pancreas,  Parathyroid  and  Chorion;  Abnorma- 
lities of  Somatic  Development  and  Growth 

7. Roy  Hertz,  D.  li,  Bergenstal.  M.  G.  LI.  Sally  B,  Fand  and  li.  M.  Graff 

PRINCIPAL  INVESTIGATORCS ) 

S .  James  A.  Plttman,  Harold  /Jtman.  I'.  W.  Tullner  and  Donald  Spencer 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Ob.jectlves; 

1.  To  learn  more  about  the  basic  metabolic  phenomena  Involved 
in  the  process  whereby  neoplasms  In  hormone-producing  organs  produce 
highly  excessive  sjnounts  of  hormone,  resulting  in  such  syndromes  as 
Gushing 's  Disease,  Acromegaly,  Hyperparathyroidism,  Hype radrenal ism, 
and  slinilar  Gndocrlnopathies. 

2.  To  maintain  in  tissue  culture  hormone-producing  tissues  and  to 
ascertain  the  factors  involved  in  quantitative  hormone  production  in 
vitro, 

3.  To  assess  the  capacity  of  new  drugs  to  alter  the, course  and 
hormonal  activity  of  hormone-producing  tumors. 

Methods  employed;  '    . 

Hormone  producing  tiimors"  from  patients  with  one  of  the  above-named 
syndromes  are  obtained  at  surgery  and  carried  in  tissue  culture.  The 
supernatant  fluid  from  the  cultures  are  tested  for  hormonal  activity 
by  appropriate  bioassay  methods  and  the  quantity  recovered  compared 
with  the  content  of  a  piece  of  tissue  exactly  equivalent  to  that  ori- 
ginally placed  in  culture. 

Patients  presenting  such  sj'ndrom-es  are  completely  characterized 
endocrinologlcally  and  metabolically  before  and  after  therapy  in  order 
to  ascertain  the  relative  effects  of  \'arlous  forras  of  therapy. 


NCI~803(G) 

SERIAL  NO. 


PROJECT  P^EPORT  FORl-I  (Cont'd). 


PAGE  2 


Patient  matorial; 


Admissions! 


Outpatient: 


No, 

Average  Sta 

y  Days 

Adult  males 

6 

11 

Adult  females 

16 

33 

Children  males 

k 

11 

Children  female  s  .. 

k 

9 

Number  of  patients 

":  :■       72 

Number  of  visits 

2S9 

I-h.l'or  findings;  -  ..„..  ,i :,,  • 

1.  Two  additional  patients  with  proven  chorio  carcinoma  have  been 
■shown  to-  lack  the  histidinuria.seen,  in  norr.ial  pregnancy, 

2.  A  patient  v/ith  proven  choriocarcinoma  has  exhibited  a  marked 
suppression  of  urinary  gonadotropin  secretion  while  on  ojaethopterin 
therapy.  This  indicates  a  specific  interference  with  the  nornr.\l 
metabolisn  of  this  hormone-producing  tumor, 

3.  Attempts  to  transplant  huiian  hormone -producing  tvimor  tissue 
to  cortisone-treated  rats  have  failed  in  the  case  of  1  islet  cell 
tum.gr,  2  adrenal  adenorxata,  1  parathyroid' adenoma  and  1  simple 
follicu3.ar  cyst, 

Lr.   An  attempt  to  grow  one  islet  cell  adenoma  in  tissue  culture 
has. failed  and  one  additional  islet  cell  tuxaor  is  surviving  in  tissue 
ciilture  2  weeks  after  oxplantation.  Its  further  course  and  possible 
in  vitro  production  of  insulin  remain  to  bo  evaluated, 

5,  Two  adrenal  carcinomr.  patients  have  been  studied  directly  by 
us  and  two  in  collaboration  with  outside-  investigators  at  other  hos- 
pitals. All  4-  have  shown  a  decisive  drop  in  urinary  corticoid  ex- 
cretion under  Amphenone  administration.  This  demonstrates  that 
Am.phenone  will  markedly  depress  the  activity  of  this  hormone-producing 
tumor.  The  duration  of  those  studies  was  too  limited  by  clinical 
circuristances  to  permit  any  evaluation  of  the  effect  of  Am.phenone 

on  the  course  of  the  m.alignancy, 

6,  In  one  patient  with  advanced  metastatic  adonocarcinomxi  of 
undetermined  origin  the  administration  of  Germanin  (Bayer  205)  has 
led  to  an  extensive  necrosis  of  the  adrenal  cortex  similar  to  that 
observed  experimentally  and  in  prior  clinical  studies  in  patients 
with  pemphigus.  This  patient  also  showed  clinical  and  biochom-ical 
evidence  of  adrena.l  insufficiency 

7,  lihdocrinological  analysis  has  porrrdtted  the  differentiation 

of  constitutional  sexual  precocity  from  that  due  to  horm.one -producing 
hyperplasia  or  tumor  in  5  additional  children. 


PAGE  3 
-       PROJECT  PilPORT  FORiM   (Cont'd.) 
NCI~803(C) 
SSRL'J.  NO. 

Ma..ior  findings  (cont'd.) 

8,  Cytological  nethods  for  the  chronosonr.1  detorr-iinr.tion  of  the 
truG  sonatic  sex  of  patients  with  adrenal  hyperplasia  or  abnomo.l 
gonadal  development  has  facilitated  the  endocrinological  evaluation 
of  10  problen  cases, 

9.  Further  observn.tions  in  acronegaly  have  been  seriously  limited 
by  paucity  of  case  iiaterial.  Nevertheless,  in  2  instances  appropriate 
specimens  for  growth  hormone  studies  have  been  obtained, 

:  10,  Conplete  endocrinological  stiid;'-  of  a  patient  with  Lawronce- 
Moon-Biedl  syndrone  and  of  another  in.th.   Sjorgen's  syndrone  revealed 
no  distinct  endocrinopathic  pattern  in  those  cases, 

11,  A  new  progestational  conpound,  19-nor  ethisterone  previously 
found  by  us  to  be  5  tines  as  active  as  oral  progesterone  in  rabbits 
and  nonkeys  has  been  assayed  in  5  patients  for  oi^l  progestational 
potency,  Endonetrial  biopsy  showed  that  this  conpound  has  the  sane 
enhanced  potency  in  the  hur:ian  as  was  shovm.  oxperirientally.  It  is 
now  available  in  anounts  sufficient  to  pemit  its  trial  in  the  therapy 
of  breast  cancer,  , 

Significance  to  cancer  research; 

Further  understanding  of  the  conditions  pernitting.  the  excessive 
horraonal  production  of  tur.iors  of  endocrine  origin  is  basic  to  otir 
understanding  of  the  netabolisr'.  of  neoplastic  tissue  generally.  In 
addition,  further  analysis  of  abnoma.lities  in'sona.tic  growth  and 
developnent  should  provide  data  pertinent  to  the  problens  of  tissue 
groTATth  generally, 

■  .  Proposed  course  of  pro.ject; 

Further  elaboration  of  these  studies  with  najor  enphasis  on  bio- 
chenical  and  phamacological  control  of  ho  mono -producing  tunors  and 
anonalous  tissue  growth  is  projected. 


11. 


BUDGET  ACTIVITY: 

RESEARCH  /^      ADMNISTR/iTION  /^ 

REVIEl'J  &  APPROVAL  £J       TECHNICAL  ASSISTANCE  £J 

12. 

COOPER/iTING  mJLTS  OF  THE  PUBLIC  HEi'iLTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUl^JDS, FACILITIES, OR  PERSONFEL  FOR  TlilS  PROJECT  IN  EITHER  1956 
or  1957: 

Dr,  Earlo's  Tissue  Culture  Unit  collaborates  in  this  , 
pro gran. 


PAGE  4. 
PROJECT  REPORT  FOPJl  (Cont'd. ) 
HCI-803(C) 

SERI/i  NO. 

NO  ENTRY  FOR  ITEI4  13, 

15. . . 

PUBLICATIONS  OTHER  TFi.N  AB3TR/.CTS  FROM  THIS  PROJECT  DURING  CALENDAR 

YEilR  1955: 

1.  Green,  Stanley,  Evans,  J.  M.  and  H^rtz,  R.,  Acyclic  nenstrual 
■  bleeding  associated  vdth  erythrocytosis,  J,  Clin, . Endocrinol.  E: 

Metab.  15*.  199,  1955. 

2.  Hertz,  R.,  Tullner,  U.  H.,  Schricker,  J.  A,,  Dhyse,  F,  G. ,  and 
Hallxian,  L,  F.,  Studies  on  Anphenone  and  related  conpounds.  Recent 
Progress  in  Homone  Research,  Vol.  11,  pp. 119-14-7,  1955. 

3.  Hertz,  R,,  l-'aite,  J.  H. ,  and  Thonas,  L.,  The  progestational 
effectiveness  of  19-nor-ethisterone  by  oral  route  in  wonen,  J, 
Clin.  Endocrinol.  &  Metab.  (In  press), 

U*   Thorn,  G.  V. , ,   Renold,  A.  E.,  Goldfien,  A,,  Nelson,  D.  H. ,  Reddy, 
¥.  J,,  and  Hertz,  R,,  Inhibition,  of  corticosteroid  secretion  by 
Anphenone  in  a  patient  with  adrenal  cortical  carcinona,  New  Eng. 
J,  Med,  (In  press), 

5.  Hertz,  R.,  Pittnian,  J.  A.,  and  Graff ,  M*  M< ,  Anphenone:  Toxicity 
and  effects  on  adrenal. and  thyroid  function  in  nan,  J,  Clin.  Endo- 
crinol. &  Metab,  (In  press). 


^6. 


■HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT  DURING  CALEIID/Jl 

YE/'iR  1955: 

Translation  of  paper  entitled  "Studies  on  Anphenone  and 
Related  Conpounds"  into  Spanish  and  published  in  "Revista  Argentina 
de  Endocrinologia  y  Metabolisno,  Btienos- Aires,.  Argentina,  1955, 


PAGE  1 
PROJECT  P.EPOP.T  FORM 

1,  National  Cancer  Institute    2,  Endocrinology  Branch 


INSTITUTE  LABORATORY  OR  BRANCH 

3,  Endocrinology  Service k» _«________________,  5.  NCI-804.(C) 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA    SERIAL  NO. 

6,  Biotin  and  the  Avidin-Biotin  Complex  in  Relation  to  Normal  and  Neoplastic 

PROJECT  TITLE 

Tissue  Growth 

7.  Roy  Hertz.  Frederick  G.  Dhyse  and  William  U«  Tullner 

PRINCIPAL  INVESTIGATOR(S )     '.  '  '.         '■ 

8,  Dr.  Lloyd  Law . 

OTHER  BIVESTIGATORS 

9.  PROJECT  DESCRIPTION  A)  Objectives;  1,  To  ascertain  the  role  of  biotin,  a 
dietary  trace  factor,  in  the  growth  of  normal  tissue'  and  in  the  development  of 
neoplasms  in  animals  and  man, 

2.  To  learn  the  normal  metabolism,  of  biotin  in  man  and  to  produce  biotin  de- 
ficiency in  cancer  patients  administering  lo^^r  biotin  diets  plus  concentrates  of 
avidin  prepared  from  egg-white, 

3.  To  develop  methods  for  the  cheap  and  practical  preparation  ox  large  amoTints 
of  avidin  for  therapeutic  trial  in  selected  cancer  cases.     i 

B)  Methods;  1.  Standard  methods  of  protein  fractionation  of  egg  white  are 
employed  to  prepare  potent  avidin  concentrates  which  are  assayed  for  growth 
suppressing  effect  on  a  biotin  requiring  yeast  organism, 

2,  Biotin  deficiency  is  produced  in  mice  by  feeding  egg  white  and  avidin 
concentrates  and  the  effect  of  this  deficiency  on  such  standardized  tumors  as 
transplantable  leukemia  is  determined  in  collaboration  v/ith  Dr.  Lloyd  Law. 

3.  Patients  are  carried  on  diets  of  knovm  and  constant  biotin  content  and  the 
urinarj"-  biotin  excretion  is  followed  microbiologically.  The  effects  of  egg  white 
feeding  and  avidin  feeding  are  observed  upon  the  patient's  clinical  nutritional 
status  and  upon  the  urinary  excretion  of  biotin, 

C)  Ma.jor  Findings;  1.  An  excellent,  economical  method  of  preparing  potent 
concentrates  of  avidin  has  been  developed  and  a  stock  pile  of  this  material  for 
clinical  and  experimental  use  has  been  prepared, 

A  large  industrial  laboratory  with  ready  access  to  abundant  supplies  of  fresh 
egg-white  has  adapted  this  method  to  mass  scale  and  is  now  providing  our  first 
adequate  supplies  of  avidin  for  extended  clinical  trial. 


PAGE  1 
PROJECT  PaiPORT  FORM 
1,     N.C.I. .     2,  Endocrinology  Branch 


INSTITUTE  LABOR/^/rORY  OR  BR/.NCH 


3,  , 4.  - 5.  NCI-805C 

SECTIOLI  OR  SERVICE  LOCATION  (IF  OTIiSR  THAN  BSTHESDA       SERLfi  NO. 


6.  Effect  of  Steroid  Therapy  on  Metabolism  of  B~Complex  Factors  in  Cancer 
PROJECT  TITLE 

Patients 


7.  Roy  Hertz  and  Frederick  G.  Dhyso 


PRINCIPAL  INVESTIGAIOR(S) 
8,  Phillip  Rayford 


OTHER  IMESTIG/^.TORS 


9.  PROJECT  DESCRIPTION  -  ■ 

Objectives;  To  determine  the  effect  of  protein  anabolic  action  of 

steroid  therapy  in  cancer  patients  upon  the  metabolism 
of  B-oonplox  factors  oonaonod  in  tho  diet. 

To  correlate  the  foregoing  with  specific  therapeutic 
effects  obtained. 

Methods  employed;  Patients  are  maintained  on  a  fixed  diet  and  their 
dietary  intake  determined  by  a  balance  procedure. 
Continuous  urine  collection  provides  specimens  for  determining  urinary 
excretion  of  nitrogen,  biotin,  riboflavin,  niacin,  folocin,  and  panto- 
thenate (See  project  SOOC). 

Patient  material; 

No,      Average  Stay  Days 

Admissions:  Adult  fern-ales  2  90 

Outpatients:      None 

Proposed  course  of  project;  It  is  proposed  to  develop  and  extend  those 

studies  as  a  new  project  largely  by  conti- 
nuing studies  already  in  progress.  Observa,tions  would  be  made  in  suit- 
able patients  with  andro gen-induced  protein-anabolism  and  in  cortisone- 
induced  protein-catabolic  states.  In  this  manner  the  relationship  of 
B-complex  balance  to  nitrogen  balance  may  be  determined  vjith  a  view  to 
clarifying  the  mechanism  of  action  of  those  steroids  on  protein  meta- 
bolism generally. 


PAGE  2 
NCI-805C  PROJECT  REPORT  FORII  (Cont'd) 

SERTixL  NO. 

Proposed  course  of  project:  (cont'd.) 

The  paired  tray  technique  has  been  replaced  by  full 
metabolic  study.  This  more  exact  technique  will  provide  necessary 
control  data  regarding  the  differences  between  androgen- induced 
nitrogen  retention  and  simple  retention  folloi.dng  increased  intake. 


11. 


12. 


BUDGET  ACTIVITY:  , 

RESEARCH  fH      ADMINISTPiiTION  /     7 

REVIEW  &  APPROVAL       /"~7       T  JCHIIIC/iL  ASSISTALICE  /~7 


COOPER/'.TING  UfllTS  OF  THE  PUBLIC  HE/lLTII  SERVICE,   OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONKSIL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957: 

Dr.  Donald  Uatkin,  General  Medicine  Branch,  is  collaborat- 
ing in  completing  the   studies  on  patients  on  the  complete  balance 
regime. 


NO  ENTRIES  FOR  ITEMS  13,..15-&  16. 


PAGE  1 
PROJECT  REPORT  FORM 


1.  N.  C.  I.  2.   Endocrinology  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3.  Endocrinology  Service     4-.  5.  NGI~806(C) 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER.  TMN  BETHESDA)  SERIAL  NO. 

6.  Endocrine  Characterization  of  Females  with  Cancer  of  the  Larynx 

PR.OJECT  TITLE 


7«  Roy  Hertz.  Morris  M,  Graff  and  Lois  F.  Hallman 

PRINCIPAL  I?]VESTIGATOR(S) 

3.     James  Pittraan  and  Harold  Altinan  

OTIiER  BTVESTIQATORS 

9.  PROJECT  DESCRIPTION 

Objectives;  1.  To  determine  the  endocrinological  characteristics  of  the 
relatively  rare  female  patient  with  cancer  of  the  larynx  in 
order  to  assess  the  role  of  endocrine  factors  in  the  pathogenesis  of  this 
disease. 

Methods  employed:  V/omen  with  proven  cancer  of  the  larynx  will  be  hospi- 
talized and  a  complete  endocrine  evaluation  made  by: 
l)  recording  a  complete  pubertal,  menstrual,  and  reproductive  history 
and  any  other  pertinent  endocrine  facts  regarding  the  patient  and  her 
famUyi  2)  complete  physical  appraisal  of  endocrine  make-up  with  emphasis 
on  obesity,  masculinity,  size  of  clitoris,  hirsutiarn,  voice  pitch,  acne, 
baldness,  vaginal  smear  and  other  endocrine  factors;  3)  determination  of 
urinary  cortlcoids,  estrogens,  ketosteroids,  and  gonadotropins;  also 
glucose  tolerance,  B.M.R.,  protein  bound  iodine,  eosinophlle  response 
test  and  other  measures  of  endocrine  functions. 

These  data  would  be  collected  in  order  to  determine  v/hether  there 
is  any  evidence  of  gonadal  or  adrenal  androgenlzation  of  the  female  who 
gets  cancer  of  the  larym:  as  compared  with  normal  females  of  the  same 
age  group. 

Ifa.ior  findings:  Preliminary  superficial  clinical  observation  of  2 

females  with  cancer  of  larynx  has  revealed  no  striking 
difference. 

Significance  to  Cancer  research:  Literature  review  indicates  that  the 

sex  ratio  for  cancer  of  the  larynx 
is  variously  estimated  as  between  16  to  1  and  200  to  1.  In  any  case 
it  is  clear  that  females  are  relatively  rare  among  patients  vrith  laryn- 
geal cancer. 


PAGE  2 
PROJECT  REPORT  FORl^i  (Cont'd.) 


NCI-306(C) 
SERIAL  NO. 


Sipinificance  to  cancer  research  (cont'd.) 

The  larynx  is  a  secondary  sex  organ  in  that  its  pubertal 
differentiation  is  homonally  dependent.  Moreover,  androf;en  ad- 
Snistration  readily  alters' the  larynx  in  the  ^man  female  but  no 
case  of  cancer  of  the  larynx  in  the  htrnian  female  has  yet  been  re- 
ported following  androgen  administration. 

ProposedcourseoL.E£OJect:  This  project  is  deferred  pending 
rroposea  cou  ,, ti_-.J additional  clinical  facilities  for 

the  admission  of  appropriate  patients. 


11. 


BUDGET  ACTI\n:TY; 

RESEARCH  •  O    ADMINISTR/iTION.        ■       O 

REVIEW  h  APPROVAL  O     TECmilGAL  ASSISTANCE  O 


NO  ENTP.ISS  FOR  ITS!1S  12,  13,  15  &  16. 


PAGE  1 
PROJECT  REPORT  FORM 
1,  N,G,I,  2,  Endocrinolo^  Branch 


INSTITUTE  LABOPivTORY  OR  BRANCH  . 

3,  Ilhdocrinology  Service    K,  5.NCI-.807(C) 

SECTION  OR  SERVICE         LXATION  (IF  OTHER  Tli/^.N  BETHESDA)  SERI/.L  NO. 

6,  Methods  of  Steroid  Analysis  in  Urine  and  Blood  from  Cancer  Patlsnts 

PROJECT  TITLE 

7.  Morris  M.  Graff 

PRINCIPAL  INVESTIGATOR(S) 

8,  Silas  Jackson,  Foster  Burnett  and  Willie  Logan 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION  - 

Objectives:  To  develop  and  iroprove  the  chonical  nethodology  for  deter- 
mination of  urinary  corticoids  and  ketosteroids  and  blood 
corticoids,  estrogens,  and  androgens. 

Methods  employed;  Classical  methods  of  reagent  and  enzymatic  hydrolysis, 
chromatographic  adsorption  and  elution,  paper  chroma- 
tography, and  spectrophotometric  determination  of  specifically  developed 
chrom.ogens.  Chemical  methods  to  be  correlated  with  equivalent  bioassays 
available  under  Project  SOo, 

Major  findings;  Methods  for  urinary  ketosteroids  and  corticoids  have 

been  developed  and  perfected  and  are  now  adaptable  for 
clinical  studies. 

Blood  corticoid  methods  have  bejn  investigated  and  an 
improved  method  devised  which  is  now  available  for  clinical  research 
studies  involving  assossm^ent  of  adrenal  corticoid  fvmction. 

Progress  in  the  developm.ent  of  a  clinically  useful 
method  for  estrogen  determination  in  body  fluids  has  been  very  liraited. 
However,  certaiji  technical  difficulties  have  been  overcome. 

Significance  to  cancer  research;  These  methods  are  basic  to  the  endo- 
crinological study  of  patients  with 
cancer  of  the  breast,  prostate,  cervix,  and  larynx. 

Proposed  course  of  project:  The  further  extension  of  methodological 

investigation  is  proposed  especial3y  for 
blood  estrogen  and  iirinary  estrogen  by  fluorom.etrie  methods.  Extended 
investigation  of  the  use  of  the  Anthrone  reagent  In  steroid  analysis 
is  planned. 

Methods  for  the  fractionation  of  alpha 
and  beta  ketosteroids  and  for  pregnandiol  detemlnrutlon  are  to  be  de- 
veloped in  relation  to  further  endocrine  characterization  of  patients 


PAGE  2 
PROJECT ■ REPORT  FORM  ( Cont ' d ) . 
NCI--807(C) 

SERI/iL  NO. 

Proposed  course  of  project  (cont'd.) 
with  timors  of  adrenal,  testis,  or  ovary. 

11,5 . , 

BUDGET  ACTIVITY: 

P.ESSARCH  /y  ADMNISTRATION  /7 

.    REVIEl«J  &  APP?.OV/iL     jT/  TECHNICAL  ASSISTANCE  ^ 


NO  ENTRIES  FOR  ITEl-K  12,   13,  14-,  &  16, 


■       .  PAGE  1 

.-  _  ,  ■  ■.PROJECT.  REPORT  FORM 

1.  N.  C.  I. 2.   Endocrinology/  Branch ■- 


mSTITUTE  LABORATORY  OR  BRANCH 

3.  Endocrinology  Service ^     U.  5.NCI-??0SC 

SECTION  OR  SERVICE         ;•   LOCATION  (IF  OTHER,  THAN  BETHESDA)  SERIAL  NO, 

6,  Endocrine  Factors  Governing  Hormone-Induced  Tissue  Growth  and  Ttrnior 

PROJECT  TITLE 

Formation  in  Animals;  Bioassay  of  Blood  and  Urine 
from  Cancer  Patients 

7.  Roy  Hertz,  Will3-an  W.  Tullner.  Donald  Spencer,  D.  M.  Bergenstal 

PRINCIPAL  INVESTIGATOR(S) 

B.   Sally  B.  Fand  and  Morris  M.  Graff 

•  OTHER  im/ESTIGATORS 

.9.  PROJECT  DESCRIPTION 

Objectives; 

^1.  To  gain  basic  information  concerning  endocrinological  and  nu- 
tritional factors  governing  homonally  conditioned  growth  and  involu- 
tion in  organs  such  as  the  uterus,  breast  and  prostate  which  are  the 
frequent  seat  of  hormone-sensitive  neoplasms. 

2,  To  develop  and  improve  bioassay  methods  for  the  quantitation 
of  hormones  in  the  blood  and  urine  of  patients  with  cancer  who  are 

.  either  receiving  various  forms  of  hormonal  therapy  or  who  present  un- 
usual hormonal  patterns, 

3.  To  determine  the  biological  properties  of  steroid  and  related 
compounds  in  terras  of  their  potential  usefulness  in  cancer  therapy. 

Methods  employed; 

Standard  methods  of  biological  study  are  employed.  These  include; 
'  l)  the  maintenance  of  animals  of  various  species  on  specified  hormonal 
or  nutritional  regimens j  2)  daily  observations  of  physical  and  beha- 
vioral changes  in  such  animals;  3)  complete  autopsy  of  test  animals 
\i±th   detailed  weighing  and  chemical  analysis  and  histochemical  study 
of  specific  organs;  J+)   surgical  alteration  of  the  endocrine  status 
of  animals  by  gonadectomy,  adrenalectomy,  hypophysectomy,  thyroidectomy, 
parabiosis,  hysterectomy  and  other  operative  procedures. 

I-fejor  findings; 

These  are  detailed  in  the  publications  listed  under  Item  15.  They 
may  be  briefly  enumerated  as  follows; 


PAGE  2 


PROJECT  REPORT  FOR^l  (Cont'd.) 

NCI~808(C) 

SERIAL  NO,  ■,,,..:.::...-■'   .  ...  -,  ,...  .  ■:.... 


Majior  flnriinprs  (cont'd.l 

1  .  The  -biolosical  properties  of  Amphenone-  in  relation ^to  adrenal 

S?.ersSpr.rsSS  trrls%rt.flXl  :L.^  to  e.c^nou.  corticoia 
is  readily  demonstrated. 

A   .series  of  analogues  of  Amphenone  have  been  synthesized  and 
studied       ?hesf  i^lX  alpha,  beta,  dmethyl-4  V  stilbenediarixne, 
?hf  dS^th^fa^ino  ^erivati^e  ^f  Mphenone,^he  o.ine  ^er-t.ve     f^ 

s:-'.iSoS:ar^-tXrs^^^^^  ~- 

If,, Axes   are  bein^^  made  with  respect  to  relative  toxicity  ana  c^nxrax 
nervous  sjstem  dlp^ssant  effect.  Twenty-five  related  compounds  have 
Serfou^d  to  be  inert,  indicating  a  relatively  l^g^f^^   °^  ^P^ 
cificity  for  these  interesting  endocrinological  eficcts. 

Extended  work  with  these  and  related  substances,  j;j2/'sScinc 
Gemanin,  is  projected  with  a  view  to  deriving  a  non-toxic,  specific 
Inhibitor  of  adrenal-cortical  function. 

Chromatographic  analysis  of  the  steroid  content  of  ^^e  a^enal 
vein  blood  of  the  Amphenone-treated  anir.ials  is  also  to  be  carried  out. 

2  Fui'ther  studies  on  potent  anti-gonadotrophic  sera  have  l^d  to 
some  de  Jee  of  purification  of  the  active  inlaibitory  material   These 
ef?orts^re  to  be  extended  by  means  of  specific  immunochemical  ad- 
sorption and  elution  techniques  now  being  developed. 

3.  Additional  steroids  have  been  screened  for  P°^?j:^^\J!;^^°°^" 
tion  of  pituitarj^  depressant  action  from  other  biological  effects. 
The  studies  on  ?he  biological  properties  of  allopregnane,  2l-ol-3, 
S  dione  and  Lichstein's  compound  S  have  been  completed  and  reported. 
Ssays  of  ?he  new  corticoids,  metacortandrosin  and  metacortandrolone 
have  indicated  no  material  dissocio.tion  of  pituitary  depressant 
action  and  enhanced  corticoid  action. 

U.   Extension  of  studies  on  the  local  catabolic  function  involved 
in  the  involution  of  hormone  sensitive  organs. fpllovanggonadectomy 
SbstanSIte  the  complete  quantit-ative  and  chronologicar  independence 
S  this  process  from  general  catabolic  effects  on  the  body  as  a  whole 

5.  Substantial  progress  has  been  made  on  a  sensitive  and  highly 
specific  assay  for  luteinizing  hormone,  A  normally  cycling  lemaie 


PAGE  3 
:PROJECT  REPORT  FORJ'I  (Cont'd.) 


NCI-808(C) 

SERIAL  MO. 


Ma.ior  findings  .(cont'd. ) 

rat  is  followed  by  vaginal  smears  until  she  is  found  to  be  in  the 
pre-ovulatory  or  pre-estrus  phase  which  is  characterized  ty  the 
appears-nce  of  very  large  numbers  of  large  rotuided,  nucleated  epi- 
thelial cells  in  the  vaginal  smears.  Our  previous  studies  had  shovm 
that  hypophysectoray  at  this  point  will  abort  the  succeeding  ovula- 
tion. Since  this  ovulation  depends  entirely  on  luteinizing  hormone, 
a  sensitive  and  specific  assay  is- based  on  the  restoration  of  ovula- 
tion by  injection  of  such  hormone  preparations.  It  is  hoped  that 
this  will  permit  us  to  assay  for  this  hormone  in  the  blood  and  urine 
of  cancer  patients  under  steroid  regimens, 

6.  A  strain  of  guinea  pigs  has  been  identified  which  has  a  large 
number  of  hypogonadal  males.  These  animals  have  normal  mating  beha- 
vior but  have  complete  aspermia  and  extremely  atrcphic  testes,  A 
complete  endocrine,  behavioral,  and  anatomical  stu.dy  of  these  animall.s 
is  being  undertaken  in  collaboration  with  Dr.  V,   C.  Yovmg,  Prof,  of 
Anatomy  at  the  University  of  Kansas. 

7.  The  hormonal  factors  involved  in  the  post-natal  differentiation 
of  the  ovary  in  the  rat  and  rabbit  have  been  investigated,   A  post- 
natal period  of  10  days  in  the  rat  and  of  10  weeks  in  the  rabbit 

has  been  characterized  as  a  period  during  which  the  ovary  is  completely 
insensitive  to  pituitary  gonadotropin  stiiaulation.  During  this  period 
the  ovary  undergoes  the  first  delineation  cf  normal  follicular 
structure,  the  final  step  being  that  of  antmmi  formation.  It  is 
only  such  antrum-containing  follicles  that  respond  to  gonadotropin 
administration.  Moreover,  the  ovary  of  a  new-born  rat  grafted  to 
its  mother's  ki'dney  vn.ll  undergo  normal,  post-natal  differentiation 
even  when  the  mother  had  been  completely  hjrpophysectortized.  Further 
studies  on  the  possible  role  of  the  hypothalamus  in  this  process 
are  under  vjay, 

8.  The  overlap  between  progestational  and  corticoid  action  of 
certain  steroids  is  well  exemplified  by  the  fact  that  desoxycorti- 
costerone  is  about  10^^  as  active  as  progesterone  for  progestational 
effect.  1'fe  nox-;  find  that  whereas,  hydrocortisone  lacks  progesta- 
tional activity,  the  introduction  of  the  fluorene  atom  in  the  9 
position  imparts  not  only  increased  corticoid  potency  but  also  renders 
the  compound  progestationally  active  at  about  5/'  the  activity  of 
progesterone  itself  in  both  the  monkey  and  rabbit.  Similarly  the 
fluorination  of  11  beta  hydroxyprogesterone  imports  progestational 
activity  to  this  compound  .at  a  level  equ?.l  to  10  percent  the  activity 
of  progesterone.  These  observr.tions  indicate  that  fluorination  has 
implications  not  only  for  corticoid  action  but  also  for  gonadal 
steroid  properties.  Moreover,  the  availability  of  com.pounds  having 
combined  corticoid  and  progestational  action  provides,  an  £ipporttmity 


NCI-808(C) 

SERIAL  NO. 


PAGE  U 
PROJECT  REPORT  FORM  (Cont'd.) 


Vh.^ov   findings  (cont'd. ) 

for  study  of  a  compound  which  vdll  proliferate  the  uterine  epithelial 
"structures  at  the  same  time  that ' stromal  resistance  is  depressed. 
Long-term  studies  are  in  progress  to  determine  what  the  final  re- 
sult of  such  tissue  changes  may  lead  to  in  the  way  of  architectural 
derangement  possibly  leading  ultimately  to  m.alignancy, 

9.  Ve  have  previously  reported  that  a  five-fold  enhancement  of 
progestational  activity  in  the  rabbit  is  effected  by  demethylation 
of  progesterone  at  the  19  position,  yielding  19-nor-progesterone, 
Similarly,  19  nor-ethisterone  was  shown  to  have  the  same  increased 
progestational  action  in  the  rabbit  when  orally  administered.  These 
studies  have  now  be on  extended  to  the  monkey,  employing  the  inhibi- 
tion of  estrogen-withdrawal  bleeding  and  the  production  of  a  secretory 
endometrium.  Both  compounds  show  comparably  enhanced  effectiveness 

in  the  monkey.  These  studies  have  led  to  siir.ilar  observations  in 
the  human  (see  project  803,  item  9  Cll),  Thus  the  quantitative 
ratios  are  similar  in  rabbit,  monkey,  and  human. 

10.  Recent  studies  on  the  effect  of  hyp":ph3r<5eotomy  on  breast 
cancer  have  implicated  the  pituitary  grov/th  ho:tT,iona  as  a  direct 
trophic  influence  on  breast  cancer.  It  becomes  iciperative  to  be 
iable  to  measure  the  level  of  growth  hormone  in  human  blood  or  urine. 
The  rat's  tibial  cartilage  and  costal  .cartilage  have  been  shox-m  to 
respond  to  administered  grov/th  hormone  by  increased  uptake  of  radio- 
active su].fur.  Preliminary  studies  suggest  that  this  may  provide 

a  sufficiently  quantitative  and  sensitive  index  of  the  action  of 
grovrbh  hormone  to  permit  its  assay  at  much  smaller  levels  than 
current  histological  methods  permit. 

11.  Detailed  histochemical  analysis  of  the  reaction  of  hormone 
■"'sensitive  tissues  and  tumors  to  altered  hormonal  states  have  been 

initiated.  Examples  are:  (a)  the  relationship  of  initial  fat 
accumulation  to  the  post-natal  differentiation  of  the  ovary;  (b) 
the  relationship  of  carbonic-anhydase  to  the  progestational  response 
of  the  rabbit  and  monkey  uterus j  (c)  the  histochemical  characteriza- 
tion of  the  lipoidal  substance  present  in  the  adrenal  of  the  Amphenone- 
■  treated  rat,  and  (d)  the  relationship  between  the  functional  state 
of  the  human  pituitary  and  specific  enzyma.tic  content  as  demonstrated 
histochemically. 

12.  The -role  of  dietary  trace  factors  in  hormonal  response  has 
been  stvidied  in  particular  relationship  to  the  activation  of  the 
adrenal  gland  of  the  hypophysectom.ized  dog  by  exogenous  ACTH,  It 
has  been  dem.onstrated  that  the  pantothenate,  riboflavin,  biotln, 
folacin,  pyrldoxine  and  ascorbic  acid  content  of  the  adrenal  gland 
remains  unchanged  vrhen  the  gland  is  at  complete  rest  as  manifested 
by  low  cortlcoid  content  of  the  adrenal  vein  effluent  or  after 


PAGE  5 
PROJECT  REPORT  FORM  (Cont'd.) 


WCI-80B(C) 
SERIAL  NO. 


Ma.jor  findings  (cont'd.) 

maximtim  activation  by  exogenous  ACTH  leading  to  enonr.ous  elevn.tion 
•  •  *  in  corticoid-  content  of  the.adjrenal  vgin  blood.  Moreover,  the 
adrenal  vein  blood  in  either  state . shows  no  change  in  -its  content 
of  these  trace  factors  despite  the  very  marked  rise  in  corticoid 
content.  Thus  it  appears  that  these  trace  factors  are  not  quanti- 
tatively critical  for  this  type  of  iimnediate  hormonr-l  response  to 
ACTH. 

Proposed  course  of  pro.ject! 

It  is  expected  to  continue  and  extend  the  foregoing  studies 
along  the  lines  already  indicated. 

11. . 

BUDGET  ACTIVITY: 

RESEARCH  /~/      ADiINISTR<1TI0N  £J 

REVIS'J  &  APPROVAL  /^       TECHI'IICAL  ASSISTANCE  jTJ 

NO  ENTRIES  FOR  ITEI^E  12  &  13 

15. 

PUBLICATIONS  OTHER  THAN  ABSTR/iCTS  FROM  THIS  PROJECT  DURING  CALEITDAR 
YE/iR  1955: 

1.  The  effect  of  17-alpha-hydroxy-ll-desoxycorticosterone  on  estrogen- 
stimulated  chick  oviduct  grovrth,  by  W.  V. .   Tullner  and  R,  Hertz. 

(Endocrinology) (In  press). 

2.  Progestatf-onal  activity  of  the  halogenated  corticosteroids  and 
related  compounds  in  the  rabbit  and  monkey,  by  R,  Hertz,  and 
W.  W.  Tullner  (Proc.  Soc.  Exper,  Biol.  &.   Med.)  (In  press). 

3.  Amphenone  inhibition  of  adrenal  corticosteroid  output  in  the  hypo- 
physectomized  dog,  by  U.  W.  Tul^lnor,  M.  M.  Graff  and  R.  Hertz 
(Endocrinology). (In  press). 

4-.  High  progestational  activity  of  nor-ethisterone  and  norprogesterone 
in  the  m.onkey,  by  R.  Hertz  and  W.  W.  Tullner.  (Endocrinology). 
(In  press). 


NCI-808(C) 
SSRI/iL  NO. 


PAGE  6 
PROJECT  REPORT  FORl'I  (Cont'd.  ) 


15.  Publications  (Cont'd.) 

5,  Vitfunin  content  of  dog  adrenals  and  adrenal  vein  blood  before 
and  after  ,ACTH,  by  R.  Hertz,  W.  W.  T-ullner.  M.  Graff  and  J.  A. 
Schricker.  (Proc,  Soc,  Exper.  Biol,  8:   Med.;  (In  press), 

6.  Studies  on  Amphenone  and  related  conpoiinds,  by  R.  Hertz,  11.   W. 
Tullner,  J.  A.  Schrieker,  F.  G.  Dhyse,  and  L.  F,  Hallnan. 
(Recent  Progress  in  Hornone  Research,  Vol.  11,  pp.  119-14-7, 
1955). 

16. ^ \ \^ 

'   HONORS  AND  AWARDS  TO  PER.'JOBIiilL  RELATING  TO  THIS  PROJECT  DURING  CALENDAR 
YEAR  1955: 

,  Mr,  William  W,  Tullner  uas  admitted  to  the  Graduate  Council  of 
George  Washington  University  as  a  candidate  for  the  degree  of 
Doctor  of  Philosophy  in  Physiology  to  be  conferred  in  June  1956, 


PAGE  1 
PROJECT  REPORT  FORM 
1.  N.  C,   1, 2.     Endocrlnolo^  Branch 


II'ISTITUTE  LABORATORY  OR  BRANCH 

3.  Endocrinology  Service      U. 5>NCI-809(c; 

SECTION  OR  SERVICE  LOCATION  (IF  OTHi^.  THAN  BETHESDA)  SERIAL  NO. 

6.  The  Preparation  of  Crude  Natxiral  Extracts  of  Hormonal  Activity  from  Ovary, 
PROJECT  TITLE 

Testis,  Adrenal  and  Pituitary  and  their  Biological  and 
Clinical  Characterization. 


7.  To  be  recruited. 


PRINCIPAL  INVESTIGATOR(S ) 


OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives!  To  prepare  for  biological  and  clinical  assay  crude 

preparations  of  hormonally  active  tissues  in  order  to 
determine  their  effect  upon  tumors  in  hormone -sensitive  organs.  We 
know  the  crystalline  steroids  now  so  widely  used  represent  only 
partial  elements  of  the  over-all  activity  of  the  various  endocrine 
organs  and  that  even  optimum  mixtures  of  them  do  not  reproduce  the 
characteristic  effect  of  the  total  internal  secretion  of  the  endocrine 
gland  itself.   Such  effects  maj   be  more  profound  and  more  desirable 
than  those  obtained  by  the  separate  synthetic  cr;'"sta.lline  factors. 

Methods  employed:  1.  Standard  chemical  methods  of  total  lipid  and 

protein  extraction  of  freshly  prepared  tissues  to 
be  collected  at  the  slaughter  house  and  processed  immediately, 

2,  The  application  of  bioassay  techniques  to  such  preparations  to 
determine  their  endocrine  effectiveness  and  their  toxicity. 

3,  Clinical  trial  of  non-toxic,  active  materials  in  suitable 
patients  with  endocrine  deficiency  or  cancer  of  breast,  prostate  or 
cervix, 

NO  ENTRIES  FOR  ITEI^IS  11,  12,  13,  15  &  16. 

N.B,  This  project  has  not  been  activated  due  to  failure  to 
recruit  properly  qualified  professional  personnel. 


PAGE  1 

PROJECT  PEPORT  FORM 


1.  N.   C.  I, «__>_  2.     Endocrinology  Branch 


INSTITUTE  LABOPJITORY  OR  BRANCH 

3,  EndocrJbiolQgv  Service  L.  _  ,     ,  ,     5,NCI-8lO(C) 

SECTION  OR  S^VICE  LOCATION  (IF  OTHER  TIIA.N  BETffiISM}     SERIAL  NO. 


6.         Studies  in  Thyrnid  Function 
■   PROJECT  TITLE 


7  KG     Sher^'f 
'principal  xlIVESTIGATOR(S) 

8  H-a-r^-Ld  Altman.  Betty  N^  Siefring  and  RiuL  liarks 


■q^FTER  INVS.STIQATORS 

,,  PROJECT  DESCRIPTION  -  A)  The  hepatic  clearance  of  plasma  protein  bound 

iodine  in  ms.n. 

Objective:  To  determine,  in  Ernn,  the  role  of  the  liver  in  the  excretion, 

conjugation,  and  utilization  of  thyroid  hormone.  Results 
obtained  are  to  be  compared  with  experimental  information  available 
from  similar  work  done  in  animals  (rat,  mouse,  cat,  dog). 

Methods  employed;  Hepatic  vein  catheterization  in  man;  serum  or  plasma 

protein  bound  iodine  determinations,  hepatic  blood 
flow  measurements  employing  the  brom-sulfaphthalein  technique, 

Mia .1  or  findings;  The  major  findings  of  this  project  during  the  past 

year  have  been  the  demonstration  of  a  significantly 
lower  plasma  protein  bound  iodine  concentration  in  blood  from  the 
hepatic  veins  of  hvimans  as  compared  to  the  concentration  of  plasma 
protein  bound  iodine  in  the  peripher8.1  veins  or  femoral  artery  blood 
of  the  same  subject.  This  enables  one  to  calculate  the  amount  of 
protein  bound  iodine  removed  by  the  liver  and  gives  a  measure  of  the 
entero-hepatic  circulation  of  thyroxine;  the  first  such  information 
available  in  man. 

Proposed  course;  See  notation  atta.ched  to  page  1, 

B)  The  effects  of  varioiis  steroids  on  thyroid  function  and  the 
metabolic  fate  of  thyroxine. 

The  effect  of  estrogens,  androgens  and  corticoids  on  thyroid 
function  and  the  metabolic  fate  of  thyroxine. 

Methods  employed;  Radioiodine  24-  hour  tracer  uptakes;  thyroid  gland 

hormone  secretion  rates;  paper  chromatography  of 
protein  bound  radioiodine  com-pounds,  serum  protein  bound  iodine  de- 
terminations; disappearance  rate  measurements  of  blood  radiothyroxinej 
serum  protein  thyroxine  binding  capacities. 


PAGE  2 
PROJECT  REPORT  FORM   (Cont'd.) 


NCI-810(C) 
SERIAL  NO. 


PROJECT  DESCRIPTION  (Cont'd.) 

Ma.lor  findings;  a)  Estrogens  cause  a  significant  rif5e  in  serum  protein 

bound  iodine  without  a  concomitant  rise  in  24-  hour 
tracer  radioiodine  uptake  of  the  thyroid  gland;  b)  secretion  rate: 
in  2  patients,  so  far,  one  has.. had  no  change  in  secretion  rate  with 
estrogen  administration;  one  lias  had  slowing  of  secretion  rate  with 
estrogen  administration;  c)  testosterone  causes  a  mp.rked  fall  in 
serum  protein  bound  iodine  and. .24.  hour  radioiodine  tracer  uptake  by 
the  thyroid  gland;  d)  Metacortandrocin  and  Metacortandrolone,  cor- 
ticoids  similar  to  hydrocortisone,  cause  a  marked  fall  in  uptake 
and  senmi  protein  bound  iodine;  g)  jxiper  partition  chromato.graphy  has 
failed  to  reveal  any  qualitative  change  in  the  iodinated  compounds 
released  by  the  thyroid  under  the  influence  of  the  steroids  mentioned 
o.bove , 

Proposed  course;  It  is  proposed,  during  the  next  calendar  year  to 

extend  these  studies  to  a  greater  number  of  cases 
and  to  employ  radioactive  iodine  labelled  thyroxine  for  tissue  uti- 
lization studies  during  control  and  steroid  administration  periods 
in  the  same  patient, 

C)  Ejctra  thyroidal  effects  of  thyroid  stir.iulating  hormone;  T3H  effect 
on  thyroxine  to  triiodo-thyronine  conversion: 

Ifethods  employed;  Measurements  of  serum  or  plasma  protein  bound  iodine, 

rate  of  radiothyroxine  utilization  in  vivo  by  scin- 
tillation gamiTia  counting  of  blood  protein  fractions  in  sequentia.l 
fashion. 

Ma.-]' or  findings:  One  case  studied  so  far  over  2  week  period.  No  change 
in  thyroxine  utilization  rate  was  found. 

Proposed  course;   ictension  of  present  study  to  several  patients  includ- 
ing radiothyroxine  utilization  studies. 

D)  In  vitro  conversion  of  thyroxine  to  triiodo-thyronine  by  surviving 
tissvie  slices  and  homogenates,  and  the  factors  influencing  this 
interconversion. 

Objectives;  To  confjjrm  and  extend  the  work  of  Albright  and  Larsen  vrho 
demonstrated  in  vitro  conversion  of  thjrroxine  to  triiodo- 
thj'ronine  by  rat  kidney  slices.  To  isolate  the  enzyme  systems  respon- 
sible for  the  deiodination  involved  and  to  ascertain  the  factors  in- 
fluencing this  enzyme's  action. 

Methods  employed;   Surviving  tissue  slices,  tissue  cultures  and  homo- 
genates incubated  with  radioiodine  labelled  1- 
thyroxine.  The  products  of  these  incubations  are  then  analyzed  by 
paper  partition  chromatography. 


PAGE  3 
PROJECT  P.EPORT  FOM  (Cont'd.) 


ECI-810(C) 

SERIAL  NO. 


PROJECT  DESCRIPTION  (Cont'd.) 

Major  findings;  No  conversion  to  triiodo-thyronine  by  mouse  liver 

tissue  cu3.tures5  slight  appearance  of  triiodo 
thyronine  after  incubation  with  monkey  surviving  kidney  slices  and 
homo ge nates  in  phosphate  buffer. 

Proposed  course;  This  project  was  discontinued  due  to  separation 
from  the  Service  of  the  principal  investigator. 
No  additional  progress  is  reported  at  this  time. 

Significance  to  cancer  research;  Further  elucidation  of  our  know- 
ledge of  the  m.echanism  of  action 
of  the  steroid  hormones  on  peripheral  tissues  may  be  expected  to 
help  explain  som.e  of  the  favorable  and  unfavorable  effects  on 
cancer  of  the  breast,  prostate  and  uterus. 


11. 


BUDGET  ACTIVITY: 

RESEARCH  /^      ADMINISTRATION  /~/ 

PJCVIEVJ  &  APPROVAL     /^     TECHNICAL  ASSISTANCE     /^ 


12.. 


COOPERATING  UNITS,  0?  THE  PUELIC  I1E/.LTH  SERVICE,  OR  OTItR  ORGANIZATIONS, 
PROVIDING  FUtlDS,  FACILITIES,  OR  PERSOMJEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957: 

Dr.  Pb.ul  Marks,  tJIAMD  cooperated  in  this  project. 


15.. 


PUBLICATIONS  OTHER  TH/iN  ABSTR/iCTS  FROM  THIS  PROJECT  DURING  CALEIvTDAR 

YEAR  1955; 

The  effect  of  meticorten  and  meticortelone  on  thyroid 
function,  by  M.   G.    Sherer  and  B,  N,   Siefring,  J.  Clin, 
Endocrinol.  &  Me tab,   (In  press). 


NO  ENTRIES  FOR  ITEt-IS  13  and  I6. 


PAGE  1 
'  PROJECT  REPORT  FORM 

1.  H.  C.  I.  2.  Endocrinolopy  Branch 


INSTITUTE  LABOR.'iTORY  OR  BRANCH 

3 ♦  Endo crinolo gy  ^e rvice 4-. ^ 5.  IICI-3ll(C^ 

SECTION  OR  SERVICE         ,  LOCATION  (IF  OTHER  Ta'.N  BETIiSSDA )  SERIAL  NO. 

6.  Studies  on  Thyroid  Adenomata  and  Related  Problems  in  Pituitary-Thyroid- 
PROJECT  TITLE 

HjTothalrjnic  Relationships 

7.  Monte  A.  Greer 

PRINCIPAL  INVESTIGATOR (S)  ' 

8.  Leslie  J.  Do  Groot,  E].eanor  Siperatein,  Howard  En-fin  and  Robert  0 .  Scow 
OTHEP.  IIWSSTIGATORS  • 

9.  PROJECT  DESCRIPTION  -  Correlation  of  histologic  morphology  and  metabolism 

of  simple  goiter  with  its  response  to  thyroid  therapy; 

Objectives:  To  determine  whether  theoo  is  any  correlation  between  the 

microscopic  appearance  and  metabolic  activity  of  various 
types  of  simple  goiter  tri.th  their  diminution  in  size  v;hen  desiccated 
thyro'id  therapy  is  administered. 

■Methods  employed;  Studies  of  the  metabolic  activity  of  simple  goiter 

are  rxido  with  1-131  tracer  studies.  A  vredge  biopsy 
is  then  made  of  the  gland  and  3  weeks  later  administratien  of  3  grains 
daily  of  desiccated  thyroid  begun.  At  the  end  of  a  4--6  month  period 
a  final  biopsy  is  performed.  In  the  case  of  single  nodules,  the  entire 
nodule  is  removed  at  the  end  of  the  therapy  period. 

Patient  material: 


Admissions:  Adult  males 

Adult  females 

No, 

u 

7 

Average  Stay  Days 
15 
29 

Outpatient:  Number  of  patients 
Number  of  visits 

15 
77 

I'la.ior  findings;  During  the  past  year,  only  U   such  patients  have  been 

studied.  These  included  cases  of  simple  goiter,  chronic 
thyroiditis,  and  single  thyroid  adenomas.  All  goiters  thus  far  studied 
have  experienced  considerable  diminution  3ji  size. 

Additional  findings  of  note  are  indicated  by  the  titles  of  publica- 
tions listed  below  under  Item.  15. 

Proposed  course;  A  continuation  of  the  study  as  outlined  above  is 
planned  for  the  coming  year. 


PAGE  2 
PROJECT  REPORT  FOM  (Cont'd.) 


NCI-811(C) 

smuij  NO. 


PROJECT  DESCRIPTION  (CONT'D.) 

B)  Isolation;  and  identification-of  precursor  of  naturally  occurring 
antithyroid  compound,  1-5  vihylthioo3cazolidone. 

Objectives;  To  isolate,  crystallize,  and  identify  the  structure  of 

the  precursor  of  the  only  known  naturally  occurring  anti- 
thjToid  substance,  venylthiooxazolidone.  The  procursor  may  be  of  a 
type  that  is  more  widespread  among  various  plant  foods  than  is  now 
believed. 

Methods  employed;  Brassica  seed  extm.cts  are  fractionated  by  various 
procedures,  including  column  chromatography,  after- 
first  heat  denaturing  the  thioglycosidase  which  specifically  hydro- 
lyzes  the  precursor.  Assay  of  the  fractions  is  greatly  facilitated 
by  following  the  UV  abortion  spectrum.  The  precursor  has  a  specific 
peak  absorption  at  2270  A°.  Crystallisation  and  identification  of 
the  pure  compound  are  by  standard  chemical  and  microchemical  procedures. 

Major  findings;  During  the  past  year,  the  precursor  has  been  purified 
and  crystallized.  It  has  been  found  to  be  a  thiogly- 
coslde  having  a  molecular  weight  of  about  1000,  m.p.  130°C.  Both 
sodiim,  potassium,  and  304.  —  have  been  identified  in  the  molecule. 
Tlie  glycoije  has  been  tentatively  identified  as  a  fructose  polymer. 

Proposed  course;  It  is  planned  to  continue  with  degradation  experiments 

to  \rork  out  the  structure  of  the  compoiind  and  to  de- 
termine T:^  animals  and  human  assay  whether  any  biologic  system  not 
containing  the  specific  thioglycosidase  foiond  in  Brassica  members  is 
capable  of  liberating  thiooxazolldone  from  it. 

C)  Stim-ulation  of  the  central  nervous  system,  of  rats  by  radiofrequency 
transmission. 

Objectives;  To  devise  apparatus  capable  of  stimulating  the  central 
nervous  system,  particularly  the  hypotlialamus  c of  rats. 
The  animals  are  to  be  completely  unrestrained  and  without  any  external 
connections  to  the  pulse  generating  equipment.  It  is  hoped  to  produce 
hyperf unction  of  the  pituitary  by  stimulation  of  those  areas  vrhich, 
when  destroyed  by  electrolytic  lesions,  result  in  decreased  hypophysial 
function.  In  this  way,  considerable  information  may  be  gained  by  neuro- 
endocrine interrelationships. 

Methods  employed;  A  technique  has  been  devised  for  fixing  radio  receivers 

to  the  skull  of  rats,  vdth  stimulating  electrodes  buried 
according  to  calculation  in  various  parts  of  the  hypothalamus,  A  ratio 
sending  and  receiving  apparatus  has  been  devised  to  carry  variable 
pulses  to  the  hypothalamic  electrodes.  The  receiver  in  the  rat  is 
entirely  subcutaneous.  It  is  possible  to  stimulate  36  or  more  rats  at 


PAGE  3 

PROJECT  REPORT  FORM  (CONT'D). 


NCI-8ll(C) 

SERIAL  NO. 


PROJECT  DESCRIPTION  (Cont'd.) 

one  time  and  with  variable  intensities  and  frequencies. 

Changes  in  hypophysial  function  are  measured  by  gross  and  cyto- 
logic changes  in  the  pituitary  and  target  endocrine  glands  and  by 
noting  alterations  in  the  functional  activity  of  the  target  glands. 

Concomitantly  observations  are  made  of  the  behavioral  changes 
■produced  in  the  animals  by  stimulat5.on. 

I>fei.1or  findings;  During  the  past  year,  the  major  emphasis  lias  been 

on  developing  equipment  and  techniques  to  a  satis- 
factory level.  It  has  been  discovered  that  stimulation  of  the  hypo- 
thalamic area  lateral  and  Just  posterior  to  the  paraventricular  nuclei 
will  induce  narked  drirJcing  behavior  in  the  rats.  Stimulation  slightly 
posterior  to  this  area  will  induce  marked  eating  behavior. 

Proposed  course t  The  major  emphasis  during  the  coming  year  will  be 

devoted  to  vrorking  out  the  "bugs"  in  the  stimulation 
system  and  develop  it  to  the  point  where'  stimulation  can  be  continued 
for  several  weeks  without  any  appreciable  loss  of  response  in  the 
stimulated  animal, 

D)  Study  of  the  structure  and  function  of  heterotopic  compared  with 
normal  pituitaries. 

Objectives:  To  determine  the  alterations  in  structure  and  function 
produced  in  the  pituitary  gland  by  transplanting 'it  to 
an  abnormal  site  and  to  determine  what  factors  are  involved  in  the 
changes  thus  produced. 

Methods  employed;  Pittiitaries  from  new  born  mice  are  transplanted  into 

the  eye  or  kidney  of  genetically  homogeneous  mature 
mice.  The  hosts  are  hjrpophyse eternized  one  week  later.  Three  v/eeks 
following  hypophysectomy  the  function  of  the  heterotopic  pituitaries 
is  measured  by  following  changes  in  body  growth  and  changes  in  the 
metabolism  and  structure  of  the  target  endocrine  glands.  Pituitary 
cytology  is  studied  by  the  various  advanced  techniques  now  available. 

Major  findings;  1,  Heterotopic  pituitaries  are  able  to  maintain  thy- 
roidal 1-131  metabolism  at  a  level  approximately  equal 
to  that  in  intact  animals.  They  are  not  able  to  m.aintain  thyroid  weight 
or  the  weight  of  other  target  endocrine  organs  significantly  above 
that  of  hypophysectomized  animals.  Raising  or  lowering  the  level  of 
circulating  thyroxin  yxU.   cause  marked  changes  in  the  secretion  of  the 
pituitary  hormone  controllijig  thyroidal  1-131  metabolism  in  these  animals 
without  having  any  significant  effect  on  thyroid  v/eight. 


PAGE  U 
PROJECT  REPORT  FORI  I   (Cont'd.) 


NCI-311(C) 
SaiLIL  NO. 


PROJECT  DESCRIPTIOII  (Cont'd.) 

2.  Glycogen  has  been  demonstrated  in  the  pituitary  for  the 
first  tine.  This  is  present  particularly  in  the  younger  glands,  and 
in  highest  concentration  in  the  pars  tuberalis. 

3,  The  differences  in  cytolog;^  of  heterotopic  compared  to  in  aitu 
pituitaries  has  been  compared  for  va.rious  ages.  One  of  the  most 
striking  differences  is  the  narked  drn.inution  in  chrom.ophiles  in  the 
heterotopic  glands.  There  is  also  a  narked  hypertrophy  of  the  pars 
intermedia  comparable  to  tkat  seen  following  hypothalamic  lesions. 

Proposed  course;  The  major  effort  during  the  coming  year  vdJ-1  be  de- 
voted to  a  study  of  whether  function  of  the  heterotopic 
pituitaries  can  be  increased  by  transplanting  -vT^-rious  p-^rts  of  the 
central  nervous  system,  particularly  the  hypothalrius ,  in  direct 
connection  with  the  pitui.tary  transplants.  A  system  has  already  been 
devised  for  making  transplants  of  considerable  size  to  the  kidney  of 
adu!l.t  mice. 

It  will  also  be  detexmined  whether  the  heterotopic  pituitaries 
secrete  the  substance  necessar:/  for  ovarian  and  uterine  response  to 
chorionic  gonadotropin,  A  further  study  vri.ll  invoj.ve  whether  they 
can  concentrate  isotopically  labelled  triiodothyronine  to  as  marked 
an  extent  as  do  normal  glands, 

E)  The  effect  of  hypothalamic  lesions  on  pituitary  function. 

Objectives;  To  deteiT'.ine  what  effect  electrolytic  lesions  in  various 

parts  of  the  h;7pothalarus  have  upon  the  regulation  and 
secretion  of  the  various  hormones  produced  by  the  pituitary  gland. 

Methods  employed;  Electrolytic  lesions  are  made  iji  the  hypothalamus 

of  the  rats  by  means  of  a  specially  designed  stereo- 
taxic instrument.  Following  a  period  of  1-3  weeks  for  reoovarj'', 
studios  are  made  of  the  ability/  of  the  pituitary  to  secrete  the 
various  trophic  hormones.  These  deterriinations  are  made  by  studying 
the  alterations  of  structure  and  function  produced  in  the  target  endo- 
crine glands  by  v^.rious  stimuli.  For  exar.iple ,  thyrotrophin  secretion 
is  studied  by  the  change  produced  in  thyroid  size  and. 1-131  metabolism 
by  chronic  administration  of  propylthiouracil, 

Ilaj or. findings;  During  the  past  year  it  has  been  found  that  the  location 
of  the  hypothalamic  area  controlling  thyrotropin  secre- 
tion in  the  rat  lies  in  the  midline  between  the  paraventricular  nuclei 
and  the  media.n  emdnence.  Destruction  of  all  or  part  of  this  area  will 
markedly  interfere  with  thyrotrophin  secretion. 


PAGE  5 
PROJECT  REPORT  F0R14  (Cont'd.) 


NCI-811(C) 

SERIAL  HO. 


PROJECT  DESCRIPTION  (Cont'd.) 

It  has  also  been  found  that  although  lesions  in  the  above  area 
will  prevent  the  goitrogenic  response  to  Anphenone  administration, 
they  ijill  not  prevent  the  usual  conconitant  adrenal  hypertrophy. 
This  provides  further  evidence  that  the  hypothalamic  areas  cohtrolling 
the  various  pituitary  fxinctions  are  discrete. 

Proposed  course;  It  is  planned  to  make  a  study  of  the  location  'of  the 
hypo  thai  pjnic  area  responsible  for  maintaining  normal 
growth  of  the  pars  intermedia  of  the  hjixiphysis.  ,  Material  from  over 
500  rats  with  hypothalrjaic  lesions  has  already  been  assembled  and  the 
remaining  requirement  is  to  determine  hc!';  far.  from  the  hj^Dophysial 
stalk  it  is  liecesaary  to  place  lesions  i:i. order  to  avoid  the  gross 
and  bizarre  hypertrophy , of  the  pars  interriiedia.  xrhich  occvirs  with  le- 
sions close  to  the  stalk, 

■  It  is  also  planned-  to  investigate  the  area  controlling  ACTH 
secretion  r.ore  thoroughly  and  to  see  if  it  is  possible  to  place 
lesions  that  will  interfere  with  ACTH  secretion  as  much  as  previous 
lesions  have  been  intarfered  with  thyrotropin  secretion, 

F)  Study  of  the  mechanism  of  action  of  stable  iodine  in  thyrotoxicosis. 

Objectives;  To  determine  the  mechanism,  of  action  of  stable  iodine 

.  in  .ameliorating  thyrotoxicosis  and,  secondarily,  to --.ga'in 
some  insight  into  the  pathogenesis  of  thyrotoxicosis. 

Methods  employed:  Thjrro toxic  and  euthyroid  subjects  are  given  a  100 

rdcrocurie  tracer  dose  of  I~131.  Twenty-four  hours 
later  the  administration  of  3O-6O  ng,  tapr.zole  every  8  houi-s  is  begun  to 
prevent  reaccumuJIation  in  the  thyroid  of  1-131  broken  doim  from  endo- 
genously  labelled  thyroxin.  In  vivo  counting  over  the  thyroid  is  ma.de 
by  means  of  an.  extema.lly  placed  scintillation  counter.  The  thyroidal 
secretion  rate  can  thus  be  deterr.iined  by  plotting  on  serdlog  paper. 

Iflhen  the  rate  has  been  established,  300  ng.  per  dr.j  of  Nal  are 
added  'to  the  regimen.  When  the  maximum  effect  from  iodine  has  been 
obtained,  10-100  units  of  thyrotrophin  are  given  daily  for  a  period 
of  several  days. 

In  the  case  of  the  control  euth;!/roid  subjects,  thyrotrophin  was 
frequentD.y  given  before  iodine  adm.inistratibn  was  begun. 

Major  findings;  It  has  been  found  that  iodine  interferes  with  the 

release  of  hormone  from,  the  thyroid  gland  in  thyro- 
toxic subjects  but  has  little,  if  any,  effect  in  euthyroid  subjects. 
It  has  also  been  foiond  that  iodine  does  not  interfere  with  the  stimu- 
lation of  euthyroid  glands  by  exogenous  thyrotropin. 


PAGE  6 
PROJECT  REPORT  FORM  (Cont'd.) 


NCI-811(C) 
SERIAL  NO. 


PROJECT  DESCRIPTION  (Cont'd.)  ' 

"\_        These  results  suggest  thnt  iodine  nets  in  hyperthyroidisn  by- 
inhibiting  the  release  of  thyrotrophin  fron  the  pituitary.  There- 
fore, hyperthyroidism  nust  be  a  supro.thyroidal  disturbance  and  not 
solely  a  disease  of  the  thyroid  gland.  .' 

Proposed  course;  The  project  is  teminr.ted  due  to  the  separation 
of  Dr.  Monte  A,  Greer, 

Significance  to  cancer  research;  Studies  of  thyroid  adononata  will 

have  a  direct  bearing  on  the  problein 
of  the  pathogenesis  of  thyroid  cri.ncer.  In  addition,  further  know- 
ledge of  the  pituitary''- thyroid  interrelationship  vjill  provide  potential 
means  of  controlling  abnormal  thyroid  grovrbh. 


11.. 


BUDGET.  ACTIVITY: 

RESEARCH  £7      ADMINISTRATION  £7 

REVIEW  &  APPROVAL  £7       TECHNICAL  ASSISTANCE     /~/ 

12. 

COOPEPATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTIiER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES ^  OR  PERSONI^IEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957; 

Dr.  Robert  0.  Scow,  NIAMD,  collaborated. 

NO  ENTRY  FOR  ITE14  13.      . 

15. ., 

PUBLICATIONS  OTHER  THAN  ABSTR/iGTS  FROM  THIS  PROJECT  DURING  CALEIMDAR  YEAR 
1955; 

1.  Denonstration  of  thyroidal  response  to  exogenous  thyrotropin 
in  irats'  with  anterior  hypotlmlanic  lesions,  by  M,  A.  Greer 
Endocrinology  (In  press). 

2.  Observations  on  the  norphology  and  histo'chenistry  of  the 
mouse  pituitr.ry  inplanted  in  the  anterior  eye  chamber,  by 
E.  R.  Siperstein  and  M.  A,  Greer,  J.  Nat,  Cancer  Inst, 
(In  preparation), 

3.  Effect  on  the  thyroid  gland  of  experimental  alteration  of  the 
level  of  circulating  thyroxine  in  mice  with  heterotopic 
pituitaries,  by  R,  0,  Scow  and  M.  A.  Greer,  Endocrinology;  56: 
590,  1955. 


PAGE  7 
PROJECT  RSPOHT  FOP^i  (Cont'd.) 


ITCI-ai(C) 
SERI/iL  NO. 


15.  FUBLICATIOITS  (Cont'd.) 

4..  Rlstocheriical  denonstration  of  glycogen  in  the  mouse 
pituitary,  by  Eleanor  R.  Siperstein,  Proc.  Soc.  Sxper, 
Biol.  ^  Med.  8S:  296,  1955. 

5,  Suggestive  evidence  of  a  prinary"drirJ<:ing  center"  in 

hypothalamus  of  the  rat,  by  M.  A.  Greer,  Proc.  Soc.  Exper. 
Biol.  &  Med.  89:59,  1955. 

6.  A  modified  Krieg  stereotaxic  instioment  for  producing  intra- 

cranial lesions  in  the  rat,  by  Monte  A.  Greer,  Proc.  Soc, 
Exper.  Biol.  &  Med.  89:  A^O,  1955. 


NO  EI'ITRY  FOR  ITEl-I  I6. 


PAGE  I 
PROJECT  REPORT  FORM 


^  •  National  Cancer  Institute  2 .  Pathologic  Anatomy  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

^  •   Surgical  Pathology  &  Post-Mortem  Service  4 . 5 ■  NCI—S^ 

SECTION  OR  SERVICE    '  LOCATION  SERIAL  NO. 

6.  No  specific  research  projects  in  this  Branch.  However,  the  diagnostic 
service  is  designed  to  aid  the  investigative  efforts  of  the  N.I.H.  staff 
responsible  for  patient  care.   In  many  instances,  investigative  study  of 

pat holcgical  tissues  is  of  great  importance  to  clinical  research. 

PROJECT  TITLE   .  ~  -       -   . 


PRINCIPAL  INVESTIGATOR (S) 


Dr.  Harold  L.  Stewart,  Chief,  Pathologic  Anatomy  Branch 

Dr.  Louis  B.  Thomas,  Head,  Surgical  Pathology  &  Post-Mortem  Service 

Dr.  Albert  W.  Hilberg,  Head,  Cytodiagnosis  Service 

Dr.  John  H.  Edgcouib,  Pathologic  Anatomy  Branch 

Dr.  Alan  S.  Rabson,  Resident,  Pathologic  Anatomy  Branch 

Dr.  Richard  L..  Swarm,  Laboratory  of  Pathology,  N.C.I. 

Dr.  Clyde  Dawe         ■  "  " 

Dr.  Eli  Nadel  "  " 

Dr.  Joseph  Leighton       "  n 

Dr.  William  Banfield      "  " 

Dr.  C.Harold  Steffee.      "  " 

Dr.  Edwin  Lerner,  Laboratory  of  Pathology  and  Histochemistry,  N.I.A.M.D. 

Dr.  Leon  Sokoloff      "  "  ''  " 

Dr.  Harold  Stanley,  Oral  Pathologist,  N.I.D.R. 


* 


These  pathologists  spend  full  time  in  the  service  and  diagnostic 
functions  of  the  Pathologic  Anatomy  Department,  Clinical  Center 
(Pathologic  Ajiatomy  Branch,  N.C.I.) 

These  associate  pathologists  spend  only  part  time  in  the  activities 
of  the  Pathologic  Anatomy  Department,  principally  in  the  performance 
of  postmortem  examinations. 


OTHER  INVESTIGATORS 


SERIAL  NO.  NCI -85^  PAGE  II 

PROJECT  REPORT  FORM  (Cont'd) 

9.  PROJECT  DESCRIPTION: 

No  specific  research  projects.  The  services  and  diagnostic  functions 
of  the  Department  during  1955  have  included: 

a.  148  autopsy  examinations. 

The  average  number  of  postmortems  performed  by  each  pathologist 
was  about  15;  the  range  was  from  21  to  8  with  the  smaller  number  being 
performed  by  those  who  were  here  less  than  one  year. 

b.  1778  surgical  pathology  accessions. 

Most  of  these  were  worked  up  by  Doctors  Thomas,  Edgcomb/  Swann, 
Rabson,  Sokoloff  and  Stanley. 

The  objectives  of  the  Surgical  Pathology  and  Post -Mortem  Service  in  the 
Clinical  Center  are  twofold:  first^  to  furnish  a  diagnostic  service  in 
autopsy  and  surgically  rei\ioved  tissues,  and  second,  to  aid  from  a  morpho- 
logical s'tatidpoint  the  various  clinical  research  problems  which  are  under 
study  in  the  Clinical  Center. 

The  methods  used  are  those  standard  methods  which  are  used  in  the 
description  of  organs  and  tissues,  the  fixing  aiid  sectioning  of  this  material 
and  the  preparation  of  histological  slides.  A  wide  variety  of  special 
staining  procedures  are  used  in  the  Histopathology  Laboratory  of  the 
Department .  The  appended  chart  shows  the  December  monthly  report  of  the 
material  studied  in  this  Department  and  includes  in  the  right  hand  column 
totals  for  the  accessions  during  the  calendar  year  of  surgical  specimens 
and  autopsies  performed.  Note  that  the  autopsy  rate  has  doubled  that  of 
1954  and  the  number  of  surgical  specimens  has  increased  by  over  50  per  cent. 

In  addition  to  the  diagnostic  services,  there  are  several  other  functions 
of  the  Department  which  have  continued  and  expanded  during  the  past  year.  At 
present  there  are  fifty  scientists  in  the  various  laboratories  at  N.I.H.  who 
have  requested  particular  types  of  tissue  from  surgical  and  postmortem 
specimens  examined  in  this  Department.  On  many  occasions  it  is  possible  to 
furnish  these  investigators  with  fresh  human  material.;.       .:  -. 

The  staff  of  the  Department  continues  to  take  active  part  at  numerous 
Clinical  Center  staff  meetings,  and  in  addition,  routinely  conducts  four 
Departmental  meetings  weekly.  These  are  the  Brain  Cutting  Conference  on 
Monday,  the  Autopsy  Conference  on  Tuesday,  the  Joint  Pathology  Staff  Con- 
ference on  Wednesday  and  the  Surgical  Pathology  Conference  on  Friday. 


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PAGE  III 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCL.S52— 


SERIAL  NO. 


II. 


BUDGET  ACT^'VITY: 


RESEARCH'         /_/ 
REVIEW  &  APPROVAL  /~7 


1 2 .   None 


ADMINISTRATION       /_/ 
TECHNICAL  ASSISTANCE  /~7 


COOPERATING  UtillTS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  i9i7 


13  .  None 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE 
ELS^T^'HERE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL, 
FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  14,  15,  or  16, 


PAGE  I 
PROJECT  REPORT  FORM 


I  .National  Cancer  Institute  2 .  Pathologic  Anatomy  Branch 

INSTITUTE  LABORATORY  OR  BRANCH 

3  .  Cytodiagnosis  Service  : :  A i-rJ.:  "'    ^ 5  .  NCI  851 

SECTION  OR  SERVICE  LOCATION (IF  OTHER  THAN  BETHESDA)   SERIAL  NO. 

6.  Exfoliative  cytology  applied  to  diagnostic  and  research  problems  in  the 

Clinical  Center,  N.I.H.   .  ^ ..__ 

PROJECT  TITLE 


7.  Albert  W.  Hilberg 


PRINCIPAL  INVESTIGATOR (S) 


8.  None 


OTHER  INVESTIGATORS 


9,  PROJECT  DESCRIPTION: 

Project:  Application  of  Exfoliative  Cytology  to  Human  Diagnostic  and 
Research  Problems. 

Objectives:  To  provide  the  service  of  exfoliative  cytology  for  the  diagnosis 

of  malignant  tumors  of  various  sites  in  the  body,  notably  the 
cervix  uteri,  lungs,  stomach,  and  body  cavities.  Evaluation  of  effects  of 
various  types  of  therapy  for  cancer  such  as  surgery,  virus  therapy,  and 
endocrine  therapy.  To  evaluate  effects  of  hormones  in  non -cancer  patients 
studied  for  endocrine  balances.  Further  objectives  involve  attempts  to 
better  evaluate  the  cytological  criteria  of  malignancy. 

Methods  Employed:  Routine  Papanicolaou  staining  and  careful  cytological 

4      screening  procedures  together  with  concurrent  tissue  and 
clinical  evaluation.  Additional  methods  involve  special  fixation  preparation 
and  staining  techniques  as  developed  in  histochemical  procedures  and  as 
needed  for  the  precise  handling  of  specimens  of  various  kinds. 

Patient  Material:   See  appended  chart. 

Major  Findings:  The  correlation  of  recurrent  cancer  at  the  site  of  surgical 

procedure  and  the  presence  of  malignant  cells  in  washings 
from  this  site  has  been  made.  Several  previously  undetected  cancers  of  the 
cervix  were  found  by  the  methods  of  exfoliative  cytology.  The  presence  or 
absence  of  estrogenic  activity  in  prepubertal  girls  or  in  cases  of  amenorrhea 
was  determined  in  many  clinical  cases.  Evaluation  of  menstrual  cycles. 
Argyrophil  secretion  patterns  indicating  estrogenic  activity  of  a  notable 
degree  past  the  menopause  was  noted  in  cases  of  carcinoma  of  the  cervix  uteri 
and  evaluation  of  this  continues. 


SERIAL  NO.  NCI  851  PAGE  11 

PROJECT  ILEPORT  FORM  (Cont'd) 

PROJECT  DESCRIPTION  (Cont'd): 

Significance  to  Cancer  Researdh;  '.T^is  project  is  a  continuing 'demdnStration 

of  the  value  of  exfoliative  cytology  as  a 
diagnostic  tool  for  the  clinician  in  the  attempt  to  detect  early  cancer^ 

The  suirgical  wound  washing  cbtitiriues  to  show  distinct  and  valuable 
correlation  of  residual  tumor  cells  and  tumor  recurrence  in  surgical  sites. 

Exfoliative  cytology  provides  an  excellent ! adjunct  to  tissue  evaluation 
of  the  effects  of  various  forms  of  therapy  on  tumors  wheri  it  is 'riot  possible 
or  practical  to  remove  tissue  for  biopsy  or  In  conjunction  with  biopsy  study. 

Proposed  Course  of  Project:  The  continuation  of  diagnostic  service  to  all 

Institutes  in  t.he  Cllttical  Center'.'  'Continued 
collaboration  in  evaluation  of  various  clinical  research  projects  such  as 
virus  therapy  of  cancer,  hormone  balance,  and  wound  washings.  Further 
development  and  refinement  of  special  preparation,  fixation  and  stashing 
techniques  as  applied  to  cytologic  material.  Expansion  of  activities  is 
planned  to  meet  increased  demands  for  diagnostic  service  and  to  further 
assistance  in  clinical  research. 


.sbsihi   'iuox .'i-K--  'it h  ftnoffi}- 


jj    iiJL    iiiiw..'    -• .  ■ 

■  .i'o   .;OJiv , 

■i'S:-^nSiZ    h-i^T; 

•  ■',"■-  J-V!V:^tflr 

.   '■>    sHT;,   ,7: 

,,  !..  :■  ^■•' 

.'J     !"')!?''] 


PAGE  m 


PROJECT  REPORT  FORM  (Cont'd) 


10.  NCI  851 


SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         M 
REVIEW  &  APPROVAL  l~l 


ADMINISTRATION       /_/ 
TECHNICAL  ASSISTANCE  l~l 


12   None  to  my  knowledge 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS, 
PROVIDING  FUNDS ,  FACILITIES ,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER 
1956  or  1957 


13.   None  to  my  knowledge 


'if  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE 
ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL, 
FACILITIES  OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


PAGE  IV 
PROJECT  REPORT  FORM  (Cont'd) 


14.  NCI  851 


SERIAL  NO. 


15  . '-^  ■  :      ■         -::r 

PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PR5(ijECT  DURING  CALENDAR  YEAR 
1955    ''     ■  ■  ■■  • 

Cancer  Cell  Seeding  of  Operative  Wounds,  Smith,  Robert  R.  and  Hilberg, 
Albert  W.,  J.  of  Nat.  Cancer  Inst.,  Dec.  1955. 

A  Ndw  Inexpensive  Marking  Deyice  for  Slides,  belVeccKio,  P.R., 

Ziegler,  E.,  and  Hilberg,  A.W. ,  J.  of  Nat.  Cancer  Inst. 

(In  Press)  , 


•v:..  HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  tO  THIS  PROJECT  DURING  CALENDAR 

YEAR  1955 


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PAGE  1 
PROJECT  REPORT 


.  National  Cancer  Institute 2»  -  Laboe^oiy  of  Pnysiolcgy 

INSTITUTE  tAW^rtJ^-Y  OR  BEA>*CH 


U  Cancer  Physiology  Section  4.  --  :..       j^-'- 

SECTION  OR  SERVICE  LOCATION  tif  other  tiun  Betoesda)      SERIAL  Na 


5.  The  "appetitiB  factor^  in  Walker  25g 
PROJECT  TITLE 


'.  Julius  White 

„  .PRINCIPAL  INVESTIGATORiS^ 


3.  

OTHER  INVESTIGATORS 


9,  PROJECT  DESCRIPTION 

Ob|edtive8;''1*6'  continue  Iftd  stoidlies  qf  the  growtij-stimulating  factor  present 
in  Walker  carcinoma  256. 

Methods  Rn\ploye<fe   Fractu-  -.:..- ^  of  th«  Walker  256  witix  the  purpose  of 
isolating  the  growth  factor. 

Major  Findings; 

1.    Verification  of  the  previous  obMrvaHMS  ol  0r.  C»  B^  MldM' 
that  supplenientijig  the  t^asal  diet  of  animal  kMUteg  )ayqe» 
plantable  tumors  at  the  point  where  the  ml—iBt  llSt  Vi^ 
Intake,  results  in  a  resumption  of  tooti  Mate  wd  ffowth  increase. 

2*    Extraction  of  lyophiUised  tumor  with  iteft|»|  tlfcuf  4M8  BOk  remove 
the  factor. 

3,  The  protein  fraction  of  the  tumor  tissue  e'er  :^     .'  :?  ':  "  tiie  ^SP^tiUto 
factor  (preliminary  observation). 

4.  Pair  (e«<ttng  tixpMrtmMili;^  cv>    ,v.     ^  ^vw^  >     ;  =  .^    .  young  r«ls 
Ingesting  a  20^  casein  and  a  20%  tumor  vsource  of  tuirvgenj  iiet,  show 
Identical  growth  curves. 


PAGE  2 


SERIAL  NO.  ■nj-x.,..i.     .>... 


5.   Animals  ingesting  the  ^0%  tumor  diet  ad  libitum  siiow  ain  ippfoxi-     ' 
mately  25  per  cent  higher  growth  increment  than  do  litter  mate  animals 
ingesting  the  20%  casein  diet  ad  libitum. 

:  y   ■''•■';  6.: ■  ^'Aniittkls, ingesting  the  20%  casein- diet  exci«^it^&^i^rii|-'^^  '' 

nitrogen  as  do  the  pair  fed  20%  tumor  diet.  '        '  ' '     '  ^' 

7,  Urea  nitrogen  excretion  of  the  tumor  fed  ^nimals  jls  approxim,a,tely..    ^ 
half  of  that  excreted  by  the  casein  fed  animals.  '    ■^''^■':;^j.  i-y^'i^ 

8,  Allantoin  excretion  from  rats  ingesting  the  casein  diet  is  approxi- 
mately half  of  that  excreted  by  the  rats  ingesting  the  tumor  diet;, 

9.  Animals  fed  the  20%  casein  diet  and  bearing  Walker  256  transplanted 
tumors  show  emaciation  and  die  sooner  than  do  litter  mate  rats 
ingesting  the  tumor  diet. 

10.  Animals  ingesting  20%  tumor  diet  and  bearing  a  Walker  256  trans- 
planted tumor  grow  massive  tumors  (50  per  cent  of  total  tumor  and 
body  weight)  without  loss  of  appetite  or  loss  in  weight. 

Significance  to  Cancer  .fieseaypti.  There  is,  undoubtedly,  a  f actor  jpresent  in 
Walker  carcinoma  256  which  stimulates  growth,  spares  hit;p(>geh  and 
has  a  high  "food  efficiency"  value. 

Proposed  dourse  of  project:  To  continue  in  seaifch  o!  tne^ active  principle    ■ 
in  tumor  tissue  which  is  responsible  for  this  "appetite  factor, " 

•■'J?;-'.' '  \ '■' - ,    ■  '.„  ^    ■'■,  \0  i^lb  hi'dui_^(>i '^iv  •         ■^'' ■■:■  ;  ;;  ' 


12, 


900  PAGE  3 

SERIAL  NO, 


BUDGET  ACTIVITY: 

RESEARCH  fT]        ADMINISTRATION  [ZJ 

REVIEW  AND  APPROVAL   /~7         TECHNICAL  ASSISTANCE   ri 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER 
ORGANIZATIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL 
FOR  THIS  PROJECT  IN  EITHER  1956  or  1957, 

None, 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS 
RESEARCH  DONE  ELSEWilERE  IN  THE  PUBLIC  HEALTH  SERVICE 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 
IDENTIFY  SUCH  RESEARCH: 

None. 


NO  ENTRIES  FOR  ITEMS  14,  15  and  16. 


■orrASTaiv^i 


T^m 


'143.  Ov4 


PAGE  1 
PiElOJECT  REPORT  J>i^i 


1.  National  Canter  Iftstitate!         :  ;>^yA  Labor atory  of  Physiology 

institute:,  •■    ^ •'■■■■■■■■  :.'■..,;  .-^i.^ ., .,. "^BaR/lTdRX ok  brangh 


3.  Cancer  Physiology  Section  4«  v^  ■■  "      ;        .       ...  ;  '  5>     '9(31       ' 

SECTION  OR  SERVICE  LOCATION  (if  other  than  Bethesda)  '  SERIAL  NO. 


6.  Probable  Sources  of  Available  Nitrogen  for  Tmior  Growth 
PROJECT  TITLE  • .  :.      .    ;         ~~^. 


7.  Julius  White 

PRINCIPAL  INVESTIGA'rOR(S) 


8.  

OTHER  INVESTIGATORS 


9.  PROJECT  DrilSCRIPTION 

Objectives;   To  determine  the  availability  of  nitrogen  for  the  rapidly  growing 
transplanted  Walker  carcinoma  250. 

Methods  Employed;   Young,  growing,  as  well  as  adult,  rats  were  placed  on  a 
20%  casein  diet  of  known  composition.    The  Walker  carcinoma  256  was 
implanted  subcutaneously  and,  at  the  same  time,  N*^  glycine  was  in- 
corporated into  the  diet.    At  various  intervals  the  animals  were  sacri- 
ficed and  the  total  nitrogen  balance  was  determined. 

Major  Findings; 

1,  The  uptake  of  N^  glycine  by  the  transplanted  tumor  was  equivalent 
to  that  taken  up  by  the  host.    This  occurred  whether  the  isotope  was  fed 
for  7  days  or  14  days  and  followed  by  a  7-day  period  on  the  basal  diet 
or  sacrificed  immediately  after  the  cessation  of  isotope  feeding, 

2,  In  all  cases  the  tumor  reached  a  maximum  size  of  8  grams.    It 
appears  that  tumors  in  their  early  stages  of  growth  can  utilize  available 
nitrogen  at  the  same  rate  as  the  host. 


;.901;  PAGE  2 

iERlAL  NO.  i-^lO'lMi  TO'M&V) 

Significance  to  Cancer  Research!  To  obtain  a  better  understanding  of  the 
tumdr-libst  relationship.    A.t  what  stage  of  tumor  growth  does  the 
metabolic  insult  appear  in  the  host?   Is  the  nitrogen  present  in  tuinor 
tissue  available  under  some  circumstances  for  growth  of  the  host? 
ii'^re  the  "building  blocks"  used  by  the  tumor  similar  to  those  used  by 
.<5^ivJ#ililie'-host?'^'    :■■    :ii^:i  limsoMM'i' '  •■        /.■-v/ua*.    -^     ; 

.'.->,,.■,?',,>.'■'  ;■■-.■  ■ . .     ■  '•       'i  \  \    ■  *\  ■  '  ■        -   .  '  .  • 

■  '     -       *'''#-    '.I  ■■■,  .  '  ■    :  i  ,   , 

Proposed  course  of  project;  To  make  a  similar  study  of  nitrogen  utilization 

in  tumor -bearing  animals  in  which  the  tumor  has  reached  a  growth 

increment  where  derinand  of  energy  exceeds  energy  intake*  To  make  , 
serial  killings  and  determine  if  a  transition  takes  place,  where  it  takes 
place,  and  the  nature  of  such  an  event. 


••ta^iVJii  >idhi' 


IfOTTq^ 


''  ■''■"'  '''•■■"■■  '■'■■■  ■  Jy9n!ttf^fJ:JW^siw-^or>te^';<i'no5ib-i5^  bf^a.  ft-s^n. 


od)9  i'/! 


lOi  901  PAGE  3 

SERIAL  NO.  T-1 


U, 

:  O  BUDGET  ACTIVITY: 


L2. 


13. 


RESEARCH  /X7        ADMINISTRATION  [ZJ 

REVIEW  AND  APPROVAL   /~7         TECHNICAL  ASSISTANCE    fl 


COOPSRATIHO  UNITS  OF  THE  PUBLIC  HEALTESSRVICE,  OR  OTHER 
ORGANIZATIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL 
FOR  THIS  PROJECT  IN  EITHER  1956  or  1957. 

None, 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  CR  PARALLELS 
RESEARCH  DONE  3LSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 
IDENTIFY  SUCH  RESEARCH: 

None. 


M 


14,         901  .   _  .-lOPAGE^; 

SERIAL  NO.  ■■ryy.rii-^/u: . 


X5. .  , ..____.-, -   ' 

I^IJBLICATIONS  other  tHAli  AfiStftACtS  t'ROM  tHISFROJECT  DURING 
CALENDAR  YEAR  1955 


None. 


16.  _^^ '  __ 

ttdl^ORS  AJiD  AWARbS  To  t>ERSONNEL  ft£LATiNG  To  tHiS  ^ftOjECf 
DURING  CALENDAR  YEAR  1955: 

Elected  to  membership  "Who's  Who"  In  Chemistry. 


PAGE  1 
PROJECT  REPORT 


1,  National  Cancer  Institute  2,    Laboratory  of  Physiology 

INSTITUTE  """  LABORATORY  OR  BRANCH 


3.  Cancer  Physiology  Section  4.  5.       902 

SECTION  OR  SERVICE  LOCATION  (if  other  than  Bethesda)      SERIAL  NO, 


6.  The  Effects  of  X-irradiation  on  the  Nucleic  and  Protein  Synthesis  of  the  Small 

Bowel  Epithelium  of  the  Rat  as  Related  to  Cell  Division 

|»ROJECT  TITLE  ~"     ^~™~        ''  '- 

7.  Julius  White '  -      .       ,'      .         

PRINCIPAL  !NVESTIGATOR(S)       '^. 

8.  Dr.  R.  Bland  Williams,  Naval  Medical  Research  Institute; 

Dr.  James  C.  Reid  and  Jane  N,  Toal. ■     .  : 

OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objectives;   To  correlate  changes  in  nucleic  acid  and  protein  synthesis  with 
histopathological  changes  in  the  small  bowel  epithelium  produced  by 
x-irradiation. 

Methods  Employed;   Rats  are  exposed  to  total  body  x-irradiation  of  varying 
concentration  (from  threshold  to  lethal  dose)  after  receiving  a  known 
quantity  of  N^°  labeled  glycine,    Histopathological  changes  in  the  small 
bowel  with  respect  to  mitotic  arrest,  acute  cell  destruction,  prolongation 
of  interphase,  overshooting  in  recovery  and  incomplete  recovery  are 
made.    Changes  in  protein  and  nucleic  acid  synthesis  are  determined  to 
parallel  the  histopathological  studies. 

Major  Findings;   It  was  previously  demonstrated  that  with  an  arrest  in  cell 
division  there  was  always  associated  an  arrest  in  DNA  synthesis  but  no 
depression  in  protein  synthesis.    This  occurred  if  the  x-irradiation 
exposure  was  450  to  2, 000  r  and  the  synthesis  studied  0-5  hours  after 
exposure.    But  if  examined  68-73  hours  after  exposure  (450  r)  there  is 
a  threefold  increase  in  protein  synthesis  and  an  eightfold  increase  in 
DNA  synthesis.    This  increased  synthesis  has  been  associated  with  the 
cells  which  were  in  interphase  and  are  now  dividing. 


902  .''HQ^i#l%l!iOft^'''  PAGE  2 

SERIAL  NO. 


In  the  preseht  ferries,  the  tdkl  dose  of  x«ir radiation  was  fractionated  in 
which  the  tiss'ie  was  allowed  ta  recover  before  repeating  the  initial  dose 
and  to  determine  the  effect  of  keeping  the  total  dose  and  total  time  con- 
stant/ bi>t  varyiag  th^  size  of  |hoip4ivi±aal  dbs^is..  Divided  doses  were 
;  mdre  ek'h'cti%'thkti  k  single  dos  3  1'^  enhancing  recovery  (prolongation  of 
interphase)  or  preventing  destn.ction  of  crypts.    Protein  and  DNA 
^^         synther^EJ  paralled  Uiese  histopjithplogical  findings.    Exposure  to  600  r 
"mB  iolloweci  by  a  slmiiir^^^^d^^^  later  repeated  the' delay  in  division, 

but  when  givsn  12  hours  altt^r  the  ifiitiai  irradiation,  there  results  a 
severe  destruction  of  large  numbers  of  crypts,  with  a  marked  arrest  in 
DNA  sjmthesis*    Nine  hundred  r  were  given  over  a  12-hour  period  or 
225  r  every  four  hours;  as  450  r  followed  12  hours  later  by  450  r;  and 
as  700  r  follcwed  12  hours  later  by ^00  r,  respectively.    The  degreie  of 
recovery  of  DHA  synthesis  was  depenient  Upbn  the  size  of  the  individual 
doses.    DMA  synthesis  (recovery)  wae  greatest  following  4  doses  of 
225  r  and  least  after  ,700  r  followed  ^2  hours  later  by  200  r. 


Significance  to  Can^^er  Research;   To  get  a  better  TihdQrstanding  of  the  hlsto- 
pathokig?cai  aid  chamicai  changes  which  take  place  in  rapidly  pro- 
liferating tissue  (epithelial  lining  of  the  small  bowel)  following 
x-irradiation,  which  may  be  useful  in  tumor  therapy  by  x-irradiatlon 
and/or  chemotherapeutic  agents. 

Propbs^d  bd^ii'se  of  prdject;  To  rbuhdoff  the  program  reported  above  and  to' 
present  the  data  obtained  for' publication. 


■'■;(■;,  .1.  '■y<{'J 


«:^,'d3'i%  ]^.riJfc9.S,i:;'^  '.     • '■     /latifOi*^'^  *:• 


10.         902 

SERIAL  NO. 


PAGE  3 


U. 


BUDGET  riCTIVITY: 

RESEARCH  [YJ      ADMINISTRATION  [^ 

REVIEW  AND  APPROVAL  [^        TECHNICAL  ASSISTANCE  [^ 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEi^XTH  SERVICE,  OR  OTHER 
ORGnNISiViIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL 
FOR  THIS  PROJECT  IN  EITHER  1956  or  1957. 

Naval  Medical  Research  Institute,  National  Naval  Medical  Center, 
Ee:hesda,  Maryland, 


13. 


IF  THIS  FRO JS C.r  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS 
RESEARCH  DO^IS  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 
IDENTIFY  SUCH  RESEARCH: 


NO  ENTRIES  FOR  ITEMS  14,  15  and  16. 


fei    •U'7-i'\''i 


MmihVk  ,sit^Ua-.;i 


4  i.  k^|U  >\ 'i; 


M:  him  U  ,1-li  SM-.'iIl  H^m  8^HTVi;-t  •')j^f; 


PAGE  1 
PROJECT  REPORT 


1.  National  Cancer  Institute 2.    Laboratory  of  Physiology 

INSTITUTE  ,  LABORATORY  OR  BRANCH 


3.  Cancer  Physiology  Section  ,4»     .  5.       903 

SECTION  OR  SERVICE  LOCATION  (if  other  than  Bethesda)      SERIAL  NO. 

■  ■    ■  '        a^ts? 

6,  To  determine  factor(s)  which  produces  cirrhosis  of  the  liver  in  rats  ingesting 

a  low  protein  diet  and  exposed  to  450-500  r  total  body  x-irradiatlon. 

PROJECT  TITLE  ~ 


7,  Julius  White 

PRINCIPAL  INVESTIGATOR(S) 


OTHER  INVESTIGATORS 


PROJECT  DESCRIPTION 

Objectives:   To  determine  whether  transmethylating  agents  or  lipotropic 

substances,  when  added  to  the  low  casein  diet,  will  have  any  effect  on 
the  production  of  cirrhosis  of  the  liver  produced  by  whole  body 
x-irradiation. 

Methods  Employed;   Rats  were  fed  one  of  four  diets  differing  from  each  other 
only  in  the  amount  of  choline,  methionine  or  both.    Four  diets  were 
employed!   (a)  6%  casein  diet,  (b)  6%  casein  supplemented  with  0, 5% 
methionine,  (c)  0. 5%  methionine  and  0. 5%  choline,  and  (d)  0, 5%  choline. 
All  animals  received  450-500  r  total  body  x-irradiation. 

Major  Findings:   As  reported  in  the  last  annual  report,  an  incidence  of 

approximately  50  per  cent  of  cirrhosis  of  the  liver  was  observed  in  the 
animals  in  group  (a).    No  cirrhosis  of  the  liver  was  observed  in  the 
other  three  groups  of  animals.    Since  methionine  and  choline  are  both 
intimately  involved  in  transmethylation,  it  may  be  that  the  utilization 
of  the  low  concentration  of  methyl  donors  present  in  the  diet  of  animals 
in  group  (a)  is  influenced  by  exposure  to  x-irradiation  and  ample 
quantities  of  methionine  or  choline  counteracts  the  irradiation  effect. 


903  "'  PAGE  2 

SERIAL  NO. 


Significance  to  Cancer  Research;  In  no  case  was  carcinonia  6t  the  liver 
produced.    Yet,  the  fact  that  cirrhosis  of  the  liver  appeared  in  50 
per  cent  of  the  animals  on  the  unsupplemented  diet  suggests  that  the 
nutritional  state  of  the  animal  can  influence  the  effect  of  total  body 
x-irradiation  on  the  liver  and;  perhaps,  other  tissueis. 


Proposed  course  of  project:  None. 


10.  9Q3  PAGE  3 

SERIAL  NO. 


U.  •  •'    •  ' 

BUDGET  ACTIVITY: 

RESEARCH  (T]     ADMINISTRATION  /~7 

REVIEW  AND  APPROVAL  [~J      TECHNICAL  ASSISTANCE   [^ 


12,  ■       '         -        ■•         '      •  • 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER 
ORGANIZATIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR 
THIS  PROJECT  IN  EITHER  1956  or  1957. 

None. 


13. ^ 

IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS 


RESE/iRCH  DONE  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 
IDENTIFY  SUCH  RESEARCH. 

None, 


14»  903  ■  v<-::i^\iAlPAOE  4 

SERIAL  NO. 


15 
1  PUBLICATIONS  6THER  THAN  ABSTRACTS  FROM  THIS  PROJECT  UUKIINU 
^   CALENDAR  YEAR  19551  'v;iad  ■ 

\ V    mks  White,  Charles  C.  Cohgdon,  PhUip  W.  Davtd,  and/ Mona  S. 

Ally:  Cirrhosis  of  the  liver  of  rats  following  total  body 
x-lrradiation.    J.  Nat.  Cancer  Inst.  15:X165-1163,  1955. 


..1. 1 


'   HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT 


DURING  CALENDAR  YEAR  1955; 
None. 


PAGE  1 
PROJECT  REPORT 


1,    National  Cancer  Institute 2,    Laboratory  of  Physiology 

INSTITUTE  [  LABORATORY  OR  BRANCH 

' :,  Departmeiit  of  Biology,  Princeton 

3.    Office  of  the  Chief  4,  University.  Princeton,  N.  J.  5.        904 


SECTION  OR  SERVICE      LOCATION  (if  other  than  Bethesda)    SERIAL  NO. 

6,  Study  of  biological  Effects  of  Ultraviolet  Irradiation, ■    

PROJECT  TITLE 

7.  Harold  F.  Blum 

PRINCIPAHNyESTIGATOR(S)         ~"~"      "  " 


8,     Drs,  Elmer  G,  Butler,  J.  J,  Chang,  R.  C,  Mawe,  S.  E.  Schmidt,  and 
A,  K,  Parpart,  of  Princeton  University;  and  Dr.  J.  W,  Green  of  Rutgers 

University 

OTHER  INVESTIGATORS  '""     ~~ 


9.      PROJECT  DESCRIPTION 

Objectives;  to  study  the  biological  effects  of  ultraviolet  irradiation  with  the 
ultimate  aim  of  better  understanding  of  the  carcinogenic  action  of  that 
agent,    these  studies  aim  to  combine  an  experimental  and  theoretical 
approach. 

Methods  Employed;  The  approach  is  a  combined  theoretical  and  experimental 
one.    Using  data  available  from  our  earlier  work  on  the  induction  of 
cancer  by  ultraviolet  light,  a  mathematical  model  has  been  constructed 
which  explains  certain  aspects.    Realizing  that  any  such  model  can  only 
"  have  meaning  if  related  to  real  biological  phenomena,  experimental 
studies  of  various  effects  of  ultraviolet  radiation  on  living  systems  have 
been  undertaken.    Our  model  would  indicate  that  the  mechanism  of  cell 
division  and  its  control  are  of  particular  importance  in  this  problem, 
and  hence  the  effect  of  ultraviolet  radiation  on  this  phenomenon  has 
;      been  a  principal  preoccupation.    Our  studies  haye  extended  into  related 
.   fields  as  this  has  seemed  indicated. 


Q04  .,^,.,  PAGE  2 

SERIAL  NO. 

Major  Findings; 

^*    Studies  of  regenerationi  after  ultraviolet  radiation  In  the  larvae  of 
the  urodele  anrtphibian.   (la  coUabora'don  with  Professor  Elmer  G» 

\i:.      Butlei'j  with  the  assistance  of  J*  J.  •  Change  R«  p.  Mawe  andS,  E, 
Schmidt,)  Ws  have  been  concerned  with  qusstions  of  dosage,  and 
relationships  to  photorecovery,  as  a  necesoary  matter  of  background, 
but  oitr  mo.fit  intsrestii^  study  has; been  a  rather  extensive,  comparison 
of  regcensratlon  cf  irradiated  and  normal  limbo  after  amputation,    A 
rather  complex  situation  is  discovered  by  these  experiments:  The  rate 
of  regeneration  is  reduced  in  the  irradiated  limb;  the  degree  of  re- 
duction varies  with  the  time  at  which  the  amputation  is  performed,  and 
doea  not  follow  the  course  of  regression,    Photorecovery  is  observed^ 
illumination  for  two  days  after  the  irradiation  greatly  Increasing  the 
regenerative  capacity  toward  normal,  and  reducing  the  development 

;  of  abnprmalities  to  a  negligible  number,.  Tiiere  .seems -to  be  no  close 
correlation  bei;v/9en  regressionof  the  limb,  regeneration  of  the  limb 
or  the  incidence  of  abnormalities  in  the  regenerate}  our  experiments 
clearly  separating  thes6  different  aspects,  apparently  for  theiirst 
time,  A  manuscript  describing  these  experiments  has  been  prepared 
for  publication  in  the  Journal  of  Cellular  and  Comparative  Physiology 
and  will  be  subm.itted  for  approval  in  the  near  future, 

^      :    II,  .  Effects  of  uLtra^violet  radiation  gn  mouse  skin,    (Begun  in  coUab- 
■    oration  with  Drr  R;,  C,  iVIawe  with  the  assistance  of  Mrs,  F, 
CorA,3tance; )   In  these  STadies  it  is  planned  to  follow,  the  changes  in 
mouf^Q  skin  resulting  from  ultraviolet  radiation,  with- particular 
atte:).tIon  to  the  course  oi  hyperplasia  and  its  regression.    Analysis  of 
our  earlier  studies  on  earcincgenesis  by  ultraviolet  radiation  indicate 
that  th-ase^  events  may  have  important  bearing  on  that  process,    A 
method  for  reproducible,  quantitative  dosing  of  mouse, ears  has  been 
.worked  ov-fc  and  some  hintological  stadias  have  been  made.    More 
precise  hietological  methods  are  naoded,  and  these  are  at  present 
being  worked  out  by  Mr,  Vincent  Gregg, .  technician  to  Professor 
■-: .  Butier, ,  '^   ■!'■'-.: :-  ^  ■  I  i-   .-     ■■■■'.   ...    .     .:■;..■.'.■ 

m.   studies  of!  henroU'sis  by  ultra  violet  radiation.    The  question  of 
■r..-  I     lysis  by  this  agent  waS'  reopened  bFo^  stu;die&"on  cytolysis  of 

Arbacia  eggs,  carried  out  at,  the  Marine  Biological  I^abpratory,  Woods 
Hole,  in  the  summer  cf  1952.    (Blum,  Cook  and  Loos,  J,  Gen,  Physiol,, 
37j  313,  1954.)   Subsequently,  John  S.  Cook  undertook  studies  of 
ultraviolet  hemolysis  as  his  thesis  for  the  Ph,  D,  degree,  under  my 
supervision.    In  an  extensive  paper,  soon  to  be  published  in  the 
J.  C.  C,  P. ,  Dr,  Cook  lias  shown  two  new  things  of  particular  interest: 


904 PAGE  3 

SERIAL  NO. 


(1)  that  hemolysis  by  this  agent  is  a  second  order  process,  and 

(2)  that  ultraviolet  radiation  of  short,  wavelengths  (0,1860jli  to  0.2500|i) 
is  much  the  iiiost  effective  part  of  thfe  mercury  arc  spectrum  for 
producing  the  hemolysis.    Dr.  Cook  is  now  in  Bern,  Switzerland,  on  a 
Public  Health  Fellowship,  studying  effects  of  ultraviolet  radiation  on 
nerve  with  Prof esjsor  Alexander  von  Miirklt. 

Dr.  Cook  and  I  began  together  a  study  of  the  O,  dependence  for  ultra- 
violet hemolysis,  whi(±  was  suggested  by  his  studies,  and  had  just 
turned  up  some  unexpected  things,  when  he  left*    I  am  just  resuming 
these  studies,  after  the  Construction  of  more  adequate  apparatus.    It 
does  not  seem  wise  to  report  our  results  to  date,  because  they  need 
repetition  and  expansion,  but  at  the  present  moment  they  seem  rather 
fundamental,  and  may  perhaps  bear  on  the  (to  my  mind)  confused 
picture  of  O^  dependence  in  effects  of  ionizing  radiations, 

IV.  Studies  of  photosensitized  erythrocytes.    (In  collaboration  with 
Professor  J.  W.  Green,  Rutgers  University,  and  Professor  A.  K. 
Parpart,  Princeton  University. )  Studies  on  permeability  of 
erythrocytes  photosensitized  with  rose  bengal  and  exposed  to  visible 
light,  have  shown  that  this  treatment  results  in  increased  permeability 
to  K"^  and  Na"*"  ions,  but  does  not  alter  the  metabolism  of  glucose  by 
these  cells.    These  studies  are  being  continued.    An  abstract  is  being 
published  in  the  Journal  of  Cellular  and  Comparative  Physiology. 

V,  Polyspermia  in  the  Arbacia  egg.    An  exploratory  study  involving 
quantitative  determinations  of  polyspermia  in  the  Arbacia  egg  was 
carried  out  with  Dr.  Murray  Eden  at  the  Marine  Biological  Laboratory, 
Woods  Hole,  in  the  summer  of  1954.    The  analysis  of  the  extensive 
data  we  obtained  has  only  now  been  completed  in  Dr.  Eden's  labora- 
tory.   It  is  hoped  to  prepare  this  material  for  publication  in  the  near 
future. 

Plans  for  work  at  the  Marine  Biological  Laboratory,  Woods  Hole, 
during  the  past  summer,  were  upset  by  my  contracting  whooping 
cough  at  a  crucial  time.    This  prevented  any  extensive  experimental 
work,  so  the  summer  was  spent  chiefly  in  reading,  writing,  and 
working  up  with  Professor  Butler  the  results  of  our  investigations  on 
amphibia,  carried  out  at  Princeton  during  the  spring. 

Significance  to  Cancer  Research!  To  throw  light  on  the  relationship  of 
ultraviolet  exposure  to  carcinogenesis. 


904  PAGE  4 

SERIAL  NO. 

PfoposBd  course  of  projfect:  Experiments  designed  to  explore  further  the 
.  effects  of  ultraviolet  radiation  ©n  rejgeneratiori  are.  now' under  way. 

.    Resumption  of  study  of  the  Oj  dependence  for  ultraviolet  hemolysis 
using  more  ade(3[uate  apparatus. 

Continuation  of  studies  of  photosensitized  erythrocytes, 

the  synthiBtic  approach  to  the  problem  of  caVcinbgiBnesis  by  ultra- 
violet radiation  will  continue^ ' 


Silfiqa  '.^iii'^ritiift  m 


10,  904  PAGE  5 

SERIAL  NO. 


11.  

:'u  BUDGET  ACTIVITY; 


RESEARCH  /Xj        ADMINISTRATION  /~7 

J;a.^.;^;  jREVIEW^AND  APPROVAL   /C7         TECHNICAL  ASSISTANCE    fZ/ 


li.  ^^      _     •  _■   -■-■  — -  •  ■  ■     ■  .-■  

-  COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER 
ORGANIZATIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR 
THIS  PROJECT  IN  EITHER  1&56  or  1957. 

Department  of  Biology,  Princeton  University, 


IF  THIS- PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS 
RESEARCH  DONE  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
^' (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 
IDENTIFY  SUCH  RESEARCH: 

:    •::;•■;  ^•:-  None, '  "■'''^•''' '•'•^■' •       "  -^iio^'-^ntm'^     ^     >   v^^ 

•     ';;•:?:;■,       ■     •■..•;     .v..  .      .  ■  ■•.V'..:  ;IA    ^  .     .    ••      '.  -  ■  ^  '•  .  ^'1      ftt-  ^  ^'i  ■ 


Uh^iJA   W  i,V' 


14,  904  PAGE  6 

SERIAL  NO.  ' 


15. ;- 

PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING 
CALENDAR  YEAR  1955: 

Butler,  E.G.,.  and  Blum,  H.  F. :   Regenerative  growth  in  the  urodele 
foriBiinib  fdiiowing  ultravlQlet  radiation.    J,  Nat.  Cancer  Inst.  15: 
,       .  .  a77-e89,  1955„  ,     ;.,  .     . 

Blum,  H.  F.:    Sunburn.    In  Radiation  Biology  (HoUaender,  A.,  ed.). 
New  York;  McGraw-lUri,  1955,  Chapter  15,  pp.  487-528. 

BIum>  Hi  F.J  Ultrd  violet  ■Miakoft' and 'Cancer,    Ig  Radiatibft  Biology 
■    (HoUaender,  A.,  e4 )/  New  Yorkj  McO^  1956,      ''^ 

Chapter  14,  pp.  529-559!*  -/  ^'  ■     •     '   '      ^^ 

Blum,  H.  F.:  'Siinlight  as  an  environmental  faictor  in  Cancer  of  the  skin. 
Military  Medicine  117:  202-208,  1955. 

Blum,  H.  F,:  Timers  Arrow  and  Evolution.    2nd  Edition.    Princeton 
University  press,  1955,  Princeton. 

-    Blum,  Hii  Fi I  i  Perspectii^efe  in  Evotation.    Ameirican' Scientist  43i 
•;  ■>  ■^■■■595-610,.  1955.^-^'-^  hI  -■  hii.rr-i-'-v  ,^.:.y:i  ■'.    ■  .fji' -  ^^.  a:-:::.x 

Papers  describing  work  carried  out  under  ;my  super  Visions  i 

Zimskin,  P.  D, ,  aEid  Schisgall,  R,  M.:   Photorecovery  from 
ultraviolet-induced  pigmentation  changes  in  anuran  larvae. 
J.  Cell.  Comp.  Physiol,  45:  167-176,  1955. 

16. 

HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT 
DURING  CALENDAR  YEAR  1955. 

1.  I  gave  the  initiation  lecture  to  the  Sigma  Xi  chapter  of  the 
University  of  Vermont,  Burlington,  on  April  26,    The  title  was 
Perspectives  in  Evolution. 

2.  I  took  part  in  a  symposium  on  Biogenesis  together  with 
H,  C,  Urey,  Philip  Abelson,  Sidney  Fox  and  George  Wald, 
as  part  of  the  Centennial  Celebration  of  the  Polytechnical 
Institute  of  Brooklyn  on  May  6.    My  position  in  the  symposium 
was  that  of  summarizer. 


904  PAGE  7 

SERIAL  NO. 


3.    I  presented  a  paper  in  a  symposium  at  the  Dedication  of  the 
Armed  Forces  Institute  of  Pathology,  Walter  Reed  Medical  Center, 
Washington,  D.  C. ,  on  May  27»    The  title  was  Sunlight  as  an 
Environmental  Factor  in  Cancer  of  the  Skin. 


PAGE  1 
PROJECT  REPORT 


1.    National  Cancer  Institute  2,    Laboratory  of  Physiology 

INSTITUTE.  ;  ';■'    ■'■"^r.'---        LABORATORY  PR  BRANCH 


3.    Cancer  Physiology  Section  4, •  5»       905 

SECTION  OR  SE;RVICE     ■       LOCATION  (if  other  than  Bethesda)    SERIAL  NO 

C     Iodide  Concentra:ting.]VJfechanism  in  the  Thyroid  Gland  .      

PROJECT  TITLE     .  j~— ^-      ,  ■       •     .■  ,  ,        — 


S.  H.  Wollman 

PRINCIPAL  INVESTIGATOR(S) 


Dr.  R,  O,  Scow  of  NIAMD 
OTHER  INVESTIGATORS^ 


PROJECT  DESCRIPTION 

Objectives!   To  learn  about  the  mechanism  by  which  the  thyroid  gland  main- 
tains a  concentration  of  radioiodide  elevated  above  that  in  the  blood  and 
about  the  way  the  thyroid  radioiodide  and  iodide  concentrations  influence 
the  uptake  of  radioiodine. 

Methods  Employed;   Kinetic  studies  will  be  made  of  the  ratio  of  radioiodide 
concentrations  in  the  thyroid  gland  and  serum  and  factors  which  control 
it. 

Major  Findings;   A  study  was  made  of  the  inhibition  of  the  iodide  concen- 
trating mechanism  by  thiocyanate.    The  dependence  of  the  ratio  of  the 
radioiodide  concentrations  in  thyroid  gland  and  serum  on  the  concen- 
trations of  iodide  and  thiocyanate  in  the  same  animal  was  examined. 
The  observed  data  can  be  fit  quantitatively  by  two  qualitatively 
different  models,  an  adsorption  model  and  an  active  transport  model. 
In  these  models  iodide  complexes  with  a  substance  in  the  thyroid,  one 
type  of  site  is  involved  with  no  interaction  between  sites;  thiocyanate 
also  complexes  with  the  same  sites  and  competes  with  iodide  for  the 
sites.    The  affinity  of  the  site  for  iodide  is  twice  as  great  as  for 
thiocyanate.    The  affinity  for  thyroid  sites  is  more  than  an  order  of 
magnitude  greater  than  that  for  serum  albumin  sites.    With  the 


905  rVvvyr.':..  .^  ■;:-,-  ;  PAGE  2 

SERIAL  NO. 


-  observed  constantsy  the  adsorption  model  requires  thiocyanate  Ije. 
concentrated^    That  it  is  not  concentrated  in  rat  and  sheep  thyroids        , 
suggests  that  the  iodide  concentrating  mechanism  Is  not  an  adsorption 
but  an  active  transport  process.  ,  :.:;, 

Significance  to  Cancer  Research:  Transplantable  tumors  of  the  thyroid  gland 
in  mice  appear  to  have  a  marked  impairment  in  or  loss  of  the  iodide 
concentrating' mechanism.    This  loss  appears  directly  .responsibl^i  for 
the  loss  of  ability  of  certain  lines  of  thyroid  tumors  to  accumulate 
radioiodine.    The  above  experimental  results  are  directeid  largely  to 
provide  an  understanding  of  the  iodide  concentrating  mechanism  and 
the  factors  controlling  it,  '   . 

Proposed  course  of  project:  Further  kinetic  studies  on  the  iodide  cohcen- 
trating  mechanism  will  be  undertaken.    In  addition,  a  more  direct 
attack  will  be  made  on  the  problem  of  deternalning  the  factors  w|iich 
most  seriously  limit  the  accumulation  of  radioiodine. 


OioTfM'J"     ■  ■         "  .  •■•  •""         •  ;,      .  ■,,       ,  5       ■•.■>«-^if?:;;-^'*..Si'':-i«Pj 


y  0h 


10.  905  PAGE  3 

SERIAL  NO. 


11. 

BUDGET  ACTIVITY; 

.V  RESEARCH  flT/      ADMINISTRATION  /      7 

REVIEW  AND  APPROVAL    /~7      TECHNICAL  ASSISTANCE/      7 


12.    ___■ 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER 
ORGANIZATIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR 
THIS  PROJECT  IN  EITHER  1956  or  1957,     , 


NIAMD,  Dr.  R.  O.  Scow 


13.    

IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS 
RESEARCH  DONE  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 
IDENTIFY  SUCH  RESEARCH! 


This  project  may  complement  research  in  the  Clinical 
Endocrinology  Branch,  NIAMD. 


14.         905  PAGE  4 

SERIAL  NO. 


15.  , ^ ....        ■  .  ■ 

PUBLICATIONS  OTtlER  TfiAN  ABSTRACTS  FROM  TdlS  PROJECT  DURING 
CALENDAR  YEAR  1955; 

S,  H.  WoUman  and  R.  O.  Scow; .  Effect  of  various  goitrogens  and  of 
dose  of  ptopylthiouracU  bn  thiB  ratio  of  radibiodlde  con- 
centrations in  the  thyroid  gland  and  serum  in  mice. 
Endocrinology  56;  448-454,  1955. 

. ;  .  S.  IL  Wolirfidii  .arid  R,  Q,  ScQ^';, Effect  of  propylthiouracil  oh  the 

'  ratio  of  the  radioibdi^/s  6pA<?^".trati^^ 

serum  in  normal  ahti  hypbphysectomlzed  rats. 

Endocrinology  56;  445-447,  1955. 


16. 


HONORS  AND  AWARDS  TO'PERSONNEL  RELATING  TO  THIS  PROJECT 
DURING  CALENDAR  YEAR  1955. 


Elected  tb  the  American  Physiological  Society. 


PAGE  1 
PROJECT  REPORT 


1.    National  Cancer  Inatitute 2.    Laboratory  of  Physiology 

INSTITUTE  LABORATORY  OR  BRANCH 


3.    Cancer  Physiology  Section  4.  . 5.       906 

SECTION  OR  SERVICE  LOCATION  (if  other  than  Bethesda)    SERIAL  NO 

131 
6.     Transplantable  Thyroid  Tumors:   Production  and  I       Metabolism, 

PROJECT  TITLE 


7.     S.  H.  Wollman  .    ;  ;;vn4 


■JuaXm"  IITt^o  ■  aTlwjT)>ii  ^ 


PRINCIPAL  INVEStlGATOR(S),::S3^^o.l--:'J--^:^'  ''^I'.-J'V^  ll'^t^:^  \:/^ 


8.     Dr.  R.  0.  Scow  of  NIAMD 
OTHER  INVESTIGATORS 


9.     PROJECT  DESCRIPTION  •  f^^.mTHiPrf^^ 


Objectives:  '^?^^^i 


^a)  To  produce  transplantable  thyroid  tumors  in  the  rat  and  the  mouse^ 
(b)  To  compare  iodine  metabolism  in  thyroid  tumors  and  normal 
thyroid. 

Methods  Employed: 

(a)  Thyroid  tumors  are  being  produced  by  chronic  feeding  of  thiouracil. 

(b)  Iodine  metabolism  was  studied  with  the  use  of  radioactive  iodine. 

Major  Findings; 

(a)  We  now  have  transplantable  thyroid  tumors  in  both  mice  and  rats. 

These  tumors  will  grow  in  hosts  fed  goitrogen  free  diets. 

(b)  No  new  results. 

Significance  to  Cancer  Research:  Tumor  material  will  now  be  available  for 
radioiodine  studies  in  two  experimental  species. 

Proposed  course  of  project;  Studies  of  the  radioiodine  metabolism  of  thyroid 
tumors  in  the  rat  will  be  undertaken  this  year. 


jQ        QQg  T^cmam  yD:;!&c.vR  page  2 

•    '    SERIAL  NO. 


BUDGET  AC'TIVITV: 

...|^...RlSEAI^Ca.-.^^.,-._.,-.^-^. m    .  ADMINISTRATK)Nyo.^  :......  O^ 

REVIEW  AND  APPROVAL    [J         TECHNICAL  ASSISTANCE  £7 

-—'—-■— —        — - -- " '"'liSlWTJMm'i 

12. 


COOPERATING  UNITS  OF  TH£  PUBLIC  HEALTH  SERVICE,  OR  OTHER 
SrOANI^SI^S,  PROVmiNG  Ji;i^,  FA^^^ 


THIS  PROJECT  IN  EITHER  1956  or  1957 
Dr.  R.  O.  Scow,  NIAMD 


RESEARCH  DONE  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SE^VICE^ 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  F^pS)^. 
IDENTIFY  SUCH  RESEARCH: 

U^m  flhWjaiprojeet  may  p^s^wiW^  .?;f Sif arch  iP;  tlie;  l^appr^xqi^  |0i . ; 

Biochemistry,  NCI,  ,;i.;ii; ,,!) 


14.  906 

SERIAL  NO, 


PAGE  3 


15. 


Place  following  wording  on  front 


""'^^'  THIS  PROJECT  DURING 


of  thyroid  lobes, 
Growth  and  I^^^ 


7,  1955. 


...ijonSj  IN- 


ACTIVITIES 
OF  HEALIH 


STIKTE 


G  TO  THIS  PROJECT 


NATIONAL  INSTimrES  OF  HEALIH 
PUBLIC  HEALTH  SERVICE 
U.  S.  DEPARIMEKT  OF  HEALffi,  EDUCATION,  AND  WELFABE 


,  „  "'     '  ^^a  "TSCB^-rflJ  tDM^Cm'i  PAGE  2 

lu.        yuo 

SERIAL  NO. 

u.      "•  '•■  • •  •-    •     •  •  •••'•• — 

BUDGET  A'JTIVITY: 
'  ^''''"  REVIEW  AND  APP^VALO  ASSISTANCE    fj 

i  t'  i 

■■^•^"^^'"'"^'  ^'"-  ■  ■•  ^-  -— --^ — ~ - '^swf''^M:ms 


^^*  -rnnpyY<ATING  UNITS  OF  TH£  PUBLIC  HEALTH  SERVICE,  OR  OTHER 


li 


THIS  PROJECT  IN  EITHER  1956  or  1957. 
Dr.  R.  O.  Scow,  NIAMD 


I 


13. 


IF  THIS  PROJECT'  KESEMiiLfi^,  ^"MPLKMi;OT^^;J^KALljEI|.^,,       , 
RESEARCH  DONE  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
fw™T  mTERCHANGE  OF  PERSONNEL,  FACILITIES  OR.,,^WpS), 
IDENTIFY  SUCH  RESEARCH: 

i^;^^.  r1?tots(ipr^e6tirnay  ij^S^w^^i^e^airph  ln:the;%a^qr^qryiO^,:; 
Biochemistry,  NCI.  ,;;;-:  ,;,ii 

jiis-i"?.  hiii;,  ^M\>->i  i\i*'xi  !,d  'y^tiotviiiKi  sua-- ,^r!.s  •v'f:7?,?«t-;i!K.i^fj.i?  rtij<.;i  %'mt  3W    In^) 


L4,  906  PAGE  3 

SERIAL  NO. 


15. 

PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING 


CALENDAR  YEAR  1955 

S.  H.  Wollman  and  R,  O.  Scow:   Comparison  of  thyroid  lobes, 
autotransplanted  and  in  situ,  in  the  rat:   Growth  and  I^^^ 
uptake,    J,  Nat.  Cancer  Inst.  15:  943-947,  1955. 


16. 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT 
DURING  CALENDAR  YEAR  1955. 


None 


,  k 


PAGE  1 
PROJECT  REPORT 


,     National  Cancer  Institute 2,      Laboratory  of  Physiology 

INSTITUTE  "  LABORATORY  OR  BRANCH 


I.    Cancer  Physiology  Section ; 4^        5.        907 

SECTION  OR  SERVICE  LOCATION  (if  other  than  Bethesda)    SERIAL  NO. 

131 
J.     Autoradiographic  Localization  of  I       in  Normal  and  Neoplastic  Thyroid  Tissue 

PROJECT  TITLE 


K     S.  H.  WoUman 

PRINCIPAL  INVESTIGATOR{S) 


J,     Mr.  1.  Wodinsky,  Laboratory  of  Chemical  Pharmacology,  NCI 
OTHER  INVESTIGATORS 


).     PROJECT  DESCRIPTION 

Objectives:  ,     ;    ^ 

(a)  To  locate  the  sites  of  high  concentration  of  radioiodide  in  the 

thyroid  gland, 

(b)  To  locate  the  site  at  which  the  binding  of  radioiodine  occurs  in  the 

thyroid  gland,  and  to  determine  changes  in  the  localization  of 
the  I^^^  after  binding. 

Methods  Employed;   High  resolution  autoradiography. 

Major  Findings;   No  new  results  this  year. 

Significance  to  Cancer  Research;   Localization  of  I^^^  by  autoradiographic. «, 
techniques  is  Important  in  understanding  of  mechanism  of  uptake  of  I 
by  thyroid  gland  and  thyroid  tumors. 

Proposed  course  of  project;   To  be  continued. 


■10-      907  .  r^mm  xo-iLOH^  page  2 

SERIAL  NO. 


BUDGET  ACTIVITY: 

RESEARCH  /S7      ADMINISTRATION  O 

REVIEW  AND  APPROVAL  £[7        TECHNICAL  ASSISTANCE    /~7 


■12.  '  '-■'.-  ■    '•      I    }  -^ 

COOPERATtNti  UtJlTS  Ot"  The  Pt)6LlC  HEALtH  SfiftVlCfi,  OR  OTHER 


ORGANIZATIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL  FOR 
THIS  PROJECT  IN  EITHER  1956  or  1957, 

Laboratory  of  Chemical  Pharmacology,  NCI, 


J3.  ^ 

If  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS       , 
RESEARCH  DONE  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUJpS),  ; 
IDENTIFY  SUCH  RESEARCH: 

None  •"      '         ...    -  j..f;^,..;  ctt-'i-j^iii 


J  ;!;■*. !u'.'.-..i    ':>••>.  ';-;.;      .^itaj,Oi..  .'   '        '   .'     -,'   V'iH. 


14.  907  PAGE  3 

SERIAL  N0» 


15. 

PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  I^ROJECT  DURING 


CALENDAR  YEAR  1955 


S,  H.  Wollman  and  I.  Wodinsky:   Localization  of  protein-bound  I  ^ 
in  the  thyroid  gland  of  the  mouse.    Endocrinology  56: 
9-20,  1955. 


16. 


HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT 
DURING  CALENDAR  YEAR  1955. 


None 


PAGE  1 
PROJECT  REPORT 


I.     National  Cancer  Institute 2.     Laboratory  of  Physiology 

INSTITUTE  LABORATORY  OR  BRANCH 


.  Cancer  Physiology  Section  4. .  '  -    ,■:,.-:  -., 5.        908 

SECTION  OR  SERVICE  LOCATION  (if  other  than  Bethesda)    SERML  NO. 


5.     Theoretical  Analysis  of  Carcinogenesis  Based  on  the  Mutation  Hypothesis 
PROJECT  TITLE  ~ 


I.     S.  H.  Wollman .  .  ■;  ■;.    ■ 

PRINCIPAL  INVESTIGATOR(S) 


OTHER  INVESTIGATORS 

).   'PROJECT  DEeJCRIPTION>     '.  ;'   ■/;;:•:  :  :  :  V    'ri-  .  f  ;     ; 

Objectives;  ^  To  aid  in  the  test  df  a-  possible  mechanism;  of  qar-cinogeneais. 

Methods  Employed;    A  simple  quantitative  model  was  designed.    Predictions 
of  the  model  were  compared  with  dose-respons.e  data  in  experimental 
carcinogenesis. 

Major  Findings;    A  model  was  designed  which  avoided  assumptions  about  the 
growth  rates  of  tumors,  and  instead  involved  total  incidence  of  tumors 
of  a  particular  type.    Tests  of  the  analysis  do  not  appear  possible  at 
present  because  of  the  absence  of  suitable  data.    The  suitability  of  most 
data  is  impaired  by  complications  which  are  a  consequence  of  the 
methods  of  administering  carcinogens,  by  difficulties  in  making  esti- 
mates of  the  number  of  tumors  produced,  and  by  dose -dependent  lethal 
effects  and  dose -dependent  indirect  effects  of  carcinogens. 

Significance  to  Cancer  Research;  Emphasizes  need  for  further  experiments 
avoiding  interpretive  difficulties  recognized  in  the  present  analysis  of 
available  data. 

Proposed  course  of  project;    This  project  has  been  completed. 


10,         908  :.•:;;:..-  PAGE  2 

SERI/vL  NO. 


li; •    -    "    '  '         -■  ■--  "■■■    ■  •■.:/. 

BUDGET  ACTIVITY: 

V    V         .       RESEARCH                              /Tk/  ADMINISTRATION                /      7 

REVIEW  AND  APPROVAL  /     7  TECHNICAL  ASSISTANCE/     7 


12. ■     ■  -     ■      '  •  ./• 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER 
ORGANIZATIONS,  PROVIDING  FUNDS,  F;.CILITIES,  OR  PERSONNEL  FOR 
THIS  PROJECT  IN  EITHER  1956  or  1957. 

None 


13.  

IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS 
RESEARCH  DONE  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
(WITHOUT  INTEjR CHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 

...  IDENTIFY  SUCH  RESEARCH: 


14.  908  PAGE  3 

SERIAL  NO. 


15*  

PUBLICATIONS  OTHER  THAN  ABSTRACTS  FROM  THIS  PROJECT  DURING 


CALENDAR  YEAR  1955 


WoUman,  S*  H,;   Comments  on  the  analysis  of  dose-response 
data  in  experimental  carcinogenesis.    J.  Nat.  Cancer 
Inst.  16:  195-204,  1955. 


16.  ^ 

HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT 
DURING  CALENDAR  YEAR  1955 

None 


>Vi\\ 


PAGE  1 
PROJECT  REPORT  _    v,> 

1,  National  Cancer  Institute        2,  Laboratory  of  Physiology 
...  .  INSTITUTE LABORATORY  OR  BRANCH 

5,  Cancer  Physlology''Section      h»     5,  909 

SECTION  OR  SERVICE  LOCATION  (IF  OTHER  THAN  BETHESDA)    SERIAL  NO 

6,  Metabolic  Fate  of  the  Histldlne  Side  Chain 

.PROJECT  TITLE  ...     .  .  "  __      [^        I    ^ 

7.  James  C .  Reid ■.■"'.'..'" . 

PRINCIPAL  INVESTIGATOR  (S)  '        "'         '■        ■   '   ' 

8.  Florence  K,  Millar  :■■■-■': 

OTHER  INVESTIGATORS 

9»   PROJECT  DESCRIPTION 

Objectives :  The  purpose  of  this  pToject  is  Ibo  acquire  more  information  on  the 
process  by  which  histldlne  is  used  f pi!'.  th6  .synthesis,  of  nucleic  acldt 

Methods  Employed:  Radioactive  carbon  is  beitig  used  as  a  tracer  to  study  the 
problem. 

Major  Findings:  Administration  of  DL-histidine  labeled  in  the  side  chain  with 
radioactive  carbon  leads  to  the  formation  of  labeled  aspartic  acid,  gluta- 
mic acid,  alanine  and  threonine.  In  addition ^  nucleotide  fractions 
derived  from  ribonucleic  acid  are  highly  radioactive.  Before  these 
results  can  be  confirmed  and  interpreted  it  is  necessary  to  resolve  the 
histldlne  so  that  the  L-form  alone  can  be  given*  A  resolution  technique 
has  been  worked  out. 

Significance  to  Cancer  Research;  The  significance  of  these  findings  to  cancer 
research  is  that  they  add  to  our  knowledge  of  the  metabolism  of  protein 
and  nucleic  acid.  Both  of  these  substances  are  important  for  the  growth 
of  cancer. 

Proposed  course  of  the  project;  During  the  coming  year  the  labeled  DL- 

histldine  will  be  resolved  and  the  L-form  will  be  used  to  confirm  and 
extend  the  findings  made  to  date.  In  particular ,  chemical  degradation 
of  labeled  metabolites  will  be  carried  out  to  gain  infoimatlon  on  the 
probable  mechanisms  of  the  transformations. 


10'   909 


SERIAL  NO . 


11. 


l^vicJ^I'jr 


PAGE  2 


BUDGET  ACTIVITY: 


RESEARCH..       .^ 
REVIEW  &  APPROVAL  £J 


ADMINISTRATION      /~J 
TECHNICAL  ASSISTANCE  £J 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  195^  or  1957. 


'^  ^'■IbjiimiAQim^vii 


None 


15. 


IF  THIS  PROJECT  RESEMBLES ,  COMPLEMENTS ,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


None 


111  •   .an 


HO  ENTRIES  FOR  ITEt©  ih,   15  and  l6« 


ifixl 


Jllw  e- 
^  aiiS  'r- 


PAGE  1 
PROJECT  REPORT 


..  National  Cancer  Institute 2.  Laboratory  of  Physiology 

INSTITUTE  LABORATORY  OR  BRANCH 

5.  Cancer  Physiology  Section  h. __^ ^.910 

SECTION  OR  SERVICE   '        LOCATION  (IF  OTHER  THAN  BETHESDAJ   SERIAL  NO, 

).  Tumor  Autoradiography. ^ ,__ 

PROJECT  TITi£ 


^  James  C.  Reid 

PRINCIPAL  INVESTIGATOR (S) 


5.  Julius  White 


OTHER  INVESTIGATORS 
).  PROJECT  DESCRIPTION 


Objectives :  It  is  desired  to  obtain  information  on  the  circulation  pattern  in 
tumors  and  the  stability  of  tumor  protein. 

Methods  Employed:  Animals  bearing  tumors  are  dosed  with  radioactive  amino 
acids  and J  after  various  periods  of  time^  the  tumors  are  removed  and 
sliced.  The  slices  are  placed  in  contact  with  photographic  film. 
After  development^  the  film  bears  an  image  vrtiich  shows  the  distribution 
of  radioactivity  through  the  slice. 

Major  Findings:  When  radioactive  glycine  is  administered  to  rats  bearing 

the  Walker  256  tumor;  isotope  is  found  in  the  tumor  tissue  within  one  hour 
but  cannot  be  demonstrated  in  10  minutes  by  the  methods  employed. 
Radioactivity  is  found  in  substantial  amount  as  late  as  11  days  after 
dosage.  Necrotic  areas  present  in  the  tumor  when  the  glycine  is  admin- 
istered do  not  become  labeled.  Conversely ^  if  an  area  becomes  labeled 
and  subsequently  becomes  necrotic  the  fixed  isotope  is  not  lost.  The 
inertness  of  the  protein  metabolism  of  necrotic  tissue  is  thus  established. 
It  is  also  desired  to  obtain  information  on  the  possibility  of  gradients 
within  the  viable  tumor  tissue.  This  is  the  major  aspect  of  the  in- 
vestigation and  bears  not  only  on  the  circulation  pattern  but  on  the 
question  of  the  degree  to  which  tumor  protein  is  in  labile  equilibrium 
with  host  protein.  No  certain  conclusions  can  be  drawn  on  this  point 
yet. 


i 


,10 

ERIAL  NO. 


PAGE  2 


Significance  to  Cancer  Research:  The  question  of  the  relationship  between 
the  nutrition  of  a  tumor  and  that  of  its  host  is  an  important  one  in 
understanding  the  uncontrolled  growth  of  tumors.  These  experiments 
represent  another  mode  of  approach  to  this  question.  The  information 
gained  is  also  of  interest  in  connection  with  the  question  of  how 
necrosis  affects  the  over-all  metabolism  of  a  tumor.  This  problem  is 
a  troublesome  one  for  investigation  of  tumor  chemistry. 

Proposed  course  of  project:  The  next  step  in  the  investigation  will  be  to 
check  the  results  obtained  with  glycine  by  repeating  the  experiments 
with  labeled  lysine.  Glycine  may  not  be  the  most  suitable  amino  acid 
for  this  work  because  it  undergoes  extensive  biochemical  side  reaction 
in  addition  to  protein  formation.  It  is  then  planned  to  extend  the 
experiments  to  other  tumors  and;  if  it  seems  necessary >  to  other  amino 
acids . 


1<^'   910  PAGE  5 

SERIAL  NO, 


11. 


BUDGET  ACTIVITY: 

RESEARCH  jT}  ADMINISTRATION       [J 

REVIEW  AND  APPROVAL  [J  TECHNICAL  ASSISTANCE  [J 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  I956  or  1957. 


None 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEIvIENTS ,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


None 


NO  ENTRIES  FOR  ITEMS  1^+,  15  and  I6. 


PAGE  1 
PROJECT  REPORT 


1.  National  Cancer  Institute 2.  Laboratory  of  Physiology 

INSTITUTE  LABORATORY  OR  BRANCH 

5,  Energy  Metabolism  Section     k-.  5'  911    

SECTION  OR  SERVICE         '    LOCATION  (IF  OTHER  THAN  BET^HESDA)   SERIAL  NO, 


6.  Energy  Metabolism  Project 


PROJECT  TITLE 

7.  A.  W.  Pratt  W.  C.  White  and  F.  K.  Putney 

PRINCIPAL  INVESTIGAl'OR(S) 

8. 

OTHER  INVESTIGATORS 

9.  PROJECT  DESCRIPTION 

Objectives :  The  overall  objective  of  this  program  remains  the  continuous,  ac- 
curate measure  of  the  energy  metabolism  of  small  experimental  animals 
under  different  experimental  conditions  for  prolonged  experimental 
periods. 

As  the  program  progresses,  certain  immediate  or  relatively  short  term 
objectives  may  be  examined  in  relation  to  accomplishment.  The  immediate 
objective  of  this  reporting  period  relates  to  the  performance  of  the 
total  proced\ires  and  specifically  the  instrumentation^  in  the  continuous 
routine  study  of  tumor-bearing  animals. 

Methods  Employed:  Not  applicable. 

Major  Findings: 

a)  Instrumentation 

At  the  end  of  the  last  reporting  period,  it  was  considered  that  the 
short  term  studies  conducted  on  the  energy  exchanges  of  rodent  experi- 
mental animals  indicated  the  need  of  further  instrumentation  on  the 
recording -measuring  equipment  if  continuous,  accurate  measureswere  to  be 
accomplished.  To  this  end,  modified  computer  techniques  were  adapted  to 
the  instrument  in  the  fom  of  an  analog  to  digital  converter  measuring 
and  recording  system.  The  new  device  (teleducer-telecomputer)  was  es- 
sentially a  multi -channel  system  which  would  allow  continuous  measure 
and  recording  of  all  the  desired  parameters, i.e.  Os  output,  COa  output, 


911 PAGE  2 

SERIAL  NO. 


radiation  chamber,  cage  and  room  temperature  comparisons,  etc.  In  addi- 
tion, we  are  afforded  the  advantage  of  being  able  to  convert  the  total 
system  to  a  fully  automatic  processing  of  the  total  data  should  such  be 
indicated.  It  is  apparent  that  the  addition  of  this  new  component 
solved  the  problem  of  continuous,  accurate  measure  of  low  value  d.c. 
potentials  over  prolonged  experimental  periods  while  affording  a  com- 
plete printed  tabulation  of  all  data.  The  new  instrument  substantially 
reduced  the  number  of  technician  man-hours  needed  for  overall  data 
handling. 

As  with  any  electromechanical  device  of  this  complexity  the  expected 
electronic,  difficulties  presented  themselves  once  the  instrument  was  in 
continuous  operation.  While  they  led  to  extensive  "down-time"  in  the 
early  application,  the  development  of  "by-pass  circuitry"  by  our  group 
has  made  the  instrument  into  a  dependable,  accurate  component  in  our 
application. 

In  summary,  it  would  appear  that  the  study  of  four  individual  animals 
for  an  average  continuous  experimental  period  of  2.5  months  each  indi- 
cate that  the  new  instruments  will  accurately  and  continuously  measure 
the  energy  exchanges  of  the  rodent  experimental  animal.  In  addition, 
three  animals  have  been  studied  in  the  Hays  instrument.  While  this  in- 
strument has  perfonned  satisfactorily,  it  is  apparent  that  only  day  to 
day  monitoring  can  be  achieved  with  this  instrument. 

b)  Animal  Findings 

The  animals  under  study  were  Osborne -Mendel  males  of  an  inbred  strain. 
The  dietary  regime  was  20^0  casein  with  added  vitamin  supplements  and  in- 
organic salts .  The  experimentaJ.  period  consisted  for  each  animal  for 
approximately  JO   to  80  days.  The  experimental  period  was  divided  into  a 
control  period,  induction  period  and  a  tumor  period.  During  the  control 
period  observations  were  made  on  the  normal  animal  and  the  energy  intake} 
energy  expenditure  was  determined  in  the  usual  fashion.  When  it  was  cer- 
tain that  the  control  period  was  adequate  in  length  of  time  to  reflect 
the  metabolic  behavior  of  the  animal,  a  Walker  256  transplant  was  ad- 
ministered by  sterile  trocar  and  the  observations  on  the  animal  con- 
tinued. The  induction  period  spanned  the  time  between  transplant  and 
the  point  when  the  tumor  was  first  palpable  in  the  animal.  After  the 
tumor  was  palpable  the  tumor  period  was  initiated  and  ended  when  the 
animal  was  sacrificed.  Maximum  growth  of  the  tumor  was  very  carefully 
regulated  in  that  no  animal  was  allowed  to  grow  a  tumor  larger  than  l8^ 
of  the  total  host-tumor  vreight. 

The  general  findings  which  are  discussed  below  reflect  the  average  be- 
havior of  tumor-bearing  hosts  studied  under  the  conditions  mentioned 
above.  Following  tvmor  transplant  the  oxygen  consumption  as  expressed 
in  liters  per  day  abruptly  rises  to  a  level  significantly  above  that 


911  PAGE  5 

SERIAL  NO. 

observed  in  the  control  period ,  reaching  a  maximum  on  the  third  or  fourth 
day.  The  maximiam  is  followed  by  a  decrease  which;  at  the  time  of  appear- 
ance of  tumor,  is  at  a  new  level  intermediate  between  the  highest  level  ob- 
served in  the  control  period  and  the  highest  level  observed  in  the  in- 
duction period.  Carbon  dioxide  production  roughly  parallels  the  oxygen 
consumption  curve.  In  the  tumor  period,  the  oxygen  consumption  level 
gradually,  but  steadily,  rises  as  the  tumor  contributes  a  greater  and 
greater  percentage  to  the  total  host-tumor  weight.  It  has  been  our 
general  observation  that  the  carbon  dioxide  production  in  the  tumor 
period  tends  to  decrease  during  the  tumor  growth  period  with  the  net 
result  of  a  fall  in  R.  Q.  and  a  significant  fall  in  the  non -protein  R.  Q. 
It  is  not,  at  the  moment,  possible  to  interpret  this  observation  in  the 
sense  of  a  real  finding.  It  isi  not  known  whether  the  fall  in  non-protein 
R.  Q.  represents  solely  an  increased  metabolic  usage  of  fat  or  whether, 
in  addition  to  the  increased  usage  of  fat,  there  is  a  superimposed 
retention  of  carbon  dioxide.  With  the  fall  in  the  value  of  the  non- 
protein R.  Q.,  the  energy  equivalent  of  calories  per  liter  of  oxygen 
consumed  falls  correspondingly,  with  the  net  result  that  the  increase 
in  oxygen  is  not  a  true  reflection  of  the  observed  increase  in  energy 
expenditure  as  a  function  of  tumor  growth.  We  have  always  observed  a 
significant  slope  in  the  increase  of  oxygen  consumption  throughout  the 
induction  and  tumor  periods.  However,  only  in  the  induction  period  does 
an  abrupt  rise  in  energy  expenditure  coincide  with  the  abrupt  rise  in 
oxygen  utilization.  In  the  tumor  period,  the  fall  in  carbon  dioxide 
produced  to  affect  a  more  gradual  or  gentle  slope  in  energy  expenditure. 

Energy  intake:  The  energy  intake  of  the  animal  during  the  control  period 
is  always  substantially  in  excess  of  the  energy  expenditure  and  the  net 
change  in  body  weight  can  be  grossly  correlated  with  the  energy  retained. 
In  the  induction  period  the  difference  between  the  energy  intake  and  the 
energy  expenditure  is  substantially  reduced,  but  the  energy  intake  remains 
in  excess  of  the  expenditure.  In  animals  growing  a  tumor,  which  at  max- 
imum is  equivalent  to  approximately  15lo   of  body  weight,  it  has  been  noted 
that  the  energy  intake  gradually  reduces  and  this  effect  can  be  seen  almost 
from  the  beginning  of  the  tumor  period. 

Energy  expenditure:  Ccsnparison  of  the  energy  expenditure  of  the  animal 
in  the  induction  period  to  the  control  period  shows  that,  following 
transplant  of  the  tumor,  an  abrupt  and  significant  increase  in  energy 
expenditure  occurs.  The  maximiim  of  this  expenditure  is  observed  on  the 
third  or  fourth  day  following  the  transplant  of  the  tvimor.  The  energy 
expenditure  then  declines,  reaching  levels  at  the  beginning  of  the 
tumor  phase  which  is  intermediate  between  the  highest  value  observed  for 
the  control  period  and  the  highest  value  observed  during  the  induction  per- 
iod. As  the  animal  experiences  progressive  tumor  growth,  the  level  of 
energy  expenditure  graduaU.y  increases.  As  the  energy  intake  decreases 
and  the  energy  expenditure  increases,  the  animal  may  achieve  a  condition 
of  isocaloric  balance.  It  has  been  our  obsei'vation  that  the  contribu- 
tion of  protein  to  the  energy  expenditure  is  roughly  constant  throughout 


911  PAGE  k 

SERIAL  WO. 


the  control  period^  induction  period  and  for  most  of  the  tumor  period. 
As  the  non -protein  energy  contribution  to  the  energy  expended  becomes 
equal  to,  or  larger  than,  the  non -protein  energy  intake,  the  slope  of 
the  protein  .energy  expended  begins  to  gradually  increase.  From  our 
observations,  it  is  clear  that  the  excess  energy  expenditure  observed 
in  the  induction  period  is  entirely  contributed  to  by  non-protein 
materialB.  In  addition,  the  increased  expenditure  during  the  tumor 
period  appears  to  be  primarily  non -protein  in  nature.  The  net  result 
being  that  isocaloric  balance  between  the  non-protein  energy  intake 
and  the  non-protein  energy  expenditure  is  achieved  very  early  after 
gross  tumor  grov/th  is,  apparent.  ,  Use  of  protein  for  energy  needs  of  the 
animal  is  sj^ared  until  a  negative  balance  in  the  non -protein  energy  oc- 
curs. It  has  been  our  observation  that  the  weight  change  of  the  tumor - 
bearing  animal  progressively  rises  as  long  as  the  protein  balance  is 
positive,  even  in  the  face  of  an  isocaloric  balance  or  perhaps  a  slightly 
negative  balance; of  the  non-protein  energy.  Pi-esvunably,  this  is  due  to 
the  retention  of  new  protein  body  weight  in  the  form  of  tumor,  and, 
in  addition,  an  extensive  hydration  of.  the  animal  body  as  the  tumor 
process  progresses.         ,,  ;     '    . 

Certain  gross  correlations  may  be  made  by  comparing  the  energy  expen- 
diture with  other  parameters.  Weight  change, in; the  non-tumor  periods  re- 
flects the  retention  of  prptein  and  non -protein  energy  in  addition  to 
■  water.  The  weight  change  during  the  tinior  period  is  primarily  dependent 
upon  the  protein  energy  balance  and  the  degree  of  hydration.  A  high  de- 
gree of  protein  sparing,  occurs  in  the  sense  that  a  significant  amount  of 
protein  will  be  retained  and  dep'osited  as  tumor,  while  the  non -protein 
energy  contributes  almost  entirely  to  the  energy  demands  of  the  animal. 
The  energy  intake  does  not. maintain  pace  vn'.th  the  increase  in- energy  ex- 
penditure observed  in  the. induction  and  tumor  periods.  On  the  contrary, 
the  anorexia  substantially  red.uces  the  animal's  energy  intake. 

Significance  to  Cancer  Research:  Study  of  the  effects  on  the  energy  metabolism 
of  a  host  animal,  follov/ing  tumor  implant  and  during  subsequent  tumor 
■  growth,  promises  to  yield  significant  information  bearing  on  host-tumor 
relationships.  The  development  of  these  new  techniques  for  these  studies 
should  allow  an  accumulation  of  whole  animal  data  which,  together  with 
the  advances  iri  intermediate  metabolism,  should  increase  our  understand- 
ing of  the  pa,thological  physiology  of  the.  tumor  process. 

Proposed  course  of  project;  We  believe  vre  Jiave  been  successful  in  accomplishing 
an  accurate  measure  of  energy  expenditure  .and  energy  XJ^ take  and,  thus,  the 
energy  retained.  Our  next  immediate  objective  will  be  the,  distribution  of 
retained  energy  into  "fat-free,  body  vreight"  and  "fat  body  weight"  for 
correlation  with  observed  weight  gain.  , 

In  addition,-  comparative  studies  on  the  body  composition  of  the  observed 
animals  at  the  time  of  sacrifice  are  being,  undertaken. 


911  PAGE  5 

SERIAL  NO. 


The  need  of  a  direct  calorimetric  measure  of  the  heat  output  of  our 
experimental  animals  during  these  prolonged  experimental  periods  is  neces- 
sary if  we  are  to  ccsupare  and  interpret  the  animal's  metabolic  behavior. 
To  that  end  we  are  beginning  pilot  construction  on  a  direct  calorimeter 
which  will  utilize  the  adiabatic  principle. 


10.   911  .  PAGE  6 

SERIAL  NO. 


11. 


BUDGET  ACTIVITY: 

RESEARCH         /xj         ADMINISTRATION       [j 
REVIEW  &  APPROVAI.  O  TECHNICAL  ASSISTANCE  [J 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUl^lDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  I956  or  1957. 


None 


15.  ^ 

IF  THIS  PROJECT  RESElvBLES,  COMPLEMEINTS ,  OR  PARALLELS  RESEARCH  DONE  ELSEl-JHERE 


IN  TI-IE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  PvESEARCH: 


None 


NO  ENTRIES  FOR  ITEMS  14,  I5  and  I6. 


PAGE  1 
PROJECT  REPORT 

1.  National  Cancer  Institute         2.  Laboratory  of  Physiology 

INSTITUTE    '  — —     .     LABORATORY  OR  BRANCH 

5»  Energy  Metabolism  Section    k,  S  912 

SECTION  OR  SERVICE  lOCATION  (IF  OTHER  THAN  BETHESDA}    SERIAL  NO . 

assiO'Lq:-: 

6,  Mass  Spectrometer'  Frggram 

PROJECT  TITl¥~        ~~'  ""  '       '  '.      '' 

7.  A.  W.  Pratt  and  Bernard  E.  Burr     .      ■  "  

PRINCIPAL  INVESTIGATOR (S) 

8. ^■_ -  ■  ■'  ■-     ■.     ■■    .      • .    .' : 

OTHER  INVESTIGATORS 


9.  PROJECT  DESCRIPTION 

Objectives;  The  instrument  is  in  continuous  routine  use  for  calibration  and 
monitoring  of  the  energy  metabolism  instruments.  Secondly,  studies  on 
diffusion  rates  of  gases  obtained  from  biological  sources  continues. 
This  study  is  particularly  concerned  with  the  deviations  that  exist  in 
the  diffusion  rate  of  these  gases  from  that  predicted  by  Graham's  Law. 
Thirdly,  pilot  experimentation  has  been  tried  out  relating  the  rate  eind 
kind  of  gases  which  are  released  from  pyrolyzed  proteins. 

Methods  Employed;  Mass  spectrometric  techniques. 

Major  Findings;  The  primary  use  of  the  mass  spectrometer  continues  as  a 

basic  reference  instrument  for  gas  analysis  of  our  energy  metabolism  in- 
struments. The  mass  spectrometer  allows  frequent  and  accurate  standardi- 
zation of  our  analytical  respirometers  and  is  primarily  responsible  for 
the  present  accuracy  and  the  continuous  performance  of  cur  new  analytical 
respirometers.  To  completely  define  the  accuracy  of  our  energy  metabolism 
measure,  it  is  necessary  that  an  extensive  performance  catalog  of  the 
mass  spectrometer  exist.  Compilation  of  these  data  will  occupy  most  of 
the  mass  spectrometer  time  for  an  extended  period. 

Studies  on  diffusion  rate  of  gases  is  progressing  very  slowly.  The  immedi- 
ate problem  at  hand  deals  with  the  deviations  that  are  observed  in  the 
diffusion  rate  of  these  gases  from  Graham's  Law.  We  are  confronted  here 
with  fundamental  problems  in  gas  behavior  and  vrhile  we  have  made  consider- 
able effort  in  relation  to  this  problem  we  have  been  unable  to  resolve  the 
questions  related  to  the  fundamental  behavior. 


PAGE  2 

912  .■r.)>H,;.  .,..;. 

SERIAL  NO. 

The  studies  from  the  pyrolysis"  products  of  P^^^^^^'^f  ,J^f  .J^^  i^""" 
Plate  to  he  discussed  at  this  moment.  It  is  apparent  that  the  study  of 
?he  diffusion  rates  and  the  study  of  pyrolysis  products  are  long-temed 
^   phJsicSche^ci.  problems  which  we  feel  will  extend  the  use^af  the  mass 
spectrometer  but  which  at  the  same -time  will  he  resolved  slowly,  •  , 

Significance  to  Cancer  Research;  It  is  hoped  hy  ^J/P^J^f  ^J^J^?J;'_f  the 
-  hiiirihj^claliSt;i?r^?rian  utilize  the  considerable  potential  of  thp 
mass  spectrometer  in  understanding  biological  functxon  and  ff-^^^^ 
composition  of  important  proteins.  The  P^?P°s^\?°^^^\°^,^f  .H^'f 
will  be  to  continue  the  pilot  experimentation  until  such  time  as  posi- 
tive findings  direct  the  development  of  the  program, 

Pro-DOsed  course  of  project:  It  is  apparent  that  the  study  of  the  diffusion 
^    rates  Slthe  study  of  pyrolysis  products  are  long-termed  physical  chem- 

ic2  pSblems  which  we  ?eel  will  extend  the  use  of  the  mass  spectrometer 

but  which  at  the  same  time  will  be  resolved  slowly. 


-:cf- 

tQ&m^  ■ 

10.    912 


SERIAL  NO. 


PAGE  3 


11. 


BUDGET  ACTIVITY: 


RESEARCH         /T/ 
REVIEl'J  &  APPROVAL  [J 


ADMINISTRATION       [J 
TECHNICAL  ASSISTANCE  [J 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHEIR  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  I956  or  195? 


None 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEM.TH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDENTIFY  SUCH  RESEARCH: 


None 


NO  ENTRIES  FOR  ITEMS  lU,  15  and  I6, 


PAGE  1 
PROJECT  REPORT 


1.  National  Cancer  Institute 2.  Laboratory  of  Physiology    

INSTITUTE  LABORATORY  OR  BRAJMCH 

5.  Energy  Metabolism  Section  k. 5,  913 

SECTION  OR  SERVICE  LOCATION "(iFOTHER  THAN  BETHESDA)"   SERIAL  NO, 

Determination  of  the  radiochemical  mechanism  of  L-raalate  decomposition 

6.  in  oxygenated,  aqueous  solutions. 

PROJECT  TITLE 


T»  A.  W.  Pratt  and  Frances  K,  Putney 

pmciPAL  investigatdrTsI 


OTHER  INVESTIGATORS 


9.  PK)JECT  DESCRIPTION 

Objectives :  To  determine  the  radiochemical  mechanism  of  L-malate  decomposi- 
tion in  oxygenated;,  aqueous  solutions. 

Methods  Employed:  Chemical  analysis  by  fluorometry,  chromatography^  and 
absorption  spectroscopy. 

Major  Findings:  It  has  been  i-eported  that  the  level  of  oxygen  tension  in  the 
ambient  air  and  thus ,  in  the  blood  of  an  experimental  animal  exerts  a 
significaoit  effect  on  the  per  cent  survival  ratio  and  survival  time  of 
irradiated  animals.  One  approach  to  the  understanding  of  this  system 
is  to  study  the  radiochemical  decomposition  of  important  biological 
compounds  in  oxygenated,  aqueous  solution.  Studies  of  x-irradiated 
malate  solutions  revealed  that  6%   of  the  decomposed  malate  was  con- 
verted to  oxalacetate  and  the  remainder  to  a-keto,  P-hydroxy  succinate 
in  the  presence  of  oxygen.  Neither  of  the  compounds  are  formed  in  non- 
oxygenated  solutions.  The  oxygen  effect  in  the  radical  reaction  is  ap- 
parent from  these  observations.  The  full  data  including  the  probable 
radiochemical  mechanisms  of  these  reactions,  which  is  now  to  be  sub- 
mitted for  publication;  clearly  indicates  the  importance  of  molecular 
oxygen  in  determining  decomposition  reactions  following  exposure  to 
ionizing  radiations. 

Significance  to  Cancer  Research:  To  aid  in  the  understanding  of  the  biologi- 
cal effect  of  ionizing  radiation. 

Proposed  course  of  project :  Completed . 


10.  £1^ PAGE  2 

SERIAL  NO . 


11. . 

BUDGET  ACTIVITY: 

RESEARCH  /x/         ADMINISTRATION       [J 

REVIEW  MD  APPROVAL  [J  TECHNICAL  ASSISTMCE  [J 


12. 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE ,  OR  OTHER  ORGANIZATIONS ,  PRO  - 
VIDING  FUNDS.  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  195^  or  1957. 


None 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS;  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  m^}FYi   SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FWros),  IDELWIi'.-  SUCH  RESEARCH. 


None 


ENTRIES  FOR  ITEMS  \\ „   15  and  l6. 


PAGE  1 
PROJECT  REPORT 


1.     National  Cancer  Institute 2.     Laboratory  of  Physiology 

INSTITUTE  LABORATORY  OR  BRANCH 


3.  Physical  Biology  Section   4.  ' '   5,        914 

SECTION  OR  SERVICE         LOCATION  (if  other  than  Bethesda)      SERIAL  NO, 


6,     In  Vivo  Measurement  of  Tumor  pH  With  the  Capillary  Glass  Electrode, 
PROJECT  TITLE 


7.     H,  Kahler 

PRINCIPAL  INVESTIGaTOR(S) 


8.     B.  Haines 


OTHER  INVESTIGATORS 


9.     PROJECT  DESCRIPTION 


Objectives;   The  object  of  this  project  is  to  determine  the  in  vivo  pH  of 
tumors  under  different  experim.ental  conditions. 

Methods  Employed:   Following  anesthesia,  three  fine-tipped  capillary 
electrodes  were  inserted  into  each  tumor  in  selected  sites,  one 
electrode  was  inserted  into  normal  leg  muscle,  and  the  pH  was 
recorded  by  automatic  equipment  over  a  period  of  18  hours.    Fine 
thermocouples  were  also  inserted  into  selected  sites  such  as  skin, 
tumor,  muscle  and  rectum,  and  the  temperatures  were  recorded  on 
another  instrument  throughout  the  experiment,    After  an  initial 
period,  an  injection  of  one  of  numerous  sugars  was  made. 

Major  Fipd'r.g3?.   The  aiid  reaction  of  tumors  follo'^^Ing  glucose  administra- 
tion if'S2m3  io  be  quite  general;  all  14  rat  tumors  studxed  chow  this 
effect 

In  a  gh'en  ";umor,  the  pH  varies  with  location  within  the  tumor, 

SignifAcanc?  to  C-m?^v  Research;  The  pH  of  a  tissue  is  one  of  its  basic 
pzc-jc^tiGf.  ana  whore  this  pH  deviates  from  tj.^.  normal  tissue  value 
ths  celxHed  etudy  of  these  changes  is  essential  to  an  understanding 
of  tumor  chemical  processes. 


914  r  ;  ;     '■.      '  i  PAGE  2 

SERIAL  NO, 


Proposed  course  of  project;   The  variation  of  pH  with  position  in  a  tumor 
suggests  it  may  be  a  function  of  the  proximity  to  blood  vessels  in 
addition  to  the  proximity  to  the  surrounding  normal  tissue.    This 
remains  to  be  seen. 

An  analysis  of  tumor  temperatures  for  information  on  energy 
characteristics  of  tumors. 


10. 


11. 


914 

SERIAL  NO. 


PAGE  3 


BUDGET  ACTIVITY:  '      ~        ~ 

RESEARCH  /T7 

REVIEW  AND  APPROVAL  .  /^ 


ADMINISTRATION  r~7 

TECHNICAL  ASSISTANCE    /"~7 


12. 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER 
ORGANIZATIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL 
FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 

None 


13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS 
RESEARCH  DONE  ELSEWHERE  IN  THE  PUBLIC  HEALTH  SERVICE 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 
IDENTIFY  SUCH  RESEARCH: 

None 


14/        914 PAGE  4 

SlmAL  NO.  ' 


15. 


PUBLICATIONS  OTHER  fHAN"ABSTlAgTS  FROM  THIS  PROJECT  DUKlJsiu 
CALENDAR  YEAR  1955: 

M.  Eden,  B.  Haines  and  H.  Kahler:   The  pH  of  rat  tumors 
measured^  vivo.    J.  Nat.  Cancer  Inst.  16t  541-556,, 
1955. 


16. 


HONORS  AND  AWARDS  TO'PEl'SONNEirRELATING  TO  THIS  PROJECT 
DURING  CALENDAR  YEAR  1955. 

None 


PAGE  1 
PROJECT  REPORT 


1.     National  Cancer  Institute 2,    Laboratory  of  Physiology 

INSTITUTE  LA BORATORy.  qk  BfeANCH        ~~~ 

-"i'X:H,iQ'i.  xy ■     ■    ■■■'■■ 

3.    Physical  Biology  S6ctiori   4. ■■   '' ^^^ '--■<■■  ^i''      915 

S E CTIO N  OR  SE RV ICE  LOCATION  (if  other  than  Bethesda)    SERIAL  NO. 

6,     Physical  Characterization  of  Cellular  Bodies;   Ultracentrifugal  Study  of 

Desoxyribonucleic  Acid  (DNA)  in  Bivalent  Ion  Solutions, 

PROJECT  TITLE  ~~~~       ~"  ■    -■  ■  ■;■  :.  .-.   ■.     '       ~T~, 


H>  Kahler 

PRINCIPAL  INVESTIGATOR(S) 


J,  Shack  and  B.  J.  Lloyd 
OTHER  INVESTIGAT(5R§" 


PROJECt  DESCRIPTION        '^  .; 

Objectives;   To  compare  the  sedimentation  characteristics  Of  DNA  in  bivalent 
and  monovalent  ion  solutions. 

Methods  Employed;   Solutions  of  different  DNA  preparations  from  normal  and 
tumor  tissue  were  centrifuged  in  various  monovalent  and  bivalent  ion 
solvents.    The  optical  photographs  were  analyzed  for  sedimentation 
velocities  and  boundary  spreading. 

Major  Findings:   DNA  has  a  higher  sedimentation  velocity  and  greater 
boundary  spreading  in  bivalent  ion  solutions  than  in  monovalent  ion 
solutions. 

Significance  to  Cancer  Research;   The  properties  of  DNA  are  of  significance 
to  genetics  and,  hence,  to  cancer.    It  is  important  to  know  not  only  the 
properties  in  monovalent  solutions  but  also  in  solutions  containing 
calcium  and  magnesium. 

Proposed  course  of  project;    Continuation  of  DNA  study,  utilizing  light- 
scattering,  ultracentrifugation,  electrophoresis,  and  electron 
microscopy. 

Continuation  of  virus  part  of  project  by  studying  morphology  of  tumor- 
inducing  viruses  sliced  with  a  microtome,  and  sectioned  viral  tissue. 


10-  915  TAm  .in  TD5i5  ■ :  ,  PAGE  2 

SERIAL  NO. 


BUDGET  ACTIVITY: 

,1:;         RESEARCH 

/x/ 

ADMINISTRATION.     ,  .^,  .. 

/  / 

REVIEW  AND  A  PPROVA  L 

n 

TECHNICAL  ASSISTANCE 

■/  / 

12. 

>;  Vu:4&rs-v/li!.-     ■:■;    .      ■,.:■, 

:  :  lM>yKtfi^O  .k'M'.?';" 

COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER 
ORGANIZATIONS,  PROVIDING  FUNDS,  FACILITIES,  OR  PERSONNEL 
FOR  THIS  PROJECT  IN  EITHER  1956  or  1957 

None 

:-■•■_■■■     •  .-..J  v-/,i  '.  ,r       ■■    .  '■■■ 

13. 


IF  THIS  PROJECT  RESEMBLES,  COMPLEMENTS,  OR  PARALLELS 
RESEARCH  DONE  ELSEWHERE  IN  THE  PUBUC  HEALTH  SERVICE  i  ^ 
(WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES  OR  FUNDS), 
IDENTIFY  SUCH  RESEARCH 


None 


^iMii^iQ  ux  B;jin£'-' 


14.  915  PAGE  3 

SERIAL  NO. 


15. 

PUBLICATIONS  OTHER  TiiAN  ABSTRACTS  FROM  THIS  PROJECT  DURING 
CALENDAR  YEAR  1955 

B.  J,  Lloyd  and  H.  Kahler:  Electron  microscopy  of  the  virus 
of  rabbit  fibroma,  J,  Nat,  Cancer  List,  15:  991-999, 
1955. 


16. 

HONORS  AND  AWARDS  TO  PERSONNEL  RELATING  TO  THIS  PROJECT 


DURING  CALENDAR  YEAR  1955 
None 


PAGE  1 
PROJECT  REPORT 

1,  National  Cancer  Institute 2.  Laboratory  of  Physiology 

"  INSTITUTE  LABORATORY  OR  BRANCH 

5.  Physical  Biology  Section ,  k,    '  ^->  ■'5>  9l6 

SECTION  OR  SERVICE  lOCATION  (IF  OTHER  THAN  BETHESDA;    SERIAL  NO 

Physical  Chemical  Characterization  and  Comparison  of  Nucleic  Aoids  .and 

6.  Nucleoproteins  of  Normal  and  Neoplastic  Tissues,   ->...   v,.  .,..  .  ,.  ;..o.,.. 

PROJECT  TITLE  ■  ,^  ii  c:.  .-.1  besB'isq:-^  Cnll 

■;..oo  "jo  aoiaJotsqo?!.,::, 

y.r  Joseph  Shack j,..^     .  .,  bt^s  ncxi-a-^-dxTss   u;- 

.:  £, PRINCIPAL  INVESTIGATOR (S}      '  i;.i:j^^>:i  ,  v.:^--.i.vV,amo-.!^uo}  • 

OTHER  INVESTIGATORS   ,  ,;j::^  o<yn   Xii>;iu-/ >;.n.t  t>'iJ-  i;:  y^-5.-  i..  e.i  sxJocfeJv 

9.  PROJECT  DESCRIPTION 

Objectives ;  The  olpjective  of  this  project  is  to  isolate  in  a  native  form  the 
nucleic  acids  and  nucleoproteins  of  various  normal  and  malignant  tissues, 
to  develop  and  apply  appropriate  methods  of  fractionating  and  characteriz- 
ing them  as  chemical  entities ^  and  to  discover  what  differences,  if  any, 
there  are  between  normal  and  malignant  tissues  with  respect  to  these 
components . 

Methods  Employed:  During  the  last  calendar  year  modified  deoxyribonucleates 
(dNA)  were  prepared  from  the  DKA  of  calf  thymus  and  strain  A  mouse 
lymphoma  L#l  by  exposure  to  heat,  acid,  alkali  and  low  salt  concentra- 
tions. The  ultraviolet  absorption  and  viscosity  behavior  of  both  the 
original  and  the  modified  DNA  were  investigated  over  a  range  of  tempera- 
tures, pH  values  and  salt  concentrations. 

Major  Findings;  The  ultraviolet  absorption  of  native  DNA  is  the  same  over  a 
wide  range  of  temperatures  and.  salt  concentrations.  The  modified  DNA's 
all  show  marked  variation  over  the  same  range  of  conditions.  These 
findings  suggest  that  the  native  nucleate  has  a  high  degree  of  structural 
rigidity  which  is  lost  on  even  mild  denaturation .  The  results  afford 
some  new  simple  criteria  for  recognition  of  the  native  state  of  DNA. 

Significance  to  Cancer  Research:  According  to  current  theories  nucleic  acids 
play  a  role  in  genetic  processes,  in  bacterial  transformations,  in  pro- 
tein synthesis,  in  virus  reproduction  and  in  the  action  of  radiation  and 
of  some  carcinogens  and  chemotherapeutic  substances.  The  importance  of 
acquiring  knowledge  of  the  nature  of  the  nucleic  acids  and  nucleoproteins 


9l6 PAGE  2 

SERIAL  NO . 


of  malignant  tissues  and  of  differences  that  may  exist  between  normal 
and  malignant  cells  is  indicated  by  these  facts. 

Proposed  course  of  project:  Recent  evidence  shows  that  the  total  nucleic  acid 
of  a  cell  consists  of  a  large  number  of  different  chemical  individuals. 
Elucidation  of  differences  among  tissues  must  be  concerned  with  study  of  .■ 
the  separated  fractions  as  well  as  with  the  total  nucleic  acid.  It  is 
planned  to  carry  out  such  fractionations  on  the  nucleic  acids  and 
nucleoproteins  of  certain  normal  and  malignant  tissues.  During  the  past 
year  exploration  and  evaluation  of  some  of  the  methods  to  be  used  in  this 
work  (chromatography,  fractional  precipitation  and  extraction)  have  been 
carried  out.  It  is  also  planned  to  carry  out  such  fractionations  on  the 
nucleic  acids  of  the  tissues  of  normal  and  tumor  bearing  animals  that 
have  received  isotoplc  tracers  in  order  to  gain  information  regarding 
the  metabolic  activity  of  the  individual  fractions  or  components  as  well 
as  of  the  total  nucleic  acid. 


®   V-  . 

s'm 

XtiTirfi- 

if^oTi^ 

s; 

*./ 

^''••- ' 

eMil.M 

-O'l'f. 

at 

■tim  '^• 

)•  J  • 

10   i- 

r^a'-:ry  ' 

10'  9X6  PAGE  5 

SERIAL  NO . 


11. 


BUDGET  ACTIVITY: 

RESEARCH         fTf  ADMINISTRATION      [J 

REVIEW  8s  APPROVAL  [J  TECHNICAL  ASSISTANCE  [J 


12, 


COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE  ,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS;  FACILITIES;  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  195^  or  1957. 


None 


15. 


IF  TEES  PROJECT  RESElyiBELS  .  COMPLEMENTS;  OR  PARALLELS  RESEARCH  DONE  ELSEV/HERE 
IN  THE  PUBLIC  HEALTH  SERVICE    (V/ITHOUT  INTERCHANGE  OF  PERSONNEL;  FACILITIES  OR 
FUNDS);  IDMTIFY  SUCH  RESEARCH: 


None 


NO  ENTRIES  FOR  ITEMS  1^^,  15  and  l6. 


VfGZ  1 

Cs-T^iiv^: .     :  -i   - .  /■.■.-.--■''  A  xblB  -project  i£  to  deteiadiie  ttos  oatore^  gjoaatltsr 
i.- :   ^'.;=:-    --;    -^    .^  i'^'.c  si'z^CDaeleasee  of  laallgnattt  ana  noiMal  tlasties 
5-1   ,:      .;.    :L.    .-:  1^     .'^,  psrtlealarly  of  ISaoee  tissoee  viioee 

'.■:--. :.-.'tz  5rT-cv^i:     I ;-. ". r ^ -. -. '.    '.r   -.-'.-..-::    '_--'!  pr€psratlOB6  of  ■body  flxiids  suit- 
iil^  f-r  5.:-:-i.-.   i::i     i.  :     -.  .zTBatic  actlcQ  OQ  pure  23ticlelc 


10.  91?  PAGE  2 

SERIAL  WO . 


11. 

BUDGET  ACTIVITY: 

RESEARCH         [TJ  ,  ADMINISTRATION       [J 

REVIEW  &  APPROVAL  [J  TECHNICAL  ASSISTA^ICE  [J 

12.  \ "      

•   COOPERATING  UNITS  OF  THE  PUBLIC  HEALTH  SERVICE,  OR  OTHER  ORGANIZATIONS,  PRO- 
VIDING FUNDS,  FACILITIES,  OR  PERSONNEL  FOR  THIS  PROJECT  IN  EITHER  195^  or  1957 


None 


15. 


IF  THIS  PROJECT  RESEi^LES,,  COMPLEMENTS,  OR  PARALLELS  RESEARCH  DONE  ELSEWHERE 
IN  THE  PUBLIC  HEALTH  SERVICE  (WITHOUT  INTERCHANGE  OF  PERSONNEL,  FACILITIES 
OR  FUNDS),  IDE]\ITIFY  SUCH  RESEAJ-'vCH: 


None 


ENTRIES  FOR  ITEMS  ll+,  15  and  l6. 


•    LIBRARY 

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