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Symposium 

twenty-five  years  of  progress 
in  mammalian  genetics 

and  cancer 


Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 


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Proceedings : 
Symposium  on 
25  Years  of  Progress  in 
Mammalian  Genetics 
and  Cancer 


I 


Koscoe  B.  Jackson  Memorial  Laboratory 
Bar  Harbor,  Maine 
June  27  to  30,  1954 


Edited  by 
Elizabeth  ShiilT  Russell 


551 


Journal    of   the   National   Cancer   Institute,    Vol.    15,   No.    3,    December    1954 
316263—54 17 


Introductory  Remarks 

Dr.  E.  B.  Wilson,  President,  Board  of  Scientific  Directors 
Office  of  Naval  Research,  Boston,  Mass. 

I  have  the  duty,  the  privilege,  and  the  pleasure  of  welcoming  you  to  this  symposium; 
I  shall  be  brief.  Let  me  say  first  that  I  am  glad  to  be  here  myself  and  glad  to  see  so 
many  of  you  in  attendance. 

Twenty-five  years  is  a  short  time  in  the  history  of  old  institutions  like  Harvard  or 
the  University  of  Cambridge  or  that  of  Bologna.  We  are  young,  but  I  venture  the 
opinion  that  in  our  25  years  we  have  discovered  new  truth  in  excess  of  that  discovered 
in  any  of  those  three  institutions  in  their  first  25  years.     And  our  future  looks  bright. 

In  the  early  days  of  this  century  when  the  subject  of  physical  chemistry  was  new, 
it  was  commonly  said  that  the  physicist,  though  making  most  precise  measurements, 
was  indifferent  to  the  constitution  of  the  materials  measured,  and  the  chemist,  while 
exercising  great  pains  to  obtain  his  materials  in  their  purest  form,  was  indifferent  to 
precise  measurements  upon  them;  but  the  physical  chemist  was  indifferent  alike  to 
the  purity  of  his  materials  and  to  the  accuracy  of  his  measurements  upon  them.  That 
period  is  now  so  far  past  that  it  is  often  the  physical  chemist  who  has  the  purest  mate- 
rials and  makes  the  most  precise  determinations  upon  them. 

Such  pejorative  comments  are  not  unfamiliar  in  the  history  of  science  in  reference 
to  new  and  interdisciplinary  fields  of  research.  At  the  beginning  of  this  century  they 
could  be  heard  about  genetics  from  old-school  biologists  of  various  sorts  when  the  sub- 
ject was  not  yet  accepted  or  even  widely  known.  But  in  especial  reference  to  the  topic 
for  this  morning's  session:  "Inbred  Strains  as  Research  Tools,"  I  may  point  out  that 
one  of  the  great  accomplishments  of  the  past  quarter  century  by  this  Laboratory  is 
its  contribution  to  a  general  recognition  that  we  must  have  pure  strains  of  animals 
with  which  to  experiment,  as  well  as  good  techniques  of  experimentation,  if  we  are  to 
get  satsifactorily  definite  biological  results. 

With  these  few  words,  I  will  stop  these  introductory  remarks  and  let  this  symposium 
get  down  to  the  business  for  which  we  are  here. 


Acknowledgment 


Grateful  acknowledgment  is  made  of  the  generosity  of  the  Rockefeller  Foundation 
in  making  this  meeting  possible. 


These  Proceedings  received  for  publication  September  10,  1954 

( 


552 


Session  I.  Inbred  Strains  as  Research 
Tools 


Chairman,  Dr.  William  S.  Murray,  Administra- 
tive Director,  Roscoe  B.  Jackson  Memorial  Labora- 
tory, Bar  Harbor,  Maine 


(This  session  is  dedicated  to  Dr.  Clarence  C. 
Little  and  Dr.  Elizabeth  Fekete) 


Speaker:  Dr.  John  W.  Gowen 

Significance  and  Utilization  of  Animal  Individuality  in  Disease  Research, 

Honoring  Dr.  Clarence  C.  Little 
Discusser:  Dr.  George  Jay 

Speaker:  Dr.  Thelma  B.  Dunn 

The  Importance  oj  Differences  in  Morphology  in  Inbred  Strains: 

Honoring  Dr.  Elizabeth  Fekete 
Discusser:  Dr.  Edwin  Murphy 


553 


Journal    of    the   National   Cancer   Institute,    Vol.    15,    No.    3,    December    1954 
316263—54 10 


Significance  and  Utilization  of  Animal 
Individuality  in  Disease  Research  *•  2 

John  W.  Gowen,  Iowa  State  College,  Ames,  Iowa 


As  the  first  speaker  on  this  program,  and  as  one  who  saw  darkly  the 
germination  of  the  ideas  that  led  to  this  notable  laboratory,  I  may  be 
pardoned  if  I  recall  some  events  which  resulted  in  its  founding.  The 
program  designates  this  symposium  as  the  celebration  of  the  25th  anniver- 
sary. But  to  me,  this  laboratory  was  conceived  and  initial  steps  were 
taken  for  its  inception  several  years  earlier  when  University  of  Maine 
students  used  to  gather  in  what  was  then  the  pine-surrounded  frame 
buildings  under  the  lee  of  Pickett  Mountain.  Here,  under  the  stimulus  of 
Dr.  Little's  personality,  these  students  became  imbued  with  an  interest  in 
biology. 

It  is  little  wonder  that,  leader  that  he  is,  this  beginning  should  have 
become  the  cradle  for,  and  the  inception  of,  a  leading  institution  in  cancer 
research.  The  wonder  is  not  in  the  inception  of  the  laboratory  but  in  its 
accomplishment.  A  well  established  laboratory  equipped  with  all  modern 
features  of  research  and  with  a  staff  of  excellent  people  is  relatively  easy 

J  to  maintain  with  able  leadership.     But  Dr.  Little  had  no  laboratory  or 

visible  means  for  one.  We  are  celebrating  the  bringing  forth  of  this 
laboratory  literally  from  the  rocks  of  Mount  Desert.  How  difficult  it  was 
to  start  from  the  bare  ground  and  build  bit  by  bit  this  magnificent 
organization,  only  Dr.  Little  and  the  group  of  scientists  who  built  with  him 
really  know.  Each  step  was  fraught  with  financial  impediments  beyond 
easy  conception  and  to  add  to  it  all,  the  laboratory  had  actually  to  rise 
from  its  own  ashes. 

The  research  emphasis  of  the  staff  is  close  to  the  theme  I  am  to  discuss — 
the  individuality  of  the  animal  and  its  significance  to  medical  research. 
The  laboratory  pioneered  the  utilization  of  this  individuality  by  the  forma- 
tion and  distribution  of  their  inbred  lines  to  the  medical  research  world. 
Their  efforts  made  the  medical  investigator  individuality  conscious.  All 
science  is  indebted  to  the  small  group  who  gathered  here  under  Dr.  Little's 
leadership  a  quarter  of  a  century  ago.  Despite  financial  depression  and 
fire,  they  contributed  much  to  science  and  to  the  opportunities  for 
research  we  now  visualize  in  this  unique  laboratory. 

The  research  I  shall  cite  deals  with  the  effects  of  this  individuality  of  host 
and  pathogen  in  infectious  disease  but  the  principles  developed  have  broad 

*  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine; 
June  27, 1954. 
2  Journal  Paper  No.  J  2570  of  the  Iowa  Agricultural  Experiment  Station,  Ames,  Iowa.    Project  No.  1187. 

555 

Journal    of    the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


\ 


556  proceedings:  symposium  on  25  years  of 

applications.     The  work  represents  the  joint  efforts  of  research  workers 
whose  investigations  have  centered  at  Iowa  State  College. 

A  wild  population,  i.e.  mice  or  poultry,  resembles  the  population  of 
human  beings  in  consisting  of  a  conglomerate  of  many  different  inheritance 
types.  The  variations  ultimately  depend  on  gene  differences  which  are 
expressed  through  differences  in  the  effects  of  allelic  series  within  loci 
and/or  in  the  interactions  of  genes  occupying  different  loci.  These  varia- 
tions among  animals  within  an  experimental  population  have  been  a  main 
cause  for  erroneous  interpretations  in  disease  research.  Yet  if  this  indi- 
viduality be  controlled  within  groups  while  allowed  full  expression  between 
groups  it  offers  a  useful  means  for  the  separation  and  analysis  of  the  factors 
pertinent  to  a  given  disease.  Several  methods  for  forming  such  popula- 
tions have  been  devised — selection  with  inbreeding,  close  inbreeding,  and 
formation  of  homozygous  strains  through  outcrossing  are  representative 
techniques.  On  the  genetic  side  homogeneity  within  groups  has  been 
emphasized  over  a  period  of  50  years  of  research.  The  early  studies 
demonstrated  differences  in  strain  reactions  to  both  spontaneous  and 
implanted  tumors  in  mice  and  rats,  and  to  such  infectious  diseases  as  those 
of  tuberculosis  in  guinea  pigs  and  typhoid,  Salmonella  typhimurium  in 
mice,  or  Salmonella  gallinarum  in  the  fowl. 

Selection  of  Species 

Selection,  at  the  species  level,  has  been  practiced  from  the  beginning  of 
active  experimental  studies  of  disease.  The  species  chosen  must  be 
receptive  to  the  disease  organism.  The  success  of  this  practice  is  known 
to  us  all.  In  recent  years  it  has  taken  another  important  step  in  the  blind 
passage  of  pathogens  through  a  series  of  animals  within  the  species  in  the 
hope  of  adapting  the  pathogen  to  the  species  so  that  the  adapted  type  may 
be  utilized  in  studies  of  the  pathology  and  immunity  connected  with  the 
disease.  This  represents  only  another  form  of  the  utilization  of  indivi- 
duality in  that  the  adaptations  observed  are  in  the  pathogen  and  are 
presumably  brought  about  by  germinal  or  somatic  segregation  and/or 
mutation. 

Selection  Within  Species 

The  selection  of  reproducible  genotypes  within  a  species  utilized  for 
experimentation  is  practiced  by  fewer  investigators.  Yet  the  variation 
between  animals  within  a  species  seriously  interferes  with  or  invalidates 
the  interpretation  of  many  experiments.  The  host  animals  vary  in  genetic 
resistance  to  constitutional  diseases,  infectious  diseases,  and  diseases  of 
unknown  antecedents.  Where  a  pathogen  is  necessary  the  individuals 
comprising  the  population  will  vary  in  their  virulences,  immunizing 
abilities,  and  other  significant  properties.  The  differences  observed  are 
hereditary.  The  factors  leading  to  the  differences  in  heredity — changes 
in  gene  frequency  in  the  contending  populations — are  mutation,  selection, 
migration,  and  random  gene  drift.  The  factors  to  be  considered  in  the 
distribution  of  the  genes  within  the  population  are  whether  the  individuals 

Journal    of    the   National   Cancer   Institute 


PKOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


557 


are  haploid,  diploid,  or  polyploid,  and  whether  sexual  fusion  occurs 
followed  by  gene  reduction,  crossing  over,  and  segregation.  Variation 
must  be  expected.  In  essence  each  animal  is  an  individual  apart  from 
every  other.  The  experiments  must  deal  with  the  individual  and  yet  have 
valid  observations  capable  of  application  to  the  whole  population. 

In  disease  research  the  pathogen  itself  may  become  the  agent  to  separate 
out  and  differentiate  between  strains.  A  population  of  mice  was  exposed 
to  intraperitoneal  inoculation  of  S.  typhimurium,  50,000  organisms  [Schott 
(i,  2),  Hetzer  (8),  Lambert  (4),  Go  wen  (5)].  Survivors  were  chosen  for 
further  breeding.  This  was  repeated  for  six  generations.  The  inoculation 
dose  was  then  raised  to  200,000  organisms  and  continued  for  eight  more 
generations.  In  the  original  population  only  18  percent  survived.  Even 
the  black  death  of  the  16th  century  was  not  that  catastrophic.  But  why 
did  18  percent  survive  when  exposure  was  uniform  throughout  the  popula- 
tion? Fourteen  generations  later,  the  population  was  uniformly  exposed 
to  an  even  greater  contagion.  Instead  of  18  percent  surviving,  93  percent 
survived.  Instead  of  survival  being  an  expression  of  individuality  sporad- 
ically appearing  in  the  population,  it  has  become  the  commonplace,  and 
susceptibility  is  even  more  the  characteristic  of  individuality.  The 
changes  in  the  resistances  of  the  successive  generations  to  S.  typhimurium 
are  shown  in  text-figure  1.  That  this  change  was  no  accident  was  shown 
by  the  fact  that  similar  experiments  with  poultry  [Lambert  (6,  7)  and 
Lambert  and  Knox  (8-10)]  led  to  like  results.  But  there  are  other  ways 
of  utilizing  the  inheritance  effects  on  disease. 


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Text-figure  1. — Changes  in  the  survival  of  successive  generations  of  mice  infected 
with  mouse  typhoid,  Salmonella  typhimurium,  as  a  consequence  of  selection  for 
resistance. 

Inbreeding  as  a  Means  of  Differentiating  and  Purifying  Types  Within 

a  Population 

A  system  of  consanguineous  matings  continued  for  a  series  of  generations 
tends  to  make  the  individuals  within  a  single  family  line  more  and  more 
alike,  the  ultimate  being  the  similarity  observed  in  truly  identical  twins, 
as  in  the  human  being.     The  pattern  of  change  is  shown  in  the  following 


Vol.   15,  No.  3,  December  1954 


558 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


GENCaTIONS 


Text-figure  2. — Direction  of  individuality  into  family  lines  through  a  system  of 
consanguineous  matings.  Dots  in  group  represent  progeny  range  in  expression  of 
character. 

diagram,  text-figure  2.  The  variation  in  reaction  of  the  individuals 
within  family  groups,  where  random  mating  is  practiced,  may  range  from 
over  the  entire  range  of  the  character  to  only  a  narrow  span  depending 
on  the  family.  There  is  no  means  of  foreseeing  what  the  family  reaction 
will  be.  This  condition  is  illustrated  in  the  families  of  the  first  generation. 
With  inbreeding  and  the  later  generations,  the  individuals  within  families 
tend  to  become  more  and  more  restricted  in  their  variations  within 
families.  The  individuals  react  alike  within  the  family  strain  so  their 
reactions  can  be  predicted.  For  all  the  families  which  may  be  developed, 
however,  the  variation  among  families  will  be  as  great  as  ever.  Nothing 
is  lost  in  this  study  of  the  disease  reaction  when  many  families  are  used. 
These  results  are  illustrated  in  the  last  generation  of  the  consanguineous 
mating  scheme  in  text-figure  2. 

Inbred  lines  of  mice  were  formed  by  20  or  more  generations  of  close 
consanguineous  matings,  brother  X  sister.  They  were  then  tested  for 
their  resistance  to  mouse  typhoid,  S.  typhimurium.  All  grades  in  typhoid 
resistance  were  observed  between  them,  but  within  each  strain  the  re- 
sistance from  test  to  test  was  fairly  uniform.  Selection  between  these 
strains  established  strains  of  known  disease  resistance  to  a  standard  dose 
of  the  disease  organism  as  shown  in  table  1. 

The  mice  of  table  1  were,  before  the  inbreeding  started,  samples  from 
the  large  population  of  laboratory  mice  probably  having  a  survival  rate 
to  S.  typhimurium  like  that  of  the  mice  shown  earlier:  15  to  20  percent. 
The  fact  that  is  striking  is  that  it  was  possible  to  select  pairs  of  individuals 


Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


559 


from  this  population  whose  25+  generation  progenies  were  differentiated 
so  markedly  in  their  natural  resistance.  The  natural  resistance  varies 
from  88  percent  in  one  strain  to  practically  zero  in  another.  Each  strain 
has  a  characteristic  level  of  resistance  which  it  has  now  maintained  over  a 
period  of  some  40  generations  without  the  parents  of  any  generation 
having  any  contact  with  the  disease.  No  selection  for  or  against  natural 
resistance  to  the  disease  was  practiced  at  any  time.  The  resistance  of  the 
strain  is  a  characteristic  of  it,  just  as  its  eye  color  or  coat  color  is  charac- 
teristic of  it,  bred  into  it  through  the  fixation  of  the  inheritance  brought 
about  by  the  genetic  breeding  methods  practiced  in  constructing  each 
strain. 

Table  1. — Natural  resistance  of  strains  of 
mice  to  BOO, 000  S.  typhi  murium  11C,  intra- 
peritoneal inoculation* 


Strain 

Total  mice 

Percent 
survived 

S 

5,179 

88 

RI 

1,623 

83 

Z 

2,958 

64 

K9 

1,732 

63 

E 

2,364 

34 

L 

2,275 

14 

Ba 

3,206 

1 

*Data  obtained  at  the  Genetics  Laboratory,  Iowa 
State  College,  over  a  period  of  15  years. 

The  sporadic  exceptions  of  the  original  population  of  mice  have  been 
expaoded  to  where  they  are  the  overwhelming  representative  types.  At 
one  end  a  major  disaster  results  from  a  few  bacteria  being  introduced  into 
the  given  mouse  strain.  At  the  other  end  the  mice  of  that  strain  receive 
the  pathogens  with  impunity.  Between  these  extremes  there  are  five 
other  strains,  each  with  a  characteristic  resistance  but  taken  together, 
spanning  the  range  from  resistance  to  susceptibility.  The  fixation  of 
these  seven  disease-resistant  types  shows  that  the  hypothesis  utilized 
was  oversimplified.  Many  gene  pairs  were  certainly  involved,  but  each 
gene  pair  was  not  equivalent.  Each  had  a  special  function  which  con- 
tributed to  resistance  or  susceptibility  to  a  greater  or  lesser  extent,  not 
uniformly  equal  extent. 

Inheritance  in  Disease  Resistance 

Crosses  of  strain  S  with  strains  L  and  Ba,  made  by  Hetzer  (3) ,  demon- 
strated again  the  individuality  of  the  strains  and  the  inheritance  of  sus- 
ceptibility and  resistance  to  this  disease,  table  2. 

The  hybrids  resemble  the  resistant  stock  in  their  reaction  to  the  typhoid 
organism,  with  the  F!  slightly  less  resistant.  The  progenies  of  crosses  of 
the  susceptible  strains  L  and  Ba  indicate  that  at  least  some  of  the  sus- 
ceptibility genes  in  these  two  strains  are  affecting  different  characters  for 
resistance.     The  results  show  the  complexity  of  the  inheritance  patterns 


Vol.    15,   No.    3,    December    1954 


560  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

that  result  in  individuality.  Individuality  in  disease  reaction  is  de- 
pendent on  many  gene  pairs.  Simple  Mendelian  ratios  are  to  be  expected 
from  hybrids  only  in  rare  cases. 

Table  2. — Survival  of  progenies  from  reciprocal 

crosses  of  resistant  strain  S  and  susceptible  strains 

L  and  Ba,  200,000  S.  typhimurium  11C 


Parents 

Progeny 
tested 

Percent 

Male 

Female 

survived 

S 

X 

L 

88 

89 

L 

X 

S 

133 

79 

S 

X 

Ba 

53 

85 

Ba 

X 

S 

40 

80 

L 

X 

Ba 

153 

20 

The  Cellular  Dependence  of  Individuality  in  Natural  Resistance  to 

Mouse  Typhoid 

Two  hypotheses  have  been  suggested  to  account  for  individuality  in 
resistance  to  a  disease.  The  hypothesis  which  has  been  followed  is  that 
inherited  genetic  factors  in  their  chance  combinations  control  the  resistant 
individuals  and  account  for  their  sporadic  appearance.  The  other  hy- 
pothesis is  that  the  individual  acquires  an  active  or  passive  immunity  or 
sensitivity.  Some  are  supposed  to  retain  a  latent  stimulus  which  is 
thought  to  keep  up  the  resistance.  These  carriers  may  spread  the  sub- 
lethal stimulus  to  their  progeny.  Evidence  indicates  that  passive  transfer 
from  mother  to  progeny  may  take  place,  but  males  are  not  able  to  transmit 
the  passive  sensitization  to  their  offspring.  The  fact  that  reciprocal 
crosses  of  the  susceptible  and  resistant  strains  react  similarly  (table  2)  is 
against  this  scheme  being  an  important  method.  Another  method  of 
testing  this  scheme  for  explaining  the  individuality  of  the  reactions  to 
disease  (11)  consists  of  crossing  two  strains  of  mice,  the  S  and  L  strains  of 
table  2,  which  have  been  differentiated  in  resistance  and  also  carry  coat 
colors  by  which  the  strains  and  their  hybrids  may  be  distinguished.  The 
S  animals  have  white,  the  L  silver,  and  the  hybrid  black  fur.  One  L 
female  is  mated  to  both  S  and  L  males  in  the  same  estrus.  Such  a  female 
may  have  both  L  and  hybrid  progeny  in  the  same  litter.  These  progeny 
are  subjected  to  all  the  common  environmental  influences  from  the 
mother  and  within  the  same  uterus.  If  the  acquired  immunity  scheme 
was  correct  the  pure  L  silver  young  and  the  black  hybrids  should  be 
equally  susceptible.  The  figures  show  that  they  are  not.  The 
silver  progeny  were  all  susceptible  while  the  hybrid  black  progeny  gave 
the  typical  hybrid  reaction  of  47  percent  resistant.  Curves  showing  the 
progress  after  infection  are  equally  distinct.  The  individuality  that  has 
been  concentrated  into  these  strains  through  genetic  techniques  represents 
rare  inheritance  patterns  observed  as  sporadic  types  in  the  original 
populations.  The  question  arises,  what  are  the  attributes  that  cause 
these  strains  to  be  so  dissimilar  in  their  susceptibility  to  this  disease? 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  561 

Biological  Characteristics  of  Significance  to  Resistance  and  Suscepti- 
bility 

The  problem  of  what  makes  one  susceptible  or  resistant  concerns  us  all. 
The  data  presented  earlier  show  that  the  differences  are  dependent  on 
many  gene  pairs.  Of  the  characters  differentiating  individuals,  some  are 
commonplace,  as  weight  and  growth. 

The  influence  of  weight  and  growth  on  resistance  to  S.  typhimurium  has 
been  investigated  for  the  strains  of  table  1  [Grahn  {12-15)].  The  weights 
and  growths  from  40  to  60  days  of  age  were  investigated.  The  mice  were 
infected  at  60  days  of  age.  The  strain  showing  the  greatest  survival  is  of 
intermediate  weight.  Below  and  above  this  strain  are  body  weights  of 
the  most  susceptible  and  the  next  most  susceptible  strains.  The  strain 
showing  second  highest  resistance  has  the  greatest  weight,  while  the  strain 
which  is  the  third  most  susceptible  is  noticeably  smallest.  About  40 
percent  of  the  total  variation  in  body  weight  is  due  to  genetic  differences. 
Another  20  percent  is  attributable  to  sex  influences.  The  weights  and 
growth  patterns  prior  to  their  infection  are  unique  attributes  of  these 
strains  but  have  little  importance  to  natural  resistance. 

Internal  organ  characteristics  may  be  related  to  individuality  of  the 
different  strains  and  to  their  disease  resistances.  Mice  were  examined  for 
strain  differences  in  heart,  kidney,  liver,  spleen,  and  testis  weights. 
Genetic  differences  account  for  some  40  percent  of  the  total  variation  in 
the  weights  of  these  organs.  For  the  kidneys  another  40  percent  was 
accounted  for  by  sex  differences.  The  livers,  spleens,  and  hearts  showed 
little  sex  effects. 

Hearts,  kidneys,  and  livers  of  larger  size  favored  survival.  Spleen  size 
was  indifferent.  The  masses  of  the  hearts  and  livers  are  more  indicative 
of  resistance  than  the  relative  sizes  of  these  organs.  For  the  kidney  both 
the  absolute  and  relative  weights  are  significant  in  the  prognosis  of  the 
disease  outcome.  The  spleen-weight  :  body-weight  ratio  has  a  high 
negative  correlation  with  the  resistance  of  five  strains  but  the  correlation 
is  broken  by  the  behavior  of  the  S  mice. 

Resistance  to  body-weight  change  during  infection  is  another  index  of  the 
general  well  being.  In  S.  typhimurium  infection  by  200,000  of  11C  or- 
ganisms, the  first  4  days  following  exposure  are  most  critical. 

During  the  course  of  the  disease  both  resistant  and  susceptible  mice  lost 
weight  during  the  4  days  following  infection  [Thompson  (16)].  Normal 
body  weight  for  age  was  regained  by  the  resistant  mice  generally  by  the 
7th  day.  These  weight  changes  were  correlated  with  the  strains'  natural 
resistances.  The  weight  changes  have  real  prognostic  value  but  may  be 
considered  a  result  rather  than  a  cause  of  the  natural  resistance. 

Change  in  strain  spleen  weights  with  the  progress  of  the  typhoid  disease 
(16)  show  changes  in  spleen  mass  which  are  characteristic  of  each  strain. 
The  course  of  the  disease  was  marked  by  enlargement  of  the  spleen  in  all 
strains.  The  E  strain  showed  least  enlargement — 1.75  times  the  original 
size.  The  Z  and  K  strains  showed  the  greatest  increase — 2.6  times. 
The  mean  spleen  weight  of  the  four  resistant  strains  doubled  by  the  4th 

Vol.    IS,   No.   3,   December    1954 


562  proceedings:  symposium  on  25  years  of 

day  and  had  quadrupled  by  the  14th  day  of  infection,  but  these  changes 
were  not  correlated  with  the  differences  in  the  strain  resistances  to  typhoid. 

Humoral  Elements  in  Individuality  of  Disease  Resistance 

Circulating  antibodies  and  the  phagocytic  cells  of  the  blood  have  become 
major  considerations  to  all  problems  of  disease,  however  without  much 
consideration  of  individuality  in  response.  The  blood  volumes  of  mice 
vary  over  a  rather  wide  range.  When  this  variation  is  partitioned  into 
that  for  the  different  strains,  the  variation  between  strains  becomes  rather 
large  and  that  between  mice  within  strains  is  much  reduced.  Plasma 
volume  showed  a  tendency  to  rise,  the  rise  being  accompanied  by  a  fall 
in  albumin  and  serum  proteins  (16). 

The  serum  proteins  of  the  different  strains  display  unique  characteristics. 
Of  the  seven  strains  analyzed  only  the  E  strain  was  observed  to  have 
beta-1  globulin  (text-fig.  3).  The  concentration  of  this  protein  was 
sufficient  to  account  for  the  increased  serum-protein  value  found  in  this 
strain.  During  mouse  typhoid,  the  beta-1  globulin  increased  (16) ,  patterns 
4  and  6. 

The  inheritance  of  this  beta-1  protein  was  studied  in  the  cross  of  the 
E  X  S  mice  infected  with  typhoid.  Sera  were  obtained  from  the  four  kinds 
of  progeny:  The  males  and  females  derived  from  the  two  reciprocal  crosses. 
The  beta-1  peak  was  present  in  all  four  sera  indicating  dominance  of  the 
gene  or  genes  responsible  for  organizing  this  globulin. 

A  new  protein  of  a  different  type,  alpha-l  globulin,  was  observed  only  in 
the  sera  from  S  mice. 

Agglutinating  antibodies  to  S.  typhimurium  were  located  in  the  gamma 
globulins  (16).  Electrophoretic  analyses  of  the  sera  after  absorption  with 
S.  typhimurium  showed  a  gramma-globulin  decrease  from  20  percent  to  5 
percent  in  the  Z  serum  and  from  8  percent  to  3  percent  in  S  serum. 

Cellular  Elements  in  Individuality  of  Disease  Resistance 

The  leukocytes  of  the  blood  have  been  considered,  since  MetchnikofTs 
observation  in  1892  of  their  phagocytic  functions,  a  primary  mechanism  of 
disease  resistance.  In  a  population  of  mice  there  is  a  pronounced  individ- 
uality in  the  number  of  leukocytes  found  in  their  blood.  These  differences 
were  directed  into  strain  differences  through  the  genetic  techniques  used  in 
forming  the  strains.  Three  sets  of  confirming  observations  have  been 
made  on  these  strains,  two  of  which  may  be  cited — one  in  the  summer  of 
1942  by  Go  wen  and  Calhoun  (17)  on  young  mice;  the  other  by  Thompson 
(16)  in  the  winter  of  1952  on  older  mice  (text-fig.  4) .  Both  sets  of  observa- 
tions show  that  the  leukocytes  found  in  the  blood  of  these  strains  are 
closely  correlated  to  natural  resistance.  The  leukocyte  numbers  in  the 
two  groups  show  a  consistent  difference,  however,  the  numbers  being 
greater  in  the  1942  observations.  This  difference  could  be  due  to  age,  to 
venous  versus  arterial  blood,  to  season  or  to  some  unknown  cause. 

There  is  a  progressive  change  in  the  strain  leukocyte  number  with  re- 
sistance in  the  two  sets  of  data.     The  two  most  resistant  strains  have  the 

Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


563 


-< Descending 


8    -i  Q/tf     or  A 
Ascending ► 


Text-figure  3. — Electrophoretic  patterns  of  6  sera  drawn  from  normal  mice  or  mice 
infected  with  Salmonella  typhimurium.  Each  sample  contained  the  pooled  sera  of 
at  least  10  female  mice. 

Pattern  1 :  Strain  E — normal  serum. 

Pattern  2:  Strain  E — serum  drawn  2  days  after  inoculation  of  2  X  105  typhoid 
bacteria. 

Pattern  3:  Strain  E — serum  drawn  3  days  after  inoculation. 

Pattern  4:  Strain  E — serum  drawn  4  days  after  inoculation. 

Pattern  5 :  Hybrid  (S  X  E)  serum  drawn  4  days  after  inoculation. 

Pattern  6:  Strain  E — serum  drawn  21  days  after  inoculation. 
The  beta-1  peak  was  observed  only  in  strain  E  and  its  hybrid  progeny.  In  normal 
serum,  the  concentration  of  this  globulin  was  low  and  its  resolution  was  variable. 
However,  during  mouse  typhoid,  this  beta-1  globulin  increased  to  twice  its  control 
level.  Electrophoretic  analyses  of  4  sera  drawn  from  hybrid  (E  X  S)  mice  at  this 
time  indicated  that  the  gene  or  genes  for  this  trait  were  dominant.  From  Thomp- 
son's figure  6. 

largest  leukocyte  numbers;  Z  and  K  strains  less,  and  the  E  strain  still  less. 
The  most  susceptible  strains,  L  and  Ba,  have  fewest  leukocytes  of  all. 

The  strain  differences  in  leukocytes  extend  into  the  changes  accompany- 
ing the  course  of  the  disease  (16).  The  leukocytes  of  the  bloods  of  all 
strains  show  a  reduction  in  number  as  the  disease  becomes  more  acute, 
the  greatest  change  coming  on  the  second  day  after  infection.  The  strains 
with  the  larger  leukocyte  numbers  retain  their  initial  advantages  through- 
out the  course  of  the  disease.  As  Thompson  points  out,  it  is  difficult  to 
reconcile  decreases  in  lymphocytes  with  differential  increases  in  the  glob- 


Vol.    15,   No.    3,   December    1954 


564 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


RELATION  BETWEEN  STRAIN  RESISTANCE 
AND  LEUCOCYTE  NUMBERS  OF  STRAIN    BLOOD 


20 


LJ 

Q. 

<A 

O 

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o 

X 


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15 


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GOWEN  AND  CALHOUN 
1942 

THOMPSON   1952 


l_l 


Bo       L  E  KZ  RIS 

STRAINS  ARRANGED  ACCORDING  TO 
THEIR  RESISTANCE  TO  S.  TYPHIMURIUM 

Text-figure  4. — Relationship  between  strain  resistance  and  number  of  leukocytes  in 
mouse  blood.  Solid  line — Gowen  and  Calhoun,  1942  (17);  dotted  line — Thompson, 
1952  (16). 

ulins  of  the  same  bloods.  Differential  rates  of  recovery  of  leukocyte 
numbers  in  the  bloods  of  the  different  strains  became  evident  after  the 
disease  had  progressed  for  1  week.  The  S  strain  followed  by  the  El  re- 
covered first.  The  strains  of  intermediate  resistance  recovered  more 
slowly.  Counts  of  the  different  kinds  of  leukocytes  were  not  important. 
It  was  as  if  the  leukocytes  came  from  one  common  stem-cell  and  that  it  was 
the  capacity  of  these  cells  to  divide  to  form  large  numbers  of  leukocytes 
when  necessary  that  was  important. 

The  interpretations  presented  above  are  subject  to  further  analysis  by 
means  of  X-ray  irradiation  of  the  animal.  These  observations  emphasize 
the  careful  consideration  that  should  be  given  to  any  X-ray  treatment. 
Gowen  and  Zelle  (18)  X-rayed  789  mice  belonging  to  our  6  different  strains 
with  dosages  ranging  from  0  to  700  roentgens  incident  to  the  body.  All 
6  strains  reacted  in  a  comparable  manner.     Radiation  decreased  the 


Journal    of    the    National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  565 

typhoid  resistance  of  the  various  strains  at  the  same  rate  per  unit  of  dosage. 
The  numbers  of  leukocytes  of  the  different  strains  were  decreased  to  the 
same  relative  amounts  by  comparable  X-ray  dosages.  It  is  known  that 
total-body  irradiation  will  affect  many  types  of  body  cells.  It  cannot, 
therefore,  be  concluded  that  the  leukocytes  are  the  only  cells  which  have 
been  affected  and  that  they  alone  are  responsible  for  the  changes  in  re- 
sistance. Rather  they  are  an  index  to  what  is  taking  place  in  cells  of  like 
X-ray  susceptibility  throughout  the  body.  The  X-ray  treatments  reduced 
the  typhoid  resistance  of  the  animal  and  the  numbers  of  its  leukocytes 
proportionally  over  the  full  dosage  range.  While  the  leukocytes  are  not 
the  only  cells  affected,  these  facts  support  the  conclusion  that  natural 
disease  resistance  is  dependent  on  cellular  function  and  on  numbers  of  the 
phagocytic  cells.  These  relations  furnish  independent  evidence  for  the 
conclusions  reached  by  other  methods. 

Parenthetically  a  large  or  small  number  of  leukocytes  does  not  surely 
lead  to  resistance  or  susceptibility  to  S.  typhimurium.  Weir  (19,  20)  has 
experiments  in  progress  in  which  he  selectively  bred  for  high-  and  low- 
leukocyte  numbers  of  the  blood.  After  several  generations  the  two  selec- 
tions had  diverged  considerably  in  leukocyte  numbers  of  the  later  genera- 
tion mice.  Tests  of  these  lines  for  resistance  to  S.  typhimurium  indicated 
that  if  they  differed  in  resistance  it  was  toward  the  lower  leukocyte  line 
being  more  resistant.  The  explanation  of  this  result  is  not  evident  but 
could  be  a  difference  in  the  quality  of  the  cells — as  appears  for  the  macro- 
phages cited  in  the  next  paragraph. 

Other  fixed  cells  of  the  body,  such  as  the  macrophages  of  the  liver  and 
spleen,  were  shown  to  play  a  significant  role  by  Oakberg  (21).  In  suscept- 
ible mice,  bacteria  are  readily  ingested  by  the  macrophages  and  large 
numbers  of  them  may  be  observed  within  these  cells.  These  bacteria 
appear  normal,  have  good  staining  properties  and  appear  to  be  reproducing 
normally.  In  genetically  fully  susceptible  mice  the  bacteria  seem  to 
increase  within  the  macrophages  to  the  point  where  they  may  break  out 
of  the  cell  and  become  new  foci  of  infection.  In  resistant  mice  it  was 
difficult  to  demonstrate  macrophages  containing  ingested  bacteria.  This 
was  only  possiWe  when  the  dose  given  was  100  times  that  received  by  the 
susceptible  mice.  In  these  cases  the  bacteria  do  not  stain  well  and  their 
cell  walls  appear  ragged .  It  appears  as  though  the  macrophages  of  the 
resistant  lines  have  a  highly  effective  digestive  enzyme  which  rapidly 
destroys  the  ingested  <S.  typhimuriums,  whereas  the  enzyme  is  in  reduced 
amounts  or  absent  in  the  macrophages  of  the  genetically  susceptible  mice. 

The  liver  cells  of  the  resistant  strains  will  perform  the  vital  functions 
in  the  glycogen  cycle  and  in  fat  synthesis  even  in  the  presence  of  large 
lesions,  whereas  the  liver  cells  of  the  susceptible  mice  will  not.  Resistant 
strains  may  show  extensive  lesions  of  the  liver  and  survive,  whereas  the 
susceptible  strains  will  die  without  any  clinically  evident  damage  to  that 
organ.  By  contrast,  the  naturally  resistant  mice  show  but  little  damage 
to  their  spleens  although  the  disease  may  be  severe.  Susceptible  mice,  on 
the  other  hand,  ordinarily  display  noticeable  lesions  in  this  organ.     These 

Vol.    IS,   No.   3,   December   1954 


566  proceedings:  symposium  on  25  years  of 

observations  again  call  attention  to  the  capacities  residing  within  the  cells 
which  are  of  significance  to  the  disease  resistance.  The  liver  cells  of  re- 
sistant mice  are  able  to  wall  off  the  large  necrotic  lesion,  block  off  the 
spread  of  the  S.  typhimurium,  and  neutralize  any  released  endotoxins  that 
may  be  formed — thus  allowing  the  remaining  tissue  to  perform  its  vital 
functions. 

Individuality  of  the  Genotypes  for  Resistance  to  Different  Diseases 

The  question  arises,  is  this  condition  one  where  the  natural  resistance 
extends  to  one  or  all  diseases?  does  the  individual  have  an  over-all  constitu- 
tion? or  is  the  constitution  specific  for  each  disease?  This  question  was 
investigated  by  Gowen  and  Schott  (22)  for  diseases  due  to  Salmonella 
typhimurium,  pseudorabies  and  the  antigenic  poison  ricin.  The  genes 
required  for  resistance  or  susceptibility  to  one  disease  were  independent  of 
those  required  for  resistance  to  another.  Webster  (23)  confirmed  this 
observation  for  the  independence  of  resistance  to  louping  ill  and  to  typhoid. 
For  bacterial  species  that  are  related  taxonomically,  the  natural  resistance 
to  one  disease  carries  over  to  that  due  to  its  close  relative.  Typhoid 
resistance  is  closely  correlated  with  Pasteurella  resistance  but  less  cor- 
related with  Klebsiella  or  pneumococcus  resistance.  Similar  conclusions 
may  be  derived  from  the  data  of  Schutze,  Gorer,  and  Finlayson  (24)  for 
S.  typhimurium,  S.  enteriditis,  louping  ill,  Pasteurella  and  pneumococcus. 

Taken  broadly,  the  results  concur  in  showing  that  a  resistant  constitu- 
tion for  one  disease  is  only  likely  to  indicate  resistance  to  another  disease  if 
the  two  diseases  are  fairly  closely  related.  When  the  diseases  are  of  differ- 
ent types,  resistance  to  one  tends  to  be  independent  of  resistance  to  the 
others.  In  this  respect  inheritance  for  disease  resistance  behaves  like  any 
other  inheritance  dependent  on  many  genes — some  genes  are  independent; 
some  appear  to  be  linked  or  to  have  more  than  one  effect;  some  have 
physiological  interactions.  But  it  would  appear  that  all  are  ultimately 
separable. 

Individuality  of  the  Agent  Initiating  the  Disease 

The  bacteria  or  other  agents  (like  the  pollens  of  a  given  species)  are 
just  as  or  more  diverse  in  their  genotypes  than  the  hosts  on  which  they  act. 
They  may  have  more  significance  to  the  disease  that  is  produced  because 
they  reproduce  in  such  large  numbers.  The  small  rate  at  which  mutations 
of  existing  genes  occur  and  the  rapidity  with  which  such  mutants  may 
replace  the  existing  population  becomes  of  great  importance  in  altering  the 
character  of  the  disease  produced.  The  difference  may  result  in  changes 
in  the  growth  pattern  of  the  colony,  in  the  color,  morphology,  or  antigenic 
properties  of  the  organism.  They  may  affect  the  cultural  requirement  of 
the  organism  making  some  strains  require  a  nutrient  for  growth  which 
other  strains  can  synthesize.  The  pathogenic  characteristics  may  be 
altered  in  either  the  direction  of  greater  or  lesser  virulence.  The  effects 
of  these  changes  on  disease  expression  have  been  under  study  in  our 
laboratory  for  some  20  years  for  such  diseases  as  tobacco  mosaic,  corn 

Journal    of   the   National   Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  567 

wilt,  and  typhoids  of  both  mouse  and  fowl.     The  results  are  concordant. 

Changes  occur  and  may  readily  be  established  in  all  of  the  pathogens 
involved.  The  new  types  may  appear  at  any  time  during  the  experiment 
and  may  cover  a  wide  range.  With  selection  it  is  possible  to  establish 
many  of  the  new  forms  as  true  breeding  types.  In  a  given  environment 
competition  may  exist  resulting  in  rapid  replacement  of  the  unfavored 
types.  Changes  in  virulence  as  dependent  upon  the  inherited  bacterial 
constitution  have  been  examined  by  searching  out  phenotypic  variants 
in  originally  pure  stocks  [Zelle  {25),  Lincoln  and  Gowen  {26),  Go  wen 
{27),  Plough,  Young,  and  Grimm  {28),  Gowen,  Stadler,  Plough,  and 
Miller  {29)].  The  mutant  pheno types  are  represented  by  changes  in  color 
or  morphology  of  the  colonies,  antigenic  types  of  the  organism,  ability  to 
synthesize  amino  acids  or  other  metabolites  from  an  energy  source  and 
simple  salts. 

The  pathogen's  ability  to  initiate  a  disease  in  a  host  is  rather  highly 
specific.  As  attained  in  nature  this  property  represents  some  chance 
combination  of  genes  which  appeared,  was  preserved,  and  improved  upon 
by  selection  during  successive  generations  of  reproduction  for  better  and 
better  gene  combinations  and  the  inclusion  of  any  of  the  more  favorable 
mutations  that  might  have  occurred  as  time  passed.  Contact  with  an 
epidemic  indicates  that  an  efficient  gene  combination  in  the  pathogen  has 
evolved.  Changes  of  a  random  sort  comparable  to  those  resulting  from 
chance  mutation  would  be  unlikely  to  improve  the  disease-producing 
ability  of  a  highly  virulent  organism.  Our  results  agree  with  this  inter- 
pretation. Mutations  of  our  highly  virulent  lines  on  the  average  led  to 
lines  of  less  virulence.  A  study  of  12  different  mutants  of  533-1 1C  re- 
quiring adenine  as  a  metabolite  illustrate  this  fact  {29).  Seven  mutants 
were  avirulent  causing  no  deaths,  three  showed  some  virulence,  two  were 
as  virulent  as  the  parent.  The  average  virulence  after  mutation  was 
less  than  the  highly  virulent  parent  type.  The  mutants  were  separated 
because  of  their  effects  on  adenine.  The  range  in  virulence  shows  that 
this  requirement  is  not  itself  the  primary  cause  of  virulence.  Rather  it  is 
some  change  which  through  chance  was  associated  with  the  change  to 
adenine-requiring  that  is  of  importance. 

Individuality  of  the  Host  and  Pathogen  in  Acquired  Immunity 

Three  factors  important  to  increasing  resistance  to  a  disease  through 
vaccination  are  common  knowledge.  The  fourth  factor  and  possibly  the 
most  important  is  not  so  generally  recognized.  In  order  of  the  emphasis 
given  them,  these  factors  are:  1)  the  dose  of  the  vaccine  administered  at 
any  one  time;  2)  the  necessity  for  vaccinations  at  successive  intervals; 
3)  the  line  of  the  bacteria  making  the  vaccine ;  and  4)  the  genotype  of  the 
host  receiving  the  vaccine. 

Clear  evidence  for  the  significance  of  the  bacterial  genotype  in  S. 
typhimurium  immunization  is  found  in  our  work  [Gowen  {SO)].  The 
typhimurium  line  which  displays  little  virulence  was  the  poorest  im- 
munizer.     The  other  two  lines  having  higher  virulence  were  also  better 

Vol.    15,  No.  3,  December   19S4 

316263—54 18 


568  proceedings:  symposium  on  25  years  of 

immunizers.  The  avirulent  line  and  one  of  the  virulent  lines  came  from 
the  third  line  by  one-step  and  by  two-step  somatic  segregation  or  muta- 
tions, respectively.  As  these  bacterial  lines  come  from  each  other  by 
mutation,  the  importance  of  even  a  gene  difference  in  the  lines'  genotypes 
is  evident. 

The  effects  of  the  hosts'  genotypes  on  their  abilities  to  immunize  like- 
wise show  individuality  [Go wen  (80,  31)].  The  6  different  strains  im- 
munize differently.  The  naturally  resistant  strains  on  first  contact  with 
the  disease  are  those  which  have  their  resistances  enhanced  most  by  im- 
munization. The  intermediate  strains,  as  measured  by  natural  resist- 
ance, are  likewise  intermediate  in  their  abilities  to  immunize.  The  most 
susceptible  strains  after  immunization  remain  more  susceptible  than  the 
other  genotypes  immunized  in  like  manner.  The  level  of  resistance  of 
each  strain  is  simply  raised  a  proportionate  amount.  In  terms  of  bacteria 
which  may  be  inoculated,  immunized  mice  can  resist  100  to  200  times  as 
many  live  virulent-strain  bacteria  as  the  unvaccinated  mice.  It  is  un- 
fortunately true  that  the  strains  most  badly  in  need  of  increased  resistance 
through  vaccination  are  the  ones  which  remain  most  susceptible. 

Summary 

The  interplay  of  two  sets  of  factors  is  important  to  determining  the  in- 
dividuality displayed  by  both  host  and  pathogen  in  disease;  those  repre- 
sented by  heredity  and  those  of  the  environment  affecting  the  expression 
of  this  inheritance.  The  inheritance  mechanism  has  been  utilized  in  the 
studies  reviewed.  From  a  population  in  which  survival  was  a  rare  spo- 
radic event,  it  was  possible  to  develop  a  population  in  which  morbidity 
and  death,  instead,  become  the  rare  circumstance.  Between  these  two  ex- 
tremes, populations  characterized  by  all  grades  of  resistance  and  suscepti- 
bility were  established.  But  little  could  be  attributed  to  either  passive 
or  active  humoral  transfer  of  resistance  from  the  parents  of  one  generation 
to  another. 

The  character  bases  for  this  range  in  disease  reaction,  as  portrayed  by 
the  correlations  of  resistance  and  susceptibility  between  inbred  lines,  give 
evidence  of  ability  to  resist  weight  change  during  the  course  of  the  disease ; 
heart,  kidney,  and  liver  size;  blood  volume  and  hematocrit  percentages; 
particular  serum  albumins  and  globulins;  leukocyte,  chiefly  lymphocyte, 
numbers;  ability  of  the  liver  cells  to  isolate  the  disease  and  allow  the  re- 
maining cells  to  function  in  normal  glycogen  and  fat  metabolism;  and 
ability  of  the  macrophages  to  ingest  and  digest  the  pathogenic  bacteria 
as  factors  of  host  constitution  significant  to  the  disease  prognosis.  On 
the  other  side,  weight  prior  to  attack  seemed  unimportant  to  natural 
resistance.  Spleen  size,  despite  its  pronounced  reaction  to  the  disease, 
could  not  be  considered  indicative  of  the  strain's  resistance.  Humoral 
elements  in  the  form  of  agglutinins  and  precipitins  were  noticeable  by  their 
absence  prior  to  infection.  The  humoral  elements  have  their  significance 
in  acquired  resistance. 

Journal   of  the  National  Cancer   Institute 


PKOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  569 

Resistance  or  susceptibility  is  shown  to  be  specific  for  the  disease  and 
host.  A  desirable  constitution  is  an  aggregation  of  many  factors  rather 
than  an  over-all  constitution  which  is  resistant  or  susceptible  to  all  dis- 
eases. Many  of  these  factors  are  hereditary,  recombining  and  segrega- 
ting as  is  expected  of  characters  under  genetic  control. 

Individuality  in  the  pathogen  may  be  generated  by  processes  compa- 
rable to  mutation.  The  breadth  of  the  effects  extend  to  varied  attributes 
some  of  which  are  basic  to  life  itself  as  invasive  power  of  the  organism  or 
metabolism  of  particular  proteins.  Basic  continuity  of  the  inheritance 
makes  the  pathogen  stable  but  mutations  can  occur,  alter  these  characters, 
and  create  new  forms.  These  mutations  may  change  the  population  of 
organisms  from  virulent  to  avirulent  or  vice  versa,  or  from  a  good  immu- 
nizer  to  a  poor,  etc. 

The  dual  nature  of  disease  reactions  is  further  clarified  in  the  template 
mechanism  observed  in  acquired  resistance.  The  genetic  constitutions 
of  host  and  of  pathogen  both  require  consideration.  Susceptible  host 
and  avirulent  pathogen  genotypes  are  required  for  repeated  attacks. 
The  interactions  involved  in  acquired  immunity  phenomena  may  explain 
many  of  the  disappointments  in  this  treatment  approach. 

This  search  for  elements  which  are  significant  to  the  expression  of  the 
typhimurium  disease  in  mice  emphasizes  that  no  one  element  is  crucial. 
Rather  resistance  is  built  upon  the  integration  of  many  elements  in  the 
physiological  well-being  of  the  organism. 

References 

(1)  Schott,  R.  G.:  The  inheritance  of  resistance  to  Salmonella  aertrycke  in  various 
strains  of  mice.     Thesis,  Iowa  State  Coll.  Libr.     1-59,  1931. 

{2)  :    The  inheritance  of  resistance  to  Salmonella  aertrycke  in  various  strains 

of  mice.     Genetics  17:  203-229,  1932. 

(3)  Hetzer,  H.  O. :  The  genetic  basis  for  resistance  and  susceptibility  to  Salmonella 

aertrycke  in  mice.     Genetics  22:  264-283,  1937. 

(4)  Lambert,  W.  V.:  Genetic  investigations  of  resistance  and  susceptibility  to  dis- 

ease in  laboratory  animals.  Rep.  Agr.  Res.,  Iowa  Agr.  Expt.  Sta.  pp.  89-90, 
1931;  91-92,  1932;  115,  1933;  142-143,  1934;  158-159,  1935;  147-148,  1936. 

(5)  Gowen,  J.  W.:  Genetic  investigations  of  resistance  and  susceptibility  to  disease 

in  laboratory  animals.  Rept.  Agr.  Res.,  Iowa  State  Coll.  Agr.  Expt.  Sta.  pp. 
158-159,  1937;  151-153,  1938;  156-160,  1939;  192-194,  1940;  171-172,  1941; 
189-190,  1942;  178-182,  1943;  204r-210,  1944;  278-283,  1945;  257-260,  1946; 
230-232,  1947. 

(6)  Lambert,  W.  V.:  Breeding  for  resistance  to  fowl  typhoid  in  poultry.     Rept.  Agr. 

Res.,  Iowa  Agr.  Expt.  Sta.  pp.  88-89,  1931;  114r-115,  1933;  142,  1934;  157-158, 
1935;  146-147,  1936. 

(7)  :  Natural  resistance  to  disease  in  the  chicken.  I.  The  effect  of  selective 

breeding  on  natural  resistance  to  fowl  typhoid.  II.  Bacteriological  studies  upon 
surviving  birds  of  the  resistant  stock  in  relation  to  progeny  resistance.  III. 
The  comparative  resistance  of  different  breeds.     J.  Immunol.  23:  229-260, 1932. 

(8)  Lambert,  W.  V.,  and  Knox,  C.  W. :  Mortality  in  chickens  following  the  feeding 

of  massive  doses  of  virulent  fowl  typhoid  bacteria.  J.  Am.  Vet.  M.A.  (new 
ser.)  73:  480-483,  1928. 

(9)  :  The  inheritance  of  resistance  to  fowl  typhoid  in  chickens.     Iowa  State 

Coll.  J.  Sc.  2:  179-187,  1928. 

Vol.   15,  No.   3,  December  1954 


570  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

(10)  :  Selection  for  resistance  to  fowl  typhoid  in  the  chicken  with  reference  to 

its  inheritance.     Iowa  Agr.  Expt.  Sta.  153:  262-295,  1932. 

(11)  Gowen,  J.  W.,  and  Schott,  R.  G.:  A  genetic  technique  for  differentiating  between 

acquired  and  genetic  immunity.     Am.  J.  Hyg.  18:  688-694,  1933. 

(12)  Grahn,  D.:  Estimation  of  the  genetic  influence  on  growth  and  organ  weight 

changes  in  mice  following  total  body  X-irradiation.     Thesis,  Iowa  State  Coll. 
Libr.,  Ames,  Iowa,  1952. 

(13)  :  Genetic  implications  of  internal  organ  weight  differences  in  inbred  mice. 

Thesis,  Iowa  State  Coll.  Libr.,  Ames,  Iowa,  1950. 

(14)  :  Genetic  variation  in  the  response  of  mice  to  total  body  X-irradiation. 

I.  Body  weight  response  of  six  inbred  strains.     J.  Exper.  Zool.  125:  39-62, 
1954. 

(15)  :  Genetic  variation  in  the  response  of  mice  to  total  body  X-irradiation. 

II.  Organ  weight  response  of  six  inbred  strains.     J.  Exper.  Zool.  125:  63-83, 
1954. 

(16)  Thompson,  S. :  Serum  proteins,  leukocytes,  and  mortality  of  seven  inbred  mouse 

strains  during  cortisone  administration  and  infection  with  Salmonella   typhi- 
murium.     Thesis,  Iowa  State  Coll.  Libr.,  Ames,  Iowa,  1952. 

(17)  Gowen,  J.  W.,  and  Calhoun,  M.  L.:  Factors  affecting  genetic  resistance  of  mice 

to  mouse  typhoid.     J.  Infect.  Dis.  73:  40-56,  1943. 

(18)  Gowen,  J.  W.,  and  Zelle,  M.  R.:  Irradiation  effects  on  genetic  resistance  of 

mice  to  mouse  typhoid.     J.  Infect.  Dis.  77:  85-91,  1945. 

(19)  Weir,  J.  A. :  The  nature  of  genetic  resistance  to  infection  in  mice.     (Abstract.) 

Rec.  Genet.  Soc.  America:  79,  1952. 

(20)  Weir,  J.  A.,  Cooper,  R.  H.,  and  Clark,  R.  D.:  The  nature  of  genetic  resistance 

to  infection  in  mice.     Science  117:  328-330,  1953. 

(21)  Oakberg,  E.  F.:  Constitution  of  liver  and  spleen  as  a  physical  basis  for  genetic 

resistance  to  mouse  typhoid.     J.  Infect.  Dis.  78:  79-98,  1946. 

(22)  Go  wen,  J.  W.,  and  Schott,  R.  G. :  Genetic  constitution  in  mice  as  differentiated 

by  two  diseases,  pseudorabies  and  mouse  typhoid.     Am.  J.  Hyg.  18:  674-687, 
1933. 

(23)  Webster,   L.   T.:   Inheritance  of  resistance  of  mice  to  enteric  bacterial  and 

neurotropic  virus  infections.     J.  Exper.  Med.  65:  261-280,  1937. 

(24)  Schutze,  R.,  Gorer,  P.  A.,  and  Finlayson,   M.  H.:  The  resistance  of  four 

mouse  lines  to  bacterial  infection.     J.  Hyg.  (Cambridge)  36:  37-49,  1936. 

(25)  Zelle,  M.  R. :  Genetic  constitutions  of  host  and  pathogen  in  mouse  typhoid. 

J.  Infect.  Dis.  71:  131-152,  1942. 

(26)  Lincoln,  R.  E.,  and  Gowen,  J.  W. :  Mutation  of  Phytomonas  stewartii  by  X-ray 

irradiation.     Genetics  27:  441-462,  1942. 

(27)  Gowen,  J.  W.:  Inheritance  of  immunity  in  animals.     Ann.  Rev.  Microbiol.  2: 

215-254,  1948. 

(28)  Plough,  H.  H.,  Young,  H.  N.,  and  Grimm,  M.  R.:  Penicillin-screened  auxo- 

trophic mutations  in  Salmonella  typhimurium  and  their  relation  to   X-ray 
dosage.     J.  Bact.  60:  145-157,  1950. 

(29)  Gowen,  J.  W.,  Stadler,  J.,  Plough,  H.  H.,  and  Miller,  H.  N.:  Virulence  and 

immunizing  capacity  of  Salmonella  typhimurium  as  related  to  mutations  in 
metabolic  requirements.     Genetics  38:  531-549,  1953. 

(30)  Gowen,  J.   W.:  Genetic  aspects  of  virulence  in  bacteria  and  viruses.     Ann. 

Missouri  Bot.  Gard.  32:  187-211,  1945. 

(SI)  :  Humoral  and  cellular  elements  in  natural  and  acquired  resistance  to 

typhoid.     Am.  J.  Human  Genet.  4:  285-302,  1952. 


Discussion 
Dr.  George  E.  Jay,  Jr.,  National  Institutes  of  Health,  Bethesda,  Md. 

I  would  like  to  take  this  opportunity  to  express  my  thanks  and  appreciation  to  the 
Jackson  Laboratory  and  to  the  arrangements  committee  for  the  invitation  to  partici- 
pate in  this  memorable  symposium.  It  is  certainly  appropriate  that  25  years  of 
mammalian  genetics  and  25  years  of  the  Jackson  Laboratory  be  commemorated  to- 
gether, for  both  have  grown  together.  These  past  25  years  have  been  fruitful  for  both; 
may  the  next  25  years  be  just  as  bountiful. 

The  role  of  a  discussant  has  always  been  a  puzzling  one  to  me.  I  have  asked  various 
people  about  it  and  have  received  various  answers,  all  of  which  contributed  to  my 
confusion.  However,  today  at  lunch  some  advice  was  offered  which  I  believe  I  will 
follow.  It  was  advised  that  a  discussant  should  comment  briefly  on  the  paper  (pro- 
foundly of  course),  raise  some  pertinent  questions,  and  sit  down.  How  profound  my 
comments  are  will  be  questionable;  I  shall  try  to  raise  at  least  one  pertinent  question, 
and  I  assure  you  that  I  am  real  good  at  sitting  down. 

A  symposium  is  defined  as  either  a  drinking  party  or  feast,  or  a  conference  at  which  a 
particular  subject  is  discussed  and  opinions  gathered.  It  seems  to  me  that  either  def- 
inition may  serve  for  this  symposium,  for  certainly  if  this  first  paper  is  any  indication, 
there  will  be  ample  opportunity  to  drink  deep  from  the  cup  of  knowledge  and  there  will 
be  an  abundance  of  food  for  thought.  For  those  of  you  more  interested  in  the  less 
intellectual  aspect  of  drinking  and  feasting,  the  proposed  extracurricular  activities  will 
provide  such  opportunities.  As  for  the  second  definition,  the  subject  of  mammalian 
genetics  is  due  for  considerable  discussion,  and  the  expression  of  opinions  will  no  doubt 
be  made. 

The  data  presented  by  Dr.  Gowen  today  are  the  results  of  a  number  of  years  of  work 
by  him  and  his  associates,  and  to  my  mind  it  is  a  classic  example  of  one  aspect  of  re- 
search in  mammalian  genetics.  Without  a  doubt  it  answers  the  original  question 
posed:  that  heredity  does  play  an  important  role  in  disease  resistance  and/or  suscepti- 
bility. Just  how  hereditary  factors  operate  is  still  not  clear,  though  Dr.  Gowen's  data 
offer  some  interesting  leads.  The  differences  between  the  strains  inbred  for  various 
levels  of  resistance  show  that  many  physiologic  and  morphologic  manifestations  enter 
into  the  creation  of  any  particular  level  of  resistance. 

These  manifestations  that  characterize  the  various  inbred  strains  are  actually  the 
genetic  results  of  individuality  expressed  in  the  original  heterogeneous  population. 
This  individuality  was  used  to  synthesize  new  groups  of  animals,  each  differing  from 
the  other  and  expressing  an  individuality  characteristic  for  that  group  or  inbred  strain. 
The  genetic  technique  of  close  inbreeding  to  fix  such  individuality  is  one  that  has 
become  a  mainstay  in  modern  mammalian  genetics,  and  is  a  technique  perhaps 
synonymous  with  the  Jackson  Laboratory.  Thus  it  is  indeed  fitting  that  Dr.  Gowen's 
paper  is  first  on  the  schedule,  for  it  properly  illustrates  the  employment  of  this  tech- 
nique in  sorting  out  the  factors  contributing  to  disease  resistance. 

These  ideas,  of  course,  have  long  existed  in  the  field  of  cancer  research.  In  fact,  it 
was  in  this  research  area  that  Dr.  Little  first  applied  these  ideas  and  practices.  Since 
most  of  you  present  are  well  acquainted  with  the  genetic  concepts  of  cancer  research, 
this  area  will  only  be  mentioned.  Within  the  past  few  years,  genetic  concepts  and 
methods  are  being  used  in  still  other  areas  of  research.  In  dental  caries,  Hunt  and  his 
associates  have  been  able  to  develop  two  strains  of  rats,  one  resistant  and  the  other 
susceptible  to  carious  lesions.  As  a  result,  material  is  now  available  in  limited  quanti- 
ties which  will  permit  more  quantitative  studies  on  the  factors  involved  in  the  elabora- 
tion of  caries. 

In  certain  aspects  of  nutrition  research,  the  individuality  of  inbred  strains  is  coming 
into  its  own.     Some  recent  work  at  the  National  Institutes  of  Health  by  Dr.  Klaus 

571 

Journal   of  the  National  Cancer  Institute,  Vol.   15,  No.  3,  December   1954 


572  PKOCEEDINGS:  SYMPOSIUM 

Schwarz  has  shown  that  one  strain  of  inbred  rats  is  particularly  suitable  for  studies  on 
dietary  liver  necrosis.  He  has  found  this  strain  (Fischer  344)  to  be  uniformly  suscep- 
tible to  dietary  liver  necrosis,  and  thus  it  probably  will  be  good  material  for  testing 
unknown  dietary  preparations  in  research  of  this  nature. 

Recent  studies  by  Dr.  E.  S.  Russell  and  her  associates,  on  differences  between  inbred 
strains  of  mice  in  blood  constituents,  offer  interesting  material  for  basic  research,  as  well 
as  possible  testing  systems  for  biologic  preparations.  Some  preliminary  work  on  drug 
metabolism  by  me  indicates  marked  strain  differences,  which  may  have  value  as  far  as 
drug  testing  is  concerned,  and  certainly  offers  possibilities  for  basic  studies  on  metabolic 
pathways.  These  and  other  examples  can  be  cited  as  new  illustrations  of  the  uses  and 
importance  of  individuality  as  expressed  between  inbred  strains  in  future  medical 
research. 

Thus  the  work  of  Dr.  Gowen,  the  work  in  cancer,  and  the  more  recent  work  in  the 
other  areas  just  mentioned,  are  all  examples  of  the  control  and  utilization  of  individu- 
ality of  inbred  strains  in  research.  The  question  I  now  raise  is  this:  Is  it  not  possible 
to  carry  this  general  concept  a  step  further  and  utilize  the  individuality  as  expressed 
by  individuals  within  these  inbred  strains?  As  you  know,  an  inbred  strain  is  highly 
uniform,  for  theoretically  most  of  the  individuals  within  the  strain  have  the  same  or 
nearly  the  same  gene  complement.  However  there  occurs  at  intervals  (sometimes 
more  often  than  the  geneticist  cares  to  admit) ,  individuals  within  a  strain  that  are  not 
like  the  others.  They  may  differ  in  morphologic  or  physiologic  aspects  to  such  an 
extent  that  they  are  obviously  different — they  exhibit  individuality.  This  individu- 
ality may  be  the  result  of  mutational  changes,  recombinations,  or  some  other  phe- 
nomenon. But  whatever  its  cause,  it  is  a  situation  that  may  be  of  considerable  value. 
For  example,  in  both  the  C57BL/6  and  C57BL/10  strains  of  mice,  Dr.  Paulo  Borges  has 
observed  and  reported  changes,  found  to  be  mutations  from  one  histocompatibility 
gene  locus  to  another.  These  mutations  resulted  in  profound  differences  in  the 
responses  to  certain  transplanted  tumors.  Thus,  the  sublines  which  now  exist  for  each 
of  these  two  parent  strains  probably  differ  by  only  one  gene  from  the  parent  strain. 
This  situation  seems  to  offer  a  unique  opportunity  for  studies  in  the  action  of  histo- 
compatibility genes.  No  doubt  many  other  differences  similar  to  this  one  have  been 
observed,  and  perhaps  many  of  you  have  speculated  on  the  possibilities  these  differences 
offer.  It  would  seem  entirely  reasonable  to  think  that  such  expressions  of  individuality 
within  an  inbred  strain  may  well  provide  additional  clues  for  further  differentiating 
the  factors  that  contribute  to  the  final  manifestation  of  a  given  characteristic.  Dr. 
Gowen's  continuing  work  with  the  inbreeding  for  differences  in  leukocyte  numbers  is 
actually  an  elaboration  of  this  point. 

Just  how  far  one  can  go  with  the  control  and  utilization  of  individuality  is  yet  to  be 
seen.  The  creation  of  inbred  strains  was  the  first  step,  the  elaboration  of  sublines  of 
inbred  strains  perhaps  the  second,  and  the  selection  out  of  individual  differences  from 
these  larger  groups  the  third.  The  future  offers  some  interesting  possibilities  for 
further  genetic  manipulations. 


The  Importance  of  Differences  in  Mor- 
phology in  Inbred  Strains  * 


Thelma    B.    Dunn,    Laboratory    oj    Pathology , 
National  Cancer  Institute,2  Bethesda,  Md. 


When  Dr.  Russell  invited  me  to  this  symposium,  I  wrote  her  that  there 
was  no  one  whom  I  would  more  delight  in  seeing  honored  than  Dr.  Eliza- 
beth Fekete,  and  no  subject  in  which  I  have  a  more  enthusiastic  interest 
than  the  one  she  suggested  for  me.  Dr.  Fekete  has  contributed  greatly  to 
our  knowledge  of  the  morphology  of  the  inbred  strains.  Whenever  a 
new  pathologist  joins  our  staff,  we  immediately  introduce  him  to  the 
chapter  by  Dr.  Fekete  on  Histology,  which  appears  in  that  much  prized 
contribution  from  this  laboratory,  The  Biology  oj  the  Laboratory  Mouse. 
This  chapter  is  like  a  compass  to  the  pathologist  trained  in  human  anatomy 
who  must  orient  himself  in  the  field  of  cancer  research.  The  intricacies 
of  morphology  in  the  mouse  are  clearly  and  accurately  described  by  one 
who  knows  firsthand.  After  this  introduction,  one  can  then  proceed 
with  the  many  other  contributions  which  Dr.  Fekete  has  made  to  mor- 
phology in  her  studies  of  inbred  strains. 

All  morphologists  should  be  grateful  to  the  laboratory  here  and  its 
staff  for  the  development  of  inbred  strains  of  mice  with  their  comforting 
uniformity.  While  we  may  encourage  individuality  for  the  best  operation 
of  a  democratic  society,  conformity  simplifies  biologic  experiments.  The 
first  requirement  in  animal  research  is  a  reliable  group  of  controls.  This 
is  especially  true  in  cancer  studies,  since  observations  must  often  be  made 
on  animals  at  an  advanced  age,  when  the  unequal  onslaughts  of  time  are 
added  to  inborn  variations.  Inbred  strains  are  our  greatest  aid,  and 
while  noninbred  mice  may  be  tolerated  for  some  studies  when  young 
animals  only  are  used,  they  are  not  acceptable  for  studies  of  cancer. 

All  the  morphologist  can  be  sure  of  is  what  he  sees  on  gross  examination 
or  in  a  microscopic  slide.  The  microscopic  slide  represents  a  single 
instant  in  a  long  series  of  events.  What  happens  in  the  intervals  before 
and  after  is  a  blank,  and  must  be  filled  in  by  conjecture.  Since  even  the 
direction  of  movement  is  sometimes  uncertain  anything  which  regulates 
the  order  of  these  still-life  views  is  a  boon.  Most  pathologists  will  agree 
as  to  what  is  actually  there,  but  in  filling  up  the  intermediate  blanks,  they 
do  not  always  see  eye  to  eye. 

»  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  27, 1954. 
2  National  Institutes  of  Health,  Public  Health  Service,  U.  S.  Department  of  Health,  Education,  and  Welfare, 

573 

Journal   of  the  National  Cancer  Institute,  Vol.    15,  No.   3,  Deeember   19S4 


574  proceedings:  symposium  on  25  years  of 

Inbred  mice  furnish  a  group  of  nearly  identical  individuals  progressing 
toward  senility  on  a  fixed  schedule.  Such  morphologic  and  chronologic 
uniformity  is  not  to  be  found  in  random-bred  animals.  An  individual 
removed  from  a  heterogeneous  group  of  mice  may  be  a  variant,  or  notably 
precocious  or  laggard  in  reference  to  the  others. 

Inbred  strains  give  animal  experiments  a  notable  advantage  in  com- 
parison with  observations  on  human  beings.  In  clinical  research  a  valiant 
effort  is  made  to  get  as  uniform  a  group  as  possible.  Except  for  a  limited 
supply  of  identical  twins,  healthy  male  medical  students,  in  their  early 
twenties  and  of  obliging  disposition,  probably  represent  the  closest  human 
approach  to  an  inbred  strain.  A  clinician  does  not  walk  out  on  the  street 
and  pick  up  the  first  100  passersby  to  determine  such  a  character  as 
normal  blood  pressure.  Yet  I  was  amazed  to  find  a  pathologist  looking 
for  a  group  of  ordinary  house  mice  which  he  regarded  as  a  ' 'basic"  or 
"universal"  mouse,  for  he  assumed  that  inbred  strains  represented  abnor- 
mal and  specialized  types. 

The  importance  of  inbred  strains  in  the  identification  of  genetic  mech- 
anisms, in  the  recognition  of  factors  related  to  bacteriologic  resistance  or 
susceptibility,  and  to  investigations  in  many  other  fields  will  be  con- 
sidered by  other  speakers. 

My  discussion  of  the  morphologic  difference  in  inbred  strains  has  been 
arranged  under  four  headings.  These  are:  1)  Development  in  early  life, 
and  the  emergence  of  pathologic  lesions  as  the  animal  ages,  2)  comparison 
of  morphologic  features  and  their  relation  to  other  factors,  3)  incidence 
of  neoplasms,  and  4)  recognition  of  precancerous  conditions  or  changes. 
Many  examples  will  overlap  and  the  purpose  of  this  grouping  is  con- 
venience rather  than  exactness.  My  observations  are  based  largely  on 
my  own  experience;  much  of  this  material  is  unpublished.  Spontaneous 
diseases  have  usually  been  selected  as  illustrations,  for  the  number  of 
induced  lesions  in  which  morphologic  differences  are  important  is  too 
extensive  to  be  reviewed  here.  Such  a  wealth  of  illustrative  material  to 
emphasize  the  desirability  of  inbred  strains  is  at  hand,  that  the  real 
difficulty  lies  in  selecting  examples. 

1.  Development   in    Early    Life,   and  Pathologic   Changes   with   Age 

The  first  example  showing  strain  differences  in  early  life  illustrates  the 
precision,  the  exact  timing,  which  inbred  strains  exhibit.  Because  of 
this  adherence  to  chronology,  inbred  strains  should  always  be  used  in 
studies  of  development.  Dr.  James  B.  Longley  (1)  of  the  National 
Institutes  of  Health  was  looking  for  a  morphologic  difference  in  portions 
of  the  proximal  convoluted  tubule  in  the  kidney,  where  it  is  known  that 
functional  differences  exist.  He  determined  by  special  stains  that  alka- 
line phosphatase  disappeared  entirely  from  the  more  distal  portion  of  the 
tubule  as  the  kidney  of  the  mouse  matured.  Animals  were  then  selected 
at  frequent  intervals  from  birth  to  maturity,  and  estimations  were  made 
of  the  amount  of  alkaline  phosphatase  in  different  segments  of  the  tubule. 

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PROGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER  575 

The  time  of  disappearance  was  found  to  be  remarkably  uniform  in  indi- 
viduals from  the  same  inbred  strains,  but  differed  in  individuals  from 
different  strains.  The  periodic  acid-Schiff  technique  also  showed  altera- 
tions as  the  kidney  matured.  Sections  of  the  kidney  from  strain  DBA 
mice,  age  29  days,  still  contained  alkaline  phosphatase  in  the  more  distal 
section  of  the  proximal  tubule.  At  31  days  the  alkaline  phosphatase 
reaction  was  fading,  and  at  36  days,  it  had  almost  completely  disappeared. 
Graphs  to  show  the  estimated  quantity  of  alkaline  phosphatase  in  the 
kidney  during  this  maturation  period  were  prepared,  using  several  inbred 
strains.  The  disappearance  was  gradual  in  strains  C3H  and  C57BL. 
In  strains  BALB/c  and  DBA,  disappearance  began  a  few  days  later,  then 
progressed  rapidly.  When  noninbred  mice  were  tested,  the  curve  was 
diphasic,  implying  a  decline,  then  an  increase,  in  the  enzyme.  It  is  certain, 
however,  that  once  alkaline  phosphatase  disappeared  it  did  not  reappear, 
so  a  false  premise  might  have  been  reached  if  observations  had  been 
confined  to  the  noninbred  mice. 

Senile  degenerative  changes  are  as  much  a  consequence  of  time  as  are 
the  developmental  changes  of  early  life.  A  kidney  lesion  of  mice  found  in 
late  life  is  due  to  amyloid  deposition.  Kidneys  of  old  mice,  especially 
of  strain  A,  often  show  scarring  of  the  surface  and  many  small  cysts. 
Study  of  the  kidneys  alone  failed  to  reveal  a  cause  for  this  alteration, 
but  other  organs  in  the  same  mice  showed  amyloid  deposition  (#).  Dr. 
Heston  and  Dr.  Deringer  (8)  crossed  strain  A  mice  with  C57L,  a  strain 
in  which  amyloidosis  is  infrequent,  and  examined  the  hybrids  and  back- 
crosses  for  amyloid.  In  strain  A,  the  incidence  was  88  percent.  In  strain 
C57L  the  incidence  was  9  percent.  The  first  generation  hybrids  between 
these  strains  had  an  incidence  of  5  percent.  When  backcrossed  to  strain 
A,  the  incidence  was  49  percent,  and  when  backcrossed  to  strain  C57L 
the  incidence  was  8  percent.  When  amyloidosis  appeared  in  strain  C57L 
it  was  secondary  to  dermatitis.  Primary  amyloidosis  and  the  accompany- 
ing renal  damage  thus  occurred  in  incidences  conformable  with  the 
inheritance  of  a  single  recessive  gene. 

The  type  of  kidney  damage  secondary  to  amyloidosis  apparently  de- 
pends upon  the  site  of  deposition  in  the  kidney.  This  varies  with  different 
strains.  In  strain  A  the  amyloidosis  was  primary  or  idiopathic  and  af- 
fected many  organs.  Amyloid  often  was  not  found  in  the  kidney  at  a  late 
stage,  but  was  found  at  an  earlier  stage  deposited  in  a  transverse  line 
across  the  papilla  (4).  This  led  to  necrosis  and  sloughing  of  the  papilla. 
Atrophy  and  cyst-formation  in  the  cortex  were  secondary  to  obstruction 
of  the  collecting  tubules.  In  strain  C57BL,  the  amyloid  was  deposited 
in  the  glomeruli,  the  tubules  were  remarkably  well  preserved,  and  the 
papilla  was  intact.  In  strain  HR,  amyloid  deposition  was  frequent 
in  the  glomeruli  and  there  was  also  calcification  of  the  tip  of  the  papilla, 
and  sloughing. 

These  observations  on  the  differences  in  amyloid  deposition  in  inbred 
strains  have  usually  been  made  on  random  groups.  Except  for  strain  A 
and  C57L  no  large  series  has  ever  been  tabulated.    A  careful  study  on 

Vol.    15,   No.   3,   December   1954 


576  proceedings:  symposium  on  25  years  of 

many  animals  from  other  strains  might  definitely  establish  characteristic 
strain  differences  in  amyloid  deposition. 

A  kidney  lesion  showing  remarkable  strain  specificity  was  produced  in 
strain  A  after  repeated  injection  or  ingestion  of  urethan  (5),  given  to 
produce  lung  tumors.  About  half  of  the  animals  developed  ascites.  On 
microscopic  examination  a  glomerular  lesion  was  found  which  had  many 
of  the  morphologic  features  of  glomerulonephritis  in  man.  An  experi- 
mental disease  of  this  type  should  be  helpful  in  studying  renal  disease,  for 
there  are  no  good  counterparts  of  human  glomerulonephritis  in  animals. 
With  this  in  mind  urethan  was  given  to  several  other  strains  of  mice,  and 
to  rats,  rabbits,  and  guinea  pigs.  All  our  attempts  to  produce  kidney 
damage  in  other  strains  or  species  were  unsuccessful.  Kirschbaum  and 
Bell  (6)  later  reported  a  similar  lesion  in  strain  NHO  mice  in  which  a 
spontaneous  disease  of  similar  type  had  previously  been  described. 

Another  example  of  a  complex  process  manifested  by  morphologic 
differences  in  the  kidney  was  produced  by  the  exposure  of  a  number  of 
mice  of  inbred  strains  to  chloroform  (7) .  The  males  were  more  seriously 
affected  than  females,  and  mice  of  some  strains  more  than  others.  Nearly 
all  strain  C3H  males  were  killed  by  a  dose  which  nearly  all  females  of 
this  strain  survived.  When  males  survived,  the  kidney  cortex  usually 
showed  calcification  when  the  mice  were  autopsied  several  months  later. 

These  observations  on  the  kidney  demonstrate  that  when  routine 
histologic  methods  are  applied  to  the  kidneys  of  normal  mice,  no  strain 
differences  may  be  detected.  By  the  use  of  special  stains  on  the  kidney 
during  development,  however,  or  from  the  study  of  a  degenerative  con- 
dition as  amyloidosis,  or  following  the  exposure  to  a  toxic  substance, 
morphologic  differences  in  the  strains  are  readily  shown. 

Let  me  now  recount  a  series  of  adventures,  or  misadventures,  where 
inbred  strains  led  me  to  believe  I  had  made  sensational  discoveries.  In 
spite  of  this  deception,  I  am  still  grateful,  for  it  was  the  presence  of  the 
same  lesions  in  control  mice  from  inbred  strains  which  corrected  the 
errors  before  there  was  time  for  publication. 

Dr.  Harold  Morris  (8)  fed  5  inbred  strains  of  mice  synthetic  diets  in 
which  pyridoxine  was  absent  or  deficient.  The  experiment  became 
exceedingly  involved,  for  each  inbred  strain  reacted  differently.  The 
effect  on  survival  time  varied  with  the  strain.  With  chronic  deficiency, 
when  a  small  but  inadequate  amount  of  pyridoxine  was  given,  some  mice 
of  strains  C58  and  C57BL  survived  about  56  weeks  while  all  strain  DBA 
mice  were  dead  by  38  weeks. 

Lesions  related  to  the  deficiency  depended  somewhat  upon  the  length 
of  time  the  deficient  diet  was  given,  and  varied  in  different  strains.  Strain 
DBA  showed  severe  anemia  and  damage  to  the  reproductive  organs. 
With  chronic  deficiency,  C57BL  developed  a  dermatitis  not  seen  in 
other  strains.  Some  C58  mice  had  a  locomotor  disturbance.  When  not 
severely  deficient,  a  few  strain  A  mice  lived  even  longer  than  the  controls, 
for  they  did  not  develop  the  characteristic  strain  A  amyloidosis.  If  one 
reasoned  from  the  particular  to  the  general,  and  considered  the  results 

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PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  577 

in  strain  A  mice  only,  it  might  be  argued  that  a  mild  pyridoxine  defi- 
ciency is  desirable  for  long  life.  A  remarkable  skeletal  lesion  developed  in 
strain  C58  mice  in  the  deficient  group.  At  first  it  was  thought  to  be  a 
hitherto  undisclosed  effect  of  pyridoxine  deficiency,  but  when  the  control 
group  on  the  synthetic  diet  was  examined  the  same  disturbance  was 
found  in  them.  Osteoid  tissue  developed  about  the  shafts  of  the  rib 
(fig.  1),  and  many  other  locations  in  the  skeleton,  especially  at  sites  sub- 
ject to  trauma.  It  appeared  in  0.83  percent  of  old  strain  C58,  which  had 
been  for  a  long  period  on  the  synthetic  diet  and  in  43  percent  of  strain 
C57BL,  which  is  related  to  strain  C58.  It  was  found  in  5  percent  of  strain 
DBA  and  never  in  strains  C3H  and  A,  which  are  related  to  each  other  but 
unrelated  to  strains  C58  and  C57BL.  We  have  not  observed  such  a 
lesion  in  our  mice  on  a  regular  diet. 

After  noting  the  bone  lesion  the  next  obvious  step  was  to  examine  the 
condition  of  the  parathyroid  glands.  It  was  found  that  in  many  of  the 
mice  with  the  bone  lesions,  the  parathyroid  glands  were  dark  in  color  (9). 
The  association  with  the  bone  lesion  appeared  significant  until  the  same 
pigmentation  was  found  in  untreated  C58  mice. 

When  the  entire  parathyroid  gland  was  cleared  in  glycerine,  and  when 
microscopic  sections  were  examined,  it  was  evident  that  the  dark  color  on 
gross  examination  was  due  to  melanocytes  in  the  parathyroid  stroma. 
Similar  dendritic  melanocytes  have  been  found  in  the  heart  valves  and  the 
meninges  of  strain  C58.  This  peculiarity  might  be  used  by  embryologists 
to  follow  the  spread  of  melanoblasts  from  the  neural  crest. 

The  melanocytes  made  the  parathyroid  gland  easily  visible,  so  that 
parathyroidectomies  could  be  performed.  The  mice  survived  this  opera- 
tion— too  well  for  our  purpose — since  none  of  them  seemed  any  the  worse 
for  the  experience.  Still  hoping  to  detect  some  endocrine  effect,  para- 
thyroid tissue  from  a  number  of  mice  was  injected  into  others  of  strain 
C58,  but  they  seemed  as  unmoved  by  a  superfluity  of  parathyroid  tissue 
as  they  were  by  deprivation.  Nevertheless,  complete  endocrine  examina- 
tions were  made  at  autopsy.  The  pituitary  gland  in  an  occasional  mouse 
was  found  to  be  cystic  and  several  times  the  normal  size  (fig.  2).  The 
pituitary  glands  of  many  untreated  C58  mice  were  then  examined,  and 
although  pituitary  cysts  were  rarely  found,  just  once  was  often  enough  to 
disprove  a  significant  relation  to  the  parathyroid.  The  lesion  was  not 
frequent  enough,  however,  for  a  controlled  experimental  study. 

2.  Comparison  of  Morphologic  Features  in  Inbred  Strains 

Another  personal  experience  will  be  given  as  an  illustration  of  this 
section.  Year-old  strain  A  mice  had  received  400  r  irradiation  at  birth. 
Damage  to  the  lens  and  severe  damage  to  the  retina  were  regularly  found 
(10)  (fig.  3C).  The  slides  were  shown  to  others  in  our  laboratory.  A  few 
days  later,  another  pathologist  found  a  similar  alteration  in  the  retina  of 
a  strain  C3H  mouse  which  had  never  been  exposed  to  X  ray.||This  was 
disconcerting.     I  consulted  Dr.  Heston,  as  I  often  do.     He  gave  me  a  refer- 

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578  proceedings:  symposium  on  25  years  of 

ence  to  retinal  anomalies  in  mice,  which  explained  the  puzzle.  We  now 
observe  that  all  our  strain  C3H  mice  at  Bethesda.have  a  secondary  degen- 
eration of  the  rod  layer  in  the  retina  (11).  We  found  the  retina  to  be 
normal  at  10  days  of  age  (fig.  3A),  but  degenerated  by  the  time  the  mouse 
reached  28  days  (fig.  3B).  Sight  must  not  be  very  important  to  a  labora- 
tory mouse.  We  had  not  suspected  before  that  all  our  strain  C3H  mice 
are  blind.  Fortunately,  for  the  validity  of  the  irradiation  experiment, 
this  anomaly  has  not  been  found  in  strain  A  mice.  Ascribing  the  retinal 
damage  to  irradiation  in  strain  A  was  justified,  and  we  did  not  have  the 
difficult  and  uncertain  task  of  separating  the  induced  lesion  from  a  spon- 
taneous retinal  anomaly. 

There  is  no  organ  which  should  be  so  lacking  in  distinction  as  the  spleen. 
In  the  mouse  the  spleen  appears  to  be  an  annex  organ,  an  overflow  tissue 
where  the  business  which  cannot  get  done  in  the  bone  marrow  or  lym- 
phatic tissue  is  carried  on.  One  would  think  a  spleen  in  any  mouse  should 
be  just  a  spleen,  and  nothing  more.  Yet  morphologic  differences  in  spleens 
from  various  strains  of  inbred  mice  are  sufficiently  distinctive  so  that  a 
shrewd  guess  as  to  the  strain  of  origin  can  often  be  made  by  an  examination 
of  the  spleen  alone.  While  examining  a  group  of  strain  A  mice  which 
had  been  treated  with  urethan  for  a  prolonged  period,  an  astonishing 
number  of  mast  cells  was  observed,  and  it  was  supposed  that  urethan 
might  be  responsible.  The  detection  of  any  drug  or  condition  which 
affects  the  number  of  mast  cells  would  be  a  real  achievement.  However, 
when  the  controls  were  examined,  mast  cells  were  equally  numerous. 
We  next  counted  the  mast  cells  in  cross  sections  of  the  spleen  taken  from 
various  strains  of  mice  at  different  ages  and  stained  with  toluidine  blue 
{12).  We  found  that  the  mast  cell  content  of  the  spleen  is  a  fairly  reliable 
strain  characteristic.  Strain  A  had  the  highest  number.  In  old  males 
the  number  was  often  above  2,000  and  could  not  be  estimated  accurately. 
Strain  C57L  had  a  smaller  number  averaging  about  255  in  males,  and  16 
in  females,  and  the  number  in  the  hybrids  of  these  strains  lay  somewhere 
between  the  parents.  Strain  I  had  almost  no  mast  cells.  The  number 
generally  increased  with  age  and  was  higher  in  males. 

Mast  cells  cannot  be  accurately  enumerated  in  any  other  organ,  so  it 
is  uncertain  whether  the  total  number  of  mast  cells  in  the  entire  body  of  a 
strain  A  mouse  greatly  exceeds  the  total  number  in  other  strains.  Old 
hybrids  with  a  strain  A  parent  have  had  more  mast  cell  neoplasms  than 
any  other  mice  in  our  laboratory.  The  function  of  the  mast  cell  has  been 
the  object  of  an  intensive  search  in  the  past  few  years.  An  investigation 
of  the  differences  in  inbred  strains  might  furnish  a  clue. 

A  more  subtle,  but  equally  dependable  difference  in  spleens  from  dif- 
ferent strains  was  found  by  Dr.  Oscar  Duque,  working  in  the  Pathology 
Laboratory  at  the  National  Cancer  Institute  (18).  Dr.  Duque  is  from 
Latin  America,  and  perhaps  it  takes  Spanish  blood  to  do  good  silver 
stains.  He  made  beautiful  preparations,  using  a  modified  del  Rio  Hortega 
method,  so  that  he  could  visualize  the  reticulum  cells  in  the  mouse  spleen. 
A  striking  difference  in  the  arrangement  and  quantity  of  the  cells  was 

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579 


found  in  inbred  strains.  For  example,  a  perifollicular  collar  formed  of 
reticulum  cells  is  well  developed  in  strain  I,  but  generally  absent  in  strain 
C58.  Reticulum  cells  are  very  numerous  in  the  red  pulp  in  strain  I.  In 
strain  C58,  the  follicles  are  small  and  numerous.  Within  the  follicles, 
the  reticulum  cells  are  prominent;  those  in  the  germinal  centers  being 
very  large.  A  difference  in  the  number  and  arrangement  of  reticulum 
cells  is  also  shown  if  stains  for  iron  are  done  on  spleens  from  old  mice. 
The  reticulum  cells  are  actively  phagocytic  for  hemosiderin,  which  is 
readily  identified  by  the  Prussian-blue  reaction,  and  the  different  strains 
show  some  of  the  same  differences  in  reticulum  that  is  seen  after  silver 
staining. 

I  have  also  observed  that  strain  C57L  has  little  hematopoietic  activity 
in  comparison  with  other  strains.  These  comparisons  might  be  thought 
a  pastime  of  academic  interest  only,  except  that  the  modification  of  X-ray 
damage  which  results  from  shielding  the  spleen  varies  in  different  inbred 
strains.  It  is  possible  that  morphologic  differences  in  the  strains  may 
explain  these  differences  and  indicate  the  tissue  or  cell  which  is  most 
important.  The  painstaking  work  from  Dr.  Russell's  laboratory  (14)  on 
differences  in  the  peripheral  blood  of  inbred  strains  also  reveals  the  slight 
but  positive  differences  which  must  exist  in  the  hematopoietic  and  lym- 
phatic tissues  of  inbred  mice. 

In  studies  of  another  system  Dr.  Fekete  described  differences  in  the 
ovaries  from  inbred  strains  which  correlate,  to  some  extent,  with  functional 
endocrine  activity  (15). 

Next  I  will  list  briefly  a  number  of  degenerative  lesions  which  are  pecul- 
iar to  some  inbred  strains.  Attention  is  called  to  them,  first,  as  a  warning 
against  supposing  that  they  are  produced  by  an  experimental  procedure, 
and,  second,  to  invite  you  to  consider  a  possible  relationship  to  physiologic 
or  metabolic  differences  among  the  strains. 

a)  Hemosiderin  is  abundant  in  the  reproductive  organs  of  old  females 
of  strain  BALB/c.  The  uterine  wall  contains  large  amounts  of  material 
stained  by  the  Prussian-blue  reaction,  and  the  epithelial  cells  of  the  mam- 
mary ducts  are  packed  with  hemosiderin.  This  degree  of  hemosiderosis 
has  not  been  observed  in  other  strains.  Can  it  be  correlated  with  any 
other  peculiarity  in  this  strain? 

b)  A  corneal  lesion  has  been  frequently  observed  in  strain  DBA.  This 
appears  in  the  living  mouse  as  opaque  patches  in  the  cornea.  It  was  first 
observed  in  experimental  mice,  and  was  suspected  of  being  an  induced 
lesion  (16).  A  degenerated,  often  calcified  area  in  the  myocardium  is  also 
commonly  found  in  strain  DBA,  and  in  old  C3H  and  C3Hf.  Are  these 
lesions  secondary  to  a  metabolic  abnormality  of  these  strains? 

c)  The  name  ' 'mesenteric  disease' '  was  applied  to  a  lesion  of  the  mesen- 
teric node  by  Simonds  (17).  It  is  common  in  old  strain  C3H  mice,  and 
is  found  in  lower  incidence  in  strain  C3H  hybrids.  An  extreme  enlarge- 
ment of  the  node  is  found,  and  microscopically  there  are  many  blood-filled 
dilatations. 


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580  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

d)  A  lesion  of  the  pancreas  appeared  a  number  of  times  in  a  group  of 
old  C3Hf  mice  from  Dr.  Heston's  colony  which  I  examined  recently. 
Cysts  were  formed  by  dilatations  of  pancreatic  ducts.  Islet  tissue  persisted, 
but  all  the  acinar  tissue  disappeared  in  large  areas.  Occasionally  these 
cysts  ruptured  and  a  condition  of  fat  necrosis,  such  as  is  found  in  human 
pancreatic  disease  appeared. 

3.  Neoplasms  in  Inbred  Mice 

When  I  first  began  working  with  mice,  I  always  consulted  an  experienced 
"mouse"  pathologist  regarding  unusual  autopsy  specimens  and  slides. 
Before  he  would  give  me  advice  or  make  a  diagnosis,  he  asked  "what 
strain?"  Depending  upon  my  answer,  the  finding  was  considered  either 
commonplace  or  remarkable.  It  took  me  some  time  to  realize  the  impor- 
tance of  this  question.  I  now  find  that  it  is  the  first  which  comes  to  mind 
when  I  am  shown  any  lesion  or  neoplasm  in  a  mouse.  Few  features  are 
more  characteristic  of  an  inbred  strain  than  its  tumors.  Many  examples 
of  this  are  familiar  to  you.  If  mammary  tumors  in  strain  C3H,  lung 
tumors  in  strain  A,  and  leukemia  in  strains  C58  and  ML  were  suddenly 
taken  away  a  good  percentage  of  cancer  research  would  be  wiped  out. 
In  many  laboratories  these  tumors  are  classified  by  site  only,  but  close 
histologic  study  of  large  groups  of  tumors  might  reveal  that  even  among 
these  much  investigated  subversives,  differences  in  morphology  may  exist 
in  inbred  strains.  For  example,  in  mammary  tumors  from  several  inbred 
strains,  various  types  of  histologic  structures  were  found  to  be  preponder- 
ant when  the  milk  agent  had  been  removed.  In  Dr.  Heston's  C3H  mice 
without  the  agent,  adenoacanthomas  were  common  (18).  In  Dr.  Ander- 
vont's  DBA  without  the  agent  (19),  but  treated  with  methylcholanthrene, 
carcinosarcomas  were  frequent  and  Dr.  Muhlbock  (20)  working  with 
agent-free  DBA  found  a  high  percentage  of  "deviating  types"  of  mammary 
tumors  many  with  marked  squamous  metaplasia.  We  have  described  a 
distinctive  type  of  mammary  tumor  that  most  often  appears  in  hybrid 
mice  of  advanced  age,  usually  without  the  milk  agent  (21).  We  have 
termed  this  adenocarcinoma  type  C.  This  tumor  is  composed  of  numerous 
small  cysts  lined  by  a  cuboidal  epithelium,  which  is  closely  invested  with 
a  fusiform  cell.  Unlike  most  other  mammary  tumors,  this  type  is  generally 
uniform  throughout  the  section.  A  van  Gieson  stain  shows  that  the  fusi- 
form cell  is  of  muscle  origin  and  not  fibroblastic,  for  no  collagen  is  devel- 
oped. This  is  the  most  distinctive  characteristic  of  this  tumor.  When 
Dr.  Foulds  from  England  was  here,  we  compared  our  experiences  with 
mammary  tumors  in  mice.  He  told  me  he  had  never  seen  the  type  just 
mentioned,  although  he  had  observed  another  pattern  combining  an 
epithelial  and  a  muscle  element.  He  showed  me  other  types  which  I  had 
not  observed  in  any  strains  used  in  our  laboratory.  These  differences 
stress  the  need  for  caution  in  transferring  information  regarding  tumors 
from  one  strain  to  another.  A  standard  mammary  tumor  is  not  to  be 
expected  in  all  strains.     One  can  say  only  that  certain  morphologic 

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PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  581 

characters  were  found  in  the  examination  of  tumors  from  a  specified  strain 
under  specified  conditions. 

Whether  other  common  neoplasms,  such  as  lymphocytic  leukemia  or 
pulmonary  tumors,  might  also  show  slight  variations  in  different  strains 
of  mice  has  not  been  determined. 

Many  remarkable  tumors  were  disclosed  in  a  survey  made  by  Dr. 
Heston  and  Dr.  Deringer,  which  Dr.  Heston  will  describe  more  fully. 
Strain  C3H  females  with  the  milk  agent  were  crossed  with  C57BL  males, 
and  then  backcrossed  to  C57BL  males  through  seven  generations,  until 
the  mice  were  largely  of  strain  C57BL  constitution.  Then  by  a  reverse 
process,  the  females  of  this  seventh  backcross  generation  were  crossed 
with  strain  C3H  males,  without  the  agent,  and  the  resulting  females  were 
backcrossed  to  C3Hf  males  through  four  generations  until  a  nearly  pure 
C3Hf  mouse  resulted.  The  following  rare  tumors  appeared,  and  were 
rather  definitely  segregated  according  to  genetic  constitution. 

a)  Osteogenic  sarcomas. — In  strain  C3Hf  and  in  hybrids  without  the 
agent  derived  from  C3H  or  C3Hf .  The  morphology  is  being  described  by 
Dr.  Hilberg  of  the  National  Cancer  Institute.  A  pattern  of  interlacing 
cords,  with  ossification  at  the  center  is  characteristic  (22) . 

b)  Harderian  gland  tumors. — Most  frequently  in  C3Hf  and  in  hybrids 
without  the  agent  derived  from  C3H  or  C3Hf .  Neoplastic  tissue  replaces 
the  Harderian  gland,  but  structural  features  of  the  normal  gland  are 
retained.  There  was  considerable  variation  in  the  degree  of  differentiation 
in  different  tumors. 

The  next  examples  show  how  the  incidence  of  tumors  characteristic  of 
an  inbred  strain  may  be  altered.  The  agent  had  been  removed  from  strain 
C3H,  and  the  mammary-tumor  incidence  was  reduced  so  that  many  mice 
survived  to  old  age.     The  following  rare  types  of  neoplasm  then  emerged. 

c)  Adenocarcinomas  of  the  uterus. — Many  of  the  adenocarcinomas  were 
polypoid,  and  closely  resembled  adenocarcinoma  of  the  uterus  in  women. 
I  have  not  found  them  previously  described. 

d)  Ovarian  tumors. — Eare  types  of  ovarian  tumors  were  found  in  strain 
C3Hf.  One,  a  papillary  cyst-adenocarcinoma  was  previously  described 
from  the  laboratory  here  (23)  and  among  noninbred  mice  of  Maude  Slye's 
colony  (24)  •  Another  very  rare  type  produces  a  mucoid  substance,  and 
somewhat  resembles  the  mucoid  neoplasms  of  the  intestine  in  man.  One 
of  these  mucoid  tumors  has  been  transplanted  successfully.  It  grows 
slowly,  it  maintains  the  same  morphology  and  produces  a  mucoid  sub- 
stance as  did  the  original.  I  have  not  found  a  previous  description  of  this 
tumor. 

Many  other  examples  of  tumors  peculiar  to  one  strain  might  be  men- 
tioned. We  have  found  localized  plasma-cell  tumors  only  in  strain  C3H 
(25).  The  majority  of  our  mast-cell  neoplasms  have  appeared  in  hybrids, 
derived  from  outcrossing  strain  A.  Tumors  of  the  central  nervous  system 
are  described  only  in  strain  NHO  (26). 

It  thus  appears  that  an  entirely  pathologic  and  destructive  process 

Vol.    15,  No.   3,   December   1954 


582  proceedings:  symposium  on  25  years  of 

a  neoplasm,  may  be  one  of  the  most  characteristic  features  of  an  inbred 
strain. 

4.  Recognition  of  Precancerous  Conditions  or  Changes 

This  involves  the  most  difficult  and  uncertain  of  all  our  endeavors. 
Investigators  working  with  mice  have  perhaps  been  overly  eager  in 
attempts  to  correlate  some  newly  discovered  morphologic  feature  or 
physiologic  difference  in  a  strain  with  cancer.  It  is  hopefully  assumed 
that  a  victim  destined  for  cancer  carries  with  him  some  distinguishing 
mark,  which  separates  him  from  others.  De  Quincey  remarking  upon 
the  frequency  of  tuberculosis  among  his  countrymen  said  that  one  destined 
for  this  disease  carried  a  sign,  like  a  tree  which  was  blazed  for  cutting. 
In  searching  for  such  a  mark  in  cancer,  the  fallacy  often  lies  in  assuming 
that  there  is  a  general  tendency  to  cancer  no  matter  at  what  site  it  develops. 
Cancer,  however,  in  mice  and  probably  in  man,  is  a  local  disease  of  a 
specific  organ.  A  high-mammary  tumor  strain  may  be  a  low-leukemia 
strain.  A  morphologic  or  physiologic  variation  promoting  cancer  should 
therefore  reside  in  or  be  closely  related  in  some  fashion  with  that  particular 
organ  or  tissue  in  which  the  disease  is  found.  Several  errors  of  correla- 
tion, or  unsuccessful  efforts  at  correlation,  to  mammary  tumor  develop- 
ment may  be  mentioned,  such  as  brown  degeneration  of  the  adrenal  (27), 
the  porphyrin  content  of  the  Harderian  gland  (28),  differences  in  blood 
groups  (29),  and  in  iron  content  of  the  mammary  tissue  (80).  Despite 
these  failures,  the  discovery  of  precancerous  conditions  is  among  our  most 
important  endeavors.  Recognition  of  them  would  give  the  experimentalist 
a  clue  to  etiology,  and  the  clinician  a  chance  at  prevention  and  early 
treatment.  Many  examples  of  precancerous  conditions  in  clinical  medi- 
cine are  available,  such  a  polyposis  of  the  colon,  xeroderma  pigmentosa, 
and  hereditary  multiple  skeletal  exostoses.  These  are  all  local  conditions 
preceding  cancer  of  the  same  site.  When  the  search  in  mice  is  directed 
toward  a  specific  organ  or  tissue  in  which  the  cancer  will  later  develop, 
some  positive  correlations  have  been  found.  We  have  for  example,  the 
hyperplastic  nodule  which  precedes  many  mammary  tumors  and  is  much 
more  frequent  in  mice  with  the  milk  agent  (31),  and  the  pathologic  altera- 
tion in  the  skin  of  hairless  mice  (82),  in  which  a  high  incidence  of  spon- 
taneous skin  cancer  develops.  Examples  of  preneoplastic  lesions  are 
numerous  if  carcinogenic  agents  are  used.  On  the  other  hand,  a  number 
of  lesions  have  been  observed  where  the  histologic  structure  strongly 
suggested  that  they  might  terminate  in  neoplasia.  Even  with  the 
stimulus  of  a  carcinogen,  many  of  these  have  proceeded  no  further  than 
hyperplasia.  For  example,  a  gastric  lesion  in  strain  I  mice  proliferates 
wildly,  but  it  does  not  progress  to  gastric  cancer  (88).  Leukemoid 
reactions,  with  granulocyte  counts  in  the  hundreds  of  thousands,  have 
never  been  successfully  transplanted  (84).  A  lesion  in  strain  C58  was 
described  as  preleukemic  (85),  but  this  also  appears  in  low  leukemia 
strains  in  our  laboratory.  It  was  noted  in  the  Jackson  Laboratory  that 
leukocyte  counts  in  young  mice  could  not  be  correlated  with  a  later  devel- 

Journal    of   the   National   Cancer   Institute 


PKOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  583 

opment  of  leukemia  in  various  inbred  strains  (36).  In  our  laboratory 
lymphatic  and  blood-forming  organs  in  high-  and  low-leukemia  strains  of 
mice  at  6  weeks  and  12  months  of  age  were  examined.  Many  interesting 
differences  were  observed  but  none  of  them  appeared  to  be  related  to  the 
development  of  neoplasms.  Altogether,  this  line  of  research  has  been 
disappointing.  We  have  usually  failed  to  discover  an  abnormality  in 
early  life  foretelling  the  later  appearance  of  cancer.  Regardless  of  these 
failures,  inbred  mice  still  offer  our  greatest  hope  in  this  search.  In  high 
tumor  strains  we  have  individuals  that  will  develop  cancer  of  a  particular 
type  at  a  particular  site  within  a  short  period  of  time.  In  a  corresponding 
low  tumor  strain  the  individual  will  not  develop  cancer  in  the  particular 
organ  within  its  normal  life  span.  The  significant  difference  in  these  mice, 
however,  has  usually  been  too  deeply  hidden  for  the  morphologist  to 
discover.  Nevertheless,  this  is  a  line  of  research  which  must  be  continued 
and,  as  always,  with  inbred  strains. 

In  closing  let  me  say  that  like  Emily  Dickinson's  bee  and  honey  the 
pedigree  of  my  human  associates  does  not  concern  me  in  the  least,  but  I 
am  very  particular,  even  snobbish,  about  having  the  family  history  of  any 
mouse  that  I  am  asked  to  work  with. 

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spontaneous  lymphatic  leukemia  in  mice.     Am.  J.  Path.  19:  239-253,  1943. 

(36)  Budds,  0.  C,  Russell,  E.  S.,  and  Abrams,  G.  E.:  Effects  of  genetics  and 

anesthesia  upon  granulocyte  and  agranulocyte  levels  in  seven  inbred  mouse 
strains.     Proc.  Soc.  Exper.  Biol.  &  Med.  84:   176-178,  1953. 


Vol.    15,   No.   3,   December    1954 


586  proceedings:  symposium  on  25  years  op 


Plate  42 

Figure  1. — Strain  C58.  Osteoid  tissue  surrounding  original  shaft  of  rib,  which  is 
more  deeply  stained.  Osteoid  encroaches  on  marrow  cavity.  Lesion  developed  in 
old  mice  on  a  synthetic  diet.     Hematoxylin  and  eosin.     X  200 

Figure  2. — Strain  C58,  cyst  of  the  pituitary,  lying  between  pars  intermedia  and 
anterior  lobe.     A  spontaneous  lesion.     Hematoxylin  and  eosin.     X  50 


JOURNAL   OF  THE   NATIONAL   CANCER  INSTITUTE,   VOL.    15 


PLATE  42 


-  ■   ■>'    ■ 


Dunn 

316263—54 20 


587 


588  proceedings:  symposium  on  25  years  of 


Plate  43 

Figure  3. — A)  Strain  C3Hf,  aged  14  days,  untreated.     All  layers  of  retina  are  present. 
Optic  nerve  fibers  emerging  at  right.     Hematoxylin  and  eosin.      X   300 

B)  Strain  C3Hf,  aged  28  days,  untreated.  Rod  layer  and  outer  nuclear 
layer  are  missing.  Optic  nerve  fibers  are  seen  beneath  retina.  Hematoxylin  and 
eosin.      X  300 

C)  Strain  A,  aged  1  year,  exposed  to  400  r  X  ray  at  birth.  Rod  layer 
and  outer  nuclear  layer  are  missing.  Optic  nerve  fibers  are  seen  beneath  retina. 
Hematoxylin  and  eosin.      X  300 


JOURNAL    OF   THE    NATIONAL    CANCER    INSTITUTE,    VOL.    15  PLATE    43 


*****  m^  *5fe\**  it  ?f  in 


*#>•*>***'"  V***e 


B  *  > 


Dunn 


Figure  3 


589 


jPPHMWW 


Discussion 
Dr.  E.  D.  Murphy,  Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 

It  is  particularly  fitting  that  Dr.  Thelma  Dunn  has  been  invited  to  speak  in  honor 
of  Dr.  Elizabeth  Fekete  and  that  she  chose  for  her  subject  the  problem  of  the  mor- 
phologic differences  between  inbred  strains,  for  these  two  investigators  clearly  share 
leadership  in  this  field.  In  fact  they  are  the  pathologists  whom  pathologists  consult 
on  problems  in  this  field. 

In  most  morphologic  work  it  is  a  comfort  to  realize  that  if  one  goes  back  in  the 
German  literature  one  can  usually  find  a  classical  morphologic  study  on  almost  any 
problem  and  one  is  saved  a  lot  of  work.  The  development  of  the  inbred  strains  has 
been  too  recent  for  that,  and  it  often  seems  to  me  that  if  Dr.  Dunn  or  Dr.  Fekete 
has  not  already  carried  out  the  basic  studies,  the  work  remains  to  be  done. 

I  was  fascinated  by  the  range  of  examples  of  morphologic  differences  between  inbred 
strains  that  Dr.  Dunn  has  shown  us.  It  is  apparent  that  cancer  workers  have  appre- 
ciated and  have  used  the  inbred  strains  extensively  in  their  work.  It  is  also  quite 
apparent  that  the  inbred  strains  are  and  certainly  will  be  of  equal  value  in  many 
other  fields  of  investigation.  Another  field  that  stands  out  to  me  would  be  the  group 
of  degenerative  diseases  as  contrasted,  let  us  say,  to  infectious  diseases  about  which 
we  had  an  example  in  the  first  paper  this  afternoon.  I  was  particularly  impressed  by 
the  number  of  problems  which  morphology  was  able  to  delineate  which  demand  further 
study.  For  example,  the  kidney  lesions  induced  by  urethan  offer  a  possible  experi- 
mental analogue  to  glomerular  lesions  in  man. 

The  term  classical,  as  applied  to  these  recent  studies,  refers  not  to  the  use  of  tradi- 
tional histologic  techniques,  but  to  the  degree  of  thoroughness  and  meticulousness. 
It  is  quite  apparent  that  modern  classical  studies  employ  the  techniques  required  to 
answer  the  questions  put,  including  the  histochemical  methods  which  are  undergoing 
such  rapid  development  at  present. 


591 

Journal   of  the   National  Cancer   Institute,   Vol.    15,   No.    3,   December   1954 


Session  II.  Mammalian  Genetics;  1929 
as  Viewed  from  1954 


Chairman,  Dr.  Clarence  C.  Little,  Director, 
Roscoe  B.  Jackson  Memorial  Laboratory,  Bar 
Harbor,  Maine 


Speaker:  Dr.  W.  E.  Castle 

The  Part  of  Mammalian  Genetics  in  Founding  The  Jackson  Memorial 
Laboratory 


593 

Journal   of   the   National   Cancer  Institute.  Vol.    15,   No.   3,   December    1954 


Introduction  to  Dr.  Castle 
Dr.  C.  C.  Little 

It  is  not  often  that  a  man  who  had  an  important  part  in  the  birth  of  a  new  science 
is  still  contributing  actively  to  its  progress  almost  55  years  later. 

After  the  rediscovery  of  Mendel's  Law,  Dr.  Castle  was  one  of  the  first  to  apply 
its  principles  to  mammalian  genetics.  Over  a  longer  period  than  anyone  else  he  has 
continued  research  in  that  field  with  fruitful  and  stimulating  results. 

In  this  room  are  many  of  his  academic  "children,"  "grandchildren,"  and  "great- 
grandchildren," who  have  in  their  lives  reflected  the  impelling  stimulus  and  the  patient 
effort  for  which  he  was  the  example. 

In  many  ways  the  Jackson  Laboratory  represents  a  happy  and  vigorous  attempt  to 
realize  the  opportunities  which  had  their  ancestry  in  the  early  days  of  his  teaching. 

From  the  basement  of  old  Lawrence  Hall  at  Harvard  to  the  Bussey  Institution  and 
out  into  the  world  of  experimental  biology  the  impetus  given  to  us  by  this  great  and 
humble  teacher  has  made  its  way  with  faith  and  strength. 

His  being  here  today  is,  for  me  personally,  the  fulfillment  of  a  dream  of  long  stand- 
ing. It  is  entirely  proper  that  this  celebration  of  our  25th  birthday  should  be  graced 
by  his  presence.  I  hope  that  both  by  word  and  by  inner  awareness,  he  will  know 
how  genuine  and  how  lasting  is  our  respect  and  affection  for  him  as  a  man,  and  as  a 
catalyst  expressed  through  his  students. 


594 


The  Part  of  Mammalian  Genetics  in 
Founding  The  Jackson  Memorial  Lab- 
oratory * 


W.  E.  Castle,  University  of  California,  Berkeley, 
Calif. 


I  have  been  reading  recently  a  little  book  about  the  life  and  work  of 
Sir  Alexander  Fleming,  the  discoverer  of  Penicillin.  He  is  therein  repre- 
sented as  holding  views  concerning  the  method  by  which  science  advances, 
which  I  find  interesting  and  worthy  of  further  consideration. 

He  thinks  the  individual  is  all-important  in  the  origination  of  new 
ideas  in  science.  These  ideas  result  from  long-continued  thought  and 
experimentation  on  the  part  of  a  gifted  and  well-trained  mind,  on  a  subject 
with  which  past  experience  has  made  it  well  acquainted. 

Fleming's  own  experience  illustrates  this.  He  had  spent  long  years  in 
the  study  of  immunity,  natural  and  acquired,  to  bacterial  infections. 
When  by  accident  a  spore  of  the  mold  Penicillium  notatum  got  into  one  of 
his  cultures  and  promptly  checked  growth  of  the  bacteria  therein,  he 
recognized  at  once  that  this  mold  was  capable  of  becoming  an  important 
agency  in  arresting  bacterial  infection. 

Here  was  the  germ  of  a  great  idea  but  it  took  10  years  of  work  on  his 
part,  and  that  of  biochemists,  to  fully  evaluate  the  idea  and  bring  it  to 
fruition. 

The  origination  of  a  revolutionary  new  idea  and  its  evaluation  are  quite 
distinct  processes.  For  the  first,  a  great  mind  alone  suffices.  For  the 
second  process  minds  of  lesser  capacity  may  be  adequate,  if  the  means 
employed  are  characterized  by  unbiased  integrity  and  sound  judgment. 

Let  us  turn  now  from  this  generalization  to  the  consideration  of  a 
specific  case,  the  status  of  experimental  biology  in  the  year  1900.  Four 
men  of  genius,  in  the  preceding  half  century,  had  formulated  ideas  of 
revolutionary  importance  in  biological  thinking.  These  men  were 
Darwin,  Weismann,  Mendel,  and  de  Vries. 

Darwin's  new  idea  was  the  origin  of  species  by  natural  selection  acting 
on  spontaneous  variations. 

Weismann's  new  idea  was  the  concept  of  germ-plasm  distinct  from 
body-plasm,  the  germ-plasm  alone  being  continuous  and  immortal, 
whereas  the  body-plasm  was  destined  to  die  when  the  life  of  the  individual 


«  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  27,  X954. 


595 


Journal    of  the  National  Cancer  Institute,   Vol.    15,  No.   3,  December   1954 


596  PKOCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 

came  to  an  end.  Weismann  conceived  the  continuity  of  life  to  depend 
upon  heredity,  the  basis  of  which  was  in  the  chromosomes  of  the  nuclei 
of  the  germ  cells. 

Mendel's  new  idea  antedated  Weismann's,  but  it  had  to  lie  dormant 
for  a  quarter  century  while  the  ideas  of  Darwin  and  Weismann  were 
conditioning  the  intellectual  soil  for  its  germination  and  fruition. 

Mendel's  idea,  which  his  careful  experiments  with  garden  peas  demon- 
strated to  be  correct,  was  that  of  the  mutual  independence  of  inherited 
characters  in  transmission.  He  did  not  attempt  to  localize  in  the  germ 
cells  the  material  basis  of  inheritance,  as  Weismann  later  did.  For  this, 
in  Mendel's  time,  knowledge  of  cytology  was  inadequate,  but  the  lack 
had  been  made  good  when  Weismann  formulated  his  ideas. 

De  Vries'  new  idea  was  that  of  mutation;  that  new  hereditary  characters 
come  into  existence  abruptly,  not  gradually  as  most  Darwinians  thought, 
and  that  thereafter  they  remain  constant. 

These  four  basic  concepts,  put  forward  by  minds  of  great  originality, 
each  working  largely  alone,  awaited  evaluation  by  biologists  in  the  year 
1900. 

At  that  time  it  was  my  good  fortune  to  be  alive  and  just  getting  a 
foothold  in  biological  investigations,  which  were  to  qualify  me  as  a  junior 
member  of  the  evaluating  team.  Senior  members  of  the  team,  who 
already  had  major  accomplishments  to  their  credit,  were:  E.  B.  Wilson, 
author  of  The  Cell,  and  T.  H.  Morgan,  experimental  zoologist,  at  Columbia 
University;  E.  G.  Conklin,  embryologist  at  Princeton  University;  and 
Ross  Harrison,  active  at  Yale  in  tissue  culture  and  transplantation.  These 
men  and  a  few  others  founded  the  Journal  of  Experimental  Zoology  for 
the  publication  of  their  findings. 

All  these  experimenters,  except  Harrison  and  me,  have  now  completed 
their  work  and  passed  off  the  stage.  Harrison  and  I,  fortunately  for  us, 
are  still  viewing  the  scene  from  the  wings. 

The  first  of  these  great  ideas  to  attain  evaluation,  nearly  unanimous  in 
its  favor,  was  the  evolution  theory  of  Darwin.  This  favorable  verdict  was 
expressed  in  a  symposium  in  1909  on  Fifty  Years  of  Darwin.  It  was  my 
privilege  to  have  a  part  in  this  symposium.  It  was  my  further  privilege 
in  1950  to  be  still  alive  and  to  join  in  the  celebration  of  Fifty  Years  of 
Validation  of  Mendel's  great  idea.  Today  I  rejoice  in  being  present  at  a 
celebration  of  the  founding  of  this  center  of  research  which  in  its  25-year 
history  has  contributed  much  to  the  validation  of  Mendel's  principles. 

The  second  basic  idea,  Weismann's  germ-plasm  theory,  received 
substantial  support  in  an  ovarian  transplantation  experiment  by  John  C. 
Phillips  and  me,  reported  in  1909.  We  showed  that  an  ovary  taken  from 
an  immature  black  guinea  pig  and  transplanted  into  the  body  of  an 
albino,  retained  there  its  distinctive  character — the  mature  ova  producing 
black  offspring  even  when  the  male  parent  as  well  as  the  foster  mother 
were  both  albinos. 

Journal   of   the   National   Canrer   Institute 


PROGEESS  IN  MAMMALIAN  GENETICS  AND  CANCER  597 

The  third  basic  idea  (that  of  Mendel)  of  the  independent  inheritance 
of  unit  characters,  was  the  prime  object  of  investigation  in  my  laboratory 
in  the  early  years  of  the  century. 

My  pupils  and  I  verified  it  first  in  the  case  of  albinism  in  mice,  later  in 
numerous  other  coat  characters  in  mice,  rats,  guinea  pigs,  and  rabbits. 
Parallel  investigations  were  at  this  time  being  made  in  England,  by  pupils 
of  Bateson,  that  furnished  additional  evidence  of  the  validity  of  Mendelian 
principles.  Indeed  it  would  be  more  correct  to  say  that  our  work  in 
America  complemented  and  extended  that  of  the  pupils  of  Bateson.  The 
initiative  was  taken  by  them. 

The  fourth  basic  idea  (that  of  de  Vries)  of  mutation  as  a  source  of  new 
and  stable  variations  received  strong  support  when  Muller,  a  pupil  of 
Morgan's,  showed  that  mutations  could  be  produced  artificially  by 
subjecting  germ  cells  to  bombardment  with  X  rays. 

What,  then,  we  may  inquire  was  the  status  of  Mammalian  Genetics  in 
1929,  when  the  Jackson  Memorial  Laboratory  was  founded? 

1)  It  had  been  shown  that  the  inheritance  of  the  coat  characters  of 
rodents  and  other  mammals  depended  upon  the  transmission  in  the  germ 
cells  of  material  bodies  which  had  come  to  be  called  genes  or  inheritance 
units. 

A  gene  might  by  mutation  suddenly  take  on  a  new  form.  The  new 
form  (termed  an  allele)  would  now  conform  with  Mendel's  law  in  relation 
to  the  antecedent  form,  being  either  dominant  over  it  or,  more  often, 
recessive  to  it.  New  combinations  of  genes  might  arise,  or  be  produced 
at  will,  as  a  result  of  mutations  in  two  or  more  different  genes.  When 
the  genes  involved  in  mutation  were  located  in  different  chromosomes, 
the  combinations  which  emerged  from  crosses  would  conform  in  their  fre- 
quencies with  the  laws  of  chance,  as  in  Mendel's  experiments  with  peas. 
But  when  two  genes  were  located  in  the  same  chromosome,  they  would 
have  a  tendency  to  stay  together  in  transmission,  a  phenomenon  called 
linkage,  not  encountered  by  Mendel,  but  first  observed  by  Bateson  and 
Punnett  in  sweetpeas,  and  later  both  observed  and  correctly  interpreted 
by  Morgan  and  his  pupils  in  Drosophila. 

In  mammals,  linkage  was  first  observed  independently  by  Haldane  and 
myself  in  mice.  Later,  cases  of  linkage  were  found  in  my  laboratory  in 
rats  and  rabbits  also.  Since  1929  the  study  of  linkage  in  rats  has  been 
one  of  my  major  interests.  Five  linkage  groups  have  now  been  identified. 
Meanwhile,  in  the  Jackson  Laboratory  the  number  of  linkage  groups  in 
mice  has  increased  to  10,  as  new  mutations  have  been  discovered.  Dr. 
George  Snell  deserves  credit  for  most  of  these  discoveries  of  linkage  in 
mice.  He  did  the  initial  work  in  this  field  at  the  Bussey  Institution, 
incorporating  the  results  in  his  doctor's  thesis. 

2)  The  genetic  interpretation  of  intermediate  or  blending  inheritance 
was  long  a  baffling  problem,  but  by  1929  it  had  been  resolved,  largely  on 
the  basis  of  experiments  with  plants,  into  a  case  of  multiple  genes  acting 
simultaneously  but  without  demonstrable  dominance  of  one  allele  over 
another. 

Vol.    15,   No.   3,   December    1954 


598  proceedings:  symposium  on  25  years  op 

An  intensive  study  had  been  made  of  size  inheritance  in  rabbits,  a 
typical  blending  character,  by  crosses  between  races  of  large  and  small 
body  size  respectively.  Attempts  to  locate  a  gene  affecting  body  size  in 
four  different  chromosomes  had  proved  fruitless.  However  P.  W. 
Gregory  and  I  were  able  to  show  from  an  embryological  study  that  genes 
located  in  the  chromosomes  must  be  assumed  to  be  involved  in  deter- 
mination of  body  size  in  rabbits,  since  size  difference  involves  a  difference 
in  rate  of  development  of  the  fertilized  eggs,  and  this  difference  is  influenced 
by  sperm  as  well  as  egg.  Large  race  egg  plus  small  race  sperm  has  sub- 
stantially the  same  rate  of  development  as  small  race  egg  plus  large  race 
sperm.  In  both  cases  intermediate  body  size  results.  Further  discoveries 
in  this  field  followed  the  founding  of  the  Jackson  Laboratory. 

3)  It  was  known  in  1929  that  close  inbreeding  of  mammals  does  not  of 
necessity  involve  racial  deterioration,  as  had  been  supposed  earlier  to  be 
true.  This  conclusion  had  been  reached  in  the  case  of  Drosophila  by  the 
50-generation  experiment  of  brother  X  sister  mating  made  in  my  labora- 
tory, the  first  breeding  experiment  in  which  this  now  famous  fly  was 
employed.  Later,  Dr.  Helen  Dean  King  verified  this  finding  for  mammals 
by  inbreeding  rats  for  a  like  extended  series  of  generations.  Theoretically 
such  prolonged  inbreeding  should  render  a  race  completely  homozygous 
and  thus  very  uniform  in  its  physiological  behavior. 

This  was  the  cornerstone  on  which  in  1929  Dr.  Little  built  the  program 
for  cancer  research  at  the  Jackson  Laboratory.  He  had  already  created 
a  long  inbred  strain  of  mice,  the  now  famous  d  br  non-agouti  race  (DBA), 
which  he  began  inbreeding  at  the  Bussey  Institution  in  1909,  and  which 
is  still  going  strong.  He  then  undertook  to  produce  other  inbred  strains 
of  different  but  uniform  genetic  constitution.  The  resulting  races  are  now 
being  used  because  of  their  known  uniform  genetic  constitution  and 
consequent  uniform  physiological  behavior  in  genetic  and  cancer  investi- 
gations throughout  the  world. 

With  this  equipment  of  generalized  ideas  about  mammalian  genetics, 
and  with  his  personal  experience  in  cancer  investigations  in  mice,  Dr. 
Little  was  ready  and  eager  in  1929  to  resume  the  investigations  which  had 
been  interrupted  to  some  extent  by  his  two  college  presidencies.  Only 
a  laboratory  with  an  assurance  of  adequate  financial  support  was  needed, 
and  this  fortunately  was  forthcoming — though  not  spontaneously,  but 
only  as  a  result  of  the  enthusiastic  personal  leadership,  which  has  always 
characterized  Dr.  Little. 

I  have  known  Dr.  Little  for  a  long  time,  longer  perhaps  than  anyone 
else  present.  We  first  came  into  contact  when  as  an  undergraduate  in 
Harvard  College  he  enrolled  in  my  course  in  genetics.  I  set  him  to 
studying  color  inheritance  in  mice  and  this  later  became  the  subject  of 
his  doctor's  thesis.  I  was  impressed  with  his  energy,  enthusiasm  and 
resourcefulness,  and  the  next  year  made  him  my  assistant  in  giving  the 
course.  He  was  then  captain  of  the  track  team,  his  own  feat  of  strength 
being,  I  believe,  putting  the  shot  farther  than  anyone  else.  His  enthu- 
siasm for  genetics  was  communicated  to  fellow  members  of  the  track 

Journal   of   the  National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


599 


team,  with  the  result  that  enrollment  in  my  course  increased  considerably. 
This  was  a  direct  evidence  of  his  gift  for  leadership.  Further  evidence  of 
it  followed  to  my  benefit. 

He  became  secretary  to  President  Lowell,  on  the  retirement  of  President 
Charles  W.  Eliot,  in  whose  administration  I  had  been  advanced  to  a 
Professorship  of  Zoology.  Little  must  have  whispered  in  the  president's 
ear  that  genetics  was  hot  stuff,  for  I  was  invited  to  give  a  course  of  lectures 
on  the  subject  at  the  Lowell  Institute  in  1910  and  these  lectures  formed 
the  basis  of  my  first  publication  in  book  form,  a  little  book  entitled 
Heredity  in  Relation  to  Evolution  and  Animal  Breeding,  New  York,  D. 
Appleton  &  Co.,  1913. 

Little's  mice,  with  the  rest  of  our  animal  colony,  were  transferred  in 
1909  to  the  Bussey  Institution  at  Forest  Hills,  alongside  of  the  Arnold 
Arboretum.  There  a  fruitful  association  was  formed  between  my  pupils 
and  those  of  a  brilliant  new  colleague,  the  late  and  lamented  E.  M.  East, 
Professor  of  Plant  Genetics. 

Little  and  I  were  joint  authors  of  several  short  publications  which  were 
outgrowths  of  his  studies  of  mice,  and  in  1913  his  full  report  on  the  subject 
was  published  (1)  with  excellent  colored  plates  in  Publication  No.  179 
of  the  Carnegie  Institution  of  Washington,  which  was  assisting  my  work 
as  a  Research  Associate. 

Little's  energy  and  enthusiasm  led  him  into  several  minor  projects, 
which  I  encouraged  him  to  undertake,  although  at  the  time  we  lacked 
adequate  facilities  for  their  completion.  These  projects  were:  crosses  of 
dogs  with  different  racial  conformation  and  instincts;  crosses  of  different 
races  of  domestic  pigeons ;  and  investigation  of  the  sterility  of  the  exception- 
ally produced  male  tortoise-shell  cat. 

The  pigeon  project,  which  we  were  unable  to  carry  out,  was  later  taken 
up  at  the  University  of  Wisconsin  with  notable  success  by  L.  J.  Cole  and 
M.  R.  Irwin. 

Little's  fondness  for  dogs  and  eagerness  to  experiment  with  them 
persisted  unrealized  until  recent  years  when  happily  facilities  for  such 
work  were  created  at  the  Jackson  Laboratory.  We  shall  hear  more  on 
this  subject  from  Dr.  Scott. 

Meanwhile,  Dr.  Tyzzer  at  Harvard  Medical  School  had  been  experi- 
menting with  crosses  of  Japanese  waltzing  mice  involving  susceptibility 
to  transplantation  of  a  tumor  peculiar  to  that  race  of  mice.  He  appealed 
to  us  at  the  Bussey  for  help  on  a  genetic  problem  that  was  baffling  him. 
I  recommended  Dr.  Little  to  him  as  the  person  best  qualified  to  render 
such  assistance,  and  this  Little  did  effectively.  So  this  is  how  he  became 
involved  in  the  study  of  cancer  in  mice — an  involvement  which  still 
continues. 

In  1917,  the  United  States  entered  World  War  I  and  both  Professor 
East  and  Dr.  Little  were  transferred  to  Washington  in  government 
service.  When  the  war  was  over,  Little  became  attached  for  a  time  to 
the  staff  of  C.  B.  Davenport,  head  of  the  department  of  genetics  of  the 
Carnegie  Institution  at  Cold  Spring  Harbor,  New  York.     But  he  presently 

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600  proceedings:  symposium  on  25  years  of 

moved  on  to  the  presidency  of  the  University  of  Maine,  and  later  to  that 
of  the  University  of  Michigan. 

But  he  was,  I  think,  homesick  for  research  in  an  institution  like  the 
Bussey  of  his  earlier  days.  And  since  time  on  the  Bussey  clock  was 
running  out,  there  was  nothing  to  do  but  found  such  an  institution  him- 
self. This  he  proceeded  to  do,  leaving  college  presidencies  to  those  more 
interested  in  academic  matters  than  in  biological  research. 

In  the  founding  and  fostering  of  the  Jackson  Memorial  Laboratory,  Dr. 
Little  has  spent  a  busy  quarter  century,  with  results  which  we  shall  learn 
more  about  in  the  next  2  days.  We  wish  for  him,  and  the  Jackson  Labo- 
ratory, continued  success  in  a  project  of  major  humanitarian  interest. 

Let  us  now  turn  from  a  consideration  of  the  state  of  mammalian  genetics 
prior  to  1929  to  a  brief  discussion  of  its  subsequent  development.  No 
revolutionary  new  discoveries  in  mammalian  genetics  have  been  made  in 
this  period,  but  progress  has  been  made  in  extending  and  clarifying 
knowledge  in  fields  already  explored  (2). 

A  greatly  increased  number  of  mutated  genes  is  now  known  in  mam- 
mals in  which  mutations  had  been  previously  reported,  as  for  example  in 
mice,  in  regard  to  which  animal  work  has  been  centered  in  the  Jackson 
Laboratory. 

Mutations  have  also  been  reported  in  many  additional  species;  for  ex- 
ample, the  mink,  which  is  now  being  reared  in  captivity  on  a  large  scale 
for  its  valuable  fur.  Thousands  of  mink  are  now  carefully  inspected  each 
year  by  breeders  and  furriers  anxious  to  discover  any  new  or  improved 
qualities  of  the  fur.  This  has  resulted  in  finding  many  mutations  that 
might  otherwise  have  been  overlooked. 

The  economic  value  of  some  of  these  mutations  has  led  to  the  formation 
of  a  Mutation  Mink  Breeders'  Association.  An  excellent  little  book  on 
Genetics  of  the  Ranch  Mink  has  been  written  by  Dr.  R.  M.  Shakelford  of 
the  University  of  Wisconsin,  a  pupil  of  the  late  lamented  Professor  L.  J. 
Cole.  Shakelford  finds  the  chromosome  number  of  the  mink  to  be  14, 
much  lower  than  that  of  laboratory  rodents,  which  is  upward  of  20.  He 
reports  on  14  different  mutations,  with  one  case  of  linkage. 

A  word  in  passing  might  be  said  about  Professor  Cole,  who  had  a  large 
share  in  the  advancement  of  mammalian  and  avian  genetics  in  his  lifetime 
through  teaching  and  research  of  distinguished  character.  While  studying 
at  Harvard  he  majored  in  a  zoological  field  which  did  not  directly  involve 
genetics,  but  became  interested  in  the  newly  developing  field  of  genetics, 
and  later  gave  to  it  his  entire  attention. 

A  similar  interest  in  genetics  was  aroused  in  the  late  Professor  H.  E. 
Walter  of  Brown  University,  who  (though  majoring  in  a  different  zoological 
field)  took  a  course  with  me  and  did  experimental  work  in  genetics  while 
studying  in  the  Graduate  School  in  Cambridge.  Later  he  became  eminent 
as  a  teacher  of  genetics  and  author  of  a  widely  used  textbook  of  genetics. 

Returning  from  this  digression,  let  us  inquire:  What  are  the  more  im- 
portant advances  in  mammalian  genetics  made  since  1929?  On  the 
theoretical  side,   these  relate  among  other  things  to  size  inheritance, 

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heterosis  and  statistics.     On  the  practical  side,  they  include  breeding  plans 
for  the  more  efficient  production  of  domestic  animals  of  the  types  desired. 

Size  Inheritance 

I  have  already  discussed  the  state  of  knowledge  of  size  inheritance  prior 
to  1929.  It  was  known  that  size  genes  existed  but  attempts  to  identify 
or  localize  them  in  linkage  systems  had  proved  fruitless. 

An  important  discovery  was  now  made  by  C.  V.  Green  (3),  a  charter 
member  of  the  staff  of  the  Jackson  Laboratory,  whose  brilliant  work  was 
brought  to  an  untimely  end  by  his  tragic  death  in  1935. 

Green  had  already  begun  at  Ann  Arbor,  under  Dr.  Little's  direction, 
the  study  of  a  cross  between  a  species  of  small  mouse  from  China,  Mus 
bactrianus,  and  Little's  much  larger  dilute-brown  inbred  strain  of  Mus 
musculus.  The  two  parental  races  differed  not  only  in  size  but  also  in 
three  color  genes,  A,  B  and  D,  of  which  the  small  race  carried  dominant 
alleles  and  the  large  race  recessive  alleles.  Green  showed  that,  in  F2  and 
backcross  populations,  individuals  which  were  homozygous  for  the  reces- 
sive alleles,  b  for  brown,  and  d  for  blue  dilution,  were  larger  than  those 
carrying  the  corresponding  dominants.  He  assumed  that  this  was  due  to 
linkage  between  a  gene,  or  genes,  for  large  size  and  color  genes  b  and  d. 

Since  Green  had  found  in  a  mouse  cross  what  I  had  previously  looked 
for  in  vain  in  rabbit-size  crosses,  I  now  undertook  to  repeat  Green's  ex- 
periment using  the  same  dilute-brown  race  used  by  Green  as  the  large 
size  parent,  stock  of  which  was  kindly  supplied  by  Dr.  Little.  For  the 
small  race  parent,  I  used,  not  the  captive  wild  race  of  Mus  bactrianus, 
but  one  of  its  supposed  domestic  derivatives,  the  Japanese  waltzing  mouse, 
of  which  W.  H.  Gates  was  making  an  intensive  study  in  my  laboratory. 
This  investigation  was  shared  by  my  pupils  at  the  Bussey  Institution — 
W.  H.  Gates,  S.  C.  Reed  and  L.  W.  Law. 

We  confirmed  Green's  findings  as  to  fact,  but  gave  them  a  different 
interpretation  (4).  Our  interpretation  was  that  color  genes,  when  they 
are  associated  with  size  differences,  owe  the  association,  not  to  linkage 
with  specific  genes  for  size,  but  to  direct  physiological  action  of  their  own. 
This  interpretation  was  verified  in  further  studies  of  size  crosses  made  by 
me  in  Berkeley,  after  my  retirement  and  years  after  Green's  death,  again 
largely  with  mouse  stocks  kindly  supplied  for  the  purpose  by  the  Jackson 
Laboratory. 

I  used  in  these  studies  three  of  the  same  criteria  of  size  used  by  Green; 
namely,  body  weight,  body  length,  and  tail  length.  I  found  that  some 
color  genes  are  without  any  discoverable  influence  on  body  size,  others 
increase  it,  and  still  others  decrease  it.  Gene  mutations  with  no  dis- 
coverable influence  on  body  size  are  non-agouti  and  albino;  those  which 
increase  body  size  by  all  three  criteria  are  brown,  blue  dilution,  and 
yellow,  the  last  being  a  dominant  mutation,  effective  only  when  heter- 
ozygous as  is  well  known  (the  homozygote  being  lethal).     Mutations 


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602  proceedings:  symposium  on  25  years  op 

which  decrease  body  size  by  all  three  criteria  are  pink-eye,  pink-eye2, 
leaden,  short  ear,  and  dwarf,  all  recessives. 

Another  finding  of  Green's  (5)  which  I  was  able  later  to  verify  was  this. 
Certain  genes  that  have  an  influence  on  general  body  size  show  greater 
influence  on  some  body  regions  than  on  others.  They  thus  show  a 
specific  local  influence  on  growth,  as  well  as  a  lesser  general  influence. 
This  is  well  illustrated  in  the  case  of  the  mutation  d,  blue  dilution,  which 
Green  in  his  original  experiment  showed  to  increase  general  body  size, 
but  to  increase  tail  length  out  of  all  proportion  to  body  weight  and  body 
length.  This  finding  of  Green's  I  was  able  fully  to  confirm.  The  muta- 
tion short  ear,  as  its  name  indicates,  has  a  specific  local  influence  in 
reducing  ear  size,  greater  than  its  effect  on  body  weight  and  body  length, 
as  had  been  shown  by  Snell. 

Both  Green  and  I,  in  our  studies  of  size  inheritance  in  mice,  were  in- 
fluenced by  the  conclusions  reached  by  Wright  in  a  statistical  analysis  of 
my  data  on  size  crosses  in  rabbits  (6).  Wright  recognized  the  existence 
of  general,  group,  and  special  size  factors.  What  we  had  shown  was 
that  general  growth  factors  (such  as  mutation  d)  may  also  function  as 
special  growth  factors  in  regard  to  tail  length  or  cranial  dimensions 
(Green),  or  ear  length  (Snell). 

Wright's  conclusions  are  unquestionably  still  valid  with  regard  to  the 
categories  of  genetic  influence  in  size  being  mostly  general,  but  to  some 
extent  exerted  regionally  on  groups  of  parts,  or  on  individual  special  parts. 
However,  it  does  not  follow  of  necessity  that  these  different  kinds  of 
influence  are  exerted  by  different  genes. 

To  find  out  whether  color  genes  influence  size  in  mammals  other  than 
mice,  I  extended  my  studies  in  Berkeley  to  rats  and  rabbits  also. 

The  color  mutation,  brown,  which  in  mice  had  been  found  to  have 
greater  influence  in  increasing  body  size  than  any  other  investigated,  was 
found  to  be  a  gene  for  increased  body  size  in  rabbits  and  in  rats  also.  I 
am  curious  to  know  whether  this  is  true  also  in  dogs,  but  have  had  no 
opportunity  to  investigate  it.  Perhaps  you  at  the  Jackson  Laboratory 
will  have. 

Heterozygous  Phenotypes 

In  animal  breeding  a  striking  difference  in  phenotype  (appearance)  be- 
tween a  heterozygote  and  a  homozygote  for  a  particular  color  gene  has 
been  well  known  ever  since  W.  Bateson  described  the  genetics  of  the  Blue 
Andalusian  fowl.  In  such  a  case,  if  breeders  happen  to  prefer  the  heter- 
ozygote, they  are  dealing  with  an  unfixable  variety,  one  which  will  breed 
true  only  to  the  extent  of  half  of  the  progeny,  which  in  turn  will  breed 
in  the  same  way.  I  discovered  man}7  years  ago,  that  this  is  true  in  the 
English  rabbit,  where  the  fanciers'  ideal  is  an  animal  with  a  maximum 
number  of  small  spots  well  distributed  over  the  body,  as  in  a  Dalmatian 
(coach)  dog. 

When  standard  English  rabbits  are  interbred,  they  produce  only  about 
50  percent  of  standard  English  young,  together  with  25  percent  of  un- 

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spotted  (self)  young,  and  25  percent  of  animals  with  too  few  spots  to 
conform  with  the  English  standard.  These  last  are  known  as  Charlie 
Chaplins,  being  regarded  as  a  joke,  so  far  as  conformation  with  the 
standard  for  English  spotting  is  concerned.  Both  the  Charlie  Chaplins 
and  the  selfs  are  homozygotes  and,  if  crossed  with  each  other,  will  produce 
100  percent  of  heterozygotes  of  the  standard  English  type. 

But  this  short-cut  in  mass  production  of  standard  English  is  not  recom- 
mended to  rabbit  fanciers  who  desire  to  produce  show  specimens,  since 
fine  points  in  the  standard  pattern,  due  to  modifying  genes,  may  be  lost 
in  the  process  of  "sight  unseen"  mass  production. 

I  have  found  a  parallel  case  to  that  of  the  English  rabbit  in  the  Palomino 
horse,  since  my  migration  to  California  where  Palominos  are  very  popular. 
The  breeder's  ideal  Palomino  has  a  golden-yellow  body  color  with  white 
mane  and  tail.  The  actual  Palomino  which  most  nearly  approximates 
this  ideal  is  heterozygous  for  a  dominant-dilution  gene  (D)  on  a  very 
specific  background  of  other  color  genes.  A  horse  homozygous  for  D  is 
too  dilute  to  be  desirable.  It  is  called  an  albino.  In  addition  to  having 
a  very  pale,  ivory-white  coat,  it  has  a  pink  skin  and  blue  eyes.  Palominos 
interbred  produce  50  percent  of  Palominos,  and  25  percent  each  of  albinos 
and  horses  of  full  color.  Both  of  these  latter  classes  are  homozygous  as 
regards  gene  D — one  having  it,  the  other  lacking  it.  Breeding  them 
together  produces  100  percent  of  heterozygotes,  the  Palomino  genotype. 
But  again,  as  in  the  case  of  the  English  rabbit,  this  practice  is  not  to  be 
recommended  to  fanciers  who  want  to  produce  show  specimens,  since  such 
can  be  produced  only  on  a  specific  background  of  color  genes. 

The  genetic  background  on  which  the  dilution  gene  acts  in  the  produc- 
tion of  the  preferred  type  of  Palomino,  is  identical  with  that  of  a  sorrel 
horse  having  a  light  mane  and  tail.  It  is  homozygous  for  the  recessive 
brown  gene  (6)  which  distinguishes  chestnut  and  liver  from  bay  and  black. 
The  distribution  of  its  brown  pigment  is  restricted  by  two  other  genes, 
the  dominant  ancestral-pattern  gene  (A)  which  makes  the  difference 
between  bay  and  black,  and  the  recessive  restriction  gene  (e)  which,  when 
homozygous,  replaces  black-brown  pigment  with  red-yellow  pigment  in 
the  coat  generally.  The  dilution  gene  (D)  when  heterozygous  reduces  the 
intensity  of  the  red  body-color  of  a  sorrel  to  the  golden  yellow  of  a 
Palomino ;  when  homozygous  it  reduces  the  red  body-color  too  much  to 
the  near  white  of  the  so-called  albino.  Its  effect,  when  heterozygous,  on 
the  light  mane  of  a  sorrel  is  to  render  it  still  lighter,  approximating  the 
"white"  of  the  Palomino  standard. 

The  net  results  of  the  interaction  of  color  genes  in  this  heterozygous 
genotype  is  a  horse  with  a  diluted  red  coat,  described  by  the  fancier  as 
"golden  yellow,  the  color  of  newly  minted  gold,"  and  an  almost  "white 
mane  and  tail." 

The  preference  of  cattle  breeders  for  a  type  of  conformation  which 
(without  their  knowing  it)  belongs  to  a  heterozygous  genotype,  may  lead 
to  disastrous  consequences.  This  has  been  shown  by  Gregory  et  at.  (7)  in 
the  case  of  cattle  which  are  heterozygous  for  a  dwarf  gene.     Homozygous 


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604  proceedings:  symposium  on  25  years  of 

dwarfs  are  either  lethal  and  so  a  total  loss,  or  of  greatly  reduced  value,  yet 
they  constitute  one  quarter  of  the  calves  produced  by  heterozygous 
parents  of  the  preferred  phenotype.  The  detection  of  heterozygotes  for 
dwarfness  among  herd  sires  thus  becomes  important  to  cattle  breeders, 
who  may  then  take  measures  to  avoid  mating  a  heterozygous  bull  to 
heterozygous  cows. 

Heterosis 

Important  advances  have  been  made  since  1929  in  regard  to  heterosis 
(hybrid  vigor).  The  basic  discoveries  in  this  field  have  been  made  in  the 
study  of  plants  or  animals  such  as  Drosophila  and  Habrobracon.  But 
these  have  found  important  applications  in  animal  breeding. 

Everyone  is  familiar  with  the  case  of  hybrid  corn,  an  astonishing  ex- 
ample of  the  improvement  in  an  important  field  crop  to  be  derived  from 
prolonged  inbreeding  followed  by  a  single  generation  of  cross  breeding. 

Breeders  of  pigs,  following  the  lead  of  the  corn  breeders  who  supply 
them  with  more  abundant  feed  for  their  pigs,  have  secured  similar  though 
less  striking  benefits  from  crossing  purebred  breeds  of  swine. 

In  the  early  days  of  the  study  in  maize  of  the  effects  of  inbreeding  and 
cross  breeding  made  by  Shull  and  East,  it  was  thought  that  the  increased 
vigor  of  hybrids  was  due  exclusively  to  the  physiological  superiority  of 
heterozygotes  over  homozygotes.  Hence  the  term  heterosis  was  coined  as 
a  short  name  for  heterozygosis. 

But  further  studies  (£),  particularly  those  of  East  and  his  pupils,  have 
given  to  heterosis  a  more  extended  significance.  It  is  now  thought  to  in- 
volve four  different  agencies. 

1)  Dominance  of  favorable  genes  over  their  unfavorable  recessive 
alleles.  This  is  ordinary  dominance.  In  this  case,  a  homozygote  has  the 
same  physiological  action  as  a  heterozygote. 

2)  Over-dominance  of  favorable  genes,  which  renders  their  action 
greater  as  heterozygotes  than  as  homozygotes.  This  is  the  original 
conception  of  hybrid  vigor. 

3)  Linkage  of  dominant  favorable  genes  with  recessive  unfavorable 
genes.  Under  prolonged  inbreeding  both  sorts  will  become  fully  operative 
and  the  net  result  will  be  deterioration  to  the  extent  that  unfavorable 
genetic  action  surpasses  favorable  genetic  action.  Crossing  of  two  differ- 
ent inbred  lines  will  suppress  the  action  of  all  unfavorable  recessive  genes 
carried  by  both  parents  and  allow  unhampered  action  of  all  favorable 
genes  whether  ordinary  dominants  or  over-dominants.  A  maximum  of 
hybrid  vigor  will  result. 

4)  Gene  interaction.  In  some  cases  favorable  action  of  a  gene  may  be 
increased  or  diminished  by  interaction  with  a  different  gene,  particularly 
with  one  located  in  a  different  chromosome. 

Opinions  differ  as  to  the  relative  importance  of  these  four  agencies. 
It  is  probable  that  linked  recessive  unfavorable  genes  are  of  commoner 
occurrence  in  maize  than  in  mammals,  and  their  suppression  of  major 
importance  in  hybrid  corn  but  not  in  cross-bred  swine,  in  which  over- 
dominance  is  more  probably  of  major  importance. 

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Linkage 

In  the  field  of  study  of  linkage  in  mammals,  in  which  I  am  currently 
much  interested,  an  important  advance  is  sure  to  result  from  a  finding  by 
Kramer  et  al.  (9)  in  barley  that  two  linkage  groups,  seemingly  independent, 
may  exist  at  opposite  ends  of  the  same  chromosome,  if  a  sufficiently  long 
portion  of  the  chromosome  devoid  of  identified  genetic  loci  intervenes 
between  them. 

That  this  principle  is  valid  for  mammals  is  shown  by  an  unpublished 
investigation  of  the  linkage  interrelations  of  three  genes  in  Chromosome  IV 
of  the  rat.  The  genes  in  question  are  wobbly,  hairless,  and  naked.  The 
gene  hairless  lies  between  the  other  two,  about  equally  distant  from  each, 
and  is  clearly  linked  with  both.  If  a  test  for  linkage  between  the  end 
genes  had  been  made,  without  knowledge  of  the  existence  of  the  inter- 
mediate gene  hairless,  the  erroneous  conclusion  would  have  been  reached 
that  these  genes  were  located  in  different  chromosomes,  since  they  give  no 
indication  of  linkage  with  each  other. 

Consequently  many  earlier  linkage  tests  that  gave  negative  results  must 
be  regarded^as  inconclusive  and  will  merit  re-examination  as  new  genes 
are  discovered  and  their  linkage  relations  studied. 


Statistics 

I  shall  not  undertake  the  discussion  of  advances  made  in  the  last  quarter 
century  in  the  field  of  statistics,  though  these  are  of  great  importance  in 
the  field  of  mammalian  genetics  in  general,  of  human  genetics  in  particular, 
and  in  discussions  of  evolution — as  well  as  in  practical  plans  for  the 
breeding  of  plants  and  animals. 

Masters  in  these  several  fields  are  with  us  here  and  we  shall  await  the 
statement  of  their  findings  in  the  programs  to  follow. 

References 

(1)  Little,  C.  C:  Experimental  studies  of  the  inheritance  of  color  in  mice.     Publ. 

No.  179,  Wash.,  D.  C,  Carnegie  Institution  of  Washington,  1913. 
{2)  Dunn,  L.  C:  Genetics  in  the  20th  Century.     New  York,  The  Macmillan  Co., 

1951,  634  pp. 
(3)   Green,  C.  V.:  Linkage  in  size  inheritance.     Am.  Nat.  65:  502-511,  1931. 
U)   Castle,  W.  E.:  Size  inheritance.     Am.  Nat.  75:  488-498,  1941. 
(5)   Green,  C.  V.:  Further  evidence  of  linkage  in  size  inheritance.     Am.  Nat.  67: 

377-380,  1933. 
{6)  Wright,   S.:   General,   group,  and  special  size  factors.     Genetics   17:  603-619, 

1932. 
(7)  Gregory,  P.   W.,   Roubicek,   C.  B.,   Carroll,  F.  D.,  Stratton,  P.   O.,  and 

Hilston,  N.  W.:  Inheritance  of  bovine  dwarfism  and  the  detection  of  heterozy- 

gotes.     Hilgardia  22:  407-450,  1953. 
(5)   Gowen,  J.  W.:  Heterosis.     Ames,  Iowa,  Iowa  State  College  Press,  1952,  552  pp. 
(9)  Kramer,  H.  H.,  Veyl,  R.,  and  Hanson,  W.  D.:  The  association  of  two  genetic 

linkage  groups  in  barley  with  one  chromosome.    Genetics  39:   159-168,  1954. 


Vol.   15,  No.   S, 


1954 


606 


proceedings:  symposium 


Comments 

Dr.  C.  C.  Little 

Dr.  Castle  was  too  modest  to  tell  you  one  of  his  most  brilliant  pieces  of  work  which 
was  early  in  the  game,  I  think  in  1903,  when  he  recognized  that  the  approximate 
equality  of  the  sexes  was  probably  due  to  the  backcross  type  of  Mendelian  one  to  one 
ratio  cross,  thereby  placing  the  theory  of  sex  determination  on  the  basis  of  Mendelism. 
The  fact  was  later  confirmed  by  McClure  and  others  by  cytological  investigations. 
Dr.  Castle  said  he  was  "in  the  wings."  Well,  the  "wings"  that  he  has  have  many 
strong  feathers  and  those  of  us  who  have  watched  his  flight  in  science  will  do  so  I  am 
sure  for  a  great  many  more  years  to  come  with  very  deep  affection  and  very  great 
admiration.     It  is  the  flight  of  a  very  kind  and  self-effacing  eagle. 


Session  III.  Genetic  Control  of  Devel- 
opmental Patterns 


Chairman,  Dr.  W.  H.  Gates,  Professor  Zoology, 
Entomology,  and  Physiology,  Louisiana  State  Uni- 
versity, Baton  Rouge,  La. 


Speaker:  Dr.  Earl  L.  Green 

Quantitative  Genetics  of  Skeletal  Variations  in  the  Mouse.     I.  Crosses 

Between  Three  Short-Ear  Strains  (P,  NB,  SEC/2) 
Discusser:  Dr.  Paul  B.  Sawin 

Speaker:  Dr.  Salome  Gluecksohn-Waelsch 

Genetic   Control  of  Embryonic   Growth  and  Differentiation 
Discusser:  Dr.  Morris  Smithberg 

Speaker:  Dr.  Meredith  N.  Runner 

Inheritance  of  Susceptibility  to  Congenital  Deformity — Embryonic  Insta- 
bility 
Discusser:  Dr.  Donald  W.  Bailey 


60; 


Quantitative  Genetics  of  Skeletal  Vari- 
ations in  the  Mouse.  I.  Crosses  Be- 
tween Three  Short-Ear  Strains  (P,  NB, 

SEC/2)  *• 2 


Earl  L.  Green,  Department  of  Zoology,  Ohio  State 
University,  Columbus,  Ohio,  and  Division  of  Biology 
and  Medicine,  U.  S.  Atomic  Energy  Commission, 
Washington,  D.  C. 


Laboratory  strains  of  the  house  mouse  are  rich  in  variations  of  skeletal 
components,  both  within  and  between  strains.  The  variable  sites  are 
distributed  over  the  appendages,  the  pelvic  and  pectoral  girdles,  and  the 
axial  skeleton  from  the  nose  to  the  tip  of  the  tail.  Some  sites  present 
a  discrete  presence  and  absence  variable,  such  as  the  presence  or  absence 
of  the  omosternal  bones;  some  present  a  continuous  measurable  variable, 
such  as  the  length  of  the  nasal  bones ;  and  some  present  a  discrete  numeri- 
cal variable,  such  as  the  number  of  digits  or  the  number  of  vertebrae. 

Variations  in  the  skeleton  of  vertebrates  have  been  the  subject  of 
numerous  investigations.  Anatomists,  embryologists,  and  geneticists 
have  recorded  and  studied  the  variations  in  the  skeleton  of  horses,  sheep, 
pigs,  dogs,  and  of  various  primates,  including  man.  It  is  fitting  to  note, 
during  this  25th  year  of  the  Jackson  Memorial  Laboratory,  that  my  own 
interest  in  the  genetic  and  nongenetic  causes  of  variation  in  the  skeleton 
of  the  mouse  started  at  the  Jackson  Laboratory.  In  the  summer  of  1938, 
following  the  lead  of  Dr.  Paul  B.  Sawin,  who  had  discovered  interesting 
variations  in  the  skeleton  of  the  rabbit,  I  visited  the  Jackson  Laboratory 
to  survey  the  available  inbred  strains  of  mice  for  skeletal  variations.  I 
found  that  there  were  marked  differences  between  inbred  strains  as  well 
as  considerable  variability  within  strains  with  respect  to  the  composition 
of  the  axial  skeleton.  Some  strains,  such  as  C57BL,  were  found  to  have 
13  thoracic  and  6  lumbar  vertebrae.  Some  other  strains,  such  as  DBA 
and  C3H,  were  characterized  by  13  thoracic  and  5  lumbar  vertebrae. 
One  strain,  P,  had  12  thoracic  and  6  lumbar  vertebrae;  and  one  strain, 
BALB/c,  had  13  or  14  thoracic  and  6  or  5  lumbar  vertebrae.  No  strain 
studied  then  or  since  has  been  absolutely  uniform  with  respect  to  the 


1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  28, 1954. 

*  This  investigation  was  supported  in  part  from  funds  granted  to  the  Ohio  State  University  by  the  Research 
Foundation  for  aid  in  fundamental  research,  and  in  part  by  research  grants  (Nos.  RG-2993  and  A-544)  from  the 
National  Institutes  of  Health,  U.  S.  Public  Health  Service. 

609 


Journal    of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    19S4 


610  proceedings:  symposium  on  25  years  of 

composition  of  the  axial  skeleton.     Usually,  5  to  10  percent  of  the  mice 
in  each  strain  have  turned  out  to  depart  from  the  principal  type. 

Questions 

The  multiplicity  of  forms  in  the  skeleton  or  any  other  organ  system 
attracts  our  attention  for  several  reasons. 

First,  what  is  the  genetic  basis  for  the  differences  between  strains? 
Do  the  differences  "mendelize"  in  any  familiar  pattern?  Are  the  differ- 
ences associated  with  any  named  genes? 

Second,  is  the  variability  within  inbred  strains  exclusively  nongenetic? 
What  is  the  relative  importance  of  genetic  and  nongenetic  causes  of  varia- 
bility within  strains?  Can  the  nongenetic  causes  of  variability  be 
identified? 

Third,  in  what  ways  are  the  developmental  patterns  different  for  mice 
of  different  skeletal  types?    How  do  the  differences  arise? 

Fourth,  what  experimental  agents,  physical  or  physiological,  applied  to 
eggs  or  to  developing  embryos  can  alter  the  course  of  development 
sufficiently  to  produce  mice  with  different  skeletal  types? 

Fifth,  what  are  the  evolutionary  implications  of  the  extensive  poly- 
morphism in  the  skeleton  of  laboratory  strains  of  mice? 

Our  present  knowledge  is  too  meager  to  answer  any  of  these  questions 
with  assurance.  Partial  answers  to  some  of  the  questions  have  been 
provided  by  several  previous  studies.  The  numerical  variations  at  the 
lumbosacral  border  in  strain  BALB/c  mice  have  been  studied  by  Green 
(1)  and  in  the  C57BL  strain  by  Searle  (2).  In  both  strains,  nongenetic 
causes  greatly  outweigh  genetic  causes  of  variation.  Crosses  between 
strains  have  shown  that  both  genetic  and  nongenetic  causes  are  concerned 
in  variation  at  the  lumbosacral  border.  In  a  cross  between  C57BL  and 
BALB/c,  it  was  found  that  3  or  more  blocks  of  genes  are  required  to 
account  for  the  difference  between  the  strains  [Green  (3)].  In  a  cross 
between  C3H  and  C57BL,  a  significant  difference  between  reciprocal 
hybrids  appeared,  with  the  hybrid  offspring  tending  to  resemble  the 
strain  of  the  female  parent  [Green  and  Russell  (4)]. 

An  attempt  to  identify  the  nongenetic  causes  of  variation  at  the  lumbo- 
sacral border  in  the  BALB/c  strain  yielded  negative  results  for  all  forces, 
among  those  tested,  acting  on  litters  as  units  except  for  age  of  mother, 
and  for  all  forces  tested  acting  on  horn-mates  as  units.  It  appeared  that 
whatever  the  agents  may  be  they  act  upon  individuals  as  units  [Green 
and  Green  (5)]. 

Named  genes  in  the  mouse  have  been  discovered  to  affect  the  numerical 
variation  in  parts  of  the  axial  skeleton.  These  include,  among  several 
others:  anury  [Dobrovolskaia-Zavadskaia  (6);  Chesley  and  Dunn  (7)], 
Danforth's  short  tail  [Gluecksohn-Schoenheimer  (8)],  stub  [Dunn  and 
Gluecksohn-Schoenheimer  (9)],  screw  tail  [MacDowell  et  al.  (10)],  short 
ear  [Green  and  Green  (11)],  luxate  [Carter  (12)],  and  luxoid  [M.  C.  Green 
(IS)}. 

Journal    of    the    National    Cancer    Institute 


PROGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


611 


The  purpose  of  this  paper  is  to  describe  some  breeding  experiments 
aimed  at  answering  the  first  question — the  genetic  basis  of  differences 
between  inbred  strains  of  mice.  One  may  approach  this  question  in 
either  of  two  ways.  The  variable  sites  may  be  studied  singly  or  in  com- 
bination. Given  that  the  time  and  effort  to  be  devoted  to  the  study  are 
in  some  way  limited,  one  may  study  a  large  number  of  variable  sites  in 
the  mice  of  the  parental,  hybrid,  and  later  segregating  generations  at  the 
expense  of  being  unable  to  study  large  numbers  of  mice.  Or  one  may 
study  a  large  number  of  mice  at  the  expense  of  reducing  the  number  of 
variable  sites  to  one,  two,  or  three.  For  the  purpose  of  this  discussion, 
one  site  only,  the  position  of  the  sacrum,  will  be  referred  to. 

The  position  of  the  sacrum  may  be  most  easily  defined  by  the  number 
of  vertebrae  which  precede  the  first  sacral  vertebra.  The  number  of 
presacral  vertebrae — cervical,  thoracic,  and  lumbar — in  the  mouse  may  be 

25,  26  or  27.  Numbers  greater  or  smaller  than  those  are  extremely  rare. 
Some  vertebrae  are  asymmetrical  so  that  they  resemble  sacral  vertebrae 
on  one  side,  lumbar  vertebrae  on  the  other.  The  position  of  the  sacrum 
may  still  be  denoted  by  the  number  of  presacral  vertebrae  as  25/26  or 
26/25,  or  as  26/27  or  27/26,  where  the  numbers  are  given  as  Right/Left. 
When  the  26th  vertebra  is  asymmetrical,  as  in  the  first  two  cases,  the 
class  of  mice  of  this  sort  may  be  denoted  as  Ax;  when  the  27th  vertebra 
is  asymmetrical,  as  in  the  latter  two  cases,  the  class  of  mice  may  be 
denoted  as  A2.  In  a  few  mice  in  crosses  between  strains,  symmetrical 
vertebrae  occur  at  the  lumbosacral  border  which  are  neither  clearly 
lumbar  nor  clearly  sacral  types.  These  vertebrae  have  transverse  proc- 
esses which  do  not  point  forward  and  downward  like  those  of  lumbar 
vertebrae,  and  which  do  not  extend  sidewise  to  articulate  with  the  in- 
nominate bones  like  those  of  sacral  vertebrae.  These  few  mice  have 
been  classed  with  the  Ai  or  A2  types,  on  the  ground  that  Ai  and  A2  repre- 
sent intermediate,  as  well  as  asymmetrical,  classes.  With  this  notation 
there  are  five  sacral  positions  with  25,  Ax,  26,  A2,  and  27  presacral  vertebrae. 

Differences  Between  Strains  in  Position  of  Sacrum 

Seven  inbred  strains  have  been  recently  sampled.  Each  strain  exhibits 
some  variation  in  the  position  of  the  sacrum.  In  no  two  of  the  strains  is 
the  variation  identical.  The  proportions  of  mice  with  25,  Ai,  26,  A2,  and 
27  presacral  vertebrae  change  from  strain  to  strain  (table  1).  There  are 
some  similarities,  however.  Three  of  the  strains  (P,  C3H,  DBA)  have 
high  percentages  of  mice  with  25  presacral  vertebrae;  two  of  the 
strains  (C57BL,  NB)  have  high  percentages  of  mice  with  26  presacral 
vertebrae.     The  other  two  strains  have  sizable  percentages  of  mice  with 

26,  A2,  and  27  presacral  vertebrae. 

Some  sources  of  variability,  both  in  inbred  strains  and  in  crossbred 
generations,  may  be  detected  at  once  when  tables  are  prepared  with  more 
detail  than  table  1.  Among  these  sources  may  be  mentioned  1)  a  differ- 
ence between  sexes  with  females  frequently  having  more  and  males 
fewer  presacral  vertebrae;  2)   a  difference  between  reciprocal  hybrids, 


Vol.    15,   No.   3,   December    1954 


612  proceedings:  symposium  on  25  years  or 

Table  1. — Distribution  of  skeletal  types  in  seven  inbred  strains  of  mice 


Strain 

Presacral  vertebrae 

Total 

25 

A, 

26 

A2 

27 

P 

Per- 
cent 
92.2 
92.6 

77.7 
3.6 

Per- 
cent 
4.4 
6.4 
9.4 
6.7 

Per- 
cent 
3.4 
1.0 
12.9 
89.7 
99.2 
52.0 
14.7 

Per- 
cent 

Per- 
cent 

384 

C3H 

203 

DBA/2L. .                                   

139 

C57BL/10.. 

419 

NB 

0.8 
24.3 
17.3 

23.' 6' 
67.9 

245 

BALB/c 

296 

SEC/2  and  2d 

0.  1 

941 

sometimes  matroclinous,  sometimes  patroclinous ;  and  3)  a  difference  in 
frequency  of  the  two  asymmetrical  types  which  make  up  Ai,  such  that  the 
25/26  type  occurs  two  or  three  times  more  frequently  than  26/25,  and  of 
the  asymmetrical  types  in  A2,  such  that  the  26/27  type  occurs  less  frequently 
than  27/26. 

Breeding  Experiments 

Nine  crosses  between  selected  pairs  of  strains  have  been  made  of  the  2 1 
possible  crosses  between  the  seven  strains  (table  2).  These  9  crosses 
may  be  arranged  in  groups  of  three,  each  group  involving  the  same  three 
strains.     To  be  specific, 


Group  1  involves 
"      2       " 
u      3 

tt  £  u 

"      5 


P,  NB,  SEC/2 
C3H,  DBA,  C57BL 
DBA,  C57BL,  BALB/c 
C3H,  DBA,  BALB/c 
C3H,  C57BL,  BALB/c 


(Crosses  1,  3,  7) 
(Crosses  2,  5,  8) 
(Crosses  0,  4,  5) 
(Crosses  4,  6,  8) 
(Crosses  0,  2,  6) 


The  production  of  mice  is  complete  in  crosses  0,  1,  2,  3,  4,  5,  7,  but  is 
still  in  progress  in  crosses  6  and  8,  and  in  crosses  2A  and  3A,  which  are 
repetitions  of  crosses  2  and  3.  The  results  of  crosses  0  and  2  have  been 
described  by  Green  (S)  and  Green  and  Russell  (4).  The  first  and  third 
groups  of  crosses  are  therefore  the  only  groups  for  which  all  three  crosses 
are  complete.     The  data  of  the  first  group  only  are  presented  in  this  paper. 

The  two  strains,  denoted  by  Pi  and  P2,  in  a  given  cross  were  mated 
reciprocally,  and  the  hybrids  so  produced  were  in  turn  mated  to  produce 
other  generations  in  accordance  with  the  scheme  in  text-figure  1.  All 
of  the  indicated  generations  were  produced  in  cross  1,  P  X  SEC/2;  Fi, 
F2,  Bi,  and  B2  were  produced  in  cross  3,  P  X  NB;  and  all  but  Bm  and  B222 
were  produced  in  cross  7,  NB  X  SEC/2.  Crosses  1  and  7  were  designed 
with  replications.  In  cross  1,  10  matings  and  their  reciprocals  made  up 
by  sibs  were  used  to  produce  the  Fx  generation,  and  4  of  these  blocks 
were  systematically  expanded  through  the  segregating  generations.  In 
cross  7,  14  blocks  were  started,  13  contributed  to  the  Fi  generation  and 
only  one  block  was  successfully  expanded  through  the  segregating  genera- 
tions.    In   cross    3,    the   block  system  was  not  used.     The    experiment 


Journal    of   the  National   Caneer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 
Table  2. — Schematic  diagram  of  crosses  between  inbred  strains 


613 


Strain 


Presacral 
vertebrae 


Cross  number 


C3H 

DBA 

C57BL 

NB 

BALB/c 

SEC/2  and  2d, 


25 
25 
25 
26 
26 
26,27 
27 


<>C> 


Cross  0     C57BL  X  BALB/c 
"IP  X  SEC/2 

"    2     C3H      X  C57BL 
"    3    P  X  NB 

"    4     DBA     X  BALB/c 


Cross  5  DBA  X  C57BL 

"    6  C3H  X  BALB/c 

"    7  NB  X  SEC/2 

"    8  C3H  X  DBA 


was  started  with  5  matings  of  type  cfP  X  9NB  and  5  matings  of  the  type 
cfNB  X  9P,  with  no  special  attention  to  matching  the  reciprocals. 

Pi P2 


Pi 


-Br 


■B, 


Pr 


P2 


B, 


F, 


B2- 


B9 


P2 


B, 


P2 


Bui  B222 

Text-figure  1. — Diagram  of  mating  system  for  identification  of  various  generations. 

All  three  of  the  strains  used  in  crosses  in  group  1  are  short-ear  strains. 

The  named  genes  of  the  three  strains  are: 

P:     aa    bb     CC        dd     pp      sese 
NB:     aa     bb    cehceb     dd      pp 


QTrrvo.  Jaa    bb    cehceh     dd      PP    sese 
tiih^/z.  |oo     bf)    ceh(,eh    Dd     pp    sese 


Vol.    15,   No.   3,   December   1954 


614 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


The  SEC/2  strain  contains  both  dense  Dd  and  dilute  dd  mice,  but  only 
dd  mice  were  used  in  the  crosses  with  P  and  NB. 

Results 

The  results  of  the  crosses  in  group  1  are  summarized  in  tables  3,  4,  and 
5.  The  percentages  of  mice  of  the  five  kinds  (25,  Ai,  26,  A2,  27)  are 
given  for  each  generation  separately.  No  distinction  is  shown  in  the 
tables  for  the  two  sexes,  for  blocks,  for  reciprocals,  for  coat  colors,  or 
for  numerous  other  variables  which  are  necessarily  a  part  of  the  experi- 
ment. The  analysis  of  the  results  of  these  crosses  is  still  in  progress.  Only 
interpretations  based  upon  the  coarse  groupings  of  the  data  as  in  tables  3 
to  5  are  available  at  this  time. 

Table  3. — Distribution  of  skeletal  types  in  various  generations  in  cross  S,  P  X   NB 


Generation 

25 

A, 

26 

A2 

27 

Total 

P1  =  P 

Per- 
cent 
92.2 

Per- 
cent 
4.4 

Per- 
cent 
3.4 
99.2 
91.2 
81.5 
36.8 
97.  1 

Per- 
cent 

Per- 
cent 

384 

P2=NB 

0.8 

245 

Fi 

3.7 

10.3 

46.0 

0.7 

5.1 

8.0 

17.2 

1.5 

215 

F2 

0.2 

627 

Bi 

383 

B2 

0.7 

411 

Table  4. — Distribution  of  skeletal  types  in  various 

generations  in  cross  7, 

NB  X  SEC/2 

Generation 

25 

A, 

26 

A2 

27 

Total 

P1=NB 

Per- 
cent 

Per- 
cent 

Per- 
cent 
99.2 
14.7 
91.  6 
80.4 
78.9 
96.8 
97.2 
54.7 
18.7 
70.4 
94.7 

Per- 
cent 
0.8 
17.3 
5.0 
6.0 
6.7 
2.6 

i6.'8* 

13.8 

14.8 

1.5 

Per- 
cent 

67.' 9' 

3.4 

13.  6 

14.4 

"i.Y 

27.9 

67.5 

14.8 

3.0 

245 

P2=SEC/2 

6.  i 

941 

Fi 

439 

F2 

317 

F3 

270 

Bx 

0.6 
0.9 
0.6 

157 

B11 

108 

B2 

161 

123 

108 

0.8 

132 

For  the  purpose  of  describing  the  results  of  the  crosses,  it  will  be  con- 
venient to  use  the  terms  low,  intermediate,  and  high  to  refer  to  the  P,  NB, 
and  SEC/2  strains,  respectively. 

Nongenetic  variation. — The  three  parental  generations  and  the  three  Fi 
generations  all  exhibit  some  phenotypic  variability.  This  variability 
appears  to  be  wholly,  or  almost  wholly  nongenetic.  The  evidence  for  this 
assertion  is  that,  in  the  strains  and  crosses  where  the  data  are  adequate  to 
make  the  test,  the  parent-offspring  correlations  are  not  significantly 
different  from  zero.  For  example,  in  the  high  SEC/2  strain,  352  mice  were 
produced  from  matings  of  the  type  27  X  27,  and  were  distributed  in  the 
classes  26,  A2,  and  27  with  frequencies  of  45,  61,  and  246.     Matings  of  the 

Journal    of    the    National    Cancer    Inslitute 


PKOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  615 

Table  5. — Distribution  of  skeletal  types  in  various  generations  of  cross  1,  P  X  SEC  1 2 


Generation 

25 

Ax 

26 

A2 

27 

Total 

P1  =  P 

Per- 
cent 
92.2 

0.  1 

1.  1 
1.4 
1.9 

21.9 
56.4 
76.5 

Per- 
cent 
4.4 

"l.3 
2.2 
1.7 
14.4 
20.  1 
13.3 

Per- 
cent 
3.4 
14.7 
97.6 
95.6 
95.  1 
63.3 
23.2 
10.2 
89.4 
62.6 
39.4 
99.0 
82.4 

Per- 
cent 

Per- 
cent 

384 

P2=SEC/2 

17.3 

67.9 

941 

Fi  .           

370 

F8 

0.3 
0.9 
0.4 
0.3 

0.5 
0.4 

634 

F3 

1,218 

Bj 

479 

Bn 

383 

255 

B2 

3.8 

13.0 

21.6 

0.3 

0.8 

6.8 
24.4 
39.0 

0.7 

555 

393 

g 

315 

g 

411 

g 

9.6 

7.2 

374 

type  26  X  27  produced  27,  36,  and  103  mice  in  the  same  three  classes. 
Altogether,  six  distinguishable  mating- types  within  the  strain  produced 
mice  in  the  three  classes  in  frequencies  which  were  approximately  propor- 
tional (x2=  10.95;  x2(10;  0.05)  =  18.31).  Similarly  the  parent-offspring 
correlations  of  Fi  parents  with  F2  or  Bi  or  B2  offspring  were  all  small  and 
nonsignificant  in  cases  which  permitted  a  test. 

Genetic  variation. — On  the  other  hand,  when  parents  were  from  a 
segregating  generation  and  the  data  permitted  a  test,  the  parent-offspring 
correlations  were  large  and  significant.  This  is  evidence  that  genes  are 
involved  in  the  differences  between  the  low,  intermediate,  and  high  strains. 
The  evidence  from  the  backcross  generations  leads  to  the  same  conclusion. 
Particularly  in  cross  1,  the  cross  of  low  by  high,  where  first,  second,  and 
third  backcrosses  to  both  parental  strains  are  available,  the  shift  of  the 
distributions  toward  the  parental  types  is  easily  seen. 

Models 

One  gene  pair  model. — The  cross  of  low  by  intermediate  (cross  3,  P  X  NB) 
produced  91.2  percent  of  mice  with  26  presacral  vertebrae  in  the  Fi  gen- 
eration. The  cross  of  intermediate  by  high  (cross  7,  NB  X  SEC/2)  pro- 
duced 91.6  percent  with  26  presacral  vertebrae.  The  cross  of  low  by  high 
(cross  1,  P  X  SEC/2)  produced  97.6  percent  with  26  presacral  vertebrae. 
These  results,  taken  alone,  suggest  that  the  intermediate  strain  contains 
genes  which  are  dominant  both  to  low  and  to  high  strain  genes. 

We  may  test  the  breeding  results  against  various  genetic  models  if  we 
may  regard  a  genotype  as  fixing  a  central  developmental  pattern  which 
is  realized  or  not  as  a  constant  phenotype,  depending  upon  the  amount  of 
"blurring"  due  to  nongenetic  causes  of  variation. 

A  model  of  one  pair  of  genes  for  the  difference  between  the  low  and  inter- 
mediate strains,  with  a  gene  for  intermediate  dominant  to  its  allele  for  low, 
indicates  that  the  percentages  of  mice  with  26  presacral  vertebrae  in  the  Pi, 
P2,  Fi,  F2,  Bx,  and  B2  generations  should  be:  0,  100,  100,  75,  50  and  100. 
The  observed  percentages  were  3,  99,  91,  82,  37,  and  97.  For  the  difference 

Vol.    15,  No.   3,  December   1954 


616  proceedings:  SYMPOSIUM  ON  25  YEARS  OF 

between  the  intermediate  and  high  strains,  with  a  gene  for  intermediate 
dominant  to  its  allele  for  high,  the  model  indicates  that  the  percentages  of 
26  presacral  vertebrae  in  Pi,  P2,  .  .  .,  B2i  should  be  100,  0,  100,  75,  75, 
100,  100,  50,  25,  75,  and  100.  The  observed  percentages  were  99,  15,  92, 
80,  79,  97,  97,  55,  19,  70,  and  95. 

These  same  two  pairs  of  genes,  each  showing  dominance  of  intermediate 
over  departures  from  intermediate  may  be  used  to  construct  a  model  for 
the  cross  of  low  by  high.  The  model  indicates  that  the  percentages  of 
26  presacral  vertebrae  in  Pi,  P2,  .  .  .,  B2i  should  be  :  0,  0,  100,  62,  62,  50, 
25,  12,  50,  25,  12,  75,  and  75.  The  observed  percentages  were  3,15,98, 
96,  95,  63,  23,  10,  90,  63,  39,  99,  and  82. 

;$|The  discrepancies  between  the  breeding  results  and  the  one  gene  pair 
model  are  conspicuous  and  numerous.  If  allowance  for  the  nongenetic 
variation  is  made,  the  agreement  is  better  but  not  satisfactory.  It  seems 
clear  that  models  based  on  one  or  two  pairs  of  genes  will  not  be  adequate. 

While  the  agreement  is  not  satisfactory,  there  is  an  unmistakable 
tendency  for  the  observed  and  theoretical  proportions  to  vary  together. 
This  suggests  that  the  number  of  pairs  of  genes  is  not  large  and  that  an 
analysis  with  a  slightly  different  approach  may  be  profitable. 

Multiple  factor  genetic  model  with  thresholds. — The  formation  of  the 
sacrum  is  the  consequence  of  a  co-ordinated  development  of  certain  dis- 
tinctive structures  on  three  or  four  vertebrae  and  on  the  two  innominate 
bones  of  the  pelvic  girdle.  The  first  vertebra  numbered  in  sequence  from 
the  cranium  to  participate  in  sacral  formation  may  be  the  26th,  27th,  or 
28th.  It  will  be  convenient  to  think  of  the  anterior  border  of  the  sacrum 
as  defined  by  two  points,  one  right,  one  left,  the  locations  of  which  vary  from 
mouse  to  mouse  and  from  side  to  side  of  the  same  mouse.  Sometimes  the 
points  rest  on  the  26th  vertebra,  sometimes  on  the  27th,  sometimes  on  the 
28th,  and  sometimes  on  different,  but  adjacent  vertebrae. 

Further,  we  may  assume  that  if  the  points  lie  anywhere  on  the  26th 
vertebra,  whether  toward  its  anterior  border  or  toward  its  posterior  border 
or  in  the  middle,  or  whether  one  point  is  in  a  slightly  different  location  from 
the  other,  the  26th  vertebra  will  participate  in  sacralization,  thereby 
leaving  25  presacral  vertebrae.  Similar  assumptions  may  be  made  about 
the  27th  and  28th  vertebrae,  leading  to  26  and  27  presacral  vertebrae, 
respectively.  However,  if  the  right  and  left  points  lie  on  different 
vertebrae,  the  vertebra  with  the  more  anterior  point  becomes,  under  this 
view,  an  asymmetrical  vertebra  since  sacralization  is  exhibited  on  one  side 
only. 

The  hypothetical  points,  of  either  the  right  or  left  side,  may  lie  anywhere 
along  the  26th,  27th,  or  28th  vertebra,  but  the  probability  for  them  to  lie 
on  a  given  position  is  a  function  of  the  position,  such  that  the  probability 
changes  from  one  position  to  another.  If  the  contrary  were  the  case, 
that  is,  if  the  probability  were  constant  from  position  to  position,  the  26th, 
27th,  and  28th  vertebrae  would  be  involved  in  sacralization  equally  often. 
This  is  clearly  not  the  case.     Rather  the  probability  for  the  points  to  lie  on 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


617 


a  given  position  is  determined  by  the  genotype  of  the  zygote  and  by  the 
environment  in  which  the  zygote  develops. 

The  location  of  the  points  for  a  collection  of  mice  of  a  given  generation 
may  be  denned  by  a  probability  distribution  with  one  or  more  parameters. 
If  the  number  of  genetic  and  nongenetic  factors  acting  upon  the  location 
of  the  points  is  large,  if  individually  they  have  relatively  small  effects,  and 
if  the  mutual  intercorrelations  of  the  effects  are  zero,  the  probability 
distribution  may  be  expected  to  be  normal  in  form  and  specifiable  by  a 
mean  and  a  variance. 

The  probability  distributions  of  the  right  and  left  side  may  both  be 
normal  in  form  but  have  different  means  and  different  variances.  The 
inequality  of  the  means,  of  the  variances,  or  of  both  may  account  for 
asymmetrical  vertebrae.  On  the  other  hand,  the  probability  distributions 
of  the  two  sides  may  have  equal  means  and  equal  variances,  but  since  the 
correlation  between  the  two  sides  is  not  perfect,  asymmetrical  sacra  may 
be  merely  an  expression  of  imperfect  side-to-side  correlation. 

The  probability  distributions  for  the  two  sides  may  be  combined  into  a 
single  distribution,  if  it  is  assumed  that  the  means  and  variances  are  equal. 
Doing  so  produces  five  categories  of  mice:  those  with  25  presacral  verte- 
brae, those  with  26,  those  with  27,  those  with  asymmetrical  combinations 
of  25  and  26,  and  those  with  asymmetrical  combinations  of  26  and  27. 
The  points  which  mark  the  boundaries  between  the  26th  and  27th,  and 
between  the  27th  and  28th  vertebrae  may  be  referred  to  as  "thresholds." 

The  four  thresholds  so  defined  may  be  denoted  as  the  T,  U,  V,  W 
thresholds: 


U 


W 


26 

(=25  presacral 

vertebrae) 


Ax  27 

(=26  presacral 
vertebrae) 


28 

(=27  presacral 

vertebrae) 


All  of  the  scale  intervals  must  of  course  be  estimated  from  data. 

Test  of  the  multiple  jactor  model. — The  multiple  gene  model  with  thres- 
holds may  be  tested  with  the  data  available  from  the  three  crosses  (tables 
3,  4,  5).  The  method  consists  of  finding  the  mean  and  variance  of  each 
generation  on  a  scale  for  which  the  unit  of  measurement  is  a  "threshold 
unit."  That  is,  the  difference  between  the  thresholds  which  separate  an 
asymmetrical  type  from  the  two  adjacent  symmetrical  types  may  be 
taken  as  unity.  This  method  was  first  used  by  Wright  (14).  Its  use  in 
connection  with  variation  in  the  position  of  the  sacrum  was  described  by 
Green  (3). 

For  the  cross  of  low  by  intermediate  (cross  3,  P  X  NB),  the  means  and 
variances  computed  from  the  data  are  given  in  table  6.  Under  the 
multiple  gene  model  with  equal  and  additive  effects  of  the  "plus"  alleles, 
with  no  linkages,  and  with  no  nonallelic  interactions,  there  are  several 
critical  relationships  between  the  means  of  various  generations.  For 
example,  using  m  to  denote  a  true  mean,  x  to  denote  a  sample  mean,  and 


Vol.   15,  No.  3,  December  1954 


618 


PEOCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


subscripts  to  denote  the  generation,  the  expectations  E  of  the  F2,  Bi,  and 
B2  means  are: 

where  M=\  (mPi+mP2)?  is  the  midparent, 

Table  6. — Computed  means  and  variances  and  expected  means  in  the  cross  of  low  by 
intermediate.     Cross  8,  P  X  NB.    xu=0,  xu—xT=l 


Generation 

Mean 

Variance 

Expected 
mean 

P1=P 

-4.4 
? 

3.2 

2.5 

-0.8 

4.7 

(2.  4)2* 
(2.  4)2* 
(2.  4)2* 
(2.  8)2 
(2.  3)2 
(2.  3)2 

P2=NB.. 

8.  6t 

Fi 

F2 

B2 

-6.  6 

B2 

5.  6 

♦Average  of  Pi  and  F!  variances. 
tEstimated  from  E{xp  )=4wF  -2mv  -w»p  . 

In  cross  3,  the  location  of  the  P2  mean  cannot  be  computed  from  the 
observations  on  the  P2  generation.  Its  location  may  be  computed  from 
E  (*p2)=4mF2— 2mFi— mPi,  provided  the  means  of  Pi,  Fi,  and  F2  are 
computable  from  their  respective  generations.  The  expected  means  of 
P2,  Bi,  and  B2  are  shown  in  table  6  for  comparison  with  the  observed 
means.  The  means  and  variance  given  in  table  6  lead  to  a  construction 
of  probability  distributions  like  those  in  text-figure  2. 

Table  7. — Computed  means  and  variances  and  expected  means  in  the  cross  of  intermediate 
by  high.     Cross  7,  NB  X  SEC/2.     xv=0,  xw-xv=l 


Generation 


Px  =  NB... 
P2  =  SEC/2 

Fx 

F2 

F3 

Bi 

B„ 

B2 

B22 

Bi2 

B2i 


Mean 


? 

2.0 
-2.6 
-3.4 
-3.2 
-4.3 
-4.5 
-0.3 

1.9 
-1.1 
-3.6 


Variance 


(1.  90)2 
(1.  90)2 
(1.  90)2 
(4.  Ol)2 
(4.  01)2 
(2.  21)2* 
(2.  14) 2 1 
(2.  21)2 
(2.  14) 2 1 
(2.  14) 2 1 
(2.  14)2t 


Expected  means 


Assump- 
tion 1 


-4.6 


2.0 
2.0 

3.  6 

4.  1 
0.3 
0.8 

2.  1 

3.  7 


Assump- 
tion 2 


10.5 


-3.4 
-6.6 
-8.5 
-0.3 
0.8 
-3.6 
-6.7 


Assump- 
tion 3 


-8.  1 


5.5 
6.8 
0.5 
0.8 
3.3 
5.8 


♦Variance  of  Bi  not  computable.  Variance  of  Bj  used  as  approximation,  which  is  satisfactory  only  if  there  is 
no  dominance. 

fVariances  of  B33  and  B21  averaged,  and  average  used  as  approximation  of  variances  of  Bu  and  Bu,  which  is 
satisfactory  only  if  there  is  no  dominance. 


Journal    of   the   National   Cancer   Institute 


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619 


For  the  cross  of  intermediate  by  high  (cross  7,  NB  X  SEC/2),  more 
generations  were  available.  The  means  and  variances  computed  from 
the  data  are  given  in  table  7.  To  compute  the  variances,  several  assump- 
tions were  used:  1)  that  the  variances  of  Pi,  P2,  and  Fx  are  equal;  2)  that 
the  variances  of  F2  and  F3  are  equal;  3)  that  the  variance  of  Bi  which 
was  not  computable  is  equal  to  the  variance  of  B2,  an  assumption  which 
is  valid  only  if  there  is  no  dominance;  4)  that  the  variances  of  B22  and  B2i, 
being  theoretically  equal,  may  be  averaged;  and  5)  that  the  variances  of 
Bn  and  Bi2,  neither  of  which  were  computable,  could  be  estimated  by  the 
average  variance  of  B22  and  B2i,  an  assumption  which  also  is  valid  only 
if  there  is  no  dominance.  The  location  of  the  mean  of  Pi  is  in  doubt, 
since  over  99  percent  of  mice  in  the  NB  strain  had  26  presacral  vertebrae. 
To  yield  this  percentage,  the  mean  should  be  at  4.6  units  below  the  V 
threshold.  This  leads  to  a  set  of  expected  means  given  under  assumption 
1  in  table  7.  From  the  means  of  P2,  Fx,  and  F2,  the  Px  mean  may  be  esti- 
mated as  lying  10.5  units  below  the  V  threshold.  This  estimate  is 
unexpectedly  large  and  leads  to  a  set  of  expected  means  given  under 

t    u 


Observed 


J 1 


MF2F,    B2 

LLLL 


Expected 

B,  B2 

Text-figure  2. — Construction  of  theoretical  distributions  for  the  cross  of  low  by 
intermediate.     Cross  3,  P  X  NB. 


Vol.  15,  No.  3,  December  1954 

316263 — 54 22 


620 


PKOCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


assumption  2  in  table  7.  A  smaller  estimate  of  8.1  units  below  V  is 
obtained  if  it  is  assumed  that  the  Fi,  F2,  and  F3  means  are  all  estimates 
of  the  same  quantity,  that  is,  if  it  is  assumed  that  there  is  no  dominance. 
This  yields  the  set  of  expected  means  given  under  assumption  3  of  table  7. 
The  computed  means  and  variances  yield  a  construction  of  probability 
distributions  such  as  in  text-figure  3. 


Observed 


BnB21F3 
P,  P,  B,  F2  F,  B12  B2 


L_L 


LU 


Expected  (1) 


mm 

p1  Bl     F2     B2   B22 


Text-figure  3. — Construction  of  theoretical  distributions  for  the  cross  of  intermediate 
by  high.     Cross  7,  NB  X  SEC/2. 

For  the  cross  of  low  by  high  (cross  1,  P  X  SEC/2),  all  of  the  generations 
through  third  backcrosses  to  both  parental  strains  were  available.  The 
means  and  variances  of  each  generation  computed  from  the  data  are  given 
in  table  8.    The  distance  between  the  V  and  W  thresholds  was  taken  as  the 


Journal    of    the   National   Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


621 


"threshold  unit."  The  distance  from  U  to  V,  estimated  from  the  com- 
bined data  of  the  F2  and  F3  generations,  is  11.7  units.  The  distance  be- 
tween the  T  and  U  thresholds  is  0.7  units.  The  following  assumptions  were 
also  made:  1)  the  variances  of  Pi,  P2,  and  Fi  are  equal;  2)  the  variances  of 
F2  and  F3  are  equal  and  estimable  from  the  combined  data  of  the  F2  and 
F3  generations;  3)  the  variance  of  B!2,  which  was  not  computable  owing  to 
the  fact  that  99  percent  of  Bi2  mice  had  26  presacral  vertebrae,  is  the  same 
as  the  B22  variance,  to  which  it  is  theoretically  equal.  These  computations 
and  assumptions  yield  the  construction  of  probability  distributions  for  the 
various  generations  of  cross  1  shown  in  text-figure  4. 

The  minimal  number  of  effective  factors  which  distinguish  the  strains 
may  be  estimated  from  the  ratio  of  the  square  of  the  difference  between  the 


Observed 


1       BmB11BlB21    Fi       F2     B: 

L  i  1  ti    LJLJ 


Expected 


*m  Bn  Bi 


FT 


B22  B222  P2 


TJ 


Text  -figure  4. — Construction  of  theoretical  distributions  for  the  cross  of  low  by  hiqh 
Cross  1,  P  X  SEC/2. 


Vol.    15,   No.   3,   December    1954 


622 


PROCEEDINGS!  SYMPOSIUM  ON  25  YEARS  OF 


Table  8. — Computed  means  and  variances  and  expected  means  in   the  cross  of  low  by 
high.     Cross  1,  P  X  SEC/2.     xv=0,  xw-xv=l,  xy-xv=11.7 


Generation 


Mean 


Variance 


Expected 
mean 


Pi  =  P 

P2  =  SEC/2 

F, 

F2 

F3 

Bi 

B„ 

Bin 

B2 

B22 

B222 

B12 

B21 


-3.2 

13.6 

3.5 

5.2 

5.2 

0.5 

-0.8 

-1.5 

6.6 

10.9 

12.2 

5.4 

1.8 


(1.  78)2 
(1.  78)2 
(1.  78) 2 
(2.  88) 2 
(2.  88) 2 
(1.  54)2 
(1.  17)2 
(1.  20)2 
(4.  15) 2 
(2.  69) 2 
(1.  85)2 
(2.  69) 2* 
(1.  91)2 


6.0 

6.0 

0.  1 

-1.6 

-2.4 

8.6 

11.  1 

12.3 

8.6 

4.4 


*Variance  of  Bi2  not  computable, 
used  as  an  approximation. 


Variance  of  B22  to  which  it  is  theoretically  equal 


parental  means  and  the  additive  genetic  variance  [Castle  (15);  Mather 
(16) ;  Wright  (17)].  The  P  and  NB  strains  appear  to  differ  by  at  least  4 
pairs  of  genes,  NB  and  SEC/2  by  at  least  1  pair,  and  P  and  SEC/2  by  at 
least  21  pairs. 

Dominance  is  indicated  by  the  failure  of  the  F2  mean  to  lie  on  the  same 
point  as  the  Fx  mean.  In  cross  3  (low  by  intermediate),  the  F2  mean  is 
slightly  below  the  Fx  mean,  indicating  possible  dominance  of  intermediate 
over  low.  In  cross  7,  there  is  evidence  of  dominance  of  high  over  inter- 
mediate. In  cross  1,  the  relationship  is  reversed,  low  being  partially 
dominant  to  high. 

Discussion 

A  multiple  gene  pair  model  is  very  flexible  because  of  the  large  number 
of  parameters  to  be  estimated  from  the  data.  These  include  the  relative 
contributions  of  genetic  and  nongenetic  factors  to  the  total  variability,  the 
number  of  pairs  of  genes  or  of  effective  blocks  of  genes,  the  average  rela- 
tionship between  alleles  (dominance) ,  and  the  average  relationship  between 
nonalleles  (epistasis) . 

For  variations  in  the  position  of  the  sacrum,  the  multiple  gene  pair 
model  has  been  used  to  provide  a  concept  of  a  continuous  underlying  vari- 
able which  is  manifested  only  in  2,  3,  4  or  5  categories  separated  by  thres- 
holds. The  scale  for  the  underlying  variable  has  been  chosen  deliberately 
to  minimize  the  nonallelic  interactions,  and  to  provide  a  scale  for  displaying 
additive  effects  of  nongenetic  and  genetic  causes  of  variation,  including 
dominance.  The  agreement  between  the  observed  results  and  the  genetic 
model  is  clearly  not  perfect,  and  at  a  few  points  is  not  even  good.  Never- 
theless a  genetic  model  based  upon  the  concepts  of  quantitative  inheritance 
appears  to  be  more  promising  than  more  elementary  models  for  explaining 
variation  in  the  position  of  the  sacrum. 

The  development  of  a  given  mouse  is  such  that  the  sacrum  forms  from 
vertebrae  following  the  25th,  26th,  or  27th  vertebra.    Many  agents  govern 


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623 


the  particular  position  of  the  sacrum.  These  agents  cannot  be  specified 
beyond  saying  that  some  are  genetic  and  some  are  nongenetic.  The  inter- 
play of  the  genetic  and  nongenetic  agents  appears  to  be  such  that  the 
position  of  the  sacrum  for  a  given  mouse  cannot  be  predicted  with  cer- 
tainty. Rather,  by  use  of  a  multiple  gene  model,  it  is  possible  to  specify 
the  probabilities  for  each  of  several  sacral  positions  for  mice  of  a  given 
strain  or  for  mice  of  a  given  generation  in  crosses  between  strains. 

The  model  also  suggests  that  the  effects  of  experimental  modifications 
of  the  embryonic  environment  will  not  be  exhibited  in  qualitative  changes 
in  the  individual  embryos.  Rather  the  detectable  effect,  short  of  the 
production  of  abnormalities,  will  be  a  change  in  the  frequency  distribution 
of  skeletal  types.  This  means  that  the  set  of  probabilities  of  the  various 
types,  rather  than  the  types  themselves,  are  changed  with  the  changing 
environment.  This  has  been  borne  out  in  experiments  with  X  irradiation 
of  embryos  [Russell  and  Russell  (18)]  and  in  experiments  using  ova 
transplantation  [Green  and  Green  (19)]. 

References 

(1)  Green,  E.  L.:  Genetic  and  non-genetic  factors  which  influence  the  type  of  the 

skeleton  in  an  inbred  strain  of  mice.     Genetics  26:   192-222,  1941. 

(2)  Searle,  A.  G.:  Genetical  studies  on  the  skeleton  of  the  mouse.     IX.  Causes 

of  skeletal  variation  within  pure  lines.     J.  Genetics  52:  68-102,  1954. 

(3)  Green,  E.  L.:  The  genetics  of  a  difference  in  skeletal  type  between  two  inbred 

strains  of  mice  (BalbC  and  C57blk).     Genetics  36:  391-409,  1951. 

(4)  Green,  E.  L.,  and  Russell,  W.  L.:  A  difference  in  skeletal  type  between  recip- 

rocal hybrids  of  two  inbred  strains  of  mice  (C57BLK  and  C3H).     Genetics  36: 
641-651, 1951. 

(5)  Green,  E.  L.,  and  Green,  M.  C.:  The  effect  of  uterine  environment  on  the 

skeleton  of  the  mouse.     Jour.  Morph.  78:   105-112,  1946. 

(6)  Dobrovolskaia-Zavadskaia,  N.:  Sur  la  mortification  spontanea   de  la  queue 

chez  la  souris  nouveau-n6e  et  sur  Texistence  d'un  caractere  (facteur)  hereditaire 
"non-viable."     Compt.  rend.  Soc.  biol.  97:   114-116,  1927. 

(7)  Chesley,  P.,  and  Dunn,  L.  C.:  The  inheritance  of  taillessness  (anury)  in  the 

house  mouse.     Genetics  21:  525-536,  1936. 

(8)  Gluecksohn-Schoenheimer,  S.:  The  morphological  manifestations  of  a  domi- 

nant mutation  in  mice  affecting  tail  and  urogenital  system.     Genetics  28: 
341-348, 1943. 

(9)  Dunn,  L.  C.,  and  Gluecksohn-Schoenheimer,  S.:  Stub,  a  new  mutation  in  the 

mouse  with  marked  effects  on  the  spinal  column.     J.  Hered.  33:  235-239,  1942. 

(10)  MacDowell,  E.  C.,  Potter,  J.  S.,  Laanes,  T.,  and  Ward,  E.  N.:  The  manifold 

effects  of  the  screw  tail  mouse  mutation.     J.  Hered.  33:  439-449,  1942. 

(11)  Green,  E.  L.,  and  Green,  M.  C.:  Effect  of  the  short  ear  gene  on  number  of  ribs 

and  presacral  vertebrae  in  the  house  mouse.     Amer.  Nat.  80:  619-625,  1946. 

(12)  Carter,  T.  C.:  The  genetics  of  luxate  mice.  I.  Morphological  abnormalities  of 

heterozygotes  and  homozygotes.     J.  Genetics  50:  277-299,  1951. 
(IS)  Green,  M.  C.:  A  new  inherited  leg  and  foot  abnormality,  luxoid,  in  the  house 
mouse.     (Abstract.)     Genetics  38:  666,  1953. 

(14)  Wright,  S.:  The  results  of  crosses  between  inbred  strains  of  guinea  pigs  differing 

in  number  of  digits.     Genetics  19:  537-551,  1934. 

(15)  Castle,  W.  E.:  An  improved  method  of  estimating  the  number  of  genetic  factors 

concerned  in  cases  of  blending  inheritance.     Science  54:  223,  1921. 


Vol.    15,  No.  3,  December   1954 


624  proceedings:  symposium 

(16)  Mather,  K.:  Biometrical  genetics.     New  York,  Dover  Pub.,  Inc.,  1949,  p.  158. 

(17)  Wright,  S.:  The  genetics  of  quantitative  variability.     In  Quantitative  Inheri- 

tance. London,  Her  Majesty's  Stationery  Office,  1952,  pp.  5-42. 

(18)  Russell,  L.  B.,  and  Russell,  W.  L.:  Changes  in  the  relative  proportions  of 

different  axial  skeletal  types  within  inbred  strains  of  mice  brought  about  by 
X-irradiation  at  critical  stages  in  embryonic  development.  (Abstract.)  Ge- 
netics 35:  689,  1950. 

(19)  Green,  E.  L.,  and  Green,  M.  C.:   Modification  of  difference  in  skeletal  types 

between  reciprocal  hybrids  by  transplantation  of  ova  in  mice.  (Abstract.) 
Genetics  38:  666,  1953. 


Discussion 
Dr.  P.  B.  Sawin,  Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 

The  author  is  to  be  commended  for  the  masterful  demonstration  of  one  of  the 
basic  attributes  of  homeotic  variations,  the  origin  of  which  has  for  more  than  three 
quarters  of  a  century  been  a  puzzle  to  many  prominent  biologists,  including  Bateson, 
Kingsley,  Kingsbury,  Bumpus,  Todd  and  Keith  et  al.  These  leading  morphologists 
of  their  time  attempted  to  solve  the  problem  from  the  phylogenetic  point  of  view,  but 
the  two  theories  proposed  have  never  been  substantiated  experimentally. 

Dr.  Green's  insight  as  to  the  best  sort  of  genetic  material  and  his  thoroughness  in 
carrying  out  established  genetic  procedures  have  led  to  the  most  clear-cut  conclusions 
yet  reached  with  regard  to  this  sort  of  variation.  It  was  Danforth,  I  believe,  who  first 
suggested  a  genetic  interpretation,  and  there  have  been  a  number  of  attempts  to  inter- 
pret the  inheritance  of  such  variation  in  terms  of  one  or  more  gene  substitutions. 
None,  however,  have  been  found  which  survive  thorough  genetic  investigation.  This 
is  particularly  significant,  I  think,  in  view  of  the  known  single-gene  effects  upon  such 
variations  in  species  such  as  Drosophila  and  Habrobracon  and  upon  the  extremities 
(limbs  and  tail)  in  mammals.  Why  single  genes  should  be  adequate  for  such  variations 
in  the  lower  forms  and  not  in  mammals,  and  why  in  the  mouse  single  genes  adequately 
explain  the  inheritance  of  the  numerous  postsacral  but  rarely  presacral  variations 
are  moot  questions. 

With  respect  to  the  first  question,  it  may  also  be  of  significance  that  although  in 
Drosophila  such  variations  tend  to  be  mono-segmental,  there  is  evidence  from  the 
work  of  Villee,  at  least,  that  in  the  case  of  certain  genes  more  than  one  adjacent  seg- 
mental part  is  frequently  affected  (pleiotropically) .  Perhaps  the  fact  that  the  gene 
effect  is  not  more  generalized  as  it  is  in  mammals  could  be  due  to  the  relatively  lower 
growth  potential  of  the  species  or  to  the  relatively  small  number  of  segments  (low 
segmental  rate).  With  regard  to  the  second,  the  question  may  well  be  asked:  "Is  it 
because  these  genes  are  terminal  in  their  effect  that  their  inheritance  is  more  precise, 
whereas  the  inheritance  of  homeotic  variations,  which  are  sub  terminal  in  development, 
may  become  complicated  by  the  very  nature  of  relations  with  their  neighboring  parts?" 
The  fact  that  Dr.  Green  has  found  the  position  of  the  pelvis  and  sacrum  can  be  governed 
by  many  agents  both  genetic  and  environmental,  plus  the  fact  that  it  can  be  further 
complicated  by  the  interplay  of  these  agents,  tremendously  increases  the  complexity 
of  the  problem.  Yet  Dr.  Green  seems  to  have  developed  a  system  from  which  pre- 
dictable results  can  be  obtained. 

In  the  rabbit  we  have  gone  about  the  same  problem  in  a  somewhat  different  manner. 
Stimulated  by  the  genetic  studies  of  Fisher  and  Kiihne  in  man  and  the  rabbit,  in 
which  they  have  stressed  the  importance  of  relationships  which  may  exist  between 
more  than  one  homeotic  variation,  we  have  focused  our  attention  upon  two  races  in 
which  there  is  a  high  correlation  between  the  variations  at  two  borders,  the  thoraco- 
lumbar and  lumbosacral,  the  one  race  having  both  extra  units  in  95  percent  of  cases, 
the  other  lacking  them.  Quantitatively  the  increase  in  relative  growth  between  these 
two  borders  is  demonstrable  in  the  adult,  as  well  as  at  birth,  and  more  recently  we  have 
found  it  in  21-day  embryos. 

In  the  left  half  of  figure  1,  a  representative  specimen  of  race  III,  we  see  the  enlarged 
lumbar  growth  area  characteristic  of  that  race.  It  is  manifested  in  the  greater  size 
and  number  of  lumbar  centra  at  21  days.  In  the  right  half  of  figure  1,  showing  a  repre- 
sentative of  race  X,  we  see  the  enlarged  anterior  growth  area  manifested  by  the  greater 
size  and  number  of  the  elements  of  the  neural  arch  at  the  same  age.  These  two  speci- 
mens, however,  are  far  from  portraying  the  entire  situation.  As  Dr.  Green  has  so  well 
shown,  one  cannot  expect  to  find  the  effects  of  experimental  modifications  of  the 
embryonic  environment  exhibited  in  individual  embroyos,  and  this  is  also  apparent 

625 


Journal   of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


626  proceedings:  symposium 

with  respect  to  the  growth  forces  acting  on  these  embryos  at  this  time.  I  have  chosen 
here  two  specimens  representative  of  the  average  pattern.  Time  will  not  permit  of 
my  demonstrating  details  of  these  points  at  this  time.  In  fact,  the  evidence  is  still  not 
complete.  What  we  have  found  can  be  summarized  in  this  way.  Considering  these 
two  races  as  a  whole  there  are  apparent  four  characteristic  differences  between  them 
which  somehow  interact  to  produce  threshold  differences  at  various  levels  of  the 
axial  skeleton.  At  this  age  they  are  1)  a  tendency  to  generalized  precosity  of  onset  of 
ossification  in  race  X  plus  certain  localized  differences  anteriorly;  2)  genetic  influences 
which  do  not  act  on  all  localized  organs  simultaneously;  for  example,  neural  arches  are 
quite  independent  of  the  centra;  3)  growth  in  width  of  centra  is  more  precocious  than 
growth  in  length,  and  especially  so  in  X.  There  is  evidence  that  it  is  changing  very 
rapidly  and  is  not  relatively  comparable  to  adult  width  at  this  time;  4)  the  regional 
growth  areas  as  a  whole  are  in  a  much  greater  state  of  flux  at  this  time  than  at  birth  or 
in  the  adult. 

Differential  growth  of  parts  in  relation  to  each  other  determines  essentially  the 
characteristics  which  define  the  kind  of  vertebra.  We  suspect  that  the  clue  to  the 
specific  gene  actions  lies  in  such  differences  which  in  turn  interact  to  determine  the 
thresholds  of  homeotic  change  described  so  clearly  by  Dr.  Green.  Whether  these 
differences  can  be  traced  back  through  cartilage  formation,  through  mesenchyme  con- 
densation and  through  still  earlier  developmental  processes  is  a  question  which  can 
only  be  answered  by  embryological  techniques  and  measured  by  statistical  methods. 
We  have  seen  them  at  three  developmental  stages.  Between  these  stages  it  would  seem 
to  be  only  a  matter  of  time  and  effort  to  describe  the  gene  influence  involved.  Here  the 
more  refined,  longitudinal  approach  now  being  used  by  the  physical  anthropologists  is 
particularly  important.  The  well  known  adolescent  spurt  in  man  is  one  example 
where  acceleration  or  retardation  of  the  growth  of  the  whole  or  the  part  in  relation  to 
the  whole  is  capable  of  profound  changes  in  the  end  result.  It  is  my  belief  that  the 
final  analysis  of  the  problem  of  the  genetic  and  environmental  basis  of  homeotic 
variations  must  ultimately  come  from  the  combined  efforts  of  the  embryologist  and  the 
geneticist  who  have  command  of  adequate  statistical  techniques  and  who  approach 
the  underlying  growth  processes  or  gradients  by  longitudinal  methods.  Since  longi- 
tudinal methods  are  not  yet  possible  embryologically,  the  importance  of  inbred  strains 
such  as  those  of  this  laboratory  cannot  be  overemphasized. 


Plate  44 

Figure  1. — Comparison  of  anterior  and  lumbar  growth  areas  of  21-day  embryos  of 
Race  III  (left)  and  Race  X  (right)  from  cleared  specimens  shown  at  the  same 
magnification. 


JOURNAL  OF  THE   NATIONAL  CANCER  INSTITUTE,   VOL.  15 


PLATE  44 


■•.. 


• 


... 
• 


- 


i  ' 


.  4*1 


> 


iv    "I 


Sawin 


Figure  1 


627 


316263—54 23 


Genetic  Control  of  Embryonic  Growth 
and  Differentiation  1,a 


Salome  Gluecksohn-Waelsch,  Department  of 
Obstetrics  and  Gynecology,  College  of  Physicians 
and  Surgeons,  Columbia  University,  New  York, 
N.  Y. 


The  identification  and  the  analysis  of  the  different  factors  which  co- 
operate intimately  with  each  other  to  produce  a  normal  organism  is  one 
of  the  most  important  goals  of  all  students  of  development.  That  these 
factors  are  of  varying  origin,  and  are  in  turn  subject  to  various  deter- 
mining influences,  is  of  course  well  known  and  does  not  need  to  be  stressed 
here.  The  recognition  and  assignment  of  developmental  factors  to 
various  categories,  e.g.,  genetic,  environmental,  etc.,  are  prerequisites 
for  a  study  of  the  mechanisms  by  which  they  exert  their  control  of  de- 
velopment. In  this  paper,  I  should  like  to  review  present  knowledge  of 
some  genetic  factors  which  have  been  found  to  control  processes  of  em- 
bryonic growth  and  differentiation.  The  organism  in  which  these  par- 
ticular factors  were  studied  is  the  house  mouse,  which  plays  such  an 
important  role  in  the  past  and  present  history  of  this  laboratory.  But 
before  I  begin  the  discussion  of  this  material,  I  should  like  to  give  an 
illustration  taken  from  a  considerably  lower  creature  than  the  mouse,  of 
how  various  factors  may  interact  in  the  operation  of  developmental 
mechanisms.  I  have  always  been  particularly  intrigued  by  the  outcome 
and  the  interpretation  of  one  of  Spemann's  most  interesting  experiments. 
Conceived  as  early  as  1921  by  Spemann  the  experiment  was  actually 
carried  out  by  Schotte  (1)  in  1932.  The  question  was  asked  if  the  re- 
acting system  of  a  certain  species  would  respond  to  an  inductive  stimulus 
of  a  different  species  in  its  own  species  specific  way  or  in  the  pattern 
of  the  inducing  species.  The  experiment  consisted  in  the  transplantation 
of  belly  ectoderm  from  an  anuran  species  to  the  mouth  region  of  a  urodele 
embryo — a  so-called  xenoplastic  transplant.  This  ectoderm  that  nor- 
mally would  have  formed  skin  of  the  abdomen  of  a  frog,  formed  structures 
of  the  mouth  region  in  response  to  the  inductive  stimulus  provided  by  the 
mouth  environment  of  the  salamander  host.  The  environment  thus 
directed   the   differentiation   of   the   transplanted   tissue   into   channels 

1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine 
June  28, 1954. 

» This  work  was  supported  in  part  by  a  research  grant  (G-3676)  from  the  National  Institutes  of  Health,  U.  S 
Public  Health  Service,  and  in  part  by  a  grant-in-aid  from  the  American  Cancer  Society  upon  recommendatio 
of  the  Committee  on  Growth  of  the  National  Research  Council. 


629 


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630  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

conforming  with  the  environment's  demands.  However,  the  hereditary 
make-up  of  the  transplanted  tissue  manifested  itself  also — namely,  in 
the  type  of  mouth  structures  formed  by  the  implant:  these  were  suckers, 
typical  of  tadpoles  of  anuran  species  in  contrast  to  the  balancers  normally 
formed  by  the  host.  The  implant  thus  retained  its  genetically  controlled 
species  characteristics  while  giving  in  to  the  demands  of  the  environment 
of  the  host  in  respect  to  regional  differentiation. 

The  interaction  of  heredity  and  environment  in  the  formation  of  a 
developmental  pattern  seems  to  me  beautifully  illustrated  by  this  example. 

For  the  detailed  analysis  of  the  control  of  developmental  patterns  by 
particular  genetic  factors  amphibians  do  not  offer  the  most  suitable 
material  mainly  for  reasons  connected  with  the  length  of  their  life  cycle. 
In  this  respect  the  mouse  is  much  more  cooperative,  as  you  all  know, 
with  the  speed  and  quantity  of  its  reproductive  activity.  It  is  because 
of  this  reason,  as  well  as  because  of  the  existence  of  a  number  of  mutations 
concerned  with  processes  of  early  growth  and  differentiation,  that  today 
we  know  perhaps  more  about  the  specific  role  of  genes  in  development  of 
the  mouse  than  of  any  other  vertebrate. 

As  mentioned  before,  developmental  patterns  owe  their  origin  to  various 
genetic  as  well  as  nongenetic  factors.  Among  the  genetic  factors  there 
are  not  only  those  more  or  less  directly  concerned  with  the  operation  of 
particular  processes  of  differentiation,  but  a] so  those  which  determine 
the  type  of  reaction  to  genetic  and  nongenetic  influences  to  which  the 
organism  is  subjected  during  its  development. 

The  example  which  I  am  about  to  mention  will  serve  to  illustrate 
typical  abnormalities  of  a  developmental  pattern  which  may  be  brought 
about  either  by  changes  of  genetic  or  of  nongenetic  factors;  the  mani- 
festation of  these  factors  in  turn  depends  to  a  large  degree  on  the  indi- 
vidual's genetically  controlled  susceptibility. 

The  abnormality  I  want  to  talk  about  is  cleft  palate  where  a  cleft 
extends  through  the  premaxilla  and  the  entire  length  of  the  hard  palate: 
this  disturbance  of  a  normal  developmental  pattern  is  found  rather 
frequently  in  mammals.  The  cleft  seems  to  be  the  consequence  of  the 
failure  to  unite  of  the  two  palatine  processes  of  the  maxillae  and  traces 
back  perhaps  to  a  reduced  growth  rate  of  the  maxillary  processes. 

It  is  interesting  that  hereditary  harelip  or  cleft  palate  has  been  re- 
ported several  times  in  the  literature  but  never  with  a  simple  genetic 
basis  (#).  Some  years  ago  we  described  the  occurrence  of  one  type  of 
cleft  palate  which  owed  its  existence  not  to  the  effect  of  one  or  more 
specific  genetic  factors  but  rather  to  the  combination  of  a  special  geno- 
type and  a  peculiar  set  of  environmental  conditions  (3).  In  this  case 
intimate  interaction  of  a  set  of  genetic  and  environmental  factors  was 
responsible  for  the  disturbance  of  the  normal  developmental  pattern  of  the 
palate.  On  the  other  hand,  cleft  palate  as  a  regular  symptom  in  a  whole 
syndrome  of  abnormalities  has  been  observed  in  our  laboratory  twice  in 
recent  years;  two  different  recessive  factors  in  homozygous  condition 
were  responsible  for  it.     Each  of  these  recessive  factors  produced  its  own 

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631 


syndrome  of  abnormalities:  in  the  case  of  one  (pc)  various  effects  were 
observed  in  other  parts  of  the  skeleton  as  well,  particularly  in  the  extremi- 
ties, while  in  the  case  of  the  other  one  (ur)  abnormal  functioning  of  the 
kidneys  resulting  in  water  retention,  in  addition  to  other  skeletal  abnor- 
malities, occurred  in  the  homozygotes.  Thus  cleft  palate  seems  to  mani- 
fest itself  more  regularly  in  combination  with  other  abnormalities. 

Cleft  palate  may  also  be  produced  experimentally  in  mice  by  the  use 
of  different  deleterious  agents.  Fraser  {4),  for  example,  reported  as 
much  as  80  percent  of  cleft  palate  in  genetically  normal  offspring  of 
certain  strains  whose  mothers  had  been  given  cortisone.  Here,  also,  the 
incidence  of  the  abnormality  depended  largely  on  the  genetic  makeup 
of  the  treated  strain. 

It  thus  seems  that  growth  and  differentiation  of  the  embryonic  material 
forming  the  hard  palate  are  subject  to  many  influences,  genetic  and 
nongenetic.  There  are  a  number  of  reasons  which  make  it  likely  that 
these  events  take  place  rather  late  in  development. 

Now  I  should  like  to  discuss  some  material  which  deals  with  develop- 
mental mechanisms  operating  considerably  earlier  in  the  life  of  the 
embryo.  One  of  the  organ  systems  of  the  developing  embryo  most  sus- 
ceptible to  all  types  of  changes,  and  reacting  to  them  with  abnormalities 
of  different  kinds  and  degrees,  is  the  nervous  system.  A  great  many 
genetic  factors,  as  well  as  several  environmental  agents,  are  known  to 
cause  disturbances  in  the  normal  development  of  this  important  organ 
system  in  the  mouse.  As  you  all  know,  the  nervous  system  shows  a 
particularly  close  relationship  to  other  developmental  systems  of  the 
embryo  during  its  morphogenesis;  its  complete  dependence  on  the  under- 
lying notochord-mesoderm  material  for  its  initial  differentiation  is  a  well 
known  fact,  at  least  in  amphibian  embryos.  Minor  variations  in 
different  embryonic  regions  during  development  therefore  easily  express 
themselves  in  abnormalities  of  the  highly  sensitive  nervous  system. 

In  the  mouse,  growth  and  differentiation  of  the  nervous  system  seem 
to  be  influenced  by  a  number  of  genetic  factors  operating  through  differ- 
ent channels.  One  type  of  brain  abnormality  resulting  from  develop- 
mental disturbances  is  known  as  pseudencephaly;  in  this  condition  the 
bony  cranial  roof  is  absent  and  parts  of  the  brain  are  inverted,  forming  a 
cap  on  top  of  the  head ;  failure  of  the  brain  folds  to  close  along  the  dorsal 
midline  is  connected  with  this  abnormality.  Genetically,  pseudencephaly 
has  been  described  as  the  result  of  a  recessive  mutation  by  Bonnevie  (5) . 
It  also  arises  in  the  presence  of  X-ray-induced  chromosomal  translocations 
as  shown  by  Snell  and  coworkers  (6) .  Abnormalities  similar  to  pseuden- 
cephaly have  been  investigated  recently  by  Auerbach  (in  press)  in  mouse 
embryos  homozygous  for  the  dominant  mutation  Splotch.  In  our  own 
laboratory  we  have  observed  a  fairly  high  incidence  of  pseudencephaly 
among  embryos  of  so-called  normal  mouse  strains. 

The  normal  inductive  relationship  between  notochord-mesoderm  ma- 
terial and  the  developing  nervous  system  has  been  shown  to  be  affected 
in  the  presence  of  the  mutations  at  the  T-locus  of  Chromosome  IX  of 


VoL    15,   No.   3,   December    1954 


632  proceedings:  symposium  on  25  years  of 

the  mouse.  Abnormalities  of  the  neural  tube  in  the  presence  of  the 
mutation  T  were  traced  back  by  Chesley  (7)  to  those  of  the  notochord. 
Duplications  of  neural  folds  in  embryos  homozygous  for  Kink,  another 
dominant  mutation  in  Chromosome  IX,  seem  to  indicate  abnormal 
functioning  of  the  material  responsible  for  formation  of  a  single  normal 
embryonic  axis.  In  our  laboratory  recently,  Dr.  Karl  Theiler  studied 
the  developmental  effects  of  embryos  homozygous  for  Fused,  another  of 
the  dominant  mutations  of  this  group,  and  found  multiple  neural  tubes  in 
different  regions  of  the  developing  embryos.  While  in  this  latter  case 
the  causal  role  of  the  notochord-mesoderm  material  is  not  clearly  indicated, 
studies  of  other  mutations  in  Chromosome  IX  point  strongly  to  a  general 
effect  of  the  factors  in  this  chromosome  on  this  important  developmental 
system.  Since  the  effects  of  these  mutations  have  been  described  and 
discussed  repeatedly  in  recent  years  (8,  9)  I  do  not  plan  to  go  into  more 
detail  about  them  here.  I  only  want  to  state  in  summary,  that  genetic 
control  of  embryonic  growth  and  differentiation  is  most  strongly  demon- 
strated by  the  existence  of  the  mutations  in  Chromosome  IX  of  the  mouse 
and  their  effects  on  notochord-mesoderm  material  and  the  developing 
nervous  system. 

The  recent  investigations  by  Auerbach  of  the  homozygous  effect  of 
Splotch,  mentioned  above,  have  shown  the  interference  of  this  mutation 
with  the  normal  functioning  of  the  neural  crest  material,  a  significant 
part  of  the  nervous  system  in  embryonic  differentiation. 

Another  example  of  a  disturbance  of  normal  inductive  relationships  in 
the  developing  nervous  system  by  a  mutant  genetic  factor  may  be  found 
in  the  effect  of  Kreisler,  a  mutation  in  the  mouse  studied  by  Paula  Hertwig 
(10) .  Here,  separation  of  the  ear  vesicle  primordium  from  the  neural  tube 
interferes  with  the  normal  differentiation  of  the  ear  vesicle. 

Numerous  further  examples  of  the  interference  of  mutations  with  normal 
processes  of  growth  and  differentiation  in  other  organ  systems  of  the 
mouse  could  be  cited.  The  question  may,  of  course,  be  asked  whether 
the  control  of  normal  growth  and  differentiation  by  genes  may  be  inferred 
from  the  demonstration  of  abnormal  processes  of  differentiation  in  the 
presence  of  mutations.  I  would  tend  to  answer  this  question  in  the 
affirmative  and  say  that  one  may  conclude  at  least  that  normal  genetic 
factors  participate  in  the  control  of  these  same  processes  shown  to  be 
abnormal  in  the  presence  of  mutations.  Of  course  the  mechanisms  by 
which  normal  or  changed  genes  exert  their  control  on  growth  and  differ- 
entiation offer  separate  problems.  It  is  the  investigation  of  these  mechan- 
isms which  occupies  us  most  at  the  present  and  I  should  like  to  mention 
briefly  a  few  of  the  possible  approaches  to  these  problems. 

One  of  them  is  based  on  a  concept  whose  origin  as  well  as  elaboration 
we  owe  to  Richard  Goldschmidt  (11).  It  is  the  concept  of  the  phenocopy, 
which  has  been  used  by  Goldschmidt  and  others  to  attack  problems  of 
gene  action.  If  it  were  possible  with  the  help  of  an  environmental  agent 
to  reproduce  the  individual  steps  in  a  chain  of  processes  begun  by  a 
mutant  gene,  a  true  phenocopy  would  result  which  would  be  of  great 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


633 


value  for  an  analysis  of  the  detailed  mechanism  by  which  a  gene  pro- 
duces its  phenotypic  effect. 

In  this  connection  the  abnormalities  in  the  rat  reported  by  Gillman 
and  coworkers  (12)  in  the  offspring  of  mothers  treated  with  trypan  blue 
seemed  to  resemble,  at  least  in  their  final  manifestations,  the  abnormalities 
caused  by  genie  action  of  mutants  of  Chromosome  IX  of  the  mouse.  In 
order  to  study  this  resemblance  more  closely,  Hamburgh  (13)  carried  out 
an  investigation  directed  towards  an  analysis  of  the  individual  steps  by 
which  the  abnormalities  induced  by  trypan  blue  arose.  His  results  showed 
that  these  were  not  mediated  by  the  notochord-mesoderm  system  and 
were  thus  different  from  the  mutant  effects,  not  being  true  phenocopies. 

Another  approach  to  the  problem  of  gene  action  in  differentiation 
which  we  are  following  up  at  the  moment  is  based  on  ideas  which  conceive 
of  the  processes  of  embryonic  growth  and  differentiation  as  essentially 
similar  to  those  operating  in  antibody  formation.  Furthermore,  since  in 
the  case  of  the  mutations  in  the  mouse  described  above  indications  exist 
that  primary  gene  action  may  involve  immune  reactions,  it  occurred  to  us 
that  the  interference  of  these  mutations  with  normal  processes  of  growth 
and  differentiation  might  be  mediated  by  a  mechanism  resembling 
immune  reactions. 

In  order  to  investigate  this  possibility  we  started  out  with  an  exami- 
nation of  the  effect  which  immunization  of  normal  mothers  against  specific 
organ  tissues  might  have  on  the  development  of  the  corresponding  organ 
system  of  their  embryos.  Indications  of  specificity  of  effect  were  obtained 
from  such  experiments;  immunization  with  brain  affected  the  developing 
nervous  system  of  embryos  more  specifically  than  did  immunization  with 
heart,  which  resulted  in  different  types  of  abnormalities,  including  those  of 
the  mesoderm. 

These  experiments  are  in  a  preliminary  state  and  require  extension  and 
corroboration.  Eventually  the  study  will  be  extended  from  normal  strains 
to  tissues  from  mutant  individuals  and  both  the  immunizing  effect  of 
mutant  tissues  and  the  reaction  of  mutant  embryos  to  immunization  of 
the  mothers  will  be  investigated. 

From  the  material  available  I  have  selected  here  for  discussion  some  of 
the  studies  which  demonstrate  the  role  of  genes  in  processes  of  embryonic 
growth  and  differentiation  in  a  mammal.  On  the  other  hand,  we  all  know 
that  these  processes  are  subject  to  the  effect  of  other  than  genetic  factors 
as  well  and  that  genetic  and  nongenetic  factors  interact  intimately  in 
mechanisms  of  growth  and  differentiation.  The  study  of  genetic  control 
of  early  embryonic  processes  has  opened  up  a  number  of  interesting  prob- 
lems in  addition  to  the  descriptive  analysis  of  gene  effects.  One  of  these 
is  the  relative  scarcity  of  genetically  caused  abnormalities  known  to  occur 
in  early  development;  this  might  possibly  be  the  result  of  the  considerable 
regulatory  power  which  the  mammalian  embryo  has  been  shown  to  possess 
like  other  vertebrates  and  which  enables  it  to  survive  unharmed  adverse 
genetic  or  environmental  effects.  It  is  interesting  to  consider  the  possible 
genetic  control  of  this  regulatory  ability  which  diminishes  as  development 
proceeds;  the  similarity  of  genetic  factors  controlling  regulatory  power 

Vol.   15,  No.  3,  December  1954 


634  proceedings:  symposium 

to  modifying  genes,  and  the  possible  mechanism  by  which  they  exert  their 
control,  have  been  discussed  by  us  recently  (9). 

Another  interesting  aspect  of  genetic  factors  controlling  growth  and 
differentiation  is  their  pleiotropic  effect.  Frequently,  the  association  of 
different  effects  as  the  result  of  abnormal  genie  action  is  not  easily  under- 
stood on  the  basis  of  existing  knowledge  of  the  interrelationships  of  de- 
velopmental structures;  such  an  association  might  point  toward 
hitherto  unknown  interdependencies  of  developmental  systems  which 
thus  may  become  revealed  by  pleiotropic  gene  effects.  However,  it  is 
necessary  here  to  remember  the  limitations  imposed  by  the  use  of  the 
purely  descriptive  approach  to  such  problems;  disregard  of  these  limita- 
tions has  frequently  produced  unwarranted  conclusions,  for  example,  in 
regard  to  gene  action  from  studies  of  pleiotropic  gene  effects. 

The  study  of  developmental  effects  of  mutations  in  the  mouse  has 
served  to  confirm  in  mammals  the  existence  of  developmental  mechanisms 
demonstrated  previously  by  experimental  means  in  other  vertebrates. 
This  fact  has  been  stressed  repeatedly  and  will  not  be  discussed  here  again. 

Of  course,  the  main  question  encountered  in  all  approaches  to  problems 
of  genetic  control  of  differentiation  is  that  of  the  mechanism  of  differentia- 
tion, knowledge  of  which  would  no  doubt  help  to  clarify  problems  of  gene 
action;  on  the  other  hand,  it  may  not  be  too  presumptious  to  expect  that 
the  study  of  genetic  factors  controlling  differentiation  will,  in  its  turn, 
contribute  to  the  clarification  of  the  very  problem  of  differentiation. 

References 

(1)  Spemann,  H.,  and  Schotte,  O.:  tJber  xenoplastische  Transplantation  als  Mittel 

zur  Analyse  der  embryonalen  Induktion.     Naturwiss.  20:  463-467,  1932. 

(2)  Steiniger,  F. :  Neuse  Beobachtungen  an  der  erblichen  Hasenscharte  der  Maus. 

Ztschr.  menschl.  Vererb.-u.  Konstitutionslehre  23:  427-462,  1939. 

(3)  Gluecksohn-Schoenheimer,  S.,  and  Dunn,  L.  C:  A  new  type  of  hereditary 

harelip  in  the  house  mouse.     Anat.  Rec.  102:  279-287,  1948. 

(4)  Fraser,  F.  C,  and  Fainstat,  T.  D.:  Production  of  congenital  defects  in  the 

offspring  of  pregnant  mice  treated  with  cortisone.     Pediatrics  8:  527-533,  1951. 

(5)  Bonnevie,   K.:  Pseudencephalie  als  spontane  recessive   (?)    Mutation  bei  der 

Hausmaus.     Norske  Vidsk.-Akad.  Oslo  Skr.  Kl.  1,  #9,  1936. 

(6)  Snell,   G.   D.,  Bodemann,   E.,  and  Hollander,   W.:  A  translocation  in  the 

house  mouse  and  its  effect  on  development.     J.  Exper.  Zool.  67:  93-104,  1934. 

(7)  Chesley,  P.:  Development  of  the  short-tailed  mutant  in  the  house  mouse.     J. 

Exper.  Zool.  70:  429-459,  1935. 

(8)  Gluecksohn-Waelsch,    S.:  Physiological   genetics    of   the    mouse.     Advances 

Genet.  4:  1-51,  1951. 

(9)  :  Lethal  factors  in  development.     Quart.  Rev.  Biol.  28:  115-135,  1953. 

(10)  Hertwig,  P.:  Die  Genese  der  Hirn- und  Gehororganmissbildungen  bei  rontgen- 

mutierten  Kreisler-Mausen.     Ztschr.  menschl.  Vererb.-u.   Konstitutionslehre 
28:  327-354,  1944. 

(11)  Goldschmidt,  R. :  Physiological  Genetics.     New  York,  McGraw-Hill  Book  Co., 

Inc.,  1938. 

(12)  Gillman,  J.,  Gilbert,  C.,  and  Gillman,  T.:  A  preliminary  report  on  hydro- 

cephalus, spina  bifida  and  other  congenital  anomalies  in  the  rat  produced  by 
trypan  blue.     South  African  J.  M.  Sc.  13:  47-90,  1948. 
(IS)  Hamburgh,   M.:  Malformations  in  mouse  embryos  induced  by  trypan  blue. 
Nature  169:  27,  1952. 


Discussion 
Dr.  Morris  Smithberg,  Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 

Dr.  Waelsch  has  brought  out  many  interesting  problems  in  her  excellent  review 
concerning  genetics  in  development,  any  one  of  which  could  be  discussed  at  con- 
siderable length.  However,  underlying  these  problems  I  believe  there  are  two 
fundamental  questions.  The  first  question  would  ask  whether  embryonic  growth  is 
at  all  under  the  control  of  genetic  factors.  Most  biologists,  if  confronted  with  this 
question  some  years  back,  would  have  said,  "Yes,  we  would  probably  expect  it  to  be." 
The  evidence  for  this  expectation  still  had  to  be  uncovered,  and  Dr.  Waelsch  has 
presented  the  affirmative  data.  However,  it  it  necessary  to  add  that  these  genetic 
factors  can  express  themselves  within  the  limits  of  the  environment  and,  likewise, 
that  environmental  factors  can  express  themselves  within  the  limits  of  the  genetic 
constitution  of  the  organism. 

The  second  question  is,  how  does  the  gene  act  in  the  control  of  developmental 
processes?  This  is  a  doubly  perplexing  situation  since  the  underlying  mechanisms  of 
both  gene  action  and  development  are  as  yet  not  completely  understood. 

There  are  many  approaches  to  these  perplexing  problems.  I  will  touch  on  just  one 
or  two.  The  use  of  mutant  strains  of  mice  in  an  attempt  to  understand  gene  action 
and  development  has  of  necessity  been  limited  to  study  of  processes  rather  late  in 
development.  Mutants  of  pre-implantation  stages  are  very  scarce.  Those  mutants 
which  find  expression  late  in  development  are  mostly  of  a  grossly  morphologic  nature 
and  eventually  are  interpreted  through  knowledge  gained  by  experimental  embryolo- 
gists  using  lower  vertebrates,  such  as  the  frog  or  chick.  For  instance,  the  action  of  a 
mutant  gene  of  the  tailless  series  can  be  simulated  by  extirpation  of  the  terminal 
portion  of  the  notochordal  anlage  of  the  frog.  Perhaps  the  most  notable  exception  to 
the  morphologic-type  mutant  deals  with  the  work  of  Dr.  Russell  of  the  Jackson 
Laboratory.  Dr.  Russell  has  traced  expression  of  a  mutant  gene  to  a  faulty  synthesis 
in  the  hemoglobin  molecule.  It  is  with  work  on  mutants  such  as  this  that  the  investi- 
gator probably  gets  closest  to  the  actual  nature  of  the  gene  action. 

The  immunological  approach  to  the  primary  nature  of  the  gene  action,  as  mentioned 
by  Dr.  Waelsch,  is  probably  the  most  promising  and  perhaps  needs  more  elaboration. 
The  impetus  for  research  of  antigen-antibody-like  reactions  in  development  results 
from  two  papers  published  simultaneously  in  1947  by  Tyler  and  Weiss.  Since  these 
stimulating  papers  many  investigators,  notably  Cooper,  Spar,  Clayton  and  Flickinger, 
using  amphibians,  and  Schechtman  and  his  students,  using  the  chick,  have  found 
ample  evidence  for  the  epigenesis  of  antigens  during  development.  In  addition, 
Ebert,  using  chick  embryos,  has  been  able  to  block  the  differentiation  of  chick-brain 
and  chick-heart  selectively  by  the  use  of  anti-heart  and  anti-brain  sera.  Ebert  has 
also  been  able  to  modify  the  growth  of  embryonic  chick  spleen  by  placing  adult 
chicken-spleen  tissue  upon  the  chorio-allantoic  membrane. 

It  was  very  interesting  indeed  to  learn  that  Dr.  Waelsch  has  been  successful  in 
producing  selectively  anomalous  embryos  in  inbred  strains  of  mice.  The  genetic 
uniformity  of  her  material  may  perhaps  give  her  a  more  defined  type  of  anomaly. 
Unfortunately,  she  is  not  here  to  give  us  the  results  in  more  detail. 

Along  these  same  lines  it  was  our  aim  to  try  to  modify  the-growth  of  the  regenerating 
liver  of  an  adult  mouse  with  the  use  of  anti-liver  serum  prepared  in  rabbits.  The 
ability  of  the  liver  to  regenerate  after  2  of  the  3  lobes  are  surgically  removed  is  one  of 
the  few  regenerative  processes  still  maintained  in  the  adult.  By  the  7th  postoperative 
day  the  lobe  left  behind  usually  increased  in  size  closely  approaching  the  total  original 
weight  at  the  time  of  surgery.  The  increase  in  weight  involved  both  cell  number  and 
ceil  size.     Anti-liver  serum  or  normal  rabbit  serum,  injected  after  surgery,  did  not 

635 


Journal    of   the   National    Cancer   Institute,    Vol.    15,   No.    3,    December    1954 


636  proceedings:  symposium 

significantly  influence  the  final  weight  of  the  growing  liver  as  we  had  expected.  How- 
ever, the  anti-serum  did  modify  the  mitotic  pattern  of  the  regenerating  liver.  In  an  un- 
treated control  animal,  or  one  in  which  normal  serum  was  injected  into  partially 
hepatectomized  animals,  one  finds  a  profound  increase  of  mitoses  at  2  to  3  days  and  a 
considerable  dropping  off  at  day  4  to  day  7  after  surgery.  On  day  7  there  are  practi- 
cally no  mitoses  to  be  found.  Partially  hepatectomized  animals  injected  with  anti- 
liver  serum  show  the  same  increase  of  mitoses  on  day  2  to  3  as  the  controls.  However, 
at  day  7  the  animals  receiving  anti-liver  serum  maintained  a  high  level  of  mitotic 
figures.  Since  the  experiment  is  only  in  a  preliminary  stage,  we  cannot  say  much  more 
about  this  discrepancy  in  mitotic  pattern. 

I  should  like  to  end  this  discussion  with  some  general  impressions.  There  was  a 
time  when  the  geneticist  camped  in  the  nucleus,  and  the  embryologists  camped  in  the 
cytoplasm.  Both  were  very  shy,  but  nevertheless  mutually  attracted.  Only  at 
metaphase,  with  the  breakdown  of  the  nuclear  membrane  did  they  temporarily  meet. 
The  embryologists  emerged  from  the  other  side  of  the  barrier  during  xenoplastic 
transplants.  But  as  nature  would  have  it,  genetics  and  embryology  have  finally 
merged,  and  a  better  knowledge  of  development  should  be  the  offspring  of  this  union. 
It  is  the  hope  of  all  geneticists  and  embryologists  that  this  offspring  will  exhibit 
extreme  hybrid  vigor. 


Inheritance  of  Susceptibility  to  Con- 
genital Deformity — Embryonic  Insta- 
bility1'2 


Meredith     N.     Runner,    Roscoe    B.    Jackson 
Memorial  Laboratory,  Bar  Harbor,  Maine 


The  proportion  of  medical  practice  and  hospital  facility  devoted  to  the 
developing  baby  (e.g.  our  children's  hospitals)  is  commensurate  with  that 
devoted  to  some  of  our  more  serious  diseases.  Infant  mortality  during 
the  past  40  years  has  decreased  from  12  to  2  percent.  Of  6  infants  that  in 
1910  would  have  died,  today  5  of  them  attain  their  first  birthday.  These 
medical  achievements,  however,  are  not  without  their  complications. 
Although  nutritional  deficiencies  and  infectious  diseases  are  almost  non- 
existent in  mothers  during  pregnancy,  the  prediabetic  condition,  for 
example,  has  become  increasingly  associated  with  production  of  abnormal 
babies.  Why  deformity  is  associated  with  diabetic  mothers  is  not  under- 
stood, but  in  general  one  can  say  that  improved  maternal,  prenatal  health 
has  increased  the  probability  of  survival  of  deformed  embryos  that 
formerly  would  have  succumbed  under  less  favorable  circumstances.  Ac- 
complishments with  prenatal  health  of  the  mother  have  progressed  to  the 
point  where  researchers  can  now  consider  factors  that  influence  the  health 
of  the  embryo. 

This  report  will  attempt  to  demonstrate  the  extent  to  which  we  can 
experimentally  unravel  interactions  of  genetic  and  environmental  factors 
that  influence  health  of  the  embryo.  My  primary  objective  will  be  to 
arrive  at  a  plan  that  will  approach  an  explanation  for  or  even  manipulation 
of  differences  between  normal  and  abnormal  morphogenesis.  Any  plan 
for  experimentation  must  be  based  upon  a  combination  of  pre-existing 
information  mixed  with  a  few  good  assumptions.  Since  no  hypothesis 
is  any  better  than  its  worst  assumption,  I  will  outline  the  mixture  of  fact 
and  assumption  that  we  may  call  principles  basic  to  this  report. 

Each  individual  is  the  resultant  of  interaction  between  effects  of  his  hereditary 
background  and  contingencies  of  his  environment.  In  other  words  each  of  us 
is  the  product  of  nature,  in  a  genetic  sense,  and  nurture,  in  the  broad 
environmental  sense.  Your  presence  here  today  is  ample  proof  that  nature 
and  nurture  have  not  had  a  major  clash  since  the  time  of  your  own  con- 
ception. 

1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  28,  1954. 

a  These  investigations  were  supported  in  part  by  a  research  grant  G-3859  from  the  National  Institutes  of  Health, 
Public  Health  Service,  U.  S.  Department  of  Health,  Education,  and  Welfare. 

637 


Journal   of  the  National  Cancer  Institute, 
316263—54 24 


Vol.   15.  No.   3,  December   1954 


638  PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 

Few  morphologic  characteristics  provide  geneticists  with  an  a  priori  basis 
with  which  to  foretell  the  respective  importance  of  heredity  and  environment. 
We  know  that  when  all  the  evidence  is  tabulated  and  lenient  extrapola- 
tions are  made,  considerable  proportions  of  individual  differences  in 
morphology  are  attributable  to  environmental,  nongenetic  factors.  Since 
no  genie  machinery  operates  in  a  vacuum,  it  becomes  essential  to  investi- 
gate the  environment  with  which  heredity  reacts.  If  we  cannot  have  good 
heredity  let  us  console  ourselves  by  the  fact  that  susceptibility  to  some 
hereditary  defects  can  be  offset  by  superior  environment. 

Infinitesimally  small  visible  variations  are  important  during  early  phases 
of  prenatal  development.  Although  these  early  minute  variations  are  often 
differences  of  degree  and  difficult  to  quantitate,  the  embryo  has  limited 
corrective  powers  and  these  variations  may  account  for  major  differences 
between  you  and  the  hopelessly  deformed  individual. 

Any  given  process  of  normal  development  is  regulated  by  numerous  genie 
units  and  the  primary  action  of  any  one  gene  is  probably  many  steps  removed 
from  direct  control  of  normal  embryology.  Embryologic  determination  may 
be  the  result  of  several  genes  acting  concomitantly  and  others  acting 
sequentially.  Grimeberg  (1)  has  suggested  an  analogy  with  evolution 
which  is  supposed  to  be  irreversible  though  it  is  brought  about  by  genie 
mutations  each  of  which  is  reversible.  High  stability  of  tissue  interactions 
during  normal  development  may  offer  a  selective  advantage.  Determina- 
tion probably  happens  in  stages  which  successively  narrow  the  develop- 
mental possibilities  of  a  region  until  it  finally  becomes  completely  deter- 
mined. The  ultimate  morphologic  results  are  determined  by  a  series  of 
concomitant  and  preceding  gene  effects  on  a  biochemical  level. 

An  evolutionary  safeguard  for  preservation  of  the  species  is  to  have  the 
fate  of  the  embryo  independent  of  any  one  single,  critical,  genetic  locus. 
On  the  other  hand,  the  more  intermediate  steps  that  exist  between  primary 
activity  of  genes  and  embryologic  differentiation,  the  larger  is  the  target 
for  environmental  influences  and  the  greater  is  the  probability  of  inter- 
ference with  normal  processes.  Ontogeny  is  probably  associated  with  an 
optimal  number  of  stepwise  processes  that  makes  the  embryo  a)  inde- 
pendent of  instability  of  single  genetic  loci  and  b)  not  overly  susceptible 
to  environmental  modification. 

A  customary  experimental  approach  for  separating  roles  of  2  variables 
(like  heredity  and  environment)  is  to  hold  one  variable  constant.  Since 
our  present  state  of  knowledge  precludes  precise  control  of  environmental 
conditions  for  mammalian  embryos  I  will  present  results  based  upon 
genetically  standardized  mammalian  embryos.  Identical  twins  would  best 
suit  our  needs  as  Dr.  Dunn  mentioned  yesterday,  for  with  such  twins  we 
could  study  identical  genotypes.  Spontaneous  or  induced  variations  in 
one  member  of  a  twin  would  be  due  to  environmental  effects.  Identical 
twinning  is  not  available  to  meet  our  experimental  requirements.  How- 
ever, we  do  have  the  next  best  mammalian  material.  Here  I  pay  homage 
to  the  vision  and  persistence  of  my  predecessors,  notably  Dr.  Little,  who 
have  contributed  inbred  mammalian  material.     For  practical  purposes 

Journal   of  the  National  Cancer  Institute 


PROGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


639 


we  have,  within  an  inbred  strain,  an  unlimited  number  of  identical  twins, 
i.e.  genetic  uniformity. 

The  manner  by  which  embryonic  differentiation  can  be  influenced  by 
environment  and  heredity,  acting  both  on  the  maternal  and  fetal  organism, 
may  be  illustrated  in  text-figure  1.  If,  in  our  experiment,  we  apply  two 
different  environmental  agents  to  the  same  genetic  strain  of  mouse,  hered- 
ity, being  common  to  both  experimental  groups,  can  be  canceled  out  as  a 
source  of  variability.  This  experiment  therefore  tests  for  difference  in 
effects  of  environment  (A)  and  (B). 

Experiments    on    prenatal    environment    in    which    hereditary    influences    are    canceled    out 


ENVIRONMENT      (a) 
siology  < A 


Maternal     physiology 

1 

Embryonic    physiology  «-- 
Morphogenesis 

i 

Phenofype       (  a  J 


ENVIRONMENT      (b) 


Maternal    physiology 

1 

Embryonic    physiology  <« 

Morphogenesis 

I 

Phenotype       (  b  ) 


Text-figubb  1. 


My  objective  to  present  evidence  for  environmental  modification  of 
morphogenesis  will  be  achieved  by  the  following  steps:  1)  Presentation  of 
characteristics  that,  in  genetically  uniform  populations,  are  susceptible 
to  modification  by  environmental  factors;  2)  demonstration  that  maternal, 
experimental  and  spontaneous  influences  do  in  fact  alter  development; 
and  3)  investigation  of  physiologic  processes  that  produce  deviation 
from  the  straight  and  narrow  path  of  normal  ontogenesis. 

A.  Teratogenic  Influences  Applied  to  Different  Genetic  Backgrounds 

Importance  of  genetic  background  upon  control  of  morphogenesis  is 
not  to  be  considered  lightly.  What  happens  when  different  genotypes  are 
subjected  to  a  common  teratogenic  influence?  Geneticists  on  a  number  of 
occasions  have  performed  such  experiments.  A  single  teratogenic  locus 
can  be  introduced  into  different  isogenic  strains  of  mice  by  backcross 
breeding  programs.  The  mutant  locus  may  be  considered  the  teratogenic 
agent  and  the  recipient  inbred  strains  will  provide  genetically  different 
backgrounds.  Often  the  phenotype  that  one  gets  will  vary  quite  un- 
predictably from  lethality  to  nonexpression,  depending  upon  the  genetic 
background  within  the  fetus  and  the  indirect  effect  of  the  genetic  back- 
ground regulating  maternal  physiology.  An  interesting  illustration  of 
this  is  expression  of  the  gene  Fu  (table  1).  The  genome  of  strain  BALB/c 
and  of  C57BH/a  permit  the  Fu  gene  to  express  itself  in  88  and  65  percent  of 
the  genotypic  offspring.  Strain  C57BE,/a  genome  contains  normalizing 
modifiers  that  protect  a  significant  number  of  embryos  from  the  teratogenic 
locus.     Strain  C57BJR,/a  mothers  carrying  the  gene  Fu  exert  an  additional 


Vol.    15,  No.   3,  December   1954 


640 


proceedings:  SYMPOSIUM  ON  25  YEARS  op 


normalizing  influence  on  their  embryos  for  the  mutant  expresses  itself  in 
only  34  percent  of  the  offspring.  Thus  the  genetic  background  of  the 
fetus,  and  in  this  case  that  of  the  mother,  has  a  permissive  effect  on  ex- 
pression of  a  teratogenic  mutant. 

Table  1. — Percent  penetrance  of  Fu  gene  in  heterozygotes 


Strain 

Mother 

Fu+ 

+  + 

BALB/c 
C57BR/a 

72 
34 

88 
65 

Effect  of  genetic  backgrounds  has  also  been  tested  against  experimen- 
tally administered  teratogenic  agents.  Ingalls,  Avis,  Curley  and  Temin 
(2)  subjected  groups  of  pregnant  mice  from  five  different  strains  to  re- 
duced atmospheric  pressure.  Their  best  example  was  induced  "mal- 
formation" of  ribs  and  vertebrae.  Four  strains  treated  (5  hours  at  27,000 
ft.  on  day  9)  were  modified  but  the  fifth  strain  of  mouse  was  not  affected. 
Although  differences  between  strains  were  apparently  observed,  the  ex- 
periment could  be  cited  with  more  confidence  if  the  authors  had  given  a 
clearer  account  of  the  criteria  by  which  fetuses  were  judged  "malformed." 
Nevertheless  genetic  factors  apparently  rendered  chondrogenesis  in  cer- 
tain strains  more  susceptible  to  modification  than  in  other  strains. 

Interaction  of  two  genotypes  with  a  teratogenic  agent  has  been  reported 
by  Fraser  and  colleagues  (3,  4).  A  given  dose  of  cortisone  (4  X  2.5  mg.) 
was  administered  to  two  strains  of  mice  and  descendents  from  these 
strains.  Text-figure  2  has  been  adapted  from  their  data  for  treatments 
beginning  on  day  11  of  pregnancy.  Cleft  palate  served  as  a  specific 
biological  end  point  to  study  inheritance  of  susceptibility  to  cortisone. 
Strains  A  and  C57BL  had  susceptibilities  of  100  and  18  percent,  respec- 
tively. (It  would  be  interesting  to  speculate  why  18  percent  of  the 
C57BL  young  had  cleft  palate  and  82  percent  completely  escaped.) 
Strain  A  females  were  mated  with  males  of  strain  C57BL  and  the  A 
mothers  were  treated  as  before.  The  anomaly,  cleft  palate,  occurred  in 
43  percent  of  the  offspring.  On  first  glance  this  might  appear  as  a  simple 
case  of  genetic  intermediacy  between  the  2  parental  frequencies.  Hybrid 
fetuses  of  the  same  genie  constitution  were  produced  within  treated 
mothers  of  strain  C57BL.  The  frequency  of  cleft  palate  was  4  percent — 
even  less  frequent  than  in  C57BL  young  gestated  within  resistant  C57BL 
mothers.  Since  the  two  groups  of  hybrid  young  were  genetically  identical, 
differences  in  the  frequencies  of  cleft  palate  (43  and  4  percent)  were  due 
to  differences  in  susceptibility  of  the  two  types  of  mothers  to  cortisone 
or  to  differences  in  tr admissibility  of  cortisone  effects  to  the  fetus.  The 
increased  resistance  of  hybrid  fetuses  in  C57BL  mothers,  as  compared 
with  C57BL  fetuses  in  C57BL  mothers,  must  be  explained  on  the  basis  of 
fetal  protection  due  to  their  own  hybrid  vigor. 

Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


641 


INCIDENCE    OF   CLEFT    PALATE    FOLLOWING  4  DAILY 
TREATMENTS    WITH    CORTISONE 


D-rO 


100% 
n*36 


18% 


□  # 


*T^ 


4% 
n=82 

D-p©     D-p-€) 


43% 
P.  F 


OS 

p, 

^J  STRAIN  A 

P2 

^fcsTRAIN  C57BL 


24% 
n»83 

P,  8C 


25% 
n=7i 


arret  Fttaseit  a  fains  tat  1931  a  k alter  1934 


55% 

nsl28 


Text-figure  2. 


Hybrid  females  (A  X  C57BL,  C57BL  X  A)  were  mated  with  strain  A 
males.  Hybrid  mothers,  treated  with  cortisone,  gave  an  incidence  of 
24  percent  cleft  palate,  a  relatively  low  incidence  considering  that  the 
mother  had  half  and  fetuses  had  three-quarters  of  the  genie  components 
of  the  susceptible  strain  A.  We  may  consider  either  partial  dominance 
of  resistance  on  the  part  of  the  fetus  or  hybrid  vigor  (delayed  maternal 
influence)  on  the  part  of  the  mother  to  account  for  the  low  incidence 
of  anomaly. 

Animals  of  another  generation  were  backcrossed  to  strain  A  males. 
These  mothers,  treated  with  cortisone,  gave  offspring  with  55  percent 
incidence  of  cleft  palate.  These  mothers  had  three-quarters  of  strain  A 
genes  and  the  young  had  seven-eighths.  Apparently  genetic  susceptibility 
had  increased  and  heterosis  decreased  to  permit  high  incidence  of  suscep- 
tibility to  the  teratogen.  Expression  of  cleft  palate  was  shown  to  be 
modifiable  by  the  genotype  expressed  through  maternal  physiology. 
Hybrid  vigor  of  both  the  mother  and  the  fetus  was  able  to  offset,  at 
least  in  part,  the  genetic  susceptibility.  Holding  constant  the  teratogen 
and  varying  the  genetic  background  has  demonstrated  the  permissive 
influence  of  the  genotype. 

Spontaneous  variations  within  isogenic  strains  reared  in  the  same  animal 
house  may  also  be  regarded  as  reactions  of  different  genotypes  to  a  com- 
mon, though  undefined,  environment.  Characterization  of  permissive 
effect  of  5  genotypes  indicates  that  polymorphism  exists  in  the  genetically 
uniform  populations  (table  2).  The  table  reports  only  those  anomalies 
that  in  each  strain  approximates  20  percent  incidence  or  more.  Each 
genotype  has  its  characteristic  pattern  of  morphologic  variability.  Poly- 
morphism is  just  as  characteristic  of  an  inbred  strain  of  mouse  as,  for 
example,  is  its  susceptibility  to  infectious  diseases  or  its  spontaneous 
patholigic  changes  that  Dr.  Gowen  (5)  and  Dr.  Dunn  (6)  reported 
yesterday. 


Vol.  15,  No.  3,  December  1954 


642  PKOCEEDINGS:   SYMPOSIUM  ON  25  YEARS  OF 

Table  2. — Frequency  percent  of  skeletal  anomalies  in  5  inbred  strains  of  mice 


Anomaly 

129 

YBL 

C57BR 

BALB/c 

A 

Interfrontal  bone 

66 

25 
80 

90 
100 

50 
100 

Parted  frontal  bones 

55 

46 

Imperfect  transverse  foramen  #4—6 

Rudimentary  ribs,  vertebra  #7 

54 
23 
38 

50" 

20 

58 

Dorsal  dyssymphysis,  #1-20 

Perforation  of  neural  plate,  #12  or  13 

Absence  of  spinous  process  #9 

21 

71 

46 

Accessory  sternebrae 

62 

50 

Sternebral  ankvlosis 

50 

100 

Xiphoid  dyssymphysis 

Lumbar  ribs,  vertebra  #21 

29 

90 
44 

Lumbar,  split  centrum.  .  . 

13 
23 
57 

90 

Lumbar  ankylosis 

Sacralization  of  vertebra  #26 

25 
22 

n — 

12 

16 

24 

Many  of  the  structural  variations  within  an  inbred  strain  are  embryo- 
logically  determined  as  demonstrated  by  Green  (7)  and  Sawin  (8).  Dr. 
Bailey  (9)  has  illustrated  how  genetic  analyses  within  and  between  strains 
can  demonstrate  developmental  forces.  A  mathematical  approach  can 
also  be  a  dynamic  one. 

Recently  we  (10)  restudied  one  of  the  earliest  reported  examples  of 
nongenetic  variability  in  the  mouse.  Strain  C57BR  was  shown  by  Murray 
and  Green  in  1933  (11)  to  have  about  half  of  its  members  with  ventral 
white.  The  size  of  the  white  patches  varied  from  a  few  white  hairs  to 
one  third  of  the  ventral  surface.  Our  survey,  20  years  and  many  genera- 
tions later,  has  substantiated  Green's  observations.  (Males  had  slightly 
more  white  than  females  but  there  was  precious  little  correlation  between 
the  pheno types  of  parents  and  offspring.)  Our  method  of  recording  white 
spots  enabled  us  to  map  the  distribution  of  white  (text-fig.  3).  The  pattern 
provided  a  V-shaped  area  pointing  posteriorly  in  the  midline.  Absence 
of  pigmentation  suggested  a  block  to  melanoblast  migration  or  a  wave 
of  degeneration  of  melanoblast  cells.  Morphology  of  ribs  and  sternum 
in  this  strain  of  mouse  (table  2)  contributes  additional  evidence  for 
similar  dynamic  phenomena  in  the  lateral  mesoderm. 

These  examples  illustrate  that  genetic  backgrounds  play  a  permissive 
role  when  environmental  influences,  induced  or  spontaneous,  modify 
development.  I  want  next  to  turn  to  the  relation  between  pattern  of 
variation  of  a  single  genotype  in  response  to  a  number  of  environmental 
influences.     What  is  the  repertoire  of  a  genotype? 

B.   Teratogenic  Agents  Applied  to  a  Single  Genotype 

Responses  of  mice  of  strain  129  to  a  number  of  environmental  influences 
have  been  studied  in  our  laboratory  (12).  Spontaneous  polymorphism  in 
strain  129  is  listed  as  control  frequencies  in  column  6  of  table  3. 

Journal    of   the   National    Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


643 


45 

> 

40 

s 

a& 

.35 

s 

S  30 

> 

IT 

/ 

fij  ma 

S  25 
o 

A 

<r  20 
8 

s 

< 

|    15 

s 

10 

s                    ^y^ 

f. 

5 

'     ^^^ 

0 

•~-    IK      3 


4*re, 


"/0/» 


Text-figure  3. — Distribution  and  frequency  of  belly  spotting  in  an  inbred  strain  of 
mouse— C57BR/cd.  n  =  230. 

Table  3. — Frequency  percent  of  skeletal  anomalies  in  strain  129* 


Experimental  groups 

Anomaly 

1 

Trypan 
blue 

2 

Reduced 
pressure 

3 

Ova 

transfer 

4 

Corti- 
sone 

5 

Old 
parents 

6 
Control 

Interfrontal  bone 

91.3 

10.8 
73.9 

32.4 

66.  1 

Rudimentary  ribs,  vertebra 
#7 

53.  8 

Dorsal  dyssymphysis  #1-20 . 
Perforation  of  neural  plate, 
#12  or  13 

93.7 

56.2 

6.2 

28.  1 

45.9 

23.0 
38.  4 

Accessory  sternebrae 

84.3 

82.4 
48.6 

0 
36.5 

5.9 
70.0 

62.  3 

Xiphoid  dyssymphysis 

29.  2 

Lumbar  ankylosis,  usually 
#  23  and  24 

47.8 
21.7 
39.  1 

23 

23.  1 

Sacralization  of  #26 

Caudal  ankylosis 

29.4 

30.6 

56.9 
0 

n= 

17 

18 

37 

55 

65 

*When  compared  to  controls  all  Items  are  below  1%  confidence  level. 

Experimental  groups:  1)  0.25  mg.  of  trypan  blue  on  day  7;  2)  2  hours  at  25,000  ft.  on  day  7;  3)  BALB/c  mother; 
4)  0.1  mg.  of  cortisone  on  day  7;  5)  litters  3-7. 

Five  other  samples  of  this  strain  were  surveyed  after  mothers  and  em- 
bryos were  subjected  to  various  treatments,  viz.,  0.25  mg.  of  trypan  blue 
on  day  7,  reduced  atmosphere  of  25,000  feet  on  day  7,  gestation  in  BALB/c 
mothers,  0.1  mg.  of  cortisone  on  day  7,  and  gestation  in  elderly  parents. 
Frequencies  of  anomalies  are  given  in  the  table  only  in  instances  that  are 
significantly  different  from  the  control  sample.  Two  points  can  be  drawn 
from  the  data,  a)  Those  structures  that  vary  in  the  controls,  i.e.,  suscep- 
tible characters,  were  altered  by  the  treatments  employed,    b)  Just  as 


Vol.   15,  No.  3,  December   1954 


644 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


did  each  genotype  have  its  characteristic  polymorphism,  so  did  each 
treatment  have  its  characteristic  pattern  of  anomalies. 

One  of  the  susceptible  characters  in  strain  129  has  been  selected  for 
presentation  in  more  detail.  It  is  more  than  coincidence  that  I  selected 
the  trait  that  Dr.  Green  (7)  has  so  skillfully  analyzed.  Differentiation  of 
vertebra  #26  in  mice  of  strain  129  finds  itself  in  an  ontogenetically  uncer- 
tain situation.  It  can  become  a)  a  typical  lumbar  as  happens  in  most 
strains  of  mice,  b)  a  typical  sacral  or  c)  something  in-between  (fig.  1). 
Strain  129  is  particularly  well  suited  for  our  study  because  the  three  types 
of  vertebrae  occur  with  about  equal  frequency.  The  sacralization  influ- 
ence of  all  treatments,  so  far  investigated,  are  listed  (table  3,  text-fig.  4). 
The  data  show  a  general  tendency  for  treatments  on  day  7,  improvement 
of  maternal  diet,  and  gestation  in  BALB/c  mothers  to  reduce  the  frequency 
of  sacralization.  Offspring  from  young  parents  were  not  significantly 
altered  but  offspring  from  older  parents  showed  a  tendency  to  increased 
incidence  of  sacralization.  Inherited  propensities  in  strain  129  were  not 
necessarily  final  judgments  for  the  embryo.  The  probability  of  occurrence 
of  anomaly  could  be  shifted  so  that  one  could  reduce  or  increase  the 
incidence  of  sacralization.  Thus  the  environmentally  susceptible  char- 
acter could  be  enhanced,  suppressed,  or  not  affected  depending  upon  the 
environmental  agent. 

Environmental  influences  applied  to  the  embryo,  so  far  presented,  have 
been  applied  empirically.  Variations  observed  have  not  indicated  path- 
ways by  which  environment  modifies  morphogenesis.  After  learning  what 
agents  can  alter  development,  an  inquiring  mind  wants  to  know  how  a 
given  agent  can  create  embryonic  imbalance.  Since  the  agents  so  far 
reported  did  not  provide  clues  about  mode  of  action  we  have  used  another 


0.25  ntq     TRYPAN 
BLUE   ON 
DAY    7 

2  HOURS  AT 
85.000  FEET 
ON  OAY    7 

OVA    TRANSFERRED 

TO   BALB/c 

MOTHER 

IMPROVED 

MATERNAL 

DIET 

0.1  m«    CORTISONE 
ON    OAY 


YOUNG    PARENTS 
LITTERS 
I   AND  2 

OLD   PARENTS 

LITTERS 

3-7 


FREQUENCY     DISTRIBUTION 

PtRCeMT  N0 

0  10         »0         30        10         SO         60        TO         SO       PROCESSES       PROBABILITY 


46  <0.0I 


54  <0 .05 


56  <0.05 


136  <0.0l 


74  <0.05 


150  >0.05 


110  <0.0l 


O  10  tO  10         40  SO  60         70         80 


Text-figure  4. — Effects  of  environmental  factors  upon  sacralization  of  vertebra  #26. 

Journal   of   the  National   Cancer   Institute 


PEOGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


645 


teratogen  on  strain  129.     Experiments  are  still  in  progress  and  interpreta- 
tions must  be  tentative  until  more  of  the  story  has  unfolded. 

Experiment  1. — Mice  of  strain  129  were  fasted  for  24  hours  during  the 
9th  day  of  pregnancy  (5  to  20  somite  period  of  the  embryo).  Anomalies 
illustrated  in  figures  2  and  3  occurred  in  32  percent  of  the  fetuses.  Using 
these  defects,  pseudencephaly  and  fused  ribs,  as  end  points,  we  have  been 
investigating  how  fasting  results  in  anomaly  (table  4) . 

Table  4 


Experiment 

No. 

Regimen 

Number 
of  fetuses 

Abnor- 
mal 

1 

Fast — 24  hours 

63 
59 

52 

99 
46 

Percent 
32 

2.. 

Folic-acid-deficient  diet  ad  libitum 

0 

3 

Folic-acid-deficient    diet    containing    X-methyl 
folic  acid 

50 

4  and  5 

8 

CHO  (0.8  gm.  of  glucose  or  0.1  gm.  of  casein) . . . 
Fast — 24  hours  force-fed  2  cc.  H2O 

4 
26 

Experiments  2  and  3. — The  types  of  congenital  defects  observed  and 
the  short  effective  period  of  treatment  suggested  similarity  to  effects  of 
folic-acid  depletion  in  rats.  A  semisynthetic  diet,  deficient  in  folic  acid, 
fed  during  the  9th  day  of  pregnancy  completely  protected  young  from  the 
effects  of  fasting.  Furthermore,  folic-acid  antagonist  administered  on 
day  9  to  fasting  mothers  did  not  appreciably  augment  the  degree  or  fre- 
quency of  anomaly.  These  observations  precluded  folic-acid  depletion 
as  a  causative  agent  for  the  teratogenic  effect  of  fasting. 

Experiments  4  and  5.- — The  semisynthetic  diet  that  protected  embryos 
from  anomaly  contained  more  than  a  dozen  ingredients.  We  were  faced 
with  the  likelihood  of  11  additional  experiments  to  eliminate  one  item 
each  time.  Instead,  we  reversed  the  approach  and  fed  by  stomach  tube 
the  mothers  that  were  otherwise  fasted.  One  group  received  a  solution 
of  vitamin-free  casein  to  offset  possible  amino  acid  depletion  and  another 
group  received  glucose  solution  by  mouth  to  ward  off  energy  depletion. 
Both  treatments  protected  the  embryos,  so  we  have  reasoned  (a)  that 
protein  and  glucose  had  a  sparing  effect  on  embryonic  tissue  protein  or 
(6)  that  protein  and  glucose  had  protected  by  preventing  depletion  of 
energy  rich  carbohydrate  in  the  form  of  glucose  directly  or  by  conversion 
from  amino  acids. 

Experiment  6. — There  exists  precedent,  from  the  work  of  Landauer  (IS) 
and  Zwilling  (14)  with  the  chick  embryo,  to  suspect  that  interruption  of 
carbohydrate  metabolism  could  induce  anomaly.  Circulating  blood 
glucose  was  measured  at  the  end  of  24  hours  of  fasting.  Sure  enough, 
the  glucose  level  was  down  75  percent.  The  hypoglycemia,  protection 
by  glucose,  and  analogy  with  the  chick  provided  a  working  hypothesis  to 
explain  the  cause  of  anomaly. 

Experiment  7. — We  turned  our  attention  to  the  embryos  to  correlate 
morphogenesis  with  the  time  of  induction  of  anomalies.     We  found  at 


Vol.    15,  No.   3,  December   1954 


646  proceedings:  symposium  on  25  years  of 

the  end  of  day  10  that  a  proportion  of  embryos  showed  retention  of  open 
brain  tubes.  Pseudencephaly  induced  by  fasting  was  a  result  of  failure 
of  the  closure  of  the  neural  tube. 

Experiment  8. — Piecing  together  odd  bits  of  observation  we  recalled 
that  fasting  mice  are  said  not  to  drink.  Mice  did  appear  shrunken  after 
fasting  24  hours.  Furthermore,  hadn't  we  protected  embryos  by  giving 
glucose  and  protein  in  solution?  Fasting  animals  were  given  isotonic 
saline  on  the  same  schedule  used  for  glucose  protection.  Anomalies  were 
observed  in  26  percent  of  the  fetuses  thereby  showing  that  the  water  in 
which  the  protein  and  glucose  was  suspended  did  not  provide  protection. 
Altered  carbohydrate  metabolism  as  a  teratogenic  influence  is  a  tempting 
hypothesis  at  the  moment  and  is  being  investigated  further. 

Conclusions 

Inbred  mammalian  material  has  offered  opportunity  for  genetic  stand- 
ardization so  that  environmental  influences  on  prenatal  life  could  be  in- 
vestigated. By  canceling  out  genetic  variability  one  learns  that  environ- 
mental factors  influence  morphogenesis  in  the  embryo. 

Geneticists  in  the  first  half  of  the  current  century  have  firmly  established 
basic  Mendelian  inheritance.  I  would  suggest  that  Mendelian  principles 
have  been  derived  from  carefully  selected  samples  of  deviants.  Most 
variations  in  man  and  his  domestic  animals  require  bulky  mathematical 
extensions  of  the  simple  Mendelian  situations.  Variations  in  normal 
development  are  most  likely  inherited  by  what  Dr.  E.  L.  Green  (7)  has 
termed  multiple-factor  situations  with  thresholds. 

For  the  sake  of  clarity  I  would  like  to  point  out  that  our  objectives 
should  be  clearly  distinguished  from  those  commonly  pursued  in  efforts 
to  study  effects  of  genie  mutants  in  embryology.  I  completely  divorce 
myself  from  the  philosophy  that  a  study  of  abnormal  development  will 
explain  its  counterpart  in  normal  physiological  embryology.  Nor  do  we 
wish  to  search  for  earliest  visible  effect  of  any  given  gene. 

"Skepticism  should  be  applied  to  the  hope  that  studies  on  the  develop- 
mental effects  of  major  mutant  genes  will  lead  to  an  understanding  of  the 
role  in  growth  and  development  of  those  genes  which  make  up  the  normal 
genotype  of  fowl  or  any  other  higher  organism.  The  damage  done  by  a 
mutation  may,  and  often  does,  find  expression  in  the  abolishment  of  a 
particular  and  well-defined  metabolic  function.  It  does  not  follow,  though 
this  assumption  is  commonly  made,  that  the  normal  allele  is  the  sole  or 
even  the  major  factor  in  this  particular  and  well-defined  function  (13)." 

Teratogenesis  is  a  function  of  background  inheritance.  Genetically 
induced  and  intangible  embryonic  imbalances  seem  to  interact  with  per- 
missive influence  of  background  inheritance  to  produce  congenital  de- 
formity. Teratogenesis  is  also  a  function  of  environmental  factors. 
Different  teratogenic  agents  (or  doses)  bring  out  characteristic  patterns 
of  polymorphism.  Mild  environmental  agents  tend  to  affect  those  char- 
acters that  are  spontaneously  variable.  Anomalies  on  a  given  genetic 
background  can  be  enhanced,  suppressed  or  not  affected.     Careful  appli- 

Journal   of   the  National   Cancer   Institute 


PROGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


647 


cation  of  teratogenic  agents  may  yield  clues  about  metabolic  pathways 
that  lead  to  deformity  in  mammals. 

References 

(1)  Gruneberg,  H.:  Embryology  of  mammalian  genes.     Revue  Swisse  de  Zoologie 

57:  129-139,  1950. 

(2)  Ingalls,  T.  H.,  Avis,  F.  R.,  Curley,  F.  J.,  and  Temin,  H.  M.:  Genetic  deter- 

minants of  hypoxia-induced  congenital  anomalies.     J.  Hered.  44:  185-194, 
1954. 

(3)  Fraser,  F.  C,  and  Fainstat,  T.  D.:  Production  of  congenital  defects  in  the 

offspring  of  pregnant  mice  treated  with  cortisone.     Pediatr.  8:  527-533,  1951. 

(4)  Kalter,  H.:  The  inheritance  of  susceptibility  to  the  teratogenic  action  of  corti- 

sone in  mice.     Genetics  39:  185-196,  1954. 
^5)  Go  wen,  J.  W.:  Significance  and  utilization  of  animal  individuality  in  disease 
research.     J.  Nat.  Cancer  Inst.  15:  555-570,  1954. 

(6)  Dunn,  T.:  The  importance  of  differences  in  morphology  in  inbred  strains.    J. 

Nat.  Cancer  Inst.  15:  573-589,  1954. 

(7)  Green,  E.  L.:  Quantitative  genetics  of  sketal  variations  in  the  mouse.  I.  Crosses 

between  three  short-ear  strains  (P,  NB,  SEC/2).       J.  Nat.  Cancer  Inst.  15: 
609-654,  1954. 

(8)  Sawin,  P.:  Discussion  following  (7).    J.  Nat.  Cancer  Inst.  15:  625-627,  1954. 

(9)  Bailey,  D.:  Discussion  to  inheritance  of  susceptibility  to  congenital  deformity — 

embryonic  instability.     J.  Nat.  Cancer  Inst.  15:  651,  1954. 

(10)  Runner,  M.  N.,  and  Morris,  N.:  Unpublished. 

(11)  Murray,  J.  M.,  and  Green,  C.  V.:  Inheritance  of  ventral  spotting  in  mice. 

Genetics  18:  481-486,  1933. 

(12)  Runner,  M.  N.:   Modified  differentiations  of  the  sacrum.     (Abstract.)  Anat. 

Rec.  115:  364,  1953. 
(18)  Landauer,   W.:  The  genetic  control  of  normal  development  in  the  chicken 

embryo.     Ann.  New  York  Acad.  Sc.  55:  172-176,  1952. 
(14)  Zwilling,  E.:  The  effects  of  some  hormones  on  development.     Ann.  New  York 

Acad.  Sc.  55:   196-202,  1952. 


Vol.   IS,  No.  3,  December   1954 


648 


proceedings:  symposium 


Plate  45 

Figure  1. — Differentiations  of  vertebra  #26  in  strain  129.  On  the  left  is  a  typical 
lumbar,  on  the  right  a  typical  sacral,  and  in  the  middle  an  asymmetrical  vertebra. 

Figure  2. — Pseudencephaly  in  mice  of  strain  129  following  treatment  (fasting)  during 
the  ninth  day  of  pregnancy.     On  the  right  is  a  normal  sibling. 

Figure  3. — Rib  fusions  in  mice  of  strain  129  following  treatment  (fasting)  during  the 
ninth  day  of  pregnancy.  These  young  were  taken  from  the  mother  at  19  days 
postcoitum.     On  the  right  is  a  normal  sibling. 


JOURNAL    OF    THE    NATIONAL    CANCER    INSTITUTE,    VOL.    15 


PLATE    45 


25 
2     26 

t*       On 

>     28 
j                 29 

W    *    V 

Runner 


649 


316263—54 25 


Discussion 
Dr.  Donald  W.  Bailey,  Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 

The  outmoded  question  of  which  is  the  more  important,  heredity  or  environment, 
has  been  replaced  by  the  more  constructive  and  reasonable  question  of  how  do  environ- 
ment and  heredity  work  together  to  produce  the  well-organized  developmental  patterns 
which  we  observe.  The  work  that  Dr.  Runner  has  just  presented  is  an  approach 
toward  answering  this  question.  Just  where  do  the  environmental  and  genetic  path- 
ways meet  during  the  course  of  development?  What  kind  of  profile  of  gene  action 
can  be  obtained  by  these  environmental  probings?  In  my  opinion,  this  approach 
should  prove  to  be  quite  fruitful. 

I  would  like  to  supplement  Dr.  Runner's  work  on  the  relationship  of  environment 
to  heredity  with  a  preliminary  polygenic  study  on  how  the  genes  and  environments 
differ  in  their  affects  on  the  shape  of  a  vertebra. 

In  this  study  the  second  cervical  vertebra  was  measured  in  most  of  its  dimensions 
as  seen  in  posterior  view.  From  these  measurements  it  was  possible  to  obtain  correla- 
tion coefficients  for  each  combination  of  measurements.  Thus  it  was  possible  to  obtain 
a  complete  set  of  such  correlations. 

In  fact,  by  the  use  of  the  methods  of  analyses  of  variance  and  covariance  it  was 
possible  to  separate  such  a  set  of  correlations  into  three  kinds,  according  to  the  source 
of  control :  Genetic,  inter-maternal  environmental,  and  intra-maternal  environmental. 
Thus  we  have  three  complete  sets  of  coefficients  showing  the  relationships  of  the 
dimensions  to  each  other. 

Now,  the  central  question  involved  in  this  investigation  was:  If  these  sets  of 
coefficients  differ,  one  from  another,  then  in  what  way?  Or  in  other  words,  do  the 
genes  and  the  two  kinds  of  environments  differ  in  their  affects  on  the  shape  of  the 
vertebra? 

In  order  to  compare  these  three  groups  of  relationships,  we  should  formulate  a  way 
to  compare  all  dimensions  of  the  vertebra  at  once  and  their  relative  changes.  The 
method  used  was  as  follows:  If  one  dimension,  arbitrarily  chosen,  is  increased  in 
size,  what  are  the  correlated  responses  in  the  other  dimensions?  For  instance,  if 
we  should  increase  the  width  of  the  neural  arch,  what  changes  would  occur  in  the 
other  dimensions?  And,  are  these  responses  different  for  the  genetic  and  environ- 
mental sources  of  control? 

Conclusions  derived  from  alternately  increasing  each  dimension  and  noting  responses : 

Genetic:  vertebra  was  not  generally  altered  in  all  dimensions  at  once;  sometimes  a 
negative  response  was  observed,  the  two  arch  dimensions  were  found  to  be  independent, 
but  the  centrum  dimensions  were  dependent. 

Inter-maternal  environment:  there  was  a  very  strong  tendency  to  alter  the  angle  of 
the  transverse  process,  arch  dimensions  were  independent,  and  centrum  dimensions 
dependent. 

Intra-maternal  environment:  the  dimensions  were  generally  all  altered  excepting  the 
centrum  dimensions  which  were  independent. 

This  study  has  shown  that  the  two  kinds  of  environments  and  the  genes  apparently 
differ  in  their  affects  on  vertebral  shape. 

The  findings  of  this  study  are  quite  in  harmony  with  those  of  Dr.  Runner's.  He  has 
shown  how  specific  alterations  in  the  environment  can  show  relatively  specific  affects 
upon  development.  This  specificity  of  environmental  action  is  perhaps  an  acceptable 
explanation  of  the  differences  in  the  environmental  and  genetic  relationships  in  the 
polygenic  study,  i.e.,  the  environmental  change  acts  disproportionally  upon  different 
portions  of  the  genie  system  determining  the  developmental  pattern.  I  think  we  should 
expect  areas  of  vulnerability  in  a  genie  system  to  a  specific  environmental  stress — both 
in  time  and  substance.  If  this  is  the  case,  then  these  environmental  probings  of 
Dr.  Runner's  should,  in  time,  provide  a  well-defined  profile  of  gene  action  in  develop- 
ment and  perhaps  eventually  a  knowledge  of  primary  gene  action  itself. 

651 


Journal    of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


Session  IV.  Genetic  Control  of  Func- 
tion 


Chairman,  Dr.  Harrison  R.  Hunt,  Professor 
Emeritus  oj  Zoology,  Michigan  State  College,  East 
Lansing,  Mich. 


Speaker:  Dr.  Herman  B.  Chase 
Some  Examples  oj  Gene-Controlled  Functional  Disturbances  in  the  Mouse 
Discusser:  Dr.  Elizabeth  S.  Russell 

Speaker:  Dr.  George  D.  Snell 

The  Enhancing  Effect  (or  Actively  Acquired  Tolerance)  and  the  Histo- 
compatibility^ Locus  in  the  Mouse 
Discusser:  Dr.  N.  A.  Mitchison 

Speaker:  Miss  Margaret  Dickie 

The  Expanding  Knowledge  oj  the  Genome  oj  the  Mouse 


653 


Journal  of  the  National  Cancer  Institute,   Vol.    15,  No.   3,   December   1954 


Introduction:   Session  IV 
Dr.  Harrison  R.  Hunt,  Chairman 

Genetics  is  an  aspect  of  physiology.  It  is  concerned  with  the  details  of  the  processes 
by  which  an  organism  acquires  its  adult  form  and  functions.  The  accomplishments  of 
a  gene  are  determined  not  only  by  its  own  activities,  but  by  the  environment  in  which 
it  operates,  and  this  environment  comprises  other  genes,  agencies  outside  the  organism 
operating  upon  it,  and  the  status  of  the  organism  at  the  time  the  gene  becomes  func- 
tional. Embryology  and  experimental  morphology  are  the  morphologic  aspects  of 
this  developmental  process.  If  the  end  result  of  such  gene-influenced  development  is 
definitely  harmful,  one  is  concerned  with  pathology,  as  in  the  appearance  of  a  malig- 
nant neoplasm. 

Rats,  mice,  and  men  are  good  subjects  for  such  investigations  in  physiological  genetics. 
For  example,  Heston's  work  on  the  bent-nosed  rat,  Jay's  and  Burrington's  on  the 
flexed-tailed  mouse,  and  Hunt's  and  Hoppert's  studies  on  the  causes  of  dental  caries 
in  rats  have  shown  that  both  the  diet  and  the  genes  are  responsible  for  the  phenotype. 
It  is  suggested  that  some  adaptation  of  Wright's  method  of  path  coefficients  might  be 
useful  in  estimating  the  relative  importance  of  heredity  and  various  environmental 
agencies  in  at  least  some  cases.  Studies  like  those  of  Dr.  Chase  and  Dr.  Snell,  using 
rodents,  should  be  undertaken  with  numerous  traits.  The  rapidly  growing  list  of 
genes  in  the  mouse,  listed  by  Miss  Dickie,  is  encouraging,  because  every  new  gene 
discovered  and  located  on  a  chromosome  is  a  potential  tool  in  investigations  of  the 
genetic  control  of  function. 
654 


Some  Examples  of  Gene- Controlled 
Functional  Disturbances  in  the 
Mouse  u  2 


Heeman  B.  Chase,    Biology   Department,   Brown 
University,  Providence,  R.  I. 


In  the  study  of  physiological  genetics,  it  becomes  evident  that  there  are 
no  clear  dividing  lines  that  separate  morphology,  physiology,  and  behavior. 
A  certain  morphologic  character  is  the  result  of  certain  gene-modified 
physiological  processes.  A  certain  behavior  or  function  is  in  turn  the 
result  of  certain  morphologic  characteristics.  The  morphologic  charac- 
teristic in  question  might  be  an  enzyme,  a  hormone,  the  number  of  types 
of  cells,  or  some  structure  of  the  skeleton  or  nervous  system.  As  employed 
in  this  paper,  then,  a  functional  disturbance  consists  of  the  description  of 
some  step  in  the  gene-controlled  process  which  leads  to  a  morphologic 
variation  or  the  description  of  some  function  resulting  from  a  morphologic 
variant.  Schematically  this  is  shown  in  text-figure  1.  Five  rather 
varied  examples  from  my  experiences  with  mice  are  presented  here. 

Insulin  tolerance. — There  is  a  strain  of  mouse  that  tolerates  200  units  of 
insulin  at  35  days  of  age,  instead  of  the  normal  one  unit  (1,  2).  This 
characteristic  is  not  due  to  a  single  gene  difference  but  probably  to  about 
three.  In  recent  experiments  we  find  that  this  mouse  has  an  extremely 
high  level  of  "insulinase"  or  some  such  substance  in  the  liver.  With  an 
injection  of  insulin  the  blood-sugar  level  of  this  mouse  begins  to  drop  as 
in  the  case  of  the  normal  but  levels  off  and  rises  again  instead  of  continuing 
to  the  critical  15-mg.  percent  level.  In  vitro  studies,  involving  extracts 
of  liver  homogenate  from  these  tolerant  mice,  indicate  the  presence  of 
"msulinase"  in  quantities  sufficient  to  counteract  800  or  more  units  of 
insulin  per  mouse  liver  in  30  minutes  (3).  The  presence  of  an  insulinase- 
inhibitor  in  intact  animals  is  indicated.  These  insulin- tolerant  mice  are 
also  more  resistant  to  alloxan-induced  diabetes  (4).  The  study  is  not 
completed,  but  it  is  possible  that  some  substance  may  be  present  which 
can  inhibit  or  destroy  alloxan  and  thus  protect  islet  cells  of  the  pancreas 
from  damage. 

Anophthalmia. — This  strain  of  eyeless  mice  was  obtained  originally 
from  Dr.  C.  C.  Little  of  the  Jackson  Memorial  Laboratory.     Anophthal- 

i  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  28, 1954. 

2  Some  of  the  work  summarized  here  was  supported,  in  part,  by  grants  from  the  U.  S.  Public  Health  Service 
and  by  grants  from  the  American  Cancer  Society  recommended  by  the  Committee  on  Growth  of  the  National 
Research  Council. 

655 

Journal   of   the  National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 
816263—54 26 


656 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


Morphology 


Physiology 


Gene 


Specific  cellular 
enzyme  or  antigen 


Differentiated  cell 


Primary  gene  action 


•Enzyme-controlled 
biochemical  processes 


Substances 

Ccellular  or  extra- cellular) 

Cdiffusible  substances, 

hormones,  granules,  etc.) 

"Gross   variant" 


roduction  of  specific 
substances 


action  of  substances 


Extra-organlsmal 
character 


Iross  function    or 
behavior 


Text-figure  1. — Each  morphologic  entity  is  preceded  and  followed  by  a  physiological 
process.  Likewise,  each  function  can  be  viewed  as  being  bracketed  by  two  levels  of 
morphology. 

mia  is  a  single  factor  recessive  in  some  crosses,  whereas  two  major  factors 
are  involved  in  other  crosses  (5-8) .  A  variety  of  pleio tropic  effects  stems 
from  the  failure  of  the  optic  vesicle  to  form  a  normal  cup  and  to  make 
contact  with  the  ectoderm  to  induce  a  lens.  Among  the  later  effects 
are  the  failure  to  develop  of  certain  layers  of  the  superior  colliculus  and 
of  the  visual  cortex  (lack  of  induction  due  to  absence  of  the  optic  nerve), 
and  the  atrophy  of  the  pioneer  fibers  of  the  trochlear  and  abducens  nerves, 
following  atrophy  of  the  extrinsic  eye  muscles  due  to  absence  of  the  eye- 
ball. Normally,  the  optic  vesicle  when  about  100  /x  in  diameter  forms  a 
cup  and  starts  to  induce  a  lens.  In  the  anophthalmic  embryos,  this 
vesicle  forms  no  cup  or  forms  several  small  abortive  cups  and  thus  fails  in 
its  initial  function  of  inducing  a  lens  and  thereby  an  eyeball,  optic  nerve 
fibers,  etc. 

Pigment  cells. — A  few  (of  the  order  of  6  or  less  in  zigzag  follicles)  den- 
dritic pigment  cells  in  the  hair  bulb  supply  pigment  granules  to  cells  of  the 
upper  bulb  which  are  to  become  the  cells  of  the  medulla  and  of  the  cortex 


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(9-18) .  Some  granules  remain  outside  the  cells  and  are  thus  carried  along 
as  the  hair  grows,  but  many  granules  are  actually  taken  up  by  the  epi- 
thelial cells  of  the  bulb.  Depending  on  the  genotype,  the  epithelial  cells 
then  modify  the  laying  down  of  additional  melanin  on  these  granule  sites. 
Some  genes  which  affect  the  dendritic  pigment  cells  themselves  and  thereby 
their  function  are:  the  gene  for  albinism  (c)  that  causes  a  failure  of  mel- 
anin formation  even  though  the  cells  are  present;  the  gene  for  white  spot- 
ting (s)  that  causes  a  failure  of  these  cells  in  whole  areas;  the  gene  for 
silver  (si)  that  causes  a  failure  of  these  cells  in  scattered  hair  follicles;  and 
the  gene  for  dilution  (d)  that  causes  a  few  of  these  cells  to  have  large 
clumps  of  pigment  rather  than  the  usual  very  small  granules.  These 
clumps  generally  do  not  become  incorporated  into  the  epithelial  cells  but 
rather  result  in  large  masses  of  pigment  along  the  hair  shaft,  so  massive 
that  the  shaft  is  often  distorted  at  these  points.  The  case  of  silver  is  of 
particular  interest  because  it  can  be  simulated  so  precisely  by  X-ray  de- 
struction of  pigment  cells,  the  X-ray  graying  effect.  With  appropriate 
modifiers  the  gene  for  silver  results  in  a  phenotype  which  we  have  called 
basal  dilution.  In  this  condition  there  are  some  all-white  and  mosaic 
hairs  as  in  the  ordinary  silver,  but  practically  all  the  remaining  hairs  have 
white  or  diluted  bases.  The  explanation  for  this  latter  condition  is  not 
yet  fully  determined  but  it  appears  possible  that  in  addition  to  there  being 
fewer  pigment  cells  there  is  also  a  shortening  of  the  period  of  melanogenic 
activity  for  these  cells. 

Except  for  skin  on  the  feet,  tail,  and  ears,  there  are  normally  no  actively 
melanogenic  pigment  cells  in  the  epidermis  of  the  adult  mouse.  Such 
cells  are  present  during  the  first  few  days  after  birth  (13),  however,  and 
can  be  made  melanogenic  again  by  irritants.  They  also  become  mildly 
active  in  old  hairless  (hr/hr)  mice. 

Yellow  obesity. — The  obesity  associated  with  the  yellow  gene  (Av)  is 
generally  lost  following  inbreeding.  With  a  high  fat  diet,  however,  such 
inbred  mice  quickly  become  obese,  whereas  their  nonyellow  litter  mates 
do  not.  The  function  of  fat  metabolism  is  thus  altered  by  the  presence  of 
the  Av  gene  (14,  15).  The  difference  is  not  due  entirely  to  the  amount  of 
food  consumed. 

Hairlessness. — The  skin  functions  in  a  precisely  integrated  cyclic  man- 
ner (11, 16).  The  corium  and  adipose  layer  become  thicker  with  the  ana- 
gen  phase  of  the  hair  growth  cycle  of  the  hair.  The  surface  epidermis  by 
increased  mitotic  activity  becomes  thicker  in  early  anagen;  but  in  later 
anagen,  when  the  hair  shaft  is  being  formed,  the  epidermis  becomes  even 
thinner  than  during  the  resting  phase  of  hair  growth,  telogen.  There  are 
simple  feed-back  mechanisms  of  control,  as  for  the  amount  of  corneum  and 
sebum,  and  in  addition,  there  are  controls,  possibly  of  a  competitive  nature, 
imposed  by  the  great  activity  of  the  hair  bulb.  In  this  system  of  cyclic 
and  interacting  processes,  a  modification  of  one  process  is  likely  to  have 
an  indirect  effect  on  many  processes.  One  such  condition  is  that  of  gene- 
controlled  hairlessness. 


Vol.    15,   No.   3,   December    1954 


658  proceedings:  symposium  on  25  years  of 

Animals  which  are  homozygous  for  this  gene  (hr/hr)  grow  a  normal  first 
coat  but  at  the  first  catagen  stage  an  abnormal  club  is  formed  and  the  fol- 
licle fails  to  shorten  for  a  normal  telogen  follicle  (11,  17,  16, 13,  18).  The 
hair  falls  out,  having  no  normal  club,  and  the  follicle  strand  becomes  at- 
tenuated and  breaks  into  isolated  portions.  These  isolated  fragments 
develop  into  sebaceous  and  later  keratinized  cysts.  The  fragment  which 
includes  the  dermal  papilla  can  later  produce  an  abortive  hair  in  a  cyst,  but 
the  continuity  of  the  follicle  with  the  surface  epidermis  is  lost.  In  normal 
catagen  the  connective-tissue  sheath  around  the  epithelial  external  sheath 
forms  a  thick  glassy  membrane  that  wrinkles  and  shortens  as  the  lower 
follicle  degenerates  and  thus  holds  the  remaining  cells  and  the  dermal 
papilla  in  contact  with  the  permanent  upper  portion  of  the  follicle.  The 
pressure  by  the  glassy  membrane  probably  also  induces  the  brushlike 
club  end  of  the  hair.  In  the  hairless  mouse  the  connective-tissue  sheath 
fails  to  produce  a  glassy  membrane  and  thus  to  perform  one  of  its  normal 
functions  (figs.  1  and  2). 

As  a  result  of  the  hairless  condition,  these  mice  have  a  greater  food  con- 
sumption and  activity  than  have  the  heterozygous  (Hr/hr)  haired  mice  of 
the  same  strain.  This  difference  is  less  pronounced  when  the  animals  are 
kept  at  a  temperature  of  78°  F.  than  when  they  are  kept  at  about  70°  F. 
Furthermore,  the  epidermis  and  associated  corneum  become  thicker  with 
each  ' 'cycle"  and  fail  to  return  to  the  normal  original  thickness.  The 
cysts  continue  to  grow  and  almost  completely  fill  the  corium.  The  adi- 
pose layer  becomes  increasingly  thin.  In  effect,  the  whole  skin  "ages" 
more  rapidly  than  would  be  the  case  if  functional  hair  follicles  were  still 
present.  Also  it  might  be  pointed  out  that  the  skin  of  young  hairless 
mice  develops  sebaceous  adenomas  very  readily,  especially  with  the 
application  of  methylcholanthrene. 

Conclusions. — These  five  cases  represent  rather  diversified  examples  of 
gene-modified  functions.  They  range  from  an  ability  to  restore  the  blood- 
sugar  level,  in  spite  of  large  injections  of  insulin,  to  the  action  of  pigment 
cells  in  producing  coat-color  characteristics;  from  a  change  in  fat  metab- 
olism to  the  inductive  action  of  the  optic  vesicle  and  cup;  from  the  action 
of  the  connective-tissue  sheath  in  the  hair  growth  cycle  and  indirectly  in 
determining  a  thermal  optimum  to  a  mechanism  that  resists  alloxan- 
induced  diabetes.  Mechanisms  which  control  embryonic  development, 
which  influence  cyclic  activities,  and  which  help  maintain  homeostasis  in 
the  organism  are  here  represented.  For  such  studies  the  mouse  is  an 
excellent  organism. 

References 

(1)  Chase,  H.  B. :  Inheritance  and  selection  of  insulin-resistance  in  mice.  (Abstract.) 
Genetics  35:  101,  1950. 

(#)  Chase,  H.  B.,  Gunther,  M.  S.,  Miller,  J.,  and  Wolffson,  D.:  High  insulin 
tolerance  in  an  inbred  strain  of  mice.     Science  107:  297-299,  1948. 

(8)  Beyer,  R.  E. :  A  study  of  insulin  metabolism  in  an  insulin  tolerant  strain  of  mice. 
Thesis,  Brown  Univ.,  1954. 

(4)  Frankenhuis,  B.:  Effects  of  alloxan  on  an  insulin  tolerant  strain  of  mice.  The- 
sis, Brown  Univ.,  1953. 

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PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  659 

(5)  Chase,  H.   B.:  Studies  on  an  anophthalmic  strain  of  mice.     III.     Results  of 

crosses  with  other  strains.     Genetics  27:  339-348,  1942. 

(6)  :  Studies  on  an  anophthalmic  strain  of  mice.  IV.  A  second  major  gene 

for  anophthalmia.     Genetics  29:  264-269,  1944. 

(7)  :  Studies  on  an  anophthalmic  strain  of  mice.  V.  Associated  cranial  nerves 

and  brain  centers.     J.  Comp.  Neurol.  83:  121-139,  1945. 

(8)  Chase,  H.  B.,  and  Chase,  E.  B.:  Studies  on  an  anophthalmic  strain  of  mice.  I. 

Embryology  of  the  eye  region.     J.  Morphol.  68:  279-301,  1941. 

(9)  Chase,  H.  B.:  Greying  of  hair.  I.  Effects  produced  by  single  doses  of  X-rays  on 

mice.     J.  Morphol.  84:  57-80,  1949. 

(10)  :  Number  of  entities  inactivated  by  X-rays  in  greying  of  hair.     Science 

113:  714-716,  1951. 

(11)  :  Growth  of  the  hair.     Physiol.  Rev.  34:  113-126,  1954. 

(12)  Chase,  H.  B.,  and  Rauch,  H.:  Greying  of  hair.  II.  Response  of  individual  hairs 

in  mice  to  variations  in  X-radiation.     J.  Morphol.  87:  381-391,  1950. 
(18)   Chase,  H.  B.,  Rauch,  H.,  and  Smith,  V.  W.:  Critical  stages  of  hair  development 
and  pigmentation  in  the  mouse.     Physiol.  Zool.  24:  1-8,  1951. 

(14)  Chase,  H.  B.,  and  Fenton,  P.  F.:  The  expression  of  obesity  in  yellow  mice. 

(Abstract).     Genetics  36:  546-547,  1951. 

(15)  Fenton,  P.  F.,  and  Chase,  H.  B.:  Effect  of  diet  on  obesity  of  yellow  mice  in 

inbred  lines.     Proc.  Soc.  Exper.  Biol.  &  Med.  77:  420-422,  1951. 

(16)  Chase,  H.  B.,  Montagna,  W.,  and  Malone,  J.  D.:  Changes  in  the  skin  in  rela- 

tion to  the  hair  growth  cycle.     Anat.  Rec.  116:  75-81,  1953. 

(17)  Chase,  H.  B.,  and  Montagna,  W.:  The  development  and  consequences  of  hair- 

lessness  in  the  mouse.     Genetics  37:  573,  1952. 

(18)  Montagna,  W.,  Chase,  H.  B.,  and  Melaragno,  H.  P.:  The  skin  of  hairless 

mice.  I.   The  formation  of  cysts  and  the  distribution  of  lipids.     J.  Invest. 
Dermat.  19:  83-94,  1952. 


Vol.    15,   No.   3,   December    1954 


660  proceedings:  symposium 


Plate  46 

Figure  1. — Normal  catagen  hair  follicle  with  normal  club  and  thickened  connective 
tissue  around  lower  follicle.     X  485 

Figure  2. — Hairless  catagen  hair  follicle  with  abnormal  club  and  no  thickened  con- 
nective-tissue sheath  around  lower  follicle.     X  485 


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PLATE  46 


Chase 


661 


316263—54 27 


Discussion 
Dr.  Elizabeth  S.  Russell,  Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 

During  the  past  half  hour  I  have  been  alternately  enjoying  Dr.  Chase's  excellent  pres- 
entation, and  worrying  for  fear  he  would  erase  a  most  intriguing  figure  which  has  given 
me  an  inspiration  for  this  discussion.  That  figure  shows  a  line  across  the  board  going 
from  morphology  to  physiology  and  back  again;  from  gene,  to  primary  gene  action,  to 
cellular  enzyme,  to  biochemical  process,  to  differentiated  cell,  and  so  on  all  the  way  out 
to  the  final  possible  stage  of  extra-organismic  character.  This  line  represents  to  me  not 
only  an  excellent  picture  of  paths  of  gene-action ;  it  is  also  a  very  clear  picture  of  a  zigzag 
hair  I  It  is  in  the  study  of  hair  growth  and  pigmentation  that  Dr.  Chase's  researches 
and  my  own  have  most  in  common.  Actually,  our  investigations  have  tended  to 
complement  each  other  in  this  field.  When  I  attempt  to  study  the  pigment  granules 
in  a  certain  hair,  and  deduce  from  them  something  of  the  story  of  gene-controlled  pig- 
ment deposition,  my  first  question  is  always,  "Just  where  are  we  along  the  hair  shaft? 
This  may  mean  something  very  different  at  the  tip  from  what  it  would  near  the  base." 
Physiological  genetics  investigation  is  very  much  like  that  zigzag  hair,  and  it  is  very 
important  to  attempt  to  trace  gene  actions  all  the  way  in  both  directions,  from  the  first 
effect  within  single  cells  to  the  last  consequence  in  organismic  behavior.  It  is  for  this 
reason  that  "major"  genes,  with  effects  easily  recognized  at  many  different  stages,  have 
been  extensively  and  intensively  studied  by  physiological  geneticists.  They  can  make 
use  of  both  qualitative  and  quantitative  differences  associated  with  genie  substitutions, 
and  can  study  both  multiple  pleiotropic  effects  of  single-gene  substitutions  and  changes 
in  single  characters  brought  about  by  the  actions  of  large  numbers  of  genes  not  in- 
dividually identifiable.  As  I  have  been  involved  in  studies  dealing  with  both  ends  of 
this  spectrum,  I  would  like  to  quote  an  example  from  each  extreme. 

Major  effects  produced  by  single-gene  substitutions  may  be  exemplified  by  studies 
of  the  anemia-producing  effects  of  the  deleterious  alleles  of  the  TF-series  in  the  mouse. 
These  effects  appear  to  be  mediated  through  an  arrest  in  the  maturation  of  hematopoi- 
etic cells  in  the  bone-marrow  (1).  It  has  further  been  shown  by  isotope  incorporation 
experiments  that  this  arrest  involves  a  great  delay  in  formation  of  one  portion  only  of 
the  hemoglobin  molecule  (2).  Protoporphyrin,  and  consequently  heme,  is  formed 
much  more  slowly  in  the  anemics  than  in  the  normals,  while  the  globin  portion  of  the 
molecule  is  formed  at  essentially  the  same  rate  in  both.  This  seems  to  me  to  be 
approaching  the  level  of  analysis  of  primary  gene  action;  at  least  we  have  gotten  inside 
the  cell.  However,  a  word  of  caution  should  be  inserted  here;  it  seems  rather  probable 
to  those  of  us  who  have  been  involved  in  these  studies  that  the  difference  in  rate  of 
protoporphyrin  formation  may  reflect  rather  than  cause  the  delayed  maturation,  and 
this  interpretation  puts  our  observations  much  further  from  primary  gene  action  than 
would  be  true  if  the  lesion  in  prophyrin  formation  caused  the  arrest. 

At  the  other  end  of  the  scale,  the  investigations  in  the  Inbred  Nucleus  of  the  Jackson 
Laboratory  have  disclosed  a  number  of  physiological  differences  known  to  be  partially 
genetically  determined  because  they  differ  much  more  among  than  within  inbred  strains 
of  mice.  Usually  it  is  not  possible  to  attribute  such  differences  to  particular  identifiable 
genes,  but  there  is  no  doubt  of  the  genetic  nature  of  strain  differences.  More  and  more 
such  differences  are  coming  to  be  recognized.  In  the  library  of  the  Jackson  Laboratory 
there  are  more  than  1,200  references  from  journals  (1951-53)  alone  to  studies  based  on 
inbred  mice.  These  have  been  catalogued  and  classified  in  a  subject-strain  bibli- 
ography (8)  which  provides  rapid  access  to  literature  pertinent  to  a  particular  strain 
or  condition.  From  this  list  I  have  gleaned  a  variety  of  newly  recognized  physiological 
strain  differences  (4)  >  These  include  differences  in  the  nature  of  disease  produced  by 
a  certain  pathogen,  in  the  survival  time  of  infected  individuals,  and  in  susceptibility 
and  resistance.     Strains  differ  in  capacity  for  antibody  production,  and  in  reaction  to 

663 

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664  proceedings:  symposium 

changes  in  environment,  for  example,  cold  tolerance  and  sensitivity  to  toxic  effects  of 
chloroform.  Strains  differ  in  content  of  particular  hormones,  sensitivity  to  particular 
hormones,  type  of  endocrine  balance,  and  in  reaction  to  extirpation  of  endocrine  organs. 
In  the  Inbred  Nucleus  we  have  particularly  studied  variations  in  blood-cell  characters 
(6,  6),  variations  in  longevity,  and  differences  in  types  of  pathology  characterizing 
different  inbred  strains  (7) . 

We  are  coming  into  a  new  stage  in  genetics,  where  an  ever-increasing  number  of 
inherited  physiological  differences  will  be  recognized.  I  hope  these  new  ones  may  be 
analyzed  with  skill  and  competence  such  as  Dr.  Chase  has  beautifully  demonstrated 
in  his  excellent  presentation  this  evening. 

Literature  Cited 

(1)  Russell,  E.  S.,  Snow,  C.  M.,  Murray,  L.  M.,  and  Cormier,  J.  P.:  The  bone 
marrow  in  inherited  macrocytic  anemia  in  the  house  mouse.  Acta  Haemat.  10: 
247-259,  1953. 

(#)  Russell,  E.  S.,  Altman,  K.  I.,  Salomon,  K.,  and  Scott,  J.  K.:  Chemical  charac- 
terization of  the  erythropoietic  defect  in  T^TF*  anemic  mice.  (Abstract.)  Ge- 
netics 38:  687,  1953. 

(3)  Staats,  J.:  A  classified  bibliography  of  inbred  strains  of  mice.     Science  119: 

295-296,  1954. 

(4)  Russell,  E.  S.:  Significance  of  the  physiological  pattern  of  animal  strains  in  bio- 

logical research.     Proc.  4th  Ann.  Meet.,  Animal  Care  Panel,  1953  pp.  140-150. 

(5)  Russell,  E.  S.,  Neufeld,  E.  F.,  and  Higgins,  C.  T.:  Comparison  of  normal  blood 

picture  of  young  adults  from  18  inbred  mouse  strains.     Poc.  Soc.  Exper.  Biol.  & 
Med.  78:  761-766,1951. 

(6)  Budds,  0.  C,  Russell,  E.  S.,  and  Abrams,  G.  E.:  Effects  of  genetics  and  anes- 

thesia upon  granulocyte  and  agranulocyte  levels  in  seven  inbred  mouse  strains. 
Proc.  Soc.  Exper.  Biol.  &  Med.  84:   176-178,  1953. 

(7)  Russell,  E.  S.,  Fekete,  E.  A.,  Borges,  P.  R.  F.,  MacFarland,  E.  K.,  and 

Collins,  J.  B.:  Longevity  and  pathology  patterns  of  mice  from  ten  major 
inbred  strains.     Anat.  Rec.  117:  547-548,  1953. 


The  Enhancing  Effect  (or  Actively  Ac- 
quired Tolerance)  and  the  Histocom- 
patibility-^ Locus  in  the  Mouse  u  2 


George  D.  Snell,3  Roscoe  B.  Jackson  Memorial 
Laboratory,  Bar  Harbor,  Maine 


At  an  anniversary  gathering  such  as  this,  more  than  at  an  ordinary 
scientific  meeting,  we  allow  ourselves  the  privilege  of  recalling  the  past 
and  questioning  the  future.  In  keeping  with  this  tradition,  I  am  going 
to  start  my  remarks  with  a  fragment  of  history  and  conclude  them  with 
a  dash  of  speculation.  In  between  these  boundaries  I  shall  present  what 
I  hope  is  a  fairly  substantial  body  of  experimentally  verified  fact. 

Historical 

The  history  begins  in  1914  at  the  Bussey  Institution.  In  that  year 
Dr.  Little,  then  just  completing  his  graduate  studies  under  Dr.  Castle, 
published  a  paper  in  Science  (1)  which  brilliantly  anticipated  the  now 
generally  accepted  genetic  theory  of  transplantation.  For  some  months 
previously  Dr.  Little  had  been  spending  part  of  his  time  in  Tyzzer's  labora- 
tory at  the  Harvard  Medical  School,  making  crosses  between  partly  inbred 
strains  of  mice,  and  inoculating  the  various  hybrid  generations  with 
transplantable  tumors.  Definite  ratios  of  susceptibility  and  resistance 
were  obtained,  but  they  simply  did  not  conform  to  any  then  recognized 
as  likely  to  result  from  segregating  Mendelian  factors.  Susceptibility 
was  manifest  in  the  Fi  in  the  manner  characteristic  of  dominants,  but 
appeared  in  an  unexpectedly  small  proportion  of  the  F2  and  backcross 
hybrids.  The  1914  paper,  while  not  mentioning  the  transplantation 
experiments,  showed  that  ratios  of  just  this  sort  were  to  be  expected  in 
the  case  of  characters  due  to  the  interaction  of  multiple  dominant  factors. 

Dr.  Little  maintained  his  interest  in  transplantable  tumors  after  his 
departure  from  the  Bussey,  and  a  program  for  the  study  of  the  genetics 
of  transplantation  which  was  initiated  at  the  University  of  Michigan,  and 
continued  at  the  Jackson  Memorial  Laboratory  after  its  founding,  led  to 

1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  28, 1954. 

3  This  investigation  was  supported  by  a  research  grant  C-1329(C2)  from  the  National  Cancer  Institute  of  the 
National  Institutes  of  Health,  U.S.  Public  Health  Service;  by  a  grant-in-aid  from  the  American  Cancer  Society 
upon  recommendation  of  the  Committee  on  Growth  of  the  National  Research  Council;  and  by  a  grant  to  the 
Roscoe  B.  Jackson  Memorial  Laboratory  from  the  Anna  Fuller  Fund. 

3  Most  of  the  experiments  here  reported  were  carried  out  by  Miss  Priscilla  Smith,  to  whom  it  is  a  pleasure  to  ex- 
press my  deep  appreciation.  Other  data  are  taken  from  published  and  unpublished  experiments  of  Dr.  Kaliss 
to  whom  I  am  indebted  for  permission  to  quote  them. 

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666  proceedings:  symposium  on  25  years  of 

our  present  understanding  of  the  essentially  genetic  basis  of  transplant 
susceptibility  and  resistance  [see  reviews  by  Little  (2)  and  Snell  (8)].  The 
work  that  I  am  going  to  describe  is  a  natural  outgrowth  of  these  early 
investigations. 

The  Experimental  Background 

The  study  of  the  mechanisms  which  determine  susceptibility  and 
resistance  to  tumors  and  normal  tissue  transplants  has  now  become  the 
concern  of  several  members  of  the  laboratory  staff,  who  share  a  common 
interest  in  this  field.  Of  the  various  methods  of  approach  now  being 
used  I  shall  be  largely  concerned  with  only  two.  All  phases  of  this  pro- 
gram, however,  form  an  interlocking  whole,  and  in  many  respects  I  am 
indebted  to  research  assistants,  visiting  fellows,  and  staff  members,  and 
most  certainly  to  Dr.  Little  himself,  for  stimulation  and  help  that  I  cannot 
acknowledge  in  detail. 

The  first  subject  which  I  wish  to  discuss  is  a  phenomenon  concerned 
with  the  abrogation  of  the  resistance  of  the  host  to  tumor  homografts 
{i.e.,  grafts  between  strains).  This  phenomenon  has  variously  been  called 
the  "XYZ  effect,"  the  "enhancing  effect/ '  and  " conditioning  the  host" 
(4~7).  The  names  " immunological  paralysis"  and  "actively  acquired 
tolerance"  have  been  applied  to  phenomena,  investigated  in  other  labora- 
tories, which  involve  different  experimental  procedures  but  probably  the 
same  fundamental  biological  mechanism  (8-10). 

As  investigated  in  this  laboratory,  the  typical  procedure  for  producing 
the  enhancing  effect  involves  the  pretreatment  of  the  host  with  prepara- 
tions of  normal  or  tumor  tissue  taken  from  the  same  strain  as  the  graft 
donor.  The  method  of  preparing  the  extract  first  employed  by  us  (11), 
and  still  one  of  the  most  satisfactory,  is  to  lyophilize  the  tumor  tissue  and 
then  to  suspend  the  resulting  dry  powder  in  distilled  water  or  saline  prior 
to  injection.  Injections,  usually  2  to  10  in  number  and  totaling  usually 
5  to  50  mg.  of  dry  tissue,  are  given  intra-abdominally,  and  the  living  tumor 
is  grafted  subcutaneously  about  a  week  after  the  last  injection. 

With  some,  but  not  all,  tumor-host  combinations,  most  of  the  injected 
or  experimental  animals  succumb  to  progressively  growing  tumors,  while 
the  controls  with  rare  exceptions  survive.  The  incidence  of  death  in 
experimental  groups  is  frequently  80  to  100  percent. 

Several  years  of  investigation  by  the  Jackson  Laboratory  group  have 
revealed  a  number  of  important  characteristics  of  the  enhancing  effect. 

Kaliss  and  Day  (12)  have  investigated  the  time  relations  of  the  pheno- 
menon. The  injections  of  tissue  preparation  are  ineffective  if  given 
more  than  one  day  after  the  tumor  graft,  and  relatively  ineffective  at  one 
day.  The  effect  is  clearly  evident,  however,  if  the  injections  precede  the 
graft  by  1  day  only.  In  evaluating  these  relationships  it  must  be  re- 
membered that  it  takes  a  tumor  implant  several  days  to  become  estab- 
lished— it  is  usually  a  week  before  there  is  a  definitely  palpable  mass — so 
that  the  effective  interval  may  be  longer  than  the  experimental  procedure 
would  imply. 

Journal    of    the   National   Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  667 

The  effect  persists.  There  is  no  diminution  in  tumor  growth  when  the 
graft  is  made  19  weeks  after  the  last  injection,  and  little  if  any  at  40  weeks. 

The  effect  can  be  passively  transferred  (13,  lJj).  Serum  from  injected 
mice  will  induce  graft  susceptibility  in  otherwise  untreated  mice  of  the 
same  host  strain.  Active  serum  can  also  be  obtained  from  rabbits  injected 
with  appropriate  preparations  of  mouse  tissue.  Further  studies  are  in 
progress  (Kaliss). 

The  effect  can  be  produced  with  preparations  of  certain  normal  mouse 
tissue  (15).  Of  several  tissues  tested,  spleen  is  apparently  the  most  active 
(at  least  in  the  tumor-host  combination  usually  employed),  kidney  the 
next,  liver  the  least.    Washed  red  cells  of  mice  produce  no  effect. 

The  effect  is  species-specific  (16) .  Tissue  from  rats,  hamsters  and  guinea 
pigs  produce  no  enhancement  of  the  growth  of  mouse  tumors  in  mice. 
Trypan  blue  is  also  nonenhancing,  though  it  may  produce  a  slight  synergis- 
tic action  given  in  conjunction  with  appropriate  lyophilized  tissues  (17). 
Other  data  on  specificity  have  been  published  by  Casey  and  co-workers  (4) . 

Studies  employing  differential  centrifugation  and  filtration  reveal  an 
association  of  the  enhancing  agent  or  agents  with  the  particulate  com- 
ponents of  the  cell  (5,  18). 

We  now  turn  to  another  phase  of  the  Jackson  Laboratory  program — the 
study  of  histocompatibility  genes.  These  are  defined  as  the  genes  which 
determine  susceptibility  and  resistance  to  tumor  and  normal  tissue  trans- 
plants (3) .  Special  methods  have  been  devised  for  this  study,  which  may 
appropriately  be  called  "mitogenetic,"  since  they  involve  tissue  grafts 
(transplantable  tumors  are  employed  in  practice  for  various  technical 
reasons),  and  such  genetic  techniques  as  the  use  of  marker  genes  and  the 
production  of  isogenic-resistant  lines.  These  methods  are  quite  distinct 
from  classical  serologic  procedures.  However,  Hoecker,  Counce  and  Smith 
(19),  using  these  procedures  in  our  laboratory,  have  fully  confirmed  our 
results  for  the  histocompatibility-2  locus,  which  is  also  a  blood-group- 
determining  locus  and  hence  amenable  to  studies  of  this  type. 

For  the  sake  of  brevity,  we  shall  give  no  description  here  of  methods, 
which  have  been  fully  discussed  elsewhere  (3,  20,  21),  but  rather  confine 
ourselves  to  a  summary  of  certain  results. 

We  are  particularly  concerned  here  with  the  histocompatibility-2  locus. 
This  locus  can  assume  at  least  seven  alternative  or  allelic  forms,  as  follows 
(some  of  the  strains  known  to  carry  each  allele  given  in  parentheses) : 
H-2a  4  (strain  A),  H-2d  (strains  BALB/c,  C57BL/6Ks,  B/10D/2,5  DBA/2), 
H-2k  (strains  C57BR/a,  CBA,  ST),  H-2*  (strains  C57BL/6,  C57BL/10, 
LP,  129/Rr),  H-2p  (strain  P),  H-2q  (strain  DBA/1),  and  H-2r  (strain 
RHI/Wy). 

These  seven  alleles  may  be  divided  into  four  groups  on  the  basis  of  the 
presence  or  absence  of  one  or  both  of  two  components  (or  histocompati- 

4Allele  H-2*  was  formerly  called  H-2dk.  The  new  designation  has  been  agreed  on  by  several  investigators  inter- 
ested in  histocompatibility  genes  and  blood  group  genes  in  the  mouse. 

6  B/10.D/2  is  an  abbreviation  for  C57BL/10.DBA/2.  This  is  a  strain  isogenic  with  C57BL/10  except  for  the  fact 
that  gene  H-2d,  introduced  from  strain  DBA/2  by  an  appropriate  series  of  crosses,  has  been  substituted  for  gene 
H-2K 

Vol.    15,   No.    3,    December    1954 


668  PKOCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 

bility  factors  or  antigenic  factors),  D  and  K.  H-2a  carries  both  D  and  K 
H-2d  the  D  factor  only,  H-2k  the  K  factor  only,  and  H-2\  H-2P,  H-#«  and 
H-2T  neither  D  nor  K.  These  factors  were  originally  demonstrated  by  his- 
togenetic  methods  (20) ,  but  they  are  equally  demonstrable  by  appropriate 
antisera  as  red  blood  cell  agglutinogens  (19).  They  are  therefore  clearly 
antigenic  in  nature;  in  fact  as  compared  with  other  isoantigens  in  the 
mouse  they  seem  to  give  particularly  high  titers. 

Experimental:  Methods  and  Results 

The  demonstration  by  Kaliss  that  the  enhancing  effect  can  be  produced 
by  injection  of  normal  tissue  preparations,  taken  together  with  our  analysis 
of  the  histocompatibility-2  locus,  has  made  it  possible  to  set  up  a  precise 
analysis  of  the  specificity  of  the  enhancing  phenomenon.  In  each  test 
three  strains  are  involved:  1)  the  host  strain;  2)  the  strain  providing  the 
normal  tissue  used  for  the  advance  injections;  3)  the  strain  providing  the 
transplantable  tumor.  These  strains  were  selected  on  the  basis  of  the 
presence  or  absence  of  the  D  and  K  factors.  The  host  strain  was  either  D 
or  K.  The  strains  providing  the  normal  tissue  were  from  all  four  allelic 
groups,  DK,  D  without  K,  K  without  D,  and  neither  D  nor  K.  The  tumor 
was  always  DK.  Host  mice  were  given  an  appropriate  series  of  lyophilized 
tissue  injections,  and  subsequently  inoculated  with  the  living  tumor. 
Uninjected  controls  were  run  with  each  experiment. 

The  results  are  presented  in  table  1 .  The  first  point  of  interest  is  that 
strain  combinations  involving  the  same  H-2  allelic  groups  produced  essen- 
tially similar  results,  particularly  in  any  one  experiment,  even  though  the 
strains  involved  were  genetically  quite  unrelated  in  other  respects.  Thus 
injection  of  D  mice  from  two  strains  with  tissue  preparations  from  four 
strains,  also  D,  (BALB/c,  C57BL/6Ks,  B/10.D/2,  and  DBA/2)  produced 
no  effect  in  any  experiment,  except  for  the  death  of  1  mouse  out  of  20  re- 
ceiving BALB/c  kidney.  Likewise,  K  mice  receiving  D  tissue  from  two 
strains  (BALB/c  and  DBA/2)  in  three  experiments  gave  deaths  in  the  pro- 
portions 12  of  20,  9  of  20,  10  of  20,  and  16  of  19,  a  relatively  homogenous 
distribution,  with  such  fluctuations  as  there  are  apparently  correlating 
with  the  relative  enhancing  potencies  of  liver,  kidney,  and  spleen. 

As  expected,  the  uninjected  controls  were  negative  or  almost  negative 
in  each  case. 

The  results  are  summarized  by  allelic  groups  in  table  2.  It  will  be  seen 
from  this  table  that  certain  donor-host  combinations  produce  clear 
enhancement  (55  to  62  percent  of  the  mice  dying),  while  others  either  do 
not  produce  it  (0  to  1  percent  dying)  or  produce  it  to  a  weak  degree  only 
(21  to  22  percent  dying).     These  distinctions  are  statistically  valid. 

The  significance  of  these  results  for  the  interpretation  of  the  enhancing 
effect  is  indicated  by  table  3.  It  will  be  seen  from  this  table  that  enhance- 
ment occurs  when  lyophilized  tissue  donor  and  tumor  transplant  donor 
share  in  common  a  histocompatibility-2  factor  which  is  lacking  in  the  host. 
Thus  where  the  tissue  shares  K  with  the  tumor,  and  the  host  is  not  K  but 

Journal    of    the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


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5 


Vol.   15,  No.  3,  December   19S4 


670 


PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 

Table  2. — Percent  mice  dying  from  tumor  {total  number  of  mice 
in  parentheses)  grouped  according  to  the  presence  or  absence  of 
histocompatibility  factors  D  and  K  in  host- and  lyophilized  tissue 
donor.      Tumor  in  every  case  was  DK 


Host  geno- 

Donor genotype 

Control 

type 

DK 

D 

K 

- 

D 

55 

(56) 

1 

(74) 

60 
(30) 

0 

(20) 

0 

(42) 

K 

60 

(78) 

62 

(76) 

22 

(139) 

21 

(38) 

3 

(59) 

D,  enhancement  occurs.  Pretreatment  with  a  foreign  H-2  factor  specifi- 
cally abrogates  resistance  to  this  factor.  This  result  is  exactly  what  would 
be  expected  in  an  immunizing  procedure,  with  the  important  and  curious 
qualification  that  the  end  result  is  not  increased  resistance  to,  but  increased 
tolerance  of,  the  test  graft. 

Table  3. — Condensed  summary  of  results* 


Host 

Donor 

DK 

D 

K 

Neither 

D 
K 

+ 
+ 

+ 

+ 

- 

♦Tumor  is  DK;  +  most  of  mice  died;  —  most  of  mice  survived. 

Discussion 

Any  discussion  of  the  enhancing  effect  in  the  light  of  our  present  know- 
ledge must  leave  many  questions  unanswered,  and  must  in  some  measure 
be  tentative.  Nevertheless,  available  data  do  suggest  certain  conclusions, 
and  this  would  appear  to  be  an  appropriate  time  to  present  them.  We 
shall  venture  three  hypotheses,  giving  them  in  order  of  what  seems  to  us 
descending  assurance  as  to  their  validity. 

Hypothesis  1. — The  {an)  enhancing  substance  is  a  product  of  the  H-2  locus. 
The  evidence  for  this  statement  is  the  almost  complete  concomitance,  in 
the  host,  tissue  donor,  and  tumor  donor  system,  as  revealed  in  tables  2 
and  3,  between  H-2  genotype  and  the  occurrence  or  nonoccurrence  of 
enhancement.  If  a  particular  H-2  allele  has  to  be  present  for  enhance- 
ment to  occur,  we  can  see  no  escape  from  the  conclusion  that  that  allele 
or  some  product  thereof  is  the  cause  of  the  enhancement.  That  it  is  a 
product  of  the  H-2  gene  and  not  the  gene  itself  is  proved  by  the  fact  that 
two  cytoplasmic  constituents,  washed  mitochondria  and  microsomes  from 
appropriate  strains  and  tissues,  are  potent  enhancing  agents  (5). 


ial    of    the   National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  671 

One  point  in  this  connection  requires  clarification.  In  the  specificity 
experiments  which  we  have  described,  a  total  of  10  different  inbred  strains 
were  employed.  A  few  of  these  have  known  common  ancestry  {e.g., 
C57BL/10  and  C57BR/a)  or  are  differentiated  by  a  single  gene  difference 
{e.g.,  C57BL/10  and  B/10.D/2),  but  most  of  them  are  quite  unrelated  and 
undoubtedly  represent  a  wide  diversity  of  genotypes.  Besides  the  known 
differences  at  the  H-2  locus,  we  can  be  sure  that  these  strains  are  char- 
acterized by  differences  at  a  variety  of  other  histocompatibility  loci, 
even  though  the  details  of  these  differences  are  as  yet  mostly  unanalyzed 
{21).  How  did  it  happen  that  virtually  the  only  effect  that  emerged  was 
the  effect  of  the  H-2  locus?  Since  the  distribution  of  other  histocom- 
patibility loci  was  in  no  way  controlled,  why  did  not  the  action  of  these 
loci  completely  confuse  the  picture? 

There  is  more  than  one  possible  answer  to  these  questions.  It  may  be 
that  there  is  some  peculiarity  of  the  products  of  the  H-2  alleles  so  that  they 
can  abrogate  host  resistance  under  appropriate  experimental  conditions, 
whereas  other  histocompatibility  gene  products  have  no  such  effect.  Or  it 
may  be  that  the  difference  is  quantitative  rather  than  qualitative,  and 
that  the  products  of  other  loci  also  enhance,  but  to  a  lesser  degree. 

There  are  two  facts  favoring  this  second  alternative.  We  have  pointed 
out  elsewhere  that  the  H-2  locus  is  a  "strong"  locus.  We  shall  not  take 
the  time  here  to  define  "strong,"  other  than  to  point  out  that  it  is  simple 
and  perhaps  correct  in  the  light  of  the  work  of  Gorer  [reviewed  in  (7)],  and 
Hoecker,  Counce  and  Smith  {19),  to  think  of  a  strong  locus  as  one  whose 
products  are  potent  producers  of  isoantibodies.  Readers  interested  in 
further  details  are  referred  to  our  earlier  publications  {20,  21).  Suffice  it 
to  say  here  that  the  clear  manifestation  of  the  enhancing  effect  of  the  H-2 
substance,  in  a  situation  where  it  must  have  been  competing  with  other 
histocompatibility  gene  products,  may  simply  be  a  consequence  of  its 
relatively  great  potency. 

The  second  point  to  be  noted  is  that  there  are  certain  irregularities  in 
our  data  which  can  be  interpreted  as  an  enhancing  action  by  other  histo- 
compatibility gene  products.  An  examination  of  table  2  will  show  that 
tissue  from  K  and  not-D-not-K  strains  tested  in  K  hosts  produced  some 
enhancement  as  compared  with  the  untreated  controls,  though  the  D 
factor  expected  to  cause  enhancement  in  this  host  genotype  was  lacking. 
The  obvious  interpretation  is  that  other  though  weaker  enhancing  sub- 
stances, presumably  also  histocompatibility  gene  products,  were  present. 

It  is  perhaps  worth  mentioning  here  that  establishment  of  isogenic 
resistant  lines  {3,  21)  makes  possible  the  design  and  execution  of  experi- 
ments in  which  enhancement,  if  it  occurs,  can  be  due  only  to  the  action  of 
single  histocompatibility  genes  or  their  products.  We  have  in  fact  pro- 
duced 100  percent  enhancement  with  strain  A  whole  blood  of  a  strain  A 
tumor  in  A.SW  mice,  where  the  only  genetic  difference  is  at  the  H-2  locus 
(unpublished  data) . 

A  significant  byproduct  of  our  first  hypothesis  is  the  inference  that  in 
our  chemical  program  for  the  isolation  of  the  enhancing  substance  (s) 

Vol.    15,    No.    3,   December    1954 


672  proceedings:  symposium  on  25  years  of 

we  should  be  able  to  substitute  for  our  present  time-consuming  assay 
based  on  the  enhancing  test  a  short  serological  assay  for  the  H-2  antigen. 
Using  either  procedure,  we  should  be  assaying  for  one  and  the  same 
compound.  I  am  indebted  to  Dr.  Mitchison  for  the  suggestion  that 
probably  the  easiest  assay  should  consist  of  the  absorption  of  an  anti- 
serum against  the  donor  H-2  allele  with  the  fraction  to  be  assayed,  fol- 
lowed by  an  agglutination  test  of  absorbed  antiserum  against  donor  red 
cells. 

Hypothesis  2. — The  enhancing  effect  is  basically  immunological.  Five 
facts,  already  cited  in  some  detail,  which  point  to  this  conclusion  are: 
1)  the  injections  have  to  be  made  before  the  period  of  active  transplant 
growth;  2)  the  effect  persists;  3)  the  effect  can  be  passively  transferred; 
4)  the  effect  is  highly  specific;  5)  the  enhancing  substance,  if  our  first 
hypothesis  is  correct,  is  a  known  isoantigen  (Gorer,  see  review  in  7). 
The  mystery  is  why  the  effect  is  manifest,  not  as  protection  against  the 
graft,  but  as  increased  tolerance  thereof. 

As  evidence  that  the  contrast  between  the  enhancing  effect  and  normal 
immune  phenomena  is  a  real  and  significant  one,  a  variety  of  observations 
may  be  cited. 

Increased  resistance  to,  rather  than  increased  tolerance  of,  tumor  and 
normal  tissue  grafts  can  be  evoked  by  appropriate  types  of  pretreatment  of 
the  host  (see  review  in  7) .  This  is  possible  with  strain  A  tumor  SAl  and 
C57BL/6Ks  and  C57BR/a  hosts,  the  combinations  of  tumor  and  host 
which  we  have  used  most  frequently  in  the  study  of  the  enhancing  effect. 
In  transplantation  studies  the  two  types  of  end  result  therefore  stand  out 
in  sharp  contrast. 

Whereas  the  enhancing  effect  is  passively  transferred  with  serum, 
graft  immunity  is  passively  transferred  only  by  cells  and  tissues,  with 
lymph  nodes  the  organs  most  clearly  implicated  (23,  24;  see  also  review  in 
7). 

While  the  enhancing  effect  is  an  unusual  phenomenon,  it  is  not  without 
several  close  parallels  in  other  areas  of  research.  As  we  have  pointed  out 
elsewhere  (10)  it  resembles  in  several  respects  Fel ton's  (8)  immunological 
paralysis  and  Anderson,  Billingham,  Lamkin  and  Medawar's  (25)  skin 
graft  susceptibility  in  twin  cattle.  We  shall  not  repeat  this  evidence  here, 
but  we  do  wish  to  point  out  that  the  argument  has  been  strengthened  by 
recent  findings.  The  demonstration  here  presented  of  high  specificity  in 
the  enhancing  effect  further  emphasizes  this  feature  of  all  three  phenomena. 
Moreover,  the  recent  brilliant  demonstration  by  Billingham,  Brent  and 
Medawar  (9)  that  mice  can  be  rendered  susceptible  to  skin  homografts  by 
injection  as  embryos  with  donor-strain  tissues  serves  to  close  the  experi- 
mental gap  between  the  enhancing  effect  and  the  situation  in  cattle  twins. 

All  these  facts  point  to  the  conclusion  that  under  certain,  as  yet  poorly 
defined,  conditions  the  antibody-producing  system  responds,  not  by  a 
reaction  of  immunity  or  resistance,  but  by  an  equally  clear  and  specific 
genesis  of  tolerance. 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  673 

If  all  these  phenomena  are  fundamentally  the  same  and  fundamentally 
immunological,  they  deserve  a  common  descriptive  name.  Billingham, 
Brent  and  Medawar  (9)  have  called  their  induced  graft  susceptibility  in 
mice  "actively  acquired  tolerance."  The  name  seems  highly  appropriate, 
and  we  propose  that  its  use  be  extended  to  the  enhancing  effect. 

Hypothesis  8. — The  enhancing  response  may  be  in  part  determined  by 
the  fact  that  the  H-2  substance  in  our  injected  lyophilized  tissue,  while  an 
effective  isoantigen,  is  also  a  native  mouse  substance  and  is  physiologically 
reacted  to  as  such.  Presumably  any  of  the  seven  or  more  variant  forms  of 
the  substance,  corresponding  to  the  seven  or  more  H-2  alleles,  can  enter 
into,  and  adequately  function  in,  some  predetermined  place  in  the  cell's 
structure.  A  foreign  variant  of  the  substance,  introduced  in  a  host  by 
experimental  procedures,  will  compete  with  the  native  variant,  just  as 
antimetabolites  of  a  vitamin  compete  with  the  structurally  related  normal 
vitamin  in  certain  physiological  processes  (26).  In  our  case,  however, 
the  competing  substance  is  functionally  adequate  rather  than  inadequate 
and  detrimental,  and  its  incorporation  in  the  cell,  with  resulting  formation 
of  a  mosaic  structure,  may  be  predicted.  An  H-2d  mouse  injected  with 
H-2k  substance  thus  becomes  partly  an  H-2k  mouse. 

A  mosaic  structure,  though  originally  at  least  entirely  at  the  inter- 
cellular rather  than  the  intracellular  level,  is  present  in  identical  twin 
cattle  and  in  Billingham,  Brent  and  Medawar's  graft-tolerant  mice. 

Possibly  also  significant  is  the  fact  that  both  the  pneumococcus  poly- 
saccharides used  to  produce  Fel ton's  immunological  paralysis,  and  the 
foreign  strains  of  cells  in  twin  cattle  and  graft-tolerant  mice,  persist, 
certainly  for  long  periods,  and  probably  throughout  the  life  of  the  host. 
Persistence  of  antigen  is  said  to  be  rare  (27,  p.  68).  TVe  have  no  evidence 
for  the  persistence  of  the  enhancing  substance  following  injection,  but  the 
postulate  that  it  is  incorporated  into  the  host's  cells  would  not  imply 
rapid  elimination.  This  question  should  be  amenable  to  experimental 
answer  in  due  course. 

Summary 

When  mice  receiving  homografts  of  certain  transplantable  tumors  are 
given  prior  injections  of  lyophilized  tumor  or  of  lyophilized  mouse  kidney, 
liver,  or  spleen  from  the  donor  strain,  progressive  growth  .of  the  tumor 
tends  to  occur.     This  phenomenon  has  been  called  the  enhancing  effect. 

A  study  of  the  specificity  of  the  effect  has  been  carried  out.  Hosts, 
normal  tissue  donors,  and  transplantable  tumor  were  selected  from  strains 
with  known  genotypes  with  respect  to  the  D  and  K  factors  of  the  histo- 
compatibility^ (H-2)  locus.  Enhancing  experiments  were  carried  out 
with  these  strains  in  various  combinations.  It  was  found  that  enhance- 
ment occurred  if,  and  only  if,  tumor  and  normal  tissue  shared  a  factor 
which  was  lacking  in  the  host  (table  3) . 

The  conclusion  is  drawn  that  the  (an)  enhancing  substance  must  be  a 
product  of  the  H-2  locus.  Keasons  are  presented  for  believing  that  the 
enhancing  effect  is  immunological  in  nature  despite  its  reversal  of  the  usual 

Vol.   15,  No.  3,  December   1954 

316263—54 28 


674  proceedings:  symposium  on  25  years  of 

immune  effect,  and  the  name  "actively  acquired  tolerance"  (after  Meda- 
war  and  co-workers)  is  proposed.  It  is  suggested  that  injected  H-2 
substance  becomes  incorporated  in  the  host's  cells  along  with  the  native 
H-2. 

References 

(1)  Little,   C.   C:  A  possible   Mendelian  explanation  for  a  type  of  inheritance 

apparently  non- Mendelian  in  nature.     Science  40:  904-906,  1914. 

(2)  :  The  genetics  of  tumor  transplantation.     In  the  Biology  of  the  Labora- 
tory Mouse,  (Snell,  G.  D.,  ed.)     Philadelphia,  The  Blakiston  Co.,  1941. 

(8)  Snell,  G.  D.:  Methods  for  the  study  of  histocompatibility  genes.     J.  Genetics 
49:  87-108,  1948. 

(4)  Casey,  A.  E.,  Ross,  G.  L.  and  Langston,  R.  R.:  Selective  XYZ  factor  in  C57 

black  mammary  carcinoma  E0771.     Proc.   Soc.   Exper.   Biol.    &    Med.    72: 
83-89,  1949. 

(5)  Snell,  G.  D. :  Enhancement  and  inhibition  of  the  growth  of  tumor  homoiotrans- 

plants  by  pretreatment  of  the  hosts  with  various  preparations  of  normal  and 
tumor  tissue.     J.  Nat.  Cancer  Inst.  13:  719-729,  1952. 

(6)  Kaliss,  N.:  Induced  alteration  of  the  normal  host-graft  relationship  in  homo- 

transplants  of  mouse  tumors.     Ann.  New  York  Acad.  Sc.     In  press,  1954. 

(7)  Snell,   G.   D.:  Transplantable  tumors.     In  The  Physiopathology  of  Cancer. 

(Homburger  and  Fishman,  eds.)     New  York,  Hoeber-Harper  Book  Co.,  1953. 

(8)  Felton,  L.  D.:  The  significance  of  antigen  in  animal  tissues.     J.  Immunol.  61: 

107-117,  1949. 

(9)  Billingham,   R.   E.,   Brent,   L.,  and   Medawar,   P.   B.:    "Actively    acquired 

tolerance"  of  foreign  cells.     Nature  172:  603-606,  1953. 

(10)  Snell,  G.  D.:  The  immunogenetics  of  tumor  transplantation.     Cancer  Res.  12: 

543-546,  1952. 

(11)  Snell,  G.  D.,  Cloudman,  A.  M.,  Failor,  E.,  and  Douglass,  P.:  Inhibition  and 

stimulation  of  tumor  homoiotransplants  by  prior  injections  of  lyophilized 
tumor  tissue.     J.  Nat.  Cancer  Inst.  6:  303-316,  1946. 

(12)  Kaliss,  N.,  and  Day,  E.  D.:  Relation  between  time  of  conditioning  of  host  and 

survival  of  tumor  homografts  in  mice.     Proc.  Soc.  Exper.  Biol.  &  Med.  86: 
115-117,  1954. 
(18)  Kaliss,  N.,  and  Molomut,  N.:  The  effect  of  prior  injections  of  tissue  antiserums 
on  the  survival  of  cancer  homoiografts  in  mice.     Cancer  Res.  12:  110-112, 
1952. 

(14)  Kaliss,  N.,  Molomut,  N.,  Harriss,  J.  L.,  and  Gault,  S.  D.:  Effect  of  pre- 

viously injected  immune  serum  and  tissue  on  the  survival  of  tumor  grafts  in 
mice.     J.  Nat.  Cancer  Inst.  13:  847-850,  1953. 

(15)  Kaliss,  N.,  and  Snell,  G.  D.:  The  effects  of  injections  of  lyophilized  normal 

and  neoplastic  mouse  tissues  on  the  growth  of  tumor  homoiotransplants  in 
mice.     Cancer  Res.  11:  122-126,  1951. 

(16)  Kaliss,  N.:  Effect  of  prior  injection  of  non-mouse  tissues  on  growth  of  tumor 

homoiografts  in  mice.     Science  116:  279-280,  1952. 

(17)  Kaliss,  N.,  and  Borges,  P.  R.  F.:  Effect  of  injected  lyophilized  tumor  and 

trypan  blue  on  host  resistance  to  tumor  grafts.     J.  Nat.  Cancer  Inst.   13: 
343-347,  1952. 

(18)  Day,  E.  D.,  and  Kaliss,  N.:  Investigations  preliminary  to  physical  and  chemical 

characterization  of  substances  in  mouse  tissues  responsible  for  the  abrogation 
of  resistance  in  mice  to  tumor  homotransplants.  J.  Nat.  Cancer  Inst.  15: 
000-000,  1954. 
(18)  Day,  E.  D.,  Kaliss,  N.,  Aronson,  I.,  Bryant,  B.  F.,  Friendly,  D..  Gabrielson, 
F.  C,  and  Smith,  P.  M.:  Investigations  of  substances  in  mouse  tissues  inducing 
alteration  of  normal  host-homograft  relationships.  J.  Nat.  Cancer  Inst.  15: 
145-159,  1954. 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  675 

{20)  Snell,  G.  D.,  Smith,  P.,  and  Gabrielson,  F.:  Analysis  of  the  histocompatibility-2 
locus  in  the  mouse.     J.  Nat.  Cancer  Inst.  14:  457-480,  1953. 

{21)  Snell,  G.  D.:  The  genetics  of  transplantation.  J.  Nat.  Cancer  Inst.  14:  691-700, 
1953. 

{22)  Snell,  G.  D.,  and  Borges,  P.  R.  F.:  Determination  of  the  histocompatibility 
locus  involved  in  the  resistance  of  mice  of  strains  C57BL/10-:c,  C57BL/6-Z  and 
C57BL/6Ks  to  C57BL  tumors.     J.  Nat.  Cancer  Inst.  14:  481-484,  1953. 

{23)  Brncic,  D.,  Hoecker,  G.,  and  Gasic,  G.:  Immunity  in  mice  against  leukemic 
cells  of  the  same  genetic  constitution.  Acta  U.  Int.  contre  le  Cancer.  7: 
761-764,  1952. 

{24)  Mitchison,  N.  A.:  Passive  transfer  of  transplantation  immunity.  Proc.  Roy. 
Soc,  s.B,  142:  72-87,  1953. 

{25)  Anderson,  D.,  Billingham,  R.  E.,  Lamkin,  G.  H.,  and  Medawar,  P.  B.:  The 
use  of  skin  grafting  to  distinguish  between  monozygotic  and  dizygotic  twins 
in  cattle.     Heredity  5:  379-397,  1951. 

{26)  Woolley,  D.  W.:  Antimetabolites.  Ann.  New  York  Acad.  Sc.  52:  (Art.  8) 
1197-1378,  1950. 

{27)  Burnet,  F.  M.,  and  Fenner,  F.:  The  Production  of  Antibodies,  2nd  ed.  Mel- 
bourne, Macmillan  and  Co.,  Ltd.,  1949. 


Vol.    15,   No.    3,   December    19S4 


. 


1 


Discussion 
Dr.  N.  A.  Mitchison,  Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 

When  Dr.  Snell  and  his  colleagues  first  described  the  effect  of  prior  injection  of 
lyophilized  tissue  on  the  growth  of  tumor  homografts  in  1946,  one  of  the  first  question 
which  came  to  mind  concerned  the  specificity  of  the  phenomenon.  Was  this  effec 
another  stimulation  or  inhibition  of  tumor  tissue  in  general,  or  was  he  interfering  with 
the  delicate  and  highly  specific  immunological  relationship  between  the  host  and  the 
graft?  Kaliss  answered  this  question  in  one  form,  but  we  now  have  evidence  of 
remarkable  elegance  from  material  of  known  genetical  constitution. 

An  answer  in  this  form  has  been  possible  only  because  Dr.  Snell  has  succeeded  in 
identifying  histocompatibility  genes  in  so  many  of  the  standard  inbred  lines;  and  this 
in  turn  has  been  possible  only  because  of  the  development  by  Dr.  Snell  of  his  isogenic 
resistant  lines.  One  can  hardly,  I  believe,  appreciate  the  labor  and  the  skill,  immuno- 
logical as  well  as  genetical,  which  has  gone  into  the  development  of  these  lines,  until 
one  has  seen  Dr.  Snell's  laboratory  in  action.  A  large-scale  program,  which  at  its 
commencement  offered  no  sure  promise  of  success,  has  been  systematically  developed, 
and  has  produced  magnificent  results.  The  identification  of  many  histocompatibility 
genes,  and  their  isolation  in  isogenic  lines,  opens  up  possibilities  in  many  directions. 
The  demonstration  of  the  specificity  of  the  enhancing  effect  that  we  have  just  heard  is, 
I  believe,  the  first  example  of  their  use.  The  evidence  that  has  been  presented  is  surely 
convincing,  but  I  think  that  Dr.  Snell  would  agree  that  the  final  proof  of  his  hypothesis 
will  come  through  the  use  of  the  isogenic  lines.  In  immunization  or  enhancing  experi- 
ments, a  difference  between  tissues  from  two  lines,  isogenic  except  at  the  H-2  locus, 
would  indicate  not  only  that  the  difference  was  due  to  the  iso-antigens  controlled  by 
the  H-2  locus,  but  also  that  no  other  iso-antigens  were  involved. 

It  is  a  surprising  observation  that,  among  the  various  more  or  less  unrelated  inbred 
lines  that  have  been  investigated  so  far,  the  H-2  locus  alone  appears  to  control  the 
specificity  of  the  enhancing  effect.  It  would  be  rash,  however,  to  conclude  that  no 
other  iso-antigenic  differences  exist  or  are  of  importance.  If  hemolytic  disease  of  the 
newborn  was  the  only  method  of  detecting  iso-antigenic  differences  in  man,  we  should 
know  about  the  Rhesus  and  Kell  groups  but  not  about  the  ABO  group! 

Dr.  Snell  has  made  it  clear  that  we  do  not  have  as  much  evidence  about  the  mechan- 
ism of  the  enhancing  process  as  about  its  specificity.  However,  what  originally  seemed 
to  be  a  rather  isolated  phenomenon  is  now  fitting  into  a  wider  biological  context.  Owen 
published  his  observations  on  the  blood  groups  of  dizygotic  cattle  twins  a  few  months 
before  the  first  observations  on  the  enhancing  effect  were  announced.  Although  at 
the  time  no  connection  was  apparent,  both  Kaliss  and  Medawar  have  pointed  out  that 
what  Medawar  and  his  co-workers  have  termed  "actively  acquired  tolerance''  probably 
involves  a  mechanism  very  similar  to  the  enhancing  effect.  Medawar  argues  that 
because  replacement  therapy  with  normal  or  immune  lymphoid  tissue  can  restore  an 
actively  tolerant  mouse  to  its  normal  condition,  actively  acquired  tolerance  must 
involve  a  specific  immunological  paralysis  of  the  antibody  producing  system.  We  now 
have  indications  that  similar  replacement  therapy  is  effective  in  the  enhanced  mouse. 
Recently,  Owen  has  produced  evidence  that  the  surprisingly  small  fraction  of  Rh- 
negative  women  who  become  immunized  against  antigen  from  their  offspring  may  be 
due  to  actively  acquired  tolerance:  the  mothers,  when  they  were  themselves  in  the 
uterus,  were  exposed  to  Rh  antigen.  All  this  will  surely  provide  fresh  stimulus  for  in- 
vestigation of  the  mechanism  of  enhancing. 

677 


Journal    of   the   National   Cancer   Institnte,    Vol.    15,   No.    3,   December    1954 


The  Expanding  Knowledge  of  the  Ge- 
nome of  the  Mouse  * 


Margaret  M.  Dickie,  Roscoe  B.  Jackson  Memo- 
rial Laboratory,  Bar  Harbor,  Maine 


Mouse  genetics,  which  began  shortly  after  the  turn  of  the  century  follow- 
ing the  rediscovery  of  Mendel's  laws,  has  come  into  a  golden  age  of  devel- 
opment. The  contributions  of  this  small  mammal  have  been  most 
impressive  in  the  study  of  development  and  physiological  genetics  which 
bear  close  relationships  with  medical  research  in  all  its  aspects. 

There  were,  until  the  first  of  June  1954,  215  named  genes  and  alleles 
in  the  mouse.  Now  according  to  latest  reports  there  are  226  named 
genes  and  alleles.  Trust  laboratories  have  been  designated  on  both  sides 
of  the  Atlantic  Ocean  which  maintain  all  useful  mutations  that  otherwise 
might  be  discarded  or  lost.  This  laboratory  now  maintains  about  100 
of  these  mutations. 

Hans  Griineberg's  The  Genetics  of  the  Mouse,  a  comprehensive  atlas  of 
the  genetic  and  morphologic  descriptions  of  all  mutations,  and  the  Mouse 
News  Letter,  a  semiannual  communication  that  is  essential  to  keep  abreast 
of  current  affairs  in  mouse  genetics,  have  been  used  as  the  sources  for  this 
presentation  of  the  chronology  of  the  mouse  genome. 

Before  scanning  the  development  of  the  genome  it  is  well  to  review 
briefly  the  kinds  of  mutations  that  have  been  recognized  over  the  years. 
One  could  go  into  the  pleiotropic  effects  or  the  developmental  action  of 
genes  that  are  under  intensive  investigation  at  the  present  time,  but  such 
a  discussion  is  not  warranted  here.  A  loose  classification  of  the  types  of 
mutations  will  suffice.  It  is  realized  that  some  mutations  overlap  these 
different  categories  but  no  final  classification  is  available  now.  In  respect 
to  color,  such  as  black  or  brown  or  dilution  of  color,  there  are  29  genes. 
In  regard  to  hair  texture  such  as  curliness  like  rex  or  wellhaarigkeit,  or 
absence  of  hair — like  hairless  and  naked — there  are  22  genes.  Spotting 
patterns  such  as  piebald,  splotch  and  belted  account  for  22  genes.  Skeletal 
defects  number  some  58  genes,  among  which  are  the  anury  series,  undulated 
and  Polydactyly.  Seventeen  genes  account  for  eye  and  brain  abnormalities 
such  as  microphthalmia  and  hydrocephalus.  The  behavior  mutants, 
although  properly  listed  as  disorders  of  the  nervous  system,  may  be 
divided  into  two  classes.  One  of  these  classes  is  the  shaker  syndrome 
or  the  choreic  mutants;  there  are  18  of  them  such  as  waltzer,  shaker  and 

1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  28, 1954. 

679 

Journal    of    the   National    Cancer    Institute,    Vol.    15,   No.    3,   December    1954 


680  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

jerker.  In  the  other  class  are  9  epileptiform  syndrome  genes;  mutations 
that  cause  convulsive  rather  than  any  circling'  behavior,  such  as  trembler, 
wabbler-lethal  and  agitans.  Four  genes  cause  anemias.  Ten  genes  act  on 
other  physiological  phenomena,  e.g.  dwarfing,  the  histocompatibility  genes 
and  obesity.  About  35  genes  are  lethal  in  the  homozygous  condition  (not 
many,  compared  with  the  557  known  in  Drosophila  by  1945). 

There  are  10  recognized  allelic  series:  albino  series  with  5  and  possibly 
6  alleles;  pink-eye  series  with  3  alleles;  dilution  series  with  3  alleles; 
hairless  series  with  4  alleles;  dominant  spotting  series  with  3  alleles;  the 
agouti  series  with  5  alleles;  the  brown  series  with  3  alleles;  anury  series 
with  23  alleles;  histocompatibility-2  series  with  10  alleles;  and  micro- 
phthalmia series  with  3  alleles. 

Remutations  have  been  reported  in  the  literature  most  notably  at  the 
agouti  and  brown  loci.  A  few  remutations  to  rex  and  caracul  have 
occurred.  Five  somatic  mutations  have  definitely  been  recognized  since 
they  involved  gonadal  mosaics,  but  it  is  assumed  that  more  have  remained 
undetected. 

These  are  merely  glimpses  at  the  present  state  of  affairs  in  mouse 
genetics  and  now  a  glimpse  at  the  history  of  its  development. 

The  records  show  that  albinism  has  been  recorded  and  described  since 
ancient  times  and  the  Japanese  waltzing  as  a  characteristic  was  known  in 
the  last  century  on  other  continents.  However,  piebald,  symbol  s,  was 
described  by  Allen  in  1903  shortly  after  the  rediscovery  of  Mendel's  laws. 
Following  that,  Bateson  described  dilution  and  in  1905  Cuenot's  classic 
studies  and  conclusions  on  the  lethality  and  inheritance  pattern  of  the 
yellow  gene  was  reported.  There  appears  to  be  a  silence  until  1912  when 
Hagedoorn  reported  a  silvering  gene,  but  it  is  known  that  at  that  time 
C.  C.  Little,  working  as  a  student  of  Dr.  W.  E.  Castle  at  Harvard,  was 
fascinated  with  the  potentials  of  different  kinds  of  mice  and  was  laying 
the  foundation  for  the  aristocrat  of  the  inbred  strains,  the  DBA.  Follow- 
ing the  announcement  of  the  silvering  gene,  Rabaud  reported  the  occur- 
rence of  a  luxate-like  character.  In  England  in  1915,  the  first  vertebrate 
linkage  was  announced  by  Haldane,  Sprunt  and  Haldane.  The  linkage 
was  between  albinism  and  pink  eye.  This  linkage  was  independently 
reported  in  the  United  States  by  Castle  and  his  co-workers.  In  the 
succeeding  years  this  linkage  was  confirmed  by  other  workers  and  as 
other  alleles  of  the  albino  series  appeared  they  were  tested,  extreme 
dilution  by  Detlefsen  in  1921  and  chinchilla  by  Feldman  in  1922.  Mean- 
while, in  1916  Little  described  the  brown  gene  and  the  light-bellied  agouti. 
Short  ear  was  reported  by  Lynch  in  1921.  In  1923,  Little  and  Bagg 
reported  the  occurrence  of  myelencephalic  blebs,  a  gene  still  independent 
of  all  known  linkage  groups.  Other  genes  reported  shortly  after  were 
rodless  retina,  dominant  spotting,  otocephaly  and  waltzing. 

During  this  first  period  that  has  been  arbitrarily  set,  the  summary 
shows  that  about  15  investigators  were  concerned  with  mouse  genes,  one 
linkage  group  had  been  set  up  and  19  genes  and  alleles  had  been  recorded 
in  the  literature  (text-fig.  1A). 

Journal    of   the   National   Cancer   Institute 


1 


PKOGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


681 


I 

1915 


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established  in  1915. 

IB. — The  physical  map  of  the  period  1926-1935.  Genes  marked  with  white 
squares  were  put  on  the  map  during  this  decade.  Dates  under  linkage  group 
number  signify  date  first  linkage  of  the  group  was  reported. 

1C—  The  decade  1936-1945. 


Vol.    15,   No.   3,   December    1954 


682 


PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


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Vol.    15,   No.   3,    December    1954 


684  proceedings:  symposium 

In  the  next  period  1926-1935,  the  number  of  workers  in  the  field 
increased  to  about  30,  and  among  them  were  Crew,  Snell,  Dobrovolskaia- 
Zavadskaia,  Dunn,  Murray  and  Griineberg.  The  second  linkage  group 
was  reported  by  Dr.  Gates  in  1927  and  by  Dr.  Snell  in  1928.  This  linkage 
was  between  dilution  and  short  ear.  The  number  of  genes  named  in- 
creased rapidly.  By  1930  there  were  31  and  by  1935  there  were  44,  of 
which  14  had  been  placed  on  five  linkage  groups.  These  linkage  groups 
were  reported  by  Snell,  Keeler  and  Roberts,  and  Quisenberry  (text-fig.  IB). 

The  next  arbitrary  period  (1936-1945)  saw  the  number  of  linkage  groups 
doubled,  genes  described  totaled  81,  and  55  were  located  on  these  10 
linkage  groups.  Investigators  pursuing  this  phase  of  research  had  risen 
to  about  50  and  included  among  others,  Fisher,  Her  twig  and  MacDowell 
(text-fig.  IB). 

Now  at  least  90  geneticists  are  actively  engaged  in  genetics  research 
with  this  rodent.  Names  added  to  our  distinguished  list  over  this  decade 
must  include  at  least  Russell,  Gluecksohn-Waelsch,  Heston,  Carter, 
Falconer,  Green,  Garber  and  Griffen.  Since  1945,  145  new  genes  have 
been  reported  which  brings  the  total  to  226.  One  hundred  and  sixteen 
of  these  named  genes  and  alleles  are  now  located  on  15  linkage  groups. 
Three  new  linkage  groups  were  established  and  31  genes  were  located  in 
the  past  year  alone  (text-fig.  2). 

The  event  which  I  am  sure  those  working  with  mouse  genes  have  been 
eagerly  awaiting  for  many  years,  became  a  fact  last  year,  i.e.,  the  identifi- 
cation of  several  sex-linked  genes  and  their  established  linkage  relation- 
ships on  the  sex  chromosome  (XX).  Mottled  was  described  by  Fraser 
in  1951,  and  brindled  by  Falconer  in  1952.  Garber  reported  on  bent  tail 
in  1952.  The  linkage  of  bent  tail  and  tabby,  and  brindled  and  mottled, 
were  recorded  last  year  and  early  this  year  jimpy  was  located  on  the 
group.  Two  other  genes,  a  sex-linked  lethal  and  tortoise  shell  are  sex- 
linked  but  no  linkage  data  to  establish  their  position  on  the  map  is  avail- 
able. 

With  increasing  numbers  of  geneticists  working  in  this  field  and  the 
number  of  mutations  being  discovered  and  studied,  it  appears  that  very 
soon  all  20  linkage  groups  will  be  identified  in  this  "golden  age"  of  mouse 
genetics. 


Session  V.  Carcinogenesis  in  Endocrine 
Organs  (Roundtable  Discussion) 


Chairman,  Dr.  Frank  E.  Adair,  Attending  Sur- 
geon Emeritus,  Memorial  Hospital,  New  York, 
N.  Y.;  President,  Board  of  Trustees,  Roscoe  B. 
Jackson  Memorial  Laboratory 


Contributors : 

Dr.  Jacob  Furth:  Thyroid-Pituitary  Tumorigenesis 
Dr.  W.  U.  Gardner:  Studies  on  Ovarian  and  Pituitary  Tumorigenesis 
Dr.  Katharine  P.  Hummel:  Induced  Ovarian  and  Adrenal  Tumors 
Dr.  George  W.  Woolley:  Carcinogenesis  in  the  Adrenal 

Floor  discussion  leaders: 
Dr.  W.  B.   Atkinson,   Dr.  E.   Elizabeth  Jones*,   Dr.   Flavia 
Richardson,  Dr.  Robert  Speirs 


•Discussion  not  submitted. 

685 


Journal    of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


Thyroid-Pituitary  Tumorigenesis  h 


Jacob  Fukth,    The   Children's   Cancer  Research 
Foundation,  Boston,  Mass. 


This  introduction  to  the  roundtable  discussion  aims  to  summarize  and 
evaluate  recent  observations  on  the  mechanism  of  induction  and  character 
of  thyrotrophic  in  comparison  with  other  types  of  pituitary  tumors. 
Most  experimental  data  have  been  or  will  be  fully  published  elsewhere; 
therefore  only  a  summary,  discussion  and  references  of  this  presentation 
will  be  printed  here. 

There  is  a  reciprocal  relationship  between  the  function  of  the  thyroid 
and  the  thyrotrophs  of  the  pituitary  and  it  is  possible  to  produce  tumors 
of  either  the  thyroid  or  the  pituitary  by  interfering  with  their  physiologic 
equilibrium.  When  the  synthesis  of  the  thyroid  hormone  (TH)  is  blocked 
without  interfering  with  growth  responsiveness  of  the  thyroid  epithelium 
(as  accomplished  by  goiterogens),  thyroid  tumors  will  result  (1-8). 
Invasive  thyroid  tumors  occurring  naturally  in  fish  (4)  and  rats  (5)  are 
probably  due  to  dietary  iodine  deficiency.  Thyroid  tumors  induced  in 
mice  by  thiouracil  have  been  shown  to  be  transplantable  to  hosts  whose 
thyroid  function  had  been  depressed  by  a  goiterogen,  but  not  to  normal 
animals.  Upon  successive  transplantation  such  dependent  thyroid 
tumors  give  rise  to  autonomous  neoplasms  (6) .  However,  thyroid  tumors 
can  also  be  produced  in  normal  hosts  with  sustained  stimulation  of  the 
normal  thyroid  by  excessive  quantities  of  the  thyroid-stimulating  hormone 
(TSH) .  This  is  easily  accomplished  by  grafting  a  thyrotrophic  pituitary 
tumor  on  a  normal  host  (7). 

Destruction  of  the  thyroid  of  mice,  best  accomplished  by  administration 
of  radioiodine  (8),  induces  pituitary  tumors.  Ionizing  radiation  was 
thought  by  Gorbman  and  Edelmann  (9)  to  be  essential  for  the  induction 
of  these  tumors,  but  experiments  of  Dent  et  al.  (10)  have  shown  that 
surgical  thyroidectomy  will  accomplish  the  same;  other  experiments  in 
the  same  laboratory  indicate  that  the  tumors  induced  by  radiothyroidec- 
tomy  are  composed  of  thyrotrophs  and  that  radiation  plays  a  minor  role, 
if  any,  in  the  induction  of  thyrotrophic  pituitary  tumors  (11).  This  idea 
is  supported  by  recent  experiments  of  Moore  et  al.  (12)  indicating  that 
long  continued  administration  of  propylthiouracil  will  also  induce  pituitary 

1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  29, 1954. 

a  The  investigations  of  the  author  are  being  supported  currently  by  the  National  Cancer  Institute:  sources  of 
support  of  earlier  work  are  indicated  in  the  original  reports. 

687 
Journal   of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


688  proceedings:  symposium  on  25  years  of 

tumors.  The  transplantability  and  character  of  the  tumors  induced  by 
this  compound  has  not  been  established  but  it  is  probable  that  they  are 
induced  by  sustained  deficiency  of  TH,  as  are  the  pituitary  tumors  which 
develop  following  thyroidectomy.  It  can  therefore  be  assumed  that  these 
pituitary  tumors  are  also  composed  of  thyrotrophs.  That  the  pituitary 
tumors  induced  by  surgical-  and  radio-thyroidectomy  are  identical  in 
character  has  been  shown  by  Dent  et  al.  (10).  The  precise  conditions 
required  for  induction  of  thyroid  tumors  by  propylthiouracil  on  the  one 
hand,  and  pituitary  tumors  on  the  other,  remain  to  be  determined.  It 
appears  that  the  determining  factor  is  the  degree  of  block  of  synthesis 
of  TSH — partial  block  yielding  thyroid  tumors;  more  complete  block, 
pituitary  and  thyroid  tumors;  and  complete  destruction  of  the  thyroid 
epithelium,  pituitary  tumors  only. 

In  the  course  of  successive  transplantations,  thyrotrophs  tumors  in- 
duced by  thyroidectomy  go  through  two  major  phases:  a)  conditioned  or 
dependent,  and  b)  autonomous  (18).  All  of  more  than  13  primary 
thyrotrophs  tumors  studied  by  serial  transplantations  in  normal  and 
athyroid  hosts  were  dependent  in  the  first  transplant  generation,  but 
after  the  second  or  later  passage,  most  of  them  turned  autonomous.  With 
one  tumor  strain  the  dependent  character  of  the  tumor  has  been  main- 
tained through  six  consecutive  passages  made  in  the  course  of  2  years. 

Evidence  for  the  occurrence  of  successive  changes  in  both  dependent 
and  autonomous  tumor  cells  is  suggested  by  the  increasing  growth  rates 
of  the  tumors,  by  the  increasing  histologic  and  cytologic  dedhTerentiation 
of  the  tumor  cells,  and  by  diminishing  hormone  production  (11,  18).  The 
fact  that  the  first  generation  grafts  never  took  in  normal  hosts  indicates 
that  the  autonomous  variants  arose  from  dependent  cells  by  a  transfor- 
mation analogous  to  a  somatic  mutation.  It  is  probable  that  the  ac- 
celerated growth  rate  occurring  in  the  course  of  successive  passages  of 
both  dependent  and  autonomous  tumors  is  caused  by  similar  modifica- 
tions of  the  tumor  cells,  but  assays  of  single-cell  progeny  are  required  to 
prove  this  supposition. 

Similarly,  it  remains  to  be  shown  whether  the  conditioned  neoplasms 
are  composed  of  unaltered  normal  cells,  growing  progressively  in  response 
to  a  tremendous  increase  of  their  normal  physiologic  stimulant,  or  of 
somewhat  altered  but  still  responsive  cells.  The  induction  of  pituitary 
tumors  can  be  prevented  by  administration  of  thyroid  hormone  (14,  15) 
and  the  take  of  a  transplanted  dependent  tumor  can  be  prevented  by  a 
similar  procedure.  Furthermore,  it  has  been  possible  to  restrain,  but  not 
to  arrest,  already  growing  dependent  pituitary  tumors  by  administration 
of  thyroid  hormone.  It  remains  to  be  determined  whether  this  failure  of 
complete  regression  is  due  to  difficulties  of  administering  large,  ' 'neutral- 
izing" quantities  of  thyroid  hormone  or  to  a  diminished  responsiveness 
of  the  dependent  tumor  cells. 

In  addition  it  would  be  desirable  to  determine  whether  or  not  the 
primary  dependent  tumor  cells  would  regress  in  the  original  thyroidec- 
tomized  hosts  following  administration  of  TH  and,  if  not,  the  exact  time 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  689 

when  responsiveness  to  TH  is  lost.  Tumorigenesis  by  derangement  of 
the  normal  hormonal  equilibrium  between  thyroid  and  pituitary  could 
serve  as  a  model  for  research  on  neoplastic  growths  of  other  cells  because 
of  the  relative  ease  and  precision  of  quantitating  the  two  opposing  forces, 
THandTSH. 

Complete  destruction  of  the  thyroid  gland  is  not  essential  for  the  induc- 
tion or  growth  of  dependent  pituitary  tumors.  Kesidual  thyroidal  epi- 
thelium, present  either  after  surgical  or  radio  thyroidectomy,  appears  to 
be  unable  to  proliferate  sufficiently  to  produce  enough  hormone  to  restrain 
a  dependent  tumor  once  the  balance  is  tilted  in  favor  of  tumor  growth  (10). 
Failure  of  thyroid  remnants  to  undergo  adequately  compensating  hyper- 
plasia at  the  site  of  radiothyroidectomy  can  be  explained  by  the  sclerotic 
changes  seen  following  irradiation,  but  it  is  puzzling  why  regeneration  of 
the  thyroid  remnants  after  subtotal  surgical  thyroidectomy  does  not  pro- 
ceed far  enough  to  restrain  the  normal  balance  between  thyroid  epithelium 
and  thyro trophic  pituitary  cells.  The  possible  explanations  include: 
1)  Regeneration  is  limited  by  the  surrounding  tissues.  The  bulk  of  the 
regenerated  thyroidal  tissue  in  surgically  thyroidectomized  animals  never 
reaches  that  of  the  normal  thyroid.  2)  An  alteration  occurs  in  the  respon- 
siveness of  the  cells  or  of  the  quality  of  the  hormone  produced  during  a 
sustained  uncompensated  period. 

Further  evidence  favoring  the  view  that  radiation  plays  no  role  in  the 
induction  of  thyrotrophs  tumors  in  radiothyroidectomized  mice  is  fur- 
nished by  the  observations  that  pituitary  tumors  induced  by  whole-body 
irradiation  are  different  in  character  from  those  induced  by  radiothyroidec- 
tomy (16).  Of  10  such  tumors  assayed  by  means  of  grafts  on  animals  of 
the  strain  of  origin  only  one  had  some  thyrotrophic  potencies  and  even 
this  was  accompanied  by  growth-promoting  potencies.  Three  of  the 
tumors  assayed  yielded  pure  adrenotrophic  transplantable  tumors  and 
three  others  appeared  to  have  only  mammary-gland  stimulating  effect. 
All  seven  tumors  induced  in  mice  by  ionizing  irradiation  were  autonomous 
at  the  start  (17). 

While  all  of  at  least  five  strains  of  mice  thus  far  tested  by  different 
investigators  readily  developed  pituitary  tumors  following  radiothyroidec- 
tomy, the  rat  proved  resistant  to  this  type  of  tumorigenesis.  It  is  not 
known  whether  this  difference  is  a  species  characteristic,  and  if  so,  whether 
normal  diet  supplies  sufficient  TH  to  thyroidectomized  rats  to  prevent  the 
development  of  pituitary  tumors. 

It  is  not  known  whether  pituitary  tumors  will  develop  in  man  in  the 
absence  of  the  thyroid;  older  records  are  inadequate  and  recently  compen- 
sation therapy  by  administration  of  TH  counteracts  such  tendencies.  On 
the  other  hand,  it  is  certain  that  sustained  excess  of  thyrotrophs,  brought 
about  by  goiterogens,  play  a  significant  role  in  the  development  of  both 
adenomas  and  carcinomas  of  the  thyroid  in  man. 

The  occurrence  of  pituitary  tumors  in  rats  and  mice  following  adminis- 
tration of  estrogen  was  reported  in  1936  independently  from  three  labora- 
tories (18).     This  has  been  amply  confirmed,  but  thus  far  the  character 

Vol.    15,   No.   3,   December    1954 
316263—54 29 


690  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

of  the  tumors  so  induced  has  not  been  ascertained.  Dunning  (19)  has 
shown  in  the  rat,  and  Gardner  (20)  in  the. mouse,  that  such  estrogen- 
induced  pituitary  tumors  are  transplantable  to  hosts  similarly  conditioned 
by  administration  of  stilbestrol  but  the  nature  of  the  hormonal  secretion 
of  these  tumors  is  a  matter  of  mere  speculation.  It  is  highly  probable 
that  they  are  gonadotrophic  but  equally  good  arguments  can  be  presented 
for  their  being  either  luteotrophic  or  follicle  stimulating  or  lactogenic. 

Experimental  material  is  now  available  to  study  mammotrophic  tumors. 
The  pituitary  tumors  developing  in  gonadectomized  mice  (21)  were  asso- 
ciated with  extensive  alveolar  hyperplasia  of  the  mammary  gland  but  this 
was  noted  also  in  mice  with  primary  thyrotrophic  pituitary  tumors  and 
the  evidence  thus  far  indicates  that  the  thyrotrophic  tumors  themselves  do 
not  secrete  prolactin.  Mammary-gland  stimulation  is  not  a  characteristic 
feature  of  grafted  thyrotrophic  tumors  (7)  and  assays  by  Bates  failed  to 
disclose  lactogenic  properties  (11). 

Three  transplantable  autonomous  pituitary  tumor  strains  derived 
from  mice  that  had  been  exposed  to  whole-body  irradiation  invariably 
stimulate  the  mammary  glands  without  causing  an  enlargement  of  other 
endocrine  organs  (17). 

The  discovery  of  a  conditioned  phase  of  tumor  growth  preceding  the 
autonomous  phase,  deserves  special  attention  because  the  alteration  in 
the  former  resides  in  the  host  and  is  controllable  by  restoration  of  hor- 
monal deficiency  even  though  the  tumor  progresses  and  metastasizes  in 
conditioned  hosts  until  death  ensues.  Since  all  cells  have  regulatory 
forces,  it  is  highly  probable  that  some  cells,  other  than  those  of  the  endo- 
crine organs,  have  a  conditioned  phase  preceding  the  autonomous  one. 
Furthermore,  it  is  of  importance  from  both  the  theoretical  and  the  prac- 
tical standpoint  that  following  or  at  the  time  of  acquisition  of  autonomy, 
the  tumor  cells  may  acquire  some  degree  of  dependency  on  the  physiologic 
agent  which  originally  restrained  them. 

The  pituitary  tumorigenesis  of  thyrotrophs  furnishes  excellent  material 
to  study  the  various  factors  involved  in  the  transformation  of  a  normal 
into  a  highly  autonomous  cancer  cell. 

References 

(1)   Purves,  H.  D.,  and  Griesbach,  W.  E.:  Studies  on  experimental  goitre.  VII. 

Thyroid  carcinomata  in  rats  treated  with  thiourea.     Brit.  J.  Exper.  Path.  27: 

294-297,  1946. 
{2)   Bielschowsky,  F.,   Griesbach,   W.  E.,  Hall,   W.  H.,  Kennedy,  T.  H.,  and 

Purves,    H.    D.:  Studies   on   experimental   goitre:  The   transplantability   of 

experimental  thyroid  tumours  of  the  rat.     Brit.  J.  Cancer  3:541-546,  1949. 

(3)  Morris,  H.  P.,  and  Green,  C.  D. :  The  role  of  thiouracil  in  the  induction,  growth, 

and  transplantability  of  mouse  thyroid  tumors.     Science  114:  44-46,  1951. 

(4)  Schlumberger,  L.  :  Personal  communication. 

(5)  Bielschowsky,  F.:  Chronic  iodine  deficiency  as  cause  of  neoplasia  in  thyroid 

and  pituitary  of  aged  rats.     Brit.  J.  Cancer  7:  203-213,  1953. 

(6)  Morris,  H.  P.,  Dalton,  A.  J.,  and  Green,  C.  D.:  Malignant  thyroid  tumors 

occurring  in  the  mouse  after  prolonged  hormonal  imbalance  during  the  inges- 
tion of  thiouracil.     J.  Clin.  Endocrinol.  11:  1281-1295,  1951. 

Journal    of    the   National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  691 

(7)  Furth,  J. :  Morphologic  changes  associated  with  thyrotrophin-secreting  pituitary 

tumors.     Am.  J.  Path.  30:  421-463,  1954. 

(8)  Gorbman,  A.:  Tumorous  growths  in  the  pituitary  and  trachea  following  radio- 

toxic  dosages  of  I131.     Proc.  Soc.  Exper.  Biol.  &  Med.  71:  237-240,  1949. 

(9)  Gorbman,  A.,  and  Edelmann,  A.:  Role  of  ionizing  radiation  in  eliciting  tumors 

of  the  pituitary  gland  in  mice.     Proc.  Soc.  Exper.  Biol.  &  Med.  81:  348-350, 
1952. 

(10)  Dent,  J.  N.,  Gadsden,  E.  L.,  and  Furth,  J.:  On  the  relation  between  thyroid 

depression  and  pituitary  tumor  induction  in  mice.     Cancer  Res.     In  press. 

(11)  Furth,  J.,  Dent,  J.  N.,  Burnett,  W.  T.,  Jr.,  and  Gadsden,  E.  L.:   The  mecha- 

nism of  induction  and  characteristics  of  pituitary  tumors  induced  by  thyroidec- 
tomy.    J.  Clin.  Endocrinol.     In  press. 

(12)  Moore,  G.  E.,  Brackney,  E.  L.,  and  Bock,  F.  G.:  Production  of  pituitary  tumors 

in   mice  by   chronic  administration  of  a  thiouracil   derivative.     Proc.   Soc. 
Exper.  Biol.  &  Med.  82:  643-645,  1953. 

(13)  Furth,  J.:  Conditioned  and  autonomous  neoplasms:  A  review.     Cancer  Res. 

13:  477-492,  1953. 

(14)  Goldberg,  R.  C,  and  Chaikoff,  I.  L.:  Development  of  thyroid  neoplasms  in 

the  rat  following  a  single  injection  of  radioactive  iodine.     Proc.  Soc.  Exper. 
Biol.  &  Med.  76:  563-566,  1951. 

(15)  Gorbman,  A.:  Factors  influencing  development  of  hypophyseal  tumors  in  mice 

after  treatment  with  radioactive  iodine.     Proc.  Soc.  Exper.  Biol.  &  Med.  80: 
538-540,  1952. 

(16)  Furth,  J.,  Gadsden,  E.  L.,  and  Upton,  A.  C:  ACTH  secreting  transplantable 

pituitary  tumors.     Proc.  Soc.  Exper.  Biol.  &  Med.  84:  253-254,  1953. 

(17)  Furth,  J.,  and  Gadsden,  E.  L.:  To  be  published. 

(18)  Gardner,  W.  U.,  Pfeiffer,  C.  A.,  Trentin,  J.  J.,  and  Wolstenholme,  J.  T.: 

Hormonal  factors  in  experimental  carcinogenesis.     In  The  Physiopathology 
of  Cancer  (Homburger,  F.,  and  Fishman,  W.  H.,  eds.).      New  York,  Hoeber, 
1953,  p.  256. 

(19)  Dunning,  W.  F.,  Curtis,  M.  R.,  and  Segaloff,  A.:  Strain  differences  in  response 

to  diethylstilbesterol  and  the  induction  of  mammary  gland  and  bladder  cancer 
in  the  rat.     Cancer  Res.  7:  511-521,  1947. 

(20)  Gardner,  W.  U. :  Studies  on  ovarian  and  pituitary  tumorigenesis.     J.  Nat.  Can- 

cer Inst.  15:  693-709,  1954. 

(21)  Dickie,  M.  M.,  and  Woolley,  G.  W.:  Spontaneous  basophilic  pituitary  tumors 

of  the  pituitary  glands  in  gonadectomized  mice.     Cancer  Res.  9:  372-384,  1949. 


Vol.    15,   No.    3,    December    1954 


Studies  on  Ovarian  and  Pituitary  Tu- 
morigenesis  lf  2 


W.  U.  Gardner,  Department  of  Anatomy,  Yale 
University  School  of  Medicine,  New  Haven,  Conn. 


Almost  21  years  ago  I  was  first  introduced  to  an  export  of  Maine,  the 
inbred  mouse.  After  he  had  moved  to  Connecticut,  Dr.  L.  C.  Strong 
introduced  me  to  about  2,000  of  these  mice  in  August  1933.  He  extolled 
their  virtues;  the  unique  value  of  having  many  almost  identical  animals 
or,  considering  different  strains,  many  animals  uniformly  unalike,  with 
which  to  perform  experiments. 

Mice  of  some  of  the  strains  had,  at  that  time,  known  differences  in 
susceptibility  to  mammary  cancer  so  the  comparative  mammary  develop- 
ment and  structure  were  first  studied.  It  was  during  the  course  of  these 
studies  that  a  most  unusual  mouse  was  found.  One  of  the  last  two  mice 
of  Dr.  Strong's  EI  strain  had  multiple  mammary  tumors,  lactating  mam- 
mary glands,  a  cystic  and  hyperplastic  endometrium,  bilateral  granulosa- 
cell  tumors,  nodular,  hyperplastic  adrenal  cortices,  and  a  chromophobic 
adenoma  of  the  pituitary  gland  (1).  It  was  really  a  "tumor"  mouse.  It 
started  a  long  series  of  investigations  on  tumors  of  the  ovaries  and  the 
pituitary  glands.  It  was  to  an  experimental  endocrinologist  interested 
in  tumors  what  the  free  martin  was  to  those  interested  in  the  determina- 
tion of  sexual  differentiation. 

For  a  number  of  years  investigations  were  undertaken  in  which  attempts 
were  made  to  reproduce  tumors  of  these  types — not  without  considerable 
success.  Multiple  tumors  could  be  obtained  in  mice  by  suitable  combina- 
tions of  hormonal  and  genetic  influences,  but  we  were  never  able  to  obtain 
the  entire  combination  of  tumors  noted  in  the  one  mouse  mentioned  above. 
Attempts  to  reproduce  by  more  controlled  experimentation  that  which 
can  occur  spontaneously  has  not  been  entirely  successful. 

We  assumed,  at  that  time,  that  the  tumors  resulted  from  an  imbalance 
of  hormones  and  gradually  devised  an  outline  for  study  of  qualitatively 
and  quantitatively  controlled  imbalances  (2).  1)  Modification  or  differ- 
ences of  rate  of  hormone  production;  2)  modifications  or  differences  of 
rates  of  hormone  inactivation,  excretion  or  utilization;  3)  modification  or 


1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  29, 1954. 

3  These  studies  have  been  reviewed,  with  more  adequate  credit  to  other  investigators  who  have  contributed  to 
this  field,  in  the  general  references  given  at  the  end  of  the  paper.  The  original  investigative  work  reported  here 
has  been  supported  by  grants  from  the  Jane  Coffin  Childs^Fund  for|Medical  Research,  the  National  Cancer 
Institute  of  the  National  Institutes  of  Health,  U.  S.  Public  Health  Service,  and  the  Anna  Fuller  Fund. 

693 

Journal    of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


694  proceedings:  symposium  on  25  years  of 

differences  of  the  sensitivity  of  end  organs,  or  4)  differences  of  the  quality 
of  hormones  produced  may  result  in  hormonal  imbalances  or  differences 
of  an  endocrine  nature  among  mice  of  diverse  strains.  These  were  not 
fictitious  possibilities;  they  all  had  been  observed,  by  interpretation  at 
least,  although  not  necessarily  all  in  any  one  animal  or  species.  In  part, 
hormonal  imbalances  have  been  reproduced  in  two  ways  under  somewhat 
carefully  controlled  conditions:  1)  The  administration  of  specific  exogen- 
ous hormones,  or  hormone-like  substances,  can  alter  or  augment  rates  or 
periodicities  of  end-organ  response  or  of  hormone  production;  and  2)  ef- 
fective production  or  destruction  of  intrinsic  hormones  may  be  modified 
through  operative  procedures. 

The  above  four  major  variables  can  in  turn  be  influenced  in  some  in- 
stances by  a)  genetic  differences,  b)  nutritional  influences,  c)  ontogenic 
influences,  d)  production  of  antagonistic  or  augmenting  substances,  e)  dis- 
ease, f)  etc.  The  total  number  of  variables  is  enormous;  their  analysis  is 
difficult,  slow,  and  tedious.  The  boundaries  between  some  of  the  quali- 
ties mentioned  above  are  difficult  to  define  sharply.  What  is  interpreted 
to  be  an  augmented  rate  of  hormone  production  in  reality  may  be  merely 
an  augmented  or  unusually  high  sensitivity  of  the  end  organ  used  to  esti- 
mate the  level  of  hormone  activity.  For  example,  Dr.  Trentin  (3)  found 
that  the  vaginal  epithelium  of  strain  C57  mice  was  about  5  times  as 
sensitive  to  injected  estrogens  as  was  the  vaginal  epithelium  of  strain  A 
mice;  mice  of  other  strains  and  hybrids  revealed  intermediate  sensitivities. 
Also,  the  mammary  glands  of  mice  of  some  strains  were  more  sensitive  to 
stimulation  by  estrogens  than  were  those  of  other  strains  (4).  In  these 
instances,  the  end  organ  itself  seemed  more  sensitive  in  mice  of  some  strains 
than  others,  but  is  this  true?  May  not  mice  of  some  strains  be  inactiva- 
ting estrogens  more  rapidly  or  counteracting  the  effects  of  these  estrogens? 
What  at  first  seems  to  be  a  simple  difference  in  sensitivity  may  not  be. 

Strain  C57BL  mice  in  our  laboratory  acquire  pituitary  tumors  when  sub- 
jected  to  prolonged  treatment  with  estrogenic  hormones,  at  least  estrogens 
such  as  estrone  and  estradiol  (and  its  esters)  and  stilbestrol  (5).  Mice 
of  only  one  other  inbred  strain  in  our  laboratory  frequently  show  such 
tumors  when  similarly  treated.  The  pituitary  glands  of  all  mice  undergo 
some  hypertrophy  when  estrogens  are  injected.  Adult  female  mice  have 
larger  pituitary  glands  than  males,  indicating  a  stimulating  effect  of  in- 
trinsic hormones.  The  differences  in  size  are  slight,  the  hypertrophic 
glands  of  estrogen-treated  mice  of  most  strains  rarely  exceeding  4  mg. 
or  approximately  twice  the  size  of  the  control  glands.  The  tumors  may 
attain  very  large  size,  are  localized  or  nodular  in  origin,  appear  to  arise  in 
the  hypertrophic  glands. 

The  tendency  for  mice  of  strain  C57BL  to  acquire  pituitary  adenomas 
when  exposed  to  extrinsic  estrogens  is  transmitted  to  their  hybrid  des- 
cendents  (6).  From  75  to  83  percent  of  the  estrogen-treated  (CBA  X 
C57BL)F!  hybrids  had,  at  death,  pituitary  glands  exceeding  12  mg. 
Smaller  glands  also  occasionally  showed  nodular  enlargements  but  since 
not  all  of  them  were  studied  histologically,  for  various  reasons,  the  term 

Journal    of    the    National    Cancer    Institute 


PEOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


695 


tumor  was  applied  only  to  the  larger  glands  that  almost  uniformly  con- 
tained extensive  areas  of,  or  were  composed  entirely  of,  abnormal,  non- 
granular chromophobic  cells  of  a  type  not  found  in  the  normal  gland. 
Both  male  and  female  mice  transmitted  the  tendency  for  pituitary  tumors 
to  appear  in  their  estrogen-treated  hybrids.  The  tumors  appeared  earlier 
in  males  than  in  females.  Some  attained  very  large  size,  almost  300 
mg.,  and  distorted  the  shape  of  their  hosts'  calvaria.  Few  tumors 
occurred  in  mice  treated  for  less  than  one  year.  Other  hybrid  mice 
(A  X  C57BL  and  C3H  X  C57BL)  also  acquired  such  tumors  (tables  1, 

2,  and  3).  The  incidence  of  pituitary  tumors  was  much  lower,  only  21 
percent,  when  hybrids  (CBA  X  C57BL)  were  backcrossed  (text-fig.  1, 
table  2)  to  mice  of  the  CBA  strain,  and  was  63  percent  when  backcrossed 
to  mice  of  the  C57BL  strain  (7).  The  tumors  also  occurred  approxi- 
mately 100  days  earlier  in  mice  of  the  latter  group  (table  2,  text-figs.  2, 

3,  and  4).  If  adequate  correction  for  ages  of  survival  had  been  made, 
mice  of  the  latter  group  would  have  had  a  relatively  much  higher  incidence 
than  mice  that  carried  less  C57BL  chromatin.     The  transmission  of  the 


Table 

1 — Pituitary  tumors  among  hybrid  and  backcross   mice 
estrogens  for  prolonged  periods 

that   received  different 

Continuous  treatment  until  death 

Treatment  stopped  before  autopsy- 

Group 

of 
mice* 

Treat- 
ment! 

Num- 
ber of 
mice 

Duration 
of  treat- 
ment 

(days) 

Size  of  pi- 
tuitary 

Num- 
ber 
of 
mice 

Age  of 

mice  at 

death 

(days) 

Period  of 
inter- 
rupted 
treat- 
ment 
(days) 

Size  of  pitu- 
itary 
(mg.) 

Cd 

SS 
DPB 

ES 

DPB 

SS 
ES 

B  16.6 
B  25 

SS 
ES 

B  16.6 
B  25 

B  25 

B  16.6 
B25 

3 
5 
1 

4 
1 
1 

9 
10 

8 
3 

2 
2 

339-458 

379-634 

20.  3 

312-482 
518 

438 

359-596 
372-602 
410-639 
431-563 

503-553 
390-394 

16.  5-120.  5 

12.  0-  42.  3 

20.  3 

20.  0-  79.  8 
59.8 
80.0 

9.  0-118.  5 
15.  0-117.  5 

21.  0-138.  0 

27.  3-281.  5 

28.  5-  44.  3 

22.  3-  47.  3 

8 

462-703 

32-130 

16.  5-111.  3 

cc2 

3 

395-555 

41-  49 

47.  5-215.  0 

HCi 

4 
1 
3 

462-703 
534 

529-668 

32-  61 

61 
62-138 

16.5-111.3 

15.0 
51.  5-  53.  5 

cc3 

2 

561-581 

90-136 

17.  3-  53.  3 

A72 

1 

568 

24 

18.  3 

cc5 

10 
3 

311-482 
263-403 

14.  0-132.  5 
12.  5-  44.  0 

*CCi(C57BL9  XCBAc?);CC2(CBA$  X  C57BW);  HCi  (C57BL9  X  C3Hd");  CC3  ( [CBA  9  X  C57BLc?] 
X  CBAo");  A72  (C57BLCJ1  X  A 9);  CC5  ( [CBA 9  X  C57BW1  X  C57BLcf). 
t  SS— 25  mg.  stilbestrol  weekly. 

DPB — 25  fig.  estradiol  dipropionate  in  sesame  oil  weekly. 

ES— pellets  of  estrone  subcutaneously. 

B  16.6 — 16.6  Mg-  estradiol  benzoate  in  sesame  oil  weekly. 

B  25—25  Mg-  estradiol  benzoate  in  sesame  oil  weekly. 


Vol.    15,    No.    3,   December    1954 


696 


PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


tendency  for  pituitary  tumors  was  compatible  with  a  dominant  genetic 
factor  or  factors  as  regulating  influences. 

Table  2. — The  incidence  of  pituitary  tumors  among  male  estrogen-treated  hybrid  and 

backcross  mice 


With 

pitui- 

Age  with  tu- 

Average 

Num- 

tary tumors 

mor 

age  at 

Group 

Origin  of  group 

Sex 

ber 
of 

death — 

nontu- 

mice 

Num- 

Per- 

Average 

Range 

morous 

ber 

cent 

(days) 

(days) 

(days) 

CCi* 

C57BL9    X  CBAd" 

M 

23 

19 

83 

513 

326-671 

320 

cc2* 

CBA?    X  C57BLcf 

M 

24 

18 

75 

480 

380-543 

360 

cc3 

(CBA  9    X  C57BLc?) 

9    X  CBAcT 

M 

38 

8 

21 

517 

390-592 

372 

cc4 

(C57BL9  X  CBAd") 

9   X  CBAcf 

M 

34 

7 

21 

525 

324-647 

429 

cc5 

(CBA  9    X  C57BLc?) 

9   X  C57BLc?) 

M 

41 

26 

63 

419 

263-577 

300 

*  Data  published  in  Cancer  Res.  1:  345, 1941. 

Table  3. —  The  incidence  of  pituitary  tumors  among  estrogen-treated  hybrid  mice  derived 
from  the  C57BL  and  CSH  strains 


Group 

Origin  of  group 

Sex 

Num- 
ber 
of 
mice 

With  pitui- 
tary tumors 

Age  with  tu- 
mor 

Average 

age  at 

death — 

Num- 
ber 

Per- 
cent 

Average 
(days) 

Range 
(days) 

nontu- 
morous 
(days) 

HCi 
HC2 

C57BL  9  X  C3H  cf 
C3H9    X  C57BW 
(B)*  C3H9    X 
C57BLd* 

M 
M 

M 

33 
38 

22 

22 
0 

11 

67.  1 
0 

50. 

514.  2 

372-703 

397.2 
332 

HC3 

508 

360-577 

464 

C3H— without  the  mammary  tumor  agent. 


P        C57£   X    CBA(?       CBA$XC57<? 


BC 


Text-figure  1. — Scheme  of  one  group  of  reciprocal  hybrid  and  backcrossed  mice. 
The  incidence  of  pituitary  gland  tumors  among  the  estrogen-treated  male  mice  of 
these  groups  is  given  in  table  2.  No  pituitary  gland  tumors  were  noted  in  the  un- 
treated controls. 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


697 


100 


90 


80 


70 

l 
fc60 


$50 


^40 


20 


!0 


CC 
50mice 

,3-(CBA? 
-16.6  or 

xc574>  X 

25.0.ug.  e 
!2$-o 

CBA<f* 
itradiol  b 

1 

enzoate  w 

1 

sekly 

38<£-  • 

• 

• 
• 

• 

• 

< 
• 

* 

o 

* 

•• 

• 

o        • 
•  •o 

•••o 

>  • 
•••  o    • 

•o       •       • 
»«e     o     • 

•    •       • 

• 

« 

300 


350  400  450 

Age  in  days  at  death 


500 


550 


600 


Text-figure  2. — Age  and  weight  distribution  of  pituitary  gland  or  tumors  of  back- 
crossed  mice.     Similar  data  for  the  hybrids  have  been  published  (6) . 


Hybrids  derived  from  reciprocal  crosses  of  mice  strains  C57BL  and  C3H, 
when  given  estrogens,  had  pituitary  tumors  when  killed  at  average  ages 
in  excess  of  500  days  (table  3,  text-figs.  5  and  6).  The  apparent  maternal 
influence  of  strain  C3H  was  indirect  rather  than  direct;  many  of  the  mice 
with  the  mammary  tumor  agent  died  early  with  mammary  tumors  and 
hence  did  not  live  long  enough  to  acquire  pituitary  tumors  (table  3). 

Pituitary  tumors,  according  to  the  definition  given  above,  were  observed 
in  none  of  the  untreated  mice  of  these  strains  or  hybrid  groups. 

The  pituitary  tumors  are  transplantable;  about  one  of  five  has  grown 
when  transplanted  subcutaneously  into  estrogen-treated  mice;  they  have 
not  grown  in  untreated  mice.  Even  those  that  did  grow  have  not  grown 
in  all  of  the  estrogen- treated  mice  into  which  they  have  been  transplanted. 
Because  a  small  number  of  hosts  were  used  it  is  difficult  to  compare  the 
relative  transplantability  of  these  tumors  with  those  of  other  endocrine 


Vol.    15,   No.   3,   December    1954 


698 


PROCEEDINGS:   SYMPOSIUM  ON  25  YEARS  OF 


100 


90 


80 


70 


,1  60 


*  40 


30 


20 


10 


«, 

<C57?X 
43mic< 

CBAd*)  ^ 
-l6.6or2 

XCBA^ 
5.0-u.g.esl 

rodiol  ber 

• 
izoate  weekly 

9?-o 
34<f-. 

• 

• 

• 

• 

• 

• 

•• 

a             o 

• 

o    _  o  •• 
1     ••  • 

•   •• 

oo           • 

•               • 
• 

• 
o 

•         •• 

•  •     • 

3 

450 


500 


550 


600 


300  350  400 

Age  tn  days  at  death 

Text-figure  3. — Age  and  weight  distribution  of  pituitary  glands  or  tumors  of  back- 
crossed  mice.     Similar  data  for  the  hybrids  have  been  published  (6). 

glands.  The  transplanted  tumors  attained  detectable  proportions  only 
after  prolonged  periods,  usually  one  year  or  more,  and  almost  always  at  a 
time  when  the  host  had  a  pituitary  tumor  developing  in  its  own  calvarium. 
After  their  appearance  they  grew  slowly  and  progressively  to  attain  diam- 
eters in  excess  of  2  cm.  No  tumor  could  be  carried  for  more  than  four 
transfer  generations.  The  very  delayed  initiation  of  palpable  growths  in 
estrogen-treated  animals  made  it  impossible  to  carry  large  numbers  of 
hosts. 

The  tumors  were  not  reversible  in  the  animals  of  origin  insofar  as  could 
be  determined,  that  is,  they  continued  to  grow  when  estrogen- treatment 
was  stopped.  Although  it  was  impossible  to  ascertain  the  sizes  of  the 
tumors,  except  by  changes  in  the  shape  of  the  calvaria,  after  the  cessation 
of  hormone  treatment,  tumors  removed  for  up  to  140  days  after  the  cessa- 
tion of  estrogen  treatment  showed  no  evidence  of  regression  and  had 
apparently  continued  to  grow  (table  1;  text-figs.  7  and  8). 

Journal    of    the    .National    Cancer    Institute 


: 


PKOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


699 


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Text-figure  4. — Age  and  weight  distribution  of  pituitary  gland  or  tumors  of  back- 
crossed  mice.     Similar  data  for  the  hybrids  have  been  published  (6). 

At  this  time  it  is  well  to  emphasize  the  possibility  that  the  environment 
promoting  tumorigenesis  may  or  may  not  be  the  same  as  that  necessary 
for  tumor  growth.  Once,  however,  the  environment  has  been  established 
to  promote  growth  of  a  dependent  tumor,  and  it  seems  that  temporal 
factors  are  of  importance,  it  is  not  reversible.  It  might  be  assumed  that 
the  foci  of  tumorigenesis  occur  spontaneously  with  advancing  age  in 
animals  with  the  proper  genetic  constitution  but  fail  to  grow  because  the 
hormonal  environment  is  inadequate.     If  this  is  true  then  tumors  should 


Vol.    15,    No.    3,    December    1954 


700 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


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Text-figure  5. — The  age  and  the  Weight  distribution  of  pituitary-gland  tumors  in 
hybrid  mice  (C57BL  $   X  C3H  <?). 

appear  more  quickly  when  old  mice  are  given  exogenous  estrogens.  This 
has  not  been  studied  adequately.  However,  if  such  an  assumption  were 
true  it  would  be  even  more  difficult  to  explain  the  prolonged  quiescence  of 
the  transplants  of  tumors  in  estrogen- treated  mice.  The  growth  of  trans- 
plants of  tumors  and  of  the  original  neoplastic  foci  tend  to  parallel  one 
another  indicating  that  the  growth-promoting  environment  is  slowly 
created;    that    it    depends    on    prolonged     exposure    to     estrogens. 

What  is  responsible  for  the  origin  of  these  tumors?  Actually  we  do 
not  know.  We  do  know  that  estrogens  do  produce  changes  in  the  func- 
tion of  the  anterior  pituitary  by  reducing  its  gonado  trophic  content  and 
growth-hormone  content.  It  is  possible  that  estrogens  prevent  the 
synthesis  of  certain  pituitary  hormones  and  the  pituitary  gland  attempts 
to  overcome  this  deficiency  by  adding  new  cells.  After  prolonged  periods 
some  of  the  cells  among  this  population  may  lose  all  regulation  by  the 
normal  restricting  influences  and  nodular  overgrowths  or  adenomas  result. 

Dr.  Furth  (8)  has  indicated  that  somewhat  analagous  circumstances 
may  predispose  to  pituitary  tumors  in  thyroid-deficient  mice  and  has 


Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


701 


1 
HC3 

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Text-figure  6. — The  age  and  weight  distribution  of  pituitary-gland  tumors  in  hybrid 
mice  [C3H  9  (without  the  mammary  tumor  agent)  X  C57BL  cF].  Hybrids  born 
of  C3H  mice  with  the  mammary  tumor  agent  did  not  live  long  enough  to  acquire 
pituitary  gland  tumors — they  died  with  mammary  tumors  when  treated  with 
estrogens. 


described  the  "feed-back"  inter-regulation  of  thyroid  and  pituitary  func- 
tion.    Essentially  this  is  the  same  hypothesis  as  that  stated  above. 

Granulosa-cell  tumors  occasionally  occur  in  untreated  mice  that  are 
not  as  involved  in  a  polyglandular  syndrome  as  indicated  above;  their 
incidence  is  low  in  mice  of  most  strains.  They  were  first  observed  at  a 
relatively  high  incidence  by  Dr.  Furth  (9)  in  mice  subjected  to  roentgen 
irradiation.  Roentgen  irradiation  destroys  the  ova  and  reduces  or  modi- 
fies the  endocrine  function  of  the  ovary.  Subsequently  ingrowths  occur 
from  the  germinal  epithelium  that  can  be  associated  with  renewed  evi- 
dence of  some  endocrine  activity  (10).  Granulosa-cell  tumors  first  ap- 
peared in  irradiated  mice  many  months  after  the  ovaries  have  been 
irradiated. 

Granulosa-cell  tumors  first  appeared  frequently  among  mice  in  our 
laboratory  after  ovaries  were  transplanted  intrasplenically  into  gonadecto- 
mized  mice  (11-13).  Dr.  M.  H.  Li,  who  had  spent  some  time  trying  to 
induce  ovarian  tumors  in  fish,  first  undertook  these  experiments  and  his 


Vol.    15,   No.    3,   December    1954 


702  proceedings:  symposium  on  25  years  of 

Pituitary   tumors   in    estrogen  -treated  .hybrid     mice     (C579     *CBA<T) 


33.3 

<l2 


35.8 


14.6 


42  3 

fc  Days  of   estrogen    treatment 


20.3 

l65  Days    subsequent     to    estrogen 

i  90.3  treatment 

————————  120.5  Numbers    represent    size    of    tumors 

ih    mg. 


—  29.3 

19.8 

7.8 


22.5 

31.0 

60.8 

18.8 


70.0 


300  400  500  600  700 

Age       in       days 

Text-figure  7. — Weights  of  some  pituitary  glands  of  estrogen-treated  hybrid  mice 
killed  during  the  course  of  prolonged  treatment  with  estrogens  or  subsequent  to 
cessation  of  estrogen  treatment  for  different  times.  Evidence  of  regression  sub- 
sequent to  discontinuance  of  treatment  was  lacking. 


observations  were  analogous  to  those  previously  observed  in  experiments 
conducted  with  rats  (14) 

We  assumed  that  these  tumors  were  caused  by  an  endocrine  imbalance. 
About  25  years  ago  it  was  shown  that  the  level  of  pituitary  gonadotrophin 
increases  after  castration;  urinary  gonadotrophins  increase  after  the  meno- 
pause. Zondek  (15)  demonstrated  that  hepatic  tissue  of  rats  destroys 
estrogens.  Subsequently,  many  investigators  have  been  concerned  with 
quantitative  and  mechanistic  aspects  of  hepatic  inactivation  of  estrogens. 
Hepatic  tissue  from  mice  is  similarly  active  in  reducing  the  biological 
activity  of  estrogens  (16).  Because  the  spleen  drains  its  blood  into  the 
liver,  an  ovary  located  at  this  site  would  be  unable  to  have  its  hormones 
effectively  by-pass  the  liver  to  reach  the  systemic  circulation.  Under 
such  conditions  the  ovary  would  exist  in  a  physiologically  castrated  host. 
Such  ovaries  are  stimulated  excessively.  By  use  of  parabiosis  it  has  been 
demonstrated  that  the  intrasplenic  ovarian  graft  does  not  prevent  aug- 
mented levels  of  circulating  gonadotrophins  in  the  host  (17).  Some  grafts 
and  the  tumors  produced  by  them  are  associated  with  evidence  that  some 
hormone  escapes  hepatic  inactivation.  Absence  of  a  complete  castration 
effect  is  indicated  further  by  the  low  incidence  of  adrenal  tumors  appearing 
in  gonadectomized  mice  with  intrasplenic  ovaries. 

Journal    of    the   National    Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 
Pituitary     tumors    in    estrogen  treated    hybrid   mice     (C  57  ox  C3  Ho*  ) 
22.8 


703 


27.0 


1)8.0 


23.8 


34.0 


72.5 
-  38.3 


29.8 


15.0 


107.0 


45.0 


93.5 


281.5 


76.0 


68.5 


138.0 


Days   of  estrogen 
treatment 


Days  subsequent 

117  5  to     estrogen 
173  treatment 


Numbers  represent 

size     of      tumors 
in    mg. 


51.5 


-    I!!. 3 


118.5 


16.5 


300 


400  500 

Age    in     days 


53.5 


Text-figure  8. — Weights  of  some  pituitary  glands  of  estrogen-treated  hybrid  mice 
killed  during  the  course  of  prolonged  treatment  with  estrogens  or  subsequent  to 
cessation  of  estrogen  treatment  for  different  times.  Evidence  of  regression  sub- 
sequent to  discontinuance  of  treatment  was  lacking. 


Mice  of  all  of  the  strains  that  have  been  studied  adequately  have  ac- 
quired ovarian  tumors  subsequent  to  roentgen  irradiation  or  when  go- 
nadectomized  mice  have  carried  intrasplenic  ovarian  grafts  (12,  13).  In 
some  strains  the  tumor  may  occur  at  earlier  ages  than  in  others  but  our 
data  are  not  adequate  to  demonstrate  this  conclusively.  It  is  the  writer's 
impression  that  mice  of  strain  C57BL  acquire  fewer  ovarian  tumors, 
except  at  great  age,  than  do  mice  of  other  strains  (C3H,  A,  CBA,  BC). 
The  strain  differences  in  the  tendency  to  acquire  ovarian  tumors  are 
not  as  decisive  as  with  tumors  of  some  other  types.  Studies  undertaken 
up  to  this  time  have  indicated  that  tumors  in  hybrids  occur  at  earlier 
ages  than  in  the  parental  strains  (Gardner,  unpublished  data) . 

The  ovarian  tumors  were  ascribed  to  hormonal  imbalances  of  excessive 
gonadotrophin;  excessive  in  amount  and/or  continuity.  The  incidence  of 
ovarian  tumors  in  control  mice  is  very  low  but  they  do  occur.  Dr.  Li 
in  our  laboratory  immediately  set  about  checking  the  hypothesis  of  a 
humoral  etiology  of  these  tumors  (13).  Under  a  variety  of  conditions, 
ovarian  tumors  did  not  occur  in  intrasplenic  grafts  (text-fig.  9).  Ovarian 
grafts  in  the  spleens  of  intact  or  of  unilaterally  gonadectomized  mice  did 


Vol.    15,   No.    3,    December    1954 


T 


704 


PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


not  become  tumorous.  Intrasplenic  grafts  in  castrated  mice  that  were 
intentionally  or  accidentally  adherent  to  the  body  wall  rarely  became 
tumorous.  The  injection  of  exogenous  estrogens  or  androgens  prevented 
tumors  in  intrasplenic  ovarian  grafts  (text-fig.  10).  Ovarian  grafts  placed 
subcutaneously  or  intratesticularly  seldom  became  tumorous.  All  of 
these  observations  indicate  that  gonadal  hormones  prevented  granulosa- 

WHEN  TUMORS  DO  NOT   APPEAR  IN   GRAFTEO  OVARIES 
I.  ADHESIONS    OF   INTRASPLENIC  GRAFT  TO  BODY  WALL 


2.  OVARIES  GRAFTED  SUBCUTANEOUSLY  OR  INTRAMUSCULARLY 


3.  INTRASPLENIC  OVARIAN  GRAFTS  IN  MICE  WITH  TESTES  OR  OVARIES 


Text-figure  9. — Examples  of  methods  of  ovarian  grafting  in  which  granulosa-cell 
tumors  do  not  appear. 


INHIBITION  OF  OVARIAN   TUMORS    IN    INTRASPLENIC    GRAFTS 
BY   EXOGENOUS   GONADAL  HORMONES 


ESTRADIOL 


TESTOSTERONE 


Text-figure  10. — Ovarian  tumors  are  inhibited  in  intrasplenic  grafts  in  castrated 
mice  by  the  injection  of  sex  hormones. 


Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  705 

cell  tumors  and  were  compatible  with  the  interpretation  of  a  tumorigenic 
effect  of  pituitary  gonadotrophins  (2). 

Two  additional  types  of  experiments  are  highly  desirable.  First,  it 
would  be  desirable  to  determine  whether  hypophysectomy  would  prevent 
the  development  of  ovarian  tumors,  and  second,  to  determine  whether 
purified  gonadotrophic  hormones  would  induce  ovarian  tumors  in  hypo- 
physectomized,  or  for  that  matter,  intact  hosts.  Attempts  in  our  labora- 
tory to  conduct  really  convincing  experiments  of  these  types  have  been 
unsuccessful  up  to  this  time. 

The  studies  on  the  influence  of  hypophysectomy  on  the  late  develop- 
ment of  ovarian  tumors  or  on  ovarian  tumorigenesis  have  been  thwarted, 
in  part  because  it  has  been  difficult  to  keep  older  mice  alive  for  long 
periods  and  in  part  because  it  is  difficult  to  predetermine  tumor  growth 
even  in  intact  animals.  The  natural  history  of  the  granulosa-cell  tumors 
reveals  extreme  variability  in  their  progressive  growth  and  behavior 
(Gardner,  unpublished  data) .  Some  tumors,  after  their  first  appearance, 
remain  small  for  periods  of  300  to  400  days;  some  begin  to  grow  rapidly 
at  the  end  of  a  long  period  of  inactivity;  some  grow  rapidly  immediately 
after  they  have  become  apparent;  and  some  grow  slowly  over  periods  of 
several  hundred  days.  The  variable  progression  of  the  tumors  in  mice 
with  intact  pituitary  glands  cannot  be  explained  adequately  at  this  time 
and  makes  it  almost  impossible  to  determine  whether  hypophysectomy 
would  affect  the  growth  of  any  one  tumor  or  of  a  small  group  of  tumors. 

What  changes  occur  in  the  intrasplenic  ovarian  grafts  and  are  these 
changes  different  from  those  in  grafts  placed  in  other  areas?  In  all  ovarian 
grafts  the  numbers  of  ova  are  depleted  more  rapidly  than  in  the  intact 
organs.  Grafts  that  persist  for  prolonged  periods  have  bursa-like  clefts 
lined  by  germinal  epithelium  investing  variable  portions  of  their  surfaces. 
Ova  and  follicles  may  be  present  for  600  days  in  grafts  placed  in  sub- 
cutaneous or  intramuscular  or  intratesticular  sites  but  these  are  rare. 
Ova  and  follicles  are  rarely  found  in  intrasplenic  grafts  in  gonadectomized 
hosts  after  200  days — they  show  a  precocious  ' 'senility.' '  We  might 
assume  that  it  is  either  the  precocious  aging  that  is  responsible  for  ovarian 
tumorigenesis  or  that  it  is  the  prolonged  exposure  to  augmented  or  un- 
interupted  gonadotrophins. 

Mice  bearing  intrasplenic  grafts  and  made  hypothyroid  by  feeding 
thouracil,  or  hyperthyroid  by  feeding  dessicated  thyroid,  showed  a  low 
incidence  of  tumors  in  intrasplenic  grafts.  Inanition  also  reduced  the 
incidence  of  ovarian  tumors  in  intrasplenic  grafts,  but  they  quickly 
appeared  when  ad  libitum  feeding  was  reinstituted  (19). 

Dr.  Fern  Smith  in  our  laboratory  a  few  years  ago  demonstrated  that  the 
amount  of  gonadotrophin  in  the  pituitary  glands  of  intact  and  castrated 
mice  decreased  with  really  advanced  age  (20).  Old  mice  have  atrophic 
ovaries.  It  is  possible  that  few  ovarian  tumors  occur  in  old  mice  because 
the  ovaries  and  pituitary  gland  decrease  in  function  simultaneoulsy,  or 
nearly  so.  Old  ovaries,  however,  can  be  transplanted  into  the  spleens  of 
young  gonadectomized  hosts.    They  do  not  become  tumorous  more  rapidly 

Vol.    15,  No.   3,  December  19S4 

316263—54 30 


706  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

than  do  ovaries  of  young  mice  (21,  22).  Irrespective  of  the  age  of  the 
ovary,  it  seems  that  the  duration  of  exposure  to  a  modified  hormonal 
environment  is  more  important  than  the  persistence  of  a  "senile"  ovary. 
The  low  incidence  of  "spontaneous"  granulosa-cell  tumors  is  probably  due 
to  the  failure  of  such  ovaries  to  persist  for  long  periods  in  a  relatively 
anovular  state  and  to  the  decline  of  pituitary  gonadotrophic  function  with 
advancing  age.  The  combination  of  precociously  senile  ovaries  in  a  mouse 
with  prolonged  effective  pituitary  function  should  promote  ovarian  tumori- 
genesis,  unless  adrenal-cortical  changes  supervene. 

When  ovaries  are  depleted  and  follicles  fail  to  form,  ingrowths  occur 
from  the  germinal  epithelium  and  extend  into  the  ovarian  stroma.  These 
tubular  or  cleftlike  ingrowths  may  extend  throughout  and  greatly  enlarge 
the  ovary.  These  ingrowths  may  form  tumors  and  are  designated  tubular 
adenomas  when  they  are  large  and  hyperplastic.  Granulosa-cell  tumors  of 
different  histological  types  also  seem  to  arise  from  the  tubular  or  cleftlike 
ingrowths  of  germinal  epithelium.  Some  of  the  areas  of  granulosa  cells 
may  store  increased  amounts  of  fatty  material  and  become  luteinized — 
luteinized  granulosa-cell  tumors.  Some  of  the  granulosa-cell  tumors  con- 
sist of  follicular  structures,  some  of  coarse  or  fine  trabecular  structures 
and  some  of  cells  that  are  arranged  in  masses  showing  no  definite  structure. 
The  histogenesis  of  the  ovarian  tumors  of  the  several  types  seems  thus  to 
be  quite  similar. 

Roentgen  rays  destroy  the  ova  of  mice  (10)  and  mice  so  treated  acquire 
ovarian  tumors  (9).  Germinal  epithelial  ingrowths  again  occur  in  these 
anovular  ovaries,  ovarian  hormonal  function  is  impaired  and  pituitary 
gonadotrophins  increase  (9,  24).  Estrogens  prevent  tumorigenesis  in  such 
ovaries  but  testosterone  propionate  (23)  does  not,  although  administered 
in  amounts  that  will  prevent  augmented  excretion  of  gonadotrophin  (24) . 
Irradiated  ovaries  also  become  tumorous  when  transplanted  into  testes  or 
when  testes  are  transplanted  to  the  irradiated  female  mouse  (Gardner, 
unpublished  data) .  With  these  exceptions  the  ovarian  tumors  in  irradiated 
mice  and  mice  bearing  intrasplenic  grafts  seem  comparable  and  seem 
to  have  a  similar  hormonal  etiology. 

Ovarian  granulosa-cell  tumors  grow  subsequent  to  transplantation  into 
other  animals  of  the  same  strain  (IS,  25).  All  that  have  been  transplanted 
have  not  grown.  They  grow  slowly  even  when  removed  and  transplanted 
subcutaneously  into  the  donor.  Some  of  the  tumors  apparently  require  a 
specific  hormonal  environment  for  their  rapid  growth  (26).  The  period  of 
dependency  is  not  too  well  known  and  only  now  are  the  histologic  character- 
istics of  dependent  and  autonomous  tumors  being  studied  in  detail.  Histo- 
logic differences  between  the  dependent  and  autonomous  tumors  are  not 
apparent  at  this  time.  The  natural  history  of  the  tumors  seems  most 
significant  prognostically;  tumors  that  are  growing  rapidly,  whether 
arising  in  the  younger  or  the  older  animals  seem  most  predisposed  to  grow 
subsequent  to  transplantation. 

Now  we  may  return  to  the  "tumor"  mouse  mentioned  earlier.  How 
can  we  account  for  the  interesting  coexistence  of  multiple  glandular  tumors 

Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  707 

and  tumors  or  hyperplasia  of  responding  end  organ?  Ovarian  granulosa- 
cell  tumors  may  develop  very  slowly;  some  have  been  followed  after 
original  detection  of  tumorous  masses,  for  almost  400  days.  These  tumors 
produce  estrogens.  Only  recently  have  we  seen  a  pituitary  tumor  in  a 
castrated  male  mouse  (A  X  C57BL)  bearing  a  subcutaneously  trans- 
planted irradiated  ovary.  This  male  had  extremely  well  developed 
mammary  glands  although  no  mammary  cancer.  The  ovarian  graft  had 
enlarged  slowly  during  7  months  to  assume  dimension  slightly  in  excess 
of  a  cubic  centimeter. 

The  695-day-old  " tumor"  mouse  of  the  EI  strain  presumably  acquired 
her  ovarian  tumors  first  by  prolonged  gonadotrophic  stimulation  of  her 
anovular  ovaries.  She  had  her  last  litter  371  days  before  death.  These 
anovular  ovaries  became  tumorous  and  produced  estrogens  which  in  turn 
induced  the  tumor  of  the  pituitary  gland,  the  latter  was  responsible  for  the 
unusual  adrenal-cortical  proliferation  and,  with  the  estrogen,  for  the 
unusual  mammary  development.  The  extent  to  which  genetic  influences 
were  concerned  can  also  only  be  surmised  or  ascertained  by  analogy  as 
this  unusual  mouse  and  her  daughter,  the  last  of  their  line,  were  both 
killed  the  same  day.  In  other  instances,  however,  the  tendency  for 
pituitary  tumors  is  strain-specific  and  presumably  this  mouse  was  from  a 
strain  susceptible  to  such  tumors. 

Summary 

Approximately  19  years  ago  one  mouse  was  found  that  had  multiple 
mammary  adenocarcinomas,  bilateral  granulosa-cell  tumors  and  a  chromo- 
phobe adenoma  of  the  pituitary  gland  together  with  nodular  hyperplastic 
adrenal  cortices  and  a  hyperplastic  endometrium. 

Subsequently  by  proper  combination  of  strains  and  hormones,  it  was 
possible  to  obtain  many  pituitary  tumors  of  large  size.  Estrogen- treated 
mice  of  the  C57BL  strain  and  their  hybrids  consistently  acquired  pituitary 
adenomas.  Prolonged  periods  of  treatment  were  necessary.  The  tumors 
were  not  reversible  in  the  animal  of  origin  and  some  grew  when  trans- 
planted subcutaneously  into  estrogen- treated  mice  of  the  same  strain  but 
the  transplants  usually  grew  only  after  a  prolonged  quiescence. 

Ovarian  granulosa-cell  tumors  arising  in  intrasplenic  ovarian  grafts  in 
castrated  mice  have  appeared  in  mice  of  all  strains  studied  and  are  pre- 
vented when  estrogens  or  androgens  are  injected  or  when  gonadal  hormones 
reach  the  systemic  circulation  in  appreciable  amounts.  Intrinsic  hor- 
mones, presumably  gonadotrophins  in  this  instance,  incite  ovarian  tumors. 

The  several  types  of  tumors  observed  in  the  one  untreated  mouse  can 
now  be  obtained  at  will,  by  proper  combination  of  transmitted  and 
hormonal  influence. 

General  References 

Gardner,  W.  U.,  Pfeiffer,  C.  A.,  Trentin,  J.  J.,  and  Wolstenholme,  J.  T.:  Hor- 
monal factors  in  experimental  carcinogenesis.  In  Physiopathology  of  Cancer. 
(Homburger,  F.  and  Fishman,  W.  H.,  eds.).     New  York,  Hoeber-Harper,  1953. 

Vol.    15,   No.   3,   December    1954 


708  proceedings:  symposium  on  25  years  of 

Gardner,  W.  U. :  Hormonal  aspects  of  experimental  tumorigenesis  (Greenstein,  J.  P., 
and  Haddow,  A.,  eds.).     In  Adv.  Cancer  Res.  1:  173-232,  1953. 

Specific  References 

(1)  Gardner,  W.  U.,  Strong,  L.  C.,  and  Smith,  G.  M.:  An  observation  of  primary- 

tumors  of  the  pituitary,  ovaries  and  mammary  glands  in  a  mouse.     Am.  J. 
Cancer  26:  541-546,  1936. 

(2)  Gardner,    W.    U.:   Hormonal   imbalances   in   tumorigenesis.     Cancer   Res.   8. 

397-411,  1948. 

(3)  Trentin,  J.  J.:  Vaginal  sensitivity  to  estrogen  as  related  to  mammary  tumor 

incidence  in  mice.     Cancer  Res.  10:  580-583,  1950. 

(4)  :  The  effect  of  the  presence  or  absence  of  the  milk  factor  and  of  castration 

on  mammary  response  to  estrogens  in  male  mice  of  strains  of  known  mammary 
tumor  incidence.     Cancer  Res.  11:  286-287,  1951. 

(5)  Gardner,  W.  U.,  and  Strong,  L.  C:  The  strain-limited  development  of  tumors 

of  the  pituitary  gland  in  mice  receiving  estrogens.     Yale  J.  Biol.  &  Med.  12: 
543-548,  1940. 

(6)  Gardner,   W.  U.:  The  effect  of  estrogen  on  the  incidence  of  mammary  and 

pituitary  tumors  in  hybrid  mice.     Cancer  Res.  1:  345-358,  1941. 

(7)  :  Hormones  in  experimental  carcinogenesis.     Acta  Unio  Contra  Cancrum 

6:  124-133,  1948. 

(8)  Furth,  J.:  Thyroid-pituitary  tumorigenesis.     J.  Nat.  Cancer  Inst.  15:  687-691, 

1954. 

(9)  Furth,  J.,  and  Butterworth,  J.  S.:  Neoplastic  diseases  occurring  among  mice 

subjected  to  general  irradiation  with  X-rays.  II.  Ovarian  tumors  and  associated 
lesions.     Am.  J.  Cancer  28:  66-95,  1936. 

(10)  Brambell,  F.  W.  R.,  and  Parkes,  A.  S.:  Changes  in  the  ovary  of  the  mouse  fol- 

lowing exposure  to  X-rays.  III.  Irradiation  of  the  non  parous  adult.     Proc. 
Roy.  Soc,  London,  s.B,  101:  316-328,  1927. 

(11)  Li,  M.  H.,  and  Gardner,  W.  U.:  Tumors  in  intrasplenic  ovarian  transplants  in 

castrated  mice,     Science  105:  13-15,  1947. 

(12)  :  Experimental  studies  on  the  pathogenesis  and  histogenesis  of  ovarian 

tumors  in  mice.     Cancer  Res.  7:  549-566,  1947. 

(IS)  :  Further  studies  on  the  pathogenesis  of  ovarian  tumors  in  mice.     Cancer 

Res.  9:  35-41,  1949. 

(14)  Biskind,  M.  S.,  and  Biskind,  G.  S.:  Development  of  tumors  in  the  rat  ovary 

after  transplantation  into  the  spleen.     Proc.  Soc.  Exper.  Biol.  &  Med.  55: 
176-179,  1944. 

(15)  Zondek,  B. :  tTber  das  Schicksal  des  Follikelhormons  (Follikulin)  im  Organismus. 

Skandinav.  Arch.  f.  Physiol.  70:  133-167,  1934. 

(16)  Rush,  B.  Jr.:  Inactivation  of  estradiol  by  the  hepatic  tissues  of  mice.     Proc.  Soc. 

Exper.  Biol.  &  Med.  74:  712-714,  1950. 

(17)  Miller,  O.  J.,  and  Pfeiffer,  C.  A.:  Demonstration  of  increased  gonadotrophic 

hormone  production  in  castrated  mice  with  intrasplenic  ovarian  grafts.     Proc. 
Soc.  Exper.  Biol.  &  Med.  75:  178-181,  1950. 

(18)  Gardner,  W.  U.:  The  effect  of  ovarian  hormones  and  ovarian  grafts  upon  the 

mammary  glands  of  male  mice.     Endocrinology  19:  656-667,  1935. 

(19)  Miller,  O.  J.,  and  Gardner,  W.  U. :  The  role  of  thyroid  function  and  food  intake 

in  experimental  ovarian  tumorigenesis  in  mice.     Cancer  Res.   14:  220-226, 
1954. 

(20)  Smith,  F.  W.,  and  Gardner,  W.  U.:  Biological  assay  of  mouse  pituitary  gonado- 

trophin.     (Abstract.)     Anat.  Rec.  106:  248,  1950. 

(21)  Li,  M.  H.,  and  Gardner,  W.  U.:  Influence  of  age  of  host  and  ovaries  on  tumori- 

genesis in  intrasplenic  and  intrapancreatic  ovarian  grafts.     Cancer  Res.  10: 
162-165,  1950. 


Journal   of   the   National   Caneer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  709 

(22)  Klein,    M.:   Ovarian   tumorigenesis  following  intrasplenic   transplantation   of 

ovaries  from  weanling,  young  adult,  and  senile  mice.     J.  Nat.  Cancer  Inst.  12: 
877-881,  1952. 

(23)  Gardner,  W.  U.:  Ovarian  and  lymphoid  tumors  in  female  mice  subsequent  to 

roentgen-ray  irradiation  and  hormone  treatment.     Proc.  Soc.  Exper.  Biol.  & 
Med.  75:  434-436,  1950. 

(24)  Chang,  C.  H.,  and  Van  Eck,  G.  J.:  Action  of  testosterone  propionate  on  the 

pituitary  activity  of  castrated  or  X-rayed  female  mice  in  parabiosis  with  normal 
females.     (Abstract.)     Cancer  Res.  12:  254,  1952. 

(25)  Bali,  T.,  and  Furth,  J.:  Morphological  and  biological  characteristics  of  X-ray 

induced  transplantable  ovarian  tumors.     Cancer  Res.  9:  449-472,  1949. 

(26)  Cliffton,  E.  E.,  and  Pan,  S.  C:  The  effect  of  progesterone  compound  on  growth 

of  a  transplanted  granulosa  cell  tumor.     Proc.  Soc.  Exper.  Biol.  &  Med.  69: 
516-518,  1948. 


Vol.    15,    No.    3,    December    1954 


Induced  Ovarian  and  Adrenal 

Tumors1'2,3 


Kathakine    P.    Hummel,    Roscoe    B.    Jackson 
Memorial  Laboratory,  Bar  Harbor,  Maine 


If  ovaries  of  strain  DBA  mice  are  subjected  to  hormonal  imbalance  by 
grafting  to  spleens  of  gonadectomized  hosts  they  become  tumorous  7 
months  later.  This  is  longer  than  the  process  takes  in  mice  of  some  other 
strains  as,  for  example,  in  strain  A  where  the  ovaries  become  tumorous 
in  3  months,  and  in  strains  C57BL  and  BALB/c  where  the  process  takes 
5  months. 

The  normal  aging  changes  in  ovaries  of  strain  DBA  mice  have  been 
described  by  Fekete  (J?).  A  characteristic  feature  of  these  ovaries  is  the 
retention  and  hyalinization  of  corpora  lutea.  Tumorous  changes  are 
rarely  found.  In  a  recent  survey,  one  tumor,  a  tubular  adenoma,  was 
found  among  13  DBA  mice  over  20  months  of  age. 

To  determine  how  long  an  ovary  must  be  subjected  to  hormonal  im- 
balance before  being  irreversibly  altered,  DBA  ovaries  were  grafted  to 
spleens  of  gonadectomized  hosts,  left  there  for  periods  of  time  ranging 
from  3  days  to  5  months  and  then  removed  and  transferred  to  the  normal 
site  or  ovarian  bursa  of  a  second  host,  this  time  a  hybrid  DBA  X  C3H. 
In  order  that  the  hormonal  environment  of  this  host  might  be  as  normal 
as  possible  only  one  ovary  was  replaced,  the  other  being  removed  4  to  6 
days  later  after  the  grafted  ovary  had  established  itself  and  could  function 
normally.  The  hybrids  bearing  grafted  pre  treated  ovaries  were  mated 
and  in  many  cases  produced  normal  young  (2).  As  has  been  generally 
observed  with  transplanted  ovaries,  considerably  fewer  litters  were  pro- 
duced by  graft-bearing  mice  than  by  unilateral  castrates  or  intact  mice. 
The  reproductive  function  of  ovaries  previously  resident  in  spleens  for 
periods  up  to  1  month  did  not  differ  essentially  from  ovaries  transplanted 
directly  without  splenic  sojourn.  Reproductive  function,  however,  did 
decrease  with  the  length  of  time  the  ovary  had  remained  in  the  spleen,  no 
young  being  produced  from  ovaries  that  had  been  in  spleens  for  over  2 
months.  There  was  no  similar  decrease  in  hormonal  function  and  no 
differences  were  noted  in  matings  as  evidenced  by  vaginal  plugs  nor  in 

1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  29, 1954. 

2  This  is  a  report  of  several  years'  work  involving  a  group  of  investigators,  notably  Mary  S.  Eddy  and  Barbara 
Rupple,  who  were  responsible  for  the  grafting  operations  in  large  part  and  Dr.  Elizabeth  Fekete,  who  kindly 
assisted  in  the  tumor  diagnoses. 

3  This  work  has  been  supported  in  part  by  a  grant-in-aid  from  the  American  Cancer  Society  upon  recommenda- 
tion of  the  Committee  on  Growth  of  the  National  Research  Council,  and  by  a  research  grant  C1073(C)  from  the 
National  Cancer  Institute  of  the  National  Institutes  of  Health,  U.  S.  Public  Health  Service. 


711 


Journal    of   the   National   Cancer   Institute.    Vol.    15,   No.    3,   December    1954 


! 

712  proceedings:  SYMPOSIUM  ON  25  YEARS  OF 

stimulation  of  mammary  glands  as  evidenced  by  the  presence  of  mammary 
gland  tumors.  In  fact,  mammary  gland  tumors  occurred  in  the  hybrid 
graft-bearing  mice  in  such  numbers,  and  at  such  an  early  age,  that  it  was 
necessary  to  use  hybrids  deprived  of  the  milk  agent  in  later  experiments. 

In  97  of  159  animals  (61%)  killed  12  to  24  months  after  receiving  the 
graft,  the  ovaries  were  found  to  be  tumorous,  31  of  these  being  grossly 
observable.  Sixteen  of  134  animals  (12%)  killed  earlier  than  12  months 
following  grafting  had  tumorous  ovaries,  all  of  these  being  microscopic 
in  size.  In  the  control  transplantation  animals  (hybrids  grafted  with 
DBA  ovaries  not  previously  resident  in  spleens),  5  of  9  (or  55%)  killed  12 
to  24  months  after  the  transplantation  had  tumors  of  the  ovaries.  No 
tumors  were  observed  in  10  controls  killed  earlier  than  12  months. 

We  concluded  from  these  results  that  the  hormonal  imbalance  imposed 
upon  the  ovaries  by  grafting  them  into  the  spleens  of  gonadectomized 
hosts  has  little  to  do  with  the  incidence  of  ovarian  tumors  and  that  trans- 
plantation per  se  must  be  an  important  factor.  The  period  of  splenic 
sojourn  apparently  hastens  tumorigenesis,  as  ovarian  tumors  were  found 
earlier  if  the  period  of  imbalance  had  lasted  at  least  2  weeks.  Ovarian 
tumors  were  found  8  months  after  ovarian  transplantation  in  these  cases, 
whereas  they  were  not  found  until  14  months  later  if  the  sojourn  in  spleen 
was  of  3  or  7  days'  duration.  Tumors  of  the  ovary  were  found  15  months 
after  direct  transplantation  in  the  control  series. 

Some  of  the  factors  that  may  have  been  operative  in  the  induction  of 
these  ovarian  tumors  following  transplantation  are  briefly  summarized. 

1)  Hybrid  environment. — Although  no  tumors  have  been  seen  in  ovaries 
of  intact  or  unilaterally  castrate  hybrid  DBA  X  C3H  mice,  it  is  well 
known  that  the  hybrid  environment  often  gives  rise  to  abnormalities  not 
seen  in  either  parent. 

2)  Transplantation  and  consequent  breakdown  of  a  large  part  oj  the  ovarian 
tissue. — The  ovary  becomes  reconstituted,  but  never  completely — as  is 
evidenced  by  an  early  decrease  in  the  number  of  ripening  follicles  and  the 
short  reproductive  life.  The  small  amount  of  functional  ovarian  tissue 
may  create  an  abnormal  ovary-pituitary  hormone  balance. 

3)  Abnormal  proportions  oj  pituitary  gonadotrophins  released. — The 
remnant  or  fragment  of  ovary  undergoes  compensatory  hypertrophy 
partly  through  accumulation  of  interstitial  cells.  These  have  been  shown 
histochemically  to  store  and  possibly  secrete  the  precursor  of  gonadal 
hormones  (cholesterol).  Mobilization  and  release  of  gonadal  hormones 
is  dependent  upon  the  proportion  of  the  gonadotrophins  F.S.H.,  L.H.  and 
L.T.H.  released  by  the  pituitary.  In  these  ovaries  there  is  an  over- 
growth of  interstitial  cells  suggesting  increased  precursor  storage,  and  in 
turn  disproportionate  gonadotrophin  release  from  the  pituitary. 

4)  Disproportionate  release  of  gonadal  hormones. — A  hypertrophied 
interstitium  is  often  associated  with  increased  androgen  production  by 
an  ovary.  The  conspicuous  interstitium  of  these  ovaries  suggests  imbal- 
ance within  the  ovary  in  the  estrogen,  androgen  and  progesterone  produc- 
tion and/or  release,  and  between  precursor  and  hormone. 

Journal    of    the   National    Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


713 


Adrenal  glands  of  many  of  the  hybrid  graft-bearing  mice  were  saved 
and  examined.  In  the  control  series  adrenals  from  14  animals  were 
studied.  Of  these,  one  in  an  animal  with  an  ovarian  tumor  killed  13 
months  after  transplantation  showed  slight  nodular  hyperplasia  of  the 
cortex.  None  of  the  others  showed  tumorous  changes  in  the  adrenals. 
In  the  experimental  series  (hybrids  with  grafted  ovaries  previously  sub- 
jected to  the  hormonal  imbalance)  many  of  the  adrenals  showed  cortical 
nodular  hyperplasias  and  carcinomas  characteristic  of  castrate  mice. 

Adrenals  of  223  hybrids  bearing  grafted  ovaries  that  had  been  previ- 
ously resident  in  spleens  were  examined.  In  46,  the  ovarian  graft  was 
absent  or  minute  and  in  these,  as  might  be  expected,  42  or  91  percent  had 
adrenal-cortical  tumors.  Table  1  summarizes  the  observations  in  the 
remaining  177  hybrids.  In  33,  or  19  percent,  killed  at  an  average  age 
after  graft  operation  of  9  months  there  were  neither  adrenal  nor  ovarian 
tumors.  One  third  of  the  animals  had  both  adrenal  and  ovarian  tumors, 
42  (or  24  percent)  had  ovarian  tumors  but  not  adrenal  tumors,  and  46 
(or  26  percent)  had  adrenal  but  not  ovarian  tumors.  There  is  a  suggestion 
that  the  adrenal  tumor  formation  preceded  the  ovarian  in  that  they  were 
found  in  animals  killed  at  an  average  of  15  months  after  grafting  of  the 
ovaries,  whereas  ovarian  tumors  were  found  at  an  average  postoperational 
age  of  18  months.  It  is  noteworthy  that  the  characteristic  retention  and 
hyalinization  of  corpora  lutea  was  not  found  in  any  ovary  of  strain  DBA 
grafted  to  a  hybrid.  Seventy-five  percent  of  the  hybrids  with  both  ovarian 
and  adrenal  tumors  had  had  functional  ovaries  in  that  they  had  either 
borne  young  or  had  vaginal  plugs  as  evidence  of  estrus  and  mating. 

Table  1. — Ovarian  and  adrenal  tumors  in  177  (DBAXC3H)  hybrids  with  grafted  DBA 
ovaries  "altered"  by  sojourn  of  8  to  5  months  in  spleens  of  castrates 


Number 
of  mice 

Average 
age 

Age 
range 

With  functional  ovaries 

Number 

without 

functional 

ovaries 

Type  of 
tumors 
present 

Total 
Number 

Number 

with 
offspring 

Number 

with 

vaginal 

plugs 

Ovarian + 
Adrenal + 

Ovarian + 
Adrenal  — 

Ovarian— 
Adrenal + 

Ovarian— 
Adrenal— 

56  (32%) 
42  (24%) 
46  (26%) 
33  (19%) 

(month) 
17 

18 

15 

9 

(month) 
9-22 

8-22 

7-23 

4-18 

42  (75%) 

20 

22 

14  (25%) 

This  finding  of  adrenal  and  ovarian  tumors  in  the  same  experimental 
animal  indicates  causation  by  a  single  type  of  hormonal  imbalance.  This 
supports  the  theory  held  by  some  that  the  basic  mechanism  of  hormonal 
imbalance  is  the  same  in  ovarian  tumors  induced  by  X  ray,  ovarian  tumors 


Vol.    15,    No.    3,    December    1954 


714  proceedings:  SYMPOSIUM  ON  25  YEARS  OF 

induced  by  implantation  to  spleens  of  gonadectomized  mice,  and  adrenal 
tumors  induced  by  neonatal  castration.  The  gonad-pituitary  balance  is 
upset.  One  widely  held  theory  is  that  a  pituitary  released  from  gonadal- 
hormone  inhibition  releases  gonadotrophins  of  abnormal  quality,  or  in 
excessive  amounts  over  a  prolonged  period  of  time. 

It  would  seem  in  the  case  of  our  experimental  animals  that  the  ovarian 
and  adrenal-cortical  tumors  have  resulted  from  an  hormonal  imbalance  im- 
posed upon  the  pituitary-gonad-adrenal  complex  by  a  fragment  or  remnant 
of  functional  ovarian  tissue.  In  support  of  this  theory,  preliminary  ob- 
servations on  a  group  of  DBA  X  C3H  hybrids  in  which  gonadectomy  was 
incomplete  (a  small  remnant  of  ovary  having  been  left)  show  adrenal- 
cortical  tumors  one  year  later — even  though  the  remnant  of  ovary  func- 
tioned to  produce  young.  Complete  gonadectomy  at  2  months  of  age, 
approximately  the  age  at  which  the  ovary  grafts  were  made,  results  in 
adrenal  tumors  12  months  later.  No  ovarian  or  adrenal  tumors  have  been 
found  in  intact  or  unilaterally  castrate  DBA  X  C3H  hybrids  up  to  24 
months  of  age. 

Morphologically  the  abnormal  adrenals  resemble  the  abnormal  ovaries. 
Large  accumulations  or  nodules  of  pale  hypertrophied  cells  which  are 
similar  in  appearance  to  the  interstitial  cells  of  the  ovary  are  present. 
There  are  masses  of  lipochrome  cells  that  in  the  ovary  are  remnants  of 
atretic  follicles.  There  are  cysts  among  the  hypertrophied  cells  into  which 
clumps  of  cells  penetrate.  In  such  clumps,  the  granulosa-cell  tumors  of  the 
ovary  seem  to  arise  and  in  similar  areas  adrenal-cortical  carcinomas  appear. 
The  adrenal  tumors  often  assume  a  folliculoid  structure  difficult  to  dis- 
tinguish from  ovarian  tissue.  Also  in  these  adrenals,  cysts  lined  with 
ciliated  epithelium,  similar  to  cysts  found  in  transplanted  DBA  ovaries,  are 
seen.  Such  cysts  are  frequently  found  at  the  hilus  of  the  ovary  in  some 
strains  of  mice,  notably  C57L  (8)  where  they  are  thought  to  be  remnants 
of  the  mesonephros.  The  presence  and  origin  of  these  cysts  in  the  adrenal 
cortex  is  puzzling  as  they  have  been  seen  only  in  DBA  X  C3H  mice  bearing 
grafted  DBA  ovaries.  The  morphologic  picture  of  the  adrenal  cortex 
leads  us  to  believe  that  it  functions  hormonally  as  a  second  ovary  under 
the  influence  of  gonadotrophins  from  a  pituitary  that,  in  turn,  is  responding 
to  a  lack  of  normal  circulating  gonadal  hormones. 

In  summary,  our  experiments  have  shown  that  transplantation  of  ovaries 
of  DBA  mice  into  DBA  X  C3H  hybrids  results  in  tumorous  changes  in  the 
grafted  ovaries  and  also  in  the  cortex  of  the  adrenal  of  the  host.  This  is 
due  to  an  upset  in  the  ovary-pituitary-adrenal  complex  possibly  occasioned 
by  deficiency  of  or  abnormal  qualities  of  gonadal  hormones  from  an  ovary 
partly  destroyed  by  transplantation.  After  transplantation  the  ovary 
does  not  fully  recover  its  reproductive  or  endocrine  function.  This 
creates  an  abnormal  and  irreversible  ovary-pituitary  balance.  Compen- 
satory mechanisms  involving  the  adrenal  cortex  set  up  a  transitory  balance 
that  can  not  be  maintained  for  any  length  of  time  and  abnormal  growth 
of  ovary  and  adrenal  cortex  is  the  end  result. 


Journal    of    the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  715 

References 

(1)  Fekete,  E.:  A  comparative  study  of  the  ovaries  of  virgin  mice  of  the  dba  and 

C57  black  strains.     Cancer  Res.  6:  263-269,  1946. 
{2)  Little,  C.  C,  Hummel,  K.  P.,  Eddy,  M.  S.,  and  Rupple,  B.:  Young  produced 

from  ovaries  subjected  to  endocrine  imbalance.     Proc.   Nat.   Acad.   Sc.   37: 

666-669,  1951. 
(5)  Fekete,  E.:  A  morphological  study  of  the  ovaries  of  virgin  mice  of  eight  inbred 

strains  showing  quantitative  differences  in  their  hormone  producing  components. 

Anat.  Rec.  117:  93-113,  1953. 


Vol.    15,    No.    3,    December    1954 


J 


T 


Carcinogenesis  in  the  Adrenal  *• 


George  W.  Woolley,  Chief,  Division  of  Steroid 
Biology,  Sloan-Kettering  Institute  for  Cancer  Re- 
search, and  Professor  of  Biology,  Sloan-Kettering 
Division,  Cornell  University  Medical  School,  New 
York21,N.Y. 


I  shall  take  the  adrenal  gland  as  an  example  for  my  discussion.  I  am 
glad  to  do  this  as  it  involves  a  site  that  has  long  been  of  particular  interest 
to  me.  I  can  well  remember  standing  at  a  table  in  the  old  Jackson  Lab- 
oratory, autopsying  a  dilute  brown  mouse  which  had  been  castrated  at 
birth  and  now  aged  and  with  breast  cancer,  and  noting  for  the  first  time 
adrenal  changes  later  described  as  nodular  hyperplasia  of  the  adrenal. 
Through  transplantation  experiments  the  ovary-like  endocrine  secretion 
present  in  this  and  other  similar  ovariectomized  animals  was  determined 
to  have  arisen  from  the  adrenal  glands. 

I  was  particularly  interested  in  the  condition  of  the  adrenal  since  as  a 
graduate  student  at  Wisconsin  I  had  heard  discussions  on  endocrines  refer- 
ring to  the  adrenal  cortex  as  "the  great  unknown"  in  endocrinology. 
How  far  knowledge  has  progressed  since  that  time!  The  adrenal  is  now 
one  of  the  best  known  glands. 

Subsequently,  a  histological  interpretation  of  the  adrenal-cortical 
changes  was  developed  with  the  aid  of  Dr.  Elizabeth  Fekete  and  Dr.  A. 
M.  Cloudman,  of  the  Jackson  Laboratory. 

Adrenal-cortical  tumors  similar  to  those  of  the  mouse  have  now  been 
described  in  such  experimental  animal  forms  as  the  guinea  pig,  the  rat, 
and  the  hamster. 

To  reminisce  a  little  further — ideas  regarding  etiological,  or  origin  rela- 
tionships of  adrenal-cortical  tumors  go  back  to  the  last  century.  In  1891, 
Marchand  described  a  case  where  a  human  female  hermaphrodite  had,  on 
autopsy,  atrophied  ovaries  and  greatly  enlarged  adrenals.  In  a  similar 
case,  Creccio  found  that  the  adrenals  had  increased  to  the  size  of  the 
kidneys. 

An  experimental  test  of  the  idea  of  ovary-adrenal  relationship  appears 
to  have  first  been  performed  by  Miss  H.  E.  Feodossiew,  at  the  University 
of  Kazan.  The  results  of  this  test  were  published  in  1906.  The  experi- 
ment seems  to  have  eluded  a  number  of  workers  in  this  country. 

i  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine 
June  29, 1954. 

8  This  investigation  was  supported  in  part  by  a  research  grant  (C-1796)  from  the  National  Cancer  Institute, 
of  the  National  Institutes  of  Health,  U.  S.  Public  Health  Service,  and  in  part  by  a  grant  from  the  American  Cancer 
Society. 

717 

Journal   of   the  National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


718  proceedings:  SYMPOSIUM  ON  25  YEARS  OF 

One  can  find  that  Professor  Lubimov  and  his  student,  Miss  Feodossiew, 
clearly  understood  that  a  relationship  existed  between  the  ovary  and  the 
adrenal  cortex.  In  exploring  this  relationship  they  used  dogs.  These  were 
castrated  and  then  autopsied  at  definite  intervals  of  months.  The  adrenal 
glands  were  studied  and  adrenal-cortical  hyperplasia  was  described.  The 
hyperplasia  started  in  the  region  of  the  glomerulosa  and  progressed  to  the 
fasciculata.  The  hyperplasia  in  both  layers  was  focal,  i.e.,  in  islands. 
The  growth  reached  and  penetrated  the  medulla  in  one  direction  and 
progressed  through  the  capsule  in  the  other,  forming  in  the  latter  case  a 
mushroom-like  growth  which  remained  united  to  the  cells  of  the  peripheral 
layer.  Increase  in  the  number  of  mitotic  figures  was  noted.  All  of  this 
sounds  remarkably  modern. 

During  the  past  few  years  we  have  come  to  understand  endocrine  rela- 
tionships more  clearly,  and  to  differentiate  these  relationships  from  other 
important  etiologic  factors — genetic  and  vascular. 

The  first  of  these  factors  to  be  mentioned  is  the  genetic  one — the  in- 
fluence of  heredity. 

We  know  that  this  influence  exists  in  the  mouse  because  of  the  presence 
and  persistance  of  strain  variations  in  the  occurrence  of  the  adrenal-cortical 
tumors.  Some  strains  have  adrenal-cortical  tumors  without  gonadectomy, 
others  only  with  gonadectomy.  Some  strains  have,  characteristically, 
only  a  few  or  no  cortical  tumors;  others,  benign  tumors;  and  still  others, 
malignant  tumors.  Tumors  of  certain  strains  produce,  characteristically, 
estrogenic  hormones;  others,  androgenic  hormones;  and  some  produce  both. 
A  few,  especially  late  in  life,  give  evidence  of  producing  progesterone-like 
hormones. 

It  is  not  known  whether  the  genetic  factors  act  most  effectively  system- 
ically  through  hormone  imbalance,  or  locally  in  the  adrenal-cortical  tissue. 
The  evidence  of  Huseby  and,  thus  far,  our  own  evidence,  indicates  that 
an  important  site  of  action  is  in  the  adrenal-cortical  tissue  itself. 

Heterosis  is  a  modifying  factor  and  acts  in  a  positive  direction  toward 
tumor-formation — as  far  as  it  has  been  studied. 

Carcinoma  tends  to  be  dominant  over  hyperplasia,  and  hyperplasia  to 
be  dominant  over  no  tumor  in  crosses  between  strains  with  different  tumor 
tendencies. 

The  second  influence  to  be  considered  in  more  detail  is  that  of  the 
endocrine  secretions. 

The  occurrence  of  adrenal-cortical  tumors  may  be  retarded  by  removal 
of  the  pituitary,  the  use  of  steroid  hormones  related  to  those  of  the  adrenal, 
ovary,  or  testes,  and  by  the  presence  of  hormone-producing  tumors. 
Both  thiouracil  and  caloric  restriction  retard  hormone  production  by  the 
tumors.  The  latter  influence  has  been  interpreted  as  being  similar  to 
partial  hypophysectomy. 

Occurrence  of  adrenal-cortical  tumors  may  be  enhanced  by  gonadectomy, 
by  excess  estrogen,  by  pituitary  hormones,  and  by  transplantation  of 
ovaries  to  the  spleen.     In  the  latter  case,  it  is  thought  that  enhancement 

Journal    of    the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  719 

occurs  because  estrogenic  hormones  are  inactivated  in  the  liver  before 
reaching  the  pituitary. 

A  third  influence  affecting  carcinogenesis,  and  the  latest  to  be  found, 
appears  to  be  a  mechanical  one  and  related  either  to  nutrition  or  to 
vascularization  of  the  adrenal.  The  influence  of  this  factor  has  been 
demonstrated  by  transplantation  of  the  adrenal  to  a  subcutaneous  site. 
It  is  a  tumor-retarding  influence. 

Finally,  the  occurrence  of  adrenal-cortical  tumors  requires  the  integra- 
tion of  genetic,  hormonal,  and  site  factors — and  possibly  many  more  as 
yet  undiscovered  factors.  Carcinogenesis  in  the  adrenal  appears  to  be 
a  complex  syndrome  but  one  capable  of  progressive  solution  and  under- 
standing. 

A  possible  theory  of  origin  might  be  the  following:  in  the  absence,  or 
deficiency,  of  gonadal  hormones — ovarian  or  testicular — a  normal  and 
proper  hormonal  brake  is  not  applied  to  the  pituitary.  The  excess  pitui- 
tary hormones  which  then  accumulate  act  on  the  adrenal  cortex,  a  tissue 
arising  embryologically  from  the  sex  ridge.  If  this  latter  tissue  has 
genetic  factors  for  tumor  susceptibility,  and  proper  vascularization  and 
nutrition,  tumors  will  develop. 


Vol.    13,   No.   3,   December    1954 


Discussion:  Carcinogenesis  in  Endocrine  Organs 

Dr.  William  B.  Atkinson,  University  of  Cincinnati  School  of  Medicine, 

Cincinnati,  Ohio 

Among  the  many  approaches  to  the  study  of  the  pathogenesis  of  neoplasms,  few 
have  been  as  productive  as  that  of  the  investigation  of  abnormal  growth  in  endocrine 
organs.  It  may  be  somewhat  trite,  but  nonetheless  valuable,  to  remind  this  group 
that  one  of  the  most  venerable  definitions  of  neoplasia  is  that  of  uncontrolled  growth 
of  a  tissue.  In  this  very  definition,  however,  we  find  the  clue  to  the  greatest  obstacle 
in  the  elucidation  of  carcinogenesis;  i.e.,  that  before  we  can  deal  successfully  with  the 
problem  of  "uncontrolled"  growth,  we  must  first  know  at  least  the  basic  physiologic 
processes  involved  in  the  normal  growth  and  differentiation  of  the  tissues  composing 
the  various  organs  of  the  body.  We  must  learn  to  manipulate  normal  growth  in 
order  to  establish  techniques  for  the  investigation  of  abnormal  growth.  In  most 
tissues  and  organs  of  the  body  our  present  knowledge,  unfortunately,  is  entirely 
inadequate  in  this  respect.  With  all  deference  to  the  excellent  work  reported  previ- 
ously in  this  conference,  insofar  as  carcinogenesis  is  concerned,  it  is  of  little  comfort  or 
ultimate  edification  to  know  simply  that  the  development  of  a  particular  tumor  may 
be  associated  with  the  action  of  a  gene  or  group  of  genes.  Likewise,  it  helps  us  but  little 
to  record  the  effects  of  X  radiation  or  chemical  carcinogens  upon  the  skin,  for  instance, 
until  we  have  a  great  deal  more  fundamental  information  concerning  this  organ  of 
the  kind  reported  by  Dr.  Chase  in  last  evening's  session. 

It  must  be  admitted  that  the  endocrinologist  has  enjoyed  several  unique  advantages 
in  his  study  of  tissue  growth.  First,  endocrine  organs  and  target  organs  depend  to 
a  considerable  extent  upon  known  humoral  agents  for  their  morphologic  and  func- 
tional maturation;  and  second,  normal  growth  and  differentiation  in  the  endocrine 
system  has  been  studied  with  ever-increasing  intensity  over  the  last  half  century. 
Another  factor  of  prime  importance  has  been  that,  in  many  instances,  the  clinical 
conditions  with  which  the  physician  has  had  to  deal  have  their  closely  parallel  counter- 
parts in  experimental  animals.  We  might  cite  the  virilizing  adrenal  carcinomas, 
adenocarcinoma  of  the  mammary  gland  and  the  uterine  hyperplasias,  to  name  but 
several.  This  has  led  to  the  ready  exchange  of  data,  and,  more  important,  ideas  that 
have  often  been  of  immediate  usefulness  in  both  the  laboratory  and  clinic. 

With  respect  to  the  several  papers  presented  this  morning,  I  have  been  intrigued 
by  two  questions  which  are  quite  general  in  scope:  1)  with  the  principal  exception  of 
the  mammary  gland,  experimentally  induced  and  even  "spontaneous"  {i.e.,  genetically 
induced)  tumors  of  the  endocrine  system  are  found  to  occur  mainly  in  the  hormone- 
producing  organs  themselves  (such  as  the  pituitary  gland,  the  gonads,  the  thyroid,  or 
the  adrenals) .  The  consistent  production  of  neoplasms  in  the  final  target  organs  (such 
as  the  uterus,  seminal  vesicles,  submaxillary  glands  or  kidneys)  is  far  less  commonly 
obtained.  Why?  2)  Dr.  Furth,  in  particular,  has  reminded  us  of  the  increasing 
autonomy  of  individual  tumors  with  respect  to  their  environment;  that  neoplasms 
which  may  survive  only  autotransplantation  in  their  early  stages  of  development, 
may  eventually  become  transplantable  even  into  unrelated  species.  I  believe  that 
this  important  biological  property  of  neoplasms  is  one  which  must  eventually  be 
taken  into  greater  account  in  studies  on  histocompatibility.  I  am  sure  we  should 
all  like  to  hear  any  comments  that  today's  speakers  may  care  to  make  on  the  fascinat- 
ing question  of  why  the  growth  of  a  tumor  transplanted  in  its  dependent  phase  of 
development  may  be  blocked  entirely  by  one  or  more  "incompatible"  genes  in  the 
host,  whereas  the  same  tumor  transplanted  in  its  later  autonomous  phase  may  grow 
with  complete  disregard  of  what  was  earlier  a  hostile  genetic  environment. 

721 


Journal   of   the   National  Cancer   Institute,   Vol.    15,   No.    3,   December    1954 
316263—54 31 


722         proceedings:  symposium  on  25  years  of 

Dr.  Flavia  L.  Richardson,  Roscoe  B.  Jackson  Memorial  Laboratory, 
Bar  Harbor,  Maine 

We  have  studied  the  effect  of  castration  and  adrenalectomy  on  the  development  of 
the  mammary  glands  of  female  mice.  Female  mice  of  strains  BALB/c  and  C57BR/cd 
were  castrated  3  days  after  birth.  These  mice  were  sacrificed  at  3  months  of  age  and 
the  mammary  glands  were  compared  with  those  of  normal  females  of  the  same  age. 
There  was  very  little  development  of  the  mammae  in  the  castrated  BALB/c  mice. 
The  ducts  were  very  narrow,  had  few  branches,  and  covered  a  much  smaller  area 
than  in  the  normal  females.  In  contrast,  the  mammary  glands  of  the  castrated 
C57BR/cd  females  showed  considerable  variation  ranging  all  the  way  from  small 
glands  similar  to  those  in  the  BALB/c  castrates  to  glands  that  very  closely  resembled 
those  of  the  normal  C57BR/cd  females.  The  ducts  were  wide,  well  branched  and 
covered  a  large  area.  The  uteri  of  all  castrated  animals  showed  no  stimulation. 
None  of  the .  castrated  animals  possessed  the  numerous  lateral  buds  or  very  small 
acini  that  were  present  in  some  of  the  glands  of  normal  animals. 

C57BR/cd  females,  castrated  at  3  days  of  age,  were  adrenalectomized  when  about 
25  days  old.  The  mammary  glands  of  animals  3  months  of  age  closely  resembled 
those  of  the  mice  that  were  castrated  only. 


Dr.  Robert  S.  Speirs,  Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 

Dr.  Gardner  has  clearly  illustrated  how  experimental  manipulations  of  genetical 
and  hormonal  factors  can  produce  growths  that  are  identical  to  spontaneously  occurring 
tumors.  His  excellent  presentation  brought  out  a  number  of  factors  that  I  would 
like  to  discuss  briefly. 

The  effects  of  hormones  and  other  substances  upon  tumor  cells  are  usually  deter- 
mined only  by  gross  measurements  of  weight  and  size.  These  are  acknowledged 
to  be  crude,  and  exceedingly  variable  from  mouse  to  mouse,  and  are  more  qualitative 
than  quantitative.  This  is  especially  true  of  tumors  that  are  transplanted  and  grown 
subcutaneously.  However,  in  recent  years  it  has  been  possible  to  adapt  many  different 
tumors  for  growth  in  the  peritoneal  cavity  (1).  In  the  peritoneal  cavity,  the  tumor 
cells  are  suspended  in  a  fluid  medium,  thus  permitting  the  application  of  modified 
hematologic  techniques.  In  this  manner,  the  total  number  of  cells  per  cubic  millimeter 
can  be  determined  by  the  chamber  method,  stained  smears  can  be  obtained  for  differ- 
ential counting  and  estimates  can  be  made  of  the  total  amount  of  fluid  in  the  peritoneal 
cavity.  Thus  the  total  number  of  cells  of  each  component  of  a  tumor,  as  well  as  the 
local  cellular  response  of  the  host  to  the  tumor,  can  be  determined. 

The  subcutaneous  air  pouch,  developed  by  Dr.  Hans  Selye  (2),  is  an  artificial  sac, 
which  is  somewhat  more  accessible  than  the  body  cavities  but  is  very  similar  in  other 
respects.  Gross  measurements  of  fluid  volume  as  well  as  quantitative  measurements 
of  cells  can  be  performed  daily  or  even  hourly  if  necessary.  Moreover,  the  whole 
sac  containing  the  tumor  cells  can  be  surgically  removed  at  any  time,  and  if  desired 
a  second  sac  can  be  produced  for  fresh  tumor  cells. 

This  procedure  of  growing  the  tumor  cells  as  a  suspension  within  the  peritoneal 
cavity  or  pouch,  permits  a  quantitative  determination  of  the  growth  rate  of  each  type 
of  cellular  component  of  a  tumor  under  normal  and  experimental  conditions.  This 
method  of  measurement  should  be  more  accurate  and  sensitive  to  experimental 
procedures  than  the  gross  measurements  of  size  and  weight. 

It  is  important  to  determine  whether  the  hormones  act  directly  upon  tumor  cells, 
or  whether  they  affect  the  defense  reactions  of  the  host  and  thereby  modify  tumori- 
genesis  and  growth  by  indirect  means.  This  type  of  problem  can  easily  be  studied 
in  the  tumors  adapted  for  growth  in  the  peritoneal  cavity  or  pouch.  It  is  commonly 
known  that  substances  injected  into  an  inflamed  peritoneal  cavity  or  air  pouch,  do 
not  pass  into  the  circulation  very  readily.  It  is  therefore  possible  to  study  the  effects 
of  a  particular  substance  acting  locally  upon  the  tumor  cells,  and  compare  it  with  the 

Journal   of   the  National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  723 

effects  of  the  same  substance  given  systemically.  Moreover,  because  these  tumor 
cells  are  growing  in  a  physiologically  isolated  type  of  tissue  culture,  it  is  possible  to 
utilize  the  peritoneal  cavity  or  air-pouch  for  culturing  viruses  or  even  for  growing 
certain  tissues  such  as  the  pituitary  or  spleen  together  with  the  tumor  cells. 

The  final  technique  which  I  would  like  to  comment  upon  is  that  of  hypophysectomy. 
In  the  clinic  there  is  a  great  deal  of  interest  in  the  effect  of  this  operation  upon  patients 
with  inoperable  cancer  and  in  some  cases  it  certainly  seems  to  have  a  striking  effect. 

The  effect  of  hypophysectomy  upon  tumorigenesis  and  growth  should  certainly 
be  investigated  more  thoroughly,  especially  in  the  mouse  where  a  wide  variety  of 
tumors  are  readily  available  for  study.  In  the  mouse,  hypophysectomy  is  somewhat 
difficult  to  learn;  however,  once  mastered  it  is  relatively  easy  and  quickly  performed. 
Using  a  modification  of  the  technique  of  Thomas  (S)  we  have  found  that  the  entire 
operation  can  be  performed  in  less  than  10  minutes  in  young  mice  and  that  the  animals 
may  live  up  to  a  year  or  more  postoperatively.  Furthermore,  very  young  mice 
weighing  as  little  as  6  grams  can  be  hypophysectomized.  A  number  of  laboratories 
have  utilized  hypophysectomized  mice  for  various  studies.  In  1946,  Korteweg  and 
Thomas  reported  on  the  outcome  of  351  hypophysectomies  (4)  and  Dr.  Gardner  has 
recently  stated  that  well  over  1,000  such  operations  have  been  performed  at  Yale. 

The  evidence  at  the  present  time  seems  to  be  that  hypophysectomy  reduces  or 
prevents  the  formation  of  mammary  cancer  in  certain  stocks  of  mice  with  a  high 
predisposition  to  that  type  of  cancer.  This  is  not  too  surprising  in  as  much  as  the 
pituitary  does  directly  or  indirectly  regulate  the  growth  of  the  mammary  tissue. 
However,  it  would  be  extremely  interesting  to  know  if  tumors  in  other  tissues  would 
also  be  inhibited  or  delayed  by  hypophysectomy.  The  rate  of  growth  of  transplantable 
or  spontaneous  tumors  can  also  be  studied  in  hypophysectomized  animals.  In  this 
way  one  can  approach  the  relation  of  the  pituitary  secretions  to  tumorigenesis  and 
tumor  growth. 

References 

(1)  Hauschka,  T.  S. :  Cell  population  studies  of  mouse  ascites  tumors.    Trans.  New 

York  Acad.  Sc.  16:  64,  1954. 

(2)  Selye,  H.:  The  diseases  of  adaption:  Rec.  Prog.  Hormone  Res.  8:  117,  1953. 

(5)  Thomas,  F.:  A  technique  for  hypophysectomy  of  the  mouse.    Endocrinology  23: 

99-102,  1938. 
(4)  Korteweg,  R.,  and  Thomas  F. :  Hypophysectomy  in  mice  with  special  reference  to 

mammary  cancer.    Cancer  Res.  8:  385-395,  1946. 


Vol.   15,  No.  3,  December   1954 


' 


Session  VI .  Genetic  Control  of  Behavior 


Chairman,  Dr.  Frank  A.  Beach,  Department  of 
Psychology,  Yale  University,  New  Haven,  Conn.; 
Board  of  Scientific  Directors,  Roscoe  B.  Jackson 
Memorial  Laboratory 


Speaker:  Dr.  Curt  P.  Richter 

The  Effects  of  Domestication  and  Selection  on  the  Behavior  of  the  Norway 
Rat 

Speaker:  Dr.  John  Paul  Scott 

The  Effects  of  Selection  and  Domestication  Upon  the  Behavior  of  the  Dog 

Speaker:  Dr.  Laurence  H.  Snyder 

The  Effects  of  Selection  and  Domestication  on  Man 

Discusser,  all  papers:  Dr.  John  L.  Fuller 


725 

Journal    of   the   National   Cancer   Institute,   Vol.    15,   No.    3,   December    1954 


The  Effects  of  Domestication  and  Selec- 
tion on  the  Behavior  of  the  Norway 
Rat  h  2 


Curt  P.  Richter,  Ph.D.,  Psychobiological 
Laboratory,  Johns  Hopkins  School  of  Medicine, 
Baltimore,  Md. 


The  Norway  rat  may  be  considered  to  be  the  first  animal  to  have  become 
domesticated  for  strictly  scientific  purposes.  It  offers  excellent  oppor- 
tunities for  studying  the  anatomical,  physiological  and  behavioral  changes 
of  domestication  for  the  following  reasons:  1)  The  live  wild  Norway  rat  is 
readily  available  in  large  numbers  throughout  the  world  in  cities,  towns, 
and  on  farms,  and  equally  large  numbers  of  domesticated  Norway  rats  are 
available  in  scientific  laboratories  throughout  the  world.  2)  Since  the 
domesticated  Norway  rat  has  been  used  in  almost  every  field  of  biological 
research,  more  is  known  about  it  than  any  other  animal,  with  the  possible 
exception  of  man.  3)  Wild  and  domesticated  Norway  rats  breed  with 
one  another.  Neither  breeds,  as  far  as  is  known,  with  any  other  rat — 
not  even  the  Alexandrine  or  roof  rat,  which  next  to  the  Norway  is  the  most 
common  rat  in  the  world.  The  fact  that  the  Norway  and  Alexandrine 
rats  do  not  breed  is  extraordinary  in  view  of  their  great  similarity  in 
appearance,  only  the  initiated  being  able  to  tell  them  apart.  4)  The  short 
life  span  of  this  rat,  and  early  age  of  maturity  make  it  possible  to  follow  the 
inheritance  of  various  characteristics  throughout  many  generations  within 
a  few  years'  time.  5)  The  Norway  rat  is  very  similar  to  man  in  many  ways, 
particularly  in  dietary  needs,  geographic  distribution,  world  population 
and  colony  formation. 

Historical  Background 

Much  of  our  knowledge  of  the  history  of  the  Norway  rat,  Mus  norvegicus, 
comes  from  the  painstaking  search  of  the  literature  by  H.  H.  Donaldson, 
who  more  than  anyone  else  is  responsible  for  the  popularity  of  the  Norway 
rat  for  scientific  research.  His  book  The  Rat  gives  an  excellent  account  of 
the  history  of  this  animal  and  many  hundred  references  (1). 

The  wild  Norway  rat  did  not  reach  Europe  until  1730  and  America 
until  sometime  later,  near  the  end  of  the  18th  century.    In  Europe,  up 

i  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  29, 1954. 

2  This  work  was  carried  out  in  part  under  contracts  between  the  Office  of  The  Surgeon  General,  Department 
of  the  Army,  and  the  Office  of  Naval  Research,  Department  of  the  Navy,  and  the  Johns  Hopkins  University; 
and  also  in  part  under  a  grant  between  the  U.  S.  Public  Health  Service  and  the  Johns  Hopkins  University. 

727 

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728  proceedings:  symposium  on  25  years  or 

until  that  time,  another  rat,  Mus  rattus,  otherwise  known  as  the  Alexan- 
drine, roof,  or  ship  rat,  had  for  many  centuries  been  the  most  common 
rat.  In  America  this  rat,  not  a  native,  was  well  settled  at  the  time  of  the 
Norway  rat's  arrival.  In  all  instances  the  Norway,  the  fiercer,  more 
aggressive  rat,  soon  largely  replaced  the  less  virile  Alexandrine  rat. 

The  Norway  rat  originally  came  to  Europe  from  somewhere  north  of 
India  by  trade  routes  and  ships.  Once  ships  became  equipped  with  guards 
for  hawsers  this  means  of  transportation  was  more  or  less  ended  for  the 
heavy  clumsy  Norway,  but  still  open  for  the  light  nimble  Alexandrine  rat. 
At  the  present  time  many  ships  still  carry  rats,  but  only  the  Alexandrine 
rat. 

When  and  how  Norway  rats  were  first  domesticated  is  not  known.  All 
we  can  do  at  this  time  is  to  venture  a  few  guesses  on  the  basis  of  experience 
and  knowledge  derived,  in  part,  from  attempts  to  tame  trapped  wild  rats 
and  first-generation  wild  rats. 

Since  there  is  no  evidence  of  the  Norway  rats'  having  made  their  ap- 
pearance in  captivity  before  their  invasion  of  Europe,  the  process  of 
domestication  must  have  started  sometime  after  their  first  arrival  in 
Europe  in  1730. 

They  did  not  make  their  appearance  in  scientific  laboratories  until  much 
later.  In  1856,  Waller  and  Philipeaux  (2)  used  them  for  experiments  on 
the  effects  of  adrenalectomy,  which  curiously  is  still  one  of  their  chief 
uses  at  the  present  time.  This  is  the  earliest  record  that  we  have  been 
able  to  find,  so  far,  of  their  use  for  scientific  purposes.  These  authors 
referred  to  the  albinos  as  Mus  rattus,  but  it  is  more  likely  that  these  rats 
were  of  the  Norway  strain,  since  there  was  a  wrong  usage  of  the  names  at 
that  time. 

Long  before  Philipeaux,  the  great  French  physiologist,  Magendieis 
reputed  to  have  recommended  the  use  of  rats  for  physiological  experiments. 
So  far,  however,  we  have  not  been  able  to  find  any  record  that  he  himself 
actually  used  them. 

After  that,  they  were  used  quite  sporadically  by  various  workers  in 
Europe,  and  also  in  America,  until  the  time  of  the  founding  of  the  Wistar 
Institute  in  1907  and  the  establishment  of  the  first  standard  strain  of 
albino  rats.  Donaldson  was  first  introduced  to  them  in  1893  by  my  former 
chief  Adolf  Meyer  (8).  Of  the  many  strains  of  Norway  rats  now  known, 
the  Wistar  is  most  widely  used.  The  source  of  the  domesticated  rats 
used  in  this  country  remains  unknown,  that  is,  we  don't  know  whether 
they  came  from  this  country  or  from  Europe. 

In  what  form  and  how  the  Norway  rat  first  made  its  appearance  in  the 
laboratory  is  not  known.  For  several  reasons  we  believe  it  came  in  its 
albino  form.  The  rats  that  Waller  and  Philipeaux  used  were  albinos, 
and  with  few  exceptions,  present-day  domesticated  Norway  rats  all  over 
the  world  are  albinos.  Their  clean  white  appearance  has  undoubtedly 
had  much  to  do  with  their  popularity.  Defects  in  vision,  owing  to  a 
lack  of  pigmentation,  may  have  tended  to  make  them  less  apt  to  escape 
and  easier  to  handle. 

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PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  729 

It  is  also  quite  likely  that  Norway  rats  came  into  captivity  as  albinos. 
We  know  that  rat-baiting  was  popular  in  France  and  England  as  early  as 
1800,  and  in  America  soon  afterwards.  This  sport  flourished  for  seventy 
years  or  more,  until  it  finally  was  stopped  by  decree.  In  this  sport  100  to 
200  recently  trapped  wild  Norways  were  placed  at  one  time  in  a  fighting 
pit.  A  trained  terrier  was  put  into  the  pit.  A  keeper  measured  the  time 
until  the  last  rat  was  killed.  Sportsmen  bet  on  the  killing  times  of  their 
favorite  terriers.  For  this  sport  many  Norway  rats  had  to  be  trapped  and 
held  in  pounds  in  readiness  for  contests.  Records  indicate  that  albinos 
were  removed  from  such  pounds  and  kept  for  show  purposes  and/or 
breeding.  It  is  thus  very  likely  that  these  show  rats,  that  probably  had 
been  tamed  by  frequent  handling,  found  their  way  at  one  time  or  another 
into  laboratories.  That  albino  rats  do  appear  with  some  frequency  is 
known  from  the  fact  that  Hatai  obtained  albinos  in  2  of  6  third-generation 
litters  of  wild  Norway  rats  that  were  bred  in  his  laboratory  (4). 

In  our  experience  wild  Norway  rats,  when  trapped  while  young,  can 
be  trained  by  constant  handling  so  that  they  can  be  held  in  the  hand 
without  danger.  Although  obviously  highly  nervous,  they  remain  tame 
as  long  as  they  are  handled  and  well  fed.  When,  however,  they  are  not 
handled  and  are  left  to  themselves,  they  generally  become  wild  and 
unmanageable  and  make  use  of  the  first  opportunity  to  escape.  For 
this  reason,  and  since  children  quickly  tire  of  them,  rats  have  never  made 
good  pets. 

Once  Norway  rats  were  brought  into  laboratories  their  obvious  virtues 
for  scientific  work  over  the  then  popular  rabbit  must  quickly  have  become 
apparent.  From  generation  to  generation  they  became  more  tractable, 
less  apt  to  escape  (as  will  be  explained  below)  and  so,  soon  changed  from 
their  role  as  captive  wild  rats  to  domesticated  rats. 

The  domesticated  Norway  rat's  advantages  for  scientific  research  may 
be  listed  in  terms  of: 

1)  Size. — It  is  just  large  enough  to  be  handled  easily.  Its  organs 
are  large  enough  to  permit  almost  any  kind  of  operation  to  be  performed. 
It  is  small  enough  that  large  numbers,  literally  hundreds,  may  be  housed 
in  the  space  that  would  hold  only  a  few  dogs. 

2)  Diet. — Its  diet  is  almost  the  same  as  man's.  For  this  reason  much 
of  our  modern  knowledge  of  nutrition  has  come  from  experiments  on  rats. 

3)  Physiology. — Its  physiology  of  nerves,  muscles  and  glands  is  much 
the  same  as  man's.  It  has  a  stable  nervous  system  so  that  results  obtained 
at  one  time  may  be  repeated  at  another.     This  is  not  true  of  the  rabbit. 

4)  Reproduction. — It  breeds  very  readily  under  conditions  of  domes- 
tication. Many  litters  can  be  obtained  in  a  short  time  and  at  a  small 
expense. 

5)  Resistance  to  infection. — It  has  a  high  degree  of  resistance  to  many 
kinds  of  infection  which  greatly  adds  to  its  value  as  an  experimental 
animal,  since  time-consuming  aseptic  technique  need  not  be  used. 

6)  Handling. — Since,  after  domestication,  the  Norway  rat  remains 
tame  even  with  only  occasional  handling,  workers  can  remove  it  from 

Vol.    15,   No.   3,   December    1954 


730  proceedings:  SYMPOSIUM  ON  25  YEARS  op 

cages,  and  inject  it,  etc.,  without  running  the  risk  of  having  it  escape. 
In  this  way  it  contrasts  sharply  with  the  Alexandrine  rat  which  because 
of  great  fleetness  and  agility  cannot  be  handled  at  all. 

My  acquaintance  with  the  wild  Norway  rat  stems  from  work  carried 
on  during  the  second  world  war.  Before  that  time  I  had  scarcely  seen 
more  than  a  dozen  or  two  wild  rats  in  my  entire  life.  The  war  work  was 
concerned  with  poisons  and  baits  for  wild  rats  to  be  used  in  a  quick  city- 
wide  extermination  campaign,  should  such  an  operation  be  needed  (5). 
For  this  work  we  had  to  capture  wild  rats  and  bring  them  to  the  laboratory 
for  toxicity  and  poison  acceptance  tests  and  for  physiological  studies  on 
the  action  of  various  poisons,  involving  investigation  of  the  endocrine, 
nervous,  and  circulatory  systems.  It  quickly  became  obvious  that 
despite  the  external  similarities  between  the  wild  and  domesticated 
Norway  rat,  many  internal  differences  existed.  A  systematic  study  was 
undertaken  to  define  these  differences  and  to  determine  what  general 
biological  significance,  if  any,  they  have. 

During  the  past  15  years  many  thousand  wild  Norway  rats — rats 
captured  from  alleys,  cellars,  and  farms — have  been  used  in  my  laboratory 
for  all  kinds  of  investigations.  Simple  methods  have  been  devised  for 
trapping  them  alive  and  for  holding  them  for  injection  and  examination 
without  the  use  of  anesthetics.  More  than  a  thousand  rats  have  been 
used  for  experiments  on  the  effects  of  such  operations  as  adrenalectomy, 
hypophysectomy,  and  pancreatectomy.  An  excellent  opportunity  was 
thus  afforded  for  becoming  well  acquainted  with  this  animal.  The  most 
important  single  instrument  in  use  for  handling  these  wild  animals  is  a 
device  designed  by  Dr.  John  T.  Emlen  for  holding  unanesthetized  rats  (6). 
It  has  made  it  possible  to  handle  the  fiercest,  wildest  rats  without  danger 
and  almost  as  readily  as  tame,  domesticated  rats.  A  modified  rabbit  trap 
is  used  in  trapping  the  wild  rats  (7).  We  have  caught  as  many  as  300 
rats  from  one  square  block  in  Baltimore  over  a  10-day  period.  The  traps 
are  inexpensive  and  are  now  being  widely  used  for  trapping  wild  rats 
in  other  cities. 

Comparisons  were  made  between  captured  wild  rats  and  domesticated 
rats  from  our  colony,  which  has  been  in  existence  for  over  30  years.  Our 
original  albino  stock  came  from  the  Wistar  colony  and  about  28  years  ago 
a  few  piebald  and  hooded  rats  were  added  from  the  colony  of  Dr.  E.  V. 
McCollum.  Since  then  no  new  strains  have  been  added  and  from  the 
beginning,  the  diet  and  external  conditions  have  remained  the  same. 
In  physiological  responses  as  well  as  in  weight  of  organs  and  glands,  our 
rats  are  very  much  like  those  from  other  laboratory  colonies.  No  con- 
scious effort  has  been  made  to  breed  the  rats  for  auy  special  character- 
istics. 

The  wild  rats  were  trapped  in  the  city  of  Baltimore  or  on  outlying 
farms.  Some  were  bred  in  the  laboratory  and  studies  were  made  of  their 
offspring. 

Part  of  the  data  presented  here  has  been  reported  in  detail  in  earlier 
papers  and  part  is  being  reported  for  the  first  time. 

Journal   of  the  National  Cancer  Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  731 

Anatomical  Differences 

Here  I  shall  pick  out  only  a  few  of  the  more  striking  differences.  Some 
organs  have  become  smaller  during  domestication.  For  instance,  as  was 
first  shown  by  Watson  (8)  and  Donaldson  (9),  and  later  by  Rogers  and 
Richter  (10),  the  adrenals  may  be  one-third  to  one-fifth  as  large  as  in  the 
wild  rat;  a  reduction  of  cortical  tissue  accounts  for  the  entire  difference. 
The  preputial  glands  have  likewise  become  much  smaller  (11)  as  have  also 
the  liver,  spleen,  brain,  heart,  kidneys  and  pancreas.  Other  glands  have 
become  larger,  for  instance  the  hypophysis  and  thymus,  and  other  organs 
have  developed  earlier,  such  as  the  gonads  (12).  The  number  of  Peyer's 
patches  in  the  intestines  of  each  rat  has  increased  from  16.3  to  18.9  (IS) 
and  the  number  of  fungi-form  papillae  on  the  tongue  of  each  rat  has  been 
markedly  reduced  from  an  average  of  217.9  to  178.3  (H).  The  changes  in 
thyroid  weights  are  not  definite. 

Physiological  Differences 

Here  again  I  shall  select  only  a  few  outstanding  differences.  The 
domesticated  rat  has  a  lower  metabolic  rate  as  is  shown  by  its  lower  food 
intake  per  kilogram  of  body  weight  and  also  by  its  lower  water  intake. 

It  has  a  lower  resistance  to  poisoning  with  the  several  compounds 
tested,  especially  thiourea  (15). 

On  a  normal  diet,  domesticated  rats  show  audiogenic  fits  when  exposed 
to  sounds  of  high  frequency  but  it  has  not  yet  been  possible  to  put  a 
single  wild  rat  into  a  fit  in  this  way  (16).  On  a  magnesium-deficient  diet, 
all  domesticated  rats  show  audiogenic  fits  and  die  within  a  few  days; 
on  the  other  hand,  although  magnesium-deficient  wild  rats  show  fits  they 
do  not  die  as  a  result  (17). 

Behavioral  Differences 

Apart  from  the  differences  in  behavior  described  above,  a  few  others 
may  be  mentioned. 

In  the  first  place,  domesticated  rats  have  lost  a  very  characteristic 
high-pitched  squeak  or  squeal  that  is  characteristic  of  the  wild  rat  when 
frightened. 

Domesticated  rats  do  not  huddle  together  as  wild  rats  do.  Wild  rats 
pack  themselves  very  closely  together  in  a  corner  against  the  walls  of  a 
cage  and  remain  motionless  literally  for  hours. 

With  few  exceptions,  wild  rats  kill  small  rats  or  mice  as  rapidly  as  they 
are  fed  into  their  living  cages.  Some  rats  repeatedly  bite  almost  any  part 
of  the  bodies  of  such  small  rats,  but  others  bite  only  once  and  then  through 
the  spinal  cord  at  the  base  of  the  brain.  Except  under  special  circum- 
stances, domesticated  rats  pay  little  or  no  attention  to  small  rats  or  mice 
that  are  thus  introduced  into  their  cages.  One  such  special  circumstance 
occurred  when  a  domesticated  lactating  mother  that  had  nursed  a  litter 
of  15  rats  for  14  days,  in  one  night,  killed  all  of  the  babies — each  one 

Vol.    15,  No.   3,  December   19S4 


732  proceedings:  symposium  on  25  years  of 

with  a  single  finely  executed  bite  through  the  spinal  cord  at  the  base  of 
the  brain. 

In  both  strains  of  rats,  fasting  increases  the  spontaneous  activity  as 
measured  in  revolving  drums,  but  the  increase  in  activity,  during  fasting, 
is  over  4  times  as  high  in  the  wild  as  in  the  domesticated  rats  (18).  It 
would  thus  appear  that  in  a  free  environment  the  wild  rats  would  have  a 
much  greater  chance  of  coming  into  contact  with  food,  through  their 
greater  activity. 

Studies  in  our  so-called  "fighting  chamber' '  brought  out  a  further 
difference.  This  chamber  consists  of  a  box  12  by  12  by  18  inches  with  a 
glass  front  and  a  floor  made  of  parallel  iron  rods  spaced  at  intervals  of  three 
quarters  of  an  inch,  and  alternately  wired  to  the  opposite  poles  of  an 
induction  coil.  A  pair  of  wild  rats  placed  in  such  a  cage  and  given  a  single 
shock  will  start  fighting  at  once,  and  only  an  occasional  shock  suffices  to 
keep  them  fighting.  Evidently  each  one  holds  the  other  responsible  for 
the  inflicted  pain.  However,  a  pair  of  domesticated  rats  will  not  fight,  no 
matter  how  severe  the  punishment,  but  each  one  simply  tries  to  escape. 

Wild  rats  are  much  more  difficult  to  poison  since  they  are  so  suspicious 
of  all  changes  in  their  food.  When  repeatedly  threatened  with  poisoning, 
they  may  ultimately  refuse  all  food  and  starve  themselves  to  death  (19). 
We  have  never  seen  a  domesticated  rat  starve  itself  to  death  in  this  way. 

These  two  strains  of  rats  have  a  very  different  reaction  to  physical 
restraint:  that  is,  being  held  in  the  experimenter's  hand  and  thus  prevented 
from  biting,  scratching,  or  escaping.  For  the  wild  rats,  as  for  most  other 
wild  animals,  this  is  obviously  a  terrifying  situation.  They  struggle 
violently  for  a  few  minutes  and  then  often  seem  to  become  limp.  A  few 
rats  react  so  violently  that  they  actually  die  within  minutes.  In  marked 
contrast,  domesticated  rats  react  only  mildly,  if  at  all,  to  this  restraint 
and  tend  to  accept  it  with  little  resistance. 

Electrocardiographic  records  taken  while  the  rats  are  being  restrained 
show,  in  the  case  of  wild  rats,  marked  slowing  of  the  heart — a  definite 
bradycardia,  and  little  or  no  change  in  the  domesticated  rats.  Thus  it 
appears  that,  as  compared  with  wild  rats,  domesticated  rats  are  much  less 
vagotonic. 

That  the  wild  rats  react  so  much  more  violently  to  physical  restraint 
and  actually  succumb  in  some  instances,  does  not  mean  that  they  are  less 
able  to  meet  stress  in  general.  It  is  merely  that  restraint  bothers  them 
much  more  than  it  does  the  domesticated  rat. 

Pattern  of  Domestication 

Do  the  changes  in  anatomy,  physiology  and  behavior  seem  to  follow  any 
definite  pattern?  We  believe  that  a  pattern  is  emerging  from  observations 
made  so  far,  particularly  on  the  adrenals,  gonads  or  hypophysis. 

We  have  found  that  during  the  process  of  domestication  the  adrenals 
apparently  are  becoming  less  important  to  the  rat,  whereas  the  gonads  are 
becoming  more  important;  and  likewise  that  the  hypophysis  may  be 
becoming  more  important  (12). 

Journal    of    the   National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  733 

What  evidence  do  we  have  for  these  statements? 

First  of  all,  the  adrenals  are  one-third  to  one-fifth  as  large  in  the  domesti- 
cated as  in  the  wild  rats.  Cortical  tissue  accounts  for  the  entire  difference. 
By  lipid  stains  it  was  shown  that  in  the  domesticated  rats  the  outer  layer 
of  the  cortex,  the  glomerulosa  or  salt-regulating  layer,  is  much  less  active; 
and  that  likewise  the  next  layer,  the  fasciculata  or  corticoid-secreting 
layer,  whose  secretion  helps  the  animal  to  meet  stress,  is  much  less  active 


Second,  as  is  known,  the  glomerulosa  layer  has  salt  regulatory  functions, 
in  that  secretions  from  this  layer  help  to  conserve  salt — preventing  salt 
from  being  washed  out  of  the  body  into  the  urine.  After  adrenalectomy 
both  domesticated  and  wild  rats  voluntarily  drink  more  salt  solution. 
However,  the  domesticated  rats  survive  and  remain  in  good  health  while 
drinking  only  slightly  increased  amounts  of  salt  solution,  but  the  wild  rats, 
on  the  contrary,  do  not  survive  adrenalectomy  after  treatment  with  any 
amount  of  salt.  They  need  in  addition  large  amounts  of  cortical  hormone, 
and  even  then  they  do  not  survive  with  any  consistency  {21). 

That  in  normal  wild  rats,  the  adrenal  functions  far  more  effectively  is 
shown  by  the  observation  that  wild  rats  are  able  to  survive  long  periods  of 
time,  even  months,  on  a  very  low  sodium  diet,  without  any  visible  effects, 
whereas  domesticated  rats  succumb  in  a  short  time. 

The  wild  rats  conserve  salt  so  well  that  salt,  when  not  accompanied  by 
adequate  amounts  of  water,  is  actually  toxic  to  them,  even  in  very  small 
amounts.  Domesticated  rats  are  able  to  take  much  larger  amounts  of  salt 
with  impunity. 

Many  workers  (22)  have  established  that  the  changes  in  the  amount  of 
ascorbic  acid  in  the  adrenals  give  a  measure  of  the  severity  of  the  reaction 
of  an  animal  to  various  forms  of  stress.  The  greater  the  drop  in  ascorbic- 
acid  levels,  the  more  severe  is  the  reaction  to  stress. 

Domesticated  and  wild  rats  were  subjected,  by  Dr.  James  Woods,  to  the 
same  form  of  stress:  either  exposure  to  cold,  fighting,  or  exhaustion  from 
swimming  {23).  In  the  wild  rat  none  of  these  forms  of  stress  had  any 
detectable  effect  on  the  amount  of  ascorbic  acid  in  the  adrenals,  whereas 
in  the  domesticated  rat  it  either  reduced  the  ascorbic-acid  content  to  a 
very  low  level  or  else  eliminated  it  altogether.  In  wild  rats,  the  ascorbic- 
acid  content  could  be  depressed  only  by  large  doses  of  ACTH.  These 
results  would  thus  indicate  that  the  wild  rat  is  better  able  to  withstand 
various  forms  of  stress — because  of  the  more  active  adrenals. 

This  evidence  indicates  that  the  adrenal  secretions  necessary  both  for 
salt  metabolism  and  for  reaction  to  stress  are  more  effective  in  the  wild 
than  in  the  domesticated  rat. 

It  has  already  been  reported  that  the  gonads  develop  earlier  in  the 
domesticated  than  in  the  wild  rat,  and  that  domesticated  rats  mate  and 
reproduce  more  freely  and  more  often.  In  the  domesticated  rat  gross 
bodily  activity,  as  measured  in  revolving  drums,  has  been  shown  to  be 
dependent  on  gonadal  secretion,  since  after  gonadectomy  the  rats  become 
very  inactive  (24).    The  average  daily  activity  may  drop  from  18,000 

Vol,    15,   No.   3,   December    1954 


734  proceedings:  symposium  on  25  years  op 

revolutions  to  only  a  few  hundred.  However,  in  the  wild  rat,  gonadectomy 
has  no  detectable  effect  on  the  level  of  running  activity,  since  the  rats  are 
quite  as  active  after  as  before  surgery  {12).  Apparently  secretions  from 
the  larger  adrenals  keep  the  wild  rats  active.  That  this  is  so,  was  shown  by 
the  results  of  experiments  in  which  domesticated  rats  were  started  on 
cortisone  immediately  after  gonadectomy.  They  remained  active  after 
gonadectomy  just  as  the  wild  rats  do  without  treatment. 

Observations  have  been  made  by  Davis  {25),  and  Davis  and  Emlen  {26), 
and  others  on  wild  rats  caught  in  the  field,  on  the  time  of  opening  of  the 
vagina,  incidence  of  pregnancy,  and  the  time  of  the  signs  of  first  pregnancy. 
Comparison  shows  that  the  domesticated  rats  mature  at  an  earlier  age  and 
show  a  higher  degree  of  fertility.  All  of  this  evidence  thus  indicates  that 
gonadal  secretions  have  become  more  important  with  domestication. 

We  have  seen  that  the  hypophysis  has  become  larger  in  the  domesticated 
rat.  David  Wood  has  found  that  hypophysectomy,  which  makes  a  domes- 
ticated rat  almost  totally  inactive,  has  a  much  less  depressive  effect  on 
some  wild  rats;  whereas  others,  especially  very  old  and  heavy  rats,  do  not 
survive  the  operation  {27). 

It  is  possible  that  in  the  domesticated  rat  the  hypophysis  has  become 
enlarged  in  an  effort  to  correct  for  the  failing  secretion  of  the  adrenal 
glands.  Thus  we  may  see  here  a  pattern  of  less  active  adrenals,  more  active 
gonads,  and  a  more  active  hypophysis.  Further  studies  are  in  progress  on 
that  relationship  by  David  Wood. 

At  this  point  I  should  like  to  say  a  word  about  the  neurology  of  domes- 
tication. What  changes,  if  any,  have  occurred  in  the  nervous  system? 
A  few  observations  on  the  effects  of  brain  lesions  in  wild  and  domesticated 
rats  have  been  made.  Removal  of  the  frontal  poles  of  the  brain  makes 
domesticated  rats  just  as  savage  as  wild  rats  {28).  This  is  best  shown  by 
their  reaction  when  confronted  with  mice  or  small  rats.  One  such  domes- 
tic rat  killed  small  rats  just  as  fast  as  they  could  be  fed  to  her  and  always 
with  the  same  clear-cut  bite — through  the  brain  stem  at  the  base  of  the 
head,  with  much  the  same  technique  used  by  wild  rats.  These  results 
would  indicate  that  during  domestication  a  change  may  have  occurred  in 
this  part  of  the  brain.  Secondly,  it  has  been  shown  by  J.  Woods  that 
wild  rats  can  be  made  just  as  tame  as  domesticated  rats  by  lesions  placed 
in  the  amygdaloid  area  of  the  brain  {29) .  We  are  now  studying  domesti- 
cated rats  with  frontal  lobectomies  and  wild  rats  with  amygdaloid  lesions 
to  determine  whether  these  operations  are  followed  by  changes  in  the 
endocrine  glands  and  other  organs,  as  well. 

Domestication  Mechanisms 

How  can  we  explain  these  various  changes  that  have  occurred  during 
the  process  of  domestication  of  the  rat? 

The  wild  Norway  rat  lives  in  an  environment  in  which  it  must  con- 
stantly be  on  the  alert  and  ready  to  fight  for  its  existence.     It  has  to 

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defend  itself  against  all  kinds  of  enemies:  other  rats,  dogs,  cats,  owls, 
and  snakes,  as  well  as  man.  It  is  a  fierce,  aggressive,  suspicious  animal 
that  attacks  at  the  least  provocation  and  in  captivity,  takes  advantage 
of  the  least  opportunity  to  escape,  remains  suspicious  and  tense,  and  breeds 
poorly. 

In  marked  contrast,  the  healthy  domesticated  Norway  rat  is  tame, 
gentle,  and  trusting,  does  not  bite  unless  frightened  or  hurt,  and  makes  no 
attempt  to  escape.  It  lives  placidly  in  the  controlled  environment  of  the 
laboratory  where  food,  water,  shelter  and  safety  are  constantly  assured. 
Its  only  contributions  to  its  own  survival  are  its  feeding,  drinking,  groom- 
ing and  mating  activities.  It  reproduces  at  an  early  age  and  with  a  rapid 
rate.  Like  other  domesticated  animals  it  has  shown  numerous  mutations. 
Castle,  a  pioneer  worker  in  this  field,  listed  23  strains  that  breed  true  (30) . 

We  believe  that  selection  has  played  the  most  important  part  in  the 
process  of  domestication.  By  selection  we  mean  here  not  the  natural 
selection  of  wild  rats  in  their  natural  habitat,  where  the  fiercest,  wildest 
and  strongest — the  fittest  for  that  type  of  environment — survive,  but 
selection  in  the  artificial  environment  of  the  laboratory  where  the  fittest 
for  this  type  of  environment  survive:  those  that  are  most  gentle  and  fertile. 
The  two  life  stages  in  which  the  selection  process  has  the  most  effect  are 
during  mating  and  nursing.  In  captivity  wild  rats  do  not  mate  well.  In 
only  a  few  out  of  many  instances  when  rats  are  put  together  does  a  preg- 
nancy result.  It  is  likely  that  only  the  tamest  of  the  rats  mate.  This 
is  probably  true  of  each  succeeding  generation  as  well — so  that  the  tamer 
rats  are  more  apt  to  propagate  their  own  characteristics,  including  pre- 
sumably the  smaller  adrenals,  etc.  After  the  young  are  born,  wild 
mothers,  with  rare  exception,  eat  their  entire  litter,  so  that  at  this  stage 
a  severe  screening  process  occurs.  The  rats  that  do  survive  these  two 
stages  must  be  the  least  apprehensive,  and  so  through  successive  genera- 
tions they  should  produce  progressively  tamer  animals. 

How  long  did  it  take  to  domesticate  the  rat?  This  we  do  not  know. 
After  25  generations  the  wild  rats  of  King  and  Donaldson  had  not  yet 
quite  reached  the  various  weight  levels  characteristic  of  domesticated 
rats  (31,  32). 

So  far  our  studies  have  been  concerned  almost  entirely  with  differences 
between  the  captured  wild  rat  and  the  tame  domesticated  rat.  Little 
or  nothing  has  been  done  with  the  underlying  genetic  problem  to  establish 
just  what  part  of  the  difference  depends  on  experience,  and  what  part  on 
inheritance.  To  fill  this  gap  1)  we  have  placed  domesticated  rats  in  a 
wild  free  environment  and  are  observing  the  effects  on  behavior,  anatomy 
etc.,  and  2)  we  are  planning  to  study  hybrids  of  the  two  strains.  Results 
of  preliminary  observations  indicate  that  the  hybrid  offspring  changes  to 
become  more  like  the  domesticated  than  the  wild  strain,  and  3)  we  are 
planning  also  to  study  rats  born  from  fertilized  eggs  of  one  strain  placed 
in  the  uteri  of  the  other  strain.  This  should  clarify  the  role  of  environ- 
mental influences. 


Vol.   IS,  No.  3,  December  1954 


736  proceedings:  symposium  on  25  years  op 

Domestication  in  the  Rat  and  Civilization  in  Man 

Finally  I  want  to  make  a  few  remarks  about  what  light,  if  any,  these 
observations  throw  on  the  domestication  of  man.  I  trust  Dr.  Snyder 
will  forgive  this  transgression  into  his  part  of  the  program. 

We  know  that  man,  like  the  wild  Norway  rat,  originally  lived  in  an 
environment  in  which  he  had  to  search  for  his  food,  provide  his  own  shelter, 
and  fight  for  his  mates — an  environment,  in  short,  in  which  his  fitness, 
hence  his  survival,  was  measured  by  his  physical  activity,  aggressiveness 
and  ability  to  withstand  violent  changes.  But  with  the  growth  of  com- 
munities and  the  consequent  increase  in  security  a  new  environment 
developed  in  which  man  was  protected  and  a  livelihood  was  almost  guaran- 
teed. Man  must  thus  have  worked  out  a  controlled  environment  for 
himself  in  which  a  transformation  occurred,  somewhat  like  that  under- 
gone by  the  Norway  rat  in  its  adaptation  to  colony  life  in  the  laboratory, 
resulting  in  an  increase  and  perhaps  even  the  predominance  of  the  so- 
called  weaker,  or  the  milder,  better  adjusted  individuals. 

Here,  just  as  in  the  domestication  of  the  rat,  a  selective  process — the 
selection  of  the  fittest  for  this  type  of  environment — must  have  played 
the  most  important  part.  This  process  has  gone  far  in  our  present-day 
society. 

It  has  not  been  possible  to  obtain  gland  weights  of  modern  and  prima- 
tive  man — especially  of  the  adrenals  and  gonads  to  determine  what  changes 
if  any  have  occurred.  There  are,  however,  indications  that  marked 
changes  may  have  occurred  in  these  glands.  At  the  present  time  it  has 
been  estimated  that  over  12  million  individuals  in  this  country  suffer 
from  hypertension,  rheumatism,  arthritis,  etc.,  all  of  which  may  have 
their  origin  in  deficient  functions  of  the  adrenal  glands  since  they  respond 
so  remarkably  to  treatment  with  cortisone  and  ACTH.  That  a  perma- 
nent deficiency  underlies  most  of  these  diseases  is  known  from  the  fact 
that  treatment  with  cortisone  and  ACTH  gives  relief  as  long  as  it  is 
continued. 

Similarly,  we  know  that  many  individuals  suffer  from  neoplastic  dis- 
eases, some  of  which  apparently  have  their  origin  in  or  are  greatly  influenced 
by  hyperactivity  of  the  gonads,  since  reduction  or  elimination  of  the 
gonadal  secretion  through  gonadectomy  so  remarkably  arrests  the  growth 
of  these  neoplasms,  and  treatment  with  sex  hormones  greatly  accelerates 
the  growth  of  this  group.  Could  this  be  an  indication  of  a  shift  in  the 
direction  of  domestication? 

Likewise,  modern  man  is  eating  more  and  more  salt,  and  in  fact,  the 
eating  of  large  amounts  of  salt  has  almost  become  a  characteristic  of  civil- 
ization. The  domesticated  rat  likewise  eats  and  is  able  to  tolerate  very 
large  amounts  of  salt.  Are  the  individuals  who  do  not  tolerate  salt  those 
who  have  not  been  so  much  affected  by  the  domestication  process,  and 
are  more  like  the  wild  rat? 

Thus,  in  summary,  the  Norway  rat,  the  first  animal  to  be  domesticated 
for  experimental  purposes,  has  during  the  course  of  its  domestication  and 
during  its  transition  from  the  free  environment  of  its  wild  habitat  to  the 

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PKOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  737 

controlled  conditions  of  the  laboratory,  undergone  marked  anatomical, 
physiological,  and  behavioral  changes.  This  study  has  offered  for  the 
first  time,  the  unique  opportunity  of  observing  a  shift  of  the  interrelations 
of  glands  rather  than  of  the  function  of  individual  organs.  The  secretion 
from  one  gland,  the  adrenal,  has  become  less  important  and  the  secretion 
from  the  gonads  more  important.  The  secretions  of  the  hypophysis  have 
become  more  important  but  their  relation  to  the  secretions  from  the  other 
two  glands  has  not  yet  been  definitely  defined.  It  is  possible  that  this 
general  shift  will  be  found  to  involve  other  glands  as  well,  and  also  that 
the  various  forms  of  changes  in  behavior  will  eventually  fall  into  relation- 
ship with  this  general  pattern  of  domestication  changes.  The  possibility 
must  thus  be  considered  that  similar  changes  and  shifts  may  have  occurred 
in  various  degrees  in  man  during  the  transition  from  his  original  free  envi- 
ronment to  the  highly  protected  and  controlled  environment  of  modern 
society. 

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The  Effects  of  Selection  and  Domesti- 
cation Upon  the  Behavior  of  the  Dog  u  2 


J.  P.  Scott,3  Division  of  Behavior  Studies,  Roscoe 
B.  Jackson  Memorial  Laboratory,  Bar  Harbor, 
Maine 


Richter  has  shown  here  and  elsewhere  (1)  how  laboratory  domestication 
has  produced  drastic  changes  in  the  behavior  of  the  Norway  rat.  These 
changes,  which  have  their  counterparts  in  almost  all  domestic  animals, 
appeared  within  a  few  generations,  and  the  rat  is  still  less  than  a  century 
old  as  a  domestic  species.  It  is  therefore  of  considerable  interest  to  com- 
pare it  with  the  dog,  which  has  probably  been  domesticated  for  something 
like  80  centuries.  Data  on  the  rat  give  us  information  regarding  the  ease 
with  which  the  changes  of  domestication  can  take  place,  and  data  on  the 
dog  give  indications  as  to  whether  or  not  the  process  is  a  progressive  one. 

Most  of  the  typical  changes  of  domestication  are  seen  in  an  exaggerated 
form  in  the  dog.  The  most  obvious  effect  is  increased  variability  com- 
pared to  the  wild  species,  both  in  anatomical  characteristics  and  in 
behavior.  Some  breeds  of  dogs  like  the  great  Danes  and  St.  Bernards 
are  larger  than  wolves;  others  like  Chihuahuas  and  Pekingese  are  much 
smaller.  Some  breeds  like  the  greyhounds  can  run  faster  than  wolves  and 
others  like  dachshunds  are  obviously  much  slower.  Most  breeds  of  dogs 
are  less  aggressive  than  wolves  but  others  are  much  more  so;  e.g.,  certain 
terriers  will  attack  bears  and  mountain  lions,  while  a  wolf  pack  will  only 
attempt  to  drive  a  bear  away  if  it  approaches  the  den. 

A  second  effect  which  is  seen  in  almost  all  domestic  animals  is  a  tendency 
toward  early  sexual  maturity  and  increased  fertility.  Wolves  do  not 
become  sexually  mature  until  the  second  year,  whereas  the  vast  majority 
of  dogs  mature  before  the  age  of  one  year.  The  average  litter  size  in 
wolves  is  about  7  and  the  largest  recorded  litter  is  14  [Young  and  Gold- 
man (#)].  The  average  litter  size  for  all  dogs  is  probably  about  6,  but 
the  average  in  a  breed  such  as  the  great  Dane  runs  9.1  and  a  litter  of  16 

i  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  29, 1954. 

*  This  paper  is  based  on  a  research  project  in  genetics  and  social  behavior  begun  in  1945. 

3  Thanks  are  due  to  the  many  individuals  who  have  contributed  to  the  paper,  particularly,  Dr.  C.  C.  Little, 
under  whose  general  direction  the  work  was  done  and  without  whose  knowledge  and  experience  of  dog  breeds, 
progress  would  have  been  slow  and  difficult,  and  Dr.  John  L.  Fuller,  who  bears  co-responsibility  for  the  project. 
The  author  ajso  wishes  to  express  his  appreciation  to  the  many  research  assistants  who  have  helped  gather  the  data, 
including  Edna  DuBuis,  Mary-'Vesta  Marston,  Margaret  Charles,  and  others  (especially  Albert  Pawlowski  and 
Ann  Causey,  who  assisted  greatly  in  the  preparation  of  this  paper).  Thanks  are  also  due  to  Dr.  Eloise  Gerry  who 
contributed  our  original  stock  of  basenji  dogs. 

739 

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740  proceedings:  symposium  on  25  years  of 

was  recorded  in  one  group  of  48  [Little  aud  DuBuis  (3)].  In  many  smaller 
breeds  of  dogs  the  average  litter  size  is  lower  than  that  in  wolves,  and 
increased  fertility  is  therefore  typical  only  of  certain  breeds. 

A  third  effect  is  a  modification  of  the  process  of  socialization  with 
respect  to  human  beings  so  that  it  is  easier  to  get  domestic  animals  to  form 
attachments  to  people.  This  may  also  be  looked  at  as  a  tendency  to 
reduce  the  flight  distance,  and  Richter  (1)  has  demonstrated  a  remarkable 
change  in  the  adrenal  glands  which  accompanies  this  effect  in  the  rat.  In 
the  dog  there  is  also  a  tendency  to  develop  individuals  that  are  positively 
responsive  to  human  beings,  and  examples  of  both  of  these  effects  will  be 
treated  in  detail  below. 

Most  widely  used  domestic  animals  belong  to  species  which  have  a 
considerable  range  of  adaptability  and  this  may  be  one  reason  why  certain 
species  rather  than  others  have  been  domesticated.  However,  there  is  a 
tendency  under  domestication  to  select  individuals  for  particular  kinds 
of  behavioral  adjustment  and  thus  limit  or  extend  the  powers  of  adaptation. 
This  process  appears  to  have  been  carried  further  in  the  dog  than  any 
other  species,  while  only  limited  experiments  have  been  done  with  rats. 

The -Ancestry  of  Dogs 

The  family  Canidae. — To  this  family  belong  the  foxes  (Vulpes,  Urocyon, 
Alopex);  the  so-called  wild  dogs  including  Lycaony  the  African  hunting 
dog;  Icticyon,  the  South  American  bush  dog;  Cuon,  the  dhole  of  India; 
and,  finally,  the  true  dogs  and  wolves  belonging  to  genus  Cams.  Accord- 
ing to  Matthew  (4)  the  foxes  and  wolves  have  a  common  ancestor  in  the 
Miocene,  but  the  relationship  with  the  wild  dogs  is  much  more  remote 
and  a  common  ancestor  is  only  found  in  Cynodictus  of  the  Oligocene. 
From  the  geological  evidence  it  is  apparent  that  the  so-called  wild  hunting 
dogs  are  less  closely  related  to  the  domestic  dog  than  are  the  foxes. 

The  genus  Canis. — This  genus  includes  three  groups  of  animals:  the  true 
dogs,  Canis  familiaris  and  Canis  dingo;  the  wolves  such  as  the  northern 
wolf,  Canis  lupus  and  the  prairie  wolf  or  coyote  Canis  latrans;  and  the 
jackals  such  as  Canis  aureus.  Matthew  (4)  traces  dogs  and  wolves  back 
to  a  common  ancestor  in  Pleistocene  times  when  wolves,  coyotes,  jackals 
and  foxes  are  found  essentially  in  their  modern  forms.  Allen  (5)  postu- 
lated a  small  species  of  wolf  which  is  now  extinct  as  the  direct  ancestor  of 
the  dog,  but,  so  far,  no  such  remains  have  been  discovered.  All  the 
anatomical  evidence  indicates  that  the  dog  is  a  domesticated  variety  of 
wolf  and  that  the  closest  related  living  species  is  probably  Canis  lupus, 
which  is  found  in  the  arctic  and  temperate  regions  of  both  Eurasia  and 
North  America. 

At  various  times  authors  have  postulated  other  species  such  as  Canis 
latrans,  the  coyote,  and  Canis  aureus,  the  jackal,  as  ancestors  either  of 
dogs  or  certain  breeds  of  dogs,  but  the  evidence  seems  to  be  based  chiefly 
on  superficial  resemblances  in  size.  Detailed  examinations  of  skulls  and 
teeth  do  not  confirm  these  ideas. 

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PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  741 

Archeological  evidence. — For  the  most  part  archeologists  have  been 
interested  chiefly  in  human  history  and  very  few  studies  have  been  made 
of  dogs  except  as  the  incidental  accompaniment  of  human  remains.  In 
Europe  the  most  extensive  recent  investigation  has  been  made  by  Degerbol 
(6)  who  studied  the  prehistoric  dogs  of  Denmark.  The  older  remains  in 
Denmark  are  found  in  the  last  part  of  the  Old  Stone  Age  in  a  transition  to 
the  Neolithic  Age  but  may  possibly  be  older  as  indicated  by  pollen  analysis. 
This  discovery  has  been  dated  as  possibly  6,000  to  8,000  years  B.  C.  and 
at  the  present  time  can  be  only  accurately  placed  with  regard  to  Azilian 
culture.  The  fragments  of  dogs  which  were  found  belong  to  two  types, 
and  Degerbol  finds  that  the  larger  one  is  quite  similar  in  measurements  to 
Eskimo  sled  dogs  and  also  to  remains  found  in  later  European  cultures 
which  have  been  called  Canis  Jamiliaris  inostranzewi.  The  smaller  form 
he  finds  closely  similar  to  a  dog  whose  bones  were  discovered  in  Lake 
Ladoga  in  Russia  and  also  associated  with  the  Swiss  lake  dwellers  and 
which  has  been  named  Canis  Jamiliaris  palustris.  Allen  (5)  considers  that 
both  these  forms  are  closely  similar  to  the  modern  Eskimo  dogs.  These 
earliest  remains  differ  from  wolves  chiefly  in  the  fact  that  the  dogs  have, 
like  modern  breeds,  smaller-sized  teeth.  Haag  (7)  points  out  that  dogs 
were  buried  as  early  as  6,000  B.  C.  in  Egyptian  tombs  and  that  these  re- 
mains may  be  as  early  as  the  Denmark  finds.  It  is  to  be  hoped  that  future 
studies  with  the  new  techniques  of  radioactive  carbons  and  pollen  analysis 
will  produce  a  clearer  picture,  but  it  is  evident  that  the  dogs  were  domesti- 
cated on  the  continent  of  Europe  at  a  very  early  age. 

In  North  America  Allen  (5)  found  historical  evidence  that  when  the 
white  man  arrived  in  North  America  dogs  were  distributed  among  the 
native  Indian  tribes  from  Alaska  to  Tierra  del  Fuego  and  that  there  was 
a  great  deal  of  variability  in  the  different  regions.  There  was  the  Eskimo 
dog,  the  common  Indian  dog  of  moderate  size,  which  was  found  in  at 
least  eight  different  varieties  or  breeds,  and  the  small  Indian  dogs  which 
included  at  least  five  different  varieties.  Of  these  only  two  have  survived ; 
the  Eskimo  dog,  which  is  very  similar  to  the  Eskimo  dog  of  Europe,  and 
the  Mexican  hairless  dog.  Haag  (7)  has  recently  studied  skeletal  re- 
mains of  dogs  from  various  regions  of  North  America  and,  using  modern 
methods  of  statistical  analysis,  comes  to  the  conclusion  that  at  least  eight 
different  populations  can  be  separated.  Those  close  together  geograph- 
ically, as  in  Kentucky  and  Alabama,  tend  to  be  similar.  It  may  be 
concluded  that  each  Indian  tribe  possessed  dogs,  sometimes  of  more  than 
one  variety,  and  that  the  dogs  tended  to  vary  from  tribe  to  tribe. 

By  contrast,  only  one  form  of  dog  was  found  in  Australia,  the  dingo. 
Canis  dingo  apparently  existed  both  in  wild  and  semidomestic  forms  and 
probably  was  brought  by  the  aborigines  to  Australia  where  it  apparently 
became  a  separate  species  by  isolation. 

The  dogs  of  Africa  have  not  been  subjected  to  extensive  scientific  study 
but  were  found  in  various  breeds  all  over  the  continent  by  recent  white 
explorers.  In  many  places,  European  breeds  have  since  been  imported 
and  mixed   with  native   populations.     One   native  breed,   the  African 

Vol.    15,   No.   3,   December    1954 


742  proceedings:  symposium  on  25  years  of 

barkless  (basenji)  was  recently  imported  into  England  without  admixture, 
and  has  been  used  in  extensive  studies  to  be  reported  here. 

All  of  the  evidence  indicates  that  the  dog  was  domesticated  probably 
from  Canis  lupus  or  a  very  similar  species  of  wolf  in  Northern  Europe  or 
Asia  about  6,000  to  8,000  years  B.  C.  Once  domesticated  the  use  of  dogs 
spread  very  rapidly  over  the  world  and  in  modern  times  there  appears  to 
have  been  no  humanly  inhabited  continent  from  which  they  were  absent. 
Each  primitive  tribe  had  its  own  population  of  dogs  the  type  of  which 
differed  from  tribe  to  tribe.  Such  conditions  provide  the  small  isolated 
breeding  populations  postulated  by  Wright  and  others  for  rapid  evolution. 
This,  combined  with  the  undoubted  existence  of  differential  selection,  will 
account  for  the  remarkable  diversity  in  modern  dog  breeds.  In  historical 
times  there  has  been  a  great  deal  of  transporting  of  dogs  from  one  area 
to  another  by  explorers,  immigrants  and  dog  fanciers  with  the  resulting 
possibility  of  mixtures  of  the  populations.  On  the  other  hand,  in  civilized 
countries,  there  has  been  an  effort  to  maintain  separate  breeds  and  over 
100  of  these  are  recognized  by  the  American  Kennel  Club. 

The  Domestication  of  Wolves 

The  social  life  of  wolves. — We  cannot,  of  course,  discover  what  actually 
happened  when  the  wolf  was  first  domesticated  but  we  can  deduce  certain 
things  from  two  sources:  the  study  of  the  behavior  of  wild  wolves  and 
the  results  of  attempting  to  tame  them.  An  excellent  study  of  wolf 
behavior  has  been  done  by  Murie  (8),  and  Young  and  Goldman  (2)  have 
collected  observations  made  by  trappers  and  hunters.  A  detailed  com- 
parison of  these  descriptions  with  various  domestic  breeds  of  dogs  shows 
that  wolves  and  dogs  exhibit  the  same  basic  traits  of  behavior  [Scott  (9)]. 
There  is  a  great  deal  more  variability  in  dogs,  but  only  one  new  trait 
seems  to  distinguish  dogs  universally  from  their  wild  relatives.  This  is 
the  tail  carriage,  which  in  dogs  varies  from  a  sickle  shape  to  a  tight  curl 
but  in  wolves  is  almost  straight  when  relaxed.  Other  differences  be- 
tween dogs  and  wolves  have  been  alleged  such  as  the  fact  that  wolves 
have  no  bark,  but  all  careful  observers  agree  that  they  do  bark,  although 
not  as  much  as  some  breeds  of  dogs. 

The  wolf  puppies  have  a  relatively  long  period  of  dependency.  They 
are  weaned  about  7  or  8  weeks  of  age,  but  continue  to  receive  food  from 
the  adults  for  a  long  period  and  first  begin  to  hunt  for  themselves  at  about 
4  months  of  age.  The  wolf  puppies  are  left  at  the  den  while  the  parents 
and  other  members  of  the  pack  go  out  to  hunt  and  large  quantities  of 
food  are  brought  back.  Some  of  this  is  vomited  for  the  young  and,  in 
addition,  large  pieces  of  meat  and  bones  may  be  brought  back  and  cached 
near  the  den.  Young  and  Goldman  (#)  recount  an  incident  where  some 
150  pounds  of  disgorged  beef  were  found  near  a  wolf  den.  The  wolves 
are  scavengers  as  well  as  hunters  and  where  they  are  allowed  to  live  near 
human  habitation  are  frequently  seen  around  garbage  dumps. 

The  social  group  of  the  wolf  is  the  pack,  which  seems  to  be  based  on  a 
litter  of  animals  which  grows  up  together.     Occasionally  the  pack  may  be 

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PROGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER  743 

founded  by  a  single  pair  and  a  litter  which  stays  on  as  adults,  but  the 
young  litters  ordinarily  move  out  of  the  territory  to  form  new  packs.  As 
described  by  Murie,  the  home  life  of  wolves  is  highly  peaceful  and  coopera- 
tive. A  certain  amount  of  dominance  is  exhibited  while  feeding,  but  the 
members  of  the  pack  get  along  well  together  even  when  more  than 
one  male  is  present.  Strange  wolves,  however,  are  violently  repulsed 
and  driven  off.  Various  persons  have  described  attempts  to  rear  wolf 
cubs  taken  at  a  very  early  age  and  Murie  found  that  such  a  female  animal 
became  a  very  tractable  pet  and  that  there  was  little  difficulty  in 
handling  it,  although  caution  had  to  be  used  with  strangers. 

On  these  bases  we  may  reconstruct  the  domestication  of  the  wolf  as 
follows:  a  primitive  hunting  tribe  in  Europe  or  Asia  may  very  easily 
have  fallen  into  a  commensal  relationship  with  wolves  with  the  latter 
frequenting  the  village  refuse  heap  and  the  human  inhabitants  finding  it 
profitable  to  rob  the  wolf  dens  at  times  when  food  was  scarce.  At  some 
point,  a  young  wolf  puppy  was  caught  and  adopted  by  the  people  and 
nursed  and  fed  by  them.  This  animal  would  consider  the  human  group 
as  its  society  and  would  be  peaceful  and  tolerant  toward  them  and  as  it 
grew  older  would  have  a  tendency  to  go  out  and  hunt  and  bring  back 
food  to  the  village.  It  would  reject  the  wild  wolves  except  at  the  time  of 
mating  and  in  due  course  other  wolf  puppies  could  be  raised  in  the  village 
and  be  socialized  with  respect  to  man.  There  would  be,  of  course,  the 
difficulty  of  telling  the  tame  and  wild  wolves  apart  but,  when  the  tail 
mutation  occurred  in  the  domestic  form,  an  easy  method  of  identification 
would  be  established  and  preserved.  There  would  also  be  a  tendency  to 
select  for  certain  other  traits,  particularly  tameness  and  fertility.  Once 
the  domestic  strain  was  established,  the  use  of  these  animals  would  tend 
to  spread  rapidly  to  other  tribes. 

The  Effects  of  Domestication  Upon  the  Behavior  of  the  Dog 

Breeds  studied. — In  order  to  study  the  effects  of  heredity  in  causing 
the  variable  behavior  seen  in  dogs,  5  different  breeds  were  selected  for 
special  study,  and  representative  individuals  were  raised  in  a  similar 
environment  which  included  a  regular  program  of  training  and  testing 
from  birth  up  to  the  age  of  1  year.  The  requirements  were  that  these 
breeds  include  as  wide  a  variety  of  behavioral  types  as  possible,  that  they 
be  of  medium  size  so  that  they  would  be  easy  to  handle,  and  that  they 
would  be  reasonably  hardy  and  fertile  so  that  reliable  genetic  results 
could  be  obtained.  It  was  felt  that  physical  characteristics  such  as  short- 
leggedness  produced  an  obvious  effect  upon  behavior  which  was  unneces- 
sary to  demonstrate,  and  the  breeds  selected  were  reasonably  normal 
with  regard  to  sensory  and  motor  development. 

Dog  fanciers  classify  the  dog  breeds  according  to  the  use  to  which  they 
are  put  and  this  functional  classification  tends  to  result  in  grouping  some 
breeds  which  are  genetically  unrelated,  particularly  if  dogs  of  widely 
different  geographical  origin  are  included.  The  final  selection  included 
4  breeds  which  originated  in  the  British  Isles  (cocker  spaniels,  beagles, 

Vol.   15,  No.  3,  December  1954 


744  proceedings:  symposium  on  25  tears  of 

Shetland  sheep  dogs,  and  wire-haired  fox  terriers,  which  consequently 
have  in  the  past  had  opportunities  for  interbreeding,  and  one  breed  from 
Central  Africa,  which  according  to  all  records  should  have  been  completely 
separated  for  many  centuries.  The  African  barkless  breed  or  basenji,  was 
selected  for  crossbreeding  with  the  cocker  spaniel.  These  two  breeds  and 
the  tasks  which  they  ordinarily  perform  may  be  briefly  described  as  follows. 
The  African  basenji  originally  came  from  the  Belgian  Congo  and  our 
animals  are  all  descended  from  5  individuals  imported  into  England  in 
1935-1940  [Williams  (10)].  Little  information  is  available  about  their 
use  in  the  native  villages.  It  is  reported  that  they  are  used  as  hunting 
dogs  chiefly  in  the  pursuit  of  game,  but  also  are  able  to  trail.  They  are 
reported  to  be  related  to  the  Egyptian  greyhound  and  are  physically 
somewhat  similar.  It  is  probable  also  that  their  ancestors  ran  loose  in  the 
villages  and  acted  as  scavengers.  They  are  long-legged,  muscular,  and 
have  considerable  skill  at  climbing  and  manipulating  objects  with  their 
paws.  They  are  specialized  in  many  respects,  but  appear  to  be  primitive 
in  that  there  is  an  annual,  seasonal  breeding  cycle  which,  however,  takes 
place  in  the  autumn  instead  of  in  the  spring  as  with  the  wolf.  Basenjis 
bark  very  seldom  but,  when  frustrated,  make  a  variety  of  howling  noises 
which  are  peculiar  to  the  breed.  As  far  as  known,  they  have  never  been 
crossed  with  European  breeds. 

The  cocker  spaniel  is  mentioned  in  early  English  historical  records. 
They  are  related  to  the  whole  group  of  bird  dogs  and  other  spaniels,  and 
particularly  to  the  setters  which  were  originally  used  for  setting  birds 
for  the  net  and  were  taught  to  crouch  down  when  the  birds  were  located, 
in  order  to  be  out  of  the  way  of  the  net.  They  have  also  been  selected 
as  retrievers  and  are  reported  to  have  been  used  in  connection  with  the 
medieval  sport  of  falconry  in  which  they  found  the  birds  and  flushed  them 
so  that  the  hawks  could  attack.  They  are  very  tolerant  in  groups  and 
in  modern  times  have  been  very  successful  as  house  pets  as  well  as  bird 
dogs. 

The  plan  of  genetic  analysis. — At  this  point  it  will  be  well  to  introduce 
a  description  of  the  experimental  methods  used.  As  stated  before,  the 
animals  were  raised  in  a  standard  environment  in  which  most  of  the  varia- 
bility should  have  reflected  hereditary  differences  and  which  has  been 
described  in  detail  elsewhere  [Scott  and  Fuller  (11)].  It  was  found  that 
all  of  the  breeds  showed  a  great  deal  of  variability,  a  large  part  of  which 
appeared  to  be  hereditary  since  offspring  of  different  matings  gave  dif- 
ferent results.  In  order  to  reduce  this  variability  somewhat,  the  animals 
chosen  from  the  parent  strains  for  the  crossbreeding  experiment  were 
descended  from  one  brother  X  sister  mating  in  the  basenjis  and  from  two 
matings  of  a  single  male  with  his  sister  and  mother  in  the  case  of  the  cocker 
spaniels.  No  selection  of  these  individuals  was  used  except  that  the 
original  pairs  were  vigorous  and  healthy  animals.  As  it  turned  out  later 
these  did  not  necessarily  illustrate  the  extremes  of  either  breed  in  all 
characteristics. 

Journal   of   the  National   Cancer  Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  745 

Reciprocal  crosses  were  made  between  these  two  groups  of  siblings  and 
an  effort  was  made  to  obtain  offspring  from  at  least  4  different  pairs  in 
each  case,  giving  two  Fx  populations.  Fx  males  were  backcrossed  to  the 
mothers  so  that  backcross  and  Fi  animals  raised  by  the  same  mother 
could  be  paired.  Finally,  F2  populations  are  being  obtained  from  both 
crosses.  It  was  planned  to  obtain  at  least  two  litters  from  each  mating 
so  that  comparisons  could  be  made  between  littermate  and  nonlittermate 
siblings.  Because  of  the  fertility  and  relatively  long  life  of  the  dog,  it 
has  been  possible  to  get  relatively  large  numbers  of  animals  from  particular 
matings. 

There  was  no  way  of  predicting  the  experimental  results  in  advance 
but  the  experiment  was  set  up  in  such  a  way  that  the  results  could  be 
subjected  to  a  variety  of  statistical  analyses.  The  cross  was  made  in  the 
form  of  a  typical  Mendelian  experiment  and  can  be  analyzed  in  the  usual 
way.  The  results  can  also  be  analyzed  in  terms  of  analysis  of  variance, 
correlation  between  relatives,  and  factorial  analysis.  Such  incidental 
factors  as  litter  size,  age  of  mother  and  season  of  the  year  can  also  be 
analyzed.  The  only  limitation  of  the  experiment  is  the  matter  of  num- 
bers, as  it  is  impossible  to  raise  and  subject  to  detailed  behavioral  analysis 
more  than  about  12  litters,  or  50  or  60  dogs  per  year.  In  many  cases  this 
results  in  the  use  of  relatively  small  samples  compared  to  many  genetic 
experiments.  We  now  have  reasonably  large  samples  in  the  Fx  and  back- 
cross  generations  and  in  some  of  the  early  experiments  have  accumulated 
fairly  large  numbers  of  F2's.  In  the  following  paragraphs  a  sample  of  the 
genetic  results  so  far  obtained  will  be  presented. 

Theoretical  considerations. — There  is  every  theoretical  reason  for  an- 
ticipating that  genetic  differences  in  behavior  should  be  affected  by 
multiple  factors  rather  than  simple  inheritance.  A  method  for  analysis 
of  multiple  factor  data  was  devised  by  Castle  and  Wright  (12)  and  Wright's 
formula  for  the  analysis  of  backcross  data  is  given  below.     The  results  of 


N= 


its  use  have  recently  been  reviewed  by  Wright  (18),  who  has  pointed  out 
the  many  difficulties  that  underlie  its  application.  The  original  formula 
was  based  on  the  genetic  assumptions  that  the  parent  stocks  were  homo- 
zygous and  that  there  was  no  dominance.  Heterozygosity  does  not 
affect  the  formula  if  there  is  no  dominance  but  it  does  affect  it  if  dominance 
is  present.  The  modified  formula  for  the  backcross  to  the  recessive, 
assuming  dominance  but  no  heterozygosis,  is  given  below. 


N= 


M<Tbx2— o-jv) 


Vol.    15,  No.  3,  December   1954 


746  proceedings:  symposium  on  25  years  of 

The  basic  mathematical  assumption  which  lies  behind  these  formulae 
is  that  the  genetic  factors  are  additive  in  theft  effects  and,  as  Wright  (14) 
has  pointed  out,  it  is  often  necessary  to  transform  the  scale  of  measure- 
ments in  order  to  bring  out  the  additive  effect.  The  formula  also  assumes 
that  the  effects  of  random  environmental  factors  are  additive.  As 
pointed  out  in  another  paper  [Scott  (15)],  this  is  not  necessarily  correct 
in  the  case  of  environmental  factors  acting  upon  behavior.  In  fact,  there 
is  every  reason  for  believing  the  opposite  to  be  true  under  many  situations 
and,  unless  the  environmental  effects  are  additive,  it  is  impossible  to  get 
a  true  estimate  of  genetic  effects  by  subtracting  the  variance  of  one 
population  from  that  of  another.  One  solution  for  this  difficulty  is  to 
find  a  situation  in  which  environmental  effects  do  seem  to  be  additive. 
If  there  is  a  threshold  of  stimulation  the  fundamental  physiological 
principle  of  summation  of  stimuli  should  hold  and  additive  effects  be 
produced  at  this  point  in  the  scale. 

There  should  be  two  general  types  of  genetic  situations.  In  one  the 
threshold  may  be  reached  by  only  one  extreme  genetic  class.  In  the 
other  situation  the  threshold  may  be  located  somewhere  near  the  center 
of  the  genetic  distribution  with  several  genetic  classes  on  either  side. 
Theoretical  results  have  been  calculated  for  both  possibilities  and  the 
following  method  of  analysis  developed. 

a)  Arbitrarily  cut  off  the  various  experimental  populations  at  two 
points:  one  which  cuts  off  a  terminal  group  and  the  other  which  most 
exactly  separates  the  Fi  into  halves. 

b)  For  each  population  calculate  the  proportion  of  individuals  found 
on  one  side  of  the  dividing  line. 

c)  Subtract  the  proportion  found  in  one  parental  population  from  that 
in  the  other.  This  gives  a  figure  which  may  be  represented  as  a  distance 
and  is  a  scale  based  on  only  one  point  in  the  original  scale  of  measurement. 
The  method  requires  only  that  environmental  effects  be  additive  at  this 
point,  which  presumably  represents  a  threshold.  This  distance  also 
represents  the  genetic  difference  between  the  two  parental  stocks,  and 
environmental  variability  has  been  eliminated. 

d)  Find  the  position  of  the  Fi  and  backcross  generations  relative  to 
one  of  the  parent  stocks  by  subtracting  the  proportion  found  in  it  from 
the  corresponding  figures  and  dividing  these  figures  by  the  distance 
between  the  parent  stocks.  This  will  give  the  relative  position  of  the 
different  populations  on  the  scale  referred  to  in  c) . 

When  the  theoretical  position  of  the  Fi  and  backcross  generations  are 
calculated  it  is  found  that  they  are  affected  both  by  heterozygosis  in  the 
parent  stocks  and  by  the  number  of  genetic  factors,  and  graphs  of  these 
two  effects  have  been  prepared  in  order  to  show  the  effects  of  heterozy- 
gosis. 

However,  one  very  interesting  and  useful  relationship  appeared  from 
this  analysis.  The  relative  distance  between  the  two  backcross  genera- 
tions is  not  affected  by  heterozygosis  in  a  one-factor  cross,  even  where 
dominance  is  involved.     Therefore  this  figure,  which  is  described  by  the 

Journal    of  the  National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


747 


following  formula,  can  be  used  as  a  simple  test  for  a  one-factor  ratio. 


Bx\ — Bx2 
Pi-ft 

It  is  affected  by  heterozygosis  when  more  than  one  factor  is  present,  but  has 
other  interesting  properties.  If  the  threshold  is  reached  only  by  the 
terminal  class,  and  there  is  no  heterozygosis,  the  distance  between  the 
backcrosses  decreases  regularly  with  the  number  of  factors  involved 
(table  1).  On  the  other  hand,  if  the  threshold  is  located  in  the  center  of 
the  genetic  distribution,  the  distance  between  backcrosses  increases 
regularly  with  the  number  of  factors.  The  effect  of  additional  factors 
is  much  greater  when  small  numbers  of  factors  are  involved  and  ac- 
curate estimates  would  be  difficult  to  make  with  more  than  four  factors. 
It  will  be  noted  that  when  heterozygosis  is  present  the  estimate  of  the 
number  of  factors  increases  (text-fig.  1),  so  that  an  assumption  of  no 
heterozygosis  will  result  in  a  minimum  estimate  of  genetic  factors  involved. 


Table  1.- 


■Relative  distance  between  2  backcrosses  when  the  data  are  arbitrarily  divided 
into  2  parts,  and  populations  are  homozygous 


Number 

of 
factors 

Threshold  at 

terminal 

class 

Threshold  mid- 
way between 
terminal  classes 

1 
2 
3 

4 

0.5 
.25 
.  125 
.0625 

0.5 
.75 
.875 
.9375 

Results 

The  above  theoretical  considerations  assume  that  only  one  trait  is 
being  measured.  It  is  very  easy,  in  measuring  behavior,  to  set  up  a  unit 
which  accidentally  measures  a  combination  of  several  traits,  and  if  these 
are  affected  by  different  genetic  mechanisms  the  results  can  be  confusing. 
For  example,  an  attempt  was  made  to  measure  the  fear  responses  of  young 
puppies  as  indicated  by  avoidance,  vocalization,  postural  and  tail  re- 
sponses, and  all  of  these  were  added  together  for  a  single  score  of  timidity. 
On  detailed  analysis  it  was  found  that  the  avoidance  and  vocalization 
scores  appeared  to  be  alternate  expressions  of  fearful  responses  and  could 
be  added  together.  The  postural  responses,  however,  were  not  directly 
related.  An  animal  might  lie  flat  as  a  timidity  response  and  he  might  also 
do  so  while  getting  ready  to  chew  on  the  shoes  and  clothing  of  the  observer. 
Consequently,  the  best  genetic  analysis  is  obtained  when  the  data  are 
divided  up  and  analyzed  in  as  small  and  discreet  segments  as  possible. 
The  following  examples  give  the  results  of  several  such  analyses. 

Avoidance  and  Vocal  Reactions  to  Human  Handlers  at  5  Weeks 

This  test  is  a  measure  of  the  process  of  socialization  of  young  puppies 
toward  their  human  handlers.     As  can  be  seen  in  table  2,  the  cocker 

Vol.    15,  No.   3,   December    1954 


748 


PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


.5 


CM 
X 

CD 
I 

X* 

CD 


OL 
I  .3 


I 

n 

m. 

/ 

O       .1      .2      .3      .4      .5      .6      .7      .8      .9      1.0 

HETEROZYGOSIS,   P2  (DOM.) 

DISTANCE     BETWEEN     BACKCROSSES 

Text-figure  1. — The  distance  between  backcrosses  relative  to  the  distance  between 
parent  strains,  in  the  case  where  a  threshold  cuts  off  a  terminal  class,  and  one  of 
the  parent  strains  shows  heterozygosis.  Roman  numerals  indicate  the  number  of 
genetic  factors  involved.  Note  that  the  figure  for  1  factor  is  unaffected  by  hetero- 
zygosis, and  that  with  increasing  heterozygosis  an  increased  number  of  factors  is 
required  to  account  for  the  difference  between  backcrosses. 

spaniel  and  basenji  breed  show  wide  differences  in  this  respect.     A  large 
proportion  of  cocker  spaniels  show  almost  no  avoidance  reactions  and 


Table  2. — Avoidance  and  vocal  reactions,  5  weeks 

Score 

B 

CS 

BCS 

CSB 

BCS  X 

CS 

CSB  X 

B 

Other 

Parent 

Other 

Parent 

49-51 

1 

46-48 

43-45 

1 

40-42 

1 

37-39 

34-36 

2 

31-33 

4 
...... 

2 
1 

4 

1 
3 

1 

28-30 

1 
1 

3 

25-27 

1 

1 

22-24 

1 

1 

19-21 

6 

1 

4 

16-18 

1 

2 

1 

2 

13-15 

3 

4 

4 

5 
3 
5 

3 

10-12 

1 
1 

2 
1 

4 

6 

4 

7-9 

4 

3 

5 

4-6 

2 

7 

3 

1 

1 

4 

2 

1-3 

1 

2 

7 

3 

3 

6 

2 

1 

0 

5 

16 

1 

3 

Total 

20 

16 

23 

26 

18 

23 

23 

27 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


749 


practically  all  basenjis  show  them  in  varying  degrees.  Inspection  of  the 
table  indicates  that  the  basenji  condition  is  dominant  in  the  first  genera- 
tion, as  the  distributions  are  quite  similar.  This  is  borne  out  by  the 
distributions  of  the  two  backcross  generations. 

If  the  data  are  divided  on  the  basis  of  a  terminal  class  in  which  there 
are  no  reactions,  a  figure  of  0.21  is  obtained  for  the  relative  distance 
between  the  backcrosses.  This  is  quite  close  to  the  theoretical  figure  of 
0.25  which  would  be  obtained  for  two  factors  with  no  heterozygosis. 
From  the  fact  that  the  parent  basenjis  show  considerably  less  variability 
than  the  rest  of  the  sample,  it  may  be  concluded  that  there  is  some 
heterozygosity  in  the  basenji  line  for  this  character  and  the  figure  of  two 
factors  must  be  considered  a  minimum. 

Aggressive  Reactions  Toward  Human  Handlers,  13  to  15  Weeks 

These  figures  are  taken  from  the  same  test  on  which  the  preceding  data 
is  based.  At  5  weeks  of  age  almost  no  aggressive  reactions  are  given,  but, 
at  13  and  15  weeks,  which  have  been  averaged  together,  biting,  chewing, 
pawing  and  some  other  aggressive  reactions  are  quite  common.  As  will 
be  seen  in  table  3,  the  distributions  are  quite  different  from  the  preceding. 
There  is  a  difference  between  the  parent  strains  but  neither  appears  to  be 
a  terminal  class.  The  first  generation  shows  greatly  increased  variability 
compared  to  the  parent  strains  which  might  be  due  to  heterozygosity  in 
one  of  the  parent  strains  or  might  be  caused  by  the  threshold  phenomenon 
described  above,  in  that  the  animals  have  to  fall  into  one  of  two  classes 
and  consequently,  if  the  Fi  is  close  to  the  threshold,  it  should  show  in- 
creased variability.  The  test  for  the  single  factor  ratio,  however,  is  not 
affected  by  heterozygosis  and  would  apply  in  either  case. 

Table  3. — Aggression,  IS  to  15  weeks 


Score 

B 

cs 

BCS 

CSB 

BCS  X 

CS 

CSB  X 

Other 

Parent 

Other 

Parent 

B 

49-52 

46-48 

1 

1 
2 
1 
3 

1 

"2" 

1 

2 
2 
2 
3 
4 
1 
3 

"2" 

1 

43-45 

40-42 
37-39 

1 

34-36 

1 
4 
1 
2 
4 
4 
3 
1 
1 
3 
1 

1 

"  i " 

3 

1 

2 
2 
2 
2 
1 

31-33 
28-30 
25-27 

""l" 

1 
1 
3 
1 
1 
1 
2 

"3" 
3 

"2 

2 

22-24 

1 
1 
1 

2 
4 
2 

4 
4 

2 

19-21 

16-18 

13-15 

10-12 

7-9 

4-6 

1-3 

3 

2 
6 
4 
8 
5 

1 
3 
3 
4 
6 
5 
3 

4 
5 
3 
2 

1 
4 

o 

Total 

28 

16 

35 

26 

18 

23 

20 

23 

Vol.    15,   No.   3,   December   1954 


750 


PROCEEDINGS:   SYMPOSIUM  ON  25  YEARS  OF 


The  relative  distance  between  the  backcrosses  gives  a  figure  of  0.73  and 
it  may  be  concluded  that  a  minimum  of  two  genetic  factors  are  involved 
and  that  the  situation  is  one  in  which  the  threshold  of  stimulation  is  near 
the  center  of  genetic  variability. 

Since  there  is  a  possibility  that  the  traits  of  avoidance  and  aggressive- 
ness may  be  related,  correlation  coefficients  were  calculated  for  the 
backcross  data.  It  was  obvious  that  in  the  backcross  to  the  basenji 
there  was  no  correlation,  whereas  the  other  backcross  showed  an  obvious 
correlation  which  would  be  estimated  on  the  basis  of  the  small  numbers 
involved  to  be  at  least  as  high  as  0.50.  The  lack  of  correlation  in  the 
basenji  backcross  can  be  explained  by  the  fact  that  the  basenji  condition 
is  dominant  for  the  avoidance  scores  and,  consequently,  there  is  no  geneti- 
cally produced  variability.  It  is  extremely  interesting  that  the  correlation 
in  the  cocker-spaniel  backcross  is  positive,  indicating  that  an  animal  which 
shows  a  high  degree  of  avoidance  early  in  life  tends  to  be  more  aggressive 
later  in  life.  This  means  either  that  the  action  of  the  genes  on  one  thresh- 
old of  behavior  may  be  quite  different  on  another  threshold,  or  that 
linkage  is  involved.  There  is  little  correlation  within  either  of  the  two 
pure  breeds,  indicating  that  this  genetic  mechanism  is  involved  chiefly 
in  the  cross. 

Heart  Bate  After  Weighing 

During  the  process  of  weekly  checkups  the  animals  are  placed  upon 
the  scale  and  an  attempt  is  made  to  keep  them  quiet  for  a  period  of  one 
minute,  after  which  the  heart  rate  is  taken.  A  resting  heart  rate  is  ob- 
tained if  the  animal  does  quiet  down,  but,  since  many  animals  do  remain 
active,  it  probably  also  reflects  the  effect  of  the  handler  on  the  behavior 
of  the  dog.  The  heart  rate  is  taken  for  15  seconds  and  the  figure  given  in 
the  table  is  the  sum  of  6  observations  taken  at  11  to  16  weeks  of  age. 
When  the  data  are  examined  (table  4)  it  will  be  seen  that  there  is  some 


Table  4. — Heart  rate  after  weighing,  11  to  16  weeks 

Sum 

B 

CS 

BCS 

CSB 

BCS  X  CS 

CSBXB 

Other 

Parent 

Other 

Parent 

350 

1 

340 

2 

330 

1 
4 
3 
3 
1 
1 

""3   ' 

2 

320 

1 
6 
3 
3 
9 
4 
1 

1 

1 
1 
3 
3 
4 
5 

1 

310 

1 
5 
4 
4 
6 
.  ....  . 

1 

1 
1 
2 
4 
4 
5 
4 
2 

1 

300 

1 

"o"  ' 
7 
5 
2 

7 

1 
5 
2 
5 
4 
3 
4 
1 

3 

290.. 

2 

280 

270 

3 
3 

260... 

3 

250... 

240 

1 

230 

1 

220 

1 

Total 

28 

16 

27 

25 

18 

23 

23 

23 

Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


751 


indication  of  a  possible  difference  between  the  reciprocal  crosses,  but 
that  in  general  the  cocker-spaniel  condition  appears  to  be  dominant  both 
in  the  Fi  and  backcross  generations.  There  is  a  great  deal  of  variability 
in  the  backcross  to  the  basenjis  as  one  would  expect  in  a  segregating  group. 
The  relative  distance  between  the  backcrosses  is  0.538,  which  is  very 
close  to  the  theoretical  figure  for  one  factor.  The  data  appear  to  be 
fairly  normally  distributed  and,  when  the  method  of  analysis  of  Castle 
and  Wright  {12)  is  used,  an  answer  of  1.25  factors  is  obtained,  thus  giving 
a  check  between  two  methods  of  calculation.  The  only  difficulty  en- 
countered in  the  application  of  the  Castle  and  Wright  formula  was  the 
great  amount  of  variability  in  the  segregating  generation  compared  with 
the  relative  distance  between  the  parent  strains.  This  had  to  be  reduced 
by  comparing  only  variance  within  litters  from  corresponding  mothers 
from  the  Fx  and  backcross  strains,  before  any  meaningful  figure  could  be 
obtained. 

The  Solution  of  a  Simple  Barrier  Problem 

Puppies  at  6  weeks  of  age  which  have  had  no  experience  with  barriers 
are  tested  on  their  ability  to  find  their  way  around  an  opaque  barrier 
beyond  which  is  a  goal  of  food  and  the  experimenter.  Successful  ex- 
perience is  given  on  the  first  day  and  on  the  second  a  longer  but  similar 
barrier  is  presented .  Some  individuals  find  their  way  around  immediately 
and  a  minimum  of  three  stops  or  turns  (errors)  is  considered  to  indicate 
an  insightful  solution;  namely,  that  the  animal  is  able  to  organize  his 
impressions  of  the  situation  to  reach  a  successful  conclusion  without 
going  through  random  exploration. 

As  can  be  seen  from  table  5,  the  great  majority  of  basenjis  are  successful 
at  this,  whereas  only  a  few  cocker  spaniels  are  as  good.  The  first  genera- 
tion appears  to  be  intermediate  between  two  strains. 


Table  5. — Errors,  first  trial,  day  2,  6-week  barrier  test 


Errors 

B 

CS 

BCS 

CSB 

BCS  X  CS 

CSBXB 

Other 

Parent 

Other 

Parent 

46  &  above. . . . 

1 

1 
1 

2 

41-45 

2 

36-40 

1 
1 
2 

1 

31-35 

1 
3 

26-30 

1 

"  i " 

3 
4 
4 
1 
2 
2 
3 
1 
3 

1 

"i" 

2 
1 
1 
1 
1 
3 
3 
4 

...... 

•y  • 

l 
l 
3 
6 
3 
1 

2 

21-25 

1 
1 
1 
2 

16-20 

3 
5 

4 
1 
1 
2 

"2" 

2 
2 
4 
2 
1 

2 

11-15 

3 

6-10 

6 

5 

2 

4 

3 

3 

3 
3 

1 
11 

"h" 

3 

7 

1 

2 

4 
2 

1 

1 

0 

4 

Total 

23 

16 

23 

26 

18 

23 

23 

27 

Vol.    15,   No.   3,   December    1954 


752 


proceedings:  SYMPOSIUM  ON  25  YEARS  op 


Both  backcrosses  show  a  great  deal  of  variability  as  one  might  expect 
in  segregating  strains  but,  on  the  average,  both  are  inferior  to  the  cocker 
spaniels.  When  the  distance  between  the  backcrosses  is  calculated,  a 
meaningless  figure  of  —0.064  is  obtained. 

No  clear  genetic  analysis  can  be  made  from  the  data  and  there  are 
two  possibilities.  One  is  that  more  than  one  trait  is  being  measured  and 
that  these  can  be  separated  by  more  detailed  analysis  of  the  data,  although 
it  is  difficult  to  see  how  this  can  be  done.  The  other  possibility  is  that 
more  than  one  trait  is  associated  with  the  process  of  organization  or 
adaptation  going  on  in  the  brain  of  the  animal.  It  would  be  expected  that 
any  animal  would  possess  many  genetically  determined  capabilities  and 
that  in  a  situation  requiring  adaptation  these  would  be  organized  in  the 
best  way  possible.  However,  in  almost  any  situation  there  are  a  variety  of 
ways  in  which  capabilities  can  be  organized  and  there  would  not  neces- 
sarily be  a  one-to-one  relationship  between  ability  and  the  resultant 
behavior.  This,  of  course,  raises  the  problem  of  whether  additive  effects 
will  ever  be  obtained  from  this  kind  of  adaptive  reaction. 

General  results. — The  data  described  above  are  summarized  together 
with  certain  other  tests  in  table  6,  and  give  a  small  but  fairly  representative 
sample  of  the  kind  of  results  which  are  being  obtained  from  the  analysis  of 
approximately  30  major  behavioral  tests.  As  will  be  seen,  the  analysis  of 
simple  behavioral  responses  in  which  thresholds  are  involved  gave  clear-cut 

Table  6. — Analysis  of  number  of  genetic  factors  affecting  each  of  9  behavior  traits 


Test 


Parent 
ratios 


B 


CS 


Relative  position  of  other  ratios 


Fi 


CSBX 
B 


BCSX 

CS 


CSB  X  B- 

BCS  X  CS 


Mini- 
mum 
No.  of 
factors 
(est.  het- 
erozygosis) 


Avoidance  &  vocal,  5  wks 

Playful    fighting,     13-15 
wks 

Activity 

Posture 

Heart  rate  before 

Heart  rate  after 

Barrier,  6  wks 

Errors 
Part  2,  #1 

Habit,  9  wks 

Time 
Part  2,  #1 

Barrier,  13  wks 

Total  errors 


0.000 

.312 
.  125 
.000 
.025 
.000 
.941 

.814 

.562 


0.615 

.808 
.880 
.560 
.680 
.320 
.346 

.600 

.760 


0.003 

.502 

.028 
.306 
.720 

.228 
.401 

*  940 

*.  367 


0.000 

.248 
-.051 
.775 
.530 
.278 
-.212 

.654 

-1.87 


0.210 

.981 
.237 
.543 
1.  240 
.816 
-.  148 

.443 

-.  312 


0.210 

.733 
.288 

-.232 

.71 

.538 

-.064 

.211 
1.558 


2(0) 

2(0) 
2(0) 

? 

2 

1 
? 


'Large  difference  between  reciprocal  crosses;  like  maternal  type. 


Journal  of  the  National  Cancer  Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  753 

genetic  results  with  the  exception  of  postural  reactions,  and  it  is  expected 
that  this  can  be  resolved  with  further  analysis.  The  minimum  number  of 
factors  is  two  except  in  one  case.  It  is  indicated  that  a  reasonably  simple 
genetic  mechanism  is  involved  in  these  traits  but  that  other  factors  would 
be  found  in  breed  populations  less  limited.  The  multiple  factor  hypothesis 
regarding  the  determination  of  behavioral  traits  is  thus  upheld. 

On  the  other  hand,  none  of  the  tests  which  involve  adaptation  and 
learning  show  such  clear-cut  results.  It  is  possible  that  more  detailed 
analysis  will  give  this  result,  but  it  is  also  likely  that  the  process  of  adapta- 
tion, in  which  the  animal  organizes  his  sensory  and  motor  capacities  to 
meet  a  new  situation,  is  nonadditive  in  nature.  The  reasons  for  this  can  be 
seen  more  clearly  in  other  tests  which  involve  motor  organization  and 
where  the  observer  can  see  what  the  animals  are  doing.  For  example,  in 
one  test  the  puppies  are  required  to  get  a  dish  of  food  out  from  under  a 
cover  and  success  largely  depends  on  the  animal's  tendency  to  move 
objects  in  some  way.  Basenjis  usually  do  well  by  pawing  the  cover  and 
the  cocker  spaniels  by  lifting  it  with  the  mouth.  The  Fi  generations  are 
intermediate  in  both  respects,  and  usually  fail.  It  is  not  possible  to  paw 
and  lift  objects  with  the  teeth  at  the  same  time,  and  the  Fi  animals  cannot 
add  these  capacities  together.  The  underlying  genetic  situation  is 
obviously  complex  and  the  effects  are  nonadditive  in  terms  of  adaptation. 

Discussion 

In  general,  the  same  types  of  effects  which  Richter  has  found  resulting 
from  selection  and  domestication  in  the  rat  are  also  found  in  greater  degree 
in  the  dog.  Variability  appears  to  be  very  great  both  in  anatomical 
characteristics  which  affect  behavior  and  in  types  of  behavior  which  have 
no  obvious  anatomical  explanation.  Early  sexual  maturation  appears  to 
be  the  rule  in  all  breeds  of  dogs.  While  the  average  fertility  is  probably 
somewhat  decreased  from  that  of  the  wolf,  certain  breeds  seem  to  show 
increased  fertility.  Traits  involving  wildness  and  early  socialization  have 
been  greatly  modified,  and  selection  for  the  power  to  learn  a  given  task 
has  proceeded  much  further  in  the  dog  than  in  the  rat. 

From  a  genetic  point  of  view  Richter  has  done  much  to  demonstrate  the 
nature  and  importance  of  behavioral  and  physiological  differences  between 
wild  and  tame  strains,  and  to  clear  up  some  of  the  problems  raised  by 
earlier  work  done  by  associates  of  Castle.  There  is  an  extensive  literature 
on  this  subject  in  the  rat  and  the  numerous  crossbreeding  experiments 
have  been  reviewed  by  Scott  and  Fredericson  (16)  and  Hall  (17).  How- 
ever, most  of  these  were  done  before  the  methods  of  analyzing  multiple 
factor  inheritance  had  been  worked  out  and  the  results  indicated  very 
little  except  that  strain  differences  existed.  Usually  only  very  few 
measurements  were  made  so  that  no  idea  was  obtained  as  to  the  nature  of 
the  traits.  Only  one  of  these  studies,  that  made  by  Dawson  (18)  on  the 
running  time  of  mice  which  were  pushed  along  an  alleyway,  gives  any 
definite  genetic  results,  and  he  found  that  there  were  between  two  and 

Vol.    15,  No.   3,   December    1954 
316263—54 33 


754  proceedings:  symposium  on  25  years  of 

three  genetic  factors  involved  in  differences  between  the  strains  that  were 
studied. 

The  work  described  in  this  paper  on  the  dog  shows  that  strain  differences 
in  timidity,  and  other  traits  which  affect  the  processes  of  socialization, 
can  be  caused  by  reasonably  simple  genetic  mechanisms,  although,  if  all 
of  the  traits  concerned  are  considered,  many  factors  may  be  involved  and 
the  multiple  factor  hypothesis  is  supported.  The  work  has  general 
importance  in  that  some  process  of  socialization  is  found  in  all  social 
animals  and  upon  it  depends  the  successful  adaptation  of  the  individual 
to  the  group  and  also  the  successful  integration  of  the  entire  group.  The 
process  of  socialization  is  important  in  human  development  and  its  modi- 
fication may  result  in  problems  of  abnormal  behavior  [Bowlby  (19)]. 
Genetic  results  on  lower  animals  indicate  that  such  modification  might  be 
produced  more  easily  in  some  individuals  than  in  others. 

An  interesting  negative  result  was  obtained  in  the  hybridization  of  two 
breeds  which  presumably  have  been  separated  for  centuries.  It  might  be 
supposed  that  modification  of  the  wild  type  had  been  achieved  by  the 
selection  of  different  genes  in  the  two  breeds  so  that  the  Fi  would  produce 
a  throwback  to  the  wild  type.  No  evidence  of  this  was  obtained  with 
regard  to  the  traits  of  wildness  or  savageness  which  affect  the  process  of 
socialization.  There  was  evidence  of  hybrid  vigor  in  some  physiological 
characteristics,  particularly  the  amount  of  milk  produced  by  Fi  mothers. 
However,  the  evidence  on  behavior  indicates  that  the  changes  toward 
tameness  were  probably  achieved  early  in  the  process  of  domestication. 
This  result  also  argues  for  the  single  ancestry  theory  which  arises  from  the 
anatomical  evidence. 

Several  experiments  on  selection  for  behavioral  differences  have  been 
done  with  the  domestic  rat  and  positive  results  achieved  in  both  emotional 
traits,  as  found  by  Hall  (17),  and  in  maze  learning,  as  found  by  Tryon  (20). 
Searle  (21),  who  studied  Tryon's  strains  with  multiple  behavioral  tests 
came  to  the  conclusion  that  the  chief  differences  between  maze-dull  and 
maze-bright  rats  was  that  the  former  were  more  afraid  of  the  maze  than 
the  latter,  as  they  perform  equally  well  or  better  on  other  types  of  mazes. 
This  work  had  the  result  of  emphasizing  the  results  of  emotional  and 
motivational  differences  as  they  affect  adaptation  and  the  consequent 
importance  of  testing  behavioral  differences  in  many  different  ways.  In 
making  crosses  between  the  two  strains,  Tryon  found  that  the  Fi  covered 
the  distribution  of  both  parent  strains  with  a  very  wide  variance.  This 
result  is,  of  course,  similar  to  that  found  in  the  test  for  aggressive  behavior 
of  the  dogs,  which  suggests  that  Tryon  may  have  been  working  with  an 
emotional  difference  in  which  the  threshold  of  stimulation  was  near  the 
center  of  the  genetic  distribution.  Unfortunately,  no  backcrosses  were 
made,  and  so  no  estimate  of  the  number  of  factors  involved  can  be  made. 

In  addition  to  the  crossbreeding  studies  described,  several  pure  breeds 
of  dogs  have  been  extensively  tested  in  a  variety  of  behavioral  characters 
[Fuller  and  Scott  (22)].  Kesults  indicate  that  each  breed  has  several  in- 
dependent capacities  and  abilities  which  affect  its  ability  to  learn  easily 

Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  755 

the  special  task  for  which  it  has  been  selected.  For  example,  the  Shet- 
land sheep  dog  has  a  tendency  to  form  fixed  and  lasting  habits,  apparently 
because  of  great  emotional  sensitivity.  The  beagle  has  the  opposite  tend- 
ency so  that  it  rarely  takes  the  same  path  twice,  which  is  of  great  advan- 
tage in  investigating  and  hunting.  Sheep  dogs  show  a  considerable 
degree  of  aggressiveness  which  results  in  their  tending  to  chase  sheep, 
while  beagles  tend  to  be  nonaggressive  so  that  dog  fights  are  infrequent 
while  they  are  living  in  packs.  Sheep  dogs  tend  to  be  indifferent  to  food 
rewards  while  beagles  are  highly  motivated  by  them.  Such  instances 
could  be  multiplied  indefinitely.  On  the  other  hand,  there  is  little  evi- 
dence that  complicated  patterns  of  behavior  will  appear  spontaneously 
without  previous  learning. 

Certain  traits  which  make  it  easy  for  an  animal  to  learn  one  type  of 
problem  may  limit  its  learning  ability  elsewhere.  The  sheep  dogs  which 
are  highly  adapted  to  learn  commands  under  close  personal  supervision, 
do  comparatively  poorly  under  kennel  conditions  and  in  situations  where 
they  are  required  to  solve  problems  independently  [Fuller  (23)].  The 
aggressiveness  of  the  fox  terriers  makes  them  easy  to  teach  to  attack  small 
game,  but  when  they  are  raised  in  litters  it  is  impossible  to  keep  more  than 
two  or  three  animals  together  because  of  physical  injuries  inflicted  on 
each  other.  The  conclusion  may  be  reached  that  the  exaggeration  of  a 
behavioral  trait  by  selection  may  make  a  particular  learning  process 
easier,  but  it  may  also  limit  the  power  of  adaptation  under  other  situations. 

A  number  of  interesting  problems  are  raised  by  nonadditive  combina- 
tion of  behavioral  traits  which  takes  place  in  adaptive  behavior.  As 
pointed  out  above,  a  single  exaggerated  trait,  such  as  excessive  timidity 
or  aggressiveness,  may  make  certain  kinds  of  adaptation  impossible.  At 
the  moment,  the  most  fruitful  line  of  research  seems  to  be  to  try  to  dis- 
cover the  nature  and  significance  of  these  components  rather  than  to 
gauge  their  combined  effect  upon  adaptation.  So  far  we  have  no  evidence 
of  general  intellectual  organizing  ability  apart  from  the  simple  components 
of  behavior.  If  such  differences  in  ability  do  exist  in  normal  individuals 
they  do  not  appear  to  be  important  compared  with  the  simple  components. 

These  tentative  conclusions  present  several  problems  to  the  dog  breeder 
who  is  interested  in  increasing  the  natural  ability  of  performance  in  his 
animals.  The  over-all  performance  as  measured  by  the  simple  speed  of 
learning  will  be  a  poor  measure  for  selection,  although  it  is  the  ultimate 
measure  of  success.  Effort  should  be  directed  toward  discovering  what 
the  simple  components  of  behavior  are  and  selecting  primarily  for  these. 
If,  as  in  the  present  experiments,  each  of  these  simple  elements  are  affected 
by  only  one  or  two  genetic  factors,  it  may  turn  out  that  the  genetic  vari- 
ability in  any  pure  breed  may  be  quickly  exhausted  as  seems  to  have  been 
the  case  where  attempts  have  been  made  to  increase  the  performance  of 
guide  dogs  by  selection. 

Any  applications  of  these  results  to  human  problems  must  be  done  with 
a  great  deal  of  caution.  As  David  and  Snyder  (24)  have  pointed  out, 
there  are  good  reasons  for  believing  that  consistent  selection  for  special 

Vol.    15,   No.   3,   December    1954 


756  proceedings:  symposium  on  25  years  of 

types  of  behavior  has  never  been  an  important  factor  in  human  evolution. 
On  the  other  hand,  the  development  of  a  high  degree  of  social  organization 
and  consequent  protection  of  the  members  permits  a  very  wide  degree  of 
variability  between  individuals  just  as  similar  protection  extended  to 
domestic  animals  allows  great  variability  among  them.  The  present 
experiments  show  that  certain  kinds  of  behavioral  traits,  particularly 
emotional  and  motivational  traits,  can  be  importantly  affected  by  heredity. 
Certain  individuals  may,  as  a  result,  have  a  very  wide  range  of  adaptation 
while  others  may  have  a  narrower  but  superior  range.  The  question  may 
be  asked,  do  these  same  conditions  exist  among  human  individuals? 

If  the  answer  is  in  the  affirmative  our  chief  practical  problems  among 
human  beings  are  to  find  means  of  recognizing  hereditary  differences  at 
an  early  age  so  that  the  proper  environment  can  be  given  to  the  individual 
and  he  can  reach  his  widest  range  of  adaptability.  From  the  animal  ex- 
periments it  would  be  indicated  that  it  would  be  most  profitable  to  search 
in  human  beings  for  the  basic  emotional  and  motivational  thresholds 
which  seem  to  be  the  most  important  determinants  of  learning  and  social 
behavior  in  normal  individuals.  Since  speech  is  so  important  a  capacity 
in  human  society  the  basic  motor  and  emotional  capacities  which  affect 
speech  should  be  another  special  object  of  search. 

It  should  also  be  remembered  that  the  animal  experiments  show  that 
there  is  a  wide  range  of  adaptability  particularly  where  a  learning  task  is 
complex.  There  are  many  different  ways  in  which  an  individual  may 
organize  his  capacities  and  equally  good  results  may  be  achieved  by  what 
appear  to  be  entirely  different  individuals.  It  is  to  be  hoped  and  expected 
that  the  recognition  and  study  of  basic  behavior  traits  affected  by  heredity 
will  greatly  aid  the  education  and  behavioral  adjustment  of  the  human 
individual. 

Summary 

1)  Typical  changes  in  behavior  of  domestic  animals  include  increased 
genetic  variability,  early  sexual  maturity,  and  modification  of  the  social- 
ization process  with  regard  to  people  (i.e.,  decreased  wildness).  In 
addition,  breed  selection  for  the  ability  to  learn  specialized  tasks  has  been 
carried  further  in  the  dog  than  any  other  animal. 

2)  According  to  the  paleontological  evidence  the  dog  was  domesticated 
from  a  single  species  of  wolf  in  northern  Eurasia. 

3)  Archeological  evidence  indicates  that  domestication  took  place 
possibly  8,000  years  ago  and  that  the  use  of  dogs  spread  rapidly  among 
primitive  peoples. 

4)  The  possession  of  dogs  by  semi-isolated  human  tribes  provided  a 
favorable  condition  for  genetic  change,  and  each  geographical  region 
tended  to  have  a  different  type  of  dog  associated  with  it. 

5)  The  social  organization  of  wolves  is  one  which  permits  easy  domesti- 
cation, and  there  are  authentic  reports  of  wolf  cubs  tamed  by  rearing  apart 
from  the  mothers. 

Journal   of   the   National    Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  757 

6)  Five  representative  breeds  were  selected  for  detailed  behavioral 
study;  beagle,  Shetland  sheep  dog,  wire-haired  fox  terrier,  cocker  spaniel, 
and  African  basenji.  The  last  two  were  chosen  for  a  crossbreeding 
program. 

7)  These  breeds  and  various  hybrid  generations  have  been  raised  under 
standard  enviromental  conditions  and  subjected  to  a  variety  of  motiva- 
tional, emotional,  social,  physiological  and  learning  tests  at  appropriate 
ages. 

8)  A  theoretical  basis  for  analysis  of  multifactorial  heredity  applied  to 
the  special  case  of  thresholds  of  behavioral  stimulation  is  described, 
including  a  simple  test  for  single  factor  inheritance.  Typical  results  are 
summarized  below. 

9)  A  measure  of  avoidance  reactions  affecting  the  process  of  socializa- 
tion toward  people  gives  a  minimum  estimate  of  two  factors  accounting 
for  the  difference  between  cockers  and  basenjis.  There  is  no  increase  in 
wildness  in  the  Fi,  indicating  a  common  genetic  mechanism  even  though 
the  two  strains  have  been  long  separated. 

10)  A  measure  of  aggressiveness  likewise  gives  an  estimate  of  two 
factors,  but  the  results  indicate  a  threshold  in  the  center  of  the  genetic 
distribution. 

11)  A  measure  of  the  resting  heart  rate  gives  an  estimate  of  a  one- 
factor    difference. 

12)  A  test  of  performance,  in  which  the  puppy  has  to  find  its  way 
around  a  barrier  on  the  basis  of  previous  experience,  does  not  give  a  clear 
picture  of  the  genetic  mechanism. 

13)  In  general,  measures  of  differences  in  the  threshold  of  stimulation  in 
simple  behavior  patterns  give  reasonably  simple  genetic  results,  although 
usually  with  more  than  one  factor  involved. 

14)  Measures  of  complex  adaptation  do  not  give  simple  genetic  results, 
and  it  is  suggested  that  this  may  result  from  the  fact  that  adaptation  con- 
sists of  organizing  the  many  capacities  of  the  individual,  and  that  this  can 
take  place  in  a  variety  of  ways  to  produce  the  same  result. 

15)  Capacities  which  increase  the  ability  to  learn  a  special  situation  may 
limit  the  range  of  adaptability  in  other  situations. 

16)  With  regard  to  human  applications,  it  is  concluded  that  the  genetic 
situations  in  the  two  species  are  quite  different,  but  that  the  dog  results 
give  an  estimate  as  to  how  great  genetic  differences  between  individuals 
may  be.  The  most  profitable  line  of  human  research  would  appear  to  be 
the  search  for  early  differences  in  basic  emotional  and  motivational  traits, 
together  with  the  types  of  environment  which  give  the  most  desirable 
expression  of  these  traits. 

References 

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{2)  Young,  S.  P.,  and  Goldman,  E.  A.:  The  Wolves  of  North  America.  Wash- 
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(3)  Little,  C.  C.  and  DuBuis,  E.:  Private  communication,  1954. 

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chology, New  York,  Wiley,  1951. 

(18)  Dawson,  W.  M.:  Inheritance  of  wildness  and  tameness  in  mice.     Genetics  17: 

296-326,  1932. 

(19)  Bowlby,  J.:   Maternal  care  and  mental  health.     World  Hlth.  Org.,  Geneva,  1951. 

(20)  Trton,    R.    C:  Individual    differences.     In    Comparative    Psychology.     New 

York,  Prentice-Hall,  1942. 

(21)  Searle,    L.    V.:  The   organization   of   hereditary   maze-brightness   and   maze- 

dullness.     Genetic  Psychol.  Mono.  39:  279-326,  1949. 

(22)  Fuller,  J.  L.,  and  Scott,  J.  P.:  Heredity  and  learning  ability  in  infra-human 

mammals.     Eugenics  Quart.  1:  28-43,  1954. 

(23)  Fuller,  J.  L.:  Cross-sectional  and  longitudinal  studies  of  adjustive  behavior  in 

dogs.     Ann.  New  York  Acad.  Sc.  56:  214-224,  1953. 

(24)  David,  P.  R.,  and  Snyder,  L.  H.:  Genetic  variability  and  human  behavior.     In 

Social  Psychology  at  the  Crossroads.     New  York,  Harper,  1951. 


The  Effects  of  Selection  and  Domesti- 
cation on  Man  x 


Laurence  H.  Snyder,   The   University  of  Okla- 
homa. Norman.  Okla. 


The  very  title  which  has  been  assigned  to  me  presents  an  enigma:  Can 
man  properly  be  said  to  be  domesticated,  or  even  to  have  been  subjected 
to  the  process  of  domestication?  To  reclaim  from  the  wild  state,  to  cicu- 
rate  or  domesticate,  logically  implies  a  reclaimer — a  domesticator.  Man 
himself  plays  this  role  in  the  usual  examples  of  reclamation  of  animals  and 
plants ;  can  he  then  wear  two  hats  in  regard  to  his  own  species?  Can  he  be 
both  domesticator  and  domes ticatee?  It  has  been  authoritatively  argued 
that  he  can  (1),  and  indeed  our  previous  speaker  has  both  here  and  else- 
where (#)  clearly  indicated  that  he  views  man  as  having  passed  through 
the  process  of  becoming  domesticated. 

For  the  purposes  of  this  discussion  we  shall  perhaps  make  more  progress 
if  we  consider  the  self-domestication  of  man  as  synonymous  with  the 
process  of  civilization.  This  process  has  been  marked  by  two  outstanding 
phenomena:  first,  the  creation  of  an  environment  of  ever-increasing  safety 
and  security;  and  second,  the  development  of  culture.  It  goes  without 
saying  that  I  use  the  word  culture  in  regard  to  the  intellectual  aspects  of 
civilization,  and  not  at  all  in  the  arrogant  and  restricted  meaning  by  which 
it  is  often  abused, 

Natural  selection  in  feral  man  probably  favored  strength,  aggressiveness 
and  freedom  from  physical  and  mental  defect.  Primitive  environments 
presented  the  necessity  of  constant  struggle  for  food,  shelter  and  mates, 
and  of  recurring  defense  against  enemies.  But  man  possessed  certain 
specializations,  which,  though  few  in  number,  were  unique.  These 
specializations  enabled  him  to  rearrange  his  environment  in  such  a  way  as 
to  provide  ever  more  efficiently  the  essentials  and  even  the  comforts  of 
life,  and  to  minimize  the  imminent  dangers,  thus  allowing  the  development 
of  high  attainments  in  art,  religion,  statecraft,  law  and  science — in  short, 
culture.  Concurrently  the  selective  advantages  of  sheer  strength  and 
aggressiveness  probably  declined. 

The  specializations  which  have  permitted  the  growth  of  cultural  patterns 
include  the  fully  upright  position  and  its  concomitants,  the  differentiation 
of  feet  and  hands ;  and  the  exceptional  size  of  the  brain,  with  the  develop- 
ment of  the  areas  involved  in  communication,  abstraction  and  symbolizing. 
Aside  from  these,  man  is  quite  generalized,  and  lacks  the  specializations 

i  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  29, 1954. 

759 

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760  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

which  are  characteristic  of  such  mammals  as  bats,  whales,  giraffes,  sloths 
or  elephants. 

Human  beings  are  most  uniform  in  those  characters  which  they  share 
with  the  greatest  variety  of  other  animals,  and  differ  one  from  another 
more  and  more  in  regard  to  traits  of  increasing  superficial ty  (3).  Thus, 
the  fundamental  chordate  pattern  is  a  remarkably  constant  characteristic 
of  human  beings;  the  ossified  skeleton  is  somewhat  more  subject  to  varia- 
tion; the  more  strictly  mammalian  characters  involving  skin,  hair,  mam- 
mary glands  and  control  of  body  temperature  are  quite  subject  to  modifi- 
cation. Mutations  involving  the  more  fundamental,  deep-seated  chordate 
traits  are  quite  likely  to  be  lethal,  but  changes  in  the  more  superficial 
characters  may  very  well  be  viable,  especially  as  those  which  might 
otherwise  be  detrimental  can  often  be  compensated  for  by  man's  ingenuity 
in  controlling  his  environment. 

It  is  difficult  to  specify  which,  if  any,  of  the  mutations  causing  vari- 
ability in  man  have  attained  their  present  population  frequencies  as  a 
direct  result  of  the  process  of  domestication.  It  is  even  more  difficult 
to  specify  which,  if  any,  of  the  human  traits  other  than  physical  peculiar- 
ities are  in  fact  the  result  of  genetic  determiners  at  all  (4,  5).  Kroeber  (1) 
lists  the  following  anatomical  characteristics  as  being  directly  associated 
with  the  domestication  of  man:  the  long  hair  on  the  head;  the  near-hair- 
lessness  of  the  body;  curly  hair,  woolly  hair,  blond  hair;  blue  eyes  and 
fair  skin.  To  these  Richter  (2)  has  added  the  possibility  that,  in  line  with 
experimental  data  from  rats,  the  adrenal  glands  of  civilized  man  may  have 
decreased  in  functional  importance,  while  the  hormonal  activity  of  the 
gonads  may  have  increased  in  salience.  Such  changes,  if  substantiated, 
might  well  be  expected  to  exert  significant  effects  on  behavior. 

In  the  course  of  man's  domestication  the  usual  evolutionary  processes 
have  acted  upon  him.  Through  geographic  or  cultural  isolation  and  the 
concomitant  effects  of  selection,  inbreeding,  mutation  and  genetic  drift, 
various  populations  have  become  more  or  less  differentiated  one  from  the 
other  in  respect  to  readily  recognizable  physical  traits,  such  as  skin  color, 
hair  form,  head  shape  and  occasionally  stature.  Whether  such  ethnic 
groups  differ  significantly  in  regard  to  mental  traits  we  shall  inquire  into 
presently. 

Within  ethnic  groups  are  found  smaller,  more  or  less  self-contained 
breeding  units,  which  we  call  isolates  (6).  These  isolates  are  delimited 
by  social  class,  religious  affiliations,  habitat,  and  other  cultural  and 
geographical  isolating  mechanisms.  They  may  in  some  instances  be 
distinguished  by  a  way  of  life.  Nomadism  and  agriculture,  for  example, 
require  for  successful  pursuance  quite  divergent  traits  of  temperament, 
and  Huxley  (7)  alleges  that  the  followers  of  these  modes  of  existence  early 
became  differentiated  in  many  such  qualities. 

In  regard  to  sets  of  alleles  whose  existence  in  man  has  been  well  estab- 
lished, the  proportions  of  the  various  alleles  can  be  shown  to  vary  not 
only  from  one  ethnic  group  to  another,  but  from  isolate  to  isolate  (8). 
Even  within  isolates  is  found  residual  genetic  variation,  some  of  it  due 

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PKOGEESS  IN  MAMMALIAN  GENETICS  AND  CANCER  761 

to  single,  major  gene  substitutions,  and  some  to  polygenic  heredity  (9). 
The  dynamics  of  major  gene  transmission  present  implications  for  human 
genetics  quite  different  from  those  evolving  from  polygenic  systems. 
These  implications  have  been  analyzed  in  some  detail  elsewhere  (10,  9), 
but  a  few  of  the  more  relevant  consequences  may  be  discussed  here.  The 
vast  majority  of  the  discontinuities  which  are  dependent  on  single  gene 
substitutions  are  pathological  in  nature.  Because  of  the  viability  impair- 
ment connected  with  them,  the  incidences  of  such  anomalies  and  diatheses, 
and  thus  of  the  genes  responsible  for  them,  are  low.  Under  the  restraining 
influence  of  natural  selection,  only  a  very  few  such  traits  have  reached 
population  incidences  above  one  in  10,000  and  the  vast  majority  are  much 
rarer  than  this. 

Conditions  of  modern  civilization,  however,  have  resulted  in  a  relaxation 
of  selection  against  some  genes,  and  in  the  subjection  of  other  genes  to  new 
selective  processes.  Some  of  the  potential  outcomes  of  these  man-made 
shifts  in  evolutionary  trends  warrant  thoughtful  scrutiny  and  deserve 
careful  analysis. 

One  of  the  many  consequences  of  man's  self-domestication  has  been 
the  phenomenal  progress  of  the  science  of  medicine.  Not  only  has  this 
progress  resulted  in  the  control  of  numerous  environmentally  conditioned 
diseases,  but  it  has  led  to  the  alleviation  of  various  genetically  determined 
disorders  and  anomalies  (11) .  The  result  is  that  some  types  of  individuals 
who  formerly  were  eliminated  before  reproducing,  or  before  completing 
their  families,  are  now  enabled  to  live  out  a  more  normal  span  of  life  and 
are  given  the  opportunity  of  more  normal  reproduction.  The  presumed 
implications  of  these  facts  have  aroused  concern  in  certain  quarters 
(12,  13).  The  concern  is,  in  my  opinion,  unjustified,  and  I  should  like  to 
point  out  once  more  the  underlying  fallacy,  to  which  attention  has  else- 
where been  directed  (14). 

Throughout  the  long  course  of  evolutionary  history,  living  organisms 
have  been  subject  to  continuing  change.  One  of  the  principal  causes  of 
genetic  change  is  mutation.  An  important  consequence  of  mutation  is 
that  the  mutated  gene  reproduces  itself  just  as  faithfully  in  its  new  mo- 
lecular arrangement  as  the  unmutated  gene  did  in  its  original  chemical 
form. 

It  is  a  familiar  observation  that  those  mutant  genes  which  produce 
conspicuous  effects  commonly  behave  as  recessive  genes,  although  these 
same  genes  may  also  produce  less  noticeable  effects  which,  by  special 
techniques,  are  detectable  in  the  heterozygote  (15).  Consequently  the 
occurrence  of  a  new  mutation  is  not  very  likely  to  be  noticed  until  two 
heterozygous  individuals  mate  and  produce  a  clearly  affected  homozygous 
offspring.  Mutation  at  a  particular  locus  may  occur  in  more  than  one 
gamete  or  in  more  than  one  individual,  and  in  fact  the  phenomenon  is 
characteristically  a  recurrent  event.  Thus,  despite  the  infrequent 
incidence  of  origin  of  any  particular  mutant  gene  by  mutation,  it  will 
tend  gradually  to  accumulate  in  the  species.  The  tendency  to  accumulate 
may  be  opposed  by  natural  selection  in  those  instances  in  which  the 

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762  proceedings:  symposium  on  25  years  of 

effects  of  the  mutant  gene  impair  to  any  degree  the  average  reproductive 
rate  of  those  who  possess  the  gene. 

Concomitant  with  long  ages  of  mutation  and  selection,  living  organisms 
are  characteristically  highly  adapted  and  delicately  adjusted  mechanisms. 
As  a  result,  fortuitous  new  mutations,  except  those  involving  only  super- 
ficial traits,  are  quite  likely  to  give  rise  to  deleterious,  even  lethal,  effects. 
Among  innumerable  fortuitous  mutations  over  the  ages,  however,  some 
few  have  been  favorable  or  beneficial,  and  from  the  selective  advantages 
conferred  upon  genes  of  this  nature  has  come  evolutionary  progress. 

Let  us  return  then  to  the  direct  consideration  of  the  concern  which 
is  being  commonly  expressed  these  days  over  the  possibility  that  the 
accumulation  even  of  spontaneous  mutations  in  man  may  eventually 
constitute  a  serious  threat  to  the  public  health.  The  argument  runs 
somewhat  as  follows:  Since  mutation  is  a  recurrent  phenomenon,  mutant 
alleles  would  gradually  accumulate  in  the  human  species  were  it  not  for 
the  fact  that  many  mutations  are  harmful.  This  fact  permits  the  tendency 
to  accumulate  to  be  opposed  by  the  process  of  natural  selection,  thus 
depressing  the  frequency  of  harmful  genes  to  an  equilibrium  value  at 
which  the  rate  of  elimination  is  balanced  by  the  rate  at  which  the  mutant 
gene  arises  by  fresh  mutation  in  any  generation. 

Now  the  progress  of  civilization,  and  especially  of  medicine  (it  is 
argued),  has  succeeded  in  ameliorating  the  effects  of  many  harmful  genes, 
so  that  selection  against  these  deleterious  genes  has  been  relaxed,  thus 
permitting  their  frequencies  to  attain  higher  and  higher  equilibrium 
levels.  Contemplation  of  the  long-range  results  of  further  successes  in 
ameliorating  the  effects  of  more  and  more  undesirable  genes  presents  an 
alarming  prospect  to  proponents  of  the  argument  as  I  have  reported  it. 

The  essential  fallacy  of  the  argument  (14)  consists  in  the  application  of 
the  epithets  "deleterious/ '  "harmful,"  or  "undesirable"  to  the  mutant 
genes  themselves  rather  than  to  their  effects.  If,  through  modern 
medical,  social  or  economic  progress,  selection  has  been  relaxed  against 
any  gene  with  harmful  effects,  this  relaxation  has  been  accomplished  only 
because  the  medical,  social  or  economic  agencies  have  provided  environ- 
ments in  which  the  effects  of  the  genes  are  rendered  less  harmful  or  quite 
innocuous. 

As  to  those  genes  that  persist  in  producing  detrimental  effects  in  all 
known  environments  and  despite  all  attempts  at  therapy,  selection  against 
them  remains  today  as  severe  and  effective  as  ever.  And  selection  will 
again  begin  to  operate  against  any  gene  for  which  it  has  been  relaxed  if 
the  burden  of  providing  the  necessary  therapeutic  conditions  begins  to 
outweigh  the  social  value  of  providing  them.  It  seems  reasonable  to 
presume,  however,  that  medical  and  social  advances  will  continue  to  be 
made  with  ever-increasing  efficiency,  and  that  therapeutic  or  preventive 
measures  which  may  now  seem  burdensome  will  be  continuously  improved 
and  will  become  ever  more  simple,  natural  and  acceptable. 

One  need  only  consider  some  of  the  many  commonplace  procedures 
which  probably  were  at  one  time  burdensome  but  which  are  now  com- 

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PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  763 

fortably  incorporated  into  our  modes  of  life.  Thus  we  compensate  for 
our  loss  of  natural  ability  to  control  adequately  the  temperature  of  our 
bodies  by  providing  ourselves  with  varying  degrees  of  clothing  and  with 
temperature-controlled  dwellings.  We  add  vitamins  to  our  diets,  and 
take  hormones  when  necessary.  We  successfully  feed  and  rear  infants 
in  the  absence  of  human  milk.  We  wear  glasses  when  indicated,  we  see 
our  dentists  at  least  twice  a  year,  and  we  are  reasonably  happy  under 
these  restrictive  derivatives  of  civilized  existence.  I  see  no  reason  to 
dread  the  genetic  effects  of  further  advances  in  medical  or  social  science. 

Let  us  turn  our  attention  now  to  the  problem  of  the  extent  to  which, 
during,  and  as  a  result  of,  the  domesticatory  process,  man's  behavior  may 
be  attributable  to  genetic  factors.  It  is  clear  from  the  foregoing  presen- 
tations in  this  symposium  that  a  genetic  basis  for  behavior  is  to  be  found 
in  some  domestic  animals,  and  that  the  process  of  domestication  has 
exerted  selective  action  on  the  gene  constellations  involved.  Indeed  the 
existence  of  polygenes  and  possibly  of  major  genes  which  affect  tempera- 
ment, motivation,  social  activity  and  other  behavioral  responses  in  experi- 
mental animals  has  been  well  established  by  various  workers  (2,  16-20). 

There  may  well  be  in  the  human  species  polygenic  systems  basic  to  the 
expression  of  organic  drives  analogous  to  those  in  animals,  but  it  is 
probable  that  as  determinants  of  social  roles,  attitudes  and  behavior, 
genetic  factors  are  of  limited  significance  in  man.  The  rationale  for  this 
point  of  view  has  been  presented  in  detail  in  an  earlier  publication  (10). 
The  salient  points  in  the  argument  may  well  be  discussed  here  anew  from 
the  standpoint  of  domestication. 

First,  there  may  be  some  doubt  as  to  the  extent  to  which  behavior  in 
animals  should  be  equated  with  that  in  man.  To  admit  "the  obvious 
and  close  analogy  between  human  and  rodent  types  of  boldness"  (21), 
for  example,  is  to  ignore  the  striking  qualitative  differences  between  the 
significance  of  organic  drives  for  animals  whose  interindividual  relation- 
ships are  on  a  physiological  or  biosocial  level,  and  their  significance  for 
human  beings,  whose  interpersonal  relationships  are  on  a  psychosocial 
level  (22,  23).  Genetic  variables  affecting  behavior  in  animals  may  fairly 
be  postulated  as  exerting  their  effects  on  neurophysiologic  mechanisms. 
While  certain  severe  (but  rare)  neurologic  aberrations  in  man  also  are 
undoubtedly  due  to  genetic  interference  with  neurophysiological  mecha- 
nisms, the  overwhelming  bulk  of  man's  behavioral  variability  must  surely 
occur  as  a  result  of  functional  disturbances  in  the  human  cerebral  cortex, 
to  which  I  have  already  pointed  as  a  specialization  unique  in  man. 

Undomesticated  animals  and  plants  have  developed  evolutionarily  by 
the  adapting  of  their  characteristics  to  existing  environments.  Domesti- 
cated organisms  have  had  their  characteristics  adapted  by  human  manip- 
ulation to  environments  which  were  themselves  at  the  same  time  being 
adapted  by  man  to  the  changing  traits  of  the  organisms.  Husbandry  is 
a  complex  art.  Man  himself,  however,  has  become  adapted  evolutionarily, 
by  consciously  and  purposefully  altering  his  own  environment  by  means 
of  his  own  inventions,  to  meet  existing  genotypes.     Moreover,  man's 

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764  proceedings:  symposium  on  25  tears  of 

ability  to  reason,  to  communicate,  and  to  record  his  thoughts  in  writing 
has  made  the  environmental  improvement  a  cumulative  process.  The 
result  is  all  too  apparently  the  gradual  outstripping  of  biological  inherit- 
ance by  social  inheritance,  or  tradition,  as  a  major  social  force. 

Various  writers  have  interpreted  in  widely  varying  ways  the  prospect  of 
the  replacement  of  the  mechanism  of  the  chromosome  and  the  gene  by  the 
forces  of  social  organization  as  the  major  determinant  of  human  behavior. 
Some,  like  Orwell  (24)  and  Seidenberg  (25),  take  pessimistic  views  indeed. 
In  clear,  sure  strokes  Seidenberg  paints  an  engrossing  verbal  picture  of 
the  long  conflict  between  instinct  and  intelligence — the  one  dominant 
because  of  its  evolutionary  priority,  the  other  offering  a  challenge  by 
virtue  of  its  inherently  cumulative  power.  He  sees,  as  the  result  of  the 
usurpation  of  the  dominant  role  by  tradition,  the  gradual  approaching  of 
fixed  perimeters,  of  a  crystallized  status  of  man  within  compelling  forms 
of  organized  procedures.  In  the  ultimate  state  of  crystallization  to 
which  the  principle  of  organization  leads,  Seidenberg  predicts  that  con- 
sciousness will  have  accomplished  its  task,  leaving  mankind  sealed,  as  it 
were,  within  patterns  of  frigid  and  unalterable  perfection. 

The  subject  is  developed  logically  and  inexorably,  with  frequent  well- 
chosen  documentation.  The  ultimate  triumph  of  organization  over 
individuality  is  presented  as  inevitable,  and  is  a  frightening  prospect 
indeed.  The  terrible  fascination  of  the  author's  logic,  which  can  be 
experienced  only  by  reading  the  volume  itself,  holds  the  interest  of  the 
reader  to  the  implacable  end. 

In  contrast  to  the  foregoing  views,  other  writers  (10,  16,  27)  take  the 
position  that,  although  man's  ability  to  control  and  alter  his  surroundings 
has  indeed  brought  about  more  and  more  uniformity  of  the  physical 
environment,  it  has  at  the  same  time  resulted  in  greater  and  greater 
heterogeneity  of  the  social  environment.  Not  only  are  human  social 
environments  different  from  place  to  place,  but  they  have  in  the  course 
of  the  history  of  mankind  been  extremely  varied,  and  in  terms  of  evo- 
lutionary time  have  succeeded  each  other  with  considerable  rapidity. 
As  a  consequence,  man  has  been  subjected  to  much  of  the  external  vari- 
ability required  for  molding  the  frequencies  of  such  alleles  as  might  be 
involved  in  various  aspects  of  behavior. 

Of  the  two  obvious  possible  responses,  namely,  the  selection  of  ever 
more  precise  specializations  which  would  fit  him  to  cope  with  specific 
environmental  situations,  and  the  development  of  increasingly  plastic 
responsiveness  of  any  of  a  variety  of  environmental  situations,  man 
apparently  followed  the  second  trail  (27).  It  is  easy  to  accept  the  sug- 
gestion (26)  that  the  one  strong  selective  pressure  to  which  mankind  has 
been  continuously  and  unremittingly  subjected  during  his  period  of 
domestication  and  in  all  social  environments  is  selection  for  educability — 
for  the  capacity  to  benefit  from  experience  and  reasoning.  Just  as  one 
of  man's  striking  physical  assets  is  the  relatively  generalized  human  hand, 
which  can  be  put  to  a  variety  of  uses,  so  plasticity  of  responsiveness, 
permitting  adjustment  to  a  variety  of  situations,  has  been  characteristic 
of  man's  psychic  evolution. 

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PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  765 

It  seems  highly  probable  that  in  all  human  social  situations  plasticity 
of  response,  and  emotional  and  temperamental  resilience,  have  been  of 
sufficient  value  as  to  have  been  at  a  selective  advantage.  If  this  be  true, 
it  is  highly  improbable  that  any  particular  population  ever  developed 
significant  genetic  differentiation  in  respect  to  specific  response  patterns, 
temperaments,  personality  types,  or  intellectual  capacities.  In  other 
words,  despite  the  exceptional  diversity  of  genotypes  among  human 
beings  (8),  the  plasticity  of  individual  response  to  the  social  environment 
is  even  more  noteworthy.  The  dynamics  of  polygenic  population  genetics 
lend  strong  support  to  such  a  view  (10).  Thus  we  arrive  logically  at  our 
original  conclusion  that,  as  determiners  of  specific  and  temporal  social 
roles  and  attitudes,  genetic  factors  are,  on  the  whole,  of  limited  signifi- 
cance. 

In  the  long-range  outlook,  however,  the  genetic  endowment  takes  its 
place  as  co-equal  with  the  social  millieu  in  the  forging  of  societies.  From 
the  vantage  point  of  history  one  may  observe  the  rhythm  of  the  growth 
and  decline  of  civilizations.  And  perhaps  it  is,  after  all,  the  historian 
who  can  successfully  bridge  the  gap  between  the  viewpoints  of  such 
writers  as  Siedenberg  and  Orwell  on  the  one  hand,  and  Dobzhansky  and 
Montagu  on  the  other.  Toynbee,  for  example  (28),  develops  the  con- 
cept of  "etherialization,"  an  overcoming  of  material  obstacles,  leading  to 
the  release  of  the  energies  of  society  to  make  responses  to  challenges 
which  are  henceforth  internal  rather  than  external,  spiritual  rather  than 
material.  Although  Toynbee  writes  with  theistic  overtones,  there  re- 
mains, when  these  are  stripped  away,  an  important  basic  historical  truth 
in  the  concept. 

Throughout  history  the  process  of  cultural  acquisition  has  resulted  in 
bursts  of  growth  and  development  of  societies,  alternating  with  their 
crystallization  and  ultimate  degeneration.  Toynbee  epitomizes  this  by 
concluding  that,  just  as  differentiation  is  the  mark  of  growth,  so  standard- 
ization is  the  mark  of  disintegration.  But  while  history  thus  furnishes 
Seidenberg  with  some  support,  in  that  standardization  has  indeed  been 
the  culmination  of  many  societies  in  the  past  and  may  well  occur  again 
in  the  future,  history  provides  at  the  same  time  the  clue  to  the  answer 
to  Seidenberg's  dilemma.  It  lies  in  the  very  fact  that  whenever  standard- 
ization of  thought  and  social  organization  has  from  time  to  time  threatened 
the  existence  of  civilization,  new  lines  of  behavior  and  of  development 
have  sprung  up  out  of  the  tremendous  potential  of  variability  of  response 
inherent  in  the  human  species.  And  with  the  increasing  ability  of  social 
psychology  to  provide  effective  techniques  for  the  management  of  inter- 
personal and  intergroup  relations  (29,  80),  we  may  look  forward  hopefully 
to  the  ever  more  efficient  use  of  the  really  enormous  constructive  poten- 
tialities which  are  biologically  characteristic  of  mankind  as  it  exists  today. 

The  preceding  considerations  carry  with  them  the  clear  implication 
that  there  is  at  present  no  great  danger  that  the  civilized  world  is  facing 
genetic  deterioration  in  regard  to  intelligence.  I  suspect  that  I  must  not 
end  this  discussion  without  presenting  my  reasons  for  this  belief,  since 

Vol.    15,   No.   3,   December    1954 


766  proceedings:  symposium  on  26  years  op 

there  are  widely  publicized  allegations  to  the  contrary  (81-38).  It  is 
claimed  that  fertility  differentials  are  today  of  such  nature  that  they 
lead  to  a  decline  in  the  average  intelligence  of  the  populations  of  Great 
Britain  and  America,  at  least,  of  from  one  to  four  I.  Q.  points  per  gener- 
ation. If  the  reduction  were  considered  to  be  merely  in  terms  of  pheno- 
typic  manifestation  there  might  seem  less  cause  for  alarm,  but  those  who 
allege  the  decline  see  it  as  involving  genotypic  deterioration  as  well.  The 
current  argument  for  the  thesis  is  the  apparent  existence  of  a  negative 
correlation  between  I.  Q.  and  fertility — correlation  said  to  be  at  least 
partly  independent  of  social  and  economic  status.  This  argument  was 
adopted  upon  the  realization  of  the  untenability  of  the  older  premises 
involving  the  reproductive  rates  in  various  socio-economic  classes  and 
their  supposed  relation  to  I.  Q.  levels  (84,  85). 

Careful  scrutiny  of  the  argument,  however,  indicates  that  the  actual 
data  on  which  the  belief  in  the  negative  correlation  between  I.  Q.  and 
fertility  is  based  merely  indicate  an  inverse  relationship  between  the  test 
intelligence  of  children  and  the  number  of  their  brothers  and  sisters  (10). 
This  relationship  does  not  necessarily  imply  a  similar  negative  correlation 
between  intelligence  and  fertility,  since  the  lower  grade  mental  defectives, 
whatever  the  size  of  their  sibships,  are  generally  not  themselves  fertile. 
Proof  that  differences  in  fertility  actually  permit  phenotypic  selection 
against  high  intelligence  must  rest  on  the  demonstration  that  any  negative 
regression  of  fertility  on  I.  Q.  over  one  portion  of  the  range  of  intelligence 
is  not  compensated  for  by  a  positive  regression  over  another  part  of  the 
range. 

Moreover,  any  statement  regarding  phenotypic  selection  based  on 
reproductive  differentials  must  take  into  account  the  demonstrable 
dependence  of  I.  Q.  development  upon  environmental  factors,  and  especial- 
ly on  such  factors  as  are  associated  with  educational  facilities  and  cultural 
stimuli  (6).  Differentials  in  I.  Q.  can  usually  be  demonstrated  between 
various  socio-economic  levels,  between  rural  and  urban  populations,  and 
between  regions  in  which  public  school  expenditures  are  at  variance. 
But  there  is  increasing  evidence  that  changes  in  these  environmental 
situations  are  reflected  in  changes  in  the  I.  Q.  In  one  isolated  population 
in  a  depressed  area,  for  example,  the  very  low  average  intelligence  test 
scores  recorded  in  1930  were  found  to  be  more  than  ten  points  higher  for 
comparable  age  groups  following  a  decade  of  considerable  improvement 
in  social  and  economic  conditions  and  in  educational  opportunities  (86). 
The  results  of  the  second  series  of  tests  in  the  comprehensive  survey  which 
has  been  conducted  in  Scotland  for  more  than  20  years  indicate  that  the 
average  test-intelligence  level  in  the  population  has  certainly  not  declined, 
and  may  have  improved  slightly  (87). 

I  do  not  doubt  that  fertility  differentials  can  be  such  as  to  produce 
phenotypic  changes  in  population  I.  Q.,  perhaps  at  a  rapid  rate,  and  in 
either  direction.  But  this  admission  is  not  to  concede  that  genotypic 
changes  are  necessarily  involved.  The  belief  that  they  are  involved 
appears  to  rest  ultimately  on  the  observable  correlation  in  I.  Q.  between 

Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  767 

parent  and  child:  a  correlation  which  may  very  easily  be  confused  with 
causation. 

It  is  possible,  for  example  (38),  that  the  deliberate  choice  of  early 
marriage  and  family  responsibilities  reduces  the  chance  of  becoming 
eminent  or  even  of  attaining  the  full  potential  of  test  intelligence,  without 
in  the  least  lowering  the  genetic  worth  of  the  individual.  The  limiting 
factors  which  determine  human  fertility  are  probably,  over  the  greater 
part  of  the  range  of  intelligence,  social  rather  than  biologic.  Moreover, 
the  factors  in  the  social  environment  which  discourage  fertility  are  in 
general  those  which  tend  to  permit  the  development  of  the  full  potential 
of  the  I.  Q.,  whereas  the  conditions  which  encourage  fertility  are  broadly 
the  same  as,  or  intimately  related  to,  those  which  tend  to  depress  the 
phenotypic  development  of  high-test  intelligence  in  many,  if  not  all 
genotypes  (10). 

It  must  be  kept  in  mind,  moreover,  that  the  genetic  background  for 
intelligence  (and  nothing  I  have  said  is  to  be  inferred  as  denying  such  a 
background)  is  undoubtedly  polygenic  in  nature.  Phenotypic  differ- 
entiation in  polygenic  traits  is  less  likely  to  result  from  genetic  drift  than 
in  characters  contingent  on  major  genes,  since  the  effects  of  individual 
polygenes  are  in  large  part  mutually  interchangeable  (39,  9) .  Neither  is 
it  probable,  as  has  already  been  pointed  out  in  regard  to  behavioral 
responses,  that  natural  selection  has  resulted  in  the  complete  fixation  of 
different  constellations  of  genes  for  intelligence  in  different  populations. 

If  the  viewpoints  just  sketched  have  any  validity  (and  I  believe  they 
have),  it  is  obvious  that  fertility  differentials  will  under  present  cultural 
conditions  have  minimal  effect  on  the  frequencies  of  genotypes  which 
may  conceivably  be  involved  in  determining  innate  potentialities  for 
intelligence-test  performance. 

Looking  to  the  future,  we  must  in  all  sincerity  pin  our  hopes  on  the 
management  of  social  relationships.  Changes  in  human  social  organiza- 
tion have  taken  place  at  unbelievably  rapid  rates  in  the  past,  and  probably 
will  continue  to  do  so  in  the  future.  On  the  other  hand,  man's  biological 
evolution  has  been,  and  will  surely  continue  to  be,  very  slow  indeed. 
Little  or  no  alteration  in  man  has  occurred  anatomically  in  the  last  half 
million  years,  and  there  is  little  to  indicate  that  intelligence  has  greatly 
changed.  I  doubt  that  the  development  of  the  atomic  bomb  is  the  mani- 
festation of  any  greater  innate  mental  capacity  than  such  creative  efforts, 
largely  buried  in  the  vaults  of  antiquity,  as  the  invention  and  employment 
of  the  wheel  or  the  bow  and  arrow.  Let  us  wish  all  success  to  our  col- 
leagues, the  social  scientists,  in  their  efforts  to  enable  man  to  use  more 
constructively  the  enormous  latent  biological  potentialities  which  exist  in 
all  peoples  everywhere. 

References 

(1)  Kroebeb,  A.  L.:  Anthropology.     New  York,  Harcourt,  Brace  and  Co.,  1948. 

(2)  Richter,  C.  P.:  Domestication  of  the  Norway  rat  and  its  implication  for  the 

study  of  genetics  in  man.     Am.  J.  Human  Genet.  4:  273-285,  1952. 
(S)  Snyder,  L.  H.:  The  genetic  approach  to  human  individuality.     Sci.  Month.  68: 
165,  1949. 

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(4)  David,  P.  R.,  and  Snyder,  L.  H.:  Genetics  and  disease.     Proc.  Second  Nat. 

Cancer  Conf.     New  York,  American  Cancer  Society,  1954,  p.  1128. 

(5)  :  Principles  of  human   genetics.     In   Genetics  and  the   Inheritance  of 

Integrated  Neurological  and   Psychiatric  Patterns    (Hooker,  D.,  ed.).     Res. 
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(6)  Snyder,  L.  H.:  The  Principles  of  Heredity,  4th  ed.     Boston,  D.  C.  Heath  and 

Co.,  1951. 

(7)  Huxley,  J.:  Genetics,  evolution  and  human  destiny.     In  Genetics  in  the  20th 

Century  (Dunn,  L.  C,  ed.).     New  York,  The  Macmillan  Co.,  1951. 

(8)  Snyder,  L.  H.:  Principles  of  gene  distribution  in  human  populations.     Yale  J. 

Biol.  &  Med.  19:  817-833,  1947. 

(9)  :  Old  and  new  pathways  in  human  genetics.     Am.  J.  Human  Genet.  3: 

1-16,  1951. 

(10)  David,  P.  R.,  and  Snyder,  L.  H.:  Genetic  variability  and  human  behavior. 

In  Social  Psychology  at  the  Crossroads  (Rohrer,  J.,  and  Sherif,  M.,  eds.).  New 
York,  Harper  and  Bros.,  1951. 

(11)  Snyder,  L.  H.:  Frontiers  in  genetics.     In  Frontiers  in  Medicine:  The  March 

of  Medicine  1950.     New  York,  Columbia  University  Press,  1951. 

(12)  Cook,  R.  C:  Lethal  genes  a  factor  in  fertility.     Eugenics  News  38:  49-55,1953. 
(IS)   Muller,  H.  J.:  Our  load  of  mutations.     Am.  J.  Human  Genet.  2:   111-176, 

1950. 

(14)  Snyder,  L.  H.,  and  David,  P.  R.:  Heredity  and  preventive  medicine.     In  Text- 

book of  Preventive  Medicine  (Leavell,  H.,  and  Clark,  E.  Gurney,  eds.).    New 
York,  McGraw-Hill  Book  Co.,  Inc.,  1953. 

(15)  Neel,  J.  V.:  The  detection  of  genetic  carriers  of  inherited  disease.     In  Clinical 

Genetics  (Sorsby,  A.,  ed.).     London,  Butterworth  and  Co.,  Ltd.,  1953. 

(16)  Fuller,  J.  L.,  and  Scott,  J.  P.:  I.  Heredity  and  learning  ability  in  infrahuman 

mammals.     Eugenics  Quart.  1:  28-43,  1954. 

(17)  Hall,  S.  C:  The  genetics  of  behavior.     In  Handbook  of  Experimental  Psy- 

chology (Stephens,  S.  S.,  ed.).     New  York,  John  Wiley,  1951. 

(18)  Heron,  W.  T.:  The  inheritance  of  brightness  and  dullness  in  maze  learning 

ability  in  the  rat.     J.  Genet.  Psychol.  59:  41-49,  1941. 

(19)  Scott,  J.  P.:  Genetic  differences  in  the  social  behavior  of  inbred  strains  of  mice. 

J.  Hered.  33:   11,  1942. 

(20)  Tryon,  R.  C:  Individual  differences.     In  Comparative  Psychology  (Moss,  F.  A., 

ed.).     New  York,  Prentice  Hall,  Inc.,  1946. 

(21)  Murphy,  G.:  Genetic  and  social  significance  of  differential  fertility;  review  of 

relevant  research  on  inheritance  of  metal  traits.     Milbank  Mem.  Fund  Quart. 
25:  373-382,  1947. 

(22)  Schneirla,  T.  C:  Problems  in  the  biopsy chology  of  social  organization.     J. 

Abn.  and  Soc.  Psychol.  41:  385-402,  1946. 

(28)  :  The  "levels"  concept.     In  Social  Psychology  at  the  Crossroads.    Rohrer, 

J.,  and  Sherif,  M.,  eds.).     New  York,  Harper  and  Bros.,  1951. 

(24)  Orwell,  G.:  "1984."     New  York,  Harcourt,  Brace  and  Co.,  1949. 

(25)  Seidenberg,  R.:  Posthistoric  Man.     Chapel  Hill,  Univ.  North  Carolina  Press, 

1950. 

(26)  Dobzhansky,  T.:  The  genetic  nature  of  differences  among  men.     In  Evolution- 

ary Thought  in  America  (Persons,  Stow,  ed.) .     New  Haven,  Yale  Univ.  Press, 
1950. 

(27)  Dobzhansky,   T.,  and  Ashley  Montagu,   M.   F.:  Natural  selection  and  the 

mental  capacities  of  mankind.     Science  105:  587-590,  1947. 

(28)  Toynbee,  A.  J.:  A  Study  of  History.     New  York  and  London,  The  Oxford 

Univ.  Press,  1947. 

(29)  Rohrer,  J.,  and  Sherif,  M.:  Social  psychology  at  the  crossroads.     In  The  Univ. 

of  Oklahoma  Lect.  in  Soc.  Psych.     New  York,  Harper  and  Bros.,  1951. 
(SO)  Sherif,  M.,  and  Wilson,  M.  O.,  eds.:  Group  relations  at  the  crossroads.     The 
Univ.  of  Oklahoma  Lect.  in  Soc.  Psychol.     New  York,  Harper  and  Bros.,  1953. 

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PEOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  769 

(31)  Burt,  C:  Intelligence  and  fertility.     Occasional  Papers  on  Eugenics.     London, 

Hamilton  Hamish  Medical  Books,  1946. 

(32)  Cattell,  R.  B.:  Effects  of  human  fertility  on  the  distribution  of  intelligence  and 

culture.     39th  Yearbook,  Nat.  Soc.  Stud.  Educ,  Part  1:  221,  1940. 

(33)  Thompson,    G.:  The    trend    of    national   intelligence.     Occasional    Papers    on 

Eugenics.     London,  Hamilton  Hamish  Medical  Books,  1947. 

(34)  Osborn,  F.:  Preface  to  Eugenics.     New  York,  Harper  and  Bros.,  1940. 

(35)  Snyder,  L.  H.:  The  genetic  and  biologic  bases  of  mental  disorders.     In  Mental 

Health.     A.  A.  A.  S.  Symposium  No.  9,  1939. 

(36)  Wheeler,  L.  R.:  A  comparative  study  of  the  intelligence  of  East  Tennessee 

mountain  children.     J.  Educ.  Psychol.  33:  321-334,  1942. 

(37)  Mental  Survey  Committee  (G.  H.  Thompson,  Chm.).     The  Trend  of  Scottish 

Intelligence.     London,  Univ.  London  Press,  1949. 

(38)  Price,  B.:  Reference  data  on  Moscow  families  of  1935.     Psychol.  Bull.  35:  696- 

697,  1938. 

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London.     Butterworth  and  Co.,  Ltd.,  1953. 


Vol.    15,   No.   3,   December   1954 

316263—54 34 


Discussion :  Session  VI 
Dr.  John  L.  Fuller,  Roscoe  B.  Jackson  Memorial  Laboratory,  Bar  Harbor,  Maine 

It  is  obvious  from  the  high  quality  of  these  three  papers  that  the  genetic  method  can 
make  a  definite  contribution  to  the  broad  area  of  behavioral  sciences.  I  shall  comment 
only  upon  certain  special  aspects  of  each  paper,  since  a  full  evaluation  would  require 
much  thought  and  time. 

Dr.  Richter  presented  an  account  of  domestication  of  the  rat  in  which  he  placed 
great  emphasis  upon  changes  in  the  relative  importance  of  the  gonads  and  adrenal 
glands.  Certainly  much  of  what  he  said  probably  applies  to  other  domestic  species. 
However,  selection  under  domestication  is  not  always  in  the  direction  of  docility. 
Certain  terrier  breeds  have  been  selected  for  aggressiveness  to  a  degree  that  appears  to 
he  detrimental  to  the  establishment  of  a  stable  social  organization  when  the  dogs  are 
living  in  small  naturalistic  groups.  Would  the  same  adrenal  changes  occur  here,  as  in 
a  strain  selected  for  docility? 

I  also  wish  to  express  some  caution  concerning  the  idea  that  the  strain  differences  in 
behavior  are  a  direct  consequence  of  endocrine-gland  differences.  Recently  we  com- 
pared adrenalectomized,  intact,  and  cortisone-injected  members  of  two  strains  of  mice 
which  were  rather  sharply  different  in  activity,  emotionality,  and  the  frequency  of 
occurrence  of  several  specific  behavior  patterns.  Drastic  changes  in  the  hormones  did 
not  modify  the  strain  differences  in  behavior.  I  believe  that  we  can  profitably  go  for- 
ward in  this  kind  of  study  viewing  the  brain  as  a  target  organ,  and  comparing  target 
organ-response  to  hormones  in  wild  and  domestic  races,  and  in  behaviorally  different 
domestic  races. 

Dr.  Scott's  paper  deals  with  two  major  topics — the  history  of  domestic  dogs,  and  an 
experiment  designed  to  learn  about  the  genetic  nature  of  the  differences  in  breed  be- 
havior which  have  been  established  by  selection.  Scott's  conclusion  that  selection  has 
not  fundamentally  altered  the  behavior  patterns  of  the  wolflike  ancestor  of  dogs  has 
obvious  implications  for  that  civilized  primate,  man. 

It  is  impossible  to  adequately  discuss  the  detailed  genetic  results  which  Dr.  Scott 
has  presented  this  afternoon  without  more  time  than  I  have  available.  He  deserves 
a  great  deal  of  credit  for  applying  a  variety  of  genetic  and  psychological  hypothetical 
models  to  his  data.  I  should  like  to  point  out  that  the  value  of  the  data  does  not 
depend  upon  the  ultimate  verification  of  each  and  every  hypothesis  which  he  has  adopted. 
My  guess  is  that  several  different  approaches  will  be  necessary  before  we  can  judge  the 
most  fruitful  model  for  a  genetics  of  behavior.  Scott's  work  is  a  pioneering  effort  in 
this  field. 

Finally  in  considering  Dr.  Snyder's  paper,  I  should  like  to  comment  on  his  hopeful 
evaluation  of  man's  genetic  future.  I  believe  he  is  correct  in  his  appraisal,  but  I  want 
to  stress  the  fact  that  this  sanguine  outlook  is  not  based  upon  a  belief  that  man  is 
superior  to  biological  forces.  Biological  and  social  conditions  at  present  are  not 
eliminating  valuable  genotypes  while  preserving  poor  ones,  but  I  suspect  that  deliberate 
selection  of  man  could  lead  to  races  of  diverse  intellectual  potentiality.  No  one  is 
seriously  proposing  this.  I  simply  mention  it  to  emphasize  the  fact  that  human  behavior, 
like  that  of  the  dog  and  rat,  has  a  biological  substratum. 

Students  of  heredity  and  of  behavior  have  sometimes  seemed  to  speak  in  different 
languages.     These  papers  indicate  that  a  new  synthesis  is  in  the  making. 

771 


Journal    of   the   National    Cancer    Institute,    Vol.    15,    No.    3,    December    1954 


Session  VII.  Genetic  Techniques  in  the 
Study  of  Cancer :  New  Approaches 


Chairman,  Dr.  Howard  B.  Andervont, 
Chief j    Laboratory    of   Biology,    National    Cancer 
Institute,  Bethesda,  Md.;  Scientific  Director,  Roscoe 
B.  Jackson  Memorial  Laboratory 


Speaker:  Dr.  Walter  E.  Heston 
Localization  of  Gene  Action  in  the  Causation  of  Lung  and  Mammary 

Gland  Tumors  in  Mice 
Discusser:  Dr.  L.  M.  Dmochowski 

Speaker:  Miss  Margaret  M.  Dickie 

The  Use  of  Ft  Hybrid  and  Backcross  Generations  to  Reveal  New  and/or 

Uncommon  Tumor  Types 
Discusser:  Dr.  Morris  Barrett* 

Speaker:  Dr.  Elizabeth  Fekete  and  Dr.  Allen  B.  Griffen 

Signiiicance  of  Recent  Developments  in  Nuclear  Cytology  and  Cytogenetics 

of  the  Mouse 
Discusser:  Dr.  Donald  F.  Jones 


'Discussion  not  submitted. 

773 

Journal   of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


Introduction :  Session  VII 
Dr.  H.  B.  Andervont,  Chairman 

Previous  sessions  of  this  symposium  presented  progress  in  mammalian  genetics  since 
the  founding  of  the  Roscoe  B.  Jackson  Memorial  Laboratory  in  1929.  This  morning 
we  shall  hear  how  the  basic  contributions  of  these  geneticists  are  assisting  in  the  fight 
against  cancer.  Thus,  our  meeting  will  demonstrate  how  a  blending  of  fundamental 
and  applied  research  is  essential  for  a  scientific  study  of  disease.  Cancer  will  serve  as  a 
prototype  of  those  diseases  open  to  attack  through  basic  research  performed  at  this 
Laboratory. 

All  participants  in  this  session  are  exceptionally  well  qualified  for  each  has  con- 
tributed to  the  basic  science  of  genetics  and  to  problems  of  practical  importance. 
They  exemplify  the  difficulty  in  drawing  a  sharp  line  of  demarcation  between  funda- 
mental and  applied  scientists.  Perhaps  the  tendency  to  perform  either  kind  of  research 
is  dependent  upon  the  genotype  of  the  individual.  We  heard  from  Dr.  Castle  how,  as  a 
young  man,  Dr.  Little  was  confronted  with  a  practical  problem  in  tumor  transplan- 
tation by  Dr.  Tyzzer.  Investigation  of  this  problem  must  have  impressed  Dr.  Little 
with  the  importance  of  inbred  animals  in  cancer  research  and  this  idea  led  to  the 
development  of  inbred  strains  of  mice  that  are  not  only  basic  research  tools  used  by 
cancer  workers  but  also  are  rapidly  affecting  other  branches  of  medical  science. 

This  morning  we  shall  hear  how  the  use  of  these  strains  has  aided  the  progress  of 
cancer  research.  It  is  reasonably  safe  to  assume  we  shall  learn  that  the  occurrence  of 
lung  tumors  in  the  inbred  mouse  is  largely  dependent  upon  hereditary  factors  and  this 
tumor  can  be  used  for  precise  determinations  of  the  interplay  of  hereditary  and  environ- 
mental factors  in  carcinogenesis.  Mammary  gland  tumors  are  more  complex  for  they 
arise  through  the  interaction  of  hereditary,  hormonal  and  environmental  influences. 
The  round-table  session  of  yesterday  emphasized  the  importance  of  hormonal  stimu- 
lation in  the  occurrence  of  a  variety  of  tumors  and  mammary  gland  tumors  belong 
in  this  group  of  neoplasms.  Now  we  shall  hear  that,  in  addition  to  hereditary  and 
hormonal  factors,  a  transmissible  agent  is  also  involved  in  the  occurrence  of  mammary 
gland  tumors  in  mice.  The  discovery  of  this  agent  is  one  of  the  outstanding  con- 
tributions of  this  Laboratory  to  cancer  research. 

After  hearing  yesterday's  discussion  of  carcinogenesis  and  today's  session  you  will 
note  that  as  we  progress  in  cancer  research  we  become  more  dependent  upon  the  use 
of  inbred  animals.  Indeed,  it  now  appears  that  these  animals  may  be  essential  for 
solution  of  the  basic  problems  of  the  difference  between  normal  and  malignant  cells 
and  the  factors  responsible  for  the  change  from  the  normal  to  the  malignant  state.  This 
does  not  imply  that  such  knowledge  is  essential  for  the  prevention  or  cure  of  cancer  for 
the  history  of  medicine  contains  many  instances  of  the  cure  or  prevention  of  disease 
without  knowledge  of  etiologic  factors  or  the  fundamental  cellular  changes  involved. 
The  Jackson  Memorial  Laboratory  has  supplied  one  excellent  example.  Discovery  of 
the  mammary  tumor  agent  made  possible  the  virtual  elimination  of  breast  tumors  in 
inbred  strains  of  mice. 

We  must  remember,  however,  that,  for  the  purpose  of  this  Symposium,  cancer  serves 
as  an  example  of  how  the  basic  investigations  from  this  Laboratory  are  applicable  to 
a  practical  and  pressing  problem  in  medical  science.  It  is  doubtful  whether  the  same 
disease  will  be  used  as  such  when  some  of  you  attend  the  fiftieth  anniversary  program. 
Regardless  of  the  problems  you  will  face,  it  is  reasonably  safe  to  predict  that  the 
genetic  constitution  of  the  hosts  will  represent  an  important  part  of  your  discussions. 
For,  under  the  inspiring  leadership  of  Dr.  Little,  the  application  of  genetic  principles 
to  biologic  problems  and  the  establishment  of  inbred  animals  as  research  tools  are 
basic  contributions  made  by  this  Laboratory. 


774 


Localization  of  Gene  Action  in  the 
Causation  of  Lung  and  Mammary 
Gland  Tumors  in  Mice  x 


W.    E.     Heston,    National    Cancer    Institute,2 
Bethesda,  Md. 


Owing  in  large  measure  to  research  carried  out  here  at  the  Jackson 
Laboratory  it  is  now  almost  universally  accepted  that  genes  are  involved 
in  the  causation  of  cancer.  The  general  pattern  of  inheritance  for  the 
various  types  of  cancer  has  been  quite  clearly  defined  as  multiple  factor 
inheritance  with  alternative  expression  of  the  character.  Primary  prob- 
lems of  the  future  lie  in  the  field  of  physiologic  genetics  where  attempts 
are  made  to  link  the  gene  to  the  character  by  describing  the  paths  through 
which  gene  action  influences  the  probability  that  the  tumor  will  occur. 

The  first  step  in  this  general  area  is  obviously  the  localization  of  the 
gene  action,  the  subject  of  this  discussion.  Such  localization  may  be  in 
respect  to  anatomy,  i.e.,  in  determining  in  what  organs  or  tissues  the  gene 
action  is  occurring;  in  respect  to  physiology,  i.e.,  what  physiologic  path- 
ways are  initiated  or  directed  by  the  gene  action;  and  in  a  broad  sense 
such  localization  may  include  the  possibility  of  gene  changes  at  the  time  of 
the  neoplastic  change  of  the  cell.  Certain  approaches  to  these  problems 
will  be  described  and  illustrated  by  projects  carried  out  with  respect  to 
mammary  gland  and  pulmonary  tumors  of  the  mouse.  There  is  no  definite 
claim  that  these  approaches  are  new  as  labeled  in  the  title  of  this  morning's 
session.  So-called  new  approaches  are  often  old  approaches  modified  to 
fit  new  situations  and  answer  new  questions.  The  chief  concern,  however, 
is  whether  or  not  the  information  obtained  is  new,  and  from  the  studies 
referred  to  herein  certain  new  information  has  been  forthcoming. 

The  transplantation  of  genetically  different  organs  or  tissues  from  two 
inbred  strains  into  a  common  host,  the  Fi  hybrid  of  those  two  strains, 
provides  a  means  for  determining  whether  or  not  certain  gene  action 
which  controls  characters  that  may  develop  later  in  these  organs  is  local- 
ized in  the  respective  organs  or  tissues.  If  while  in  a  common  host  the 
genetically  different  organs  or  tissues  retain  their  difference  in  respect  to 
the  later  occurrence  of  the  character,  one  could  assume  that  the  action  of 
the  genes  controlling  this  character  is  being  manifest  within  the  trans- 
planted organ  or  tissue  or  that  some  characteristic  relative  to  the  later 
occurrence  of  the  character  has  been  established  in  the  organ  or  tissue  and 

i  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  30, 1954. 
2  National  Institutes  of  Health,  Public  Health  Service,  U.S.  Department  of  Health,  Education,  and  Welfare. 

775 

Journal   of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December   1954 


776  proceedings:  symposium  on  25  years  of 

has  persisted  after  transplantation.  On  the  other  hand,  if  the  difference 
is  eliminated  and  the  occurrence  of  the  character  is  characteristic  of  the 
host  one  would  assume  that  the  action  of  the  genes  is  being  manifest 
through  some  general  systemic  mechanism.  Transplantation  of  the  organs 
or  tissues  at  various  ages  could  give  information  on  sequence  of  events 
in  the  gene-action  path. 

This  type  of  study  can  be  illustrated  by  an  experiment  we  have  carried 
out  in  regard  to  pulmonary  tumors  (1).  It  had  been  estimated  earlier 
that  the  susceptible  strain  A  and  the  resistant  strain  C57L  differed  by  at 
least  four  pairs  of  genes  controlling  the  occurrence  of  pulmonary  tumors 
(2).  Then,  the  question  to  be  answered  was:  Did  these  genes  have  their 
primary  action  in  the  tissues  of  the  lung  or  was  their  action  manifest 
through  some  general  systemic  mechanism?  Transplants  of  small  por- 
tions of  the  lungs  of  adult  strain  A  mice  and  of  adult  strain  C57L  mice 
were  made  subcutaneously  in  the  axillae  of  a  common  host,  the  (A  X  C57L) 
Fi  hybrid.  After  allowing  time  for  the  transplants  to  become  vascularized 
the  host  was  injected  intravenously  with  a  carcinogen  1,  2,  5,  6-dibenz- 
anthracene.  Twelve  to  fifteen  months  following  the  injection,  the  Fi 
animals  were  necropsied  and  the  transplants  were  recovered.  Serial  sec- 
tioning of  the  transplants  revealed  that  whereas  many  of  the  susceptible 
strain  A  lung  transplants  had  developed  typical  lung  tumors,  very  few  of 
the  genetically  resistant  strain  L  transplants  had  done  so,  although  they 
were  in  a  common  host.  Thus,  instead  of  the  genotype  of  the  host  govern- 
ing the  occurrence  of  the  tumors,  their  occurrence  was  controlled  by  the 
genotype  of  the  donors  acting  within  the  transplanted  tissue  or  by  having 
fixed  a  characteristic  of  the  tissue  that  persisted  following  transplantation. 

This  latter  possible  explanation  was  later  tested  by  transplantation  of 
lobes  of  fetal  lungs  into  adult  Fx  hosts.3  These  fetal  transplants  proved 
to  be  very  interesting  in  their  growth  patterns  in  that,  although  very 
small  when  transplanted,  many  proceeded  to  grow  until  in  many  cases 
they  were  approximately  the  size  of  lobes  of  the  lungs  of  adults,  at  which 
time  growth  ceased.  Furthermore,  there  were  many  interesting  histo- 
logic changes  in  these  transplants  which  Dr.  C.  H.  Steffee  plans  to  study 
in  detail  later  and  which  will  therefore  not  be  discussed  here.  The 
general  outcome  of  this  experiment,  however,  was  similar  to  that  in  which 
adult  tissues  were  used.  The  occurrence  of  tumors  in  the  transplants 
was  characteristic  of  the  donor  strains  and  not  of  the  Fi  host.  This  fact 
would  give  further  support  to  the  idea  that  the  primary  action  of  these 
genes  controlling  the  occurrence  of  lung  tumors  is  in  the  lung  tissue. 

More  tumors  were  found  in  these  susceptible  fetal  transplants  than  in 
the  adult  transplants,  evidently  just  because  the  transplants  had  grown 
and  had  provided  more  tissue  in  which  tumors  could  arise.  Many  trans- 
plants had  multiple  nodules.  Furthermore,  some  of  the  tumors  occurring 
in  the  fetal  transplants  were  larger  than  those  found  in  the  adult  trans- 
plants. A  number  of  the  larger  tumors  were  transplanted  into  the  strain 
of  origin  of  the  original  transplant  and  all  grew  progressively.  They  are 
still  being  carried  after  many  transplant  generations. 

» Unpublished  data. 

Journal    of    the   National   Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  777 

In  this  second  experiment  some  of  the  hosts  were  injected  with  the 
carcinogen,  whereas  others  were  not.  The  difference  in  response  of  the 
transplants  due  to  the  carcinogen  was  similar  to  the  response  in  the  lungs 
of  the  donor  strains. 

This  type  of  experiment  can  be  and  has  been  adapted  to  many  different 
situations.  Shapiro  and  Kirschbaum  (3)  have  carried  out  such  an  ex- 
periment using  the  susceptible  Bagg  albino  strain  and  the  resistant  strain 
DBA.  Lung  transplants  from  day-old  mice  were  inoculated  into  the  ears 
of  the  Fi  hybrids,  and  after  receiving  the  transplants  the  Fi  hybrid  hosts 
were  injected  with  urethan.  The  results  of  this  study  indicated  that  the 
genes  controlling  the  occurrence  of  lung  tumors,  and  by  which  these 
strains  differed,  also  had  their  primary  action  in  the  lung  tissue.  It  has 
not  been  estimated  by  how  many  such  genes  these  two  strains  differ,  nor 
is  it  known  which,  if  any,  are  the  same  genes  as  those  by  which  strains 
A  and  C57L  differ. 

The  same  technique  can,  however,  be  used  in  respect  to  specific  genes, 
as  is  being  done  in  our  laboratory  with  the  lethal  yellow  gene  that  is 
known  to  increase  the  occurrence  of  pulmonary  tumors.  This  experiment 
has  not  been  completed.  It  would  appear  that  significant  results  may 
be  more  difficult  to  obtain  here,  owing  to  the  relatively  small  difference 
effected  by  only  one  specific  gene.  The  outcome  of  this  test,  however,  will 
be  of  particular  interest  since  in  this  case  we  are  dealing  with  a  gene  known 
to  have  effects  elsewhere  in  the  body,  i.e.,  controlling  pigmentation  of  the 
hair,  causing  obesity,  and  in  some  unknown  way  having  a  lethal  effect  in 
embryos  homozygous  for  the  gene. 

Such  techniques  can  be  readily  applied  to  other  organs.  For  example, 
Huseby  and  Bittner  (4)  showed  by  transplantation  of  adrenal  glands  that 
the  genotype  of  the  transplanted  adrenal  gland  governed  whether  the 
adrenals  became  carcinomatous  or  merely  hyperplastic  following  castra- 
tion. In  his  discussion  yesterday,  Dr.  Woolley  (5)  described  similar 
results  using  strains  CE  and  DBA.  This  is  particularly  interesting  in 
this  system  where  physiological  interorgan  relationships  are  so  prominent. 
Law  (6)  applied  similar  techniques  in  his  study  of  the  occurrence  of 
leukemia  in  Fi  hybrids  bearing  thymic  transplants  from  the  parent  strains. 
He  encountered  a  very  interesting  situation  here  in  that  only  the  stroma 
of  the  transplant  persisted,  whereas  the  transplanted  thymocytes  were 
apparently  replaced  by  invasion  of  those  from  the  host.  The  resulting 
leukemias  were  not  transplantable  back  into  the  donor  strain,  but  were 
transplantable  to  other  of  these  Fi  hybrids.  Similar  techniques  have 
been  applied  to  the  problems  of  mammary  gland  tumors,  and  these  will 
be  described  later.  Approaches  in  which  tissue  culture  is  substituted  for 
the  common  Fx  host  would  be  of  interest.  Strains  differ  in  respect  to 
occurrence  of  subcutaneous  sarcomas.  It  would  be  interesting  to  see 
whether  or  not  this  genetic  difference  would  persist  in  cultured  fibroblasts 
from  the  different  mouse  strains. 

In  the  problem  of  mammary  gland  tumors  in  mice,  attempts  at  locali- 
zation of  gene  action  have  been  more  toward  physiologic  pathways  through 

Vol.    15,   No.   3,   December   1954 


778  proceedings:  symposium  on  25  years  op 

which  the  action  becomes  manifest.  At  the  present  time  one  can  visualize 
three  such  paths:  1)  the  control  of  the  propagation  and  transmission  of 
the  mammary  tumor  agent;  2)  the  control  of  production  of  the  hormonal 
stimulation;  and  3)  the  control  of  the  response  of  the  mammary  tissue 
either  to  the  mammary  tumor  agent  or  to  the  hormonal  stimulation. 

Prehn  (7)  transplanted  mammary  glands  from  both  resistant  strain 
C57BL  and  susceptible  strain  BALB/c  females  into  (C57BL  X  BALB/c)  Fx 
hybrids.  Following  the  introduction  of  the  mammary  tumor  agent, 
tumors  and  hyperplastic  nodules  arose  in  the  transplanted  BALB/c  glands 
but  not  in  the  transplanted  C57BL  glands,  indicating  that  there  was  some 
genetic  control  over  the  response  of  the  mammary  tissue.  But  with 
these  strains  it  is  not  certain  whether  this  response  is  to  the  stimulation 
by  the  agent  or  to  the  hormonal  stimulation.  In  an  experiment  we  now 
have  in  progress,  mammary-gland  transplants  have  been  made  from 
strain  C3H  with  a  high  incidence  of  mammary  tumors  in  virgin  females, 
and  strain  A  with  a  low  incidence  in  virgin  females,  into  their  (C3H  X  A)Fi 
hybrids  which  were  kept  as  virgins.  Thus  far  only  one  mammary  tumor 
has  arisen  in  a  transplanted  gland  and  it  arose  in  a  gland  from  strain  C3H. 
The  fact  that  the  tumor  was  transplantable  back  to  strain  C3H  added 
evidence  of  its  C3H  origin.  Since  the  genetic  difference  between  these 
strains  is  in  respect  to  the  hormonal  stimulation  the  study  should  give 
evidence  as  to  whether  the  genes  were  controlling  the  response  of  the 
gland  to  the  hormonal  stimulus  or  controlling  the  output  of  the  stimulus. 

Huseby  and  Bittner  (8)  approached  this  problem  through  transplanta- 
tion of  ovaries.  They  found  that  (C3H  X  A)Fi  spayed  females  bearing 
either  C3H  or  F]  ovaries  had  a  higher  incidence  of  mammary  tumors  with 
a  lower  tumor  age  than  did  the  same  type  of  F!  females  bearing  A  ovaries. 
This  would  indicate  that  the  action  of  certain  genes  occurs  within  the 
ovaries,  or  some  other  organ  of  the  endocrine  system,  thus  controlling  the 
hormonal  stimulation.  It  was  also  indicated,  however,  that  other  genes 
may  be  acting  within  the  cells  of  the  mammary  glands  in  controlling  their 
response  to  the  hormonal  stimulation,  for  there  were  more  tumors  in  Ft 
females  bearing  A  ovaries  than  in  A  females  bearing  transplanted  A 
ovaries. 

Of  major  interest  to  us,  however,  is  the  relationship  between  the  gene 
or  genes  and  the  mammary  tumor  agent.  Definite  control  of  the  genotype 
over  the  propagation  and  transmission  of  the  agent  was  demonstrated 
through  comparison  of  backcross  females  from  the  original  cross  of  high- 
tumor  strain  C3H  females  with  low-tumor  strain  C57BL  males  (9). 
Females  resulting  from  backcrossing  the  Fi  females  to  C3H  males  not 
only  had  a  higher  incidence  of  mammary  tumors  than  did  the  females 
resulting  from  backcrossing  the  Fx  females  to  C57BL  males,  but  they  also 
transmitted  the  agent  to  test  females  more  effectively  than  did  the  C57BL 
backcross  females.  BC3H  F!  test  females  that  nursed  upon  the  C3H 
backcross  females  had  a  higher  incidence  of  tumors  than  did  those  that 
nursed  upon  the  C57BL  backcross  females.  Thus,  genetic  control  over 
the  agent  was  indicated.     It  was  suggested  that  multiple  genes  were 

Journal    of    the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  779 

involved  since  variation  of  the  C57BL  backcross  appeared  to  be  con- 
tinuous. 

Subsequently  a  more  extensive  analysis  of  this  relationship  between 
the  genes  and  the  mammary  tumor  agent  has  been  made.4  This  was  the 
study  to  which  Dr.  Dunn  made  reference  in  her  paper.  It  will  be  reported 
in  detail  in  the  near  future.  In  this  study  again  strain  C3H  females 
were  outcrossed  to  C57BL  males  and  this  outcrossing  was  followed  by  a 
series  of  7  generations  of  backcrossing  to  C57BL  males.  By  this  pro- 
cedure a  C57BL  genetic  background  was  built  up,  while  at  the  same  time 
a  continuous  line  of  females  through  which  the  agent  might  be  transmitted 
was  maintained.  Not  only  was  the  tumor  incidence  for  each  generation 
tabulated,  but  the  females  of  each  generation  were  tested  for  their  ability 
to  transmit  the  agent  to  foster-nursed  C3Hf  test  females  without  the 
agent.  From  the  earlier  study  it  was  anticipated  that  such  procedure 
would  probably  eliminate  the  agent.  Furthermore,  Murray  and  Little 
(10)  had  reported  earlier  that  the  agent  was  rendered  noneffective  in  the 
eighth  generation  of  backcrossing  to  C57BL  males  subsequent  to  out- 
crossing strain  DBA  females  to  C57BL  males.  By  testing  each  generation 
as  was  done  in  the  present  experiment  it  was  possible  to  determine  just 
how  readily  the  agent  could  be  eliminated. 

By  the  second  backcross  generation  the  incidence  of  mammary  tumors 
had  been  reduced  to  5  percent  in  the  breeding  backcross  females  and  zero 
in  the  virgins.  The  5  percent  represented  one  animal,  and  its  tumor 
arose  at  21  months  of  age.  These  data  themselves  would  have  suggested 
that  by  this  generation  the  agent  had  been  eliminated  had  not  19  percent 
of  the  C3Hf  test  females  foster  nursed  by  these  second-backcross-genera- 
tion  females  developed  tumors  at  an  average  age  of  13  months.  Further 
inspection  of  the  data  revealed  that  two  of  the  second  backcross  females 
that  did  not  themselves  develop  tumors  were  transmitting  the  agent  to 
their  test  females.  Thus,  it  could  not  be  considered  that  the  agent  had 
been  completely  eliminated  by  the  second  backcross  generation. 

In  the  third  backcross  generation,  however,  none  of  the  breeding 
females  developed  tumors.  Only  one  of  the  virgins  developed  a  tumor 
and  it  arose  when  the  animal  was  26  months  of  age.  Furthermore,  of 
the  93  C3Hf  test  females  foster-nursed  by  these  third  backcross  females 
only  one  developed  a  tumor  and  it  arose  when  the  female  was  21  months 
old.  One  tumor  occurred  in  a  later  backcross  generation  and  a  few  oc- 
curred in  the  C3Hf  females  used  to  test  the  later  generations  but  all  of 
these  occurred  when  the  females  were  very  old.  Thus,  it  was  evident 
that  the  agent  had  been  eliminated  or  rendered  inactive  by  the  third 
backcross  generation. 

The  fact  that  the  agent  was  eliminated  by  as  early  as  the  third  back- 
cross  generation  is  highly  significant  in  that  it  indicates  that  not  many 
genes  could  be  involved  in  the  control  of  the  agent,  and  even  suggests  the 
possibility  that  this  relationship  may  be  between  but  one  gene  and  the 

*  Unpublished  data. 


Vol.    15,   No.    3,   December    1954 


780  proceedings:  symposium  on  25  years  op 

agent.  This  would  be  in  line  with  the  gene-Kappa  relationship  described 
by  Sonneborn  and  co-workers  in  Paramecia.  If  there  were  delay  in 
elimination  of  the  agent  in  animals  without  the  gene,  such  as  has  been 
observed  with  respect  to  Kappa,  this  could  explain  the  failure  in  the  earlier 
study,  to  observe  single  gene  segregation  in  the  first  backcross  generation. 
Andervont  (11)  found  that  whereas  strain  C57BL  females  that  received 
the  agent  could  transmit  it  to  susceptible  foster-nursed  mice,  their  C57BL 
female  progeny  could  not  transmit  it.  This  would  suggest  one-generation 
delay  in  the  elimination  of  the  agent  in  mice  without  the  genetic  factors 
necessary  for  its  survival.  In  such  a  situation  the  genotype  of  a  female 
could  not  be  judged  as  well  by  the  occurrence  of  tumors  in  her  foster- 
nursed  test  females,  as  by  the  occurrence  of  tumors  in  the  test  females  that 
nursed  upon  her  female  progeny.  Examination  of  the  present  data  from 
this  viewpoint  gave  further  suggestion  that  a  single-gene  difference  might 
be  involved.  It  is  hoped  that  this  possibility  of  but  a  single-gene  relation- 
ship to  the  agent  can  be  tested  further. 

To  determine  whether  or  not  through  alteration  in  genotype  the  agent 
had  been  eliminated  or  merely  rendered  inactive,  females  of  the  seventh 
backcross  generation  were  outcrossed  to  strain  C3Hf  males  without  the 
agent  and  this  was  followed  by  a  series  of  backcrosses  to  C3Hf  males 
through  four  generations.  By  restoring  the  C3H  genotype  one  could 
expect  to  activate  an  agent  that  had  been  inactivated  by  the  reverse 
procedure.  An  interesting  assortment  of  mammary  tumors  occurred  in 
the  females  of  this  series  but  the  incidence  of  tumors  was  low  and  did  not 
increase  as  proportion  of  C3H  chromatin  background  was  increased  in 
successive  generations.  Furthermore,  the  tumors  came  up  when  the 
females  were  very  old,  so  there  was  no  suggestion  that  the  agent  had  been 
activated.  It  was,  thus,  evident  that  by  eliminating  the  necessary  gene, 
or  genes,  the  agent  likewise  had  been  eliminated  and  not  caused  to  mutate 
to  a  latent  or  inactive  state. 

At  the  time  this  experiment  was  begun  the  possibility  that  the  agent 
might  have  arisen  through  mutation  of  some  normal  cell  component  was 
a  rather  popular  consideration,  despite  the  fact  that  this  was  based  in 
large  measure  upon  speculative  thinking.  To  test  this  possibility  recip- 
rocal series  of  backcrosses  were  produced.  Strain  C57BL  females  were 
outcrossed  to  C3H  males  and  this  outcross  was  followed  by  seven 
generations  of  backcrossing  to  C3H  males.  Because  of  the  possible 
introduction  of  the  agent  by  the  C3H  males  a  second  series  was  produced 
using  C3Hf  males. 

Although  a  few  tumors  occurred  in  the  C3Hf  backcross  series,  in  no 
generation  was  the  incidence  high  but  remained  low  despite  the  increase 
in  proportion  of  C3H  chromatin  built  up  in  the  successive  backcross 
generations.  Furthermore,  in  all  cases  the  tumors  arose  in  females  of 
advanced  age  indicating  that  the  tumors  had  resulted  from  causes  other 
than  the  milk  agent.  In  the  C3H  backcross  series,  however,  more  mam- 
mary tumors  arose,  the  incidence  increasing  and  average  tumor  age 
decreasing  in  successive  generations  until,  with  the  exception  of  one  line, 

Journal    of    the   National    Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  781 

the  seventh  generation  was  comparable  to  strain  C3H  in  both  incidence 
and  tumor  age.  This  was  the  result  of  introduction  of  the  agent  by  the 
males.  In  the  pedigree  chart  each  point  at  which  the  agent  was  intro- 
duced was  evident,  for  once  introduced  the  female  progeny  then  developed 
tumors  as  females  of  any  high-tumor  line  would  develop  them. 

Some  interesting  observations  were  made  in  the  classification  of  the 
mammary  tumors  that  arose  in  these  series.  These  were  classified  by 
Dr.  Thelma  Dunn  according  to  the  classification  she  has  been  using  (12) . 
The  greater  proportion  of  tumors  were  adenocarcinomas  of  Type  A  and 
Type  B.  For  the  most  part  these  were  the  types  that  arose  in  the  presence 
of  the  agent  and  in  the  younger  females.  As  the  agent  was  eliminated 
in  the  C57BL  backcross  series  the  proportions  of  these  two  types  were 
diminished;  the  proportion  of  the  adenocarcinomas  of  Type  C,  of  the 
adenoacanthomas,  of  the  molluscoid  variation  of  the  adenoacanthoma, 
and  of  the  carcinosarcomas  increased.  These  latter  four  unusual  types 
were  particularly  numerous  in  the  series  resulting  from  outcrossing  the 
seventh  generation  C57BL  backcross  females  to  C3Hf  and  backcrossing 
to  C3Hf  males.  In  the  C3Hf  backcross  series  the  unusual  types  were 
scattered  through  the  series  along  with  a  few  adenocarcinomas  of  Type  A 
and  Type  B.  In  the  C3H  backcross  series  the  proportion  of  adenocar- 
cinomas of  Type  A  and  Type  B  was  low  in  the  early  generations  but 
increased  as  the  mammary  tumor  agent  was  introduced  and  the  tumor 
age  was  decreased.  An  extremely  interesting  study  is  thus  opened  in 
respect  to  relationship  between  the  various  causative  factors  and  the 
resultant  histologic  type  of  tumor.  It  is  assumed  that  genes  influencing 
occurrence  of  mammary  tumors  through  control  over  the  milk  agent  have 
little  or  nothing  to  do  with  the  occurrence  of  these  unusual  types  that 
develop  in  the  absence  of  the  agent.  It  would  be  of  interest  to  know 
whether  action  of  other  specific  genes  might  be  associated  specifically 
with  some  of  these  unusual  histologic  types  of  tumors. 

The  question  of  whether  or  not  the  malignant  change  is  the  direct  result 
of  a  change  in  a  gene  of  the  cell  that  becomes  malignant,  i.e.,  a  somatic 
mutation,  has  been  studied  through  many  approaches.  These  have  all 
been  indirect  approaches,  however,  as  they  will  continue  to  be  until  some 
means  of  observing  segregation  of  genes  in  somatic  cells  is  devised.  One 
of  the  approaches  in  which  we  have  been  particularly  interested  has  been 
through  analysis  of  dose  response.  The  shape  of  the  response  curve 
should  give  some  indication  of  events  at  the  time  of  the  malignant  change. 
If  the  curve  were  exponential  one  could  assume  that  more  than  one  in- 
dependent event  were  necessary  for  the  change,  whereas  a  linear  response 
would  indicate  the  result  of  only  one  event.  If  the  malignant  change 
were  due  to  a  recessive  mutation,  two  mutations  at  the  locus  concerned 
would  be  necessary  and  thus  the  curve  would  be  exponential.  If  the 
malignant  change  were  due  to  a  dominant  mutation  only  one  mutation 
at  the  locus  concerned  would  be  necessary,  and  one  would  expect  the 
dose-response  relationship  to  be  linear. 

Charles  and  Luce-Clausen  (13)  analyzed  the  dose-response  data  ob- 

Vol.    15,   No.   3,   December    1954 


782  proceedings:  symposium  on  25  years  of 

tained  by  Morton,  Luce-Clausen,  and  Mahoney  in  inducing  papillomas 
in  strain  DBA  mice  by  successive  paintings  of  the  skin  with  methyl- 
cholanthrene.  When  the  square  root  of  the  average  number  of  papillomas 
was  plotted  against  time,  in  this  case  a  measure  of  dose,  a  straigho  line 
was  observed,  and  it  was  pointed  out  that  this  was  what  could  be  expected 
if  the  papillomas  arose  from  a  recessive  mutation  in  the  treated  cells  of 
the  skin,  thus  requiring  two  independent  events  at  the  locus  concerned. 

Induced  pulmonary  tumors  offer  excellent  material  for  such  a  study. 
The  multiple  independent  tumors  arising  following  the  injection  of  a 
carcinogen  provide  an  excellent  quantitative  measure  of  response.  In  an 
early  experiment  (lJf)  we  injected  groups  of  strain  A  mice  with  graded 
doses  of  1,2,5,6-dibenzanthracene  ranging  from  0.1  mg.  to  0.5  mg.  in  an 
aqueous  colloidal  dispersion.  Six  months  after  the  injection  the  animals 
were  killed  and  the  number  of  tumor  nodules  appearing  on  the  surface  of 
the  lungs  of  each  animal  was  recorded.  When  average  number  of  nodules 
in  each  group  was  plotted  against  dosage,  a  linear  relationship  was  ob- 
served. Extension  of  this  line  did  not,  however,  pass  through  the  average 
number  in  untreated  animals,  but  intersected  the  response  scale  at  a  point 
below  zero.  Investigation  of  response  between  zero  and  0.1  mg.  dose 
was  therefore  indicated. 

This  area  of  the  dose  range  has  now  been  investigated.5  Groups  of 
strain  A  mice  were  injected  with  0.01  mg.,  0.02  mg.,  0.04  mg.,  0.06  mg., 
0.08  mg.,  0.1  mg.,  and  0.5  mg.  of  1,2,5,6-dibenzanthracene  respectively.  It 
was  necessary,  however,  to  use  a  new  aqueous  colloidal  dispersion  of  the 
dibenzanthracene  for  these  injections.  Because  of  variation  in  ages  of 
the  animals,  two  series  were  injected  at  an  interval  of  approximately  2 
months.  When  the  mice  of  the  first  series  were  killed  at  6  months  after 
the  injection,  the  dose  response  relationship  was  again  found  to  be  linear 
within  these  dose  limits.  Mice  of  the  second  series  have  now  been  killed 
after  the  same  time  interval  and  again  a  linear  relationship  was  observed 
between  response  and  dose.  Combination  of  the  two  series  even  improved 
the  approximation  to  a  straight  line. 

In  the  second  experiment,  as  in  the  first,  the  average  number  of  nodules 
at  zero  dose  deviated  significantly  above  the  extension  of  the  dose- 
response  curve.  This  deviation  in  both  experiments  was  probably  due 
to  some  of  the  carcinogen  of  each  dose  passing  through  the  lungs.  It 
would  seem  logical  to  assume  that  some  of  the  smaller  crystals  would  not 
be  lodged  in  the  capillaries  of  the  lung.  The  deviation  at  the  zero  dose  in 
the  second  experiment,  however,  was  not  as  great  as  that  in  the  first. 
This  was  probably  because  two  dispersions  were  used  and  the  particles 
of  the  second  dispersion  were  probably  larger  than  were  those  of  the  first 
dispersion.  With  the  colloidal  mill  it  is  difficult  to  get  two  dispersions 
with  the  same  particle  size.  Variation  in  particle  size  was  also  indicated 
by  the  fact  that  a  considerably  greater  response  was  noted  at  the  0.1  mg. 
and  0.5  mg.  doses  in  the  second  experiment  than  in  the  first.  Shim  kin 
and  Lorenz  (15)  have  shown  that  a  greater  response  does  result  when  the 
size  of  the  particles  is  larger. 

» Unpublished  data. 

Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  783 

These  observations  include  responses  throughout  the  practical  dose 
range.  Higher  doses  would  give  so  many  nodules  that  counting  could  not 
be  accurate.  Furthermore,  the  number  of  nodules  is  limited  by  the 
amount  of  lung  tissue,  and  thus,  extension  of  the  line  would  be  expected 
to  curve  downward.  Whether  or  not  a  linear  relationship  would  be 
observed  for  other  time  intervals  could  be  ascertained  only  by  further 
experimentation.  It  is  interesting  that  within  this  practical  dosage  range 
and  at  this  time  interval  a  linear  dose-response  relationship  was  observed. 
This  is  in  line  with  a  mechanism  involving  a  single  happening  in  the  cell 
transforming  to  the  malignant  cell.  If  this  happening  were  a  change  in 
a  gene  it  would  thus  be  a  dominant  mutation. 

References 

(1)  Heston,  W.  E.,  and  Dunn,  T.  B.:  Tumor  development  in  susceptible  strain  A 

and  resistant  strain  L  lung  transplants  in  LA  Fi  hosts.     J.  Nat.  Cancer  Inst. 
11:  1057-1071,  1951. 

(2)  Heston,  W.  E.:  Genetic  analysis  of  susceptibility  to  induced  pulmonary  tumors 

in  mice.     J.  Nat.  Cancer  Inst.  3:  69-78,  1942. 
(8)  Shapiro,  J.  R.,  and  Kirschbaum,  A.:  Intrinsic  tissue  response  to  induction  of 
pulmonary  tumors.     Cancer  Res.  11:  644-647,  1951. 

(4)  Husebt,  R.  A.,  and  Bittner,  J.  J.:  Differences  in  adrenal  responsiveness  to  post- 

castrational  alteration  as  evidenced  by  transplanted  adrenal  tissue.     Cancer 
Res.  11:  954-961,  1951. 

(5)  Woolley,  G.  W.:  Carcinogenesis  in  the  adrenal.     J.  Nat.  Cancer  Inst.     15: 

717-719,  1954. 

(6)  Law,  L.  W.:  Increase  in  incidence  of  leukemia  in  hybrid  mice  bearing  thymic 

transplants  from  a  high  leukemic  strain.     J.  Nat.  Cancer  Inst.  12:  789-806, 
1952. 

(7)  Prehn,   R.   T\:  Tumors  and  hyperplastic  nodules  in  transplanted  mammary 

glands.     J.  Nat.  Cancer  Inst.  13:  859-872,  1953. 

(8)  Huseby,  R.  A.,  and  Bittner,  J.  J.:  Studies  on  the  inherited  hormonal  influence. 

Acta  Unio  Internat.  Contra  Cancrum  6:  197-205,  1948. 

(9)  Heston,  W.  E.,  Deringer,  M.  K.,  and  Andervont,  H.  B.:   Gene-milk  agent 

relationship    in    mammary-tumor    development.     J.    Nat.    Cancer    Inst.    5: 
289-307,  1945. 

(10)  Murray,    W.   S.,    and   Little,    C.    C:  Chromosomal   and    extrachromosomal 

influence  in  relation  to  the  incidence  of  mammary  tumors  in  mice.     Am.  J. 
Cancer  37:  536-552,  1939. 

(11)  Andervont,  H.  B.:  Fate  of  the  C3H  milk  influence  in  mice  of  strains  C  and  C57 

black.     J.  Nat.  Cancer  Inst.  5:  383-390,  1945. 

(12)  Dunn,  T.  B. :  Morphology  of  mammary  tumors  in  mice.  In  The  Physiopathology 

of  Cancer,  (Hamburger,  F.,  and  Fishman,  W.  H,  eds.),  New  York,  Hoeber-Harper, 
1953,  Ch.  8. 

(13)  Charles,  D.  R.,  and  Luce-Clausen,  E.  M.:  The  kinetics  of  papilloma  formation 

in  benzpyrene-treated  mice.     Cancer  Res.  2:  261-263,  1942. 

(14)  Heston,  W.  E.,  and  Schneiderman,  M.  A.:  Analysis  of  dose-reponse  in  relation 

to  mechanism  of  pulmonary  tumor  induction  in  mice.     Science  117:  109-111, 
1953. 

(15)  Shimkin,  M.  B.,  and  Lorenz,  E.:  Factors  influencing  the  induction  of  pulmonary 

tumors  in  strain  A  mice  by  carcinogenic  hydrocarbons.     J.  Nat.  Cancer  Inst. 
2:499-510,  1942. 


Vol.    15,   No.   3,   December    1954 


Discussion 

Dr.  Leon  Dmochowski,1  College  of  Physicians  and  Surgeons,  Columbia  University, 

New  York,  N.  Y. 

It  is  an  honor  and  pleasure  for  me  to  participate  in  this  meeting  of  the  Roscoe  B. 
Jackson  Memorial  Laboratory  and  I  should  like  to  express  my  sincere  thanks  to  Dr. 
C.  C.  Little  and  Dr.  Elizabeth  S.  Russell  for  their  kind  invitation,  and  to  all  who  made 
it  possible  for  me  to  be  present  at  this  memorable  meeting. 

I  would  like  to  congratulate  Dr.  W.  E.  Heston  on  his  penetrating  and  stimulating 
analysis  of  the  localization  of  gene  faction  in  the  origin  of  lung  and  mammary  cancer 
of  mice.  There  is  indeed  little,  if  anything,  I  can  add  to  what  he  has  presented  to  us. 
Of  necessity,  dictated  by  lack  of  personal  experience,  I  shall  limit  myself  in  what  I 
will  say  to  observations  on  mammary  cancer.  In  the  few  observations  I  propose  to 
discuss,  I  will  attempt  to  show  the  possible  part  played  by  genes  in  the  origin  of  mam- 
mary cancer,  and  will  be  grateful  to  hear  Dr.  Heston's  and  others'  comments  on  the 
possible  action  of  genes  in  the  development  of  breast  cancer  recorded  in  these 
observations. 

As  you  have  heard,  one  of  the  paths  along  which  the  action  of  genes  may  manifest 
itself,  is  the  control  of  the  propagation  and  transmission  of  the  mammary-tumor 
inciter.  You  have  also  heard  that  there  may  even  be  a  single-gene  agent  relationship, 
in  spite  of  the  variation  in  ability  to  transmit  the  agent  in  the  backcross  generations. 
Here,  I  should  like  to  mention  the  following  observation  (1-4)-  After  the  mating  of 
low-cancer-strain  females  with  high-cancer-strain  males,  combined  with  brother  X 
sister  matings  of  their  hybrid  progeny  and  subjecting  this  progeny  to  increased  hor- 
monal stimulation,  a  variable  number  of  mammary  tumors  has  been  observed  [by 
ourselves  and  also  by  others  (9-11)]  to  develop  in  successive  generations  of  this 
hybrid  progeny.  Biological  tests  of  these  tumors,  which  we  have  carried  out,  revealed 
the  agent  in  some  very  young  as  well  as  in  very  old  tumors,  while  in  other  young  and 
old  tumors  the  same  tests  failed  to  demonstrate  the  agent.  These  tests,  showed  that 
under  the  same  experimental  conditions  among  hybrids  of  the  same  derivation  some 
developed  tumors  which  either  harbored  the  agent  or  which  failed  to  reveal  the  agent, 
while  other  hybrids  even  their  litter  mates  died  without  tumors.  Some  of  the  progeny 
of  tumorous  Fi  hybrids  failed  to  develop  breast  cancer,  while  other  progeny  of  the 
same  hybrids  developed  cancer  in  which  the  agent  was  either  demonstrated  or  could 
not  be  shown.  Some  of  the  progeny  of  tumor-free  Fi  hybrids  developed  breast  tumors 
that  either  harbored  the  agent  or  did  not  reveal  the  agent.  Examination  of  family 
charts  of  the  progeny  of  both  tumorous  and  nontumorous  females  reveals,  perhaps, 
the  variation  in  the  susceptibility  to  tumor  development  on  one  hand,  and  on  the 
other,  the  variation  in  propagation  and  transmission  of  the  agent  in  hybrid  progeny 
of  identical  origin.  It  also  appears  that  the  different  behavior  of  tumors  in  the 
bioassays  may  be  an  expression  of  the  gene  control  over  the  relationship  of  the  agent 
to  the  substrate,  that  is,  the  mammary-gland  cell  as  a  whole,  or  the  known  and  as  yet 
perhaps  unknown  components  of  the  cell.  In  other  words,  assuming  the  presence  of 
similar  amounts  of  the  agent  in  the  tumors  tested,  the  difference  in  the  bioassay  be- 
havior of  the  tumors  may  be  based  on  variation  in  the  integration  of  the  agent  with 
cellular  constituents,  an  integration  controlled  by  genetic  factors  either  directly  or 
indirectly  through  their  control  of  the  response  of  the  mammary-gland  cell  to  hormonal 
stimulation  or  both. 

A  somewhat  more  puzzling  observation  is  the  development  of  breast  cancer  in  the 
hybrid  progeny  of  two  agent-free,  low-cancer  strains  which  had  been  maintained  by 
brother  X  sister  matings  and  subjected  to  increased  hormonal  stimulation.     These 

i  On  leave  of  absence  from  Leeds  University,  Leeds,  England. 

785 

Journal   of   the  National   Cancer   Institute,   Vol.    15,  No.   3,   December   1954 

316263—54 35 


786  proceedings:  symposium  on  25  years  of 

tumors  have  been  found  to  contain  the  agent  {12).  Although  the  incidence  of  tumors 
observed  was  much  lower  than  that  in  the  hybrid  progeny  of  low-  and  high-breast- 
cancer  strains,  the  question  that  still  remains  to  be  answered  is,  how  does  the  agent 
originate  in  these  tumors?  Is  it  possible  that  crossing  of  genes  from  two  different 
strains  known  to  be  agent-free  results  in  a  substrate  more  sensitive  to  hormonal 
stimulation  than  the  genetic  constitution  of  either  of  the  parental  strains?  Can, 
therefore,  the  combination  of  a  suitable  genetic  background  with  appropriate  hormonal 
environment  result  in  the  appearance  of  the  agent?  I  hesitate  to  answer  this  in  the 
affirmative.  Even  so,  I  do  not  know  if  this  would  not  be  better  than  the  acceptance 
of  a  universal  agent,  latent  or  otherwise,  to  be  revealed  by  some  suitable  genetic 
combination  and  suitable  internal  and/or  external  environments. 

To  continue  on  this  puzzling  trend,  I  would  like  to  mention  the  observation  of  an 
increased  incidence  of  breast  cancer  in  a  subline  of  strain  C57BL  mice  and  the  eventual 
demonstration  of  the  agent  in  tumors  of  mice  of  later  generations  of  this  subline  (IS). 
This  may  be  seen  in  a  simplified  version  on  a  chart,  part  of  which  is  known  to  some 
of  you.  When  three  years  ago  I  discussed  this  observation  with  Dr.  Heston,  he  urged 
me  to  study  the  behavior  of  virgin  females  of  this  subline.  We  have  now  found  that 
these  females  do  not  develop  breast  cancer,  thus  revealing  the  importance  of  the 
hormonal  stimulation  in  the  origin  of  breast  cancer  in  this  subline.  However,  this 
still  does  not  answer  the  problem  of  the  origin  of  the  agent  unless  we  assume  either 
its  latency  in  the  preceding  generations — where  again  we  may  stumble  on  the  agent's 
ubiquity  or  that  a  mutation  of  gene  or  genes  had  taken  place.  This  latter  possibility 
could  not  be  verified  by  transplantation  as  the  tumors  grew  in  mice  of  all  other  sub- 
lines of  strain  C57BL,  which,  of  course,  does  not  exclude  the  possibility  of  a  genetic 
change.  Finally,  we  may  think  of  endogenous  origin  based  on  a  change  in  a  normal 
cell  component,  either  independently  or  following  a  genetic  change  directly  or  indirectly 
through  hormonal  factors.  What  the  answer  is  I  don't  know,  but  it  may  possibly 
lend  itself  to  a  discussion  on  the  probable  part  played  by  genes  in  the  appearance  of 
the  agent. 

There  is  one  more  observation  made  recently  by  us  in  the  study  of  the  appearance 
of  breast-tumor  cells  in  thin  sections  seen  in  the  electron  microscope  (14).  Following 
the  observation  of  characteristic  bodies  in  the  cytoplasm  of  tumors  from  high-cancer 
strains,  obtained  at  first  from  our  own  and  then  also  from  several  widely  separated 
laboratories  (fig.  1) ,  tumors  from  low-cancer  strains  have  been  studied.  In  the  absence 
of  these  bodies  in  breast  tumors  of  a  low-cancer  strain  in  our  own  laboratory,  we 
obtained  a  number  of  tumors  from  an  agent-free  strain,  the  so-called  C3Hf,  from  Dr. 
Heston  and  also  tumors  from  agent-free  strain  C3H  mice  born  from  transplanted  ova 
and  supplied  by  Dr.  Fekete.  Similar  bodies  to  those  present  in  high-cancer-strain 
tumors,  were  found  in  4  of  8  tumors  obtained  from  Dr.  Fekete  and  also  in  7  of  14  tumors 
supplied  by  Dr.  Heston.  This  seemed  to  be  disappointing  as  far  as  any  conclusions 
about  the  significance  of  these  bodies  were  concerned.  However,  each  of  these  tumors 
studied  in  the  electron  microscope  was  also  tested  biologically.  Three  of  the  4  tumors 
from  Dr.  Fekete  have  already  been  found  to  harbor  the  agent,  and  2  of  the  7  containing 
the  characteristic  bodies  and  obtained  from  Dr.  Heston  have  so  far  been  found  to  be 
active  in  the  bioassay.  It  is  too  early  as  yet  to  judge  the  bioassays  of  the  remaining 
tumors.  As  to  the  nature  of  these  bodies,  the  first  question  which  we  must  ask  our- 
selves is,  are  they  normal  cell  constituents?  It  appears  to  us  on  the  basis  of  all  the 
available  evidence  that  they  are  not.  The  next  problem  confronting  us  is  the  possible 
connection  between  the  presence  of  these  bodies  and  the  presence  of  the  agent.  If 
there  is  a  connection,  are  they  the  agent  itself  or  are  they  an  expression  of  a  functional 
state  of  the  cell  which  harbors  the  tumor-inducing  activity?  Admittedly,  these 
particles  do  look  like  viruses  reported  by  others  in  various  cells.  Before  fulfilling 
Koch's  postulates  and  combining  them  with  suitable  electron-microscope  studies  it 
is  impossible  to  answer  the  question  whether  these  bodies  are  the  agent  itself.  There 
is,  however,  the  problem  of  the  undoubted  tumor-inducing  activity  of  some  of  the  tu- 
mors as  shown  in  the  biological  tests.     This  may  well  be  the  result  of  a  genetic  change, 


Journal    of   the   National    Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  787 

which  may  lead  to  a  functional  state  of  the  cell  which  in  turn  is  manifested  biologically 
by  the  presence  of  tumor-inducing  activity  in  these  cells.  The  connection  between 
the  tumor-inducing  activity  and  these  bodies — that  is,  between  the  two  properties 
of  a  cell,  the  morphological  and  biological — although  as  yet  not  finally  established  by 
us,  appears  to  be  at  least  interesting  to  contemplate  as  a  possible  basis  for  correlation 
of  the  gene  action,  in  the  morphological  and  physiological  sense. 

References 

(1)  Dmochowski,  L.:  In  27th  Ann.  Rep.  Brit.  Emp.  Cancer  Campaign  27:  162,  1949. 
In  27th  Ann.  Rep.  Brit.  Emp.  Cancer  Campaign  28:  169-170,  1950. 
In  27th  Ann.  Rep.  Brit.  Emp.  Cancer  Campaign  29:  150-151,  1951. 
A  study  of  the  development  of  mammary  tumours  in  hybrid  mice.     Brit. 


(3)  

(4)  

J.  Cancer  7:  73-119,  1953. 

(5)  Andervont,  H.  B.,  and  Dunn,  T.  B.:  Mammary  tumors  in  mice  presumably 

free  of  the  mammary-tumor  agent.     J.  Nat.  Cancer  Inst.  8:  227-233,  1948. 

(6)  :  Efforts  to  detect  a  mammary- tumor  agent  in  strain  C  mice.     J.  Nat. 

Cancer  Inst.  8:  235-240,  1948. 

(7)  :  Further  studies  on  the  relation  of  the  mammary-tumor  agent  to  mam- 
mary tumors  of  hybrid  mice.     J.  Nat.  Cancer  Inst.  9:  89-104,  1949. 

(8)  :  Attempt  to  detect  a  mammary-tumor  agent  in  strain  C  mice  by  X- 

radiation.     J.  Nat.  Cancer  Inst.  10:  1157-1190,  1950. 

(9)  Bittner,  J.  J.:  Transfer  of  the  agent  for  mammary  cancer  in  mice  by  the  male. 

Cancer  Res.  12:  387-398,  1952. 

(10)  Foulds,  L.:  Mammary  tumors  in  hybrid  mice:  the  presence  and  transmission 

of  the  mammary  tumor  agent.     Brit.  J.  Cancer  3:  230-239,  1949. 

(11)  MtiHLBOCK,  O. :  Studies  on  the  transmission  of  the  mouse  mammary  tumor  agent 

by  the  male  parent.     J.  Nat.  Cancer  Inst.  12:  819-837,  1952. 

(12)  Dmochowski,  L.:  Unpublished  data. 

(13)  :  Studies  on  spontaneous  mammary  tumors  in  a  low  breast-cancer  strain 

of  mice.     (Abstract.)     Proc.  Am.  Assn.  Cancer  Res.  1:  11-12,  1954. 

(14)  Dmochowski,  L.,  Haagensen,  C.  D.,  and  Moore,  D.  H.:  Electron  microscope 

studies  of  thin  sections  of  normal  and  malignant  mammary  cells  of  some  high 
and  low  cancer-strain  mice.  (Abstract.)  Proc.  Am.  Assn.  Cancer  Res. 
1:  12,  1954. 


Vol.    15,   No.    3,    December    1954 


788 


proceedings:  symposium 


Plate  47 


Figure  1. — Section  of  a  high-cancer-strain  mammary  tumor.  Characteristic  bodies 
present  in  a  duct  and  showing  an  internal  structure  composed  of  an  inner  dense  region 
surrounded  by  an  outer  paler  zone,  which  in  turn  is  surrounded  by  a  dense  outer 
region.     X  67,000 


JOURNAL  OF  THE  NATIONAL  CANCER  INSTITUTE,  VOL.   15 


PLATE  47 


67,000 


*-* 


K" 


a    * 


•  ■  •   I 


# 


' 


1 


..   . 


** 


Dmochowski 

316263—54- 


Figure  1 


The  Use  of  Fi  Hybrid  and    Backcross 
Generations  to  Reveal  New  and/or 

Uncommon  Tumor  Types  h  2 


Margaret  M.  Dickie,3  Roscoe  B.  Jackson 
Memorial  Laboratory,  Bar  Harbor, 
Maine 


In  celebrating  25  years  of  progress  in  mammalian  genetics  and  cancer 
one  must  pay  tribute  to  the  laboratory  and  to  its  founders,  who  have 
played  a  major  role  in  the  development  of  the  inbred  strains  of  mice 
whose  biological  uniformity  have  contributed  so  much  to  this  progress. 
The  search  for  biological  material  to  meet  the  needs  of  present-day  re- 
search is  never  ending.  Perhaps  one  of  our  greatest  assets  to  date  is  this 
inbred-strain  mouse.  Here  is  an  example  of  biological  material  with  dif- 
fering potentials  that  have  been  so  fixed  by  inbreeding  that  they  can  be 
reproduced  in  unlimited  quantities,  each  one  as  identical  to  the  next  as  it 
is  possible  for  biological  material  to  be. 

Take  for  example  strain  C3H:  here  is  a  strain  that  for  some  30  to  40 
generations  has  produced  98  percent  incidence  of  mammary  tumors. 
Strain  A  has  produced  a  very  high  incidence  of  lung  tumors  over  many 
generations.  Why  these  incidences  have  not  been  100  percent  still  re- 
mains a  genetic  problem,  in  part  at  least. 

To  go  on  with  a  reverse  example:  strain  C57BL  is  resistant  to  mammary- 
tumor  development  and  to  the  milk  agent,  a  perfect  control  for  strain 
C3H. 

Our  list  can  be  increased  by  including  such  things  as  strains  which 
differ  in  the  numbers  of  presacral  vertebrae  (1),  and  in  resistance  or  sus- 
ceptibility to  specific  viruses  and  bacteria. 

So  the  values  of  our  inbred  strains  need  no  further  elaboration.  How 
do  we  develop  these  inbred  strains?  Haphazard  mating  of  mice  would  be 
to  no  avail  but  a  logical  procedure  of  crossing  one  inbred  strain  with 
another  inbred  strain,  of  which  the  genetic  background  is  known,  is  an 
organized  method  of  arriving  at  the  creation  of  a  new  strain. 


1  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor, 
Maine,  June  30, 1954. 

*  This  investigation  has  been  supported  by  grants  to  the  Roscoe  B.  Jackson  Memorial  Laboratory  from  the 
American  Cancer  Society  upon  recommendation  of  the  Committee  on  Growth  of  the  National  Research  Council 
and  by  research  grant  C-362  from  the  National  Cancer  Institute  of  the  National  Institutes  of  Health,  U.  S.  Public 
Health  Service. 

3  Grateful  acknowledgment  is  made  of  the  valuable  assistance  of  Mrs.  P.  W.  Lane  in  collecting  the  data  for  this 
presentation. 

791 

Journal    of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


792  proceedings:  symposium  on  25  years  of 

Hybridization  is  known  to  produce  conditions  unknown  in  either  parent 
or  conditions  more  extreme  than  those  found  in  either  parent  (2) .  Utilizing 
these  facts  it  may  then  be  possible  to  develop  strains  of  mice  showing 
perhaps  a  new  tumor  type  or  pathologic  condition.  In  addition,  the  Fi 
animal  may  show  dominance  or  lack  of  dominance  of  a  condition  already 
apparent  in  the  parental  strains.  This  is  the  same  technique  used  to 
establish  proof  that  new  characters  are  mutations  and  to  provide  a  clue 
to  the  manner  of  their  inheritance. 

Bearing  these  facts  in  mind,  the  first  step  that  the  geneticist  must  take 
is  to  create  the  Fx  hybrid.  Because  most  of  these  phenomena  described 
above  do  not  occur  in  100  percent  of  the  animals,  the  possibility  that 
cxtrachromosomal  factors  like  maternal  influence,  as  well  as  multiple 
genie  effects,  can  play  a  part  makes  it  necessary  to  create  reciprocal 
hybrid  animals.  This  in  effect  tests  the  maternal  influence  upon  the 
occurrence  of  any  phenomena.  An  excellent  example  of  this  was  the 
demonstration  of  the  presence  of  the  milk  agent  using  reciprocal  hybrid 
mice  (3). 

If  the  geneticist,  once  started  on  such  a  program,  records  the  results 
of  the  Fi  hybrid  animals  and  wishes  to  establish  the  inheritance  more 
clearly  and  ascertain  the  genes  involved  in  the  occurrence  of  the  response, 
he  must  proceed  one  step  further  and,  for  most  efficient  utilization  of  the 
mice,  backcross  the  Fi  hybrids  to  the  parental  strains.  Again  reciprocal 
backcrosses  may  be  used  for  the  same  reasons  reciprocal  Fx  hybrids  were 
created.  If  a  single-gene  difference  were  responsible  for  the  genetics  of 
the  character  in  question,  the  result  of  this  backcross  would  be  its  linkage 
data  with  the  genes  against  which  it  was  tested.  In  most  instances  when 
inbred  strains  are  being  tested  for  a  response  however,  the  genetic  analysis 
is  far  more  complicated  since  several  genes  are  probably  concerned  with 
many  physiological  responses.  By  application  of  certain  known  formulae 
to  the  data  an  estimate  of  the  minimum  number  of  genes  involved  in 
producing  any  given  response  may  be  calculated.  The  need  for  tagging 
such  "in visible  gene  action"  is  apparent  so  that  linkage  of  physiological 
responses  with  phenotypic  characteristics  is  very  desirable.  The  linkage 
of  two  genes,  fused  tail  and  histocompatibility-2,  is  an  excellent  demon- 
stration of  a  phenotypic  tag  for  a  physiologic  characteristic  (4). 

The  values  of  the  hybrid  animal  have  long  been  recognized  by  the 
geneticist  and  therefore  the  use  of  hybrids  is  not  really  a  new  approach  to 
the  study  of  cancer  problems.  For  example,  Dr.  Myron  Gordon  (5)  has 
demonstrated  this  with  his  hybrid  fish  and  melanoma  tumor  production. 
Both  the  hybrids  and  the  backcross  mice  should  be  exploited  more  fully 
not  only  for  the  information  they  can  give  on  expected  responses  but  for 
the  information  they  can  furnish  as  to  conditions  occurring  de  novo, 
which  may  be  the  basis  for  creating  new  inbred  strains  if  the  condition  is 
not  a  result  of  hybridization  alone  and  disappears  in  the  next  generation. 

This  last  statement  is  based  on  the  results  of  an  investigation  begun 
several  years  ago  by  Drs.  Fekete,  Woolley  and  Little  (6).  Although  the 
original  experiment  was  designed  for  an  entirely  different  purpose,  as  often 

Journal    of    the   National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  793 

happens  in  this  business  of  research,  the  results  showed  that  inbred  strains 
varied  in  their  response  to  neo-natal  gonadectomy.  It  is  this  particular 
response  and  its  ramifications  that  will  be  used  to  demonstrate  the  truth 
of  the  statements  just  presented. 

Strain  DBA/2Wy,  following  gonadectomy,  developed  nodular  hyper- 
plasia of  the  adrenal  cortex  and  this  was  accompanied  by  feminization  of 
the  accessory  reproductive  organs  (6,  7).  Strain  CE/Wy,  however,  pro- 
duced adrenal-cortical  carcinomas  and  concomitant  masculinization  and/or 
feminization  of  the  accessory  reproductive  organs  following  gonadectomy 
(8-10).  Hybrids  of  these  two  inbred  strains  always  developed  adrenal- 
cortical  carcinomas  after  neo-natal  gonadectomy,  thus  demonstrating 
that  the  carcinoma  response  predominated  over  the  hyperplasia  response 
in  these  animals  (11).  The  time  of  occurrence  of  these  changes  seemed  to 
be  dependent  upon  extrachromosomal  factors ;  offspring  of  the  CE  females 
produced  carcinomas  earlier  than  those  with  DBA  mothers.  Backcrosses 
to  both  parental  strains  have  been  made  but  results  for  gonadectomized 
animals  are  not  yet  available. 

Strain  C3H/Di,  following  gonadectomy  responded  in  a  manner  similar 
to  that  of  strain  DBA,  i.e.,  nodular  hyperplasia  of  the  adrenal  and  feminiz- 
ing changes  (12).  Strain  A/Wy  showed  little  change  in  the  adrenal  and 
the  accessory  reproductive  organs  remained  permanently  immature  after 
gonadectomy  (12).  Hybrids  of  these  strains  responded  to  gonadectomy 
by  production  of  adrenal-cortical  carcinomas  (IS).  In  this  combination 
the  carcinoma  response  may  have  been  the  result  of  hybridization  per  se. 
Backcrosses  to  these  parental  strains  have  also  been  made  but  data  on  the 
gonadectomized  mice  are  too  incomplete  to  have  any  report  at  this  time. 

Using  strain  DBA  in  a  cross  with  another  stain,  DE/Wy  (which  although 
developed  from  strain  CE  responded  to  gonadectomy  like  strain  A,  i.e., 
no  changes  in  the  adrenal  or  accessory  reproductive  organs)  another  hybrid 
was  created  and  responded  to  gonadectomy  by  producing  not  only  adrenal- 
cortical  carcinomas  but  also  by  consistently  producing  basophil  adenomas 
of  the  pituitary.  These  basophil  adenomas  are  physiologically  active, 
i.e.,  diagnosis  for  their  presence  is  the  abundance  of  secretion  and  alveolar 
development  of  the  mammary  gland  (14).  The  adrenal-cortical  carci- 
nomas stimulate  growth  of  the  mammary  glands,  but  they  alone  never 
produce  the  extreme  development  found  in  the  mice  carrying  basophil 
tumors  as  well.  In  this  instance,  then,  both  abnormalities  appear  to  be 
the  result  of  hybridization  since  neither  response  has  been  observed  in 
either  parent  strain. 

Thus  the  three  different  sets  of  hybrids  have  produced  similar  responses 
from  different  backgrounds  and  for  different  reasons  (straight  inheritance 
in  one  instance  and  hybridization  in  the  other  two  cases).  Factors  in 
addition  to  the  gonadectomy  response  have  been  found  in  the  hybrids 
and  have  caused  this  investigation  to  take  on  an  even  wider  scope. 

One  hundred  percent  of  the  intact  or  control  hybrid  DBA  X  CE  virgin 
females  developed  a  pathologic  condition  of  the  uteri  that  resembled 
eventually  the  "Swiss  cheese"  endometrium  found  frequently  in  the  clinic 

Vol.    15,   No.    3,   December    1954 


794  proceedings:  symposium  on  25  years  of 

(15).  This  condition  began  with  cystic  glandular  hyperplasia  that  ap- 
peared about  8  months  of  age  and  was  followed  by  adenomyosis  and 
adenomatous  hyperplasia.  The  same  condition  of  the  uterus  was  apparent 
in  the  gonadectomized  females  and  occurred  at  about  the  same  age  as  in 
the  intact  animals.  Studies  carried  out  by  Atkinson  and  others  showed 
that  in  the  intact  mice  the  ovary  and  not  the  adrenal  was  responsible  for 
this  hyperestrogenic  or  "hyperovarian"  syndrome  (16).  Recent  studies 
have  shown  that  development  of  this  condition  may  be  prevented  by 
breeding  these  mice  (17).  Under  such  conditions  the  uteri  have  remained 
normal. 

Investigation  of  these  particular  Fi  animals  is  continuing  in  an  attempt 
to  elucidate  the  physiological  factors  involved  in  the  development  of  this 
syndrome.  Its  pattern  of  occurrence  is  being  observed  in  the  backcross 
animals  so  that  the  inheritance  of  this  condition  may  possibly  be  clarified. 
Data  tabulated  so  far,  showed  that  in  the  backcrosses  of  the  DBA  X  CE 
hybrids  to  strain  CE,  only  19  of  114  females  or  16.66  percent  showed  this 
condition.  Since  only  two  color  types  were  involved  in  this  backcross, 
data  were  tabulated  according  to  this  factor  and  in  both  types  the  occur- 
rence was  16.66  percent.  Comparing  the  data  of  the  offspring  of  the  inbred 
strain  mother  with  that  of  the  Fi  mother  showed  that  the  incidence  was 
very  slightly  higher  in  offspring  of  Fi  mothers.  In  the  backcrosses  of 
these  DBA-CE  hybrids  to  strain  DBA,  of  the  205  females  thus  far  ex- 
amined only  39  or  19.02  percent  have  been  classified  as  hyperestrogenic. 
Data  are  too  incomplete  to  be  analyzed  according  to  color  type  but 
occurrence  was  much  lower  when  the  DBA  was  the  mother  (12.28%)  than 
when  the  Fi  was  the  mother  (21 .62%).  This  indicates  that  inheritance  is 
a  factor  in  the  occurrence  of  this  condition  since  it  has  been  transmitted 
beyond  the  hybrid  generation.  Incidence  in  both  sets  of  backcrosses 
indicates  that  maternal  factors  also  play  a  role  in  the  manifestatiou  of  this 
condition. 

The  pituitary  basophil  adenomas,  which  occurred  consistently  in  the 
DE-DBA  gonadectomized  hybrids,  occurred  in  about  60  percent  of  the 
DBA-CE  hybrids,  and  occasionally  in  other  hybrid  crosses  that  were 
studied  (14).  This  phenomenon,  too,  appears  to  be  a  new  condition 
hitherto  unknown  in  any  of  the  parent  strains  and  is  thus  a  product  of 
hybridization.  Very  preliminary  data  collected  on  DE  and  DBA  back- 
cross  gonadectomized  mice  indicated  that  basophil  adenomas  occurred 
in  approximately  30  percent  of  the  DBA  backcross  animals  and  in  approxi- 
mately 29  percent  of  the  DE  backcross  mice.  This  too  shows  that  inherit- 
ance must  be  an  important  factor  in  its  occurrence  since  the  condition  has 
appeared  in  another  generation.  No  genetic  analysis  is  yet  possible  and 
no  other  tabulation  of  the  data  has  been  made. 

Incidental  information  was  collected  on  these  hybrids  concerning  the 
occurrence  of  mammary  tumor,  lung  tumor  or  appearance  of  any  other 
tumor  types.  One  other  hybrid  group,  studied  for  an  entirely  different 
reason,  is  included  in  the  list  given  in  table  1 . 


Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 
Table  1. — Types  oj  tumors  found  in  F\  reciprocal  hybrid  mice 


795 


CEXDBA 

32  mice 
2  lung  tumors 
6  lymphoid  tumors 

AXC3H 

16  mice 

14  lung  tumors  (87.50%) 

7  mammary  tumors  (100%) 

1  hepatoma 

DBAXCE 

29  mice 

5  lung  tumors 

6  mammary  tumors  (42.85%) 
1  papilloma 

1  interstitial-cell  tumor  testis 

C3HXA 

20  mice 

11  lung  tumors  (55.00%) 

2  hepatomas 

2  lymphoid  tumors 

DEXDBA 

34  mice 

14  lung  tumors  (41.17%) 
2  adenomas  Harderian  gland 

2  epidermoid  carcinomas 

3  fibrosarcomas 

1  lymphatic  leukemia 

2  papillomas 

1  myoepithelioma    salivary 
gland 

3  hepatomas 

1  giant-cell  sarcoma 

CXC3H 

111  mice 

7  lung  tumors 
3  ovarian  tumors 
6  uterine  sarcomas 

1  hepatoma 

2  sarcomas 

The  occurrence  of  hyperestrinism  in  the  CE  and  DBA  backcrosses  has 
been  discussed.  Ovarian- tumor  incidence  in  these  same  backcross  series 
gives  an  excellent  example  of  a  condition  appearing  to  a  greater  extent 
in  the  backcross  generation  than  in  either  of  the  parent  strains  or  in  the 
Fi  hybrids.  Strain  CE  females  had  the  greatest  number  of  ovarian 
tumors  found  in  any  strain.  After  20  months  of  age  the  incidence  was 
34  percent.  Such  tumors  did  not  occur  at  all  in  strain  DBA  mice.  The 
incidence  was  33.33  percent  in  DBA-CE  hybrid  females  and  19.04  percent 
in  CE-DBA  hybrid  females.  Totaling  all  females  of  the  CE  backcrosses 
the  incidence  was  52.00  percent  after  20  months  of  age,  an  enhanced 
incidence  over  the  34  percent  available  from  strain  CE.  When  examined 
according  to  color  type,  the  data  showed  that  58.62  percent  of  the  extreme 
dilute  mice  and  42.85  percent  of  the  black  agouti  mice  in  this  age  group 
had  ovarian  tumors.  When  data  were  tabulated  according  to  parental 
type  44.00  percent  of  the  offspring  of  CE  mothers  had  these  tumors  and 
60.00  percent  of  the  offspring  of  the  Fi  mothers  had  tumors.  In  the  back- 
crosses  to  DBA  only  three  ovarian  tumors  were  observed.  All  three 
tumors  were  papuliferous  cystadenomas.  A  diagram  of  the  occurrence 
of  ovarian  tumors  in  the  three  generations  is  presented  in  text-figure  1 . 

Not  only  did  the  numbers  of  ovarian  tumors  show  an  increase  over  that 
observed  in  the  hybrid  and  in  the  parent  strain  CE,  but  the  tumors  in 
many  instances  appeared  to  be  atypical  granulosa-cell  tumors  and  tubular 
adenomas.4    Histologic  study  of  these  tumors  is  not  complete. 

The  adrenal  glands  of  these  various  types  of  intact  backcross  mice 
have  been  examined  and  present  some  interesting  possibilities  for  further 
investigation.  In  some  groups  25  percent,  or  more,  intact  mice  had 
pathologic  changes  in  the  adrenal  glands.  Their  incidence  was  highest 
in  the  backcrosses  to  strain  CE.    These  have  not  all  been  classified  as 

*  Diagnoses  courtesy  of  Dr.  Hummel  and  Dr.  Murphy. 
Vol.   15,  No.  3,  December   1954 


796 


PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


O/m, 

279     9  tu. 
34.81% 


T 


Tig 


CE9  i  DBAC? 
219  4  tu. 
19.04* 


DBA9  x  CEtf 
95        3  tu. 

33.33% 


DBVa»y 

959     0  tu. 

o.oox 


I 


CE9  x   (CE-DBAjd1 

139  6  tu. 

46. 19* 

7e«  3  tu.  42.85% 
6AB  3  tu.   50.00* 


CE9  x   (DBA-CE)cf 

1^  5   tu. 

41.66% 

7ce  4  tu.   57.14% 
5AB   1    tu.    20.00* 


(CE-DBA)9   x  CEO" 

139  7  tu. 

53.84% 

7ce   3  tu.   42.85% 
6AB  4   tu.   66.66* 


(DBA-CE)9 

X 

CEtf 

12? 

8 

tu. 

66.66% 

8ce  7   tu. 

87 

.50% 

4AB  1    tu. 

25 

.00* 

BC 

DBA9   x   Fjtf 

59     1   tu. 

20.00% 

FX9  x  DBJtf 
219     2  tu. 
9.52% 

26  9         3  tu. 
11.53% 


50  9     *26  tu. 
52.00% 

(CE   299  17  tu.  58.62%  I  AS  219  9  tu.  42.85%) 

Text-figure  1. — Ovarian  tumor  occurrence  after  20  months  o)  age. 

yet  and  their  physiological  action,  if  any,  is  not  clearly  understood. 
Many  of  the  adrenals  exhibited  nodular  lesions  that  were  composed  of  the 
small  subcapsular  cells  which  extended  over  a  large  portion  of  the  cortex. 
In  some  of  these  nodules,  areas  of  clear  hyperplastic  cells  were  seen  and 
such  areas  closely  resembled  the  nodular  hyperplasia  seen  in  gonadecto- 
mized  DBA  mice.  Another  adrenal  tumor  of  quite  a  different  type  was 
found  and  has  been  provisionally  diagnosed  as  a  differentiated  adrenal- 
cortical  carcinoma.  Medullary  disturbances  were  also  found.  Medul- 
lary nodules  arose  but  did  not  expand  greatly  in  size.  One  pheochromocy- 
toma  was  also  observed.5 

Other  rare  tumors  were  found  in  the  backcross  animals.  One  thyroid 
adenocarcinoma  was  found  in  a  CE-DBA  backcross  animal  and  3 
ganglioneurofibromas  of  the  optic  tract  were  discovered  in  DE-DBA 
backcross  mice.5 

A  list  of  the  various  types  of  tumors  in  these  different  backcross  groups 
serves  to  illustrate  again  the  value  not  only  of  hydridization  but  of  the 
backcross  animals  (table  2). 

The  value  of  the  inbred  strain,  the  hybrid  and  the  backcross  mouse  in 
their  role  of  revealing  new  conditions  and  providing  information  on  the 
inheritance  of  conditions  already  described|is  thus  apparent. 

Using  this  technique  of  hybrid  and  backcross  generations  in  an  experi- 
ment originally  designed  to  demonstrate  the  inheritance  of  the  gonadec- 
tomy  response,  far-reaching  results  have  occurred  and  are  thus  sum- 
marized: 1)  New  conditions  have  occurred  in  hybrids  which  show  evidence 
of  their  inheritance  by  their  appearance  in  the  backcross  generations. 
They  are  a)  hyperestrinism  in  100  percent  of  intact  hybrid  DBA-CE 
virgin  females  (an  excellent  experimental  tool  for  the  uterine  pathologist) 
and  b)  basophil  adenomas  of  the  pituitary  in  100  percent  of  gonadecto- 

» Diagnoses  courtesy  of  Dr.  Edwin  Murphy. 


Journal    of    the   National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  797 

Table  2. —  Types  of  tumors  found  in  reciprocal  backcross  series  of  mice 


Backcross  to  CE 

329 
1 

49 
2 
6 

mice 

epidermoid   car- 
cinoma 
lung  tumors 
papillomas 
uterine  fibrosar- 

Backcross  to  A 

373 

189 
25 

12 
6 

mice 

lung  tumors 
mammary  tu- 
mors, 
hepatomas 
lymphoid  tu- 

comas 

1  cervical  sarcoma 

2  interstitial-cell 

tumors,  testis. 
26  lymphoid  tu- 

mors. 

9 
3 

mors. 
hepatomas 
hemangiomas 

Backcross  to  DE 

346  mice 
40  lung  tumors 
2  lymphoid  tumors 
1  hepatoma 
8  uterine  fibrosar- 
comas 
1  cervical  sarcoma 

1  hemangioma 

2  chromophobe  ad- 

enomas pitui- 
tary 

Backcross  to  C3H 
(with  A) 

377 

77 
149 

1 

16 

1 

7 

mice 

lung  tumors 
mammary  tu- 
mors 
papilloma 
hepatomas 
lymphoid  tumor 
hemangiomas 

Backcross  to  DBA 

440 
43 
20 

1 

2 

mice 

lung  tumors 
mammary  tu- 
mors 
papilloma 
uterine  fibrosar- 

Backcross to  C3H 

(with  C) 

271  females 
67  lung  tumors 
12  ovarian  tumors 
8  cervical     sarco- 
mas 
8  uterine  fibrosar- 

4 

42 

17 

3 

1 

comas 
cervical  sarcomas 
lymphoid  tumors 
hepatomas 
hemangiomas 
thyroid     adeno- 
carcinoma 

comas 

10  hepatomas 

19  lymphoid  tu- 
mors 

2  osteogenic  sar- 

comas 

3  adrenal-cortical 

1 

carcinomas 
pheochromocy- 
toma 

Backcross  to  DBA 
(with  DE) 

455 
21 

7 

1 
5 

7 

11 

4 

3 

mice 

lung  tumors 

mammary  tu- 
mors 

papilloma 

uterine  fibrosar- 
comas 

cervical  sarcomas 

lymphoid  tumors 

hepatomas 

ganglioneurofi- 
bromas 

mized  DE-DBA  mice  (another  experimental  tool  for  endocrine  research) . 
2)  A  20-percent  increase  in  ovarian  tumor  incidence  over  that  available 
in  inbred  strains  has  occurred  in  certain  backcrosses.  3)  Several  rare 
tumor  types  have  been  observed  in  some  of  the  backcross  mice,  and  4) 
adrenal-cortical  abnormalities  have  been  noted  in  intact  backcross  mice. 

Vol.    15,   No.   3,   December    1954 


798 


PROCEEDINGS!  SYMPOSIUM  ON  25  YEARS  OF 


Needless  to  say,  the  occurrence  of  these  particular  phenomena  may  well 
complicate  the  analyses  of  the  gonadectomy  response. 

No  attempt  can  be  made  to  go  into  detailed  genetic  analysis  of  any  of 
these  phenomena  at  this  time,  but  a  few  general  statements  can  be  made 
about  these  various  animals.  Not  all  hybrids  produce  exciting  and  un- 
expected pathologic  conditions  or  physiologic  changes  and  when  these 
hybrids  are  backcrossed  to  the  parental  strains  this  situation  continues 
to  be  true.  Such  is  the  case  with  the  A  and  C3H  mice.  Therefore  it 
may  be  said  that  the  potential  of  these  two  strains  in  such  combination 
does  not  yield  indications  for  the  development  of  any  new  strains.  How- 
ever, strains  CE,  DBA,  DE  and  C  produce  a  wider  variety  of  tumor  types, 
(new  phenomena  not  known  in  the  parental  strains  and  enhancement  of 
tumor  incidence'  in  one  instance)  so  that  these  particular  types  of  mice 
will  be  valuable  in  attempting  to  develop  new  strains  known  for  some  of 
these  characteristics. 

Such  then  is  one  method  whereby  leads  may  be  found  in  the  continued 
search  for  valuable  new  types  of  mice  that  may  be  of  help  not  only  in 
meeting  the  needs  of  research  but  also  as  an  aid  to  the  clinician  in  the 
problems  with  which  he  is  concerned. 

References 

(1)  Green,  E.  L.:  The  genetics  of  a  difference  in  skeletal  type  between  two  inbred 

strains  of  mice  (BalbC  and  C57blk).     Genetics  36:  391-409,  1951. 

(2)  Little,  C.  C.:  Hybridization  and  tumor  formation  in  mice.     Proc.  Nat.  Acad. 

Sc.  25:  452-455,  1939. 
(8)   Murray,  W.  S.,  and  Little,  C.  C.:  The  genetics  of  mammary  tumor  incidence 
in  mice.     Genetics  20:  466-496,  1935. 

(4)  Gorer,  P.  A.,  Lyman,  S.,  and  Snell,  G.  D.:  Studies  on  the  genetic  and  antigenic 

basis  of  tumor  transplantation.  Linkage  between  a  histocompatibility  gene 
and  "fused"  in  mice.     Proc.  Roy.  Soc.  s.B,  London,  135:  499-505,  1948. 

(5)  Gordon,  M.:  Effects  of  five  primary  genes  on  the  site  of  melanomas  in  fishes  and 

the  influence  of  two  color  genes  on  their  pigmentation.     Spec.  Publ.  New  York 
Acad.  Sc.  4:  216-268,  1948. 

(6)  Fekete,  E.,  Woolley,  G.  W.,  and  Little,  C.  C:  Histological  changes  following 

ovariectomy  in  mice.  I.  dba  high  tumor  strain.     J.  Exper.  Med.  74:  1-8,  1941. 

(7)  Woolley,  G.  W.,  Fekete,  E.,  and  Little,  C.  C.:  Effect  of  castration  in  the 

dilute  brown  strain  of  mice.     Endocrinol.  28:  341-343,  1941. 

(8)  Woolley,  G.  W.,  and  Little,  C.  C:  The  incidence  of  adrenal  cortical  carcinoma 

in  gonadectomized  female  mice  of  the  extreme  dilution  strain.  I.  Observations 
on  the  adrenal  cortex.     Cancer  Res.  5:  193-202,  1945. 

(9)  :  The  incidence  of  adrenal  cortical  carcinoma  in  gonadectomized  female 

mice  of  the  extreme  dilution  strain.  II.  Observations  on  the  accessory  sex 
organs.     Cancer  Res.  5:  203-210,  1945. 

(10)  :  The  incidence  of  adrenal  cortical  carcinoma  in  male  mice  of  the  extreme 

.  dilution  strain  over  one  year  of  age.     Cancer  Res.  5:  506-509,  1945. 

(11)  Woolley,  G.  W.,  Dickie,  M.  M.,  and  Little,  C.  C:  Adrenal  tumors  and  other 

pathological  changes  in  reciprocal  crosses  in  mice.  I.  Strain  DBA  X  Strain  CE 
and  the  reciprocal.     Cancer  Res.  12:  142-152,  1952. 

(12)  Unpublished  data. 

(18)  Woolley,  G.  W.,  Dickie,  M.  M.,  and  Little,  C.  C:  Adrenal  tumors  and  other 
pathological  changes  in  reciprocal  crosses  in  mice.  II.  An  introduction  to  results 
of  four  reciprocal  crosses.     Cancer  Res.  13:  231-245,  1953. 


Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  799 

(14)  Dickie,  M.  M.,  and  Woolley,  G.  W.:  Spontaneous  basophilic  tumors  of  the 

pituitary  glands  in  gonadectomized  mice.     Cancer  Res.  9:  372-384,  1949. 

(15)  Christy,. N.  P.,  Dickie,  M.  M.,  Atkinson,  W.  B.,  and  Woolley,  G.  W.:  The 

pathogenesis  of  uterine  lesions  in  virgin  mice  and  in  gonadectomized  mice 
bearing  adrenal  cortical  and  pituitary  tumors.    Cancer  Res.  11:  413-422,  1951. 

(16)  Atkinson,  W.  B.,  and  Dickie,  M.  M.:  Further  studies  on  the  pathogenesis  of 

uterine  lesions  in  DBA  X  CE  and  reciprocal  hybrid  mice.     Cancer  Res.  13: 
165-167,  1953. 

(17)  Atkinson,  W.  B.,  Dickie,  M.  M.,  and  Fekete,  E.:  Effects  of  breeding  on  the 

development  of  ovarian,   adrenal,   and  uterine  lesions  in  DBA  X  CE  and 
reciprocal  hybrid  mice.     Endocrinol.     55:  316-325,  1954. 


Vol.    15,   No.   3,   December   1954 


Significance  of  Recent  Developments  in 
Nuclear  Cytology  and  Cytogenetics  of 
the  Mouse  u  2 


Elizabeth  Fekete  and  Allen  B.  Griffen,3 
Roscoe  B.  Jackson  Memorial  Laboratory,  Bar 
Harbor,  Maine 


I.  Histopathological  Studies 

Studies  on  a  transplantable  ovarian  teratoma  of  a  mouse  which  main- 
tained its  pluripotency  and  is  still  capable  of  giving  rise  to  differentiated 
tissues  have  been  reported  from  this  laboratory  (1). 

In  this  paper  we  are  presenting  studies  on  another  transplantable 
ovarian  teratoma.  This  neoplasm,  originally  composed  of  both  differ- 
entiated and  undifferentiated  embryonal  elements,  lost  its  power  of 
differentiation  after  four  subcutaneous  transplant  generations  and  at 
present  is  composed  only  of  embryonic  immature  tissue.  The  sub- 
cutaneously  transplanted  tumor  was  successfully  transformed  into  an 
ascites  tumor  by  the  method  described  by  George  Klein  {2).  It  has  been 
carried  both  subcutaneously  as  nodules  and  intraperitoneally  as  ascites 
tumor  through  several  transplant  generations. 

The  neoplasm  occurred  in  a  193-day-old  C3H/Ks  female  in  Dr.  Kaliss' 
colony.  The  mouse  had  given  birth  to  two  litters.  She  was  killed  July 
16,  1952,  at  which  time  the  abdomen  was  greatly  distended.  The  left 
ovary  was  very  large,  measuring  40  X  20  X  10  mm.  and  the  right  ovary 
was  about  twice  normal  size.  Many  small  nodules  were  scattered  through 
the  abdominal  cavity,  attached  to  different  parts  of  the  viscera,  causing 
numerous  adhesions.  Some  of  the  nodules  had  infiltrated  the  abdominal 
wall  and  were  adhered  to  the  skin;  others  penetrated  the  diaphragm  and 
were  attached  to  and  infiltrated  the  lungs.  The  large  left  ovary  had 
hemorrhagic  areas  and  the  central  part  was  necrotic.  Some  of  the  small 
nodules  contained  black  pigment,  some  were  dense  and  solid,  while  others 
contained  cysts  and  alveolar  areas. 

Part  of  each  ovary  and  several  of  the  nodules  were  fixed,  sectioned,  and 
examined  microscopically.  The  left  ovary  had  only  a  narrow  margin  of 
non-necrotic  tissue  composed  of  several  different  types  of  tissues  as  well  as 

i  The  second  section  of  this  paper  was  presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian 
Genetics  and  Cancer,  Bar  Harbor,  Maine,  June  30, 1954. 

2  These  investigations  were  aided  by  grants  from  the  National  Cancer  Institute  of  the  National  Institutes  of 
Health,  U.  S.  Department  of  Health,  Education,  and  Welfare. 

»  The  authors  wish  to  express  their  gratitude  to  Miss  Hope  Otis  and  to  Mr.  Merrill  C.  Bunker  for  their  capable 
assistance  in  all  parts  of  this  work. 

801 

Journal    of    the   National    Cancer    Institute,    Vol.    15,   No.    3,   December    1954 


802  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

undifferentiated  embryonic  tissue.  The  right  ovary  contained  some 
normal  follicles  and  in  the  medullary  part  elements  of  the  neoplasm  which 
were  embryonal  (fi?.  1).  The  other  nodules  were  composed  of  mixtures  of 
well  differentiated  tissues  and  embryonal  elements  (figs.  2  and  3).  The 
differentiated  tissues  included:  nervous  tissues  composed  mostly  of 
neuroepithelial  and  neuroblastic  elements,  cartilage,  bone,  smooth  muscle 
fibers  and  different  types  of  connective  tissues.  Small  cavities  lined  by 
ciliated  respiratory  epithelium,  or  tall  columnar  epithelium  and  goblet 
cells,  resembling  intestinal  epithelium,  or  stratified  squamous  epithelium 
or  pigmented  epithelium  were  also  present.  Large  cells  and  giant  cells 
resembling  trophoblasts  composed  part  of  a  small  nodule.  Most  of  the 
tissues  were  richly  cellular  and  mitotic  figures  were  numerous. 

The  neoplasm  was  designated  E8156  and  was  diagnosed  as  a  teratoma. 
As  the  left  ovary  contained  the  largest  tumor  this  was  considered  to  be  the 
site  of  origin  and  the  other  nodules  to  be  the  result  of  dissemination  and 
metastasis. 

Several  nodules  of  the  neoplasm  were  cut  into  small  pieces,  mixed,  and 
transplanted  subcutaneously  into  five  C3H/Fe  mice  at  weaning  age. 
Three  of  these  were  negative  and  two  had  large  tumors  at  the  site  of  trans- 
plantation within  3  months.  These  were  retransplanted  subcutaneously 
and  the  tumor  has  been  carried  this  way  through  about  25  transplant 
generations.  The  rate  of  growth  increased  so  that  at  present  trans- 
plantation has  to  be  performed  about  once  in  3  weeks.  The  tumor  grows 
almost  100  percent  in  males  and  females  of  strain  C3H/Fe  and  their  Fi 
hybrids. 

Grossly  the  transplanted  tumors  were  dense  and  hard  for  about  three 
generations,  after  which  they  became  very  soft  and  hemorrhagic.  Parallel 
with  the  gross  changes  there  were  alterations  in  microscopic  appearance. 
There  was  a  decrease  in  the  amount  of  differentiated  tissues;  after  the 
fourth  transplant  generation  only  undifferentiated  embryonal  elements 
were  present,  and  the  blood  supply  was  very  rich  (figs.  4  and  5).  Areas  of 
giant  cells  resembling  trophoblasts  were  often  present  (fig.  6). 

In  an  attempt  to  grow  the  neoplastic  cells  in  the  ascitic  fluid,  part  of  a 
subcutaneous  growth  of  the  fifth  transplant  generation  was  homogenized, 
diluted  with  saline  and  injected  into  the  peritoneal  cavity  of  C3H/Fe  mice 
at  weaning  age.  Parts  of  the  same  tumor  were  also  transplanted  sub- 
cutaneously. The  intraperitoneal  injection  produced  both  numerous 
solid  neoplastic  nodules  and  a  great  increase  of  ascitic  fluid  in  4  weeks. 
About  2cc.  of  bloody  ascitic  fluid  was  drawn  out  with  a  syringe  and  re- 
transplanted  intraperitoneally  into  new  hosts.  In  these  animals  large 
numbers  of  neoplastic  cells  were  present  in  the  ascitic  fluid  but  no  solid 
nodules  were  found.  The  tumor  has  been  carried  by  reinjecting  ascitic 
fluid  intraperitoneally  through  about  10  generations.  The  rate  of  growth 
increased  and  it  is  necessary  now  to  reinject  the  ascitic  fluid  into  new 
hosts  about  every  15  to  20  days.  Ascitic  fluid  of  C3H/Fe  mice  has  been 
injected  into  the  peritoneal  cavity  of  6  AKK/Fe  mice  and  has  produced  an 

Journal   of   the  National   Cancer  Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  803 

increase  in  ascitic  fluid  containing  tumor  cells  in  all  of  them.     It  is  planned 
to  test  the  growth  in  several  other  strains. 

Ascitic  fluid  of  C3H/Fe  mice  injected  subcutaneously  into  the  same 
strain  produces  local  growth  which  is  similar  morphologically  to  the  sub- 
cutaneously transplanted  tumors.  Any  of  the  subcutaneously  growing 
transplanted  neoplasms  can  be  readily  converted  into  ascites  form  by 
homogenizing  them  and  injecting  them  intraperitoneally.  Cytologic 
studies  of  the  tumor  cells  present  in  the  ascitic  fluid  have  been  conducted 
on  smear  preparations  by  Dr.  Griffen. 

II.  Cytological  Studies 

The  successful  completion  of  preliminary  chromosome  maps  of  the 
mouse  by  Slizynski  (8,  4)  and  by  Griffen  (5)  has  shown  that  mouse 
cytogenetics,  though  difficult,  has  great  promise  of  application  in  classical 
genetics  and  in  tumor  research.  The  present  paper  indicates  that  familiar- 
ity with  the  germinal  chromosomes  may  enable  the  investigator  to  handle 
and  to  understand  the  nuclear  phenomena  of  neoplastic  cells  with  greater 
ease  than  has  been  possible  in  the  past.  The  teratoma  which  has  been 
described  has  provided  some  very  good  material  for  such  studies. 

Both  sections  and  teased  smear-preparations  of  the  nodular  transplants, 
as  carried  by  Dr.  Fekete,  revealed  an  abundance  of  cells  in  whose  frequent 
mitotic  figures  the  chromosomes  were  large  and  sharply  defined.  No 
satisfactory  chromosome  counts  were  obtained  from  these  preparations, 
but  the  frequent  appearance  of  polyploid  cells  and  of  multipolar  spindles 
was  observed.  Upon  the  successful  growth  of  the  neoplastic  cells  in  the 
ascitic  fluid,  samples  of  the  material  were  prepared  for  high-magnification 
microscopy  by  the  smear  or  squash  method.  The  procedures  may  be 
summarized  as  follows :  a)  a  drop  of  fluid,  consisting  of  a  rich  cell  suspen- 
sion, was  placed  upon  each  of  10  to  20  slides;  6)  a  drop  of  Sudan  Black  B 
stain-fixer,  prepared  according  to  a  formula  given  by  Cohen  (6),  was 
added  to  each  slide  and  thoroughly  mixed  with  the  cell  suspensions  by 
stirring  with  a  needle;  c)  cover  glasses  were  placed  on  the  preparations 
and  each  cover  was  firmly  pressed  and  blotted  with  filter  paper;  d)  all 
slides  were  made  permanent  by  the  vapor  dehydration  method  of  Bridges 
(7),  followed  by  mounting  in  Euparal. 

Sudan  Black  smear  preparations  of  the  first  ascitic  generation  revealed 
great  numbers  of  cells  as  single  bodies,  2-,  4-,  and  8-celled  groups,  and 
other  clusters  (presumably  clones)  containing  higher  numbers  of  cells. 
Careful  chromosome  counts  made  from  camera  lucida  drawings  yielded  a 
constant  number  of  38,  or  2N  minus  2  in  terms  of  the  normal  mouse 
chromosome  complement,  for  diploid  cells.  Polyploid  cells,  easily  classi- 
fied as  tetraploid  and  octoploid  by  means  of  direct  chromosome  counts, 
were  of  frequent  occurrence.  In  all  cases  the  ploidy  was  based  on  a 
haploid  complement  of  19,  or  N  minus  1,  so  that  all  countable  tetraploid 
cells,  for  example,  contained  no  more  than  76  chromosomes  whereas  80 
should  normally  be  expected. 

Vol.    15,  No.   3,   December   1954 
316263—54 37 


804  proceedings:  symposium  on  25  years  of 

As  further  ascitic  transplants  were  made,  permanent  smears  were 
prepared;  however,  not  all  generations  were  included  in  the  collection  of 
material.  Since  the  study  of  these  slides  is  still  in  progress,  comprehensive 
results  cannot  be  presented  at  this  time ;  but  the  seventh  ascitic  generation 
has  been  given  thorough  analysis,  revealing  several  striking  characteristics. 
Countable  diploid  cells  regularly  showed  more  than  38  chromosomes.  In 
all  cases  in  which  the  metaphase  plates  were  complete  beyond  doubt  and 
showed  no  possibility  of  occlusions  by  superimpositions,  the  chromosome 
number  was  42,  indicating  a  gain  of  four  chromosomes  by  some  means. 
All  polyploid  nuclei  were  apparently  based  upon  the  new  haploid  number, 
N  =  21,  for  all  countable  tetraploid  and  octoploid  cells  regularly  showed 
more  than  80  and  more  than  160  chromosomes,  respectively.  In  addition 
to  diploid,  tetraploid  and  octoploid  cells,  the  unexpected  classes  haploid 
(N  =  21),  triploid  (3N  =  63)  and  hexaploid  (6N  =  126)  were  found. 
The  frequency  of  all  cell  types,  as  determined  from  a  total  metaphase 
count  in  one  sample  smear,  was  as  follows: 

Degree  of      Chromosome  Cell 

ploidy  number  count  Percent 


N 

21 

11 

1.7 

2N 

42 

503 

78.2 

3N 

63 

35 

5.4 

4N 

84 

66 

10.2 

6N 

126 

7 

1.08 

8N 

168 

21 

3.2 

643 

Tetraploid  cells  were  found  to  have  unusual  reproductive  features,  which 
have  not  been  observed  in  other  cells,  except  for  a  single  hexaploid  cell  to  be 
mentioned  below.  Through  normal  bipolar  mitosis  a  4N  cell  may  give 
rise  to  two  4N  daughters  with  no  unusual  features  observable  in  the  spindle 
mechanism  at  anaphase.  But  in  addition,  4N  cells  may  form  tripolar 
spindles  and  divide  reductionally  to  produce  one  diploid  (2N)  and  two 
haploid  (N)  daughters;  or  by  means  of  tetrapolar  spindles,  4N  cells 
may  divide  reductionally  into  four  haploid  (N)  daughters.  While  the 
old  term  '  'reduction  division"  is  hardly  suitable  for  anything  other  than 
the  meiotic  phenomena  seen  in  primary  germ  cells,  there  is  nevertheless 
in  these  tumor-cell  activities  a  very  real  reduction  of  chromosome  number 
which  is  brought  about  through  a  separatory  type  of  division  without 
chromosome  reduplication.  It  has  not  been  possible  to  determine  to 
what  extent  the  reduced  daughter  nuclei  may  contain  genetically  com- 
plementary members  of  the  chromosome  complement,  since  the  meta- 
phase chromosome  morphology  of  the  various  daughters  should  need  to 
be  studied  at  great  length;  however,  since  haploid  cells  are  frequently 
seen  dividing,  the  haploid  chromosome  sets  may  be  to  some  extent  com- 
plete. In  addition  to  the  tetraploids,  a  single  hexaploid  (6N)  cell  has 
been  seen  dividing  by  means  of  a  tripolar  spindle  to  produce  three  polar 
chromosome  clusters  which  were  3N,  2N,  and  N  in  approximate  count. 

In  meiosis  the  physical  basis  for  chromosome  segregation  is  synapsis. 
Since  the  phenomenon  of  synapsis  has  been  observed  frequently  in  tetra- 

Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  805 

p]oid  cells  of  the  teratoma  the  haploid  and  other  cells  resulting  from 
reduction  may  indeed  approach  genetic  perfection  in  chromosome  distri- 
bution, similar  to  that  resulting  from  meiotic  activities  in  germ  cells. 
While  the  synapsis  is  apparently  weak,  it  is  at  least  strong  enough  to 
provide  opportunity  for  segmental  exchanges  between  chromosomes,  for 
typical ' 'groups  of  four"  are  often  found,  sometimes  several  in  a  single  cell. 
It  has  not  yet  been  possible  to  determine  whether  the  exchange  figures 
result  from  crossing  over  or  from  translocation.  Either  of  these  phenom- 
ena would  provide  additional  means  for  genetic  variation  in  the  tumor. 
Even  though  the  tumor  originated  as  tissue  from  the  inbred  C3H  strain, 
there  is  no  conceivable  hindrance  to  gene  mutation;  crossing  over  can 
serve  effectively  in  making  new  combinations  of  any  mutants  which  may 
arise.  Translocations,  on  the  other  hand,  would  provide  directly  for 
gene  changes  through  position  effects,  and  also  would  provide  opportunity 
for  segmental  aneuploidy  {i.e.,  duplication-deficiency  phenomena  involv- 
ing parts  of  chromosomes). 

From  these  observations  it  is  apparent  that  the  tumor,  as  presently 
grown  in  ascitic  form,  is  capable  of  undergoing  variation  in  the  manner 
seen  in  sexually  reproducing  organisms.  Mutation,  synapsis,  segmental 
exchanges,  and  segregation  through  reduction  are  in  operation,  without 
the  necessity  of  sexual  reproduction  for  survival.  In  the  saprophytic  or 
parasitic  conditions  of  the  ascites  growth,  variant  cells  are  doubtless 
subject  to  selection  as  is  indicated  by  the  changes  in  chromosome  number. 
The  growth  of  the  C3H  tumor  in  AKR  hosts  further  suggests  evolution 
within  the  material. 

Hauschka  and  Levan  (8)  have  presented  extensive  evidence  that,  in  a 
number  of  tumor  types,  modifications  of  host-tumor  specificities  are 
based  upon  the  predominance  of  4N  and  other  polyploid  cells  in  the  tumors. 
The  activities  of  tetraploid  cells  in  the  teratoma  suggest  several  possible 
mechanisms  for  such  modifications. 

Summary 

A  spontaneously  occurring  ovarian  teratoma  which  produced  many 
secondary  nodules  in  the  peritoneal  and  pleural  cavities  is  described. 

Transplanted  subcutaneously  this  neoplasm  showed  a  decrease  in  the 
amount  of  differentiated  tissues;  after  the  fourth  transplant  generation 
only  undifferentiated  embryonal  elements  were  present. 

A  homogenized  subcutaneous  tumor  injected  intraperitoneally  produced 
an  increase  in  ascitic  fluid,  containing  neoplastic  cells. 

The  tumor  is  being  maintained  by  subcutaneous  transplantation  and 
by  reinjection  of  ascitic  fluid  intraperitoneally. 

The  first  ascitic  generation,  studied  in  Sudan  Black  smear  preparations, 
had  a  diploid  chromosome  number  of  38  or  2N-2.  In  the  seventh  ascitic 
generation  the  number  had  changed  to  42. 

Polyploidy  is  common  in  the  ascitic  cells,  tetraploid  bodies  appearing 
to  be  more  numerous  than  others. 

Vol.    15,  No.   3,   December    1954 


806  PKOCEEDINGS:   SYMPOSIUM 

Tetraploid  cells,  and  presumably  other  polyploids,  frequently  show 
synapsis  of  chromosomes  and  figures  which  indicate  segmental  exchanges 
resulting  from  either  crossing  over  or  translocation. 

Through  the  formation  of  multipolar  spindles,  tetraploid  cells  may 
divide  reductionally  to  produce  diploid  and  haploid  cells;  the  latter  are 
cable  of  survival  and  are  often  found  dividing  normally. 

The  teratoma  exhibits  the  mechanisms  for  genetic  variation  and  evolu- 
tion such  as  are  found  in  sexually  reproducing  free-living  organisms. 

References 

(1)  Fekete,   E.,  and  Ferrigno,   M.   A.:  Studies  on  a  transplantable  teratoma  of 

the  mouse.     Cancer  Res.  12:  438-440,  1952. 
(#)   Klein,  G.:  Comparative  studies  of  mouse  tumors  with  respect  to  their  capacity 

for  growth  as  "ascitic  tumors"  and  their  average  nucleic  acid  content  per  cell. 

Exper.  Cell  Res.  2:  518-573,  1951. 
(5)  Slizynski,    B.    M.:  A  preliminary   pachytene   chromosome   map   of  the   house 

mouse.     J.  Genetics  49:  242-245,  1949. 

(4)  :  Pachytene  analysis  of  Snell's  T(5;  8) a  translocation  in  the  mouse.     J. 

Genetics  50:  507-511,  1952. 

(5)  Griffen,  A.   B.:  A   late  pachytene  chromosome  map  of  the  male  mouse.     J. 

Morphol.,  1954.     In  press. 

(6)  Cohen,  I.:  Sudan  Black  B — a  new  stain  for  chromosome  smear  preparations. 

Stain  Technol.  24:  177-184,  1949. 

(7)  Bridges,  C.  B.:  The  vapor  method  of  changing  reagents,  and  of  dehydration. 

Stain  Technol.  12:  51-52,  1937. 

(8)  Hauschka,   T.   S.,  and  Levan,   A.:  Inverse  relationship  between  chromosome 

ploidy    and   host-specificity    of   sixteen    transplantable    tumors.     Exper.    Cell 
Res.  4:  457-467,  1953. 


Plate  48 

Figure  1. — The  right  ovary  of  the  mouse  in  which  the  teratoma  occurred.  The 
cortex  contains  normal  ovarian  follicles,  the  neoplastic  medullary  part  is  composed 
of  embryonal  tissue.     X  200 

Figure  2. — A  neoplastic  nodule  in  the  peritoneum  of  the  same  mouse  showing  heavily 
pigmented  epithelium,  neuroblasts,  connective  tissue  and  cartilage.     X  150 

Figure  3. — Another  neoplastic  peritoneal  nodule  of  the  same  mouse  showing  a  small 
cyst  lined  by  ciliated  epithelium  and  another  lined  by  stratified  squamous  epithe- 
lium.    X  200 

Figure  4. — Subcutaneous  growth  of  the  sixth  transplant  generation  showing  well 
vascularized  embryonal  tissue.     X  200 

Figure  5. — Subcutaneous  growth  of  the  fourth  transplant  generation  showing 
embryonal  tissue.     X  425 

Figure  6. — Another  subcutaneous  growth  of  the  fourth  transplant  generation  show- 
ing large  cells  resembling  trophoblasts.     X  425 


PLATE  48 


Fekete  and  Griff  en 
316263—54 — —38 


807 


808  proceedings:  symposium 


Plate  49 

Figure  7. — Smear  of  ascitic  fluid  showing  neoplastic  cells.  Stained  with  Wright 
stain.     X  500 

Figure  8. — Camera-lucida  drawings  of  diploid  metaphases  from  first  ascitic  genera- 
tions, showing  the  characteristic  38  chromsomes.      X  650 

Figure  9. — Diploid  metaphase  from  first  ascitic  generation;  Sudan  Black  smear 
photographed  with  phase  contrast.      X   1,900 

Figure  10. — Octoploid  metaphase  from  seventh  ascitic  generation,  showing  more 
than  160  chromosomes;  Sudan  Black,  phase  contrast.      X  1,600 

Figure  11.— Tetraploid  anaphase  from  seventh  ascitic  generation,  dividing  by  means 
of  a  tripolar  spindle  into  two  haploid  chromosome  clusters  (above)  and  one  diploid 
(below);  fresh  Sudan  Black  smear,  phase  contrast.      X   1,200 

Figure  12. — Tetraploid  metaphase  from  seventh  ascitic  generation  showing  three 
exchange  configurations  (arrows);  Sudan  Black  smear,  phase  contrast.      X  1,900 


JOURNAL  OF  THE  NATIONAL  CANCER  INSTITUTE,    VOL.   15 


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PLATE  49 


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809 


Discussion 

Dr.  Donald  F.  Jones,  The  Connecticut  Agricultural  Experiment  Station  and 
Yale  University,  New  Haven,  Conn. 

Long  before  the  rediscovery  of  Mendelism,  chromosome  aberration  had  been  asso- 
ciated with  abnormal  growth.  It  is  now  generally  considered  that  these  irregularities 
in  chromosome  behavior  are  the  result  and  not  the  cause  of  the  changes  in  growth. 
Visible  chromosomal  aberrations  have  obscured  minute  changes  that  initiate  the  first 
departure  from  normal.  So  far,  these  small  rearrangements  have  been  detected  only 
by  the  use  of  genetic  markers  that  produce  alterations  visible  in  single  cells.  Plant 
tissues  are  generally  more  favorable  material  in  which  to  see  these  changes,  as  plants 
have  cell  walls  and  many  genes  produce  nondiff usible  cell  products.  Breaks  and  realign- 
ments of  chromatin  have  been  shown  to  initiate  growth  changes  (1) . 

Recent  studies  by  McClintock,  Brink  and  others  have  shown  position  effects  result- 
ing from  migrating  pieces  of  chromatin  in  maize  and  other  plant  material.  These 
result  in  unstable  gene  effects  clearly  visible  in  variegated  colors.  So  far  these  mutable 
genes  have  not  been  associated  with  growth  changes.  Since  there  is  definite  evidence 
that  genes  control  growth  in  many  ways,  unstable  effects  would  be  expected  when 
migrating  chromatin  pieces  come  in  contact  with  growth-regulating  regions. 

The  excellent  cytologic  and  cytogenetic  studies  that  Dr.  Griffen  has  reported  give 
additional  evidence  for  the  importance  of  chromosome  rearrangements  in  abnormal 
growth.     The  detailed  chromosome  maps  will  be  an  important  aid  to  future  studies. 

Evidence  from  all  sources  combines  to  show  that  organisms  are  a  product  of  the 
genes,  the  cytoplasm  and  the  environment,  both  external  and  internal.  A  normal 
genome-plasmone-environ  interaction  is  dependent  upon  a  well  balanced  system  re- 
sulting from  countless  generations  of  selecting  under  all  possible  environmental  condi- 
tions. There  is  good  evidence  that  a  failure  of  this  genome-plasmone  relation  in  the 
normal  environ  is  the  beginning  of  species  separation  (2) . 

Abnormal  growth  that  is  irreversible  has  much  in  common  with  species  formation, 
since  it  is  a  new  kind  of  tissue  that  has  been  removed  from  normal  control.  There  is 
every  reason  to  expect  that  the  same  factors  involved  in  species  separation  will  also  be 
found  to  operate  in  the  initiation  of  tumor  formation. 

References 

(1)  Jones,  D.  F.:  Nuclear  changes  affecting  growth.     Am.  J.  Botany  27:   149-155, 

1940. 

(2)  :  The    cytoplasmic   separation    of   species.     Proc.    Nat.    Acad.    Sc.     37: 

408-410,  1951. 


811 

Journal   of   the   National    Cancer  Institute,    Vol.    15,   No.    3,   December    1954 


Session  VIII.  Genetic  Techniques  in 
the  Study  of  Cancer:  New  Approaches 
(cont.) 


Chairman,  Dr.  Clarence  C.  Little,  Director, 
Roscoe  B.  Jackson  Memorial  Laboratory,  Bar 
Harbor,  Maine 


Speaker:  Dr.  Lloyd  W.  Law 

Studies  on  Transformations  in  Leukemic  Cells  of  the  Mouse 
Discusser:  Dr.  Arthur  G.  Steinberg 

Speaker:  Dr.  Sewall  Wright 

Summary  of  Patterns  of  Mammalian  Gene  Action 


813 

Journal   of   the   National   Cancer   Institute,    Vol.    15,   No.    3,   December   1954 


Introduction:  Session  VIII 
Dr.  C.  C.  Little,  Chairman 

It  might  be  of  interest  to  mention  at  this  time,  one  or  two  principles  or  guiding 
"lines  of  thought"  that  I  have  found  helpful  as  reference  points  to  which  one  might 
from  time  to  time  possibly  return  during  studies  on  growth. 

The  first  of  these  principles  is  the  great  truth  that  animal  cells  in  the  organized  body 
possess  a  far  greater  latent  power  of  growth  than  the  student  of  a  particular  problem  of 
normal  or  abnormal  growth  is  likely  to  recognize.  The  most  eloquent  and  enduring 
effect  of  the  latent  power  of  growth  is  the  unbroken  history  of  the  protozoa,  which  has 
shown  us  that  the  division  rate  of  the  healthy,  free  animal  cell  has  not  been  slowed 
down  through  the  geological  periods.  The  germ  cells  of  mammals,  as  another  example, 
are  liable  to  be  underestimated.  Also  the  organs  that  give  rise  to  these  germ 
cells  should,  I  think,  be  viewed  with  more  respect  as  definite  centers  of  latent-growth 
power.  The  process  of  regeneration  is  an  extraordinary  evidence  that  many  cells 
otherwise  controlled  can,  when  challenged  by  trauma  or  by  other  experience,  rise  to 
the  occasion  without  the  addition  of  any  chemical  substance,  without  any  experimental 
change.  That  they  are  able  to  replace  structures  is  striking  evidence  of  the  latent 
power  of  growth.  The  ordinary  repair  processes  of  the  body  which  we  take  for  granted 
are  really  evidence  of  immense  latent  power  of  growth  in  the  differentiated  cells. 
Because  we  see  these  cells  so  definite,  so  diversified,  we  are  apt  to  think  of  them  as  being 
permanently  limited.  This  is  not  the  case.  In  other  words,  a  great  latent  power  of 
growth  is  submerged  in  the  higher  organism,  but  is  not  lost.  The  power  of  replacement 
of  tissue  is  still  less  sensational,  but  is  tremendously  impressive.  It  is  the  expression 
of  enormous  latent  power  of  growth.  A  very  unhappy  but  striking  demonstration  of 
latent  power  of  growth  is  the  origin  of  neoplasia  from  cells  in  differentiated  organisms. 
Latent  power  of  growth  is  therefore  one  great  principle  which  we  have  largely  neglected 
in  our  own  thinking. 

It  is  well  to  realize  that  what  we  call  the  "normal"  cell  of  the  mammal  is  really  quite 
"abnormal,"  from  the  point  of  view  of  successful  primitive  animal  cells.  The  protozoa 
are  much  more  typical;  cancer  cells  are  much  more  efficient.  They  outlive,  and  out- 
last the  differentiated  environment  around  them.  The  differentiated  cell  of  the 
mammal  is  "usual,"  but  it  is  not  a  normal  self-sufficient  biological  cell  in  comparison 
with  those  that  are  expressing  the  birthright  of  every  healthy  animal  cell,  which  is 
unlimited  growth. 

The  second  great  principle  is  that  of  balance  and  unbalance.  This  principle  has  also 
been  largely  overlooked  because  we  are  all  much  more  comfortable  intellectually  if  we 
deal  with  end  products  of  a  reaction,  rather  than  the  reaction  itself.  If  we  are  geneti- 
cists we  like  the  finished  character  or  the  chromosome;  if  we  are  pathologists  we  like 
to  have  microscopic  cells  to  study  and  classify.  We  all  like  definite  things,  because 
our  intellects  are  definite;  and  yet  the  whole  process  of  life,  the  characteristic  thing 
that  makes  it  baffling  and  bewildering,  is  that  it  is  a  dynamic  function.  It  is  something 
which  is  moving  and  it  tends  to  move  on  a  basis  of  balance.  It  can  move  around  and 
from  the  "center"  of  balance  to  meet  various  "challenges."  The  body  does  this 
normally  in  every  body  function  which  has  any  cyclic  phase  whatever.  Any  sequence 
of  buildup  and  destructive  phases  is  a  cycle.  It  is  a  question  of  balance,  and  the  cell 
must  come  back,  or  the  tissue,  or  the  organ,  must  come  back  to  a  "resting"  or  balanced 
stage  which  is  health,  which  is  normal,  which  is  in  a  very  real  sense  a  balance.  Now  the 
question  of  unbalance  becomes  critical.  When  the  pendulum  is  swung  so  far  by  a 
challenge  that  it  sticks  at  a  new  center  of  function,  a  new  center  of  balance  is  established. 
Sometimes  this  is  to  the  advantage,  and  sometimes  to  the  disadvantage,  of  the  com- 
plicated organism  in  which  the  shift  occurs.  We  know  that  we  can  upset  balance  in 
a  great  many  ways.     By  irradiation,  by  temperature  changes,  for  example,  we  can 

815 


Journal   of   the  National   Cancer   Institute,    Vol.    15,   No.    3,   December    1954 


816  proceedings:  symposium 

challenge  the  balance  of  the  organism.  We  also  know  it  is  challenged  by  various 
carcinogens  and  also  as  you  heard  today  by  the  process  of  hybridization. 

When  you  bring  together  in  a  cross,  two  germ  cells  that  are  alike,  or  as  nearly  alike 
as  you  can  get  them  by  the  process  of  inbreeding,  it  is  obvious  that  the  resulting 
systems  of  physiology  and  morphological  structures  worked  out  during  development 
tend  to  coincide.  The  two  parental  tendencies  maintain  a  balance,  repeat  each 
other's  rates  and  tempos,  so  that  the  organism  is  not  greatly  challenged.  But  when 
you  bring  together  two  systems  that  differ  in  tempo,  either  the  total  growth  tempo  or 
periodic  or  local  tempo,  they  challenge  each  other.  There  follows  an  increase  in 
tumors,  an  increase  in  mutation  after  hydridization.  This  may  have  an  important 
bearing  on  the  process  of  heterosis.  If  one  admits  a  latent  power  of  growth  bringing 
together  two  unlike  cell-potential  systems,  one  system  may  have  the  gene  to  "stop"  a 
certain  growth  process  at  a  certain  time  and  the  other  may  lack  it.  One  then  has 
produced  the  maximum  challenge  to  growth  control,  the  maximum  weakening  of  the 
"control"  of  this  latent  power  of  growth.  Heterosis  may  be  due  to  more  unbalances 
and  less  ability  to  stop  growth  processes  at  the  same  time  and  in  the  same  manner. 
One  gets  the  summation  of  the  growth  potentials  of  both  parents,  and  because  they 
don't  coincide  and  are  different,  they  compete  with  each  other.  Heterosis  might  then 
depend  upon  the  unbalance  between  the  tempos  of  the  control  of  growth  in  the  con- 
flicting systems. 

The  principle  of  unbalance  applies  all  the  way  down  the  size  scale  between  organs, 
between  tissues,  between  cells  in  the  tissue,  between  the  nucleus  and  cytoplasm  of  the 
cell,  between  chromosomes  within  the  nucleus,  and  between  genes,  within  the  chromo- 
some, and  finally  in  the  conformation  of  the  genes  themselves. 

We  have  wondered  why  the  same  agents  produce  neoplasia  and  mutation.  It 
would  seem  probable  that  it  is  the  unbalance  produced  by  these  agents  which,  in 
cellular  terms,  produces  a  break  in  control  within  the  cells  as  a  unit.  This  results 
in  more  rapid  cell  division.  In  the  gene,  the  agent  breaks  the  conformation  or  organi- 
zation of  the  gene,  producing  unbalance  and  later  the  establishment  of  a  new  balance 
in  the  chemical  structure  of  that  gene,  a  new  relationship  between  the  atoms.  It  is 
probable  that  mutation  is  not  neoplasia,  or  neoplasia  mutation,  but  that  both  processes 
depend  upon  establishment  of  a  new  balance,  one  intracellular,  and  the  other  intra- 
genic. This  consideration  of  latent  growth  potential  and  balance  versus  unbalance 
may  help  to  bring  certain  scientific  disciplines  together,  and  may  make  us  able  to 
exchange  a  great  deal  of  evidence  on  dynamic  processes  which  though  different  in 
their  end  products,  may  have  a  common  origin,  a  common  basic  philosophy  back  of 
them. 

This  afternoon  we  are  indeed  very  privileged  to  hear  two  papers.  The  first  of  these 
is  going  to  deal  with  transformations  in  leukemic  cells  by  Dr.  Lloyd  Law  of  the  National 
Cancer  Institute.  The  second  one  is  going  to  be  a  summary  and  digest  of  the  subject 
matter  of  this  symposium  by  Dr.  Wright.  First,  then,  I  should  like  to  call  on  Dr.  Law 
to  speak  on  the  transformations  of  leukemic  cells. 


Studies   on   Transformations   in    Leu- 
kemic Cells  of  the  Mouse  * 

L.  W.  Law,  National  Cancer  Institute,2  Bethesda, 
Md. 

Two  groups  of  compounds,  classed  as  antimetabolites,  have  been  used 
to  a  great  extent  in  the  treatment  of  acute  lymphocytic  leukemias  of 
children  and  in  laboratory  investigations  employing  this  morphologic 
form  of  leukemia  in  mice:  1)  folic-acid  antagonists,  particularly  those 
with  a  4-amino  substituent,  and  2)  purine  antagonists.  Folic-acid  antag- 
onists have  been  found,  in  our  laboratory,  to  be  antileukemic  agents  to 
each  and  every  lymphocytic  leukemia  tested.  On  the  other  hand,  purine 
antagonists  are  effective  antileukemic  agents  in  some,  but  not  other, 
leukemias. 

These  metabolic  antagonists  are  of  interest  for  several  reasons:  1)  They 
are  the  most  effective  of  known  antileukemic  agents,  2)  selectivity  of 
action  is,  in  particular  cases,  striking,  and,  3)  the  inhibitory  effect  can 
be  shown  to  be  of  a  competitive  nature,  in  certain  instances,  affording  a 
better  means  of  elucidating  the  metabolic  reactions  involved  in  therapeutic 
control. 

The  failure,  after  a  period  of  time,  to  achieve  remissions  in  patients 
with  A-methopterin  and  6-mercaptopurine  is  a  common  observation.  In 
the  experimental  leukemias  it  has  been  shown  that  these  failures  result 
from  the  development  of  transformations  in  the  population  of  leukemic 
cells  to  resistance  and/or  dependence  of  various  degrees. 

It  is  the  purpose  of  this  report  to  consider  the  experimental  production 
of  these  transformations,  some  characteristics  of  the  transformed  leukemic 
cells,  and  to  investigate  the  manner  of  origin  of  these  variant  cells.  Some 
basic  information  obtained  involving  the  mechanisms  of  these  phenomena 
will  be  given. 

Folic-Acid  Analogs 

Both  types  of  transformation,  to  resistance,  wherein  leukemic  cells 
grow  optimally  either  in  the  presence  or  absence  of  antagonist  or,  to 
dependence,  wherein  the  cells  grow  optimally  only  in  the  presence  of 
antagonist,  have  been  obtained  using  the  4-amino-substituted  folic 
analogs,  4-amino  PGA  (aminopterin) ,  4-amino-N10-methyl  PG  (A-methop- 
terin), 4-amino-9-methyl  PGA  (A-ninopterin),  and  4-amino-9,N10-methyl 
PGA  (A-denopterin) 3  (1-3).     See  text-figure  1  and  table  1. 

i  Presented  at  the  Symposium  on  25  Years  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  30, 1954. 

2  National  Institutes  of  Health,  Public  Health  Service,  U.  S.  Department  of  Health,  Education,  and  Welfare. 

3  All  folic-acid  analogs  have  been  supplied  through  the  courtesy  of  the  Lederle  Laboratories  Division,  American 
Cyanamid  Co.  Purine  analogs  have  been  supplied  by  Dr.  George  H.  Hitchings,  Wellcome  Research  Labora- 
tories. 

817 

Journal    of   the   National   Cancer    Institute,    Vol.    15,   No.    S,    December    1954 


818 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


LEUKEMIA  L  1210 


AN-R  AO-R 

— Ts~l Til 


(40)  (40) 

DISC.  DISC. 


TRANSFER 
GENERATION 


00 
65 
70 
100 
110 
116 
175 
181 


200 
205 


Text-figure  1. — Showing  origin  of  various  transformed  sublines  discussed  in  text: 
AN-R  (resistant  to  A-ninopterin) ,  AD-R  (dependent  on  A-denopterin) ,  AM-D 
(dependent  on  A-methopterin),  8-AG-D  (dependent  on  8-azaguanine) ,  AM-R 
(resistant  to  A-methopterin),  8-AG-R  (resistant  to  8-azaguanine),  6-M-R  (resistant 
to  6-mercaptopurine) .  Hatched  squares  represent  resistance;  darkened  squares, 
dependence.  The  control  line,  represented  by  circles,  has  been  carried  through  240 
consecutive  transfers  and  remains  sensitive  to  the  antifolic  and  antipurine  com- 
pounds. Certain  transfer  lines  have  been  discontinued  as  shown;  others  have  been 
carried  serially  through  the  number  of  transfers  designated  below  the  square. 

Table  1. — Transformations  in  leukemic  cells  of  several  transplantable  lines 


Line  of 

Strain  of 
mouse 

Type  of  transformation 

Antagonist  used 

leukemia 

Resistant 

Dependent 

L1210 

DBA 

+ 

+ 

A-methopterin 

L1210 

DBA 

+ 

A-methopterin 

L1210 

DBA 

+ 

+ 

A-denopterin 

L1210 

DBA 

+ 

— 

A-ninopterin 

L3054 

C58 

+ 

— 

A-denopterin 

HE8186 

A 

+ 

? 

A-methopterin 

L4946 

AKR 

+ 

4- 

A-methopterin 

L1210 

DBA 

+ 

+ 

8-Azaguanine 

L1210 

DBA 

+ 

— 

8-Azaguanine 

L1210 

DBA 

+ 

6-Mercaptopurine 

Variant  sublines  of  transplantable  acute  lymphocytic  leukemias  are 
obtained  usually  with  ease  following  consecutive  serial  transfers  in  mice 


Journal    of   the   National   Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


819 


receiving  either  1)  near-maximum  tolerable  levels  of  antagonist,  or  2) 
periodic  increases  in  the  level  of  antagonist.  Once  transformation  has 
been  achieved  the  variant  lines  retain  their  characteristics;  no  reversion 
to  sensitivity  has  been  observed  or  changes  from  one  type  of  transforma- 
tion to  another.  These  characteristics  are  maintained  in  the  absence  of 
antagonist  used  to  achieve  the  transformation.  The  character  is  thus 
shown  to  be  stable,  irreversible,  and  heritable. 

One  particular  subline  of  an  acute  lymphocytic  leukemia,  L1210,  the 
AM-D  subline,  dependent  on  folic  antagonists  for  optimal  growth,  has 
been  of  interest  in  determining  some  of  the  physiologic  characteristics  of 
these  transformed  cells. 

Characteristics  of  Leukemic  Cells  Dependent  on  Folic  Analogs  jor  Optimal 

Growth 

The  AM-D  variant  line  has  now  been  carried  through  more  than  200 
consecutive  serial  transfers  in  strain  DBA/2  mice.  Optimal  growth  is 
obtained  in  the  presence  of  4-amino-N10-methyl  PGA  (A-methopterin) , 
the  analog  used  to  develop  dependence.  The  behavior  of  these  trans- 
formed cells  is  shown  in  table  2  in  comparison  with  the  sensitive,  control 
line,  using  the  criterion  of  localized  growth  of  lymphomatous  tissue,  and 
in  table  3  the  behavior  in  the  development  of  generalized  leukemia  follow- 
ing transfer  of  a  standard  dose  of  leukemic  cells  in  Locke's  solution  (8  X  105 
cells) .  It  is  of  interest  to  note  that  even  at  a  dosage  level  of  A-methopterin 
as  high  as  12  mg.  per  kg.  (total  dose,  48  mg./kg.)  an  inhibition  of  localized 
growth  of  lymphomatous  tissue  of  50  percent  occurred  but  infiltration  into 
lymph  nodes  and  spleen  was  moderate.  This  indicates  at  least  a  50-fold 
increase  in  tolerance  to  this  antagonist,  since  it  requires  0.25  mg.  per  kg. 
(total  dose  1.0  mg./kg.)  of  antagonist  to  inhibit  localized  growth  of  the 
sensitive  cells  to  a  similar  degree. 

A  specific  cross-dependence  which  is  characteristic  for  all  such  trans- 
formations is  shown  in  table  4.  Any  4-amino-substituted  folic  antagonist 
is  capable  of  providing  for  optimal  growth.  Some  of  the  so-called  "weak" 
antagonists,  notably  N10-methyl  PGA  and  9-methyl  PGA,  though  lacking 


Table  2. — Dependence  in  leukemic  cells*  of  the  AM-D 
subline  oj  leukemia  LI 2 10 


Number 
of  mice 

A-methopterin 
dosage  (mg./kg.) 

Mean  weight 
lymphoma- 
tous tissue 
(mg.) 

Individual 

Total 

Dependent 

20 

78 
29 

12 

3 

None 

48 
12 

685.5 
833.3 
212.  7 

Sensitive 

20 
30 
55 

12 
3 
None 

48 
12 

0 

6.0 
1,190.  0 

•Transfer  generations  98-108  of  sensitive  cells  and  33-39  of  dependent  cells  used. 
Vol.   13,  No.  3,  December   1954 


820 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


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Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


821 


Table  4. — Comparative  sensitivity  of  A-methopterin-dependent  (AM-D)  and  sensitive 
leukemic  cells  to  several  antileukemic  agents 


Compound 


Dosage 
(mg./kg.) 


Individual        Total 


Inhibition  index* 


Sensitive     Dependent 


8-Azaguanine , 

a-Peltatin 

TEM 

6-Mercaptopurine 
A-ninopterin 

A-denopterin 

Amino-an-f  ol 

Aminopterin 


50 

5.0 

0.75 
75 

3 

3 
30 

0.2 


400 

15 

3.0 

525 

12 

12 

120 

0.8 


13 
06 
27 
10 
04 
01 
20 
01 


.  17 
.06 
.08 
.  10 
.94 

1.03 
.78 

1.0 


♦Inhibition  index- 


Mean  wt.  tumor-treated 


Mean  wt.  tumor-controls 

antileukemic  activity,  are  also  able  to  provide  for  approximately  50  percent 
optimal  growth  of  these  dependent  cells.  Such  antileukemic  compounds 
as  8-azaguanine,  aZpAa-peltatin,  TEM  and  6-mercaptopurine,  on  the  other 
hand,  show  independence  of  action,  in  being  carcinostatic  for  either  the 
dependent  or  sensitive  sublines.  Two  compounds,  cortisone  and  a 
purine  antagonist,  2,6-diaminopurine,  were  ineffective  in  either  the 
dependent  or  sensitive  subline  in  this  study. 

The  4-amino-substituted  folic  analogs  appear  to  inhibit  leukemic-cell 
growth  by  antagonism  of  folic  acid  (PGA)  or  citrovorum  factor  (CF) 
since  either  of  these  compounds  will  prevent  antileukemic  action  of  this 
class  of  agents  (4) .  Since  it  appears  that  CF,  on  a  weight  basis,  is  more 
effective  than  PGA  in  reversing  the  effects  of  folic  analogs,  it  was  of 
interest  to  study  the  effects  of  this  metabolite  on  the  growth  characteris- 
tics of  the  sensitive  and  dependent  lines  of  leukemia  L1210  and  to  deter- 
mine the  blocking  effect  of  CF  on  1)  the  antileukemic  action  of  A-methop- 
terin  on  sensitive  leukemic  cells  and  2)  the  optimal  growth-promoting 
capacity  of  this  antagonist  on  dependent  leukemic  cells.  It  may  be  seen 
by  reference  to  table  5  that  partial  reversals  of  both  the  antileukemic 
effect  in  the  sensitive  line  and  of  the  growth-promoting  effect  in  the 
dependent  line  were  obtained.  Folic  acid  (PGA)  was  also  found  to  give 
similar  reversals.  The  ratio  of  analog  to  metabolite  (PGA)  for  maximum 
effect  was  approximately  1:15  and  the  ratio  was  the  same  for  sensitive 
or  dependent  cells,  provided  that  PGA  was  always  given  prior  to  ad- 
ministration of  analog.  PGA  and  CF  were  found  not  to  influence  the 
growth  characteristics  of  either  sensitive  or  dependent  cells,  under  the 
conditions  of  these  experiments. 

No  changes  in  morphology,  antigenicity  or  transplantability  have  been 
observed  in  the  several  variant  forms  of  resistant  or  dependent  cells 
developed  in  leukemia  L1210  or  the  other  transplantable  leukemias  em- 
ployed, although  it  is  to  be  noted  that  specific  differences  in  growth  rates 
do  exist.  Leukemic  cells  of  the  sensitive  and  of  the  transformed  sublines, 
as  observed  in  localized  growth  or  in  infiltrations  into  spleen,  liver, 
lymph  nodes  or  in  the  peripheral  blood  are  morphologically  indistin- 


Vol.    15,  No.   3,  December   1954 


822 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


guishable.     Attempts  to   detect   antigenic   differences  by  complement- 
fixation  tests  have  been  unsuccessful. 

Possible  Mechanisms  of  Resistance  and  Dependence 

Resistance  of  leukemic  cells  to  folic  analogs  may  be  the  result  of  1)  a 
lowered  PGA  requirement  accompanied  by  a  much  greater  capacity  to 
convert  PGA  to  utilizable  CF,  2)  an  increased  ability  to  detoxify  the  PGA 
antagonist,  3)  a  more  efficient  utilization  of  CF  due  to  changes  in  permea- 
bility of  the  cell,  or,  4)  the  ability  of  transformed  cells  to  convert  antagonist 
to  PGA  or  CF  by  one  of  several  methods:  deamination,  demethylation, 
etc.  In  the  case  of  Streptococcus  jaecalis,  resistant  to  folic  antagonist,  it 
appears  that  this  strain  has  a  lowered  requirement  for  PGA  and  a  much 


Table  5. 


-Citrovorum  factor  (CF)   and  the  effects  of  A-methopterin  on  sensitive  and 
dependent  (AM-D)  leukemic  cells  of  leukemia  LI 2 10* 


Experiment 


Number 
of  mice 


Dosagef 


Daily 


Total 


Relative 

mean 
weights  f 


Dependent  (AM-D) 


A-methopterin 
A-methopterin 

+  CFJ 

CF 

Controls 

A-methopterin 
A-methopterin 

+  CFJ 

CF 

Controls 


"[Data  from  Law  (2).] 

tFor  convenience,  optimal  growth  in  both  groups  considered  as  1.0. 

JDosage  of  A-methopterin  in  mg./kg.:  citrovorum  factor  dosage  in  units  per  kg. 


71 
35 
31 


Sensitive 


greater  capacity  to  convert  PGA  to  CF  than  the  antagonist-sensitive 
strain  so  that  significant  inhibitions  of  growth  are  obtained  only  with  very 
high  concentrations  of  antagonist  (5).  Evidence  is  now  at  hand  to  indi- 
cate that  resistance  to  A-methopterin  by  S.  jaecalis  also  involves  an  altered 
permeability  of  the  cells  to  antagonists  which  greatly  reduces  the  acces- 
sibility of  the  susceptible  enzyme  system  to  antagonist. 

It  has  been  reported  recently  (6,  7)  that  a  folic-antagonist-resistant 
strain  of  S.  jaecalis  was  able  to  use  aminopterin  and  related  analogs  for 
growth,  in  contradistinction  to  the  sensitive  strain,  by  converting  the 
analogs  to  PGA  or  CF.  Similarly,  Kidder  et  al.  (8)  observed  that  the 
protozoon,  Tetrahymena,  was  able  to  use  aminopterin  (and  methopterin) 
in  growth  processes,  suggesting  that  this  organism  also  possessed  enzymes 
capable  of  deaminating  and  demethylating  these  antagonists.     It  has  been 


Journal    of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  823 

found  by  Nichol  et  al.  (5),  however,  employing  paper  chromatographic 
techniques,  that  the  folic-acid  antagonists,  which  are  presently  available, 
contain  sufficient  PGA  and  pteroic  acid  as  impurities  to  account  for  the 
apparent  utilization  of  these  analogs  by  S.faecalis  and  Tetrahymena  and 
that  the  explanations  given  above  are  not  valid. 

In  considering  explanations  for  the  dependence  characteristic,  it  is  ap- 
parent that  many  of  the  suggestions  pertaining  to  resistance  are  unlikely. 
For  example,  the  ability  of  leukemic  cells  to  detoxify  the  antagonist  or 
to  convert  antagonist  to  PGA  or  CF  would  explain  the  phenomenon  of 
resistance  but  not  dependence.  Preliminary  trials  by  Nichol  (9)  on  the 
ability  of  our  L1210  A-methopterin-dependent  (AM-D)  leukemic  cells  to 
convert  PGA  to  CF  indicate  that  the  dependent  leukemic  cells  are  less 
active  than  sensitive  cells  in  this  respect,  in  contrast  to  the  results  ob- 
tained with  S.  faecalis. 

Also,  it  is  quite  likely  that  differential  absorption  of  folic  analog  or  PGA 
is  not  related  to  the  dependence  phenomenon.  Preliminary  trials  by 
Skipper  et  al.  (10)  using  Cu-labeled  PGA  and  A-methopterin  show  C1* 
contents  of  sensitive  and  dependent  L1210  leukemic  cells  to  be  of  the 
same  order.  Recent  evidence  by  Kieler  and  Kieler  (11)  on  the  action  of 
A-methopterin  on  leukemic  cells  in  vitro  indicates  the  possibility  of  differ- 
ences in  intracellular  distribution  of  PGA  and  its  antagonists.  There 
are  no  definitive  data  available  at  present  relating  to  such  distribution. 

It  is  possible  that  the  dependent  leukemic  cells  described  here  have 
acquired  the  ability  to  use  folic  analogs  without  conversion  to  PGA  or  CF, 
employing  a  different  mechanism  for  the  synthesis  of  nucleic  acids  than 
that  suggested  as  occurring  normally.  Certain  preliminary  data  are 
available  relating  to  this  interpretation.  As  mentioned  previously,  it  is 
evident  that  the  4-aminopteroylglutamic  acids  act  by  competing  with 
PGA  or  CF.  It  appears  that  PGA  is  converted  to  CF  which  is  associated 
with  enzymes  concerned  with  transfer  of  single  carbon  units.  Folic  an- 
tagonists compete  with  formed  CF,  the  end  result  of  this  antagonism  is  an 
inhibition  of  nucleic  acid  synthesis  as  well  as  other  biochemical  processes. 
It  has  been  found  (12),  using  NaFormate-C14  (a  precursor  of  the  2-  and 
8-carbon  atoms  of  nucleic  acid  purines)  that  the  folic-acid  antagonist, 
A-methopterin,  inhibits  de  novo  synthesis  of  DNA  and  UNA  purines  of  the 
sensitive  cells  and  viscera  of  mice  bearing  these  cells  while  the  analog 
more  than  doubles  the  rate  of  de  novo  DNA  and  RNA  synthesis  in  the 
dependent  leukemic  cells,  profoundly  inhibiting  the  nucleic  acid  synthesis 
in  the  viscera  of  mice  bearing  these  transplanted  cells.  These  results  are 
shown  in  table  6. 

Similar  results  were  obtained  from  in  vitro  studies  of  sensitive  and 
dependent  leukemic  cells  from  the  same  sources.  A-methopterin  at  con- 
centrations of  0.01  mg.  per  ml.  strongly  inhibited  the  incorporation  of  C14- 
formate  into  the  protein  and  purine  pentose  nucleotides  of  sensitive  cells. 
A-methopterin  at  much  higher  levels  was  found  to  be  ineffective  on  the 
in  vitro  incorporation  of  P3204  in  sensitive  and  transformed  cells.  These 
results  indicate  that  the  effect  of  folic  analogs  is  not  an  over-all  inhibition 

Vol.    15,  No.  3,  December   1954 
316263—54 39 


824 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


Table    6. — Incorporation   of   Cu-formate   into    nucleic   acid   moieties   of   viscera   and 
A-methopterin-sensitive  and  dependent  (AM-D)  tumor  masses  of  leukemia  L1210* 


Leukemia 

Tissue 

Treatment 

Orig- 
inal 
tissue 

Specific  activities  (nc/mole  C) 

Group 
No 

DNA 

RNA 

Gua- 
nine 

Ade- 
nine 

Thy- 
mine 

Gua- 
nine 

Ade- 
nine 

1) 

Sensitive.  . 
Sensitive.  . 

Sensitive.  . 
Sensitive .  . 

Dependent . 
Dependent . 

Dependent . 
Dependent . 

Viscera . 
Tumor . 

Viscera . 
Tumor . 

Viscera . 
Tumor . 

Viscera . 
Tumor . 

None 

None 

6.9 
42 

5.6 
1.8 

7.3 
1.8 

4.9 
4.8 

171 
206 

26 
34 

113 
55 

15 
127 

280 
227 

16 
39 

99 
51 

10 
115 

ioo' 

4 
10 

"24' 
3 

51 

199 
309 

70 
54 

156 

85 

37 
162 

248 
308 

2) 
3) 

A-methopterinf . 
A-methopterinj . 

None 

90 
112 

140 

4) 

None 

A-methopterinJ . 
A-methopterinj . 

86 

35 

156 

*Data  from  Skipper,  Bennett  and  Law  {12). 

fA-methopterin  (3  mg./kg.)  immediately  before  HC14OONa  injection  on  7th  post- 
inoculation  days. 

JA-methopterin  (3  mg./kg.)  on  3d,  5th,  and  7th  days.     HC14OONa  on  7th  day. 

of  tissue  metabolism  (18),  but  is  probably  directed  against  limited  enzyme 
systems. 

It  should  be  pointed  out  that,  although  the  available  folic  antagonists 
contain  certain  contaminants  which  are  growth  factors,  it  is  not  neces- 
sarily a  complicating  factor  in  the  production  and  development  of  trans- 
formations to  resistance  and  dependence.  Contrariwise,  it  would  appear 
that  the  presence  of  PGA  in  the  antagonist  used  in  S.  faecalis  experiments 
aided  in  the  selection  of  resistant  mutants.  It  should  be  recognized,  how- 
ever, that  some  confusion  in  the  interpretation  of  results  has  arisen. 

Though  the  mechanism  of  dependence,  in  biochemical  terms,  is  far 
from  a  solution,  some  definite  leads  have  been  established.  Additional 
evidence,  which  tends  to  support  the  thesis  that  dependent  leukemic  cells 
employ  a  different  mechanism  for  the  synthesis  of  nucleic  acids  than  that 
suggested  as  occurring  normally,  is  to  be  found  in  the  use  of  weak  folic 
antagonists.  Although  N10-methyl  PGA  is  found  not  to  inhibit  the  growth 
of  sensitive  leukemic  cells  it  does  provide  for  approximately  50  percent 
optimal  growth  of  dependent  cells  (L1210  AM-D).  It  has  been  de- 
termined that  lymphomatous  tissue  or  spleen  obtained  from  (L1210 
AM-D)  mice  apparently  does  not  utilize  this  compound  as  a  precursor  for 
CF  (9). 

Purine  Analogs 

Since  the  report  of  Kidder  et  al.  in  1949  (14)  showing  the  cancerostatic 
effect  of  a  triazolopyrimidine  analog  of  guanine,  8-azaguanine,  on  certain 
adenocarcinomas  and  a  leukemia  in  mice,  this  compound  has  been  studied 
rather  extensively.  It  has  proved  to  be  a  useful  and  interesting  tool  in 
investigations  of  cellular  biochemical  reactions.  Although  specific  and 
definite  inhibitory  action  has  been  noted  for  a  fairly  wide  range  of  mor- 


Journal    of    the   National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


825 


phologic  forms  of  neoplasm  (15-17),  it  is  equally  clear  that  it  is  entirely 
inactive  against  other  neoplasms  in  the  mouse  and  rat  (15,  16).  This 
dichotomy  of  response  to  6-mercaptopurine  and  thioguanine  has  been 
observed  also  in  a  study  of  a  spectrum  of  acute  lymphocytic  leukemias  of 
the  mouse  (23).  An  explanation  for  response  or  lack  of  response  to  8- 
azaguanine  has  been  given  by  Gellhorn  and  co-workers. 

In  certain  acute  lymphocytic  leukemias  of  the  mouse  (17),  particularly 
the  transplantable  leukemia  L1210,  a  definite,  regular  and  reproducible 
inhibition  of  growth  results  following  parenteral  administration  of  the 
guanine  antagonist  at  nontoxic  levels  far  below  the  maximum  tolerable 
dose. 

Transformations  to  resistance  and  to  dependence  have  been  obtained 
in  leukemic  cells  of  Line  L1210  by  consecutive  serial  passage  in  DBA/2 
mice  receiving  near  MTD  levels  of  8-azaguanine  (see  text-fig.  1).  The 
dependent  line  (8-AG-D)  was  developed  from  the  100th  transfer  of  sensitive 
cells  and  a  resistant  line  from  the  175th  transfer  (18). 

Transformations  to  Resistance  and  Dependence  Using  8-Azaguanine 

Table  7  shows  the  characteristic  of  dependence  in  the  transformed  cells. 
Optimal  growth,  as  determined  by  localized  growth  of  lymphomatous 
tissue,  was  obtained  at  dosage  levels  of  8-azaguanine  as  high  as  150  mg.  per 
kg.  (total  dose,  1,200  mg.  per  kg.).  At  this  level  complete  inhibition  of 
growth  is  observed  in  the  sensitive  line.  Leukemic  death,  following 
intraperitoneal  transfer  of  cells,  also  strikingly  reflects  the  dependence 
characteristic.  The  mean  survival  time  of  mice  bearing  the  dependent 
subline  was  15.8  ±  0.45  days.  If  these  mice  are  given  8-azaguanine 
(75  mg.  per  kg.  X  8)  they  die  earlier,  12.1  ±  0.23  days  with  a  florid 
leukemia  (see  table  9).  In  contrast  DBA/2  mice  bearing  the  control, 
sensitive  subline  die  at  7.9  ±  0.06  days  and  when  given  8-azaguanine 
parenterally,  at  12.4  ±  0.20  days. 

This  dependent  subline  has  now  been  carried  through  100  transfer 
generations,  since  emergence  of  the  trait,  and  has  retained  its  characteristic 
response  without  evidence  of  reversion. 

Table  7. — Effect  of  8-azaguanine  on  sensitive  and  8-azaguanine- 
dependent  (8-AG-D)  lines  of  leukemia  LI 210* 


Transfer  line 

Number 
of  mice 

Dosage 
(mg./kg.) 

Tumor  wt.  at 
9  days 
(mg.) 

Daily 

Total 

Dependent 

f         24f 

183 
[         89 

150 
75 

None 

1,200 
600 

591.  1 

538.  6   ±  42.  1 

240.4  ±   21.8 

Sensitive 

f         10 

53 

[         54 

150 
75 

None 

1,200 
600 

0 

16.8  ±     2.2 

775.6  ±   30.2 

•[Data  from  Law  (18).] 

fTransfer  generations  107-144  of  the  sensitive  line  and  7-44  of  the  dependent  line  used. 


Vol.    15,  No.  3,  December   1954 


826 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


Cross-dependence  on  other  purine  analogs  has  been  demonstrated  in 
this  8-azaguanine-dependent  line.  6-Mercaptopurine  and  thioguanine 
(both  moderately  carcinostatic) ,  8-azaxan thine,  and  2,6-diaminopurine 
(ineffective  for  sensitive  leukemic  cells)  all  provide  for  50  percent  or  more 
optimal  growth  of  the  dependent  line.  The  folic  antagonist,  A-methop- 
terin  and  TEM  are  inhibitory  for  the  dependent  cells  as  well  as  the 
sensitive.  A  striking  sensitivity  to  folic  analogs  of  the  dependent  cells, 
as  well  as  of  other  transformations  produced  by  purine  analogs  has  been 
noted  and  will  be  discussed  later. 

L1210  leukemic  cells  transformed  to  resistance,  using  8-azaguanine, 
grow  optimally  in  DBA/2  mice  with  or  without  this  antagonist,  succumbing 
from  leukemia  at  10.1  ±0.15  days.  Cross-resistance  to  all  other  purine 
analogs  has  been  demonstrated  (see  table  8)  but  these  resistant  cells 
remain  sensitive  to  other  unrelated  compounds  such  as  folic  analogs  and 
TEM.  This  line  has  now  been  carried  through  85  consecutive  serial 
passages  in  DBA/2  mice,  retaining  its  characteristics. 

Resistance  to  an  Adenine  Antagonist,  6-Mercaptopurine 


The  adenine  analog,  6-mercaptopurine,  has  been  shown  to  act  as  a 
purine  antagonist  in  the  metabolism  of  Lactobacillus  casei  (19).  It  has 
also  been  shown  to  be  a  unique  inhibitor  of  Sarcoma  180  (20)  and  of 
certain  mammary  adenocarcinomas  (10).  Limited  clinical  trials  of  this 
compound  in  advanced  leukemia  of  children  have  been  encouraging  (21). 
As  with  8-azaguanine  this  compound  has  been  shown  to  give  definite, 
regular  and  reproducible  inhibition  of  leukemic-cell  growth  in  certain 
lymphocytic  leukemias  but  not  others  (22,  23).  Striking  increases  in 
survival  time  occur  in  leukemia  L1210.  The  mean  survival  time  of  mice 
bearing  the  sensitive  subline  of  leukemic  L1210  was  7.9  ±0.06  days  and 
an  increase  in  survival  of  87.3  percent,  to  14.8 ±0.18  days  was  obtained 
using  this  antagonist  within  the  total  dosage  range  of  250  to  1,200  mg.  per 
kg.  The  effects  obtained  at  the  higher  dosage  levels  (near  MTD)  were 
within  the  same  range  as  those  obtained  at  lower  levels,  300  to  600 
mg.  per  kg. 

A  resistant  line  was  procured  starting  with  the  200th  transfer  of  the 
sensitive  line.     The  resistance  characteristic  was  apparent  after  5  consecu- 

Table  8. — Influence  of  several  purine  antagonists  on  variant  sublines 
of  leukemia  LI 210* 


Antagonist 

Sensitive* 

8-AG-Df 

8-AG-RJ 

6-M-R§ 

8-Azaguanine 

6-Mercaptopurine 

2,6-Diaminopurine 

8-Azaxanthine 

+  (100%) 

+  (  80%) 

0 

0 

+  (  80%) 

-(100%) 
-(  60%) 
-<  50%) 
-(  50%) 
-(  40%) 

0 
0 
0 
0 
0 

0 
0 
0 

0 

Thioguanine 

0 

*  +=inhibition;  — -support  of  growth  (cross-dependence);  0=no  influence  (cross-resistance). 
t  8-Azaguanine-dependent. 
J  8-Azaguanine-resistant. 
§  6-Mercaptopurine-resistant. 

Journal    of    the   National   Cancer    Institute 


PROGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER  827 

tive  transfers  in  DBA/2  mice  receiving  75  mg.  per  kg.  X  7  dosage  levels. 
No  influence  of  6-mercaptopurine  could  then  be  demonstrated  in  this  line, 
test  mice  with  and  without  antagonist  dying  at  9.9  ±0.10  days.  Cross- 
resistance,  similar  to  that  observed  in  the  8-azaguanine-resistant  line,  was 
found  using  other  purine  analogs  (table  8)  but  sensitivity  to  TEM  and 
A-methopterin  was  evident.  A  considerably  increased  sensitivity  to  the 
folic  antagonist  was  characteristic. 

Mode  of  Action  of  Purine  Antagonists 

There  appears  to  be  little  doubt  that  the  purine  antagonist  8-azaguanine 
is  incorporated  into  nucleic  acids.  This  has  been  demonstrated  by 
Heinrich  et  al.  (24)  in  the  protozoon,  Tetrahymena,  by  Mitchell  et  al.  (25) 
for  viscera  of  mice  and  more  recently,  using  finer  techniques,  for  mouse 
viscera  and  tumor  tissue  by  Skipper  (10).  The  incorporation  is  for  the 
most  part  in  RNA  and  in  relatively  small  amounts.  There  appears  to  be 
little  doubt  also  in  Tetrahymena,  which  requires  preformed  guanine,  that 
the  guanine  antagonist  acts  strictly  as  an  antimetabolite;  physiological 
guanine,  or  its  nucleotide,  reversing  in  competitive  fashion  the  growth- 
inhibiting  capacity  of  8-azaguanine.  Evidence  for  a  clear-cut  metabolite- 
antimetabolite  relationship  in  mice  or  other  mammals  is  not  yet  at  hand. 
Guanine  or  guanylic  acid  has  been  shown  to  reverse  the  carcinostatic 
effect  of  8-azaguanine,  as  determined  by  leukemic  deaths  in  mice  (17,  26). 
In  our  own  observations  with  the  8-azaguanine-dependent  leukemia, 
guanylic  acid  regularly  interferes  with  the  growth-promoting  capacity  of 
8-azaguanine  more  effectively  than  another  ribotide,  adenylic  acid.  It 
has  not  been  determined  whether  this  is  done  competitively.  On  the 
other  hand,  Gellhorn  et  al.  (27)  have  observed,  in  rabbits  bearing  the 
Brown-Pearce  carcinoma,  that  the  carcinostatic  effects  of  8-azaguanine 
are  more  easily  reversed  by  the  nucleosides  and  nucleotides  of  adenine. 
This  suggests  that  8-azaguanine  is  converted  first  to  adenine  prior  to 
conversion  to  a  riboside. 

Extensive  attempts  in  our  laboratory  (23)  to  reverse  the  carcinostatic 
activity  of  6-mercaptopurine  by  physiological  purine  bases  have  been 
relatively  unsuccessful,  although  on  occasion  reversals  have  been  obtained 
particularly  with  hypoxanthine.  In  Lactobacillus  casei  any  of  the  four 
physiological  purines  will  prevent  the  inhibition  produced  by  this  com- 
pound, easily  and  competitively  (19).  The  negative  outcome  of  reversal 
experiments,  using  6-mercaptopurine,  does  not  necessarily  mean  that  its 
mode  of  action  is  different  in  these  experimental  animals  as  compared  with 
Lactobacillus  but  that  the  techniques  employed  in  the  complicated  system 
in  experimental  animals  are  not  adequate  or  that  some  metabolite  other 
than  the  four  physiologic  purines  must  be  supplied. 

Some  suggestive  preliminary  data  obtained  through  a  study  of  nucleic 
acid  metabolism  of  sensitive  and  dependent  (8-azaguanine)  leukemic  cells 
of  leukemia  L1210  are  at  hand.  8-Azaguanine  2-C14  has  been  found  to  be 
incorporated  into  the  RNA  of  sensitive  leukemic  cells  at  levels  100  times 
the  incorporation  of  this  purine  antagonist  into  dependent  cells  (27). 

Vol.    15,  No.  3,  December   19S4 


828  proceedings:  SYMPOSIUM  ON  25  YEARS  of 

This  may  be  considered  as  evidence  that  fixation  of  8-azaguanine  in  nucleic 
acids  may  be  related  to  the  carcinostatic  activity  of  the  compound.  Low 
levels  of  incorporation  of  the  purine  antagonist  2,6-diaminopurine  (as  2,6 
DAP-2-C14)  in  dependent  cells  has  also  been  found  (10)  paralleling  the 
results  with  8-azaguanine,  whereas  the  utilization  of  thymine  and  guanine 
(as  2-C14  products)  are  of  the  same  order  of  magnitude  in  sensitive  and 
dependent  cells,  a  relatively  low  incorporation  of  guanine  being  observed. 
Since  2,6-diaminopurine  is  known  to  be  readily  converted  to  nucleic  acid 
guanine  (28)  these  results  indicate  a  difference  in  capacity  of  the  two 
types  of  cells  to  utilize  this  compound  as  a  source  of  guanine. 

The  observations  discussed  here  concerning  metabolism  of  sensitive  and 
transformed  leukemic  cells  suggest  that  differences  in  nucleic  acid  metabo- 
lism may  exist.  Attempts  at  characterization  of  the  differences  are  now 
being  made. 

It  has  been  reported  by  Hirschberg  et  at.  (29)  and  by  Gellhorn  (SO)  that 
experimental  tumors  most  sensitive  to  8-azaguanine  have  a  low  concen- 
tration of  an  enzyme,  deaminase,  capable  of  converting  8-azaguanine  to 
8-azaxanthine,  a  noncarcinostatic  agent,  in  contradistinction  to  those 
tumors  not  influenced  by  the  compound.  Thus,  it  is  suggested  that  varia- 
tion in  response  to  neoplastic  tissues  results  from  their  ability  to  metabo- 
lize 8-azaguanine  to  an  inactive  form.  In  preliminary  studies  comparing 
deaminase  concentrations  of  Ll210-sensitive  and  8-azaguanine-dependent 
cells  this  does  not  appear  to  be  the  case,  since  enzyme  levels  obtained, 
although  relatively  high,  were  the  same  level  in  both  types  of  cell  (81). 
It  has  been  shown  also  (32)  that  a  combination  of  8-azaguanine  and  4- 
amino-5-imidazole  carboxamide  is  ineffective  in  increasing  survival  of  mice 
bearing  resistant  variants  of  lymphocytic  leukemia  L1210,  indicating  that 
factors  other  than  deaminase  content  distinguish  sensitive  from  resistant 
leukemic  cells. 

Origin  of  Resistance  in  Leukemic  Cells  to  Antimetabolites 

It  appears  extremely  likely  that  the  transformations  observed  in  leu- 
kemic cells  of  the  mouse,  to  resistance  or  dependence,  occur  spontaneously 
and  rather  generally  among  populations  of  leukemic  cells ;  and  the  role  of 
the  antimetabolite  is  merely  that  of  a  selective  agent  (S3) .  Increases  in 
resistance  have  been  shown  to  occur  in  a  discrete  stepwise  fashion  resem- 
bling in  this  respect  the  development  of  resistance  in  bacteria  to  penicillin 
(34).  This  observation,  along  with  results  obtained  by  the  "fluctuation 
test"  and  the  stability  of  variant  forms  in  the  absence  of  antagonist, 
would  seem  to  favor  the  assumption  that  mutation  and  selection  constitute 
the  mechanism  by  which  variant  cells  arise.  It  is  impossible  to  determine, 
with  these  somatic  cells,  whether  the  observed  transformations  are  genetic. 
In  Escherichia  coli,  strain  K12,  a  sexually  fertile  strain,  it  has  been  shown 
that  the  numerous  changes  to  resistance  and  dependence,  using  strepto- 
mycin, appear  to  have  arisen  by  change  at  a  single  gene  locus  (35) .  Thus, 
these  traits  in  bacteria  behave  as  if  controlled  by  allelic  forms  of  the  same 
gene  locus. 

Journal    of   the   National    Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  829 

Therapeutic  Considerations 

Two  different  approaches  to  the  chemotherapy  of  leukemia  which  appear 
to  be  of  considerable  significance  are  suggested  by  these  studies.  The 
first  relates  to  changes  in  sensitivity  to  folic  analogs  of  leukemic  cells  trans- 
formed through  the  use  of  purine  antagonists;  the  second  relates  to  a  use 
of  combinations  of  antileukemic  agents  in  an  attempt  to  suppress  the 
selection  of  spontaneous  mutations  to  resistance  and  dependence. 

All  three  variant  sublines  of  leukemia  L1210,  dependent  upon  8-aza- 
guanine  (8-AG-D),  resistant  to  8-azaguanine  (8-AG-R)  and  resistant  to 
6-mercaptopurine  (6-M-R)  show  a  striking  increase  in  sensitivity  to  the 
folic  antagonist  A-methopterin.  This  change  in  response  is  similar  to  that 
recorded  by  Elion  and  Hitchings  (19)  in  a  6  mercaptopurine-resistant 
strain  of  Lactobacillus  casei  which  shows  a  significantly  increased  require- 
ment for  folic  acid. 

Table  9  shows  this  striking  difference  in  response  of  the  three  variant 
lines  of  leukemic  cells  contrasted  with  the  usual  response  of  the  sensitive 
leukemia  {23). 

The  data  of  table  10  appear  to  provide  a  clear  rationale  for  the  use  of 
two  or  more  antileukemic  agents  acting  independently.  The  two  most 
effective  compounds  used  in  the  laboratory,  A-methopterin  and  8-aza- 
guanine have  been  shown  to  act  as  selective  agents  in  the  isolation  of 
resistant  and  dependent  mutants.  Each  also  has  been  shown  to  act  in- 
dependently of  the  other.  Since  there  appears  to  be  no  known  method 
for  decreasing  mutation  rates,  and  it  is  unlikely  that  the  host  can  alter 
the  process  of  spontaneous  mutation,  the  approach  appears  to  be  an 
attempt  to  suppress  the  selection  of  spontaneously  occurring  transforma- 
tions to  resistance  or  dependence.  The  principle  of  combined  therapy 
with  two  or  more  agents,  acting  independently,  has  been  used  success- 
fully in  infectious  diseases,  particularly  in  the  treatment  of  tuberculosis. 
If  the  frequency  of  mutation  to  resistance  of  a  cell,  bacterial  or  cancerous, 
to  drug  A  is  1  X  10~6  and  a  frequency  of  mutation  to  drug  B  is  1  X  10~5, 
only  one  cell  in  1011  will  simultaneously  develop  both  mutations.  Thus, 
doubly  resistant  mutants  have  a  negligible  probability  of  emerging  in  a 
sensitive  tumor  or  bacterial  population  in  the  presence  of  two  or  more 
effective  agents  which  exhibit  different  mechanisms  of  action.  It  may  be 
seen  from  the  table  that  these  two  antimetabolites  given  singly,  in  com- 
bination (one  followed  by  the  other),  at  dosage  levels  below  the  MTD 
are  very  effective  in  increasing  survival;  when  given  simultaneously,  in 
combination,  they  exhibit  a  striking  potentiation  of  effect.  Many  of 
these  mice,  though  receiving  a  standard  dose  (8  X  105  cells)  of  leukemic 
cells  live  beyond  a  90-day  period  and  show  no  evidence  of  leukemia. 
These  survivors,  in  all  probability,  represent  cases  in  which  all  or  most 
leukemic  cells  were  killed  and  doubly  resistant  forms  completely  sup- 
pressed (36). 

The  examples  discussed  here  of  transformations  to  resistance  and 
dependence  involve  changes  in  the  cells  of  the  neoplasm.  It  is  conceivable 
that  adaptation  could  occur  in  cells  of  the  host  so  that  a  drug  is  rendered 

Vol.    15,  No.  3,  December   1954 


830 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


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Vol.    15,  No.   3,   December   1954 


832 


PROCEEDINGS*.  SYMPOSIUM  ON  25  YEARS  OF 


ineffective.  The  development  of  an  efficient  hepatic  detoxication  mech- 
anism or  an  efficient  urinary  excretion  mechanism,  for  example,  may 
render  a  compound  ineffective  against  neoplastic  cells.  A  known  example 
is  that  by  Pollak  et  al.  {87) ,  who  presented  evidence  which  indicates  that 
refractoriness  of  leukemia  in  mice  to  potassium  arsenite  has  been  con- 
tributed by  the  host,  although  other  mechanisms  appear  quite  likely  in 
this  case. 

Recent  observations  indicate  the  development  of  resistance  in  neo- 
plasms other  than  leukemia:  Sarcoma  180  to  6-mercaptopurine  {20), 
the  Ehrlich  carcinoma  to  colchicine  {38),  and  the  Walker  rat  sarcoma  to 
an  acetyl-nitrogen-mustard  compound  {89) .  In  this  latter  case  resistance 
is  accompanied  by  a  lowered  peptidase  level.  The  compound  studied 
is  believed  to  act  after  hydrolysis  by  a  peptidase. 

A  recent  report  {40)  is  of  interest  for  it  indicates  the  development  of 
resistance  in  normal  cells  which  may  prove  to  be  of  a  distinct  advantage 
to  the  host.  Resistance  in  cells  of  the  intestinal  mucosa  of  the  rat  to 
aminopterin  appears  to  have  developed.  The  occurrence  of  marrow 
depression  and  mucosal  damage  in  patients  treated  with  folic  analogs  are 
of  serious  concern  to  the  clinician.  These  observations  suggest  the 
possibility  of  control  of  those  toxic  reactions. 

Summary 

Transformations  to  resistance  and  to  dependence  are  found  to  occur 
rather  generally  among  acute  lymphocytic  leukemias  of  the  mouse.  Two 
types  of  antimetabolites  have  been  used  in  the  development  of  these 
variant  forms:  folic-acid  antagonists  and  purine  antagonists. 

Leukemic  cells  resistant  to,  or  dependent  on,  folic  analogs  are  inhibited 
by  other  nonrelated  antileukemic  agents,  but  show  a  characteristic  cross- 
resistance  (or  cross-dependence)  to  all  other  4-amino-substituted  folic 
antagonists.  Folic  acid  and  citrovorum  factor  were  found  not  to  influence 
the  growth  of  variant  cells  but  both  compounds  reversed  the  growth- 
promoting  action  of  A-methopterin  in  dependent  leukemic  cells. 

Leukemic  cells  resistant  to,  or  dependent  on,  purine  antagonist  (8-aza- 
guanine  and  6-mercaptopurine)  show  cross-resistance  (or  cross-depend- 
ence) to  all  other  purine  analogs  tested,  but  other  nonrelated  compounds 
remain  carcinostatic.  A  striking  increase  in  sensitivity  to  folic  analogs 
of  all  antipurine  variants  was  observed. 

The  changes  discussed  are  shown  to  be  stable,  irreversible  and  heritable. 
No  reversions  to  sensitivity,  or  from  one  form  to  another,  have  occurred 
among  the  various  lines  carried  in  transplant. 

Experimental  evidence  favors  the  assumption  that  the  variant  cells 
arise  by  spontaneous  mutation,  which  occurs  constantly  in  populations 
of  leukemic  cells,  the  antimetabolites  acting  as  selective  agents  in  the 
isolation  and  propagation  of  the  variant  forms. 

Certain  preliminary  metabolic  studies  relating  to  mechanisms  of  resist- 
ance and  dependence  have  been  given. 

Some  considerations  of  therapeutic  interest  relating  to  suppression  of 


Journal    of    the   National    Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  833 

resistant  leukemic  cells  and  the  use  of  altered  sensitivity  to  folic  analogs 
have  been  discussed. 

References 

(1)  Law,  L.  W.:  Observations  on  properties  of  leukemic  cells  resistant  to  folic  acid 

antagonists.    J.  Nat.  Cancer  Inst.  11:  849-865,  1951. 

(2)  :  Response  of  a  resistant  variant  of  leukemic  cells  to  an  antagonist  of 

pteroylglutamic  acid.    Proc.  Soc.  Exper.  Biol.  &  Med.  77:  340-344,  1951. 

(3)  Law,  L.  W.,  and  Boyle,  P.  J.:  Development  of  resistance  to  folic  acid  antagonists 

in  a  transplantable  lymphoid  leukemia.  Proc.  Soc.  Exper.  Biol.  &  Med.  74: 
599-602,  1950. 

(4)  Burchenal,  J.  H.,  Babcock,  G.  M.,  Beoquist,  H.  P.,  and  Jukes,  T.  H.:  Pre- 

vention of  chemotherapeutic  effects  of  4-amino-N10-methyl-pteroylglutamic 
acid  on  mouse  leukemia  by  citrovorum  factor.  Proc.  Soc.  Exper.  Biol.  &  Med. 
74:  735-737,  1950. 

(5)  Nichol,  C.  A.,  Zakrzewski,  S.  F.,  and  Welch,  A.  D.:  Studies  on  the  resistance 

to  folic  acid  analogues  in  a  strain  of  Streptococcus  faecalis.  Proc.  Soc.  Exper. 
Biol.  &  Med.  83:  272-277,  1953. 

(6)  Hutchison,  D.  J.,  and  Burchenal,  J.  H.:  Observations  on  the  mechanisms  of 

resistance  to  folic  acid  antagonists.  (Abstract.)  Proc.  Am.  Assn.  Cancer  Res. 
1:  26,  1953. 

(7)  Broquist,  H.  P.:  Role  of  folic  acid  and  citrovorum  factor  in  metabolic  processes 

and  their  relation  to  cancer.    Texas  Rep.  Biol.  &  Med.  10:  953-960,  1952. 

(8)  Kidder,  G.  W.,  Dewey.  V.  C,  and  Parks,  R.  E.,  Jr.:  Growth  promotion  in 

Tetrahymena  by  folic  acid  analogs.  Proc.  Soc.  Exper.  Biol.  &  Med.  78:88-91, 
1951. 

(9)  Nichol,  C.  A.:  Studies  of  the  mechanism  of  resistance  to  folic  acid  antagonists 

by  leukemic  cells.     Cancer  Res.  14:522-526,  1954. 

(10)  Skipper,  H.  E.:  Progress  Report,  1953.    Southern  Research  Institute,  Birming- 

ham, Alabama. 

(11)  Kieler,  J.,  and  Kieler,   E.:  The  effect  of  A-methopterin  on  sensitive  and 

resistant  leukemic  cells  in  vitro.    Cancer  Res.  14:  428-432,  1954. 

(12)  Skipper,  H.  E.,  Bennett,  L.  L.,  Jr.,  and  Law,  L.  W.:  Effects  of  A-methopterin 

on  formate  incorporation  into  the  nucleic  acids  of  susceptible  and  resistant  leu- 
kemic cells.    Cancer  Res.  12:  677-679,  1952. 

(13)  Williams,  A.  D.,  Winzler,  R.  J.,  and  Law,  L.  W.:  The  effects  of  A-methopterin 

on  the  in  vitro  incorporation  of  P32(>4  into  normal  and  leukemic  mouse  tissues. 
Cancer  Res.  14:  135-138,  1954. 

(14)  Kidder,  G.  W.,  Dewey,  V.  C,  Parks,  R.  E.,  and  Woodside,  G.  L.:  Purine 

metabolism  in  Tetrahymena  and  its  relation  to  malignant  cells  in  mice.  Science 
109:  511-514,  1949. 

(15)  Gellhorn,  A.,  Engelman,  M.,  Shapiro,  D.,  Graff,  S.,  and  Gillespie,  H.: 

The  effect  of  5-amino-7-hydroxy-lH-v-triazolo  (d)  pyrimidine  (guanzaolo)  on 
a  variety  of  neoplasms  in  experimental  animals.  Cancer  Res.  10:  170-177, 
1950. 

(16)  Sugiura,  K,  Hitchings,  G.  H.,  Cavalieri,  L.  F.,  and  Stock,  C.  C:  The  effect 

of  8-azaguanine  on  the  growth  of  carcinoma,  sarcoma,  osteogenic  sarcoma, 
lymphosarcoma  and  melanoma  in  animals.    Cancer  Res.  10:  178-185,  1950. 

(17)  Law,  L.  W.:  Studies  on  the  effects  of  a  guanine  analog  on  acute  lymphoid 

leukemias  of  mice.    Cancer  Res.  10:  186-190,  1950. 

(18)  :  Resistance  in  leukemic  cells  to  a  guanine  analog,  8-azaguanine.    Proc. 

Soc.  Exper.  Biol.  &  Med.  78:  499-502,  1951. 

(19)  Elion,  G.  B.,  and  Hitchings,  G.  H.:  The  mechanism  of  action  of  6-mercapto- 

purine  as  revealed  by  microbiological  studies.  (Abstract.)  Proc.  Am.  Assn. 
Cancer  Res.     1:  13-14,  1953. 

Vol.    15,   No.   3,   December    1954 


834 


proceedings:  symposium 


(20)  Clarke,  D.  A.,  Philips,  F.  S.,  Sternberg,  S.  S.,  Stock,  C.  C,  Elion,  G.  B., 

and  Hitchings,  G.  H.:  6-Mercaptopurine:  effects  in  mouse  sarcoma  180  and  in 
normal  animals.     Cancer  Res.  13:  593-604,  1953. 

(21)  Burchenal,  J.   H.,   Karnofsky,   D.   A.,    Murphy,   L.,   Ellison,  R.   R.,  and 

Rhoads,  C.  P.:  Effects  of  6-mercaptopurine  in  man.  (Abstract.)  Proc.  Am. 
Assn.  Cancer  Res.  1:  7-8,  1953. 

(22)  Law,  L.  W.:  Resistance  in  leukemic  cells  to  an  adenine  antagonist,  6-mercapto- 

purine.    Proc.  Soc.  Exper.  Biol.  &  Med.  84:  409-412,  1953. 

(23)  Law,  L.  W.,  Taormina,  V.,  and  Boyle,  P.  J.:  Response  of  acute  lymphocytic 

leukemias  to  the  purine  antagonist,  6-mercaptopurine.  Ann.  New  York 
Acad.  Sc.     In  press,  1954. 

(24)  Heinrich,   M.  R.,   Dewey,   V.   C,   Parks,   R.  E.,  Jr.,  and  Kidder,    G.    W.: 

The  incorporation  of  8-azaguanine  into  the  nucleic  acids  of  Tetrahymena  geleii. 
J.  Biol.  Chem.  197:   199-204,  1952. 

(25)  Mitchell,  J.  H.,  Jr.,  Skipper,  H.  E.,  and  Bennett,  L.  L.,  Jr.:  Investigations 

of  the  nucleic  acids  of  viscera  and  tumor  tissue  from  animals  injected  with 
radioactive  8-azaguanine.     Cancer  Res.  10:  647-649,  1950. 

(26)  Goldin,    A.,     Greenspan,    E.     M.,    and    Schoenbach,    E.    B.:  Studies    on 

the  mechanism  of  action  of  chemotherapeutic  agents  in  cancer.  IV.  Relation- 
ship of  guanine  and  guanylic  acid  to  the  action  of  guanazolo  on  lymphoid 
tumors  in  mice  and  rats.    J.  Nat.  Cancer  Inst.  11:  319-338,  1950. 

(27)  Gellhorn,  A.,  Hirschberg,  E.,  and  Kells,  A.:  The  effect  of  purines,  nucleosides, 

and  nucleotides  on  the  carcinostatic  action  of  8-azaguanine.  J.  Nat.  Cancer 
Inst.  14:  935-939,  1954. 

(28)  Bendich,  A.,  Furst,  S.  S.,  and  Brown,  G.  B.:  On  the  role  of  2,6-diaminopurine 

in  the  biosynthesis  of  nucleic  acid  guanine.    J.  Biol.  Chem.  185:  423-433,  1950. 

(29)  Hirschberg,  E.,  Kream,  J.,  and  Gellhorn,  A.:  Enzymatic  deamination  of 

8-azaguanine  in  normal  and  neoplastic  tissues.    Cancer  Res.  12:  524-528,  1952. 

(30)  Gellhorn,   A.:  Laboratory  and  clinical  studies  on  8-azaguanine.     Cancer  6: 

1030-1033,  1953. 

(31)  :  Unpublished  data. 

(32)  Mandel,  H.  G.,  and  Law,  L.  W.:  The  effect  of  4-amino-5-imidazole  carboxamide 

on  the  carcinostatic  action  of  8-azaguanine.    Cancer  Res.    In  press,  1954. 

(33)  Law,  L.  W.:  Origin  of  the  resistance  of  leukemic  cells  to  folic  acid  antagonists. 

Nature  169:  628-629,  1952. 

(34)  Demerec,  M.:  Origin  of  bacterial  resistance  to  antibiotics.    J.  Bact.  56:  63-74, 

1948. 

(35)  Newcombe,    H.    B.,   and   Nyholm,    M.    H.:  The  inheritance  of  streptomycin 

resistance  and  dependence  in  crosses  of  Escherichia  coli.  Genetics  35:  603-611, 
1950. 

(36)  Law,  L.  W.:  Effects  of  combinations  of  antileukemic  agents  on  an  acute  lym- 

phocytic leukemia  of  mice.    Cancer  Res.  12:  871-878,  1952. 

(37)  Pollak,  M.  J.,  Kirschbaum,  A.,  and  Wagner,  J.:  Refractoriness  in  the  therapy 

of  transplanted  mouse  leukemia.     Cancer  Res.  13:  39-44,  1953. 

(38)  Lettre,      H.:    Eigenschaftsanderungen      von      Tumorzellen.       Zeitschr.      fur 

Krebsforschung  59:  568-580,  1953. 

(89)  Danielli,  J.  F.:  The  designing  of  selective  drugs.  Ciba  Foundation  Symposium 
on  Leukaemia  Research.    London,  J.  &  A.  Churchill,  Ltd.,  pp.  263-274,  1954. 

(40)  Vitale,  J.  J.,  Zamechek,  N.,  Hagsten,  D.  M.,  and  Di  Georgi,  J.: Effect  of 
antifolics  (aminopterin)  on  the  morphology  and  metabolism  of  gastro- 
intestinal mucosa.     In  press,  1954. 


Discussion 

Dr.  Arthur  G.   Steinberg,  Children's  Cancer  Research  Foundation,  Children's 
Medical  Center,  Boston,  Mass. 

You  see  before  you  a  very  sad  man.  Sad  because  he  has  lived  so  long  and  only  now 
has  he  had  the  opportunity  to  meet  Dr.  Little  and  his  group  here  at  Bar  Harbor. 
Sad  because  now  he  realizes  what  he  has  been  missing  over  the  course  of  the  years. 
I  hope,  somewhat  selfishly,  that  the  Jackson  Laboratory  will  have  a  long,  long  future 
so  that  I  can  catch  up  on  at  least  some  part  of  what  I  have  missed. 

As  I  listened  to  Dr.  Law's  presentation  I  became  more  and  more  enthusiastic  about 
it  as  long  as  I  could  forget  that  I  would  be  expected  to  discuss  his  paper.  Then  I 
became  resentful.  I  felt  that  by  his  excellent  presentation  of  a  series  of  very  well 
planned  and  executed  experiments,  he  was  complicating  my  task  immeasurably. 
How  can  a  discussant  show  his  mettle  when  he  agrees  with  the  speaker  and  has  no 
material  of  his  own  to  present?  I  must  congratulate  Dr.  Law  for  a  beautiful  job, 
beautifully  presented.  Certainly  one  cannot  take  serious  exception  to  Dr.  Law's 
interpretation  of  his  excellently  designed  experiments  or  to  his  suggestions  for  applying 
in  the  clinic  the  conclusions  he  has  drawn  from  them.  I  would,  however,  like  to  draw 
some  parallels  and  indicate  some  possible  cautions. 

I  wonder  if  Dr.  Law  really  insists  that  the  "  .  .  .  transformations  (to  resistance) 
are  .  .  .  irreversible."  The  design  of  his  experiments  was  such  that  reversion  to 
sensitivity  could  not  be  detected.  It  would  be  surprising  if  such  reversions  did  not 
occur  because,  in  general,  the  ability  to  undergo  reverse  mutation  appears  to  be  the 
rule  rather  than  the  exception. 

There  are  interesting  parallels  to  Dr.  Law's  work  in  the  studies  of  mutations  to 
resistance  to  radiation  and  to  antibiotics  in  Escherichia  coli.  Witkin  selected  50 
mutants  which  were  resistant  to  the  killing  action  of  ultraviolet  radiation.  Thirty-one 
of  these  were  also  resistant  to  penicillin  and  sulfathiazole;  8  were  resistant  to  penicillin 
but  not  to  sulfathiazole;  5  were  resistant  to  sulfathiazole  but  not  to  penicillin;  and  6 
were  resistant  to  neither.  The  data  make  it  extremely  likely  that  each  resistant  mu- 
tant was  the  result  of  a  single  mutation.  The  last  6  mutants  show  no  cross  resistance; 
the  remainder  show  cross  resistance  to  at  least  one  of  the  antibiotics.  Szybalski  and 
Bryson  showed  that  the  presence  or  absence  of  cross  resistance  to  antibiotics  depended 
to  some  extent  upon  the  method  of  selection.  For  example,  selection  for  resistance 
to  viomycin  increased  the  resistance  to  this  drug  tenfold  over  that  of  the  parent  strain 
and  simultaneously  increased  the  resistance  to  streptomycin  fivefold.  However, 
selection  of  a  strain  with  greater  than  a  thousandfold  increase  in  resistance  to  strep- 
tomycin did  not  result  in  increased  resistance  to  viomycin.  Many  other  similar  ex- 
amples may  be  found  in  their  paper.  We  must  consider  on  the  basis  of  the  fore- 
going examples  that  cross  resistance  to  antileukemic  drugs  may  be  observed  in  mice  if 
sufficient  tests  are  run. 

The  experience  of  clinicians  who  treat  children  suffering  from  acute  leukemia 
suggests  that  cross  resistance  may  not  be  infrequent  in  humans.  Some  patients  who 
suffer  a  relapse  after  experiencing  a  remission  induced  by  one  of  the  antifolic  com- 
pounds are  resistant  to  ACTH,  to  cortisone  and  to  the  antip urines;  others  are  not. 
Similarly  some  patients  who  suffer  relapse  after  a  remission  induced  by  ACTH  or 
cortisone  are  resistant  to  the  antifolics  and  to  the  antipurines,  while  others  may 
respond  to  these  drugs  and  so  on. 

Some  clinicians  stop  the  administration  of  the  antileukemic  agent  when  a  remission 
is  induced.  Such  patients  may  remain  in  remission  for  weeks  or  months  before  suffer- 
ing a  relapse.  I  emphasize  that  during  the  period  of  remission  the  patients  do  not 
receive  any  antileukemic  agents.  Nevertheless,  some  have  been  found  to  be  re- 
sistant not  only  to  the  agent  that  induced  the  remission  but  also  to  all  other  available 
agents. 

835 

Journal    of   the   National   Cancer  Institute,    Vol.    15,  No.    3,   December   1954 


836 


proceedings:  symposium 


Finally,  it  should  be  noted  that  a  considerable  proportion  of  children  with  acute 
leukemia  fail  to  respond  to  chemotherapy,  i.e.,  are  resistant  to  all  antileukemic 
agents. 

These  illustrations  indicate  that  cross  resistance  to  antileukemic  agents  does  occur 
in  humans.  Furthermore,  they  raise  the  possibility  that  resistance  in  humans,  in 
some  cases  at  least,  may  not  be  determined  by  mutation  in  the  same  way  that  it  is 
in  mice. 

I  have  one  more  point  to  offer  for  consideration.  In  humans  leukemia  is  treated 
in  the  host  of  origin,  while,  in  the  mouse,  leukemia  is  treated  in  a  host  which  has  ac- 
quired leukemia  via  the  transplantation  of  leukemic  cells.  I  wonder  what  bearing 
this  may  have  on  the  difference  in  response  of  human  leukemia  and  mouse  leukemia 
to  chemotherapy.     I  should  like  to  hear  the  comments  of  a  pathologist  on  this  point. 


Introduction  to  Dr.  Wright 

Dr.  C.  C.  Little 

We  move  on  to  the  next  paper  of  the  program  which  is  a  summary  of  patterns  of 
mammalian  gene  action  by  Dr.  Wright.  No  one  that  I  know  of  is  as  well  qualified 
to  give  the  last  formal  paper  here.  Dr.  Wright,  I  am  sure  in  the  opinion  of  most  of 
us  both  in  the  scope  of  his  mind  and  its  penetrating  power,  is  the  ranking  geneticist 
alive  today.  Dr.  Wright's  scientific  relationship  to  Dr.  Castle  and  his  membership 
on  our  Board  of  Trustees  seem  to  bring  together  the  component  elements  that  make 
it  particularly  happy  to  have  him  in  this  position  on  the  program  at  the  present 
time.     Dr.  Sewall  Wright  of  the  University  of  Chicago. 


Summary  of  Patterns  of  Mammalian 
Gene  Action  1 


Sewall  Wright,  The  University  of  Chicago, 
Chicago,  111. 


The  Gene  Concept 

There  has  been  so  much  questioning  of  the  validity  of  the  whole  concept 
of  the  gene  in  recent  years  (1)  that  it  seems  necessary  for  anyone  who 
proposes  to  discuss  patterns  of  gene  action  to  begin  with  a  statement  of 
his  understanding  of  what  a  gene  is. 

The  controversy  probably  arises  from  the  increasing  divergence  of  two 
primary  concepts.  One  of  these  has  been  that  of  the  gene  as  a  physio- 
logical unit,  concerned  with  a  single  elementary  physiological  process,  a 
unit  character.  Alleles  are  supposed  to  differ  only  quantitatively.  The 
other  concept  is  that  of  a  morphologic  unit,  a  component  of  the  chromo- 
some that  is  self-duplicating  with  respect  to  its  specificity,  and  that  is  not 
resolvable  into  two  or  more  self-duplicating  components  by  crossing  over 
or  chromosome  rearrangement.  Under  this,  alleles  are  alternative  forms 
of  a  gene,  due  to  mutation  within  its  structure,  irrespective  of  the  sort  of 
change  in  physiological  effect. 

To  a  considerable  extent  there  is  agreement  in  practice.  Morphologic 
alleles  usually  affect  the  same  character  and  often  in  an  apparently  graded 
fashion.  Heterozygotes  of  two  recessive  alleles  are  usually  within  the 
range  of  the  homozygous  recessives,  rather  than  of  the  dominant  type, 
contrary  to  the  case  of  heterozygotes  of  most  similar  nonalleles. 

It  has,  however,  become  increasingly  apparent  that  this  agreement  is 
far  from  complete.  Careful  study  usually  reveals  pleio tropic  effects  on 
single  characters  that  differ  qualitatively  rather  than  quantitatively.  In 
extreme  cases  the  major  effects  may  seem  to  have  nothing  in  common 
(e.g.  aristopedia  and  spineless,  dumpy-oblique  and  vortex  in  Drosophila 
melanogaster) .  Heterozygotes  reconstitute  type  in  these  extreme  cases 
and  may  even  do  so  where  the  effects  are  on  the  same  character  [e.g.  pro- 
tanopia  and  deuteranopia  in  man  (&)].  On  the  other  hand,  a  considerable 
number  of  cases  are  now  known  in  which  genes  that  are  not  alleles  in  the 
morphologic  sense,  since  separable  by  crossing  over,  have  such  inter- 
dependent effects  physiologically  that  heterozygotes  (of  recessives)  do 
not  reconstitute  the  dominant  type  [pseudoalleles  (8)]. 

It  has  seemed  to  me,  as  I  think  to  most  geneticists,  that  the  morphologic 
concept  must  continue  to  be  the  primary  one.     We  could  not  get  along 

»  Presented  at  the  Symposium  on  25  Tears  of  Progress  in  Mammalian  Genetics  and  Cancer,  Bar  Harbor,  Maine, 
June  30, 1954. 

837 

Journal    of   the   National   Cancer    Institute,    Vol.    15,   No.    3,   December    1954 


838  proceedings:  symposium  on  25  years  op 

very  well  at  the  level  of  the  organism  without  such  morphologic  terms  as 
pituitary,  adrenal,  ovary,  etc.,  even  though  we  learn  that  each  of  these  is 
heterogeneous  in  function  and  that  they  are  physiologically  interde- 
pendent. Similarly,  whatever  are  the  most  convenient  blocks  in  the 
linkage  systems  must  be  named  for  reference  irrespective  of  physiology. 

The  physiological  concept  suffers  from  the  lack  of  sharp  criteria.  No 
one  would  consider  that  mutations  that  are  far  apart  in  the  same  chromo- 
some are  alleles  merely  because  they  affect  the  same  character  (though 
they  may  be  alleles  of  duplicates).  Yet  position  effects  due  to  a  rearrange- 
ment that  brings  heterochromatin  close,  but  not  necessarily  very  close, 
to  a  locus  are  sometimes  treated  as  alleles.  There  seems  to  be  no  evidence 
of  any  change  of  pattern  at  the  locus  itself  and  thus  no  mutation  there, 
but  merely  a  fluctuating  effect  of  the  heterochromatin  on  its  functioning 
(and  often  on  that  of  its  allele  in  the  homologous  chromosome).  The 
situation  is  similar  with  the  remarkable  phenomena  described  by  McClin- 
tock  (4)  and  by  Brink  and  Nilan  (5)  in  connection  with  what  had  pre- 
viously been  considered  high  mutability  at  certain  loci  of  corn.  Their 
demonstration  of  a  hitherto  unrecognized  sort  of  agent,  that  resembles 
the  heterochromatin  of  Drosophila  in  inhibiting  the  functioning  of  genes 
while  in  their  proximity,  but  which  differs,  in  frequently  shifting  by 
itself  (under  certain  conditions)  from  one  position  in  the  genome  to 
another,  removes  this  phenomenon  from  the  category  of  gene  mutation. 
What  had  been  considered  a  single  mutable  gene,  has  been  resolved  into 
two  elements  that  require  separate  designations. 

The  genes  as  morphologic  units  are,  of  course,  always  provisional  in 
character.  This  leads  to  a  logical  difficulty  from  the  lack  of  assurance 
that  there  are  any  blocks  in  the  chromosomes  that  are  wholly  exempt  from 
crossing  over  or  dissociation  by  rearrangement,  short  of  single  nucleotides. 
Ephrussi-Taylor  (6)  has  indeed  shown  that  many  exchanges  can  occur 
between  DNA  molecules  of  pneumococcus  of  molecular  weight  about 
500,000  which  otherwise  one  would  tend  to  compare  to  single  genes. 
On  the  other  hand,  the  very  appearance  of  the  salivary  chromosomes  of 
Drosophila  and  of  the  prophase  chromosomes  of  many  species  especially 
in  electron  micrographs  (7)  indicates  a  heterogeneity  of  structure  in 
which  there  are  blocks  that  are  not  wholly  arbitrary.  More  significant 
is  chemical  evidence  [Mazia  (8)]  that  the  continuity  of  the  chromosomes 
depends  on  two  different  types  of  bond.  It  has  long  been  known  that 
Drosophila  chromosomes  may  be  dissolved  by  enzymatic  splitting  of 
peptide  bonds.  This  has  suggested  a  continuous  protein  backbone.  It 
has  now  been  shown,  however,  that  these  chromosomes  can  be  broken 
into  nucleoprotein  particles  some  4,000  A°  long  X  200  A°  wide  by  agents 
capable  of  binding  Ca  or  Mg.  ions  (e.g.  citrate)  followed  by  a  medium  of 
sufficiently  low  ionic  strength  (distilled  water)  to  permit  strong  electrical 
repulsion. 

It  is  thus  becoming  probable  that  the  genes  correspond  to  natural 
physical  units  (nucleoprotein  macromolecules) ,  bound  together  to  form 
chromosomes  by  divalent  ions.     Mazia  suggests  that  this  may  give  a 

Journal    of   the   National   Cancer   Institute 


PKOGKESS  IN  MAMMALIAN  GENETICS  AND  CANCER  839 

physical  basis  for  a  sharp  distinction  betweec  intragenic  phenomena  (point 
mutation)  and  intergenic  phenomena  (crossing  over,  rearrangement) . 

Even,  however,  if  the  continuity  of  the  chromosome  rested  on  only  one 
type  of  bond  throughout  its  length,  it  would  still  be  desirable  to  name 
regions  on  as  natural  a  basis  as  possible.  It  would  hardly  be  satisfactory 
from  the  physiological  standpoint  to  limit  discussion  to  the  effects  of 
mutations.  There  is  undoubtedly  differentiation  along  the  type  chromo- 
some and  different  specific  physiological  effects  must  be  attributed  to  the 
regions. 

Gene  Duplication 

The  most  important  thing  that  any  gene  can  do  is,  of  course,  to  dupli- 
cate itself.  The  most  plausible  hypothesis  with  any  substantial  factual 
basis  has  come  only  very  recently.  Watson  and  Crick  (9)  interpret  the 
X-ray  diffraction  pattern  of  DNA  as  indicating  a  two-strand  helix  in 
which  each  strand  is  a  polynucleotide  with  10  (or  11)  nucleotides  per  gyre, 
phosphate  and  pentose  backbone  on  the  outside,  and  cross-connections 
between  strands,  each  consisting  of  a  pyrimidine  linked  by  H  bonds  with 
a  particular  purine:  cytosine  with  guanine,  thymine  with  adenine,  in 
accordance  with  spatial  relations.  There  is  basis  for  specificity  in  the 
order  within  either  one  of  the  strands,  that  of  the  other  being  necessarily 
exactly  complementary.  They  suppose  that  under  certain  cell  conditions 
there  is  untwisting  and  separation,  followed  by  separate  recoiling  and 
synthesis  by  each  of  its  complement.  This  does  not  explain  the  relation 
to  the  polypeptide  chains  which  are  always  associated  with  the  DNA  in 
the  duplicating  chromosome  and  which,  as  noted,  seem  to  give  the  firmest 
basis  for  physical  continuity  within  the  nucleoprotein  macromolecule,  but 
is  not  inconsistent  with  there  being  such  a  relation. 

Antigens 

Among  the  most  important  contributions  of  the  genetics  of  mammals 
and  birds  are  those  from  the  study  of  antigens,  especially  of  red-blood 
corpuscles.  Each  antigenic  property  requires  in  general  only  a  single 
gene  for  its  determination,  and  the  red  cells  of  an  individual,  exhibit,  in 
general,  all  of  the  antigens  inherited  from  both  parents  without  dominance 
or  interaction.  As  noted  by  Haldane  (10),  we  seem  closer  to  the  one-to-one 
relation  of  preformation  here  than  in  any  other  known  character,  suggest- 
ing an  unusually  direct  relation  between  gene  and  character. 

On  the  other  hand,  no  other  class  of  loci  is  known  which  exhibits  such 
complex  systems  of  multiple  alleles,  each  determining  a  different  array  of 
antigenic  properties.  The  most  complex  known  seems  to  be  the  B  locus 
of  cattle  [Stormont  et  al.  (11)]  in  which  some  80  alleles  determine  various 
patterns  of  21  antigenic  properties,  each  demonstrable  by  its  appropriate 
test  serum.  Similar  cases  are  known  in  many  other  organisms  including: 
man  (12),  fowl  (18),  and  duck  (14,  15). 

Those  who  make  physiological  considerations  primary  in  the  definition 
of  the  gene  would  split  these  loci  up  into  at  least  as  many  loci  as  there  are 
sets  of  antigenic  properties  that  are  not  alternative  in  occurrence.     Thus 

Vol.    15,  No.   3,   December   1954 
316263—54 40 


840 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


M  and  N  in  man  seem  to  be  strictly  alternative  and  so  are  S  and  s  but  the 
combinations  MS,  Ms,  NS  and  Ns  all  occur  and  are  taken  to  indicate  at 
least  two  loci,  even  though  no  crossing-over  has  been  detected.  The 
frequencies  of  these  four  types  in  populations  are  often,  however,  very  far 
from  those  of  random  combination.  It  was  shown  many  years  ago  by 
Robbins  (16)  that  any  initial  deviation  from  random  combination  falls 
off  in  each  generation  of  random  mating  by  the  mean  of  the  recombination 
percentages  in  oogenesis  and  spermatogenesis.  The  observed  deviation 
from  random  combination  thus  implies  very  close  linkage,  very  recent 
origin  from  mixtures  of  populations  of  different  origin,  or  very  strong 
local  selection  for  certain  combinations.  In  the  case  of  the  II  series  in 
man  in  which  subdivision  into  at  least  C,  D  and  E  components  has  been 
suggested  (17)  the  frequencies  of  combinations  are  almost  as  far  as  possible 
from  random  throughout  Western  Europe.  In  Czechoslovakia,  for  ex- 
ample, there  is  39.6  percent  CDe,  16.9  percent  cDE,  40.0  percent  cde, 
each  two  steps  from  each  of  the  others,  and  only  3.5  percent  of  all  other 
combinations.  The  situation  is  closely  similar  in  England  (18).  Even 
if  this  population  traced  to  mixture  of  three  populations  absolutely  hom- 
allelic  in  CDe,  cDE  and  cde,  respectively,  as  recently  as  300  generations 
ago,  which  is  very  improbable  in  view  of  the  blood  groups  in  the  rest  of  the 
world,  crossing-over  must  be  supposed  to  be  less  than  0.01  percent  per 
generation  to  account  for  the  rarity  of  the  supposed  crossovers.  If, 
instead,  there  is  equilibrium  between  crossing-over  and  a  possible  but 
unknown  selection  against  the  recombinants,  crossing-over  must  be  less 
than  10  percent  of  the  selective  differential. 

The  hypothesis  of  multiple  loci,  moreover,  does  not  account  for  the  many 
cases  of  asymmetrical  occurrence  of  combinations.  In  the  human  ABO 
series,  the  allele  Ai  determines  two  antigenic  properties  of  which  one 
never  seems  to  occur  except  in  association  with  one  that  occurs  by  itself 
in  A2.  There  are  many  such  cases  in  cattle.  Thus  properties  B  and  G 
are  found  frequently  in  all  of  the  possible  combinations  (BG,  Bg,  bG, 
bg)  suggesting  separate  loci  but  K,  which  is  very  common  in  BGK  seems 
never  to  be  found  otherwise. 

It  is,  of  course,  very  probable  that  some  supposedly  single  loci  will  later 
be  shown  to  be  compound,  in  view  of  the  frequency  of  apparent  gene 
duplication  indicated  by  the  known  cases  of  pseudoalleles.  The  criteria 
for  splitting  loci  must,  however,  be  separation  by  crossing-over  or  rear- 
rangement, not  quality  of  effect. 

The  simplest  hypothesis  is  that  the  antigenic  properties  reflect  overlap- 
ping aspects  of  a  single  varying  macromolecular  pattern,  imposed  on  the 
antigenic  substrate  (mucoid  carbohydrate  in  some  cases  at  least)  by  a 
corresponding  pattern  in  the  gene  (19,  12,  15,  and  11). 

It  should  be  added  that  even  in  this  class  of  characters  the  relation  of 
gene  to  character  is  not  always  one  to  one.  Irwin  and  his  associates  have 
shown,  especially  in  hybrids  of  pigeons  and  doves,  that  some  of  the 
antigens  which  one  would  expect  to  find  inherited  from  the  parents  are 
absent  and  in  their  place  are  new  hybrid  antigens. 


Journal    of    the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  841 

Reaction  Against  Grafts 

Mouse  genetics  has  contributed  especially  to  the  understanding  of 
another  aspect  of  gene-controlled  specificity,  viz.,  the  reaction  against 
transplanted  tissues.  The  subject  had  two  principal  roots.  Leo  Loeb 
(20) }  who  began  its  study  in  the  1890's,  found  that  success  of  a  graft  was 
correlated  with  closeness  of  relationship:  100  percent  success  in  the  case 
of  autotransplants,  a  usually  hostile  reaction  and  little  success  between 
Utter  mates  (syngenesio transplants) ,  a  more  violent  reaction  between 
unrelated  members  of  the  species  (homoio transplants) .  He  considered 
that  he  was  dealing  with  the  heredity  of  protoplasmic  individuality  but 
arrived  at  no  detailed  genetic  interpretation.  Little  and  Tyzzer  (21) 
obtained  definite  Mendelian  results  which  indicated  that  susceptibility 
depended  on  the  simultaneous  presence  of  a  considerable  number  of 
dominant  genes  just  as  the  agouti  color  of  the  wild  mouse  depends  on  the 
simultaneous  presence  of  numerous  dominant  color  factors.  This  first 
formulation  implied  that  susceptibility  itself  was  the  significant  character 
that  was  involved,  but  later  Little  and  Strong  (22)  combined  the  Mendelian 
description  with  the  concept  of  hereditary  specificity.  The  revised  theory 
was  that  each  pertinent  gene  determines  something  that  tends  to  induce  a 
hostile  reaction  in  a  host  lacking  it.  This  principle  was  found  to  apply  to 
the  success  or  failure  of  transplants  of  normal  tissues  as  well  as  of  tumors 
[Little  and  Johnson  (23);  Loeb  and  Wright  (24)].  Gorer  (25)  clinched 
this  viewpoint  years  later  by  showing  that  one  of  the  genes  (H-2)  con- 
cerned in  resistance  to  tumor  grafts  was  the  same  as  one  that  determined  a 
red-cell  antigen. 

Rejection  or  acceptance  of  a  tumor  graft  has,  however,  turned  out  to  be 
a  much  less  reliable  indicator  of  the  presence  or  absence  of  "histocom- 
patability"  genes  than  serologic  reactions  of  red  cells.  If  there  were  no 
complications,  the  number  of  such  genes  (x)  in  a  donor  strain,  absent  in 
the  host,  could  be  estimated  by  finding  the  proportion  of  susceptibles  in 
F2,  or  a  backcross,  and  equating  to  (3/4) x  or  (1/2) x,  respectively.  It  soon 
became  apparent,  however,  that  different  tumors  from  the  same  strain  or 
even  the  same  mouse,  might  give  very  different  results  on  the  same  F2  or 
backcross  individuals.  One  of  the  most  thorough  studies  of  this  point 
was  that  of  Cloudman  (26)  who  used  8  tumors  from  the  same  closely 
inbred  strain  (Bagg  albinos)  in  tests  of  Fi,  F2  and  backcrosses  to  another 
such  strain  (dilute  browns) .  His  application  of  the  above  formulae  gave 
results  that  indicated  that  one  of  the  tumors  had  about  12  genes  foreign 
to  the  dilute  browns,  while  the  others  indicated  only  4,  2  or  1.  The  results 
were  consistent  for  each  tumor,  except  for  one  that  shifted  from  two  to 
one  apparent  factor  in  the  course  of  the  investigation.  It  seems  clear 
that  something  tends  to  happen  in  the  tumor  cell  that  prevents  most  of  the 
antigens  from  inducing  a  sufficiently  hostile  reaction  to  lead  to  rejection. 
This  could  be  either  loss  of  the  genes  or  overriding  of  their  effects.  Somatic 
mutation  is  not  a  plausible  answer  where  so  many  homozygous  dominant 
genes  are  involved. 

There  are  various  possibilities  with  respect  to  the  number  of  genes 

Vol.    15,   No.    3,   December    1954 


842 


PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


affected.  Cloudman's  calculations  were  based  on  the  assumption  that 
genes  are  either  wholly  effective  or  wholly  ineffective.  Another  possi- 
bility is  that  all  are  equally  reduced  in  the  probability  of  preventing 
acceptance.  Finally,  penetrance  may  be  reduced  unequally.  The  last 
seems  to  have  been  the  case.  Snell  and  Higgins  {27)  have  shown  that 
there  is  one  gene  (H-2dk)  that  is  much  the  strongest  and  was  the  one  that 
remained  effective  in  the  Cloudman  tumors  in  which  the  apparent  number 
was  reduced  to  one. 

The  contrast  between  the  expectations  under  different  hypotheses  can 
be  illustrated  by  considering  one  of  Cloudman's  experiments.  He  grafted 
the  same  two  tumors  (II,  V)  on  each  of  105  F2  mice.  The  observed 
pattern  of  acceptances  (+)  and  rejections  (— )  was  as  follows:  Expecta- 
tions are  given  under  three  hypotheses. 


Expectations 

V=AB            11=  V= 

11=  V= 

V 

// 

Observed 

11= AC             A  (BC) 

A  (BCD) 

+ 

+ 

50 

44.  2                   49.  6 

46.  8 

+ 

— 

8 

14.  8                    7.  9 

10.7 

— 

+ 

7 

14.  8                     7.  9 

10.7 

— 

— 

40 

31.  2                   39.  6 

36.8 

105 


105.0 


105.0 


105.0 


The  percentage  of  acceptances  was  nearly  the  same  for  both  tumors 
(54.7%  and  52.6%),  or  approximately  (3/4) 2=  56.25  percent,  implying 
only  two  dominant  factors  if  penetrance  is  an  all-or-none  matter.  Under 
this  hypothesis,  one  factor  must  clearly  be  in  common  and  the  other 
different  to  account  for  the  strong  but  not  perfect  correlation.  The 
observed  numbers  do  not,  however,  agree  at  all  well  with  expectation  on 
this  basis  (probability  from  x2  less  than  0.01).  If  however,  one  factor 
(A,  actually  SnelTs  H-2dk)  has  100  percent  penetrance,  and  two  or  three 
others  are  assumed  to  have  the  same  lower  degree,  and  the  rest  none,  we 
obtain  expectations  that  agree  almost  perfectly  (B,  C  both  with  58 
percent  penetrance)  or  sufficiently  well  (B,  C,  D  all  with  40  percent 
penetrance,  probability  0.20). 

The  mode  of  calculation  in  the  former  case  is  shown  below. 


** 


ABC. 


ABcc 
AbbC 
Abbcc 


Frequency  +      + 
27/64              1 

9/64           p2 
9/64           p2 
3/64           p* 

16/64             0 

Probability 

+      - 
or  —      + 

0 
2p  (1-p) 

2p  a-p) 
P2  (1-P2) 

0 

0 

(1-p)2 
(1-P)2 
(1-P2)2 

1 

Probability  arrays  of 
reactions 

No  hostile  reaction  from 

A,  B,  or  C. 
(pC+  +  (1-P)  c-)» 
(pB++  (1-p)  B-)* 
[p>  (B+O)  +  (1-P2) 

(BO-P 
Hostile  reaction  from  A 

1       J2 

105 

15 
105 

40 
105 

All  ABC  mice  are  expected  to  accept  both  tumors.     It  is  assumed  that 
C  fails  to  produce  a  hostile  reaction  in  cc  mice  in  the  proportion  #,  but 


Journal    of   the   National   Cancer    Institute 


PKOGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  843 

does  so  in  the  proportion  (1—  p)  in  each  tumor,  independently  of  the 
other.  The  same  array  of  probabilities  is  assumed  for  the  action  of  B 
in  bb  mice.  If  both  B  and  C  are  absent  in  a  mouse,  the  chance  of  failure 
of  penetrance  of  both  is  p2  and  of  penetrance  of  at  least  one  (1—  p2). 

The  equation  — -  -| — —~-  +  -rrr  =  tttf  from  the  cases  of  double  accept- 
64         64  o4        105 

ance  leads  to  the  solution  p  =  .418  indicating  about  58  percent  penetrance 

of  either  B  or  C  by  itself. 

The  recognition  that  penetrance  is  often  incomplete  at  the  level  of  the 

observed  character  (rejection  or  acceptance)  has  opened  the  way  to  studies 

of  the  physiological  factors  involved  in  penetrance,  which  are  being 

actively  pursued  here  under  the  leadership  of  Dr.  Snell. 

Elementary  Metabolic  Processes,  Enzymes 

There  is  another  class  of  characters  which  approaches  the  antigens  in 
the  apparent  directness  of  its  determination  by  genes.  This  is  the  class 
of  elementary  metabolic  processes,  and  back  of  this  presumably  (and  in 
some  cases  demonstrably)  the  specific  enzymes.  A  distinction  must  of 
course  be  made  between  those  cases  in  which  the  molecular  pattern  of 
the  enzyme  is  altered  by  an  allelic  change  and  those  in  which  enzyme 
activity  is  merely  modified  by  gene-controlled  changes  in  the  conditions 
in  the  cell. 

Mammalian  genetics  made  the  earliest  contribution  to  this  field  in  the 
work  of  Garrod  {28)  on  "Inborn  Errors  of  Metabolism"  in  man.  Many 
other  cases  have  been  studied  since.  Dr.  Sawin  of  this  Laboratory,  for 
example,  in  association  with  Glick  {29)  demonstrated  that  presence  or 
absence  of  atropinesterase  in  the  blood  of  rabbits  depends  on  a  single 
locus.  Dr.  Chase  has  described  here  differences  in  insulinase  in  strains 
of  mice,  but  in  this  case  the  mode  of  inheritance  seems  to  be  complex. 

The  metabolic  processes  of  mammals  are  difficult  to  isolate.  For  a 
systematic  analysis  of  the  genetics  of  metabolic  chains  we  must  turn  to 
the  studies  of  micro-organisms,  initiated  by  those  of  Beadle  and  Tat um 
on  Neurospora.  These  have  borne  out,  with  some  qualifications,  the 
concept  of  a  one-to-one  relation  between  gene  and  elementary  metabolic 
step,  on  a  grand  scale. 

The  usual  one-to-one  relation  of  genes  to  antigens  and  enzymes  has 
suggested  that  in  these  cases  the  genes  either  determine  directly  the  pat- 
tern of  synthesis  of  these  macromolecules  or  at  least  impose  a  specific 
steric  pattern  on  generalized  polysaccharide  or  protein  molecules,  in 
either  case  in  direct  relation  to  their  own  patterns  as  templates. 

The  physiologist  traces  the  properties  of  cells  to  the  array  of  specific 
enzymes  that  they  contain  and  the  stimuli  which  they  receive  from  their 
cellular  and  humoral  environments.  Differences  in  cellular  environment 
may  trace  in  part  to  differences  in  external  environment,  but  depend 
primarily  on  the  products  of  other  cells  and  hence  trace  to  enzymes  and 
antigens  produced  in  these  cells. 

Vol.    15,  No.   3,   December    1954 


844 


proceedings:  SYMPOSIUM  ON  25  YEARS  of 


Such  physiological  considerations,  as  well  as  those  of  degrees  of  genetic 
complexity,  suggest  that  primary  action  of  a  gene  (including  self-duplica- 
tion) may  be  wholly  restricted  to  imposing  a  reflection  of  its  own  specific 
pattern  on  various  sorts  of  macromolecules. 

Secondary  Characters 

Text-figure  1  is  intended  to  bring  out  diagrammatically  these  ideas  on 
the  relation  of  genes  to  characters  of  various  sorts  (30-32).  At  each 
level,  physiology  is  registered  in  structure  which  gives  a  firm  basis  for 
physiology  or  behavior  at  the  next  higher  level.  There  is  no  fundamental 
difference  between  the  relation  of  genes  to  hereditary  extraorganic  struc- 
ture (such  as  that  of  the  webs  characteristic  of  each  species  of  spider), 
and  to  organic  structure.     Both  are  indirect. 


Behavior 


Extraorganic 
Structure 


External 
Environment 


Histogenesis 


Genome 


Gene  Duplication 


r Homeostasis  vs.  Disease 


Organic 
Structure 


Cell 
Constitution 


Morphogenesis 


Cell 
Product 


\ 

N       ) 

\     t 

V 

Metabolism 


Genome 


Mocromolecular    Pattern     Na 


Enzyme 


Antigen 


Text-figure  1. — Diagram  of  relations  of  genome    and    external   environment   to 
observed  characters  at  various  levels  of  organization. 


Under  this  viewpoint,  we  leave  the  field  of  gene  action  (strictly  speaking) 
with  the  determination  of  macromolecular  pattern,  and  pass  into  the  fields 
of  physiology  and  behavior.  Nevertheless,  it  is  convenient  to  consider 
the  types  of  relation  between  gene  and  character  at  these  higher  levels, 
skipping  over  the  intermediate  physiology.  It  is  not  always  appreciated 
that  the  quantitative  study  of  all  possible  combinations  of  the  genes  that 
affect  a  particular  character  provides  a  remarkably  delicate  technique 
of  analysis  because  of  the  absence  of  disturbances  due  to  experimental 
intervention.  It  is,  however,  a  method  that  must  be  associated  with 
physiological  experiment  as  far  as  practicable  for  adequate  interpretation. 


Journal    of    the    National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  845 

Cellular  Characters 

The  coat  color  of  mammals  is  a  character  at  the  cellular  level  since  it 
concerns  the  quality  and  quantity  of  certain  intracellular  products,  pig- 
ment granules.  In  the  guinea  pig,  combinations  of  alleles  at  10  loci 
theoretically  make  possible  more  than  a  million  genotypes.  What  are 
probably  the  most  significant  have  been  studied  quantitatively  (83). 
Nonadditive  interactions  are  encountered  in  nearly  all  cases.  The  situa- 
tion is  similar  in  the  mouse  but  with  an  even  greater  number  of  possible 
genotypes  from  known  loci. 

Some  of  the  color  genes  of  mammals  have  been  shown  to  be  related 
directly  or  indirectly  to  enzyme  differences.  There  are  reaction  chains, 
somewhat  like  the  metabolic  chains  worked  out  in  Neurospora.  The  situa- 
tion is,  however,  more  complex  in  accordance  with  the  greater  complexity 
of  the  mammal.  The  genes  that  affect  pigment  production  in  a  particular 
cell  are  in  large  part  those  of  its  own  nucleus,  but  also  include  those  of 
neighboring  cells  (responsible  for  tract  specificity  in  coat  patterns)  and  in 
some  cases  of  remote  endocrine  cells.  Migration,  differentiation  and 
differential  viability  play  roles  that  require  analysis.  Thus  even  here  we 
pass  beyond  the  strictly  cellular  level  to  that  of  the  organism  as  a  whole. 

The  frequency  of  pleiotropic  effects  of  color  factors  on  seemingly  unre- 
lated characters  indicate  that  in  many  cases  changes  in  pigmentation  are 
by-products  of  more  fundamental  physiological  effects  of  the  genes.  The 
classical  example  is  Darwin's  reference  to  the  correlation  between  blue 
eyes  and  deafness  in  cats.  Many  heterozygous  whites  or  near  whites  in 
mammals  and  birds  are  lethal  when  homozygous.  Dr.  Eussell  (3$.),  here, 
has  made  a  notable  study  of  a  multiple  allelic  series  of  this  sort  in  the  mouse 
in  which  dilution  or  absence  of  pigment  is  associated  with  effects  on 
hemoglobin.  Another  example  is  the  effect  of  gene  A7  in  the  mouse  in 
producing  yellow  color  and  a  disturbed  fat  metabolism  in  heterozygotes, 
lethality  in  homozygotes,  referred  to  by  Dr.  Chase. 

The  Problem  of  Differentiation 

The  strong  tendency  toward  persistence  of  the  type  of  differentiation  of 
cells  when  removed  to  indifferent  sites,  or  to  tissue  culture  implies  differ- 
ences among  cells  of  the  same  individual  that  are  hereditary  in  a  broad 
sense,  at  the  level  of  the  cell  as  a  reproducing  organism.  The  nature 
of  these  self -perpetuating  patterns  that  arise  regularly  from  a  single  geno- 
type in  each  generation  is  one  of  the  most  obscure  problems  of  biology. 

There  are  a  number  of  alternative  possibilities.  Differentiation  might 
depend  either  on  persistent  changes  in  the  genome  or  in  the  cytoplasm. 
Under  the  former  head  comes  Weismann's  hypothesis  of  nonequational 
mitosis,  now  generally  discredited,  except  for  certain  apparently  very 
special  cases  [e.g.  germ  line  determining  chromosomes  in  Sciara,  (35)].  A 
second  possibility  is  mutation  of  a  class  of  diphasic  genes,  under  the  con- 
trol of  local  conditions.  Some  support  can  again  be  found  in  special  cases 
[e.g.  the  mutable  gene  miniature  gamma  in  Drosophila  virilis,  (36)]  but 

Vol.    15,   No.    3,    December    1954 


846  proceedings:  symposium  on  25  years  of 

no  evidence  has  yet  been  obtained  to  indicate  that  this  is  a  general 
mechanism.  A  third  possibility  is  differential  multiplication  of  genes  in 
the  qualitatively  identical  genomes  of  different  tissues,  again  under 
stimulus  of  local  conditions,  for  which  Huskins  found  some  support  in 
plant  tissues  and  Kosswig  and  Shengiin  [sic]  (37),  and  Sengiin  (38)  in 
the  giant  somatic  chromosomes  of  different  tissues  of  Chironomus  larvae. 
If  on  the  other  hand,  the  basis  is  in  the  cytoplasm,  the  alternative  possi- 
bilities of  self-duplicating  particles  (plasmagenes)  and  of  a  repertoire  of 
self-regulatory  stable  states  of  the  cells  as  wholes,  must  be  considered.  A 
form  of  the  latter  appears  in  Weiss'  (39)  hypothesis  that  there  is  determina- 
tion of  the  cell  type  by  the  type  of  molecule  that  comes  to  prevail  in  its 
surface  under  the  influence  of  local  conditions,  including  especially  the 
surface  conditions  in  adjacent  cells,  and  that  once  established  this  tends  to 
perpetuate  itself.  He  and  others  have  found  evidence  that  the  growth  of 
the  differentiated  cell  is  regulated  in  some  way  by  the  concentration  of 
serologic  products  of  its  own,  or  other  kinds  of  cell,  in  the  blood  stream. 
The  doubling  in  the  size  of  one  kidney  after  removal  of  the  other  is  a 
familiar  illustration  of  such  regulation. 

Dr.  Dunn's  studies  of  differences  in  cellular  morphology  in  inbred  strains 
of  mice  bear  on  this  subject.  In  addition  to  such  primary  defects  as 
rodless  retina  in  one  of  the  strains,  she  finds  an  abundance  of  character- 
istically different  pathologic  lesions  appearing  in  tissues  (kidney,  bone, 
parathyroid,  pituitary,  etc.)  of  aging  mice  of  the  different  strains. 

Cancer  in  Relation  to  Genetics 

A  cancer  cell  may  be  looked  upon  as  a  cell  in  which  the  pattern  of 
differentiation  has  changed  to  a  new  self-perpetuating  type  of  such  a 
nature  that  the  normal  regulation  of  growth  by  the  rest  of  the  organism 
is  upset.  If  this  is  true,  the  papers  on  carcinogenesis  bear  directly  on  the 
subject  of  the  nature  of  differentiation.  The  round-table  discussion  by 
Doctors  Furth,  Gardner,  Hummel  and  Woolley  dealt  with  the  transforma- 
tion of  normal  endocrine  tissue  cells  into  cancerous  ones  by  mere  excess 
or  defect  of  hormones  from  other  endocrine  glands.  Dr.  Furth  described 
the  induction  of  thyroid  tumors  by  sustained  stimulation  of  the  normal 
thyroid  by  excess  TSH  from  grafted  pituitary  tumors  and  conversely  the 
induction  of  pituitary  tumors  by  removal  of  the  thyroid,  whether  by 
radioiodine  or  thyroidectomy.  Dr.  Hummel  and  Dr.  Woolley  discussed 
the  induction  of  ovarian  and  adrenal-cortical  tumors,  respectively,  by 
various  means  that  prevent  estrogenic  hormones  from  reaching  and  reg- 
ulating the  pituitary. 

Dr.  Woolley  has  stressed  the  importance  of  strain  genotype  as  a  factor 
in  the  susceptibility  to  such  processes.  Dr.  Dunn  described  strain  differ- 
ences in  the  types  of  tumors  that  appeared  spontaneously  with  especial 
reference  to  rare  types  peculiar  to  a  strain  of  hybrids.  Miss  Dickie  brought 
out  especially  the  importance  of  both  Fx  hybrids  and  backcrosses  to 
inbred  strains  in  producing  genotypes  that  in  some  way  lead  to  the  appear- 
ance of  rare  or  hitherto  unknown  types  of  tumor. 

Journal   of   the   National   Cancer   Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  847 

Dr.  Heston  investigated  the  question  whether  a  genotypic  difference 
between  two  strains  with  high  and  low  incidence  of  lung  cancer,  respec- 
tively, acts  through  local  or  general  physiological  channels,  by  transplant- 
ing lung  tissue  from  the  two  strains  to  Fi  hybrids  (which  accept  both, 
since  the  grafts  bring  in  no  foreign  genes) .  The  retention  of  the  character- 
istic differences  in  rates  indicated  local  action.  In  experiments  on  the 
induction  of  lung  tumors  by  dibenzanthracene,  he  found  a  linear  relation 
of  number  of  independent  tumor  nodules  to  dosage,  indicating  dependence 
on  a  single  random  event  in  each  case.  He  noted  that  this  is  consistent 
with  the  hypothesis  of  dominant  mutation. 

Dr.  Law  discussed  carcinogenesis  as  a  reversible  phenomenon  in  the 
case  of  transformation  of  leukemic  cells  to  apparent  normality  by  various 
chemical  agents. 

Morphogenesis 

Turning  now  to  morphogenesis,  Doctors  Gluecksohn-Waelsch  and  Chase 
went  into  a  considerable  number  of  cases  in  the  mouse  in  which  mutant 
genes  bring  about  consistent  and  in  some  cases  drastic  changes  in  the 
pattern  of  embryonic  development.  They  showed  how  histologic  study 
at  successive  stages  might  throw  light  on  the  nature  of  the  primary  effect 
(e.g.,  Gluecksohn-Waelsch's  account  of  abnormalities  of  the  notochord 
mesoderm  with  correlative  effects  on  the  nervous  system  and  Chase's 
analysis  of  the  chain  of  events  in  hairless  mice,  tracing  to  failure  of  the 
connective-tissue  sheath  of  the  follicle  to  form  a  glassy  membrane  that 
holds  the  latter  together  at  a  certain  stage) . 

Dr.  Gluecksohn-Waelsch  referred  to  the  value  of  what  Goldschmidt  has 
called  phenocopies — environmentally  induced  abnormalities,  identical  with 
genetic  ones,  in  analysis  of  the  detailed  mechanism  by  which  a  gene 
produces  its  phenotypic  effect.  Considerable  caution  is  necessary.  The 
very  fact  that  identical  abnormalities  may  be  produced  by  such  diverse 
means  (often  including  several  very  different  environmental  agents) 
implies  that  neither  gene  nor  environmental  agent  has  anything  to  do 
with  the  pattern  as  such.  The  physiology  of  development  has  remarkable 
self-regulatory  aspects  by  which  normality,  or  a  close  approach,  may  be 
arrived  at  in  spite  of  rather  serious  disturbances  on  the  way.  Some  aspects 
are  more  vulnerable  than  others.  Any  disturbance  of  physiology  at  a 
critical  time  may  start  a  chain  of  processes  that  results  in  one  of  the  limited 
number  of  types  of  mammalian  abnormality  determined  by  the  points  of 
vulnerability.  There  is  no  real  copying  of  gene  action  by  an  environ- 
mental agent  or  the  reverse,  but  a  mere  triggering  by  each  of  a  chain  of 
processes  characteristic  of  the  species.  The  point  on  which  light  may  be 
thrown  is  the  nature  of  the  initial  abnormal  physiological  condition,  but 
even  this  may  be  induced  in  such  widely  different  ways  that  little  light 
may  be  thrown  on  the  actual  primary  action  of  the  gene. 

In  most  cases,  no  sharp  cleavage  can  be  made  between  genetic  and 
environmental  determination  of  an  abnormality.  Any  given  combination 
of  genotype  and  environment,  both  controlled  as  far  as  possible,  leads 
merely  to  the  occurrence  of  a  certain  percentage  of  the  abnormality  in 

Vol.    15,   No.   3,   December    1954 


848 


PROCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 


question  [e.g.,  otocephalic  monsters  of  the  guinea  pig,  Wright  and  Eaton, 
(40) ;  Wright,  (£1)\.  It  is  convenient  to  speak  of  a  probability  distribution 
of  physiological  states,  due  to  uncontrolled  factors,  that  is  cut  by  the 
threshold  for  the  abnormality  in  such  a  way  that  the  area  above  it  is  the 
observed  percentage.  Timofeeff-Ressovsky's  term  penetrance  is  a  con- 
venient one  for  this  percentage.  We  have  already  used  the  concept  in 
connection  with  the  percentage  of  resistance  to  grafts  of  a  tumor  that 
carries  a  foreign  antigen. 

Dr.  Green  has  made  use  of  this  concept  in  describing  the  genetics  of 
the  number  of  presacral  vertebrae  in  mice.  Each  of  several  inbred 
strains  had  its  own  characteristic  array  of  frequencies,  clearly  not  due  to 
Mendelian  segregation,  but  to  a  probability  distribution  with  respect  to 
nongenetic  factors,  cut  by  thresholds.  In  F2  and  backcrosses,  this  non- 
genetic  variability  was  of  course  supplemented  by  segregation  of  multiple 
factors.  His  mathematical  analysis  was  along  a  line  found  useful  in  de- 
scribing the  genetics  of  number  of  digits  in  guinea  pigs  (42). 

Dr.  Runner  varied  both  genotype  (by  use  of  different  inbred  strains  of 
mice)  and  the  environment  (by  use  of  different  doses  of  such  teratogenic 
agents  as  cortisone)  in  a  study  of  the  incidence  of  developmental  anomalies. 
There  was  always  residual  nongenetic  variability  and  a  threshold.  His 
results  brought  out  admirably  the  symmetry  in  the  roles  of  heredity  and 
environment  in  the  genesis  of  abnormalities. 

Reaction  to  Infection 

We  come  now  to  a  class  of  characters  still  more  remote  from  primary 
gene  action  than  morphogenesis — the  reactions  of  the  organism  to  its 
external  environment.  A  subclass  is  the  resistance  to  infection.  Gowen 
notes  the  tremendous  value  of  inbred  strains  in  revealing  genotypic 
differences  in  this  complicated  sort  of  character:  reaction  to  tuberculosis 
in  strains  of  guinea  pig  (43)  and  to  typhoid  (Salmonella  typhimurium)  in 
mice  and  (Salmonella  gallinarum)  in  fowls.  In  his  studies  of  mouse 
typhoid,  he  found  an  extraordinary  variety  of  underlying  characters 
correlated  with  resistance,  morbidity,  and  death;  e.g.,  ability  to  maintain 
weight  during  the  course  of  the  disease,  size  of  heart,  kidneys  or  liver, 
blood  volume,  hematocrit  and  leukocyte  number,  ability  of  the  liver  cells 
to  isolate  the  disease  and  allow  the  remaining  normal  cells  to  function  in 
glycogen  and  fat  metabolism,  and  ability  of  the  macrophages  to  ingest 
and  digest  the  pathogenic  bacteria.  Differences  in  humoral  elements — 
albumin  and  globulin — were  significant  in  acquired  resistance. 

Dr.  Heston  analyzed  the  genetic  control  of  the  transmission  of  the  milk 
agent  for  mammary  cancer  on  the  assumption  of  normal  variability  and  a 
threshold.  The  percentages  of  eliminatiou  in  repeated  backcrosses 
indicated  that  in  this  case  a  very  small  number  of  loci  differentiated  the 
strains  used,  possibly  only  one.  The  relative  simplicity  of  strain  diff- 
erences in  the  response  to  an  agent  that  is  almost  part  of  the  heredity  of 
the  mouse  is  not  too  surprising. 


Journal    of    the    National    Cancer    Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER  849 

Behavior 

The  session  on  genetical  control  of  behavior  dealt  with  a  very  different 
aspect  of  the  reaction  of  the  organism  to  its  environment.  Dr.  Richter's 
paper  analyzed  some  of  the  physiologic  and  morphologic  changes 
that  mediate  between  genotype  and  the  marked  behavior  differences  of 
domestic  and  wild  rats.  He  finds  evidence  of  extensive  shifting  in 
physiology  and  anatomy  of  the  endocrine  glands,  probably  brought  about 
by  conscious  and  unconscious  selection  for  fertility  and  tameness. 

Dr.  Scott  showed  how  the  complex  differences  in  behavior  among  widely 
diverse  breeds  of  dogs  could  be  separated  into  elements  and  analyzed 
genetically  on  the  hypothesis  of  multiple,  but  not  necessarily  very  num- 
erous, factors. 

Dr.  Snyder  discussed  the  supposed  selection  for  lower  intelligence  in 
man,  based  on  the  observed  negative  correlation  between  I.Q.  and  family 
size,  in  a  relatively  optimistic  vein. 

Formal  Genetics  of  the  Mouse 

While  most  of  the  authors  stressed  the  complexity  of  the  genotypic 
differences  underlying  characters  at  all  levels  and  the  consequent  im- 
portance of  inbred  strains,  the  current  advantages  of  the  mouse  for  analysis 
by  conventional  genetics  were  also  brought  out  in  the  impressive  list  of 
known  genes  and  their  arrangement  in  linkage  systems,  presented  by 
Miss  Dickie.  Dr.  Griff  en  showed  how  far  study  of  the  patterns  of  the 
individual  chromosomes  and  their  identification  with  linkage  systems 
has  gone. 

Conclusion 

We  may  refer  finally  to  Dr.  Castle's  interesting  account  of  the  early 
history  of  mammalian  genetics,  from  its  beginnings  in  his  own  laboratory 
at  Harvard  almost  immediately  after  the  rediscovering  of  Mendelian 
heredity  in  1900.  Dr.  Castle's  early  studies  laid  the  foundation  for  the 
great  expansion  in  which  the  Jackson  Laboratory  has  played  the  major 
role  in  the  25  years  since  its  establishment. 

The  results  of  the  Conference  gave  ample  demonstration  of  the  wisdom 
of  the  program  of  the  Jackson  Laboratory  in  supporting  a  systematic 
attack  on  the  fundamental  genetics  of  mammals  at  all  levels — from  antigen 
to  behavior — along  with  its  attack  on  the  problem  of  cancer. 

References 

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Quant.  Biol.,  16:  1-11,  1951. 
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von  angeborener  Rotgrunblindheit.     Zeit.  ind.  Abst.  Ver.  45:  279-333,  1927. 
(5)  Lewis,  E.  B.:  Pseudoallelism  and  gene  evolution.     Cold  Spring  Harbor  Symp. 

Quant.  Biol.  16:  159-174,  1951. 
(4)  McClintock,  B. :  Chromosome  organization  and  genie  expression.     Cold  Spring 

Harbor  Symp.  Quant.  Biol.  16:  13-47,  1951. 

Vol.    15,   No.   3,   December    1954 


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850  PKOCEEDINGS:  SYMPOSIUM  ON  25  YEARS  OF 

(5)  Brink,  R.  A.,  and  Nilan,  R.  A.:  The  relation  between  light  variegated  and 

medium  variegated  pericarp  in  maize.     Genetics  37:  519-544,  1952. 

(6)  Ephrussi-Taylor,    H.:  Genetic    aspects    of    transformation    of    pneumococci. 

Cold  Spring  Harbor  Symp.  Quant.  Biol.  16:  445-456,  1951. 

(7)  Yazumi,  G.,  and  Keiko,  I. :  Electron  microscopy  of  salivary  gland  chromosomes. 

J.  Hered.  45:  135-142,  1954. 

(8)  Mazia,  D. :  The  particulate  organization  of  the  chromosome.     Proc.  Nat.  Acad. 

Sc.  40:  521-527,  1954. 

(9)  Watson,  J.  D.,  and  Crick,  F.  H.  C:  The  structure  of  DNA.     Cold  Spring 

Harbor  Symp.  Quant.  Biol.  18:  123-132,  1953. 

(10)  Haldane,  J.  B.  S.:  In  Perspectives  in  Biochemistry  (Needham,  J.  and  Green, 

D.  E.,  eds.).     New  York  and  London,  Cambridge  Univ.  Press,  1938,  pp.  361. 

(11)  Stormont,  C,  Owen,  R.  D.,  and  Irwin,  M.  R. :  The  B  and  C  systems  of  bovine 

blood  groups.     Genetics  36:  134-161,  1951. 

(12)  Wiener,  A.  S.,  Sonn-Gordon,  E.  B.,  and  Hardman,  L.:  Heredity  of  the  Rh 

blood  types.  VI.  Additional  family  studies  with  special  reference  to  the  theory 
of  multiple  allelic  genes.     J.  Immunol.  37:  203-214,  1947. 
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effecting  cellular  antigens  in  the  chicken.     Genetics  35:  633-652,  1950. 

(14)  McGibbon,  W.  H.:  Further  division  of  contrasting  antigens  in  species  hybrids 

in  ducks.     Genetics  30:  252-265,  1945. 

(15)  Irwin,  M.  R.:  Immunogenetics.     Advances  in  Genetics  1:  133-159,  1947. 

(16)  Robbins,  R.  B.:  Applications  of  mathematics  to  breeding  problem:  II.     Genetics 

3:  73-92,  1918. 

(17)  Fisher,  R.  A.:  Population  genetics.     Proc.  Roy.  Soc,  s.B,  141:  510-523,  1953. 

(18)  Race,  R.  R.,  Mourant,  A.  E.,  Lawler,  S.  D.,  and  Sanger,  R. :  The  Rh  chromo- 

some frequencies  in  England.     Blood  3:  689-695,  1948. 

(19)  Irwin,  M.  R.,  and  Cole,  L.  J. :  Immunogenetic  studies  of  species  and  of  species 

hybrids  in  doves  and  the  separation  of  species-specific  substances  in  the  back- 
cross.     J.  Exper.  Zool.  73:  85-108,  1936. 

(20)  Loeb,  L.:  Transplantation  and  individuality.     Biol.  Bull.  40:  143-180,  1921. 

(21)  Little,  C.  C,  and  Tyzzer,  E.  E.:  Further  experimental  studies  on  the  inherit- 

ance of  susceptibility  to  a  transplantable  tumor,  carcinoma  (JWA)  of  the 
Japanese  waltzing  mouse.     J.  M.  Res.  33:  393-453,  1916. 

(22)  Little,  C.  C,  and  Strong,  L.  C:  Genetic  studies  on  the  transplantation  of  two 

adenocarcinomata.    J.  Exper.  Zool.  41:  53-114,  1924. 

(23)  Little,  C.  C,  and  Johnson,  B.  W.:  The  inheritance  of  susceptibility  to  implants 

of  splenic  tissue  in  mice.     1.  Japanese  waltzing  mice,  albinos  and  their  Fj  gen- 
eration hybrids.    Proc.  Soc.  Exper.  Biol.  &  Med.  19:   163,  1922. 

(24)  Loeb,  L.,  and  Wright,  S.:  Transplantation  and  individuality  differentials  in 

inbred  families  of  guinea  pigs.     Am.  J.  Path.  3:  251-283,  1927. 

(25)  Gorer,  P.  A.:  The  genetic  and  antigenic  basis  of  tumour  transplantation.     J. 

Path.  &  Biol.  44:  691-697,  1937. 

(26)  Cloudman,  A.  M.:  A  comparative  study  of  the  transplantability  of  eight  mam- 

mary gland  tumors  arising  in  inbred  mice.     Am.  J.  Cancer  16:  568-630,  1932. 
Snell,  G.  D.,  and  Higgins,  G.  F.:  Alleles  at  the  histocompatibility  locus  in  the 

mouse  as  determined  by  tumor  transplantation.     Genetics  36:  306-310,  1951. 
Garrod,  A.  E.:  Inborn  errors  of  metabolism.     Lancet  Jan.  4,  11,  18,  25,  1908. 
Sawin,  P.  B.,  and  Glick,  D.:  Atropinesterase,  a  genetically  determined  enzyme 

in  the  rabbit.     Proc.  Nat.  Acad.  Sc.  29:  55-59,  1943. 

(30)  Wright,  S.:  Physiological  and  evolutionary  theories  of  dominance.     Am.  Nat. 

68:  24-33,  1934. 

(31)  :  The  physiology  of  the  gene.     Physiol.  Rev.  21:  487-527,  1941. 

(32)  :   Genes  as  physiological  agents:  general  considerations.     Am.  Nat.  79: 

289-303,  1945. 

(83)  :  Estimates  of  the  amounts  of  melanin  in  the  hair  of  diverse  genotypes 

of  the  guinea  pig  from  transformation  of  empirical  grades.     Genetics  34:  245- 
251,  1949. 

Journal  of  the  National  Cancer  Institute 


PROGRESS  IN  MAMMALIAN  GENETICS  AND  CANCER 


851 


(34)  Russell,  E.  S.:  Analysis  of  pleiotropism  at  the  W-locus  in  the  mouse:  relation- 

ship between  the  effects  of  W  and  WJ  substitution  on  hair  pigmentation  and  on 
erythrocytes.     Genetics  34.  708-723,  1949. 

(35)  Metz,    C.    W.:  Chromosome  behavior  inheritance   and   sex   determination  in 

Sciara.     Amer.  Nat.  72:  485-520,  1938. 

(36)  Demerec,    M.:  Unstable   genes   in    Drosophila.     Cold   Spring   Harbor   Symp. 

Quant.  Biol.  9:   145-149,  1941. 

(37)  Kosswig,  C,  and  Shengun,  A.:  Intraindividual  variability  of  chromosome  IV  of 

Chironomus.     J.  Hered.  38:  235-239,  1947. 

(38)  Sengun,   A.:  Variability  of  the  banding  patterns   of   giant   chromosomes.     J. 

Hered.  45:   119-122,  1954. 

(39)  Weiss,  P.:  Some  introductory  remarks  on  the  cellular  basis  of  differentiation. 

J.  Embryol.  &  Exper.  Morphol.  1:   181-211,  1953. 

(40)  Wright,  S.,  and  Eaton,  O.  N.:  Factors  which  determine  otocephaly  in  guinea 

pigs.     J.  Agr.  Res.  26:   161-182,  1923. 

(41)  Wright,  S.:  On  the  genetics  of  subnormal  development  of  the  head  (otocephaly) 

in  the  guinea  pig.     Genetics  19:  471-505,  1934. 

(42)  :  The  results  of  crosses  between  inbred  strains  of  guinea  pigs,  differing  in 

number  of  digits.     Genetics  19:  537-551,  1934. 

(43)  Wright,  S.,  and  Lewis,  P.  A.:  Factors  in  the  resistance  of  guinea  pig  to  tuber- 

culosis with  especial  reference  to  inbreeding  and  heredity.     Am.   Nat.  55: 
20-50,  1921. 


Vol.    15,   No.   3,   December    1954 


f  \ 


Announcement 


Summer  Research  Awards  for  College  Faculty  Members 


The  Lalor  Foundation  recently  announced  a  new  program  for  1955  to  include  20 
summer  or  interim  awards  to  college  and  university  faculty  members  for  study  and 
research  in  which  chemistry  or  physics  is  used  to  attack  problems  in  any  of  the  bio- 
logical sciences.  Each  award  will  normally  not  exceed  $900  to  single  men  and  women 
and  $1,100  to  married  persons,  but  is  subject  to  circumstances.  The  place  of  work 
may  be  at  the  faculty  member's  own  institution  or  elsewhere,  as  may  fit  the  best 
interests  of  the  program. 

It  is  the  hope  of  the  Foundation  that  not  only  significant  research,  but  also  more 
dynamic  teaching  of  science  may  result  from  this  new  program  and  that  younger 
faculty  members  may  find  opportunity  by  this  means  to  advance  in  their  profession. 

Also,  the  Foundation  continues  with  its  eighth  year  of  underwriting  of  awards  of 
postdoctoral  summer  fellowships  administered  by  the  Marine  Biological  Laboratory 
at  Woods  Hole,  Massachusetts. 

The  Foundation  is  discontinuing  its  previous  program  of  full-year  predoctoral  and 
postdoctoral  fellowship  awards. 

Inquiries  respecting  the  new  faculty  summer  awards  should  be  directed  to  C.  L. 
Burdick,  Director  of  the  Lalor  Foundation,  4400  Lancaster  Pike,  Wilmington  5, 
Delaware.     Applications  for  these  awards  must  be  filed  before  January  15,  1955. 

Inquiries  regarding  Marine  Biological  Laboratory  postdoctoral  summer  awards 
should  be  directed  to  the  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts. 
The  final  filing  date  for  these  applications  is  February  1,  1955. 

853 


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of  the  National  Cancer  Institute 


U.  S.  DEPARTMENT  OF  HEALTH,  EDUCATION,  AND  WELFARE 
Public  Health  Service National  Institutes  of  Health