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
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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
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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.
Journal of the National Cancer Institute
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
Journal of the National Cancer Institute
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-
Vol. 15, No. 3, December 1954
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
Journal of the National Cancer Institute
PROGRESS IN MAMMALIAN GENETICS AND CANCER
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.
Vol. 15, No. 3, December 1954
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
Journal of the National Cancer Institute
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.
References
(1) Longlet, J. B., and Fisher, E. R.: Alkaline phosphatase and periodic acid-
Schiff reaction in proximal tubule of vertebrate kidney. A study in segmental
differentiation. Anat. Rec. 120: 1954. In press.
(2) Dunn, T. B.: Relationship of amyloid infiltration and renal disease in mice.
J. Nat. Cancer Inst. 5: 17-28, 1944.
(3) Heston, W. E., and Deringer, M. K.: Hereditary renal disease and amyloidosis
in mice. Arch. Path. 46: 49-58, 1948.
(4) Dunn, T. B.: Some observations on the normal and pathologic anatomy of the
kidney of the mouse. J. Nat. Cancer Inst. 9: 285-301, 1949.
(5) Dunn, T. B., and Larsen, C. D.: Hyalinization of glomeruli produced in strain
A mice by the administration of urethan (ethyl carbamate). (Abstract.)
Fed. Proc. 5: 220, 1946.
(6) Kirschbaum, A., and Bell, E. T.: Induction of renal glomerular lesions by
urethan in inbred mice susceptible to spontaneous glomerulonephritis. Proc.
Soc. Exper. Biol. & Med. 64: 71-72, 1947.
(7) Deringer, M. K., Dunn, T. B., and Heston, W. E.: Results of exposure of
strain C3H mice to chloroform. Proc. Soc. Exper. Biol. & Med. 83: 474r-479,
1953.
(8) Morris, H. P., and Dunn, T. B.: Pyridoxine deficiency. Nutritional and
histological observations on five strains of mice. 12th Congr. Pure and Applied
Chem. 13: 164-165, 1951.
(9) Dunn, T. B.: Melanoblasts in the stroma of the parathyroid glands of strain
C58 mice. J. Nat. Cancer Inst. 10: 725-733, 1949.
(10) Lorenz, E., and Dunn, T. B.: Ocular lesions induced by acute exposure of the
whole body of newborn mice to roentgen radiation. Arch. Ophth. 43: 742-749
1950.
(11) Tansley, K.: Hereditary degeneration of the mouse retina. Brit. J. Ophth.
35: 573-582, 1951.
(12) Deringer, M. K., and Dunn, T. B.: Mast-cell neoplasia in mice. J. Nat.
Cancer Inst. 7: 289-298, 1947.
(IS) Duqu£, O.: Unpublished.
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584 proceedings: symposium on 25 years of
(14) Russell, E. S., Neufeld, E. F., and Higgins, C. T.: Comparison of normal
blood picture of young adults from 18 inbred strains of mice. Proc. Soc.
Exper. Biol. & Med. 78: 761-766, 1951.
(15) Fekete, E.: A morphological study of the ovaries of virgin mice of eight inbred
strains showing quantitative differences in their hormone producing compo-
nents. Anat. Rec. 117: 93-113, 1953.
(16) Morris, H. P., Dunn, T. B., and Wagner, B. P.: Influence of gonadotrophin
on pyridoxine-deficient and diet-restricted female mice. J. Nat. Cancer Inst.
14: 493-511, 1953.
(17) Simonds, J. P.: Leukemia, pseudoleukemia, and related conditions in the Slye
stock of mice. J. Cancer Res. 9: 329-373, 1925.
(18) Heston, W. E., Deringer, M. K., Dunn, T. B., and Levillain, W. D.: Factors
in the development of spontaneous mammary gland tumors in agent-free
strain C3Hb mice. J. Nat. Cancer Inst. 10: 1139-1155, 1950.
(19) Andervont, H. B., and Dunn, T. B.: Response of mammary-tumor-agent-free
strain DBA female mice to percutaneous application of methylcholanthrene.
J. Nat. Cancer Inst. 10: 895-925, 1950.
(20) Muhlbock, O., Tengbergen, W. van E., and Rijssel, Th. G. van: Studies on
the development of mammary tumors in dilute-brown (DBAb) mice without
the agent. J. Nat. Cancer Inst. 13: 505-531, 1952.
(21) Andervont, H. B., and Dunn, T. B.: Attempt to detect a mammary tumor-
agent in strain C mice by X-radiation. J. Nat. Cancer Inst. 10: 1157-1189,
1950.
Hilberg, A. W.: Osteogenic sarcoma of mice. (Abstract.) Proc. Am. Assoc.
Cancer Res. 1: 20, 1954.
Cloudman, A. M.: Spontaneous neoplasms in mice. In Biology of the Labora-
tory Mouse (Snell, G. D., ed.). Philadelphia, The Blakiston Co., pp. 168-233,
1941.
(24) Slye M., Holmes, H. F., and Wells, H. G.: Tumors of the ovary in mice.
J. Cancer Res. 5: 205-226, 1920.
(25) Dunn, T. B.: Transplantable plasma cell neoplasm in strain C3H mice. (Ab-
stract.) Proc. Am. Assoc. Cancer Res. 1: 13, 1954.
(26) Stewart, H. L., Kaplan, H. S., and Bennett, J. G.: Report of two cases of
identical primary tumors involving spinal nerve roots and meninges in strain
NHO mice. J. Nat. Cancer Inst. 11: 177-197, 1950.
(27) Cramer, W., and Horning, E. S.: On the association between brown degenera-
tion of the adrenals and the incidence of mammary cancer in inbred strains of
mice. Am. J. Cancer 37: 343-354, 1939.
(28) Figge, F. H. J., Strong, L. C, Strong, L. C, Jr., and Shanbrom, A.: Fluores-
cent porphyrins in harderian glands and susceptibility to spontaneous mam-
mary carcinoma in mice. Cancer Res. 2: 335-342, 1942.
(29) Davidsohn, I., and Stern, K.: Further studies on natural antisheep agglutinins
in mice of inbred strains. Cancer Res. 10: 571-576, 1950.
(30) Rawlinson, H. E., and Hankinson, H. W.: Stainable iron deposits in the
epithelium of the mammary glands of mice. Anat. Rec. 102: 55-61, 1948.
(81) Huseby, R. A., and Bittner, J. J.: A comparative morphological study of the
mammary glands with reference to the known factors influencing the develop-
ment of mammary carcinoma in mice. J. Cancer Res. 6: 240-255, 1946.
(82) Deringer, M. K.: Spontaneous and induced tumors in haired and hairless
strain HR mice. J. Nat. Cancer Inst. 12: 437-445, 1951.
(88) Stewart, H. L. and Andervont, H. B.: Pathologic observations on the adeno-
matous lesion of the stomach in mice of strain I. Arch. Path. 26: 1009-1022,
1938.
Journal of the National Cancer Institute
PROGRESS IN MAMMALIAN GENETICS AND CANCER 585
(34) Barnes, W. A., and Sisman, I. E.: Myeloid leukemia and non-malignant extra-
medullary myelopoiesis in mice. Am. J. Cancer 37: 1-35, 1939.
(35) Potter, J. S., Victor, J., and Ward, E. N.: Histological changes preceding
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
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Dunn
Figure 3
589
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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
Vol. 15, No. 3, December 1954
—
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,
Journal of the National Cancer Institute
PROGRESS IN MAMMALIAN GENETICS AND CANCER
601
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
Vol. 15, No. 3, December 1954
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-
Journal of the National Cancer Institute
PROGRESS IN MAMMALIAN GENETICS AND CANCER
603
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
Vol. 15. No. 3, December 1954
316263—54 21
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
PROGRESS IN MAMMALIAN GENETICS AND CANCER
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
Journal of the National Cancer Institute
PROGEESS IN MAMMALIAN GENETICS AND CANCER
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
Journal of the National Cancer Institute, Vol. 15, No. 3, December 1954
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
Journal of the National Cancer Institute
PROGRESS IN MAMMALIAN GENETICS AND CANCER
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
Journal of the National Cancer Institute
PROGRESS IN MAMMALIAN GENETICS AND CANCER
657
(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.
Journal of the National Cancer Institute
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
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 15
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
Journal of the National Cancer Institute, Vol. 15, No. 3, December 1954
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.
665
Journal of the National Cancer Institute, Vol. 15, No. 3, December 1954
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
669
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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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Text-figure 1A. — The chromosome map of 1903-1925. One linkage group was
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
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fc60
$50
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20
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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*) ^
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••
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• ••
oo •
• •
•
•
o
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• • •
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
"
O 80
Q.
O 50
30
—
cc5
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•
•
•
•
uvc
- «.
• 4
1
0
250 300 350 400 450 500 550 600 650
Age in days at death
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
180
160
140
45 m'
1 1
HC,
ce - 16.6 or 25 }jg. estradiol benzoate weekly
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1
400 450 500 550 600
Age in days at death
650
700
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
160
22
mice —
16.6 pg. estradiol benzoate weekly
140
lo-o
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20
•
-«
o«
•
• •
••
■*
300
350
400
450
500
550
600
Age in days at death
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
Journal of the National Cancer Institute, Vol. 15, No. 3, December 1954
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.
Journal of the National Cancer Institute
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
Journal of the National Cancer Institute
PROGRESS IN MAMMALIAN GENETICS AND CANCER 735
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
Journal of the National Cancer Institute
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.
References
(1) Donaldson, H. H.: The Rat. Memoirs of the Wistar Institute, No. 6, 2d
ed., 1924.
(2) Philipeaux, J. M.: Note sur l'extirpation des capsules surrSnales chez les rats
albinos (Mus rattus). Compt. rend. Acad. sc. 43: 904-906, 1856.
(3) Conklin, E. G.: Biographical Memoirs of H. H. Donaldson (1857-1938). Nat.
Acad. Sc, 1939.
(4) Hatai, S. : On the appearance of albino mutants in litters of the common Norway-
rat, Mus norvegicus. Science 35: 875-876, 1912.
(5) Richter, C. P.: The development and use of alpha-naphthyl thiourea (Antu)
as a rat poison. J. A. M. A. 129: 927-931, 1945.
(6) Emlen, J. T., Jr.: Device for holding live wild rats. J. Wildlife Mgt. 8: 264-
265, 1944.
(7) Richter, C. P., and Emlen, J. T., Jr.: A modified rabbit box trap for use in
catching live wild rats for laboratory and field studies. Pub. Health Rep. 60:
1303-1308, 1945.
(8) Watson, C: A note on the adrenal gland in the rat. J. Physiol. 35: 230-232,
1907.
(9) Donaldson, J. C: Adrenal gland in wild gray and albino rat: Cortico-medullary
relations. Proc. Soc. Exper. Biol. & Med. 25: 300-301, 1928.
(10) Rogers, P. V., and Richter, C. P.: Anatomical comparison between the ad-
renal glands of wild Norway, wild Alexandrine and domestic Norway rats.
Endocrinology 42: 46-55, 1948.
(11) Hall, C. E.: Comparison of the preputial glands in the Alexandrine, the wild
and the domestic Norway rat. Proc. Soc. Exper. Biol. & Med. 69: 233-237,
1948.
(12) Richter, C. P., and Uhlenhuth, E. H.: Comparison of the effects of gonad-
ectomy on spontaneous activity of wild and domesticated Norway rats. En-
docrinology 54: 311-322, 1954.
(13) Richter, C. P., and Hall, C. E. : Comparison of intestinal lengths and Peyer's
patches in wild and domestic Norway and wild Alexandrine rats. Proc. Soc.
Exper. Biol. & Med. 66: 561-566, 1947.
(14) Fish, H. S., and Richter, C. P.: Comparative number of fungiform and foliate
papillae on tongues of domestic and wild Norway rats. Proc. Soc. Exper.
Biol. & Med. 63: 352-355, 1946.
(15) Dieke, S. H., and Richter, C. P.: Acute toxicity of thiourea to rats in relation
to age, diet, strain and species variation. J. Pharmacol. & Exper. Therap. 83:
195-202, 1945.
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316263—54 32
738 PKOCEEDINGS: SYMPOSIUM
(16) Griffiths, W. J., Jr.: Absence of audiogenic seizures in wild Norway and Alex-
andrine rats. Science 99: 62-63, 1944.
(17) : Audiogenic fits produced by magnesium deficiency in tame domestic
Norway rats and in wild Norway and Alexandrine rats. Am. J. Physiol. 149:
135-141, 1947.
(18) Richter, C. P., and Rice, K. K.: Comparison of the effects produced by fasting
on gross bodily activity of wild and domesticated Norway rats. In press.
(19) Richter, C. P.: Experimentally produced behavior reactions to food poisoning
in wild and domesticated rats. Ann. New York Acad. Sc. 56: 225-239, 1953.
(20) Mosier, H. D., Jr.: Histological study of the adrenals of wild and domesticated
Norway rats. In preparation.
(21) Richter, C. P., Rogers, P. V., and Hall, C. E.: Failure of salt replacement
therapy in adrenalectomized recently captured wild Norway rats. Endocri-
nology 46: 233-242, 1950.
(22) Sayers, G., and Sayers, M. A.: The pituitary-adrenal system. Ann. New York
Acad. Sc. 50: 522-529, 1949.
(23) Woods, J. W.: Some observations on adrenal cortical functions in wild and dom-
esticated Norway rats. Doctoral Thesis, Johns Hopkins University, 1954.
(24) Wang, G. H.: The relation between "spontaneous" activity and oestrous cycles
in the white rat. Comp. Psychol. Mon. 2: 1-27, 1923.
(25) Davis, D. E.: The weight of wild brown rats at sexual maturity. J. Mamm. 30:
125-130, 1949.
(26) Davis, D. E., and Emlen, J. T., Jr.: The placental scar as a measure of fertility
in rats. J. Wildlife Mgt. 12: 162-167, 1948.
(27) Wood, D. E.: Behavioral and metabolic studies on the hypophysectomized wild
and domesticat'ed Norway rat. In preparation.
(28) Richter, C. P., and Hawkes, C. D.: Increased spontaneous activity and food
intake produced in rats by removal of the frontal poles of the brain. J. Neurol.
& Psychiat. 2: 231-242, 1939.
(29) Woods, J. W.: Taming effect of amygdaloid lesions in wild Norway rats. In
press.
(30) Castle, W. E.: The domestication of the rat. Proc. Nat. Acad. Sc. 33: 109-
117, 1947.
(31) King, H. D.: Life processes in gray Norway rats during fourteen years of cap-
tivity. Am. Anat. Memoirs No. 17, 1939.
(32) King, H. D., and Donaldson, H. H.: Life processes and size of the body and
organs of gray Norway rat during ten generations in captivity. Am. Anat.
Memoirs No. 14, 1929.
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
Journal of the National Cancer Institute, Vol. IS, No. 3, December 1954
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.
Journal of the National Cancer Institute
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
Journal of the National Cancer Institute
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
{1) 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.
{2) Young, S. P., and Goldman, E. A.: The Wolves of North America. Wash-
ington, D. C., American Wildlife Institute, 1944.
(3) Little, C. C. and DuBuis, E.: Private communication, 1954.
(4) Matthew, W. D.: The phylogeny of dogs. J. Mamm. 11: 117-138, 1930.
Vol. 15, No. 3, December 3951
758 proceedings: symposium
(5) Allen, G. M.: Dogs of the American aborigines. Bull. Mus. Comp. Zool.
Harvard 63: 431-517, 1920.
(6) Degerbol, M.: tlber prahistorische danische Hunde. Vidensk. Meddel.
Dansk. Naturhist. For. Kobenhavn 84: 17-72, 1927.
(7) Haag, W. G.: An osteometric analysis of some aboriginal dogs. Univ. of
Kentucky Rep. Anthropol. 7: 107-264, 1948.
(8) Murie. A.: The Wolves of Mount McKinley. U. S. D. I. Fauna Ser., No. 5,
Washington, D. C., Government Printing Office, 1944.
(9) Scott, J. P.: The social behavior of dogs and wolves: an illustration of socio-
biological systematics. Ann. New York Acad. Sc. 51: 1009-1021, 1950.
(10) Williams, V. T.: Basenjis, the Barkless Dogs (revised ed.). London, Wat-
moughs, 1954.
(11) Scott, J. P., and Fuller, J. L.: Manual of dog testing techniques (mimeo-
graphed). Roscoe B. Jackson Memorial Laboratory, Bar Harbor, 1950.
(12) Castle, W. E.: An improved method of estimating the number of genetic factors
concerned in cases of blending inheritance. Science 54: 223, 1921.
(18) Wright, S.: The results of crosses between inbred strains of guinea pigs, differ-
ing in number of digits. Genetics 19: 537-551, 1934.
(14) * The genetics of quantitative variability. In Quantitative Inheritance.
London Agricultural Res. Council, 1952.
(15) Scott, J. P.: Biomathematical problems in animal behavior. In Statistics and
Mathematics in Biology. Ames, Iowa, Iowa State College Press, 1954.
(16) Scott, J. P., and Fredericson, E.: The causes of fighting in mice and rats.
Physiol. Zool. 24: 273-309, 1951.
(17) Hall, C. S.: The genetics of behavior. In Handbook of Experimental Psy-
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
Journal of the National Caneer Institute, Vol. 15, No. 3, December 1954
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
Journal of the National Cancer Institute
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
Vol. 15, No. 3, December 1954
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-
Journal of the National Cancer Institute
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
Vol. 15, No. 3, December 1954
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.
Journal of the National Caneer Institute
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.
Publ. Assn. Res. Nerv. and Ment. Dis., No. 33, 1954.
(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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(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.
(39) Mather, K.: Polygenic inheritance. In Clinical Genetics (Sorsby, A., ed.).
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
i
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.
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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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Due
Date Due
Returned Due
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of the National Cancer Institute
U. S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
Public Health Service National Institutes of Health