DIALOGUE ON THE RELATIONS OF GENETICS AND
EXPERIMENTAL EMBRYOLOGY TO NEOPLASIA*
C. C. LITTLE AND STANLEY P. REIMANN
Dr. Little: Why do you suppose the Society of Clinical Pathologists has chosen a question of this type for discussion?
Dr. Reimann: Because pathologists realize that the basis of
comparison for tumor growth problems is normal growth. Because they realize that many of their every-day problems in
tumor diagnosis run into questions of inheritance, potencies,
the way cells differentiate, the way they organize; in general in
the way cells behave. They realize too that their specimens
show cells only as of the instant when they are removed and
fixed. They like to figure out how the cells arrived where they
are—and what would have happened had the tissue not been
removed when it was. In other words, in addition to the statics
of pathology, they like to add the dynamic; in addition to cellular pathology, they want to think in terms of cellular physiology
and when possible, even in terms of cellular chemistry. All of
these and other questions, they realize, make it important that
they know as much as possible of what has been learned and is
being learned in such dynamic subjects as genetics and experimental embryology.
Dr. Little: This is interesting, Dr. Reimann, because in genetics
we too are increasingly interested in the dynamic phase of that
field. We find that experimental embryology is in one way the
road over which genetics must travel in order to understand the
relationship between a sub-microscopic structure known as the
* Read before the Fifteenth Annual Convention of The American Society
of Clinical Pathologists, Philadelphia, June 2-6,1937. Received for publication
November 1, 1937.
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gene and the finished product of the adult character on which all
of us clinical pathologists and biologists must base our classification.
Dr. Reimann: But Doctor Little, pathologists for many years
have had the feeling that the genes alone will not solve some of
their problems and it is with the greatest of interest that we learn
from you that the tendency to tumor formation, at least in certain cases, can now be ascribed to transmission by way of the
cytoplasm of cells. I believe pathologists would be interested
in knowing how you determined that fact. You know that
quite similar anatomic pictures, so far as chromosomes are concerned, are found in inflammatory conditions as in those that
the pathologist calls real tumors. If healthy tumor cells are
used for study, no change in the chromosome anatomy has been
found in them specific of malignancy and no change which has
not also been found in conditions other than malignancy, such
as inflammations.
Dr. Little: I should be glad to talk about that briefly, Dr. Reimann, with the understanding that I am quite conscious of the
fragmentary and preliminary nature of our knowledge of the
topic. In the first place, the relationship between chromosomes
and cytoplasm is one in which the chromosomes have certain
definite advantages. Their characteristic structure, number,
and distribution make a strong appeal to the natural desire of
scientists to focus attention on what can be relatively easily seen
and measured. The cytoplasm suffers because of its relatively
obscure and variable type of organization and structure.
Geneticists naturally first described and have largely studied
these characters which trace back to some real or hypothetical
unit which can be correlated with the chromosome. They have
found, however, in mammals that the vast majority of variations
in form or structure cannot be wholly accounted for by that
type of influence. Indefiniteness frequently appears in the picture. Many of us have therefore come to look upon the inherited or transmitted material other than the chromosomes as a
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potential seat of new elements to be discovered. One such case
appeared very strikingly in crosses made between strains of mice
which differed greatly in the amount of cancer of the breast
which they formed spontaneously. It was found that the influence of the mother upon the incidence of breast cancer in her
female progeny was from seven to ten times as great as was that
of the father. Since the female progeny inherited chromosomes
equally from both mother and father it is evident that the
greater influence of the mother depends upon something outside
of the chromosomes. The simplest working hypothesis for the
present is that this something may be inherent in or transmitted
by the cytoplasm.
How about cases, Dr. Reimann, in which growth characters
other than those influencing tumor formation show a cytoplasmic basis? Are there any such?
Dr. Reimann: There occurs to me immediately the question of
sex and while this, strictly speaking, cannot be ascribed to cytoplasmic influence, nevertheless, it has been shown that sex
destiny may be determined by factors other than chromosomes.
In some animals there is a cychc change of sex; thus the oyster
is a she one year and a he the next year. It cannot be because
the arrangement of chromosomes changes but because of some
other factor. Then there is sex reversal. Thus, the famous
rooster of Basle who started life as a perfectly good hen and after
several years as an egg-layer and good mother began to grow a
comb and spurs. Also he, she or it, greeted the morning like
chanticleer. You will remember that this hen-rooster or roosterhen was arrested, solemnly tried for witchcraft, and publicly
burned at the stake. Surely in this beast and others which
spontaneously change their sex, and in those in which it has been
changed experimentally, the chromosome x-y complex has not
been altered. The interest of pathologists in this lies in the fact
that certain tumors exert definite influence on sex characteristics,
such as the granulosa cell tumor and the arrhenoblastoma.
Breast conditions are also influenced, such as in gynecomastia
and certain benign tumors in both sexes.
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It is plain, therefore, that chromosomes are not the sole determiners of those processes which determine this fate. The cell is
a physiological unit and this in itself means that the cytoplasm
also must enter actively into determination, because the functional unity of a cell depends on active reaction between nucleus
and cytoplasm. It depends apparently on the viewpoint of the
observer which part appears to predominate. Furthermore, we
cannot ignore the localization problem. Inherited factors exert
effects on development and differentiation, but this effect is
often applied at a localized place. The individual is not a
conglomeration of chemically different matter but is, in all
senses of the word, an organism. Thus, if cytoplasm is indifferent in development, and if material particles of the chromosomes
are the sole determiners of development, it is extremely difficult,
if not impossible, to understand the typical localization of their
action. But apart from such general considerations, experiments
with bastardization have shown that twins result, for instance,
in triton when half blastomeres are experimentally constricted
and separated. If the plane of cleavage is in certain positions
one blastomere develops into a new organism and the other into
only a fragment. Notwithstanding the fact that the triton egg
has great capacity to regulate itself, i.e., compensate, the nucleus
of one of the half blastomeres cannot maintain itself even though,
according to the genes idea, it contains all of the anlage material.
This demonstrates activity on the part of the cytoplasm as well
as a determining effect. Similar experiments have shown that
the same thing holds true in certain insects. Perhaps, Dr. Little,
you could give another example or two.
Dr. Little: Indeed I can, Dr. Reimann. I can mention the
famous experiments of Wettstein on mosses in which leaf-shape
inheritance and the growth habit inheritance is purely cytoplasmic. This is so well known that it is even in standard text
books of botany. Then again chromosomes of one species of
Crepis, one of the compositae, have been transferred to the
cytoplasm of another without the second losing its cytoplasmic
character. Similar experiments have been done on Epilobium,
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another plant. Finally no one has as yet demonstrated chromatin, let alone chromosomes, in the so-called blue-green algae and
yet they breed true to form. The pollen tube of Pelargonium
carries no chloroplasts and the color of the leaf is therefore
inherited maternally. I need not call your attention to the fact
that bacteria have no nucleus in the ordinary sense of the word
and no chromosomes. While they are said to mutate with considerable ease, nevertheless it is possible to breed them true.
Dr. Reimann: I am very glad to hear about this because, after
all, a strict chromosomal theory of development is preformation.
We no longer think in terms of the picture of the little man inside of the spermatozoon, he in turn having little men inside of
his spermatozoa and so on ad infinitum as per the old idea of
preformation, or mere unfolding out of preformed structures.
I am afraid that complete dependence on the chromosomes is
just another of these preformation ideas. If development is
anything, it is certainly epigenetic; which means that as one part
is produced, it influences and exercises effects on the oncoming
parts as they are developed. Furthermore, the idea that both
nuclear and cytoplasmic constituents play r61es in development
gives more opportunity for fitting in the facts of potency to our
theories. You know, Dr. Little, that pathologists for many
years have worked very industriously attempting to trace back
the origin of tumors. They have not been content to say that a
tumor arises in such and such an anatomical situation, such as,
let us say, the side of the tongue, but they must trace it back
to a microscopic origin identifying such and such a cell or few
cells as having been the point from which the tumor arose. As
you know, an enormous amount of work has been devoted to this
subject, but since neoplasia is a dynamic consideration and the
material on which these studies have been made has been fixed
and stained, it does not surprise you that there are differences of
opinion and endless squabbles about the points of origin. I am
so glad to hear you say that indefiniteness frequently appears
in the picture, especially in mammals.
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Dr. Little: The existence of indefiniteness does not by any
means necessarily mean defeat. It really should be considered
a stimulus to further study in many different fields. A good
example of the value of such studies is the work done by Dr.
D. F. Jones at New Haven. Dr. Jones is a plant geneticist.
He works chiefly with Indian corn—or maize. In this plant
there are many mendelian characters known to depend primarily
upon genes. The relationship of these genes to one another has
been carefully analyzed. An excellent place to study the variation in such genes and in other chromosomal characters is in the
endosperm of the plant. As you know, the endosperm is derived from gametic tissue—that is to say, tissue in which changes
in the chromosomes will at once be evident. Dr. Jones found
that minor variations in the distribution of chromatin material
were fairly frequent. These small changes in chromatin distribution often lead to defects or as geneticists call them, deficiencies. Usually they produced only pigment changes but in a
few cases actual morphological variations appeared. These were
either overgrowth, under development, or abnormal growth of
tissues usually controlled or orderly. The bearing of these experiments on the theory of tumor origin in mammals is indirect
but highly suggestive.
Dr. Jones' work is also of interest in its bearing on Cohnheim's
theory of embryonic rests. What do you think, Dr. Reimann,
is the present status of that theory—or rather, how do you see
its application to the experimental work which you and your
associates are doing?
Dr. Reimann: Suppose I put it this way: The fertilized ovum
contains all the potencies necessary for the production of a whole
new individual. As the descendants of this fertilized ovum
display these potencies they turn into different types of cells
and then organize together into different kinds of tissues, parts
and organs. This indefiniteness which you mention can be correlated with several principles in experimental embryology, first,
and very important, every cell which has not used up all of its
potencies still has more than it normally expresses. For ex-
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ample, ordinarily but one new organism arises from a fertilized
ovum, but under certain circumstances two or even more may
appear. Thus the quintuplets. And by separating the two
cells of a divided fertilized sea urchin ovum it has been possible
to obtain at least eight sea urchins from one single cell. This
cell therefore has the potency of producing not one, but at least
eight complete, competent sea urchins. Then there are the regulatory phenomena whereby in typical, so-called regulatory eggs,
cells can be removed from very young embryos and the completed organism shows no defect. Thus, the potency of the cells
remaining would normally have been exerted to produce a certain given part; nevertheless, when necessary, other parts can be
produced. Even in the completed organism regenerative and
similar phenomena show that more potency exists than is normally expressed, for regenerates themselves can regenerate, and
regenerated regenerates can again regenerate in various organisms in various parts. This means, as far as the origin of
tumors is concerned, that when they arise from cells which have
not lost their potencies, different kinds of potency may be expressed.
Experimental embryology has shown also that when differentiation of a cell has proceeded to a certain point it can no longer
retrace its steps, that is, it can no longer dedifferentiate. It is
only in very primitive organisms and in primitive cells that dedifferentiation has been found.
A third point is that after a cell has reached a certain degree of
differentiation it can no longer divide. Unfortunately degrees of
differentiation have not as yet been measured quantitatively
with any degree of accuracy. In the very nature of the problem you can see what a complicated task this measurement is,
for each cell has its own type of differentiation and its degree.
Is there anything contrary to the findings of genetics when, from
the point of view of experimental embryology, we say (1) tumors
arise from incompletely differentiated cells; (2) if incompletely
differentiated they have more potencies than they express and
(3) it is possible to have developed all sorts of differentiations
from any particular, incompletely differentiated cell or a group
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of them. Consequently instead of searching as was done in the
past for the origin of this, that and the other tumor, may we
not say that they arose from division-capable, therefore incompletely differentiated, cells, therefore with multiple potencies,
and therefore with the possibility of producing different anatomic
pictures? This, at least, focuses attention on what protoplasm
can do and makes so much more interesting the newer experiments in transplantation, with organizers and so on.
Dr. Little: I see nothing contrary to genetics in the situation
that you outline. Of course, we must remember that we have
no real idea of the number or extent of the earliest stages of
processes which, if continued, may give rise to neoplastic growth.
It is entirely conceivable that there may be hundreds of centers
of abnormal growth which become abortive before a critical or
even a discernible disturbance in morphology takes place. Our
methods of observation as commonly employed are very rudimentary and primitive. It would perhaps be reasonable to
change the major premises which we make and query whether it
is not remarkable that more centers of abnormal growth do not
appear rather than that so many do. From a biologist's point
of view the importance of "malignancy" as such is not so great.
The real question is what can start growth and cell development
independent of centralized control? What causes or allows cells
to resume or to assume a type of growth which characterizes
lower forms of life or the very early stages of mammalian ontogeny?
Dr. Reimann: The crux of the situation in malignancy from
the pathologist's point of view is that while the cells may differentiate and thus produce good anatomic pictures of, let us say,
glands, and physiologically, may differentiate even to the point
of producing mucus or hormones as from particular testicular
tumors, they nevertheless do not organize. From many considerations such as the fact that ofttimes in the very same environment, malignant cells and normal cells display differences when
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growing side by side, the latter organizing but the former not,
it appears that some sort of change has taken place within the
cell which destroys its ability to organize and incidentally to
differentiate properly. Since this change occurs in a somatic and
not a sex cell it has been called somatic mutation. Of course we
all realize that the word "mutation," as ordinarily used, means
a change occurring within the cell which leads to the production
of descendants differing from their forebears. Nevertheless in
ordinary mutation, these changed cells do organize. There is
this difference. In ordinary mutation organization takes place,
in malignancy it does not. Could you tell us, Dr. Little, a few
things about what has been learned in genetics about somatic
mutation?
Dr. Little: The reference to Jones' work, already made, bears
on this question and need not be repeated. It may be helpful
to remember that the definition of a mutation is that it is a sudden and self perpetuating genetic change. It is obvious that a
cell or cells which give rise to a neoplasm meet this definition.
That is not the whole question, however. Such a genetic change
may theoretically occur in (1) a gene, (2) a portion or block of a
chromosome larger than a gene, (3) in a whole chromosome, (4)
in a whole set of chromosomes, (5) in the cytoplasm. If it occurs
in a cell other than a germ cell it is said to be "somatic" i.e. of the
body. It is evident that the influences which cause such a
change may be solely initiated by the introduction of external
agents such as radiation or application of carcinogenic substances. Since the degree or extent of such external stimuli
can be changed experimentally it is evident that they comprise
one variable in the situation. There must also be considered
the reaction of the cell to such stimuli. This reaction may be
largely determined by genetics as expressed in the material
composition and type of organization of the cell. It may also
be modified by the relative proportion of various chemical secretions and stimuli produced in different types of internal environments within the animal's body. These in turn may depend
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C. C. LITTLE AND S. P. KEIMANN
partly upon different genetic influences as expressed by various
degrees of activity of enzyme activity or hormonal secretion.
The situation is therefore complex but lends itself to and demands much more extensive investigation than it has yet been
able to receive.
Dr. Reimann: This is indeed very interesting, Dr. Little, and
so we may consider somatic mutation as a possible working
hypothesis. Might I imagine a state of affairs such as this:
I would like to picture a cell which can still do something, that
is, which has not exhausted its potencies, as having in it chemical compounds with numerous open bonds. It has actually
been shown that immature tissue has in it much more general
and more labile groups of compounds than mature tissue. As a
cell matures may we picture that those open bonds are satisfied
in certain directions, but that the satisfaction of these bonds is
not definitely fixed but merely guided along general lines by
inherited characteristics? Thus, for instance, the cell must
remain species specific, but within this wall it may do numerous
things, and what it does depends on the environment, in the
broad sense of the term, in which it finds itself; in other words,
what building blocks are offered to it, and what physical forces
are exerted upon it? Now when a cell becomes malignant it
cannot utilize these building blocks nor does it obey the physical
forces which ordinarily make it organize.
Dr. Little: Such a conception would be perfectly consistent
with what geneticists have found. In fact the degree of elasticity, chemically speaking, possessed by its cells may well be the
fundamental basis for such evolutionary differences as one finds
between such fixed and inelastic types as insects and such versatile and adaptable types as mammals. It may not be an accident, therefore, that theoretical genetics have been chiefly advanced by studies in insects while the practical relationship
between the fixed inherited elements and the more unpredictable
phases of growth and development remain very largely a mystery
in mammals and similar forms.
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Dr. Reimann: It seems, therefore, Dr. Little, that we are face
to face with the organization problem in cancer. Biology has
gone through periods in which certain large problems have occupied the center of attention such as descriptive morphology,
evolution and genetics. Do you not think that in the future
the organization problem will come increasingly to the fore?
Just why do organisms organize the way they do into replicas of
their parents? After all, geneticists are hard at work on this,
and experimental embryologists also. The more we pathologists
see of malignancy the more subtle do the differences appear
between normal and cancer cells and the more insistent does the
organization problem become. A few words from you on this
would surely be appreciated, and then perhaps you will be good
enough to sum up a bit and indicate in a general way the direction in which this biological problem should be attacked and
wherein we pathologists can help and wherein we can keep our
eyes open for contributions from geneticists and others in this
field.
Dr. Little: To sum up I should advocate the general adoption
by all active in experimental pathology and other branches of
experimental medicine of the principle of using known genetic
stocks of animals. These will be homogeneous and predictable
to the highest degree possible in mammalian material. Second
I should advocate the variation under experimental conditions
of the factors of internal environment such as sex, age, amount
of internal secretion, nutrition, various degrees of exposure to
stimuli, etc. Then, keeping these latter factors constant, I
should begin to vary the genetic types of animals used. One
could thus gain a much more accurate picture of the relative
importance of genetic and other agents in the etiology of all
growth phenomena including cancer.