Chromosomal Enlargement in Neoplastic Rabbit
Tissues
john J. Biesele, Ph.D.*
(From the Department o/Zoology, University o/Pennsylvania, Philadelphia 4, Pennsyh'ania)
(Received for publication October 19, 1944)
A study of the chromosomes of rabbit neoplasms
was undertaken for several reasons. The primary
object was to determine whether the enlargement of
chromosomes to double or quadruple their original
size occurred in malignant tissues of yet another
organism, the rabbit, as it had been observed to occur in cancers of the mouse (8), goldfish (2)., man
(7), and the rat (3). The secondary aim was to
learn whether chromosomal enlargement was possibly of value as a morphological indicator of autonomy
in tumors.
absolute alcohol. Several days later small bits of
the tissues were stained in acetocarmine, spread out
under coverglasses, dehydrated in alcohol, and mounted
in diaphane.
The acetocarmine preparations were studied under
oil immersion, and camera lucida drawings at a
magnification of 3,000 times were made of 25 metaphase figures of each specimen in most of the cases.
In the mammary tumor at autopsy mitoses were rare
and only 10 were drawn; and 30 metaphases were
drawn from the adenocarcinoma of animal X1800-2.
The average volume of the chromosomes of: each
metaphase was calculated as previously described
MATERIALS AND METHODS
(7, 8).
The tissue specimens used were kindly provided
after fixation by Dr. Harry S. N. Greene, of the Yale
University School of Medicine. In addition to the
small intestine of an adult female (obtained elsewhere), the normal material included the liver, kidney,
and lung of a 24 day old.fetus. Among the tumors
examined was a transplant of an adenocarcinoma
of the uterus of an animal designated X1800-2. This
adenocarcinoma was of proved growth autonomy in
the anterior chamber of the eye (10). Two other
uterine tumors studied, from animals M101-3 and
X16072-2, were of doubtful or negative autonomy.
That from the latter animal was an adenoma of the
endometrium. Examinations were made of the chromosomes of specimens of a breast tumor of animal
HA672-2 taken at biopsies on November 11, 1942,
on January 7, 1943, and on April 22, 1943, as well
as at autopsy after sacrifice on June 16, 1943. According to Dr. Greene (10), this mammary tumor
appeared histologically to be cancerous, but it was of
negative growth autonomy and autopsy revealed no
invasion by it or metastases from it. In the uterus
of animal HA672-2, however, there was found at
autopsy a primary adenocarcinoma, which grew slowly
in some of the anterior chambers to which it was
transplanted. The chromosomes of this adenocarcinoma were Mso investigated.
The specimens were fixed in Carnoy's fluid, composed of 1 part of glacial acetic acid and 3 parts of
* Fellow of The International Cancer Research Foundation,
1942-4; now at the Carnegie Institution of Washington, Department of Genetics, Cold Spring Harbor, New York.
An attempt was made to analyze the nucleoli of
resting nuclei in order to determine the probable
initial number of plasmosomes before fusion set in.
While some data were obtained, t h e acetocarmine
preparations of rabbit tissues unfortunately did not
prove to be favorable material for the study of
plasmosomes.
OBSERVATIONS
Table I sets forth the frequencies of metaphase
figures containing chromosomes of certain average
volumes. The 3 embryonic tissues (Figs. 1 and 2)
had average chromosome volumes of about 0.4 and
0.5 cubic micron, while the chromosomes of the adult
small intestine were slightly larger. The 4 normal
tissues had unimodal distributions of metaphase~
according to average chromosome volume. The
maximum number of plasmosomes in the resting
nuclei of these normal organs seemed to be 6.
In the uterine adenocarcinoma of animal X1800-2,
on the other hand, 3 groups of metaphases according
to average chromosome volume could be distinguished
(Figs. 7 to 10). Their respective means fell at about
0.5, 1.2, and 2.4 cubic microns. The second group
was numerically predominant. It was composed
chiefly of diploid division figures, although the 2
chromatids of each metaphasic chromosome were
often so widely separated that at first glance many
of the figures appeared to be polyploid. Since most
of the resting nuclei of the adenocarcinoma had a
dozen or slightly fewer plasmosomes, it is probable
that the chromosomes of the predominant second
179
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Cancer Research
180
group of metaphases had twice the normal number
of strands. It is also likely that the chromosomes
of the one figure in the third group, which averaged
2.4 cubic microns in volume, were constituted of
4 times the normal number of chromonemata. This
metaphase is illustrated in Fig. 10. The uterine
adenocarcinoma of HA672-2 also seems to have contained chromosomes of double the normal complexity,
although in a minority of its dividing cells (Fig. 6).
The apparently nonautonomous uterine tumors of
animals M101-3 and X16072-2 did not give evidence
of a chromosomal change from the normal condition
TABLE I:
Average
chromosome
volume,
~3
0.2-0.3
0.3-0.4
0.4-0.5
0.5-O.6
0.6-0.7
0.7-0.8
0.8-0.9
0.9-1.0
1.0-1.1
1.1-1.2
1.2-1.3
1.3-1.4
1.4-1.5
Polyploidy was found in some of the neoplasms.
The diploid number of rabbit chromosomes, it will
be recalled, is 44 (14). The breast tumor of animal
HA672-2 had 2 approximately tetraploid figures
among 85 metaphases, and the uterine adenocarcinoma
of this animal had one octoploid figure with small
chromosomes among 25 figures examined. Of the
30 metaphases drawn from the adenocarcinoma of
X1800-2, 8 were in the tetraploid range with about
70 to 90 chromosomes, and 2 had about 120 chromosomes. One of the latter metaphases is illustrated
in Fig. 8. Polyploidy in the cancer of X1800-2 was
FREQUENCIES OF METAPHASES BY AVERAGE CHROMOSOME VOLUME IN RABBIT TISSUES
Normal organs
24 (lay fetus
Adult Ut. adenoearcinomas
Ut. adenomas
laver Kidney l.ung Sin. int. X1800-2 HA672-2 M101-3 X16072-2 Nov.
4
2
6
1
9
2
8
4
1
1
8
8
7
3
1
3
4
3
3
3
9
7
11
4
3
6
9
I
4
1
10
2
9
7
9
7
1
3
2
4
3
3
1
1
3
1
1
7
1
3
1
6
5
Mammary tumor
HA672-2
Jan.
Apr.
1
1
1
9
6
3
13
6
1
1
6
1
1
0.6_6
1.04
0.65
0.99
June
3
2
3
1
1
1
1
2.4-2.5
NIEAN CHROMOSONIE \'OLU~IES IN CUB1C NI1CRONS
Simple
Double
Quadruple
0.40
0.4{)
0.38
0.56
0.51
1.17
0.56
1.02
0.61
0.59
0.65
1.11
0.49
2.44
(Fig. 3). Their average chromosome volume, 0.6 cubic
micron, can be regarded as essentially normal, and in
t h e absence of a normal control specimen it can be
used as a basis for estimating the expected volume
of double chromosomes in the adenocarcinomas.
The last 3 of the 4 specimens taken from the probably regressing mammary tumor of animal HA672-2
showed a progressive decrease in frequency of metephases interpreted as containing double chromosomes.
The specimen taken at autopsy was notable for the
smallness of its chromosomes. In the first 2 biopsy
specimens the bimodality of frequency of metaphases
according to average chromosome volume provided
additional evidence of the presence of double chromosomes, although no nuclei were noted with more than
6 plasmosomes.
The data on chromosomal enlargement and autonomy in the various rabbit tumors are assembled in
Table II. The 4 entries under the percentage of
metaphases with enlarged chromosomes in the mammary tumor are those for the 3 biopsy and the 1
autopsy specimens.
largely or perhaps entirely confined to metaphases
with enlarged chromosomes. We do not consider
polyploidy to be of especial significance in cancer,
although our earlier concept (8) of endomitosis that
could give rise either to polyploidy or to polyteny,
depending on the completeness of the reduplication
and separation of sister strands, might be construed
as suggesting that a tissue in which polyteny was
found was also liable to contain instances of mitoses
with multiples of the normal number of chromosomes.
DISCUSSION
The rabbit appears to have been among the first
organisms in which chromosomal changes of the type
under discussion were noted. Barratt (1) in 1907
injected Scharlach R dissolved in olive oil subcutaneously in the rabbit, and in the subsequent extreme
hypertrophy of the Malpighian layer of the skin
there occurred altered chromosomes. They were
coarser than the chromosomes found in the testis, and
in a minority of the dividing epidermal cells the
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Biesele--Chromosomal Enlargement in Neoplastic Rabbit Tissues
chromosomes were so large that some of them: no
longer preserved the form of curved rods. In these
cells the chromosomes appeared to be in pairs or
in undivided pairs. Although the epidermal pro-
181
as well as those of carcinomas descended from such
skin, probably contained a greater number of strands
than is normally found (5).
T h e 4 normal rabbit tissues examined showed sonmwhat different sizes of chromosomes, and Painter
(14), moreover, has found spermatogonial chromosomes of the rabbit to be considerably smaller than
amniotic chromosomes. Hence it is possible that
the rabbit has much the same variability in chromosome size from organ to organ as has been described
in the rat (3). Since there was no difference in maxim u m number of plasmosomes per resting nucleus
from one rabbit organ to another in our small series,
it may be inferred that the observed differences in
size of normal chromosomes were not expressions of
polyteny. The larger series of organs studied in the
rat (3) permitted the conclusion to be drawn that
"FABLE II: CHROMOSOMAL ENLAR(;EMENT AND AUTONOMY 1X
RABBIT TUMORS
Neoplasm
8
Fro. 1.--Metaphase from fetal rabbit liver; 44 chromosomes
of an average volume of 0.4 cubic micron.
Fic. 2.--Metaphase from fetal kidney; 44 chromosomes
averaging 0.5 cubic micron in volume.
Fie. 3.--Metaphase f r o m uterine adenoma of rabbit
X16072-2; 44 chromosomes averaging 0.6 cubic micron in
volume.
Fro. 4.--Metaphase from biopsied inammary tumor of
HA672-2; 44 chromosomes averaging 0.8 cubic micron in
volume.
Fro. 5.---Metaphase from biopsied Inammary tumor of
HA672-2: 44 chromosomes averaging 0.7 cubic inicron in
volume.
Fic. 6.--Metaphasc from uterine tumor of HA672-2 :it
autopsy; 42 chromosomes, possibly double, avcraging 1.1 cubic
micron in volume.
Fro. 7.--Metaphase from uterine adenocarcinoma of X180(/-2:
47 simple chromosomes averaging 0.5 cubic micron in volume.
Flo. 8.--Metaphase from uterine adenocarcinolna of X18011-2:
about 117 doublc chromosomes averaging 1.1 cubic micron in
VO] U n l e .
Fie,. 9.--Metaphase from uterine adenocarcinoma of X1800-2;
49 double chromosomes averaging 1.2 cubic micron in volume.
FIG. l 0,--Metaphase from uterine adenocarcinoma of X 1800-2"
51 quadruple chromosomes averagin~ 2.4 cubic microns in
volume.
liferation subsided when the Scharlach R disappeared,
it may be conjectured that Barratt observed a process
similar to that later noted (6) in mouse skin painted
with a benzene solution of methylcholanthrene. In
the latter case, application of the carcinogen was followed in several days by the appearance of enlarged
chromosomes in about one-tenth of the dividing cells.
A further cytological analysis has indicated that the
enlarged chromosomes of the treated mouse skin,
Metaphases
with enlarged
Animal
~.~
ell rOlllOSOllles~
Uterine adcnocarcinoma X1800-2
"
HA672-2
87
12
"
adclaoma
X16072-2
0
"
tumor
M101-3
0
Manunary "
HA672-2 16, 32, 4, 0
+ From 11. S. N. Greene (10).
AtltOllOnly ~
Strongly positive
Weakly positive
Negative
lc
within a species a certain average chromosome volume
was characteristic of a given cell type under given
conditions. Chromosomes of several organs in the
adult rat, notably the liver and kidney, were considerably larger than their counterparts in the newborn rat. The enlargement of the chromosomes in
the adult organs was again not accompanied by
changes in plasmosome number, although there were
such nucleolar alterations concomitant with the chromosomal changes in neoplastic tissues.
Most of the cells in the adenocar.cinoma of animal
X1800-2 contained double chromosomes of the type
found in cancers of other organisms. Apparently such
chromosomes were also present in some of the cells
in the uterine adenocarcinoma of HA672-2, and in
about one-fourth of the dividing cells of the early
specimens taken from the m a m m a r y tumor. In the
2 other new growths examined no enlarged chromosomes were to be seen. T h a t these 2 adenomas were
also lacking in growth autonomy is of interest.
By reference to Table II it may be seen that although our small selection of tumors yielded little
evidence to the contrary, the data are hardly sufficient to justify the assumption of a connection between
autonomy of a tumor and the structural condition
of the chromosomes in the majority of the cells. This
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Research.
182
Cancer Research
may likewise have been indicated by the studies of
Greene. A consistent relation was not found, for
instance, between autonomy and the degree of anaplasia in tumors (12). T h e relation between anaplasia and cancerous chromosomal change of the sort
involved here is itself poorly defined, although
Ehrich (9) regarded the generalized doubling and
quadrupling of nuclear volume in cancers as an
anatomical expression of anaplasia, and we have shown
this nuclear volume doubling probably to result from
doubling of chromosome strands (7). Furthermore,
the positive growth autonomy of embryonic tissues
(11) would demand a chromosomal similarity between embryonic and autonomous malignant tissues,
if the chromosomes were intimately concerned in autonomy. The embryonic tissues of the rabbit, mouse,
and rat subjected to examination have contained uniformly simple chromosomes, which obviously do not
agree in point of structure with the double chromosomes of malignant tumors. Although a functional
similarity has been suggested for chromosomes of
embryonic tissues and cancers of the rat (4), the
question of the relation of chromosomes to autonomous
growth remains undecided.
W e wish to lay greater stress in this paper on the
finding of enlarged chromosomes, of the double type
seen in other cancers, in the rabbit's uterine adenocarcinoma. The rabbit now becomes the fifth organism proved to have such chromosomes in neoplastic tissue. Three of the 5 organisms are rodents,
and it may be expected that cancers of other rodents
will be found to be marked by the possession of similar
enlarged chromosomes. Four of the 5 organisms are
mammals, and all 5 are vertebrates. A m o n g invertebrates, polytene chromosomes are often of normal
occurrence, especially in insects. If the structural
differem-e between chromosomes of normal and malignant tissues in the vertebrates is of causal significance
in malignancy, then it may be expected that the enlarged chromosomes of vertebrate cancers will prove
to be different in some respect from the polytene
chromosomes of insect tissues. Possibly chromosomal
alterations are also involved in the abnormality of
Mottram's carcinogen-treated ciliate protozoa (13).
Mottram has found the abnormal ciliates to have a
multiple constitution in the possession of 2 or more
micronuclei, which are sometimes double or quadruple the normal size, instead of 1 small micronucleus as in the normal controls.
SUMMARY
An adenocarcinoma of the rabbit uterus was found
to have a high frequency of metaphases, the majority
of which were diploid, containing chromosomes about
twice as large as those in normal tissues, and this
neoplasm also had about 12 plasmosomes in most
of its resting nuclei. Since the 4 normal tissues examined seemed to have a m a x i m u m plasmosome nun>
ber of 6 per nucleus, it is probable that the enlarged
chromosomes of the adenocarcinoma contained twice
as many strands as the chromosomes of normal tissues. Similar enlarged chromosomes were present in
lower frequency in another uterine adenocarcinoma
and a m a m m a r y tumor, but none was found in 2
apparently benign tumors of the uterus.
Although the evidence is suggestive, the series of
tumors examined is too small for definite conclusions
to be drawn concerning the relation of chromosomal
conditions to growth autonomy.
The rabbit becomes the fifth organism to show a
chromosomal enlargement by doubling of strands
in its malignant cells.
ACKNOWLEDGMENT
I am indebted to the late Dr. Edgar Allen and to I)r. Harry
S. N. Greene for initiating this study.
REFERENCES
1. BAR~ATT,J. O. W. On Mitosis in Proliferating Epitheliun3.
Proc. Roy. Soc., London, s. B, 79:372-377. 191)7.
2. BIESELE, J. J. l)iplochromosomes in a Goldfish Tumor.
Cancer Research, a:411-412. 1943.
3. BIESELE,J. J. Chromosome Size in Normal Rat Organs in
Relation to B Vitamins, Ribonucleic Acid, and Nuclear
Volume. Cancer Research, 4:529-539. 1944.
4. BIESELt, J. J. Size and Synthetic Activity of the Chromosomes of Two Rat Neoplasms. Cancer Research, 4:540546. 1944.
5. BI~-SELE, J. I. Ribonucleic Acid and Heterochromatin in
Epidermal Carcinogenesis. Cancer Research, 4:737-750.
1944.
6. BIFSVLE, J. J., and CownRv, E. V. Chromosomal Changes
in Epidermal Carcinogenesis. J. Nat. Cancer Inst., 4:373384. 1944.
7. BIESELE, J. l., and Pov.',q:R, H. Polytene Chromosomes in
Two Mammary Carcinomas of the Human Subject.
Cancer Research, 3:779-783. 1943.
8. BI~SELE, J. J., POVNER, H., and P;UXa'ER, T. S. Nuclear
Phenomena in Mouse Cancers. Austin: University of
Texas Publication No. 4243. 1942.
9. EnRmn, W. E. Nuclear Sizes in Growth Disturbances.
With Special Reference to the Tumor Cell Nucleus. Am.
1- M. Sc., 192:772-790. 1936.
10. GREEXV,H. S. N. Personal communication.
11. GREEXE, H. S. N. The Heterologous Transplantation of
Embryonic Mammalian Tissues. Cancer Research, ,3:809822. 1943.
12. Ga~v.XE, H. S. N., and Luxn, P. K. The Heterologous
Transplantation of Human Cancers. Cancer Research,
4:352-363. 1944.
13. MOTTRAM, I. C. The Multiple Constitution of Abnormal
Ciliates Produced by Blastomatogenic Agents. Cancer
Research, 4:241-244. 1944.
14. PAIYWER, T. S. Studies in Mammalian Spermatogenesis.
VI. The Chromosomes of the Rabbit. 1. Morph., 4 a :
1-44. 1926.
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Chromosomal Enlargement in Neoplastic Rabbit Tissues
John J. Biesele
Cancer Res 1945;5:179-182.
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