[CANCER RESEARCH
28, 82ÃŽ-830,
May 1968]
Karyotypes
of Rats from Strains
Mammary Cancer Induction1
of Different
Susceptibility
to
E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, and Joe R. Pugh
Departments
o¡Medicine and Pharmacology,
University
o¡Kentucky,
SUMMARY
The female Sprague-Dawley rat is quite vulnerable to the
induction of mammary carcinomas by 3-methylcholanthrene
and by 7,12-dimethylbenz(a)anthracene,
a majority of the car
cinomas induced are sex hormone dependent. The Long-Evans
rat is relatively resistant to mammary carcinoma induction but
is more susceptible to leukemia. Karyologic studies were per
formed on cells from rats of both strains. No difference was
noted in the X or Y chromosome. In the Sprague-Dawley,
both members of the #3 pair were subterminal, whereas the
members were generally heteromorphic (one subterminal and
one terminal) in cells from Long-Evans rats. In inbred LongEvans rats in particular, the #12 chromosome was interesting
in that one of the members frequently had larger upper arms
than the other, and, not infrequently, one of the homologs had
large satellites on prominent upper arms. Study of diploid cells
of both strains indicated a tendency, especially after adminis
tration of 3-methylcholanthrene
or 7,12-dimethylbenz(a)anthracene, for a chromosome of subterminal morphology to be
missing and a terminal chromosome to be gained, apparently
through loss of upper arms of the former. Karyotypes of inbred
Fischer, Marshall, and Osborne-Mendel rats were also estab
lished. The present data do not indicate a correlation between
karyotype and susceptibility to mammary cancer induction.
INTRODUCTION
In a study of the karyotypes of three strains of laboratory
rats, Hungerford and Nowell (6) noted polymorphism of the
X chromosome in the noninbred Lewis and Wistar (Shay)
strains. In these two strains, the Y chromosome was the small
est terminal chromosome, whereas in the BN strain, the Y
chromosome could not be distinguished from medium-sized
terminal autosomes. Fitzgerald (2) previously determined a
Wistar karyotype and found that the Y chromosome was the
smallest terminal chromosome and the X chromosome was one
of the largest terminal chromosomes (but less distinctive than
the Y). The results were in accord with those obtained earlier
on the laboratory rat at Lund by Tjio and Levan (14). In each
1Supported by a grant from the AMA-ERF. J. C. C. and J. R. P.
were summer student fellows supported by institutional funds
provided by the American Cancer Society and the National Can
cer Institute.
Received September 7, 1967; accepted January 13, 1968.
Lexington,
Kentucky
40506
of these rat strains, there were 5 subterminal, 8 terminal, and
7 median autosomal pairs. More recently, Yosida and Amana
(16) reported similar findings in their karyologic studies of
several strains of laboratory and wild rats; however, polymor
phism of the #3 chromosome pair was noted in some strains.
Bianchi and Molina have described polymorphism of the small
est subterminal chromosome in their strain of laboratory rat
(1).
Sydnor et al. (13; personal communication) have demon
strated differences between rat strains in susceptibility to mam
mary cancer induction by oral administration of the polycyclic
aromatic hydrocarbon carcinogens, o-methylcholanthrene and
7,12-dimethylbenz(a)anthracene.
In view of (a) the sex-hor
mone dependency of these induced mammary cancers (3), (b)
the differential susceptibility of rats of different strains to form
these cancers (13), and (c) the X chromosome polymorphism
noted in at least one rat strain (6), a study of the karyotypes
of rat strains of differing susceptibility to mammary cancer
induction was initiated. The susceptible Sprague-Dawley strain
and the relatively resistant Long-Evans strain were primarily
emphasized in the present study, but the karyotypes of Fischer,
Marshall, and Osborne-Mendel rats were also established.
MATERIALS AND METHODS
Sprague-Dawley rats were obtained from the Holtzman Com
pany (Madison, Wisconsin). Long-Evans rats were provided
by Dr. Katherine Sydnor and were all descended from a single
mating pair (obtained from Diablo Farms, Inc., Berkeley,
California). The animals were maintained in stainless steel
cages with wire bottoms in an air-conditioned room. Water
and chow pellets were available ad libitum. Liver was provided
once a week and lettuce twice a week. Most of the animals
were 50-100 days old at the time of the study, but younger
and older animals were also studied; however, no variation in
karyotype was noted with age, though older animals generally
did not provide as many good spreads. Chromosomes were
prepared from marrow of the femur and tibia by the method
of Tjio and Whang (15) and stained with aceto-orcein. Photo
micrographs of 22 suitable spreads were obtained and enlarged
to about X 5500 for chromosome measurements. At the time
of karyotype construction, each chromosome in a photograph
was checked against the preparation by microscopic exam
ination. Karyotypes (Figs. 1-5) were arranged according to
Hungerford and Nowell (6). The mean relative length of
chromosomes and the accompanying standard deviations and
standard errors were also calculated.
MAY 1968
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823
E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, and Joe R. Pugh
In the latter part of this study, chromosome spreads were
prepared using the fixation procedure of Moorhead et al. (9),
since a greater number of good spreads were obtained. These
animals were injected intraperitoneally with 0.75 mg of colchicine in 1 ml of isotonic saline a half hour before the animal
was decapitated, and the femoral and tibial marrows were re
moved for study. The slides were stained with aceto-orcein or
with Giemsa stain. In order to minimize statistical bias in
evaluating the distribution of chromosome number per cell,
counts of chromosome number were limited to no more than
10 cells per rat (225 cells from 23 Sprague-Dawley rats and
137 from 14 Long-Evans rats). For the Sprague-Dawley strain,
90% of all cells were diploid; for Long-Evans, 95% were diploid. With few exceptions, deviations from diploidy were
numbers smaller than 42 that probably represented an artifact
of the technic mainly. The study of both rat strains was con
current.
Since a karyotypic difference between the Long-Evans and
Sprague-Dawley strains was found, additional strains of inbred
rats of the Fischer, Marshall, and Osborne-Mendel strains were
obtained for study from Dr. Katherine Sydnor, who had deter
mined their susceptibility to mammary cancer induction. The
karyotypic studies of these three strains were not as extensive
as for the Long-Evans and Sprague-Dawley animals; but at
least 10 good preparations from each of at least 5 rats of each
strain were carefully examined. Also, the influence of polycyclic
hydrocarbons on marrow cell chromosomes of 50-day-old fe
male Sprague-Dawley rats was determined by studying prep
arations 1-16 days after intragastric administration of a single
dose of either 3-methylcholanthrene (100 mg) or 7,12-dimethylbenz(a)anthracene
(20 mg).
RESULTS
It is evident by microscopic examination, as well as by sta
tistical analysis of karyologic measurements, that certain pairs
of chromosomes are distinguishable and others are not. Al
though the largest median chromosomes are significantly larger
than the shortest terminal autosomes, the karyotype system
of Hungerford and Nowell (6) was followed for reasons of
simplicity and in recognition of the limitation (10) of pairing
by length (adjoining chromosomes in the 4-10 and 14-20 group
could not be distinguished from one another). The two largest
subterminal (#1 and #3) pairs, the largest terminal (#2)
pair, and generally the Y chromosome were readily distinguish
able in both Sprague-Dawley and Long-Evans rats. The X
chromosome seemed to be the second largest terminal chromo
some, but often could not be distinguished definitely. Poly
morphism was not recognized in the X chromosome of either
the Sprague-Dawley or the Long-Evans rats. On plotting the
arm ratios of the six smallest subterminal chromosomes against
their relative lengths in the manner of Patau (10), the #11
and #13 chromosomes of both strains fell into completely dis
tinct groups, but the group of #12 chromosomes overlapped
somewhat the margins of the #11 and #13 groups. Due to
shorter upper arms, the #12 subterminal pairs had greater
long arm/short arm ratios than did the #11 and #13 pairs.
One of the #12 homologs not infrequently had larger upper
arms than did the other, especially in cells from Long-Evans
824
rats. In good preparations, each subterminal chromosome could
generally be classified on careful microscopic examination. It
should be mentioned that, according to the criteria and nomen
clature of Levan et al. (8), the centromere of the #11 and
#13 chromosomes is in a submedian rather than subterminal
position; more specificially, these authors suggest that what
we specify for convenience as subterminal chromosomes in the
rat should be designated as smst (submedian-subterminal)
chromosomes.
Most of the variability in gross chromosome morphology
observed in both strains was in the subterminal chromosomes.
In the Long-Evans rats, almost invariably one of the #3
homologs was terminal and the other was either satellited or
had definite upper arms (Figs. 6, 7). Occasionally both mem
bers were terminal. Generally both members of the #3 pair
in the Sprague-Dawley rat were subterminal ; but it was not
unusual for satellites, rather than definite upper arms, to be
present on one or both members (Fig. 8). Rarely one of the
homologs appeared to be terminal. Both members of the #11
pair were subterminal in both strains, and, in some cells of
some Long-Evans rats, at least one of the homologs possessed
satellites. One or both of the members of the #12 pair often
had satellites rather than definite upper arms in both SpragueDawley and Long-Evans rats (Fig. 6). In both strains the
upper arms of the #12 chromosomes were shorter, less plump,
and less spread apart than those of the #11 chromosomes
(Figs. 6-9). Not infrequently a single morphologically unique
#12 (Fig. 7) was seen in cells from the inbred Long-Evans
rats but not Sprague-Dawley rats. The general appearance of
this chromosome was one of large satellites extending from
prominent upper arms; sometimes a secondary constriction of
the lower arms was suggested instead. We have not seen this
sort of chromosome in cells from noninbred Long-Evans rats
obtained from several commercial sources. Occasionally satel
lites were seen on one or both of the #13 sub terminal chromo
somes in the Sprague-Dawley rats (Fig. 9) ; this was noted
quite often in the Long-Evans rats (Fig. 7). More recently,
we have carried out karyologic studies on Sprague-Dawley rats
obtained from commercial sources other than Holtzman, and
we have detected no definite difference in Sprague-Dawley
karyotype in these rats.
Only minor differences were observed in the karyotypes of
the Osborne-Mendel, Fischer, and Marshall strains. The X
chromosome(s) could frequently be distinguished in the Mar
shall and Fischer strain but very seldom in the Osborne-Mendel
animals. The Y chromosome also was quite distinctive in the
Marshall and Fischer rats, though appearing somewhat more
globular in the latter. In Osborne-Mendel males, the Y chro
mosome could only occasionally be definitely recognized. In
all three strains, both members of the #3 chromosome pair
were subterminal; they were frequently satellited in the Mar
shall and Fischer rats. Only occasionally was a satellited chro
mosome noted in the marrow cells of the Osborne-Mendel rats
(generally on a #11 chromosome). Satelliting was also com
mon on #13 chromosomes in the Fischer and Marshall rats.
Differences in the size of the upper arm of the #12 chromosome
was not nearly so marked or frequent in these strains as in the
Long-Evans animals.
CANCER
RESEARCH
VOL. 28
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Karyotypes of Rats of Different Cancer Susceptibility
Table 1
chromosomes/cell1090.7«5.795271.684.121.6701.1t00
strainHoltzmanLong-EvansCellsanalyzed9788Subterminal
Rat
Distribution of number of subterminal
cells.
0 Percent of cells.
chromosomes
in diploid
DISCUSSION
The present study and that of Yosida and Amano (16) have
only two rat strains in common, the Long-Evans and Fischer.
With respect to the Fischer, the observations on the #3 chro
mosome pair were the same: both members were subterminal.
In the case of their inbred Long-Evans strain, both members
of the #3 pair were subterminal, whereas the strain we used
had a heteromorphic pair. Yosida and Amano (16) felt that
the X chromosome is intermediate in size between the fourth
and fifth largest chromosome pairs; our observations are more
in accord with those of Hungerford and Nowell (6), who placed
the X between the #3 and #4 pairs. It is difficult to be certain
on this point, however, for in many instances the X chromo
some cannot be distinguished definitely.
In most good chromosome preparations it is possible to
identify the morphology of each chromosome and to determine
the number of chromosomes in each morphologic group. Count
ing the number of chromosomes in each morphologic group
permits a convenient analysis for gross chromosome alterations,
since the procedure can be done directly under the microscope
and the results can be expressed quantitatively. Variation of
the number of chromosomes in each morphologic group can
be due to actual karyologic differences, to an artifact of prep
aration, and to an error in assigning a morphologic classifica
tion. In any case, it is important to know for comparative pur
poses the magnitude of variation in normal cells. Approximately
10 percent of the diploid cells in Sprague-Dawley rats showed
an alteration in the morphologic grouping, and generally this
involved a missing subterminal chromosome (Table 1) with cor
responding gain of a terminal chromosome. Intragastric admin
istration of 3-methylcholanthrene or 7,12-dimethylbenz(a)anthracene more than doubled the incidence of morphologic
alterations in diploid cells (Table 2), and, again, the primary
alteration was loss of a chromosome of subterminal morphology
and gain of a terminal chromosome. Presumably this represents
loss of the upper arms of a subterminal chromosome with conTable 2
lacking
subterminal
analyzed165 chromosome(s)
(%)8.5
Controls, untreated
3-MC, i.g.
DMBA, i.g.Cells
115
107Cells
27.0
23.3
Proportion of diploid cells lacking one or more chromosomes of
subterminal morphology (Sprague-Dawley females). 3-MC, i.g.,
3-methylcholanthrene
given intragastrically ; DMBA, i.g., 7,12-dimethylbenz( a) anthracene given intragastrically.
version to terminal morphology. In the untreated rats, a #3
chromosome was predominately involved in this change, whereas
with 3-methylcholanthrene or 7,12-dimethylbenz(a)anthracene
treatment, the other subterminal chromosomes were mainly
affected. Although the technic of chromosome preparation for
cultured cells differs from that for marrow cells, it is of interes't
that diploid cells cultured from induced mammary carcinomas
were found to have a diminished number of subterminal chro
mosomes (11; unpublished studies). A nonrandom representa
tion of chromosome types in human tumor stemlines has been
noted by Levan (7) and Steenis (12).
The female Long-Evans rat is relatively resistant (13) to
induction of mammary cancer by intragastric instillation of
3-methylcholanthrene
or 7,12-dimethylbenz(a)anthracene
as
compared to the marked susceptibility of the female SpragueDawley rat. On the other hand, the incidence of leukemia is
much higher in Long-Evans rats than in Sprague-Dawley rats
after intravenous administration of 7,12-dimethylbenz(a)anthracene (4, 5). A relationship between heteromorphism of the
#3 chromosome pair in the Long-Evans rat and its response
to the polycyclic hydrocarbon carcinogens was considered,
though it did not seem likely. Any simple relationship is ruled
out, however, by the study of the Fischer, Marshall, and
Osborne-Mendel karyotypes and the observation that intra
gastric instillation of a single dose of 7,12-dimethylbenz(a)anthracene (100 mg/kg body weight) induces mammary cancers
in 90-100% of female Sprague-Dawley and Osborne-Mendel
rats but in only about 10% of Long-Evans, Fischer, and Mar
shall rats (Katherine Sydnor, personal communication). The
only other karyotypic difference noted between the strains was
the presence of a morphologically unique #12 chromosome
which was seen not infrequently in cells from Long-Evans rats.
No apparent difference in the sex chromosomes of these two
rat strains was noted. On the basis of present data there seems
to be no direct relationship between strain karyotype and tu
mor susceptibility.
ACKNOWLEDGMENTS
We express our thanks to Dr. Katherine Sydnor who generously
supplied the inbred rats and provided data on the tumor sus
ceptibilities of the different rat strains. Dr. Peter C. Nowell kindly
reviewed some of our slides and offered helpful advice.
REFERENCES
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825
E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, and Joe R. Pugh
6. Hungerford, D. A., and Nowell, P. C. Sex Chromosome Poly
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CANCER
RESEARCH
VOL. 28
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Karyotypes of Rats of Different Cancer Susceptibility
Holtzman
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827
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CANCER RESEARCH VOL. 28
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Karyotypes of Rats of Different Cancer Susceptibility
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Fig.
Fig.
Fig.
Fig.
Fig.
1. Karyotype
2. Karyotype
3. Karyotype
4. Karyotype
5. Karyotype
of
of
of
of
of
male
male
male
male
male
Sprague-Dawley (Holtzman)
Long-Evans rat.
Fischer rat.
Marshall rat.
Osborne-Mendel rat.
rat.
MAY 1968
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g29
E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, and Joe R. Pugh
Fig. 6. Metaphase chromosome
Fig. 7. Metaphase chromosomes
nent upper arms, and lower arrow
Fig. 8. Metaphase chromosomes
Fig. 9. Metaphase chromosomes
830
of female Long-Evans rats arrow indicates satellited #12 chromosome. Giemsa, X 2000.
of female Long-Evans rats upper arrow indicates a #12 chromosome with large satellites on promi
indicates a satellited #13 chromosome. Giemsa, X 2400.
of female Sprague-Dawley rat; a satellited #3 chromosome is indicated by arrow. Giemsa, X 1700.
of female Sprague-Dawley rat; arrow points to satellited #13 chromosome. Giemsa, X 1800.
CANCER
RESEARCH
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VOL. 28
Karyotypes of Rats from Strains of Different Susceptibility to
Mammary Cancer Induction
E. Douglas Rees, Amy Eversole Shuck, Joseph C. Christian, et al.
Cancer Res 1968;28:823-830.
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