[CANCER RESEARCH 46, 1199-1202,
March 1986]
Inheritance and Site of Expression of Genes Controlling Susceptibility to
Mammary Cancer in an Inbred Rat Model1
Michael N. Gould
Department of Human Oncology, Wisconsin Clinical Cancer Center, University of Wisconsin, Madison, Wisconsin 53792
ABSTRACT
Sprague-Dawley rats and inbred Fischer F344 rats were resistant
to carcinogen-induced mammary cancer (1).
An inbred rat model for genetically controlled susceptibility to
chemically induced mammary cancer has been established. Wistar-Furth (W/Fu) rats were found to be more susceptible to 7,12dimethylbenz(a)anthracene-induced mammary tumors than were
Fischer (F344) rats. The susceptibilities of various Fi, F2, and
backcross generations of these strains were examined for sus
ceptibility to 7,12-dimethylbenz(a)anthracene-induced
mammary
The pattern of inheritance of susceptibility in the Huggins
outbred rat model was one of dominance that seemed to be
controlled by a single autosomal gene (1). Using our inbred rat
model, we also found susceptibility to be a dominant character
istic (5). Here it is shown that in our inbred model this trait is not
controlled by a single locus.
The rat mammary gland has been shown to be a direct target
to DMBA2-induced carcinogenesis (6). Furthermore, rat mam
tumors. The data suggest that susceptibility is inherited as a
dominant trait. Both a single locus autosomal model and an Xlinked model have been ruled out. However, the data support
the hypothesis that complete susceptibility is controlled by any
one of a group of independently segregating genes; i.e., any one
gene of this group is both necessary and sufficient to induce
maximal susceptibility. It is not known if these genes are identical
or different. In order to identify the role of these genes we asked
if they were expressed in the mammary epithelial cells them
selves or elsewhere in the rat. Chimeric animals were produced
by transplanting mammary cells from either W/Fu or F344 rats
into the white interscapular fat pad of female W/Fu x F344 F,
rats. One month after transplantation the animals were treated
with 7,12-dimethylbenz(a)anthracene and then palpated weekly
for tumor development at the graft site. Tumors developed more
rapidly and in greater total frequency at sites grafted with W/Fu
mammary cells. This result suggests that the genes controlling
inherited susceptibility are expressed in the mammary cells. The
role of these genes is now under investigation. We have thus far
shown that they do not control carcinogen metabolism or acti
vation.
INTRODUCTION
Women are heterogeneous for their propensity to develop
breast cancer. Susceptibility to this major form of cancer is
controlled by both genetic and physiological factors, both of
which have been studied using rodent models (e.g., Refs. 1 and
2). An early model for studying genetic susceptibility was pro
posed by Muggins (3), who demonstrated that outbred SpragueDawley rats were more susceptible to chemically induced mam
mary cancers than were outbred Long-Evans rats. Inbred strains
of rats were also shown to have distinct susceptibilities to
chemically induced cancers. For example, Dunning ef al. (4)
demonstrated that the Copenhagen rat was resistant to hormonally induced breast cancer, while we previously reported that
inbred Wistar-Furth rats were as susceptible as outbred
Received 9/9/85; revised 11/19/85; accepted 11/22/85.
The costs of publication of this article were defrayed in part by the payment of
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' Supported by Grant CA28954 from the National Cancer Institute, NIH, Depart
ment of Health and Human Services.
mary parenchymal cells are capable of activating DMBA (7).
However, there are also many data supporting the concept that
the direct effects of DMBA on the mammary gland can be
modulated by systemic factors such as ovarian and pituitary
hormones (8, 9). It is thus important to determine if the gene(s)
controlling susceptibility are active at the level of mammary
epithelial cell or at the systemic level. Chimeric rats are used
here to address this question.
MATERIALS AND METHODS
Animals. W/Fu and F344 rats were purchased from HarÃ-anSpragueDawley (Madison, Wl). All genetic crosses of these animals were bred in
our local animal facility.
Tumor Induction for Genetic Analyses. DMBA (Sigma Chemical Co.,
St. Louis, MO) of greater than 95% purity as determined by highperformance liquid chromatography was dissolved in sesame oil. Female
rats age 55 ±5 days were intubated with DMBA (130 mg/kg). Starting
3 weeks after DMBA administration each rat was palpated weekly for
the detection of mammary tumors. Tumors were confirmed by histopathological examination. Greater than 95% of tumors were mammary car
cinomas. The end point used in the genetic analysis experiments was
time to the appearance of first carcinoma. This end point was chosen
over tumor multiplicity because in previous experiments in our laborato
ries we found that multiple mammary tumors in rats did not follow a
Poisson distribution (10, 11), making the validity of this end point ques
tionable.
Chimeric Rat Construction. Mammary glands were removed from W/
Fu or F344 female rats age 30 ±5 days. After the lymph nodes were
removed the glands were minced and enzymatically monodispersed. The
monodispersed mammary cells were counted and diluted. Cells were
transplanted into the interscapular white fat pads of recipient rats (female
W/Fu x F344 Fi rats, age 30 ±5 days). Each rat received two injections
of a cell suspension. Each injection contained 9 x 10s cells. Details of
methods of cell preparation and transplantation have been published
(12). We have previously shown that when animals are grafted with this
number of cells, 100% of the graft sites will develop morphologically and
functionally normal mammary tissue (12,13).
When the grafted rats were 55-60 days old they were intubated with
DMBA as described above. All tumors arising in the host mammary gland
were resected to preserve the animals' health. Tumors arising at the
graft site were followed by palpation, confirmed by surgical inspection,
2The abbreviation used is: DMBA, 7,12-dimethylbenz(a)anthracene.
CANCER RESEARCH VOL. 46 MARCH 1986
1199
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SUSCEPTIBILITY
TO MAMMARY
and removed for histopathological examination. Rats were necropsied
11 months after DMBA administration. This time was chosen because
of the declining health status of the rats due to multiple and nonresectable
tumors in the in situ mammary glands.
RESULTS
CANCER
and (c) only the F, males from this cross were used in the above
backcrosses. In order to test this X-linked hypothesis, F, rats
were produced by reversing the strains of the parents; i.e.,
female F344 rats were crossed with male W/Fu rats. Males from
this F! cross were backcrossed to F344 females or used to
produce F2 rats. This change drastically alters the predictions of
an X-linked model (Table 1). The results of studies with these
Genetic Analysis. Mammary tumors were found to develop new crosses (Fig. 3) were found not to be compatible with the
X-linked single locus model (Table 1).
more rapidly in W/Fu than ¡nF344 rats (Fig. 1), confirming our
The total data obtained from testing rats of eight different
previously published findings (1). When W/Fu females were
crosses presented in Figs. 1-3 are compatible with an inheritance
crossed with F344 males, the F! rats had a susceptibility to
pattern in which susceptibility to DMBA-induced breast cancer
DMBA-induced mammary tumors that could not be distinguished
is controlled by a group of independently segregating dominant
from their sensitive W/Fu parent (Fig. 1). The simplest model
genes where the presence of one or more of these genes confers
compatible with these results is a single locus autosomal domi
complete susceptibility to the induction of mammary tumors by
nant inheritance pattern (Table 1). This model of inheritance was
DMBA (Table 1).
tested by examining the susceptibility of: the backcross of W/Fu
The number of independently segregating genes in this group
x F344 F, males with W/Fu females; the backcross of the same
can only be approximated from the current data. The data in
F, males to F344 females; and F2 rats produced by crossing
Figs. 1-3 show that only groups of resistant F344 rats, F344 x
similar Ff females and males. Female rats produced from these
Fi backcrosses, and the F2 crosses fail to develop mammary
three crosses had tumor latencies that were indistinguishable
tumors
in 100% of their rats by 20 weeks post-DMBA feeding.
from those of the W/Fu and W/Fu x F344 F, females (Fig. 2).
In fact, at the termination of these backcross and F2experiments
While the results obtained with the F344 x Fi cross were
(23 weeks post-DMBA) several rats remain tumor free. Specifi
compatible with the single locus autosomal model, the results of
cally in the F344 x (W/Fu x F344 F,) crosses, two rats of 55
the W/Fu x F, cross and the F2 cross were not. These results
(3.6%) are tumor free while in the F344 x (F344 x W/Fu F,)
were, however, compatible with a single locus X-chromosome
model (Table 1), because: (a) only female rats were tested; (£»)crosses two rats of 50 (4%) are tumor free. In the two F2crosses,
only a single rat was tumor free at this time (1 of 49, 2%). It
the F, rats were a cross of sensitive females to resistant males;
should also be noted that in the F344 rats at 23 weeks postDMBA 13 of 47 (27.7%) of rats were tumor free. For the purposes
of analysis, the raw data presented in Table 2, Column 2, can
be normalized. Since the inbred F344 strain is defined as resist
ant we can normalize the data by multiplying 13 of 47 by a
correction factor of 3.6 to obtain a value of 100%; i.e., all rats
are resistant. If the fraction of tumor-free rats in the F344 x FT
backcross (i.e., 4 of 105) is multiplied by the same correction
factor, 13.8% (8-20%, exact binomial 95% confidence intervals)
of the offspring of this cross are found to be "resistant." A similar
o
ir
normalization can be done for the F2 rats when the fraction of
rats without tumors at 23 weeks, i.e., 1 of 49, is multiplied by
the correction factor, and 7.4% (3-11%) are found to be "resist
ant." These approximations of resistance can be compared to
8
I2
I6
20
24
28
32
Weeks Post DMBA
Fig. 1. The time to first mammary tumor of rats given DMBA (130 mg/kg) at
zero time. Rat strains incude F344 (A), W/Fu (•),
and W/Fu x F344 F, (»).See
text for details.
prediction of a mendelian model which assumes: (a) that suscep
tibility is controlled by a number of independently segregating
genes; and (b) that the presence of any of these genes will confer
complete susceptibility. As shown in Table 2, the data of these
Table 1
Rat strains, crosses, and predicted sensitivities to DMBA mammary cancer induction based on several models of inheritance
predictionSingle
type1.
Cross no. and rat
2.
3.
4.
5.
6.
7.
8.
F344
W/Fu
F, (9 W/Fu x i F344)
Backcross (9 W/Fu x d F,a)
Backcross (9 F344 x d F,a)
F2(9 F, x <5F,a)
Backcross6(9 F-344 x d F, ")
F2(F,°xF, )Single
locus autosomal
dominantAll
locus X-chromosomeAll
locus
autosomal
dominantAll
sensitive
All sensitive
'/2 sensitive, Vi resistant
% sensitive, Vi resistant
Vi sensitive, Vi resistant
% sensitive, Vi resistantModel
sensitive
All sensitive
All sensitive
All sensitive
All resistant
Vi sensitive, Vi resistantMultiple
Sensitive
sensitive
All sensitive
All sensitive
All sensitive
Most sensitive
Most sensitive
Most sensitive
All sensitive
Most sensitive
Most sensitive
Most sensitiveObservedResistant
Most sensitive
8 F, = (W/Fu 9 x F-344 <5).
" F, = (F344 9 x W/Fu d).
CANCER
RESEARCH
VOL. 46 MARCH
1986
1200
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SUSCEPTIBILITY
TO MAMMARY
crosses are most compatible with a model predicting three
independent genes present in the W/Fu rat which can confer
susceptibility to chemically induced mammary cancer. It is
stressed that this latter analysis is preliminary.
Chimeric Rats. Chimeric rats were produced by grafting either
mammary cells from F344 (resistant) or W/Fu (susceptible) rats
into the mammary-free interscapular white fat pads of W/Fu x
F344 F! animals. These rats were followed for 11 months for
CANCER
tumor development at the graft site following intubation with
DMBA or oil alone. None of the oil-fed control rats developed
mammary tumors at the site of the graft irrespective of the strain
of origin of the grafted cells. Tumors did develop at the graft site
in animals fed DMBA. As shown in Fig. 4, the percentage of
carcinomas at the graft sites varied with the strain of origin of
the grafted cells. F, rats grafted with W/Fu cells developed
mammary carcinomas at the graft site in greater frequency and
at shorter latency than those receiving F344 cells. At 11 months
post-DMBA feeding, 12 of 39 (31%) of the rats with W/Fu grafts
had develped carcinomas whereas only 3 of 43 (7%) rats with
F344 grafts developed tumors. It should be noted that three of
the W/Fu carcinomas were found at final necropsy while one of
the F344 carcinomas was found at this time.
In addition to the carcinomas, a few benign mammary tumors
(fibroadenomas) were also found. These fibroadenomas were
mainly discovered at the final necropsy (11 months post-DMBA).
Unlike the findings with carcinomas, no major difference in fibro
adenoma frequency could be related to strain of origin of the
graft. Four of 39 (10%) rats with W/Fu and 6 of 43 (14%) rats
with F344 grafts had fibroadenomas at the transplant site.
8
12
Weeks Post
16
20
24
28
32
DISCUSSION
DMBA
Fig. 2. The time to first mammary tumor of rats given DMBA (130 mg/kg) at
zero time. Rat strains include the parent strains W/Fu (
) and F344 (
)
and specific crosses: W/Fu x F344 F, (»);backcross F344 x (W/Fu x F344 F,)
(•);backcross W/Fu x (W/Fu x F344 F,) (•);and F2 (F, x F,, F, = W/Fu x F344)
(A).
IOO
8
I2
16
20
24
28
32
Weeks Post DMBA
Fig. 3. The time to first tumor of rats given DMBA (130 mg/kg) at zero time.
Rat strains include the parent strains W/Fu (
) and F344 (
) and specific
crosses: backcross F344 x (F344 x W/Fu F,) (»)and F2 cross (F, x F,, F, = F344
x W/Fu) (•).
typeF344W/Fu
Rat
We have shown that susceptibility to DMBA-induced mam
mary cancer in the W/Fu-F344 rat model is controlled as a
dominant trait. Complete susceptibility can be conferred by any
of a number of independently segregating genes. A preliminary
analysis suggests that approximately three loci may be involved.
The pattern of inheritance found in this inbred model is different
than that reported for the Huggins Sprague Dawley-Long Evans
outbred model in which susceptibility was found to be controlled
by a single dominant gene (3). It is most likely that this difference
in inheritance is real. However, it should be noted that at least
two major differences exist in addition to the strains used: (a)
our model uses inbred rats whereas Huggins' model used
outbreds, which may complicate genetic analysis; (b) our end
point is a latency measurement, i.e., time to first tumor, whereas
Huggins used incidence, i.e., frequency of tumors.
The difference between the pattern of inheritance of suscep
tibility of the SD-LE model and the W/Fu-F344 model may be
the result of different susceptibility-conferring genes or a result
of different numbers of the same gene. While this question
cannot yet be answered certain available information may be
relevant. The SD and W/Fu rats have almost identical suscepti
bilities to DMBA-induced mammary carcinogenesis. This is com-
Table 2
Results of tumor induction experiments and predictions of various multicene experiments
on1-gene
of % of "resistant" rats based
%
without
of "resistant"
of "resistant"
tumors (23
rats/total
rats"47/47
rats100
model100
model100
wk)/total
rats13/47
0/48
0/48
F,
W/Fu x F,
0/49
F344 x F,
4/105
1/49No.
FzRats
a Method of normalization calculations given in text.
0 See text for details.
c Exact binomial 95% confidence interval.
0/48
0/48
0/49
14.5/105
3.6/49Observed
0
0
0
13.8(8-20)"
7.4(3-11)cPredictions"
CANCER RESEARCH VOL. 46 MARCH
1201
0
0
0
0
0
0
0
0
0
50
25
12.5
252-genemodel100
12.53-genemodel100
6.24-gene
0
0
06.2
3.1
1986
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SUSCEPTIBILITY
TO MAMMARY
CANCER
larger number of "target" cells in the susceptible mammary gland
both in situ and following grafting. We have presented evidence
of an inverse correlation of target cell number and latency in
radiation-induced mammary carcinomas.3 Evidence in this report
10
20
Weeks Post
30
40
also supports this hypothesis for chemically induced cancers. If
the latency is compared for in situ cancers (Figs. 1-3) versus
cancer in grafted glands (Fig. 4) for W/Fu mammary cells, it is
clear that a much longer latency exists at the graft site. It is also
clear that the graft site contains far fewer mammary epithelial
cells and thus presumably fewer target cells than in situ glands.
We plan to directly test this hypothesis of a differential number
of target cells in each strain by using a quantitative transplanta
tion model that we have developed that allows us to both
measure clonogenic mammary stem cell population and estimate
neoplastic transformation per surviving exposed mammary clon
ogenic cell (12).3
50
DMBA
Fig. 4. Chimeric rats with interscapular grafts of W/Fu (•)
or F344 (•)
mammary
glands were fed DMBA at zero time. The time and percentages of rats that
developed mammary carcinomas at the graft site are plotted. See text for details.
ACKNOWLEDGMENTS
patible with a hypothesis of susceptibility genes with identical
function in these two rat strains. This hypothesis is strengthened
by the fact that the SD rat originates from stock that included
the Wistar rat (14). Furthermore the possible amplification of this
gene in the W/Fu rat might have arisen during its inbreeding by
Furth and colleagues, whose aim in breeding this strain was to
develop rats with a high leukemia incidence (15). While this aim
was not achieved, it is possible that this selective breeding led
to multiple copies of the susceptibility gene. This latter hypoth
esis is being examined by determining if other inbred Wistar
strains have single or multiple susceptibility genes.
In an initial attempt to begin to understand the function of
these genes, it was asked if they acted at the level of the
mammary cells or elsewhere in the rat. Chimeric rats were
constructed by grafting either W/Fu or F344 mammary cells into
the mammary cell-free interscapular white fat pad of W/Fu x
F344 F, rats. After complete mammary glands developed at
these graft sites, DMBA was administered. If the susceptibility
genes to DMBA-induced mammary cancer were functioning
primarily in the mammary cells themselves then one would
expect a differential response at the graft site. However, if the
genes acted abscopally then no differential response would be
expected. Our results support the former possibility in that a
much greater number of carcinomas was found at sites grafted
with W/Fu mammary cells. It is interesting to note that there was
no difference in the number of fibroadenomas at the two types
of graft sites. It is not yet clear, however, if the genes being
studied influence the induction of mammary fibroadenomas.
The action of this gene(s) at the level of the mammary cell is
under investigation. Thus far, we have ruled out the possibility
that this gene(s) is controlling carcinogen metabolism or activa
tion. Specifically, we have shown that mammary epithelial cells
from both strains, when assayed by a mediated mutagenesis
assay, activate DMBA to its mutagenic metabolites to an equal
extent (1). Other postactivation stages of mammary carcinogen-
I thank Dr. Martin Tanner for statistical analysis of the data and Dr. Kelly Clifton
for many very valuable discussions during the course of this work. I also thank
Joan Mitchen. Holli Joslin, Jami Boston, and Beth King for excellent technical
assistance and Peggy Shager for preparation of the manuscript.
REFERENCES
1. Moore, C. J., Bachhuber, A. J., and Gould, M. N. Relationship of polycyclic
hydrocarbon metabolism in rat mammary cells to tumor susceptibility in vivo.
J. Nati. Cancer Inst., 4: 777-784,1983.
2. Moore, C. J., and Gould, M. N. Differences in mediated mutagenesis and
polycyclic aromatic hydrocarbon metabolism in mammary cells from pregnant
and virgin rats. Carcinogenesis (Lond.), 5: 103-108, 1984.
3. Muggins, C. B. Experimental Leukemia and Mammary Cancer. Chicago. The
University of Chicago Press, 1979.
4. Dunning, W. F., Curtis, M. R., and Segloff, A. Strain differences in response
to diethylstilbestrol and the induction of mammary gland and bladder cancer
in the rat. Cancer Res., 7: 511 -521,1947.
5. Gould, M. N. Genetic susceptibility to DMBA induced cancer at the cellular
level. Proc. Am. Assoc. Cancer Res., 25: 153, 1984.
6. Dao, T. C., and Sinha, D. Mammary adenocarcinoma induced in organ culture.
J. Nati. Cancer Inst., 49.- 591-597, 1972.
7. Gould, M. N. Mammary gland cell mediated mutagenesis of mammalian cells
by organ specific carcinogens. Cancer Res., 40:1836-1841,1980.
8. Dao, T. C. The role of ovarian steroid hormones in mammary carcinogenesis.
Banbury Rep., 8: 281-298, 1981.
9. Welsch, C. W. Prolactin and growth hormone in the development, progression,
and growth of murine mammary tumors. Banbury Rep., 8: 299-315,1981.
10. Gould, M. N., Clifton, K. H., and Crowley, J. Effects of endocrinological
conditions associated with acute versus chronic lactation on the incidence of
mammary carcinomas in irradiated rats. J. Nati. Cancer Inst., 60: 469-471,
1978.
11. Clifton, K. H., and Crowley, J. Effects of radiation type and dose and the role
of glucocorticoids, gonadectomy and thyroidectomy in mammary tumor induc
tion in mammotropin-secreting pituitary tumor-grafted rats. Cancer Res., 38:
1507-1513,1978.
12. Gould, M. N., Biel, W. F., and Clifton, K. H. Morphological and quantitative
studies of gland formation from inocula of monodispersed rat mammary cells.
Exp. Cell Res., 708: 405-416, 1977.
13. Gould, M. N., and Clifton, K. H. The survival of rat mammary gland cells
following irradiation in vivo under different endocrinological conditions. J.
Radiât.Oncd. Biol. Phys., 4: 629-632,1978.
14. Altman, P. L., and Katz, D. D. (eds.). Inbred and Genetically Defined Strains of
Laboratory Animals. Part I, p. 234. Bethesda, MD: Federation of American
Societies for Experimental Biology, 1979.
15. Kim, U., Clifton, K. H., and Furth, J. A highly inbred line of Wistar rats yielding
spontaneous mammo-somatotropic pituitary and other tumors. J. Nati. Cancer
Inst., 24: 1031-1055,1960.
3 K. H. Clifton, M. A. Tanner, and M. N. Gould. Assessment of radiogenic cancer
esis are currently under investigation in these rat strains.
Another possibility that must also be explored is that of a
CANCER
RESEARCH
initiation frequency per clonogenic rat mammary cells in vivo, submitted for publi
cation.
VOL.
46
MARCH
1986
1202
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Inheritance and Site of Expression of Genes Controlling
Susceptibility to Mammary Cancer in an Inbred Rat Model
Michael N. Gould
Cancer Res 1986;46:1199-1202.
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