Genetic Control of Resistance to Chemically ... - Cancer Research

Genetic Control of Resistance to Chemically Induced Mammary
Adenocarcinogenesis in the Rat
John T. Isaacs
Cancer Res 1986;46:3958-3963.
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(CANCER RESEARCH 46, 3958-3963, August 1986]
Genetic Control of Resistance to Chemically Induced Mammary
Adenocarcinogenesis in the Rat1
John T. Isaacs
Oncology Center and Department of Urology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
ABSTRACT
Fifty-day-old female rats of a series of outbred (i.e., SD) and inbred
(i.e., NSD, WF, LEW, F344, ACI, and COP) strains were exposed to a
single dose of either of two highly effective mammary chemical carcino
gens, 7,12-dimethylbenzfa|anthracene (DMBA) or 1-methyl-1-nitrosourea
(MM), to determine the characteristic number of mammary adenocarcinomas
induced/rat for each strain. Female rats of the inbred NSD,
WF, and LEW strains were found to be as highly susceptible to DMBA
exposure as the randomly outbred SD strain (i.e., >2 mammary adenocarcinomas/rat
develop). Inbred female F344 and ACI rats were found to
be much less susceptible to DMBA induced mammary adenocarcinogenesis
(i.e., 2 years).
Inbred female BN and F344 rats are less susceptible, with only
6% of either of these strains developing mammary cancer during
their lifetimes (6-8). The inbred ACI female is even less suscep
tible with only 4% of these animals developing mammary cancer
if untreated (9). Inbred female COP rats are the most resistant
to developing mammary cancer with not a single mammary
cancer appearing among over 1500 of these female rats allowed
to live out their normal lifetimes without any exogenous treat
ment (10).
This same gradation in susceptibility is observed with regard
to mammary cancer induction by AAF2 (11, 12). When SD,
inbred OM, or inbred BUF female rats are chronically fed AAF,
more than two-thirds of the animals develop mammary cancer
with less than 10% developing liver cancer (11). When
F344 (11) or inbred AUG (12) rats are fed AAF, 10-20% of
animals develop mammary cancer and 10-20% develop liver
cancer. Only 8% of ACI rats (11, 12) fed AAF develop mam
mary cancer while liver cancer develops in 34% of these animals.
Female COP rats were completely resistant to AAF induction
Received 12/10/85; revised 4/11/86; accepted 5/2/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
'Supported by Grants from the American Cancer Society (IN-I1U) and
Department of Health and Human Services (CA 42954).
1The abbreviations used are: AAF, 2-acetylaminofluorene; DMBA, dimethylbenz[a]anthracene;
MNU,l-methyl-l-nitrosourea.
of mammary cancer (12). Female COP rats did develop, how
ever, a 20% incidence of liver cancer demonstrating that not all
tissues in this strain are resistant to AAF.
Chronic treatment of female rats with exogenous estrogens,
like AAF feeding, is an efficient inducer of mammary cancer
development in some strains of rats and not others (13, 14).
Chronic (i.e., >1 year) high dose estrogen treatment of SD (13),
AUG (14), or ACI (14) rats results in more than 40% of all
treated animals developing mammary cancers. Inbred LEW
(13) and F344 (13) rats are less susceptible to exogenous
estrogen treatment, developing only a 31 and 16% incidence of
mammary cancer, respectively. Again, the mammary glands of
the female COP are completely resistant (i.e., 0% incidence) to
such high dose estrogen treatment (14). This chronic estrogen
treatment does produce, however, a 20% incidence of bladder
cancer in these female COP rats (14), again demonstrating that
not all tissues in this strain are of the resistant phenotype.
These background data demonstrate that various strains of
female rats are not equally susceptible to the induction of
mammary cancer and that the female COP rat might be genet
ically unique in its resistance to mammary adenocarcinogenesis,
regardless of what type of carcinogenic treatment is used. There
are a series of chemical carcinogens (e.g., DMBA and MNU)
which are known to be highly effective in inducing the devel
opment of mammary cancers in female rats of certain strains
following a single exposure (5, 15). Therefore, female rats from
a series of inbred strains including the COP were separately
treated with DMBA and MNU to determine the resistance
versus susceptibility of each strain to mammary adenocarcinoma
development.
MATERIALS AND METHODS
Animals. Randomly outbred Sprague-Dawley (SD/HsdBr), inbred
ACI/Seg (ACI/SegHsdBR), inbred Fischer (F344/NHsdBr), inbred
Wistar-Furth (WF/NHsd3R), inbred COP (COP/NHsdBr), and inbred
Lewis (LEW/NHsdBr) rats were obtained from HaríanSprague-Daw
ley, Inc. (Indianapolis, IN). The inbred Sprague-Dawley (NSD/N)
animals were derived from an in-house colony established from a
breeding nucleus of NSD rats obtained at the 54th brother x sister
generation through the generosity of the Veterinary Resources Branch
of the NIH Genetic Resource. Following exposure to carcinogen, as
described below, all animals were housed in a Vickers Total Contain
ment Isolation Unit (London, United Kingdom) for 1 month before
being returned to normal animal rooms. Animals were housed in a
room lighted 12 h/day and maintained at a temperature of 23°C.All
rats were palpated for mammary tumors twice weekly with the time of
detection post-carcinogen exposure being individually recorded for each
tumor which developed in each rat.
The rats in each grouping underwent a complete autopsy at the time
of spontaneous death or at 365 days post-carcinogen exposure. At the
time of autopsy all grossly detectable mammary tumors (i.e., >2 mm
in diameter) were removed and fixed in 10% buffered formalin, and
paraffin sections were prepared and stained with hematoxylin and eosin.
Each mammary tumor was individually classified histopathologically
according to the criteria of Van Zwieten (16). This histológica!evalu
ation thus allowed the number of detectable mammary adenocarcino
mas, sarcomas, and fibroadenomas for each rat to be individually
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assigned; therefore, the mean number of mammary adenocarcinomas
per rat for each experimental treatment group could be determined at
various times post-carcinogen exposure. The mean values ±SE for the
mammary adenocarcinomas per animal for each group were determined
by dividing the number of new mammary adenocarcinomas observed
within the group in each 10-day interval by the number of rats at risk
in that interval and then summing these values by the method of
McCormick et al. (5). This calculation thus takes into consideration
intercurrent mortality.
Carcinogenic Exposure. For the systemic exposure to DMBA, groups
of animals were given either a single or multiple doses of sesame oil
containing varying amounts of DMBA by means of gastric intubation
according to the method of Huggins et al. (15). For the direct in vivo
exposure of mammary glands to DMBA. the technique of Sinha and
Dao (17) was used. This involves anesthetizing rats with Metofane
(Pittman-Moore, Inc., Washington Crossing, NJ), exposing the right
inguinal mammary gland, dusting 3mg of a 2:1 mixture of cholesterol
and DMBA (i.e., 1 mg DMBA total) directly onto the exposed gland,
and then closing the skin incision with stainless steel autoclips.
For the MNU experiments, groups of rats were given either a single
or multiple injections of MNU via the femoral vein according to the
method of Cullino et al. (18). Some rats were also given s.c. injections
of a single dose of MNU according to the method of Thompson and
Meeker (19). For i.v. or s.c. injections, MNU was dissolved in 0.85%
NaCl solution adjusted to pH 5.0 with 3% acetic acid. DMBA and
MNU were obtained from Sigma Chemical Co. (St. Louis, MO).
RESULTS
DMBA Experiments. When 20 mg of DMBA are adminis
tered systemically via a single stomach tube feeding to 50-dayold
female SD rats, greater than 90% of the animals develop
multiple mammary adenocarcinomas within 1 year (Fig. 1).
Like the randomly outbred SD rat, 50-day-old inbred NSD,
inbred WF, and inbred LEW female rats are all equally highly
susceptible to multiple mammary adenocarcinoma development
following DMBA exposure (Fig. 1). Following a single feeding
of 20 mg of DMBA, female F344 rats are less susceptible to
development of mammary adenocarcinoma and female ACI
rats are even less susceptible (Fig. 1). In direct contrast to the
other strains, the mammary glands of female COP rats are
resistant to p.o. DMBA feeding. None of a series of twenty five
50-day-old female COP rats fed 20 mg of DMBA developed
any detectable mammary adenocarcinomas even if these treated
rats were allowed to live for more than 2 years following dosing.
At 50 days of age, there are differences in the body weights
3.0!
5O 100 150 200 250 300 350
GENETIC RESISTANCE TO MAMMARY CANCER
FISCHER
Doys Posi DMBA Feeding
Fig. 1. Temporal pattern of mammary adenocarcinoma development in var
ious strains of female rats following a single p.o. feeding of 20 mg of DMBA. A
total of twenty-five 50-day-old female rats were dosed for each strain.
between the various strains of female rats tested. Since each
strain of rat received a single feeding of 20 mg of DMBA, this
resulted in differences in the dose of DMBA (mg/kg body
weight) when normalized on a body weight basis (i.e., SD, 133;
NSD, 133; WF, 160; LEW, 148; F344, 200; ACI, 200; COP,
200 mg). The fact that the high susceptibility SD, NSD, WF,
and LEW strains actually received a lower dose of DMBA,
when expressed on a body weight basis, as compared to the low
susceptibility F344 and ACI and resistant COP strains, dem
onstrates that the rank order of susceptibility or resistance to
DMBA cannot be trivially explained by differences in body
weights at time of carcinogen exposure.
When female SD rats are exposed multiple times to p.o.
feeding of DMBA, the number of mammary adenocarcinomas
induced per rat increases synergistically, not merely additively,
as compared to a single dose exposure (20). Therefore, 10
female COP rats were fed 10 mg of DMBA 3 times, at weekly
intervals, starting at 36 days of life. The animals were followed
for up to 1 year of life, or until moribund, during which time
no mammary adenocarcinomas developed although 50% of the
treated groups did develop leukemia, which was transplantable
by means of inoculation of donor blood into recipient untreated
COP rats. An additional group of 10 COP female rats was fed
5 mg of DMBA four times at weekly intervals starting at 36
days of life with the same result (i.e., no mammary cancers,
60% incidence of leukemia). As a point of comparison, NSD
rats given the same 5-mg, four-dose feeding schedule developed
4.0 ±0.5 mammary adenocarcinomas/rat within 200 days.
Since pregnancy commencing after carcinogen exposure has
been shown to sharply increase mammary cancer development
and growth in certain strains (21), 20 female COP rats were fed
20 mg of DMBA p.o. at 50 days of life and then 1 week later
were bred. These animals were allowed to deliver their litters
and then were rebred for a minimum of four pregnancies. These
animals were followed for over 1 year and only 1 mammary
adenocarcinoma developed in the 20 treated rats (i.e., average
of 0.05 ±0.05 mammary adenocarcinomas/rat).
If 1 mg of DMBA is directly applied to an exposed inguinal
mammary gland of SD rat, mammary adenocarcinomas develop
in 100% of the directly exposed glands within 200 days but in
0% of the unexposed mammary glands of the treated hosts
(17). A series of inbred rat strains were tested using this direct
application technique. The right inguinal mammary gland of
10 rats/strain were directly exposed to 1 mg of DMBA. In the
NSD group, all exposed glands (i.e., 100%) developed mam
mary adenocarcinomas within 200 days; for the WF strain, also
100% incidence; for the F344 strain, 40% incidence; for the
ACI, 25% incidence; and for the COP, 10% incidence of mam
mary adenocarcinoma with a 40% incidence of fibrosarcoma
formation.
If 50-day-old SD rats are given ¡.p.injections of an oil
emulsion containing 5 mg of DMBA, all animals develop
mammary adenocarcinomas (22). Therefore, ten 50-day-old
female NSD rats were given injections of an oil emulsion of 5
mg of DMBA and all animals developed mammary adenocar
cinomas (i.e., 1.2 ±0.1 mammary adenocarcinomas/rat) and
one animal (i.e., 10%) developed an abdominal sarcoma. In
contrast, when ten 50-day-old female COP rats were similarly
given i.p. injections of DMBA, no animal developed mammary
cancer; however, 40% of these COP animals developed i.p.
sarcomas.
MNU Experiments. DMBA is known to require metabolic
conversion to its ultimate carcinogen (23). The mammary epi
thelial cells of female COP rats might be unable to properly
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GENETIC RESISTANCE TO MAMMARY CANCER
activate DMBA, thus explaining the resistance of this tissue to
DMBA. In order to eliminate this complication, another known
direct acting mammary carcinogen, MNU, was tested [i.e.,
MNU does not require metabolic activation to be carcinogenic
(24)]. Therefore, ten 50-day-old female NSD, F344, and COP
rats were given i.v. injections of MNU (50 mg/kg body weight).
Within 6 months, all of the NSD rats developed multiple
mammary adenocarcinomas (i.e., average, 3.0 ±0.4 mammary
adenocarcinomas/rat). In contrast, within 1 year, only 1.2 ±
0.3 mammary adenocarcinomas developed/F344 rat while none
of the COP rats developed any mammary cancers. MNU treat
ment of COP rats did induce the development of leukemia
(10%) and kidney cancers (20%) at an incidence essentially
identical with that of the NSD and F344 strains.
MNU injected s.c. at a dose of 50 mg/kg body weight can
also induce a high yield of mammary cancer in 50-day-old
female SD rats (19). Therefore, ten 50-day-old female COP and
NSD rats were inoculated s.c. with MNU (50 mg/kg). The
incidence of mammary adenocarcinoma for the NSD group was
2.4 ±0.3 mammary adenocarcinomas/rat within 1 year, while
only a single mammary adenocarcinoma developed in 1 of the
10 COP rats (i.e., 0.1 ±0.1 mammary adenocarcinomas/rat).
When MNU treatment of female SD rats is given, starting at
50 days of age, as either two i.v. injections of 50 mg/kg l week
apart (25) or as three i.v. injections of 50 mg/kg at monthly
intervals (18), little toxicity is produced and a very high inci
dence of multiple mammary adenocarcinomas develop per
treated rat. Therefore, similar MNU treatments were performed
on inbred 50-day-old female NSD and COP rats (i.e., 10 rats/
strain). When two injections of MNU were given, the NSD rats
developed an average of 3.9 ±0.5 mammary adenocarcinomas/
rat and the COP rats developed 0 mammary adenocarcinomas/
rat. When the three-injection schedule was used, an average of
4.8 ±0.4 mammary cancers developed/NSD rat, while again
no mammary cancers developed in any of the COP rats.
Genetic Analysis of Resistance to Chemically Induced Mam
mary Adenocarcinogenesis. Genetic breeding techniques were
used to determine how the resistance of the female COP rat to
DMBA induced mammary adenocarcinogenesis is inherited
[i.e., is it due to one or a series of dominant or recessive genetic
alíeles,or is it instead due to polygenic (i.e., blended) inherit
ance?]. Parental inbred (i.e., homozygous) NSD and COP rats
were cross-bred to produce heterozygous F, hybrid animals.
Both NSD x COP F, and COP x NSD F, hybrids were bred
and at 50 days of age were fed 20 mg of DMBA p.o. Regardless
of which type of F! hybrid was used, both were equally resistant
to DMBA induced mammary adenocarcinoma development
(Table 1). If resistance was due to multiple genetic alíeles,none
of which were dominant (i.e., polygenic inheritance), or to a
single sex chromosome linked alíele,then the FI hybrids should
have been exactly intermediate in their susceptibility between
the NSD and COP strains (i.e., F, hybrids should have had
approximately 1.2 mammary1 carcinomas/rat, not 0.3). If re
sistance was due to either one or a series of recessive autosomal
alíeles,then the FI hybrid should have been just as susceptible
as the highly susceptible NSD parental strain. The fact that
both types of FI hybrids were resistant, like their COP parental
strain, demonstrates that the differential resistance of COP
versus NSD female rats is genetically controlled by either one
dominant or several codominant autosomal alíelesand that no
material factors (e.g., milk agent) is involved. Since no material
factor was demonstrated, all subsequent FI hybrid animals were
produced by breeding NSD females to COP males.
In order to determine if a single dominant autosomal alíele
Table 1 incidence of mammary adenocarcinomas induced by p.o. DMBA feeding
in parental and hybrid crosses between NSD and COP rats
adenocarcinomas/ratType
of female
dosedNSD rat
COPFI
hybrids
NSD x COP
COP x NSD
Total
F2 hybrids'
COP x F, backcrosses''
No. of mammary
(0/25)0.20 0.00
±0.30°(68/25)*
based upon
a single dominant
autosomal
resistance alíele0.77
±0.13 (2/10)
0.40 ±0.16 (4/10)
0.3 ±0.12 (6/20)
0.80 + 0.27(12/15)
0.06 + 0.03(1/17)
NSD x F, backcrosses'Observed2.48
1.00 ±0.17 (17/17)Expected
" Mean ±SE.
0.15
1.39
* Numbers in parentheses, ratio of the total number of mammary cancers
which developed to the total number of female animals exposed to DMBA per
grouping.
f Produced by breeding NSD x COP F, hybrids.
' COP female x (NSD x COP) F, male hybrid backcrosses.
' NSD female x (NSD x COP) F, male hybrid backcrosses.
or several codominant autosomal alíelescontrol this DMBA
resistance of the female COP rat, F, hybrids were cross-bred to
themselves to produce F2 hybrids or separately cross-bred to
female rats of either the NSD or COP parental strains to
produce the appropriate backcrossed animals. At 50 days of
age, these additional animals were given 20 mg of DMBA p.o.
and their responses were determined (Table 1). If the resistance
of the female COP rat to DMBA is due to a single dominant
autosomal resistance alíele,then 0.77 mammary adenocarcinoma/rat
would be expected in the F2 generation (see "Appen
dix" for theoretical calculations). In contrast, if this resistance
is due to multiple codominant autosomal alíeles(i.e., several
independently segregating autosomal genes, the presence of any
one of which can confer complete resistance), then the number
of mammary cancers present in the F2 generation should be
even lower (e.g., 0.42 and 0.33 mammary adenocarcinomas/rat
expected if 2 or 3 codominant alíeleswere involved, respec
tively; see "Appendix" for theoretical calculations). The results
of the F2 generation studies are thus at odds with a codominant
model of inheritance and instead are consistent with the resist
ance being due to the inheritance of a single dominant autoso
mal alíele.Additional support for a single dominant alíelebeing
responsible for this resistance is provided by the data on the
mean number of mammary adenocarcinomas induced by
DMBA in the COP x F, and NSD x F, backcross animals
(Table 1).
Examination of the distribution of the number of mammary
adenocarcinomas/individual rat (Fig. 2) also supports this con
clusion. In the F2 segregating generation, both the parental
homozygous phenotypes (i.e., COP-RR type with 0 mammary
cancers per rat, and NSD-rr type with >1 mammary cancer/
rat), as well as the hybrid heterozygous phenotype (i.e., Rr with
< 1 mammary cancer/rat), are observed with the expected fre
quency if the resistance of the female COP rat to DMBA is due
to a single dominant autosomal alíele.In order to determine
the generality of this dominant autosomal type of mendelian
inheritance, similar studies were performed using MNU as the
carcinogen. At 50 days of age, groups of female NSD, COP,
FI, and F2 rats were given a single 50-mg/kg i.v. injection of
MNU. The mean number of mammary adenocarcinomas per
rat for these various groups again demonstrated that resistance
of the female COP rat to MNU is due to a dominant autosomal
alíele(Table 2).
Additional genetic analyses were performed on FI hybrid
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lOO-i SPRAGUE- DAWLEY
2345
00-5O-75250COPENHAGEN12345F
O I 2
Number of Mammary Adenocarcinomas/Rat
Fig. 2. Distribution of the number of mammary adenocarcinomas per rat
which developed in parental and hybrid crosses between NSD and COP female
rats following a single p.o. feeding of 20 mg of DMBA. Based upon the animals
presented in Table 1.
Table 2 Incidence of mammary adenocarcinomas induced by i.v. MNU injection
in parental and hybrid crosses between NSD and COP rats
adenocarcinomas/ratType
of female
dosedNSD rat
No. of mammary
±0.40°(30/10)*
COP
0.00 (0/10)
F, hybrids'
0.40 ±0.13 (4/10)
F2 hybrids'*Observed3.00
1.00 ±0.35 (12/1 2)Expected
' Mean ±SE.
* Numbers in parentheses, ratio of the total number of mammary adenocarci
nomas which developed to the total number of animals exposed to MNU per
grouping.
c NSD x COP F, hybrids.
rfProduced by breeding NSD X COP F, hybrids.
female rats produced by cross-breeding WF females (i.e., an
other high susceptibility inbred strain), to male COP rats. At
50 days of age, groups of 10 rats each of the WF, COP, and
their FI hybrid strains were given 20 mg of DMBA p.o. Within
1 year, the parental WF strain developed 2.7 ±0.26 mammary
adenocarcinomas/rat while the parental COP strain developed
no mammary cancers. The FI hybrids resulting from these two
parental strains developed only 0.5 ±0.15 mammary adenocarcinoma/rat.
These results are consistent with the inheritance of
a single dominant autosomal resistance alíelefrom the COP
genome being able to suppress DMBA induced mammary carcinogenesis
in WF X COP heterozygous hybrids, although
without the data on the I-• and backcross generations for this
particular pair of rat strains this point is not in the strictest
sense proven.
DISCUSSION
Previous studies have demonstrated that there is a definitive
genetic component in the susceptibility to DMBA and MNU
GENETIC RESISTANCE TO MAMMARY CANCER
induced mammary adenocarcinogenesis. For example, Sydnor
et al. (26) demonstrated that randomly outbred female SD rats
are highly susceptible to a single feeding of DMBA (i.e., 3.8
mammary cancers develop/rat), while randomly outbred and
genetically different female Long-Evans (LE) rats are much less
susceptible to a comparable feeding of DMBA (i.e., only 0.2
mammary cancer/rat develop following a single exposure). This
differential susceptibility, however, can be overcome if female
LE rats are dosed multiple times with DMBA (i.e., 3.0 mam
HYBRID1
mary cancers/rat developed following 4 exposures). Chan et al.
(27) likewise demonstrated that randomly outbred female SD
I 2345
rats are highly susceptible to MNU with 6 to 9 mammary
cancers developing/rat depending on the type of fat diet (i.e.,
F2 HYBRID
high versus low) fed following a single injection of MNU. In
contrast, this group demonstrated that the genetically different
2345
inbred female F344 rat is much less susceptible to a comparable
F, »COP
exposure to MNU with only 0.5 to 2.5 mammary cancers
BACKCROSS
developing/rat depending on the type of fat diet fed.
The studies reported in the present paper have extended these
12345
previous genetic findings and have demonstrated that female
F, i SO
BACKCROSS
NSD, WF, and LEW rats are as highly susceptible to DMBA
exposure as the randomly outbread female SD rat. These con
clusions agree with the studies of Shellabarger (28) and Moore
et al. (29) which compared the DMBA susceptibility of SD to
LEW and WF female rats, respectively. The fact that the
outbred SD, inbred NSD, inbred WF, and inbred LEW strains
are all equally highly susceptible to DMBA could be due to the
fact that all of these 4 strains are genetically related through a
common derivation from the original breeding stock of the
Wistar Institute, Philadelphia, PA (1). Female F344 and ACI
rats, which are not derived from Wistar background, were found
in the present study to be much less susceptible to DMBA
induced mammary carcinogenesis than the previously cited 4
based upon
a single dominant
autosomal
resistance alíele0.00
Wistar related strains. Again, the data on the reduced suscep
tibility of the F344 rat to DMBA agree with the reported
findings of Moore et al. (29) and Gould (30). The reduced
susceptibility of inbred Fischer was not restricted to DMBA,
since essentially identical results were found using MNU as the
0.95
carcinogen as shown in the present study and as reported by
Chan et al. (27).
In contrast to all the other inbred strains of female rats tested,
the COP rat was unique in its resistance to all attempts to
induce mammary adenocarcinomas by either DMBA or MNU
exposure. The female COP rat is also unique in that, in direct
contrast to the LE rat, attempts to overcome the resistance of
the female COP rat to DMBA induced mammary adenocarcin
ogenesis by using multiple exposures to the carcinogen were
completely unsuccessful. Treatment with the direct acting car
cinogen, MNU, regardless of the route of administration or the
number of exposures, was no more successful in inducing
mammary cancers in COP rats than the indirect carcinogen,
DMBA. This strongly suggests that a more complicated expla
nation than merely carcinogen activation is responsible for the
resistance of female COP rats to carcinogen treatment. The
fact that female COP rats are also resistant to spontaneous
(10), AAF (12), and estrogen (14), as well as DMBA and MNU
induced mammary carcinogenesis suggests that the mechanism
for the resistance of the mammary epithelium of the female
COP rat must be a rather effective and general process for a
large series of chemically unrelated carcinogens.
Not all tissues in the female COP rat are resistant to DMBA
induced malignant transformation (i.e., sarcomas and leukemias
are inducible), the mammary epithelium of the female
COP rat thus appears rather unique. In fact, COP mammary
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GENETIC RESISTANCE TO MAMMARY CANCER
connective tissue appears to have a higher susceptibility to
fibrosarcomatous transformation, when carcinogen is directly
applied to the gland, than mammary connective tissue of other
rat strains more susceptible to mammary adenocarcinogenesis
than the COP rat (i.e., NSD, WF, F344, ACI). This increased
sensitivity of the connective tissue of COP rats is not restricted
to the mammary gland, however. For example, i.p. injection of
DMBA into COP females resulted in 40% of the animals
developing i.p. sarcomas while only 10% of the NSD rats
similarly treated developed sarcomas. The connective tissue of
the female COP may be inherently more sensitive to malignant
transformation than similar tissue in NSD rats; however, there
may be alternative reasons for these findings. When 50-day-old
female COP or NSD rats are given s.c. injections in the flank
of 2.5 mg of DMBA dissolved in an oil emulsion in order to
produce a slow release of the carcinogen, >70% of the injected
animals, independent of rat strain, develop sarcomas at the site
of inoculation (data not shown).
These results could mean that the connective tissue cells of
the female COP are no more sensitive than the NSD but that
when injected i.p. or directly applied to the mammary gland,
DMBA might not be removed from these sites as rapidly in the
COP as in the NSD female. This possibility is based upon the
observation of Muggins and Fukunishi (22) demonstrating that
when SD rats are given i.p. injections of DMBA no sarcomas
develop. In contrast, if DMBA pellets are placed in the perito
neum to maintain contact between DMBA and the connective
tissue cells for an extended period of time, then peritoneal
tumors of the connective tissue were universally induced. Fur
ther support for this possibility is the observation that when
female rats of both COP and NSD strains are given s.c. injec
tions of MNU which very rapidly decomposes spontaneously
[i.e., biological half-life approximately l h (23)], neither strain
develops any sarcomas at the site of injection.
Regardless of whether the observed differences in the suscep
tibility of the connective tissue between COP and NSD females
are due to differences in the cells themselves or to differences
in clearance rates for the carcinogen in different connective
tissues of these strains, there still remains the important obser
vation that there is a unique dichotomy of resistance versus
susceptibility in the mammary epithelium versus connective
tissue in the female COP rat. Genetic breeding analysis dem
onstrated that the resistance of the mammary epithelium of the
female COP rat to DMBA and MNU is due to the mendelian
inheritance of a dominant autosomal genetic alíele.The inher
itance of a single copy of this resistance alíeleis able to prevent
both the DMBA and MNU induced development of mammary
adenocarcinomas in F, hybrids produced by cross-breeding
COP rats to either of the highly susceptible NSD or WF strains.
While resistance of the mammary epithelium of the female
COP rat is controlled by a single dominant autosomal alíele,
susceptibility to DMBA induced mammary adenocarcinomas
in the highly susceptible WF rats has been demonstrated by
Gould (30) to be due to a series of codominant autosomal
alíeles.Since F, hybrids between the WF and COP rats are
resistant to DMBA induced mammary adenocarcinogenesis,
this suggests that the COP resistance alíelemay be an epistatic
alíelecapable of suppressing the expression of the several
normally codominant susceptibility alíeles.This would be anal
ogous to the ability of the albino alíele,when present as the
homozygous recessive ce alíeles,to suppress the expression of
a series of dominant (e.g., agouti) and recessive (e.g., hooded)
alíelesresponsible for coat color in the rat (31). Studies are
presently under way to determine if the genetically controlled
resistance of the female COP rat to chemically induced mam
mary carcinogenesis is due to an intrinsic resistance of the
mammary epithelial cells themselves or to some general host
systemic factor (e.g., systemic hormonal environment, etc.)
which secondarily induces the mammary epithelial cells to
become resistant.
ACKNOWLEDGMENTS
The studies reported in this paper would not have been possible
without the continuous expert assistance of Susan P. Dalrymple. The
gifted and enlightened help of Ruth Middleton in the completion of
this manuscript is likewise gratefully acknowledged.
APPENDIX
If a single dominant autosomal alíeleis responsible for the resistance
of the COP mammary epithelial cell to DMBA, then the genotype with
regard to this resistance alíeleshould be RR for the COP and rr for the
NSD rat. Thus the female F, rat produced by crossing NSD and COP
rats would have the heterozygous Rr genotype. The gametes of these
Fi hybrid rats would have 2 possible configurations of alíeles(i.e., R or
r) and thus there are 4 possible genotypes in the F2generation; however,
since two of the F2 genotypes are equivalent phenotypically to the FI
phenotype (i.e., Rr and rR) there are only 3 possible phenotypes. Thus
by mendelian principles, the genotypes of the F¡generation will segre
gate into a 1 RR:2Rr.l rr ratio. Since the RR and Rr genotypes should
both show the phenotype of resistance if resistance is controlled by a
single dominant autosomal alíele,0.77 mammary adenocarcinoma/rat
would be expected in the F2 generation [i.e, of four F2 rats, one rat
should have 0 mammary cancers since it has the COP RR genotype,
one rat should have 2.48 mammary cancers since it has the NSD rr
genotype, and two rats should have 0.3 cancer each since they are of
the F, hybrid Rr genotype; 0 + 2.49 + (2 x 0.3 mammary cancers) =
3.08/4 rats = 0.77 mammary adenocarcinoma/rat].
If two codominant autosomal alíelesare involved in COP mammary
epithelial cell resistance to DMBA, then the genotype with regard to
these resistance alíeleshould be R,R¡R2R2 for the COP and r,r, r2r2
for the NSD rat. Thus the female Ft rat produced by crossing NSD
and COP rats would have the heterozygous R,rt R2r2 genotype. The
gametes of these I-, hybrid rats would have 4 possible combinations of
alíeles(i.e., R,R2, R\r2, rtR2, or r,r2) and thus there are 16 possible
genotypes in the F2 generation. All of these genetic combinations except
for the NSD rtrt r2r2genotype would be phenotypically resistant if the
resistance alíeleswere codominant and thus only 1 of 16 animals in the
1: generation should be as highly susceptible as the NSD parental line.
Thus the F2 generation should develop 0.42 mammary adenocarci
noma/rat if the resistance were due to 2 codominant autosomal alíeles
(i.e., of 16 rats, 1 should have 0 cancers since it would have the COP
R¡R,R2R2 genotype, one rat should have 2.48 cancers since it would
have the NSD rtr} r2r2genotype, and 14 rats should have no more than
0.3 cancer each since they are of the heterozygous genotype; 0 + 2.48
= [14 x 0.3 mammary cancers] = 6.68/16 rats = 0.42 mammary
adenocarcinoma/rat.
If three codominant autosomal alíelesare involved, the genotype
should be R,R, R2R2 RiR] for the COP and r,r, r2r2 r,r, for the NSD
rats. The FI hybrid would thus be R,r, R2r2R}r}and the gametes of the
FI hybrid would have 8 possible combinations of the alíelesand thus
there are 64 possible genotypes in the F2 generation. All of the genetic
combinations except for the r¡r,r2r2r3r3genotype would be phenotyp
ically resistant if the alíeleswere all codominant and thus only 1 of 64
female rats in the F2 should be as highly sensitive as the NSD parental
line. Thus the F2 generation should develop 0.33 mammary adenocar
cinoma/rat if 3 codominant autosomal alíelesare involved (i.e., of 64
rats, 1 should have 0 cancers since it would have the COP RtRi R2R2
R)Ri genotype, 1 rat should have 2.48 cancers since it would have the
NSD r,r, r2r2r}r} genotype, and 62 rats should have no more than 0.3
cancer each since they are of the heterozygous genotype; 0 + 2.48 +
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[62 x 0.3 mammary cancers] = 21.08/64 rats = 0.33 mammary adenocarcinoma/rat).
Similar theoretical calculations can be made for the expected number
of mammary adenocarcinomas per rat for the F2 generation following
exposure to MNU if one, two, or three dominant autosomal alíelesare
involved using the data in Table 2 concerning the number of mammary
cancers per rat induced in the parental lines and in the F, hybrids
following MNU exposure.
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