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

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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 Â±0.4 mammary adenocarcinomas/rat). In contrast, within 1 year, only 1.2 Â± 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 Â±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 Â±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 Â±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 Â±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 alÃeles,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 alÃeles,none of which were dominant (i.e., polygenic inheritance), or to a single sex chromosome linked alÃele,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 alÃeles,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 alÃelesand 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 alÃele 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 Â±0.30Â°(68/25)* based upon a single dominant autosomal resistance alÃele0.77 Â±0.13 (2/10) 0.40 Â±0.16 (4/10) 0.3 Â±0.12 (6/20) 0.80 + 0.27(12/15) 0.06 + 0.03(1/17) NSD x F, backcrosses'Observed2.48 1.00 Â±0.17 (17/17)Expected " Mean Â±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 alÃelescontrol 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 alÃele,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 alÃeles(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 alÃeleswere 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 alÃele.Additional support for a single dominant alÃelebeing 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 alÃele.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 alÃele(Table 2). Additional genetic analyses were performed on FI hybrid 3960 Downloaded from cancerres.aacrjournals.org on February 22, 2013 Copyright © 1986 American Association for Cancer Research
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 Â±0.40Â°(30/10)* COP 0.00 (0/10) F, hybrids' 0.40 Â±0.13 (4/10) F2 hybrids'*Observed3.00 1.00 Â±0.35 (12/1 2)Expected ' Mean Â±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 Â±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 Â±0.15 mammary adenocarcinoma/rat. These results are consistent with the inheritance of a single dominant autosomal resistance alÃelefrom the COP genome being able to suppress DMBA induced mammary carcinogenesis in WF X COP heterozygous hybrids, although without the data on the I-â€¢ 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, Â»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 alÃele0.00 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 3961 Downloaded from cancerres.aacrjournals.org on February 22, 2013 Copyright © 1986 American Association for Cancer Research
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Genetic Control of Resistance to Chemically ... - Cancer Research

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