Mammary Carcinomas Induced in Human c-Ha-ras Proto-oncogene Transgenic Rats Are Estrogen-independent, but Responsive to d-Limonene Treatment

We have previously shown that transgenic rats carrying three copies of the human c-Ha- ras proto- oncogene (Hras128) are highly susceptible to N -methyl- N -nitrosourea (MNU) mammary carcino- genesis. All transgenic rats treated with 50 mg/kgMNU, i.v. at 50 days of age, were found to rapidly develop multiple, large mammary carcinomas within as short a period as 8 weeks. In the present study, the effects of ovariectomy and treatment with d -limonene, known to inhibit mammary carcinogenesis in non-transgenic female rats, were investigated in Hras128 animals treated withMNU to clarify the role of the human c-Ha- ras proto-oncogene and to characterize the induced mammary carcinomas. Although ovariectomy completely inhibited development of mammary carcinomas in their wild-type counterparts, it did not affect either the incidence or the multiplicity of the mammary carcinomas in the Hras128 rats. On the other hand, treatment with d -limonene, an inhibitor of ras protein isoprenylation, inhibited the breast tumor development. These results indicate that aberrant c-Ha- ras gene expression is involved in ovarian hormone-independent growth and c-Ha- ras protein isoprenylation plays an important role in mammary carcinogenesis     

Mammary carcinomas induced in human c-Ha-ras proto-oncogene transgenic rats are estrogen-independent, but responsive to d-limonene treatment.

We have previously shown that transgenic rats carrying three copies of the human c-Ha-ras proto-oncogene (Hras128) are highly susceptible to N-methyl-N-nitrosourea (MNU) mammary carcinogenesis. All transgenic rats treated with 50 mg / kg MNU, i.v. at 50 days of age, were found to rapidly develop multiple, large mammary carcinomas within as short a period as 8 weeks. In the present study, the effects of ovariectomy and treatment with d-limonene, known to inhibit mammary carcinogenesis in non-transgenic female rats, were investigated in Hras128 animals treated with MNU to clarify the role of the human c-Ha-ras proto-oncogene and to characterize the induced mammary carcinomas. Although ovariectomy completely inhibited development of mammary carcinomas in their wild-type counterparts, it did not affect either the incidence or the multiplicity of the mammary carcinomas in the Hras128 rats. On the other hand, treatment with d-limonene, an inhibitor of ras protein isoprenylation, inhibited the breast tumor development. These results indicate that aberrant c-Ha-ras gene expression is involved in ovarian hormone-independent growth and c-Ha-ras protein isoprenylation plays an important role in mammary carcinogenesis.     

Susceptibility of lean and obese Zucker rats to tumorigenesis induced by N-methyl-N-nitrosourea

To address the possible involvement of hyperinsulinemia in breast cancer development, we have examined the susceptibility of lean and obese Zucker rats to N-methyl-N-nitrosourea (MNU)-induced mammary cancer. Fifty-day-old female lean or obese Zucker rats received intraperitoneal (i.p.) injections of 37.5 or 20 mg/kg MNU, respectively. We showed in separate experiments that these doses produce similar levels of DNA methylation in the mammary epithelial cells of the lean and obese animals. Over the course of 29 weeks following MNU treatment, half of the lean rats developed carcinomas of the mammary gland, demonstrating that they are of intermediate susceptibility to mammary tumorigenesis. During this period, the obese rats developed hyperinsulinemia and insulin resistance as expected. Although palpable tumors developed at a similar rate in the lean and obese rats, only 10% of the obese animals developed mammary carcinomas. The obese rats, however, developed a high incidence (63.3%) of epidermal cysts that occurred mainly in the region of the mammary glands. A 13.3% incidence of colon carcinomas was also found in the obese rats. These results suggest that the development of hyperinsulinemia does not render the obese Zucker rats more susceptible to mammary gland carcinogenesis. Our observation of colon carcinomas in obese, but not lean rats, however, is consistent with evidence that hyperinsulinemia promotes colon cancer in rodents and humans.     

Dose-responsive induction of mammary gland carcinomas by the intraperitoneal injection of 1-methyl-1-nitrosourea

Dose-response relationships for the induction of mammary tumors by a single i.p. injection of 1-methyl-1-nitrosourea (MNU) were studied. Groups of 30 female Sprague-Dawley rats were given i.p. injections of 50, 37.5, 25, 12.5, or 0 mg MNU/kg body weight at 50 days of age. Animals were palpated for tumor detection twice weekly throughout a 28-week observation period. Administration of MNU i.p. caused no acute toxicity. Both benign and malignant mammary tumors were induced by MNU, but malignant tumors appeared earlier and at a faster rate than benign tumors. The incidence and numbers of mammary carcinomas increased whereas median cancer-free time decreased with increasing dose of MNU. Approximately twice as many mammary cancers were observed in the cervical-thoracic as in the abdominal-inguinal mammary gland chains irrespective of carcinogen dose, while the frequency of tumor occurrence in the left versus right chains was similar. Tumor latency decreased with increasing dose of MNU, but the quartiles for time to detection of all tumors within each carcinogen dose group were similar irrespective of anatomical region in which the tumors occurred. The mammary tumor response attained via i.p. injection was similar but the coefficient of variation for tumor multiplicity within a carcinogen dose group was lower in comparison to that observed when MNU was administered i.v. or s.c. Among these techniques for carcinogen injection, the i.p. route is the most rapid method and offers an added advantage of ease of administration with quantitative, reproducible delivery of the desired amount of carcinogen and a decrease in variability of tumor response among animals within a treatment group. This method is well suited for the technically less experienced investigator and for those in need for a rapid method of injecting MNU to large numbers of animals.     

Effect of carcinogen dose and age at administration on induction of mammary carcinogenesis by 1-methyl-1-nitrosourea.

The objective of the work reported in this paper was to determine if the tumorigenic response to 1-methyl-1-nitrosourea (MNU) in the mammary gland varied with age of administration and was dose dependent when the carcinogen was injected prior to 50 days of age. Using a recently developed method for mammary tumor induction, MNU was injected i.p. at doses ranging from 25 to 75 mg/kg at 35 days of age or 50 mg/kg at 28, 35 or 42 days of age. Treatment with MNU resulted in induction of both benign and malignant mammary tumors. The incidence of mammary gland adenocarcinomas was 100% at and above the 50 mg/kg dose of MNU, irrespective of the age at which carcinogen was administered. The number of cancers increased in proportion to carcinogen dose, whereas cancer latency decreased as the MNU dose increased. In rats injected with 50 mg MNU/kg body weight at 28, 35 or 42 days of age, differences among groups in cancer incidence, number or latency were not statistically significant. Metastases of mammary neoplasms to lung, liver and spleen were observed in rats injected with MNU at 35 or 42 days of age. These data indicate (i) the dose responsiveness of MNU-induced mammary carcinogenesis in rats initiated prior to 50 days of age; (ii) the lack of effect of age at initiation if prior to 50 days on final tumor outcome; and (iii) that the age at which MNU is injected may affect the metastatic potential of the mammary carcinomas that are induced.     

Molecular cloning of rat Krev-1 cDNA and analysis of the mRNA levels in normal and NMU-induced mammary carcinomas

Human Krev-1 has been reported to reverse the transformed phenotype in a variety of cell types containing activated ras. Most mammary carcinomas induced by N-nitrosomethylurea in the rat have been reported to exhibit activated ras. Here we report that rat Krev-1 has a very high degree of homology to human Krev-1. Furthermore, Krev-1 expression is high in both normal and neoplastic mammary glands. No difference was found in expression levels in tumors with and without ras activation. These data argue against a tumor suppressor function for Krev-1 in the in vivo rat mammary gland.     

Analysis of allelic imbalances at multiple cancer-related chromosomal loci and microsatellite instability within the same tumor using a single tumor gland from colorectal carcinomas

Genetic changes related to colorectal carcinomas are accumulated in individual tumor glands during disease progression. Microsatellite allelic analysis of individual tumor glands from 30 colorectal carcinomas using a polymerase chain reaction (PCR) assay coupled with crypt isolation was used to detect intratumoral genetic heterogeneity, the sequence of allelic imbalances (AIs) and the microsatellite instability status of single tumor glands during neoplastic progression. In addition, the CpG islands methylated phenotype (CIMP) status was examined using a methylation-specific PCR method. The specimens were divided into 2 groups: a pooled gland sample, which was composed of more than 50 tumor glands, and a single tumor gland sample. The latter consisted of 10 single tumor glands, which were obtained from the same tumor separately. Most colorectal carcinomas (27 of 30 tumors) examined were heterogeneous for at least one genetic alteration, with from 2 to 7 genotypically different subclones detected per tumor. In 12 of the 27 heterogeneous tumors, it was possible to define the order of genetic alterations during the tumor progression. By analyzing multiple single tumor glands within the same tumor, we found that various subclonal expansions were seen within the same tumors. Finally, the AI pattern of single tumor glands was not correlated with CIMP status. Most carcinomas appeared to have a heterogeneous composition. This may have resulted from the successful progression of one clone that had different AIs in many chromosomal regions. This suggests that knowledge of the different genotypes of multiple single tumor glands may help clarify the process of tumor progression.     

Parous mammary glands exhibit distinct alterations in gene expression and proliferation responsiveness to carcinogenic stimuli in Lewis rats

Early full-term pregnancy affords lifetime protection against the development of breast cancer. Parity-induced protection can be reproduced in a carcinogen-induced rat mammary carcinoma model, but the molecular mechanisms of this protection against carcinogenic stimuli in rat mammary glands have not been fully characterized. To gain a better understanding of these molecular mechanisms, we used an oligonucleotide microarray to examine gene expression in parous and age-matched virgin (AMV) mammary glands of Lewis rats before and after carcinogen (N-methyl-N-nitrosourea; MNU) treatment. Parous mammary glands before MNU treatment showed up-regulation of multiple differentiation-related genes, such as whey acidic protein (Wap), casein beta (Csn2), casein gamma (Csng), lipopolysaccharide binding protein (Lbp), secreted phosphoprotein 1 (Spp1) and glycosylation-dependent cell adhesion molecule 1 (Glycam1). Also, parous mammary glands before MNU treatment exhibited down-regulation of growth-related genes such as regenerating islet-derived 3 alpha (Reg3a), mesothelin (Msln), insulin-like growth factor 2 (Igf2) and insulin-like growth factor binding protein 4 (Igfbp4). After MNU treatment, AMV mammary glands exhibited up-regulation of growth-related genes, such as Msln, cell division cycle 2 homolog A (Cdc2a), Igf2, Igfbp4, stathmin 1 (Stmn1) and homeobox, msh-like 1 (Msx1), whereas expression of these genes remained low in parous mammary glands. AMV mammary glands also exhibited marked up-regulation of Cdc2a and Stmn1 in response to MNU. After MNU treatment, the PCNA labeling index increased significantly in AMV mammary epithelial cells (13.7+/-1.1%), but remained low in parous mammary glands (3.6+/-0.4%). The response of AMV mammary glands to carcinogenic stimuli includes up-regulation of growth-related genes and increased cell proliferation. The lack of a similar response in parous mammary glands may explain parity-induced protection against mammary tumor development.     

H-ras oncogene mutations during development of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary gland cancer

Laser capture microdissection, polymerase chain reaction-restriction fragment length polymorphism analysis, and DNA sequencing was used to detect H-ras codon 12 and 13 mutations during the stages of mammary gland cancer development in rats exposed to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen found in cooked meat. Ten oral doses of PhIP (75 mg/kg, p.o., once per day) were administered to adolescent female Sprague-Dawley rats and mammary glands examined histologically for intraductal proliferations (IDPs), carcinoma in situ and carcinomas 7-14 weeks later. Mammary gland epithelial cells from normal tissue and distinct lesions were collected from glass slides and analyzed for mutations. H-ras codon 12/13 mutations were detected in 73%, 75%, 100%, and 100% of normal mammary glands, IDPs, carcinoma in situ, and carcinoma, respectively, after PhIP treatment. The spectrum of activating mutations included G(35) to A or C base substitution mutations in codon 12, and G(37) to T or A base substitution mutations in codon 13. The spectrum of H-ras mutations was similar among normal mammary gland from PhIP treated rats, preneoplastic lesions, and carcinomas. Furthermore, the spectrum of mutations was consistent with the involvement of PhIP-guanine adduct formation. The results support the notion that mutations in H-ras codons 12 and 13 are largely PhIP-DNA adduct-induced and involved in the initiation and development of mammary gland cancer in rats exposed to PhIP.     

K-ras oncogene mutations in rat colon tumors induced by N-methyl-N-nitrosourea.

We have been studying a rat model of colon cancer in which tumors are induced by direct application of N-methyl-N-nitrosourea (MNU) to discrete areas of the colonic mucosa for a limited period of time. Activation of the ras genes by point mutation has been observed in many experimental tumors, including tumors induced by MNU. To detect potential activating point mutations in the H-ras and K-ras oncogenes in MNU-induced rat colon tumors, DNA samples from 40 adenomas, nine carcinomas, and 14 histologically normal tissue samples from 14 rats--as well as from 16 foci induced on NIH3T3 cells by tumor DNAs--were amplified by the polymerase chain reaction and hybridized with allele-specific oligonucleotide probes. No H-ras point mutations were observed in any of these samples. We did detect K-ras point mutations, however, in four primary tumours--one adenoma (2.5%) and three carcinomas (33%); these mutations were all G----A transitions at the second nucleotide of codons 12 and 13. The absence of detectable ras mutations from the majority of tumors suggests that, in contrast to other animal models utilizing MNU, tumorigenesis in MNU-induced rat colon tumors may predominantly involve activation of genes other than ras.     

Induction of mammary carcinomas by N-methyl-N-nitrosourea in ovariectomized rats treated with epidermal growth factor

The importance of epidermal growth factor (EGF) in both normal and malignant mammary gland development are presented in these studies. Initial findings demonstrated that in the absence of ovarian hormones, EGF had a significant proliferative effect on mammary epithelial cells. To determine whether mammary epithelial cells grown with EGF, in the absence of ovarian hormones, could be transformed by N-methyl-N-nitrosourea (MNU), female ovariectomized Lewis rats were implanted with pellets containing EGF for 1 week and then treated with MNU for initiation. Two days after MNU treatment, ovaries were implanted and EGF pellets were removed from all ovariectomized groups in order to promote carcinogenesis. The mammary carcinoma incidence of the EGF-stimulated group (90%) was not significantly different from the intact group (100%). The mammary cancer morphology of EGF-treated carcinomas was either ductal carcinoma or cribriform adenocarcinoma, whereas intact animals developed mainly papillary and occasional cribriform carcinomas. Fifty-eight percent of the carcinomas from the EGF group were ovarian hormone-independent compared with 10% of carcinomas from the intact group. These results demonstrate that EGF-induced proliferation during initiation with MNU was sufficient to induce the transformation of mammary carcinomas in the absence of ovarian hormones. The hormonal dependency of these EGF-induced carcinomas were different compared with MNU-initiated mammary carcinomas in intact rats.     

Loss of chromosomal integrity drives rat mammary tumorigenesis

Breast cancer incidence varies with diet and other environmental influences, including carcinogen exposure. However, the effects of carcinogens on cell growth control pathways are poorly understood. Here, we have examined processes that are activated in the mammary glands of rats treated with 1-methyl-1-nitrosourea (MNU). This synthetic carcinogen was used to study events occurring during mammary tumor initiation and development. In female Wistar-Furth rats, given 1 dose of MNU beginning at 50 days of age, 84% of the rats developed tumors by 46 weeks of age (latency 13-15 weeks). Changes in the gland occurred as early as 1-day post-MNU. Cells exhibited DNA damage, leading to chromosomal instability, supernumerary centrosomes and higher levels of Aurora A; these events correlated with the appearance of preneoplasia in the glands. In mammary tumors, elevated numbers of centrosomes coincided with genomic instability. Tumors were transplanted into syngeneic hosts and subsequent tumor generations displayed the same marker chromosomes in mostly aneuploid metaphases with hyperdiploid numbers of chromosomes, suggesting that clonality and aneuploidy were passed on from one generation to the next. Collectively, these data suggest that the carcinogen MNU induces changes resulting in genetic instability detectable before hyperplasia and tumors develop in the rat mammary gland.     

ras gene alterations in invasive and non-invasive rat bladder carcinomas induced by N-methyl-N-nitrosourea

We have established a reliable method to induce invasive and non-invasive carcinomas in the heterotopically transplanted urinary bladder of rats by repeated injection of N-methyl-N-nitrosourea (MNU), and examined the alterations of the ras oncogenes and ras oncogene product (p21) in the induced tumours. The incidence of muscle-invasive carcinomas was proportional to the total dose of MNU. When 5, 6 or 12 doses of MNU were used, muscle invasive carcinomas developed in 22, 58 or 45% of animals, respectively, after a mean observation period, respectively, of 54 +/- 9, 45 +/- 13 and 38 +/- 3 weeks. Whereas activated H-ras gene was detected in only one non-invasive carcinoma by DNA transfection assay, seven of 18 non-invasive and invasive carcinomas showed activated ras p21 when examined by immunoblot analysis. Amplification or rearrangement of myc or epidermal growth factor (EGF) receptor gene was not observed. The results indicate that alterations of ras gene may be involved in the development of rat bladder carcinomas but not of invasiveness.     

4-(hydroxyphenyl)retinamide selectively inhibits the development and progression of ductal hyperplastic lesions and carcinoma in situ in mammary gland.

In most previous chemoprevention studies on inhibition of mammary carcinogenesis, the formation of palpable tumors has been used as an end-point. Little is known about whether chemopreventive agents may similarly or selectively suppress hyperplastic and premalignant stages of the neoplastic process. In this study, we evaluated the effect of 4-(hydroxyphenyl)retinamide (4-HPR) on the development and progression of hyperplastic lesions and carcinoma in situ (CIS) in the N-methyl-N-nitrosourea (MNU) mammary carcinogenesis model in rats. 4-HPR was used as the chemopreventive agent because of its proven inhibitory effect on both the early and late phases of mammary carcinogenesis. Treatment with 4-HPR (2.0 mM/kg diet), beginning 2 days after MNU administration and administered continuously for 10 weeks, suppressed all mammary gland lesions (hyperplasia, CIS and invasive carcinoma) in 35% of animals. In the remaining 65%, 4-HPR allowed the development of hyperplastic lesions, alone or combined with CIS, and/or invasive carcinomas (CA). 4-HPR also increased by 2-fold the ratio between CIS and CA (0.75 per animal in control versus 1.5 in 4-HPR-treated animals), suggesting that it may also suppress the transition of CIS into CA. 4-HPR, when administered beginning 4 weeks after MNU administration [when hyperplastic and premalignant (CIS) lesions are present in the mammary gland], inhibited the frequency of terminal end bud hyperplasia (TEBH) and CA but did not significantly suppress ductal hyperplasia, ductal alveolar hyperplasia, alveolar hyperplasia and CIS. In these animals, 4-HPR induced partial disintegration of mostly peripheral areas of lesions, including carcinomas. Taken together, our data indicate that 4-HPR selectively suppresses the development and progression of hyperplastic lesions and CIS in TEBs. Furthermore, it appears that, in addition to mammary carcinomas, TEBH and CIS could also be used as end-point biomarkers in breast cancer chemoprevention studies.     

Protective effects of pregnancy and lactation against N-methyl-N-nitrosourea-induced mammary carcinomas in female Lewis rats

The role of parity before and after N-methyl-N-nitrosourea (MNU) treatment in protection against mammary carcinogenesis was investigated. The effect of lactation on reduction in the incidence of mammary carcinoma was also examined. Parous rats were compared with respective age-matched virgins (AMVs). Pregnancy and lactation prior to MNU exposure significantly reduced both the incidence of mammary carcinoma (22 versus 72%) and the average number of mammary carcinomas per rat (0.22 versus 0.86) and significantly prolonged the latency of the carcinomas (247 versus 215 days). Pregnancy and lactation following MNU exposure also significantly reduced both the incidence of mammary carcinoma (25 versus 94%) and the average number of mammary carcinomas per rat (0.25 versus 1.50) and significantly prolonged the latency (240 versus 155 days). Lactation showed an additive effect on the reduction in mammary cancer. Pregnancy suppressed the number of estrogen receptor (ER)- and progesterone receptor (PgR)-positive cells and lowered the cell proliferation rate in the non-tumoral mammary glands. Since the majority (>76%) of the mammary carcinomas was hormone dependent in both the parous and AMV rats, pregnancy and lactation appear to decrease the ER- and/or PgR-positive cells presumed to be the progenitors of hormone-dependent carcinomas and they lowered the cell turnover necessary for tumor promotion in parous rats, resulting in a lower mammary carcinoma yield.     

Induction of invasive carcinomas of the seminal vesicles and coagulating glands of F344 rats by administration of N-methylnitrosourea or N-nitrosobis(2-oxopropyl)amine and followed by testosterone propionate with or without high-fat diet

The potential modifying effects of testosterone propionate (TP) and high-caloric high-fat diet (20% corn oil, HF) on rat accessory sex gland carcinogenesis were investigated. Male F344 rats were treated five times at 4-week intervals with N-methylnitrosourea (MNU) i.v. or N-nitrosobis(2-oxopropyl)amine (BOP) s.c., each injection following 2 weeks pretreatment with dietary ethinyl estradiol. After completion of this carcinogen administration stage, animal groups received subcutaneous implantation of Silastic tubes filled with 40 mg TP with or without HF for 40 weeks. Carcinomas of the seminal vesicles and/or coagulating glands were induced in 5, 39 and 56% of rats given MNU alone, MNU and TP, and MNU and HF plus TP respectively. No equivalent tumors were found in rats given MNU and HF. In the BOP-treated groups, 11% of animals receiving TP but no HF diet demonstrated seminal vesicle carcinomas and 6% of rats receiving TP plus HF diet had coagulating gland carcinoma. Thus while TP exerted a strong enhancing effect on tumor growth in the seminal vesicles and coagulating glands, high caloric HF did not manifest any significant influence.     

Temporal sequence of mammary intraductal proliferations, ductal carcinomas in situ and adenocarcinomas induced by 1-methyl-1- nitrosourea in rats

An experimental model for mammary carcinogenesis has been described in which intraductal proliferations, ductal carcinomas in situ and adenocarcinomas can be readily detected and the frequency of their occurrence quantified. The objective of the experiment reported in this study was to determine the latency period between carcinogen administration and the occurrence of each of these types of lesion. A total of 150 female Sprague-Dawley rats were injected i.p. with 50 mg 1- methyl-1-nitrosourea (MNU)/kg body wt at 21 days of age. Groups of 30 rats each were killed at 7, 14, 21, 28 and 35 days post-carcinogen. Mammary intraductal proliferations were the first detected lesions and were observed in 20% of the animals at 14 days following carcinogen administration. At 21 days post-carcinogen ductal carcinomas in situ and adenocarcinomas were observed. The number of each type of lesion increased with time post-carcinogen, but the temporal pattern of occurrence was different among lesion types. The pattern of lesion occurrence was consistent with intraductal proliferations being a precursor lesion for ductal carcinomas in situ and adenocarcinomas. Furthermore, the data imply that ductal carcinomas in situ represent one pathway of morphological progression by which intraductal proliferations evolve into invasive carcinomas, but that this lesion type, as currently defined histologically, may not be an obligatory intermediate in morphologic progression. These findings are consistent with emerging evidence of multiple but distinct pathogenetic pathways leading to mammary carcinomas that display different morphological patterns and biological activities.      

Decreased susceptibility to NMU-induced mammary carcinogenesis in transgenic rats carrying multiple copies of a rat ras gene driven by the rat Harvey ras promoter

Ras protein over-expression has been observed in human breast cancers although the significance of Ras over-expression in the etiology of breast cancer is unknown and its contribution to breast cancer prognosis is still debated. In this study, the over-expression of both wild-type Harvey and Kirsten Ras proteins as contributors to rat mammary carcinogenesis were examined using a transgenic rat model. Three rat transgenic lines (designated HrHr transgenics) carrying three to six copies of wild-type rat Harvey ras driven by the wild-type rat Harvey ras promoter were produced. In addition, transgenic lines carrying either three or seven copies of the Kirsten ras gene under the same promoter (HrKr) were produced. No pathological changes in the mammary gland were observed in any of the HrHr or HrKr transgenic rat line heterozygotes. Two of the Ras transgenic lines, HrHr (R8) and HrKr (4334), had a significant reduction in NMU-induced rat mammary cancer when compared to their non-transgenic littermates. All five Ras transgenic lines developed fewer carcinomas than their non-transgenic littermates following NMU exposure. The percentage of NMU-induced G35 to A35 activating mutations in the endogenous Harvey ras gene in mammary carcinomas from the HrHr, HrKr transgenic rats and their non-transgenic littermates was similar ( approximately 50%). In contrast, less than 1% of the NMU-induced carcinomas in these Ras transgenic rats had an activating ras mutation in their transgenes. These findings highlight the potential of Ras to function as a modifier gene in repressing mammary carcinogenesis.