Characterizing a rat Brca2 knockout model.

Evidence exists that BRCA2 carriers may have an elevated risk of breast, ovarian, colon, prostate, and pancreatic cancer. In general, carriers are defined as individuals with protein truncating mutations within the BRCA2 gene. Many Brca2 knockout lines have been produced and characterized in the mouse. We previously produced a rat Brca2 knockout strain in which there is a nonsense mutation in exon 11 between BRC repeats 2 and 3, and a truncated protein is produced. Interestingly, while such a mutation in homozygous mice would lead to limited survival of approximately 3 months, the Brca2-/- rats are 100% viable and the vast majority live to over 1 year of age. Brca2-/- rats show a phenotype of growth inhibition and sterility in both sexes. Aspermatogenesis in the Brca2-/- rats is due to a failure of homologous chromosome synapsis. Long-term phenotypes include underdeveloped mammary glands, cataract formation and lifespan shortening due to the development of tumors and cancers in multiple organs. The establishment of the rat Brca2 knockout model provides a means to study the role of Brca2 in increasing cancer susceptibility and inducing a novel ocular phenotype not previously associated with this gene.

The Mcs7 quantitative trait locus is associated with an increased susceptibility to mammary cancer in congenic rats and an allele-specific imbalance.

Identification of high-penetrance breast cancer genes such as Brca1 has been accomplished by analysing familial cases. However, these genes occur at low frequency and do not account for the majority of genetic risk. Identification of low-penetrance alleles that occur commonly in populations may benefit from unbiased genome-wide screening. One such approach uses linkage studies in rodent models to identify homologous human candidates. The Wistar Kyoto (WKy) rat is resistant to mammary carcinomas induced with 7,12-dimethybenz[a]anthracene (DMBA), whereas the Wistar Furth (WF) strain is susceptible. Previous genome-wide linkage studies in crosses of these strains identified three WKy resistance quantitative trait loci, Mcs5, Mcs6 and Mcs8, and one predicted to increase susceptibility, Mcs7. The Mcs7 region on rat chromosome 10 (RNO10) is orthologous to human 17q, a common site of genetic aberrations in breast cancer. Here, we establish the independent phenotype conferred by Mcs7 using congenic rats carrying the WKy Mcs7 locus on a WF background. Tumor multiplicity was significantly higher ( approximately 50%) in DMBA-treated congenics homozygous and heterozygous for the WKy allele at the Mcs7 locus, compared to controls. We also investigated allelic imbalance (AI) in mammary carcinomas from (WKy x WF)F1 rats and Mcs7 heterozygous congenics. Of the four known WKy Mcs loci tested, only Mcs7 displayed AI. The pattern of AI in carcinomas from both F1 and Mcs7 congenic rats was similar, suggesting a WF allelic loss. Together, these data suggest that one or more breast cancer-related genes are located within the dominantly acting WKy allele at the Mcs7 locus.

Pharmacologic, but not dietary, genistein supports endometriosis in a rat model.

Endometriosis is a disease in which uterine tissue proliferates in extrauterine sites. Using a surgical model to simulate endometriosis, we explored the potential for the phytoestrogen genistein, by injection and diet, to sustain endometriosis in rats. Uterine tissue was attached to intestinal mesentery of 8-week-old Sprague Dawley rats. After 3 weeks, the rats were ovariectomized and the implants measured. Following 3 weeks of daily injections or exposure to dietary genistein, animals were necropsied and implants located and measured. Injections of genistein (50 and 16.6 microg/g BW) or estrone (1 microg/rat) sustained the implants; injection of sesame oil (vehicle for estrone), dimethylsulfoxide (DMSO; vehicle for genistein), or genistein at 5.0 microg/g BW did not sustain implants. Dietary genistein (250 or 1000 mg genistein/kg AIN-76A diet) did not support the implants. In ovary-intact rats exposed to 250 mg genistein/kg AIN-76A diet, implant size was not altered, compared to control-fed animals. To assess estrogenic actions of genistein, we measured uterine estrogen receptor alpha (ER-alpha) and progesterone receptor (PR) isoforms A and B by Western blot analyses. Injections of estrone or genistein (50 or 16.6 microg/g BW) significantly reduced uterine ER-alpha compared to vehicle-treated animals. PR (B) was significantly increased by all injected doses of genistein or estrone and by the higher dietary dose (1000 mg genistein/kg AIN-76A). PR (A) was significantly increased by injected doses of genistein (16.6 and 5.0 microg/g BW). We conclude that pharmacologic injections, but not dietary physiological concentrations of genistein, support surgically induced endometriosis in rats. Our results suggest a critical role for ER modulation and genistein bioavailability in the maintenance of the implants.

Sex steroid receptor regulation by genistein in the prepubertal rat uterus.

We evaluated the mechanism of action by the phytoestrogen genistein in the prepubertal rat uterus, when administered pharmacologically or physiologically. Female rats were injected with genistein (500 microg/g body weight), estradiol benzoate (EB) (500 ng/g body weight) or vehicle, dimethylsulfoxide (DMSO), on days 16, 18, and 20 postnatal. In 21-day-old rats, both compounds increased circulating estradiol and decreased progesterone concentrations. Uterine estrogen receptor alpha (ER-alpha) and androgen receptor (AR) proteins were reduced, and progesterone receptors (PR) were increased, as measured by western blot analyses. Immunohistochemistry for ER-alpha was confirmatory. Reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated a decrease in ER-alpha, but not in ER-beta, PR and AR mRNA levels following genistein treatment. In prepubertal rats exposed perinatally to 250 mg genistein per kg AIN-76A diet or 250 microg estradiol per kg diet, uterine ER-alpha, AR, and PR proteins were not altered significantly. We conclude that pharmacologic, but not physiologic concentrations of genistein can modulate sex steroid receptor expression in the rat uterus.

Genistein studies in rats: potential for breast cancer prevention and reproductive and developmental toxicity.

Asian women and men who consume a traditional diet high in soy products have low incidences of breast and prostate cancers, respectively. Yet Asians who immigrate to the United States and adopt a Western diet lose this protection. We investigated the potential of genistein, a component of soy, to protect against breast cancer and to cause reproductive and developmental toxicity. Our study showed that injections of genistein in rats during the prepubertal period resulted in a 50% reduction of chemically induced mammary tumorigenesis. Studies in mammary whole mounts revealed that prepubertal genistein exposure resulted in fewer terminal end buds and more lobules type II. Cell proliferation in the terminal end buds of adult rats treated prepubertally with genistein was less than that in animals treated with the vehicle (dimethyl sulfoxide). Reproductive and developmental toxicity studies did not find significant alterations to fertility, number of male and female offspring, body weight, anogenital distance, vaginal opening, testes descent, estrus cycle, or follicular development. We concluded that pharmacologic doses of genistein given to immature rats enhance mammary gland differentiation, resulting in a significantly less proliferative gland that is not as susceptible to mammary cancer. We speculate that breast cancer protection in Asian women consuming traditional soy-containing diets is, in part, derived from early exposure to genistein-containing soy. We believe that early programming events are essential for cancer protection benefits.

Prepubertal genistein treatment modulates TGF-alpha, EGF and EGF-receptor mRNAs and proteins in the rat mammary gland.

We have previously demonstrated that exposure to genistein early in life protects against chemically-induced mammary cancer in rats. To gain insight into the mechanism of action, we have investigated the expression of the EGF-signaling pathway in the mammary glands of 21 and 50 day old rats treated on days 16, 18, and 20 postpartum with 500 microg genistein/g body weight (B.W.) or an equivalent volume of the vehicle, dimethylsulfoxide (DMSO). This prepubertal genistein treatment up-regulated TGF-alpha and the EGF-receptor (EGFR), but not EGF, in mammary terminal ductal structures at day 21 postpartum. TGF-alpha, EGF and EGFR mRNA levels were similar in 21 day old control- and genistein-treated animals. At day 50 postpartum, mammary glands of genistein treated rats had more lobules and fewer terminal end buds (TEBs) and terminal ducts (TDs), i.e. they were more differentiated. TGF-alpha mRNA levels were down-regulated in TEB of proestrous and estrous females; EGF mRNA levels were down-regulated in TDs of proestrous, but not in estrous females; and EGFR mRNA levels were not altered in 50 day old proestrous or estrous female rats. EGFR immunostaining intensity was decreased in TEBs, but not in the total gland. EGF was increased in TEBs and TDs. TGF-alpha, EGF and EGFR were also observed in the stroma and fat pad, but genistein treatment did not alter the expression of these proteins in those locations. TGF-alpha, but not EGF and EGFR, immunostaining was observed in cell nuclei (not modulated by genistein), suggesting that this growth factor may act directly on nuclear events such as transcription and DNA replication. For comparative purposes, prepubertal diethylstilbestrol treatment was investigated and found to decrease EGFR immunostaining intensity and total IHC staining in all terminal ductal structures. We conclude that prepubertal genistein treatment directly stimulates TGF-alpha and EGFR to enhance mammary gland differentiation. This programs the differentiated cells for a down-regulated EGF-signaling pathway in TEBs and TDs of adult mammary glands. Reduced EGFR expression at time of carcinogen exposure may account for genistein programming against mammary cancer.