L. Michelle Bennett

Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice

The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-specific patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.

A targeted mouse Brca1 mutation removing the last BRCT repeat results in apoptosis and embryonic lethality at the headfold stage.

A mouse model with a targeted mutation in the 3′ end of the endogenous Brca1 gene, Brca11700T, was generated to compare the phenotypic consequences of truncated Brca1 proteins with other mutant Brca1 models reported in the literature to date. Mice heterozygous for the Brca11700T mutation do not show any predisposition to tumorigenesis. Treatment of these mice with ionizing radiation or breeding with Apc, Msh-2 or Tp53 mutant mouse models did not show any change in the tumor phenotype. Like other Brca1 mouse models, the Brca11700T mutation is embryonic lethal in homozygous state. However, homozygous Brca11700T embryos reach the headfold stage but are delayed in their development and fail to turn. Thus, in contrast to Brca1null models, the mutant embryos do not undergo growth arrest leading to a developmental block at 6.5 dpc, but continue to proliferate and differentiate until 9.5 dpc. Homozygous embryos die between 9.5–10.5 dpc due to massive apoptosis throughout the embryo. These results indicate that a C-terminal truncating Brca1 mutation removing the last BRCT repeat has a different effect on normal cell function than does the complete absence of Brca1.

Mammary Tumor Induction and Premature Ovarian Failure in ApcMin Mice Are Not Enhanced by Brca2 Defi ciency

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7 ± 2.8 and 7.2 ± 2.7, respectively, compared to wild-type and Brca2-mutant females, which had mean mammary tumor multiplicities of 0.1 ±0.4 and 0.3 ± 0.5, respectively. Female ENU-treated Apc-mutant and Brca2/Apc double heterozygotes were also susceptible to premature ovarian failure. Thus, the inheritance of an Apc mutation predisposes ENU-treated female and male mice to mammary tumors and, in the case of female mice, to ovarian failure. These results indicate that mammary tumor development in Apc-mutant mice can progress independently of ovarian hormones. The Apc mutation-driven phenotypes were not modified by mutation of Brca2, perhaps because Brca2 acts in a hormonally dependent pathway of mammary carcinogenesis.

Distinct Effects of Calorie Restriction and Exercise on Mammary Gland Gene Expression in C57BL/6 Mice

Energy balance, including diet, weight, adiposity, and physical activity, is associated with carcinogenesis. Epidemiologic studies indicate that obesity and sedentary and/or active behavior are risk factors for breast cancer in postmenopausal women and survival in both premenopausal and postmenopausal breast cancer patients. Thus, understanding the influence of energy balance modulation on changes in gene expression patterns in the normal mammary gland is important for understanding mechanisms linking energy balance and breast cancer. In a 6-week-long study, female C57BL/6 mice (9-week-old) were randomized into four groups: (a) food consumed ad libitum (AL), (b) AL with access to running wheels (AL+EX), (c) 30% calorie restricted (CR), and (d) 30% CR with access to running wheels (CR+EX). CR mice received 70% of calories but 100% of all other nutrients compared with AL mice. Diet and exercise treatments, individually and combined, had significant effects on body composition and physical activity. Affymetrix oligomicroarrays were used to explore changes in gene expression patterns in total RNA samples from excised whole mammary glands. Contrasting AL versus CR resulted in 425 statistically significant expression changes, whereas AL versus AL+EX resulted in 45 changes, with only 3 changes included among the same genes, indicating that CR and EX differentially influence expression patterns in noncancerous mammary tissue. Differential expression was observed in genes related to breast cancer stem cells, the epithelial-mesenchymal transition, and the growth and survival of breast cancer cells. Thus, CR and EX seem to exert their effects on mammary carcinogenesis through distinct pathways.

BRCA2-null embryonic survival is prolonged on the BALB/c genetic background.

Women who inherit mutations in the BRCA2 cancer susceptibility gene have an 85% chance of developing breast cancer. The function of the BRCA2 gene remains elusive, but there is evidence to support its role in transcriptional transactivation, tumor suppression, and the maintenance of genomic integrity. Individuals with identical BRCA2 mutations display a different distribution of cancers, suggesting that there are low‐penetrance genes that can modify disease outcome. We hypothesized that genetic background could influence embryonic survival of a Brca2 mutation in mice. Brca2‐null embryos with a 129/SvEv genetic background (129B2−/−) died before embryonic day 8.5. Transfer of this Brca2 mutation onto the BALB/cJ genetic background (BALB/cB2−/−) extended survival to embryonic day 10.5. These results indicate that the BALB/c background harbors genetic modifiers that can prolong Brca2‐null embryonic survival. The extended survival of BALB/cB2−/− embryos enabled us to ask whether transcriptional regulation of the Brca1 and Brca2 genes is interdependent. The interdependence of Brca1 and Brca2 was evaluated by studying Brca2 gene expression in BALB/cB1−/− embryos and Brca1 gene expression in BALB/cB2−/− embryos. Nonisotopic in situ hybridization demonstrated that Brca2 transcript levels were comparable in BALB/cB1−/− embryos and wild‐type littermates. Likewise, reverse transcriptase–polymerase chain reactions confirmed Brca1 mRNA expression in embryonic day 8.5 BALB/cB2−/− embryos that was comparable to Brca2‐heterozygous littermates. Thus, the Brca1 and Brca2 transcripts are expressed independently of one another in Brca1‐ and Brca2‐null embryos. Mol. Carcinog. 28:174–183, 2000.

Sequence analysis of the rat Brca1 homolog and its promoter region

Abstract. Since the identification of the human breast and ovarian cancer gene, BRCA1, a large spectrum of germline mutations has been characterized that predispose women to developing these diseases. We have determined the complete coding sequence for the rat BRCA1 homolog and compared it with those of the mouse, dog, and human to help identify the important functional domains of the BRCA1 protein. The overall rat Brcal amino acid identity compared with the predicted mouse, dog, and human gene products is 81%, 69%, and 58%, respectively. In spite of this low overall homology, the amino terminal RING finger domain and one of two nuclear localization signals are highly conserved among these species. In addition, two BRCT domains at the carboxy terminus and a highly acidic region are relatively well conserved. We have also identified several putative regulatory elements through comparison of the bidirectional BRCA1 promoter regions among the rat, mouse, and human genes. These include motifs for CCAAT and G/C boxes, as well as potential SP1, CREB, and NFkB transcription factor binding sites. Finally, analysis of splice variants from rat mammary gland, ovary, testis, spleen, and liver tissues revealed that, while alternative transcripts are detectable, full-length transcripts are the predominant steady-state form.

Spontaneous and irradiation-induced tumor susceptibility in BRCA2 germline mutant mice and cooperative effects with a p53 germline mutation.

Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the loss of both genes enhances tumor development. To elucidate this interaction in an animal model, mice lacking the carboxy terminal domain of Brca2 were crossed with p53 heterozygous mice. Females from this intercross were then irradiated with an acute dose of 5 Gy ionizing radiation at 5 weeks of age and compared to nonirradiated controls. We found decreased survival and timing of tumor onsets, and significantly higher overall tumor incidences and prevalence of particular tumors, including stomach tumors and squamous cell carcinomas, associated with the homozygous loss of Brca2, independent of p53 status. The addition of a p53 mutation had a further impact on overall survival, incidence of osteosarcomas and stomach tumors, and tumor latency. The spectrum of tumors observed for this Brca2 germline mouse model suggest that it faithfully recapitulates some human disease phenotypes associated with BRCA2 loss. In addition, these findings include extensive in vivo data demonstrating that germline Brca2 and p53 mutations cooperatively affect animal survivals, tumor susceptibilities, and tumor onsets.

Breast cancer: Genetic predisposition and exposure to radiation

The identification of breast cancer susceptibility genes, such as BRCA1, BRCA2, ATM, and p53, has been accompanied by the examination of the effects of radiation in combination with genetic mutations at these loci. Women at high risk for developing breast cancer may respond differently than the general population to low‐ and high‐dose radiation exposures associated with screening and treatment. Epidemiologic studies are being performed to investigate the effects of radiation on subsequent breast cancer development in genetically predisposed individuals. Mouse strains with specific genetic modifications are being created to study the consequence of both inherited mutations and radiation on mammary gland carcinogenesis. Finally, studies investigating DNA damage–response pathways after radiation exposure are being performed. Recent work on the effects of several known or suspected breast cancer susceptibility genes, alone or in combination with radiation, is presented here, and directions for future research are considered. Mol. Carcinog. 26:143–149, 1999.

Flor-Essence herbal tonic does not inhibit mammary tumor development in Sprague Dawley rats.

Background: Women who are diagnosed with breast cancer often self-administer complementary and alternative medicines to augment their conventional treatments, improve health, or prevent recurrence. Flor-Essence® tonic is a complex mixture of herbal extracts used by cancer patients because of anecdotal evidence that it can treat or prevent disease. Methods: Female Sprague-Dawley rats were given water or exposed to 3 or 6% Flor-Essence® beginning at 1 day of age. Mammary tumors were induced with a single oral 40 mg/kg/bw dose of dimethyl-benz[a]anthracene at 50 days of age and sacrificed at 23 weeks. Rats were maintained on AIN-76A diet. Results: Control rats had palpable mammary tumor incidence of 51.0% at 19 weeks of age compared to 65.0 and 59.4% for the 3 and 6% Flor-Essence® groups respectively. Overall, no significant difference in time until first palpable tumor was detected among any of the groups. At necropsy, mammary tumor incidence was 82.5% for controls compared to 90.0 and 97.3% for rats consuming 3 and 6% Flor-Essence®, respectively. Mean mammary tumor multiplicity (±SES) for the controls was 2.8 (±0.5) and statistically different from the 3 or 6% Flor-Essence® groups with 5.2 (±0.7), and 4.8 (±0.6), respectively (p £ 0.01). As expected, the majority of isolated tumors were diagnosed as adenocarcinomas. Conclusions: Flor-Essence® can promote mammary tumor development in the Sprague-Dawley rat model. This observation is contrary to widely available anecdotal evidence as well as the desire of the consumer that this commercially available herbal tonic will suppress and/or inhibit tumor growth

Pregnancy and protection from hormonally associated tumor development

Why is full-term pregnancy protective for breast cancers, ovarian cancers, and as described in this issue of EPIDEMIOLOGY, uterine leiomyomas?1 Is there a single mechanism or are there multiple mechanisms underlying the protective effects of parity? Are there shared or separate mechanisms that reduce the risk of tumors of the breast, ovary and uterus? The answers to these important questions remain unknown, but the observations of Rostgaard et al.2 on ovarian cancers and Baird and Dunson1 on uterine fibroids provide some insights and potential new directions of study. Although parity is consistently protective for all these tumors, the extent of the protective effect varies with age at pregnancy. For breast cancer, pregnancy before the age of 20 results in the greatest protection.3,4 For ovarian cancer, older age at first pregnancy is more protective.5,6 Baird and Dunson show that pregnancies at an early age ( 25 years) or a late age ( 30 years) have no effect on uterine leiomyomas, whereas a first pregnancy between age 25 and 29 years is protective.1 The differing effects of age at pregnancy suggest different mechanisms of protection. For breast cancer, there are numerous hypotheses to explain the protective effect of pregnancy, including decreasing responsiveness to carcinogens and mitogens and alterations in cell fate associated with mammary gland differentiation.7 In the ovary, interruption of incessant ovulation by pregnancy is one possible mechanism. However, pregnancy-associated clearance of premalignant cells is an alternate hypothesis supported by Rostgaard et al.8 The authors carefully note that these hypotheses are not mutually exclusive. Using the simple concept of premalignant cell clearance, Rostgaard et al. provide a model that statistically fits the data from a cohort of 1.5 million Danish women relating reproductive history and ovarian cancer. Baird and Dunson base the explanation of their results, showing only pregnancies at a certain age to be protective, on the hypothesis that postpartum involution of the uterus clears small fibroids during the dramatic remodeling of the tissue.1 At an early age of pregnancy small fibroids have not formed, whereas at a later age of pregnancy fibroids are larger, making them resistant to the effects of remodeling after parturition.