The markedly elevated risk of breast cancer observed in women carrying germline mutations in the BRCA2 breast cancer susceptibility gene strongly suggests that this gene plays a critical role in the regulation of mammary epithelial cell growth. We have determined the developmental pattern of expression of the murine homologue of BRCA2 in fetal and adult tissues. Our results indicate that Brca2 expression is highest in proliferating cellular compartments, particularly those undergoing differentiation. Specifically, we have found that Brca2 expression is developmentally regulated in the breast during puberty and pregnancy, and in mammary epithelial cells in vitro during proliferation and differentiation. Recent evidence has also implicated Brca2 in the response to ionizing radiation in embryonic cells. We hypothesize that Brca2 plays a critical role in the regulation of proliferation, differentiation and the response to DNA damage in the mammary epithelium. In this application, we propose to determine the mechanisms by which BRCA2 normally controls these processes in mammary epithelial cells.
The specific aims of this proposal are to: I. Determine the role of Brca2 in mammary epithelial proliferation. We will test the hypothesis that Brca2 plays central role in regulating mammary epithelial cell proliferation by conditionally overexpressing Brca2, and inducibly blocking Brca2 expression, in nontransformed mammary epithelial cells. The ability of mutant forms of Brca2 to promote or block cell proliferation will be used to identify and analyze functional domains involved in the regulation of cell growth. The interaction of Brca2 with known cell cycle-regulatory pathways will be analyzed. Transgenic mice inducibly overexpressing wild-type Brca2 will be used to analyze the effect of Brca2 overexpression on mammary epithelial proliferation in vivo. II. Determine the role of Brca2 in mammary epithelial differentiation. We will test the hypothesis that Brca2 upregulation is required for the differentiation of mammary epithelial cells by conditionally blocking Brca2 expression in mammary epithelial cells induced to differentiate by lactogenic hormones. In order to determine whether Brca2 upregulation is sufficient to induce mammary epithelial cell differentiation, we will conditionally overexpress wild- type Brca2 in mammary epithelial cells. The ability of mutant forms of Brca2 to promote or block the differentiation of mammary epithelial cells will be used to identify and analyze functional domains involved in the regulation of mammary epithelial differentiation. Transgenic mice inducibly overexpressing Brca2 will be used to analyze the effect of Brca2 overexpression on the normal differentiation and development of the mammary gland in vitro. III. Determine the role of Brca2 in DNA damage response. We will test the hypothesis that Brca2 plays a role in the DNA damage response by determining whether alterations in Brca2 expression levels affect the response to DNA damage in mammary epithelial cells. We will also determine whether Brca2 is posttranslationally modified following DNA damage in a manner similar to that observed for Brca1. Finally, we will determine whether Brca2 and Brca1 interact during these processes.
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