Identification of the hereditary breast and ovarian cancer susceptibility gene, BRCA1, has led to increased awareness regarding the disease, increased surveillance in women with a family history, and the advent of genetic testing for mutations. However, our knowledge regarding the role of this gene in sporadic cancers and its function in normal and neoplastic growth and development is sparse. This proposal is a continuation of studies performed over the past two years designed to investigate these issues. The following experiments are outlined in this proposal: 1) Since BRCA1 is rarely a target for somatic mutation breast cancer, is it inactivated by loss of expression? Using microdissected normal epithelium, carcinoma in situ, and invasive cancers, both expression and promoter methylation will be evaluated employing PCR based methodology. 2) BRCA1 mRNA expression is tightly regulated through the cell cycle. Alterations in expression, either the levels of temporal nature, may find its function. Therefore, the basal and cell cycle regulated promoter elements will be identified. Using the minimal promoter fragment, the identity of trans-acting factors that complex with specific promoter sequences will be established. These experiments will complement and extend those proposed in the first aim. 3) It has been difficult to derive cell lines that over- express BRCA1 in order to study phenotypic and molecular events associated with the protein. In order to overcome this problem, tetracycline inducible cell lines have been established. BRCA1 under the test-inducible promoter will be introduced into these cells to study phenotypic effects of BRCA1 over-expression including cell cycle parameters, apoptosis, response to DNA damage, and response to differentiating agents. 4) Using these same cell lines, genes that are induced by BRCA1 expression will be identified using representational difference analysis (RDA). From the promoter elements of these genes, common BRCA1 response elements will be distinguished.
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