Women carrying germ-line mutations in the BRCA1 gene are virtually certain to develop either breast cancer, ovarian cancer, or both. The BRCA1 coding sequence predicts a novel protein of over 200 kd. Near the NH2 terminus lies a consensus Zn2+ finger DNA binding domain strongly suggesting that this is a new transcription factor. Several germ-line nonsense mutations have been found indicating that loss of BRCA1 function is tumorigenic, analogous to other recessive oncogenes. The BRCA1 message is expressed at low levels in normal breast, ovary, and a wide variety of other tissues. This raises the question of why carriers of the mutant gene are specifically susceptible to breast and ovarian cancer. Preliminary studies from our laboratory indicate that expression of BRCA1 is regulated by estrogen, thereby providing a clue to its tissue-specific effects. The experiments proposed in this project will begin by producing basic reagents to analyze BRCA1 function and expression. Wild-type and mutant BRCA1 cDNAs will be constructed and cloned into expression vectors. A baculovirus fusion protein will be made to initiate antibody production. Mammalian expression vectors will be constructed to test whether expression of BRCA1 is growth inhibitory in breast cancer cells, analogous to other recessive oncogenes. Mutant BRCA1 vectors will also be tested in these assays to determine if they have lost the ability to inhibit growth. Expression of BRCA1 mRNA and protein will be analyzed in primary cancers and correlates made with established clinico-pathologic features, particularly hormone receptor status. In situ methods will be used to study localization of BRCA1 expression. Using the Core for Genetic Susceptibility Testing, patients that are potential BRCA1 carriers will be identified. Consent will be obtained and the BRCA1 gene will be sequenced. Age matched controls with no evidence of family history will also be sequenced to determine the prevalence of these mutations in familial and sporadic cancers. Estrogen regulation of the BRCA1 gene will be examined. A profile of response to estrogens and antagonists will be determined. Regulation of BRCA1 will also be studied in vivo by examining the hormone sensitive endometrium at different times during the menstrual cycle. Finally, the estrogen response element will be identified from the BRCA1 promoter region. Other consensus promoter sequences will also be identified and regulation via these DNA binding sites will be explored.
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