Loss of function of several known 'tumor suppressor' genes plays an important role in transformation to malignancy. We have shown that the retinoblastoma (RB) gene is one of these, and its dysfunction is found in many cancers besides retinoblastoma, including some breast cancers. Furthermore, normal Rb protein inhibits progression through the cell cycle, and in tumors with dysfunctional Rb, we have shown that restoration of functional Rb can reverse malignancy. We have now identified several new proteins which interact with Rb and which regulate the cell cycle at distinct points; clearly these could also function as tumor suppressors. In addition, the genes BRCA-1 and brush-1 (which may be BRCA-2) which are associated with familial breast cancer appear to be tumor suppressor genes, though their prevalence in non-familial breast cancer has not been established. We therefore propose to investigate the roles of all of these genes in human breast cancer, demonstrating prevalence, biological activity, relationship with progression and clinical outcome, and potential for therapeutic reversal of their dysfunction. Our specific aims are: 1) To carry out genetic analyses of our newly characterized Rb-associated genes H-nuc, mitosin, and E2F-1 in breast cancer and paired normal tissues. For genes found to be mutated in breast cancer, we will define their biological actions in a tumor cell culture system. 2) To determine the biological significance of BRCA-1 and brush-1 mutations in the same system. 3) To prepare effective monoclonal antibodies against the H-nuc, mitosin, E2F-1, and BRCA-1 proteins for use in immunohistochemistry. 4) To detect these proteins and their mutations by immunostaining in a large series of human breast cancers and premalignant breast lesions of defined stages, in order to evaluate their association with known biomarkers, with probability of recurrence of primary breast cancer, and with evolutionary stage and cancer risk of premalignant lesions. 5) To explore the therapeutic power of an adenovirus vector carrying the normal RB gene and potentially some of the other tumor suppressor genes studied here to inhibit growth of tumors in which these genes are dysfunctional, using tumor xenograft models of both local (mammary fat pad) and locally advanced (malignant ascites) human breast cancer.