BRCA1 encodes a tumor suppressor that is mutated in familial breast and ovarian cancers. Here, it is shown that BRCA1 interacts in vitro and in vivo with hRad50, which forms a complex with hMre11 and p95/nibrin. Upon irradiation, BRCA1 was detected in discrete foci in the nucleus, which colocalize with hRad50. Formation of irradiation-induced foci positive for BRCA1, hRad50, hMre11, or p95 was dramatically reduced in HCC/1937 breast cancer cells carrying a homozygous mutation in BRCA1 but was restored by transfection of wild-type BRCA1. Ectopic expression of wild-type, but not mutated, BRCA1 in these cells rendered them less sensitive to the DNA damage agent, methyl methanesulfonate. These data suggest that BRCA1 is important for the cellular responses to DNA damage that are mediated by the hRad50-hMre11-p95 complex.

To define a mechanism by which retinoblastoma protein (Rb) functions in cellular differentiation, we studied primary fibroblasts from the lung buds of wild-type (RB + / +) and null-mutant (RB-/-) mouse embryos. In culture, the RB +/+ fibroblasts differentiated into fat-storing cells, either spontaneously or in response to hormonal induction; otherwise syngenic RB -/-fibroblasts cultured in identical conditions did not. Ectopic expression of normal Rb, but not Rb with a single point mutation, enabled RB-/-fibroblasts to differentiate into adipocytes.

The BRCA2 gene was identified based on its involvement in familial breast cancer. The analysis of its sequence predicts that the gene encodes a protein with 3,418 amino acids but provides very few clues pointing to its biological function. In an attempt to address this question, specific antibodies were prepared that identified the gene product of BRCA2 as a 390-kDa nuclear protein. BRCA2 was identified (1, 2) based on its initial mapping to chromosome 13q12-13 by linkage analysis of families with inherited breast cancer not attributed to mutations in BRCA1 (3). Germ-line mutations in BRCA2 account for the same percentage of familial breast cancers as BRCA1 (1). Together, these two breast cancer susceptibility genes are responsible for a large percentage of familial cases. In addition to breast cancer, BRCA2 mutations are also linked to other cancers including ovarian (4, 5), hepatocellular (6), pancreatic (5, 7), and prostate (4-6) tumors. However, mutations in BRCA2, like BRCA1, are mainly found in familial breast cancer but seldom occur in sporadic cases (8, 9). There have been over 100 distinct mutations spanning the sequence of this large gene (Breast Cancer Information Core). The majority of these mutations lead to truncation of the gene product.BRCA2 has 27 exons and expresses an mRNA 11 kb in size (1). The expression pattern of BRCA2 mRNA is similar to that of BRCA1, with highest levels in the testis, thymus, and ovaries (10). During mouse development, Brca2 mRNA is first detected on embryonic day 7.5, a time of rapid proliferation (11). At the cellular level, expression is regulated in a cell cycledependent manner with peak expression of BRCA2 mRNA in S phase (12). These results suggest BRCA2 may have a role in proliferating cells. Using a gene knockout method to create mice with BRCA2 mutations, homozygous mutant mice with BRCA2 truncated from the 5Ј half of exon 11 cannot survive embryogenesis (refs. 11, 13, and 14, and our unpublished results), suggesting that Brca2, like Brca1, plays an essential role in early embryonic development. Similar to Brca1, Brca2 heterozygotes are phenotypically normal and fertile. Although they are predicted to be genetically predisposed to cancer, they show no evidence to date of increased tumor formation.The identification of the BRCA2 gene was accomplished, quickly giving rise to the hope that the function(s) of the gene product would soon become clear. However, BRCA2 presents dilemmas similar to BRCA1, as very little insight in its function has been determined. To address this question systematically, we have identified the cellular BRCA2 protein as a nuclear protein and determined the domain responsible for interactions with human RAD51. Furthermore, BRCA2 apparently has a critical role in response to DNA damage. These results provide a molecular basis that begins to explain how mutations of BRCA2 contribute to carcinogenesis.

Mutations of the gene encoding p53, a 53-kilodalton cellular protein, are found frequently in human tumor cells, suggesting a crucial role for this gene in human oncogenesis. To model the stepwise mutation or loss of both p53 alleles during tumorigenesis, a human osteosarcoma cell line, Saos-2, was used that completely lacked endogenous p53. Single copies of exogenous p53 genes were then introduced by infecting cells with recombinant retroviruses containing either point-mutated or wild-type versions of the p53 cDNA sequence. Expression of wild-type p53 suppressed the neoplastic phenotype of Saos-2 cells, whereas expression of mutated p53 conferred a limited growth advantage to cells in the absence of wild-type p53. Wild-type p53 was phenotypically dominant to mutated p53 in a two-allele configuration. These results suggest that, as with the retinoblastoma gene, mutation of both alleles of the p53 gene is essential for its role in oncogenesis.

The BRCA1 gene product was identified as a 220-kilodalton nuclear phosphoprotein in normal cells, including breast ductal epithelial cells, and in 18 of 20 tumor cell lines derived from tissues other than breast and ovary. In 16 of 17 breast and ovarian cancer lines and 17 of 17 samples of cells obtained from malignant effusions, however, BRCA1 localized mainly in cytoplasm. Absence of BRCA1 or aberrant subcellular location was also observed to a variable extent in histological sections of many breast cancer biopsies. These findings suggest that BRCA1 abnormalities may be involved in the pathogenesis of many breast cancers, sporadic as well as familial.