Much of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in the BRCA1 tumour-suppressor gene. The nuclear protein BRCA1 has the properties of a transcription factor, and can interact with the recombination and repair protein RAD51. Young women with germline alterations in BRCA1 develop breast cancer at rates 100-fold higher than the general population, and BRCA1-null mice die before day 8 of development. However, the mechanisms of BRCA1-mediated growth regulation and tumour suppression remain unknown. Here we show that BRCA1 transactivates expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in a p53-independent manner, and that BRCA1 inhibits cell-cycle progression into the S-phase following its transfection into human cancer cells. BRCA1 does not inhibit S-phase progression in p21-/- cells, unlike p21+/+ cells, and tumour-associated, transactivation-deficient mutants of BRCA1 are defective in both transactivation of p21 and cell-cycle inhibition. These data suggest that one mechanism by which BRCA1 contributes to cell-cycle arrest and growth suppression is through the induction of p21.

Breast carcinoma is the most common malignancy among women in developed countries. Because family history remains the strongest single predictor of breast cancer risk, attention has focused on the role of highly penetrant, dominantly inherited genes in cancer-prone kindreds (1). BRCA1 was localized to chromosome 17 through analysis of a set of high-risk kindreds (2), and then identified four years later by a positional cloning strategy (3). BRCA2 was mapped to chromosomal 13q at about the same time (4). Just fifteen months later, Wooster et al. (5) reported a partial BRCA2 sequence and six mutations predicted to cause truncation of the BRCA2 protein. While these findings provide strong evidence that the identified gene corresponds to BRCA2, only two thirds of the coding sequence and 8 out of 27 exons were isolated and screened; consequently, several questions remained unanswered regarding the nature of BRCA2 and the frequency of mutations in 13q-linked families. We have now determined the complete coding sequence and exonic structure of BRCA2 (GenBank accession #U43746), and examined its pattern of expression. Here, we provide sequences for a set of PCR primers sufficient to screen the entire coding sequence of BRCA2 using genomic DNA. We also report a mutational analysis of BRCA2 in families selected on the basis of linkage analysis and/or the presence of one or more cases of male breast cancer. Together with the specific mutations described previously, our data provide preliminary insight into the BRCA2 mutation profile.

Mutations of the BRCA1 tumor suppressor gene are the most commonly detected alterations in familial breast and ovarian cancer. Although BRCA1 is required for normal mouse development, the molecular basis for its tumor suppressive function remains poorly understood. We show here that BRCA1 increases p53-dependent transcription from the p21 WAF1/CIP1 and bax promoters. We also show that BRCA1 and p53 proteins interact both in vitro and in vivo. The interacting regions map, in vitro, to aa 224 ± 500 of BRCA1 and the C-terminal domain of p53. Tumor-derived transactivation-de®cient BRCA1 mutants are defective in co-activation of p53-dependent transcription and a truncation mutant of BRCA1 that retains the p53-interacting region acts as a dominant inhibitor of p53-dependent transcription. BRCA1 and p53 cooperatively induce apoptosis of cancer cells. The results indicate that BRCA1 and p53 may coordinately regulate gene expression in their role as tumor suppressors.

Inherited mutations in BRCA1 confer susceptibility to breast and ovarian neoplasms. However, the function of BRCA1 and the role of BRCA1 in noninherited cancer remain unknown. Characterization of alternately spliced forms of BRCA1 may identify functional regions; thus, we constructed expression vectors of BRCA1 and a splice variant lacking exon 11, designated BRCA1⌬672-4095. Immunofluorescence studies indicate nuclear localization of BRCA1 but cytoplasmic localization of BRCA1⌬672-4095. Two putative nuclear localization signals (designated NLS1 and NLS2) were identified in exon 11; immunofluorescence studies indicate that only NLS1 is required for nuclear localization. RNA analysis indicates the expression of multiple, tissue-specific forms of BRCA1 RNAs; protein analysis with multiple antibodies suggests that at least three BRCA1 isoforms are expressed, including those lacking exon 11. The results suggest that BRCA1 is a nuclear protein and raise the possibility that splicing is one form of regulation of BRCA1 function by alteration of the subcellular localization of expressed proteins.BRCA1 was isolated by positional cloning methods as a gene linked to breast cancer in families with a pattern of autosomal dominant inheritance of the disease (21). Single dominant susceptibility alleles are thought to account for 5 to 10% of all breast cancers, and BRCA1 germ line mutations are widely held to be responsible for approximately 50% of all inherited breast cancers. Inherited BRCA1 mutations are also thought to be responsible for the disease in 80 to 90% of all families with breast-ovarian cancer syndrome (9). Women inheriting mutations in BRCA1, most of which are truncating mutations that result in nonfunctional or unstable proteins (8), have an 85% chance of developing breast cancer in their lifetime (9). An on-line database now provides a listing of known BRCA1 mutations (http:// www. nchgr. nih. gov/ Intramural_research/ Lab_transfer/Bic/index.html), but little is known about the regulation of this gene or the function of its protein product. Unfortunately, mutations in BRCA1 are distributed evenly over the gene, providing little in the way of clues for localizing critical functional regions.BRCA1 is a large gene, with a coding region of 5.5 kb and a total mRNA of approximately 8.0 kb. There is little identifiable homology to known genes, with the exception of a short region in the 5Ј end (spanning exons 2 to 5) that encodes a RING finger with a typical Cys 3 -His-Cys 4 structure. This is a zincbinding motif that is found in a family of transcription factors and may be a protein-protein interaction site. Initial reports also provided evidence for a complex pattern of alternate splicing and the potential for translation of a number of BRCA1 protein isoforms (21). BRCA1 fits the model of a classic tumor suppressor gene, a hypothesis supported by recent work demonstrating that expression of BRCA1 inhibits growth of breast and ovarian cancer cell lines and MCF7-based tumor development in nude mice (16). Additional da...

The breast cancer susceptibility gene, BRCA2 on chromosome 13q12-13, was recently isolated. Mutations in BRCA2 are thought to account for as much as 35% of all inherited breast cancer as wall as a proportion of inherited ovarian cancer. Many BRCA2-linked families also contain cases of male breast cancer. We have analysed germline DNA from 50 males with breast cancer (unselected for family history) and 26 individuals from site-specific female breast and breast-ovarian cancer families for mutations in BRCA2. All 17 breast-ovarian cancer families have been screened for BRCA1 coding region mutations and none were detected. Conformation-sensitive gel electrophoresis (CSGE) analysis of PCR-amplified DNA followed by direct sequencing was used to detect sequence variants. Three of eleven individuals carry the same mutation, all are of Ashkenazi Jewish descent, supporting the observation by Neuhausen et al. in this issue that there is a common mutation in this population. Eleven truncating mutations and nine polymorphisms were identified -- all were coding region variants. No loss-of-transcript mutations were identified in the sixteen samples for which this analysis was possible. Seven of the nine disease-associated mutations were detected in the 50 men with breast cancers; for thus in our series, BRCA2 mutations account for 14% of male breast cancer, all but one of which had a family history of male and/or female breast cancer.