DSS1 is required for RAD51 focus formation and genomic stability in mammalian cells

BRCA2 is a breast cancer susceptibility gene implicated in the repair of double-strand breaks by homologous recombination with RAD51. BRCA2 associates with a 70-amino-acid protein, DSS1, but the functional significance of this interaction has remained unclear. Recently, deficiency of a DSS1 orthologue in the fungus Ustilago maydis has been shown to cause a defect in recombinational DNA repair. Here we have investigated the consequences of DSS1 depletion in mammalian cells. We show that like BRCA2, DSS1 is required for DNA damage-induced RAD51 focus formation and for the maintenance of genomic stability, indicating a function conserved from lower eukaryotes to humans. However, DSS1 seems to be not required for BRCA2 or RAD51 stability or for BRCA2 and RAD51 to interact, raising the possibility that DSS1 may be required for the BRCA2-RAD51 complex to become associated with sites of DNA damage.     

Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.     

Centrobin与BRCA2蛋白间相互作用及其细胞定位

为分析乳腺癌易感基因2（breast cancer susceptibility gene 2, BRCA2）蛋白与中心体BRCA2相互作用蛋白（centromal BRCA2 interacting protein, centrobin）间相互作用及其细胞定位的关系,探讨二者功能上的联系,本研究采用哺乳细胞双杂交实验检测体内结合并初步判定BRCA2分子上的结合区域;免疫共沉淀实验进一步验证其体内结合活性,GST-pulldown法检测其体外结合活性,免疫组织化学染色观测BRCA2蛋白的细胞定位及在有丝分裂各期centrobin的细胞定位.结果显示,BRCA2与centrobin间存在体内和体外结合,且BRCA2分子的结合区域主要位于2　393～2　952氨基酸残基处;外源表达BRCA2定位于中心体,在有丝分裂各时相centrobin均定位于中心体. BRCA2与centrobin在体内形成复合物,并存在直接物理结合作用,二者存在细胞空间定位的一致性.该结果为进一步研究BRCA2在中心体复制中的调控作用提供了线索.     

酵母双杂交技术研究与人孕酮受体B相互作用的蛋白质

应用酵母双杂交技术研究与人孕酮受体B（hPRB）发生相互作用的蛋白质,有助于进一步阐明其在乳腺癌的发生发展中发挥重要作用的调控机制。应用酵母双杂交系统3,以hPRB不同结构域为诱饵,筛选人乳腺cDNA文库,寻找能与之相互作用的蛋白质,并运用X—α—Gal等实验提供的信息,筛除假阳性克隆。最终以AF1-DBD结构域作为诱饵最终筛出了1个阳性克隆,经测序及生物信息学分析,这个克隆所编码的蛋白为PIAS3（活化的STAT3的蛋白抑制剂）。结果表明,孕激素受体可以和PIAS3发生相互作用,它们的相互作用有可能参与乳腺癌的生长调控。     

BRCA1 plays a role in the hypoxic response by regulating HIF-1alpha stability and by modulating vascular endothelial growth factor expression

A recent study of breast cancer patients with and without BRCA1 gene mutations found significantly lower levels of VEGF in serum from patients with BRCA1 mutations (Tarnowski, B., Chudecka-Glaz, A., Gorski, B., and Rzepka-Gorska, I. (2004) Breast Cancer Res. Treat. 88, 287-288). Here, we describe a possible mechanistic explanation for this correlation. Because hypoxia in tumors stimulates VEGF expression and secretion we hypothesized that altered BRCA1 protein levels in breast tumors could affect hypoxia-stimulated VEGF promoter activity. This possibility was tested in cells transfected with various combinations of expression plasmids for BRCA1, BRCA1 specific inhibitory RNAs (BRCA1-siRNAs), HIF-1alpha, and a VEGF promoter-reporter and then incubated in normoxia (21%, O2) or hypoxia (0.1%, O2). As predicted, increased BRCA1 levels enhanced both hypoxia-stimulated VEGF promoter activity and the amounts of VEGF mRNA, as determined by semiquantitative RT-PCR and quantitative real time PCR. Using the ChIP assay, we discovered that BRCA1 could be recruited to the endogenous human VEGF promoter along with HIF-1alpha following hypoxia. An interaction between BRCA1 and HIF-1alpha was found in human breast cancer cells. We also found that hypoxia-stimulated VEGF promoter activity and secretion was reduced in cells containing reduced amounts of endogenous BRCA1 protein (obtained by transfecting with BRCA1 siRNAs). A mechanistic explanation for these effects is provided by our finding a reduced half-life and reduced accumulation of HIF-1alpha in hypoxic cells transfected with BRCA1-siRNAs and that proteasome inhibitors blocked these effects of BRCA1-siRNAs. Thus, our results suggest that normal amounts of BRCA1 function in hypoxia to regulate HIF-1alpha stability, probably by interacting with HIF-1alpha.     

BRCA1在人类肿瘤细胞中的表达

人类乳癌易感基因1（BRCA1）是乳癌,卵巢癌和前列腺癌的危险因素之一,而且表现出许多的生物功能,采用Western Blotting和半定量RT－PCR的方法,我们检测了内源性BRCA1蛋白质和mRNA在从十一种人类肿瘤组织中建立的四十三种肿瘤细胞系的表达水平。在不同的肿瘤细胞中BRCA1的表达水平是各不一样的。而且并没有发现BRCA1的表达和细胞的内源性p53基因状况有明显的相关性。通过采用细胞转染乳头状瘤病毒－E6致癌基因或采用畸变的p53基因（143Ala→Val)而导致的p53基因功能失活并不对内源性BRCA1本底表达水平产生任何的影响,胆两种与p53功能有关p21(-/-)和Gadd45基因剔除则轻微地增加BRCA1蛋白质的表达。因此,虽然我们目前还不清楚BRCA1在人类肿瘤细胞中不同表达的功能意义,但本文的结果为进一步研究BRCA1在不同种瘤细胞系的生物功能提供了有价值的背景资料。     

Expression of EMSY gene in sporadic ovarian cancer

The majority of familial breast and ovarian cancers arise from mutations in the BRCA1 and BRCA2 genes. Amplification at the 11q13.5 locus is commonly observed in breast and ovarian cancers. In 2003, Hughes-Davies et al. identified a novel gene (EMSY) at this locus which binds BRCA2 within a region deleted in some cancers. Although little is known about the cellular role of EMSY, indirect evidence suggests that this nuclear protein is capable of silencing the activation potential of BRCA2. In this study we aimed to investigate expression of the EMSY gene and its protein product in sporadic ovarian cancer. Real-time quantitative RT-PCR was performed in 50 ovarian cancer and 17 normal ovarian tissue samples. Overexpression of the EMSY gene was found in 6/50 cases (12%), but in none of the control samples. To determine the EMSY protein by Western blotting, semi-quantitative analysis of the EMSY protein was performed using the Scion Image Gel Analysis Program. Statistical analysis was performed using SPSS 11.5. All patients having EMSY overexpression also displayed increased levels of the EMSY protein. Sporadic ovarian cancer shows overexpression of EMSY at a prevalence similar to that found in breast cancer and the overexpression is correlated with the protein level.     

Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

Mammary tumors are the most common tumor type in both human and canine females. Mutations in the breast cancer susceptibility gene, BRCA2, have been found in most cases of inherited human breast cancer. Similarly, the canine BRCA2 gene locus has been associated with mammary tumors in female dogs. However, deleterious mutations in canine BRCA2 have not been reported, thus far. The BRCA2 protein is involved in homologous recombination repair via its interaction with RAD51 recombinase, an interaction mediated by 8 BRC repeats. These repeats are 26-amino acid, conserved motifs in mammalian BRCA2. Previous structural analyses of cancer-associated mutations affecting the BRC repeats have shown that the weakening of RAD51''s affinity for even 1 repeat is sufficient to increase breast cancer susceptibility. In this study, we focused on 2 previously reported canine BRCA2 mutations (T1425P and K1435R) in BRC repeat 3 (BRC3), derived from mammary tumor samples. These mutations affected the interaction of canine BRC3 with RAD51, and were considered deleterious. Two BRC3 mutations (K1440R and K1440E), reported in human breast cancer patients, occur at amino acids corresponding to those of the K1435R mutation in dogs. These mutations affected the interaction of canine BRC3 with RAD51, and may also be considered deleterious. The two BRC3 mutations and a substitution (T1430P), corresponding to T1425P in canine BRCA2, were examined for their effects on human BRC3 function and the results were compared between species. The corresponding mutations and the substitution showed similar results in both human and canine BRC3. Therefore, canine BRCA2 may be a good model for studying human breast cancer caused by BRCA2 mutations.     

Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (∼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD’s helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign – DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction.     

Prolactin-dependent up-regulation of BRCA1 expression in human breast cancer cell lines.

We report experimental evidence that BRCA1, a breast and ovarian cancer susceptibility gene, is up-regulated in response to prolactin (PRL) stimulation. Expression of the BRCA1 gene was monitored in 2 human breast cancer cell lines (MCF-7 and T-47D) and in the normal mammary epithelial cell line MCF10a. Using competitive RT-PCR, we have shown that PRL induced an increase in BRCA1 mRNA level in MCF-7 and T-47D cell lines at a dose resulting in the maximal enhancement of cell proliferation. The up-regulation was 12-fold in MCF-7 cells and 2-fold in T-47D cells. No increase in BRCA1 mRNA level was observed in the MCF10a cell line. The level of BRCA1 protein was quantified using an affinity chromatography strategy. At the protein level, PRL treatment induced a 4-fold increase of BRCA1 protein expression in MCF-7 and a 6-fold increase in T-47D cells, whereas BRCA1 protein expression was not affected by PRL in MCF10a.