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Current Research: Breast cancer is the most common malignancy in women. Mutations in BRCA1 and BRCA2 genes play an important role in the development of early-onset familial breast cancer. Our goal is to carry out functional dissection of these genes using a mouse model system. These proteins interact with RAD51 and other proteins involved in DNA repair. Such interactions suggest that BRCA1 and BRCA2 are involved in detecting and repairing double-strand breaks, thereby controlling cell cycle progression. Loss of such a repair/monitor system results in accumulation of mutations, leading to tumorigenesis. Since complete loss of function mutations in mice result in embryonic lethality, we are engaged in generating an array of specific mutations along the length of proteins encoded by these genes. To accomplish this, we have established humanized mouse models for functional analysis of human BRCA1 and BRCA2 genes. Although transgenic humanized mice using BACs are excellent in vivo models for understanding the functional significance of the human mutations, considering the fact that hundreds of different mutation have been identified, they are not ideally suited for characterizing large numbers of mutations. Hence, we have also developed an embryonic stem (ES) cell-based functional assay to rapidly screen large numbers of mutations. The assay is based on the observation that mouse ES cells that lack both copies of either Brca1 or Brca2 genes do not survive. Unlike human breast or ovarian cells, embryonic stem cells fail to proliferate in the absence of BRCA1/2 due to stringent cell cycle regulation. We have generated ES cell lines that have a single functional allele of the Brca2 gene, which can be disrupted by using an inducible system like Cre-loxP mediated recombination. We are now using this assay system to study large numbers of mutations identified in BRCA2 by testing their ability to rescue the ES cell lethality. Those mutations that can rescue the lethality are being examined for defects in various DNA repair processes. We will soon have ES cell lines that can be used to study mutations in BRCA1.
Proposed Project: One of the key questions that we are interested in understanding is why mutation in genes like BRCA1/2 that are involved in DNA repair, a function affecting all cell types, results in predominantly breast and ovarian cancer. There are several hypotheses to explain the tissue specific phenotype associated with BRCA1/2 mutation. One of these is the presence of redundant repair pathways in other tissues that are not functional in breast and ovarian epithelial cells, making breast and ovarian tissues more susceptible to loss of BRCA1/2 function. Alternatively, the genes involved in activating the apoptotic machinery in response to failed DNA repair may not be very efficient in these tissues. Consequently, cells with damaged DNA continue to proliferate and acquire additional mutations, some likely to be in genes involved in growth control. We hope to identify such genes that trigger apoptosis or cell cycle arrest in response to loss of BRCA1 or BRCA2 function. Identification of such genes is likely to improve our understanding of the tissue-specific phenotype associated with BRCA1 and BRCA2 mutations. We will identify genes that can rescue the lethality of BRCA1 or BRCA2-deficient embryonic stem cells. We will accomplish this by undertaking a retrovirus-mediated insertional mutagenesis approach in ES cells. Once BRCA1/2-deficient ES cells carrying retroviral insertion are identified, we will clone the insertion sites by using the inverse PCR method. After sequence analysis, we will map the tagged sites to the mouse genome using BLAT analysis. We will examine the mouse genomic sequence to identify candidate genes that may be over-expressed due to the presence of viral LTR. Next, we will determine the ability of the candidate genes to rescue ES cell lethality by over-expressing them in ES cells. We will test the in vivo relevance by generating transgenic mice over-expressing the candidate genes in embryos as well as in adult tissues. We will generate these transgenic mice in a Brca1 or Brca2 mutant genetic background and determine whether the transgene can rescue the lethality of the homozygous mutant embryos, partially or completely. If the lethality is completely rescued then we will monitor the adults for tumor development. We anticipate that the genes identified in our screen will also be involved in tumorigenesis in humans and may be amplified/mutated in BRCA1 or BRCA2-deficient tumors. We will test this by determining whether these genes are amplified in human tumors.
