| BRCA1 (BReast CAncer susceptibility gene 1) has been implicated in cellular processes such as cell-cycle control, centrosome duplication, DNA-damage repair/recombination, and transcription but the exact mechanism is still under investigation. In this thesis dissertation, I report two BRCA1-associated proteins, IFI16 and BAT1, which were studied to further understand the mechanism of breast carcinogenesis induced by the loss of BRCA1 function.; We identified IFI16 as a BRCA1-associated protein involved in p53-mediated apoptosis. IFI16 contains the Pyrin/PAAD/DAPIN domain, commonly found in cell death-associated proteins. BRCA1 (aa 502-802) interacted with the IFI16 Pyrin domain (aa 1-130). We found that IFI16 was localized in both nucleoplasm and nucleoli in normal mammary epithelial cells. In response to IR (ionizing radiation), a transient decrease in nucleoplasmic IFI16 was detected. This localization was not observed in HCC1937 BRCA1 mutant cells, but re-introduction of wild-type BRCA1 restored nuclear distribution of IFI16 as well as loss of nucleoplasmic IFI16 after IR. Co-expression of IFI16 and BRCA1 enhanced DNA damage-induced apoptosis in mouse embryonic fibroblasts from BRCA1 mutant mice expressing wild-type p53, although mutant IFI16 deficient in binding to BRCA1 did not induce apoptosis. Furthermore, tetracycline-induced IFI16 caused apoptosis when adenovirus p53 was co-expressed in p53-deficient EJ cells when treated with DNA damage. These results indicate a BRCA1-IFI16 role in p53-mediated transmission of DNA damage signals and apoptosis.; Recent studies have shown that stimulation of cells with ionizing radiation (IR) results in immediate phosphorylation of Ser1981 of ATM, A taxia Telangiectasia Mutated, leading to the catalytic activation of the protein. Here we report a BRCA1-associated protein, BAT1 (BRCA1-associated protein required for ATM activation-1), which was isolated by yeast two-hybrid screening using the C-terminal BRCA1 protein as bait. Co-localization of BRCA1 and BAT1 increases after IR treatment. Interestingly, BAT1 also binds to ATM and the enhanced interaction was demonstrated by co-immuprecipitation and immunocytochemical assay when treated with IR. Expression of adenovirus BRCA1 in SNU-251 cells, an ovarian cancer cell line carrying mutant BRCA1, revealed that co-localization of BAT1 and ATM requires wild type BRCA1. Down-regulation of BAT1 via small interfering RNA attenuated the activation of ATM and CHK2 post IR treatment. Expression of BAT1 in HEK 293T cells protected dephosphorylation of Ser1981 of ATM by calf intestine alkaline phosphatase in vitro, but not Ser15 of p53, suggesting the specific role of BAT1 in regulating ATM activation. At the level of the cell, BAT1 down-regulation resulted in growth retardation, delayed S-phase progression and sensitivity to IR damage. Taken together our above results suggest a possible role for BAT1 in the DNA damage response. |
