Regulation of the Topoiomerase II-Dependent G2 Decatenation Checkpoint

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DESCRIPTION (provided by applicant): Breast cancer is the most common type of cancer occurring among women in the United States and the second highest cause of cancer-related deaths. Recent evidence has suggested that several different subtypes of breast cancer exist and that each type presents itself as an individual disease with a specific course of treatment. The subtype with the least favorable prognosis is the basal-like subtype which includes those harboring a BRCA1 mutation. BRCA1 is thought to function in almost every aspect of cell cycle control and plays a pivotal role in maintaining genomic stability, suggesting that its signaling mechanisms are required to prevent malignant progression. BRCA1 is an integral part of the decatenation checkpoint, which prevents entry into mitosis until entangled sister chromatids are sufficiently decatenated, a process mediated by topoisomerase Ila enzymatic activity. Failure of this checkpoint can result in chromatid breakage and/or aneuploidy and thus contribute to the generation of cancer cells. The long term goal of this research is to define the major signaling molecules responsible for the human decatenation checkpoint and topoisomerase Ila regulation. Specific aims of this application include the development of a genetic system in which the decatenation checkpoint can be studied in human mammary epithelial cells and subsequent investigation of the role of the BRCA1/BARD1 E3 ubiquitin ligase complex in decatenation checkpoint signaling. Our hypothesis states that a failure of the decatenation checkpoint may lead to aneuploidy and chromatid breakage, and that the topoisomerase lla protein and BRCA1/BARD1 complex are essential components of this process. Immunofluorescence microscopy, metaphase preparations, western immunoblotting, flow cytometry, and siRNA depletion techniques will be employed to analyze nuclear structure, chromosomal structure, and mitotic entry to determine the effects of these signaling proteins on the decatenation checkpoint. These experiments will provide a genetic system for the study of the signaling pathways involved in the decatenation checkpoint and generate solid genetic evidence regarding the mechanisms of the decatenation checkpoint with a specific focus on basal-like breast cancers. RELEVANCE: Breast cancer is the most common type of cancer afflicting women in the United States. This proposal seeks to understand signaling mechanisms that may be responsible for initiating molecular changes leading to breast cancer and contribute to the design of specific chemotherapeutic drugs that may help to prevent breast cancer progression.