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    Centrosome Replication and the Role of BRCA1 in Breast Cancer

    Scientific Abstract:
    SCIENTIFIC ABSTRACT Centrosome replication and the role of BRCA1 in breast cancer Background: In cancer cells, alteration of centrosome homeostasis by deregulation of cell cycle check points leads to centriole overduplication and multipolar mitoses, thereby increasing the rate of genomic instability. Initial studies suggested that the supernumery centrosomes are a downstream consequence of p53 nullizygosity, though alternate pathways independent of the p53 pathway have also been demonstrated. Several studies have shown that the cyclin-dependant kinases and ubiquitin ligases have a fundamental role in regulating the centrosome cycle. Breast Cancer Suppressor gene (BRCA1) mutations have been shown to lead to centrosome amplification. BRCA1 contains a RING zinc finger domain, and a heterodimeric complex of BRCA1 and BARD1 has been shown to have ubiquitin ligase activity. BRCA1 associates with the centrosome during mitosis. Whether the ubiquitin ligase activity of BRCA1 is required at the centrosome for regulation of the centrosome replication and genome stability is an important question that remains to be answered. Additionally, recent data suggest that BRCA1 function with regard to the centrosome appears to be breast cell specific. Objective/Hypothesis: We hypothesize that an array of different proteins associate with the centrosome, and the composition of the centrosome complex is cell type specific. We predict that the breast cell specific effect of BRCA1 is mediated by its role in regulating the centrosome replication in these cells. This regulation is probably brought about by the differential ubiquitination of specific targets in the breast cells by BRCA1. Specific aims: (1) Purify, identify, and compare components of the centrosome from breast and non-breast cells. (2) Study dynamic changes in the centrosomal components through the different stages of cell cycle, in response to DNA damage, and in response to drugs affecting microtubule stability such as Taxol, vincristine and nocodazole. (3) Study modification of centrosomal components with special emphasis on ubiquitination. We will identify the centrosomal targets for ubiquitination by BRCA1. Study design: Centrosomes would be partially purified from cell-lines (Hela S3, non breast cells and Hs578T, breast cancer cell line), after different treatments, using discontinuous sucrose gradient. We have developed a technique for the purification of centrosomes via a special affinity matrix containing centrin. This method would further enrich the centrosomal complex from the fractionated sample. The purified complex will be analyzed by protein gel electrophoresis, and polypeptides will be identified by mass spectroscopy. Drugs affecting microtubule stability such as Taxol, vincristine and nocodazole will be used to check changes in subunit composition. Cells in culture will be synchronized by double thymidine block, followed by treatment with bleomycin to introduce double stranded DNA breaks. Tagged centrosomal proteins (gamma tubulin, epsilon tubulin and centrin 1), will be stably expressed in cell lines of interest. Centrosomes partially purified from these stable cell lines would be used to study modifications specific to these proteins under the various conditions. Modifications of the various subunits would be studied with special emphasis on BRCA1 mediated ubiquitination. Potential outcomes and benefits of the Research: It is known that breast cancer tumors acquire extra centrosomes. Studying the differences in its subunit composition and their modification, and their variation through various stages of cell cycle will help in understanding the basic mechanism of centrosome replication in breast cells. In addition, this project will identify those crucial components that are involved in promoting abnormal centrosome replication and tumor progression. These observations, and the assays we will develop, will help in finding new drug targets for treatment of breast cancer.

    Lay Abstract:
    LAY ABSTRACT Centrosome replication and the role of BRCA1 in breast cancer Breast cancer cells have abnormal DNA, called aneuploidy, and an important reason for the aneuploidy in breast cancer is that tumor cells fail to divide the DNA appropriately during cell division. The component of the cell that regulates the division of the DNA is called the centrosome, and it is vital that each cell has exactly two centrosomes during cell division. Extra centrosomes (greater than two) have been observed as a common change in breast cancer, and new work suggests that the Breast Cancer Tumor suppressor gene, BRCA1 protein regulates centrosome number. Recent data from our lab suggest that BRCA1 function with regard to the centrosome appears to be breast cell specific. We propose to understand the basic mechanism of the centrosome replication cycle by identifying the various subunits of the centrosome complex and understand how BRCA1 may regulate this process in a breast cell specific manner. In order to accomplish this goal, we will purify centrosome complexes and identify the different subunits. This technique would also help to identify how the centrosome changes during the different stages of the cell cycle. This project is fundamental basic research aimed at identifying an etiologic cause of breast cancer. The results generated by this study will not only be crucial for understanding the basic mechanisms of centrosome replication in breast cell, but will also identify those components that cause abnormal centrosome replication and tumor progression. Moreover, understanding the mechanism of action of microtubule stabilizing drugs such as Taxol and vincristine may help in designing better chemotherapeutic drugs that would specifically target the centrosome replication and treat this disease.