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This dissertation, MAD2 Inactivation on Chromosomal Instability and Tumorigenesis in Prostate Epithelial Cells by Kit-wa, To, 杜潔華, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MAD2 inactivation on chromosomal instability and tumorigenesis in prostate epithelial cells Submitted by To Kit Wa For the degree of Doctor of Philosophy at the University of Hong Kong in October 2007 Mitotic checkpoint is a surveillance mechanism for accurate chromosome segregation to two daughter cells during mitosis and is crucial for the maintenance of genome fidelity and integrity. The mitotic arrest deficient 2 (MAD2) play an important role in a functional mitotic checkpoint and the maintenance of chromosomal stability. Binding of MAD2 to mitotic checkpoint regulators MAD1 and CDC20 in unattached kinetochores inhibits anaphase-promoting complex/ cyclosome (APC/C) activity and delays the onset of anaphase until all of the kinetochores are bipolarly attached to spindle microtubules. However, little is known about the biological significance of MAD2 inactivation in human cells. In this study, a dominant negative C-terminal deleted MAD2 gene, MAD2ΔC was stably transfected into an immortalized human prostate epithelial cell line, Hpr-1 and its effects on chromosomal instability, cell proliferation, mitotic checkpoint control, centrosome amplification and anchorage-independent growth ability were studied. I found that MAD2ΔC induced aneuploidy through promoting chromosomal duplication as a result of an impaired mitotic checkpoint and cytokinesis, suggesting a critical role of MAD2-mediated mitotic checkpoint in chromosome stability in human cells. In addition, the MAD2ΔC expressing cells displayed anchorage-independent growth in soft agar after treatment of 7,12-dimethylbenz[A]anthracene (DMBA) and 12-O-tetradecanoylphorbol-3-acetate (TPA), demonstrating a cancer-promoting effect of MAD2 inactivation and defective mitotic checkpoint in human cells. The anchorage-independent MAD2ΔC cells acquire additional structural aberrations of chromosomes, suggesting that accumulation of chromosome alternations may provide survival advantages for anchorage-independent growth. Furthermore, the DMBA-induced transformation was accompanied by a complete loss of DNA damage-induced p53 response and activation of MAPK pathway in MAD2ΔC cells. These results indicate that MAD2 inactivation or defective mitotic checkpoint alone is not a direct cause of tumorigenesis, but it may predispose human cells to carcinogen-induced malignant transformation. The evidence presented here provides a linkage between MAD2 inactivation and malignant transformation of epithelial cells. To further investigate the molecular mechanism of MAD2 inactivation in promoting anchorage independent growth, two-dimensional gel electrophoresis was used to explore the differential protein expression profiles among control, MAD2ΔC cells and anchorage independent MAD2ΔC cells. Twenty-five protein spots were found to be differentially expressed among these cell lines and 14 proteins were identified to be responsible for different biological mechanisms. Among these proteins, calmodulin-like protein (CLP) was found to be down-regulated during anchorage-independent transformation of MAD2ΔC cells. Meanwhile, over-expression of calmodulin-like protein in prostate cancer cell lines, PC3 and DU145 resulted in moderate reduction of cell proliferation rate, ability to grow in soft agar and cell migration rate. These results suggest that loss of calmodulin-like protein may be one of the factors in prom

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