Disorazole C1 (DZ) is a synthesized polyene macrodiolide, originally identified as a minor metabolite from the myxobacterium fermentation broth. I examined the cellular responses to DZ in noncancer and cancer cells, and compared the results to vinblastine and taxol. In noncancer cells, DZ induced a prolonged mitotic arrest, followed by mitotic slippage, which was associated with cyclin B degradation, but did not require p53. Four apoptosis detection assays, including examination of poly(ADP-ribose) polymerase cleavage, cytochrome C release, mitochondrial depolarization, and annexin V staining, were conducted and demonstrated little induction of apoptosis in noncancer cells treated with DZ. However, I observed an activated apoptotic pathway in cancer cells, suggesting that normal and malignant cells respond differently to DZ, and indicating a potential therapeutic application for this compound.
GpIba is a transmembrane subunit of the von Willebrand factor receptor on platelet surfaces, functioning in platelet adhesion and activation. Recent research has revealed that GpIba also plays roles in transformation and genomic instability as an oncoprotein. In my studies, GpIba colocalized with F-actin and filamin A, an actin-binding protein, at the cleavage furrow in anaphase cells, suggesting a novel role for GpIba in cytokinesis. However, when GpIba was overexpressed in noncancer cells with p53 knockdown, GpIba, F-actin, and filamin A became mislocalized. RhoA, a cytokinesis regulator, was localized asymmetrically, although Aurora B, an important cytokinesis kinase, retained its correct localization. Additionally, anaphase bridges were observed in these GpIba-overexpressing cells, along with elevated percentages of cytokinesis failure and binucleation, indicating that GpIba overexpression led to cytokinesis defects/failure and tetraploidization. Conversely, in cancer cells that endogenously overexpress GpIba, I observed decreases in the percentages of binucleation and mitotic defects upon GpIba knockdown. Furthermore, down-regulation of Aurora B was demonstrated to mediate the mechanism through which GpIba overexpression led to cytokinesis failure, and the filamin A-binding domain and the signal peptide of GpIba were shown to be indispensible for this mechanism. These results add a new component of the existing cancer cell genetic instability pathways, by suggesting that overexpression of GpIba in cancer cells disrupts normal cytokinesis and causes tetraploidization through down-regulation of Aurora B.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04152010-160048 |
| Date | 24 June 2010 |
| Creators | Xu, Fengfeng |
| Contributors | Billy W. Day, William S. Saunders, Kirill Kiselyov, James M. Pipas, Lewis A. Jacobson |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-04152010-160048/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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