Ectopic expression of BCL2 in MCF10A cells : differential BCL-2 family protein expression and response to staurosporine-induced apoptosis / by W. Samuel Fagg.
| Author/creator | Fagg, W. Samuel author. |
| Other author | Farwell, Mary A., degree supervisor. |
| Other author | East Carolina University. Department of Biology. |
| Format | Theses and dissertations |
| Production | 2008. |
| Description | 75 leaves : illustrations ; 29 cm |
| Supplemental Content | Access via ScholarShip |
| Subjects |
| Summary | Over-expression of the B-cell lymphoma 2 (BCL2) proto-oncogene has been implicated in a variety of cancers. The precise mechanism by which dysregulated BCL2 expression contributes to carcinogenesis in unknown, however it is known that BCL2 expression contributes to resistance to programmed cell death. In order to better understand these observed changes in phenotype, BCL2 has been ectopically expressed in MCFIOA cells. This immortalized, non-tumorigenic cell line has been used as a model of "normal" breast epithelial tissue in numerous previous studies. The current study seeks to better understand a model of Bcl-2 family protein expression and apoptotic resistance when BCL2 is ectopically expressed in MCFIOA cells (MCFlOA/Bcl-2). We hypothesize that a differential expression pattern of Bcl-2 family proteins shall emerge, as well as a staurosporine-resistant phenotype characterized by a slower death kinetic and an up-regulation of the Bad and Bimer proteins. By using western blotting we observed a differential pattern of Bcl-2 protein expression. characterized by up-regulation of pro-apoptotic and down-regulation of anti-apoptotic proteins in MCFlOA/Bcl-2. Cell-based and biochemical analyses showed a delay in cell death induced by staurosporine in MCFlOA/Bcl-2. Additionally, upon staurosporine introduction, the Bcl-2 family member Bad was up-regulated in MCFlOA/Bcl-2 and control MCFIOA (MCFIOA/Neo), whereas BimEL was up-regulated in MCFIOA/Neo and down-regulated in MCFlOA/Bcl-2. It was determined by immimoprecipitation that Bad directly bound to Bcl-2, and this interaction increased when staurosporine was introduced. Finally, through RT-PCR it was shown that neither Bim nor Bad were regulated at the transcriptional level, suggesting that the observed changes in protein levels were due to post-translational events. In conclusion, it was determined that Bad was the major BH3-only Bcl-2 family protein with a role in inducing death triggered by staurosporine in MCFlOA/Bcl-2 ceils. |
| General note | Presented to the faculty of the Department of Biology. |
| General note | Advisor: Mary A. Farwell |
| Dissertation note | M.S. East Carolina University 2008 |
| Bibliography note | Includes bibliographical references (leaves 70-75). |
| Genre/form | dissertations. |
| Genre/form | Academic theses. |
| Genre/form | Academic theses. |
| Genre/form | Thèses et écrits académiques. |