SCREENING ASSAY FOR SELECTIVE ESTROGEN RECEPTOR MODULATORS
Elena Sirbu, BS
University of Pittsburgh, 2006
Estrogen influences the development and progression of breast cancer and of other types of cancer, such as ovarian and lung cancer. The best strategy for prevention and treatment of estrogen dependent cancers is to selectively block estrogen activity in the affected estrogen dependent tissues. The beneficial role of estrogen in the other tissues should be preserved. One of the most common methods to prevent the harmful effects of estrogen is to block the estrogen receptor signaling. The intense research in the breast cancer treatment and prevention field produced a number of estrogen related compounds. The existing screening assays to test the selectivity and potency of these compounds have major limitations. I propose here the development and validation of a rapid screening assay for selective estrogen receptor modulators. This assay is based on the use of an ERE (estrogen response elements) to drive expression of a fluorescent protein that can be visualized directly in living cells. I presented here the first step in developing the screening assay, the generation and evaluation of two fluorescent clones, ERE-GFP and ERE-DsRed. The clones were introduced in CV-1 cells, together with ER, using transient transfection in order to test whether they are under tight estrogenic control. The cells were further treated with know ER ligands. These results predict that the clones function as expected. A robust signal resulted in the presence of estradiol, while with a pure antiestrogen such as ICI 182,780 resulted in very little red/green fluorescence. The vehicle control (ethanol) also elicited very little response (fluorescence). Further, these clones can be stably integrated in CV-1 cells together with either ER alpha or ER beta in order to develop a high content screening assay for SERMs. The new SERMs identified using this assay can be used eventually in therapy of breast or lung cancers or as hormone replacement. In addition, compounds that differentiate ER¦Á and ER¦Â will be valuable tools to further dissect ER signaling pathways. It is important to know more about coactivator recruitment, gene expression profile or about the response with ER mutations. This will lead to a better understanding of estrogen related cancers and will help designing new therapeutic approaches.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04112006-111635 |
| Date | 25 April 2006 |
| Creators | Sirbu, Elena |
| Contributors | Donald DeFranco, Mark Nichols, Lin Zhang, Billy W. Day, Daniel Johnson |
| 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-04112006-111635/ |
| Rights | unrestricted, 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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