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Screening Assay for Selective Estrogen Receptor Modulators

Sirbu, Elena (2006) Screening Assay for Selective Estrogen Receptor Modulators. Master's Thesis, University of Pittsburgh. (Unpublished)

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SCREENING ASSAY FOR SELECTIVE ESTROGEN RECEPTOR MODULATORSElena Sirbu, BSUniversity of Pittsburgh, 2006Estrogen 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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDeFranco, Donalddod1@pitt.eduDOD1
Committee MemberDay, Billy Wbday@pitt.eduBDAY
Committee MemberJohnson, Danieljohnsond@pitt.eduJOHNSOND
Committee MemberZhang, LinZhangLx@upmc.eduLIZ22
Committee MemberNichols, Markmnichols@pitt.eduMNICHOLS
Date: 25 April 2006
Date Type: Completion
Defense Date: 30 March 2006
Approval Date: 25 April 2006
Submission Date: 11 April 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Pharmacology
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: breast cancer; estrogen receptor; GFP; screening assay; selective estrogen receptor modulators; SERM
Other ID:, etd-04112006-111635
Date Deposited: 10 Nov 2011 19:35
Last Modified: 19 Dec 2016 14:35


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