Stable transfection of an estrogen receptor beta cDNA isoform into MDA-MB-231 breast cancer cells
AuthorTonetti, D. A.
Pappas, S. G.
Bentrem, D. J.
Constantinou, Andreas I.
Jordan, V. C.
SourceJournal of Steroid Biochemistry and Molecular Biology
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We previously reported stable transfection of estrogen receptor alpha (ERα) into the ER-negative MDA-MB-231 cells (S30) as a tool to examine the mechanism of action of estrogen and antiestrogens [J. Natl. Cancer Inst. 84 (1992) 580]. To examine the mechanism of ERβ action directly, we have similarly created ERβ stable transfectants in MDA-MB-231 cells. MDA-MB-231 cells were stably transfected with ERβ cDNA and clones were screened by estrogen response element (ERE)-luciferase assay and ERβ mRNA expression was quantified by real-time RT-PCR. Three stable MDA-MB-231/ERβ clones were compared with S30 cells with respect to their growth properties, ability to activate ERE- and activating protein-1 (AP-1) luciferase reporter constructs, and the ability to activate the endogenous ER-regulated transforming growth factor alpha (TGFα) gene. ERβ6 and ERβ27 clones express 300-400-fold and the ERβ41 clone express 1600-fold higher ERβ mRNA levels compared with untransfected MDA-MB-231 cells. Unlike S30 cells, 17β-estradiol (E2) does not inhibit ERβ41 cell growth. ERE-luciferase activity is induced six-fold by E2 whereas neither 4-hydroxytamoxifen (4-OHT) nor ICI 182, 780 activated an AP-1-luciferase reporter. TGFα mRNA is induced in response to E2, but not in response to 4-OHT. MDA-MB-231/ERβ clones exhibit distinct characteristics from S30 cells including growth properties and the ability to induce TGFα gene expression. Furthermore, ERβ, at least in the context of the MDA-MB-231 cellular milieu, does not enhance AP-1 activity in the presence of antiestrogens. In summary, the availability of both ERα and ERβ stable breast cancer cell lines now allows us to compare and contrast the long-term consequences of individual signal transduction pathways. © 2003 Elsevier Ltd. All rights reserved.
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