Abstract
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.
Original language | English (US) |
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Pages (from-to) | 47-55 |
Number of pages | 9 |
Journal | Journal of Steroid Biochemistry and Molecular Biology |
Volume | 87 |
Issue number | 1 |
DOIs | |
State | Published - Oct 2003 |
Funding
This work was supported in part by the Avon Foundation, NIH CA96517, and the NIH SPORE in Breast Cancer CA 89018-02. We are extremely grateful to Dr. Benita Katzenellenbogen of the University of Illinois for the generous gift of the ERβ antibody CWK-F12. We also thank Dr. Laird Madison and Dr. Eun Jig Lee, both from Northwestern University, for the generous gifts of the ERβ cDNA expression plasmid and the AdERELuc adenovirus, respectively. A special thanks to Dr. Kyung-Hee Lee for her hard work optimizing the ERβ Western blotting technique.
Keywords
- AP-1
- Breast cancer
- Estrogen receptor beta
- MDA-MB-231
- Stable clones
- TGF alpha
- Tamoxifen
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Endocrinology
- Clinical Biochemistry
- Cell Biology