FIG. 2.

Estrogen agonists, but not antagonists, inhibit GR-mediated transactivation. (A) MCF-7 cells were pretreated for 48 h with estrogen agonists 10 nM E2 (E), 10 nM DES (D), and 100 nM genistein (G), or antagonist 100 nM raloxifene (R) or tamoxifen (T), followed by treatment with 1 nM DEX or ER ligands plus DEX for 16 h. Lysates were harvested and analyzed for luciferase activity in triplicate samples for each treatment condition. Data are reported as RLU normalized for total protein. Results are expressed as means ± standard errors for three determinations per treatment condition. (B) Estrogen agonists inhibit glucocorticoid-induced MMTV-LUC mRNA. MCF-7 cells were pretreated for 48 h with estrogen agonists E2 and DES (10 nM) (lanes 3 to 6) or genistein (100 nM) (lanes 7 and 8) or antagonists raloxifene or tamoxifen (100 nM) (lanes 9 to 12), followed by treatment with 1 nM DEX (lane 2) or ER ligands plus DEX (lanes 4, 6, 8, 10, and 12) for 16 h. Total RNA was harvested and analyzed by primer extension with primers specific for MMTV or 18S rRNA. Levels of labeled PCR transcripts were analyzed on 8% polyacrylamide denaturing gels and exposed to phosphorimager screens for quantification. (C) The estrogen antagonist ICI attenuates E2-mediated inhibition of the MMTV promoter activity. Cells were pretreated for 48 h with 1 or 10 nM E2 (E) (lanes 3, 4, 13, 14, and 17) or 10 or 100 nM ICI (lanes 7 and 8) or ICI and E2 (lanes 11 to 18), followed by treatment with 1 nM DEX (lanes 2, 5, 6, 9, 10, 12, 15, 16, and 18) for 16 h. Lysates were harvested and analyzed for luciferase activity as described above. Data are reported as RLU normalized for total protein. The procedure was done in triplicate for each treatment condition, and data are expressed as means ± standard errors for three determinations per treatment condition.