Fig. 2.
G1T48 is a complete estrogen receptor antagonist. a G1T48 inhibits ER target gene expression in breast cancer cells. MCF7 cells were treated with ER antagonists (10–10–10–7 M) plus estradiol (E2; 10–9 M) for 18 h. TFF1 mRNA expression was analyzed by real-time PCR. GAPDH was used to normalize real-time PCR data. b G1T48 competes for estrogen binding to ER. MCF7 cells were treated with 10–10 M 3H-17β-E2 and competitor ligand (10–12–10–6 M) for 2 h. Cells were collected and radioactive counts were monitored on a Beckman LS 6000SC Scintillation counter. Error bars indicate the SD of duplicate samples. c G1T48 regulates ER target gene pharmacology similar to other SERDs. MCF7 breast cancer cells were treated with ER ligands (E2, fulvestrant, RU, RAL @ 100 nM; G1T48, 810, 9496, Laso, 4OHT, 7604, BZA @ 1.0 μM; 5638, Tam @ 10 μM) for 24 h. mRNA expression was analyzed by real-time PCR. GAPDH was used to normalize real-time PCR data. Heatmaps were generated from real-time PCR data after analysis with JMP pro software (SAS) using the Ward hierarchical clustering algorithm. d G1T48 blocks estrogen-dependent recruitment of ER to the TFF1 promoter. MCF7 cells were treated with ligand (E2: 5 × 10−10 M; ER antagonists: 10–6 M) as indicated for 45 min. Cells were fixed with formaldehyde and chromatin was immunoprecipitated with anti-ER antibody. Real-time PCR was used to assess the relative amount of ER bound to the TFF1 gene promoter. Error bars indicate the SD of triplicate samples