Superfund Research Program
Project Leader: Stephen H. Safe
Grant Number: P42ES004917
Funding Period: 2000-2008
Deletion analysis of several 17β-estradiol (E2)-responsive genes have identified GC-rich sites that are associated with hormone-induced transactivation in MCF-7 breast cancer cells. However, the role of individual specificity proteins (Sps) in mediating hormone-induced gene expression has not been unequivocally determined. In transient transfection studies using E2-responsive GC-rich promoters from the E2F1, carbamoylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), and retinoic acid receptor α (RARα) genes, RNA interference using small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3) and Sp4 (iSp4) decreased both basal and E2-induced transactivation. The contributions of individual Sp proteins to basal and E2-induced activity were promoter dependent. iSp1, iSp3 and iSp4 also significantly inhibited hormonal induction of E2F1, CAD and RARα mRNA levels; however, the enhanced inhibitory effects of the latter two small inhibitory RNAs suggest that Sp3 and Sp4 play a major role in estrogen receptor α/Sp-mediated gene expression in MCF-7 cells. Dr. Safe has investigated the effects of E2, diethylstilbestrol (DES), antiestrogens, the phytoestrogen resveratrol, and the xenoestrogens octylphenol (OP), nonylphenol (NP), endosulfan, kepone, 2,3,4,5-tetrachlorobiphenyl-4-ol (HO-PCB-Cl4), bisphenol-A (BPA), and 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on induction of luciferase activity in breast cancer cells transfected with a construct (pSp13) containing three tandem GC-rich Sp binding sites linked to luciferase and wild-type or variant ERα. The results showed that induction of luciferase activity was highly structure-dependent in both MCF-7 and MDA-MB-231 cells. Moreover, RNA interference assays using small inhibitory RNAs for Sp1, Sp3 and Sp4 also demonstrated structure-dependent differences in activation of ERα/Sp1, ERα/Sp3 and ERα/Sp4. These results demonstrate for the first time that various structural classes of ER ligands differentially activate wild-type and variant ERα/Sp-dependent transactivation, selectively use different Sp proteins, and exhibit selective ER modulator (SERM)-like activity.