Expression of novel ERE‐binding proteins in human breast & uterine cells

Abstract

While investigating ERE-binding properties of estrogen receptor-α (hERα) in human breast cancer cytosols, lower molecular weight ERE-binding proteins (ERE-BP) were observed. Recognition properties of ERE-BP were evaluated by electrophoretic mobility shift (EMSA) and super-shift assays with ERE sequences of estrogen responsive genes. Cytosols, prepared from human breast cancer and uterine reference samples in 50 mM K2HPO4/KH2PO4 buffer, pH 7.4 with 1.5 mM EDTA, 10 mM Na2MoO4, 1 mM PMSF & 10 μM monothioglycerol, were incubated 16 hr, 4° C with [32P]ERE sequences (VitA2, CathD, pS2, jun, h-fos and prolactin) and separated by EMSA. Four ERE-BP expression profiles were detected exhibiting different ERE recognition and affinity. No ERE-BP super-shifted when incubated with ERα monoclonal antibodies AER 314, 320, 611, 1D5 and 5D11 (NeoMarkers®) or a polyclonal antibody (PanVera®/Invitrogen). Cytosols from breast cancer, uterus and fibroids, but not from myometrium exhibited ERE-BP. Neither rat nor calf uterine cytosol exhibited ERE-BP, but ververt monkey uterine cytosol expressed two distinct ERE-BP. To ascertain if ERE-BP influenced ERE recognition by ERα, competition experiments, performed with increasing [cytosol] and constant [hERα] and [ERE], revealed breast cancer and uterine cytosols inhibited hERα binding in a concentration dependent manner with simultaneous ERE-BP appearance. The biological significance and distribution of these unique proteins are being evaluated in a wider selection of estrogen and non-estrogen responsive tissues. Supported in part by grants from the NIH/NCI 1 R43 CA106059-01A1& Phi Beta Psi Sorority Res. Fund.