Mechanisms of transcriptional activation of bcl-2 gene expression by 17β-estradiol in breast cancer cells

bcl-2 gene expression is induced by 17 beta-estradiol (E2) in T47D and MCF-7 human breast cancer cells, and the mechanism of E2 responsiveness was further investigated by analysis of the bcl-2 gene promoter. The -1602 to -1534 distal region (bcl-2j) of the promoter was E2-responsive; however, in gel mobility shift assays, the estrogen receptor alpha (ERalpha) did not bind [P-32]bcl-2j, whereas Spl protein formed a retarded band complex. Further analysis demonstrated that the upstream region (-1603 to -1579) of the bcl-2 gene promoter contained two GC/GA-rich sites at -1601 (5'-GGGCTGG-3') and -1588 (3'-GGAGGG-5') that bound Spl protein. Subsequent studies confirmed that transactivation by E2 was dependent on ERalpha/Sp1 interactions with both GC-rich sites, and this was confirmed by in uitro footprinting. In contrast, a 21-base pair E2-responsive downstream region (-1578 to -1534) did not bind Spl or ER, protein; however, analysis of a complex binding pattern with nuclear extracts showed that ATF-1 and CREB-1 bound to this motif, These data coupled,with results of transient transfection studies demonstrated that transcriptional activation by E2 of the -1578 to -1534 region of the bcl-2 gene promoter was dependent on induction of cAMP and subsequent activation through a cAMP response element, Thus, hormone regulation of bcl-2 gene expression in breast cancer cells involves multiple enhancer elements and E2-mediated transactivation does not require direct binding of the estrogen receptor with promoter DNA.