Regulation of E2F-1 gene expression in human breast cancer cells

17?-Estradiol induces E2F-1 gene expression in ZR-75 and MCF-7 human breast cancer cells. Analysis of the E2F-1 gene promoter in MCF-7 cells previously showed that hormone-induced transactivation required interactions between estrogen receptor ? (ER?)/Sp1 bound to upstream GC-rich sites and NFYA bound to downstream CCAAT sites within the -169 to -54 promoter region. This promoter region was also E2-responsive in ER?-positive ZR-75 cells; however, further analysis of the promoter showed that cooperative ER?/Sp1/NFY interactions were not necessary for hormone-induced transactivation in ZR-75 cells. The upstream GC-rich motifs are activated independently by ER?/Sp1 in ZR-75 but not MCF-7 cells, and the downstream CCAAT sites were also E2-responsive. E2 also induced reporter gene activity in ZR-75 cells transfected with an expression plasmid containing the yeast GAL4 DNA binding domain fused to pM-NFYA and a construct containing five tandem GAL4 response elements. Subsequent studies showed that hormonal activation of pE2F-1jm1 and pM-NFYA are dependent on non-genomic pathways in which E2 activates cAMP/protein kinase A. Hormone-dependent regulation of E2F-1 gene expression in ZR-75 and MCF-7 involves different mechanisms, demonstrating the importance of cell context on transactivation pathways, even among ER-positive breast cancer cell lines. TCDD inhibited ER?-mediated responses in MCF-7 and ZR-75 cells. E2- induced E2F-1protein and mRNA levels in MCF-7 and ZR-75 cells and this response was inhibited by TCDD. Constructs containing GC-rich sites alone or in combination with the downstream NFY sites were used in transactivation studies to investigate the mechanism of inhibitory AhR-ER? crosstalk. Although TCDD inhibited E2-induced mRNA, protein and reporter gene actitivity, it was not possible to determine if the inhibitory response was due to limiting ER? protein levels due to proteasome degradation since proteaome inhibitors alone blocke hormone-dependent responses. TCDD also inhibited the cAMP/PKA pathway by inhibiting adenyl cyclase activity. In Drosophila SL-2 cells cotransfected with the GC-rich -169 to -54 region, ER? and Sp1 plasmids E2 induced transactivation in cells cotransfected with AhR/Arnt expression plasmids suggesting that the AhR complex suppressed ER?/Sp1 action. These results demonstrate that TCDD inhibits E2-dependent activation of both non-genomic and genomic pathways of ER-mediated E2F-1 gene expression. 17?-Estradiol induces E2F-1 gene expression in ZR-75 and MCF-7 human breast cancer cells. Analysis of the E2F-1 gene promoter in MCF-7 cells previously showed that hormone-induced transactivation required interactions between estrogen receptor ? (ER?)/Sp1 bound to upstream GC-rich sites and NFYA bound to downstream CCAAT sites within the -169 to -54 promoter region. This promoter region was also E2-responsive in ER?-positive ZR-75 cells; however, further analysis of the promoter showed that cooperative ER?/Sp1/NFY interactions were not necessary for hormone-induced transactivation in ZR-75 cells. The upstream GC-rich motifs are activated independently by ER?/Sp1 in ZR-75 but not MCF-7 cells, and the downstream CCAAT sites were also E2-responsive. E2 also induced reporter gene activity in ZR-75 cells transfected with an expression plasmid containing the yeast GAL4 DNA binding domain fused to pM-NFYA and a construct containing five tandem GAL4 response elements. Subsequent studies showed that hormonal activation of pE2F-1jm1 and pM-NFYA are dependent on non-genomic pathways in which E2 activates cAMP/protein kinase A. Hormone-dependent regulation of E2F-1 gene expression in ZR-75 and MCF-7 involves different mechanisms, demonstrating the importance of cell context on transactivation pathways, even among ER-positive breast cancer cell lines. TCDD inhibited ER?-mediated responses in MCF-7 and ZR-75 cells. E2- induced E2F-1protein and mRNA levels in MCF-7 and ZR-75 cells and this response was inhibited by TCDD. Constructs containing GC-rich sites alone or in combination with the downstream NFY sites were used in transactivation studies to investigate the mechanism of inhibitory AhR-ER? crosstalk. Although TCDD inhibited E2-induced mRNA, protein and reporter gene actitivity, it was not possible to determine if the inhibitory response was due to limiting ER? protein levels due to proteasome degradation since proteaome inhibitors alone blocke hormone-dependent responses. TCDD also inhibited the cAMP/PKA pathway by inhibiting adenyl cyclase activity. In Drosophila SL-2 cells cotransfected with the GC-rich -169 to -54 region, ER? and Sp1 plasmids E2 induced transactivation in cells cotransfected with AhR/Arnt expression plasmids suggesting that the AhR complex suppressed ER?/Sp1 action. These results demonstrate that TCDD inhibits E2-dependent activation of both non-genomic and genomic pathways of ER-mediated E2F-1 gene expression.