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

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Title: Regulation of E2F-1 gene expression in human breast cancer cells
Author: Ngwenya, Sharon Khethiwe
Abstract: 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 .
URI: http : / /hdl .handle .net /1969 .1 /2302
Date: 2005-08-29

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Regulation of E2F-1 gene expression in human breast cancer cells. Available electronically from http : / /hdl .handle .net /1969 .1 /2302 .

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