17β-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 α (ERα)/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 ERα-positive ZR-75
cells; however, further analysis of the promoter showed that cooperative
ERα/Sp1/NFY interactions were not necessary for hormone-induced
transactivation in ZR-75 cells. The upstream GC-rich motifs are activated
independently by ERα/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 ERα-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-ERα 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 ERα
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, ERα and Sp1 plasmids E2
induced transactivation in cells cotransfected with AhR/Arnt expression plasmids
suggesting that the AhR complex suppressed ERα/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.
17β-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 α (ERα)/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 ERα-positive ZR-75
cells; however, further analysis of the promoter showed that cooperative
ERα/Sp1/NFY interactions were not necessary for hormone-induced
transactivation in ZR-75 cells. The upstream GC-rich motifs are activated
independently by ERα/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 ERα-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-ERα 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 ERα
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, ERα and Sp1 plasmids E2
induced transactivation in cells cotransfected with AhR/Arnt expression plasmids
suggesting that the AhR complex suppressed ERα/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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/2302 |
| Date | 29 August 2005 |
| Creators | Ngwenya, Sharon Khethiwe |
| Contributors | Safe, Stephen H. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 2866238 bytes, electronic, application/pdf, born digital |
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