Mechanisms of estrogen-regulated expression of insulin-like growth factor binding protein-4, ornithine decarboxylase, and p53 in human breast cancer cells /
17β-Estradiol (E2) induces insulin-like growth factor binding protein-4 (IGFBP-4), p53 tumor suppressor and ornithine decarboxylase (ODC) gene expression and luciferase activity in MCF-7 human breast cancer cells transfected with constructs containing promoter inserts from these genes. This study id...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=726103301&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | 17β-Estradiol (E2) induces insulin-like growth factor binding protein-4 (IGFBP-4), p53 tumor suppressor and ornithine decarboxylase (ODC) gene expression and luciferase activity in MCF-7 human breast cancer cells transfected with constructs containing promoter inserts from these genes. This study identified the enhanceosomes in the gene promoters that mediate E2 action and explored the pathways employed for E2 transactivation in MCF-7 cells. Analysis of the IGFBP-4 gene promoter identified two GC-rich sequences (at -559 and -83) that were associated with E2-responsiveness. Both sequences formed complexes with Sp1 protein, and coincubation with ER enhanced intensities of the Sp1-DNA retarded bands. These results demonstrated that interaction of a transcriptionally-active ER/Sp1 complex with GC-rich motifs are required for hormone-inducibility of the IGFBP-4 gene promoter. Analysis of the p53 gene promoter indicated that binding of NF[]B and CTF-1 to the -106 to -30 region was required and sufficient for E2-responsiveness. However, E2 did not affect NF[]B-DNA binding, but directly activated NF[]B p65 C-terminal domain and this was inhibited by intracellular Ca²⁺ chelator and an inhibitor of calmodulin kinae IV (CaMKIV). Moreover, E2 induced phosphorylation of CaMKIV in vivo and constitutively active CaMKIV activates p65, suggesting that induction of p53 gene expression in MCF-7 cells by E2 is dependent on CaMKIV-dependent non-genomic activation of p65. In contrast, CTF-1 was not activated by E2, but functionally and physically interacted with p65, suggesting that DNA-bound CTF-1 acts as cofactor of NF[]B for activation of p53. A CAAT-like motif (at -93) and a "core" enhancer element (at -72) in the ODC gene promoter were identified as E2-responsive motifs that interacted with NF-Y and LSF transcription factors, respectively. However, no physical and functional interactions between NF-Y and LSF were observed using a mammalian two-hybrid assay or a coimmunoprecipitation assay with in vitro translated proteins. Studies using protein kinase inhibitors were unable to identify a specific inhibitor of E2-induced activation of ODC gene expression. This study showed for the first time that NF-Y and LSF are key transcription factors required for E2-induced ODC gene expression. |
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| Item Description: | Vita. "Major Subject: Toxicology". In title numerals are used. |
| Physical Description: | xii, 268 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 163-267). |