| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Arylhydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxicological and bio-logical effects of 2,3,7,8-tetrachlorodiben.zo-p-dioxine (TCDD) and related compounds. AhR is included in a family of basic-helix-loop-helix/PAS domain-containing transcription factors. AhR expression is associated with the development of human lung carcinoma. In fact, we found that the overexpression of AhR accelerated the cell proliferation of A549 cells, established from a human alveolar cell carcinoma. We also demonstrated that the increased cell proliferations in A549 cells were associated with the activation of E2F gene expression, a transcription factor related in carcinogenesis, by the expression of heterodimer complex of AhR and AhR nuclear translocator (Arnt). In this study, we investigated the mechanisms of relationship of E2F gene activation and expression of AhR/Arnt complex.
We obtained the deleted cell lines of AhR, Arnt and E2F genes expression by s
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iRNA transfection technique in A549 cells. E2F-dependent transcription activity was decreased by the suppression of AhR, Arnt and E2F expression. To identify the domains of AhR and Arnt genes responsible for E2F-dependent transcription activity, a series of deletion constructs linked the luciferase activity were transfected into A549 cells. The PAS regions of AhR and Arnt sequences were required for the activation of E2F transcription. Next, an effect of E2F expression to the transcription activity of AhR and Arnt was determined by using of reporter assay contained XRE sequences, as AhR/Arnt-binding regions. The AhR/Arnt complex-dependent transcription activity on XRE sequences was increased by the suppression of E2F expression. The Rb protein-binding regions in E2F sequences were required on the interaction of AhR/Arnt complex. Rb protein is a repressive factor of carcinogenesis. Furthermore, we demonstrated that AhR/Arnt complex was complexed with E2F protein using a immuno-precipitation assay. These results suggested that AhR/Arnt and E2F complex binding on E2F-binding sequences was accelerated the cell proliferation, while the complex binding on XRE sequences was affected as a repressor in gene transcription. Less
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