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Xu, Chuanli
Transcriptional suppression of cytochrome P450 1A1 gene is under redox regulation - Ah receptor-mediated processes with distinct mechanisms.

Order No. 9726319

Oxidative stress in a cell is defined as an unusually high level of reactive oxygen species which can be caused by a number of stimuli. We have investigated the molecular mechanism whereby transcriptional expression of CYP1A1 gene was regulated by redox potential. XRE was found to be the response element by which H(sub 2) O(sub 2) exhibited its inhibitory effect on the transcription of CYP1A1 gene in Hepa 1 cell line using transient transfection technique. However, H(sub 2) O(sub 2) did not alter the DNA binding activity of the Ah receptor. Further study demonstrated that modulation of XRE enhancer strength by various means could modify H(sub 2) O(sub 2)-dependent suppression of CAT expression. The results from this study suggest the presence of a protein that inhibits transactivation by the Ah receptor without influencing its DNA binding ability. In search for the candidate protein(s) which mediated H(sub 2) O(sub 2) action on Ah receptor function, we first demonstrated that overexpression of the product of the retinoblastoma susceptibility gene (RB) downregulated transcription of the CYP1A1 gene. XREs alone were sufficient to mediate RB action. Results from coimmunoprecipitation assays indicated that the Ah receptor coprecipitated with the RB protein or its family member p107 and vice versa. Similar to other RB binding proteins, the Ah receptor only bound to the hypophosphorylated form of RB or p107 protein. To further characterize regulation of CYP1A1 by redox potential, more powerful and more specific oxidants were used to oxidize vicinal sulfhydryl groups in intact Hepa 1 cells. Pretreatment with diamide or phenylarsine oxide for 20 minutes rapidly prevented the formation of ligand-dependent Ah receptor/XRE complex and thus inhibited XRE-mediated luciferase expression. Direct oxidization of the Ah receptor by PAO was further demonstrated by the experiments in which DTT, a reducing agent, could restore the Ah receptor DNA binding activity. Finally, a one hundred-fold difference in the effectiveness between dithiol 2,3-dimercaptopropanol and monothiol 2- mercaptoethanol in reversing PAO-dependent inhibition of Ah receptor DNA binding activity suggests that vicinal sulfhydryl residues may be involved in the DNA binding of Ah receptor. Source: DAI, 58, no. 03B, (1997): 1130

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