NAD(P)H:Quinone Oxidoreductases (NQO1 and NQO2) catalyze metabolic detoxification of quinones and, thus, protect the cells against redox cycling, oxidative stress and neoplasia. To investigate the regulation of expression of these enzymes, the genes encoding human NQO1 and NQO2 were cloned and sequenced. These genes are highly expressed in liver tumors and induced in response to xenobiotics, antioxidants, and dioxin (TCDD). They are also expressed in tissue specific manner. Transfection assays using deletion mutants of the NQO1 gene promoter identified: 1) a 24-bp antioxidant response element (ARE) between nucleotides -470 and -447 and nuclear transcription factors Nrf1 and Nrf2, which are required both for the high level of NQO1 gene transcription in tumor cells and for induction in response to beta-naphthoflavone (beta-NF) and t-butyl hydroquinone (t- BHQ), 2) a TCDD response region (-780 to -365) containing one copy each of a xenobiotic response element (XRE) and an ARE, 3) an AP-2 binding site at -157 and a unique basal region (-130 to -47). Analysis of the NQO2 gene promoter showed one ARE at position -936. We plan to elucidate the molecular mechanisms that control ARE-mediated expression and coordinated induction of the NQO1 gene and other detoxifying enzyme (NQO2 and GST Ya) genes. We will also examine signal transduction from xenobiotics and antioxidants to the NQO1 gene ARE and tissue specific expression of NQO1 gene. To this end, we will perform bandshift, supershift, and transfection assays to determine if the Nrf1 and Nrf2 proteins, which bind to the NQO1 gene ARE and regulate its expression and induction, also bind the AREs from other detoxifying enzyme genes and regulate their expression and induction. Signal transduction studies will include analysis of beta-NF induced transcription and post-transcriptional modifications of Nrf1 and Nrf2 proteins. In vitro bandshift and supershift assays and in vivo transfection assays are planned to study the role of redox factor Ref- l protein in redox regulation of Nrf1 and Nrf2 proteins in response to beta-NF. Deletion mutagenesis, transfection, bandshift, supershift and DNase I footprinting experiments will be performed to determine the role of XRE and ARE in the regulation of TCDD induction of the NQO1 gene expression. Northern and Western blotting, bandshift, supershift and transfection assays will be used to determine the role of ARE and Nrf1 and Nrf2 proteins in tissue specific expression of NQO1 gene among various human tissues.
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