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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047466-08
Application #
2649426
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1991-07-01
Project End
2000-11-30
Budget Start
1997-12-01
Budget End
1998-11-30
Support Year
8
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Khatri, Raju; Shah, Preeti; Guha, Rupa et al. (2015) Aromatase Inhibitor-Mediated Downregulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer. Mol Cancer Ther 14:1728-37
Niture, Suryakant K; Khatri, Raju; Jaiswal, Anil K (2014) Regulation of Nrf2-an update. Free Radic Biol Med 66:36-44
Shelton, Phillip; Jaiswal, Anil K (2013) The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene? FASEB J 27:414-23
Niture, Suryakant K; Jaiswal, Anil K (2013) Nrf2-induced antiapoptotic Bcl-xL protein enhances cell survival and drug resistance. Free Radic Biol Med 57:119-31
Niture, Suryakant K; Jaiswal, Anil K (2012) Nrf2 protein up-regulates antiapoptotic protein Bcl-2 and prevents cellular apoptosis. J Biol Chem 287:9873-86
Niture, Suryakant K; Jain, Abhinav K; Shelton, Phillip M et al. (2011) Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression. J Biol Chem 286:28821-32
Niture, Suryakant K; Jaiswal, Anil K (2011) Inhibitor of Nrf2 (INrf2 or Keap1) protein degrades Bcl-xL via phosphoglycerate mutase 5 and controls cellular apoptosis. J Biol Chem 286:44542-56
Kaspar, James W; Jaiswal, Anil K (2011) Tyrosine phosphorylation controls nuclear export of Fyn, allowing Nrf2 activation of cytoprotective gene expression. FASEB J 25:1076-87
Kaspar, James W; Jaiswal, Anil K (2010) Antioxidant-induced phosphorylation of tyrosine 486 leads to rapid nuclear export of Bach1 that allows Nrf2 to bind to the antioxidant response element and activate defensive gene expression. J Biol Chem 285:153-62
Niture, Suryakant K; Kaspar, James W; Shen, Jun et al. (2010) Nrf2 signaling and cell survival. Toxicol Appl Pharmacol 244:37-42

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