Regulation of human NAD(P)H:quinone oxidoreductase gene. Role of AP1 binding site contained within human antioxidant response element.
Deletion mutagenesis and transfection studies into hepatic (mouse hepatoma (Hepa-1) and human hepatoblastoma (Hep-G2)) and nonhepatic (HeLa) cells indicated that high levels of expression of the human NAD(P)H:quinone oxidoreductase gene in tumor cells and its induction by beta-naphthoflavone and 3-(2)-tert-butyl-4-hydroxyanisole are mediated by human antioxidant response element (hARE) located in the region between -470 and -445. The hARE, when attached to the thymidine kinase promoter and transfected into several mammalian cells, expressed high levels of the chloramphenicol acetyltransferase gene that was inducible by beta-naphthoflavone and 3-(2)-tert-butyl-4-hydroxyanisole. Nucleotide sequence analysis of the hARE revealed the presence of a recognition site for binding to the AP1 protein. Mutation of the AP1 binding site located within the hARE resulted in the loss of expression and induction upon transfection into various cell types. Band shift and competition assays with hARE and nuclear extracts from control and beta-naphthoflavone-treated Hepa-1, Hep-G2 and HeLa cells indicated specific interaction of regulatory protein(s) to the hARE. The supershift assays using antibodies against specific proteins of the AP1 family identified Jun-D and c-Fos as two members in the hARE-protein complex observed in band shift assays.
Li
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Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Jaiswal
A K
AK
D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants; D001571:Benzoflavones / Q000494:pharmacology; D001665:Binding Sites; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D019324:beta-Naphthoflavone
D000975:Antioxidants; D001571:Benzoflavones; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D002083:Butylated Hydroxyanisole; D019324:beta-Naphthoflavone; D016660:NAD(P)H Dehydrogenase (Quinone); D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase