Activation of NF-kappa B and elevation of MnSOD gene expression by thiol reducing agents in lung adenocarcinoma (A549) cells.
The effect of reducing agents, including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on nuclear transcription factor-kappa B (NF-kappa B) activation and manganese superoxide dismutase (MnSOD) expression was investigated in a pulmonary adenocarcinoma (A549) cell line. NAC, DTT, and 2-ME each activated the transcription factor NF-kappa B and increased steady-state levels of MnSOD mRNA and enzyme activity in these cells. In addition, NAC, DTT, and 2-ME increased chloramphenicol acetyltransferase (CAT) activity in cells transfected with a construct containing the CAT gene under the control of the rat MnSOD promoter. SOD and catalase (500 U/ml) plus ethanol (1 mM) did not inhibit activation of NF-kappa B or elevation of steady-state MnSOD mRNA levels by NAC, DTT, or 2-ME. Controls in which comparable amounts of O2-. to those produced by thiols were generated by hypoxanthine and xanthine oxidase, or in which H2O2 was added directly, had neither activated NF-kappa B nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates, which may be formed during autooxidation, may not contribute to activation of NF-kappa B. Because the MnSOD promoter also contains potential binding sites for other transcription factors, such as promoter-selective transcription factor-1 (SP-1), activator protein-1 (AP-1), AP-2, adenosine 3',5'-cyclic monophosphate-regulator element binding factor (CREB), and transcription factor IID complex (TFIID), the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kappa B, there was only minor activation of AP-1 by thiols, and none of the other transcription factors were activated by thiols. AP-1 activation was inhibited by catalase (500 U/ml) plus SOD plus ethanol (1 mM). Addition of 700 microM H2O2 also activated AP-1, and catalase at 500 U/ml prevented this activation. This indicates that H2O2 produced as a result of autooxidation of thiols can activate AP-1 but not NF-kappa B. Thus a close association between exposure to reducing agents, activation of NF-kappa B, and elevation of MnSOD gene expression is demonstrated.
D000111:Acetylcysteine / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D004229:Dithiothreitol / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008623:Mercaptoethanol / Q000378:metabolism; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000502:physiology*; D010084:Oxidation-Reduction; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000502:physiology; D014407:Tumor Cells, Cultured
D015335:Molecular Probes; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D018808:Transcription Factor AP-1; D008623:Mercaptoethanol; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D004229:Dithiothreitol; D000111:Acetylcysteine