Activation of nf-kb and elevation of manganese superoxide dismutase gene expression by thiol reducing agents in lung adenocarcinoma (a549) cells. Pediatrics, Department Of, National Jewish Center For Immunology & Respiratory Medicine, Denver, Co 80206. Kumuda C. Das, Yvette Lewis-Molock and Carl W. White
APStracts 2:0113L, 1995.
The effect of reducing agents including N-acetylcysteine (NAC), dithiothreitol (DTT), and 2-mercaptoethanol (2-ME) on NF-kB 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-kB 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 (500u/ml) plus ethanol (1 mM) did not inhibit activation of NF-kB 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-kB nor elevated MnSOD mRNA. This shows that reactive oxygen intermediates which may be formed during auto-oxidation may not contribute to activation of NF-kB. Because the MnSOD promoter also contains potential binding sites for other transcription factors such as SP-1, AP-1, AP-2, CREB and TFIID, the effect of thiols on their activation also were evaluated. In contrast to findings with NF-kB, 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 [mu]M H2O2 also activated AP-1, and catalase at 500 u/ml prevented this activation. This indicates that H2O2 produced as a result of auto-oxidation of thiols can activate AP-1 but not NF-kB. Thus, a close association between exposure to reducing agents, activation of NF-kB and elevation of MnSOD gene expression is demonstrated. 

Received 7 September 1994; accepted in final form 9 June 1995.
APS Manuscript Number L264-4.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 11 July 1995.