The metal chelator and anti-oxidant pyrollidine dithiocarbamate (PDTC) has been used extensively in studies implicating reactive oxygen intermediates in the activation of nuclear factor κB (NFκB). In agreement with other studies, we have shown that PDTC inhibits NFκB activation in response to the pro-inflammatory cytokines interleukin 1 (IL1) and tumour necrosis factor (TNF). However, we have found that the inhibition was reversed by treatment of inhibited nuclear extracts with the reducing agent 2-mercaptoethanol. This was observed in extracts prepared from IL1-treated EL4.NOB-1 thymoma cells and TNF-treated Jurkat E6.1 lymphoma cells. These results suggested that the inhibition was caused by oxidation of NFκB on a sensitive thiol, possibly on the p50 subunit (which was detected in NFκB complexes in both cell types), and not by inhibition of the activation pathway. The possibility that PDTC was acting as a pro-oxidant was therefore investigated. PDTC caused an increase in oxidized glutathione, suggesting that it acts as an oxidizing agent in the cells tested rather than as an anti-oxidant. Similar results were obtained with diamide, a compound designed to oxidize glutathione. Finally, an increase in the ratio of oxidized to reduced glutathione was shown to inhibit NFκB–DNA binding in vitro. On the basis of these results we suggest that, while NFκB activation is unaffected by PDTC, DNA binding is inhibited through a mechanism involving a shift towards oxidizing conditions, and that this is the mechanism of action of both PDTC and diamide in the cells tested here.
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December 1996
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Research Article|
December 15 1996
2-mercaptoethanol restores the ability of nuclear factor κB (NF κB) to bind DNA in nuclear extracts from interleukin 1-treated cells incubated with pyrollidine dithiocarbamate (PDTC). Evidence for oxidation of glutathione in the mechanism of inhibition of NFκB by PDTC
Paul BRENNAN;
Paul BRENNAN
*
1Department of Biochemistry, Trinity College, Dublin 2, Ireland
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Luke A. J. O'NEILL
Luke A. J. O'NEILL
†
2Department of Biochemistry, Trinity College, Dublin 2, Ireland
†To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
March 19 1996
Revision Received:
August 05 1996
Accepted:
August 21 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 320 (3): 975–981.
Article history
Received:
March 19 1996
Revision Received:
August 05 1996
Accepted:
August 21 1996
Citation
Paul BRENNAN, Luke A. J. O'NEILL; 2-mercaptoethanol restores the ability of nuclear factor κB (NF κB) to bind DNA in nuclear extracts from interleukin 1-treated cells incubated with pyrollidine dithiocarbamate (PDTC). Evidence for oxidation of glutathione in the mechanism of inhibition of NFκB by PDTC. Biochem J 15 December 1996; 320 (3): 975–981. doi: https://doi.org/10.1042/bj3200975
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