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2-Mercaptoethanol restores the ability of nuclear factor kB (NFkB) to bind DNA in nuclear extracts from interleukin 1-treated cells incubated with pyrollidine dithiocarbamate (PDTC)

Brennan, Paul and O'Neill, L. A. J. 1996. 2-Mercaptoethanol restores the ability of nuclear factor kB (NFkB) to bind DNA in nuclear extracts from interleukin 1-treated cells incubated with pyrollidine dithiocarbamate (PDTC). Biochemical Journal -London- 320 , pp. 975-981.

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Abstract

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 kB (NFkB). In agreement with other studies, we have shown that PDTC inhibits NFkB 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 NFkB on a sensitive thiol, possibly on the p50 subunit (which was detected in NFkB 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 NFkB–DNA binding in vitro. On the basis of these results we suggest that, while NFkB 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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Publisher: Portland Press
ISSN: 0264-6021
Last Modified: 04 Jun 2017 06:12
URI: http://orca-mwe.cf.ac.uk/id/eprint/57258

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