2-mercaptoethanol restores the ability of nuclear factor kappa B (NF kappa B) 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 NF kappa B by PDTC.
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 kappa B (NF kappa B). In agreement with other studies, we have shown that PDTC inhibits NF kappa B 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 NF kappa B on a sensitive thiol, possibly on the p50 subunit (which was detected in NF kappa B 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 NF kappa B-DNA binding in vitro. On the basis of these results we suggest that, while NF kappa B 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.
Brennan
P
P
Department of Biochemistry, Trinity College, Dublin 2, Ireland.
O'Neill
L A
LA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D008623:Mercaptoethanol / Q000494:pharmacology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D013859:Thiocarbamates / Q000494:pharmacology*; D013945:Thymoma; D014407:Tumor Cells, Cultured
D000975:Antioxidants; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D009687:Nuclear Proteins; D013859:Thiocarbamates; D003958:Diamide; C066229:prolinedithiocarbamate; D008623:Mercaptoethanol; D011392:Proline; D005978:Glutathione