Tags

Type your tag names separated by a space and hit enter

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.
Biochem J. 1996 Dec 15; 320 (Pt 3):975-81.BJ

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 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.

Authors+Show Affiliations

Department of Biochemistry, Trinity College, Dublin 2, Ireland.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9003388

Citation

Brennan, P, and L A. O'Neill. "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 Biochemical Journal, vol. 320 (Pt 3), 1996, pp. 975-81.
Brennan P, O'Neill LA. 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. Biochem J. 1996;320 (Pt 3):975-81.
Brennan, P., & O'Neill, L. A. (1996). 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 Biochemical Journal, 320 (Pt 3), 975-81.
Brennan P, O'Neill LA. 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. Biochem J. 1996 Dec 15;320 (Pt 3):975-81. PubMed PMID: 9003388.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Brennan,P, AU - O'Neill,L A, PY - 1996/12/15/pubmed PY - 1996/12/15/medline PY - 1996/12/15/entrez SP - 975 EP - 81 JF - The Biochemical journal JO - Biochem J VL - 320 (Pt 3) N2 - 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. SN - 0264-6021 UR - https://www.unboundmedicine.com/medline/citation/9003388/2_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_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/bj3200975 DB - PRIME DP - Unbound Medicine ER -