diamide and Thymoma

Stimulation of the Interleukin-1 receptor type I (IL-1-RI) with IL-1 activates an associated serine/threonine kinase, IRAK, which phosphorylates downstream targets, resulting in NFkappaB activation. The signaling cascade is accompanied by oxidative processes and contains putative targets for redox regulation. Preincubation of the murine T cell line EL-4 and the human umbilical cord vein endothelial cell line ECV 304 with thiol modifying compounds like diamide, menadione or phenylarsine oxide inhibited the IL-1-induced phosphorylation of an endogenous substrate with a molecular mass of 60 kD. In the endothelial cell line, a second target of about 85 kD was phosphorylated after IL-1 stimulation, which was also inhibited by thiol modification. These data suggest that IL-1 signal transduction depends on free thiols which might be targets for redox regulation not only in lymphocytes, but also in endothelial cells.

Topics: Animals; Arsenicals; Cells, Cultured; Diamide; Endothelium, Vascular; Enzyme Inhibitors; Humans; Interleukin-1; Mice; Molecular Weight; NF-kappa B; Oxidation-Reduction; Phosphorylation; Receptors, Interleukin-1; Receptors, Interleukin-1 Type I; Signal Transduction; Sulfhydryl Compounds; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Umbilical Veins; Vitamin K

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.

Topics: Animals; Antioxidants; Diamide; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Glutathione; Humans; Interleukin-1; Mercaptoethanol; Mice; NF-kappa B; Nuclear Proteins; Oxidation-Reduction; Proline; Protein Binding; Thiocarbamates; Thymoma; Tumor Cells, Cultured

Topics: Acetylcysteine; Animals; Cell Line; Cell Nucleus; Diamide; Dinitrochlorobenzene; Ethacrynic Acid; Humans; Hydrogen Peroxide; Interleukin-1; Mice; NF-kappa B; Sulfhydryl Reagents; T-Lymphocytes; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured