vitamin-k-semiquinone-radical 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 interleukin-1 receptor type I (IL-1RI) is associated with other proteins thus forming a complex system by which IL-1 exerts its various signals. The initiating event is still uncertain, but activation of a recently described receptor-associated protein kinase is one of the earliest events detectable (Martin et al., Eur. J. Immunol. 1994. 24: 1566). IL-1 signaling is commonly accompanied by oxidative processes and is thought to be subject to redox regulation. We therefore investigated whether the activation of the IL-1RI-associated protein kinase could be a target for redox regulation and whether an altered activity of the kinase could influence IL-1-mediated NF-kappa B activation. A murine T cell line, EL4, was stimulated with IL-1 with and without pretreatment with different compounds known to influence the cellular redox status. Thiol modifying agents like diamide, menadione, pyrrolidine dithiocarbamate (PDTC), diethyl dithiocarbamate or phenylarsine oxide inhibited the IL-1-induced activation of the IL-1RI-associated protein kinase. N-Acetylcysteine, alpha,alpha'-dipyridyl, aminotriazole or nitrofurantoin did not show any effect. The inhibition by PDTC was reversible unless glutathione synthesis was blocked by buthionine sulfoximine. The described conditions which inhibited or prevented the activation of the IL-1RI-associated kinase similarly impaired the activation of NF-kappa B in EL4 cells. From these observations we conclude that free thiols in the IL-1RI complex are essential for the activation of the IL-1RI-associated protein kinase and that this process is mandatory for IL-1 signaling leading to NF-kappa B activation.

Topics: Animals; Ditiocarb; Enzyme Activation; Glutathione; Humans; Interleukin-1; Interleukin-1 Receptor-Associated Kinases; Mice; NF-kappa B; Oxidation-Reduction; Protein Kinase Inhibitors; Protein Kinases; Pyrrolidines; Receptors, Interleukin-1; Sulfhydryl Compounds; T-Lymphocytes; Thiocarbamates; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Vitamin K

The stress-induced redistribution of cellular proteins between Triton (X-100)-soluble and Triton-insoluble fractions was studied in EL-4 thymoma and Ehrlich carcinoma cells. It was shown by electrophoresis and immunoblotting that a common sign of the cells which in vitro underwent such different harmful influences as ATP depletion, heat shock, oxidative stress, and SH reagent treatment was significant insolubilization of actin and 70-kDa heat-shock protein (hsp70). At the same time, each type of injury caused the specific insolubilization of some major cellular proteins. The transient ATP deprivation alone (without protein denaturation) induced a rapid insolubilization of myosin that was the earliest manifestation of ATP deficiency in the cells. Thermal stress without sharp decrease in ATP level induced a transition of 90-kDa heat-shock protein (hsp90) and 47-kDa polypeptide in Triton-insoluble fraction. The insolubilization of myosin and 47- and 35-kDa proteins was typical for the cells subjected to oxidative stress or SH reagent treatment, both of which caused damage of cellular proteins as well as ATP loss. The redistribution of the above proteins was intrinsic in the stressed cells of either ascites cell line, allowing one to consider it as characteristic and stress-specific cellular response to various injuries.

Topics: Actins; Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Heat-Shock Proteins; Hot Temperature; Hydrogen Peroxide; Neoplasm Proteins; Octoxynol; Oxidation-Reduction; Rotenone; Solubility; Thymoma; Tumor Cells, Cultured; Vitamin K

Resting ascites tumour cells (Ehrlich and EL-4 thymoma) treated with menadione (50 microM) died (up to 80% cell death over 2 h) without dividing (i.e. interphase). Glucose (25 mM) added to the cell suspensions partially protected these cells from menadione action. During incubation of the cells with menadione, the rates of free oxidation and lipid peroxidation were elevated, cellular ATP and nonprotein SH-group levels were much decreased, and [Ca2+]i was moderately increased. From a comparison of these effects and cell survival rates with those seen with rotenone, KCN (both inhibitors of oxidative phosphorylation), and H2O2 (an inducer of oxidative stress), it is concluded that ATP depletion is the main factor leading to the death of cells treated with menadione. The level of cellular ATP dropped to less than 10% of its initial value after 1 h incubation with menadione and may have resulted in irreversible damage to cytoskeletal structures, bleb formation, and changes in plasma membrane permeability that are incompatible with cell viability.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Energy Metabolism; Glucose; Glycolysis; Hydrogen Peroxide; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Oxidation-Reduction; Potassium Cyanide; Rotenone; Superoxide Dismutase; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Vitamin K