Protein kinase C-dependent phosphorylation is involved in resistance to tumour necrosis factor-alpha-induced cytotoxicity in a human monocytoid cell line.
To investigate the mechanism underlying resistance to tumour necrosis factor-alpha (TNF alpha)-induced cytotoxicity, we have developed a human hybrid cell line, designated A10, derived from the fusion of human U-937 monocytoid cells and human monocytes, which expressed large numbers of TNF alpha receptors and yet remained highly resistant to TNF alpha. However, in the presence of the protein kinase C (PKC) inhibitors RO-31-7549 or RO-31-8220 (donated by Roche), these cells became sensitive to TNF alpha-induced cytotoxicity, suggesting that PKC activity is required for protective mechanisms. On investigation of protein phosphorylation in TNF alpha-stimulated permeabilized A10 cells, a rapid increase in serine/threonine phosphorylation of phosphoproteins of molecular masses 130, 90, 80, 65 and 42 kDa was found. Subsequently, we found a similar pattern of increased phosphorylation following stimulation of A10 cells with mezerein, a phorbol ester derivative which activates PKC, a serine/threonine kinase. The theory that activation of PKC was responsible for increased phosphorylation was confirmed by a dose-dependent inhibition of the TNF alpha-induced protein phosphorylation by the PKC inhibitors RO-31-7549 and RO-31-8220. The possible link between the TNF alpha-stimulated early protein phosphorylation events and the maintenance of protective mechanisms against TNF alpha-induced cytotoxicity is discussed.
Division of Immunobiology, National Institute for Biological Standards and Control, Potters Bar, Herts, U.K.
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Sampson
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Mire-Sluis
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Meager
D002470:Cell Survival / Q000187:drug effects; D004351:Drug Resistance; D006801:Humans; D006822:Hybrid Cells; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D007211:Indoles; D008301:Maleimides; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D011493:Protein Kinase C; C064758:Ro 31-8220; C064755:Ro 31-7549