PROTEIN-KINASE C-DEPENDENT PHOSPHORYLATION IS INVOLVED IN RESISTANCE TO TUMOR NECROSIS FACTOR-ALPHA-INDUCED CYTOTOXICITY IN A HUMAN MONOCYTOID CELL-LINE

To investigate the mechanism underlying resistance to tumour necrosis factor-alpha (TNFalpha)-induced cytotoxicity, we have developed a huma n hybrid cell line, designated A10, derived from the fusion of human U -937 monocytoid cells and human monocytes, which expressed large numbe rs of TNFalpha receptors and yet remained highly resistant to TNFalpha . However, in the presence of the protein kinase C (PKC) inhibitors RO -31-7549 or RO-318220 (donated by Roche), these cells became sensitive to TNFalpha-induced cytotoxicity, suggesting that PKC activity is req uired for protective mechanisms. On investigation of protein phosphory lation in TNFalpha-stimulated permeabilized A10 cells, a rapid increas e 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 PK C, a serine/threonine kinase. The theory that activation of PKC was re sponsible for increased phosphorylation was confirmed by a dose-depend ent inhibition of the TNFalpha-induced protein phosphorylation by the PKC inhibitors RO-31-7549 and RO-31-8220. The possible link between th e TNFalpha-stimulated early protein phosphorylation events and the mai ntenance of protective mechanisms against TNFalpha-induced cytotoxicit y is discussed.