TNF-MEDIATED CYTOTOXICITY OF L929 CELLS - ROLE OF STAUROSPORINE IN ENHANCEMENT OF CYTOTOXICITY AND TRANSLOCATION OF PROTEIN-KINASE-C ISOZYMES

The role of protein kinase C (PKC) in tumour necrosis factor (TNF)-med iated cytotoxicity was investigated, using the L929 cell line, The PKC activator phorbol-12-myristate 13-acetate (PMA) increased proliferati on and inhibited TNF-induced cytotoxicity of L929 cells, A range of sp ecific PKC inhibitors had no effect on TNF-mediated killing, suggestin g that PKC does not play a direct role in this response, However, stau rosporine enhanced cytotoxicity by TNF in the presence of actinomycin D, a protein synthesis inhibitor, In view of this finding, the authors investigated the role of specific PKC isozymes in both TNF-mediated c ytotoxicity and staurosporine-induced sensitization to killing. PKC-al pha, beta, epsilon and zeta were detected in L929 cells, PKC-beta was only weakly detected in the cytoplasm, PKC alpha, epsilon and zeta wer e all found in resting cytoplasm and membrane, Stimulation with PMA ca used a strong translocati alpha of PKC-ar but not zeta to the membrane , TNF had no effect on these isozymes but interestingly caused a trans location of PKC-epsilon, which also occurred in response to PMA, Staur osporine caused a translocation of PKC-zeta to the plasma membrane and a loss of PKC-epsilon from the cytosol, Although TNF induced PKC-epsi lon translocation, this is unlikely to be involved in cytotoxicity as this effect was also induced by PMA which protected against TNF-mediat ed cytotoxicity. Staurosporine also induced translocation of PKC-zeta, an isozyme whose activity was previously found to be resistant to inh ibition by staurosporine. These findings suggest the possibility that the mechanism by which staurosporine potentiates TNF action does not i nvolve inhibition of PKC, but in contrast may involve modulation of PK C-zeta. (C) 1997 Academic Press Limited.