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)-mediated cytotoxicity was investigated using the L929 cell line. The PKC activator phorbol-12-myristate 13-acetate (PMA) increased proliferation and inhibited TNF-induced cytotoxicity of L929 cells. A range of specific PKC inhibitors had no effect on TNF-mediated killing, suggesting that PKC does not play a direct role in this response. However, staurosporine enhanced cytotoxicity by TNF in the presence of atinomycin D, a protein synthesis inhibitor. In view of this finding, the authors investigated the role of specific PKC isozymes in both TNF-mediated cytotoxicity and staurosporine-induced sensitization to killing. PKC-α, β, ϵ and ζ were detected in L929 cells. PKC-β was only weakly detected in the cytoplasm, PKC α, ϵ and ζ were all found in resting cytoplasm and membrane. Stimulation with PMA caused a strong translocation of PKC-α but not ζ to the membrane. TNF had no effect on these isozymes but interestingly caused a translocation of PKC-ϵ, which also occurred in response to PMA. Staurosporine caused a translocation of PKC-ζ to the plasma membrane and a loss of PKC-ϵ from the cytosol. Although TNF induced PKC-ϵ translocation, this is unlikely to be involved in cytotoxicity as this effect was also induced by PMA which protected against TNF-mediated cytotoxicity. Staurosporine also induced translocation of PKC-ζ, an isozyme whose activity was previously found to be resistant to inhibition by staurosporine. These findings suggest the possibility that the mechanism by which staurosporine potentiates TNF action does not involve inhibition of PKC, but in contrast may involve modulation of PKC-ζ.