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 actinomycin 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-alpha, beta, epsilon and zeta were detected in L929 cells. PKC-beta was only weakly detected in the cytoplasm, PKC alpha, epsilon and zeta were all found in resting cytoplasm and membrane. Stimulation with PMA caused a strong translocation of PKC-alpha but not zeta to the membrane. TNF had no effect on these isozymes but interestingly caused a translocation of PKC-epsilon, which also occurred in response to PMA. Staurosporine caused a translocation of PKC-zeta to the plasma membrane and a loss of PKC-epsilon from the cytosol. Although TNF induced PKC-epsilon 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-zeta, 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-zeta.
O'Connell
M A
MA
Department of Clinical Medicine, Trinity College, Dublin, Ireland.
Kelleher
D
D
Liskamp
R M
RM
Hall
N
N
O'Neill
L A
LA
Long
A
A
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D003602:Cytotoxicity, Immunologic; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D014409:Tumor Necrosis Factor-alpha; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; C064758:Ro 31-8220