1C). This is most likely due to the ability of ionomycin to weakly activate the PKC pathway 44. However, Nur77 levels were significantly enhanced when PMA or the DAG-lactone, HK434, were added (Fig. 1C and data not shown). Nur77 levels dropped at the highest HK434 concentrations, presumably due to extensive apoptosis. The same results were found with Nor-1 mitochondria translocation (data not shown and Fig. 1C). We conclude that Nur77 and Nor-1 induction

and mitochondrial targeting are dependent on two intracellular signals, the PKC and the calcium pathways. It is well established that activation of PKC by phorbol esters such as PMA triggers an apoptotic selleckchem response in thymocytes 35, 45, 46. In LNCaP cells, the PKC activator, HK434, was shown to mimic the action of PMA with respect to apoptosis. In thymocytes, the level and kinetics of apoptosis induced by HK434 and ionomycin were similar to that induced by PMA and ionomycin

(Fig. 2A). To confirm that the apoptotic effect of PMA and the DAG-lactone in thymocytes is mediated by activation of PKC, we assessed the affect of HK434 and PMA in the presence of pharmacological inhibitors that specifically block classical or novel PKC isoforms. The classical PKC inhibitor, Gö6976 sufficiently abrogated HK434-induced death (Fig. 2B) as well as the cytotoxic affects of anti-CD3/CD28 antibody treatment (Fig. 2B). CHIR-99021 chemical structure The inhibitory effect of Gö6976 on PMA/ionomycin-induced thymocyte cell death is controversial. One group found that it could block PMA/ionomycin death although the effect was modest at best 28 while another group could not see any effect 46. In our hands, Gö6976 could not block thymocyte death induced by PMA, even at subnanomolar concentrations of the phorbol ester. However, the classical and novel PKC isoform inhibitor, GF109203X, almost completely this website blocked cell death induced by all treatments (Fig. 2B). Pre-treatment

with GF109203X effectively blocked activation induced by all stimulation conditions, as assessed by CD69 staining (data not shown). Interestingly, though 1 μM Gö6976 had no affect on PMA-induced thymocyte apoptosis; the inhibitor was sufficient in blocking thymocyte activation mediated by PMA as assessed by CD69 staining. These results suggest that cPKC isozymes are responsible for the death induced by the PKC ligand, HK434 and anti-CD3/CD28 antibodies. Yet, nPKC but not cPKC isoforms play a role in thymocyte apoptosis induced by PMA. Inhibition of conventional PKC isozymes with Gö6976 was effective in blocking cell death induced by HK434/ionomycin but not PMA/ionomycin signals; therefore, we wanted to examine Nur77 localization in the presence of this cPKC-specific inhibitor as well as the PKC general inhibitor. Inhibition of cPKC with Gö6976 is sufficient in blocking Nur77 and Nor-1 translocation to the mitochondria mediated by HK434/ionomycin (Fig. 3A).