Fas-mediated apoptosis in human prostatic carcinoma cell lines occurs via activation of caspase-8 and caspase-7

We previously demonstrated that treatment with cycloheximide (CHX) converte d the phenotype of Fas-resistant human prostatic carcinoma cell lines to Fa s-sensitive and that resistance to Fas-mediated apoptosis was due to a domi nant-negative protein(s), In this study, we investigated the sequential act ivation of caspase family members, to gain insight into the likely site of action of the suppressor protein(s), We did not find Tyr-Val-Ala-Aspase act ivity in any of the cell lines examined. Time-dependent Asp-Glu-Val-Aspase activity was detected during Fas-mediated apoptosis in Fas-sensitive cell l ines PC3 and ALVA31. Asp-Glu-Val-Aspase activity in Fas-resistant cell line s DU145 and JCA1, was detected only under combined treatment with CHX and a nti-Fas agonistic mAb, In experiments with caspase inhibitors we show that Fas-mediated apoptosis in PC3 Is mainly executed by the caspase-3 subfamily , but another member(s) of the caspase family may be involved in Fas mediat ed apoptosis in ALVA31, DU145, and JCA1, Western blot analysis revealed tha t Fas-ligation activated caspase-7, but not caspase-3. The activated form o f caspase-8 was detected in DU145 only after 4 h of simultaneous treatment with CHX and anti-Fas mAb, whereas In PC3 caspase-8 was found to be activat ed after 1 h of Fas-ligation. We have also found that treatment with stauro sporin did not activate caspase-8, whereas staurosporin induced apoptosis a t the same levels in both Fas-resistant and Fas-sensitive cell lines. These results suggest that an inhibitory protein(s), which suppresses apoptosis in Fas-resistant cell lines, presumably acts at the apex of apoptotic casca de by preventing the activation of caspase-8.