A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis

Granulysin is a cytolytic molecule released by CTL via granule-mediated exo cytosis. In a previous study we showed that granulysin induced apoptosis us ing both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granuly sin-induced apoptosis of tumor cells. Granulysin-induced death is significa ntly inhibited by Bcl-2 overexpression and is associated with a rapid (1-5 h) loss of mitochondrial membrane potential, which is not mediated by ceram ide generation and is not inhibited by the general caspase inhibitor benzyl oxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C-18) ceramide is truly associa ted with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketon e. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexp ression. The nuclear morphology of cells dying after granulysin treatment i n the presence of caspase inhibitors suggested the involvement of mitochond rial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We d emonstrate using confocal microscopy that AIF is translocated from mitochon dria to the nucleus during granulysin-induced apoptosis. The majority of Bc l-2 transfectants are protected from granulysin-induced cell death, mitocho ndrial membrane potential loss, and AIF translocation, while a small percen tage are not protected. In this small percentage the typical nuclear apopto tic morphology is delayed, being of the AIF type at 5 h time, while at long er times (12 h) the normal apoptotic morphology is predominant. These and p revious results support a key role for the mitochondrial pathway of apoptos is, and especially for AIF, during granulysin-induced tumoral cell death.