Temporal relationships between ceramide production, caspase activation andmitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis

To clarify the chronology of events leading to anti-Fas-induced apoptosis, and the mechanisms of resistance to this death effector, we compared the re sponse kinetics of three tumour cell lines that display varying sensitivity to anti-Fas (based on levels of apoptosis), in terms of ceramide release, mitochondrial function and the caspase-activation pathway. In the highly se nsitive Jurkat cell line, early caspase-8 activation, observed from 2 h aft er treatment, was chronologically associated with an acute depletion of glu tathione and the cleavage of caspase-3 and poly-ADP ribosyl polymerase (PAR P), followed by a progressive fall in the mitochondrial transmembrane poten tial (Delta psi (m)), between 4 and 48 h after treatment. Ceramide levels b egan to increase 2 h after the addition of anti-Fas (with no increase durin g the first hour), and increased continuously to 640% of control cells at 4 8 h. In the moderately sensitive SCC61 adherent cells, comparable results w ere observed, though with lower levels of ceramide and a delay in the respo nse kinetics, with apoptotic cells becoming flotant. Finally, despite early cleavage of caspase-8 at 2 h, and a sustained level of activation until 48 h, no apoptotic response was observed in anti-Fas-resistant SQ20B cells. T his was confirmed by a lack of ceramide generation and mitochondrial change s, and by the absence of any detectable cleavage of caspase-3 or PARP. Inhi bition of caspase processing, and amplification of endogenous ceramide sign alling by pharmacological agents, allowed us to establish the order of cell ular events, locating ceramide release after caspase-8 activation and befor e caspase-3 activation, and demonstrating a direct involvement for ceramide release in mitochondrial dysfunction. Furthermore, these experiments provi de strong arguments for the role of endogenous ceramide as a key executor o f apoptosis, rather than as a consequence of membrane alterations.