Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinomacells

Cytotoxic lymphocytes may induce apoptosis in their target cells by the Fas L (Fas ligand) pathway or the perforin/granzyme B pathway. It has been show n that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-medi ated apoptosis. The aims of this study were to determine whether CC cells a re also resistant to perforin/granzyme B and whether the FasL resistance li es upstream of caspase-3 activation. The resistance of the Fas-expressing r at CC531s cells to the FasL pathway was confirmed by treating them with rec ombinant human soluble FasL, using rat hepatocytes as a positive control. T he intracellular delivery of granzyme B by sublytic concentrations of perfo rin, on the other hand, resulted in many features of apoptosis (chromatin c ondensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn wh ether the FasL resistance was due to failure to activate this crucial execu tioner. Caspase-3 activation occurred in CC531s cells after perforin/granzy me B treatment, but not after the addition of recombinant FasL. Furthermore , we showed that caspase-3 activity is involved in the execution of perfori n/granzyme-B-induced apoptosis in CC531s cells, since the cell-permeable ca spase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these r esults suggest that CC cells are sensitive to perforin/granzyme-B-induced a poptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase.