Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells.
Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, 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 whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531 s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase.
Vermijlen
D
D
Laboratory for Cell Biology and Histology, Free University Brussels (VUB), Belgium. dvermijl@cyto.vub.ac.be
Froelich
C J
CJ
Luo
D
D
Suarez-Huerta
N
N
Robaye
B
B
Wisse
E
E
D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology*; D004273:DNA, Neoplasm / Q000032:analysis; D019008:Drug Resistance, Neoplasm; D053222:Fas Ligand Protein; D053804:Granzymes; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism* / Q000494:pharmacology*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011419:Propidium; D012697:Serine Endopeptidases / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D001562:Benzimidazoles; D004273:DNA, Neoplasm; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D011419:Propidium; C509213:GZMB protein, human; D053804:Granzymes; C509215:Gzmb protein, rat; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C017807:bisbenzimide ethoxide trihydrochloride