Bcl-2 expression in target cells leads to functional inhibition of caspase-3 protease family in human NK and lymphokine-activated killer cell granule-mediated apoptosis.
In the granule exocytosis pathway of cell-mediated cytotoxicity, rapid apoptotic nuclear damage in target cells has been unequivocally linked to granzyme B activity. Direct cleavage and activation of caspase-3 and related proteases by granzyme B have been identified as a central event in apoptosis induction by cytotoxic granules. The Bcl-2 oncoprotein has been recently shown to act at the level or upstream of caspase-3 family activation to inhibit apoptosis induced by various stimuli including Fas ligation, an alternative cell-mediated lytic pathway. In this study, we have investigated whether activation of this caspase family by granzyme B, during human NK and lymphokine-activated killer cell granule-mediated apoptosis, could be influenced by Bcl-2 expression. Bcl-2-overexpressing clones were generated from parental K562 and U937 cell lines (K6 and U4 clones, respectively). Bcl-2 expression abrogated early 125I-DNA release and DNA fragmentation, these defects being compensated for by extended incubation times. Cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 family substrate, was detected in parental K562 cells exposed to lymphokine-activated killer effectors but not in K6 targets, indicating that caspase-3 and related proteases function was inhibited by Bcl-2. Functional inhibition of caspase-3 family with benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe) fluoromethylketone led to similar consequences on apoptotic nuclear events as for Bcl-2 expression. Thus, Bcl-2 antagonizes granzyme B-mediated apoptosis by a mechanism that interferes with caspase-3 activity. Finally, Bcl-2 expression or the Asp-Glu-Val-Asp peptide was much less efficient in preventing phosphatidylserine externalization, suggesting that despite impaired nuclear apoptosis, immediate recognition and elimination of Bcl-2-expressing cells by tissue phagocytes should remain partly unaffected.
Renvoizé
C
C
INSERM Unit 461, Receptors and Signalization of Interleukins, Faculty of Pharmacy, University of Paris XI, Châtenay-Malabry, France.
Roger
R
R
Moulian
N
N
Bertoglio
J
J
Bréard
J
J
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000276:immunology; D003594:Cytoplasmic Granules / Q000473:pathology; D005089:Exocytosis / Q000187:drug effects; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D053804:Granzymes; D006801:Humans; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology / Q000473:pathology*; D007694:Killer Cells, Natural / Q000276:immunology / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000276:immunology; D012697:Serine Endopeptidases / Q000494:pharmacology
D019253:Proto-Oncogene Proteins c-bcl-2; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases