Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage.
Nitric oxide (NO) promotes apoptotic cell death in the mouse macrophage cell line RAW 264.7 and in the human promyelocytic leukaemia cell line U937, which exemplifies p53-dependent and p53-independent executive death pathways. Here, we followed the cleavage of two caspase substrates during NO-intoxication, assaying poly(ADP-ribose) polymerase and U1-70kDa small ribonucleoprotein (U1-70kDa) degradation. By using pharmacological inhibitors, we found that Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB; 100 microM), a caspase-like protease inhibitor, completely blocked S-nitrosoglutathione (GSNO)-induced apoptosis in both RAW 264.7 and U937 cells (IC50 = 50 microM for RAW 264.7 macrophages vs. IC50 = 33 microM for U937 cells). Notably, a characterized caspase-3 (Ac-DEVD-CHO) inhibitor left NO-induced DNA fragmentation and the appearance of an apoptotic morphology unaltered, although completely blocking caspase-3 activity. However, Z-Asp-CH2-DCB suppressed protease-mediated U1-70kDa cleavage and DNA fragmentation in parallel. In contrast, poly(ADP-ribose) polymerase cleavage in U937 cells was only delayed by Z-Asp-CH2-DCB, while poly(ADP-ribose) polymerase digestion in RAW 264.7 macrophages proceeded unaltered. We further compared U1-70kDa and poly(ADP-ribose) polymerase cleavage in stably Bcl-2 transfected RAW 264.7 macrophages. Rbcl2-2, a Bcl-2 overexpressing clone, suppressed DNA fragmentation and U1-70kDa digestion in response to GSNO, although allowing delayed but complete poly(ADP-ribose) polymerase degradation. Conclusively, poly(ADP-ribose) polymerase cleavage not causatively coincided with the appearance of other apoptotic parameters. Our results suggest that NO-induced apoptosis demands a Z-Asp-CH2-DCB inhibitable caspase activity, most likely distinct from caspase-3 and caspase-1. NO-mediated executive apoptotic signaling results in U1-70kDa and poly(ADP-ribose) polymerase cleavage. Whereas U1-70kDa digestion closely correlates to the occurrence of apoptotic parameters such as DNA fragmentation or an apoptotic morphology, poly(ADP-ribose) polymerase-breakdown does not.
University of Erlangen-Nürnberg, Faculty of Medicine, Department of Medicine IV, Erlangen, Germany.
U K
UK
Messmer
D M
DM
Reimer
B
B
Brüne
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D008264:Macrophages / Q000187:drug effects* / Q000502:physiology; D051379:Mice; D009569:Nitric Oxide / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000494:pharmacology; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D019253:Proto-Oncogene Proteins c-bcl-2; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human; C490687:Snrnp70 protein, mouse; D016159:Tumor Suppressor Protein p53; C093260:benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene; D001224:Aspartic Acid; D009569:Nitric Oxide; D011065:Poly(ADP-ribose) Polymerases; D003546:Cysteine Endopeptidases