Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage

Eur J Pharmacol. 1998 May 22;349(2-3):333-43. doi: 10.1016/s0014-2999(98)00189-7.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / physiology*
  • Aspartic Acid / analogs & derivatives
  • Aspartic Acid / pharmacology
  • Cell Line
  • Cysteine Endopeptidases / metabolism*
  • Enzyme Activation
  • Macrophages / drug effects*
  • Macrophages / physiology
  • Mice
  • Nitric Oxide / pharmacology*
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Proto-Oncogene Proteins c-bcl-2 / pharmacology
  • Ribonucleoprotein, U1 Small Nuclear / metabolism*
  • Tumor Suppressor Protein p53 / metabolism


  • Proto-Oncogene Proteins c-bcl-2
  • Ribonucleoprotein, U1 Small Nuclear
  • SNRNP70 protein, human
  • Snrnp70 protein, mouse
  • Tumor Suppressor Protein p53
  • benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene
  • Aspartic Acid
  • Nitric Oxide
  • Poly(ADP-ribose) Polymerases
  • Cysteine Endopeptidases