Macrophages resistant to endogenously generated nitric oxide-mediated apoptosis are hypersensitive to exogenously added nitric oxide donors: dichotomous apoptotic response independent of caspase 3 and reversal by the mitogen-activated protein kinase kinase (MEK) inhibitor PD 098059.
Nitric oxide (NO) induction through the inducible NO synthase has been demonstrated to cause cell death in macrophages. We demonstrate that, in macrophages that have been rendered resistant to apoptosis induced by inducible NO synthase (RES cells), exposure to exogenous NO donors results in a hypersensitive apoptosis reaction when compared with the parental RAW 264.7 cells. The apoptosis induced via exogenous NO donors was found to be caspase 3-independent. Although caspase 3 activity was stimulated in the apoptotic macrophages, inhibition of caspase 3 by the inhibitor DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde) did not reverse the apoptosis induced by the NO donor S-nitrosoglutathione (GSNO). This suggests that although caspase 3 activity is stimulated during apoptosis in macrophages, this signal is not sufficient to induce apoptosis. Cleavage of the enzyme poly(ADP ribose) polymerase mirrors our results of the caspase activity. Interestingly, we show that exogenous NO donation results in an accumulation of cells at the G2/M-phase border. Here, we demonstrate that the mitogen activated protein kinase kinase (MEK) inhibitor PD 098059 can be used to reverse the G2/M-phase block and show that this treatment also inhibits the observed apoptosis in RES macrophages. Treatment with the MEK inhibitor also reversed both the caspase 3 activity and poly(ADP ribose) polymerase cleavage in cells treated with GSNO. This result indicates that the mitogen-activated protein kinase pathway may be involved in regulation of the caspase cascade. Alternatively, it may suggest an activity for the MEK inhibitor heretofore not observed, that of a cyclin kinase inhibitor. Our results suggest that selection of macrophages by resistance to endogenously generated NO may cause hypersensitivity to exogenous NO donors. These findings have relevant implications for the treatment of apoptotic-resistant cell populations that may occur in both cancer and atheroma.
Mohr
S
S
Case Western Reserve University, School of Medicine and University Hospitals of Cleveland, Molecular Cardiovascular Research Center, Biomedical Research Building 440, 2109 Adelbert Road, Cleveland, OH 44106-4958, USA.
McCormick
T S
TS
Lapetina
E G
EG
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D048369:MAP Kinase Kinase 1; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D009569:Nitric Oxide / Q000502:physiology*; D009603:Nitroso Compounds / Q000494:pharmacology; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D026422:S-Nitrosoglutathione
D004791:Enzyme Inhibitors; D005419:Flavonoids; D009603:Nitroso Compounds; D011506:Proteins; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D005978:Glutathione; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one