MACROPHAGES RESISTANT TO ENDOGENOUSLY GENERATED NITRIC OXIDE-MEDIATEDAPOPTOSIS 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 dono rs was found to be caspase 3-independent. Although caspase 3 activity was stimulated in the apoptotic macrophages, inhibition of caspase 3 b y the inhibitor DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde) did not r everse the apoptosis induced by the NO donor S-nitrosoglutathione (GSN O), This suggests that although caspase 3 activity is stimulated durin g apoptosis in macrophages, this signal is not sufficient to induce ap optosis, Cleavage of the enzyme poly(ADP ribose) polymerase mirrors ou r results of the caspase activity. Interestingly, we show that exogeno us NO donation results in an accumulation of cells at the G(2)/M-phase border. Here, we demonstrate that the mitogen activated protein kinas e kinase (MEK) inhibitor PD 098059 can be used to reverse the G(2)/M-p hase block and show that this treatment also inhibits the observed apo ptosis in RES macrophages, Treatment with the MEK inhibitor also rever sed both the caspase 3 activity and poly(ADP ribose) polymerase cleava ge 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 en dogenously generated NO may cause hypersensitivity to exogenous NO don ors. These findings have relevant implications for the treatment of ap optotic-resistant cell populations that may occur in both cancer and a theroma.