Nitric oxide-induced apoptosis: p53-dependent and p53-independent signalling pathways.

Nitric oxide (NO) generation initiates apoptotic cell death in different experimental systems. In RAW 264.7 macrophages the appearance of typical apoptotic markers is linked to inducible NO synthase induction. Mechanistically, accumulation of tumour suppressor p53 precedes apoptotic DNA fragmentation. With the use of S-nitroglutathione (GSNO) we correlated a dose-dependent p53 up-regulation to DNA fragmentation measured after 4 h and 8 h, respectively. Our studies revealed a linear correlation between the potency of five different NO donors with respect to apoptosis induction and p53 accumulation. Furthermore, we probed for NO-induced apoptosis after stable transfection of RAW 264.7 macrophages with plasmids encoding p53 antisense RNA. Clones with down-regulated p53 levels in response to GSNO exhibited a marked reduction in DNA fragmentation. Expression of the inducible NO synthase in response to lipopolysaccharide and interferon-gamma caused apoptosis in RAW 264.7 macrophages and neomycin-vector controls within 24 h. In contrast, p53 antisense RNA-expressing clones appeared highly resistant towards endogenous NO, although inducible NO synthase induction with concomitant nitrite production remained unchanged. For RAW 264.7 macrophages our results established a functional role of the tumour suppressor p53 during NO-induced apoptotic cell death. However, p53 antisense experiments and the use of the p53-negative cell line U937 substantiated p53-independent signalling pathways operative during NO-mediated apoptosis.