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

Abstract

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.

p53 accumulation in apoptotic macrophages is an energy demanding process that precedes cytochrome c release in response to nitric oxide

“…In corroboration with previous studies (Messmer and BruÈ ne, 1996a) we noticed roughly 80% decreased p53 accumulation in response to 0.5 mM GSNO in RDp53asn-11 compared to parent macrophages (Figure 2a). Extending examinations allowed to position p53 upstream of subsequent events.…”

“…p53 antisense transfection and Bcl-2 overexpression in RAW 264.7 macrophages Construction of p53 antisense expressing RAW 264.7 cells (RDp53asn-11) and Bcl-2 overexpressing RAW 264.7 cells (Rbcl2-14) were previously described (Messmer and BruÈ ne, 1996a;Messmer et al, 1996b).…”

“…evoked typical morphological apoptotic alterations (Messmer et al, 1995) and upregulation of the tumor suppressor p53 as well as activation of caspases . Attenuating p53 accumulation by p53 antisense encoding plasmids (Messmer and BruÈ ne, 1996a) or enforced Bcl-2 overexpression abrogated NO .…”

Roles of CHOP/GADD153 in endoplasmic reticulum stress

“…NO was reported to activate ryanodine receptors or to inhibit SERCA activity by protein nitration. [82][83][84] Overexpression of calreticulin, which is a major Ca 2 þ -binding protein in the ER, prevents b-cells from NO-mediated apoptosis. 56 It should be noted that the expression of SERCA2b and calreticulin is induced by ER stress, which means that increased ER Ca 2 þ stores are likely to be required to adapt ER stress conditions.…”

Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis

“…p300/CBP activates several genes, including some associated with apoptosis, of which p53 is particularly well characterized 10-12 . Moreover, a major role for p53 in cell death induced by oxidative stress is well established [21][22][23] . GSNO treatment of GAPDH-transfected HEK293 cells enhanced p53 acetylation (Fig.…”