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

Abstract

Apoptosis in response to stress signals activates e ector caspases known to be regulated by the release of cytochrome c (Cyt c) from mitochondria and the subsequent ATP-dependent activation of the death regulator apoptotic protease-activating factor 1 (Apaf-1). Experiments were carried out to determine whether the release of Cyt c is evoked by NO . in RAW 264.7 macrophages and to position signaling components relative to mitochondria. S-nitrosoglutathione and spermine-NO caused a fast p53 accumulation, followed by Bcl-x L downregulation, Cyt c release, and caspase activation. These alterations were absent in p53 antisense expressing macrophages (RDp53asn-11). In Bcl-2 overexpressing cells (Rbcl2-14) Cyt c relocation and caspase activation were abrogated although p53 accumulation remained intact. The use of caspase inhibitors revealed Cyt c release and decreased Bcl-x L expression to be caspase independent. ATP-depleted cells showed a shift from apoptosis towards necrosis and no p53 accumulation or caspase activation upon NO . addition. Conclusively, NO . -mediated apoptosis in macrophages is entirely controlled by the mitochondrial pathway with the implication that Cyt c relocation demands p53 accumulation. Moreover, pulse-chase-experiments in combination with the ATP-depletion protocol identi®ed p53 accumulation and stabilization as an energy requiring process. This allowed to dissect two ATPdependent steps, one is in association with Apaf-1 formation, while the other resides in p53 accumulation.

Sodium nitroprusside enhances TRAIL-induced apoptosis via a mitochondria-dependent pathway in human colorectal carcinoma CX-1 cells

“…At this time, we can only speculate how SNP promotes TRAILinduced apoptosis. Several studies have shown that NO stimulates apoptosis through activation of caspases (Tamatani et al, 1998), cytochrome c release (Ushmorov et al, 1999), changes in the expression of pro-and anti-apoptotic Bcl-2 family members (Tamatani et al, 1998; Brockhaus and Brune, 1999), and upregulation of the tumor suppressor p53 (Brockhaus and Brune, 1999). Mitochondrial damage (lipid degradation, lipid peroxidation, and cytochrome c release) induced by NO plays a crucial role in the subsequent activation of caspase and apoptosis (Ushmorov et al, 1999;Ghafourifar et al, 1999a).…”

Nitric oxide (NO): an effector of apoptosis

“…In addition, ATP-depleted cells showed a shift from apoptosis towards necrosis upon NO addition, together with attenuated p53 accumulation and caspase activation. 21 We suggest that NO-mediated apoptosis in macrophages is entirely controlled by the mitochondrial pathway with the implication that p53 accumulation is upstream of cytochrome c release. This assumption is supported by the observation that thymocytes from p53 knockout-mice are protected against irradiation-induced apoptosis together with suppressed mitochondrial transmembrane depolarization.…”

“…41,42 Demonstrating that a caspase-3-like or a pan-caspase inhibitor left cytochrome c and p53 accumulation unaltered implied that a caspase activity is not required for both events. 21 In close analogy to the macrophage system the ability of NO to initiate cell death with the occurrence of apoptotic parameters was identified in vascular smooth muscle cells of murine and human origin. 43,44 In these cells apoptosis was characterized by p53 accumulation, decreased Bcl-2 expression, and caspase-3 activation with the further notion that inhibition of caspase-3 significantly prevented apoptosis.…”

“…19,20 One might speculate that in these systems NO evoked a necrotic rather than an apoptotic response which is fully compatible with our observations that ATP-depletion shifted NO-elicited apoptosis to necrosis. 21 The p53 response, cytochrome c relocation, and caspase activation One pivotal target that responds to DNA damage is the tumor suppressor p53. The cellular level of p53 is kept at a low level due to a short protein half-life but in response to a variety of stimuli it is increased steeply by stabilization.…”

Radiation-induced genomic instability and bystander effects: inter-related nontargeted effects of exposure to ionizing radiation

“…However, the findings are remarkably similar to the neutrophil infiltration observed in the thymus after irradiation (Uchimura et al, 2000) and in vitro studies have shown that phagocytosis of apoptotic cells results in the production of both pro-and anti-inflammatory cytokines (Giles et al, 2000;Gregory, 2000). Additionally, nitric oxide can be either pro-or antiapoptotic, can either downregulate or upregulate p53 activity (Brune et al, 1996;Brockhaus and Brune, 1999) and may be pro-or anti-inflammatory (Nathan and Shiloh, 2000) depending on the context. Thus, it is possible that nonspecific inflammatory-type responses to radiation-induced stress and injury may contribute to the wide variety of bystander-mediated effects and to genomic instability.…”