p53 accumulation in apoptotic macrophages is an energy demanding process that precedes cytochrome c release in response to nitric oxide.
Apoptosis in response to stress signals activates effector 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-xL downregulation, Cyt c release, and caspase activation. These alterations were absent in p53 antisense expressing macrophages (R delta p53asn-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-xL 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 identified p53 accumulation and stabilization as an energy requiring process. This allowed to dissect two ATP-dependent steps, one is in association with Apaf-1 formation, while the other resides in p53 accumulation.
Brockhaus
F
F
University of Erlangen-Nürnberg, Faculty of Medicine, Department of Medicine IV-Experimental Division, Germany.
Brüne
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D009569:Nitric Oxide / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051020:bcl-X Protein
C494812:Bcl2l1 protein, mouse; D003574:Cytochrome c Group; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051020:bcl-X Protein; D009569:Nitric Oxide; D000255:Adenosine Triphosphate