Formation of caspase-3 complexes and fragmentation of caspase-12 during anisomycin-induced apoptosis in AKR-2B cells without aggregation of Apaf-1.
Treatment of AKR-2B fibroblasts with anisomycin (10 microM) led to a rapid disintegration of the cells (t1/2 = 5 h) which was complete after 24 h. Cell death was associated with typical hallmarks of apoptosis like membrane blebbing, exposure of phophatidylserine on the cell surface, nuclear condensation and specific cleavage of rRNA. However, there was no dissipation of the mitochondrial potential and no intranucleosomal fragmentation. By affinity labeling with YVK(-bio)D.aomk in combination with immunostaining against activated caspase-3 analyzed by 2-D gel electrophoresis it was shown that caspase-3 is the dominant executioner caspase. Gel filtration experiments of cytosolic extract analyzed by Western blotting revealed the formation of high-molecular-weight complexes of caspase-3 (600 kDa and 250 kDa, respectively), but there was no complex formation of Apaf-1. Anisomycin treatment led to a strong activation of the stress kinases p38 kinases and the jun kinases, that was not sufficient for the activation of caspase-3 which required much higher concentrations. By using the selective inhibitors SB 203580 for p38 kinases and SP 600125 for c-jun kinases, respectively, it is shown that activation of these kinases is not necessary for cell death induced by anisomycin in AKR-2B cells. Furthermore, we disclose the activation of caspase-12 in AKR-2B cells following the addition of anisomycin. Caspase-12 zymogen present as a cytosolic complex (> 600 kDa) is activated by anisomycin leading to an uncomplexed cleaved enzyme. Since anisomycin treatment did neither lead to stress of the endoplasmic reticulum nor to a breakdown of intracellular Ca(2+)-stores, alternative pathways involved in the activation of caspases are discussed.
Hoppe
Jürgen
J
Department of Physiological Chemistry, Biozentrum, University of Würzburg, Germany. hoppe@biozentrum.uni-wuerzburg.de
Kilic
Mehtap
M
Hoppe
Vivianne
V
Sachinidis
Agapios
A
Kagerhuber
Ulrike
U
D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D053454:Caspase 12; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D005057:Eukaryotic Cells / Q000187:drug effects / Q000201:enzymology*; D005347:Fibroblasts; D046911:Macromolecular Substances; D051379:Mice; D008806:Mice, Inbred AKR; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D015388:Organelles / Q000187:drug effects / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000187:drug effects / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000187:drug effects / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D046911:Macromolecular Substances; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D000841:Anisomycin; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010447:Peptide Hydrolases; C506440:Casp12 protein, mouse; C505409:Casp3 protein, mouse; D053454:Caspase 12; D053148:Caspase 3; D020169:Caspases; C108711:DEVDase