Formation of caspase-3 complexes and fragmentation of
caspase-12 during anisomycin-induced apoptosis in AKR-2B cells without
aggregation of Apaf-1.
Hoppe J, Kilic M, Hoppe V, Sachinidis A, Kagerhuber U.
Department of Physiological Chemistry, Biozentrum, University of Wurzburg,
Germany. hoppe@biozentrum.uni-wuerzburg.de
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.
PMID: 12437191
