Activation of caspase-9 and -3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation.
The treatment of PC12 cells with H2O2 (100-500 microM) resulted in typical apoptotic changes including fragmentation and condensation of nuclei, and DNA fragmentation observed as DNA ladder. H2O2-induced apoptosis was associated with activation of caspase-3 as assessed by cleavage of specific fluorogenic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) polymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibited activation of caspase-3 and apoptotic cell death. However, the relatively specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activation of caspase-3, induction of cytochrome c release followed by processing of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PC12 cells overexpressing Bcl-2, the number of apoptotic cells was markedly decreased and activation of both caspases-9 and -3 was prevented. These results suggest that cytochrome c and caspase-9 initiate the activation of executor caspase-3 in H2O2-treated PC12 cells, and that Bcl-2 inhibits H2O2-induced release of cytochrome c from mitochondria and then proteolytic processing of procaspase-9.
Yamakawa
H
H
Department of Neurosurgery, Gifu University School of Medicine, Japan. nohgeka@cc.gifu-u.ac.jp
Ito
Y
Y
Naganawa
T
T
Banno
Y
Y
Nakashima
S
S
Yoshimura
S
S
Sawada
M
M
Nishimura
Y
Y
Nozawa
Y
Y
Sakai
N
N
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002467:Cell Nucleus / Q000648:ultrastructure; D002518:Ceramides / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D016716:PC12 Cells; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D002518:Ceramides; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D004792:Enzyme Precursors; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases