Characterization of distinct consecutive phases in non-genotoxic p53-induced apoptosis of Ewing tumor cells and the rate-limiting role of caspase 8.
To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets first observable after 1 h at 32 degrees C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was first observed 4 h after p53 induction. Using peptide inhibitors we identified activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 specific inhibitor z-IETD.fmk did not affect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the N-terminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. However, the VP16 - p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process. Oncogene (2000) 19, 4096 - 4107
Kovar
H
H
Children's Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria.
Jug
G
G
Printz
D
D
Bartl
S
S
Schmid
G
G
Wesierska-Gadek
J
J
D017209:Apoptosis; D015152:Blotting, Northern; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D005434:Flow Cytometry; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D006801:Humans; D020287:In Situ Nick-End Labeling; D047908:Intracellular Signaling Peptides and Proteins; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012512:Sarcoma, Ewing; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D017405:Herpes Simplex Virus Protein Vmw65; D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; C411296:TP53I3 protein, human; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; D011065:Poly(ADP-ribose) Polymerases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases