Ceramide-induced apoptosis is mediated by caspase activation independently from retinoblastoma protein post-translational modification.
Recent evidence suggests that untimely retinoblastoma protein (RB) dephosphorylation and/or proteolytic degradation might provide key events down-stream cysteine protease (caspase) activation in apoptosis induction. We have dealt with this issue by studying apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells, maintained in complete growth medium. We report that C6-Cer-induced apoptosis occurred predominantly in G1/S phases of the cycle and was associated with RB dephosphorylation, in the setting of negligible Bcl-2 expression. Apoptosis was also associated with poly(ADP-ribose) polymerase (PARP) cleavage, thus indicating activation of CPP32/Yama/apopain (caspase-3); however, while the tripeptide caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone was able to prevent both C6-Cer-induced PARP cleavage and apoptosis, it was ineffective in preventing RB dephosphorylation. Moreover proteolytic RB cleavage occurred only to a marginal extent after C6-Cer treatment. These results indicate that apoptosis induced by ceramide in CHP-100 cells is caspase-mediated, but RB post-translational modification does not provide a key step, downstream caspase activation, in apoptosis execution.
Spinedi
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Department of Biology, University of Rome Tor Vergata, Italy. spinedi@seneca.ccd.utovrm.it
Amendola
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Di Bartolomeo
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Piacentini
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D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases; D002518:Ceramides / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D016193:G1 Phase; D006801:Humans; D018241:Neuroectodermal Tumors, Primitive, Peripheral / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016160:Retinoblastoma Protein / Q000235:genetics*; D016196:S Phase; D014407:Tumor Cells, Cultured
D002518:Ceramides; D016160:Retinoblastoma Protein; C101954:N-caproylsphingosine; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases