Ordering ceramide-induced cell detachment and apoptosis in human neuroepithelioma.
We studied whether cell detachment from the matrix, observed during ceramide-induced apoptosis, is secondary to completion of the apoptotic program. CHP-100 neuroepithelioma cells exposed to N-hexanoylsphingosine (C(6)-Cer) underwent detachment from the substrate and apoptosis with slow kinetics. Apoptotic cells were fairly completely recovered in the detached fraction, that, differently from the adherent counterpart, displayed the hallmarks of caspase 3 activation, as well as poly-(ADP)ribose polymerase (PARP) cleavage and focal adhesion kinase (FAK) downregulation. A key role for caspase 3 in apoptosis execution was suggested by the evidence that its selective inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde inhibited cell death. However, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (targeting not only caspase 3 but also caspases 1, 5, 7, 8 and 9) did not prevent ceramide-induced cell detachment, although apoptosis, caspase 3 processing, PARP cleavage and FAK downregulation were suppressed in floating cells. These results demonstrate that ceramide-induced cell detachment is upstream activation of effector caspases. We discuss the possibility that ceramide-induced cell detachment might be instrumental to apoptosis execution.
Di Bartolomeo
Sabrina
S
Dipartimento di Biologia, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy.
Spinedi
Angelo
A
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002518:Ceramides / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D018241:Neuroectodermal Tumors, Primitive, Peripheral / Q000378:metabolism / Q000473:pathology*; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000187:drug effects / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D002518:Ceramides; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; C108637:aspartyl-glutamyl-valyl-aspartal; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011065:Poly(ADP-ribose) Polymerases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases