Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid.
The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 - 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 microM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 - 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 - 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 microM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 microM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 - 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.
Spinedi
A
A
Department of Biology, University of Rome 'Tor Vergata', Italy. spinedi@uniroma2.it
Di Bartolomeo
S
S
Di Sano
F
F
Rodolfo
C
C
Ambrosino
A
A
Piacentini
M
M
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002264:Carboxylic Acids / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D037341:Fumonisins; D006801:Humans; D018241:Neuroectodermal Tumors, Primitive, Peripheral / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D019319:Okadaic Acid / Q000008:administration & dosage / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
D002264:Carboxylic Acids; D002518:Ceramides; D004791:Enzyme Inhibitors; D037341:Fumonisins; D019319:Okadaic Acid; C056933:fumonisin B1; D010749:Phosphoprotein Phosphatases; D020169:Caspases