benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Neuroectodermal-Tumors--Primitive--Peripheral

We have previously shown that the protein phosphatase inhibitor okadaic acid (OA) induces caspase-3 activation and apoptosis in CHP-100 human neuroepithelioma cells. Herein we provide a more general picture of the effects brought about by OA in this system, also investigating whether caspase activation is necessary for apoptosis induction. We report that incubation for 24 h with 10 nM OA induced a large fraction of the cell population to undergo premature chromosome condensation (PCC) or mitotic arrest, but not apoptosis. The former two effects were also observed after cell treatment with 20 nM OA; however, at this concentration, typical apoptotic cells were also detected, characterized by pycnotic and fragmented nuclei. Occurrence of the above-mentioned apoptotic figures turned extensive at 100 nM OA. The pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk, 100 microM) fully prevented apoptosis induced by 20 nM OA, increasing PCC incidence. Conversely, 100 nM OA induced an apoptotic-like phenotype, even in the presence of Z-VAD.fmk: in this case, however, nuclei, albeit pycnotic, displayed morphological characteristics distinct from those of typical apoptotic cells; moreover, as assessed by flow cytometry, they were largely unfragmented. The reported OA effects occurred in a setting in which neither p53 nor p21(Cip1/Waf1) was upregulated, thus ruling out a role for these proteins in apoptosis induction. On the other hand, apoptotic doses of OA induced a shift of the retinoblastoma gene product to the hypophosphorylated state and its downregulation by a caspase-dependent mechanism.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Cell Death; Cell Nucleus; Cysteine Proteinase Inhibitors; DNA, Neoplasm; Flow Cytometry; Humans; Neuroectodermal Tumors, Primitive, Peripheral; Okadaic Acid; Thymidine; Tumor Cells, Cultured

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.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Caspases; Ceramides; Cysteine Proteinase Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Neuroectodermal Tumors, Primitive, Peripheral; Oligopeptides; Poly(ADP-ribose) Polymerases; Protein-Tyrosine Kinases; Time Factors; Tumor Cells, Cultured

Caspase-dependent apoptosis induced by okadaic acid (OA) in CHP-100 neuroepithelioma cells has previously been shown to associate with a rapid and sustained elevation in intracellular ceramide concentration. We now report that treatment of CHP-100 cells with OA also evoked a rapid elevation in glucosylceramide levels that was maintained at steady state as cells underwent apoptosis; moreover, as observed for ceramide, OA-induced glucosylceramide accumulation was not blocked by fumonisin B1. Remarkably, when cell death was prevented by caspase inhibition, glucosylceramide accumulation was potentiated and ceramide elevation reduced, thus suggesting that, during apoptosis completion, accumulation of ceramide was partly driven by impairment of its glucosylation through a caspase-dependent mechanism. We studied whether ceramide glucosylation provided a mechanism for negative modulation of OA-induced apoptosis. We observed that the blocking of glucosylceramide synthesis markedly potentiated OA-induced ceramide elevation, but neither accelerated apoptosis onset nor potentiated the apoptotic response. These results indicate that modulation of ceramide glucosylation does not affect the apoptotic response to okadaic acid and suggest that caution must be exercised concerning the possibility that ceramide plays a key role in apoptosis induction.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Ceramides; Cysteine Proteinase Inhibitors; Drug Synergism; Enzyme Inhibitors; Glucosyltransferases; Humans; Neuroectodermal Tumors, Primitive, Peripheral; Okadaic Acid; Tumor Cells, Cultured

Doxorubicin (0.5 microgram/ml) induced caspase-dependent apoptosis in SH-SY5Y neuroblastoma and CHP-100 neuroepithelioma cells. The apoptotic response started to be evident approximately 15 h after drug administration and, as monitored over a 48-h period, was more pronounced in CHP-100 than in SH-SY5Y cells. In both systems, apoptosis was accompanied by elevation of intracellular ceramide levels. Ceramide accumulation was blocked by the ceramide synthase inhibitor fumonisin B(1) (25 microM); this compound, however, did not prevent drug-induced apoptosis. Untreated cells from both lines expressed negligible p53 levels; on the other hand, whereas p53 and p21(Cip1/Waf1) were rapidly up-regulated in doxorubicin-treated SH-SY5Y cells, such a response was not observed in CHP-100 cells. Doxorubicin induced a G(2)/M phase block in both cell lines, but whereas the G(1) phase was markedly depleted in CHP-100 cells, it was substantially retained in SH-SY5Y cells. In the latter system, double G(1) and G(2)/M block largely preceded cell death; however, as apoptosis underwent completion, it selectively targeted late S and G(2)/M cells. Moreover, apoptosis suppression by caspase inhibition did not result in a recovery of the G(1) cell population. These results support the notion that doxorubicin-induced apoptosis and ceramide elevation are divorced events in neuroectodermal tumors and that p53 function is at least dispensable for apoptosis completion. Indeed, as G(1) cells appear to be refractory to doxorubicin-induced apoptosis, p53 up-regulation and p21(Cip1/Waf1) expression may provide an unfavorable setting for the apoptotic action of the drug.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Brain Neoplasms; Caspase Inhibitors; Caspases; Cell Cycle; Ceramides; Doxorubicin; Enzyme Activation; G1 Phase; Humans; Kinetics; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Neuroprotective Agents; Tumor Cells, Cultured