Caspase inhibition shifts neuroepithelioma cell response to okadaic acid from apoptosis to an apoptotic-like form of death

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Abstract

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 μM) 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 p21Cip1/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.

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Materials and methods

Materials. Material for cell culture was from Gibco BRL (MD, USA). OA and the pan-caspase inhibitor benzyloxycarbonyl–Val–Ala–Asp–fluoromethylketone (Z-VAD.fmk) were from Calbiochem Novachem (La Jolla, CA, USA). The anti-p53 mouse monoclonal antibody was from Upstate Biotechnology (Lake Placid, NY, USA). The anti-p21Cip1/Waf1 and the anti-retinoblastoma protein (Rb) rabbit polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-β-actin mouse monoclonal antibody

OA dose-dependently affects the DNA histogram of CHP-100 cells: effects of the caspase inhibitor Z-VAD.fmk

Figs. 1A–D shows representative DNA histograms of propidium iodide-stained nuclei from CHP-100 cells exposed for 24 h to increasing OA concentrations (0–100 nM). In comparison with untreated cells (Fig. 1A), it can be observed that OA dose-dependently induced accumulation of a hypodiploid population, namely a fraction generally assumed to include apoptotic elements, due to the fact that apoptotic chromatin condensation reduces DNA staining and nuclei may undergo fragmentation [20]. Figs. 1B and C

Discussion

We have shown that OA exerts pleiotropic effects in CHP-100 cells, evoking, in a dose-dependent fashion, not only caspase-dependent apoptosis but also PCC and mitotic arrest. Notably, microscopy analysis showed that, under the experimental conditions used, the latter two effects were already outstanding at the lowest OA concentration employed (10 nM), at which apoptosis was not observed. In this respect, it is feasible that accumulation of hypodiploid elements monitored by flow cytometry at this

Acknowledgements

This work was supported by MURST grants (60% funds) to A.S.

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  • Cited by (3)

    1

    Present address: Department of Experimental Medicine and Pathology, University of Rome “La Sapienza,” Viale Regina Elena 324, 00161 Rome, Italy.

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