Caspase inhibition shifts neuroepithelioma cell response to okadaic acid from apoptosis to an apoptotic-like form of death
Section snippets
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.
References (31)
- et al.
Mitotic arrest and enhanced nuclear protein phosphorylation in human leukemia K562 cells by okadaic acid, a potent protein phosphatase inhibitor and tumor promoter
J. Biol. Chem.
(1991) - et al.
Okadaic acid overrides the S-phase check point and accelerates progression of G2-phase to induce premature mitosis in HeLa cells
Exp. Cell Res.
(1996) - et al.
The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells
Exp. Cell Res.
(1991) - et al.
The protein phosphatase inhibitors okadaic acid and calyculin A induce apoptosis in human osteoblastic cells
Exp. Cell Res.
(1997) - et al.
Caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis
Exp. Cell Res.
(2001) - et al.
Apoptosis and mitotic arrest are two independent effects of the protein phosphatases inhibitor okadaic acid in K562 leukemia cells
Biochem. Biophys. Res. Commun.
(1999) - et al.
Inhibition of protein phosphatase activity induces p53-dependent apoptosis in the absence of p53 transactivation
J. Biol. Chem.
(1997) - et al.
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
(1951) - et al.
Difficulties and pitfalls in analysis of apoptosis
Methods Cell Biol.
(2001) - et al.
Regulation of protein kinase cascades by protein phosphatase 2A
Trends Biochem. Sci.
(1999)
High molecular weight protein phosphatase type 1 dephosphorylates the retinoblastoma protein
J. Biol. Chem.
Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics
Biochem. J.
Serine/threonine protein phosphatases
Biochem. J.
Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells
EMBO J.
Cell cycle-independent regulation of p21Cip1/Waf1 and retinoblastoma protein during okadaic acid-induced apoptosis is coupled with induction of Bax protein in human breast carcinoma cells
Cell Growth Differ.
Cited by (3)
Morphological and biochemical changes associated with apoptosis induced by okadaic acid in human amniotic FL cells
2009, Environmental ToxicologyPhosphatase-1 and -2A inhibition modulates apoptosis in human osteoarthritis chondrocytes independently of nitric oxide production
2005, Annals of the Rheumatic Diseases
- 1
Present address: Department of Experimental Medicine and Pathology, University of Rome “La Sapienza,” Viale Regina Elena 324, 00161 Rome, Italy.