ABSTRACT
OBJECT: Okadaic acid (OA), a potent protein phosphatase inhibitor, has been known to induce apoptosis in a variety of cell types. The authors attempted to characterize further this model by identifying proteins involved in this form of programmed cell death. METHODS: Cellular proliferation was assessed using a colorimetric nonradioactive proliferation assay and cell counts. Apoptosis was determined by fluorescent microscopy. Activation of the mitogen-activated protein kinase (MAPK) pathways was determined by immunoprecipitation of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38 followed by in vitro kinase assays. Western blot analyses were conducted to show inhibitory-kappaB (IkappaB) phosphorylation and degradation as well as Bax upregulation. The binding of nuclear factor-kappaB (NFkappaB) was shown by electrophoretic mobility shift assay. Okadaic acid induced cell death in T98G human malignant cell lines (50% inhibiting concentration = 20-25 nM). In T98G cells YO-PRO fluorescent staining was identified, thus indicating an apoptotic mechanism with a smaller percentage of cells undergoing necrotic cell death. Additionally OA induced JNK and MAPK activities in a time-dependent manner, increased the expression of Bax, and increased IkappaB phosphorylation and NFkappaB activation. There was a temporal correlation between these subcellular events and the detection of apoptosis morphology in glioma cells. CONCLUSIONS: The authors believe that OA acts by blocking dephosphorylation events, thus activating apoptotic pathways through ERK and JNK activity. Additionally Bax, IkappaB and NFkappaB may also play a role in regulating these pathways.
