Okadaic-acid-induced apoptosis in malignant glioma cells.
OBJECTIVE
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.
Rami
Bimal G
BG
Department of Neurosurgery and Greenbaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA.
Chin
Lawrence S
LS
Lazio
Barbara E
BE
Singh
Satyendra K
SK
D021581:Active Transport, Cell Nucleus; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000473:pathology*; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects / Q000473:pathology; D024202:Electrophoretic Mobility Shift Assay; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005910:Glioma / Q000473:pathology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D020672:I-kappa B Proteins; D016328:NF-kappa B; D009363:Neoplasm Proteins; D051028:bcl-2-Associated X Protein; D019319:Okadaic Acid; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases