Abstract
Reversible protein phosphorylation regulates the biological activities of many human proteins involved in crucial cellular processes, e.g., protein-protein interactions, cell signaling, gene transcription, cell growth, and death. A malfunction of cellular homeostasis in retinal pigment epithelial (RPE) cells is involved in the age-related retinal degeneration. In this study, we examined cytotoxicity in human RPE cells subjected to the protein phosphatase inhibitor, okadaic acid (OA). Moreover, the influence of Hsp90 inhibitor geldanamycin (GA), a benzoquinone ansamycin, in cytoprotection was assessed. Hsp70 protein levels were analyzed by Western blot. Cellular viability was determined by LDH and MTT assays. To study apoptotic cell death, caspase-3 enzyme activity was measured by assaying the cleavage of a fluorescent peptide substrate and Hoechst dye was used to visualize nuclear morphology. OA treatment caused morphological changes and induced cytotoxicity by caspase-3-independent manner in the RPE cells. No evidence of nuclear fragmentation was observed in response to OA. Interestingly, GA treatment accumulated Hsp70 protein and attenuated OA-induced cytotoxicity. This study suggests that Hsp70 and Hsp90 are closely related to cytoprotection of RPE cells in response to protein phosphatase inhibition.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Benzoquinones
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Caspase 3
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Caspases / drug effects
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Caspases / metabolism
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Cell Death / drug effects
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Cell Death / physiology
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Cell Line
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Cell Nucleus / drug effects
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Cell Nucleus / metabolism
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Cell Survival / drug effects
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Cell Survival / physiology
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Cysteine Proteinase Inhibitors / pharmacology
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Cytoprotection / drug effects
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Cytoprotection / physiology
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Enzyme Activation / drug effects
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Enzyme Activation / physiology
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Enzyme Inhibitors / toxicity
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Epithelial Cells / drug effects*
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Epithelial Cells / metabolism
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HSP70 Heat-Shock Proteins / drug effects*
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HSP70 Heat-Shock Proteins / metabolism
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HSP90 Heat-Shock Proteins / drug effects
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HSP90 Heat-Shock Proteins / metabolism
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Humans
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Lactams, Macrocyclic
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Okadaic Acid / antagonists & inhibitors*
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Okadaic Acid / toxicity
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Phosphoprotein Phosphatases / antagonists & inhibitors*
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Phosphoprotein Phosphatases / metabolism
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Phosphorylation / drug effects
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Pigment Epithelium of Eye / drug effects*
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Pigment Epithelium of Eye / metabolism
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Quinones / pharmacology*
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Retinal Degeneration / enzymology
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Retinal Degeneration / physiopathology
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Up-Regulation / drug effects
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Up-Regulation / physiology
Substances
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Benzoquinones
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Cysteine Proteinase Inhibitors
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Enzyme Inhibitors
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HSP70 Heat-Shock Proteins
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HSP90 Heat-Shock Proteins
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Lactams, Macrocyclic
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Quinones
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Okadaic Acid
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Phosphoprotein Phosphatases
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CASP3 protein, human
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Caspase 3
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Caspases
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geldanamycin