Abstract
Kainic acid (KA)-induced neuronal damage and the protective effects of flavopiridol were studied in primary cultures of rat cerebellar granule cells (CGNs). When neurons were treated with 500 μM KA, the percentage of cells with condensed nuclei measured by nuclear counting increased by up to 55%. After flavopiridol treatment, an antitumoral drug that is a broad inhibitor of cyclin-dependent kinases, the percentage of condensed nuclei decreased by up to 26%. Furthermore, this KA-mediated cell death was only partially dependent on the activation of the initiator caspase-9 and the effector caspases-3 and -6. This argues for a minor role of caspases in the intracellular pathway leading to KA-induced programmed cell death in CGNs. We examined the possible implication of cell cycle proteins in KA-induced neurotoxicity. We found an increase in the expression of proliferating cell nuclear antigen and E2F-1, two proteins implicated in S-phase, by Western blot. KA increased bromodeoxyuridine incorporation in CGNs, a marker of cell proliferation, and flavopiridol attenuated this effect. These results indicated that flavopiridol decreased the expression of cell cycle markers in CGNs after KA treatment. Flavopiridol might thus be used as a preventive agent against neurodegenerative diseases associated with cell cycle activation.
Footnotes
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This study was supported by SAF2002-00790, FISS G03/137, FISS G03/167, and Collegi de Farmacèutics de Barcelona (2002-2003). E.V. and E.G.J. are recipients of a fellowship from the University of Barcelona.
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DOI: 10.1124/jpet.103.057497.
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ABBREVIATIONS: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ANOVA, analysis of variance; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone; BdrU, bromodeoxyuridine; BSA, bovine serum albumin; Ac-DEVD-pNA, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide; Ac-LEHD-pNA, N-acetyl-Leu-Glu-His-Asp-p-nitroanilide; Cdk, cyclin-dependent kinase; CGN, cerebellar granule neuron; KA, kainic acid; LDH, lactate dehydrogenase; LH-BSA, Locke-HEPES buffer; PBS, phosphate-buffered saline; PCNA, proliferating cellular nuclear antigen; PI, propidium iodide; pRb, retinoblastoma protein; TBS-T, Tris-buffered saline/Tween 20; GYKI 52466, 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride.
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↵1 These two authors contributed equally to this work.
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↵2 Senior coauthors.
- Received July 24, 2003.
- Accepted October 2, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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