Glutathione depletion induces giant DNA and high-molecular-weight DNA fragmentation associated with apoptosis through lipid peroxidation and protein kinase C activation in C6 glioma cells.
Glutathione (GSH) depletion caused by l-buthionine-(S,R)-sulfoximine (BSO) induced apoptosis that was recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endo-labeling (TUNEL), nuclear DNA staining with fluorescence dye, and internucleosomal DNA fragmentation in C6 rat glioma cells. The BSO-induced cell death was associated with caspase-3 activation. Lipid peroxidation and protein kinase C (PK-C) activation were observed during the apoptosis of C6 cells, and these events were inhibited by antioxidants and iron chelators without affecting BSO-induced GSH depletion. Furthermore, approximately 2 Mbp giant DNA fragments were observed in the BSO-treated cells. The giant DNA fragmentation were followed by approximately 30-700 kbp and then less than 100 kbp, including internucleosomal DNA fragmentations. Such serial DNA degradation was prevented by the antioxidants, the iron chelators, and the PK-C inhibitors. These results suggest that during apoptosis induced by GSH-depletion caused by BSO, reactive oxygen species endogenously produced cause lipid peroxidation and that the lipid peroxidation induced PK-C activation, processes which are thought to be involved in the giant DNA, high-molecular-weight DNA, and the internucleosomal DNA fragmentations.
Department of Pharmacology, Kanazawa University School of Medicine, Ishikawa, Kanazawa, 920-8640, Japan.higuchi@kenroku.kanazawa-u.ac.jp
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Higuchi
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Matsukawa
D000818:Animals; D017209:Apoptosis; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D004247:DNA / Q000378:metabolism*; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000172:deficiency* / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D007424:Intracellular Fluid / Q000378:metabolism; D015227:Lipid Peroxidation / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014810:Vitamin E / Q000494:pharmacology
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D014810:Vitamin E; D019328:Buthionine Sulfoximine; D004247:DNA; D011493:Protein Kinase C; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione