Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells.
Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.
Ray
S K
SK
Department of Neurology, Medical University of South Carolina (MUSC), 600 MUSC Complex, Charleston, SC 29425, USA.
Wilford
G G
GG
Crosby
C V
CV
Hogan
E L
EL
Banik
N L
NL
D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D000001:Calcimycin / Q000627:therapeutic use*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005910:Glioma / Q000188:drug therapy* / Q000378:metabolism; D017665:Hydroxyl Radical; D007371:Interferon-gamma / Q000627:therapeutic use*; D007476:Ionophores / Q000627:therapeutic use*; D051381:Rats; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D007476:Ionophores; C071482:calpeptin; D017665:Hydroxyl Radical; D000001:Calcimycin; D007371:Interferon-gamma; D002154:Calpain