Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptos is of immune cells. Since central nervous system (CNS) is abundant in calpa in, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells expos ed to reactive hydroxyl radical (. OH) [formed via the Fenton reaction (Fe2 + + H2O2 + H+ --> Fe3++ H2O + . OH)], interferon-gamma (IFN-gamma), and cal cium ionophore (A23187). Cell death, cell cycle, calpain expression, and ca lpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) a nd biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-E nd Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 ce lls at the G(2)/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) expr ession, but increased calpain expression, and the upregulated bar (pro-apop totic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in e xpression of calpastatin (endogenous calpain inhibitor). Western blot analy sis showed an increase in calpain content and degradation of myelin-associa ted glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpressi on, MAG degradation, and DNA fragmentation. We conclude that calpain overex pression due to . OH stress, IFN-gamma stimulation, or Ca2+ influx is invol ved in C6 cell death, which is attenuated by a calpain-specific inhibitor. (C) 1999 Elsevier Science B.V. All rights reserved.