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.