When grown for seven days in a medium containing nerve growth factor (
100 ng/ml), 10% horse serum and 5% fetal bovine serum PC12 cells stopp
ed dividing, extended neurites and assumed a neuronal phenotype. Withd
rawal of nerve growth factor from these cells resulted in loss of neur
ites and apoptotic changes in many cells. The apoptotic changes were e
xacerbated if the cells were also exposed to 1-2 mu M S-100, a calcium
binding protein purified from bovine brain. After exposure to S-100,
the PC12 cells underwent characteristic apoptotic changes. Within 2 h
neurites retracted, the cell body shrunk and submembranous accumulatio
n of condensed cytoplasmic material was observed. DNA ladders were pre
sent after 24-48 h and 60% of the cells became hypodiploid after 72 h.
S-100 induced apoptosis by binding to specific sites (K-d=189 nM) on
PC12 cells and this caused a rise in [Ca2+](i) due to a transmembrane
capacitative flux followed by the depletion of internal stores. This i
ncrease was reversed if 5 mu M nifedipine, a specific L-type Ca2+ chan
nel inhibitor, was added to the medium after S-100 and completely abol
ished if the cells were pretreated with 5 mM thapsigargin, an inhibito
r of endoplasmic reticulum Ca2+-ATPase. The presence of nerve growth f
actor in the culture medium completely blocked the apoptotic changes i
nduced by S-100, probably due to interaction of nerve growth factor an
d S-100 at the same binding sites. These data indicate that nerve grow
th factor not only prevents apoptosis during cell development, but als
o apoptosis induced by endogenous substances such as S-100. Copyright
1996 IBRO.