We investigated whether altered Ca2+ homeostasis is causally involved in apoptosis. We employed cells that responded differently to apoptotic induction following growth factor removal (i.e. low serum). Early stage preneoplastic, immortal cells (sup+I), show a high susceptibility to induction of apoptosis, whereas late stage preneoplastic sup-II are relatively resistant to apoptosis following serum reduction to 0.2%. To compare differences in ER calcium between cells that undergo apoptosis at a high rate (sup+I) with those with a low rate of apoptosis (sup-II), thapsigargin releasable Ca2+ was measured in the presence of either 10% or 0.2% serum. Sup+I cells had less thapsigargin releasable Ca2+ than sup-II cells in 0.2% serum, consistent with the hypothesis that depletion of ER calcium plays a role in apoptosis. ER calcium depletion was observed consistently with induction of apoptosis, regardless of the agent or cell line used. A direct role for calcium in apoptosis was demonstrated by showing that raising extracellular Ca2+ to 3 mM blocked both the decrease in ER calcium and DNA fragmentation. To measure directly whether Ca2+ entry was decreased in sup+I cells in 0.2% serum, Mn2+ uptake was used to monitor Ca2+ influx. These data show that in 0.2% serum the rate of thapsigargin induced Mn2+ entry in sup+I cells is approximately 50% less than that of sup-II cells, suggesting that capacitative entry is reduced in sup+I cells in low serum and this could be responsible for the observed decrease in ER calcium. In further support of this hypothesis, we find decreased Ca2+ entry in sup+I cells (0.2% serum) following addition of extracellular Ca2+ (2 mM) to thapsigargin treated cells. The data show that the sup+I cells in low serum are deficient in Ca2+ influx that is normally coupled with ER depletion, thus providing insight into the mechanism responsible for decreased ER calcium and apoptosis.
