The overall goal of this project is to investigate the signaling pathways, particularly ion signaling pathways, that regulate apoptosis. We studied cells that respond differently to apoptotic induction following growth factor removal (i.e. low serum). Early stage preneoplastic, immortal cells (sup+), show a high susceptibility to induction of apoptosis, whereas late stage preneoplastic cells (sup-) are relatively resistant to apoptosis following serum reduction to 0.2%. We investigated whether altered Ca2+ homeostasis is causally involved in apoptosis. We showed that a sustained increase in cytosolic free Ca2+ (Cai) does not precede apoptosis. Differences in endoplasmic reticulum (ER) calcium between sup+ cells and sup- were determined by measuring thapsigargin releasable Ca2+ in the presence of 10% and 0.2% serum. Sup+ cells in low serum exhibit decreased endoplasmic reticulum calcium levels and subsequent DNA laddering, indicative of apoptosis. The decrease in endoplasmic reticulum calcium in these cells appears to be due, at least in part, to reduced capacitative calcium entry at the plasma membrane, and treatment with either SKF96365 (30-100 uM), an inhibitor of capacitative calcium entry, or cell-impermeant BAPTA (10 mM), to reduce extracellular calcium, is able to induce apoptosis of sup+ cells in 10% serum, a condition where apoptosis does not normally occur. Because previous work has implicated vesicular trafficking as a mechanism of regulating capacitative calcium entry, we investigated whether disruption of vesicular trafficking could lead to decreased capacitative calcium entry and subsequent apoptosis in sup+ cells. Coincident with low serum-induced apoptosis, we observed an accumulation of vesicles within the cell, suggesting deregulated vesicle trafficking. Treatment of sup+ cells with bafilomycin, an inhibitor of the endosomal proton ATPase, produced an accumulation of vesicles within cells without measurably changing cytoplasmic pH. Treatment with 50 nM bafilomycin decreased capacitative entry by ~ 30% and induced apoptosis in sup+ cells in 10% serum. These data suggest that deregulation of vesicular transport results in reduced capacitative calcium entry which in turn results in apoptosis. We are also investigating signals that are downstream of decreased ER calcium (ceramide and NF-kB). A 60-70% increase in ceramide is observed at 16 hours after serum reduction in sup+ but not in sup- cells. No change in ceramide is observed after 4 hours of serum reduction, a time in which a decrease in ER calcium is noted. Thus the change in ceramide occurs after the decrease in ER calcium. Depletion of ER calcium with thapsigargin also caused an increase in ceramide in both sup+ and sup- cells. Furthermore if we blocked the decrease in ER calcium in low serum, by raising extracellular calcium we blocked the rise in ceramide. Thus alterations in ER calcium per se can affect generation of ceramide. Also there are reports that a decrease in ER calcium can lead to activation of NF-kB. We therefore measured NF-kB, but we found that sup+ cells showed little activation of NF-kB in either 10% or 0.2% serum. Thus, in spite of the decreased ER Ca2+ in sup+ cells in low serum we did not observe an elevation of NF-kB. In contrast, sup- cells showed considerable NF-kB activation in 10% and 0.2% serum (~5-10x higher than sup+). The increase in basal NF-kB in sup- compared to sup+ cells could be related to the increase in basal Cai in sup- cells, consistent with reports that calcium can enhance degradation of I-kB and thus activate NF-kB. To test this hypothesis we lowered basal calcium in sup- cells in 10% serum by addition of the intracellular calcium chelator BAPTA-AM. We confirmed using fura-2 and fluorescent microscopy that BAPTA lowered basal calcium by 31%; this reduction in calcium was accompanied by a 36% reduction in NF-kB activation. - Calcium, Apoptosis, Endoplasmic reticulum, Fluorescent microscopy, pH
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