Effects of alterations in calcium homeostasis on apoptosis during neoplastic progression.

Our previous studies showed that early, stage I preneoplastic cells (sup+ I) are highly susceptible to apoptosis, whereas the later, stage II preneoplastic cells (sup- II) are relatively resistant. To examine possible mechanisms that might explain these differences in the regulation of apoptosis, Ca2+ homeostasis was analyzed and comparisons were made between these two Syrian hamster embryo cell lines. The Ca2+ indicator, fura-2, and fluorescent microscopy were used to measure intracellular free calcium concentrations, [Ca2+]i. The results indicated that the [Ca2+]i level in logarithmically growing sup+ I cells (approximately 100 nM) was considerably lower than that observed in sup- II cells (approximately 260 nM). Serum removal resulted in a reduction of [Ca2+]i in the sup+ I cells (approximately 82 nM), whereas the [Ca2+]i level in sup- II cells did not change. Endoplasmic reticulum (ER) calcium levels were determined by measuring thapsigargin-releasable Ca2+. Reduced ER calcium was consistently observed in cells induced to undergo apoptosis. Specifically, thapsigargin-releasable Ca2+ was greatly reduced in sup+ I cells (45 nM) as compared to sup- II cells (190 nNM) after 4 h in low serum. When sup- II cells were placed under conditions that resulted in apoptosis (thapsigargin or okadaic acid), decreased ER calcium was observed. To determine whether reduced ER calcium had a causative effect in apoptosis, ER calcium levels were exogenously increased in sup+ I cells by raising extracellular Ca2+ to 3 mM; ER calcium levels were maintained, and apoptosis was blocked. Studies were performed to determined whether the decrease in ER calcium could be attributed to reduced Ca2+ influx at the plasma membrane. To measure directly whether Ca2+ entry was decreased in sup+ I cells in 0.2% serum, Mn2+ uptake was used to monitor Ca2+ influx. The data show that in low serum, the rate of thapsigargin-induced Mn2+ entry in sup+ I cells was approximately 50% lower than that of sup- II cells, demonstrating that capacitative entry is reduced in sup+ I cells. In further support of this hypothesis, thapsigargin-treated sup+ I cells (0.2% serum) showed decreased Ca2+ entry upon raising extracellular Ca2+ from 0 to 2 mM. We report the novel finding that early preneoplastic cells, which exhibit a high propensity to undergo apoptosis, have decreased calcium entry at the plasma membrane, resulting in decreased ER calcium pools. This study provides new insight into mechanisms that can be involved in the regulation/dysregulation of apoptosis during neoplastic progression. Furthermore, the data imply that preneoplastic cells, which have developed a mechanism to maintain ER calcium, would be less susceptible to apoptosis and would thus have an increased potential for becoming transformed.