EFFECTS OF ALTERATIONS IN CALCIUM HOMEOSTASIS ON APOPTOSIS DURING NEOPLASTIC PROGRESSION

Our previous studies showed that early, stage I preneoplastic cells (s up(+)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 regul ation of apoptosis, Ca2+ homeostasis was analyzed and comparisons were made between these two Syrian hamster embryo cell lines, The Ca2+ ind icator, fura-2, and fluorescent microscopy were used to measure intrac ellular free calcium concentrations, [Ca2+](i), The results indicated that the [Ca2+](i) level in logarithmically growing sup(+)I cells (sim ilar to 100 nM) was considerably lower than that observed in sup(-)II cells (similar to 260 nM), Serum removal resulted in a reduction of [C a2+](i) in the sup(+)I cells (similar to 82 nM), whereas the [Ca2+](i) level in sup(-)II cells did not change, Endoplasmic reticulum (ER) ca lcium levels were determined by measuring thapsigargin-releasable Ca2. Reduced ER calcium was consistently observed in cells induced to und ergo apoptosis, Specifically, thapsigargin-releasable Ca2+ was greatly reduced in sup(+)I cells (45 nM) as compared to sup(-)II cells (190 n M) after 4 h in low serum, When sup(-)II cells were placed under condi tions that resulted in apoptosis (thapsigargin or okadaic acid), decre ased ER calcium was observed, To determine whether reduced ER calcium had a causative effect in apoptosis, ER calcium levels were exogenousl y increased in sup(+)I cells by raising extracellular Ca2+ to 3 mM; ER calcium levels were maintained, and apoptosis was blocked, Studies we re 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% ser um, 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 cel ls was approximately 50% lower than that of sup(-)II cells, demonstrat ing that capacitative entry is reduced in sup(+)I cells, In further su pport of this hypothesis, thapsigargin-treated sup(+)I cells (0.2% ser um) 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 c alcium entry at the plasma membrane, resulting in decreased ER calcium pools, This study provides new insight into mechanisms that can be in volved in the regulation/dysregulation of apoptosis during neoplastic progression, Furthermore, the data imply that preneoplastic cells, whi ch have developed a mechanism to maintain ER calcium, would be less su sceptible to apoptosis and would thus have an increased potential for becoming transformed.