A supramicromolar elevation of intracellular free calcium ([Ca2+]i) is consistently required to induce the execution phase of apoptosis

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Abstract
Many agents, such as the endoplasmic reticulum Ca2+ ATPase inhibitor, thapsigargin, or the ionophore, ionomycin, induce apoptosis by transiently elevating [Ca2+]i. The role of [Ca2+]i in apoptosis induced by agents that do not immediately increase [Ca2+]i, such as 5-FdUr, TGFβ-1, doxorubicin, or radiation, is far more controversial. In the present paper, [Ca2+]i was measured continuously for 120 h. in prostate and bladder cancer cell lines exposed to these four agents: 5-FdUR, TGFβ-1, doxorubicin, or radiation. Each of them consistently induced a delayed [Ca2+]i rise associated with the morphological changes that characterize the execution phase of apoptosis (i.e. rounding, blebbing). This [Ca2+]i rise occurred in two consecutive steps (10 μM and >10 μM) and resulted from a Ca2+ influx from the extracellular medium. This delayed supramicromolar [Ca2+]i rise was also observed previously in breast, prostate and bladder cancer cell lines exposed to thapsigargin. This influx regulated transcriptional reprogramming of Gadd153 and is required to activate cytochrome c release, caspase-3 activation, loss of clonal survival and DNA fragmentation. When cells were maintained in low extracellular Ca2+ media, these phenomena were temporarily delayed but occurred on return to normal Ca2+ medium. Similarly, apoptosis could be delayed by overexpressing the Ca2+-binding proteins, Calbindin-D28K and parvalbumin. As this delayed 10 μM [Ca2+]i elevation was observed in a number of cell lines exposed to a variety of different agents, we conclude that such elevation constitutes a key and general event of apoptosis in these malignant cells.