INDUCTION OF A CASPASE-3-LIKE ACTIVITY BY CALCIUM IN NORMAL CYTOSOLICEXTRACTS TRIGGERS NUCLEAR APOPTOSIS IN A CELL-FREE SYSTEM

Calcium is involved in several steps of the apoptotic process. In nucl ei, endonucleases are presumed to be the main targets of calcium; howe ver, little is known about its role during the cytosolic phase of apop tosis. We used a cell-free system to address this question. Our result s show that CaCl2 triggered nuclear apoptosis (i.e. typical morphologi cal change and DNA fragmentation) at concentrations of 5 mM. This conc entration was lowered 10-fold by the co-incubation with cytosolic extr acts from nonapoptotic cells. Apoptotic changes induced by the incubat ion of nuclei with CaCl2 in the presence of these cytosols were strong ly reduced in the presence of an inhibitor of caspase-3 and to a lesse r extent by an inhibitor of caspase-1. We also show that calcium-induc ed apoptosis is affected by protease inhibitors such as N-tosyl-L-phen ylalanine chloromethyl ketone, but not by calpain or several lysosomal protease inhibitors. The addition of CaCl2 to the cell-free system in creased a caspase-3 activity in nonapoptotic cytosols as shown by spec ific antibodies and an enzymatic assay. No activation of a caspase-3-l ike activity by the addition of cytochrome c was observed in these ext racts under similar conditions. The enhanced caspase-3 activity induce d by calcium was inhibited by protease inhibitors affecting morphologi cal nuclear apoptosis except for those responsible for the degradation of lamin A. These results suggest that CaCl2 could trigger, in normal cells, an apoptotic cascade through the activation of cytosolic caspa se-3 activity.