Induction of a caspase-3-like activity by calcium in normal cytosolic extracts triggers nuclear apoptosis in a cell-free system.
Calcium is involved in several steps of the apoptotic process. In nuclei, endonucleases are presumed to be the main targets of calcium; however, little is known about its role during the cytosolic phase of apoptosis. We used a cell-free system to address this question. Our results show that CaCl2 triggered nuclear apoptosis (i.e. typical morphological change and DNA fragmentation) at concentrations of 5 mM. This concentration was lowered 10-fold by the co-incubation with cytosolic extracts from nonapoptotic cells. Apoptotic changes induced by the incubation of nuclei with CaCl2 in the presence of these cytosols were strongly reduced in the presence of an inhibitor of caspase-3 and to a lesser extent by an inhibitor of caspase-1. We also show that calcium-induced apoptosis is affected by protease inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone, but not by calpain or several lysosomal protease inhibitors. The addition of CaCl2 to the cell-free system increased a caspase-3 activity in nonapoptotic cytosols as shown by specific antibodies and an enzymatic assay. No activation of a caspase-3-like activity by the addition of cytochrome c was observed in these extracts under similar conditions. The enhanced caspase-3 activity induced by calcium was inhibited by protease inhibitors affecting morphological 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 caspase-3 activity.
Juin
P
P
Unité INSERM 419, Institut de Biologie, 9 Quai Moncousu 44035, Nantes Cedex 01, France.
Pelletier
M
M
Oliver
L
L
Tremblais
K
K
Grégoire
M
M
Meflah
K
K
Vallette
F M
FM
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D002122:Calcium Chloride / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases; D002457:Cell Extracts; D002467:Cell Nucleus / Q000187:drug effects* / Q000201:enzymology; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000096:biosynthesis*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects* / Q000201:enzymology; D004790:Enzyme Induction; D005609:Free Radicals; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats
D002457:Cell Extracts; D015853:Cysteine Proteinase Inhibitors; D005609:Free Radicals; D010447:Peptide Hydrolases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; C108711:DEVDase; D002122:Calcium Chloride