A selective requirement for elevated calcium in DNA degradation, but not early events in anti-Fas-induced apoptosis.
Jurkat cells undergo apoptosis in response to anti-Fas antibody through a caspase-dependent death cascade in which calcium signaling has been implicated. We have now evaluated the role of calcium during this death cascade at the single cell level in real time utilizing flow cytometric analysis and confocal microscopy. Fluo-3 and propidium iodide were employed to evaluate calcium fluxes and to discriminate between viable and non-viable cells, respectively. Anti-Fas treatment of Jurkat cells resulted in a sustained increase in intracellular calcium commencing between 1 and 2 h after treatment and persisting until subsequent loss of cell membrane integrity. The significance of this rise in calcium was evaluated by buffering intracellular calcium with BAPTA and/or removing calcium from the extracellular medium and monitoring the effects of these manipulations on calcium signaling and components of the apoptotic process. Complete inhibition of the anti-Fas induced rise in intracellular calcium required both chelation of [Ca(2+)](i) and removal of extracellular calcium. Interestingly, this condition did not abrogate several events in Fas-induced apoptosis including cell shrinkage, mitochondrial depolarization, annexin binding, caspase activation, and nuclear poly(A)DP-ribose polymerase cleavage. Furthermore, calcium-free conditions in the absence of anti-Fas antibody weakly induced these apoptotic components. In marked contrast, calcium depletion did not induce DNA degradation in control cells, and inhibited apoptotic DNA degradation in response to anti-Fas. These data support the concept that the rise in intracellular calcium is not a necessary component for the early signal transduction pathways in anti-Fas-induced apoptosis in Jurkat cells, but rather is necessary for the final degradation of chromatin via nuclease activation.
Scoltock
A B
AB
Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Bortner
C D
CD
St J Bird
G
G
Putney
J W
JW
Cidlowski
J A
JA
D017209:Apoptosis; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000648:ultrastructure; D048429:Cell Size; D002614:Chelating Agents / Q000494:pharmacology; D002843:Chromatin / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D008563:Membrane Lipids / Q000032:analysis; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000502:physiology; D010718:Phosphatidylserines / Q000494:pharmacology; D015398:Signal Transduction; D019014:fas Receptor / Q000378:metabolism*
D002614:Chelating Agents; D002843:Chromatin; D004273:DNA, Neoplasm; D008563:Membrane Lipids; D010718:Phosphatidylserines; D019014:fas Receptor; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002118:Calcium