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 c aspase-dependent death cascade in which calcium signaling has been implicat ed. 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 c onfocal microscopy. Fluo-3 and propidium iodide were employed to evaluate c alcium fluxes and to discriminate between viable and non-viable cells, resp ectively. Anti-Fas treatment of Jurkat cells resulted in a sustained increa se in intracellular calcium commencing between 1 and 2 h after treatment an d persisting until subsequent loss of cell membrane integrity. The signific ance of this rise in calcium was evaluated by buffering intracellular calci um with BAPTA and/or removing calcium from the extracellular medium and mon itoring the effects of these manipulations on calcium signaling and compone nts of the apoptotic process. Complete inhibition of the anti-Fas induced r ise in intracellular calcium required both chelation of [Ca2+](i) and remov al of extracellular calcium. Interestingly, this condition did not abrogate several events in Fas-induced apoptosis including cell shrinkage, mitochon drial depolarization, annexin binding, caspase activation, and nuclear poly (A)DP-ribose polymerase cleavage. Furthermore, calcium-free conditions in t he absence of anti-Fas antibody weakly induced these apoptotic components. In marked contrast, calcium depletion did not induce DNA degradation in con trol 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 ant i-Fas-induced apoptosis in Jurkat cells, but rather is necessary for the fi nal degradation of chromatin via nuclease activation.