A Selective Requirement for Elevated Calcium in DNA Degradation, but Not Early Events in Anti-Fas-induced Apoptosis

Alyson B Scoltock,Carl D Bortner,Gary St J Bird,James W Putney,John A Cidlowski

doi:10.1074/jbc.m004058200

Abstract

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.

Highlights

Tion pathways that eventually lead to a central cascade of effectors that carry out the final stages of cell death the precise signaling pathways that regulate apoptosis are not yet fully understood
They showed that T-lymphocytes with a mutated inositol 1,4,5-trisphosphate receptor, which are incapable of mobilizing intracellular calcium, were resistant to Fas-mediated apoptosis when DNA degradation was used as the sole criterion for apoptosis
Anti-Fas Antibody Promotes Calcium Mobilization in Jurkat Cells—We initially examined Jurkat cells treated in the presence or absence of anti-Fas for changes in their intracellular calcium concentration

Summary

Introduction

Tion pathways that eventually lead to a central cascade of effectors that carry out the final stages of cell death the precise signaling pathways that regulate apoptosis are not yet fully understood. Recent studies from several laboratories have indicated that the Fas-induced death pathway may effectively activate lymphocyte apoptosis via mechanisms dependent on ionic changes in the cell, such as those involving cell volume regulation [2] or calcium mobilization [3,4,5,6,7]. The precise role of calcium in the cell death process remains controversial with some studies suggesting that it is essential [3, 5,6,7] whereas others suggest that it is not [11, 12] These differences may reflect differences in the model systems under investigation or perhaps the end points used to evaluate apoptosis. Perhaps the best evidence for the involvement of calcium in Fas-mediated apoptosis in T-lymphocytes comes from the work of Jayaraman and Marks [18]. Where and how calcium is involved in lymphocyte apoptosis remains unclear

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Conclusion