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

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.

Characterization of Fortilin, a Novel Antiapoptotic Protein

“…In addition, thapsigargin, a Ca 2ϩ -ATPase inhibitor that increases intracellular calcium by inhibiting the uptake of calcium into the endoplasmic reticulum, also induces apoptosis in certain cell types (56). Furthermore, the rise in intracellular calcium has been shown to be necessary for DNA degradation in anti-Fas-induced apoptosis of Jurkat cells (57). On the other hand, antiapoptotic proteins have been shown to modulate intracellular calcium levels.…”

Role of Ca(2+) in diallyl disulfide-induced apoptotic cell death of HCT-15 cells

“…Indeed, the elevation of cytosolic Ca 2+ by exposure to Ca 2+ ionophores or thapsigargin is sufficient to induce apoptosis in many different cell types (Takadera and Ohyashiki, 1997;Nakamura et al, 2000), and agents that suppress Ca 2+ influx or buffer intracellular Ca 2+ can prevent apoptosis in several different systems (Scoltock et al, 2000;Shen et al, 2001;Zhang et al, 2001). However, despite the accumulation of data, the link between intracellular Ca 2+ homeostasis and the activation of the apoptotic program remains unknown.…”

An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: Antagonism by physiological concentrations of K+ ions

“…An increase of Ca 2+ is observed when glucocorticoids induce apoptosis in lymphocytes [5,9] and primary thymocytes [10]. Treatment of cells in vitro with multiple apoptotic agents also induces an increase in intracellular Ca 2+ (ionomycin, A23187 or thapsigargin) resulting in apoptosis morphologically and biochemically similar to glucocorticoid treatment of lymphatic cells [5,9,11].…”

“…Treatment of cells in vitro with multiple apoptotic agents also induces an increase in intracellular Ca 2+ (ionomycin, A23187 or thapsigargin) resulting in apoptosis morphologically and biochemically similar to glucocorticoid treatment of lymphatic cells [5,9,11]. This phenomenon is thought to result from the apoptotic stimulus inducing Ca 2+ influx into the cytoplasm from the extra-cellular environment, as well as release of Ca 2+ from the endoplasmic reticulum pools.…”

“…Alterations in cellular Ca 2+ also occur during cell growth, differentiation, survival and apoptosis [3]. The regulation of cellular Ca 2+ concentration is strongly associated with apoptotic states and the initiation of apoptosis [4,5]. During the early phase of apoptosis, the Ca 2+ concentration increases in the cytoplasm in various apoptotic model systems, reaching a level much higher than in normal cells [6].…”