Abstract
Persistent cell shrinkage is a major hallmark of apoptotic cell death. The early-phase shrinkage, which starts within 30−120 min after apoptotic stimulation and is called apoptotic volume decrease (AVD), is known to be accomplished by activation of K+ channels and volume-sensitive outwardly rectifying (VSOR) Cl− channels in a manner independent of caspase-3 activation. However, it is controversial whether AVD depends on apoptotic dysfunction of mitochondria and activation of initiator caspases. Here, we observed that AVD is induced not only by a mitochondrial apoptosis inducer, staurosporine (STS), in mouse B lymphoma WEHI-231 cells, but also by ligation of the death receptor Fas in human B lymphoblastoid SKW6.4 cells, which undergo Fas-mediated apoptosis without involving mitochondria. Overexpression of Bcl-2 failed to inhibit the STS-induced AVD in WEHI-231 cells. These results indicate that AVD does not require the mitochondrial pathway of apoptosis. In human epithelial HeLa cells stimulated with anti-Fas antibody or STS, the AVD induction was found to precede activation of caspase-8 and caspase-9 and to be resistant to pan-caspase blockers. Thus, it is concluded that the AVD induction is an early event independent of the mitochondrial apoptotic signaling pathway and initiator caspase activation.
Highlights
A major hallmark of apoptotic cell death is normotonic persistent shrinkage of cells [1]
When a Clг channel blocker, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or 4-acetamido-4'-isothiocyanostilbene (SITS), was simultaneously applied with anti-Fas antibody, apoptotic volume decrease (AVD), sustained cell shrinkage, caspase-3 activation and cell death were all completely prevented (Figure 1). These results suggest that the mechanism of AVD induction is independent of the mitochondrial pathway and that actions of the Clг channel blocker were not mediated by the mitochondrial voltage-dependent anion channel (VDAC)
The AVD induction was observed by Fas-ligation in type-I SKW6.4 cells, which undergo Fas-mediated apoptosis without involving mitochondria
Summary
A major hallmark of apoptotic cell death is normotonic persistent shrinkage of cells [1]. Late-phase apoptotic cell shrinkage observed at around 1 to 12 h after apoptotic stimulation was reported to be preceded by alterations in mitochondrial functions, including changes in the mitochondrial membrane potential and generation of mitochondrial ROS [11,12]. It is not known whether the early-phase apoptotic shrinkage, that is AVD, is dependent on the mitochondrial apoptotic signaling pathway. The first aim of the present study is to answer this question
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