Plagiochin E,an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway

Background

Plagiochin E (PLE) is an antifungal active macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. To elucidate the mechanism of action, previous studies revealed that the antifungal effect of PLE was associated with the accumulation of ROS, an important regulator of apoptosis in Candida albicans. The present study was designed to find whether PLE caused apoptosis in C. albicans.

Methods

We assayed the cell cycle by flow cytometry using PI staining, observed the ultrastructure by transmission electron microscopy, studied the nuclear fragmentation by DAPI staining, and investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V staining. The effect of PLE on expression of CDC28, CLB2, and CLB4 was determined by RT-PCR. Besides, the activity of metacaspase was detected by FITC-VAD-FMK staining, and the release of cytochrome c from mitochondria was also determined. Furthermore, the effect of antioxidant L-cysteine on PLE-induced apoptosis in C. albicans was also investigated.

Results

Cells treated with PLE showed typical markers of apoptosis: G₂/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. The expression of CDC28, CLB2, and CLB4 was down-regulated by PLE, which may contribute to PLE-induced G₂/M cell cycle arrest. Besides, PLE promoted the cytochrome c release and activated the metacaspase, which resulted in the yeast apoptosis. The addition of L-cysteine prevented PLE-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, indicating the ROS was an important mediator of PLE-induced apoptosis.

Conclusions

PLE induced apoptosis in C. albicans through a metacaspase-dependent apoptotic pathway.

General significance

In this study, we reported for the first time that PLE induced apoptosis in C. albicans through activating the metacaspase. These results would conduce to elucidate its underlying antifungal mechanism.     

持续性细胞皱缩在人上皮细胞凋亡过程中的必要性

持续性细胞皱缩是凋亡发生的一个主要标志。近期研究发现细胞皱缩在细胞凋亡过程中并不是一个被动的次要事件。在各种细胞中,包括人上皮细胞,凋亡因子（apoptogen）刺激后马上发生全细胞皱缩,又称为凋亡性容积减小（apoptotic　volumede　crease,AVD）,继而发生caspase激活、DNA片段化、细胞破裂死亡。K^＋和Cl^-通道的激活导致KCl外流,诱导AVD发生。抑制AVD发生可以抑制细胞凋亡。AVD与调节性容积增加（regulatory　volume　increase,RVI）异常相伴发生时,人上皮性HeLa细胞发生持续性细胞皱缩。RVI功能受损时,高渗本身就能诱导HeLa细胞持续性细胞皱缩,继而凋亡。即使在正常渗透压、无凋亡因子刺激的情况下,将HeLa细胞置于缺乏Na^＋或Cl。的溶液也会导致细胞持续性皱缩,继而凋亡。因此,AVD诱导和RVI异常所导致的持续性细胞皱缩是人上皮细胞发生凋亡的首要条件。     

Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation

Cell shrinkage is an incipienthallmark of apoptosis and is accompanied by potassium releasethat decreases the concentration of intracellular potassium andregulates apoptotic progression. The plasma membrane K⁺channel recruited during apoptosis has not been characterized despite its importance as a potential therapeutic target. Here weprovide evidence that two-pore domain K⁺ (K_2P)channels underlie K⁺ efflux during apoptotic volumedecreases (AVD) in mouse embryos. These K_2P channels areinhibited by quinine but are not blocked by an array of pharmacologicalagents that antagonize other K⁺ channels. TheK_2P channels are uniquely suited to participate in theearly phases of apoptosis because they are not modulated bycommon intracellular messengers such as calcium, ATP, and arachidonic acid, transmembrane voltage, or the cytoskeleton. A K⁺channel with similar biophysical properties coordinates regulatory volume decreases (RVD) triggered by changing osmotic conditions. Wepropose that K_2P channels are the pathway by whichK⁺ effluxes during AVD and RVD and that apoptosisco-opts mechanisms more routinely employed for homeostatic cell volume regulation.

     

The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans

We show that the antifungal plant defensin Raphanus sativus antifungal protein 2 (RsAFP2) from radish induces apoptosis and concomitantly triggers activation of caspases or caspase-like proteases in the human pathogen Candida albicans. Furthermore, we demonstrate that deletion of C. albicans metacaspase 1, encoding the only reported (putative) caspase in C. albicans, significantly affects caspase activation by the apoptotic stimulus acetic acid, but not by RsAFP2. To our knowledge, this is the first report on the induction of apoptosis with concomitant caspase activation by a defensin in this pathogen. Moreover, our data point to the existence of at least two different types of caspases or caspase-like proteases in C. albicans.     

Apoptosis, cell volume regulation and volume-regulatory chloride channels

Apoptosis occurs in response to various stimuli under physiological and pathological circumstances. A major hallmark of the programmed cell death is normotonic shrinkage of cells. Induction of the apoptotic volume decrease (AVD) was found to precede cytochrome c release, caspase-3 activation and DNA laddering. A broad-spectrum caspase inhibitor blocked these biochemical apoptotic events but failed to block the AVD. The normotonic AVD induction was coupled to facilitation of the regulatory volume decrease (RVD), which is attained by parallel operation of Cl- and K+ channels, under hypotonic conditions. Both the AVD induction and RVD facilitation were prevented by application of a blocker of volume-regulatory Cl- or K+ channels. Furthermore, apoptotic cell death was rescued by channel blocker-induced prevention of AVD. Thus, it is concluded that the AVD is produced under normotonic conditions by a mechanism similar, though without preceding swelling, to RVD and represents an early prerequisite to apoptotic events leading to cell death. It was previously reported that hypertonic stress triggers apoptosis in cell types that lack the regulatory volume increase (RVI) mechanism. Taken together, it is suggested that 'disordered' or altered cell volume regulation is associated with apoptosis.     

Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl⁻ transport inhibitor DIDS and the K⁺ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl⁻ flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl⁻ fluxes rather than blocking cell shrinkage or K⁺ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.     

Stimulation of Kv1.3 potassium channels by death receptors during apoptosis in Jurkat T lymphocytes

The loss of intracellular potassium is a pivotal step in the induction of apoptosis but the mechanisms underlying this response are poorly understood. Here we report caspase-dependent stimulation of potassium channels by the Fas receptor in a human Jurkat T cell line. Receptor activation with Fas ligand for 30 min increased the amplitude of voltage-activated potassium currents 2-fold on average. This produces a sustained outward current, approximately 10 pA, at physiological membrane potentials during Fas ligand-induced apoptosis. Both basal and Fas ligand-induced currents were blocked completely by toxins that selectively inhibit Kv1.3 potassium channels. Kv1.3 stimulation required the expression of Fas-associated death domain protein and activation of caspase 8, but did not require activation of caspase 3 or protein synthesis. Furthermore, Kv1.3 stimulation by Fas ligand was prevented by chronic stimulation of protein kinase C with 20 nm phorbol 12-myristate 13-acetate during Fas ligand treatment, which also blocks apoptosis. Thus, Fas ligand increases Kv1.3 channel activity through the same canonical apoptotic signaling cascade that is required for potassium efflux, cell shrinkage, and apoptosis.     

钾通道在培养大鼠海马神经元凋亡性容积减少中的作用

为探讨钾通道参与神经元凋亡的可能机制,在星形孢菌素（STS)诱导的培养海马神经元凋亡模型上,研究了凋亡时神经细胞容积的动态变化及钾通道在其中的作用.实验结果显示,钾通道阻断剂四乙铵或升高细胞外K⁺均能够明显抑制STS诱导的神经元凋亡,并且大电导钙激活钾通道（BK）选择性阻断剂iberiotoxin和paxilline具有同样程度的抗细胞凋亡作用,表明钾通道（可能主要是BK通道）参与了STS诱导的培养海马神经元凋亡.在STS诱导神经元凋亡的早期就出现了细胞容积的显著减少,而钾通道阻断剂或升高细胞外K⁺均可阻断该细胞容积减少.研究结果提示细胞内钾离子的外流可能参与了凋亡性细胞容积减少,这也可能是钾通道介导细胞凋亡的重要机制之一.     

Cell shrinkage and monovalent cation fluxes: role in apoptosis

The loss of cell volume or cell shrinkage has been a morphological hallmark of the programmed cell death process known as apoptosis. This isotonic loss of cell volume has recently been term apoptotic volume decrease or AVD to distinguish it from inherent volume regulatory responses that occurs in cells under anisotonic conditions. Recent studies examining the intracellular signaling pathways that result in this unique cellular characteristic have determined that a fundamental movement of ions, particularly monovalent ions, underlie the AVD process and plays an important role on controlling the cell death process. An efflux of intracellular potassium was shown to be a critical aspect of the AVD process, as preventing this ion loss could protect cells from apoptosis. However, potassium plays a complex role as a loss of intracellular potassium has also been shown to be beneficial to the health of the cell. Additionally, the mechanisms that a cell employs to achieve this loss of intracellular potassium vary depending on the cell type and stimulus used to induce apoptosis, suggesting multiple ways exist to accomplish the same goal of AVD. Additionally, sodium and chloride have been shown to play a vital role during cell death in both the signaling and control of AVD in various apoptotic model systems. This review examines the relationship between this morphological change and intracellular monovalent ions during apoptosis.     

Role of TASK2 in the control of apoptotic volume decrease in proximal kidney cells

Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor alpha. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-alpha-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production.     

(+)-Medioresinol leads to intracellular ROS accumulation and mitochondria-mediated apoptotic cell death in Candida albicans

The phytochemical (+)-Medioresinol, a furofuran type lignan identification and isolation on the stem bark of Sambucus williamsii, which is a folk medicinal plant used in traditional medicine. (+)-Medioresinol is known to possess a lesishmanicidal activity and cardiovascular disease risk reduction but its antifungal effects have not yet been identified. In this study, to confirm (+)-Medioresinol's antifungal properties and mode of action, we observed morphological and physiological change in Candida albicans. In cells exposed to (+)-Medioresinol, arrested the cell cycle and intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased. The increase of ROS induced oxidative stress and the mitochondria dysfunction which causes release of pro-apoptotic factors. We investigated a series of characteristic cellular changes of apoptosis by using various apoptosis detection methods. We report here for the first time that (+)-Medioresinol has effects on mitochondria and induced the accumulation of ROS in C. albicans cells. We demonstrated that one of the important features of apoptosis, mitochondrial membrane depolarization is caused by ROS. Substantially, we investigated the release of cytochrome c, which is one of the factors of metacaspase activity. We also show that the effects of (+)-Medioresinol are mediated at an early stage in apoptosis acting on the plasma membrane phosphatidylserine externalization. In addition, (+)-Medioresinol induced apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC). In late stage of confirmation of diagnostic markers in yeast apoptosis include the effects of nucleus morphological change, DNA fragmentation and condensation by influence of oxidative stress. These apoptotic phenomena represent that oxidative stress and mitochondria dysfunctions by inducing the phytochemical (+)-Medioresinol must be an important factors of the apoptotic process in C. albicans. These results support the elucidation of the underlying antifungal mechanisms of (+)-Medioresinol.     

Staurosporine-induced apoptotic water loss is cell- and attachment-specific

Apoptotic volume decrease (AVD) is a characteristic cell shrinkage observed during apoptosis. There are at least two known processes that may result in the AVD: exit of intracellular water and splitting of cells into smaller fragments. Although AVD has traditionally been attributed to water loss, direct evidence for that is often lacking. In this study, we quantified intracellular water in staurosporine-treated cells using a previously described optical microscopic technique that combines volume measurements with quantitative phase analysis. Water loss was observed in detached HeLa and in adherent MDCK but not in adherent HeLa cells. At the same time, adherent HeLa and adherent MDCK cells exhibited visually similar apoptotic morphology, including fragmentation and activation of caspase-3. Morphological changes and caspase activation were prevented by chloride channel blockers DIDS and NPPB in both adherent and suspended HeLa cells, while potassium channel blocker TEA was ineffective. We conclude that staurosporine-induced dehydration is not a universal cell response but depends on the cell type and substrate attachment and can only be judged by direct water measurements. The effects of potassium or chloride channel blockers do not always correlate with the AVD.     

Effects of dihydroxy gymnemic triacetate (DGT) on expression of apoptosis associated proteins in human prostate cancer cell lines (PC3)

Abstract

Prostate cancer is the most common malignancies among men. The present study is aimed at the investigation of dihydroxy gymnemic triacetate (DGT) from Gymnema sylvestre on mitochondrial apoptotic pathway and cell cycle arrest. Treatment of DGT resulted in a dose-dependent inhibition of growth of PC-3 cells. The cell cycle arrest was observed at the G2/M phase and accumulation of apoptotic cells was observed in DGT-treated prostate cancer cell lines. The occurrence of apoptosis in these cells was observed by DNA fragmentation. These events were associated with increased levels of pro-apoptotic proteins Bax, Bad and reduced levels of the antiapoptotic proteins Bcl-2, Bcl-xL and Mcl-1. DGT also induces the activation of caspase-9 and caspase-3. The above results, clearly, suggest that DGT induces apoptosis by the intrinsic pathways which could be very useful for the treatment of prostate cancer.     

Granzyme M targets topoisomerase II alpha to trigger cell cycle arrest and caspase-dependent apoptosis

Cytotoxic lymphocyte protease granzyme M (GrM) is a potent inducer of tumor cell death. The apoptotic phenotype and mechanism by which it induces cell death, however, remain poorly understood and controversial. Here, we show that GrM-induced cell death was largely caspase-dependent with various hallmarks of classical apoptosis, coinciding with caspase-independent G2/M cell cycle arrest. Using positional proteomics in human tumor cells, we identified the nuclear enzyme topoisomerase II alpha (topoIIα) as a physiological substrate of GrM. Cleavage of topoIIα by GrM at Leu¹²⁸⁰ separated topoIIα functional domains from the nuclear localization signals, leading to nuclear exit of topoIIα catalytic activity, thereby rendering it nonfunctional. Similar to the apoptotic phenotype of GrM, topoIIα depletion in tumor cells led to cell cycle arrest in G2/M, mitochondrial perturbations, caspase activation, and apoptosis. We conclude that cytotoxic lymphocyte protease GrM targets topoIIα to trigger cell cycle arrest and caspase-dependent apoptosis.     

Abieslactone Induces Cell Cycle Arrest and Apoptosis in Human Hepatocellular Carcinomas through the Mitochondrial Pathway and the Generation of Reactive Oxygen Species

Abieslactone is a triterpenoid lactone isolated from Abies plants. Previous studies have demonstrated that its derivative abiesenonic acid methyl ester possesses anti-tumor-promoting activity in vitro and in vivo. In the present study, cell viability assay demonstrated that abieslactone had selective cytotoxicity against human hepatoma cell lines. Immunostaining experiments revealed that abieslactone induced HepG2 and SMMC7721 cell apoptosis. Flow cytometry and western blot analysis showed that the apoptosis was associated with cell cycle arrest during the G₁ phase, up-regulation of p53 and p21, and down-regulation of CDK2 and cyclin D1. Furthermore, our results revealed that induction of apoptosis through a mitochondrial pathway led to upregulation of Bax, down-regulation of Bcl-2, mitochondrial release of cytochrome c, reduction of mitochondrial membrane potential (MMP), and activation of caspase cascades (Casp-9 and -3). Activation of caspase cascades also resulted in the cleavage of PARP fragment. Involvement of the caspase apoptosis pathway was confirmed using caspase inhibitor Z-VAD-FMK pretreatment. Recent studies have shown that ROS is upstream of Akt signal in mitochondria-mediated hepatoma cell apoptosis. Our results showed that the accumulation of ROS was detected in HepG2 cells when treated with abieslactone, and ROS scavenger partly blocked the effects of abieslactone-induced HepG2 cell death. In addition, inactivation of total and phosphorylated Akt activities was found to be involved in abieslactone-induced HepG2 cell apoptosis. Therefore, our findings suggested that abieslactone induced G₁ cell cycle arrest and caspase-dependent apoptosis via the mitochondrial pathway and the ROS/Akt pathway in HepG2 cells.     

Caspase- and p53-dependent apoptosis in breast carcinoma cells induced by a synthetic selenadiazole derivative

Selenadiazole derivative is one kind of synthetic organoselenium compounds with potent and broad-spectrum antitumor activity. In this study, we showed that anthrax [1,2-c] [1,2,5] selenadiazolo-6,11-dione (ASDO), an novel selenadiazole derivative, induced time- and dose-dependent apoptotic cell death in MCF-7 human breast carcinoma cells, as indicated by accumulation of sub-G1 cell population, DNA fragmentation, nuclear condensation, caspase activation and PARP cleavage. ASDO-induced apoptosis was significantly inhibited by a general caspase inhibitor z-VAD-fmk, demonstrating the important role of caspases in ASDO-induced apoptotic pathway. Treatment of MCF-7 cells with ASDO resulted in a rapid depletion of mitochondrial membrane potential and release of cytochrome c and Smac/Diablo through up-regulation of Bax, Bad and PUMA expression and down-regulation of Bcl-xl expression. Moreover, ASDO treatment up-regulated the expression levels of total p53 and its target gene p21Waf1. Silencing of p53 activation with RNA interference effectively blocked the ASDO-induced cell PARP cleavage, DNA fragmentation and caspase activation. Furthermore, ASDO-induced apoptosis was interestingly found to be independent of reactive oxygen species production. Taken together, we conclude that ASDO induces MCF-7 cell apoptosis through a p53-dependent and mitochondria-mediated pathway.     

Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease

Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation, and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C₆-ceramide. Overexpression of AQP-1 in Chinese hamster ovary (CHO-AQP-1) cells enhanced their rate of apoptosis. The AVD is driven by loss of K⁺ from the cell, and we hypothesize that after the AVD, AQPs become inactive, which halts further water loss and allows K⁺ concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes, and CHO-AQP-1 cells revealed that indeed, the shrunken (apoptotic) subpopulation has very low water permeability compared with the normal-sized (nonapoptotic) subpopulation. In thymocytes, AQP-1 is present and was shown to colocalize with the plasma membrane receptor tumor necrosis factor receptor-1 (TNF-R1) both before and after the AVD, which suggests that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis, and that inactivation after the AVD may help create the low K⁺ concentration that is essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane. water movement; major intrinsic protein; channel; enzyme     

Induction of Apoptosis and Antiproliferative Activity of Naringenin in Human Epidermoid Carcinoma Cell through ROS Generation and Cell Cycle Arrest

A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G₀/G₁ phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G₀/G₁ phase and caspase-3 activation.     

Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis.

The loss of cell volume is a fundamental feature of apoptosis. We have previously shown that DNA degradation and caspase activity occur only in cells which have shrunken as a result of potassium and sodium efflux (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). Furthermore, maintaining a normal intracellular potassium concentration represses the cell death process by inhibiting the activity of apoptotic nucleases and suppressing the activation of effector caspases (Hughes, F. M., Jr., Bortner, C. D. Purdy, G. D., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 30567-30576). We have now investigated the relationship between cell shrinkage, ion efflux, and changes in the mitochondrial membrane potential, in addition to the role of caspases in these apoptotic events. Treatment of Jurkat cells with a series of inducers which act via distinct signal transduction pathways, resulted in all of the cell death characteristics including loss of cell viability, cell shrinkage, K(+) efflux, altered mitochondrial membrane potential, and DNA fragmentation. Interestingly, only cells which shrunk had a loss of mitochondrial membrane potential and the other apoptotic characteristics. Treatment of Jurkat cells with an anti-Fas antibody in the presence of the general caspase inhibitor z-VAD, abrogated these features. In contrast, when Jurkat cells were treated with either the calcium ionophore A23187 or thapsigargin, z-VAD failed to prevent cell shrinkage, K(+) efflux, or changes in the mitochondrial membrane potential, while effectively inhibiting DNA degradation. Treatment of Jurkat cells with various apoptotic agents in the presence of either the caspase-3 inhibitor DEVD, or the caspase-8 inhibitor IETD also blocked DNA degradation, but failed to prevent other characteristics of apoptosis. Together these data suggest that the cell shrinkage, K(+) efflux, and changes in the mitochondrial membrane potential are tightly coupled, but occur independent of DNA degradation, and can be largely caspase independent depending on the particular signal transduction pathway.     

Cationic gradient reversal and cytoskeleton-independent volume regulatory pathways define an early stage of apoptosis

Cell shrinkage, or apoptotic volume decrease (AVD), is a ubiquitous characteristic of programmed cell death that is independent of the death stimulus and occurs in all examples of apoptosis. Here we distinguished two specific stages of AVD based on cell size and a unique early reversal of intracellular ions that occurs in response to activation of both intrinsic and extrinsic cell death signal pathways. The primary stage of AVD is characterized by an early exchange of the normal intracellular ion distribution for sodium from 12 to 113.6 mm and potassium from 139.5 to 30 mm. This early ionic reversal is associated with a 20-40% decrease in cell volume, externalization of phosphatidylserine, loss of mitochondrial membrane potential, and caspase activation and activity along with nuclear condensation that occurs independent of actin cytoskeleton disruption. Disruption of the actin cytoskeleton, however, prevents a secondary stage of AVD in apoptotic cells, characterized by a loss of both potassium and sodium that results in an 80-85% loss in cell volume, DNA degradation, and apoptotic body formation. Together these studies demonstrate that AVD occurs in two distinct stages with the earliest stage reflecting a cellular cationic gradient reversal.