SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis

Apoptosis is characterized by the activation of specific biochemical pathways that lead to the organized demise of cells. Intracellular GSH depletion has been observed during apoptosis; however, neither the mechanisms involved in the reduction of the intracellular GSH concentration, [GSH](i), nor its link to the progression of apoptosis have been elucidated. We have studied this issue using Fas ligand (FasL)-induced apoptosis in Jurkat cells where changes in [GSH](i) can be analyzed biochemically and at the single cell level by flow cytometry. A reduction in the total [GSH](i) in response to FasL occurs in two distinct stages prior to the loss of membrane integrity. Jurkat cells express several members of the multidrug resistance protein (ABCC/MRP), and the organic anion-transporting polypeptide protein (SLCO/OATP) families of GSH efflux pumps at the mRNA level. Glutathione loss and its accumulation in the extracellular medium, induced by FasL, was trans-stimulated by the organic substrates MK571, probenecid, taurocholic acid, estrone sulfate, and bromosulfophthalein and inhibited by high concentrations of extracellular GSH. Single cell analysis demonstrated that intracellular GSH loss was paralleled by the activation of an organic anion uptake process, supporting the role of an anion exchange mechanism (SLCO/OATP-like transport) in GSH efflux induced by FasL. Additionally, high extracellular GSH inhibited the activation of the execution caspases, the cleavage of their substrates poly(ADP-ribose) polymerase (PARP) and alpha-fodrin, and DNA degradation. In contrast, the trans-stimulation of GSH efflux by MK571 increased the cleavage of the execution caspases and their substrates. Together these results suggest that GSH efflux during FasL-induced apoptosis is mediated by a SLCO/OATP-like transport mechanism that modulates the progression of the execution phase of apoptosis.     

Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation

Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via a GSH transporter. We now evaluate the relationship between GSH depletion, the generation of reactive oxygen species (ROS), and the progression of apoptosis. Simultaneous single cell analysis of changes in GSH content and ROS formation by multiparametric FACS revealed that loss of intracellular GSH was paralleled by the generation of different ROS including hydrogen peroxide, superoxide anion, hydroxyl radical, and lipid peroxides. However, inhibition of ROS formation by a variety of antioxidants showed that GSH loss was independent from the generation of ROS. Furthermore, GSH depletion was observed to be necessary for ROS generation. Interestingly, high extracellular thiol concentration (GSH and N-acetyl-cysteine) inhibited apoptosis, whereas, inhibition of ROS generation by other non-thiol antioxidants was ineffective in preventing cell death. Finally, GSH depletion was shown to be a necessary for the progression of apoptosis activated by both extrinsic and intrinsic signaling pathways. These results document a necessary and critical role for GSH loss in apoptosis and clearly uncouple for the first time GSH depletion from ROS formation.     

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells placed into hyperosmotic medium

Time course of changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium with addition of 200 mM sucrose was studied. Ouabain-sensitive and ouabain-resistant Rb+ (K+) influxes were measured during regulatory cell volume increase (RVI) and apoptotic volume decrease (AVD). Microscopy of cells stained by Acrydine orange, Ethydium bromide, APOPercenrage Dye and polycaspase marker FLICA was performed. We found that initial osmotic cell shrinkage induced both RVI and AVD responses. RVI dominated at the early stage whereas AVD prevailed at the later stage. In view of the data obtained in U937 cells the current opinion that RVI "dysfunction" is a prerequisite for apoptosis and AVD (Subramanyam et al., 2010) should be revised. U937 cells are capable to trigger of apoptosis and AVD in spite of the unimpaired RVI response. It is concluded that AVD plays a significant role in preventing osmotic lysis of apoptotic cells rather than in the initiation of apoptosis.     

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.     

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.     

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.     

Reactive oxygen species are related to ionic fluxes and volume decrease in apoptotic cerebellar granule neurons: role of NOX enzymes

Reactive oxygen species (ROS) are produced early during apoptosis of cerebellar granule neurons induced by low potassium (K5) and staurosporine (Sts). In addition, K5 and Sts activate NADPH oxidases (NOX). Recently, we described that K5 and Sts induce apoptotic volume decrease (AVD) at a time when ROS generation and NOX activity occur. In the present study, we evaluated the relationship between ROS generation and ionic fluxes during AVD. Here, we showed that K5- and Sts-induced AVD was inhibited by antioxidants and that direct ROS production induced AVD. Moreover, NOX inhibitors eliminated AVD induced by both K5 and Sts. Sts, but not K5, failed to induce AVD in cerebellar granule neurons from NOX2 knockout mice. These findings suggest that K5- and Sts-induced AVD is largely mediated by ROS produced by NOX. On the other hand, we also found that the blockage of ionic fluxes involved in AVD inhibited both ROS generation and NOX activity. These findings suggest that ROS generation and NOX activity are involved in ionic fluxes activation, which in turn could maintain ROS generation by activating NOX, leading to a self-amplifying cycle.     

Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl- conductance

We show that dysregulation of the Cl- homeostasis mediates the staurosporine-induced apoptotic cell death in human ECV304 cells. A pronounced apoptotic volume decrease (AVD), and an increase in plasma membrane Cl- conductance were early (<1 h) events following staurosporine challenge. Both processes were involved in apoptotic death, as demonstrated by the observation that the Cl- channel blocker phloretin inhibited both the staurosporine-evoked Cl- current and AVD, and preserved cell viability. Prolonged incubation (>2 h) with staurosporine caused a decrease in intracellular pH, which, however, was not required for the progression of the apoptotic process, because inhibitors of proton extrusion pathways, which lowered cytoplasmic pH, failed to inhibit both caspase-3 activation and DNA laddering. Moreover, clamping the cytosolic pH to an alkaline value did not prevent the apoptotic cell death. Collectively, these data demonstrate that staurosporine-mediated apoptosis of ECV304 cells is caused by the upregulation of Cl- channel activity and subsequent AVD, but is independent of intracellular acidification.     

Glutathione depletion regulates both extrinsic and intrinsic apoptotic signaling cascades independent from multidrug resistance protein 1

Glutathione (GSH) depletion is an important hallmark of apoptosis. We previously demonstrated that GSH depletion, by its efflux, regulates apoptosis by modulation of executioner caspase activity. However, both the molecular identity of the GSH transporter(s) involved and the signaling cascades regulating GSH loss remain obscure. We sought to determine the role of multidrug resistance protein 1 (MRP1) in GSH depletion and its regulatory role on extrinsic and intrinsic pathways of apoptosis. In human lymphoma cells, GSH depletion was stimulated rather than inhibited by pharmacological blockage of MRP1 with MK571. GSH loss was dependent on initiator caspases 8 and 9 activity. Genetic knock-down (>60 %) of MRP1 by stable transfection with short hairpin small interfering RNA significantly reduced MRP1 protein levels, which correlated directly with the loss of MRP1-mediated anion transport. However, GSH depletion and apoptosis induced by both extrinsic and intrinsic pathways were not affected by MRP1 knock-down. Interestingly, stimulation of GSH loss by MK571 also enhanced the initiator phase of apoptosis by stimulating initiator caspase 8 and 9 activity and pro-apoptotic BCL-2 interacting domain cleavage. Our results clearly show that caspase-dependent GSH loss and apoptosis are not mediated by MRP1 proteins and that GSH depletion stimulates the initiation phase of apoptosis in lymphoid cells.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

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。     

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     

N-acetylcysteine increases apoptosis induced by H(2)O(2) and mo-antiFas triggering in a 3DO hybridoma cell line

N-Acetylcysteine (NAC) has been used as an antioxidant to prevent apoptosis triggered by different stimuli in different cell types. It is common opinion that cellular redox, which is largely determined by the ratio of oxidized and reduced glutathione (GSH), plays a significant role in the propensity of cells to undergo apoptosis. However, there are also contrasting opinions stating that intracellular GSH depletion or supplemented GSH alone are not sufficient to lead cells to apoptosis or conversely protect them. Unexpectedly, this study shows that NAC, even if it maintains the peculiar characteristics of an agent capable of reducing cell proliferation and increasing intracellular GSH content, increases apoptosis induced by H(2)O(2) treatment and mo-antiFas triggering in a 3DO cell line. We found that 24 h of NAC pre-treatment can shift cellular death from necrotic to apoptotic and determine an early expression of FasL in a 3DO cell line treated with H(2)O(2).     

Life and death of lymphocytes: a volume regulation affair

The loss of cell volume, termed apoptotic volume decrease (AVD) has been a hallmark feature of apoptosis. However the role of this characteristic attribute of programmed cell death has always been questioned as to whether it plays an active or passive factor during apoptosis. Here we review studies that suggest that AVD plays an active role during apoptosis and the underlying flux of ions that results in this morphological event regulates the programmed cell death process.     

The potential role of caveolin-1 in inhibition of aquaporins during the AVD

BACKGROUND INFORMATION: During apoptosis, the first morphological change is a distinct cell shrinkage known as the AVD (apoptotic volume decrease). This event is driven by a loss of intracellular K(+), which creates an osmotic gradient, drawing water out of the cell through AQPs (aquaporins). Loss of water in balance with K(+) would create a shrunken cell with an equivalent intracellular concentration of K(+) ([K(+)](i) = 140 mM). However, we have previously shown that the [K(+)](i) of the shrunken apoptotic cell is 35 mM, and this level is absolutely essential for the activation of apoptotic enzymes. We have recently found that AQPs are inactivated following the AVD, so that continued loss of K(+) will reduce the intracellular concentration to this critical level. Using thymocytes, we have investigated the expression profile and regulation of the AQP family members. RESULTS: In the present study, we have found that AQP1, AQP8 and AQP9 are present in non-apoptotic thymocytes and localized primarily to the plasma membrane. Expression and localization did not change when these cells were induced to undergo apoptosis by growth factor withdrawal for 24 h. To explore other possible mechanisms by which these water channels are inactivated, we investigated their association with CAV-1 (caveolin-1), binding to which is known to inactivate a variety of proteins. We found that CAV-1 is present in thymocytes and that this protein co-localizes with a portion of AQP1 in normal (non-apoptotic) thymocytes. However, thymocytes induced to undergo apoptosis greatly increase their AQP1/CAV-1 association. CONCLUSIONS: Taken together, these results indicate that AQPs are localized to the plasma membrane of shrunken apoptotic thymocytes where increased binding to CAV-1 potentially inactivates them. AQP inactivation, coupled with continued K(+) efflux, then allows the [K(+)](i) to decrease to levels conducive for the activation of downstream apoptotic enzymes and the completion of the apoptotic cascade.     

Upregulation of Fas ligand expression by human immunodeficiency virus in human macrophages mediates apoptosis of uninfected T lymphocytes.

Apoptosis has been proposed to mediate CD4+ T-cell depletion in human immunodeficiency virus (HIV)-infected individuals. Interaction of Fas ligand (FasL) with Fas (CD95) results in lymphocyte apoptosis, and increased susceptibility to Fas-mediated apoptosis has been demonstrated in lymphocytes from HIV-infected individuals. Cells undergoing apoptosis in lymph nodes from HIV-infected individuals do not harbor virus, and therefore a bystander effect has been postulated to mediate apoptosis of uninfected cells. These data raise the possibility that antigen-presenting cells are a source of FasL and that HIV infection of cells such as macrophages may induce or increase FasL expression. In this report, we demonstrate that HIV infection of monocytic cells not only increases the surface expression of Fas but also results in the de novo expression of FasL. Interference with the FasL-Fas interaction by anti-Fas blocking antibodies abrogates HIV-induced apoptosis of monocytic cells. Human monocyte-derived macrophages from healthy donors contain detectable FasL mRNA, which is further upregulated following HIV infection with monocytotropic strains. HIV-infected human macrophages result in the apoptotic death of Jurkat T cells and peripheral blood T lymphocytes. Interruption of the FasL-Fas interaction abrogates the HIV-infected macrophage-dependent death of T lymphocytes. These results provide evidence that human macrophages can provide a source of FasL, especially following HIV infection, and can thus participate in lymphocyte depletion in HIV-infected individuals.     

Glutathione regulates caspase-dependent ceramide production and curcumin-induced apoptosis in human leukemic cells

Depletion of intracellular glutathione (GSH) is the prime hallmark of the progression of apoptosis. Previously, we reported that curcumin induces reactive oxygen species (ROS)-mediated depletion of GSH, which leads to caspase-dependent and independent apoptosis in mouse fibroblast cells (F. Thayyullathil et al., Free Radic. Biol. Med.45, 1403-1412, 2008). In this study, we investigated the antileukemic potential of curcumin in vitro, and we further examined the molecular mechanisms of curcumin-induced apoptosis in human leukemic cells. Curcumin suppresses the growth of human leukemic cells via ROS-independent GSH depletion, which leads to caspase activation, inhibition of sphingomyelin synthase (SMS) activity, and induction of ceramide (Cer) generation. Pretreatment of leukemic cells with carbobenzoxy-Val-Ala-Asp fluoromethylketone, a universal inhibitor of caspases, abrogates the SMS inhibition and Cer generation, and in turn prevents curcumin-induced cell death. Curcumin treatment of leukemic cells also downregulates the expression of the inhibitor of apoptosis proteins (IAPs), phospho-Akt, c-Myc, and cyclin D1. Extracellular supplementation with GSH attenuates curcumin-induced depletion of GSH, caspase-dependent inhibition of SMS, Cer generation, and downregulation of IAPs, whereas, L-D-buthionine sulfoximine, a widely used inhibitor of GSH synthesis, potentiates GSH depletion, Cer generation, and apoptosis induced by curcumin. Taken together, our findings provide evidence suggesting for the first time that GSH regulates caspase-dependent inhibition of SMS activity, Cer generation, and apoptosis induced by curcumin in human leukemic cells.     

Dysfunction of regulatory volume increase is a key component of apoptosis

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non-apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF-alpha or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase-3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1-deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity-induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.     

Effects of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid on 4-hydroxy-2-nonenal-induced apoptotic T cell death

4-Hydroxy-2-nonenal (HNE), the aldehydic product of lipid peroxidation, is associated with multiple immune dysfunctions, such as HIV and hepatitis C virus infection. HNE-induced immunosuppression could be due to a decrease in CD4+ T lymphocyte activation or proliferation. Glutathione (GSH) is the most abundant endogenous antioxidant in cells, and an adduct between HNE and GSH has been suggested to be a marker of oxidative stress. Our earlier studies showed that HNE induced cytotoxicity and Akt inactivation, which led to the enhancement of FasL expression and concomitantly decreased cellular FLICE-like inhibitory protein (c-FLIP(S)) levels. In this study, we found that HNE caused intracellular GSH depletion in Jurkat T cells, and we further investigated the role of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA), a GSH prodrug, in attenuating HNE-induced cytotoxicity in CD4+ T lymphocytes. The results show that PTCA protected against HNE-induced apoptosis and depletion of intracellular GSH. PTCA also suppressed FasL expression through increasing levels of Akt kinase as well as antiapoptotic c-FLIP(S) and decreasing the activation of type 2 protein serine/threonine phosphatase. Taken together, these data demonstrate a novel correlation between GSH levels and Akt activation in T lymphocyte survival, which involves FasL down-regulation and c-FLIP(S) expression through increasing intracellular GSH levels. This suggests that PTCA could potentially be used in the treatment of oxidative stress-induced immunosuppressive diseases.