A role for polyploidy in the tumorigenicity of Pim-1-expressing human prostate and mammary epithelial cells

Background

Polyploidy is a prominent feature of many human cancers, and it has long been hypothesized that polyploidy may contribute to tumorigenesis by promoting genomic instability. In this study, we investigated whether polyploidy per se induced by a relevant oncogene can promote genomic instability and tumorigenicity in human epithelial cells.

Principal Findings

When the oncogenic serine-threonine kinase Pim-1 is overexpressed in immortalized, non-tumorigenic human prostate and mammary epithelial cells, these cells gradually converted to polyploidy and became tumorigenic. To assess the contribution of polyploidy to tumorigenicity, we obtained sorted, matched populations of diploid and polyploid cells expressing equivalent levels of the Pim-1 protein. Spectral karyotyping revealed evidence of emerging numerical and structural chromosomal abnormalities in polyploid cells, supporting the proposition that polyploidy promotes chromosomal instability. Polyploid cells displayed an intact p53/p21 pathway, indicating that the viability of polyploid cells in this system is not dependent on the inactivation of the p53 signaling pathway. Remarkably, only the sorted polyploid cells were tumorigenic in vitro and in vivo.

Conclusions

Our results support the notion that polyploidy can promote chromosomal instability and the initiation of tumorigenesis in human epithelial cells.     

AIF suppresses chemical stress-induced apoptosis and maintains the transformed state of tumor cells

Apoptosis-inducing factor (AIF) exhibits reactive oxygen species (ROS)-generating NADH oxidase activity of unknown significance, which is dispensable for apoptosis. We knocked out the aif gene in two human colon carcinoma cell lines that displayed lower mitochondrial complex I oxidoreductase activity and produced less ROS, but showed increased sensitivity to peroxide- or drug-induced apoptosis. AIF knockout cells failed to form tumors in athymic mice or grow in soft agar. Only AIF with intact NADH oxidase activity restored complex I activity and anchorage-independent growth of aif knockout cells, and induced aif-transfected mouse NIH3T3 cells to form foci. AIF knockdown in different carcinoma cell types resulted in lower superoxide levels, enhanced apoptosis sensitivity and loss of tumorigenicity. Antioxidants sensitized AIF-expressing cells to apoptosis, but had no effect on tumorigenicity. In summary, AIF-mediated resistance to chemical stress involves ROS and probably also mitochondrial complex I. AIF maintains the transformed state of colon cancer cells through its NADH oxidase activity, by mechanisms that involve complex I function. On both counts, AIF represents a novel type of cancer drug target.     

Reactive oxygen species mediate IL-8 expression in Down syndrome candidate region-1-overexpressed cells

Reactive oxygen species (ROS) have been considered to mediate inflammation in Down syndrome (DS). The present study is purposed to examine the mechanism of increased ROS levels and inflammatory cytokine IL-8 expression in Down syndrome candidate region-1 (DSCR1)-transfected cells, by determining ROS levels, IL-8 expression, NF-κB activation, and SOD1 levels in human embryonic kidney (HEK) 293 cells. The cells were treated with an antioxidant N-acetyl cysteine (NAC) or a calcium chelator BAPTA and stimulated with or without IL-1β. As a result, basal levels of ROS, IL-8, and NF-κB-DNA binding activity were higher, and basal SOD1 levels were higher in DSCR1-transfected cells than pcDNA-transfected cells. BAPTA and NAC inhibited increase in ROS (intracellular and mitochondrial levels) in DSCR-1-transfected cells without treatment of IL-1β. DSCR1 transfection-induced changes were increased by treatment with IL-1β, which was suppressed by NAC and BAPTA. Transfection of SOD1 inhibited ROS levels in DSCR1-transfected cells. In conclusion, ROS activate NF-κB and IL-8 induction in DSCR1-transfected cells in a calcium-dependent manner, which is augmented by IL-1β since IL-1β increases calcium and ROS levels in the cells. Reducing ROS levels by treatment of antioxidants may be beneficial for preventing DS-associated inflammation by suppressing cytokine expression.     

Impaired mitochondrial function protects against free radical-mediated cell death

Free radical damage can have fatal consequences. Mitochondria carry out essential cellular functions and produce high levels of reactive oxygen species (ROS). Many agents also generate ROS. Using the yeast Saccharomyces cerevisiae as a eukaryotic model, the role of functional mitochondria in surviving free radical damage was investigated. Respiratory-deficient cells lacking mitochondrial DNA (rho(0)) were up to 100-fold more resistant than isogenic rho(+) cells to killing by ROS generated by the bleomycin-phleomycin family of oxidative agents. Up to approximately 90% of the survivors of high oxidative stress lost mitochondrial function and became "petites." The selective advantage of respiratory deficiency was studied in several strains, including DNA repair-deficient rad52/rad52 and blm5/blm5 diploid strains. These mutant strains are hypersensitive to lethal effects of free radicals and accumulate more DNA damage than related wild-type strains. Losses in mitochondrial function were dose-dependent, and mutational alteration of the RAD52 or BLM5 gene did not affect the resistance of surviving cells lacking mitochondrial function. The results indicate that inactivation of mitochondrial function protects cells against lethal effects of oxygen free radicals.     

Adenosine induces apoptosis in human liver cancer cells through ROS production and mitochondrial dysfunction

Mitochondria are the most important sensor for apoptosis. Extracellular adenosine is well reported to induce apoptosis of tumor cells. Here we found that extracellular adenosine suppresses the cell growth by induction of apoptosis in BEL-7404 liver cancer cells, and identified a novel mechanism that extracellular adenosine triggers apoptosis by increasing Reactive Oxygen Species (ROS) production and mitochondrial membrane dysfunction in the cells. We observed that adenosine increases ROS production, activates c-Caspase-8 and -9 and Caspase effectors, c-Caspase-3 and c-PARP, induces accumulation of apoptosis regulator Bak, decreases Bcl-xL and Mcl-1, and causes the mitochondrial membrane dysfunction and the release of DIABLO, Cytochrome C, and AIF from mitochondria to cytoplasm in the cells; ROS inhibitor, NAC significantly reduces adenosine-induced ROS production; it also shows the same degree of blocking adenosine-induced loss of mitochondrial membrane potential (MMP) and apoptosis. Our study first observed that adenosine increases ROS production in tumor cells and identified the positive feedback loop for ROS-mediated mitochondrial membrane dysfunction which amplifies the death signals in the cells. Our findings indicated ROS production and mitochondrial dysfunction play a key role in adenosine-induced apoptosis of 7404 cells.     

Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.     

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone,from buds of Cleistocalyx operculatus,induces apoptosis in human hepatoma SMMC-7721 cells through a reactive oxygen species-dependent mechanism

Nowadays, much effort is being devoted to detect new substances that not only significantly induce the death of tumor cells, but also have little side effect on normal cells. Our previous study showed that 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC) exhibited significant cytotoxic potential with an IC₅₀ value of 32.3 ± 1.13 μM against SMMC-7721 cells and could induce SMMC-7721 cells apoptosis. In the present study, we found that DMC was almost nontoxic to human normal liver L-02 and human normal fetal lung fibroblast HFL-1 cells as their IC₅₀ values (111.0 ± 4.57 and 152.0 ± 4.83 µM for L-02 and HFL-1 cells, respectively) were much higher. To further explore the apoptotic mechanism of DMC, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by DMC in SMMC-7721 cells. Our results suggested that the cytotoxicity and the generation of intracellular ROS were inhibited by N-acetylcysteine (NAC). Reversal of apoptosis in NAC pretreated cells indicated the involvement of ROS in DMC-induced apoptosis. The loss of mitochondrial membrane potential (ΔΨm) induced by DMC was significantly blocked by NAC. NAC also prevented the decrease of Caspase-3 and -9 activities, the increase of Bcl-2 protein expression and the decrease of p53 and PUMA protein expressions. Together, these results indicated that ROS played a key role in the apoptosis induced by DMC in human hepatoma SMMC-7721 cells.     

药物诱导玉米孤雌生殖植株的倍性变异

实验结果表明,孤雌生殖植株根尖体细胞以二倍体细胞最多,占６８．９％,其次为非整倍体细胞,占２８．７％,其他异倍体和单倍体细胞极少（２．４％）。Ｐａ１植株可分为二倍体和混倍体两类,以二倍体细胞占绝对多数的混倍体植株最多,为８３．５％,这些植株生长发育和结实均正常。在花粉母细胞中正常二倍体频率比根尖体细胞明显提高,提高频率为３５．３一５９．６％,不同材料之间趋势一致。讨论了体细胞染色体变异的来源及其能否延续到生殖细胞。     

远缘杂交形成的二倍体鱼和多倍体鱼生殖细胞染色体研究

本文采用性腺染色体制片及组织学切片方法,系统地研究了不同发育时期的鲫鲤杂交第二代(F2) (2n=100)、异源四倍体鲫鲤(4n=200)、三倍体鲫鱼(3n=150))、雌核发育二倍体鲫鲤第二代(G2)(2n=100)及鲤鱼(Cypninus carpio L)(2n=100)(对照组)生殖细胞的染色体特征.研究结果表明,对照组中鲤鱼精原细胞染色体数与体细胞染色体数一致,为二倍体精原细胞(2n=100),而远缘杂交形成的二倍体鱼和多倍体鱼的生殖细胞中则观察到明显的染色体数加倍现象,其中,鲫鲤杂交第二代(F2)精巢生殖细胞染色体数加倍现象特别丰富,占检测的染色体分裂相的21.6%,为其产生不减半的二倍体配子提供了直接的细胞学证据,同时也说明远缘杂交是导致生殖细胞染色体数加倍的一个重要因素.该研究在探讨多倍体鱼的发生及鱼类遗传育种方面具有重要意义.     

Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells

Hexokinase type II (HK II) is the key enzyme for maintaining increased glycolysis in cancer cells where it is overexpressed. 3-bromopyruvate (3-BrPA), an inhibitor of HK II, induces cell death in cancer cells. To elucidate the molecular mechanism of 3-BrPA-induced cell death, we used the hepatoma cell lines SNU449 (low expression of HKII) and Hep3B (high expression of HKII). 3-BrPA induced ATP depletion-dependent necrosis and apoptosis in both cell lines. 3-BrPA increased intracellular reactive oxygen species (ROS) leading to mitochondrial dysregulation. NAC (N-acetyl-l-cysteine), an antioxidant, blocked 3-BrPA-induced ROS production, loss of mitochondrial membrane potential and cell death. 3-BrPA-mediated oxidative stress not only activated poly-ADP-ribose (PAR) but also translocated AIF from the mitochondria to the nucleus. Taken together, 3-BrPA induced ATP depletion-dependent necrosis and apoptosis and mitochondrial dysregulation due to ROS production are involved in 3-BrPA-induced cell death in hepatoma cells.     

Regulation of the mitochondrial ATP-sensitive potassium channel in rat uterus cells by ROS

In previous study we demonstrated the presence of ATP-sensitive potassium current in the inner mitochondrial membrane, which was sensitive to diazoxide and glybenclamide, in mitochondria isolated from the rat uterus. This current was supposed to be operated by mitochondrial ATP-sensitive potassium channel (mitoK(ATP)). Regulation of the mitoK(ATP) in uterus cells is not studied well enough yet. It is well known that the reactive oxygen species (ROS) can play a dual role. They can damage cells in high concentrations, but they can also act as messengers in cellular signaling, mediating survival of cells under stress conditions. ROS are known to activate mitoK(ATP) during the oxidative stress in the brain and heart, conferring the protection of cells. The present study examined whether ROS mediate the mitoK(ATP) activation in myometrium cells. Oxidative stress was induced by rotenone. ROS generation was measured by 2',7'-dichlorofluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochondrial membrane was also detected with the use of the potential-sensitive dye DiOC6 (3,3'-dihexyloxacarbocyanine iodide). Diazoxide, a selective activator of mitoK(ATP), depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoK(ATP) blocker glybenclamide effectively restored mitochondrial potential in rat myocytes. Estimated value for diazoxide to mitoK(ATP) under normoxia was four times higher than under oxidative stress conditions: 5.01 +/- 1.47-10(-6) M and 1.24 +/- 0.21 x 10(-6) M respectively. The ROS scavenger N-acetylcysteine (NAC) successfully eliminates depolarization of mitochondrial membrane by diazoxide under oxidative stress. These results suggest that elimination of ROS by NAC prevents the activation of mitoK(ATP) under oxidative stress. Taking into account the higher affinity of diazoxide to mitoK(ATP) under stress conditions than under normoxia, we conclude that the oxidative stress conditions are more favourable than normoxia for the activation of mitoK(ATP). Thus we hypothesize that the ROS regulate the activity of the mitoK(ATP) in myocytes.     

植物同源多倍体耐盐性研究进展

多倍化是高等植物进化最重要的动力之一,多倍体植物由于基因组组成以及基因表达方面的变化,通常会表现出不同的生理现象,多倍体的抗性优于其同源二倍体祖先。土壤盐碱化和次生盐渍化是影响农作物生产的重要因素,严重制约着我国农业的可持续发展。同源多倍体植物耐盐能力较强,是作物遗传改良的重要种质资源,了解其耐盐机理对培育耐盐品种具有重要意义。本文从与盐胁迫相关的耐盐性进化、生理生化水平、细胞结构和分子层面等多角度总结了植物同源多倍体盐胁迫研究进展,并以作者所在研究团队培育出的多倍体西瓜为例讨论了多倍体抗逆性研究存在的问题及未来的发展方向,以期为多倍体抗逆优势机理研究提供参考。     

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Down-Regulation of Deacetylase HDAC6 Inhibits the Melanoma Cell Line A375.S2 Growth through ROS-Dependent Mitochondrial Pathway

Previous studies have shown that histone deacetylase 6 (HDAC6) plays critical roles in many cellular processes related to cancer. However, its biological roles in the development of melanoma remain unexplored. Our aim was to investigate whether HDAC6 has a biological role in human melanoma development and to understand its underlying mechanism. In the present study, HDAC6 expression was up-regulated in melanoma tissues and cell lines. Knockdown of HDAC6 significantly inhibited the proliferation and colony formation ability of A375.S2 cells, promoted cell arrest at G0/G1 phase and apoptosis. Additionally, western blotting assay showed that HDAC6 silencing suppressed Bcl-2 level and enhanced Bax level, then activated caspase-9 and caspase-3, and further activated the release of cytochrome c from mitochondria to cytoplasm, finally induced cell apoptosis involving the mitochondrial pathway. Knockdown of HDAC6 triggered a significant generation of ROS and disruption of mitochondrial membrane potential (MMP). Furthermore, ROS inhibitor, NAC reduced HDAC6 siRNA-induced ROS production, and blocked HDAC6 siRNA-induced loss of MMP and apoptosis. NAC also significantly blocked HDAC6 siRNA-induced mtDNA copy number decrease and mitochondrial biogenesis and degradation imbalance. In conclusion, the results showed that knockdown of HDAC6 induced apoptosis in human melanoma A375.S2 cells through a ROS-dependent mitochondrial pathway.     

Reactive oxygen species are involved in eosinophil extracellular traps release and in airway inflammation in asthma

In asthma, there are high levels of inflammatory mediators, reactive oxygen species (ROS), and eosinophil extracellular traps (EETs) formation in airway. Here, we attempted to investigate the ROS involvement in EETs release and airway inflammation in OVA-challenged mice. Before the intranasal challenge with ovalbumin (OVA), animals were treated with two ROS inhibitors, N-acetylcysteine (NAC) or diphenyleneiodonium (DPI). We showed that NAC treatment reduced inflammatory cells in lung. DPI and NAC treatments reduced eosinophil peroxidase (EPO), goblet cells hyperplasia, proinflammatory cytokines, NFκB p65 immunocontent, and oxidative stress in lung. However, only the NAC treatment improved mitochondrial energy metabolism. Moreover, the treatments with DPI and NAC reduced EETs release in airway. This is the first study to show that ROS are needed for EETs formation in asthma. Based on our results, NAC and DPI treatments can be an interesting alternative for reducing airway inflammation, mitochondrial damage, and EETs release in asthma.     

Comparative evaluation of N-acetylcysteine and N-acetylcysteineamide in acetaminophen-induced hepatotoxicity in human hepatoma HepaRG cells

Acetaminophen (N-acetyl-p-aminophenol, APAP) is one of the most widely used over-the-counter antipyretic analgesic medications. Despite being safe at therapeutic doses, an accidental or intentional overdose can result in severe hepatotoxicity; a leading cause of drug-induced liver failure in the U.S. Depletion of glutathione (GSH) is implicated as an initiating event in APAP-induced toxicity. N-acetylcysteine (NAC), a GSH precursor, is the only currently approved antidote for an APAP overdose. Unfortunately, fairly high doses and longer treatment times are required due to its poor bioavailability. In addition, oral and intravenous administration of NAC in a hospital setting are laborious and costly. Therefore, we studied the protective effects of N-acetylcysteineamide (NACA), a novel antioxidant, with higher bioavailability and compared it with NAC in APAP-induced hepatotoxicity in a human-relevant in vitro system, HepaRG. Our results indicated that exposure of HepaRG cells to APAP resulted in GSH depletion, reactive oxygen species (ROS) formation, increased lipid peroxidation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase release. Both NAC and NACA protected against APAP-induced hepatotoxicity by restoring GSH levels, scavenging ROS, inhibiting lipid peroxidation, and preserving mitochondrial membrane potential. However, NACA was better than NAC at combating oxidative stress and protecting against APAP-induced damage. The higher efficiency of NACA in protecting cells against APAP-induced toxicity suggests that NACA can be developed into a promising therapeutic option for treatment of an APAP overdose.     

Glucose suppresses superoxide generation in metabolically responsive pancreatic beta cells

High rates of glucose metabolism and mitochondrial electron transport have been associated with increased mitochondrial production of reactive oxygen species (ROS). This mechanism was also proposed as a possible cause for dysfunction and death of pancreatic beta cells exposed to high glucose levels. We examined whether high rates of glucose metabolism increase ROS production in purified rat beta cells. Glucose up to 20 mm did not stimulate H(2)O(2) or superoxide production, whereas it dose-dependently increased cellular NAD(P)H and FADH(2) levels with an EC(50) around 8 mm. On the contrary, glucose concentration-dependently suppressed H(2)O(2) and superoxide formation, with a major effect between 0 and 5 mm, parallel to an increase in cellular NAD(P)H levels. This suppressive effect was more marked in beta cells with higher NAD(P)H responsiveness to glucose; it was not observed in glucagon-containing alpha cells, which lacked a glucose-induced increase in NAD(P)H. Suppression was also induced by the mitochondrial substrates leucine and succinate. Experiments with electron transport chain inhibitors indicate a role of respiratory complex I in ROS production at low mitochondrial activity and low NADH levels. Superoxide production at low glucose is potentially cytotoxic, because scavenging by the superoxide dismutase mimetic agent manganese(III)tetrakis(4-benzoic acid)porphyrin was found to reduce the rate of beta cell apoptosis. Analysis of islets cultured at 20 mm glucose confirmed that this condition does not induce ROS production in beta cells as a result of their increased rates of glucose metabolism. Our study indicates the need of beta cells for basal nutrients maintaining mitochondrial NADH production at levels that suppress ROS accumulation from an inadequate respiratory complex I activity and thus inhibit a potential apoptotic pathway.     

NAC Attenuates LPS-Induced Toxicity in Aspirin-Sensitized Mouse Macrophages via Suppression of Oxidative Stress and Mitochondrial Dysfunction

Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC), an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.     

Role of calcium and cyclophilin D in the regulation of mitochondrial permeabilization induced by glutathione depletion

The mitochondrial permeability transition (MPT) is a calcium and oxidative stress sensitive transition in the permeability of the mitochondrial inner membrane that plays a crucial role in cell death. However, the mechanism regulating the MPT remains controversial. To study the role of oxidative stress in the regulation of the MPT, we used diethyl maleate (DEM) to deplete glutathione (GSH) in human leukemic CEM cells. GSH depletion increased mitochondrial calcium and reactive oxygen species (ROS) levels in a co-dependent manner causing loss of mitochondrial membrane potential (deltapsi(m)) and cell death. These events were inhibited by the calcium chelator BAPTA-AM and the antioxidants N-acetylcysteine (NAC) and the triphenyl phosphonium-linked ubiquinone derivative MitoQ. In contrast, the MPT inhibitor cyclosporine A (CsA) and small interference RNA (siRNA) knockdown of cyclophilin D (Cyp-D) were not protective. These results indicate that mitochondrial permeabilization induced by GSH depletion is not regulated by the classical MPT.