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.     

Does the redox status of cytochrome C act as a fail-safe mechanism in the regulation of programmed cell death?

It has now become recognized that one of the key events in the induction of apoptosis, or programmed cell death, in both plants and animals is the release of cytochrome c from mitochondria. It is also known that oxidative stress imposed on cells can have a profound effect on the onset or progression of apoptosis. Here, we discuss how the redox status of cytochrome c, and thus its structure, can be altered by the presence of reactive oxygen species (ROS) and reduced glutathione (GSH). We suggest that cytochrome c will only induce programmed cell death if present in the cytoplasm in the oxidized state, and that the presence of high levels of cytoplasmic GSH maintain cytochrome c in an inactive (reduced) state, thus behaving as a fail-safe mechanism if cytochrome c is released by mitochondria when programmed cell death is not the required outcome. If the redox status of the cell is disturbed however, perhaps in the presence of hydrogen peroxide, GSH concentrations will drop, the cellular E(h) will rise, and cytochrome c will tend towards the oxidized state, allowing programmed cell death to proceed. Therefore, we propose that the redox state of cytoplasmic cytochrome c may be a key regulator of programmed cell death.     

Reduced glutathione prevents nitric oxide-induced apoptosis in vascular smooth muscle cells

The control of medial and neointimal growth, in which vascular smooth muscle (VSM) plays a central role, is most important to the development of hypertension and atherosclerosis, respectively. Growth of vascular smooth muscle cells is regulated by a number of factors, including the vasodilator nitric oxide (NO). In addition, NO modulates intracellular thiol redox states and the thiol redox state of the cell influences NO production. We, therefore, examined the nature of the effect of NO on growth of VSM cells and its modulation by cellular glutathione content. Here, we report that NO, either generated by NO donors or synthesized by iNOS in VSM cells, inhibited DNA synthesis and induced apoptosis in this cell type. NO-induced apoptosis was associated with a significant decrease in the intracellular concentration of reduced glutathione and with an increase in the level of the tumor suppressor gene p53 mRNA. Moreover, addition of glutathione monoethylester to the culture restored the level of reduced glutathione in VSM cells, and prevented the NO-induced increase in p53 expression and programmed cell death. Our findings suggest a role for reduced glutathione in protecting VSM cells exposed to NO from apoptosis.     

Mechanisms of BSO (L-buthionine-S,R-sulfoximine)-induced cytotoxic effects in neuroblastoma

Glutathione (GSH) depletion is widely used to sensitize cells to anticancer treatment inducing the progression of programmed cell death and overcoming chemoresistance. It has been reported that neuroblastoma cells with MYCN amplification are unable to start TRAIL-dependent death and MYCN, in concert with cytotoxic drugs, efficiently induces the mitochondrial pathway of apoptosis through oxidative mechanisms. In this study, we show that GSH loss induced by L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis, leads to overproduction of reactive oxygen species (ROS) and triggers apoptosis of MYCN-amplified neuroblastoma cells. BSO susceptibility of SK-N-BE-2C, a representative example of MYCN-amplified cells, has been attributed to stimulation of total SOD activity in the absence of changes in the level and the activity of catalase. Therefore, the unbalanced intracellular redox milieu has been demonstrated to be critical for the progression of neuroblastoma cell death that was efficiently prevented by antioxidants and rottlerin. These results describe a novel pathway of apoptosis dependent on ROS formation and PKC-delta activation and independent of p53, bcl-2, and bax levels; the selective redox modulation of PKC-delta might be suggested as a potential strategy for sensitizing MYCN-amplified cells to therapeutic approaches.     

Redox Regulation of Programmed Cell Death in Lymphocytes

A redox imbalance caused by an over-production of prooxidants or a decrease in antioxidants seems to play a role in the programmed cell death that occurs in various developmental programs. Such a physiological function for oxidative stress is particularly applicable to the immune system, wherein individual lymphocytes undergo continuous scrutiny to determine if they should be preserved or programmed to die. Following activation, lymphocytes produced increased levels of reactive oxygen species (ROS) which may serve as intracellular signaling molecules. The ultimate outcome of this increased ROS formation, i.e., lymphocyte proliferation versus programmed cell death, may be dictated by macrophage-derived costimulatory molecules that bolster or diminish lymphocyte antioxidant defenses. HIV-1-infected individuals display multiple symptoms of redox imbalance consistent with their being in oxidative stress, and lymphocytes from such individuals are more prone to undergo apoptosis in vitro. It is suggested that oxidative stress is a physiological mediator of programmed cell death in lymphoid cells, and that HIV disease represents an extreme case of what can happen when regulatory safeguards are compromised.     

N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death.

Gamma-irradiation, glucocorticoid hormones, and calcium ionophores stimulate a suicide process in thymocytes, known as apoptosis or programmed cell death, that involves internucleosomal DNA fragmentation by a Ca(2+)- and Mg(2+)-dependent nuclear endonuclease. In this study we report that N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) blocked DNA fragmentation and cell death in thymocytes exposed to gamma-radiation, dexamethasone, or calcium ionophore A23187. WR-1065 protected the thymocytes from radiation-induced apoptosis when incubated with cells after irradiation but not before and/or during irradiation. WR-1065 inhibited Ca(2+)- and Mg(2+)-dependent DNA fragmentation in isolated thymocyte nuclei. Our results suggest that WR-1065 protects thymocytes from apoptosis by inhibiting Ca(2+)- and Mg(2+)-dependent nuclear endonuclease action.     

Adenoviral SERCA1 overexpression triggers an apoptotic response in cultured neonatal but not in adult rat cardiomyocytes

Although apoptosis and necrosis have been considered different pathways to cell death, only one compound induces both types of cell death. Diethyldithiocarbamate (DDC) has been shown to have antioxidant or prooxidant effects in several different systems. We observed in our present study that DDC induced not only apoptosis but also necrosis depending on its dosage in HL60 premyelocytic leukemia cells. Moreover, in hypoxia cell culture conditions, DDC-induced necrotic cells decreased but DDC-induced apoptosis continued. We investigated the DDC-induced different cell death mechanisms as they are correlated with reactive oxygen species (ROS). High-dose DDC-induced necrotic cell death is thought to depend on the increase of intracellular ROS, while low-dose DDC-induced apoptosis is thought to depend on changes of the intracellular redox state by the transporting of external metal ions. There was no sequential or quantitative change of Bcl-2 family proteins in DDC-induced apoptotic or necrotic pathways. However, the mitochondrial transmembrane potential was remarkably decreased in the DDC-induced necrosis. Finally, duration of c-Jun N-terminal kinase (JNK) activation resulted in different types of cell death.     

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.     

Higher plant antioxidants and redox signaling under environmental stresses

Main antioxidants in higher plants include glutathione, ascorbate, tocopherol, proline, betaine, and others, which are also information-rich redox buffers and important redox signaling components that interact with biomembrane-related compartments. As an evolutionary consequence of aerobic life for higher plants, reactive oxygen species (ROS) are formed by partial reduction of molecular oxygen. The above enzymatic and non-enzymatic antioxidants in higher plants can protect their cells from oxidative damage by scavenging ROS. In addition to crucial roles in defense system and as enzyme cofactors, antioxidants influence higher plant growth and development by modifying processes from mitosis and cell elongation to senescence and death. Most importantly, they provide essential information on cellular redox state, and regulate gene expression associated with biotic and abiotic stress responses to optimize defense and survival. An overview of the literature is presented in terms of main antioxidants and redox signaling in plant cells. Special attention is given to ROS and ROS-antioxidant interaction as a metabolic interface for different types of signals derived from metabolism and from the changing environment, which regulates the appropriate induction of acclimation processes or, execution of cell death programs, which are the two essential directions for higher plants.     

Primary antioxidant free radical scavenging and redox signaling pathways in higher plant cells

Antioxidants in plant cells mainly include glutathione, ascorbate, tocopherol, proline, betaine and others, which are also information-rich redox buffers and important redox signaling components that interact with cellular compartments. As an unfortunate consequence of aerobic life for higher plants, reactive oxygen species (ROS) are formed by partial reduction of molecular oxygen. The above enzymatic and non-enzymatic antioxidants in higher plant cells can protect their cells from oxidative damage by scavenging ROS. In addition to crucial roles in defense system and as enzyme cofactors, antioxidants influence higher plant growth and development by modifying processes from miotosis and cell elongation to senescence and death. Most importantly, they provide essential information on cellular redox state, and regulate gene expression associated with biotic and abiotic stress responses to optimize defense and survival. An overview of the literature is presented in terms of primary antioxidant free radical scavenging and redox signaling in plant cells. Special attention is given to ROS and ROS-anioxidant interaction as a metabolic interface for different types of signals derived from metabolisms and from the changing environment. This interaction regulates the appropriate induction of acclimation processes or execution of cell death programs, which are the two essential directions for higher plant cells.     

植物中参与活性氧调控的基因网络

植物体内活性氧（reactive oxygen species,ROS）是氧化还原反应的必然副产物,具极高的活性和毒性,从而对细胞产生毒害。同时,活性氧作为信号分子对很多生理过程诸如植物生长发育、细胞程序化死亡及生物和非生物胁迫应答起调控作用。植物中ROS双重作用的协调机制目前尚不明确,确定的是细胞中ROS维持于稳定水平需要精细的调节。拟南芥中至少包括152个基因组成的网络参与ROS的调控,该网络具高度的灵活性和互补性。本文综述了ROS网络中鉴定的一些关键基因及细胞学定位和协同作用,ROS信号转导,尤其是叶绿体中ROS信号的调控。     

TCR and immunophenotype changes in dimethyl sulfoxide-dependent programmed cell death

In the thymus most deleted cells are immature thymocytes and the high rate of cell death within the thymus is involved in the development of the initial T-cell receptor repertoire. Functional T-cell receptor recognition units are created by somatic rearrangements of gene segments, and the expression of successfully assembled TCR complex is the key to molecular events that culminate in T-cell activation, growth and differentiation. Previously, we reported that DMSO induces apoptosis in RPMI-8402 human pre-T cells. Here we examine the fate of pre-T cells undergoing negative selection analysing the responsiveness to DMSO-enforced TCR expression and immunophenotype modulation. Our results demonstrate that DMSO induces cell growth inhibition, cell phenotype changes, with down-regulation of CD2 and CD7, and increases in alpha/beta or gamma/delta TCR chains led by TdT, RAG-1 and RAG-2 activity. These modifications are associated with an apoptotic program. Taken together, these data suggest the existence of an early checkpoint that ensures in vivo the effective intrathymic differentiation supported from another point of view, the linkage between immunophenotypes and TCR regulation in T-cell differentiation and programmed cell death.     

Spermine prevents endonuclease activation and apoptosis in thymocytes.

Glucocorticoid hormones, Ca2+ ionophores, and some toxic chemicals activate a suicide process in thymocytes, known as apoptosis or programmed cell death. A crucial event in apoptosis is the activation of a Ca(2+)- and Mg(2+)-dependent endonuclease that promotes extensive DNA fragmentation. In this study, we investigated the effect of various polyamines on endonuclease activation leading to thymocyte apoptosis. We found that both glucocorticoid- and Ca2+ ionophore-induced DNA fragmentation and apoptosis were prevented by spermine. Other polyamines such as putrescine or spermidine had moderate or no effect. Moreover, spermine, and to a lesser extent spermidine, but not putrescine, prevented endonuclease activation in permeabilized liver nuclei incubated in the presence of Ca2+ and Mg2+, indicating that spermine efficiency in blocking DNA fragmentation was related to the interaction of this polyamine with the endonuclease or its substrate, DNA. Experiments with the fluorescent dye, ethidium bromide, and a purified preparation of liver endonuclease revealed that the protective effect of spermine on DNA fragmentation was related to its ability to modify the chromatin arrangement. Thymocytes incubated with methyl glyoxal bis(guanylhydrazone) to deplete intracellular spermine exhibited spontaneous DNA fragmentation, which suggests that modulation of the intracellular polyamine content and regulation of chromatin structure may play a critical role in the early phases of apoptosis. Finally, these results demonstrate that inhibition of DNA fragmentation also prevents the onset of apoptosis, directly linking endonuclease activation and cell death.     

Effects of heterologous expression of thioredoxin reductase on the level of reactive oxygen species in COS-7 cells

Thioredoxin reductase (TrxR), a component of the redox control system involving thioredoxin (Trx), is implicated in defense against oxidative stress, control of cell growth and proliferation, and regulation of apoptosis. In the present study a stable transfectant was made by introducing the vector pcDNA3.0 harboring the fission yeast TrxR gene into COS-7 African green monkey kidney fibroblast cells. The exogenous TrxR gene led to an increase in TrxR activity of up to 3.2-fold but did not affect glutathione (GSH) content, or glutaredoxin and caspase-3 activities. Levels of reactive oxygen species (ROS), but not those of nitric oxide (NO), were reduced. Conversely, 1-chloro-2,4-dinitrobezene (CDNB), an irreversible inhibitor of mammalian TrxR, enhanced ROS levels in the COS-7 cells. After treatment with hydrogen peroxide, the level of intracellular ROS was lower in the transfectants than in the vector control cells. These results confirm that TrxR is a crucial determinant of the level of cellular ROS during oxidative stress as well as in the normal state.     

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.     

A critical role of redox state in determining HL-60 cell granulocytic differentiation and apoptosis via involvement of PKC and NF-κB

The modifications of intracellular redox balance leads to important cellular changes in many cell types. Here, a causal relationship among redox state, granulocytic differentiation induced by all-trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) and apoptosis have been studied in the human acute promyelocytic leukaemia HL-60 cells. The modulation of intracellular reactive oxygen species levels by d, l-buthionine-(S, R) sulfoximide (BSO), and n-acetyl-l-cysteine (NAC) caused inducer- and time-dependent or stage-specific effects on HL-60 cell growth inhibition, differentiation and subsequent apoptosis. The presence of BSO during the commitment stage suppressed RA—but not dbcAMP-mediated differentiation, while NAC inhibited both. BSO alone and in combination with RA or dbcAMP-induced apoptosis, which was prevented by NAC in dbcAMP—but not in RA-treated cells. Using protein kinase C inhibitor, calphostin C, cross-talk effects between the intracellular redox state and PKC signalling was identified by demonstrating inducer-dependent changes in cell differentiation or apoptosis, which were associated with the changes in DNA-NF-κB binding activity. These observations suggest a critical role of redox state in determining HL-60 cell behaviour and provide new insights into the complex effects of redox perturbations on the intracellular signalling network via the involvement of PKC and NF-κB.     

Different roles of spermine in glucocorticoid- and Fas-induced apoptosis

Two experimental systems representative of the mitochondrial and death receptor apoptotic pathways are the dexamethasone-induced programmed cell death in mouse thymocytes and the antibody-mediated cross-ligation of the Fas receptor in the human leukemic T-cell line Jurkat, respectively. In both cell systems, caspase-9, -8, and -3 were activated upon induction of apoptosis and a sub-G(1) peak appeared as a sign of ongoing DNA fragmentation. Addition of 1 mM spermine together with dexamethasone inhibited caspase activation and the appearance of the sub-G(1) peak in mouse thymocytes. In contrast, Fas-induced cell death was totally unaffected by spermine addition. Spermine addition significantly elevated the spermine concentration in both thymocytes and Jurkat cells. Thus, spermine per se did not inhibit the caspases but rather their activation. The fact that spermine inhibited caspase activation only in the thymocytes implies that spermine inhibited dexamethasone-induced apoptosis upstream of caspase-9 activation.     

Screening of dietary antioxidants against mitochondria-mediated oxidative stress by visualization of intracellular redox state

Mitochondrial impairment and the resulting generation of reactive oxygen species (ROS) have been associated with aging and its related pathological conditions. Recently, dietary antioxidants have gained significant attention as potential preventive and therapeutic agents against ROS-generated aging and pathological conditions. We previously demonstrated that food-derived antioxidants prevented intracellular oxidative stress under proteasome inhibition conditions, which was attributed to mitochondrial dysfunction and ROS generation, followed by cell death. Here, we further screened dietary antioxidants for their activity as redox modulators by visualization of the redox state using Redoxfluor, a fluorescent protein redox probe. Direct alleviation of ROS by antioxidants, but not induction of antioxidative enzymes, prevented mitochondria-mediated intracellular oxidation. The effective antioxidants scavenged mitochondrial ROS and suppressed cell death. Our study indicates that redox visualization under mitochondria-mediated oxidative stress is useful for screening potential antioxidants to counteract mitochondrial dysfunction, which has been implicated in aging and the pathogenesis of aging-related diseases.