Old yellow enzymes, highly homologous FMN oxidoreductases with modulating roles in oxidative stress and programmed cell death in yeast

In a genetic screen to identify modifiers of Bax-dependent lethality in yeast, the C terminus of OYE2 was isolated based on its capacity to restore sensitivity to a Bax-resistant yeast mutant strain. Overexpression of full-length OYE2 suppresses Bax lethality in yeast, lowers endogenous reactive oxygen species (ROS), increases resistance to H(2)O(2)-induced programmed cell death (PCD), and significantly lowers ROS levels generated by organic prooxidants. Reciprocally, Delta oye2 yeast strains are sensitive to prooxidant-induced PCD. Overexpression and knock-out analysis indicate these OYE2 antioxidant activities are opposed by OYE3, a highly homologous heterodimerizing protein, which functions as a prooxidant promoting H(2)O(2)-induced PCD in wild type yeast. To exert its effect OYE3 requires the presence of OYE2. Deletion of the 12 C-terminal amino acids and catalytic inactivation of OYE2 by a Y197F mutation enhance significantly survival upon H(2)O(2)-induced PCD in wild type cells, but accelerate PCD in Delta oye3 cells, implicating the oye2p-oye3p heterodimer for promoting cell death upon oxidative stress. Unexpectedly, a strain with a double knock-out of these genes (Delta oye2 oye3) is highly resistant to H(2)O(2)-induced PCD, exhibits increased respiratory capacity, and undergoes less cell death during the adaptive response in chronological aging. Simultaneous deletion of OYE2 and other antioxidant genes hyperinduces endogenous levels of ROS, promoting H(2)O(2)-induced cell death: in Delta oye2 glr1 yeast high levels of oxidized glutathione elicited gross morphological aberrations involving the actin cytoskeleton and defects in organelle partitioning. Altering the ratio of reduced to oxidized glutathione by exogenous addition of GSH fully reversed these alterations. Based on this work, OYE proteins are firmly placed in the signaling network connecting ROS generation, PCD modulation, and cytoskeletal dynamics in yeast.     

Hydrogen peroxide as a signal controlling plant programmed cell death

Hydrogen peroxide (H2O2) has established itself as a key player in stress and programmed cell death responses, but little is known about the signaling pathways leading from H2O2 to programmed cell death in plants. Recently, identification of key regulatory mutants and near-full genome coverage microarray analysis of H2O2-induced cell death have begun to unravel the complexity of the H2O2 network. This review also describes a novel link between H2O2 and sphingolipids, two signals that can interplay and regulate plant cell death.     

Oxidative stress and mitochondrial dysfunctions are early events in narciclasine-induced programmed cell death in tobacco Bright Yellow-2 cells

Narciclasine (NCS) is a plant growth inhibitor isolated from the secreted mucilage of Narcissus tazetta bulbs. It is a commonly used anticancer agent in animal systems. In this study, we provide evidence to show that NCS also acts as an agent in inducing programmed cell death (PCD) in tobacco Bright Yellow-2 (TBY-2) cell cultures. NCS treatment induces typical PCD-associated morphological and biochemical changes, namely cell shrinkage, chromatin condensation and nuclear DNA degradation. To investigate possible signaling events, we analyzed the production of reactive oxygen species (ROS) and the function of mitochondria during PCD induced by NCS. A biphasic behavior burst of hydrogen peroxide (H(2)O(2)) was detected in TBY-2 cells treated with NCS, and mitochondrial transmembrane potential (MTP) loss occurred after a slight increase. Pre-incubation with antioxidant catalase (CAT) and N-acetyl-L-cysteine (NAC) not only significantly decreased the H(2)O(2) production but also effectively retarded the decrease of MTP and reduced the percentage of cells undergoing PCD after NCS treatment. In conclusion, our results suggest that NCS induces PCD in plant cells; the oxidative stress (accumulation of H(2)O(2)) and the MTP loss play important roles during NCS-induced PCD.     

Active oxygen and cell death in cereal aleurone cells

The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.     

Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner

Active oxygen species (AOS), especially hydrogen peroxide, play a critical role in the defence of plants against invading pathogens and in the hypersensitive response (HR). This is characterized by the induction of a massive production of AOS and the rapid appearance of necrotic lesions is considered as a programmed cell death (PCD) process during which a limited number of cells die at the site of infection. This work was aimed at investigating the mode of cell death observed in cultures of BY-2 tobacco cells exposed to H(2)O(2). It was shown that H(2)O(2) is able to induce various morphological cell death features in cultured tobacco BY-2 cells. The hallmarks of cell death observed with fluorescent and electron microscopy differed greatly with the amount of H(2)O(2) added to the cell culture. The appearance of nuclear fragmentation similar to 'apoptotic bodies' associated with a fragmentation of the nuclear DNA into small fragments appear for almost 18% of the cells treated with 12.5 mM H(2)O(2). The early stages of the induction of this PCD process consisted in cell shrinkage and chromatin condensation at the periphery of the nucleus. Above 50 mM, H(2)O(2) induces high necrotic cell death. These data suggest that H(2)O(2)-induced cell damage is associated with the induction of various cell death processes that could be involved differently in plant defence reactions.     

Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells.

Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO(4). Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2-3 days which indicates the existence of an adaptation mechanism. Cadmium-induced cell death was alleviated by the addition of sub muM concentrations of peptide inhibitors specific to human caspases indicating that cell death proceeds through a mechanism with similarities to animal programmed cell death (PCD, apoptosis). Cadmium-induced cell death was accompanied by an increased production of hydrogen peroxide (H(2)O(2)) and simultaneous addition of antioxidants greatly reduced cell death. Inhibitors of phospholipase C (PLC) and phospholipase D (PLD) signalling pathway intermediates reduced cadmium-induced cell death. Treatment with the G-protein activator mastoparan and a cell permeable analogue of the lipid signal second messenger phosphatidic acid (PA) induced cell death. Ethylene, while not inducing cell death when applied alone, stimulated cadmium-induced cell death. Application of the ethylene biosynthesis inhibitor aminoethoxy vinylglycine (AVG) reduced cadmium-induced cell death, and this effect was alleviated by simultaneous treatment with ethylene. Together the results show that cadmium induces PCD exhibiting apoptotic-like features. The cell death process requires increased H(2)O(2) production and activation of PLC, PLD and ethylene signalling pathways.     

The oleate-stimulated phospholipase D, PLDdelta, and phosphatidic acid decrease H2O2-induced cell death in Arabidopsis

Hydrolysis of common membrane phospholipids occurs in response to various environmental stresses, but the control and cellular function of this hydrolysis are not fully understood. Hydrogen peroxide (H2O2) is a pivotal signaling molecule involved in various stress responses. Here, we show that the plasma membrane-bound phospholipase D, PLDdelta, is activated in response to H2O2 and that the resulting phosphatidic acid (PA) functions to decrease H2O2-promoted programmed cell death. The Arabidopsis genome has 12 PLD genes, and knockout of PLDdelta abolishes specifically the oleate-stimulated PLD activity. H2O2 treatment of Arabidopsis cells activates PLD enzyme activity, and ablation of PLDdelta abolishes that activation. PLDdelta-null cells display increased sensitivity to H2O2-induced cell death. The addition of PA to PLDdelta-null cells mitigates the H2O2 effect, whereas suppression of the H2O2-induced PA formation in wild-type cells increases the effect. PLDdelta-ablated plants exhibit increased susceptibility to stress. These results demonstrate that activation of oleate-stimulated PLDdelta constitutes an important step in the plant response to H2O2 and increasing plant stress tolerance.     

Catalase T and Cu,Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae

To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA-PCD), we compared Saccharomyces cerevisiae cells (C-Y) and cells individually over-expressing catalase T (CTT1-Y) and Cu,Zn-SOD (SOD1-Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA-PCD does not occur in CTT1-Y, where H2O2 levels were lower than in C-Y and the over-expressed catalase activity decreased with time. In SOD1-Y, AA-PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA-PCD, but catalase activity was strongly reduced.     

Programmed cell death in Giardia

Programmed cell death (PCD) has been observed in many unicellular eukaryotes; however, in very few cases have the pathways been described. Recently the early divergent amitochondrial eukaryote Giardia has been included in this group. In this paper we investigate the processes of PCD in Giardia. We performed a bioinformatics survey of Giardia genomes to identify genes associated with PCD alongside traditional methods for studying apoptosis and autophagy. Analysis of Giardia genomes failed to highlight any genes involved in apoptotic-like PCD; however, we were able to induce apoptotic-like morphological changes in response to oxidative stress (H2O2) and drugs (metronidazole). In addition we did not detect caspase activity in induced cells. Interestingly, we did observe changes resembling autophagy when cells were starved (staining with MDC) and genome analysis revealed some key genes associated with autophagy such as TOR, ATG1 and ATG 16. In organisms such as Trichomonas vaginalis, Entamoeba histolytica and Blastocystis similar observations have been made but no genes have been identified. We propose that Giardia possess a pathway of autophagy and a form of apoptosis very different from the classical known mechanism; this may represent an early form of programmed cell death.     

Peroxiredoxin I and II inhibit H2O2-induced cell death in MCF-7 cell lines

Apoptosis is known to be induced by direct oxidative damage due to oxygen-free radicals or hydrogen peroxide or by their generation in cells by the actions of injurious agents. Together with glutathione peroxidase and catalase, peroxiredoxin (Prx) enzymes play an important role in eliminating peroxides generated during metabolism. We investigated the role of Prx enzymes during cellular response to oxidative stress. Using Prx isoforms-specific antibodies, we investigated the presence of Prx isoforms by immunoblot analysis in cell lysates of the MCF-7 breast cancer cell line. Treatment of MCF-7 with hydrogen peroxide (H2O2) resulted in the dose-dependent expressions of Prx I and II at the protein and mRNA levels. To investigate the physiologic relevance of the Prx I and II expressions induced by H2O2, we compared the survivals of MCF10A normal breast cell line and MCF-7 breast cancer cell line following exposure to H2O2. The treatment of MCF10A with H2O2 resulted in rapid cell death, whereas MCF-7 was resistant to H2O2. In addition, we found that Prx I and II transfection enabled MCF10A cells to resist H2O2-induced cell death. These findings suggest that Prx I and II have important functions as inhibitors of cell death during cellular response to oxidative stress.     

Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes

Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are "immature," in contrast to "mature" mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 microM for 15 min) hydrogen peroxide (H(2)O(2)) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H(2)O(2) induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.     

Protective effect of the octadecaneuropeptide on hydrogen peroxide-induced oxidative stress and cell death in cultured rat astrocytes

Oxidative stress, resulting from accumulation of reactive oxygen species (ROS), plays a critical role on astrocyte death associated with neurodegenerative diseases. Astroglial cells produce endozepines, a family of biologically active peptides that have been implicated in cell protection. Thus, the purpose of the present study was to investigate the potential protective effect of one of the endozepines, the octadecaneuropeptide ODN, on hydrogen peroxide (H(2) O(2) )-induced oxidative stress and cell death in rat astrocytes. Incubation of cultured astrocytes with graded concentrations of H(2) O(2) for 1 h provoked a dose-dependent reduction of the number of living cells as evaluated by lactate dehydrogenase assay. The cytotoxic effect of H(2) O(2) was associated with morphological modifications that were characteristic of apoptotic cell death. H(2) O(2) -treated cells exhibited high level of ROS associated with a reduction of both superoxide dismutases (SOD) and catalase activities. Pre-treatment of astrocytes with low concentrations of ODN dose-dependently prevented cell death induced by H(2) O(2) . This effect was accompanied by a marked attenuation of ROS accumulation, reduction of mitochondrial membrane potential and activation of caspase 3 activity. ODN stimulated SOD and catalase activities in a concentration-dependent manner, and blocked H(2) O(2) -evoked inhibition of SOD and catalase activities. Blockers of SOD and catalase suppressed the effect of ODN on cell survival. Taken together, these data demonstrate for the first time that ODN is a potent protective agent that prevents oxidative stress-induced apoptotic cell death.     

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H₂O₂) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H₂O₂ accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H₂O₂ production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H₂O₂ accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells.     

A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and nuclear and DNA fragmentation that are commonly associated with apoptosis in animal systems. These effects of camptothecin can effectively be blocked by inhibitors of animal caspases, indicating that, in tomato suspension cells, camptothecin induces a form of programmed cell death (PCD) with similarities to animal apoptosis (A.J. De Jong et al. (2000) Planta 211:656-662). Camptothecin induced cell death was employed to study processes involved in plant PCD. Camptothecin induced a transient increase in H2O2 production starting within 2 h of application. Both camptothecin-induced cell death and the release of H2O2 were effectively blocked by application of the calcium-channel blocker lanthanum chloride, the caspase-specific inhibitor Z-Asp-CH2-DCB, or the NADPH oxidase inhibitor diphenyl iodonium, indicating that camptothecin exerts its effect on cell death through a calcium- and caspase-dependent stimulation of NADPH oxidase activity. In addition, we show that ethylene is an essential factor in camptothecin-induced PCD. Inhibition of either ethylene synthesis or ethylene perception by L-alpha-(2-aminoethoxyvinyl)glycine or silver thiosulphate, respectively, blocked camptothecin-induced H2O2 production and PCD. Although, in itself, insufficient to trigger H2O2 production and cell death, exogenous ethylene greatly stimulated camptothecin-induced H2O2 production and cell death. These results show that ethylene is a potentiator of the camptothecin-induced oxidative burst and subsequent PCD in tomato cells. The possible mechanisms by which ethylene stimulates cell death are discussed.     

Nitric oxide and protein S-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H(2)O(2)) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H(2)O(2)-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H(2)O(2)-induced leaf cell death in rice.     

Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells

Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)-mediated cell death is unknown. We examined H(2)O(2)-induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)-scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time- and concentration-dependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)-mediated cell death.