Overexpression Of Antioxidant Enzymes Research Articles

Potentiation of Cisplatin Cytotoxicity in Resistant Ovarian Cancer SKOV3/Cisplatin Cells by Quercetin Pre-Treatment.

Previously, we demonstrated that the overexpression of antioxidant enzymes (SOD-1, SOD-2, Gpx-1, CAT, and HO-1), transcription factor NFE2L2, and the signaling pathway (PI3K/Akt/mTOR) contribute to the cisplatin resistance of SKOV-3/CDDP ovarian cells, and treatment with quercetin (QU) alone has been shown to inhibit the expression of these genes. The aim of this study was to expand the previous data by examining the efficiency of reversing cisplatin resistance and investigating the underlying mechanism of pre-treatment with QU followed by cisplatin in the same ovarian cancer cells. The pre-incubation of SKOV-3/CDDP cells with quercetin at an optimum dose prior to treatment with cisplatin exhibited a significant cytotoxic effect. Furthermore, a long incubation with only QU for 48 h caused cell cycle arrest at the G1/S phase, while a QU pre-treatment induced sub-G1 phase cell accumulation (apoptosis) in a time-dependent manner. An in-depth study of the mechanism of the actions revealed that QU pre-treatment acted as a pro-oxidant that induced ROS production by inhibiting the thioredoxin antioxidant system Trx/TrxR. Moreover, QU pre-treatment showed activation of the mitochondrial apoptotic pathway (cleaved caspases 9, 7, and 3 and cleaved PARP) through downregulation of the signaling pathway (mTOR/STAT3) in SKOV-3/CDDP cells. This study provides further new data for the mechanism by which the QU pre-treatment re-sensitizes SKOV-3/CDDP cells to cisplatin.

Do Antioxidants Extend Longevity in Invertebrate and Vertebrate Animals?

In the 1950’s Denham Harman proposed the Free Radical Theory of Aging whereby species lifespan and individual longevities are the consequence of free radical driven damage to biomolecules. This led to decades of research to ascertain the effect of altered antioxidant defense systems on aging and mortality in an array of species using reverse genetics and dietary manipulation. Within invertebrates, the data generally support the Free Radical Theory in that overexpression of antioxidant enzymes or dietary supplementation with antioxidant compounds increases longevity and resistance to oxidative damage. Likewise, genetic knockdown of antioxidant defenses generally shortens longevity within invertebrates. On the other hand, for endothermic vertebrates (i.e., birds and mammals) the results have been equivocal. Downregulation of antioxidant enzymes typically results in an increased oxidative burden, but without an appreciable effect on longevity, while dietary supplementation with antioxidants has little-to-no effect, at least at the concentrations used. Upregulation of antioxidant enzyme genes also fails to increase longevity in vertebrates most of the time. Interestingly, manipulating antioxidant defenses in fishes increases longevity in conjunction with reduced oxidative damage akin to what is seen in invertebrates. Since invertebrates and fishes are both exothermic this raises the possibility that the evolution of endothermy interferes with the ability of antioxidants to slow the aging process.

Crucial Roles of Ubiquitin Carboxy-Terminal Hydrolase L1 in Motor Neuronal Health by Drosophila Model.

Aims: Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) plays an important role in the ubiquitin-proteasome system and is distributed mostly in the brain. Previous studies have shown that mutated forms or reduction of UCH-L1 are related to neurodegenerative disorders, but the mechanisms of pathogenesis are still not well understood. To study its roles in motor neuronal health, we utilized the Drosophila model in which dUCH, a homolog of human UCH-L1, was specifically knocked down in motor neurons. Results: The reduction of Drosophila ubiquitin carboxyl-terminal hydrolase (dUCH) in motor neurons induced excessive reactive oxygen species production and multiple aging-like phenotypes, including locomotive defects, muscle degeneration, enhanced apoptosis, and shortened longevity. In addition, there is a decrease in the density of the synaptic active zone and glutamate receptor area at the neuromuscular junction. Interestingly, all these defects were rescued by vitamin C treatment, suggesting a close association with oxidative stress. Strikingly, the knockdown of dUCH at motor neurons exhibited aberrant morphology and function of mitochondria, such as mitochondrial DNA (mtDNA) depletion, an increase in mitochondrial size, and overexpression of antioxidant enzymes. Innovation: This research indicates a new, possible pathogenesis of dUCH deficiency in the ventral nerve cord and peripheral nervous systems, which starts with abnormal mitochondria, leading to oxidative stress and accumulation aging-like defects in general. Conclusion: Taken together, by using the Drosophila model, our findings strongly emphasize how the UCH-L1 shortage affects motor neurons and further demonstrate the crucial roles of UCH-L1 in neuronal health. Antioxid. Redox Signal. 37, 257-273.

Modification of Chloroplast Antioxidant Capacity by Plastid Transformation.

As immobile organisms, green plants must be frequently challenged by a broad range of environmental stresses. During these constantly adverse conditions, reactive oxygen species (ROS) levels can rise extremely in plants, leading to cellular dysfunction and cell death presumably due to irreversible protein overoxidation. Once considered merely as deleterious molecules, cells seek to remove them as efficiently as possible. To enhance ROS scavenging capacity, genes encoding antioxidative enzymes can be directly expressed from the genome of plastid (chloroplast), a major compartment for ROS production in photosynthetic organisms. Thus, overexpression of antioxidant enzymes by plastid engineering may provide an alternative to enhance plant's tolerance to stressful conditions specifically related with chloroplast-derived ROS. Here, we describe basic procedures for expressing glutathione reductase, a vital component of ascorbate-glutathione pathway, in tobacco via plastid transformation technology.

Exercise as a calorie restriction mimetic. Implications for the treatment of age associated frailty

The free radical theory of aging has provided a theoretical framework for an enormous amount of work leading to advances in our understanding of aging. A critical blow to the free radical theory of aging came from epidemiological studies showing that antioxidant supplementation did not lower the incidence of age-associated diseases. However, work from many laboratories supports the theory, for instance showing that overexpression of antioxidant enzymes results in increases not only in life-span but also in health-span. Improving healthy aging means delaying disability. Disability is often preceded by a state characterized by reduced capacity to respond to stressors, caused by a decline in functional reserves. This condition, frailty, might precede by several years the development of disability. We have recently found that a mouse model with overexpression of G6PD have lower levels of ROS-derived damage and this is accompanied by a protection from frailty. Two interventions with translational potential to improve healthy aging are caloric restriction and exercise training. Both of them share a common protective molecular mechanism, a reduction in oxidative damage through the induction of the antioxidant defense.

Oxidant/antioxidant effects of chronic exposure to predator odor in prefrontal cortex, amygdala, and hypothalamus.

The incidence of anxiety-related diseases is increasing these days, hence there is a need to understand the mechanisms that underlie its nature and consequences. It is known that limbic structures, mainly the prefrontal cortex and amygdala, are involved in the processing of anxiety, and that projections from prefrontal cortex and amygdala can induce activity of the hypothalamic–pituitary–adrenal axis with consequent cardiovascular changes, increase in oxygen consumption, and ROS production. The compensatory reaction can include increased antioxidant enzymes activities, overexpression of antioxidant enzymes, and genetic shifts that could include the activation of antioxidant genes. The main objective of this study was to evaluate the oxidant/antioxidant effect that chronic anxiogenic stress exposure can have in prefrontal cortex, amygdala, and hypothalamus by exposition to predator odor. Results showed (a) sensitization of the HPA axis response, (b) an enzymatic phase 1 and 2 antioxidant response to oxidative stress in amygdala, (c) an antioxidant stability without elevation of oxidative markers in prefrontal cortex, (d) an elevation in phase 1 antioxidant response in hypothalamus. Chronic exposure to predator odor has an impact in the metabolic REDOX state in amygdala, prefrontal cortex, and hypothalamus, with oxidative stress being prevalent in amygdala as this is the principal structure responsible for the management of anxiety.

Oxidized low-density lipoprotein alters endothelial progenitor cell populations

Oxidized low-density lipoprotein (ox-LDL) is critical to atherosclerosis in hyperlipidemia. Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are important to preventing atherosclerosis, and significantly decreased in hyperlipidemia. This study was to demonstrate ox-LDL and hyperlipidemia could exhibit similar effect on EPC population and the role of reactive oxygen species (ROS). ROS production in BM and blood was significantly increased in male C57BL/6 mice with intravenous ox-LDL treatment, and in hyperlipidemic LDL receptor knockout mice with 4-month high-fat diet. ROS formation was effectively blocked with overexpression of antioxidant enzymes or N-acetylcysteine treatment. In hyperlipidemic and ox-LDL-treated mice, c-Kit(+)/CD31(+) cell number in BM and blood, and Sca-1(+)/Flk-1(+) cell number in blood, not in BM, were significantly decreased, which were not affected by inhibiting ROS production, while blood CD34(+)/Flk-1(+) cell number was significantly increased that was prevented with reduced ROS formation. However, blood CD34(+)/CD133(+) cell number increased in ox-LDL-treated mice, while decreased in hyperlipidemic mice. These data suggested that ox-LDL produced significant changes in BM and blood EPC populations similar (but not identical) to chronic hyperlipidemia with predominantly ROS-independent mechanism(s).

Oxidative Imbalance and Anxiety Disorders

The oxidative imbalance appears to have an important role in anxiety development. Studies in both humans and animals have shown a strong correlation between anxiety and oxidative stress. In humans, for example, the increased malondialdehyde levels and discrepancies in antioxidant enzymes in erythrocytes have been observed. In animals, several studies also show that anxiety-like behavior is related to the oxidative imbalance. Moreover, anxiety-like behavior can be caused by pharmacological-induced oxidative stress. Studies using knockout or overexpression of antioxidant enzymes have shown a relationship between anxiety-like behavior and oxidative stress. Related factors of oxidative stress that could influence anxious behavior are revised, including impaired function of different mitochondrial proteins, inflammatory cytokines, and neurotrophic factors. It has been suggested that a therapy specifically focus in reducing reactive species production may have a beneficial effect in reducing anxiety. However, the neurobiological pathways underlying the effect of oxidative stress on anxiety symptoms are not fully comprehended. The challenge now is to identify the oxidative stress mechanisms likely to be involved in the induction of anxiety symptoms. Understanding these pathways could help to clarify the neurobiology of the anxiety disorder and provide tools for new discovery in therapies and preventive strategies.

Energy and Redox Homeostasis in Tumor Cells

Cancer cells display abnormal morphology, chromosomes, and metabolism. This review will focus on the metabolism of tumor cells integrating the available data by way of a functional approach. The first part contains a comprehensive introduction to bioenergetics, mitochondria, and the mechanisms of production and degradation of reactive oxygen species. This will be followed by a discussion on the oxidative metabolism of tumor cells including the morphology, biogenesis, and networking of mitochondria. Tumor cells overexpress proteins that favor fission, such as GTPase dynamin-related protein 1 (Drp1). The interplay between proapoptotic members of the Bcl-2 family that promotes Drp 1-dependent mitochondrial fragmentation and fusogenic antiapoptotic proteins such as Opa-1 will be presented. It will be argued that contrary to the widespread belief that in cancer cells, aerobic glycolysis completely replaces oxidative metabolism, a misrepresentation of Warburg's original results, mitochondria of tumor cells are fully viable and functional. Cancer cells also carry out oxidative metabolism and generally conform to the orthodox model of ATP production maintaining as well an intact electron transport system. Finally, data will be presented indicating that the key to tumor cell survival in an ROS rich environment depends on the overexpression of antioxidant enzymes and high levels of the nonenzymatic antioxidant scavengers.

Catalase and Superoxide Dismutase Conjugated with Platelet-Endothelial Cell Adhesion Molecule Antibody Distinctly Alleviate Abnormal Endothelial Permeability Caused by Exogenous Reactive Oxygen Species and Vascular Endothelial Growth Factor

Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of specific ROS in vascular pathology and may be translated into remedies for these ROS-induced abnormalities.

Reactive oxygen species and the brain in sleep apnea

Rodents exposed to intermittent hypoxia (IH), a model of obstructive sleep apnea (OSA), manifest impaired learning and memory and somnolence. Increased levels of reactive oxygen species (ROS), oxidative tissue damage, and apoptotic neuronal cell death are associated with the presence of IH-induced CNS dysfunction. Furthermore, treatment with antioxidants or overexpression of antioxidant enzymes is neuroprotective during IH. These findings mimic clinical cases of OSA and suggest that ROS may play a key causal role in OSA-induced neuropathology. Controlled production of ROS occurs in multiple subcellular compartments of normal cells and de-regulation of such processes may result in excessive ROS production. The mitochondrial electron transport chain, especially complexes I and III, and the NADPH oxidase in the cellular membrane are the two main sources of ROS in brain cells, although other systems, including xanthine oxidase, phospholipase A2, lipoxygenase, cyclooxygenase, and cytochrome P450, may all play a role. The initial evidence for NADPH oxidase and mitochondrial involvement in IH-induced ROS production and neuronal injury unquestionably warrants future research efforts.

Modulation of cellular effects of gamma-rays by overexpression of antioxidant enzymes in human cells

Modulation of cellular effects of gamma-rays by overexpression of antioxidant enzymes in human cells

ECTOPIC EXPRESSION OF A SPINACH SOD GENE IN YOUNG APPLE TREES ENHANCES ABIOTIC STRESS RESISTANCE

Reactive oxygen species (ROS) are induced during both biotic and abiotic stress, either as signaling molecules or as result of stress injury. ROS are highly destructive to cell components and the injury resulting from these compounds is referred to as oxidative stress. Antioxidant enzymes, such as superoxide dismutase (SOD), scavenge oxygen radicals preventing the injury resulting from oxidative stress. The objective of the present research was to produce transgenic apple plants (Malus x domestica 'Royal Gala') with enhanced production of a cytosolic SOD. A full-length SOD cDNA was isolated from spinach by a combination of RT-PCR and conventional plaque lift screening of a pea cDNA library. The SOD gene was mobilized into a binary vector consisting of pBINPLUSARS and pRTL2 for Agrobacterium-mediated transformation of apple. The resulting SOD-overexpression (SOD-OX), blank-cassette, and un-transformed lines were evaluated for resistance to acute and prolonged exposure to high temperature, and freezing temperatures in non-acclimated and acclimated plants, by ion leakage assays of leaves and bark from one-year-old trees. Results indicated that SOD-OX leaves exhibited improved resistance to both acute (30 min) and longer-term exposure (2h to 24h) to elevated temperatures compared to the non SOD-OX lines. Cold tolerance of non-cold-acclimated SOD-OX tissues did not differ from the control plants. Cold acclimated (2 weeks exposure to a short day photoperiod at 4°C) leaves of SOD-OX trees, however, were more cold tolerant compared to the other lines, while bark was not. The overexpression of antioxidant enzymes is believed to help cells recover from post-injury ROS rather than directly increase stress tolerance. Therefore, the differences observed in increased stress tolerance in the transgenic apple plants may be a reflection of the type and extent of injury caused by heat vs. freezing stress.

Glutathione peroxidase protein expression and activity in human islets isolated for transplantation

Overexpression of antioxidant enzymes has been reported to protect rodent beta cells from oxidative stress. However, very little is known about protein expression and activity of antioxidant enzymes in human islets. Human islet protein levels by Western analysis and enzymatic activity for the key antioxidant enzymes superoxide dismutases (SODs), catalase, and glutathione peroxidase-1 (GPx) were examined. Enzyme protein expression and activity were in the order SODs > catalase > GPx. Human islet GPx protein expression was significantly less than that found for catalase (p < 0.0001) and levels of GPx activity were virtually undetectable. As glucose and estrogens have been proposed to alter antioxidant enzyme levels, we examined islet data from male and female donors separately and under varying glucose concentrations. We found significantly less (p < 0.001) GPx protein expression in islets from females compared to males, but no significant regulation by glucose in either gender. Human islets have very low protein and activity levels for GPx, the essential enzyme for protection against excessive levels of intracellular lipid peroxides. GPx mimetics may be especially valuable in providing human islets with the broadest spectrum of protection against oxidative stress during isolation and transplantation.

Genetic variation for life span, resistance to paraquat, and spontaneous activity in unselected populations of Drosophila melanogaster: Implications for transgenic rescue of life span

Genetic variation in adult life span, resistance to paraquat, resistance to DDT, and spontaneous flying activity were measured in 138 recombinant inbred lines of Drosophila melanogaster. We find that the phenotypic correlation between life span and resistance to an exogenous oxidizing agent is positive, though weak, and that there is little correlation between the two traits at the level of quantitative trait loci (QTLs). The sign of the life span–resistance correlation is haplotype-specific, suggesting a high degree of statistical interaction and dependence on genetic background. Because of the genotype-specificity in the relationship between life span and resistance phenotypes, interventions to extend life span by overexpression of antioxidant enzymes are likely to produce strain-specific results. These observations are in general agreement with the “genetic rescue” hypothesis of Sohal et al. [Sohal, R.S., Mockett, R.J., Orr, W.C., 2002. Mechanisms of aging: an appraisal of the oxidative stress hypothesis. Free Radic. Biol. Med. 33, 575–586.], though we emphasize that such statistical interaction is a normal feature of standing genetic variation, and does not imply that some genotypes are pathological. Ad hoc observation of spontaneous flying activity 5 days after emergence proved to be a much better predictor of life span than resistance to an exogenous oxidant in these populations.

Antioxidant Enzyme Induction: A New Protective Approach Against the Adverse Effects of Diesel Exhaust Particles

Exposure to airborne particulate pollutants such as diesel exhaust particles (DEPs) has been associated with allergic respiratory disorders, including asthma and allergic rhinitis. In this communication, we review recent advances in the mechanism by which DEPs elicit their harmful effects and the protective role of antioxidants. Reactive oxidative species (ROS) are believed to play a key role in cellular damage after exposure to DEPs. Numerous reports demonstrate that both proinflammatory and anti-inflammatory products are induced by DEPs via the activation of transcription factors. DEPs trigger multiple signaling pathways, which lead to DNA damage and cell apoptosis, inflammatory response, and antioxidant defense. Recent studies both in vitro and in mice show that antioxidants could alleviate the allergic inflammatory effects of DEPs. Human in vivo models suggest that the important phase II enzymes GSTM1 and GSTP1 modify the adjuvant effect of diesel exhaust particles on allergic inflammation. We have shown that the induction of phase II enzymes by the chemical sulforaphane can block DEP-induced enhanced immunoglobulin (Ig) E production in B cells and DEP-induced proinflammatory cytokine production in epithelial cells. These findings suggest that overexpression of antioxidant enzymes could constitute a powerful potential chemopreventive approach against adverse effects induced by oxidant pollutants such as DEPs.

Metabolomic, proteomic and biophysical analyses of Arabidopsis thaliana cells exposed to a caesium stress. Influence of potassium supply

The incorporation and localisation of 133Cs in a plant cellular model and the metabolic response induced were analysed as a function of external K concentration using a multidisciplinary approach. Sucrose-fed photosynthetic Arabidopsis thaliana suspension cells, grown in a K-containing or K-depleted medium, were submitted to a 1 mM Cs stress. Cell growth, strongly diminished in absence of K, was not influenced by Cs. In contrast, the chlorophyll content, affected by a Cs stress superposed to K depletion, did not vary under the sole K depletion. The uptake of Cs was monitored in vivo using 133Cs NMR spectroscopy while the final K and Cs concentrations were determined using atomic absorption spectrometry. Cs absorption rate and final concentration increased in a K-depleted external medium; in vivo NMR revealed that intracellular Cs was distributed in two kinds of compartment. Synchrotron X-ray fluorescence microscopy indicated that one could be the chloroplasts. In parallel, the cellular response to the Cs stress was analysed using proteomic and metabolic profiling. Proteins up- and down-regulated in response to Cs, in presence of K + or not, were analysed by 2D gel electrophoresis and identified by mass spectrometry. No salient feature was detected excepting the overexpression of antioxidant enzymes, a common response of Arabidopsis cells stressed whether by Cs or by K-depletion. 13C and 31P NMR analysis of acid extracts showed that the metabolome impact of the Cs stress was also a function of the K nutrition. These analyses suggested that sugar metabolism and glycolytic fluxes were affected in a way depending upon the medium content in K +. Metabolic flux measurements using 13C labelling would be an elegant way to pursue on this line. Using our experimental system, a progressively stronger Cs stress might point out other specific responses elicited by Cs.

1062. Potential Utility of Heat Shock Protein 70 Promoter (HSP70-P) as a Stress Responsive Promoter in Gene Delivery: HSP70-P Regulated Expression of Transgenes SOD1 and GPX1

Cells respond to heat stress by synthesis and accumulation of several members of functionally distinct family of heat shock (or stress) proteins (HSP27, HSP60, HSP70 and HSP90). HSPs confer protection against a large array of cytotoxic reagents and are implicated in thermo tolerance development. Heat shock protein70-promoter (HSP70-P) is a stress responsive promoter, which is highly responsive to a variety of stresses, including heat stress. We hypothesized that HSP70-P or other stress-responsive promoter-driven antioxidant expression could provide reasonable sensitivity of transgene expression to heat and other stresses, while avoiding constitutive overexpression of anti-oxidant enzymes. Our eventual goal is to evaluate protection of neurons from oxidant injury such as is elicited by exposure to HIV-1 gp120. In the present study we tested HSP70-P for gene delivery. We first performed a series of transfection studies using luciferase as a reporter gene. Expression of luciferase driven by HSP70-P was triggered maximally at 8Hrs post heat shock at 410C when tested in 293 and NT-2 derived neurons. Having established the likelihood that the HSP70-P could be used as a stress-responsive promoter to drive transgene expression, we then used it to test expression of antioxidant transgenes SOD1 and GPX1 in response to heat stress. HSP-70 promoter driven expression of SOD1 and GPX1 was studied in 293 cells and in terminally differentiated NT-2 derived neuronal cells by heat stress over a range of 0-72Hr. Cells, when stressed for an optimal 4-8Hr period, induced maximal expression of these transgenes. The increase in expression was about 4-fold at the optimal time period with no further increase in expression at later time intervals. These studies using a stress responsive promoter may help to better understand the ability of heat-shock promoter to regulate expression of enzymes needed to provide anti-oxidant induce protection.

Adenovirus-Mediated Delivery of Catalase to Retinal Pigment Epithelial Cells Protects Neighboring Photoreceptors from Photo-Oxidative Stress

Oxidative stress is involved in the pathogenesis of many diseases. Overexpression of antioxidant enzymes by gene therapy may protect tissues from oxidative damage. Because the reactive oxygen species hydrogen peroxide can diffuse across cell membranes, we hypothesized that overexpression of the antioxidant catalase within certain cells might protect neighboring cells. To test this hypothesis, we transduced retinal pigment epithelial (RPE) cells in vitro and in vivo with adenovirus carrying the catalase gene (Ad.CMV.catalase). After transduction of only a subset of RPE cells in vitro, all cells in the culture were protected from exogenous hydrogen peroxide. Similarly, in vivo, eyes injected with Ad. CMV. catalase had high catalase levels in the RPE, which protected the adjacent photoreceptors from light damage and reduced photoreceptor oxidative stress as measured by the markers 4-hydroxynonenal and nitrotyrosine. Both in vitro and in vivo, gene therapy with Ad. CMV. catalase protected neighboring cells from oxidative stress-induced terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) positivity. The data provide a paradigm for antioxidant gene therapy with catalase, designed to protect not only transduced cells, but also neighboring cells.

Fas-stimulated generation of reactive oxygen species or exogenous oxidative stress sensitize cells to Fas-mediated apoptosis.

Inhibition of Fas-mediated apoptosis in B cell lymphomas by thiol antioxidants (glutathione and N-acetylcysteine) supported previous studies, suggesting that Fas-stimulated ROS generation may play a role in Fas-mediated apoptosis. Thus, the goal of the current study was to determine if Fas stimulation could induce ROS generation and what role, if any, it played in apoptosis. Fas crosslinking induced rapid generation of ROS (within 15 min) well before the appearance of characteristic apoptotic changes. Overexpression of catalase or superoxide dismutase suggested that Fas induced production of both superoxide anion and hydrogen peroxide. ROS generation was only observed, however, in cells that were sensitive to apoptosis and not in B cells inherently resistant to anti-Fas or in those in which resistance was induced by B cell receptor crosslinking. The exogenous addition of 250 microM hydrogen peroxide could reverse the resistant phenotype and sensitize cells to Fas-induced apoptosis. In Fas-sensitive cells, depletion of endogenous antioxidant defenses with buthionine sulfoximine increased the sensitivity to Fas-induced apoptosis, while overexpression of antioxidant enzymes and antiapoptotic proteins suggested a role for Fas-induced production of hydrogen peroxide in apoptosis. Further analysis suggested a redox-sensitive step early in Fas signaling at the level of initiator caspase (caspase-8) activation. Thus, the data suggest that the level of oxidative stress, either from exogenous sources or generated endogenously upon receptor stimulation, regulates the sensitivity to Fas-mediated apoptosis.