Oxidative stress to human lymphocytes by xanthine oxidoreductase activity

The in vitro toxicity of the reactive oxygen species generating enzyme xanthine oxidoreductase (XOR) to human peripheral blood lymphocytes was studied after stimulation with phytohaemoagglutinin or anti-CD3/CD28 antibodies. Apoptosis and necrosis were induced by the XOR/hypoxanthine system in a time- and concentration-dependent manner. CD8+ lymphocytes showed a higher sensitivity than CD4+ cells to the XOR/hypoxanthine system. The occurrence of apoptosis was demonstrated by annexin-V binding to injured cell membrane, which was the most precocious alteration observed, followed by the increment of transglutaminase activity, which was significant at the lowest XOR concentration used. Nuclear damage was assessed by the increased hypodiploid nuclei and by DNA migration on gel electrophoresis, which turned to an apoptotic pattern before the occurrence of cell membrane necrotic lesions. Apoptosis was induced by XOR activity proportionally to substrate concentration and was prevented by the competitive enzyme inhibitor, allopurinol. The hydrogen peroxide scavenging enzyme, catalase, gave a higher protection than superoxide dismutase from the toxicity caused by the XOR/hypoxanthine system. Necrosis occurs in a variable percentage indicating that reactive oxygen species may trigger both apoptosis and necrosis in proliferating human lymphocytes, mostly depending on XOR concentration.     

Toxicity of xanthine oxidoreductase to malignant B lymphocytes

Reactive oxygen species (ROS) generated by xanthine oxidoreductase (XOR) were toxic to B lymphoma-derived Raji cells (positive for 8A monoclonal antibody, mAb). The sensitivity of these malignant cells to the hypoxanthine/XOR system was higher than that observed in peripheral human lymphocytes. The understanding of the mechanisms of cytotoxicity induced by XOR-produced ROS is essential in view of a possible clinical application. Cell death mostly had the feature of apoptosis and post-apoptotic necrosis and depended on the activity of XOR. Catalase, but not superoxide dismutase, protected cells from the toxicity of XOR, thus indicating that cell damage depended on the production of hydrogen peroxide. The toxicity of ROS was selectively targeted to malignant Raji cells by antibody-XOR conjugation, either directly, with an 8A-XOR conjugate, or indirectly, with an 8A mAb plus an anti-mouse IgG-XOR. Both direct and indirect immunotoxins induced apoptotic death to target cells in a dose-dependent manner. These conjugates showed no aspecific cytotoxicity in conditions very similar to the ex vivo treatment of cell suspension for bone marrow transplantation. Moreover, the prevalence of apoptotic death over necrosis may reduce the in vivo inflammatory response and its local and systemic consequences, thus becoming relevant in the construction of immunotoxins with therapeutic potential.     

Reactive oxygen species induce signals that lead to apoptotic DNA degradation in primary CD4+ T cells

Reactive oxygen species are toxic to cells but they may also have active roles in transducing apoptotic events. To study the role of reactive oxygen species in growth factor depletion induced apoptosis of human primary CD4+ T cells, we used a synthetic manganese porphyrin superoxide dismutase mimetic to detoxify superoxide anions formed during apoptosis. Apoptosis of primary CD4+ T cells was characterized by generation of superoxide anions, plasma membrane phosphatidyl-serine translocation, loss of mitochondrial membrane potential, activation of caspase 3, condensation of chromatin, as well as DNA degradation. The detoxification of superoxide anions did not influence plasma membrane phosphatidyl-serine translocation, or chromatin condensation, and only marginally inhibited the loss of mitochondrial membrane potential and the formation of DNA strand breaks. In contrast, the detoxification of superoxide anions significantly reduced caspase 3 activity and almost completely inhibited the apoptotic decrease in total cellular DNA content as measured by propidium iodide staining. Our results indicate that reactive oxygen anions induce signals leading to efficient DNA degradation after the initial formation of DNA strand breaks. Thus, reactive oxygen anions have active roles in signaling that lead to the apoptotic events.     

Reactive oxygen species produced by the mitochondrial respiratory chain are involved in Cd-induced injury of rat ascites hepatoma AS-30D cells

Using AS-30D rat ascites hepatoma cells, we studied the modulating action of various antioxidants, inhibitors of mitochondrial permeability transition pore and inhibitors of the respiratory chain on Cd²⁺-produced cytotoxicity. It was found that Cd²⁺ induced both necrosis and apoptosis in a time- and dose-dependent way. This cell injury involved dissipation of the mitochondrial transmembrane potential, respiratory dysfunction and initial increase of the generation of reactive oxygen species (ROS), followed by its decrease after prolonged incubation. Inhibitors of the mitochondrial permeability transition pore, cyclosporin A and bongkrekic acid, and inhibitors of respiratory complex III, stigmatellin and antimycin A, but not inhibitor of complex I, rotenone, partly prevented necrosis evoked by exposure of the cells to Cd²⁺. Apoptosis of the cells was partly prevented by free radical scavengers and by preincubation with N-acetylcysteine. Stigmatellin, antimycin A and cyclosporin A also abolished Cd²⁺-induced increase in ROS generation. It is concluded that Cd²⁺ toxicity in AS-30D rat ascites hepatoma, manifested by cell necrosis and/or apoptosis, involves ROS generation, most likely at the level of respiratory complex III, and is related to opening of the mitochondrial permeability transition pore.     

Vanadate induces apoptosis in epidermal JB6 Pplus cells via hydrogen peroxide-mediated reactions

Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P⁺ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 M vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H₂O₂ as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H₂O₂ production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H₂O₂ induced by vanadate plays a major role in this process.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O2*-) and hydrogen peroxide (H2O2), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H2O2-eliminating enzyme, catalase, but not in the presence of an O2*--eliminating enzyme, superoxide dismutase. Treatment with H2O2 itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H2O2 treatment decreased in H2O2-resistant cells. Although both UVB and H2O2 are known to induce DNA damage, other DNA damaging agents, like gamma-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H2O2 was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Glutathione depletion in fibroblasts is the basis for apoptosis-induction by endogenous reactive oxygen species

Our study aimed at clarifying the role of the intracellular concentration of reduced glutathione for induction of apoptosis in fibroblasts. Treatment of fibroblasts with buthionine sulfoximine (BSO) caused efficient depletion of intracellular reduced glutathione which was followed by substantial cell death. Based on the induction of membrane blebbing, chromatin condensation and DNA strand breaks, cell death was characterized as apoptosis. Apoptosis after glutathione depletion seemed to be induced by endogenous reactive oxygen species (ROS), as it was antagonized by the antioxidant catechol and the hydroxyl radical scavenger DMSO. Paracrine interaction between cells prevented ROS-induced apoptosis and therefore points to the existence of extracellular survival factors. Our data show that the apoptosis-inducing potential of endogenous ROS is controlled by the intracellular glutathione concentration and by paracrine survival factors.     

Differential effect of l-histidine in human lymphocytes damaged by different oxygen radical producing systems

The effect of histidine on damage induced by oxygen radicals was studied in peripheral blood lymphocytes treated with free oxygen radical-inducing agents: hydrogen peroxide, xanthine oxidase plus hypoxanthine, bleumycin and γ-rays. l-Histidine, at a concentration of 1 mM, was found to potentiate both cell killing and inhibition of PHA-stimulated cell division brought about by hydrogen peroxide or xanthine oxidase plus hypoxanthine. In contrast, l-histidine did not affect γ-ray- or bleomycin-induced cell killing and inhibition of PHA-stimulated cell division. We suggest that l-histidine potentiation of cell damage is mainly mediated by interaction of the amino acid with hydrogen peroxide and/or iron rather than with other reactive oxygen species. In addition, these results also indicate that hydrogen peroxide produced by γ-radiation- or bleomycin-treated cells plays no role in the toxic effects elicited by these agents.     

Evaluation of the toxicity effects of silk fibroin on human lymphocytes and monocytes

Silk fibroin nanoparticles (SFNPs) as a natural polymer have been utilized in biomedical applications such as suture, tissue engineering‐based scaffolds, and drug delivery carriers. Since there is little data regarding the toxicity effects on different cells and tissues, we aimed to determine the toxicity mechanisms of SFNPs on human lymphocytes and monocytes based on reliable methods. Our results showed that SFNPs (0.5, 1, and 2 mg/mL) induced oxidative stress via increasing reactive oxygen species production, mitochondrial membrane potential (?Ψ) collapse, which was correlated to cytochrome c release and Adenosine diphosphate (ADP)/Adenosine tri phosphate (ATP) ratio increase as well as lysosomal as another toxicity mechanism, which led to cytosolic release of lysosomal digestive proteases, phosphor lipases, and apoptosis signaling. Taken together, these data suggested that SFNPs toxicity was associated with mutual mitochondrial/lysosomal cross‐talk and oxidative stress on human lymphocytes and monocytes with activated apoptosis signaling.     

Insulin Secretion, DNA Damage, and Apoptosis in Human and Rat Islets of Langerhans Following Exposure to Nitric Oxide, Peroxynitrite, and Cytokines

Cytokine-induced damage may contribute to destruction of insulin-secreting beta-cells in islets of Langerhans during autoimmune diabetes. There is considerable controversy (i) whether human and rat islets respond differently to cytokines, (ii) the extent to which cytokine damage is mediated by induction of nitric oxide formation, and (iii) whether the effects of nitric oxide on islets can be distinguished from those of reactive oxygen species or peroxynitrite. We have analyzed rat and human islet responses in parallel, 48 h after exposure to the nitric oxide donor S-nitrosoglutathione, the mixed donor 3-morpholinosydnonimine, hypoxanthine/xanthine oxidase, peroxynitrite, and combined cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma). Insulin secretory response to glucose, insulin content, DNA strand breakage, and early-to-late stage apoptosis were recorded in each experiment. Rat islet insulin secretion was reduced by S-nitrosoglutathione or combined cytokines, but unexpectedly increased by peroxynitrite or hypoxanthine/xanthine oxidase. Effects on human islet insulin secretion were small; cytokines and S-nitrosoglutathione decreased insulin content. Both rat and human islets showed significant and similar levels of DNA damage following all treatments. Apoptosis in neonatal rat islets was increased by every treatment, but was at a low rate in adult rat or human islets and only achieved significance with cytokine treatment of human islets. All cytokine responses were blocked by an arginine analogue. We conclude: (i) Reactive oxygen species increased and nitric oxide decreased insulin secretory responsiveness in rat islets. (ii) Species differences lie mainly in responses to cytokines, applied at a lower dose and shorter time than in most studies of human islets. (iii) Cytokine effects were nitric oxide driven; neither reactive oxygen species nor peroxynitrite reproduced cytokine effects. (iv) Rat and human islets showed equal susceptibility to DNA damage. (v) Apoptosis was not the preferred death pathway in adult islets. (vi) We have found no evidence of human donor variation in the pattern of response to these treatments.     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

Analysis of cytotoxic effects of chlorhexidine gluconate as antiseptic agent on human blood lymphocytes

The aim of this study was to assess the cytotoxicity of chlorhexidine gluconate (CHG) on human blood lymphocytes as a useful ex vivo model for accelerated human toxicity studies. Using biochemical and flow cytometry assessments, we demonstrated that addition of CHG at 1 μM concentration to human blood lymphocytes induced cytotoxicity following 6 h. The CHG‐induced cytotoxicity on human blood lymphocytes was associated with intracellular reactive oxygen species generation, mitochondrial membrane potential collapse, lysosomal membrane injury, lipid peroxidation, and depletion of glutathione. According to our results, CHG triggers oxidative stress and organelles damages in lymphocytes which are important cells in defense against foreign agents. Finally our findings suggest that using of antioxidants and mitochondrial/lysosomal protective agents could be of benefit for the people in the exposure with CHG.     

Inhibition of human lymphocyte function by organic solvents

We studied the direct effect of reactive hydroxyl precursors and inhibitors on CD4+ T-cell function. We used hydrogen peroxide plus ferrous chloride as the hydroxyl radical-generating system and di-methyl sulphourea, di-methyl sulfoxide, pyrrolidine dithiocarbonate, methanol, and ethanol, at a noncytotoxic concentration, as inhibitors. The immune parameter studies were proliferation and interleukin-2 production by peripheral blood lymphocytes stimulated with anti-CD3 antibody, phytohemagglutinin and alloantigens; proliferation, interleukin-2 production and mRNA expression of interleukin-4 and interferon gamma by allogeneic CD4+ T-cell clones stimulated with alloantigens. The results show that lymphocytes produce significant amounts of reactive oxygen species as measured by malondialdehyde produced in cultures. The hydroxyl radical-generating system did not change any of the cellular responses studied although it doubled Malondialdehyde production. Hydroxyl radical scavengers signi tly inhibited all responses at doses that didn't significantly decrease malondialdehyde production. DNA analysis failed to show evidence for apoptosis. Conclusion: Hydroxyl radical scavengers inhibit lymphocyte mitogenesis by a process that is independent of scavenging hydroxyl radicals.     

Xanthine oxidoreductase is a critical mediator of cigarette smoke-induced endothelial cell DNA damage and apoptosis

Cigarette smoke (CS) exposure is unquestionably the most frequent cause of emphysema in the United States. Accelerated pulmonary endothelial cell (EC) apoptosis is an early determinant of lung destruction in emphysema. One of the pathogenic causes of emphysema is an alveolar oxidant and antioxidant imbalance. The enzyme xanthine oxidoreductase (XOR) has been shown to be a source of reactive oxygen species (ROS) in a multitude of diseases (S. Sakao et al., FASEB J. 21, 3640–3652; 2007). The contribution of XOR to CS-induced apoptosis is not well defined. Here we demonstrate that C57/bl6 mice exposed to CS have increased pulmonary XOR activity and protein levels compared to filtered-air-exposed controls. In addition, we demonstrate that primary pulmonary human lung microvascular endothelial cells exposed to cigarette smoke extract undergo increased rates of caspase-dependent apoptosis that are reliant on XOR activity, ROS production, and p53 function/expression. We also demonstrate that exogenous XOR is sufficient to increase p53 expression and induce apoptosis, suggesting that XOR is an upstream mediator of p53 in CS-induced EC apoptosis. Furthermore, we show that XOR activation results in DNA double-strand breaks that activate the enzyme ataxia telangiectasia mutated, which phosphorylates histone H2AX and upregulates p53. In conclusion, CS increases XOR expression, and the enzyme is both sufficient and necessary for p53 induction and CS-induced EC apoptosis.     

Diphenyleneiodonium suppresses apoptosis in cerulein-stimulated pancreatic acinar cells

NADPH oxidase has been considered a major source of reactive oxygen species in phagocytic and non-phagocytic cells. Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently, we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells, which are activated by cerulein, a cholecystokinin analogue. Cerulein induces an acute and edematous form of pancreatitis. We investigated whether inhibition of NADPH oxidase by diphenyleneiodonium suppresses the production of reactive oxygen species and apoptosis by determining viable cell numbers, DNA fragmentation, TUNEL staining, caspase-3 activity, and the expression of apoptosis-inducing factor in pancreatic acinar AR42J cells stimulated with cerulein. Inhibition on NADPH oxidase by diphenyleneiodonium was assessed by the alterations in NADPH oxidase activity and translocation of the cytosolic subunits p67phox and p47phox to the membrane. Intracellular Ca2+ level was monitored to investigate the relationship between NADPH oxidase and Ca2+ in cells stimulated with cerulein. As a result, cerulein induced the activation of NADPH, increased production of reactive oxygen species, and apoptotic indices determined by the expression of apoptosis-inducing factor, caspase-3 activation, TUNEL staining, DNA fragmentation, and cell viability. Treatment with DPI inhibited cerulein-induced activation of NADPH oxidase, the production of reactive oxygen species, and apoptosis, but not the increase of intracellular Ca2+ levels in pancreatic acinar cells. These results demonstrate that the cerulein-induced increase in intracellular Ca2+ level may be an upstream event of NADPH oxidase activation. Diphenyleneiodonium, an NADPH oxidase inhibitor, inhibits the expression of apoptosis-inducing factor and caspase-3 activation, and thus apoptosis in pancreatic acinar cells.     

Reactive oxygen species produced by the mitochondrial respiratory chain are involved in Cd2+-induced injury of rat ascites hepatoma AS-30D cells

Using AS-30D rat ascites hepatoma cells, we studied the modulating action of various antioxidants, inhibitors of mitochondrial permeability transition pore and inhibitors of the respiratory chain on Cd(2+)-produced cytotoxicity. It was found that Cd(2+) induced both necrosis and apoptosis in a time- and dose-dependent way. This cell injury involved dissipation of the mitochondrial transmembrane potential, respiratory dysfunction and initial increase of the generation of reactive oxygen species (ROS), followed by its decrease after prolonged incubation. Inhibitors of the mitochondrial permeability transition pore, cyclosporin A and bongkrekic acid, and inhibitors of respiratory complex III, stigmatellin and antimycin A, but not inhibitor of complex I, rotenone, partly prevented necrosis evoked by exposure of the cells to Cd(2+). Apoptosis of the cells was partly prevented by free radical scavengers and by preincubation with N-acetylcysteine. Stigmatellin, antimycin A and cyclosporin A also abolished Cd(2+)-induced increase in ROS generation. It is concluded that Cd(2+) toxicity in AS-30D rat ascites hepatoma, manifested by cell necrosis and/or apoptosis, involves ROS generation, most likely at the level of respiratory complex III, and is related to opening of the mitochondrial permeability transition pore.     

Ceramide-induced formation of ROS and ATP depletion trigger necrosis in lymphoid cells

In lymphocytes, Fas activation leads to both apoptosis and necrosis, whereby the latter form of cell death is linked to delayed production of endogenous ceramide and is mimicked by exogenous administration of long- and short-chain ceramides. Here molecular events associated with noncanonical necrotic cell death downstream of ceramide were investigated in A20 B lymphoma and Jurkat T cells. Cell-permeable, C6-ceramide (C6), but not dihydro-C6-ceramide (DH-C6), induced necrosis in a time- and dose-dependent fashion. Rapid formation of reactive oxygen species (ROS) within 30 min of C6 addition detected by a dihydrorhodamine fluorescence assay, as well as by electron spin resonance, was accompanied by loss of mitochondrial membrane potential. The presence of N-acetylcysteine or ROS scavengers like Tiron, but not Trolox, attenuated ceramide-induced necrosis. Alternatively, adenovirus-mediated expression of catalase in A20 cells also attenuated cell necrosis but not apoptosis. Necrotic cell death observed following C6 exposure was associated with a pronounced decrease in ATP levels and Tiron significantly delayed ATP depletion in both A20 and Jurkat cells. Thus, apoptotic and necrotic death induced by ceramide in lymphocytes occurs via distinct mechanisms. Furthermore, ceramide-induced necrotic cell death is linked here to loss of mitochondrial membrane potential, production of ROS, and intracellular ATP depletion.     

Amelioration of experimental acute pancreatitis with Dachengqi Decoction via regulation of necrosis-apoptosis switch in the pancreatic acinar cell

Severity of acute pancreatitis contributes to the modality of cell death. Pervious studies have demonstrated that the herb medicine formula "Dachengqi Decoction" (DCQD) could ameliorate the severity of acute pancreatitis. However, the biological mechanisms governing its action of most remain unclear. The role of apoptosis/necrosis switch within acute pancreatitis has attracted much interest, because the induction of apoptosis within injured cells might suppress inflammation and ameliorate the disease. In this study, we used cerulein (10(-8) M)-stimulated AR42J cells as an in vitro model of acute pancreatitis and retrograde perfusion into the biliopancreatic duct of 3.5% sodium taurocholate as an in vivo rat model. After the treatment of DCQD, cell viability, levels of apoptosis and necrosis, reactive oxygen species positive cells, serum amylase, concentration of nitric oxide and inducible nitric oxide syntheses, pancreatic tissue pathological score and inflammatory cell infiltration were tested. Pretreatment with DCQD increased cell viability, induced apoptosis, decreased necrosis and reduced the severity of pancreatitis tissue. Moreover, treatment with DCQD reduced the generation of reactive oxygen species in AR42J cells but increased the concentration of nitric oxide of pancreatitis tissues. Therefore, the regulation of apoptosis/necrosis switch by DCQD might contribute to ameliorating the pancreatic inflammation and pathological damage. Further, the different effect on reactive oxygen species and nitric oxide may play an important role in DCQD-regulated apoptosis/necrosis switch in acute pancreatitis.     

Generation of reactive oxygen species and radiation response in lymphocytes and tumor cells

Several types of lymphoid and myeloid tumor cells are known to be relatively resistant to radiation-induced apoptosis compared to normal lymphocytes. The intracellular generation of reactive oxygen species was measured in irradiated spleen cells from C57BL/6 and BALB/c mice and murine tumor cells (EL-4 and P388) by flow cytometry using dichlorodihydrofluoresceindiacetate and dihydrorhodamine 123 as fluorescent probes. The amount of reactive oxygen species generated per cell was low in the tumor cells compared to spleen cells exposed to 1 to 10 Gy of gamma radiation. This could be due to the higher total antioxidant levels in tumor cells compared to normal cells. Further, the changes in mitochondrial membrane potential and cytoplasmic Ca2+ content were appreciable in lymphocytes even at a dose of 1 Gy. In EL-4 cells, no such changes were observed at any of the doses used. About 65% of spleen cells underwent apoptosis 24 h after 1 Gy irradiation. However, under the same conditions, EL-4 and P388 cells failed to undergo apoptosis, but they accumulated in G2/M phase. Thus the intrinsic radioresistance of tumor cells may be due to a decreased generation of reactive oxygen species after irradiation and down-regulation of the subsequent events leading to apoptosis.