L-carnitine enhances resistance to oxidative stress by reducing DNA damage in Ataxia telangiectasia cells

In this study, the modulating effect of L-carnitine on tert-butyl-hydroperoxide-induced DNA damage was compared with that of mannitol, a well known scavenger of hydroxyl radicals, both in normal and Ataxia telangiectasia mutated (ATM)-deficient lymphoblastoid cell lines established from A. telangiectasia (A-T) patients. The alkaline version of the comet assay was employed to measure the frequency of single-strand breaks (SSBs) and alkali-labile sites induced by t-butyl-OOH immediately after treatment and at different recovery times in normal and A-T cell lines, with and without pre-treatment with L-carnitine. In addition, both the yield of induced chromosomal damage and the effect on cell proliferation were evaluated. Our results show that pre-treatment of cells with L-carnitine produced an enhancement of the rate and extent of DNA repair in A-T cell lines at early recovery time; furthermore, in samples pre-treated with L-carnitine a reduction of all types of chromosomal aberration was observed, both in A-T and in wild-type cell lines. The reducing effect of L-carnitine pre-treatment on oxidative DNA damage was more prominent than that of pre-treatment with mannitol. In conclusion, we demonstrated a protective effect of L-carnitine on oxidative stress-induced DNA damage in A-T cells, suggesting its possible role in future pharmacological applications in A-T therapy.     

Oxidative damage induced genotoxic effects in human fibroblasts from Xeroderma Pigmentosum group A patients

Xeroderma Pigmentosum A protein plays a pivotal role in the nucleotide excision repair pathway. Through site-directed binding of rigidly kinked double-stranded DNA, it verifies damaged DNA for subsequent excision and incision. Although Xeroderma Pigmentosum A-deficient cells have shown to be defective in oxidative base-lesion repair, the effects of oxidative assault on such cells have not been fully explored. Therefore, we sought to determine the involvement of Xeroderma Pigmentosum A in oxidative DNA damage-repair by treating primary fibroblasts from a patient suffering from Xeroderma Pigmentosum A with sodium arsenite and hydrogen peroxide. Our results show dose-dependent increase in genotoxicity with little change in cytotoxicity with both arsenite and H2O2 in Xeroderma Pigmentosum A-deficient cells compared to control cells. Xeroderma Pigmentosum A-deficient cells displayed increased susceptibility and reduced repair capacity when subjected to DNA damage induced by oxidative stress. Superarray results of apoptotic genes revealed differential expression of approximately 10 genes between Xeroderma Pigmentosum A-deficient and normal cells following arsenite treatment. Interestingly, we noted that arsenite did not inflict as much damage in the cells compared to H2O2. Lack of functional Xeroderma Pigmentosum A seems to increase the susceptibility of oxidative stress-induced genotoxicity while retaining cell viability posing as a potential cancer risk factor of Xeroderma Pigmentosum A patients.     

L-carnitine protects C2C12 cells against mitochondrial superoxide overproduction and cell death

AIM To identify and characterize the protective effect that L-carnitine exerted against an oxidative stress in C2C12 cells.METHODS Myoblastic C2C12 cells were treated with menadione, a vitamin K analog that engenders oxidative stress, and the protective effect of L-carnitine(a nutrient involved in fatty acid metabolism and the control of the oxidative process), was assessed by monitoring various parameters related to the oxidative stress, autophagy and cell death. RESULTS Associated with its physiological function, a muscle cell metabolism is highly dependent on oxygen and may produce reactive oxygen species(ROS), especially under pathological conditions. High levels of ROS are known to induce injuries in cell structure as they interact at many levels in cell function. In C2C12 cells, a treatment with menadione induced a loss of transmembrane mitochondrial potential, an increase in mitochondrial production of ROS; it also induces autophagy and was able to provoke cell death. Pre-treatment of the cells with L-carnitine reduced ROS production, diminished autophagy and protected C2C12 cells against menadione-induced deleterious effects. CONCLUSION In conclusion, L-carnitine limits the oxidative stress in these cells and prevents cell death.     

Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression.

To analyze the effects of high concentrations of zinc ions on oxidative stress protection, we developed an original model of zinc-resistant HeLa cells (HZR), by using a 200 microM zinc sulfate-supplemented medium. Resistant cells specifically accumulate high zinc levels in intracellular vesicles. These resistant cells also exhibit high expression of metallothioneins (MT), mainly located in the cytoplasm. Exposure of HZR to Zn-depleted medium for 3 or 7 d decreases the intracellular zinc content, but only slightly reduces MT levels of resistant cells. No changes of the intracellular redox status were detected, but zinc resistance enhanced H2O2-mediated cytotoxicity. Conversely, zinc-depleted resistant cells were protected against H2O2-induced cell death. Basal- and oxidant-induced DNA damage was increased in zinc resistant cells. Moreover, measurement of DNA damage on zinc-depleted resistant cells suggests that cytoplasmic metal-free MT ensures an efficient protection against oxidative DNA damage, while Zn-MT does not. This newly developed Zn-resistant HeLa model demonstrates that high intracellular concentrations of zinc enhance oxidative DNA damage and subsequent cell death. Effective protection against oxidative damage is provided by metallothionein under nonsaturating zinc conditions. Thus, induction of MT by zinc may mediate the main cellular protective effect of zinc against oxidative injury.     

H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis.

H2O2 treatment on U937 cells leads to the block of glycolytic flux and the inactivation of glyceraldehyde-3-phosphate-dehydrogenase by a posttranslational modification (possibly ADP-ribosylation). Glycolysis spontaneously reactivates after 2 h of recovery from oxidative stress; thereafter cells begin to undergo apoptosis. The specific ADP-ribosylation inhibitor 3-aminobenzamide inhibits the stress-induced inactivation of glyceraldehyde-3-phosphate-dehydrogenase and the block of glycolysis; concomitantly, it anticipates and increases apoptosis. Exogenous block of glycolysis (i.e., by culture in glucose-free medium or with glucose analogs or after NAD depletion), turns the transient block into a stable one: this results in protection from apoptosis, even when downstream cell metabolism is kept active by the addition of pyruvate. All this evidence indicates that the stress-induced block of glycolysis is not the result of a passive oxidative damage, but rather an active cell reaction programmed via ADP-ribosylation for cell self-defense.     

Resveratrol increases vascular oxidative stress resistance

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its vasculoprotective effects are not completely understood. Because oxidative stress and endothelial cell injury play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits oxidative stress-induced endothelial apoptosis. We found that oxidized LDL and TNF-alpha elicited significant increases in caspase-3/7 activity in endothelial cells and cultured rat aortas, which were prevented by resveratrol pretreatment (10(-6)-10(-4) mol/l). The protective effect of resveratrol was attenuated by inhibition of glutathione peroxidase and heme oxygenase-1, suggesting a role for antioxidant systems in the antiapoptotic action of resveratrol. Indeed, resveratrol treatment protected cultured aortic segments and/or endothelial cells against increases in intracellular H(2)O(2) levels and H(2)O(2)-mediated apoptotic cell death induced by oxidative stressors (exogenous H(2)O(2), paraquat, and UV light). Resveratrol treatment also attenuated UV-induced DNA damage (comet assay). Resveratrol treatment upregulated the expression of glutathione peroxidase, catalase, and heme oxygenase-1 in cultured arteries, whereas it had no significant effect on the expression of SOD isoforms. Resveratrol also effectively scavenged H(2)O(2) in vitro. Thus resveratrol seems to increase vascular oxidative stress resistance by scavenging H(2)O(2) and preventing oxidative stress-induced endothelial cell death. We propose that the antioxidant and antiapoptotic effects of resveratrol, together with its previously described anti-inflammatory actions, are responsible, at least in part, for its cardioprotective effects.     

Nitroxides block DNA scission and protect cells from oxidative damage.

The protective effect of cyclic stable nitroxide free radicals, having SOD-like activity, against oxidative damage was studied by using Escherichia coli xthA DNA repair-deficient mutant hypersensitive to H2O2. Oxidative damage induced by H2O2 was assayed by monitoring cell survival. The metal chelator 1,10-phenanthroline (OP), which readily intercalates into DNA, potentiated the H2O2-induced damage. The extent of in vivo DNA scission and degradation was studied and compared with the loss of cell viability. The extent of DNA breakage correlated with cell killing, supporting previous suggestions that DNA is the crucial cellular target of H2O2 cytotoxicity. The xthA cells were protected by catalase but not by superoxide dismutase (SOD). Both five- and six-membered ring nitroxides, having SOD-like activity, protected growing and resting cells from H2O2 toxicity, without lowering H2O2 concentration. To check whether nitroxides protect against O2.(-)-independent injury also, experiments were repeated under hypoxia. These nitroxides also protected hypoxic cells against H2O2, suggesting alternative modes of protection. Since nitroxides were found to reoxidize DNA-bound iron(II), the present results suggest that nitroxides protect by oxidizing reduced transition metals, thus interfering with the Fenton reaction.     

Expression of human telomerase (hTERT) does not prevent stress-induced senescence in normal human fibroblasts but protects the cells from stress-induced apoptosis and necrosis

Cells subjected to sub-lethal doses of stress such as irradiation or oxidative damage enter a state that closely resembles replicative senescence. What triggers stress-induced premature senescence (SIPS) and how similar this mechanism is to replicative senescence are not well understood. It has been suggested that stress-induced senescence is caused by rapid telomere shortening resulting from DNA damage. In order to test this hypothesis directly, we examined whether overexpression of the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS. We therefore analyzed the response of four different lines of normal human fibroblasts with and without hTERT to stress induced by UV, gamma-irradiation, and H(2)O(2). SIPS was induced with the same efficiency in normal and hTERT-immortalized cells. This suggests that SIPS is not triggered by telomere shortening and that nonspecific DNA damage serves as a signal for induction of SIPS. Although telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were more resistant to stress-induced apoptosis and necrosis. We hypothesize that healing of DNA breaks by telomerase inhibits the induction of cell death, but because healing does not provide legitimate DNA repair, it does not protect cells from SIPS.     

The Drosophila S3 multifunctional DNA repair/ribosomal protein protects Fanconi anemia cells against oxidative DNA damaging agents

Cells harvested from Fanconi anemia (FA) patients show an increased hypersensitivity to the multifunctional DNA damaging agent mitomycin C (MMC), which causes cross-links in DNA as well as 7,8-dihydro-8-oxoguanine (8-oxoG) adducts indicative of escalated oxidative DNA damage. We show here that the Drosophila multifunctional S3 cDNA, which encodes an N-glycosylase/apurinic/apyrimidinic (AP) lyase activity was found to correct the FA Group A (FA(A)) and FA Group C (FA(C)) sensitivity to MMC and hydrogen peroxide (H2O2). Furthermore, the Drosophila S3 cDNA was shown to protect AP endonuclease deficient E. coli cells against H(2)O(2) and MMC, and also protect 8-oxoG repair deficient mutM E. coli strains against MMC and H2O2 cell toxicity. Conversely, the human S3 protein failed to complement the AP endonuclease deficient E. coli strain, most likely because it lacks N-glycosylase activity for the repair of oxidatively-damaged DNA bases. Although the human S3 gene is clearly not the genetic alteration in FA cells, our results suggest that oxidative DNA damage is intimately involved in the overall FA phenotype, and the cytotoxic effect of selective DNA damaging agents in FA cells can be overcome by trans-complementation with specific DNA repair cDNAs. Based on these findings, we would predict other oxidative repair proteins, or oxidative scavengers, could serve as protective agents against the oxidative DNA damage that occurs in FA.     

胶原蛋白肽经由Nrf2信号通路对H2O2诱导的肝细胞氧化损伤的保护作用

目的：氧化应激在肝脏疾病中扮演着重要的角色。胶原蛋白肽是天然的抗氧化剂,其在动物实验中已经被证实有抑制氧化应激的作用。最新研究证实胶原蛋白肽将有可能被应用在肝脏疾病的预防中,但是很少有研究报道其分子作用机制。因此本研究在胶原蛋白肽是对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用的基础上,并探索其分子作用机制。方法：实验设空白对照组,H2O2模型组,胶原蛋白肽低、中、高剂量组（10,100,200μg/ml）。胶原蛋白肽各组加入相应浓度的药物预处理12 h后,与模型组一起加入300μM H2O2的H2O2共同培养12 h,空白对照组正常培养。细胞毒性是由CCK8和乳酸脱氢酶（LDH）的释放检测。抗氧化试剂盒检测细胞内活性氧的水平,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性和丙二醛（MDA）含量的变化。Western blot检测细胞内Nrf2蛋白的表达水平。结果：胶原蛋白肽对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用。胶原蛋白肽能够及时清除细胞内的活性氧,增加Nrf2的蛋白表达水平,提高超氧化物歧化酶（SOD）、过氧化氢酶（CAT）的活性,减轻脂质过氧化反应,从而保护正常人的肝细胞系HL7702。结论：总之,胶原蛋白肽通过增加Nrf2的蛋白表达水平,提高抗氧化活性,对H2O2诱导损伤的肝细胞发挥保护作用。本研究为胶原蛋白肽的分子作用机制提供了新的证据,将有助于预防氧化应激所致的肝损伤。     

Contribution of mitochondrial DNA repair to cell resistance from oxidative stress

Numerous studies have revealed that a part of the cellular response to chronic oxidative stress involves increased antioxidant capacity. However, another defense mechanism that has received less attention is DNA repair. Because of the important homeostatic role of mitochondria and the exquisite sensitivity of mitochondrial DNA (mtDNA) to oxidative damage, we hypothesized that mtDNA repair plays an important role in the protection against oxidative stress. To test this hypothesis mtDNA damage and repair was evaluated in normal HA1 Chinese hamster fibroblasts and oxidative stress-resistant variants isolated following chronic exposure to H2O2 or 95% O2. Reactive oxygen species were generated enzymatically using xanthine oxidase and hypoxanthine. When treated with xanthine oxidase reduced levels of initial mtDNA damage and enhanced mtDNA repair were observed in the cells from the oxidative stress-resistant variants, relative to the parental cell line. This enhanced mtDNA repair correlated with an increase in mitochondrial apurinic/apyrimidinic endonuclease activity in both H2O2- and O2-resistant HA1 variants. This is the first report showing enhanced mtDNA repair in the cellular response to chronic oxidative stress. These results provide further evidence for the crucial role that mtDNA repair pathways play in protecting cells against the deleterious effects of reactive oxygen species.     

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.     

胶原蛋白肽经由Nrf2信号通路对H_2O_2诱导的肝细胞氧化损伤的保护作用(英文)

目的:氧化应激在肝脏疾病中扮演着重要的角色。胶原蛋白肽是天然的抗氧化剂,其在动物实验中已经被证实有抑制氧化应激的作用。最新研究证实胶原蛋白肽将有可能被应用在肝脏疾病的预防中,但是很少有研究报道其分子作用机制。因此本研究在胶原蛋白肽是对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用的基础上,并探索其分子作用机制。方法:实验设空白对照组,H2O2模型组,胶原蛋白肽低、中、高剂量组(10,100,200μg/ml)。胶原蛋白肽各组加入相应浓度的药物预处理12 h后,与模型组一起加入300μM H2O2的H2O2共同培养12 h,空白对照组正常培养。细胞毒性是由CCK8和乳酸脱氢酶(LDH)的释放检测。抗氧化试剂盒检测细胞内活性氧的水平,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量的变化。Western blot检测细胞内Nrf2蛋白的表达水平。结果:胶原蛋白肽对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用。胶原蛋白肽能够及时清除细胞内的活性氧,增加Nrf2的蛋白表达水平,提高超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性,减轻脂质过氧化反应,从而保护正常人的肝细胞系HL7702。结论:总之,胶原蛋白肽通过增加Nrf2的蛋白表达水平,提高抗氧化活性,对H2O2诱导损伤的肝细胞发挥保护作用。本研究为胶原蛋白肽的分子作用机制提供了新的证据,将有助于预防氧化应激所致的肝损伤。     

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.     

Mitochondrial DNA damage is sensitive to exogenous H(2)O(2) but independent of cellular ROS production in prostate cancer cells

Intrinsic oxidative stress through enhanced production of reactive oxygen species (ROS) in prostate and other cancers may contribute to cancer progression due to its stimulating effect on cancer growth. In this study, we investigate differential responses to exogenous oxidative stimuli between aggressive prostate cancer and normal cell lines and explore potential mechanisms through interactions between cytotoxicity, cellular ROS production and oxidative DNA damage. The circular, multi-copy mitochondrial DNA (mtDNA) is used as a sensitive surrogate to oxidative DNA damage. We demonstrate that exogenous H(2)O(2) induces preferential cytotoxicity in aggressive prostate cancer than normal cells; a cascade production of cellular ROS, composed mainly of superoxide (O(2)(-)), is shown to be a critical determinant of H(2)O(2)-induced selective toxicity in cancer cells. In contrast, mtDNA damage and copy number depletion, as measured by a novel two-phase strategy of the supercoiling-sensitive qPCR method, are very sensitive to exogenous H(2)O(2) exposure in both cancer and normal cell lines. Moreover, we demonstrate for the first time that the sensitive mtDNA damage response to exogenous H(2)O(2) is independent of secondary cellular ROS production triggered by several ROS modulators regardless of cell phenotypes. These new findings suggest different mechanisms underpinning cytotoxicity and DNA damage induced by oxidative stress and a susceptible phenotype to oxidative injury associated with aggressive prostate cancer cells in vitro.     

A modified alkaline Comet assay for in vivo detection of oxidative DNA damage in Drosophila melanogaster

Modifications to the alkaline Comet assay by using lesion-specific endonucleases, such as formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (ENDOIII, also known as Nth), can detect DNA bases with oxidative damage. This modified assay can be used to assess the genotoxic/carcinogenic potential of environmental chemicals. The goal of this study was to validate the ability of this modified assay to detect oxidative stress-induced genotoxicity in Drosophila melanogaster (Oregon R(+)). In this study, we used three well known chemical oxidative stress inducers: hydrogen peroxide (H(2)O(2)), cadmium chloride (CdCl(2)) and copper sulfate (CuSO(4)). Third instar larvae of D. melanogaster were fed various concentrations of the test chemicals (50-200μM) mixed with a standard Drosophila food for 24h. Alkaline Comet assays with and without the FPG and ENDOIII enzymes were performed with midgut cells that were isolated from the control and treated larvae. Our results show a concentration-dependent increase (p<0.05-0.001) in the migration of DNA from the treated larvae. ENDOIII treatment detected more oxidative DNA damage (specifically pyrimidine damage) in the H(2)O(2) exposed larvae compared to FPG or no enzyme treatment (buffer only). In contrast, FPG treatment detected more oxidative DNA damage (specifically purine damage) in CuSO(4) exposed larvae compared to ENDOIII. Although previously reported to be a potent genotoxic agent, CdCl(2) did not induce more oxidative DNA damage than the other test chemicals. Our results show that the modified alkaline Comet assay can be used to detect oxidative stress-induced DNA damage in D. melanogaster and thus may be applicable for in vivo genotoxic assessments of environmental chemicals.     

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.     

Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports aging-related processes.

The ongoing DNA damage caused by reactive oxygen species generated during oxidative metabolism is considered a key factor contributing to cell aging as well as preconditioning cells to neoplastic transformation. We postulated before that a significant fraction of constitutive histone H2AX phosphorylation (CHP) and constitutive activation of ATM (CAA) seen in untreated normal and tumor cells occurs in response to such DNA damage. In the present study, we provide further evidence in support of this postulate. The level of ATM activation and H2AX phosphorylation, detected immunocytochemically, has been monitored in WI-38, A549, and TK6 cells treated with H2O2 as well as growing under conditions known or suspected to affect the level of endogenous oxidants. Thirty- to 60-min exposure of cells to 100 or 200 microM H2O2 led to an increase in the level of H2AX phosphorylation and ATM activation, particularly pronounced (nearly fivefold) in S-phase cells. Cell growth for 24-48 h under hypoxic conditions (3% O2) distinctly lowered the level of CHP and CAA while it had minor effect on cell cycle progression. Treatment (4 h) with 0.1 or 0.3 mM 3-bromopyruvate, an inhibitor of glycolysis and mitochondrial oxidative phosphorylation, reduced the level of CHP (up to fourfold) and also decreased the level of CAA. Growth of WI-38 cells in 2% serum concentration for 48 h led to a 25 and 30% reduction in CHP and CHA, respectively, compared with cells growing in 10% serum. The antioxidant vitamin C (2 mM) reduced CHP and CAA by 20-30% after 24 h of treatment, while the COX-2 inhibitor celecoxib (5 microM) had a minor effect on CHP and CAA, though it decreased the level of H2O2-induced H2AX phosphorylation and ATM activation. In contrast, dichloroacetate known to shift metabolism from anaerobic to oxidative glycolysis through its effect on pyruvate dehydrogenase kinase enhanced the level of CHP and CAA. Our present data and earlier observations strongly support the postulate that a large fraction of CHP and CAA occurs in response to DNA damage caused by metabolically generated oxidants. Cytometric analysis of CHP and CAA provides the means to measure the effectiveness of exogenous factors, which either through lowering aerobic metabolism or neutralizing radicals may protect DNA from such damage.     

DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.     

Overexpression of phospholipase Cbeta-1 protects NIH3T3 cells from oxidative stress-induced cell death

Oxidative stress has been implicated in a wide range of cellular damage which includes DNA oxidation, membrane lipid peroxidation, and apoptosis. In our study, we found that overexpression of PLC-beta1 in NIH3T3 fibroblasts protected them from cell death occuring in response to oxidative stress. Cell death caused by treatment with prooxidant tert-butylhydroperoxide (TBH), H2O2, or CdCl2 was considerably suppressed in PLC-beta1 overexpressed NIH/beta1-14 cells in comparison to control NIH/neo cells. However, overexpression of PLC-beta1 failed to protect the cells from toxicity by diamide or KCN. In addition, while accumulation of c-fos mRNA was observed within 30 min of TBH treatment in vector transfected NIH/neo cells, TBH-induced c-fos mRNA generation was completely suppressed in NIH/beta1-14 cells, while that of c-jun and GAPDH was not affected. These findings suggest that PLC-beta1 may play a role in process that can protect cells from oxidative stress-induced cell death.