Preconditioning regulation of bcl-2 and p66shc by human NOS1 enhances tolerance to oxidative stress.

Preconditioning stress induced by a transient ischemia may increase brain tolerance to oxidative stress, and the underlying neuroprotective mechanisms are not well understood. In a series of experiments, we found that endogenous nitric oxide (NO), S-nitrosoglutathione (GSNO), and antioxidants blocked serum deprivation-induced oxidative stress and apoptosis in human neuroblastoma cells. Similar to nuclear redox factor-1 (Ref-1), mRNA of human neuronal nitric oxide synthase (hNOS1) was maximally up-regulated within 2 h after oxidative stress and down-regulated by NO/GSNO and hydroxyl radical (OH) scavenger. A brief preconditioning stress induced by serum deprivation for 2 h caused a delayed increase in the expression of hNOS1 protein and the associated formation of NO and cGMP, which in turn decreased OH generation and stress-related cell death. In addition to inhibiting caspase-3 through a dithiothreitol-sensitive S-nitrosylation process, preconditioning stress concomitantly up-regulated the expression of the anti-apoptotic bcl-2 protein and down-regulated the p66shc adaptor protein. This beneficial cytoprotective process of preconditioning stress is mediated by newly synthesized NO because it can be suppressed by the inhibition of hNOS1 and guanylyl cyclase. Therefore, the constitutive isoform of hNOS1 is dynamically redox-regulated to meet both functional and compensatory demands of NO for gene regulation, antioxidant defense, and tolerance to oxidative stress.     

p66shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severe oxidative stress in mouse renal proximal tubule cells

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66(shc) isoform can inhibit this p46/52(shc) function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser(36) phosphorylation of p66(shc). Isoform-specific knockdown of p66(shc) or mutation of Ser(36) to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66(shc) to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser(36) phosphorylation of p66(shc) and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser(36) phosphorylation of p66(shc) or knocking down p66(shc) expression in vivo.     

Mitochondrial matrix P53 sensitizes cells to oxidative stress

A mitochondrial matrix-specific p53 construct (termed p53-290) in HepG2 cells was utilized to determine the impact of p53 in the mitochondrial matrix following oxidative stress. H₂O₂ exposure reduced cellular proliferation similarly in both p53-290 and vector cells, and p53-290 cells demonstrating decreased cell viability at 1 mM H₂O₂ (~ 85% viable). Mitochondrial DNA (mtDNA) abundance was decreased in a dose-dependent manner in p53-290 cells while no change was observed in vector cells. Oximetric analysis revealed reduced maximal respiration and reserve capacity in p53-290 cells. Our results demonstrate that mitochondrial matrix p53 sensitizes cells to oxidative stress by reducing mtDNA abundance and mitochondrial function.     

Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: role in Rac1-induced oxidative stress

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-induced apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.     

Propofol attenuates high glucose-induced P66shc expression in human umbilical vein endothelial cells through Sirt1

Perioperative hyperglycemia is a common metabolic disorder in clinic settings.Hyperglycemia leads to endothelial inflammation,endothelial cell apoptosis,and dysfunction,thus resulting in endothelial injury.Propofol(2,6-diisopropylphenol)is a widely used intravenous anesthetic in clinic settings.Our previous study indicated that propofol inhibits mitochondrial reactive oxygen species(ROS)production via down-regulation of phosphatase A2(PP2A)expression,inhibition of Ser36-p66shc dephosphorylation and mitochondrial translocation,thus improving high glucose-induced endothelial injury.The expression of p66shc was inhibited by propofol in hyperglycemic human umbilical vein endothelial cells(HUVECs).However,the mechanism by which propofol inhibits p66shc expression in hyperglycemic HUVECs is still obscure.In the present study,we mainly examined how propofol inhibited high glucose-induced p66shc expression in HUVECs.Compared with 5 mM glucose treatment,high glucose increased p66shc expression and decreased sirt1 expression,which was inhibited by propofol treatment.Moreover,EX527(a sirt1 inhibitor)reversed the effect of propofol against high glucose-induced p66shc expression.However,EX527 did not reverse the effects of propofol against high glucose-induced ROS accumulation,endothelial inflammation,and apoptosis.Furthermore,when cells were incubated with propofol,EX527,and FTY720(a PP2A activator)simultaneously,the effects of propofol against high glucose-induced ROS accumulation,inflammation,and apoptosis were reversed.Our results suggested that propofol inhibited high glucose-induced p66shc expression via upregulation of sirt1 expression in hyperglycemic HUVECs.Moreover,propofol protects against high glucose-mediated ROS accumulation and endothelial injury via both inhibition of p66shc expression and dephosphorylation of Ser36-p66shc.     

Specificity of resistance to oxidative stress

Two clonal nerve-like cell lines derived from HT22 and PC12 have been selected for resistance to glutamate toxicity and amyloid toxicity, respectively. In the following experiments it was asked if these cell lines show cross-resistance toward amyloid beta peptide (Abeta) and glutamate as well as toward a variety of additional neurotoxins. Conversely, it was determined if inhibitors of oxytosis, a well-defined oxidative stress pathway, also protect cells from the neurotoxins. It is shown that both glutamate and amyloid resistant cells are cross resistant to most of the other toxins or toxic conditions, while inhibitors of oxytosis protect from glutathione and cystine depletion and H2O2 toxicity, but not from the toxic effects of nitric oxide, rotenone, arsenite or cisplatin. It is concluded that while there is a great deal of cross-resistance to neurotoxins, the components of the cell death pathway which has been defined for oxytosis are not used by many of the neurotoxins.     

alpha1,2Fucosyltransferase increases resistance to apoptosis of rat colon carcinoma cells

Accumulation of histo-blood group antigens such as Lewis b, Lewis Y and H in colon cancer is indicative of poor prognosis. It is accompanied by increase in alpha1,2fucosyl-transferase activity, a key enzyme for synthesis of these antigens. Using a model of colon carcinoma, we previously showed that alpha1,2fucosylation increases tumorigenicity. We now show that tumorigenicity inversely correlates with the cells' sensitivity to apoptosis. In addition, poorly tumorigenic REG cells independently transfected with three different alpha1,2fucosyltransferase cDNAs, the human FUT1, the rat FTA and FTB were more resistant than control cells to apoptosis induced in vitro by serum deprivation. Inversely, PRO cells, spontaneously tumorigenic in immunocompetent syngeneic animals and able to synthesize alpha1,2fucosylated glycans, became more sensitive to apoptosis after transfection with a fragment of the FTA cDNA in the antisense orientation. Expression of alpha1,2fucosyl-transferase in poorly tumorigenic REG cells dramatically enhanced their tumorigenicity in syngeneic rats. However, in immunodeficient animals, both control and alpha1,2fuco-syltransferase transfected REG cells were fully tumorigenic and metastatic, indicating that the presence of alpha1,2fucosylated antigens allowed REG tumor cells to escape immune control. Taken together, the results show that increased tumorigenicity mediated by alpha1,2fucosyl-ation is associated to increased resistance to apoptosis and to escape from immune control.     

Iron chelators increase the resistance of Ataxia telangeictasia cells to oxidative stress

Ataxia telangeictasia (A-T) is an autosomal recessive disorder characterized by immune dysfunction, genomic instability, chronic oxidative damage, and increased cancer incidence. Previously, desferal was found to increase the resistance of A-T, but not normal cells to exogenous oxidative stress in the colony forming-efficiency assay, suggesting that iron metabolism is dysregulated in A-T. Since desferal both chelates iron and modulates gene expression, we tested the effects of apoferritin and the iron chelating flavonoid quercetin on A-T cell colony-forming ability. We demonstrate that apoferritin and quercetin increase the ability of A-T cells to form colonies. We also show that labile iron levels are significantly elevated in Atm-deficient mouse sera compared to syngeniec wild type mice. Our findings support a role for labile iron acting as a Fenton catalyst in A-T, contributing to the chronic oxidative stress seen in this disease. Our findings further suggest that iron chelators might promote the survival of A-T cells and hence, individuals with A-T.     

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology.

Methods We have measured H₂O₂-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H₂O₂ and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H₂O₂-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage.

Results A dose-response relationship was found for the H₂O₂-induced dye decomposition in NIH3T3 cells (7.8–125 μM H₂O₂) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62–1000 μM H₂O₂). Fluorescence was significantly increased at 62 μM H₂O₂ in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H₂O₂ than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells.

Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H₂O₂-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H₂O₂.     

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology.

Methods We have measured H₂O₂-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H₂O₂ and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H₂O₂-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage.

Results A dose-response relationship was found for the H₂O₂-induced dye decomposition in NIH3T3 cells (7.8-125 μM H₂O₂) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62-1000 μM H₂O₂). Fluorescence was significantly increased at 62 μM H₂O₂ in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H₂O₂ than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells.

Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H₂O₂-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H₂O₂.     

Expression profiles of p53 and p66shc during oxidative stress-induced senescence in fetal bovine fibroblasts

Somatic cells undergo a permanent cell cycle arrest, called cellular senescence, after a limited number of cell divisions in vitro. Both the tumor suppressor protein p53 and the stress-response protein p66(shc) are suggested to regulate the molecular events associated with senescence. This study was undertaken to investigate the effect of different oxygen tensions and oxidative stress on cell longevity and to establish the role of p53 and p66(shc) in cells undergoing senescence. As a model of cellular senescence, primary fetal bovine fibroblasts were cultured in either 20% O(2) or 5% O(2) atmospheres until senescence was reached. Fibroblasts cultured under 20% O(2) tension underwent senescence after 30 population doublings (PD), whereas fibroblasts cultured under 5% O(2) tension did not exhibit signs of senescence. Oxidative stress, as measured by protein carbonyl content, was significantly elevated in senescent cells compared to their younger counterparts and to fibroblasts cultured under 5% O(2) at the same PD. p53 mRNA gradually decreased in 20% O(2) cultured fibroblasts until senescence was reached, whereas p53 protein levels were significantly increased as well as p53 phosphorylation on serine 20, suggesting that p53 might be stabilized by posttranslational modifications during senescence. Senescence was also associated with high levels of p66(shc) mRNA and protein levels, while the levels remained low and stable in dividing fibroblasts under 5% O(2) atmosphere. Taken together, our results show an effect of oxidative stress on the replicative life span of fetal bovine fibroblasts as well as an involvement of p53, serine 20-p53 phosphorylation and p66(shc) in senescence.     

Habitual exercise induced resistance to oxidative stress

We investigated whether habitual exercise (HE) modulates levels of oxidative DNA damage and responsiveness to oxidative stress induced by renal carcinogen Fe-nitrilotriacetic acid (Fe-NTA). During a ten week protocol, two groups of rats either remained sedentary or underwent swimming for 15-60 min per day, 5 days per week, with or without a weight equivalent to 5% of their body weight. Then we injected Fe-NTA and sacrificed the rats 1 h after the injection. We determined the activity of superoxide dismutase (SOD) in diaphragm and kidney, evaluated levels of 8-hydroxydeoxyguanosine (8OHdG), catalase, and glutathione peroxidase, and assayed OGG1 protein levels in kidney. SOD activity in the diaphragm and kidney was increased in HE rats. By itself, HE had no effect on the level of 8OHdG, but it did significantly suppress induction of 8OHdG by Fe-NTA, and the amount of suppression correlated with intensity of exercise. These results suggest that HE induces resistance to oxidative stress and, at least at the initiation stage, inhibits carcinogenesis.     

Habitual exercise induced resistance to oxidative stress

We investigated whether habitual exercise (HE) modulates levels of oxidative DNA damage and responsiveness to oxidative stress induced by renal carcinogen Fe-nitrilotriacetic acid (Fe-NTA). During a ten week protocol, two groups of rats either remained sedentary or underwent swimming for 15–60?min per day, 5 days per week, with or without a weight equivalent to 5% of their body weight. Then we injected Fe-NTA and sacrificed the rats 1?h after the injection. We determined the activity of superoxide dismutase (SOD) in diaphragm and kidney, evaluated levels of 8-hydroxydeoxyguanosine (8OHdG), catalase, and glutathione peroxidase, and assayed OGG1 protein levels in kidney. SOD activity in the diaphragm and kidney was increased in HE rats. By itself, HE had no effect on the level of 8OHdG, but it did significantly suppress induction of 8OHdG by Fe-NTA, and the amount of suppression correlated with intensity of exercise. These results suggest that HE induces resistance to oxidative stress and, at least at the initiation stage, inhibits carcinogenesis.     

Iron-induced oxidative damage in colon carcinoma (Caco-2) cells

Intestinal epithelial cells have an active apical iron uptake system that is involved in the regulated absorption of iron. By the action of this system, intestinal cells acquire increasing amounts of iron with time. Since intracellular reactive iron is a source of free radicals and a possible cause of colon carcinoma, this study analyzed the oxidative damages generated by iron accumulation in Caco-2 cells. Cells cultured with increasing concentrations of iron increased both total intracellular iron and the reactive iron pool, despite an active IRE/IRP system, which regulates intracellular iron levels. Increasing concentrations of iron resulted in increased protein oxidative damage, as shown by the immunoreactivity for 4-hydroxy-2-nonenal-modified proteins, and markedly induced DNA oxidation determined by 8-hydroxy-2'-deoxyguanidine production. Iron also impaired cell viability, resulting in increased cell death after 6 days of culture. In summary, iron accumulation by intestinal Caco-2 cells correlated with oxidative damage to proteins and DNA. Oxidative damage finally resulted in loss of cell viability. The Fe-induced oxidative damage observed may be relevant in understanding the cascade of events associated with iron-mediated colon carcinogenesis.     

Influence of polyphenols on Escherichia coli resistance to oxidative stress

Among all polyphenols tested (tannic acid and flavonoids belonging to different subclasses) only tannin and quercetin significantly enhanced resistance of Escherichia coli to peroxide stress. Pretreatment of the cells with quercetin and tannin resulted in a decrease in the growth arrest duration under moderate H₂O₂ concentration (2 mM) and an increase in survival under high (10 mM) doses. The shorter growth recovery period in pretreated cells was connected with more rapid H₂O₂ elimination because of induced activity of scavenging enzymes. This effect was absent in the ΔoxyR mutant, which was unable to induce genes responding to peroxide stress. The data obtained suggest that the observed protection was a result of two overlapping effects: induction of OxyR regulon by low concentrations of H₂O₂, accumulated during extracellular autoxidation of quercetin and tannin, and protection of synthesis of OxyR-regulated antioxidant enzymes during H₂O₂ stress because of intracellular binding of iron by quercetin and tannin and suppressing Fenton chemistry.     

Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway.

Shc proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to Ras. The p46shc and p52shc isoforms share a C-terminal SH2 domain, a proline- and glycine-rich region (collagen homologous region 1; CH1) and a N-terminal PTB domain. We have isolated cDNAs encoding for a third Shc isoform, p66shc. The predicted amino acid sequence of p66shc overlaps that of p52shc and contains a unique N-terminal region which is also rich in glycines and prolines (CH2). p52shc/p46shc is found in every cell type with invariant reciprocal relationship, whereas p66shc expression varies from cell type to cell type. p66shc differs from p52shc/p46shc in its inability to transform mouse fibroblasts in vitro. Like p52shc/p46shc, p66shc is tyrosine-phosphorylated upon epidermal growth factor (EGF) stimulation, binds to activated EGF receptors (EGFRs) and forms stable complexes with Grb2. However, unlike p52shc/p46shc it does not increase EGF activation of MAP kinases, but inhibits fos promoter activation. The isolated CH2 domain retains the inhibitory effect of p66shc on the fos promoter. p52shc/p46shc and p66shc, therefore, appear to exert different effects on the EGFR-MAP kinase and other signalling pathways that control fos promoter activity. Regulation of p66shc expression might, therefore, influence the cellular response to growth factors.     

SOD2-mediated effects induced by WR1065 and low-dose ionizing radiation on micronucleus formation in RKO human colon carcinoma cells

RKO36 cells exposed to either WR1065 or 10 cGy X rays show elevated SOD2 gene expression and SOD2 enzymatic activity. Cells challenged at this time with 2 Gy exhibit enhanced radiation resistance. This phenomenon has been identified as a delayed radioprotective effect or an adaptive response when induced by thiols or low-dose radiation, respectively. In this study we investigated the relative effectiveness of both WR1065 and low-dose radiation in reducing the incidence of radiation-induced micronucleus formation in binucleated RKO36 human colon carcinoma cells. The role of SOD2 in this process was assessed by measuring changes in enzymatic activity as a function of the inducing agent used, the level of protection afforded, and the inhibitory effects of short interfering RNA (SOD2 siRNA). Both WR1065 and 10 cGy X rays effectively induced a greater than threefold elevation in SOD2 activity 24 h after exposure. Cells irradiated at this time with 2 Gy exhibited a significant resistance to micronucleus formation (P < 0.05; Student's two-tailed t test). This protective effect was significantly inhibited in cells transfected with SOD2 siRNA. SOD2 played an important role in the adaptive/delayed radioprotective response by inhibiting the initiation of a superoxide anion-induced ROS cascade leading to enhanced mitochondrial and nuclear damages.