Sublethal Oxidative Stress Research Articles

The relationship between p53/p21/Rb and MAPK signaling pathways in human endometrium-derived stem cells under oxidative stress

Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H2O2 activate both p53/p21/Rb and p38MAPK/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However the mutual relations between p53/p21/Rb and MAPK signaling pathways, including ERK, p38 and JNK remain unexplored as yet. Here, we used the specific inhibitors--pifithrin-α (PFT), U0126, SB203580 and SP600125 to "switch off" one of the proteins in these cascades and to evaluate the functional status alterations of the rest proteins. Suppression each of the MAPK significantly increased the p53 phosphorylation levels, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation compared with p38 and JNK. These results suppose the existence of the reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. Analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as the ERK antagonists: JNK is capable to activate ERK, while p38 may block the ERK activation. Together, these results demonstrate complex links between different signaling cascades in stressed hMESC, implicating ERK, p38 and JNK in regulation of the premature senescence via p53/p21/Rb pathway.

The Natural Polyphenol Epigallocatechin Gallate Protects Intervertebral Disc Cells from Oxidative Stress.

Oxidative stress-related phenotypic changes and a decline in the number of viable cells are crucial contributors to intervertebral disc degeneration. The polyphenol epigallocatechin 3-gallate (EGCG) can interfere with painful disc degeneration by reducing inflammation, catabolism, and pain. In this study, we hypothesized that EGCG furthermore protects against senescence and/or cell death, induced by oxidative stress. Sublethal and lethal oxidative stress were induced in primary human intervertebral disc cells with H2O2 (total n = 36). Under sublethal conditions, the effects of EGCG on p53-p21 activation, proliferative capacity, and accumulation of senescence-associated β-galactosidase were tested. Further, the effects of EGCG on mitochondria depolarization and cell viability were analyzed in lethal oxidative stress. The inhibitor LY249002 was applied to investigate the PI3K/Akt pathway. EGCG inhibited accumulation of senescence-associated β-galactosidase but did not affect the loss of proliferative capacity, suggesting that EGCG did not fully neutralize exogenous radicals. Furthermore, EGCG increased the survival of IVD cells in lethal oxidative stress via activation of prosurvival PI3K/Akt and protection of mitochondria. We demonstrated that EGCG not only inhibits inflammation but also can enhance the survival of disc cells in oxidative stress, which makes it a suitable candidate for the development of novel therapies targeting disc degeneration.

Streptomyces natalensis programmed cell death and morphological differentiation are dependent on oxidative stress.

Streptomyces are aerobic Gram-positive bacteria characterized by a complex life cycle that includes hyphae differentiation and spore formation. Morphological differentiation is triggered by stressful conditions and takes place in a pro-oxidant environment, which sets the basis for an involvement of the oxidative stress response in this cellular process. Characterization of the phenotypic traits of Streptomyces natalensis ΔkatA1 (mono-functional catalase) and ΔcatR (Fur-like repressor of katA1 expression) strains in solid medium revealed that both mutants had an impaired morphological development process. The sub-lethal oxidative stress caused by the absence of KatA1 resulted in the formation of a highly proliferative and undifferentiated vegetative mycelium, whereas de-repression of CatR regulon, from which KatA1 is the only known representative, resulted in the formation of scarce aerial mycelium. Both mutant strains had the transcription of genes associated with aerial mycelium formation and biosynthesis of the hyphae hydrophobic layer down-regulated. The first round of the programmed cell death (PCD) was inhibited in both strains which caused the prevalence of the transient primary mycelium (MI) over secondary mycelium (MII). Our data shows that the first round of PCD and morphological differentiation in S. natalensis is dependent on oxidative stress in the right amount at the right time.

Abstract 816: Persistent effects of radiation on intestinal stem cells: implications for colorectal carcinogenesis

Abstract Adult stem cells, similar to somatic cells, are also subject to a similar array of insults and are therefore presumed to accrue radiation damage. Unlike somatic cells, however, lesions arising in the stem cell compartment are propagated both to daughter stem cells and to downstream lineages through the processes of self-renewal and differentiation. The impact of damage accrued in individual stem cells can thus have major ramifications for all levels of the developmental hierarchy leading to cancer initiation and promotion in human as well as in animal models of human colorectal cancer (CRC) such as in APCMin/+. Differentiation of intestinal stem cell (ISC) and ordered migration of differentiated cells along crypt-villus axis is essential for maintenance of intestinal homeostasis and provides a model system to study stem cell alterations after radiation. Furthermore, considering that tumors in animal models of CRC almost exclusively develop in the small intestine and increased intestinal tumor frequency was radiation quality dependent, we investigated persistent effects of radiation quality on ISC as well as on differentiated intestinal epithelial cells in mice. Mice (6 to 8 week; C57BL/6J) were exposed to γ (0.5 and 2 Gy) and 56-Fe (0.5 and 1.6 Gy) radiation, samples were collected 7 d and 2 m after radiation exposure, and carcinogenic precursor events such as persistent oxidative stress and DNA damage were assessed. Higher persistently increased oxidative stress and oxidative DNA damage was observed after 56-Fe relative to γ radiation. However, increased oxidative stress did not increase cell death implying that the oxidative stress was sub-lethal. Autophagy, a self-cannibalistic process of cell involved in removing damaged cell and cell constituents, was consistently downregulated in a radiation quality dependent manner even 2 m after exposure suggesting persistence of damage-bearing cells. We believe radiation-induced autophagy downregulation and sub-lethal oxidative stress is working in tandem to trigger proliferative signaling such as PI3K/Akt and mTOR in intestinal cells. Taken together our results provide insight into radiation quality dependent persistent cancer-related molecular events, which could have altered phenotypic behavior of ISC such as proliferation, differentiation, and migration. Note: This abstract was not presented at the meeting. Citation Format: Kamal Datta, Shubhankar Suman, Santosh Kumar, Albert J. Fornace. Persistent effects of radiation on intestinal stem cells: implications for colorectal carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 816. doi:10.1158/1538-7445.AM2015-816

Beneficial Effects of Nitric Oxide Induced Mild Oxidative Stress on Post-Thawed Bull Semen Quality.

Cryopreservation of semen requires optimized conditions to minimize the harmful effects of various stresses. The main approach for protection of sperm against stress is based on the use of antioxidants and cryoprotectants, which are described as defensive methods. Recently, the application of controlled mild stressors has been de- scribed for activation of a temporary response in oocyte, embryo and somatic cells. In this study a sub-lethal oxidative stress induced by precise concentrations of nitric oxide (NO) has been evaluated for sperm during cryopreservation. In this experimental study, we used different concentrations of NO [0 µM (NO-0), 0.01 µM (NO-0.01), 0.1 µM (NO-0.1), 1 µM (NO-1), 10 µM (NO-10) and 100 µM (NO-100)] during cryopreservation of bull semen. Their effects on post-thawed sperm quality that included motility and velocity parameters, plasma mem- brane functionality, acrosome integrity, apoptosis status, mitochondrial activity and lipid peroxidation after freezing-thawing were investigated. Exposure of sperm before freezing to NO-1 significantly increased total motility (88.4 ± 2.8%), progressive motility (50.4 ± 3.2%) and average path velocity (VAP, 53.8 ± 3.1 µm/s) compared to other extenders. In addition, NO-1 significantly increased plasma mem- brane functionality (89.3 ± 2.9%) compared to NO-0 (75.3 ± 2.9%), NO-0.01 (78.3 ± 2.9%), NO-0.1 (76.4 ± 2.9%), NO-10 (64 ± 2.9%) and NO-100 (42 ± 2.9%). Sperm exposed to NO-1 produced the highest percentage of viable (85.6 ± 2.3%) and the lowest percentage of apoptotic (10.8 ± 2.4%) spermatozoa compared to the other extenders. Also, NO-100 resulted in a higher percentage of dead spermatozoa (27.1 ± 2.7%) compared to the other extenders. In terms of mitochondrial activity, there was no significant difference among NO-0 (53.4 ± 3.2), NO-0.01 (52.1 ± 3.2), NO-0.1 (50.8 ± 3.2) and NO-1 (53.1 ± 3.2). For acrosome integrity, no significant different was observed in sperm exposed to different concentrations of NO. Induction of sub-lethal oxidative stress with 1 µM NO would be beneficial for cryopreservation of bull semen.

2-deoxy-D-Glucose Synergizes with Doxorubicin or L-Buthionine Sulfoximine to Reduce Adhesion and Migration of Breast Cancer Cells.

Cancer metastasis depends on cell motility which is driven by cycles of actin polymerization and depolymerization. Reactive oxygen species (ROS) and metabolic oxidative stress have long been associated with cancer. ROS play a vital role in regulating actin dynamics that are sensitive to oxidative modification. The current work aimed at studying the effects of sub-lethal metabolic oxidative stress on actin cytoskeleton, focal adhesion and cell migration. T47D human breast cancer cells were treated with 2-deoxy- D-glucose (2DG), L-buthionine sulfoximine (BSO), or doxorubicin (DOX), individually or in combination, and changes in intracellular total glutathione and malondialdehyde (MDA) levels were measured. The expression of three major antioxidant enzymes was studied by immunoblotting, and cells were stained with fluorescent- phalloidin to evaluate changes in F-actin organization. In addition, cell adhesion and degradation ability were measured. Cell migration was studied using wound healing and transwell migration assays. Our results show that treating T47D human breast cancer cells with drug combinations (2DG/BSO, 2DG/DOX, or BSO/DOX) decreased intracellular total glutathione and increased oxidized glutathione, lipid peroxidation, and cytotoxicity. In addition, the drug combinations caused a reduction in cell area and mitotic index, prophase arrest and a decreased ability to form invadopodia. The formation of F-actin aggregates was increased in treated T47D cells. Moreover, combination therapy reduced cell adhesion and the rate of cell migration. Our results suggest that exposure of T47D breast cancer cells to combination therapy reduces cell migration via effects on metabolic oxidative stress.

Interaction between ROS dependent DNA damage, mitochondria and p38 MAPK underlies senescence of human adult stem cells.

Human endometrium-derived mesenchymal stem cells (hMESCs) enter the premature senescence under sublethal oxidative stress, however underlying mechanism remains unknown. Here, we showed that exogenous H2O2 induces a rapid phosphorylation and co-localization of ATM, H2A.X, 53BP1 leading to DNA damage response (DDR) activation. DDR was accompanied with nuclear translocation of p-p53 followed by up-regulation of p21Waf1 and the permanent hypophosphorylation of pRb. Additionally, the increased p38MAPK/MAPKAPK-2 activation persisted in H2O2-treated cells. We suggest that both p53/p21/pRb and p38MAPK/MAPKAPK-2 pathways are responsible for establishing an irreversible cell cycle arrest that is typical of senescence. The process of further stabilization of senescence required prolonged DDR signaling activation that was provided by the permanent ROS production which in turn was regulated by both p38MAPK and the increased functional mitochondria. To reverse senescence, the pharmacological inhibition of p38MAPK was performed. Cell treatment with SB203580 was sufficient to recover partially senescence phenotype, to block the ROS elevation, to decrease the mitochondrial function, and finally to rescue proliferation. Thus, suppression of the p38MAPK pathway resulted in a partial prevention of H2O2-induced senescence of hMESCs. The current study is the first to reveal the molecular mechanism of the premature senescence of hMESCs in response to oxidative stress.

Oxidative stress effects of titanium dioxide nanoparticle aggregates in zebrafish embryos

There is limited data on the sub-lethal oxidative stress effects of titanium dioxide nanoparticle aggregates (NM-TiO2) and its modulation by simulated solar radiation (SSR) to aquatic organisms. This study aimed to examine sublethal oxidative stress effects of aqueous exposure to three different types of NM-TiO2 differing in their coating or crystal structure but of similar primary size (20nm) plus a micron-sized bulk material to zebrafish embryos without and with SSR. Oxidative stress responses of known model prooxidant (tert-Butyl hydroperoxide) and photoprooxidant (fluoranthene) compounds were also studied. Results evidenced a low bio-availability of NM-TiO2 to embryos with detrimental effects on growth at 1mgml−1. Phototoxicity increased moderately, by 3 and 1.5 fold, under co-exposures to fluoranthene (100μgl−1) and to the NM-TiO2 P25 (1mgml−1), respectively, being unchanged in the other TiO2 aggregates. In vitro exposures under SSR confirmed that the NM-TiO2 P25 had the highest potential to generate reactive oxygen species (ROS). Antioxidant enzyme activities of superoxide dismutase increased shortly after exposure to the studied materials, whereas the levels of glutathione tend to be altered after longer exposures. All compounds were able to produce oxidative stress enhancing the senescence-associated β galactosidase pigment (SA-β-gal). Under SSR radiation the NM-TiO2 P25 affected antioxidant and oxidative stress responses as the phototoxic compound fluoranthene. These results indicated that despite the low bio-availability of NM-TiO2 to zebrafish embryos, P25 was phototoxic due to the production of reactive oxygen species. Nevertheless, overall our results indicated that fish development may not be at high risk in the face of NM-TiO2, even when combined with prooxidant conditions.

Acute and potentially persistent effects of scuba diving on the blood transcriptome of experienced divers

During scuba diving, the circulatory system is stressed by an elevated partial pressure of oxygen while the diver is submerged and by decompression-induced gas bubbles on ascent to the surface. This diving-induced stress may trigger decompression illness, but the majority of dives are asymptomatic. In this study we have mapped divers' blood transcriptomes with the aim of identifying genes, biological pathways, and cell types perturbed by the physiological stress in asymptomatic scuba diving. Ten experienced divers abstained from diving for >2 wk before performing a 3-day series of daily dives to 18 m depth for 47 min while breathing compressed air. Blood for microarray analysis was collected before and immediately after the first and last dives, and 10 matched nondivers provided controls for predive stationary transcriptomes. MetaCore GeneGo analysis of the predive samples identified stationary upregulation of genes associated with apoptosis, inflammation, and innate immune responses in the divers, most significantly involving genes in the TNFR1 pathway of caspase-dependent apoptosis, HSP60/HSP70 signaling via TLR4, and NF-κB-mediated transcription. Diving caused pronounced shifts in transcription patterns characteristic of specific leukocytes, with downregulation of genes expressed by CD8+ T lymphocytes and NK cells and upregulation of genes expressed by neutrophils, monocytes, and macrophages. Antioxidant genes were upregulated. Similar transient responses were observed after the first and last dive. The results indicate that sublethal oxidative stress elicits the myeloid innate immune system in scuba diving and that extensive diving may cause persistent change in pathways controlling apoptosis, inflammation, and innate immune responses.

Epigenetics and the Environment: In Search of the “Toleroasome” Vital to Execution of Ischemic Preconditioning

Activation and repression of gene expression are key features of ischemic tolerance. Converging lines of inquiry from several groups suggests that epigenetic proteins may transduce sublethal stresses, including bioenergetic or oxidative stress into durable (2-3 days) changes in gene expression that mediate ischemic tolerance. Here we discuss the potential mechanisms by which changes in cell state (e.g., ATP, NAD+, and oxygen) can modify specific targets including polycomb complexes, jumonji domain histone demethylases, and zinc and NAD-dependent histone decetylases and thus trigger an adaptive program. A major unanswered question is whether these proteins work in parallel or convergently as part of a "tolerosome" (tolero is the Latin word for tolerance), a multiprotein complex recruited to promoters or enhancers of specific genes, to mediate preconditioning. Whatever the case may be, epigenetic proteins are fertile targets for the treatment of stroke.

Sublethal Oxidative Stress Induces the Premature Senescence of Human Mesenchymal Stem Cells Derived from Endometrium

The specific responses of mesenchymal stem cells to oxidative stress may play a crucial role in regulation of tissue homeostasis as well as regeneration of organs after oxidative injury. The responses of human endometrium-derived mesenchymal stem cells (hMESCs) to oxidative stress remain still unknown. Herein, we examined the impact of H2O2 on cell viability, induction of premature senescence, and apoptosis. hMESCs were highly resistant to H2O2 compared with human diploid fibroblasts. To test a hypothesis whether hMESCs may undergo oxidative stress-induced premature senescence, cells were briefly exposed to the sublethal H2O2 doses. H2O2-treated cells were permanently arrested, lost Ki67 proliferation marker, and exhibited a senescent phenotype including cell hypertrophy and increased SA-β-Gal activity. Additionally, in stressed cells the expression levels of p21Cip1, SOD1, SOD2, and GPX1 were elevated. hMESCs survived under stress were not able to resume proliferation, indicating the irreversible loss of proliferative potential. While the low H2O2 doses promoted senescence in hMESCs, the higher H2O2 doses induced also apoptosis in a part of the cell population. Of note, senescent hMESCs exhibited high resistance to apoptosis. Thus, we have demonstrated for the first time that hMESCs may enter a state of premature senescence in response to sublethal oxidative stress.

Effects of chronic sub-lethal oxidative stress on biofilm formation by Azotobacter vinelandii

This work showed that perturbations of the physiological steady-state level of reactive oxygen species (ROS) affected biofilm genesis and the characteristics of the model bacterium Azotobacter vinelandii. To get a continuous endogenous source of ROS, a strain exposed to chronic sub-lethal oxidative stress was deprived of the gene coding for the antioxidant rhodanese-like protein RhdA (MV474). In this study MV474 biofilm showed (i) a seven-fold higher growth rate, (ii) induction of catalase and alkyl-hydroxyl-peroxidase enzymes, (iii) higher average thicknesses due to increased production of a polysaccharide-rich extracellular matrix and (iv) less susceptibility to hydrogen peroxide than the wild-type strain (UW136). MV474 showed increased swimming and swarming activity and the swarming colonies experienced a higher level of oxidative stress compared to UW136. A continuous exogenous source of ROS increased biofilm formation in UW136. Overall, chronic sub-lethal oxidative events promoted sessile behavior in A. vinelandii.

Increase in antioxidant gene transcripts, stress tolerance and biocontrol efficacy of Candida oleophila following sublethal oxidative stress exposure

A pretreatment of the yeast, Candida oleophila, with 5 mM H(2)O(2) for 30 min (sublethal) increased yeast tolerance to subsequent lethal levels of oxidative stress (50 mM H(2)O(2)), high temperature (40 °C), and low pH (pH 4). Compared with non-stress-adapted yeast cells, stress-adapted cells exhibited better control of apple fruit infections by Penicillium expansum and Botrytis cinerea and had initially higher growth rates in apple wounds. Suppression subtractive hybridization analysis was used to identify genes expressed in yeast in response to sublethal oxidative stress. Transcript levels were confirmed using semiquantitative reverse transcription-PCR. Seven antioxidant genes were upregulated. The elevated expression of these genes was associated with less accumulation of reactive oxygen species and a lower level of protein and lipid oxidation under subsequent stresses. These data support the premise that induction of abiotic stress tolerance in biocontrol yeast can improve biocontrol efficacy by upregulation of genes involved in the amelioration of oxidative stress.

Cells Exposed to Sublethal Oxidative Stress Selectively Attract Monocytes/Macrophages via Scavenger Receptors and MyD88-Mediated Signaling

The innate immune system responds to endogenous molecules released during cellular stress or those that have undergone modifications normally absent in healthy tissue. These structures are detected by pattern-recognition receptors, alerting the immune system to "danger." In this study, we looked for early signals that direct immune cells to cells undergoing stress before irreversible damage takes place. To avoid detecting signals emanating from apoptotic or necrotic cells we exposed fibroblasts to sublethal oxidative stress. Our results indicate that both nonenzymatic chemical reactions and aldehyde dehydrogenase-2-mediated enzymatic activity released signals from fibroblasts that selectively attracted CD14(+) monocytes but not T, NK, and NKT cells or granulocytes. Splenocytes from MyD88(-/-) mice did not migrate, and treatment with an inhibitory peptide that blocks MyD88 dimerization abrogated human monocyte migration. Monocyte migration was accompanied by downmodulation of CD14 expression and by the phosphorylation of IL-1R-associated kinase 1, a well-known MyD88-dependent signaling molecule. The scavenger receptor inhibitors, dextran sulfate and fucoidan, attenuated monocyte migration toward stressed cells and IL-1R-associated kinase 1 phosphorylation. Surprisingly, although monocyte migration was MyD88 dependent, it was not accompanied by inflammatory cytokine secretion. Taken together, these results establish a novel link between scavenger receptors and MyD88 that together function as sensors of oxidation-associated molecular patterns and induce monocyte motility. Furthermore, the data indicate that MyD88 independently regulates monocyte activation and motility.

Roles of αvβ5, FAK and MerTK in oxidative stress inhibition of RPE cell phagocytosis

Efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelial cells (RPE) plays a key role in biological renewal of these highly peroxidizable structures and in maintenance of retina health. Here, we used an in vitro RPE cell phagocytosis assay to investigate how sub-lethal oxidative stress modifies the key components of the cell phagocytic machinery leading to severe impairment of phagocytosis. Sub-lethal oxidative treatment, induced by hydrogen peroxide (H 2O 2), significantly inhibited binding and uptake of POS by RPE cells. However, sub-lethal oxidative stress did not affect cell surface expression of αvβ5 or RPE cell adhesion to αvβ5. Similarly, the enzymatic activity of mature cathepsin D was not altered upon challenge by oxidative stress. In contrast, studies of signaling molecules in the RPE cell phagocytic machinery revealed that sub-lethal oxidative stress inhibits POS-induced activation of FAK and MerTK. Our data demonstrate that sub-lethal oxidative treatment with H 2O 2 inhibits phagocytic activity of ARPE-19 cells, in part by inhibiting FAK and MerTK.

Oxidative stress induces senescence in chondrocytes

Cellular senescence is a program activated during diverse situations of cell stress. Chondrocytes differ from other somatic cells as articular cartilage is an avascular tissue. The effects of oxidative stress on chondrocytes are still unknown. Our studies were to investigate into the proliferation potential, cytological features and the telomere linked stress response system of human osteoarthritic chondrocytes, subjected to acute or prolonged oxidant challenge with hydrogen peroxide. Telomere length was measured using the telomere restriction fragment assay, gene expression was determined by RT-PCR. Sub-lethal doses of oxidative stress induced cell-cycle arrest, senescent-morphological features and senescence-associated β-galactosidase positivity. Prolonged oxidative treatment had no effects on cell proliferation or morphology. Sub-lethal and prolonged low doses of oxidative stress considerably accelerated telomere attrition. The effects of sub-lethal oxidative stress regarding proliferation and telomere biology were more distinct in senescent cells. Acute oxidant insult caused up-regulation of p21 expression to levels comparable to senescent cells. TRF2 protects telomere ends and showed elevated expression levels. SIRT1 and XRCC5 enable cells to cope with unfavorable growing conditions. Both were up-regulated after oxidant insult, but expression levels decreased in aging cells. Taken together, oxidative stress considerably accelerated telomere shortening and cellular aging in chondrocytes. Senescent cells showed a reduced tolerance to oxidative stress.

TRAF2 phosphorylation promotes NF-κB–dependent gene expression and inhibits oxidative stress-induced cell death

Tumor necrosis factor α (TNF-α) receptor-associated factor 2 (TRAF2) regulates activation of the c-Jun N-terminal kinase (JNK)/c-Jun and the inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling cascades in response to TNF-α stimulation. Gene knockout studies have revealed that TRAF2 inhibits TNF-α-induced cell death but promotes oxidative stress-induced apoptosis. Here we report that TNF-α and oxidative stress both induce TRAF2 phosphorylation at serines 11 and 55 and that this dual phosphorylation promotes the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation. Prolonged IKK activation trigged by TNF-α plays an essential role in efficient expression of a subset of NF-κB target genes but has no substantial role in TNF-α-induced cell death. On the other hand, TRAF2 phosphorylation in response to oxidative stress significantly promotes cell survival by inducing prolonged IKK activation and by inhibiting the prolonged phase of JNK activation. Notably, stable expression of phospho-null mutant TRAF2 in cancer cells leads to an increase in the basal and inducible JNK activation and B-cell lymphoma 2 (Bcl-2) phosphorylation. In addition, exposure of cells expressing phospho-null mutant TRAF2 to sublethal oxidative stress results in a rapid degradation of Bcl-2 and cellular inhibitor of apoptosis 1 as well as significantly increased cell death. These results suggest that TRAF2 phosphorylation is essential for cell survival under conditions of oxidative stress.

Novel mechanism of increased Ca2+ release following oxidative stress in neuronal cells involves type 2 inositol-1,4,5-trisphosphate receptors

Dysregulation of Ca2+ signaling following oxidative stress is an important pathophysiological mechanism of many chronic neurodegenerative disorders, including Alzheimer's disease, age-related macular degeneration, glaucomatous and diabetic retinopathies. However, the underlying mechanisms of disturbed intracellular Ca2+ signaling remain largely unknown. We here describe a novel mechanism for increased intracellular Ca2+ release following oxidative stress in a neuronal cell line. Using an experimental approach that included quantitative polymerase chain reaction, quantitative immunoblotting, microfluorimetry and the optical imaging of intracellular Ca2+ release, we show that sub-lethal tert-butyl hydroperoxide-mediated oxidative stress result in a selective up-regulation of type-2 inositol-1,4,5,-trisphophate receptors. This oxidative stress mediated change was detected both at the transcriptional and translational level and functionally resulted in increased Ca2+ release into the nucleoplasm from the membranes of the nuclear envelope at a given receptor-specific stimulus. Our data describe a novel source of Ca2+ dysregulation induced by oxidative stress with potential relevance for differential subcellular Ca2+ signaling specifically within the nucleus and the development of novel neuroprotective strategies in neurodegenerative disorders.

Endoplasmic reticulum stress inducers provide protection against 6-hydroxydopamine-induced cytotoxicity

6-Hydroxydopamine (6-OHDA) is a neurotoxin used to establish experimental models of Parkinson's disease. Exposure to 6-OHDA results in cell death associated with oxidative stress. Pretreatments with sublethal oxidative stress and some pharmacological drugs have been shown to exert preconditioning effects on cytotoxicity caused by 6-OHDA. In this study, we investigated whether endoplasmic reticulum (ER) stress exerts preconditioning effects on 6-OHDA-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Pretreatment with ER stress inducers, thapsigargin (Tg) and tunicamycin (Tm), promoted GRP78 mRNA induction and ATF4 translation, which are ER stress markers, under our experimental conditions and protected against the cytotoxicity. The protective effect of Tg was more potent than that of Tm. We also found that Tg induced the expression of the antioxidant gene heme oxygenase-1 (HO-1) in a dose-dependent manner, whereas Tm had a weak effect on HO-1 induction. Flow cytometric analysis revealed that reactive oxygen species generated by 6-OHDA were more effectively suppressed in cells pretreated with Tg than with Tm. Therefore, it is likely that Tg enhances antioxidative defenses in SH-SY5Y cells compared with Tm. Because actinomycin D inhibited HO-1 induction by Tg, the induction of HO-1 may be regulated at the transcriptional level. Moreover, the specific eIF2α phosphatase inhibitor salubrinal augmented Tg-induced HO-1 expression. Therefore, the downstream signaling pathway of eIF2α might be involved in Tg-induced HO-1 expression. On the other hand, the reporter assay revealed that Tg stimulated the antioxidant response element (ARE) that is located in regulatory regions of antioxidant genes such as HO-1. Taken together, our data suggest that preconditioning effects induced by Tg mediate an adaptive response to 6-OHDA-induced cytotoxicity via phosphorylation of eIF2α and activation of the ARE.

Abstract 1692: TRAF2 phosphorylation plays a critical role in cell adaptation to chronic oxidative stress

Abstract The tumor microenvironment is characterized by hypoxia, low glucose and oxidative stress. Cancer-cell adaptation to such chronic stress has profound consequences for malignant progression and the response to therapy. Numerous studies have demonstrated that the NF-κB signaling pathway promotes tumor progression and metastasis, whereas its inhibition sensitizes cancer cells to stress-induced apoptosis. TNF receptor associated factor 2 (TRAF2) is an adaptor protein that regulates the activation of the JNK and NF-κB signaling cascades in response to TNFα stimulation. We mapped two phosphorylation sites (Ser-11 and Ser-55) on the N-terminal RING domain of TRAF2, and reported that phosphorylation at these sites increases basal and TNFα-induced NF-κB activation. Recently we found that oxidative stress induces TRAF2 phosphorylation at both Ser-11 and Ser-55, and that this phosphorylation plays a critical role in cell survival in the context of chronic oxidative stress: cells that expressed a TRAF2 mutant in which both Ser-11 and Ser-55 are replaced by Ala (TRAF2-S11/55A) underwent apoptosis even when the cells were subjected to sublethal oxidative stress. Analysis of both JNK and IKK pathways revealed that oxidative stress induces strong and prolonged JNK, but not IKK, activation in cells expressing TRAF2-S11/55A, suggesting that TRAF2 phosphorylation promotes cell survival by inhibiting the prolonged phase of JNK activation. Importantly, TRAF2 was found to be constitutively phosphorylated in some malignant cancer cell lines, as well as in some human tumor tissues. In addition, basal TRAF2 phosphorylation is elevated in H-Ras-V12-transformed cells, and its inhibition significantly sensitized the cells to oxidative stress-induced apoptosis. These results unveil a new layer of mechanism for cancer cell adaptation to oxidative stress modulated by TRAF2 phosphorylation, and suggest that inhibiting TRAF2 phosphorylation could be a new target for anti-cancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1692.