An evolutionarily conserved Rit GTPase-p38 MAPK signaling pathway mediates oxidative stress resistance

Ras-related small GTP-binding proteins control a wide range of cellular processes by regulating a variety of effector pathways, including prominent roles in the control of mitogen-activated protein kinase (MAPK) cascades. Although the regulatory role(s) for many Ras family GTPases are well established, the physiological function for the Rit/Rin subfamily has been lacking. Here, using both knockout mice and Drosophila models, we demonstrate an evolutionarily conserved role for Rit subfamily GTPases (mammalian Rit and Rin, and the Drosophila RIC homologue) in governing survival in response to oxidative stress. Primary embryonic fibroblasts derived from Rit knockout mice display increased apoptosis and selective disruption of MAPK signaling following reactive oxygen species (ROS) exposure but not in response to endoplasmic reticulum stress or DNA damage. These deficits include a reduction in ROS-mediated stimulation of a p38-MK2-HSP27 signaling cascade that controls Akt activation, directing Bad phosphorylation to promote cell survival. Furthermore, D-RIC null flies display increased susceptibility to environmental stresses and reduced stress-dependent p38 signaling, extending the Rit-p38 survival pathway to Drosophila. Together, our studies establish the Rit GTPases as critical regulators of an evolutionarily conserved, p38 MAPK-dependent signaling cascade that functions as an important survival mechanism for cells in response to oxidative stress.     

p38 MAPK: A dual role in hepatocyte proliferation through reactive oxygen species

Abstract

p38 MAPKs are important mediators of signal transduction that respond to a wide range of extracellular stressors such as UV radiation, osmotic shock, hypoxia, pro-inflammatory cytokines, and oxidative stress. The most abundant family member is p38α, which helps to couple cell proliferation and growth in response to certain damaging stimuli. In fact, increased proliferation and impaired differentiation are hallmarks of p38α-deficient cells. It has been reported that reactive oxygen species (ROS) play a critical role in cytokine-induced p38α activation. Under physiological conditions, p38α can function as a mediator of ROS signaling and either activate or suppress cell cycle progression depending on the activation stimulus. The interplay between cell proliferation, p38 MAPK activation, and ROS production plays an important role in hepatocytes. In fact, low levels of ROS seem to be needed to activate several signaling pathways in response to hepatectomy and to orchestrate liver regeneration. p38 MAPK works as a sensor of oxidative stress and cells that have developed mechanisms to uncouple p38 MAPK activation from oxidative stress are more likely to become tumorigenic. So far, p38α influences the redox balance, determining cell survival, terminal differentiation, proliferation, and senescence. Further studies would be necessary in order to clarify the precise role of p38 MAPK signaling as a redox therapeutical target.     

Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

Background

Genotoxic stress, such as by exposure to bromodeoxyuridine (BrdU) and cigarette smoke, induces premature cell senescence. Recent evidence indicates that cellular senescence of various types of cells is accelerated in COPD patients. However, whether the senescence of airway epithelial cells contributes to the development of airway diseases is unknown. The present study was designed to test the hypothesis that premature senescence of airway epithelial cells (Clara cells) impairs repair processes and exacerbates inflammation after airway injury.

Methods

C57/BL6J mice were injected with the Clara-cell-specific toxicant naphthalene (NA) on days 0, 7, and 14, and each NA injection was followed by a daily dose of BrdU on each of the following 3 days, during which regenerating cells were allowed to incorporate BrdU into their DNA and to senesce. The p38 MAPK inhibitor SB202190 was injected 30 minutes before each BrdU dose. Mice were sacrificed at different times until day 28 and lungs of mice were obtained to investigate whether Clara cell senescence impairs airway epithelial regeneration and exacerbates airway inflammation. NCI-H441 cells were induced to senesce by exposure to BrdU or the telomerase inhibitor MST-312. Human lung tissue samples were obtained from COPD patients, asymptomatic smokers, and nonsmokers to investigate whether Clara cell senescence is accelerated in the airways of COPD patients, and if so, whether it is accompanied by p38 MAPK activation.

Results

BrdU did not alter the intensity of the airway epithelial injury or inflammation after a single NA exposure. However, after repeated NA exposure, BrdU induced epithelial cell (Clara cell) senescence, as demonstrated by a DNA damage response, p21 overexpression, increased senescence-associated β-galactosidase activity, and growth arrest, which resulted in impaired epithelial regeneration. The epithelial senescence was accompanied by p38 MAPK-dependent airway inflammation. Senescent NCI-H441 cells impaired epithelial wound repair and secreted increased amounts of pro-inflammatory cytokines in a p38 MAPK-dependent manner. Clara cell senescence in COPD patients was accelerated and accompanied by p38 MAPK activation.

Conclusions

Senescence of airway epithelial cells impairs repair processes and exacerbates p38 MAPK-dependent inflammation after airway injury, and it may contribute to the pathogenesis of COPD.     

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.     

RAC2-P38 MAPK-dependent NADPH oxidase activity is associated with the resistance of quiescent cells to ionizing radiation

Our recent study showed that quiescent G0 cells are more resistant to ionizing radiation than G1 cells; however, the underlying mechanism for this increased radioresistance is unknown. Based on the relatively lower DNA damage induced in G0 cells, we hypothesize that these cells are exposed to less oxidative stress during exposure. As a catalytic subunit of NADPH oxidase, Ras-related C3 botulinum toxin substrate 2 (RAC2) may be involved in the cellular response to ionizing radiation. Here, we show that RAC2 was expressed at low levels in G0 cells but increased substantially in G1 cells. Relative to G1 cells, the total antioxidant capacity in G0 phase cells increased upon exposure to X-ray radiation, whereas the intracellular concentration of ROS and malondialdehyde increased only slightly. The induction of DNA single- and double-stranded breaks in G1 cells by X-ray radiation was inhibited by knockdown of RAC2. P38 MAPK interaction with RAC2 resulted in a decrease of functional RAC2. Increased phosphorylation of P38 MAPK in G0 cells also increased cellular radioresistance; however, excessive production of ROS caused P38 MAPK dephosphorylation. P38 MAPK, phosphorylated P38 MAPK, and RAC2 regulated in mutual feedback and negative feedback regulatory pathways, resulting in the radioresistance of G0 cells.     

Exposure to heavy ion radiation induces persistent oxidative stress in mouse intestine

Ionizing radiation-induced oxidative stress is attributed to generation of reactive oxygen species (ROS) due to radiolysis of water molecules and is short lived. Persistent oxidative stress has also been observed after radiation exposure and is implicated in the late effects of radiation. The goal of this study was to determine if long-term oxidative stress in freshly isolated mouse intestinal epithelial cells (IEC) is dependent on radiation quality at a dose relevant to fractionated radiotherapy. Mice (C57BL/6J; 6 to 8 weeks; female) were irradiated with 2 Gy of γ-rays, a low-linear energy transfer (LET) radiation, and intestinal tissues and IEC were collected 1 year after radiation exposure. Intracellular ROS, mitochondrial function, and antioxidant activity in IEC were studied by flow cytometry and biochemical assays. Oxidative DNA damage, cell death, and mitogenic activity in IEC were assessed by immunohistochemistry. Effects of γ radiation were compared to (56)Fe radiation (iso-toxic dose: 1.6 Gy; energy: 1000 MeV/nucleon; LET: 148 keV/μm), we used as representative of high-LET radiation, since it's one of the important sources of high Z and high energy (HZE) radiation in cosmic rays. Radiation quality affected the level of persistent oxidative stress with higher elevation of intracellular ROS and mitochondrial superoxide in high-LET (56)Fe radiation compared to unirradiated controls and γ radiation. NADPH oxidase activity, mitochondrial membrane damage, and loss of mitochondrial membrane potential were greater in (56)Fe-irradiated mice. Compared to γ radiation oxidative DNA damage was higher, cell death ratio was unchanged, and mitotic activity was increased after (56)Fe radiation. Taken together our results indicate that long-term functional dysregulation of mitochondria and increased NADPH oxidase activity are major contributing factors towards heavy ion radiation-induced persistent oxidative stress in IEC with potential for neoplastic transformation.     

Impact of HMG-CoA reductase inhibition on oxidant-induced injury in human retinal pigment epithelium cells

In addition to cholesterol-lowering effect, HMG-CoA reductase inhibition by statins has been shown to have protective effect in many cells type. The loss of vision in retinal degeneration disease associates with oxidative stress and apoptosis in retinal pigment epithelium (RPE) cell. This study was designed to examine the effect of statins on oxidant-induced damage in human RPE cells. Cultured human ARPE-19 (ARPE) cells were challenged with hydrogen peroxide (H(2) O(2) ) plus tumor necrosis factor alpha (TNFα) in the presence or absence of statins or various stress signaling inhibitors, including anti-oxidants N-acetyl cysteine (NAC), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI), and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580. Apoptosis was evaluated by TUNEL analysis and cell viability was determined by MTT assay. Reactive oxygen species (ROS) were detected by 2',7'-dichlorodihydrofluorescein diacetate (H(2) DCFH-DA). Expression of p-p38 MAPK protein was measured by Western blot analysis. Our findings indicate that statins treatment significantly suppressed oxidant-induced ROS accumulation and RPE apoptosis. Statins increased cell viability in a dose-dependent manner. In addition, statins treatment prevented the activation of NADPH oxidase and p38 MAPK signaling induced by oxidative stress. These results suggest that statins protects ARPE cells from oxidative stress via an NADPH oxidase and/or p38 MAPK-dependent mechanisms, which may contribute to statins-induced beneficial effects on RPE function.     

Double-stranded RNA activates a p38 MAPK-dependent cell survival program in biliary epithelia

Double-stranded RNA (dsRNA) is produced during replicative viral infection or genotoxic stress. Thus knowledge of the cellular response to dsRNA is necessary to understand the effects of DNA damage or viral infection in biliary epithelia. We assessed the effect of dsRNA on biliary epithelial cell proliferation and apoptosis and the role of the stress-activated p38 MAPK signaling pathway in these responses. dsRNA did not induce apoptosis or proliferation in Mz-ChA-1 human malignant cholangiocytes, but decreased cytotoxicity induced by camptothecin or tumor necrosis factor-related apoptosis inducing ligand and decreased activity of caspases 3, 8, and 9. Furthermore, dsRNA increased p38 MAPK and JNK kinase active site phosphorylation but had no effect on either MAPK kinase (MEK)1/2 or protein kinase R phosphorylation. Inhibition of p38 MAPK with SB-203580 increased basal caspase activity. Thus dsRNA stimulates a p38 MAPK-dependent cell-survival pathway in biliary epithelial cells that may modulate the response of the biliary epithelia to dsRNA produced during genotoxic injury or virus infection.     

Accumulation of the cell cycle regulators TP53 and CDKN1A (p21) in human fibroblasts after exposure to low- and high-LET radiation

The accumulation of the cell cycle regulators TP53 and CDKN1A (p21/CIP1/WAF1) was investigated after exposure to X rays and carbon ions (170 keV microm(-1)) and xenon, bismuth and uranium ions (8900-15,000 keV microm(-1)) in normal human fibroblasts. The influence of the overall dose and the LET of these radiation types was studied systematically and the kinetics of the cell response was followed up to 24 h after exposure. The accumulation of TP53 protein was dependent on the dose and the LET, and TP53 levels declined to lower levels for all radiation types within 24 h after exposure. CDKN1A levels increased and peaked at 3 to 6 h after exposure. The persisting level of this protein at 24 h was strongly dependent on the dose and the LET for X rays and carbon ions. The exposure to very high-LET ions (8900-15,000 keV microm(-1)) did not lead to a further increase in CDKN1A, suggesting a saturation effect for the induction of this protein. The cellular effects of elevated CDKN1A after particle irradiation are discussed.     

Ketone bodies alter dinitrophenol-induced glucose uptake through AMPK inhibition and oxidative stress generation in adult cardiomyocytes

In aerobic conditions, the heart preferentially oxidizes fatty acids. However, during metabolic stress, glucose becomes the major energy source, and enhanced glucose uptake has a protective effect on heart function and cardiomyocyte survival. Thus abnormal regulation of glucose uptake may contribute to the development of cardiac disease in diabetics. Ketone bodies are often elevated in poorly controlled diabetics and are associated with increased cellular oxidative stress. Thus we sought to determine the effect of the ketone body beta-hydroxybutyrate (OHB) on cardiac glucose uptake during metabolic stress. We used 2,4-dinitrophenol (DNP), an uncoupler of the mitochondrial oxidative chain, to mimic hypoxia in cardiomyocytes. Our data demonstrated that chronic exposure to OHB provoked a concentration-dependent decrease of DNP action, resulting in 56% inhibition of DNP-mediated glucose uptake at 5 mM OHB. This was paralleled by a diminution of DNP-mediated AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation. Chronic exposure to OHB also increased reactive oxygen species (ROS) production by 1.9-fold compared with control cells. To further understand the role of ROS in OHB action, cardiomyocytes were incubated with H(2)O(2). Our results demonstrated that this treatment diminished DNP-induced glucose uptake without altering activation of the AMPK/p38 MAPK signaling pathway. Incubation with the antioxidant N-acetylcysteine partially restored DNP-mediated glucose but not AMPK/p38 MAPK activation. In conclusion, these results suggest that ketone bodies, through inhibition of the AMPK/p38 MAPK signaling pathway and ROS overproduction, regulate DNP action and thus cardiac glucose uptake. Altered glucose uptake in hyperketonemic states during metabolic stress may contribute to diabetic cardiomyopathy.     

Non-thermal activation of the hsp27/p38MAPK stress pathway by mobile phone radiation in human endothelial cells: molecular mechanism for cancer- and blood-brain barrier-related effects

Abstract We have examined whether non-thermal exposures of cultures of the human endothelial cell line EA.hy926 to 900 MHz GSM mobile phone microwave radiation could activate stress response. Results obtained demonstrate that 1-hour non-thermal exposure of EA.hy926 cells changes the phosphorylation status of numerous, yet largely unidentified, proteins. One of the affected proteins was identified as heat shock protein-27 (hsp27). Mobile phone exposure caused a transient increase in phosphorylation of hsp27, an effect which was prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38MAPK). Also, mobile phone exposure caused transient changes in the protein expression levels of hsp27 and p38MAPK. All these changes were non-thermal effects because, as determined using temperature probes, irradiation did not alter the temperature of cell cultures, which remained throughout the irradiation period at 37 ± 0.3 °C. Changes in the overall pattern of protein phosphorylation suggest that mobile phone radiation activates a variety of cellular signal transduction pathways, among them the hsp27/p38MAPK stress response pathway. Based on the known functions of hsp27, we put forward the hypothesis that mobile phone radiation-induced activation of hsp27 may (i) facilitate the development of brain cancer by inhibiting the cytochrome c/caspase-3 apoptotic pathway and (ii) cause an increase in blood-brain barrier permeability through stabilization of endothelial cell stress fibers. We postulate that these events, when occurring repeatedly over a long period of time, might become a health hazard because of the possible accumulation of brain tissue damage. Furthermore, our hypothesis suggests that other brain damaging factors may co-participate in mobile phone radiation-induced effects.     

WR-1065, the active metabolite of amifostine, mitigates radiation-induced delayed genomic instability

Compounds that can protect cells from the effects of radiation are important for clinical use, in the event of an accidental or terrorist-generated radiation event, and for astronauts traveling in space. One of the major concerns regarding the use of radio-protective agents is that they may protect cells initially, but predispose surviving cells to increased genomic instability later. In this study we used WR-1065, the active metabolite of amifostine, to determine how protection from direct effects of high- and low-LET radiation exposure influences genomic stability. When added 30 min before irradiation and in high concentrations, WR-1065 protected cells from immediate radiation-induced effects as well as from delayed genomic instability. Lower, nontoxic concentrations of WR-1065 did not protect cells from death; however, it was effective in significantly decreasing delayed genomic instability in the progeny of irradiated cells. The observed increase in manganese superoxide dismutase protein levels and activity may provide an explanation for this effect. These results confirm that WR-1065 is protective against both low- and high-LET radiation-induced genomic instability in surviving cells.     

WR-1065, the active metabolite of amifostine,mitigates radiation-induced delayed genomic instability

Compounds that can protect cells from the effects of radiation are important for clinical use, in the event of an accidental or terrorist-generated radiation event, and for astronauts traveling in space. One of the major concerns regarding the use of radio-protective agents is that they may protect cells initially, but predispose surviving cells to increased genomic instability later. In this study we used WR-1065, the active metabolite of amifostine, to determine how protection from direct effects of high- and low-LET radiation exposure influences genomic stability. When added 30 min before irradiation and in high concentrations, WR-1065 protected cells from immediate radiation-induced effects as well as from delayed genomic instability. Lower, nontoxic concentrations of WR-1065 did not protect cells from death; however, it was effective in significantly decreasing delayed genomic instability in the progeny of irradiated cells. The observed increase in manganese superoxide dismutase protein levels and activity may provide an explanation for this effect. These results confirm that WR-1065 is protective against both low- and high-LET radiation-induced genomic instability in surviving cells.     

Japanese encephalitis virus down-regulates thioredoxin and induces ROS-mediated ASK1-ERK/p38 MAPK activation in human promonocyte cells

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes severe neurological disease with high mortality. Molecular mechanisms of JEV pathogenesis such as upstream apoptotic processes and pathways are not yet completely resolved or understood. In this study, JEV replication in human promonocyte cells induced time-dependent apoptosis and activated virus dose-dependent caspases 3, 8 and 9. Proteomic analysis demonstrated up- and down-regulated (more or less than 1.5-fold) proteins in JEV-infected promonocyte cells. Biological process categorization showed processes of antioxidation, free radical removal, and sulfur redox metabolism entailed many identified up- and down-regulated proteins. Down-regulation of thioredoxin, confirmed by using Western blotting, was involved in the apoptosis process of the oxidative stress response pathway. JEV infection caused increased intracellular ROS production and activation of ASK1-ERK/p38 MAPK signaling. ERK/p38 MAPK inhibitor PD98059 treatment definitely suppressed this apoptosis. Down-regulation of thioredoxin, increased intracellular ROS, and activation of ASK1-ERK/p38 MAPK signaling all were associated with JEV-induced apoptosis. These results are suggestive of an oxidative stress-pathway as a key element of JE pathogenesis.     

Evidence that age-related changes in p38 MAP kinase contribute to the decreased steroid production by the adrenocortical cells from old rats

The current studies were initiated to investigate whether excessive oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. Western blot analysis indicated that aging caused increased phosphorylation and activation of rat adrenal p38 MAPK, but not the ERK1/2 or JNK1/2. Lipid peroxidation measurements (an index of cellular oxidative stress) indicated that adrenal membranes from young animals contained only minimal levels of endogenous thiobarbituric acid-reactive substances (TBARS), and exposure of membranes to enzymatic and non-enzymatic pro-oxidants enhanced TBARS formation approximately 12- and 20-fold, respectively. The adrenal membranes from old animals showed much more susceptibility to lipid peroxidation and exhibited roughly 4- to 6-fold higher TBARS formation than young controls both under basal conditions and in response to pro-oxidants. Qualitatively similar results were obtained when lipid peroxide formation was measured using a sensitive FOXRS (ferrous oxidation-xylenol orange-reactive substances) technique. We next tested whether aging-induced excessive oxidative insult alters steroidogenesis through modulation of MAPK signaling pathway. Treatment of adrenocortical cells from old rats with specific p38 MAPK inhibitors restored Bt(2)cAMP-stimulated steroidogenesis approximately 60-70% of the value seen in cells of young animals. Likewise, pretreatment of cells with reactive oxygen species (ROS) scavengers MnTMPyP and N-acetyl cysteine also partially rescued age-induced loss of steroid production. In contrast, simultaneous treatment of cells with ROS scavengers and p38 MAPK inhibitor did not produce any additional effect suggesting that both types of inhibitors exert their stimulatory action through inhibition of p38 MAPK activation. Collectively, these results indicate that p38 MAPK functions as a signaling effector in oxidative stress-induced inhibition of steroidogenesis during aging.     

Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes

The p38 MAPK pathway controls critical premitochondrial events culminating in apoptosis of UVB-irradiated human keratinocytes, but the upstream mediators of this stress signal are not completely defined. This study shows that in human keratinocytes exposed to UVB the generation of reactive oxygen species (ROS) acts as a mediator of apoptosis signal regulating kinase-1 (Ask-1), a redox-sensitive mitogen-activated protein kinase kinase kinase (MAP3K) regulating p38 MAPK and JNK cascades. The NADPH oxidase antagonist diphenylene iodonium chloride and the EGFR inhibitor AG1487 prevent UVB-mediated ROS generation, the activation of the Ask-1-p38 MAPK stress response pathway, and apoptosis, evidencing the link existing between the early plasma membrane-generated ROS and the activation of a lethal cascade initiated by Ask-1. Consistent with this, Ask-1 overexpression considerably sensitizes keratinocytes to UVB-induced mitochondrial apoptosis. Although the JNK pathway is also stimulated after UVB, the killing effect of Ask-1 overexpression is reverted by p38 MAPK inhibition, suggesting that Ask-1 exerts its lethal effects mainly through the p38 MAPK pathway. Moreover, p38alpha(-/-) murine embryonic fibroblasts are protected from UVB-induced apoptosis even if JNK activation is fully preserved. These results argue for an important role of the UVB-generated ROS as mediators of the Ask-1-p38 MAPK pathway that, by culminating in apoptosis, restrains the propagation of potentially mutagenic keratinocytes.     

AICAR stimulates IL-6 production via p38 MAPK in cardiac fibroblasts in adult mice: a possible role for AMPK

Though known as a sensor of energy balance, AMP-activated protein kinase (AMPK) was recently shown to limit damage and apoptotic activity and contribute to the late preconditioning in heart. Interleukin-6 was also reported to involve in anti-apoptosis and cardio-protection in myocardium. Interestingly, both AMPK activity and IL-6 level were increased in response to ischemia, hypertrophy and oxidative stress. To determine whether AMPK activation will promote IL-6 production, cardiac fibroblasts (CFs) from mice were incubated with AMPK activator, 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR). The results demonstrated that AICAR time and dose-dependently stimulated IL-6 production by ELISA and immunofluorescence. Pretreatment with p38 mitogen-activated protein kinase (MAPK) inhibitor blocked AICAR-induced IL-6 production; furthermore, AICAR-activated p38 MAPK phosphorylation by Western blot. To confirm that the increase in IL-6 production is ascribed to AMPK activation, we used another known AMPK activator, metformin. It also dose-dependently potentiated IL-6 production in CFs, and this potentiation could be reversed by p38 MAPK inhibitor. In conclusion, AMPK activation promoted IL-6 production in CFs via p38 MAPK-dependent pathway.     

Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation

Several recent studies have suggested that the reactive oxygen species (ROS) generated from mitochondria contribute to genomic instability after exposure of the cells to ionizing radiation, but the mechanism of this process is not yet fully understood. We examined the hypothesis that irradiation induces mitochondrial dysfunction to cause persistent oxidative stress, which contributes to genomic instability. After the exposure of cells to 5 Gy gamma-ray irradiation, we found that the irradiation induced the following changes in a clear pattern of time courses. First, a robust increase of intracellular ROS levels occurred within minutes, but the intracellular ROS disappeared within 30 min. Then the mitochondrial dysfunction was detected at 12 h after irradiation, as indicated by the decreased activity of NADH dehydrogenase (Complex I), the most important enzyme in regulating the release of ROS from the mitochondrial electron transport chain (ETC). Finally, a significant increase of ROS levels in the mitochondria and the oxidation of mitochondrial DNA were observed in cells at 24 h or later after irradiation. Although further experiments are required, results in this study support the hypothesis that mitochondrial dysfunction causes persistent oxidative stress that may contribute to promote radiation-induced genomic instability.     

Non-Classical P38 Map Kinase Functions: Cell Cycle Checkpoints and Survival

The p38 MAPK kinase pathway is activated in response to a wide range of cellular stress stimuli and cytokines. Our understanding of the important functions of p38 MAPK in the process of differentiation and cell death has grown considerably in the recent years and is now relatively established. Here we discuss the role of p38 MAPK in the mediation of cell cycle checkpoints and cell survival, processes that have received less attention. We describe how p38 MAPK regulates both the G2/M as well as a G1/S cell cycle checkpoint in response to cellular stress such as DNA damage. While p38 MAPK has classically been associated with the induction of apoptosis, we discuss that p38 MAPK can also mediate cell survival in specific situations, such as in response to DNA damage. It is important to recognize these less appreciated functions of p38 MAPK when considering the potential use of pharmacological inhibitors of p38 MAPK in therapeutic treatments for disease.