Scavenging of extracellular H2O2 by catalase inhibits the proliferation of HER-2/Neu-transformed rat-1 fibroblasts through the induction of a stress response

High levels of reactive oxygen species (ROS) are associated with cytotoxicity. Alternatively, nontoxic levels of ROS like hydrogen peroxide (H(2)O(2)) can mediate the transmission of many intracellular signals, including those involved in growth and transformation. To identify pathways downstream of endogenous cellular H(2)O(2) production, the response of Rat-1 fibroblasts exhibiting differential HER-2/Neu receptor tyrosine kinase activity to removal of physiological H(2)O(2) concentrations was investigated. The proliferation of all cells was abolished by addition of the H(2)O(2) scavenger catalase to the culture medium. HER-2/Neu activity was not significantly affected by catalase treatment, suggesting that the target(s) of the H(2)O(2) signal lie downstream of the receptor in our model. ERK1/2 phosphorylation was blocked by catalase in fibroblasts expressing wild type Neu, however such a response did not occur in cells possessing activated mutant Neu. This indicates that the ERK1/2 response contributes little to the growth inhibition observed. By contrast, JNK1 activity increased following the addition of catalase or H(2)O(2), regardless of Neu activity or level of cell transformation. Phosphorylation of p38 MAPK was induced by H(2)O(2) but not by catalase. These observations suggest that scavenging of H(2)O(2) from the cellular environment blocks Rat-1 proliferation primarily through the activation of stress pathways.     

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.     

Catalase regulates cell growth in HL60 human promyelocytic cells: evidence for growth regulation by H(2)O(2)

Reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)) are generated constitutively in mammalian cells. Because of its relatively long life and high permeability across membranes, H(2)O(2) is thought to be an important second messenger. Generation of H(2)O(2) is increased in response to external insults, including radiation. Catalase is located at the peroxisome and scavenges H(2)O(2). In this study, we investigated the role of catalase in cell growth using the H(2)O(2)-resistant variant HP100-1 of human promyelocytic HL60 cells. HP100-1 cells had an almost 10-fold higher activity of catalase than HL60 cells without differences in levels of glutathione peroxidase, manganese superoxide dismutase (MnSOD), and copper-zinc SOD (CuZnSOD). HP100-1 cells had higher proliferative activity than HL60 cells. Treatment with catalase or the introduction of catalase cDNA into HL60 cells stimulated cell growth. Exposure of HP100-1 cells to a catalase inhibitor resulted in suppression of cell growth with concomitant increased levels of intracellular H(2)O(2). Moreover, exogenously added H(2)O(2) or depletion of glutathione suppressed cell growth in HL60 cells. Extracellular signal regulated kinase 1/2 (ERK1/2) was constitutively phosphorylated in HP100-1 cells but not in HL60 cells. Inhibition of the ERK1/2 pathway suppressed the growth of HP100-1 cells, but inhibition of p38 mitogen-activated protein kinase (p38MAPK) did not affect growth. Moreover, inhibition of catalase blocked the phosphorylation of ERK1/2 but not of p38MAPK in HP100-1 cells. Thus our results suggest that catalase activates the growth of HL60 cells through dismutation of H(2)O(2), leading to activation of the ERK1/2 pathway; H(2)O(2) is an important regulator of growth in HL60 cells.     

c-Jun N-terminal kinase is essential for growth of human T98G glioblastoma cells

The c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway is activated by numerous cellular stresses. Although it has been implicated in mediating apoptosis and growth factor signaling, its role in regulating cell growth is not yet clear. Here, the influence of JNK on basal (unstimulated) growth of human tumor glioblastoma T98G cells was investigated using highly specific JNK antisense oligonucleotides to inhibit JNK expression. Transient depletion of either JNK1 or JNK2 suppressed cell growth associated with an inhibition of DNA synthesis and cell cycle arrest in S phase. The growth-inhibitory potency of JNK2 antisense ((JNK)2 IC(50) = 0.14 micrometer) was greater than that of JNK1 antisense ((JNK)1 IC(50) = 0.37 micrometer), suggesting that JNK2 plays a dominant role in regulating growth of T98G cells. Indeed, JNK2 antisense-treated populations exhibited greater inhibition of DNA synthesis and accumulation of S-phase cells than did the JNK1 antisense-treated cultures, with a significant proportion of these cells detaching from the tissue culture plate. JNK2 (but not JNK1) antisense-treated cultures exhibited marked elevation in the expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1) accompanied by inhibition of Cdk2/Cdc2 kinase activities. Taken together, these results indicate that JNK is required for growth of T98G cells in nonstress conditions and that p21(cip1/waf1) may contribute to the sustained growth arrest of JNK2-depleted T98G cultures.     

Maintenance of higher H(2)O(2) levels, and its mechanism of action to induce growth in breast cancer cells: Important roles of bioactive catalase and PP2A

We assessed the catalase bioactivity and hydrogen peroxide (H(2)O(2)) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H(2)O(2) steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H(2)O(2) which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O(2)(-)) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O(2)(-) produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H(2)O(2) levels and led to apoptosis of these cells. The H(2)O(2) produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O(2)(-) leads to an increase in steady-state levels of H(2)O(2) in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.     

Catalase, but not MnSOD, inhibits glucose deprivation-activated ASK1-MEK-MAPK signal transduction pathway and prevents relocalization of Daxx: hydrogen peroxide as a major second messenger of metabolic oxidative stress

Overexpression of catalase, but not manganese superoxide dismutase (MnSOD), inhibited glucose deprivation-induced cytotoxicity and c-Jun N-terminal kinase 1 (JNK1) activation in human prostate adenocarcinoma DU-145 cells. Suppression of JNK1 activation by catalase overexpression resulted from inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation by preventing dissociation of thioredoxin (TRX) from ASK1. Overexpression of catalase also inhibited relocalization of Daxx from the nucleus to the cytoplasm as well as association of Daxx with ASK1 during glucose deprivation. Taken together, hydrogen peroxide (H(2)O(2)) rather than superoxide anion (O(2) (*-)) acts as a second messenger of metabolic oxidative stress to activate the ASK1-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-mitogen-activated protein kinase (MAPK) signal transduction pathway.     

Angiopoietin-1 attenuates H2O2-induced SEK1/JNK phosphorylation through the phosphatidylinositol 3-kinase/Akt pathway in vascular endothelial cells

Oxidative stress activates various signal transduction pathways, including Jun N-terminal kinase (JNK) and its substrates, that induce apoptosis. We reported here the role of angiopoietin-1 (Ang1), which is a prosurvival factor in endothelial cells, during endothelial cell damage induced by oxidative stress. Hydrogen peroxide (H2O2) increased apoptosis of endothelial cells through JNK activation, whereas Ang1 inhibited H2O2-induced apoptosis and concomitant JNK phosphorylation. The inhibition of H2O2-induced JNK phosphorylation was reversed by inhibitors of phosphatidylinositol (PI) 3-kinase and dominant-negative Akt, and constitutively active-Akt attenuated JNK phosphorylation without Ang1. These data suggested that Ang1-dependent Akt phosphorylation through PI 3-kinase leads to the inhibition of JNK phosphorylation. H2O2-induced phosphorylation of SAPK/Erk kinase (SEK1) at Thr261, which is an upstream regulator of JNK, was also attenuated by Ang1-dependent activation of the PI 3-kinase/Akt pathway. In addition, Ang1 induced SEK1 phosphorylation at Ser80, suggesting the existence of an additional signal transduction pathway through which Ang1 attenuates JNK phosphorylation. These results demonstrated that Ang1 attenuates H2O2-induced SEK1/JNK phosphorylation through the PI 3-kinase/Akt pathway and inhibits the apoptosis of endothelial cells to oxidative stress.     

Oxygen toxicity in control and H2O2-resistant Chinese hamster fibroblast cell lines

Following exposure to 95% oxygen, clonogenic cell survival was assayed and qualitative morphologic changes were observed in a Chinese hamster fibroblast cell line (HA-1). The time in 95% O2 necessary to clonogenically inactivate 90% of the cells was inversely related to the cell density of the cultures at the beginning of hyperoxic exposure (from 1 to 6 X 10(4) cells/cm2). The O2-induced loss in clonogenicity and evidence of morphologic injury were shown to be significantly delayed (17-22 h) in an H2O2-resistant variant of the parental HA-1 cell line. After the delay in onset of clonogenic cell killing or morphologic injury, the process of injury proceeded in a similar fashion in both cell lines. The H2O2-resistant cell line demonstrated significantly greater catalase activity (20-fold), CuZn superoxide dismutase activity (2-fold), and Se-dependent glutathione peroxidase activity (1.5-fold). The greater activities of CuZn superoxide dismutase and catalase were accompanied by similarly greater quantities of immunoreactive protein as determined by immunoblotting. These data demonstrate that the cells adapted and/or selected for growth in a highly peroxidative environment also became refractory to O2-induced toxicity, which may be related to increased expression of antioxidant enzymes. However, the magnitude of this cross-resistance to O2 toxicity was less than the magnitude of the cellular resistance to the toxicity of exogenous H2O2, suggesting that in this system the toxicity of 95% oxygen is not identical to H2O2-mediated cytotoxicity.     

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.     

Transient and sustained oxidative stress differentially activate the JNK1/2 pathway and apoptotic phenotype in H9c2 cells

The aim of this study was to investigate the activation of JNK1/2 signalling pathway and the respective cellular phenotype of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation of JNK1/2 pathway by exogenous H₂O₂, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation of c-Jun was observed, coincidently with the activation of nuclear JNK1/2; under sustained stress, these phosphorylation levels remained elevated above basal for up to 6 h, whereas under transient stress they declined to basal ones within 4 h of withdrawal. Furthermore, the JNK1/2 selective inhibitor SP600125 abolished the c-jun phosphorylation induced by oxidative stress. Our results using cell viability assays and light microscopy revealed that sustained H₂O₂ stimulation significantly and time-dependently decreased H9c2 viability, in contrast to transient stimulation; SP600125 (10 μM) abolished cell death induced by sustained as well as cell survival induced by transient oxidative stress. Hoechst staining showed an increase in DNA condensation during sustained, but not during transient stimulation. Moreover, from the antioxidants tested, catalase and superoxide dismutase prevented oxidative stress-induced cell death. Flow cytometry studies reconfirmed that sustained oxidative stress induced apoptosis, whereas transient resulted in the recovery of cardiac myoblasts within 24 h. We conclude that in H9c2 myoblasts, sustained activation of JNK1/2 signalling pathway during oxidative stimulation is followed by an apoptotic phenotype, while transient JNK1/2 activation correlates well with cell survival, suggesting a dual role of this signalling pathway in cell fate determination.     

Stable H2O2-resistant variants of Chinese hamster fibroblasts demonstrate increases in catalase activity

Hydrogen peroxide (H2O2)-resistant variants of the Chinese hamster ovary HA-1 line have been derived by culturing cells in progressively higher concentrations of H2O2 (greater than 200 days, in 50-800 microM H2O2). The H2O2-resistant phenotype has been stable for over 60 passages (240 days) following removal from the H2O2 stress. The resistant cells demonstrate both increased capacity to deplete exogenously added H2O2 from the growth medium and increased catalase activity. H2O2 resistance correlates well with catalase activity. An increase in chromosome number occurred in the cells adapted to 200-800 microM H2O2, but increases in aneuploidy and tetraploidy were not necessary for resistance. These results suggest that adaptation to chronic oxidative stress mediated by H2O2 in mammalian cells is accompanied by a stable heritable change in expression of catalase activity.     

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.     

Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species

c-Jun NH(2)-terminal kinase (JNK) is activated by a number of cellular stimuli such as inflammatory cytokines and environmental stresses. Reactive oxygen species also cause activation of JNK; however, the signaling cascade that leads to JNK activation remains to be elucidated. Because recent reports showed that expression of Cas, a putative Src substrate, stimulates JNK activation, we hypothesized that the Src kinase family and Cas would be involved in JNK activation by reactive oxygen species. An essential role for both Src and Cas was demonstrated. First, the specific Src family tyrosine kinase inhibitor, PP2, inhibited JNK activation by H(2)O(2) in a concentration-dependent manner but had no effect on extracellular signal-regulated kinases 1 and 2 and p38 activation. Second, JNK activation in response to H(2)O(2) was completely inhibited in cells derived from transgenic mice deficient in Src but not Fyn. Third, expression of a dominant negative mutant of Cas prevented H(2)O(2)-mediated JNK activation but had no effect on extracellular signal-regulated kinases 1 and 2 and p38 activation. Finally, the importance of Src was further supported by the inhibition of both H(2)O(2)-mediated Cas tyrosine phosphorylation and Cas.Crk complex formation in Src-/- but not Fyn-/- cells. These results demonstrate an essential role for Src and Cas in H(2)O(2)-mediated activation of JNK and suggest a new redox-sensitive pathway for JNK activation mediated by Src.     

Iron-mediated H2O2 production as a mechanism for cell type-specific inhibition of tumor necrosis factor alpha-induced but not interleukin-1beta-induced IkappaB kinase complex/nuclear factor-kappaB activation

Coordinated and specific regulation of tumor necrosis factor (TNF) and interleukin (IL)-1 signaling pathways and how and whether they are modified by different agents are key events for proper immune responses. The IkappaB kinase complex (IKK)/NF-kappaB and JNK/AP-1 pathways are central mediators of TNF and IL-1 during inflammatory responses. Here we show that l-mimosine, a toxic non-protein amino acid that has been shown to reduce serum TNFalpha levels and affect inflammatory responses, specifically inhibits TNF-induced IKK but not JNK in a cell type-specific manner. l-Mimosine did not affect IKK and NF-kappaB activation by IL-1beta. l-Mimosine caused cell cycle arrest at G(1)-S phase, but inhibition of IKK was found to be independent of cell cycle arrest. Treatment of cells with l-mimosine resulted in production of H(2)O(2). Addition of FeSO(4) restored IKK activation by TNFalpha as did ectopic expression of catalase or pretreatment of cells with N-aceltyl-l-cysteine, indicating a role for intracellular H(2)O(2) as a mediator of inhibition. Cleavage and degradation of TNF pathway components TNFR1, RIP, and Hsp90 were observed in l-mimosine and H(2)O(2) treated cells indicating a putative mechanism for selective inhibition of TNF but not IL-1beta-induced IKK activation.     

N-acetyl-L-cysteine counteracts oxidative stress and prevents H2O2 induced germ cell apoptosis through down-regulation of caspase-9 and JNK/c-Jun

The mechanism of H(2)O(2) induced oxidative stress leading to male germ cell apoptosis was earlier reported from our laboratory. In the present study, we investigated the mechanisms by which N-acetyl-L-cysteine (NAC, which is highly cell specific with strong antioxidant and anti-genotoxic properties), stimulated cell survival under such conditions. Co-incubation with 5 mM NAC significantly (P<0.001) reduced the germ cell apoptosis induced by 10 μM H(2)O(2). Lipid peroxidation was brought down with significant restoration of activities of antioxidant enzymes, SOD, GST, and catalase. Expression of pro-apoptotic marker, Bax up-regulated following H(2)O(2) exposure, was reversed back to control levels. In contrast, expression of anti-apoptotic Bcl-2 and phospho-Akt revealed a completely opposite trend. While caspase-8 activity remained unaffected, NAC successfully attenuated the increased activities of caspase-3 and -9 in the H(2) O(2) treated cells. Simultaneously, the increased expression of caspase-9, phospho-JNK, and phospho-c-Jun after H(2)O(2) treatment was down-regulated by NAC. The above findings indicate that the mechanism of inhibition of H(2)O(2) induced male germ cell apoptosis by NAC is mediated through regulation of caspase-9 and JNK.     

H(2)O(2) signals 5-HT-induced ERK MAP kinase activation and mitogenesis of smooth muscle cells

Our previous studies have shown that 5-hydroxytryptamine (5-HT) induces cellular hyperplasia/hypertrophy through protein tyrosine phosphorylation, rapid formation of superoxide (O(2)(-)), and extracellular signal-regulated kinase (ERK)1/ERK2 mitogen-activated protein (MAP) kinase activation. Intracellularly released O(2)(-) is rapidly dismuted by superoxide dismutase (SOD) to H(2)O(2), another possible cellular growth mediator. In the present study, we assessed whether H(2)O(2) participates in 5-HT-induced mitogenic signaling. Inactivation of cellular Cu/Zn SOD by copper-chelating agents inhibited 5-HT-induced DNA synthesis of bovine pulmonary artery smooth muscle cells (BPASMCs). Infection of BPASMCs with an adenovirus containing catalase inhibited both ERK1/ERK2 MAP kinase activation and DNA synthesis induced by 5-HT. Although we could not find evidence of p38 MAP kinase activation by 5-HT, SB-203580 and SB-202190, reported inhibitors of p38 MAP kinase, inhibited the 5-HT-induced growth of BPASMCs. However, these inhibitors also inhibited 5-HT-induced O(2)(-) release. Thus quenching of O(2)(-) may be their mechanism for inhibition of cellular growth unrelated to p38 MAP kinase inhibition. These data indicate that generation of O(2)(-) in BPASMCs in response to 5-HT is followed by an increase in intracellular H(2)O(2) that mediates 5-HT-induced mitogenesis through activation of ERK1/ERK2 but not of p38 MAP kinase.     

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.     

转录因子HBP1调控H2O2诱导的骨肉瘤细胞生长阻滞过程

为探讨氧化应激对人骨肉瘤细胞增殖的影响及其作用机理,首先用H2O2处理U2OS细 胞,采用Western印迹和real-time PCR检测HMG盒转录因子1 (HBP1)及其下游靶基因 DNMT1和p16表达水平的变化. 用荧光素酶报告基因实验检测在H2O2诱导下, HBP1对于DNMT1 和p16启动子的影响. 用细胞增殖试验（BrdU掺入,细胞生长曲线）检测 H2O2对细胞增殖的影响以及HBP1的作用. 用衰老相关β半乳糖苷酶(SA-β-Gal)染色 检测在H2O2诱导的细胞衰老中HBP1所起的作用. Western 印迹, real-time PCR及荧光素酶报告基因实验结果显示,细胞经H2O2处理后,明显增高HBP1表达水平,转录抑制DNMT1的表达, 促进p16蛋白的表达. 细胞增殖实验结果显示, H2O2显著抑制了细胞的增殖,HBP1 knockdown可部分逆转这种抑制作用. SA-β-Gal染色实验说明, H2O2可诱导HBP1表达正常的U2OS细胞衰老,而HBP1 knockdown使这种促衰老作用减弱. 研究结果说明, H2O2可抑制人骨肉瘤细胞增殖,诱导细胞衰老. 其作用机制是通过上调转录因子HBP1的表达,转录抑制或促进其下游靶基因DNMT1或p16的表达来抑制细胞增殖,促进细胞衰老.     

Glycogen synthase kinase 3beta is a negative regulator of growth factor-induced activation of the c-Jun N-terminal kinase

The c-Jun N-terminal kinase (JNK)/stress activated protein kinase is preferentially activated by stress stimuli. Growth factors, particularly ligands for G protein-coupled receptors, usually induce only modest JNK activation, although they may trigger marked activation of the related extracellular signal-regulated kinase. In the present study, we demonstrated that homozygous disruption of glycogen synthase kinase 3beta (GSK-3beta) dramatically sensitized mouse embryonic fibroblasts (MEFs) to JNK activation induced by lysophosphatidic acid (LPA) and sphingosine-1-phosphate, two prototype ligands for G protein-coupled receptors. To a lesser degree, a lack of GSK-3beta also potentiated JNK activation in response to epidermal growth factor. In contrast, the absence of GSK-3beta decreased UV light-induced JNK activation. The increased JNK activation induced by LPA in GSK-3beta null MEFs was insufficient to trigger apoptotic cell death or growth inhibition. Instead, the increased JNK activation observed in GSK-3beta-/- MEFs was associated with an increased proliferative response to LPA, which was reduced by the inhibition of JNK. Ectopic expression of GSK-3beta in GSK-3beta-negative MEFs restrained LPA-triggered JNK phosphorylation and induced a concomitant decrease in the mitogenic response to LPA compatible with GSK-3beta through the inhibition of JNK activation, thus limiting LPA-induced cell proliferation. Mutation analysis indicated that GSK-3beta kinase activity was required for GSK-3beta to optimally inhibit LPA-stimulated JNK activation. Thus GSK-3beta serves as a physiological switch to specifically repress JNK activation in response to LPA, sphingosine-1-phosphate, or the epidermal growth factor. These results reveal a novel role for GSK-3beta in signal transduction and cellular responses to growth factors.