bcl-2高表达细胞株的构建

利用基因转染技术,将人的bcl-2基因cDNA克隆于逆转录病毒载体pLXSN,通过PA317细胞包装成病毒后感染HeLa细胞。经PCR及蛋白质印迹证明转染成功,得到bcl-2稳定高表达株Hbc17。用顺铂诱导凋亡,Hbc17比对照细胞株H1具有明显的抗凋亡能力。为进一步研究顺铂诱导肿瘤细胞凋亡的机理奠定基础。     

Retroviral overexpression of bcl-2 in the embryonic chick lens influences denucleation in differentiating lens fiber cells

During the course of their differentiation, embryonic lens fibers undergo loss of their cytoplasmic organelles and nuclei. The denucleation process bears similarities to the nuclear breakdown that occurs during apoptosis. This has given rise to the hypothesis that this denucleation is analogous to apoptosis, but without the plasma membrane changes characteristic of apoptotic cell death. Previous work has shown that several members of the apoptotic cascade are active during denucleation. Here, we have overexpressed the anti-apoptotic molecule bcl-2 in developing lenses of the 8-day-old chick embryo in ovo, using the replication-competent retrovirus RCAS. We find that lenses overexpressing bcl-2 show varying degrees of distortion in comparison with untreated and negative insert controls, including a more spherical shape and disorganized fiber cells. All overexpressing lenses showed significantly higher numbers of smaller nuclei in the lens core, where denucleation begins. There was no change in cell size or pattern of proliferation. These in vivo results were confirmed in vitro using lens epithelial cell cultures, which differentiate into lentoids. The lentoids in treated cultures showed the same effect on nuclear number and size. We further found that in lenses overexpressing bcl-2 there was a reduction in the activation of caspase-9 and the cleavage of the caspase substrate DFF45, and, in the lens core, a failure of the nuclear chromatin to condense. These results provide strong support for the view that embryonic lens fiber cell denucleation is analogous to the nuclear degradation that occurs during apoptosis, and that similar control pathways are involved in both these phenomena.     

Iron toxicity in organotypic cultures of hippocampal slices: role of reactive oxygen species

Free iron has been assumed to potentiate oxygen toxicity by generating reactive oxygen species (ROS) via the iron-catalyzed Haber-Weiss reaction, leading to oxidative stress. ROS-mediated iron cytotoxicity may trigger apoptotic cell death. In the present study, we used iron treatment of organotypic cultures of hippocampal slices to study potential mechanisms involved in iron-induced neuronal damage. Exposure of mature hippocampal slices to ferrous sulfate resulted in concentration- and time-dependent cell death. After iron treatment, markers of ROS formation and lipid peroxidation, i.e. intensity of dichlorofluorescein (DCF) fluorescence and levels of thiobarbiturate reactive substances (TBARS), were significantly increased. Levels of cytochrome c were increased while levels of pro-caspase-9 and pro-caspase-3 were decreased in cytosolic fractions of iron-treated hippocampal slice cultures. Treatment of cultured slices with a synthetic catalytic ROS scavenger, EUK-134, provided between 50 and 70% protection against various parameters of cell damage and markers of oxidative stress. In addition, inhibition of caspase-3 activity by Ac-DEVDcho partially protected cells from iron toxicity. The combination of EUK-134 and Ac-DEVDcho resulted in an almost complete blockade of iron-induced damage. These results indicate that iron elicits cellular damage predominantly by oxidative stress, and that ROS-mediated iron toxicity may involve cytochrome c- and caspase-3-dependent apoptotic pathways.     

Insulin on hydrogen peroxide-induced oxidative stress involves ROS/Ca2+ and Akt/Bcl-2 signaling pathways

Abstract

Oxidative stress is induced by excess accumulation of reactive oxygen and nitrogen species (RONS). Astrocytes are metabolically active cells in the brain and understanding astrocytic responses to oxidative stress is essential to understand brain pathologies. In addition to direct oxidative stress, exogenous hydrogen peroxide (H₂O₂) can penetrate biological membranes and enhance formation of other RONS. The present study was carried out to examine the role of insulin in H₂O₂-induced oxidative stress in rat astrocytic cells. To measure changes in the viability of astrocytes at different concentrations of H₂O₂ for 3 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay was used and 500 μM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 3 h of 500 μM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and calcium ion (Ca²⁺) in C6 cells, with insulin able to effectively diminish H₂O₂-induced oxidative damage to C6 cells. Western blotting studies showed that insulin treatment of astrocytes increased the levels of phosphorylated Akt and magnified the decrease in total Bcl-2 protein. The protective effect of insulin treatment on H₂O₂-induced oxidative stress in astrocytes by reducing apoptosis may relate to the PI3K/Akt pathway.     

Mitochondrial apoptotic cell death and moderate superoxide generation upon selective activation of non-desensitizing AMPA receptors in hippocampal cultures

In the present work we investigated the effect of selective stimulation of non-desensitizing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the intracellular processes leading to hippocampal neuronal death and production of reactive oxygen species (ROS). Activation of AMPA receptors in the presence of cyclothiazide (CYZ), a blocker of AMPA receptor desensitization, resulted in the death of approximately 25% of neurones, which was prevented by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline (NBQX), an AMPA-preferring receptor antagonist. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) protected the neurones from necrotic death induced by AMPA or NMDA receptor activation. Neurodegeneration caused by selective activation of non-desensitizing AMPA receptors, in the presence of AMPA, CYZ and MK-801, significantly decreased the number of Co2+-positive neurones, used as a cytochemical marker of Ca2+-permeable AMPA receptors, but maintained intracellular ATP/ADP. The AMPA-mediated apoptotic cell death involved mitochondrial cytochrome c release and the activation of caspases-1 and -3, which was prevented by NBQX. Interestingly, although selective activation of AMPA receptors was not associated with production of intracellular peroxides, a moderate increase in superoxide production was observed upon exposure to antimycin A (AA). Furthermore, increased activity of Mn- superoxide dismutase (SOD) was observed on selective activation of non-desensitizing AMPA receptors. Taken together, these data make important contributions to the elucidation of the downstream pathways activated in AMPA receptor-mediated excitotoxicity in cultured rat hippocampal neurones.     

Autophagy: a cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.     

Bid-mediated mitochondrial damage is a key mechanism in glutamate-induced oxidative stress and AIF-dependent cell death in immortalized HT-22 hippocampal neurons

Glutamate toxicity involves increases in intracellular calcium levels and enhanced formation of reactive oxygen species (ROS) causing neuronal dysfunction and death in acute and chronic neurodegenerative disorders. The molecular mechanisms mediating glutamate-induced ROS formation are, however, still poorly defined. Using a model system that lacks glutamate-operated calcium channels, we demonstrate that glutamate-induced acceleration of ROS levels occurs in two steps and is initiated by lipoxygenases (LOXs) and then significantly accelerated through Bid-dependent mitochondrial damage. The Bid-mediated secondary boost of ROS formation downstream of LOX activity further involves mitochondrial fragmentation and release of mitochondrial apoptosis-inducing factor (AIF) to the nucleus. These data imply that the activation of Bid is an essential step in amplifying glutamate-induced formation of lipid peroxides to irreversible mitochondrial damage associated with further enhanced free radical formation and AIF-dependent execution of cell death.     

Mitochondrial peroxiredoxin-3 protects hippocampal neurons from excitotoxic injury in vivo

Mitochondria are involved in excitotoxic damage of nerve cells. Following the breakdown of the calcium-buffering ability of mitochondria, mitochondrial calcium overload induces reactive oxygen species (ROS) bursts that produce free radicals and open permeability transition pores, ultimately leading to neuronal cell death. In the present study, we focused on a mitochondrial antioxidant protein, peroxiredoxin-3 (Prx-3), to investigate the mechanism by which toxic properties of ROS were up-regulated in mitochondria of damaged nerve cells. Immunohistochemical analysis revealed that Prx-3 protein exists in mitochondria of rat hippocampus, whereas we found a significant decrease in Prx-3 mRNA and protein levels associated with an increase in nitrated proteins in the rat hippocampus injured by microinjection of ibotenic acid. Furthermore, in vivo adenoviral gene transfer of Prx-3 completely inhibited protein nitration and markedly reduced gliosis, a post-neuronal cell death event. Since mitochondrial Prx-3 seems to be neuroprotective against oxidative insults, our findings suggest that Prx-3 up-regulation might be a useful novel approach for the management of neurodegenerative diseases.     

The mechanisms of oxidative DNA damage and apoptosis induced by norsalsolinol, an endogenous tetrahydroisoquinoline derivative associated with Parkinson's disease

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N -acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using ³²P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H₂O₂ with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.     

小鼠成纤维细胞凋亡与bcl-2结合蛋白

为检测细胞凋亡过程中bcl-2基因的结合蛋白,用PCR技术扩增小鼠bcl-2基因(mbcl-2)调控区,经亚克隆后的序列分析证实其准确性.用5-氟尿嘧啶(5-Fu)诱导小鼠成纤维细胞(C3H 10 T1/2 Cl 8)凋亡,以此PCR产物作探针,对上述细胞的核蛋白质粗提物进行迁移位移法(EMSA)与DNA-蛋白质印迹分析.结果表明,细胞核内一分子质量为53 ku的结合蛋白与mbcl-2调控区的结合信号在5-Fu刺激12 h后明显增强,而一种100 ku的DNA结合蛋白与该区的结合信号在5-Fu刺激12 h后明显减弱.因而,p53蛋白有可能是bcl-2的负转录因子,100 ku的DNA结合蛋白有可能是bcl-2的正转录因子.     

Reinforcing apoptosis-resistance of COS and myeloma cells by transfecting with bcl-2 gene

COS, myeloma and HeLa cells, which are commonly used for protein production by cell culture, were transfected with human bcl-2 gene encoded on the shuttle vector BCMGS. Expression of human bcl-2 improved survival of cells remarkably, mildly, or negligibly for COS, myeloma, and HeLa, respectively. Four clones were obtained from the human bcl-2 expressing cell population of COS cells. They expressed human bcl-2 almost at the same level. The viable cell numbers were 6, 2.5, 2.5, and 0.8 times as many for the clones #8, #5, #6, and #7, respectively, as for the control COS cells, when they were cultured at low (0.2%) serum concentration for 9 days. The bcl-2 overexpressing COS cells showed morphology different from that of the control COS cells in serum limited condition. When transfected with mouse lambda protein gene carried by an SV40-derived vector, clone #8 of the bcl-2 transfected COS cells continued the transient expression of lambda protein longer than the control COS cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

JNK介导的信号转导途径以及活性氧在其中的作用

JNK是一个受外界应激因素调控的信号分子,调节包括凋亡在内的一系列细胞内的反应,但目前越来越多的报道证实了JNK信号途径具有促凋亡和抗凋亡的双重功能,这种双重功能受到细胞类型、刺激物的种类、剂量和持续时间以及胞内其他信号途径的影响。活性氧作为一种常见的外界应激因素也部分参与了JNK信号途径的激活,对细胞的生死产生了重要的影响。本文将主要总结JNK介导的信号转导途径及活性氧在这一途径中所发挥的作用。     

Disruptive effects of glucocorticoids on glutathione peroxidase biochemistry in hippocampal cultures

Glucocorticoids (GCs), the adrenal steroids secreted during stress, compromise the ability of hippocampal neurons to survive various necrotic insults. We have previously observed that GCs enhance the hippocampal neurotoxicity of reactive oxygen species and, as a potential contributor to this, decrease the activity of the antioxidant enzyme, glutathione peroxidase (GSPx). In this report, we have studied the possible mechanisms underlying this GC effect upon GSPx in primary hippocampal cultures and have observed several results. (i) Corticosterone (the GC of rats) decreased glutathione levels; this was predominately a result of a decrease in levels of reduced glutathione (GSH), the form of glutathione which facilitates GSPx activity. (ii) Corticosterone also decreased levels of NADPH; this may help explain the effect on GSH as NADPH is required for regeneration of GSH from oxidized glutathione. (iii) However, the corticosterone effect on total glutathione levels could not just be caused by the NADPH effect, as there were also reduced levels of oxidized glutathione. (iv) Corticosterone caused a small but significant decrease in GSPx activity over a range of glucose concentrations; this occurred under circumstances of an excess of glutathione as a substrate, suggesting a direct effect of corticosterone on GSPx activity. (v) This corticosterone effect was likely to have functional implications, in that enhancement of GSPx activity (to the same magnitude as activity was inhibited by corticosterone) by GSPx overexpression protected against an excitotoxin. Thus, GCs have various effects, both energetic and non-energetic in nature, upon steps in GSPx biochemistry that, collectively, may impair hippocampal antioxidant capacity.     

Doxorubicin-induced cardiomyocyte apoptosis: Role of mitofusin 2

BackgroundDoxorubicin (DOX) is an anti-tumor agent that is widely used in clinical setting for cancer treatment. The application of the DOX, however, is limited by its cardiac toxicity which can induce heart failure through an undefined mechanism. Mitofusin 2 (Mfn2) is a mitochondrial GTPase fusion protein that is located on the outer membrane of mitochondria (OMM). The Mfn2 plays an important role in mitochondrial fusion and fission. The aim of this study is to identify the role of the Mfn2 in DOX-induced cardiomyocyte apoptosis.MethodsCultured neonatal rat cardiomyocytes were used in this study. Mfn2 expression in cardiomyocytes was determined after the cardiomyocytes were challenged with DOX. Cardiomyocyte mitochondrial fission, mitochondrial reactive oxygen species (ROS) production was assessed with mitochondrial fragmentation and MitoSOX fluorescence probe, respectively. Cardiomyocyte apoptosis was determined with caspase3 activity and TUNEL staining.ResultsChallenging of the cardiomyocytes with DOX resulted in increasing in cardiomyocyte oxidative stress and apoptosis. In addition, levels of Mfn2 in cardiomyocytes were decreased after the cells were challenged with DOX which was associated with increased mitochondrial fission (fragmentation) and mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 attenuated the DOX-induced increase in mitochondrial fission and prevented cardiomyocyte mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 or pretreatment of cardiomyocytes with an anti-oxidant, Mito-tempo, also prevented the DOX-induced cardiomyocyte apoptosis.ConclusionOur results indicate that DOX results in a decreased cardiomyocyte Mfn2 expression which promotes mitochondrial fission and ROS production further leads to cardiomyocyte apoptosis.     

Protective effects of Alpha-lipoic acid on MeHg-induced oxidative damage and intracellular Ca2+ dyshomeostasis in primary cultured neurons

Methylmercury (MeHg) is one of the ubiquitous environmental toxicant that leads to long-lasting neurological deficits in animals and humans. However, the mechanisms of MeHg-induced neuronal cell death are incompletely understood. Treatment of neuronal cells with MeHg (0-2?μM) for 0.5-12?h, or pretreated with LA (12.5-100?μM) for 0.5-6?h resulted in toxic effects of primary cultured neurons concentration- and time-dependently. For further experiments, 12.5, 25, and 50?μM of LA pretreatment for 3?h followed by 1?μM MeHg for 6?h were performed for the examination of the responses of neurons. Exposure of MeHg resulted in damages of neurons, which were shown by a loss of cell viability, and supported by high levels of lactate dehydrogenase (LDH) release, apoptosis, and morphological changes. In addition, neurons were sensitive to MeHg-mediated oxidative stress, a finding that is consistent with ROS over-production, leading to decrease Ca²⁺-ATPase activity and increase intracellular free calcium. Moreover, expressions of NMDA receptor subunits in neurons were down-regulated after MeHg exposure, and expression of NR2A mRNA and protein were much more sensitive to MeHg than those of NR1 and NR2B. On the contrary, pretreatment with LA presented a concentration-dependent prevention against MeHg-mediated cytotoxic effects of neurons. In conclusion, present results showed that oxidative stress and intracellular Ca^2+?dyshomeostasis resulting from MeHg exposure contributed to neuronal injury. LA could attenuate MeHg-induced neuronal toxicity via its antioxidant properties in primary cultured neurons.     

Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line

Glucose metabolism plays a pivotal role in many physiological and pathological conditions. To investigate the effect of hypoglycemia (obtained by glucose deprivation) on PC12 cell line, we analyzed the cell viability, mitochondrial function (assessed by MTT reduction, cellular ATP level, mitochondrial transmembrane potential), and the level of reactive oxygen species (ROS) after glucose deprivation (GD). Upon exposure to GD, ROS level increased and MTT reduction decreased immediately, intracellular ATP level increased in the first 3 hours, followed by progressive decrease till the end of GD treatment, and the mitochondrial transmembrane potential (ΔΨ_m) dropped after 6 hours. Both necrosis and apoptosis occurred apparently after 24 hours which was determined by nuclei staining with propidium iodide(PI) and Hoechst 33342. These data suggested that cytotoxity of GD is mainly due to ROS accumulation and ATP depletion in PC12 cells.     

菟丝子提取物对活性氧引起已分化的PC12细胞损伤的保护作用

以常用的神经嗜铬细胞瘤PC12细胞株为实验模型,通过比较活性氧(ROS)作用细胞后的细胞活力、凋亡相关蛋白(p53、Bax)水平以及细胞中SOD、GSH、MDA的差异,发现菟丝子提取物不仅能提高ROS损伤的已分化PC12细胞活力,调节细胞中凋亡相关基因的表达,而且还能提高细胞中SOD和GSH的含量,降低MDA水平。由此表明,菟丝子提取物对ROS造成的PC12细胞损伤有一定的保护作用。