Role of Bcl-2 expression for productive herpes simplex virus 2 replication

Herpes simplex viruses infect a variety of cells in vitro. However, not all infected cells sustain a fully productive replication of these viruses. We have shown that, in U937 monocytoid cells, herpes simplex virus 2 (HSV-2) causes a low-productive infection characterized by apoptosis as cytopathic effect at a late stage of infection. This effect was associated with a down-regulation of the Bcl-2 protein. We therefore asked whether destabilization of Bcl-2 expression could act as a limiting factor for the productive HSV-2 infection. We found that overexpression of Bcl-2 in U937 cells dramatically increased the capability of these cells to sustain a fully productive infection, while protecting against apoptosis induced by HSV-2. Overall, our data indicate that Bcl-2 expression acts as a regulator of HSV-2 replication.     

Fibroblast growth factor-2 inhibits endothelial cell apoptosis by Bcl-2-dependent and independent mechanisms.

Intact endothelium acts as a sensor and transducer of signals and also provides a nonthrombogenic surface at the blood-vascular wall interface. Hence, mechanisms that maintain the integrity of the endothelium are of interest in physiological and pathological states. In this study we show that apoptosis induced by growth factor and serum deprivation of endothelial cells occurs at all phases of the cell cycle and can be blocked by fibroblast growth factor-2 (FGF-2) independently of its mitogenic activity. As the Bcl-2 family of proteins plays a prominent role in regulating cell survival, we attempted to identify Bcl-2 homologues expressed in endothelial cells. Here we demonstrate that, in addition to the previously identified A1, four other members of the Bcl-2 family, Bcl-2, Mcl-1, Bcl-X(L), and Bax, are expressed in endothelial cells. Of these family members, only Bcl-2 is induced by FGF-2. Overexpression of Bcl-2, using a retroviral vector, protects endothelial cells from serum and growth factor deprivation. There is no difference in FGF-2-induced proliferation between Bcl-2-overexpressing cells and those transduced with the empty retroviral vector. At early time points Bcl-2 is not up-regulated, but FGF-2 still has a protective effect. However, FGF-2 protects only adherent endothelial cells but not those that are cultured in suspension. The early effect of FGF-2 is dependent on tyrosine phosphorylation but not on activation of the MAP kinase pathway. Thus, FGF-2 inhibits endothelial cell apoptosis by Bcl-2-dependent and independent mechanisms.     

姜黄素抑制顺铂诱导人肾小管上皮细胞系HK-2凋亡

目的探讨姜黄素对顺铂诱导人肾小管上皮细胞系HK-2凋亡的影响。方法用MTT法观察HK-2增殖;用Western blot检测HK-2的凋亡蛋白。结果 1)顺铂呈剂量依赖性诱导HK-2凋亡(P<0.05)。2)在姜黄素和顺铂联合作用下,凋亡的HK-2明显减少,细胞存活率提高(P<0.05);HK-2中Bax蛋白表达降低(P<0.05);Bcl-2蛋白表达增加(P<0.05)。结论姜黄素可以通过调控Bax和Bcl-2蛋白的表达抑制顺铂引起的细胞凋亡。     

SARS-CoV nucleocapsid protein induced apoptosis of COS-1 mediated by the mitochondrial pathway

To investigate the apoptosis effect of SARS coronavirus nucleocapsid protein on cultured cell lines and to explore the possible pathway of apoptosis. pCDNA3.1(-)/his-myc vector containing the SARS coronavirus nucleocapsid gene (N), matric gene (M), spike gene (S) were transfected into COS-1, Huh-7 and HepG2 cells. Apoptosis induced by SARS coronavirus N protein under starvation of serum of COS-1 cells was monitored by Annexin V and electron microscopy assays. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (DeltaPsim) were determined by flow cytometric assay. Cytochrome C, cleaved caspase (cysteine aspartic acid protease)-3, 9, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. After removal of serum in COS-1 cells, we observed the loss of DeltaPsim, the increase of ROS and cytochrome C release into cytosol and subsequent activation of caspase-3 and PARP cleavage. The pan-caspase inhibitor z-VAD-fmk can block the activation of caspase 3, 9 and PARP cleavage. In conclusion, SARS coronavirus N protein can induce apoptosis of COS-1 cells by activating mitochondrial pathway. SARS coronavirus M, S protein can not induce apoptosis in COS-1, HepG2 and Huh-7 and SARS coronavirus N protein can not induce apoptosis in HepG2 and Huh-7 by methods used in this study.     

Coordinate induction of IFN-alpha and -gamma by SARS-CoV also in the absence of virus replication

BACKGROUND: Severe acute respiratory syndrome (SARS) is an emerging infection caused by a novel coronavirus known as SARS-CoV, characterized by an over-exuberant immune response with lung lymphomononuclear cells infiltration and proliferation that may account for tissue damage more than the direct effect of viral replication. This study is aimed at investigating the capability of SARS-CoV to activate IFN-alpha and -gamma expression in lymphomonocytes (PBMC) from healthy donors, evaluating whether viral replication is necessary for this activation. RESULTS: SARS-CoV virus is able to induce both IFN-alpha and -gamma mRNA accumulation and protein release in a dose-dependent manner, MOI 10 being the most effective. The time course curve indicated that IFN-alpha mRNA induction peaked at 24 h.p.i,. whereas IFN-gamma mRNA was still increasing at 48 h.p.i. Released IFN (both types) reached a plateau after 24-48 h.p.i. and remained rather stable over a 5-day period. A transient peak of negative strand viral RNA was detected after 1-2 days of infection, but neither infectious virus progeny yield nor newly produced viral genomic RNA could be evidenced in infected cultures, even after prolonged observation time (up to 13 days). Cocultivation of PBMC with fixed SARS-CoV-infected Vero cells was even more efficient than exposure to live virus in eliciting IFN-alpha and -gamma induction. A combination of IFN-alpha and -gamma strongly inhibited SARS-CoV replication in Vero cells, while the single cytokines were much less effective. CONCLUSIONS: This study provides evidence that SARS-CoV is able to induce in normal PBMC a coordinate induction of IFN-alpha and -gamma gene expression. Virus replication is not necessary for IFN induction since efficient IFN expression could be obtained also by the cocultivation of normal PBMC with fixed SARS-CoV-infected cells. Concomitant activation of IFN-alpha and -gamma gene expression by SARS-CoV in vivo may be relevant for the pathogenesis of the disease, both for the possible involvement in immunomediated damage of the tissues and for the strong inhibition of SARS-CoV replication as a result of combined cytokine action.     

H2O2诱导骨骼肌卫星细胞凋亡及法舒地尔的保护作用

背景：骨骼肌卫星细胞是成体骨骼肌中位于肌细胞膜和基膜之间具有增殖分化潜能的肌源性干细胞,研究表明骨骼肌卫星细胞的有效性及安全性,但是移植后的干细胞成活率极低,极大的限制了骨骼肌卫星细胞的应用。 目的：观察过氧化氢(H₂O₂)对大鼠骨骼肌卫星细胞凋亡的影响以及法舒地尔的保护作用。 方法：取体外培养的骨骼肌卫星细胞,随机分为正常对照组,H₂O₂组,H₂O₂⁺法舒地尔组(法舒地尔组),采用流式细胞仪检测细胞凋亡率,ELISA法检测白细胞介素4、肿瘤坏死因子a的浓度,Western blot检测Bax蛋白的表达。 结果与结论：同H₂O₂组相比,法舒地尔组凋亡发生率明显下降(P < 0.05),Bax蛋白水平表达明显降低(P < 0.05),白细胞介素4、肿瘤坏死因子a的分泌显著减少(P < 0.05)。结果提示,法舒地尔抑制Rho激酶信号途径发挥抗凋亡保护作用,其机制可能与减少Bax蛋白的表达有关。      

Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70

Heat-shock protein 70 (HSP70) is highly expressed in Toxoplasma gondii-infected cells. However, the role of this protein is not well understood, especially during apoptosis. This study addresses the mechanism behind the antiapoptotic chaperone activity of HSP70 in Toxoplasma-infected host cells using a human macrophage cell line, THP-1 by Western blot, DNA fragmentation assay, immunoprecipitation, and a caspase-3/7 activity assay based on cleavage of the colorimetric substrate DEVD-pNA. Apoptosis induced by arsenic trioxide (As₂O₃) was inhibited in T. gondii-infected THP-1 cells, but not in uninfected cells. Without As₂O₃ induction of apoptosis, T. gondii infection caused increased expression of Bcl-2 and HSP70, but not caspase-3. However, active form caspase-3 levels were lower in As₂O₃-treated infected cells as compared with As₂O₃-treated uninfected cells. Bcl-2 expression in As₂O₃-treated infected cells was similar to that in cells infected with T. gondii. Translocation of apoptosis-inducing factor (AIF) and release of cytochrome c from mitochondria were inhibited in As₂O₃-treated infected cells as compared with As₂O₃-treated uninfected cells. Increased parasite loads in Toxoplasma-infected macrophages caused higher HSP70 and Bcl-2 expression in whole-cell extracts and fractionated components, respectively. However, expression of AIF and cytochrome c was unaffected. Toxoplasma dose-dependently inhibited caspase-3 activation, thus revealing an anti-apoptotic parasite activity on cytochrome c-mediated caspase activation in subcellular components. In addition, immunoprecipitation analysis suggested that HSP70 is capable of binding to the pro-apoptotic factors AIF and Apaf-1, but not to cytochrome c or procaspase-9. Taken together, these data demonstrate that T. gondii infection inhibits mitochondrial apoptosis through overproduction of anti-apoptotic Bcl-2 as well as HSP70, which are increased parasite loads dependently.     

苦参碱通过调节Bcl-2/Bax的平衡抑制大鼠缺血再灌注引起的肝细胞凋亡

目的肝脏缺血再灌注损伤是肝脏切除手术中最常见的并发症,由于激发细胞异常凋亡,常导致肝功能不全甚至引起死亡。本研究旨在揭示肝脏缺血再灌注损伤发生过程中苦参碱参与调节凋亡蛋白Bcl-2/Bax平衡的分子机制。方法健康成年SD大鼠45只,随机分为3组:一组作为空白对照组(SO组,n=15),不阻断肝脏血流;另外2组大鼠行夹闭门静脉和肝动脉缺血70 min后进行再灌注方法建立HIRI模型(缺血再灌注损伤模型),以缺血/再灌注+生理盐水组为对照组(NS组),缺血/再灌注+苦参碱组为实验组(MT组)。MT组在夹闭门静脉和肝动脉前经门静脉主干注入苦参碱。NS组注入生理盐水,阻断供血1h后恢复灌注,在恢复灌注2 h后采集标本行mRNA及Western blot检测。结果 qRT-PCR结果显示,正常对照SO组Bcl-2 mRNA表达水平显著高于Bax,在肝脏缺血再灌注模型组NS组中,高Bcl-2表达水平受到抑制,Bax表达水平显著高于Bcl-2。当行苦参碱干预后,Bax mRNA水平显著下降(MT组),基本接近SO组Bax水平。Western blot验证结果与mRNA水平结果相似。结论苦参碱显著抑制大鼠缺血再灌注引起的肝细胞凋亡,该途径可能由于苦参碱参与调节Bcl-2/Bax的平衡所致。     

姜黄素抑制ox-LDL诱导HUVECs凋亡

目的研究姜黄素对氧化低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)凋亡的保护作用及其可能机制。方法体外培养HUVECs,实验分为:对照组、ox-LDL组、ox-LDL加内质网应激(ERS)抑制剂PBA组、姜黄素组、ox-LDL加姜黄素组和ox-LDL加姜黄素加PI3K抑制剂LY294002组。CCK-8法检测细胞存活率;流式细胞仪检测细胞凋亡;激光共聚焦显微镜观察活化转录因子6(ATF6)转位;Western bolt检测ERS相关蛋白:糖调节蛋白78(GRP78)、蛋白激酶样内质网激酶(PERK)和肌醇激酶-1(IRE-1)以及相关通路蛋白:LOX-1、AKT和p-AKT的表达。结果与对照组相比,ox-LDL可增加细胞凋亡,提高ERS相关蛋白的表达(P0.01),促使ATF6向核内转位,以及提高LOX-1(P0.01)和降低p-AKT的表达(P0.01);与ox-LDL组相比,PBA可抑制ox-LDL诱导的细胞凋亡(P0.01),姜黄素可抑制ox-LDL诱导的ERS相关蛋白和LOX-1的表达(P0.01),ATF6的核转位,内皮细胞的凋亡(P0.01),同时它还可提高ox-LDL引起的p-AKT表达下调(P0.01);LY294002可部分削弱姜黄素抑制ox-LDL诱导ERS相关蛋白表达的作用(P0.05)。结论姜黄素可降低ox-LDL诱导HUVECs的凋亡,其可能机制是通过抑制LOX-1的表达和激活AKT通路减轻细胞ERS来实现的。     

白藜芦醇对乳腺癌MDA-MB-231细胞凋亡及Bcl-2蛋白表达的影响

目的探讨白藜芦醇(resveratrol,Res)对人乳腺癌MDA-MB-231细胞凋亡及相关蛋白Bcl-2表达的影响。方法不同浓度白藜芦醇处理人乳腺癌MDA-MB-231细胞,MTT法检测白藜芦醇对细胞增殖抑制率,流失细胞技术检测细胞凋亡,Western blot法检测细胞凋亡相关蛋白Bcl-2、Bcl-xl、Mcl-1和Caspase-3的表达。结果白藜芦醇25、50、100、200μmol/L处理细胞,增殖抑制率分别为(24.22±1.66)%、(30.79±1.60)%、(45.72±1.25)%和(53.31±1.94)%。白藜芦醇0、50、100、200μmol/L处理细胞,凋亡率分别为(3.53±0.25)%、(28.80±1.34)%、(38.78±1.19)%和(58.96±1.26)%。Western blot结果显示,白藜芦醇处理细胞后,Caspase-3蛋白裂解片段表达增加,凋亡相关蛋白Bcl-2和Bcl-xl的表达减低。结论白藜芦醇能诱导MDA-MB-231细胞凋亡增加,这可能与白藜芦醇激活Caspase-3蛋白同时抑制凋亡相关蛋白Bcl-2和Bcl-xl表达有关。     

Point mutations in the C-terminus of HIV-1 gp160 reduce apoptosis and calmodulin binding without affecting viral replication

One hallmark of AIDS progression is a decline in CD4+ T lymphocytes, though the mechanism is poorly defined. There is ample evidence that increased apoptosis is responsible for some, if not all, of the decline. Prior studies have shown that binding of cellular calmodulin to the envelope glycoprotein (Env) of HIV-1 increases sensitivity to fas-mediated apoptosis and that calmodulin antagonists can block this effect. We show that individual mutation of five residues in the C-terminal calmodulin-binding domain of Env is sufficient to significantly reduce fas-mediated apoptosis in transfected cells. The A835W mutation in the cytoplasmic domain of gp41 eliminated co-immunoprecipitation of Env with calmodulin in studies with stably transfected cells. Four point mutations (A835W, A838W, A838I, and I842R) and the corresponding region of HIV-1 HXB2 were cloned into the HIV-1 proviral vector pNL4-3 with no significant effect on viral production or envelope expression, although co-immunoprecipitation of calmodulin and Env was decreased in three of these mutant viruses. Only wild-type envelope-containing virus induced significantly elevated levels of spontaneous apoptosis by day 5 post-infection. Fas-mediated apoptosis levels positively correlated with the degree of calmodulin co-immunoprecipitation, with the lowest apoptosis levels occurring in cells infected with the A835W envelope mutation. While spontaneous apoptosis appears to be at least partially calmodulin-independent, the effects of HIV-1 Env on fas-mediated apoptosis are directly related to calmodulin binding.     

Multiple defects in the genome of pseudorabies virus can affect virulence without detectably affecting replication in cell culture

Several independently isolated vaccine strains of pseudorabies virus were studied to identify the functions that play a role in the expression of virulence of this virus. All the strains that were studied grew well in three different cell types. No differences that could be correlated with avirulence could be detected either in the virus yield produced by the cells or in the length of the eclipse phases. All the attenuated strains, however, had lost their ability to replicate efficiently in the brains of day-old chickens. The defects leading to the decrease in the virulence for day-old chickens varied in the different vaccine strains. The Tatarov vaccine strain is defective in the thymidine kinase (TK) gene; restoration of a functional TK gene restores to this strain its virulence for day-old chickens and for pigs. Three out of four different, independently isolated avirulent strains were found to be defective in different loci, as determined by their ability to generate virulent recombinants. Two strains, Bartha and Buk Z300, however, yielded few virulent recombinants, indicating that they were defective in at least one closely linked function. Furthermore, all the virulent recombinants obtained from cells coinfected with different pairwise combinations of the vaccine strains had higher LD50 values than virulent wild-type virus, indicating that the recombinants had not acquired all the functions necessary for optimum expression of virulence. Partial virulence was also restored to Buk Z900 by marker rescue with sequences originating from three different regions of the wild-type pseudorabies virus genome. All three of these regions were different from those that had previously been shown to rescue virulence of the Bartha strain (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1987, J. Virol. 61, 796-801). Our results thus show that (1) defects in several different loci of the pseudorabies virus genome can affect virulence without detectably affecting growth in cell culture and (2) most vaccine strains have multiple defects contributing to their lack of virulence.     

Bcl-2 delays macrophage engulfment of human B cells induced to undergo apoptosis

The capacity to be recognized and engulfed by phagocytes is an important characteristic of cells dying by apoptosis. Phagocytosis of apoptotic cells occurs rapidly in vivo, probably prior to plasma membrane breakdown. While the molecular mechanisms mediating phagocytosis of apoptotic cells are beginning to be defined, little is yet known of the relationship between the cell-death program itself and the surface changes on the dying cells that signal for engulfment. Here, we investigate to what extent the apoptosis repressor Bcl-2 can modulate the recognition and phagocytosis of human B cells exposed to triggers of apoptosis. Burkitt lymphoma (BL)-derived, Bcl-2⁻ B cells were induced into apoptosis either by the Ca²⁺-ionophore ionomycin or by the inhibitor of protein synthesis cycloheximide. Apoptotic BL cells, but not viable BL cells, were recognized and phagocytosed by monocyte-derived macrophages. bcl-2-transfected BL populations showed a reduced capacity both to undergo apoptosis in response to these inducing agents and to interact with macrophages. Like their Bcl-2⁻ counterparts, Bcl-2⁺ BL cells interacted with macrophages only after activation of their apoptotic program as assessed by changes in nuclear morphology. These results demonstrate not only that continued protein synthesis in B cells undergoing apoptosis is not essential for their recognition by macrophages, but also that macrophage recognition of apoptotic B cells cannot be uncoupled from the cell-death program that is controlled by Bcl-2. In this respect, the behavior of B cells contrasts markedly with that of neutrophils in which Bcl-2 has been reported to inhibit apoptosis without affecting phagocytic clearance (Lagasse and Weissman, J. Exp. Med. 1994. 179: 1047).     

Induction of Bad-mediated apoptosis by Sindbis virus infection: involvement of pro-survival members of the Bcl-2 family

It is known that infection with Sindbis virus (SNV) induces apoptosis, which is inhibited by two pro-survival members of the Bcl-2 family, Bcl-2 and Bcl-xL. However, the mechanism of involvement of the other members of the Bcl-2 family in SNV-induced apoptosis remains unclear. In this study we report that Bad protein, one of the pro-apoptotic Bcl-2 family members, mediates apoptosis in the mammalian cells infected with SNV. Expression of Bad was shown to promote SNV-induced apoptosis in human embryonic kidney 293T and baby hamster kidney cells. SNV infection also induced translocation of endogenous Bad into mitochondria and heterodimerization of Bad with Bcl-xL. On the other hand, the structurally most similar pro-survival members, Bcl-2, Bcl-xL, and Bcl-w, suppressed SNV-induced apoptosis in the absence of Bad, whereas Mcl-1 and A1 did not. Bcl-w could inhibit SNV-induced apoptosis in the presence of Bad, but Bcl-xL could not. Bad could be coimmunoprecipitated with Bcl-xL or Bcl-2, but not with Bcl-w. Two viral Bcl-2 homologs, E1B19K and BHRF1, also suppressed SNV-induced apoptosis irrespective of the presence of Bad and no physical association with Bad was observed. These results suggest that direct interaction of Bad with pro-survival members of the Bcl-2 family contributes to the progress of SNV-induced apoptosis and that nonbinding members restrain SNV-induced apoptosis irrespective of Bad expression.     

Bluetongue virus induces apoptosis in cultured mammalian cells by both caspase-dependent extrinsic and intrinsic apoptotic pathways

Summary Bluetongue virus (BTV) causes haemorrhagic disease in sheep and induces death in cultured mammalian cells. In the present study, BTV-induced apoptotic pathways in Vero cells were elucidated. Cells infected with BTV at 0.1 m.o.i underwent DNA fragmentation and membrane blebbing within 48 h postinfection. BTV-induced apoptosis was blocked by the pan-caspase inhibitor, z-VAD-FMK. Immuno-blotting using anti-caspase-8 and -9 antibodies detected the activation of the respective caspases. Flow cytometry analyses following 3, 3′ dihexyloxacarbocyanine iodide staining revealed the loss of mitochondrial membrane potential. Our study confirms the involvement of both caspase-dependent extrinsic and intrinsic pathways of apoptosis in BTV-infected cells.     

血管内皮生长因子拮抗H2O2诱导的血管内皮细胞凋亡

目的：探讨血管内皮生长因子（VEGF）预防血管成形术后再狭窄的机制。方法：构件含人VEGF₁₆₅基因的重组腺病毒载体并感染体外培养的血管平滑肌细胞，72 h后收集含VEGF的条件培养液，将对数生长期的人脐静脉内皮细胞（HUVEC）分成对照组、H₂O₂处理组和H₂O₂+VEGF处理组，12 h后采用原位末端标记法和流式细胞术检测各组细胞的凋亡发生情况。结果：H₂O₂处理组细胞凋亡明显多于对照组和H₂O₂+VEGF处理组（P<0.01）。结论：H₂O₂能诱导HUVEC凋亡，而VEGF能部分拮抗上述作用。     

Mycophenolic acid inhibits dengue virus infection by preventing replication of viral RNA

Dengue fever is a mosquito-borne viral disease of global importance with no available antiviral therapy. We assessed the ability of mycophenolic acid (MPA), a drug currently used as an immunosuppressive agent, to inhibit dengue virus (DV) antigen expression, RNA replication, and virus production. Pharmacological concentrations of MPA effectively blocked DV infection, decreasing the percentage of infected cells by 99% and the levels of secreted virus by up to a millionfold. Results were reproduced with four hepatoma cell lines and different flaviviruses, including a recent West Nile virus isolate. Experiments were performed to define the stage in the viral lifecycle at which MPA abrogates infection. Early steps in viral infection, such as viral entry and nucleocapsid uncoating, were not the primary targets of MPA action since its inhibitory effect was retained when naked DV RNA was transfected directly into cells. Biosynthetic labeling experiments showed that MPA did not block the initial phase of viral translation but did interfere with viral protein synthesis in the amplification phase. Quantitative RT-PCR demonstrated that MPA prevented the accumulation of viral positive- and negative-strand RNA as the infection proceeded. We conclude that MPA inhibits flavivirus infection by preventing synthesis and accumulation of viral RNA.