NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition

Ribonucleases, antibiotics, bacterial toxins, and viruses inhibit protein synthesis, which results in apoptosis in mammalian cells. How the BCL-2 family of proteins regulates apoptosis in response to the shutoff of protein synthesis is not known. Here we demonstrate that an Escherichia coli toxin, MazF, inhibited protein synthesis by cleavage of cellular mRNA and induced apoptosis in mammalian cells. MazF-induced apoptosis required proapoptotic BAK and its upstream regulator, the proapoptotic BH3-only protein NBK/BIK, but not BIM, PUMA, or NOXA. Interestingly, in response to MazF induction, NBK/BIK activated BAK by displacing it from anti-apoptotic proteins MCL-1 and BCL-X(L) that sequester BAK. Furthermore, NBK/BIK- or BAK-deficient cells were resistant to cell death induced by pharmacologic inhibition of translation and by virus-mediated shutoff of protein synthesis. Thus, the BH3-only protein NBK/BIK is the apical regulator of a BAK-dependent apoptotic pathway in response to shutoff of protein synthesis that functions to displace BAK from sequestration by MCL1 and BCL-X(L). Although NBK/BIK is dispensable for development, it is the BH3-only protein targeted for inactivation by viruses, suggesting that it plays a role in pathogen/toxin response through apoptosis activation.     

PARP inhibition induces BAX/BAK-independent synthetic lethality of BRCA1-deficient non-small cell lung cancer

Evasion of apoptosis contributes to both tumourigenesis and drug resistance in non-small cell lung carcinoma (NSCLC). The pro-apoptotic BCL-2 family proteins BAX and BAK are critical regulators of mitochondrial apoptosis. New strategies for targeting NSCLC in a mitochondria-independent manner should bypass this common mechanism of apoptosis block. BRCA1 mutation frequency in lung cancer is low; however, decreased BRCA1 mRNA and protein expression levels have been reported in a significant proportion of lung adenocarcinomas. BRCA1 mutation/deficiency confers a defect in homologous recombination DNA repair that has been exploited by synthetic lethality through inhibition of PARP (PARPi) in breast and ovarian cells; however, it is not known whether this same synthetic lethal mechanism exists in NSCLC cells. Additionally, it is unknown whether the mitochondrial apoptotic pathway is required for BRCA1/PARPi-mediated synthetic lethality. Here we demonstrate that silencing of BRCA1 expression by RNA interference sensitizes NSCLC cells to PARP inhibition. Importantly, this sensitivity was not attenuated in cells harbouring mitochondrial apoptosis block induced by co-depletion of BAX and BAK. Furthermore, we demonstrate that BRCA1 inhibition cannot override platinum resistance, which is often mediated by loss of mitochondrial apoptosis signalling, but can still sensitize to PARP inhibition. Finally we demonstrate the existence of a BRCA1-deficient subgroup (11-19%) of NSCLC patients by analysing BRCA1 protein levels using immunohistochemistry in two independent primary NSCLC cohorts. Taken together, the existence of BRCA1-immunodeficient NSCLC suggests that this molecular subgroup could be effectively targeted by PARP inhibitors in the clinic and that PARP inhibitors could be used for the treatment of BRCA1-immunodeficient, platinum-resistant tumours.     

Cloning of the herpes simplex virus ICP4 gene in an adenovirus vector: effects on adenovirus gene expression and replication

To assess the ability of the herpes simplex virus ICP4 protein to complement adenovirus E1a mutants we have constructed an adenovirus type 5 vector containing a temperature-sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in cells which are permissive (293) or nonpermissive (KB and R970-5) for viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus-encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus; however, it is incapable of complementing adenovirus E1a mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. Our results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators.     

Complementation of adenovirus early region 1a and 2a mutants by Epstein-Barr virus immortalized lymphoblastoid cell lines.

Human B-lymphocytes may be infected by both adenoviruses and the Epstein-Barr virus (EBV). Some of the immediate early and early proteins in the two viruses are similar in function even though their primary structures are different. As these viruses might infect the same B-cells in man, we asked if complementation could take place. The adenovirus mutant H5ts125 has a thermolabile DNA-binding protein and is defective in DNA replication at 39 degrees. Several EBV-transformed human lymphoblastoid cell lines and a tamarin cell line B95-8 were infected with H5ts125 and incubated at either the nonpermissive or the permissive temperatures. Adenoviral DNA replication and assembly of new virions were observed at both temperatures, suggesting complementation by the resident EBV gene products. The adenovirus E1a region is deleted in the mutant d1312. Complementation of this mutant was only obtained in the EBV producer B95-8 cells. Immortalization by EBV was apparently not sufficient for effective complementation. This supports an earlier observation that one of the EBV early proteins (MS-EA) behaves like adenovirus E1a and can transactivate the E4 promoter in a CAT assay. The complementation of mutant adenoviruses in EBV-transformed lymphocytes may help the rescue of new adenovirus serotypes in immunosuppressed patients.     

Down-regulation of multiple cell survival proteins in head and neck cancer cells by an apoptogenic mutant of adenovirus type 5

Head and neck squamous cell carcinomas (HNSCC) are one of the leading causes of cancer deaths world wide. Up-regulation of the epidermal growth factor receptor (EGFR) and BCL-2 family anti-apoptosis proteins in these cancers is linked to aggressive tumor growth, metastasis and chemoresistance. Infection of two HNSCC cell lines, SCC25 and CAL27 by an Ad5 mutant (lp11w) defective in coding for the viral anti-apoptosis protein, E1B-19K efficiently induced apoptotic cell death. In cells infected with lp11w there was a dramatic down-regulation of EGFR by apoptosis-dependent and -independent mechanisms. The levels of the anti-apoptotic proteins BCL-2, BCL-xL and MCL-1 were also down-regulated in lp11w-infected cells compared to uninfected or Ad5-RM infected cells. Infection with lp11w also enhanced sensitivity of the HNSCC cells to the chemotherapeutic drug cisplatin. Our results suggest that adenoviral vectors defective in E1B-19K would be valuable for efficient down-regulation of cell survival proteins and EGFR in epithelial cancers and could be exploited as oncolytic agents to treat HNSCCs.     

Rescue of AAV by antibody-induced Fas-mediated apoptosis from viral DNA integrated in HeLa chromosome

Adenoassociated virus (AAV) provirus in latently infected cells is rescued by superinfection with adenovirus. We examined helper-independent rescue of AAV by Fas-mediated apoptosis from the HeLa lines with AAV DNA integrated in chromosome (HeLa/AAV). In HeLa/AAV, anti-Fas antibody was found to induce low-level production of AAV virions, as detected by the presence of AAV DNA protected from DNase digestion. The antibody induced higher virion production in Fas-enriched HeLa/AAV, which was generated by transfecting HeLa/AAV with an expression plasmid for Fas, than in HeLa/AAV, and the rescued virions were shown to be infectious as assayed along with adenovirus. The antibody also induced apoptotic DNA fragmentation, as detected by staining intracellular fragmented DNA and electrophoresis, in HeLa/AAV and, more markedly, in Fas-enriched HeLa/AAV. Furthermore, an inhibitor for caspase-8 suppressed both the AAV virion production and the DNA fragmentation. Thus, the integrated AAV DNA is likely to be induced to initiate a low level of replication when Fas-mediated apoptosis is activated in HeLa cells.     

Induction of apoptosis in Vero cells by Newcastle disease virus requires viral replication, de-novo protein synthesis and caspase activation

Newcastle disease virus causes (NDV) apoptotic death of infected cells. In the present study, the stimulus that provoked the induction of apoptosis in infected cells was examined. Vero cells infected with NDV developed apoptosis as characterized by DNA fragmentation and decreased DNA content. In presence of ammonium chloride, infected cells did not show reduced DNA content indicating the requirement of virus entry for the induction of apoptosis. UV-inactivated NDV did not induce apoptosis in cells suggesting the need of virus replication. Although cycloheximide blocked NDV-induced apoptosis, actinomycin-D did not, suggesting that de-novo viral protein synthesis was critical for the induction of apoptosis. In addition, activation of caspases was also detected by flowcytometry, indirect fluorescent and colorimetric assays. Based on the results, it was concluded that NDV-induced apoptosis in Vero cells required virus replication, de-novo protein synthesis and caspase activation.     

TR基因重组腺病毒载体的构建和鉴定

目的 :构建含人硫氧还蛋白还原酶 (TR)基因的重组腺病毒载体 ,探讨TR的抗氧化功能与神经退行性疾病的相关性。方法 :从重组质粒pGEM TR上用内切酶切下编码 5 0 0个氨基酸的全长TRcDNA片段 ,并连接穿梭质粒pShuttle,再双酶切pShuttle TR。将带有CMV启动子的目的片段 ,插入E1、E3缺失的Adeno X病毒DNA中 ,以Adeno TRDNA通过脂质体转染HEK2 93细胞 ,获得重组腺病毒Adeno TR进行PCR鉴定及病毒滴度测定。用重组腺病毒感染CV1细胞 ,通过免疫荧光染色与Westernblot分别检测重组腺病毒感染的细胞上和裂解液中TR蛋白的表达。结果 :重组腺病毒Adeno TR的病毒滴度为 4 .4× 10 11pfu/L。PCR、荧光显微镜证实 ,以及Westernbolt分析 ,在相对分子质量 (Mr)约 5 5 0 0 0处均出现特异性条带。结论 :成功地构建了重组腺病毒载体 ,并能介导外源基因TR表达 ,为进一步研究TR的功能及其与疾病的相关性奠定了基础。     

BCL-2 family proteins in peripheral T-cell lymphomas: correlation with tumour apoptosis and proliferation

The present study investigated expression levels of the anti-apoptotic proteins BCL-2, BCL-XL and MCL-1 and the pro-apoptotic proteins BAX and BCL-XS in a series of 112 peripheral T-cell lymphomas (PTCLs) classified according to the WHO classification. Using immunohistochemical methods and a 10% cut-off, each protein was detected in a subset of PTCLs: BCL-2 in 46%, BCL-XS in 49%, BAX in 57%, BCL-XL in 57%, and MCL-1 in 65%. The mean percentage of positive cells for these proteins varied significantly among the PTCL types. Only two types of PTCL, ALK-positive anaplastic large cell lymphoma (ALCL) and enteropathy-type T-cell lymphoma, had a distinctive pattern of expression; all were BCL-2-negative and MCL-1-positive. The mean percentage of BAX-positive and BCL-XS-positive tumour cells was higher in ALK-positive ALCL than in ALK-negative ALCL or other types of PTCL (p = 0.06 and p = 0.01, respectively, Kruskal-Wallis test). MCL-1 was detected significantly more frequently (p = 0.01, chi-square test) and at higher levels (p = 0.0001, Kruskal-Wallis test) in ALK-positive ALCL and ALK-negative ALCL than in other PTCL types. The apoptotic rate, evaluated by the TUNEL assay, correlated inversely with BCL-2 expression (p = 0.035). The proliferation index, assessed by the MIB-1 antibody, correlated with expression levels of MCL-1 (R = 0.42, p = 0.003), BCL-2 (R = 0.32, p = 0.027), BAX (R = 0.33, p = 0.014), and BCL-XL (R = 0.34, p = 0.015) (Spearman rank). In conclusion, BCL-2 family proteins are expressed by a subset of PTCLs and their levels correlate with some histological types, apoptotic rate, and proliferation index. Expression of these proteins may explain the poor response of many types of PTCL to standard chemotherapy. These proteins may also provide novel targets for experimental therapy.     

Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells.     

Expression of cellular proteins Bcl-X(L), XIAP and Bax involved in apoptosis in cells infected with herpes simplex virus 1 and effect of pavine alkaloid (-)-thalimonine on virus-induced suppression of apoptosis

The expression of three cellular proteins involved in the modulation of apoptosis, namely antiapoptotic Bcl-X(L) and XIAP and proapoptotic Bax, was investigated in cells infected with Herpes simplex virus 1 (HSV-1). To assess whether the regulation of apoptosis in virus-infected cells depends on strain specificity the wild-type (wt) strain Victoria and the mutant R-100 resistant to acyclovir (ACV) were used. In addition, the expression of Bcl-X(L), XIAP and Bax was studied in cells infected with HSV-1 and treated with pavine alkaloid (-)-thalimonine. Our previous work has demonstrated that (-)-thalimonine irreversibly inhibits the replication of wt HSV-1 in cultured cells. Our data showed that (-)-thalimonine down-regulates the expression of viral proteins U(L)17, VP11-12, VP22, VP24 and gamma1 34.5, and affects negatively the posttranslational processing of glycoproteins D (gD) and G (gG). As both gamma1 34.5 and glycoprotein D possess antiapoptotic activity, we investigated whether the antiviral effect of the alkaloid could also be due to its ability to suppress the antiapoptotic activity of the virus. Our results demonstrated that: (i) the virus induced overexpression of antiapoptotic proteins Bcl-X(L) and XIAP; (ii) (-)-thalimonine reduced their overexpression, and (iii) this effect was stronger with the acyclovir resistant mutant R-100 than with the wt virus. Taken together, these data suggest that: (i) the virus abolishes apoptosis by means of virus-induced up-regulation of cell-specific prosurvival proteins Bcl-X(L) and XIAP, and (ii) (-)-thalimonine, apart from affecting essential viral targets, inhibits the infectious progeny production via restoration of apoptosis during viral replication.     

表达GFP基因的溶瘤腺病毒对大肠癌细胞的体外杀伤作用

目的： 研究在端粒酶启动子驱动下表达绿色荧光蛋白（GFP）及 5型腺病毒早期基因（E1A）基因的腺病毒Ad/hTERT-GFP-E1对大肠癌细胞的杀伤作用及其可能的机制。方法:将不同滴度Ad/hTERT-GFP-E1感染大肠癌及正常细胞,以巨细胞病毒（CMV）启动子驱动下表达GFP基因的腺病毒Ad/CMV-GFP作为载体对照,通过半数组织培养感染量（TCID50）法测定病毒滴度及体外复制能力,然后用MTT法及细胞克隆形成试验评价该病毒在体外对肿瘤细胞及正常细胞的杀伤作用,并对病毒感染后的细胞进行原位凋亡检测。结果:Ad/hTERT-GFP-E1能够在DLD1细胞内持续复制,GFP的表达能够对病毒的感染和复制起到监测作用;MTT结果显示该病毒对于结直肠肿瘤细胞有显著杀伤作用,而对于正常细胞没有明显的杀伤作用;对病毒感染后的DLD1进行细胞凋亡检测发现Ad/hTERT-GFP-E1所引起的凋亡率显著高于对照组(P＜0.01)。结论:溶瘤腺病毒Ad/hTERT-GFP-E1能够选择性地在肿瘤细胞内复制并杀伤肿瘤细胞,其机制与细胞凋亡的途径有关。     

Adeno-associated virus induces apoptosis during coinfection with adenovirus

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that efficiently replicates in the presence of adenovirus (Ad). Exogenous expression of the AAV replication proteins induces caspase-dependent apoptosis, but determining if AAV infection causes apoptosis during viral infection is complicated by Ad-mediated programmed cell death. To eliminate Ad-induced cytolysis, we used an E3 adenoviral death protein (ADP) mutant, pm534. AAV and pm534-coinfected cells exhibited increased cell killing compared to pm534 alone. Relative to cells infected with Ad alone, AAV and wild-type Ad-infected cells displayed decreased ADP expression, increased cytolysis until the third day of the infection, and decreased cytolysis thereafter. Biochemical and morphological characteristics of apoptosis were observed during coinfections with AAV and pm534 or Ad, including a moderate degree of caspase activation that was not present during infections with pm534 or Ad alone. AAV coinfection also increased extracellular pH. These studies suggest that AAV induces caspase-dependent and caspase-independent apoptosis.     

Contributions of E1A to mouse adenovirus type 1 pathogenesis following intranasal inoculation

We investigated the role of mouse adenovirus type 1 (MAV-1) early region 1A (E1A) protein in adenovirus respiratory infection. Intranasal (i.n.) inoculation of mice with wild type (wt) virus induced chemokine and cellular inflammatory responses in the lung. We observed similar responses in mice infected with an E1A-null mutant virus at the same dose, although the magnitude of these responses was lower. Levels of viral hexon gene expression were lower in the lung following infection with E1A-null virus than with wt virus. When input doses were adjusted so that equivalent viral loads were present following infection with varying doses of wt and E1A-null virus, we observed equivalent chemokine upregulation in the lung. Dissemination to the brain occurred following i.n. inoculation with equal doses of wt or E1A-null virus, but viral gene expression and viral loads were lower and the magnitude of chemokine responses was lower in brains of E1A-null virus-infected mice. CD4 and CD8 T cells and neutrophils were recruited to the brains of mice infected with either wt or E1A-null virus. Together, these data suggest that MAV-1 E1A makes important contributions to viral replication in the lung and the brain following i.n. inoculation. However, E1A is not essential for the induction of inflammatory responses in the lung or for viral dissemination out of the lung.     

Caspase activation is required for permissive replication of Aleutian mink disease parvovirus in vitro

Aleutian mink disease parvovirus (ADV) is distinct among the parvoviruses as infection in vivo is persistent, restricted, and noncytopathic. In contrast, infections with other more prototypic parvoviruses, like mink enteritis virus (MEV), are acute, cytopathic, and characterized by permissive replication in vivo. Although apoptosis results in the death of cells acutely infected by parvoviruses, the role of apoptosis in ADV infections is unknown. Permissive infection of ADV resulted in apoptosis of Crandell feline kidney (CrFK) cells as indicated by TUNEL staining, Annexin-V staining, and characteristic changes in cell morphology. Pretreatment of infected cells with caspase 3 or broad-spectrum caspase inhibitors prevented apoptosis. In addition, treatment of infected cells with these inhibitors caused a 2 log(10) reduction in the yield of infectious virus compared to untreated cultures. This block in replication preceded substantial viral DNA amplification and gene expression. However, inhibitors of caspases 1, 6, and 8 did not have this effect. MEV also induced caspase-dependent apoptosis following infection of CrFK cells, although production of infectious progeny was not affected by inhibition of apoptosis. Thus, permissive replication of ADV in vitro depended upon activation of specific caspases. If ADV infection of cells in vivo fails to initiate caspase activation, the requirement of caspase activity for replication may not be met, thus providing a possible mechanism for persistent, restricted infection.     

Adenovirus E4 34k and E4 11k inhibit double strand break repair and are physically associated with the cellular DNA-dependent protein kinase.

The adenovirus oncoproteins E4 34k and E4 11k, the products of E4 open reading frames 6 and 3, respectively, individually prevent the formation of concatemers of the linear viral genome in infected cells. We show here that genome concatenation in E4 mutant-infected cells requires the cellular DNA-dependent protein kinase (DNA PK) and that E4 34k inhibits V(D)J recombination, a normal cellular process that is also dependent on DNA PK. We further show that both E4 34k and E4 11k coimmunoprecipitate with DNA PK. These observations indicate that E4 products block formation of concatemers of the viral genome by inhibiting DNA PK-dependent double strand break repair and suggest that they act by forming a physical complex with DNA PK. DNA PK also participates in activation of p53 DNA-binding activity by DNA damage. By inhibiting DNA PK function, E4 products may block p53 activation in response to the products of viral DNA replication and thus provide a new mechanism to prevent apoptosis of infected cells.     

Adenovirus-based gene therapy during clevudine treatment of woodchucks chronically infected with woodchuck hepatitis virus

Interferon-alpha (IFN-alpha) is a potent suppressor of hepatitis B virus (HBV) replication in the HBV-transgenic mouse, depleting virus replication intermediates from infected hepatocytes via pathways mediated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). It has also been hypothesized that cytokines induce curing of infected hepatocytes via non-cytolytic pathways during resolution of transient hepadnavirus infections. We have therefore evaluated therapy of chronic woodchuck hepatitis virus (WHV) infections using treatment with the nucleoside analog clevudine [L-FMAU; 1-(2-fluoro-5-methyl-b-L-arabinofuranosyl) uracil] and therapy with adenovirus vectors expressing INF-gamma, TNF-alpha, and beta-galactosidase. Before their use in vivo, expression of IFN-gamma and TNF-alpha from the adenovirus vectors was evaluated in vitro. Conditioned media from adenovirus-infected WC-3 cells was shown to inhibit WHV replication in baculovirus-transduced cells. Adenovirus super-infection of the liver in woodchucks led to declines in the percentage of hepatocytes with detectable core antigen and nucleic acids, and in levels of covalently closed circular DNA (cccDNA) and total WHV DNA, but a major long-term benefit of adenovirus super-infection during clevudine treatment was not demonstrated. Moreover, the effect took at least 2 weeks to develop suggesting that the declines in the percentage of WHV-infected cells, ccc, and total WHV DNA resulted from induction of the adaptive immune response by the adenovirus super-infection, and only indirectly from the expression of cytokines by the vectors.     

Adenovirus DNA synthesis is coupled to virus assembly

The relationship between viral DNA synthesis and virion assembly was studied in adenovirus type 2 infected HEp2 cells. When cells were infected at the restrictive temperature with ts3, an assembly-negative mutant which permits normal viral DNA and protein synthesis, labeled and shifted to the permissive temperature, only de novo synthesized nonradioactive viral DNA was encapsidated. This suggested that only concurrently synthesized DNA is encapsidated. Blocking protein or DNA synthesis with cycloheximide or hydroxyurea after the temperature shift inhibited virus assembly. Therefore efficient virus assembly requires both concurrent protein and DNA synthesis. When DNA synthesis was arrested by shifting ts125 infected cells to the restrictive temperature, protein synthesis continued but assembly was completely blocked. Sucrose gradient sedimentation analysis of nuclear extracts of wt and ts3 infected cells provided evidence in support of a physical coupling between replication complexes and virus assembly complexes. Further evidence of coupling was also shown by preferential pulse labeling of the molecular right end of the genome isolated from reversibly cross-linked assembly intermediate particles. While DNA replication is not dependent on concurrent virion assembly, at least some significant proportion of replication complexes appear to be coupled to and are prerequisite for virion assembly.