Lack of apoptosis in Sendai virus-infected HEp-2 cells without participation of viral antiapoptosis gene.

Sendai virus (SeV) has been reported to induce apoptosis in many types of cells. In HEp-2 cells, however, it did not induce apoptosis in most of the infected cells under the conditions in which vesicular stomatitis virus induced massive apoptosis. The use of a novel technique, which allows the detection of viral antiapoptotic activity in the infected cells, showed that SeV does not have any antiapoptotic activity to interfere with the induction of apoptosis. Consistently, vesicular stomatitis virus-induced apoptosis was not interfered with by preinfection with SeV. These results indicate that the observed lack of apoptosis in these SeV-infected cells does not result from the suppression of apoptosis by viral antiapoptotic activity in the infected cells and suggest that, without activating a signaling pathway for the induction of apoptotic response in the infected cells, SeV can escape apoptosis of the cells, allowing long-term survival of the infected cells.     

Cytotoxicity and apoptosis induction of some selected marine bacteria metabolites

AIMS: To study the potential apoptosis effects of cytotoxic marine bacterial metabolites on human HeLa cell line. METHODS AND RESULTS: After HeLa cells were routinely cultured, tetrazolium-based colorimetric assay for cytotoxicity was performed to screen the marine bacteria extracts showing 12 strains active. To find the potential active strain with apoptosis mechanism, a battery of apoptosis assays, including AO/EB staining, TUNEL assay (terminal-deoxynucleotidyl transferase mediated nick end labelling), gel electrophoresis and flow cytometry, were used to determine whether apoptosis was involved in HeLa cell cytotoxicity of marine bacterial extracts. The results indicated that four strains could induce cell shrinkage, cell membrane blebbing, formation of apoptotic body and DNA fragmentation. CONCLUSIONS: Crude extracts of 12 of 153 strains of marine bacteria showed cytotoxic effects with ID50 ranged from 77.20 to 199.84 microg ml(-1), in which eight strains of bacteria were associated bacteria. The metabolites in the strains of QD1-2, NJ6-3-1, NJ1-1-1 and SS6-4 were able to induce HeLa cells apoptosis. Furthermore, the assessment by flow cytometry indicated that the hypodiploid apoptotic cells increased in a time-dependent manner, suggesting that induced apoptosis occurred from 24 h to 48 h after the extracts treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggested that the compounds from fermentation in these four marine bacterial strains could be candidates for developing apoptosis specific anti-tumour agents with lower toxicity. This study indicated that associated marine bacteria could be good source to find cytotoxic metabolites, and some cytotoxic marine bacterial metabolites could have apoptosis mechanisms.     

Effects of <Emphasis Type="Italic">Helicobacter hepaticus</Emphasis> on the proteome of HEp-2 cells

Helicobacter hepaticus infects the bowel and biliary tree of several animals, producing inflammation. Colonisation of mouse livers can induce hepatocellular carcinomas. The effects of H. hepaticus on the proliferation and global protein expression of human HEp-2 cells were studied by examining the changes in the protein profiles of cells exposed to the bacterium. HEp-2 cells were grown for four days under a microaerobic atmosphere or under the same conditions in co-cultures with H. hepaticus at various inoculum densities. Enlargement, distension and elongation of HEp-2 cells were observed in co-cultures with H. hepaticus. The number of live cells declined by only an order of magnitude at bacterial inocula of approximately 10(9)cfu/ml, but were reduced to less than 10(3)cells/ml at approximately 10(10)cfu/ml bacteria inocula. Protein expression by HEp-2 cells was investigated employing two-dimensional gel electrophoresis. In cells grown with or without bacteria, 17 differentially expressed proteins were identified by tandem mass spectrometry. These proteins participated in several biological functions including amino acid metabolism, cell growth and proliferation, stress response, protein translation and modification, etc. The onset of a catastrophic killing of HEp-2 cells at a bacterial density of approximately 10(9)cfu/ml suggested a multimodal action for H. hepaticus infection, and the modulation of the expression of proteins involved in different biological functions showed that the presence of H. hepaticus has broad effects on the physiology of HEp-2 cells.     

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.     

Enzymic detection of adhesion of enteropathogenic Escherichia coli to HEp-2 cells

We established a new method for detecting enteropathogenic Escherichia coli adhering to HEp-2 cells. An essential part of the method is an assay of beta-galactosidase activity of adhered bacterial cells. It consisted of the following steps: (1) culture of bacterial cells in a medium containing isopropyl-thio-beta-D-galactoside, an inducer of beta-galactosidase; (2) incubation of a bacterial culture with monolayered HEp-2 cells in a 96-well culture plate; (3) washing wells to remove bacterial cells which did not adhere to HEp-2 cells, and (4) enzymic reaction for beta-galactosidase activities. However, a calibration curve for the enzyme activity, obtained from each bacterial sample, showed that 10(5) bacteria per well permitted an accurate estimation. The enzyme activity of adhered bacteria to the monolayered cells showed that 10(7) bacteria were appropriate for the adherence assay. The number of adhered bacteria thus obtained was in good agreement with a viable cell count. The result indicates that the new method is more reliable than a widely used method, counting the number of bacteria under a microscope. The present method also makes it easy to detect adherent strains of E. coli in large numbers of specimens.     

Lack of effective anti-apoptotic activities restricts growth of Parachlamydiaceae in insect cells

The fundamental role of programmed cell death in host defense is highlighted by the multitude of anti-apoptotic strategies evolved by various microbes, including the well-known obligate intracellular bacterial pathogens Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae. As inhibition of apoptosis is assumed to be essential for a successful infection of humans by these chlamydiae, we analyzed the anti-apoptotic capacity of close relatives that occur as symbionts of amoebae and might represent emerging pathogens. While Simkania negevensis was able to efficiently replicate within insect cells, which served as model for metazoan-derived host cells, the Parachlamydiaceae (Parachlamydia acanthamoebae and Protochlamydia amoebophila) displayed limited intracellular growth, yet these bacteria induced typical features of apoptotic cell death, including formation of apoptotic bodies, nuclear condensation, internucleosomal DNA fragmentation, and effector caspase activity. Induction of apoptosis was dependent on bacterial activity, but not bacterial de novo protein synthesis, and was detectable already at very early stages of infection. Experimental inhibition of host cell death greatly enhanced parachlamydial replication, suggesting that lack of potent anti-apoptotic activities in Parachlamydiaceae may represent an important factor compromising their ability to successfully infect non-protozoan hosts. These findings highlight the importance of the evolution of anti-apoptotic traits for the success of chlamydiae as pathogens of humans and animals.     

Applications for green fluorescent protein (GFP) in the study of hostpathogen interactions

The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity. To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens. The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm). Both expression vectors were found to be stable and to direct high levels of GFP synthesis. Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells. Mm expressing gfp was also visualized in infected animal tissues, gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages. In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages. Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.     

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.     

Involvement of C-jun NH2-terminal kinase and apoptosis induced factor in apoptosis induced by deglycosylated bleomycin in laryngeal carcinoma cells

In our previous studies, we demonstrated that the deglycosylation of bleomycin-A2 (BLM-A2) does not affect the capacity of this drug to induce cell death by apoptosis in a caspase-independent manner in laryngeal cancer cells (HEp-2), but suppresses the ability of BLM-A2 to induce ROS formation. We have now investigated the consequence of BLM-A2 deglycosylation in terms of the involvement of apoptotic pathways in HEp-2 cells. Apoptosis induced by bleomycin-A2 and deglyco-BLM-A2 is associated with the release of cytochrome c and AIF. Only Bax was oligomerized with BLM-A2-induced HEp-2 cell death. BLM-A2 and deglyco-BLM-A2-induced apoptosis depended on JNK activation but was independent of death receptors expression. In contrast, both of these drugs would sensitize HEp-2 cells to death receptor ligand-induced cell death. These observations indicate that the deglycosylation of BLM does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway by the release of AIF responsible for mitochondrial permeability and chromatin condensation independent of caspases activation.     

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

Apoptosis-inducing human-origin Fcepsilon-Bak chimeric proteins for targeted elimination of mast cells and basophils: a new approach for allergy treatment

During the past few years, many chimeric proteins have been developed to specifically target and kill cells expressing specific surface molecules. Generally these molecules carry a bacterial or plant toxin to destroy the unwanted cells. The major obstacle regarding these molecules in their clinical application is the immunogenicity and nonspecific toxicity associated with bacterial or plant toxins. We lately reported a new approach for construction of chimeric proteins: we successfully replaced bacterial or plant toxins with human apoptosis-inducing proteins. The resulting chimeras were shown to specifically induce apoptosis in the target cells. Taking advantage of the human apoptosis inducing proteins Bak and Bax as novel killing components, we have now constructed new chimeric proteins targeted against the human FcepsilonRI, expressed mainly on mast cells and basophils. These cells are the main effectors of the allergic response. Treatment of the target cells with the new chimeric proteins, termed Fcepsilon-Bak/Bax, had a dramatic effect on cell survival, causing apoptosis. The effect was specific to cells expressing the FcepsilonRI of both human and, very unexpectedly, also of mouse origin. Moreover, interaction of the chimeric proteins with the mast cells did not cause degranulation. Fcepsilon-Bak/Bax are new chimeric proteins of human origin and, as such, are expected to be both less immunogenic and less toxic and, thus, may be specific and efficient reagents for the treatment of allergic diseases.     

Outer membrane protein 38 of Acinetobacter baumannii localizes to the mitochondria and induces apoptosis of epithelial cells

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial infection. Despite considerable clinical and epidemiological data regarding the role of A. baumannii in nosocomial infection, the specific virulence factor or pathogenic mechanism of this organism has yet to be elucidated. This study investigated the molecular mechanism of apoptosis on the infection of human laryngeal epithelial HEp-2 cells with A. baumannii and examined the contribution of outer membrane protein 38 (Omp38) on the ability of A. baumannii to induce apoptosis of epithelial cells. A. baumannii induced apoptosis of HEp-2 cells through cell surface death receptors and mitochondrial disintegration. The Omp38-deficient mutant was not as able to induce apoptosis as the wild-type A. baumannii strain. Purified Omp38 entered the cells and was localized to the mitochondria, which led to a release of proapoptotic molecules such as cytochrome c and apoptosis-inducing factor (AIF). The activation of caspase-3, which is activated by caspase-9, degraded DNA approximately 180 bp in size, which resulted in the appearance of a characteristic DNA ladder. AIF degraded chromosomal DNA approximately 50 kb in size, which resulted in large-scale DNA fragmentation. These results demonstrate that Omp38 may act as a potential virulence factor to induce apoptosis of epithelial cells in the early stage of A. baumannii infection.     

Binding of bacteria to HEp-2 cells infected with influenza A virus

Epidemiological studies indicate influenza virus infection increases susceptibility to bacterial respiratory pathogens and to meningococcal disease. Because density of colonisation is an important factor in the development of bacterial disease, the objectives of the study were to use flow cytometry methods for assessment of bacterial binding and detection of cell surface antigens to determine: (1) if HEp-2 cells infected with human influenza A virus bind greater numbers of bacteria than uninfected cells; (2) if influenza infection alters expression of cell surface antigens which act as receptors for bacterial binding; (3) if neuraminidase affects binding of bacteria to HEp-2 cells. There was significantly increased binding of all isolates tested regardless of surface antigen characteristics. There were no significant differences between virus-infected and -uninfected Hep-2 cells in binding of monoclonal antibodies to Lewisb, Lewisx or H type 2. There were significant increases in binding of monoclonal antibodies to CD14 (P < 0.05) and CD18 (P < 0.01). Treatment of cells with monoclonal antibodies significantly reduced binding of Neisseria meningitidis strain C:2b:P1.2, CD14 (P < 0.001) and CD18 (P < 0.001). No reduction in binding of a strain of Streptococcus pneumoniae (12F) was observed in these experiments. Neuraminidase treatment of HEp-2 cells increased binding of monoclonal antibodies to CD14 (P < 0.01) and CD18 (P < 0.01). In three experiments, the increase in binding of meningococcal strain C:2b:P1.2 to neuraminidase-treated cells was not significant, but binding of Staphylococcus aureus strain NCTC 10655 was significant (P < 0.05).     

肿瘤坏死因子相关的凋亡诱导配体(TRAIL)cDNA的克隆、表达和活性测定

肿瘤坏死因子相关的凋亡诱导配体 (TRAIL)能选择性诱导肿瘤细胞凋亡 .为利用基因工程技术获得重组TRAIL蛋白可溶性片段 (sTRAIL) ,设计 1对引物 .利用PCR技术特异性扩增出sTRAIL的cDNA ,克隆于质粒pGEM 3Zf( )的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后克隆于表达质粒pBV2 2 0的EcoRⅠ和PstⅠ位点 ,转化大肠杆菌DH5α .转化菌株经温度诱导 ,SDS PAGE检测和Western印迹鉴定 ,获得重组sTRAIL的高水平非融合表达菌株 .表达量占菌体总蛋白的 2 0 % .对其表达产物进行了初步纯化 ,SDS PAGE结果显示纯度可达 90 %以上 .用L92 9细胞测定其生物学活性表明 ,重组蛋白在体外能明显诱导肿瘤细胞凋亡     

迟缓爱德华氏菌对Hep-2细胞的侵袭特性

用细胞裂解计数法及超薄切片电镜观察法分析了迟缓爱德华氏菌侵袭ＨＥｐ－２细胞的基本特性。在１５株来源各异的迟缓爱德华氏菌中,有６株细菌具有对ＨＥｐ－２细胞的侵袭能力。细菌侵入细胞后,主要位于空泡内。侵入细胞内的迟缓爱德华氏菌不仅可在细胞内增殖,而且可从细胞内释放出来。用细胞松弛素破坏微丝后可抑制其侵袭作用,而且表现出剂量依赖关系,而在秋水仙素破坏微管后不影响其侵袭力。这表明在迟缓爱德华氏菌对ＨＥｐ－     

Carboplatin induces apoptotic cell death through downregulation of constitutively active nuclear factor-kappaB in human HPV-18 E6-positive HEp-2 cells

Because the role of nuclear factor kappaB (NF-kappaB) is in cellular growth control and neoplasia, we explored the status of NF-kappaB and investigated its role in survival of human HPV-18 E6-positive HEp-2 cells. We observed accumulation of p65 in the nucleus. Moreover, without any external stimulus constitutive NF-kappaB DNA binding and transactivation activity were detected in HEp-2 cells. Treatment with NF-kappaB inhibitor curcumin (diferuloylmethane) and transient transfection of the mutant form of IkappaBalpha, IkappaBalpha super repressor (IkappaBalpha-SR), suppressed constitutive NF-kappaB activity as well as proliferation, suggesting that constitutive NF-kappaB activity is required for the survival of HEp-2 cells. Carboplatin treatment downregulated constitutive NF-kappaB activity and prevented nuclear retention of p65. Further, carboplatin also suppressed the constitutive IkappaBalpha phosphorylation leading to stabilization of IkappaBalpha protein in the cells. Carboplatin inhibited NF-kappaB binding to its response element present in Bcl-2 promoter resulting in downregulation of antiapoptotic Bcl-2 protein. Thus, our results for the first time indicate that constitutive NF-kappaB has a significant role in the survival of HPV-18 E6-positive HEp-2 cells. Moreover, inactivation of NF-kappaB is one of the mechanisms underlying the induction of carboplatin-mediated apoptosis in HPV-18 E6-positive cancer cells.     

Effect of viral preinfection upon cell adherence in some staphylococcus strains from specific infections or carriers

The adherence of bacteria to eukaryote cells has been largely investigated as an essential step in the occurrence of bacterial infection. Some clinical and epidemiological studies have revealed the frequent association of certain viral infections with bacterial infections originating in the same ecological niche. Therefore, we investigated the effect of the viral preinfection (ADV4) of some cultivated cells (HEp-2 and IC.SK-27) upon the adherence of staphylococcus to these cells. The analysis of cell adherence within the mentioned conditions, estimated by flow cytometry, allowed of the following conclusions: 1. bacterial adherence to cultivated and virally preinfected cells is augmented by the viral preinfection, and its value on a given cell substrate may characterize a bacterial strain; 2. bacterial adherence to the investigated cell substrates does not correlate with the origin of the tested staphylococcus strains (infections or carriers) and some cell lines can differentiate bacterial strains depending upon the ecological niche or inside it.