Epithelial cells release proinflammatory cytokines and undergo c-myc-induced apoptosis on exposure to filarial parasitic sheath protein-Bcl2 mediates rescue by activating c-H-ras

Summary Circulating filarial proteins elicit strong immunologic reactions in humans leading to the chronic manifestations in human lymphatic filariasis such as lymphatic occlusion, fibrosis, edema, and in some cases, tropical pulmonary eosinophilia. Our earlier studies, in vitro, conclusively prove that filarial parasitic sheath proteins induce apoptosis in HEp2 cells, an epithelial cell line, by a pathwa inhibitable by bcl2. The present findings provide evidence that c-myc activation triggers apoptosis in HEp2 cells and that it is also responsible for the burst of abortive proliferation at 6 d of treatment of HEp2 bcl2 cells that overexpress bcl2, with filarial parasitic sheath protein, demonstrating the interplay between the two genes c-myc and bcl2, wherein bcl2 acts by restoring the prosurvival signal to c-myc and keeping its apoptotic tendency in check. This study also indicates that bcl2 upregulates c-H-ras, engaring ras to bring about the suppression of apoptosis through protein tyrosine kinase elevation, thus promoting the survival of the HEp2 bcl2 cells. In addition to the activation of these “signal switches,” we also observe that these cells release cytokines like IL-6 and IL-8 through the upregulation of c-fos, when exposed to filarial parasitic sheath protein, reflecting on the immunomodulatory capacity of the epithelium to elicit a host immune response by setting up a chemotactic gradient, attracting inflammatory cells to the site of infection.     

Stiffness changes of tumor HEp2 cells correlates with the inhibition and release of TRAIL-induced apoptosis pathways

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) is a promising apoptotic agent that can selectively act on tumor cells. However, some cancer cells are resistant to TRAIL mediated apoptosis. In specific type of cells, sensitization by chemotherapeutic drugs may overcome the resistance to TRAIL induced apoptosis. In this work, atomic force microscopy (AFM) nanoindentation spectroscopy combined with fluorescence methods were used to investigate the biomechanical aspects of the resistance and unblocking of apoptosis in larynx carcinoma HEp2 cells treated with TRAIL. It is shown that there is a direct correlation between the increase in mechanical cell stiffness and the inhibition of apoptosis induced by TRAIL in HEp2 cells. Conversely, unblocking of apoptosis by sensitization of HEp2 cells with a chemotherapeutic drug Actinomycin D is related to the depolymerization of F-actin and to the decrease in the cell stiffness. Both effects, that is, changes in the mechanical stiffness of the cell and the inhibition of apoptotic pathway, are closely related to the Bcl-2 activity. Most probably, the depolymerization of F-actin results from downregulation of Rho protein, which in turn is accompanied by a lower activity of Bcl-2 and in consequence releases the intrinsic apoptotic channel. The presented results reveal a promising application of nanoindentation spectroscopy with an AFM tip as a novel tool for monitoring the processes of apoptosis inhibition.     

Differences in adhesiveness among type 1 fimbriate strains of Enterobacteriaceae revealed by an in vitro HEp2 cell adhesion model

Ten type 1 fimbriate strains of Enterobacteriaceae were examined in an in vitro adhesion assay with HEp2 epithelial cells. The range of HEp2 cell adhesiveness, which was characteristic for each strain, was affected by motility, type 1 fimbriation and production of mannose sensitive haemagglutinin. Nevertheless, not all type 1 fimbriate strains adhered well in this model. The findings are discussed with regard to the possibility that different type 1 fimbriate enterobacteria, though all are mannose sensitive, recognize different mannose-containing receptors present or available on the surfaces of the HEp2 cells.     

Differences in adhesiveness among type 1 fimbriate strains of Enterobacteriaceae revealed by an in vitro HEp2 cell adhesion model

Ten type 1 fimbriate strains of Enterobacteriaceae were examined in an in vitro adhesion assay with HEp2 epithelial cells. The range of HEp2 cell adhesiveness, which was characteristic for each strain, was affected by motility, type 1 fimbriation and production of mannose sensitive haemagglutinin. Nevertheless, not all type 1 fimbriate strains adhered well in this model. The findings are discussed with regard to the possibility that different type 1 fimbriate enterobacteria, though all are mannose sensitive, recognize different mannose-containing receptors present or available on the surfaces of the HEp2 cells.     

Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2.

The product of the c-myc proto-oncogene is an important positive regulator of cell growth and proliferation. Recently, c-Myc has also been demonstrated to be a potent inducer of apoptosis when expressed in the absence of serum or growth factors. To further examine Myc-induced apoptosis, we coexpressed the proto-oncogene bcl2, which has been shown to block apoptosis in other systems, with c-myc in serum-deprived Rat 1a fibroblasts. Here we report that ectopic expression of bcl2 specifically blocks apoptosis induced by constitutive c-myc expression. Constitutive c-myc expression in serum-deprived Rat 1a cells caused a > 15-fold increase in the number of dead cells, accompanied by DNA fragmentation. However, coexpression of bcl2 with c-myc in these cells led to a 10-fold increase in the number of live cells and a significant decrease in DNA fragmentation. Thus, Bcl-2 effectively inhibits Myc-induced apoptosis in serum-deprived Rat 1a fibroblasts without blocking entry into the cell cycle. These results imply that apoptosis serves as a protective mechanism to prevent tumorigenicity elicited by deregulated Myc expression. This protective mechanism is abrogated, however, by Bcl-2 and therefore may explain the synergism between Myc and Bcl-2 observed in certain tumor cells.     

Infection of HEp2 epithelial cells with Candida albicans: adherence and postadherence events

We have previously observed that the infection of HEp2 epithelial cells with Candida albicans results in HEp2 cell actin rearrangement, and that a culture filtrate of C. albicans (Candida metabolite) caused the same changes and reduced membrane ruffling and motility. It was found that the Candida metabolite consisted of several proteins and nonproteinaceous components. In this study we report on the identity of three of the main proteins in the Candida metabolite, namely a secretory aspartate protease (Sap), an agglutinin-like adhesion sequence (Als) and a glucan 1,3-beta-glucosidase. The effect on HEp2 cells caused by the Candida metabolite, an inhibitor of the PKC MAP kinase signal pathway - bisindolylmaleimide (BIM), or the actin polymerization inhibitor - cytochalasin D (CyD) were studied alone and in combination. Exposure of HEp2 cells to the Candida metabolite, together with the BIM or CyD, had profound effects on HEp2 cell morphology, as compared to individually treated cells, and also reduced the adherence of the organisms to HEp2 cells. Our results show that the interaction of C. albicans with HEp2 cells is, not unexpectedly, complex, and involves changes in the host cell that may be related to the effect of Candida-secreted biomolecules.     

Phosphorylation of cytokeratin 17 by herpes simplex virus type 2 US3 protein kinase

We previously reported the establishment of an HEp2 cell line which expresses the US3 protein kinase (PK) of herpes simplex virus type 2 (HSV-2) upon induction with IPTG. Here we report that expression, phosphorylation and ubiquitination of cytokeratin 17 (CK17) are enhanced in US3-expressing HEp2 cells. In vitro kinase and co-immunoprecipitation assays provided evidence that US3 PK directly phosphorylates CK17. Expression of US3 PK caused a significant decrease in filamentous staining of CK17, suggesting that phosphorylation of CK17 by US3 PK causes a disruption of intermediate filaments. Our observations suggest a role for US3 in the regulation of CKs and intermediate filaments in cells. Moreover, we found that infection of a keratinocyte-derived cell line, A431, with a US3-deficient virus, results in cytopathic effects that are morphologically distinct from those induced by wild-type and revertant viruses, suggesting that US3 PK may be important for interaction between HSV-2 and peripheral epithelial cells.     

NECROSIS OF LUNG EPITHELIAL CELLS BY FILARIAL PARASITIC PROTEIN VIA AN EARLY INDUCTION OF c-H-rasAND TNFα EXPRESSION

The direct interaction of filarial proteins with lung epithelial cells was examined to determine the possible mechanism of inducing cell death, an event that is observed in patients with tropical pulmonary eosinophilia. Exposure of lung epithelial cells to filarial parasitic proteins,Brugia malayi(BmA),Setaria digitata(Sd), and recombinant filarial protein (pGT 7)in vitrofor more than 2 days, causes the appearance of DNA fragments both in the cytoplasm and culture supernatants, while no fragmentation was observed in the untreated controls. The release of DNA fragments both in the cytoplasm and the culture supernatants simultaneously, indicates that cell death is induced by a necrotic event rather than apoptosis. Fluorescent-labelled studies also indicate the fragmentation of DNA increasing in a time-dependent manner. Normal cellular function is controlled through several oncogenes. The modulation of specific proto-oncogenes like myc, ras and TNFα during exposure to filarial parasitic proteins reveal elevated levels of expression of ras and TNFα as early as 2 hours, implicating their involvement prior to DNA fragmentation leading to pathogenesis.     

Flavonoid compound breviscapine suppresses human osteosarcoma Saos‐2 progression property and induces apoptosis by regulating mitochondria‐dependent pathway

This study was aimed to investigate the ability of a flavonoid compound breviscapine (BVP) to suppress growth and elicit apoptosis in human osteosarcoma (OS) Saos‐2 cells. The cells were cultured in vitro and treated with three concentrations of BVP (80, 160, and 320 μg/ml). Moreover, C57 mice were injected with Saos‐2 cells to establish a subcutaneous xenograft model, and they were subsequently treated with three doses of BVP via intraperitoneal injection. The viability of the cells was examined by the Cell Counting Kit‐8 method. The apoptotic cells were assessed by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The tumor volume and weight were monitored from day 3 through day 21 after the last injection. The expression of bax, bcl‐2, and cytochrome c (cyt c) mRNA was detected by a real‐time polymerase chain reaction. The protein levels of bax, bcl‐2, cyt c, caspase 3, and caspase 9 were evaluated by Western blot. The expression and distribution of bcl‐2 and bax in tissues were detected by immunohistochemistry. Compared with the control group, BVP treatment inhibited cell proliferation and induced apoptosis of Saos‐2 cells in vitro. Consistently, treatment of mice bearing transplanted tumors with BVP suppressed the growth of OS tumors and promoted cell apoptosis; it also reduced tumor volume and weight. Mechanistically, BVP‐induced apoptosis was mediated by the mitochondria‐dependent pathway, as evidenced by the increased expression of bax and cyt c and the decreased expression of bcl‐2, as well as activation of caspase 9 and caspase 3 in vitro and in vitro. Collectively, BVP inhibits growth and promotes apoptosis of OS by activating the mitochondrial apoptosis pathway.     

Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions

TB/C3 hybridoma cells were transected with either pEF-MClneopA or pEF bcl2-MClneopA vectors to produce a control cell line (TB/C3 pEF) and a cell line that overexpresses the "antiapoptotic" human bcl-2 protein (TB/C3 bcl2). Flow cytometry analysis of intracellular bcl-2 protein levels enabled near on-line monitoring of the stability of bcl-2 expression in the absence of drug selection. It was possible to maintain spontaneous selection of cells with the overexpression of bcl-2 protein during semicontinuous cultures at very low dilution rates, where cells were subjected to the selective conditions of nutrient limitation and high toxic metabolite concentrations. Interestingly, cells that overexpressed bcl-2 were adapted to suspension culture conditions significantly faster than control cells. Dual fluorescence staining with acridine orange and propidium iodide allowed for discrimination between viable, apoptotic, secondary necrotic, and necrotic cells, respectively. Compared with the usual trypan blue method of establishing culture viability, dual staining demonstrated that under stressful conditions a significant proportion of cells that excluded trypan blue were also undergoing cell death through apoptosis. In batch cultures the overexpression of bcl-2 more than doubled the membrane intact (MI) cell productive period (the integral of Ml cell density with respect to culture time) and increased the monoclonal antibody (mAb) production by approximately 40% when compared with the control cell line. The overexpression of bcl-2 protein also significantly extended the cell integrity and viability by the suppression of apoptosis in conditions of hypoxia, hyperoxia, glutamine deprivation, glucose deprivation, and serum limitation. The suppression of apoptosis in anaerobic conditions suggests that bcl-2 exerts its antiapoptotic activity by a mechanism that does not involve an oxidative reactive pathway. In conditions of excess thymidine, which suppressed cell proliferation, Ml cell density and specific mAb productivity were further enhanced by the overexpression of bcl-2, which suggests the possibility of accomplishing a controlled proliferation in immortalized cell lines without invoking cell death. Cell size and intracellular mAb were increased for TB/C3 bcl2 cells compared with TB/C3 pEF control cells when analyzed by flow cytometry. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 1-16, 1997.     

Apoptotic effect of methanol extract of Picrasma quassioides by regulating specificity protein 1 in human cervical cancer cells

In the present study, we examined the effects of methanol extracts of Picrasma quassioides (MEPQ) on apoptosis in human cervical cancer cells. The results showed that MEPQ decreased the viability and induced caspase‐dependent apoptosis in HEp‐2 cells. MEPQ decreased specificity protein 1 (Sp1) in HEp‐2 cells, whereas Sp1 mRNA was not changed. We found that MEPQ reduced Sp1 protein through proteasome‐dependent protein degradation, but not the inhibition of protein synthesis. Also, MEPQ increased the expressions of Bad and truncated Bid (t‐Bid) but did not alter other Bcl‐2 family members. The knock‐down of Sp1 by both Sp1 interfering RNA and Mithramycin A, Sp1 specific inhibitor clearly increased Bad and t‐Bid expression to decrease cell viability and induce apoptosis. In addition, MEPQ inhibited cell viability and induced apoptotic cell death through the modulation of Sp1 in KB cells. These results suggest that MEPQ may be a potential anticancer agent for human cervical cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

Targeting apoptotic signalling pathway and pro-inflammatory cytokine expression as therapeutic intervention in TPE induced lung damage

Tropical pulmonary eosinophilia (TPE) is an occult manifestation of filariasis, brought about by helminth parasites Wuchereria bancrofti and Brugia malayi. Treatment of patients suffering from TPE involves the administration of diethyl carbamazine and Ivermectin. Although the drugs are able to block acute inflammation, they are not able to alleviate chronic basal inflammation. We have attempted to examine the disease by targeting two important components; namely filarial parasitic sheath proteins (FPP) induced apoptosis and pro-inflammatory cytokine response in human laryngeal carcinoma cells of epithelial origin (HEp-2) cells an epithelial cell line. Earlier studies by us have shown that FPP exposure induced apoptosis in these cells. In this study with hydrocortisone, calpain inhibitor (ALLN) and phorbol myristate acetate (PMA) treatments we demonstrate that apoptosis is inhibited as shown by [3H] thymidine incorporation studies, propidium iodide staining and Annexin V staining. Hydrocortisone at a dose, which inhibits cell death also down regulated, the expression of pro-inflammatory cytokines IL-6 and IL-8. These findings give us insights into the multifaceted approach one may adopt to target critical signalling molecules using appropriate inhibitors, which could eventually be used to reduce lung damage in TPE.     

Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.     

Chloride intracellular channel 1 identified using proteomic analysis plays an important role in the radiosensitivity of HEp‐2 cells via reactive oxygen species production

The nature of the molecules underlying the radioresistance phenotype of laryngeal cancer cells remains to be established. We initially generated radioresistant laryngeal cancer cell lines from human HEp‐2 cells with fractionated radiation. These RR‐HEp‐2 cells and isolated clones displayed more radioresistant and anti‐apoptotic phenotypes than parental HEp‐2 cells after radiation. Characteristics of RR‐Hep‐2 cell lines were confirmed by upregulation of radioresistance‐related genes, such as epidermal growth factor receptor, Hsp90, and Bcl‐xl. Subsequently, we examined proteome changes between HEp‐2 and RR‐HEp‐2 cells and identified 16 proteins showing significantly altered expression levels. Interestingly, protein expression of chloride intracellular channel 1 (CLIC1) was markedly suppressed in RR‐HEp‐2 cells, compared with non‐irradiated control cells. Suppression of CLIC1 with an indanyloxyacetic acid‐94 or small interfering RNA led to radioresistance in HEp‐2 cells by suppressing the radiation‐induced cellular ROS level. However, ectopic overexpression of CLIC1 induced radiosensitivity in RR‐HEp‐2 cells via induction of ROS level after radiation, suggesting that the protein acts as a positive regulator of ROS production. Our results collectively indicate that suppression of CLIC1 contributes to acquisition of the radioresistance phenotype of laryngeal cancer cells via inhibition of ROS production, implying that this protein is an important candidate molecule for radiotherapy in radioresistant laryngeal cancer cells.     

Adhesion of Salmonella dublin to HEp2 epithelial cells

Two strains of Salmonella dublin , grown serially in brain heart infusion broth, were motile and produced mannose sensitive (MS) but not mannose resistant (MR) haemagglutinins; grown on phosphate buffered agar, the strains were poorly motile and phenotypically MSHA^- MRHA ⁺. In adhesion tests with HEp2 epithelial cells, broth grown bacteria that were motile and MSHA⁺ MRHA^- adhered better than agar grown bacteria that were poorly motile and MSHA^- MRHA⁺. Thus, in adhesion tests with HEp2 epithelial cells, strains of S. dublin behaved like S. typhimurium strains in that their HEp2 cell adhesiveness was associated with motility and production of MSHA.     

bcl-2核酶(Ribozyme)促进紫杉醇诱导的细胞凋亡

用核酶技术阻断或降低抗凋亡蛋白 Bcl- 2的表达以促进化疗药物紫杉醇诱导的食管癌细胞凋亡 ,探索克服耐药、提高紫杉醇疗效的新途径 .将特异性切割 Bcl- 2 m RNA的核酶克隆至含MTII启动子并可为 Zn SO4 诱导表达的真核表达载体中 ,通过脂质体转入食管癌鳞状上皮细胞系Eca 1 0 9中 ,经 G41 8筛选得到稳定抗性细胞株 X1 0 9R,挑取单细胞株扩大培养 ,1 40μmol/L Zn SO4诱导 3d,用 Northern- blot、免疫荧光、流式细胞仪鉴定核酶及 Bcl- 2蛋白表达情况 ,用 TUNEL标记及流式细胞术检测凋亡细胞的比例 .bcl- 2核酶在不同单细胞株中有不同程度的表达 ,其中一株X1 0 9R1 4表达最高 .测定其中 Bcl- 2蛋白含量 ,发现 Bcl- 2蛋白表达大为降低 .加入紫杉醇后 ,TUNEL标记及凋亡峰测定结果都表明同一条件下凋亡率升高 .结果提示 ,转入特异性切割 bcl- 2m RNA的核酶可有效地阻断 Bcl- 2蛋白合成 .Bcl- 2蛋白表达降低可明显促进紫杉醇诱导的细胞凋亡 .说明 Bcl- 2蛋白在细胞产生耐药过程中起着重要作用     

光化学法脑缺血后细胞凋亡及其相关基因bcl-2表达的变化

采用ＴＵＮＥＬ染色及免疫组织化学技术对光化学法脑缺血后细胞凋亡及其相关基因ｂｃｌ－２表达的变化进行了研究。结果发现，缺血后１２ｈ，损伤侧皮层缺血区内凋亡细胞数及ｂｃｌ－２免疫反应阳性细胞数明显增加，一直持续至缺血后７２ｈ；并呈现下列时程变化：在缺血后３ｈ每张切片上几乎无凋亡细胞出现，以后逐渐增加，缺血后１２ｈ达到峰值，缺血后２４ｈ和缺血后７２ｈ逐渐减少，但仍高于假手术组水平。凋亡相关基因ｂｃｌ－２的表达在缺血后３ｈ以前不明显，缺血后１２ｈ逐渐增加，缺血后２４ｈ最多，以后逐渐下降。上述结果提示，缺血后凋亡细胞的时程变化可能与缺血后梗塞灶的发生和发展有关，而ｂｃｌ－２表达的变化可能与抑制细胞凋亡、发挥内源性细胞保护作用有关。     

猪水肿病毒素Stx2e的致Vero细胞凋亡作用

摘要：【目的】研究猪水肿病的致病因子志贺毒素2e（Shiga toxin 2e, Stx2e）的致病机理。【方法】以AO/EB荧光染色法、琼脂糖凝胶电泳法和Western blot等方法研究Stx2e对Vero细胞的致凋亡作用及其信号途径。【结果】从细胞形态学和染色质水平证明,Stx2e 能诱导Vero细胞凋亡,并表现出时间和浓度依赖性;同时引起caspase-3表达量明显上调,Bax、caspase-9的表达量没有明显变化。【结论】Stx2e对Vero细胞的致凋亡作用主要通过膜受体通路引起,线粒体信号通路所起的作用较小。     

Bcl-2 inhibits apoptosis and extends recombinant protein production in cells infected with Sindbis viral vectors

Viruses carrying foreign genes are often used for the production of recombinant proteins in mammalian cells and other eukaryotic expression systems. Though high levels of gene expression are possible using viral vectors, the host cell generally responds to the infection by inducing apoptotic cell death within several days, abruptly ending protein production. It has recently been demonstrated, however, that apoptosis can be suppressed in virally infected cells using anti-apoptotic genes, such as bcl-2. In this study, stably transfected rat carcinomal cell lines, AT3-bcl2 and AT3-neo, were infected with a Sindbis virus carrying the gene for chloramphenicol acetyltransferase (CAT) in an effort to determine the effect of bcl-2 on cell viability and recombinant protein production. Infected AT3-bcl2 cells consistently maintained viabilities close to 100% and a growth rate equivalent to that of uninfected cells (0.040 h^-1). In contrast, the Sindbis viral vector induced apoptosis in the AT3-neo cells, which were all dead by three days post-infection. Though infected AT3-neo cells generated higher levels of heterologous protein, over 1000 mUnits per well, CAT activity fell to zero by two days post-infection. In contrast, chloramphenicol acetyltransferase was present in AT3-bcl2 cells for almost a week, reaching a maximum level of 580 mUnits per well. In addition, recombinant protein production in AT3-bcl2 cells was extended and amplified by the regular addition of virus to the culture medium, a process which resulted in expression for the duration of the cell culture process.Abbreviations BHK Baby Hamster Kidney - CAT chloramphenicol acetyltransferase - dsSV-CAT double subgenomic Sindbis viral vector containing the gene for CAT - MOI multiplicity of infection