Apoptosis in dengue virus infected liver cell lines HepG2 and Hep3B

While both in vivo and in vitro evidence has suggested that liver cells undergo apoptosis in response to dengue virus infection, little is known about the mechanism of induction. Given that the p53 tumour suppressor gene is a key mediator of apoptosis, we sought to define the role of this gene in response to dengue virus infection. After infection, a p53 wild type liver cell line (HepG2) showed changes consistent with apoptosis including alterations of cell morphology, cellular detachment and DNA laddering. However, p53 was neither up-regulated, nor showed any evidence of complexing with dengue virus proteins as determined by immunoprecipitation. Infection of a p53 null liver cell line (Hep3B) also produced changes consistent with the induction of apoptosis. While the profile of the cells undergoing apoptosis in each cell line was similar as determined by flow cytometry, the absolute levels were markedly different with up to 90% of Hep3B cells undergoing apoptosis compared to only 20% of HepG2 cells at day 5 post infection. By day 7, all Hep3B infected cells were dead. In contrast, it proved possible to culture dengue virus infected HepG2 cells for 3 months. Viral progeny released from the p53 null cell line were nine-fold higher per attached cell than from the p53 wild type cell line. These results suggest that, while induction of apoptosis in liver cells is mediated by a non-p53 regulated pathway, p53 may play a role in restricting the level of viral progeny to below a critical level at which apoptosis is triggered.     

Gal‐3 is stimulated by gain‐of‐function p53 mutations and modulates chemoresistance in anaplastic thyroid carcinomas

Galectin‐3 (Gal‐3) is an anti‐apoptotic molecule of the β‐galactoside‐binding lectin family. Gal‐3 is down‐regulated by wt‐p53 and this repression is required for p53‐induced apoptosis. Since poorly differentiated thyroid carcinomas (PDTCs) and anaplastic thyroid carcinomas (ATCs) frequently harbour p53 mutations, we asked whether Gal‐3 expression and activity could be influenced by such mutations in these tumours. We found a positive correlation between Gal‐3 expression and p53 mutation in human thyroids and in thyroid carcinoma cell lines (TCCLs) harbouring different p53 mutations. Gal‐3 was over‐expressed in most ATCs and TCCLs, especially those with the most frequently detected p53 mutation (p53^R273H). Over‐expression of p53^R273H in two p53‐null cells (SAOS‐2 and SW‐1736) as well as in two wt‐p53‐carrying TCCLs (TPC‐1 and K1), stimulated Gal‐3 expression, while interference with p53^R273H endogenous expression in ARO cells down‐regulated Gal‐3 expression. Conversely, over‐expression of wt‐p53 in ARO cells restored the inhibitory effect on Gal‐3 expression. ARO cells are highly resistant to apoptosis and express both p53 and Gal‐3, which are increased upon cisplatin treatment. Interference with Gal‐3 expression in these cells stimulated their chemosensitivity. In conclusion, gain‐of‐function p53 mutant acquires the de novo ability to stimulate Gal‐3 expression and to increase chemoresistance in ATCs. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

miR-183 promotes radioresistance of lung adenocarcinoma H1299 cells via epithelial-mesenchymal transition

Lung adenocarcinomas are usually sensitive to radiation therapy, but some develop resistance. Radiation resistance can lead to poor patient prognosis. Studies have shown that lung adenocarcinoma cells (H1299 cells) can develop radioresistance through epithelial-mesenchymal transition (EMT), and this process is regulated by miRNAs. However, it is unclear which miRNAs are involved in the process of EMT. In our present study, we found that miR-183 expression was increased in a radioresistant lung adenocarcinoma cell line (H1299R cells). We then explored the regulatory mechanism of miR-183 and found that it may be involved in the regulation of zinc finger E-box-binding homeobox 1 (ZEB1) expression and mediate EMT in lung adenocarcinoma cells. qPCR results showed that miR-183, ZEB1, and vimentin were highly expressed in H1299R cells, whereas no difference was observed in E-cadherin expression. Western blot results showed that ZEB1 and vimentin were highly expressed in H1299R cells, while E-cadherin expression was decreased. When miR-183 expression was inhibited in H1299R cells, radiation resistance, proliferation, and cell migration were decreased. The expression of ZEB1 and vimentin in H1299R cells was decreased, while the expression of E-cadherin was increased. Moreover, miR-183 overexpression in H1299 cells enhanced radiation resistance, proliferative capacity, and cell migration ability. The expression of ZEB1 and vimentin in H1299 cells was increased, while that of E-cadherin was decreased. In conclusion, miR-183 may promote EMT and radioresistance in H1299 cells, and targeting the miR-183-ZEB1 signaling pathway may be a promising approach for lung cancer treatment.     

Cell cycle arrest and apoptosis induced by the coronavirus infectious bronchitis virus in the absence of p53

Manipulation of the cell cycle and induction of apoptosis are two common strategies used by many viruses to regulate their infection cycles. In cells infected with coronaviruses, cell cycle perturbation and apoptosis were observed in several reports. However, little is known about how these effects are brought out, and how manipulation of the functions of host cells would influence the replication cycle of coronavirus. In this study, we demonstrate that infection with coronavirus infectious bronchitis virus (IBV) imposed a growth-inhibitory effect on cultured cells by inducing cell cycle arrest at S and G(2)/M phases in both p53-null cell line H1299 and Vero cells. This cell cycle arrest was catalyzed by the modulation of various cell cycle regulatory genes and the accumulation of hypophosphorylated RB, but was independent of p53. Proteasome inhibitors, such as lactacystin and NLVS, could bypass the IBV-induced S-phase arrest by restoring the expression of corresponding cyclin/Cdk complexes. Our data also showed that cell cycle arrest at both S- and G(2)/M-phases was manipulated by IBV for the enhancement of viral replication. In addition, apoptosis induced by IBV at late stages of the infection cycle in cultured cells was shown to be p53-independent. This conclusion was drawn based on the observations that apoptosis occurred in both IBV-infected H1299 and Vero cells, and that IBV infection did not affect the expression of p53 in host cells.     

FasL/Fas pathway is involved in dengue virus induced apoptosis of the vascular endothelial cells

The hallmark of the dengue hemorrhagic fever/dengue shock syndrome is hematologic abnormality. The pathogenesis of dengue hemorrhagic fever/dengue shock syndrome remains unknown. Our work showed that the dengue virus serotype‐2 induced apoptosis in human umbilical vein endothelial cells. Fas (CD95), Tumor Necrosis Factor receptors, and Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors are the most common death receptors, which can induce apoptosis. Compared with the untreated human umbilical vein endothelial cells, Fas expression was increased both in the mRNA level and on the surface of infected human umbilical vein endothelial cells. FasL was expressed at similar levels on human umbilical vein endothelial cells over a course of dengue virus serotype‐2 infection, but the expression in mRNA level was increased in infected human umbilical vein endothelial cells. It is possible that there is soluble FasL secreted from human umbilical vein endothelial cells in the supernatant. Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptor 1 and Tumor Necrosis Factor receptors 1–2 were constantly very low, whereas Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors 2–4 decreased after dengue virus serotype‐2 infection. This result suggested that dengue virus serotype‐2 may inhibit Tumor Necrosis Factor‐related apoptosis‐inducing ligand receptors‐induced apoptosis. The apoptotic rates in human umbilical vein endothelial cells were decreased upon the addition of caspase family inhibitors. In addition, activated caspase 8 and caspase 3 were also observed by Western blot following dengue virus serotype‐2 infection. Thus, it is shown that the Fas/FasL pathway may participate in dengue virus‐induced apoptosis of vascular endothelial cells in vitro. J. Med. Virol. 82:1392–1399, 2010. © 2010 Wiley‐Liss, Inc.     

Primary human endothelial cells support direct but not antibody‐dependent enhancement of dengue viral infection

Microvascular plasma leakage is the hallmark of dengue hemorrhagic fever and dengue shock syndrome. The precise molecular mechanisms leading to microvascular leakage are yet to be determined, but dengue virus (DENV) infection and consequent endothelial cell death has been suggested as its major cause. However, the extent of endothelial cell permissiveness to DENV infection and the magnitude of cell death following DENV infection are controversial. To clarify this issue, we analyzed the kinetics and consequences of DENV infection of human umbilical vein endothelial cells (HUVEC) using a novel molecularly cloned DENV2‐16681 virus. Viral replication was detected as early as 24 hr post‐infection by RT‐PCR and plaque assays. However, merely 2% of HUVEC were DENV antigen‐positive even after 96 hr of infection as measured by the FACS indirect immunofluorescence assays. Unlike monocytes/macrophages, HUVEC did not support antibody dependent enhancement of dengue viral infection due to a lack of FcγRI and FcγRII. Furthermore, DENV infection did not increase HUVEC apoptosis as compared to mock‐infected cells. Because in vitro only a small percentage of endothelial cells were productively infected in vitro with no significant apoptosis occurring in either infected or bystander cells, it would be important to re‐examine whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage observed in patients with dengue hemorrhagic fever and dengue shock syndrome. J. Med. Virol. 81:519–528, 2009. © 2009 Wiley‐Liss, Inc.     

Genome‐wide transcriptome analyses reveal p53 inactivation mediated loss of miR‐34a expression in malignant peripheral nerve sheath tumours

Malignant peripheral nerve sheath tumours (MPNSTs) are aggressive soft tissue tumours that occur either sporadically or in patients with neurofibromatosis type 1. The malignant transformation of the benign neurofibroma to MPNST is incompletely understood at the molecular level. We have determined the gene expression signature for benign and malignant PNSTs and found that the major trend in malignant transformation from neurofibroma to MPNST consists of the loss of expression of a large number of genes, rather than widespread increase in gene expression. Relatively few genes are expressed at higher levels in MPNSTs and these include genes involved in cell proliferation and genes implicated in tumour metastasis. In addition, a gene expression signature indicating p53 inactivation is seen in the majority of MPNSTs. Subsequent microRNA profiling of benign and malignant PNSTs indicated a relative down‐regulation of miR‐34a in most MPNSTs compared to neurofibromas. In vitro studies using the cell lines MPNST‐14 (NF1 mutant) and MPNST‐724 (from a non‐NF1 individual) show that exogenous expression of p53 or miR‐34a promotes apoptotic cell death. In addition, exogenous expression of p53 in MPNST cells induces miR‐34a and other miRNAs. Our data show that p53 inactivation and subsequent loss of expression of miR‐34a may significantly contribute to the MPNST development. Collectively, our findings suggest that deregulation of miRNAs has a potential role in the malignant transformation process in peripheral nerve sheath tumours. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

重组腺病毒介导的p^16与p^53基因联合转移对人肺癌细胞系 …

目的 探讨ｐ＾１６和ｐ＾５３基因对肺癌细胞的协同抑制效应及凋亡诱导作用。方法 首先用同源重组技术构建重组ｐ＾１６和ｐ＾５３腺病毒载体,然后单独或联合感染人肺癌细胞系Ｈ３５８,用免疫组织法及Ｗｅｓｔｅｒｎ ｂｌｏｔ检测腺病毒介导的基因转移效率与表达水平,用克隆形成实验、原位末端标记及流式术观察它们对Ｈ３５８生长特性及凋亡的影响,结果 免疫组织化学染色结果表明重组腺病毒载体可高效地将外源基因ｐ＾５３转     

Dramatic caspase-dependent apoptosis in antibody-enhanced dengue virus infection of human mast cells

Severe forms of dengue virus disease, known as dengue hemorrhagic fever and dengue shock syndrome, result from an aberrant immune response involving antibody-dependent enhancement of infection, thrombocytopenia, and a loss of vascular integrity, culminating in hemorrhage, shock, and in some cases, death. Several studies have indicated that dengue virus infection results in the induction of apoptosis of certain cells believed to be contributory players in dengue pathogenesis. However, none have specifically examined the role of antibody enhancement in the context of induction of apoptosis. Here, we show that antibody-enhanced dengue virus infection of the FcR-bearing mast cell/basophil KU812 cell line results in a massive induction of apoptosis. Confocal microscopy and flow cytometry indicate two distinct subpopulations consisting of productively infected cells and apoptotic-uninfected bystanders. Apoptosis was found to be caspase-dependent, involving global caspase activation and cleavage of poly-ADP-ribose polymerase (PARP) and D4-guanosine diphosphate dissociation inhibitor (D4-GDI). Additional FcR-bearing cells, including K562, U937, and human mast cell 1 (HMC-1), were analyzed for apoptosis induction following infection. Although all cells displayed high susceptibility to antibody-enhanced dengue virus infection, only cells of a mast cell phenotype (KU812 and HMC-1) were found to undergo apoptosis. Dengue-induced apoptosis of KU812 cells was shown to require antibody-enhanced dengue virus infection by blockade of FcgammaRII. Transfection of KU812 cells with L-SIGN/DC-SIGNR was able to overcome the requirement for antibody enhancement with regard to dengue virus infection and apoptosis.     

Reversal of P‐glycoprotein‐mediated multi‐drug resistance by the E3 ubiquitin ligase Cbl‐b in human gastric adenocarcinoma cells

P‐glycoprotein (P‐gp)‐mediated multi‐drug resistance (MDR) is a major barrier to the effective chemotherapy of many cancers. Recent studies have shown that inhibition of the PI3K/Akt signalling pathway can reverse P‐gp‐mediated MDR. We investigated the expression of activated Akt (p‐Akt) in 124 human gastric carcinoma tissue samples. Ubiquitous p‐Akt expression was recorded in the majority (88/124). There was a significant correlation between p‐Akt expression and the expression of P‐gp. In the adriamycin‐resistant MDR gastric carcinoma cell line SGC7901/ADR, p‐Akt expression was increased in comparison with the parental cell line SGC7901. Treatment of SGC7901/ADR cells with the PI3K inhibitor LY294002 reduced the expression of both p‐Akt and P‐gp. To explore the role of ubiquitin ligase Cbl‐b in this regulatory pathway, SGC7901/ADR cells were transfected with a plasmid overexpressing wild‐type Cbl‐b. This down‐regulated the expression of both p‐Akt and P‐gp. Furthermore, resistance to chemotherapeutic drugs was partially reversed. These results demonstrate an important role for Cbl‐b in reversing P‐gp‐mediated gastric cancer MDR through suppression of the PI3K/Akt signalling pathway and the down‐regulation of P‐gp expression. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Regulation of cathepsin X overexpression in H. pylori‐infected gastric epithelial cells and macrophages

Cathepsin X (CTSX) is strongly up‐regulated in Helicobacter pylori‐infected gastric mucosa and intestinal‐type gastric cancer. The overexpression of CTSX is mediated predominantly by associated macrophages; depends on a functional type IV‐secretion system; and leads to increased migration of gastric epithelial cells. In the present study, we analysed the role of CagA in CTSX overexpression and identified H. pylori‐induced inflammatory factors and signalling pathways required for stimulating CTSX expression by H. pylori. Gastric epithelial cells were co‐cultured with macrophages in Transwell chambers of 0.4 µm pore size, enabling exchange of fluids but retracting H. pylori. N87 gastric epithelial cells were infected with H. pylori P1 wild‐type strain in the presence of inhibitors for p38, JNK, and ERK1/2 signal transduction pathways. Furthermore, cytokines and growth factors were tested for their regulatory function using inhibitory antibodies, and their gene expression was studied by quantitative RT‐PCRs and western blots. CTSX is strongly up‐regulated at both the mRNA and the protein levels by TNF‐α, IL‐1β, IL‐6, and IL‐8, depending on cell type. All these cytokines were found to be increased by five‐ to ten‐fold in macrophages by H. pylori infection of co‐cultured N87 gastric epithelial cells. In macrophages, H. pylori up‐regulated CTSX via ERK1/2 signalling pathways, and in N87 cells via JNK irrespective of p38 signalling. Our results suggest that H. pylori induced overexpression of CTSX in macrophages and epithelium through specific cytokines that are initiated by CagA‐dependent pathways in a cell type‐dependent manner. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Heterogeneity of Nef proteins in cells infected with human immunodeficiency virus type 1

Human T-lymphocytic cell line H9 infected with the HTLV-IIIB isolate of human immunodeficiency virus type 1 (HIV-1) synthesizes two forms of the Nef protein (p25 and p27) that differ both in molecular weight and charge. Different subpopulations of viruses were isolated from the HTLV-IIIB stock which induce expression of only p25 or p27. Cells infected with HIV-1 derived from the HXB3 clone of the HTLV-IIIB isolate made only the p25 species, whereas the 8E5/LAV cell line which harbors a single defective LAV provirus produces only the p27 species. These findings are consistent with the notion that the HTLV-IIIB isolate consists of at least two distinct variants with different nef genes, one specifying p25 and the other encoding p27. After a considerable number of passages in culture, H9 cells chronically infected with the HTLV-IIIB isolate produced high levels of p25 and lower levels of p27. Passages in culture appear to select for a subpopulation of virus variants that specify high levels of p25 Nef expression.     

Influenza and dengue virus co‐infection impairs monocyte recruitment to the lung,increases dengue virus titers,and exacerbates pneumonia

Co‐infections of influenza virus and bacteria are known to cause severe disease, but little information exists on co‐infections with other acute viruses. Seasonal influenza and dengue viruses (DENV) regularly co‐circulate in tropical regions. The pandemic spread of influenza virus H1N1 (hereafter H1N1) in 2009 led to additional severe disease cases that were co‐infected with DENV. Here, we investigated the impact of co‐infection on immune responses and pathogenesis in a new mouse model. Co‐infection of otherwise sublethal doses of a Nicaraguan clinical H1N1 isolate and two days later with a virulent DENV2 strain increased systemic DENV titers and caused 90% lethality. Lungs of co‐infected mice carried both viruses, developed severe pneumonia, and expressed a unique pattern of host mRNAs, resembling only partial responses against infection with either virus alone. A large number of monocytes were recruited to DENV‐infected but not to co‐infected lungs, and depletion and adoptive transfer experiments revealed a beneficial role of monocytes. Our study shows that co‐infection with influenza and DENV impairs host responses, which fail to control DENV titers and instead, induce severe lung damage. Further, our findings identify key inflammatory pathways and monocyte function as targets for future therapies that may limit immunopathology in co‐infected patients.     

TRX is up‐regulated by fibroblast growth factor‐2 in lung carcinoma

Deng Z‐H, Cao H‐Q, Hu Y‐B, Wen J‐F, Zhou J‐H. TRX is up‐regulated by fibroblast growth factor‐2 in lung carcinoma. APMIS 2010. We have previously shown that exogenous fibroblast growth factor‐2 (FGF‐2) inhibits apoptosis of the small‐cell lung cancer (SCLC) cell line NCI‐H446, but the underlying mechanism remains unknown. In this study, the protein profiles of FGF‐2‐treated and untreated NCI‐H446 cells were determined by 2‐D gel electrophoresis combined with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and bioinformatics. Differential expression analysis of the protein profiles after FGF‐2 treatment identified a total of 24 protein spots, of which nine were up‐regulated and 15 were down‐regulated. Four proteins were identified by MALDI‐TOF‐MS: thioredoxin (TRX), visfatin, ubiquitin carboxyl‐terminal hydrolase L1 (UCHL1) and Cu/Zn superoxide dismutase (CuZn‐SOD). Western blotting revealed that TRX was up‐regulated in NCI‐H446 and A549 cells treated with FGF‐2. Furthermore, immunohistochemical staining confirmed that both FGF‐2 and TRX were overexpressed in lung cancer tissues and could be correlated with both lymph node metastasis and clinical stage. These data indicate that TRX may be involved in the FGF‐2 signaling pathway.     

Caveolin‐1 is an Important Factor for the Metastasis and Proliferation of Human Small Cell Lung Cancer NCI‐H446 Cell

Caveolin‐1 (CAV‐1) has been reported to play an important role in the development of a variety of human cancers. CAV‐1 expression is revealed to be reduced or absent in the malignant tumor cells of small cell lung cancers (SCLC). This study was performed to investigate the influences of the stable expression of CAV‐1 on the metastasis and proliferation of SCLC in vitro. The wild‐type CAV‐1 gene was successfully transfected into the NCI‐H446 cells and was stably expressed in the NCI‐H446 cells. The effects of CAV‐1 on the morphology, proliferation, and metastasis potential for NCI‐H446 cell were evaluated by crystal violet staining, MTT analysis, transwell assay, and scratch wound assay, respectively. Western blot and gelatin zymography were used to examine the expression changes of the metastasis‐related MMP‐3 and E‐cadherin. Stable expression of CVA‐1 was observed in the H446‐CAV‐1 cells, which enlarged the cell shape with filopodia. The proliferation of H446‐CAV‐1 was inhibited, while its migration and invasion abilities were promoted in vitro. The re‐expression of CAV‐1 reduced the expression of E‐cadherin, while it increased the protein expression and enzyme activity of MMP‐3. Taken together, the cellular proliferation of the NCI‐H446 could be inhibited by the re‐expression of CAV‐1. CAV‐1 might increase the cell metastasis potential through the interaction with E‐cadherin and MMP‐3 genes. These in vitro findings confirm the involvement of CAV‐1 in the proliferation and metastasis of SCLC. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.