Role of p38 MAPK and RNA-dependent protein kinase (PKR) in hepatitis C virus core-dependent nuclear delocalization of cyclin B1

Some hepatitis C virus (HCV) proteins, including core protein, deregulate the cell cycle of infected cells, thereby playing an important role in the viral pathogenesis of HCC. Thus far, there are only few studies that have deeply investigated in depth the effects of the HCV core protein expression on the progression through the G1/S and G2/M phases of the cell cycle. To shed light on the molecular mechanisms by which the HCV core protein modulates cell proliferation, we have examined its effects on cell cycle in hepatocarcinoma cells. We show here that HCV core protein perturbs progression through both the G1/S and the G2/M phases, by modulating the expression and the activity of several cell cycle regulatory proteins. In particular, our data provided evidence that core-dependent deregulation of the G1/S phase and its related cyclin-CDK complexes depends upon the ERK1/2 pathway. On the other hand, the viral protein also increases the activity of the cyclin B1-CDK1 complex via the p38 MAPK and JNK pathways. Moreover, we show that HCV core protein promotes nuclear import of cyclin B1, which is affected by the inhibition of both the p38 and the RNA-dependent protein kinase (PKR) activities. The important role of p38 MAPK in regulating G2/M phase transition has been previously documented. It is becoming clear that PKR has an important role in regulating both the G1/S and the G2/M phase, in which it induces M phase arrest. Based on our model, we now show, for the first time, that HCV core expression leads to deregulation of the mitotic checkpoint via a p38/PKR-dependent pathway.     

Complex formation between hepatitis C virus core protein and p21Waf1/Cip1/Sdi1

The core protein (Core) of hepatitis C virus (HCV) has been known to play an important role in hepatocarcinogenesis. By using glutathione S-transferase (GST) pull-down assay, we show here that Core formed a complex with p21Waf1/Cip1/Sdi1 (p21) cell cycle regulator. The deletion-mapping analysis revealed that a portion near the N-terminus of Core (amino acids 24-52) and a C-terminal portion of p21 (amino acids 139-164) were involved in the complex formation. The complex formation was not impaired by point mutations of p21 at residues 147, 149, and 150, which have been reported to abrogate interaction of p21 with proliferating cell nuclear antigen (PCNA), discriminating the Core-binding sequence from the PCNA-binding sequence. Due to the close vicinity of the binding sites, however, Core and PCNA competed with each other when interacting with p21. The distinct interaction between Core and p21 may provide a new aspect to the studies of HCV pathogenesis.     

The Akt/GSK-3β pathway mediates flurbiprofen-induced neuroprotection against focal cerebral ischemia/reperfusion injury in rats

The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.     

Cell cycle-dependent expression of phosphorylated histone H2AX: reduced expression in unirradiated but not X-irradiated G1-phase cells

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (gammaH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in gammaH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced gammaH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of gammaH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of gammaH2AX. With the exception of xrs5 cells, gammaH2AX level was approximately 3 times lower in unirradiated G(1)-phase cells than S- and G(2)-phase cells, and the slope of the G(1)-phase dose-response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G(1)-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5-10-fold higher levels of gammaH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of gammaH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G(2)-phase cells.     

Cell cycle progression is associated with distinct patterns of phosphorylation of Op18.

Op18 is a highly conserved major cytosolic phosphoprotein which has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. Following mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study we have characterized Op18 phosphorylation during progression of freshly isolated PBL through the cell cycle. Transition from G0 to G1 following activation with OKT3 was associated with an increase in a phosphorylated form designated Op18c. Progression of cells through G1 into S resulted in an increase in phosphorylated Op18 forms, designated Op18a and Op18b, which paralleled new Op18 synthesis. Transition of cells into G2 + M resulted in the appearance of the more acidic phosphorylated forms Op18d and Op18e. Calphostin C, a specific inhibitor of protein kinase C, dramatically decreased all forms of phosphorylated Op18 in OKT3 treated Jurkat cells. Our results suggest that Op18 phosphorylation is mediated in part by PKC activation as well as by other kinases yielding different phosphorylated forms at specific stages of the cell cycle.     

Caffeine inhibits cell proliferation and regulates PKA/GSK3β pathways in U87MG human glioma cells

Caffeine is the most commonly ingested methylxanthine and has anti-cancer effects in several types of cancer. In this study, we examined the anti-cancer effects of caffeine on gliomas, both in vitro and in vivo. In vitro, caffeine treatment reduced glioma cell proliferation through G(0)/G(1)-phase cell cycle arrest by suppressing Rb phosphorylation. In addition, caffeine induced apoptosis through caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. Caffeine also phosphorylated serine 9 of glycogen synthase kinase 3 beta (GSK3β). Pretreatment with H89, a pharmacological inhibitor of protein kinase A (PKA), was able to antagonize caffeine-induced GSK3β(ser9) phosphorylation, suggesting that the mechanism might involve a cAMP-dependent PKA-dependent pathway. In vivo, caffeine-treated tumors exhibited reduced proliferation and increased apoptosis compared with vehicle-treated tumors. These results suggest that caffeine induces cell cycle arrest and caspase-dependent cell death in glioma cells, supporting its potential use in chemotherapeutic options for malignant gliomas.     

丙型肝炎病毒功能基因在CHO细胞中的表达研究

丙型肝炎病毒(HCV)与宿主细胞因子的相互作用已经成为国内外研究的热点和难点。近期研究已经证实HCV的感染对宿主多种途径中基因的转录均能产生影响。为了进一步研究究竟是HCV中的哪些功能基因在与特定细胞因子的相互作用中起主导作用,构建了分别含有HCV Core、E1、E2、p7、NS2、NS3、NS4A、NS4B、NS5A和NS5B基因的真核表达质粒,分别转入宿主细胞CHO-K1中,在G418的选择压力下筛选获得稳定表达HCV单个蛋白的细胞系(10株)。PCR和RT-PCR可分别从稳定细胞系中检测到相应的HCV基因的DNA和mRNA,冻存和复苏不会造成HCV基因的丢失。Western-blot检测到稳定细胞系中表达E1,E2和NS5B蛋白,说明HCV基因在CHO-K1中已经形成稳定表达。薄层层析(TLC)结果显示,含有不同HCV基因的稳定传代细胞系中,UDP-葡萄糖神经酰胺葡萄糖基转移酶(UGCG)活性均发生了不同程度的变化,其中E2和p7的表达使胞内UGCG的活性提高了约1倍,NS2和NS5A则使UGCG的酶活提高了约0.6倍。该稳定细胞系的建立为研究病毒与宿主因子的相互作用及药物筛选奠定了基础。     

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.     

Bone morphogenetic protein-7 and interferon-alpha synergistically suppress hepatitis C virus replicon

Various cytokines contribute to control hepatitis C virus (HCV) viral replication. HCV subgenomic replicon systems have been developed, and cell-cycle-dependent replication has been reported. But the molecules involved in this processes is not totally elucidated. The aim of this study is to investigate the involvement of the bone morphogenetic protein (BMP)-7, a member of TGF-beta superfamily, to the in vitro HCV replication. BMP-7 dose-dependently suppressed the replication and protein expression from the HCV replicon in Huh7/Rep-Feo cells and was associated with cell-cycle arrest at the G1 phase. These results were consistent with the effect of TGF-beta in a previous study. Combination of BMP-7 and interferon-alpha showed a synergic decrease of HCV replication, and was more effective compared to the treatment with interferon-alpha alone. This synergistic effect was also present in HCV-JFH1 virus cell culture. While BMP-7 alone did not stimulate expression of the interferon-stimulated genes (ISGs), it augmented interferon-induced expression of the ISGs independently of the interferon-induced Jak/STAT pathway. Taken together, BMP-7 may constitute a novel molecule to suppress HCV replication.     

Hyaluronan suppresses prostate tumor cell proliferation through diminished expression of N-cadherin and aberrant growth factor receptor signaling

Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and β-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell–cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.     

Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells

Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1 mM) of butyrate had no effect on cell proliferation. However, at 5 and 10 mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10 mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10 mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.     

Different responses of two highly permissive cell lines upon HCV infection

The construction of the first infectious clone JFH-1 speeds up the research on hepatitis C virus (HCV). However, Huh7 cell line was the only highly permissive cell line for HCV infection and only a few clones were fully permissive. In this study, two different fully permissive clones of Huh7 cells, Huh7.5.1 and Huh7-Lunet-CD81 (Lunet-CD81) cells were compared for their responses upon HCV infection. The virus replication level was found slightly higher in Huh7.5.1 cells than that in Lunet-CD81 cells. Viability of Huh7.5.1 cells but not of Lunet-CD81 cells was reduced significantly after HCV infection. Further analysis showed that the cell cycle of infected Huh7.5.1 cells was arrested at G1 phase. The G1/S transition was blocked by HCV infection in Huh7.5.1 cells as shown by the cell cycle synchronization analysis. Genes related to cell cycle regulation was modified by HCV infection and gene interaction analysis in GeneSpring GX in Direct Interactions mode highlighted 31 genes. In conclusion, the responses of those two cell lines were different upon HCV infection. HCV infection blocked G1/S transition and cell cycle progress, thus reduced the cell viability in Huh7.5.1 cells but not in Lunet-CD81 cells. Lunet-CD81 cells might be suitable for long term infection studies of HCV.     

Hepatitis C Virus Core Protein Inhibits Interferon Production by a Human Plasmacytoid Dendritic Cell Line and Dysregulates Interferon Regulatory Factor-7 and Signal Transducer and Activator of Transcription (STAT) 1 Protein Expression

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.