Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatitis C virus (HCV) is a positive-stranded RNA virus that causes severe liver diseases, such as cirrhosis and hepatocellular carcinoma. HCV uses an RNA-dependent RNA polymerase to replicate its genome and an internal ribosomal entry site to translate its proteins. HCV infection is characterized by an increase in the concentrations of reactive oxygen species (ROS), the effect of which on HCV replication has yet to be determined. In this report, we investigated the effect of ROS on HCV replication, using a bicistronic subgenomic RNA replicon and a genomic RNA that can replicate in human hepatoma cells. The treatment with peroxide at concentrations that did not deplete intracellular glutathione or induce cell death resulted in significant decreases in the HCV RNA level in the cells. This response could be partially reversed by the antioxidant N-acetylcysteine. Further studies indicated that such a suppressive response to ROS was not due to the suppression of HCV protein synthesis or the destabilization of HCV RNA. Rather, it occurred rapidly at the level of RNA replication. ROS appeared to disrupt active HCV replication complexes, as they reduced the amount of NS3 and NS5A in the subcellular fraction where active HCV RNA replication complexes were found. In conclusion, our results show that ROS can rapidly inhibit HCV RNA replication in human hepatoma cells. The increased ROS levels in hepatitis C patients may therefore play an important role in the suppression of HCV replication.
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PMID:Reactive oxygen species suppress hepatitis C virus RNA replication in human hepatoma cells. 1475 26

Chronic infection by hepatitis C virus (HCV) can lead to severe hepatitis and cirrhosis and is closely associated with hepatocellular carcinoma. The replication cycle of HCV is poorly understood but is likely to involve interaction with host factors. In this report, we show that NS5B, the HCV RNA-dependent RNA polymerase (RdRp), interacts with a human RNA helicase, p68. Transient expression of NS5B alone, as well as the stable expression of all the nonstructural proteins in a HCV replicon-bearing cell line (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Science 285:110-113), causes the redistribution of endogenous p68 from the nucleus to the cytoplasm. Deletion of the C-terminal two-thirds of NS5B (NS5BDeltaC) dramatically reduces its coimmunoprecipitation (co-IP) with endogenous p68, while the deletion of the N-terminal region (NS5BDeltaN1 and NS5BDeltaN2) does not affect its interaction with p68. In consistency with the co-IP results, NS5BDeltaC does not cause the relocalization of p68 whereas NS5BDeltaN1 does. With a replicon cell line, we were not able to detect a change in positive- and negative-strand synthesis when p68 levels were reduced using small interfering RNA (siRNA). In cells transiently transfected with a full-length HCV construct, however, the depletion (using specific p68 siRNA) of endogenous p68 correlated with a reduction in the transcription of negative-strand from positive-strand HCV RNA. Overexpression of NS5B and NS5BDeltaN1, but not that of NS5BDeltaC, causes a reduction in the negative-strand synthesis, indicating that overexpressed NS5B and NS5BDeltaN1 sequesters p68 from the replication complexes (thus reducing their replication activity levels). Identification of p68 as a cellular factor involved in HCV replication, at least for cells transiently transfected with a HCV expression construct, is a step towards understanding HCV replication.
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PMID:Cellular RNA helicase p68 relocalization and interaction with the hepatitis C virus (HCV) NS5B protein and the potential role of p68 in HCV RNA replication. 1511 10

Parvovirus B19 (B19 virus) can persist in multiple tissues and has been implicated in a variety of diseases, including acute fulminant liver failure. The mechanism by which B19 virus induces liver failure remains unknown. Hepatocytes are nonpermissive for B19 virus replication. We previously reported that acute fulminant liver failure associated with B19 virus infection was characterized by hepatocellular dropout. We inoculated both primary hepatocytes and the hepatocellular carcinoma cell line Hep G2 with B19 virus and assayed for apoptosis by using annexin V staining. Reverse transcriptase PCR analysis and immunofluorescence demonstrated that B19 virus was able to infect the cells and produce its nonstructural protein but little or no structural capsid protein. Infection with B19 virus induced means of 28% of Hep G2 cells and 10% of primary hepatocytes to undergo apoptosis, which were four- and threefold increases, respectively, over background levels. Analysis of caspase involvement showed that B19 virus-inoculated cultures had a significant increase in the number of cells with active caspase 3. Inhibition studies demonstrated that caspases 3 and 9, but not caspase 8, are required for B19 virus-induced apoptosis.
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PMID:Parvovirus B19-induced apoptosis of hepatocytes. 1522 Apr 51

To determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3' noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3'NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3.
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PMID:Role of La autoantigen and polypyrimidine tract-binding protein in HCV replication. 1582 7

Vascular endothelial growth factor (VEGF) activity is correlated with a progressive tumor disease in patients with hepatocellular carcinoma (HCC). In spite of the well-recognized role of VEGF in HCC, there are few data available regarding therapeutic strategies to block VEGF activity. Therefore, we employed a recombinant adenoviral vector encoding a soluble dominant negative fragment of VEGF receptor 2 (Flk-1), AdsFlk-1, to control pre-established murine orthotopic and metastatic hepatomas. Vector function was confirmed via reverse-transcriptase polymerase chain reaction and ELISA, and angiostatic effects were analyzed in vitro and in vivo. Antitumoral effects of systemic AdsFlk-1 application were studied in a subcutaneous and orthotopic Hepa129 HCC model. Cell supernatant containing the truncated form of Flk-1 had no direct effect on cell proliferation of Hepa129 cells in vitro but reduced endothelial tube formation on matrigel matrix by approximately 80% in vitro. Endothelial-like cell infiltration into matrigel plugs in vivo was also decreased by 80%. Systemic treatment of tumor-bearing mice inhibited tumor growth by 84% compared with the corresponding control group within 16 days after vector application. Likewise, the survival rate was significantly improved in the AdsFlk-1 group compared with control. Orthotopic tumor growth was reduced by 82%, and development of malignant ascites was also retarded. In conclusion, systemic adenoviral-mediated gene transfer of an Flk-1 fragment significantly inhibited tumor growth in orthotopic and metastatic murine HCC. The data support the value of VEGF blockade as an effective target for HCC treatment.
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PMID:Effective angiostatic treatment in a murine metastatic and orthotopic hepatoma model. 1591 56

The retinoblastoma tumor-suppressor protein (Rb) plays a critical role in controlling cellular proliferation and apoptosis by regulating E2F transcription factors. Rb is a key target of oncoproteins expressed by DNA tumor viruses, but RNA viruses are not known to regulate Rb function. Here, we show that Rb abundance is negatively regulated in cells containing replicating genomic RNA from hepatitis C virus, a human virus strongly associated with hepatocellular carcinoma. The viral RNA-dependent RNA polymerase NS5B forms a complex with Rb, targeting it for degradation and resulting in reduction of Rb abundance, activation of E2F-responsive promoters, and cell proliferation. NS5B contains a conserved Leu-x-Cys/Asn-x-Asp motif that is homologous to Rb-binding domains in the oncoproteins of DNA viruses. This domain overlaps the polymerase active site, and mutations within it abrogate Rb binding and reverse the effects of NS5B on E2F promoter activation and cell proliferation. These findings suggest a unique link between an oncogenic RNA virus implicated in the development of liver cancer and a critically important tumor-suppressor protein.
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PMID:Down-regulation of the retinoblastoma tumor suppressor by the hepatitis C virus NS5B RNA-dependent RNA polymerase. 1633 62

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide-and is the main cause of adult liver transplants in developed nations. We have identified a class of novel and specific inhibitors of HCV NS5B RNA-dependent RNA polymerase (RdRp) activity in vitro. Characterization of two such inhibitors, COMPOUND1 (5-(4-chlorophenylmethylene)-3-(benzenesulfonylamino)-4-oxxo-2-thionothiazolidine) and COMPOUND2 (5-(4-bromophenylmethylene)-3-(benzenesulfonylamino)-4-oxxo-2-thionothiazolidine), is reported here. With IC(50) values of 0.54muM and 0.44muM, respectively, they are reversible and non-competitive with nucleotides. Biochemical and structural studies have suggested that these compounds can inhibit the initiation of the RdRp reaction. Interestingly, these inhibitors appear to form a reversible covalent bond with the NS5B cysteine 366, a residue that is not only conserved among all HCV genotypes and a large family of viruses but also required for full NS5B RdRp activity. This may reduce the potential resistance of the viruses to this class of inhibitors.
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PMID:Novel inhibitors of hepatitis C virus RNA-dependent RNA polymerases. 1647 48

Cruciferous vegetables contain a series of relatively unique secondary metabolites of amino acids, called glucosinolates. Sinigrin, the predominant aliphatic glucosinolate in cruciferous vegetables, is hydrolyzed to yield allyl isothiocyanate (AITC), which, after absorption and metabolism in humans, is excreted in the urine as an N-acetylcysteine (NAC) conjugate. We have determined the inhibitory effects of AITC and its NAC conjugate on cell proliferation, the expression of matrix metalloproteinases (MMPs), adhesion, invasion, and migration in SK-Hep 1 human hepatoma cells. Our results demonstrate that AITC and NAC-AITC suppress SK-Hep 1 cell proliferation in a dose-dependent manner; by 25% and 30% for 10 microM AITC and 10 microM NAC-AITC, respectively. We examined the influence of AITC and NAC-AITC on the gene expression of MMPs and tissue inhibitors of metalloproteinase (TIMPs). Gelatin zymography also revealed a significant downregulation of MMP-2/-9 expression in SK-Hep1 cells treated with 0.1-5 microM AITC and NAC-AITC compared with controls. Reverse transcriptase polymerase chain reaction revealed dose-dependent decreases in MMP-2/-9 messenger RNA levels in both AITC-treated and NAC-AITC-treated cells. TIMP-1/-2 activities were unaffected by treatment with AITC or NAC-AITC in our experiments. NAC-AITC inhibited cancer cell adhesion and invasion much more potently than its parent compound. NAC-AITC at 5 microM caused excellent inhibition of cell migration for 48 hrs. These results demonstrate the potential of AITC and NAC-AITC as chemopreventive agents.
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PMID:Allyl isothiocyanate and its N-acetylcysteine conjugate suppress metastasis via inhibition of invasion, migration, and matrix metalloproteinase-2/-9 activities in SK-Hep 1 human hepatoma cells. 1656 38

To compare gene expression patterns between the poorly-differentiated (HA22T/VGH) and well-differentiated (HepG2) hepatocellular carcinoma cells, messenger RNA was isolated form both kinds of cells and subjected to differential displays reversing transcriptase-polymerase chain reaction (DDRT-PCR) technology. Gene fragments showing difference in the expression were recovered, reamplified, cloned and sequenced. Anion exchanger 2 (AE2), an isoform of band 3 protein, was identified and chosen for further characterization. AE2 was strongly expressed in HA22T/VGH cells, while it was little expressed in the well-differentiated hepatocellular carcinoma cells (PLC/PRF5 and HepG2) or little in the normal liver cell (Chang liver). In the 28 pairs of HCC and adjacent non-tumor tissues, levels in the cancer tissue (32.7 +/- 5.0) were significantly higher than that in the adjacent non-tumor tissue (9.1 +/- 2.4) (P < 0.01). Moreover, 19 cases (68%) showed over-expression of AE2 in HCC tissues, 3 cases were similar in both tissues, and 6 cases exhibited little or undetectable signals. Twenty cases (71%) of adjacent normal tissue showed little or undetectable signals. The results indicated that overexpression of AE2 may be involved in the development of human HCC.
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PMID:Overexpression of anion exchanger 2 in human hepatocellular carcinoma. 1705 51

The aim of this study was to assess the value of alphafeto protein (AFP) mRNA-expressing cells detected in peripheral blood for predicting tumor recurrence after living donor liver transplantation (LDLT) in patients with hepatocellular carcinoma (HCC). The test group consisted of 25 patients who underwent LDLT for end-stage liver disease with HCC while the control group consisted of 37 living donors. Quantitative real-time reverse-transcriptase polymerase chain reaction was used for detection of AFP mRNA-expressing cells in peripheral blood. Nine (36%) of 25 patients developed tumor recurrences (four lung; one liver; one peritoneum; two bone; one adrenal gland) during the follow-up period. Perioperatively, AFP mRNA was positive in peripheral blood of eight patients (32.0%) but only in 1 (2.7%) of the control. Preoperative AFP mRNA was positive in three cases. Univariate analyses revealed that preoperative and perioperative AFP mRNA and microscopical vascular invasion were the significant predictors for HCC recurrence (P = .007, .037, and .005, respectively). In the patients with HCC exceeding Milan criteria (n = 15), the presence of AFP mRNA-positive cells in the peripheral blood correlated significantly with HCC recurrence (P = .033). We concluded that the presence of AFP mRNA-expressing cells could be a useful predictor of HCC recurrence in liver transplant patients.
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PMID:Alpha-fetoprotein mRNA detection in peripheral blood for prediction of hepatocellular carcinoma recurrence after liver transplantation. 1717 54


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