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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Based on the fact that aberrant overexpression of some growth factor receptors was observed in a variety of human cancer cells, a novel nonviral gene delivery system GE7, which contains a 16-amino-acid ligand for identifying EGF receptor was constructed for tumor-targeted gene therapy. Intravenous administration of GE7 system revealed that it has the ability to target beta-galactosidase (beta-gal) reporter gene into murine hepatoma (Hepa) cells. Owing to the limited antitumor effects elicited by a single-gene transfer, recent efforts to treat malignancy using combined gene therapy have been accomplished with varying degrees of success. In this study, the human cyclin-dependent kinase inhibitor gene p21(WAF-1) and the murine cytokine gene granulocyte-macrophage colony-stimulating factor (GM-CSF) were used simultaneously for in vivo gene therapy through systemic injection of the EGF R targeted GE7/DNA complex into murine hepatoma-bearing mice. The results demonstrated that combined administration of p21(WAF-1) and GM-CSF could remarkably inhibit the growth of subcutaneously transplanted hepatoma Hepa cells, and significantly increase the survival rate of tumor-bearing mice. The activities of natural killer (NK) cells and specific cytotoxic T lymphocytes (CTL) were clearly enhanced after combined gene therapy. In vitro experiments showed that p21(WAF-1) gene transfer exhibited a suppressive function on the growth of Hepa cells and the expression of H-2K(b) and B7-1 molecules on Hepa cells increased significantly after combined genes delivery. All these results suggested that the GE7 system was able to target therapeutic genes efficiently to cancer cells, which showed high EGF R expression. The cotransfer of p21(WAF-1) and GM-CSF genes apparently inhibited the growth of tumors through (a) the arrest of tumor cell growth and (b) the enhancement of systemic antitumor immunity.
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PMID:Systemic genetic transfer of p21WAF-1 and GM-CSF utilizing of a novel oligopeptide-based EGF receptor targeting polyplex. 1283 33

The effect of interferon alpha (IFN alpha) and the progression of the cell cycle on translation mediated by the 5' untranslated region (5'UTR) of hepatitis C virus (HCV) was evaluated in a transgenic mouse model containing the beta-galactosidase (beta-gal) gene under the control of the mouse albumin promoter and HCV 5'UTR. The transgene was exclusively expressed in the liver and specifically in hepatocytes around the periportal area. IFN alpha significantly suppressed the expression of both the beta-gal gene product and its enzymatic activity at 6 h after the treatment of the mice. The mRNA level of the transgene and endogenous albumin gene expression were not affected, so this suppression was considered to be specific to 5'UTR-directed translation. Phosphorylation of the Stat1 protein was observed in the liver extract 20 min after the treatment, thus confirming a specific known effect of IFN alpha in vivo. We suggest that suppression of 5'UTR-directed translation may be one of the mechanisms whereby IFN alpha exerts its anti-viral activity. We further investigated whether the restriction of 5'UTR-directed translation in periportal hepatocytes may be explained by the proliferative state of the cell. Transgene expression was slightly enhanced in the liver 48 h after partial hepatectomy when a substantial number of hepatocytes entered cell cycle progression. However, 5'UTR-directed translation could not be detected in hepatocellular carcinoma lesions in transgenic mice that were induced to develop such tumours. We suggest that the state of differentiation of the cell, and not its proliferative capacity, is important for supporting HCV expression. This animal model may be a useful tool to dissect the control of HCV expression and to search for ways to block viral replication.
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PMID:Effect of interferon alpha and cell cycle progression on translation mediated by the hepatitis C virus 5' untranslated region: a study using a transgenic mouse model. 1473 56

Prostanoids have been implicated in the transcriptional control of several genes. Since prostanoid synthesis inhibitors are commonly used in subjects with coronary heart disease we studied the effect of cyclooxygenase (COX) inhibition on apolipoprotein AI (apoAI) expression in a human hepatoma cell line (HepG2) transfected with full-length apoAI promoter attached to the chloramphenicol acetyl transferase (CAT) reporter gene. To control for transfection efficiency, the cells were cotransfected with the plasmid pCMV.SPORT-beta-gal containing the beta-galactosidase gene driven by the cytomegalovirus promoter. Treatment of these cells with varying concentrations of indomethacin (INDO, 0, 50, 100, and 300 micromol/L) resulted in a dose-dependent decrease in apoAI promoter activity (% acetylation corrected for beta-galactosidase activity: were 46.1 +/- 2.6, 29.9 +/- 1.2, 25.2 +/- 2.9, and 17.2 +/- 2.8, respectively, P <.001). INDO treatment did not cause significant changes in beta-galactosidase activity. A similar reduction in apoAI promoter activity was found after treating the cells with 50 micromol/L acetylsalicylic acid (ASA) (31.8 +/- 1.8%, P <.001), suggesting that the effect of INDO is related to COX inhibition rather than a peculiar effect of INDO. Nuclear run-off assays indicated that treatment of cells with 50 micromol/L INDO resulted in 31.4% reduction in apo A1 transcription rate (P <.0002). Northern blot analysis of RNA from HepG2 cells treated with 50 micromol/L of INDO for 72 hours showed that the apoAI mRNA concentration relative to G3PDH mRNA was 4,043.0 +/- 84.6 and 3,064.0 +/- 49.8 in control and INDO-treated cells, respectively (P <.0006). Kinetic studies of apoAI mRNA in HepG2 cells indicated that the half-life of apoAI mRNA was not significantly altered with 50 micromol/L INDO treatment. Apo AI mRNA half-life was 25.3 hours in control cells and 26.9 hours in INDO-treated cells. Western blot analysis of culture media of HepG2 cells treated with 50 micromol/L of INDO for 72 hours showed a significant reduction in apoAI protein (6,760.0 +/- 318.1 v 4,773.0 +/- 112.0 arbitrary units, P <.004). Treatment of cells with either arachidonic acid (COX substrate) or various prostanoids including prostaglandin I(2), thromboxane B(2), (+/-)5-HETE, or (+/-)12-HETE did not significantly alter apoAI promoter activity. However, prostaglandin E(1) and E(2) at the highest concentration tested (50 nmol/L) significantly repressed apoAI promoter activity. COX activity measurements in HepG2 cells verified the efficacy of COX inhibition by INDO. It is concluded that COX inhibition with INDO or ASA downregulates apoAI expression at the transcriptional level. This effect could not be attributed to either arachidonic acid excess or to a deficiency in various prostanoids tested.
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PMID:Cyclooxygenase inhibition is associated with downregulation of apolipoprotein AI promoter activity in cultured hepatoma cell line HepG2. 1476 68

Hepatocellular carcinoma (HCC) is a lethal malignancy with poor prognosis and few effective treatments, as well as ever-increasing frequencies in the Western world. Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to significantly attenuated antiviral responses in many tumor cells. The aim of this study was to evaluate the potential of VSV, administered via the hepatic artery, as an effective and safe therapeutic agent for treating "multifocal" HCC in the rat liver. Recombinant VSV vector expressing beta-galactosidase (rVSV-beta-gal) was generated by reverse genetics and infused into the hepatic artery of Buffalo rats bearing orthotopically implanted multifocal HCC. Access by the virus to multifocal HCC lesions in the liver, as well as the kinetic profiles of intratumoral viral replication and spread, was established by X-gal staining of liver and tumor sections. Plaque assays were also performed to determine the infectious viral yields in tumor and normal liver tissues. Pharmacotoxicology studies, including serum chemistries and proinflammatory cytokine production, as well as organ histopathology, were performed. Buffer- or vector-treated tumor-bearing rats were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. Hepatic arterial infusion of rVSV-beta-gal at the maximum tolerated dose in tumor-bearing rats resulted in efficient viral transduction of multifocal HCC lesions in their livers, tumor-selective viral replication, and extensive oncolysis. Importantly, no significant vector-associated toxicities were noted and, in particular, no damage to the hepatic parenchyma was seen. Finally, survival of vector-treated rats was substantially prolonged over that of animals in the control treatment group (p < 0.028). Thus, hepatic arterial administration of VSV is both effective and safe in an orthotopic animal model of multifocal HCC. The results suggest that oncolytic VSV can be developed into an effective and safe therapeutic modality for patients with multifocal HCC in the future.
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PMID:Oncolysis of multifocal hepatocellular carcinoma in the rat liver by hepatic artery infusion of vesicular stomatitis virus. 1500 3

The E1 protein of hepatitis C virus (HCV) shows the ability to induce cell lysis by the alteration of membrane permeability when expressed in Escherichia coli cells. This function seems to be an intrinsic property of a C-terminal hydrophobic region of E1 as permeability changes and cell lysis can be blocked by mutagenesis of specific amino acids in this domain. To establish whether the expression of E1 protein and its C-terminal domain was able to induce cell death also in eukaryotic cell, we cloned HCV sequences expressing the full-length E1 (E383), the C-terminal domain (SVP) and a mutant lacking the C-terminal region (E340) in the pRC/CMV expression vector. HepG2 cell line was co-transfected with empty vector or HCV expression plasmids and a reporter vector that expressed beta-galactosidase (beta-gal) to visualize co-transfected blue cells. At 60 h after transfection, the loss of blue cells, considered as a measure of cell death, was 31.5 and 64.3% for the E1 and SVP clones. On the contrary, the number of blue cells after transfection with E340 plasmid was similar to that observed with the control vector. The analysis by the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay revealed an increased number of apoptotic cells at 48 h after transfection with E1 and SVP clones. Furthermore, cells transfected with SVP revealed a typical internucleosomal DNA fragmentation and the activation of caspase-3-like proteases as the specific inhibitor Ac-DEVD-CHO peptide partially blocked SVP apoptosis. These data indicate that the intracellular expression of HCV E1 protein and its C-terminal domain induces an apoptotic response in human hepatoma cell line.
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PMID:The transmembrane domain of hepatitis C virus E1 glycoprotein induces cell death. 1517 86

Plasminogen activator inhibitor type 1 (PAI-1) has been shown to be an independent risk factor for coronary artery disease, myocardial infarction, and cerebrovascular events. Previous studies on variations in plasma PAI-1 levels and associations between PAI-1 levels and PAI-1 genotypes have suggested that PAI-1 expression maybe regulated in a genotype-specific manner by insulin, hypertriglyceridemic very low-density lipoprotein, and lipoprotein. We investigated whether basal transcription of the PAI-1 gene also is regulated in a genotype-specific manner. Allele-specific polymerase chain reaction-amplified fragments containing a 4G/5G polymorphism of the PAI-1 gene promoter were ligated into the chloramphenicol acetyltransferase (CAT) reporter gene. The constructs of p4G-CAT or pSG-CAT and pSV-beta-galactosidase as an internal control were transiently cotransfected into human HepG2 hepatoma cells. Electrophoresis mobility shift assays (EMSA) employed a fragment from positions -687 to -664 (4G allele) or from -688 to -664 (5G allele) labeled with adenosine triphosphate tagged with phosphorous 32 in the gamma position and used nuclear extracts of HepG2 cells. Analysis of CAT produced by constructs containing the PAI-1 4G or 5G allele showed similar 3-fold increases in CAT activity in the PAI-1 4G/4G and PAI-1 5G/5G constructs, compared with the CAT activity in the pCAT3-Basic construct. Analyses using the probes containing the 4G or 5G allele site in the EMSA assay revealed no difference in the binding of nuclear protein. Our in vitro assay of basal transcription suggests no difference in the transcriptional activities of the alleles of the PAI-1 4G/5G polymorphism.
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PMID:No association of the plasminogen activator inhibitor-1 promoter 4G/5G polymorphism with inhibitor level during basal transcription in vitro. 1521 74

Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21(Waf1), p27(Kip1) and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21(Waf1) was increased, while p27(Kip1) and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21(Waf1) over-expression.
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PMID:Involvement of mitogen-activated protein kinases and p21Waf1 in hydroxyurea-induced G1 arrest and senescence of McA-RH7777 rat hepatoma cell line. 1555 22

Transcatheter hepatic arterial chemoembolization using emulsions composed of anticancer agents and gelatin sponges (GS) has been an efficient and safe palliative treatment for inoperable hepatocellular carcinoma (HCC). We employed catheter-mediated left hepatic arterial embolization (CHAE) to increase transduction efficiency of adenoviral vector in canine hepatocytes. The emulsion was prepared by mixing pieces of GSP and adenoviral vectors expressing recombinant beta-galactosidase (Ad.LacZ) or human hepatocyte growth factor (Ad.hHGF). After the left hepatic artery was catheterized under angiography, CHAE with Ad.LacZ or Ad.hHGF was performed. Livers were removed and stained for LacZ activity on day 7. The expression pattern of LacZ staining was either scarce or patchy around the central hilum of the hepatic artery, or was homogeneously distributed in whole lobes, depending on whether large or small pieces of GSP were used. Hematological and serum biochemical changes during CHAE exhibited only a few effects. The chronological measurement of serum HGF concentration showed that the duration of transgene expression was greater after CHAE with Ad.hHGF. A similar pattern of transgene expression was observed in a rat model after hepatic arterial embolization with differential doses of Ad.hHGF soaked in GSP. These results suggest that hepatic arterial embolization by transcatheter mediated infusion with a mixture of adenovirus-GSP could be used for human HCC.
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PMID:Vascular administration of adenoviral vector soaked in absorbable gelatin sponge particles (GSP) prolongs the transgene expression in hepatocytes. 1557 67

In the course of malignant growth processes in patients with lung cancer, a decrease of natural cytotoxic activity of peripheral blood lymphocytes was observed. This process was accompanied by changes of activities of two lysosomal enzymes, arylsulfatase and acid phosphatase, suggesting participation of these enzymes in manifestation of effector functions of lymphocytes in cancer patients. The level of activity of granular enzyme, beta-glucuronidase, remained unchanged at all stages of disease. A study of natural killer activity of C3HA mice splenocytes after inoculation of transplantable hepatoma 22-a cells revealed a relative stability of the level of their cytotoxicity, and of the activities of lysosomal enzymes--arylsulfatase, acid phosphatase, alpha-mannosidase, acid lipase, N-acetyl-beta-D-glucosidase, and beta-galactosidase, beginning from the 3rd day after hepatoma implantation.
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PMID:[The influence of tumor growth on natural cytotoxicity and activity of some lysosomal enzymes of human effector cells and the C3HA mouse splenocytes]. 1559 12

An objective of designing molecular vehicles exhibiting virus-like transgene delivery capabilities but with low toxicity and immunogenicity continues to drive synthetic vector development. As no single step within the gene delivery pathway represents the critical limiting barrier for all vector types under all circumstances, improvements in synthetic vehicle design may be aided by quantitative analysis of the contributions of each step to the overall delivery process. To our knowledge, however, synthetic and viral gene delivery methods have not yet been explicitly compared in terms of these delivery pathway steps in a quantitative manner. As a first address of this challenge, we compare here quantitative parameters characterizing intracellular gene delivery steps for an E1/E3-deleted adenoviral vector and three polyethylenimine (PEI)-based vector formulations, as well as the liposomal transfection reagent Lipofectamine and naked DNA; the cargo is a plasmid encoding the beta-galactosidase gene under a CMV promoter, and the cell host is the C3A human hepatocellular carcinoma line. The parameters were determined by applying a previously validated mathematical model to transient time-course measurements of plasmid uptake and trafficking (from whole-cell and isolated nuclei lysates, by real-time quantitative PCR), and gene expression levels, enabling discovery of those for which the adenoviral vector manifested superiority. Parameter-sensitivity analysis permitted identification of processes most critically rate-limiting for each vector. We find that the adenoviral vector advantage in delivery appears to reside partially in its import to the nuclear compartment, but that its vast superiority in transgene expression arises predominantly in our situation from postdelivery events: on the basis of per-nuclear plasmid, expression efficiency from adenovirus is superior by orders of magnitude over the PEI vectors. We find that a chemical modification of a PEI-based vector, which substantially improves its performance, appears to do so by enhancing certain trafficking rate parameters, such as binding and uptake, endosomal escape, and binding to nuclear import machinery, but leaves endosomal escape as a barrier over which transgene delivery could be most sensitively increased further for this polymer.
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PMID:Quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes. 1581 3


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