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)

The transcription factor CCAAT/enhancer binding protein (C/EBP alpha) is expressed predominantly in differentiated tissues and is able to induce growth arrest and differentiation in preadipocytes. C/EBP alpha expression is high in non-dividing hepatocytes, but decreases during liver regeneration. These observations suggest that C/EBP alpha is inversely related to cell proliferation. To investigate the mechanism of growth inhibition by C/EBP alpha, the response of immortal human cells to cotransfection of a C/EBP alpha expression vector (CMV alpha) and a CMV beta-galactosidase expression vector was examined. Hep3B2, a hepatoma; Saos2, an osteosarcoma deficient for p53 and Rb; and 639, a fibroblast expressing SV40 T-antigen, were examined. Transiently transfected cells were stained for beta-gal activity to monitor their ability to undergo division. The ability of stable transformants to form colonies was also assessed for each cell line. Cells transfected with CMV alpha remained as non-dividing cells while control cells divided to form colonies. Mutations of the C/EBP alpha sequence demonstrated that only a small, previously uncharacterized activation domain was required for antimitotic activity. Our results suggest that C/EBP alpha may play a role in maintaining the quiescent state of hepatocytes and other cells. Furthermore, it appears that the effects of C/EBP alpha are not mediated through p53 or Rb and are not altered by T-antigen.
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PMID:Inhibition of cell proliferation by C/EBP alpha occurs in many cell types, does not require the presence of p53 or Rb, and is not affected by large T-antigen. 852 67

Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled beta-galactosidase fusion protein carrying amino acids 111-135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 degrees C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 degrees C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 degrees C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import.
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PMID:Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport. 867 Jan 27

Attainment of cell type-specific cytotoxicity with minimal side effects is the ultimate goal of cancer therapy. By employing the prostate-specific antigen promoter (PSAP), we investigated (1) whether PSAP-driven antisense genetic constructs targeting DNA polymerase-alpha and topoisomerase II alpha (Top II alpha), designated PSAP-antipol and PSAP-antitop respectively, could induce death of prostate cancer cells, and (2) whether the cytotoxicity is restricted to cells of prostate origin. A PSAP-driven beta-galactosidase gene, PSAP-LacZ, was also used to estimate the expression of the PSAP-driven transcripts. Lipofection-mediated gene transfers were performed with these 3 constructs and a control plasmid, pCDNA3, in 3 human prostate cancer cell lines (LNCaP, DU-145, PC-3) and 5 other cell lines (Cos-1 [monkey kidney], HL-60 [human myeloid leukemia], Hep G2 [human hepatoma], NCI H460 [human lung cancer] and SW 480 [human colon cancer]). On transfection with PSAP-LacZ, LNCaP, DU-145, and PC-3 showed a 10.8, 1.8, and 1.6 fold increase in beta-galactosidase activity, respectively. The remaining 5 cell lines showed no changes after transfection. Corresponding to the levels of the induced beta-galactosidase activity, LNCaP showed the strongest growth inhibition by the antisense constructs: 36% by PSAP-antipol, 39% by PSAP-antitop and 80% by PSAP-antipol+PSAP-antitop. DU-145 and PC-3 had minimal growth inhibition with PSAP-antipol alone or PSAP-antitop alone. However, when cotransfected with PSAP-antipol and PSAP-antitop, DU-145 and PC-3 displayed 42% and 55% growth inhibition, respectively. In contrast, no cytotoxicity was observed in the remaining 5 cell lines when transfected with PSAP-antipol, PSAP-antitop or both. Therefore, PSAP-driven antisense gene therapy targeting DNA polymerase-alpha and Top II alpha inhibits the growth of human prostate cancer cells and the cytotoxic effect is restricted in cells of prostate origin.
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PMID:Prostate-specific antigen promoter driven gene therapy targeting DNA polymerase-alpha and topoisomerase II alpha in prostate cancer. 871 4

Many approaches exist for hepatic gene delivery, including viral vectors and non-viral vectors. In this study, we tested a panel of liposomes to transfer pAGO, a plasmid containing one copy of herpes simplex virus (HSVtk) gene, and pYED11, a plasmid containing two copies of the HSVtk gene, into a murine hepatocarcinoma cell line (Hepa 1-6) and a human hepatocarcinoma cell line (Hep-G2). The efficiency of gene delivery and expression was characterized by beta-galactosidase staining, flow cytometric analysis and quantitative lacZ activity. Different combinations of liposomes and DNA and the ratio of the concentration of liposome to DNA were tested. The efficient transfer was shown with DOTAP followed by transfectam and lipofectamine. Under these conditions, we tested the cytotoxicity of ganciclovir (GCV) exposure on Hepa 1-6 and Hep-G2 transfected separately with liposome-pAGO and liposome-pYED11 complexes. This study demonstrates the in vitro efficacy of each liposome tested to transduce the HSVtk gene into hepatocarcinoma cell lines. The transfer of two copies of the HSVtk gene rendered cells 1.5 times more sensitive to GCV than cells transduced by pAGO as compared to controls. This was achieved most efficiently by the DOTAP-pYED11 complex. Thus, pYED11 may be considered as an alternative to pAGO as a gene transfer vector.
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PMID:Gene transfer in hepatocarcinoma cell lines: in vitro optimization of a virus-free system. 878 97

Cationic liposomes are considered to be safe vectors for gene transfer, but they are less efficient at delivering DNA to cells when compared with retroviral vectors. Cationic liposomes complexed with DNA were targeted to specific cells in vitro by means of monoclonal antibodies (mAbs) or ligands associated with the liposomes. Significant increases in expression of a beta-galactosidase reporter gene were observed in vitro in mAb-targeted liposomes, compared with non-targeted liposomes, in both an adherent tumor cell line (human adenocarcinoma) and a suspension cell line (human T-lymphoma). Also, use of asialofetuin as a targeting ligand significantly increased expression of the reporter gene in human hepatoma cells. Our results suggest that site-specific targeting of cationic liposomes is a good strategy for increasing both the selectivity and the efficiency of DNA delivery to cells and with further development may lead to targeted DNA delivery in vivo.
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PMID:Use of targeted cationic liposomes in enhanced DNA delivery to cancer cells. 885 50

Recombinant adenoviruses are widely used for the transfer of foreign genes into various mammalian cells. However, the utilization of these vectors for cancer gene therapy requires the specific and efficient expression of the transferred gene in tumor cells. To obtain targeted expression in hepatoma cells, we constructed recombinant adenoviral vectors containing transcriptional elements from either the rat alpha-fetoprotein (AFP) or the human insulin-like growth factor II (IGFII) genes driving expression of the nuclear beta-galactosidase gene (nls lacZ). In vitro infection revealed that the AFP but not the IGFII transcriptional regulatory sequence controlled nls lacZ expression specifically in hepatoma cells. The same specificity was obtained in vivo in subcutaneous human hepatic tumors generated by engraftment of Huh7 hepatoma cells in nude mice as well as in primary liver tumors developed in rats and mice. No marker gene expression was detectable after AFP-nls lacZ gene transfer to normal rat liver parenchyma despite evidence for the presence of DNA encoding the nls lacZ gene. However, in vivo experiments with primary liver tumors in rats and mice also revealed that primary hepatoma cells were poorly infected by adenoviral vectors. Peritumoral and normal tissues were infected efficiently by adenoviral vectors. We conclude that hepatoma cell-specific expression of a transgene can be achieved with AFP regulatory sequences but that adenoviral vectors may not be the preferable vector for transferring genes in vivo in primary liver tumors.
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PMID:In vitro and in vivo hepatoma cell-specific expression of a gene transferred with an adenoviral vector. 886 51

Serum alpha-fetoprotein (AFP) is a glycoprotein of which the sugar chain is considered to show structural changes with malignancies. Microheterogeneity of the serum AFP carbohydrate structure was studied in samples from 35 patients with benign and malignant diseases. Sera were digested directly, extensively, and sequentially with sialidase. beta-galactosidase and beta-N-acetylhexosaminidase. Before and after digestion, sera were examined by means of lectin affinity electrophoresis using eight lectins. Relationships between AFP carbohydrate structures and liver diseases were elucidated by the lectin-reactive profiles and the effect of glycosidase digestion. More than 94% of the AFP carbohydrate structures found in patients with benign and malignant liver diseases were biantennary complex-type oligosaccharides. Changes in the AFP carbohydrate structures at the early stage of hepatocellular carcinoma revealed the addition of alpha 1-->6 fucose to the reducing terminal N-acetylglucosamine and monosialylated AFPs. In both advanced hepatocellular carcinoma and AFP producing extrahepatic malignancies, AFP carbohydrate structures were characterized as the further addition of beta 1-->4 N-acetylglucosamine and heterogeneity in the galactose and N-acetylglucosamine residues. Sequential glycosidase digestion and lectin affinity electrophoresis is useful for analysing the carbohydrate structures of serum glycoprotein.
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PMID:Comparison of carbohydrate structures of serum alpha-fetoprotein by sequential glycosidase digestion and lectin affinity electrophoresis. 889 7

The human apolipoprotein B (apoB) gene resides in a 47.5 kb DNasel-sensitive chromosomal domain in hepatic and intestinal cells, flanked by the 5' distal matrix association region (MAR) and the 3' proximal MAR. A third MAR, the 5' proximal MAR, is found only in transcriptionally active hepatic (HepG2) cells. Hepatic expression of the apoB gene requires a tissue-specific promoter (-898 to +121) and an enhancer from the second intron of the gene (+360 to +1064). A vector containing this portion of the gene linked to the beta-galactosidase reporter is sufficient for low level expression in the livers of transgenic mice. Expression in transgenic mice was increased when the promoter-enhancer beta-gal vector was flanked by MARs. The results were similar whether the 5' distal, the 5' proximal or the 3' proximal MARs were placed at both ends of the construct, or whether the construct was flanked by the 5' distal and the 3' MAR, suggesting that the apoB MARs play a role in gene expression in vivo. When the MAR-containing constructs were transiently transfected into HepG2 cells, the resulting beta-gal activities were similar to that of the construct lacking MARs, thus demonstrating that the MARs do not exhibit any enhancer activity. Recent experiments (Kalos, M., and R. E. K. Fournier. 1995. Mol. Cell. Biol. 15: 198-207) examining stable integration of some of our constructs into human and rat hepatoma transfectants suggest that in single and double copy transfectants, the apoB MARs behave as boundary "insulators", protecting the integrated transgenes against position effects regardless of their site of integration. However, multicopy transfectants are transcriptionally inactive and when the MARs are absent, expression of the transgenes drops to background levels. Our results to date with single and low-copy number transgenes do not support an insulator function for the apoB MARs, although they appear to be required to increase the levels of expression.
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PMID:Evaluation of the function of the human apolipoprotein B gene nuclear matrix association regions in transgenic mice. 890 89

Production of autologous tumor vaccines would be facilitated by the development of a rapid and efficient method for the transfer of genes into freshly isolated cells. To evaluate the potential of replication defective herpes simplex viral (HSV) amplicon vectors as gene transfer vehicles for tumor vaccine generation, a vector that expresses the human interleukin-2 (IL-2) gene (HSV-IL2) and one that expresses Escherichia coli beta-galactosidase (HSVlac) were tested in hepatoma cells of both murine and human origin. Gene transfer into murine hepatoma cells (HEPA 1-6) was both rapid and highly efficient: greater than 50% of cells expressed beta-Gal when infected at a multiplicity of infection (m.o.i.) of 1 with an exposure period of 20 min. Moreover, gene transfer was as efficient in tumor cells after irradiation with 10,000 rads as in nonirradiated tumor cells. Irradiated HEPA 1-6 cells infected with HSV-IL2 for 20 min secreted IL-2 at a rate of 1,200 +/- 160 ng/10(6) cells per day. C57B1/6J mice immunized with irradiated, HSV-IL-2-transduced tumor cells produced in this way demonstrated specific tumor immunity by in vitro splenocyte tumoricidal activity and by in vivo protection against tumor challenge. Human hepatobiliary tumor specimens harvested at the time of operation, irradiated, and infected with HSV-IL-2 also produced nanogram quantities of IL-2/10(6) cells per 24 hr. These results indicate that the HSV amplicon vector is a good candidate vehicle for gene transfer in the production of autologous tumor vaccines. By allowing rapid gene transfer to freshly harvested tumor specimens, these vectors bypass the requirement for cell culture and make feasible reinfusion of genetically modified and irradiated autologous cells within hours of tumor harvest.
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PMID:Rapid production of interleukin-2-secreting tumor cells by herpes simplex virus-mediated gene transfer: implications for autologous vaccine production. 895 12

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in human hepatocellular carcinoma (HCC). Here, we demonstrate that replication defective recombinant adenoviral vectors, containing the human AFP promoter/enhancer, can be used to express the Escherichia coli cytosine deaminase (CD) gene (AdAFPCD) and the beta-galactosidase gene (AdAF-PlacZ) in AFP-producing HCC cell lines. Expression of the CD gene by adenovirus from the AFP promoter/enhancer (AdAFPCD) induced cells sensitive to 5-fluorocytosine (5FC) in the AFP-producing cells but not in the AFP-nonproducing cells. Transduction by an adenoviral vector harboring an ubiquitous strong promoter and CD gene showed enzymatic activity and 5FC killing in all cell lines. When AdAFPlacZ was injected into the s.c. established hepatoma in vivo, expression of the beta-galactosidase gene was confined to AFP-producing HCC xenografts. Moreover, HCC xenografts regressed by transduction with AdAFPCD and subsequently with 5FC treatment in vivo. These findings suggest that utilization of the AFP promoter/enhancer in an adenoviral vector can confer selective expression of a heterologous suicide gene in hepatocellular carcinoma cells in vitro and in vivo.
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PMID:In vivo gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene. 901 74


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