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)

We made several generic plasmids for construction of recombinant vaccinia virus (rvv) expressing foreign proteins in high yield. Rvvs expressing biologically active Escherichia coli beta-galactosidase (rvv-lacZ) and the cytokine murine GM-CSF (rvv-mGM-CSF) were constructed by using these plasmids. To obtain attenuated rvv, cDNA for these proteins was inserted in the thymidine kinase gene of vaccinia virus. Their expression was controlled by vaccinia early/late promoter, 7.5 K so that these proteins could be expressed in the infected cells throughout the life cycle of the virus. Female C57BL/6 mice were immunized subcutaneously with B16-F10 melanoma cells infected with rvv, and 2 weeks later challenged with viable B16 cells. Mice immunized with rvv-mGM-CSF showed delay in tumor development, smaller tumor volumes and longer survival time compared with unimmunized mice, as well as mice immunized with rvv-lacZ. Mice immunized with rvv-mGM-CSF followed by a booster injection after 1 week responded slightly better than those immunized once, but this difference was not statistically significant. These results suggested that rvv-mGM-CSF could be a promising vaccine for cancer therapy.
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PMID:Construction of recombinant vaccinia virus expressing GM-CSF and its use as tumor vaccine. 892 12

One potential avenue for future cancer therapy involves the specific targeting of effector genes to cancer cells throughout the body, including distant metastatic sites. As a first step toward this goal, we tested the ability of the transcriptional regulatory elements of the human and mouse tyrosinase genes to promote high levels of pigment cell-specific transcription. A construct consisting of 209 bp of the human tyrosinase promoter linked to two enhancer elements was demonstrated to drive high-level, melanoma-specific expression of a beta-galactosidase (beta-gal) reporter gene in transient transfection assays. In studies of the murine tyrosinase promoter region, constructs containing up to 2500 bp of the 5' regulatory region were found to have very low transcriptional activity in murine melanoma cells. However, as with the human system, addition of two tandem repeats of an upstream enhancer element resulted in high levels of lineage-specific transcriptional activation. The murine tyrosinase promoter-enhancer expression cassette was introduced into the E1 region of a recombinant adenovirus to generate the virus AdmTyr-beta gal. This virus grows to high titer and maintains transcriptional specificity for pigment cell lineages. Strikingly, AdmTyr-beta gal is extremely active in human melanoma cells, in some cases exceeding the transcriptional activity of a cytomegalovirus promoter-driven recombinant beta-gal virus. Tissue specificity of gene expression is maintained, with very low levels observed in tumors and primary human cells derived from other lineages. These data provide evidence that it is possible to target human melanoma cells with great efficiency and specificity using high-titer recombinant adenovirus vectors.
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PMID:Transcriptional targeting of recombinant adenoviruses to human and murine melanoma cells. 897 Nov 69

Because metallothionein (MT) is elevated and may be protective in UV-irradiated skin, we have studied the effects of UV and other agents on MT transcription using the sheep MT 1A promoter, linked to the beta-galactosidase gene and stably transfected into human cell lines. beta-galactosidase reporter activity was inducible by adding Zn2+ ions to the medium (100 microM for 2-4 h). Two differentiating agents, butyric acid and azelaic bishydroxamic acid (ABHA), significantly increased the response to Zn2+ in a melanoma cell line (MM96L-gal). UVB (280-315 nm) had two distinct, time-dependent effects. During the first 4 h after irradiation, high doses of UVB inhibited induction by Zn2+, an effect that was made more acute by simultaneous exposure to the differentiating agents. These changes in reporter activity were not due to alterations in Zn2+ transport into the cell. The UVB-depressed MT response subsequently recovered and by 24 h was double the control, yet remained sensitive to ABHA. Reporter activity in transfected HeLa cells differed from that in MM96L, being depressed 4 and 24 h after UVB and insensitive to ABHA at both times. Galactosidase reporter activity driven by non-MT promoters was not affected by these treatments. Dependence of MT transcriptional activity on UV-related DNA damage could be inferred because equitoxic UVC (254 nm) affected the response to Zn2+ in a similar fashion, whereas UVA, cisplatin and a methylating agent had no effect. The MT response was partly dependent on the PKC signal transduction pathway because it was inhibited by phorbol ester in HeLa, and by bisindolyl maleimide in HeLa and MM96L. The biphasic MT transcriptional response may model a signal transduction pathway that gives an early, depressed response to acute UV damage, with exacerbation by concurrent differentiation stimuli, but switches to a positive, cell-specific and potentially protective response at later times.
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PMID:Biphasic response of the metallothionein promoter to ultraviolet radiation in human melanoma cells. 907 40

A flow cytometric method has been developed for the rapid analysis of lacZ transduced cells. The method described is based on an indirect immunofluorescence staining procedure using a monoclonal antibody which binds specifically to beta-galactosidase from E. coli and to beta-galactosidase fusion proteins. This technique was used for the quantification in vitro as well as in vivo of beta-galactosidase expression in B16 melanoma cells. The described method is appropriate for a variety of cell types (species, lineage), is simple, quantitative, reliable, rapid and applicable to all constructs containing the lacZ selectable markers. It should prove to be very helpful (1) for the quantification of cells expressing the lacZ reporter gene and (2) for studying gene regulation, including transfection modality, promoter efficacy, enhancer activity, and other regulatory factors.
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PMID:A flow cytometric method for the rapid detection of beta-galactosidase transfected cells: an in vitro and in vivo study. 920 9

Lipid-based DNA transfer formulations are typically selected on the basis of in vitro transfection studies where the activity of specific formulations is defined by transgene expression. It is unclear, however, whether expression is directly related to the efficiency of DNA transfer. In an attempt to correlate DNA transfer with transgene expression, we used a simple assay consisting of measuring DNA (3H-plasmid encoding for beta-galactosidase) binding to murine (B16/BL6) and human (KZ) melanoma cells in vitro at 4 and 37 degrees C. The difference in cell association at these temperatures was assumed to be a consequence of DNA uptake, an assumption that was confirmed by protease removal of cell surface-associated DNA. DNA associated with B16/BL6 melanoma cells (up to 30 ng or 12% of the added DNA) following incubation with dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (DOPE) liposome-DNA aggregates was comparable to that achieved with 1,2-dioleoyloxypropyl-3-trimethylammonium bromide/DOPE or dimethyldioctadecylammonium bromide/DOPE liposomes; however, transgene expression was 2- and 5-fold less for the latter two formulations, respectively. Similarly, equivalent amounts of DNA delivery were achieved with B16/BL6 and KZ melanoma cells, yet the level of transgene expression in the KZ cells was undetectable. It was demonstrated that the lack of transgene expression was not a consequence of cell-specific differences in DNA degradation.
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PMID:Analysis of cationic liposome-mediated interactions of plasmid DNA with murine and human melanoma cells in vitro. 923 50

The efficient genetic modification of solid tumors in situ to stimulate therapeutic immune responses against them is currently under active investigation, but is not yet possible using existing gene transfer technologies. Thus, ex vivo/in vivo vaccination strategies have been proposed in which the patient's tumor is surgically excised, single cell suspensions are prepared, the therapeutic genes are introduced and then the gene-modified cells, after being gamma-irradiated, are injected back into the patient. However, even with high-efficiency gene delivery systems, this is a labor-intensive process. Moreover, it is often difficult to obtain sufficient numbers of gene-modified primary tumor cells during short-term culturing. On the other hand, extended in vitro passaging of primary tumor explants may alter their immunophenotypic properties. One approach to overcome these limitations would be to design universal vaccines consisting of standardized gene-transduced neoplastic cell lines or mixtures of gene-transduced cell lines to be combined with autologous tumor samples if available. Melanoma, which is notable for being one of the most immunogenic human malignancies, represents a cancer where shared tumor-associated antigens have been identified. We developed and analyzed several different retroviral vectors for their ability to stably express exogenous genes at high levels in a panel of melanoma cell lines. All vectors contained a reporter gene (nlslacZ) encoding beta-galactosidase with a nuclear localization signal and the neomycin phosphotransferase (neo) gene as selectable marker. One vector, DCCMV, which carried a bicistronic nlslacZ-neo transcriptional unit under the control of the human cytomegalovirus immediate-early promoter in the U3 region of its 3' LTR, was found to perform consistently better than the other vectors. The DCCMV vector, which is an extreme example of the double-copy class of retroviral vectors, was subsequently used to generate melanoma cell lines constitutively secreting human interleukin-6 or a soluble form of the human interleukin-6 receptor for potential use in a phase II clinical vaccine trial for the treatment of melanoma patients. The DCCMV vector design may also be useful in gene therapy applications where the intent is to implant polymer-encapsulated cell lines genetically engineered to stably express high levels of bioactive proteins.
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PMID:Double-copy bicistronic retroviral vector platform for gene therapy and tissue engineering: application to melanoma vaccine development. 941 12

The context in which an antigen is presented shapes the nature of the immune response to that antigen and can result in B cell activation, T cell activation, or immune tolerance. To elicit anti-tumor immune responses, various cell types have been employed as cellular adjuvants with tumor antigens, and recently several groups have shown that dendritic cells (DCs), cultured with tumor lysates, tumor antigens, or peptides eluted from tumor cells, induced significant anti-tumor immunity in vivo. In all of these approaches, the DCs were pulsed with an exogenous source of antigen. An alternative method is to engineer DCs to express tumor antigens. We genetically modified DCs to express beta-galactosidase (beta-gal) as a surrogate tumor antigen and then tested the anti-tumor activity of the beta-gal+ DCs in mice against a beta-gal+ murine melanoma cell line. A single vaccination with the gene-modified DCs protected mice against a lethal dose of beta-gal-B16 melanoma cells and induced beta-gal-specific cytotoxic T lymphocytes. These results demonstrate that expression of tumor antigens by DCs is a potent method of inducing tumor antigen-specific responses in vivo.
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PMID:Induction of protective anti-tumor immunity by gene-modified dendritic cells. 945 35

We show that efficient permeabilization of murine melanoma can be obtained in vivo by applying electric pulses. More than 80% of the cell population is affected as shown by the penetration of propidium iodide. A protein, beta-galactosidase, can be transferred and expressed into the cells by incorporating either the protein or a plasmid carrying the reporter gene with respective efficiencies of 20% and 4%. This is obtained by a direct injection of either the protein or the plasmid in the tumor, followed by the application of electric pulses with surface electrodes in contact with the skin. This approach is simple and safe to use, reproducible, and specific; moreover, it is potentially applicable to a wide variety of tissues, cell types, and animals.
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PMID:In vivo electrically mediated protein and gene transfer in murine melanoma. 948 24

To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.
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PMID:Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells. 960 16

The efficacy of adenovirus-mediated gene therapy for treatment of metastatic B16 melanomas, established in syngeneic C57BL/6 mice, was assessed via an ex vivo cytokine vaccine approach or via an in vivo strategy utilizing combination cytokine/herpes simplex virus-thymidine kinase (HSV-tk) suicide gene delivery and treatment with ganciclovir (GCV). In the ex vivo tumor vaccine approach, B16 melanoma cells, transduced in vitro by adenovirus containing either interleukin (IL)-2, granulocyte-macrophage colony stimulating factor (GM-CSF), or tumor necrosis factor-alpha cytokine genes and gamma irradiated, were subcutaneously injected into the flank and a distant subcutaneous challenge injection of unmodified B16 melanoma cells was performed 15 d later. Significant reductions in challenge tumor volume were observed in the IL-2 group (75% reduction; p = 0.02) and in the GM-CSF group (88% reduction; p = 0.0006), whereas the effect for tumor necrosis factor-alpha was not statistically significant. In the in vivo treatment of established melanomas, this cytokine approach was combined with a suicide gene therapy and subcutaneous B16 melanomas were directly injected with (i) IL-2/recombinant, replication-deficient adenovirus (adv) and thymidine kinase (tk)/adv, (ii) GM-CSF/adv, IL-2/adv, and tk/adv, or (iii) control beta-galactosidase (beta-gal)/adv and tk/adv. After intraperitoneal application of GCV (10 mg per kg) for 6 d, the residual tumor masses were excised and the animals challenged with unmodified B16 cells. Challenge tumor growth was reduced by 56% for the IL-2/tk/adv/GCV treatment (p = 0.041) and by 77% for the GM-CSF/IL-2/tk/adv/GCV treatment p (p = 0.037), in comparison with the beta-gal/tk/GCV control group. These data may hold significant promise for the development of effective ex vivo and in vivo gene therapy modalities to counter the highly metastatic nature of human melanoma.
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PMID:Ex vivo and in vivo adenovirus-mediated gene therapy strategies induce a systemic anti-tumor immune defence in the B16 melanoma model. 962 Feb 91


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