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:3.5.4.1 (cytosine deaminase)
747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Escherichia coli codBA operon encodes cytosine permease (CodB) and cytosine deaminase (CodA). CodB mediates uptake of exogenously supplied cytosine, and CodA catalyses the hydrolytic deamination of cytosine to uracil and ammonia. The hydropathic profile of CodB indicates that it is an integral cytoplasmic membrane protein possessing several transmembrane-spanning domains. The membrane topology of CodB was investigated by using gene fusions containing varying lengths of the amino-terminus of CodB fused to either alkaline phosphatase (AP) or beta-galactosidase (BG). The AP activities expressed by the CodB-AP fusions are consistent with a topological model in which the amino- and the carboxy-termini of CodB are located in the cytoplasm, and in which CodB possesses 12 membrane-spanning segments. The enzyme activities of most of the CodB-BG fusions support the model. However, the results obtained with some of the CodB-BG fusions illustrate the limitations of using BG as a reporter protein in studies of membrane protein topology.
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PMID:Membrane topology analysis of the Escherichia coli cytosine permease. 853 18

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

Although replication-deficient adenovirus (Ad) vectors are efficient vehicles for in vivo gene transfer, persistence of expression of the Ad genome is limited in immunocompetent hosts by cellular immunity directed against the gene product of the vector. While most attention has been focused on cytotoxic T lymphocytes (CTL) directed against the low-level early and late Ad gene expression in the Ad vector-infected target cells, significant cellular immunity is likely also directed against the product of heterologous transgenes. To evaluate this concept, in vivo generation of CTL was evaluated in C57B1/6 and BALB/c mice with Ad vectors expressing a variety of heterologous transgenes, including Escherichia coli chloramphenicol acetyl transferase (CAT), beta-galactosidase (beta-Gal), cytosine deaminase, and human thrombopoietin (hTPO), with an Ad vector expressing no transgene ("null") as a control. Following intravenous administration of Ad vectors, spleen cells were harvested 2 weeks later, stimulated for 5 days with syngeneic cells infected with various Ad vectors, and then evaluated for CTL activity using 51Cr-release from syngeneic Ad vector-infected targets. In all cases, CTL directed against the heterologous transgene products was observed, although there were differences in the amounts of transgene-specific CTL. CTL directed against the transgene were also observed with other routes of administration, including intratracheal, subcutaneous, and intraperitoneal administration. These observations suggest that inclusion of a heterologous transgene in Ad vectors enhances the elimination of vector-infected cells, a circumstance that will be partially circumvented using autologous genes. For some applications, specific immune responses to products of transgenes delivered by Ad vectors might be exploited for therapeutic purposes.
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PMID:Cytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors. 921 38

Previously, we reported that adenoviral vectors carrying the carcinoembryonic antigen (CEA) promoter sequences to direct the Echerichia coli beta-galactosidase gene (AdCEA-lacZ) or cytosine deaminase (CD) gene (AdCEA-CD) confer selective gene expression on a CEA-positive gastric cancer cell line (MKN45) in vitro. Here, adenovirus-mediated tumor-specific gene therapy for CEA-positive gastric carcinoma in vivo was investigated. Using an animal model with i.p. disseminated MKN45 tumors, adenovirus-mediated tumor-specific transgene expression and therapeutic efficacy were analyzed. After an i.p. injection of AdCEA-lacZ, beta-galactosidase activity was confined to tumor xenografts. Moreover, CD mRNA was expressed exclusively in MKN45 tumor xenografts after infection with AdCEA-CD, despite the fact that an adenovirus-mediated transfer of CD DNA was detected in all tissues tested. In contrast, CD mRNA was detected not only in tumor xenografts but also in other organs of mice infected with AdCA-CD, in which CD gene expression is governed by an ubiquitous promoter. Suppression of tumor growth and prolongation of survival were noted in tumor-bearing mice treated with AdCEA-CD and 5-fluorocytosine (5FC) without observable adverse effects. In contrast, significant hepatic toxicity was noted in animals treated with AdCA-CD. These results reveal that the CEA promoter restricts CD gene expression to CEA-positive tumor cells in the adenoviral context in vivo, along with the beneficial therapeutic effects of 5FC treatment, suggesting the i.p. AdCEA-CD/5FC system may provide a novel approach to treatment of i.p. disseminated gastric cancer.
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PMID:In vivo selective gene expression and therapy mediated by adenoviral vectors for human carcinoembryonic antigen-producing gastric carcinoma. 933 Oct 89

Cholangiocarcinoma is a virtually incurable tumor, resistant to current surgical, chemotherapy, and radiotherapy interventions. We applied the gene therapy strategy of toxin gene conversion of nontoxic prodrug to chemotherapeutic drug in combination with radiation therapy to the treatment of cholangiocarcinoma. In this regard, 5-fluorouracil (5-FU) is an accepted radiosensitizing and chemotherapeutic agent presently used in cancer therapy. The Escherichia coli enzyme cytosine deaminase (CD) converts the prodrug 5-fluorocytosine (5-FC) to 5-FU. Therefore, our goal was to express the CD gene in the human cholangiocarcinoma cell line, SK-ChA-1, assess the cytotoxicity of intracellular production of 5-FU, and determine any enhanced cell killing by the addition of external beam radiation. The susceptibility of SK-ChA-1 cells to recombinant adenoviral infection was determined by fluorescence-activated cell sorting analysis. We used the recombinant adenoviral vector AdCMVLacZ, encoding the E. coli beta-galactosidase reporter gene under control of the human cytomegalovirus (CMV) promoter, to infect SK-ChA-1 and HeLa cells at 10 and 100 plaque forming units (pfu)/cell, followed by FACS analysis. To evaluate CD-mediated conversion of 5-FC to 5-FU and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes CD. Cells were then plated in 96-well microtiter plates and exposed to varying concentrations of 5-FC. Cell proliferation assays (tetrazolium salt conversion to formazan colorimetric assay) were performed beginning 2-8 days after plating. We evaluated the effects of external beam radiation using a single 8 Gy 60Co dose to AdCMVCD infected cells, with prior exposure to 5-FC for 2-3 days. MTS assays were performed following radiation treatment. Radiation dose-response analysis, via clonogenic assay, was used as a more sensitive assay to confirm the interaction of the treatment conditions. s.c. SK-ChA-1 tumors in athymic nude mice were established, which then received three intratumoral injections of 1 x 10(9) pfu AdCMVCD. Mice received i.p. injections of 400 mg/kg of 5-FC twice daily for 7 days beginning the day of initial AdCMVCD injection (day -2). The radiation treatment group received 10 Gy of 60Co exposure to their tumor on day 0. SK-ChA-1 cells were efficiently transduced (48.7 and 99.2%) by 10 and 100 pfu/cell of AdCMVLacZ, respectively. From 37.9 to 84.4% of SK-ChA-1 cells were killed following infection with 10 pfu/cell AdCMVCD and 8 days of exposure to various concentrations of 5-FC (5, 10, 30, 50, and 100 microg/ml). Higher 5-FC concentrations and longer duration of exposure resulted in greater cell killing. Radiation treatment (8 Gy) enhanced cell killing by greater than 70% when combined with 10 or 20 microg/ml of 5-FC. Radiation dose-response analysis with clonogenic assay confirmed enhanced SK-ChA-1 cell cytotoxicity as a result of radiation treatment following AdCMVCD infection and 5-FC exposure, with radiobiological parameters alpha = 0.44 and D0 = 0.96. Combined treatment of SK-ChA-1 tumors with AdCMVCD, 5-FC, and radiation in animals resulted in significantly greater survival, time to tumor regrowth, and doubling time compared to the nonradiation treatment group (P = 0.03, 0.015, and 0.002, respectively). Significantly greater change in tumor size, smaller ratio of final tumor size to original tumor size, and smaller final tumor size were observed in the radiation treatment group compared to the no radiation treatment group (P = 0.02, 0.03, and 0.03, respectively). Human cholangiocarcinoma cells were transduced with a recombinant adenovirus in vitro at high efficiency and were susceptible to CD-mediated intracellular 5-FU production. Radiobiological survival curve parameters confirmed an interactive cytotoxic effect when viral infection and prodrug therapy were combined with external beam radiation exposure. (ABSTRACT TRUNCATED)
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PMID:Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo. 933 Oct 94

The antitumor effect of the combined transfer of a suicide gene and a cytokine gene was evaluated in the present study. Adenoviruses expressing Escherichia coli cytosine deaminase (AdCD) and adenoviruses expressing murine interleukin-2 (AdIL-2) were utilized for the treatment of established tumors. The mice were inoculated s.c. with FBL-3 erythroleukemia cells and 3 days later received an intratumoral injection of AdCD in the presence or absence of AdIL-2 followed by intraperitoneal 5-fluorocytosine (5-FC) administration. The results demonstrated that tumor-bearing mice treated with AdCD/5-FC in combination with AdIL-2 showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdCD/5-FC, AdIL-2, adenovirus expressing beta-galactosidase/5-FC or phosphate-buffered saline. The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD4+ and CD8+ T cells infiltrating the tumor after combined therapy. The splenic natural killer and cytotoxic T lymphocyte activities increased significantly in the mice after combined therapy with AdCD/5-FC/AdIL-2. Our results demonstrate that therapy combining a suicide gene and IL-2 gene can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently.
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PMID:Adenovirus-mediated combined suicide gene and interleukin-2 gene therapy for the treatment of established tumor and induction of antitumor immunity. 987 29

The Fab fragment of monoclonal antibody B4G7 against human epidermal growth factor (EGF) receptor was conjugated with cationic poly-L-lysine and the resulting conjugate was further complexed with reporter genes or therapeutic genes. This Fab/DNA complex was designated as "Fab immunogene." The Fab immunogene transfer in vitro was mediated through the EGF receptors in two melanoma cell lines. The frequency of cells expressing beta-galactosidase (beta-Gal) reporter gene was approximately 1%. The induction of suicide effects after Fab immunogene transfer of herpes simplex virus thymidine kinase (TK) or Escherichia coli cytosine deaminase (CD) gene was quite remarkable, and the growth of melanoma cells was inhibited for over 7 days in the presence of ganciclovir (GCV) or 5-fluorocytosine (5-FC). Similarly, when melanoma cells treated in vitro with the Fab immunogene carrying TK or CD were transplanted into the back of nude mouse, subsequent systemic administration of GCV or 5-FC effectively suppressed the growth of tumors, indicating the occurrence of in vivo suicide effects.
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PMID:Ex vivo delivery of suicide genes into melanoma cells using epidermal growth factor receptor-specific Fab immunogene. 1036 86

The potential of gene therapy to treat premalignant disease or recurrent cancer has not been investigated. The goal of the present investigation was to explore the efficacy of pro-drug-mediated, suicide gene therapy as a strategy to treat incipient neoplasia in stratified squamous epithelium. To test this strategy, a tissue model of premalignancy was generated by mixing normal human keratinocytes (NHK) that express the bacterial cytosine deaminase gene (CD) with premalignant keratinocytes which have been genetically marked with the bacterial gene for beta-galactosidase (II-4-beta-gal) in skin-like organotypic cultures. Preliminary studies in monolayer cultures demonstrated that CD-transduced NHK (NHK/CD) efficiently expressed the transgene and deaminated the pro-drug 5-fluorocytosine (5FC) to the toxic product 5-fluorouracil (5FU). The capacity of NHK/CD to kill II-4-beta-gal cells through bystander effect was assayed in both submerged culture and in the organotypic model of premalignancy. In submerged cultures, it was found that CD-mediated killing of II-4-beta-gal cells did not require cell-cell contact and that the LD(50) of 5FC for efficient bystander killing of II-4-beta-gal was 0.5 mM. When this concentration of pro-drug was used in organotypic cultures, a significant number of dysplastic II-4-beta-gal cells were eliminated from the tissue. Bystander killing of II-4-beta-gal cells was related to the number of NHK/CD present. These findings demonstrated that potentially malignant keratinocytes could be eliminated from a dysplastic tissue through activation of pro-drug and killing of adjacent cells through the bystander effect. By establishing an in vitro model to eliminate premalignant cells using suicide gene therapy, these studies provide a new approach for the treatment of incipient cancer as it develops, thereby preventing invasive disease.
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PMID:Suicide gene therapy for premalignant disease: a new strategy for the treatment of intraepithelial neoplasia. 1131 95

Adenovirus (Ad) gene transfer vectors traffic to regional lymph nodes (RLNs) after footpad injections in mice, resulting in localized production of interferon gamma (IFN-gamma). With this background, we evaluated the hypothesis that Ad vector administration may inhibit RLN tumor metastasis independent of the transgene in the expression cassette. Tumors of MM48, a cell line with a propensity toward lymphogenous metastasis, were established in the footpads of syngeneic C3H mice, and E1(-)E3(-) Ad vectors encoding no transgene (AdNull) or encoding an irrelevant transgene (AdCD; Escherichia coli cytosine deaminase with no 5-fluorocytosine administration) were administered (10(10) particles) in a peritumoral location. Both vectors suppressed the growth of tumor in the regional (popliteal) lymph node. This effect was localized to the regional, but not distant, lymph nodes (p < 0.05). Heat inactivation of the vector or decreasing the dose of the vector to 10(9) particles did not suppress RLN growth of the tumor when compared with 10(10) particles of active AdNull (p < 0.05 and p < 0.01, respectively). The ability of an E1(-)E4(-) vector expressing beta-galactosidase (AdRSVbetagal.11) to suppress RLN tumor growth showed that the E4 region of the Ad vector was not responsible for the effect. Blocking either IFN-gamma or natural killer (NK) cells with systemic antibody treatment in immunocompetent mice allowed rapid growth of RLN metastases despite Ad vector administration, and Ad vector injection into the footpads of tumor-free mice induced the accumulation of NK cells in the RLN. These data demonstrate that, in a metastatic murine tumor model, a low dose (10(10) particles) of replication-deficient Ad vectors inhibits RLN metastases independent of a therapeutic transgene, an effect that is mediated, at least in part, by IFN-gamma and NK cells.
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PMID:Adenovirus gene transfer vectors inhibit growth of lymphatic tumor metastases independent of a therapeutic transgene. 1153 67

A major potential limitation to the success of enzyme prodrug gene therapy is the toxicity that could result from gene expression in normal tissues. In this study, we investigated the use of an enhanced human carcinoembryonic antigen (CEA) promoter for yeast cytosine deaminase (yCD), which converts 5-fluorocytosine to 5-fluorouracil, to increase targeting while maintaining activity both in cell culture and in nude rats bearing intrahepatic xenografts. We found that an enhanced CEA-yCD adenoviral vector can achieve significantly greater yCD expression in CEA-expressing colon carcinoma cell lines (LoVo, HT29, and CaCo2) compared with a nonspecific Rous sarcoma virus-yCD virus. In contrast, infection with CEA-yCD led to lower or equivalent yCD expression in normal hepatocytes or fibroblasts compared with that produced by the RSV-yCD. Adenovirus administered in the portal vein or the hepatic artery of nude rats bearing intrahepatic LoVo colon carcinomas could mediate beta-galactosidase expression equally in liver and tumors under the control of cytomegalovirus, a nonspecific promoter. However, infusion of CEA-yCD virus markedly increased yCD expression in tumors over normal liver (>4-fold) measured both by levels of mRNA and yCD activity. Moreover, the efficiency of 5-fluorocytosine conversion into 5-fluorouracil in tumors was significantly higher than that in normal liver ( approximately 3-fold) in rats receiving portal venous viral infusion of CEA-yCD and subsequent 5FC treatment. Thus, an enhanced CEA promoter can preferentially stimulate yCD gene expression in CEA-expressing cells in vivo. Such tumor-specific expression should prove useful in colorectal cancer gene therapy to achieve selective prodrug conversion in tumors.
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PMID:Regional delivery and selective expression of a high-activity yeast cytosine deaminase in an intrahepatic colon cancer model. 1256 11


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