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)

To evaluate the hypothesis that regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene (AdCMV.CD) together with systemic 5-FC could suppress the growth of metastatic colon cancer in the liver, the AdCMV.CD vector was injected 0.8-1 cm from the site of a human colon cancer tumor in the livers of nude mice. The growth of the human colon cancer cells was quantified by dot blot analysis of genomic DNA extracted from tumor-bearing liver, hybridized with a human-specific Alu probe. The combination of regional AdCMV.CD plus systemic 5-fluorocytosine (5-FC) suppressed the growth of the metastatic tumors over the 21 days of evaluation following vector administration. Histologic evaluation showed necrosis at the site of the tumor in the livers of mice treated with AdCMV.CD/5-FC, but not in control groups. Evaluation of the potential toxicity of AdCMV.CD plus 5-FC on the normal liver showed only mild, self-limited dose-related inflammation, with no deaths. These data suggest that the regional administration of AdCMV.CD together with systemic 5-FC may be a safe and effective strategy to suppress the growth of metastases of colorectal carcinoma in the liver.
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PMID:Regional delivery of an adenovirus vector containing the Escherichia coli cytosine deaminase gene to provide local activation of 5-fluorocytosine to suppress the growth of colon carcinoma metastatic to liver. 886 57

Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.
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PMID:Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia. 886 65

The magnitude of the proton gradient (delta mu H+) driving solute accumulation in Saccharomyces cerevisiae has long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (delta psi). In the present work, the size of the cytosine gradient (delta mu cyt) that the yeast generated was used as a measure of the driving gradient (delta mu H+). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H+/cytosine system. delta mu cyt, assayed in washed cell suspensions fermenting glucose and containing 0.5 or 50 mM KCl, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, -delta mu H+ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered delta mu cyt to a value roughly equal to the magnitude of the pH gradient (delta pH). A mathematical model was used to correct the first estimates of delta mu H+ for the effect of cytosine leakage outside the symport. In such a system, delta mu cyt cannot exceed the equivalent ratio Vmax/KmL, where Vmax and Km are kinetic parameters of the symport and L is the rate coefficient for leakage. The feasibility of assaying delta mu H+ depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, -delta mu H+ during fermentation was estimated to be up to 25 mV larger than delta mu cyt and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of delta pH, delta psi was in turn deduced and compared with the maximum methylamine gradient (delta mu M) the yeast formed. The results supported the claim in the literature that, at acid pH, delta mu M is a measure of delta psi.
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PMID:Cytosine accumulation as a measure of the proton electrochemical gradient acting on the overexpressed cytosine permease of Saccharomyces cerevisiae. 886 19

Viral vector-mediated transfer of chemosensitization genes represents a promising new approach to the treatment of cancer. Previous reports have demonstrated that transfection of the bacterial cytosine deaminase (cd) gene into mammalian cells can sensitize them to the otherwise nontoxic nucleoside, 5-fluorocytosine (5-FC). We now report that a replication-deficient adenovirus vector that transduces the cd gene (Ad.CMV-cd) highly sensitizes 9L gliosarcoma cells to 5-FC, and that gene transduction is associated with a potent bystander effect that is not dependent on direct cell-to-cell contact. Stereotactic injection of Ad.CMV-cd into established rat gliomas, followed by systemic administration of 5-FC in vivo, results in prolongation of survival.
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PMID:In vivo replication-deficient adenovirus vector-mediated transduction of the cytosine deaminase gene sensitizes glioma cells to 5-fluorocytosine. 891 93

Epstein-Barr virus (EBV)-positive lymphomas are frequent among immunosuppressed patients. We have examined the feasibility of killing EBV-immortalized B lymphocytes by gene transfer involving the use of "suicide" genes whose expression in target cells renders them susceptible to killing by a prodrug. We examined two gene/prodrug pairs: the Escherichia coli cytosine deaminase (CD) gene with the prodrug 5-fluorocytosine (5-FC), and the herpes simplex virus thymidine kinase (HSV-TK) gene with the prodrug ganciclovir. Retroviral vectors and drug selection were used to obtain CD or HSV-TK expression in cells. Both the CD/5-FC and the HSV-TK/ganciclovir combinations yielded substantial killing of EBV-immortalized B lymphocytes in vitro, although the CD/5-FC regimen had a significantly greater therapeutic margin than the HSV-TK/ganciclovir combination. The CD/5-FC pair, but not the HSV-TK/ganciclovir pair, was shown to have a "bystander killing effect" in vitro. When only 30% of the cells expressed the suicide gene, scid mouse tumors regressed in both the CD/5-FC regimen and the HSV-TK/ganciclovir regimen, documenting an in vivo bystander effect with both regimens. However, a greater percentage of tumors completely regressed with the CD/5-FC regimen. Overall, the sum of our data indicates that the CD/5-FC combination is the more promising regimen for treatment of EBV-associated lymphomas in vivo.
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PMID:Killing Epstein-Barr virus-positive B lymphocytes by gene therapy: comparing the efficacy of cytosine deaminase and herpes simplex virus thymidine kinase. 895 14

By functional complementation of a fcy1 null mutant of Saccharomyces cerevisiae, we have cloned and characterized the FCY1 gene, encoding cytosine deaminase in Saccharomyces cerevisiae, and its homologue FCA1, encoding cytosine deaminase in Candida albicans. Disruption of FCY1 resulted in high resistance to 5-fluorocytosine (10(-2) M) and in total loss of cytosine deaminase activity. By contrast the transformation by FCY1 or FCA1 of the haploid FCY1-disrupted host strain restored sensitivity to 5-fluorocytosine and allowed growth on cytosine, as a source of pyrimidine, or ammonium. FCA1 as opposed to FCY1 contains an intron. FCA1 and FCY1 encode respectively 150- and 158- residue proteins of 60% identity. Both Fcy1p and Fca1p share common motifs with cytidine and CMP deaminases, but homology with cytosine deaminase of E. coli could not be detected.
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PMID:Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans. 900 Mar 74

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

An attempt was made to use simple cationic liposomes DC-Chol/DOPE and DDAB/DOPE (DC-Chol is 3 beta (N(N',N-dimethylaminoethane) carbamoyl) cholesterol, DDAB is dimethyldioctadecyl ammonium bromide and DOPE is dioleoylphosphatidylethanolamine) for transfer of Escherichia coli cytosine deaminase 'suicide' gene under the control of tissue-specific tyrosinase gene promoter directly into the murine melanoma B16(F10) tumor. Several repeated intratumoral injections of DNA-liposome complexes followed by intraperitoneal administrations of 5-fluorocytosine, which is converted to 5-fluorouracil, caused strong retardation of murine melanoma B16(F10) tumor growth and, in some cases, rejection of the pre-established tumor. The inhibition of tumor growth expressed as the increased survival of mice is better seen in the case of using DNA-DDAB/DOPE complexes as compared to DNA-DC-Chol/DOPE ones. It seems that the observed therapeutic effect appears to result from several factors: 5-fluorouracil generation by transfected cells, liposome toxicity (DDAB is more toxic than DC-Chol and hence more tumor cells are killed), increased transfection efficiency of surviving cancer cells (in this case DDAB is a better transfection agent than DC-Chol) and, finally, the bystander effect which causes destruction of cells untransfected with CD gene by easily diffusible 5-fluorouracil.
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PMID:The use of cationic liposomes DC-CHOL/DOPE and DDAB/DOPE for direct transfer of Escherichia coli cytosine deaminase gene into growing melanoma tumors. 904 44

A recombinant adenovirus expressing Escherichia coli cytosine deaminase (AdCD) was constructed with the purpose of exploring its utility for the treatment of breast cancer. Infection of the human breast cancer cell line, MDA-MB-231, with AdCD resulted in high levels of cytosine deaminase enzyme activity. MDA-MB-231 cells infected with AdCD were 1000-fold more sensitive to 5-fluorocytosine (5-FC) than cells infected with a control adenovirus. Cell mixing experiments indicated that only 10% of AdCD-infected cells in a population were needed to induce complete cytotoxicity of noninfectious cells exposed to 5-FC. This suggests that bystander effects play an important role in AdCD-mediated cytotoxicities. Direct injection of AdCD into human breast MDA-MB-231-derived tumors grown as xenografts in nude mice, followed by daily intraperitoneal injection 5-FC was sufficient to inhibit tumor growth. These results suggest that in vivo gene therapy for breast cancer using AdCD is feasible.
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PMID:Enzyme/prodrug gene therapy approach for breast cancer using a recombinant adenovirus expressing Escherichia coli cytosine deaminase. 908 Jan 20

To develop a model system for studies of homologous recombination in plants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia lines were generated harbouring a single target T-DNA containing the negative selective codA gene encoding cytosine deaminase (CD) and the beta-glucuronidase (GUS) gene. Subsequently, the target lines were transformed with a replacement-type T-DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin-resistance gene. For both Nicotiana species kanamycin-resistant lines were selected which had lost the CD gene and the GUS activity. One tobacco line was the result of a precise gene targeting event. However, most other lines were selected due to a chromosomal deletion of the target locus. The deletion frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf protoplasts. T-DNA transfer was not required for induction of the deletions, indicating that the target loci were unstable. A few lines were obtained in which the target locus had been deleted partially. Sequence analysis of the junctions revealed deletion of DNA sequences between microhomologies. We conclude that T-DNAs, which are stable during plant development as well as in transmission to the offspring, may become unstable during propagation in callus tissue. The relationships between callus culture, genetic instability and the process of T-DNA integration and deletion in the plant genome are discussed.
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PMID:Gene targeting and instability of Agrobacterium T-DNA loci in the plant genome. 916 Oct 32


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