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)

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogeneous. The molecular weight of cytosine deaminase was determined to be approx. 230000 containing four identical subunits with each subunit having a molecular weight 54000. Cytosine was deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50 degrees C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The Km and V values for cytosine were 0.74 mM and 47.16 mumole/min, respectively. As effectors of enzyme activity, PPi stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171-1174).
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PMID:Purification and some properties of cytosine deaminase from Salmonella typhimurium. 675 59

The synthesis of cytosine deaminase in Salmonella typhimurium is repressed by pyrimidines. This repression is mediated by both a uridine and a cytidine compound, indicating a distinct difference in the regulation of synthesis of cytosine deaminase from the regulation of the de novo pyrimidine pathway enzymes. A salvage role for the enzyme in pyrimidine metabolism is postulated.
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PMID:Repression of cytosine deaminase by pyrimidines in Salmonella typhimurium. 703 40

For many years, antibodies have been examined as means to deliver cytotoxic proteins to kill target cells (immunotoxins). More recently, there have been studies on enzymes that convert prodrugs to active drugs to kill target cells. The advances in gene therapy strategies now allow one to deliver the gene for the protein or enzyme as an alternative. This technique, although in its infancy, promises to overcome some of the problems associated with antibody-mediated delivery. Thymidine kinase and cytosine deaminase are some of the enzymes currently being exploited in this way, but more are on the horizon. However, more research is still needed to enable full exploitation of the transcriptional differences between tumour and normal cells so that more existing cancers can be treated in this way.
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PMID:Genetic delivery of enzymes for cancer therapy. 755 83

Pyrimidine synthesis in the food spoilage agent Burkholderia cepacia ATCC 25416 was investigated. The five de novo pathway enzymes of pyrimidine biosynthesis were found to be active in B. cepacia ATCC 25416 and growth of this strain on uracil had an effect on the de novo enzyme activities. The in vitro regulation of aspartate transcarbamoylase activity in B. cepacia ATCC 25416 was studies and its activity was inhibited by PP(i), ATP, GTP, CTP and UTP. The enzymes cytidine deaminase, uridine phosphorylase and cytosine deaminase were found to be active in the salvage of pyrimidines in ATCC 25416. Overall, de novo pyrimidine synthesis in B. cepacia ATCC 25416 was regulated at the level of enzyme activity and its pyrimidine salvage enzymes differed from those found in B. cepacia ATCC 17759.
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PMID:Pyrimidine synthesis in Burkholderia cepacia ATCC 25416. 757 30

The 5' sequences from the human carcinoembryonic antigen gene (CEA) were analyzed using luciferase reporter gene assays. This analysis identified important cis-acting sequences needed for selective expression in CEA-positive cells. Over 50 CEA/luciferase reporter clones were constructed and analyzed in two CEA-positive and two CEA-negative cell lines. The CEA sequences analyzed extended from the translational start to 14.5 kb 5' of the CEA gene. A 408-bp region from the CEA 3' untranslated region was also examined for its effect on reporter gene activity. The CEA promoter was located between bases -90 and +69 of the transcriptional start site. Sequences between -41 and -18 were essential for expression from the CEA promoter. Multimerization of sequences between -89 and -40 resulted in copy number-related increases in both expression level and selectivity for CEA-positive cells. Two upstream regions of CEA, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the multimerized promoter led to high-level, selective expression in CEA-positive cell lines. Several CEA/luciferase constructs demonstrated 80- to 120-fold higher expression in CEA-positive cell lines compared to expression in CEA-negative Hep3B cells. The expression from these constructs was quite strong in CEA-positive cells, being two- to four-fold higher than an SV40 enhancer/promoter construct. The most promising CEA transcriptional regulatory sequences were used to regulate the expression of cytosine deaminase (CD) in stable cell lines. The expression of CD was assessed directly by an enzymatic assay and indirectly by determining the in vitro IC50 to 5-fluorocytosine (5FC). The chimeric gene pCEA/CD-145 displayed the desired expression spectrum--high-level expression in the CEA-positive cells and low-level expression in CEA-negative cells. CD expression from this chimera correlated well with the expression of the endogenous CEA gene. Treatment of mice bearing NCI H508 pCEA/CD-145 tumor xenografts with 5FC lead to significant antitumor effects in vivo. The CEA/CD chimeric gene should be useful for tumor-specific suicide gene therapy of CEA-positive tumors.
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PMID:Transcriptional regulatory sequences of carcinoembryonic antigen: identification and use with cytosine deaminase for tumor-specific gene therapy. 757 7

To evaluate the concept that in vivo transfer of the Escherichia coli cytosine deaminase gene will confer sensitivity of a solid tumor to the prodrug 5-fluorocytosine (5FC), we constructed an adenovirus vector (AdCMV.CD) carrying the cytosine deaminase gene driven by the cytomegalovirus (CMV) promoter, infected HT29 colon carcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CD produced a functional cytosine deaminase protein in HT29 cells in vitro as evidenced by the ability of lysates from the infected cells to convert [3H]5FC to its active metabolite 5-fluorouracil (5FU). The AdCMV.CD vector effectively suppressed HT29 cell growth in vitro in the presence of 5FC in a dose-dependent manner. Infection with AdCMV.CD, when as few as 10% of cells expressed the cytosine deaminase gene, was associated with a bystander effect when combined with 5FC in cell mixing studies. Further, this bystander effect was not dependent on cell-to-cell contact as demonstrated by suppression of [3H]thymidine incorporation in HT29 cells when supernatant from AdCMV.CD-infected cells treated with 5FC was transferred cells. Consistent with these in vitro observations, when AdCMV.CD was directly injected into established subcutaneous HT29 tumors in nude mice receiving 5FC, there was a four-fold reduction in tumor size at day 15 compared to controls, and a five-fold reduction at day 28. These observations suggest that adenovirus-mediated gene transfer of the E. coli cytosine deaminase gene and concomitant administration of 5FC may have potential as a strategy for local control of the growth of tumor cells susceptible to 5FU.
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PMID:In vivo adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma-derived tumors induces chemosensitivity to 5-fluorocytosine. 757 18

Current treatments for metastatic malignant disease are often ineffective. One of the most promising of the selective genetic strategies against cancer is VDEPT (virally directed enzyme prodrug therapy). This uses a viral vector to carry a prodrug-activating enzyme gene into both tumour and normal cells. By linking the foreign gene downstream of tumour-specific transcription units, tumour-specific expression of the foreign enzyme gene can be achieved. We have developed a genetic therapy strategy using VDEPT against cancers that overexpress the oncogene ERBB2. This occurs in approximately one-third of breast and pancreatic tumours (and in a smaller proportion of other tumours) and involves transcriptional up-regulation of the ERBB2 gene with or without gene amplification. We have constructed a chimeric minigene consisting of the proximal ERBB2 promoter linked to the gene encoding cytosine deaminase, an enzyme that can deaminate the prodrug 5-fluorocytosine (5-FC) to form cytotoxic 5-fluorouracil (5-FU). We have constructed a double-copy recombinant retrovirus to deliver the enzyme gene under the control of the ERBB2 promoter into a panel of ERBB2 expression-positive (ERBB2+) and -negative (ERBB2-) pancreatic and breast cell lines. Cytosine deaminase activity was high in ERBB2+ transduced cells but was not detected in ERBB2- transduced cells. Significant cell death was observed in ERBB2+ transduced cells treated with 5-FC whereas ERBB2- cells were not affected. Hence we present a novel gene therapy strategy that is potentially tumour-specific and could be used against a range of tumour types that overexpress the ERBB2 oncogene.
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PMID:Gene therapy for cancer using tumour-specific prodrug activation. 758 78

We have been developing an enzyme/prodrug gene therapy approach for the treatment of primary and metastatic tumors in the liver. This system uses the cytosine deaminase/5-fluorocytosine (CD/5-FCyt) enzyme/prodrug combination. Another system that has received considerable attention is the herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) enzyme/prodrug combination. The purpose of the present study was to compare these two enzyme/prodrug systems. The human colorectal tumor cell line, WiDr, was genetically modified to express either the CD gene (WiDr/CD) or the HSV-TK gene (WiDr/TK). The IC50 (concentration of drug producing 50% inhibition of cell growth) for GCV was approximately 3.4 microM in WiDr/TK cells, while the IC50 for 5-FCyt was approximately 27 microM in WiDr/CD cells. In vivo antitumor studies were conducted using high but nontoxic levels of GCV (50 mg/kg/day) or 5-FCyt (500 mg/kg/day). When tumor xenografts were composed of 100% of cells expressing either HSV-TK or CD, 100% tumor-free animals were observed after GCV or 5-FCyt treatment, respectively. However, when only 10% of the tumor cells expressed HSV-TK, no antitumor effect by GCV treatment could be observed. In contrast, when tumors were composed of 4% of the cells expressing CD, 60% of the animals were tumor-free after 5-FCyt treatment. Transmission electron microscopy of the WiDr solid tumors revealed the presence of desmosomes but no gap junctions.
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PMID:Enzyme/prodrug gene therapy: comparison of cytosine deaminase/5-fluorocytosine versus thymidine kinase/ganciclovir enzyme/prodrug systems in a human colorectal carcinoma cell line. 758 11

A yeast strain lacking cytosine deaminase activity and over-expressing the cytosine proton symport has been used to study three aspects of symport function. (1) The proton flow during cytosine uptake after depletion of cellular ATP implies that the distribution of cytosine eventually approaches equilibrium with the proton gradient, one proton being absorbed with each molecule of cytosine. After correction for the presence of a minor leak pathway for cytosine, the cytosine distribution during energy metabolism was used to assay the magnitude of delta microH. Values of about 280 mV at pH 5 were obtained in this way. (2) Certain other substrates of the cytosine symport (hypoxanthine and especially fluorocytosine) cause the uptake of more than one equivalent of protons, but nevertheless accumulate to the same extent as cytosine. This phenomenon appears to be distinct from that of proton slip and is termed pseudochannelling. (3) The recycling of symported protons through the proton pump is an ill-defined process in plants and fungi. It usually occurs only after a distinct time lag during which the change in bulk intracellular pH may be relatively small. We have found conditions where there is no apparent time lag before protons entering yeast with glycine or histidine are recycled. This behaviour is discussed in relation to the possible voltage characteristics of the proton pump, its putative regulation by delta microH and the metabolic consequences of ATP hydrolysis being accelerated.
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PMID:Proton and charge circulation through substrate symports in Saccharomyces cerevisiae: non-classical behaviour of the cytosine symport. 759 38

Pyrimidine nucleoside catabolism in the human pathogen Sphingomonas paucimobilis was studied. It was observed that S. paucimobilis was only capable of utilizing cytidine or deoxycytidine as a sole nitrogen source when glucose served as the carbon source. Thinlayer chromatographic analyses of cytidine and uridine catabolic products revealed that the enzymes cytidine deaminase and uridine phosphorylase were active in the extracts prepared from S. paucimobilis cells. The levels of cytidine deaminase and cytosine deaminase activities were lowered after growth on cytidine or deoxycytidine as a nitrogen source instead of ammonium sulfate. Uridine phosphorylase activity increased more than 4-fold after growth on deoxycytidine as a nitrogen source while growth on the nitrogen source cytidine caused a depression in phosphorylase activity.
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PMID:Pyrimidine nucleoside catabolism in Sphingomonas paucimobilis: role of cytidine deaminase and uridine phosphorylase. 760 8


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