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)

Mode of action of 5-fluorocytosine (5-FC) and mechanisms of resistance to the drug are discussed on the basis of experiments performed with Candida albicans ATCC 26790 and with 50 selected clinical isolates of C. albicans belonging to serological type A or B and representing various degrees and models of 5-FC resistance (sensitivity). Incorporation of 5-fluorouridylic acid into RNA appeared as a prerequisite to antifungal activity, although at a given incorporation rate, growth inhibition varied considerably from one strain to the other. The amino acid pool was unbalanced, and there was evidence for disturbance of protein synthesis. These dysfunctions of RNA probably account for growth inhibition and cell death, whereas up to the present, there was no proof of formation of 5-fluorodeoxyuridylic acid nor of subsequent inhibition of thymidylate synthetase. Incorporation of fluorinated pyrimidine into RNA was lower in normally sensitive type B strains than in normally sensitive ones of type A, whereas the frequency of 5-FC-resistant mutants was the same. The two serological types did not differ in the activity of cytosine permease nor in that of cytosine deaminase. Among 29 clinical isolates with 6-FC resistance (or impaired sensitivity) no instance of cytosine permease deficiency was found. Two isolates (belonging to the serological type A) were deficient in cytosine deaminase, whereas the majority was probably deficient in uridine monophosphate pyrophosphorylase or had a surplus of de novo synthesis of pyrimidines. Relative 5-FC resistance was more common than complete resistance.
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PMID:Mode of action of 5-fluorocytosine and mechanisms of resistance. 109 64

Incorporation of labelled 14C-pyrimidines and 5-fluoropyrimidines (5-FC and 5-FU) in four different phenotypes of wild strains of Candida isolated from man showed comparable results to those obtained by the minimal inhibition concentration (MIC) test. Kinetics studies demonstrated significant rates of incorporation after 24 hours of culture in each case. It was also possible to infer the biochemical mechanisms of resistance to 5-FC, namely a defect in UMP pyrophosphorylase, cytosine deaminase and 5-FU permease in the ease of the following phenotypes: 5-FCR 5-FUR, 5-FCR 5-FUS and 5-FCS 5-FUR (R = resistant; S = sensitive). In this study, the permeation process was approached by a consumption assay which determined the rate of labelled substrates into the medium before and after 24 hours of culture. Thus, it was found that the consumption levels of the phenotype 5-FCS 5-FUS were very high, while those of the phenotype 5-FCR 5-FUR were minimal. The 5-FCS 5-FU5 phenotype had no detectable consumption of 5-FU. With regard to the 5-FC5 5-FUS phenotype, it seems that the non-incorporation of 5-FC into the RNAs after the consumption by the yeasts has a feed back effect on the permeation process.
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PMID:Sensitivity and resistance of pathogenic yeasts to 5-fluoropyrimidines. II.--Mechanisms of resistance to 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU) (author's transl). 121 21

Flucytosine (5-FC)-resistant strains were isolated from the haploid opportunistic pathogen Candida glabrata by UV-induced mutation and fluoropyrimidine selection. These strains were characterized biochemically, and the metabolism of fluorinated pyrimidines was studied by 19F nuclear magnetic resonance spectroscopy. No evidence was obtained from these studies for degradative metabolism of the fluorinated derivatives. In the parental susceptible strain of C. glabrata, 5-fluorouracil but not 5-FC was detected within the cells. 5-Fluorouracil was also present in the culture supernatant after incubation of the cells with 5-FC. The distribution of fluorinated derivatives within the 5-FC-resistant strains was consistent with their genotype. Two strains of C. glabrata which had only a partial loss of cytosine deaminase and UMP pyrophosphorylase activity had high levels of resistance to 5-FC. Both C. glabrata and Candida albicans were susceptible to 5-fluorouridine. This compound but not the anticancer drug 5-fluoro-2-deoxyuridine was shown to be transported into susceptible cells by a specific uridine permease.
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PMID:19F nuclear magnetic resonance study of fluoropyrimidine metabolism in strains of Candida glabrata with specific defects in pyrimidine metabolism. 229 66

In terms of genetically determined susceptibility to the clinical antifungal agent 5-fluorocytosine (5-FC), Candida albicans may be homozygous sensitive (FCY/FCY), homozygous resistant (fcy/fcy), or heterozygous (fcy/FCY). Although heterozygotes are only slightly resistant, they occur at significant frequency among clinical strains and carry preexisting resistance determinants which may be responsible, following homozygosis, for treatment failures. There are two resistance genes (FCY1 and FCY2) known. Resistance in fcy1/fcy1 strains was associated with decreased UMP pyrophosphorylase activity, whereas resistance in fcy2/fcy2 strains was associated with decreased cytosine deaminase activity. These results were confirmed and extended in a 19F nuclear magnetic resonance study of 5-FC uptake and metabolism in genetically defined strains. By means of hybridization via spheroplast fusion, a complementation test was devised to test allelism of resistance determinants. Resistance to 5-FC was employed as a useful genetic marker in basic studies. In tetraploid hybrids which bore appropriate fcy markers, it was possible to select for reduction in ploidy by selecting for increased resistance to 5-FC; a novel parasexual system was thus generated (2n x 2n----4n----2n). In linkage studies, the gene FCY1 was shown to be linked to the gene HIS. Reciprocal mitotic recombination was demonstrated repeatedly with fcy1 and his alleles in cis and in trans configurations and evidence for nonreciprocal recombination (mitotic gene conversion) was also obtained. In Cryptococcus neoformans, mutation in either of two genes (FCY1, FCY2) is sufficient to confer resistance. These genes behave as simple Mendelian determinants which recombine freely. Diploid C. neoformans heterozygous for resistance (FCY/fcy) provided useful strains in which to develop genetic mapping methodology based on mitotic recombination.
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PMID:The genetic basis of resistance to 5-fluorocytosine in Candida species and Cryptococcus neoformans. 331 20

A complementation test was devised to study allelism among the genetic determinants of resistance to 5-fluorocytosine in Candida albicans. Complementation was demonstrated in control hybrids produced by crossing a resistant strain that was deficient in cytosine deaminase activity with four other resistant strains deficient in UMP pyrophosphorylase activity. This complementation test was used to test allelism of the resistance determinants present in five clinical isolates. All were found to bear recessive alleles of the locus (FCY1) that determined 5-fluorocytosine resistance associated with low levels of UMP pyrophosphorylase activity.
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PMID:Complementation analysis of resistance to 5-fluorocytosine in Candida albicans. 352 27

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.
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PMID:Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine. 978 50

Transduction of the cytosine deaminase (CD) gene into tumor cells followed by administration of 5-fluorocytosine (5-FC), called 5-FC/CD gene therapy, was created as suicide gene therapy for various cancers. The uracil phosphoribosyltransferase (UPRT) gene, which is absent from mammalian cells, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine 5'-monophosphate. We evaluated whether the coexpression of CD and UPRT genes could generate a synergistic antitumor effect on experimental brain tumors. In vitro study showed that 9L cells, transduced with the UPRT gene by an adenovirus, were 16 times more sensitive to 5-FU, and CD + UPRT-transduced cells were 6,000 times more sensitive to 5-FC than parent cells, indicating that the acquisition of CD and UPRT further increased the 5-FC sensitivity of 9L cells compared with cells transduced with CD alone. In a rat brain tumor model, decreased amounts of CD and UPRT vectors were inoculated into the tumors to detect any additional effect of UPRT. CD and UPRT coexpression followed by 5-FC administration showed an antitumor effect as detected by sequential magnetic resonance imaging. This therapy significantly prolonged animal survival. These results suggest that 5-FC/CD + UPRT gene therapy can enhance the antitumor effect of 5-FC/CD gene therapy. Consequently, this approach might be a more feasible modality for the treatment of malignant brain tumors.
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PMID:Experimental gene therapy for brain tumors using adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene with 5-fluorocytosine. 1064 41

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.
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PMID:Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine. 1091 4

Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.
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PMID:In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene. 1091 55

Virus-directed enzyme prodrug therapy (VDEPT) utilising cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) into the chemotherapy agent, 5-fluorouracil (5-FU), and has entered into a clinical trial for metastatic colon cancer. To improve this system, a replication-deficient adenovirus, containing a bifunctional fusion gene, CD:uracil phosphoribosyltransferase (UPRT), was constructed (AdCDUPRT). UPRT enhances the conversion of 5-FU into its active metabolites, which inhibit DNA and RNA synthesis. In vitro, AdCDUPRT infection of colon cancer cells resulted in a marked increase in sensitisation to 5-FU, compared with AdCD-infected or uninfected cells. The corollary is a approximately 100-fold and approximately 10 000-fold increase in sensitisation to 5-FC in AdCDUPRT-infected cells, compared to AdCD-infected and uninfected cells, respectively. There was a strong bystander effect in vitro, 70% of tumour cells were killed by 5-FC when only 10% of cells expressed CDUPRT. In vivo, athymic mice with colon cancer xenografts treated with intratumoral AdCDUPRT and intraperitoneal 5-FC, significantly reduced tumour growth rates compared with untreated controls (P = 0.02), whereas AdCD/5-FC treated mice did not. At higher AdCDUPRT virus doses, 5-FC and 5-FU were equally effective at delaying tumour growth compared with controls. In summary, VDEPT for colon cancer utilising AdCDUPRT is more effective than AdCD and the bifunctional CDUPRT gene enables the use of either 5-FC or 5-FU as prodrugs.
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PMID:In vivo gene therapy for colon cancer using adenovirus-mediated, transfer of the fusion gene cytosine deaminase and uracil phosphoribosyltransferase. 1170 15


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