Resistance to 5-fluorouracil associated with increased cytidine triphosphate levels in V79 Chinese hamster cells.
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Abstract:
Clones stably resistant to the toxic effects of 5-fluorouracil have been isolated from V79.5 Chinese hamster fibroblast cells by a single-step selection procedure. The 5-fluorouracil-resistant lines were found to (a) have an auxotrophic requirement when grown in dialyzed fetal calf serum that was satisfied by the addition of either thymidine, deoxyuridine, or deoxycytidine to the medium, (b) be cross-resistant to the toxic effects of 1-beta-D-arabinofuranosylcytosine and to high concentrations of thymidine, (c) have increased intracellular levels of cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate and decreased levels of uridine 5'-triphosphate, (d) also be resistant to 5-fluorouridine but not to 5-fluorodeoxyuridine, and (e) incorporate less 5-fluorouracil into RNA than do the wild-type cells. The primary lesion in these mutant appears to be an altered CTP synthetase activity which is no longer sensitive to negative regulation by CTP. The resulting increased CTP levels appear to be responsible for the various phenotypic characteristics of these mutants, including the resistance to 5-fluorouracil.Keywords:
Chinese hamster
Cytidine
Deoxyuridine
Uridine triphosphate
Thymidine
Uracil
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The nucleoside triphosphate pools of two cytidine auxotrophic mutants of Salmonella typhimurium LT-2 were studied under different conditions of pyrimidine starvation. Both mutants, DP-45 and DP-55, are defective in cytidine deaminase and cytidine triphosphate (CTP) synthase. In addition, DP-55 has a requirement for uracil (uridine). Cytidine starvation of the mutants results in accumulation of high concentrations of uridine triphosphate (UTP) in the cells, while the pools of CTP and deoxy-CTP drop to undetectable levels within a few minutes. Addition of deoxycytidine to such cells does not restore the dCTP pool, indicating that S. typhimurium has no deoxycytidine kinase. From the kinetics of UTP accumulation during cytidine starvation, it is concluded that only cytidine nucleotides participate in the feedback regulation of de novo synthesis of UTP; both uridine and cytidine nucleotides participate in the regulation of UTP synthesis from exogenously supplied uracil or uridine. Uracil starvation of DP-55 in presence of cytidine results in extensive accumulation of CTP, suggesting that CTP does not regulate its own synthesis from exogenous cytidine. Analysis of the thymidine triphosphate (dTTP) pool of DP-55 labeled for several generations with 32 P-orthophosphate and 3 H-uracil in presence of 12 C-cytidine shows that only 20% of the dTTP pool is derived from uracil (via the methylation of deoxyuridine monophosphate); 80% is apparently synthesized from a cytidine nucleotide.
Cytidine
Uracil
Nucleotide salvage
Uridine triphosphate
Thymidine
Pyrimidine metabolism
Deoxyuridine
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Schistosoma
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Cooperativity
Cytidine
Chinese hamster
Uridine triphosphate
Wild type
Adenosine triphosphate
Cooperative binding
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8 uridine-requiring pyr mutants were isolated from Aspergillus nidulans under nitrosoguanidine treatment. All the mutants are capable to grow on the medium containing 20 mkg/ml of uridine or cytidine, or 100 mkg/ml of uracil, and they do not utilize thymidine, thymine, cytosine and deoxyuridine. Their ability to grow in the presence of orotic acid demonstrates that the pyrimidine synthesis in all the mutants is blocked at stages preceding the conversion of orotic acid into orotidine monophosphate. All the pyr mutants are of nuclear nature, they are recessive and represent three complementation groups located in the VIII chromosome. Unlike U. maydis mutant, the requirement in pyrimidines does not increase the sensitivity of A. nidulans pyr mutants to UV-irradiation.
Aspergillus nidulans
Uracil
Orotic acid
Cytidine
Cytosine
Thymine
Pyrimidine metabolism
Deoxyuridine
Thymidine
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Clones stably resistant to the toxic effects of 5-fluorouracil have been isolated from V79.5 Chinese hamster fibroblast cells by a single-step selection procedure. The 5-fluorouracil-resistant lines were found to (a) have an auxotrophic requirement when grown in dialyzed fetal calf serum that was satisfied by the addition of either thymidine, deoxyuridine, or deoxycytidine to the medium, (b) be cross-resistant to the toxic effects of 1-beta-D-arabinofuranosylcytosine and to high concentrations of thymidine, (c) have increased intracellular levels of cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate and decreased levels of uridine 5'-triphosphate, (d) also be resistant to 5-fluorouridine but not to 5-fluorodeoxyuridine, and (e) incorporate less 5-fluorouracil into RNA than do the wild-type cells. The primary lesion in these mutant appears to be an altered CTP synthetase activity which is no longer sensitive to negative regulation by CTP. The resulting increased CTP levels appear to be responsible for the various phenotypic characteristics of these mutants, including the resistance to 5-fluorouracil.
Chinese hamster
Cytidine
Deoxyuridine
Uridine triphosphate
Thymidine
Uracil
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ABSTRACT. Pyrimidine salvage enzyme activities in cell‐free extracts of Toxoplasma gondii were assayed in order to determine which of these enzyme activities are present in these parasites. Enzyme activities that were detected included phosphoribosyltransferase activity towards uracil (but not cytosine or thymine), nucleoside phosphorylase activity towards uridine, deoxyuridine and thymidine (but not cytidine or deoxycytidine), deaminase activity towards cytidine and deoxycytidine (but not cytosine, cytidine 5′‐monophosphate or deoxycytidine 5′‐monophosphate), and nucleoside 5′‐monophosphate phosphohydrolase activity towards all nucleotides tested. No nucleoside kinase or phosphotransferase activity was detected, indicating that T. gondii lack the ability to directly phosphorylate nucleosides. Toxoplasma gondii appear to have a single non‐specific uridine phosphorylase enzyme which can catalyze the reversible phosphorolysis of uridine, deoxyuridine and thymidine, and a single cytidine deaminase activity which can deaminate both cytidine and deoxycytidine. These results indicate that pyrimidine salvage in T. gondii probably occurs via the following reactions: cytidine and deoxycytidine are deaminated by cytidine deaminase to uridine and deoxyuridine, respectively; uridine and deoxyuridine are cleaved to uracil by uridine phosphorylase; and uracil is metabolized to uridine 5′‐monophosphate by uracil phosphoribosyltransferase. Thus, uridine 5′‐monophosphate is the end‐product of both de novo pyrimidine biosynthesis and pyrimidine salvage in T. gondii.
Nucleotide salvage
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Deoxyuridine
Uracil
Thymidine
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The metabolism of pyrimidine nucleosides present in the plasma of the mouse has been examined.Uridine and cytidine are rapidly cleared from the circulation with tn of <5 min.Uracil, deoxycytidine, deoxyuridine, and thymidine are cleared more slowly with tu of 9 to 13 min.Various tissues differed markedly in the extent of nucleotide formation from circulating nucleosides.Cytidine and uridine are predominantly converted to nucleotides (>50%) rather than catabolized, whereas uracil is almost entirely degraded.Thymidine, deoxyuridine, and deoxycytidine are intermediate in the extent of their conversion to nucleotides: 8.9 to 21% of these nucleosides are salvaged in the mouse.Both anabolic and catabolic routes are important in the metabolism of pyrimidine nucleosides in vivo.
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Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10(-5) M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [3H]thymidine incorporation. Proliferation is completely restored by 5 X 10(-5) M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 X 10(-5) M each, fail to re-establish proliferation but produce an isotropic dilution of [3H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (less than 0.001 nmol X min-1 X 10(-6) cells).
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Uracil
Thymidine
Thymine
Deoxyuridine
De novo synthesis
Cytosine
Pyrimidine metabolism
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Growth (dry weight accumulation) of Sordaria fimicola in standing liquid culture (sucrose-nitrate-salts-vitamins) is inhibited by the presence of 5 μM 5-fluorouracil in the medium. This inhibition is completely prevented by uracil, deoxyuridine, and 5-bromouracil, partly prevented (40 to 90% of growth observed without 5-fluorouracil) by uridine, thymidine, and 5-bromodeoxyuridine, and slightly prevented by trifluorothymine, cytosine, cytidine, deoxycytidine, and 5-methylcytosine (all at 0.5 to 1 mM). Thymidine and thymine riboside were without any apparent effect. Growth is also inhibited by 0.2 mM 6-azauracil, and this inhibition was completely prevented by uracil and uridine, partly prevented by deoxyuridine, 5-bromouracil, cytidine, and 5-methylcytosine, and slightly prevented by thymine, thymidine, 5-bromodeoxyuridine, cytosine, and deoxycytidine. The data suggest that the observed inhibition of growth by 5-fluorouracil is due to inhibition of both ribonucleic acid and deoxyribonucleic acid synthesis. The data also allow inferences concerning pyrimidine interconversions in S. fimicola; i.e., thymine can be anabolized to thymidylic acid without first being demethylated, although demethylation appears to occur also.
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Cytidine
Thymine
Cytosine
Thymidine
Deoxyuridine
Bromodeoxyuridine
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Cytidine
Thymidine
Deoxyuridine
Chinese hamster
Uridine triphosphate
Cytosine
Auxotrophy
Nucleotide salvage
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