Biochemical phenotype of 5-fluorouracil-resistant murine T-lymphoblasts with genetically altered CTP synthetase activity.

Abstract

From wild type mouse lymphoma cells, a clone, (FURT-1A), was isolated by virtue of its resistance to 1 microM 5-fluorouracil and 10 microM thymidine. In comparative growth rate experiments, FURT-1A cells were also less sensitive than parental cells to the growth inhibitory effects of thymidine, deoxyguanosine, 5-fluorouridine, and arabinosylcytosine. The altered growth sensitivity of FURT-1A cells to cytotoxic nucleosides was directly related to their decreased ability to accumulate the corresponding triphosphate from exogenous nucleoside. FURT-1A cells contained elevated cytidylate nucleotide pools which prevented normal growth sensitivity and interfered with the salvage of nucleosides by inhibiting nucleoside kinase activities, by stimulating nucleotide dephosphorylating activities, and by overcoming certain allosteric inhibitions imposed on ribonucleotide reductase. Metabolic flux experiments with [3H]uridine in situ indicated that FURT-1A cells had a 2-fold enhanced rate of conversion of UTP to CTP. Kinetic analyses indicated that the CTP synthetase activity in extracts of FURT-1A cells was refractory to inhibition by CTP. The genetic loss of normal allosteric inhibition of the CTP synthetase activity in FURT-1A cells could account for the unusual phenotypic properties of these cells and conferred a high spontaneous mutator phenotype to cells possessing this mutation.