Uracil nucleotide synthesis in a human breast cancer cell line (MCF-7) and in two drug-resistant sublines that contain increased levels of enzymes of the de novo pyrimidine pathway

Abstract

Cultured wild-type MCF-7 human breast cancer cells and two MCF-7 sublines that overproduce enzymes of the de novo pyrimidine biosynthetic pathway were compared with regard to: rate of de novo biosynthesis of uracil nucleotides, sensitivity of the de novo and salvage pathways to the concentration of intracellular uracil nucleotides, and potential of exogenous uridine at concentrations equivalent to plasma levels to affect de novo pyrimidine biosynthesis. The PALAR MCF-7 subline, which is resistant to N-(phosphonacetyl)-L-aspartate and has 5.2 times the activity of the first de novo enzyme as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 5.8 times that of the wild type MCF-7 cells. The PYRR MCF-7 subline, which is resistant to pyrazofurin and has 15.1 times the activity of orotate phosphoribosyltransferase as the wild-type MCF-7 cells, synthesizes uracil nucleotides via the de novo pathway at a rate that is 1.4 times that of wild-type MCF-7 cells. These results are consistent with carbamyl phosphate synthetase being the rate-controlling step of de novo pyrimidine biosynthesis. In the presence of exogenous uridine at concentrations equivalent to that found in plasma (4.4-8.6 microM), the uracil nucleotide pool of wild-type MCF-7 cells was expanded by 20% and de novo synthesis was inhibited by 55%. Incubation of PALAR MCF-7 cells with uridine at concentrations between 7.3 and 16.8 microM caused a 40% increase in the uracil nucleotide pool and a 30% inhibition of de novo synthesis. De novo synthesis of uracil nucleotides in PYRR MCF-7 cells was not affected by a greater than 10-fold increase in the uracil nucleotide pool. Salvage of [14C] uridine was inhibited by an expanded uracil nucleotide pool in the wild-type and PYRR MCF-7 cells but was not inhibited in the PALAR MCF-7 cell line. These results demonstrate that, although the overproduced enzymes exhibit substrate affinities and specificities in cell-free preparations similar to those of the wild-type enzymes, in intact cells the resistant cell lines exhibit marked differences in the control of de novo and salvage pyrimidine biosynthetic pathways by intracellular uracil nucleotides.