Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin‐inhibited dna synthesis

Abstract

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.

Nucleotide Biosynthesis Links Glutathione Metabolism to Ferroptosis Sensitivity

“…To test this hypothesis, we first treated cells with small molecule inhibitors of pyrimidine and purine metabolism, which generate the ribonucleotide precursors that RNR reduces to dNTPs in a GSH-dependent manner. Cells were pretreated with mycophenolic acid (MPA), to inhibit the purine synthetic enzyme inosine-5′-monophosphate dehydrogenase (IMPDH), or with pyrazofurin (Pyr), to inhibit the pyrimidine synthetic enzyme orotidine 5'-monophosphate decarboxylase (UMPS) (Fleming et al, 1996;Ringer et al, 1991). Nucleotide depletion itself triggers p53 stabilization, and we confirmed that both MPA and Pyr caused this effect in HT-1080 Control cells (Figure 4A).…”

Nucleotide biosynthesis links glutathione metabolism to ferroptosis sensitivity

“…To test this hypothesis, we first treated cells with small molecule inhibitors of pyrimidine and purine metabolism, which generate the ribonucleotide precursors that RNR reduces to dNTPs in a GSH-dependent manner. Cells were pretreated with mycophenolic acid (MPA), to inhibit the purine synthetic enzyme inosine-59-monophosphate dehydrogenase, or with pyrazofurin (Pyr), to inhibit the pyrimidine synthetic enzyme orotidine 5'-monophosphate decarboxylase (UMPS) (Ringer et al, 1991;Fleming et al, 1996). Nucleotide depletion itself triggers p53 stabilization, and we confirmed that both MPA and Pyr caused this effect in HT-1080 Control cells (Fig 4A).…”

Targeting Pyrimidine Metabolism in the Era of Precision Cancer Medicine

“…Pyrazofurin was found to inhibit the orotidine 5′monophosphate decarboxylase activity of UMPS as a nucleoside analogue in the last century with high efficacy especially in acute myelogenous leukemia patients (162). Nevertheless, the resistance to pyrazofurin appeared early in clinical (163)(164)(165).…”