Phase and Cell Cycle Specificity of Pyrazofurin Action

Abstract Pyrazofurin killed hepatoma 3924A cells in tissue culture with a 50% lethal dose of 0.4 µm, as determined by the colony-forming ability of the cells after 7-day incubation with the drug. Only in the logarithmic phase of growth were the hepatoma cells sensitive to the cytotoxic action of Pyrazofurin which had no detectable effect on plateau-phase cells. In the logarithmic growth phase, treatment of the culture with Pyrazofurin for 1 or 2 hr resulted in a biphasic dose-response curve. In synchronized tumor cell cultures, it was shown that the cells sensitive to Pyrazofurin were those in early G 1 and early and mid-S phases of the cycle. Uridine and cytidine protected hepatoma 3924A cells from the cytotoxic action of Pyrazofurin. In the presence of 0.5 µm Pyrazofurin for 7 days, an equimolar concentration of uridine resulted in 70% survival, and 5 to 10 µm uridine provided complete protection. In the presence of 1 µm Pyrazofurin, cytidine (1.0 mm) provided 80% protection, and uridine (0.1 mm) resulted in 90% survival; hypoxanthine and uracil were ineffective. In hepatoma 3924A cells, uridine and cytidine were rapidly incorporated into trichloroacetic acid insoluble material. Uracil was incorporated at a slower rate. Pyrazofurin enhanced the incorporation of uridine into nucleic acids 2-fold and that of uracil 3-fold. In the logarithmically growing hepatoma cells, the activity of orotidine 5′-monophosphate decarboxylase (EC 4.1.1.23), the target of Pyrazofurin 5′-monophosphate, was increased 3.3-fold over that observed in the plateau-phase tumor cells. In the lethal effect of Pyrazofurin, the growth phase and the increased activity of orotidine 5′-monophosphate decarboxylase play an important role. In the protection of the tumor cells from the drug, the rapid uptake of uridine and cytidine and the high activity and strong affinity of uridine-cytidine kinase towards uridine and cytidine are significant. In the failure of protection by uracil, the slow incorporation of this nucleoside in these hepatoma cells is relevant.