The isolated perfused pat liver exports the purines adenine and hypoxanthine at a rate such that 0.5-1.0 MuM of each compound is maintained in the perfusate. It was found that this concentration of either purine completely reversed the growth inhibition of L1210 and HL-60 cells caused by DON, an inhibitor of de novo purine biosynthesis. Also, 1 MuM adenine (in the absence of DON) completely inhibited de novo purine synthesis. Thus, physiologic concentrations of these purines can support cell growth in the absence of de novo synthesis. An in vivo tumor model was established to study the actual utilization of circulating purines and pyrimidines by a solid tumor. Studies with 15NH4CL and 15N-glutamine demonstrated extensive involvement of theurea cycle in the formation of pyrimidines, de novo, in isolated rat hepatocytes. 3-Deazauridine and dipyridamole were evaluated as inhibitors of uridine salvage in vivo. 3-Deazauridine produced a marked, but short-lived, inhibition of uridine salvage in liver, kidney, and L1210 tumor cells. Dipyridamole did not significantly reduce salvage. A series of 29 analogues of uridine was examined as inhibitors of uridine kinase in vitro and of uridine salvage by intact L1210 cells. Of these compounds, cyclopentenyl uracil is most promising since it selectively blocks the salvage of uridine by L1210 cells, is resistant to phosphorolysis, and is non-cytotoxic at biochemically active concentrations. Uridine phosphorylase was purified to homogeneity and tested in vivo for its ability to reduce the concentration of circulating uridine that would be available for salvage. Uridine phosphorylase alone produced a modest (20%) decrease in uracil nucleotides of L1210 ascites cells, but greatly depleted (greater than 95%) pools in combination with PALA, an inhibitor of pyrimidine synthesis de novo. A series of uridine analogues modified in the 5'-position of the ribosyl moiety was evaluated as inhibitors and substrates of uridine phosphorylase. Replacement or esterification of the 5'-hydroxyl group led to nearly complete loss of susceptibility to phosphorolysis.
