Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency is thought to cause T-lymphocyte depletion by accumulation of dG and
dGTP, resulting in feedback inhibition of
ribonucleotide reductase (RR; EC 1.17.4.1) and hence
DNA synthesis. To test for additional toxic mechanisms of dG, we selected a double mutant of the mouse T-
lymphoma S-49 cell line, dGuo-L, which is deficient in PNP and partially resistant to
dGTP feedback inhibition of RR. The effects of dG on dGuo-L cells (concn. causing 50% inhibition, IC50 = 150 microM) were compared with those on the wild-type cells (IC50 = 30 microM) and the NSU-1 mutant with
PNP deficiency only (IC50 = 15 microM). Fluorescence flow cytometry showed that equitoxic dG concentrations arrested wild-type and NSU-1 cells at the G1-S interface while allowing continued
DNA synthesis in the S-phase, whereas the double mutant dGuo-L cells progressed through the cell cycle normally. dGuo-L cells accumulated high levels of
dGTP in G1-phase, but not in S-phase cells, because of the utilization of
dGTP for
DNA synthesis and limited capacity to synthesize
dGTP from dG. These results support the hypothesis that dG/
dGTP toxicity occurs in the G1-phase or at the G1-S interface. Failure of dG to arrest the double mutant dGuo-L cells at the G1-S interface allows these cells to escape into S-phase, with an accompanying drop in
dGTP levels. Thus the partial resistance of dGuo-L cells to dG toxicity may result from their shorter residence time in G1, allowing them to sustain higher
dGTP levels. Hence RR inhibition by dGuo may not be the primary toxic mechanism in S-49 cells; rather, it may serve as an accessory event in dG toxicity by keeping the cells in the sensitive phase of the cell cycle. Among the possible targets of dG toxicity is
RNA synthesis, which was inhibited at an early stage in dGuo-L cells.