Characterization of analog resistance and purine metabolism of adult rat-liver epithelial cell 8-azaguanine-resistant mutants

Adult rat-liver epithelial cultures were sensitive to the lethal effects of 8-azaguanine (AG), but lines contained variants resistant to AG. The frequency of retrievable AG-resistant colonies varied with both the concentration of AG used and the seeding density of the population under selection. Cells resistant to AG were also cross-resistant to 6-thioguanine and unable to grow in medium containing hypoxanthine, aminopterin and thymidine. Resistance was stable. AG resistance was due to a deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) activity which was not caused by an inhibitor. In the assay for HGPRTase, a substantial amount of product appeared as inosine (In) in addition to inosine monophosphate (IMP). Purine nucleoside phosphorylase will generate In from hypoxanthine and, indeed, the cells did possess this activity. However, several findings indicated that the In was derived from IMP by catabolism by 5'-nucleotidase (NTase): (1) IMP decreased as In increased and (2) the inhibitors of NTase, adenosine monophosphate and thymidine triphosphate, reduced the generation of In by over 90% without inhibiting purine nucleoside phosphorylase. The cells possessed substantial NTase activity, 35% of which was located in the cytosol along with 69% of HGPRTase. Several lines of evidence suggested that the NTase activity limited the amount of 8-azaguanylic acid presented to the cells by catabolising the nucleotide and, thereby, reducing the toxicity of available AG.