Isolation of a Chinese Hamster Cell Mutant with Low Intracellular Phosphoribosylpyrophosphate Concentration

Abstract

A tritium-adenine suicide procedure was used to select for mutants with reduced uptake of adenine from a population of Chinese hamster V79 cells mutagenized with ethyl methane sulfonate. In one of the mutant lines isolated, designated KC62, the uptake of adenine, hypoxanthine, and guanine was reduced by approximately 70%. The specific activities, Km values, and Vma values of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase were the same in extracts from KC62 and from the parental cell line. Metabolic fate studies of incorporated [3H]adenine and [3H]hypoxanthine revealed a metabolic block at the level of phosphoribosylation. Determination of phosphoribosylpyrophosphate pool size showed that the mutant contained only 25% of the phosphoribosylpyrophosphate found in the parent. Its reduced availability in KC62 appears to result in a decreased ability to salvage adenine, hypoxanthine, and guaninine via phosphoribosylation. Phosphoribosylpyrophosphate synthetase from KC62 was shown to have an increased sensitivity to inhibition by a variety of nucleotides.In recent years, numerous variant cell lines have been isolated which are resistant to the toxic effects of adenine analogs. Cell lines resistant to 2,6-diaminopurine have been isolated from Chinese hamster ovary (CHO) lines (7, 33), from human and mouse fibroblasts (19,24), and from human and mouse lymphocytes (4, 30). Other adenine analog-resistant lines have been selected with 2-fluoroadenine and 8-azaadenine. Lines resistant to 2-fluoroadenine have been reported for human and mouse fibroblasts (3,17). Mutants resistant to 8-azaadenine in CHO cell lines (15) and human lymphocytes (30) have also been reported.Adenine analog resistance in all of the cases above was shown to result from decreased activity of the adenine salvage enzyme adenine phosphoribosyltransferase (APRT). Decreased APRT activity affords these cell lines resistance to toxic analogs since phosphoribosylation is necessary for the toxic effects of adenine analogs to be expressed. The requirement for metabolic activation, generally by phosphoribosylation in the case of purines and pyrimidines, conceivably could restrict the variety of mutant cell types which can be isolated with structural analogs.It should be possible to obtain a wider variety of mutant phenotypes with the selection procedure known as tritium suicide. The killing effects of tritium depend upon radiolytic decay of an incorporated compound and not upon metabolic activation of a potential metabolite.