Isolation and characterization of purine-nucleoside phosphorylase-deficient T-lymphoma cells and secondary mutants with altered ribonucleotide reductase: Genetic model for immunodeficiency disease

Abstract

The inherited deficiency of purine-nucleoside phosphorylase (PNPase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) in humans is associated with a severe deficiency of the T [thymus-derived] lymphocytes of the immune system. Because of the unsatisfactory nature of previously described model systems, a mutant mouse T cell lymphoma (S49) completely deficient in PNPase was selected, cloned and characterized. Of the 4 substrates of PNPase, only deoxyguanosine [dG] at low concentrations is toxic to the PNPase-deficient (NSU-1) cells. To delineate the biochemical processes necessary for the sensitivity of the NSU-1 cells to dG, a series of secondary mutants resistant to dG was isolated from the PNPase-deficient line. Of these mutants, one is defective in its ability to transport dG into the cell. A 2nd type of mutant cannot phosphorylate the dG and is totally deficient in dC kinase activity. A 3rd type of mutant (NSU-1-dGuo-L) can transport and phosphorylate dG and accumulates dGTP. Unlike its parent, NSU-1-dGuo-L does not become depleted of dCTP and TTP when exposed to exogenous dG. The reduction of CDP to dCDP by the ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2''-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2''-oxidoreductase EC 1.17.4.1) of NSU-1-dGuo-L cells is not normally sensitive to feedback inhibition by dGTP. To exert its toxicity dG must be transported into the cell, be phosphorylated by dC kinase, and be accumulated as dGTP. By inhibiting ribonucleotide reductase, dGTP depletes the cell of dCTP and to some extent TTP, thus preventing the synthesis of DNA, a process necessary for any proliferation-dependent function of T cells.