Abstract

The uptake, phosphorylation, and biological half-life of the purine nucleoside analogue 9-β-D-xylofuranosyladenine (xyl-A) was studied in wild-type Chinese hamster ovary cells and in nucleoside kinase-deficient mutants. It was found that [³H]xyl-A and [³H]adenosine were readily phosphorylated to the triphosphate level in both the wild-type and deoxycytidine kinase (EC 2.7.1.74)-deficient mutant, but neither of these adenine nucleosides was phosphorylated by the adenosine kinase (EC 2.7.1.20)-deficient cells. The reproductive capacity of wild-type and deoxycytidine kinase deficient cells was inhibited 50% by 3 and 4 µM xyl-A, respectively, whereas cells deficient in adenosine kinase were resistant to 100 µM xyl-A. Cellular uptake of xyl-A into the wild-type cells was followed through 6 hr of incubation. Values for the apparent K_m and V_max of this uptake process were 43.9 µM and 118.7 nmoles/min/10⁹ cells, respectively. The major intracellular metabolite of xyl-A, the 5'-triphosphate xyl-ATP, accumulated to a 3.6-fold higher concentration than xyl-ADP, with very little xyl-AMP detected. The biological half-life of xyl-ATP was 5.1 hr, significantly longer than the congener analogue, 9-β-D-arabinofuranosyladenine 5'-triphosphate, in the same cell line. These results demonstrate in a single cell line that xyl-A does not produce cytotoxicity as a free nucleoside; phosphorylation to the nucleoside 5'-triphosphate, an activating pathway initiated by adenosine kinase, is required for activity of the compound.