The
prodrug of 9-beta-D-arabinosylguanine (
ara-G),
nelarabine, demonstrated efficacy against T-cell
acute lymphoblastic leukemia, and its effectiveness correlated with the accumulation of the
triphosphate form (
ara-GTP). Although in vitro investigations using purified
deoxycytidine kinase (dCK) or
deoxyguanosine kinase (dGK) suggested that
ara-G is a substrate for both
enzymes, controversy exists in regard to the role of these
enzymes in whole cells. In this work, we used a CEM mutant cell line containing low endogenous levels of dGK and deficient in dCK (dCK-) to assess the role of these
kinases in
ara-G phosphorylation. Using a retroviral vector system, we infected the dCK- mutant cell line to obtain cell lines with overexpression of dCK (dCK+) or dGK (dGK+). Only the dCK+ cell line phosphorylated 1-beta-D-arabinofuranosylcytosine (used as a substrate for dCK) in a cell-free system; phosphorylation of this compound by dGK+ was below the limit of detection. Again in in vitro assays, the dCK-and dCK+ cell lines phosphorylated dGuo to similar levels (0.91 +/- 0.15 and 0.93 +/- 0.19 pmol/mg/min, respectively), whereas dGK+ phosphorylated dGuo more efficiently (150 pmol at 60 min). When
ara-G was used as a substrate in a cell-free system, the maximum accumulation of phosphorylated product was observed in dGK+ extracts at low
ara-G levels (10 microM) and in dCK+ extracts at high concentrations of
ara-G (100 microM). Thus, both dCK and dGK can phosphorylate
ara-G, but at low
ara-G concentrations, dGK seems to predominate, whereas at higher
ara-G concentrations, dCK seems to be the preferred
enzyme. In whole-cell systems after a 3-h incubation with 10 microM
ara-G, both dCK+ and dGK+ cells accumulated
ara-GTP; however, the levels were significantly (P = 0.0008) higher in dGK+ cells. In contrast, at 100 microM
ara-G, intracellular
ara-GTP accumulated to similar levels (P = 0.5529) in these cell types; 25 +/- 3.7 microM in dCK+, and 27.8 +/- 2.7 microM in the dGK+ cells. These results from whole-cell experiments are consistent with those from the cell-free system and strongly suggest that
ara-G is phosphorylated by both
kinases, and at low substrate concentrations, dGK is preferred
enzyme. Evaluation of the expression of each of these
kinases in primary
leukemia cells may reveal a biochemical basis for the pharmacological differences in the accumulation of
ara-GTP.