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Arabinosylguanine Is Phosphorylated by Both Cytoplasmic Deoxycytidine Kinase and Mitochondrial Deoxyguanosine Kinase¹

Departments of Experimental Therapeutics [C. O. R., M. A., V. G.] and Leukemia [V. G.], The University of Texas M. D. Anderson Cancer Center, and The Graduate School of Biomedical Sciences, Houston, Texas 77030-4095; Departments of Pharmacology and Medicine, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599 [B. S. M.]; and Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, S-751 23 Uppsala, Sweden [S. E.]

The prodrug of 9-ß-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-ß-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 µM) and in dCK⁺ extracts at high concentrations of ara-G (100 µM). 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 µM 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 µM ara-G, intracellular ara-GTP accumulated to similar levels (P = 0.5529) in these cell types; 25 ± 3.7 µM in dCK⁺, and 27.8 ± 2.7 µM 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.

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