title: Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase.
abstract: 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.
authors.affiliation: Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030-4095, USA.
authors.forename: Carlos O
authors.initials: CO
authors.lastname: Rodriguez
authors.forename: Beverly S
authors.initials: BS
authors.lastname: Mitchell
authors.forename: Mary
authors.initials: M
authors.lastname: Ayres
authors.forename: Staffan
authors.initials: S
authors.lastname: Eriksson
authors.forename: Varsha
authors.initials: V
authors.lastname: Gandhi
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism*; D001087:Arabinonucleosides / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D003842:Deoxycytidine Kinase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013601:T-Lymphocytes / Q000201:enzymology; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D001087:Arabinonucleosides; C028771:9-arabinofuranosylguanine; D017853:Phosphotransferases (Alcohol Group Acceptor); C019165:deoxyguanosine kinase; D003842:Deoxycytidine Kinase