Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase

Carlos O. Rodriguez, Beverly S. Mitchell, Mary Ayres, Staffan Eriksson, Varsha Gandhi

Research output: Contribution to journalArticle

62 Scopus citations

Abstract

The prodrug of 9-β-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-β-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.

Original languageEnglish (US)
Pages (from-to)3100-3105
Number of pages6
JournalCancer Research
Volume62
Issue number11
StatePublished - Jun 1 2002
Externally publishedYes

    Fingerprint

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Rodriguez, C. O., Mitchell, B. S., Ayres, M., Eriksson, S., & Gandhi, V. (2002). Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Cancer Research, 62(11), 3100-3105.