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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.
AuthorsCarlos O Rodriguez Jr, Beverly S Mitchell, Mary Ayres, Staffan Eriksson, Varsha Gandhi
JournalCancer research (Cancer Res) Vol. 62 Issue 11 Pg. 3100-5 (Jun 01 2002) ISSN: 0008-5472 [Print] United States
PMID12036920 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • Antineoplastic Agents
  • Arabinonucleosides
  • 9-arabinofuranosylguanine
  • Phosphotransferases (Alcohol Group Acceptor)
  • deoxyguanosine kinase
  • Deoxycytidine Kinase
Topics
  • Antineoplastic Agents (metabolism)
  • Arabinonucleosides (metabolism)
  • Cell Line
  • Cell-Free System
  • Cytosol (enzymology)
  • Deoxycytidine Kinase (biosynthesis, genetics, metabolism)
  • Humans
  • Kinetics
  • Mitochondria (enzymology)
  • Phosphorylation
  • Phosphotransferases (Alcohol Group Acceptor) (biosynthesis, genetics, metabolism)
  • T-Lymphocytes (enzymology)
  • Transfection

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