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 Citations (Scopus)

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

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deoxyguanosine kinase
Deoxycytidine Kinase
Cell-Free System
Cell Line
Phosphotransferases
Enzymes
9-arabinofuranosylguanine

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.

Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. / Rodriguez, Carlos O.; Mitchell, Beverly S.; Ayres, Mary; Eriksson, Staffan; Gandhi, Varsha.

In: Cancer Research, Vol. 62, No. 11, 01.06.2002, p. 3100-3105.

Research output: Contribution to journalArticle

Rodriguez, CO, Mitchell, BS, Ayres, M, Eriksson, S & Gandhi, V 2002, 'Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase', Cancer Research, vol. 62, no. 11, pp. 3100-3105.
Rodriguez CO, Mitchell BS, Ayres M, Eriksson S, Gandhi V. Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Cancer Research. 2002 Jun 1;62(11):3100-3105.
Rodriguez, Carlos O. ; Mitchell, Beverly S. ; Ayres, Mary ; Eriksson, Staffan ; Gandhi, Varsha. / Arabinosylguanine is phosphorylated by both cytoplasmic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. In: Cancer Research. 2002 ; Vol. 62, No. 11. pp. 3100-3105.
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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.",
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AU - Rodriguez, Carlos O.

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N2 - 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.

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