Abstract
BackgroundNine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is then phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis.MethodsIn the present study, we developed a novel ara-G-resistant variant (CEM/ara-G) of human T-lymphoblastic leukemia cell line CCRF-CEM, and elucidated its mechanism of ara-G resistance. The cytotoxicity was measured by using the growth inhibition assay and the induction of apoptosis. Intracellular triphosphate concentrations were quantitated by using HPLC. DNA synthesis was evaluated by the incorporation of tritiated thymidine into DNA. Protein expression levels were determined by using Western blotting.ResultsCEM/ara-G cells were 70-fold more ara-G-resistant than were CEM cells. CEM/ara-G cells were also refractory to ara-G-mediated apoptosis. The transcript level of human Equilibrative Nucleoside Transporter 1 was lowered, and the protein levels of deoxycytidine kinase and deoxyguanosine kinase were decreased in CEM/ara-G cells. The subsequent production of intracellular ara-GTP (21.3 pmol/107 cells) was one-fourth that of CEM cells (83.9 pmol/107 cells) after incubation for 6 h with 10 μM ara-G. Upon ara-G treatment, ara-G incorporation into nuclear and mitochondrial DNA resulted in the inhibition of DNA synthesis of both fractions in CEM cells. However, DNA synthesis was not inhibited in CEM/ara-G cells due to reduced ara-G incorporation into DNA. Mitochondrial DNA-depleted CEM cells, which were generated by treating CEM cells with ethidium bromide, were as sensitive to ara-G as CEM cells. Anti-apoptotic Bcl-xL was increased and pro-apoptotic Bax and Bad were reduced in CEM/ara-G cells.ConclusionsAn ara-G-resistant CEM variant was successfully established. The mechanisms of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.
Highlights
Nine-beta-D-arabinofuranosylguanine, an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is phosphorylated to an intracellular active metabolite ara-G triphosphate by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase
Establishment of ara-G-resistant CEM (CEM/ara-G) cells The Sodium 3′-(1-[(phenylamino)-carbonyl3 (XTT) proliferation assay demonstrated that CEM/ ara-G cells were 70-fold more resistant to ara-G than CEM cells (Figure 1a, Table 1)
Cleavage of caspase 3 and caspase 9 was demonstrated in CEM cells treated with ara-G, suggesting that mitochondria-mediated apoptosis was induced by ara-G (Figure 2)
Summary
Nine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Ara-G is phosphorylated to ara-G monophosphate by cytoplasmic deoxycytidine kinase (dCK) and mitochondrial deoxyguanosine kinase (dGK) [9]. This phosphorylation is the rate-limiting step of the intracellular activation of nelarabine. Ara-GTP is an intracellular active metabolite, which is subsequently incorporated into both nuclear and mitochondrial DNA, thereby terminating DNA elongation. Incorporation of the drug into DNA is critical for its cytotoxicity [8,9,10]
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