A new high-performance liquid chromatography method determines low production of 9-β-D-arabinofuranosylguanine triphosphate, an active metabolite of nelarabine, in adult T-cell leukemia cells

Abstract

Abstract. The 9-ß-D-arabinofuranosylguanine (ara-G), an active compound of nelarabine, demonstrates potent cytotoxicity specifically on T-cell malignancies. In cells, ara-G is phosphorylated to ara-G triphosphate (ara-GTP), which is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Because ara-GTP is crucial to ara-G's cytotoxicity, the determination of ara-GTP production in cancer cells is informative for optimizing nelarabine administration. Here, we developed a new, sensitive isocraticelution HPLC method for quantifying ara-GTP. Samples were eluted isocratically by using phosphate buffer at a constant flow rate. Ara-GTP was clearly separated from other nucleotides by using an anion-exchange column and it was quantitated by its peak area at 254 nm. The standard curve was linear with low variability and a sensitive detection limit (10 pmol). Furthermore, due to ara-G's specificity to T-cells we hypothesized that nelarabine might be effective against adult T-cell leukemia (ATL). The ara-GTP production was compared between T-lymphoblastic leukemia CCRF-CEM and ATL cell lines in vitro. When CEM cells were incubated with ara-G, the ara-GTP production increased in a concentration-and time-dependent manner. In contrast, 5 ATL cell lines accumulated lower ara-GTP in the same condition. While ara-G inhibited the growth of CEM cells with a 50% growth inhibition concentration of 2 μM, the inhibitory-concentration values were >1 mM in 8 of the 12 ATL cell lines. This ineffectiveness appeared to correspond with the low ara-GTP production. The present study is the first to evaluate the potential of ara-G against ATL cells; our results suggest that nelarabine would not be effective against ATL.

High ENT1 and DCK gene expression levels are a potential biomarker to predict favorable response to nelarabine therapy in T‐cell acute lymphoblastic leukemia

“…First, to evaluate the antileukemic activity of nelarabine, we investigated ara-G sensitivity of 23 T-ALL cell lines using alamarBlue cell viability assay. The representative dose-response curves are shown in Figure 1B 9 Consistent with above observation, intracellular ara-GTP levels in the three ara-G-sensitive cell lines (DND-41, HPB-ALL, and RPMI-8402) were significantly higher than those in the three ara-G-resistant cell lines (BE-13, LOUCY, and L-KAW) ( Figure 1E). We next investigated the association of cytogenetic abnormalities with ara-G sensitivity in T-ALL cell lines.…”

“…These results suggested that combined ENT1 and DCK gene expression levels are highly associated with ara‐G sensitivity in T‐ALL cell lines. We also quantified intracellular ara‐GTP levels in six representative cell lines after a 5‐hour exposure to 10 μM of ara‐G using high‐performance liquid chromatography analysis . Consistent with above observation, intracellular ara‐GTP levels in the three ara‐G‐sensitive cell lines (DND‐41, HPB‐ALL, and RPMI‐8402) were significantly higher than those in the three ara‐G‐resistant cell lines (BE‐13, LOUCY, and L‐KAW) (Figure E).…”

Combination of guanine arabinoside and Bcl‐2 inhibitor YC137 overcomes the cytarabine resistance in HL‐60 leukemia cell line

“…The acid‐soluble fraction (nucleotide pool) was extracted from HL‐60 cells or HL‐60/ara‐C60 cells (1 × 10 6 /mL, 10 mL), treated or untreated. High performance liquid chromatography was then used to determine intracellular ara‐CTP and ara‐GTP as described in previous studies …”

“…High performance liquid chromatography was then used to determine intracellular ara-CTP and ara-GTP as described in previous studies. (12,15,25) Nucleoside transport capacity. To evaluate the capacity of membrane nucleoside transporters, ara-C uptake was quantified using the method of Wiley et al with slight modifications.…”

Camptothecin induces DNA strand breaks and is cytotoxic in stimulated normal lymphocytes

“…To evaluate the cell proliferation, a sodium 3'-{1-[(phenylamino)-carbonyl-3,4-tetrazolium]}-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay was performed according to the manufacturer's instructions (Roche, Indianapolis, IN, USA) with slight modifications (19). In brief, 1 ml of a cell suspension (5x10 4 /ml) was incubated for 24 h in a 24-well plate, followed by the addition of a 10-μl aliquot of CPT solution at various concentrations.…”