Fludarabine-Mediated Circumvention of Cytarabine Resistance Is Associated with Fludarabine Triphosphate Accumulation in Cytarabine-Resistant Leukemic Cells

Abstract

The combination of cytarabine (ara-C) with fludarabine is a common approach to treating resistant acute myeloid leukemia. Success depends on a fludarabine triphosphate (F-ara-ATP)-mediated increase in the active intracellular metabolite of ara-C, ara-C 5'-triphosphate (ara-CTP). Therapy-resistant leukemia may exhibit ara-C resistance, the mechanisms of which might induce cross-resistance to fludarabine with reduced F-ara-ATP formation. The present study evaluated the effect of combining ara-C and fludarabine on ara-C-resistant leukemic cells in vitro. Two variant cell lines (R1 and R2) were 8-fold and 10-fold more ara-C resistant, respectively, than the parental HL-60 cells. Reduced deoxycytidine kinase activity was demonstrated in R1 and R2 cells, and R2 cells also showed an increase in cytosolic 5'-nucleotidase II activity. Compared with HL-60 cells, R1 and R2 cells produced smaller amounts of ara-CTP. Both variants accumulated less F-ara-ATP than HL-60 cells and showed cross-resistance to fludarabine nucleoside (F-ara-A). R2 cells, however, accumulated much smaller amounts of F-ara-ATP and were more F-ara-A resistant than R1 cells. In HL-60 and R1 cells, F-ara-A pretreatment followed by ara-C incubation produced F-ara-ATP concentrations sufficient for augmenting ara-CTP production, thereby enhancing ara-C cytotoxicity. No potentiation was observed in R2 cells. Nucleotidase might preferentially degrade F-ara-A monophosphate over ara-C monophosphate, leading to reduced F-ara-ATP production and thereby compromising the F-ara-A-mediated potentiation of ara-C cytotoxicity in R2 cells. Thus, F-ara-A-mediated enhancement of ara-C cytotoxicity depended on F-ara-ATP accumulation in ara-C-resistant leukemic cells but ultimately was associated with the mechanism of ara-C resistance.

Characterization of cytarabine-resistant leukemic cell lines established from five different blood cell lineages using gene expression and proteomic analyses

“…To evaluate the proliferative activity of each cell line, the sodium 3'-[1-(phenylaminocarbonyl)-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 (24).…”

“…To evaluate the capacity of membrane nucleoside transporters, nucleoside analog uptake was quantified in all cell lines using the method of Wiley et al with slight modifications (6,24). Non-facilitated drug uptake was determined in the presence of 3 µM nitrobenzylthioinosine (Sigma), which interferes with the function of membrane nucleoside transporters.…”

“…The activity of dCK was assayed as previously described with slight modifications (24). Briefly, crude enzyme was obtained by sonication of cells suspended in 100 µl of 50 mM Tris-HCl (pH 8.0) containing 50% glycerol, and the solution was then clarified by centrifugation (100,000 x g for 60 min at 4˚C).…”

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

“…The parameter for predicting the clinical efficacy of a given nucleoside analog is not the plasma drug concentration but its triphosphate form concentration in cancer cells (9)(10)(11). Pharmacokinetic evaluation of ara-GTP will provide crucial information to optimize ara-G therapy (3)(4)(5).…”

Leukemia cells are sensitized to temozolomide, carmustine and melphalan by the inhibition of O6-methylguanine-DNA methyltransferase

“…To evaluate the growth-inhibitory effect of each agent on the two cell lines, the trypan blue exclusion assay was performed (17,18). Briefly, the cells were incubated with various concentrations of TMZ, BCNU or MEL (10 nM, 100 nM, 1 µM or 10 µM), alone or in combination with BG (10 µM), for 72 h. Subsequently, the samples were stained with trypan blue (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the viable cells, which exhibited negative staining, were counted.…”