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.2011 Mar 1;71(5):1781-91.
doi: 10.1158/0008-5472.CAN-10-1919. Epub 2011 Jan 18.

Deoxycytidine kinase modulates the impact of the ABC transporter ABCG2 on clofarabine cytotoxicity

Affiliations

Deoxycytidine kinase modulates the impact of the ABC transporter ABCG2 on clofarabine cytotoxicity

Shinjiro Nagai et al. Cancer Res..

Abstract

Purine nucleoside antimetabolites, such as clofarabine, are effective antileukemic agents. However, their effectiveness depends on an initial activation step in which they are monophosphorylated by deoxycytidine kinase (dCK). Some purine nucleoside antimetabolites and their monophosphate derivatives are exported by the ABC transporter ABCG2. Because clofarabine is a dCK substrate, and we show substantial variation in dCK and ABCG2 in myeloid leukemia, we hypothesized that the activity of dCK may modulate ABCG2-mediated resistance to clofarabine by regulating the formation of clofarabine monophosphate. We show that ABCG2 influence on clofarabine cytotoxicity was markedly influenced by dCK activity. When dCK expression was reduced by siRNA, clofarabine cytotoxicity was strongly reduced by enhanced ABCG2-mediated efflux. Conversely, dCK overexpression blunted ABCG2-mediated efflux of clofarabine by increasing the formation of clofarabine nucleotides. The use of an ABCG2 inhibitor confirmed that ABCG2 export of clofarabine is maximal when dCK levels are minimal. Analysis of intracellular clofarabine metabolites suggested that ABCG2 exported clofarabine more readily than clofarabine monophosphate. That ABCG2 primarily effluxes clofarabine, but not chlorfarabine-monophosphate, was confirmed by HPLC analysis of drug exported from ABCG2-overexpressing cells. Because the level and function of dCK and ABCG2 vary substantially among other types of cancer, these findings have important implications not only for clofarabine therapy but for purine nucleoside therapy in general. Therefore, we propose that addition of ABCG2 inhibitors would effectively increase the antitumor efficacy of purine nucleosides by blocking drug efflux that may be a significant mode of resistance when dCK levels are low.

©2011 AACR.

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Figures

Figure 1
Figure 1. Expression and function of dCK and ABCG2
A, Model illustrating dCK and ABCG2 interplay in the cellular metabolism of clofarabine.B, Expression of ABCG2 and dCK mRNA in prototypical myeloid leukemia cell lines as determined by real-time PCR (see Supplementary Information).C, Cells were incubated with 10 μM [3H] clofarabine for the indicated intervals (left); Cells were incubated for 60min with10 μM [3H] clofarabine.D, Immunoblot analysis of Saos2 and OCI-AML3 cells after transduction with an ABCG2 expression vector. The immunoblot was compiled by cropping a single immunoblot to remove unnecessary space.
Figure 2
Figure 2. The cell context differentially affects ABCG2-mediated clofarabine resistance
A, Efflux of the fluorescent ABCG2 substrates Hoechst 33342 and mitoxantrone is almost identical in Saos2 and OCI-AML3 cells. This is a representative experiment repeated several times with almost identical results.B, Clofarabine cytotoxicity was determined in Saos2 cells (left panel) and OCI-AML3 cells (right panel) in the absence and presence of fumitremorgin C (FTC) and is the average of 3 independent experiments conducted using triplicate determinations at each concentration. C, Saos2 and OCI-AML3 cells were exposed to mitoxatrone and cytotoxicity determined as described in the “Materials and Methods” the values are the average of a triplicate determination from a representative experiment repeated twice with similar results. The error bars are smaller than the symbol size.D, Intracellular accumulation of [3H]-clofarabine in Saos2 cells and OCI-AML3 cells. The values are the mean from four independent experiments with each point in duplicate. The bars represent one standard deviation.E, A representative dCK immunoblot of 100 μg of cytosol performed as described in the “Materials and Methods”. In panels A-D, the left hand column represents experiments conducted in Saos2 and the right hand column, OCI-AML3.
Figure 3
Figure 3. dCK function affects intracellular clofarabine metabolism
A, Diagram of dCK expression vector and P-loop mutation.B, Immunoblot analysis of Saos2 cells expressing either no dCK, wild-type dCK, or mutant dCK (left panel), the ratio of phosphorylated clofarabine metabolites to unchanged clofarabine is substantially greater in parental Saos2 cells expressing wild-type dCK than in cells containing the mutant dCK (right panel). MP, monophosphate; DP, diphosphate; TP, triphosphate. C, Saos2 cells overexpressing wild-type dCK are more sensitive to clofarabine while cells harboring either mutant dCK or empty vector are of similar sensitivity. Cytotoxicity was assessed as described in the “Materials and Methods”. Each value is an average of three-independent determinations from a representative experiment repeated three times with similar results.
Figure 4
Figure 4. ABCG2-mediated efflux is greater in cells expressing mutant dCK than in cells with enhanced dCK expression
A, A representative immunoblot analysis of wild-type (WT) and mutant (MT) dCK in vector-control (SV) and ABCG2-expressing (SB) Saos2 cells.B, Survival of Saos2 vector- or ABCG2-transduced cells expressing mutant (MT) or wild-type (WT) dCK. The values are the average of three-independent experiments performed in triplicate. The bars represent one standard deviation.C, Clofarabine metabolites were determined by HPLC. Arabic numerals (#) indicate individual cell pools; D, Efflux of clofarabine was determined from the ratio of the proportion of drug in the media after a 60 min efflux period and the proportion of drug within the cell. This is a representative of an experiment repeated three times with similar results. E, Various concentrations of clofarabine were added to vesicles expressing either no ABCG2 or ABCG2 and 10μM [3H] methotrexate the values are means of triplicate determinations from a representative experiment.
Figure 5
Figure 5. Knockdown of dCK enhances ABCG2-mediated clofarabine resistance by reducing clofarabine phosphorylation in ABCG2-overexpressing cells
A, Immunoblot analysis of dCK protein after transfection with dCK siRNA. This is a representative blot from an experiment repeated three separate times.B, Intracellular clofarabine metabolites after siRNA treatment. This is a representative HPLC analysis of clofarabine intracellular metabolites t from average values in this experiment repeated three times with similar results.
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