Expression of ABCC-type nucleotide exporters in blasts of adult acute myeloid leukemia: relation to long-term survival

Abstract

Purpose: Successful treatment of acute myeloid leukemia (AML) remains a therapeutic challenge, with a high percentage of patients suffering from persistent or relapsed disease. Resistance to drug therapy can develop from increased drug export and/or altered intracellular signaling. Both mechanisms are mediated by the efflux transporters ABCC4 (MRP4), ABCC5 (MRP5), and ABCC11 (MRP8), which are involved in cellular efflux of endogenous signaling molecules (e.g., cyclic adenosine 3', 5'-monophosphate and cyclic guanosine 3',5'-monophosphate) and nucleoside analogues. The nucleoside analogue cytosine arabinoside (AraC) is administered to all patients with AML.

Experimental design: Expression of ABCC transporters MRP4, MRP5, and MRP8 in blast samples from 50 AML patients was investigated by real-time reverse transcription-PCR analysis and correlated with clinical outcome measures. Accumulation of radiolabeled AraC, transport of AraC metabolites, and AraC cytotoxicity were analyzed in MRP8-transfected LLC-PK1 cells.

Results: Regression analysis revealed that high expression of MRP8 is associated with a low probability of overall survival assessed over 4 years (P<0.03). MRP8-transfected LLC-PK1 cells accumulated reduced intracellular levels of AraC (63% of the parental vector-transfected LLC-PK1 control cells) as well as AraC metabolites. Furthermore, AraC monophosphate was transported by MRP8-enriched membrane vesicles (116+/-6 versus 65+/-13 pmol/mg/10 minutes by control vesicles), and MRP8-transfected cells were resistant to AraC.

Conclusion: These data suggest that MRP8 is differentially expressed in AML blasts, that expression of MRP8 serves as a predictive marker for treatment outcome in AML, and that efflux of AraC metabolites by MRP8 is a mechanism that contributes to resistance of AML blasts.

Fig. 1

MRP8 mRNA expression in blasts of AML patients predicts long-term survival. Overall survival (A) and relapse-free survival (B) in adults diagnosed with AML subdivided into two groups of low and high MRP8 expression. mRNA of blast samples of at least 85% purity was prepared and MRP8 mRNA expression was quantified as described in Materials and Methods. The median of MRP8 mRNA expression was used as a cutoff for low and high expression. The Kaplan-Meier method was used to estimate the distribution of overall survival, and CI estimation for the survival curves was based on the cumulative hazard function using Greenwood’s formula for the SE estimation. Survival distributions were compared using the log-rank test.

Fig. 2

Cellular accumulation of AraC. Time course of accumulation of [³H]AraC in parental vector-transfected (LLC-PK1-pcDNA, ○) and MRP8-transfected (LLC-PK1-MRP8-1, •) LLC-PK1 cells. Cells were incubated in 1 μmol/L [³H]AraC and intracellular radioactivity was measured at various time points. Points, mean of a representative experiment done in triplicate; bars, SE.

Fig. 3

Analysis of AraC metabolites in MRP8-transfected cells. A, chemical structure and metabolic pathway of intracellular AraC phosphorylation. B, parental vector-transfected (white columns, LLC-PK1-pcDNA) and MRP8-transfected (black columns, LLC-PK1-MRP8-1) cells were incubated with 10 μmol/L [³H]AraC and 50 nmol/L tetrahydrouridine for 3 h. Intracellular AraC metabolites were extracted by the perchloric acid method, and AraCMP, AraCDP, and AraCTP were separated and quantitated using high-performance liquid chromatography as described in Materials and Methods. Columns, mean of three or more separate experiments; bars, SD. *, P < 0.05. C, membrane vesicles (10 μg) prepared from parental vectortransfected (LLC-PK1-pcDNA) or MRP8-transfected (LLC-PK1-MRp8-1 and LLC-PK1-MRP8-2) cells were incubated for 10 min at 37°C in uptake medium containing 4 mmol/L ATP or 4 mmol/L AMP and 40 μmol/L [³H]AraCMP. MgATP-dependent uptake was calculated by subtracting the values obtained in transport medium containing AMP from the values obtained in medium containing MgATP. Columns, mean of a representative experiment; bars, SE.

Fig. 4

Sensitivity of MRP8-transfected and parental vector-transfected LLC-PK1 cells to AraC. The sensitivity of parental vector-transfected (○, LLC-PK1-pcDNA) and MRP8-transfected LLC-PK1 cells (▴, LLC-PK1-MRP8-1; ▾, LLC-PK1-MRP8) to AraC was analyzed using the tetrazolium salt microtiter plate assay as described in Materials and Methods. Points, mean of a representative experiment done in triplicate; bars, SE.