Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines☆

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Abstract

Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were investigated in human leukemia cell lines. We developed two 6-MP- and 6-TG-resistant cell lines from the human T-lymphoblastic cell line (MOLT-4) by prolonged exposure to these drugs. The resistant cells were highly cross resistant to 6-MP and 6-TG, and exhibited marked reduction in cellular uptake of 6-MP (70% and 80%, respectively). No significant modification of the activities of hypoxanthine–guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase was observed. Real-time PCR of concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2) of resistant cells showed substantial reductions in expression of messenger RNAs. Small interfering RNA designed to silence the CNT3 and ENT2 genes down-regulated the expression of these genes in leukemia cells. These decreases were accompanied by reduction of transport of 6-MP (47% and 21%, respectively) as well as its cytocidal effect (30% and 21%, respectively). Taken together these results show that CNT3 and ENT2 play a key role in the transport of 6-MP and 6-TG by leukemia cells. From a clinical point of view determination of CNT3 and ENT2 levels in leukemia cells may be useful in predicting the efficacy of thiopurine treatment.

Section snippets

Material and methods

Chemicals. 6-Mercaptopurine, 6-thioguanine, and their metabolites methylmercaptopurine riboside, S-adenosyl-l-methionine, 5-phosphoribose-1-pyrophosphate, hypoxanthine, inosine 5′-monophosphate (IMP), xanthosine 5′-monophosphate, S-(4-nitrobenzyl)-6-thioinosine, and dipyridamole (Sigma–Aldrich, Stockholm, Sweden); Tris–HCl, ammonium dihydrogen phosphate (NH4H2PO4), and ethylenediaminetetraacetic acid (EDTA) (Merck, Darmstadt, Germany); [8-14C]6-mercaptopurine (51 mCi/mmol); [8-14C]inosine

Selection and characterization of 6-MP- and 6-TG-resistant cell lines

In order to assess the mechanism underlying resistance to thiopurines, we selected two resistant cell lines by exposing the parental MOLT4 cells during 12–18 cycles (72 h each) to stepwise increasing concentrations of 6-MP or 6-TG (from 0.05 to 5 μM). The resulting cell lines were similar to the parental cells with respect to growth rate and various cell cycle parameters (data not shown). Characterization of employing a 72-h exposure to the drugs revealed that the cells selected for resistance to

Discussion

Although the thiopurine analogs, 6-MP and 6-TG, are widely used in cancer chemotherapy, the mechanisms of their action and the mechanisms by which cells become resistant to these drugs remain to be elucidated in detail. One of the most extensively characterized mechanisms for in vitro resistance to 6-MP and 6-TG is the absence of HGPRT activity, which transfers a phosphoribosyl moiety to these molecules in the first step of their intracellular metabolism (Fig. 1). In a previous clinical study,

Acknowledgments

This study was supported by grants from the Swedish Childhood Cancer Foundation and the Swedish Cancer Foundation.

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    Abbreviations: 6-MP, 6-mercaptopurine; 6-TG, 6-thioguanine; HGPRT, hypoxanthine–guanine phosphoribosyl transferase; IC50, the drug concentration inhibiting cell growth by 50%; IMPDH, inosine monophosphate dehydrogenase; GMPS, guanosine monophosphate synthetase; TPMT, thiopurine methyltransferase; meMPR, methyl mercaptopurine riboside; CNT3, concentrative nucleoside transporter 3; ENT2, equilibrative nucleoside transporter 2; TGMP, 6-thioguanosine 5′-monophosphate; TIMP, 6-thioinosine 5′ monophosphate; MRP(s), multi-drug resistance-associated protein(s); NT(s), human nucleoside transporter(s).

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