Ecto-5′-nucleotidase and thiopurine cellular circulation: Association with cytotoxicity

Thiopurine drugs such as 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used to treat acute lymphoblastic leukemia of childhood. To test the hypothesis that variation in the expression of genes within the "thiopurine pathway" might influence 6-MP and 6-TG sensitivity, we generated basal gene expression profiles and IC₅₀ values for both of these thiopurine drugs using a model system consisting of 194 Human Variation Panel lymphoblastoid cell lines. Association analysis showed that thiopurine S-methyltransferase, ecto-5′-nucleotidase (NT5E), and multidrug resistance protein 4 (ABCC4) expression were correlated with thiopurine cytotoxicity. Those observations suggested the possible existence of a "thiopurine cellular circulation" involving nucleotide efflux by ABCC4, hydrolysis of thiopurine nucleotide monophosphates outside of the cell by NT5E, and subsequent transport of thiopurine nucleosides back into the cell by nucleoside transporters. The existence of this cellular circulation was confirmed by a series of functional experiments performed with cultured cells stably or transiently transfected with ABCC4 and/or NT5E. Because of the central role of NT5E in this cellular circulation, the NT5E gene was resequenced using 287 DNA samples from three ethnic groups, with the identification of 68 single nucleotide polymorphisms (SNPs), 46 of which were novel. Several SNPs in the 5′-flanking region of NT5E were highly correlated with expression, rs9450278 having the lowest p value (p = 2.4 × 10^-10, R = -0.376). The thiopurine cellular circulation and genetic polymorphisms for genes encoding the proteins involved should be incorporated into future studies of thiopurine drug therapy and effect.