Ecto-5′-Nucleotidase and Thiopurine Cellular Circulation: Association with Cytotoxicity

Abstract

ABSTRACT: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

Thiopurine Pharmacogenomics: Association of Snps with Clinical Response and Functional Validation of Candidate Genes

“…Functional validation of thiopurine pathway genes showed significant shifts of cytotoxicity curves in the anticipated direction (p < 0.05) for TPMT and ITPA (Figure 3) as well as ABCC4 , ADA and SLC28A3 in HeLa cells (Supplementary Figure 1). The anticipated increase in sensitivity was observed after TPMT knockdown, while increased resistance was observed after SLC28A3 knockdown – as anticipated based on our previous LCL-expression studies [30]. The overall results of the functional analyses are summarized in Supplementary table 5.…”

“…The statistical methods for LCL analysis, including adjusting for ethnicity, have been previously described [30]. For the functional validation studies best fit curves, before and after gene knockdown, were fitted using GraphPad Prism Software (CA, USA).…”

“…Specifically, we associated 6-MP and 6-TG cytotoxicity (i.e., IC 50 ) values with genome-wide expression array data. As a result of those studies we reported a novel ‘cellular circulation’ of thiopurines that involved extrusion of nucleotide monophosphates by ABCC4, hydrolysis of the nucleotides outside of the cell by the ecto-enzyme NT5E and transport of the resultant nucleosides back into the cell by SLC29 [30]. The LCL model system used to perform those studies has repeatedly proven to be a powerful tool for both generating pharmacogenomic hypotheses and for pursuing SNP signals identified during clinical GWAS [28,29,31–34].…”

“…Specifically we have applied a ‘three-tiered’ experimental strategy to investigate candidate genes associated with clinical response in patients with childhood ALL. We began with studies of 176 LCLs from the ethnically diverse Coriell ‘Human Variation Panel’ [30,35] for which we had performed GWAS of the association of SNPs and gene expression with 6-TG and 6-MP cytotoxicity (IC 50 values) [30]. As the second step, we genotyped SNPs identified during the SNP GWAS performed with the LCLs as well as thiopurine pathway gene tagSNPs using DNA from Caucasian children with ALL who were enrolled in the UK ALL97 clinical trial.…”

The Pharmacogenetic Basis of Individual Variation in Thiopurine Metabolism

“…In vitro, ABCC4 transports TGMP across the cell membrane, where it is dephosphorylated by NT5E to form a TG-riboside. The extracellular circulation is completed by transfer of TG-riboside back into the cell via the SLC28/29 nucleoside transporters [53]. Identification of other cellular efflux pumps may additionally explain differences in thiopurine metabolism.…”