Functional Characterization and Haplotype Analysis of Polymorphisms in the Human Equilibrative Nucleoside Transporter, Ent2

Abstract

ABSTRACT:The equilibrative nucleoside transporter 2 (ENT2; SLC29A2) is a bidirectional transporter that is involved in the disposition of naturally occurring nucleosides as well as a variety of anticancer and antiviral nucleoside analogs. The goal of the current study was to evaluate the function of genetic variants in ENT2 in cellular assays and to determine the haplotype structure of the coding and flanking intronic region of the gene. As part of a large study focused on genetic variation in membrane transporters (Leabman et al., 2003), DNA samples from ethnically diverse populations (100 AfricanAmericans, 100 European-Americans, 30 Asians, 10 Mexicans, and 7 Pacific Islanders) were screened for variants in membrane transporters, including SLC29A2. Fourteen polymorphic sites in SLC29A2 were found, including 11 in the coding region. Five protein-altering variants were identified: three nonsynonymous variants, and two deletions. Each of the protein-altering variants was found at a very low frequency, occurring only once in the sample population. The nonsynonymous variants and the deletions were constructed via site-directed mutagenesis and were subsequently characterized in Xenopus laevis oocytes. All variants were able to take up inosine with the exception of ENT2-⌬845-846, which resulted in a frameshift mutation that prematurely truncated the protein. ENT2 showed very infrequent variation compared with most other transporter proteins studied, and it was found that five haplotypes were sufficient to describe the entire sample set. The low overall genetic diversity in SLC29A2 makes it unlikely that variation in the coding region contributes significantly to clinically observed differences in drug response.Synthetic nucleoside analogs are widely used to treat a variety of diseases, including various types of cancer, HIV, hepatitis C, and other illnesses (Barreiro et al., 2004;Byrd et al., 2004;Li et al., 2004;Pearlman, 2004). Although nucleoside analogs are often the best available therapy, some common problems with nucleoside analog therapies occur, including lack of an initial response, or the development of resistance to therapy. One potential hypothesis for the ineffectiveness of some nucleoside analogs is genetic variation in nucleoside transporters, which function in the uptake of these compounds into cells. Genetic variation could lead to reduced function, or nonfunctional transporter proteins, which in turn could reduce the amount of drug that enters the cell and, therefore, the intracellular levels of the drug. Genetic variation in both concentrative and equilibrative nucleoside transporter members has been previously examined including CNT1 (Gray et al., 2004), CNT2 (Owen et al., 2005, CNT3 (Badagnani et al., 2005), and ENT1 (Osato et al., 2003).In this report, we describe the functional characteristics of genetic variants of ENT2 that were identified previously in a large DNA sample set. ENT2 is thought to play a role in nucleoside analog therapy, since it has broad substrate specificity and is able to...

Molecular Basis of Nucleobase Transport Systems in Mammals

“…The substrate specificity of ENBT1/SLC43A3 is similar to that of Na + -independent purine nucleobase transport systems that are different from those of ENT1 and ENT2, which include carrier-mediated transport systems characterized in many types of cells, including human erythrocytes, 52) COR-L239, 40) LLC-PK1, 17) PK15NTD, 53) rat cortical neurons, 54) human and mouse primary microvascular endothelial cells, 34,45) and rabbit cornea epithelial cells. 55) ENBT1/ SLC43A3 selectively mediates the transport of purine nucleobases such as guanine, hypoxanthine, xanthine, and adenine, but not that of nucleosides.…”

“…This raises the possibility that ENT2 expressed in the lung plays a role in the regulation of extracellular adenosine levels, rather than salvaging the intracellular purine. Genomic analysis revealed that single-nucleotide polymorphism (SNP) frequency in ENT2 is extremely low, 45) suggesting that it plays a crucial role in nucleotide synthesis and metabolism. Further studies are needed to elucidate the role of ENT2 as a nucleobase transporter.…”

Pharmacogenomic Discovery Using Cell-Based Models

“…This approach has been applied to many pharmacogenetic investigations of membrane transporters, which are important mediators of drug absorption and elimination and are also targets of many drugs. Significant effort has been invested in resequencing transporter genes in LCLs from the Studies of Pharmacogenetics in Ethnically Diverse Populations participants, followed by functional characterization using in vitro assays in Xenopus laevis oocytes or human cell lines such as human embryonic kidney 293 cells (Osato et al, 2003;Gray et al, 2004;Badagnani et al, 2005Badagnani et al, , 2006Fujita et al, 2005;Owen et al, 2005Owen et al, , 2006Urban et al, 2006bUrban et al, , 2007Abla et al, 2008;Huang et al, 2008a;Sorani et al, 2008;Chen et al, 2009;Miller et al, 2009;Yee et al, 2009). More recently, these LCLs, along with HapMap LCLs, have been used in functional studies as well, particularly involving allelic expression imbalance.…”

The role of nucleoside transporters in cancer chemotherapy with nucleoside drugs

“…These results suggest that coding region variants of hENT1 do not contribute to inter-individual differences in response to nucleoside drugs. Fourteen polymorphic sites, including 11 in the coding region, were found in hENT2 at very low frequencies [112]. Five of the altered hENT2 variants (three nonsynonymous variants and two deletions) were able to transport inosine; one variant, which resulted in a premature truncation of hENT2, was not.…”