Brief Report

Assignment of the Human Equilibrative Nucleoside Transporter (hENT1) to 6p21.1–p21.2☆

Worldwide, alcohol is the third leading risk factor for disease burden, while its harmful use leads to 2.5 million deaths every year. Alcohol dependence (AD) is a complex disease, with devastating effects on individuals, families, and society. It is estimated that 76.3 million people worldwide have suffered from alcohol use disorders (AUD), including alcohol abuse and AD. Alcohol withdrawal or alcohol withdrawal symptom (AWS) refers to a cluster of symptoms that may occur when a heavy drinker suddenly stops or significantly reduces their alcohol intake. These symptoms can start as early as 2 h after the last drink, persist for weeks, and range from mild anxiety and shakiness to severe complications, such as seizures and delirium tremens. Family, twin, and adoption studies have indicated that genetic and environmental factors and their interactions contribute to the development of AD and related phenotypes, with a heritability coefficient of more than 0.5 for AD. Whole-genome linkage and candidate gene association studies have successfully identified several chromosome regions and genes that are related to AD and AWS. Furthermore, gene expression analysis, epigenetic studies, and genome-wide association studies (GWAS) have provided regions and loci for AWS. This chapter reviews the recent findings in genetic studies of AWS.

Responses to nucleoside analog drugs used in the treatment of cancers and viral infections can vary considerably between individuals. Genetic variability between individuals in their ability to transport drugs may be a contributory factor. Nucleoside transporters (NTs) move nucleosides and analog drugs across cell membranes. Four human NTs have been cloned: hENT1, hENT2, hCNT1, and hCNT2. Human NT expression profiles are not well defined; therefore, we undertook a comprehensive quantitative analysis of the differential expression of NTs within normal and tumor tissue. Results show tissue specific expression of the different NTs in normal tissue while matched normal/tumor tissue cDNA array data show considerable variability in all NT expression profiles from different individuals, in particular decreased expression in tumor tissue. Decreased NT expression in tumor tissue may contribute to reduced drug uptake and the development of resistance. These data suggest that nucleoside analog drug therapies may be optimized by determining individual NT expression profiles.

We have isolated a mouse cDNA clone corresponding to a novel isoform of the NBMPR-sensitive equilibrative nucleoside transporter (ENT1). The cDNA contains a 6 bp deletion in the open reading frame that changes the amino acid composition in a consensus casein kinase II (CKII) phosphorylation site at Ser-254. The clone containing Ser-254 is termed mENT1.1 and the clone lacking the serine termed mENT1.2. The deduced amino acid sequence of mENT1.1 corresponds to the previously cloned human and rat ENT1 proteins at Ser-254. Tissue distribution studies show that mRNA for both ENT1 isoforms are ubiquitously co-expressed in mouse. Analysis of genomic DNA corresponding to mouse ENT1 indicates the isoforms can be produced by alternative splicing at the end of exon 7. CEM/C19 cells stably expressing mENT1.1 and mENT1.2 show similar dose response curves for NBMPR and dipyridamole inhibition of [³H]adenosine uptake as well as exhibiting comparable selectivity for both purine and pyrimidine nucleosides but not the corresponding nucleobases.

We have cloned and characterized the genomic structure of the mouse gene for the NBMPR-sensitive equilibrative nucleoside transporter (mENT1), which is located on chromosome 17C. About 12-kb of genomic DNA was sequenced including the promoter region, 12 exons, 11 introns, and the 3′-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. Primer extension analysis demonstrated a transcription initiation site located 252 bp upstream of the translation start site. Analysis of the 2.5-kb 5′-flanking sequence shows putative binding sites for several transcription factors, including GATA-1, IRF-2, Pit-1, myogenin, CREB, Sp-1, Ap-2, MAZ, and GR. We demonstrated that mouse ENT1 mRNA was highly expressed in heart, spleen, lung, liver, and testis. Lower levels of expression were detected in brain and kidney. Functional analysis of the 5′-flanking region showed that the nucleotide sequence from −652 to −111 contains cis-regulatory elements that promote gene expression. We found two Sp-1 binding sites (−296/−303, −307/−313) and two MAZ binding sites (−353/−359, −522/−528) in this region. Luciferase assay results suggest that MAZ and Sp-1 transcription factors are important positive regulators of transcription for the ENT1 gene in NG108-15 cells.

This chapter describes the molecular mechanisms of nucleoside and nucleoside drug transport. Specialized nucleoside transporter (NT) proteins are required for translocation across cell membranes and transportability is a critical determinant of intestinal absorption, intracellular metabolism, and, for nucleoside drugs, pharmacological actions. NTs also regulate adenosine concentrations in the vicinity of their cell surface receptors and have profound effects on vascular tone, ion secretion, neuromodulation, and other processes. Nucleosides are important precursors of nucleic acids and energy-rich cellular metabolites and have functions as local hormones in a variety of tissues, including the gastrointestinal system. Cells obtain nucleosides from the breakdown of dietary and endogenous nucleotides. The former are important human nutrients and are absorbed as nucleosides by the enterocytes of the intestinal mucosa. Enterocytes appear to have a limited capacity for de novo nucleotide synthesis and require both dietary and endogenous nucleosides for their own metabolism and differentiation. Nucleoside analogs are important antiviral and anticancer agents and a number are administered orally. Nucleosides and most nucleoside drugs enter and leave cells by transporter-mediated processes located in the plasma membrane. Nucleosides and most nucleoside drugs enter and leave cells by transporter-mediated processes located in the plasma membrane. Intestinal transport of nucleosides involves both inwardly directed, concentrative and bidirectional, equilibrative mechanisms.

This article discusses the role of nucleoside transport processes in the cytotoxicity of clinically important anticancer nucleosides. This article summarizes recent advances in the molecular biology of nucleoside transport proteins, review the current state of knowledge of the transportability of therapeutically useful anticancer nucleosides, and provide an overview of the role of nucleoside transport deficiency as a mechanism of resistance to nucleoside cytotoxicity are summarized. Several strategies for utilization of nucleoside transport processes to improve the therapeutic index of anticancer therapies, including the use of nucleoside-transport inhibitors to modulate toxicity of both nucleoside and non-nucleoside antimetabolite drugs are also presented.