Human Equilibrative Research Articles

O095 hCNT (human concentrative nucleoside transporter) and hENT (human equilibrative nucleoside transporter) in human gastric cancer and response to chemotherapies

Abstract Introduction hCNT (Human Concentrative Nucleoside Transporter, also known as SLC28A1) and hENT (Human Equilibrative Nucleoside Transporter, SLC29A1) belong to the large solute carrier (SLC) family, a large membrane transport molecule group for the influx and efflux across cell membrane of various solutes including some of the anticancer drugs. hCNT and hENT are suggested to be involved in the transportation of certain chemotherapeutic drugs. The present study investigated the role of both transporters in gastric cancer and the response of gastric cancer cell and patients to chemotherapeutic drugs. Methods The expression levels of hCNT and hENT gene transcripts and proteins were determined in two independent gastric cancer cohorts and assessed against patient's responses to neoadjuvent chemotherapies. Their value in assessing patient's survival, clinical and pathological indicators was also assessed. Results Gastric tumour tissues expressed significantly higher levels of hCNT and hENT than normal stomach tissues (p=0.022 and p=0.0071, respectively for hCNT and hENT). Expression levels of the hCNT and hENT gene transcripts presented significant correlation with the overall survival (OS) (Hazard Ration (HR)=2.885, p<0.001 for hCNT; HR=2.078, p<0.001 for hENT) and disease-free survival of the patients (HR=2.434, p=0.004 for hCNT; HR=2.072, p<0.001). Patients with high levels of hCNT in their gastric tumours were more resisted to neoadjuvant therapies (HR=3.437, p=0.013, by ROC model), although the same was not seen with hENT. Conclusion hCNT and hENT have significant prognostic factors for patients with gastric cancer and theranostic factor for response to neoadjuvant therapies.

Functional Comparison of Human and Murine Equilibrative Nucleobase Transporter 1

BackgroundThiopurines are a key component for the immunosuppressive therapy of various leukemias, inflammatory bowels disease, and other autoimmune disorders. Thiopurine therapy discontinuation/interruption is associated with a higher risk of relapse, but is common due to the prevalence of severe and potentially fatal adverse events, such as hepatotoxicity.Drug transporter expression is a known factor for patient variability in drug response and toxicity. We have recently established that the SLC43A3‐encoded transporter, equilibrative nucleobase transporter 1 (ENBT1), is the primary mechanism by which the thiopurine, 6‐mercaptopurine (6‐MP) enters cells. ENBT1 is known to be highly expressed in human hepatocytes, however, the relationship between ENBT1 and thiopurine‐induced hepatotoxicity has not been explored in the literature.To investigate this paradigm, our lab has proposed developing a novel SLC43A3 knockout mouse model. However, the functional differences between human and murine ENBT1, in mediating 6‐MP transport, has equally not been explored. Evidence that suggests ENBT1 is functionally similar between species, would be an essential foundation to assist bridging the gap between our proposed animal model and clinical studies within the literature.HypothesisWe hypothesize that hSLC43A3‐encoded hENBT1 and mslc43a3‐encoded mENBT1 are functionally similar and will have non‐significantly different 6‐MP transport kinetics and resulting cytotoxicity.MethodsThe ENBT1‐deficient human embryonic kidney 293 (HEK293) cell line was stably transfected with either hSLC43A3 or mslc43a3. [3H]Adenine and [3H]6‐MP were used in an oil‐stop centrifugation assay to assess ENBT1‐mediated transport activity in wildtype and transfected HEK293 cell lines. To determine cytotoxicity, wildtype and transfected HEK293 cell lines were incubated for 48 hours with a range of 6‐MP concentrations (78 nM – 1.28 mM) before being assessed via MTT cell viability assay.ResultsOil stop centrifugation assay of ENBT1‐mediated [3H]adenine and [3H]6‐MP transport revealed that hENBT1 and mENBT1 have similar transport kinetics (Km and Vmax), where resulting Michaelis‐Menten curves were not significantly different (unpaired T‐test: Adenine ‐ t16=0.32, p=0.76, n=5 & 6‐MP ‐ t16=0.64, p=0.53, n=4). Adenine inhibition of ENBT1‐mediated [3H]6‐MP transport and 6‐MP inhibition of ENBT1‐mediated [3H]adenine transport also showed that hENBT1 and mENBT1 have concurring inhibition kinetics (Ki), inhibition curves were not significantly different (unpaired T‐test: Adenine: t17=0.29, p=0.77, n=5 & 6‐MP: t14=0.18, p=0.86, n=4). Subsequent MTT cell viability assay showed that wildtype HEK293 are relatively 6‐MP resistant, while the transfected‐HEK293s are 6‐MP sensitive and have non‐significantly different cell viability curves (unpaired T‐test: t28=0.13, p=0.89, n=5).ConclusionOur results show that hENBT1 and mENBT1 are functionally similar in regards to ENBT1‐mediated adenine and 6‐MP transport in a stably transfected cell line. Additional work is necessary to investigate whether this is the case in human and murine hepatic cell lines/primary hepatocytes that have endogenous SLC43A3/ENBT1 expression and a larger range of xenobiotic metabolizing enzymes that may alter the equilibrium of 6‐MP within cells.

"Open Sesame?": Biomarker Status of the Human Equilibrative Nucleoside Transporter-1 and Molecular Mechanisms Influencing its Expression and Activity in the Uptake and Cytotoxicity of Gemcitabine in Pancreatic Cancer.

Simple SummaryDespite the enormous advance in biomarker discovery, many potential biomarkers of drug activity are unable to satisfy the clinical need due to inadequate sensitivity and specificity. The nucleoside transporter hENT-1 has been studied as a potential biomarker to predict the effect of the widely used anticancer drug gemcitabine in pancreatic cancer. However, several studies showed controversial results regarding the predictive value of hENT-1, prompting new analyses with larger cohorts of patients and standardized methodologies. Improved insights on molecular mechanisms underlying hENT-1 expression and activity should also help in the identification of subsets of patients who are more likely to benefit from specific treatments and improve their clinical outcome. The establishment of such biomarker is especially valuable in pancreatic cancer, which is frequently characterized by complex disease biology and high mortality.Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive tumor characterized by early invasiveness, rapid progression and resistance to treatment. For more than twenty years, gemcitabine has been the main therapy for PDAC both in the palliative and adjuvant setting. After the introduction of FOLFIRINOX as an upfront treatment for metastatic disease, gemcitabine is still commonly used in combination with nab-paclitaxel as an alternative first-line regimen, as well as a monotherapy in elderly patients unfit for combination chemotherapy. As a hydrophilic nucleoside analogue, gemcitabine requires nucleoside transporters to permeate the plasma membrane, and a major role in the uptake of this drug is played by human equilibrative nucleoside transporter 1 (hENT-1). Several studies have proposed hENT-1 as a biomarker for gemcitabine efficacy in PDAC. A recent comprehensive multimodal analysis of hENT-1 status evaluated its predictive role by both immunohistochemistry (with five different antibodies), and quantitative-PCR, supporting the use of the 10D7G2 antibody. High hENT-1 levels observed with this antibody were associated with prolonged disease-free status and overall-survival in patients receiving gemcitabine adjuvant chemotherapy. This commentary aims to critically discuss this analysis and lists molecular factors influencing hENT-1 expression. Improved knowledge on these factors should help the identification of subgroups of patients who may benefit from specific therapies and overcome the limitations of traditional biomarker studies.

Su1473 Human Equilibrative Transporter 1 (hENT1) Expression is not Predictive for Gemcitabine Outcome in Patients With Advanced Biliary Tract Cancer

Su1473 Human Equilibrative Transporter 1 (hENT1) Expression is not Predictive for Gemcitabine Outcome in Patients With Advanced Biliary Tract Cancer

Prognostic Value of Excision Repair Cross-Complementing Gene 1, Dihydropyrimidine Dehydrogenase, and Human Equilibrative Nucleotide Transporter 1 Expression and Their Implications for Adjuvant Treatment in Patients With Ampullary Carcinoma.

The purpose of this study was to characterize the intratumoral expression profiles of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), and human equilibrative nucleotide transporter 1 (hENT1) in ampullary carcinomas (ACs) to evaluate their prognostic values and better tailor adjuvant chemotherapy for individual patients with AC after surgery. This study included 49 patients with AC who underwent a curative pancreaticoduodenectomy. Various clinicopathological factors, including ERCC1, DPD, and hENT1, were analyzed in relation to postoperative disease recurrence and the patients' survival. The median recurrence-free survival and overall survival were 24.5 months and 32.4 months, respectively. Multivariate Cox regression analysis of recurrence-free survival identified a DPD expression (hazard ratio [HR], 8.18; 95% confidence interval [CI], 2.00-34.8; P = 0.003) and combined ERCC1/DPD expression (HR, 134.8; 95% CI, 11.8-1920; P < 0.001) as independent predictors of disease recurrence. Multivariate Cox regression analysis of overall survival also identified a DPD expression (HR, 8.48; 95% CI, 1.71-46.3; P = 0.008) and combined ERCC1/ DPD expression (HR, 135.6; 95% CI, 11.8-1940; P < 0.001) as independent predictors of survival. The DPD and ERCC1 expression profile could potentially serve as a useful prognostic biomarker and therapeutic target for surgically resected patients with AC.

Expression and activity of matrix metalloprotease-2 in canine early placenta

Expression and activity of matrix metalloprotease-2 in canine early placenta

Prognostic Value of Human Equilibrative NucleosideTransporter1 in Pancreatic Cancer Receiving Gemcitabin-Based Chemotherapy: A Meta-Analysis

BackgroundThe potential prognostic value of human equilibrative nucleoside transporter1 in pancreatic cancer receiving gemcitabine-based chemotherapy is variably reported.ObjectiveThe objective of this study was to conduct a systematic review of literature evaluating human equilibrative nucleoside transporter1 expression as a prognostic factor in pancreatic cancer receiving gemcitabine-based chemotherapy and to conduct a subsequent meta-analysis to quantify the overall prognostic effect.MethodsRelated studies were identified and evaluated for quality through multiple search strategies. Only studies analyzing pancreatic cancer receiving gemcitabine-based chemotherapy were eligible for inclusion. Data were collected from studies comparing overall, disease-free and progression-free survival (OS, DFS and PFS) in patients with low human equilibrative nucleoside transporter1 levels and those having high levels. The hazard ratio (HR) and its 95% confidence interval (95%CI) were used to assess the strength of associations. Hazard ratios greater than 1 reflect adverse survival associated with low human equilibrative nucleoside transporter1 levels.ResultsA total of 12 studies (n = 875) were involved in this meta-analysis (12 for OS, 5 for DFS, 3 for PFS). For overall and disease-free survival, the pooled HRs of human equilibrative nucleoside transporter1 were significant at 2.93 (95% confidence interval [95% CI], 2.37–3.64) and 2.67 (95% CI, 1.87–3.81), respectively. For progression-free survival, the pooled HR in higher human equilibrative nucleoside transporter1 expression in pancreatic cancer receiving gemcitabine-based chemotherapy was 2.76 (95% CI, 1.76–4.34). No evidence of significant heterogeneity or publication bias was seen in any of these studies.ConclusionThese results support the case for a low human equilibrative nucleoside transporter1 level representing a significant and reproducible marker of adverse prognosis in pancreatic cancer receiving gemcitabine-based chemotherapy.

Cysteine Residues in the Transmembrane (TM) 9 to TM11 Region of the Human Equilibrative Nucleoside Transporter Subtype 1 Play an Important Role in Inhibitor Binding and Translocation Function

Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. A previous study from our laboratory established Cys222 in transmembrane (TM) 6 as the residue responsible for methyl methanethiosulfonate (a membrane-permeable sulfhydryl modifier)-mediated enhancement of the binding of the ENT1 inhibitor nitrobenzylmercaptopurine riboside (NBMPR) in intact cells. However, the capacity of charged sulfhydryl reagents to inhibit the binding of NBMPR in broken cell preparations (allowing cytoplasmic access) was not affected by mutation of any of the cysteines (Cys87, 193, 213, or 222) in the N-terminal half of the protein. We thus hypothesized that the inhibitory effects of the modifiers were due to the one or more of the six cysteine residues in the C-terminal half of ENT1, particularly one or both of those in the fifth intracellular loop (Cys414 and Cys416). Each of the cysteines were mutated to serine or alanine and expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate sulfhydryl-modifying reagents. Transporter function was assessed by the site-specific binding of [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. These studies established that Cys378 is an extracellular-located residue modified by [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET) to inhibit the binding of NBMPR to intact cells. Mutation of Cys414 led to an enhancement of the ability of MTSET to inhibit NBMPR binding, and this enhancement was eliminated by the comutation of Cys378, indicating that disruption of the fifth intracellular loop modifies the conformation of TM10 and its extracellular extension. Mutation of Cys416 led to the loss of the ability of the charged sulfhydryl reagents to inhibit NBMPR binding in isolated membranes and also led to the loss of transport function. This finding further supports an allosteric interaction between the fifth intracellular loop and the extracellular NBMPR binding domain and implicates this region in the translocation function of human ENT1.

Su1546 Which is More Useful as a Predictive Marker of Adjuvant Gemcitabine-Based Chemotherapy for Pancreatic Carcinoma After Surgical Resection, Human Equilibrative Nucleoside Transporter 1 or Ribonucleotide Reductase Regulatory Subunit M1 Expression?

Background: Given that the life expectancy is increasingly becoming longer, safety and efficacy of pancreaticoduodenectomy for periampullary cancers in elderly patients is a great clinical concern. Over the last decademany reports have described outcome of pancreaticoduodenectomy in elderly patients, but the results are still inconsistent. Methods: From a database of all the patients with periampullary cancers undergoing pancreaticoduodenectomy between 2001 and 2009, the patients over 80 years were identified. Perioperative characteristics, postoperative complications, mortality, and a long-term survival were retrospectively compared between the patients aged 80 and older, and the patients younger than 80 years. Results: Among a total of 561 patients undergoing pancreaticoduodenectomy, 22 patients (3.9%) were over 80 years. The pathological diagnosis in the elderly patients consisted of as follows; pancreatic cancer (n=8), bile duct cancer (n=5), ampullary neoplasm (n=5), intraductal papillary mucinous neoplasm (n=3), and gallbladder cancer (n=1). The elderly patients had a higher ASA score (P=0.02) than the younger patients, but there was no significant difference in operative time (455 vs 520 minutes; P=0.08), and blood loss (654 vs 838 ml; P=0.38). The mortality rate was 4.5% in the elderly patients and 0.9% in the younger patients, though the difference was not statistically significant (p=0.106). Although the incidence of postoperative pancreatic fistula and delayed gastric emptying were similar between the two groups, that of intraabdominal abscess, intraabdominal hemorrhage, pneumonia and ascites in the elderly patients (22.7%, 18.2%, 9.1% and 4.5%, respectively) were higher than in the younger patients (6.9%, 2.2%, 0.6% and 0.2%, respectively). The rate of grade IIIa or higher complication (Clavien-Dindo classification) was 27.3% in the elderly patients and 6.3% in the younger patients (P=0.008). There was no significant difference in overall survival between the two groups. Among the elderly patients, the median survival for those with pancreatic cancer was significantly shorter than that with non-pancreatic cancer. (P=0.003) Conclusions: Pancreaticoduodenectomy in patients aged 80 and older should be indicated carefully, because of the higher incidence of the severe postoperative complications. Pancreaticoduodenectomy for pancreatic cancer in elderly patients could not provide a satisfactory outcome in terms of a long-term survival compared with that for nonpancreatic cancers.

Combined Analysis of Dihydropyrimidine Dehydrogenase and Human Equilibrative Nucleoside Transporter 1 Expression Predicts Survival of Pancreatic Carcinoma Patients Treated with Adjuvant Gemcitabine Plus S-1 Chemotherapy after Surgical Resection

Although postoperative adjuvant chemotherapy for pancreatic ductal adenocarcinoma (PDAC) improves survival in some patients, the effectiveness varies by individual, and the results remain unsatisfying. The aim of this study was to investigate whether intratumoral dihydropyrimidine dehydrogenase (DPD) and human equilibrative nucleoside transporter 1 (hENT1) expression can predict the survival of PDAC patients treated with adjuvant gemcitabine plus S-1 (GEM+S-1) chemotherapy. Intratumoral DPD and hENT1 expression were examined by immunohistochemistry in 86 PDAC patients who received adjuvant GEM+S-1 chemotherapy after surgical resection (all R0 or R1). Relationships between clinicopathologic factors, including DPD and hENT1 expression, and disease-free or overall survival were evaluated by univariate and multivariate analyses. DPD and hENT1 expression had no significant relationship with any other clinicopathologic factors. A multivariate disease-free survival analysis revealed that lymph node metastasis (hazard ratio [HR], 2.90: 95% confidence interval [CI], 1.51-5.90; P = 0.001), DPD expression (HR 2.47; 95% CI 1.37-4.44; P = 0.003), and hENT1 expression (HR 2.55; 95% CI 1.37-4.64; P = 0.004) as independent factors. Multivariate overall survival analysis also identified pT factor (HR 3.47; 95% CI 1.08-15.8; P = 0.03), lymph node metastasis (HR 2.08; 95% CI 1.01-4.57; P = 0.04), DPD expression (HR 1.98; 95% CI 1.06-3.71; P = 0.03), and hENT1 expression (HR 2.18; 95% CI 1.10-4.19; P = 0.02) as independent factors. Combined analysis of DPD and hENT1 expression predicts the survival of PDAC patients treated with adjuvant GEM+S-1 chemotherapy.

Prognostic Impact of Dihydropyrimidine Dehydrogenase and Human Equilibrative Nucleoside Transporter 1 Expression in Adjuvant Gemcitabine Plus S-1 Chemotherapy After Surgical Resection for Pancreatic Adenocarcinoma

Prognostic Impact of Dihydropyrimidine Dehydrogenase and Human Equilibrative Nucleoside Transporter 1 Expression in Adjuvant Gemcitabine Plus S-1 Chemotherapy After Surgical Resection for Pancreatic Adenocarcinoma

Characterization and Functional Analysis of the Promoter for the Human Equilibrative Nucleoside Transporter Gene, hENT1

Equilibrative nucleoside transporters (ENTs) are membrane proteins that transport nucleosides, nucleobases and analogs across membranes. ENT genes and the regulation of their expression are poorly understood. Therefore, we isolated and functionally characterized the promoter of the prototypic human ENT, hENT1. A single transcriptional initiation site 58 bp downstream of the TATA box and 272 bp upstream of the translation initiation site is present. Limited sequence similarity exists between the hENT1 and mouse ENT1 (mENT1) promoters suggesting conservation of ENT1 transcriptional regulators in mammals. Putative consensus sites for transcription factors exist within the hENT1 promoter. Reporter assays revealed similar but not identical transcriptional activity profiles in human cells.

Identification and Mutational Analysis of Amino Acid Residues Involved in Dipyridamole Interactions with Human and Caenorhabditis elegans Equilibrative Nucleoside Transporters

The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile429 in TM 11 of CeENT1 and Met33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport.

Equilibrative nucleoside transporter 2 is expressed in human umbilical vein endothelium, but is not involved in the inhibition of adenosine transport induced by hyperglycaemia

Human equilibrative, Na(+)-independent nucleoside transport is mediated by membrane proteins sensitive (system es, hENT1) or insensitive (system ei, hENT2) to nitrobenzylthioinosine (NBMPR). Gestational diabetes and elevated extracellular concentrations of D-glucose reduce adenosine transport in human umbilical vein endothelium (HUVEC). We studied hENT2 and hENT1 expression in HUVEC, and the effect of D-glucose on their activity and expression in HUVEC preincubated with 25 mM D-glucose (24 h). hENT2 and hENT1 mRNA were quantified by real-time reverse transcription polymerase chain reaction, and their proteins were detected by Western blotting. hENT2 and hENT1 proteins are co-expressed in HUVEC and are located at the plasma membrane, however, hENT2 was mainly cytoplasmatic and perinuclear in location. D-Glucose reduced hENT1 and hENT2 mRNA expression, but only hENT1 protein abundance at the plasma membrane. Adenosine transport was inhibited by D-glucose and NMBPR (1 microM) in intact cells and membrane vesicles. Hypoxanthine inhibited adenosine transport in the absence or in the presence of 1 microM NBMPR. D-Glucose reduced NBMPR maximal binding in intact cells, membrane vesicles, and plasma membrane fractions. In conclusion, the present study demonstrates that hENT2 and hENT1 are co-expressed in HUVEC, and even when adenosine transport is also mediated by hENT2, the hENT2-mediated transport activity is not involved in the d-glucose-induced down-regulation of total adenosine transport.

Use of a small reporter molecule to determine cell-surface proteins by capillary electrophoresis and laser-induced fluorescence: use of 5-SAENTA-x8f for quantitation of the human equilibrative nucleoside transporter 1 protein.

The human equilibrative nucleoside transporter 1 protein (hENT1) is a major mediator of cellular entry of nucleosides and anticancer nucleoside drugs; its assay is important in understanding and diagnosing chemotherapy resistance. Here we present a novel assay for quantifying hENT1 using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A cellular population is treated with 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine-x8-fluorescein (5-SAENTA-x8f), which binds with high affinity and specificity to the hENT1 protein. The cells are washed to remove excess reagent, lysed, and centrifuged, and the supernatant is analyzed by CE-LIF with the use of an internal standard. Accuracy was evaluated by comparing the capillary electrophoresis results with those obtained by flow cytometry; the results were highly correlated, r = 0.96. The relative standard deviation of the hENT1 assay was 10%, determined from nine independent assays of the same cell line, which is 3 times superior to results obtained in a flow cytometry assay. The detection limit for 5-SAENTA-x8f was 4300 molecules injected into the capillary.

Topology of a Human Equilibrative, Nitrobenzylthioinosine (NBMPR)-sensitive Nucleoside Transporter (hENT1) Implicated in the Cellular Uptake of Adenosine and Anti-cancer Drugs

The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in Xenopus oocytes.

In the present study, one has determined the relative role of plasma membrane equilibrative (Na+-independent) ENT nucleoside transport proteins (particularly ENT2) in the uptake of antiviral nucleoside analogues for comparison with the previously reported drug transport properties of concentrative (Na+-dependent) CNT nucleoside transport proteins. The human and rat nucleoside transport proteins hENT1, rENT1, hENT2 and rENT2 were produced in Xenopus oocytes and investigated for their ability to transport three 3'-deoxy-nucleoside analogues, ddC (2'3'-dideoxycytidine), AZT (3'-azido-3'-deoxythymidine) and ddI (2'3'-dideoxyinosine), used in human immunodeficiency virus (HIV) therapy. The results show, for the first time, that the ENT2 transporter isoform represents a mechanism for cellular uptake of these clinically important nucleoside drugs. Recombinant h/rENT2 transported ddC, ddI and AZT, whilst h/rENT1 transported only ddC and ddI. Relative to uridine, h/rENT2 mediated substantially larger fluxes of ddC and ddI than h/rENT1. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 transport-positive for AZT and enhanced the uptake of both ddC and ddI, identifying this region as a major site of 3'-deoxy-nucleoside drug interaction.

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced inXenopusoocytes

Transport of antiviral 3'-deoxy-nucleoside drugs by recombinant human and rat equilibrative, nitrobenzylthioinosine (NBMPR)-insensitive (ENT2) nucleoside transporter proteins produced in<i>Xenopus</i>oocytes

Cloning of the Human Equilibrative, Nitrobenzylmercaptopurine Riboside (NBMPR)-insensitive Nucleoside Transporter ei by Functional Expression in a Transport-deficient Cell Line

Mammalian cells obtain nucleic acid precursors through the de novo synthesis of nucleotides and the salvage of exogenous nucleobases and nucleosides. The first step in the salvage pathway is transport across the plasma membrane. Several transport activities, including equilibrative and concentrative mechanisms, have been identified by their functional properties. We report here the functional cloning of a 2.6-kilobase pair human cDNA encoding the nitrobenzylmercaptopurine riboside (NBMPR)-insensitive, equilibrative nucleoside transporter ei by functional complementation of the transport deficiency in a subline of CEM human leukemia cells. Expression of this cDNA conferred an NBMPR-insensitive, sodium-independent nucleoside transport activity to the cells that exhibited substrate specificity and inhibitor sensitivity characteristic of the ei transporter. The cDNA contained a single open reading frame that encoded a 456-residue protein with 11 potential membrane-spanning regions and two consensus sites for N-glycosylation in the first predicted extracellular loop. The predicted protein was 50% identical to the recently cloned human NBMPR-sensitive, equilibrative nucleoside transporter ENT1 and thus was designated ENT2. Surprisingly, the carboxyl-terminal portion of the ENT2 protein was nearly identical to a smaller protein in the GenBankTM data base (human HNP36, 326 residues) that has been identified as a growth factor-induced delayed early response gene of unknown function. Comparison of the ENT2 and HNP36 nucleotide sequences suggested that HNP36 was translated from a second start codon within the ENT2 open reading frame. Transient expression studies with the full-length ENT2 and a 5'-truncated construct that lacks the first start codon (predicted protein 99% identical to HNP36) demonstrated that only the full-length construct conferred uridine transport activity to the cells. These data suggest that the delayed early response gene HNP36 is a truncated form of ENT2 and that the full-length open reading frame of ENT2 is required for production of a functional plasma membrane ei transporter.

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

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