Nucleobase transport by human equilibrative nucleoside transporter 1 (hENT1)

The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938-24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of V(max) (pmol/oocyte · min(-1)):K(m) (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C-) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter.     

PKC regulation of the human equilibrative nucleoside transporter,hENT1

Regulation of nucleoside transporters is poorly understood. We show that acute stimulation of protein kinase C (PKC) causes a rapid increase in S-(4-nitrobenzyl)-6-thioinosine-sensitive (human equilibrative nucleoside transporter 1, hENT1) nucleoside uptake, in human cultured cells, which is not due to increased metabolism and which can be blocked by PKC inhibitors. Use of isoform-specific inhibitors indicates that PKC delta and/or epsilon (but not alpha, beta or gamma) are responsible for the acute effects. Down-regulation of PKC decreases hENT1-dependent uridine uptake. These are the first data to show rapid PKC delta/epsilon-dependent stimulation of hENT1 transport by a mechanism that may involve activation of transporters at the membrane possibly by post-translational modification of the protein.     

Extended exposure to substrate regulates the human equilibrative nucleoside transporter 1 (hENT1)

ABSTRACT

Human equilibrative nucleoside transporter 1 (hENT1) is a major route of entry of nucleosides and nucleoside analog drugs. The regulation of hENT1 is poorly understood in spite of its clinical importance as a drug transporter. Immunofluorescence microscopy and fluorescence-activated cell sorting suggested that cytidine pre-treatment (40 μM, 6 h) promotes hENT1 internalization in a way that does not affect either hENT1-mediated nucleoside uptake or gemcitabine-induced cytotoxicity. The Scatchard plot analyses of our NBTI binding data support previous speculations that hENT1 proteins exist as two sub-populations, and suggest that cytidine pre-treatment leads to the internalization of one population.     

A single glycine mutation in the equilibrative nucleoside transporter gene, hENT1, alters nucleoside transport activity and sensitivity to nitrobenzylthioinosine

The human equilibrative nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (NBMPR), is expressed in a wide variety of tissues. hENT1 is involved in the uptake of natural nucleosides, including regulation of the physiological effects of extracellular adenosine, and transports nucleoside drugs used in the treatment of cancer and viral diseases. Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of nucleosides. We have hypothesized that amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of nucleosides. Therefore, we explored the role of point mutations of two conserved glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast nucleoside transporter assay system. Mutations of glycine 179 to leucine, cysteine, or valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as glycine to alanine or serine preserved both targeting to the plasma membrane and transport activity. Similar point mutations at glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity. Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM). Based on these data, we conclude that when hENT1 is expressed in yeast, glycine 179 is critical not only to the ability of hENT1 to transport uridine but also as a determinant of hENT1 sensitivity to NBMPR. In contrast, glycine 184 is likely important in targeting the transporter to the plasma membrane. This is the first identification and characterization of a critical amino acid residue of hENT1 that is important in both nucleoside transporter function and sensitivity to inhibition by NBMPR.     

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.     

Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs

Many antiviral drugs (e.g. fialuridine; FIAU) produce clinically significant mitochondrial toxicity that limits their dose or prevents their use in the clinic. Because the majority of nucleoside drugs is too hydrophilic to cross the highly impermeable mitochondrial membrane, we have hypothesized that they must be transported into the mitochondria to produce their toxicity. To test this hypothesis, we have sought to determine whether the nucleoside transporters, human equilibrative nucleoside transporter 1 (hENT1) or human concentrative nucleoside transporter 1 (hCNT1), when stably expressed in Madin-Darby canine kidney cells as yellow fluorescent fusion protein (YFP), are localized to the mitochondria. By using organelle-selective dyes and confocal microscopy, we have found that hENT1-YFP is localized to the mitochondria as well as the plasma membrane, whereas hCNT1-YFP was found predominantly on the plasma membrane. hENT1-YFP was not localized to the nuclear envelope, endosomes, lysosomes, or Golgi complex. Western blotting confirmed the presence of hENT1-YFP or endogenous hENT1 in mitochondria isolated from hENT1-YFP-expressing cells and human livers, respectively. In agreement with these localization data, [14C]FIAU was efficiently transported into the mitochondria of cells expressing hENT1-YFP but not of cells expressing hCNT1-YFP. The mitochondrial toxicity of FIAU to Madin-Darby canine kidney cells was enhanced by hENT1-YFP, even when hENT1 activity on the plasma membrane was selectively blocked by 10 nm nitrobenzylthioinosine. Moreover, FIAU (50 microm) produced significant mitochondrial toxicity ( approximately 70% decrease in mitochondrial DNA synthesis) when it was directly incubated with mitochondria isolated from hENT1-expressing cells. In conclusion, we have identified for the first time that hENT1 is expressed on the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of nucleoside drugs such as FIAU. Mitochondrial expression of hENTs may explain the clinically significant mitochondrial toxicity caused by the anti-HIV nucleoside drugs such as zidovudine, stavudine, and didanosine.     

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.     

New role of the human equilibrative nucleoside transporter 1 (hENT1) in epithelial-to-mesenchymal transition in renal tubular cells

Epithelial-to-mesenchymal transition (EMT) is an important pro-fibrotic event in which tubular epithelial cells are transformed into myofibroblasts. Nucleoside transporters (NT) are regulated by many factors and processes, some of which are involved in fibrosis, such as cytokines, inflammation, and proliferation. Equilibrative nucleoside transporter 1 (ENT1) has been proved to be the most widely expressed adenosine transporter. In that sense, ENT1 may be a key player in cell damage signaling. Here we analyze the role of human ENT1 (hENT1) in the EMT process in proximal tubular cells. Addition of the main inducer of EMT, the transforming growth factor-β1, to HK-2 cells increased hENT1 mRNA and protein level expression. ENT1-mediated adenosine uptake was also enhanced. When cells were incubated with dipyridamole to evaluate the potential contribution of ENT1 to EMT by blocking its transport activity, EMT was induced. Moreover, the knock down of hENT1 with siRNA induced EMT and collagen production in HK-2 cells. Kidneys isolated from ENT1 knockout mice showed higher levels of interstitial collagen and α-SMA positive cells than wild-type mice. Our results point to a new potential role of hENT1 as a modulator of EMT in proximal tubular cells. In this sense, hENT1 could be involved in renal protection processes, and the loss or reduced expression of hENT1 would lead to an increased vulnerability of cells to the onset and/or progression of renal fibrosis.     

CoMFA and CoMSIA 3D-QSAR studies on S6-(4-nitrobenzyl)mercaptopurine riboside (NBMPR) analogs as inhibitors of human equilibrative nucleoside transporter 1 (hENT1)

3D-QSAR (CoMFA and CoMSIA) studies were performed on human equlibrative nucleoside transporter (hENT1) inhibitors displaying K_i values ranging from 10,000 to 0.7 nM. Both CoMFA and CoMSIA analysis gave reliable models with q² values >0.50 and r² values >0.92. The models have been validated for their stability and robustness using group validation and bootstrapping techniques and for their predictive abilities using an external test set of nine compounds. The high predictive r² values of the test set (0.72 for CoMFA model and 0.74 for CoMSIA model) reveals that the models can prove to be a useful tool for activity prediction of newly designed nucleoside transporter inhibitors. The CoMFA and CoMSIA contour maps identify features important for exhibiting good binding affinities at the transporter, and can thus serve as a useful guide for the design of potential equilibrative nucleoside transporter inhibitors.     

Constrained NBMPR analogue synthesis, pharmacophore mapping and 3D-QSAR modeling of equilibrative nucleoside transporter 1 (ENT1) inhibitory activity

Conformationally constrained analogue synthesis was undertaken to aid in pharmacophore mapping and 3D-QSAR analysis of nitrobenzylmercaptopurine riboside (NBMPR) congeners as equilibriative nucleoside transporter 1 (ENT1) inhibitors. In our previous study [J. Med. Chem. 2003, 46, 831-837], novel regioisomeric nitro-1,2,3,4-tetrahydroisoquinoline conformationally constrained analogues of NBMPR were synthesized and evaluated as ENT1 ligands. 7-NO(2)-1,2,3,4-Tetrahydroisoquino-2-yl purine riboside was identified as the analogue with the nitro group in the best orientation at the NBMPR binding site of ENT1. In the present study, further conformational constraining was introduced by synthesizing 5'-O,8-cyclo derivatives. The flow cytometrically determined binding affinities indicated that the additional 5'-O,8-cyclo constraining was unfavorable for binding to the ENT1 transporter. The structure-activity relationship (SAR) acquired was applied to pharmacophore mapping using the PHASE program. The best pharmacophore hypothesis obtained embodied an anti-conformation with three hydrogen-bond acceptors, one hydrophobic center, and two aromatic rings involving the 3'-OH, 4'-oxygen, the NO(2) group, the benzyl phenyl and the imidazole and pyrimidine portions of the purine ring, respectively. A PHASE 3D-QSAR model derived with this pharmacophore yielded an r(2) of 0.916 for four (4) PLS components, and an excellent external test set predictive r(2) of 0.78 for 39 compounds. This pharmacophore was used for molecular alignment in a comparative molecular field analysis (CoMFA) 3D-QSAR study that also afforded a predictive model with external test set validation predictive r(2) of 0.73. Thus, although limited, this study suggests that the bioactive conformation for NBMPR at the ENT1 transporter could be anti. The study has also suggested an ENT1 inhibitory pharmacophore, and established a predictive CoMFA 3D-QSAR model that might be useful for novel ENT1 inhibitor discovery and optimization.     

Prognostic role of human equilibrative transporter 1 (hENT1) in patients with resected gastric cancer

Nucleoside transporter proteins are specialized proteins that mediate the transport of nucleosides and nucleoside analog drugs across the plasma membrane. The human equilibrative nucleoside transporter 1 (hENT1) is a member of these proteins and mediates cellular entry of gemcitabine, cytarabine, and fludarabine. The hENT1 expression has been demonstrated to be related with prognosis and activity of gemcitabine‐based therapy in breast, ampullary, lung, and pancreatic cancer. We investigated the immunohistochemical expression of hENT in tumor samples from 111 patients with resected gastric adenocarcinoma, correlating these data with clinical parameters and disease outcomes. None of the patients received chemotherapy or radiation therapy before or after surgery as a part of an adjuvant or neoadjuvant program. On univariate survival analysis, the hENT1 expression was associated with overall survival (OS) and disease free survival (DFS). Specifically, those patients with overexpression of hENT1 showed a shorter OS (P = 0.021) and a shorter DFS (P = 0.033). Considering only the node positive patients, higher hENT levels were associated with significantly shorter median DFS (21.7 months; 95% CI 11.1–32.4) compared with patients with low expression of hENT1. The hENT1 expression was defined, in the lymph‐node positive patients, as an independent prognostic factor (P = 0.019). Furthermore, considering only patients with diffuse or mixed tumors and lymph‐node positive, the expression of hENT1 was strongly related with DFS and OS. Immunohistochemistry for the hENT1 protein carries prognostic information in patients with resected gastric cancer and holds promise as a predictive factor in chemotherapy decisions. J. Cell. Physiol. 223: 384–388, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1): implications for interspecies differences in mitochondrial toxicity of fialuridine

We have previously shown that the human equilibrative nucleoside transporter 1 (hENT1) is expressed and functional in the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of the nucleoside drug, fialuridine (FIAU) (Lai, Y., Tse, C. M., and Unadkat, J. D. (2004) J. Biol. Chem. 279, 4490-4497). Here we report on identification of the mitochondrial targeting sequence of hENT1. Using confocal microscopy and different truncated and point mutants of hENT1-YFP (yellow fluorescent protein) expressed in Madin-Darby canine kidney cells, we identified amino acid residues Pro(71),Glu(72), and Asn(74) (the PEXN motif) of hENT1 as important in mitochondrial targeting of hENT1. Identification of this mitochondrial targeting sequence provides a possible explanation for the dramatic difference in mitochondrial toxicity of FIAU between humans and rodents. Although the mouse ENT1 (mENT1), expressed in Madin-Darby canine kidney cells, can transport FIAU, confocal microscopy showed that mENT1-GFP (green fluorescent protein) was not localized to the mitochondria. Consistent with this observation, mitochondria isolated from mouse livers did not transport FIAU. Sequence alignment of hENT1, mENT1, and rat ENT1 (rENT1) showed that the PEXN motif of hENT1 was substituted with a PAXS motif in both mENT1 and rENT1. Substitution of PAXS in mENT1 with PEXN (to create mENT1-PEXN-GFP) and of PEXN in hENT1 with PAXS (to create hENT1-PAXS-YFP) resulted in partial mitochondrial localization of mENT1-PEXN-GFP and loss of mitochondrial localization of hENT1-PAXS-YFP. This is the first time that the mitochondrial targeting signal of hENT1 has been identified. Our data suggest that the lack of mitochondrial toxicity of FIAU in mice is due to the lack of mENT1 targeting to and expression in the mitochondria.     

Comparison of the interaction of uridine, cytidine, and other pyrimidine nucleoside analogues with recombinant human equilibrative nucleoside transporter 2 (hENT2) produced in Saccharomyces cerevisiae.

The human equilibrative nucleoside transporters I and 2 (hENT1, hENT2) share 50% amino acid identity and exhibit broad selectivities, accepting purine and pyrimidine nucleosides as permeants. The permeant selectivity of hENT2 is less well understood because of the low abundance of the native transporter in cells amenable to functional analysis. Recent studies of hENT2 produced in recombinant form in functional expression systems have shown that it differs from hENT1 in that it transports nucleobases. To further understand the structural requirements for permeant interaction with hENT2, we compared the relative abilities of uridine, cytidine, and their analogues to inhibit transport of [3H]uridine by recombinant hENT1 and hENT2 produced in yeast. hENT1 and hENT2 tolerated halogen modification at the 5 position of the base and the 2' and 5' positions of the ribose moieties of uridine whereas removal of the hydroxyl group at the 3' position of the ribose moiety of uridine eliminated interaction with both transporters. hENT2 displayed a lower ability, compared with hENT1, to interact with cytidine and cytidine analogues, suggesting a low tolerance for the presence of the amino group at the 4 position of the base.     

Expression of human equilibrative nucleoside transporter 1 in mouse neurons regulates adenosine levels in physiological and hypoxic-ischemic conditions

Activation of adenosine A(1) receptors inhibits excitatory synaptic transmission. Equilibrative nucleoside transporters (ENTs) regulate extracellular adenosine levels; however, the role of neuronal ENTs in adenosine influx and efflux during cerebral ischemia has not been determined. We used mice with neuronal expression of human ENT type 1 and wild type (Wt) littermates to compare responses to in vitro hypoxic or ischemic conditions. Extracellular recordings in the CA1 region of hippocampal slices from transgenic (Tg) mice revealed increased basal synaptic transmission, relative to Wt slices, and an absence of 8-cyclopentyl-1,3-dipropyl-xanthine mediated augmentation of excitatory neurotransmission. Adenosine (10-100 μM) had a reduced potency for inhibiting synaptic transmission in slices from Tg mice; inhibitory concentration 50% values were approximately 25 and 50 μM in Wt and Tg slices, respectively. Potency of the A(1) receptor agonist N(6) -cyclopentyladenosine (1 nM-1 μM) was unchanged. Transient hypoxia or oxygen-glucose deprivation produced greater inhibition of excitatory neurotransmission in slices from Wt than Tg, mice. The ENT1 inhibitor S-(4-nitrobenzyl)-6-thioinosine abolished these differences. Taken together, our data provide evidence that neuronal ENTs reduce hypoxia- and ischemia-induced increases in extracellular adenosine levels and suggest that inhibition of neuronal adenosine transporters may be a target for the treatment of cerebral ischemia.     

Mutation of Trp29 of human equilibrative nucleoside transporter 1 alters affinity for coronary vasodilator drugs and nucleoside selectivity

hENT1 (human equilibrative nucleoside transporter 1) is inhibited by nanomolar concentrations of various structurally distinct coronary vasodilator drugs, including dipyridamole, dilazep, draflazine, soluflazine and NBMPR (nitrobenzylmercaptopurine ribonucleoside). When a library of randomly mutated hENT1 cDNAs was screened using a yeast-based functional complementation assay for resistance to dilazep, a clone containing the W29G mutation was identified. Multiple sequence alignments revealed that this residue was highly conserved. Mutations at Trp29 were generated and tested for adenosine transport activity and inhibitor sensitivity. Trp29 mutations significantly reduced the apparent V(max) and/or increased the apparent K(m) values for adenosine transport. Trp29 mutations increased the IC50 values for hENT1 inhibition by dipyridamole, dilazep, NBMPR, soluflazine and draflazine. NBMPR and soluflazine displayed remarkably similar trends, with large aromatic substitutions at residue 29 resulting in the lowest IC50 values, suggesting that both drugs could interact via ring-stacking interactions with Trp29. The W29T mutant displayed a selective loss of pyrimidine nucleoside transport activity, which contrasts with the previously identified L442I mutant that displayed a selective loss of purine nucleoside transport. W29T, L442I and the double mutant W29T/L442I were characterized kinetically for nucleoside transport activity. A helical wheel projection of TM (transmembrane segment) 1 suggests that Trp29 is positioned close to Met33, implicated previously in nucleoside and inhibitor recognition, and that both residues line the permeant translocation pathway. The data also suggest that Trp29 forms part of, or lies close to, the binding sites for dipyridamole, dilazep, NBMPR, soluflazine and draflazine.     

Electrogenic uptake of nucleosides and nucleoside-derived drugs by the human nucleoside transporter 1 (hCNT1) expressed in Xenopus laevis oocytes

The concentrative pyrimidine-preferring nucleoside transporter 1 (hCNT1), cloned from human fetal liver, was expressed in Xenopus laevis oocytes. Using the two-electrode voltage-clamp technique, it is shown that translocation of nucleosides by this transporter generates sodium inward currents. Membrane hyperpolarization (from -50 to -150 mV) did not affect the K(0.5) for uridine, although it increased the transport current approximately 3-fold. Gemcitabine (a pyrimidine nucleoside-derived drug) but not fludarabine (a purine nucleoside-derived drug) induced currents in oocytes expressing the hCNT1 transporter. The K(0.5) value for gemcitabine at -50 mV membrane potential was lower than that for natural substrates, although this drug induced a lower current than uridine and cytidine, thus suggesting that the affinity binding of the drug transporter is high but that translocation occurs more slowly. The analysis of the currents generated by the hCNT1-mediated transport of nucleoside-derived drugs used in anticancer and antiviral therapies will be useful in the characterization of the pharmacological profile of this family of drug transporters and will allow rapid screening for uptake of newly developed nucleoside-derived drugs.