Expression and purification of human and Saccharomyces cerevisiae equilibrative nucleoside transporters.

Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis.

Equilibrative nucleoside transporter 1 expression in primary human hepatocytes is highly variable and determines uptake of ribavirin.

Aims Ribavirin is a nucleoside analogue and remains a necessary component of both interferon-based and directly acting anti-viral regimens for the treatment of hepatitis C virus infection. The achievable concentration of ribavirin within hepatocytes is likely to be an important determinant of therapeutic outcome. In vitro expression levels of equilibrative nucleoside transporter 1 (ENT1) has been shown to be a predictor of treatment response in patients receiving nucleoside-based chemotherapeutic agents. We therefore investigated whether a similar relationship existed between ENT1 expression and ribavirin uptake in freshly isolated primary hepatocytes. Methods Primary hepatocytes were cultured on collagen-coated plates and exposed to ribavirin. Parallel samples were taken for high-performance liquid chromatography to assess ribavirin uptake and for quantitative polymerase chain reaction to evaluate ENT1 expression. Similar assays were performed on the human hepatoma cell line (Huh7). ENT1 gene sequence was analysed by cloning of polymerase chain reaction amplified complementary DNA followed by direct sequencing. Results There was a strong direct correlation between expression of ENT1 in primary hepatocytes and ribavirin uptake at 24 hr. Huh7 cells expressed ENT1 at similar levels to the majority of primary hepatocytes, but did not take up ribavirin. Sequencing revealed that ENT1 in Huh7 cells is wild type. Conclusions In this study, we clearly demonstrate that ribavirin uptake in primary human hepatocytes is variable and correlates with ENT1 expression. This variation in ENT1 expression may account for differences in response rate in patients receiving ribavirin-based anti-hepatitis C virus therapy.

Verapamil inhibits tumor progression of chemotherapy-resistant pancreatic cancer side population cells.

Tumor side population (SP) cells display stem-like properties that can be modulated by treatment with the calcium channel blocker verapamil. Verapamil can enhance the cytotoxic effects of chemotherapeutic drugs and multidrug resistance by targeting the transport function of the P-glycoprotein (P-gp). This study focused on the therapeutic potential of verapamil on stem-like SP tumor cells, and further investigated its chemosensitizing effects using L3.6pl and AsPC-1 pancreatic carcinoma models. As compared to parental L3.6pl cells (0.9±0.22%), L3.6pl gemcitabine-resistant cells (L3.6plGres) showed a significantly higher percentage of SP cells (5.38±0.99%) as detected by Hoechst 33342/FACS assays. The L3.6plGres SP cells showed stable gemcitabine resistance, enhanced colony formation ability and increased tumorigenicity. Verapamil effectively inhibited L3.6plGres and AsPC-1 SP cell proliferation in vitro. A pro-apoptotic effect of verapamil was observed in L3.6pl cells, but not in L3.6plGres cells, which was linked to their differential expression of P-gp and equilibrative nucleoside transporter-1 (ENT-1). In an orthotopic pancreatic cancer mouse model, both low and high dose verapamil was shown to substantially reduce L3.6plGres-SP cell tumor growth and metastasis, enhance tumor apoptosis, and reduce microvascular density.

Human Equilibrative Nucleoside Transporter 1 Expression in Endoscopic Ultrasonography-Guided Fine-Needle Aspiration Biopsy Samples Is a Strong Predictor of Clinical Response and Survival in the Patients With Pancreatic Ductal Adenocarcinoma Undergoing Gemcitabine-Based Chemoradiotherapy.

OBJECTIVES: This study aimed to clarify whether pretreatment human equilibrative nucleoside transporter (hENT1) expressions in endoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS-FNAB) specimens obtained from resectable, borderline resectable, and locally advanced unresectable pancreatic ductal adenocarcinoma (PDAC) are concordant with those in the resected specimen after gemcitabine-based chemoradiotherapy (Gem-CRT) and to validate the utility of hENT1 expression using EUS-FNAB samples as a prognostic marker. METHODS: We evaluated the relationship between hENT1 expressions assessed by immunohistochemical staining and clinical outcomes in 51 of 76 patients with PDAC who were diagnosed by EUS-FNAB and received preoperative Gem-CRT. RESULTS: The concordance rate of hENT1 expressions was 89.2% (K = 0.681). Median survival time (month) in the 51 whole patients and 37 patients with resection was significantly longer in hENT1 positive than in hENT1 negative: 25.0 and 30.0 versus 9.0 and 9.0, respectively. A multivariate analysis confirmed that hENT1 expression was an independent prognostic factor in both whole patients and those with resection. Regardless of T3 and T4, hENT1-positive patients with resection had significantly better prognosis than hENT1-negative patients, whose prognosis was similar to those without resection. CONCLUSIONS: The assessment of hENT1 expression using EUS-FNAB samples before Gem-CRT provides important information on patients with PDAC who can benefit from curative-intent resection.

Effect of Hypoxanthine on Functional Activity of Nucleoside Transporters ENT1 and ENT2 in Caco-2 Polar Epithelial Intestinal Cells.

We studied regulation of hypoxanthine transport depending on its concentration in the culture medium. Caco-2 cells were differentiated on membrane filters to create a model of the intestine. Different hypoxanthine uptake on the apical and basolateral cell membranes was observed. The expression of SLC29 family genes encoding passive nucleoside transporters increased upon changes in hypoxanthine concentration in the medium Localization of the transporters and their influence on the effect of pharmacological preparations are discussed.

Zidovudine, an anti-viral drug, resensitizes gemcitabine-resistant pancreatic cancer cells to gemcitabine by inhibition of the Akt-GSK3ß-Snail pathway.

Pancreatic cancer is one of the most difficult malignancies to treat owing to the rapid acquisition of resistance to chemotherapy. Gemcitabine, a first-line treatment for pancreatic cancer, prolongs patient survival by several months, and combination treatment with gemcitabine and other anti-cancer drugs in the clinic do not show any significant effects on overall survival. Thus, identification of a drug that resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine and a better understanding of the molecular mechanisms of gemcitabine resistance are critical to develop new therapeutic options for pancreatic cancer. Here, we report that zidovudine resensitizes gemcitabine-resistant pancreatic cancer to gemcitabine as shown by screening a compound library, including clinical medicine, using gemcitabine-resistant cells. In analyzing the molecular mechanisms of zidovudine effects, we found that the epithelial-to-mesenchymal transition (EMT)-like phenotype and downregulation of human equilibrative nucleoside transporter 1 (hENT1) are essential for the acquisition of gemcitabine resistance, and zidovudine restored these changes. The chemical biology investigations also revealed that activation of the Akt-GSK3ß-Snail1 pathway in resistant cells is a key signaling event for gemcitabine resistance, and zidovudine resensitized resistant cells to gemcitabine by inhibiting this activated pathway. Moreover, our in vivo study demonstrated that co-administration of zidovudine and gemcitabine strongly suppressed the formation of tumors by gemcitabine-resistant pancreatic cancer and prevented gemcitabine-sensitive pancreatic tumors from acquiring gemcitabine-resistant properties, inducing an EMT-like phenotype and downregulating hENT1 expression. These results suggested that co-treatment with zidovudine and gemcitabine may become a novel therapeutic strategy for pancreatic cancer by inhibiting chemoresistance-specific signaling.

RNA expression of genes involved in cytarabine metabolism and transport predicts cytarabine response in acute myeloid leukemia.

BACKGROUND: Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. METHODS: Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. RESULTS: Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69-11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. CONCLUSION: This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity. Original submitted 22 December 2014; Revision submitted 9 April 2015.

The hENT1 and DCK genes underlie the decitabine response in patients with myelodysplastic syndrome.

Decitabine is approved for the treatment of MDS, but resistance to this agent is common. To determine the mechanisms underlying decitabine resistance, we measured the mRNA expression of metabolism (hENT1, DCK, CDA) and apoptosis (BCL2L10) genes and found that the hENT1 mRNA level was significantly higher in response compared with non-response patients (P=0.004). Furthermore, the DCK level was significantly reduced for relapse (P=0.012) compared with those with continued marrow CR (P=0.222). These findings indicate that the decitabine metabolic pathway affects its therapeutic effects, lower hENT1 expression may induce primary resistance and down-regulated DCK expression may be related to secondary resistance.

Contribution of CNT1 and ENT1 to ribavirin uptake in human hepatocytes.

The objective of this study was to investigate the contributions of a sodium-dependent concentrative nucleoside transporter (CNT) 1 and an equilibrative nucleoside transporter (ENT) 1 to ribavirin uptake in human hepatocytes. The initial studies in oocytes expressing CNT1 and ENT1 showed increases in ribavirin uptake, indicating that ribavirin was a substrate for both CNT1 and ENT1. The CNT1- and ENT1-mediated ribavirin uptake showed concentration dependency with the following kinetics parameters: Km 26.3 µM and Vmax 426.2 fmol/min/oocyte for CNT1; Km 70.5 µM and Vmax 134.3 fmol/min/oocyte for ENT1. Ribavirin uptake clearance in six human hepatocytes ranged from 21.3 to 300.7 µL/min. Estimation of the contributions of CNT1 and ENT1 to the hepatic uptake of ribavirin by using a relative activity factor method indicated that the relative contribution of ENT1 to the ribavirin uptake was 82.8 ± 3.9%. Real-time polymerase chain reaction analysis of CNT1 and ENT1 expressions in the hepatocytes showed that ENT1 mRNA expression was closely correlated with ribavirin uptake (R = 0.95, P = 0.003) while CNT1 was not. The findings indicated that ENT1 was the major transporter controlling the hepatic uptake of ribavirin.

Human equilibrative nucleoside transporter 1 is not predictive for gemcitabine efficacy in advanced pancreatic cancer: translational results from the AIO-PK0104 phase III study with the clone SP120 rabbit antibody.

BACKGROUND: The role of human equilibrative nucleoside transporter 1 (hENT1) as a predictive biomarker for gemcitabine efficacy in advanced pancreatic cancer remains unclear to date. PATIENTS AND METHODS: AIO-PK0104 was a German multicenter phase III trial comparing gemcitabine/erlotinib followed by capecitabine (GEC) with capecitabine/erlotinib followed by gemcitabine (CEG) in advanced pancreatic cancer. Archival tumour tissue from 169 of the 274 eligible study patients was available for a central and standardised immunohistochemistry staining for hENT1 expression using the SP120 rabbit monoclonal anti-hENT1 antibody. Within a retrospective translational subgroup analysis, biomarker data were correlated with efficacy end-points. RESULTS: Thirty-nine out of 130 fresh-cut slides were scored as hENT1(high) (30%), whereas 91 samples were classified as hENT1(low) (70%). For the 62 patients randomised to CEG median overall survival was estimated with 6.4 months in the hENT1(low) compared to 6.9 months in the hENT1(high) subgroup (Hazard Ratio (HR) 0.88, 95% confidence interval (CI) 0.48-1.61, p=0.67). For the 68 patients randomised to GEC survival was 5.7 months in the hENT1(low) compared to 4.4 months in the hENT1(high) subgroup (HR 1.16, 95% CI 0.69-1.96, p=0.57). In 101 patients receiving gemcitabine at any time during study treatment (either within the 1st- or 2nd-line setting) hENT1(low) cases had a median overall survival of 7.5 months and hENT1(high) patients an overall survival of 4.4 months (HR 1.30, 95% CI 0.84-2.03, p=0.24), respectively. CONCLUSION: Within this subgroup analysis from Arbeitsgemeinschaft Internistische Onkologie-pancreatic cancer (AIO-PK0104), no evidence supporting the use of hENT1 as a predictive biomarker for gemcitabine efficacy in patients with advanced pancreatic cancer was found.

Expression of nucleoside transporter in freshly isolated neurons and astrocytes from mouse brain.

Nucleoside transporters comprise equilibrative ENT1-4 and concentrative CNT1-3. CNTs transport against an intracellular/extracellular gradient and are essential for transmitter removal, independently of metabolic need. ENT1-4 mediate transport until intracellular/extracellular equilibrium of the transported compound, but are very efficient, when the accumulated nucleoside or nucleobase is rapidly eliminated by metabolism. Most nucleoside transporters are membrane-bound, but ENT3 is mainly intracellular. This study uses freshly isolated neurons and astrocytes from two adult mouse strains. In one transgenic strain the neuronal marker Thy1 was associated with a compound fluorescing at one wavelength, and in the other the astrocytic marker GFAP was associated with a compound fluorescent at a different wavelength. Highly purified astrocytic and neuronal populations (as determined by presence/absence of cell-specific genes) were obtained from these mice by fluorescence-activated cell sorting. In each population mRNA analysis was performed by reverse-transcription polymerase chain reaction. CNT1 was absent in both cell types; all other nucleoside transporters were expressed to at least a similar degree (in relation to applied amount of RNA and to a house-keeping gene) in astrocytes as in neurons. Astrocytic ENT3 enrichment was dramatic, but it was not up-regulated after fluoxetine-mediated increase in DNA synthesis. A comparison with results obtained in cultured astrocytes shows that the latter are generally compatible with the present findings and suggests that many observations obtained in intact tissue, mainly by in situ hybridization (which also determines mRNA expression) may underestimate astrocytic nucleoside transporter expression.

Repression of the equilibrative nucleoside transporters dampens inflammatory lung injury.

Acute lung injury (ALI) is a devastating disorder of the lung that is characterized by hypoxemia, overwhelming pulmonary inflammation, and a high mortality in the critically ill. Adenosine has been implicated as an anti-inflammatory signaling molecule, and previous studies showed that extracellular adenosine concentrations are increased in inflamed tissues. Adenosine signaling is terminated by the uptake of adenosine from the extracellular into the intracellular compartment via equilibrative nucleoside transporters (ENTs). However, their role in controlling adenosine signaling during pulmonary inflammation remains unknown. After inflammatory in vitro experiments, we observed a repression of ENT1 and ENT2 that was associated with an attenuation of extracellular adenosine uptake. Experiments using short, interfering RNA silencing confirmed a significant contribution of ENT repression in elevating extracellular adenosine concentrations during inflammation. Furthermore, an examination of the ENT2 promoter implicated NF-κB as a key regulator for the observed ENT repression. Additional in vivo experiments using a murine model of inflammatory lung injury showed that the pharmacological inhibition of ENT1 and ENT2 resulted in improved pulmonary barrier function and reduced signs of acute inflammation of the lung. Whereas experiments on Ent1(-/-) or Ent2(-/-) mice revealed lung protection in LPS-induced lung injury, an examination of bone marrow chimeras for ENTs pointed to the nonhematopoetic expression of ENTs as the underlying cause of dampened pulmonary inflammation during ALI. Taken together, these findings reveal the transcriptional repression of ENTs as an innate protective response during acute pulmonary inflammation. The inhibition of ENTs could be pursued as a therapeutic option to ameliorate inflammatory lung injury.

Effects of dipyridamole coadministration on the pharmacokinetics of ribavirin in healthy volunteers.

  Ribavirin (RBV), a guanosine analog for treatment of hepatitis C, is a substrate of a nucleoside transporter, solute carrier family 29 member 1 (SLC29A1). To clarify the impact of SLC29A1 on the pharmacokinetics of RBV, an open-label, crossover study of single-dose RBV (200 mg, p.o.) with and without coadministration of dipyridamole (DP), an inhibitor of SLC29A1, was performed. Plasma and erythrocyte concentrations of RBV in the control phase and DP phase (25 mg, 3 times daily for 4 days) were compared in 10 healthy volunteers. SLC29A1 mRNA expression in peripheral blood mononuclear cells was also determined. In the DP phase, area under the concentration-time curves (AUC) of RBV in plasma and erythrocytes showed reductions of 23% and 17%, respectively (p < 0.05), with increases in apparent oral clearance of 18% and 25%, respectively (p < 0.05). The reduction rate of the AUC of erythrocyte RBV in the DP phase was associated with SLC29A1 mRNA expression: higher mRNA expression showed greater AUC reduction. The elimination half-life of both plasma and erythrocyte RBV did not differ between the 2 phases. These results suggest that RBV/DP coadministration reduces the concentration of RBV in blood by inhibiting an important role of SLC29A1 in gastrointestinal absorption of RBV.

Variability of gemcitabine accumulation and its relationship to expression of nucleoside transporters in peripheral blood mononuclear cells.

The concentrative nucleoside transporter CNT1 and equilibrated nucleoside transporter ENT1 mediate the cellular uptake of naturally occurring pyrimidine and purine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs, thereby regulating drug responses or toxicity at the target site. The objectives of this study were to analyze interindividual variations in the cellular accumulation of gemcitabine and to examine the correlation between the uptake of gemcitabine and expression levels of CNT1 and ENT1 transporters. Gemcitabine was a substrate for both CNT1 and ENT1 with higher affinity to CNT1 than to ENT1. The difference in gemcitabine uptake was 4.8-fold in peripheral blood mononuclear cells (PBMCs) from 10 subjects. Among these, the CNT1- and ENT1-mediated uptake of gemcitabine was 14.3- and 16.5-folds, respectively. CNT1-mediated gemcitabine uptake showed a higher correlation with the CNT1 expression level than did ENT1-mediated uptake with ENT1 expression level. In conclusion, CNT1 seemed to be a major contributing factor to gemcitabine uptake in PBMCs and showed 14.3-fold inter-individual variations. However, ENT1-mediated uptake of gemcitabine might compensate for the total uptake of gemcitabine; therefore, the variation in the apparent accumulation of gemcitabine was smaller than that of the individual transporters.

Nucleoside transporters are widely expressed in ovarian carcinoma effusions.

OBJECTIVE: Equilibrative and concentrative nucleoside transporters (ENTs and CNTs) mediate the cellular uptake of anticancer nucleosides and sensitivity to such compounds. We studied the expression of ENTs and CNTs in ovarian carcinoma effusions. METHODS: ENT1, ENT2, ENT4 and CNT3 expression was analyzed in 66 ovarian carcinoma effusions (61 peritoneal, 5 pleural) from 64 ovarian carcinoma patients by flow cytometry. The majority of patients received platinum-based chemotherapy. Results were analyzed for association with clinicopathologic parameters and survival. RESULTS: With the exception of one ENT2-negative effusion, ENT1, ENT2, ENT4 and CNT3 protein was detected on carcinoma cells in all effusions, with expression observed in 1-95% of tumor cells. Nucleoside transporter expression was comparable between peritoneal and pleural effusions and was unrelated to age, tumor grade, International Federation of Gynecology and Obstetrics (FIGO) stage, residual tumor volume after surgery, previous exposure to chemotherapy and response to chemotherapy at diagnosis (P > 0.05). No correlation was found between ENT or CNT expression and overall survival or progression-free survival, although higher ENT2 expression was associated with a trend for longer overall (45 vs. 23 months; P = 0.055) and progression-free (17 vs. 5 months; P = 0.087) survival. CONCLUSION: Nucleoside transporters are frequently expressed in ovarian carcinoma effusions, but their expression generally appears to be unrelated to chemoresponse in this cancer in a cohort of patients treated by platinum-based chemotherapy. The role of ENT2 as a prognostic marker in this disease, as well as the role of these molecules in determining chemoresponse in patients treated by nucleoside analogs, merits further research.

Identification of cysteines involved in the effects of methanethiosulfonate reagents on human equilibrative nucleoside transporter 1.

Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. Given that selective ENT1 inhibitors, such as nitrobenzylmercaptopurine riboside (NBMPR), bind to the N-terminal half of the ENT1 protein, we hypothesized that one or more of the four cysteine residues in this region were contributing to the effects of the sulfhydryl modifiers. Recombinant human ENT1 (hENT1), and the four cysteine-serine ENT1 mutants, were expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate (MTS) sulfhydryl-modifying reagents. Transporter function was assessed by the binding of [(3)H]NBMPR and the cellular uptake of [(3)H]2-chloroadenosine. The membrane-permeable reagent methyl methanethiosulfonate (MMTS) enhanced [(3)H]NBMPR binding in a pH-dependent manner, but decreased [(3)H]2-chloroadenosine uptake. [2-(Trimethylammonium)ethyl] methane-thiosulfonate (MTSET) (positively charged, membrane-impermeable), but not sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) (negatively charged), inhibited [(3)H]NBMPR binding and enhanced [(3)H]2-chloroadenosine uptake. Mutation of Cys222 in transmembrane (TM) 6 eliminated the effect of MMTS on NBMPR binding. Mutation of Cys193 in TM5 enhanced the ability of MMTS to increase [(3)H]NBMPR binding and attenuated the effects of MMTS and MTSET on [(3)H]2-chloroadenosine uptake. Taken together, these data suggest that Cys222 contributes to the effects of MTS reagents on [(3)H]NBMPR binding, and Cys193 is involved in the effects of these reagents on [(3)H]2-chloroadenosine transport. The results of this study also indicate that the hENT1-C193S mutant may be useful as a MTSET/MTSES-insensitive transporter for future cysteine substitution studies to define the extracellular domains contributing to the binding of substrates and inhibitors to this critical membrane transporter.

Design, synthesis, and evaluation of 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate (8MDP-fluor), as a novel equilibrative nucleoside transporter probe.

Nucleoside transporters are integral membrane glycoproteins that play critical roles in physiological nucleoside and nucleobase fluxes, and influence the efficacy of many nucleoside chemotherapy drugs. Fluorescent reporter ligands/substrates have been shown to be useful in the analysis of nucleoside transporter (NT) protein expression and discovery of new NT inhibitors. In this study, we have developed a novel dipyridamole (DP)-based equilibrative nucleoside transporter 1 (ENT1) fluorescent probe. The potent ENT1 and ENT2 inhibitor analogue of dipyridamole, 2,6-bis(diethanolamino)-4,8-diheptamethyleneiminopyrimido[5,4-d]pyrimidine (4, 8MDP), was modified to replace one ß-hydroxyethyl group of the amino substituent at the 2-position with a ß-aminoethyl group and then conjugated through the amino group to 6-(fluorescein-5-carboxamido)hexanoyl moiety to obtain a new fluorescent molecule, 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate, designated 8MDP-fluorescein (8MDP-fluor, 6). The binding affinities of 8MDP-fluor at ENT1 and ENT2 are reflected by the uridine uptake inhibitory K(i) values of 52.1 nM and 285 nM, respectively. 8MDP-fluor was successfully demonstrated to be a flow cytometric probe for ENT1 comparable to the nitrobenzylmercaptopurine riboside (NBMPR) analogue ENT1 fluorescent probe SAENTA-X8-fluorescein (SAENTA-fluor, 1). This is the first reported dipyridamole-based ENT1 fluorescent probe, which adds a novel tool for probing ENT1, and possibly ENT2.

Human equilibrative nucleoside transporter 1 (hENT1) levels predict response to gemcitabine in patients with biliary tract cancer (BTC).

BACKGROUND AND AIM: Translational data suggest that nucleoside transporters, in particular human equilibrative nucleoside transporter 1 (hENT1), play an important role in predicting clinical outcome after gemcitabine chemotherapy for several types of cancer. The aim of this study was to retrospectively determine patients' outcome according to the expression of hENT1 in tumoral cells of patients receiving gemcitabine-based therapy. MATERIALS AND METHODS: The immunohistochemistry analysis was performed on samples from thirty-one patients with unresectable biliary tract cancer (BTC) consecutively treated with first line gemcitabine-based regimens. RESULTS: Positive hENT1 staining patients were 21 (67.7%); negative hENT1 staining patients were 10 (32.3%). Statistical analysis revealed no association between baseline characteristics, toxicities and tumor response to gemcitabine and hENT1 levels. In the univariate analysis, HENT1 expression was significantly correlated with time to progression (TTP) (p=0.0394; HR 2.902, 95%CI 1.053-7.996). The median TTP was 6.33 versus 2.83 months, respectively in patients with positive versus negative hENT1 staining. Moreover, patients with positive hENT1 expression showed a longer median overall survival when compared with patients with low hENT1 expression (14 versus 7 months, respectively), but this difference did not reach the statistical significance (p=0.128). CONCLUSIONS: Therefore, hENT1 may be a relevant predictive marker of benefit from gemcitabine-based therapies in patients with advanced BTC.

Differential expression of functional nucleoside transporters in non-differentiated and differentiated human endothelial progenitor cells.

Extracellular adenosine removal is via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) in the endothelium, thus regulating adenosine-induced revascularization and angiogenesis. Since human endothelial progenitor cells (hEPCs) promote revascularization, we hypothesize differential expression of nucleoside transporters in hEPCs. hEPCs were cultured 3 (hEPC-3d) or 14 (hEPC-14d) days. RT-PCR for prominin 1, CD34, octamer-4, kinase insert domain receptor, oxidized low-density lipoprotein (lectin-like) receptor 1 and tyrosine endothelial kinase was used to evaluate phenotypic differentiation. Flow cytometry was used to estimate CD34(+)/KDR(-) (non-differentiated), CD34(-)/KDR(+) (differentiated) or CD34(+)/KDR(+) (mixed) cell populations. Adenosine transport was measured in absence or presence of sodium, S-(4-nitrobenzyl)-6-thio-inosine (NBTI, 1-10 µM), inosine, hypoxanthine or guanine (0.1-5 mM), hENTs protein abundance by western blot, and hENTs, hCNT1, hCNT2 and hCNT3 mRNA expression by real time RT-PCR. hEPC-3d cells were CD34(+)/KDR(-) compared with hEPC-14d cells that were CD34(-)/KDR(+). hEPC-3d cells exhibit hENT1-like adenosine transport (NBTI-sensitive, Na(+)-independent), which is absent in hEPC-14d cells. hEPC-14d cells exhibit two transport components: component 1 (NBTI insensitive, Na(+)-independent) and component 2 (NBTI insensitive, Na(+)-dependent, Hill coefficient ∼1.8), the latter resembling CNT3-like transport. hEPC-3d cells express hENT1 protein and mRNA, which is reduced (∼90%) in hEPC-14d cells, but instead only hCNT3 mRNA is expressed in this cell type. hENT2, hCNT1 and hCNT2 were undetectable in hEPCs. Thus, hEPCs exhibit a differential expression of hENT1 and hCNT3 functional nucleoside transporters, which could be related with its differentiation stage.