Fluorescent Nucleoside Derivatives as a Tool for the Detection of

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FLUORESCENT NUCLEOSIDE DERIVATIVES AS TOOL FOR THE DETECTION OF CONCENTRATIVE NUCLEOSIDE TRANSPORTERS (CNTs) ACTIVITY USING CONFOCAL MICROSCOPY AND FLOW CYTOMETRY Ana Claudio-Montero, Itziar Pinilla-Macua, Paula Fernandez-Calotti, Carlos SanchoMateo, María Pilar Lostao, Dolors Colomer, Anna Grandas, and Marçal Pastor-Anglada Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00142 • Publication Date (Web): 29 Apr 2015 Downloaded from http://pubs.acs.org on May 4, 2015

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Molecular Pharmaceutics

FLUORESCENT NUCLEOSIDE DERIVATIVES AS TOOL FOR THE DETECTION OF CONCENTRATIVE NUCLEOSIDE TRANSPORTERS (CNTs) ACTIVITY USING CONFOCAL MICROSCOPY AND FLOW CYTOMETRY Ana Claudio-Montero,†,¥ Itziar Pinilla-Macua,†,∥ Paula Fernandez-Calotti,† Carlos Sancho-Mateo,‡ Maria Pilar Lostao,‡ Dolors Colomer,§ Anna Grandas,¥,∀ Marçal Pastor-Anglada,*,†,∀. †

Department of Biochemistry and Molecular Biology, University of Barcelona, Institute

of Biomedicine (IBUB) and Oncology Programme, National Biomedical Research Institute on Liver and Gastrointestinal Diseases (CIBER ehd), Instituto de Salud Carlos III, Barcelona, Spain. ‡

Department of Nutrition, Food Science and Physiology, University of Navarra,

Pamplona 31008, Spain ¥

Department of Organic Chemistry, University of Barcelona, Institute of Biomedicine

(IBUB). §

Hematopathology Unit, Hospital Clínic and Institut d’Investigacions Biomèdiques

August Pi i Sunyer (IDIBAPS), Barcelona 08036, Spain.

∥Present

address: Department of Cell Biology, School of Medicine, University of

Pittsburgh, Pittsburgh, PA.

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ABSTRACT The abundance and function of transporter proteins at the plasma membrane are likely to be crucial in drug responsiveness. Functional detection of Human Concentrative Nucleoside Transporters (hCNTs) is of interest to predict drug sensitivity due to their ability to transport most nucleoside-derived drugs. In the present study two fluorescent nucleoside analogues, Uridine-furan and Etheno-cytidine, were evaluated as tools to study in vivo nucleoside transporter-related functions. These two molecules showed high affinity interactions with hCNT1 and hCNT3 and were also shown to be substrates of both transporters. Both fluorescent microscopy and flow cytometry experiments showed that Uridine-furan uptake was better suited for distinguishing cells that express or not hCNT1 or hCNT3. These data highlight the usefulness of fluorescent nucleoside derivatives as long as they fulfil the requirements of confocal microscopy and flow cytometry for in vivo analysis of hCNT-related function.

KEYWORDS Fluorescent nucleosides, drug transporters, confocal microscopy, flow cytometry


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INTRODUCTION Natural nucleosides are fairly hydrophilic molecules that require a transporter to mediate their uptake across the plasma membrane. Two nucleoside transporter (NT) families, SLC28 and SLC29, have been identified so far. SLC28 genes encode human Concentrative Nucleoside Transporters (hCNTs).1 This family consists of three members: hCNT1, hCNT2 and hCNT3, which differ in their substrate selectivities. While hCNT1 prefers pyrimidine nucleosides, hCNT2 prefers purine nucleosides and hCNT3 transports both pyrimidine and purine nucleosides. All three mediate the unidirectional uptake of nucleosides into cells in an active process coupled to the transmembrane sodium gradient, although their Na+/nucleoside stoichiometry is also variable, 1/1 for hCNT1 and hCNT2 and 2/1 for hCNT3. SLC29 genes encode human Equilibrative Nucleoside Transporters (hENTs).2 Four members have been identified within this family: hENT1, hENT2, hENT3 and hENT4. These transporters facilitate the down-hill concentration passive diffusion of nucleosides, being potentially bidirectional carriers. In general terms, hENT proteins exhibit broader selectivity than hCNTs but they are low affinity transporters when compared to hCNTs. The analysis of NT’s expression, localization and function is a relevant issue in pharmacology because these proteins are able to recognize and transport most nucleoside-derived drugs used in anticancer and antiviral therapies.3 The role of NT proteins in nucleoside-derived drug cytotoxicity has been suggested in many studies with cultured cell models4 and clinical validation of this type of correlation has increased over the past few years.4,5 Absence of NT expression contributes to gemcitabine (dFdC, 2’,2’-difluorodeoxycytidine) resistance.4 Variable transporter protein expression patterns in solid tumors, such as pancreatic adenocarcinoma, relates to patient survival after gemcitabine chemotherapy.4,6,7



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The use of radiolabeled (often tritiated) nucleosides is routine in the functional analysis of NT proteins, however it does not allow either real-time in vivo studies of intracellular trafficking of nucleosides or quantification of NT proteins. Fluorescent nucleoside analogues may be suitable for these approaches, thereby allowing the measurement of cellular uptake and accumulation using both fluorescence microscopy and flow cytometry. In this regard fluorescent nucleoside analogues with similar structure to natural nucleosides are required, but even more importantly, these molecules must show absorption wavelength peaks at equal or higher than 350 nm, which is the minimal excitation wavelength required by those techniques. Many fluorescent nucleoside analogues developed so far have been designed for structural studies of DNA and RNA.8,9 Most derivatives may not be useful for the goals mentioned above because either their structure is not compatible for NT protein interaction or their spectrophotometric properties rule out their use within the wavelength range required for the above-mentioned techniques. Several studies have used fluorescent derivatives of NBTI (S-(4-nitrobenzyl)-6-thioinosine), an inhibitor of ENT proteins.10,11 Data suggest that these derivatives can be used to determine cell-surface abundances of hENT1 in cell lines using flow cytometry. For that reason, these nucleoside analogues could provide a new approach to quantitatively analyze nucleoside transporter-related functions that in turn would be helpful in the prediction of responsiveness to therapy. In contrast, at this moment no nucleoside analogues have been described that allow the quantification of CNT proteins and in vivo analysis of CNT function. Therefore, the aim of this study was to identify nucleoside analogues suitable to be used as tools to monitor CNT protein expression, as well as CNT-related functions in in vivo approaches involving confocal microscopy and flow cytometry. In the present work we have focused on two pyrimidine nucleoside analogues, Uridine-furan and Etheno-


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cytidine, a commercially available compound (Figure 1). Similar structures (two and three extra carbon compared to their related natural nucleosides, respectively) and maximum absorption peaks at wavelengths near 350 nm make them optimal candidates for this purpose. The transportability of these molecules has previously been described, the former being a good analogue to quantify hENT1 transporter proteins in living cells using fluorescent microscopy.12 Nevertheless, these derivatives have not been used for the study of hCNTs.

EXPERIMENTAL SECTION Chemical Synthesis. General Procedures. Etheno-cytidine was purchased from Berry&Associates. All reactions were monitored by TLC on silica gel plates 60 F254 from Merck. Mass spectra were obtained on an LC/MSD-TOF spectrometer from Agilent Technologies or on a 4800 MALDI-TOF from ABSciex. Reversed-phase HPLC purification was performed using semipreparative Waters or Shimadzu systems and Jupiter C18 column (10µm, 300Å, 250×10.0mm) from Phenomenex. 1H and 13C NMR analyses were carried out in a Varian Mercury 400MHz spectrometer. 2',3',5'-tri-O-acetyl-uridine (1). To a solution of uridine (4 g, 16.4 mmol) in anhydrous pyridine (20 mL), catalytic DMAP (dimethylaminopyridine) and acetic anhydride (15.5 mL, 164 mmol) were added maintaining the mixture at 4 ºC. The mixture was stirred at room temperature for 24 h under an Ar atmosphere. The reaction mixture was dried thoroughly under reduced pressure. AcOEt (60 mL) was added, and the resulting solution was washed with brine (2×) and water. The organic fraction was dried over anhydrous MgSO4 and filtered, and the solvent was removed under vacuum. The resulting crude was purified by silica gel column chromatography eluting with DCM and increasing amounts of MeOH (up to 4%). Product 1 was obtained as a white solid 5 ACS Paragon Plus Environment


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(5.7 g, 95%). M.P: 122-123 ºC. 1H NMR (400 MHz, CDCl3): δ (ppm) 2.10 (s, 3H), 2.13 (s, 3H), 2.14 (s, 3H), 4.30 (m, 3H), 5.30 (m, 2H), 5.79 (dd, J= 8.4, 2.0 Hz, 1H), 6.04 (d, J= 5.2 Hz, 1H), 7.39 (d, J= 8.4 Hz, 1H), 8.76 (s, 1H). 13C NMR (101 MHz, CDCl3): δ (ppm) 20.41, 20.50, 20.77, 63.11, 70.17, 72.7, 79.94, 87.44, 103.40, 139.22, 150.04, 162.44, 169.6, 170.08. HRMS (ESI, positive mode) m/z 392.9 [M+Na+], M calcd for C15H18N2O9 370.3. 2',3',5'-tri-O-acetyl-5-iodo-uridine (2). In a 50 mL round-bottomed flask, 1 (2.2 g, 5.9 mmol) and ICl (1.45 g, 8.9 mmol) were taken up in anhydrous DCM (20 mL) under an Ar atmosphere. The mixture was heated to 40 ºC for 6 h. The mixture was diluted with DCM (20 mL) and was washed with a 2% NaHSO3 solution (3×30 mL) and brine. The organic fraction was dried over anhydrous MgSO4, filtered, and concentrated to dryness. The resulting crude was purified by silica gel column chromatography using 030% AcOEt in DCM to yield 2 (2.5 g, 95%) as white solid which was thoroughly dried in a desiccator before its use in the following reaction. M.P: 147-147.5 ºC. 1H NMR (400 MHz, CDCl3): δ (ppm) 2.10 (s, 3H), 2.13 (s, 3H), 2.25 (s, 3H), 4.30 (m, 3H), 5.30 (m, 2H), 6.07 (d, J= 4.8 Hz, 1H), 7.89 (s, 1H), 8.57 (s, 1H).

13

C NMR (101 MHz,

CDCl3): δ (ppm) 20.39, 20.50, 21.10, 63.01, 69.53, 70.19, 73.08, 80.34, 87.13, 143.68, 149.71, 159.30, 169.57, 169.60, 170.04. HRMS m/z (ESI, positive mode) 518.8 (MNa+), M calcd for C15H17N2O9I 496.2. 2',3',5'-tri-O-acetyl‐5‐[2‐(trimethylsilyl)ethynyl]-uridine (3). All material used for this reaction was dried previously at 130 ºC overnight and reagent 2 was previously coevaporated with anhydrous MeCN (3×10 mL). In a 100 mL round-bottomed flask, 2 (2.3 g, 4.6 mmol) was taken up in a 1:1 (v/v) anhydrous MeCN:TEA mixture under an Ar atmosphere. Dichlorobis(triphenylphosphine)palladium(II) (650 mg, 0.9 mmol), CuI


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(44 mg, 0.2 mmol) and trimethylsilylacetylene (2.6 mL, 18.5 mmol) were added to the flask. After 5 h the reaction was completed. Solvent was removed under reduced pressure, and the resulting crude was dissolved in AcOEt (50 mL) and washed with brine (2×30 mL) and water (30 mL). The organic fraction was dried over anhydrous MgSO4, filtered, and concentrated to dryness. The resulting crude was purified by silica gel column chromatography using 0-60% AcOEt in hexanes to yield 3 (1.4 g, 64%) as pale yellowish solid. M.P: 79-80 ºC. 1H NMR (400 MHz, CDCl3): δ (ppm) 0.22 (s, 9H), 2.10 (s, 3H), 2.13 (s, 3H), 2.22 (s, 3H), 4.30 (m, 3H), 5.30 (m, 2H), 6.10 (d, J=4.8Hz, 1H), 7.77 (s, 1H), 8.25 (s, 1H).

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C NMR (101MHz, CDCl3): δ (ppm) -0.24, 20.37,

20.48, 20.94, 62.91, 70.11, 73.15, 80.22, 87.07, 94.77, 100.24, 101.42, 141.94, 148.99, 160.36, 169.47, 169.5, 169.93. HRMS m/z (ESI, positive mode) 490.2 (MNa+), M calcd for C20H26N2O9Si 466.5. 2',3',5'-tri-O-acetyl‐5‐ethynyl-uridine (4). Potassium fluoride (425 mg, 7.3 mmol) and tetrabutylammonium bromide (1.5 g, 7.3 mmol) were added to a solution of 3 in anhydrous MeCN. The mixture was left to react at room temperature until TLC analysis (AcOEt:hexanes 6:4, Rf= 0.27) showed complete disappearance of 3 (12 h reaction time). The reaction mixture was dried under vacuum and the resulting crude was dissolved in chloroform (40 mL), and washed with water (2×30 mL) and brine (30 mL). The organic fraction was dried over anhydrous MgSO4, filtered, and concentrated to dryness under reduced pressure. The resulting crude was purified by silica gel column chromatography using 50-100% AcOEt in hexane to yield 4 (1.25 g, 87%) as white solid. The product had to be protected from light. 1H NMR (400 MHz, CDCl3): δ (ppm) 2.11 (s, 3H), 2.13 (s, 3H), 2.21 (s, 3H), 3.22 (s, 1H), 4.30 (m, 3H), 5.30 (m, 1H), 6.08 (d, J= 4.4 Hz, 1H), 7.84 (s, 1H). HRMS m/z (ESI, positive mode) 417.1 (MNa+), M calcd for C17H18N2O9 394.3. 7 ACS Paragon Plus Environment


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3‐(β-D-ribofuranos-1'-yl)furo[2,3-d]pyrimidin-2(3H)-one (6). A solution of 4 (200 mg, 0.51 mmol) in a 2:1 (v/v) anhydrous MeCN:TEA mixture (20 mL) was bubbled with Ar for 1h. CuI (97 mg, 0.51 mmol) was added and the resulting mixture was left to react at reflux temperature for 8 h. Solvent was removed under vacuum, and the resulting crude was purified by a rapid silica gel column chromatography using 80100% AcOEt in hexanes to yield a pale yellowish solid (5) that was reacted immediately due to its high instability. This product (150 mg, 0.38 mmol) was treated with concentrated aqueous ammonia overnight at room temperature. Ammonia was eliminated under reduced pressure and the resulting crude purified by HPLC (solvent A: 0.01 M aqueous AcONH4 and solvent B: (1:1) water/MeCN; linear gradient from 0 to 30% of B in 30 min; flow: 3 mL·min-1; detection wavelength: 350nm; tR= 11,1 min) to yield 6. 1H NMR (400MHz, D2O): δ (ppm) 3.92 (dd, J= 12.8, 3.6 Hz, 1H), 4.07 (d, J= 13.4 Hz, 1H), 4.22 (m, 2H), 4.34 (dd, J= 4, 2.4 Hz, 1H), 6.07 (d, J= 2 Hz, 1H), 6.45 (d, J= 4 Hz, 1H), 7.18 (d, J= 3.6 Hz, 1H), 8.76 (s, 1H).

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C NMR (101 MHz, CDCl3) δ

(ppm) 61.63, 69.86, 76.32, 84.98, 93.58, 103.02, 129.54, 138.98, 157.17, 158.61. HRMS m/z (ESI, positive mode) 290.8 (MNa+), M calcd for C11H12N2O6 268.2.

Isolation and culture of primary cells. Six untreated patients diagnosed with chronic lymphocytic leukemia (CLL) using World Health Organization criteria25 were included in the present study. Informed consent was obtained from each patient in accordance with guidelines of the Institutional Ethics Committee of our hospital and the Declaration of Helsinki. Peripheral blood mononuclear cells were isolated by Ficoll/Hypaque sedimentation (Seromed, Berlin, Germany). Cells were cryopreserved in liquid nitrogen with 10% (v/v) dimethyl sulfoxide and 60% (v/v) heat-inactivated Fetal Bovine Serum


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(FBS). Previous studies have shown that freezing has no effect on cell response compared to that of freshly isolated CLL cells.26 After thawing, mononuclear cells from CLL patients were cultured in RPMI medium (Roswell Park Memorial Institute medium, Invitrogen) at a density of 2x106 cells/mL, in a humidified atmosphere at 37 ºC containing 5% CO2. MEC1 cell line and mononuclear cells from CLL patients were cultured in RPMI medium supplemented with 10% FBS, 2 mM glutamine, 50 µg/mL penicillin/streptomycin (Invitrogen), and 100 µg/mL normocin (Amaxa, Khöl, Germany). All cell lines were maintained at 37 ºC in a humidified atmosphere containing 5% CO2.

Cell culture and transfection. The HeLa cell line was routinely cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS, 1% mixture of antibiotics (100 U/mL penicillin G, 0.1 mg/mL streptomycin and 0.25 µg/mL Fungizone) and 2 mM glutamine. HeLa cells were transiently transfected with the hCNT1/2/3-cHApcDNA3.1 or hCNT1/3-pEGFP-C1 plasmids, developed in our laboratory,27 using calcium phosphate precipitation or Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Nucleoside transport and confocal microscopy were carried out 48 h after transfection.

Nucleoside Transport Assay. Nucleoside transport activity was measured in HeLa cells as previously described.28 The uptake of [3H]-labeled uridine (1µM, 1µCi/mL) was measured in the presence of either 137mM NaCl or 137mM choline chloride. The uptake medium also contained 5.4mM KCl, 1.8nM CaCl2, 1.2mM MgSO4 and 10mM HEPES (pH 7.4). Incubation was stopped after 1 minute (thus maintaining initial 9 ACS Paragon Plus Environment


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velocity conditions) by washing the monolayers twice in 2mL of cold STOP buffer (137mM NaCl and 10mM Tris - HEPES (pH 7.4)). Cells were then lysated with 100µL of 100mM NaOH, 0.5% Triton-X100. Aliquots were taken for protein determination (BCA Protein Assay Reagent, Thermo scientific Pierce, IL, USA) and radioactivity measurements.

Expression of CNTs in Xenopus laevis oocytes. cDNAs encoding human CNTs were subcloned into the pBluescript vector.29,30 The plasmid was linearized with Xbal and cRNA was transcribed and capped in vitro using the T3 RNA promoter (MEGAscript Kit, Ambion, Austin, TX). Mature X. laevis oocytes (Xenopus Express) were injected with 50 ng of cRNA coding for each protein as previously described29 and maintained at 18ºC in Barth’s medium (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3 and 10 mM HEPES-Tris, pH 7.4) supplemented with gentamicin (50 mg/l) for 3-5 days. Noninjected oocytes served as controls.

Electrophysiology. The electrophysiology experiments were performed to examine substrate-induced Na+ inward currents using the two-electrode voltage-clamp method.29,30 Oocytes expressing hCNT1, hCNT2 or hCNT3 were clamped at -50 mV and continuously perfused with Na+ buffer in the absence of the each nucleosides. The Na+-buffer contained: 100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES-Tris, pH 7.5. Continuous current data were recorded using Axoscope V1.1.1.14 (Molecular Devices, Sunnyvale, CA).


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Confocal microscopy assay. To perform the in vivo experiments, confocal microscopy with green fluorescent protein (GFP) fused proteins was performed on subconfluent monolayers of transfected HeLa cells cultured on 8-well chamber slide from Thermo Scientific. Images were obtained using a Leica TCS SL laser scanning confocal spectral microscope (Leica Microsystems) with argon and HeNe lasers attached to Leica SPII inverted microscope lasers. Images were captured by excitation at 488 nm and 350 nm.

Intracellular accumulation detection by flow cytometry. 5·105 MEC1 cells were incubated for 15 minutes with transforming growth factor β1 (TGFβ1, 1 ng/ml, SigmaAldrich) and NBTI (100 µM, Sigma-Aldrich) to block equilibrative transporters before adding 500 µM of Uridine-furan. After 30 minutes of incubation, ten thousand cells were analysed immediately using Gallios cytometer (Beckman Coulter Inc.) and the proportions of fluorescent cells were analysed using FlowJo software (Treestar). Controls were performed for each experiment without the addition of Uridine-furan which permitted us to delimit a negative region which is shown as a line in each histogram.

RESULTS AND DISCUSSION

Chemistry. Synthesis of Uridine-furan. There is no publication that fully describes the total synthesis of Uridine-furan. Nevertheless, many years ago 2’-deoxyuridine nucleosides with alkynyl groups at the 5 position were prepared and evaluated as anticancer and antiviral drugs, and our synthesis of 5-ethynyl-uridine was based on this methodology.13 The optimization and refinement of the individually described reactions allowed us a proper total synthesis starting from uridine (Scheme 1). After protection of



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the hydroxyls with acetyl groups using standard methodology14, the treatment of 1 with ICl afforded product 2 in high yield. The Sonogashira coupling reaction15 of 5iodouridine 2 with an excess of terminal alkyl ethynyltrimethylsilane and catalysed by Pd(II) resulted in 5-alkynylderivative 3, which was deprotected by treatment with KF to yield compound 4. The subsequent reaction was an intramolecular cyclization16 promoted by CuI to give derivative 5. Due to the poor stability of derivative 5, final hydroxyl groups deprotection had to be performed immediately after. The reaction of 5 with aqueous ammonia afforded Uridine-furan 6, which was purified by HPLC and characterized by NMR and mass spectrometry.

Biological Assays. Inhibitory effects of Uridine-furan and Etheno-cytidine on hCNT function. Interaction of pyrimidine nucleoside derivatives with hCNT proteins was evaluated by determining their ability to inhibit [3H]Uridine uptake, using HeLa cells transiently transfected with cDNAs encoding either hCNT1, hCNT2 or hCNT3. Uridine is a common substrate to the three isoforms, being the only pyrimidine nucleoside translocated by the purine-preferring nucleoside transporter hCNT2. Inhibition of hCNT2-mediated uridine uptake was not observed even when using high concentrations of derivatives (400 µM). Neither Uridine-furan nor Etheno-cytidine could interact with hCNT2 (data not shown), which is consistent with purine nucleoside selectivity of this transporter and thereby compromises its ability to interact with pyrimidine nucleosides other than uridine. Figure 2 shows the concentration-dependent inhibition of uridine uptake, mediated by hCNT1 and hCNT3, triggered by increasing concentrations of the fluorescent nucleosides of choice. Etheno-cytidine showed high-affinity interactions with both hCNT1 and hCNT3 proteins, with EC50 values of 1.5 ± 0.5 µM and 7.8 ± 2.3 µM (mean


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± SEM, n=5) for hCNT1 and hCNT3, respectively. Uridine-furan also inhibited uridine uptake mediated by hCNT1 and hCNT3, although full inhibition was not reached at the highest concentrations tested and, in this case, EC50 values were 0.5 ± 0.1 µM and 1.6 ± 0.5 µM, for hCNT1 and hCNT3, respectively. Cass and coworkers previously described the interaction of these fused pyrimidine nucleoside17 with NTs, but using recombinant nucleoside transporters produced in non-mammalian cell background (yeast). Transport of Uridine-furan and Etheno-cytidine in Xenopus laevis oocytes expressing hCNT

proteins.

Concentrative

nucleoside

transporters

are

electrogenic,

this

characteristic being a way of direct measurement of substrate transport.18,19,20 In order to determine whether these two derivatives can not only interact with hCNT1 and hCNT3 but also be translocated by these transporters, the two-electrode voltage clamp technique was used to ascertain whether these molecules could induce Na+ currents coupled to the transport of the derivative. Figure 3 shows a continuous record from a representative experiment, in which currents were measured in the presence of 50 µM uridine, cytidine, Etheno-cytidine or Uridine-furan. Currents induced by uridine in oocytes expressing hCNT1 and hCNT3 reached 60 nA (Figure 3), whereas those induced by cytidine reached 25 nA and 75 nA for hCNT1 and hCNT3, respectively, in agreement with previous studies.19,20 Addition of Etheno-cytidine generated an inward current of 10 nA and 20 nA in oocytes expressing hCNT1 and hCNT3, respectively. Currents generated by the Uridine-furan derivative were higher than those induced by Etheno-cytidine, reaching 15 nA for hCNT1 and 50 nA for hCNT3. These observations were consistent with Uridine-furan and Etheno-cytidine being poorer hCNT1 and hCNT3 substrates than their natural counterparts and are consistent with previous work by Cass and co-workers.17 Nevertheless, these data also unequivocally proved that these fluorescent derivatives are translocated by both transporter proteins. In particular,



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inward currents generated by Uridine-furan in hCNT3 expressing oocytes were comparable to those generated by the natural nucleoside uridine, thereby proving that this derivative can be transported with high efficiency by hCNT3. In line with the inhibitory effects, none of the fluorescent derivatives induced inward current through hCNT2 (Figure 3). Based on these results we chose Uridine-furan to perform the confocal microscopy experiments summarized below. Confocal microscopy studies. Confocal uptake studies, performed as described in the experimental section, showed the highest accumulation of fluorescent nucleoside in cells expressing hCNT1 or hCNT3 (Figure 4). Consistently with the expected function of these membrane proteins, it was observed that control cells treated with NBTI did not show any fluorescent signal throughout the incubation time, as a result of the inhibition of the endogenous equilibrative transporters and the inability of Uridine-furan to freely diffuse across the plasma membrane (Figure 4A-B). In the absence of NBTI, intracellular fluorescent accumulation was clearly observed. In fact, Uridine-furan was shown to interact with hENT type proteins albeit with much lower affinity than with hCNT1 and hCNT3 (Supplementary S7). In hCNT1 expressing cells with the endogenous hENTs inhibited (by NBTI) fluorescence intensity decreased considerably as compared to control cells, although it was still significant. In any case it seems that in this particular cell line Uridine-furan accumulation via endogenous hENT proteins is higher than that mediated by hCNT1. The major role for hENTs in Uridine-furan accumulation may be the result of different phenomena contributing to substrate uptake and its intracellular trapping. First, based upon the interaction affinity constants mentioned above, we can assume that hCNT proteins are saturated at the fluorescent nucleoside concentration used. In fact, hENTs are in general high capacity low affinity transporter proteins, thereby playing a major


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role in Uridine-furan accumulation at saturating concentrations. Second, and even more important, nucleosides may be channelled into the intracellular milieu to particular metabolic fates depending upon their entry route, as suggested by previous work from our own laboratory.21 Unfortunately intracellular channelling was not evident from the confocal microscopy approach, even though Cass and colleagues reported some targeting of extracellular nucleosides into the mitochondria.22 Moreover, it is important to keep in mind that although hENTs are equilibrative transporters showing low affinity for substrates, if their translocation function is tightly linked to substrate phosphorylation by appropriate kinases, this type of metabolic trapping can explain the significant accumulation of both analogues in control cells. In essence, the intracellular fluorescent signal is likely to correspond to phosphorylated forms of the trapped nucleoside which, on the other hand, are not expected to alter its spectrofluorimetric properties. The fluorescence increase rate of cells expressing hCNT3 treated with NBTI was similar to that of untreated control cells suggesting that substrate accumulation mediated by hCNT3 was similar to that mediated by hENTs. hCNT3-mediated accumulation of Uridine-furan could be markedly blocked by the presence of phloridzin, an inhibitor of hCNT function (Supplementary S8). A major biochemical difference between hCNT3 and hCNT1 is the fact, as mentioned above, that the former is potentially much more concentrative than the latter, due to its 2 to 1 Na+/nucleoside stoichiometry. This would explain why hCNT3-related intracellular fluorescence was higher than the one associated with hCNT1 function. Based upon all these observations, we assume that the determination of Uridine-furan intracellular accumulation will allow the identification of cells expressing functional hCNT1 and hCNT3 proteins, thereby anticipating the ability of fluorescent nucleoside



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derivatives, and the Uridine-furan analogue in particular, to label hCNT-related functions in vivo. This would be feasible under conditions of hENT inhibition using well known hENT blockers. Flow cytometry. To further assess the usefulness of these tools in the analysis of the cellular regulation of hCNT type proteins we decided to monitor Uridine-furan accumulation in a physiological set. A previous study from our laboratory proved that all-trans-retinoic acid (ATRA) was able to promote the trafficking of hCNT3-enriched vesicles from intracellular compartments to the plasma membrane of the Chronic Lymphocytic Leukaemia (CLL) derived cell line, MEC1.23 This phenomenon is mediated by TGF-β1.23 It was suggested that this mechanism could be of pharmacological relevance as long as both ATRA and TGF-β1 were similarly able to increase fludarabine uptake and enhance cytotoxicity in primary CLL cells.24 We used this regulatory mechanism to further assess the usefulness of Uridine-furan as an hCNT3 biomarker. To do so, we monitored the nucleoside concentrative capacity of MEC1 cells, either untreated or treated with TGF-β1, by determining the Na+-coupled cytidine uptake of these cells, as well as the intracellular fluorescent nucleoside accumulation. As expected, MEC1 cells exposed to TGF-β1 showed an increase in hCNT3-related activity (Figure 5A). The evidence that this regulatory mechanism of hCNT3 resulted in in vivo Uridine-furan accumulation was obtained by treating MEC1 cells with 1 ng/mL TGF-β1 and 100 µM NBTI for 15 minutes prior to the addition of Uridine-furan. Cells were acquired by flow cytometry after 30 minutes of incubation thereby ensuring a plateau value in fluorescent nucleoside accumulation (as defined in Figure 4). Under these conditions, TGF-β1 induced a significant increase in intracellular fluorescent accumulation (Figure 5B-C), thereby showing that Uridine-furan was targeting the in vivo function of up-regulated hCNT3. In practice, this fluorescent 16 ACS Paragon Plus Environment

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analogue appears to be a useful tool to detect different levels of hCNT3 function in MEC1 cells. More importantly, this approach was also applied to primary CLL cells from 6 patients. Interestingly each patient showed different basal levels of intracellular fluorescent accumulation (data not shown). This might have been the result of variable hCNT3 expression levels, as previously shown by our own laboratory.24 These CLL cases had previously been reported to be sensitive to TGF-β1 treatment24 and, accordingly, this cytokine should promote in all of them an increase in Uridine-furan accumulation as it was indeed the case as shown in Figure 6. Moreover, these observations anticipate the possibility of modifying well known anticancer/antiviral nucleoside-derived drugs for similar purposes, but its usefulness might be limited by their spectroscopic properties.

CONCLUSIONS Overall this contribution highlights the need of getting fluorescent nucleoside derivatives that effectively interact with NT proteins and fulfil the requirements of confocal microscopy and flow cytometry for in vivo analysis of hCNT-related function. Uridine-furan was shown to be useful for these purposes and its accumulation within cells appears to be a good indicator of hCNT-related activity. Even more interestingly, it also proves to be suitable for monitoring in vivo modulation of transporter function, both in cell lines and primary cells from cancer (CLL) patients.

ASSOCIATED CONTENT Supporting information 1

H NMR,

13

C NMR spectra of compounds synthesized according to described

procedures and HPLC chromatogram of Uridine-furan (S1-S6); inhibition of



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[3H]Uridine uptake by Uridine-furan in HeLa cells using Na+ free medium (S7); transfected HeLa cells images in presence of Uridine-furan and phloridzin (S8). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Phone: (34) 934021543 (office), (34) 934021544 (lab), E-mail: [email protected] Author Contributions ∀

These authors contributed equally. All authors have given approval to the final

version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This work has been funded by Ministerio de Economía y Competitividad (MINECO), Spain (SAF2011-23660, SAF2014-52067-R, and IPT-2012-0673-010000 to M.P.-A., and CTQ2010-21567-C02-01 and CSD2009-00080 to A.G.), and Generalitat de Catalunya (2009SGR624 to M.P.-A., and 2009SGR-208 to A.G.). The MPET laboratory is a member of the Oncology Program of the National Biomedical Research Institute of Liver and Gastrointestinal Diseases (CIBER ehd). CIBER ehd is an initiative of Instituto de Salud Carlos III (Spain). A.C.-M. was a fellow funded by the Institut de Biomedicina de la Universitat de Barcelona (IBUB). I.P.-M. hold a Formación de Personal Investigador fellowship (FPI, MINECO, Spain). The authors thank Ingrid Iglesias for her technical support. 18 ACS Paragon Plus Environment

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ABREVIATIONS AcOEt, ethyl acetate; Ar, argon; ATRA, all-trans-retinoic acid; CLL, chronic lymphocytic leukemia; DCM, dichloromethane; DMAP, dimethylaminopyridine; FBS, fetal bovine serum; GFP, green fluorescent protein; hCNTs, human concentrative nucleoside transporters; hENTs, human equilibrative nucleoside transporters; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HPLC, high pressure liquid chromatography; MeCN, acetonitrile; MeOH, methanol; NBTI, S-(4-Nitrobenzyl)-6thioinosine; NMR, nuclear magnetic resonance; NT, nucleoside transporter; SEM, standard error mean; TEA, triethylamine; TGFβ1, transforming growth factor β1; TLC, thin layer chromatography; RPMI, Roswell Park Memorial Institute medium.

FIGURE LEGENDS

Figure 1. Structures of the fluorescent nucleoside derivatives Etheno-cytidine and Uridine-furan and their related natural nucleoside, cytidine and uridine respectively.

Scheme 1. Synthesis of Uridine-furan. Reagents and conditions: (i) Ac2O, DMAP, anh pyridine, rt, 24 h; (ii) ICl, anh DCM, 40 ºC, 6 h; (iii) HC2Si(CH3)3, CuI, (Ph3P)2PdCl2, MeCN/TEA (1:1), rt, 5 h; (iv) KF, Bu4NBr, anh MeCN, rt, 12 h; (v) MeCN:TEA (2:1), CuI, reflux, 6 h; (vi) conc. aq. NH3, rt, 16 h.

Figure 2. Inhibition of [3H]Uridine uptake by Uridine-furan and Etheno-cytidine in transiently transfected HeLa cells transiently expressing either hCNT1 ( ) or hCNT3 ( ). Uptake was measured at room temperature over 1 minute in the presence of



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increasing concentrations of Uridine-furan (up) or Etheno-cytidine (bottom). Uptake values are given as the percentage of uptake values versus control values determined in the absence of derivative. Points represent the mean ± SEM of 4 to 6 independent experiments.

Figure 3. Two voltage clamp analysis of nucleoside and nucleoside uptake by hCNT1, hCNT2 and hCNT3. Representative sodium currents generated by perfusing either nucleoside analogues or uridine (as a control) in oocytes expressing each of the hCNT isoforms. Concentrations used and magnitude of currents are displayed in the figure.

Figure 4. (A) Representative live cell images corresponding to the UV channel, after 10, 20 and 30 minutes of the exposure to 500 µM Uridine-furan. HeLa cells transfected with GFP were taken as a control. Fluorescence accumulation was measured either in the presence or absence of 100 µM NBTI. (B) Representative time course of intracellular fluorescence after treatment with Uridine-furan. Data are normalized to maximum uptake rates and expressed as mean ± SEM.

Figure 5. (A) Cytidine uptake into MEC1 cells. The uptake of 1 µM Cytidine was measure at room temperature over 1 minute either in the presence or absence of 1 ng/mL TGF-β1 as described in the Experimental Section. Data are expressed as mean ± SEM of three independent experiments. (B) Uridine-furan uptake by hCNT3 determined the intracellular fluorescence intensity in MEC1 cells treated or not (control) with 1 ng/mL TGF-β1 by flow cytometry as described in the Experimental Section. Data are normalized to the control cells and expressed as mean ± SEM of three independent experiments. (C) Representative graphs of number of MEC1 cells versus intracellular 20 ACS Paragon Plus Environment

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fluorescent intensity obtained by flow cytometry. Control cells (grey graph) and cells treated with 1ng/mL TGF-β1 (red graph) are superposed in the third panel to see clearly the differences between the intracellular fluorescence accumulation.

Figure 6. (A) Uridine-furan uptake by hCNT3 determined the intracellular fluorescence intensity in 6 different patient cells treated or not (control) with 1 ng/mL TGF-β1, assessed by flow cytometry as described in the Experimental Section. Data are normalized to the control cells and expressed as mean ± SEM of three independent experiments. (B) Two representative graphs of number of patient cells versus intracellular fluorescence intensity were obtained by flow cytometry. Control cells (grey graph) and cells treated with 1ng/mL TGF-β1 (red graph) are superposed in the third panel to see clearly the differences between the intracellular fluorescent accumulation.

REFERENCES

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(5) Pastor-Anglada, M.; Perez-Torras S. Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets. Front. Pharmacol. Submitted. (6) Giavannetti, E.; Del Tacca, M.; Mey, V.; Funel, N.; Nannizzi, S.; Ricci, S.; Orlandini, C.; Boggi, U.; Campani, D.; Del Chiaro, M.; Bevilacqua, G.; Mosca, F.; Danesi, R. Transciption analysis of human equilibrative nucleoside transporter-1 predicts survival in pancreas cancer patients treated with gemcitabine. Cancer Res. 2006, 66, 3928-3935. (7) Spratlin, J.; Sangha, R.; Glubrecht, D.; Dabbagh, L.; Young, J. D.; Dumontet, C.; Cass, C.; Lai, R.; Mackey, J. R. The absence of human equilibrative nucleoside transporter 1 is associated with reduced survival in patients with gemcitabine-treated pancreas adenocarcinoma. Clin. Cancer Res. 2004, 10, 6956-6961. (8) Sinkeldam, R. W.; Greco, N. J.; Tor, Y. Fluorescent analogs of biomolecular building blocks: design, properties and applications. Chem. Rev. 2010, 110, 2579-2619. (9) Tanpure, A. A.; Pawar, M. G.; Srivatsan, S. G. Probes for investigating nucleic acid structure and function. Isr. J. Che., 2013, 53, 366-378. (10) Robins, M. J.; Peng, Y.; Damaraju, V. L.; Mowles, D.; Barron, G.; Tackaberry, T.; Young, J. D.; Cass, C. E. Improved synthesis of 5’-S-(2-Aminoethyl)-6-N-(4nitrobenzyl)-5’-thioadenosine (SAENTA), analogues, and fluorescent probe conjugates: analysis of cell-surface human equilibrative nucleoside transporter 1 (hENT1) levels for prediction of the antitumor efficacy of gemcitabine. J. Med. Chem., 2010, 53, 60406053. (11) Sen, R. P.; Delicado, E. G.; Alvarez, A.; Brocklebank, A. M.; Wiley, J. S.; MirasPortugal, M. T. Flow cytometric studies of nucleoside transport regulation in single chromaffin cells. FEBS Lett., 1998, 422, 368-372.


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(12) Zhang, J.; Sun, X.; Smith, M.; Visser, F.; Carpenter, P.; Barron, G.; Peng, Y.; Robins, M. J.; Baldwin, S. A.; Cass, C. E. Studies of nucleoside transporters using autofluorescent nucleoside probes. Biochemistry. 2006, 45, 1087-1098. (13) De Clercq, E.; Descamps, J.; Balzarini, J.; Giziewicz, J.; Barr, P. J.; Robins, M. J. Nucleic acid related compounds. 40. Syntheis and biological activities of 5Alkynyluracil nucleosides. J. Med. Chem. 1983, 26, 661-666. (14) Robins, M. J.; Manfredini, S.; Wood, S. G.; Wanklin, R. J.; Rennie, B. A.;Sacks, S.

L. Nucleic acid related compounds. 65. New syntheses of 1-(β-D-Arabinofuranosyl)-5(E)-(2-iodovinyl)uracil (IVAraU) from vinylsilane precursors. Radioiodine uptake as a marker for thymidine kinase positive Herpes viral infections. J. Med. Chem. 1991, 34, 2275-2280. (15) Sonogashira, K.; Tohhda, Y.; Hagihara, N. A convenient synthesis of acetylenes: catalytic substitutions of acetylenic hydrogen with bromoalkenes, iodoarenes and bromopyridines. Tetrahedron Lett. 1975, 16, 4467-4470. (16) Robins, M. J.; Miranda, K.; Rajwanski, V. K.; Peterson, M. A.; Andrei, G.; Snoeck, R.; Clercq, E.; Balzarini, J. Synthesis and biological evaluation of 6-(Alkyn-1yl)furo[2,3-d]pyrimidin-2(3H)-one base and nucleoside derivatives. J. Med. Chem. 2006, 49, 391-398. (17) Damaraju, V. L.; Smith, K. M.; Mowles, D.; Nowak, I.; Karpinski, E.; Young, J. D.; Robins, M. J.; Cass, C. E. Interaction of fused-pyrimidine nucleoside analogs with human concentrative nucleoside transporters: high-affinity inhibitors of human concentrative nucleoside transporter 1. Biochem. Pharmacol. 2011, 81, 82-90. (18) Young, J. D.; Yao, S. Y.; Baldwin, J. M.; Cass, C. E.; Baldwin, S. A. The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29. Mol. Aspects Med. 2013, 34 (2-3), 529-547.



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(19) Larráyoz, I. M.; Casado, F. J.; Pastor-Anglada, M.; Lostao M. P.; Electrophysiological characterization of the human Na(+)/nucleoside cotransporter 1 (hCNT1) and role of adenosine on hCNT1 function. J. Biol. Chem. 2004. 279 (10). 8999-9007. (20) Gorraitz, E.; Pastor-Anglada, M.; Lostao, M. P. Effects of Na+ and H+ on steadystate and presteady-stade currents of the human concentrative nucleoside transporter 3 (hCNT3). Pflugers. Arch. 2010. 460 (3). 617-32. (21) Soler, C.; García-Manteiga, J.; Valdés, R.; Xaus, J.; Comalada, M.; Casado, F.J.; Pastor-Anglada, M.; Celada, A.; Felipe, A. Macrophages require different nucleoside transport system for proliferation and activation. FASEB J. 2001, 15, 1979-1988. (22) Zhang, J.; Sun, X.; Smith, K. M.; Visser, F.; Carpenter, P.; Barron, G.; Peng, Y.; Robins, M. J.; Baldwin, S. A; Young, J. D.; Cass, C. E. Studies of nucleoside transporter using novel autofluorescent nucleoside probes. Biochemistry. 2006, 45, 1087-1098. (23) Fernández-Calotti, P.; Pastor-Anglada, M. All-trans-retinoic acid promotes trafficking of human concentrative nucleoside transporter-3 (hCNT3) to the plasma membrane by a TGF-β1-mediated mechanism. J. Biol. Chem. 2010, 285, 13589-13598. (24) Fernández-Calotti, P.; Lopez-Guerra, M.; Colomer, D.; Pastor-Anglada, M. Enhancement of fludarabine sensitivity by all-trans-retinoic acid in chronic lymphocytic leukemia cells. Haematologica. 2012, 97, 943-951. (25) Ghia, P.; Stamatopoulos, K.; Belessi, C.; Moreno, C.; Stilgenbauer, S.; Stevenson, F.; et al. ERIC recommendations on IGHV gene mutational status analysis in chronic lymphocytic leukemia. Leukemia. 2007, 21(1), 1-3. (26) Bellosillo, B.; Villamor, N.; López-Guillermo, A.; Marcé, S.; Bosch, F.; Campo, E.; Montserrat, E.; Colomer, D. Spontaneous and drug-induced apoptosis is mediated by


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conformational changes of Bax and Bak in B-cell chronic lymphocytic leukemia. Blood. 2002, 100 (5), 1810-1816. (27) Errasti-Murugarren, E.; Molina-Arcas, M.; Casado, F. J.; Pastor-Anglada, M. A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside transporter-3 (hCNT3) protein localized in the endoplasmic reticulum. FASEB J. 2009, 23, 172–182. (28) Duflot, S.; Riera, B.; Fernández-Veledo, S.; Casado, V.; Norman, R. I.; Casado, F. J.; Lluís, C.; Franco, R.; Pastor-Anglada, M. Mol. Cell. Biol. 2004, 24, 2710–2719.(29) Birnir, B.; Loo, D. D.; Wright, E. M. Voltage-clamp studies of the Na+/glucose cotransporter cloned from rabbit small intestine. Pflügers Arch.1991, 418, 79–85. (30) Hirayama, B. A.; Lostao, M. P.; Panayotova-Heiermann M.; Loo, D. D.; Turk, E.; Wright, E. M. Kinetic and specificity differences between rat, human, and rabbit Na+glucose cotransporters (SGLT-1). Am. J. Physiol. Gastrointest. Liver Physiol. 1996, 270, G919–G926.



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Table of Contents Graphic

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Figure 1.

NH

NH 2

N N

HO

N O

O

N

HO O

OH OH

OH OH

Etheno-cytidine

Cytidine O

O

NH

N N

HO

O

O

N

HO O

O OH OH

Uridine-furan

OH OH

Uridine


O


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Scheme 1.

O

O

N

O

O

N

AcO

i

O

O

ii

N

NH

O

iii

O

N

AcO

O

O OAc OAc

OAc OAc

1

O

NH

AcO

OAc OAc

OH OH

Si

O I

NH

NH HO

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3

2

iv O

O

N O OH OH

6

O

N

N HO

H

O

vi

N

AcO

NH O

O OAc OAc

5


v

N

AcO O

OAc OAc

4

O

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Figure 2.



250s!

Cytidine! Eth-Cyt! 50 μM! 50 μM!

250s!

Eth-Cyt! 1 mM!

250s!

Cytidine! Eth-Cyt! 50 μM! 50 μM! Uridine! 50 μM!

20nA!

hCNT1!

Ur-Fur! 50 μM!

Uridine! 1 mM!

hCNT2!

Ur-Fur! 1 mM!

Uridine! 50 μM!

hCNT3!

50nA!

Ur-Fur! 50 μM!

Figure 3.

50nA!

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Time (min)!

30! 0! 20!

40!

60!

80!

100!

20 min! 20 min!

30 min!

10 min! 10 min!

60!

1! NT hC

10! 0! 20!

40!

60!

80!

100!

B)!

30 min!

co

A)!

Figure 4. !

% of intracellular UV ! fluorescence!


Time (min)!

30! 20!

co

ol ntr

+

! TI B N

ol ntr

!

hCNT1!

hC

40!

1 NT

50!

B +N

! TI

co

ol ntr

+

10!

! TI B N

20!

l tro n co

!

hC

hCNT3!

3 NT

40!

B +N

! TI

N hC

50!

! T3

60!

Figure 4.

% of intracellular UV ! fluorescence!

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Figure 5. ! !

Fluorescent Intensity! mediated by hCNT3!

B)!

pmol Cyt/mg prot·min!

A)!

Control!

C)!

TGF-β1!

Control!

Overlay!

TGF-β1!

cell number!

Control!

Fluorescence intensity!

TGF-β1!

cell number!

Figure 5.

cell number!

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A)!

Fluorescent Intensity! mediated by hCNT3!

Figure 6.

Control!

TGF-β1! Overlay!

cell number!

cell number!

TGF-β1! cell number!

Control!

B)!




cell number!


cell number!

cell number!

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