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The goal of this study was to understand roles of nucleoside and nucleobase transport processes in capecitabine pharmacology in cells derived from human renal proximal tubule cells (hRPTCs) and three human renal cell carcinoma (RCC) cell lines, A498, A704, and Caki-1. Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) mediated activities and a sodium-independent nucleobase activity were present in hRPTCs. In hRPTCs, uptake of 5'-deoxy-5-fluorouridine (DFUR), a nucleoside metabolite of capecitabine, was pH dependent with highest uptake seen at pH 6.0. In RCC cell lines, hENT1 was the major nucleoside transporter. Nucleobase transport activity was variable among the three RCC cell lines, with Caki-1 showing the highest and A498 showing the lowest activities. Treatment of RCC cell lines with interferon alpha (IFN-α) increased thymidine phosphorylase levels and prior treatment of RCC cell lines with IFN-α followed by 5-FU or DFUR resulted in enhanced sensitivity of all cell lines to 5-FU and two of three cell lines to DFUR. We report for the first time a nucleobase transport activity in hRPTCs and RCC cell lines. In addition, our in vitro cytotoxicity results showed that RCC cell lines differed in their response to 5-FU and DFUR and prior treatment with IFN-α potentiated cytotoxic response to metabolites of capecitabine. Key words: nucleosides, membrane transport, renal cancer, nucleobases and cytotoxicity. Cette etude visait a comprendre les roles qu'exercent les processus de transport des nucleosides et des bases nucleiques dans la pharmacologie de la capecitabine, chez des cellules derivees des tubules proximaux renaux humains (hRPTC) et trois lignees cellulaires humaines de carcinome du rein (RCC), les A498, A704 et Caki- 1. Des activites dependantes des transporteurs equilibratifs de nucleosides 1 et 2 chez l'humain (hENTl et hENT2) et une activite nucleobasique independante du sodium etaient presentes chez les hRPTC. La captation de la 5'-deoxy-5-fluorouridine (DFUR), un nucleoside metabolite de la capecitabine, etait dependante du pH chez les hRPTC, la captation maximale etant observee a pH 6.0. Chez les RCC, hENTl etait le principal transporteur de nucleoside. L'activite de transport des bases nucleiques variait en fonction des trois lignees cellulaires, les cellules Caki-1 etant celles qui presentaient l'activite la plus elevee et les A498, la plus faible. Un traitement des lignees de RCC avec de l'interferon-alpha (IFN-α) augmentait les niveaux de thymidine phosphorylase, et un traitement prealable des lignees de RCC avec de l'IFN-α suivi du 5-FU ou du DFUR resultait en un accroissement de la sensibilite de toutes les lignees cellulaires au 5-FU et de deux des trois lignees, au DFUR. Nous rapportons pour la premiere fois la presence d'une activite de transport des bases nucleiques chez les hRPTC et les lignees de RCC. De plus, les resultats de l'etude de cytotoxicite realisee in vitro ont revele que les lignees de RCC repondaient differemment au 5-FU et au DFUR, et qu'un traitement prealable a l'IFN-α potentialisait la reponse cytotoxique aux metabolites de la capecitabine. [Traduit par la Redaction] Mots-cles: nucleosides, transport membranaire, cancer renal, bases nucleiques, cytotoxicite., Introduction Capecitabine (Xeloda®) is a prodrug of 5'-deoxy-5-fluorouridine (DFUR) and 5-fluorouracil (5-FU) (for reviews see (Judson et al. 1999; Reigner et al. 2001)). Carboxyl esterase in the liver hydrolyses capecitabine [...]

Owing to the overlapping and redundant roles of the seven mammalian nucleoside transporters (NTs), which belong to two protein families (ENTs and CNTs), the physiological importance of individual NTs has been difficult to assess. Mice that have NT genes knocked out can be a valuable tool in gaining an understanding of the NT proteins. We have generated a strain of mice that is homozygous for a disruption mutation between exons 2 and 3 of the mouse equilibrative nucleoside transporter, mENTL We have undertaken a quantitative survey of NT gene expression in 10 tissues, as well as microarray analysis of heart and kidney, from wild-type and mENT1 knockout mice. Rather than a consistent change in expression of NT genes in all tissues of mENT1 knockout mice, a complex pattern of changes was found. Some genes, such as those encoding mCNT1 and mCNT3 in colon tissue, exhibited increased expression, whereas other genes, such as those encoding mCNT2 and mENT4 in lung tissue, exhibited decreased expression. Although mCNT3 has been shown to be important in human and rat kidney tissue, we were unable to detect mCNT3 transcripts in the kidney of either the wild-type or mENT1 knockout mice, suggesting differences in renal nucleoside resorption between species. Key words: mENT1, nucleoside transporter, gene expression, OmniBank. Le chevauchement et la redondance des roles joues par les sept transporteurs de nucleosides (NT) chez les mammiferes, lesquels appartiennent a deux familles de proteines (ENT et CNT), rendent difficile revaluation de l'importance physiologique des NT individuels. Des souris ayant subi une invalidation geenique des NT peuvent etre d'une grande utilite pour mieux comprendre ces NT. Nous avons genere une lignee de souris homozygotes portant une mutation entre les exons 2 et 3 qui interrompt le transporteur equilibratif des nucleosides, mENTl. Nous avons entrepris une etude quantitative de l'expression des genes NT dans dix tissus, ainsi qu'une analyse sur puce d'ADN du coeur et du rein des souris sauvages et knockout. Plutot que regulier, le patron des changements d'expression des genes NT dans tous les tissus des souris knockout mENTl est complexe. L'expression de certains genes, notamment ceux qui codent mCNTl et mCNT3 dans le colon, etait augmenteee, alors que pour d'autres, dont ceux qui codent mCNT2 et mCNT4 dans le poumon, elle etait diminuee. Meme si mCNT3 s'est avere important dans les tissus renaux chez l'humain et le rat, nous avons ete incapables de detecter des transcrits de mCNT3 dans le rein des souris sauvages et knockout mCNTl, ce qui suggere que la resorption renale des nucleosides est differente selon les especes. Mots-cles: mENTl, transporteur des nucleosides, expression genique, OmniBank. [Traduit par la Redaction], Introduction Specialized nucleoside transporter (NT) proteins are required for efficient translocation of purine and pyrimidine nucleosides across biological membranes. Nucleosides are hydrophilic, and transportability is often a critical determinant of [...]

This study examined the roles of human nucleoside transporters (hNTs) in mediating transepithelial fluxes of adenosine, 2'-deoxyadenosine, and three purine nucleoside anti-cancer drugs across polarized monolayers of human renal proximal tubule cells (hRPTCs), which were shown in previous studies to have human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) activities (11). Early passage hRPTCs were cultured on transwell inserts under conditions that induced formation of polarized monolayers with experimentally accessible apical and basolateral domains. Polarized hRPTC cultures were monitored for inhibitor sensitivities and sodium-dependence of the following: 1) transepithelial fluxes of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine (9-[beta]-D-arabinosyl-2-fluoroadenine), cladribine (2-chloro-2'-deoxyadenosine), and clofarabine (2-chloro-2'-fluoro-deoxy-9-[beta]-D-arabinofuranosyladenine); 2) mediated uptake of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine, cladribine, and clofarabine from either apical or basolateral surfaces; and 3) relative apical cell surface hCNT3 protein levels. Transepithelial fluxes of adenosine were mediated from apical-to-basolateral sides by apical hCNT3 and basolateral bENT2, whereas transepithelial fluxes of 2'-deoxyadenosine were mediated from basolateral-to-apical sides by apical hENT1 and basolateral human organic anion transporters (hOATs). The transepithelial fluxes of adenosine, hCNT3-mediated cellular uptake of adenosine, and relative apical cell surface hCNT3 protein levels correlated positively in polarized hRPTCs. The purine nucleoside anti-cancer drugs fludarabine, cladribine, and clofarabine, like adenosine exhibited apical-to-basolateral fluxes. Collectively, this evidence suggested that apical hCNT3 and basolateral hENT2 are involved in proximal tubular reabsorption of adenosine and some nucleoside drugs and that apical hENT1 and basolateral hOATs are involved in proximal tubular secretion of 2'-deoxyadenosine. kidney; fludarabine; cladribine; clofarabine

Nucleoside transporters in kidney mediate renal reabsorption and secretion of nucleosides. Using RT-PCR, we demonstrated mRNAs encoding hENT1, hENT2, hCNT1, hCNT2, and hCNT3 in both cortex and medulla. Immunoblotting with crude membrane preparations revealed abundant hENT1 and hCNT3 in both cortex and medulla, and little, if any, hENT2, hCNT1, or hCNT2, indicating that the latter were either absent or below limits of detection of immunoassays, hENT1 immunostaining was observed on apical surfaces of proximal tubules and on both apical and basal surfaces of thick ascending loops of Henle and collecting ducts. Prominent hCNT3 immunostaining was observed on apical surfaces of proximal tubules and thick ascending loops of Henle in addition to some cytoplasmic staining. Equilibrium binding of [[sup.3]H]nitrobenzylmercaptopurine ribonucleoside (NBMPR), a high-affinity inhibitor of hENT1, to brush-border membrane vesicles from cortex confirmed the presence of hENT1 on apical surfaces of proximal tubules. Uptake of [[sup.3]H]uridine by polarized renal proximal tubule cells exhibited a sodium-dependent component that was inhibited by thymidine and inosine as well as a sodium-independent component that was partially inhibited by NBMPR and completely inhibited by dilazep, indicating high levels of hENT1 and hCNT3 and low levels of hENT2 activities. The presence of 1) transcripts for hENT1/2 and hCNT1/2/3 and the hENT1 and hCNT3 proteins in human kidneys and 2) hENT1, hENT2, and hCNT3 activities in cultured proximal tubule cells suggest involvement of hENT1, hCNT3, and possibly also hENT2 in renal handling of nucleosides and nucleoside drugs. renal transport of nucleosides: tissue distribution of nucleoside transporters doi:10.1152/ajprenal.00007.2007

Abstract: Renal handling of physiological and pharmacological nucleosides is a major determinant of their plasma levels and tissue availabilities. Additionally, the pharmacokinetics and normal tissue toxicities of nucleoside drugs are [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tips.2006.06.004 Byline: Anne E. King (1), Michael A. Ackley (2), Carol E. Cass (3)(4), James D. Young (5), Stephen A. Baldwin (1) Abstract: Hydrophilic purine and pyrimidine nucleosides rely on specialized carrier proteins for their membrane translocation. The recent identification of two gene families encoding equilibrative and concentrative nucleoside transporters in mammals and other organisms has provided the essential breakthrough to a more complete understanding of the biological significance of nucleoside transport. Although nucleoside salvage is a primary function of these proteins, recent data indicate functions beyond metabolic recycling. In brain and spinal cord, for example, nucleoside transporters have the potential to regulate synaptic levels of neuroactive purines such as adenosine and, thereby, indirectly modulate physiological processes through G-protein-coupled purine P1 receptors. As described in this review, recent research indicates novel putative functions for CNS nucleoside transporters in sleep, arousal, drug and alcohol addiction, nociception and analgesia. The therapeutic use of nucleoside analogue drugs and nucleoside transporter inhibitors in viral, neoplastic, cardiovascular and infectious disease is also described. Author Affiliation: (1) Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK (2) Pfizer Global R&D, Sandwich, Kent CT13 9NJ, UK (3) Membrane Protein Research Group, Department of Oncology, University of Alberta, Alberta T6G 2H7, Canada (4) Cross Cancer Institute, Edmonton, Alberta T6G 2H7, Canada (5) Membrane Protein Research Group, Department of Physiology, University of Alberta, Alberta T6G 2H7, Canada

Conformational changes in purified human deoxycytidine kinase (dCK) in solution before and after substrate binding by employing fluorescence energy transfer experiments is examined to resolve the discrepancy between the kinetic results and the structural model. Kinetic studies showed that the addition of probe N(1-pyrene)-maleimide (PM) did not have any influence on the enzymatic activity of Cys-185-Ala, although there were changes in substrate specificity.

A description of a novel methodology for real-time visualization of nucleoside transport processes in living cells is presented. Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake, a fast first stage that represents the initial uptake rate, followed by a slow long-lasting second stage.

The first biophysical examination of full-length XRCC1 is represented. Sedimentation equilibrium measurements indicated that the human DNA repair protein, hXRCC1 exists as a monomer at lower protein concentrations but forms a dimer at higher protein concentrations with a K(sub d) of 5.7 x 10(super -7) M.

An ancient prevertebrate [Na.sup.+]-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti). Am J Physiol Cell Physiol 283: C155-C168, 2002. First published February 20, 2002; 10.1152/ajpcell.00587.2001.The human concentrative ([Na.sup.+]-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively. All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematodes, pathogenic yeast, and bacteria. Here, we report the cDNA cloning and functional characterization of a CNT family member from an ancient marine prevertebrate, the Pacific hagfish (Eptatretus stouti). This [Na.sup.+]-nucleoside symporter, designated hfCNT, is the first transport protein to be characterized in detail in hagfish and is a 683-amino acid residue protein with 13 predicted transmembrahe helical segments (TMs). hfCNT was 52, 50, and 57% identical in sequence to hCNT1, hCNT2, and hCNT3, respectively. Similarity to hCNT3 was particularly marked in the TM 4-13 region. When produced in Xenopus oocytes, hfCNT exhibited the transport properties of system cib, with uridine, thymidine, and inosine apparent [K.sub.m] values of 10-45 [micro]M. The antiviral nucleoside drugs 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, and 2',3'-dideoxyinosine were also transported. Simultaneous measurement of uridine evoked currents and radiolabeled uridine uptake under voltage-clamp conditions gave a [Na.sup.+]-to-uridine coupling ratio of 2:1 (cf. 2:1 for hCNT3 and 1:1 for hCNT1/2). The apparent [K.sub.50] value for [Na.sup.+] activation was > 100 mM. A 50:50 chimera between hfCNT and hCNT1 (TMs 7-13 of hfCNT replaced by those of hCNT1) exhibited hCNT1-like cation interactions, establishing that the structural determinants of cation stoichiometry and binding affinity were located within the carboxy-terminal half of the protein. The high degree of sequence similarity between hfCNT and hCNT3 may indicate functional constraints on the primary structure of the transporter and suggests that cib-type CNTs fulfill important physiological functions. Craniata; Xenopus oocyte; 3'-azido-3'-deoxythymidine; 2',3'-dideoxycytidine; 2',3'-dideoxyinosine.

The human equilibrative nucleoside transporter 1 protein (hENT1) is a major mediator of cellular entry of nucleosides and anticancer nucleoside drugs; its assay is important in understanding and diagnosing chemotherapy resistance. Here we present a novel assay for quantifying hENT1 using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). A cellular population is treated with 5'-S-(2-aminoethyl)-[N.sup.6]-(4-nitrobenzyl)-5'-thioadenosine-x8-fluorescein (5-SAENTA-x8f), which binds with high affinity and specificity to the hENT1 protein. The cells are washed to remove excess reagent, lysed, and centrifuged, and the supernatant is analyzed by CE-LIF with the use of an internal standard. Accuracy was evaluated by comparing the capillary electrophoresis results with those obtained by flow cytometry; the results were highly correlated, r = 0.96. The relative standard deviation of the hENT1 assay was 10%, determined from nine independent assays of the same cell line, which is 3 times superior to results obtained in a flow cytometry assay. The detection limit for 5-SAENTA-x8f was 4300 molecules injected into the capillary.

Szkotak, Artur J., Amy M. L. Ng, Jolanta Sawicka, Stephen A. Baldwin, S. F. Paul Man, Carol E. Cass, James D. Young, and Marek Duszyk. Regulation of [K.sup.+] current in human airway epithelial cells by exogenous and autocrine adenosine. Am J Physiol Cell Physiol 281: C1991-C2002, 2001.--The regulatory actions of adenosine on ion channel function are mediated by four distinct membrane receptors. The concentration of adenosine in the vicinity of these receptors is controlled, in part, by inwardly directed nucleoside transport. The purpose of this study was to characterize the effects of adenosine on ion channels in A549 cells and the role of nucleoside transporters in this regulation. Ion replacement and pharmacological studies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated [K.sup.+] channels, most likely [Ca.sup.2+]-dependent intermediate-conductance [K.sup.+] ([I.sub.k]) channels. [A.sub.1] but not [A.sub.2] receptor antagonists blocked the effects of adenosine. RTPCR studies showed that A549 cells expressed mRNA for [I.sub.k]-1 channels as well as [A.sub.1], [A.sub.2]A, and [A.sub.2b] but not As receptors. Similarly, mRNA for equilibrative (hENT1 and hENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleoside transporters was detected, a result confirmed in functional uptake studies. These studies showed that adenosine controls the function of [K.sup.+] channels in A549 cells and that hENTs play a crucial role in this process. A549 cells; nitrobenzylthioinosine; human concentrative nucleoside transporter; human equilibrative nucleoside transporter; calcium-dependent intermediate-conductance potassium channels Received 8 May 2001; accepted in final form 17 August 2001

Research shows that human polynucleotide kinase, a DNA repair enzyme, exists as a monomer, is asymmetric, and possesses approximately 50% beta structure and 25% alpha-helix. Data suggest that ATP induced conformational changes in the kinase are due to an increase in beta-structure of the protein with an enhanced exposure of aromatic acids.

Research has been conducted on the rat equilibrative nucleoside transporters belonging to the integral membrane protein family. The creation of chimeras between nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive nucleoside transporters has been carried out.