Nucleoside Transport Processes Research Articles

Nucleobase and nucleoside transport and integration into plant metabolism.

Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides, i.e., nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level.

Comparative in vitro evaluation of transportability and toxicity of capecitabine and its metabolites in cells derived from normal human kidney and renal cancers

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.

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na(+) and H(+) to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na(+)/nucleoside and H(+)/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H(+) dependence (all mutants), shift in Na(+):nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na(+)-specific hCNT1, uncoupled Na(+) currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.

Conserved Glutamate Residues Are Critically Involved in Na+/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter 1 (hCNT1)

Human concentrative nucleoside transporter 1 (hCNT1), the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of 10 conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the 10 residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q, and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q, and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, whereas E308D, E308Q, E322D, E322Q, and E498D displayed increased K50(Na+) and/or Km(uridine) values and diminished Vmax(Na+) and Vmax(uridine) values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for Glu-308, Glu-322, and Glu-498 in Na+/nucleoside cotransport and suggest locations within a common cation/nucleoside translocation pore. Glu-322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate and 2-aminoethyl methanethiosulfonate when converted to cysteine.

The role of nucleoside transporters in cancer chemotherapy with nucleoside drugs

Nucleoside analogs are important components of treatment regimens for various malignancies. Nucleoside-specific membrane transporters mediate plasma membrane permeation of physiologic nucleosides and most nucleoside analogs, for which the initial event is cellular conversion of nucleosides to active agents. Understanding of the roles of nucleoside transporters in nucleoside drug toxicity and resistance will provide opportunities for potentiating anticancer efficacy and avoiding resistance. Because transportability is a possible determinant of toxicity and resistance of many nucleoside analogs, nucleoside transporter abundance might be a prognostic marker to assess drug resistance. Elucidation of the structural determinants of nucleoside analogs for interaction with transporter proteins as well as the structural features of transporter proteins required for permeant interaction and translocation will lead to "transportability guidelines" for the rational design and therapeutic application of nucleoside analogs as anticancer drugs. It should eventually be possible to develop clinical assays that predict sensitivity and/or resistance to nucleoside anti-cancer drugs and thus to identify those patient populations that will most likely benefit from optimal nucleoside analog treatments. This review discusses recent results from structure/function studies of human nucleoside transporters, the role of nucleoside transport processes in the cytotoxicity and resistance of several anticancer nucleoside analogs and strategies to improve the nucleoside transporter-related anticancer effects of nucleoside analogs.

Studies of Nucleoside Transporters Using Novel Autofluorescent Nucleoside Probes

To better understand nucleoside transport processes and intracellular fates of nucleosides, we have developed a pair of fluorescent nucleoside analogues, FuPmR and dFuPmR, that differ only in the sugar moiety (ribofuranosyl versus 2'-deoxy, respectively), for real-time analysis of nucleoside transport into living cells by confocal microscopy. The binding and transportability of the two compounds were assessed for five recombinant human nucleoside transporters (hENT1/2, hCNT1/2/3) produced in Saccharomyces cerevisiae and/or oocytes of Xenopus laevis. The ribosyl derivative (FuPmR) was used to demonstrate proof of principle in live cell imaging studies in 11 cultured human cancer cell lines with different hENT1 activities. The autofluorescence emitted from FuPmR enabled direct visualization of its movement from the extracellular medium into the intracellular compartment of live cells, and this process was blocked by inhibitors of hENT1 (nitrobenzylmercaptopurine ribonucleoside, dipyridamole, and dilazep). Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake: a fast first stage that represented the initial uptake rate (i.e., transport rate) followed by a slow long-lasting second stage. The accumulation of FuPmR and/or its metabolites in nuclei and mitochondria was also visualized by live cell imaging. Measurements of fluorescence intensity increases in nuclei and mitochondria revealed rate-limited processes of permeant translocation across intracellular membranes, demonstrating for the first time the intracellular distribution of nucleosides and/or nucleoside metabolites in living cells. The use of autofluorescent nucleosides in time-lapse confocal microscopy is a novel strategy to quantitatively study membrane transport of nucleosides and their metabolites that will provide new knowledge of nucleoside biology.

Distribution of CNT2 and ENT1 transcripts in rat brain: selective decrease of CNT2 mRNA in the cerebral cortex of sleep-deprived rats.

Nucleoside transport processes regulate the levels of adenosine available to modulate neurotransmission, vascular tone and other physiological events. However, although equilibrative transporter transcripts or proteins have been mapped in the central nervous system of rats and humans, little is known about the presence and distribution of the complete family of nucleoside transporters in brain. In this study, we analysed the distribution of the transcript encoding the high affinity adenosine-preferring concentrative transporter CNT2 in the rat central nervous system and compared it with that of the equilibrative transporter ENT1. Furthermore, we evaluated the changes in expression of these two transporters in a situation of increased extracellular levels of adenosine, such as sleep deprivation. CNT2 mRNA was widespread in rat brain, although most prevalent in the amygdala, the hippocampus, specific neocortical regions and the cerebellum. The distribution of CNT2 mRNA only partially overlapped that of ENT1. Most of the cells labelled were neurones. Total sleep deprivation dramatically diminished the amounts of CNT2 mRNA, whereas ENT1 mRNA remained unchanged. This specific decrease in CNT2 transcript suggests a new physiological role for the transporter in the modulation of extracellular adenosine levels and the sleep/wakefulness cycle.

Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes.

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.

Cytotoxicity of 2-chlorodeoxyadenosine and arabinosylcytosine in leukaemic lymphoblasts from paediatric patients: significance of cellular nucleoside transporter content.

2-chlorodeoxyadenosine (2-CdA) and arabinosylcytosine (araC) are nucleoside drugs that are used to treat various leukaemias, although 2-CdA has not been tested extensively in children with acute lymphoblastic leukaemia (ALL). Nucleoside cytotoxicity depends on the conversion of these agents to 5'-phosphate derivatives, following drug entry into cells via nucleoside transport (NT) processes. This study compared es nucleoside transporter content, determined using a flow cytometric assay with SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] fluorescein, and cytotoxicities of 2-CdA and araC in fresh lymphoblasts from previously untreated paediatric ALL patients and the human T-lymphoblast cell line, CCRF-CEM. Lymphoblast samples from individual patients ranged widely in sensitivity to both 2-CdA (IC50, 6 nmol/l to > 5 micromol/l; mean = 418 nmol/l; n = 8) and araC (IC50, 59 nmol/l to > 5 micromol/l; mean = 1050 nmol/l; n = 7), although IC50 values for the two drugs were correlated (r = 0.78, P = 0.032, n = 7). Cellular es nucleoside transporter content varied more than 35-fold among samples from 10 patients. The correlation between es nucleoside transporter content and drug sensitivity was statistically significant for araC (r = -0.93, P = 0.023, n = 5), but not for 2-CdA (r = -0.57, P = 0.23, n = 6). Exposure of CCRF-CEM cells to araC resulted in a substantial araC concentration-dependent increase in the relative survival of es transporter-deficient cells, whereas the increase was slight following exposure to 2-CdA. We conclude that, in ALL lymphoblasts, es nucleoside transporter content is a determinant of araC sensitivity and that a deficiency in NT may impart resistance to araC.

Distinct regional distribution of human equilibrative nucleoside transporter proteins 1 and 2 (hENT1 and hENT2) in the central nervous system

Nucleoside transport processes play an important role in human cells in salvage of nucleosides used in the biosynthesis of nucleic acids and in regulating endogenous adenosine concentrations in the human central nervous system (CNS). By altering the levels of adenosine available to interact with cell–surface receptors, nucleoside transporters have profound effects on the ability of adenosine to modulate neurotransmission, vascular tone and other physiological events. Although the human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) are believed to play a crucial role in modulating brain function, their distribution within the major divisions of the human CNS is not known. In this work, antibodies specific for hENT1 and hENT2 were produced against fragments of the transporter proteins and used for immunoblot analysis of enriched membrane fractions prepared from several regions of the human brain. While hENT1 was most prevalent in the frontal and parietal lobes of the cerebral cortex, thalamus, midbrain and basal ganglia, hENT2 was concentrated in the cerebellum and brainstem regions, particularly the pons. The apparent reciprocal distribution of hENT1 and hENT2 in human brain suggests that these nucleoside transporter proteins are produced in distinct regions of the CNS where they function in nucleoside salvage and/or regulation of exogenous adenosine. Within the brain regions that were investigated, the pattern of hENT1 distribution correlated well with adenosine A 1 receptor abundance. The regional co-localization of hENT1 and A 1 receptor protein suggests an important role of hENT1-mediated transport process in the control of neuromodulatory actions mediated by adenosine A 1 receptors in human brain.

Selective loss of nucleoside carrier expression in rat hepatocarcinomas

Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity. (Hepatology 2000;32:239-246.)

Nucleoside transporter proteins: emerging targets for drug discovery

Nucleosides are central salvage metabolites and, as precursors of nucleotides, play an essential role in intermediary metabolism and biosynthesis, as well as signal transduction through interaction with purinergic receptors. Nucleoside analogues are used to treat haematological malignancies, certain solid tumours and many viral diseases. Because most nucleosides are relatively hydrophilic molecules, the presence of specialised transporter proteins in plasma membranes is essential for cellular uptake. Nucleoside transport is mediated by either bi-directional equilibrative processes, driven by chemical gradients, or inwardly directed concentrative processes, driven by sodium gradients. Recent advances in the identification of nucleoside transporter proteins from humans and lower organisms, using molecular cloning and functional expression of cDNAs, have transformed our understanding of nucleoside transport processes. Most of the nucleoside transporters identified thus far are members of two previously unrec...

Enhancement of retention and cytotoxicity of 2-chlorodeoxyadenosine in cultured human leukemic lymphoblasts by nitrobenzylthioinosine, an inhibitor of equilibrative nucleoside transport.

In leukemic cells exposed to 2-chlorodeoxyadenosine (2-CdA), levels of the nucleoside drug and its phosphate metabolites decay with time in the absence of external 2-CdA; an intrinsic part of this process is the efflux of 2-CdA. The effects of nitrobenzylthioinosine (NBMPR) and of dipyridamole (DPM), both potent inhibitors of es (e, equilibrative; s, sensitive to NBMPR) nucleoside transport processes, were studied in four lines of cultured leukemic lymphoblasts. Suspensions of 2-CdA-loaded cells were diluted 10-fold with 2-CdA-free medium to initiate the cellular 2-CdA decay processes, which followed a biexponential time course. When diluting media contained NBMPR or DPM, intracellular levels of 2-CdA and its metabolites were substantially increased (P < 0.001) compared with cells in media lacking the transport inhibitors, and 2-CdA loss followed a monoexponential time course. As a consequence, the AUCs (area under time-course plots of intracellular 2-CdA and its metabolites) were significantly (P < 0.001) lower in untreated control cells compared to inhibitor-treated cells. These results suggest that nucleoside transport processes contribute to the efflux of 2-CdA from the cultured lymphoblasts. The cytotoxicity of 1-h exposure to 2-CdA of Reh-A2 and CCRF-CEM cells was enhanced three-fold by subsequent exposure to 0.5 microM NBMPR relative to that of control cells subjected to the same manipulations without NBMPR exposure. However, before such a strategy may be considered to have a therapeutic application, careful examination of effects in normal lymphocytes and ex vivo leukemic lymphoblasts must first be undertaken. Leukemia (2000) 14, 52-60.

Adenosine transport: Recent advances in the molecular biology of nucleoside transporter proteins

Adenosine is a hydrophilic molecule that requires specialized transport proteins for permeation of cell membranes. Functional studies have identified two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are widely distributed among various cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelial Using molecular cloning techniques and functional expression of cDNAs in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding integral membrane proteins of the four major nucleoside transport processes of rat and human cells (es, ei, cit, cif). The equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the Equilibrative Nucleoside Transporter (ENT) and the Concentrative Nucleoside Transporter (CNT) protein families. This review summarizes the current state of knowledge of the molecular biology of the ENT and CNT protein families, focusing on the role of these proteins (r/hENT1, r/hENT2, r/hCNT1, r/ hCNT2) in the transport of adenosine.

Recent advances in the molecular biology of nucleoside transporters of mammalian cells

Nucleosides are hydrophilic molecules and require specialized transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are found in most mammalian cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelia. Using a variety of cloning strategies and functional expression in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding the rat and human nucleoside transporter proteins of the four major nucleoside transport processes of mammalian cells (es, ei, cit, cif). From the sequence relationships of these proteins with each other and with sequences in the public data bases, we have concluded that the equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the equilibrative nucleoside transporter (ENT) and the concentrative nucleoside transporter (CNT) protein families. This review summarizes the current state of knowledge in the molecular biology of the ENT and CNT protein families, focusing on the characteristics of the four human (h) and rat (r) nucleoside transport proteins (r/hENT1, r/hENT2, r/hCNT1, r/hCNT2).Key words: nucleoside transporter, equilibrative, concentrative, ENT, CNT.

Effect of adenosine receptor agonists on release of the nucleoside analogue [ 3H]formycin B from cultured smooth muscle DDT 1 MF-2 cells

Adenosine has receptor-mediated effects in a variety of cell types and is predominantly formed from ATP by a series of nucleotidase reactions. Adenosine formed intracellularly can be released by bidirectional nucleoside transport processes to activate cell surface receptors. We examined whether stimulation of adenosine receptors has a regulatory effect on transporter-mediated nucleoside release. DDT 1 MF-2 smooth muscle cells, which possess nitrobenzylthioinosine-sensitive (ES) transporters as well as both adenosine A 1 and A 2 receptors, were loaded with the metabolically stable nucleoside analogue [ 3H]formycin B. N 6-cyclohexyladenosine (CHA), a selective adenosine A 1 receptor agonist, produced a concentration-dependent inhibition of [ 3H]formycin B release with an IC 50 value of 2.7 μM. Further investigation revealed CHA interacts directly with nucleoside transporters with a K i value of 3.3 μM. Neither 5′- N-ethylcarboxamidoadenosine (NECA), a mixed adenosine A 1 and A 2 receptor agonist, nor CGS 21680, a selective adenosine A 2A receptor agonist, affected nucleoside release. We conclude that release of the nucleoside formycin B from DDT 1 MF-2 cells is not regulated by adenosine A 1 or A 2 receptor activation.

Recent advances in the molecular biology of nucleoside transporters of mammalian cells.

Nucleosides are hydrophilic molecules and require specialized transport proteins for permeation of cell membranes. There are two types of nucleoside transport processes: equilibrative bidirectional processes driven by chemical gradients and inwardly directed concentrative processes driven by the sodium electrochemical gradient. The equilibrative nucleoside transport processes (es, ei) are found in most mammalian cell types, whereas the concentrative nucleoside transport processes (cit, cif, cib, csg, cs) are present primarily in specialized epithelia. Using a variety of cloning strategies and functional expression in oocytes of Xenopus laevis, we have isolated and characterized cDNAs encoding the rat and human nucleoside transporter proteins of the four major nucleoside transport processes of mammalian cells (es, ei, cit, cif). From the sequence relationships of these proteins with each other and with sequences in the public data bases, we have concluded that the equilibrative and concentrative nucleoside transport processes are mediated by members of two previously unrecognized groups of integral membrane proteins, which we have designated the equilibrative nucleoside transporter (ENT) and the concentrative nucleoside transporter (CNT) protein families. This review summarizes the current state of knowledge in the molecular biology of the ENT and CNT protein families, focusing on the characteristics of the four human (h) and rat (r) nucleoside transport proteins (r/hENT1, r/hENT2, r/hCNT1, r/hCNT2).

Nucleoside transport and its significance for anticancer drug resistance

This article discusses the role of nucleoside transport processes in the cytotoxicity of clinically important anticancer nucleosides. This article summarizes recent advances in the molecular biology of nucleoside transport proteins, review the current state of knowledge of the transportability of therapeutically useful anticancer nucleosides, and provide an overview of the role of nucleoside transport deficiency as a mechanism of resistance to nucleoside cytotoxicity are summarized. Several strategies for utilization of nucleoside transport processes to improve the therapeutic index of anticancer therapies, including the use of nucleoside-transport inhibitors to modulate toxicity of both nucleoside and non-nucleoside antimetabolite drugs are also presented.

Molecular Cloning and Functional Characterization of Nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) Equilibrative Nucleoside Transporter Proteins (rENT1 and rENT2) from Rat Tissues

Equilibrative nucleoside transport processes in mammalian cells are either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Previously, we isolated a cDNA from human placenta encoding the 456-residue glycoprotein hENT1. When expressed in Xenopus oocytes, hENT1 mediated es-type transport activity and was inhibited by coronary vasoactive drugs (dipyridamole and dilazep) that may compete with nucleosides and NBMPR for binding to the substrate binding site. We now report the molecular cloning and functional expression of es and ei homologs of hENT1 from rat tissues; rENT1 (457 residues) was 78% identical to hENT1 in amino acid sequence, and rENT2 (456 residues) was 49-50% identical to rENT1/hENT1 and corresponded to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in truncated form. rENT1 was inhibited by NBMPR (IC50 = 4.6 nM at 10 microM uridine), whereas rENT2 was NBMPR-insensitive (IC50 > 1 microM). Both proteins mediated saturable uridine influx (Km = 0.15 and 0.30 mM, respectively), were broadly selective for purine and pyrimidine nucleosides, including adenosine, and were relatively insensitive to inhibition by dipyridamole and dilazep (IC50 > 1 microM). These observations demonstrate that es and ei nucleoside transport activities are mediated by separate, but homologous, proteins and establish a function for the HNP36 gene product.

Potential signalling roles for UTP and UDP: sources, regulation and release of uracil nucleotides.

Increasing evidence for receptors for uracil nucleotides has focused interest on specific signalling mechanisms involving UTP and UDP. At least three metabotropic P2 receptors are stimulated by uracil nucleotides with equal or greater potency than by adenine nucleotides, and there might be ionotropic receptors as well. Regulation of uridine and uracil nucleotide levels is important when considering the receptor-mediated effects of these compounds. Cells can synthesize uracil nucleotides de novo or by salvage of uridine. UTP made from salvage might be preferentially used for RNA synthesis in the nucleus, while UTP synthesized de novo seems to be used for UDP-sugar and CDP-phospholipid production. UTP from both pathways can enter a free UTP pool, from which UTP can be released from cells. UTP and UDP can stimulate pyrimidinoceptors, but metabolism by ecto-nucleotidases limits their effects. Alternatively, UTP might be a substrate for ecto-protein kinases, and this could contribute to its extracellular regulation. Cells can reclaim uridine, using nucleoside transport processes, following dephosphorylation of UTP, UDP and UMP. In this article Christopher Anderson and Fiona Parkinson discuss how understanding the processes that regulate uridine and uracil nucleotide concentrations will enhance our ability to manipulate UTP/UDP signalling pathways for pharmacological effect.