Nucleoside transport and its significance for anticancer drug resistance (original) (raw)
Related papers
Journal of Biological Chemistry, 2005
The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent proteintagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and in Xenopus oocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.
Physiology of Nucleoside Transporters: Back to the Future
Physiology, 2008
Nucleoside transporters (NTs) are integral membrane proteins responsible for mediating and facilitating the flux of nucleosides and nucleobases across cellular membranes. NTs are also responsible for the uptake of nucleoside analog drugs used in the treatment of cancer and viral infections, and they are the target of certain compounds used in the treatment of some types of cardiovascular disease. The important role of NTs as drug transporters and therapeutic targets has necessarily led to intense interest into their structure and function and the relationship between these proteins and drug efficacy. In contrast, we still know relatively little about the fundamental physiology of NTs. In this review, we discuss various aspects of the physiology of NTs in mammalian systems, particularly noting tissues and cells where there has been little recent research. Our central thesis is reference back to some of the older literature, combined with current findings, will provide direction for f...
Neuropharmacology, 1997
Equilibrative nucleoside transport processes in mammalian cells are categorized as either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). inhibition of the es process arises from binding of NBMPR to a high-affinity site(s) on the es transporter that can be identified by photoaffinity labeling with [3H]NBMPR. This study examined the equilibrative nucleoside transport processes of cultured human erythroleukemia (K562) cells. The presence of NBMPR binding sites (4.8 + 0.9 x 105/cell, & = 0.3 nM), together with the identification of polypeptides by specific photolabeling of membranes with [3H]NBMPR, indicated that K562 cells possess es nucleoside transporters (ca 500000 copies/cell). The photolabeled polypeptides of K562 cells migrated with lower relative mobility (peak Mt value, 63 000) than did those of human erythrocytes (peak M, value, 53000). This difference in apparent M, was abolished by prolonged treatment of membrane proteins with N-glycosidase F, suggesting that equilibrative nucleoside transport in K562 cells and erythrocytes is mediated by the same, or a closely related, es isoform. A cDNA encoding the es nucleoside transporter of human placenta (termed hENT1) was recently isolated by a strategy based on the N-terminal sequence of the es transporter of human erythrocytes. hENT-like n-RNA species were detected in K562 cells, as well as in several other human cell lines of neoplastic origin (A459, G361, HeLa, HL-60, Molt-4, Raji., SW480), by high-stringency northern analysis with a placental hENT1 probe. A cDNA that encoded a prote:in identical to hENT1 was isolated by reverse transcriptase polymerase chain reaction with primers specific for hENT1. NBMPR inhibited zero-trans influx of 3H-labeled adenosine, uridine and thymidine by 50% (1~50 values) at 0.4-1.0 nM, confirming the presence of an NBMPR-sensitive (es) transport process, which a.ccounted for 80-90% of total transport activity. The remaining component was identified as the equilibrative NBMPR-insensitive (ei) transport process since it: (i) exhibited low (1~+=1.0 PM) sensitivity to NBMPR; (ii) was not concentrative; and (iii) was unchanged by elimination of the sodium gradient. The kinetic parameters (determined at 37°C) for the es-and ei-mediated processes differed markedly. Values for transport of uridine by the es-and ei-mediated processes were, respectively: K,,, = 229 f 39 and 1077 f 220 PM; V,,, 186 f 31 and 40 + 5 pmol/pl cell water/set. Values for transport of adenosine by the es and ei-mediated processes were, respectively, 61 f 9 and 133 + 17 PM; V,,,,, 70 + 5 and 23 + 8 pmol/pl cell water/set. The ei-mediated process, although small, was of pharmacologic importance since K562 cells could not be protected by NBMPR (10 PM) from the cytotoxic effects of tubercidin (7deazaadenosine). 0 1997 Elsevier Science Ltd.
Differential Expression of Human Nucleoside Transporters in Normal and Tumor Tissue
Biochemical and Biophysical Research Communications, 2001
Responses to nucleoside analog drugs used in the treatment of cancers and viral infections can vary considerably between individuals. Genetic variability between individuals in their ability to transport drugs may be a contributory factor. Nucleoside transporters (NTs) move nucleosides and analog drugs across cell membranes. Four human NTs have been cloned: hENT1, hENT2, hCNT1, and hCNT2. Human NT expression profiles are not well defined; therefore, we undertook a comprehensive quantitative analysis of the differential expression of NTs within normal and tumor tissue. Results show tissue specific expression of the different NTs in normal tissue while matched normal/tumor tissue cDNA array data show considerable variability in all NT expression profiles from different individuals, in particular decreased expression in tumor tissue. Decreased NT expression in tumor tissue may contribute to reduced drug uptake and the development of resistance. These data suggest that nucleoside analog drug therapies may be optimized by determining individual NT expression profiles.
Molecular Pharmacology, 2012
SLC28 genes encode three plasma membrane transporter proteins, human concentrative nucleoside transporter (CNT)1, CNT2, and CNT3, all of which are implicated in the uptake of natural nucleosides and a variety of nucleoside analogs used in the chemotherapy of cancer and viral and inflammatory diseases. Mechanisms determining their trafficking toward the plasma membrane are not well known, although this might eventually become a target for therapeutic intervention. The transporter regulator RS1, which was initially identified as a short-term, post-transcriptional regulator of the high-affinity, Na ϩ -coupled, glucose transporter sodium-dependent glucose cotransporter 1, was evaluated in this study as a candidate for coordinate regulation of membrane insertion of human CNTtype proteins. With a combination of studies with mammalian cells, Xenopus laevis oocytes, and RS1-null mice, evidence that RS1 down-regulates the localization and activity at the plasma membrane of the three members of this protein family (CNT1, CNT2, and CNT3) is provided, which indicates the biochemical basis for coordinate regulation of nucleoside uptake ability in epithelia and probably in other RS1-expressing cell types.
2011
SLC28 genes encode three plasma membrane transporter pro-teins, human concentrative nucleoside transporter (CNT)1, CNT2, and CNT3, all of which are implicated in the uptake of natural nucleosides and a variety of nucleoside analogs used in the chemotherapy of cancer and viral and inflammatory dis-eases. Mechanisms determining their trafficking toward the plasma membrane are not well known, although this might eventually become a target for therapeutic intervention. The transporter regulator RS1, which was initially identified as a short-term, post-transcriptional regulator of the high-affinity, Na-coupled, glucose transporter sodium-dependent glucose cotransporter 1, was evaluated in this study as a candidate for coordinate regulation of membrane insertion of human CNT-type proteins. With a combination of studies with mammalian cells, Xenopus laevis oocytes, and RS1-null mice, evidence that RS1 down-regulates the localization and activity at the plasma membrane of the three members ...
Biochemical Journal, 2000
Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human mouse rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1 has 88 % and 78 % amino acid identity with rat ENT1 and human ENT1 respectively ; mENT2 is more highly conserved, with 94 % and 88 % identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1 ; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids Abbreviations used : ei, equilibrative inhibitor-insensitive nucleoside transport ; ENT, equilibrative nucleoside transporter (the prefixes m, r and h denote mouse, rat and human respectively) ; es, equilibrative inhibitor-sensitive nucleoside transport ; EST, expressed sequence tag ; GSP, gene-specific primer ; NBMPR, nitrobenzylthioinosine o6-[(4-nitrobenzyl)thio-9-β-D-ribofuranosylpurineq ; PKC, protein kinase C ; RACE, rapid amplification of cDNA ends ; RT-PCR, reverse transcriptase-PCR ; TM, transmembrane segment ; UTR, untranslated region. 1 To whom correspondence should be addressed (e-mail jhammo!julian.uwo.ca). The nucleotide sequence data reported will appear in the GenBank2, EMBL, GSDB and DDBJ Nucleotide Sequence Databases under the following accession numbers : mENT1, AF131212 ; mENT2, AF183397 ; mENT1b, AF305501. (designated mENT1b) that adds a potential protein kinase CK2 phosphorylation site in the central intracellular loop of the transporter, and is similar, in this regard, to the human and rat ENT1 orthologues. The other cDNA has a 5h-untranslated region sequence that is distinct from that of full-length mENT1. Microinjection of mENT1, mENT1b or mENT2 cRNA into Xenopus oocytes resulted in enhanced uptake of [$H]uridine by the oocytes relative to that seen in water-injected controls. mENT1-mediated, but not mENT2-mediated, [$H]uridine uptake was inhibited by nitrobenzylthioinosine and dilazep. Dipyridamole inhibited both mENT1 and mENT2, but was significantly more effective against mENT1. Adenosine inhibited both systems with a similar potency, as did a range of other purine and pyrimidine nucleosides. These results are compatible with the known characteristics of the native mENT1 and mENT2 transporters.
MOL#71837 1 Expression and Distribution of Nucleoside Transporter Proteins in the
2016
The plasma membrane distribution and related biological activity of nucleoside transporter proteins (NT) have been investigated in human syncytiotrophoblast from term placenta using a variety of approaches, including nucleoside uptake measurements into vesicles from selected plasma membrane domains, NT immunohistochemistry and subcellular localization (basal, heavy and light apical membranes as well as raft enriched membranes from the apical domain). Conversely to other epithelia, we have identified the high-affinity pyrimidine-preferring concentrative nucleoside transporter hCNT1 as the only hCNT-type protein expressed at both the basal and apical membranes of this epithelium. hCNT1 localization in lipid rafts is also dependent upon its subcellular localization in the apical plasma membrane, suggesting a complex cellular and regional expression. Overall, this favours the view that the placenta is a pyrimidine-preferring nucleoside sink from both maternal and fetal sides and hCNT1 plays a major role in promoting pyrimidine salvage and placental growth. This finding may be of pharmacological relevance, because hCNT1 is known to interact with anticancer nucleoside-derived drugs and other molecules, such as nicotine and caffeine, for which a great variety of harmful effects on placental and fetal development, including IUGR, have been reported.