Incomplete nucleoside transport deficiency with increased hypoxanthine transport capability in mutant T-lymphoblastoid cells (original) (raw)
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Journal of Biological Chemistry
Cultured mouse leukemia L1210 cells express the nucleoside-specific membrane transport processes designated es, ei, and cif. The es and ei processes are equilibrative, but may be distinguished by the high sensitivity of the former to 6-[(4-nitrobenzyl)thio1-9-8-0-ribofuranosylpurine (NBMPR); the cif process is mediated by a Na+/nucleoside cotransporter of low sensitivity to NBMPR. Cells of an ei-deficient clonal line, L12 10/MC5-1, were mutagenized, and clones were selected in soft agar medium that contained (i) NBMPR (an inhibitor of es processes), (ii) erythro-9-(2-hydroxy-3-nony1)adenine (an inhibitor of adenosine deaminase), and (iii) arabinofuranosyladenine (a cytotoxic substrate for the three nucleoside transporters). The selection medium did not allow es activity and selected against cells that expressed the Na+-linked cif process. Cells of the L1210jB23.1 clonal isolate were deficient in cif transport activity, and inward fluxes of formycin B, a poorly metabolized analog of inosine, were virtually abolished by NBMPR in these cells. In the mutant cells, nonisotopic formycin B behaved as a countertransport substrate during influx of ['Hlformycin B, and inward fluxes of the latter were competitively inhibited by purine and pyrimidine nucleosides. The transport behavior of L121OjB23.1 cells indicates that (i) the mutationjselection procedure impaired or deleted the Na+-linked cif process and (ii) es nucleoside transport activity is expressed in the mutant cells.
1986
The single nucleoside transport function of mouse S49 lymphoblasts also transports purine bases (B. Aronow and B. Ullman, J. Biol. Chem. 261:2014-2019). This transport of purine bases by S49 cells is sensitive to inhibition by dipyridamole (DPA) and 4-nitrobenzylthioinosine, two potent inhibitors of nucleoside transport. Therefore, wild-type S49 cells cannot salvage low hypoxanthine concentrations in the presence of 10 ,uM DPA and 11 ,uM azaserine; the latter is a potent inhibitor of purine biosynthesis. Among a mutagenized wild-type population, a cell line, JPA2, was isolated which could proliferate in 50 ,M hypoxanthine-11 ,uM azaserine-10 M DPA. The basis for the survival of JPA2 cells under these selective conditions was expression of a unique, high-affinity purine nucleobase transport function not present in wild-type cells. JPA2 cells could transport 5 ,uM concentrations of hypoxanthine, guanine, and adenine 15to 30-fold more efficiently than parental cells did. Kinetic analyses revealed that the affinity of the JPA2 transporter for all three purine bases was much greater than that of the wild-type nucleobase transport system. Moreover, nucleobase transport in JPA2 cells, unlike that in parental cells, was insensitive to inhibition by DPA, 4-nitrobenzylthioinosine, sulfhydryl reagents, and nucleosides. No alterations in nucleoside transport capability, phosphoribosylpyrophosphate levels, or purine phosphoribosyltransferase enzymes were detected in JPA2 cells. Thus, JPA2 cells express a novel nucleobase transport capability which can be distinguished from the nucleoside transport function by multiple biochemical parameters.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
Cr.1 cells, LK35.2 mouse B-cell hybridoma cells and cultured mouse peritoneal macrophages express both Na+-depe ndent, active and nonconcentrative, facilitated nucleoside transport systems. In the mouse cell lines, active transport represented only a minor nucleoside transport component and was detected only by measuring formycin B uptake in the presence of dipyridamole or nitrobenzylthioinosine, strong inhibitors of facilitated, but not of active, nucleoside transport. Inhibition of facilitated transport resulted in the concentrative accumulation of formycin B in cells expressing active nucleoside transport. Concentrative formycin B accumulation was abolished by treatment of the cells with gramicidin or absence of Na + in the extracellular medium and strongly inhibited by ATP depletion or ouabain treatment. Mouse macrophages accumulated formycin B to 70-times the extracellnlar concentration in the absence of dipyridamole during 90 min of incubation at 37 ° C. Thus active transport represents a major nucleoside transport system of these cells, similarly as previously reported for mouse spleen lymphocytes. In contrast to the various types of mouse cells, active formycin B transport was not detected in human HeLa cells, human H9, Jurkat and CEM T lymphoidal cells and pig spleen lymphocytes. These cells expressed only facilitated nucleoside transport with kinetic properties similar to those of the facilitated transporters of other mammalian cells.
Nucleoside transport and its significance for anticancer drug resistance
Drug Resistance Updates, 1998
AbstractThis 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 nonnucleoside antimetabolite drugs are also presented.
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.
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...