title: Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line.
abstract: Previous studies of nucleoside transport in mammalian cells have identified two types of activities: the equilibrative nucleoside transporters and concentrative, Na+-nucleoside cotransporters. Characterization of the concentrative nucleoside transporters has been hampered by the presence in most cells and tissues of multiple transporters with overlapping permeant specificities. With the recent cloning of cDNAs encoding rat and human members of the concentrative nucleoside transporter (CNT) family, it is now possible to study the concentrative transporters in isolation by use of functional expression systems. We report here the isolation of a nucleoside transport-deficient subline of L1210 mouse leukemia (L1210/DNC3) that is a suitable recipient for stable expression of cloned nucleoside transporter cDNAs. We have used L1210/DNC3 as the recipient in gene transfer studies to develop a stable cell line (L1210/DU5) that produces the recombinant concentrative nucleoside transporter with selectivity for pyrimidine nucleosides (CNT1) that was initially identified in rat intestine (Q.Q. Huang, S.Y. Yao, M.W. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. 1994. J. Biol. Chem. 269: 17,757-17,760). L1210/DU5 was used to examine the permeant selectivity of recombinant rat CNT1 by comparing a series of nucleoside analogs with respect to (i) inhibition of inward fluxes of [3H]thymidine, (ii) initial rates of transport of 3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2'-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2'-deoxyadenosinedeoxyadeno-2'-deoxyadenosine, 7-deazaadenosine) were potent inhibitors of CNT1, they were poor permeants when uptake was measured directly by analysis of isotopic fluxes or indirectly by comparison of cytotoxicity ratios. We conclude that comparison of analog cytotoxicity to L1210/DU5 versus L1210/DNC3 is a reliable indirect predictor of transportability, suggesting that cytotoxicity assays with a panel of such cell lines, each with a different recombinant nucleoside transporter, would be a valuable tool in the development of antiviral and antitumor nucleoside analogs.
authors.affiliation: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: C R
authors.initials: CR
authors.lastname: Crawford
authors.forename: C E
authors.initials: CE
authors.lastname: Cass
authors.forename: J D
authors.initials: JD
authors.lastname: Young
authors.forename: J A
authors.initials: JA
authors.lastname: Belt
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D017136:Ion Transport; D007938:Leukemia / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D009705:Nucleosides / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D009705:Nucleosides; D011993:Recombinant Fusion Proteins; C065171:cif nucleoside transporter