Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line.
Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line.
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重组钠依赖性嘧啶选择性核苷转运蛋白 (CNT1) 在转运缺陷型小鼠白血病细胞系中的稳定表达。
DOI：
10.1139/bcb-76-5-843
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Belt,JA
中科院分区：
文献类型：
--
作者：
Crawford,CR;Cass,CE;Young,JD;Belt,JA
以前的研究核苷转运在哺乳动物细胞中已经确定了两种类型的活动：平衡的核苷转运和集中，Na+-核苷共转运。由于大多数细胞和组织中存在具有重叠渗透特异性的多种转运蛋白，因此集中核苷转运蛋白的表征受到阻碍。随着编码大鼠和人的浓缩核苷转运蛋白（CNT）家族成员的cDNA的克隆，现在有可能通过使用功能表达系统来研究分离的浓缩转运蛋白。我们在这里报告分离的核苷转运蛋白缺陷的亚系L1210小鼠白血病（L1210/DNC 3），是一个合适的受体稳定表达克隆的核苷转运蛋白cDNA。我们已经在基因转移研究中使用L1210/DNC 3作为受体，以开发稳定的细胞系（L1210/DU 5），其产生最初在大鼠肠中鉴定的对嘧啶核苷（CNT 1）具有选择性的重组浓缩核苷转运蛋白（Q. Q.黄世义Yao，M.W.作者：A. R. P. Paterson，C.E.卡斯和J.D.年轻1994. 269：17 757 - 17 760）。L1210/DU 5用于检查重组大鼠CNT 1的渗透选择性，通过比较一系列核苷类似物与亲本转运缺陷细胞系对L1210/DU 5的（i）[3 H]胸苷内向通量的抑制，（ii）3 H-类似物的初始转运速率，和（iii）细胞毒性。根据所有三个标准，重组CNT 1转运5-氟-2-引物-脱氧尿苷和5-氟尿苷良好，而阿糖胞苷差。虽然一些嘌呤核苷（2 prime-deoxyadenosine，2-chloro-2 prime-deoxyadenosine，7-deazaadenosine）是CNT 1的有效抑制剂，但当通过同位素通量分析直接或通过比较细胞毒性比率间接测量摄取时，它们的渗透性较差。我们得出结论，类似物细胞毒性对L1210/DU 5与L1210/DNC 3的比较是可转运性的可靠间接预测因子，这表明使用一组这样的细胞系进行细胞毒性测定，每个细胞系具有不同的重组核苷转运蛋白，将是开发抗病毒和抗肿瘤核苷类似物的有价值的工具。核苷转运体，CNT 1，大鼠，钠依赖性，重组，克隆基因，转染，稳定转染子。
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 of3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2prime-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2prime-deoxyadenosine, 2-chloro-2prime-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.Key words: nucleoside transporter, CNT1, rat, sodium-dependent, recombinant, cloned gene, transfection, stable transfectant.