Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells
Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells
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DOI：
10.1016/s0005-2736(02)00516-3
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发表时间：
2002-09-20
影响因子：
3.4
通讯作者：
Endou, H
中科院分区：
文献类型：
--
作者：
Kim, D;Kanai, Y;Endou, H
系统L是一个主要的营养转运系统，负责包括几种必需氨基酸在内的大型中性氨基酸的Na+非依赖性转运。在恶性肿瘤中，系统L转运蛋白L型氨基酸转运蛋白1（LAT 1）被上调以支持肿瘤细胞生长。LAT 1对于氨基酸和氨基酸相关药物透过血脑屏障也至关重要。为了寻找检测化合物与LAT 1相互作用的体外测定系统，我们研究了T24人膀胱癌细胞中系统L转运蛋白的表达和[C-14]（L）-亮氨酸转运的性质。北方印迹、实时荧光定量PCR和免疫荧光分析显示，T24细胞在质膜上表达LAT 1及其相关蛋白4F 2 hc，而T24细胞不表达另一种系统L亚型LAT 2。T24细胞摄取[C-14]（L）-亮氨酸不依赖于Na+，几乎完全被L系统选择性抑制剂BCH抑制。T24细胞中氨基酸和氨基酸相关化合物对[C-14]（L）-亮氨酸摄取的抑制作用与非洲爪蟾卵母细胞中表达的LAT 1相当。因此，在T24细胞中，大多数[14 C]（L）-亮氨酸摄取是由LAT 1介导的。与非洲爪蟾卵母细胞中的LAT 1一致，预加载的[C-14]（L）-亮氨酸的流出由T24细胞中的LAT 1的细胞外施加的底物诱导。由于三碘甲腺原氨酸、甲状腺素和美法仑能诱导T24细胞中预先装载的[C-14]（L）-亮氨酸的外排，因此这种外排测量已被证明比爪蟾卵母细胞中的外排测量更敏感，而爪蟾卵母细胞表达系统未检测到这种外排。因此，T24细胞被认为是研究化合物与LAT 1相互作用的极好工具。(C)2002 Elsevier Science B. V.保留所有权利。
System L is a major nutrient transport system responsible for the Na+ -independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L-type amino acid transporter 1 (LAT1) is up-regulated to support tumor cell growth. LAT1 is also essential for the permeation of amino acids and amino acid-related drugs through the blood-brain barrier. To search for in vitro assay systems to examine the interaction of chemical compounds with LAT1, we have investigated the expression of system L transporters and the properties of [C-14](L)-leucine transport in T24 human bladder carcinoma cells. Northern blot, real-time quantitative PCR and immumofluorescence analyses have reveled that T24 cells express LAT1 in the plasma membrane together with its associating protein 4F2hc, whereas T24 cells do not express the other system L isoform LAT2. The uptake of [C-14](L)-leucine by T24 cells is Na+ -independent and almost completely inhibited by system L selective inhibitor BCH. The profiles of the inhibition of [C-14](L)-leucine uptake by amino acids and amino acid-related compounds in T24 cells are comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [14C](L)-leucine uptake is, therefore, mediated by LAT1 in T24 cells. Consistent with LAT1 in Xenopus oocytes, the efflux of preloaded [C-14](L)-leucine is induced by extracellularly applied substrates of LAT1 in T24 cells. This efflux measurement has been proven to be more sensitive than that in Xenopus oocytes, because triiodothyronine, thyroxine and melphalan were able to induce the efflux of preloaded [C-14](L)-leucine in T24 cells, which was not detected for Xenopus oocyte expression system. T24 cell is, therefore, proposed to be an excellent tool to examine the interaction of chemical compounds with LAT1. (C) 2002 Elsevier Science B.V. All rights reserved.