title: Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells.
abstract: 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 [14C]L-leucine transport in T24 human bladder carcinoma cells. Northern blot, real-time quantitative PCR and immunofluorescence 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 [14C]L-leucine by T24 cells is Na(+)-independent and almost completely inhibited by system L selective inhibitor BCH. The profiles of the inhibition of [14C]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 [14C]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 [14C]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.
authors.affiliation: Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Tokyo 181-8611, Mitaka, Japan.
authors.forename: Do Kyung
authors.initials: DK
authors.lastname: Kim
authors.forename: Yoshikatsu
authors.initials: Y
authors.lastname: Kanai
authors.forename: Hye Won
authors.initials: HW
authors.lastname: Choi
authors.forename: Sahatchai
authors.initials: S
authors.lastname: Tangtrongsup
authors.forename: Arthit
authors.initials: A
authors.lastname: Chairoungdua
authors.forename: Ellappan
authors.initials: E
authors.lastname: Babu
authors.forename: Kittipong
authors.initials: K
authors.lastname: Tachampa
authors.forename: Naohiko
authors.initials: N
authors.lastname: Anzai
authors.forename: Yuji
authors.initials: Y
authors.lastname: Iribe
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Endou
mesh_terms: D000598:Amino Acids, Cyclic / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D015152:Blotting, Northern; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000032:analysis / Q000378:metabolism; D006801:Humans; D027283:Large Neutral Amino Acid-Transporter 1 / Q000032:analysis / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism*; D018613:Microscopy, Confocal; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured / Q000378:metabolism*; D001749:Urinary Bladder Neoplasms
chemical_list: D000598:Amino Acids, Cyclic; D002250:Carbon Radioisotopes; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027283:Large Neutral Amino Acid-Transporter 1; C006504:2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; D007930:Leucine