Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells

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.     

The apoptotic process of human bladder carcinoma T24 cells induced by retinoid

Breakdown of the cytoskeletal network and redistribution of cytoplasmic organelles are early events of programmed cell death. Previous studies showed that retinoic acid induces programmed cell death in many tumor cell lines and that cytokeratins, particularly cytokeratin 18, are affected in the early events of apoptosis. In this study, patterns of cytoplasmic intermediate filaments (cytokeratin 18), actin filaments, and microtubules, as well as Bax and Bcl-2 proteins in human bladder carcinoma T24 cells were examined before and after retinoic acid treatment by immunocytochemistry and conventional electron microscopy. Our results demonstrate that the redistribution of Bax and Bcl-2 proteins in the subcellular compartment of T24 cells is correlated with reorganization of the cytoplasmic intermediate filament network and that cytokeratins are cleaved by caspases, as revealed by the M30 antibody which recognizes a specific caspase cleavage site within cytokeratin 18. The cytoskeletal architectures of microtubules are not significantly affected in the early apoptotic process, from our observations. We suggest that the breakdown in the intermediate filament network associated with the aggregation of mitochondria and lysosome may be a crucial event in the apoptotic process and that aggregation of cytoplasmic Bax may accelerate apoptotic death.     

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter

Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.     

Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters

A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

Expression of the mammalian Na+-independent L system amino acid transporter in Xenopus laevis oocytes

System L is primarily responsible for the Na+-independent transport of neutral amino acids, those with bulky chains such as leucine, isoleucine, phenylalanine, etc., into mammalian cells. mRNA from rat kidney and human lymphoid cells, when microinjected into Xenopus laevis oocytes, induced expression of this transport system. The expressed transport exhibits characteristics similar to those reported for the System L amino acid transporter from a variety of mammalian cells. Injection of size-fractionated mRNA indicates that the System L transporter in both the rat kidney and human lymphoid cells is encoded by mRNA of about 3 to 4 kb.     

Characterization of the preprotein and amino acid transporter gene family in Arabidopsis

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.     

Identification of LAT4, a novel amino acid transporter with system L activity

System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now three proteins that induce system L activity have been identified: LAT1, LAT2, and LAT3. The former two proteins belong to the solute carrier family 7 (SLC7), whereas the latter belongs to SLC43. In the present study we present a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chloride- and pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide but not that with high affinity. Mutation in LAT4 of the SLC43 conserved serine 297 to alanine abolishes sensitivity to N-ethylmaleimide. LAT4 activity is detected at the basolateral membrane of PCT kidney cells. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of the distal tubule and the collecting duct in the kidney. In the intestine, LAT4 is mainly present in the cells of the crypt.     

SNAT4 isoform of system A amino acid transporter is expressed in human placenta

The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6-10 wk) and late (10-13 wk) first-trimester and full-term (38-40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.     

Beta-eleostearic acid induce apoptosis in T24 human bladder cancer cells through reactive oxygen species (ROS)-mediated pathway

Beta-eleostearic acid (β-ESA, 9E11E13E-18:3), a linolenic acid isomer with a conjugated triene system, is a natural and biologically active compound. Herein, we investigated effects of β-eleostearic acid on T24 human bladder cancer cells. In this study, results showed that β-eleostearic acid had strong cytotoxicity to induce cell apoptosis, which was mediated by reactive oxygen species (ROS) in T24 cells. The cell viability assay results showed that incubation with β-eleostearic acid concentrations of 10-80μmol/L caused a dose- and time-dependent decrease of T24 cell viability, and the IC(50) value was 21.2μmol/L at 24h and 13.1μmol/L at 48h. Annexin V/PI double staining was used to assess apoptosis with flow cytometry. Treatment with β-eleostearic acid caused massive ROS accumulation and GSH decrease, which lead to activation of caspase-3 and down-regulation of Bcl-2 indicating induction of apoptosis. Subsequently, N-acetyl-l-cysteine (NAC) and PEG-catalase effectively blocked the ROS elevated effect of β-eleostearic acid, which suggested that β-eleostearic acid-induced apoptosis involved ROS generated. Additionally, we found that treating T24 cells with β-eleostearic acid induced activation of PPARγ. A PPARγ-activated protein kinase inhibitor was able to partially abrogate the effects of β-eleostearic acid. These results suggested that β-eleostearic acid can induce T24 cells apoptosis via a ROS-mediated pathway which may be involved PPARγ activation.     

CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration

CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCR and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy.     

Transport of l-proline by the proton-coupled amino acid transporter PAT2 in differentiated 3T3-L1 cells

Mechanism and substrate specificity of the proton-coupled amino acid transporter 2 (PAT2, SLC36A2) have been studied so far only in heterologous expression systems such as HeLa cells and Xenopus laevis oocytes. In this study, we describe the identification of the first cell line that expresses PAT2. We cultured 3T3-L1 cells for up to 2 weeks and differentiated the cells into adipocytes in supplemented media containing 2 μM rosiglitazone. During the 14 day differentiation period the uptake of the prototype PAT2 substrate l-[³H]proline increased ~5-fold. The macro- and microscopically apparent differentiation of 3T3-L1 cells coincided with their H⁺ gradient-stimulated uptake of l-[³H]proline. Uptake was rapid, independent of a Na⁺ gradient but stimulated by an inwardly directed H⁺ gradient with maximal uptake occurring at pH 6.0. l-Proline uptake was found to be mediated by a transport system with a Michaelis constant (K_t) of 130 ± 10 μM and a maximal transport velocity of 4.9 ± 0.2 nmol × 5 min^?1 mg of protein^?1. Glycine, l-alanine, and l-tryptophan strongly inhibited l-proline uptake indicating that these amino acids also interact with the transport system. It is concluded that 3T3-L1 adipocytes express the H⁺-amino acid cotransport system PAT2.     

The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66 h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, ¹⁴C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of ¹⁴C-MeAIB uptake revealed two distinct transport systems; system 1: K_m = 0.38 ± 0.12 mM, V_max = 27.8 ± 9.0 pmol/mg protein/20 min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K_m = 45.4 ± 25.0 mM, V_max = 1190 ± 291 pmol/mg protein/20 min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific siRNA significantly reduced system A activity (median 75% knockdown, n = 7). Conclusion: These data enhance our limited understanding of the relative importance of the system A subtypes for amino acid transport in human placental trophoblast by demonstrating that SNAT1 is a key contributor to system A activity at term.