Overexpression of LAT1/CD98 Light Chain Is Sufficient to Increase System l-Amino Acid Transport Activity in Mouse Hepatocytes but Not Fibroblasts
2001; Elsevier BV; Volume: 276; Issue: 20 Linguagem: Inglês
10.1074/jbc.m008248200
ISSN1083-351X
AutoresWilliam A. Campbell, Nancy L. Thompson,
Tópico(s)Epigenetics and DNA Methylation
Resumol-amino acid transporter-1 (LAT1) is a highly conserved gene identified as a light chain of the CD98 amino acid transporter and cellular activation marker. In our previous studies we found increased expression of LAT1 in primary human cancers. We have demonstrated also that LAT1 response to arginine availability is lost in transformed and tumorigenic cells such that expression is constitutively high. System l-amino acid transport activity correlates with changes in LAT1. To assess the functional relevance of increased LAT1 expression and the requirement for 4F2 heavy chain, we overexpressed these CD98 subunits together and separately in nontransformed hepatocytes and fibroblasts. Antigen tags in the expression constructs confirmed that expressed proteins were localized to both cytoplasmic and plasma membrane locations within the cells. Overexpression of LAT1 alone in mouse hepatocytes, but not fibroblasts, was sufficient to increase system l transport, and these cells displayed a growth advantage in conditions of limited arginine. Our results suggest that loss of regulation leading to constitutive expression of LAT1 can contribute to oncogenesis. We hypothesize that the altered LAT1 expression observed in hepatocarcinogenesis gives cells a growth or survival advantage through increased transport activity in a tumor microenvironment of limited amino acid availability. l-amino acid transporter-1 (LAT1) is a highly conserved gene identified as a light chain of the CD98 amino acid transporter and cellular activation marker. In our previous studies we found increased expression of LAT1 in primary human cancers. We have demonstrated also that LAT1 response to arginine availability is lost in transformed and tumorigenic cells such that expression is constitutively high. System l-amino acid transport activity correlates with changes in LAT1. To assess the functional relevance of increased LAT1 expression and the requirement for 4F2 heavy chain, we overexpressed these CD98 subunits together and separately in nontransformed hepatocytes and fibroblasts. Antigen tags in the expression constructs confirmed that expressed proteins were localized to both cytoplasmic and plasma membrane locations within the cells. Overexpression of LAT1 alone in mouse hepatocytes, but not fibroblasts, was sufficient to increase system l transport, and these cells displayed a growth advantage in conditions of limited arginine. Our results suggest that loss of regulation leading to constitutive expression of LAT1 can contribute to oncogenesis. We hypothesize that the altered LAT1 expression observed in hepatocarcinogenesis gives cells a growth or survival advantage through increased transport activity in a tumor microenvironment of limited amino acid availability. Chee's essential medium green fluorescent protein phosphate-buffered saline Krebs Ringer phosphate 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid hemagglutinin LAT1/TA1 and its homologs have been identified by multiple investigators as one of several alternate light chains of the CD98 amino acid transporter and cellular activation marker (1Mannion B.A. Kolesnikova T.V. Lin S.H. Wang S. Thompson N.L. Hemler M.E. J. Biol. Chem. 1998; 273: 33127-33129Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 2Tsurudome M. Ito M. Takebayashi S. Okumura K. Nishio M. Kawano M. Kusagawa S. Komada H. Ito Y. J. Immunol. 1999; 162: 2462-2466PubMed Google Scholar, 3Nakamura E. Sato M. Yang H. Miyagawa F. Harasaki M. Tomita K. Matsuoka S. Noma A. Iwai K. Minato N. J. Biol. Chem. 1999; 274: 3009-3016Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 4Prasad P.D. Wang H. Huang W. Kekuda R. Rajan D.P. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1999; 255: 283-288Crossref PubMed Scopus (211) Google Scholar, 5Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar, 6Mastroberardino L. Spindler B. Pfeiffer R. Skelly P.J. Loffing J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (470) Google Scholar). TA1 was cloned in our laboratory on the basis of its differential expression between rat hepatoma cells and normal adult rat liver and is identical to the C terminus (amino acids 272–512) of LAT1 (7Sang J. Lim Y.-P. Panzica M. Finch P. Thompson N.L. Cancer Res. 1995; 55: 1152-1159PubMed Google Scholar). The CD98 complex consists of an 80-kDa heavy chain (4F2hc) and a 40–45-kDa light chain (8Hemler M.E. Strominger J.L. J. Immunol. 1982; 129: 623-628PubMed Google Scholar). 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The CD98 complex can mediate various amino acid transport systems in Xenopusoocytes depending on which CD98 light chain (CD98lc) is associated with the CD98 heavy chain (CD98hc) (3Nakamura E. Sato M. Yang H. Miyagawa F. Harasaki M. Tomita K. Matsuoka S. Noma A. Iwai K. Minato N. J. Biol. Chem. 1999; 274: 3009-3016Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 4Prasad P.D. Wang H. Huang W. Kekuda R. Rajan D.P. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1999; 255: 283-288Crossref PubMed Scopus (211) Google Scholar, 5Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar, 6Mastroberardino L. Spindler B. Pfeiffer R. Skelly P.J. Loffing J. Shoemaker C.B. Verrey F. Nature. 1998; 395: 288-291Crossref PubMed Scopus (470) Google Scholar, 13Torrents D. Estevez R. Pineda M. Fernandez E. Lloberas J. Shi Y.-B. Zorzano A. Palacin M. J. Biol. Chem. 1998; 273: 32437-32445Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Co-injection of cRNA for TA1/LAT-1 and 4F2hc has been shown to mediate system ltransport of large neutral amino acids with branched or aromatic side chains in Xenopus oocytes (3Nakamura E. Sato M. Yang H. Miyagawa F. Harasaki M. Tomita K. Matsuoka S. Noma A. Iwai K. Minato N. J. Biol. Chem. 1999; 274: 3009-3016Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 5Kanai Y. Segawa H. Miyamoto K. Uchino H. Takeda E. Endou H. J. Biol. Chem. 1998; 273: 23629-23632Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar). We have found increased levels of LAT1 mRNA in both rat and human carcinomas and in the carbon tetrachloride model of liver injury/regeneration (7Sang J. Lim Y.-P. Panzica M. Finch P. Thompson N.L. Cancer Res. 1995; 55: 1152-1159PubMed Google Scholar, 22Wolf D.A. Wang S. Panzica M.A. Bassily N.H. Thompson N.L. Cancer Res. 1996; 56: 5012-5022PubMed Google Scholar, 23Shultz V.D. Degli Esposti S. Panzica M.S. Abraham A. Finch P. Thompson N.L. Pathobiology. 1997; 65: 14-25Crossref PubMed Scopus (4) Google Scholar, 24Shultz V.D. Campbell W. Karr S. Hixson D.C. Thompson N.L. Toxicol. Appl. Pharmacol. 1999; 154: 84-96Crossref PubMed Scopus (11) Google Scholar). Adaptive regulation of message for LAT1 but not 4F2hc was observed in response to arginine levels in normal hepatic cells (25Campbell W.A. Sah D.E. Medina M.M. Albina J.E. Coleman W.B. Thompson N.L. J. Biol. Chem. 2000; 275: 5347-5354Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Loss of LAT1 regulation with corresponding increased system l transport was associated with malignant progression, suggesting that up-regulation of LAT1 or functional CD98 complex contributes to the neoplastic phenotype. Hara et al.(26Hara K. Kudoh H. Enomoto T. Hashimoto Y. Masuko T. Biochem. Biophys. Res. Commun. 1999; 262: 720-725Crossref PubMed Scopus (65) Google Scholar) have shown that NIH3T3 cells transfected to overexpress 4F2hc demonstrated classic properties of malignant cells including higher saturation density, growth in soft agar, and tumor formation in athymic mice. Researchers from the same laboratory demonstrated more recently that transformation of BALB3T3 cells by overexpression of 4F2hc requires its association with an unidentified light chain (27Shishido T. Uno S. Kamohara M. Tsuneoka-Suzuki T. Hashimoto Y. Enomoto T. Masuko T. Int. J. Cancer. 2000; 87: 311-316Crossref PubMed Scopus (42) Google Scholar). We are interested in the regulation and role of LAT1 and CD98-related molecules in transformation and carcinogenesis. To examine the relationship of cell type to overexpression of these molecules and associated transport properties, we transiently overexpressed LAT1 and 4F2hc alone or together in nontransformed mouse hepatocyte and fibroblast cell lines. We found that overexpression of LAT1 alone was sufficient to increase system l transport activity significantly in the epithelial cells but not in the fibroblasts. In contrast, increases in transport activity in fibroblasts required coexpression of both LAT1 and 4F2hc. Hepatic cells overexpressing LAT1 displayed a growth advantage relative to control cells under conditions of limited arginine. We postulate that differences in LAT1 expression and response to amino acid availability influence cell growth and survival properties and that the LAT1-CD98 pathway may represent a unique therapeutic target for cancer intervention. AML12 cells, a normal mouse hepatocyte line, were provided kindly by Dr. Nelson Fausto (Department of Pathology, University of Washington) and maintained in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Life Technologies, Inc.) containing 10% fetal bovine serum and supplemented with insulin, transferrin, and selenium (ITS+) at 1 ml/liter medium (Becton Dickinson) and 0.1 μm dexamethasone. NIH3T3 cells were cultured routinely in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. A custom formulation of Chee's essential medium (CEM)1 that was demonstrated to maintain long term differentiated cell functionin vitro was provided kindly by Dr. Hugo Jauregui (Department of Pathology, Rhode Island Hospital) and prepared as described previously (28Waxman D.J. Morrissey J. Naik S. Jauregui H. Biochemistry. 1990; 271: 113-119Crossref Scopus (168) Google Scholar). Arginine was not present in this custom formulation and was added from stock solutions. For experiments in which gene expression was assayed as a function of arginine availability, cells were seeded into T-75 flasks with CEM containing 5% fetal bovine serum with and without arginine (25Campbell W.A. Sah D.E. Medina M.M. Albina J.E. Coleman W.B. Thompson N.L. J. Biol. Chem. 2000; 275: 5347-5354Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Previous experiments have shown no difference in results with dialyzed or nondialyzed serum (25Campbell W.A. Sah D.E. Medina M.M. Albina J.E. Coleman W.B. Thompson N.L. J. Biol. Chem. 2000; 275: 5347-5354Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Medium was changed every day. Viability was at least 90% in all cases. Rat LAT1 cDNA was subcloned into the pFLAG-5a expression vector (Sigma) and sequenced to confirm the correct sequence and the addition of the FLAG epitope tag. pL(CD98-HA)IZ expression construct and monoclonal antibody HA.11 (Babco) were provided kindly by Dr. Tatiana Kolesnikova and Dr. Martin Hemler (Dana Farber Cancer Institute, Harvard Medical School). pL(CD98-HA)IZ expresses human 4F2hc/CD98 heavy chain with a hemagglutinin epitope tag. pEGFP-F (CLONTECH) encodes farnesylated enhanced green fluorescent protein. For transport assays, AML12 cells and NIH3T3 cells were seeded, respectively, at 2 × 105 and 7.5 × 104 cells/well of a 24-well plate 24 h prior to transfection. For immunofluorescence, AML12 and NIH3T3 cells were seeded, respectively, at 4 × 105 and 1.5 × 105 cells/chamber of a slide chamber 24 h prior to transfection. Transfection was carried out with LipofectAMINE Plus reagent (Life Technologies, Inc.). For a 24-well plate, 0.6 μg of DNA, 3 μl of Plus reagent, and 1.5 μl of LipofectAMINE were used per well. For a chamber slide, 1.2 μg of DNA, 6 μl of Plus reagent, and 3 μl of LipofectAMINE were used per chamber. The functional expression of each cDNA was analyzed 48 h post-transfection by measuring radiolabeled amino acid uptake and by immunofluorescence in AML12 and NIH3T3 cells. Cotransfection with pEGFP-F was used for every experiment to monitor transfection efficiency by counting fluorescent cells expressing GFP. Transfection efficiency routinely approached 40 and 50% for NIH3T3 and AML12, respectively. Expression of exogenous LAT1 and 4F2 was monitored by indirect immunofluorescence microscopy using antibodies directed to the epitope tag present on each protein. M2 antibody (Sigma) was used to detect FLAG-tagged LAT1, whereas HA.11 antibody (Babco) was used to detect hemagglutinin-tagged 4F2hc. Briefly, AML12 and NIH3T3 cells were transfected with pFLAG-LAT1 or pL(CD98-HA)IZ, and at 48 h after transfection, slides were fixed in cold acetone. Slides were blocked with 1% normal goat serum in PBS and then incubated with either M2 (mouse anti-FLAG) antibody at 10 μg/ml or HA.11 (mouse anti-hemagglutinin) antibody at 1:100 dilution in 1% normal goat serum in PBS. Slides were washed with PBS, blocked again, and incubated with goat anti-mouse secondary antibody (Sigma) at 1:100 dilution in 1% normal goat serum in PBS. After a subsequent wash and coverslipping, slides were examined on a Nikon Microphot-EPI-FL fluorescence microscope. Expression of endogenous LAT1 was detected using affinity-purified anti-peptide antibody to the NH2 terminus of LAT1 at 12 μg/ml. MacVector sequence analysis of the rat LAT1 coding region was used to select a region with little or no homology to other CD98 light chains and with high conservation to human LAT-1 and high hydrophilicity. Peptide 20–30, EERQAREKML, near the NH2terminus scored highest for these qualities and was synthesized commercially with an NH2-terminal cysteine for conjugation to keyhole lympet hemocyanin and used to immunize two rabbits following a conventional immunization schedule (Sigma-Genosys). The specificity of this antibody was demonstrated by absorption with the immunizing peptide (data not shown). Expression of endogenous 4F2hc was detected using an anti-4F2hc rabbit anti-peptide antibody (1:100) kindly provided by Dr. Suresh S. Tate (Cornell University Medical College, NY). This polyclonal antiserum was raised against a 92-amino acid peptide fragment (Lys-114–Tyr-205) of the rat glioma cell 4F2hc (29Broer S. Broer A. Hamprecht B. Biochem. J. 1995; 312: 863-870Crossref PubMed Scopus (63) Google Scholar). Normal rabbit serum at the same concentration or dilution was used as a negative control (data not shown). Normal liver and GP7TB tumor cells were used as negative and positive controls, respectively, for LAT1 antibody reactivity (data not shown). Total RNA was isolated using the guanidinium isothiocyanate/cesium chloride method (30Chirgwin J.M. Przbyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar) for rat placenta and a modification of that method for cultured cell lines (Totally RNA Kit™, Ambion, Austin, TX). RNA was isolated from cultured cells after subculture in CEM for the relevant time points. Aliquots (12 μg) of total RNA were size-fractionated on 1% agarose/formaldehyde gels as described previously (22Wolf D.A. Wang S. Panzica M.A. Bassily N.H. Thompson N.L. Cancer Res. 1996; 56: 5012-5022PubMed Google Scholar). After electrophoresis, gels were equilibrated in 1 m ammonium acetate, and RNA was transferred to Nytran nylon membranes (Schleicher and Schüll). Blots were baked for 2 h at 80 °C and hybridized at 65 °C according to Church and Gilbert (31Church G. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7266) Google Scholar). RatTA1 p900, a fragment corresponding to nucleotides 816–1536 of the full-length rat LAT-1 inserted into Bluescript SK vector, was labeled with [32P]dCTP (3000 Ci/mmol, PerkinElmer Life Sciences) by random primed labeling (Roche Molecular Biochemicals) for use as a probe. Blots wrapped in plastic wrap were exposed to x-ray film (Eastman Kodak Co.) at −70 °C in the presence of intensifying screens. Blots were stripped and rehybridized to an 1800-base pairEcoRI fragment of human 4F2hc (provided by Dr. Martin Hemler) (32Quackenbush E. Clabby M. Gottesdiener K.M. Barbosa J. Jones N.H. Strominger J.L. Speck S. Leiden J.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6526-6530Crossref PubMed Scopus (100) Google Scholar). Densitometry using the Quantity One™ IBM software package was used to quantify differences in RNA levels with normalization to 18 S ribosomal RNA. The transport of radiolabeled amino acids by cell monolayers was performed using a modification of the cluster-tray method developed by Gazzola et al. (33Gazzola G.C. Dall'Asta V. Franchi-Gazzola R. White M.F. Anal. Biochem. 1981; 115: 368-374Crossref PubMed Scopus (209) Google Scholar) and described by Kilberg (34Kilberg M.S. Methods Enzymol. 1989; 173: 564-575Crossref PubMed Scopus (47) Google Scholar). All 3H-labeled amino acids were purchased from PerkinElmer Life Sciences and unlabeled amino acids were purchased from Sigma. Cells were near confluent for the transport assay. Transport assays were performed as described previously (25Campbell W.A. Sah D.E. Medina M.M. Albina J.E. Coleman W.B. Thompson N.L. J. Biol. Chem. 2000; 275: 5347-5354Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Briefly, before the transport assays, cells were rinsed with warm Na+-free KRP buffer, in which the sodium-containing salts were iso-osmotically replaced with choline, to remove extracellular Na+ and amino acids. Cells were incubated in warm Na+-free KRP buffer for 10 min to deplete intracellular amino acids. The uptake of radiolabeled amino acids (5 μCi of [3H]amino acid/ml) at 50 μmol/liter in either 200 μl of Na+-free KRP buffer or sodium KRP was measured for 30 s at 37 °C. Preliminary experiments indicated that uptake of each 3H-labeled amino acid was linearly dependent on incubation time up to at least 3 min; therefore, uptake was measured for 30 s (data not shown). In inhibition experiments, excess cold 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) was added to the uptake buffer at 10 mm final concentration. Uptake was terminated by washing the cells rapidly four times with 1 ml/well of ice-cold Na+-free KRP buffer. After the trays were allowed to dry, the cells were incubated for 1 h with 0.2 ml/well of 0.2% (w/v) SDS plus 0.2 N NaOH to release intracellular radioactivity. A 0.1-ml aliquot from each well was neutralized with 0.1 ml of 0.2n HCl and quantified in a Beckman LS 6000SC liquid scintillation counter. The remaining 0.1 ml was analyzed for protein content using the BCA protein assay reagent (Pierce). Transport velocities were calculated from radioactive counts, specific activities of uptake mixes, and protein absorbance values and were expressed as picomoles amino acid transported per mg of protein per min (averages ± S.E. of ≥ 4 separate determinations). All data were normalized for transfection efficiency. Data comparing two experimental results were analyzed statistically by Student'st test using the InStat Macintosh statistics program. Two experimental results were considered to be statistically significantly different when p < 0.05. Each experiment was repeated with independent transfections at least twice to show qualitatively the same results. Averages of three independent experiments are shown for both AML12 and NIH3T3 cells. Cells were seeded into 6-well plates, transfected with plasmid DNA, and then cultured in CEM containing 5% fetal bovine serum without arginine. The medium was changed every day. Transfection efficiency was monitored by GFP expression and immunofluorescence. Cells were counted by the trypan dye exclusion test at the beginning of the experiment as well as 24 and 48 h after culture in CEM without arginine. Cell number was expressed as averages ± S.E. of three or more separate determinations. The experiment was repeated with independent transfections to show qualitatively the same results. An aliquot of cells from each condition was saved for cell cycle analysis. These cells were stained with 1× propidium iodide stain (50 μg/ml propidium iodide, 0.1% Nonidet P-40, and 0.1% sodium citrate in H2O) for ∼10 min. Cell cycle analysis was performed using a Becton Dickinson FACSORT flow cytometer, and data were analyzed with Cell Quest software. AML12 cells, an immortalized, nontransformed, nontumorigenic, well differentiated hepatocyte cell line, was established from the liver of transgenic mice overexpressing transforming growth factor-α (35Wu J.C. Merlino G. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 674-678Crossref PubMed Scopus (250) Google Scholar). This cell line was established because hepatocytes cannot be maintained for long periods of time as replicating, differentiated cells while remaining nontumorigenic. These cells have been used extensively as a model to study tissue-specific gene regulation and hepatocarcinogenesis because they are easily transfectable, do not display anchorage-independent growth or form tumors in nude mice, and maintain expression of differentiated hepatocyte markers after extensive passaging (35Wu J.C. Merlino G. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 674-678Crossref PubMed Scopus (250) Google Scholar, 36Dumenco L. Oguey D. Wu J. Messier N. Fausto N. Hepatology. 1995; 22: 1279-1288Crossref PubMed Google Scholar, 37Tarn C. Bilodeau M.L. Hullinger R.L. Andrisani O.M. J. Biol. Chem. 1999; 274: 2327-2336Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). NIH3T3 cells have been utilized by other investigators (26Hara K. Kudoh H. Enomoto T. Hashimoto Y. Masuko T. Biochem. Biophys. Res. Commun. 1999; 262: 720-725Crossref PubMed Scopus (65) Google Scholar) to examine the functional significance of CD98 heavy chain overexpression. AML12 and NIH3T3 cells were cotransfected with pEGFP-F and pFLAG-5a, and transfection efficiency was monitored 48 h post-transfection by fluorescence of GFP (Fig. 1). Transfection efficiencies routinely approached 50 and 40% for AML12 and NIH3T3, respectively. Cotransfection with pEGFP-F was used to monitor transfection efficiency in all amino acid transport experiments. NIH3T3 and AML12 cells were transfected with either pFLAG-LAT1 or pL(CD98-HA)IZ to verify expression of exogenous protein by immunofluorescence in addition to transport activity (Fig. 2, Aand B). Both FLAG-tagged LAT1 and hemagglutinin-tagged 4F2hc were detected in transfected NIH3T3 and AML12 cells. The staining pattern indicated significant colocalization in both cell types. Staining was observed at the cell surface as well as within the cytoplasm. Anti-peptide antibodies were used to detect endogenous 4F2hc and LAT1 staining in both AML12 and NIH3T3 cells (Fig.2, C and D). The LAT1 epitope is on the NH2 terminus of the molecule, which is predicted to be cytoplasmic (38Verrey F. Jack D.L. Paulsen I.T. Saier Jr., M.H. Pfeiffer R. J. Membr. Biol. 1999; 172 (. H.): 181-192MCrossref PubMed Scopus (146) Google Scholar). The antibody to 4F2hc is directed to a predicted ectodomain (38Verrey F. Jack D.L. Paulsen I.T. Saier Jr., M.H. Pfeiffer R. J. Membr. Biol. 1999; 172 (. H.): 181-192MCrossref PubMed Scopus (146) Google Scholar). Intense staining was observed for 4F2hc in AML12 and NIH3T3 cells and for LAT1 in NIH3T3 cells. Staining for LAT1 was very weak in AML12 cells. The staining pattern for endogenous 4F2hc and LAT1 was very similar to staining for exogenous 4F2hc and LAT1 with both cytoplasmic and cell membrane staining. We observed similar immunofluorescent staining for all transfections. In previous studies using a panel of hepatic cell lines with specific differences in transformation and tumorigenicity, we found that LAT1 but not 4F2hc correlated with transformation, tumorigenicity, and increases in systeml transport activity (25Campbell W.A. Sah D.E. Medina M.M. Albina J.E. Coleman W.B. Thompson N.L. J. Biol. Chem. 2000; 275: 5347-5354Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). To determine the functional relevance of overexpression of LAT1 and requirement for 4F2hc in nontransformed hepatocytes, we overexpressed LAT1 and/or 4F2hc in AML12 cells and measured transport activity. Transport activity for systemsl and y+ were found to be sodium-independent (data not shown). As shown in Fig.3 a, overexpression of LAT1 increased system l-specific transport activity 2-fold (significant at p < 0.005) relative to 4F2hc or empty vector transfection. Overexpression of LAT1 and 4F2hc did not increase transport activity above that of LAT1 alone. As expected, addition of excess cold BCH inhibited leucine transport, consistent with systeml activity. Arginine transport (system y+) was not significantly different in any of the transfection conditions (Fig.3 b) and served as a control for any nonspecific influence of the transfection conditions. Other investigators have demonstrated that overexpression of CD98 heavy chain/4F2hc transforms NIH3T3 cells and that transformation depends on association with an unidentified light chain (26Hara K. Kudoh H. Enomoto T. Hashimoto Y. Masuko T. Biochem. Biophys. Res. Commun. 1999; 262: 720-725Crossref PubMed Scopus (65) Google Scholar, 27Shishido T. Uno S. Kamohara M. Tsuneoka-Suzuki T. Hashimoto Y. Enomoto T. Masuko T. Int. J. Cancer. 2000; 87: 311-316Crossref PubMed Scopus (42) Google Scholar). To determine whether LAT1 transfection can increase transport activity in NIH3T3 cells and to compare activity in hepatocytes versusfibroblasts, we overexpressed LAT1 alone or with 4F2hc in NIH3T3 cells. We found that overexpression of both LAT1 and 4F2hc increased systeml-specific transport activity approximately 2-fold (significant at p < 0.005) relative to LAT1, 4F2hc, or empty vector transfection (Fig.4 a). In contrast to the studies in hepatocytes, however, overexpression of LAT1 alone had no significant effect on transport activity. As expected, the addition of excess cold BCH inhibited leucine transport, which is consistent with system l activity. Arginine transport (system y+) was not significantly different after any of the transfections (Fig. 4 b). Interestingly, system lamino acid transport activity was 3–6-fold higher in NIH3T3 cells compared with AML12 cells on a transport activity per cell basis (Table I).Table IRelative System l amino Acid transport activity in transfected cellsTransfected DNAAML12NIH3T3LAT1264F2hc16LAT1 and 4F2hc211Empty vector16 Open table in a new tab In previous studies we have seen adaptive regulation of message for LAT1 but not CD98 heavy chain/4F2 in response to arginine levels in normal but not transformed or tumorigenic hepatic cells (25Campbell W.A. Sah D.E. Medina M.M. Albina J.E. Coleman W.B. Thompson N.L. J. Biol. Chem. 2000; 275: 5347-5354Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We were interested in whether the response to arginine of LAT1 and 4F2hc message in AML12 and NIH3T3 cells resembled the response of normal
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