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RAGE Expression in Rhabdomyosarcoma Cells Results in Myogenic Differentiation and Reduced Proliferation, Migration, Invasiveness, and Tumor Growth

2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2007.070049

ISSN

1525-2191

Autores

Francesca Riuzzi, Guglielmo Sorci, Rosario Donato,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEΔcyto) (TE671/RAGEΔcyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH2 terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEΔcyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential. Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEΔcyto) (TE671/RAGEΔcyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH2 terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEΔcyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential. At a certain stage during myogenesis, myoblasts, ie, the precursors of skeletal muscle cells, cease to proliferate and start to differentiate once they attain a critical density.1Andrés V Walsh K Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis.J Cell Biol. 1996; 132: 657-666Crossref PubMed Scopus (503) Google Scholar, 2Arnold HH Winter B Muscle differentiation: more complexity to the network of myogenic regulators.Curr Opin Genet Dev. 1998; 8: 539-544Crossref PubMed Scopus (247) Google Scholar, 3Chargé SB Rudnicki MA Cellular and molecular regulation of muscle regeneration.Physiol Rev. 2004; 84: 209-238Crossref PubMed Scopus (1987) Google Scholar, 4Buckingham M Myogenic progenitor cells and skeletal myogenesis in vertebrates.Curr Opin Genet Dev. 2006; 16: 525-532Crossref PubMed Scopus (333) Google Scholar Extracellular factors acting via cell surface receptors as well as adhesion molecules control proliferation arrest and differentiation of myoblasts by regulating via signaling pathways the expression of muscle-specific transcription factors. 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24: 4880-4894Crossref PubMed Scopus (109) Google Scholar In this context, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun NH2 terminal protein kinase (JNK) need to be inactivated, and p38 mitogen-activated protein kinase (MAPK) and Akt need to be activated for myoblasts to exit the cell cycle and for differentiation to proceed.12Bennett AM Tonks NK Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases.Science. 1997; 278: 1288-1291Crossref PubMed Scopus (305) Google Scholar, 13Winter B Arnold H-H Activated raf kinase inhibits muscle cell differentiation through a MEF2-dependent mechanism.J Cell Sci. 2000; 113: 4211-4220Crossref PubMed Google Scholar, 14Perry RL Parker MH Rudnicki MA Activated MEK1 binds the nuclear MyoD transcriptional complex to repress transactivation.Mol Cell. 2001; 8: 291-301Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15Wu Z Woodring PJ Bhakta KS Tamura K Wen F Feramisco JR Karin M Wang JYJ Puri PL p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps.Mol Cell Biol. 2000; 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it is present in rat embryo skeletal muscle fibers and in rat postnatal myofibers until ∼11 days after birth and absent from adult muscle fibers,11Sorci G Riuzzi F Arcuri C Giambanco I Donato R Amphoterin stimulates myogenesis and counteracts the antimyogenic factors basic fibroblast growth factor and S100B via RAGE binding.Mol Cell Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar suggesting that it might play a role in embryonic myogenesis. RAGE can be activated by several ligands, including HMGB1 (amphoterin), and effects of RAGE engagement depend on the cell type considered.19Schmidt AM Yan SD Yan SF Stern D The biology of the receptor for advanced glycation end products and its ligands.Biochim Biophys Acta. 2000; 1498: 99-111Crossref PubMed Scopus (607) Google Scholar Thus, RAGE has been shown to transduce trophic and toxic stimuli in neurons depending on the nature and concentration of the ligand,20Yan SD Chen X Fu J Chen M Zhu H Roher A Slattery T Zhao L Nagashima M Morser J Migheli A Nawroth P Stern D Schmidt AM RAGE and amyloid-β peptide neurotoxicity in Alzheimer's disease.Nature. 1996; 382: 685-691Crossref PubMed Scopus (1819) Google Scholar, 21Huttunen HJ Kuja-Panula J Rauvala H Receptor for advanced glycation end products (RAGE) signaling induces CREB-dependent chromogranin expression during neuronal differentiation.J Biol Chem. 2002; 277: 38635-38646Crossref PubMed Scopus (150) Google Scholar, 22Huttunen HJ Kuja-Panula J Sorci G Agneletti AL Donato R Rauvala H Coregulation of neurite outgrowth and cell survival by amphoterin and S100 proteins through RAGE activation.J Biol Chem. 2000; 275: 40096-40105Crossref PubMed Scopus (513) Google Scholar to take part in the inflammatory response in monocytes/macrophages/microglia,23Hofmann MA Drury S Fu C Qu W Taguchi A Lu Y Avila C Kambham N Bierhaus A Nawroth P Neurath MF Slattery T Beach D McClary J Nagashima M Morser J Stern D Schmidt AM Receptor for advanced glycation end products (RAGE) signaling induces CREB-dependent chromogranin expression during neuronal differentiation.Cell. 1999; 97: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar and to be involved in neoplastic transformation and metastasis of neuroepithelial tumor cells.24Taguchi A Blood DC del Toro G Canet A Lee DC Qu W Tanji N Lu Y Lalla E Fu C Hofmann MA Kislinger T Ingram M Lu A Tanaka H Hori O Ogawa S Stern DM Schmidt AM Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases.Nature. 2000; 405: 354-360Crossref PubMed Scopus (1107) Google Scholar, 25Takada M Koizumi T Toyama H Suzuki Y Kuroda Y Differential expression of RAGE in human pancreatic carcinoma cells.Hepatogastroenterology. 2001; 48: 1577-1578PubMed Google Scholar, 26Huttunen HJ Fages C Kuja-Panula J Ridley AJ Rauvala H Receptor for advanced glycation end products-binding COOH-terminal motif of amphoterin inhibits invasive migration and metastasis.Cancer Res. 2002; 62: 4805-4811PubMed Google Scholar, 27Kuniyasu H Chihara Y Kondo H Differential effects between amphoterin and advanced glycation end products on colon cancer cells.Int J Cancer. 2003; 104: 722-727Crossref PubMed Scopus (132) Google Scholar Besides, RAGE engagement by HMGB1 in myoblasts results in stimulation of differentiation via a Cdc42-Rac1-MKK6-p38 MAPK pathway,11Sorci G Riuzzi F Arcuri C Giambanco I Donato R Amphoterin stimulates myogenesis and counteracts the antimyogenic factors basic fibroblast growth factor and S100B via RAGE binding.Mol Cell Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar supporting the possibility that the HMGB1/RAGE pair might contribute to embryonic myogenesis and potentially muscle regeneration. Moreover, RAGE ligation by HMGB1 results in a decreased proliferation and increased apoptosis in wild-type (wt) myoblasts, whereas myoblasts stably overexpressing a RAGE mutant lacking the cytoplasmic and transducing domain (RAGEΔcyto) show enhanced pro-liferation, migration, and invasiveness in vitro and increased tumor formation in vivo compared with wt myoblasts.28Riuzzi F Sorci G Donato R The amphoterin/RAGE pair modulates myoblast proliferation, apoptosis, adhesiveness, migration and invasiveness: functional inactivation of RAGE in L6 myoblasts results in tumor formation in vivo.J Biol Chem. 2006; 281: 8242-8253Crossref PubMed Scopus (107) Google Scholar These latter findings suggest RAGE engagement in myoblasts, besides activating the myogenic program, might contribute to the proliferation arrest required for myoblast differentiation and that functional inactivation or repression of expression of RAGE in myoblasts might contribute to rhabdomyosarcomagenesis. Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue sarcoma, arising from muscle precursor cells. The two most common histological subtypes are embryonal RMS, which has a more favorable prognosis, and alveolar RMS, which has a poor prognosis. The embryonal-type TE671 RMS cells, which are similar if not identical to the RMS cells, RD,29Stratton MR Darling J Pilkington GJ Lantos PL Reeves BR Cooper CS Characterization of the human cell line TE671.Carcinogenesis. 1989; 10: 899-905Crossref PubMed Scopus (126) Google Scholar are unable to complete the differentiation program despite the expression of muscle-specific regulatory proteins.30Tapscott SJ Thayer MJ Weintraub H Deficiency in rhabdomyosarcomas of a factor required for MyoD activity and myogenesis.Science. 1993; 259: 1450-1453Crossref PubMed Scopus (164) Google Scholar, 31Otten AD Firpo EJ Gerber AN Brody LL Roberts JM Tapscott SJ Inactivation of MyoD-mediated expression of p21 in tumor cell lines.Cell Growth Differ. 1997; 8: 1151-1160PubMed Google Scholar However, persistent activation of p38 MAPK in a panel of RMS cells resulted in proliferation arrest and terminal differentiation,32Puri PL Wu Z Zhang P Wood LD Bhakta KS Han J Feramisco JR Karin M Wang JYJ Induction of terminal differentiation by constitutive activation of p38 MAP kinase in human rhabdomyosarcoma cells.Genes Dev. 2000; 14: 574-584PubMed Google Scholar implying that defective activation of p38 MAPK might be one of the causes of the inability of RMS cells to exit the cell cycle and initiate the myogenic program. We show here that the embryonal-type TE671 RMS cells do not express RAGE and that enforced expression of RAGE in TE671 cells results in myogenic differentiation and reduced proliferation, migration, and invasiveness in vitro and reduced tumor growth in vivo. We also show that the RMS cell lines CCA (embryonal-type) and RMZ-RC2 (alveolar-type), which do express RAGE albeit to a different extent from one another, show an inverse relationship between the level of RAGE expression and migration/invasiveness in vitro and exhibit a reduced myogenic potential and enhanced invasive properties in vitro when transfected with the signaling-deficient RAGE mutant RAGEΔcyto and an increased myogenic potential and reduced invasive properties when transfected with full-length RAGE. TE671 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA), 100 U/ml penicillin, and 100 μg/ml streptomycin, in a H2O-saturated 5% CO2 atmosphere at 37°C. Transfection of TE671 cells to obtain clones stably expressing human RAGE33Neeper M Schmidt AM Brett J Yan SD Wang F Pan YC Elliston K Stern D Shaw A Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.J Biol Chem. 1992; 267: 14998-15004Abstract Full Text PDF PubMed Google Scholar or RAGEΔcyto34Huttunen HJ Fages C Rauvala H Receptor for advanced glycation end products (RAGE)-mediated neurite outgrowth and activation of NF-κB require the cytoplasmic domain of the receptor but dif-ferent downstream signaling pathways.J Biol Chem. 1999; 274: 19919-19924Crossref PubMed Scopus (573) Google Scholar was performed exactly as described for L6 myoblasts.9Sorci G Agneletti AL Riuzzi F Marchetti C Donato R S100B inhibits myogenic differentiation and myotube formation in a RAGE-independent manner.Mol Cell Biol. 2003; 23: 4870-4881Crossref PubMed Scopus (72) Google Scholar TE671/RAGE cells, TE671/RAGEΔcyto cells, and TE671/wt cells were used in experiments described below, in media containing Geneticin (G-418) (125 μg/ml), in the presence of HMGB1, a polyclonal anti-HMGB1 antibody (BD Phar-Mingen, San Diego, CA), or an anti-RAGE extracellular domain antibody (N16; Santa Cruz Biotechnology, Santa Cruz, CA) where required. Neutralization of culture medium HMGB1 using an anti-HMGB1 antibody and neutralization of RAGE in TE671/RAGE cells were performed as described previously.11Sorci G Riuzzi F Arcuri C Giambanco I Donato R Amphoterin stimulates myogenesis and counteracts the antimyogenic factors basic fibroblast growth factor and S100B via RAGE binding.Mol Cell Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar Transient transfections were performed using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. In brief, cells cultured in 5% FBS without antibiotics were transfected with expression plasmids MKK6AA (an inactive mutant of the p38 MAPK upstream kinase MKK6),35Wang X McGowan CH Zhao M He L Downey JS Fearns C Wang Y Huang S Han J Involvement of the MKK6–p38γ cas-cade in γ-radiation-induced cell cycle arrest.Mol Cell Biol. 2000; 20: 4543-4552Crossref PubMed Scopus (238) Google Scholar N17Rac1 or N17Cdc42 (inactive forms of Rac1 and Cdc42, respectively),36Caron E Hall A Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases.Science. 1998; 282: 1717-1721Crossref PubMed Scopus (805) Google Scholar MKK6EE (a constitutively active form of MKK6),37Raingeaud J Whitmarsh AJ Barrett T Derijard B Davis RJ MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway.Mol Cell Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1147) Google Scholar or empty vector and with muscle creatine kinase (MCK)-luc reporter gene,38Vincent CK Gualberto A Patel CV Walsh K Different regulatory sequences control creatine kinase-M gene expression in directly injected skeletal and cardiac muscle.Mol Cell Biol. 1993; 13: 1264-1272Crossref PubMed Scopus (87) Google Scholar myogenin-luc reporter gene,39Gerber AN Klesert TR Bergstrom DA Tapscott SJ Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis.Genes Dev. 1997; 11: 436-450Crossref PubMed Scopus (233) Google Scholar or p21WAF1-luc reporter gene.40el-Deiry WS Tokino T Velculescu VE Levy DB Parsons R Trent JM Lin D Mercer WE Kinzler KW Vogelstein B WAF1, a potential mediator of p53 tumor suppression.Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7936) Google Scholar After 6 hours in the cases of MCK, myogenin, and p21WAF1 and after 24 hours in the cases of MKK6AA, MKK6EE, N17Rac1, and N17Cdc42, the cells were washed with DMEM and cultivated in 2% FBS. After another 24 hours, the cells were harvested to measure luciferase activity. Transient transfection of TE761/wt, TE671/RAGE, CCA, and RMZ-RC2 cells with RAGE or RAGEΔcyto was done as described for MKK6AA, MKK6EE, N17Rac1, or N17Cdc42. Where used, the p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA), the phosphatydilinositol-3-kinase (PI3-K) inhibitor LY294002 (Calbiochem), the mitogen-activated protein kinase kinase inhibitor PD98059 (Calbiochem), and the JNK inhibitor SP600125 (Alexis, Lausen, Switzerland) were used at a final concentration of 2, 10, 30, and 10 μmol/L, respectively, in dimethylsulfoxide. For reverse transcriptase-polymerase chain reaction (RT-PCR), TE671/RAGE, TE671/RAGEΔcyto, TE671/wt, CCA, and RMZ-RC2 cells were processed as described previously.28Riuzzi F Sorci G Donato R The amphoterin/RAGE pair modulates myoblast proliferation, apoptosis, adhesiveness, migration and invasiveness: functional inactivation of RAGE in L6 myoblasts results in tumor formation in vivo.J Biol Chem. 2006; 281: 8242-8253Crossref PubMed Scopus (107) Google Scholar Immunofluorescence was performed as using a polyclonal anti-RAGE antibody (1:25, N16; Santa Cruz Biotechnology), and myosin heavy chain (MHC) was detected by immunocytochemistry, as described previously.11Sorci G Riuzzi F Arcuri C Giambanco I Donato R Amphoterin stimulates myogenesis and counteracts the antimyogenic factors basic fibroblast growth factor and S100B via RAGE binding.Mol Cell Biol. 2004; 24: 4880-4894Crossref PubMed Scopus (109) Google Scholar For detection of F-actin, TE671 cells were fixed for 10 minutes in 3.7% formaldehyde in phosphate-buffered saline (PBS), extensively washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 2 minutes, washed again, and incubated with fluorescein isothiocyanate-phalloidin (Sigma, St. Louis, MO) (1:250 in PBS) for 1 hour in a humid chamber at room temperature. After three washes in PBS, the cells were mounted in mounting medium (Meridian Bioscience Inc., Cincinnati, OH) and viewed on a DM Rb fluorescence microscope equipped with a digital camera (Leica, Wetzlar, Germany). To detect RAGE, RAGEΔcyto, myogenin, MHC, cyclin D1, tubulin, phosphorylated and total p38 MAPK, phosphorylated and total Akt, phosphorylated and total ERK1/2, phosphorylated and total retinoblastoma suppressor protein (Rb), and phosphorylated JNK by Western blotting, cells were cultivated as detailed in the figure legends, washed twice with PBS, and solubilized with 2.5% sodium dodecyl sulfate, 10 mmol/L Tris-HCl, pH 7.4, 0.1 mol/L dithiothreitol, and 0.1 mmol/L N-tosyl-l-phenylalanine chloromethyl ketoneprotease inhibitor (Roche, Basel, Switzerland). The following antibodies were used: monoclonal anti-developmental MHC antibody (1:300; Biogenesis, Poole, Dorset, UK), monoclonal anti-myogenin antibody (1:1000; PharMingen), monoclonal anti-α-tubulin antibody (1:10,000; Sigma), polyclonal anti-RAGE extracellular domain (1:2000; Santa Cruz Biotechnology), monoclonal anti-RAGE antibody (1:2000; Chemicon International, Temecula, CA), polyclonal anti-phosphorylated (Thr180/Tyr182) p38 MAPK (1:1000; New England BioLabs, Ipswich, MA), polyclonal anti-phosphorylated (Ser473) Akt (1:1000; New England BioLabs), polyclonal anti-p38 MAPK antibody (1:2000; New England BioLabs), polyclonal anti-Akt antibody (1:2000; New England BioLabs), polyclonal anti-phosphorylated (Thr202/Tyr204) ERK1/2 (1:2000; New England BioLabs), polyclonal anti-ERK1/2 antibody (1:20,000; Sigma), polyclonal anti-phosphorylated (Ser-807/811) Rb antibody (1:1000; Cell Signaling Technology, Danvers, MA), polyclonal anti-Rb antibody (1:1000; Cell Signaling Technology), polyclonal anti-phosphorylated JNK (Thr183/Tyr185) antibody (1:1000; Cell Signaling Technology), and monoclonal anti-cyclin D1 antibody (1:200; Santa Cruz Biotechnology). The immune reaction was developed by ECL [SuperSignal West Femto Maximum (Pierce, Rockford, IL) for RAGE; SuperSignal West Pico (Pierce) for all other antigens]. To characterize basal release of HMGB1 from mouse C2C12 myoblasts and TE671/wt, TE671/RAGE, TE671RAGEΔcyto, CCA, and RMZ-RC2 RMS cells, confluent or near-confluent cells were transferred to serum-free medium for 24 hours. The next steps were as described previously.41Charoonpatrapong K Shah R Robling AG Alvarez M Clapp DW Chen S Kopp RP Pavalko FM Yu J Bidwell JP HMGB1 expression and release by bone cells.J Cell Physiol. 2006; 207: 480-490Crossref PubMed Scopus (61) Google Scholar In brief, individual culture media were clarified by centrifugation, added with 1/100 volume of 2% sodium deoxycholate and subjected to precipitation with 1/10 volume of 100% trichloroacetic acid. The resultant pellets were resuspended in sodium dodecyl sulfate buffer and titrated with 1 N NaOH to obtain the normal blue color of the sample buffer, boiled for 5 minutes, and subjected to Western blotting using an anti-HMGB1 antibody (BD PharMingen). Purified HMGB1 was used as a marker. HMGB1 in culture media was measured semiquantitatively by densitometry relative to known amounts of purified HMGB1. HMGB1 was also detected by Western blotting in lysates from cells, and its levels were normalized to tubulin. Culture media were also analyzed for levels of LDH to document the contribution of cell necrosis to released HMGB1. Under the present experimental conditions, negligible amounts of LDH were measured, pointing to absence of significant cell necrosis (data not shown). To measure [3H]thymidine incorporation, TE671 cells (25 × 103 cells/well) were cultivated in 10% FBS for 24 hours in 24-multiwell plates, washed with DMEM, serum-starved for 24 hours, washed with DMEM, and cultivated in DMEM for 24 hours in the presence of 1 μCi of [3H]thymidine/ml. Parallel TE671 cells treated in the same manner in the absence of [3H]thymidine were used to count cell numbers. For cell number measurements, TE671 cells were plated in 96-multiwell plates at a density of 4 × 103 cells/well and processed by a tetrazolium-based [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] colorimetric assay. To measure apoptosis and analyze cell cycle, TE671 cells were seeded onto 35-mm plastic dishes (18 × 104 cells/dish) for 24 hours, washed with DMEM, and cultivated for 72 hours in DMEM containing varying concentrations of FBS (see figure legends). Cells were stained with propidium iodide and subjected to fluorescence-activated cell sorting (FACS).42Nicoletti I Migliorati G Pagliacci MC Grignani F Riccardi C A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.J Immunol Methods. 1991; 139: 271-279Crossref PubMed Scopus (4426) Google Scholar FACS analysis was also used to measure cell size.43Ahmadzadeh M Hussain SF Faber DL Effector CD4 T cells are biochemically distinct from the memory subset: evidence for long-term persistence of effectors in vivo.J Immunol. 1999; 163: 3053-3063PubMed Google Scholar For adhesion experiments, TE671/wt, TE671/RAGE, and TE671/RAGEΔcyto cells (3 × 104 cells in 0.1 ml of DMEM containing 10% FBS) were seeded in 96-multiwell plates and incubated for 3 hours. The supernatant with nonadherent cells was removed by two washes with warmed culture medium. Attached cells were fixed with 30% methanol/ethanol for 15 minutes at room temperature, stained with 0.1% crystal violet (Sigma) in PBS, extensively washed with distilled water, and dried at room temperature. The dye was resuspended with 50 μl of 0.2% Triton X-100/well, and color yield was measured using an enzyme-linked immunosorbent assay reader at 590 nm. For migration assay, we used Boyden chambers (pore size, 8 μm) (BD Biosciences). Individual TE671 clones and CCA and RMZ-RC2 cells (5 × 104 cells in 0.5 ml of DMEM) were placed in the upper chamber, and 0.75 ml of DMEM containing 10% FBS was placed in the lower chamber. After 20 hours in culture, cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were fixed in methanol for 2 minutes and stained with 0.05% crystal violet in PBS for 15 minutes. Cells on the underside of the filters were viewed and counted under a microscope. For invasion assay, conditions were as described for migration assay except that BioCoat Matrigel invasion chambers (pore size, 8 μm) (BD Biosciences) were used. For tumor growth in vivo, female (nonobese diabetic/severe combined immunodeficient) mice weighing ∼20 g were inoculated subcutaneously with 4 × 106 TE671/wt, TE671/RAGEΔcyto, or TE671/RAGE cells and monitored for 4 to 6 weeks. The mice were sacrificed by cervical dislocation. Consent was obtained by the Ethics Committee of the University of Perugia. Tumor masses were excised and weighed, and tumor volume was calculated by the equation: tumor volume = x2y/2, where x and y correspond to the width and thickness of the tissue, respectively. Tumors were then fixed with 4% paraformaldehyde in PBS (2 days at 4°C), extensively washed in PBS, and paraffin-embedded. Sections were either stained with hematoxylin or subjected to immunohistochemistry using an anti-Ki-67 antibody (anti-MIB1, 1:10; Dako, Carpinteria, CA) to determine semiquantitatively cell proliferation. Before fixation, samples of individual tumors were subjected to RT-PCR to confirm lack of expression of RAGE in tumors arising from injected TE671/wt cells and expression of RAGEΔcyto and RAGE in tumors arising from injected TE671/RAGEΔcyto cells and TE671/RAGE cells, respectively (data not shown). Histopathology was performed by an independent pathologist. Each experiment was repeated at least three times. Representative experiments are depicted in the figures unless stated otherwise. The data were subjected to analysis of variance wi

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