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• W2092804562 abstract "The integrin αvβ6 is a fibronectin receptor whose expression is not detectable on normal oral epithelium but is increased significantly in healing and in oral epithelial dysplasia and oral squamous cell carcinoma, suggesting it may promote changes associated with tumor development. To study whether αvβ6 may drive invasive behavior we have used transfection and retroviral infection to create a panel of epithelial cell lines expressing various levels of αvβ6. We report that increased expression of αvβ6 in malignant keratinocytes promotes invasion and leads to an increased capacity for migration towards fibronectin. αvβ6 expression may have a significant role in contributing to the malignant behavior of epithelial cells. The integrin αvβ6 is a fibronectin receptor whose expression is not detectable on normal oral epithelium but is increased significantly in healing and in oral epithelial dysplasia and oral squamous cell carcinoma, suggesting it may promote changes associated with tumor development. To study whether αvβ6 may drive invasive behavior we have used transfection and retroviral infection to create a panel of epithelial cell lines expressing various levels of αvβ6. We report that increased expression of αvβ6 in malignant keratinocytes promotes invasion and leads to an increased capacity for migration towards fibronectin. αvβ6 expression may have a significant role in contributing to the malignant behavior of epithelial cells. Increased epithelial mobility and migration play a fundamental role in normal and pathologic processes such as development, wound healing, and malignant transformation. Most dramatically this is seen in carcinogenesis, where epithelial cells cross the basement membrane and proliferate and migrate within the connective tissues. The process of invasion involves altered cell-cell and cell–matrix interactions, loss of growth control, and acquisition of the ability to degrade and invade stromal tissues. Frequently these changes are accompanied by altered expression of cell adhesion molecules, of which the integrins are the most important extracellular matrix (ECM) receptors (Juliano and Varner, 1993Juliano R.L. Varner J.A. Adhesion molecules in cancer: the role of integrins.Curr Opin Cell Biol. 1993; 5: 812-818Crossref PubMed Scopus (256) Google Scholar;Giancotti and Mainiero, 1994Giancotti F.G. Mainiero F. Integrin-mediated adhesion and signalling in tumorigenesis.Biochim Biophys Acta. 1994; 1198: 47-64PubMed Google Scholar;Ben-Ze'Ev, 1997Ben-Ze'Ev A. Cytoskeletal and adhesion proteins as tumor suppressors.Curr Opin Cell Biol. 1997; 9: 99-108Crossref PubMed Scopus (163) Google Scholar). Worldwide, oral squamous cell carcinoma (SCC) is the sixth most common cancer, representing about 5.5% of all malignancies (Parkin et al., 1993Parkin D.M. Pisani P. Ferlay J. Estimates of the worldwide incidence of eighteen major cancers in 1985.Int J Cancer. 1993; 54: 594-606Crossref PubMed Scopus (1589) Google Scholar). In the U.K. there are over 3000 new cases each year with a mortality rate that has remained at over 50% for decades and is not improving (Johnson and Warnakulasuriya, 1993Johnson N.W. Warnakulasuriya K.A.A.S. Epidemiology and aetiology of oral cancer in the United Kingdom.Comm Dent Health. 1993; 10: 12-29Google Scholar;Hindle et al., 1996Hindle I. Downer M.C. Speight P.M. The epidemiology of oral cancer.Br J Oral Maxillofac Surg. 1996; 34: 471-476Abstract Full Text PDF PubMed Scopus (81) Google Scholar). One integrin heterodimer, αvβ6, is not expressed in normal epithelium but is consistently found in oral epithelial dysplasia and oral SCC, suggesting it may play a role in the development and progression of such tumors. Integrins are transmembrane cell surface receptors, composed of noncovalently linked heterodimers of α and β chains (Hynes, 1992Hynes R.O. Integrins versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9002) Google Scholar;Sonnenberg, 1993Sonnenberg A. Integrins and their ligands.Curr Top Microbiol Immunol. 1993; 184: 7-35Crossref PubMed Scopus (176) Google Scholar). Integrins mediate adhesion to the ECM and function as coordinators of signaling pathways that regulate a diverse range of cell functions including motility, proliferation, differentiation, and apoptosis (Hynes, 1992Hynes R.O. Integrins versatility, modulation, and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9002) Google Scholar;Sonnenberg, 1993Sonnenberg A. Integrins and their ligands.Curr Top Microbiol Immunol. 1993; 184: 7-35Crossref PubMed Scopus (176) Google Scholar;Frisch and Francis, 1994Frisch S.M. Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis.J Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2768) Google Scholar;Clark and Brugge, 1995Clark E.A. Brugge J.S. Integrins and signal transduction pathways: the road taken.Science. 1995; 268: 233-239Crossref PubMed Scopus (2812) Google Scholar;Yamada, 1997Yamada K.M. Integrin signaling.Matrix Biol. 1997; 16: 137-141Crossref PubMed Scopus (89) Google Scholar). These intracellular signals are generated as a result of ligand binding and in this way cells may respond to ECM-specific interactions (Damsky and Werb, 1992Damsky C.H. Werb Z. Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information.Curr Opin Cell Biol. 1992; 4: 772-781Crossref PubMed Scopus (487) Google Scholar). Different integrins generate different signals and some of these may have profound effects on the biologic behavior of tumors. De novo expression of αvβ6 is seen in a number of epithelial malignancies, particularly oral SCC, which may be correlated with a downregulation of αvβ5 (Agrez et al., 1996Agrez M.V. Bates R.C. Mitchell D. Wilson N. Ferguson N. Anseline P. Sheppard D. Multiplicity of fibronectin-binding alpha V integrin receptors in colorectal cancer.Br J Cancer. 1996; 73: 887-892Crossref PubMed Scopus (37) Google Scholar;Jones et al., 1997Jones J. Watt F.M. Speight P.M. Changes in the expression of αv integrins in oral squamous cell carcinoma.J Oral Pathol Med. 1997; 26: 63-68Crossref PubMed Scopus (113) Google Scholar). More recently,Hamidi et al., 2000Hamidi S. Salo T. Kainulainen T. Epstein J. Lerner K. Larjava H. Expression of alpha (v) beta 6 integrin in oral leukoplakia.Br J Cancer. 2000; 82: 1433-1440Crossref PubMed Scopus (81) Google Scholar found that αvβ6 was expressed in a high percentage of oral epithelial dysplasias where it correlated with disease progression, suggesting that it may promote transition to a malignant phenotype. Intriguingly, increased αvβ6 expression also is seen on wound keratinocytes suggesting that αvβ6 normally plays a role in tissue repair and/or remodeling (Haapasalmi et al., 1996Haapasalmi K. Zhang K. Tonnesen M. et al.Keratinocytes in human wounds express αvβ6 integrin.J Invest Dermatol. 1996; 106: 1-7Crossref Scopus (137) Google Scholar). To address the potential role of αvβ6 in oral SCC, we have examined the functional significance of high αvβ6 expression using a range of SCC cell lines, which have been created by transfection and retroviral infection. We report that increased expression of αvβ6 promotes invasion and leads to an increased capacity for migration towards fibronectin, perhaps suggesting that αvβ6 may have a role in tumor progression and may enhance the aggressive behavior of carcinoma. Eight monoclonal antibodies (all of murine origin unless stated) were used in this study. L230 (antihuman αv) was prepared in our laboratory from hybridoma cells obtained from the American Type Culture Collection (Rockville, MD) (Weinacker et al., 1994Weinacker A. Chen A. Agrez M. et al.Role of the integrin alpha v beta 6 in cell attachment to fibronectin. Heterologous expression of intact and secreted forms of the receptor.J Biol Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar). E7P6 and R6G9 against the β6 subunit (Weinacker et al., 1994Weinacker A. Chen A. Agrez M. et al.Role of the integrin alpha v beta 6 in cell attachment to fibronectin. Heterologous expression of intact and secreted forms of the receptor.J Biol Chem. 1994; 269: 6940-6948Abstract Full Text PDF PubMed Google Scholar) and 10D5 against αvβ6 (Huang et al., 1998Huang X. Wu J. Spong S. Sheppard D. The integrin αvβ6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin.J Cell Sci. 1998; 111: 2189-2195Crossref PubMed Google Scholar) were prepared in the laboratories of Dean Sheppard. P1F6 against αvβ5 was obtained from Life Technologies, Paisley, U.K. P1D6 against α5β1 was purchased from Chemicon International (Harrow, U.K.). Fluorescein isothiocyanate (FITC) conjugated rabbit antimouse antibodies were purchased from Dako (High Wycombe, U.K.). Geneticin (neomycin analog G418), puromycin, type I collagen, cellular fibronectin, and bovine serum albumin (BSA) were purchased from Sigma Chemical (Poole, Dorset, U.K.). Magnetic beads (Dynabeads) conjugated to antimouse antibody were purchased from Dynal (Merseyside, U.K.). Matrigel was purchased from Becton Dickinson (Oxford, U.K.). We created a panel of cell lines expressing various levels of αvβ6. The V3 cell line was generated from an αv-negative oral SCC line (H357;Prime et al., 1990Prime S.S. Nixon S.V.R. Crane I.J. et al.The behaviour of human oral squamous cell carcinoma in culture.J Pathol. 1990; 160: 259-269Crossref PubMed Scopus (154) Google Scholar;Sugiyama et al., 1993Sugiyama M. Speight P.M. Prime S.S. Watt F.M. Comparison of integrin expression and terminal differentiation capacity in cell lines derived from oral squamous cell carcinoma.Carcinogenesis. 1993; 14: 2171-2176Crossref PubMed Scopus (55) Google Scholar) by transfection of αv cDNA (Jones et al., 1996Jones J. Sugiyama M. Speight P.M. Watt F.M. Restoration of αvß5 integrin in neoplastic keratinocytes results in increased capacity for terminal differentiation and suppression of anchorage independent growth.Oncogene. 1996; 12: 119-126PubMed Google Scholar). V3 cells predominantly express the αvβ5 heterodimer. Cells were grown in standard keratinocyte growth medium (KGM) as described previously (Sugiyama et al., 1993Sugiyama M. Speight P.M. Prime S.S. Watt F.M. Comparison of integrin expression and terminal differentiation capacity in cell lines derived from oral squamous cell carcinoma.Carcinogenesis. 1993; 14: 2171-2176Crossref PubMed Scopus (55) Google Scholar;Jones et al., 1996Jones J. Sugiyama M. Speight P.M. Watt F.M. Restoration of αvß5 integrin in neoplastic keratinocytes results in increased capacity for terminal differentiation and suppression of anchorage independent growth.Oncogene. 1996; 12: 119-126PubMed Google Scholar). KGM comprised α modified Eagle's medium (α-MEM) containing 10% fetal bovine serum (FBS; Globepharm, Surrey, U.K.) supplemented with 100 IU per l penicillin, 100 µg per l streptomycin, 2.5 µg per l amphotericin B (Gibco BRL), 1.8 × 10–4 M adenine, 5 µg per ml insulin, 1 × 10–10 M cholera toxin, 0.5 µg per ml hydrocortisone, and 10 ng per ml epidermal growth factor (Sigma). G418 was added to the transfected cells during routine culture (2 mg per ml) but removed for experimental procedures. All cells were tested routinely for mycoplasma. β6 cDNA was polymerase chain reaction subcloned from the plasmid pcDNA1neoβ6, containing the complete cDNA sequence of the human β6 subunit, into Pcr2.1 (In Vitrogen), from where it was excised with EcoR1 and ligated into the EcoR1 site of the retroviral plasmid pBabe puro (Morgenstern and Land, 1990Morgenstern J.P. Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.Nucl Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1898) Google Scholar). Maxiprep DNA of pBabe puro/β6 and also (as control) pBabe puro was transfected into the AM12 amphotrophic retroviral packaging cell line, using Promega Tfx transfection kit. Transfected cells were selected in puromycin (1.75 µg per ml). Prior to harvesting retroviruses, puromycin-free growth medium was added to the AM12-puro and AM12-puroβ6 cells for 24 h. To retrovirus-containing medium, hexadimethrine bromide (Sigma) was added (4 µg per ml) and the mixture was filtered through a 0.45 µm sterile filter. Target cells (V3) were exposed to the filtered, retrovirus-containing conditioned medium for 16–20 h. Medium was replaced with fresh for 24 h before selecting cells by adding puromycin (0.75 µg per ml). Resistant cells expressing β6 were evident 3–4 d after puromycin selection. As controls, null-transfectants (C1 cells) were created by infection with the retroviral vector (pBabe puro) alone. A population of β6-expressing cells were selected by two rounds of magnetic bead sorting according to manufacturer's instructions (Dynabeads, Dynal) using mouse anti-β6 antibody E7P6. These cells were called VB6. Subconfluent cells were washed twice with phosphate-buffered saline (PBS) and harvested by trypsin/ethylene diamine tetraacetic acid (EDTA) (0.25% wt:vol, 5 mM). Cells were washed once in PBS containing 10% FBS. Cells were incubated with primary antibody for 40 min at 4°C and washed twice with PBS. FITC-conjugated secondary antibody was applied to the cells for 30 min at 4°C. Briefly, cells were washed twice with PBS and resuspended in 0.5 ml PBS with 10% FBS. Labelled cells were scanned on a FACSCalibur cytometer (Becton Dickinson) and analyzed using Cellquest software, acquiring 1 × 104 events. Cells were surface-iodinated with [125I] using the lactoperoxidase method as described previously (Marshall et al., 1991Marshall J.F. Nesbitt S.A. Helfrich M.H. Horton M.A. Polakova K. Hart I.R. Integrin expression in human melanoma cell lines: heterogenicity of vitronectin receptor composition and function.Int J Cancer. 1991; 49: 924-931Crossref PubMed Scopus (75) Google Scholar). Cells were washed in cold PBS supplemented with 1 mM Ca2+ and 0.5 mM Mg2+ and lyzed with Nonidet P-40 lysis buffer (20 mM HEPES, 1% Nonidet P-40, 50 mM NaCl, 1 mM CaCl2, 3 mM MgCl2, 0.3 M sucrose, 0.1% sodium azide), supplemented with protease inhibitors (leupeptin 100 µg per ml, phenylmethylsulfonyl fluoride 100 µg per ml, aprotinin 100 µg per ml). Lysates were cleared by centrifugation at 13,000g for 10 min. Protein-incorporated 125I was determined by trichloroacetic acid (TCA) precipitation of 2 µl samples of lysates which were then diluted with lysis buffer to give equal TCA-precipitable [125I] per unit volume. To equal volumes of lysate was added TS2/16 (anti-β1, 6 µg), L230 (anti-αv, 9 µg), or P2W7 (anti-αv, 10 µg), followed by rabbit antimouse IgG (Dako Z259). Protein A-sepharose (50 µl of 1:1 suspension in lysis buffer; Amersham Pharmacia Biotech, Little Chalfont, U.K.) was added and samples were tumbled overnight at 4°C. Precipitated complexes were boiled for 10 min in nonreducing buffer (0.5 M Tris-HCl pH 6.8, 10% sodium dodecyl sulfate, 10% glycerol, 0.4% bromophenol blue), and separated on 10% acrylamide gels. Gels were fixed and dried before exposure to film (Kodak XAR-5) at -80°C for up to 6 d. Ninety-six-well plates (Falcon 3912; Becton Dickinson) were coated with plasma fibronectin (Sigma). A 50 µl solution at a concentration of 10 µg per ml (fibronectin) was added to the wells and incubated at 37°C for 1 h. After incubation wells were washed with PBS and then blocked with 0.1% BSA at 37°C for 30 min. Control wells were incubated with 0.1% BSA. Cells were chromium [51Cr] labeled (Brunner et al., 1976Brunner K.T. Enger H.D. Cerottini J.-C. The 51Cr-release assay as used for the quantitative measurement of cell-mediated cytolysis in vitro.in: Bloom B.R. David J.H.R. In Vitro Methods in Cell-Mediated and Tumour Immunity. Academic Press, New York1976: 423-436Google Scholar), washed, and resuspended in α-MEM (1.5 × 104 cells per well). For blocking experiments, cells were incubated with specific antibodies (as described in Results) for 10 min on ice in each well. Plates were incubated at 37°C for 1 h. Non-adherent cells were removed by flooding plates with PBS (supplemented with 1 mM CaCl2 and 0.5 mM MgCl2). After two washes, the plates were cut into individual wells and the radioactivity associated with each well was determined in a gamma counter (1261 Multigamma; LKB Wallac, Bromma, Sweden). The percent adhesion was expressed as the adherent cell radioactivity as a proportion of the total cell input. The nonspecific adhesion (attachment to wells coated with BSA) was subtracted. Experiments were repeated on three occasions in quadruplicate, with similar results. 2 ×104 cells were plated onto 13 mm glass coverslips coated with fibronectin (10 µg per ml) and blocked with 0.1% BSA in PBS. Coverslips were incubated for 6, 9, and 24 h at 37°C in 5% CO2. Cells were rinsed twice in PBS, fixed for 15 min in 10% formalin, and permeabilized with 0.1% Triton X-100 for 10 min, followed by incubation for 60 min in PBS containing 0.1% BSA. L230 (18 µg per ml) or R6G9 (1:10 dilution of supernatant:wash buffer) diluted in PBS containing 0.1% BSA with 0.1% azide was added for 60 min at 4°C. Bound antibody was detected with FITC-conjugated rabbit antimouse secondary antibody (Dako; 1:40). Actin was visualized with TRITC-conjugated phalloidin (Sigma: 5 ng per ml). Coverslips were washed three times for 5 min in wash buffer and mounted with MOWIOL 4–88 (Novabiochem, Nottingham, U.K.: 0.1 g per ml of Citifluor mounting medium) and viewed with a confocal laser scanning microscope (Zeiss LSM510; Welwyn Garden City, U.K.). Cells were grown on uncoated or matrigel-coated six-well plates for 160 h. Prior to plating, wells were coated with matrigel (1:20 dilution in α-MEM) for 1 h at 37°C and blocked with α-MEM containing 0.5% BSA for a further 30 min. Cells were also grown within matrigel gels (1:2 dilution in α-MEM). 2 × 104 cells were plated into each well and fed every 3 d. Cells were removed from triplicate wells by trypsinization or gel digestion with Matrisperse (Becton Dickinson, Bedford, U.K.) and counted on a Casy 1 counter (Sharfe System, Germany). Readings were taken every day until day 10. Experiments were repeated a minimum of four times in triplicate. Haptotactic cell migration assays were performed using matrix-coated polycarbonate filters (8 µm pore size, Transwell; Becton Dickinson). The membrane undersurface was coated with fibronectin (10 µg per ml) or collagen I (50 µg per ml) in PBS for 1 h at 37°C and blocked with migration buffer (0.5% BSA in α-MEM) for 30 min at 37°C. For blocking experiments, cells were incubated with antibody for 30 min at 4°C prior to seeding. The lower chamber was filled with 500 µl of migration buffer, following which cells were plated in the upper chamber of triplicate wells, at a density of 1 × 105 in 100 µl of migration buffer, and incubated at 37°C for 3 h. Following incubation, Transwell inserts were fixed in 10% formalin, stained with 0.5% crystal violet in 10% ethanol for 10 min, and washed. Cells in the upper compartment were removed using a cottonwool swab and the filter was mounted in DPX on a microscope slide. Cells, which had migrated to the lower surface of the filter, were counted by microscopy, using multiple random high-powered fields (at least six fields per filter as determined by cumulative frequency analysis). Between 200 and 800 cells were counted routinely per filter. The experiments were repeated four times in triplicate. Cell invasion assays were performed using matrigel-coated polycarbonate filters (8 µm pore size, Transwell; Becton Dickinson). Matrigel (70 µl; 1:2 dilution in α-MEM) was added to the upper membrane and allowed to gel for 1 h at 37°C. For blocking experiments, cells were incubated with anti-integrin antibody for 30 min at 4°C prior to seeding. Additional antibody was added to the assay at 24 h intervals. To act as a chemoattractant, 500 µl of KGM was placed in the lower chamber. Cells were plated in the upper chamber of quadruplicate wells at a density of 5 × 104 in 200 µl of α-MEM and incubated at 37°C for 72 h. The cells in the lower chamber (including those attached to the undersurface of the membrane) were then trypsinized and counted on a Casy 1 counter (Sharfe System). Experiments were repeated six times in quadruplicate. Adhesion assays on matrigel were also carried out at this time to ensure that any reduction of invasion produced by anti-integrin blocking antibodies was not simply due to inhibition of initial cell attachment. Data are expressed as the mean ±SD of a given number of observations. Where appropriate, one way analysis of variance was used to compare multiple groups. Comparisons between groups were by Tukey's pairwise comparison (set at 5% significance). A p-value of <0.05 was considered to be significant. V3 cells were infected with a retrovirus containing β6 cDNA to create VB6. Flow cytometry confirmed that the C1 null transfectants showed unaltered αv (or other integrin) expression compared with the V3 parental cells Table I. Although a low level of αvβ6 was expressed in C1 and V3 cells, the predominant αv-containing heterodimer was αvβ5, which we have shown previously to be a functional vitronectin receptor (Jones et al., 1996Jones J. Sugiyama M. Speight P.M. Watt F.M. Restoration of αvß5 integrin in neoplastic keratinocytes results in increased capacity for terminal differentiation and suppression of anchorage independent growth.Oncogene. 1996; 12: 119-126PubMed Google Scholar). In contrast, αvβ6 expression on VB6 cells showed a 100-fold increase compared with the V3 and C1 cells Table I. The level of αvβ5 expression in VB6 cells remained unaltered, as did the levels of the fibronectin receptor α5β1 and other integrins Table I. Fluorescence-activated cell sorter analysis also confirmed that these cells do not express αvβ3 Table I.Table IFlow cytometric analysis of keratinocyte cell lines derived from oral SCCaCell lines H357, V3, VB6, and C1 were detached by trypsin/EDTA and immunolabeled with antibodies to α1 (TS2/7), α2 (P1E6), α3 (P1B5), α4 (7.2), α5 (P1D6), α6 (GOH3), β1 (TS2/16), β4 (3E1), αv (L230), αvβ3 (LM609), αvβ5 (P1F6), or αvβ6 (E7P6). Bound antibody was detected by FITC-conjugated rabbit antimouse antisera. Negative control had secondary antibody only and has been subtracted from the results. The table shows a representative experiment (ND = not detected). Flow cytometry confirmed high αvβ6 in the VB6 cells and showed that V3 and C1 cells express predominantly αvβ5. The null transfectant control C1 cells are the same as the V3 parentals confirming that the transfection and infection processes had not altered levels of other integrins expressed by the cells. The integrin subunits α1, α4, and the αvβ3 heterodimer were not detectable in the cell lines. The αv subunit or αv heterodimers were not detectable in the original H357 cells.α1α2α3α4α5α6αvβ1β4αvβ3αvβ5αvβ6H357ND142.4181.8ND8.7581.0ND270.1199.0NDNDNDV3ND170.5211.3ND8.18662.312.6346.3201.0ND5.31.8VB6ND163.4165.5ND9.1689.7157.1383.2229.7ND6.8174.6C1ND146.4210.9ND10.9542.07.2338.1218.0ND2.42.5a Cell lines H357, V3, VB6, and C1 were detached by trypsin/EDTA and immunolabeled with antibodies to α1 (TS2/7), α2 (P1E6), α3 (P1B5), α4 (7.2), α5 (P1D6), α6 (GOH3), β1 (TS2/16), β4 (3E1), αv (L230), αvβ3 (LM609), αvβ5 (P1F6), or αvβ6 (E7P6). Bound antibody was detected by FITC-conjugated rabbit antimouse antisera. Negative control had secondary antibody only and has been subtracted from the results. The table shows a representative experiment (ND = not detected). Flow cytometry confirmed high αvβ6 in the VB6 cells and showed that V3 and C1 cells express predominantly αvβ5. The null transfectant control C1 cells are the same as the V3 parentals confirming that the transfection and infection processes had not altered levels of other integrins expressed by the cells. The integrin subunits α1, α4, and the αvβ3 heterodimer were not detectable in the cell lines. The αv subunit or αv heterodimers were not detectable in the original H357 cells. Open table in a new tab In order to determine whether V3, VB6, and C1 cells expressed αvβ1, another potential fibronectin receptor, cells were analyzed by immunoprecipitation. Fig 1 shows that immunoprecipitation with two different anti-αv antibodies failed to coprecipitate β1. As a comparison, β1 integrins were immunoprecipitated with a β1-specific antibody (TS2/16) and electrophoresed on the same gel. Thus the combined results from flow cytometry and immunoprecipitation show that the only αv integrins expressed by V3, C1, and VB6 cells are αvβ5 and αvβ6 and that VB6 cells express high levels of αvβ6. To confirm that αvβ6 in VB6 cells was functional, the cell lines were plated onto fibronectin in the presence or absence of blocking antibodies against the αv subunit (L230), αvβ6 (10D5), α5β1 (P1D6), or an irrelevant antibody against α6 (GOH3). Combinations of these antibodies were also used. Adhesion of C1 cells Fig 2a and V3 cells (data not shown) to fibronectin could be blocked using P1D6 alone. Maximal inhibition of VB6 adhesion to fibronectin, however, required a combination of 10D5 and P1D6 (or L230 and P1D6) indicating that αvβ6 is functional and that the VB6 cells bind to fibronectin using both α5β1 and αvβ6 Fig 2b. Immunofluorescence for β6 in VB6 cells showed that β6 colocalized with actin in focal adhesions. This was confirmed by colocalization of β6 with the focal adhesion protein talin (data not shown). The growth rates of the V3, VB6, and C1 lines were compared on uncoated versus matrigel-coated tissue culture plastic. Cell proliferation was also measured within matrigel gels. G418 and puromycin were removed from the medium for the duration of the experiment. αvβ6 had no effect on cell growth on uncoated or matrigel-coated tissue culture plastic, or within matrigel gels (data not shown).Fig 3 To determine whether αvβ6 is involved in keratino cyte migration, haptotactic migration assays were performed using fibronectin-coated Transwell filters. Over four separate experi ments, migration towards fibronectin was significantly increased in VB6 cells compared with either V3 (p <0.001) or C1 cells (p < 0.001). Fig 4(a) shows a representative experiment. No difference in migration was observed between the V3 parental cells and C1 null transfectants. The cell lines showed no significant differences when migrating towards collagen I, confirming that the differences in migration potential were specific for fibronectin Fig 4b. The migration towards fibronectin of V3 and C1 cells was inhibited by 79% and 68%, respectively, using antibodies against α5β1 (P1D6) Fig 4c, d but not by antibodies against the αv integrins (L230, P1F6, 10D5). In marked contrast, antibodies against αv (L230), αvβ6 (10D5), or α5β1 (P1D6) inhibited migration of the VB6 cells (19%, 52%, and 31%, respectively) Fig 4e. To block migration completely, however, a combination of 10D5 and PID6 (or L230 and P1D6) was necessary. Migration towards fibronectin in the presence of P1D6 (anti-α5β1) in VB6 cells shows that αvβ6 alone is capable of mediating migration towards fibronectin, although to a lesser extent than when both receptors are available. To determine whether αvβ6 is involved in cell invasion, cells resuspended in α-MEM were added to matrigel-coated Transwell filters and allowed to invade for 72 h toward KGM placed in the lower chamber. In six separate experiments, invasion consistently was increased significantly in VB6 cells compared with V3 (p =0.001) or C1 cells (p <0.001). This increased invasion was not due to differences in growth rates of cells in matrigel, which were similar for all the SCC cell lines (data not shown). No significant difference in invasion was observed between the V3 parental cell line and C1 null transfectants. Fig 5(a) shows a representative experiment. The invasion of V3 and C1 cells was inhibited by 61% and 65%, respectively, using antibodies against α5β1 (P1D6) Fig 5b, c but not by antibodies against the αv integrins (L230, P1F6, 10D5). In contrast, antibodies against αv (L230) and αvβ6 (10D5) dramatically inhibited invasion of VB6 cells (77% and 73%, respectively) whereas antibodies against α5 (P1D6) had no significant effect Fig 5d. Invasion of matrigel by all cell lines could be blocked with antibody against β1 (P4C10). Adhesion to matrigel in these lines also, however, is blocked completely by anti-β1 (data not shown) and it is probable that P4C10 inhibits cell attachment to matrigel rather than invasion (10D5 has no effect on VB6 adhesion to matrigel - data not shown). To date there are few data available on the biologic role of αvβ6 in oral SCC. As de novo expression of αvβ6 in oral epithelial dysplasia and SCC has been implicated in disease progression we tested whether induced increases in this heterodimer affected biologic behavior of transformed keratinocytes. Using transfection and retroviral infection we generated a series of SCC cell lines, derived from a single αv-negative precursor, which express varying levels of αvβ6. We show that high αvβ6 expression is associated with a more invasive and more migratory phenotype. As these processes are fundamental to epithelial malignancy this may explain, in part, how upregulation of αvβ6 in oral SCC (Breuss et al., 1995Breuss J.M. Gallo J. Delisser H.M. et al.Expression of the β6 subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling.J Cell Sci. 1995; 108: 2241-2251Crossref PubMed Google Scholar;Jones et al., 1997Jones J. Watt F.M. Speight P.M. Changes in the expression of αv integrins in oral squamous cell carcinoma.J Oral Pathol Med. 1997; 26: 63-68Crossref PubMed Scopus (113) Google Scholar) could" @default.
• W2092804562 created "2016-06-24" @default.
• W2092804562 creator A5002426532 @default.
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• W2092804562 date "2001-07-01" @default.
• W2092804562 modified "2023-10-11" @default.
• W2092804562 title "Expression of the αvβ6 Integrin Promotes Migration and Invasion in Squamous Carcinoma Cells" @default.
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