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• W79277247 abstract "Hepatocyte growth factor (HGF) plays important roles in tumor development and progression. It is currently thought that the main action of HGF is of a paracrine nature: HGF produced by mesenchymal cells acts on epithelial cells that express its receptor c-MET. In this investigation, we explored the significance of c-MET expression in myofibroblasts, both in culture and in patients with lung adenocarcinoma. We first showed that human myofibroblasts derived from primary lung cancer expressed c-MET mRNA and protein by reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of myofibroblasts was stimulated in a dose-dependent manner by exogenously added recombinant human HGF whereas it was inhibited in a dose-dependent manner by neutralizing antibody to HGF. The addition of HGF in the culture medium stimulated tyrosine phosphorylation of c-MET. The c-MET protein was immunohistochemically detected in myofibroblasts in the invasive area of lung adenocarcinoma. Finally, the prognostic significance of c-MET expression in stromal myofibroblasts was explored in patients with small-sized lung adenocarcinomas. c-MET-positive myofibroblasts were observed in 69 of 131 cases (53%). A significant relationship between myofibroblast c-MET expression and shortened patient survival was observed in a whole cohort of patients including all pathological stages (two-sided P = 0.0089 by log-rank test) and in patients with stage IA disease (two-sided P = 0.0019 by log-rank test). These data suggest that the HGF/c-MET system constitutes an autocrine activation loop in cancer-stromal myofibroblasts. This autocrine system may play a role in invasion and metastasis of lung adenocarcinoma. Hepatocyte growth factor (HGF) plays important roles in tumor development and progression. It is currently thought that the main action of HGF is of a paracrine nature: HGF produced by mesenchymal cells acts on epithelial cells that express its receptor c-MET. In this investigation, we explored the significance of c-MET expression in myofibroblasts, both in culture and in patients with lung adenocarcinoma. We first showed that human myofibroblasts derived from primary lung cancer expressed c-MET mRNA and protein by reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of myofibroblasts was stimulated in a dose-dependent manner by exogenously added recombinant human HGF whereas it was inhibited in a dose-dependent manner by neutralizing antibody to HGF. The addition of HGF in the culture medium stimulated tyrosine phosphorylation of c-MET. The c-MET protein was immunohistochemically detected in myofibroblasts in the invasive area of lung adenocarcinoma. Finally, the prognostic significance of c-MET expression in stromal myofibroblasts was explored in patients with small-sized lung adenocarcinomas. c-MET-positive myofibroblasts were observed in 69 of 131 cases (53%). A significant relationship between myofibroblast c-MET expression and shortened patient survival was observed in a whole cohort of patients including all pathological stages (two-sided P = 0.0089 by log-rank test) and in patients with stage IA disease (two-sided P = 0.0019 by log-rank test). These data suggest that the HGF/c-MET system constitutes an autocrine activation loop in cancer-stromal myofibroblasts. This autocrine system may play a role in invasion and metastasis of lung adenocarcinoma. Hepatocyte growth factor (HGF) is a heterodimeric polypeptide exhibiting pleiotropic biological functions as a mitogen, motogen, and morphogen.1Nakamura T Structure and function of hepatocyte growth factor.Prog Growth Factor Res. 1991; 3: 67-85Abstract Full Text PDF PubMed Scopus (295) Google Scholar HGF is secreted as an inactive single-chain glycoprotein, and inactive HGF is converted to its active form (mature HGF) by a serine protease HGF activator.2Gak E Taylor WG Chan AM Rubin JS Processing of hepatocyte growth factor to the heterodimeric form is required for biological activity.FEBS Lett. 1992; 311: 17-21Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 3Mizuno K Tanoue Y Okano I Harano T Takada K Nakamura T Purification and characterization of hepatocyte growth factor (HGF)-converting enzyme: activation of pro-HGF.Biochem Biophys Res Commun. 1994; 198: 1161-1169Crossref PubMed Scopus (38) Google Scholar, 4Jiang W Hiscox S Matsumoto K Nakamura T Hepatocyte growth factor/scatter factor, its molecular, cellular and clinical implications in cancer.Crit Rev Oncol Hematol. 1999; 29: 209-248Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar The mature HGF molecule is a heterodimer consisting of a 69-kd α-chain and a 34-kd β-chain.1Nakamura T Structure and function of hepatocyte growth factor.Prog Growth Factor Res. 1991; 3: 67-85Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 5Nakamura T Nishizawa T Hagiya M Seki T Shimonishi M Sugimura A Tashiro K Shimizu S Molecular cloning and expression of human hepatocyte growth factor.Nature. 1989; 342: 440-443Crossref PubMed Scopus (1982) Google Scholar The mRNA expression and protein synthesis/secretion of HGF have been detected primarily in a variety of mesenchymal cells including fibroblasts, smooth muscle cells, and endothelial cells.4Jiang W Hiscox S Matsumoto K Nakamura T Hepatocyte growth factor/scatter factor, its molecular, cellular and clinical implications in cancer.Crit Rev Oncol Hematol. 1999; 29: 209-248Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar Its receptor c-MET proto-oncogene product consists of an α-chain of 50 kd and a β-chain of 145 kd. The α-chain is exposed at the cell surface, whereas the β-chain spans the cell membrane and possesses an intracellular tyrosine kinase domain.6Park M Dean M Kaul K Braun MJ Gonda MA Vande Woude G Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors.Proc Natl Acad Sci USA. 1987; 84: 6379-6383Crossref PubMed Scopus (483) Google Scholar, 7Naldini L Weidner KM Vigna E Gaudino G Bardelli A Ponzetto C Narsimhan RP Hartmann G Zarnegar R Michalopoulos GK Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor.EMBO J. 1991; 10: 2867-2878Crossref PubMed Scopus (589) Google Scholar, 8Bottaro DP Rubin JS Faletto DL Chan AM Kmiecik TE Vande Woude GF Aaronson SA Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.Science. 1991; 251: 802-804Crossref PubMed Scopus (2090) Google Scholar, 9Giordano S Zhen Z Medico E Gaudino G Galimi F Comoglio PM Transfer of motogenic and invasive response to scatter factor/hepatocyte growth factor by transfection of human MET protooncogene.Proc Natl Acad Sci USA. 1993; 90: 649-653Crossref PubMed Scopus (144) Google Scholar The c-MET protein is expressed in epithelial cells of a variety of organs,10Sonnenberg E Meyer D Weidner KM Birchmeier C Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development.J Cell Biol. 1993; 123: 223-235Crossref PubMed Scopus (643) Google Scholar melanocytes,11Halaban R Rubin JS White W Met and HGF-SF in normal melanocytes and melanoma cells.EXS. 1993; 65: 329-339PubMed Google Scholar and nonepithelial cells including endothelial cells,12Bussolino F Di Renzo MF Ziche M Bocchietto E Olivero M Naldini L Gaudino G Tamagnone L Coffer A Comoglio PM Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.J Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1204) Google Scholar microglial cells,13Di Renzo MF Bertolotto A Olivero M Putzolu P Crepaldi T Schiffer D Pagni CA Comoglio PM Selective expression of the Met/HGF receptor in human central nervous system microglia.Oncogene. 1993; 8: 219-222PubMed Google Scholar, 14Yamada T Tsubouchi H Daikuhara Y Prat M Comoglio PM McGeer PL McGeer EG Immunohistochemistry with antibodies to hepatocyte growth factor and its receptor protein (c-MET) in human brain tissues.Brain Res. 1994; 637: 308-312Crossref PubMed Scopus (73) Google Scholar neurons,15Honda S Kagoshima M Wanaka A Tohyama M Matsumoto K Nakamura T Localization and functional coupling of HGF and c-Met/HGF receptor in rat brain: implication as neurotrophic factor.Brain Res Mol Brain Res. 1995; 32: 197-210Crossref PubMed Scopus (171) Google Scholar hepatic stellate cells,16Ikeda H Nagoshi S Ohno A Yanase M Maekawa H Fujiwara K Activated rat stellate cells express c-met and respond to hepatocyte growth factor to enhance transforming growth factor beta1 expression and DNA synthesis.Biochem Biophys Res Commun. 1998; 250: 769-775Crossref PubMed Scopus (37) Google Scholar and muscle cells.17Epstein JA Shapiro DN Cheng J Lam PY Maas RL Pax3 modulates expression of the c-Met receptor during limb muscle development.Proc Natl Acad Sci USA. 1996; 93: 4213-4218Crossref PubMed Scopus (316) Google Scholar Studies suggest a possible role of HGF/c-MET in tumor development and progression.18Weidner KM Behrens J Vandekerckhove J Birchmeier W Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells.J Cell Biol. 1990; 111: 2097-2108Crossref PubMed Scopus (580) Google Scholar, 19Rong S Bodescot M Blair D Dunn J Nakamura T Mizuno K Park M Chan A Aaronson S Vande Woude GF Tumorigenicity of the met proto-oncogene and the gene for hepatocyte growth factor.Mol Cell Biol. 1992; 12: 5152-5158Crossref PubMed Scopus (291) Google Scholar, 20Nakamura T Matsumoto K Kiritoshi A Tano Y Induction of hepatocyte growth factor in fibroblasts by tumor-derived factors affects invasive growth of tumor cells: in vitro analysis of tumor-stromal interactions.Cancer Res. 1997; 57: 3305-3313PubMed Google Scholar HGF stimulates the proliferation and migration of tumor cells in vitro. Transgenic mice that overexpress c-MET develop tumors.21Liang TJ Reid AE Xavier R Cardiff RD Wang TC Transgenic expression of tpr-met oncogene leads to development of mammary hyperplasia and tumors.J Clin Invest. 1996; 97: 2872-2877Crossref PubMed Scopus (89) Google Scholar HGF is expressed mainly by stromal myofibroblasts, whereas c-MET is expressed by cancer cells.4Jiang W Hiscox S Matsumoto K Nakamura T Hepatocyte growth factor/scatter factor, its molecular, cellular and clinical implications in cancer.Crit Rev Oncol Hematol. 1999; 29: 209-248Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar Although an HGF/c-MET autocrine loop has recently been reported in nonepithelial malignancies,22Ferracini R Di Renzo MF Scotlandi K Baldini N Olivero M Lollini P Cremona O Campanacci M Comoglio PM The Met/HGF receptor is over-expressed in human osteosarcomas and is activated by either a paracrine or an autocrine circuit.Oncogene. 1995; 10: 739-749PubMed Google Scholar, 23Harvey P Warn A Newman P Perry LJ Ball RY Warn RM Immunoreactivity for hepatocyte growth factor/scatter factor and its receptor, met, in human lung carcinomas and malignant mesotheliomas.J Pathol. 1996; 180: 389-394Crossref PubMed Scopus (71) Google Scholar, 24Borset M Hjorth-Hansen H Seidel C Sundan A Waage A Hepatocyte growth factor and its receptor c-met in multiple myeloma.Blood. 1996; 88: 3998-4004Crossref PubMed Google Scholar, 25Rao UN Sonmez-Alpan E Michalopoulos GK Hepatocyte growth factor and c-MET in benign and malignant peripheral nerve sheath tumors.Hum Pathol. 1997; 28: 1066-1070Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 26Laterra J Nam M Rosen E Rao JS Lamszus K Goldberg ID Johnston P Scatter factor/hepatocyte growth factor gene transfer enhances glioma growth and angiogenesis in vivo.Lab Invest. 1997; 76: 565-577PubMed Google Scholar the HGF/c-MET system is currently thought to function mainly in a paracrine manner. Of the various malignant tumors, lung carcinoma is the most common cause of cancer death in the world. The prognosis of the patients is primarily dependent on the stage of the disease. However, even in patients with pathological stage I non-small-cell lung cancer, the overall survival is 64.6% (range, 55 to 72%).27Nesbitt JC Putnam Jr, JB Walsh GL Roth JA Mountain CF Survival in early-stage non-small cell lung cancer.Ann Thorac Surg. 1995; 60: 466-472Abstract Full Text PDF PubMed Scopus (338) Google Scholar Approximately 30 to 40% of pathological stage I patients have disease recurrence and die after curative resection.28Strauss GM Kwiatkowski DJ Harpole DH Lynch TJ Skarin AT Sugarbaker DJ Molecular and pathologic markers in stage I non-small-cell carcinoma of the lung.J Clin Oncol. 1995; 13: 1265-1279PubMed Google Scholar It has been reported that tumor cells with thick fibrovascular stroma are more likely to metastasize in lung adenocarcinoma.29Nomori H Hirohashi S Noguchi M Matsuno Y Shimosato Y Tumor cell heterogeneity and subpopulations with metastatic ability in differentiated adenocarcinoma of the lung. Histologic and cytofluorometric DNA analyses.Chest. 1991; 99: 934-940Crossref PubMed Scopus (22) Google Scholar Similarly, in small-sized lung adenocarcinoma (maximum dimension 2 cm or less) the presence of active fibroblast proliferation correlates with poor prognosis.30Noguchi M Morikawa A Kawasaki M Matsuno Y Yamada T Hirohashi S Kondo H Shimosato Y Small adenocarcinoma of the lung. Histologic characteristics and prognosis.Cancer. 1995; 75: 2844-2852Crossref PubMed Scopus (1202) Google Scholar Active fibroblasts are also commonly referred to as myofibroblasts because they show morphological characteristics of both fibroblasts and smooth muscle cells.31Gabbiani G Ryan GB Majne G Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction.Experientia. 1971; 27: 549-550Crossref PubMed Scopus (1186) Google Scholar, 32Gabbiani G Hirschel BJ Ryan GB Statkov PR Majno G Granulation tissue as a contractile organ. A study of structure and function.J Exp Med. 1972; 135: 719-734Crossref PubMed Scopus (660) Google Scholar The presence of myofibroblasts has been documented in several types of cancers, such as those of the uterine cervix,33Cintorino M Bellizzi de Marco E Leoncini P Tripodi SA Xu LJ Sappino AP Schmitt-Graff A Gabbiani G Expression of alpha-smooth-muscle actin in stromal cells of the uterine cervix during epithelial neoplastic changes.Int J Cancer. 1991; 47: 843-846Crossref PubMed Scopus (54) Google Scholar colon,34Sappino AP Dietrich PY Skalli O Widgren S Gabbiani G Colonic pericryptal fibroblasts. Differentiation pattern in embryogenesis and phenotypic modulation in epithelial proliferative lesions.Virchows Arch A Pathol Anat Histopathol. 1989; 415: 551-557Crossref PubMed Scopus (78) Google Scholar ovary,35Czernobilsky B Shezen E Lifschitz-Mercer B Fogel M Luzon A Jacob N Skalli O Gabbiani G Alpha smooth muscle actin (alpha-SM actin) in normal human ovaries, in ovarian stromal hyperplasia and in ovarian neoplasms.Virchows Arch B Cell Pathol Incl Mol Pathol. 1989; 57: 55-61Crossref PubMed Scopus (67) Google Scholar and breast.36Tsukada T McNutt MA Ross R Gown AM HHF35, a muscle actin-specific monoclonal antibody. II. Reactivity in normal, reactive, and neoplastic human tissues.Am J Pathol. 1987; 127: 389-402PubMed Google Scholar, 37Sappino AP Skalli O Jackson B Schurch W Gabbiani G Smooth-muscle differentiation in stromal cells of malignant and non-malignant breast tissues.Int J Cancer. 1988; 41: 707-712Crossref PubMed Scopus (272) Google Scholar Myofibroblasts occur in a variety of other conditions including hypertrophic scars,38Eddy RJ Petro JA Tomasek JJ Evidence for the nonmuscle nature of the “myofibroblast” of granulation tissue and hypertropic scar. An immunofluorescence study.Am J Pathol. 1988; 130: 252-260PubMed Google Scholar healing wound,39Doillon CJ Hembry RM Ehrlich HP Burke JF Actin filaments in normal dermis and during wound healing.Am J Pathol. 1987; 126: 164-170PubMed Google Scholar granulation tissue,38Eddy RJ Petro JA Tomasek JJ Evidence for the nonmuscle nature of the “myofibroblast” of granulation tissue and hypertropic scar. An immunofluorescence study.Am J Pathol. 1988; 130: 252-260PubMed Google Scholar idiopathic pulmonary fibrosis,40Kuhn C McDonald JA The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis.Am J Pathol. 1991; 138: 1257-1265PubMed Google Scholar, 41Broekelmann TJ Limper AH Colby TV McDonald JA Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis.Proc Natl Acad Sci USA. 1991; 88: 6642-6646Crossref PubMed Scopus (688) Google Scholar pulmonary hypertension,42Kapanci Y Burgan S Pietra GG Conne B Gabbiani G Modulation of actin isoform expression in alveolar myofibroblasts (contractile interstitial cells) during pulmonary hypertension.Am J Pathol. 1990; 136: 881-889PubMed Google Scholar scleroderma lesions,43Sappino AP Masouye I Saurat JH Gabbiani G Smooth muscle differentiation in scleroderma fibroblastic cells.Am J Pathol. 1990; 137: 585-591PubMed Google Scholar anterior capsular cataract,44Schmitt-Graff A Pau H Spahr R Piper HM Skalli O Gabbiani G Appearance of alpha-smooth muscle actin in human eye lens cells of anterior capsular cataract and in cultured bovine lens-forming cells.Differentiation. 1990; 43: 115-122Crossref PubMed Scopus (86) Google Scholar heart pressure overload,45Leslie KO Taatjes DJ Schwarz J vonTurkovich M Low RB Cardiac myofibroblasts express alpha smooth muscle actin during right ventricular pressure overload in the rabbit.Am J Pathol. 1991; 139: 207-216PubMed Google Scholar bleomycin-injured lung,46Mitchell J Woodcock-Mitchell J Reynolds S Low R Leslie K Adler K Gabbiani G Skalli O Alpha-smooth muscle actin in parenchymal cells of bleomycin-injured rat lung.Lab Invest. 1989; 60: 643-650PubMed Google Scholar and postradiation fibrosis of the breast.47Brouty-Boye D Raux H Azzarone B Tamboise A Tamboise E Beranger S Magnien V Pihan I Zardi L Israel L Fetal myofibroblast-like cells isolated from post-radiation fibrosis in human breast cancer.Int J Cancer. 1991; 47: 697-702Crossref PubMed Scopus (32) Google Scholar By secreting various growth factors and cytokines including HGF, these myofibroblasts may modulate the behavior of adjacent cancer cells. In the present study, we demonstrate that stromal myofibroblasts of lung adenocarcinoma express c-MET both in vitro and in vivo. We then present evidence that the HGF/c-MET system functions as an autocrine stimulatory loop in cultured myofibroblasts derived from primary lung cancer. Finally, we show that c-MET expression in stromal fibroblasts is correlated with poor prognosis in small-sized lung adenocarcinoma. Human myofibroblast cell lines (MRC5, WI38) and human gastric cancer cell lines (MKN28, MKN45) were purchased from the Human Science Research Resources Bank. Human lung myofibroblasts (N421, T421, N425, T425, T501, N515, T5062, N5162, T5162) were obtained by subculturing fibroblastic cells that grew from enzymatically digested lung specimens. T421, T425, T501, T5062, and T5162 were grown from lung carcinoma tissues, whereas N421, N425, N515, and N5162 were from nontumor lung tissues. A human umbilical vein endothelial cell line (HUVEC.SV) was established by immortalization of human umbilical vein endothelial cells with SV40 large T antigen. Human myofibroblast cell lines, lung myofibroblasts, and HUVEC.SV were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Human gastric cancer cell lines were cultured in RPMI1640 containing 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured at 37°C in humidified atmosphere of 5% CO2 and 95% air. The culture was maintained by subculturing every 3 or 4 days. Recombinant human HGF, transforming growth factor β (TGF-β), epidermal growth factor (EGF), interleukin 1β (IL-1β), acidic fibroblast growth factor (aFGF), basic FGF (bFGF), FGF-4, keratinocyte growth factor (KGF), platelet-derived growth factor (PDGF)-AB, PDGF-BB, monoclonal anti-human HGF antibody (neutralizing antibody to HGF, clone no. 24612.111), and mouse IgG1 isotype control (clone no. 11711.11) were purchased from R & D Systems (Minneapolis, MN). The ND50 of anti-human HGF antibody is ∼0.1 to 0.3 μg/ml in the presence of 100 ng/ml of recombinant human HGF, according to the manufacturer. Polyclonal rabbit anti-c-MET antibody (no. 18321) was purchased from IBL Laboratories (Gumma, Japan). Rabbit immunoglobulin fraction (for negative control, no. X0936), mouse monoclonal anti-human α-smooth muscle actin (no. M0851), mouse monoclonal anti-human desmin (no. M0760), and mouse monoclonal anti-human cytokeratin AE1/AE3 (no. M3515) were purchased from DAKO. The anti-human cytokeratin AE1/AE3 is a mixture of AE1 and AE3 at a ratio of 4:1. Monoclonal antibody AE1 recognizes the 56.5-, 50-, 48-, and 40-kd keratins of the acidic subfamily, whereas monoclonal antibody AE3 reacts with the basic keratins of molecular weights 65 to 67, 64, 59, 58, 56, and 52 kd. Formalin-fixed, paraffin-embedded tumor specimens were obtained from a series of 131 patients with small-sized lung adenocarcinomas (maximum tumor dimension 2 cm or less; median age, 59 years; range, 26 to 80 years) who underwent complete resection of tumors and mediastinal lymphadenectomy at the National Cancer Center Hospital, Japan, between 1984 and 1993. The pathological stage was determined by the new TNM staging system that had been recognized by the Union Internationale Contre le Cancer and the American Joint Committee on Cancer.48Mountain CF Revisions in the International System for Staging Lung Cancer [see comments].Chest. 1997; 111: 1710-1717Crossref PubMed Scopus (4542) Google Scholar The median duration of follow-up was 5.7 years (range, 0.6 to 12.0 years). Patient and tumor characteristics are listed in Table 1.Table 1Characteristics of Small-Sized Lung AdenocarcinomaClinical featureNo. of casesClinical featureNo. of casesAll cases131Nodal involvement (pN)Age (years)Negative (pN0)107Range26–80Positive (pN1, 2)24Median59Pleural invasionSexNegative95Male73Positive36Female58Pathological stageTumor size (mm)IA96Range7.0–20.0IB7Mean16.9IIA7Vascular invasionIIB1Negative83IIIA11Positive48IIIB2Lymphatic invasionIV7Negative77Years follow-upPositive43Range0.591–11.992Equivocal11Median5.744 Open table in a new tab The primers and probes used for reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR were as follows: c-MET primer, forward 5′-CATGCCGACAAGTGCAGTA-3′, reverse 5′-TCTTGCCATCATTGTCCAAC-3′; c-MET probe, 5′-TCCAGGCAGTGCAGCATGTAGTGAT-3′; HGF primer, forward 5′-GAGTTATCGAGGTCTCATGGATC-3′, reverse 5′-CCAACGCTGACATGGAATT-3′; HGF probe, 5′-TCAGACACCACACCGGCACAAAT-3′; E-cadherin primer, forward 5′-CTTCTCTCACGCTGTGTCATC-3′, reverse 5′-CTCCTGTGTTCCTGTTAATGGT-3′; E-cadherin probe, 5′-TACAATGCCGCCATCGCTTACAC-3′; CD31 primer, forward 5′GCAGATAATTGCCATTCCCATG-3′, reverse 5′-CTTCATTCACAGCAGCACATTGCAG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer, forward 5′-GAAGGTGAAGGTCGGAGTC-3′, reverse 5′-GAAGATGGTGATGGGATTTC-3′; GAPDH probe 5′-CAAGCTTCCCGTTCTCAGCC-3′. The probes for c-MET, HGF, and E-cadherin were labeled with the fluorescent dyes 6-carboxyfluorescein (FAM) at the 5′ end and N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) at the 3′ end. The GAPDH probe was labeled with the fluorescent dyes 6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein (JOE) at the 5′ end and N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) at the 3′ end. The primers and probes were synthesized at Greiner Labortechnik Co. Ltd. and Applied Biosystems/Perkin Elmer, respectively. Total RNA was isolated from frozen tissues and cultured cells using an RNeasy mini kit (Qiagen, Basel, Switzerland) following the manufacturer’s protocol. All RNA samples were treated with deoxyribonuclease I, amplification grade (Gibco/BRL), followed by a clean-up with an RNeasy Mini Kit (Qiagen). We performed a real-time quantitative one-step RT-PCR assay49Heid CA Stevens J Livak KJ Williams PM Real time quantitative PCR.Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (5040) Google Scholar, 50Gibson UE Heid CA Williams PM A novel method for real time quantitative RT-PCR.Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1781) Google Scholar to quantify c-MET, HGF, E-cadherin, and GAPDH mRNAs in microdissected samples following the manufacturer’s protocol (Applied Biosystems/Perkin Elmer). The real-time one-step RT-PCR reactions were performed using the TaqMan EZ RT-PCR core reagents (no. N808-0236, Applied Biosystems/Perkin Elmer) in an ABI PRISM 7700 sequence detection system (Applied Biosystems/Perkin Elmer). The RT-PCR reactions were performed using 96-well optical tubes and caps in a 50-μl final reaction volume consisting of 200 nmol/L each primer, 100 nmol/L probe, 1× TaqMan EZ buffer, 3 mmol/L manganese acetate, 300 μmol/L each deoxyATP, deoxyCTP, deoxyGTP, 600 μmol/L deoxyUTP, 0.1 U/μl rTth DNA polymerase, 0.01 U/μl AmpErase uracil-N-glycosylase, 21.5 μl RNase-free water, and 1 μl RNA samples. The thermal cycling conditions consisted of an initial denaturation step with one cycle of 50°C for 2 minutes, one cycle of 60°C for 30 minutes, one cycle of 95°C for 5 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each RT-PCR run included fivefold serial dilutions of a standard RNA, a no-template control, and test samples. We quantified transcripts of GAPDH as endogenous RNA controls, and each value was normalized on the basis of the GAPDH mRNA content. Experiments were performed in triplicate and the results were expressed as mean values. For ordinary RT-PCR analysis, we used a GeneAmp PCR System 9600 (Applied Biosystems/Perkin Elmer). All RT-PCR products were separated by 3% Nusieve 3:1 agarose gel (FMC BioProducts) and visualized by ethidium-bromide staining. Contamination was routinely checked by RT-PCR assay of RNA-free samples (water control). Tumor tissues were snap-frozen in OCT Compound (Sakura Finetechnical Co., Ltd., Japan). They were sliced into 15-μm thick sections, mounted on glass slides 0.17 mm thick (very thin slides were needed to prevent laser energy from being dispersed before reaching the tissue section), and stained with hematoxylin and eosin. We microdissected the specimens with an UV-laser microscope system (PALM UV-laser microbeam; Wolfratshausen, Germany) to separate cancer and stromal cells.51Cerroni L Minkus G Putz B Hofler H Kerl H Laser beam microdissection in the diagnosis of cutaneous B-cell lymphoma.Br J Dermatol. 1997; 136: 743-746Crossref PubMed Scopus (29) Google Scholar, 52Schutze K Posl H Lahr G Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine.Cell Mol Biol. 1998; 44: 735-746PubMed Google Scholar Total RNA was extracted from these microdissected specimens for real-time quantitative RT-PCR analysis. Cells were lysed in a lysis buffer containing 1% Triton X-100 (Sigma Chemical Co.) and 1% Nonidet P-40 (Sigma Chemical Co.) and a cocktail of proteinase inhibitors. Equal amounts of protein samples were size-separated on discontinuous 6% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were immersed for 20 minutes in a blocking solution containing 5% skim milk and 0.1% Tween, and then incubated for 2 hours at room temperature with rabbit anti-cMET antibody or rabbit immunoglobulin IgG as a negative control. After washing, the membranes were incubated for 1 hour at room temperature with peroxidase-linked secondary antibody. The antigen was detected using enhanced chemiluminescence Western blotting detection reagents (Amersham, Arlington Heights, IL) following the instructions of the manufacturer. For immunoprecipitation, cells were lysed in a buffer containing 1% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 50 mmol/L NaF, 1 mmol/L sodium orthovanadate, and a cocktail of proteinase inhibitors. Equal amounts of samples (500 μg) were diluted with lysis buffer, and precleared with 90 μl of protein-A Sepharose (Pharmacia, Uppsala, Sweden) (50% slurry) for 1 hour. After the addition of anti-c-MET antibody (5 μg), samples were incubated overnight at 4°C. Immune complexes were precipitated by incubating the samples for 1 hour with 90 μl of protein-A Sepharose. Sepharose beads were then washed three times with lysis buffer and once with water. After boiling in loading buffer, samples were subjected to Western blot analysis using anti-phosphotyrosine antibody (clone 4G10, dilution ×1/1000; Upstate Biotechnology). Western blot was performed essentially the same as described above except that 2% bovine serum albumin was used as a blocking solution. Subconfluent myofibroblasts were washed twice with serum-free DMEM and incubated for 24 hours with DMEM containing 1% FCS and 2 μg heparin. The latter was included in the media to facilitate the release of matrix-bound HGF from the cell layer. The conditioned media were then subjected to the enzyme-linked immunosorbent assay (ELISA) analysis using Quatikine human HGF (R & D systems) following the manufacturer’s protocol. The results were corrected for the protein content of the cell layer. We analyzed the effect of HGF/c-MET signaling in cultured myofibroblasts by 5-bromo-2′-deoxyuridine (BrdU) incorporation into DNA, using a Cell Proliferation ELISA BrdU kit (catalog no. 1647229, Boehringer Mannheim, Mannheim, Germany).53Williamson K Halliday I Hamilton P Ruddell J Varma M Maxwell P Crockard A Rowlands B In vitro BrdUrd incorporation of colorectal tumour tissue.Cell Prolif. 1993; 26: 115-124Crossref PubMed Scopus (4) Google Scholar, 54H" @default.
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• W79277247 date "2001-04-01" @default.
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• W79277247 title "c-MET Expression in Myofibroblasts" @default.
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