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Expression of the C-Met Proto-Oncogene and Its Possible Involvement in Liver Invasion in Adult T-Cell Leukemia1

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Expression of the C-Met Proto-Oncogene and Its Possible Involvement in Liver Invasion in Adult T-Cell Leukemia1 Vol. 9, 181–187, January 2003 Clinical Cancer Research 181 Expression of the c-Met Proto-Oncogene and Its Possible Involvement in Liver Invasion in Adult T-cell Leukemia1 Yoshitaka Imaizumi, Hiroyuki Murota, mRNA was not detected in c-Met mRNA-expressed cell Shigeru Kanda, Yoshitaka Hishikawa, lines. From freshly isolated peripheral blood mononuclear Takehiko Koji, Takashi Taguchi, Yuetsu Tanaka, cells, the expression of c-Met mRNA was detected in 10 of 16 ATL patients but not from healthy individuals. Finally, Yasuaki Yamada, Shuichi Ikeda, Tomoko Kohno, serum transaminase levels were significantly increased in Kazuo Yamamoto, Naoki Mori, c-Met-positive ATL cases, and all of the infiltrated c-Met- Masao Tomonaga, and Toshifumi Matsuyama2 positive cells into liver were shown to be multilobularly Divisions of Cytokine Signaling [Y. I., H. M., To. K., K. Y., T. M.] nucleated phenotype. Taken together, these data suggest for and Endothelial Cell Biology [S. K.], Department of Molecular the first time that c-Met is involved in the liver invasive Microbiology and Immunology, Nagasaki University Graduate School phenotype of ATL. of Medical Sciences and Departments of Laboratory Medicine [Y. Y.], Hematology, Atomic Disease Institute [Y. I., M. T.], and Histology and Cell Biology [Y. H., Ta. K.] and Second Department of INTRODUCTION Pathology [T. T.], Nagasaki University School of Medicine, Nagasaki ATL3 is a mature helper T cell-derived neoplasia caused by 852-8523; Departments of Infectious Disease and Immunology, HTLV-I (1, 2). One of the frequent manifestations of ATL is Okinawa-Asia Research Center of Medical Science [Y. T.], and Virology [N. M.], Faculty of Medicine, University of the Ryukyus, invasion of leukemic cells into various tissues. In general, T-cell Okinawa 903-0215; and Department of Medicine, City of Sasebo infiltration into tissue depends on a cascade of rapid and selec- General Hospital, Sasebo 857-8511 [S. I.], Japan tive adhesive interactions with endothelium. A step in this cascade requires a triggering to activate T-cell integrins. Thus, several studies have focused on the mechanism of ATL cell ABSTRACT invasion from its expression of integrins and associated mole- c-Met is a tyrosine kinase receptor for hepatocyte cules (3, 4). Involvement of other molecules such as chemokine growth factor and suggested to be involved in oncogenesis or receptors CCR7/EBI1, heparin sulfate, and leukotactic factor metastatic phenotypes in many malignancies. Adult T-cell activity-1 has also been reported in ATL (5–10). Hence, various leukemia (ATL) is a neoplasia characterized by massive molecules required for adhesive interactions and migrations invasion of the leukemic cells into various organs. Recently, seem to be involved in the invasion step of ATL cells. we have reported frequent hepatic involvement and the HGF, also known as scatter factor, was identified as a relationship between liver invasion and the poor prognosis chemoattractant for a variety of cells. HGF is produced by cells in ATL. In the present study, we investigated the expression of mesenchymal origin, including liver, but not by epitherial of c-Met in ATL cells and its relation to liver dysfunction. In origin and has a pleiotropic function, such as liver regeneration. three of four human T-cell lymphotrophic virus-I-positive It also has mitogenic, morphogenic, and motogenic effects on T-cell lines, c-Met was expressed both at mRNA and protein epithelial cells, as well as endotherial cells (11). The receptor for levels, whereas it was not expressed in human T-cell lym- HGF is encoded by met proto-oncogene (c-Met). The c-Met phoma virus-I-negative T-cell lines. The expressed c-Met protein belongs to a tyrosine kinase cell surface receptor and should be functional, because hepatocyte growth factor consists of an extracellular ␣- and a transmembrane ␤ chain. could induce the autophosphorylation of c-Met. Although The ␤ chain contains the tyrosine kinase domain as well as the the viral-transactivating protein Tax has been shown to be site for tyrosine autophosphorylation. Ligation of HGF causes involved in the deregulated expression of cellular genes, Tax the autophosphorylation of c-Met, followed by a variety of signaling cascade (12). Although normal HGF/c-Met signaling is involved in many aspects of embryogenesis, abnormal HGF/ c-Met signaling has been implicated in both tumor development Received 3/26/02; revised 8/16/02; accepted 8/23/02. and progression (13). In particular, HGF/c-Met signaling has The costs of publication of this article were defrayed in part by the been shown to play a significant role in promoting tumor cell payment of page charges. This article must therefore be hereby marked invasion and metastasis (14). Furthermore, HGF and/or c-Met advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by a grant from N. D. R. Corp., Gifu, Japan. 2 To whom requests for reprints should be addressed, at Division of 3 The abbreviations used are: ATL, adult T-cell leukemia; HTLV-I, Cytokine Signaling, Department of Molecular Microbiology and Immu- human T-cell lymphoma virus-I; PBMC, peripheral blood mononuclear nology, Nagasaki University Graduate School of Medical Sciences, cell; FBS, fetal bovine serum; GPT, glutamic pyruvic transaminase; Nagasaki 852-8523, Japan. Phone: 81-95-849-7079; Fax: 81-95-849- GOT, glutamic oxaloacetic transaminase; MoAb, monoclonal antibody; 7083. RT-PCR, reverse transcription-PCR; HGF, hepatocyte growth factor. Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2003 American Association for Cancer Research. 182 c-Met in ATL and Liver Invasion expression/overexpression has been documented in a wide va- TATCC-3Ј (40); for ␤-actin, sense 5Ј-AAGAGAGGCATCCT- riety of human tumors (15–20). CACCCT-3Ј, antisense 5Ј-TACATCGCTGGGGTGTTGAA-3Ј. c-Met is predominantly expressed in epithelial cells but has Product sizes were 536, 505, 145, and 218 bp, respectively. Cycling been detected in various hematopoietic cells as well (21–29). conditions were as follows: denaturing at 94°C for 60 s, annealing Furthermore, lymphoid malignancies, such as multiple myeloma at 60°C for 60 s, and extension at 72°C for 60 s. The PCR products and several B cell lymphomas, were found to express c-Met, were fractionated on 2% agarose gel and visualized by ethidium suggesting that c-Met is involved in the pathogenesis of these bromide staining. diseases (30–34). Regarding T cells, it was reported that HGF Detection of c-Met Protein on the Cell Surface by Flow could trigger a subset of T-cell adhesion and migration; how- Cytometric Analysis. The expression of c-Met on the cell ever, the expression of c-Met was not detectable by surface surface was analyzed by flow cytometry. Briefly, 3–5 ϫ 105 staining, PCR, or antiphosphotyrosine staining (35). On the cells were washed twice with PBS containing 2% FBS (FBS/ other hand, thymic lymphoid cells were found to express c-Met PBS). Cells were incubated at 4°C with a mouse antihuman and respond to HGF to generate mature T cells expressing c-Met MoAb (Do-24; Upstate Biotechnology, Lake Placid, NY) antigen receptors (36). for 60 min. After being washed twice with FBS/PBS, cells were Recently, we have reported frequent hepatic involvement incubated with FITC-labeled antimouse IgG MoAb (PharMin- and the relationship between liver invasion and poor prognosis gen), washed twice with FBS/PBS, suspended in FBS/PBS, and in ATL (37). To clarify this issue, we investigated the expres- analyzed by FACScan using CellQuest software (Becton Dick- sion of c-Met on ATL cells and found for the first time that inson). c-Met is expressed in HTLV-I-positive T-cell lines. The expres- Detection of c-Met Protein by Western Blot Analysis. sion of c-Met in the ATL cell line was functional, because we Cellular lysates were fractionated by 10% SDS-PAGE gel, detected the autophosphorylatiojn of c-Met in response to HGF. electrophoretically transferred to polyvinylidine difluoride In addition, the expression of c-Met mRNA is documented in membranes (Immobilon-P; Millipore Corp., Bedford, MA), and PBMCs freshly isolated from ATL patients. Furthermore, its then analyzed for immunoreactivity with a mouse antihuman expression is correlated with the liver dysfunction of ATL c-Met polyclonal antibody (Santa Cruz Biotechnology, Santa patients. Thus, we propose the possible involvement of HGF/c- Cruz, CA) and horseradish peroxidase-conjugated sheep anti- Met signaling pathway in the invasion into tissues, particularly mouse IgG (Amersham Life Science, Inc., Arlington Heights, liver, in ATL. IL) with an enhanced chemiluminescence detection system (Amersham Life Science, Inc.). Immunohistochemistry. Using a formalin-fixed, paraffin- MATERIALS AND METHODS embedded section of liver from an autopsy specimen, we per- Cell Lines. Jurkat and MOLT4 are HTLV-I-negative T- formed immunohistochemical analysis. The antibody used was cell lines. HUT102, MT-4, ST-1, and KK-1 are HTLV-I-posi- the same as flowcytometric analysis at a dilution of 1:500. The tive T-cell lines (38, 39). ST-1 and KK-1 are cell lines derived protein was visualized using the DAKO LSAB kit (DAKO A/S, from ATL patients (37). All of these cell lines are grown in Glostrup, Denmark) that uses a biotinylated second antibody RPMI 1640 supplemented with 10% heat-inactivated FBS, pen- complexed with horseradish peroxydase and a diaminobenzi- icillin G (50 units/ml), and streptomycin (50 ␮g/ml) in a hu- done-based stain. midified incubator containing 5% CO2 in air. HeLa cells were Statistical Analysis. Statistical significance was deter- maintained in Dulbecco’s medium supplemented with 10% FBS. mined by Student’s t test, Fisher’s exact probability test, and the Patient Samples.
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