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.

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. The diagnosis of ATL was based on Mann-Whitney test. Differences were considered to be statisti- clinical features, hematological findings, and serum anti- cally significant at P Ͻ 0.05. HTLV-I antibodies. Monoclonal HTLV-I provirus integration into the genomic DNA of leukemic cells was confirmed by Southern blot hybridization in all cases (data not shown). Mono- RESULTS nuclear cells from patients with ATL were obtained by density Expression of c-Met and HGF in HTLV-I-positive T- gradient separation from the peripheral blood, before chemo- cell Lines. The expression of c-Met mRNA in T-cell lines was therapy, after informed consent. Serum transaminase levels at analyzed by RT-PCR using c-Met-specific primers. In four the sampling point for each case were analyzed. HTLV-I-positive T-cell lines, HUT102, MT-4, and KK-1 ex- RT-PCR. Total RNA was extracted by using RNeasy mini pressed c-Met mRNA, whereas ST-1 did not (Fig. 1A, Lanes kit (Qiagen, Hilden, Germany), according to the protocol provided 3–6). In HTLV-I-negative T-cell lines Jurkat and MOLT4, the by the manufacturer. First-strand cDNA was synthesized by using expression could not be detected (Fig. 1A, Lanes 1 and 2). RT-PCR kit (Stratagene, La Jolla, CA) with oligodeoxythymidylic Western blot analyses showed the same expression pattern of acid primers. Thereafter, cDNA was amplified for 30 cycles for c-Met protein as that of mRNA (Fig. 1B). In KK-1, which is an c-Met, HGF, and Tax and for 23 cycles for ␤-actin, respectively. ATL patients-derived cell line, c-Met protein is definitely de- The oligo nucleotide primers used were as follows: for c-Met, sense tected, whereas the expression of the viral transactivating pro- 5Ј-ACT CCCCCTGAAAACCAAAGCC-3Ј, antisense 5Ј-GGC- tein Tax could hardly be detected (Fig. 1B, Lane 6), suggesting TTACACTTCGGGCACTTAC-3Ј; for HGF, sense 5Ј-ACT- the existence of the Tax-independent mechanism of c-Met ex- GGCTCTTTTAGGCACTGACTC-3Ј, antisense 5Ј-TGTTCCC- pression in ATL cells. The cell surface expression of c-Met TTGTAGCTGCGTCCTTT-3Ј, for Tax, sense 5Ј-ATCCCGT- protein was confirmed in HTLV-I-positive T-cell lines by flow GGAGACTCCTCAA-3Ј, antisense 5Ј-AACACGTAGACTGGG- cytometric analyses (Fig. 1C). These data indicate that c-Met is

Fig. 2 c-Met expressed in ATL cells is autophosphorylated in response to HGF. In A, ATL-derived cell lines were incubated in the presence (ϩ) or absence (Ϫ) of HGF for 12 h; then whole cell extracts were analyzed with antiphosphotyrosine antibody (anti-PY, 4G10). KK-1 is a c-Met- positive and ST-1 is a c-Met-negative cell line. Arrows, the bands representing newly developed phosphotyrosine proteins after treatment with HGF. * indicates the putative band of tyrosine-phosphorylated c-Met. In B, HeLa, KK-1, and ST-1 cells were incubated in the presence (ϩ) or absence (Ϫ) of HGF, lysed, immunoprecipitated with anti-c-Met antibody, and blotted with the same antibody or phosphotyrosine (anti- PY) antibody.

sion level of HGF was strong in HUT102 but weak in ST-1 and KK-1. Interestingly, coexpression of HGF and c-Met was dem- Fig. 1 HGF and c-Met are expressed in HTLV-I-positive T-cell lines. onstrated in HUT102 and KK-1, suggesting possible auto-stim- In A, HGF and c-Met mRNA were detected by RT-PCR analysis in ulatory loop in these cells. HTLV-I-positive T-cell lines. Lane 1, Jurkat RNA; Lane 2, MOLT4 Autophospholyration of c-Met Was Induced in HTLV- RNA; Lane 3, HUT102 RNA; Lane 4, MT-4 RNA; Lane 5, ST-1 RNA; Lane 6, KK-1 RNA. Top, c-Met; middle, HGF; bottom, ␤-actin. In B, I-positive T-cell Lines by HGF Treatment. To examine c-Met protein is expressed in HTLV-I-positive T-cell lines. The expres- whether c-Met expressed in HTLV-I-positive T-cell lines is sion of c-Met and Tax protein in human T-cell lines was analyzed by functional, we examined the autophosphorylation of c-Met in immunoblotting using anti-c-Met antibody or anti-Tax antibody. The response to HGF treatment. When KK-1, a c-Met-positive cell same blots were reprobed with antiactin antibody for loading standard. line, was incubated with HGF, several signals representing In C, c-Met protein is expressed on the cell surface of HTLV-I-positive T-cell lines. T-cell lines were incubated with anti-c-Met MoAb or tyrosine phosphorylation were newly detected (Fig. 2A, Lanes 1 ϳ isotype control antibody, followed by staining with FITC-conjugated and 2). One of these signals was Mr 140,000, which corre- antimouse immunogloblin antibody. Histograms were shown for anti- sponds to the autophosphorylation of c-Met. On the other hand, c-Met MoAb (solid lines) and isotype control antibody (dotted line). in ST-1, a c-Met-negative cell line, no difference could be seen with or without HGF treatment, suggesting that no signal could be transduced into ATL cells without c-Met expression (Fig. 2A, expressed in some of the HTLV-I-positive T-cell lines, includ- Lanes 3 and 4). To further confirm the autophosphorylation of ing one established from an ATL patient. In other lymphoid c-Met in response to HGF treatment, c-Met was immunopre- malignancies, such as multiple myeloma or primary effusion cipitated with c-Met-specific antibody, and Western blotting lymphoma, coexpression of c-Met and HGF was reported, and was performed using c-Met or phosphotyrosine-specific anti- an auto-stimulatory loop of HGF/c-Met signaling has been bodies (Fig. 2B). A signal of Mr 140,000 representing autophos- suggested (30, 31, 34, 41). Thus, we examined the expression of phorylation of c-Met was detected in HeLa, a positive control HGF mRNA in T-cell lines. HGF mRNA was also expressed in cell line, and KK-1, only when they were incubated in the some of the HTLV-I-positive cell lines (Fig. 1A). The expres- presence of HGF (Fig. 2B, Lanes 1–4). In KK-1, the autophos-

Fig. 3 c-Met is expressed in PBMCs from ATL patients. The expression of c-Met and HGF mRNA in freshly isolated PBMCs from ATL patients and normal healthy donors was analyzed by RT-PCR. Lanes 1 and 2, PBMC RNA from healthy donors (HD). Lanes 3–13, PBMC RNA from ATL patients. Lane 14, Jurkat RNA as negative control (J). Lane 15, HUT102 RNA as positive control (H). The expression of ␤-actin mRNA was shown as control. Fig. 4 Serum transaminase levels were significantly higher in c-Met- positive ATL patients. Serum GOT and GPT levels were compared between c-Met mRNA-positive and c-Met mRNA-negative ATL patients (for GOT, 26.2 ϩ/Ϫ 23.8 versus 102 ϩ/Ϫ 29.7; for GPT, 17.8 Table 1 Clinical features of ATL patients at the time of sample ϩ/Ϫ 8.2 versus 74.2 ϩ/Ϫ 24.6; means ϩ/Ϫ SE, c-Met negative versus collection c-Met positive, respectively). Horizontal bars, the mean of the measured WBC values; statistical analyses were performed using Mann-Whitney test; *, Patient count Ab-Ly GOT GPT c-Met P Ͻ 0.05; **, P Ͻ 0.02. no. (/mm3) (%)a (IU/liter)b (IU/liter)c expressiond 1 10 300 20 16 11 Ϫ 2 41 800 68 15 7 Ϫ Ϫ 3 17 600 23 10 4 c-Met expression. On the other hand, the expression of HGF 4 6 400 31 23 14 Ϫ 5 23 400 53 17 4 ϩ could not be detected in PBMCs from ATL patients. 6 51 900 80 91 48 Ϫ Expression of c-Met Correlates with Liver Dysfunction 7 98 900 82 74 58 ϩ in ATL. To evaluate the correlation between the expression of 8 33 800 37 65 81 ϩ c-Met and liver dysfunction, serum transaminase level was ϩ 9 35 200 66 84 36 examined in ATL patients. Interestingly, in 10 of c-Met-positive 10 70 200 87 64 46 ϩ 11 36 700 55 87 64 ϩ cases, 8 cases showed increased levels of serum transaminase, 12 18 300 38 15 8 Ϫ whereas only 1 of 6 c-Met-negative cases showed increased 13 73 600 94 19 13 Ϫ transaminase levels (Fisher’s exact probability test, P Ͻ 0.05). ϩ 14 18 100 61 47 48 The levels of serum GOT and GPT were higher in c-Met- 15 66 500 71 316 127 ϩ 16 31 300 78 27 15 ϩ positive patients (Fig. 4), and there was a statistically significant difference in serum transaminase levels between the c-Met- a Ab-Ly indicates the percentage of abnormal lymphocytes in WBCs. positive and -negative patients (Mann-Whitney test: for GOT, b Normal range of GOT is 13–35 IU/liter. P Ͻ 0.02; for GPT, P Ͻ 0.05). c Normal range of GPT is 8–42 IU/liter. As liver transaminase is a nonspecific marker of liver cell d c-Met expression in PBMCs was examined by RT-PCR. damage, liver autopsy specimens from a patient suffering with liver dysfunction (GOT, 94; GPT, 46) were analyzed. The patient died of leukemic crisis, associated with massive hepa- phorylation of c-Met was not observed in the absence of HGF tosplenomegaly as a result of tumor progression. Immuno- treatment. In contrast, no signal representing c-Met or phospho- staining revealed most but not all infiltrated atypical lympho- tyrosine could be detected in ST-1, regardless of the treatment cytes were c-Met-positive cells (Fig. 5). On the other hand, all with HGF (Fig. 2B, Lanes 5 and 6). These results indicate that c-Met-positive lymphocytes showed multilobularly nucleated c-Met expressed in ATL cell lines is actually functional and phenotype. Hepatocytes were stained weak but homogenous transduces the signals in response to HGF stimulation. with anti-c-Met antibody as described previously (17). Similar Expression of c-Met mRNA in Clinical Samples from pathological findings were seen in another autopsy case of ATL ATL Patients. To examine the expression of c-Met and HGF associated with liver dysfunction (data not shown). mRNA in primary ATL cells, RT-PCR analyses were performed using PBMCs freshly isolated from ATL patients. We could DISCUSSION observe a definite band of c-Met in 10 of 16 samples from ATL In ATL, coetaneous involvement and hypercalcemia are patients, whereas no band could be detected in PBMCs from frequently demonstrated. In addition, hepatosplenomegaly is healthy donors (Fig. 3 and data not shown). As the number of often experienced, and this is one of the diagnostic criterion of atypical cells in each patient does not reflect the result of the ATL (42). Importantly, invasive characters of ATL cells into c-Met mRNA amplification (Table 1), we regarded that the liver and consequent impaired general conditions seemed to be failure of the detection of c-Met mRNA was not caused by a factors affecting poor prognosis (37). Cell migrations are a lower input of tumor-derived RNA. As reported previously, the critical step in invasion of leukocytes. It has been reported that expression of Tax could not be detected in primary ATL cells, ATL cells adhere to endothelial cells through an adhesion cas- suggesting the existence of the Tax-independent mechanism in cade similar to normal leukocytes; thus, it is reasonable that

Fig. 5 Invasion of c-Met-positive ATL cells in liver. A paraffin-embedded section of liver specimen from a patient suffering with liver dysfunction was stained with anti-c-Met antibody, then H&E counterstaining. A massive invasion of lymphocytes was seen in the perivascular area, and cells strongly stained with anti-c-Met antibody are multinucleated lobular phenotype, a typical finding of ATL cells (inset).

several molecules found in usual pathological process have been found that induced Tax in a Jurkat cell line, JPX9, could not required to infiltrate ATL. HGF has been proposed to be a induce c-Met but several Tax-induced genes (data not shown). physiologically relevant trigger in T-cell migration, although the Thus, the Tax-independent mechanism seems to operate the expression of c-Met was not demonstrated (35). Indeed, c-Met c-Met expression in ATL. expression on cells of T-cell origin has not been clearly recog- In some lymphoid malignancies, HGF and c-Met were nized, although its expression has been documented in various coexpressed, and an autocrine mechanism is suggested to be tissue origins, including B cells. There was a report that the involved in the pathogenesis (30, 31, 34). Concomitant expres- expression of c-Met was not detected in PBMCs but induced by sion of HGF and c-Met is also found in some ATL cell lines in reagents such as phorbol 12-myristate 13-acetate, Con A, and our experiment. However, in primary ATL cells, the expression HGF in B cell-rich fractions (33). In this study, the expression of HGF mRNA was not detected (Fig. 3). Although HGF is not of c-Met in ATL-derived cells was clearly detectable in PBMCs expressed on primary ATL cells, HGF is induced in various from ATL patients, and HTLV-I-infected T-cell lines, indicating cells by proinflammatory cytokines, such as interleukin-1 and that c-Met is expressed on cells of T-cell origin. In addition, the tumor necrosis factor-␣ (43, 44), which were produced by ATL expressed c-Met was functional, because HGF could induce cells (45). Thus, HGF secreted in papracrine rather than auto- autophosphorylation of c-Met in the c-Met-positive ATL cell crine mechanism seems to be involved in the pathogenesis of line KK-1. Although HGF was reported to induce cytoskeletal ATL. Furthermore, it was shown that HGF/c-Met signaling not changes and migration of memory T cells on which c-Met was only stimulates the growth of cells but also stimulates the not detectable (35), this cytokine could not induce autophos- release of chemokines in monocytes and other cell types (22, phorylation and chemotaxis in the c-Met-negative ATL cell line 46). Therefore, a part of stimulated ATL cells with HGF further ST-1. Thus, putative receptor(s) other than c-Met does not seem augments the migratory activity of leukemic T cells through the to be involved to mediate HGF signaling in ST-1. Alternatively, liberation of chemokines. This probably accounts for the fact molecule(s) other than c-Met responsible for HGF signaling was that not every invasive lymphocyte in liver expressed c-Met in concomitantly defective in this cell line. our clinical specimens (Fig. 5). We speculate that monoclonally It was reported that c-Met was expressed in Hodgkin’s developed ATL cells become more aggressive phenotypes by Reed-Sternberg cells, and the expression of c-Met was strongly the acquisition of c-Met expression on its surface. correlated with the presence of EBV (33). In addition, c-Met was expressed in human herpes virus-8-positive primary effu- ACKNOWLEDGMENTS sion lymphoma (34). Although the molecular mechanisms in- We thank Drs. A. Koda, H. Ichinose, and M. Miyazaki for encour- volved in the expression of c-Met in these virus-infected cells agement. remain unknown, HTLV-I has the viral-transactivating protein Tax, which is involved in the deregulated expression of cellular genes; hence, Tax could induce the expression of c-Met in ATL REFERENCES cells. However, the expression of c-Met was not correlated with 1. Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. D., and Gallo, R. C. Detection and isolation of type C retrovirus the expression of Tax in KK-1 (Fig. 1B, Lane 6). Similarly, particles from fresh and cultured lymphocytes of a patient with cutane- c-Met mRNA could be definitely detected but not Tax mRNA in ous T-cell lymphoma. Proc. Natl. Acad. Sci. USA, 77: 7415–7419, freshly isolated PBMCs (Fig. 3 and data not shown). Finally, we 1980.

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Yoshitaka Imaizumi, Hiroyuki Murota, Shigeru Kanda, et al.

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