Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth

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Abstract

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has a significant role in initiating EBV-associated lymphoproliferative disease and EBV-related malignancies. In view of clinical features related to the type of EBV latency, LMP1 may influence invasiveness of EBV associated tumors categorized as types II and III as represented on nasopharyngeal carcinoma (NPC). To screen for genes associated with invasion of epithelial cells transformed by LMP1, Madin-Darby canine kidney (MDCK) epithelial cells were transformed by LMP1. Stable transfection of a LMP1 gene into MDCK cells induced morphological change from cobblestone to a long spindle-shape, reduced cell-cell adhesion and caused high cell motility. Parental MDCK cells, which form spherical cysts in three-dimensional collagen gel matrix, form branching tubules following exposure to hepatocyte growth factor (HGF). MDCK cells transformed by LMP1 showed invasive growth to form branching tubules into collagen gel without HGF-treatment. mRNA differential display and Northern hybridization identified plasminogen activator inhibitor-1 (PAI-1), urokinase type plasminogen activator (uPA) and ets1 as genes upregulated during transformation by LMP1. Expression of a dominant negative type of Ets1 in LMP1-transformed cells downregulated uPA expression and cell motility. Deletion of LMP1 cytoplasmic carboxy-terminal activating region 1 (CTAR1) domain abolished transformation, but a deletion mutant lacking CTAR2 domain still retained transforming and uPA-inducing ability. Expression of Ets1 was immunolocalized in tumor cells of NPC tissue which frequently express LMP1. Taken together, it is suggested that LMP1 induces expression of Ets1 which may contribute to invasion of NPC by stimulating cell motility and uPA expression.

Introduction

Epstein-Barr virus (EBV) is a human herpesvirus associated with a number of malignancies including Burkitt's lymphoma and nasopharyngeal carcinoma (NPC) (Young and Rowe, 1992; Rickinson and Kieff, 1996). EBV transforms resting B lymphocytes in vitro resulting in the outgrowth of immortalized lymphoblastoid cell lines (Kieff, 1996; Rickinson and Kieff, 1996). Among the viral proteins expressed in immortalized cells, the nuclear antigens EBNA2, EBNA3A and EBNA3C and the latent membrane protein 1 (LMP1) are essential for growth transformation and immortalization of resting B lymphocyte (Henderson et al., 1991; Liebowitz and Kieff, 1993; Kieff, 1996). LMP1 is of particular interest as it resembles a classical oncogene in its ability to induce growth transformation of certain rodent fibroblast cell lines (Baichwal and Sugden, 1988; Wang et al., 1990). The transforming effects of LMP1 are likely due to its profound effects on cellular gene expression. Expression of this viral oncogene in B-cell lines can induce many of the phenotypic changes reminiscent of the activation process initiated by EBV infection, including upregulation of CD23, CD40, CD54, bcl-2 and A20 genes (Young et al., 1988; Dawson et al., 1990; Wang et al., 1990; Henderson et al., 1991). In epithelical cells LMP1 induces expression of the epidermal growth factor receptor (EGFR) and A20 genes (Miller et al., 1995; Fries et al., 1996).

LMP1 is a 62-kDa integral membrane protein containing six transmembrane segments, 24 and 200 amino acid cytoplasmic domains at the amino and carboxy terminus, respectively. The cytoplasmic carboxy terminal region contains two signaling domains that are essential for directly mediating the gene expression changes described above. The distal domain located between amino acids 352 and 386 C-terminal activating region (CTAR)-2 is the dominant NF-κB and c-Jun N-terminal kinase (JNK) activating region of this protein. The proximal domain located between amino acids 187 and 231 (CTAR1) induces low levels of NF-κB and an effect which could be attributed to its ability to interact with the TNF-α receptor associated factors (TRAFs) (Mosialos et al., 1995; Devergne et al., 1996; Kaye et al., 1996). The proximal domain (TRAF domain) also induces expression of the EGFR through a pathway distinct from NF-κB activation alone (Miller et al., 1997), and the signaling from this domain is sufficient to drive initial proliferation of EBV-infected resting B lymphocytes (Kaye et al., 1995; Izumi et al., 1997).

LMP1 and EBNAs are needed for immortalization of the B cell. On the other hand, there are distinctive clinical features related to LMP1 expression. Tumors, which express LMP1 and thus are categorized as type II or III latency, show invasive and metastatic phenotype (Miller, 1990). Therefore, it was hypothesized that LMP1 may also influence tumor invasion and metastasis.

Ets1 is a member of Ets transcription factor family and recognizes specific nucleotides sequences with a GGAA/T core sequence (Dittmer and Nordheim, 1998). Physiologically, Ets1 is expressed in various mesodermal derivatives, such as endothelial cells and mesenchymal cells (Vandenbunder et al., 1989; Queva et al., 1993; Kola et al., 1993), but is also induced in dissociating epithelial cells, during epithelial-mesenchymal transitions occurring during early development and oncogenic transformation to acquire invasive features (Gilles et al., 1996; Fafeur et al., 1997). Expression of Ets1 in tumor cells has been found to correlate with the grade of invasiveness. Sixty four per cent of gastric adenocarcinomas and 80% of those with more invasive features showed immunostaining for Ets1, whereas normal gastric epithelium or gastric adenomas were Ets1-negative (Nakayama et al., 1996). Ets1 expression in these tumors correlated with the presence of lymph node metastasis. Ets1 expression is also associated with the invasion of astrocytic tumors (Nakada et al., 1999).

In this study we demonstrated that expression of LMP1 in Madin-Darby canine kidney (MDCK) epithelial cells reduced cell-cell adhesion and enhanced motility which resulted in an invasive growth to form branching tubules into collagen gel matrix. Ets1 expression, which is induced by LMP1, contributes in part to invasion by stimulating motility and urokinase type plasminogen activator (uPA) expression.

Results

Transformation of MDCK cells with LMP1

MDCK cells were transfected with the LMP1 gene and stable clones were selected (Figure 1). Parental (MDCK) and MDCK cells transfected with control plasmid (neo) exhibited the characteristic cobblestone morphology. Most LMP1 transfectants lost cell-to-cell contacts and acquired long spindle-shape morphology, and two representative clones were isolated (LMP1-2 and LMP1-3) Both transformed cells grew almost equally with parental MDCK cells (data not shown). Expression of 63 kDa LMP1 proteins in transformed cells was detected by Western blotting with a monoclonal antibody against LMP1 (Figure 2). Densitometric analysis showed 1.5 and 0.7-fold levels of LMP1 expression in clone LMP1-2 and LMP1-3 as compared with that of B95-8 cells, respectively.

Figure 1
figure1

Morphological changes of MDCK cells induced by transformation with LMP1. MDCK cells (MDCK) and MDCK cells transfected with a control plasmid (neo), and two clones of MDCK cells transformed by LMP1 gene transfection (LMP1-2 and LMP1-3, respectively) (2×105 cells) were plated onto a 35 mm plastic dish, and were observed 24 h after plating. Magnification=200×

Figure 2
figure2

Western analysis for LMP-1 expression. MDCK cells (Lane MDCK), MDCK cells transfected with control plasmid (Lane neo), and two clones of LMP1-transformed cells (lanes, LMP1-2 and LMP1-3, respectively) were solubilized to SDS–PAGE. EBV-infected B95-8 cells were also solubilized and electropholesed (lane, B95-8). After blotting to a nitrocellulose filter, LMP1 protein was detected with a monoclonal antibody against LMP1

Tubular formation of MDCK-LMP1 cells

MDCK cells transfected with control plasmid (Figure 3a, neo) formed spheroidal cysts in the collagen gel matrix and generated branching tubules following exposure to HGF (neo+HGF) as usually seen with parental MDCK cells (Montesano et al., 1991a,b). MDCK cells transformed by LMP1 spontaneously formed branching tubules through collagen gel in the absence of HGF. The LMP1 high producer clone (LMP1-2) showed more invasive growth and formed thinner tubules than moderate producer clone (LMP1-3).

Figure 3
figure3

Enhanced motility of LMP1 transformants. (a) Invasion assay in 3-dimensional collagen gel. MDCK cells transfected with control plasmid (panel neo), and two clones of LMP1-transformed cells (panels: LMP1-2 and LMP1-3, respectively) were cultured in a 3-dimensional collagen gel matrix for 7 days. HGF (50 ng/ml) was added to the culture of MDCK cells transfected with control plasmid (panel: neo+HGF). Magnification=100×. (b) Scrape-wound migration assay. Confluent monolayers of MDCK cells transfected with control plasmid (panel: neo), and two clones of LMP1-transformed cells (panels: LMP1-2 and LMP1-3, respectively) were scraped by a plastic pipet, and wound-induced migration of cells was observed after 5 h. Magnification=150×

Enhanced motility of MDCK-LMP1 cells

To compare the motility of LMP1 clones with that of control MDCK cells, a wound-induced migration assay was performed (Figure 3b). Confluent monolayers of each cell clone were scrape-wounded with plastic pipet, and cells migrating into a wounded area were observed after 5 h. Control MDCK cells did not migrate away from the monolayers and the monolayer edge soon became smooth due to tight cell-cell adhesion (neo). In contrast, LMP1-transformed cells migrated into a wounded area away from the monolayer edges, and the high LMP1-producer clone (LMP1-2) migrated more extensively than the moderate clone (LMP1-3).

Genes induced by LMP1

To screen genes differentially expressed between control and LMP1-transformed MDCK cells (clone LMP1-3), mRNA differential display was performed. Plasminogen activator inhibitor-1 (PAI-1) was identified as one of the genes upregulated in transformed cells, and enhanced expression of PAI-1 mRNA was confirmed by Northern blotting (Figure 4, panel PAI-1). As a gene co-expressed with PAI-1, urokinase type plasminogen activator (uPA) mRNA expression was examined by Northern hybridization, and also seen to be upregulated in transformed cells compared with parental and neo-transfected MDCK cells (Figure 4, panel uPA) (Grondaho-Hansen et al., 1993; Foekens et al., 1994). Ets1 is known to be one of the major factors regulating transcription of the uPA gene (Stacey et al., 1995; D'Orazio et al., 1997; Watabe et al., 1998), and thus ets1 mRNA expression was compared between control and LMP1-transformed MDCK cells by Northern hybridization (panel ets-1). Expression of ets1 mRNA, which was very low in parental and neo-transfected MDCK cells, was also upregulated in proportion to LMP1 mRNA level in MDCK cells transformed by LMP1.

Figure 4
figure4

Northern blotting analysis of genes induced by transformation with LMP1. Total RNA (5 μg) from MDCK cells (lane: MDCK), MDCK cells transfected with control plasmid (lane: neo), and two clones of LMP1-transformed cells (lanes: LMP1-2 and LMP1-3, respectively) were electrophoresed in a gel, stained with ethidium bromide (bottom), and then examined by Northern blotting probed with 32P-labeled LMP1 (panel: LMP1), PAI-1 (panel: PAI-1), uPA (panel: uPA) and ets-1 (panel: ets-1) cDNA fragments

Ets1 is associated with cell motility

To examine the role of Ets1 in transformation of MDCK cells by LMP1, a dominant negative form of Ets1 (Ets-DN) was expressed in transformed cells. As expectedly, stable expression of Ets-DN downregulated synthesis of uPA mRNA in the LMP1-transformed MDCK cells (Figure 5). However, LMP1-transformant expressing Ets-DN still retained a long spindle-shape morphology and showed reduced cell-cell adhesion (Figure 6). In the wound-induced migration assay, expression of Ets-DN downregulated migration of transformed cells into wounded area, but cells at leading edge did not adhere to each other tightly as seen in parental MDCK cells (see Figure 3).

Figure 5
figure5

Effect of Ets-DN on uPA gene expression. Total RNA from control MDCK cells (lanes: neo) and LMP1-transformed cells (lanes: LMP1) which were transfected with empty plasmid (lanes: +pCEP4) and expression plasmid for Ets-DN (lanes: +ets-DN) were electrophoresed in a gel, stained with ethidium bromide (bottom), and then examined by Northern blotting probed with 32P-labeled ets-1 and uPA cDNA fragments

Figure 6
figure6

Effects of Ets-DN on wound-induced migration. Left panels; a subconfluent culture of LMP1-transformed MDCK cells (clone LMP1-2) transfected with control plasmid (panel: pCEP4) and expression plasmid for Ets-DN (panel: ets-DN). Magnification=200×. Right panels; confluent monolayer of LMP1-transformed MDCK cells (clone LMP1-2) transfected with control plasmid (panel: pCEP4) and expression plasmid for Ets-DN (panel: ets-DN) were scraped by a plastic pipet, and wound-induced migration of cells was observed after 5 h. Magnification=150×

Transformation requires LMP1 CTAR1 region

Previously two domains in the carboxy-terminus of LMP1 have been identified to be important for NF-κB activation, the CTAR-1 (residues 194–232) and CTAR-2 (residues 351–386) (Huen et al., 1995; Kaye et al., 1995). To explore the mechanism of transformation by LMP1, LMP1 deletion mutants were stably transfected into MDCK cells and their transforming ability was compared (Figure 7). Expression of LMP1 mutant lacking CTAR1 domain (Δ187–351) failed to transform MDCK cells, but cells expressing a mutant lacking CTAR2 domain (1–231) showed transformed morphology and high motility in wound-induced migration assay.

Figure 7
figure7

Transformation by a LMP1 deletion mutant. (a) Total RNA (5 μg) from MDCK cells (lane: MDCK), MDCK cells transfected with control plasmid (lane: neo), expression plasmid for LMP1 (lane: LMP1), LMP1 deletion mutants lacking carboxy-terminal a.a. 238–386 region and a.a. 187–351 region (lanes: 1–231 and Δ187–351, respectively) were electrophoresed in a gel, stained with ethidium bromide (bottom panel), and then examined by Northern blotting probed with 32P-labeled LMP1 and uPA cDNA fragments (upper and middle panels, respectively). (b) Left panels: subconfluent cultures of MDCK cells transfected with control plasmid and expression plasmid for LMP1 deletion mutants lacking carboxy-terminal a.a. 232–386 region and a.a. 187–351 region (panels: 1–231 and Δ187–351, respectively) are shown. Right panels: confluent monolayer of MDCK cells transfected with control plasmid and expression plasmid for LMP1 deletion mutants lacking carboxy-terminal a.a. 232–386 region and a.a. 187–351 region (panels: 1–231 and Δ187–351, respectively) were scraped by a plastic pipet, and wound-induced migration of cells was observed after 5 h. Magnification=150×

Overexpression of Ets1 in NPC

Expression of Ets1 in NPC tissue, in which LMP1 was frequently expressed, was analysed by immunostaining with a monoclonal antibody against Ets1 (Figure 8). Tumor cells in a NPC tissue were positively immunostained with a monoclonal antibody against Ets1. Ets1 antigen was not restricted to nuclei but whole cells were stained. LMP1 was immunolocalized primarily on cell membrane of tumor cells in the NPC tissue.

Figure 8
figure8

Immunolocalization of Ets1 and LMP1 in the NPC tissues. Immunostaining was performed with mouse monoclonal antibodies against Ets1 and LMP1 (panels: Ets1 and LMP1, respectively). Arrows indicate intense staining. Magnification=200×

Discussion

LMP1 has a significant role in initiating EBV-associated lymphoproliferative disease and EBV-related malignancies (Young and Rowe, 1992; Liebowitz and Kieff, 1993). In view of its association with clinical features related to type of latency, LMP1 may influence invasiveness of EBV associated tumors categorized as types II and III. Tumor invasion consists of three major steps, e.g. adhesion, degradation and motility (Liotta, 1992). In this study we demonstrated that transformation of MDCK cells by LMP1 resulted in morphological change, reduced cell-cell adhesion and high motility. MDCK cells, which form spherical cysts in three-dimensional collagen gel matrix, form branching tubules following exposure to HGF (Montesano et al., 1991a,b). MDCK cells forming branching tubules share some of the properties of metastatic tumor cells, such as reduced cell-cell adhesion, high motility and degradation of extracellular matrix components including type I collagen. MDCK cells transformed by LMP1 spontaneously formed branching tubules in collagen gel without HGF treatment. Transformation of MDCK cells by LMP1 resulted in morphological changes similar to that observed with HGF treatment. Thus, expression of LMP1 gene in MDCK cells has a similar effect to HGF treatment. In this study we observed induction of PAI-1, uPA and ets1 gene expression during transformation of MDCK cells by LMP1, and interestingly HGF-treatment of MDCK cells also stimulated expression of these genes (Fafeur et al., 1997). In response to HGF, the intracellular portion of the c-Met proto-oncogene receptor becomes autophosphorylated and binds to a number of proteins, including phosphatidylinositol 3-kinase, phospholipase, Cγ, pp60c-src, Grb-2, SHC, and GAB1 (Bardelli et al., 1992; Ponzetto et al., 1994; Pelicci et al., 1995). The CTAR1 domain of LMP1, which was reported to induce expression of EGFR through the TRAF signaling pathway (Mosialos et al., 1995; Devergne et al., 1996; Kaye et al., 1996), was responsible for transformation of MDCK cells. However, the CTAR2 domain, which is the dominant NF-κB and JNK activating region (Huen et al., 1995; Kaye et al., 1995; Eliopoulos and Young, 1998), was not essential for transformation of MDCK cells. The signaling from CTAR1 domain is also essential and sufficient to induce initial proliferation of EBV-infected resting B lymphocytes (Kaye et al., 1995; Izumi et al., 1997). It still remains to be solved which transduction pathway is activated by the CTAR1 domain of LMP1, which shares a common effect with signaling from c-Met HGF receptor.

LMP1 reportedly reduces expression of E-cadherin in a keratinocyte cell line (Fahraeus et al., 1990), however, the cell dissociation caused by HGF-treatment is not due to downregulation of E-cadherin expression but due to counteraction of junctional assembly through tyrosine-phosphorylation of the E-cadherin-catenin complex (Nabeshima et al., 1998; Grisendi et al., 1998; Davies et al., 1999; Hiscox and Jiang, 1999). Considering the similar morphological effects caused by HGF-treatment and LMP1 transformation, reduced cell-cell adhesion caused by the expression of LMP1 is more likely due to phosphorylation of E-cadherin-catenin complex.

Expression of Ets-DN in MDCK cells transformed by LMP1 resulted in downregulation of uPA expression and cell motility. These results suggest that among various phenotypical changes induced by LMP1 expression, regulation of uPA gene expression and enhanced cell motility are mediated at least in part by Ets1. Since the uPA gene promoter contains a binding site for Ets, and Ets1 is a major transcription factor regulating uPA gene expression, enhanced expression of uPA gene in transformed cells is largely attributed to overexpression of Ets-1 (Watabe et al., 1998; D'Orazio et al., 1997; Stacey et al., 1995). uPA is a serine protease and contributes to tumor invasion by degrading extracellular matrix components, and thus its expression is widely correlated with tumor metastasis (Testa and Quigley, 1990; Bindal et al., 1994). PAI-1 is an inhibitor of uPA, but paradoxically expression of PAI-1 also correlates with tumor metastasis (Sappino et al., 1991; Grondaho-Hansen et al., 1993; Foekens et al., 1994). As described above, expression of PAI-1, uPA and ets1 genes is induced in MDCK cells by HGF treatment, however, induction of PAI-1 gene expression takes place earlier than that of uPA and ets1 genes (Fafeur et al., 1997). This suggests that Ets1 does not directly regulate expression of PAI-1 gene.

Although Ets1 has oncogenic potential in mice, there is no direct evidence that it plays a role in oncogenesis in human. However, Ets1 expression in tumor cells has been found to correlate with the grade of invasiveness in human tumor tissues (Nakayama et al., 1996; Nakada et al., 1999) and correlates with a degree of invasiveness in breast cancer cell lines (Gilles et al., 1996). A high level of expression of Ets1 was detected by immunostaining in NPC tissue specimen, where it may stimulate expression of genes associated with invasion. The mechanism of regulating cell motility by Ets1 is not fully understood. Ets family members were reportedly involved in the control of cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells (Delannoy et al., 1998). Recently we found that metastasis associated mts1/S100A4 calcium binding protein is implicated in motility of MDCK cells during tubulogenesis and oncogenic transformation (submitted for publication) and will examine whether expression of mts1 gene is controlled by Ets1 or not.

Previously, we reported that LMP1 stimulated expression of matrix metalloproteinase-9 (MMP-9), which is implicated in tumor invasion and metastasis by degrading type IV collagen, a major component of basement membrane (Yoshizaki et al., 1998; Takeshita et al., 1999). Expression of MMP-9 gene was at a very high level in parental MDCK cells and was not further stimulated by transformation with LMP1 (data not shown). However, the MMP-9 gene promoter contains a binding site for Ets in addition to AP-1 and NF-κB (Sato et al., 1992), and thus Ets1 may positively regulate MMP-9 gene expression in different cell lines (Watabe et al., 1998). Therefore, Ets1 may stimulate transcription of many genes associated with tumor invasion and metastasis, and would be an effective target in preventing invasion of EBV-associated tumors.

Materials and methods

Plasmids and expression vectors

The pcDNA3 eukaryotic expression vector (Invitrogen, Carlsbad, USA) which contains the CMV immediate early promoter and neomycin resistant gene was used for the LMP1 expression constructs. Expression plasmids for LMP1 mutants were described previously (Takeshita et al., 1999).

The cDNA sequence encoding Ets-DN, which lacks transcription activation domain and corresponds to amino acids residues 306–441, was obtained by PCR amplification of human Ets-1 cDNA. The cDNa fragment thus obtained was inserted behind the ATG start codon introduced into pCEP4 expression plasmid, which contains hygromycin resistant gene (Invitrogen) (Nakada et al., 1999).

Cell lines and stable transfection

MDCK cells were obtained from Health Science Resources Bank (Osaka, Japan) and cultured in Dulbecco's MEM (DMED) supplemented with 5% fetal calf serum (FCS). MDCK cells stably expressing LMP1 and its mutants were generated by transfecting pcDNA3 plasmid containing each cDNA fragment as described previously (Kadono et al., 1998a). Transfected MCDK cells were cultured in the presence of 600 μg/ml of G418, and several clones were isolated using stainless Peniciline cups 10 days after G418 selection. MDCK cells expressing LMP1 were transfected with pCEP4 plasmid containing Ets-DN cDNA fragment, and were selected under 800 μg/ml hygromycin B for 10 days.

Western blotting

Western blotting to detect LMP1 protein was carried out as described previously using a monoclonal antibody against LMP1 (gift from LA Young) (Kadono et al., 1998a). Densitometric scanning of the data was analysed by 1NIH Image.

Differential display

Total RNA was extracted from MDCK cells transfected with control plasmid or pcDNA3-LMP1 plasmid by the guanidine isothiocyanate method. PolyA RNA was selected by oligo dT cellulose column chromatography. cDNA was synthesized using FITC-labeled GT12N oligo DNA as primers, where N represents A, C or G, and then PCR amplification of cDNA was done using FITC-labeled GT12N and 10 mer arbitrary oligo primers (Noritake et al., 1999). PCR products were separated on 4 and 6% polyacrylamide gels and were analysed by FluorImager 585 (Molecular Dynamics, Sunnyvale, USA). PCR fragments differentially amplified were picked up from the gels and were further PCR amplified with unlabeled primers. Amplified fragments were labeled with 32P and used as probes for Northern hybridization to confirm the differential expression of the gene.

cDNA cloning

A cDNA library was constructed using a Uni-ZAP XR vector kit according to the manufacturer's instruction (Stratagene, La Jolla, USA). Screening of the library was performed using 32P-labeled PCR fragments obtained as above. Nucleotide sequences were determined by automated sequencer Model 373A (Applied Biosystems, Foster City, USA).

Northern hybridization

Total RNA samples (5 μg) extracted from control and LMP1 MDCK cells were separated by electrophoresis on a denaturing 1.0% agarose gel and transferred onto to nylon membrane Hybond-N (Amersham, Buckinghamshire, UK). Hybridization and preparation of 32P-labeled probes were carried out as described previously (Kadono et al., 1998). Radioactivity was analysed by a Bioimage Analyzer BAS 1000 (Fuji Photo Film Co., Tokyo, Japan).

Collagen gel culture

To make the collagen mixture, seven volumes of ice cold type I collagen stock solution (Nitta Gelatin, Osaka, Japan) as mixed with two volumes of 5× concentrated DMEM and one volume of 0.05 N NaOH containing 2.2% NaHCO3, and 200 mM HEPES. Cells were suspended in the collagen mixture at 5×104 cells/ml and incubated at 37°C for 30 min until the mixture was allowed to gel, and was then overlayed with standard medium. Cells were incubated with 50 ng/ml recombinant human HGF (TOYOBO, Osaka, Japan).

Scrape-wound migration assay

Confluent monolayers of control LMP1 MDCK cells were scraped by plastic pipet and cells migrating into the scraped area were observed after 5 h.

Immunolocalization of Ets1 and LMP1 in the NPC tissues

Tissue samples of NPC were fixed with paraformaldehyde, and the paraffin sections were immunostained using the monoclonal antibodies to Ets1 (Santa Cruz, Santa Cruz, USA) and LMP1.

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Acknowledgements

We thank Dr EW Thompson for critical reading of the manuscript, Dr N Raab-Traub for expression plasmids for truncated LMP1, and Dr LA Young for the monoclonal antibody against LMP1. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan and by a grant from NOVARTIS Foundation (Japan) for the Promotion of Science.

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Correspondence to Hiroshi Sato.

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Keywords

  • EB virus
  • LMP1
  • MDCK
  • Ets1
  • transformation
  • tubulogenesis

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