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[Cancer Research 61, 7985-7991, November 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Expression of Dominant-negative Form of Ets-1 Suppresses Fibronectin-stimulated Cell Adhesion and Migration Through Down-Regulation of Integrin {alpha}5 Expression in U251 Glioma Cell Line1

Daisuke Kita, Takahisa Takino, Mitsutoshi Nakada, Tomoya Takahashi, Junkoh Yamashita and Hiroshi Sato2

Department of Molecular Virology and Oncology [D. K., Ta. T., M. N., To. T., H. S.], Center for the Development of Molecular Target Drugs, Cancer Research Institute [H.S.], Department of Neurosurgery, School of Medicine [D. K., M. N., To. T., J. Y.] Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ets transcription factors are associated with tumor malignancy. We reported previously that the stable transfection of the dominant-negative form of Ets-1 (Ets-DN) in the glioma cell line U251 induced down-regulation of urokinase-type plasminogen activator mRNA expression and invasiveness (M. Nakada et al., J. Neuropathol. Exp. Neurol., 58: 329–334, 1999). Here we analyzed effects of Ets-DN expression on cell adhesion, migration, and phosphorylation of focal adhesion kinase. U251 cells expressing Ets-DN (U251-DN) showed reduced cell adhesion, spreading, and extension of actin stress fibers on dishes coated with fibronectin but not on dishes coated with collagen. Migration of U251-DN cells was found to be significantly inhibited compared with that of parental cells when examined by wound-induced migration assay on fibronectin-coated dishes. Phosphorylation levels of focal adhesion kinase in U251-DN cells were also attenuated on dishes coated with fibronectin. Reduced expression level of integrin {alpha}5 subunit in U251-DN cells was demonstrated by semiquantitative reverse transcription-PCR analysis. Semiquantitative reverse transcription-PCR of surgical samples of brain tumors revealed that the expression level of Ets-1 mRNA correlated with that of integrin {alpha}5 mRNA in glioma. The experimental metastatic ability of U251-DN cells examined in chick embryo was considerably lower than that of parental cells. These results suggest that Ets-1 contributes to glioma malignancy by up- regulating expression of the integrin {alpha}5 subunit, which composes integrin {alpha}5ß1 and mediates intracellular signaling and the subsequent acceleration of the invasive process, including cell adhesion and migration.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ets transcription factors regulate many genes associated with tumor invasion, angiogenesis, cell adhesion, and organ development (1) . The Ets family comprises >30 members sharing a highly conserved DNA binding motif termed the ETS domain (2) . The Ets-DNA binding site has been reported to be contained within the functional promoter site of many genes, including those encoding ECM3 -degrading enzymes (uPA; Ref. 3 ); MMP-1 (4) , MMP-3 (5) , MMP-9 (6) , and their inhibitor (tissue inhibitor of MMP-1; Ref. 7 ); cell adhesive molecules (integrin {alpha}IIb subunit; Ref. 8 ), integrin {alpha}v subunit (9) , integrin ß2 subunit (10) , integrin ß3 subunit (11) , and integrin ß4 subunit (12) ; VE-cadherin (13) ; growth factors and their receptors (platelet-derived growth factor-ß chain (14) ; flt-1 (15) ; polysaccharides (ß1,6-GlcNAc-bearing N-glycans; Ref. 16 ); and transcription factors Ets-1 (17) and Egr (18) .

Ets-1, part of the Ets family of transcription factors and highly expressed in malignant tumors and angiogenic vessels, controls the invasive process. In malignant glioma, overexpression of uPA correlated with that of Ets-1. In addition, expression of the Ets-DN in the glioma CL U251 resulted in down-regulation of uPA expression and less invasiveness, suggesting that Ets-1 contributes to the invasion of astrocytic tumors through up-regulation of uPA (19) . Tumor invasion involves adhesive and migratory events in addition to proteolytic degradation of ECM (20) , all of which can be induced by interactions between tumor cells and ECM components, such as fibronectin, collagen, and laminin. ECM-integrin interaction generates intracellular signaling, which induces focal adhesion, actin cytoskeleton formation, cell migration, cell growth, and various gene expressions.

In this study, we investigated the effect of Ets-DN expression in U251 glioma cells on cell adhesion, migration, and phosphorylation of FAK, which initiates intracellular signaling. The results suggest that Ets-1 regulates expression of integrin {alpha}5, which mediates intracellular signaling through interaction with fibronectin and accelerates subsequent invasive events in glioma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Cell Culture.
The human glioma CL U251 was obtained from American Type Culture Collection and cultured in DMEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FBS.

Construction of Ets-dominant-negative Plasmid and Cell Transfection.
The cDNA-encoding Ets-DN, which lacks a transcription activation domain and corresponds to amino acid residues 306–441, was inserted into pCEP4 expression plasmid (Invitrogen, Groningen, The Netherlands; pCEP4-Ets-DN) as described (21) . U251-DN cells were obtained by transfecting pCEP4-Ets-DN into U251 cells by the calcium phosphate method. Transfected U251 cells were cultured in the presence of 800 µg/ml hygromycin B, and resistant cells were cloned.

RNA Extraction and Northern Blotting.
Total RNA was isolated by ISOGEN (Nippon Gene, Toyama, Japan), according to the manufacturer’s instruction. Northern blotting was carried out as described previously (22) . Briefly, the RNA samples (5 µg/lane) were electrophoresed on 1% agarose gels containing 2.2 M formaldehyde and transferred onto Hybond N+ membranes (Amersham Pharmacia Biotech). The membranes were hybridized with 32P-labeled probes for uPA, ets-1, and GAPDH as reported previously (19) . Radioactivity was analyzed by a Bioimage Analyzer BAS 1000 (Fuji Photo Film, Tokyo, Japan).

Three-dimensional Cell Culture.
Type-I collagen gel for three-dimensional cell culture (Nitta Gelatin, Osaka, Japan) was prepared as described previously (21) . Briefly, 1 ml of collagen gel containing 5 x 104 cells was polymerized in each well of a 24-well plate and then covered with 1 ml of DMEM containing 10% FBS. These cultures were maintained for 7 days and then observed light microscopically.

Cell Adhesion Assay.
Cell adhesion assay was carried out as described previously (23) . Briefly, 96-well plastic plates were coated with either 10 µg/ml human fibronectin (Asahi Techno Glass, Tokyo, Japan) or 30 µg/ml Type-I collagen (Nitta Gelatin) overnight at 4°C and then blocked with 1 mg/ml BSA dissolved in PBS for an additional 1 h at 37°C. Control dishes were prepared by blocking with BSA alone. Cells incubated in DMEM containing 0.5% FBS for 12 h were harvested as single cell suspensions by treatment with EDTA and trypsin. Then, they were immediately treated with trypsin inhibitor and washed with Hanks’ solution. The cells were suspended for 30 min at 37°C in DMEM containing 1 mg/ml BSA and plated at 1 x 104 cells/well. Cells were allowed to adhere to the dishes for 2 h at 37°C and then stained with 1% crystal violet. After the plate had been washed with PBS, the crystal violet bound to the cells was eluted with 10% acetic acid and measured by absorption at 590 nm using an ImmunoMini NJ-2300 spectrophotometer (System Instruments, Tokyo, Japan).

Immunofluorescence Staining of Actin Filaments.
Glass coverslips of 12-mm diameter (Carolina Biological Supply Co., Burlington, NC) were coated with fibronectin or Type-I collagen as described above. The cells were plated onto the coverslips in 35-mm dishes and cultured for 12 h in DMEM supplemented with 10% FBS. They were fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.5% Triton X-100 and 4% paraformaldehyde in PBS for 5 min. Actin filaments were stained with rhodamine-labeled phalloidin (Molecular Probes, Inc., Eugene, OR).

Wound-induced Migration Assays.
Wound-induced migration assay was performed on ECM-coated dishes as described elsewhere (24) . Cells suspended in DMEM with 10% FBS (2 x 105 cells/well) were plated onto 12-well plastic plates coated with either fibronectin or collagen plates and incubated for 24 h. Then, subconfluent monolayers of the cells were scraped with a plastic pipette tip and washed with Hanks’ solution twice, and the medium was replaced with serum-free DMEM. Cells were photographed at 0 and 6 h after scraping, and the distance between migrating cell fronts was measured (n = 20/CL).

Immunoprecipitation and Immunoblotting.
U251 cells were harvested by trypsinization, washed twice with DMEM supplemented with 10% FBS, kept in suspension for 30 min, and then replated onto culture dishes coated with either 10 µg/ml fibronectin or 30 µg/ml Type-I collagen. One or 3 h after plating, cells were washed twice with ice-cold PBS and dissolved in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, 1% NP-40, 0.25% sodium deoxycholate, and protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany). Cell lysates were centrifuged at 20,000 x g for 30 min at 4°C to remove insoluble materials. The protein concentration of each cell lysate was determined using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL) and was made equal with cell lysis buffer. Polyclonal antibody against FAK (Santa Cruz Biotechnology, Santa Cruz, CA) was added, and incubation carried out for 2 h at 4°C followed by sedimentation with GammaBind Plus Sepharose (Amersham Pharmacia Biotech). The immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blocked with 3% BSA in Tris-buffered saline-T (50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature, then probed with horseradish peroxidase-conjugated antiphosphotyrosine antibody (BD Transduction Laboratories, Lexington, KY) for 2 h at room temperature. Binding of horseradish peroxidase-conjugated antiphosphotyrosine antibody was detected by exposure to Hyperfilm ECL (Amersham Pharmacia Biotech) after treatment with SuperSignal (Pierce Chemical). The membranes were then stripped for reprobing with 2% SDS and 100 mM ß-mercaptoethanol in 62.5 mM Tris-HCl (pH 6.8) for 20 min at 70°C. Stripped membranes were washed extensively with Tris-buffered saline, placed in 3% BSA-blocking buffer overnight, and then reprobed with anti-FAK monoclonal antibody (BD Transduction Laboratories).

Clinical Samples and CLs.
Fresh human brain tumor tissues were obtained from 15 patients with astrocytic tumor (5 LGAs, 5 AAs, and 5 GBs) and 5 patients with Meta brain tumor who underwent therapeutic removal of brain tumors. The classification of human brain tumors used in this study is based on the revised WHO criteria for tumors of the central nervous system. All of the tumor tissues were obtained at primary resection, and none of the patients had been subjected to chemotherapy or radiation therapy previously. NB tissues were obtained from 4 patients undergoing temporal lobectomy for epilepsy. The samples were snap frozen in liquid nitrogen to obtain total mRNA. All of the clinical samples were obtained with the informed consent of the patient.

U251, U87, and T98 glioma CLs were also used in this analysis. They were obtained from American Type Culture Collection and cultured in DMEM supplemented with 10% FBS.

Semiquantitative RT-PCR.
Semiquantitative RT-PCR was performed as described previously (25) . Briefly, 5 µg of total RNA was converted to a single-stranded cDNA using SuperScript reverse transcription cDNA synthesis kit (Life Technologies, Inc.) in a 20-µl reaction volume. cDNA was amplified by Taq Ex polymerase (TAKARA, Otsu, Japan; 1 µl of single-stranded cDNA solution/50 µl of reaction volume) using specific human primers for uPA (forward primer: 5'-AGAATTCACCACCATCGAGA-3', reverse primer: 5'-ATCAGCTTCACAACAGTCAT-3'), integrin ß1 (5'-ACTTCGGATCTGTACACTTA-3', 5'-AGTAGAGGTTATTCTTCAGT-3'), integrin ß3 (5'-CGGCCAGATGATTCGAAGAA-3', 5'-TCAGTTAGCGTCAGCACGTGTTT-3'), integrin {alpha}2 (5'-TGGTCTCATCAATCTCATCT-3', 5'-TGACATCAGTTGTAATGCAG-3'), integrin {alpha}5 (5'-CCAGCAACAAAGTCTTCTGTGTCAT-3', 5'-TGCTACCTCTCCACAGATAACTT-3'), integrin {alpha}v (5'-GGAACATGCTTTCTTCAAGATGG-3', 5'-AAGGAGCTATGGCACTGCCAAAC-3'), and GAPDH (5'-CCACCCATGGCAAATTCCATGGCA-3', 5'-TCTAGACGGCAGGTCAGGTCCACC-3').

From the 24th to 32nd cycle of PCR, 6 µl of PCR sample were taken every two cycles and electrophoresed on a 1% agarose gel containing 1% Synergel (Diversified Biotech, Boston, MA), which is equivalent to a 3% agarose gel. After electrophoresis, the gels were stained with 1/10,000 concentration of SYBR Green I (FMC, Rockland, ME), and fluorescence intensity was measured using a FluoroImager SI (Molecular Dynamics, Sunnyvale, CA). In the exponential amplification phase of the PCR, the fluorescence intensities of each PCR product were quantified after normalization to the efficiency of GAPDH cDNA amplification.

Metastasis Assay in Chick Embryos.
Metastasis assay in chick embryos was performed as described previously (26) . Briefly, cells (1 x 106 cells/egg) were injected into the chorioallantoic membrane vein of 10-day chick embryos (Plymouth Rock x White Leghorn) and incubated at 37°C for 7 days. Then, embryonic livers were dissected, and the total DNA was extracted to detect human ß-grobin gene by PCR using species-specific primers. The number of cells that metastasized to the liver was evaluated by the semiquantitative PCR technique described above. Histological sections of dissected livers were also examined light microscopically after staining with H&E.

Statistical Analysis.
Differences among the groups were examined by one-way ANOVA for multiple comparison followed by Fisher’s test in all of the experiments.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
U251 Cells Expressing Ets-DN.
U251 cells were transfected with pCEP4-Ets-DN, and stable clones were selected. The expression of 0.4-kb mRNA encoding Ets-DN in these clones was confirmed by Northern blotting. Endogenous ets-1 mRNA (6.8 kb) was also detected in parental U251 cells and U251 cells transfected with control plasmid and pCEP4-Ets-DN plasmid. The mRNA level of Ets-DN in U251-DN1, U251-DN2, and U251-DN3 was higher than that of endogenous ets-1 by 2.57-, 2.93-, and 2.80-fold, respectively. To confirm the effect of Ets-DN, expression of uPA mRNA, which is known to be regulated by Ets-1 in glioma CLs, was investigated by Northern blotting (Fig. 1A)Citation . Expression of uPA mRNA (2.5 kb) was down-regulated by ~50% in U251-DN cells compared with U251-Mock. In three-dimensional collagen gels, parental U251 cells and U251-Mock cells showed scattered and invasive growth with spindle-shaped morphology; however, U251-DN cells exhibited a round morphology and did not spread into the gel (Fig. 1B)Citation .



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  		Fig. 1. Effects of Ets-DN expression in U251 cells. In A, total RNA extracted from parental U251 cells (U251), U251 cells transfected with control plasmid (Mock), or pCEP4-Ets-DN plasmid (DN1, DN2, and DN3) was subjected to Northern blotting with 32P-labeled cDNA fragments specific for ets-1, uPA, and GAPDH. These probes detected mRNA of expected size for ets-1 (6.8 kb), ets-DN (0.4 kb), uPA (2.5 kb), and GAPDH (1.2 kb). In B, U251, U251-Mock, or U251-DN cells were cultured in three-dimensional collagen gel for 7 days as described in "Materials and Methods."

 
Ets-DN Reduced Cell Adhesion and Spreading on Fibronectin-coated Dishes.
The morphology of parental U251, U251-Mock, and U251-DN cells cultured on dishes coated with fibronectin or Type-I collagen was compared. Both parental U251 and U251-Mock cells adhered and spread well on dishes coated with collagen and fibronectin. U251-DN cells also showed adhesion and spreading on collagen coated-dishes; however, they exhibited a round morphology or poor spreading 2 h after being plated on dishes coated with fibronectin (Fig. 2A)Citation .



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  		Fig. 2. Cell adhesion and cytoskeleton formation. In A, U251, U251-Mock, or U251-DN cells were plated onto dishes coated with fibronectin (top panels) or collagen (bottom panels) and incubated for 2 h at 37°C. In B, cells were incubated on dishes coated as above for 12 h and then stained with rhodamine-labeled phalloidin. In C, cells were plated onto dishes coated with Type-I collagen (panel a), fibronectin (panel b), or control BSA alone (panel c) and incubated for 2 h at 37°C. Crystal violet, which stained cells attached to dishes, was eluted with 10% acetic acid and was measured by spectrophotometer at 590 nm. Mean absorbance value from U251-Mock cells attached to dishes coated with fibronectin is shown as 1. Error bars, SE; **, P < 0.01; n.s., not significant, respectively, by Fisher’s exact test.

 
Actin filaments were visualized with rhodamine-labeled phalloidin staining 12 h after plating onto dishes coated with either fibronectin or Type-I collagen. Both parental U251 and U251-Mock cells formed well-developed actin stress fibers on dishes coated with fibronectin or collagen. In contrast, U251-DN cells failed to form a complete cytoskeleton on fibronectin-coated dish, while they developed actin stress fibers on collagen-coated dishes (Fig. 2B)Citation .

To investigate whether Ets-DN expression impairs cell adhesion to fibronectin, the number of cells adhered to dishes coated with fibronectin or collagen was examined. Parental U251, U251-Mock, and U251-DN cells adhered equally to dishes coated with collagen (mean ± SE; 0.97 ± 0.02 for U251, 1.01 ± 0.03 for U251-Mock, 0.95 ± 0.07 for U251-DN1, 0.93 ± 0.09 for U251-DN2, 0.86 ± 0.09 for U251-DN3, arbitrary value; Fig. 2CCitation , Panel a). However, the adhesion of U251-DN cells to fibronectin-coated dishes was significantly reduced (mean ± SE; 1 ± 0.05 for U251, 1 ± 0.10 for U251-Mock, 0.39 ± 0.02 for U251-DN1, 0.52 ± 0.06 for U251-DN2, 0.49 ± 0.09 for U251-DN3, arbitrary value; P < 0.01; Panel b). All of the cells lost some of their adhesive capability when plated onto BSA-coated dishes (mean ± SE; 0.39 ± 0.02 for U251, 0.38 ± 0.02 for U251-Mock, 0.41 ± 0.05 for U251-DN1, 0.42 ± 0.06 for U251-DN2, 0.44 ± 0.08 for U251-DN3, arbitrary value; Panel c). These results demonstrate that the expression of Ets-DN specifically reduced cell adhesion and spreading on fibronectin.

Ets-DN Reduced Cell Motility.
The effect of Ets-DN expression on cell motility was examined by wound-induced migration assay. Cell migration during 6 h after wounding was measured on dishes coated with fibronectin or collagen. Migration distance for U251-DN cells on fibronectin-coated dish was ~60% of that for parental U251 and U251-Mock cells (mean ± SE; 49.9 ± 3.40 for U251, 48.2 ± 4.27 for U251-Mock; 27.8 ± 2.84 for U251-DN1, 30.9 ± 7.22 for U251-DN2, 26.3 ± 6.88 for U251-DN3; µm/6 h; Fig. 3Citation , Panel a). However, there was no significant difference among these cells in migration distance on collagen-coated dishes (mean ± SE; U251, 26 ± 2.73; U251-Mock, 25.4 ± 4.43; U251-DN1, 23.9 ± 5.22; U251-DN2, 23 ± 5.12; U251-DN3, 22.7 ± 5.01 µm/6 h; Panel b).



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  		Fig. 3. Wound-induced migration assay. Confluent monolayers of parental, mock-transfected, and Ets-DN U251 cells cultured on dishes coated with fibronectin (panel a) or Type-I collagen (panel b) were scraped by plastic pipette tip, and wound-induced migration of cells was measured after 6 h. Error bars, SE; *, P < 0.05, n.s., not significant, respectively, by Fisher’s exact test.

 
Ets-DN Diminished mRNA Expression of Integrin {alpha}5 and ß3 Subunits.
The alteration of cell adhesion and migration on fibronectin- coated dishes caused by expressing Ets-DN suggested that Ets-DN down-regulates the expression of receptors for fibronectin. Semiquantitative RT-PCR was carried out to compare the expression level of integrin subunits which interact with fibronectin. mRNA expression levels of integrin {alpha}5 and ß3, which compose heterodimeric fibronectin receptors, {alpha}5ß1 and {alpha}vß3, respectively, were markedly reduced in U251-DN cells. Expression of the integrin ß1 subunit, which is a part of receptor for both collagen and fibronectin, was not affected by expression of Ets-DN. No significant difference was detected in the mRNA expression of integrin {alpha}2 and {alpha}v subunits among these cells (Fig. 4)Citation .



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  		Fig. 4. Semiquantitative RT-PCR analysis. mRNA expression levels of GAPDH, uPA, and integrin ß1, ß3, {alpha}2, {alpha}5, and {alpha}v subunit genes in parental U251, U251-Mock, and U251-DN cells were analyzed by RT-PCR as described in "Materials and Methods." The products from the 28th cycle of PCR amplification are shown. The relative mRNA expression level of each gene, which was normalized by amplification of the GAPDH gene, is shown.

 
Decreased Phosphorylation of FAK in U251-DN.
FAK is associated with the intracellular domain of the integrin ß subunit and involved in signaling transduction for cytoskeleton formation, cell adhesion, and cell migration (27) . We examined whether Ets-DN expression in U251 cells affects phosphorylation of FAK on stimulation with fibronectin (Fig. 5)Citation . Phosphorylated levels of FAK in U251-Mock and U251-DN cells in suspension before plating were equally low. On incubation on dishes coated with fibronectin for 3 h, FAK was phosphorylated at its tyrosine to a significantly greater extent in U251-Mock cells than U251-DN cells. In contrast, FAK was phosphorylated to the same extent in U251-Mock and U251-DN cells cultured on collagen-coated dishes.



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  		Fig. 5. Tyrosine phosphorylation of FAK. Mock and Ets-DN-transfected U251 cells were plated onto dishes coated with either fibronectin (panel a) or Type-I collagen (panel b) and harvested at the time point for immunoprecipitation with anti-FAK antibody. Immunoprecipitated FAK was immunoblotted with antiphosphotyprosin antibody (top panels) and then reprobed with anti-FAK antibody (bottom panels).

 
High-level Expression of Integrin {alpha}5 mRNA in Malignant Glioma Tissues.
mRNA expression levels of the integrin {alpha}5 and ß3 subunits and ets-1 in surgical samples of brain tumors, NBs, and glioma CLs were analyzed by semiquantitative RT-PCR (Fig. 6)Citation . Expression of integrin {alpha}5 mRNA was detected in AAs (four of five cases), all GBs (five of five cases), Meta brain tumors (five of five cases), and glioma CLs (three of three CLs) but only one of five cases of LGA and none of four cases of NB. Statistical analyses of the results of quantitative RT-PCR revealed that the mRNA expression level is significantly higher in the GB and AA samples than in the NB and LGA samples (Panel a). Expression of integrin ß3 mRNA was seen in AA (one of five cases), GB (four of five cases), Meta (four of five cases), and all cases of CL (three of three cells) but not in LGA (zero of five cases) and NB (zero of four cases). The expression level of integrin ß 3 mRNA was significantly higher in the GB samples than NB, LGA, and AA samples (Panel b). Expression of ets-1 mRNA was also observed in AA (one of five cases), GB (four of five cases), Meta (four of five cases), and CL (three of three CLs) but not in LGA or NB (Panel c). Expression of ß1 subunit mRNA was detected in all of the surgical samples and CLs, with AA, GB, Meta, and CL showing higher expression levels than NB and LGA (data not shown). When the expression level of mRNA for the {alpha}5 or ß3 subunit was plotted against that for ets-1 in each case of AA and GB, mRNA levels of ets-1 significantly correlated with those of {alpha}5 (correlation coefficient; r = 0.818, P < 0.01, n = 10; Panel d) and ß3 integrin (correlation coefficient; r = 0.649, P < 0.01, n = 10; Panel e).



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  		Fig. 6. Integrin {alpha}5, ß3, and ets-1 mRNA expression in surgical samples. In A, mRNA levels of integrin {alpha}5, ß3 subunits, and ets-1 in NBs (n = 4), LGAs (n = 5), AAs (n = 5), GBs (n = 5), Meta brain tumors (n = 5), and glioma CLs (n = 3) were analyzed by RT-PCR. The amounts of PCR amplification product for each gene at the 28th cycle were compared. The relative mRNA levels of the integrin {alpha}5 subunit (panel a), the integrin ß3 subunit (panel b), and ets-1 (panel c) are shown. Bars, the mean value; *, P < 0.05, respectively, by Fisher’s exact test. In B, either integrin {alpha}5 (panel d) or ß3 subunit (panel e) mRNA levels were plotted against those of ets-1 in surgical samples of glioma (AA and GB, n = 10). The correlation coefficients for the plots are 0.818 (panel d) and 0.649 (panel e), respectively (P < 0.01).

 
Meta Ability of U251-DN in Chick Embryos.
The Meta ability of U251-DN cells was compared with that of parental or U251-Mock cells in chick embryos. Histological examination of liver sections demonstrated that apparently fewer Meta colonies were formed by U-251-DN cells compared with parental or U251-Mock cells (Fig. 7A)Citation . PCR amplification of the human ß-globin gene fragment from embryonic liver samples also showed that both parental and U251-Mock cells had undergone liver metastasis. In contrast, U251-DN-1 or U251-DN-2 cells inoculated into chick embryo did not form metastasized foci in liver as effectively as parental or Mock-U251 cells (number of metastasized cell per 104 liver cells, mean ± SE; 7.60 x 102 ± 4.47 x 102 for U251, 1.34 x 103 ± 0.62 x 103 for U251-Mock, 3.57 ± 0.32 for U251-DN1, and 2.90 x 101 ± 0.93 x 101 for U251-DN2; Fig. 7BCitation ). These results indicate that the abilities of U-251-DN cells to form Meta colonies in the liver were suppressed.



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  		Fig. 7. Detection of metastasis in embryonic liver. Parental U251, U251-Mock, U251-DN1, and U251-DN2 cells (1 x 106 cells/egg) were inoculated into the chorioallantoic membrane vein and incubated for 7 days. In A, histological sections were prepared from dissected livers. H&E, x200. White arrows, the foci of metastasized U251 glioma cells. In B, the number of metastasized U251 cells was measured by PCR as described in "Materials and Methods."

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We reported previously that the expression level of ets-1 mRNA was correlated with malignancy of astrocytic tumors, and the expression of Ets-DN in U251 glioma cells reduced uPA expression and invasiveness (19) . In the present study, we demonstrated that the expression of Ets-DN in U251 glioma cells induced a marked decrease in their adhesive property, a reorganization of actin stress fibers, and motility on dishes coated with fibronectin but not on dishes coated with Type-I collagen. Fibronectin has been identified among ECM proteins as a potent mediator of cell adhesion, migration, and growth. In glioma tissues, fibronectin was mainly immunolocalized to the basement membrane of the tumor vessels and the ECM surrounding the tumors (28) . In vitro, fibronectin stimulates glioma cell adhesion and migration (28 , 29) . Thus, the interaction of glioma cells with fibronectin is a key step in glioma cell invasion. Actually, expression of Ets-DN in U251 cells down-regulated experimental metastasis in chick embryos.

Among integrins which are cell surface receptors for ECM, the {alpha}5 integrin subunit recognizes only fibronectin as its ligand, composing an {alpha}5ß1 heterodimer, and correspondingly, the ß3 subunit heterodimerized with the {alpha}v integrin subunit binds fibronectin and vitronectin but not collagen (30) . However, it was reported that neutralizing antibodies against {alpha}5, ß1, and {alpha}5ß1 integrin inhibited attachment of U251 glioma cells to fibronectin, but an antibody against {alpha}vß3 integrin did not, suggesting that {alpha}5ß1 integrin plays an important role in adhesion to fibronectin in U251 cells (31) . It was also reported that variants of Chinese hamster ovary cells, which are deficient in the expression of the {alpha}5ß1 integrin, reduced normal haptotactic motility on fibronectin, regardless of the sufficient expression of the {alpha}vß3 integrin (32) . These indicated that the {alpha}5ß1 integrin plays a more important role in interaction with fibronectin than {alpha}vß3 integrin.

In this study, we demonstrated that Ets-DN down-regulated the expression of integrin {alpha}5 and ß3 in U251 cells. This suggested that the Ets family of transcription factors is involved in transcriptional regulation of the {alpha}5 subunit gene, the product of which mediates adhesion to fibronectin and subsequent fibronectin-stimulated migratory events. Actually, malignant gliomas display high-level expression of the integrin {alpha}5 subunit, which is well correlated with both pathological malignancy and expression of Ets-1. Both the integrin {alpha}5 and ß3 gene promoters contain a binding site for Ets adjacent to the AP-1 site and are regulated by Ets transcription factors (33 , 34) . In U251 cells, the expression of Ets-DN down-regulated transcription from the {alpha}5 gene promoter, as demonstrated by luciferase reporter assay (data not shown).

Although there is evidence that the expression of integrin {alpha}5 stimulates adhesion and migration of tumor cells on ECM, it was reported that neutralizing antibodies to the {alpha}5 subunit enhanced glioma migration (35) . Similarly, the overexpression of the {alpha}5ß1 heterodimer reduced tumorigenicity in K562 erythroleukemia cells (36) , Chinese hamster ovary cells (37 , 38) , and HT29 colon carcinoma cells (39) . The roles of integrin {alpha}5, which has either a facilitatory or an inhibitory effect on cell migration, may depend on cell type and ECM components.

As described above, Ets-1 is a major Ets transcription factor and associated with the malignant phenotype of tumor cells. Expression of antisense RNA for ets-1 mRNA in endothelial cells down-regulated the expression of integrin ß3, adhesion to vitronectin, and phosphorylation of FAK (11) . Expression of the Ets-DN in endothelial cells also impaired cell adhesion to vitronectin, cell proliferation, cell migration, formation of the cytoskeleton, and the organization of the capillary-like structure in collagen gel (40) . Stable expression of Ets-DN has also been shown previously to block the invasiveness of breast and prostate tumor CLs (41, 42, 43) . Collectively, Ets family members are involved in the transcriptional regulation of genes for not only ECM receptors but also ECM-degrading enzymes (4, 5, 6 , 19 , 41) , growth factors and their receptors (14, 15, 16) , and transcription factors (17 , 18) , which are all associated with the malignancy of tumors.

In conclusion, we have shown that the expression of Ets-DN in glioma cells down-regulated integrin {alpha}5 expression, resulting in reduced interaction with fibronectin, which abrogates intracellular signals mediating focal adhesion, actin cytoskeleton formation, cell migration, and experimental metastasis in chick embryos. These "benign" transformations by Ets-DN indicate that the suppression of Ets family members is a therapeutic option for malignant glioma, one of the most lethal of brain tumors.


   ACKNOWLEDGMENTS
 
We thank Y. Endo and T. Sasaki for supplying PCR primers for the amplification of uPA and integrin subunit genes, M. Tanaka for his advice on experimental metastasis assay in chick embryos, and H. Miyamori for his valuable discussion.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant-in-aid for scientific research from Ministry of Education, Culture, Sports, Science and Technology, Japan (B2-11470286 to J. Y. and B2-11240203 to H. S., respectively) and a grant from Japanese Foundation for Multidisciplinary Treatment of Cancer (to H. S.). Back

2 To whom requests for reprints should be addressed, at Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan. Phone: 81-76-265-2750; Fax: 81-76-234-4505; E-mail: vhsato{at}kenroku.kanazawa-u.ac.jp Back

3 The abbreviations used are: ECM, extracellular matrix; Ets-DN, dominant-negative form of Ets-1; AA, anaplastic astrocytoma; BSA, bovine serum albumin; FAK, focal adhesion kinase; FBS, fetal bovine serum; GB, glioblastoma; LGA, low-grade astrocytoma; MMP, matrix metalloproteinase; NB, normal brain; RT-PCR, reverse transcription- polymerase chain reaction; uPA, urokinase-type plasminogen activator; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Meta, metastatic; CL, cell line; VE, vascular endothelial. Back

Received 12/13/00. Accepted 9/ 4/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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