Epidermal Growth Factor Receptor Signaling and the Invasive Phenotype of Ovarian Carcinoma Cells

Özge Alper, Elke S. Bergmann-Leitner, Teresa A. Bennett, Neville F. Hacker, Kurt Stromberg, William G. Stetler-Stevenson

Affiliations of authors: Ö. Alper, Department of Oncology, Georgetown University, Washington, DC; E. S. Bergmann-Leitner (Laboratory of Tumor Immunology and Biology), T. A. Bennett, W. G. Stetler-Stevenson (Extracellular Matrix Pathology Section, Laboratory of Pathology, Center for Cancer Research), National Cancer Institute, Bethesda, MD; N. F. Hacker, Gynaecological Cancer Center, Royal Hospital for Women, Randwick, Australia; K. Stromberg, Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, National Institutes of Health, Bethesda.

Correspondence to: William G. Stetler-Stevenson, M.D., Ph.D., National Institutes of Health, Bldg. 10, Rm. 2A33, MSC 1500, Bethesda, MD 20892–1500 (e-mail: sstevenw{at}mail.nih.gov).

Dedicated to ovarian cancer victim Georgina Emslie Stevenson (December 3, 1925–April 22, 1982) by W. G. Stetler-Stevenson.


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Most (70%–100%) ovarian carcinomas express high levels of the epidermal growth factor receptor (EGFR). To examine the relationship between EGFR and the invasive phenotype, we assessed integrin expression, adhesion, matrix metalloproteinase (MMP) activity, and migration in ovarian cancer cells in which EGFR expression was modified. Methods: NIH:OVCAR-8 human ovarian carcinoma cells were transfected with an expression vector containing the human EGFR complementary DNA in an antisense orientation (EGFR-antisense cells) or the vector alone (vector control cells). We compared vector control and EGFR-antisense cells for cell morphology and adhesion by light microscopy, expression of {alpha}6- and {alpha}3-integrin subunits by flow cytometry, MMP and tissue inhibitor of MMP (TIMP) activity by zymography, and migration by a wound migration assay. In some experiments, EGFR kinase activity in parental cells was inhibited by treatment with PD153035. All statistical tests were two-sided. Results: EGFR-antisense cells were morphologically distinct from vector control cells and had a selective decrease in adhesion to laminin-1 that was not observed with vector control cells (P = .008) or on other extracellular matrix substrates. Compared with vector control cells, cell surface {alpha}6-integrin expression decreased by approximately 80% (difference = 78.7%; 95% confidence interval [CI] = 77.8% to 79.6), MMP-9 activity decreased by approximately 50%, and TIMP activity increased by approximately 50% in EGFR-antisense cells. Vector control cells were highly motile (5.51 arbitrary distance unit; 95% CI = 4.98 to 6.04), whereas the EGFR-antisense cells were not (0.99 arbitrary distance units; 95% CI = 0.38 to 1.60). The morphology and integrin profile of NIH:OVCAR-8 parental cells treated with PD153035 were similar to those of the EGFR-antisense cells. Conclusions: Reduced EGFR expression in ovarian carcinoma cells decreased their adhesion to laminin-1, expression of the {alpha}6-integrin subunit (a well-characterized laminin-1 receptor), and MMP-9 activity. These data support the hypothesis that EGFR overexpression in ovarian cancer cells results in multiple phenotypic changes that enhance the invasive phenotype.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ligand binding and subsequent activation of the epidermal growth factor receptor (EGFR) initiate a variety of cellular changes, including the rapid alteration in cell morphology, cytoskeletal organization, and EGFR redistribution (1). Increased levels of EGFR messenger RNA (mRNA) and protein have been observed in a variety of human cancers (2). The epidermal growth factor (EGF)-related growth factors play a role in human cancer growth through autocrine and paracrine mechanisms (3). High levels of EGFR expression in primary tumors are associated with the presence of metastatic disease, an increased probability of tumor recurrence, and poor patient survival (4). Relative to normal ovarian epithelium, tissue extracts of more than one third of ovarian carcinoma tissues have elevated levels of factors that competed for binding 125I-EGF to EGFR (5). This finding and the observation of increased EGFR expression in approximately 70% of ovarian carcinomas suggest that the EGF-ligand–EGFR axis is an important mechanism for supporting the autocrine growth of these tumors (2).

Although 17 ovarian carcinoma cell lines examined expressed substantial-to-high levels of EGFR and the majority were growth stimulated by the EGF-related ligand, transforming growth factor-{alpha} (TGF-{alpha}) (6), little is known about the mechanistic role of EGFR in the pathogenesis and progression of ovarian carcinoma. Decreased levels of EGFR expression by antisense oligonucleotides or antisense complementary DNA transfection, with concomitant suppression of the malignant phenotype, have been reported for a number of human cancer cell lines, including ovarian cancer (7,8). We have found previously that EGFR-antisense transfection of human NIH:OVCAR-8 ovarian cancer cells decreases EGFR expression and activation (autophosphorylation) in response to EGF stimulation (8). EGFR-antisense expression was found to have little effect on erbB-2 but did increase erbB-3 expression (8). EGFR-antisense cells also showed reduced proliferation and tumor formation in nude mice. A morphologic alteration in EGFR antisense cells was associated with a decrease in the expression of E-cadherin, {alpha}-catenin, and {beta}-catenin. We hypothesized that EGFR-regulated expression of these cell-adhesion proteins may enhance cell growth and adhesiveness. However, the direct effects of EGFR antisense-mediated alterations in cell-matrix adhesion, integrin expression, protease expression, and migration have not been addressed.

Cell invasion depends on the integration of cellular adhesion events with extracellular matrix proteolysis and migration (9). Proliferating carcinoma cells traverse the subepithelial basement membrane by altering their attachment and secreting proteolytic enzymes that degrade the extracellular matrix. Both alterations in tumor cell adhesiveness and tumor-associated protease activity facilitate migration to allow invasive tumor cells access to the surrounding stroma and vascular compartment, where they can metastasize to other sites. Invasion through the extracellular matrix is an active process that is accompanied by the destruction of some extracellular matrix structural components and modification of other components to reveal cryptic sites, such as those present in laminins and type IV collagen, that modify cell growth and migration (9,10).

During the progression of epithelial cancers of the ovary, tumor cells invade the basement membrane to reach the underlying ovarian stroma. In addition, the sloughing of ovarian cancer cells from the ovarian surface can lead to formation of a malignant ascites. Ovarian cancer frequently spreads by seeding of tumor cells onto the mesothelial lining of the peritoneal cavity, from which they invade organs of the peritoneal cavity (11). This process of tumor cell dissemination and metastasis also depends on interactions of the tumor cells with normal stromal cells (i.e., fibroblasts, endothelia cells, and mesothelial cells) and the local tissue microenvironment (9,12).

Tumor cell migration and invasive behavior are the net result of activation of both GFR signaling pathways and integrating altered cell–extracellular matrix adhesive events. The response of tumor cells to EGFR signaling can also be modulated by changes in cell adhesion to the extracellular matrix. EGFR signaling increases cellular adhesion to a variety of extracellular matrix proteins, and these adhesive interactions can be inhibited by blocking either intracellular tyrosine kinase activity or ligand-induced EGFR activity with monoclonal antibodies (13,14). Integration of GFR signaling with signals from integrin-mediated extracellular matrix adhesion to coordinate cell behavior is an emerging field of study (9,12). The convergence of these signaling pathways can influence cell proliferation, differentiation, and migration. Some of these events are accomplished via modulation of integrin function (i.e., "inside out" integrin signaling) (1517).

Like EGFR signaling, integrin signaling events are implicated in tumor progression by cell biologic, biochemical, and molecular genetic data (9,1218). Although a number of integrins have been implicated in tumor progression, the role of {alpha}6 subunit-containing receptors is particularly relevant to epithelial cell malignancies (18). The {alpha}6-integrin subunit associates with {beta}1 or {beta}4 subunits to form functional heterodimers that selectively increase cell adhesion to laminins. The {alpha}6-containing receptors are well-characterized laminin-1 receptors as demonstrated by the loss of cell adhesion to laminin-1-containing substrates in the presence of {alpha}6-neutralizing antibodies or by the competitive binding of laminin-1 peptide fragments (18). The {alpha}6{beta}4 integrin is essential for the organization and maintenance of normal epithelial structures, where it mediates formation of hemidesmosomes that link the intermediate filaments with the extracellular matrix (1921). The {alpha}6{beta}4 integrin also plays a pivotal role in the pathogenesis of invasive carcinomas by stimulating cell migration (22,23). The association of {alpha}6-containing integrins, {alpha}6{beta}4 and {alpha}6{beta}1, with EGFRs has been demonstrated in human carcinoma cell lines (24). Moreover, monoclonal anti-{alpha}6 antibodies can activate the EGFR as demonstrated by coprecipitation and EGFR phosphorylation experiments (24). The integrin {alpha}3{beta}1 also has a novel role in laminin-mediated cell adhesion and in the establishment and/or maintenance of focal contacts and basement membrane integrity (19,21,23,25,26).

EGFR activation increases cell motility, the production of proteolytic enzymes, and the expression of extracellular matrix proteins (2730). Increased EGFR signaling has been associated with tumor invasion and metastasis (29,30). For example, cells with forced high-level expression of full length, wild-type EGFR invaded through an in vitro reconstituted extracellular matrix to a greater extent than control cells (31,32). Migration of these cells was inhibited by a monoclonal antibody that prevents EGF binding and EGFR activation. On the basis of these findings (31,32), it was proposed that signaling domains in the carboxyl terminus of the EGFR are required for EGFR-mediated invasiveness.

EGFR activation of human carcinoma cell lines also increases matrix metalloproteinase-9 (MMP-9) (also known as gelatinase B or 92-kd type IV collagenase) activity, which is associated with increased in vitro cell invasion (3335). The increased invasive activity after EGF-mediated induction of MMP-9 could be blocked by an anticatalytic MMP-9 antibody or by synthetic (low-molecular-weight) or endogenous MMP inhibitors (known as the tissue inhibitors of MMPs or TIMPs). These findings indicate that EGFR activation can result in enhanced MMP-9 expression, which, in turn, facilitates removal of extracellular matrix barriers to tumor invasion.

Thus, because the EGFR can regulate adhesive, proteolytic, and migratory events, we test, in the present study, the hypothesis that decreased expression (with an EGFR-antisense construct) or activation (via treatment with the EGFR kinase inhibitor PD153035) of EGFR in NIH:OVCAR-8 human ovarian carcinoma cells results in loss of phenotypic changes, such as cellular morphology, cell adhesion, integrin subunit, and protease expression and migration that are associated with tumor progression and aggressive tumor behavior.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Culture

The NIH:OVCAR-8 human ovarian cancer cell line (parental) was obtained from the Division of Cancer Therapy Tumor Repository (National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD). The construction, cloning, and selection of the vector control, and EGFR-antisense vector transfected (EGFR-antisense cells) NIH:OVCAR-8 cells, as well as their responses to exogenous EGF stimulation and protein kinase A regulatory subunit expression, have been reported (8,36). NIH:OVCAR-8 parental, vector control and EGFR-antisense cells were cultured in Iscove's modified Dulbecco's medium, supplemented with 10% fetal bovine serum (FBS) (Life Technologies Inc. [GIBCO BRL], Rockville, MD) in a humidified incubator (95% air–5% CO2) at 37 °C. HT1080 fibrosarcoma cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle medium (DMEM) with 10% FBS.

EGFR Activation Status by Western Blot Analyses

The EGFR tyrosine kinase inhibitor PD153035 (37) was obtained from Calbiochem Corp. (San Diego, CA). NIH:OVCAR-8 parental cells were grown in the presence of EGFR kinase inhibitor PD153035 (50–100 nM) (37) in serum-free medium for 24 hours before the basal level of EGFR activation (phosphorylation) or integrin subunit expression analyses. Basal levels of EGFR activation in response to autocrine stimulation were assessed by immunoprecipitation with an anti-EGFR antibody (antibody AB-1 [528], 2 µg/mL; Neomarkers, Fremont, CA) and by western blot analysis as described previously (8). NIH:OVCAR-8 cell lysates were normalized for total protein concentration before immunoprecipitation with anti-EGFR antibodies. The immunoprecipitates were then analyzed on 10% polyacrylamide gel electrophoresis–sodium dodecyl sulfate (SDS) gels before electroblotting to polyvinylidene difluoride membranes. Western blotting was performed with antiphosphotyrosine monoclonal antibody (4G10, 2 µg/mL; Upstate Biologicals, Lake Placid, NY). Western blots were developed with the use of a chemiluminesence kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ).

EGFR and Integrin Subunit Analysis by Fluorescence-Activated Cell Sorting (FACS) Analysis

NIH:OVCAR-8 parental, vector control, and EGFR-antisense cells, grown on plastic tissue culture dishes, were detached from the culture dishes by treatment with 0.25% trypsin (Life Technologies, Inc.), collected by low-speed centrifugation at 1500g for 5 minutes at 4 °C, and suspended at 1 x 106 cells/mL in Ca2+- and Mg2+-free phosphate-buffered saline (PBS). One milliliter of each cell line suspension, as well as parental NIH:OVCAR-8 cells treated with PD153035 as described above, was incubated for 30 minutes at 4 °C with either anti-EGFR monoclonal antibody (Ab-3; Oncogene Research Products, Cambridge, MA) or murine isotype control antibody (Pharmingen, San Diego, CA). Antibody concentrations were 5 µg/mL as suggested by the manufacturer (Oncogene Research Products). The cells were washed twice with ice-cold PBS and suspended on ice for 30 minutes in 1 mL of Ca2+- and Mg2+-free PBS containing fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (Becton Dickinson Immunocytometry Systems, San Jose, CA). Unbound FITC-conjugated secondary antibody was removed by two further washes in Ca2+- and Mg2+-free PBS. The cells were suspended at 2 x 106 cells/mL in Ca2+- and Mg2+-free PBS and analyzed with an FACS analyzer (FACScan; Becton Dickinson Immunocytometry Systems). For integrin labeling, FITC-labeled anti-human CD49f (clone GoH3; Pharmingen), which recognizes the {alpha}6-integrin subunit, and FITC-labeled anti-human {alpha}3 integrin (clone P1B5; Life Technologies, Inc.) were used at the manufacturer's recommended concentrations. Additional controls included incubating the cells with the primary or FITC-labeled secondary antibodies (Becton Dickinson Immunocytometry Systems) alone.

Positive cell staining for analysis of both the EGFR and integrin-subunit staining was defined as fluorescence intensity greater than 99% of that for the corresponding isotype control antibody staining. In these experiments, the gating of fluorescence intensity for positive cells is greater than or equal to the staining intensity of the top 1% of cells observed in the isotype (negative) control.

Cellular Morphology, Adhesion, and Spreading

To examine cell morphology, NIH:OVCAR-8 parental, vector control, or EGFR-antisense cells on plastic tissue culture dishes were seeded and grown to 80% confluence in serum-containing medium. In experiments using the EGFR kinase inhibitor PD153035 (37), NIH:OVCAR-8 parental cells were first grown to 80% confluence in serum-containing medium before treatment with PD135035 for 24 hours in serum-free medium. All cell monolayers were washed with PBS, fixed with 70% (vol/vol) methanol for 5 minutes at room temperature, and then stained with Giemsa (Bio-Rad Laboratories, Richmond, CA) for 15 minutes at room temperature. Stained cells were visualized with the use of a Nikon–Diaphot inverted microscope.

To examine the effects of the extracellular matrix on adhesion and cell spreading, we plated vector control or EGFR-antisense cells (5 x 104 cells/well) on plastic 24-well tissue culture dishes coated with either 0.5 mL of Matrigel (10 mg/mL; provided by Dr. Hynda Kleinman, National Institute of Dental and Craniofacial Research, Bethesda, MD) or laminin-1 (0.5 mg/mL; Collaborative Research Inc., Bedford, MA). The plates were incubated at 37 °C in Iscove's modified Dulbecco's medium containing 10% FBS for various times. Nonadherent cells were removed by being washed gently three times with PBS. Adherent cells were visualized and counted with the use of an inverted scope.

Quantitative Analysis of Adhesion

Cell adhesion was quantified by measuring dye uptake as described previously (38). Flat-bottomed, 96-well plastic tissue culture plates were coated with extracellular matrix components (100 µL/well) diluted in PBS for 2 hours at room temperature. Extracellular matrix components tested included the following: Matrigel (undiluted), laminin-1 (0.5 mg/mL), fibronectin (4 µg/mL; Collaborative Research Inc.), and gelatin (1 µg/mL; Sigma Chemical Co., St. Louis, MO). The extracellular matrix solutions were then removed, and the wells were blocked at room temperature with 1 mg/mL fatty acid-free bovine serum albumin (BSA) (Sigma Chemical Co.) in PBS for 30 minutes. The EGFR-antisense and vector control cells were detached with trypsin, resuspended in Iscove's modified Dulbecco's medium containing 10% FBS, counted, and allowed to recover in suspension for 1 hour at 37 °C with gentle rocking. After recovery, the cells were collected by low-speed centrifugation at 1500g for 5 minutes at 4 °C and again suspended at a concentration of 4 x 105 cells/mL in Iscove's modified Dulbecco's medium containing 1 mg/mL of fatty acid-free BSA. One hundred microliters of the cell suspensions was plated per well in triplicate, and the plates were incubated at 37 °C for 3 hours. Unattached cells were removed by gently washing the wells three times with PBS. Attached cells were fixed for 5 minutes in Diff-Quik Fixative (Baxter Scientific, McGaw Park, IL) and then stained for 5 minutes in Diff-Quik Solution II. After staining, the cells were washed gently with PBS, and then 100 µL of a solution of 10% methanol–5% glacial acetic acid was added to each well to extract the dye. The plates were analyzed at 650 nm on an enzyme-linked immunosorbent assay plate reader (Molecular Devices, Mountain View, CA).

Zymogram and Reverse Zymogram Analyses

MMP activities were measured by zymography, an extremely sensitive technique that can detect picogram quantities, as described previously (39). Briefly, cells were grown to 80% confluence in complete medium and then incubated for 24 hours in serum-free medium. Aliquots of serum-free conditioned medium were normalized on the basis of total cell number, diluted in sample buffer (i.e., 50 mM Tris–HCl [pH 6.8], 2% SDS, 0.1% bromophenol blue, and 10% glycerol), applied to 10% (wt/vol) polyacrylamide gels containing 1 mg/mL gelatin as a substrate (Novex, San Diego, CA), and subjected to electrophoresis at 20 mA/gel. The gels were then incubated in 2.5% (vol/vol) Triton X-100 for 60 minutes to remove the SDS and incubated overnight in developing buffer (i.e., 50 mM Tris–HCl, 200 mM NaCl, 5 mM CaCl2, and 0.02% [wt/vol] Brij-35 [Sigma Chemical Co.], pH 7.6). Gels were stained for 3 hours in 30% methanol, 10% glacial acetic acid, and 0.5% Coomassie blue G-250 (Bio-Rad Laboratories), destained for 2 hours in 30% methanol–10% glacial acetic acid, and dried overnight. Dried gels were scanned by use of a flat-bed scanner (Arcus DUOSCAN; Agfa, Agfa-Gevaert, NV), and the data were integrated with the use of National Institutes of Health (NIH) Image 1.6 software (Bethesda, MD).

Gelatin reverse zymography has been shown to reproducibly detect picogram quantities of TIMPs (40). TIMP activity was assayed in polyacrylamide gels containing gelatin as a substrate copolymerized with recombinant pro-MMP-2 as described previously (39). Aliquots of conditioned medium were treated as described above for zymography, with the exception that the samples were separated on 15% polyacrylamide gels containing 0.1% SDS, 2.5 mg/mL gelatin, and 160 ng/mL recombinant pro-MMP-2 (39).

Migration Assay

Cell migration was quantified in the in vitro wound-healing assay as described previously (41). Equal numbers of vector control and EGFR-antisense cells (1 x 105) in Iscove's modified Dulbecco's medium with 10% FBS were seeded into the 10-cm plastic tissue culture dishes. After the cells reached 70% confluence, a single wound was created in the center of the cell monolayer by the gentle removal of the attached cells with a sterile plastic pipette tip. The migration of the cells into the wound was then observed at different time points and in the absence or presence of a synthetic hydroxamate inhibitor of MMPs, BB-94 (100 nM; British Biotechnology, Oxford, U.K.), to determine the effect of MMP inhibition on cell migration. Three or 72 hours after creating the monolayer wound, the cells were fixed and stained with Giemsa as described above. Cells in the wound and at the wound margin were visualized and photographed with the use of an inverted microscope. Cell migration from the border of the wounded area was measured in eight separate high-power fields for both the vector control and EGFR-antisense cells with the use of the NIH Image 1.6 software. The mean migration in arbitrary migration units and 95% confidence intervals (CIs) were determined for both cell lines in the presence and absence of the MMP inhibitor.

Statistical Analysis

All statistical analyses were performed by use of the Prism software package for Macintosh (version 3.0a; GraphPad Software Inc., San Diego, CA). All tests for statistical significance were two-sided and reported as the mean with 95% CIs.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
EGFR Cell Surface Expression and Activation

To assess the role of the autocrine stimulation of EGFR in invasive ovarian carcinoma, we compared the phenotypic characteristics of NIH:OVCAR-3 parental cells with those of EGFR-antisense cells without the addition of exogenous growth factors. FACS analysis by use of anti-EGFR monoclonal antibody revealed that EGFR expression was positive in greater than 95% of vector control cells but in less than 17% of EGFR-antisense cells (Fig. 1Go, A), demonstrating that EGFR-antisense transfection successfully reduced EGFR expression by approximately 80% (difference = 78.7%; 95% CI = 77.8% to 79.6%).



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Fig. 1. Epidermal growth factor receptor (EGFR) cell surface expression and effect of PD153035 on EGFR activation in NIH:OVCAR cells. Panel A: EGFR expression by NIH:OVCAR-8 vector control (Vector) and on EGFR-antisense cells (Antisense) was determined by fluorescent-labeled anti-EGFR antibody (solid lines) staining and fluorescent-activated cell sorting analysis. Isotype antibody-control staining (dotted line) was used to establish limits for negative staining (M1). The threshold for positive staining for EGFR (M2) was set at the upper 99th percentile of the negative control. Panel B: NIH:OVCAR-8 cells were treated with the EGFR kinase inhibitor PD153035 (50–100 nM) for 24 hours before EGFR activation was assessed by immunoprecipitation and western blot analysis. Cell lysates were normalized for total protein concentration, and the EGFR was immunoprecipitated with an anti-EGFR antibody. Immunoprecipitates were analyzed on 10% polyacrylamide gel electrophoresis–sodium dodecyl sulfate gel electrophoresis, transferred to polyvinylidene difluoride by standard techniques, and immunoblotted with an antiphosphotyrosine antibody. Protein bands were visualized by chemiluminescence. Arrow indicates phosphorylated EGFR (P-EGRF). The lower band is nonspecific and is used as a loading control. Band intensity was determined by densitometric analysis.

 
We have demonstrated previously that EGFR-antisense cells have reduced EGFR mRNA expression and reduced EGFR tyrosine phosphorylation both in the basal state and after stimulation by exogenous EGF (8). To determine if subsequent phenotypic changes in EGFR-antisense cells were also related to decreased basal (autocrine) EGFR activation, we examined NIH:OVCAR-8 parental control cells treated with the EGFR kinase inhibitor PD153035 to prevent autophosphorylation of the receptor (37). Treatment of NIH:OVCAR-8 parental cells with PD153035 (50–100 nM) for 24 hours resulted in greater than 80% inhibition of EGFR phosphorylation as determined by immunoprecipitation of the EGFR and western blot analysis with antiphosphotyrosine antibodies (Fig. 1Go, B). In these experiments, the integrated density of the band corresponding to phosphorylated EGFR was decreased from a basal level of 1.0 relative density units to less than 0.15 with PD153035 at all concentrations tested. These results demonstrate that EGFR antisense and PD153035 can alter EGFR expression or activation (phosphorylation), respectively, to a similar degree (>80%).

Analysis of Cell Morphology, Adhesion, and Spreading

To evaluate the morphologic changes associated with the loss of EGFR expression and/or basal activation, EGFR-antisense cells, vector control cells, and parental NIH:OVCAR-8 cells were stained with Giemsa and examined with the use of an inverted microscope (Fig. 2Go). In confluent cultures, NIH:OVCAR-8 parental (Fig. 2Go, Parental) and vector control (Fig. 2Go, Vector) cells formed overlapping cell clusters and cell aggregates (Fig. 2Go, dark [purple] stellate-shaped clusters) that were composed of large polygonal cells, with interconnecting bands of cells that surround small acini of monolayer cells. By contrast, EGFR-antisense cells (Fig. 2Go, Antisense) showed no acinar organization, formed few cell clusters and aggregates, and had a flattened, round morphology in a single-cell monolayer (Fig. 2Go, Antisense; see also Fig. 3Go).



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Fig. 2. Morphology of NIH:OVCAR-8 vector control and epidermal growth factor receptor (EGFR)-antisense cells grown in monolayer culture. NIH: OVCAR-8 parental (Parental), vector control (Vector), and EGFR-antisense cells (antisense) were grown to confluence on tissue culture plastic. Cells were photographed on an inverted microscope (original magnification x150) after Giemsa staining. Note the overlapping bands of cells, cell clusters, and aggregates surrounding acini of large polygonal cells in the NIH:OVCAR-8 parental and vector control cells that are absent in the EGFR-antisense cell cultures.

 


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Fig. 3. Comparison of morphology and adhesion to extracellular matrix components between NIH:OVCAR-8 vector control and epidermal growth factor receptor (EGFR)-antisense cells. Upper panel: NIH:OVCAR-8 vector control (Vector) and EGFR-antisense cells (Antisense) were grown on Matrigel (left panels, original magnification x400) and laminin-1 (right panels, original magnification x400). NIH:OVCAR-8 vector control cells grown on Matrigel and on laminin-1 have a similar polygonal morphology with filopodial membrane extensions and an array of cell membrane protrusions that interconnect cell clusters. By contrast, EGFR-antisense cells have rounded cell morphology on both Matrigel and laminin-1, with isolated single cells and small cell clusters without extensive cell membrane protrusions or connections. Lower panel: Cells were adhered to tissue culture plastic, laminin-1, fibronectin, and gelatin for 3 hours in serum-free medium. Adhered cells were fixed and stained with Diff-Quik Fixative. The dye was extracted in 10% methanol–5% glacial acetic acid and quantified in a plate reader at 650 nm. Bars represent the mean of triplicate determinations and standard deviations and are representative of two independent experiments. Filled bars represent EGFR-antisense cells. Open bars represent NIH:OVCAR-8 vector control cells. O.D. = optical density.

 
NIH:OVCAR-8 cells treated for 24 hours with the EGFR kinase inhibitor PD153035 (37) (50–100 nM) had an altered cell morphology that was identical to that of NIH:OVCAR EGFR-antisense cells (data not shown). These findings suggest that disrupting EGFR function, either by reducing EGFR expression in the antisense cells or by inhibiting EGFR kinase activity in the parental cells, alters cell morphology to produce a flattened and rounded cell appearance, without acinar formation or dense three-dimensional cell clusters.

The ability of certain cancer cells to form cell extensions that interconnect cell clusters while proliferating on Matrigel in vitro is associated with their metastatic potential in vivo (42). Because we observed decreased formation of cell extensions and interconnections of EGFR antisense cells in culture on plastic, we next examined their growth on Matrigel. When grown on Matrigel-coated dishes, NIH:OVCAR-8 vector control cells were polygonal in shape, with filopodial membrane extensions and cylindrical-appearing cell extensions that interconnected cell clusters (Fig. 3Go, Vector Matrigel). When grown on purified, laminin-1-coated tissue culture dishes, NIH:OVCAR-8 vector control cells had a similar morphology (Fig. 3Go, Vector Laminin). EGFR-antisense cells had an altered morphology on both Matrigel or laminin-1-coated tissue culture dishes (Fig. 3Go, Antisense Matrigel and Antisense Laminin). EGFR antisense cells remained viable as isolated, rounded single and small multicell aggregates that did not form tubular extensions on either Matrigel or laminin-1 (Fig. 3Go). These findings suggest that EGFR-antisense cells did not readily spread or form filopodial extensions on laminin-1 or Matrigel, a laminin-containing extracellular matrix.

Changes in the morphology of cells grown on laminin or Matrigel are possibly the result of previously characterized changes in cell–cell adhesion protein expression (8) or of decreased cell adhesion to laminin-1. To examine the latter hypothesis, we tested NIH:OVCAR-8 vector control and EGFR-antisense cells for their ability to adhere to several extracellular matrix substrates, including laminin-1, fibronectin, and gelatin, in a rapid, quantitative in vitro assay. Cell adhesion to these substrates is mediated by different integrin subunits. NIH:OVCAR-8 vector control and EGFR-antisense cells had markedly different adhesion properties when cultured on laminin-1, in contrast with other extracellular matrix substrates (Fig. 3Go, graph). NIH:OVCAR-8 vector control cells adhered to laminin-1 better than EGFR-antisense cells (0.417 optical density [OD] units [95% CI = 0.352 to 0.482] versus 0.289 OD units [95% CI = 0.255 to 0.323], respectively) (Fig. 3Go, graph). These results were statistically significant (P = .008), with a difference between means of 0.128 (95% CI = 0.05 to 0.205). The differences between the adhesion of NIH:OVCAR-8 vector control cells and EGFR-antisense cells to other matrix components or to tissue culture plastic were not statistically significant (Fig. 3Go, graph). These results suggest that the reduction in EGFR expression by EGFR antisense selectively disrupts cell adhesion to laminin-1 but not to collagen or fibronectin.

{alpha}6- and {alpha}3-Integrin Cell Surface Expression

EGFR-antisense cell adhered to fibronectin and gelatin as well as to vector control cells, suggesting that EGFR antisense did not globally disrupt integrin-mediated cell adhesion. For this reason, we investigated altered expression only of integrin subunits that are specifically associated with cell adhesion to laminin-1, the {alpha}6- and {alpha}3-integrin subunits (18). Integrin {alpha}6 and {alpha}3 expression on NIH:OVCAR-8 vector control, EGFR-antisense cells, and PD153035-treated parental control cells (Fig. 4Go) was assessed by FACS analysis. Approximately 93% of NIH:OVCAR-8 vector control cells stained positive for the {alpha}6 subunit, whereas only 14% of EGFR-antisense cells and 67% of PD153035-treated parental control cells stained positive. In EGFR-antisense cells, {alpha}6-subunit expression was reduced in approximately 80% (difference = 78.7%; 95% CI = 72.4% to 84.9%) of cells, which is similar in magnitude to the reduction of EGFR expression observed initially. The smaller degree of reduction in the levels of {alpha}6-integrin subunit expression following PD153035 treatment of parental control cells (difference = 26.2%; 95% CI = 22.3% to 30.1%) may be due to the fact that PD153035 treatment was only for the 24 hours before FACS analysis. By contrast, EGFR-antisense cells continuously inhibit EGFR expression and signaling over an extended period in cell culture or in vivo (8). The findings of reduced {alpha}6 expression in both EGFR-antisense and PD153035-treated cells are consistent with one another and suggest that either reduction in EGFR expression and/or activation results in diminished expression of this laminin-1 receptor subunit.



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Fig. 4. {alpha}6-Integrin subunit expression in NIH:OVCAR-8 vector control, epidermal growth factor receptor (EGFR)-antisense, and PD153035-treated cells. Expression of {alpha}6-integrin subunit (solid line) in NIH:OVCAR-8 vector control cells (Vector), EGFR-antisense cells (Antisense), and PD153035-treated parental cells (50 nM, 24 hours) cells (PD153035) was determined by fluorescence-activated cell sorting analysis. Isotype antibody control staining (dotted line) was used to establish limits for negative staining (M1). The threshold for positive staining for {alpha}6-integrin subunit (M2) was set at the upper 99th percentile of the negative control.

 
We also assessed the expression of the {alpha}3-integrin subunit because the {alpha}3{beta}1 integrin is another receptor for laminin-1 (25,26). Compared with NIH:OVCAR-8 vector control cells, the percentage of EGFR-antisense cells with high levels of cell surface expression of the {alpha}3-integrin subunit was reduced by less than 20%, from 92% to 76% (difference = 16.5%; 95% CI = 14.3% to 18.7%). There was no difference between PD153035-treated parental cells and vector control in the percentage of cells with high levels of {alpha}3-integrin subunit expression (data not shown).

These results demonstrate that the EGFR-antisense reduction of EGFR expression selectively decreases {alpha}6-integrin subunit levels, but not {alpha}3-integrin subunit levels, and this selective reduction in integrin subunit expression is consistent with the change in adhesion only to laminin-1.

Expression of MMPs and TIMPs

Expression of MMPs, especially MMP-2 and MMP-9, can promote cell migration and invasion by the selective proteolysis of extracellular matrix components (810,33,43). Gelatin zymography and reverse zymography analyses were carried out because both are highly sensitive techniques for the detection of picogram amounts of proteases and protease inhibitors (39,40). To examine the relationship between EGFR expression and MMP activity in NIH:OVCAR-8 cells, we analyzed the activity of MMP-2 and MMP-9 and their endogenous inhibitors, TIMPs (Fig. 5Go). Integrated densities for the MMP and TIMP activities were normalized to those in the conditioned medium from HT1080 fibrosarcoma control cells. NIH:OVCAR-8 vector control cell-conditioned medium had high levels of MMP-9 activity relative to that of MMP-2, similar to that observed in conditioned medium from the HT1080 control cells. Culture medium conditioned by EGFR-antisense cells had approximately 50% less activity associated with MMP-9 (Fig. 5Go, Antisense) than culture medium conditioned by NIH:OVCAR-8 vector control cells (Fig. 5Go, Vector), but almost 2.4-fold more MMP-2 activity. It is clear from the zymogram that the levels of MMP-2 activity are much less than those of MMP-9 for all cells examined.



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Fig. 5. Zymogram and reverse zymogram analyses of matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) activity in epidermal growth factor receptor (EGFR)-antisense and vector control cells. Zymogram analysis of MMP-9 and MMP-2 activity (upper panel) and reverse zymogram analysis of TIMP-1 and TIMP-2 activity (lower panel) were carried out by use of conditioned medium from EGFR-antisense (Antisense), vector control (Vector), and the matrix MMP standard HT1080 (Control) cells. Integrated densities, determined by densitometric analysis, were normalized to MMP activity in the control sample and are shown immediately below bands of activity.

 
Reverse zymogram analysis was performed to assess the levels of endogenous TIMPs also present in the NIH:OVCAR-8 cell-conditioned medium. This analysis revealed clearly detectable levels of TIMP-1 and TIMP-2 (Fig. 5Go, lower panel) but not of other TIMPs (data not shown). Integrated densities of the TIMP-1 and TIMP-2 bands were normalized to the levels of inhibitors present in HT1080 fibrosarcoma cell-conditioned medium. The integrated densities of TIMP-1 and TIMP-2 activities increased approximately 50% and 70%, respectively, in the EGFR antisense-conditioned medium relative to those in vector control cells (ratio of integrated densities antisense/vector control = 1.47 and 1.74, respectively). These findings suggest that a reduction in EGFR expression is accompanied by a loss of MMP-9 activity and a gain in TIMP activity as well as an imbalance in MMP/protease inhibitor levels that favors the inhibitors.

Analysis of Migration

Because both {alpha}6 integrins and MMP-9 have been implicated in the migration and invasion of tumor cells, including ovarian cancer cells (22,23,33,44), we examined the migration of NIH:OVCAR-8 vector control and EGFR-antisense cells by using a monolayer-wounding assay (41). In this assay, cells are grown to confluence in monolayers, and their ability to migrate into and across a denuded area of the monolayer is followed over time. NIH:OVCAR-8 vector control cells readily migrated into the wounded area within 3 hours (Fig. 6Go, A). EGFR-antisense cells, which exhibited greatly reduced filopodial membrane extensions and flattened, rounded, nonpolarized morphology, did not migrate into the wounded area within 3 hours (Fig. 6Go, A). Even after 72 hours of continued culture and observation, there was no apparent migration of the EGFR-antisense cells into the wounded area. The mean distance of migration was 5.51 arbitrary distance units (95% CI = 4.98 to 6.04) for the NIH:OVCAR-8 control cell cultures and 0.99 arbitrary distance units (95% CI = 0.38 to 1.60) for the EGFR-antisense cells (Fig. 6Go, B), a statistically significant difference (P<.001) of 4.52 arbitrary distance units (95% CI = 3.62 to 5.42).



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Fig. 6. Monolayer wounding assay for cell migration. Monolayers of vector control (Vector) or epidermal growth factor receptor (EGFR) antisense (Antisense) NIH:OVCAR-8 cells were grown to confluence on tissue culture plastic, and a single, linear wound in the monolayer was created with a sterile pipette tip as described in the "Materials and Methods" section. Panel A: Cells were photographed on an inverted microscope (original magnification x400) after staining with Giemsa. Panel B: Migration into the wounded area was quantified by measuring the migration distance of cells from the wound edge. Net migration was determined as the mean (95% confidence intervals) of at least eight independent measurements from each culture. Student's t test analysis revealed that the difference between vector control and antisense cells was statistically significant (P<.001). Relative unit = arbitrary unit.

 
To determine if MMPs are important in NIH:OVCAR-8 cell migration, we examined the migration of vector control cells treated with BB-94, a synthetic, hydroxamate inhibitor of MMPs. Treatment with BB-94 decreased NIH:OVCAR-8 vector control cell migration (mean, 5.51 relative distance units) to the same levels observed for EGFR-antisense cells (<1.0 relative distance units, data not shown). These findings suggest a requirement for MMP activity during in vitro migration of NIH:OVCAR-8 ovarian cancer cells.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulation of gene expression by EGF results in the modulation of cell adhesion, migration, and differentiation, under both physiologic and pathologic conditions (3,8,3032,45). Aberrant, high expression levels of EGFR and EGFR ligands, such as EGF and TGF-{alpha}, by tumor cells are associated with increased cell proliferation and migration, progression to a transformed phenotype, and an enhanced aggressive tumorigenic phenotype in vivo (3,8,3032,45). These effects are mediated by both autocrine and paracrine mechanisms (3,30). To study the relationship between EGFR expression and the aggressive phenotype of ovarian cancer cells, we transfected NIH:OVCAR-8 human ovarian cancer cells with an EGFR-antisense expression vector to substantially reduce cell surface EGFR expression. Previously, we examined the effects of a decrease in EGFR in NIH:OVCAR-8 cells on parameters associated with cellular proliferation under conditions of exogenous EGF exposure, cell–cell attachment (cadherin and catenin expression), and tumorigenicity (8). We undertook the present study to understand why EGFR-antisense cells were less tumorigenic (8) by assessing in vitro changes associated with the tumorigenic and invasive phenotype under conditions of autocrine (basal) stimulation (i.e., without exogenous EGFR ligands). Under these conditions, we examined cell morphology, adhesion, integrin subunit expression, MMP expression, and migration.

Rapid changes in the morphology of adherent cells occur within minutes after EGF exposure (2,30). We found that reduced surface EGFR expression associated with transfection of the EGFR-antisense vector resulted in marked changes in the cell culture behavior and morphology. The EGFR-antisense cells did not form overlapping cell clusters or acinar structures (Fig. 2Go), were rounded, and did not show filopodial membrane extensions either on tissue culture plastic or on laminin-1 (Figs. 2 and 3GoGo). These findings are consistent with previous reports of altered morphology and adhesion of tumor cells treated with EGFR function-blocking antibodies or kinase inhibitors (2,5,6,13,14, 3033,44) and led us to further investigate the adhesion of EGFR-antisense cells to various extracellular matrix components.

EGF stimulation of tumor cell adhesion to extracellular matrix components can be either enhanced or reduced, depending on the level of EGFR expression (13,14). Furthermore, the opposing effects of increased or decreased adhesion to extracellular matrix components can be blocked by inhibiting EGFR signaling, suggesting that events downstream of the EGFR are responsible for the observed changes in cell adhesion (13,14). Our findings confirm these observations. Compared with NIH:OVCAR-8 vector control cells, adhesion to laminin-1 or laminin-containing extracellular matrix substrates was specifically and statistically significantly decreased in the EGFR-antisense cells. Laminin-1 is a major component of basement membranes. Because interacting with basement membranes is important in malignant epithelial cell invasion, the disruption of EGFR signaling in EGFR-antisense cells, which alters their ability to adhere to laminin-1-containing basement membranes, may at least, in part, account for the reduced invasive and metastatic potential of EGFR-antisense cells.

Altering the expression of specific integrin subunits appears to be one mechanism by which EGFR-antisense cells altered interactions with laminin-1. The {alpha}6-integrin subunit is a principal cell surface receptor for laminin-1. In many epithelial cell types, the {alpha}6-integrin subunit forms a functional heterodimer with the {beta}4-integrin subunit. The {alpha}6{beta}4 integrin is essential for the formation of hemidesmosome structures and for the organization and maintenance of epithelial cell polarity (19,21). However, expression of {alpha}6{beta}4 integrins persists in many tumor cells that have a motile and invasive phenotype and that no longer form stable, adhesive cell–cell contacts (22,23). Thus, there is an apparent shift in {alpha}6{beta}4-integrin function associated with malignant transformation. Expression of {alpha}6{beta}4 integrins dramatically increases the invasive potential of a {beta}4 integrin-deficient colon carcinoma cell line and mediates colon carcinoma cell line migration on laminin-1 by promoting filopodia formation and stabilization (23). Although one study (22) demonstrated that activation of phosphatidylinositol-3 kinase (PI-3K) mediates {alpha}6{beta}4 integrin-induced cell migration on laminin-1 and that the small G protein Rac is required downstream of PI-3K for cell invasion, changes in cell migration were not associated with altered expression of MMP activity.

A previous report (46) has also observed an association between EGFR and {alpha}6{beta}4-integrin expression. Various ovarian cancer cell lines with high EGFR expression levels, such as OVCAR-3, were positive for {alpha}6{beta}4 integrins by immunofluorescence (46), suggesting that increased expression of EGFR may result in a concomitant change in {alpha}6{beta}4-integrin expression and function. This relationship is supported in part by our present study, in that decreased expression of EGFR in NIH:OVCAR-8 cells by EGFR antisense resulted in reduced levels of cell surface {alpha}6-integrin expression. Collectively, these findings are consistent with the hypothesis that increased expression of {alpha}6-integrin subunits, mediated by EGFR-induced signals, is at least one of the mechanisms responsible for altered cell adhesion and enhanced cell migration and invasion. Further examination of the relationship between EGFR and {alpha}6{beta}4 integrins awaits decreased expression of the integrin in NIH:OVCAR-8 cells and analysis of EGFR signaling.

Our results also suggest that the effect of reduced EGFR signaling on integrin-subunit expression is selective. In EGFR-antisense cells, reduction in EGFR expression was not associated with reduced {alpha}3-integrin subunit surface expression. This observation suggests that {alpha}6 subunit-containing integrins are the principal laminin-1 receptors on NIH:OVCAR-8 cells. Furthermore, cell-adhesion functions of integrins may require cooperative functioning with other integrins as well as with nonintegrin receptors (18). For example, {alpha}6{beta}4 integrins interact with laminin to form dynamic adhesive interactions under conditions of laminar flow in vitro, whereas {alpha}2{beta}1 integrins interact with laminin under static conditions (18). That different integrins interact with the same substrate under different conditions is plausible in light of the growing understanding of differences in downstream signaling from integrin receptors, as well as the role of inside-out signaling in regulating integrin affinity (1517). Similarly, our findings of selective reduction of {alpha}6-integrin subunit expression and function in response to decreased EGFR expression, but not global disruption of integrin function, suggest that signaling responses specific to the EGFR pathway control {alpha}6-integrin expression.

Previous studies (12,47) have demonstrated both synergism and cross-talk between signaling pathways activated by GFRs and integrins. To our knowledge, the present study is the first to demonstrate a relationship between EGFR and {alpha}6-subunit expression in ovarian cancer cells. Furthermore, our findings suggest that NIH:OVCAR-8 ovarian cancer cells may require EGFR expression and/or function to maintain {alpha}6 expression and laminin-1 adhesion.

MMP activity has also been implicated in the acquisition of a migratory and invasive phenotype (9,10,29,33,44). Both GFR and integrin-mediated receptor signaling pathways regulate MMP expression (9,10,29,33,44,48). It has been demonstrated that EGF stimulation of ovarian carcinoma cell lines enhances cell invasion through reconstituted basement membranes, such as Matrigel (33,44). In two of four cell lines (33,44), enhanced invasive activity was associated with an induction of MMP-9 and could be inhibited by an anticatalytic MMP-9 antibody. In another study (48), the invasive potential and production of MMP-9 were induced in ovarian cancer cells by fibronectin that was present in human peritoneal cell-conditioned medium. In the present study, we showed that MMP-9 activity was decreased and that TIMP activity was increased in NIH:OVCAR-8 EGFR-antisense cells compared with vector control cells, which retained their migratory phenotype on laminin. By contrast, reconstituted {alpha}6{beta}4-integrin expression in human breast carcinoma cells resulted in the cells acquiring a motile and invasive phenotype that was independent of altered protease activity or localization (23). Although the results reported in this previous publication (23) appear to conflict with our findings of diminished MMP-9 activity in NIH:OVCAR-8 EGFR-antisense cells, these differences may be cell type-specific effects of the pathways that diverge and induce different phenotypes.

In our experiments, EGFR-antisense cells had concomitant, but divergent, changes in the levels of both protease (MMP-9) and protease inhibitor (TIMPs). These findings are similar to changes reported for the protein encoded by the von Hippel–Lindau (VHL) tumor suppressor gene, which is mutated in familial and most sporadic renal cell carcinoma (49). Expression of the wild-type VHL gene in renal cell carcinoma cells with mutant VHL genes resulted in increased expression of TIMP-2, decreased expression of MMP-2, and loss of invasive behavior and branching morphogenesis induced by hepatocyte growth factor/scatter factor (49). The findings in our study concur with this previous report (49) and suggest that the loss of a tumor suppressor gene function (i.e., VHL) or the acquisition of an oncogene or oncogenic function (i.e., high level expression of a GFR, such as EGFR) may result in a shift in the balance of protease and protease inhibitor activities that favors tumor cell invasion.

The human ovarian surface epithelium or ovarian mesothelium is functionally complex, with an ability to proliferate, migrate, and contribute to ovulation and ovulatory repair in response to cyclic hormonal and environmental changes. It has been reported that increased EGFR signaling increased ovarian and prostate tumor cell motility and increased invasiveness. To our knowledge, our study is the first to suggest that loss of EGFR expression negatively regulates both {alpha}6-subunit expression and MMP-9 activity. The use of the EGFR kinase inhibitor suggests that loss of EGFR function, concomitant with reduced EGFR expression in the antisense cells results in reduction of these markers for cell invasion. Confirmation of these findings awaits further detailed analysis of the EGFR signaling pathway through use of selective inhibitors and/or dominant negative strategies. We propose that analysis of {alpha}6-integrin subunit and/or MMP-9 expression may provide useful clinical endpoints in ovarian cancer for monitoring emerging anti-EGFR therapeutic strategies.


    NOTES
 
Supported in part by the Alexander C. and Tillie S. Speyer Foundation (to Özge Alper).

We acknowledge the generous postdoctoral support of the Gynecological Cancer Fund of the Royal Hospital for Women Foundation, Sydney, Australia.


    REFERENCES
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received November 22, 2000; revised July 2, 2001; accepted July 19, 2001.


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