Molecular Human Reproduction, Vol. 5, No. 2, 168-174,
February 1999
© 1999 European Society of Human Reproduction and Embryology
CD9 is involved in invasion of human trophoblast-like choriocarcinoma cell line, BeWo cells
1 Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, 2 Department of Gynecology and Obstetrics, Ohtsu Municipal Hospital, Ohtsu, 3 Institute for Virus Research, Kyoto University, and 4 Institute for Frontier Medical Science, Kyoto University, Sakyo-ku, Kyoto 606, Japan
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Abstract |
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The CD9 molecule is expressed on human extravillous trophoblasts, which invade the endometrium during implantation and placentation. To elucidate the role of CD9 in trophoblastic function, we investigated the expression of CD9 protein and mRNA in BeWo cells, a human trophoblast-like choriocarcinoma cell line, using immunohistochemistry, Western blotting and reverse transcription–polymerase chain reaction (RT–PCR). When BeWo cells were cultured with anti-CD9 monoclonal antibodies (mAb), their invasion through the extracellular matrices was significantly enhanced in a dose-dependent manner. Cell proliferation and human chorionic gonadotrophin production were unaffected. On the other hand, culture in the presence of mAb against integrins
3, 5 and ß1, which partially block the interaction with the extracellular matrices, inhibited BeWo cell invasion. Anti-CD9 monoclonal antibody had a stimulatory effect on BeWo cell invasion in the presence of anti-integrin 3 antibody. In contrast, it had no effect in the presence of mAb against integrins 5 and ß1, which were also highly expressed on BeWo cells. These findings suggest that CD9 has a function connected with the invasive properties of BeWo cells, which is partially mediated by integrin 5ß1. This may relate to the involvement of CD9 in trophoblastic invasion. BeWo cell/CD9/invasion/trophoblast
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The cluster of differentiation antigen (CD) 9 molecule, which was originally reported to be expressed on a pre-B cell line (Kersey et al., 1981
), has been implicated in Schwann cell migration and adhesion (Anton, 1995; Hadjiargyrou and Patterson, 1995). Using immunohistochemiistry, we have found that CD9 is expressed on the extravillous trophoblasts, especially those localized at the feto–maternal interface (Hirano et al., 1999). These cells had ceased invasion (Aplin, 1991). The specific expression of CD9 at the feto–maternal interface suggests that it may have a physiological role in the function of extravillous trophoblasts.
To elucidate the function of CD9 in the extravillous trophoblasts, we examined the effect of anti-CD9 monoclonal antibody (mAb) on the cell proliferation and human chorionic gonadotrophin (HCG) secretion of BeWo cells, a human trophoblast-like choriocarcinoma cell line. The expression of CD9 by these cells was confirmed by immunohistochemistry, Western blotting and reverse transcriptase–polymerase chain reaction (RT–PCR) analysis. We also investigated the effect of anti-CD9 mAb on the invasive ability of BeWo cells. CD9 is known to be associated with ß1-related integrins (Rubinstein et al., 1994; Nakamura et al., 1995). Since BeWo cells have been shown immunohistochemically to express integrins 3, 5, and ß1, we examined the relationship between CD9 and these integrins in the regulation of the invasion by BeWo cells, using anti-integrin 3 and 5 mAb.
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Materials and methods |
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Cells and culture conditions
BeWo, a continuous cell line established from a human choriocarcinoma (Patillo and Gey, 1968
), was obtained from the Japanese Cancer Research Resources Bank and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Dainippon Pharmaceutical Co, Osaka, Japan), 100 IU/ml of penicillin and 100 µg/ml of streptomycin. The cells were maintained as monolayers in 25 ml flasks at 37°C in a humidified atmosphere of 5% CO2 in air.
Antibodies
Two mouse anti-human CD9 mAb [TP-82 and ALB-6, immunoglobulin G1 (IgG1) class] were purchased from Nichirei Co Ltd (Tokyo, Japan) and Cosmo Bio Co Ltd (Tokyo, Japan) respectively (Boucheix et al., 1983; Higashihara et al., 1985). Anti-CD9 mAb, SYB-1, IgG1 (Rubinstein et al., 1994), was a generous gift from Dr C.Boucheix, INSERM U268, Hospital Paul Brousse (Villejuif, France). The mouse anti-human integrin 3 (11G5, IgG1) and 5 (SAM 1, IgG2b) were purchased from Serotec (Oxford, UK) and mouse anti-human integrin ß1 (K20, IgG2a) was obtained from DAKO A/S (Glostrup, Denmark) (Morimoto et al., 1985). Mouse anti-human integrin 3 (P1B5, IgG1), 5 (P1D6, IgG3), and ß1 (P4C10, IgG1) mAbs (Carter et al., 1990) were purchased from Life Technologies Inc (Gaithersburg, MD, USA). In immunohistochemical and Western blot analyses, the anti-trinitrophenyl (TNP) mouse mAb (unrelated mAb, IgG1 class) was used as a negative control (Tsujimura et al., 1990). The fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulins (Dakopatts, Glostrup, Denmark) were used as secondary antibodies for immunohistochemistry. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulins (Dakopatts) were used as the second antibody in Western blotting.
Immunohistochemistry
BeWo cells were grown to confluence on 4-chamber slides (Lab Tec, Chamber Slide, Nunc Inc, Naperville, IL, USA). The cultured slides were gently washed three times with phosphate-buffered saline (PBS), thoroughly dried, and fixed with acetone at –20°C. The slides were incubated with the anti-CD9 mAb (5 µg/ml), anti-integrin 3 (5 µg/ml), anti-integrin 5 (5 µg/ml), anti-integrin ß1 (5 µg/ml), or the anti-TNP mAb (5 µg/ml) for 60 min at room temperature. The antibodies were diluted with RPMI culture medium (Gibco) containing 10% FCS (Dainippon Pharmaceutical Co) and 0.1% NaN3. After washing in PBS, the slides were incubated with FITC-conjugated rabbit anti-mouse immunoglobulins (diluted 1:40) for 30 min at room temperature in the dark. The slides were washed extensively, mounted with an anti-fade agent (Perma Fluor Aqueous Mounting Medium; Immunon, Pittsburgh, PA, USA) and then examined under a fluorescent microscope (Nikon, Tokyo, Japan). Three independent experiments were performed.
Indirect immunofluorescence staining and flow cytometry
The dispersed BeWo cells (2x105) were centrifuged and incubated at 4°C for 30 min with 10 µl of an anti-CD9 mAb (clone ALB-6, 100 µg/ml), an anti-integrin mAb (11G5 for 3, SAM-1 for 5, or K20 for ß1 subunit; 100 µg/ml each), or the anti-TNP mAb (negative control, 100 µg/ml). The cells were washed twice with Hank's balanced salt solution (HBSS), and incubated with 20 µl of FITC-conjugated rabbit anti-mouse immunoglobulins (diluted to 1:40) at 4°C for 30 min in the dark. The cells were then washed twice with HBSS, resuspended in glycerin/ PBS (1:1), mounted on glass slides, and examined by fluorescence microscopy.
For flow cytometry, the cells were washed three times and resuspended in HBSS after the second antibody incubation, and the number of viable cells was determined (FACScan; Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA). Three independent experiments were performed.
RNA isolation
BeWo cells (1x106 cells) were washed three times with PBS and immediately frozen in liquid nitrogen and stored at –80°C until RNA extraction. Total RNAs of these tissues were isolated using a commercial kit (TRIzol; Gibco BRL, Gaithersburg, MD, USA).
RT–PCR analysis of CD9 mRNA in the BeWo cells
Total RNA (5 µg) from BeWo cells were reverse-transcribed with random primers using a commercial kit (First Strand cDNA Synthesis Kit; Pharmacia Inc, Piscataway, NJ, USA). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification with oligonucleotides from the human CD9 cDNA as primers (Boucheix et al., 1991) (sense primer 5'-ACTGTTCTTCGGCTTCCTCT-3': position 321–340; antisense primer 5'-AAAATCCCAAAAATCTTCAT-3': position 774–793) or with human S26 primers (Vincent et al., 1993) (sense primer 5'-GGTCCGTGCCTCCAAGATGA-3': position 8–27; antisense primer 5'-TAAATCGGGGTGGGGGTGTT-3': position 308–327). After PCR amplification, 10 µl from each PCR product was electrophoresed on a 1% agarose gel, and amplified bands were detected by ethidium bromide staining. The resultant PCR fragment was cloned into a pBluescript SK(–) plasmid. DNA sequencing was performed using a commercial kit (BcaBEST Dideoxy Sequencing Kit; Takara, Ohtsu, Japan).
Western blotting
BeWo cells (1x106 cells) were lysed in sample buffer [20 mM Tris–HCl pH 8.6, 1% sodium dodecyl sulphate (SDS), 20% glycerol, Bromophenol Blue], and the lysed proteins were separated under non-reducing condition by 12% SDS–PAGE. The bands were then electrically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, USA) in a buffer containing 25 mM Tris–HCl, 192mM glycine and 20% methanol. The membranes were soaked in a blocking solution [PBS containing 1% bovine serum albumin (BSA)] for 3 h at room temperature, and were then incubated for 1 h with the anti-CD9 mAb (SYB-1, diluted ascites 1:10 000 in PBS containing 0.1% BSA) or the control mAb (anti-TNP, 1 µg/ml in PBS containing 0.1% BSA). The membranes were washed several times with PBS, and incubated for 1h with HRP-conjugated rabbit anti-mouse IgG (diluted 1:1,000 in PBS containing 0.1% BSA). After several washes, the binding of the antibodies was visualized by incubation with 0.5mg/ml of diaminobenzidine and 0.02% H2O2 in PBS.
HCG secretion and cell growth
BeWo cells (2x104) were plated into each well of a 24-well plate in 1 ml of RPMI 1640 with 10% FCS culture medium. The anti-CD9 mAb (ALB-6; 0, 0.5, 1.0, and 5 µg/ml) and the control mAb (5 µg/ml) were then added to the culture medium, and the cells were cultured in triplicate. The media were changed or collected on the next day (day1), day 3, and day 5, centrifuged, and frozen at –20°C until HCG assay. For the assessment of cell growth, the cultured cells were dispersed with 0.05% trypsin (Difco Laboratories, Detroit, MI, USA) and 0.05% EDTA, and the number of cell nuclei was determined by the citric acid–Crystal Violet method (Patterson, 1979).
The concentrations of HCG in the culture media were determined by radioimmunoassay using a commercial kit (DPC Co Ltd, Los Angeles, CA, USA). The detection threshold was 0.3 mIU/ml. The values for the HCG concentration were normalized on the basis of the number of cells.
Invasion assay
The invasion assay was carried out as previously described, with slight modifications (Katsuragawa et al., 1997). BeWo cells were cultured on cell culture inserts (6.4 mm in diameter; Becton Dickinson Labware, Bedford, MA, USA) containing polyethylene terephthalate membranes with 8 µm diameter pores, which were placed in each well of a 24-well tissue culture plate (Corning Costar Co, Cambridge, MA, USA). The upper surface of the filters was coated with cold Matrigel (100 µg/cm2; Collaborative Research Co, Bedford, MA, USA) and air-dried aseptically. Prior to use, the inserts were rehydrated with 100 µl of warm RPMI 1640 for 2 h. The BeWo cells (2x105 in 500 µl of RPMI 1640) were added to the top of the filter, and 1 ml of medium was added to the culture well. The anti-CD9 mAb (ALB-6; 0, 0.5, 1.0, and 5 µg/ml), the anti-integrin mAb (P1B5 for 3, P1D6 for 5, or P4C10 for ß1 subunit, diluted 1:50 000, 1:10 000, and 1:5000 from an ascites preparation respectively) or the control antibody (anti-TNP, 5 µg/ml) was then added to the culture medium (RPMI 1640 with 10% FCS). After 24 h of culture, the upper surface of the filter was `scrubbed' three times with a cotton swab. Cells remaining on the lower surface of the `scrubbed' filter were fixed in methanol for 10 min at room temperature, and stained with haematoxylin and eosin. For quantification, the cells that had migrated to the lower surface were counted under a microscope in 5 pre-determined fields at a magnification of x200. The assay was performed in triplicate chambers.
The effects of the ALB-6 mAb on BeWo cell invasion was also examined in the presence of the anti-integrin mAb (P1B5 for 3, P1D6 for 5, or P4C10 for ß1 subunit, diluted 1:50 000, 1:10 000, and 1:5000 from an ascites preparation respectively).
Statistical analysis
The data were expressed as the mean ± SEM, and were analysed by a two-tailed paired t-test or an one-way analysis of variance, followed by Scheffé's F test for multiple comparisons.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Detection of CD9 and integrins
3, 5 and ß1 in cultured BeWo cells by indirect immunofluorescence staining and flow cytometryUsing indirect immunofluorescence staining, CD9 expression was observed in acetone-fixed cultured BeWo cells (Figure 1
) and on the cell surface of freshly dispersed BeWo cells (data not shown). The expression of integrins 3, 5 and ß1 on BeWo cells was also observed by immunohisto-chemical analysis (data not shown). Flow cytometry showed 70.1 ± 1.7% immunopositivity for CD9 expression on dispersed BeWo cells. The BeWo cells also expressed 3, 5, and ß1 integrins on their cell surfaces (92.1 ± 2.4, 54.8 ± 4.9, and 76.4 ± 3.4% immunopositivity respectively) (Figure 2).
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Detection of CD9mRNA in cultured BeWo cells by RT–PCR
The expression of CD9 mRNA was observed in the BeWo cells (Figure 3
). The nucleotide sequence of the PCR product, 473 bp in length was analysed by DNA sequencer and confirmed to be identical to that of CD9 cDNA as previously reported (Boucheix et al., 1991). The expected PCR product of S26 was also detected in BeWo cells.
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Detection of CD9 protein in cultured BeWo cells by Western blotting
Using Western blotting, the antigen which reacted with the anti-CD9 mAb was detected as a single 27 kDa protein band, which was compatible with the reported molecular mass of CD9 (Figure 4
) (Rubinstein, et al. 1994).
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Effect of anti-CD9 mAb on HCG secretion and proliferation of BeWo cells
Neither HCG secretion nor cell proliferation were affected by the anti-CD9 mAbs (Figures 5 and 6
). In the presence of forskolin, HCG secretion by BeWo cells was enhanced, as reported by Wice et al. (1990) and Taylor et al. (1991). Under these conditions, the anti-CD9 mAb again had no effect on HCG secretion (data not shown).
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Effects of anti-CD9 mAb on the invasion by BeWo cells
In the presence of the anti-CD9 mAb (ALB-6), the number of invading BeWo cells increased significantly in a dosedependent manner (Figure 7
). In contrast, the control anti-TNP mAb had no effect on the invasion by BeWo cells. Another anti-CD9 mAb, clone TP-82, did not affect the invasion by BeWo cells (data not shown).
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Effects of anti-CD9 mAb on the invasion by BeWo cells in the presence of blocking mAbs against ß1-related integrins
The blocking mAbs against integrins
3 (P1B5), 5 (P1D6), and ß1 (P4C10) inhibited BeWo cell invasion in a dose-dependent manner. The anti-CD9 mAb ALB-6 enhanced BeWo cell invasion in the presence of the anti-integrin 3 mAb, but had no effect on BeWo cell invasion following treatment with the anti-integrin 5 and ß1 mAbs (Figure 8).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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CD9 was initially reported to be specific for acute lymphoblastic leukaemia cells (Kersey et al., 1981
). Later, it was found to be expressed on other cells, including pre-B cells, activated T cells, platelets and Schwann cells (Jennings et al., 1990; Masellis-Smith et al., 1990; Miyake et al., 1991; Anton et al., 1995). Recent studies have shown that anti-CD9 mAb induce the migration of Schwann cells (Anton et al., 1995), and that CD9 regulates the adhesion of pre-B cells to bone marrow fibroblasts (Masellis-Smith and Shaw, 1994).
Recently, we observed the intense expression of CD9 in the extravillous trophoblasts of the placental bed and chorion laeve. In order to elucidate the functional role of CD9 on trophoblasts, we used the human trophoblast-like choriocarcinoma cell line BeWo, which has the characteristics of both villous and extravillous trophoblasts (Aplin and Charlton, 1990; Ellis et al., 1990; Charnock-Jones et al., 1994); e.g. this cell line secretes HCG following stimulation by forskolin. Using flow cytometry and Western blotting, we confirmed that ~70% of the BeWo cells expressed CD9 on their cell surface. An invasion assay demonstrated that the binding of the anti-CD9 mAb (ALB-6) to CD9 enhanced the number of invading BeWo cells. Since this mAb did not affect cell proliferation, its stimulatory effect in the invasion assays must have been due to an enhancement of the invasive property itself, rather than to enhanced cell proliferation. This suggests that the CD9 molecule may be related to the regulation of BeWo cell invasion. In other CD9-positive cells, this mAb has been reported to have stimulatory effects on cell aggregation, migration, and adhesion (Jennings et al., 1990; Masellis-Smith and Shaw, 1994; Anton et al., 1995). Although another anti-CD9 mAb, TP-82, has been reported to have a stimulatory effect on platelet aggregation, it had no effect on BeWo cell invasion. This suggests that the binding sites of the mAbs to the CD9 epitopes are crucial in their ability to affect BeWo cell invasion. On the other hand, neither anti-CD9 mAb had any affect on either basal or forskolin-stimulated HCG secretion, which is the main function of villous trophoblasts. Based on the results obtained with the BeWo cells, we conclude that the physiological role of CD9 in trophoblasts is related to their invasive property rather than to hormonal production, which is compatible with our observation that CD9 is expressed on the extravillous trophoblast cell lineage.
Extracellular matrices (ECM) and integrins are considered to be concerned with regulation of normal trophoblast invasion (Damsky et al., 1992, 1994; Tabibzadeh and Babaknia, 1995; Burrows et al., 1996). Since CD9 is known to be associated with ß1-related integrins (Rubinstein et al., 1994; Nakamura et al., 1995), it may modulate signal transduction mediated by ß1-related integrins and thus regulate trophoblast invasion. We have found that the integrins 3ß1 and 5ß1 in the extravillous trophoblasts are associated with CD9 (Hirano et al., 1999). In this study, we confirmed the expression of integrins 3ß1 and 5ß1 on the cell surface of BeWo cells using immunocytology. Previous work by Aplin et al. (1992) had shown that integrin 3 was not detected in BeWo cells. This discrepancy may be caused by the differences among cell sublines. To investigate the functional relationship between integrins and CD9, we examined the effects on BeWo cell invasion of the anti-CD9 mAb combined with mAbs against integrins. Anti-integrin mAb inhibited BeWo cell invasion in a dose-dependent manner, suggesting that both integrins 3ß1 and 5ß1 facilitate BeWo cell invasion. These effects may be exerted by interactions with the ECM. However, this remains to be clarified because Matrigel contains small amounts of fibronectin, which acts as a ligand for integrin 5ß1. The anti-CD9 mAb, ALB-6, enhanced BeWo cell invasion in the presence of the anti-integrin 3 mAb, but had no effect on BeWo cell invasion in the presence of anti-integrin 5 and ß1 mAb. This indicates the involvement of integrin 5ß1 in the regulation of BeWo cell invasion by the CD9 molecule. On the other hand, integrin 3ß1 may not be necessary.
In this study, we did not determine whether the binding of ALB-6 mAb activates or inhibits CD9 function. The transfection of the CD9 gene into several cell lines was reported to suppress cell motility and metastasis (Ikeyama et al., 1993). The expression of CD9 in breast cancer was inversely correlated with its metastatic potential, implying that CD9 is involved in the suppression of cell invasion (Miyake et al., 1995). These reports suggest that CD9 plays an inhibitory role in the invasiveness of BeWo cells.
In conclusion, this study has demonstrated that expression of the CD9 molecule by BeWo cells is related to cell invasion, in the absence of any effect on cell proliferation or HCG secretion. Although the precise mechanism of CD9 in invasion is unknown, CD9 may be implicated in the function of integrins. At present, the factors which limit trophoblast invasion within the uterus, and which induce invading trophoblasts to become non-invasive, are still unknown. The clarification of the role of CD9 in BeWo cell invasion will contribute to understanding the regulatory mechanism of extravillous trophoblast invasion, especially the protecting mechanisms against excessive trophoblast invasion.
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Acknowledgments |
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This work was supported in part by Grants-in-Aid for Scientific Research (no. 09671673, 09671674, 09671676).
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Notes |
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5 To whom correspondence should be addressed at: Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan
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Submitted on June 18, 1998; accepted on November 5, 1998.
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 C. M. Longhurst, J. D. Jacobs, M. M. White, J. T. Crossno Jr., D. A. Fitzgerald, J. Bao, T. J. Fitzgerald, R. Raghow, and L. K. Jennings Chinese Hamster Ovary Cell Motility to Fibronectin Is Modulated by the Second Extracellular Loop of CD9. IDENTIFICATION OF A PUTATIVE FIBRONECTIN BINDING SITE J. Biol. Chem., August 30, 2002; 277(36): 32445 - 32452. [Abstract] [Full Text] [PDF]
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 W. Xiang and L. A. MacLaren Expression of Fertilin and CD9 in Bovine Trophoblast and Endometrium During Implantation Biol Reprod, June 1, 2002; 66(6): 1790 - 1796. [Abstract] [Full Text] [PDF]
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H. Egawa, H. Fujiwara, T. Hirano, T. Nakayama, T. Higuchi, K. Tatsumi, T. Mori, and S. Fujii Peripheral blood mononuclear cells in early pregnancy promote invasion of human choriocarcinoma cell line, BeWo cells Hum. Reprod., February 1, 2002; 17(2): 473 - 480. [Abstract] [Full Text] [PDF]
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 J. B. Welsh, P. P. Zarrinkar, L. M. Sapinoso, S. G. Kern, C. A. Behling, B. J. Monk, D. J. Lockhart, R. A. Burger, and G. M. Hampton Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer PNAS, January 30, 2001; 98(3): 1176 - 1181. [Abstract] [Full Text] [PDF]
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K. R. Park, T. Inoue, M. Ueda, T. Hirano, T. Higuchi, I. Konishi, H. Fujiwara, and S. Fujii Anti-CD9 monoclonal antibody-stimulated invasion of endometrial cancer cell lines in vitro: possible inhibitory effect of CD9 in endometrial cancer invasion Mol. Hum. Reprod., August 1, 2000; 6(8): 719 - 725. [Abstract] [Full Text] [PDF]
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K. R. Park, T. Inoue, M. Ueda, T. Hirano, T. Higuchi, M. Maeda, I. Konishi, H. Fujiwara, and S. Fujii CD9 is expressed on human endometrial epithelial cells in association with integrins {alpha}6, {alpha}3 and {beta}1 Mol. Hum. Reprod., March 1, 2000; 6(3): 252 - 257. [Abstract] [Full Text] [PDF]
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Y. Takao, H. Fujiwara, S. Yamada, T. Hirano, M. Maeda, S. Fujii, and M. Ueda CD9 is expressed on the cell surface of human granulosa cells and associated with integrin {alpha}6ß1 Mol. Hum. Reprod., April 1, 1999; 5(4): 303 - 310. [Abstract] [Full Text] [PDF]
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