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Stimulation of Syncytium Formation in vitro in Human Trophoblast Cells by Galectin-1
Placenta 31 (2010) 825e832
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Stimulation of Syncytium Formation in vitro in Human Trophoblast Cells by Galectin-1 I. Fischer a, S. Redel a, S. Hofmann a, C. Kuhn a, K. Friese a, H. Walzel b, U. Jeschke a, * a b
Ludwig Maximilians University of Munich, Department of Obstetrics and Gynecology, Maistrasse 11, 80337 Munich, Germany University of Rostock, Department of Biochemistry and Molecular Biology, Schillingallee 70, 18059 Rostock, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 24 June 2010
Background: Galectin-1 (gal-1), a member of the mammalian b-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In trophoblast cells, gal-1 binds to cell surface glycoproteins (e.g., Mucin1). It has been demonstrated that gal-1 recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast from second trimester human placenta and choriocarcinoma cells BeWo, which reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype. So the aim of this study was to investigate the effect of gal-1 on syncytium formation in BeWo and human villous trophoblasts (HVT) cells. Materials and methods: The effect of gal-1 on syncytium formation was investigated with immunocytochemical and double immunofluorescence stainings, cell-labelling and Real-time RT-PCR. BeWo choriocarcinoma and HVT cells were incubated in vitro for 24 and 48 h in the absence (controls) and presence of gal-1 and forskolin and stained with antibodies against Ki67, b-catenin, E-cadherin and syncytin. BeWo and HVT cells were incubated with 60 mg/ml gal-1 for 48 h (BeWo) or 96 h (HVT) and cell fusion was detected by fluorescent cell-labelling solution. Finally, BeWo cells were incubated for 1 h or 48 h in the absence and presence of 60 mg/ml gal-1 and Real-time RT-PCR was performed. Results: We showed with immunocytochemical staining a downregulation of b-catenin expression in the 24 h BeWo cell culture and with double immunofluorescence staining an inhibition of the b-catenin and E-cadherin expression in the 48 h BeWo cell culture stimulated with gal-1 or forskolin. The inhibition of E-cadherin was demonstrated on mRNA level in the 1 h BeWo cell culture too. Increased cell fusion was also showed with DiO and DiI fluorescent cell-labelling solution in the 48 h BeWo cell culture. In addition, we demonstrated the downregulation of Ki67 protein expression in the 24 h BeWo cell culture and on mRNA level in the 1 h BeWo cell culture. We also showed the upregulation of syncytin protein and mRNA expression after incubation of the 48 h BeWo cell culture with gal-1 or forskolin. Similar results were obtained with HVT cells: the amount of cell fusion was significantly increased in the gal-1 treated 48 h HVT cell culture in vitro compared to untreated cells as demonstrated with b-catenin and E-cadherin double immunofluorescence staining. This increase was also shown by fluorescent celllabelling with DiO and DiI in the 96 h HVT cell culture compared to untreated cells. Conclusion: Our data suggest that gal-1 stimulates the syncytium formation in choriocarcinoma cells BeWo and HVT cells in vitro and inhibits the expression of b-catenin, E-cadherin and in addition Ki67 in BeWo cells. Therefore gal-1 may be a major trigger for the process of trophoblast cell fusion. Ó 2010 Published by Elsevier Ltd.
Keywords: Galectin-1 BeWo Primary trophoblast cells Syncytium formation
1. Introduction Galectins, a growing family of carbohydrate proteins, are defined by an affinity for b-galactosides [1]. Although LacNAc (NAcetyl-Lactosamine) is the basic ligand recognized by gal-1, the
* Corresponding author. Tel.: þ49 89 5160 4266; fax: þ49 89 5160 4916. E-mail address: [email protected] (U. Jeschke). 0143-4004/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.placenta.2010.06.016
proto-type galectin binds with increased avidity to multiple Galb14GlcNAc sequences presented on branched N-linked or on repeating LacNAc-residues on N- and O-linked glycans. Extracellularly, gal-1 exerts biological effects by the recognition of glycan ligands, including those involved in cell adhesion [2] metastasis [3], cell growth regulation [4,5] and apoptosis [6]. In addition, it has been demonstrated that gal-1 induces cell differentiation processes in syncyciotrophoblast and extravillous trophoblast layer and on BeWo choriocarcinoma cells [7].
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Table 1 Antibodies used for this study. Antibody
Isotype
Clone
Dilution
Source
Immunocytochemistry b-catenin Ki67
Rabbit IgG Mouse IgG
polyclonal Mib-1
1:600 in PBS 0.1 M 1:500 in PBS 0.1 M
Diagnostic Biosystems, DAKO Glastrup
Double immunofluorescence stainings of BeWo cells after incubation with recombinant and placental gal-1 and forskolin E-cadherin Mouse IgG1 HECD-1 1:80 in DAKO S3022 Pan-Keratin Cy3 Mouse IgG A45-B/B3 1:1000 in DAKO S3022 Rabbit-Anti-Mouse Cy 2 Rabbit IgG polyclonal 1:50 in DAKO S3022
Calbiochem Micromet Dianova
Syncytin stainings of BeWo cells after incubation with recombinant gal-1 and forskolin Syncytin Rabbit IgG polyclonal Goat-Anti-Rabitt IgG Cy2 Goat IgG polyclonal
1:20 in DAKO S3022 1:100 in DAKO S3022
Abnova Dianova
Double immunofluorescence stainings of BeWo and HVT cells after incubation with recombinant gal-1 E-cadherin Mouse IgG1 HECD-1 b-Catenin Rabbit IgG polyclonal Goat-Anti-Mouse IgG Cy2 Goat IgG polyclonal Goat-Anti-Rabbit IgG Cy3 Goat IgG polyclonal
1:50 in DAKO S3022 1:50 in DAKO S3022 1:500 in DAKO S3022 1:100 in DAKO S3022
Calbiochem Diagnostic Biosystems Dianova Dianova
Expression of gal-1 has been well documented in placental trophoblast cells, decidual cells [8] and in immune privileged organs [9]. In addition, previous studies have shown that gal-1 decreases cellular hCG and progesterone production as well as hCG b gene transcription [10]. Furthermore, there is an evidence of a role for gal-1 in promoting murine fetomaternal tolerance in allogenetic matings, suggesting a potential approach for therapeutic intervention aimed at restoring immune cell homeostasis in failing pregnancies [11]. Gal-1 is defined by a conserved amino acid sequence motif in the carbohydrate recognition domain (CRD). Gal-1 with a single CRD forms a non-covalently associated homodimer to become functionally bivalent under physiological conditions. The bivalent nature of gal-1 facilitates glycan-mediated cell surface receptor cross-linking believed to be essential in inducing signalling events [6,12]. This information led to the question if gal-1, via its two carbohydrate binding sites, could influence the fusion formation in trophoblast cells. It has already been shown that gal-1 was strongly expressed by syncytiotrophoblasts in term and first trimester placenta, whereas villous cytotrophoblasts were negative [8,13]. The invading cytotrophoblast was weakly stained and BeWo cells expressed gal-1 particularly strongly [14]. It has also been demonstrated that gal-1 binds to BeWo cells, which form a syncytium in vitro, but does not bind to fresh isolated trophoblast cells and to choriocarcinoma cells Jeg-3, which cannot form a syncytium [10]. In a recent study we could demonstrate that the concentration of 60 mg/ml gal-1 induced a significant alteration of signal intensity in only 3 from 71 different Receptor tyrosine kinases, showing the specificity of gal-1 binding to cell surface glycoproteins at the concentration used for this experiment [15]. To demonstrate the effect of gal-1 on the syncytium formation in trophoblast cells, choriocarcinoma cell cultures BeWo, derived from human gestational choriocarcinoma and permanently established in 1968 by Pattillo et al. offer an attractive model because they reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype [16]. In addition, primary trophoblast cells can be used as a culture model for cells that are able to fuse in vitro but are unable to proliferate. Together, these two cell culture models might explain aspects of the complex situation of the placenta in vivo. Because fusion of trophoblast cells is accompanied by downregulation of Ecadherin and b-catenin [17,18] these proteins can be used for the analysis of trophoblast cell fusion. In addition, Ki67 as a proliferation marker [19] can serve as indicator for trophoblast differentiation.
2. Methods 2.1. Immunocytochemistry The choriocarcinoma cell line BeWo was obtained from the European Collection of Cell Cultures (ECACC, UK). BeWo cell suspensions at 1 105 cells/ml DMEM medium (Dulbecco’s Modified Eagle Medium, Biochrom, Germany, 3.7 g/l NaHCO3, 4.5 g/l D-Glucose, 1.028 g/l stable glutamine, Na-Pyruvate, supplemented with 10% heat-inactivated FCS (fetal calf serum) without antibiotics and antimycotics) were incubated for 24 and 48 h in chamberslides with 10, 30 and 60 mg/ml galectin-1 (Recombinant Human Galectin-1, 250 mg, Sigma Missouri, USA). Untreated cell cultures were used as controls. The experiments were done in quadruplicates. Slides were fixed with Ethanol/Methanol (1/1) for 15 min, washed with PBS (phosphate buttered saline, pH 7.4, 5 min) and stained with horse serum (30 min, room temperature). Then, the slides were stained with the primary antibodies b-catenin (1:500) and Ki67 (1:600) (see Table 1) for 18 h at 4 C. Sections were stained with the biotinylated secondary antibodies (30 min, room temperature). The VectastainÒ Elite ABC-Kit (Vector Laboratories, UK) was used for visualization according to the manufacturer’s instructions. Between each step, the sections were washed with PBS three times. Then, the slides were stained with AEC plus (3-Amino-9-Ethylcarbazole, Dako, Denmark) (12 min, room temperature) and washed in tap water. Finally, slides were counterstained with hemalaun (2 min, room temperature), washed in tap water and then covered. Sections were examinated using a Leica photomicroscope (Solms, Germany). Images were obtained with a digital camera system (JVC, Victor Company of Japan, Japan). 9 fields were analysed with the size of 1000 mm 1000 mm. For the Ki67 staining, the intensity and distribution patterns of specific immunohistochemical staining were evaluated using the semi-quantitative assay as previously described [20]. The IRS score was calculated by multiplication of optical staining intensity (graded as 0 ¼ no, 1 ¼ weak, 2 ¼ moderate and 3 ¼ strong staining) and the percentage of positive stained cells (0 ¼ no staining, 1 % of the cells, 2 ¼ 11e50% of the cells, 3 ¼ 51e80% of the cells and 4 81% of the cells). The slides were examined by two independent observers, including a gynecological pathologist. For the b-catenin staining, the intensity of antigen expression was evaluated by examinating the percental changing of the membrane-staining compared to the cell number (100% ¼ all membranes are positive, 0% ¼ no membrane is positive). The SPSS/PC software package, version 15.01 (SPSS, Germany), was used for collection, processing, and statistical data analysis. Statistical analysis was performed using the non-parametric Wilcoxon’s signed rank tests for comparison of the means. P < 0.05 values were considered statistically significant.
2.2. Double immunofluorescence staining of BeWo cells after incubation with recombinant and placental gal-1 and forskolin BeWo cell suspensions (ECACC, UK) at 1 105 cells/ml DMEM medium were incubated for 48 h in chamberslides with 10, 30 and 60 mg/ml galectin-1 (Recombinant Human Galectin-1, 250 mg, Sigma Missouri, USA, placental gal-1 (Dr. Walzel, Institut for Biochemie and Molecularbiologie, Germany) or 60 mg/ml forskolin (Sigma Aldrich, USA). The placental gal-1 was used in previous investigations. Due to the limited availability of placental gal-1, we used commercial available gal-1. To prove the homology of both proteins, placental and recombinant gal-1 were both used in this experiment. Untreated cell cultures were used as controls. The experiments were done in quadruplicates. Slides were fixed with Ethanol/Methanol (1/1) for 15 min, washed
I. Fischer et al. / Placenta 31 (2010) 825e832 with PBS and stained with Ultra V Block (Labvision, USA, 15 min, room temperature). Then, the slides were stained with the primary antibody E-cadherin Mouse IgG (1:80) (Table 1) overnight at 4 C. After washing, chamberslides were stained with Cy2 labelled antibody RabbitAnti-Mouse IgG (1:50; room temperature, 30 min) which will appear green. After washing, chamberslides were stained with Cy3 labelled Mouse anti-cytokeratin IgGAntibody (1:1000; room temperature, 45 min), which will appear red. The slides were finally covered with mounting buffer containing 40 ,6-diamino-2-phenylindole (DAPI, Denmark) and examined with a photomicroscope (Zeiss Axiophot, Germany). Images were obtained with a digital camera system (Axiocam, Zeiss, Germany). 9 fields were analysed with the size of 1000 mm 1000 mm. The intensity of the green E-cadherin expression was evaluated by examinating the percentage changing of the membrane-staining compared to the cell number (100% ¼ all membranes are positive, 0% ¼ no membrane is positive). Statistical analysis was performed as mentioned in Section 2.1.
2.3. Syncytin expression of BeWo cells after incubation with recombinant gal-1 and forskolin BeWo cell suspensions (ECACC, UK) at 1 105 cells/ml DMEM medium were incubated for 48 h in chamberslides with 60 mg/ml gal-1 (Recombinant Human Galectin-1, 250 mg, Sigma Missouri, USA) or 60 mg/ml forskolin (Sigma Aldrich, USA). Untreated cell cultures were used as controls. The experiments were done in quadruplicates. Slides were fixed with Aceton for 10 min, washed with PBS and stained with Ultra V Block (Labvision, USA, 15 min, room temperature). Then, the slides were stained with the primary antibody syncytin Rabbit IgG (1:20) (Table 1) overnight at 4 C. After washing, chamberslides were stained with Cy2 labelled antibody GoatAnti-Rabbit IgG (1:100; room temperature, 30 min) which will appear green. The slides were finally covered with mounting buffer containing 40 ,6-diamino-2-phenylindole (DAPI, Denmark) and examined with a photomicroscope (Zeiss Axiophot, Germany). Images were obtained with a digital camera system (Axiocam, Zeiss, Germany). 9 fields were analysed with the size of 1000 mm 1000 mm. The intensity of the expression was evaluated as mentioned in Section 2.2.
2.4. Double immunofluorescence staining of BeWo cells and HVT cells after incubation with recombinant gal-1 BeWo cells (ECACC, UK) and human villous trophoblasts (HVT) (ScienCell Researchlab., USA, HVT were isolated from human placental villi and cryopreserved at passage primary culture and delivered frozen. Each vial contains >1 106 cells in 1 ml volume. HVT were characterized by immunofluorescent method with antibodies to alpha- and beta-hCG. HVT are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HVT are guaranteed to further culture condition provided by ScienCell Research Laboratories.) (5 104 cells in 0.5 ml supplemented DMEM medium) were incubated for 48 h in chamberslides with 60 mg/ml gal-1 (recombinant human gal-1, 250 mg, Sigma Missouri, USA). Untreated cell cultures were used as control. Slides were fixed with Ethanol/Methanol (1/1) for 15 min, washed with PBS and stained with Ultra V Block (Labvision, USA; 15 min, room temperature). Then, the slides were stained with the primary antibodies E-cadherin Mouse IgG (1:50) and b-catenin Rabbit IgG (1:50) (Table 1) overnight at 4 C. After washing, chamberslides were stained with Cy2 labelled Mouse IgG-Antibody (1:500, room temperature, 30 min) and Cy3 labelled Rabbit IgG-Antibody (1:100, room temperature, 30 min). After washing, slides were finally embedded in mounting buffer containing 40 ,6-diamino-2-phenylindole (Dapi, Denmark) and examined with a fluorescent microscope (Axiophot, Zeiss, Germany). Images were obtained with a digital camera system (Axiocam, Zeiss, Germany). 5 independent experiments for both BeWo and HVT cells were performed. The amount of cell fusion was evaluated by the following equation: S ¼ noC1 1=m þ noC2 1=m þ noC3 1=m þ noC4 1=m þ . S ¼ sum, noC ¼ number of cell cluster, m ¼ number of cells forming the cell cluster. S is the sum of all clusters. Each cluster was divided by the number of nuclei in each cluster. 10 fields were analysed with the size of 1000 mm 1000 mm. Because fusion experiments were done on living cell cultures, it is not possible to induce toxic substances like DAPI to the cells. The advantage of both fluorescence dyes is that they are not toxic and have no effect on cell proliferation as described in literature. The formula was developed in our research team in cooperation with an engineer (MS) of orthopaedics from LMU in Großhadern, Munich. Statistical analysis was performed as mentioned in Section 2.1.
2.5. Detection of cell fusion by cell-labelling Both the choriocarcinoma cell line BeWo (ECACC, UK) and the human villous trophoblasts (HVT) (ScienCell Researchlab., USA) were treated according to following procedure:
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2.5 105 cells were labelled with 8 mg/ml DiO (1,10 -dioctadecyl-indocarbocyanine perchlorate) fluorescent cell-labelling solution (Vybrant Cell-Labelling Solutions, Molecular Probes, Scotland) in serum-free DMEM medium for 20 min at 37 C without CO2. Further 2.5 105 cells were labelled with 4 mg/ml DiI (1,10 -dioctadecyl-3,3,30 -tetramethylindocarbocyanine perchlorate) fluorescent cell-labelling solution (Vybrant Cell-Labelling Solutions, Molecular Probes, Scotland). Cells were washed with serum-free DMEM medium three times. After washing, cells were resuspended in DMEM medium supplemented with 10% heat-inactivated FCS, 250 mg/m Amphotericin B and 10 000 U/10 000 mg/ml Pencillin/Streptomycin. DiO and DiI labelled cell suspensions were mixed in one well of a 24-well plate. Finally, cells were incubated with 60 mg/ml gal-1 (recombinant human gal-1, 250 mg, Sigma Missouri, USA) for 48 h (BeWo) or 96 h (HVT) at 37 C. Cell suspensions without application of gal-1 were used as control. 5 independent experiments for both BeWo and HVT cells were performed. The amount of cell fusion was evaluated in ten randomly chosen fields of each well using Zeiss Axiovert 40 CFL fluorescent microscope (Zeiss, Germany). Images were obtained with a digital camera system (Power Shot A620, Canon, Japan). Statistical analysis was performed as mentioned in Section 2.1.
2.6. RNA extraction, reverse transcription, real-time RT-PCR 2.6.1. RNA extraction BeWo cells (ECACC, UK) (1 106 cells in 1.5 ml supplemented DMEM medium) were incubated in 25 ml cell culture bottles for 1 h or 48 h in the absence (controls) and presence of 60 mg/ml gal-1 (Recombinant Human Galectin-1, 250 mg, Sigma Missouri, USA) or 60 mg/ml forskolin (Sigma Aldrich, USA). Untreated cell cultures were used as controls. Total RNA was investigated by NucleoSpinÒ RNAII Kit (Machery-Nagel, Germany), according to the manufacturer’s protocol. 2.6.2. Reverse transcription Reverse transcription (RT) was carried out with the ‘High Capacity cDNA Reverse Transcription Kit’ (Applied Biosystems, Germany) (in 6 independant experiments) according to the protocol in a mastercycler gradient (Eppendorf, Germany). RT conditions were: 10 min 25 C, 2 h 37 C, 5 s 85 C and 4 C on hold). 2.6.3. Real-time RT-PCR Real-time reverse transcriptase-polymerase chain reactions (RT-PCRs) were performed in triplicates (in 6 independant experiments) in optical 96-well reaction microtiter plates covered with optical caps, in a volume of 20 ml containing 1 ml TaqManÒ Gene Expression Assay 20 (Order number Hs00170423_m1 for E-cadherin, Hs00606991_m1 for Ki67, Hs00170025_m1 for b-Catenin and Hs 00205893_m1 for syncytin all Applied Biosystems), 10 ml TaqManÒ Fast Universal PCR Master Mix 2 (Applied Biosystems), 1 ml template and 8 ml H2O. Thermal cycling conditions were: 20 s at 95 C, followed by 40 cycles of amplification with 3 s at 95 C and 30 s at 60 C. The ABI PRISM 7500 Fast (Applied Biosystems) was used to perform the PCR assays. Quantification was carried out by the ΔΔCt method using glycerinaldehyd phosphate dehydrogenase (GAPDH) as house-keeping gene (Order Number Hs99999905_m1, Applied Biosystems). Statistical analysis was performed as mentioned in Section 2.1.
3. Results 3.1. Immunocytochemistry As demonstrated in Fig. 1, Ki67 expression was significantly reduced in the gal-1 treated 24 h BeWo cell culture compared to the non-treated cells (IRS Score ¼ 9, SEM ¼ 0.35) (1A): 10 mg/ml (p ¼ 0.025) (IRS Score ¼ 6, SEM ¼ 0.35) (1B), 30 mg/ml (p ¼ 0.039) (IRS Score ¼ 4, SEM ¼ 0.35) (1C) and 60 mg/ml (p ¼ 0.041) (IRS Score ¼ 3.8, SEM ¼ 0.41) (1D). Incubation of 48 h BeWo cell culture with gal-1 did not further reduce the expression of Ki67 (data not shown). As demonstrated in Fig. 2, b-catenin expression was significantly reduced in the gal-1 treated 24 h BeWo cell culture compared to the non-treated cells (¼80%, SEM ¼ 3.53) (2A): 10 mg/ml (¼54%, SEM ¼ 5.70) (p ¼ 0.042), 30 mg/ml (¼34%, SEM ¼ 5.70) (p ¼ 0.042) (data not shown) and 60 mg/ml (¼12%, SEM ¼ 4.18) (p ¼ 0.041) (2B). Incubation of 48 h BeWo cell culture with gal-1 did not further reduce the expression of b-catenin, compared to the data shown for the 24 h cell culture.
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Fig. 1. BeWo cells were treated with gal-1 (24 h), and Ki67 positive cells numbers were assessed. A: control; B: gal-1 (10 mg/ml); C: gal-1 (30 mg/ml); D: gal-1 (60 mg/ml) (20). Positive cells are marked with an arrow.
3.2. Double immunofluorescence staining E-cadherin expression was significantly reduced from 70% (in the untreated BeWo cells SEM ¼ 4.18) to 30% in the 48 h BeWo cell culture treated with 60 mg/ml placental gal-1 (p ¼ 0.025, SEM ¼ 4.18) and to 33% with 60 mg/ml recombinant gal-1 (p ¼ 0.042, SEM ¼ 4.47). As demonstrated in Fig. 3AeD, the number of positive cells was significantly increased in the gal-1 treated 48 h BeWo cell culture (S ¼ 26.2) compared to untreated cells (S ¼ 95.5; p ¼ 0.005). As showed in Fig. 3E, the E-cadherin expression was significantly reduced from 70% (in the untreated BeWo cells SEM ¼ 4.18) to 33% in the 60 mg/ml forskolin treated 48 h BeWo cell culture (p ¼ 0.025, SEM ¼ 4.47). Syncytin protein expression was significantly increased from 29% (in the untreated BeWo cells SEM ¼ 3.95) to 78% in the 60 mg/ ml gal-1 treated cells (p ¼ 0.011, SEM ¼ 2.85) and to 85% in the
60 mg/ml forskolin treated cells (p ¼ 0.012, SEM ¼ 3.95), as demonstrated in Fig. 3FeH. The number of positive cells was significantly increased in the gal-1 treated 48 h HVT cell culture (S ¼ 38.7) compared to untreated cells (S ¼ 125.3; p ¼ 0.005), as demonstrated in Fig. 4.
3.3. Detection of cell fusion by cell-labelling As demonstrated in Fig. 5, the number of positive cells (mean SEM) was significantly increased in the gal-1 treated 48 h BeWo cell culture (21.0% 5.3%) compared to untreated cells (6.6% 1.7%; p ¼ 0.042). The number of positive cells (mean SEM) was significantly increased in the gal-1 treated 96 h HVT cell culture (23.2% 2.3%) compared to untreated cells (13.3% 1.7%; p ¼ 0.034), as demonstrated in Fig. 6.
Fig. 2. BeWo cells were treated with gal-1 (24 h), and b-catenin positive cells numbers were assessed. A: control; B: gal-1 (60 mg/ml) (20).
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Fig. 3. BeWo cells were treated with gal-1 (48 h), and b-catenin (red) positive cells numbers were assessed. A: control; B: gal-1 (60 mg/ml) E-cadherin (green) positive cell numbers were assessed C: control; D: gal-1 (60 mg/ml) (40). E-cadherin (green) stimulated with 60 mg/ml forskolin, 48 h (E). Syncytin (green) control (F), stimulated with 60 mg/ml forskolin (G) and stimulated with 60 mg/ml gal-1, 48 h (H) (all 20). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.4. Reverse transcriptase-polymerase chain reaction The treatment of the 1 h BeWo cell culture with 60 mg/ml gal-1 significantly decreased the E-cadherin mRNA expression to 78.8% (p < 0,01) and the Ki67 mRNA expression to 93.4% (p < 0,01) compared to the control. b-catenin mRNA expression was reduced
to 82.6% (p ¼ 0.043) compared to the control. The treatment of the 48 h BeWo cell culture with 60 mg/ml gal-1 significantly increased the syncytin mRNA expression to 121% (p ¼ 0.026) compared to the control. The treatment of the 48 h BeWo cell culture with 60 mg/ml forskolin non e significantly increased the syncytin mRNA expression to 110% (p ¼ 0.063) compared to the control.
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Fig. 4. HVT cells were treated with gal-1 (48 h), E-cadherin (green) positive cell numbers were assessed. A: control; B: gal-1 (60 mg/ml). b-catenin positive cell numbers were assessed C: control; D: gal-1 (60 mg/ml), a single non-fused cell is marked with an arrow (40). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 5. The number of positive cells was significantly increased in the gal-1 treated 48 h BeWo cell culture in vitro compared to untreated cells (p ¼ 0.005). BeWo cells were either stained with DiO (green) or Dil (red) and mixed. Both cell populations are seen in BeWo cells, 0 h cell culture in vitro, (A), and in BeWo cells stimulated with 60 mg/ml gal-1, 0 h (B), fusion of BeWo cells appears in yellow, 48 h (C), significantly higher numbers fused BeWo cells stimulated with 60 mg/ml gal-1, 48 h (D) (40). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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Fig. 6. The number of positive cells was significantly increased in the gal-1 treated 96 h HVT cell culture in vitro compared to untreated cells (p ¼ 0.005). HVT cells were either stained with DiO (green) or Dil (red) and mixed. Both cell populations are seen in BeWo cells, 0 h cell culture in vitro, (A), and in HVT cells stimulated with 60 mg/ml gal-1, 0 h (B), fusion of HVT cells appears in yellow, 96 h (C), significantly higher numbers fused HVT cells stimulated with 60 mg/ml gal-1, 96 h (D) (40). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
4. Discussion Galectins are members of a family of soluble animal lectins defined by shared characteristic amino acid sequences and an affinity for b-galactosides [21]. These human proteins have been shown to be involved in cellematrix adhesion, resistance to apoptosis, and angiogenesis. In addition, the involvement of galectins in cellular processes that are crucial for cancer progression and metastasis has been reported [22,23]. It has already been demonstrated that gal-1 is expressed by syncytiotrophoblasts in term and first trimester placenta [8,13] and binds to BeWo cells, which can form a syncytium. Furthermore, expression of gal-1 has been documented in decidual cells [8]. Syncytium formation is an exceptional process that takes place only in a few cell types, e.g., in myoblast, osteoclast [24] and trophoblast cells. The effect of cell fusion by gal-1 has been demonstrated in other cell types. It has been shown that gal-1 has a role in myoblast fusion in vivo and in vitro. The lectin has been implicated in the development of skeletal muscle, being maximally expressed at the time of myofiber formation [25,26]. It is located within the sarcoplasm of mononuclear myoblasts, but is exported to the extracellular compartment when muscle cells differentiate to their multinucleate stage of development [25,26]. In absence of gal1, myoblast cells cannot fuse as effectively as without. The exact mechanism is not fully understood [24]. In addition, the molecular mechanisms of the trophoblast membrane fusion are poorly investigated. However it is known that proteins implicated in cell adhesion (cadherin11) [27] and cellecell communication (connexin 43) [28] are directly involved. It has been demonstrated that the product of the HERV-W env gene (Env-W) is a fusogenic membrane glycoprotein (syncytin) that induces the formation of syncytia on interaction with the D-mammalian retrovirus receptor RDR [29].
Because it is also expressed in trophoblastic cells of the column, interstitial and endovascular trophoblast and thus in not-fusing cells, Malassine et al. suggest, that it appears to be required but not sufficient for trophoblastic cell fusion [29]. In this study we were now able to demonstrate that gal-1 stimulates fusion: in the 24 h BeWo cell culture we demonstrated the downregulation of b-catenin expression (Fig. 2), which is an indicator for cell fusion [17]. To underline our results, we investigated the effect of forskolin, which is known as an inducer of syncylization [30]. The staining results of E-cadherin after incubation with forskolin on BeWo cells were identical to the pictures of BeWo cells after incubation with gal-1 (Fig. 3AeE). Furthermore, we demonstrated an inhibition of the b-catenin and E-cadherin expressions in the 48 h BeWo cell culture with double immunofluorescence staining (Fig. 3A and B). The disappearance of E-cadherin from the cell-borders, parallel to the downregulation of the mRNA is an indicator for the syncytium formation in BeWo cells [18]. Although the inhibition of E-cadherin expression on protein level was observed after 48 h, the effect of gal-1 on mRNA level could be measured already 1 h after application of gal-1 to the BeWo cells. This rapid downregulation of mRNA level can be explained with the nature of signal transduction and transcription induction induced by gal-1 which were shown in former studies [15]. Increased cell fusion was also demonstrated with DiO and DiI cell labelling in the 48 h BeWo cell culture in vitro compared to untreated cells (Fig. 5). To show that this cascade is a de novo event induced by gal-1, we used syncytin as a fusion marker [29]: syncytin was upregulated on protein and mRNA level in the 48 h gal-1 stimulated BeWo cell culture (Fig. 3FeH). Similar results were obtained with the HVT cell culture: the amount of cell fusion was significantly increased in the gal-1 treated 48 h HVT cell culture in vitro compared to untreated cells as
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demonstrated with b-catenin and E-cadherin double immunofluorescence staining on protein level (Fig. 4). This increase was also demonstrated by cell-labelling with DiO and DiI in the 96 h HVT cell culture in vitro compared to the control (Fig. 6). Due to the nature of HVT cells which do not proliferate and migrate as fast as BeWo cells, cell fusion was significantly induced after 96 h, whereas cell fusion in BeWo cells was already upregulated after 48 h. In this study we were also able to confirm previous studies, which showed that the proliferation of BeWo cells is inhibited by gal-1. This was demonstrated with a 5-bromo-20 -deoxy-uridine (BrdU) incorporation and ELISA [7]. We now demonstrated the downregulation of Ki67 expression (Fig. 1), which is an indicator for proliferation inhibition [19], in the 24 h BeWo cell culture and on mRNA level in the 1 h BeWo cell culture. Syncytium formation and decreased proliferation are linked processes. Because only non-fusing BeWo cells proliferate, Ki67, as a proliferation marker, is already downregulated in the 24 h cell culture, whereas E-cadherin, as a syncytium indicator, is altered in the 48 h BeWo cell culture. The association with b-catenin, which interacts with the cytoplasmic domain of the transmembrane-adhesion-molecule E-cadherin [31] and links it to the cytoskeleton via a-catenin [32], is essential for the cadherin mediated cell-to-cell adhesion [33]. This is in line with the results of our study, because the b-catenin expression is already inhibited after 24 h and the E-cadherin only after 48 h. In a previous study we could demonstrate that an increase of apoptosis in BeWo cells by gal-1 was only shown by additional stimuli like hyperthermia, removal of CO2 and FCS. So the downregulation of Ki67 in this study is not due to death of the cells [34]. In summary, we could show in this study that gal-1 is an inducer of syncytium formation both in isolated trophoblast cells and choriocarcinoma cells BeWo. This effect was studied with special fluorescence labelling of individual cell populations and with immunocytochemistry labelling of cell membrane bound adhesion proteins. Downregulation of adhesion molecule expression as the result of cell fusion was measured on protein and mRNA level. Fusion of BeWo cells is accompanied by reduced proliferation: we detected this inhibition of proliferation in gal-1 treated BeWo cells. Acknowledgement We thank S. Kunze and S. Schulze for excellent assistance. The study was supported by the “Deutsche Forschungsgemeinschaft” (DFG). References [1] Barondes SH, Castronovo V, Cooper DN, Cummings RD, Drickamer K, Feizi T, et al. Galectins: a family of animal beta-galactoside-binding lectins. Cell 1994;76(4):597e8. [2] Hughes RC. Galectins as modulators of cell adhesion. Biochimie 2001; 83(7):667e76. [3] Raz A, Lotan R. Endogenous galactoside-binding lectins: a new class of functional tumor cell surface molecules related to metastasis. Cancer Metastasis Rev 1987;6(3):433e52. [4] Wells V, Mallucci L. Identification of an autocrine negative growth factor: mouse beta-galactoside-binding protein is a cytostatic factor and cell growth regulator. Cell 1991;64(1):91e7. [5] Adams L, Scott GK, Weinberg CS. Biphasic modulation of cell growth by recombinant human galectin-1. Biochim Biophys Acta 1996;1312(2):137e44. [6] Perillo NL, Pace KE, Seilhamer JJ, Baum LG. Apoptosis of T cells mediated by galectin-1. Nature 1995;378(6558):736e9. [7] Jeschke U, Karsten U, Wiest I, Schulze S, Kuhn C, Friese K, et al. Binding of galectin-1 (gal-1) to the Thomsen-Friedenreich (TF) antigen on trophoblast cells and inhibition of proliferation of trophoblast tumor cells in vitro by gal-1 or an anti-TF antibody. Histochem Cell Biol 2006;126(4):437e44. [8] Vicovac L, Jankovic M, Cuperlovic M. Galectin-1 and -3 in cells of the first trimester placental bed. Hum Reprod 1998;13(3):730e5.
[9] Rabinovich GA. Galectins: an evolutionarily conserved family of animal lectins with multifunctional properties; a trip from the gene to clinical therapy. Cell Death Differ 1999;6(8):711e21. [10] Jeschke U, Reimer T, Bergemann C, Wiest I, Schulze S, Friese K, et al. Binding of galectin-1 (gal-1) on trophoblast cells and inhibition of hormone production of trophoblast tumor cells in vitro by gal-1. Histochem Cell Biol 2004;121 (6):501e8. [11] Blois SM, Ilarregui JM, Tometten M, Garcia M, Orsal AS, Cordo-Russo R, et al. A pivotal role for galectin-1 in fetomaternal tolerance. Nat Med 2007;13 (12):1450e7. [12] Walzel H, Blach M, Hirabayashi J, Kasai KI, Brock J. Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells. Glycobiology 2000;10(2):131e40. [13] Walzel H, Neels P, Bremer H, Kohler H, Raab N, Barten M, et al. Immunohistochemical and glycohistochemical localization of the beta-galactosidebinding S-type lectin in human placenta. Acta Histochem 1995;97(1):33e42. [14] Jeschke U, Mayr D, Schiessl B, Mylonas I, Schulze S, Kuhn C, et al. Expression of galectin-1, -3 (gal-1, gal-3) and the Thomsen-Friedenreich (TF) antigen in normal, IUGR, preeclamptic and HELLP placentas. Placenta 2007; 28(11e12):1165e73. [15] Fischer I, Schulze S, Kuhn C, Friese K, Walzel H, Markert UR, et al. Inhibiton of RET and JAK2 signals and upregulation of VEGFR3 phosphorylation in vitro by galectin-1 in trophoblast tumor cells BeWo. Placenta 2009;30(12):1078e82. [16] Pattillo RA, Gey GO, Delfs E, Mattingly RF. Human hormone production in vitro. Science 1968;159(3822):1467e9. [17] Getsios S, Chen GT, MacCalman CD. Regulation of beta-catenin mRNA and protein levels in human villous cytotrophoblasts undergoing aggregation and fusion in vitro: correlation with E-cadherin expression. J Reprod Fertil 2000;119(1):59e68. [18] Coutifaris C, Kao LC, Sehdev HM, Chin U, Babalola GO, Blaschuk OW, et al. E-cadherin expression during the differentiation of human trophoblasts. Development 1991;113(3):767e77. [19] Zabaglo L, Ormerod MG, Dowsett M. Measurement of proliferation marker Ki67 in breast tumour FNAs using laser scanning cytometry in comparison to conventional immunocytochemistry. Cytometry B Clin Cytom 2003;56 (1):55e61. [20] Remmele W, Stegner HE. [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe 1987;8(3):138e40. [21] Hirabayashi J, Kasai K. Human placenta beta-galactoside-binding lectin. Purification and some properties. Biochem Biophys Res Commun 1984; 122(3):938e44. [22] van den Brule F, Califice S, Garnier F, Fernandez PL, Berchuck A, Castronovo V. Galectin-1 accumulation in the ovary carcinoma peritumoral stroma is induced by ovary carcinoma cells and affects both cancer cell proliferation and adhesion to laminin-1 and fibronectin. Lab Invest 2003;83(3):377e86. [23] Rabinovich GA, Rubinstein N, Matar P, Rozados V, Gervasoni S, Scharovsky GO. The antimetastatic effect of a single low dose of cyclophosphamide involves modulation of galectin-1 and Bcl-2 expression. Cancer Immunol Immunother 2002;50(11):597e603. [24] Georgiadis V, Stewart HJ, Pollard HJ, Tavsanoglu Y, Prasad R, Horwood J, et al. Lack of galectin-1 results in defects in myoblast fusion and muscle regeneration. Dev Dyn 2007;236(4):1014e24. [25] Cooper DN, Barondes SH. Evidence for export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism. J Cell Biol 1990;110(5):1681e91. [26] Harrison FL, Wilson TJ. The 14 kDa beta-galactoside binding lectin in myoblast and myotube cultures: localization by confocal microscopy. J Cell Sci 1992;101 (Pt3):635e46. [27] Getsios S, MacCalman CD. Cadherin-11 modulates the terminal differentiation and fusion of human trophoblastic cells in vitro. Dev Biol 2003;257 (1):41e54. [28] Frendo JL, Cronier L, Bertin G, Guibourdenche J, Vidaud M, Evain-Brion D, et al. Involvement of connexin 43 in human trophoblast cell fusion and differentiation. J Cell Sci 2003;116(Pt 16):3413e21. [29] Malassine A, Handschuh K, Tsatsaris V, Gerbaud P, Cheynet V, Oriol G, et al. Expression of HERV-W Env glycoprotein (syncytin) in the extravillous trophoblast of first trimester human placenta. Placenta 2005;26(7):556e62. [30] Wice B, Menton D, Geuze H, Schwartz AL. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp Cell Res 1990; 186(2):306e16. [31] Ozawa M, Baribault H, Kemler R. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J 1989;8(6):1711e7. [32] Hinck L, Nathke IS, Papkoff J, Nelson WJ. Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly. J Cell Biol 1994;125(6):1327e40. [33] Polakis P. Wnt signaling and cancer. Genes Dev 2000;14(15):1837e51. [34] Wiest I, Seliger C, Walzel H, Friese K, Jeschke U. Induction of apoptosis in human breast cancer and trophoblast tumor cells by galectin-1. Anticancer Res 2005;25(3A):1575e80.
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Stimulation of Syncytium Formation in vitro in Human Trophoblast Cells by Galectin-1 I. Fischer a, S. Redel a, S. Hofmann a, C. Kuhn a, K. Friese a, H. Walzel b, U. Jeschke a, * a b
Ludwig Maximilians University of Munich, Department of Obstetrics and Gynecology, Maistrasse 11, 80337 Munich, Germany University of Rostock, Department of Biochemistry and Molecular Biology, Schillingallee 70, 18059 Rostock, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 24 June 2010
Background: Galectin-1 (gal-1), a member of the mammalian b-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In trophoblast cells, gal-1 binds to cell surface glycoproteins (e.g., Mucin1). It has been demonstrated that gal-1 recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast from second trimester human placenta and choriocarcinoma cells BeWo, which reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype. So the aim of this study was to investigate the effect of gal-1 on syncytium formation in BeWo and human villous trophoblasts (HVT) cells. Materials and methods: The effect of gal-1 on syncytium formation was investigated with immunocytochemical and double immunofluorescence stainings, cell-labelling and Real-time RT-PCR. BeWo choriocarcinoma and HVT cells were incubated in vitro for 24 and 48 h in the absence (controls) and presence of gal-1 and forskolin and stained with antibodies against Ki67, b-catenin, E-cadherin and syncytin. BeWo and HVT cells were incubated with 60 mg/ml gal-1 for 48 h (BeWo) or 96 h (HVT) and cell fusion was detected by fluorescent cell-labelling solution. Finally, BeWo cells were incubated for 1 h or 48 h in the absence and presence of 60 mg/ml gal-1 and Real-time RT-PCR was performed. Results: We showed with immunocytochemical staining a downregulation of b-catenin expression in the 24 h BeWo cell culture and with double immunofluorescence staining an inhibition of the b-catenin and E-cadherin expression in the 48 h BeWo cell culture stimulated with gal-1 or forskolin. The inhibition of E-cadherin was demonstrated on mRNA level in the 1 h BeWo cell culture too. Increased cell fusion was also showed with DiO and DiI fluorescent cell-labelling solution in the 48 h BeWo cell culture. In addition, we demonstrated the downregulation of Ki67 protein expression in the 24 h BeWo cell culture and on mRNA level in the 1 h BeWo cell culture. We also showed the upregulation of syncytin protein and mRNA expression after incubation of the 48 h BeWo cell culture with gal-1 or forskolin. Similar results were obtained with HVT cells: the amount of cell fusion was significantly increased in the gal-1 treated 48 h HVT cell culture in vitro compared to untreated cells as demonstrated with b-catenin and E-cadherin double immunofluorescence staining. This increase was also shown by fluorescent celllabelling with DiO and DiI in the 96 h HVT cell culture compared to untreated cells. Conclusion: Our data suggest that gal-1 stimulates the syncytium formation in choriocarcinoma cells BeWo and HVT cells in vitro and inhibits the expression of b-catenin, E-cadherin and in addition Ki67 in BeWo cells. Therefore gal-1 may be a major trigger for the process of trophoblast cell fusion. Ó 2010 Published by Elsevier Ltd.
Keywords: Galectin-1 BeWo Primary trophoblast cells Syncytium formation
1. Introduction Galectins, a growing family of carbohydrate proteins, are defined by an affinity for b-galactosides [1]. Although LacNAc (NAcetyl-Lactosamine) is the basic ligand recognized by gal-1, the
* Corresponding author. Tel.: þ49 89 5160 4266; fax: þ49 89 5160 4916. E-mail address: [email protected] (U. Jeschke). 0143-4004/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.placenta.2010.06.016
proto-type galectin binds with increased avidity to multiple Galb14GlcNAc sequences presented on branched N-linked or on repeating LacNAc-residues on N- and O-linked glycans. Extracellularly, gal-1 exerts biological effects by the recognition of glycan ligands, including those involved in cell adhesion [2] metastasis [3], cell growth regulation [4,5] and apoptosis [6]. In addition, it has been demonstrated that gal-1 induces cell differentiation processes in syncyciotrophoblast and extravillous trophoblast layer and on BeWo choriocarcinoma cells [7].
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Table 1 Antibodies used for this study. Antibody
Isotype
Clone
Dilution
Source
Immunocytochemistry b-catenin Ki67
Rabbit IgG Mouse IgG
polyclonal Mib-1
1:600 in PBS 0.1 M 1:500 in PBS 0.1 M
Diagnostic Biosystems, DAKO Glastrup
Double immunofluorescence stainings of BeWo cells after incubation with recombinant and placental gal-1 and forskolin E-cadherin Mouse IgG1 HECD-1 1:80 in DAKO S3022 Pan-Keratin Cy3 Mouse IgG A45-B/B3 1:1000 in DAKO S3022 Rabbit-Anti-Mouse Cy 2 Rabbit IgG polyclonal 1:50 in DAKO S3022
Calbiochem Micromet Dianova
Syncytin stainings of BeWo cells after incubation with recombinant gal-1 and forskolin Syncytin Rabbit IgG polyclonal Goat-Anti-Rabitt IgG Cy2 Goat IgG polyclonal
1:20 in DAKO S3022 1:100 in DAKO S3022
Abnova Dianova
Double immunofluorescence stainings of BeWo and HVT cells after incubation with recombinant gal-1 E-cadherin Mouse IgG1 HECD-1 b-Catenin Rabbit IgG polyclonal Goat-Anti-Mouse IgG Cy2 Goat IgG polyclonal Goat-Anti-Rabbit IgG Cy3 Goat IgG polyclonal
1:50 in DAKO S3022 1:50 in DAKO S3022 1:500 in DAKO S3022 1:100 in DAKO S3022
Calbiochem Diagnostic Biosystems Dianova Dianova
Expression of gal-1 has been well documented in placental trophoblast cells, decidual cells [8] and in immune privileged organs [9]. In addition, previous studies have shown that gal-1 decreases cellular hCG and progesterone production as well as hCG b gene transcription [10]. Furthermore, there is an evidence of a role for gal-1 in promoting murine fetomaternal tolerance in allogenetic matings, suggesting a potential approach for therapeutic intervention aimed at restoring immune cell homeostasis in failing pregnancies [11]. Gal-1 is defined by a conserved amino acid sequence motif in the carbohydrate recognition domain (CRD). Gal-1 with a single CRD forms a non-covalently associated homodimer to become functionally bivalent under physiological conditions. The bivalent nature of gal-1 facilitates glycan-mediated cell surface receptor cross-linking believed to be essential in inducing signalling events [6,12]. This information led to the question if gal-1, via its two carbohydrate binding sites, could influence the fusion formation in trophoblast cells. It has already been shown that gal-1 was strongly expressed by syncytiotrophoblasts in term and first trimester placenta, whereas villous cytotrophoblasts were negative [8,13]. The invading cytotrophoblast was weakly stained and BeWo cells expressed gal-1 particularly strongly [14]. It has also been demonstrated that gal-1 binds to BeWo cells, which form a syncytium in vitro, but does not bind to fresh isolated trophoblast cells and to choriocarcinoma cells Jeg-3, which cannot form a syncytium [10]. In a recent study we could demonstrate that the concentration of 60 mg/ml gal-1 induced a significant alteration of signal intensity in only 3 from 71 different Receptor tyrosine kinases, showing the specificity of gal-1 binding to cell surface glycoproteins at the concentration used for this experiment [15]. To demonstrate the effect of gal-1 on the syncytium formation in trophoblast cells, choriocarcinoma cell cultures BeWo, derived from human gestational choriocarcinoma and permanently established in 1968 by Pattillo et al. offer an attractive model because they reveal two coexisting phenotypes, the cytotrophoblast-like and the syncytiotrophoblast-like phenotype [16]. In addition, primary trophoblast cells can be used as a culture model for cells that are able to fuse in vitro but are unable to proliferate. Together, these two cell culture models might explain aspects of the complex situation of the placenta in vivo. Because fusion of trophoblast cells is accompanied by downregulation of Ecadherin and b-catenin [17,18] these proteins can be used for the analysis of trophoblast cell fusion. In addition, Ki67 as a proliferation marker [19] can serve as indicator for trophoblast differentiation.
2. Methods 2.1. Immunocytochemistry The choriocarcinoma cell line BeWo was obtained from the European Collection of Cell Cultures (ECACC, UK). BeWo cell suspensions at 1 105 cells/ml DMEM medium (Dulbecco’s Modified Eagle Medium, Biochrom, Germany, 3.7 g/l NaHCO3, 4.5 g/l D-Glucose, 1.028 g/l stable glutamine, Na-Pyruvate, supplemented with 10% heat-inactivated FCS (fetal calf serum) without antibiotics and antimycotics) were incubated for 24 and 48 h in chamberslides with 10, 30 and 60 mg/ml galectin-1 (Recombinant Human Galectin-1, 250 mg, Sigma Missouri, USA). Untreated cell cultures were used as controls. The experiments were done in quadruplicates. Slides were fixed with Ethanol/Methanol (1/1) for 15 min, washed with PBS (phosphate buttered saline, pH 7.4, 5 min) and stained with horse serum (30 min, room temperature). Then, the slides were stained with the primary antibodies b-catenin (1:500) and Ki67 (1:600) (see Table 1) for 18 h at 4 C. Sections were stained with the biotinylated secondary antibodies (30 min, room temperature). The VectastainÒ Elite ABC-Kit (Vector Laboratories, UK) was used for visualization according to the manufacturer’s instructions. Between each step, the sections were washed with PBS three times. Then, the slides were stained with AEC plus (3-Amino-9-Ethylcarbazole, Dako, Denmark) (12 min, room temperature) and washed in tap water. Finally, slides were counterstained with hemalaun (2 min, room temperature), washed in tap water and then covered. Sections were examinated using a Leica photomicroscope (Solms, Germany). Images were obtained with a digital camera system (JVC, Victor Company of Japan, Japan). 9 fields were analysed with the size of 1000 mm 1000 mm. For the Ki67 staining, the intensity and distribution patterns of specific immunohistochemical staining were evaluated using the semi-quantitative assay as previously described [20]. The IRS score was calculated by multiplication of optical staining intensity (graded as 0 ¼ no, 1 ¼ weak, 2 ¼ moderate and 3 ¼ strong staining) and the percentage of positive stained cells (0 ¼ no staining, 1 % of the cells, 2 ¼ 11e50% of the cells, 3 ¼ 51e80% of the cells and 4 81% of the cells). The slides were examined by two independent observers, including a gynecological pathologist. For the b-catenin staining, the intensity of antigen expression was evaluated by examinating the percental changing of the membrane-staining compared to the cell number (100% ¼ all membranes are positive, 0% ¼ no membrane is positive). The SPSS/PC software package, version 15.01 (SPSS, Germany), was used for collection, processing, and statistical data analysis. Statistical analysis was performed using the non-parametric Wilcoxon’s signed rank tests for comparison of the means. P < 0.05 values were considered statistically significant.
2.2. Double immunofluorescence staining of BeWo cells after incubation with recombinant and placental gal-1 and forskolin BeWo cell suspensions (ECACC, UK) at 1 105 cells/ml DMEM medium were incubated for 48 h in chamberslides with 10, 30 and 60 mg/ml galectin-1 (Recombinant Human Galectin-1, 250 mg, Sigma Missouri, USA, placental gal-1 (Dr. Walzel, Institut for Biochemie and Molecularbiologie, Germany) or 60 mg/ml forskolin (Sigma Aldrich, USA). The placental gal-1 was used in previous investigations. Due to the limited availability of placental gal-1, we used commercial available gal-1. To prove the homology of both proteins, placental and recombinant gal-1 were both used in this experiment. Untreated cell cultures were used as controls. The experiments were done in quadruplicates. Slides were fixed with Ethanol/Methanol (1/1) for 15 min, washed
I. Fischer et al. / Placenta 31 (2010) 825e832 with PBS and stained with Ultra V Block (Labvision, USA, 15 min, room temperature). Then, the slides were stained with the primary antibody E-cadherin Mouse IgG (1:80) (Table 1) overnight at 4 C. After washing, chamberslides were stained with Cy2 labelled antibody RabbitAnti-Mouse IgG (1:50; room temperature, 30 min) which will appear green. After washing, chamberslides were stained with Cy3 labelled Mouse anti-cytokeratin IgGAntibody (1:1000; room temperature, 45 min), which will appear red. The slides were finally covered with mounting buffer containing 40 ,6-diamino-2-phenylindole (DAPI, Denmark) and examined with a photomicroscope (Zeiss Axiophot, Germany). Images were obtained with a digital camera system (Axiocam, Zeiss, Germany). 9 fields were analysed with the size of 1000 mm 1000 mm. The intensity of the green E-cadherin expression was evaluated by examinating the percentage changing of the membrane-staining compared to the cell number (100% ¼ all membranes are positive, 0% ¼ no membrane is positive). Statistical analysis was performed as mentioned in Section 2.1.
2.3. Syncytin expression of BeWo cells after incubation with recombinant gal-1 and forskolin BeWo cell suspensions (ECACC, UK) at 1 105 cells/ml DMEM medium were incubated for 48 h in chamberslides with 60 mg/ml gal-1 (Recombinant Human Galectin-1, 250 mg, Sigma Missouri, USA) or 60 mg/ml forskolin (Sigma Aldrich, USA). Untreated cell cultures were used as controls. The experiments were done in quadruplicates. Slides were fixed with Aceton for 10 min, washed with PBS and stained with Ultra V Block (Labvision, USA, 15 min, room temperature). Then, the slides were stained with the primary antibody syncytin Rabbit IgG (1:20) (Table 1) overnight at 4 C. After washing, chamberslides were stained with Cy2 labelled antibody GoatAnti-Rabbit IgG (1:100; room temperature, 30 min) which will appear green. The slides were finally covered with mounting buffer containing 40 ,6-diamino-2-phenylindole (DAPI, Denmark) and examined with a photomicroscope (Zeiss Axiophot, Germany). Images were obtained with a digital camera system (Axiocam, Zeiss, Germany). 9 fields were analysed with the size of 1000 mm 1000 mm. The intensity of the expression was evaluated as mentioned in Section 2.2.
2.4. Double immunofluorescence staining of BeWo cells and HVT cells after incubation with recombinant gal-1 BeWo cells (ECACC, UK) and human villous trophoblasts (HVT) (ScienCell Researchlab., USA, HVT were isolated from human placental villi and cryopreserved at passage primary culture and delivered frozen. Each vial contains >1 106 cells in 1 ml volume. HVT were characterized by immunofluorescent method with antibodies to alpha- and beta-hCG. HVT are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HVT are guaranteed to further culture condition provided by ScienCell Research Laboratories.) (5 104 cells in 0.5 ml supplemented DMEM medium) were incubated for 48 h in chamberslides with 60 mg/ml gal-1 (recombinant human gal-1, 250 mg, Sigma Missouri, USA). Untreated cell cultures were used as control. Slides were fixed with Ethanol/Methanol (1/1) for 15 min, washed with PBS and stained with Ultra V Block (Labvision, USA; 15 min, room temperature). Then, the slides were stained with the primary antibodies E-cadherin Mouse IgG (1:50) and b-catenin Rabbit IgG (1:50) (Table 1) overnight at 4 C. After washing, chamberslides were stained with Cy2 labelled Mouse IgG-Antibody (1:500, room temperature, 30 min) and Cy3 labelled Rabbit IgG-Antibody (1:100, room temperature, 30 min). After washing, slides were finally embedded in mounting buffer containing 40 ,6-diamino-2-phenylindole (Dapi, Denmark) and examined with a fluorescent microscope (Axiophot, Zeiss, Germany). Images were obtained with a digital camera system (Axiocam, Zeiss, Germany). 5 independent experiments for both BeWo and HVT cells were performed. The amount of cell fusion was evaluated by the following equation: S ¼ noC1 1=m þ noC2 1=m þ noC3 1=m þ noC4 1=m þ . S ¼ sum, noC ¼ number of cell cluster, m ¼ number of cells forming the cell cluster. S is the sum of all clusters. Each cluster was divided by the number of nuclei in each cluster. 10 fields were analysed with the size of 1000 mm 1000 mm. Because fusion experiments were done on living cell cultures, it is not possible to induce toxic substances like DAPI to the cells. The advantage of both fluorescence dyes is that they are not toxic and have no effect on cell proliferation as described in literature. The formula was developed in our research team in cooperation with an engineer (MS) of orthopaedics from LMU in Großhadern, Munich. Statistical analysis was performed as mentioned in Section 2.1.
2.5. Detection of cell fusion by cell-labelling Both the choriocarcinoma cell line BeWo (ECACC, UK) and the human villous trophoblasts (HVT) (ScienCell Researchlab., USA) were treated according to following procedure:
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2.5 105 cells were labelled with 8 mg/ml DiO (1,10 -dioctadecyl-indocarbocyanine perchlorate) fluorescent cell-labelling solution (Vybrant Cell-Labelling Solutions, Molecular Probes, Scotland) in serum-free DMEM medium for 20 min at 37 C without CO2. Further 2.5 105 cells were labelled with 4 mg/ml DiI (1,10 -dioctadecyl-3,3,30 -tetramethylindocarbocyanine perchlorate) fluorescent cell-labelling solution (Vybrant Cell-Labelling Solutions, Molecular Probes, Scotland). Cells were washed with serum-free DMEM medium three times. After washing, cells were resuspended in DMEM medium supplemented with 10% heat-inactivated FCS, 250 mg/m Amphotericin B and 10 000 U/10 000 mg/ml Pencillin/Streptomycin. DiO and DiI labelled cell suspensions were mixed in one well of a 24-well plate. Finally, cells were incubated with 60 mg/ml gal-1 (recombinant human gal-1, 250 mg, Sigma Missouri, USA) for 48 h (BeWo) or 96 h (HVT) at 37 C. Cell suspensions without application of gal-1 were used as control. 5 independent experiments for both BeWo and HVT cells were performed. The amount of cell fusion was evaluated in ten randomly chosen fields of each well using Zeiss Axiovert 40 CFL fluorescent microscope (Zeiss, Germany). Images were obtained with a digital camera system (Power Shot A620, Canon, Japan). Statistical analysis was performed as mentioned in Section 2.1.
2.6. RNA extraction, reverse transcription, real-time RT-PCR 2.6.1. RNA extraction BeWo cells (ECACC, UK) (1 106 cells in 1.5 ml supplemented DMEM medium) were incubated in 25 ml cell culture bottles for 1 h or 48 h in the absence (controls) and presence of 60 mg/ml gal-1 (Recombinant Human Galectin-1, 250 mg, Sigma Missouri, USA) or 60 mg/ml forskolin (Sigma Aldrich, USA). Untreated cell cultures were used as controls. Total RNA was investigated by NucleoSpinÒ RNAII Kit (Machery-Nagel, Germany), according to the manufacturer’s protocol. 2.6.2. Reverse transcription Reverse transcription (RT) was carried out with the ‘High Capacity cDNA Reverse Transcription Kit’ (Applied Biosystems, Germany) (in 6 independant experiments) according to the protocol in a mastercycler gradient (Eppendorf, Germany). RT conditions were: 10 min 25 C, 2 h 37 C, 5 s 85 C and 4 C on hold). 2.6.3. Real-time RT-PCR Real-time reverse transcriptase-polymerase chain reactions (RT-PCRs) were performed in triplicates (in 6 independant experiments) in optical 96-well reaction microtiter plates covered with optical caps, in a volume of 20 ml containing 1 ml TaqManÒ Gene Expression Assay 20 (Order number Hs00170423_m1 for E-cadherin, Hs00606991_m1 for Ki67, Hs00170025_m1 for b-Catenin and Hs 00205893_m1 for syncytin all Applied Biosystems), 10 ml TaqManÒ Fast Universal PCR Master Mix 2 (Applied Biosystems), 1 ml template and 8 ml H2O. Thermal cycling conditions were: 20 s at 95 C, followed by 40 cycles of amplification with 3 s at 95 C and 30 s at 60 C. The ABI PRISM 7500 Fast (Applied Biosystems) was used to perform the PCR assays. Quantification was carried out by the ΔΔCt method using glycerinaldehyd phosphate dehydrogenase (GAPDH) as house-keeping gene (Order Number Hs99999905_m1, Applied Biosystems). Statistical analysis was performed as mentioned in Section 2.1.
3. Results 3.1. Immunocytochemistry As demonstrated in Fig. 1, Ki67 expression was significantly reduced in the gal-1 treated 24 h BeWo cell culture compared to the non-treated cells (IRS Score ¼ 9, SEM ¼ 0.35) (1A): 10 mg/ml (p ¼ 0.025) (IRS Score ¼ 6, SEM ¼ 0.35) (1B), 30 mg/ml (p ¼ 0.039) (IRS Score ¼ 4, SEM ¼ 0.35) (1C) and 60 mg/ml (p ¼ 0.041) (IRS Score ¼ 3.8, SEM ¼ 0.41) (1D). Incubation of 48 h BeWo cell culture with gal-1 did not further reduce the expression of Ki67 (data not shown). As demonstrated in Fig. 2, b-catenin expression was significantly reduced in the gal-1 treated 24 h BeWo cell culture compared to the non-treated cells (¼80%, SEM ¼ 3.53) (2A): 10 mg/ml (¼54%, SEM ¼ 5.70) (p ¼ 0.042), 30 mg/ml (¼34%, SEM ¼ 5.70) (p ¼ 0.042) (data not shown) and 60 mg/ml (¼12%, SEM ¼ 4.18) (p ¼ 0.041) (2B). Incubation of 48 h BeWo cell culture with gal-1 did not further reduce the expression of b-catenin, compared to the data shown for the 24 h cell culture.
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Fig. 1. BeWo cells were treated with gal-1 (24 h), and Ki67 positive cells numbers were assessed. A: control; B: gal-1 (10 mg/ml); C: gal-1 (30 mg/ml); D: gal-1 (60 mg/ml) (20). Positive cells are marked with an arrow.
3.2. Double immunofluorescence staining E-cadherin expression was significantly reduced from 70% (in the untreated BeWo cells SEM ¼ 4.18) to 30% in the 48 h BeWo cell culture treated with 60 mg/ml placental gal-1 (p ¼ 0.025, SEM ¼ 4.18) and to 33% with 60 mg/ml recombinant gal-1 (p ¼ 0.042, SEM ¼ 4.47). As demonstrated in Fig. 3AeD, the number of positive cells was significantly increased in the gal-1 treated 48 h BeWo cell culture (S ¼ 26.2) compared to untreated cells (S ¼ 95.5; p ¼ 0.005). As showed in Fig. 3E, the E-cadherin expression was significantly reduced from 70% (in the untreated BeWo cells SEM ¼ 4.18) to 33% in the 60 mg/ml forskolin treated 48 h BeWo cell culture (p ¼ 0.025, SEM ¼ 4.47). Syncytin protein expression was significantly increased from 29% (in the untreated BeWo cells SEM ¼ 3.95) to 78% in the 60 mg/ ml gal-1 treated cells (p ¼ 0.011, SEM ¼ 2.85) and to 85% in the
60 mg/ml forskolin treated cells (p ¼ 0.012, SEM ¼ 3.95), as demonstrated in Fig. 3FeH. The number of positive cells was significantly increased in the gal-1 treated 48 h HVT cell culture (S ¼ 38.7) compared to untreated cells (S ¼ 125.3; p ¼ 0.005), as demonstrated in Fig. 4.
3.3. Detection of cell fusion by cell-labelling As demonstrated in Fig. 5, the number of positive cells (mean SEM) was significantly increased in the gal-1 treated 48 h BeWo cell culture (21.0% 5.3%) compared to untreated cells (6.6% 1.7%; p ¼ 0.042). The number of positive cells (mean SEM) was significantly increased in the gal-1 treated 96 h HVT cell culture (23.2% 2.3%) compared to untreated cells (13.3% 1.7%; p ¼ 0.034), as demonstrated in Fig. 6.
Fig. 2. BeWo cells were treated with gal-1 (24 h), and b-catenin positive cells numbers were assessed. A: control; B: gal-1 (60 mg/ml) (20).
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Fig. 3. BeWo cells were treated with gal-1 (48 h), and b-catenin (red) positive cells numbers were assessed. A: control; B: gal-1 (60 mg/ml) E-cadherin (green) positive cell numbers were assessed C: control; D: gal-1 (60 mg/ml) (40). E-cadherin (green) stimulated with 60 mg/ml forskolin, 48 h (E). Syncytin (green) control (F), stimulated with 60 mg/ml forskolin (G) and stimulated with 60 mg/ml gal-1, 48 h (H) (all 20). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.4. Reverse transcriptase-polymerase chain reaction The treatment of the 1 h BeWo cell culture with 60 mg/ml gal-1 significantly decreased the E-cadherin mRNA expression to 78.8% (p < 0,01) and the Ki67 mRNA expression to 93.4% (p < 0,01) compared to the control. b-catenin mRNA expression was reduced
to 82.6% (p ¼ 0.043) compared to the control. The treatment of the 48 h BeWo cell culture with 60 mg/ml gal-1 significantly increased the syncytin mRNA expression to 121% (p ¼ 0.026) compared to the control. The treatment of the 48 h BeWo cell culture with 60 mg/ml forskolin non e significantly increased the syncytin mRNA expression to 110% (p ¼ 0.063) compared to the control.
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Fig. 4. HVT cells were treated with gal-1 (48 h), E-cadherin (green) positive cell numbers were assessed. A: control; B: gal-1 (60 mg/ml). b-catenin positive cell numbers were assessed C: control; D: gal-1 (60 mg/ml), a single non-fused cell is marked with an arrow (40). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 5. The number of positive cells was significantly increased in the gal-1 treated 48 h BeWo cell culture in vitro compared to untreated cells (p ¼ 0.005). BeWo cells were either stained with DiO (green) or Dil (red) and mixed. Both cell populations are seen in BeWo cells, 0 h cell culture in vitro, (A), and in BeWo cells stimulated with 60 mg/ml gal-1, 0 h (B), fusion of BeWo cells appears in yellow, 48 h (C), significantly higher numbers fused BeWo cells stimulated with 60 mg/ml gal-1, 48 h (D) (40). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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Fig. 6. The number of positive cells was significantly increased in the gal-1 treated 96 h HVT cell culture in vitro compared to untreated cells (p ¼ 0.005). HVT cells were either stained with DiO (green) or Dil (red) and mixed. Both cell populations are seen in BeWo cells, 0 h cell culture in vitro, (A), and in HVT cells stimulated with 60 mg/ml gal-1, 0 h (B), fusion of HVT cells appears in yellow, 96 h (C), significantly higher numbers fused HVT cells stimulated with 60 mg/ml gal-1, 96 h (D) (40). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
4. Discussion Galectins are members of a family of soluble animal lectins defined by shared characteristic amino acid sequences and an affinity for b-galactosides [21]. These human proteins have been shown to be involved in cellematrix adhesion, resistance to apoptosis, and angiogenesis. In addition, the involvement of galectins in cellular processes that are crucial for cancer progression and metastasis has been reported [22,23]. It has already been demonstrated that gal-1 is expressed by syncytiotrophoblasts in term and first trimester placenta [8,13] and binds to BeWo cells, which can form a syncytium. Furthermore, expression of gal-1 has been documented in decidual cells [8]. Syncytium formation is an exceptional process that takes place only in a few cell types, e.g., in myoblast, osteoclast [24] and trophoblast cells. The effect of cell fusion by gal-1 has been demonstrated in other cell types. It has been shown that gal-1 has a role in myoblast fusion in vivo and in vitro. The lectin has been implicated in the development of skeletal muscle, being maximally expressed at the time of myofiber formation [25,26]. It is located within the sarcoplasm of mononuclear myoblasts, but is exported to the extracellular compartment when muscle cells differentiate to their multinucleate stage of development [25,26]. In absence of gal1, myoblast cells cannot fuse as effectively as without. The exact mechanism is not fully understood [24]. In addition, the molecular mechanisms of the trophoblast membrane fusion are poorly investigated. However it is known that proteins implicated in cell adhesion (cadherin11) [27] and cellecell communication (connexin 43) [28] are directly involved. It has been demonstrated that the product of the HERV-W env gene (Env-W) is a fusogenic membrane glycoprotein (syncytin) that induces the formation of syncytia on interaction with the D-mammalian retrovirus receptor RDR [29].
Because it is also expressed in trophoblastic cells of the column, interstitial and endovascular trophoblast and thus in not-fusing cells, Malassine et al. suggest, that it appears to be required but not sufficient for trophoblastic cell fusion [29]. In this study we were now able to demonstrate that gal-1 stimulates fusion: in the 24 h BeWo cell culture we demonstrated the downregulation of b-catenin expression (Fig. 2), which is an indicator for cell fusion [17]. To underline our results, we investigated the effect of forskolin, which is known as an inducer of syncylization [30]. The staining results of E-cadherin after incubation with forskolin on BeWo cells were identical to the pictures of BeWo cells after incubation with gal-1 (Fig. 3AeE). Furthermore, we demonstrated an inhibition of the b-catenin and E-cadherin expressions in the 48 h BeWo cell culture with double immunofluorescence staining (Fig. 3A and B). The disappearance of E-cadherin from the cell-borders, parallel to the downregulation of the mRNA is an indicator for the syncytium formation in BeWo cells [18]. Although the inhibition of E-cadherin expression on protein level was observed after 48 h, the effect of gal-1 on mRNA level could be measured already 1 h after application of gal-1 to the BeWo cells. This rapid downregulation of mRNA level can be explained with the nature of signal transduction and transcription induction induced by gal-1 which were shown in former studies [15]. Increased cell fusion was also demonstrated with DiO and DiI cell labelling in the 48 h BeWo cell culture in vitro compared to untreated cells (Fig. 5). To show that this cascade is a de novo event induced by gal-1, we used syncytin as a fusion marker [29]: syncytin was upregulated on protein and mRNA level in the 48 h gal-1 stimulated BeWo cell culture (Fig. 3FeH). Similar results were obtained with the HVT cell culture: the amount of cell fusion was significantly increased in the gal-1 treated 48 h HVT cell culture in vitro compared to untreated cells as
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demonstrated with b-catenin and E-cadherin double immunofluorescence staining on protein level (Fig. 4). This increase was also demonstrated by cell-labelling with DiO and DiI in the 96 h HVT cell culture in vitro compared to the control (Fig. 6). Due to the nature of HVT cells which do not proliferate and migrate as fast as BeWo cells, cell fusion was significantly induced after 96 h, whereas cell fusion in BeWo cells was already upregulated after 48 h. In this study we were also able to confirm previous studies, which showed that the proliferation of BeWo cells is inhibited by gal-1. This was demonstrated with a 5-bromo-20 -deoxy-uridine (BrdU) incorporation and ELISA [7]. We now demonstrated the downregulation of Ki67 expression (Fig. 1), which is an indicator for proliferation inhibition [19], in the 24 h BeWo cell culture and on mRNA level in the 1 h BeWo cell culture. Syncytium formation and decreased proliferation are linked processes. Because only non-fusing BeWo cells proliferate, Ki67, as a proliferation marker, is already downregulated in the 24 h cell culture, whereas E-cadherin, as a syncytium indicator, is altered in the 48 h BeWo cell culture. The association with b-catenin, which interacts with the cytoplasmic domain of the transmembrane-adhesion-molecule E-cadherin [31] and links it to the cytoskeleton via a-catenin [32], is essential for the cadherin mediated cell-to-cell adhesion [33]. This is in line with the results of our study, because the b-catenin expression is already inhibited after 24 h and the E-cadherin only after 48 h. In a previous study we could demonstrate that an increase of apoptosis in BeWo cells by gal-1 was only shown by additional stimuli like hyperthermia, removal of CO2 and FCS. So the downregulation of Ki67 in this study is not due to death of the cells [34]. In summary, we could show in this study that gal-1 is an inducer of syncytium formation both in isolated trophoblast cells and choriocarcinoma cells BeWo. This effect was studied with special fluorescence labelling of individual cell populations and with immunocytochemistry labelling of cell membrane bound adhesion proteins. Downregulation of adhesion molecule expression as the result of cell fusion was measured on protein and mRNA level. Fusion of BeWo cells is accompanied by reduced proliferation: we detected this inhibition of proliferation in gal-1 treated BeWo cells. Acknowledgement We thank S. Kunze and S. Schulze for excellent assistance. The study was supported by the “Deutsche Forschungsgemeinschaft” (DFG). References [1] Barondes SH, Castronovo V, Cooper DN, Cummings RD, Drickamer K, Feizi T, et al. Galectins: a family of animal beta-galactoside-binding lectins. Cell 1994;76(4):597e8. [2] Hughes RC. Galectins as modulators of cell adhesion. Biochimie 2001; 83(7):667e76. [3] Raz A, Lotan R. Endogenous galactoside-binding lectins: a new class of functional tumor cell surface molecules related to metastasis. Cancer Metastasis Rev 1987;6(3):433e52. [4] Wells V, Mallucci L. Identification of an autocrine negative growth factor: mouse beta-galactoside-binding protein is a cytostatic factor and cell growth regulator. Cell 1991;64(1):91e7. [5] Adams L, Scott GK, Weinberg CS. Biphasic modulation of cell growth by recombinant human galectin-1. Biochim Biophys Acta 1996;1312(2):137e44. [6] Perillo NL, Pace KE, Seilhamer JJ, Baum LG. Apoptosis of T cells mediated by galectin-1. Nature 1995;378(6558):736e9. [7] Jeschke U, Karsten U, Wiest I, Schulze S, Kuhn C, Friese K, et al. Binding of galectin-1 (gal-1) to the Thomsen-Friedenreich (TF) antigen on trophoblast cells and inhibition of proliferation of trophoblast tumor cells in vitro by gal-1 or an anti-TF antibody. Histochem Cell Biol 2006;126(4):437e44. [8] Vicovac L, Jankovic M, Cuperlovic M. Galectin-1 and -3 in cells of the first trimester placental bed. Hum Reprod 1998;13(3):730e5.
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