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
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- Jeschke, U., Karsten, U., Wiest, I. et al. Histochem Cell Biol (2006) 126: 437. doi:10.1007/s00418-006-0178-1
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Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, recognizes preferentially Galβ1-4GlcNAc sequences of several cell surface oligosaccharides. We demonstrate histochemically that the lectin recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast layer from second trimester human placenta and on BeWo chorion carcinoma cells. Gal-1 binding to BeWo cells was diminished by the Thomsen–Friedreich (TF)-disaccharide (Galβ1-3GalNAc-) conjugated to polyacrylamide (TF–PAA). Gal-1 also inhibited BeWo cell proliferation in a concentration-dependent manner. Similar antiproliferative effects were also observed with an anti-TF monoclonal antibody (mAb, A78-G/A7). Therefore, we conclude that ligation of Galβ1-4GlcNAc and Galβ1-3GalNAc epitopes on BeWo cells may have regulatory effects on cell proliferation.
The Thomsen–Friedenreich glycotope (Galβ1-3GalNAc-), briefly called TF-antigen, is expressed in more than 85% of human carcinomas (Springer 1984; Goletz et al. 2003). Beside its remarkable tumor specificity, the TF disaccharide is a blood group-related stage-specific oncofetal antigen present during the early fetal phase. In normal adult human tissues TF epitopes are only expressed in limited amounts and are restricted to a few immunologically privileged sites (Cao et al. 1996). The TF antigen is, however, expressed on fetal epithelia and mesothelia (Barr et al. 1989), on transferrin from human amniotic fluid (van Rooijen et al. 1998) and on trophoblast cells (Richter et al. 2000). The TF antigen and its carrier protein mucin 1 (MUC1) are expressed by syncytiotrophoblasts at the materno-fetal interphase and by extravillous trophoblast cells invading the decidua. It is also found on BeWo trophoblastic tumor cells forming a syncytium in vitro (Jeschke et al. 2002). Therefore it is conceivable that the TF antigen is involved in homotypic and heterotypic adhesion processes leading to the formation of multicellular aggregates. Recent experimental evidence demonstrated a key role for galectin-3 (gal-3) in initiating the adhesion of human breast and prostate carcinoma cells to vasculatory endothelium (Glinsky et al. 2000, 2001; Lehr and Pienta 1998). By interaction with the cancer-associated TF antigen gal-3 mediates the adhesion of human breast carcinoma cells to the endothelium under static and flow conditions by a unique mechanism that qualitatively distinguishes their homo- and hetero-typic adhesive behavior from leukocytes (Khaldoyanidi et al. 2003). Lactulosyl-l-leucine that competes with the TF antigen for gal-3 binding caused a strong reduction of homo- and hetero-typic adhesion of highly metastatic MDA-MB-435 breast carcinoma cells expressing high levels of both gal-3 and TF-antigen. Several TF-binding proteins influence epithelial tumor cell proliferation (Yu et al. 1997). TF-disaccharide binding proteins, that stimulate the proliferation of epithelial tumor cell lines, recognize mainly the terminal β-Gal region of β-linked TF antigen, whereas proliferation inhibition is mainly associated by binding to subterminal GalNAc of α-anomeric TF antigen (Irazoqui et al. 2001). On tumor cells MUC1 is post translationally modified resulting in incomplete O-glycosylation and presentation of the TF-disaccharide. Natural antibodies of TF-specificity may provide an early barrier against TF-carrying tumor cells (Butschak and Karsten 2002). Autoantibody recognition of endometrial and serum sialylated TF-antigen bearing proteins are a common feature of endometriosis (Yeaman et al. 2002). Autoantibody targeting to TF-antigen may be indicative of an underlying defect or in the control of glycosylation by steroid hormones. By contrast with the restricted expression of TF-disaccharide, N-acetyllactosamine (LacNAc, Galβ1-4GlcNAc) is found on the termini of N-linked and O-linked oligosaccharides on numerous glycoproteins. The galectin family members are defined by a conserved amino acid sequence motif in the carbohydrate recognition domain (CRD) and an affinity for β-galactosides (Barondes et al. 1994a, b). Although LacNAc is the basic ligand recognized by gal-1, the proto-type galectin binds with increased avidity to multiple Galβ1-4GlcNAc sequences presented on branched N-linked or on repeating LacNAc-residues on N- and O-linked glycans. Gal-1 having 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 signaling events (Perillo et al. 1995; Walzel et al. 2000). Extracellularly, gal-1 exerts distinct biological effects in various tissues and on cells by the recognition of glycan ligands, including cell adhesion (Hughes 2001; Hafer-Macko et al. 1996), metastasis (Raz and Lotan 1987), cell growth regulation (Wells and Mallucci 1991; Adams et al. 1996), immunosuppression (Offner et al. 1990), and apoptosis (Perillo et al. 1995).
In this article we describe the binding of gal-1 on TF antigen expressing trophoblast cells and demonstrate that binding of appropriate glycotopes by gal-1 and a TF-specific antibody negatively regulates the proliferation of BeWo cells in vitro.
Materials and methods
DMEM-medium and fetal calf serum (FCS) were purchased from Biochrom AG (Berlin, Germany). FITC-labelled streptavidin and Cy-3 labelled secondary antibodies were obtained from Dianova (Hamburg, Germany). Cytokeratin 7 monoclonal antibody (mAb, Ks7.18) was from Progen Biotech (Heidelberg, Germany), the mAbs A78-G/A7 and the A63-C/A9 (both IgM) from Glycotope (Berlin, Germany). The TF–PAA conjugate was obtained from Lectinity Holding Inc. (Moscow, Russia), biotin-N-hydroxysuccinimide and the BrdU cell proliferation ELISA kit were from Roche Diagnostics (Mannheim, Germany), and Bio-Gel P6 was from Bio-Rad (Munich, Germany). Frozen placental tissues were obtained from women who underwent delivery at the first Department of Obstetrics and Gynaecology of the LMU Munich. Specimens were collected immediately after delivery from eight patients following a normal course of pregnancy (mean date of delivery: 38.2±3 weeks of gestation).
The chorionic carcinoma cell line BeWo was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were cultured in DMEM medium supplemented with 10% heat-inactivated FCS without antibiotics and antimycotics.
Purification and biotinylation of gal-1 from human placenta
Gal-1 was prepared from term placental tissue by lactose extraction with EDTA-MePBS (20 mM sodium phosphate, pH 7.2, 150 mM NaCl, 4 mM 2-mercaptoethanol, 2 mM EDTA, all BioRad, Munich, Germany) and purified by sequential affinity chromatography on asialofetuin Sepharose 4B (Hirabayashi and Kasai 1984) followed by affinity chromatography on lactosyl agarose (GE healthcare, Freiburg, Germany). Then the protein was purified to homogeneity by anion exchange chromatography on a Resource Q column (GE Healthcare) (Walzel et al. 1999).
The lectin (1 mg/ml) was biotinylated in phosphate buffered saline (PBS, pH 8.0) by the addition of 40 μl 10 mM biotinyl-N-hydroxysuccinimide in dimethyl sulfoxide (Boorsma et al. 1986). After incubation at room temperature for 1 h, biotinylated gal-1 was affinity-purified on lactosyl agarose (GE healthcare, Freiburg, Germany). The bound fraction was eluted with 50 mM lactose in EDTA-MePBS. Buffer change was performed on a Bio-Gel P6 column equilibrated with PBS, pH 7.4 (Jeschke et al. 2004).
Glyco- and immuno-histochemical staining
Cells of the trophoblast tumor cell line BeWo were grown on three-well multitest slides to subconfluency, dried, wrapped, and stored at −80°C. In addition, frozen tissue sections of third trimester placentas (39th week of gestation) were used. After thawing, slides were fixed with 5% formalin in PBS, pH 7.4, for 5 min. Then the slides were incubated overnight at 4oC with biotinylated gal-1 at 0.3 μg/ml in PBS, pH 7.4, in the absence and presence of up to 40 μg/ml TF-α disaccharide coupled to polyacrylamide (TF–PAA). After washing, Cy2-labelled streptavidin (1:200 dilution) served as fluorochrome. For immunostaining the slides were incubated with the antibodies at 2 μg/ml (cytokeratin 7, mouse IgG, Novocasta, Newcastle, UK, or CD7, mouse IgG, Serotec, Düsseldorf, Germany) overnight at 4°C. Cy3-labelled goat anti-mouse IgG (1:200 dilution) served as second antibody conjugate. The slides were washed and finally embedded in mounting buffer containing 4′,6′-diamino-2-phenylindole (DAPI) resulting in blue staining of the nuclei (Jeppesen and Nielsen 1989) and examined with a Zeiss (Jena, Germany) Axiophot photomicroscope. Images were obtained with a digital camera system (Axiocam; Zeiss, Jena, Germany).
Treatment of BeWo cell cultures with gal-1, TF antibodies and glycophorin antibodies
Cell suspensions at 1×106 cells/ml culture medium were incubated in 24-well cell culture plates with 10, 20, 30, and 60 μg/ml gal-1, with 10, 20, 30, and 60 μg/ml anti-TF mAb (A78-G/A7), or with 10 20, 30, and 60 μg/ml anti-glycophorin mAb (A63-C/A9) as isotype control for up to 72 h. Untreated cell cultures were also used as controls. Cell proliferation of non-treated cultures (controls) and the effects of gal-1-, TF mAb- and of glycophorin mAb-treated cell cultures were studied simultaneously and carried out in triplicates.
Cell proliferation assay
Cell proliferation was analyzed with a 5-bromo-2’-deoxy-uridine (BrdU) labelling and detection kit according to the instructions of the manufacturer. BeWo cells (1×105 in 0.1 ml supplemented cell culture medium) were grown in 96-well tissue culture plates for 72 h in the absence (controls) and presence of gal-1, the TF mAb (A78-G/A7), or the glycophorin IgM mAb. After labelling with BrdU for 3 h, the cells were fixed and BrdU incorporation into DNA was measured by an ELISA technique. Cellular proliferation is expressed as percent compared to the control.
The SPSS/PC software package, version 12.01 (SPSS, Munich, Germany), was used for collection, processing, and statistical data analysis. Statistical analysis was performed using the non-parametrical Wilcoxon´s signed rank tests for comparison of the means. P<0.05 values were considered statistically significant.
Glyco- and immuno-histochemical staining
Inhibition of BeWo cell proliferation induced by gal-1
Inhibition of BeWo cell proliferation induced by IgM TF-mAb
BeWo cell proliferation is non-significantly changed by the glycophorin IgM mAb as an isotype control
The data presented here demonstrate the binding of gal-1 to TF-antigen expressing placental cells. Gal-1 binding to TF-antigen presenting BeWo trophoblast tumor cells induces anti-proliferative effects. The TF epitope is expressed on syncytiotrophoblasts at all stages of pregnancy and on extravillous trophoblasts of the decidua in the first and second trimester of pregnancy (Richter et al. 2000; Jeschke et al. 2002). The TF-antigen is considered to be an oncodevelopmental marker and TF expression resembles changes in glycosylation associated with neoplastic transformation (Cao et al. 1997). BeWo chorionic carcinoma cell cultures reveal two coexisting phenotypes: a cytotrophoblast-like and a syncytiotrophoblast-like phenotype (Grummer et al. 1994). We found strong expression of TF-antigen on these cells. TF-positive cells very likely represent the syntiotrophoblast-like phenotype because the syncytiotrophoblast in vivo also strongly express TF-antigen (Jeschke et al. 2002).
Galectins are members of a family of soluble animal lectins defined by shared characteristic amino acid sequences and an affinity for β-galactosides (Hirabayashi and Kasai 1993). Gal-1, a member of the family of soluble β-galactoside binding proteins, is initially synthesized in the trophectoderm of the expanded blastocyst immediately prior to implantation, suggesting a role in the attachement of the embryo to the uterine epithelium (Poirier et al. 1992). It has been demonstrated that gal-1 was strongly expressed by syncytiotrophoblasts in term and first trimester placenta, whereas villous cytotrophoblasts were negative (Walzel et al. 1995; Vicovac et al. 1998). The invading cytotrophoblast was weakly stained and BeWo cells expressed gal-1 particularly strongly (Vicovac et al. 1998). This expression scheme is in striking conformity with the TF-antigen expression in the placenta (Richter et al. 2000; Jeschke et al. 2002). The presented data confirm the hypothesis that gal-1 binds to the TF-antigen on syncytiotrophoblast, extravillous trophoblast cells, and on BeWo cells. Gal-1 binding to tissue sections could be inhibited by the TF–PAA conjugate.
Galectins have been shown to be involved in cellular processes that are crucial for cancer progression and metastasis (Rabinovich et al. 2002). Both, gal-1 and gal-3 have been reported to induce the homotypic adhesion of isolated tumor cells by interacting with membrane-associated ligands (Glinsky et al. 2000; Tinari et al. 2001). In addition, the involvement of galectins in cell-matrix adhesion, resistance to apoptosis, and angiogenesis have been reported (van den Brule et al. 2004). Binding of gal-1 to the TF-antigen on trophoblast cells could play a role during implantation of the embryo in the endometrium. On the other hand, gal-1 also induces apoptosis of activated T cells through binding to CD45 (Perillo et al. 1995; Walzel et al. 1999) and CD7 (Pace et al. 2000). Extracellular gal-1 also induces death of T cells and thymocytes through binding to CD7 (Stillman et al. 2006). We identified CD7 positive cells that bound gal-1 in the placenta but these cells were not of trophoblast origin and BeWo cells expressed no CD7. The formation of the placenta resembles in many respects the invasion of malignant tumors. At the implantation site fetus-derived trophoblast cells invade into the maternal decidua and the contact was found to be intimate in the first trimester of pregnancy (Hammer et al. 1999; Hammer and Dohr 1999). Because expression of the TF-antigen on extravillous trophoblast cells is restricted to the first and second trimester, gal-1 could act as a suppressor of maternal T-cells within the decidua.
It has been demonstrated that recombinant gal-1 has a biphasic effect on cell proliferation (Adams et al. 1996). Studies with ovarian cancer cell lines showed decreasing effects on cell proliferation and modulation of cell adhesion to laminin-1 (van den Brule et al. 2003). Consistent results were obtained in our study. Gal-1 inhibited the proliferation of BeWo cells in a concentration-dependent manner. Whereas gal-1 at concentrations of 10 and 20 μg/ml non-significantly decreases cell proliferation, higher concentrations (30 and 60 μg/ml) significantly inhibited BeWo cell proliferation. Furthermore, the TF-specific mAb (A78-G/A7), (Karsten et al. 1995) also displayed comparable inhibitory effects, whereas the addition of an unrelated IgM mAb did not induce proliferation inhibition. These results indicate an involvement of the TF-antigen in regulation of cell proliferation.
We have previously shown that gal-1 non-significantly increased the apoptosis rate of MCF7 and BeWo cells. Under stress conditions (hyperthermia, removal of CO2 and FCS) however, gal-1 significantly increased the apoptosis rate in both cell lines when compared to control cultures (Wiest et al. 2005). Together with the results of this study we speculate that inhibition of BeWo cell proliferation by gal-1 is not due to induction of apoptosis in these cells.
In summary, our data suggest that gal-1 is localized at the border between fetal trophoblast and maternal stroma could modulate these interactions and promote invasion and maintenance of pregnancy.
We thank D.U. Richter and G. Gaede for excellent technical assistance. The study was supported by the “Deutsche Forschungsgemeinschaft” (DFG) for U. Jeschke and H. Walzel.