Histochem Cell Biol (2004) 121:501–508 DOI 10.1007/s00418-004-0660-6

ORIGINAL PAPER

Udo Jeschke · Toralf Reimer · Claudia Bergemann · Irmi Wiest · Sandra Schulze · Klaus Friese · Hermann Walzel

Binding of galectin-1 (gal-1) on trophoblast cells and inhibition of hormone production of trophoblast tumor cells in vitro by gal-1 Accepted: 6 May 2004 / Published online: 8 June 2004
Springer-Verlag 2004

Abstract Galectin-1 (gal-1), a member of the mammalian b-galactoside-binding proteins, recognizes preferentially Galb1–4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. As demonstrated histochemically, the lectin recognizes appropriate glycoepitopes on the syncytiotrophoblast and on chorionic carcinoma cells (BeWo). Freshly isolated trophoblast cells and trophoblast tumor cells Jeg3 did not bind gal-1. BeWo cells in contrast to Jeg3 form a syncytium in vitro and synthesize progesterone as well as hCG. BeWo cells were used as an approach to study the effects of gal-1 on hormone production. The lectin decreased cellular hCG and progesterone production as well as hCGb gene transcription as measured by real-time RT-PCR. Gal-1 mediated inhibition of cellular progesterone production was reduced in the presence of a Thomsen-Friedenreich (TF)– polyacrylamide conjugate. Inhibition of cellular hCG and progesterone production was also induced by anti-TF monoclonal antibodies. The results demonstrate that ligation of Galb1–4GlcNAc and Galb1–3GalNAc (TF) epi-topes on BeWo cells may have regulatory effects on hCG and progesterone production. Keywords Galectin-1 · Glycotopes · Hormone production · BeWo cells U. Jeschke ()) · I. Wiest · S. Schulze · K. Friese Department of Obstetrics and Gynaecology, Ludwig Maximilians University of Munich, Maistrasse 11, 80337 Munich, Germany e-mail: [email protected] Tel.: +49-89-51604266 Fax: +49-89-51604916 T. Reimer · C. Bergemann Department of Obstetrics and Gynaecology, University of Rostock, Doberaner Strasse 142, 18055 Rostock, Germany H. Walzel Institute of Medical Biochemistry and Molecular Biology, University of Rostock, Schillingallee 70, 18057 Rostock, Germany

Introduction The galectin family members are defined by a conserved amino acid sequence motif in the carbohydrate recognition domain (CRD) and an affinity for b-galactosides. Galectin-1 (gal-1), the earliest described member of the family, is a homodimeric protein with two CRDs (Barondes et al. 1994). Expression of gal-1 has been specially identified in lymphoid organs such as thymus, lymph nodes, in activated macrophages, T cells, and in immuneprivileged sites such as placenta and cornea, suggesting an important role in generating and maintaining immune tolerance (La et al. 2003). Although N-acetyllactoseamine (LacNAc) is the basic ligand recognized by gal-1, the prototype galectin binds with increased avidity to multiple Galb1–4GlcNAc 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 crosslinking believed to be essential in inducing signaling events (Perillo et al. 1995; Walzel et al. 2000). Extracellularly, by recognition of glycan ligands, gal-1 exerts distinct biological effects in various tissues and on cells, including cell adhesion (Hughes 1992; 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). Gal-1 is expressed in the placenta. The placenta plays a key role in the maintenance of local tolerance and allows the mother to accept the embryo until completion of pregnancy. The complex process of tolerance accompanying the survival of the fetus is controlled at the maternofetal interface by factors deriving from decidualized endometrium and from the trophoblast itself. Trophoblasts develop various strategies to evade the damaging attack of the maternal immune response including expression of non-classical MHC class I antigens and of complement regulatory proteins (Bulla et al. 2003). Cho-

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riocarcinoma cell lines were evaluated as an experimental model of trophoblast-derived immunoregulation (Bennett et al. 1995). The cell line BeWo was established by Pattillo et al. (1968). In contrast to Jeg3, cultures of BeWo choriocarcinoma cells contain two coexisting phenotypes: a cytotrophoblast-like and a syncytiotrophoblastlike phenotype (Grmmer et al. 1990). We found with these cells a moderate staining for mucin 1 (MUC 1) but a strong expression of the Thomsen-Friedenreich (TF) tumor antigen (Jeschke et al. 2002). This pattern very likely represents the syncytiotrophoblast-like phenotype because the syncytiotrophoblast in vivo also strongly expresses TF and MUC 1 (Richter et al. 2000). In this article we describe the binding of placental gal-1 on trophoblast cells and demonstrate that ligation of appropriate glycoepitopes by gal-1 and a TF-monoclonal antibody (mAb) negatively regulates the hCG and progesterone production of BeWo cells in vitro.

1 ml acetone, 25 ml 50 mM sodium acetate, pH 5.0, and 15 ml H2O2 (30%). Preparation and culture of villous cytotrophoblast cells (CTB) The CTBs were prepared according to an already published protocol (Jeschke et al. 2002). Briefly, first trimester placental tissue was minced and digested three times with trypsin and DNase I in HBSS-HEPES (all obtained from Sigma-Aldrich Fine Chemicals, Taufkirchen, Germany). The first purification step was performed on a Percoll gradient. After centrifugation, CTBs were aspirated and immunopurified using antibodies to CD45 and CD9 (Dianova, Hamburg, Germany) to eliminate contaminating leukocytes and extravillous trophoblasts. Trophoblast identity of the isolated cells was determined morphologically by light microscopy and by immunostaining with cytokeratin 7 mAb (Progen Biotechnik, Heidelberg, Germany). Cells were cultured in DMEM medium with 10% inactivated FCS and penicillin/streptomycin (100 U/ml; Biochrom) and amphotericin B (2.5 mg/ml; Biochrom). Immunocytochemistry

Materials and methods Purification of gal-1 Gal-1 was obtained 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) and purified by sequential affinity chromatography on asialofetuin Sepharose 4B (Hirabayashi and Kasai 1984) followed by lactosyl agarose. Then the protein was purified to homogeneity by anion exchange chromatography on a Resource Q column (Walzel et al. 1999).

BeWo and Jeg3 trophoblast tumor cells and isolated first trimester trophoblast cells were grown on three-well multitest slides (Roth, Karlsruhe, Germany) to subconfluency, dried, wrapped, and stored at 80C. After thawing, cells were briefly fixed with formalin (Merck, Darmstadt, Germany; 5% in PBS, 5 min). Biotinylated gal1 used for the experiments was diluted to 0.3 mg/ml with PBS and incubated with the slides overnight at 4C. After washing, Cy3labeled streptavidin, diluted 1:200, served as fluorochrome. The slides were finally embedded in mounting buffer containing 40 ,6diamino-2-phenylindole resulting in blue staining of the nucleus (Jeppesen and Nielsen 1989) and examined with a Zeiss (Jena, Germany) Axiophot photomicroscope. Digital images were obtained with a digital camera system (CF20DXC; KAPPA Messtechnik, Gleichen, Germany).

Biotinylation of gal-1 The lectin (1 mg/ml) was biotinylated in phosphate-buffered saline (PBS, pH 8.0) by the addition of 40 ml 10 mM biotinyl-N-hydroxysuccinimide in dimethylsulfoxide (Boorsma et al. 1986). After incubation at room temperature for 1 h, biotinylated gal-1 was purified by affinity chromatography on lactosyl agarose. The binding fraction was released with 50 mM lactose in EDTAMePBS. Buffer change was performed on a Bio-Gel P6 column equilibrated with PBS, pH 7.4. Cells The chorionic carcinoma cell lines BeWo and Jeg3 were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). Cells were cultivated in DMEM medium (Biochrom, Berlin, Germany), with 10% heat-inactivated fetal calf serum (FCS; Biochrom) and without antibiotics and antimycotics. Immunochemical detection of gal-1 in BeWo cell lysates on blots BeWo cells (3107) were lysed in 400 ml Laemmli sample buffer for 5 min at 100C. Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 4% BSA in TBS/Tween (20 mM TRIS/HCl, pH 7.2, 1 M NaCl, 0.05% Tween 20), blots were incubated with 3 mg/ml gal-1 polyclonal antibodies (pAb), 3 mg/ml rabbit IgG as a control, or TBS/ Tween (blank) in TBS/Tween with 1% BSA for 16 h at 6C. Blots were washed and incubated with a goat anti-rabbit IgG-HRP conjugate (diluted 1:500 in TBS/Tween with 1% BSA) for 2 h. Staining was performed with 10 mg 3-amino-9-ethylcarbazole in

Treatment of trophoblast cultures with gal-1, TF mAb, and a TF-disaccharide-polyacrylamide (PAA) conjugate Cell suspensions were incubated at 1106 cells/ml supplemented cell culture medium in 24-well plates (Greiner Labortechnik, Solingen, Germany) in the presence of 15, 30, 60, and 100 mg/ml gal-1 or 10 mg/ml TF mAb (A78-G/A7; NEMOD, Berlin, Germany) or 30 mg/ml TF-PAA conjugates (Synthesome, Moscow, Russia). Untreated cell cultures were used as controls. The effect of gal-1 on hormone secretion was studied by comparing the kinetics of hCG mRNA or protein and progesterone production of untreated (controls) and gal-1-treated cell cultures simultaneously. All experiments were carried out at least in triplicate. Identification of secreted hormones The secretion of hCG and progesterone was determined by means of the analyzer SR1 (BioChemImmunoSystems, Freiburg, Germany). Progesterone was analyzed by competitive enzyme immunoassay, and hCG by a direct method (immunoenzymometric assay). Specific antibodies were labeled with fluorescein. After reaction with antifluorescein antibody and binding to magnetic particles, separation was performed. Color intensity was measured at three different wave lengths. Cell culture supernatants for hCG were used undiluted, but samples for progesterone determination were diluted 1:3 with hormone-free diluent (BioChemImmunoSystems) because of the intensively colored culture medium. Sensitivity (<6.4 nMol/l) and the intra-assay coefficient of variation of progesterone EIA (6.8% at a medium value of 11.5 nMol/l or 5.4% at 65.6 nMol/l), and of hCG IEMA (1 IU/l,

503 related to 1 IRP 75/537; 6.4% at 11.1 IU/l or 4.2% at 104 IU/l, respectively) demonstrate the accuracy of the applied methods. Identification of hCGb-mRNA transcripts (real-time RT-PCR) Total RNA was prepared using the acid guanidium thiocyanatephenol-chloroform protocol (Chomczynski and Sacchi 1987). The quality of the RNA samples was determined by electrophoresis through denaturing agarose gels and staining with ethidium bromide. The RNA was quantified and evaluated for purity by UV spectrophotometry. To further assess the quality of RNA, all specimens were tested by analysis of housekeeping gene expression using conventional RT-PCR. The hCG-b primers and probe were designed using the Primer Express 1.0 program (PE Applied Biosystems, Foster City, CA). Oligonucleotide hybridization probe and primers with the following sequences were synthesized as follows: TaqMan probe 50 -CTG CTG AGC ATG GGC GGG ACA T (exon 2), forward primer 50 CCA AGG ATG GAG ATG TTC CAG, and reverse primer 50 GCA CGC GGG TCA TGG T. The reverse primer was designed to span an exon/intron junction in order to avoid amplification of DNA sequences, whereas the forward primer was complementary to the 30 -end of exon 1. Primers and probe were obtained from Applied Biosystems (Weiterstadt, Germany). The primers yielded an RT-PCR product of 199 nucleotides. Preparation of RNA standard and quantification of hCG-b mRNA expression by ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) were published in detail previously (Reimer et al. 2000a, b). The TaqMan EZ RT-PCR kit (PE Applied Biosystems) was used for reverse transcription and amplification of both targets and standards. All RT-PCR reactions were performed in triplicate with a final volume of 25 ml. RT-PCR conditions were optimized for primers (300 nM), probe (100 nM), template (100 ng), and manganese acetate concentrations (3 mM). The reaction conditions were 2 min at 50C, 30 min at 60C, 2 min at 95C, 40 cycles with 20 s at 94C and 1 min at 60C.

Fig. 1 a Fluorescence microscopy of biotinylated galectin-1 (gal-1; 0.3 mg/ml) binding to the syncytiotrophoblast layer of a third trimester placenta (39th week of gestation). Gal-1 binding was visualized with streptavidin-Cy3. 40 ,6-Diamino2-phenylidole was added producing the blue reaction in the nucleus; 40 lens. b Binding of biotinylated gal-1 (0.3 mg/ml) to trophoblast tumor cells of the cell line BeWo; 63 lens. c Biotinylated gal-1 (0.3 mg/ml) did not bind to trophoblast tumor cells of the cell line Jeg3; 63 lens. d Biotinylated gal-1 (0.3 mg/ml) did not bind to freshly isolated trophoblast cells from a first trimester placenta (10th week of gestation); 63 lens

Statistical analysis The SPSS/PC software package, version 6.01 (SPSS, Munich, Germany), was used for collection, processing, and statistical analysis of all data. Statistical analysis was performed using the non-parametric Wilcoxon signed rank tests for comparison of the means. P<0.05 values were considered statistically significant.

Results Immunocytochemistry We observed strong binding of gal-1 to the syncytiotrophoblast layer of a third trimester placenta (39th week of pregnancy; Fig. 1a). In addition, we found strong binding of gal-1 to BeWo cells (Fig. 1b). Gal-1 did not bind either to Jeg3 cells (Fig. 1c) or to freshly isolated trophoblast cells from the first trimester of pregnancy (Fig. 1d). Immunochemical detection of gal-1 in BeWo cell lysates on blots Results of immunochemical detection (western blots) of gal-1 in BeWo cell lysates are shown in Fig. 2. The gal-1 pAb generates a main protein band in the 14-kDa molecular mass range (lane 1). Non-specific binding reactions were excluded by probing the blots with rabbit IgG followed by incubation with goat anti-rabbit IgG-HRP (lane 2) or with the HRP conjugate in the absence of rabbit IgG (lane 3).

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Fig. 2 Western blot of gal-1 detection in BeWo cell lysates. Lane 1 gal-1 pAb; lane 2 normal rabbit IgG as control; lane 3 TBS/Tween as blank

Effects of gal-1 on hCG and progesterone secretion of BeWo, Jeg3, and isolated trophoblast cells To test the effect of gal-1 on hCG secretion of BeWo cells in vitro, the cells were incubated with increasing concentrations of the lectin. Analysis of hCG showed reduced secretion into cell culture supernatants from gal-1-treated BeWo cells compared to non-treated cells after 48 h culture (Fig. 3). There was a significant difference between hCG production of gal-1-treated and untreated cell cultures (P=0.02 for 100 mg gal-1). The inhibitory effect of gal-1 is dose dependent. Cells treated with 100 mg/ml gal-1 showed a lower hCG secretion compared to cells

Fig. 3 Effects of gal-1 and Thomsen-Friedenreich (TF) monoclonal antibody (mAb) on inhibition of hCG production of BeWo cells in vitro. Data (mean € SD) represent hCG concentration in aliquots of the culture media at 48 h after addition of gal-1 (n=4 for every group, P=0.02, 100 mg/ml gal-1, *) and asterisks indicate signifi-

treated with 60 mg/ml gal-1. In BeWo cells treated with 60 mg/ml gal-1, hCG production is lower than that in unstimulated controls, albeit not statistically significant (P>0.05). Incubation of BeWo cells for 48 h with the mAb (A78-G/A7; Karsten et al. 1995) directed against the TF epitope resulted in inhibition of hCG secretion. BeWo cell cultures did respond with increased inhibitory effects on hCG secretion when co-treated with gal-1 and the TF mAb (P=0.028). In contrast to BeWo cells, gal-1 and/or TF mAb had no significant effects on hCG secretion of Jeg3 cells and freshly isolated trophoblast cells (data not shown). Treatment of BeWo cell cultures with gal-1 for 48 h reduced progesterone secretion into medium when compared with non-treated cells (Fig. 4). There was a significant difference between progesterone production by gal-1-treated and untreated cell cultures (P=0.03 for 60 mg gal-1 and P=0.028 for 100 mg gal-1). The inhibitory effect of gal-1 on progesterone secretion is dose dependent. Cells treated with 100 mg/ml gal-1 showed a higher inhibition compared to cells treated with 60 mg/ml gal-1. In BeWo cells treated with 60 mg/ml gal-1, progesterone production is reduced to 79%, whereas 100 mg/ml gal-1 decreased progesterone production to 68% relative to unstimulated controls. Incubation of BeWo cells with the TF mAb also resulted in decreased progesterone secretion after 48 h culture. Treatment with gal-1 and TF mAb further decreased hormone secretion (P=0.043). In contrast to BeWo cells, incubation of Jeg3 and freshly isolated trophoblast cells with gal-1 and/or TF mAb did not significantly change progesterone secretion.

cant levels between controls and treated cultures. In addition, we found similar effects of the TF mAb on inhibition of cellular hCG secretion, whereas co-treatment of BeWo cells with gal-1 and TFmAb further decreased hCG secretion significantly (P=0.028)

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Fig. 4 Effect of gal-1 and TF mAb on inhibition of progesterone production of BeWo cells in vitro. Data (mean € SD) represent progesterone concentration in aliquots of the culture media at 48 h after addition of gal-1 (n=4 for every group). P=0.03 (60 mg/ml gal1, *) and P=0.028 (100 mg/ml gal-1, **); asterisks indicate sig-

nificant levels between controls and treated cultures. In addition, co-treatment of BeWo cell cultures with gal-1 and TF mAb further significantly decreased progesterone secretion of BeWo cells (P=0.043)

Fig. 5 Effect of gal-1 on inhibition of hCG production of BeWo cells in vitro could be partially abolished by TF-polyacrylamide (PAA) conjugate

Co-incubation of BeWo cells with gal-1 and TF-PAA conjugates The TF-PAA conjugate did not significantly reduce the gal-1 induced inhibitory effect on cellular hCG production, as demonstrated in Fig. 5 illustrating the inhibitory effect of gal-1 on cellular hCG production. Progesterone secretion of BeWo cell control cultures (81 ng/ml) was reduced by 60 mg/ml gal-1 to 47.4 ng/ml. The gal-1-mediated inhibitory effect on hormone secretion was reduced to 59.3 ng/ml in the presence of 30 mg/ ml TF-PAA conjugate (Fig. 6; P=0.026).

Gal-1 inhibits mRNA transcription of the hCGb gene In order to investigate whether gal-1 affects the expression of hCGb gene on the level of transcription, we determined the concentration of transcripts in BeWo cells treated with 30 mg/ml and 60 mg/ml gal-1 as well as in non-treated cells by the TaqMan assay. As demonstrated in Fig. 7, a significant inhibition of hCGb mRNA expression was measured in BeWo cells treated with gal-1 for 30, 60, and 90 min when compared with non-treated BeWo cells. Differences in hCGb copy numbers for the control incubations without gal-1 at 30, 60, and 90 min were statistically non-significant compared to 30 mg/ml gal-1 but significant compared to 60 mg/ml gal-1 (P=0.037).

506 Fig. 6 Effect of gal-1 on inhibition of progesterone production of BeWo cells in vitro could be partially abolished by TF-PAA conjugate (P=0.026)

Fig. 7 Time-dependent quantification of hCGb mRNA copy numbers (mean values € SD) per 200 ng total mRNA in gal-1 (30 mg/ml, 60 mg/ml)-treated or untreated BeWo cell cultures. Quantification was performed using the external hCGb mRNA as a

standard that runs in parallel (n=4 for every group). P=0.203 (30 mg/ml gal-1) and P=0.037 (60 mg/ml gal-1, *); asterisks indicate significant different levels between controls and treated cultures

Discussion

immune response such as the induction and release of gal1 upon T cell activation as well as its inhibitory effect on antigen-induced proliferation of T cells (Blaser et al. 1998). Furthermore, gal-1 can rapidly induce apoptosis of activated, but not naive, peripheral T cells (Perillo et al. 1995). Despite these mechanisms, maternal alloreactive lymphocytes can be detected. Naturally occurring murine regulatory CD4+ CD25+ T cells in lymph nodes and spleen are markedly increased during pregnancy. These maternal regulatory T cells suppress the proliferation of alloreactive cells in a mixed lymphocyte reaction and an allogenic response directed against the fetus (Aluvihare et al. 2004). The data presented here demonstrate the binding of gal-1 to the syncytiotrophoblast layer and to the trophoblastic tumor cell line BeWo forming a syncytium in vitro. Freshly isolated trophoblast cells from the first trimester of pregnancy and the trophoblastic tumor cell line Jeg-3 did not bind gal-1. Furthermore, immunoblotting experiments revealed that gal-1 is strongly expressed by BeWo cells. Therefore, the expression of both, gal-1

Gal-1 is initially synthesized in the trophoectoderm of the expanded blastocyst immediately prior to implantation, suggesting a role in the attachment of the embryo to the uterine epithelium (Poirier et al. 1992). Immunohistochemical studies revealed the localization of a b-dgalactoside-binding lectin in the cytotrophoblast of the middle and distal cell columns and in the syncytiotrophoblast (Walzel et al. 1995; Vicovac et al. 1998); gal-1 was also localized in decidual and endometrial stromal cells (Bevan et al. 1994). During pregnancy, the maternal immune response tolerates the persistence of paternal alloantigens. Several localized mechanisms contribute to fetal evasion from immune attack such as the expression of HLA-G and FAS ligand (CD95L) by fetal tissues leading to inhibition of natural killer cells (Karre 2002) and inducing apoptosis of activated maternal lymphocytes (Hammer et al. 1999). Several lines of evidence support the involvement of gal-1 in suppression the maternal

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and appropriate glycan recognition structures for the lectin prompted us to study the effects of exogenously added gal-1 on hormone production of BeWo cells. The cell lines Jeg3 and BeWo are trophoblastic tumor cells at various stages of differentiation. Whereas Jeg3 cells compose from invasive and cytotrophoblast cells, BeWo cells contain two coexisting phenotypes: a cytotrophoblast-like and a syncytiotrophoblast-like phenotype (Bergemann et al. 2003). The commitment of cytotrophoblasts to syncytiotrophoblasts is associated with activation of -a and hCGb subunit genes. These intermediates are transient and they differentiate to syncytiotrophoblasts (Hoshina et al. 1984). Furthermore chorion carcinoma cells consisting of clusters of cytotrophoblast like and large multinucleated cells express -a and hCGb mRNA (Hoshina et al. 1985). Additional to cell binding effects, gal-1 inhibited the production of hCG and progesterone in BeWo cells. Results of hCG inhibition were obtained on protein and mRNA levels. Similar inhibitory effects were observed on hormone production by an mAb directed against the TF antigen, whereas co-incubation with gal-1 and the TFPAA conjugate reduced the gal-1-induced inhibition of hCG and progesterone secretion. To study the effects of gal-1 on hCG production on the transcription level, BeWo trophoblast tumor cells were treated with different amounts of the lectin and the expression of hCGb mRNA was evaluated. A decrease of hCGb mRNA expression was measured after treatment of BeWo cells with 30 and 60 mg/ml gal-1 applying the TaqMan assay. Kinetic studies showed decreased hCGb mRNA levels at 30, 60, and 90 min after gal-1 treatment, with significant differences for 60 mg/ml gal-1, compared to untreated controls. The results suggest that exogenously added gal-1 has the potency to regulate hormone production in trophoblast cells and the effects seem to be related to the expression of the TF antigen on the surface of these cells. In summary, Jeg3 and BeWo cells are trophoblast tumor cell lines at various stages of differentiation. The Jeg3 contain invasive and cytotrophoblast cells whereas BeWo cells display a cytotrophoblast- and syncytiotrophoblast-like phenotype. Progesterone and hCG release was found to be decreased in gal-1-treated BeWo trophoblast tumor cells compared to untreated cells. The inhibitory effects of gal-1 on cellular hCG production were found on both hCGb mRNA and on protein. The observed decrease in hCGb mRNA copy numbers after gal-1 treatment leads to a lower hCG protein production. Gal-1 did not bind to Jeg3 cells. Similar results were obtained by incubation with anti-TF antibodies. The results support the hypothesis that the TF antigen is involved in gal-1-induced inhibition of hormone production in BeWo cells. Acknowledgements We thank Mrs. G. Geade and Mrs. D.U. Richter for excellent technical assistance and Dr. rer. nat. habil. U. Karsten for helpful discussion. Supported by the Deutsche Forschungsgemeinschaft (DFG) for U. Jeschke (Je 181/4-1).

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