Hum. Reprod. Advance Access originally published online on October 6, 2005
Human Reproduction 2006 21(1):193-201; doi:10.1093/humrep/dei272
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Differential effects of inducers of syncytialization and apoptosis on BeWo and JEG-3 choriocarcinoma cells
1 Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
2 To whom correspondence should be addressed: e-mail: salwan.al-nasiry{at}med.kuleuven.ac.be
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Abstract |
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BACKGROUND: The interactions of trophoblasts with the cytokine network at the fetomaternal interface determine the pathway the cell undertakes, e.g. proliferation, differentiation and apoptosis. METHODS: We used cultures of fusigenic BeWo and non-fusigenic JEG-3 choriocarcinoma cells to study the effects of inducers of syncytialisation (forskolin) and apoptosis [tumour necrosis factor-
(TNF)] on differentiation, viability, proliferation and apoptosis. RESULTS: E-cadherin immunostaining showed that syncytium formation was confined to BeWo and not JEG-3 cells, while secretion of hCG was promoted by forskolin in both cell types implying a ‘dissociation’ between morphological and biochemical differentiation. Forskolin also had differential effects on cell viability (MTT reduction test) and proliferation (Ki67 immunostaining with MIB-1 monoclonal antibody), both decreasing in BeWo and increasing in JEG-3 cells. TNF increased apoptosis (cytokeratin neo-epitope immunostaining with M30 monoclonal antibody) in both cell types, an effect which was blocked by epidermal growth factor selectively in JEG-3 cells. CONCLUSION: Our results suggest that the differential responses of BeWo and JEG-3 cells to inducers of syncytialization and apoptosis might be related to their fusigenic capacity. Caution is needed when extrapolating results obtained by these models to normal trophoblast populations. However, we speculate that these models can help identify key factors involved in trophoblast differentiation at the placental bed.
Key words:
choriocarcinoma/differentiation/EGF/orskolin/TNF
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Introduction |
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A successful pregnancy is largely dependent on co-ordinated events taking place very early after implantation at the fetomaternal interface in which trophoblast cells are key players. These cells differentiate according to one of two distinct pathways. In the villous pathway, mononuclear cytotrophoblasts fuse to form a specialized multinuclear syncytium on the outer layer of placental villi with synthetic, immune and transport functions. In the extravillous pathway, cytotrophoblasts proliferate and differentiate into an invasive phenotype that penetrates the maternal decidua and myometrium, and ultimately fuse into multinuclear giant cells.
While there is much literature on how the process of fusion is regulated in villous trophoblast, little is known about the regulation of extravillous trophoblast fusion. Fusion of extravillous trophoblasts, as well as induction of apoptosis, has been suggested to restrict extravillous trophoblast invasion (Pijnenborg et al., 1981
; Reister et al., 2001). Fusion and apoptosis in both villous and extravillous trophoblasts are regulated by different components of the cytokine network. Indeed, the cytokine tumour necrosis factor- (TNF) has been implicated in the control of apoptosis of the invasive trophoblast of the placental bed (Pijnenborg et al., 1998; Reister et al., 2001) as well as trophoblast proliferation and differentiation (Yang et al., 1993). Epidermal growth factor (EGF) on the other hand has been shown to induce proliferation and differentiation into syncytiotrophoblast (Morrish et al., 1987), and to inhibit TNF induced apoptosis (Garcia-Lloret et al., 1996). Yet, the effects of any cytokine may differ according to the specific type of trophoblastic cell and may be modulated by the presence of other cytokines and growth factors.
We therefore hypothesized that trophoblastic cells with different fusigenic capacity respond differently to inducers of syncytialization and apoptosis. Although primary trophoblast cultures would be an obvious choice, they were not used in this study because of the high variability between samples reflecting heterogeneous populations of isolated cyto- and syncytiotrophoblasts. Instead, we used two choriocarcinoma cell lines (Ringler and Strauss, 1990; King et al., 2000; Shiverick et al., 2001; Sullivan, 2004): (i) the fusigenic BeWo cells (Pattillo and Gey, 1968; Wice et al., 1990); and (ii) the non-fusigenic JEG-3 cells (Kohler and Bridson, 1971; Coutifaris et al., 1991).
We studied the effects of forskolin, an inducer of syncytialization (Wice et al., 1990), of TNF, an inducer of apoptosis, and of the combination of both (Fk/TNF) on morphological differentiation (E-cadherin expression), biochemical differentiation (hCG levels in culture media), viability (MTT reduction test), proliferation (Ki67 immunostaining using MIB-1 monoclonal antibody) and apoptosis (cytokeratin immunostaining using M30 monoclonal antibody). Secondly, since pretreatment with EGF has been reported to protect trophoblast against TNF-induced cytotoxicity (Garcia-Lloret et al., 1996), we evaluated whether pretreatment with EGF would modulate the cellular responses to TNF and forskolin in the chosen model system.
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Materials and methods |
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Cell culture
BeWo and JEG-3 choriocarcinoma cells were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and were maintained in 75 cm2 Falcon culture flasks (BD Biosciences, Erembodegem, Belgium) under standard culture conditions of 5% CO2 in air (containing 20% O2) at 37°C with medium renewal every 2–3 days. The culture media used were Ham F12 (Sigma-Aldrich, Bornem, Belgium) for BeWo cells and Dulbeco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies, Belgium) for JEG-3 cells containing 25 mM glucose, 25 mM HEPES, 200 mM glutamine, 1/100 penicillin-streptomycin solution and 1/1000 gentamycin solution, and supplemented with 10% heat-inactivated fetal calf serum (FCS).
Cell concentrations were adjusted to 4 x 104 cell/ml (BeWo) and 2 x 104 cell/ml (JEG-3) after counting the cells with a haemocytometer. For the experiments, cells passaged for 10–16 times after purchase were used. Cell suspensions (500 µl) were seeded onto eight-well LabTek slides (Nunc, VEL, Leuven, Belgium) and 250 µl suspensions onto 96-well Nunc plates (Nunc). Cells were kept for 4 days in standard culture conditions of 5% CO2 in air (containing 20% O2) at 37°C. After the initial 4 h of culture, which allowed for cell attachment, the medium was changed with the addition of EGF (5 ng/ml) (Gibco BRL, Life Technologies, Merelbeke, Belgium) into half the wells. After 24h, the medium was removed and kept at –20°C for hCG determination and fresh serum-free medium was added containing 100 µM of forskolin (Sigma-Aldrich, Bornem, Belgium) with or without 1000 IU/ml of human recombinant TNF (Invitrogen, Life Technologies). After preliminary testing, it was decided to fix forskolin treatment at 100 µM concentration. Serum-free medium was used to avoid possible stimulatory effects of growth factors present in FCS. Cultures were stopped after 48 and 72 h, viability was tested by an MTT test on the 96-well Nunc plates, and LabTek slides were fixed for immunohistochemistry. Conditioned media were collected from LabTek slides for protein and hCG quantification.
MTT cell viability assay
An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test was performed on cultures at 48 h and 72 h after initial plating as described by Mosmann (1983). The MTT test is based on the ability of viable cells to produce formazan from the cleavage of the tetrazolium salt by functional mitochondria. After a 2-hour incubation with MTT (Sigma-Aldrich, Bornem, Belgium) at 37°C, the cells were lysed with dimethyl sulphoxide (DMSO) in Sorensen’s glycine buffer and the formazan crystals solubilized. Absorbance was read at 550 nm using a spectrophotometric microplate reader. The experiments were performed in triplicate wells and repeated six times. The intraassay and interassay coefficients of variation were 3.8% (SD 1.5) and 8.2% (SD 3.1), respectively.
Immunohistochemistry
LabTek slide cultures were stopped at 48 h and 72 h after initial plating by washing in phophsate-buffered saline (PBS) solution and fixation in 50/50 (v/v) ethanol/paraformaldehyde solution for 1 min. Cells were kept in 70% ethanol at 4°C until immunostaining. All incubations were carried out at room temperature. Briefly, the cells were washed twice with 0.01M Tris-buffered saline pH 7.6 (TBS) and blocked with bovine serum albumin (BSA) 2% containing Tween 80 (0.5%) for 15 min. The following primary antibodies were used:
- monoclonal mouse anti-human E-cadherin, clone HECD-1 (R&D Systems, Abingdon, UK) in a concentration of 2 µg/ml for 30 min;
- monoclonal mouse Ab (MIB-1 clone) against Ki67 epitope (DakoCytomation, Glostrup, Denmark) in a concentration of 4 µg/ml for 30 min
- monoclonal mouse Ab against the M30 epitope of cytokeratin 18 (Boehringer Mannheim Gmbh, Mannheim, Germany) in a concentration of 4 µg/ml for 30 min.
After another blocking step, peroxidase-conjugated goat antimouse IgG 1/100 (v/v) in TBS (Jackson ImmunoResearch, West Grove, Pennsylvannia, USA), with the addition of normal human serum 1/25 (v/v) in TBS, was used as secondary antibody. Peroxidase activity was demonstrated by adding 3-amino-9-ethylcarbazole (AEC) for 15 min in a dark chamber. The cells were counterstained with Mayer’s haematoxylin. Finally, the slides were mounted with glycerine jelly. As negative controls, we used irrelevant mouse antibody as the primary antibody.
Slides were evaluated by light microscopy at 20x magnification. In different slides, the number of entities (nuclei or cells) positive for M30 or MIB-1, or negative for E-cadherin was counted in 16 central fields. This number was normalized to a total number of 500 nuclei per slide and the mean of values from three experiments was calculated. The quality of the staining was generally adequate for cytological evaluation with <10% of tested areas showing unsatisfactory cell attachment or fixation/staining errors, which were excluded from evaluation.
hCG and protein measurement in culture media
Conditioned media were collected from LabTek wells at the time intervals specified and were kept at 4°C until the time of assay. Protein concentration in culture media was quantified based on the Biuret reaction using the BCA kit (Pierce, Rockford, Illinois, USA) according to the manufacturer’s instructions. For hCG quantification, an enzyme immunoassay kit was used (DRG Diagnostics, Marburg, Germany), which is based on the sandwich principle and detects both whole hCG molecule and the free b-hCG subunit present in spent media. The assay was carried out according to the manufacturer’s instructions. To minimize the effects of medium colour on photometric reading, samples were diluted 1/10 (v/v) in the sample diluent solution provided with the kit. The optical density was read at 450 nm with a microplate reader. The intensity of colour developed from the peroxidase-substrate reaction is proportional to the concentration of hCG in the sample. The measurement was carried out in duplicate wells from three experiments. The intraassay and interassay coefficients of variation were 6.6% (SD 3.0) and 7.8% (SD 4.9), respectively. The results are expressed as hCG concentrations (after correction for background optical density) in mIU/100 mg protein in culture media.
Statistical analysis
The experiments were done in duplicate or triplicate wells and repeated 3–6 times, depending on the analysis. We used one-way ANOVA (analysis of variance) with either Bonferroni’s post hoc test to measure statistical differences between the means of treatment conditions versus control conditions, or with Dunnett’s post hoc test for EGF pretreatment versus non-pretreatment. P values of < 0.05 were considered significant.
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Results |
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Morphological differentiation assay: E-cadherin immunostaining
E-cadherin is a cell adhesion molecule involved in trophoblast differentiation and invasion. During the process of differentiation of isolated trophoblast cells (as well as BeWo cells treated with cAMP agonists), E-cadherin mRNA and protein are down-regulated in association with loss of E-cadherin staining from the surface of fusing cells (Coutifaris et al., 1991
; Getsios et al., 2000). E-cadherin staining was evaluated by two methods: (i) by percentage of E-cadherin-negative cells in a total of 500 nuclei; and (ii) by the number of nuclei per syncytium. Cells were considered E-cadherin-negative when total disappearance of E-cadherin from cell surfaces was present, indicating syncytializing cells. Cells still in the process of syncytialization exhibiting partial E-cadherin staining were not considered as E-cadherin-negative.
In BeWo cells
Under control conditions, >90% cells aggregated and showed strong E-cadherin staining at cell boundaries (Fig. 1C). Occasionally cells underwent spontaneous syncytialization at 72h—manifested by formation of small multinuclear masses with enlarged clear cytoplasm. Fk alone or in combination with TNF resulted in disappearance of E-cadherin from cell–cell contacts, i.e. higher number of E-cadherin-negative nuclei (P < 0.01 versus control conditions) (Table IA), together with formation of large, sometimes giant, syncytia containing up to 35 nuclei per syncytium (Table IB). Neither TNF (alone) nor EGF pretreatment had any effect on the number of E-cadherin-negative cells or number of nuclei per syncytium.
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In JEG-3 cells
In contrast to BeWo cells, JEG-3 cells remained mononuclear with strong expression of E-cadherin at cell boundaries under all conditions tested (Fig. 1D). Data relating to E-cadherin-negative cells or number of nuclei per syncytium are not shown.
Biochemical differentiation assay: hCG secretion in culture medium
Trophoblast differentiation is associated both in vitro and in vivo with increased synthesis and secretion of hCG (Kliman et al., 1986; Morrish et al., 1987; Kao et al., 1988). Choriocarcinoma cells secrete increasing amounts of hCG into culture medium in response to cAMP agonists (Wice et al., 1990; Hohn et al., 1998). We measured both whole hCG molecule and the free b-hCG subunit released in culture media. Results are expressed in mIU of hCG per 100 mg protein in collected media.
In BeWo cells
Culture of BeWo cells over time resulted in almost two-fold increase in hCG secretion (Fig. 2A and B). Addition of Fk significantly raised hCG levels 2.6 and 3.7 fold at 48 and 72h, respectively, compared with control conditions (P < 0.01). TNF alone had no effect on hCG secretion, while the combination of Fk/TNF had a less pronounced and less sustained effect than Fk alone. EGF pretreatment had a noticeable stimulatory effect on hCG secretion in all treatment groups (versus EGF non-pretreatment).
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P < 0.05,
In JEG-3 cells
Secretion of hCG increased over time in JEG-3 cell cultures (1.6 fold) (Fig. 2C and D). Similar to its effect on BeWo cells, Fk increased hCG secretion in JEG-3 cells, as did the combination Fk/TNF. TNF alone had no effect. These effects were noticed in the group not pretreated with EGF at 48 and 72 h. EGF pretreatment increased hCG levels (versus EGF non-pretreated cells) in all treatment groups, but no additional stimulatory effect was noticed with TNF, Fk or the combination Fk/TNF.
Viability assay: MTT test
In a homogenous sample of cells like the choriocarcinoma cell lines, MTT reduction should be proportional to the number of metabolically active cells, but is frequently used as a crude indicator of cell proliferation. However, this does not take into account the spontaneous rates of proliferation and apoptosis, and the possible effects of cytokines on mitochondrial function. Therefore, we prefer to use the term ‘viability’ and not ‘proliferation’ in interpreting results obtained with the MTT test.
In BeWo cells
When BeWo cells are kept in control conditions (i.e. serum-free medium is added), a significant increase in MTT absorbance is seen over culture time (72 h versus 48 h). Conversely, MTT absorbance remained basically unchanged over culture time when Fk, TNF or the combination Fk/TNF was added to cells (data not shown). Fk alone or in combination with TNF decreased MTT absorbance compared with control conditions both at 48 and 72 h (Fig. 3A and B). TNF alone had a similar deleterious effect only at 72 h. In all tested conditions, MTT absorbance was unaffected by EGF– pretreatment at either points in time.
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In JEG-3 cells
In contrast to BeWo cells, JEG-3 cells showed important differences in their response to the same effectors. Treatment of JEG-3 cells with Fk alone or in combination with TNF increased MTT absorbance compared with cells receiving control medium. This effect was very significant both at 48 and 72h (Fig. 3C and D). Addition of TNF alone did not significantly affect cell viability, neither did pretreatment with EGF in all tested conditions.
Proliferation assay: MIB-1 immunostaining
The use of the monoclonal antibody MIB-1 to determine cell proliferation is well established. This antibody reacts with recombinant parts of the Ki-67 nuclear antigen present in all actively proliferating cells including trophoblasts (Cheung et al., 1994). Positive cells showed generally a red nuclear staining together with frequent occurrence of mitotic figures (Fig. 1E and F).
In BeWo cells
In control conditions, 4% of cells showed MIB-1 staining (Fig. 4A and B). Treatment with TNF reduced the number of MIB-1 positive cells (P < 0.01 versus control conditions). The number of MIB-1 positive cells was also severely reduced with Fk and Fk/TNF treatment with <0.7% and 0.4%, respectively, of cells still showing MIB-1 staining at 72 h. EGF pretreatment had no effect on the number of MIB-1 positive cells.
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In JEG-3 cells
In sharp contrast to its effects on the BeWo cells, Fk increased the number of MIB-1 positive cells in JEG-3 cells (Fig. 4C and D). However, this effect was only evident at 72 h of culture. Neither treatment with TNF or Fk/TNF nor pretreatment with EGF appeared to alter the proliferation of JEG-3 cells.
Apoptosis assay: M30 immunostaining
During the early stages of apoptosis in epithelial cells, a neo-epitope on cytokeratin 18 is formed by caspase 3 cleavage and reacts with the monoclonal antibody M30 (Caulin et al., 1997; Leers et al., 1999; Morsi et al., 2000). M30 immunostaining is superior to the TUNEL method in detecting apoptotic cells (Kadyrov et al., 2001; Austgulen et al., 2002). Non-epithelial and necrotic cells show a negative M30 staining (Morsi et al., 2000; Kadyrov et al., 2001). M30-positive cells showed a homogeneous red staining of the cytoplasmic filaments (Fig. 1A and B). They also showed other morphological signs of apoptosis including chromatin condensation and nuclear fragmentation.
In BeWo cells
Under control conditions, the cells showed 
0.4% rate of apoptosis, based on the number of M30-positive cells (Fig. 5A and B). Apoptosis rates were slightly increased over culture time (72 h versus 48 h). When Fk alone or in combination with TNF was added to the cells, the apoptosis rates increased up to 2.3 and 6.6%, respectively, at 72 h (P < 0.01 versus control conditions). TNF alone increased apoptosis rates only at 72 h (2.6%). EGF had no effect.
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In JEG-3 cells
JEG-3 cells had 10x higher apoptosis rates (4%) than BeWo cells (Fig. 5C and D). At 72 h of culture, TNF increased apoptosis rates to 6.4% (P < 0.05 versus control conditions). The combination Fk/TNF increased apoptosis rates further to 10% (P < 0.01 versus control conditions). Fk alone had no effect. Unlike BeWo cells, EGF pretreatment completely blocked the TNF- and Fk/TNF-induced apoptosis at both time points (P < 0.001 versus EGF non-pretreated).
The results are summarized in Table II.
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Discussion |
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Placental cytokines and growth factors acting at the fetomaternal interface including EGF, TNF
, transforming growth factor (TGF)-
, insulin-like growth factors (IGF) and placental growth factor (PlGF) are suggested to play a role in regulating various trophoblast functions. The pathways of differentiation followed by placental trophoblasts are at least partly regulated by these cytokines (Morrish et al., 1998). Our knowledge on the subject has been largely based on experiments with isolated trophoblast cells from early and term placentas.
However, this widely used model has certain limitations. The obtained cells represent a heterogeneous population of cyto- and syncytiotrophoblasts with possible contamination with fibroblasts and macrophages. The use of established cell lines in the study of trophoblast function have been advocated by many researchers (Ringler and Strauss, 1990; King et al., 2000; Shiverick et al., 2001) and recently reviewed by Sullivan (2004). The homogeneity of these cells and the practicality of in vitro propagation procedures are important advantages. Particularly in the study of apoptosis, immortalized cell lines could be preferred to primary trophoblasts because of their low spontaneous rate of apoptosis (Gaus et al., 1997). Here, we utilized two choriocarcinoma cell lines with differing fusigenic capacity as a model to study trophoblast proliferation, differentiation and apoptosis in response to various biological inducers.
The disappearance of E-cadherin protein from cell–cell boundaries, parallel to down-regulation of its mRNA, has been used as an indication of syncytium formation in normal trophoblasts and in BeWo cells (Wice et al., 1990; Coutifaris et al., 1991). This has been confirmed by immunoneutralization studies using function-perturbing antibodies to E-cadherin. In concordance with these studies, E-cadherin immunostaining showed that BeWo cells differentiate morphologically to form multinuclear syncytia in response to forskolin. Forskolin treatment also increased hCG secretion in culture medium indicating biochemical differentiation. In contrast, JEG-3 cells showed no signs of morphological differentiation with forskolin treatment, although they showed biochemical differentiation evident by increased hCG secretion. This dichotomous effect of forskolin on differentiation suggests that at least some aspects of trophoblast differentiation can be dissociated. A similar dissociation was also obtained in trophoblast cultured in the absence of serum (Kao et al., 1988). It is interesting to speculate that analogous mechanisms for this dissociation exist in the two models.
Induction of cAMP is associated with decreased cell proliferation in normal trophoblast as well as BeWo cells (Taylor et al., 1991). The decreased viability of BeWo cells in our study could also be explained by rapid syncytialization induced by high dose (100 µM) forskolin, which would imply acceleration of the life cycle ending by apoptosis of syncytialized BeWo nuclei. The presented data show that forskolin, an inducer of cAMP, had also differential effects on cell viability (MTT reduction) and proliferation (MIB-1 immunostaining), both decreasing in BeWo and increasing in JEG-3 cells. Our data suggest that the divergent effect of cAMP on BeWo and JEG-3 cell lines could be related to their fusigenic capacity. It is tempting to speculate that the lack of fusigenic capacity in JEG-3 cells may be partly responsible for the altered response to cAMP observed in our study and may result in switching the cellular programmes to a more proliferative type.
TNF is a pro-inflammatory cytokine with a wide range of cellular actions affecting proliferation, apoptosis and differentiation of various cell types. In trophoblast cell lines, these effects depend on cell type, dose of TNF and culture conditions. In JEG-3 cells, TNF had a less wide-spectrum effect than in BeWo cells, confined to increasing apoptosis in the EGF non-pretreated group. This concurs with findings by Yang et al. (1993) that JEG-3 cells produce less TNF and are less reliant on this cytokine for their growth than other choriocarcinoma cells. On the other hand, BeWo cells showed decreased viability and proliferation with TNF treatment in addition to increased apoptosis rates resembling in this manner the response to TNF in the normal trophoblast culture (Garcia-Lloret et al., 1996). Since both normal trophoblast and BeWo cells are fusigenic, one can speculate that the full-range response to TNF particularly on viability and proliferation may be related to the fusigenic capacity. Likewise, the fusigenic capacity of invasive trophoblasts in the placental bed might determine their response to TNF induced-apoptosis as a possible mechanism of restricting their invasion. Currently, we are studying the role of fusion proteins in formation of trophoblast giant cells and the interaction with TNF at the placental bed of normal and pathological pregnancies.
Pretreatment of cell cultures with EGF increased hCG secretion in culture media in both cell lines implying biochemical differentiation. However, EGF pretreatment was able to block the TNF-induced apoptosis only in JEG-3, but not BeWo cells, resembling in this aspect its effect on normal trophoblasts (Pijnenborg et al., 2000). Our experimental design did not allow us to investigate whether this block was a direct effect of EGF on TNF binding/signal transduction or secondary to its differentiation-enhancing effect.
The lack of protective effects of EGF against TNF-induced cytotoxicity in BeWo cells is a novel finding. Although both normal and malignant trophoblasts produce and express receptors for EGF (Duello et al., 1994), BeWo cells have at least 10-fold higher levels of EGF receptor (EGF-R) than benign trophoblasts with functional tyrosine kinase activity (Filla and Kaul, 1997). The over-expression of EGF-R in BeWo cells can render them unresponsive to exogenous EGF (Filla and Kaul, 1997), which might explain the lack of protective effect of EGF in our BeWo model.
The differential response of BeWo and JEG-3 cell to other inducers has been described previously (Mandl et al., 2002). In addition, differences in gene expression profiles between these choriocarcinoma cell lines and normal trophoblasts cells have been described recently using the technique of differential display RT-PCR (Garcia and Castrillo, 2004). These findings support the notion that these cell lines can serve as ‘specific’ models to study certain defects in trophoblast function and that caution is necessary in extrapolating results obtained with one trophoblast cell line to other cell lines or to the ‘general’ trophoblast population.
It is tempting to view the observed differential responses of choriocarcinoma cell lines in the context of trophoblast aberrations described in pre-eclampsia. The behaviour of BeWo cells in our model resembles that of cultured placental villi from pre-eclamptic pregnancies described by Crocker et al. (2004) in at least two aspects. First, both BeWo cells and pre-eclamptic villi showed higher hCG secretion and exaggerated apoptotic response to TNF. compared with JEG-3 cells and normal villi. Secondly, while both JEG-3 cell in our model and villi from normotensive placentas showed increased proliferation rates in response to forskolin and hypoxia respectively, BeWo cells and preeclamptic villi showed restricted proliferation response to forskolin and hypoxia respectively. The link between fusion defects in the trophoblast and the pathology of pre-eclampsia is still speculative. Further studies are needed to clarify mechanisms of altered cell kinetics in choriocarcinoma cell lines before relating the observed changes to clinical situations.
In conclusion, this study demonstrates the differential responses of two choriocarcinoma cell lines with different fusigenic capacity to inducers of syncytialization and apoptosis, suggesting that fusigenic capacity might play a role in determining the responses to biological inducers in the selection of cellular pathways. Whilst highlighting the need for caution when extrapolating results obtained by these models to normal trophoblast populations, this study endorses the use of these cell lines as specific models in the search for novel defects in syncytialization. This could give us insight into the mechanisms controlling the selection of different pathways of trophoblast differentiation in normal and pathologic pregnancies.
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Submitted on May 9, 2005; resubmitted on June 27, 2005; accepted on July 20, 2005.
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