Differentiation Markers and Invasiveness: Discordant Regulation in Normal Trophoblast and Choriocarcinoma Cells

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

244, 249 –258 (1998)

EX984184

Differentiation Markers and Invasiveness: Discordant Regulation in Normal Trophoblast and Choriocarcinoma Cells Hans-Peter Hohn,*,1 Manuela Linke,* Bernhard Ugele,† and Hans-Werner Denker* *Institut fu¨r Anatomie, Universita¨tsklinikum,Universita¨t-GH Essen, Essen, Germany; and †I. Frauenklinik, Klinikum Innenstadt, Ludwig-Maximilians-Universita¨t, Mu¨nchen, Germany

In tumor cells, malignant (invasive) behavior and differentiation tend to be correlated inversely, although it is not clear to what extent this can be generalized and whether it may also apply to normal invasive cell types. We have modulated differentiation of normal trophoblast cells from first trimester or term placenta as well as choriocarcinoma cells (BeWo, Jeg-3, and JAr) with retinoic acid (RA), methotrexate (MTX), dibutyryl-cAMP (dbcAMP), or phorbol-[12-myristoyl-13-acetyl]-diester (PMA). The secretion of the differentiation marker chorionic gonadotrophin was stimulated by nearly all substances in all cell types. The activity of cellular sterylsulfatase showed a tendency to be increased (decreased by RA and dbcAMP in normal trophoblast; not detected in JAr). Invasiveness was decreased by all effectors in normal trophoblast (both types) and in BeWo. In Jeg-3 and JAr, however, PMA treatment (in JAr also RA treatment) increased invasion rates. These results suggest that only in normal trophoblast and in BeWo (but not in other choriocarcinoma cells, i.e., Jeg-3 and JAr) invasiveness and differentiation tend to be correlated inversely. When extrapolating to the various subpopulations of cells within a tumor, induction of differentiation—as intended in certain strategies for tumor therapy (“differentiation therapy”)–may have the unwanted effect of stimulating invasiveness in certain subpopulations of tumor cells. © 1998 Academic Press Key Words: cell biology; differentiation; invasion; tumor; trophoblast; choriocarcinoma.

INTRODUCTION

The malignant behavior of cancer cells is mainly based on the unrestrained invasiveness of these cells or at least of cell subpopulations in a tumor and not so much on extensive proliferation which is also found in benign tumors. However, radio-, photo-, and chemotherapy affect cells mainly because of their high rates 1

To whom correspondence and reprint requests should be addressed at Universita¨tsklinikum Essen, Institut fu¨r Anatomie, Hufelandstr. 55, D-45122 Essen, Germany. Fax: 149 201 723-5916.

of proliferation (as a rule by nonspecific damage of DNA) while invasiveness may remain unaffected [1–5]. In metastasizing tumors such antiproliferative therapy is the only possible strategy so far, despite its inevitable side effects, particularly in the digestive tract, the immune system, and the hematopoietic system. In the past decade “differentiation therapy” has been discussed as a new type of therapeutic approach [6 –15]. This is based on the observation that malignancy and the degree of cell differentiation are often correlated inversely in a given tumor. However, tumor cells are known to be heterogeneous and cannot be regarded generally as undifferentiated, highly proliferative, and highly invasive cells. In addition to the last two properties, tumor cells may indeed display various degrees of differentiation. It has been shown in many investigations in vitro that the differentiation of tumor cells can be induced, increased, or modulated by treatment with different physiological or nonphysiological factors [4, 6, 9 –19]. Whether or not manipulation of differentiation may result in modification and potentially in reduction of invasiveness in tumor cells has not been studied intensively so far (cf. [20 –22]). The effects of induction of differentiation (maybe leading to terminal differentiation) can be expected to include reduced invasiveness, arrest of proliferation, and possibly apoptosis of cancer cells. Successful application of differentiation therapy has been demonstrated in a few cases, particularly in certain types of leukemia, so far [23, 24], but first clinical trials with other types of cancer appeared not to be very successful [25]. Cell differentiation is a very complex process depending on many factors which can often be expected to vary locally so that a number of cell subpopulations may form after application of only one single compound; i.e., a uniform response in the sense of reduction of properties that are relevant for invasiveness and metastasis cannot necessarily be expected. Therefore, the relevance of differentiation therapy in oncology is still a matter of debate. In the present investigation the correlation between induction of differentiation and invasive potential was

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assessed using normal and malignant trophoblast cells as a model. While choriocarcinoma cells are highly malignant, normal trophoblast cells also display (physiological) invasiveness which is, however, subject to very effective control mechanisms limiting invasion locally and temporally during normal pregnancy (see Discussion). In the present experiments, three choriocarcinoma cell lines (BeWo, Jeg-3, and JAr) and also normal trophoblast cells isolated from first trimester or from term placenta were used. In our experiments, the cells were treated with retinoic acid (RA),2 methotrexate (MTX), dibutyryl-cAMP (dbcAMP), or phorbol-[12myristoyl-13-acetyl]-diester (PMA). The secretion of chorionic gonadotrophin (hCG) and the activity of cellular sterylsulfatase (STS), which plays a role in placental estrogen synthesis, were monitored as differentiation parameters. Invasiveness was examined in a Matrigel penetration assay. The results suggest that differentiation and invasiveness are subject to different regulatory mechanisms. Both phenomena are correlated inversely in some but not all trophoblastic cell types, but can be stimulated in parallel in others. It is concluded that induction of differentiation (as attempted in “differentiation therapy”) may thus have the unwanted effect of stimulating invasiveness and possibly metastasis in certain subpopulations of tumor cells (at least in trophoblast tumors). MATERIALS AND METHODS Cell culture. Choriocarcinoma cell lines (BeWo, JAr, and Jeg-3) were obtained from the American Type Culture Collection (ATCC, Rockland, MD). BeWo cells (ATCC CCL 98) were maintained in Ham’s F12 (Gibco, Eggenstein, Germany) medium containing 15% fetal bovine serum (FBS, Gibco). JAr cells (ATCC HTB 144) were routinely cultured in RPMI 1640 (Gibco) with 10% FBS. Jeg-3 cells (ATTC HTB 36) were kept in MEM with Earle’s salts (Gibco) and 10% FBS. Normal trophoblast cells were isolated from term placenta after spontaneous delivery or after planned cesarean section as described previously by Hohn et al. [26] using a protocol established by Kliman et al. [27] based on trypsin and DNAse treatment and application of a preformed continuous gradient of Percoll (70%, Pharmacia, Freiburg, Germany) in order to separate trophoblast cells from red blood cells and other cells. The cells were cultured in MEM D-valine medium (Gibco) supplemented with 20% FBS, 50 IU/ml penicillin (Gibco), and 50 mg/ml streptomycin (Gibco). Cytotrophoblast cells from first trimester placenta (6 to 12 weeks gestation) were obtained after legal abortion following the protocol used by Fisher et al. [28, 29] including incubation with collagenase, hyaluronidase, and trypsin. In addition, the cells were also fractionated on a Percoll gradient. The culture medium for first trimester cells consisted of MEM D-valine medium (Gibco), 10% FBS, and 40 mg/ml gentamycin (Gibco). According to cytokeratin immunofluorescence analysis, cells obtained from first trimester and term placentas were about 95% trophoblast (cf. [28]). All cells were cultured at 37°C in

2 Abbreviations used: dbcAMP, dibutryl-cAMP; DHEA-S, dehydroepiandrosterone sulfate; EtOH, ethanol; hCG, human chorionic gonadotrophin; MTX, methotrexate; PMA, phorbol-[12-myristoyl-13acetyl]-diester; RA, all-trans-retinoic acid; STS, sterylsulfatase.

humidified atmosphere of air with 5% CO2. While choriocarcinoma cells were detached from monolayer cultures in tissue culture flasks (Greiner, Frickenhausen, Germany) using trypsin-EDTA (Gibco) for subculturing (once a week) and for experiments, normal trophoblast cells were used for experiments immediately after isolation. In routine cultures and during experiments, media were always exchanged every second day if not mentioned. Cell treatment for modulation of differentiation markers. For experiments, the media were supplemented with substances that are well known to affect cell behavior (differentiation modulators): alltrans-retinoic acid (Sigma, Deisenhofen, Germany) at 1 mM, methotrexate (Sigma) at 1 mM, dibutryl-cAMP (Sigma) at 1 mM, and phorbol-[12-myristoyl-13-acetyl]-diester (Serva, Heidelberg, Germany) at 200 nM. The concentrations used either were found to be most effective in preliminary experiments in which the secretion of hCG (see below) was monitored or/and were chosen in agreement with the literature when saturation curves were not obtained as with dbcAMP and PMA (RA [30, 31], MTX [32–35], dbcAMP [36 –38], PMA [39, 40]). Since RA and PMA were diluted from stock solutions in ethanol, media containing equivalent amounts of ethanol (0.1% final concentration) were used for control cultures as well as media without any additions. hCG measurement. Cells were grown on tissue culture plastic for 2 days with or without differentiation modulators and for an additional period of 24 h (until Day 3 of culture) in fresh media. Then the accumulation of human chorionic gonadotrophin (hCG) in supernatant media was assessed by an established immunoenzymetric test (Tandem-E hCG, Hybritech, Cologne, Germany). The concentrations were normalized against cellular DNA which was determined in homogenates of the cells in a fluorometer (Aminco-Bowman Spectrofluorometer, Model J4-8931AE, American Instruments Company, Silver Spring, MD) using 49,6-diamidino-2-phenylindol-dihydrochloride, Serva), as fluorescent, dye according to [41]. In previous studies, increased secretion of hCG was observed not to result from intracellular pools in BeWo cells or in normal trophoblast cells from term placenta [26, 42]. Therefore, in this publication the terms production, secretion, and release of hCG are used synonymously. Determination of sterylsulfatase activity. Cells were grown in the presence or absence of differentiation modulators in plastic dishes (10 cm diameter) for 3 days to confluence. Then they were rinsed twice with PBS (phosphate-buffered saline), incubated with 200 to 400 ml Tris-acetate buffer (100 mM, pH 7.0) for 10 min on ice, scraped off with a rubber policeman, and frozen at 220°C. The activity of sterylsulfatase was determined in homogenates of the cells as described previously [43, 44] using 10 mmol/L 35S-labeled dehydroepiandrosterone sulfate (DHEA-S) as the substrate. Protein concentrations of homogenates were measured according to [45]. Invasion assay. Invasiveness of normal and malignant trophoblastic cells was estimated in a Boyden chamber system according to [46] and [47]. This assay is based on penetration of a matrix barrier by the cells followed by transmigration across the filter on which the matrix was layered. The filter membranes (6.5-mm-diameter polyvinylpyrrolidone-free polycarbonate filters with 8-mm-pore size) of Transwell inserts (Costar, Cambridge, MA) for 24-well tissue culture plates were covered with 100 ml of a 1:20 dilution of Matrigel (Collaborative Biomedical Products distributed by Becton Dickinson, Heidelberg, Germany; about 200 mg/cm2) in serum-free media which was air-dryed under a laminar flow hood overnight. Before use the deposit was rehydrated with 100 ml of sterile distilled water. After at least 1 h 200 ml of cell suspension was transferred into the upper chamber. Per filter 50,000 cells for choriocarcinoma cell lines or 200,000 cells for normal trophoblast, respectively, were seeded in order to compensate for differences in proliferation rates between trophoblast cells and choriocarcinoma cells. After different periods of incubation (dependent on the cell type: 2 days for Jeg-3, 3 days for BeWo and JAr, 3 days for first trimester trophoblast, and 7 days for term trophoblast; cf. “Modulation of Invasiveness” under Results) at

DIFFERENTIATION AND INVASIVENESS 37°C in moist atmosphere with 5% CO2 the upper surface of the filter was wiped clean with a cotton swap under visual control at high magnification. After the cells on the lower surface had been fixed with ice-cold methanol and stained with hematoxylin and eosin the filters were cut out with scalpels and mounted for microscopic evaluation. For reliability, only cells with well-visible nuclei were counted. Since invasive cells were found to be distributed inhomogeneously (see Results) all cells on the lower surface had to be counted. In order to allow this strenuous and time-consuming visual quantitation, experiments had to be terminated at times when the cell layer on the lower surface of the filters was subconfluent to prevent inaccurate cell counts due to nuclear overlap. As a result cell numbers in the controls were rather low (see Results) while almost incountably high numbers (. 5000) were sometimes found with PMA treatment. An alternative approach, i.e., using radioactively labeled cells after [3H-thymidine or 35S-methionine incorporation, did not yield reliable data in our hands, due to unfavorable signal-to-noise ratios (high radioactive background). In our chosen approach we consider it an advantage to be able to detect low cell numbers at termination of the experiment; these cells can be regarded to represent the invasion front (i.e., actively invading cells and not merely migratory cells that may use gaps in the matrix created through proteolysis by the invasive front “pioneer” cells). Before an invasion assay was started choriocarcinoma cells had been grown with or without media additives for 3 days. The respective substances were also supplemented to the media in the upper and in the lower chamber throughout invasion experiments with choriocarcinoma cells or with trophoblast cells. Some of the substances that were used for manipulating the cells had also been found to influence cell proliferation. Therefore, invasion rates were determined by referring the numbers of cells found on the lower filter surface to total cell numbers. Total cell numbers were obtained in parallel experiments in which the same numbers of the respective cells as transferred into invasion chambers were seeded and grown for the same time under the same conditions on Matrigel as in the invasion assay but in 96-well plates (diameter 5 6.5 mm). For counting in a hemocytometer the cells were suspended with trypsin/EDTA and dispase (Sigma). Significance analysis was performed using Student’s paired twotailed t test.

RESULTS

Modulation of Differentiation Markers When normal trophoblast cells and choriocarcinoma cells in monolayer cultures were treated with RA, MTX, dbcAMP, or PMA, the effects on the release of hCG differed between the studied cell types (Figs. 1 and 2). The release of hCG was substantially increased by dbcAMP and PMA in first trimester trophoblasts and in BeWo cells, by dbcAMP and MTX in Jeg-3 cells and by all substances in JAr cells. Weak stimulation was obtained with RA in BeWo and in Jeg-3 cells. When choriocarcinoma cells were grown as spheroids in suspension culture the same trends were observed with the exceptions that hCG production was also stimulated by RA and MTX in BeWo cells and by PMA in Jeg-3 cells (data not shown). The effects on hCG secretion by trophoblast cells from term placenta were comparable to those obtained with first trimester trophoblasts (data not shown). The activity of STS was detected only at very low

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FIG. 1. Modulation of hCG production in normal trophoblast cells. After isolation from first trimester placenta cells were cultured in the presence or absence of either EtOH, RA, MTX, dbcAMP, or PMA for 3 days. Cultures were then continued with fresh media (with or without differentiation modulators) for 24 h before cellular DNA and hCG concentrations in the media were determined. Media containing 0.1% EtOH served as control for cultures in the presence of RA or of PMA. Values represent averages 6 SD from three experiments with two dishes in each. a, P # 0.1; b, P , 0.001 as compared to control for MTX and dbcAMP or to EtOH for RA and PMA.

levels (around 5 pmol/min 3 mg protein) in first trimester trophoblast cells (Fig. 3) while term trophoblast cells displayed about 60-fold higher activity (around 300 pmol/min 3 mg protein; Fig. 3). In BeWo and Jeg-3 cells the activities of this enzyme were found at intermediate levels (around 50 or 120 pmol/min 3 mg protein, respectively). STS was not detected in JAr cells and the activity from enzyme levels was not raised detectably by any of the substances used for inducing differentiation (data not shown). In general, enzyme activities were only weakly influenced by the various differentiation modulators. In both types of normal trophoblast cells RA and dbcAMP reduced the activity while MTX and PMA had no effect (Fig. 3). Treating trophoblast cells from term placenta with the same substances gave similar results except that activities were reduced from 331.1 6 43.6 pmol/min 3 mg protein in controls to 252.6 6 19.1 pmol/min 3 mg protein in RA-treated cells or to 219.8 6 30.9 pmol/min 3 mg protein after dbcAMP treatment (Fig. 3). In contrast, in BeWo and Jeg-3 cells all substances used tended to increase the enzyme activity to minor degrees (Fig. 4). Only MTX treatment in Jeg-3 and PMA in BeWo cells resulted in more substantial increases in STS activity. Proliferation as derived from DNA determination was not affected significantly by RA, dbcAMP, or PMA in JAr and Jeg-3 cells, while it was inhibited by MTX in these cells resulting in decreasing DNA content with culture time (data not shown). In BeWo cells and in first trimester trophoblast, proliferation rates were slightly decreased by all substances (proliferation rates: PMA , dbcAMP # MTX , RA # control, data

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above, the absolute numbers of penetrated cells represented a very small portion of the total population, i.e., about 3.5 ‰ for JAr cell, 9 ‰ for Jeg-3 cells, 7 ‰ for BeWo cells, 3 ‰ for first trimester trophoblast cells, and about 1.5 ‰ for normal trophoblast cells. In all cases the cells did not transmigrate through the gels homogeneously (Fig. 5, shown for JAr cells), the main mass of cells forming a circle in the periphery (outer half) of the filter. A minor portion of cells was found concentrated at the center. This distribution was not affected by any of the differentiation modulators. Also changing the protocol for preparation of the Matrigelcovered filters or using commercially available invasion assay kits (Becton Dickinson, Heidelberg, Germany) did not change these patterns. Treatment of normal trophoblast cells with RA, MTX, dbcAMP, and PMA concordantly reduced invasion rates in both early and late trophoblast (Fig. 6). Choriocarcinoma cells were affected heterogeneously (Fig. 7). Invasiveness of BeWo cells was reduced by all substances (with very strong inhibition by MTX and dbcAMP but only a weak effect of RA). In JAr and Jeg-3 cells only MTX elicited comparably strong suppressive effects. Treatment of JAr with dbcAMP and of Jeg-3 with RA resulted in about 50% reduction. dbcAMP caused no response in Jeg-3 cells while it did in the

FIG. 2. Modulation of hCG secretion in choriocarcinoma cells. Monolayer cultures were treated as described in Fig. 1. Averages 6 SD from three to five experiments with two parallel wells. a, P # 0.01.

not shown). Term trophoblast did not proliferate at all (not shown). Modulation of Invasiveness The various cell types used needed notably different times to penetrate the Matrigel barrier. While choriocarcinoma cells were detected on the lower surface of the filters already after about 15 h penetration took 2 days for first timester trophoblast cells and 4 days for term trophoblast. For reliable quantitation (cf. Materials and Methods), Jeg-3 invasion cultures were incubated for 2 days and BeWo, JAr, and first trimester trophoblast cells for 3 days. Penetrated term trophoblast cells could be quantitated only after 7 days. Thus different time points had to be chosen for terminating experiments with the different cell types in order to allow a count of penetrated cells under all conditions of treatments. Depending on the type of cells, numbers of penetrated cells in untreated controls were found ranging from about 300 to 1200 for choriocarcinoma and first trimester trophoblast cells and around 100 to 200 cells for term trophoblast. At the times indicated

FIG. 3. Modulation of STS in normal trophoblast cells. After isolation, first trimester or trophoblast cells, respectively, were maintained for 3 days in the absence or presence of either of the different substances before enzyme activity (hydrolysis of DHEA-S) and cellular protein were measured in cell homogenates. Averages of enzyme activities from five parallel wells 6 SD. Note different scales for first trimester and term trophoblast. a, P # 0.05.

DIFFERENTIATION AND INVASIVENESS

FIG. 4. Modulation of STS in choriocarcinoma cells. Experimental design with BeWo, JAr, and Jeg-3 cells was as described for normal trophoblast cells in Fig. 3. The enzyme was not detected in JAr cells. Averages of enzyme activities from three different experiments with two parallel wells for each condition. Note different scales for BeWo and Jeg-3. a, P # 0.01; b, P # 0.02.

other choriocarcinoma cells. However, treatment of JAr and of Jeg-3 cells with PMA stimulated Matrigel penetration. In JAr cells invasiveness was also increased by RA. DISCUSSION

In the present investigation, invasion rates of normal and malignant trophoblast cells were recorded while differentiation was modulated by treatment with

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various agents. In BeWo cells MTX and dbcAMP reduced invasiveness significantly (only slight reduction with PMA $ RA). On the other hand, hCG production was considerably increased by dbcAMP and PMA (slight stimulation by RA; increase by MTX only in spheroid cultures, not shown). Likewise the other differentiation marker, STS, tended to be stimulated in the presence of the different substances. In BeWo cells, therefore, invasiveness seems to be negatively correlated with differentiation. A similar negative correlation for hCG expression and invasiveness was observed in normal trophoblast cells, although in this case STS activity was not affected by MTX and PMA and reduced by RA and dbcAMP. In JAr and Jeg-3 cells the picture was different. All compounds increased the production of hCG (PMA did so in spheroid cultures, not shown, but not in monolayer cultures). A tendency to respond with an increase was also found for STS activity in Jeg-3 cells, particularly in treatments with MTX or PMA (STS was not detected in JAr cells under any condition). Remarkably, however, RA and PMA also increased the invasive activity of JAr cells as did PMA in Jeg-3 cells. These cells thus showed evidence of simultaneous stimulation of both differentiation and invasive activity. Therefore, our results suggest that differentiation and invasiveness are under different control and not directly linked in normal and malignant trophoblast cells as has also been discussed for tumor cells in general [48]. These different control mechanisms may lead to either direct or inverse correlations between invasiveness and parameters of differentiation depending on the cell type/subpopulations and the conditions (i.e., microenvironment and differentiation-inducing agents). Concerning the hCG expression response, all three choriocarcinoma cell lines are comparable to normal trophoblast cells. With respect to invasiveness, BeWo cells appear to resemble normal trophoblasts more closely in their response

FIG. 5. Patterns of Matrigel penetration in JAr cells. A total of 50,000 cells was seeded onto the layer of Matrigel in the upper compartment of the Boyden chamber assembly. After 3 days the cells on the lower surface of the filters were stained. (a) Control, (b) in the presence of PMA, or (c) of MTX. In the experiment exemplified here cells in the control (a) and in the presence of PMA (b) had penetrated the filters in such high numbers that quantitation was impossible. During treatment with MTX (c), in contrast, only 37 cells were counted on the lower side of the filter. Diameter of filters 5 6.5 mm.

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mary tumors and to form metastases. Induction of differentiation may, therefore, not necessarily always result in loss of invasiveness. It has been shown in vitro for RA and for phorbol esters that agents that are known to be able to induce differentiation eventually can even stimulate invasiveness in some cell types [51, 52]. In those studies, however, the correlation between invasiveness and differentiation was not examined. An inverse relationship between differentiation and invasive potential has also been discussed previously for the normal invasive cell type studied in the present experiments, the human trophoblast [26, 53, 54]. In

FIG. 6. Treatment of normal trophoblast cells with differentiation modulators reduces invasiveness. Invasion assay assemblies were cultured for 3 days with first timester trophoblast cells or for 7 days with term trophoblast cells in the absence or presence of differentiation-inducing agents before cells on the lower filter surfaces were stained and counted. Averages 6 SD from four to five experiments with two parallel wells for each condition in each experiment. *Invasion represents the portion of cells that had reached the lower surface of the filters referred to total cell numbers determined in parallel under the same conditions in regular tissue culture wells. a, P # 0.05; b, P # 0.02; c, P # 0.01; d, P , 0.001 as compared to control.

while JAr cells appear to be more aberrant, although invasiveness could be stimulated with insulin-like growth factor (data not shown) as Lysiak et al. [49] have also observed in normal trophoblast. Induction of differentiation in cancer cells has been discussed as a new therapeutic strategy in oncology for more than a decade now (see Introduction). Many substances, and in particular perhaps the most often used model drug for differentiation inducing therapy, retinoic acid and its derivatives, have been shown to reduce invasion and metastasis of a number of tumor cells in vivo and in vitro [15, 20, 25]. In the meantime, differentiation therapy has been applied successfully in certain types of leukemia by administration of RA [23, 24]. However, there are only few reports of favorable results in neoplastic diseases other than leukemia, and even acceleration of the disease was observed in clinical studies with RA or other drugs [12, 15, 25]. This opens the question whether differentiation and invasiveness may be under different control mechanisms. In fact, there are highly differentiated squamous cell carcinomas that are nevertheless highly malignant [50]. Malignancy of tumor cells is mainly based on their invasive behavior enabling them to evade pri-

FIG. 7. Modulation of invasiveness by induction of differentiation in choriocarcinoma cells. Invasion assays were carried out for 2 days (Jeg-3 cells) or for 3 days (BeWo and JAr cells) in the absence or presence of RA, MTX, dbcAMP, or PMA. Then the cells on the lower filter surfaces were stained and counted. Values are averages 6 SD from three to five experiments per cell line with two or three parallel wells for each condition in each experiment. *Invasion represents the portion of cells that had reached the lower surface of the filters referred to total cell numbers determined in parallel under the same conditions in regular tissue culture wells. a, P # 0.001; b, P # 0.05; c, P # 0.1; d, P # 0.2 as compared to control.

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general, histology suggests that the trophoblast loses its invasiveness while placentation goes on and assumes new, nutritional, and endocrinological, functions. However, as close examination reveals there are a number of trophoblast cell subpopulations in addition to villous cyto- and syncytiotrophoblast, and in particular various types of extravillous trophoblast that are only very incompletely characterized and may have quite different functions and patterns of behavior [53, 55, 56]. The invasive subpopulations may even be regarded as representing a peculiar state of differentiation. The mechanisms controlling trophoblast invasiveness and differentiation are poorly understood so far. The most favored hypothesis proposes an intrinsic program that may be modulated by external factors [53]. It appears to be closely related to the program of epithelial-mesenchymal transition also seen in other systems (embryology, cancer) [57, 58]. Differentiation and invasion of trophoblast cells in vitro are affected by many different factors [53, 59]. Particularly TGF-b appears to reduce invasiveness [60] while EGF may stimulate it [61]. Also insulin-like growth factor stimulates trophoblast invasion [49]. However, the relationship between invasiveness and differentiation still remains unclear, although understanding their regulation will have clinical relevance for placenta accreta (trophoblast invasion too deep) and preeclampsia (invasion into maternal blood vessels defective). Malignant trophoblast (i.e., choriocarcinoma) cells are a well-accepted and frequently used model for studying trophoblast behavior. The different choriocarcinoma cell lines used in this study are all of placental origin [62– 66], display different patterns of invasiveness [67], and have been shown to respond to induction of differentiation quite well (e.g. [30 – 40, 42, 68, 69]). Also normal trophoblast cells respond to induction of differentiation in vitro [26, 53, 59]. In contrast to first trimester trophoblast cells which are highly invasive, term trophoblast displays only a very low level of invasiveness [54, 70, present communication]. It appears reasonable, therefore, to compare the differentiation and invasion behavior of the two types of trophoblast cells and of choriocarcinoma cells since the pattern of responses obtained may, in approximation, give an idea about the behavior of trophoblast subpopulations with different degrees of invasiveness and inducibility in vivo. In most of such studies the expression of hCG has been used as the most reliable marker for trophoblastic differentiation. hCG has been documented to be characteristic for early trophoblast differentiation with highest production in intermediate stages during transition from cytotrophoblast to syncytiotrophoblast [71]. In contrast, STS appears to be associated with the late stages of morphological differentiation with peak production found in syncytiotrophblast [44, 72, 73]. Ac-

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cordingly, STS activity is much higher in term placenta and in agreement with this, trophoblast cells isolated from term placenta displayed much higher enzyme levels than first trimester cells in the experiments presented here. Invasion has been observed to be very closely linked to protease expression. For human trophoblast invasion Matrix Metalloproteinase-9 (MMP-9) has been strongly implicated as a key role player [54]. In other cell types phorbol esters like PMA have been shown to upregulate MMP-9 through a TRE site present on its promotor [74], and phorbol ester-stimulated MMP-9 expression was correlated with invasiveness in many cell types (e.g. [75–77]). It may, therefore, be surprising to find that PMA treatment of first trimester and term trophoblast cells as well as of BeWo cells leads to a decrease in invasive ability. However, simultaneous stimulation of MMP-9 expression and invasion has usually been studied in malignant cells but not in normal invasive cells like trophoblast (as suggested above, BeWo cells may resemble normal trophoblasts more closely than JAr or Jeg-3 cells). So far, it cannot be excluded that normal cells respond differently to PMA treatment provoking an increase of differentiation associated with reduced invasiveness rather than with an increase as observed in malignant cell types. This is supported by the fact that MMP-9 expression does not respond to phorbol ester treatment in all cell types analyzed [75]. In trophoblast cells MMP-9 secretion and invasive potential in vitro have been observed to be downregulated in the course of gestation along with trophoblast differentiation to different subpopulations [54]. It is well known that tumors usually consist of many heterogeneous cell subpopulations. BeWo, Jeg-3, and JAr cells as well as the two types of trophoblast cells studied may be considered representative of possible trophoblast phenotypes with more or less differing potential for differentiation and invasion. From the present results, it cannot be determined whether invasiveness of all cells or of only a minority of cells in a culture was reduced or stimulated. Although stimulated invasiveness was observed in JAr or Jeg-3 cells along with increased expression of differentiation markers, it is not known whether the invasive population was still proliferative (in contrast to the dogma that differentiation leads to arrest of proliferation). While reduced invasion was correlated with reduced proliferation during MTX treatment, particularly in JAr and Jeg-3 cells, increased invasion was not correlated with elevated proliferation rates after PMA or RA treatment in JAr or Jeg-3 cells (proliferation data not shown). Stimulation of invasiveness would be particularly hazardous for the patient if the invasive compartment still contained proliferative cells. When extrapolating our results to the various subpopulations of cells

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within a tumor it can be expected that treatment with differentiation-inducing drugs may affect different subpopulations differently. It could be difficult, therefore, to force most or even all cells in a tumor into terminal differentiation, thereby exhausting the proliferative compartment. Our observations on JAr cells treated with RA or with PMA and Jeg-3 cells with PMA suggest that “differentiation” drug treatment can also stimulate invasiveness of some subpopulations of cells. In conclusion, differentiation and invasiveness of trophoblastic cell types appear to be under different control mechanisms, although they may be correlated inversely in some cell subtypes and experimental situations. Differentiation-inducing treatment of cancer may in some cases even have the adverse effect of stimulating invasiveness in certain subpopulations, thereby favoring metastasis formation (if invasive cells are still proliferative). Since it appears nonpractical to characterize the differentiation and invasion response of the cancer cells of each individual patient in vitro, before chosing a therapeutic strategy, the present results suggest the formulation of a caveat with respect to the chances of finding drugs suited for general, broad-spectrum differentiation therapy. It may be reasonable to conduct investigations comparable to those presented here with many more types of tumor cells. The authors thank Ms. U. Steih and Ms. K. Regemann for excellent technical assistance. The darkroom work of D. Kittel and his crew is highly appreciated. The support from Dr. U. Feldmann and Prof. Dr. Lamberti (Essen) and their staff have been essential for this work. We also like to express our gratitude to the Dr. Mildred Scheel Stiftung fu¨r Krebsforschung for supporting this study (Grants 10186 and 10-299). Parts of the data have been presented previously in abstract form (Eur. J. Cell Biol. 60, Suppl. 37, 17, 1993; Placenta 14, A44, 1993; Ann. Anat. 176, Suppl., 226 –227, 1994; Cell Biol. Int. 18, 576, Th-153, 1994; Clin. Exp. Metastas. 14, Suppl. 1, 36, 1996).

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