[CANCER RESEARCH 52, 3713-3717. July 1. 1992]

Differentiation of Choriocarcinoma Cell Line (JAr)Abraham Hochberg,1 Jacob Rachmilewitz, Talia Eldar-Geva, Tamar Salant, 'l'amar Schneider, and Nathan de Groot

Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel

ABSTRACT

Choriocarcinoma, a highly malignant tumor arising from the tropho-blast. comprises a heterogenous population of cells including cytotroph-oblasts, intermediate trophoblasts, and syncytiotrophoblasts. In order toinvestigate trophoblast differentiation, we used centrifugal elutriation toseparate cells from the JAr Choriocarcinoma cell line according to theirsize and to further show that the resultant cell populations differ in theirstage of differentiation. Two % of the cell population consists of large,multinuclear cells, which display the highest level of chorionic gonado-tropin (CG) mRNAs. The increase in the CGßmRNA with cell size is aconsequence of the transcriptional mechanism, since agents which inducedifferentiation in JAr cells, i.e., methotrexate, increase the level of CGaand CG0 transcripts, cause a shift in cell size, and result in the formationof multinuclear cells. The multinuclear cells in the JAr population arise,at least partly, from kariokinesis without cytokinesis.

INTRODUCTION

Choriocarcinoma, a highly malignant tumor arising from thetrophoblast, comprises a heterogenous population of cells thatare apparently in different stages of differentiation. The nativeChoriocarcinoma is a mixture of cytotrophoblasts, intermediatetrophoblasts, and syncytiotrophoblasts (1) and lacks the presence of chorionic villi. Such tumors produce high amounts ofCG,2 and some synthesize hPL or pregnancy-specific ßi-glyco-

protein (1). In situ hybridization studies of sections from placentae and trophoblast tumors suggest that expression of theseplacenta! proteins is linked to particular stages of trophoblastdifferentiation (2-4). Previous reports suggested that both invivo and cultured Choriocarcinoma cells apparently undergo acell fusion and differentiate into CG-synthesizing cells (1, 5).

Cell lines derived from Choriocarcinoma such as JEG-3,BeWo, and JAr were intensively used in order to investigatetrophoblast differentiation. Although factors causing cytotro-phoblast stem cells either to divide or to terminally differentiateare not known, adenosine derivatives (5, 6), MTX (7, 8), andcoculturing of cytotrophoblasts with Choriocarcinoma cell lines(9) were used to facilitate trophoblast differentiation. Friedmanand Skehan (7) were the first to show that BeWo cells withcytotrophoblast-like features could be induced to acquire syn-cytiotrophoblast ultrastructure and behavior, when cultured inthe presence of MTX.

Throughout differentiation of trophoblasts, a multistagepathway was proposed. It was suggested that defined intermediates could be identified according to the specific proteinsproduced (2). These intermediate cells were proposed morethan once as an obligatory step in cytotrophoblast differentiation both in vivo and in vitro for normal placenta! cells as wellas for Choriocarcinoma (1,2, 9).

Here we show that, by using centrifugal elutriation, one canfractionate and isolate trophoblast cells according to their stageof differentiation. We follow the biological behavior of each

Received 12/26/91; accepted 4/20/92.' To whom requests for reprints should be addressed.2The abbreviations used are: CG, chorionic gonadotropin; hPL, human pla

centa! lactogen; MTX, methotrexate; HEPES, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid; HBSS, Hanks' balanced salt solution; cDNA, complementary

DNA; SDS, sodium dodecyl sulfate; CAT, Chloramphenicol Acetyltransferase.

separate fraction in culture, in order to study the mechanism ofthe differentiation pathway in Choriocarcinoma.

MATERIALS AND METHODSCell Culture. The JAr Choriocarcinoma cell line was maintained in

Medium 199 containing 10% fetal calf serum, 25 HIM HEPES (pH7.4), penicillin (180 units/ml), streptomycin (100 ng/ml). and ampho-tericin B (0.2 Mg/ml). Four x IO4cells/cnr were plated in polystyrene

culture dishes (Nunc). The medium was changed every 24 h. Every 3days the cells were trypsinized with 0.05% trypsin-EDTA solution (BeitHaemek) for 5 min and plated again at the same initial density or asdescribed.

For JAr-Kpn, the CGßCAT construct was stably integrated in theJAr cells now designated JAr-Kpn, containing the 5' region of the

CGßSgene extending to the Kpn\ site (10). This corresponds to adistance of 3.5 kilobases from the mRNA Cap site.

Centrifugal Elutriation. Two to 2.5 x 10s JAr cells were concentrated

into 10 ml of Medium 199 containing 25 mM HEPES (pH 7.4) andloaded into a Beckman J2-21M elutriator rotor (Beckman. Palo Alto.CA), using a standard chamber and a Masterflex peristaltic pump (Cole-Parmer Instruments, Chicago, IL). Constant rotor speed was maintained at 2000 rpm. The elutriation buffer was HBSS containing 2%newborn calf serum at 4°C.The cells were loaded at a flow rate of 10

ml/min. The first 150 ml served as a washing step and were discarded.The flow rate was increased from 15 to 65 ml/min, in 5-ml steps, and11 fractions (100 ml each) were collected. At the last flow rate, thecentrifuge was stopped. The fractions were spun at 2000 rpm. and thepellets were resuspended in incubation medium. The first fraction (at aflow rate of 15 ml/min) from JAr cells contained mostly cell debris andwas discarded.

Cell Size Determination. Cells from each of the fractions were suspended in HBSS, and their number and size distribution were determined using a Coulter Counter (Coulter Electronics. Harpenden, Herts,England).

Flo»Cytometry. Five x IO5 cells from each of the fractions after

elutriation were prepared for flow cytometric DNA analysis (11). Determination of the DNA content of the nuclei in each fraction wasevaluated by FACS 440 cell sorter (Becton Dickinson. Sunnyvale, CA).

RNA Isolation and Northern Blot Hybridization. Total cellular RNAwas isolated from the cells in each fraction immediately after elutriationby the guanidinium-thiocyanate/cesium-chloride method (12). Ten Mgof each RNA sample were separated by 1% agarose-formaldehyde gelelectrophoresis and transferred to a Hybond-N nylon filter (Amcrsham,England). The blots were hybridized with specific cDNA probes at 42°C

in 50% formamide. 5x SSPE (sodium chloride, sodium phosphate,EDTA) 5x Denhart's solution, 0.1% SDS. and 0.1 mg/ml of herring

testis DNA. The probes used for hybridization were CO«(13), CGß(14), and hPL (15). The blots were washed twice in O.lx standardsaline citrate (0.15 M NaCl:0.05 M sodium citrate):0.1% SDS at 65°Cand exposed to AGFA Curix film at -80°C.

Determination of DNA Synthesis. JAr cells ( 10') were cultured in 12-

well multidishes. in 1 ml of Medium 199 containing HEPES. fetal calfserum, and antibiotics as described above.

To determine incorporation of ['Hjthymidine into DNA, 10 ^1 of[methyl-3H]tiiymidine (1 pCi/pi; 81 Ci/mmol; ICN Biomedicals, Irvine,

CA) were added to the culture medium in each well. After 24 h oflabeling, the medium was removed, the cells were washed twice withHBSS, 1 ml of distilled water was added, and the cells were scrapedfrom the plate and frozen. After thawing, aliquots of the lysate weretaken for protein determination by the Bio-Rad protein assay dyereagent (Bio-Rad Laboratories GmbH, Munich, Germany; CatalogueNo. 500-0006). ['H]Thymidine incorporation was determined by tri-

chloroacetic acid precipitation (16).3713

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JAr DIFFERENTIATION

RESULTS

Using centrifugal elutriation we attempted to separate andisolate the various intermediate cells which were in differentstages of the differentiation pathway. Cells were incubated for72 h, trypsinized, and separated according to their sedimentation coefficient by centrifugal elutriation. We have separated11 fractions and measured the physical, morphological featuresand the gene expression of each cell population. Fraction 1contained mostly cell debris and was not analyzed. The percentage size distribution of cells is shown in Fig. \A. The sumof the cells in Fractions 2 to 6 contained more than 80% of thewhole cell population. The other fractions contained 1 to 5%each.

Centrifugal elutriation is used to obtain cell cycle stage-specific populations (17-19), by virtue of the increase in cellsize as cells progress from Go-Gi to G2-M. The DNA contenthistograms obtained from flow cytometric analysis for the cell

fraction 10 11

diameter (jim) 12.2 12.4 15.116.2 17.2 17.7 19.5 20.5 23.6 25.6

A

2n 4n 6n 8n 6n on 2n 4n 6n 8n

567

FRACTION

Fig. I. Percentage distribution and the DNA content histograms of elutriatedJAr cells. Cells were separated using centrifugal elutriation. In A. ten fractionswere collected, the cells in each fraction were counted, and their average size wasdetermined using a Coulter Counter. B. DNA content histogram of the elutriatedcells. Numbers in the right corner of each histogram represent the number of theelutriated fraction: U. unfractionated cells. In C, the percentage of nuclei withmore than 4 times the haploid DNA content was calculated from the DNAcontent histograms. The average recovery of cells after elutriation was 72% (n =20).

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Fig. 2. Phase microscopy of elutriated JAr cells 90 min after plating. ElutriatedFraction 2 (A). Fraction 5 (B), and Fraction 11 (C). Elutriated Fraction 11 72 hafter plating (D). Arrows indicate some of the multinuclear cells. The bar indicates25 |jm.

fractions are shown in Fig. IB. The nuclei in the elutriatedfractions display increasing DNA content; indeed, JAr cells areexponentialy growing cells. Cells in different stages of the cellcycle are represented in Fractions 2 to 6 From G0-Gi in Fraction2 (2 times the haploid DNA content, 2n) through S in Fractions3 and 4 to G^-M in Fractions 5 and 6 (4n). In successive nucleifractions, more than 4n DNA was detected and at least 6 to 8nin Fractions 9 to 11. From the flow cytometric analysis (Fig.1Ä),the percentage of nuclei containing more than 4n DNA ineach fraction was calculated (Fig. 1C). In Fractions 2 to 4, lessthan 1% of the nuclei contained more than 4n DNA, thoughfrom Fraction 5, there is a gradual increase in the percentageof nuclei with more than 4n DNA, reaching up to 43% inFraction 11. Using the method applied for flow cytometricanalysis, the DNA content in the nuclei and not in the intactcells was determined, as the cells were treated with trypsin,RNase, and detergent (11). The polyploid nuclei phenomenon(i.e., nuclei containing 8n) can be explained in two ways: eitherthe nucleus contains 8n DNA; or there exist 4 tightly connectednuclei, each containing 2n DNA and not separated by thetreatment.

Phase microscopy of JAr cells immediately after elutriationrevealed a gradual increase in cell size in the different cellpopulations accompanied by an increase in the number of nucleiper cell (Fig. 2). Most of the cells in Fractions 2 to 7 aremononuclear (Fig. 2, A and A), but a profound increase in thepercentage of multinuclear cells is revealed in Fractions 8 to11. At least 50% of the cells in Fraction 11 are multinuclear(between 2 and 6 nuclei per cell; Fig. 2C). Phase microscopy ofcells from Fraction 11, 96 h after plating (Fig. 2D), revealed atleast 6 nuclei tightly connected. This phenomenon was observedin the majority of the cells in Fraction 11. Among unfractionated cells plated for 96 h, few cells with the multinuclei appearance can be seen (not shown). Cells from Fraction 11 werecompared after 24 or 96 h of incubation, and an increase in the

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JAr DIFFERENTIATION

70-

n-

FRACTION

FRACTION

Fig. 3. Percentage distribution of reelutriated JAr cells. Cells were separatedusing centrifugal elutriation, and each fraction was plated separately. After 72 hthe cells were trypsinized, reelutriated. and counted. The percentage distributionsof the reelutriated cells from Fraction 5 (A) and Fraction 11 (B) were determined.

number of nuclei per cell was observed.We compared multinuclear JAr cells to multinuclear human

placental syncytiotrophoblasts. Trophoblasts isolated fromterm placenta were separated using centrifugal elutriation. Fraction 11 contained multinuclear syncytiotrophoblasts. After flowcytometric analysis of syncytiotrophoblasts, only 2n nuclei wereobserved (not shown), indicating that each nucleus in the syn-cytiotrophoblast cell is separate from the other.

We addressed the question of whether the cells in Fraction11 are in a terminal stage of differentiation or can reenter thecell cycle. Cells from Fractions 5 and 11 were cultured separately and, after 96 h, were trypsinized and reelutriated. Thepercentage of cells in each fraction was determined (Fig. 3).While the distribution of reelutriated cells from Fraction 5 (Fig.3/1) resembled the distribution of cells elutriated at time zero(Fig. 1/1), most of the cells in Fraction 11 have not changed insize and were reisolated in the same fraction (i.e., Fraction 11;Fig. 3B). We conclude that the cells in Fraction 11 are notdividing and have exited the cell cycle.

DNA synthesis in the different fractions of JAr cells (asdetermined by measuring ['H]thymidine incorporation into

DN A/Vg of protein) was the same in all the cell fractions whenmeasured between 0 and 24 or 72 and 96 h after plating (datanot shown). Despite the fact that cells in Fraction 11 are notdividing, their nuclei maintain an active incorporation of thy-midine into DNA to the same extent as do the cells in the otherfractions.

The expression of CG and other pregnancy-specific proteinsis linked to particular stages of trophoblast differentiation (2,20). CG is a placental specific protein and an establisheddifferentiation marker of trophoblast cells (20-22). We followed the expression of the CG genes in the different elutriatedcells. RNA was isolated from the different elutriation fractionsand hybridized with CG«and CG/3 probes (Fig. 4). CG«(Fig.4A) and CG/3 (Fig. 4B) mRNAs were not detected in Fractions2 to 4, but gradually increased in RNA from Fractions 5 to 10

23456789 10 11Fig. 4. Abundance of CG«and CG/i mRNA in RNA of the elutriated fractions

of JAr cells. Autoradiogram of Northern blots containing RNA isolated fromelutriated cells. The blots were hybridized with CG<«(A) and CGrf (B) cDNAprobes. Numbers represent cell fractions.

reaching their highest level in Fraction 11.Although the total population of JAr cells does not seem to

express hPL, this does not exclude the possibility that only asmall fraction of the total cell population does express thisgene. We, therefore, isolated RNA from the different elutriationfractions and hybridized it to the hPL probe. None of theelutriated fractions expressed hPL mRNA.

In order to investigate whether the increase of CG/3 mRNAlevel as a function of the cell differentiation state is a consequence of the transcriptional mechanism, we cultured JAr cellsstably transfected with a plasmid containing the CAT reportergene linked to the promoter of the CGßfgene, designated JAr-Kpn (10). After 72 h of incubation the cells were subjected tocentrifugal elutriation. The CAT activity was measured in thecell extracts from each fraction (Fig. 5/1). The spots corresponding to the acetylated chloramphenicol were excised and counted(Fig. 5B). A gradual increase in the promoter activation in thedifferent cell fractions is seen from Fractions 2 to 10, with asharp increase in Fraction 11 (5-fold as compared with Fraction2).

Following MTX treatment, the majority of cells within a

^r ^f ^F ^r ^f ^r

fraction 234567 9 1011

FRACTION

Fig. 5. Expression of CAT reporter gene in elutriated fractions of JAr-Kpncells. Ten fractions of JAr-Kpn cells were separated using centrifugal elutriation.and the CAT assay was performed as described (24) except for the use of ethyl-acetate as the Chromatographie solvent. Arrow denotes the position of acetylatedchloramphenicol (A). Spots corresponding to acetylated chloramphenicol wereexcised and counted (B).

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Mr DIFFERENTIATION

23456789 10 11

FRACTION

B50

«

I0

FRACTION

Fig. 6. Percentage distribution of elutriated JAr cells grown in the presence ofMIX. Cells were treated with 1 ^M MTX for 72 (A) and 120 h (B). Afterincubation, cells were trypsinized and separated using centrifugal elutriation. Tenfractions were collected, and the cells in each fraction were counted.

BeWo culture transform morphologically, acquiring many ofthe same ultrastructural characteristics that have been reportedfor the differentiated in utero syncytiotrophoblast (7). Following72 and 120 h of incubation in the presence of 1 /¿MMTX, JArcells were subjected to centrifugal elutriation, and the percentage distribution of the cell fractions was determined (Fig. 6).MTX treatment induced a shift in cell size in the majority ofcells in culture toward the size of cells in Fraction 11. Thisobservation is also based on phase microscopy (not shown).While the majority of the cells in the untreated cultures arecollected in Fractions 2 to 5 (Fig. IA), most of the cells incultures treated for 72 h with MTX are collected in Fractions5 to 8 (Fig. 6A) and in Fractions 8 to 11 in cultures treated for120 h (Fig. 6B). MTX did not change thymidine incorporationinto DNA (not shown) as was previously reported (7, 8).

Arbiser et al. (8) reported an induction of synthesis of themRNA encoding CG«and CGßby MTX in BeWo and JEG-3

cell lines. They have also shown that hydroxyurea induces theactivation of the CG«promoter. Similarly incubation of JAr-Kpn cells for 72 h with MTX resulted in a 5-fold increase in

the activity of the CAT reporter gene driven by the CGß5promoter, compared to control (Fig. 7).

In order to elucidate whether the induced expression of CG«by MTX is linked to an increase in cell size, cells were grownfor 96 h in the presence of MTX. After incubation, cells weretrypsinized and elutriated. RNA was isolated from the differentelutriation fractions and hybridized with the CGa probe (Fig.8). The level of CG«mRNA in Fractions 5 to 11 in the MTX-

treated cells (Fig. 8B) was higher than the control (Fig. 8/4),though no gradual increase of CG«mRNA is observed withincreasing cell size. We suggest that MTX activates the CG«promoter regardless of the change in cell size. Similar resultswere seen with CG/3 mRNA (not shown).

DISCUSSION

During differentiation, pluripotent stem cells give rise tocommitted precursor cells which lose their capacity to divide asthey reach the terminal stages. As differentiation progresses,cell division is reduced and eventually lost. Cancer cells fail todifferentiate normally, though many tumors grown in tissueculture can be induced to differentiate (23).

The trophoblast differentiation pathway includes morphological intermediate or transitional cells, with distinctive biochemical and cytological characteristics, which are the direct precursors of syncytial trophoblasts. The above observations implythat multiple intermediates are formed during the trophoblastdifferentiation pathway (2,6). Normal placentae, native chorio-carcinoma, and choriocarcinoma cell lines comprise a heterog-enous population of intermediate cells in different stages ofdifferentiation, the isolation of which facilitates the elucidationof the differentiation pathway. Isolation of such cells has previously not been described.

Centrifugal elutriation is currently a widely used preparativecell separation technique, that under optimal conditions canseparate cells that differ only slightly in their size or theirsedimentation velocity. This is the method of choice for theseparation of cells according to their cell cycle stage (17-19).

Here we report on the separation of JAr cells according totheir cell size and show that the various cell populations differin their stage of differentiation. Based on morphological andbiochemical characteristics observed in the different cell fractions obtained by centrifugal elutriation, we propose that JArcells are dividing cells, but very few exit the cell cycle anddifferentiate. Those cells that do differentiate express both CG«and CGß,the expressions of which are closely coupled. TheJAr differentiation includes an increase in cell size accompaniedwith polyploidity. However, further differentiation is limited,and hPL synthesis is not seen. The polynuclear cells (Fraction11) are in a terminal stage of JAr differentiation, as these cellsexit the cell cycle and express the highest level of CG«andCGß,as was evident in the level of CG«and CGßprotein (5)

JAr-Kpn JAr-Kpn+MTX

Fig. 7. Activation of the CAT reporter gene derived by CG/i, promoter byMTX treatment. JAr-Kpn cells were grown for 96 h in the presence or absenceof MTX. The CAT assay was performed as described (24). The arrow denotes theposition of acetylated chloramphenicol. Spots corresponding to the acetylatedchloramphenicol were excised and counted: JAr-Kpn. 4.380 ±400; JAr-Kpn +MTX. 20.030 ±2.000 (n = 5).

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JAr DIFFERENTIATION

5 6 7 8 9 10 11B

Fig. 8. Abundance of CG«mRNA in elutriated fractions of JAr cells aftertreatment with MTX. Cells were grown for 72 h in the absence (.-() and presence(B) of MTX. After incubation, the cells were trypsinized and separated usingcentrifugal elutriation. Northern blots were hybridized with CG<r cDNA probes.Numbers indicate cell fractions.

and mRNA. Furthermore, the increase in the CG/J mRNA withcell size is a consequence of an increase in the rate of transcription as shown through the CAT reporter gene. Agents whichinduce differentiation in choriocarcinoma cell lines, i.e., MTX(7, 8) and hydroxyurea (8), increased the CG«and CGft transcripts level and also caused a shift in cell size and the formationof multinuclear cells.

The multinuclear cells may arise from either cell fusion (aswas shown for placenta! cytotrophoblasts) or from karyokinesiswhich is not followed by cytokinesis. We suggest that themultinuclear cells observed in JAr cultures are formed by karyokinesis without cytokinesis, since active DNA synthesis ismeasured despite the fact that they have differentiated and littleif any cell division is seen. Further investigation in nativechoriocarcinoma can provide the answer to whether the multi-nuclear cells are formed by the same mechanism as was described here for JAr cells.

ACKNOWLEDGMENTS

We thank Dr. Averell Gnatt for his many helpful discussions. Wealso wish to thank Dr. I. Boime for the JAr-Kpn cell line.

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1992;52:3713-3717. Cancer Res   Abraham Hochberg, Jacob Rachmilewitz, Talia Eldar-Geva, et al.   Differentiation of Choriocarcinoma Cell Line (JAr)

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