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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2458
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 8 4873-4879
Copyright © 2005 by The Endocrine Society

Transfection of Antisense Chorionic Gonadotropin ß Gene into Choriocarcinoma Cells Suppresses the Cell Proliferation and Induces Apoptosis

Anna Lissa Hamada, Koji Nakabayashi, Asomi Sato, Kenji Kiyoshi, Yukou Takamatsu, Jovelle B. Laoag-Fernandez, Noriyuki Ohara and Takeshi Maruo

Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan

Address all correspondence and requests for reprints to: Dr. Takeshi Maruo, Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: maruo{at}kobe-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Choriocarcinoma cells not only synthesize human chorionic gonadotropin (hCG), but also express LH/CG receptors on the cell membrane. This suggests that the hCG and LH/CG receptors may play a role in regulating the biological function of choriocarcinoma cells in an autocrine/paracrine manner.

Objective and Methods: The objective of this study was to ascertain whether the inhibition of CGß gene expression in choriocarcinoma cells affects their proliferation and apoptosis. Expression vector bearing antisense CGß gene was transfected into the choriocarcinoma cell line, JAr. CGß protein synthesis was monitored by Western immunoblot, and CGß mRNA expression was determined by RT-PCR. Cell proliferation was assessed by 3-[4,5-dimethlthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and nuclear incorporation of 5-bromo-2'-deoxyuridine, and the apoptosis-positive rate was assessed by terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling analysis and nuclear staining with Hoechst 32258.

Results: JAr cells transfected with antisense CGß gene (JAr-aCGß cells) showed a significant decrease in hCG production and cell proliferation compared with untransfected and mock-transfected cells. The apoptosis-positive rate of the JAr-aCGß cells significantly increased compared with that of the controls. LH/CG receptor expression in JAr-aCGß cells decreased compared with that in controls. By contrast, supplementation of exogenous hCG significantly increased the LH/CG receptor expression and viability of JAr-aCGß cells.

Conclusions: These results suggest that hCG, through its binding to the LH/CG receptor, may augment proliferation and inhibit apoptosis in choriocarcinoma JAr cells, and that the introduction of an antisense gene may be a potential approach to the inhibition of choriocarcinoma cell growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHORIOCARCINOMA IS A highly anaplastic and invasive tumor derived from trophoblastic cells. Just like normal placental trophoblastic cells, choriocarcinoma cells produce human chorionic gonadotropin (hCG). The glycoprotein hormone hCG is required for luteal support in early pregnancy. It is a noncovalent dimer consisting of {alpha}- and ß-subunits. The {alpha}-subunit is identical with that of FSH, LH, and TSH, whereas the ß-subunit is unique and confers the biological and immunological specificity to each hormone (1, 2). The production of hCG has been described to be dependent on one hCG{alpha} gene and six hCGß genes (3, 4). It has been suggested that the expression of the {alpha}- and ß-subunits of hCG is transcriptionally regulated (5). Although there is an imbalance in the synthesis and secretion of {alpha}- and ß-subunits, it has been proposed that ß-subunit synthesis controls glycoprotein hormone expression (6, 7, 8).

hCG is produced in excessive amounts in choriocarcinoma and other gestational trophoblastic tumors, and free hCGß has been used as a tumor marker to monitor gestational trophoblastic diseases (9, 10). The serum levels of free hCGß correlate not only with the size of the tumor, but also with the malignant potential of the gestational trophoblastic diseases, because it has been shown that more free hCGß is secreted by malignant trophoblastic cells than by normal placental trophoblasts. Occasionally, malignant trophoblastic tumors secrete mostly free CGß and almost no intact hCG (11, 12, 13, 14).

Only the intact heterodimer form of hCG is biologically active, whereas the free subunits lack any hormonal effect (15, 16). The exact biological function of the hCG {alpha}- and ß-subunits in trophoblastic neoplasia, however, remains unexplained. It is important to understand the roles of hCG and its subunits in the growth of choriocarcinoma cells, because in vitro investigations involving different cultured cells have shown that hCG dimer and hCGß stimulate tumor growth (17, 18, 19).

The hCG biosynthesis in choriocarcinoma cells differs from that in primary placental trophoblastic cells, whereby choriocarcinoma cells cannot self-regulate hCG production (20). Several investigators have shown that the choriocarcinoma cells not only contain LH/CG receptors, but also overexpress these receptors compared with normal trophoblastic cells (21). The presence of these receptors and the enormous hCG production would lead us to presume that some biological function of hCG-LH/CG receptor binding may exist in choriocarcinoma cells, and that hCG may be essential in the continued growth of those cells.

The introduction of recombinant plasmid expression vectors that direct synthesis of antisense mRNA has been performed to inhibit the translation of sense mRNA in different cells in vitro (22, 23, 24, 25, 26, 27). Using this procedure, either reduction of endogenous protein synthesis (22, 23, 24, 25) or cellular growth inhibition was achieved in certain cells (26, 27). In this context, the present study was conducted to ascertain whether the transfection of antisense CGß gene into choriocarcinoma cells affects proliferation and apoptosis in those cells. By interfering with hCG synthesis through the introduction of an antisense CGß gene, we evaluated whether the resultant suppression of hCG production could be a novel strategy for controlling the growth of choriocarcinoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of expression vector pM2AH bearing antisense CGß

The recombinant plasmid pM2HA with the sense CGß (gift from Dr. Irving Boime, St. Louis, MO) was digested with restriction enzymes XhoI and NotI (Promega Corp., Madison, WI), releasing the sense CGß. This was followed by ligation of this CGß to another recombinant plasmid, pM2AH (gift from Dr. Irving Boime), resulting in antisense CGß, as previously described (28).

DNA transfection and clone selection

Antisense CGß was subsequently transfected into JAr cells (American Type Culture Collection, Manassas, VA) by the calcium phosphate precipitation method (29). A simultaneous transfection was also performed in JAr cells using an empty pM2AH plasmid (JAr-pM2AH) as a control. Stable colonies were selected 14 d after transfection using 62.5 µg/ml of the neomycin analog G418 (Sigma-Aldrich Corp., St. Louis, MO). The transfected colonies were screened for the expression of hCGß by Western dot-blot analysis. Untransfected JAr cells together with JAr-pM2AH cells were used as positive controls throughout the experiment.

Cell culture

JAr-aCGß cells and JAr-pM2AH cells were maintained in DMEM (Invitrogen Life Technologies, Inc., Carlsbad, CA), containing 5% fetal bovine serum and 62.5 µg/ml, and G418, supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM glutamine at 37 C in a humidified atmosphere of 5% CO2. For hCG measurement in these transfected cells, the cells were grown until confluence, washed with PBS, and then replaced with serum-free medium. After 24 h, the spent media were collected, and hCG secreted by cultured cells was determined with an enzyme immunoassay test kit (ICN Pharmaceuticals, Orangeburg, NY). For LH/CG receptor expression analysis, spent maintenance medium of each sample was replaced with fresh medium containing different concentrations of hCG (30, 100, 300, 1,000, 3,000, and 10,000 mIU/ml). After the designated time period had elapsed, the cells were washed with PBS and immediately frozen at –80 C until use.

Protein extraction and Western immunoblot analysis

The transfected JAr cells were homogenized in 50 mM Tris, pH 7.6, containing 5 mM EDTA, 150 mM NaCl, 0.6% sodium dodecyl sulfate, and 0.1 mM phenylmethylsulfonylfluoride. Equal amounts of cell lysates were resolved on 12.5% sodium dodecyl sulfate-polyacrylamide gels without heating under nonreducing conditions and were blotted onto Hybond-P membranes (Amersham Biosciences, Little Chalfont, UK). After blocking of nonspecific binding sites, the membranes were probed with rabbit polyclonal antisera against either ß-subunit (1:1,000; Takara Shuzo Ltd., Shiga, Japan) or {alpha}-subunit (1:10,000; gift from Dr. Irving Boime). The membranes were visualized using Western Light Chemiluminescent Detection System (Tropix, Inc., Bedford, MA). Molecular weight marker proteins (Bio-Rad Laboratories, Hercules, CA) were loaded into adjacent lanes to determine molecular sizes. The signal of specific protein (e.g. hCG dimer, free hCGß, and free hCG {alpha}) was scanned and determined by dividing its signal intensity by that of the corresponding total protein to correct for any loading difference between lanes (30). The intensity of the bands was quantified using spot densitometry software (Alpha Innotech, Inc., San Leandro, CA).

RT-PCR

Total RNA was isolated from the cultured JAr cells using an RNeasy Mini Kit (Qiagen, Max Volmer Strausse, Hilden, Germany), then cDNA was synthesized from 2 µg total RNA using a reverse transcriptase system (Promega Corp.), and amplification of the cDNA was performed with a PerkinElmer Thermal Cycler (GeneAmp PCR Instruments Systems, Roche, Branchburg, NJ).

The primers used for CGß were: upstream primer, 5'-CCTCAGGTGGTGTGCAACTA-3'; and downstream primer, 5'-ATTGTGGGAGGATCGGGGTG-3'. Those used for LH/CG receptor were: upstream primer, 5'-GCCTCTGAATGGACTCTAGG-3'; and downstream primer, 5'-TCTCAGATTGATTCCCTGGA-3'. Amplification conditions for the CGß gene were as follows: denaturation at 94 C for 1 min, annealing at 60 C for 1 min, and extension at 72 C for 1 min. The reactions were subjected to 24 cycles. For LH/CG receptor gene, denaturation was performed at 94 C for 1 min, annealing at 58 C for 1 min, and extension at 72 C for 2 min, for 35 cycles. The neomycin-resistant gene was used as an internal control to check the transfection efficiency. For the neomycin resistance gene, the primers were: upstream primer, 5'-CGGTGCCCTGAATGAACT-3'; and downstream primer, 5'-ACCGGCTTCCATCCGA. Denaturation was performed at 95 C for 1 min, annealing at 60 C for 1 min, and extension at 72 C for 1 min, for 30 cycles. All amplified products were separated in 2.0% agarose gels, and the bands were visualized with ethidium bromide staining.

Determination of cell viability and proliferation

The number of viable cells was determined on d 2, 4, and 6 of culture using a 3-[4,5-dimethlthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) Cell Growth Assay Kit (Chemicon International, Inc., Temecula, CA). The assay is based on the cleavage of tetrazolium salt MTT to a blue formazan product by mitochondrial dehydrogenase present in viable cells. At the end of the designated periods, 10 µl MTT (5 mg/ml) was added to the various samples in each well of a 96-well culture dish and incubated at 37 C for 4 h. Then, 100 µl isopropanol was added to each well and mixed thoroughly by repeated pipetting using a multichannel pipettor. The absorbance was measured on an MTP-120 ELISA plate reader (Corona Electric Co., Osaka, Japan) with a test wavelength of 570 nm and a reference wavelength of 630 nm.

Replicating DNA of the cultured JAr cells were simultaneously monitored using a BrdU cell proliferation kit (Amersham Biosciences). Briefly, 103 cells of the various groups of JAr cells were cultured in chamber slides. At the end of the designated days of culture, the spent media were removed, and the cells were labeled with fresh medium containing 1:1000 aqueous BrdU. The JAr cells were allowed to incubate for 30 min at 37 C, washed with PBS, and then fixed. Rehydration was performed with PBS, followed by addition of 100 µl anti-BrdU antibody to the cells. After 1 h of incubation at room temperature, the cells were washed with PBS, and 100 µl peroxidase-conjugated antimouse IgG2a was added to the cells for 30 min at room temperature. The JAr cells were then washed and immersed in staining solution containing 3'3-diaminobenzidine. PBS was used in place of BrdU antibody as a negative control.

Determination of apoptosis

Apoptotic cells were identified by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA (ApopTag In Situ Apoptosis Detection Kit, Intergen Co., Purchase, NY) using terminal deoxynucleotidyl transferase deoxyuridine triphosphate (TdT) nick end labeling (TUNEL) following the manufacturer’s instructions. JAr cells cultured in the chamber slides on d 2, 4, and 6 were treated with TdT enzyme and washed to terminate the reaction, then two drops of antidigoxigenin peroxidase were added, and peroxidase activity was detected with 3,3-diaminobenzidine substrate. H2O2 (2%) in PBS was used to quench endogenous peroxidase activity. Distilled water was used to replace TdT enzyme for the negative control. A section of rat mammary glands obtained 4 d after weaning was included as a positive control. More than 1000 JAr nuclei were counted for each experimental group in determining the mean percentage of TUNEL-positive nuclei in JAr cells.

At the end of the designated culture period, the JAr cells were treated with trypsin (0.05% trypsin and 0.53 mM EDTA) for 1.5 min, fixed in 4% neutral buffered formalin, and then washed in PBS. The cells were resuspended in Hoechst 33258 nuclear staining solution (0.1 µg/ml; Sigma-Aldrich Corp., St. Louis, MO) for 10 min and placed onto slides for microscopic determination of apoptotic bodies. Nuclear staining was observed using an LSM 5 Pascal Laser Scanning microscope (Carl Zeiss AIM GmbH, Jena, Germany) under x200 magnification. Typical apoptotic nuclei with nuclear condensation, fragmentation, and shrinkage in the JAr cells were identified and counted. A minimum of 200 cells/group were counted using randomly selected fields.

Statistical analysis

Results were expressed as the mean ± SEM of three independent experiments. Statistical analyses were carried out by one- or two-way ANOVA and post hoc Tukey test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Screening of the transfected JAr cells expressing hCGß was performed using Western dot-blot analysis (results not shown). The cells revealing high suppression of hCGß synthesis by introduction of antisense CGß cDNA were selected (JAr-aCGß1, -5, and -6) and used in the subsequent experimental procedures.

hCG secreted for 24 h by JAr-aCGß cells, untransfected JAr cells, and JAr-pM2AH cells was measured by enzyme immunoassay (Table 1Go). Compared with the untransfected JAr cells and JAr-pM2AH cells, the levels of hCG secreted by JAr-aCGß cells were significantly lower.


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  		TABLE 1. The levels of hCG secreted for 24 h by untransfected JAr, JAr-pM2AH and JAr-aCGß cells

 
To confirm the presence of hCGß and hCG{alpha} in the cell lysates, Western immunoblot analysis was performed using JAr-aCGß cells, untransfected JAr cells, and JAr-pM2AH cells. Immunoreactive bands with molecular masses of approximately 34 and 50 kDa, corresponding to free hCGß and hCG dimer, respectively, were observed in all samples (Fig. 1AGo). Lower signal intensities of both free hCGß and hCG dimer were noted in JAr-aCGß cells compared with those in the controls, including untransfected JAr cells and JAr-pM2AH cells, despite loading equal amounts of protein in each lane. Densitometric analyses of the bands indicated that the hCGß secretion was significantly lower in JAr-aCGß cells than in the controls. Regarding the effect of transfection of antisense CGß on the production of hCG{alpha} from these JAr cells, no significant differences in signal intensities were observed among the JAr-aCGß cells when free hCG{alpha} (~22 kDa) was determined, whereas the signal intensities of the hCG dimer decreased (Fig. 1BGo).



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  		FIG. 1. Effect of antisense CGß transfection on the expression of hCG dimer and of free hCGß (A) and free hCG{alpha} (B) in JAr cells. JAr cells were transfected with either antisense CGß (JAr-aCGß) or an empty vector (JAr-pM2AH). The cells were collected for Western immunoblot analysis for hCGß and hCG{alpha}. Representative filters (upper panels) and densitometric scans (lower panels) are shown. Data represent the mean ± SEM of three independent experiments. *, P < 0.05 (compared with untransfected JAr).

 
When RT-PCR with specific primers for either hCGß or LH/CG receptor was performed using RNA extracted from JAr-aCGß cells, untransfected JAr cells, and JAr-pM2AH cells, a predicted 280-bp PCR product for hCGß was observed in each sample (Fig. 2AGo). For the expression of hCGß mRNA, the band intensities in JAr-aCGß cells were lower than those in untransfected JAr cells and JAr-pM2AH cells. In contrast, a 501-bp product was seen in each sample, corresponding to the predicted cDNA for LH/CG receptor (Fig. 2BGo). Human embryonic kidney 293t cells expressing the LH/CG receptor (293-LHr) were used as a positive control during determination of LH/CG receptor. When reverse transcriptase was omitted from all samples, no band was apparent. The JAr-aCGß cells without hCG showed lower intensities of the band compared with the controls. However, treatment with increasing amounts of hCG resulted in significant increases in LH/CG receptor mRNA expression in JAr-aCGß cells compared with untreated JAr-aCGß cells, beginning at a dose of 1000 mIU/ml hCG, in a dose-dependent manner (Fig. 3Go). The same findings were observed after all designated days of culture.



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  		FIG. 2. Expression of CGß mRNA (A) and LH/CG receptor mRNA (B) in JAr-aCGß cells. RT-PCR products for hCGß (280 bp) and LH/CG receptor (501 bp), respectively, are shown in each lane (lanes 2–6) of the upper panels. The lower panels show RT-PCR products for ß-actin (590 bp) obtained from the same RNA preparations. MW, Molecular weight (lane 1), pBR322 was used as a size marker. Untransfected JAr cells and JAr-pM2AH cells were used as the controls in A and B; 293-LHr was also used as a control in B.

 


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  		FIG. 3. Effects of increasing doses of exogenous hCG on the expression of LH/CG receptor mRNA in JAr-aCGß cells. This representative figure shows d 2 of culture. Data represent the mean ± SEM of three independent experiments. *, P < 0.05 (compared with untreated JAr-aCGß).

 
Analysis of RT-PCR products of the neomycin-resistant gene (neo) showed that the amplified DNA bands were about 292 bp, as expected (results not shown). The amplified PCR products of the neo gene were seen only in JAr-aCGß and JAr-pM2AH cells, whereas no such bands were amplified in untransfected JAr cells.

MTT assay of JAr-aCGß cells showed an increase in viable cell number from d 2 to 4 of culture, which then rapidly declined on d 6 of culture (Fig. 4AGo). Compared with untransfected JAr cells and JAr-pM2AH cells, JAr-aCGß cells showed a significant decrease in cell viability on d 6 of culture.



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  		FIG. 4. Effects of exogenous hCG on cell viability and proliferation of JAr-aCGß cells. Cell viability and proliferation were determined by MTT assay (A, C, and D) and BrdU incorporation (B), respectively, on d 2 ({blacksquare}), d 4 ({square}), and d 6 ( {permzspch022}) of culture. A, Without hCG; C, with 300 mIU/ml hCG; D, with 1000 mIU/ml hCG. Values represent the mean ± SEM of three separate experiments. + and *, P < 0.05 (compared with untransfected JAr on d 4 and 6 of culture, respectively).

 
To assess the effects of transfection of antisense CGß on cell proliferation, BrdU incorporation into the nuclei in the different groups of JAr cells was observed. Compared with untransfected JAr cells and JAr-pM2AH cells, JAr-aCGß cells demonstrated a significant decrease in BrdU incorporation in the nuclei on d 4 and 6 of culture (Fig. 4BGo). Addition of exogenous hCG at doses of 300 mIU/ml (Fig. 4CGo) and 1000 mIU/ml (Fig. 4DGo) resulted in an increase in the cell viability of JAr-aCGß cells in a dose-dependent manner, and at a dose of 1000 mIU/ml, the cell viability of JAr-aCGß cells was not significantly different from that of JAr-pM2AH cells.

TUNEL analysis showed that a significant increase in the TUNEL-positive rate of JAr-aCGß cells was observed on d 2, 4, and 6 of culture compared with the controls (Fig. 5AGo). Nuclear staining using Hoechst 32258 also revealed an increase in apoptotic nuclei-positive rate in JAr-aCGß cells compared with that in untransfected JAr cells and JAr-pM2AH cells (Fig. 5BGo). A significant increase in the apoptotic nuclei-positive rate in JAr-aCGß cells was noted beginning on d 4 of culture and thereafter, compared with that in either of the controls.



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  		FIG. 5. Effect of antisense CGß transfection on the apoptosis-positive rate. A, TUNEL-positive rate of JAr-aCGß cells assessed by TUNEL analysis. B, Apoptotic nuclei-positive rate assessed by Hoechst nuclear staining. The number of apoptosis-positive nuclei in JAr-aCGß cells was greater than those in untransfected JAr cells and JAr-pM2AH cells. Values represent the mean ± SEM of three separate experiments. {diamondsuit}, +, and *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
hCG belongs to a family of a glycoprotein hormones characterized by a heterodimeric structure with a common {alpha}-subunit noncovalently bound to the ß-subunit, the latter being hormone specific (11). Only the heterodimer is functional, and genes 5, 3, and 8 are transcriptionally active (31, 32). Cystine knots are scaffolds in the subunits that play a role in heterodimer formation (28), and the biologically active form of cystine knot-containing proteins is either a homo- or heterodimer (33). Subunits of the cystine knot family, which exist as homodimers, such as brain-derived neurotropic factor and neurotrophin-3, had been found to form biologically active heterodimers (34), suggesting that common {alpha}- or ß-subunits may form dimers with subunits of other proteins that have a cystine knot motif. This may explain why the free ß-subunit exhibits a growth factor effect on a bladder cancer cell line (19), where it could form a dimer with monomeric growth factors and then bind to a receptor existing on those tumor cells. The ß-subunit homodimer molecule binds the hLH/CG receptor with an affinity approximately 3 orders of magnitude lower than urinary or recombinant hCG, and cAMP response studies indicate that the ß-subunit homodimer can elicit as high a cAMP response as hCG at concentrations at which it can bind the receptor (35). They also observed the spontaneous formation of ß-subunit homodimer molecules in vitro from free ß-subunit. There is a possibility that high hCG ß-subunit concentrations could result in the formation of homodimers that could potentially mimic heterodimers due to conformation similarity. A reduction in hCG synthesis was found to have no effect on cell proliferation in human choriocarcinoma JAr cells (24). Furthermore, Lei et al. (36) demonstrated that the tumorigenic effects of hCG did not include an increase in cell proliferation, but, rather, decreased apoptosis and increased the ability of cell invasion of JAr cells in nude mice.

Our investigation has shown that stable transfection of the antisense CGß gene into JAr choriocarcinoma cells suppressed both hCGß mRNA expression and hCGß protein synthesis. Introduction of the plasmid-bearing antisense CGß mRNA successfully led to the production of double-stranded RNA that cannot transcribe the CGß protein. Efficient assembly of the free hCG ß- and {alpha}-subunits was inhibited due to a decrease in the level of hCGß in JAr-aCGß cells, which subsequently led to an expected decrease in the synthesis of the hCG dimer. No decrease in free hCG{alpha} mRNA expression or hCG{alpha} protein synthesis, however, was noted.

The transfection of the antisense CGß gene resulted in a significant inhibition of cell proliferation and a significant increase in the apoptosis-positive rate of JAr cells. JAr-aCGß cells showed diminished cell growth and more abundant apoptotic nuclei compared with those in the controls. However, full recovery of viable cells in JAr-aCGß cells was obtained by the addition of exogenous hCG to the medium starting at a dose of 1000 mIU/ml in a dose-dependent manner. The concentrations of hCG used in this investigation to stimulate these transfected JAr cells were similar to those found in the sera of patients with malignant trophoblastic tumors (37). This suggests that the production of enormous amounts of hCG in choriocarcinoma may be an adaptive mechanism by which the malignant tumor cells ensure their continued growth and survival. The transfection procedure alone was not responsible for the decreased proliferation of JAr-aCGß cells, because simultaneous transfection of empty pM2AH was performed. No significant differences in hCG secretion, hCGß and LH/CG receptor mRNA expression, cell proliferation, or apoptosis-positive rates were noted between untransfected JAr cells and JAr-pM2AH cells throughout the present study.

To complete the circuit of the proposed theory based on an autocrine/paracrine action of hCG on choriocarcinoma cell growth, the role of the LH/CG receptor was investigated by observing the effect of transfection of antisense CGß on LH/CG mRNA expression in JAr cells. A previous study showed that JAr choriocarcinoma cells were unable to self-regulate hCG biosynthesis (20). The investigators showed that the addition of highly purified hCG at concentrations greater than 1000 mIU/ml decreased hCG subunit mRNA expression as well as hCG dimer protein content in cultured normal placental trophoblasts and term placental tissues, whereas no effect was observed when JAr cells were exposed to the same concentrations of hCG. Consistent with their findings, the present study showed that the addition of increasing doses of exogenous hCG to untransfected JAr cells had no effect on LH/CG receptor mRNA expression. However, in JAr-aCGß cells, the addition of hCG resulted in a significant increase in LH/CG receptor mRNA expression compared with that in untreated JAr-aCGß cells beginning at a dose of 1000 mIU/ml hCG. Addition of the same doses of hCG had no effect on the expression of LH/CG receptor mRNA in the controls, including 293-LHr cells, untransfected JAr cells, and JAr-pM2AH cells. With concentrations greater than 1000 mIU/ml hCG, cell viability as well as LH/CG receptor mRNA expression in the JAr-aCGß cells reached a plateau.

It is therefore likely that enormous amounts of hCG produced by choriocarcinoma cells may contribute to the uncontrolled growth of those cells due to the intrinsic lack of self-regulation and the high expression of LH/CG receptors in those cells. It seems that 1000 mIU/ml hCG may be a critical threshold concentration for cell viability via the interaction between hCG and LH/CG receptors.

Lei et al. (36) demonstrated that antisense hCG {alpha}-subunit cDNA transfection resulted in an reduction in hCG {alpha}-subunit mRNA and dimer hCG protein levels, an increase in apoptosis, a decrease in cell invasion, and a decreased incidence of tumor formation in female nude mice. In this context, a meaningful suggestion was made that the loss of tumorigenic properties of JAr cells after the inhibition of their hCG synthesis could be restored by the addition of exogenous hCG. This approach should theoretically work, provided that hCG can get to its relevant cellular sites of action (36).

Our present study showed that inhibition of the CGß gene by transfection of antisense CGß interfered with the production of hCG dimer as well as free hCGß by JAr choriocarcinoma cells and significantly decreased the cell viability and induced apoptosis in those cells. Moreover, our study suggests that the antisense CGß transfection down-regulates LH/CG receptor transcription in JAr cells, and that hCG concentrations between 300-1000 mIU/ml up-regulate LH/CG receptor mRNA expression in those cells. It is, therefore, likely that the introduction of antisense CGß may be a novel strategy for controlling choriocarcinoma cell proliferation.


   Acknowledgments
 
We thank Dr. Irving Boime (Washington University, St. Louis, MO) for his kind gifts of the recombinant plasmids and rabbit antiserum used in the present studies. We also thank Prof. Benjamin Tsang (University of Ottawa, Ottawa, Canada) for his valuable suggestions on this research.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research 12770913 and 16790953 from the Japan Society for the Promotion of Science and by the Ogyaa-Donation Foundation of the Japan Association of Maternal Welfare.

First Published Online May 10, 2005

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; hCG, human chorionic gonadotropin; MTT, 3-[4,5-dimethlthiazol-2-yl]-2,5-diphenyltetrazolium bromide; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling.

Received December 15, 2004.

Accepted May 2, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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