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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 2 427-431
Copyright © 1998 by The Endocrine Society

Original Studies

Evidence for Tight Coupling of Gonadotropin-Releasing Hormone Receptor to Stimulated Fas Ligand Expression in Reproductive Tract Tumors: Possible Mechanism for Hormonal Control of Apoptotic Cell Death1

Atsushi Imai, Atsushi Takagi, Shinji Horibe, Hiroshi Takagi and Teruhiko Tamaya

Department of Obstetrics and Gynecology, Gifu University School of Medicine, Gifu 500, Japan

Address all correspondence and requests for reprints to: Atsushi Imai, M.D., Department of Obstetrics and Gynecology, Gifu University School of Medicine, Tsukasamachi, Gifu 500, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fas ligand induces cell death by means of apoptosis in a variety of cell types when cross-linked with its natural receptor, Fas. GnRH receptor-bearing tumors undergo apoptosis in vivo and in vitro with GnRH agonists. To provide a potential association of the Fas system with the antiproliferative signaling process of GnRH receptor, we have evaluated the regulation of Fas ligand expression in GnRH receptor-positive tumors and cloned cell lines known to have substantial GnRH receptors. Surgically removed uterine endometrial carcinomas and ovarian carcinomas had been screened for GnRH receptor expression before analysis. Fas ligand protein was characterized by immunoblotting of membrane proteins with the specific antibody. Fas ligand messenger RNA was determined by RT-PCR using oligonucleotide primers synthesized according to the published Fas ligand sequence. Incubation with a GnRH analog (1 µmol/L) induced the expression of Fas ligand messenger RNA and immunoreactive Fas ligand with a lag time of 48 h in cloned cell lines (endometrial carcinoma HHUA cells, and ovarian carcinoma SK-OV-3 and Caov-3 cells). There was no detectable Fas ligand expression within 24 h. The stimulatory effect of GnRH on Fas ligand protein expression revealed a dose dependency; a half-maximal effect occurred with 10 nmol/L GnRH analog (P < 0.01). The stimulated Fas ligand expression could be neutralized by displacement of GnRH from its receptor by GnRH antagonist antide. Cells isolated from GnRH receptor-bearing ovarian carcinomas and uterine endometrial carcinomas gave identical results to those obtained in cloned cell lines. These data demonstrate the functional coupling of stimulated Fas ligand expression to GnRH receptor activation. Increased Fas ligand level within the GnRH receptor-bearing tumors might promote apoptotic cell death through attack on intratumoral Fas-positive cells that could, at least in part, account for the antiproliferative action of the hormone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SYNTHETIC agonists of GnRH have direct antiproliferative effects on certain hormone-sensitive tumors, including carcinoma of the endometrium, the ovary, the breast, and the prostate in vitro and in vivo (1, 2). GnRH receptor has been demonstrated in the GnRH-sensitive tumors arising in reproductive organs (3, 4, 5), supporting the concept that GnRH receptor might mediate the direct antitumor effects of GnRH analog. Molecular relationship of its receptor to the growth-inhibiting activity, however, remains unclear.

The GnRH-induced antiproliferation may proceed by stimulated apoptotic (programmed) cell death (6, 7). A cell surface receptor protein, Fas, triggers apoptosis in a variety of cell types when cross-linked with its natural ligand, Fas ligand (8, 9). The similar characteristics in apoptosis of different cell types suggest that the mechanism of apoptosis may be common to all cell types. GnRH receptor reveals characteristics of the family of guanosine triphosphate (GTP)-binding protein-coupled receptors that possess seven putative transmembrane domains (10), whereas Fas is a single-chain polypeptide with a single transmembrane domain (11). However, cellular responses mediated by GnRH receptor or Fas include enhanced apoptotic cell death, and thus, these two may share the same intracellular signal transduction pathway. In fact, recent data have demonstrated the involvement of the tyrosine kinase/phosphatase system in signal transduction in the activation of the Fas ligand gene through the T cell receptor (8), as well as the action of GnRH on GnRH receptor-bearing tumors (12, 13, 14). These findings prompted us to examine the possible role of the Fas-Fas ligand system on the antiproliferative signaling process of GnRH receptor.

In the course of characterization of Fas (Fas ligand receptor) distribution (15), we found that GnRH induces intratumoral expression of Fas ligand in the GnRH-sensitive carcinoma cells. The local Fas ligand expression, responsive to a certain hormonal stimulus, may provide a potential mechanism through which tumors arising in reproductive organs undergo apoptosis with the hormone manipulation. Here we describe tight coupling of intratumoral production of Fas ligand to GnRH receptor activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

GnRH agonists, buserelin and leuprolide, were gifts from Hoechst Japan (Tokyo, Japan) and Takeda Pharmaceutical (Osaka, Japan), respectively. GnRH antagonist, antide, and TRH were purchased from Sigma Chemical (St. Louis, MO). Antibody PC78 against Fas ligand was obtained from Calbiochem (Cambridge, MA). The messenger RNA (mRNA) purification kit and nitrocellulose membranes were products of Pharmacia-LKB (Uppsala, Sweden). Moloney murine leukemia RT and Taq polymerase were from Takara-Shuzo (Tokyo, Japan). Hybond-N+ membranes, ECL-direct nucleic acid labeling and detection system, and sequence kit were from Amersham (Amersham, UK). Dispase was from Boehringer Mannheim (Indianapolis, IN). All other chemicals were of reagent grade.

Tissue collection and cell cultures

Reproductive tract tumor specimens were placed in ice-cold PBS immediately after surgical removal, and representative portions were excised to prepare the materials for histological frozen sections. These tissue samples were washed and immediately minced in RPMI 1640 medium with dispase, as previously described (12), and the cells were plated in RPMI 1640 medium with 10% FBS in the presence of agents to be tested. Only tissues obtained at the initial surgery were used for analyses. We screened the surgical samples for the presence of GnRH binding sites and GnRH receptor mRNA, as described previously (3, 12). Of a total of six samples (three endometrial carcinomas and three ovarian carcinomas), five (three endometrial carcinomas and two ovarian carcinomas) had obvious GnRH receptor. Clinical data and the results of GnRH receptor analyses of all tumors submitted to the experiments in this study are summarized in Table 1Go. The investigation had the approval of the Gifu University Research Ethics Committee, and all patients gave informed consent to the disposition of their surgically removed tissues. Aside from the diagnosis of gynecological tumor, these patients were free of endocrine or systemic diseases.


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  		Table 1. Summary of reproductive tract tumors screened for Fas ligand expression

 
Human endometrial carcinoma cell line (HHUA) and ovarian carcinoma cell lines (SK-OV-3 and Caov-3) were obtained from American Type Culture Collections (Rockville, MD); these cell lines are GnRH receptor-positive (3, 12). The cells were grown at 37 C in an appropriate medium containing FBS in a humidified atmosphere of 5% CO2/95% air. To examine hormonal effects, all cell lines and cells from surgically removed tumors were grown to confluence in multiwell plates and were treated with the indicated concentrations of test agents for the desired time intervals.

Plasma membrane isolation

The procedure described previously (12, 16) was used to obtain highly purified plasma membrane fractions from the specimens. The isolated plasma membranes were immediately submitted to the following experiments. The SA of a marker enzyme 5'-nucleotidase in the plasma membrane fraction was increased approximately 6- to 9-fold, when compared with the homogenate (1–3 µmol/mg protein·h).

Immunoblotting

Immunoblotting was carried out, as described previously (13, 14), using the PC78 antibody raised against extracellular regions of Fas ligand. The primary antiserum was used at 1:1,000 dilution for 1 h at room temperature. Biotin-labeled antimouse whole antibody from sheep was used as a second antibody to develop the immunoblots.

RT-PCR amplification

Total RNA was extracted from cell pellets, according to the guanidine isothiocyanate/acid phenol method (17), and poly(A) RNA was selected by oligo(dT) cellulose column chromatography, according to the manufacturer’s instructions.

A random primed complementary DNA (cDNA) library was obtained from 200 ng of each of the poly(A) RNA prepared, using the Moloney murine leukemia RT under the conditions recommended by the supplier. The cDNA reaction (25 µL) was diluted with 300 µL water, was heat-denatured at 95 C for 5 min, and was quickly chilled on ice. The cDNA (1 µL) was amplified in a 50-µL reaction buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2 mmol/L MgCl2, 50 µmol/L each deoxynucleotide triphosphates, 1.25 U Taq polymerase, and 0.5 µmol/L primers (3, 18). The sequences of oligonucleotide primers, synthesized according to the published human Fas ligand sequence (19), were as followed: primer a (sense): 5'-TTCTTCCCTGTCCAACCTCT-3' (150–169), primer b (sense): 5'-CGCCACCACTGCCTCCACTA-3' (243–262), primer c (antisense): 5'-CTCATCATCTTCCCCTCCAT-3' (737–756), primer d (antisense): 5'-CTTCCCCT-CCATCATCACCA-3' (729–748). One tube contained each of the upstream and downstream primers, with a predicted DNA fragment of 607 bp (primers a and c), 599 bp (a and d), 514 bp (b and c), or 506 bp (b and d), respectively. Each primer set was designed to flank the entire transmembrane domain. We carried out 35 cycles of amplification; denaturation at 95 C for 20 sec, annealing at 55 C for 20 sec, and extension at 72 C for 20 sec followed by a final extension for 10 min at 72 C. The DNA product (10 µL) was run on 2% agarose gels, and bands were visualized by ethidium bromide staining on an ultraviolet transilluminator. Sequence of the PCR products was analyzed and confirmed manually by the dideoxy chain termination method (20) using Sequenase II according to the manufacturer’s protocols.

Statistics

Statistical analysis was performed by t test. Differences were considered significant if P was less than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH-induced Fas ligand mRNA expression

PCR amplification of first-strand cDNA from human endometrial and ovarian carcinoma cells and their cloned cell lines was conducted with four sets of oligonucleotide primers, as described in Materials and Methods. As shown in Fig. 1Go, A and C, when incubated with a GnRH agonist buserelin (1 µmol/L) for 48 h, ovarian carcinoma cell line SK-OV-3 yielded an expected amount of products, whereas there was no detectable product at 24 h. Similar results were obtained with another GnRH analog leuprolide (data not shown). Control peptide, TRH, had no effect (Fig. 1AGo).



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  		Figure 1. PCR amplification of first-strand cDNA from endometrial carcinomas and ovarian carcinomas and their cloned cell lines. A, SK-OV-3 (ovarian carcinoma) cells were incubated for indicated intervals with a GnRH agonist buserelin (1 µmol/L) or for 48 h with TRH (1 µmol/L) (lane C). B, Caov-3 (ovarian carcinoma) cells, HHUA (endometrial carcinoma) cells, cells isolated from surgically removed ovarian carcinomas (patients 4 and 5 of Table 1Go), and from endometrial carcinoma (patient 2 of Table 1Go) were incubated for 48 h with a GnRH agonist, buserelin (1 µmol/L). Oligonucleotide primers a and c, described in Materials and Methods, were used. C, Amplification profiles of first-strand cDNA from SK-OV-3 cells, incubated for 48 h with buserelin (1 µmol/L), using primers a and d, b and c, and b and d. Gels were stained with ethidium bromide, and bands were visualized with ultraviolet light.

 
The identical results also were obtained in two cell lines, including ovarian carcinoma (Caov-3) cells and endometrial carcinoma (HHUA) cells, and all surgically removed specimens, including three endometrial carcinomas and two ovarian carcinomas. Clinical data of these tumors and their expression of Fas ligand are summarized in Table 1Go; GnRH had no effects in cells from GnRH receptor-negative tumor. A representative profile of these is given in Fig. 1BGo.

GnRH-induced Fas ligand protein expression

The GnRH receptor-bearing tumors were then examined for a presence of immunoreactive Fas ligand. Immunoblotting with the specific antibody, PC78, detected a band of 40 kDa in SK-OV-3 cells exposed to GnRH analog buserelin (1 µmol/L) with a lag time of 48 h, as shown in Fig. 2AGo. No significant Fas ligand protein was expressed within 24 h. Figure 3Go shows the dose-response characteristics of these effects of buserelin on SK-OV-3, endometrial carcinoma HHUA cells, and isolated ovarian carcinoma cells of surgically removed specimen (patient 4 of Table 1Go). The stimulatory response to buserelin of Fas ligand protein expression was dose dependent; a half-maximal effect occurred at approximately 10 nmol/L in SK-OV-3 and HHUA cells. The representative profile of the dose-related immunoblotting was illustrated in Fig. 2BGo. The GnRH’s effect was abolished by the addition of 10 µmol/L GnRH antagonist antide (Fig. 2BGo), a dose that displaces virtually all of the bound GnRH from its receptor under these conditions (21). An identical result was obtained with another GnRH agonist, leuprolide.



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  		Figure 2. Effects of exposure to a GnRH analog on Fas ligand protein level in ovarian carcinoma SK-OV-3 Cells. A, The cells were incubated for indicated intervals with buserelin (1 µmol/L), and plasma membranes were isolated; B, the cells were incubated for 48 h with increasing concentrations of buserelin (1 µmol/L) alone or buserelin plus antide (10 µmol/L). Fas ligand was resolved by SDS-PAGE and subsequent immunoblotting with specific antibody PC78. Molecular sizes are shown at the left (in kilodaltons). Shown are representative profiles of three separate experiments that gave similar results.

 


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  		Figure 3. Dose effect of GnRH analog buserelin on Fas ligand protein expression in HHUA cells, SK-OV-3 cells, and isolated ovarian carcinoma cells. Plasma membranes were isolated from HHUA (endometrial carcinoma) cells, SK-OV-3 (ovarian carcinoma) cells, or ovarian carcinoma cells (patient 4 of Table 1Go) that had been incubated for 48 h with increasing concentrations of buserelin. Fas ligand was resolved by SDS-PAGE and subsequent immunoblotting with specific antibody PC78. Each point is expressed as percent of maximal densitometric presentation and represents means ± SD of four separate experiments. *, P < 0.01 vs. control point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Physiologic cell death occurs primarily through an evolutionarily conserved form of cell suicide, apoptosis. Hormone-responsive tumors undergo apoptosis in vivo and in vitro with the appropriate stimulus (7, 8, 9). Increased expression of Fas ligand, an intrinsic inducer of cell death, by a certain hormone in the hormone-sensitive tumors could impact on the promoting tumor cell apoptosis. In the current study, we demonstrated that GnRH distinctly stimulated intratumoral Fas ligand expression in GnRH-responsive tumors by two separate approaches: 1) immunoreactive Fas ligand protein expression in the isolated plasma membranes; and 2) the detection of Fas ligand mRNA.

The Fas ligand has four potential N-glycosylation sites, which seem to be variably used (22, 23). Because of the variation in glycosylation, the full-length protein migrates as several distinct molecular-weight species. The highest degree of homology with Fas ligand forms occurs in the extracellular domain corresponding to the carboxylterminal portion (22). Anti-Fas ligand antibody (PC78) used in the present study is raised against the extracellular domain corresponding to amino acid residues 261–277. Using immunodetection with the specific antibody, we identified a substantial level of Fas ligand in isolated membranes of the endometrial carcinoma and ovarian carcinoma specimens and their cell lines that had been exposed to a GnRH analog for 48 h. Fas ligand mRNA was detected by RT-PCR amplification, based on the generation of identical PCR-amplified products to authorized Fas ligand cDNA. For amplification, four separate sets of primers were used; each set is designed to flank the nucleotide sequence encoding the entire transmembrane domain. The GnRH-responsive products had the predicted sizes, and they included complementary sequences confirmed by direct sequencing of the PCR products. Although surgically removed tumor specimens may contain some T cells, Fas ligand was not detected by PCR amplification 24 h after GnRH treatment. The expression of Fas ligand in T cells could be induced only by activation with a certain stimulus or T cell receptor engagement (24), and it is likely that resting T cells present in tumor specimens express no Fas ligand. In experiments with antide, the GnRH-stimulated Fas ligand expression was neutralized by the competitive antagonist; a dose of antide used in this study can replace the previously bound GnRH from its receptor under these conditions (21). These findings allow us to draw two important conclusions: 1) the hormonal activation of Fas ligand expression was mediated through GnRH receptor; and 2) GnRH-responsive Fas ligand was characterized as membrane protein (expressed in plasma membrane), although the Fas ligand is also cleaved from the surface of the cell and released as a soluble form (23).

Among the various human cell types examined, only activated T cells (8) and cytotrophoblasts (25) express Fas ligand, whereas Fas is detected in a variety of normal and neoplastic cells (8, 11). Our present results show that a GnRH agonist can induce Fas ligand production in GnRH receptor-bearing ovarian carcinoma cells and endometrial carcinoma cells. Apoptotic cell death via Fas requires the cross-linking of Fas, either with antibodies to Fas (with cell-expressing Fas ligand) or with purified Fas ligand (26). We recently have demonstrated the frequent expression of Fas in such GnRH receptor-positive carcinoma cells but not in the GnRH receptor-negative tumors (15). The intratumoral production of Fas ligand could be suggested to prevent the cellular proliferation through apoptotic attack on the Fas-bearing cells present in GnRH receptor-positive tumors.

There are differences in the signal transduction pathways activated by GnRH receptors in the peripheral tumor vs. the anterior pituitary. We have demonstrated that GnRH receptor is coupled to pertussis toxin-sensitive GTP-binding proteins of the Gi family in reproductive tract tumors (13, 14). Recent studies (27, 28) have identified that members of the pertussis toxin-insensitive GTP-binding proteins of the Gq/G11 subfamily mediate the message from GnRH receptor in pituitary gonadotrophs. The difference of GTP-binding protein coupled to GnRH receptor may assess that the cellular responses (including Fas ligand expression) to GnRH in the peripheral tumor are distinct from that in the anterior pituitary.

Lastly, the present results suggest that Fas ligand appears in endometrial carcinoma and ovarian carcinoma on GnRH stimulation. It cannot be ruled out that the Fas ligand represents only a phylogenetic residue without functional importance, but the sensitivity of tumors to physiological control is strongly associated with the possession of receptors for the ligand. The Fas ligand expression linked to GnRH receptor activation may, at least partially, mediate the antiproliferative action of GnRH agonists by increasing apoptotic cell death within the tumor. Although our data were derived from experiments that used surgically removed reproductive tract tumors and cell lines, our findings of a hormone-induced Fas ligand expression may be applicable to all hormone-dependent carcinomas responding to stimuli that lead to apoptosis.


   Footnotes
 
1 This study was supported in part by Research Grant 07457383 from the Ministry of Education, Culture and Science, Japan. Back

Received August 29, 1997.

Revised October 7, 1997.

Accepted October 14, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Emons G, Schally AV. 1994 The use of luteinizing hormone releasing hormone agonists and antagonists in gynecological cancers. Human Reprod Update. 9:1364–1379.
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals