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Endocrinology, doi:10.1210/en.2003-0551
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Endocrinology Vol. 145, No. 1 194-204
Copyright © 2004 by The Endocrine Society

Gonadotropin-Releasing Hormone Receptor-Mediated Growth Suppression of Immortalized LßT2 Gonadotrope and Stable HEK293 Cell Lines

Lauren E. C. Miles, Aylin C. Hanyaloglu, Jasmin R. Dromey, Kevin D. G. Pfleger and Karin A. Eidne

Western Australian Institute for Medical Research, Center for Medical Research, Sir Charles Gairdner Hospital, and Animal Science Group, University of Western Australia, Nedlands, Perth, Western Australia 6009, Australia

Address all correspondence and requests for reprints to: Prof. Karin A. Eidne, Western Australian Institute for Medical Research, B Block, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Perth, Western Australia 6009, Australia. E-mail: keidne{at}waimr.uwa.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Continuous administration of GnRH analogs results in an inhibition of tumor growth that may be mediated in part by direct activation of GnRH receptors (GnRHRs) expressed on tumor cells. However, it is not fully understood how the GnRHR mediates these growth effects. This study aimed to determine how the presence or absence of this receptor in different cell types might affect the ability of GnRH to directly mediate growth effects. We demonstrate that continuous treatment with GnRH or a GnRH agonist (GnRHA) induces an antiproliferative effect in a gonadotrope-derived cell line (LßT2) and also in HEK293 cells stably expressing either the rat or human GnRHR. The antiproliferative effect was time and dose dependent and was verified using [3H]thymidine incorporation, light microscopy, and analysis of cell number. Inhibition was specifically mediated via the GnRHR, as cotreatment of the GnRHR-expressing cell lines with a GnRH antagonist blocked the growth-suppressive effect induced by GnRHA treatment. Cell cycle analysis revealed that GnRHA-treated HEK/GnRHR cell lines induced an accumulation of cells in the G2/M phase, whereas a G0/G1 arrest was observed in LßT2 cells. GnRHA treatment also caused a small, but significant, increase in apoptotic cells. This study provides evidence for a direct role for the GnRHR in mediating antiproliferative events in two cell systems, neither of which was derived from extrapituitary reproductive tumors. The ability to induce these effects, regardless of the cell system involved, has implications regarding the use of GnRH analogs for the treatment of endocrine-related disorders and tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH RECEPTORS (GnRHRs) belong to the G protein-coupled receptor (GPCR) superfamily and in the pituitary signal via G{alpha}q/11 G proteins and phospholipase C (1). The function of the hypothalamic decapeptide, GnRH and the GnRHR in human cancers has been the subject of intensive research. In addition to its classical role in reproduction, where it stimulates the release of both LH and FSH from pituitary gonadotropes, GnRH and its analogs have been postulated to affect cellular growth in cancers of the ovary, endometrium, prostate, and breast (2). The primary route of GnRH analog therapy for hormone-dependent cancers is via down-regulation of pituitary GnRHRs, where the tumor is effectively starved of the steroid support required for growth as a result of this hormonal castration (3). Our initial identification of GnRHR-binding sites on breast cancer cells (4, 5) led to the view that a more direct pathway via GnRHRs expressed in extrapituitary tissue could also contribute to this antiproliferative effect. Since then, GnRHRs have been detected in a wide variety of cancer cells, including those derived from breast, prostate, ovary, and endometrium, and numerous studies have documented growth inhibitory effects after treatment with GnRH analogs on a range of cancer cell lines (6, 7, 8, 9, 10). Such findings are controversial, as other studies have reported GnRH analogs to either have no effect on or to promote the growth of tumor cell lines previously documented to be inhibited by GnRH analog treatment (11, 12, 13, 14, 15).

As indicated above, research investigating direct growth effects of GnRH analogs and GnRHR activation has primarily focused on cell lines derived specifically from cancers or metastases of extrapituitary reproductive tissues. Although the gene encoding the GnRHR in extrapituitary reproductive tissues and tumor cells is structurally identical to that expressed in pituitary gonadotrope cells (16), there is sufficient evidence to suggest that the different functional effects on cellular growth could be a consequence of the different cellular environment (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Therefore, it is important to establish whether this antiproliferative effect is unique to these extrapituitary tumor cells. In particular, does a GnRHR-mediated antiproliferative effect occur in pituitary-derived gonadotrope cells despite the different cellular environment? Furthermore, is the phenomenon sufficiently noncell type dependent that it is exhibited in a totally unrelated cell type such as a human embryonic kidney cell line exogenously expressing GnRHRs? We have generated human embryonic kidney (HEK293) cells exogenously expressing either rat or human GnRHRs. These are particularly useful because they provide the opportunity to compare cell populations that only differ on the basis of GnRHR expression. Thus, any functional differences observed can be attributed to the presence or absence of this one protein.

This study has characterized a specific time- and dose-dependent GnRH agonist-induced antiproliferative effect in both an immortalized pituitary cell line (LßT2) and HEK293 cells stably expressing either rat or human GnRHRs. Looking for possible mechanisms that might be able to mediate these antiproliferative effects, we examined whether GnRH agonist had an effect on cell cycle arrest and/or apoptosis. Our findings show that both cell cycle arrest and apoptosis are likely to have a role in the growth-suppressive effects of GnRH agonists.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
GnRH and GnRH agonist (GnRHA; [D-Leu6,Pro9-Et]GnRH, also known as leuprolide acetate, Lupron, or Lucrin) were supplied by Abbott Australasia (Sydney, Australia). [D-Trp6,Pro9-Et]GnRH and the GnRH antagonist (GnRHAnt; [Phe(4Cl)2,D-Pal(3)3,Lys(Nic)5,D-Lys(Nic)6,Lys(iPr)8,D-Ala10]GnRH, known as antide) were obtained from Sigma-Aldrich Corp. (Sydney, Australia). Cyclohexamide and TNF{alpha} were purchased from Sigma-Aldrich Corp. 125I was purchased from Amersham Pharmacia Biotech (Sydney, Australia).

Cell culture
All cell lines were maintained in DMEM containing glutamine (0.3 mg/ml) and penicillin-streptomycin (100 U/ml; complete DMEM) supplemented with 10% fetal calf serum (FCS; Invitrogen Technologies, Perth, Australia) at 37 C in a humidified atmosphere of 5% CO2 in air. HEK293 cells stably transfected with the rat GnRHR (HEK/rGnRHR, receptor expressed at 0.96 pM/106 cells) (17) or modified human GnRHR (HEK/hGnRHR; patent WO 99/67292) have been previously characterized. Modification of hGnRHR does not alter receptor binding characteristics (compared with wild-type receptor), but increases receptor expression upon stable transfection (13.76 pM/106 cells; patent WO 99/67292). The immortalized mouse pituitary-derived cell line LßT2 (18) was a gift from Prof. Pamela Mellon (University of California, San Diego, CA) and exhibits GnRHR expression at 0.12 pM/106 cells (19).

Confocal imaging
The GnRHRs were epitope-tagged [either hemagglutinin (HA)-tagged at the NH2 terminal region or enhanced green fluorescence protein (EGFP)-tagged at the COOH terminus], and this allowed us to visualize receptor distribution in GnRHR-expressing cells before and after treatment with GnRHA. Cells were plated onto poly-L-lysine-coated eight-well chamber slides and treated 24 h later with GnRHA (1 h at 1 µM). Post treatment, cells were prepared for confocal microscopy, and the HA-tagged GnRHR or EGFP-tagged GnRHR was visualized as previously described (17, 20). Cells were examined using an MRC 1000/1024 UV confocal laser scanning microscope (Bio-Rad Laboratories, Richmond, CA) using a Nikon x60 NA 1.4 oil immersion objective (Melville, NY).

Whole cell binding assays
Dose displacement receptor binding assays using 125I-labeled [D-Trp6,Pro9-Et]GnRH (100,000 cpm/well) were performed in whole cells as previously described (20). Iodinated [D-Trp6,Pro9-Et]GnRH was prepared as previously described (21) using the lactoperoxidase method and was purified by chromatography on a Sephadex G-25 column in 0.01 M acetic acid/0.1% BSA. The specific activity was approximately 50 µCi/µg and was calculated as described previously (21). Cells were plated into 24-well plates and incubated with 125I-labeled [D-Trp6,Pro9-Et]GnRH (100,000 cpm/well) in DMEM containing HEPES buffer with 0.1% BSA at 4 C for 2 h. Cells were washed twice with PBS and solubilized in 0.2 M NaOH/1% sodium dodecyl sulfate. Nonspecific binding for each time point was determined under the same conditions in the presence of 10 µM unlabeled agonist. Radioactivity was determined using a Cobra II {gamma}-counter (Packard Instruments, Meriden, CT).

Receptor internalization assay
GnRHR internalization assays were performed as described previously (17). Briefly, cells in 24-well plates were incubated with 125I-labeled [D-Trp6Pro9-Et]GnRH (100,000 cpm/well) for 2 h at 37 C. Surface-bound radioactivity was removed by washing with acid solution (50 mM acetic acid and 150 mM NaCl, pH 2.8). Internalized radioactivity was determined after solubilizing cells in 0.2 M NaOH/1% sodium dodecyl sulfate. Nonspecific binding was determined under the same conditions in the presence of 1 µM unlabeled GnRHA. After subtraction of nonspecific radioactivity, internalized radioactivity was expressed as a percentage of the total binding. All time points were performed in duplicate in at least three separate experiments.

Total inositol phosphate (IP) assays
Total IPs were extracted and separated as described previously (22). Briefly, cells were plated into 24-well plates in complete DMEM supplemented with 10% FCS. Twenty-four hours later, medium was replaced with 0.5 ml inositol-free DMEM containing 1% dialyzed FCS/well and incubated for 24 h with [3H]myo-inositol (2 µCi/ml; Amersham Pharmacia Biotech). Medium was then removed, and cells were washed twice with buffer A (1 mg/ml fatty acid-free BSA, 140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM D-glucose, 1 mM MgCl, and 1 mM CaCl2), followed by incubation for 10 min with buffer A containing 10 mM LiCl with or without addition of GnRHA at 37 C for 60 min. The assay buffer was removed, and the cells were incubated at 4 C for 30 min with 10 mM formic acid. After transfer to tubes containing Dowex (AG 108) anion exchange resin (Bio-Rad Laboratories, Sydney, Australia), total IPs were eluted, and the amount of radioactivity was counted. All treatments were performed in triplicate in at least three separate experiments.

[3H]Thymidine incorporation assays
Cell proliferation was analyzed using [3H]thymidine incorporation assays. Cells were seeded in poly-L-lysine-coated, 24-well culture plates in complete DMEM supplemented with 10% FCS at approximately 10% confluence. After cells had adhered to the culture plate (4–6 h), medium was replaced with complete DMEM containing 5% FCS, with or without the appropriate peptides. Medium (containing fresh peptide) was replaced every 24 h unless specified otherwise. For transient transfection experiments, HEK293 cells were transfected with rGnRHR in pcDNA3 (23) using SuperFect or PolyFect (Qiagen, Melbourne, Australia) and plated onto poly-L-lysine-coated, 24-well culture plates 24 h posttransfection. Transfected cells were then treated for an additional 24 h with GnRHA (1 µM) in complete DMEM containing 5% FCS before being assessed for [3H]thymidine incorporation. [3H]Thymidine (0.5 µCi/ml; Amersham Pharmacia Biotech) was added to each well and incubated for at least 3 h unless specified otherwise. Cells were washed twice with PBS and three times with 10% trichloroacetic acid and then were solubilized with 0.1 M NaOH. The NaOH was neutralized with 10% acetic acid, and 4 ml Optiphase Hisafe 2 scintillation cocktail (PerkinElmer, Melbourne, Australia) were added to each sample. Samples were measured using a Packard 2000 Tri-Carb ß-counter.

Photomicrographs of cells
Cells were plated and treated as described for [3H]thymidine assays, except that after 96 h of treatment (medium and peptide replaced daily), cells were visualized using an IMT-2 inverted microscope (Olympus, New Hyde Park, NY), and images were captured using an Olympus DPII digital camera.

Analysis of cell number
Changes in cell number as a consequence of GnRH analog treatment were detected using flow cytometry. Cells were seeded at approximately 10% confluence onto poly-L-lysine-coated, six-well culture plates in complete DMEM containing 10% FCS. This was replaced with complete DMEM containing 5% FCS (with or without peptide) for the duration of the experiments (medium and peptide changed every 24 h). Briefly, cells were harvested, washed, and resuspended in PBS containing 3% FCS and 0.5 µg/ml propidium iodide (PI; Sigma-Aldrich Corp., Sydney, Australia). Each sample was added to a TruCount tube containing fluorescent beads (BD Biosciences, Perth, Australia) and processed using a FACSCalibur (BD Biosciences, San Jose, CA). Analysis was carried out using CellQuest software (version 3.1f, BD Biosciences). A minimum of 10,000 cells were counted per sample.

Detection of apoptotic cells by annexin V-FLUOS/PI
Apoptotic cells were detected via annexin V-FLUOS (Roche, Perth, Australia) combined with PI staining. Annexin V-FLUOS staining detects the early stages of apoptosis before membrane disruption and DNA fragmentation. Cells were seeded, treated, and harvested as described for analysis of cell number. One hundred microliters of labeling solution comprising 1% annexin V-FLUOS dye and 0.5 µg/ml PI in incubation buffer [10 mM HEPES (pH 7.5), 140 mM NaCl, and 5 mM CaCl2] were added to each sample. The cells were incubated on ice for 15 min, after which 400 µl incubation buffer were added. Samples were then analyzed by flow cytometry. All samples were gated on forward and side scatter measurements to exclude cellular debris and aggregates. Apoptotic cells were defined as those cells that were annexin V-FLUOS positive and PI negative, whereas necrotic cells were defined as those that were both annexin V-FLUOS positive as well as PI positive.

Analysis of cell cycle
Cell cycle phase distributions of control and treated cell cultures were analyzed by measuring relative DNA contents of individual cells using flow cytometry. Cells were seeded into 60-mm dishes in complete DMEM supplemented with 10% FCS for approximately 5 h, and treatments were carried out in complete DMEM supplemented with 5% FCS for 96 h with medium and peptide changes every 24 h. Samples were prepared according to the methods described previously (24). Briefly, cells were harvested, washed with PBS, and fixed in ice-cold 70% ethanol for 1 h at 4 C. Cells were washed and incubated in PBS containing ribonuclease A (0.2 mg/ml) and Triton X-100 (0.2%) at 37 C for 30 min. After a final PBS wash, cells were incubated with PI (50 mg/ml) for a minimum of 10 min at 4 C. Cells were filtered through 0.3-µm pore size mesh before measurements were taken. Samples were then processed using a FACSCalibur (BD Biosciences) and analyzed using CellQuest software (version 3.1f, BD Biosciences). A minimum of 10,000 cells were counted per sample.

Statistical analysis and data presentation
Data are presented as the mean ± SEM and are the combined results of at least three independent experiments unless otherwise specified. Data were analyzed using PRISM graphing software (GraphPad, San Diego, CA). Statistical significance was determined using two-tailed t tests. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Functional characteristics of the hGnRHR
The generation of cell lines stably expressing the hGnRHR has been problematic, as levels of receptor expression are very low, and cell lines do not retain expression after repeated passage. We have not experienced this problem with the generation of rGnRHR stable cell lines (17). We generated a stable cell line expressing a modified version of the human GnRHR that overcomes this problem by expressing high levels of receptor without significant loss of receptor over time (patent WO 99/67292). The modified hGnRHR retains function with regard to ligand binding, internalization, and IP production, possessing functional characteristics similar to those of the rGnRHR (Table 1Go). We have previously shown that epitope tagging of GnRHRs with either HA or EGFP tags does not affect receptor function (17, 20, 22). Confocal microscopy was used to demonstrate that epitope-tagged modified hGnRHR expressed in HEK293 cells localized to the plasma membrane (Fig. 1Go) and that a proportion of receptor was internalized after treatment with GnRHA. Similar distribution and internalization were observed for the epitope-tagged rGnRHRs (Fig. 1Go).


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  		TABLE 1. Functional characterization of GnRHRs

 


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  		FIG. 1. Localization of GnRHRs in HEK293 cells. HEK293 cells expressing the HA-tagged rGnRHR were plated into eight-well, poly-L-lysine-coated chamber slides. The following day, cells were treated with GnRHA (1 µM, 1 h), fixed, permeabilized, and probed to detect HA-tagged receptors as previously described (17 ). HEK 293 cells expressing the hGnRHR/EGFP fusions were seeded into eight-well, poly-L-lysine-coated chamber slides. After an additional 24 h, cells were treated with GnRHA (1 µM, 1 h), fixed, mounted, and analyzed by confocal microscopy. Confocal microscopy demonstrated that both the HA-tagged rGnRHR and hGnRHR/EGFP were correctly localized at the plasma membrane. GnRHA treatment resulted in receptor internalization, with a substantial percentage of GnRHR remaining at the cell surface.

 
Antiproliferative effect of GnRH and GnRH agonist is dose dependent
To address the mitogenic role of the GnRHR in gonadotrope cells that endogenously express the GnRHR, we first used the immortalized mouse pituitary-derived cell line, LßT2. The effect of GnRH on cell proliferation was determined by measuring [3H]thymidine incorporation into the DNA of LßT2 cells cultured in the presence of increasing concentrations of either GnRH or the GnRH agonist, [D-Leu6,Pro9-Et]GnRH (GnRHA) (Fig. 2Go). Maximal inhibition occurred at nanomolar concentrations of GnRHA. Native GnRH was less potent than the agonist and was required at a higher concentration to induce maximal inhibition (50% effective concentration values are shown in Table 2Go).



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  		FIG. 2. Dose-dependent effect of GnRH and GnRHA on [3H]thymidine incorporation in LßT2, HEK/rGnRHR, and HEK/hGnRHR cell lines. Cells were plated at approximately 10% density in 24-well plates. After 6 h, either GnRH or GnRHA ([D-Leu 6,Pro9-Et]GnRH) was added to each well at the final concentration indicated, and cells were incubated for an additional 96 h (medium and peptide replenished daily) before assessment of [3H]thymidine incorporation. Data were normalized by obtaining measurements of [3H]thymidine incorporation in samples of each cell line incubated in the absence of peptide. Data were expressed as a percentage of the control value. Each graph represents the combined results of at least three independent experiments (mean ± SEM), each of which was performed in triplicate.

 

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  		TABLE 2. EC50 values of [3H]thymidine incorporation in LßT2-, HEK/rGnRHR-, and HEK/hGnRHR-expressing cells after 4-d treatment with either GnRH or GnRHA (10-12–10-6 M)

 
In addition to the pituitary cell line, HEK293 cells stably expressing either rat (HEK/rGnRHR), or human (HEK/hGnRHR) GnRHRs were used to assess the ability of the GnRHR to mediate proliferative events in a cell type that does not usually express this receptor. Using these cell lines alongside control HEK293 cells (untransfected), which have no GnRHRs, has the advantage that any observed differences in proliferation as a result of GnRHA treatment can be associated with the presence or absence of the receptor. Similar to our observations in the LßT2 cell line, GnRH and GnRHA inhibited [3H]thymidine incorporation in a dose-dependent fashion (Fig. 2Go). As observed in the LßT2 cell line, GnRHA was more effective than GnRH in inducing an antiproliferative effect in both HEK/rGnRHR and HEK/hGnRHR cell lines (Table 2Go).

Time course of the antiproliferative effect of GnRHA
Cell lines were treated continuously with 1 µM GnRHA and assayed for [3H]thymidine incorporation. Cells were harvested every 6 h for 24 h and then daily for 4 consecutive days. A significant (P < 0.05) inhibition of [3H]thymidine incorporation was observed at 6 h in both LßT2 and HEK/hGnRHR cell lines (31 ± 2.7% and 37 ± 8.1%, respectively). Maximal inhibition of 68 ± 1.3% in the LßT2 and 65 ± 2.2% in the HEK/hGnRHR cell lines was achieved by 24 and 12 h, respectively, and was observed for the remainder of the experiment. The HEK/rGnRHR cell line exhibited a significant (P < 0.05) inhibition (41 ± 0.9%) after 12 h, which continued to increase with time, reaching a maximum inhibition of 68 ± 6.7% after 72 h. No inhibition of [3H]thymidine incorporation was observed in HEK293 cells similarly treated with GnRHA at any of the time points tested (Fig. 3AGo).



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  		FIG. 3. A, Effect of GnRHA on [3H]thymidine incorporation in LßT2, HEK293, HEK/rGnRHR, and HEK/hGnRHR cell lines is time dependent. Cells were treated with GnRHA (1 µM) for 6, 12, 18, 24, 48, 72, and 96 h, and assessment of [3H]thymidine incorporation was undertaken at each of these time points. Cells were incubated with 0.5 µCi/ml [3H]thymidine for at least 3 h. Fresh medium and peptide were added daily to all experiments lasting longer than 24 h. Data points are expressed as a percentage of the control (samples of each cell line incubated in the absence of GnRHA) and are the combined results of three independent experiments (mean ± SEM), each of which was performed in triplicate. B, Effect of GnRHA on [3H]thymidine incorporation in LßT2, HEK293, HEK/rGnRHR, and HEK/hGnRHR cell lines is blocked by GnRHAnt. Cells were treated with GnRHA (10 nM), GnRHAnt (100 nM), or their combination for 96 h. Medium containing peptides was removed and replenished daily. Cells were assayed for [3H]thymidine incorporation after 96 h. [3H]Thymidine incorporation was only significantly inhibited in GnRHR-expressing cell lines after GnRHA treatment (*, P < 0.05 compared with untreated cells). Data are expressed as a percentage of the control (samples of each cell line incubated in the absence of GnRHA) and represent the combined results of three independent experiments (mean ± SEM), each of which was performed in triplicate.

 
To determine that the effect of the GnRHA on [3H]thymidine incorporation observed in the GnRHR-expressing cell lines was a GnRHR-mediated event, agonist-treated cells were cultured in the presence or absence of GnRHAnt for 4 consecutive days. The GnRHA-induced antiproliferative effect could be successfully blocked in all GnRHR-expressing cell lines by cotreatment with GnRHAnt (Fig. 3BGo). GnRHAnt alone had no effect on [3H]thymidine incorporation. The ability of GnRHAnt to block the agonist-induced antiproliferative effect suggests that GnRHA is not cytotoxic, and growth suppression is mediated specifically by GnRHR activation.

Inhibition of [3H]thymidine incorporation as a consequence of GnRHA treatment was also observed in HEK293 cells transiently expressing the GnRHR (receptor expression was confirmed using whole cell binding assays; data not shown). Cells were transfected with rGnRHR and 24 h later were treated with GnRHA. At 24 h post treatment, the degree of inhibition in HEK293 cells transiently expressing the rGnRHR was similar (44 ± 4.1%) to that in the rGnRHR/HEK stable cell line (39 ± 9.5%; Fig. 4Go). Neither sham-transfected (with pcDNA3 only) nor untransfected HEK293 cells exhibited an inhibition of [3H]thymidine incorporation after GnRHA treatment.



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  		FIG. 4. Effect of GnRHA on [3H]thymidine incorporation in HEK293 cells either stably or transiently expressing GnRHRs. Transiently transfected cells (transfected with either 2 µg rat GnRHR cDNA in pcDNA3 vector or 2 µg pcDNA vector only) or a stable cell line expressing rat GnRHRs (HEK/rGnRHR) were seeded into 24-well plates and treated for 24 h with GnRHA (1 µM). After GnRHA treatment, cells were assayed to determine whether the presence (+) or absence (-) of the GnRHR affected [3H]thymidine incorporation. Data are expressed as a percentage of the control (cell samples of each cell line incubated in the absence of peptide). GnRHA treatment significantly inhibited [3H]thymidine incorporation only in cells expressing GnRHRs (*, P < 0.05). The graph represents the combined results of three independent experiments (mean ± SEM), each of which was performed in triplicate.

 
Effects of GnRHA on cell number
A visible reduction in cell density was observed after 96 h treatment with GnRHA in HEK/rGnRHR, but not in untransfected HEK293, cells using phase contrast microscopy (Fig. 5AGo). Addition of GnRHAnt alone had no visible effect on cell density. The absence of any reduction in the cell density of HEK/rGnRHR cells cotreated with GnRHA and GnRHAnt suggests that GnRHAnt was able to successfully block the antiproliferative effect of GnRHA. A reduction in cell density was also seen microscopically in the HEK/hGnRHR stable cell line as well as the endogenously GnRHR-expressing LßT2 cell line when treated with GnRHA, and this effect was again blocked by cotreatment with GnRHAnt (data not shown).



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  		FIG. 5. A, Effects of GnRHA and GnRHAnt on cell density of HEK and HEK/rGnRHR cell lines. HEK293 cells (untransfected or stably expressing rat GnRHRs; HEK/rGnRHR) were treated with GnRHA (10 nM) in the presence or absence of GnRHAnt (100 nM) for 96 h, with medium and peptide replaced daily. GnRHA treatment (ii) induced a reduction in cell density compared with untreated (i) GnRHAnt either alone (iii) or in combination with GnRHA (iv), which had no effect on cell density. Bar, 200 µm. B, Effects of GnRHA and GnRHAnt on cell number in LßT2, HEK293, HEK/rGnRHR, or HEK/hGnRHR cell lines. Upon agonist (10 nM) treatment, cell number was significantly (*, P < 0.05) reduced in all GnRHR-expressing cell lines compared with untreated cells. Treatment of LßT2 cell lines with GnRHAnt (100 nM) alone or in combination with GnRHA induced a small, but significant, decrease in cell number compared with untreated cells (*, P < 0.05). Cells were treated as described for [3H]thymidine incorporation, except that after 96 h, each cell sample was stained with PI and added to a TruCount tube. Cell number was assessed using flow cytometry. Results are expressed as a percentage of the control (samples of each cell line incubated in the absence of GnRHA). Data are presented as the mean ± SEM and are the combined results of three independent experiments.

 
To quantitate the observed loss in cell density, flow cytometry was used to determine whether GnRHA treatment caused a reduction in cell number (Fig. 5BGo). All cell lines were treated with GnRHA (as for the [3H]thymidine incorporation assay), and a significant (P < 0.05) reduction in cell number (LßT2, 48 ± 1.6%; HEK/rGnRHR, 51 ± 5.8%, HEK/hGnRHR, 84 ± 3.1%; compared with each of these cell lines incubated in the absence of GnRHA) was only observed in those cell lines expressing the GnRHR (Fig. 5BGo). The dramatic reduction in cell number upon GnRHA treatment could be blocked by cotreatment with GnRHAnt (Fig. 5BGo). Cells treated with GnRHAnt alone did not show the large reduction in cell number, although a small, but significant, decrease in cell number was observed in the LßT2 cell line (this reduction is minimal compared with that induced by GnRHA treatment). The reduction in cell number combined with the [3H]thymidine incorporation results verify that continuous GnRHA treatment induces an antiproliferative effect mediated specifically by GnRHR activation in both the exogenous and endogenous cell lines studied.

GnRHA-induced increase in apoptosis
To study possible mechanisms by which the inhibition of [3H]thymidine incorporation and the reduction in cell number occurred, we undertook experiments to examine apoptotic events after treatment with GnRHA and GnRHAnt in our cell lines. Cell lines were treated with GnRHA in the presence or absence of GnRHAnt for 96 h and were analyzed by flow cytometry using annexin V-FLUOS/PI staining to detect apoptotic cells. GnRHA treatment induced a small, but significant (P < 0.05), increase in apoptotic cells in all cell lines expressing the GnRHR (Fig. 6Go). However, the magnitude of the increase in apoptotic cells varied among the cell lines. The LßT2 cell line demonstrated a higher basal level of apoptotic cells (6.2 ± 1.4%), which increased to 12 ± 1.5% (1.9-fold increase) after agonist treatment (Fig. 6Go). GnRHA treatment increased the percentage of apoptotic cells from 0.43 ± 0.18% to 2.2 ± 0.6% in the HEK/rGnRHR cell line and from 1.2 ± 0.6% to 3.0 ± 0.5% in the HEK/hGnRHR cell line (5- and 2.5-fold increases, respectively; Fig. 6Go). All increases in apoptotic cells were significant in comparison with both untreated and GnRHAnt-treated cells (P < 0.05). GnRHAnt successfully blocked the GnRHA-induced increase in apoptotic cells. A positive control was included in each annexin V-FLUOS experiment (LßT2 cells treated with 5 µg/ml cycloheximide and 10 ng/ml TNF{alpha} for 48 h) and induced an increase in apoptotic cells of 20.4 ± 2.5% compared with untreated cells.



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  		FIG. 6. Effects of GnRHA and GnRHAnt on apoptosis in LßT2, HEK293, HEK/rGnRHR, and HEK/hGnRHR cell lines. Cells were plated at 10% density in six-well plates and were treated with either GnRHA (10 nM) or GnRHAnt (100 nM) alone or in combination for 96 h (medium and peptides replenished daily). GnRHA treatment significantly (*, P < 0.05) increased the percentage of apoptotic cells in all GnRHR-expressing cell lines compared with untreated cells. Combined annexin V/FLUOS/PI staining was used to detect apoptotic (annexin V/FLUOS-positive, PI-negative) cells by flow cytometry. The numbers of apoptotic cells identified are presented as percentages of the cells analyzed (mean ± SEM) and represent the combined data of three independent experiments. A positive control (LßT2 cells treated with 5 µg cycloheximide and 10 ng TNF{alpha} for 48 h) was included in each annexin V/FLUOS/PI experiment.

 
Influence of GnRHA on cell cycle arrest
As only a small increase in apoptotic cells was observed in GnRHR-expressing cell lines upon GnRHA treatment, flow cytometry of PI-stained cells was used to determine the contribution of cell cycle arrest to the antiproliferative effect observed in the cell lines studied. Continuous GnRHA treatment induces a small amount of cell cycle arrest in the GnRHR-expressing cell lines (Fig. 7AGo). However, the phase of arrest varied according to cell line. GnRHA treatment of LßT2 cells induced a significant (P < 0.05) increase in the proportion of cells in the G0/G1 phase of the cell cycle from 43 ± 7.2% to 55 ± 4% (Fig. 7BGo). This G0/G1 increase was concordant with a significant (P < 0.05) decrease in S phase cells (40 ± 7.9% to 24 ± 7%; Fig. 7BGo). Changes in G2/M were negligible in these cells after GnRHA treatment. This suggests that the cell cycle blockade in these cells occurs at G1 before DNA synthesis.



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  		FIG. 7. A, Flow cytometric analysis of GnRHA-induced alteration in cell cycle distribution in LßT2, HEK293, HEK/rGnRHR, and HEK/hGnRHR cell lines. Cells were treated with GnRHA (10 nM) for 96 h (medium and peptides replenished daily), fixed, permeabilized, and stained with PI for analysis by flow cytometry. GnRHA treatment (lower panels) induced shifts in cell cycle distribution in all GnRHR-expressing cell lines compared with untreated cells (upper panels). B, Effect of GnRHA and GnRHAnt treatment on cell cycle events in LßT2, HEK/rGnRHR, and HEK/hGnRHR cell lines. Cells were treated with GnRHA (10 nM) in the presence or absence of GnRHAnt (100 nM) for 96 h and were processed for analysis by flow cytometry as described in A. GnRHA treatment induced a significant increase (*, P < 0.05) in the percentage of cells in G0/G1 and a significant decrease ({dagger}, P < 0.05) in the percentage of cells in S phase compared with untreated cells in the LßT2 cell line. Both the HEK/rGnRHR and HEK/hGnRHR cell lines experienced a significant decrease (*, P < 0.05) in the percentage of cells in G0/G1 as a consequence of GnRHA treatment compared with untreated cells. Cotreatment with GnRHAnt completely blocked the GnRHA-induced cell cycle effects in the LßT2 and HEK/rGnRHR cell lines. GnRHAnt partially blocked the effects of GnRHA in the HEK/hGnRHR cell line. Medium and peptides were replenished daily. Data are presented as a percentage of cells per stage of the cell cycle (mean ± SEM) and are the combined data of at least three independent experiments.

 
In contrast, no G1 block was observed after GnRHA treatment of HEK/rGnRHR and HEK/hGnRHR cell lines (Fig. 7AGo). Indeed, the proportion of cells in G0/G1 decreased significantly (P < 0.05) from 59 ± 0.4% to 46 ± 2.4% in the HEK/rGnRHR cell line and from 57 ± 2.3% to 40 ± 3.6% in the HEK/hGnRHR cell line (Fig. 7BGo). The proportion of cells in the G2/M phase also increased from 19 ± 1.4% to 26 ± 2.2% in HEK/rGnRHR cells and from 19 ± 1% to 29 ± 3.8% in the HEK/hGnRHR cell line. No change in the proportion of cells in each phase of the cell cycle was observed after GnRHA treatment of untransfected HEK293 cells (Fig. 7AGo). Thus, these increases combined with the decrease in cells in G0/G1 suggest that GnRHA treatment may block cell cycle progression through mitosis at G2/M in both HEK/rGnRHR and HEK/hGnRHR-expressing cell lines (Fig. 7Go, A and B). Cotreatment with GnRHAnt blocked agonist-induced cell cycle arrest in all three cell lines, albeit incompletely in HEK/hGnRHR cells (Fig. 7BGo). GnRHAnt alone had no effect on cell cycle distribution (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vitro evidence supports the idea that GnRH analogs used to inhibit the growth of hormone-dependent cancers may exert their effects directly via GnRHRs located on the surface of the tumor cell (6, 7, 8, 9, 10, 25). However, the majority of studies have used extrapituitary tumor-derived cell lines, in which inconsistent effects of GnRH analogs have been reported despite the receptor being structurally identical to that expressed in pituitary gonadotrope cells (5, 6, 8, 10, 12, 13, 14, 15, 26, 27, 28, 29). This study aimed to characterize the effect of GnRH on cellular proliferation in a gonadotrope-derived cell system (the LßT2 cell line), which endogenously expresses the GnRHR. In addition, we used a cell system (HEK293 cells) in which we could directly compare the growth effects of GnRHR activation in the same cell line that either does or does not express the GnRHR. Both HEK293 and LßT2 are immortalized cell lines, generated via adenovirus 5 and simian virus 40 T antigen targeting, respectively (18, 30). However, unlike the majority of studies investigating the effects of GnRH on proliferation, these cell lines have not been generated directly from cancers or metastases of reproductive tissues and, as such, may more clearly elucidate the role of the GnRHR in mediating growth effects.

Our studies show that treatment with either GnRH or GnRHA results in dose-dependent growth suppression in both the gonadotrope-derived LßT2 cell line and HEK293 cells stably expressing either rat or human GnRHR. A number of studies measuring GnRH analog effects in cell lines derived specifically from extrapituitary reproductive cancers were only able to show an influence when micromolar concentrations of either ligand was administered, and this raised questions regarding the specificity of GnRH action in these cell systems (9, 13, 27). In this study we were able to demonstrate that both GnRH and GnRH agonists exerted their inhibitory effects on cell growth at very low concentrations. GnRHA demonstrated a similar potency in all GnRHR-expressing cell lines used in this study and was effective at inducing an antiproliferative effect at lower doses than GnRH. GnRH exhibited a higher potency in the HEK/hGnRHR cell line compared with the other cell lines, which may reflect the interspecies differences. The GnRHA used in this study contains modifications that increase its potency and has been shown previously to be 50–100 times more biologically active than GnRH (3). The increased potency is likely to result from a combination of increased binding affinity, due to conformational constraint of the bioactive conformation, and increased resistance to enzymatic degradation (3, 31, 32).

The antiproliferative effect of GnRHA on LßT2 and stably transfected HEK293 cells was also shown to be time dependent. Significant reductions in [3H]thymidine incorporation were observed by 6–12 h in the GnRHR-expressing cell lines. This inhibitory effect was maximal by 48–72 h and was maintained for the duration of the experiment. The apparent variation in time required to reach maximal inhibition may reflect the different receptor species or receptor expression.

To support the GnRHR-mediated effects on [3H]thymidine incorporation, cell number was monitored (by both light microscopy and flow cytometry) in GnRHR-expressing cell lines, and we observed dramatic reductions in cell density. The antiproliferative effect visualized by microscopy and confirmed by both flow cytometry and [3H]thymidine incorporation was only seen in GnRHR-transfected HEK293 cells (both stable and transient) and was not observed in untransfected HEK293 cells or in HEK293 cells expressing another GPCR, the TRH receptor (data not shown). Therefore, this effect can only be attributed to the presence of GnRHR in these cells. Everest et al. (28) and Franklin et al. (29) recently demonstrated that transient adenovirus infection of MCF-7 breast and PC3 prostate cancer-derived cell lines with GnRHR can induce an antiproliferative effect upon stimulation of the receptor. Our results demonstrate that such an antiproliferative effect can be achieved upon transient transfection of the GnRHR in HEK293 cells and may not necessarily be restricted to transfection of hormone-dependent, cancer-derived cell lines.

Overall, these results are the first to demonstrate antiproliferative activity in LßT2 cells, supporting previous findings in another gonadotrope-derived cell line, {alpha}T3-1 (33, 34). Kakar et al. (33) demonstrated an antiproliferative effect of GnRH agonist treatment in {alpha}T3-1 cells in a time and dose-dependent manner and suggested that this is a result of receptor down-regulation. The theory implies that GnRHR activation has a positive role in cellular proliferation or survival, and thus, receptor down-regulation causes an antiproliferative effect. Supporting this idea is the report of an antiproliferative effect of the antagonist antide (GnRHAnt) in HEK293 cells stably expressing GnRHRs (33). However, our studies did not show an antiproliferative effect with the identical GnRHAnt in the same cell line using three independent techniques (light microscopy, [3H]thymidine incorporation, and flow cytometry), suggesting that down-regulation of the GnRHR may not be the mechanism by which the growth-suppressive effects are occurring in our cell lines. Furthermore, cotreatment with GnRHAnt blocked the antiproliferative effect of GnRHA, demonstrating that activation of the GnRHR is required for growth-suppressive effects. Our results agree with those reported by Everest et al. (28) and Franklin et al. (29), who were unable to demonstrate an antiproliferative effect with the same GnRHAnt (antide) used in this study with the identical dose of 100 nM. Furthermore, Everest et al. (28) and Franklin et al. (29) demonstrated that this GnRHAnt effectively blocked the antiproliferative effect of the GnRH agonist buserelin in both MCF-7 and PC3 cell lines, which had been infected with adenovirus to induce GnRHR expression.

Upon establishing that continuous GnRHA treatment was indeed causing an inhibition of [3H]thymidine incorporation and a reduction in cell number, we investigated the mechanism or pathway by which this was occurring, apoptosis and/or cell cycle arrest. This study showed that GnRHA treatment induces a significant increase in apoptotic cells in both LßT2 and HEK293 cell lines stably expressing the GnRHR, which could be completely abrogated by cotreatment with GnRHAnt. The gonadotrope cells demonstrated a higher basal level of apoptosis than the HEK/GnRHR-expressing cells, the reason for which remains unclear.

A number of studies point to the involvement of GnRH and its analogs in mediating apoptosis via the GnRHR both in vivo and in vitro. Billig et al. (35) demonstrated an induction of apoptosis in granulosa cells obtained from HX rats as a consequence of GnRH administration. The induction of apoptosis has also been demonstrated in cell lines derived from extrapituitary reproductive tumors upon GnRH analog treatment (3, 36, 37). However, evidence also exists that GnRH and its analogs may provide a protective effect, inhibiting apoptosis. Yin and Arita (38) demonstrated that GnRH administration was able to prevent estrus-induced apoptosis of anterior pituitary cells in cycling female rats. Furthermore, Gründker et al. (39) provided evidence that the apoptotic action of doxorubicin could be reduced by cotreatment with the GnRH agonist triptorelin. Thus, the pro- or antiapoptotic nature of GnRH remains to be clarified. In addition, the specific role of the GnRHR in mediating such GnRH-induced apoptotic events remains unclear.

The evidence provided in this study suggests that apoptosis mediated specifically by the GnRHR may play only a minor role in the antiproliferative events induced by activation of this receptor. Despite being statistically significant, the small increase in apoptosis observed in this study as a consequence of GnRHA treatment could not account for the dramatic reduction in cell number. As such, we investigated other means by which the antiproliferative effect of GnRHA administration could be explained.

GnRHA treatment increased the number of cells in G0/G1, whereas decreasing cells in S phase in the LßT2 cell line suggesting that a cell cycle blockade occurs at G1 before DNA synthesis in this cell line. Similar treatment of HEK/rGnRHR and HEK/hGnRHR cell lines resulted in reduced cells in the G0/G1 phase of the cell cycle. A corresponding increase in cell number in G2/M was observed, however, this was not significant in either of the two HEK/GnRHR cell lines. The effects of GnRHA on cell cycle events could be prevented by the presence of the GnRHAnt in both the LßT2 and the HEK/rGnRHR cell lines, providing further evidence of the specific involvement of the GnRHR in the different cell types. Although the same concentration of GnRHAnt was able to reduce the effects of GnRHA on the cell cycle events measured in HEK/hGnRHR cells, it was unable to completely block these effects. To obtain a complete block, higher doses of GnRHAnt may be needed (GnRHR expression is >10-fold greater in HEK/hGnRHR cells than in HEK/rGnRHR cells), but this then raises questions regarding nonspecific effects of micromolar concentrations of GnRHAnt. Our findings indicate that cell cycle arrest after GnRHR activation may occur at either G0/G1 or possibly G2/M depending on the cell type. The differences in the cell cycle responses of HEK293 and LßT2 cells may reflect different genome alterations present in these cell lines and/or different cell-specific responses. HEK293 cells have been immortalized by adenovirus 5 oncoproteins that target and disrupt the function of the pRB (retinoblastoma) family of proteins (40, 41). Therefore, HEK293 cells have virtually no active G1/S checkpoint control, and so cell cycle perturbations usually result in G2/M arrest or delay (42). Cell cycle checkpoints in LßT2 cells have not been well characterized. These cells were derived through simian virus 40 T antigen transformation in vivo (18, 30), which should compromise the G1/S checkpoint owing to disruption of the pRB pathway (40, 41). However, the ability to delay/arrest in G1/S suggests that the G1/S checkpoint retains some function. Like the LßT2 cells, endometrial and ovarian cancer cell lines also undergo arrest at the G0/G1 cell cycle phase upon treatment with GnRH agonists (36, 43). The ovarian cancer cells also exhibited minimal apoptosis (36). Our study has shown for the first time that GnRHA treatment of a pituitary-derived cell line (LßT2) can concurrently induce apoptosis and cell cycle arrest. Despite the HEK/rGnRHR, HEK/hGnRHR and LßT2 cell lines being transformed such that their ability to undergo cell cycle arrest is impaired, GnRHA treatment is still able to cause cell cycle arrest/delay. The G0/G1 arrest demonstrated in the LßT2 cell line is likely to more accurately reflect the natural system. However, the effect in cells that have not been transformed, such as in the physiological situation, may well be more dramatic. A recent publication revealed that treatment with the GnRH agonist [des-Gly10,D-Ala6]GnRH affected the expression of genes encoding proteins involved in cell proliferation, including cell cycle and apoptotic events (44). This research combined with our own data support the ability of the GnRHR to mediate growth-related events.

In conclusion, we have demonstrated that GnRH and GnRH agonist can induce an antiproliferative effect in both a gonadotrope-derived cell line and HEK293 cells exogenously expressing either the rat or human GnRHR. This inhibition, which is blocked when a GnRHAnt is present, can be attributed at least in part to both induction of apoptosis and cell cycle arrest. As such, this study helps to clarify the specific role of the GnRHR in mediating growth events in two cell systems that have not been specifically derived from tumors of reproductive tissues, providing evidence that the GnRHR-mediated antiproliferative effect is not limited to the extrapituitary cancer cell system alone. The ability to induce such an effect regardless of the cell system involved (endogenous or exogenous expression of the GnRHR) has implications regarding the extensive use of GnRH analogs for the treatment of a broad spectrum of endocrine-related disorders and hormone-dependent tumors. Consequently, the potential effects this current therapy may have on nontumor cell populations expressing the GnRHR, such as pituitary gonadotrope cells, may need to be reassessed.


   Acknowledgments
 
We are grateful to the 7TM Laboratory for support and advice and thank E. Faccenda for generation of the HEK/hGnRHR stable cell line. We also thank M. Wikstrom for his support with FACS technology, and G. Worth for his assistance with radioisotopes. We thank P. Rigby for his invaluable assistance with confocal microscopy. In addition, we thank the Lotteries Commission of Western Australia for support of the Flow Cytometry and Biological Confocal Microscopy Research Center.


   Footnotes
 
This work was supported by the Keogh Institute for Medical Research, the Raine Foundation, and the National Health and Medical Research Council (Grant 116501), Australia.

L.E.C.M. is the recipient of a University Postgraduate Award from the University of Western Australia.

K.D.G.P. is the recipient of a Western Australian Institute for Medical Research postdoctoral fellowship.

Abbreviations: EGFP, Enhanced green fluorescence protein; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; GnRHA, GnRH agonist; GnRHAnt, GnRH antagonist; GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; HA, hemagglutinin; hGnRHR, human GnRH receptor; IP, inositol phosphate; PI, propidium iodide; rGnRHR, rat GnRH receptor.

Received May 2, 2003.

Accepted for publication September 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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