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Endocrinology Vol. 142, No. 11 4663-4672
Copyright © 2001 by The Endocrine Society

ARTICLES

Signaling and Antiproliferative Effects Mediated by GnRH Receptors After Expression in Breast Cancer Cells Using Recombinant Adenovirus

Helen M. Everest, James N. Hislop, Tom Harding, James B. Uney, Andrea Flynn, Robert P. Millar and Craig A. McArdle

University Research Center for Neuroendocrinology, University of Bristol, Bristol, United Kingdom BS2 8HW; Medical Research Council Human Reproductive Science Unit, Center for Reproductive Biology (R.P.M.), Edinburgh, United Kingdom EH3 9ET

Address all correspondence and requests for reprints to: Dr. Craig A McArdle, University Research Center for Neuroendocrinology, University of Bristol, Bristol, United Kingdom BS2 8HW. E-mail: craig.mcardle{at}bris.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
GnRH receptors (GnRH-Rs) are found in human cancers, including those of the breast, and GnRH can inhibit the growth of cell lines derived from such cancers. Although pituitary and extrapituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extrapituitary GnRH-Rs have low affinity for GnRH analogs and may not activate PLC or discriminate between agonists and antagonists in the same way as pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in breast cancer cells differ from those in gonadotropes. We found no evidence for endogenous GnRH-Rs in MCF7 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at a multiplicity of infection of 10 or greater, at least 80% expressed GnRH-Rs. These had high affinity (Kd for [125I]buserelin, 1.4 nM) and specificity (rank order of potency, buserelin>GnRH>>chicken GnRH-II) and mediated stimulation of [3H]IP accumulation. Increasing viral titer [from multiplicity of infection, 3–300] increased receptor number (10,000–225,000 sites/cell) and [3H]IP responses. GnRH stimulated ERK2 phosphorylation in Ad GnRH-R-infected cells, and this effect, like stimulation of [3H]IP accumulation, was blocked by GnRH-R antagonists. GnRH also inhibited [3H]thymidine incorporation into Ad GnRH-R-infected cells (but not control cells). This effect was mimicked by agonist analogs and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at density comparable to that in gonadotropes, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotropes, and increasing expression of high affinity GnRH-Rs can dramatically enhance the direct antiproliferative effect of GnRH agonists on breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH REGULATES THE secretion of LH and FSH from the pituitary (1). GnRH-stimulated gonadotropin secretion can be blocked with antagonists or mimicked by agonists, but in the latter case sustained stimulation causes desensitization. Thus, both agonists and antagonists ultimately reduce the circulating levels of gonadotropins and gonadal steroids. This effect, termed medical castration, is exploited to treat sex hormone-dependent neoplasms such of those of the prostate, ovary, endometrium, or breast (2, 3). At the pituitary, GnRH acts via G protein-coupled receptors (GPCRs) that act via Gq/11 to stimulate PLC, thereby causing an IP3-mediated mobilization of Ca2+ from intracellular stores. This Ca2+ mobilization along with the entry of Ca2+ across the plasma membrane and the concomitant activation of PKC, are thought to mediate GnRH-stimulated gonadotropin secretion (1, 4, 5, 6). GnRH also activates four MAPK signaling modules in pituitary cells, and PKC plays a key role in mediating such activation (5, 7, 8, 9). Although there is no direct evidence that GnRH can activate any G protein other than Gq/11 in gonadotrophs or {alpha}T3–1 cells, it is apparently able to do so in other cell types. In heterologous expression systems, GnRH has been shown to stimulate cAMP accumulation via GnRH receptors (GnRH-Rs) expressed stably in GGH3 cells (10) or transiently in COS-7 (11) and Sf9 cells (12). Interestingly, the coupling of GnRH-Rs to adenylyl cyclase is apparently density dependent in GGH3 cells, where GnRH-stimulated cAMP accumulation increases as GnRH-R number is reduced (13). Accordingly, GnRH-R signaling may be qualitatively and quantitatively dependent upon cellular context and receptor density.

GnRH-Rs are also found (often along with GnRH) in some mammary, prostatic, endometrial, and ovarian cancers (3, 14, 15, 16, 17). Interest in these extrapituitary GnRH-Rs stems primarily from the fact that GnRH analogs (or cytotoxic derivatives of GnRH analogs) can inhibit the proliferation of cell lines derived from such cancers and that direct antiproliferative effects on cancer cells may therefore contribute to therapeutic effects of GnRH analogs in cancer treatment (14, 15, 16, 17, 18). Although GnRH-R transcripts detected in breast and ovarian cancers are identical to those in pituitary (19), the receptors may differ functionally. In binding studies (16) pituitary GnRH-Rs have high affinity for agonists such as buserelin (nanomolar Kd values), whereas the majority of GnRH-Rs in extrapituitary sites have low affinity (micromolar Kd values). There are also apparent differences in signaling. Whereas GnRH-Rs in gonadotropes are positively coupled to PLC and MAPK activation, those in ovarian and endometrial cancer cell lines appear not to activate PLC and, in the presence of epidermal growth factor (EGF), actually inhibit ERK phosphorylation (3, 16). Similar inhibition was observed in prostatic cancer cell lines (20), and it has been suggested that a GI-mediated activation of protein phosphatase activity underlies the antiproliferative effect of GnRH in human cancer cells (21, 22). Moreover, the antiproliferative effects of GnRH-R agonists in some cancer cell models can be mimicked by analogs such as cetrorelix, which are competitive antagonists at pituitary GnRH-Rs (16), leading to the suggestion that the agonist/antagonist dichotomy established for pituitary GnRH-Rs may not apply in extrapituitary sites (23). This issue is controversial, however, because endogenously produced GnRH may also stimulate the proliferation of some cancer cell lines, so that antiproliferative effects of endogenous agonists and antagonists could reflect GnRH-R down-regulation and blockade, respectively (24).

Until recently it was generally thought that any given GPCR would have one active conformation and couple to one G protein-coupled (e.g. Gs or Gq) with one effector (e.g. PLC or adenylyl cyclase), but recent studies have revealed diversity of coupling for a number of receptors. Some GPCRs act via multiple G proteins to control multiple effectors or to directly activate proteins other than heterotrimeric G proteins, and GPCR function can be dramatically altered by accessory proteins (25, 26, 27). The activation of multiple effector proteins by a single receptor can reflect the existence of multiple active conformations preferentially coupled to distinct effectors, in which case specific ligands may preferentially stabilize specific active conformations such that different ligands can target signaling to distinct effectors (28). Accordingly, ligand specificity and signaling can depend not only on GPCR structure, but also on receptor number, cell type, and ligand, such that the reported differences between pituitary and extrapituitary receptors could reflect an extreme degree of context dependence by this receptor. If this is the case, GnRH-Rs expressed exogenously in extrapituitary sites would be expected to display functional characteristics distinct from those of GnRH-Rs in pituitary gonadotropes. To test this possibility we have developed recombinant adenovirus-expressing GnRH-Rs (Ad GnRHs) and used these to express receptors in a human breast cancer-derived cell line at a density that would be physiological for pituitary GnRH-Rs. We show that these receptors are essentially indistinguishable (in terms of binding, signaling, and agonist/antagonist discrimination) from those in pituitary cells. This procedure also facilitates a pronounced antiproliferative effect of GnRH agonists on these cells, implying that GnRH-R number is limiting for this direct antiproliferative effect and that manipulation increasing GnRH-R number in breast cancer (for example) may therefore increase the effectiveness of direct GnRH-R-targeted therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and cell culture
GnRH and chicken GnRH-II (cGnRH-II) were purchased from Peninsula Laboratories, Inc. (Merseyside, UK) or Sigma (Poole, UK). Buserelin and [125I]buserelin (2000 Ci/mmol) were provided by Prof. J. Sandow (Aventis Pharma GmbH, Frankfurt, Germany). Culture media, sera, and plasticware were obtained from Life Technologies, Inc. (Paisley, UK), or Falcon (Becton Dickinson and Co., Oxford, UK). The GnRH antagonists, antide and cetrorelix, were purchased from Sigma or provided by Asta Medica (Frankfurt am Rhein, Germany), respectively. 3-[4,5-Dimethylthiazol-2yl]-diphenyltetrazolium bromide (MTT) was also obtained from Sigma, and all other reagents were from standard commercial suppliers. MCF-7 cells were purchased from the European Collection of Cell Cultures (Salisbury, UK) and routinely cultured in DMEM supplemented with 10% FCS, penicillin, and streptomycin. For experiments they were harvested by trypsinization and then incubated for 1–3 d in flasks or culture plates as described in the figure legends. For Ca2+ imaging, cells were cultured in 12-well plates (2 ml/well) containing untreated round glass coverslips.

Generation of recombinant adenovirus
Recombinant, E1-deleted adenovirus-expressing sheep GnRH receptors (Ad GnRH-R) were prepared as previously described (29, 30, 31). Briefly, DNA encoding sheep GnRH-R was excised from pcDNA1/Amp plasmids (Invitrogen, Nu Leek, The Netherlands), and the insert was ligated into an identically digested (BamHI/XbaI) pXCXCMV shuttle vector. After transformation and growth in Escherichia coli, this was purified and used for homologous recombination with the pJM17vector after CaPO4 transfection of HEK-293 cells (Microbix Systems, Inc., Toronto, Canada). The cells were overlayed in medium with 0.5% agarose, and individual recombinant Ad plaques were amplified. After sequence confirmation, Ad stocks were expanded by infection of HEK-293 cells, followed by extraction and CsCl2 gradient purification. Viral titer was determined using a plaque assay and is reported as multiplicity of infection (moi) where an moi of 1 represents 1 plaque forming unit/plated cell. In some experiments control Ad (empty Ad lacking the GnRH-R insert) was used, and in others transfection efficiency was assessed using an Ad expressing enhanced green fluorescent protein (Ad EGFP). These had been previously prepared (31).

Accumulation of [3H]IPs and Ca2+ imaging
[3H]IP accumulation was used as a measure of PLC activity using cells labeled by preincubation with [3H]inositol and stimulated in the presence of LiCl (31, 32). Cells were cultured in 24-well plates in 1 ml medium, and 2 µCi [2-3H]inositol (14–16 Ci/mmol) were added to each well for the final 16 h of incubation. After two washes in physiological salt solution (PSS; 127 mM NaCl, 1.8 mM CaCl2, 5 mM KCl, 2 mM MgCl2, 0.5 mM NaH2PO4, 5 mM NaHCO3, 10 mM glucose, 0.1% BSA, and 10 mM HEPES, pH 7.4), each well was stimulated with 200–250 µl PSS containing 10 mM LiCl and the indicated concentration of stimulatory peptide. The stimuli were terminated by adding 1 ml water at 95 C. IPs were then extracted and separated from free [3H]inositol using anion exchange chromatography (31, 32). In preliminary experiments in which cells were stimulated for 5–60 min with 0 or 10-7 M GnRH in medium with 10 mM LiCl, GnRH-stimulated [3H]IP accumulation remained approximately linear for 30–40 min. Accordingly, a 30-min stimulation time was used for all experiments shown here. For video imaging, fura-2-loaded cells were loaded for 30 min in 1 ml PSS containing 2 µM fura-2 (33). Image capture was performed within 10–25 min of loading at 37 C in approximately 500 µl PSS or in Ca2+-free PSS (containing 250 µM EGTA instead of CaCl2) using MagiCal hardware and Tardis software from Applied Imaging (Newcastle-upon-Tyne, UK) and a Nikon Diaphot microscope (Melville, NY). The cells were excited alternately at 340 and 380 nm, and emitted light was collected at 510 nm. The ratio of fluorescence at 340 and 380 nm was calculated on a pixel by pixel basis and used to determine the Ca2+ concentration, assuming a dissociation constant of 225 nM for fura-2 and Ca2+. Calibration was performed as previously described (33).

Radioligand binding
GnRH-R expression was assessed using whole cell binding assays in which approximately 50,000 cells were incubated at 21 C in 100 µl PSS containing 1 mg/ml bacitracin with approximately 10-10 M [125I]buserelin (a high affinity GnRH receptor agonist) and 0 or 10-11–10-5 M unlabeled competitor. Free and bound peptide were separated by centrifugation through oil (31, 32), and nonlinear regression (PRISM, GraphPad Software, Inc., San Diego, CA) was used to determine Kd and binding capacity (Bmax) values, assuming that the tracer and competitor bind with identical affinity to a single class of receptor. Cell counts performed in parallel enabled calculation of the number of receptors per cell. In preliminary experiments the time course of association and dissociation of [125I]buserelin was determined, and the results obtained were essentially indistinguishable from those seen in pituitary cells (34). Binding was maximal and reversible at 30 min, and this incubation time was therefore used in all subsequent experiments.

MAPK activation
MAPK activation was measured using standard techniques (31). Briefly, MCF-7 cells were plated in 60-mm dishes at 300,000 cells/dish and infected at moi values between 50–100. After 48 h they were stimulated (see below) before being washed twice in ice-cold PBS and then lysed on ice for 10 min in 400 µl extraction buffer [10 mM Tris (pH 7.6), 5 mM EDTA, 1 mM EGTA, 50 mM NaCl, 30 mM Na+ pyrophosphate, 50 mM NaF, 1 mM dithiothreitol, 100 µM Na+ orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml antipain, 2 µg/ml leupeptin, and 2 µg/ml pepstatin]. The samples were then centrifuged (13,000 x g, 10 min, 4 C), and 40 µl supernatant were boiled with 40 µl sample buffer. Proteins were separated by SDS-PAGE (8% gel), transferred to polyvinylidene difluoride membrane, and blocked with 5% milk/Tris-buffered saline/Tween. ERK2 was detected with anti-ERK2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK). The appearance of a retarded (phosphorylated) band was taken as evidence of ERK2 activation. This was quantified by densitometry, and the intensity of the phosphorylated band was normalized as a percentage of the summed densities of both (providing an internally controlled measure of ERK2 activation).

[3H]Thymidine incorporation and MTT assays
Incorporation of [3H]thymidine into newly synthesized DNA was used as an index of cell proliferation (35, 36). Cells were plated in 96-well plates in 250 µl DMEM with 10% FCS at a density of 5000 cells/ml. After 24 h they were transferred to DMEM containing 1% FCS and incubated with test adenovirus at varied moi 3–300(3–300, or 0 in control cells). After 6 h the cells were transferred to fresh DMEM (1% FCS) and incubated for approximately 24 h before addition of test peptides. After a further 6 or 7 d, 0.5 µCi [3H]thymidine was added to each well and left to incorporate for 4 h. The cells were incubated in 100 µl trypsin/EDTA at 37 C for 30 min. The cells were then frozen and thawed, and incorporated [3H]thymidine was collected on A filter papers (Wallac, Inc., Gaithersburg, MD) using a 96-well harvester and quantified by ß-counting (l450 Microbeta Plus, Wallac, Inc.). In some experiments the rate of MTT hydrolysis was also determined by incubating cells cultured in 96-well plates for 4 h at 37 C in culture medium with 0.5 mg/ml MTT (Sigma). The incubation medium was then replaced with 50 µl acidified isopropanol (10 mM HCl in isopropanol), and the colored product was quantified by absorbance spectroscopy at 515 nm. Standard curves were also generated by measuring MTT activity in known numbers of cells (5,000–120,000), enabling calculation of cell number from MTT activity.

Statistical analysis and data presentation
The figures show data from a single representative experiment or the mean ± SEM of data pooled from n independent experiments (data normalized as described in the figure legends). Data are typically reported in the text as the mean ± SEM, and statistical analysis was performed using two-tailed t tests, accepting P < 0.05 as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Our first attempts at expression of GnRH-Rs in MCF-7 cells were unsuccessful because of the low transfection efficiency achieved with conventional strategies. When MCF-7 cells were transfected with an EGFP reporter using lipofectin, LipofectAMINE Plus (Life Technologies, Inc.), Fugene (Boehringer Mannheim, Lewes, UK), and CaPO4 as previously described (31), flow cytometry revealed transfection efficiencies of less than 20% (not shown). In contrast, infection with Ad EGFP yielded a transfection efficiency approaching 100% (at moi values of >=10) and much higher levels of fluorescence per cell (not shown). We therefore constructed sheep Ad GnRH-R, confirmed the identity of the insert by restriction digestion and sequencing (not shown), and measured cell surface expression of GnRH-Rs after infection of MCF-7 cells with Ad GnRH-R. We were unable to detect specific binding of [125I]buserelin to control (uninfected) MCF-7 cells, but after 2 d of infection with Ad GnRH-R at varied viral titer (moi, 3–300), there was a clear titer-dependent increase in specific binding (Fig. 1Go). This binding was blocked in a concentration-dependent manner by unlabeled buserelin, and curve fitting revealed these to be high affinity sites, with Kd values of 0.9–2.0 nM. As the Kd was not dependent upon viral titer, Bmax values were calculated by fitting the curves with the mean Kd value of 1.4 nM, and this revealed that increasing the Ad GnRH-R titer from 3 to 300 increased the number of binding sites from 10,000 to 225,000 sites/cell (Fig. 1Go, inset). To assess whether these sites were indeed functional receptors, possible activation of PLC was assessed using an [3H]IP accumulation assay. As shown (Fig. 2Go), GnRH failed to activate PLC in control MCF-7 cells, and infection with Ad GnRH-R did not increase basal [3H]IP accumulation in the absence of GnRH. However, infection with Ad GnRH-R facilitated a clear concentration- and titer-dependent stimulation of [3H]IP accumulation by GnRH.



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  		Figure 1. Titer dependence of receptor expression in MCF-7 cells infected with Ad GnRH-R. MCF-7 cells cultured in 60-mm petri dishes were infected with sGnRH-Ad at moi of 0, 10, 30, 100, or 300 as indicated, then cultured for 2 d before being scraped from the culture vessels and used for suspension binding assays using approximately 0.25 nM [125I]buserelin and the indicated concentration of unlabeled buserelin. The pooled Kd was 1.4 ± 0.3 nM (n = 5), and the values shown are the mean ± SEM (n = 3), normalized as a percentage of the binding seen without competitor in cells infected at an moi of 300. The insets show the numbers of receptors per cell calculated from Bmax values derived by fitting curves through the pooled Kd value and normalization according to cell number determined in parallel experiments (mean ± SEM; n = 3).

 


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  		Figure 2. Titer dependence of [3H]IP accumulation in MCF-7 cells infected with Ad GnRH-R. MCF-7 cells cultured in 24-well plates were infected with Ad GnRH-R at moi of 0, 10, 30, 100, or 300 as indicated, then cultured for 2 d. [3H]Inositol was added to the medium for the final 16 h of culture, after which the cells were washed and stimulated for 30 min with the indicated concentration of GnRH in the presence of 10 mM LiCl. Data shown are the mean ± SEM (n = 3) from three experiments, each having duplicate or triplicate determinations. For data pooling, they were normalized as a percentage of the [3H]IP concentration in maximally stimulated cells within each internally controlled experiment.

 
Ligand specificity was next examined in cells infected with Ad GnRH-R (moi, 100) by constructing competition binding curves with [125I]buserelin and increasing concentrations of buserelin, GnRH, or cGnRH-II and by stimulating [3H]IP accumulation with the same peptides. As shown (Fig. 3Go), all three peptides inhibited binding and stimulated [3H]IP accumulation, with a rank order of potency (buserelin>GnRH>cGnRH-II) identical to that for endogenous mammalian GnRH-Rs in pituitary cells (1, 4, 32) and for human and sheep GnRH-Rs expressed using recombinant Ad in {alpha}T4 pituitary cells (31) (data not shown). Together, these data demonstrate that infection of MCF-7 cells with Ad GnRH-R causes expression of functional GnRH-Rs with fundamental pharmacological characteristics (affinity, potency, and relative potency for ligands in binding and functional assays) indistinguishable from those of mammalian GnRH-Rs in gonadotrophs. Preliminary experiments were also performed using recombinant Ad expressing the human GnRH-R, and although similar results were obtained, higher expression levels were achieved with the Ad-expressing sheep GnRH (not shown). As no major differences are known between the signaling or pharmacological characteristics of these receptors, the sheep GnRH-R was used throughout the following experiments.



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  		Figure 3. Concentration dependence of GnRH, buserelin, and cGnRH-II on [125I]buserelin binding and [3H]IP accumulation. Upper panel, MCF-7 cells were infected with Ad GnRH-R (moi, 100), then cultured for 1–2 d before being scraped from the culture vessels and used for suspension binding assays using approximately 0.25 nM [125I]buserelin (GnRH-Rs) and the indicated concentration of unlabeled GnRH, buserelin, or cGnRH-II. Lower panel, MCF-7 cells cultured in 24-well plates were infected with Ad GnRH-R (moi, 100) and then cultured for 1–2 d. [3H]Inositol was added to the medium for the final 16 h of culture, after which the cells were washed and stimulated for 30 min with the indicated concentration of peptide as indicated in the presence of 10 mM LiCl. The data shown in both panels are the mean ± SEM (n = 3) from repeated experiments (each having duplicate or triplicate observations). Binding data were normalized as a percentage of that seen without competitor, and [3H]IP responses were normalized as a percentage of the maximum response seen in each internally controlled experiment.

 
We next determined the sensitivity of these [3H]IP responses to blockade by GnRH-R antagonists and, because GnRH-stimulated PLC activation mediates Ca2+ mobilization in pituitary cells, examined possible effects on the cytosolic Ca2+ concentration ([Ca2+]i). As shown (Fig. 4Go), GnRH failed to influence [Ca2+]i in control MCF-7 cells or in MCF-7 cell infected with control Ad, but caused a pronounced increase in [Ca2+]i in cells infected with Ad GnRH-R (moi, 100). The [Ca2+]i response consisted of a gradual increase to a sustained plateau elevation, in stark contrast to the characteristic biphasic (spike-plateau) response to GnRH seen with the endogenous GnRH-Rs of pituitary cells or with Ad GnRH-R-infected {alpha}T4 pituitary cells (not shown). The [Ca2+]i response of Ad GnRH-R-infected cells to GnRH was abolished when cells were treated with GnRH in the presence of antide, a GnRH analog that acts as a competitive antagonist at GnRH-Rs in pituitary cells. Antide and a second receptor antagonist, cetrorelix, both also completely blocked GnRH-stimulated [3H]IP accumulation in Ad GnRH-R-infected MCF-7 cells (Fig. 5Go).



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  		Figure 4. Effects of GnRH on [Ca2+]i in MCF-7 cells infected with Ad GnRH-R. MCF-7 cells were infected with Ad GnRH-R (moi, 100) or with control (empty) Ad or were not infected (as indicated) and then cultured for 1 d before being loaded with fura-2 and used for Ca2+ imaging. During imaging, control uninfected cells, control cells infected with empty Ad, and cells infected with Ad GnRH-R were stimulated with 10-7 M GnRH. In addition, cells infected with Ad GnRH-R were stimulated first with antide (10-7 M; vertical arrow) and then with GnRH (10-7 M) in the presence of antide. The traces each show the mean ± SEM from seven individual cells imaged in a single representative experiment. Note that GnRH failed to increase [Ca2+]i in either of the control groups or in the presence of antide, so these three traces are superimposed and indistinguishable.

 


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  		Figure 5. Effects of GnRH, antide, and cetrorelix on [3H]IP accumulation in MCF-7 cells infected with Ad GnRH-R. MCF-7 cells were cultured, infected, and treated exactly as described in Fig. 2Go, except that the stimulation was with 0 or 10-8 M GnRH in the presence or absence of 10-6 M antide or cetrorelix, as indicated. For these experiments the antagonist was added 5 min before GnRH, and the figure shows data pooled from four separate experiments after normalization as a percentage of the response to GnRH alone.

 
As GnRH and a GnRH-R antagonist both inhibit EGF-stimulated MAPK signaling in human ovarian and endometrial cancer cell lines (16), we next explored this possibility in MCF-7 cells infected with Ad GnRH-R. As shown (Fig. 6Go, upper panel), GnRH and EGF both caused a rapid activation of MAPK signaling (as measured by increased phosphorylation of ERK2) in Ad GnRH-R MCF-7 cells. The responses to GnRH and EGF had similar time courses (both maximal at ~15 min, with a gradual reduction thereafter), although the response to EGF was greater than that to GnRH. Using a single time point (15 min; Fig. 6Go, lower panel), we found that cetrorelix failed to stimulate MAPK in control or Ad GnRH-R-infected cells, and that the stimulatory effect of GnRH was only observed in Ad GnRH-R-infected cells. Moreover, the stimulatory effect of EGF was similar in control and Ad GnRH-R-infected cells and was not inhibited by GnRH or cetrorelix.



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  		Figure 6. Effects of EGF, GnRH, and cetrorelix on MAPK in MCF-7 cells. Activation of the ERK1/ERK2 MAPK signaling cascade was assessed by Western blotting to detect and distinguish unphosphorylated and phosphorylated ERK2 by the retarded mobility of the latter in SDS-PAGE gels. Upper panel, The time course of ERK2 activation was assessed by stimulating Ad GnRH-R-infected MCF-7 cells for 5–60 min with 10-7 M EGF (•), 10-7 M GnRH ({blacktriangleup}), or no additional stimulus ({circ}; controls at 0 and 30 min), as indicated. The inset shows the gel from a single representative experiment, and the main panel shows data pooled from three experiments (mean ± SEM) after densitometry and normalization of the P-ERK2 band as a percentage of the total ERK (P-ERK2 plus ERK2). Lower panel, Effects of GnRH (10-7 M) and cetrorelix (10-7 M) were assessed in the presence and absence of 10-7 M EGF in control (uninfected) cells and in cells infected with Ad GnRH-R (moi, 100) as indicated. Data were pooled from five experiments (as described above) in which GnRH and EGF significantly increased ERK2 phosphorylation (P < 0.05) whereas cetrorelix did not (P > 0.05), and neither had any measurable effect in the presence of EGF (P > 0.05).

 
As GnRH analogs can directly inhibit the proliferation of some breast cancer cell lines, we examined possible peptide effects on [3H]thymidine incorporation and MTT hydrolysis in Ad GnRH-R-infected MCF-7 cells. As shown (Fig. 7Go), GnRH caused a dose-dependent inhibition of [3H]thymidine incorporation and MTT activity in MCF-7 cells infected with Ad GnRH-R at an moi of 100, but failed to inhibit either in control cells (below and unpublished data). As [3H]thymidine incorporation was assessed during a brief incubation after 5–6 d of exposure to the tested peptide, its inhibition could reflect a reduction in proportional entry into the S phase at the time of [3H]thymidine exposure and/or a reduction in proliferation or survival of cells during the preceding 5–6 d. Preliminary experiments, using release of lactate dehydrogenase as a measure of cytotoxicity, revealed no cytotoxic effect of GnRH (10-11–10-6 M) in control or Ad GnRH-R-infected cells. As the inhibition of [3H]thymidine incorporation is more pronounced than the reduction in MTT activity (which is proportional to cell number), GnRH-R activation apparently reduced the rate of DNA synthesis (per cell), thereby reducing cell number.



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  		Figure 7. Effects of GnRH on [3H]thymidine incorporation and MTT activity in Ad GnRH-R-infected MCF-7 cells. Cells plated at low density in 96-well plates were infected with Ad GnRH-R (moi, 100) and then maintained for 5–6 d in the presence of the indicated concentration of GnRH before assessment of [3H]thymidine incorporation and MTT activity on the final day of culture, as described in Materials and Methods. The figure shows data pooled from five separate experiments (mean ± SEM; n = 4–5), each of which had three to six replicate observations. Pooling was achieved by normalizing the data as a percentage of [3H]thymidine incorporation or MTT activity seen in control cells without GnRH. The control value for [3H]thymidine incorporation without GnRH was 4.2 ± 2.7 x 103 cpm/well. In parallel experiments a standard curve was generated by measuring MTT activity in known numbers of cells (~5,000–120,000). Using this, we estimate that the control MTT activity (0.25 ± 0.06 arbitrary absorbance units/well in cells receiving no GnRH) is equivalent to approximately 75,000 cells/well. Similar experiments were performed in control cells receiving no adenovirus, and these revealed no measurable effect of GnRH on either parameter (not shown; see also Fig. 8Go) and no measurable effect of Ad GnRH-R on control [3H]thymidine incorporation or MTT activity (not shown).

 
To test for dependence on the transfected receptors, we next assessed GnRH effects on [3H]thymidine incorporation in control and Ad GnRH-R-infected cells. As shown (Fig. 8Go, upper panel), there was no inhibitory effect of GnRH in control uninfected cells or in cells infected with a control (empty) Ad (moi, 100). In contrast, GnRH caused a clear dose-dependent inhibition of [3H]thymidine incorporation in cells infected with Ad GnRH-R. In other experiments (not shown) this inhibitory effect of GnRH was dependent upon Ad titer (25 ± 12%, 81 ± 5%, and 96 ± 1% inhibition by 10-7 M GnRH in cells infected at moi values of 30, 100, and 300, respectively). However, the highest Ad titer (moi, 300) inhibited [3H]thymidine incorporation alone (an effect also seen with the control Ad at high titer), so subsequent experiments were performed using an moi of 100 (the highest titer at which the Ad alone did not measurably reduce [3H]thymidine incorporation). Using this Ad titer, comparison of three GnRH receptor agonists revealed that buserelin, GnRH, and cGnRH-II all inhibit [3H]thymidine incorporation into Ad GnRH-R-infected MCF-7 cells (Fig. 8Go, lower panel), although none had any measurable effect in control (uninfected) cells (not shown). The rank order of potency (buserelin>GnRH>cGnRH-II) was identical to that observed in the [3H]IP accumulation and radioligand binding assays (see above). Similar inhibitory effects were seen with zoladex and triptorelin (not shown). These agonists had comparable potency to buserelin, and none of the peptides tested measurably altered [3H]thymidine incorporation in control (uninfected) cells (not shown). In contrast to the concentration-dependent inhibitory effect of buserelin, antide and cetrorelix failed to measurably alter [3H]thymidine incorporation into Ad GnRH-R-infected MCF-7 cells (Fig. 9Go) or into uninfected control cells (not shown). Indeed, when the effect of buserelin was determined in the presence of varied concentrations of antide or cetrorelix, both behaved as competitive inhibitors, shifting the buserelin concentration-response curve rightward in a concentration-dependent manner (Fig. 10Go).



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  		Figure 8. Effects of GnRH on [3H]thymidine incorporation into control and Ad GnRH-R-infected MCF-7 cells. Upper panel, Cells plated at low density in 96-well plates were either not infected (control) or were infected with empty adenovirus or with Ad GnRH-R (each at an moi of 100) and used for assessment of [3H]thymidine incorporation as described in Fig. 7Go. The figure shows data pooled from five separate experiments (mean ± SEM; n = 4–5), each of which had four to six replicate observations. Pooling was achieved by normalizing the data as a percentage of [3H]thymidine incorporation seen in control cells without GnRH. The control value of 9.3 ± 1.6 x 103 cpm/well (in cells without Ad or peptide) did not differ (P > 0.1) from the control value in cells treated with Ad GnRH-R or with empty adenovirus. Lower panel, [3H]Thymidine incorporation was assessed in cells that had been infected with Ad GnRH-R (moi, 100) and cultured as described above, except that the culture medium contained the indicated concentration of GnRH, buserelin, or cGnRH-II. The figure shows data pooled from three separate experiments (mean ± SEM; n = 3), each of which had quadruplicate observations. Pooling was achieved by normalizing the data as a percentage of the [3H]thymidine incorporation seen in control cells without peptide for each curve, and the control values did not differ significantly from one another.

 


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  		Figure 9. Effects of buserelin, antide, and cetrorelix on [3H]thymidine incorporation into control and GnRH-R-infected MCF-7 cells. Cells were plated, incubated, infected with Ad GnRH-R, and used for assessment of [3H]thymidine incorporation (on the last day of culture) as described in Fig. 7Go, except that the cells were cultured in the presence of the indicated concentration of buserelin, antide, or cetrorelix. The figure shows data pooled from four separate experiments (mean ± SEM; n = 3–4), each of which had quadruplicate observations. Pooling was achieved by normalizing the data as a percentage of the [3H]thymidine incorporation seen in control cells without GnRH, and the three control values did not differ significantly from one another. In similar experiments 10-9 M buserelin reduced MTT activity to 76 ± 2% of the control (0.135 ± 0.028 arbitrary fluorescence units, equivalent to approximately 50,000 cells/well), whereas no such effect was seen with 10-7 M cetrorelix or antide.

 


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  		Figure 10. Inhibition of the effect of buserelin on [3H]thymidine incorporation by antide and cetrorelix. Cells were plated, incubated, infected with Ad GnRH-R, and used for assessment of [3H]thymidine incorporation (on the last day of culture) as described in Fig. 7Go, except that the cells were cultured in the presence of buserelin with antide (upper panel) or cetrorelix (lower panel), as indicated. The figure shows data pooled from three separate experiments (mean; n = 3), each of which had quadruplicate observations. SEMs were usually less than 5% and have been omitted for clarity. Pooling was achieved by calculating the percent inhibition of [3H]thymidine incorporation caused by each concentration of buserelin compared with the control rate of incorporation with each concentration of antagonist. These data were generated in parallel with those shown in Fig. 8Go, so the control buserelin responses (shown as a percentage of the control in Fig. 8Go and as the percent inhibition of the control in Fig. 10Go) are identical. As noted in Fig. 9Go, 10Go-9 M buserelin reduced MTT activity to 76 ± 2% (from a control value equivalent to ~50,000 cells/well). However, no measurable inhibition occurred in the presence of 10-7 M cetrorelix or antide (not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GnRH or its analogs can exert direct antiproliferative effects on numerous types of steroid-dependent and steroid-independent cancer cells, and for several of these there is good evidence for expression of GnRH-Rs and/or GnRH itself (3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). This has raised the possibility that in addition to reducing gonadal steroid levels (and thereby reducing the steroid-dependent proliferation of tumor cells), GnRH analogs may directly inhibit the proliferation of these cancers, and this direct effect may be further exploited to improve the effectiveness of GnRH analogs. With such a wide range of cell types and model systems explored, it is difficult to generalize about mechanisms of action, but there are three main schools of thought. The first is that GnRH-R activation is inhibitory, such that activation of this receptor slows cancer cell proliferation. From the pharmacological perspective, this is perhaps the most intriguing possibility because of the major differences between GnRH-Rs and response characteristics in pituitary and extrapituitary sites (predominant expression of low affinity receptors that do not discriminate between agonists and antagonists, do not activate PLC or MAPK, and actually inhibit EGF-stimulated MAPK activity in extrapituitary sites). This interpretation essentially requires that the GnRH-R display an extreme example of context or density dependence, begging the question of the molecular determinants of such dependence. The second possibility is that the GnRH-R activation is stimulatory, such that locally produced GnRH supports cancer cell proliferation, and this is prevented by GnRH-R antagonists or by down-regulation with agonists. This provides the simplest explanation for inhibition of cancer cell proliferation by antagonists, where endogenous agonist can be shown to be supporting proliferation. The third possibility is that effects of GnRH analogs are mediated by as yet uncharacterized additional forms of the GnRH-R (or related receptors). Several nonmammalian vertebrates express two or three forms of GnRH-R with distinct pharmacological characteristics (37, 38), and this may well hold true for mammals, including humans (39, 40).

In this study we sought to address the fundamental question of whether breast cancer cells are capable of expressing exogenous high affinity GnRH-Rs and, if so, whether they differ functionally from GnRH-Rs expressed in the pituitary when expressed at density that would be physiological for gonadotrophs. To do so it was necessary to generate recombinant Ad-expressing GnRH-Rs, because conventional transfection strategies based upon CaPO4, Fugene, lipofectin, or Lipofectamine Plus were found to be inefficient (<20% of cells transfected as revealed by flow cytometry when tested using an EGFP-expressing vector). In contrast, relatively high expression levels were achieved using an EGFP-expressing Ad, and flow cytometry revealed that the vast majority of cells (>90%) expressed the protein after infection at an moi of 10 or above (31).

When radioligand binding studies were performed to characterize cell surface GnRH-Rs, we were unable to detect any specific binding of [125I]buserelin. This is in contrast to earlier studies demonstrating antiproliferative effects of GnRH agonists in MCF-7 cells (17, 41), but such effects are not always seen (42), and it seems likely that the use of different subclones or passages of MCF-7 or the use of different cell culture conditions and bioassays can determine responsiveness to GnRH analogs (42). In our hands there was no evidence for endogenous GnRH-Rs (as judged by radioligand binding or functional assays) in MCF-7 cells, although we did find endogenous [125I]buserelin-binding sites and observe inhibition of proliferation in a second human breast cancer cell line (T47D cells; data not shown). MCF-7 cells from the European Tissue Culture Collection were selected for these studies, specifically to avoid the possible activation of endogenous GnRH-Rs.

Using radioligand binding studies we established that MCF-7 cells, which do not express measurable endogenous GnRH-Rs, are indeed capable of expressing exogenous high affinity GnRH-Rs, and these are essentially indistinguishable from the endogenous GnRH-Rs of pituitary cells and exogenous GnRH-Rs transfected into gonadotroph progenitor cells using recombinant adenovirus (31). Using [3H]IP accumulation as a measure of PLC activation, we were unable to detect any effect of GnRH in control (uninfected) cells, but observed a clear titer-dependent stimulation in cells infected with Ad GnRH-R. This establishes that the [125I]buserelin binding sites are functional GnRH-Rs and that these high affinity receptors do indeed activate PLC. These receptors were also found to mediate elevation of [Ca2+]i, because GnRH had no measurable effect in control cells, but caused a robust and sustained increase in Ad GnRH-R-infected cells. Interestingly, GnRH caused a gradual increase to a sustained plateau level of [Ca2+]i in these cells, compared with the characteristic biphasic spike-plateau increase seen with high concentrations of agonist acting at endogenous GnRH-Rs in pituitary cells (43) and with Ad-mediated transfection of GnRH-Rs into gonadotroph progenitor cells (31). As the spike phase of Ca2+ elevation in pituitary cells is due to IP3-mediated mobilization from intracellular stores, the implication is that in MCF-7 cells such stores are mobilized less rapidly or that the mobilized Ca2+ is cleared less rapidly from the cytoplasm.

Using Ca2+ responses to assess transfection efficiency (31), we found that approximately 90% of cells responded to GnRH at an moi of 10 (not shown), implying that the vast majority of cells express GnRH-Rs at this titer and that the increase in binding caused by increasing titer from 10–300 therefore reflects an increase in receptors per cell (rather than an increase in the proportion of cells expressing the receptor). This conclusion was reenforced using flow cytometry to assess EGFP expression in cells infected with an Ad EGFP. Again, the vast majority of cells expressed the protein at an moi of 10, and increasing the titer above this increased the amount of protein per cell without measurably altering the proportion of cells expressing the protein (not shown). The dynamic range of receptor number achieved by increasing the Ad GnRH-R titer from 3 to 300 (10,000 to 225,000 sites/cell) encompasses the range of GnRH-R densities seen in rat pituitaries (~20,000–75,000 sites/gonadotroph) (44, 45) and {alpha}T3–1 gonadotrophs (~65,000–85,000 sites/cell) (35). Accordingly, manipulation of viral titer provides a simple means of controlling GnRH-R expression in MCF-7 cells at density that would be physiological for gonadotropes.

Activation of these receptors was also found to stimulate MAPK (ERK2 phosphorylation), but not to inhibit EGF-stimulated MAPK activation. Cetrorelix also failed to activate MAPK or influence EGF-stimulated MAPK activity, whereas cetrorelix (and antide) blocked GnRH-stimulated [3H]IP accumulation in Ad GnRH-R-infected cells. Accordingly, these studies performed with GnRH-Rs expressed at a density that would be physiological for pituitary gonadotrophs have not revealed any major difference between the endogenous GnRH-Rs of pituitary cells and exogenous GnRH-Rs in MCF-7 cells. Both have high affinity (nanomolar Kd) for buserelin, both are positively coupled to PLC, Ca2+ mobilization, and MAPK activation, and both show identical ligand specificity, as judged by rank orders of potency and agonist/antagonist discrimination.

When the possible effects of GnRH analogs on the proliferation MCF-7 cells were investigated using a [3H]thymidine incorporation assay, there was no inhibitory effect of GnRH in control uninfected cells or in cells infected with a control (empty) adenovirus. However, infection of these cells with Ad GnRH-R facilitated a clear concentration-dependent inhibition of [3H]thymidine incorporation. We were unable to detect any cytotoxic effect of GnRH (in control or Ad GnRH-R-infected cells), and the inhibition of [3H]thymidine incorporation was associated with a less marked reduction in cell number. Thus, it appears that GnRH acts via transfected receptors to inhibit proliferation and thereby to reduce cell number. This effect was dependent upon adenovirus titer and was mimicked by buserelin and cGnRH-II, with a rank order of potency (buserelin > GnRH > cGnRH-II) identical to that seen for radioligand binding and [3H]IP accumulation. Two further agonists (zoladex and triptorelin) had similar inhibitory effects, with potencies comparable to that of buserelin (data not shown), whereas antide and cetrorelix (both of which are competitive antagonists at pituitary GnRH-Rs) had no such effect. Indeed, both peptides caused competitive inhibition of the response to buserelin (shifting the buserelin concentration-response curve rightward in a concentration-dependent manner). None of these peptides measurably altered [3H]thymidine incorporation or MTT activity in control (uninfected) cells.

Together the data presented above establish that direct activation of GnRH-Rs can indeed dramatically inhibit the proliferation of these breast cancer cells and that the pharmacological characteristics of this (exogenous) GnRH-R-mediated response are indistinguishable (in terms of ligand recognition and agonist/antagonist discrimination) from those of pituitary GnRH-Rs. Mechanistically, it is unlikely that this effect is due to inhibition of EGF-stimulated ERK2 phosphorylation, because we saw no such inhibition, and GnRH actually stimulated ERK2 phosphorylation. It is also unlikely to be due to GnRH-R down-regulation, because the antiproliferative effect was increased as receptor number increased. As sustained elevation of [Ca2+]i is antiproliferative and/or proapoptotic for many cells, inhibition of [3H]thymidine incorporation may be mediated by Ca2+ mobilization in this model, and this issue is currently under investigation.

It is important to recognize that the conclusions drawn above pertain to the characteristics of GnRH-Rs expressed exogenously in mammary cancer cells and that these do not necessarily reflect the characteristics of any endogenous GnRH-Rs in such cells. It remains conceivable, for example, that accessory proteins dramatically alter the binding and functional characteristics of GnRH-Rs in extrapituitary sites, but that such proteins are present in limiting amounts, and their effects are therefore not evident at high levels of expression of high affinity receptors. Alternatively, it is possible that glyosylation influences GnRH-R function and that glycosylation patterns differ between endogenous receptors expressed at low levels and high level expression of exogenous receptors. The possibility therefore remains that there are genuine functional differences between the endogenous and exogenous GnRH-Rs in mammary cancer cells. Nevertheless, interest in extrapituitary GnRH-Rs stems primarily from their possible use as targets for cancer therapy using agonists, antagonists, or cytotoxic derivatives thereof (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Regardless of the mechanism of action, our data unambiguously establish that direct activation of GnRH-Rs can indeed inhibit the proliferation of mammary cancer cells and that receptor number is a limiting determinant of this effect. There is currently considerable interest in the improvement and/or development of endocrine and gene therapies for cancer and in the possible combination of these approaches. It has been suggested, for example, that adenovirus-mediated expression of somatostatin receptors may aid in vivo visualization of transgene expression (46). It has also been shown that Ad-mediated transfection with type 2 somatostatin receptors can inhibit the growth of pancreatic tumors and improve the effectiveness of somatostatin receptor-targeted therapy in vivo (47). Such data imply that a vector delivering receptor DNA can be considered as a pro-drug, used to increase the effectiveness of subsequently administered receptor ligand. Although this approach remains highly speculative, the GnRH-R is of particular interest in this context not only because GnRH analogs are already routinely used in hormone-dependent cancer therapy, but also because mammalian GnRH-Rs (unlike all other known GPCRs) do not express C-terminal tails and therefore do not rapidly desensitize (31, 32, 48).

In summary, we have developed Ad GnRH-Rs and shown that these provide an efficient means of expressing GnRH-Rs in mammary cancer cells. These receptors are indistinguishable from pituitary GnRH-Rs (in terms of ligand recognition, signaling, and agonist/antagonist discrimination) and facilitate a pronounced and potent direct antiproliferative effect of GnRH agonists (but not antagonists) on these cells. As the low number of endogenous high affinity GnRH-Rs may limit the therapeutic effects of GnRH analogs on mammary cancer in vivo, endocrine manipulations or gene therapy used to increase the expression of GnRH-Rs in these cells could increase the effectiveness of subsequent therapies in which the GnRH-R is targeted with agonists or their cytotoxic derivatives.


   Acknowledgments
 
We are grateful to Prof. J. Sandow (Aventis Pharma GmbH, Frankfurt, Germany) for providing the buserelin and [125I]buserelin, and to Asta Medica (Frankfurt am Main, Germany), AstraZeneca (Macclesfield, UK), and Ferring Pharmaceuticals Ltd. (Kiel, Germany) for the cetrorelix, Zoladex, and Triptorelin, respectively.


   Footnotes
 
This work was supported in part by the Wellcome Trust (Grant 054949), the Neuroendocrine Charitable Trust (PMS/VW-98/99-139) the Medical Research Council (G78/6046), the South African Research Council, and the National Research Foundation.

Abbreviations: Ad EGFP, Adenovirus expressing enhanced green fluorescent protein; Ad GnRH-R, recombinant adenovirus-expressing GnRH-R; Bmax, binding capacity; [Ca2+]i, cytosolic Ca2+ concentration; cGnRH-II, chicken GnRH-II; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; GnRH-R, GnRH receptor; GPCR, G protein-coupled receptor; moi, multiplicity of infection; MTT, 3-[4,5-dimethylthiazol-2yl]-diphenyltetrazolium bromide; PSS, physiological salt solution.

Received February 20, 2001.

Accepted for publication July 30, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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R. Lopez de Maturana, A. J. Pawson, Z.-L. Lu, L. Davidson, S. Maudsley, K. Morgan, S. P. Langdon, and R. P. Millar
Gonadotropin-Releasing Hormone Analog Structural Determinants of Selectivity for Inhibition of Cell Growth: Support for the Concept of Ligand-Induced Selective Signaling
Mol. Endocrinol., July 1, 2008; 22(7): 1711 - 1722.
[Abstract] [Full Text] [PDF]

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A. R Finch, K. R Sedgley, C. J Caunt, and C. A McArdle
Plasma membrane expression of GnRH receptors: regulation by antagonists in breast, prostate, and gonadotrope cell lines
J. Endocrinol., February 1, 2008; 196(2): 353 - 367.
[Abstract] [Full Text] [PDF]

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 J EndocrinolHome page
K. R Sedgley, A. R Finch, C. J Caunt, and C. A McArdle
Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells
J. Endocrinol., December 1, 2006; 191(3): 625 - 636.
[Abstract] [Full Text] [PDF]