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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
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
). 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. 1, 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. 2), 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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), 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 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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), 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 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. 5).
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, 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. 6, 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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), 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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, 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. 8, 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. 9) 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. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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.
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Acknowledgments |
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Footnotes |
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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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References |
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  K. Morgan, A. J. Stewart, N. Miller, P. Mullen, M. Muir, M. Dodds, F. Medda, D. Harrison, S. Langdon, and R. P. Millar Gonadotropin-Releasing Hormone Receptor Levels and Cell Context Affect Tumor Cell Responses to Agonist In vitro and In vivo Cancer Res., August 1, 2008; 68(15): 6331 - 6340. [Abstract] [Full Text] [PDF]
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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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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]
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 J. N Hislop, C. J Caunt, K. R Sedgley, E. Kelly, S. Mundell, L. D Green, and C. A McArdle Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization? J. Mol. Endocrinol., August 1, 2005; 35(1): 177 - 189. [Abstract] [Full Text] [PDF]
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 E. Levens, X. Luo, L. Ding, R. S. Williams, and N. Chegini Fibromodulin is expressed in leiomyoma and myometrium and regulated by gonadotropin-releasing hormone analogue therapy and TGF-{beta} through Smad and MAPK-mediated signalling Mol. Hum. Reprod., July 1, 2005; 11(7): 489 - 494. [Abstract] [Full Text] [PDF]
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 C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
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 C. J. Caunt, J. N. Hislop, E. Kelly, A.-L. Matharu, L. D. Green, K. R. Sedgley, A. R. Finch, and C. A. McArdle Regulation of Gonadotropin-Releasing Hormone Receptors by Protein Kinase C: Inside Out Signalling and Evidence for Multiple Active Conformations Endocrinology, August 1, 2004; 145(8): 3594 - 3602. [Abstract] [Full Text] [PDF]
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R. P. Millar, Z.-L. Lu, A. J. Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley Gonadotropin-Releasing Hormone Receptors Endocr. Rev., April 1, 2004; 25(2): 235 - 275. [Abstract] [Full Text] [PDF]
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 A. R. Finch, L. Green, J. N. Hislop, E. Kelly, and C. A. McArdle Signaling and Antiproliferative Effects of Type I and II Gonadotropin-Releasing Hormone Receptors in Breast Cancer Cells J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1823 - 1832. [Abstract] [Full Text] [PDF]
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L. E. C. Miles, A. C. Hanyaloglu, J. R. Dromey, K. D. G. Pfleger, and K. A. Eidne Gonadotropin-Releasing Hormone Receptor-Mediated Growth Suppression of Immortalized L{beta}T2 Gonadotrope and Stable HEK293 Cell Lines Endocrinology, January 1, 2004; 145(1): 194 - 204. [Abstract] [Full Text] [PDF]
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 N. Chegini, J. Verala, X. Luo, J. Xu, and R. S. Williams Gene Expression Profile of Leiomyoma and Myometrium and the Effect of Gonadotropin Releasing Hormone Analogue Therapy Reproductive Sciences, April 1, 2003; 10(3): 161 - 171. [Abstract] [PDF]
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N. Chegini, C. Ma, X.M. Tang, and R.S. Williams Effects of GnRH analogues, `add-back' steroid therapy, antiestrogen and antiprogestins on leiomyoma and myometrial smooth muscle cell growth and transforming growth factor-{beta} expression Mol. Hum. Reprod., December 1, 2002; 8(12): 1071 - 1078. [Abstract] [Full Text] [PDF]
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E. Levens, X. Luo, L. Ding, R. S. Williams, and N. Chegini Fibromodulin is expressed in leiomyoma and myometrium and regulated by gonadotropin-releasing hormone analogue therapy and TGF-{beta} through Smad and MAPK-mediated signalling Mol. Hum. Reprod., July 1, 2005; 11(7): 489 - 494. [Abstract] [Full Text] [PDF]
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), or
no additional stimulus (
; 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).
