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Center for Endocrinological Oncology, Department of Endocrinology (P.L., R.M.M., M.M.M., D.D., M.M.), and Department of Medical Pharmacology (M.P.), University of Milano, 20133 Milano, Italy
Address all correspondence and requests for reprints to: Dr. P. Limonta, Center for Endocrinological Oncology, Department of Endocrinology, Via Balzaretti 9, 20133 Milano, Italy. E-mail: limonta{at}mailserver.unimi.it
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i protein. Finally, LHRH-A
significantly counteracted the forskolin-induced increase of
intracellular cAMP levels in both cell lines. These data demonstrate
that the LHRH receptor, which is present in prostate cancer cells,
independently of whether they are androgen-dependent or not,
corresponds to the pituitary receptor, in terms of messenger RNA
expression and protein molecular size. However, at variance with the
receptor of the gonadotrophs, prostate cancer LHRH receptor seems
to be coupled to the Gi protein-cAMP signal transduction
pathway, rather than to the Gq/11-phospholipase C
signaling system. This might be responsible for the different actions
of LHRH in anterior pituitary and in prostate cancer. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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q/G11 protein and
to the phospholipase C signaling pathway (5, 6, 7). In recent years, evidence has been accumulated indicating that LHRH might be present in a variety of extrapituitary tissues, both normal (8, 9, 10, 11) and tumoral (12, 13, 14, 15, 16, 17), where it might act in an autocrine/paracrine fashion.
In line with these observations, we have previously demonstrated that LHRH agonists exert a significant and dose-dependent inhibitory action on the proliferation of prostate cancer cells, either androgen-dependent or androgen-independent, through the binding to specific receptors (18, 19). Moreover, we have found that a messenger RNA (mRNA) coding for LHRH is expressed in these cells, suggesting that the peptide is locally synthesized (19, 20). Taken together, these data indicate that an autocrine/paracrine LHRH system is expressed also in prostate cancer tissue and that this system may participate in the local regulation of tumor growth by inhibiting cell proliferation (18, 19, 20).
At the level of the tumoral prostate, LHRH receptors have been studied, so far, mainly in terms of binding parameters (i.e. affinity constant for LHRH analogs); however, divergent results have been reported by the different authors (18, 19, 21, 22, 23, 24, 25).
The present experiments have been performed to better characterize the LHRH receptor in prostate cancer. To this purpose, we have analyzed the mRNA expression, the molecular size, and the signal transduction pathway of these receptors in two cell lines [androgen-dependent (LNCaP) and androgen-independent (DU 145)] derived from prostate tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell cultures
The cell lines LNCaP-FGC (Lymph Node Carcinoma of the
Prostate-Fast Growing Colony) and DU 145 were obtained from
American Type Culture Collection (Rockville, MD). Both
cell lines (passages 27–35 for LNCaP and 60–70 for DU 145) were
routinely grown in RPMI-1640 medium (Seromed, Biochrom KG, Berlin,
Germany) supplemented with FBS (Life Technologies,
Paisley, Scotland) (10% for LNCaP and 5% for DU 145 cells), glutamine
(1 mM) and antibiotics (100 IU/ml penicillin G sodium, 100
µg/ml streptomycin sulfate) in a humidified atmosphere of 5%
CO2-95% air. In these culture conditions, the duplication
period is 48 h for LNCaP and 36 h for DU 145 cells,
respectively.
Animals
Because of the impossibility of obtaining normal human pituitary
in our country, male rats have been used as positive (pituitary) and as
negative (skeletal muscle) controls in Western blot analysis of LHRH
receptors.
Adult male Sprague Dawley rats were obtained from Charles River Laboratories, Inc. (Calco, Como, Italy). Animals were maintained on a 14-h light, 10-h dark schedule, with standard pellet food and water available ad libitum. All rats were killed by decapitation; tissues were quickly collected and frozen at -70 C until Western blot analysis.
RT-PCR analysis of LHRH receptor mRNA
Total RNA from LNCaP and DU 145 cell lines was prepared
according to a modification of the guanidium thiocyanate/cesium
chloride method (26). RNA (2 µg) from each sample was used in a RT
reaction. Complementary DNA (cDNA) synthesis was performed using the
Gene AMP kit (Perkin-Elmer Corp. Cetus, Norwalk, CT), with
an oligo(dT)16 as a primer for the reverse transcriptase.
Samples containing cDNAs obtained from prostate tumor cells were then
amplified in a 100-µl solution containing PCR buffer (50
mM KCl, 10 mM Tris-HCl), 2 mM
MgCl2, 0.15 µM of a pair of specific primers,
and 2.5 U Taq polymerase. Amplification was carried out for
35 cycles (1-min denaturation at 94 C, 1-min primer annealing at 50 C,
and 3-min primer extension at 72 C). The primers were synthesized,
based on the reported sequence of the human pituitary LHRH receptor
cDNA (27). The primers used were: sense 5'-GCTTGAAGCTCTGTCCTGGGA-3'
(-25 to -5) and antisense 5'-CCTAGGACATAGTAGGG-3' (844–860). These
primers have been previously used by Kakar et al. (14) to
detect the presence of LHRH receptor mRNA in human extrapituitary
tissues. After PCR, the amplified cDNA products were separated on a
1.5% agarose gel and stained with ethidium bromide. Southern blot
analysis was performed as described (28). Blots were hybridized with a
synthetic 32P-labeled oligonucleotide probe (19 mer)
specific for a sequence (nucleotides 392/410) of the human pituitary
LHRH receptor cDNA.
Western blot analysis of the LHRH receptor
Membrane fractions were prepared from rat pituitaries and
skeletal muscle, from LNCaP, and from DU 145 cells, according to the
protocol reported by Karande et al. (29), with some
modifications. Samples were homogenized in 10 mM Tris-HCl
(pH 7.6) buffer containing 1 mM dithiothreitol on ice. The
homogenates were centrifuged two times for 10 min each at 800 x
g to remove cellular debris, and the resulting supernatants
were centrifuged at 18,000 x g to pellet down the
membrane fractions. The pellets were solubilized in RIPA buffer [50
mM Tris-HCl (pH 7.7), 150 mM NaCl, 0.8% Triton
X-100, 0.8% sodium deoxycholate, 0.08% SDS, 10 mM
ethylendiamine tetraacetate, 100 µM
Na3VO4, 50 mM NaF, 0.3
mM phenylmethylsulfonylfluoride, and 5 mM
iodoacetic acid] and electrophoresed on 10% polyacrylamide gel under
reducing conditions. Proteins were transferred onto a nitrocellulose
filter, in 25 mM Tris-HCl (pH 8.3), 92 mM
glycine, and 20% methanol at 30 V overnight. Filters were probed with
the F1G4 monoclonal antibody raised against the human pituitary LHRH
receptor (kindly provided by Dr. A. A. Karande, Department of
Biochemistry, Indian Institute of Science, Bangalore, India), followed
by incubation with an antimouse IgG. Antobody bound to the LHRH
receptor was detected with the ECL-Western blotting detection system
after a 5- to 10-min exposure to a Hyperfilm-ECL x-ray film
(Amersham Pharmacia Biotech, Milan, Italy), at room
temperature (30). The specificity of F1G4 antibody for the human
pituitary LHRH receptor has been previously demonstrated (29).
Cell proliferation studies
LNCaP and DU 145 cells were plated at a density of 5,000 and 500
cells/cm2, respectively, in 6-mm dishes in standard culture
medium. Cells were allowed to attach and start growing for 3 days; the
seeding media were then changed to experimental media. Cells were
treated, for 7 days, with LHRH-A (1 µM) either in the
absence or in the presence of PTX (25 ng/ml for LNCaP and 50 ng/ml for
DU 145 cells); the medium was changed every 2 days. At the end of the
treatment, cells were collected and counted by hemocytometer. The dose
of LHRH-A selected for this and the following experiments has been
chosen on the basis of previous papers from our laboratory analyzing
the interaction between LHRH and stimulatory growth factors in prostate
cancer cells (31, 32).
ADP-ribosylation
ADP-ribosylation was carried out as described (33). Briefly,
isolated plasma membranes (0.5 mg/ml) from LNCaP and DU 145 cells were
incubated with PTX (2 µg/ml) in 20 mM Tris-HCl, pH 7.5,
containing 1 mM ATP, 0.1 mM GTP, 1
mM EDTA, 1 mM dithiothreitol, 10 mM
thymidine, 10 µM 32P-NAD (5 x
106 cpm/nmol), either in the absence or in the presence of
LHRH-A (1 µM), in a final vol of 200 µl. After
incubation for 30 min at 37 C, the reaction was terminated by the
addition of 1 ml ice-cold 20 mM Tris-HCl, pH 7.5. Membranes
were pelleted by centrifugation and washed twice with the same buffer.
Membrane proteins were solubilized in Laemmli’s SDS sample buffer and
resolved by 12% PAGE. After electrophoresis, gels were dried, and
ADP-ribosylated proteins were detected by autoradiography.
Western blot analysis of Gi1 subunit protein
The polyclonal rabbit antiserum I1C, directed toward a synthetic
peptide corresponding to aminoacids 160–169 (only present in the
Gi1 subunit) was kindly donated by G. Milligan (Division
of Biochemistry and Molecular Biology, University of Glasgow, Glasgow,
Scotland, UK) (34). Membrane preparations, protein separation, and
electrophoretic transfer to nitrocellulose filters were performed as
described under Western blot analysis of LHRH receptors.
Blots were incubated overnight at 4 C in TBST buffer (20 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing
5% powdered skim milk. After five washes with TBST, membranes were
incubated for 3 h at room temperature with I1C primary antibody
diluted in milk/TBS and for 1.5 h with horseradish
peroxidase-conjugated goat antirabbit IgG. Proteins were detected using
the SuperSignal detection kit (Pierce Chemical Co.,
Rockford, IL).
cAMP determination
LNCaP and DU 145 cells were plated at a density of 30,000
cells/cm2 in 24-multiwell plates. After 2 days, cells were
washed with 1 ml serum-free medium, pretreated with
3-isobutyl-1-methylxantine (0.5 mM) (Sigma Chemical Co.) for 15 min at 37 C and then treated with forskolin
(FSK, 5 µM), either alone or in the presence of LHRH-A (1
µM), for 15 min at 37 C. After the treatment, cells were
extracted with ethanol 65% at 4 C for 5 min, two times, and
centrifuged for 3 min (10,000 x g). The supernatants
were collected, dried, and stored at -20 C. cAMP content in each
sample was determined by the 3H-cAMP assay system
(Amersham Pharmacia Biotech), according to the
manufacturer’s instructions.
Statistical analysis
The data from experiments involving cell proliferation and cAMP
determination were analyzed according to Dunnett’s test (35) after
one-way ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) was obtained both in LNCaP (lane 1)
and in DU 145 (lane 2) cells. Negative control samples without RT did
not give rise to any detectable product (lane 3), ruling out the
possibility of genomic DNA contamination. Gels were Southern blotted
and hybridized with a synthetic 32P-labeled oligonucleotide
probe specific for the pituitary LHRH receptor cDNA. The cDNA fragments
obtained from LNCaP and DU 145 cells specifically hybridized with the
labeled probe (Fig. 1B, lanes 1 and 2). No hybridization signal was
found in the negative control (Fig. 1B, lane 3).
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, a major protein band of approximately
64 kDa molecular mass is identified by the antibody both in LNCaP and
in DU 145 cells (lanes 1 and 2, respectively). This molecular size
corresponds to that reported for the human pituitary LHRH receptor (29, 36). Fig. 3 (lanes 3 and 4) confirms the
presence of a 64-kDa protein binding the F1G4 antibody in prostate
cancer cell lines. Moreover, Fig. 3 (lane 1) also shows that, in rat
pituitary protein preparations, the antibody identified a band of
approximately 60 kDa; this molecular mass corresponds to that
previously described by Wormald et al. (36) for the rat
pituitary LHRH receptor. No binding of the F1G4 antibody to protein
preparations from rat skeletal muscle samples could be detected (Fig. 3, lane 2).
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q/11-phospholipase C system as occurs at the
pituitary level. Therefore, we hypothesized that, in these cells, the
antiproliferative action of LHRH agonists might be mediated by the
Gi-cAMP signal transduction pathway. It is known that
PTX, through ADP-ribosylation of Gi proteins, impairs
the receptor-effector interaction (37). Therefore, we have studied
whether PTX might interfere with the antimitogenic action of LHRH-A. As
expected, the LHRH agonist significantly inhibited LNCaP cell growth
(Fig. 4A), around 30% in the experiments
here reported. In previous studies, we have demonstrated that this
antiproliferative action is specific, because it can be blocked by a
second-generation antagonist (Nal-Arg-LHRH) (18, 19). More recently,
third-generation antagonists have been reported to act as agonists in
this and in similar experiments (38, 39). Fig. 4 also shows that the
treatment with PTX alone did not affect cell proliferation. On the
other hand, when PTX and LHRH-A were given together, PTX completely
prevented the antiproliferative action of the LHRH agonist (Fig. 4A).
Similar results were obtained in DU 145 cells (Fig. 4B).
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i protein. To further confirm this
hypothesis, we investigated whether LHRH-A might affect PTX-induced
ADP-ribosylation of the Gi protein. As expected,
incubation of prostate cancer cell membranes with PTX, in the presence
of 32P-NAD, brought about ADP-ribosylation of a 41-kDa
Gi protein in both LNCaP and DU 145 cells (Fig. 5, A and B; lane 1). LHRH-A substantially
counteracted the transfer of 32P-ADP-ribose to the
Gi protein in the two cell lines (Fig. 5, A and B; lane
2).
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i proteinsi subunit proteins in prostate
cancer cells was evaluated by Western blot analysis. Fig. 6 shows that, after immunoblotting with
the specific antibody, the 41-kDa Gi1 subunit could be
detected in LNCaP as well as in DU 145 cells and suggests that this
specific Gi protein subunit might mediate LHRH activity
in prostate cancer cells. In these studies, Western blot analysis has
been performed with an antibody specifically recognizing the 41-kDa
Gi1 protein subunit. Obviously, the possibility that
additional Gi subunits might be present (and/or coupled
to the LHRH receptor) in prostate cancer cells cannot be ruled out.
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i1
protein seem different in LNCaP and in DU 145 cells. This and other G
proteins are not exclusively linked to the LHRH receptor; they may
represent the beginning of the signaling pathways for other growth
regulatory mechanisms that have not been investigated in the present
study.
Effects of LHRH-A on cAMP accumulation
Activation of Gi subunit proteins is negatively
correlated with cAMP production. To further confirm that, in prostate
cancer cells, LHRH receptors might be coupled to Gi
proteins, we studied the effects of LHRH-A on FSK-induced cAMP
accumulation, both in LNCaP and in DU 145 cells. In LNCaP cells (Fig. 7A), LHRH-A, when given alone, did not
affect cAMP levels. FSK, as expected, substantially stimulated cAMP
accumulation. LHRH-A significantly counteracted the increase in cAMP
levels induced by FSK, an effect which could be blocked by a
second-generation antagonist (Nal-Arg-LHRH, 10-8
M) (data not shown). Similar results were obtained in DU
145 cells (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data obtained show, by RT-PCR, that a mRNA coding for the LHRH receptor can be detected in LNCaP as well as in DU 145 cells. In line with this observation, the expression of the mRNA for LHRH receptors has been previously reported in some peripheral tumors, such as breast, endometrial, and ovarian cancers (13, 14, 15, 16, 17).
In LNCaP and in DU 145 cells, the mRNA is further translated into a receptor protein. This is based on the observation that both cell lines, when analyzed by Western blot using a monoclonal antibody raised against the human pituitary LHRH receptor, reveal the presence of a protein of the same molecular size of the receptor found in normal human gonadotrophs (64 kDa) (29, 36).
The present data, obviously, do not exclude that the gene coding for this protein might have undergone a mutation and that, consequently, the protein, even with the same molecular size, might present some deviation from the classical receptor. This would be in line with the reports from us and from others showing that the binding affinity of the receptor in prostate and in other tumoral tissues might be significantly lower than that observed in the pituitary (18, 19, 22, 40, 41). Because of the findings by Kakar et al. (14) and those here reported, which have used the same methodology, one would expect the eventual mutation to reside outside the 885-bp fragment amplified by Kakar et al. (14) and in the present work.
Some doubts have been raised on the ability of the antibody used in the experiments here reported to recognize the authentical LHRH receptor protein. This argument may be counteracted by the following considerations. First, this antibody fully recognizes the 29-amino acid peptide, corresponding to the N-terminal portion of the LHRH receptor protein, which has been used to raise it (29). Moreover, in immunohistochemical studies, the antibody stains clusters of cells in the human pituitary. This staining is specific, because the reaction can be stopped by a preincubation of the antibody with an excess of the antigenic peptide before incubation with the tissue section (29). By Western blot analysis, the antibody will recognize the typical 64-kDa band of the LHRH receptor in human pituitary (29), as well as in human placenta and in breast carcinoma (A. A. Karande, personal communication) membrane proteins. The same band will also bind a 125I-labeled LHRH, showing its ability to act as a full receptor. Finally, by immunohistochemistry, COS-7 cells transfected with the LHRH receptor cDNA show a specific positive staining to the monoclonal antibody (A. A. Karande, personal communication).
At the pituitary level, LHRH receptors are coupled, via the
Gq/G11 group of G proteins, to PLC (5, 6, 7).
The data here reported indicate that, in prostate tumor cells, the LHRH
receptor is linked to Gi proteins which, through the
inhibition of cAMP accumulation, probably mediate the antiproliferative
action of the peptide. These conclusions are based on the following
observations: 1) treatment with PTX counteracts the antiproliferative
action of LHRH agonists; 2) LHRH agonists cause a profound reduction in
the PTX-mediated ADP-ribosylation of a membrane protein of relative
molecular mass 41 kDa corresponding to Gi1, as revealed
by Western blot analysis, performed using a specific antibody; 3) LHRH
agonists significantly counteract FSK-induced increase of intracellular
cAMP levels. In the authors’ opinion, this is the first demonstration
of the coupling of the LHRH receptor to the Gi-cAMP
signal transduction pathway in prostate cancer, in addition to the
identification of the molecular size of the LHRH receptor protein (see
above). In line with the present data, Imai and co-workers (42) have
recently reported that, in tumors of the human female reproductive
tract, LHRH receptors are coupled to a 41-kDa Gi
protein; these authors suggested that this protein might mediate the
LHRH-induced phosphotyrosine phosphatase activity in tumor cells (42, 43).
We failed to observe any change in phosphoinositide metabolism and/or
Ca2+ levels in prostate cancer cells after the treatment
with LHRH agonists (unpublished observations), which inhibit cell
proliferation; this suggests that the LHRH receptor might not be linked
to the Gq/11-PLC system in these cells. In agreement
with our observations, Emons and co-workers (44) have shown that, in
human ovarian and endometrial cancer cells, the LHRH agonist
triptorelin, at concentrations that are clearly inhibitory on cell
proliferation, does not affect PLC or PKC activity. However, at
variance with these observations, LHRH agonists have been reported to
stimulate PLC activity in other types of tumors, such as rat (45) and
human (46) mammary tumors, as well as human ovarian cancers (47).
Therefore, the possible involvement of the Gq/11
signaling system in the antimitogenic action of LHRH still remains an
open question and, certainly, needs further studies.
Taken together, the present data strongly indicate that, in prostate
tumor cells, the LHRH receptor is expressed and corresponds to the
pituitary LHRH receptor, in terms of molecular weight. Interestingly,
the signal transduction pathway of this receptor
(Gi-cAMP pathway) seems to be different from that of the
same receptors at pituitary level (Gq/11-PLC system)
(Fig. 8). This fact might be
responsible for the different actions of LHRH in peripheral tumors and
in the anterior pituitary. In gonadotrophs, LHRH receptor-coupled
Gq/11 proteins mediate the stimulatory action of the
hypothalamic hormone on gonadotropin synthesis and release (5, 6, 7). In
prostate cancer cells, on the contrary, LHRH may behave as an
inhibitory autocrine/paracrine factor, which exerts its antimitogenic
action through the activation of Gi proteins, negatively
coupled to the c-AMP intracellular signaling pathway. It is noteworthy,
in this context, that LHRH receptor mRNA, molecular size, and signal
transduction pathway seem to be the same in androgen-dependent and
androgen-independent prostate cancer cells.
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Acknowledgments |
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Footnotes |
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Received February 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Biochem
Biophys Res Commun 189:601–609[CrossRef][Medline]
subunits. Methods Enzymol 237:268–283[Medline]
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K. Morgan, D. Conklin, A. J. Pawson, R. Sellar, T. R. Ott, and R. P. Millar A Transcriptionally Active Human Type II Gonadotropin-Releasing Hormone Receptor Gene Homolog Overlaps Two Genes in the Antisense Orientation on Chromosome 1q.12 Endocrinology, February 1, 2003; 144(2): 423 - 436. [Abstract] [Full Text] [PDF]
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R. M. Moretti, M. Montagnani Marelli, J. C. Van Groeninghen, and P. Limonta Locally Expressed LHRH Receptors Mediate the Oncostatic and Antimetastatic Activity of LHRH Agonists on Melanoma Cells J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3791 - 3797. [Abstract] [Full Text] [PDF]
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 A. Wells, J. C. S. Souto, J. Solava, J. Kassis, K. J. Bailey, and T. Turner Luteinizing Hormone-releasing Hormone Agonist Limits DU-145 Prostate Cancer Growth by Attenuating Epidermal Growth Factor Receptor Signaling Clin. Cancer Res., April 1, 2002; 8(4): 1251 - 1257. [Abstract] [Full Text] [PDF]
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B. Straub, M. Muller, H. Krause, M. Schrader, C. Goessl, R. Heicappell, and K. Miller Increased Incidence of Luteinizing Hormone-releasing Hormone Receptor Gene Messenger RNA Expression in Hormone-refractory Human Prostate Cancers Clin. Cancer Res., August 1, 2001; 7(8): 2340 - 2343. [Abstract] [Full Text] [PDF]
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C. Grundker, P. Volker, and G. Emons Antiproliferative Signaling of Luteinizing Hormone-Releasing Hormone in Human Endometrial and Ovarian Cancer Cells through G Protein {{alpha}}I-Mediated Activation of Phosphotyrosine Phosphatase Endocrinology, June 1, 2001; 142(6): 2369 - 2380. [Abstract] [Full Text] [PDF]
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N. Auersperg, A. S. T. Wong, K.-C. Choi, S. K. Kang, and P. C. K. Leung Ovarian Surface Epithelium: Biology, Endocrinology, and Pathology Endocr. Rev., April 1, 2001; 22(2): 255 - 288. [Abstract] [Full Text]
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L. Z. Krsmanovic, N. Mores, C. E. Navarro, M. Tomic, and K. J. Catt Regulation of Ca2+-Sensitive Adenylyl Cyclase in Gonadotropin-Releasing Hormone Neurons Mol. Endocrinol., March 1, 2001; 15(3): 429 - 440. [Abstract] [Full Text]
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S. K. Kang, C.-J. Tai, P. S. Nathwani, K.-C. Choi, and P. C. K. Leung Stimulation of Mitogen-Activated Protein Kinase by Gonadotropin-Releasing Hormone in Human Granulosa-Luteal Cells Endocrinology, February 1, 2001; 142(2): 671 - 679. [Abstract] [Full Text] [PDF]
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K. W. Cheng, P. S. Nathwani, and P. C. K. Leung Regulation of Human Gonadotropin-Releasing Hormone Receptor Gene Expression in Placental Cells Endocrinology, July 1, 2000; 141(7): 2340 - 2349. [Abstract] [Full Text] [PDF]
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