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I-Mediated Activation of Phosphotyrosine Phosphatase1
Department of Gynecology and Obstetrics, Georg August University, D-37070 Gottingen, Germany
Address all correspondence and requests for reprints to: Prof. Dr. Günter Emons, Department of Gynecology and Obstetrics, Robert Koch Street 40, D-37075 Gottingen, Germany. E-mail: emons{at}med.uni-goettingen.de
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Abstract |
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The present experiments were performed to analyze in endometrial and ovarian cancer cells: 1) whether mutations or splice variants of the LHRH receptor are responsible for differences in LHRH signaling, 2) the coupling of G protein subtypes to LHRH receptor, 3) the phosphotyrosine phosphatase (PTP) activation counteracting growth factor receptor tyrosine kinase activity. For these studies, the well characterized human Ishikawa and Hec-1A endometrial cancer cell lines and human EFO-21 and EFO-27 ovarian cancer cell lines were used, which express LHRH and its receptor.
1) Sequencing of the complementary DNA of the LHRH receptor from
position 31 to position 1204, covering the complete coding region
(position 56 to position 1042) showed that there are neither mutations
nor splice variants of the LHRH receptor transcript in Ishikawa and
Hec-1A endometrial cancer cells or in EFO-21 and EFO-27 ovarian cancer
cells. 2) All analyzed cell lines except for the ovarian cancer cell
line EFO-27 expressed both G proteins, 
i and
q, as shown by RT-PCR and Western blotting. In the
EFO-27 cell line only G protein i, not G protein
q, expression was found. Cross-linking experiments using
disuccinimidyl suberate revealed that in the cell lines expressing G
protein i and G protein q, both G
proteins coupled to the LHRH receptor. Inhibition of epidermal growth
factor (EGF)-induced c-fos expression by LHRH, however,
was mediated through pertussis toxin (PTX)-sensitive G protein
i. Moreover, LHRH substantially antagonized the
PTX-catalyzed ADP-ribosylation of G protein i. 3) Using
a phosphotyrosine phosphatase assay based on molybdate-malachite green,
treatment of quiescent EFO-21 and EFO-27 ovarian cancer cells and
quiescent Ishikawa and Hec-1A endometrial cancer cells with 100
nM of the LHRH agonist triptorelin resulted in a 4-fold
increase in PTP activity (P < 0.001). This effect
was completely blocked by simultaneous treatment with PTX, supporting
the concept of mediation through G protein i. As shown
by quantitative Western blotting, EGF-induced tyrosine
autophosphorylation of EGF receptors was reduced 45–63% after LHRH
(100 nM) treatment (P < 0.001). This
effect was completely blocked using the PTP inhibitor vanadate
(P < 0.001).
These results demonstrate that mutations or splice variants of the LHRH
receptor in human endometrial and ovarian cancer cells are not
responsible for the different signal transduction compared with that in
pituitary gonadotrophs. We provide evidence that the tumor LHRH
receptor couples to multiple G proteins, but the antiproliferative
signal transduction is mediated through the PTX-sensitive G protein
i. The tumor LHRH receptor activates a PTP counteracting
EGF-induced tyrosine autophosphorylation of EGF receptor, resulting in
down-regulation of mitogenic signal transduction and cell
proliferation.
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Introduction |
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q/11 and to the
phospholipase C signaling pathway (4, 5, 6 ). In addition to
this well documented classic hypophysiotropic actions, LHRH is present
in the brain and a variety of peripheral organs, both normal and
tumoral, where it might act in an autocrine/paracrine fashion
(7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Expression of LHRH and its receptor has been
detected in most endometrial and ovarian cancer cell lines and in over
80% of biopsy specimens of these cancers (11). The
proliferation of the cancer cell lines that express LHRH receptors, was
inhibited by both agonistic and antagonistic analogs of LHRH,
indicating that the dichotomy of LHRH agonists and antagonists does not
exist in tumor cells (11). These antiproliferative effects
were evident at nanomolar concentrations of the LHRH analogs,
suggesting that they are mediated through the LHRH receptors in the
tumor cells (11). The classical LHRH receptor signal transduction mechanisms known to operate in the pituitary (6, 17) are not involved in the mediation of antiproliferative effects of LHRH analogs in cancer cells (18). LHRH analogs instead interfere with the mitogenic signal transduction of growth factor receptors and related oncogene products associated with tyrosine kinase activity, resulting in a down-regulation of growth factor-induced c-fos expression (18, 19). The reasons for LHRH inducing this specific signaling in cancer cells, however, are still obscure.
The present experiments were performed to clarify the reasons for the differences in LHRH signal transduction in endometrial and ovarian cancer cells compared with that acting in pituitary gonadotrophs. Both endometrial and ovarian epithelial cancers develop from the Müllerian epithelium and have several features in common, in particular regarding their LHRH system (11). As this system has been well characterized in these cancers (11) we choose them as a model for our present experiments.
Experimentally induced mutations of the LHRH receptor have altered LHRH
binding, G protein-receptor interaction, or proper membrane
incorporation (20, 21, 22, 23, 24, 25). Some normal and neoplastic human
tissues were found to express differential splice variants of the LHRH
receptor gene in a tissue-dependent manner (26).
Therefore, it seemed reasonable to check whether mutations or splice
variants of LHRH receptors expressed in human cancers are responsible
for the signaling mechanisms different from that acting in pituitary
gonadotrophs. If this was not the case, it might be possible that a
different coupling to G proteins is responsible for the distinct LHRH
signal transduction in endometrial and ovarian cancer cells. To check
whether a different cellular equipment of G proteins exists in the
tumor cells, we analyzed which subtypes of G proteins are expressed in
ovarian and endometrial cancer cells. To show directly which subtype of
G proteins couples to LHRH receptor in the tumor cells, we performed
cross-linking experiments using disuccinimidyl suberate (DSS). To
confirm the hypothesis that LHRH analogs act through G protein
i, we investigated whether LHRH affects
PTX-induced ADP-ribosylation of the G protein
i. In addition, we analyzed whether PTX
inhibits LHRH-induced down-regulation of epidermal growth factor
(EGF)-induced c-fos expression.
It has been speculated that LHRH activates a phosphotyrosine
phosphatase (PTP) and thus antagonizes growth factor-induced tyrosine
phosphorylation (11). The antiproliferative effects of
LHRH analogs might be directly mediated through inhibition of growth
factor signaling on its first step, the autophosphorylation of tyrosine
residues of growth factor receptors. Some indirect evidence that LHRH
activates a PTP was found by Imai et al. (27),
showing that LHRH reduces the net tyrosine phosphorylation of membrane
proteins. Direct proof of the G protein
i-mediated activation of a PTP and the
reduction of the EGF receptor tyrosine phosphorylation by LHRH has not
been provided to date. To assess whether LHRH activates a PTP that
counteracts EGF receptor tyrosine kinase activity in endometrial and
ovarian cancer cells, we analyzed whether LHRH activates a PTP and
reduces EGF receptor phosphorylation. In addition, we analyzed whether
activation of PTP is mediated through the PTX-sensitive G protein
i.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LHRH analogs
The LHRH agonist
[D-Trp6]LHRH (triptorelin) was
provided by Ferring Pharmaceuticals Ltd. Arzneimittel
(Kiel, Germany).
Isolation of RNA and complementary DNA (cDNA) synthesis
Total RNA was prepared from cells grown in monolayer using the
RNeasy protocol (QIAGEN, Hilden, Germany). The
concentration of RNA in each sample was determined by
photospectroscopy. First strand cDNA was generated by RT of 4 µg
total RNA using p(deoxythymidine)15 primers
(Roche Molecular Biochemicals, Mannheim, Germany) with
Moloney murine leukemia virus reverse transcriptase according to the
instructions of the suppliers (Life Technologies, Inc.,
Karlsruhe, Germany). After determining the concentrations of the cDNAs,
the samples were used for PCR analysis. The integrity of the samples
was tested by RT-PCR of the housekeeping gene GAPDH (forward primer,
5'-CAT CAC CAT CTT CCA GGA GCG AGA-3'; backward primer, 5'-GTC TTC TGG
GTG GCA GTG ATG G-3').
RT-PCR of G proteins
The cDNAs (2 ng) were amplified in a 50-µl reaction volume
containing 10 mM Tris-HCl (pH 8.3), 50 mM
potassium chloride, 1.5 mM magnesium chloride, 200
µM of each of the deoxy-NTPs, 1 µM of the
appropriate primers (G protein i: forward
primer, 5'-CAG TCC ATC ATT GCA ATC ATA AGA-3'; backward primer, 5'-CTC
AGC CAG AAC AAG GTC ATA ATC-3'; G protein q:
forward primer, 5'-ATG ACT TGG ACC GTG TAG CCG ACC-3'; backward primer,
5'-CCA TGC GGT TCT CAT TGT CTG ACT-3'), and 1.25 U Taq
polymerase (Roche Molecular Biochemicals) in a
Perkin-Elmer Corp. DNA thermal cycler 2400 (Weiterstadt,
Germany). Twenty-five cycles of amplification were carried out:
denaturation at 94 C for 30 sec, annealing at 55 C (G protein
i) or 60 C (G protein
q) for 30 sec, followed by extension at 72 C
for 60 sec. The PCR product amplified with the G protein
i primers has a total length of 474 bp. The
PCR product amplified with the G protein q
primers has a total length of 260 bp. The respective DNA products were
run on 1.5% agarose gels, and bands were visualized by ethidium
bromide staining on an UV transilluminator.
Sequence analysis of LHRH receptor messenger RNA (mRNA)
For LHRH receptor sequencing and splice variant analysis, the
following oligonucleotide primers (Fig. 1
) were designed according the sequence
found by Kakar et al. (32): LHRH-R-1: forward
primer, 5'-GCT TGA AGC TCT GTC CTG GG-3'; backward primer, 5'-CAG GCT
GAT CAC CAC CAT CAT-3' (positions 31–466); LHRH-R-2: forward primer,
5'-AGT CCA ATG GTA TGC TGG AGA-3' (positions 367–777); backward
primer, 5'-ACC CGT GTC AGG GTG AAG AT-3'; and LHRH-R-3: forward primer,
TCA TGC TGA TCT GCA ATG CAA-3'; backward primer, AAT TGA GGC TCT GAA
GAC TGA GT-3' (positions 732-1204). The PCR runs were carried out under
the same conditions as those described above: denaturation at 94 C for
30 sec, annealing at 60 C for 30 sec, followed by extension at 72 C for
60 sec. The PCR product amplified with the LHRH-R-1 primers (positions
31–466) has a total length of 436 bp, the PCR product amplified with
the LHRH-R-2 primers (positions 367–777) has a total length of 411 bp,
and the PCR product amplified with the LHRH-R-3 primers (positions
732-1204) has a total length of 473 bp.
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Plasma membrane isolation
Cells were collected by centrifugation at 200 x
g and washed twice with PBS/BSA. After counting aliquots,
cells were suspended and homogenized using an all-glass Potter
homogenizer (Braun, Melsungen, Germany) in 10 mmol/liter Tris-HCl
buffer, pH 7.6, containing 2 g BSA/liter, 2 g
NaN3/liter, and 1 mmol/liter dithiothreitol (DTT;
Merck & Co., Darmstadt, Germany). After removing nuclei
and debris by centrifugation at 200 x g, plasma
membranes were collected at 70,000 x g. Aliquots of
the membrane preparations, equivalent to 300,000–400,000 cells, were
resuspended in lysis buffer (1 mmol/liter EGTA, 1 mmol/liter DTT, and
10 mmol/liter Tris-HCl, pH 7.4).
Western blotting of G proteins
Cell membranes were electrophoresed on SDS-PAGE (7.5%) under
reducing conditions and transferred to nitrocellulose. The
nitrocellulose membranes were blocked in 3% BSA (Sigma,
St. Louis, MO) in TBST [10 mM Tris (pH 8), 500
mM NaCl, and 0.1% Tween 20] for 2 h, incubated with
polyclonal rabbit antihuman G protein i or G
protein q antibodies (a gift from Dr. Hinsch,
Giessen, Germany) in a 1:1,000 dilution in 1% BSA in TBST for 1
h, and then, after washings, incubated with horseradish
peroxidase-conjugated antirabbit IgG in an 1:10,000 dilution in 1% BSA
in TBST (Amersham Pharmacia Biotech, Aylesbury, UK) for
1 h. After washings, specifically bound antibody was detected
using the enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech).
ADP-ribosylation
ADP-ribosylation was carried out as described previously
(21). Briefly, isolated plasma membranes (0.5 mg/ml) were
incubated with PTX (2 µg/ml) in 20 mmol/liter Tris-HCl, pH 7.5,
containing 1 mmol/liter ATP, 1 mmol/liter EDTA, 1 mmol/liter DTT, 10
mmol/liter thymidine, 10 µmol/liter [32P]NAD
(5 x 106 cpm/nmol), and the ligand tested
in a final volume of 200 µl. After incubation for 30 min at 37 C, the
reaction was quenched by adding 1 ml ice-cold 20 mmol/liter Tris-HCl,
pH 7.5, containing 1 mmol/liter EDTA. Membranes were pelleted by
centrifugation and washed twice in the same buffer. Membrane proteins
were solubilized in Laemmli’s SDS sample buffer and resolved by 15%
PAGE as described above (Western blotting). ADP-ribosylated proteins
were detected by autoradiography.
Cross-linking analysis
After washing in PBS containing 0.1% BSA (three times, 10 min
each time), the cells were placed in PBS containing 0.75 mM
disuccinimidyl suberate (DSS), diluted from a 25-mM stock
in dimethylsulfoxide. Cross-linking was allowed to proceed at 25 C for
15 min and was terminated by placing the cells in 10 mM
Tris-HCl, pH 7.4, containing 1 mM EDTA and 0.15
M NaCl for 30 min at 4 C. The cells were then lysed as
described above. Protein separation was performed by SDS-PAGE in 7.5%
gels. The mol wt standard was obtained from Pharmacia Biotech (Freiburg, Germany). After electrophoresis, the proteins
were transferred to nitrocellulose. The nitrocellulose membranes were
blocked in 3% bovine serum as described above and then, after
washings, were incubated with a monoclonal antibody raised against the
human pituitary LHRH receptor (provided by Dr. A. A. Karande,
Bangalore, India) in a 1:500 dilution in 1% BSA in TBST for 1 h
or with polyclonal rabbit antihuman G protein
i or G protein q
antibodies in a 1:1,000 dilution in 1% BSA in TBST for 1 h, and
then, after washings, incubated with horseradish peroxidase-conjugated
antimouse IgG (LHRH receptor) or antirabbit IgG (G proteins) in a
1:10,000 dilution in 1% BSA in TBST (Amersham Pharmacia Biotech) for 1 h. After washings, specifically bound
antibody was detected using the ECL kit (Amersham Pharmacia Biotech).
Quantification c-fos mRNA expression
Semiquantitative RT-PCR of c-fos was carried out as
described in detail previously (19). Briefly, a 161-bp
internal standard was generated by PCR containing synthetic DNA and
c-fos-specific primer sites.
The PCR product amplified with the c-fos primers (forward primer, 5'-GAG ATT GCC AAC CTG CTG AA-3'; backward primer, 5'-AGA CGA AGG AAG ACG TGT AA-3') has a total length of 483 bp. For determining the optimal concentration of internal standard used in semiquantitative PCR, internal standard and target cDNA were added to the PCR tubes in inverse serial dilutions. PCR products were separated by gel electrophoresis in 1.5% agarose. PCR reactions yielding standard and target signals of identical intensity were used for PCR analysis for determination of c-fos expression levels. The respective DNA products were run on 1.5% agarose gels, and bands were visualized by ethidium bromide staining on an UV transilluminator. The bands were quantified using the Kodak 1D image system (Kodak, New Haven, CT) in comparison with basal c-fos expression levels.
PTP assay
The cells were plated at a density of 106
cells in 100-mm dishes and grown under standard conditions. After 2
days, the cells (
90% confluence) were incubated in serum-free
medium for 24 h before treatment with or without 100
nM of the LHRH agonist triptorelin with or without PTX for
15 min. The stimulation reactions were stopped by washing with ice-cold
PBS. After washing the cells were detached immediately with 1 ml of a
solution containing 0.5 g trypsin (Biochrom, Berlin,
Germany) and 5 mmol EDTA in 1 liter PBS/BSA, counted, and lysed
in 50 mM ß-glycerophosphate (pH 7.3), 2 mM
EDTA, 1 mM EGTA, 5 mM ß-mercaptoethanol,
1% Triton X-100, 1.0 mM benzamidine, 0.1 mM
phenylmethylsulfonylfluoride, 20 µg/ml leupeptin, 1 µM
pepstatin A, and 1 µg/ml aprotinin. Endogenous phosphate was removed
using Sephadex G-25 spin columns (Promega Corp., Madison,
WI). Tyrosine phosphatase activity was measured using the malachite
green detection assay (Promega Corp.), initially described
by Harder et al. (33). The synthetic
PTP-specific phosphopeptide used in this assay was END(pY)INASL
(34). The assay was performed with 5–15 µg protein,
initiated by the addition of enzyme sample, and incubated at 23 C for
15 min. Reaction was stopped by adding molybdate-malachite green. Data
resulting from this assay are expressed as a percentage of the
untreated controls (=100%).
Quantification of phosphorylated EGF receptor
The cells were plated at a density of 106
cells in 100-mm dishes and grown under standard conditions. After 2
days, culture media were changed to FCS-free and phenol red-free medium
for 72 h. The quiescent cells were incubated with 100
nM bovine EGF (Sigma) for 30 min with or
without 10 µM of the LHRH agonist triptorelin in the
absence or presence of 100 µM sodium vanadate
(Sigma). After incubation the cells were detached
immediately with 1 ml of a solution containing 0.5 g trypsin
(Biochrom) and 5 mmol EDTA in 1 liter PBS/BSA and counted. Plasma
membranes were isolated as described above and than lysed using a
buffer containing 9.5 M urea, 2.0% Nonidet P-40, and 5.0%
ß-mercaptoethanol. The cell lysates were electrophoresed on SDS-PAGE
(7.5%) under reducing conditions and transferred to nitrocellulose.
The nitrocellulose membranes were blocked in 3% BSA
(Sigma) in TBST [10 mM Tris (pH 8), 500
mM NaCl, and 0.1% Tween 20] for 2 h, incubated with
polyclonal rabbit antihuman phosphotyrosine (Promega Corp., Mannheim, Germany) in a 1:100 dilution in 1% BSA in TBST
for 1 h, and then, after washings, incubated with horseradish
peroxidase-conjugated antirabbit IgG in a 1:1500 dilution in 1% BSA in
TBST (Amersham Pharmacia Biotech) for 1 h. After
washings, specifically bound antibody was detected using the ECL kit
(Amersham Pharmacia Biotech). The bands were analyzed
using the Kodak 1D image system.
Statistical analysis
All experiments were reproduced three times in different
passages of the respective cell lines. Data were tested for significant
differences using the Mann-Whitney U test. The data from the
phosphatase experiments were tested for significant differences by
one-way ANOVA, followed by Student-Newman-Keuls test for comparison of
individual groups, after a Bartlett test had shown that variances were
homogenous.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the
oligonucleotide primers pairs used for LHRH receptor sequence analysis
and the respective PCR products. The primers were designed according to
the sequence found by Kakar et al. (32), taking
care that the cDNA fragments overlapped. Figure 2 shows the nucleotide sequence of the
human LHRH receptor mRNA in EFO-21 and EFO-27 ovarian cancer cells and
in Hec-1A and Ishikawa endometrial cancer cells. Sequencing of the cDNA
of the LHRH receptor from position 31 to position 1204, covering the
complete coding region (positions 56–1042) showed no point mutations,
deletions, or insertions in any of the analyzed cell lines (Figs. 1 and 2).
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i, G protein
q, or both, we assessed the presence of G
protein i and G protein
q mRNA (Fig. 3)
and immunoreactivity (Fig. 4). Both G
proteins were expressed in the ovarian cancer cell line EFO-21 and the
endometrial cancer cell lines Ishikawa and Hec-1A, but not in the
ovarian cancer cell line EFO-27. In this cell line only the G protein
i was detectable.
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i. To further confirm this hypothesis, we
investigated whether LHRH agonists might affect PTX-induced
ADP-ribosylation of the G protein i in the
ovarian cancer cell lines EFO-21 and EFO-27 and the endometrial cancer
cell lines Ishikawa and Hec-1A (Fig. 5).
G Proteins in the cell membranes, which are isolated in the absence of
GTP, GTP analogs, or other activators, are in their inactive trimeric
form and are good substrates for PTX-catalyzed ADP-ribosylation.
Incubation of membranes of the above-mentioned cell lines with
[32P]NAD and PTX in the absence of GTP, GTP
analogs, or other activators resulted in a marked PTX-catalyzed
ADP-ribosylation in the 41-kDa protein (Fig. 5, lane 1). G protein
i becomes a poor substrate for PTX-catalyzed
ADP-ribosylation when the trimeric G protein has become dissociated
upon LHRH receptor activation. Incubation with the LHRH analog
triptorelin in the presence of GTP remarkably reduced ADP-ribosylation
in the 41-kDa protein (Fig. 5, lane 4). Incubation with triptorelin and
a nonhydrolysable GTP analog, GTP-
-S, produced a further
reduction of ribosylation of this protein, bringing about a loss of
greater than 90% of the 32P count (Fig. 5, lane
2). The inhibitory action of triptorelin on the transfer of
[32P]ADP-ribose to 41-kDa protein revealed a
dose dependency (not shown). The G protein
i-subunit is a better substrate for
ADP-ribosylation when the nucleotide binding site is free of GDP and
GTP, and GDP dissociation is stimulated by triptorelin. In the absence
of GTP, but in the presence of triptorelin, ADP-ribosylation of the G
protein i was increased (Fig. 5, lane 3). GTP
alone had no effect (Fig. 5, lane 5). These results support the concept
that the G protein i is activated by the tumor
LHRH receptor.
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q
in addition to G protein i, we performed
cross-linking experiments (Fig. 6). Using
a G protein i antibody, two protein complexes
could be distinguished, migrating at about 41 and 103 kDa. Assuming a
stoichiometric binding of 1:1, subtraction of the molecular mass of G
protein i (41 kDa) yielded a protein of about
62 kDa. This corresponds to the molecular mass of LHRH receptor. The
band of 41 kDa corresponds to the molecular mass of G protein
i. Using the G protein
q antibody, the results were similar, except
for membranes from the ovarian cancer cell line EFO-27, which does not
express G protein q. In this cell line neither
the 41-kDa band (G protein q) nor the 103-kDa
band (G protein q plus LHRH receptor) could be
detected (Fig. 6). Using a LHRH receptor antibody, we could also detect
the 103-kDa band, indicating that the 62-kDa part of the 103-kDa
complex after subtraction of the molecular mass of the G proteins seems
to be the LHRH receptor protein. Additional use of LHRH receptor
antibody lead to the detection of a lower band at about 62 kDa,
representing the unbound LHRH receptor protein. In addition, some other
inconsistent bands were observed that we consider to be nonspecific
artifacts. These findings demonstrate in addition to the findings of
the ADP-ribosylation experiments, that the LHRH receptor couples to G
protein i. Furthermore LHRH receptor couples
to G protein q.
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q. Therefore, we hypothesized that in these
cells the effects induced by LHRH agonists might be mediated by G
protein i. PTX impairs the receptor-effector
interaction through ADP-ribosylation of G protein
i. Therefore, we examined LHRH-induced
inhibition of EGF-induced c-fos expression in the cell
lines treated with PTX (Fig. 7). The
LHRH-induced inhibition of EGF-induced c-fos expression was
blocked completely by PTX in ovarian cancer cell lines EFO-21 and
EFO-27 and in endometrial cancer cell lines Ishikawa and Hec-1A (Fig. 7). This demonstrates that LHRH-induced inhibition of EGF-induced
c-fos expression, in these cell lines is mediated through
the G protein i. As mentioned, PTX also
impaired LHRH-induced reduction of proliferation (not shown).
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i, the next set of experiments was performed.
Treatment of quiescent EFO-21 and EFO-27 ovarian cancer cells and
quiescent Ishikawa and Hec-1A endometrial cancer cells with 100
nM of the LHRH agonist triptorelin resulted in a
significant increase in PTP activity (Fig. 8). This effect was completely blocked by
simultaneous treatment with PTX. PTP activity is expressed as a
percentage of the control value (no treatment = 100%). In the
ovarian cancer cell lines EFO-21 (Fig. 8A) and EFO-27 (Fig. 8B), PTP
activity was increased to 397.5 ± 47.2% or 372.5 ± 36.1%,
respectively (P < 0.001 vs. control).
Simultaneous treatment with PTX resulted in a decrease to 77.5 ±
7.5% or 80.2 ± 9.1% of the control value, respectively
(P < 0.001 vs. triptorelin). In the
endometrial cancer cell lines Ishikawa (Fig. 8C) and Hec-1A (Fig. 8D),
PTP activity was increased to 442.5 ± 27.8% or 417.5 ±
26.3%, respectively (P < 0.001 vs.
control). Simultaneous treatment with PTX resulted in a decrease to
91.3 ± 10.1% or 90.1 ± 10.8%, respectively
(P < 0.001 vs. triptorelin).
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i, we now assessed whether this leads to a
relevant reduction of EGF-induced autophosphorylation of EGF receptors.
The amount of phosphorylated EGF receptors in quiescent cells was below
the detection limit of our assay (not shown). Treatment of quiescent
EFO-21 (Fig. 9, A and B) and EFO-27 (Fig. 9C) ovarian cancer cells and quiescent Ishikawa (Fig. 9D) and Hec-1A
(Fig. 9E) endometrial cancer cells with 100 nM EGF resulted
in a dramatic increase in tyrosine-phosphorylated EGF receptors
(=100%). This effect of EGF was antagonized by simultaneous treatment
with LHRH agonist triptorelin (10 µM). In the ovarian
cancer cell lines EFO-21 and EFO-27 the amount of phosphorylated EGF
receptors was decreased to 63 ± 2.1% or 44.5 ± 9.6% of
the control value, respectively (P < 0.001). In the
endometrial cancer cell lines Ishikawa (Fig. 9D) and Hec-1A (Fig. 9E),
the amount of tyrosine-phosphorylated EGF receptors was decreased to
58 ± 6.8% or 59 ± 2.4% of the control value, respectively
(P < 0.001). When the PTP inhibitor vanadate (100
µM) was present in these experiments, this
antagonism was completely blocked (P < 0.001). Figure 9 shows the relative changes in the amount of phosphorylated EGF
receptors in the absence or presence of vanadate in terms of the
percentage of phosphorylated EGF receptors after EGF treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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q,
leading to activation of PLC, rapid hydrolysis of membrane
phospholipids, liberation of inositol phosphates, subsequent
mobilization of intracellular Ca2+, and
activation of PKC (5, 6, 17). We clearly demonstrated in
earlier studies the activation of phospholipase C, PKC, and adenylyl
cyclase in the tumor cells by pharmacological stimuli. The LHRH agonist
triptorelin, at concentrations that are clearly inhibitory to
proliferation, however, had no effect on the activities of these
signaling systems (18). Instead, we found that the
antiproliferative effects of LHRH analogs are mediated through
interaction with growth factor-induced mitogenic signaling, as LHRH
analogs antagonized growth factor-induced proliferation,
mitogen-activated protein kinase activity, and c-fos
expression (18, 19). Comparable data were obtained by
Moretti et al. (38) in the human
prostatic cancer cell lines LNCaP and DU 145. In addition, the
proliferation of endometrial and ovarian cancer cells was
inhibited by both agonistic and antagonistic analogs of LHRH,
indicating that the dichotomy of LHRH agonists and antagonists does not
exist in tumor cells (11). Looking for reasons for the differences between LHRH signaling in pituitary gonadotrophs and human cancers, we first focused on putative mutations or splice variant formation as a possible explanation. We could not find any mutations or splice variants of the LHRH receptor transcripts in the endometrial and ovarian cancer cell lines analyzed. The LHRH receptor sequence found in Ishikawa and Hec-1A endometrial cancer cell lines and in EFO-21 and EFO-27 ovarian cancer cells lines was identical to that found in the pituitary gonadotrophs by Kakar et al. (32). In agreement with our results, Kakar and co-workers (39) have shown that in a not well characterized ovarian tumor biopsy, composed of malignant cells and desmoplastic fibrous tissue, that the LHRH receptor transcript was identical to that found in pituitary gonadotrophs. In contrast to this biopsy we show here for the first time that in well characterized human epithelial ovarian cancer cells lines obtained from a poorly differentiated serous ovarian adenocarcinoma and a mucinous papillary ovarian adenocarcinoma of intermediate differentiation, respectively, the LHRH receptor transcript was identical to that found in pituitary gonadotrophs. LHRH receptor sequence data obtained from endometrial cancer cells are shown for the first time here. It cannot be ruled out, however, that the LHRH receptor in tumors might differ in its posttranslational processing compared with the pituitary LHRH receptor.
In the next step we studied whether the G proteins might be responsible
for the different LHRH signal transduction pathway. Imai et
al. (40) speculated that the G protein
i that possibly couples the LHRH receptor to
the effector may be responsible for difference in response in
peripheral tumors and the anterior pituitary. Coupling of LHRH receptor
to G protein q in cancer cells was not shown
(40). As we found both G protein subunits
i and q to be highly
expressed in endometrial and ovarian cancer cells, this theory cannot
be explained by a lack of G protein q
expression. Our study provides evidence for LHRH receptor coupling to
both G protein i and
q in human endometrial and ovarian cancer
cells. Our data demonstrate that tumor cell LHRH receptor couples to
both G proteins in a single cell type. However, the antiproliferative
actions of LHRH analogs were predominantly mediated through the
PTX-sensitive G protein i. EGF-induced
expression of the immediate early gene c-fos is reduced by
binding of LHRH to its receptors in endometrial and ovarian cancer cell
lines (19). This effect was completely blocked by PTX,
indicating that it is mediated by G protein i.
Recently and in the present paper we showed that in the ovarian cancer
cell line EFO-27, which does not express G protein
q, the EGF-induced c-fos expression
(19) as well as the EGF-induced cell proliferation
(18) were antagonized by LHRH agonist as effectively as in
other tumor cell lines expressing both G proteins,
q and i. These
findings support the concept that the antiproliferative effects of LHRH
agonists in endometrial and ovarian cancer cells are mediated through G
protein i. We recently found that LHRH induces
activation of nuclear factor 
B in ovarian cancer cells
(41). This effect was inhibited by PTX, indicating that
LHRH-induced activation of nuclear factor B is also mediated through
PTX-sensitive G protein i (unpublished
results). Moreover, the LHRH agonist triptorelin substantially
antagonized the PTX-catalyzed ADP-ribosylation of G protein
i. Limonta et al. (42)
found a comparable mechanism in prostate cancer cells, suggesting that
LHRH receptor seems to be coupled to the G protein
i-cAMP signal transduction pathway rather than
to the G protein q/11-PLC signaling system
(42). In endometrial and ovarian cancer cells, however,
cAMP is not involved in LHRH signaling (18). It is true
that the LHRH receptor in endometrial and ovarian cancer cells couples
additionally to G protein q as in the
pituitary gonadotrophs; the signaling mechanisms induced by G protein
q in the pituitary, however, are not induced
by LHRH receptor in these cancer cells (18). In addition,
the antiproliferative effects of LHRH analogs are not mediated through
G protein q. It might be speculated that minor
mutations of the G protein q to which LHRH
receptors are coupled in the endometrial and ovarian cancer cells could
be responsible for the phenomenon that G protein
q is able to couple the LHRH receptor, but
that a signal transduction comparable to that in pituitary gonadotrophs
is not activated. In addition, it is possible that the
three-dimensional structures of the LHRH receptor might be different in
pituitary gonadotrophs and peripheral cancers. This speculation will be
the subject of further investigations.
The ability of the LHRH receptor to couple to multiple G proteins seems
not to be specific to endometrial and ovarian cancer cells. Stanislaus
and co-workers recently reported that the LHRH receptor in pituitary
gonadotrophs and in the lactotroph cell line GGH3
couples to multiple G proteins (43). The ability of
seven-transmembrane domain receptors to couple to multiple G proteins
is documented for other members of this family (44, 45).
In contrast, Grosse et al. (46) reported that
in gonadotropic T3–1 cells and in CHO-K1 cells transfected with the
human LHRH receptor cDNA LHRH receptors couple exclusively to G
proteins of the Gq/11 family. However, in
endometrial and ovarian cancer cells the picture is different. The
mechanism through which multiple G proteins interact with the LHRH
receptor is unknown. The second and third intracellular loops appear to
be involved in signal transduction, suggesting that multiple sites on
the receptor may interact with G proteins.
Having shown that LHRH agonists inhibit EGF-induced net tyrosine
phosphorylation in endometrial and ovarian cancer cells
(18), we checked whether this effect might be due to
LHRH-induced activation of a PTP. In addition, we analyzed whether this
effect might be mediated through PTX-sensitive G protein
i. To specify PTP activity we used the
enzyme-specific phosphopeptide END(pY)INASL (34) to
exclude interference with other phosphatase enzymes. The system used
determines the amount of free phosphate generated in a reaction by
measuring the absorbance of molybdate-malachite green-phosphate
complex (33). LHRH treatment resulted in a significant
increase in PTP activity. This effect was completely blocked by
simultaneous treatment with PTX, indicating the involvement of
PTX-sensitive G protein i in LHRH-induced PTP
activation. As a final step we checked, whether the LHRH-induced
activation of PTP really results in a reduction of EGF-induced tyrosine
autophosphorylation of EGF receptors. LHRH treatment resulted in a
marked decrease in tyrosine-phosphorylated EGF receptors. When the
experiments were performed in the presence of vanadate, an inhibitor of
PTP, the reduction of EGF-induced tyrosine autophosphorylation of EGF
receptors by treatment with triptorelin was completely blocked. Both
activation of PTP and reduction of EGF-induced phosphorylation of EGF
receptors were induced in EFO-27 cells, which do not express G protein
q as effectively as other cell lines
expressing this G protein-coupled receptor. These data also
support the concept that the antiproliferative activity of LHRH in
endometrial and ovarian cancer cells is not mediated through G protein
q. These findings suggest that the link
between LHRH receptor activity and growth factor-induced cell
proliferation is a PTP. PTP activation by LHRH analogs counteracts
EGF-induced tyrosine autophosphorylation of EGF receptor. This
results in down-regulation of mitogenic signal transduction and cell
proliferation.
In summary, this study provides evidence that in human endometrial and
ovarian cancers, LHRH receptor gene mutations as well as splice
variants are not responsible for the signaling different from that
acting in the pituitary. We could show that the tumor LHRH receptor
instead activates a PTP mediated through the PTX-sensitive G protein
i counteracting EGF-induced tyrosine
autophosphorylation of EGF receptors and resulting in inhibition of
mitogenic signal transduction and reduction of cell proliferation. In
addition, the present data indicate that in endometrial and ovarian
cancer cells, the LHRH receptor couples to G protein
i and G protein q in
a single cell type in endometrial and ovarian cancer cells.
Nevertheless, the antiproliferative signal transduction seems to be
mediated predominantly through PTX-sensitive G protein
i. Whether the coupling of G protein
q to the LHRH receptor, as in the pituitary
gonadotrophs, induces any intracellular activities in tumor cells is
not known and will be the subject of further investigations.
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Acknowledgments |
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Footnotes |
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Received November 27, 2000.
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References |
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B-activation and inhibits apoptosis in ovarian cancer cells.
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