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Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5
Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß) in human ovarian surface epithelial (hOSE)
cells and the ovarian cancer cell line, OVCAR-3, and provided novel
evidence that estrogen may have a growth regulatory effect in these
cells. Expression levels of ER messenger RNA (mRNA) were 1.5-fold
higher in OVCAR-3 cells than in hOSE cells, as revealed by
semiquantitative RT-PCR and Southern blot analysis. A significant
increase (3.3-fold) in ERß mRNA levels was observed in OVCAR-3 cells
compared with hOSE cells. In parallel with mRNA levels, expression
levels of ER and ERß proteins were also higher in OVCAR-3 cells
compared with hOSE cells. We recently proposed that GnRH and its
receptor may have an autocrine role in hOSE and ovarian cancer cells.
To determine whether estrogen regulates GnRH and GnRH receptor (GnRHR),
hOSE and OVCAR-3 cells were treated with various concentrations of
17ß-estradiol for 24 h. Expression levels of GnRH and GnRHR mRNA
were examined using quantitative and competitive RT-PCR, respectively.
Treatment with 17ß-estradiol induced a significant down-regulation of
GnRH mRNA in OVCAR-3 cells, but not in hOSE cells and of GnRHR mRNA in
both hOSE and OVCAR-3 cells. Tamoxifen, an estrogen antagonist,
prevented the effects of 17ßestradiol, suggesting that estradiol
action is mediated via the ER. Finally, the effect of estrogen on the
growth of hOSE and OVCAR-3 cells was investigated. The cells were
treated with various concentrations of 17ß-estradiol, and the
proliferative index of cells was measured using
[3H]thymidine incorporation and DNA fluorometric assays.
17ß-Estradiol stimulated the growth of OVCAR-3 cells in a dose- and
time-dependent manner. In contrast, 17ß-estradiol failed to stimulate
the growth of hOSE cells. As estrogen down-regulated GnRH and GnRHR
mRNA, we investigated whether estrogen treatment blocks the growth
inhibitory effect of a GnRH agonist in OVCAR-3 and hOSE cells. Cells
were treated with 17ß-estradiol (10-7
M) together with (D-Ala6)-GnRH
(10-7 M), and the proliferative
index of cells was measured. Pre- or cotreatment of cells with
17ß-estradiol significantly attenuated the growth inhibitory effect
of the GnRH agonist in OVCAR-3 cells, whereas no effect of
17ß-estradiol treatment was observed in hOSE cells. To our knowledge,
these results provide the first demonstration of a potential
interaction between the estradiol/ER and GnRH/GnRHR systems, which may
be important in the growth regulation of normal and neoplastic hOSE
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biological effects of estrogen are mediated through an interaction with
its intracellular receptor, a member of the steroid/thyroid/retinoid
receptor gene superfamily (reviewed in Ref. 15). Until
recently, the classical estrogen receptor (ER; now referred to as
ER) was thought to be the only form of nuclear receptor able to bind
estrogen and mediate its hormonal effects in their target tissues.
However, the recent cloning of a gene encoding a second type of ER
(ERß) in the rat (16), mouse (17), and
human (18) has prompted further investigations on the
estrogen signaling system. Recent studies have revealed different
tissue distributions and expression levels of ER and ERß in the
ovary, suggesting different biological roles of ER and ERß in
these tissues (16, 17, 18, 19, 20, 21). In addition, the existence of
ER and ERß in normal ovarian epithelial (OSE) cells and ovarian
cancers has been reported (22, 23). Other epidemiological
and clinical observations have implicated estrogen in the pathogenesis
and growth regulation of carcinomas arising from ovary
(24, 25, 26, 27, 28). For instance, experimental ovarian tumors could
be induced by diethylstilbestrol (25), and estrogen
stimulated the growth of several ER-positive ovarian carcinoma cell
lines in vitro (26, 27, 28). However, the precise
relationship between estrogen and the regulation of OSE or ovarian
cancer cell growth remains unknown.
Considering that GnRH has recently been proposed to be a potential
autocrine regulator in normal OSE and ovarian cancer cells (14, 29), we sought to investigate the relationship between
estrogen/ER and the GnRH/GnRHR system in these cells. In the present
study experiments were designed to investigate 1) the expression of
ER and ERß at the mRNA and protein levels in hOSE and ovarian
cancer cells, 2), the regulation of GnRH and its receptor gene by
estrogen, and 3) the growth regulatory effect of estrogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR amplification of ER and ERß
Total RNA was prepared from cultured cells using the RNaid kit
(Bio/Can Scientific, Mississauga, Canada) according to the
manufacturer’s suggested procedure. The RNA concentration was measured
based on absorbance at 260 nm, and its integrity was confirmed by
agarose-formaldehyde gel electrophoresis. Total RNA (2.5 µg) was
reverse transcribed into complementary DNA (cDNA) using first strand
cDNA synthesis kit (Amersham Pharmacia Biotech, Oakville,
Canada), following the manufacturer’s procedure. The primers were
designed to amplify ER and ERß based on the published sequences of
human ER and ERß (18, 31). Primers for ER were:
sense, 5'-ATGACCATGACCCTCAACACCAA-3' (F1); and antisense,
5'-CTTGGCAGATTCCATAGCCATAC-3' (R1). Primers for ERß were: sense,
5'-TACAGCATTCCCAGCAATGTCAC-3' (F2); and antisense,
5'-GAAGTGAGCATCC-CTCTTTGAAC-3' (R2). The cDNA was
amplified in a 50-µl PCR reaction containing 2.5 U Taq
polymerase (Life Technologies, Inc.) and its buffer, 1.5
mM MgCl2, 2
mM deoxy-NTP, and 50 pmol of specific primers.
For semiquantitative PCR amplifications for ER and ERß, the
conditions under which PCR amplification was in the logarithmic phase
were determined. Total RNA (2.5 µg) was reverse transcribed, and
aliquots (2 µl) were amplified using different numbers of cycles. The
semiquantitative RT-PCR amplification were carried out with denaturing
for 1 min at 94 C, annealing for 35 sec at 50-60 C depending on the
primers employed, extension for 90 sec at 72 C, and a final extension
for 15 min at 72 C. Primers for ß-actin were designed based on
published sequences (32), as previously described
(33). PCR for ß-actin was carried out to rule out the
possibility of RNA degradation and was used to control the variation in
mRNA concentration in the RT reaction, as previously described
(33). A linear relationship between PCR products and
amplification cycles was observed in ER, ERß, and ß-actin in
OVCAR-3 cells (Fig. 1
) and hOSE cells
(data not shown). Thirty cycles for ER and ERß and 18 cycles for
ß-actin were employed for semiquantitative RT-PCR amplification.
Amplified PCR products were subjected to Southern blot analysis. Ten
microliters of PCR products were fractionated on a 1.5% agarose gel
and stained with ethidium bromide. The PCR products were transferred to
a nylon membrane and hybridized with digoxigenin-labeled cDNA probe for
human GnRH, GnRHR, and ß-actin (3, 14, 33) following the
manufacturer’s recommended procedure (Roche Molecular Biochemicals, Laval, Canada). After washing, the membranes were
exposed to Kodak X-Omat x-ray film (Eastman Kodak Co., Rochester, NY). PCR products isolated from gel were cloned
into pCRII vector using the TA Cloning Kit (Invitrogen,
San Diego, CA) and were sequenced by the dideoxy nucleotide chain
termination method using the T7 DNA polymerase sequencing kit
(Amersham Pharmacia Biotech). Amplified PCR products were
quantified by densitometry (NIH Image ß3) after Southern blot
analysis. The expression levels of ER and ERß were normalized
against ß-actin levels. Data are presented as the mean ±
SD.
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(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a goat polyclonal
antibody for ERß (Santa Cruz Biotechnology, Inc.). After
washing, the signals were detected with horseradish
peroxidase-conjugated secondary antibody and visualized using the ECL
chemiluminescent system (Amersham Pharmacia Biotech),
followed by autoradiography. The levels of ER and ERß protein were
quantitated by densitometry (NIH Image ß3). Data are presented as the
mean ± SD.
[3H]Thymidine incorporation assay
To investigate the effect of 17ß-estradiol on the growth of
hOSE and OVCAR-3 cells, a [3H]thymidine
incorporation assay was performed as previously described with some
modifications (14). hOSE cells (n = 3; passage 2) and
OVCAR-3 cells were plated in 24-well plates at 2 x
104 cells/well in 0.5 ml medium 199 (phenol red
free) supplemented with 2% charcoal-stripped FBS, 100 U/ml penicillin
G, and 100 µg/ml streptomycin. After a preincubation period of
24 h, the cells were cultured for 18 h and incubated with
medium containing 1 µCi [3H]thymidine (5.0
Ci/mmol; Amersham Pharmacia Biotech) for 6 h. The
cells were then collected and served as day 0 controls. On the day of
treatment, the cells were treated with various concentrations
(10-8,
10-7, or
10-6 M) of
17ß-estradiol for 2, 4, and 6 days. To investigate the effect of
17ß-estradiol on the growth inhibitory effect of a GnRH agonist,
OVCAR-3 cells were pretreated with 17ß-estradiol
(10-7 M) and
vehicle for 24 h and then treated with
(D-Ala6)-GnRH
(10-7 M),
17ß-estradiol (10-7
M), and (D-Ala6)-GnRH
(10-7 M) for
2, 4, and 6 days. The same treatment was applied to hOSE cells for 6
days. Control cultures were treated with vehicle. Before the day of
collection, the cells were treated with hormone for 18 h and
incubated with medium containing 1 µCi
[3H]thymidine for 6 h. After incubation,
the culture medium was removed, and the cells were washed three times
with PBS and precipitated with 0.5 ml 10% trichloroacetic acid for 15
min at 4 C. After a methanol wash, the precipitate was solubilized in
0.5 ml 0.1 N sodium hydroxide, and the incorporated
radioactivity was measured in a 1217 Rackbeta liquid scintillation
counter (LKB Wallac, Inc., Turku, Finland).
Each experiment was repeated three times with triplicate samples.
Values are expressed as the percentage of growth compared with the
control value and are the mean ± SD of three
experiments with triplicate samples.
DNA fluorometric assay
In addition to the [3H]thymidine
incorporation assay, the effect of 17ß-estradiol on the growth of
OVCAR-3 cells was determined by measuring the DNA content as described
previously with some modifications using a 24-well plate
(35). To validate the experimental conditions, serial
dilutions of calf thymus DNA (Sigma-Aldrich Corp.) were
prepared in TNE buffer (10 mM Tris, 1 mM EDTA,
and 2 M NaCl, pH 7.4). A 125-µl sample of each dilution
was pipetted into wells for a DNA content of 500, 2,000, 5,000, or
10,000 ng/well, and 125 µl TNE buffer and 250 µl (20 µg) Hoechst
33258 DNA dye (Sigma-Aldrich Corp.) were added to each
well. DNA was measured using an automated microplate fluorescence
reader (model FL600, Bio-Tek Instruments, Inc., Winooski,
VA) at an excitation wavelength of 350 nm and an emission wavelength of
460 nm (sensitivity, 80). A linear relationship between the amount of
DNA and fluorescence units was observed (Fig. 2A). Serial dilutions of OVCAR-3 cells
(10,000–150,0000 cells/well) were prepared and seeded into 24-well in
0.5 ml medium 199 (phenol red free) supplemented with 2%
charcoal-stripped FBS, 100 U/ml penicillin G, and 100 µg/ml
streptomycin. The cells were cultured for 24 h, washed with TNE
three times, and stored at -70 C. On the day of assay, 250 µl
distilled water were added to the wells, and the plates were incubated
for 1 h at room temperature. The plates were frozen for 1 h
at -70 C and thawed until reaching room temperature. A 250-µl
(20-µg) aliquot of Hoechst 33258 DNA dye (Sigma-Aldrich Corp.) was added, and fluorescence was measured as described
above. A linear relationship between the number of cells and
fluorescence units was observed (Fig. 2B). For estrogen treatments,
OVCAR-3 cells were plated in 24-well plates at 2 x
104 cells/well in 0.5 ml medium 199 (phenol red
free) supplemented with 2% charcoal-stripped FBS, 100 U/ml penicillin
G, and 100 µg/ml streptomycin. After a preincubation period of
24 h, the cells were cultured for 24 h, collected, and served
as day 0 controls. On the day of treatment, the cells were treated with
various concentrations
(10-8,
10-7, or
10-6 M) of
17ß-estradiol for 2, 4, and 6 days. To investigate the effect of
17ß-estradiol on the growth inhibitory effect of a GnRH agonist,
OVCAR-3 cells were pretreated with 17ß-estradiol
(10-7 M) and
vehicle for 24 h and then treated with
(D-Ala6)-GnRH
(10-7 M),
17ß-estradiol (10-7
M), and (D-Ala6)-GnRH
(10-7 M) for
2, 4, and 6 days. Control cultures were treated with vehicle. The
amount of DNA was measured as described above. Each experiment was
repeated three times with triplicate samples. The number of cells was
calculated from inserting the fluorescence unit into a standard curve
(Fig. 2B). Cell proliferation was expressed as the percentage of growth
compared with the control value. The values are the mean ±
SD of three experiments with triplicate samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß mRNA and ERß mRNA in hOSE and
OVCAR-3 cells, two sets of primers were designed, and semiquantitative
RT-PCR was performed. As shown in Fig. 3A, a 540-bp DNA fragment for ER and a
279-bp DNA fragment for ERß were obtained from hOSE cells and OVCAR-3
cells. These fragments were validated as ER and ERß by
hybridization with a specific probe for ER and ERß cDNA (Fig. 3A)
and sequence analysis (data not shown). The possibility of genomic DNA
or cross-contamination was ruled out, because no PCR products were
observed or detected in negative controls (without template and without
reverse transcriptase in the RT reaction) by ethidium bromide staining
and Southern blot analysis, respectively (data not shown). Quantitative
analysis of the present study showed different expression levels of
ER and ERß mRNA. The expression levels of ER mRNA were 1.5-fold
higher in OVCAR-3 cells compared with hOSE cells (Fig. 3B). A
significant increase (3.3-fold) in ERß mRNA was observed in OVCAR-3
cells compared with hOSE cells (Fig. 3B).
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and ERß protein and ERß protein in hOSE
and OVCAR-3 cells, immunoblot analysis was performed using a mouse
monoclonal antibody for ER and a goat polyclonal antibody for ERß.
As shown in Fig. 4A, ER protein (68
kDa) and ERß (55 kDa) were observed in both cell types. Quantitative
analysis of the present study showed a slight, but significant,
increase in ER protein in OVCAR-3 cells compared with hOSE cells
(Fig. 4B). In contrast, a highly significant increase (2.5-fold) in
ERß protein was observed in OVCAR-3 cells (Fig. 4B).
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). In contrast, no significant
down-regulation of GnRH mRNA was observed in hOSE cells (Fig. 5A).
Treatment with 17ß-estradiol
(10-9 and
10-7 M)
resulted in a significant down-regulation of GnRHR mRNA in OVCAR-3
cells. Maximum down-regulation (to 43% of control levels) was observed
at 10-7 M
(Fig. 5B). In hOSE cells, 17ß-estradiol induced a downregulation
of GnRHR mRNA in a dose-dependent manner, with maximum down-regulation
(to 60% of control levels) at
10-9 M (Fig. 5B). To determine whether estradiol action is mediated through the
estrogen receptor, the cells were treated with 17ß-estradiol together
with tamoxifen, an estrogen antagonist. Cotreatment with tamoxifen
abolished the down-regulation of GnRH in OVCAR-3 cells and GnRH and
GnRHR mRNA in both cells (Fig. 6).
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, 17ß-estradiol stimulated the growth
of OVCAR-3 cells in a dose-dependent manner when measured by a
[3H]thymidine incorporation assay. A
significant increase in proliferation was detected as early as the
second day of treatment at concentrations of
10-8 and
10-7 M. At a
higher concentration (10-6
M), stimulation of cell growth was significant on days 4
and 6. The growth effect of 17ß-estradiol was also time dependent, as
the stimulatory effect increased with time of treatment (Fig. 7A).
Stimulation of growth continued to the fourth day and was further
evident on the sixth day of treatment. On day 6, 17ß-estradiol
(10-7 M)
induced cell proliferation to 195% of the control value, and
10-8 and
10-6 M
17ß-estradiol stimulated cell growth to 148% and 139% of the
control level, respectively. The growth stimulatory effect of
17ß-estradiol in OVCAR-3 cells was also observed when assessed by DNA
fluorometric assay (Fig. 7B). In contrast, 17ß-estradiol failed to
significantly affect the growth of hOSE cells (Fig. 7C), as measured by
a [3H]thymidine incorporation assay.
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, GnRHa induced a
significant growth inhibition of OVCAR-3 cells as early as the second
day of treatment when measured by a
[3H]thymidine incorporation assay. Inhibition
of growth continued through the fourth day and was further evident on
the sixth day of treatment (to 50% of the control level). Pretreatment
with 17ß-estradiol for 24 h partially blocked the growth
inhibitory effect of GnRHa on day 2, but failed to block the effect of
GnRHa on days 4 and 6 of treatment. Pretreatment for 24 h and
cotreatment with 17ß-estradiol on a daily basis induced a significant
attenuation of growth inhibitory effect of GnRHa throughout the
treatment. The effect of 17ß-estradiol on the growth inhibitory
effect of GnRHa in OVCAR-3 cells was also observed when determined by
the amount of DNA (Fig. 8B). In contrast, neither pre- nor cotreatment
with 17ß-estradiol blocked the growth inhibitory effect of GnRHa in
hOSE cells in a [3H]thymidine incorporation
assay (Fig. 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biological effects of estrogens are mediated through an interaction
with its intracellular receptor (reviewed in Ref. 15).
Recent studies have revealed different tissue distributions and
expression levels of ER and ERß in the ovary
(16, 17, 18, 19, 20, 21). It has been demonstrated that ER is localized
primarily in the ovarian stromal and thecal cells, whereas ERß is
predominantly detected in the granulosa cells of small, developing, and
preovulatory follicles (18, 19, 20). In the present study, we
demonstrated the expression of ER and ERß mRNA in normal hOSE
cells and epithelial cancers of the ovary, i.e. OVCAR-3
cells. Quantitative analysis showed different expression levels of
ER and ERß mRNA. Expression levels of ER and ERß mRNA were
higher in OVCAR-3 cells than in hOSE cells. Our data are in agreement
with recently demonstrated ER and ERß expression in cultured hOSE
cells and primary cultures of ovarian cancer cells (22, 23). Despite the abundance of information on ERs mRNA, there is
little characterization of the ER protein in the ovary. The open
reading frame predicted from the ERß cDNAs encodes a protein of
approximately 54 kDa, which contrasts with the size of ER (
67
kDa) detected by Western blotting (16, 31). In the present
study, we demonstrated the predicted size of ER and ERß protein in
hOSE and OVCAR-3 cells using immunoblot analysis. Our results are
consistent with previous reports that demonstrated the existence of ER
in normal OSE and ovarian cancer cells using ligand binding and
immunocytochemical studies (38, 39). In parallel with mRNA
levels, different expression levels of ER and ERß protein were
observed in hOSE and OVCAR-3 cells. The different expression levels of
ER and ERß suggest that these receptors may have different
biological roles in regulating the functions of normal hOSE and OVCAR-3
cells.
In addition to its well documented role in the regulation of
gonadotropin synthesis and secretion, GnRH has direct effects on the
gonads. At the hypothalamus-pituitary levels (1, 2),
estrogen is thought to be a key regulator of GnRH and its receptor
system (1, 10). During the rat estrous cycle, GnRH mRNA
levels in the hypothalamus were inversely correlated to plasma estrogen
levels (11). However, other groups have documented that
estrogen increases GnRH gene expression in the rat (40),
which may lead to the gonadotropin surge before ovulation. Like GnRH,
estrogen regulates GnRHR in the pituitary. In the rat pituitary,
estradiol negatively regulates GnRHR mRNA (13). However,
other groups have shown that estrogen increased GnRHR mRNA levels
(41, 42, 43). Differences in steroid-induced regulation of
GnRH and GnRHR may be explained by different effects of estrogen on the
hypothalamus and pituitary depending on the duration and dosage of
hormone exposure, time points of measurement, differences in
sensitivity of techniques, and perhaps species differences. Recently,
we proposed that GnRH may be an autocrine/paracrine regulator in normal
hOSE and ovarian cancer cells (14, 30). However, no
information is available on the role of 17ß-estradiol in regulating
GnRH and GnRHR in these cells. In the present study, we demonstrated
that estrogen significantly decreased the GnRH mRNA level in OVCAR-3
cells and GnRHR mRNA levels in hOSE and OVCAR-3 cells. The exact
mechanism by which estrogen regulates GnRH and its receptor mRNAs in
the ovary and in ovarian cancer remains unclear. After binding to its
nuclear receptor, estrogen may modulate GnRH and GnRHR directly or
indirectly. Even though no consensus sequence for an estrogen response
element is found in the promoter region of the human GnRH and GnRHR
gene (44, 45), estrogen can directly modulate
transcription of the GnRH gene (44) and GnRHR promoter
activity (our unpublished data). Alternatively, estrogen may act
through indirect pathways to regulate GnRH and its receptor. It has
been demonstrated that estrogen can mobilize intracellular
Ca2+ (46), which may lead to
activation of the protein kinase C pathway. Phorbol ester-induced
activation of protein kinase C has been shown to modulate GnRHR gene
expression (47). In the present study, the effect of
estrogen appears to be a receptor-mediated event, as cotreatment with a
competitive estrogen antagonist, tamoxifen, abolished the effect of
estradiol. It remains to be determined which type of receptor mediates
the effect of estrogen in these cells. Other studies have demonstrated
that homodimers ER/ER and ERß/ERß or heterodimers ER/ERß
can be formed in vitro, bind to the estrogen response
elements, and stimulate the transcription of a reporter gene (48, 49). Therefore, the relative expression of ER and ERß and
the difference in DNA-binding activity between heterodimers and
homodimers could determine the tissuespecific effects of estrogen
action.
Estrogen has been implicated in the pathogenesis and growth regulation
of carcinomas arising from the ovary (24, 25, 26, 27, 28). In this
study, 17ß-estradiol stimulated the growth of an ovarian cancer cell
line, OVCAR-3, in a time- and dose-dependent manner. The growth
stimulation was detected after 2 days of estradiol treatment and was
further evident at 6 days. The requirement for a long-term treatment
suggests that the growth stimulatory effect of estrogen may be
indirect. This idea is supported by findings that estrogen has been
shown to stimulate the growth of other ovarian cancer cells through
stimulating expression of transforming growth factor-
(50) and epidermal growth factor (51). In
contrast to OVCAR-3 cells, estrogen had no effect on the growth of hOSE
cells. Our data are in agreement with the previous finding that
estrogen has no effect on the growth of hOSE cells (38).
The exact mechanisms of estrogen insensitivity in ER-positive hOSE
cells are not known. The responses to estrogen may be dependent on the
expression levels of ERs in the target tissues. This idea is supported
by findings that only ovarian cancer cell lines with high levels of ER
expression were responsive to estrogen treatment (26, 28).
The different levels of ER and ERß expression between hOSE and
OVCAR-3 cells observed in the present study corroborate this view.
Alternatively, the regulation of epithelial cell growth by estrogen in
reproductive organs may occur through common paracrine mechanisms
mediated by stromal hormone receptors. This possibility is well
demonstrated in uterine and vaginal epithelium, where their growth is
dependent on stromal ER that may induce epithelial cell proliferation
by producing a variety of growth factors (reviewed in Ref.
52). In this regard, it is likely that the OVCAR-3 cells
have acquired the capacity to respond mitogenically to estrogen, a
capacity not expressed by normal hOSE cells. Furthermore, it is
possible that normal OSE may express splice variant forms of ER and
ERß, which negatively affect normal ER signaling. This suggestion is
supported by the finding of a mutation in the exon of ER, which may
explain why an ovarian cancer cell line, SKOV3, was reported to be ER
positive but estrogen insensitive (22, 53).
It has been demonstrated that GnRH agonists and antagonists inhibited the growth of GnRHR-bearing tumors (7, 8, 9). Our previous studies have demonstrated that long-term treatment of GnRHa induced significant growth inhibition in hOSE and OVCAR-3 cells (14, 30). As GnRH actions are mediated through a G protein-coupled receptor, and estrogen down-regulates GnRH and GnRHR levels, we further explored the possibility that estrogen may block the growth inhibitory effect of GnRHa in OVCAR-3 and hOSE cells. Our data demonstrate that pre- or cotreatment with 17ß-estradiol induced a significant attenuation of the growth inhibitory effect of GnRHa in OVCAR-3 cells. These results suggest that estrogen may function as a growth regulatory factor as well as a mitogen in OVCAR-3 cells. However, we cannot rule out the possibility that the mitogenic activity of estrogen may override the growth inhibitory activity of GnRHa independent of down-regulation of GnRHR in OVCAR-3 cells. Despite down-regulation of GnRHR mRNA levels, neither pre- nor cotreatment with 17ß-estradiol blocked the growth inhibitory effect of GnRHa in hOSE cells. These results suggest that estrogen may have no growth regulatory effect in hOSE cells, but may mediate other functions in hOSE cells. Considering that hOSE cells are reported to produce cytokines, cell adhesion molecules, and proteolytic enzymes (30, 54, 55), it is possible that estrogen may play a role in regulating these factors in hOSE cells.
In summary, we have demonstrated that 1) OVCAR-3 and hOSE cells express
ER and ERß at the mRNA and protein levels; 2) estrogen through its
receptors down-regulates the GnRH mRNA level in OVCAR-3 cells and GnRHR
mRNA levels in OVCAR-3 and hOSE cells; 3) GnRH has an inhibitory effect
on OVCAR-3 and hOSE cell proliferation; 4) estradiol has a stimulatory
effect on proliferation of OVCAR-3, but not hOSE, cells; and 5)
concomitant treatment of estradiol and GnRHa antagonizes the growth
inhibitory effect of GnRHa on OVCAR-3, but not on hOSE cells.
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Acknowledgments |
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Footnotes |
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2 Recipient of a studentship award from the British Columbia Research
Institute of Children’s and Women’s Health.
3 Recipient of a career investigatorship from the British Columbia
Research Institute of Children’s and Women’s Health.
Received September 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J.-H. Choi, C. B. Gilks, N. Auersperg, and P. C. K. Leung Immunolocalization of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Type I GnRH Receptor during Follicular Development in the Human Ovary J. Clin. Endocrinol. Metab., November 1, 2006; 91(11): 4562 - 4570. [Abstract] [Full Text] [PDF]
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 Y. S. Choi, S.-Y. Ku, B.-C. Jee, C.-S. Suh, Y. M. Choi, J. G. Kim, S. Y. Moon, and S. H. Kim Comparison of follicular fluid IGF-I, IGF-II, IGFBP-3, IGFBP-4 and PAPP-A concentrations and their ratios between GnRH agonist and GnRH antagonist protocols for controlled ovarian stimulation in IVF-embryo transfer patients Hum. Reprod., August 1, 2006; 21(8): 2015 - 2021. [Abstract] [Full Text] [PDF]
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 J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C K Leung Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid by gonadotropins in human immortalized ovarian surface epithelium and ovarian cancer cells. Endocr. Relat. Cancer, June 1, 2006; 13(2): 641 - 651. [Abstract] [Full Text] [PDF]
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K.-Y. Kim, K.-C. Choi, N. Auersperg, and P. C K Leung Mechanism of gonadotropin-releasing hormone (GnRH)-I and -II-induced cell growth inhibition in ovarian cancer cells: role of the GnRH-I receptor and protein kinase C pathway. Endocr. Relat. Cancer, March 1, 2006; 13(1): 211 - 220. [Abstract] [Full Text] |
