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Department of Obstetrics and Gynaecology, British Columbia Women’s Hospital, University of British Columbia, Vancouver, British Columbia, V6H 3V5, Canada
Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynaecology, University of British Columbia, 2H30-4490 Oak Street, British Columbia Women’s Hospital, 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 ß were expressed in IOSE cell lines.
No difference was observed in normal OSE and IOSE-29 cells, whereas
treatment with 17ß-estradiol (E2;
10-8–10-6
M) resulted in an increased thymidine incorporation and DNA
content per culture in IOSE-29EC cells. This effect of E2
was attenuated with tamoxifen treatment (10-6
M), the estrogen antagonist, suggesting that the effect of
E2 is mediated through specific ERs. There was no
stimulatory effect on thymidine incorporation before day 6, but after 6
days of E2 treatment, thymidine incorporation was
significantly increased. Because the ratio of thymidine
incorporation to DNA content per culture did not change, this
E2 effect does not appear to indicate stimulation of
proliferation but, rather, inhibition of apoptosis. In addition,
treatment with tamoxifen (10-6
M) induced apoptosis up to 3-fold in IOSE-29EC cells,
whereas cotreatment with E2
(10-8–10-6
M) plus tamoxifen attenuated tamoxifen-induced apoptosis in
a dose-dependent manner. Both proapoptotic bax and antiapoptotic bcl-2
at messenger RNA (mRNA) and protein levels were expressed in IOSE cell
lines. Interestingly, treatments with E2 resulted in a
significant increase of bcl-2 mRNA and protein levels (2- and
1.7-fold, respectively), whereas no difference was observed in bax
mRNA level. Thus, E2 may enhance survival of IOSE-29EC by
up-regulating bcl-2, and antiapoptotic bcl-2 may be a dominant
regulator of apoptotic pathway in these cells. In conclusion, the
present study indicates that early neoplastic (IOSE-29), tumorigenic
(IOSE-29EC), and late neoplastic (IOSE-29EC/T4 and T5) OSE cells
expressed both ER and ERß at the mRNA and protein levels. In
addition, E2 prevented tamoxifen induced-apoptosis through
ERs. The mechanism of E2 action may be associated with
up-regulation of bcl-2 gene at mRNA and protein levels.
These results suggest that estrogen may play a role in ovarian
tumorigenesis by preventing apoptosis in tumorigenic OSE cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The common epithelial ovarian tumors appear to arise from the ovarian
surface epithelium (OSE), which is a simple squamous-to-cuboidal
mesothelium covering the ovary (18). The exact mechanism
of ovarian tumorigenesis is not well known even though this disease is
the most frequent cause of cancer death in gynecological malignancies
(19). Repeated ovulation contributes to neoplastic
transformation of OSE, indicating that the process of healing ruptured
OSE may contribute to the disease (19, 20). Therefore, it
has been suggested that endocrine and autocrine factors may influence
the occurrence of ovarian tumors in women (19, 20, 21, 22, 23, 24).
Actions of estrogen are mediated through an interaction with its
intracellular receptor, a member of the steroid/thyroid/retinoid
receptor gene superfamily (reviewed in Ref. 25). 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
cloning of a second form of ER, now referred to as ERß, has caused a
reexamination of the estrogen signaling system (26).
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 this tissue
(26, 27, 28). In addition, the existence of ER and ERß in
normal OSE and ovarian cancers has been demonstrated (29, 30). Although 17ß-estradiol (E2) is not
a mitogen for normal OSE (31), treatments with exogenous
estrogen resulted in a growth stimulation of several ER-positive
ovarian carcinoma cell lines in vitro (32, 33, 34).
However, the role of estrogen in ovarian tumorigenesis and regulation
of apoptosis by estrogen in neoplastic OSE cells remains uncertain.
The present study was performed to investigate the role of
E2 in the regulation of apoptosis in normal,
early neoplastic, tumorigenic, and late neoplastic OSE cells. Recently,
nontumorigenic [immortalized OSE (IOSE)-29] and tumorigenic
(IOSE-29EC) immortalized OSE cells were generated from normal OSE
directly by transfection with simian virus 40-large T antigen and
subsequent E-cadherin (35, 36). These IOSE-29EC cells were
found to be anchorage independent and formed transplantable, invasive
sc and ip adenocarcinomas in SCID mice (37). Two
additional cell lines, designated IOSE-29EC/T4 and IOSE-29EC/T5, were
established from tumors that arose in IOSE-29EC-inoculated SCID mice
(37). The characteristics of these cell lines resemble
those of ovarian cancer (36, 37). The expression of ER
and ERß was determined in these OSE cell lines to investigate the
effect of E2. Furthermore, cell proliferation and
prevention of apoptosis by E2 were examined in
these generated immortalized OSE cell lines. Finally, to elucidate the
mechanism of E2 in the prevention of apoptosis,
the regulation of proapoptotic bax and antiapoptotic bcl-2 was
investigated following treatments with E2 and/or
antiestrogen, tamoxifen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As outlined in Table 1
, we recently
developed a culture system with cells representing several stages in
the neoplastic progression of OSE. The IOSE-29 cell line (referred to
as IOSE-Mar in some previous publications) was generated by
transfection with the immortalizing simian virus 40 early genes into
normal human OSE at passage 5 (35). The IOSE-29EC cell
line was made from IOSE-29 at passage 11 by transfecting the
full-length mouse E-cadherin complementary DNA (cDNA) under the control
of the ß-actin promoter (36). The IOSE-29EC/T4 and
IOSE-29EC/T5 were established from tumors that arose in
IOSE-29EC-inoculated SCID mice, and they formed tumors on mesenteries
and omentum, invaded the liver and thigh musculature, and produced
ascites (37). For monolayer culture, all cell lines were
maintained on tissue culture dishes (Corning Costar Corp.,
Cambridge, MA) in a 1:1 mixture of medium 199/MCDB 105 medium
supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml
streptomycin. To study the regulation of proapoptotic bax and
antiapoptotic bcl-2 messenger RNA (mRNA) by E2
(Sigma-Aldrich Corp.), 2 x 105
IOSE-29EC cells were plated onto 35-mm culture dishes. After a
preincubation of 48 h, the cells were treated with
E2 at concentrations of
10-8,
10-7, and
10-6 M in
phenol-red-free medium 199 (Sigma-Aldrich Corp.) with 2%
charcoal/dextran-treated FBS (HyClone Laboratories, Inc.,
Logan, UT) for 24 h. To confirm the specificity of
E2, the cells were treated with
E2
(10-7 M) plus
tamoxifen (10-6
M, Sigma-Aldrich Corp.) for 24 h. Control
cultures were treated with vehicle (absolute ethyl alcohol).
Furthermore, to investigate the regulation of bax and bcl-2 apoptotic
proteins by E2, 2 x
105 IOSE-29EC cells were plated onto 35-mm
culture dishes and cultured for 48 h. Subsequently, the cells were
treated with E2
(10-8,
10-7, and
10-6 M) plus
tamoxifen (10-6
M) for 48 h.
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based on
the published sequences. The PCR were performed in a 25-µl PCR
mixture containing 1x PCR buffer, 0.2 mM each
deoxynucleotide triphosphate, 1.6 mM
MgCl2, 50 pmol specific primers, each cDNA
template, and 0.25 U Taq polymerase. The PCR was performed
for two or three independent cDNA preparations of each RNA sample. PCR
products (12 µl) were analyzed by agarose (1%) gel electrophoresis
and visualized by ethidium bromide staining, and the sizes were
estimated by comparison to DNA molecular weight markers.
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and ERß isolated from human granulosa cells 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.). The cDNA
clones for bax and bcl-2 were subcloned from ovarian cancer cell line
(OVCAR-3) and verified by sequence analysis already mentioned. The cDNA
probes for ERs ( and ß) and apoptotic genes (bax and
bcl-2) were labeled with DIG cDNA labeling kit
(Roche Molecular Biochemicals, Laval, Canada) for
hybridization. The hybridized membranes were detected with luminescence
method (Roche Molecular Biochemicals) and exposed to x-ray
film for 1–10 min at room temperature. The specific bands were
scanned and quantified using a computerized visual light densitometer
(model 620; Bio-Rad Laboratories, Inc., Richmond, CA).
Immunoblot analysis
Immortalized OSE cell lines (IOSE-29, IOSE-29EC, IOSE-29EC/T4,
and IOSE-29EC/T5) were seeded at a density of 2 x
105 cells in 35-mm culture dishes and cultured in
a humidified atmosphere of 5% CO2-95% air at 37
C. Cells were washed twice with ice-cold PBS and lysed in ice-cold RIPA
buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate,
0.1% SDS, 50 mM Tris (pH, 7.5), 1 mM
phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 100 µg/ml
aproteinin). The extracts were placed on ice for 15 min and centrifuged
to remove cellular debris. Protein concentrations in the supernatants
were determined using a Bradford assay (Bio-Rad Laboratories, Inc.). Thirty micrograms of total protein were run on 10%
SDS-PAGE gels and electrotransferred to a nitrocellulose membrane
(Hybond-C; Amersham Pharmacia Biotech). The membrane was
immunoblotted using a mouse monoclonal antibody for ER (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a goat polyclonal
antibody for ERß (Santa Cruz Biotechnology, Inc.). To
determine whether the in vitro treatments affected the
expression of the genes involved in apoptosis, the membranes were
immunoblotted using mouse monoclonal antibodies of bax (BD
PharMingen Inc., Mississauga, Ontario, Canada) and bcl-2
(Santa Cruz Biotechnology, Inc.). The loaded amount of
protein was normalized with actin protein in the same membrane. After
washing, the signals were detected with horseradish
peroxidase-conjugated secondary antibody, and visualized using the
enhanced chemiluminescence system (Amersham Pharmacia Biotech).
[3H]Thymidine incorporation assay
[3H]Thymidine incorporation assay was
performed to analyze the effect of E2 on DNA
synthesis in normal and neoplastic OSE cells. The cells were plated in
24-well plates at 2 x 104 cells/well in 0.5
ml medium 199:MCDB105 supplemented with 10% FBS and antibiotics, and
incubated for 48 h. On the day of treatment, the cells were
incubated with increasing concentrations
(10-8,
10-7, or
10-6 M) of
E2 in phenol-red-free medium 199 with
charcoal/dextran-treated FBS for 2–6 days. On the days indicated in
the results, during the last 6 h of the incubations to be
harvested, the medium was changed to include the same concentration of
E2 and 1 µCi
[3H]thymidine (5.0 Ci/mmol; Amersham Pharmacia Biotech). At the end of the incubation period, the
culture medium was removed and the cells were washed three times with
PBS, followed by precipitation with 0.5 ml 10% trichloroacetic acid
for 20 min at 4 C. The precipitate was washed in methanol twice and
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).
DNA fluorometric assay
In addition to the [3H]thymidine
incorporation assay, the effect of E2 on the
growth of IOSE-29 and IOSE-29EC was determined by measuring the DNA
content as previously described with some modifications in 24-well
plates (42). The cells were treated with various
concentrations (10-8,
10-7, or
10-6 M) of
E2 and/or tamoxifen
(10-6 M) in
phenol-red-free medium 199 with charcoal/dextran-treated FBS for 6
days. After treatment, the cells were washed with TNE buffer (10
mM Tris, 1 mM EDTA, 2 M NaCl, pH
7.4) three times and stored at -70 C. On the day of assay, 250 µl
distilled water was added in the wells and incubated for 1 h at
room temperature. The plates were frozen then for 1 h at -70 C
and thawed until reaching room temperature. The amount of DNA was
measured using an automated microplate fluorescence reader (Model
FL600; Bio-Tek Instruments, Inc., Winooski, VA) at
excitation wavelength 350 nm and emission wavelength 460 nm
(sensitivity = 90). The amount of DNA in the culture was
calculated from inserting the fluorescence unit into a standard
curve.
Quantification of apoptotic cells
To quantify the induction of apoptosis, DNA fragmentation was
measured using the cell death detection enzyme-linked immunosorbent
assay (ELISA; Roche Molecular Biochemicals) as previously
described (4). Briefly, the cells (1 x
104) were plated in each well of 24-well plates.
After treatments with E2 and tamoxifen for 6
days, the used media were collected and stored during the treatments.
The cells were washed with PBS, and 0.1 ml lysis buffer was added.
Following a 15-min incubation on ice, apoptotic cells in cell lysates
and used medium were assayed for DNA fragments according to the
manufacturer’s protocol. The same amount (1 µg) of cell lysate was
used for the procedure of cell death ELISA. DNA fragmentation was
measured at 405 nm against untreated control.
Statistical analysis
Data are shown as the means of two or three individual
experiments with triplicate samples, and are presented as the mean
± SD. In the [3H]thymidine
incorporation and DNA fluorometric assays, values are expressed as the
percentage of growth compared with the control value and are the
mean ± SD of three individual experiments with
triplicate samples. In the quantification of apoptotic cells, values
are expressed as the percentage of DNA fragmentation compared with
untreated control and are the mean ± SD of three
individual experiments with duplicate samples. The data were analyzed
by one-way ANOVA followed by Tukey’s multiple comparison test or
Dunnett’s test. P less than 0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß mRNAs and ERß in IOSE-29 (passages 13–16),
IOSE-29EC (passages 15–17), IOSE-29EC/T4, and IOSE-29EC/T5 were
investigated by RT-PCR and Southern blot analysis. The possibility of
cross-contamination was ruled out because no PCR products were observed
and detected in the negative control [TmA(-); without template
in the room temperature reaction] by ethidium bromide (data not
shown) and Southern blot analysis (Fig. 1). A linear relationship between PCR
products and amplification cycles was obtained in all cell types (data
not shown). Predicted PCR products of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), ER, and ERß were obtained as 373 bp, 540
bp, and 279 bp, respectively, and confirmed by Southern blot analysis
using DIG-labeled probes (Fig. 1) and sequence analysis (data not
shown). This result indicates that the mRNAs of ER and ERß are
expressed in IOSE-29, IOSE-29EC, IOSE-29EC/T4, and IOSE-29EC/T5.
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and ERß proteins and ERß proteins in
immortalized OSE cell lines, immunoblot analysis was performed using
the mouse monoclonal antibody for ER and a goat polyclonal antibody
for ERß. As shown in Fig. 2, ER
protein (68 kDa) was observed in all cell types. OVCAR-3 cell line was
used for positive control of the expression of ER expression. ERß
protein was also observed as 55 kDa in immortalized OSE cell lines.
Immunoblot analysis of the present study demonstrated that ER and
ERß proteins were observed in IOSE-29, IOSE-29EC, IOSE-29EC/T4, and
IOSE-29EC/T5.
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),
whereas E2 treatment for 2–6 days resulted in an
increase of the growth in OVCAR-3 cells in a time-dependent manner as
positive control (Fig. 3B). Highest proliferative effect of
E2 was observed at
10-7
M, whereas the effect of E2
was less increased at 10-6
M in OVCAR-3 cells (Fig. 3B). No difference was
observed following E2 treatment for 6 days in
IOSE-29 cells by thymidine incorporation (Fig. 3C) and fluorometric
assay (Fig. 3D). In contrast, treatment with E2
(10-8–10-6
M) before day 6 had no effect on thymidine
incorporation, but it resulted in a significant increase on day 6 (Fig. 3F; 100.0 ± 8.16% vs. 134.7 ± 5.37%,
156.8 ± 12.23%, or 132.8 ± 6.85%) in IOSE-29EC cells.
Similarly, DNA content in culture also increased significantly on day 6
(Fig. 3G; 100.0 ± 6.15% vs. 138.4 ± 4.08%,
176.3 ± 22.02%, or 147.6 ± 24.00%). Cotreatment with
E2
(10-7
M) plus tamoxifen
(10-6
M) attenuated the effect of
E2 (100.0 ± 8.16% vs. 74.2
± 6.50% in the thymidine and 100.0 ± 6.15% vs.
77.7 ± 7.94% in the DNA content) in IOSE-29EC cells. The ratio
of thymidine incorporation to DNA content per culture did not change
following E2 and/or tamoxifen treatments in
IOSE-29 (Fig. 3E) and IOSE-29EC cells (Fig. 3H), suggesting that
E2 effect does not include stimulation of
proliferation. Treatment with tamoxifen only also caused inhibitory
effect in IOSE-29 (Fig. 3, C and D) and IOSE-29EC cells (Fig. 3, F and
G). In addition, a significant increase of thymidine incorporation by
E2
(10-7
M) was also observed in IOSE-29EC/T4 and
IOSE-29EC/T5 cells, whereas cotreatment with tamoxifen attenuated the
E2 effect (Fig. 3, I and J).
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, treatment with
tamoxifen resulted in a significant increase of DNA fragmentation in
IOSE-29EC cells (100.0 ± 5.89% vs. 287.7 ±
11.26%). Cotreatments with E2
(10-8,
10-7, and
10-6
M) plus tamoxifen attenuated tamoxifen-induced
apoptosis in a dose-dependent manner (Fig. 4, and 287.7 ± 11.26%
vs. 219.8 ± 21.47%, 175.8 ± 12.02%, or
174.9 ± 16.50%).
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) and sequence analysis (data not
shown). No difference was observed in the expression level of bax mRNA
between IOSE-29 and IOSE-29EC cells. In contrast, the mRNA expression
level of bcl-2 was higher in IOSE-29EC cells than IOSE-29 cells (Fig. 5A). In parallel of mRNA level, protein level of bcl-2 was investigated
in these cell lines. As shown in Fig. 5B, the expression level of bcl-2
protein was higher in IOSE-29EC cells than IOSE-29 cells.
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, A and B; 100.0 ± 7.19%
vs. 172.9 ± 14.47%, 190.9 ± 22.03%, or
171.8 ± 17.55%). Cotreatment with tamoxifen
(10-6
M) plus E2 attenuated this
E2 effect (190.9 ± 22.03% vs.
116.8 ± 10.92%). However, no significant difference in the mRNA
level of bax was observed by E2 treatment (Fig. 6A).
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.
Consistent with the mRNA levels, treatments with
E2
(10-8–10-6
M) significantly up-regulated bcl-2 protein level
in these cells (Fig. 7, A and B; 100.0 ± 9.92% vs.
162.05 ± 12.68%, 166.7 ± 19.61%, or 154.4 ±
20.86%). Cotreatment with tamoxifen
(10-6
M) plus E2 attenuated this
E2 effect (166.7 ± 19.61% vs.
109.9 ± 10.22%). In contrast, no significant difference in the
protein level of bax was observed by E2 treatment
in IOSE-29EC cells (Fig. 7A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß are expressed in IOSE cell lines by
semiquantitative RT-PCR and immunoblot analysis. No difference in the
expression levels of ERs was observed among early neoplastic OSE,
tumorigenic OSE, late neoplastic OSE, and OVCAR-3 cells. In the
previous studies, it has been demonstrated that human normal OSE cells
express both mRNAs and proteins of ER and ERß (Refs.
29, 30). In addition, the expression levels of ER were
enhanced when compared with those in normal ovaries, whereas ERß
levels were significantly decreased in ovarian tumors, suggesting that
ER and ERß mRNAs are differentially expressed in normal and
neoplastic OSE cells (30, 43). These results suggest that
overexpression of ER relative to ERß mRNA may be a marker of
ovarian tumorigenesis. Consistent with mRNA levels, ER protein is also
highly expressed in ovarian carcinomas when compared with normal or
benign ovarian tumors (44). The open reading frame
predicted from the ERß cDNAs encodes a protein of molecular mass of
approximately 54 kDa, which contrasts with the size of ER
(approximately 67 kDa) detected by Western blotting (27, 39). ER and ERß can homodimerize ( or ßß)
or heterodimerize (ß) upon binding to the ERE (45).
Thus, it is hypothesized that differential expression of ER and
ERß in ovarian tumors may alter a responsiveness of estrogen or
antiestrogen treatment. Recently, a mutation involving a 32-bp deletion
in exon 1 of ER transcripts was detected in SKOV-3 cell line, which
is not responsive to estrogen treatment even though this cell line is
ER-positive (29).
In addition to its well-documented role in reproductive organs, it has
been suggested that estrogen, especially E2, may
be associated with ovarian tumorigenesis. Treatments with exogenous
E2 resulted in a growth stimulation of several
ER-positive ovarian carcinoma cell lines in vitro
(32, 33, 34). Some cultures of human epithelial ovarian cancer
cells have been demonstrated to produce E2 and
progesterone (46). The present studies demonstrated that
exogenous E2
(10-8–10-6
M) resulted in an increased thymidine
incorporation and DNA content per culture in IOSE-29EC cells but not in
IOSE-29. The effect of E2 was attenuated by the
estrogen antagonist tamoxifen
(10-6
M). This observation suggests that the effect of
E2 is mediated through specific receptors.
Because there was no stimulatory effect on thymidine incorporation
before day 6, and because the ratio of thymidine incorporation to DNA
content per culture did not change, the E2 effect
does not include stimulation of proliferation. The growth of
ER-positive ovarian tumors that are responsive to
E2 is also attenuated by antiestrogen, such as
tamoxifen and the pure antiestrogen ICI 164,384 (47, 48).
It is not known yet which ERs (, ß, or both) are blocked by
tamoxifen treatment.
In the present study, E2 does not appear to be mitogenic for IOSE-29EC cells even though E2 resulted in a significant increase of thymidine after 6-day treatment, because the increase in thymidine incorporation was paralleled by an increase of DNA content per culture. In addition, no difference in proliferative index was obtained after E2 treatment for 1 or 2 days (data not shown). These observations suggest that the increase in thymidine incorporation and DNA content may be due to reduced apoptosis. This increase in thymidine incorporation could reflect the stimulation of proliferation by E2 (i.e. an increase in the proportion of dividing cells per total cell number), or it could be the result of an unchanged rate of proliferation in cell populations that had increased in size because apoptosis was inhibited. The observation that a significant increase in thymidine incorporation was only observed on day 6 of E2 treatment supports the latter possibility. To define the basis for the increase in thymidine incorporation more definitely, we carried out total DNA determinations on the cell populations. These determinations showed an increase in DNA content that paralleled the changes in thymidine incorporation, i.e. the ratio of thymidine incorporation over total cell number did not change. Therefore, it appears that the increase in cell number on day 6 was the result of inhibited apoptosis rather than enhanced proliferation. Further, in the work presented here, treatment with tamoxifen (10-6 M) only resulted in a growth-inhibitory effect in both IOSE-29 and IOSE-29EC cells, regardless of E2 treatment. Clinically relevant concentrations of tamoxifen (10-7–10-5 M) have been shown to inhibit the growth of the ER-negative ovarian cancer cell line, A2780, and induce apoptosis (49), This estrogen-independent role of tamoxifen in ER-negative ovarian and breast cancer cells was well documented in previous studies (49, 50, 51), suggesting that tamoxifen (10-6 M) may have dual functions, prevention of estrogen effect by blocking ER and inhibition of growth by itself through estrogen-independent manner in this experiment. Our results confirm others (31), which indicated that E2 does not affect the growth of normal OSE. The role of ERs in OSE and IOSE-29 remains to be elucidated, but our results suggest that the introduction of E-cadherin resulted in altered ER-elicited effect and responsiveness to E2 or tamoxifen resembling that of ovarian cancer lines.
Dysregulation of proliferation and/or cell death plays a critical role in tumorigenesis. The present study demonstrates that tamoxifen (10-6 M) can induce apoptosis in IOSE-29EC cells, whereas E2 can attenuate the effect of tamoxifen on these cells. Only IOSE-29EC cells were further used for apoptosis study because this cell line expressed both ERs and responded to E2/tamoxifen treatments. Cotreatment with E2 (10-8–10-6 M) plus tamoxifen attenuated tamoxifen-induced apoptosis in a dose-dependent manner. Among proapoptotic and antiapoptotic genes in the bcl-2 family, bax and bcl-2 genes are dominant regulators for apoptosis. The ratio of bcl-2 to bax is important in determining susceptibility to apoptosis (52). The present study has demonstrated that bax and bcl-2 are expressed at both mRNA and protein levels in neoplastic OSE cells. No difference was observed in the expression level of bax mRNA between IOSE-29 and IOSE-29EC cells. Interestingly, the expression level of bcl-2 mRNA and protein is higher in IOSE-29EC cells than IOSE-29 cells, suggesting that IOSE-29EC cells may be more resistant to apoptosis. In addition, treatments with E2 resulted in a significant increase of bcl-2 mRNA level (up to 2-fold), whereas E2 was attenuated with tamoxifen treatment, suggesting that the up-regulation of bcl-2 mRNA by E2 is mediated through specific receptors, ERs. These findings are in agreement with a previous report where estrogen up-regulated antiapoptotic bcl-2 gene, whereas bax level was not affected by E2 in breast cancer cells (4, 5, 53, 54). The up-regulation of bcl-2 by E2 in this series of experiments indirectly suggests that E2 affects the survival of IOSE-29EC cells through bcl-2, which is known to be a dominant regulator of apoptosis in other tissues (4, 5, 53, 54). It has been shown that estrogen down-regulated proapoptotic bak and antiapoptotic bcl-XL mRNA and protein in a dose-dependent manner, suggesting different members of bcl-2 family may be regulated through different pathway by estrogen (53). In parallel with the mRNA level, E2 caused a significant induction of bcl-2 protein level (up to 1.7-fold), whereas no difference was observed in bax mRNA level. This induction of bcl-2 protein by E2 was attenuated with tamoxifen treatment (10-6 M). Thus, the mechanism of estrogen on regulation of apoptotic pathway may be related with up-regulation of bcl-2 gene. Recently, it has been found that the bcl-2 major promoter does not contain cis-acting elements directly involved in transcriptional control by E2 and that E2 induces bcl-2 expression via two estrogen-responsive elements located within its coding region (5).
In conclusion, the present study indicates that early neoplastic
(IOSE-29), tumorigenic (IOSE-29EC), and late neoplastic (IOSE-29EC/T4
and T5) OSE cell lines, which were generated from normal OSE, express
both ER and ERß at the mRNA and protein levels.
E2 has been demonstrated to prevent tamoxifen
induced-apoptosis through ERs. The mechanism of action of
E2 may be associated with up-regulation of
bcl-2 gene at the mRNA and protein levels. These results
suggest that estrogen may play a role in the prevention of apoptosis in
tumorigenic OSE cells for ovarian tumorigenesis.
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Footnotes |
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2 Career investigator of the British Columbia Research Institute of
Children’s and Women’s Health.
Received October 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the rat ovary.
Endocrinology 140:963–971 and -ß, progesterone receptor, and androgen
receptor mRNA in normal and malignant ovarian epithelial cells. Proc
Natl Acad Sci USA 96:5722–5727) and beta (ER-ß ) mRNAs in normal ovary:
ovarian serous cystadenocarcinoma and ovarian cancer cell lines:
down-regulation of ER-in neoplastic tissues. J Clin Endocrinol
Metab 83:1025–1028 and ß-messenger RNAs as a
potential marker of ovarian carcinogenesis. Cancer Res 58:5367–5373This article has been cited by other articles:
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 F. C.-K. CHEN, G. OSKAY-OZCELIK, K. J. BUHLING, U. KOPSTEIN, M. MENTZE, W. LICHTENEGGER, and J. SEHOULI Prognostic Value of Serum and Ascites Levels of Estradiol, FSH, LH and Prolactin in Ovarian Cancer Anticancer Res, May 1, 2009; 29(5): 1575 - 1578. [Abstract] [Full Text] [PDF]
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 G. N. Armaiz-Pena, L. S. Mangala, W. A. Spannuth, Y. G. Lin, N. B. Jennings, A. M. Nick, R. R. Langley, R. Schmandt, S. K. Lutgendorf, S. W. Cole, et al. Estrous Cycle Modulates Ovarian Carcinoma Growth Clin. Cancer Res., May 1, 2009; 15(9): 2971 - 2978. [Abstract] [Full Text] [PDF]
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 J.-H. Choi, C.-L. Chen, S. L. Poon, H.-S. Wang, and P. C K Leung Gonadotropin-stimulated epidermal growth factor receptor expression in human ovarian surface epithelial cells: involvement of cyclic AMP-dependent exchange protein activated by cAMP pathway Endocr. Relat. Cancer, March 1, 2009; 16(1): 179 - 188. [Abstract] [Full Text] [PDF]
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 S.-H. Park, L. W. T. Cheung, A. S. T. Wong, and P. C. K. Leung Estrogen Regulates Snail and Slug in the Down-Regulation of E-Cadherin and Induces Metastatic Potential of Ovarian Cancer Cells through Estrogen Receptor {alpha} Mol. Endocrinol., September 1, 2008; 22(9): 2085 - 2098. [Abstract] [Full Text] [PDF]
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 A. Vasconsuelo, L. Milanesi, and R. Boland 17{beta}-Estradiol abrogates apoptosis in murine skeletal muscle cells through estrogen receptors: role of the phosphatidylinositol 3-kinase/Akt pathway J. Endocrinol., February 1, 2008; 196(2): 385 - 397. [Abstract] [Full Text] [PDF]
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 J.-H. Choi, A. S. T. Wong, H.-F. Huang, and P. C. K. Leung Gonadotropins and Ovarian Cancer Endocr. Rev., June 1, 2007; 28(4): 440 - 461. [Abstract] [Full Text] [PDF]
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 A. Zheng, A. Kallio, and P. Harkonen Tamoxifen-Induced Rapid Death of MCF-7 Breast Cancer Cells Is Mediated via Extracellularly Signal-Regulated Kinase Signaling and Can Be Abrogated by Estrogen Endocrinology, June 1, 2007; 148(6): 2764 - 2777. [Abstract] [Full Text] [PDF]
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 P. C.K. Leung and J.-H. Choi Endocrine signaling in ovarian surface epithelium and cancer Hum. Reprod. Update, March 1, 2007; 13(2): 143 - 162. [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] [PDF]
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 J. S. Lewis, K. Meeke, C. Osipo, E. A. Ross, N. Kidawi, T. Li, E. Bell, N. S. Chandel, and V. C. Jordan Intrinsic Mechanism of Estradiol-Induced Apoptosis in Breast Cancer Cells Resistant to Estrogen Deprivation J Natl Cancer Inst, December 7, 2005; 97(23): 1746 - 1759. [Abstract] [Full Text] [PDF]
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J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C K Leung Gonadotropins upregulate the epidermal growth factor receptor through activation of mitogen-activated protein kinases and phosphatidyl-inositol-3-kinase in human ovarian surface epithelial cells Endocr. Relat. Cancer, June 1, 2005; 12(2): 407 - 421. [Abstract] [Full Text] [PDF]
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 K.-Y. Kim, K.-C. Choi, S.-H. Park, N. Auersperg, and P. C. K. Leung Extracellular Signal-Regulated Protein Kinase, But Not c-Jun N-Terminal Kinase, Is Activated by Type II Gonadotropin-Releasing Hormone Involved in the Inhibition of Ovarian Cancer Cell Proliferation J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1670 - 1677. [Abstract] [Full Text] [PDF]
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 C. M. Grimaldi, R. Hicks, and B. Diamond B Cell Selection and Susceptibility to Autoimmunity J. Immunol., February 15, 2005; 174(4): 1775 - 1781. [Abstract] [Full Text] [PDF]
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 D. A. Symonds, D. Tomic, K. P. Miller, and J. A. Flaws Methoxychlor Induces Proliferation of the Mouse Ovarian Surface Epithelium Toxicol. Sci., February 1, 2005; 83(2): 355 - 362. [Abstract] [Full Text] [PDF]
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 M. E. Jung, M. B. Gatch, and J. W. Simpkins Estrogen Neuroprotection Against the Neurotoxic Effects of Ethanol Withdrawal: Potential Mechanisms Experimental Biology and Medicine, January 1, 2005; 230(1): 8 - 22. [Abstract] [Full Text] [PDF]
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J.-H. Choi, K.-C. Choi, N. Auersperg, and P. C. K. Leung Overexpression of Follicle-Stimulating Hormone Receptor Activates Oncogenic Pathways in Preneoplastic Ovarian Surface Epithelial Cells J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5508 - 5516. [Abstract] [Full Text] [PDF]
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 O Gubbay, W Guo, M T Rae, D Niven, A F Howie, A S McNeilly, L Xu, and S G Hillier Anti-inflammatory and proliferative responses in human and ovine ovarian surface epithelial cells Reproduction, November 1, 2004; 128(5): 607 - 614. [Abstract] [Full Text] [PDF]
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A Bardin, N Boulle, G Lazennec, F Vignon, and P Pujol Loss of ER{beta} expression as a common step in estrogen-dependent tumor progression Endocr. Relat. Cancer, September 1, 2004; 11(3): 537 - 551. [Abstract] [Full Text] [PDF]
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 R. I. Fernando and J. Wimalasena Estradiol Abrogates Apoptosis in Breast Cancer Cells through Inactivation of BAD: Ras-dependent Nongenomic Pathways Requiring Signaling through ERK and Akt Mol. Biol. Cell, July 1, 2004; 15(7): 3266 - 3284. [Abstract] [Full Text] [PDF]
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K.-Y. Kim, K.-C. Choi, S.-H. Park, C.-S. Chou, N. Auersperg, and P. C. K. Leung Type II Gonadotropin-Releasing Hormone Stimulates p38 Mitogen-Activated Protein Kinase and Apoptosis in Ovarian Cancer Cells J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 3020 - 3026. [Abstract] [Full Text] [PDF]
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S. Mabuchi, M. Ohmichi, A. Kimura, Y. Nishio, E. Arimoto-Ishida, N. Yada-Hashimoto, K. Tasaka, and Y. Murata Estrogen Inhibits Paclitaxel-Induced Apoptosis via the Phosphorylation of Apoptosis Signal-Regulating Kinase 1 in Human Ovarian Cancer Cell Lines Endocrinology, January 1, 2004; 145(1): 49 - 58. [Abstract] [Full Text] [PDF]
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S. M. Raza, G. N. Fuller, C. H. Rhee, S. Huang, K. Hess, W. Zhang, and R. Sawaya Identification of Necrosis-Associated Genes in Glioblastoma by cDNA Microarray Analysis Clin. Cancer Res., January 1, 2004; 10(1): 212 - 221. [Abstract] [Full Text] [PDF]
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 A. Evangelou, M. Letarte, I. Jurisica, M. Sultan, K. J. Murphy, B. Rosen, and T. J. Brown Loss of Coordinated Androgen Regulation in Nonmalignant Ovarian Epithelial Cells with BRCA1/2 Mutations and Ovarian Cancer Cells Cancer Res., May 15, 2003; 63(10): 2416 - 2424. [Abstract] [Full Text] [PDF]
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 P. Dan, J. C. Y. Cheung, D. R. L. Scriven, and E. D. W. Moore Epitope-dependent localization of estrogen receptoralpha , but not -beta , in en face arterial endothelium Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1295 - H1306. [Abstract] [Full Text] [PDF]
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 M. E. Marin-Castano, S. J. Elliot, M. Potier, M. Karl, L. J. Striker, G. E. Striker, K. G. Csaky, and S. W. Cousins Regulation of Estrogen Receptors and MMP-2 Expression by Estrogens in Human Retinal Pigment Epithelium Invest. Ophthalmol. Vis. Sci., January 1, 2003; 44(1): 50 - 59. [Abstract] [Full Text] [PDF]
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 K.-C. Choi, C.-J. Tai, C.-R. Tzeng, N. Auersperg, and P. C.K. Leung Adenosine Triphosphate Activates Mitogen-Activated Protein Kinase in Pre-Neoplastic and Neoplastic Ovarian Surface Epithelial Cells Biol Reprod, January 1, 2003; 68(1): 309 - 315. [Abstract] [Full Text] [PDF]
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 K. Burry, J. M. Cain, L. Gilbert, S. Krishnamurthy, S. L. Tan, E. L. Franco, J. V. Lacey, Jr, P. J. Mink, J. H. Lubin, M. E. Sherman, et al. Estrogen Replacement Therapy and Risk of Ovarian Cancer in Postmenopausal Women JAMA, November 27, 2002; 288(20): 2538 - 2539. [Full Text] [PDF]
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C. Borgeest, D. Symonds, L. P. Mayer, P. B. Hoyer, and J. A. Flaws Methoxychlor May Cause Ovarian Follicular Atresia and Proliferation of the Ovarian Epithelium in the Mouse Toxicol. Sci., August 1, 2002; 68(2): 473 - 478. [Abstract] [Full Text] [PDF]
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