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Endocrinology Vol. 142, No. 6 2351-2360
Copyright © 2001 by The Endocrine Society

ARTICLES

Estradiol Up-Regulates Antiapoptotic Bcl-2 Messenger Ribonucleic Acid and Protein in Tumorigenic Ovarian Surface Epithelium Cells1

Kyung-Chul Choi, Sung Keun Kang, Chen-Jei Tai, Nelly Auersperg and Peter C. K. Leung2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Most epithelial ovarian tumors appear to arise from the ovarian surface epithelium (OSE). Even though it has been suggested that estrogen may be associated with ovarian tumorigenesis, the exact role of estrogen in the regulation of apoptosis in neoplastic OSE cells remains uncertain. Immortalized OSE (IOSE) cell lines were generated from human normal OSE. These cell lines represent early neoplastic (IOSE-29), tumorigenic (IOSE-29EC), and late neoplastic (IOSE-29EC/T4 and IOSE-29EC/T5) transformation stages from human normal OSE. The present studies demonstrated that both mRNAs and proteins of estrogen receptor (ER) {alpha} 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{alpha} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOSIS, which is documented as programmed cell death, is an important phenomenon in normal physiological processes. Morphologically, this phenomenon is characterized by chromatin condensation, membrane blebbing, and loss of cell volume (1, 2, 3). It has been suggested that physiological and pathological stimuli, such as steroids (4, 5, 6) or peptide hormones (7, 8), growth factors (9, 10), cytokines (11), radiation (12), and anticancer drugs (13, 14, 15) may regulate apoptotic pathways in ovarian or breast cancer cells. The bcl-2 gene family is widely accepted as regulators of cell death (reviewed in Refs. 1 and 16), and bax and bcl-2 are considered dominant regulators of apoptosis. The ratio of bcl-2 to bax is critical in determining susceptibility to apoptosis (16). Furthermore, it has been demonstrated that steroid hormones may regulate proapoptotic or antiapoptotic genes in breast and ovarian cancer cells (4, 5, 6, 17).

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{alpha}) 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{alpha} and ERß in the ovary, suggesting different biological roles of ER{alpha} and ERß in this tissue (26, 27, 28). In addition, the existence of ER{alpha} 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{alpha} 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatments
Normal human OSE cells were scraped from the ovarian surface during laparoscopies for nonmalignant disorders and cultured as previously described (38) in medium 199:MCDB 105 (Life Technologies, Inc., Burlington, Canada; and Sigma-Aldrich Corp., Oakville, Canada, respectively) containing 10% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc.) in a humidified atmosphere of 5% CO2-95% air and passaged with 0.06% trypsin (1:250)/0.01% EDTA in Mg2+/Ca2+-free HBSS when confluent.

As outlined in Table 1Go, 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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  		Table 1. Development of early neoplastic, tumorigenic and late neoplastic OSE cells from human normal OSE {texf}

 
RNA extraction and RT-PCR procedure
Total RNA was prepared from cultured cells using the RNaid kit (Bio/Can Scientific, Mississauga, Canada) according to the manufacturer’s suggested procedure. RNA integrity was confirmed by agarose gel electrophoresis and ethidium bromide staining. The total RNA concentration was determined from spectrophotometric analysis at A260/280. cDNA was synthesized from 2.5 µg total RNA by RT at 37 C for 2 h using a first strand cDNA synthesis kit (Pharmacia Ltd., Uppsala, Sweden). The synthesized cDNA was used as template for PCR amplification. The amplification was achieved using a thermal cycler (DNA Thermal Cycler; Perkin-Elmer Corp., Norwalk, CT). Total RNA (2.5 µg) was reverse transcribed into first strand cDNA (Amersham Pharmacia Biotech, Oakville, Canada) following the manufacturer’s procedure. Synthetic oligonucleotides used for PCR primers and PCR conditions are listed in Table 2Go 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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  		Table 2. Oligonucleotide sequences of PCR primers for human

 
Southern blot analysis
Following electrophoresis, PCR products were transferred to nylon membranes (Hybond-N; Amersham Pharmacia Biotech) and fixed by UV irradiation. The blotted membranes were prehybridized for 3 h at 42 C in prehybridization solution containing 50% formamide, 5x SSC, 0.1% N-lauroyl sarcosine, 0.02% SDS, and 2% blocking solution. The prehybridized membranes were hybridized overnight at 42 C with digoxigenine (DIG)-labeled probes. PCR products of ER{alpha} 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 ({alpha} 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{alpha} (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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of ER{alpha} and ERß mRNAs
The mRNA levels of ER{alpha} 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. 1Go). 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{alpha}, and ERß were obtained as 373 bp, 540 bp, and 279 bp, respectively, and confirmed by Southern blot analysis using DIG-labeled probes (Fig. 1Go) and sequence analysis (data not shown). This result indicates that the mRNAs of ER{alpha} and ERß are expressed in IOSE-29, IOSE-29EC, IOSE-29EC/T4, and IOSE-29EC/T5.



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  		Figure 1. The mRNA levels of ER{alpha} and ERß in IOSE cell lines. The mRNA levels of ER{alpha} and ERß in IOSE-29 (passages 13–16), IOSE-29EC (passages 15–17), IOSE-29EC/T4 (T4), and IOSE-29EC/T5 (T5) were investigated by RT-PCR and Southern blot analysis. Expected PCR products of GAPDH, ER{alpha}, and ERß were obtained as 373 bp, 540 bp, and 279 bp, respectively, and confirmed by Southern blot analysis using DIG-labeled probes and sequence analysis (data not shown).

 
Expression of ER{alpha} and ERß proteins
To investigate the expression of ER{alpha} and ERß proteins in immortalized OSE cell lines, immunoblot analysis was performed using the mouse monoclonal antibody for ER{alpha} and a goat polyclonal antibody for ERß. As shown in Fig. 2Go, ER{alpha} 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{alpha} and ERß proteins were observed in IOSE-29, IOSE-29EC, IOSE-29EC/T4, and IOSE-29EC/T5.



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  		Figure 2. The protein levels of ER{alpha} and ERß in IOSE cell lines. In parallel of mRNA expression of ER{alpha} and ERß, immunoblot analysis was carried out using specific antibodies for ER{alpha} and ERß in IOSE cell lines. OVCAR-3 cell line was used for positive control of the expression of ERs. ER{alpha} protein (68 kDa) was observed in all cell types. ERß protein was also observed as 55 kDa in IOSE cell lines. T4, IOSE-29EC/T4; T5, IOSE-29EC/T5.

 
Effects of E2 on cell proliferation
To evaluate the role of E2 in normal and immortalized OSE cell lines, the cells were treated with increasing concentrations (10-8, 10-7, and 10-6 M) of E2 for 2–6 days. The [3H]thymidine incorporation and DNA fluorometric assays were performed as previously described in Materials and Methods. Tamoxifen (10-6 M), which is an estrogen antagonist, was used to block the action of E2 in the cell proliferation study. Treatment with E2 did not affect the growth of normal OSE (Fig. 3AGo), 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. 3BGo). 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. 3BGo). No difference was observed following E2 treatment for 6 days in IOSE-29 cells by thymidine incorporation (Fig. 3CGo) and fluorometric assay (Fig. 3DGo). 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. 3FGo; 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. 3GGo; 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. 3EGo) and IOSE-29EC cells (Fig. 3HGo), suggesting that E2 effect does not include stimulation of proliferation. Treatment with tamoxifen only also caused inhibitory effect in IOSE-29 (Fig. 3Go, C and D) and IOSE-29EC cells (Fig. 3Go, 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. 3Go, I and J).




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  		Figure 3. Effect of E2 on cell proliferation/apoptosis. To evaluate the role of E2 in IOSE cell lines, the cells were treated with increasing concentrations (10-8, 10-7, and 10-6 M) of E2 and/or tamoxifen (Txf; 10-6 M) for 2–6 days as previously described in Materials and Methods. [3H]Thymidine incorporation was analyzed following E2 treatment for 2–6 days in normal OSE (A) and OVCAR-3 cells (B). [3H]Thymidine incorporation and DNA fluorometic assays were performed following E2/tamoxifen treatment for 6 days in IOSE-29 (C and D) and IOSE-29EC cells (F and G). The ratio of thymidine incorporation to DNA content per culture did not change following E2 and/or tamoxifen treatments in IOSE-29 (E) and IOSE-29EC cells (H). In addition, thymidine incorporation by E2/tamoxifen was also analyzed in IOSE-29EC/T4 and IOSE-29EC/T5 cells (I and J) after 6-day treatment. Data are shown as the means of three individual experiments in triplicate, and are presented as the mean ± SD. a, P < 0.05 vs. untreated control; b, P < 0.05 vs. E2 (10-7 M) treatment.

 
Effects of E2 on apoptosis
To examine the role of E2 in the prevention of apoptosis, DNA fragmentation was measured using the cell death detection ELISA. To quantify the induction of apoptosis, IOSE-29EC cells were treated with tamoxifen (10-6 M) and/or E2 (10-8, 10-7, and 10-6 M) for 6 days. As shown in Fig 4Go, 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. 4Go, and 287.7 ± 11.26% vs. 219.8 ± 21.47%, 175.8 ± 12.02%, or 174.9 ± 16.50%).



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  		Figure 4. Effect of E2 on apoptosis. To examine the role of E2 in the prevention of apoptosis, DNA fragmentation was measured using the cell death detection ELISA. Data are shown as the means of three individual experiments with duplicate, and are presented as the mean ± SD. a, P < 0.05 vs. untreated control; b, P < 0.05 vs. tamoxifen (10-6 M) treatment; c, P < 0.05 vs. E2 (10-8 M) plus tamoxifen (10-6 M) treatment.

 
Expression of proapoptotic and antiapoptotic gene mRNAs and proteins
The mRNA levels of proapoptotic bax and antiapoptotic bcl-2 in IOSE-29 and IOSE-29EC were investigated by RT-PCR and Southern blot analysis. Predicted PCR products of GAPDH, bax, and bcl-2 were obtained as 373 bp, 323 bp, and 459 bp, respectively, and confirmed by Southern blot analysis using DIG-labeled probes (Fig. 5Go) 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. 5AGo). In parallel of mRNA level, protein level of bcl-2 was investigated in these cell lines. As shown in Fig. 5BGo, the expression level of bcl-2 protein was higher in IOSE-29EC cells than IOSE-29 cells.



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  		Figure 5. Expression of bax and bcl-2 mRNAs and proteins. The mRNA and protein levels of bax and bcl-2 in IOSE-29 and IOSE-29EC were investigated by RT-PCR, Southern blot, and immunoblot analysis. Predicted PCR products of GAPDH, bax, and bcl-2 were obtained as 373 bp, 323 bp, and 459 bp, respectively, and confirmed by Southern blot analysis using DIG-labeled probes and sequence analysis (data not shown). A, The mRNA level of bax and bcl-2 in IOSE-29 and IOSE-29EC cells. B, The protein levels of bcl-2 in IOSE-29 and IOSE-29EC cells. The protein amount was normalized by actin protein (41 kDa).

 
Effect of E2 on proapoptotic and antiapoptotic mRNAs
To investigate the mechanism of E2 action in the prevention of apoptosis, the regulation of bax and bcl-2 was examined using RT-PCR in IOSE-29EC cells. The cells were treated with E2 and/or tamoxifen for 24 h. The expected sizes of PCR products for bax and bcl-2 were obtained as 323 bp and 459 bp, respectively. The mRNA expression of bax and bcl-2 was normalized with GAPDH (373 bp) to quantify the mRNA levels. Treatments with E2 (10-8–10-6 M) up-regulated bcl-2 mRNA up to 2-fold in these cells (Fig. 6Go, 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. 6AGo).



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  		Figure 6. Effect of E2 on bax and bcl-2 mRNA levels. To investigate the mechanism of E2 in the prevention of apoptosis, the regulation of bax and bcl-2 was examined using RT-PCR in IOSE-29EC cells. The cells were treated with E2 and/or tamoxifen (Txf) for 24 h. The expected sizes of PCR products for bax and bcl-2 were obtained as 323 bp and 459 bp, respectively. The mRNA expression of bax and bcl-2 was normalized with GAPDH (373 bp) to quantify the mRNA levels. A, The expression of bax and bcl-2 mRNA after treatment with E2 and/or tamoxifen. B, Relative bcl-2 mRNA expression after treatment E2 and/or tamoxifen. Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P < 0.05 vs. untreated control; b, P < 0.05 vs. E2 (10-7 M) treatment. Lane 1, Untreated control; lane 2, E2 (10-8 M) treatment; lane 3, E2 (10-7 M) treatment; lane 4, E2 (10-6 M) treatment; lane 5, tamoxifen (10-6 M) treatment; lane 6, E2 (10-7 M) plus tamoxifen (10-6 M) treatment.

 
Effect of E2 on proapoptotic and antiapoptotic proteins
To investigate the protein levels of bax and bcl-2 by E2, immunoblot analysis was performed following E2 and/or tamoxifen treatments. The cells were treated with E2 and/or tamoxifen for 48 h as previously described in Materials and Methods. Specific signals for bax and bcl-2 protein were detected at 21 and 26 kDa, respectively, as shown in Fig. 7Go. Consistent with the mRNA levels, treatments with E2 (10-8–10-6 M) significantly up-regulated bcl-2 protein level in these cells (Fig. 7Go, 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. 7AGo).



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  		Figure 7. Effect of E2 on bax and bcl-2 proteins. In parallel of mRNA level, protein level of bax and bcl-2 was investigated by immunoblot analysis. The cells were treated with E2 and/or tamoxifen (Txf) for 48 h as previously described in Materials and Methods. Specific signals for bax and bcl-2 protein were detected at 21 kDa and 26 kDa, respectively. A, The expression of bax and bcl-2 protein after E2/tamoxifen treatment. B, Relative bcl-2 protein level after E2/tamoxifen treatment. The protein amount in the groups was normalized by actin protein (41 kDa). Data are shown as the means of three individual experiments, and are presented as the mean ± SD. a, P < 0.05 vs. untreated control; b, P < 0.05 vs. E2 (10-7 M) treatment. Lane 1, Untreated control; lane 2, E2 (10-8 M) treatment; lane 3, E2 (10-7 M) treatment; lane 4, E2 (10-6 M) treatment; lane 5, tamoxifen (10-6 M) treatment; lane 6, E2 (10-7 M) plus tamoxifen (10-6 M) treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The characteristics of IOSE-29EC resemble to those of ovarian cancer (35, 36, 37). The previous reports have suggested that the IOSE-29 derived cell lines represent early neoplastic (IOSE-29), tumorigenic (IOSE-29EC), and late neoplastic (IOSE-29EC/T4 and T5) transformation stages of human normal OSE. Thus, these cell lines should be a useful in vitro model to understand ovarian tumorigenesis because these cell lines were generated by introducing only two genes (simian virus 40 large T antigen and E-cadherin genes) into normal OSE. The present studies demonstrated that both mRNAs and proteins of ER{alpha} 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{alpha} and ERß (Refs. 29, 30). In addition, the expression levels of ER{alpha} were enhanced when compared with those in normal ovaries, whereas ERß levels were significantly decreased in ovarian tumors, suggesting that ER{alpha} and ERß mRNAs are differentially expressed in normal and neoplastic OSE cells (30, 43). These results suggest that overexpression of ER{alpha} 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{alpha} (approximately 67 kDa) detected by Western blotting (27, 39). ER{alpha} and ERß can homodimerize ({alpha}{alpha} or ßß) or heterodimerize ({alpha}ß) upon binding to the ERE (45). Thus, it is hypothesized that differential expression of ER{alpha} 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{alpha} 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 ({alpha}, 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{alpha} 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.


   Footnotes
 
1 This research was supported by grants from the Canadian Institutes of Health Research and National Cancer Institute of Canada. Back

2 Career investigator of the British Columbia Research Institute of Children’s and Women’s Health. Back

Received October 17, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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