Program in Toxicology and Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
Received September 3, 2004; accepted October 25, 2004
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ABSTRACT |
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Key Words: methoxychlor; ovarian surface epithelium; proliferation; cell cycle; apoptosis; estrogen receptor.
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INTRODUCTION |
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Since the OSE is known to possess estrogen receptor (ER) (Pelletier et al., 2000
), and MXC has been shown to bind to estrogen receptors (Gaido et al., 1999
), it is possible that MXC affects the OSE via an estrogen receptor pathway. This possibility is supported by literature indicating that the OSE is a modified coelemic derivative with specialized characteristics that enable it to respond to hormones and hormone-like agents (Auersperg et al., 2001
). Specifically, some studies have shown a proliferative response of the OSE to estrogen in the rabbit (Bai et al., 2000
) and rat (Stewart et al., 2004
). While estrogen increases proliferation of the OSE in these species, it is unclear to what extent MXC and other xenoestrogens reproduce the effects of estrogen or to what extent they operate through an ER pathway. Hodges et al. (2000)
found that 2,2-bis[p-hydroxyphenyl]-1,1,1,-trichloroethane (HPTE), the active metabolite of MXC, induced proliferation in uterine smooth muscle cultures, and that the proliferation was diminished by the estrogen receptor blocker ICI 182,780, suggesting an ER-mediated mechanism for MXC in the uterus. In contrast, Ghosh et al. (1999)
found that uterine lactoferrin and glucose-6-phosphate dehydrogenase mRNA are increased by both estrogen and MXC, and that the estrogen-induced increases in these mRNAs, but not the MXC-induced increases in these mRNAs, were blocked by ICI 182,780, suggesting a non-ER mechanism for MXC.
Thus, one objective of this study was to test the hypothesis that MXC alters cell kinetics of the OSE, resulting in increased proliferation, either through stimulation of the cell cycle and its regulators, or through inhibition of apoptosis and its regulators. Parallel studies were performed with HPTE because this is considered to be the most active metabolite of MXC (Cummings, 1997). Since the OSE has ER (Pelletier et al., 2000
) and MXC binds to ER (Gaido et al., 1999
), a second objective was to test the hypothesis that MXC alters cell growth through an ER pathway. A third goal of this work was to isolate mouse OSE and to develop an in vitro culture system that could be used to examine the effects of MXC on the OSE. While established cell lines have been reported with SV40 large T antigen transfection (Jiang et al., 2003
) and p53-deficient mice (Kido et al., 1998
), we desired to obtain an epithelial component in a native state. Therefore, we adapted a method originally described to isolate rabbit OSE (Nicosia et al., 1984
) to obtain sufficient cells for studies in mice.
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MATERIALS AND METHODS |
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Primary cultures of OSE cells. Ovaries were excised from mice aseptically and individually placed in 0.5 ml of Medium 199(E) (BioSource, Rockville, MD) with 500 units of crude collagenase (Type XI, Sigma-Aldrich Inc., St. Louis, MO). After 60 min incubation at 37°C, each ovary was vortexed for 2 min, ovaries were removed, and the suspension was vortexed again for 2 min to disaggregate cell clusters. The suspension was diluted with additional Medium 199(E) containing 15% fetal calf serum and an anti-microbial solution (10 µl/ml) containing 10 mg/ml streptomycin, 10 mU/ml penicillin G, and 25 µg/ml amphotericin (Sigma-Aldrich Inc., St. Louis, MO). Aliquots containing 200500 cells/100 µl from a single ovary were pipetted into 96-well plates with frequent mixing during aliquoting. Control and treatment wells were from the same suspension. Cultures were incubated at 37°C with 5% CO2 for 3 days to allow for attachment. The residual ovarian tissue was histologically examined for the extent of removal of the OSE. To confirm that the cell suspensions were epithelial, they were evaluated with a monoclonal, mouse ascitic fluid pan cytokeratin cocktail as primary antibody at 1:100 dilution (Sigma Aldrich Inc., St. Louis, MO) and with a fluorescein-labeled secondary antibody (Sigma Aldrich Inc., St. Louis, MO) at 1:1000 dilution. To confirm that the attached cells were epithelial, they were evaluated with immunohistochemistry using a pan cytokeratin cocktail as primary antibody (Sigma Aldrich Inc., St. Louis, MO) at 1:100 dilution and visualization with the HistomouseTM-SP system (Zymed Laboratories, San Francisco, CA) according to manufacturer's specifications. To confirm that isolated cells still contained ER, the cells were subjected to real-time polymerase chain reaction for ER
mRNA expression as described below.
Treatment of cultures. After cell attachment, cell cultures were incubated with 150 µl of Medium 199(E) containing 15% fetal calf serum, anti-microbial solution, and one of the following: vehicle (dimethylsulfoxide; DMSO), MXC (3, 30, 300 µM), HPTE (3 µM), ICI 182,780 (1 µM), ICI 182,780 (1 µM) plus MXC (3 µM), or ICI 182,780 (1 µM) plus HPTE (3 µM). Incubation was carried out for 14 days with renewal of incubation solution every third day. Purified MXC was obtained from ChemService (West Chester, PA), HPTE was obtained from Cedra Corp. (Austin, TX), and ICI 182,780 was obtained from Tocris-Cookson (Ellisville, MO). The doses of MXC, HPTE, and ICI 182,780 were selected because they have been shown to affect either proliferation or gene expression in ER-regulated tissues (Chedres et al., 2001; Hodges et al., 2000
).
Measurement of cell growth. After treatment, cell cultures adherent to the microtiter plates were washed twice with phosphate buffer and subjected to the CyQuantTM Assay (Molecular Probes, Eugene, OR). Briefly, cells were frozen at 70°C for 30 min, thawed, and treated with 200 µl of lysis buffer containing CyQuant GR dye, which binds stoichiometrically with nucleic acids and, thus, measures cell number (Jones et al., 2001). Quantification was performed with a microtiter plate fluorimeter (CytoFluor 4000, PerSeptive Biosystems) with excitation at 485 nm and emission detection at 530 nm.
Measurement of cell proliferation. Cell cultures adherent to the microtiter plates were fixed for 30 min with 70% ethanol at 4°C. Mouse anti-PCNA (EMD Biosciences, San Diego, CA) was used at a dilution of 1:100 for 30 min incubation in combination with the HistomouseTM-SP system (Zymed Laboratories, San Francisco, CA) and DAB chromogen. Nuclei with dense, granular staining were enumerated as positive for proliferating cell nuclear antigen (PCNA). Over one thousand cells were counted for each treatment group in at least three independent experiments.
Measurement of apoptosis. The ApopTag Peroxidase system (Chemicon Int., Temecula, CA) was used to identify apoptotic cells by labeling free 3'-hydroxy DNA termini via enzymatic reaction and immunohistochemical demonstration of labeled nucleotides. Cell cultures adherent to the microtiter plates were treated according to manufacturer's instructions for adherent cultured cells and evaluated directly without dehydration or mounting. Densely dark stained cells were enumerated as positive and recorded as a percentage of total cells.
RNA isolation for real-time polymerase chain reaction (PCR). Cells grown for 14 days with the above treatment schedule were used for RNA isolation. Total RNA was isolated using the RNeasy procedure from Qiagen (Valencia, CA) according to the manufacturer's protocol. Cells were detached in lysis buffer by scraping with an Eppendorf pipette tip, and lysates from six wells were pooled. Homogenization was performed with a QiShredder column and a syringe containing a 21-gauge needle. Reverse-transcriptase generation of cDNA was performed with 0.4 µg of total RNA using an Omniscript RT kit (Qiagen, Inc., Valencia, CA) with random primers according to the manufacturer's protocols. Subsequent PCR analysis was carried out on 3 µl of the cDNA as described below.
Real-time PCR analysis of ER, bcl-2, bax, cyclinD2, and cdk4. Real-time PCR analysis was performed as previously described (Borgeest et al., 2004
) using a MJ Research (OPTICON) Real Time PCR machine and accompanying software according to the manufacturer's instructions (MJ Research Inc., Waltham, MA). The OPTICON quantifies the amount of PCR product generated by measuring a dye (SYBR green) that fluoresces when bound to double-stranded DNA. A standard curve was generated from five serial dilutions of purified PCR product. Primer sequences for each product were as follows: ER
(forward) 5'-AATTCTGACAATCGACGCCAG-3', (reverse) 5'-GTGCTTCAACATTCTCCCTCCTC-3'; bcl-2 (forward) 5'-CTCGTCGCTACCGTCGTGACTTCG-3', (reverse) 5'-CAGATGCCGGTTCAGGTACTCAGTC-3'; bax (forward) 5'-ACCAGCTCTGAACAGATCATG-3', (reverse) 5'-TGGATCCAGACAAG-3'; cyclinD2 (forward) 5'-ATCTGTCCCTGATCCGCAAG-3', (reverse) 5'-GTCAACATCCCGCACGTCTG-3' (Piatelli et al., 2002
); cdk4 (forward) 5'-TGGCTGCCACTCGATATGAAC-3', (reverse) 5'-CCTCAGGTCCTGGTCTATATG-3' (Perez de Castro et al., 1999
). Primers specific for mouse ß-actin were used as an internal control as previously described (Weihua et al., 2000
). For each primer, a melting curve was performed. An initial incubation of 95°C for 10 min was followed by 40 cycles at 94°C for 10 s, 55°C for 1020 s, and 72°C for 1030 s, with final extension at 72°C for 10 min. Arbitrary numbers were assigned for each standard. Values were calculated for the experimental samples from the standard curve. ß-Actin mRNA was measured in each sample and used to normalize ratios between samples. Data were collected from at least three independent experiments.
Statistical analysis. Data were analyzed using SPSS statistical software (SPSS, Inc., Chicago). ANOVA was used with Tukey's test in the post hoc analysis for cell growth studies to ascertain differences between each group. An independent t test was used to compare controls to treatment groups in the other studies. Data are presented as means ± standard error of the mean. A p value 0.05 was considered significant.
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RESULTS |
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Effect of MXC and HPTE on Expression of bcl-2, bax, cyclinD2 and cdk4
Real-time PCR demonstrated a significant increase in the anti-apoptotic factor bcl-2 in treated versus control cells: normalized bcl-2/ß-actin ratios were 0.78 ± 0.23 for DMSO-treated cells, 2.0 ± 0.45 for MXC-treated cells (n = 3, p 0.05), and 1.56 ± 0.12 for HPTE-treated cell (n = 3, p
0.05) (Fig. 5A). Expression of bax for MXC- and HPTE-treated cells was decreased compared to controls (Fig. 5B; bax/ß-actin-normalized ratios of 1.25 ± 0.26 for DMSO-treated cells, 0.67 ± 0.08 for MXC-treated cells, p
0.05; and 0.69 ± 0.12 for HPTE-treated cells, n = 3, p
0.07).
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DISCUSSION |
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The present study demonstrates that the OSE responds to MXC and its major metabolite HPTE with increased cellular growth, as quantified by total nucleic acid content, through an ER-dependent mechanism (see Fig. 3). It appears to do so by stimulating cell cycle progression, as manifested by increased PCNA staining (see Fig. 4) and up-regulation of cyclinD2 (see Fig. 5C) and cdk4 (see Fig. 5D) compared to controls. It also appears to do so to a lesser extent by dampening apoptotic cell removal, as manifested by decreased apoptosis and up-regulation of bcl-2 in MXC- and HPTE-treated cells (see Fig. 5A) compared to controls. To our knowledge, this is the first study to report the effects of MXC and HPTE on proliferation and apoptosis in the OSE. Previous studies have shown that estradiol is mitogenic for granulosa cells by up-regulating cyclinD2 transcription and enabling progression through G1 phase of the cell cycle (Robker et al., 1998). Estradiol has also been demonstrated to up-regulate bcl-2 transcription in both OSE neoplastic cells (Choi et al., 2001
) and normal breast cells (Somai et al., 2003
). Thus, our study suggests that MXC and HPTE appear to reproduce the dual action of estradiol by regulation of both the cell cycle and apoptosis.
While the exact mechanisms of the estrogenic activity of MXC and HPTE are unknown, the suppression of OSE proliferation by ICI 182,780 in our study suggests an ER-mediated process. Estrogenic compounds stimulate or repress transcription through a variety of genes by means of two different receptors, ER and ERß (Harrington et al., 2003
). MXC, via its major metabolite HPTE, has been shown to stimulate ER
, but to suppress ERß-mediated activity in transfected hepatoma cells (Gaido et al., 1999
). This differential action of MXC in different ERs could explain the simultaneous inhibitory and stimulatory effects upon growth regulation in the ovary. For example, MXC has been shown to up-regulate pro-apoptotic bax and induce cell death of granulosa cells, an ovarian cell type that predominantly contains ERß (Borgeest et al., 2002
). This current study shows that decreased apoptosis in the ER
-controlled OSE may be due to up-regulation of anti-apoptotic bcl-2 and may contribute to increased cell density. Apoptosis is a complex process in which Bcl-2 is suppressive by forming heterodimers with pro-apoptotic proteins, ultimately inhibiting the final stage in the apoptotic cascade, caspase-3 activation (Somai et al., 2003
). The importance of disturbed apoptosis as a pathway for allowing genetically damaged cells to initiate carcinogenesis has been recognized (Wyllie, 1997
), and anti-apoptotic mechanisms have been suggested as a potential source of neoplastic OSE transformation (Rodriguez et al., 1998
). Thus, understanding the basis for the actions of xenoestrogens such as MXC could have significant implications for understanding the progression of ovarian surface neoplasia.
The current study also raises the question of whether the OSE functions as a major processing site for ovarian detoxification. Most biologic effects of MXC are thought to be the result of its transformation to HPTE (Cummings, 1997). In OSE proliferation, we found MXC to be nearly as active as HPTE, raising the possibility that the OSE is capable of metabolizing MXC to its active form. In this respect, it is interesting that in a study of another ER
tissue, uterine smooth muscle leiomyoma cells, HPTE stimulated proliferation, but MXC did not do so (Hodges et al., 2000
). The role of the OSE as a detoxifying tissue merits further study because of the importance of this function in impacting reproduction and possibly ovarian neoplasia.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at University of Maryland School of Medicine, Department of Epidemiology and Preventive Medicine, 660 W. Redwood Street, HH 133B, Baltimore, MD 21201. Fax: (410) 706-1503. E-mail: jflaws{at}epi.umaryland.edu.
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