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Endocrinology Vol. 143, No. 6 2198-2207
Copyright © 2002 by The Endocrine Society

CANCER

Proliferation of Rhesus Ovarian Surface Epithelial Cells in Culture: Lack of Mitogenic Response to Steroid or Gonadotropic Hormones

Jay W. Wright, SuEllen Toth-Fejel, Richard L. Stouffer and Karin D. Rodland

Departments of Cell and Developmental Biology (J.W.W., K.D.R.), Surgical Oncology (S.T.-F.), and Physiology and Pharmacology (R.L.S.), Oregon Health Sciences University, Portland, Oregon 97201; Division of Reproductive Sciences (R.L.S.), Oregon Regional Primate Research Center, Beaverton, Oregon 97006; and Molecular Biosciences (K.D.R.), Pacific Northwest National Laboratory, Richland, Washington 99352

Address all correspondence and requests for reprints to: Karin Rodland, Pacific Northwest National Laboratory, Department of Cell and Developmental Biology, 902 Battelle Boulevard, P.O. Box 999, Richland, Washington 99353. E-mail: . Karin.Rodland{at}pnl.gov


   Abstract
Top
 Abstract
Introduction
 Methods and Materials
 Results
 Discussion
 References
 
Ovarian cancer is the most lethal gynecological cancer, and approximately 90% of ovarian cancers derive from the ovarian surface epithelium (OSE), yet the biology of the OSE is poorly understood. Factors associated with increased risk of nonhereditary ovarian cancer include the formation of inclusion cysts, effects of reproductive hormones and the number of ovulations experienced in a woman’s lifetime. Distinguishing between these factors is difficult in vivo, but cultured OSE cells are viable tools for some avenues of research. Here we establish rhesus macaque OSE cultures and demonstrate that these cells express cytokeratin, vimentin, N-cadherin, ER-{alpha}, and PR but are negative for E-cadherin. We show that these cells activate MAPK and proliferate in response to extracellular calcium, as do human and rat OSE. In contrast, the gonadotropic hormones FSH (4–400 IU/liter), LH (8.5–850 IU/liter), and human CG (10–1000 IU/liter) fail to stimulate proliferation. We find that concentrations of progesterone and estrogen normally present in follicles just before ovulation (~1000 ng/ml) significantly decrease the number of mitotically active rhesus macaque OSE cells as determined by PCNA labeling, total cell count, and 3H-thymidine uptake, whereas lower steroid concentrations have no effect.


   Introduction
Top
 Abstract
 Introduction
Methods and Materials
 Results
 Discussion
 References
 
THE OVARIAN SURFACE epithelium (OSE) surrounding the ovary is a sheet of squamous to cuboidal mesothelial cells with pluripotential capacities, retaining both epithelial and mesenchymal potential. The functional biology of the OSE is poorly understood, with OSE apparently acting as a simple mesothelium (1). However, approximately 90% of human ovarian carcinomas are derived from the OSE, and the survival rate after diagnosis of late stage ovarian cancer is only 5–10%, making this the most lethal gynecological cancer (2, 3, 4). These observations have stimulated substantial research interest in the biology of OSE and the detection, prevention, and treatment of ovarian cancer (for a recent comprehensive review on OSE and ovarian cancer, see Ref. 5). Factors contributing to the incidence of ovarian cancer include age, heredity, and pollutant exposure (5, 6, 7), and epidemiological studies have shown a positive correlation between the number of times a woman has ovulated and her risk of ovarian cancer (1, 8). Pregnancy and use of oral contraceptives have both been associated with protection against ovarian cancer (9), whereas controlled ovarian stimulation by exogenous gonadotropins during in vitro fertilization (IVF) procedures has been correlated to increased risk in some studies, but not in others (10, 11). Even if the association between IVF treatment and increased risk of ovarian cancer proves to be statistically valid, it is not clear whether the increased risk can be attributed to controlled ovarian stimulation per se, to preexisting factors underlying the need for infertility treatment, or to direct action of the hormones themselves (12, 13). The mechanistic relationship between increased ovulation and carcinogenic changes in the OSE cannot be elucidated without a better understanding of the mechanisms regulating proliferation of OSE cells in response to normal hormonal and proliferative stimuli.

The underlying factors linking the biology of the OSE to the development of ovarian cancer are poorly understood, in part due to the lack of suitable animal models. Ovulation itself has been linked to ovarian cancer, possibly as a consequence of the proliferation and wound repair that occurs in the OSE after each ovulation. The inclusion cyst hypothesis (14) implicates the formation of epithelium-lined inclusion cysts as premalignant lesions (15, 16, 17) because OSE cells found within such cysts often display neoplastic morphologies (18). Hormonal effects may also contribute to the development of ovarian cancer, as there is evidence that hormone replacement therapy and superovulatory drug treatments may increase the risk of ovarian cancer (19, 20).

This study presents a nonhuman primate alternative to normal human OSE cell (HOSE) culture. Here we establish normal rhesus monkey OSE cells (RhOSE) in culture, showing the expression of appropriate cell type-specific intermediate filaments and cadherins. We show that these cells respond to calcium mitogenically through activation of the MAPK pathway, demonstrating that the RhOSE cells resemble both rat and human OSE cells in their sensitivity to changes in extracellular calcium concentration (21, 22). We also compare the use of PCNA and Ki-67 antibodies to thymidine incorporation as measures of the proliferative response, and we apply these techniques to study the effects on RhOSE proliferation of gonadotropic and steroid hormones, some of which are used in oral contraception and IVF protocols. We find that levels of estrogen and progesterone found in preovulatory follicles are inhibitors of RhOSE proliferation. Under no conditions did we observe a mitogenic response to estrogen or progesterone.


   Methods and Materials
Top
 Abstract
 Introduction
 Methods and Materials
Results
 Discussion
 References
 
Cell lines and culture conditions
RhOSE were obtained from rhesus macaques through the Tissue Distribution Program (Oregon Regional Primate Research Center) under a protocol approved by the ORPRC Institutional Animal Care and Use Committee. Cells were scraped from the ovarian surface with a sterile scalpel, into Chang Medium C (Irvine Scientific, Santa Ana, CA), and cultured at 37 C in 5%CO2/95% air. Cells were grown to confluence in 60 mm dishes, upon which time they were passaged and subsequently grown in DMEM containing 5% calf serum, 1.8 mM CaCl2, but no phenol red. Penicillin-streptomycin (100 U/ml, 100 µg/ml, Life Technologies, Inc., Gaithersburg, MD) and tetracycline (10 µg/ml, Calbiochem, San Diego, CA) were included in the media. Purity of the cultures was assessed by staining for cytokeratin expression, and no cytokeratin-negative cells were observed in these cultures. Cells were routinely split 1:3, and extra cells were frozen for later use. In the described experiments, no cultures were used beyond passage 12, even though the morphology, immunohistochemical profiles, and mitogen responsiveness indicated cells that grew as late as passage 20 were essentially indistinguishable from early passage cells (measured as early as passage 4; data not shown). Other cell lines used include a normal HOSE line transfected with the SV40 large T antigen to increase the proliferative life of the cells (IOSE-29, from Dr. N. Auersperg), a human epithelial ovarian cell line derived from a stage IV adenocarcinoma (SKOV-3, from ATCC, Manassas, VA), and Rat-1 fibroblasts. For most experiments, cells were sent into quiescence by overnight incubation in serum-free DMEM with 0.05 mM Ca2+. In these experiments, cells were then stimulated by increasing calcium to 1.8 mM. Compounds added to culture experiments included the MEK inhibitors PD98059 (New England Biolabs, Inc., Beverly, MA) and UO126 (Calbiochem), the steroid hormones progesterone and E2, and the human (h) glycoprotein hormones hCG, LH, and FSH. All hormones were obtained from Calbiochem. FSH (3150 IU/mg) was used at 4–400 U/liter, LH (8550 IU/mg) was used at 8.5–850 U/liter, hCG (3450 IU/mg) was used at 10–1,000 U/ml, and progesterone and estrogen were each used at 10–1,000 ng/ml, equivalent to 0.032–3.2 or 0.037–3.7 µM, respectively.

Immunohistochemistry and immunoblotting
Activation of the MAPK pathway was measured as an increase in phosphorylated ERK-1 and ERK-2 (phospho-p42/44). Immunoblotting was performed as described (21), with 40 µg of protein applied in each lane. The antiphospho-ERK 1/2 antibody (Transduction Laboratories, Inc., Lexington, KY) was visualized using either a peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:1,000) followed by chemiluminescent analysis, or a phosphatase-conjugated secondary antibody (Kirkegaard \|[amp ]\| Perry Laboratories, Gaithersburg, MD; 1:1,000) followed by densitometric analysis of the scanned blot using NIH Image 1.61. Both methodologies yielded similar results.

Immunohistochemical (IHC) analysis was performed on cells grown on chambered glass slides (Nalge Nunc International, Rochester, NY; Lab Tek II slides). As some of the primary antibodies are sensitive to antigen masking following acetone or typical formaldehyde fixations, we used a fixation protocol that enhances the reactivity of some antigens and was described previously (23). Stock formaldehyde (38% solution, Fisher Scientific, Pittsburgh, PA) was diluted 1:1 in PBS-Triton (0.6% Triton X-100, 1.8 mM NaH2PO4, 8.4 mM Na2HPO4, and 175 mM NaCl) and adjusted to pH 9 with NaOH. Cells were fixed for 10 min and then washed in PBS-Triton. All subsequent blocking, washing, and antibody incubations were carried out in PBS-Triton. Phosphatase-conjugated secondary antibodies were used (Kirkegaard \|[amp ]\| Perry Laboratories), and signal was detected using a NBT/BCIP premixed solution (Kirkegaard \|[amp ]\| Perry Laboratories). Cells were examined using light microscopy and differential interference contrast (DIC) optics. The following primary antibodies were applied overnight at 4 C: Ki-67 (mouse), cytokeratin (rabbit), PR (rabbit), and ER (mouse), from DAKO Corp. (Carpinteria, CA); phospho-p42/p44 (rabbit), N-cadherin (mouse), E-cadherin (mouse), ß-catenin (mouse), and {alpha}-catenin (mouse), from Transduction Laboratories; PCNA (mouse) from Chemicon (Pittsburgh, PA); and vimentin (mouse) from Zymed Laboratories, Inc. (South San Francisco, CA). To control for nonspecific antibody labeling, secondary antibodies were applied in the absence of primary antibodies and reacted.

Assessment of proliferation
The proliferative capacity of RhOSE in culture was assayed as the amount of 3H-thymidine incorporated in 24 h, or immunohistochemically as a count of the total number of PCNA- or Ki-67-positive nuclei, or as the total number of nuclei seen after hematoxylin staining in each experimental condition. 3H-thymidine incorporation was performed as previously described (24), using cells rendered quiescent by serum deprivation for 18 h, followed by a 24 h exposure to agonist in the presence of 3H-thymidine at a concentration of 1 µCi/ml. For IHC analysis, cells were examined by light microscopy following colorimetric reaction or nuclear staining. Counting of nuclei or labeled cells was performed using a 4x or 10x lens, and sufficient fields within each chamber were photographed to image the majority of each chamber. Slides were scanned and printed and blind counts were conducted manually. SD is expressed as variance among images within each chamber to control for heterogeneity of cellular distribution (generally low) or clustering of proliferatively active cells (generally higher). Statistical significance was calculated using unpaired, one-tailed t tests.


   Results
Top
 Abstract
 Introduction
 Methods and Materials
 Results
Discussion
 References
 
Antibody labeling demonstrates RhOSE retain epithelial characteristics
Cultured RhOSE expressed the epithelial-associated intermediate filament cytokeratin (Fig. 1AGo), and no cells were seen to be cytokeratin-negative, even in late passage, demonstrating the purity of culture and the retention of epithelial characteristics. Immortalized OSE (IOSE)-29 cells, derived from HOSE cells, also expressed cytokeratin (Fig. 1BGo), whereas the human ovarian carcinoma line SKOV-3 showed exceedingly low levels of cytokeratin expression (Fig. 1CGo), consistent with their tumor derivation. The intermediate filament vimentin, normally associated with mesenchymal cells and with normal HOSE (25, 26) is expressed in all three lines, at similar levels (Fig. 1Go, D–F).



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  		Figure 1. Immunohistochemical analysis of three primate OSE cell lines. RhOSE in the left column, a normal human line (IOSE-29), transfected with the large T antigen in the center column, and the tumor-derived human line, SKOV-3 in the right column. Cells were labeled with antibodies against: cytokeratin (A–C), vimentin (D–F), E-cadherin (G–I), N-cadherin (J–L), or ß-catenin (M–O). Secondary antibody was alkaline phosphatase-conjugated, and was also applied in the absence of primary antibody to confirm specificity of staining (data not shown). Scale bar, 25 µm.

 
E-cadherin is expressed in some early stage tumors and in HOSE adopting aberrant ovarian epithelial morphology (18) and has been shown to contribute to HOSE transformation in culture (27), yet it is not found in normal HOSE or in later stage tumors (28, 29). RhOSE, IOSE-29, and SKOV-3 (from a stage IV ovarian tumor, see Ref. 30), were all negative for E-cadherin expression.

N-cadherin is expressed in normal HOSE, and we found this cadherin expressed in all three cell lines (Fig. 1Go, J–L). Interestingly, the pattern of N-cadherin expression was markedly different in the RhOSE line, compared with the IOSE-29 and SKOV-3 lines. In RhOSE, N-cadherin was appropriately localized to regions of membrane contact between cells. It was not distributed across the surface of the cells, nor was it seen at membrane edges that were not in contact with sister cells (Fig. 1JGo). IOSE-29 cells did not show such clear localization of N-cadherin to sites of cell-cell contact and had the lowest apparent expression of N-cadherin. In this line, diffuse staining was visible at cell-cell junctures, yet significant labeling was also found in perinuclear regions (Fig. 1KGo). SKOV-3 cells also did not display any apparent localization of N-cadherin, and in these cultures N-cadherin seemed concentrated where SKOV-3 cells were growing in multilayered clusters (Fig. 1LGo). This might simply reflect accumulation of antigen from vertical stacking of cell membrane, as opposed to localization.

Antibodies against ß-catenin (Fig. 1Go, M–O) and {alpha}-catenin (not shown), which necessarily associate with cadherins to mediate signaling and cytoskeletal attachment, recapitulate N-cadherin expression: RhOSE showed localization to regions of cell-cell contact, whereas IOSE-29 and SKOV-3 did not. The functional significance of altered N-cadherin and ß-catenin localization in these cell types is unclear.

Analytic methodologies to assess mitogenic response
We wished to examine RhOSE mitogenic response to elevated extracellular calcium, using 3H-thymidine incorporation or quantifiable IHC analysis. Extracellular calcium levels of 1.4 mM or higher had previously been shown to stimulate proliferation of human and rat OSE cells via activation of the calcium-sensing receptor (21, 22). RhOSE that had been incubated overnight in serum-free DMEM containing 0.05 mM Ca2+ and then transferred into serum-free DMEM adjusted to 0.3 or 1.8 mM Ca2+ for 24 h showed a marked increase in 3H-thymidine incorporation (Fig. 2AGo), consistent with findings that HOSE grow optimally when external calcium is above 0.8 mM (22, 29). IHC analysis with antibodies against PCNA or the Ki-67 antigen also showed an increase in the number of mitotically active cells in serum-free media containing 1.8 mM Ca2+ compared with 0.05 mM Ca2+ (Fig. 2Go, B and C). These data show agreement between the readily quantifiable 3H-thymidine incorporation assay and IHC using cell cycle markers.



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  		Figure 2. Proliferation of RhOSE in response to extracellular calcium. Following overnight incubation in serum-free DMEM containing 0.05 mM Ca2+, RhOSE were placed in serum-free DMEM containing 0.05, 0.3, or 1.8 mM Ca2+ for 24 h. Proliferation was measured as the amount of 3H-thymidine incorporated (A), or as the number of PCNA-positive nuclei (B) or Ki-67-positive nuclei (C) counted. B and C also show examples of staining. Filled arrowheads show positive cells. Unfilled arrowheads show negative nuclei, visible due to the differential interference contrast used. *, P < 0.05; **, denotes P < 0.01. Scale bar, 15 µm. Error bars represent one SD from the mean. The results shown are typical of five replicate experiments.

 
ERK activity in RhOSE proliferation
The MAPK signaling pathway is a major component associated with proliferation and differentiation in a number of cell types (31). To determine the role of ERK activation in both acute and chronic growth response, we measured ERK activation in RhOSE under a variety of conditions.

RhOSE were incubated in serum-free DMEM containing 0.05 mM Ca2+ overnight and were then either maintained in serum-free DMEM at 0.05 mM Ca2+ or transferred to 1.8 mM Ca2+ DMEM in the presence or absence of the MEK inhibitors PD98059 or UO126, and grown overnight in 1 µCi of 3H-thymidine. We observed a 2- to 3-fold increase in the amount of 3H-thymidine incorporated in the presence of elevated Ca2+, and this increase was dependent on ERK activity as it was blocked by treatment with either PD98059 or U0126, two chemical inhibitors of MEK, an activator of ERK (Fig. 3AGo, left panel). In parallel experiments, using the PCNA antibody, we found approximately twice as many S-phase cells present following calcium elevation to 1.8 mM. This increase was abrogated by MEK inhibitors (Fig. 3AGo, right panel). MEK inhibition also decreased 3H-thymidine incorporation and PCNA labeling in cultures that had not been synchronized by serum starvation before addition of 1.8 mM Ca 2+ (data not shown).



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  		Figure 3. ERK activation in RhOSE in response to extracellular calcium. RhOSE were placed in serum-free DMEM containing 0.05 mM Ca2+ overnight, then moved into serum-free DMEM containing either 0.05 or 1.8 mM Ca2+ for 24 h. The MEK inhibitors PD98059 (PD) or UO126 (UO) were added 4 h before Ca2+ elevation, and were used at 20 µM and 15 µM, respectively. A, 3H-Thymidine incorporation and PCNA expression. B, Immunoblot of phosphoErk in stimulated cells. C, Quantification of ERK phosphorylation in immunoblots. D, Immunohistochemistry of phosphoERK in RhOSE cells. Not all cells respond the same to Ca2+ stimulation, and unresponsive cells can be seen (arrow). *, P < 0.05; **, P < 0.01. Scale bar, 15 µm. Error bars represent one SD from the mean. The results shown are typical of five replicate experiments for panels A–C and three replicate experiments for panel D.

 
RhOSE cultures also responded to acute activation of the MAPK pathway by Ca2+. Stimulation with Ca2+ following serum deprivation typically resulted in a 3-fold increase in the level of phospho-ERK, with maximal levels occurring at roughly 20 min after stimulation (Fig. 3Go, B and C). This transient activation was inhibited by the MEK inhibitors PD98059 and UO126. Alternatively, RhOSE were stimulated and then fixed in 18% buffered alkali formaldehyde. The ensuing IHC analysis using the {alpha}-phospho-p42/44 antibody showed an accumulation of phospho-ERK within stimulated cells, primarily in the cytoplasm, but also within the nucleus. Blocking ERK activation with MEK inhibitors prevented the accumulation of phospho-ERK (Fig. 3DGo). It is interesting to note that not all cells respond identically to mitogen stimulation, as some cells showed robust phospho-ERK labeling, whereas others contained little or no phospho-ERK (arrow in Fig. 3DGo). The combined PCNA, phospho-ERK, and 3H-thymidine incorporation data indicate that MEK inhibitors not only block mitogenic activation of ERK, but also inhibit DNA synthesis and cell cycle progression.

Effect of reproductive hormones on the proliferation of cultured RhOSE
The effects of gonadotropins and steroid hormones on the proliferative behavior of RhOSE were examined. Cells were grown in 1.8 mM Ca2+ in the presence or absence of hormones and 3H-thymidine overnight (Fig. 4Go, A and B), in a number of concentrations spanning systemic levels found in humans during the ovulatory cycle. a = low, b = mid, c = high dose. hCG was used at 10, 100, and 1000 IU/liter; LH was used at 8.5, 85, and 850 IU/liter; and FSH was used at 4, 40, and 400 IU/liter. Progesterone or estrogen were used at 10, 100, and 1000 ng/ml. Only the highest dose of progesterone (3.2 µM) or estrogen (3.7 µM) is shown because lower doses had no effect. hCG, LH, and FSH had neither positive nor negative effects on proliferation (Fig. 4AGo). Progesterone and estrogen also had no effect at concentrations equivalent to maximal systemic levels (100 ng/ml, data not shown), consistent with previous findings in HOSE (32). Replicate experiments were performed in 0.3 and 0.05 mM Ca2+, to test for calcium-dependent effects. No hormonal effects were observed in submaximal Ca2+ (data not shown).



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  		Figure 4. Effects of reproductive hormones on RhOSE proliferation. 3H-Thymidine incorporation was measured as described in Fig. 2Go in the presence of gonadotropic hormone concentrations spanning systemic levels found in humans during the ovulatory cycle. a, Low; b, mid; c, high dose. RhOSE were exposed to hCG at 10, 100, or 1,000 IU/liter, LH at 8.5, 85, or 850 IU/liter, or FSH at 4, 40, or 400 IU/liter (panel A). Progesterone or estrogen were used at 10, 100, and 1000 ng/ml. Conditions shown here are: P = 1000 ng/ml progesterone, e = 1000 ng/ml estrogen, p/e= 1000 ng/ml of each. *, P < 0.05; **, denotes P < 0.01. Results shown are typical of five replicate experiments. C, Expression of ER and PR by RhOSE and IOSE-29 cells, but not by Rat-1 fibroblasts. Scale bar, 15 µm. Error bars represent one SD from the mean.

 
In vivo, the OSE are in close apposition to maturing follicles. In rhesus macaque, the follicular levels of progesterone and estrogen are 100-fold higher than systemic levels, following a hormonal surge associated with ovulation, and reach 1–5 µg/ml (33). When progesterone or estrogen were used at 1 µg/ml, proliferation of RhOSE and IOSE-29 cells was greatly reduced, but Rat-1 fibroblasts were unaffected (Fig. 4BGo). In combination, these steroid hormones further reduced RhOSE and IOSE-29 proliferation significantly below the level seen when each hormone was applied singly. Although these steroid hormones had no significant effect on Rat-1 proliferation individually, pooling data from three experiments in which the high dose estrogen and progesterone treatments were combined gave an average reduction in thymidine incorporation of 12% (minimally statistically significant at P = 0.05; Fig. 4BGo). Results in individual experiments were somewhat variable, ranging from no effect of combined E+P to 14% decrease. IHC staining with antibodies to PR and ER-{alpha} revealed that RhOSE and IOSE-29 cells expressed these receptors in culture, whereas Rat-1 did not (Fig. 4CGo). Applying the secondary antibody alone resulted in no detectable signal (not shown). The correlation of steroid responsiveness with receptor expression suggests that the effects of high progesterone and estrogen is receptor-dependent and requires follicular, as opposed to systemic levels of hormone. The minor response of Rat-1 cells to the addition of progesterone and estrogen together suggests some level of nonspecific steroid effects, perhaps via cross-reactivity with other steroid receptors. Although a nonspecific cytotoxic effect of combined estrogen and progesterone at µM concentrations cannot be rigorously excluded, examination of RhOSE cells for signs of apoptosis after exposure to follicular steroid levels for up to 72 h showed no apoptotic response (not shown).

Follicular levels of progesterone and estrogen inhibit entrance into the cell cycle
Although high levels of progesterone and estrogen reduced 3H-thymidine incorporation in 24-h asynchronous RhOSE culture, we failed to note a parallel reduction in PCNA-positive nuclei or in the total number of nuclei present (Fig. 5AGo). Discrepancies between the highly sensitive 3H-thymidine incorporation assay and other, less sensitive, colorimetric assays have been reported previously (34). We determined to resolve the differences between these two methodologies by extending the time of culture.



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  		Figure 5. RhOSE cells do not immediately exit the cell cycle in response to estrogen or progesterone. A, Comparison of thymidine incorporation, PCNA immunostaining, and total cell counts in RhOSE cells exposed to combined estrogen and progesterone for 24 or 72 h. Over a 24-h treatment period, progesterone and estrogen induce a rapid decline in 3H-thymidine uptake, but a slower exit from the cell cycle, as judged by IHC PCNA-analysis. After 72 h in the presence of steroid hormones, PCNA labeling and cell counts show an inhibition of RhOSE proliferation. B, Comparison of thymidine incorporation and PCNA staining in quiescent RhOSE cells that have been serum deprived and then exposed to steroid hormones and elevated Ca2+. RhOSE cells were placed in 0.05 mM Ca2+ overnight, then exposed to progesterone and estrogen (p/e) for 4 h before Ca2+ elevation to 1.8 mM for 24 h. 3H-Thymidine incorporation is reduced and the number of PCNA-positive nuclei is diminished. *, P < 0.05; **, P < 0.01. Error bars represent one SD from the mean. Results shown are typical of five replicate experiments.

 
We maintained RhOSE under high progesterone and estrogen for 72 h, a time more closely relating to in vivo exposure. We observed an even greater decrease in 3H-thymidine incorporation into hormonally treated RhOSE. We also detected significant decreases in the number of PCNA-positive nuclei and the total number of cells (Fig. 5AGo). Thus, given sufficient time in culture, 3H-thymidine incorporation and IHC analysis are in agreement that progesterone and estrogen inhibit RhOSE proliferation.

The growth-inhibitory effects of progesterone and estrogen were more pronounced in cultures that were synchronized by incubation in serum-free DMEM containing 0.05 mM Ca2+. These cells were then treated with gonadotropic or steroid hormones for 4 h before Ca2+ stimulation, and maintained in the presence of these hormones for 24 h. After 24 h in culture, we detected estrogen- and progesterone-dependent decreases in both 3H-thymidine incorporation and PCNA-positive nuclei. No effect from LH/hCG or FSH treatment was seen. This suggests that, once RhOSE have exited the cell cycle, high levels of progesterone and estrogen inhibit future cell cycle progression, but that progesterone and estrogen receptor-mediated events do not induce exit from the cell cycle. No effect from LH/hCG or FSH was seen (not shown).


   Discussion
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
References
 
Establishing primary RhOSE culture
Methods for culturing primary OSE from mammalian species have been previously described, and include cow, pig, and rat, in addition to human OSE (e.g. Refs. 34, 35, 36, 37). In using OSE to investigate ovarian cancer, the previously described nonhuman models suffer some shortcomings that raise questions concerning their use. For example, E-cadherin is expressed in neoplastic but not normal HOSE, and transfection of HOSE with E-cadherin in culture induces some dysplasic behavior (27); however, E-cadherin is in fact expressed by normal rat and pig OSE (36, 38, 39), making analysis of any potential role for E-cadherin in the progression of ovarian cancer difficult in these model systems. Most significantly, though, is the failure to detect OSE-derived carcinomas in vivo in these nonhuman species, because they simply do not occur or are very rare (40). While normal HOSE have been successfully cultured, severe limitations in their proliferative life span in culture have been reported (35, 37), and attempts to extend their life in culture via transfection with the SV40 large T antigen or papilloma virus E6/E7 proteins cause the loss of some primary characteristics including the elimination of normal cell cycle regulation, which raises questions about the usefulness of such lines for some avenues of investigation (41).

We established RhOSE cultures in much the same way as reported for HOSE cultures, although with some modifications. We found that the initial survival of the culture benefited from a high-serum medium, but that high levels of serum (over 5%) slowed the rate of proliferation. Once established, RhOSE exhibited variable life spans in culture, sometimes becoming senescent within only a few passages, but commonly remaining proliferatively active much longer. Importantly, even the late passage RhOSE retained a normal cell morphology, and did not display a fibroblast-like shape that has been associated with transformation of OSE. We saw no change in the proliferative responsiveness of RhOSE cultures over time in culture ranging from early (passage 4) to the latest observed passage (passage 20). RhOSE responded well to lateral contact inhibition, growing as a monolayer in culture, unlike IOSE-29 or SKOV-3, which are not growth-inhibited by lateral contact and grow in multilayered aggregates. Thus RhOSE cells offer some distinct advantages for in vitro investigations of normal OSE function.

Immunohistochemical analysis of RhOSE
IHC analysis of RhOSE showed that they express markers of both mesenchymal and epithelial nature, reflecting the dual potential of normal OSE in vivo (see Ref. 5). Unlike most other mesothelial tissues, OSE is required to behave as a stationary epithelium surrounding a quiescent ovary, but must acquire mesenchymal features following follicular rupture and wound closure.

Intermediate filament expression has been used for histological identification of normal and transformed OSE (16) and can be used to assay the purity of a primary culture. It has been observed that the mesenchymal (25) intermediate filament vimentin is retained by tumor-derived HOSE, but the more epithelial filament cytokeratin is lost in many of these lines and is associated with the regression of these cells toward a less differentiated state seen in tumors. In rat OSE culture, cytokeratin expression is lost after several passages, as these cells lose characteristics associated with their primary identity (26). In contrast, cultured RhOSE did not lose cytokeratin expression, even after many passages, suggesting they maintain their dual mesenchymal-epithelial potential.

E-cadherin has been suggested to play a role in ovarian cancer progression, as E-cadherin is expressed only by HOSE found in inclusion cysts, OSE neoplasias, or early stage carcinomas, but not in normal HOSE (16, 35, 42) or in primary RhOSE culture. Transfection of the T-antigen-transfected IOSE-29 cells with E-cadherin promotes transformation in culture and confers tumorigenic potential when these cells are introduced into SCID mice (27, 43). Expression of E-cadherin in the early stages of progression is atypical of most epithelial neoplasms, which become less differentiated as transformation proceeds. In neoplastic progression, E-cadherin could be involved in promoting aggregation of OSE that have been trapped within the ovary to form inclusion cysts, widely believed to be precursors to ovarian cancers (17, 44); however, expression of E-cadherin by normal pig and rat OSE suggests that E-cadherin expression alone is insufficient for OSE transformation.

N-cadherin is expressed by neuroectodermally and mesodermally derived tissues (45) and is characteristic of mesenchymal cell types. In the normal ovary of human, pig, rat, and mouse, N-cadherin is expressed in the OSE (38, 39, 46). When we compared N-cadherin expression in RhOSE vs. IOSE-29 and SKOV-3, we observed a unique pattern of spatial regulation of N-cadherin by RhOSE cells. In RhOSE, N-cadherin was found only at sites of cell-cell contact, with staining patterns clearly outlining the membrane of individual RhOSE cells, whereas N-cadherin was less clearly localized in IOSE-29 cells and generally unlocalized in SKOV-3 cells. The differences in expression patterns of N-cadherin among these cell lines is consistent with them having differing degrees of progression toward full transformation. The RhOSE cell strains, derived from true primary cultures, retain strong epithelial morphology, whereas the tumor-derived SKOV-3 are mesenchymal and fibroblast-like. IOSE-29, which are derived from normal HOSE but express the SV40 large T antigen, show variability in cell shape, displaying both epithelial and fibroblastic morphologies.

Cadherin-mediated adhesion provides structural support to tissues, but also participates in cell signaling events, and in fact both E-cadherin and N-cadherin have been shown to induce differentiation of ectodermal and mesodermal tissues (47, 48). If the aberrant distribution of N-cadherin reflects a dysfunction of this molecule in SKOV-3 and, to a lesser extent IOSE-29, then it is possible that normal N-cadherin function is required to prevent OSE from adopting morphologic and proliferative changes associated with transformation.

Ca2+-dependent proliferative activity of RhOSE
The MAPK pathway is a major signaling cascade associated with cellular proliferation, differentiation, and tumorigenesis, and requires receptor-mediated activation of ERK (49, 50). We show here that Ca2+ acts as a mitogenic cue in RhOSE that have been calcium deprived (0.05 mM) overnight and then presented with 1.8 mM Ca2+. In this regard, RhOSE cells resemble human and rat OSE, which also proliferate in response to elevated extracellular calcium (21, 22). Using immunoblotting and IHC, we show transient activation of ERK, and this activation is prevented by MEK inhibitors. ERK activation is also required for continued proliferation. Growing RhOSE in elevated Ca2+ (1.8 mM vs. 0.05 mM) increases both DNA synthesis, measured by 3H-thymidine incorporation, and the number of cells in S-phase, defined as PCNA-positive nuclei. DNA synthesis and the number of S-phase RhOSE are decreased by MEK inhibitors.

The confirmation of IHC data with immunoblot, showing ERK phosphorylation, and with 3H-thymidine incorporation (compared with PCNA expression), makes the IHC approach attractive for future experiments because it consumes far less material than the alternative techniques and because IHC has the power to reveal mitogenic responsiveness at the level of individual cells, illustrating variability within populations of cells.

Gonadotropic and steroid hormone effects on RhOSE proliferation
In distinguishing among the probable causes of ovarian cancer, the gonadotropic theory can be juxtaposed against two other leading theories: the incessant ovulation theory, first proposed by Fathalla (1) in 1971, and the inclusion cyst theory, proposed by Cramer and Welch (14) in 1983. Each model is supported by available data, yet they ascribe different factors to the most salient risks of ovarian cancer.

The gonadotropic theory postulates a direct tumor-promoting activity of reproductive hormones on OSE and is supported by studies indicating that women undergoing IVF procedures are at increased risk for ovarian cancer (51), by the documented effects of estrogen in promoting growth of breast and some ovarian cancer cells (52, 53), and by studies suggesting that estrogen treatment in hormone replacement therapy might increase the risk of ovarian cancer (54). However, the epidemiological studies supporting this model have not been consistently replicated (10, 11).

The incessant ovulation theory proposed that repeated wounding of the OSE due to ovulation increases the likelihood of neoplastic progression in cells undergoing repeated proliferation. Thus, the protective effects of pregnancy and oral contraceptives on ovarian cancer incidence could be attributed to decreased lifetime ovulations.

The inclusion cyst theory is based on the observation that epithelium-lined bodies within the ovary are precancerous lesions. Here, clefts form on the ovarian surface and involute into the underlying stroma, thereby entrapping OSE cells (14). Both ovulation and age are contributors to cyst formation, as these create the potential for cleft formation. Proliferative or differentiative effects of hormones or other factors on trapped OSE cells are considered to participate in tumor formation.

Determining the relative weight of contribution to risk from ovulation, proliferation, age, and hormonal influences is impossible in vivo, but some of these factors can be singled out in vitro. In this study, we examined the effects of certain hormones on OSE proliferation.

Both FSH and LH/hCG are used in IVF treatment, and recent work in human (55) and macaque (56) using recombinant human FSH suggests that FSH and LH/hCG have considerable overlapping function in promoting fertility. HOSE reportedly express FSH and LH/hCG receptors (51, 57). LH/hCG stimulated OSE proliferation in some studies (58, 59, 60), but not others (61), whereas high levels of FSH were recently reported to slightly reduce HOSE proliferation (61). When we tested the effects of hCG, LH, or FSH on RhOSE proliferation, we found no statistically significant effect, even at very high concentrations. It should be pointed out that even though the RhOSE used in this study were derived from reproductively active primates, the age of these animals (5–9 yr, equivalent to approximately 13–30 human years) was much lower than the average age of women supplying HOSE for experimentation. Ivarsson et al. (61) observed the most significant effects of high levels of FSH on HOSE, which were derived from postmenopausal women (mean age 62 yr). Thus, the relatively younger age of the RhOSE cultures compared with the human OSE cultures used by Ivarsson et al. may account for our failure to observe an inhibitory effect of FSH treatment on RhOSE cells.

We show here that RhOSE in culture express PR and ER-{alpha}, and these receptors have previously been observed in RhOSE in vivo at all phases of the ovulatory cycle (62). Despite the presence of these receptors, progesterone and estrogen had no effect on proliferation of RhOSE in culture, when these steroids were used at concentrations near maximal plasma levels, consistent with observations in HOSE cultures (32). Follicular concentrations of progesterone and estrogen in response to the LH surge reach or exceed 1 µg/ml in rhesus macaque (33). In humans and other animals, peak concentrations of these steroids in ovarian follicles have been observed to reach 3–5 µM (63, 64). When we presented RhOSE with 1 µg/ml progesterone or estrogen (3.2 and 3.7 µM, respectively), a potent inhibition of proliferation was observed. Furthermore, we found that the human IOSE-29 line was also growth inhibited by follicular concentrations of progesterone and estrogen. Rat-1 fibroblasts, which do not express the PR or ER-{alpha}, were unaffected by these steroids except when both were used simultaneously. The effect in these cells, though statistically significant, was slight (~12% inhibition, compared with ~70% inhibition in RhOSE and IOSE-29) and might indicate nonspecific interactions between such high steroid levels and other steroid receptors. It is also possible that the combination of estrogen and progesterone at micromolar concentrations might have some nonspecific cytotoxic effect, operating independent of ER and PR. However, no signs of apoptosis were observed in RhOSE, IOSE-29, or Rat-1 cells treated with high doses of estrogen and progesterone, even after 72 h of exposure. As OSE cells in vivo are in the immediate vicinity of maturing follicles and corpora lutea, it is likely that the elevated concentrations of progesterone and estrogen used in this study are physiologically relevant.

The inhibitory effects of progesterone and estrogen are somewhat surprising, given that OSE proliferation is required for repair following ovulation, as well as the observation that estrogen promotes cell cycle progression through G1 in breast cancer cells (see Refs. 52 and 65 for reviews). Our data do not support a gonadotropic model of ovarian cancer. On the contrary, it could be argued that elevated levels of estrogen and progesterone at the time of ovulation could act in a protective manner against ovarian cancer, by delaying proliferation of OSE in the vicinity of a newly ruptured follicle and decreasing the likelihood that OSE might be enclosed in epithelium-lined structures, precursors to neoplastic inclusion cysts. Other data indicate that contraceptive levels of the progestin levonorgestrel increase apoptosis of RhOSE in vivo (66), providing further evidence for a nonmitogenic role for progestins.

The growth inhibitory effects of progesterone and estrogen on RhOSE as compared with observations of a mitogenic effect in breast and other cancers (67) make it interesting to determine what differences might exist between effector molecules associated with steroid receptor signaling between these cell types. The potential importance of determining whether progesterone and/or estrogen responsiveness correlates to ovarian cancer risk is great, and the findings of Ivarsson et al. (61), showing a greater sensitivity to FSH in postmenopausal HOSE, as well as the contribution of age to ovarian cancer risk, suggest that age might also be important in hormone response and OSE transformation.

Although the gonadotropic model of ovarian cancer is not supported here, IVF treatment could contribute to the development of neoplastic inclusion cysts by the simple fact of increased ovulation. If so, then the growth-inhibitory effects of progesterone and estrogen might act as significant protectants against ovarian cancer. We can use RhOSE culture in the future to examine in more detail the proliferative effects of peptide and steroid hormones, including downstream effectors of the relevant signaling pathways. In addition, we will seek to determine whether there are age-specific influences on hormone responsiveness, which could serve to suggest what manner of IVF treatment is best suited to women in specific age groups, to promote fertilization without increasing the risk of ovarian cancer.


   Acknowledgments
 


   Footnotes
 
This work was supported in part by NIH Grants RR-00163, HD-20869 (to R.L.S.), and CA-78722 (to K.D.R.).

Abbreviations: HOSE, Human OSE cells; h, human; IHC, immunohistochemical; IOSE, immortalized OSE cells; IVF, in vitro fertilization; OSE, ovarian surface epithelium; RhOSE, rhesus macaque OSE.

Received January 17, 2002.

Accepted for publication February 22, 2002.


   References
Top
 Abstract
 Introduction
 Methods and Materials
 Results
 Discussion
 References
 

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