Estrogen Mediates Phosphorylation of Histone H3 in Ovarian Follicle and Mammary Epithelial Tumor Cells via the Mitotic Kinase, Aurora B
Z. Tatiana Ruiz-Cortés,
Sarah Kimmins,
Lucia Monaco,
Kathleen H. Burns,
Paolo Sassone-Corsi and
Bruce D. Murphy
Grupo de Fisiología y Biotecnología de la Reproducción (Z.T.R.-C.), Facultad de Ciencias Agrarias, Universidad de Antioquia, Medellín, Colombia; Centre de recherche en reproduction animale (Z.T.R.-C., B.D.M.), Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Quebec, Canada J2S 7C6; Institut de génétique et biologie moléculaire et cellulaire (S.K, L.M., P.S.-C.), Université Louis Pasteur, 67404 Illkirch-Strasbourg, France; and Department of Pathology (K.H.B.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Bruce D. Murphy, Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Quebec, Canada J2S 7C6.
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ABSTRACT
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Cells of the ovarian follicle undergo extensive proliferation and differentiation from the time that the follicle escapes from the primordial state to its acquisition of ovulatory capacity. We examined the dynamic modification of the phosphorylation state of the histone H3 N-terminal tail in granulosa cells during follicular development. In rodent follicles, the granulosa cell H3 phosphorylation on Ser10 peaks during proestrus. This epigenetic mark is induced by both FSH and 17ß-estradiol (E2), acting independently. E2-induced H3 phosphorylation fails to occur in mice with inactivated
-isoform of the nuclear estrogen receptor. E2 induction of histone phosphorylation is attenuated by cell cycle inhibition. Further, E2 induces the activity of the mitotic kinase, Aurora B, in a mammary tumor cell model where mitosis is estrogen receptor-
dependent. These results provide evidence for mitotic regulation in follicle development by estrogen and demonstrate a previously undiscovered mechanism for induction of cell proliferation in ovarian and mammary gland cells.
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INTRODUCTION
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EPIGENETIC MECHANISMS EXERT effects that are essential to the regulation of gene expression in eukaryotes. One class of epigenetic regulation is the covalent modification of the N-terminal tails of core histones by acetylation, phosphorylation, methylation, poly(ADP-ribosyl)ation, and ubiquitination (1). Covalent modifications of histones reshape nucleosomal structure to alter accessibility of associated DNA sequences to regulatory factors. They are believed to be important in transduction of extracellular signals that regulate gene expression. Histone tail modifications correlate with transcriptional, mitogenic, and mitotic events in somatic cells (2) and play important roles in gene silencing (3).
A coordinated program integrating both genomic and epigenetic elements regulates the complex process of folliculogenesis in the mammalian ovary. Endocrine and paracrine ligands orchestrate proliferation and differentiation of somatic cells to bring about development of follicles with the capacity to ovulate. FSH (4, 5) and estrogens, particularly 17ß-estradiol (E2) (6) are the essential regulators of folliculogenesis. Both exert their primary influence on granulosa cells after the appearance of the follicular antrum. They are active under pathological conditions, in that human granulosa cell tumors express estrogen receptors (ERs) and respond to FSH (7). Signaling pathways for both ligands have been investigated in the ovary, and it has been shown that gonadotropins, acting through the protein kinase A pathway, induce histone acetylation associated with expression of steroidogenic genes (8, 9). Further, FSH directly induces phosphorylation of histone H3 on Ser10, again via the protein kinase A pathway (10, 11).
Estrogens have paracrine and autocrine effects on their tissue of origin, the ovarian follicle (12), and are believed to contribute to the pathogenesis of human granulosa cell tumors (7). They have the potential to modify histones, e.g. estrogen-induced hypoacetylation of histone H3 and H4 tails represses transcription (13). Nonetheless, there is a dearth of information on effects of estrogen in epigenetic modification of ovarian cells. The current investigation provides novel insight into the estrogen induction of phosphorylation of the highly conserved Ser10 on the N-terminal tail of histone H3 in granulosa cells of the mammalian ovary and in an estrogen-sensitive tumor cell model, MCF-7 breast cancer cells. We found that cell cycle-dependent phosphorylation of histone H3 in both ovarian granulosa and breast cancer cells is driven by estrogen, acting through the oncogenic kinase, Aurora B.
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RESULTS
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FSH and E2 Independently Induce H3 Phosphorylation (P-H3) on Ser10 in Ovarian Cells
Immunohistochemical analysis of postpubertal mouse and rat ovaries, using antisera detecting H3 phosphorylated on Ser10, demonstrated numerous nuclei positive for the P-H3 signal (Fig. 1A
), most prominently in antral follicles, but also present in both primary and secondary follicles. Incubation with normal rabbit serum in place of the P-H3 antibody resulted in no cell-specific fluorescence. Nuclei of granulosa cells displayed either punctate or condensed distribution of the P-H3 signal, the latter associated with the mitotic phase of the cell cycle (Fig. 1B
). Counting of all positive nuclei in five fields from three ovarian sections per animal demonstrated a pattern of frequency of occurrence of P-H3 that varied through the rat estrous cycle, with highest values at proestrus (Fig. 1C
). Because proestrus coincides with maximal ovarian secretion of estrogens, under the influence of FSH (14), we examined the direct effects of these two hormones on P-H3. Immunoblots revealed that granulosa cells from immature rats treated in vitro with FSH displayed a 2-fold elevation in the P-H3 signal over 24 h, whereas E2 alone caused a 4-fold increase over the same time period (Fig. 2A
). There was an apparent additive effect of the two ligands (Fig. 2A
). This result was recapitulated in ovaries of intact, immature rats treated with E2, FSH, or their combination for 24 h (Fig. 2B
). To determine whether the effects of FSH and E2 are separate and independent, we treated hypophysectomized, immature rats for a similar period, and counted P-H3-positive cells in the ovary. In this context, FSH, E2 and their combination induced similar 2.5- to 3-fold increases in signal frequency over ovaries in hypophysectomized, untreated rats (Fig. 2B
). We then treated mice bearing a targeted inactivation of the FSH receptor (15) with FSH, E2, or their combination. Ovaries of animals treated with E2, but not FSH, displayed increased P-H3 signal frequency, providing strong confirmation of direct E2 effects (Fig. 2C
). Because one of the roles of FSH in the ovary is induction of estrogen synthesis (16), we sought to establish whether FSH could induce P-H3 independent of E2. Treatment with either of two pharmaceutical inhibitors of estrogen synthesis, aminoglutethimide and anastrazole (17), prevented accumulation of E2 in rat granulosa cells treated with FSH (Fig. 3A
). The inhibitors caused nonsignificant reduction in FSH-induced P-H3 expression in rat granulosa cells in vitro (Fig. 3B
) and in the frequency of the P-H3 signal in the ovaries of intact, immature rats (Fig. 3C
). Together, these findings demonstrate a direct FSH effect, independent of its influence on estrogen synthesis.

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Fig. 1. Expression of Phosphorylated H3 (P-H3) Is Dependent on the Estrous Cycle
A, P-H3 occurs in the nuclei of granulosa cells of the ovarian follicles of mature, WT mice as revealed by immunohistochemistry employing antibodies directed against phosphorylated Ser10 on H3. B, The P-H3 signal is found in a punctate configuration (left panel) as well as in condensed chromatin associated with metaphase (middle panel) and anaphase (right panel) of the granulosa cell cycle. C, The mean (±SD) of the number of P-H3 positive cells through the estrous cycle of the rat (PE, proestrus; E, estrus; ME, metestrus; and DE, diestrus). All positive nuclei in five fields from three ovarian sections per animal were counted. Highest expression was observed during PE. Different superscripts (ac) indicate significant differences among means at stages of the estrous cycle (P < 0.05).
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Fig. 2. P-H3 Is Induced Independently by FSH and E2 in Rat and Mouse Granulosa Cells
A, Western analysis of P-H3 in rat granulosa cells that were untreated (C) or treated with FSH (100 ng/ml), E2 (20 µM), or their combination for 12 h. The Ponceau-stained band that migrated at approximately 17 kDa indicates total H3 abundance (loading) in this and subsequent figures. B, FSH and E2 stimulate P-H3 in granulosa cells of rats, and E2 effects are independent of FSH (middle panel). Immature intact or hypophysectomized (Hypox) rats (n = 3/group) were treated with E2 or FSH and killed 24 h later, and expression of P-H3 in the ovary was quantified by enumeration of signal abundance in five fields from three ovarian sections per animal. C, Mice bearing a targeted mutation of the FSH receptor respond to E2 but not FSH. WT and FSH receptor knockout mice were treated with E2 or FSH for 24 h. Ovaries were sampled as described above. In all three panels, data are presented as mean (±SD). Superscripts indicate statistical differences between groups, whereas asterisks indicate differences between means and corresponding controls (P < 0.05). C, Control.
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Fig. 3. FSH Induces P-H3 Independent of Its Capacity to Provoke Estrogen Synthesis
Primary cultures of rat granulosa cells were treated with anastrazole (AN) or aminogluthimide (AG), inhibitors of estrogen synthesis, followed by treatment with FSH or E2 for 12 h. A, Mean (±SD) estrogen content in medium of cell cultures (n = 3). B, Western analysis. C, Mean (±SD) abundance of positive cells in five nonoverlapping fields from wells of three replicate experiments. Superscripts indicate statistical differences at P < 0.05. kD, Kilodaltons.
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E2 Induces P-H3 via ER
and ERß Isoforms
Estrogen action is mediated by its cognate nuclear receptors, ER
and ERß (18). We interfered with E2 interaction with its receptors by treatment of intact, immature rats or rat granulosa cells in vitro with the high-affinity ER
and ERß receptor antagonist, ICI 182,780 (19). The absence of P-H3 response to E2 in vitro (Fig. 4A
) and in vivo (data not shown) in the presence of the antagonist established that E2 stimulation of P-H3 depends on ERs. Using mice bearing specific inactivating mutations of either ER
or ERß, we examined receptor specificity of the E2 effects on P-H3. Follicles develop in ovaries of both gene deletion models (20), but follicles fail to form an antrum in ER
knockout mice (Fig. 4B
). ERß knockout mice have antral follicles of lesser diameter than wild type (WT, Fig. 4B
) and are subfertile (20). Immunohistochemical analysis revealed that ovaries from WT, ER
, and ERß knockout mice displayed the P-H3 signal. The WT response after treatment with E2 was eliminated in ER
mutant animals and nonsignificantly reduced in those lacking ERß (Fig. 4
, B and C). Together, these observations demonstrate that E2 induction of P-H3 in ovarian follicles depends absolutely on the presence of the ER
and may also require the ERß isoform.

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Fig. 4. Estrogen Acts Primarily through the ER in Inducing P-H3
A, The effects of estrogen are blocked by pretreatment of rat granulosa cells with the estrogen antagonist ICI 182,780 followed by E2 for 12 h. Upper panel is a Western blot; lower panel presents the mean (±SD) density of three immunoblots normalized to control. B, Photomicrographs of frozen sections of ovaries from mice treated with E2 for WT and mice with mutation of the receptor subtypes ER or ERß treated with 1 mg/d E2. C, Western analysis of ovarian expression of P-H3 in untreated WT, ER , or ERß mutant mice untreated, or treated with E2 (n = 3/group). The superscripts indicate means that are significantly different from each other in panel A, whereas asterisks indicate difference from the WT in panel B (P < 0.05). KO, Knockout; ICI, ICI 182,780.
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E2 Induction of P-H3 Is Cell Cycle Related
The phosphorylation of H3 on Ser10 associates with gene transcription, mitogenesis, and mitosis in eukaryotic cells (2). P-H3 correlates with chromosome condensation, beginning in early G2 in pericentromeric heterochromatin (21). To investigate the P-H3 dynamics relative to the cell cycle, we first determined the time course of the estrogen response in rat granulosa cells. We detected an estrogen-induced increase in the former cell type within 1 h of treatment with E2 that became asymptotic from 412 h (Fig. 5A
). Control cultures displayed an increase at 2 h and remained likewise constant through 12 h (Fig. 5B
).

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Fig. 5. The Effects of E2 Are Time and Cell Cycle Dependent
A, Time course indicates that that the E2 response is rapid in rat granulosa cells (panel A) and MCF-7 cells (panel B), with definable effects within 1 h. Data are expressed as mean (±SD) immunoblot density relative to 0 time control. B, Western analysis demonstrating that pretreatment of rat granulosa cultures for 6 h with the cell cycle inhibitor, Roscovitine (R), abolishes the ability of E2, but not FSH, to induce P-H3. C, Western analysis of P-H3 in rat granulosa cells treated with E2, E2+Roscovitine (R), or FSH+R. D, Signal frequency (mean ± SD) of P-H3 in cultures of rat granulosa cells treated with E2, E2+R, or FSH+R. E, Roscovitine attenuates cell proliferation and enriches the G2/M component of the cell population in MCF-7 cells. Open bars represent the percentage of cells in G2/M by fluorescence-activated cell sorting analysis, whereas black bars display the percentage of viable cells/ml of culture medium. T-0 indicates initiation of the experiment; T-24C indicates 24 h without treatment; and T-24 R indicates 24-h treament with Roscovitine. Asterisks indicate differences from control or T-0 at P < 0.05.
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To determine whether the observed effects of estrogen were related to the cell cycle, we studied MCF-7 breast cancer cells, a cell line in which the ER
isoform is predominantly expressed (22) and in which estrogen induction of proliferation is mediated through ER
(23). There was demonstrable increase in P-H3 induced by E2 with differences present as early as 1 h after treatment (Fig. 5B
), mimicking the pattern in granulosa cells. The signal in control cultures increased at a lesser rate over the 24 h of culture (Fig. 5B
). Treatment of granulosa cells for periods of 824 h with Roscovitine, a cell cycle inhibitor (24), abrogated the E2-mediated, but not the FSH-mediated, induction of P-H3 evaluated by Western blot (Fig. 5C
) and by signal frequency (5D), suggesting the effects of estrogen are cell cycle mediated. To demonstrate that Roscovitine attenuated the cell cycle, we treated MCF-7 cells at 25 µM for 24 h and analyzed cell populations by fluorescence-activated cell sorting. The ratio of total to viable cells ranged from 7589% and was unaffected by treatment. Cell division was attenuated, as indicated by number of viable cells in cultures treated with Roscovitine, and cell populations treated with the inhibitor displayed significant enrichment of the percentage of cells G2/M (Fig. 5E
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The Mitotic Kinase, Aurora B, Is the Effector of E2-Mediated P-H3
The mitotic kinase, Aurora B, associates with P-H3 during the M phase of the cell cycle in HeLa and NIH3T3 cells (25). We found that Aurora B and P-H3 colocalize, both in histological sections from the ovaries of WT mice, and in MCF-7 cells in culture (Fig. 6A
). Immunoblots confirmed that P-H3 expression in MCF-7 cells increased by 5-fold or more at 4, 8, and 12 h after initiation of treatment with 20 µM E2 (Figs. 5B
and 6B
), with little or no concurrent increase in Aurora B protein (Fig. 6B
). In contrast, Aurora B expression underwent 3-fold elevation by 24 h and later in E2-treated MCF-7 cells (data not shown). We postulated that the early (14 h) increase in E2 induction of P-H3 is due to steroid effects on Aurora B kinase activity. To test this hypothesis, Aurora B was immunoprecipitated from MCF-7 cells that had been treated with 20 µM E2 for 12 h. Subsequent in vitro activity determination (26) indicated that E2-treated cultures had a 2.4-fold increase in Aurora B kinase activity (Fig. 6C
). We further demonstrated that E2 did not activate protein kinase A under the same conditions, indicating specificity of the effect (data not shown).

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Fig. 6. Estrogen Directly Stimulates P-H3 via Activation of the Mitotic Kinase, Aurora B
A, Aurora B kinase and P-H3 colocalized in granulosa cells of the mouse ovary and in MCF-7 cells in vitro. B, Western analysis showing that E2 stimulates expression of P-H3 but not Aurora B over 12 h of treatment of MCF-7 cells. C, Immunoprecipitation employing antibodies against Aurora B (IP-Aurora B) or preimmune serum (IP-IgG) of MCF-7 cells treated for 12 h with 20 µM E2. The band at 45 kDa represents immunoprecipitated Aurora B protein, whereas that at 17 kDa indicates that equal amounts of the substrate were employed. Analysis of the autoradiogram (box) demonstrates a 2.4-fold increase in phosphorylation of substrate in cells treated with E2. kD, Kilodaltons; DAPI, 4',6-Diamidino-2-phenylindole.
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To further establish effects of E2 on Aurora B induction of phosphorylation of H3, we employed Hesperadin, an inhibitor of Aurora B kinase activity (27) in dose response experiments. Hesperadin at 0.210 nM did not interfere with basal phosphorylation of H3, whereas reduction in basal activity was observed at concentrations of 50 nM and greater (Fig. 7
, A and B). The stimulatory effects of 20 µM E2 on P-H3 in MCF-7 cells were completely reversed by doses of Hesperadin of 0.210 nM (Fig. 7
, A and B). In vitro kinase assay revealed that a dose of 10 nM Hesperadin interfered with both basal and E2-induced Aurora B kinase activity (Fig. 7C
). Together these findings provide convincing evidence that effects of E2 on short-term induction of P-H3 can be attributed to activation of Aurora B kinase.

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Fig. 7. Aurora B Kinase Inhibitor, Hesperadin, at Concentrations of 0.210 nM, blocks E2 Induction of P-H3 over 12 h in MCF-7 Cells
Basal and E2-stimulated P-H3 were both reduced in the presence of 50 or 100 nM Hesperadin. A, Western blot of P-H3. B, Graphic representation of mean (±SD) signal density in Western blots from three independent experiments. C, In vitro kinase assay demonstrating that Hesperadin (Hes) blocks E2-induced Aurora B kinase activity in MCF-7 cells. The upper box represents phosphorylation of Aurora B immunoprecipitates, whereas the lower box demonstrates Coomassie staining of gels.
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DISCUSSION
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Herein we provide the first demonstration of a direct role for estrogen in chromatin remodeling in ovarian granulosa and other estrogen-sensitive cells, as well as the first comprehensive demonstration of a direct effect of a steroid hormone on histone phosphorylation (28). The results indicate that estrogen has an effect that is independent of FSH, as E2-mediated increases in P-H3 occur in hypophysectomized animals, where no endogenous FSH is present, as well as in mice with null mutation of the gene encoding the FSH receptor, and thus incapable of responding to endogenous FSH (12, 15). Likewise, experiments employing two blockers of estrogen synthesis with different sites of action provide convincing evidence that FSH can induce P-H3, independent of its induction of estrogen synthesis.
Estrogens are essential regulators of ovarian follicle development. Targeted disruption of the cytochrome P450aromatase gene (Cyp19) and, consequently, estrogen synthesis causes an age-dependent reduction in the number of both secondary and antral follicles (29, 30). More significantly, a large proportion of the follicles that reach the antral stage are abnormal and apparently atretic, and no corpora lutea ensue, indicating interdiction of ovulation (30). This implicates estrogens in both proliferation and differentiation of granulosa cells of periantral and postantral follicles. Salvador et al. (11) linked P-H3 to FSH-induced granulosa cell differentiation. The extent to which estrogen induces differentiation-related changes in P-H3 remains undiscovered.
The phosphorylation of H3 on Ser10 correlates with chromosome condensation in mitosis and meiosis (31). Our investigation ties E2 induction of P-H3 to proliferation, as Roscovitine-induced attenuation of the cell cycle eliminated the E2 effect on P-H3. Receptor antagonist experiments indicate that E2 is acting through its receptors, concurring with studies in MCF-7 cells, where transfection of an inactivating dominant negative or antisense ER reduced the E2-induced transition to G2/M (32). Transgenic ablation of ER
and ERß (33), as well as inactivating mutation of the Cyp19 gene (29), severely compromises granulosa cell proliferation. In the current study, reduction in response to E2 in ERß knockout mice (albeit nonsignificant), and its complete absence in the ER
mutant mouse suggest that both receptor isoforms subserve proliferation-associated P-H3 in the ovary. This view concurs with conclusions by Dupont et al. (33) and with the postulate of Britt and Findlay (6) that interaction of estrogens with ER
induces granulosa cell proliferation.
Effects of estrogens on target tissues are believed to engage diverse signaling pathways. Indeed, in addition to two separate nuclear receptor pathways (20), there are membrane ERs that have the potential for activation of the MAPK (34) and Akt/protein kinase B (35) intracellular pathways. The current experiments demonstrate rapid effects of estrogen on P-H3 in two estrogen-sensitive cell models, rat granulosa cells and MCF-7 breast cancer, suggestive of nongenomic actions (36, 37). The signaling pathways and mechanisms that mediate these rapid effects will require further investigation.
To define upstream effectors of E2 induction of P-H3, we investigated the mitotic kinase, Aurora B, known to be required for completion of the M phase of the cell cycle (38) and implicated in mitotic phosphorylation of H3 (25). We showed that Aurora B colocalizes with P-H3 in both ovarian granulosa and MCF-7 cells and that its activity is increased by E2 treatment in the latter cell model. We show that the indolinone, Hesperadin, employed at low doses, eliminates Aurora B kinase activity in MCF-7 cells, consistent with its effects in HeLa cells (27). Further, Hesperadin entirely prevents the effects of E2 on P-H3 in MCF-7 cells at doses that are orders of magnitude below the effective dose in HeLa cells (20100 nM) (27). Together these results argue strongly for estrogen induction of P-H3 via the mitotic kinase, Aurora B.
A current view is that estrogen induces cell proliferation by action early in the cell cycle (39). Our finding that E2 stimulates both Aurora B kinase and the occurrence of histone phosphorylation suggests that E2 acts at the G2/M transition. Upstream events that mediate the activation of Aurora B kinase are not known, although there is evidence for constitutive inhibition of its activity in interphase nuclei by the phosphatase, PP1 (40). In neural tissue, estrogen inhibits PP1activity (41). Given that, at least in the short term, E2 did not increase the expression of Aurora B, interference with its inhibition by attenuation of PP1 is an attractive mechanism for rapid E2 induction of Aurora B kinase activity and consequent mitotic phosphorylation in target tissues. Confirmation requires further study.
In summary, the current investigation demonstrates a novel and specific effect of E2 on the induction of phosphorylation of histone H3 on Ser10 in ovarian granulosa cells and in MCF-7 cells. This effect is mediated through ER
and -ß, and is cell cycle-dependent. Inhibition of Aurora B kinase prevents E2 induction of P-H3 in estrogen-dependent cells, suggesting that activation of this enzyme is required for estrogen-induced mitosis. Abnormally elevated expression of Aurora B correlates with oncogenesis (42), highlighting the importance of our finding that this kinase is stimulated by estrogen to induce proliferation of cell types that can undergo estrogen-driven oncogenic transformation. A role for Aurora B kinase in breast and granulosa cell cancer is not yet known, but in light of the data presented here, this possibility warrants investigation.
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MATERIALS AND METHODS
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In Vivo Studies
Animal studies were approved by the Comité de déontologie de la Faculté de Médecine Vétérinaire de lUniversité de Montréal, accredited by the Canadian Council on Animal Care. We followed the estrous cycle in adult Wistar rats (Simonsen Laboratories, Gilroy, CA) by vaginal cytology and collected ovaries from three animals at diestrus, proestrus, estrus, and metestrus. To determine the occurrence and independence of FSH and E2 effects, intact and hypophysectomized immature Sprague Dawley rats (21 d of age, Charles River Canada, Saint-Constant, Quebec, Canada) were treated with ip doses of FSH (10 µg; Sigma, Oakville, Ontario, Canada) on d 2122, E2 (1.5 µg; Sigma) on d 23d 25 or the E2+FSH combination on d 21d 25 (n = 5/treatment). Further confirmation was undertaken by treating mice bearing null mutation of the FSH receptor (15) with FSH (10 µg) or E2 (1 mg). To determine the ER isoform mediating the response, mice with inactivating mutations of ER
or -ß were treated with 1 mg/d E2 in sesame oil, whereas control mice received oil alone (n = 3/treatment). Ovaries were collected and frozen at 70 C for immunohistochemistry or Western blotting.
In Vitro Studies
Granulosa cells were isolated from ovaries of 26-d-old Sprague Dawley rats primed with 1.5 mg E2 on d 2325 (10, 11), plated on dishes or coverslips at a density of approximately 1.53 x 106 cells per well in DMEM-Hams F-12 culture medium (GIBCO, Burlington, Ontario, Canada) supplemented with 0.1% BSA (Sigma), 100 U/ml penicillin (GIBCO), and 100 µg/ml streptomycin (GIBCO) and incubated at 37 C in 5% CO2. In addition, MCF-7 cells, maintained in DME H-21 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin, were employed to provide an alternate model for estrogen action. Beginning 24 h after plating, granulosa cells were incubated for 12 h with E2 (20 µM) or porcine FSH (100 ng/ml). To study the independence of FSH from E2, we treated cells with nonsteroidal aromatase inhibitors, aminoglutethimide (Sigma, 1.9 µM) (43) or anastrozole (Arimidex, 15 ng/ml; AstraZeneca, Mississauga, Ontario, Canada) (44), followed by treatment with FSH. Inhibition of estrogen synthesis was confirmed in a single RIA as previously described (45). The intraassay coefficient of variation, calculated among triplicates ranged from 0.27.1%. Cell cycle inhibition was effected with Roscovitine (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) at 25 µM for 824 h, followed by E2, FSH, and E2+FSH treatment for periods of 112 h. The effects of Roscovitine on the cell cycle were investigated by fluorescence-activated cell sorting. To verify that E2 affects P-H3 via its nuclear receptors in granulosa cells, we added ICI 182,780 (Tocris, Ellisville, MO), a high-affinity ER
and ERß antagonist, at 1.5 or 10 µM, with or without E2 (20 µM). The inhibitor of Aurora B kinase, Hesperadin (STGOO183), a gift of Boehringer Ingelheim Inc. (Vienna, Austria), was employed at doses of 0.2100 nM in MCF-7 cells.
Western Blot Analysis
Protein extracts were resolved by SDS-PAGE and electroblotted to Trans-Blot Transfer Medium nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were incubated overnight at 4 C in 0.1% (vol/vol) Tween 20 in Tris-buffered saline (100 mM Tris; 0.9% sodium chloride, pH 7.5) containing rabbit anti-P-H3 on Ser10 (Upstate Biotechnology, Inc., Waltham, MA) or rabbit anti-Aurora B (25). Donkey antirabbit antibodies (1:1000; Amersham Pharmacia Biotech, Arlington, Heights, IL) conjugated to horseradish peroxidase revealed immunocomplexes by enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL). To control for loading, blots were stained with Ponceau or Coomassie, and the band corresponding to histone H-3 (17 kDa) was visualized.
Immunofluorescence
Tissue sections or cells fixed in 100% methanol and permeabilized with 0.2% Triton-1x PBS were blocked for 1 h in 5% BSA in 1x PBS Tween 20, and incubated with rabbit or rat anti-P-H3 or rabbit anti-Aurora B (1:200) for 2 h, and then with Cy3-conjugated antirabbit serum or fluorescein isothiocyanate-conjugated antirat serum (Jackson ImmunoResearch, West Grove, PA), and counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (Roche Clinical Laboratories, Indianapolis, IN). Normal serum (rabbit or rat) was employed as negative control. To quantify signal frequency, five fields from three ovarian sections per animal, or five fields from each of three replicate in vitro plates were counted.
Immunoprecipitation and in Vitro Kinase Assays
MCF-7 cells were lysed in 0.5 ml kinase lysis buffer (50 mM HEPES, pH 8.0; 600 mM KCl; 0.5% Nonidet P-40; 1 mM Na3V04; 1 mM dithiothreitol, 1 mM microcystine protease inhibitor cocktail), centrifuged and precleared with protein A-Sepharose (25). Lysates were incubated with Aurora B antibody or, as a control, protein kinase A antibody (Euromedex; Souffelweyersheim, France) at concentrations of 1:50 for 3 h at 4 C, and immunoprecipitates were collected by centrifugation after adding Protein A-Sepharose. Kinase assays were performed in 30 µl of kinase buffer (20 mM HEPES, pH 7.4; 150 mM MnCl; 5 mM NaF; 0.5% Nonidet P-40; 1 mM Na3V04; 1 mM dithiothreitol, 50 µM ATP; 1 mM microcystine protease inhibitor cocktail) containing 0.5 mg myelin basic protein (Boehringer Ingelheim, Inc.) or an equivalent amount of recombinant histone H3 (Kimmins, S., and P. Sassone-Corsi, submitted), and 20 mM 32P-labeled ATP and incubated 20 min at 37 C. Reactions were terminated by addition of 30 µl Laemmli sodium dodecyl sulfate buffer, and products were denatured for 10 min at 95 C, separated by SDS-PAGE, and visualized by autoradiography and Western blotting.
Sampling and Statistical Analyses
Signal density was evaluated by the ImageJ 131v program (National Institutes of Health, Bethesda, MD). Data were subjected to Shapiros test for normality and Bartletts test for homogeneity of variance. In its absence, analysis was performed on square root-transformed data. Each experiment was repeated at least three times, and, in the presence of significant F value by ANOVA, means were compared by the Student-Newman-Keuls procedure. Significance was P < 0.05.
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ACKNOWLEDGMENTS
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We thank Dr. A. Dierich for FSH receptor knockout mice, Drs. M. Matzuk and K. Korach for ER knockout mice, and E. Heitz and M. Dobias for technical assistance.
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FOOTNOTES
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This work was supported by grants from Canadian Institutes for Health Research (to B.D.M.) and to P.S.C. from Centre nationale de la recherche scientifique, Institut National de la Santé et de la Recherche Médicale, Centre hospitalier universitaire régionale, Fondation de la recherche médicale, Ligue contre le Cancer, and LAssociation pour la recherche contre le cancer. S.K. is supported by the Fondation pour la recherche medicale.
First Published Online July 14, 2005
Abbreviations: E2, 17ß-Estradiol; ER, estrogen receptor; P-H3, H3 phosphorylation; WT, wild type.
Received for publication November 1, 2004.
Accepted for publication July 7, 2005.
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