Effects of Histone Deacetylase Inhibitors on Estradiol-Induced Proliferation and Hyperplasia Formation in the Mouse Uterus

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539 Effects of histone deacetylase inhibitors on estradiol-induced proliferation and hyperplasia formation in the mouse uterus

Andrei G Gunin, Irina N Kapitova and Nina V Suslonova Department of Obstetrics and Gynecology, Medical School Chuvash State University, PO Box 86, 428034, Cheboksary, Russia (Requests for offprints should be addressed to A G Gunin; Email: [email protected])

Abstract It is suggested that estrogen hormones recruit mechanisms of mitotic and bromodeoxyuridine-labelled cells in luminal controlling histone acetylation to bring about their effects and glandular epithelia, in stromal and myometrial cells. in the uterus. However, it is not known how the level of Levels of estrogen receptor- and progesterone receptors histone acetylation affects estrogen-dependent processes in in uterine epithelia, stromal and myometrial cells were the uterus, especially proliferation and morphogenetic decreased in mice treated with estradiol and trichostatin A changes. Therefore, this study examined the effects of or sodium butyrate. Expression of -catenin in luminal histone deacetylase blockers, trichostatin A and sodium and glandular epithelia was attenuated in mice treated butyrate, on proliferative and morphogenetic reactions in with estradiol with trichostatin A or sodium butyrate. Both the uterus under long-term estrogen treatment. Ovari- histone deacetylase inhibitors have similar unilateral effects; ectomized mice were treated with estradiol dipropionate however the action of trichostatin A was more expressed (4 µg per 100 g; s.c., once a week) or vehicle and tricho- than that of sodium butyrate. Thus, histone deacetylase statin A (0·008 mg per 100 g; s.c., once a day) or sodium inhibitors exert proliferative and morphogenetic effects of butyrate (1% in drinking water), or with no additional estradiol. The effects of trichostatin A and sodium butyrate treatments for a month. In animals treated with estradiol are associated with changes in expression of estrogen and trichostatin A or sodium butyrate, uterine mass was receptor-, progesterone receptors and -catenin in the increased, and abnormal uterine glands and atypical endo- uterus. metrial hyperplasia were found more often. Both histone Journal of Endocrinology (2005) 185, 539–549 deacetylase inhibitors produced an increase in the numbers

Introduction factors and others (Couse & Korach 1999, Gunin et al. 2001). Recent data showed that estrogen also recruits Estrogen hormones have a variety of effects in the uterus. intracellular regulatory systems to bring about its action Proliferation and changes in structure and architecture of (Couse & Korach 1999, Deroo et al. 2004, Gunin et al. uterine tissues are estrogen-dependent events (Martin et al. 2004b). The scientific literature gives some direct and 1973, Bigsby 2002). Endometrial cancer is also an estrogen- indirect evidence that the system that maintains the status dependent disease, and it can be regarded as a consequence of histone acetylation interacts with estrogen signalling of estrogen-induced alteration in proliferation and mor- (Alao et al. 2004, Kurtev et al. 2004, Margueron et al. phogenesis (Emons et al. 2000, Archer 2004). Endometrial 2004). hyperplasia and cancer can easily be induced in laboratory Several types of histone deacetylases are present in the rodents by continuous estrogen exposure (Akhmedkhanov nucleus of cells, and together with histone acetyltrans- et al. 2001). Administration of estrogen hormones can lead ferases they determine the acetylation status of histone to endometrial cancer formation in women (Deligdisch proteins (Margueron et al. 2004). Shifts in the level of 2000). Numerous cases of endometrial cancer are regis- histone acetylation are accompanied by changes in the tered each year in every country (Archer 2004). Therefore, activity of the transcription process (Riester et al. 2004). regulation of estrogen action and the interactions between Histone deacetylase activity keeps chromatin in a tran- estrogens and target tissues must be investigated more scriptionally inactive state (Riester et al. 2004). Data have intensively in order to achieve more effective prevention been published showing interactions between estrogen and treatment of estrogen-dependent pathology. signalling and acetylation of histones (Jang et al. 2004, It is known that the action of estrogen is regulated by a Margueron et al. 2004). It has also been reported that variety of extracellular factors – such as hormones, growth estrogen hormones affect the level of histone acetylation in

Journal of Endocrinology (2005) 185, 539–549 DOI: 10.1677/joe.1.06118 0022–0795/05/0185–539 2005 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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target tissues (Sun et al. 2001). There is some evidence that allowed to drink tap water with 1% (w/v) sodium butyrate histone acetylation is involved in processes that are also (Sigma) for 30 days. These groups also received s.c. regulated by estrogens, for example proliferation and cell injections of saline (0·1 ml per mouse) once a day for differentiation (Sakai et al. 2003). It is therefore supposed 30 days. that shifts in histone acetylation can affect the action of The fourth (n=5), fifth (n=5) and sixth (n=5) groups estrogen on the uterus, such as proliferation and morpho- received s.c. injections of the vehicle of estradiol (olive oil; genetic alterations. However, it is not known how the 0·1 ml per mouse) once a week and saline or trichostatin A histone acetylation status influences estrogen-dependent or sodium butyrate, respectively, once a day for 30 days. processes in the uterus, such as proliferation and changes in Our previous observations clearly showed that treat- structure of tissues. Therefore, the aim of this research ment with estradiol for 30 days was quite enough to was to examine estrogen-induced processes in the uterus produce expressed estrogen-dependent changes in uterine and their response to substances that change the level of morphology and to induce hyperplastic changes in the histone acetylation. uterus (Gunin et al. 2001, 2002, 2004a,b). This estradiol Trichostatin A, an antifungal antibiotic, has a potent and dose and treatment regime produced estradiol levels in the specific inhibitory effect on histone deacetylase activity blood that were close to the normal physiologic values (Riester et al. 2004). Butyric acid and its salts are also (Gunin et al. 2004a). well-known blockers of the activity of histone deacetylases The uteri were removed 48 h after the last estradiol or (Riester et al. 2004). Therefore two reagents, trichostatin vehicle injection. All animals were injected i.p. with A and sodium butyrate, were chosen for use in our bromodeoxyuridine (BrdU; 5 mg per 100 g of body mass; experiments to block histone deacetylases; this was fol- Sigma) dissolved in 0·15 M sodium chloride 2 h before the lowed by induction of a hyperacetylated histone state. tissues were removed. Organs were removed under deep ether anesthesia. Uteri were weighed and middle seg- ments of uterine horns were then placed in modified Material and Methods Bouin’s fixative (Gunin et al. 2000) for 6 h at room temperature, and were then dehydrated and embedded Animals in paraffin. Uteri were transversely oriented and cut at 5–7 µm. All procedures were performed in accordance with the UFAW Handbook on the Care and Management of Laboratory Animals and with the Chuvash State Univer- Uterine histology sity Rules for work with laboratory animals. White out- Histological changes in the uterus were analyzed and bred CFW female mice (19·1 0·3 g, mean S.E.M.) diagnosed according to Scully et al. (1994). To estimate were used. Animals were obtained from the Animal the extent of any hyperplastic or neoplastic changes in the Department of Chuvash State University (Cheboksary, endometrium, uterine glands were subdivided into four Russia) and were housed with free access to water and morphological types: (1) normal glands (simple tubular food. Mice were ovariectomized 2 weeks before the glands which can appear in section as round, oval, or experiments were started. All surgical procedures were elongated with a narrow lumen; this type has no branches performed under anesthesia with ketamine and diazepam or daughter glands); (2) cystic glands (round-shaped glands (75 and 0·12 mg/kg respectively, i.p.; Gedeon-Richter, of more than average or large size); (3) glands with Budapest, Hungary). daughter glands (these glands have various shapes (round, elongate, tortuous) and sizes and have forming or formed daughter gland or glands inside the epithelium or inside Treatments the mother gland lumen, or on the outer surface of the Ovariectomized mice were divided into several groups mother gland); (4) conglomerate of glands (this type has a according to the treatments, as follows. The first group very complex architecture in which individual glands are (n=15) was treated with s.c. injections of estradiol dipro- closely disposed to each other almost without intervening pionate in olive oil (Minmedprom, Rostov-Don, Russia) stroma and have multiple interconnecting lumens – this at a dose of 4 µg per 100 g of body mass once a week and type may develop from glands with daughter glands), as received s.c. injections of saline (0·15 M sodium chloride) described in Gunin et al. 2001). The numbers of each type at a dose of 0·1 ml per mouse once a day for 30 days. The of gland were calculated in randomly selected sections. No second group (n=15) was treated with s.c. injections of less than three sections from each animal were examined. estradiol once a week and received s.c. injections of Results were expressed as the percentage of each type of trichostatin A (Sigma) at a dose 0·008 mg per 100 g once gland. The epithelium of all glands in randomly selected a day for 30 days. Trichostatin A was dissolved in 0·15 M sections was examined and typed as simple, pseudostrati- sodium chloride. The third group of mice (n=15) was fied or stratified (multilayered) epithelia. The percentage treated with injections with estradiol once a week and of glands with each type of epithelium was calculated.

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The number of mitotic and BrdU-labelled cells stained components of a tissue (Gunin et al. 2002, 2004a). Proliferative processes were assessed from the number of The photometric procedure was performed using Sigma mitotic and BrdU-labelled cells as described previously Scan Pro software (SPSS Inc., Chicago, IL, USA). Ini- (Gunin et al. 2001). Mitoses were counted in sections tially, uterine sections immunohistochemically stained for stained with iron haematoxylin. BrdU was detected estrogen or progesterone receptors were photographed immunohistochemically. Anti-BrdU mouse monoclonal using an Olympus light microscope (objective magnifica- antibody conjugated with biotin (catalog number, MO tion 40), an Olympus C3040-ADU camera adapter and an 5215; Caltag Laboratories, Burlingame, CA, USA) diluted Olympus Camedia 4040z 4 megapixel digital camera. At 1:50 in Tris-buffered saline (TBS) (pH 7·2–7·6) was used least three randomly selected sections were photographed as the primary antibody. Streptavidin conjugated with for each mouse, and three to five randomly selected fields alkaline phosphatase (catalog number, SA 1008; Caltag were photographed from one section. Photographs were Laboratories) diluted 1:50 in TBS with 0·1% (v/v) Triton then loaded in Sigma Scan Pro software and analyzed to X-100 was then applied. Alkaline phosphatase activity was find the optical density. It was performed by measuring revealed through the use of naphtol AS-BI-phosphate the intensity of staining of positive-stained structures (F) ). The intensify of and new fuchsin as chromogens. Control sections were and structures with no staining (F0 staining was measured from equal areas of tissue image (19 stained in a similar manner, except the primary antibody pixels). Positive staining for estrogen and progesterone was replaced with normal mouse serum. All results were receptors was detected in the nuclei of all uterine tissues expressed as the percentage of mitotic or labelled cells. (luminal epithelium, glandular epithelium, stromal cells, myometrial cells). Therefore, the intensity of nuclear Estrogen receptor-, progesterone receptors and -catenin staining (F; positive staining) and the intensity of stain- Estrogen receptor-, progesterone receptors and -catenin ing of the internuclear space in the endometrial stroma were detected using routine indirect immunohisto- (F0; negative staining) were measured for estrogen and chemical staining. Rabbit anti-estrogen receptor- poly- progesterone receptors. clonal antibody (catalog number, sc-542; Santa Cruz -Catenin was detected in luminal and glandular epi- Biotechnology Inc., Santa Cruz, CA, USA) diluted 1:50, thelia. Therefore, the intensity of staining of cytoplasm of rabbit anti-progesterone receptors antiserum (catalog these epithelial cells (F; positive staining) and intensity of number, sc-538; Santa Cruz Biotechnology Inc.) diluted staining of the internuclear space in the endometrial 1:50 and rabbit anti--catenin antiserum (catalog number, stroma (F0; negative staining) was measured for -catenin. C2206; Sigma) diluted 1:50 were used as primary anti- Optical density (light absorption) was calculated from the bodies. For detection of estrogen receptors, goat anti-rabbit formula D=l g(F0/F). The level of expression was consid- immunoglobulin G antibody conjugated with alkaline ered as the value of optical density (Gunin et al. 2002, phosphatase (catalog number 111–055–045; Jackson 2004a). At least 100 nuclei were analyzed for each ImmunoResearch Laboratories Inc., West Grove, PA, structure in each mouse. USA) was used as secondary antibody, and alkaline phosphatase activity was then revealed using naphtol Statistics AS-BI-phosphate and new fuchsin as chromogens. For progesterone receptors and -catenin, goat anti-rabbit Arithmetic means and standards errors were calculated for immunoglobulin G antibody conjugated with peroxidase each data group. The significance of differences was (catalog number 111–035–045; Jackson ImmunoResearch determined by Student’s t-test (uterine mass, proliferation, Laboratories Inc.) was used as the secondary antibody, estrogen and progesterone receptors, -catenin) and by the 2 peroxidase activity was then developed by the use of use of the observed versus expected test (gland types, hydrogen peroxide and diaminobenzidine (Sigma) tech- epithelium types, pathology). Values of P<0·05 were niques, slides were also preincubated in 0·1% hydrogen considered significant. peroxide in distilled water for 10 min to block endogenous peroxidase activity. Control sections were stained in a Results similar manner, except the primary antibody was replaced with normal rabbit serum. To avoid possible differences in Uterine mass the intensity of staining, sections from all mice were processed simultaneously for each antigen, so that all The uterine mass of ovariectomized mice receiving olive sections were incubated in exactly the same TBS, the same oil instead of estradiol and saline for 30 days (group 4) was mixtures of primary and secondary antibodies, the same 115·222·4 mg per 100 g body mass (meanS.E.M.; mixture for development of enzyme activity, for the n=5); the addition of trichostatin A (group 5; 120·6 same times, at the same temperature. 24·1 mg per 100 g body mass; n=5) or sodium butyrate Intensity of immunostaining was quantified by photo- (group 6; 116·819·7 mg per 100 g body mass; n=5) for metric measurement of optical density (D) for positive- 30 days had no effect on uterine mass. www.endocrinology-journals.org Journal of Endocrinology (2005) 185, 539–549

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In the uteri of mice treated with estradiol and sodium butyrate for 30 days (group 3), glands with daughter glands, conglomerates of glands and glands with atypical epithelium were also found in a greater percentage of cases (Figs 2 and 3). Atypical endometrial hyperplasia was found in 64·3% of cases (Figs 2 and 3).

Proliferation Proliferation in the uterus was estimated by two parameters: the number of mitotic cells and the number of BrdU-labelled cells. Treatment with estradiol and trichostatin A for 30 days (group 2) led to an increase in the percentage of mitotic and BrdU-labelled cells in all uterine tissues (Fig. 4). Treatment with estradiol and sodium Figure 1 The data on uterine mass (mg per 100 g body mass of butyrate for 30 days (group 3) also produced an increase in ovariectomized mice treated with estradiol and saline (open bars) the number of mitotic and BrdU-labelled cells in all or with trichostatin A (grey bars) or sodium butyrate (black bars) for 30 days. Values are meansS.E.M. *P<0·05; ***P<0·001; structures (Fig. 4). Mice that received the estradiol vehicle Student’s t-test. (olive oil) with no additional treatments (group 4) or with trichostatin A (group 5) or sodium butyrate (group 6) for 30 days had no changes in proliferative parameters in all uterine tissues (Fig. 4b and d). Treatment with estradiol and trichostatin A for 30 days (group 2) resulted in a 34% increase in uterine mass as compared with data for control mice receiving estradiol Estrogen receptor- alone (group 1). Administration of estradiol and sodium Using immunohistochemistry, estrogen receptor- was butyrate (group 3) also produced a 17% increase in uterine found in luminal and glandular epithelia, in stromal and mass (Fig. 1). myometrial cells of the uterus. Treatment with estradiol and trichostatin A for 30 days (group 2) reduced the level of estrogen receptor- in all uterine compartments, as Uterine histology compared with that in control animals (group 1) treated All uteri of ovariectomized mice, which were not treated with estradiol (Figs 2 and 5). Treatment with estradiol and with estradiol and received trichostatin A (group 5) or sodium butyrate also led to a reduction in estrogen sodium butyrate (group 6) or no additional treatments receptor levels in all uterine tissues. (group 4) for 30 days, were diagnosed with atrophic Mice that received the estradiol vehicle (olive oil) and endometrium (Fig. 2). All endometrial glands in all these trichostatin A or sodium butyrate, or with no additional uteri had a narrow lumen, and had a round, oval, or treatments for 30 days (groups 4 to 6) had no changes in elongated shape (a microscopical reflection of simple estrogen receptor expression (Fig. 5). tubular glands), which were regarded as normal. All glands were lined with simple cuboidal epithelium. In control mice receiving estradiol for 30 days (group 1), Progesterone receptors abnormal glands, especially glands with daughter glands Using immunohistochemistry, progesterone receptors and glands forming conglomerates, were observed (Figs 2 were detected in luminal and glandular epithelia, stromal and 3). Glands lined with pseudostratified or atypical and myometrial cells of the uteri of mice in all treatments stratified epithelium were also found in these uteri (Figs 2 group. Treatment with estradiol and trichostatin A for 30 and 3). Atypical endometrial hyperplasia was diagnosed in days (group 2) led to marked reduction in the level of 36·5% of control mice treated with estradiol for a month. progesterone receptors in all uterine compartments, as In animals treated with estradiol and trichostatin A for compared with that in control animals (group 1) treated 30 days (group 2), glands with daughter glands, glands with estradiol (Figs 2 and 6). Treatment with estradiol and forming conglomerates and glands with atypical stratified sodium butyrate (group 3) also resulted in a marked columnar epithelium were observed more often (Figs 2 decrease in progesterone receptor expression in all uterine and 3). Atypical endometrial hyperplasia was found in tissues. 78·6% of cases, and there were no cases of simple endo- The data from mice treated with estradiol vehicle only metrial hyperplasia. Normal proliferative endometrium or with trichostatin A, or with sodium butyrate for a month was also not diagnosed in mice receiving estradiol and (groups 4 to 6) are shown in Fig. 6b. There are no differ- trichostatin A (Figs 2 and 3). ences in progesterone receptor levels among these groups.

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Figure 2 Photomicrographs demonstrating histological ﬁndings (row a), immunohistochemical staining for estrogen receptor- (row b), progesterone receptors (row c) and -catenin (row d) in the uterus of mice treated with estradiol with no additional treatments (column i), estradiol and trichostatin A (column ii), estradiol and sodium butyrate (column iii) for 30 days. Multiple enlarged glands with small daughter glands and conglomerates of glands are seen in the uterus of mice receiving estradiol and trichostatin A (a-ii) or sodium butyrate (a-iii). In control mice treated with estradiol only (a-i), glands with daughter glands are also present, but in a lower percentage of cases. However, normal glands with small dimensions and lined with simple columnar or cuboidal epithelia are also seen in the uterus of control mice (a-i). There is a marked decrease in the levels of estrogen receptor- (b-ii, b-iii), progesterone receptors (c-ii, c-iii) and -catenin (d-ii, d-iii) in uterine tissues of mice treated with estradiol and trichostatin A (ii) or sodium butyrate (iii) for 30 days, as compared with control (b-i, c-i). Panels in column 0 represent the general histology (a-0), expression of estrogen receptor- (b-0), progesterone receptors (c-0) and -catenin (d-0) in the uterus of control mice treated with olive oil (the estradiol vehicle) for 30 days. le, luminal epithelium; g, endometrial glands; s, endometrial stroma. Scale bar, 100 m.

-Catenin Discussion

Immunohistochemical staining for -catenin showed that A group of parameters was employed to estimate estradiol this protein was clearly detected in luminal and glandular action in the uterus. Uterine mass is a well-known epithelia of the uteri of mice in all treatment groups. indicator of estrogen action (Emons et al. 2000, Bigsby Treatment with estradiol and trichostatin A (group 2) or 2002). Proliferation, which was determined by the num- with sodium butyrate (group 3) for 30 days led to a bers of mitotic and BrdU-labelled cells, also depends on reduction in the level of -catenin in both epithelia (Figs estrogen action (Martin et al. 1973, Gunin et al. 2000). 2 and 7). The effect of trichostatin A was more expressed. In addition, morphogenetic alterations – such as shape The data from mice treated with estradiol vehicle only of glands, type of glandular epithelium and pathology or with trichostatin A, or with sodium butyrate for a findings – appear in the uterus under long-term estrogen month (groups 4 to 6) are shown in Fig. 7b. There are no action (Martin et al. 1973, Gunin et al. 2002). Moreover, differences in -catenin level among these groups. hyperplastic changes in the endometrium are often found. www.endocrinology-journals.org Journal of Endocrinology (2005) 185, 539–549

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Figure 3 Types of endometrial glands, types of glandular epithelium, and pathology diagnosis in uteri of ovariectomized mice treated with estradiol with no additional treatments, estradiol and trichostatin A, estradiol and sodium butyrate for 30 days. PE, proliferative endometrium; SH, simple hyperplasia; CoH, complex hyperplasia; AH, atypical hyperplasia; A, normal glands; B, cystic glands; C, glands with daughter glands; D, glands forming conglomerate; 1, simple columnar epithelium; 2, pseudostratiﬁed columnar epithelium; 3, stratiﬁed columnar epithelium. Values 2 are means+S.E.M. P<0·001; test).

Some hyperplastic changes have nonfavorable prognosis, supposition that histone deacetylase inhibitors affect some especially complex and atypical hyperplasies. Complex steps in the mechanism of estrogen action. hyperplasia is characterized by architectural disarray in In our previous experiments, treatments sometimes had gland shape and glandular epithelium. Glands may have an effect on proliferation but had no action on morpho- multiple lumens which interconnect. Glandular epi- genetic changes in the uterus, and vice versa (Gunin et al. thelium may be tall and columnar or pseudostratified. 2001, 2004a,b). The present results show that histone However, epithelial cells in general do retain their deacetylase blockers affect both proliferation and morpho- orientation to the lumen. Atypical hyperplasia has approxi- genetic alterations. It is known that proliferation and mately the same characteristics, but hyperplastic endo- control of cell shape, differentiation, adhesion and apop- metrium shows cytologic atypia including large nuclei of tosis are regulated by different mechanisms and depend on variable size and shape that have lost polarity. Of all the work of different genes (Bigsby 2002, Klotz et al. endometrial hyperplasies, atypical hyperplasia has the most 2002). Hence, histone deacetylases are involved in the increased risk for progression to endometrial cancer (Scully control of all these estrogen-dependent processes, and et al. 1994, Deligdisch 2000). inhibition of these deacetylases intensifies proliferative and Results clearly showed that both histone deacetylase morphogenetic estrogen actions. One more well-known blockers had unilateral effects and led to an increase in estrogen effect in the uterus is the induction of progester- uterine mass and proliferation and to more expressed one receptors (Couse & Korach 1999). Our current results morphogenetic alterations than in control mice. The effect showed that both histone deacetylase blockers led to a of trichostatin A is more expressed than that of butyrate. decrease in progesterone receptor expression in all uterine This situation is probably associated with the fact that tissues. Hence, it can be concluded that histone deacetyl- trichostatin A has a more specific and a stronger action in ases also manage this part of estrogen action, but their blocking histone deacetylase activity (Riester et al. 2004). blockade attenuates the estrogen effect on progesterone For all parameters tested, the effects of histone deacetylase receptor expression. inhibitors were found only in estrogen-treated mice and To define some possible mechanisms involved in the were not documented in control animals receiving olive action of histone deacetylases on estrogen-induced effects, oil instead of estradiol. Hence, these results support the the expression of estrogen receptor- and progesterone

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Figure 4 The numbers of mitotic (a) and BrdU-labelled cells (c) in the compartments of the uteri of ovariectomized mice treated with estradiol alone (open bars) or with trichostatin A (grey bars) or sodium butyrate (black bars) for 30 days. The numbers of mitotic (b) and BrdU-labelled cells (d) of mice treated with olive oil (vehicle of estradiol) only (thin-hatched bars, vehicle of estradiol) or with trichostatin A (thick-hatched bars) or sodium butyrate (parallel-line-filled bars) for 30 days. No mitoses were found in stromal and myometrial cells of mice treated with olive oil only or with trichostatin A or with sodium butyrate for 30 days (panels b and d). Values are means+S.E.M. *P<0·05; **P<0·01; ***P<0·001; Student’s t-test. receptors in uterine tissues was examined. Results showed acceleration of their degradation. Other researchers also that both blockers reduced the level of estrogen receptor- reported that trichostatin A decreased the levels of proges- in all uterine compartments, as compared with control. terone receptor coactivators and impaired progesterone Other data support our observation and also show that receptor function (Wilson et al. 2002, Condon et al. 2003). histone deacetylase blockers decrease estrogen receptor- It is interesting to note that in mice treated with histone levels in ovarian, endometrial and mammary gland cancer deacetylase blockers and estradiol, the more intensive cell lines (Alao et al. 2004, Margueron et al. 2004). estrogen-dependent processes (increase in mass, prolifer- In general, the level of receptors in a tissue depends on ation, morphogenesis) in the uterus proceed with lower a balance between their synthesis and degradation (Ing levels of estrogen and progesterone receptors. Other data & Ott 1999, Nephew et al. 2000). Histone deacetylase also showed that more malignant and less-differentiated blockers probably attenuate estrogen receptor synthesis or endometrial tumors had low levels of estrogen and proges- activate their degradation. The effect of histone deacetyl- terone receptors (Sivridis et al. 2001, Ali et al. 2004). It is ase blockers on progesterone receptor levels may also be possible that a diminished level of estrogen and progester- caused by a decrease in the speed of their synthesis or by one receptors does not allow estrogens to adequately www.endocrinology-journals.org Journal of Endocrinology (2005) 185, 539–549

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Figure 6 (a) Expression of progesterone receptors in uterine tissues of mice treated with estradiol alone (open bars) or together Figure 5 (a) Expression of estrogen receptor- in uterine tissues of with trichostatin A (grey bars) or with sodium butyrate (black bars) mice treated with estradiol alone (open bars) or together with for 30 days. (b) Data from mice treated with olive oil (vehicle of trichostatin A (grey bars) or with sodium butyrate (black bars) for estradiol) alone (thin-hatched bars) or with trichostatin A 30 days. (b) Data from mice treated with olive oil (vehicle of (thick-hatched bars) or sodium butyrate (parallel-line-ﬁlled bars) for estradiol) alone (thin-hatched bars) or with trichostatin A 30 days. Quantitation of immunostaining was performed by (thick-hatched bars), or sodium butyrate (parallel-line-ﬁlled bars) photometric determination of optical density (light absorption) of for 30 days. Quantitation of immunostaining was performed by positively stained components of a tissue. The value of optical photometric determination of optical density (light absorption) of density was used as the level of expression. Values are positively stained components of a tissue. The value of optical means+S.E.M. **P<0·01; ***P<0·001; Student’s t-test. density was used as the level of expression. Values are means+S.E.M. **P<0·01; ***P<0·001; Student’s t-test.

-Catenin content in a tissue is also a result of the control the processes managing the morphogenesis that balance between its synthesis and degradation. It has leads to atypical hyperplasia formation. previously been shown that changes in -catenin expres- -Catenin is implicated in cell adhesion and is a sion can be caused by the work of glycogen-synthase component of the Wnt-pathway (Cong et al. 2003). kinase-3, an enzyme which takes part in -catenin -Catenin provides intercellular adhesion and it is possible degradation (Gunin et al. 2003). Other data showed that that if its concentration is high, cell–cell connection is estrogen hormones can attenuate -catenin biosynthesis in more stable and that this protects from the formation of the uterus (Fujimoto et al. 1996). Direct interactions precancerous changes. In the case of low -catenin con- between -catenin and histone deacetylases were docu- centration, cell–cell interactions are less solid, which mented (Billin et al. 2000). It was also shown that provides a foundation for cancer development. It has been -catenin led to a loss of activity of histone deacetylases reported that the level of -catenin in the uterus was (Henderson et al. 2002, Baek et al. 2003). A function of decreased following estrogen action and cancer formation -catenin is compromised by fusion to a transcriptional (Fujimoto et al. 1998, Nei et al. 1999, Miyamoto et al. repressor domain from histone deacetylase (Cong et al. 2000, Gunin et al. 2004b). There is a decrease in -catenin 2003). However, further studies are needed to elucidate expression in uterine epithelia in mice treated with the roles of histone deacetylases in the regulation of estradiol and histone deacetylase blockers compared with -catenin content in the uterus, as well as to deﬁne the that of mice treated with estradiol alone. Our results role of -catenin in uterine morphogenesis. showed that more expressed morphogenetic shifts, which There are reports showing that histone deacetylase were found in estradiol and trichostatin A or sodium blockers – trichostatin A, sodium butyrate and others – led to butyrate treated mice, are accompanied by decreased a decrease in proliferation in endometrial and mammary levels of -catenin. Hence, -catenin is involved in gland cancer cell lines (Takai et al. 2004a,b). Our results changes in estrogen-dependent uterine morphology which showed that trichostatin A or sodium butyrate enhanced are aﬀected by histone deacetylase blockers. proliferation and hyperplasia formation in the murine uterus.

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used in vitro (Deschner et al. 1990, Lupton 2004) and accelerates cancer formation in vivo (Freeman 1986, Lup- ton 2004). There are, however, observations documenting an increase in colon cancer formation under sodium butyrate treatment in vitro and in vivo (Deschner et al. 1990). Finally, all published data were obtained on already formed cancer cells, but not on normal cells that are transforming to cancerous cells. Our experiments were done on normal, nonmalignant, uterine tissues which were then exposed to estrogen followed by hyperplasia formation. One more interesting remark, valproic acid, which is used for treatment of epilepsy and has a mechanism of action which involves the blockade of histone deacetylases, revealed expressed teratogenic effects in humans (Phiel et al. 2001, Kultima et al. 2004). Trichostatin A also has teratogenic effects in vertebrate embryos (Phiel et al. 2001). However, further studies are needed to explain some contradictory points and to elucidate the exact mechanisms involved in the interactions between estrogen signalling and histone deacetylases. Thus, this research provides evidence that histone deacetylase blockers, trichostatin A and sodium butyrate, enhance proliferative and morphogenetic estrogen action and support the development of estrogen-dependent endometrial hyperplasia. However, the exact mechanisms of the actions of histone deacetylase blockers on estrogen- dependent changes in uterine tissues remain unclear. It is possible that histone deacetylase inhibitors affect the Figure 7 (a) Expression of -catenin in uterine tissues of mice treated with estradiol alone (open bars) or together with activity of estrogen-regulated genes in the uterus and this trichostatin A (grey bars), or with sodium butyrate (black bars) for is an important avenue for further research. We hope that 30 days. (b) Data from mice treated with olive oil (vehicle of this research will lead to a better understanding of the estradiol) alone (thin-hatched bars) or with trichostatin A origin and progression of estrogen-dependent cancer of the (thick-hatched bars), or sodium butyrate (parallel-line-filled bars) female reproductive system. for 30 days. Quantitation of immunostaining was performed by photometric determination of optical density (light absorption) of positively stained components of a tissue. The value of optical density was used as the level of expression. Values are Funding means+S.E.M. *P<0·05; **P<0·01; Student’s t-test. This work was supported by grants from the Russian Foundation for Basic Research (03–04–48000) and the What are the sources of these contradictions? First, most of Ministry of Education and Science of Russia (E02–6·0– the studies examining the effects of changes in the level of 136; yp. 11·01·026). The authors declare that there is no histone acetylation on proliferation and differentiation in conflict of interest that would prejudice the impartiality of uterine and mammary gland cell lines were performed in this scientific work. the absence of estrogen administration (Adhikari et al. 2000, Terao et al. 2001). Secondly, most works that showed the antiproliferative and anticancer potency of References histone deacetylase blockers were performed in vitro. However, in vitro and in vivo conditions are principally Adhikari D, Feroz F, Liefshitz A, Barakat RR, Bertino JR & Banerjee different. Concentrations of reagents are constant in vitro, D 2000 Pretreatment of endometrial carcinoma cell lines with butyrate results in upregulation of Bax and correlates with but in vivo, concentrations of acting substances are sub- potentiation of radiation induced cell kill. In Vivo 14 603–609. jected to fluctuations depending on the activity of meta- Akhmedkhanov A, Zeleniuch-JacquotteA&TonioloP2001Roleof bolic processes. There is a report showing a rapid increase exogenous and endogenous hormones in endometrial cancer: review in proliferation of human endometrial adenocarcinoma of the evidence and research perspectives. Annals of the New York Academy of Sciences 943 296–315. cells after removal of sodium butyrate from a medium Alao JP, Lam EW, Ali S, Buluwela L, Bordogna W, Lockey P, (Saito et al. 1991). Other works demonstrated that in large Varshochi R, Stavropoulou AV, Coombes RC & Vigushin DM intestine sodium butyrate has an antineoplastic effect when 2004 Histone deacetylase inhibitor trichostatin A represses estrogen www.endocrinology-journals.org Journal of Endocrinology (2005) 185, 539–549

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Journal of Endocrinology (2005) 185, 539–549 www.endocrinology-journals.org

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Sakai N, Maruyama T, Sakurai R, Masuda H, Yamamoto Y, Shimizu Takai N, Kawamata N, Gui D, Said JW, MiyakawaI&Koeffler HP A, Kishi I, Asada H, Yamagoe S & Yoshimura Y 2003 2004b Human ovarian carcinoma cells: histone deacetylase inhibitors Involvement of histone acetylation in ovarian steroid-induced exhibit antiproliferative activity and potently induce apoptosis. decidualization of human endometrial stromal cells. Journal of Cancer 101 2760–2770. Biological Chemistry 278 16675–16682. Terao Y, Nishida J, Horiuchi S, Rong F, Ueoka Y, Matsuda T, Kato Scully RE, Bonfiglio TA, Kurman RJ, Silverberg SG & Wilkinson EJ H,FurugenY,YoshidaK,KatoK&WakeN2001Sodium 1994 Histological typing of female genital tract tumours. In butyrate induces growth arrest and senescence-like phenotypes in International Histological Classification of Tumours, 2nd edn, pp. 13–18. gynecologic cancer cells. International Journal of Cancer 94 257–267. Geneva: WHO. Wilson MA, Ricci AR, Deroo BJ & Archer TK 2002 The histone Sivridis E, Giatromanolaki A, Koukourakis M & Anastasiadis P 2001 deacetylase inhibitor trichostatin A blocks progesterone receptor- Endometrial carcinoma: association of steroid hormone receptor mediated transactivation of the mouse mammary tumor virus expression with low angiogenesis and bcl-2 expression. Virchows promoter in vivo. Journal of Biological Chemistry 277 15171–15181. Archiv 438 470–477. Sun JM, Chen HY & Davie JR 2001 Effect of estradiol on histone acetylation dynamics in human breast cancer cells. Journal of Received 28 February 2005 Biological Chemistry 276 49435–49442. Received in final form 9 March 2005 Takai N, Desmond JC, Kumagai T, Gui D, Said JW, Whittaker S, Accepted 10 March 2005 ffl MiyakawaI&Koe er HP 2004a Histone deacetylase inhibitors Made available online as an have a profound antigrowth activity in endometrial cancer cells. Clinical Cancer Research 10 1141–1149. Accepted Preprint 18 March 2005

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