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Long-Term Estradiol Exposure Is a Direct Mitogen for Insulin/EGF-Primed Endometrial Cells and Drives PTEN Loss-Induced Hyperplasic Growth

2013; Elsevier BV; Volume: 183; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2013.03.008

ISSN

1525-2191

Autores

Núria Eritja, Cristina Mirantes, David Llobet‐Navàs, Andrée Yeramian, Laura Bergadà, María Dosil Santamaría, Mónica Domingo, Xavier Matías‐Guiu, Xavier Dolcet,

Tópico(s)

TGF-β signaling in diseases

Resumo

Loss of tumor-suppressor PTEN is the most common alteration in endometrial carcinoma. However, the relationship between loss of PTEN, growth factors [eg, insulin/insulin-like growth factor (IGF)-1], epidermal growth factor (EGF), and hyperestrogenism in the development of endometrial carcinoma is still controversial. By using three-dimensional (3D) cultures of PTEN+/+ and PTEN+/− endometrial epithelial cells, we investigated the effects of EGF, insulin/IGF, and estradiol in endometrial cell proliferation. We have previously demonstrated that 3D cultures of endometrial cells require EGF and insulin/IGF to proliferate. Herein, we demonstrate that, in the presence of EGF and insulin/IGF, long-term estradiol treatment directly induces proliferation of 3D cultures. Moreover, we show that the mitogenic effects of estradiol require the presence of insulin/IGF and EGF, because withdrawal of such factors completely abolishes estradiol-induced proliferation. In the presence of EGF and insulin/IGF, PTEN+/− and PTEN+/+ spheroids display a similar rate of proliferation. However, the addition of estradiol causes an exaggerated proliferation of PTEN+/− cultures, leading to formation of complex structures, such as those observed in endometrial hyperplasia or carcinoma. In summary, we demonstrate that EGF and insulin/IGF prime endometrial epithelial cells to direct the mitogenic effects of estradiol. Furthermore, PTEN deficiency results in enhanced responsiveness to this combination, leading to the development of hyperplasia of endometrial cells in culture. Loss of tumor-suppressor PTEN is the most common alteration in endometrial carcinoma. However, the relationship between loss of PTEN, growth factors [eg, insulin/insulin-like growth factor (IGF)-1], epidermal growth factor (EGF), and hyperestrogenism in the development of endometrial carcinoma is still controversial. By using three-dimensional (3D) cultures of PTEN+/+ and PTEN+/− endometrial epithelial cells, we investigated the effects of EGF, insulin/IGF, and estradiol in endometrial cell proliferation. We have previously demonstrated that 3D cultures of endometrial cells require EGF and insulin/IGF to proliferate. Herein, we demonstrate that, in the presence of EGF and insulin/IGF, long-term estradiol treatment directly induces proliferation of 3D cultures. Moreover, we show that the mitogenic effects of estradiol require the presence of insulin/IGF and EGF, because withdrawal of such factors completely abolishes estradiol-induced proliferation. In the presence of EGF and insulin/IGF, PTEN+/− and PTEN+/+ spheroids display a similar rate of proliferation. However, the addition of estradiol causes an exaggerated proliferation of PTEN+/− cultures, leading to formation of complex structures, such as those observed in endometrial hyperplasia or carcinoma. In summary, we demonstrate that EGF and insulin/IGF prime endometrial epithelial cells to direct the mitogenic effects of estradiol. Furthermore, PTEN deficiency results in enhanced responsiveness to this combination, leading to the development of hyperplasia of endometrial cells in culture. 17β-Estradiol (E2) is required for epithelial cell proliferation of endometrial epithelial cells. Although it is well known that E2 is a potent mitogen for endometrial epithelial cells, the molecular mechanisms by which E2 triggers its proliferation are still under active research. It has been extensively demonstrated that E2 alone is unable to induce proliferation of endometrial epithelial cells.1Fukamachi H. McLachlan J.A. Proliferation and differentiation of mouse uterine epithelial cells in primary serum-free culture: estradiol-17 beta suppresses uterine epithelial proliferation cultured on a basement membrane-like substratum.In Vitro Cell Dev Biol. 1991; 27A: 907-913Crossref PubMed Scopus (17) Google Scholar, 2Uchima F.D. Edery M. Iguchi T. Bern H.A. Growth of mouse endometrial luminal epithelial cells in vitro: functional integrity of the oestrogen receptor system and failure of oestrogen to induce proliferation.J Endocrinol. 1991; 128: 115-120Crossref PubMed Scopus (38) Google Scholar, 3Mutter G.L. Lin M.C. Fitzgerald J.T. Kum J.B. Baak J.P. Lees J.A. Weng L.P. Eng C. Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers.J Natl Cancer Inst. 2000; 92: 924-930Crossref PubMed Google Scholar, 4Mutter G.L. Ince T.A. Baak J.P. Kust G.A. Zhou X.P. Eng C. Molecular identification of latent precancers in histologically normal endometrium.Cancer Res. 2001; 61: 4311-4314PubMed Google Scholar Because E2 itself is not a direct mitogen for epithelial cells in the uterus, different models to explain how E2 induces proliferation have been proposed.5Koos R.D. Minireview: putting physiology back into estrogens' mechanism of action.Endocrinology. 2011; 152: 4481-4488Crossref PubMed Scopus (59) Google Scholar The most widely accepted hypothesis is that growth factors, such as insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and transforming growth factor α, mediate E2-induced proliferation.6DiAugustine R.P. Petrusz P. Bell G.I. Brown C.F. Korach K.S. McLachlan J.A. Teng C.T. Influence of estrogens on mouse uterine epidermal growth factor precursor protein and messenger ribonucleic acid.Endocrinology. 1988; 122: 2355-2363Crossref PubMed Scopus (263) Google Scholar, 7Nelson K.G. Takahashi T. Lee D.C. Luetteke N.C. Bossert N.L. 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Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene.Mol Endocrinol. 1995; 9: 1441-1454Crossref PubMed Scopus (406) Google Scholar Classic experiments demonstrated that stromal ERα mediates the mitogenic effects of E2 on epithelial cells.13Cooke P.S. Buchanan D.L. Young P. Setiawan T. Brody J. Korach K.S. Taylor J. Lubahn D.B. Cunha G.R. Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium.Proc Natl Acad Sci U S A. 1997; 94: 6535-6540Crossref PubMed Scopus (481) Google Scholar It has also been demonstrated that ERα is required for the expression of IGF-1 in the stromal compartment.14Klotz D.M. Hewitt S.C. Korach K.S. Diaugustine R.P. Activation of a uterine insulin-like growth factor I signaling pathway by clinical and environmental estrogens: requirement of estrogen receptor-alpha.Endocrinology. 2000; 141: 3430-3439Crossref PubMed Scopus (91) Google Scholar IGF-1 secreted from stromal cells acts on epithelial cells to mediate the mitogenic effects of E2.10Zhu L. Pollard J.W. Estradiol-17beta regulates mouse uterine epithelial cell proliferation through insulin-like growth factor 1 signaling.Proc Natl Acad Sci U S A. 2007; 104: 15847-15851Crossref PubMed Scopus (114) Google Scholar More recently, ERα ablation on epithelial cells demonstrates that ERα is dispensable for the mitogenic effects of E2 in endometrial epithelial cells.15Winuthayanon W. Hewitt S.C. Orvis G.D. Behringer R.R. Korach K.S. Uterine epithelial estrogen receptor α is dispensable for proliferation but essential for complete biological and biochemical responses.Proc Natl Acad Sci U S A. 2010; 107: 19272-19277Crossref PubMed Scopus (180) Google Scholar These results contrast with the observation that ERα is required to drive the mitogenic effects of a variety of growth factors, including IGF-1/insulin16Klotz D.M. Hewitt S.C. Ciana P. Raviscioni M. Lindzey J.K. Foley J. Maggi A. DiAugustine R.P. Korach K.S. Requirement of estrogen receptor-alpha in insulin-like growth factor-1 (IGF-1)-induced uterine responses and in vivo evidence for IGF-1/estrogen receptor cross-talk.J Biol Chem. 2002; 277: 8531-8537Crossref PubMed Scopus (252) Google Scholar, 17Bartella V. De Marco P. Malaguarnera R. Belfiore A. Maggiolini M. New advances on the functional cross-talk between insulin-like growth factor-I and estrogen signaling in cancer.Cell Signal. 2012; 24: 1515-1521Crossref PubMed Scopus (60) Google Scholar or EGF,18Lupien M. Meyer C.A. Bailey S.T. Eeckhoute J. Cook J. Westerling T. Zhang X. Carroll J.S. Rhodes D.R. Liu X.S. Brown M. Growth factor stimulation induces a distinct ER(alpha) cistrome underlying breast cancer endocrine resistance.Genes Dev. 2010; 24: 2219-2227Crossref PubMed Scopus (147) Google Scholar, 19Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase.Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1731) Google Scholar, 20Kato S. Estrogen receptor-mediated cross-talk with growth factor signaling pathways.Breast Cancer. 2001; 8: 3-9Crossref PubMed Scopus (74) Google Scholar, 21Bunone G. Briand P.A. Miksicek R.J. Picard D. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (859) Google Scholar, 22Smith C.L. Cross-talk between peptide growth factor and estrogen receptor signaling pathways.Biol Reprod. 1998; 58: 627-632Crossref PubMed Scopus (265) Google Scholar in endometrium and other tissues. In the normal uterus, the mitogenic effects of E2 are crucial to complete estrous cycles or to support pregnancy, but excessive E2 exposure is a major risk factor for the development of endometrial carcinoma (EC).23Yeramian A. Moreno-Bueno G. Dolcet X. Catasus L. Abal M. Colas E. Reventos J. Palacios J. Prat J. Matias-Guiu X. Endometrial carcinoma: molecular alterations involved in tumor development and progression.Oncogene. 2013; 32: 403-413Crossref PubMed Scopus (138) Google Scholar, 24Tinelli A. Vergara D. Martignago R. Leo G. Malvasi A. Tinelli R. Hormonal carcinogenesis and socio-biological development factors in endometrial cancer: a clinical review.Acta Obstet Gynecol Scand. 2008; 87: 1101-1113Crossref PubMed Scopus (40) Google Scholar, 25Shang Y. Molecular mechanisms of oestrogen and SERMs in endometrial carcinogenesis.Nat Rev Cancer. 2006; 6: 360-368Crossref PubMed Scopus (210) Google Scholar The most important signaling pathway in the regulation of uterine epithelial cells is the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, and alterations of phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha isoform (PI3KCA) or pentaerythritol tetranitrate (PTEN) lead to development of EC.23Yeramian A. Moreno-Bueno G. Dolcet X. Catasus L. Abal M. Colas E. Reventos J. Palacios J. Prat J. Matias-Guiu X. Endometrial carcinoma: molecular alterations involved in tumor development and progression.Oncogene. 2013; 32: 403-413Crossref PubMed Scopus (138) Google Scholar, 26Bussaglia E. del Rio E. Matias-Guiu X. Prat J. PTEN mutations in endometrial carcinomas: a molecular and clinicopathologic analysis of 38 cases.Hum Pathol. 2000; 31: 312-317Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 27Velasco A. Bussaglia E. Pallares J. Dolcet X. Llobet D. Encinas M. Llecha N. Palacios J. Prat J. Matias-Guiu X. PIK3CA gene mutations in endometrial carcinoma: correlation with PTEN and K-RAS alterations.Hum Pathol. 2006; 37: 1465-1472Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar PTEN inactivation is an early event in endometrial carcinogenesis,28Hecht J.L. Mutter G.L. Molecular and pathologic aspects of endometrial carcinogenesis.J Clin Oncol. 2006; 24: 4783-4791Crossref PubMed Scopus (442) Google Scholar, 29Lacey Jr., J.V. Mutter G.L. Ronnett B.M. Ioffe O.B. Duggan M.A. Rush B.B. Glass A.G. Richesson D.A. Chatterjee N. Langholz B. Sherman M.E. PTEN expression in endometrial biopsies as a marker of progression to endometrial carcinoma.Cancer Res. 2008; 68: 6014-6020Crossref PubMed Scopus (64) Google Scholar, 30Mutter G.L. Zaino R.J. Baak J.P. Bentley R.C. Robboy S.J. Benign endometrial hyperplasia sequence and endometrial intraepithelial neoplasia.Int J Gynecol Pathol. 2007; 26: 103-114Crossref PubMed Scopus (128) Google Scholar and it has been demonstrated that sporadic endometrial mutations in PTEN are frequent in the histologically normal endometrium of women of reproductive age.4Mutter G.L. Ince T.A. Baak J.P. Kust G.A. Zhou X.P. Eng C. Molecular identification of latent precancers in histologically normal endometrium.Cancer Res. 2001; 61: 4311-4314PubMed Google Scholar Therefore, it has been hypothesized that those PTEN-null latent precancerous cells progress through endometrial hyperplasia and carcinoma on exposition to risk factors, such as excessive estrogen exposure.3Mutter G.L. Lin M.C. Fitzgerald J.T. Kum J.B. Baak J.P. Lees J.A. Weng L.P. Eng C. Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers.J Natl Cancer Inst. 2000; 92: 924-930Crossref PubMed Google Scholar, 4Mutter G.L. Ince T.A. Baak J.P. Kust G.A. Zhou X.P. Eng C. Molecular identification of latent precancers in histologically normal endometrium.Cancer Res. 2001; 61: 4311-4314PubMed Google Scholar The role of PTEN in EC is evidenced by PTEN knockout mice. Nearly 100% of PTEN+/− female mice develop endometrial hyperplasia, of which approximately 30% progresses to EC.31Di Cristofano A. Pesce B. Cordon-Cardo C. Pandolfi P.P. Pten is essential for embryonic development and tumour suppression.Nat Genet. 1998; 19: 348-355Crossref PubMed Scopus (1321) Google Scholar, 32Podsypanina K. Ellenson L.H. Nemes A. Gu J. Tamura M. Yamada K.M. Cordon-Cardo C. Catoretti G. Fisher P.E. Parsons R. Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems.Proc Natl Acad Sci U S A. 1999; 96: 1563-1568Crossref PubMed Scopus (844) Google Scholar The role of PTEN in endometrial neoplastic growth has been further demonstrated by the recent generation of mice with conditional deletion of both PTEN alleles.33Daikoku T. Hirota Y. Tranguch S. Joshi A.R. DeMayo F.J. Lydon J.P. Ellenson L.H. Dey S.K. Conditional loss of uterine Pten unfailingly and rapidly induces endometrial cancer in mice.Cancer Res. 2008; 68: 5619-5627Crossref PubMed Scopus (183) Google Scholar However, the role of ERα in PTEN-driven EC is not fully understood. Although it has been demonstrated that inhibition of ERα expression hampers the development of EC in PTEN+/− mice,34Vilgelm A. Lian Z. Wang H. Beauparlant S.L. Klein-Szanto A. Ellenson L.H. Di Cristofano A. Akt-mediated phosphorylation and activation of estrogen receptor alpha is required for endometrial neoplastic transformation in Pten+/− mice.Cancer Res. 2006; 66: 3375-3380Crossref PubMed Scopus (79) Google Scholar a recent work has demonstrated that PTEN+/− ERα−/− mice show an even higher incidence of in situ and invasive carcinoma, suggesting that endometrial tumorigenesis can progress in the absence of ERα.35Joshi A. Wang H. Jiang G. Douglas W. Chan J.S. Korach K.S. Ellenson L.H. Endometrial tumorigenesis in Pten(+/−) mice is independent of coexistence of estrogen and estrogen receptor alpha.Am J Pathol. 2012; 180: 2536-2547Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar The complex interplay between E2 and growth factors, such as EGF or insulin/IGF-1, to promote endometrial cell proliferation in normal and PTEN-deficient endometrial epithelial cells is not completely understood. Herein, by using a three-dimensional (3D) culture of isolated endometrial epithelial cells, we have investigated the effects of the major players in the regulation of endometrial proliferation, which are E2 and the growth factors insulin/IGF-1 and EGF. Finally, we have assessed the effects of PTEN alterations in the response to E2 and growth factors and its role in endometrial development of hyperplasia/carcinoma. The recombinant basement membrane Matrigel was purchased from BD Biosciences (San Jose, CA). Epidermal growth factors, ICI182170 (ICI) and LY 294002, were obtained from Sigma (St. Louis, MO); insulin-transferrin-sodium selenite supplement was obtained from Invitrogen (Carlsbad, CA). Antibody to E-cadherin was from BD Biosciences, zonula occludens protein-1 was from Zymed (San Francisco, CA), and bisBenzimide H33342 trihydrochloride (Hoechst), rhodamine-conjugated phalloidin, and antibodies to laminin and tubulin were obtained from Sigma. Alexa-Fluor–conjugated anti-rabbit and anti-mouse antibodies were from Invitrogen. Anti–phophorylated-Akt and phophorylated-extracellular signal–regulated kinase antibodies were from Cell Signaling Technology (Beverly, MA). Anti-ERα antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Jackson ImmunoResearch Europe Ltd (Suffolk, UK). All other reagents were obtained from Sigma, unless otherwise specified. PTEN knockout mice (strain B6.129-PTENtm1Rps) were obtained from the National Cancer Institute (Frederick, MD) mouse repository. The C57BL6 and PTEN knockout mice used to isolate endometrial cells were maintained in temperature- and light-controlled conditions and fed ad libitum. The Institutional Animal Care Committee of the IRB-Lleida Institute approved all experimental procedures. The isolation of endometrial epithelial cells was processed as previously described.36Eritja N. Llobet D. Domingo M. Santacana M. Yeramian A. Matias-Guiu X. Dolcet X. A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis.Am J Pathol. 2010; 176: 2722-2731Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar In brief, uterine horns were dissected from 3- to 4-week-old C57BL6 mice. Uteri were washed with Hank's balanced salt solution (HBSS) and digested with trypsin (Invitrogen). After trypsin digestion, epithelial sheets were squeezed out of the uterine pieces. Epithelial sheets were washed twice with PBS and resuspended in 1 mL of Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen) supplemented with 1 mmol/L HEPES (Sigma), 1% penicillin/streptomycin (Sigma), and fungizone (Invitrogen) (basal medium). Epithelial sheets were mechanically disrupted in basal medium. Cells were diluted in basal medium containing 2% dextran-coated charcoal-stripped serum (Hyclone, Logan, UT) and plated in culture dishes (BD Falcon, Bredford, MA). Cells were cultured for 24 hours in an incubator at 37°C with saturating humidity and 5% CO2. Growth of endometrial epithelial cells in 3D cultures was performed as previously described.36Eritja N. Llobet D. Domingo M. Santacana M. Yeramian A. Matias-Guiu X. Dolcet X. A novel three-dimensional culture system of polarized epithelial cells to study endometrial carcinogenesis.Am J Pathol. 2010; 176: 2722-2731Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Twenty-four hours after plating in plastic, cells were washed with HBSS and incubated with trypsin/EDTA solution (Sigma) for 5 minutes at 37°C. Trypsin activity was stopped by adding DMEM containing 10% fetal bovine serum, and clumps of two to eight cells were obtained. Cells were centrifuged at 18 × g for 3 minutes and diluted in basal medium containing 3% Matrigel to obtain 4 × 104 cell clumps/mL. For immunofluorescence, cells were seeded in a volume of 40 μL per well in 96-well black plates with a microclear bottom (Greiner Bio-one). For Western blot analysis, cells were placed in a volume of 200 μL in 24-well plates (BD Biosciences). In all cases, 24 hours after plating, medium was replaced by basal medium supplemented with 5 ng/mL EGF and a 1:100 dilution of insulin-transferrin-sodium selenite supplement (Invitrogen) and 3% fresh Matrigel [this medium is referred to as bullous ichthyosiform erythroderma (BIE)]. Medium was replaced every 2 to 3 days. 3D cultures were fixed with formalin for 5 minutes at room temperature, washed twice with PBS. Depending on primary antibody, cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes or permeabilized with 100% methanol for 2 minutes. Next, cultures were incubated overnight at 4°C with the indicated dilutions of antibodies: anti-laminin (1:500), rhodamine-conjugated phalloidin (1:500), E-cadherin (1:250), zonula occludens protein-1 (1:250), and anti-GM130 (1:100). After 1 day, cells were washed twice with PBS and incubated with PBS containing 5 μg/mL of Hoechst 33342 and a 1:500 dilution of Alexa Fluor secondary anti-mouse or anti-rabbit antibodies for 2 hours at room temperature. For double-immunofluorescence staining, cells were incubated with the second round of primary and secondary antibodies. In all double-immunofluorescence stains, first and second primary antibodies were from a different isotope. Immunofluorescence staining was visualized and analyzed using confocal microscopy (model FV1000; Olympus, Tokyo, Japan) with the ×10 and the oil-immersion ×60 magnification objectives. Analysis of images was obtained with Fluoview FV100 software (Olympus). Images of endometrial epithelial spheroids were captured and digitized with a confocal microscope (Fluoview FV1000). Epithelial perimeter analysis was processed by image analysis software (ImageJ version 1.46r; NIH, Bethesda, MD), generating binary images of the spheroids as previously described.37Baak J.P. Kurver P.H. Overdiep S.H. Delemarre J.F. Boon M.E. Lindeman J. Diegenbach P.C. Quantitative, microscopical, computer-aided diagnosis of endometrial hyperplasia or carcinoma in individual patients.Histopathology. 1981; 5: 689-695Crossref PubMed Scopus (53) Google Scholar, 38Mutter G.L. Kauderer J. Baak J.P. Alberts D. Group G.O. Biopsy histomorphometry predicts uterine myoinvasion by endometrial carcinoma: a Gynecologic Oncology Group study.Hum Pathol. 2008; 39: 866-874Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 39Gakunga P. Frost G. Shuster S. Cunha G. Formby B. Stern R. Hyaluronan is a prerequisite for ductal branching morphogenesis.Development. 1997; 124: 3987-3997Crossref PubMed Google Scholar The presence of one lumen was revealed by phalloidin immunostaining. For each experiment, we quantified at least 100 spheroids. Cell polarity of epithelial cells forming spheroid structures was evidenced by double immunostaining, as indicated in each figure. Spheroid endometrial 3D cultures stimulated for the indicated periods of time were washed with HBSS and incubated with trypsin/EDTA solution for 5 minutes at 37°C. Incubation with trypsin was done to allow us to separate the spheroid structures from Matrigel. Trypsin activity was stopped by adding DMEM containing 10% fetal bovine serum, and the cells were lysed with lysis buffer [2% SDS and 125 mmol/L Tris-HCL (pH 6.8)]. Relative protein concentrations were determined by loading an 8% acrylamide gel, transferred to polyvinylidene difluoride membranes, and blotted with anti-tubulin antibody. Band density was determined using Quantity One software version 4.5.2 (Bio-Rad, Richmond, CA). Equal amounts of proteins were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Non-specific binding was blocked by incubation with TBST [20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 0.1% Tween-20] containing 5% nonfat milk. Membranes were incubated with the primary antibodies overnight at 4°C, followed by a 1-hour incubation with secondary antibody, 1:10,000, in TBST. Signal was detected with electrochemiluminescence Advance (Amersham-Pharmacia, Buckinghamshire, UK). Total RNA was prepared using the Rneasy mini kit (Qiagen, Germantown, MD), according to the manufacturer's protocol. Reverse transcription reactions were performed using 1 μg total RNA with a TaqMan Reverse Transcription Kit from Applied Biosystems. Quantitative real-time PCR detection of gene expression was performed with the ABI Prism 7000 Sequence Detection System using the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA). Primers and probes for real-time PCR analysis were from Applied Biosystems. Expression Products: (Ccnd1) Mm00432359_m1, c-fos Mm00432359_m1 and glyceraldehyde-3-phosphate dehydrogenase (Gadph) Mm99999915_g1. Relative expression was determined from CT values, which were normalized to Gadph as the endogenous control. Experiments were performed at least three times, and statistical significance was determined by Student's t-test with P value. The bromodeoxyuridine protocol was performed as previously described, with minor modifications.40Pallares J. Llobet D. Santacana M. Eritja N. Velasco A. Cuevas D. Lopez S. Palomar-Asenjo V. Yeramian A. Dolcet X. Matias-Guiu X. CK2beta is expressed in endometrial carcinoma and has a role in apoptosis resistance and cell proliferation.Am J Pathol. 2009; 174: 287-296Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar 3D cultures were incubated with 3 ng/mL 5-bromodeoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO) for 15 hours and then fixed with 4% paraformaldehyde. After DNA denaturing with 2 mol/L HCl for 30 minutes and neutralization with 0.1 mol/L Na2B4O7 (pH 8.5) for 2 minutes, cells were blocked in PBS solution containing 5% horse serum, 5% fetal bovine serum, 0.2% glycine, and 0.1% Triton X-100 for 1 hour (Sigma-Aldrich). Subsequently, cells were subjected to indirect immunofluorescence with a mouse 1:100 dilution of anti-BrdU monoclonal antibody (Dako, Carpentaria, CA) and Alexa Fluor–conjugated anti-mouse secondary antibody. Nuclei were counterstained with 5 μg/mL Hoechst 33258, and cells were visualized under a confocal microscope. BrdU-positive nuclei were scored and divided by the total number of cells (visualized by Hoechst staining). The results are expressed as a percentage of BrdU-positive cells. Experiments were performed at least three times, and statistical significance was determined by the Student's t-test. Although E2 is essential for endometrial physiological characteristics, hyperestrogenism is a major risk factor for the development of EC.23Yeramian A. Moreno-Bueno G. Dolcet X. Catasus L. Abal M. Colas E. Reventos J. Palacios J. Prat J. Matias-Guiu X. Endometrial carcinoma: molecular alterations involved in tumor development and progression.Oncogene. 2013; 32: 403-413Crossref PubMed Scopus (138) Google Scholar, 24Tinelli A. Vergara D. Martignago R. Leo G. Malvasi A. Tinelli R. Hormonal carcinogenesis and socio-biological development factors in endometrial cancer: a clinical review.Acta Obstet Gynecol Scand. 2008; 87: 1101-1113Crossref PubMed Scopus (40) Google Scholar, 25Shang Y. Molecular mechanisms of oestrogen and SERMs in endometrial carcinogenesis.Nat Rev Cancer. 2006; 6: 360-368Crossref PubMed Scopus (210) Google Scholar It is still unclear whether E2 can induce endometrial proliferation by direct binding to the ERα receptor in epithelial cells. The culture of isolated endometrial epithelial cells as glandular structures (spheroids) provides a suitable scenario to investigate the effects of E2 on epithelial cells, without interference of paracrine factors derived from stromal cells. To mimic a long-term hyperestrogenic situation, spheroids were grown for 12 days in the presence of DMEM/F12 basal medium supplemented with EGF and insulin (BIE) or BIE supplemented with increasing doses of E2 (see protocol for E2 treatments depicted in Figure 1A). Medium was replaced every 3 days for four consecutive times. After four stimulations, we analyzed cell proliferation by BrdU incorporation and spheroid size by measuring its perimeter. The addition of E2 caused a decrease in BrdU incorporation after the first stimulation, but the following treatments progressively increased the number of BrdU-positive cells (Figure 1B). Such an increase was visualized as an increase in the size of spheroids and an increase of the spheroid perimeter (Figure 1, C and D). To demonstrate that E2 increased the size of spheroids but did not cause alterations in its morphological characteristics, we performed double immunofluorescence with phalloidin (to evidence an apical actin cytoskeleton) and cytokeratin (Figure 1E). To assess whether E2 was able to stimulate transcriptional activity of ERα in epithelial spheroids, we measured the induction of cyclin D1 and c-fos expression by real-time PCR. Consistently, E2 did not increase either cyclin D1 or c-fos expression after the first E2 stimulation, but triggered a marked up-regulation of cyclin D1 and c-fos mRNA after the four

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