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Disrupted Cell Cycle Control in Cultured Endometrial Cells from Patients with Endometriosis Harboring the Progesterone Receptor Polymorphism PROGINS
2009; Elsevier BV; Volume: 175; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080966
ISSN1525-2191
AutoresPaulo D’Amora, Thiago Trovati Maciel, Rodrigo Tambellini, Marcelo A. Mori, João Bosco Pesquero, Hélio Sato, Manoel Joâo Batista Castello Girão, Ismael Dale Cotrim Guerreiro da Silva, Eduardo Schor,
Tópico(s)Uterine Myomas and Treatments
ResumoPresently, little is understood about how endometriosis is established or maintained, or how genetic factors can predispose women to the disease. Because of the crucial role that the progesterone receptor polymorphism PROGINS plays in predisposing women to the development of endometriosis, we hypothesized that this variant may influence critical steps during endometrial cell metabolism that are involved in the pathogenesis of endometriosis. Eutopic endometria were collected from three sources: women with endometriosis who had a single PROGINS allele (from the progesterone receptor gene); women with endometriosis who had the wild-type progesterone receptor allele; and women without endometriosis who had the wild-type allele. Cells prepared from the eutopic endometria of these women were stimulated with both estradiol and progesterone, and then examined for cell proliferation, viability, and apoptosis. The cells from women with endometriosis that carried the PROGINS allele demonstrated increased proliferation, greater viability, and decreased apoptosis following progesterone treatment. In general, these parameters were very different as compared with those of women with endometriosis but without the PROGINS allele and women in the control group. This result indicates there is a reduced level of progesterone responsiveness in women who carry the PROGINS polymorphism. Because progesterone responsiveness is known to be an important characteristic of women with endometriosis, these data support the contention that the PROGINS polymorphism enhances the endometriosis phenotype. Presently, little is understood about how endometriosis is established or maintained, or how genetic factors can predispose women to the disease. Because of the crucial role that the progesterone receptor polymorphism PROGINS plays in predisposing women to the development of endometriosis, we hypothesized that this variant may influence critical steps during endometrial cell metabolism that are involved in the pathogenesis of endometriosis. Eutopic endometria were collected from three sources: women with endometriosis who had a single PROGINS allele (from the progesterone receptor gene); women with endometriosis who had the wild-type progesterone receptor allele; and women without endometriosis who had the wild-type allele. Cells prepared from the eutopic endometria of these women were stimulated with both estradiol and progesterone, and then examined for cell proliferation, viability, and apoptosis. The cells from women with endometriosis that carried the PROGINS allele demonstrated increased proliferation, greater viability, and decreased apoptosis following progesterone treatment. In general, these parameters were very different as compared with those of women with endometriosis but without the PROGINS allele and women in the control group. This result indicates there is a reduced level of progesterone responsiveness in women who carry the PROGINS polymorphism. Because progesterone responsiveness is known to be an important characteristic of women with endometriosis, these data support the contention that the PROGINS polymorphism enhances the endometriosis phenotype. Endometriosis is a chronic inflammatory disease characterized by implantation and growth of endometrial tissue outside of the uterus.1Bulun SE Endometriosis.N Engl J Med. 2009; 360: 268-279Crossref PubMed Scopus (1495) Google Scholar It affects 10% to 15% of all women of reproductive age, and it is significantly associated with infertility,2Gupta S Goldberg JM Aziz N Goldberg E Krajcir N Agarwal A Pathogenic mechanisms in endometriosis-associated infertility.Fertil Steril. 2008; 90: 247-257Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar chronic pain,3Ozawa Y Murakami T Terada Y Yaegashi N Okamura K Kuriyama S Tsuji I Management of the pain associated with endometriosis: an update of the painful problems.Tohoku J Exp Med. 2006; 210: 175-188Crossref PubMed Scopus (24) Google Scholar and morbidity,4Gao X Outley J Botteman M Spalding J Simon JA Pashos CL Economic burden of endometriosis.Fertil Steril. 2006; 86: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar making endometriosis a significant problem for public health. In 1925, Sampson et al5Sampson JA Peritoneal endometriosis due to menstrual dissemination of the endometrial tissue into the peritoneal cavity.Am J Obstet Gynecol. 1927; 14: 422-469Abstract Full Text PDF Google Scholar suggested that the transtubarian reflux of viable endometrial cells represents the origin of endometriosis. However, several subsequent studies reported that approximately 90% of women have viable endometrial cells in the peritoneal cavity,6Halme J Hammond MG Hulka JF Raj SG Talbert LM Retrograde menstruation in healthy women and in patients with endometriosis.Obstet Gynecol. 1984; 64: 151-154PubMed Google Scholar, 7Liu DT Hitchcock A Endometriosis: its association with retrograde menstruation, dysmenorrhoea and tubal pathology.Br J Obstet Gynaecol. 1986; 93: 859-862Crossref PubMed Scopus (199) Google Scholar disputing the notion that retrograde menstruation theory could explain the cause of the disease. It is also noteworthy that only a small portion of women with retrograde menstruation develops endometriosis. Environmental, endocrine, immune, and genetic factors have all been related to the pathogenesis of endometriosis. Of note, genetic studies of close relatives suggest that there is a 6% increase in the risk of developing endometriosis.8Kennedy S The genetics of endometriosis.Eur J Obstet Gynecol Reprod Biol. 1999; 82: 129-133Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Several studies also suggest that women with endometriosis present abnormalities in the eutopic endometrium, raising questions about whether the uterine mucosa is involved in the pathogenesis of the disease. In that context, modifications of cell cycle control, with increased levels of cell proliferation9Schor E Silva IDCG Sato H Baracat EC Girão MJBC Freitas V P27Kip1 is down-regulated in the endometrium of women with endometriosis.Fertil Steril. 2008; 90: 2086-2090Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar and decreased apoptosis,10Harada T Kaponis A Iwabe T Taniguchi F Makrydimas G Sofikitis N Paschopoulos M Paraskevaidis E Terakawa N Apoptosis in human endometrium and endometriosis.Hum Reprod Update. 2004; 10: 29-38Crossref PubMed Scopus (195) Google Scholar emerge as major mechanisms responsible for endometriosis development. Likewise, enhanced cell adhesion and invasion via increased expression of matrix metalloproteinases and the simultaneous down-regulation of their inhibitors,11Collete T Maheux R Mailloux J Akoum A Increased expression of matrix metalloproteinase-9 in the eutopic endometrial tissue of women with endometriosis.Hum Reprod. 2006; 21: 3059-3067Crossref PubMed Scopus (97) Google Scholar, 12Collete T Bellehumecr C Kats R Maheux R Mailloux J Villeneuve M Akoum A Evidence for an increased release of proteolytic activity by the eutopic endometrial tissue in women with endometriosis and for involvement of matrix metalloproteinase-9.Hum Reprod. 2004; 19: 1257-1964Crossref PubMed Scopus (77) Google Scholar, 13Chung HW Wen Y Chun SH Nezhat C Woo BH Lake Polan M Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 mRNA expression in ectopic and eutopic endometrium in women with endometriosis: a rationale for endometriotic invasiveness.Fertil Steril. 2001; 75: 152-159Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar as well as abnormal sex hormone metabolism,14Kitawaki J Kusuki I Koshiba H Tsukamoto K Fushiki S Honjo H Detection of aromatase cytochrome P-450 in endometrial biopsy specimens as a diagnostic test for endometriosis.Fertil Steril. 1999; 72: 1100-1106Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Zeitoun K Takayama K Sasano H Suzuki T Moghrabi N Andersson S Johns A Meng L Putman M Carr B Bulun SE Deficient 17ß-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize 17ß-estradiol.J Clin Endocrinol Metab. 1999; 83: 4474-4480Crossref Google Scholar are additional hallmarks of the disease. Progesterone plays a major role in the processes mentioned above,16Fang Z Yang S Lydon JP DeMayo F Tamura M Gurates B Bulun SE Intact progesterone receptors are essential to counteract the proliferative effect of estradiol in a genetically engineered mouse model of endometriosis.Fertil Steril. 2004; 82: 673-678Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar reinforcing the theory that impaired progesterone function could facilitate the genesis and development of endometriosis.17Gazvani R Templeton A New considerations for the pathogenesis of endometriosis.Int J Gynaecol Obstet. 2002; 76: 117-126Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 18Vinatier D Cosson M Dufour P Is endometriosis an endometrial disease?.Eur J Obstet Gynecol Reprod Biol. 2000; 91: 113-125Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar Interestingly, several studies demonstrated that progesterone is able to induce the expression of a large number of genes in the eutopic endometrium. Microarray analysis reported by Burney et al19Burney RO Talbi S Hamilton AE Vo KC Nyegaard M Nezhat CR Lessey BA Giudice LC Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis.Endocrinology. 2007; 148: 3814-3826Crossref PubMed Scopus (576) Google Scholar demonstrated that FOX10, MIG6, and CYP26A1 expression was significantly modified in the uterine mucosa. In another study, Wang et al used a baboon model of endometriosis and demonstrated that the expression of progesterone responsive factors is altered during the secretory stage of the menstrual cycle, suggesting that progesterone resistance plays a major role in the genesis of endometriosis.20Wang C Mavrogianis PA Fazleabas AT Endometriosis is associated with progesterone resistance in the baboon (Papio anubis) oviduct: evidence based on the localization of oviductal glycoprotein 1 (OVGP1).Biol Reprod. 2009; 80: 272-278Crossref PubMed Scopus (27) Google Scholar Several studies have addressed the question of whether genetic mutations contribute to the development of endometriosis. In these studies, several candidate genes and polymorphisms were associated with the development of endometriosis.21Tempfer CB Simoni M Destenaves B Fauser BCJM Functional genetic polymorphisms and female reproductive disorders: part II – endometriosis.Hum Reprod Update. 2009; 15: 97-118Crossref PubMed Scopus (85) Google Scholar One particular candidate, the progesterone receptor (PR) gene variant named PROGINS (NCBI Data Bank accession numbers AF016381 and Z49816), has emerged as an important disease component of endometriosis. PROGINS is characterized by a 306-bp insertion in intron G, which exists in linkage disequilibrium with point mutations in exons 4 and 5.22Rowe SM Coughlan SJ McKenna NJ Garrett E Kieback DG Carney DN Headon DR Ovarian carcinoma-associated TaqI restriction fragment length polymorphism in intron G of the progesterone receptor gene is due to an Alu sequence insertion.Cancer Res. 1995; 55: 2743-2745PubMed Google Scholar Epidemiological studies have shown that women carrying the PROGINS polymorphism have an increased risk for the development of hormone-dependent gynecological disorders, such as endometrial and ovarian carcinomas, recurrent abortions, and uterine fibroids,23Junqueira MG da Silva ID Nogueira-de-Souza NC Carvalho CV Leite DB Gomes MT Baracat EC Lopes LA Nicolau SM Goncalves WJ Progesterone receptor (PROGINS) polymorphism and the risk of endometrial cancer development.Int J Gynecol Cancer. 2007; 17: 229-232Crossref PubMed Scopus (28) Google Scholar, 24Pijnenborg JM Romano A Dam-de Veen GC Dunselman GA Fischer DC Groothuis PG Kieback DG Aberrations in the progesterone receptor gene and the risk of recurrent endometrial carcinoma.J Pathol. 2005; 205: 597-605Crossref PubMed Scopus (38) Google Scholar, 25Leite DB Junqueira MG de Carvalho CV Massad-Costa AM Gonçalves WJ Nicolau SM Lopes LA Baracat EC da Silva ID Progesterone receptor (PROGINS) polymorphism and the risk of ovarian cancer.Steroids. 2008; 73: 676-680Crossref PubMed Scopus (23) Google Scholar, 26Romano A Lindsey PJ Fischer DC Delvoux B Paulussen AD Janssen RG Kieback DG Two functionally relevant polymorphisms in the human progesterone receptor gene (C331G/A and progins) and the predisposition for breast and/or ovarian cancer.Gynecol Oncol. 2006; 101: 287-295Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 27Schweikert A Rau T Berkholz A Allera A Daufeldt S Wildt L Association of progesterone receptor polymorphism with recurrent abortions.Eur J Obstet Gynecol Reprod Biol. 2004; 113: 67-72Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 28Gomes MT Castro Rde A Villanova FE da Silva ID Baracat EC de Lima GR Girao MJ The progesterone receptor gene polymorphism. PROGINS, may be a factor related to the development of uterine fibroids.Fertil Steril. 2007; 87: 1116-1121Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar conditions in which progesterone plays a critical role. Several researchers, including those in our laboratory, have reported that patients carrying a single PROGINS allele have an increased risk for endometriosis development.29De Carvalho CV Nogueira-De-Souza NC Massad-Costa AM Baracat EC Girão MJBC D'Amora P Schor E Silva IDCG Genetic polymorphisms of cytochrome P450c17a (CYP17) and progesterone receptor genes (PROGINS) in the assessment of endometriosis risk.Gynecol Endocrinol. 2007; 23: 29-33Crossref PubMed Scopus (41) Google Scholar, 30Lattuada D Somigliana E Viganò P Candiani M Pardi G Di Blasio AM Genetics of endometriosis: a role for the progesterone receptor gene polymorphism PROGINS?.Clin Endocrinol (Oxf). 2004; 61: 190-194Crossref PubMed Scopus (59) Google Scholar, 31Wieser F Schneeberger C Tong D Tempfer C Huber JC Wenzl R PROGINS receptor gene polymorphism is associated with endometriosis.Fertil Steril. 2002; 77: 309-312Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 32van Kaam KJ Romano A Schouten JP Dunselman GA Groothuis PG Progesterone receptor polymorphism +331G/A is associated with a decreased risk of deep infiltrating endometriosis.Hum Reprod. 2007; 22: 129-135Crossref PubMed Scopus (41) Google Scholar In addition, in vitro data from Romano et al demonstrated that the PROGINS variant of the PR gene is less responsive to progestins, as compared with wild-type PR, resulting in reduced mRNA stability and protein activity, as well as a diminished ability to efficiently inhibit cell proliferation in rodent ovarian cell lines.33Romano A Delvoux B Fischer D-M Groothuis P The PROGINS polymorphism of the human progesterone receptor diminishes the response to progesterone.J Mol Endocrinol. 2007; 38: 331-350Crossref PubMed Scopus (79) Google Scholar Yet the effects of the PROGINS polymorphism on the phenotype of eutopic endometrial cells have never been described. The goal of the present study was to evaluate the influence of the PR variant PROGINS on cell viability, apoptosis, and proliferation in cell cultures of eutopic endometrium from women with and without endometriosis. Endometrial tissues were obtained from informed volunteers in the Endometriosis Unit/Department of Gynecology at the Federal University of São Paulo (UNIFESP). The Institutional Ethics Review Board (CEP0571/06) had previously approved the study. Women undergoing laparoscopy for routine evaluation of infertility, pelvic pain, or elective tubal sterilization were recruited. All patients had a history of regular menses and were not taking any sex steroids or steroid-modulating medications 3 months before surgery. After general anesthesia and just before the surgical procedure, endometrial tissues were collected using the Nowak's curette. During laparoscopy, a systematic observation of the pelvis was conducted and the patients were assigned to either the endometriosis or the control group. The collected endometrial tissue was separated into two parts: one for histological analysis according to the criteria of Noyes et al34Noyes RW Hertig AT Rock J Dating the endometrial biopsy.Am J Obstet Gynecol. 1975; 122: 262-263PubMed Scopus (703) Google Scholar and the other for cell culture processing. Data for the patients' laparoscopic diagnosis, age, menstrual cycle date, and stage of endometriosis were also recorded (Table 1).Table 1Characteristics of Samples Used in the StudySample selected for an experiment [+]/Passage no. usedGroupsSamplesAge (yr)Cycle dateStage of endometriosis*Revised American Society for Reproductive Medicine classification of endometriosis.Laparoscopic diagnosePROGINS genotypeIFqPCRB/ABCtTmCCV/AA5CMEndometriosis (E-Alu)141ProliferativeStage IEndometriosisT1/1+/1, 4–7+/5+/5−−+/6+/7+/7−234SecretoryStage IEndometriosisT1/1+/1, 4–7−−−−+/6−+/7−323ProliferativeStage IIEndometriosisT1/1+/1, 4–7+/5+/5+/5+/5+/6+/7+/7−426ProliferativeStage IVEndometriosisT1/1+/1, 4–7+/5+/5−−+/6+/7+/7+/5542SecretoryStage IEndometriosisT1/1+/1, 4–7−+/5−−−+/7−−633ProliferativeStage IEndometriosisT1/1+/1, 4–7−−−−−+/7−−733ProliferativeStage IIIEndometriosisT1/1+/1, 4–7−+/5+/5+/5−−−+/5835ProliferativeStage IIIEndometriosisT1/1+/1, 4–7+/5−+/5+/5+/6+/7+/7−933SecretoryStage IIEndometriosisT1/1+/1, 4–7+/5+/5+/5+/5+/6+/7+/7+/51022SecretoryStage IEndometriosisT1/1+/1, 4–7+/5−+/5+/5+/6−+/7+/5Endometriosis (E+Alu)1124ProliferativeStage IEndometriosisT1/2+/1, 4–7−+/5+/5+/5+/6+/7+/7+/51236SecretoryStage IVEndometriosisT1/2+/1, 4–7−+/5+/5+/5+/6+/7+/7+/51335SecretoryStage IEndometriosisT1/2+/1, 4–7−+/5+/5+/5+/6+/7+/7+/51437ProliferativeStage IEndometriosisT1/2+/1, 4–7−+/5+/5+/5+/6+/7+/7+/51528SecretoryStage IEndometriosisT1/2+/1, 4–7−+/5+/5+/5+/6+/7+/7+/51629ProliferativeStage IVEndometriosisT1/2+/1, 4–7−+/5−−−−−−Control (CP-Alu)1731SecretoryN/ANormal pelvisT1/1+/1, 4–7−−+/5+/5−+/7−+/51838SecretoryN/AOvarian cystT1/1+/1, 4–7−+/5+/5+/5−−−+/51930SecretoryN/APelvic adhesionsT1/1+/1, 4–7+/5+/5+/5+/5+/6+/7+/7−2026ProliferativeN/AAdnexal cystT1/1+/1, 4–7+/5+/5+/5+/5+/6−+/7−2136ProliferativeN/ANormal pelvisT1/1+/1, 4–7−−+/5−−+/7−−2233ProliferativeN/AAdnexal cystT1/1+/1, 4–7+/5+/5−−+/6+/7+/7−2331ProliferativeN/APelvic adhesionsT1/1+/1, 4–7+/5−−−+/6+/7+/7−2425SecretoryN/AOvarian cystT1/1+/1, 4–7−−−−−+/7−+/52528ProliferativeN/ANormal pelvisT1/1+/1, 4–7−−−−−+/7−+/52634ProliferativeN/ANormal pelvisT1/1+/1, 4–7+/5+/5−−+/6+/7+/7−Eutopic endometrium was obtained from control patients (CP-Alu) without the PROGINS allele, and from women with endometriosis with (E+Alu) and without (E-Alu) the single PROGINS allele.N/A = Not applicable.PROGINS genotype: T1/1 = homozygous major allele, T1/2 = PROGINS heterozygous.[+] = sample selected in an experiment, [−] = sample not used.IF = immunofluorescence, qPCR = time and dose response quantitative real-time PCR for PR; B/AB = PRB/AB mRNA expression ratio; Ct = Cell Counting; Tm = thymidine incorporation assay; CC = cell cycle analysis; V/A = viability and apoptosis (Viacount assay); A5 = Annexin-V labeling; and CM = chromatin morphology analysis. Experiments were performed between passages 4 and 7.* Revised American Society for Reproductive Medicine classification of endometriosis. Open table in a new tab Eutopic endometrium was obtained from control patients (CP-Alu) without the PROGINS allele, and from women with endometriosis with (E+Alu) and without (E-Alu) the single PROGINS allele. N/A = Not applicable. PROGINS genotype: T1/1 = homozygous major allele, T1/2 = PROGINS heterozygous. [+] = sample selected in an experiment, [−] = sample not used. IF = immunofluorescence, qPCR = time and dose response quantitative real-time PCR for PR; B/AB = PRB/AB mRNA expression ratio; Ct = Cell Counting; Tm = thymidine incorporation assay; CC = cell cycle analysis; V/A = viability and apoptosis (Viacount assay); A5 = Annexin-V labeling; and CM = chromatin morphology analysis. Experiments were performed between passages 4 and 7. Before the endometrial biopsy procedure, peripheral blood samples were obtained from the patients. Genomic DNA (gDNA) was extracted and purified using the GFX DNA extraction kit (GE Healthcare, Little Chalfont Buckinghamshire, UK). Genotyping of the PROGINS polymorphism was performed by PCR as described previously.29De Carvalho CV Nogueira-De-Souza NC Massad-Costa AM Baracat EC Girão MJBC D'Amora P Schor E Silva IDCG Genetic polymorphisms of cytochrome P450c17a (CYP17) and progesterone receptor genes (PROGINS) in the assessment of endometriosis risk.Gynecol Endocrinol. 2007; 23: 29-33Crossref PubMed Scopus (41) Google Scholar Endometriosis and control groups were subdivided based on detection of PROGINS: control (CP-Alu), endometriosis wild-type homozygous (E-Alu), and endometriosis PROGINS heterozygous (E+Alu). Specimens were transported to the laboratory in culture medium supplemented with antibiotics and antimycotics. Tissues were minced into small pieces and treated with collagenase IA (Life Technologies, Inc., Grand Island, NY), as previously described.35Sharpe KL Zimmer RL Griffin WT Penney LL Polypeptides synthesized and released by human endometriosis differ from those of the uterine endometrium in cell and tissue explant culture.Fertil Steril. 1993; 60: 839-851PubMed Scopus (69) Google Scholar No procedures were performed to isolate epithelial or stromal cells. Cells from each individual specimen were plated in 100-mm diameter culture dishes (TPP, Trasadingen, Switzerland). After 16 hours of incubation, non-adherent cells were washed away. The typical yield of stromal cells using this technique is 90%.36Piva M Horowitz GM Shape-Timms KL Interleukin-6 differentially stimulates haptoglobin production by peritoneal and endometriotic cells in vitro: a model for endometrial-peritoneal interaction in endometriosis.J Clin Endocrinol Metab. 2001; 86: 2553-2561Crossref PubMed Scopus (54) Google Scholar Cells were grown in phenol-free Dulbecco's Modified Eagle's Medium (Sigma-Aldrich, St. Louis, MO) containing 10% heat-inactivated fetal bovine serum (Life Technologies, Inc., Rockville, MD), 100 IU/ml penicillin, 100 mg/ml streptomycin (Sigma-Aldrich), and 250 μg/ml amphotericin-B (Cultilab, Campinas, Brazil) in a humidified incubator at 37°C with 5% CO2. Adherent cells were characterized by immunofluorescence using specific monoclonal antibodies against cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Dako Corp., CA). The proportions of cytokeratin-positive cells (endometrial glandular cells) and vimentin-positive cells (endometrial stromal cells) were assessed in each cell culture, as described previously.37Sharpe KL Zimmer RL Khan RS Penney LL Proliferative and morphogenic changes induced by the coculture of rat uterine and peritoneal cells: a cell culture model for endometriosis.Fertil Steril. 1992; 58: 1220-1229Abstract Full Text PDF PubMed Google Scholar Normal goat serum (Sigma-Aldrich) was used instead of the primary antibody as a negative control. Because the cultures presented high percentages of vimentin-positive cells, human breast ductal carcinoma cells (T47D cell line), which constitutively express cytokeratin in the form of intermediate filaments, were used as positive controls for cytokeratin to ensure the specificity of the method. A time-course and dose-response study was conducted with 17β-estradiol (Sigma-Aldrich) to induce optimal progesterone receptor mRNA expression for the cytometric experiments with progesterone.38lng NH Tornesi MB Estradiol up-regulates estrogen receptor and progesterone receptor gene expression in specific ovine uterine cells.Biol Reprod. 1997; 56: 1205-1215Crossref PubMed Scopus (137) Google Scholar Total RNA was extracted from the endometrial cells (RNeasy kit, Qiagen Inc, Hilden, Germany), and the reverse transcriptase reaction (Superscript-III, Invitrogen) was performed with 1 μg of DNase I (G&E Healthcare Biosciences, Upsala, Sweden)-treated RNA. qPCR was performed using the ABI 7700 thermocycler (Applied Biosystems, Foster City, CA) and the Power SYBr Green Mastermix PCR kit (Applied Biosystems), according to the manufacturer's instructions. PCR primers (IDT, Coralville, IA) were synthesized with the following sequences: total progesterone receptor PR (A+B): forward primer 5′-ACACAAAACCTGACACCTCC-3′ and reverse primer 5′-TACAGCATCTGCCCACTGAC-3′; progesterone receptor subunit B (PR-B): forward primer 5′-GCCAGAGAAAAAGTCGGGAG-3′ and reverse primer 5′-TGGGGAGCGCAAGAAAAAG-3′39Zhang W Mazella J Kloosterboer HJ Tseng L Effects of tibolone on nuclear receptors in human endometrial cells.Am J Obstet Gynecol. 2006; 195: 97-102Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar; and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH): forward primer 5′-ACCACAGTCCATGCCATCAC-3′ and reverse primer 5′-TCCACCACCCTGTTGCTGTA-3′.40Maciel TT Melo RS Schor N Campos AH Gremlin promotes vascular smooth muscle cell proliferation and migration.J Mol Cell Cardiol. 2008; 44: 370-379Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar The polymerase chain reaction was performed with the following conditions: one cycle at 95°C for 15 minutes and 35 cycles at 94°C for 15 seconds, 60°C for PR (A+B) and PR-B or 58°C for GAPDH for 30 seconds, and 72°C for 30 seconds. Cycle numbers obtained at the log-linear phase of the reaction were plotted against a standard curve prepared with serially diluted control samples. The expression of target PR (A+B) and PR-B genes was normalized to GAPDH mRNA levels, which were measured concurrently.41Pfaffl MW A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: 2002-2007Crossref Scopus (25597) Google Scholar The specificity of each reaction was confirmed by melting curve analysis and agarose gel electrophoresis. Endometrial cells (2 × 104) seeded in 6-well plates at 50% to 70% confluence were treated with 100 nmol/L 17β-estradiol for 24 hours, followed by 100 nmol/L progesterone for 24 hours. Cells were mixed with Guava Viacount reagent and allowed to stain for 10 minutes (Guava Technologies, Hayward, CA). The Guava System differentiates viable from non-viable cells by detecting fluorescence signals from two fluorescent DNA-binding dyes: one membrane-permeable dye stains all nucleated cells while the second dye enters cells when membrane integrity has been compromised, ie, non-viable cells.42Hill RP Wheeler P MacNeil S Haycock JW α-Melanocyte stimulating hormone cytoprotective biology in human dermal fibroblast cells.Peptides. 2005; 26: 1150-1158Crossref PubMed Scopus (27) Google Scholar Viable cells were quantified using a Guava Personal Analyzer (PCA) flow cytometer (Guava Technologies), according to the manufacturer's specifications. Endometrial cells (2 × 104) seeded in 6-well plates at 50% to 70% confluence were treated with 100 nmol/L 17β-estradiol for 24 hours, followed by 100 nmol/L progesterone for an additional 24 hours. To detect apoptosis, the cells were double stained with annexin V and nexin 7-AAD, according to the manufacturer's recommendations (Guava Nexin Method; Guava Technologies). Cell-associated fluorescence was analyzed using a Guava PCA flow cytometer (Guava Technologies). Results were expressed as the percentage of apoptotic-positive cells. Both early apoptotic (annexin V-positive) and late apoptotic (annexin V and 7-AAD-positive) cells were included in the analysis.43Voisin T Firar AE Avondo V Laburthe M Orexin-induced apoptosis: the key role of the seven-transmembrane domain orexin type 2 receptor.Endocrinology. 2006; 147: 4977-4984Crossref PubMed Scopus (47) Google Scholar Endometrial cells (3 × 104) were seeded in 6-well plates and cultured until subconfluence (50% to 70%), at which time the cells were serum-deprived for 24 hours. After treating the cells with 100 nmol/L 17β-estradiol for 24 hours followed by 100 nmol/L progesterone stimulation for 24 hours, 0.5 μg/ml Hoescht 33342 (Sigma-Aldrich) was added to the medium for 30 minutes at 37°C. Attached and non-attached cells were collected and analyzed by UV fluorescence microscopy (×400). Apoptosis was evaluated based on chromatin morphology.44Pollman MJ Yamada T Horiuchi M Gibbons GH Vasoactive substances regulate vascular smooth muscle cell apoptosis: countervailing influences of nitric oxide and angiotensin II.Circ Res. 1996; 79: 748-756Crossref PubMed Scopus (313) Google Scholar Two hundred cells per sample were counted. Results are expressed as the percentage of apoptotic cells in the solution. Endometrial cells (2 × 104) were grown in 24-well plates until 50% to 70% confluence was attained, and then quiescence of the cells was achieved by addition of phenol/serum-free Dulbecco's Modified Eagle's Medium for 24 hours. Cells were treated with 100 nmol/L 17β-estradiol for 24 hours, followed by 100 nmol/L progesterone for 20 hours. After 14 hours of progesterone exposure, cells were labeled with 1 μCi/ml [3H]-methyl-thymidine (Amersham Biosciences, Piscataway, NJ) for 6 hours. At the end of this period, the cells were washed in saline solution and methanol, and precipitated with trichloroacetic acid. Samples were dissolved with NaOH and diluted in scintillation buffer. Radioactivity was measured in a Packard Tri-Carb LS β-counter (PerkinElmer, Wellesley, MA). Changes in the number of cells were determined by flow cytometry using the Guava Viacount kit (Guava Technologies). Briefly, 1.5 × 104 endometrial cells were plated and grown until a confluence of 50% to 70% was attained. Synchronization of the cell cycle was then induced by serum-starva
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