Normal Human Endometrium Research Articles

Endometrial cancer is a receptor-mediated target for Mullerian Inhibiting Substance.

Mullerian Inhibiting Substance (MIS), a 140-kDa homodimer glycoprotein member of the TGF-beta superfamily of biological-response modifiers, causes regression of the Mullerian ducts in developing male embryos. MIS also can induce growth arrest and apoptosis in ovarian and cervical cancer cell lines. The embryonic progenitor of the ovarian and cervical epithelium is the coelomic epithelium, the same tissue that regresses under the direction of MIS in the male. The endometrium and uterus also arise from the coelomic epithelium and the Mullerian ducts. Here, we show that both normal human endometrium and endometrial cancers express the receptor for MIS and that MIS can inhibit the proliferation of a number of human endometrial cancer cell lines that express the MIS type II receptor. In the representative endometrial cancer cell line AN3CA, MIS affects the expression of key cell-cycle regulatory proteins. This work broadens the scope of tumors that MIS can potentially control and, by elucidating the MIS signaling pathway, identifies other potential avenues for intervention.

Coexpression of Fractalkine and Its Receptor in Normal Human Endometrium and in Endometrium from Users of Progestin-Only Contraception Supports a Role for Fractalkine in Leukocyte Recruitment and Endometrial Remodeling

Leukocytes are critical mediators of endometrial remodeling, but the mechanisms by which leukocyte subpopulations enter the uterus are currently unknown. Endometrial leukocytes have no genomic progesterone receptors; thus, we hypothesized that leukocyte migration is induced indirectly by progesterone-regulated chemokines. Fractalkine (CX3CL1), a chemotactic membrane-bound adhesion factor, and its receptor (CX3CR1) were assessed by immunohistochemistry in endometrial samples across the menstrual cycle, in early pregnancy, and in women using progestin-only contraceptives. Fractalkine was localized predominantly to glandular epithelial and decidualized stromal cells, with the highest staining intensity in the secretory phase and early pregnancy. It was also detected in subpopulations of endometrial leukocytes (macrophages and uterine NK cells), with maximal numbers during the proliferative phase and early pregnancy. CX3CR1 was similarly colocalized to the glandular epithelium and decidualized stromal cells, with the highest expression in the secretory phase. CX3CR1-positive leukocytes (macrophages and neutrophils) were in greatest abundance during the menstrual phase. In the endometrium of women using progestin-only contraceptives, immunoreactive fractalkine was markedly reduced in the glandular epithelium, but was increased in decidualized stroma and infiltrating leukocytes. These findings support a number of roles for fractalkine in the endometrium, in the secretory phase, in early pregnancy, and when influenced by progestin-only contraceptives.

Expression of DCC and netrin-1 in normal human endometrium and its implication in endometrial carcinogenesis

Objective. Although DCC has been considered as a candidate tumor suppressor, the roles it plays in the uterine endometrium and in the carcinogenic process remains unclear. To define these roles more clearly, we examined the expression of DCC and its ligand, netrin-1, in the normal endometrium and in endometrial cancer. Methods. The expression of DCC and netrin-1 in normal endometrial glands and in cancer cell lines was examined by RT-PCR and immunohistochemistry. The effects of exogenous DCC and netrin-1 expression were observed together with the respective expression vector transfection. Results. Endometrial glands in the proliferative and early secretory phase expressed both DCC and netrin-1, but glands in the late-secretory phase tended to silence DCC expression. In addition, all of the endometrial cancer cell lines lost normal DCC expression. Restored DCC expression in the cancer cell lines in the absence of netrin-1 induced apoptosis. However, no changes were observed in the presence of netrin-1. Conclusion. Our observations suggest that DCC/netrin-1 signaling may commit cells to the transition of endometrial gland architecture or function from a proliferating to a secretory phase. In addition, the silencing of DCC expression may contribute to the escape of endometrial cancer cells from a DCC-regulated apoptotic program, thereby promoting malignant phenotypes.

Human endometrium mRNA profile assessed by third dimensional oligonucleotide microarray analysis

ObjectiveIn order to further comprehend the genetic events underlying the endometrial steroids response we undertook a 10.000 genes microarray analysis between human normal endometrium in the proliferative and secretory phases of the menstrual cycle.DesignThird dimensional microarray analysis.Materials and methodsHuman normal endometrial samples were obtained at the Gynecology Department, Pérola Byigton Hospital in São Paulo, Brazil. The patients were 80 women who were diagnosed as having leiomyomata in their uterus. Neither patient had received any hormonal treatment during the six months before the diagnosis of leiomyomata, endometrial fragments were divided in proliferative and secretory after histological analysis. Total RNA was extracted from samples by using TRIZOL (Invitrogen) according to manufacture`s instructions and further purified with Qiagen collums. RNA integrity was controlled on denaturing formaldehyde/agarose gel electrophoresis. Prior to reverse transcription, two pools of RNA were obtained by mixing 5 μg of total RNA from each individual case in two separated tubes representing endometrial tissues in proliferative (group 1) and secretory phase (group 2). These two pools were then hybridized two times each to four CodeLink UniSet Human I Bioarrays (Amersham Biosciences, Piscataway, NJ).Results257 genes were found differentially expressed between secretory and proliferative phases when a 5× difference was used as a cut-off criteria. Among them, genes involved in immune response, cell cycle control and calcium metabolism were the most strikingly differentially expressed.ConclusionGenes involved in immune response and calcium metabolism were found differentially expressed specially in the secretory phase. Authors hypothesize that overexpression of calcium related and immune responsive genes might be one of the hallmarks of the second phase of menstrual cycle. ObjectiveIn order to further comprehend the genetic events underlying the endometrial steroids response we undertook a 10.000 genes microarray analysis between human normal endometrium in the proliferative and secretory phases of the menstrual cycle. In order to further comprehend the genetic events underlying the endometrial steroids response we undertook a 10.000 genes microarray analysis between human normal endometrium in the proliferative and secretory phases of the menstrual cycle. DesignThird dimensional microarray analysis. Third dimensional microarray analysis. Materials and methodsHuman normal endometrial samples were obtained at the Gynecology Department, Pérola Byigton Hospital in São Paulo, Brazil. The patients were 80 women who were diagnosed as having leiomyomata in their uterus. Neither patient had received any hormonal treatment during the six months before the diagnosis of leiomyomata, endometrial fragments were divided in proliferative and secretory after histological analysis. Total RNA was extracted from samples by using TRIZOL (Invitrogen) according to manufacture`s instructions and further purified with Qiagen collums. RNA integrity was controlled on denaturing formaldehyde/agarose gel electrophoresis. Prior to reverse transcription, two pools of RNA were obtained by mixing 5 μg of total RNA from each individual case in two separated tubes representing endometrial tissues in proliferative (group 1) and secretory phase (group 2). These two pools were then hybridized two times each to four CodeLink UniSet Human I Bioarrays (Amersham Biosciences, Piscataway, NJ). Human normal endometrial samples were obtained at the Gynecology Department, Pérola Byigton Hospital in São Paulo, Brazil. The patients were 80 women who were diagnosed as having leiomyomata in their uterus. Neither patient had received any hormonal treatment during the six months before the diagnosis of leiomyomata, endometrial fragments were divided in proliferative and secretory after histological analysis. Total RNA was extracted from samples by using TRIZOL (Invitrogen) according to manufacture`s instructions and further purified with Qiagen collums. RNA integrity was controlled on denaturing formaldehyde/agarose gel electrophoresis. Prior to reverse transcription, two pools of RNA were obtained by mixing 5 μg of total RNA from each individual case in two separated tubes representing endometrial tissues in proliferative (group 1) and secretory phase (group 2). These two pools were then hybridized two times each to four CodeLink UniSet Human I Bioarrays (Amersham Biosciences, Piscataway, NJ). Results257 genes were found differentially expressed between secretory and proliferative phases when a 5× difference was used as a cut-off criteria. Among them, genes involved in immune response, cell cycle control and calcium metabolism were the most strikingly differentially expressed. 257 genes were found differentially expressed between secretory and proliferative phases when a 5× difference was used as a cut-off criteria. Among them, genes involved in immune response, cell cycle control and calcium metabolism were the most strikingly differentially expressed. ConclusionGenes involved in immune response and calcium metabolism were found differentially expressed specially in the secretory phase. Authors hypothesize that overexpression of calcium related and immune responsive genes might be one of the hallmarks of the second phase of menstrual cycle. Genes involved in immune response and calcium metabolism were found differentially expressed specially in the secretory phase. Authors hypothesize that overexpression of calcium related and immune responsive genes might be one of the hallmarks of the second phase of menstrual cycle.

Expression and regulation of vascular endothelial growth factor and its receptors flt-1, KDR in normal human endometrium

Expression and regulation of vascular endothelial growth factor and its receptors flt-1, KDR in normal human endometrium

Endometrial cancer expresses the Mullerian Inhibiting Substance (MIS) type II receptor and is growth inhibited by MIS

Introduction: In female embryos, the reproductive organs arise from the Mullerian ducts and associated epithelium; these structures regress under the influence of MIS produced by male embryos. The MIS type II receptor (MISIIR) can mediate MIS-induced growth inhibition of malignant ovarian and cervical cells. We propose that endometrial cancers will express the MISIIR and MIS will inhibit endometrial cancer growth. Methods: Human samples were obtained under IRB approval (2002-P-001324). Rat tissue was collected under institutional protocol 1999-N-00138-Rats. Cells lines were purchased from American Tissue and Cell Culture. Tissue and cell samples were analyzed by Western, PCR, growth inhibition assays, and FACS. Results: Western detected MISIIR protein in adult rat uterus; reverse transcriptase PCR detected MISIIR RNA in normal and malignant human endometrium. AN3CA and KLE, two human endometrial cancer cell lines, expressed MISIIR. MIS inhibited AN3CA and KLE cell growth 67.3% (p < 0.0001, n = 4) and 52.5% (p = 0.0041, n = 3), respectively. MIS-treated AN3CA cells demonstrated G1 arrest, increased Caspase 3 cleavage indicating apoptosis, increased levels of cell cycle proteins p107 and p130 and transcription factor E2F3, and decreased levels of transcription factor E2F1. Conclusions: MISIIR is expressed in normal endometrium and in human endometrial cancer. MIS inhibits the growth of two human endometrial cancer cell lines, induces cell cycle arrest and apoptosis in AN3CA cells, and affects proteins important to the control of cell cycle progression. In the future, treatment targets for MIS may broaden to include advanced endometrial cancer

Expression of the inhibin/activin subunits alpha (alpha), beta-A (betaA) and beta-B (betaB) in benign human endometrial polyps and tamoxifen-associated polyps.

Inhibins (INH) are dimeric glycoproteins, composed of an alpha subunit (INH-alpha) and one of two possible beta subunits (INH-betaA or INH-betaB). They have substantial roles in human reproduction and in endocrine-responsive tumours. Therefore, the aims of this study were to determine the frequency and tissue distribution of INH-alpha, INH-betaA and INH-betaB in normal human endometrium and glandular-cystic endometrial polyps, and polyps caused by tamoxifen use. Tissue samples were obtained from women in the proliferative, early secretory and late secretory phase as well as glandular-cystic polyps and endometrial polyps associated with tamoxifen use (n = 5 each). Immunohistochemistry with specific monoclonal antibodies, a semi-quantitative analysis and statistical evaluation was performed. INH-alpha, INH-betaA and INH-betaB were primarily observed in glandular and luminal epithelial cells, with a variant staining intensity in stromal cells. INH-alpha in glands was significantly higher during the early secretory phase (p < 0.05) and the late secretory phase (p < 0.01) than in the proliferative phase with a significant difference between the early secretory and the late secretory phases (p < 0.01). INH-betaA expression was significantly higher during the late secretory than the proliferative phase (p < 0.05) and the late secretory than the early secretory phase (p < 0.05), with no significant differences for INH-betaB. Glandular-cystic polyps showed significantly lower expression of INH-alpha and INH-betaA than the late secretory endometria (p < 0.05 and p < 0.01 respectively). Additionally, tamoxifen-associated polyps also demonstrated a significantly lower expression of INH-alpha and INH-betaA than late secretory endometria (p < 0.01 and p < 0.01 respectively). No statistical differences were observed between tamoxifen-associated and glandular-cystic polyps. INH-alpha, INH-betaA and INH-betaB were expressed in normal endometrium and endometrial polyps. A cyclical expression of INH-alpha and INH-betaA in normal glands may reflect a functional and hormone-dependent role in human endometrium. Significant differences in staining reaction between the late secretory endometria and polyps suggest that this tissue remains in the proliferating state rather than the secretory state. Therefore, endometrial polyps may be tumours of dysregulation with mainly proliferating characteristics, being unable to synchronise with normal endometrium.

Evaluation of mRNA expression of estrogen receptor beta and its isoforms in human normal and neoplastic endometrium.

Endometrial cancer is well known to be estrogen-dependent. Two estrogen receptor types, ERalpha and ERbeta, are major mediators of a diversity of biologic functions of estrogen and play an important role in estrogen-dependent tissues and cancers. Cloning of ERbeta was followed by the discovery of a variety of its isoforms. Using real-time RT-PCR, the relative expression levels of ERbeta1, ERbeta2 (ERbetacx), ERbeta3, ERbeta4 and ERbeta5 were studied. We observed coexpression of ERbeta isoforms in the endometrium and upregulation of the ERbeta5 transcript in malignant endometrium. We also observed downregulation of ERbeta2Delta5 transcript in neoplastic endometrium, using a semiquantitative method. Our results suggest that analyzing the changes in ERbeta and its isoforms may be important in the diagnosis, prognosis and treatment of endometrial cancer.

Immunohistochemical analysis of estrogen receptor alpha, estrogen receptor beta and progesterone receptor in normal human endometrium

The endometrium expresses estrogen (ER) and progesterone receptors (PR), which are involved in autocrine and paracrine regulation processes in response to estrogen and progesterone. The aim of the present study was to evaluate immunohistochemical distribution patterns of estrogen receptor alpha (ER α), estrogen receptor beta (ER β) and PR in normal human endometrial tissue with the use of monoclonal antibodies. Human endometria were obtained from 17 premenopausal patients undergoing surgery for non-malignant diseases and were classified to be in proliferative, early secretory and late secretory phases by histological and anamnestical means. Distribution patterns of the steroid receptors were evaluated using the IRS-score and the Mann–Whitney rank-sum test was used to compare the means. Correlation was assessed with the Spearman factor and linear regression analysis. ER α and PR expression decreased significantly ( p<0.05) in glandular epithelium from the proliferative to the late secretory phase. ER β expression showed a similar significant decrease ( p<0.05), although staining intensity was lower than that of ER α. A significant correlation between expression of all three steroid receptors was observed ( p<0.001). Distribution patterns of ER α, ER β and PR in normal human endometrium showed a cyclic variation during the menstrual cycle. A significant correlation between expression of ER α, ER β and PR was also demonstrated using regression analysis, indicating dependence of expression of these three steroid receptors. The present study shows the presence of steroid receptors in human endometrial epithelium, indicating that these cells respond to estrogen and progesterone and thus playing a significant role in endometrial physiology.

H19 and IGF2 gene expression in human normal, hyperplastic, and malignant endometrium.

We examined H19 and insulin-like growth factor 2 (IGF2) gene expression in normal endometrium (12 cases), hyperplasia (27 cases), and cancer (27 cases) by non-radioactive in situ hybridization. H19 was not expressed in the epithelium of normal endometrium, but its frequency of expression was 15% in hyperplastic and 60% in neoplastic epithelium. In stroma cells, H19 frequency of expression was 75% in normal endometrium, 55% in hyperplasia, and 37% in carcinoma. According to the grade of endometrial cancer cell differentiation, H19 showed increased frequency and level of expression in the epithelium from well to moderately and poorly differentiated tissues. Our results indicate that H19 expression in epithelial cells of endometrial hyperplasia and cancer merits further investigation and could be useful as a complementary histopathologic and prognostic marker among other modalities in endometrial cancer. IGF2 expression did not appear useful for diagnostic or prognostic purposes.

H19 and IGF2 gene expression in human normal, hyperplastic, and malignant endometrium

We examined H19 and insulin-like growth factor 2 (IGF2) gene expression in normal endometrium (12 cases), hyperplasia (27 cases), and cancer (27 cases) by non-radioactivein situhybridization. H19 was not expressed in the epithelium of normal endometrium, but its frequency of expression was 15% in hyperplastic and 60% in neoplastic epithelium. In stroma cells, H19 frequency of expression was 75% in normal endometrium, 55% in hyperplasia, and 37% in carcinoma. According to the grade of endometrial cancer cell differentiation, H19 showed increased frequency and level of expression in the epithelium from well to moderately and poorly differentiated tissues. Our results indicate that H19 expression in epithelial cells of endometrial hyperplasia and cancer merits further investigation and could be useful as a complementary histopathologic and prognostic marker among other modalities in endometrial cancer. IGF2 expression did not appear useful for diagnostic or prognostic purposes.

Immunohistochemical analysis of estrogen receptor alpha, estrogen receptor beta and progesterone receptor in normal human endometrium

The endometrium expresses estrogen (ER) and progesterone receptors (PR), which are involved in autocrine and paracrine regulation processes in response to estrogen and progesterone. The aim of the present study was to evaluate immunohistochemical distribution patterns of estrogen receptor alpha (ERα), estrogen receptor beta (ERβ) and PR in normal human endometrial tissue with the use of monoclonal antibodies. Human endometria were obtained from 17 premenopausal patients undergoing surgery for non-malignant diseases and were classified to be in proliferative, early secretory and late secretory phases by histological and anamnestical means. Distribution patterns of the steroid receptors were evaluated using the IRS-score and the Mann–Whitney rank-sum test was used to compare the means. Correlation was assessed with the Spearman factor and linear regression analysis. ERα and PR expression decreased significantly (p<0.05) in glandular epithelium from the proliferative to the late secretory phase. ERβ expression showed a similar significant decrease (p<0.05), although staining intensity was lower than that of ERα. A significant correlation between expression of all three steroid receptors was observed (p<0.001). Distribution patterns of ERα, ERβ and PR in normal human endometrium showed a cyclic variation during the menstrual cycle. A significant correlation between expression of ERα, ERβ and PR was also demonstrated using regression analysis, indicating dependence of expression of these three steroid receptors. The present study shows the presence of steroid receptors in human endometrial epithelium, indicating that these cells respond to estrogen and progesterone and thus playing a significant role in endometrial physiology.

In vivo and in vitro regulation of Akt activation in human endometrial cells is estrogen dependent.

Estrogen-bound estrogen receptors (ER) alpha and beta classically activate gene expression after binding to the estrogen response element in the promoter regions of target genes. Estrogen also has rapid, nongenomic effects. It activates several membranous or cytoplasmic kinase cascades, including the phosphatidylinositol 3-phosphate (PI3K/Akt) cascade, a signaling pathway that plays a key role in cell survival and apoptosis. Normal human endometrium is exposed to variable levels of steroid hormones throughout the menstrual cycle. We hypothesized that Akt phosphorylation in human endometrium may vary with the menstrual cycle and in early pregnancy and that fluctuations in estrogen level may play a role in Akt activation in endometrial cells. We analyzed Akt phosphorylation using in vivo and in vitro techniques, including Western blot, immunohistochemistry, and immunocytochemistry. Estradiol significantly increased Akt phosphorylation in endometrial cells. Rapid stimulation of Akt activation in cultured stromal cells was observed. Akt phosphorylation by estradiol was inhibited by the PI3K inhibitor, wortmannin, but not by the ER antagonist, ICI 182 780. The maximal effect on Akt activity was observed following 5-15 min of estradiol treatment. Our results suggest that estradiol may directly affect PI3K-related signaling pathway by increasing the phosphorylation of Akt in endometrial cells. Thus, estradiol may exert part of its proliferative and antiapoptotic effects by a nongenomic manner through the Akt signaling pathway.

Reduced expression of death-associated protein kinase in human uterine and ovarian carcinoma cells

The expression of the death-associated protein kinase (DAPK) protein and promoter methylation in 11 human uterine and ovarian carcinoma cell lines originally established from histopathologically-different carcinoma tissues were examined to investigate the relationship between DAPK and carcinogenesis in female reproductive tissues. The 11 cell lines included three cervical carcinomas, three endometrial carcinomas and five ovarian carcinomas. Western blot analysis showed no detectable expression of DAPK protein in 4 cell lines (ME180, HOKUG, MCAS and OVK-18) while moderate levels of DAPK protein were readily detected in normal human endometrium, and normal murine uterus and ovary. Methylation-specific PCR of the 11 cell lines revealed that 5 carcinoma cell lines (ME180, HOKUG, MCAS, OVK-18 and HEC-1) expressed hypermethylated promoters in the DAPK genes, while DAPK promoters in the other 6 carcinoma cell lines remained unmethylated. These results indicate that DAPK protein expression is reduced or silenced in some human uterine and ovarian carcinoma cells by methylation of the DAPK gene promoter region. Therefore, reduced DAPK expression and methylation of the DAPK promoter may be involved in carcinogenesis of human uterine and ovarian tissues.

Identification of c-Jun as bcl-2 transcription factor in human uterine endometrium.

We describe the application of the biomolecular interaction (BIA) technique to detection of the interaction between protein (e.g., c-Jun) and DNA (e.g., two AP-1 motifs from bcl-2 promoter), compared with immunohistochemistry (IHC) of c-Jun. The specific binding assay for the interaction of c-Jun and activating protein-1 (AP-1) motifs was performed using a Biacore 2000 system. Intense immunoreactivity of c-Jun in glandular cells of the human uterine endometrium was observed in the proliferative phase, while c-Jun in stromal cells was expressed throughout the menstrual cycle. In contrast to the IHC of c-Jun, the specific binding of c-Jun to two separate AP-1 motifs in the bcl-2 promoter region was detected only in nuclear extracts of glandular cells, but not in stromal cells, during the proliferative phase. These results indicate that, while transmitting various signals, c-Jun enhances the transcription level of bcl-2, which in turn keeps glandular cells alive and proliferating in normal human endometrium during the proliferative phase. Moreover, the method involving real-time biomolecular interactions such as DNA-protein binding is novel for the study of transcription factors when combined with IHC.

Expression of estrogen receptor coregulators in normal and malignant human endometrium

Objectives. To compare the expression of nuclear receptor coregulators in normal and malignant human endometrium and to identify any relationship to grade, stage, age, depth of myometrial invasion, estrogen receptor α (ERα), or progesterone receptor (PR) expression. Methods. Gene expression of SRC-1, SRC-2, SRC-3, N-CoR, SMRT, ERα, and PR was measured in 26 samples of normal endometrium and 30 primary endometrial carcinomas using real-time RT-PCR. ERα protein expression of each tissue was also measured by Western blot. Results. All coregulators showed significantly increased mRNA expression in endometrial carcinoma as compared to normal endometrium. The mRNA expression of each coregulator showed a high correlation with ERα mRNA, PR mRNA, and with the other coregulators in both normal and malignant endometrium. In the normal endometrium, SRC-1 mRNA expression was positively correlated with ERα protein expression and SRC-3 mRNA expression was positively correlated with patient age. No relationship was found between coregulator mRNA expression and grade, stage, or depth of myometrial invasion. Conclusion. The nuclear receptor coregulators SRC-1, SRC-2, SRC-3, N-CoR, and SMRT were found to be up-regulated in malignant endometrium. Our findings suggest that these proteins may have a role in the development of endometrial carcinoma.

Immunohistochemical expression of inhibin-alpha in human endometrium and the in vitro secretion of inhibin, estradiol and cortisol in cultured human endometrial glandular cells.

Inhibins are multipotent dimeric glycoproteins, composed of an alpha-subunit and one of two possible beta-subunits (betaA or betaB). Aims of this study were (a): the immunohistochemical characterisation of normal human endometrium for the inhibin-alpha subunit; (b) the assessment of the secretion and metabolism of inhibin, E2 and cortisol; (c) the evaluation of any relationship between these three substances in cell culture medium of isolated and cultivated normal human endometrial glandular cells. Samples of human endometrium were obtained from 34 premenopausal patients. Nineteen endometrial specimen (proliferative [PP] n=8; early secretory [ES] n=7; late secretory phase [LS] n=4) were brought into cell culture. Fifteen endometrial specimen (PP n=5; ES n=5; LS n=5) were paraffin-fixed and used for the immunohistochemical analysis for inhibin-alpha. Stromal and epithelial cells were separated by collagenase digestions, filtrations, sedimentations and Ficoll-gradient centrifugation. E2 and cortisol were measured with radioimmunoassay (RIA) and inhibin with enzyme-immunoassay (EIA). Statistical analysis was performed with the non-parametric Mann-Whitney rank-sum test and linear regression analysis. Inhibin-alpha showed a weak (positive) expression during proliferative phase, which increased significantly as the menstrual cycle continued. In secretory glands the mean inhibin concentration was higher than that from proliferative samples. A significant correlation was observed between inhibin and E2 (p<0.001) as well as cortisol and inhibin (p<0.0001) in glands from proliferative phase. Between inhibin and E2 (p<0.05) as well as inhibin and cortisol (p<0.002) a significant correlation in early secretory glands was also noted. In late secretory phase inhibin and E2 (r2=0.78650; p<0.0001), inhibin and cortisol (r2=0.58326; p<0.001) and E2 and cortisol (r2=0.52880; p<0.001) showed a significant correlation. In conclusion, we found a cyclical expression of inhibin-alpha subunit in the endometrium demonstrated by immunohistochemical means. A higher in vitro secretion of inhibin from secretory glands was also observed. In addition, a significant correlation between inhibin with E2 and cortisol in PP and ES glands and a significant correlation between inhibin, E2 and cortisol in LS glands could also be demonstrated. We conclude that inhibin can be associated with E2 and cortisol metabolism, playing an important role in paracrine/autocrine mechanisms in the endometrium and possibly exerting its function through cortisol and E2. The cortisol concentration also correlates with E2, suggesting a link between these steroids in the endometrial function. The correlation of inhibin, E2 and cortisol suggest complex autocrine/ paracrine mechanisms in human endometrial glands, modulated and controlled by all these three substances.

Cyclic changes in the expression of steroid receptor coactivators and corepressors in the normal human endometrium.

To examine the sex steroid-dependent growth mechanisms of the human endometrium, the expression of steroid receptor coactivators [steroid receptor coactivator-1 (SRC-1) and p300/CREB-binding protein (p300/CBP)] and corepressors (nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptors) was examined by immunohistochemistry, using 50 samples of normal endometria, and was compared with that of estrogen receptors (ER), progesterone receptors (PR), and proliferation marker Ki-67. In addition, actual binding of the coactivators to ER or PR was analyzed by immunoprecipitation. The expression of SRC-1 was diffusely observed in glandular and stromal cells in the proliferative phase and drastically decreased in the secretory phase. Such change in the expression pattern of SRC-1 resembled that of ER, PR, and Ki-67. On the other hand, p300/CBP expression was relatively constant throughout the menstrual cycle, with slight predominance in the proliferative phase. The expression of corepressors nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptors was focal in the endometrium. Immunoprecipitation, using tissue samples of both proliferative and secretory phases, revealed the complex formation between the coactivators and receptors. Binding of SRC-1 to ER was observed in the proliferative (but not in the secretory) endometrium. In contrast, binding p300/CBP to ER was noted in the endometria of both phases. Complex formation between p300/CBP and PR was noted in the secretory endometrium, whereas that between SRC-1 and PR was not apparent. Accordingly, we showed the expression pattern of steroid receptor coactivators and corepressors in the normal endometrium. Cyclic change in the expression of SRC-1 during the menstrual cycle might be important in the estrogen-action for the glandular and stromal cells.

Activation of the receptor protein tyrosine kinase EphB4 in endometrial hyperplasia and endometrial carcinoma

Members of the Eph family of tyrosine kinases have been implicated in embryonic pattern formation and vascular development; however, little is known about their role in the adult organism. We have observed estrogen-dependent EphB4 expression in the normal breast suggesting its implication in the hormone-controlled homeostasis of this organ. Since the endometrium is a similarly hormone dependent organ and endometrial carcinoma is thought to result from estrogenic stimulation, we have investigated EphB4 expression in normal human endometrium and during its carcinogenesis. EphB4 expression was analyzed immunohistochemically in 26 normal endometrium specimens, 15 hyperplasias and 102 endometrioid adenocarcinomas and correlated with clinical and prognostic tumor characteristics. In normal endometrial tissue no EphB4 protein was detected. Strikingly, we observed a drastic increase (P <0.0001) in the number of EphB4 protein-expressing glandular epithelial cells in the majority of hyperplasias and carcinomas. Moreover, we found a statistically highly significant positive correlation between EphB4 expression and post-menopausal stage of the patient (P = 0.007). These findings indicate that in the endometrium, EphB4 is an early indicator of malignant development and, thus, EphB4 may represent a potent tool for diagnosis and therapeutic intervention.

Subcellular Localization of Galactosyltransferase Associated with Tumors in Endometrial and Ovarian Cancer Cells.

Frozen or resin sections of normal human endometrium, endometrial adenocarcinoma, and ovarian adenocarcinoma were examined with two monoclonal antibodies for galactosyltransferase associated with tumor (GAT) by immunofluorescence and immunoelectron microscopy. One antibody (MAb8628) stained the trans-cisternae of the Golgi apparatus, the trans-Golgi network, and the intracytoplasmic vesicles more intensely in cancer cells than in normal endometrial glandular cells. The other antibody (MAb8513) stained intracytoplasmic vesicles more intensely in cancer cells than in normal cells. MAb8513 also showed moderate staining of the trans-cisternae of the Golgi apparatus and trans-Golgi network in cancer cells, but only faint staining of these structures in normal glandular cells, indicating that GAT was overexpressed in the cancer cells. An immunofluorescence study using serial semithin cryosections (1 μm) demonstrated that the staining pattern of each antibody was different inside a single cancer cell, conforming to the two patterns mentioned above. The reason for this difference in staining remains unclear. GAT is a soluble form of β1,4-galactosyltransferase, so a difference in the cleavage site of the membrane-bound peptide may cause changes in immunoreactivity after its conversion to the soluble form (i.e., GAT) in endometrial or ovarian adenocarcinoma cells.