RESUMEN
The possible adverse effects of the so-called environmental estrogens have raised considerable concern. Developmental, endocrine and reproductive disorders in wildlife animals have been linked to high exposure to persistent environmental chemicals with estrogen-like activity (xenoestrogens); yet, the potential impact of environmental estrogens on human health is currently under debate also due to lack of data. A battery of in vitro assays exist for identifying compounds with estrogenic activity, but only a few models are available to assess estrogenic potency in a multiparametric analysis. We have recently established the endometrial adenocarcinoma cell line RUCA-I; it enables us to compare estrogenic effects both in vitro and in vivo as these cells are estrogen responsive in vitro and grow estrogen sensitive tumors if inoculated in syngeneic animals in vivo. Here we report in vitro data concerning (a) the relative binding affinity of the selected synthetic chemicals Bisphenol A, nonylphenol, p-tert-octylphenol, and o,p-DDT to the estrogen receptor of RUCA-I cells and (b) the relative potency of these compounds in inducing increased production of complement C3, an endogenous estrogen-responsive gene. Competitive Scatchard analysis revealed that xenoestrogens bound with an at least 1000-fold lower affinity to the estrogen receptor of RUCA-I cells than estradiol itself, thereby exhibiting the following affinity ranking, estradiol>>>nonylphenol>bisphenol A approximately p-tert-octylphenol>o,p-DDT. Despite these low binding affinities, bisphenol A, nonylphenol and p-tert-octylphenol increased production of complement C3 in a dose dependent manner. Compared with estradiol, only 100-fold higher concentrations were needed for all the compounds to achieve similar levels of induction, except o,p-DDT which was by far less potent. Northern blot analyses demonstrated that the increased production of complement C3 was mediated by an increased transcription. In summary, cultured RUCA-I cells represent a valuable endometrial derived model system to assess the relative potencies and the molecular mode of action of environmental estrogens in vitro. Our results further show that no intimate correlation exists between the relative binding affinity and the biological response of these compounds. Therefore, data obtained from single-parametric analyses may result in misleading conclusions. On the other hand, the presented in vitro data will provide us with tools to study the activity of xenoestrogens in vivo and thus carry risk assessment one step further.
Asunto(s)
Endometrio/efectos de los fármacos , Congéneres del Estradiol/metabolismo , Congéneres del Estradiol/farmacología , Receptores de Estrógenos/metabolismo , Xenobióticos/metabolismo , Xenobióticos/farmacología , Adenocarcinoma/metabolismo , Animales , Sitios de Unión , Complemento C3/biosíntesis , Complemento C3/genética , Relación Dosis-Respuesta a Droga , Endometrio/metabolismo , Endometrio/patología , Contaminantes Ambientales , Estradiol/metabolismo , Estradiol/farmacología , Congéneres del Estradiol/antagonistas & inhibidores , Congéneres del Estradiol/química , Moduladores de los Receptores de Estrógeno/farmacología , Femenino , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de Estrógenos/antagonistas & inhibidores , Medición de Riesgo , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Células Tumorales Cultivadas , Xenobióticos/antagonistas & inhibidores , Xenobióticos/químicaRESUMEN
Serum neuron-specific enolase (s-NSE) and s-100 protein (s-100) are sensitive markers of various brain diseases. We investigated both of these markers in nine patients within 5 min, 6 h, 12 h, and 48 h after a single tonic-clonic seizure. The mean peak s-NSE level was significantly higher after 5 min (11.97 +/- 8.56 microg/l) and 48 h (10.31 +/- 8.92 microg/l, P < 0.05) than the levels of seizure-free, age-matched controls. Five patients had increased s-NSE levels regarding the upper limit of normal as mean + 3 SD. s-100 was not detected either in controls or epileptic patients. These data indicate that s-NSE in contrast to s-100 may be an in vivo marker after generalized seizures in some patients.
Asunto(s)
Epilepsia Tónico-Clónica/sangre , Fosfopiruvato Hidratasa/sangre , Proteínas S100/sangre , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Epilepsia Tónico-Clónica/diagnóstico , Epilepsia Tónico-Clónica/enzimología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Severe developmental and reproductive disorders in wild animals have been linked to high exposure to persistent environmental chemicals with hormonal activity. These adverse effects of environmental estrogens have raised considerable concern and have received increasing attention. Although numerous chemicals with the capacity to interfere with the estrogen receptor (ER) have been identified, information on their molecular mechanism of action and their relative potency is rather limited. For the endometrium, the lack of information is due to the lack of a suitable experimental model. We investigated the functions of phytoestrogens in an endometrial-derived model, RUCA-I rat endometrial adenocarcinoma cells. The cells were cultured on a reconstituted basement membrane to preserve their functional differentiation and estrogen responsiveness. We assessed the relative binding affinity to the estrogen receptor of the selected phytoestrogens coumestrol, genistein, daidzein, and the putative phytoestrogen mangostin compared to estradiol by a competitive Scatchard analysis. The following affinity ranking was measured: 17beta-estradiol >>> coumestrol > genistein > daidzein >>> mangostin. In addition, we investigated the capacity of these compounds to promote the increased production of complement C3, a well-known estradiol-regulated protein of the rat endometrium. All substances tested increased the production of complement C3, although different concentrations were necessary to achieve equivalent levels of induction compared to estradiol. Mechanistically we were able to demonstrate that the increase of complement C3 production was mediated by primarily increasing its steady-state mRNA level. These findings indicate that RUCA-I cells represent a sensitive model system to elucidate relative potencies and functions of environmental estrogens in an endometrium-derived model.
Asunto(s)
Endometrio/metabolismo , Estrógenos no Esteroides/metabolismo , Isoflavonas , Receptores de Estrógenos/metabolismo , Adenocarcinoma , Animales , Complemento C3c/genética , Complemento C3c/metabolismo , Cumestrol/metabolismo , Cumestrol/farmacología , Neoplasias Endometriales , Endometrio/efectos de los fármacos , Estradiol/metabolismo , Estradiol/farmacología , Estrógenos no Esteroides/farmacología , Femenino , Genisteína/metabolismo , Genisteína/farmacología , Fitoestrógenos , Preparaciones de Plantas , Plantas , ARN Mensajero/análisis , Ratas , Receptores de Estrógenos/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Clusterin is a heterodimeric, 80kDa, glycoprotein that is synthesized in a wide variety of tissues in response to a number of diverse stimuli, including hormone ablation. We have investigated the regulation of clusterin expression by estradiol and anti-estrogens in RUCA-I rat endometrial adenocarcinoma cells in vitro and in vivo. We have also compared clusterin expression in endometrial tumors and in normal uterine tissue. Estradiol treatment significantly increases the steady state mRNA levels of clusterin in RUCA-I cells cultured on a reconstituted basement membrane, with a maximal induction 24 hr after estradiol treatment. The inductive effects of estrogen on clusterin mRNA steady state levels in vitro are significantly more pronounced than the effects on fibronectin mRNA levels, an estrogen-repressed gene in RUCA-I. In vivo, induction of clusterin expression in primary and metastatic endometrial adenocarcinoma is also dependent on the presence of estradiol, in marked contrast to expression of clusterin in the normal endometrium of the same animals. These data suggest that clusterin mRNA expression in rat endometrial adenocarcinoma cells is tightly regulated by estrogens and anti-estrogens in vitro and in vivo, and that there is a complex mechanism of regulation of clusterin expression in the normal and cancerous endometrium.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Endometriales/metabolismo , Endometrio/metabolismo , Estrógenos/fisiología , Glicoproteínas/biosíntesis , Chaperonas Moleculares , ARN Mensajero/metabolismo , Animales , Clusterina , Endometrio/efectos de los fármacos , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Femenino , Ratas , Ratas Endogámicas , Sensibilidad y Especificidad , Tamoxifeno/farmacología , Células Tumorales CultivadasRESUMEN
Estrogens are believed to play a crucial role in growth regulation and differentiation of the normal endometrial tissue as well as in the carcinogenesis of the endometrium. Therefore, the influence of estrogens and antiestrogens on gene expression in the estrogen receptor-positive rat endometrial adenocarcinoma cell line RUCA-I was investigated. Differentially expressed genes were detected by differential display PCR of RNA of untreated, estradiol-treated and antiestrogen-treated RUCA-I cells. By means of the PCR technique, 14 differentially expressed fragments could be detected. Three of these 14 differentially expressed fragments were confirmed by Northern blotting. The steady state mRNA levels of the three gene fragments named AH41, AH42 and AH44 were downregulated by the antiestrogen ICI 164384. Further characterization revealed that the fragment AH41 is not expressed in stromal cells but in the human and rodent epithelial cell lines, BG-1 and RUCA-II. A comparison of the cDNA sequence of fragment AH41 with the EMBL database showed no high homology to known genes. Therefore, fragment AH41 has to be regarded as a fragment of a novel, estradiol-sensitive gene.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Receptores de Estrógenos/metabolismo , Adenocarcinoma/tratamiento farmacológico , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN de Neoplasias/genética , Neoplasias Endometriales/tratamiento farmacológico , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Reacción en Cadena de la Polimerasa , Ratas , Células Tumorales CultivadasRESUMEN
Localization of tenascin-C in vivo and cell culture experiments in vitro have provided evidence for stromal production of tenascin-C in malignant tumors of a variety of organs. Here we raised the question of whether the mesenchymal stroma in the case of endometrial adenocarcinoma is the unique source of tenascin-C. Therefore, the expression of tenascin-C mRNA by human endometrial adenocarcinoma cells and endometrial stroma cells was investigated. Several preparations of endometrial stroma cells produced tenascin-C mRNA. Using a serum-free defined cell culture medium, production of tenascin-C mRNA could be increased by adding either serum or 20 ng TGF-beta/mL to the cell culture medium. Reverse transcriptase polymerase chain reaction analysis revealed that five out of six endometrial adenocarcinoma cell lines produced tenascin-C mRNA. Northern blot experiments and ribonuclease protection assays provided evidence that the number of copies of tenascin-C mRNA was small. Analysis of expressed splice variants by reverse transcriptase polymerase chain reaction analysis revealed the abundance of one major splice variant that lacked all potential alternatively spliced fibronectin type-III-like repeats. Regarding larger splice variants, all fragment sizes that could theoretically originate from seven alternatively spliced fibronectin type-III-like repeats were observed. Evaluating relative signal intensities, the splice variants containing a single fibronectin type-III-like repeat and the variant possessing all but one alternatively spliced repeats were most frequent. In summary, evidence is provided that tenascin-C can originate from both tissue compartments of the human endometrium stroma and (tumor) epithelium. Splice variant analysis revealed a high number of splice variants and a relative high proportion of variants that have so far been regarded as minor constituents of expressed tenascin-C.
Asunto(s)
Adenocarcinoma/metabolismo , Empalme Alternativo/efectos de los fármacos , Neoplasias Endometriales/metabolismo , Endometrio/citología , Tenascina/biosíntesis , Tenascina/genética , Factor de Crecimiento Transformador beta/farmacología , Adenocarcinoma/genética , Animales , Northern Blotting , Bovinos , Cartilla de ADN , Neoplasias Endometriales/genética , Endometrio/metabolismo , Femenino , Sangre Fetal/fisiología , Humanos , Reacción en Cadena de la Polimerasa , Ribonucleasas , Células del Estroma/metabolismo , Tenascina/efectos de los fármacosRESUMEN
We recently established and characterized two rat endometrial adenocarcinoma cell lines which we called RUCA-I and RUCA-II. Despite high estrogen receptor levels neither cell line responded to estradiol in conventional cell culture conditions on plastic and in the presence of charcoal stripped fetal calf serum. We further demonstrated that culturing of these cells on a reconstituted basement membrane induced the estrogen responsiveness for both proliferation and gene expression. Particularly, the expression of components of the complement C3 system, which represent major estradiol inducible proteins in the rat uterus in vivo, were found to be under the control of estrogens and antiestrogens. In this paper the search for estrogen repressed proteins is reported. For this purpose secretory proteins of RUCA-I cells were metabolically labelled with 35S-methionine and tested for the presence of estrogen-repressed, antiestrogen-inducible protein species. Analyzing cell culture supernatants of RUCA-I cells by polyacrylamide gel electrophoresis under reducing conditions a protein with an apparent size of approx. 250-270 kDa became conspicuous. The formation and secretion of this protein was suppressed by estradiol and induced by the antiestrogen ICI 164384. Gel electrophoresis performed under non-reducing conditions and hyaluronidase digestion showed that this estrogen-repressed protein represents a dimeric glycoprotein. By immunoprecipitation this glycoprotein was identified as fibronectin. Investigations of steady state mRNA levels of fibronectin by rtPCR suggested a post-transcriptional regulation of this molecule by estradiol. This is the first report on repression of components of the extracellular matrix by estradiol and induction by the complete antiestrogen ICI 164384. The consequences of this finding in regard to growth and invasion of endometrial tumors are discussed.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias Endometriales/metabolismo , Estradiol/farmacología , Fibronectinas/biosíntesis , Animales , Secuencia de Bases , Estradiol/metabolismo , Femenino , Fibronectinas/antagonistas & inhibidores , Datos de Secuencia Molecular , Neoplasias Experimentales/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Células Tumorales CultivadasRESUMEN
We recently described the establishment and the characterization of two rat endometrial adenocarcinoma cell lines which we called RUCA-I and RUCA-II. Despite fairly high estrogen receptor levels neither cell line responded to estradiol in conventional cell culture conditions on plastic and in the presence of serum. A limited hormonal response to the antiestrogen tamoxifen was detectable in RUCA-I but not in RUCA-II cells. To advance our cell culture conditions we plated RUCA-I cells on a layer of reconstituted basement membrane (Harbor Matrix) in the presence of a serum-free defined medium. These cell culture conditions induced hormone responsiveness of RUCA-I cells and permitted a stimulation of proliferation by estradiol. Further, two estradiol-induced secretory proteins with an apparent molecular weight of 115 kD and 60 kD could be identified by SDS-gelelectrophoresis if analyzed under reducing conditions. These proteins migrated as a single band in a non-reducing electrophoresis gel and were identified as components of the complement C3 system. Additionally, our results suggest that the effects of extracellular matrix and hormones on the expression of these proteins are additive. We conclude that processes of functional differentiation are most likely to occur in this in vitro model, particularly since the expression of components of the complement C3 system was under estrogenic control. Complement C3 proteins represent major estradiol-inducible secretory protein of the immature rat uterus in vivo. Culturing RUCA-I cells on top of a layer of reconstituted basement membrane provides a novel tool to study the importance of the extracellular environment on the hormone-induced gene expression in endometrial carcinogenesis in vitro.
Asunto(s)
Complemento C3/biosíntesis , Endometrio/metabolismo , Estradiol/farmacología , Matriz Extracelular/fisiología , Adenocarcinoma , Animales , Secuencia de Bases , Membrana Basal/fisiología , Diferenciación Celular/efectos de los fármacos , Complemento C3/química , Endometrio/citología , Endometrio/fisiología , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Femenino , Expresión Génica , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/metabolismo , Alcamidas Poliinsaturadas , ARN Mensajero/biosíntesis , Ratas , Receptores de Estrógenos/genética , Receptores de Progesterona/genética , Células Tumorales CultivadasRESUMEN
The catechol estrogens (CE), 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) were analyzed for their binding affinity to the estrogen receptor of MCF-7 cells. Applying a competitive binding assay to cytosols prepared from MCF-7 breast cancer cells, we measured a relative binding affinity of 23% (2-OH-E2) and 26% (4-OH-E2) compared to E2. Nuclear binding assays with the same cell line demonstrated a high specific binding with Kd's of 0.31 nM (2-OH-E2) and 0.21 nM (4-OH-E2). The relative binding affinity measured was 25% and 42% for 2-OH-E2 and 4-OH-E2, respectively. Based on this nuclear binding it can be concluded that the estrogen receptor occupied by CE is bound within the nucleus and might therefore be transcriptionally active.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Estradiol/análogos & derivados , Receptores de Estrógenos/metabolismo , Unión Competitiva , Neoplasias de la Mama/ultraestructura , Núcleo Celular/química , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Citosol/química , Citosol/metabolismo , Citosol/ultraestructura , Estradiol/análisis , Estradiol/aislamiento & purificación , Estradiol/metabolismo , Estrógenos de Catecol/análisis , Estrógenos de Catecol/aislamiento & purificación , Estrógenos de Catecol/metabolismo , Humanos , Prolactina/metabolismo , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Receptores de Progesterona/metabolismo , Células Tumorales CultivadasRESUMEN
The binding affinity and relative estrogenic potency of 2-bromo-, 4-bromo-, 2-methyl- and 4-methylestradiol was evaluated in MCF-7 breast cancer cells. The relative binding affinities compared to estradiol were 47% for 2-methyl-, 25% for 4-methyl-, 37% for 4-bromo- and 17% for 2-bromoestradiol. However, both 2- and 4-methyl- as well as 2- and 4-bromoestradiol were able (a) to translocate the cytosolic estrogen receptor into the nucleus and (b) to induce the progesterone receptor in a concentration dependent manner. Finally, all ring-A substituted estrogens used in this study induced the pS2 mRNA as demonstrated by Northern-blotting. From these findings we conclude that 2-bromo-, 4-bromo-, 2-methyl- and 4-methylestradiol are agonistic ligands for the estrogen receptor in MCF-7 breast cancer cells.
Asunto(s)
Estradiol/análogos & derivados , Receptores de Estrógenos/metabolismo , Northern Blotting , Neoplasias de la Mama , Regulación hacia Abajo , Estradiol/metabolismo , Humanos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores de Progesterona/metabolismo , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
An improved radioreceptor assay of unfixed cryostat sections of human target tissues has been developed. Sections collected on glass coverslips were immediately incubated with 5 nM concentrations of either tritiated estradiol-17 beta for estrogen receptor (ER) or ORG 2058 for progesterone receptor (PR) determination. For quantitation, receptor-bound and free hormone were separated by isoelectric focusing (IEF). The assay allows the determination of steroid hormone receptors and comparative histological examinations in immediately neighbouring serial sections of a single piece of tissue. Biochemically, the validity of the assay procedure was evidenced by Scatchard analysis, by ligand and tissue specificities, by the linear relations of receptor and protein concentrations and the number of sections per test tube. Diagnostically, we compared the routine (6 point DCC-Scatchard) procedure for breast cancer analysis with the section method. A good correlation for ER and a less pronounced correlation for PR was found. Statistically, the precision of the method was verified by low deviations of duplicate determinations, low day-to-day variations and low inter-assay variations.
Asunto(s)
Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Neoplasias de la Mama/análisis , Endometrio/análisis , Estradiol/metabolismo , Femenino , Humanos , Focalización Isoeléctrica , Cinética , Neoplasias Ováricas/análisis , Pregnenodionas/metabolismo , Especificidad por SustratoRESUMEN
The activity of the oestradiol-17 beta hydroxysteroid dehydrogenase in human endometrial and breast cancer specimens was determined by the NAD-dependent conversion of oestradiol-17 beta to oestrone. The sensitivity of the determination was improved by the separation of the hormones by HPLC. We are now able to determine oestradiol-17 beta hydroxysteroid dehydrogenase quantitatively in cryostat sections. A clear correlation of serum progesterone levels and oestradiol-17 beta hydroxysteroid dehydrogenase activity in the endometrium was demonstrated, and we found a more than 30-fold increase in enzyme activity after the progesterone surge. In contrast, in breast cancer samples, we found no correlation between oestradiol-17 beta hydroxysteroid dehydrogenase and the measured serum parameters.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/análisis , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/diagnóstico , Estradiol Deshidrogenasas/análisis , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Cromatografía Líquida de Alta Presión , Pruebas Enzimáticas Clínicas , Endometrio/enzimología , Endometrio/patología , Estradiol/aislamiento & purificación , Estradiol/metabolismo , Estradiol Deshidrogenasas/metabolismo , Estrona/aislamiento & purificación , Femenino , Humanos , Menstruación , Progesterona/sangreRESUMEN
All living children suffering from myelomeningoceles and being 1 to 12 years old were registered by means of a questionnaire. The death certificates of all those children who came to death from myelomeningoceles between 1973 and 1977 were also evaluated. Ratio of morbidity: 6 to 10000 live born children. In the first year of life the mortality rate amounted to 34%.