RESUMEN

This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is predominantly confined to a single cell population, i.e. the choanocytes, and in this process apoptosis only plays a minor role. To our knowledge, such fast cell kinetics under steady-state conditions, with high turnover by shedding in the absence of apoptosis, has not been observed previously in any other multicellular organism. The duration of the cell cycle in vivo resembles that of unicellular organisms in culture. Morphological and histochemical studies demonstrate compartmentalization of choanocytes in the sponge tissue, which corresponds well with its remarkable cellular kinetics. Coral reef cavity sponges, like H. caerulea, inhabit low nutrient tropical waters, forcing these organisms to filter large volumes of water and to capture the few nutrients efficiently. Under these oligotrophic conditions, a high cell turnover may be considered as a very useful strategy, preventing permanent damage to the sponge by environmental stress. Halisarca caerulea maintains its body mass and keeps its food uptake system up to date by constantly renewing its filter system. We conclude that studies on cell kinetics and functional morphology provide new and essential information on the growth characteristics and the regulation of sponge growth in vivo as well as in vitro and the role of choanocytes in tissue homeostasis.

Asunto(s)

Ciclo Celular/fisiología , Proliferación Celular , Poríferos/citología , Animales , Apoptosis/fisiología , Bromodesoxiuridina , Inmunohistoquímica , Antillas Holandesas , Poríferos/fisiología

RESUMEN

BACKGROUND: To study how caretaker gene silencing relates to gatekeeper mutations in colorectal cancer (CRC), we investigated whether O6-methylguanine DNA methyltransferase (MGMT) and Human Mut-L Homologue 1 (MLH1) promoter hypermethylation are associated with APC, KRAS and BRAF mutations among 734 CRC patients. METHODS: We compared MGMT hypermethylation with G:C > A:T mutations in APC and KRAS and with the occurrence of such mutations in CpG or non-CpG dinucleotides in APC. We also compared MLH1 hypermethylation with truncating APC mutations and activating KRAS and BRAF mutations. RESULTS: Only 10% of the tumors showed both MGMT and MLH1 hypermethylation. MGMT hypermethylation occurred more frequently in tumors with G:C > A:T KRAS mutations (55%) compared with those without these mutations (38%, P < 0.001). No such difference was observed for G:C > A:T mutations in APC, regardless of whether mutations occurred in CpG or non-CpG dinucleotides. MLH1 hypermethylation was less common in tumors with APC mutations (P = 0.006) or KRAS mutations (P = 0.001), but was positively associated with BRAF mutations (P < 0.001). CONCLUSIONS: MGMT hypermethylation is associated with G:C > A:T mutations in KRAS, but not in APC, suggesting that MGMT hypermethylation may succeed APC mutations but precedes KRAS mutations in colorectal carcinogenesis. MLH1-hypermethylated tumors harbor fewer APC and KRAS mutations and more BRAF mutations, suggesting that they develop distinctly from an MGMT methylator pathway.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Colorrectales/genética , Metilación de ADN/genética , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Proteínas Nucleares/genética , Mutación Puntual , Proteínas Supresoras de Tumor/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anciano , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Islas de CpG/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Femenino , Silenciador del Gen , Genes APC , Genes ras/genética , Humanos , Masculino , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Países Bajos , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Estudios Prospectivos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Supresoras de Tumor/metabolismo , Proteínas ras/genética

RESUMEN

Since a KRAS oncogene mutation is an early event in colorectal cancer development and cigarette smoking is thought to have an effect on early stages of colorectal tumorigenesis, smoking, especially long-term smoking, may be associated with the risk for colorectal cancer with KRAS oncogene mutations. In the Netherlands Cohort Study on diet and cancer (n=120,852 men and women), using a case-cohort design, adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) were computed for colorectal tumors with wild-type and with mutated KRAS gene, and with specific G:C-->T:A or G:C-->A:T point mutations in KRAS, according to cigarette smoking status, frequency, duration, pack years, age at first exposure, years since cessation, inhalation and filter usage. After 7.3 years and excluding the first 2.3 years, 648 cases and 4083 sub-cohort members were included in the analyses. Ex-smokers, but not current smokers, were at increased risk for colorectal cancer with wild-type KRAS gene tumors when compared with never smokers, albeit not statistically significant (RR 1.26, 95% CI 0.96-1.66). This was not observed for KRAS mutated tumors when comparing ex-smokers with never smokers (RR 1.15, 95% CI 0.79-1.66). The highest category of smoking frequency (>20 cigarettes/day) and inhalation of smoke were associated with an increased risk for colorectal cancer with wild-type KRAS gene tumors, though not statistically significant, when compared with never smoking (frequency: RR 1.24, 95% CI 0.90-1.71 and inhalation: RR 1.25, 95% CI 0.94-1.67). These associations were strongest in men (ex-smokers: RR 1.79, 95% CI 1.00-3.20; frequency: RR 1.91, 95% CI 1.03-3.52; inhalation: RR 1.69, 95% CI 0.94-3.04). No associations were observed between any of the smoking characteristics and the risk for colorectal cancer with mutated KRAS gene tumors, nor where there any clear associations with tumors with specific G:C-->A:T transitions or G:C-->T:A transversions. These results suggest that, in contrast to the hypothesis, smoking does not increase the risk for colorectal tumors with a mutated KRAS gene. Some smoking characteristics, i.e. being an ex-smoker, frequency and inhalation, may be associated with risk for colorectal cancer characterized by the wild-type KRAS gene, especially in men.

Asunto(s)

Adenocarcinoma/genética , Neoplasias Colorrectales/genética , Genes ras , Mutación Puntual , Fumar/efectos adversos , Adenocarcinoma/inducido químicamente , Adenocarcinoma/epidemiología , Anciano , Consumo de Bebidas Alcohólicas/epidemiología , Estudios de Cohortes , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/epidemiología , Femenino , Estudios de Seguimiento , Ligamiento Genético , Humanos , Exposición por Inhalación , Masculino , Persona de Mediana Edad , Mutación Puntual/efectos de los fármacos , Estudios Prospectivos , Factores de Riesgo , Caracteres Sexuales , Fumar/epidemiología , Cese del Hábito de Fumar , Factores de Tiempo

RESUMEN

In this study, we assessed the effects of tibolone and its metabolites on the production of a progesterone sensitive parameter, prolactin, in human endometrium stroma cells in vitro. In addition, the metabolism of the compounds by isolated stromal and epithelial cells was evaluated. The reference compounds, progesterone, Org 2058, and DHT all induced prolactin production. Oestradiol also slightly induced prolactin production and enhanced the response to Org 2058. Tibolone and Delta4-tibolone were similar with regard to potency to induce prolactin levels in the culture supernatant. Their potency was lower than that of Org 2058, similar to that of progesterone and higher than that of DHT. The efficacies of tibolone, Delta4-tibolone and Org 2058 were similar (approximately 200-fold induction). The estrogenic tibolone metabolites 3alpha- and 3beta-OH tibolone also significantly stimulated prolactin production. Their potency, however, was low since significance was reached only at the highest concentrations tested. The PR antagonist Org 31710 inhibited both tibolone- and Delta4-tibolone-induced prolactin production. The responses of tibolone and Delta4-tibolone were not affected by co-incubation with the androgen receptor antagonist OH-flutamide. The effect of tibolone, but not Delta4-tibolone, was antagonized approximately 50% in combination with the highest dose (1 microM) estrogen receptor antagonist, ICI 164384. The induction of prolactin by 3alpha- and 3beta-OH tibolone was antagonized most potently by Org 31710, but also by ICI 164384 and OH-flutamide. Tibolone is metabolized differently in epithelial and stromal cells of the human endometrium. The epithelial cells mostly produce the progestagenic/androgenic Delta4-tibolone. The stromal cells produce predominantly the 3beta-OH tibolone, and some Delta4-tibolone, but the net effect observed with regard to prolactin production is progestagenic. When the metabolites 3alpha-OH, 3beta-OH, and Delta4-tibolone were added to the cultures no conversions were observed. The HPLC analyses showed no evidence for the production of sulfated metabolites. In conclusion, the net effects on endometrial stromal cells are predominantly progestagenic. Tibolone is converted by epithelial cells into Delta4-tibolone which displays progestagenic and androgenic activities, whereas in stromal cells also the estrogenic metabolites 3alpha- and 3beta-OH tibolone are formed.

Asunto(s)

Endometrio/citología , Moduladores de los Receptores de Estrógeno , Norpregnenos , Prolactina/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Células Cultivadas , Dihidrotestosterona/metabolismo , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Moduladores de los Receptores de Estrógeno/metabolismo , Moduladores de los Receptores de Estrógeno/farmacología , Femenino , Humanos , Norpregnenos/metabolismo , Norpregnenos/farmacología , Pregnenodionas/química , Pregnenodionas/metabolismo , Progesterona/química , Progesterona/metabolismo , Células del Estroma/citología

RESUMEN

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1alpha and CA-IX in the menstrual and early proliferative phases. HIF1alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed by estrogen during the late proliferative phase.

Asunto(s)

Endometrio/química , Menstruación/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto , Western Blotting , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Endometrio/citología , Endometrio/metabolismo , Femenino , Expresión Génica/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inmunohistoquímica , Ciclo Menstrual/genética , Ciclo Menstrual/metabolismo , Menstruación/genética , Persona de Mediana Edad , Neuropilina-1/genética , Neuropilina-1/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Case-cohort analyses were performed on meat and fish consumption in relation to K-ras mutations in 448 colon and 160 rectal cancers that occurred during 7.3 years of follow-up, excluding the first 2.3 years, and 2948 subcohort members of The Netherlands Cohort Study on diet and cancer. Adjusted incidence rate ratios and 95% confidence intervals were computed for colon and rectal cancer and for K-ras mutation status subgroups. Total fresh meat, most types of fresh meat and fish were not associated with colon or rectal cancer, neither overall nor with K-ras mutation status. However, several weak associations were observed for tumours with a wild-type K-ras, including beef and colon tumours, and an inverse association for pork with colon and rectal tumours; for meat products, an increased association was observed with wild-type K-ras tumours in the colon and possibly with G>A transitions in rectal tumours.

Asunto(s)

Neoplasias del Colon/etiología , Neoplasias del Colon/genética , Dieta , Genes ras , Carne , Neoplasias del Recto/etiología , Neoplasias del Recto/genética , Anciano , Estudios de Casos y Controles , Estudios de Cohortes , Neoplasias del Colon/epidemiología , Neoplasias del Colon/prevención & control , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Productos de la Carne , Persona de Mediana Edad , Países Bajos/epidemiología , Neoplasias del Recto/epidemiología , Neoplasias del Recto/prevención & control , Factores de Riesgo , Alimentos Marinos

RESUMEN

Endometrial carcinoma, generally, has a good prognosis. However, in some patients, the tumor appears to behave very aggressively, a course that cannot be explained with histopathological characteristics. More insight into the molecular background can be valuable to clarify these differences in tumor behavior. The three components associated with the Wnt pathway--i.e., adenomatous polyposis coli (APC), beta-catenin, and E-cadherin--were evaluated in a case-control study of 28 patients with stage-I endometrial carcinomas to determine their involvement in the development of recurrent disease. Mutation analysis of the mutation cluster region of the APC gene, determination of gene promoter methylation status of the APC-1A and E-cadherin genes, and immunohistochemical analysis of APC, E-cadherin, and beta-catenin were performed using paraffin-embedded tumor tissue. Twenty-one APC gene mutations were detected in 12 of 28 (43%) patients. Only three mutations would result in a stopcodon in the APC gene. APC gene promoter methylation was assessed in 12 of 28 (43%) patients. APC immunostaining was absent in two of 24 (8.3%) patients. The occurrence of APC mutations, APC gene promoter methylation, and APC immunostaining were not predictive for recurrence. No E-cadherin expression was observed in four of 24 patients (17%). E-cadherin gene promoter methylation could not be detected in any of the patients. The absence of E-cadherin expression was predictive for distant metastases, but not for local recurrence. Nuclear localization of beta-catenin was present in nine of 24 (38%) patients and was not predictive for recurrent disease. Involvement of epigenetic and genetic aberrations in APC and beta-catenin genes seems to be of minor importance for the development of local recurrences and distant metastases. Although the number of patients is limited, E-cadherin expression appears to be predictive for the development of distant metastases in endometrial carcinoma.

Asunto(s)

Cadherinas/genética , Carcinoma/genética , Carcinoma/patología , Proteínas del Citoesqueleto/genética , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Genes APC , Recurrencia Local de Neoplasia/genética , Transactivadores/genética , Poliposis Adenomatosa del Colon/genética , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Metilación de ADN , Análisis Mutacional de ADN , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Metástasis de la Neoplasia/genética , Pronóstico , Factores de Riesgo , beta Catenina

RESUMEN

BACKGROUND: Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). METHODS: Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. RESULTS: Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and alpha-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. CONCLUSIONS: In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Asunto(s)

Menstruación , Epiplón/patología , Actinas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Comunicación Celular , Movimiento Celular , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Genisteína/farmacología , Humanos , Indoles/farmacología , Queratinas/metabolismo , Epiplón/efectos de los fármacos , Epiplón/fisiopatología , Fenotipo , ARN Mensajero/metabolismo , Factores de Transcripción de la Familia Snail , Azida Sódica/farmacología , Sulfonamidas/farmacología , Distribución Tisular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tubulina (Proteína)/metabolismo , Regulación hacia Arriba , Vimentina/genética , Vimentina/metabolismo

Asunto(s)

Biomarcadores de Tumor/sangre , Neoplasias Colorrectales/genética , Proteínas Proto-Oncogénicas/sangre , Genes ras , Humanos , Estudios Multicéntricos como Asunto , Mutación Puntual , Proteínas Proto-Oncogénicas p21(ras) , Proteínas ras

Asunto(s)

Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales/genética , Dieta , Predisposición Genética a la Enfermedad , Mutación , Anciano , Consumo de Bebidas Alcohólicas/efectos adversos , Estudios de Cohortes , Neoplasias Colorrectales/epidemiología , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales Hereditarias sin Poliposis/epidemiología , Neoplasias Colorrectales Hereditarias sin Poliposis/etiología , Femenino , Estudios de Seguimiento , Genes APC , Predisposición Genética a la Enfermedad/genética , Mutación de Línea Germinal/genética , Humanos , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Polimorfismo Genético , Estudios Prospectivos , Factores de Riesgo , Verduras

RESUMEN

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Asunto(s)

Neoplasias Colorrectales/genética , Bases de Datos Factuales , Genes ras/genética , Mutación Puntual , Sistema de Registros , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Codón/genética , Neoplasias Colorrectales/mortalidad , Supervivencia sin Enfermedad , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Mutación Missense , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Análisis de Supervivencia , Valina/genética

RESUMEN

BACKGROUND: The timing and mechanisms of new blood vessel formation in the endometrium during the menstrual cycle are still largely unknown. In the present study we used the chick embryo chorioallantoic membrane (CAM) as an in-vivo assay for angiogenesis to assess the angiogenic potential of endometrium obtained at different stages of the menstrual cycle. METHODS: Endometrial fragments were explanted onto the CAM and, after 4 days of incubation, slides of the treated area were taken in ovo through a microscope for computerized image analysis. The vascular density index (VDI), a stereological estimate of vessel number and length, was obtained by counting the intersections of vessels with five concentric circles of a circular grid superimposed on the computerized image. RESULTS: We demonstrated that human endometrium has angiogenic potential throughout the menstrual cycle. Furthermore, there was a significant difference in angiogenic response between the stages of the menstrual cycle (P = 0.01). The VDIs of the early proliferative, early and late secretory stage were significantly higher than the VDI of the late proliferative phase. CONCLUSIONS: Elongation of existing vessels during the early proliferative phase as well as growth and coiling of the spiral vessels during the secretory phase may demand far higher angiogenic activity than outgrowth and maintenance of vessels during the late proliferative phase.

Asunto(s)

Endometrio/irrigación sanguínea , Ciclo Menstrual , Neovascularización Fisiológica , Alantoides/irrigación sanguínea , Animales , Vasos Sanguíneos/anatomía & histología , Embrión de Pollo , Corion/irrigación sanguínea , Técnicas de Cultivo , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador

RESUMEN

Sampson's transplantation theory for the pathogenesis of peritoneal endometriosis is widely accepted. The events that take place, however, on the cellular and subcellular level during the transition of endometrial tissue in the abdominal cavity into peritoneal endometriosis remain controversial. The mesothelium plays a central role in the debate on this subject. The interaction between endometrium and peritoneum has been studied in an in-vitro model using amnion, peritoneum and mesothelial cells in culture on the one hand and cyclic and menstrual endometrium on the other hand. The results of these studies indicate that (i) an intact mesothelial lining prevents adhesion of shed endometrial tissue, (ii) shed endometrial tissue adheres to the peritoneal extracellular matrix and (iii) menstrual effluent creates its own adhesion sites by damaging the mesothelial lining thus exposing the extracellular matrix. Therefore we conclude that the mesothelium has the properties of Teflon, while the extracellular matrix resembles Velcro.

Asunto(s)

Endometriosis/patología , Endometriosis/fisiopatología , Epitelio/patología , Femenino , Humanos , Menstruación , Peritoneo/citología , Peritoneo/fisiología

RESUMEN

The chick embryo chorioallantoic membrane (CAM) bioassay was used to investigate the early pathogenesis of endometriosis. Endometrial fragments were explanted onto the CAM. The grafts including the surrounding CAM were excised at 24, 48 or 72 h after explantation, fixed and embedded in paraffin. Immunohistochemical analysis was used to distinguish endometrial cells. To identify cells of human origin, in-situ hybridization was performed using a probe specific for human chromosome 1. After 24 h, direct contact between endometrial stromal as well as epithelial cells and the mesenchymal layer of the CAM was observed. Invasion of both stromal cells and intact endometrial glands into the mesenchymal layer was observed after 48 h. At 72 h, endometriosis-like lesions were observed in the mesenchymal layer. Positive staining with antibodies to vimentin and pan-cytokeratin was observed in the invading cells as well as in the lesions. In the lesions these positively stained cells showed in-situ hybridization signals for human chromosome 1, confirming their human origin. In conclusion, after 3 days of incubation, endometriosis-like lesions consisting of human endometrial glands and stromal cells were found in the mesenchymal layer of the CAM. These lesions apparently resulted from the invasion of intact human epithelial structures and stromal cells.

Asunto(s)

Corion/patología , Corion/trasplante , Endometriosis/patología , Endometrio/trasplante , Alantoides/trasplante , Animales , Embrión de Pollo , Corion/metabolismo , Femenino , Trasplante de Tejido Fetal , Humanos , Queratinas/metabolismo , Trasplante Heterólogo , Vimentina/metabolismo

RESUMEN

OBJECTIVE: To assess the angiogenic activity of peritoneal fluid in women with minimal to mild endometriosis and to investigate the relationship between this activity and the concentration of macrophage-derived angiogenic factors and clinical variables, such as phase of menstrual cycle, type of lesion, and revised American Society for Reproductive Medicine classification. DESIGN: In vivo bioassay. SETTING: Tertiary-care university medical center. PATIENT(S): Fifty-two female volunteers with laparoscopic findings indicating minimal to mild endometriosis. INTERVENTION(S): Peritoneal fluid was collected at the start of laparoscopy. A standard amount of peritoneal fluid was applied to a chick embryo chorioallantoic membrane assay. MAIN OUTCOME MEASURE(S): Angiogenic response was assessed by determining the vascular density index. RESULT(S): 85% of the peritoneal fluid samples induced angiogenesis in the chick embryo chorioallantoic membrane bioassay. Tumor necrosis factor-alpha and total protein were significantly related to the vascular density index, whereas interleukin-1beta, interleukin-8, and clinical variables appeared to not affect the angiogenic response. CONCLUSION(S): The results confirms previous findings of peritoneal fluid angiogenic activity in women with minimal to mild endometriosis and indicate involvement of tumor necrosis factor-alpha.

Asunto(s)

Líquido Ascítico , Endometriosis/metabolismo , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Neovascularización Patológica/inducido químicamente , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Alantoína/metabolismo , Animales , Embrión de Pollo , Corion/metabolismo , Femenino , Humanos , Laparoscopía , Membranas/metabolismo , Neovascularización Patológica/patología , Proteínas/metabolismo , Análisis de Regresión

RESUMEN

AIM: To investigate whether mutations in the STK11/LKB1 gene and genes implicated in the colorectal adenoma-carcinoma sequence are involved in Peutz-Jeghers syndrome (PJS) related tumorigenesis. METHODS: Thirty nine polyps and five carcinomas from 17 patients (from 13 families) with PJS were analysed for loss of heterozygosity (LOH) at 19p13.3 (STK11/LKB1 gene locus), 5q21 (APC gene locus), 18q21-22 (Smad4 and Smad2 gene locus), and 17p13 (p53 gene locus), and evaluated for immunohistochemical staining of p53. In addition, mutational analysis of K-ras codon 12, APC, and p53 and immunohistochemistry for Smad4 expression were performed on all carcinomas. RESULTS: LOH at 19p was seen in 15 of the 39 polyps and in all carcinomas (n = 5). Interestingly, six of the seven polyps from patients with cancer had LOH, compared with nine of the 31 polyps from the remaining patients (p = 0.01). In one polyp from a patient without a germline STK11/LKB1 mutation, no LOH at 19p or at three alternative PJS candidate loci (19q, 6p, and 6q) was found. No LOH at 5q was observed. However, mutational analysis revealed an APC mutation in four of the five carcinomas. LOH at 17p was not seen in polyps or carcinomas; immunohistochemistry showed expression of p53 in one carcinoma and focal expression in three polyps. At subsequent sequence analysis, no p53 mutation was found. One carcinoma had an activating K-ras codon 12 mutation and another carcinoma showed 18q LOH; however, no loss of Smad4 expression was seen. CONCLUSIONS: These results provide further evidence that STK11/LKB1 acts as a tumour suppressor gene, and may be involved in the early stages of PJS tumorigenesis. Further research is needed to see whether LOH in PJS polyps could be used as a biomarker to predict cancer. Differences in molecular genetic alterations noted between the adenoma-carcinoma sequence and PJS related tumours suggest the presence of a distinct pathway of carcinogenesis.

Asunto(s)

Hamartoma/genética , Pérdida de Heterocigocidad , Síndrome de Peutz-Jeghers/genética , Proteínas Serina-Treonina Quinasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Progresión de la Enfermedad , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/metabolismo , Genes APC , Genes p53 , Genes ras , Hamartoma/metabolismo , Humanos , Técnicas para Inmunoenzimas , Proteínas de Neoplasias/metabolismo , Síndrome de Peutz-Jeghers/metabolismo , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

In previous studies, we have shown that menstrual endometrium preferentially adheres to the subepithelial lining of the peritoneum. It remains to be elucidated, however, whether this damage is preexisting or inflicted by the menstrual tissue itself. We hypothesized that the menstrual tissue itself damages the peritoneum. To investigate this, the viability of menstrual endometrial tissue in peritoneal fluid (PF) was evaluated and the morphologic changes in the mesothelial cells were studied by in vitro cocultures of menstruum with mesothelial cell monolayers. Menstruum was collected with a menstrual cup. Endometrial tissue was isolated from the menstruum, resuspended in culture medium or in the cell-free fraction of PF and cultured for 24, 48 or 72 h. A 3(4, 5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to obtain a relative measure of viable adhered endometrial cells. Mesothelial cells isolated from human omental tissue were cultured on Matrigel or uncoated plastic. At confluence, overnight cocultures were performed and scanning electron microscopy was used to evaluate the morphologic changes. The viability of endometrial fragments was 84% (n = 36, p < 0.05), 82% (n = 27, not significant) and 104% (n = 14, not significant) when cultured in the cell-free fraction of PF for 24, 48 and 72 h, respectively, when compared to medium with 10% fetal calf serum. Menstrual endometrial fragments or menstrual serum added to and cocultured with mesothelial cells induced severe morphologic alterations of the latter, including retraction, shrinking and gap formation. Similar morphologic changes were observed when mesothelial cells were cocultured with menstrual endometrial fragments in PF or in culture inserts. Incubation with conditioned medium from cultured menstrual endometrium induced similar but less pronounced changes in morphology. In conclusion, menstrual endometrial fragments remain viable in PF in vitro for at least 72 h. Antegradely shed menstruum induces changes in mesothelial cell morphology, including retraction and shrinking with exposure of the underlying surface. These findings suggest that menstruum is harmful to the peritoneal lining. Therefore, by local destruction of the mesothelial layer, menstrual endometrium is able to create sites for adhesion.

Asunto(s)

Endometrio/citología , Endometrio/fisiología , Menstruación/fisiología , Adipocitos , Adulto , Líquido Ascítico , Adhesión Celular , Tamaño de la Célula , Supervivencia Celular , Técnicas de Cocultivo , Colágeno , Medios de Cultivo Condicionados , Combinación de Medicamentos , Células Epiteliales/citología , Femenino , Humanos , Laminina , Microscopía Electrónica de Rastreo , Epiplón , Proteoglicanos

RESUMEN

In a previous study on the pathogenesis of endometriosis, we observed that constituents of menstrual effluent induce morphological alterations in human mesothelial cells. In this study, we investigated whether these alterations were associated with apoptosis or necrosis or were the result of cellular remodelling. After overnight incubation of confluent monolayers of human omental mesothelial cells (HOMEC) with conditioned media prepared from menstrual effluent shed anterogradely, severe alterations in morphology were observed. Typical polygonal mesothelial cell cultures at confluency acquired elongated spindle morphology, resulting in gaps between the cells. In contrast, mesothelial cells from the control groups receiving culture medium only, retained a normal morphology. Immunofluorescence staining revealed that cytokeratin, vimentin and actin filaments were still present, homogeneously distributed in the cell cytoplasm following changes in morphology. To evaluate whether the morphological alterations were associated with apoptosis and/or necrosis, the cells were stained with the M30 CytoDeath antibody or annexin V with propidium iodide and analysed using flow cytometry. The results showed that only a small percentage (1-7%) of the affected HOMEC were undergoing apoptosis or necrosis. We conclude that the profoundly altered morphology of HOMEC is a result of cellular remodelling and that the role of apoptosis and necrosis is negligible. Soluble paracrine factors released by cells isolated from menstrual effluent shed anterogradely may induce a reorganization of the cytoskeleton. As a result, the underlying basement membrane will be exposed and the mesothelium may no longer prevent implantation of endometrium shed retrogradely into the peritoneum, thus facilitating the development of endometriosis.

Asunto(s)

Endometrio/fisiología , Menstruación/fisiología , Epiplón/citología , Anexina A5/metabolismo , Apoptosis , Línea Celular , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , ADN/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Etopósido/farmacología , Femenino , Citometría de Flujo , Humanos , Necrosis , Epiplón/efectos de los fármacos , Epiplón/ultraestructura

RESUMEN

Previous in-vitro studies have shown that the endometrium preferentially adheres to the extracellular matrix (ECM) of the amnion and peritoneum. This interaction probably involves adhesion molecules, e.g. integrins. We evaluated the expression of integrins in naturally shed menstrual endometrium and the adhesion pattern of this tissue to different components of the ECM. To identify integrins and matrix components involved, blocking studies were performed. Most of the 15 menstrual tissue samples showed positive staining for each of the integrins investigated, except alpha(4)beta(1). Compared with binding to collagen IV, which was set at 100%, adhesion to collagen I was 93% (not significant), to fibronectin 87% (P < 0.05), and to laminin 74% (P < 0.05). Scanning electron micoscopy showed that endometrium adhered to laminin but hardly spread, whereas spreading was observed when layered on the other coatings. Compared with the control (which was set at 100%), incubation with 4B4, a monoclonal antibody against the integrin beta(1) subunit, showed a significant reduction of adhesion (to approximately 50%; P < 0.05) when layered on laminin and a smaller reduction (to 82-86%; P < 0.05) when layered on the other three coatings. Incubation with antibody GOH3 against integrin alpha(6)beta(1) resulted in a similar reduction in adhesion to laminin. Incubation with an RGD peptide significantly reduced adhesion (to 84%; P < 0.05) when plated on fibronectin. In conclusion, antegradely shed menstrual endometrium expresses various integrins. It shows preferential attachment to collagen IV and collagen I, when compared with fibronectin and laminin. Blockage of the integrin beta(1) subunit resulted in greatest disruption to adhesion when layered on laminin, implying that the interaction was mediated by the alpha(6)beta(1) integrin. Since this adhesion was not completely blocked, other mechanisms are likely to be involved.

Asunto(s)

Adhesión Celular/fisiología , Endometrio/fisiología , Endometrio/ultraestructura , Matriz Extracelular/fisiología , Integrinas/fisiología , Laminina/fisiología , Menstruación/fisiología , Adulto , Anticuerpos Monoclonales , Endometriosis/etiología , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Integrina alfa6beta1 , Integrinas/antagonistas & inhibidores , Microscopía Electrónica de Rastreo , Oligopéptidos/inmunología

RESUMEN

OBJECTIVE: To investigate the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in antegradely shed menstruum and peritoneal fluid. DESIGN: A cell biological and immunohistochemical study. SETTING: Tertiary care university medical center. INTERVENTION(S): Immunohistochemistry was performed on cryostat sections and cultures of menstrual endometrium. Zymography was used to characterize MMP activity in peritoneal fluid, in menstrual serum, and in conditioned medium. Western blot analysis was used to further identify the MMPs in these fluids. MAIN OUTCOME MEASURE(S): Staining of MMPs and TIMPs in cryostat sections and cultures and MMP expression and activity in peritoneal fluid and menstrual blood serum. RESULT(S): Strong staining for MMP-1 and MMP-3 was observed in stroma and for MMP-7 in epithelium. Matrix metalloproteinase-2 and MMP-9 were weakly expressed in stroma. Both TIMP-1 and TIMP-2 were expressed in menstrual endometrium. Menstrual serum showed a pattern of MMP activity on zymography different from peritoneal fluid. Western blot analysis showed the presence of MMP-7 and MMP-9 in menstrual serum. CONCLUSION(S): Antegradely shed menstrual endometrium expresses several MMPs and TIMPs, even after culturing for 24 hours. MMP activity in menstrual serum is different from and more intense than MMP activity in peritoneal fluid. These enzymes may be involved in the early invasion of menstrual endometrium into the extracellular matrix of the peritoneum.

Asunto(s)

Líquido Ascítico/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Menstruación/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Western Blotting , Células Cultivadas , Endometrio/metabolismo , Femenino , Técnicas Histológicas , Humanos , Inmunohistoquímica , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/análisis