RESUMEN
Estrogen receptor-α (ER) is an important target both for therapeutic compounds and endocrine disrupting chemicals (EDCs); however, the mechanisms involved in chemical modulation of regulating ER transcriptional activity are inadequately understood. Here, we report the development of a high content analysis-based assay to describe ER activity that uniquely exploits a microscopically visible multi-copy integration of an ER-regulated promoter. Through automated single-cell analyses, we simultaneously quantified promoter occupancy, recruitment of transcriptional cofactors and large-scale chromatin changes in response to a panel of ER ligands and EDCs. Image-derived multi-parametric data was used to classify a panel of ligand responses at high resolution. We propose this system as a novel technology providing new mechanistic insights into EDC activities in a manner useful for both basic mechanistic studies and drug testing.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente/métodos , Compuestos de Bencidrilo , Western Blotting , Línea Celular Tumoral , Análisis por Conglomerados , Disruptores Endocrinos/farmacología , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/genética , Fulvestrant , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Fenoles/farmacología , Análisis de Componente Principal , Regiones Promotoras Genéticas/genética , Clorhidrato de Raloxifeno/farmacología , Reproducibilidad de los Resultados , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologíaRESUMEN
BACKGROUND: Previously, proteomic methods were applied to characterise differentially expressed proteins in microdissected pancreatic ductal adenocarcinoma cells. AIMS: To report that CapG and a related protein, gelsolin, which have established roles in cell motility, are overexpressed in metastatic pancreatic cancer; and to describe their pattern of expression in pancreatic cancer tissue and their effect on cell motility in pancreatic cancer cell lines. METHODS: CapG was identified by mass spectrometry and immunoblotting. CapG and gelsolin expression was assessed by immunohistochemical analysis on a pancreatic cancer tissue microarray and correlated with clinical and pathological parameters. CapG and gelsolin levels were reduced using RNA interface in Suit-2, Panc-1 and MiaPaCa-2 cells. Cell motility was assessed using modified Boyden chamber or wound-healing assays. RESULTS: Multiple isoforms of CapG were detected in pancreatic cancer tissue and cell lines. Immunohistochemical analysis of benign (n = 44 patients) and malignant (n = 69) pancreatic ductal cells showed significantly higher CapG staining intensity in nuclear (p<0.001) and cytoplasmic (p<0.001) compartments of malignant cells. Similarly, gelsolin immunostaining of benign (n = 24 patients) and malignant (n = 68 patients) pancreatic ductal cells showed higher expression in both compartments (both p<0.001). High nuclear CapG was associated with increased tumour size (p = 0.001). High nuclear gelsolin was associated with reduced survival (p = 0.01). Reduction of CapG or gelsolin expression in cell lines by RNAi was accompanied by significantly impaired motility. CONCLUSIONS: Up regulation of these actin-capping proteins in pancreatic cancer and their ability to modulate cell motility in vitro suggest their potentially important role in pancreatic cancer cell motility and consequently dissemination.
Asunto(s)
Movimiento Celular/fisiología , Gelsolina/análisis , Proteínas de Microfilamentos/análisis , Proteínas de Neoplasias/análisis , Proteínas Nucleares/análisis , Neoplasias Pancreáticas/química , Adenocarcinoma/química , Adenocarcinoma/genética , Adenocarcinoma/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Femenino , Humanos , Isomerismo , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Páncreas/metabolismo , Páncreas/patología , Conductos Pancreáticos/metabolismo , Conductos Pancreáticos/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Interferencia de ARN/fisiología , ARN Neoplásico/metabolismo , Regulación hacia ArribaRESUMEN
Epithelial organization is maintained by cell proliferation, migration, and differentiation. In the case of the gastric epithelium, at least some of these events are regulated by the hormone gastrin. In addition, gastric epithelial cells are organized into characteristic tubular structures (the gastric glands), but the cellular mechanisms regulating the organization of tubular structures (sometimes called branching morphogenesis) are uncertain. In the present study, we examined the role of the gastrin-cholecystokinin(B) receptor in promoting branching morphogenesis of gastric epithelial cells. When gastric cancer AGS-G(R) cells were cultured on plastic, gastrin and PMA stimulated cell adhesion, formation of lamellipodia, and extension of long processes in part by activation of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase. Branching morphogenesis was not observed in these circumstances. However, when cells were cultured on artificial basement membrane, the same stimuli increased the formation of organized multicellular arrays, exhibiting branching morphogenesis. These effects were reversed by inhibitors of PKC but not of PI-3 kinase. We conclude that, in the presence of basement membrane, activation of PKC by gastrin stimulates branching morphogenesis.
