RESUMO
INTRODUCTION: The Epithelial Cell Adhesion Molecule (EpCAM) has been shown to be strongly expressed in human breast cancer and cancer stem cells and its overexpression has been supposed to support tumor progression and metastasis. However, effects of EpCAM overexpression on normal breast epithelial cells have never been studied before. Therefore, we analyzed effects of transient adenoviral overexpression of EpCAM on proliferation, migration and differentiation of primary human mammary epithelial cells (HMECs). METHODS: HMECs were transfected by an adenoviral system for transient overexpression of EpCAM. Thereafter, changes in cell proliferation and migration were studied using a real time measurement system. Target gene expression was evaluated by transcriptome analysis in proliferating and polarized HMEC cultures. A Chicken Chorioallantoic Membrane (CAM) xenograft model was used to study effects on in vivo growth of HMECs. RESULTS: EpCAM overexpression in HMECs did not significantly alter gene expression profile of proliferating or growth arrested cells. Proliferating HMECs displayed predominantly glycosylated EpCAM isoforms and were inhibited in cell proliferation and migration by upregulation of p27(KIP1) and p53. HMECs with overexpression of EpCAM showed a down regulation of E-cadherin. Moreover, cells were more resistant to TGF-ß1 induced growth arrest and maintained longer capacities to proliferate in vitro. EpCAM overexpressing HMECs xenografts in chicken embryos showed hyperplastic growth, lack of lumen formation and increased infiltrates of the chicken leukocytes. CONCLUSIONS: EpCAM revealed oncogenic features in normal human breast cells by inducing resistance to TGF-ß1-mediated growth arrest and supporting a cell phenotype with longer proliferative capacities in vitro. EpCAM overexpression resulted in hyperplastic growth in vivo. Thus, we suggest that EpCAM acts as a prosurvival factor counteracting terminal differentiation processes in normal mammary glands.
Assuntos
Antígenos de Neoplasias/genética , Mama/metabolismo , Mama/patologia , Moléculas de Adesão Celular/genética , Células Epiteliais/metabolismo , Expressão Gênica , Animais , Antígenos de Neoplasias/metabolismo , Caderinas/genética , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Molécula de Adesão da Célula Epitelial , Feminino , Perfilação da Expressão Gênica , Humanos , Hiperplasia , Cultura Primária de Células , Fator de Crescimento Transformador beta1/farmacologia , Transplante HeterólogoRESUMO
Glucocorticoids (GC) induce apoptosis in lymphoblasts and are thus essential in the treatment of acute lymphoblastic leukemia (ALL). Their effects result from gene regulations via the GC receptor (NR3C1/GR), but it is unknown how these changes evolve, what the primary GR targets are, and to what extent responses differ between ALL subtypes and nonlymphoid malignancies. We delineated the transcriptional response to GC on the exon level in a time-resolved manner in a precursor B- and a T childhood ALL model employing Exon microarrays and combined this with genome-wide NR3C1-binding site detection using chromatin immunoprecipitation-on-chip technology. This integrative approach showed that the response was strongly influenced by kinetics and extent of GR autoinduction in both models. Although remarkable differences between the ALL systems were apparent, we defined a set of common response genes enriched in apoptosis-related processes. Globally, GR binding was higher for GC-induced vs. -repressed genes, suggesting that GR mediates gene repression by interaction with distant enhancers or by cross talk with other transcription factors. Exon level analysis defined several new GC-regulated transcript variants of genes, including ATP4B, GPR98, TBCD, and ZBTB16. Our study provides unprecedented insight into the transcriptional response to GC in ALL cells, essential to understand this biologically and clinically important phenomenon. We found evidence of cell type-specific as well as common responses, possibly related to apoptosis induction, and detected induction of novel transcript variants by GC in the investigated systems. Finally, we implemented a bioinformatic framework that might be useful for high-density microarray analyses to identify alternative transcript variant expression.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Leucemia de Células T/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Pré-Escolar , Imunoprecipitação da Cromatina , DNA/genética , DNA/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia de Células T/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de Glucocorticoides/genéticaRESUMO
A rapidly learned odor discrimination task based on spontaneous foraging behavior of the rat was used to evaluate the role of N-methyl-D-aspartate (NMDA) receptors (NMDARs) in ongoing memory consolidation. Rats were trained in a single session to discriminate among three odors, one of which was associated with palatable food reward. Previous experiments showed that the NMDAR antagonist DL-APV induced amnesia for this task when injected immediately after training. In the present study, memory was reactivated 24 h after training by exposure to the rewarded odor within the experimental context after which rats received an intracerebroventricular injection of APV. Combined reactivation-drug treatment induced profound amnesia when tested 48 h later. Animals receiving drug alone, in absence of reactivation, showed perfect retention. It is concluded that NMDARs support a consolidation process taking place after memory reactivation.
