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1.
Cell Mol Life Sci ; 72(8): 1577-83, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25563489

RESUMEN

Identification and characterization of the normal epithelial lineages in the mammary gland is a fundamental step in understanding both development and cellular origin of cancer. In contrast to other tissues where lineage tracing has been widely accepted as a method of choice for dissecting the stem cell hierarchy, mammary gland has long remained a challenge due to its unique developmental and topological features. Recent advances in high-resolution single-cell imaging, combined with the use of inducible Cre-recombinase and in situ cell ablation, have provided unprecedented insight into mammary epithelial cell composition and function. Here, we briefly summarize and compare different mammary gland lineage tracing strategies, examine associated caveats and discuss future challenges and opportunities.


Asunto(s)
Glándulas Mamarias Animales/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Linaje de la Célula , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Colorantes Fluorescentes/química , Imagenología Tridimensional , Integrasas/genética , Integrasas/metabolismo , Glándulas Mamarias Animales/química , Glándulas Mamarias Animales/patología , Modelos Animales
2.
Proc Natl Acad Sci U S A ; 107(11): 5012-7, 2010 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-20194747

RESUMEN

Aberrant activation of Notch receptors has been implicated in breast cancer; however, the mechanisms contributing to Notch-dependent transformation remain elusive because Notch displays dichotomous functional activities, promoting both proliferation and growth arrest. We investigated the cellular basis for the heterogeneous responses to Notch pathway activation in 3D cultures of MCF-10A mammary epithelial cells. Expression of a constitutively active Notch-1 intracellular domain (NICD) was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. Interestingly, we found that these heterogeneous phenotypes reflect differences in Notch pathway activation levels; high Notch activity caused down-regulation of multiple matrix-adhesion genes and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. Moreover, microarray analyses implicated NICD-induced p63 down-regulation in loss of matrix adhesion. In addition, a reverse-phase protein array-based analysis and subsequent loss-of-function studies identified STAT3 as a dominant downstream mediator of the NICD-induced outgrowth. These results indicate that the phenotypic responses to Notch are determined by the dose of pathway activation; and this dose affects the balance between growth-stimulative and growth-suppressive effects. This unique feature of Notch signaling provides insights into mechanisms that contribute to the dichotomous effects of Notch during development and tumorigenesis.


Asunto(s)
Células Epiteliales/metabolismo , Glándulas Mamarias Humanas/citología , Receptor Notch1/metabolismo , Transducción de Señal , Adhesión Celular , Proliferación Celular , Células Cultivadas , Células Epiteliales/citología , Matriz Extracelular/metabolismo , Femenino , Humanos , Fenotipo , Estructura Terciaria de Proteína , Receptor Notch1/química , Factor de Transcripción STAT3/metabolismo , Transactivadores/metabolismo , Factores de Transcripción , Proteínas Supresoras de Tumor/metabolismo
3.
Drug Discov Today Dis Models ; 3(2): 149-154, 2006 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-19337569

RESUMEN

Mice have mainly been used in ovarian cancer research as immunodeficient hosts for cell lines derived from the primary tumors and ascites of ovarian cancer patients. These xenograft models have provided a valuable system for pre-clinical trials, however, the genetic complexity of human tumors has precluded the understanding of key events that drive metastatic dissemination. Recently developed immunocompetent, genetically defined mouse models of epithelial ovarian cancer represent significant improvements in the modeling of metastatic disease.

4.
Mol Cancer Ther ; 1(3): 215-25, 2002 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12467216

RESUMEN

The goal of this study was to determine the prevalence of sequence variants in the class I beta-tubulin (clone m40) gene and their occurrence in human tumors and cancer cell lines. DNA was isolated from 93 control individuals representing a wide variety of ethnicities, 49 paclitaxel-naive specimens (16 ovarian cancers, 17 non-small cell lung cancers, and 16 ovarian cancer cell lines), and 30 paclitaxel-resistant specimens (9 ovarian cancers, 9 ovarian cancer cell lines, and 12 ovarian cancer xenografts in nude mice). Denaturing high-performance liquid chromatography and direct sequence analysis detected two silent polymorphisms in exon 4, Leu217Leu (CTG/CTA) and Gly400Gly (GGC/GGT), with minor allele frequencies of 17 and 0.5%, respectively. Five nucleotide substitutions and one single-base deletion were detected in introns 1, 2, and 3 and in the 3' untranslated region. Analysis of 49 paclitaxel-naive and 30 paclitaxel-resistant specimens revealed no additional polymorphisms in the coding region. In addition, no amino acid replacements were found in chimpanzee, gorilla, and orangutan in comparison to human. Our data demonstrate a very high degree of sequence conservation in class I beta-tubulin, suggesting that all residues are important in tubulin structure and function. Individual variation in response to treatment with paclitaxel is not likely to be caused by genetic variations in the beta-tubulin drug target. Moreover, acquired mutations in class I beta-tubulin are unlikely to be a clinically relevant cause of drug resistance.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN de Neoplasias/metabolismo , Resistencia a Antineoplásicos , Neoplasias Pulmonares/genética , Mutación/genética , Neoplasias Ováricas/genética , Paclitaxel/farmacología , Tubulina (Proteína)/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/etnología , Estudios de Casos y Controles , Secuencia Conservada , Análisis Mutacional de ADN , Cartilla de ADN , Femenino , Variación Genética , Haplotipos , Heterocigoto , Humanos , Neoplasias Pulmonares/etnología , Ratones , Neoplasias Ováricas/etnología , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Tubulina (Proteína)/clasificación , Células Tumorales Cultivadas/efectos de los fármacos
5.
Nat Cell Biol ; 15(5): 451-60, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23604318

RESUMEN

Notch signalling is implicated in stem and progenitor cell fate control in numerous organs. Using conditional in vivo genetic labelling we traced the fate of cells expressing the Notch2 receptor paralogue and uncovered the existence of two previously unrecognized mammary epithelial cell lineages that we term S (Small) and L (Large). S cells appear in a bead-on-a-string formation and are embedded between the luminal and basal/myoepithelial layers in a unique reiterative pattern, whereas single or paired L cells appear among ductal and alveolar cells. Long-term lineage tracing and functional studies indicate that S and L cells regulate ipsi- and contralateral spatial placement of tertiary branches and formation of alveolar clusters. Our findings revise present models of mammary epithelial cell hierarchy, reveal a hitherto undescribed mechanism regulating branching morphogenesis and may have important implications for identification of the cell-of-origin of distinct breast cancer subtypes.


Asunto(s)
Linaje de la Célula , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/metabolismo , Receptor Notch2/genética , Coloración y Etiquetado/métodos , Factores de Edad , Animales , Biomarcadores/metabolismo , Antígeno CD24/metabolismo , Diferenciación Celular , Tamaño de la Célula , Citoplasma/genética , Citoplasma/metabolismo , Células Epiteliales/citología , Femenino , Técnica del Anticuerpo Fluorescente , Lactancia/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Mucina-1/genética , Mucina-1/metabolismo , Fenotipo , Embarazo , Receptor Notch2/metabolismo , Transducción de Señal , Tamoxifeno/análogos & derivados , Tamoxifeno/química , beta-Galactosidasa/metabolismo
7.
PLoS One ; 6(10): e25785, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21991352

RESUMEN

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFP(SAT), demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues.


Asunto(s)
Linaje de la Célula , Técnicas de Sustitución del Gen , Intestinos/citología , Receptores Notch/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Células Clonales , Enterocitos/citología , Enterocitos/metabolismo , Marcación de Gen , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Integrasas/metabolismo , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microvellosidades/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Factor de Transcripción HES-1 , Transcripción Genética
8.
Curr Protoc Pharmacol ; Chapter 14: Unit14.11, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22294391

RESUMEN

Ovarian cancer is the fourth most common cancer in women and the most lethal gynecological malignancy. The high mortality rate is attributable to the asymptomatic nature of the early stage of the disease, the lack of reliable screening tests, and the development of drug resistance. Approximately 90% of ovarian cancers are thought to originate from the ovarian surface epithelia (OSE). Development of in vivo experimental models that accurately recapitulate genetic events that occur during human epithelial ovarian cancer (EOC) initiation and progression is crucial for a better understanding of EOC pathogenesis, identification of early disease markers, and development of more effective therapy. Historically, one of the most challenging problems in developing genetically engineered mouse models (GEMMs) of EOC has been the lack of tissue-specific promoters that regulate transgene expression exclusively in adult OSE cells. Recent improvements in gene delivery technology have greatly accelerated development of GEMMs of EOC. This unit describes two distinct methods of transforming OSE cells in GEMMs and the potential applications of these models in oncology drug discovery and development.


Asunto(s)
Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Ingeniería Genética/métodos , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Animales , Carcinoma Epitelial de Ovario , Transformación Celular Neoplásica/genética , Femenino , Técnicas de Transferencia de Gen , Ratones , Ratones Transgénicos , Trasplante de Neoplasias/métodos , Manejo de Especímenes/métodos
9.
Proc Natl Acad Sci U S A ; 102(49): 17763-8, 2005 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-16306258

RESUMEN

Germ cell tumors (GCTs) of the testis are the predominant cancer among young men. We analyzed gene expression profiles of 50 GCTs of various subtypes, and we compared them with 443 other common malignant tumors of epithelial, mesenchymal, and lymphoid origins. Significant differences in gene expression were found among major histological subtypes of GCTs, and between them and other malignancies. We identified 511 genes, belonging to several critical functional groups such as cell cycle progression, cell proliferation, and apoptosis, to be significantly differentially expressed in GCTs compared with other tumor types. Sixty-five genes were sufficient for the construction of a GCT class predictor of high predictive accuracy (100% training set, 96% test set), which might be useful in the diagnosis of tumors of unknown primary origin. Previously described diagnostic and prognostic markers were found to be expressed by the appropriate GCT subtype (AFP, POU5F1, POV1, CCND2, and KIT). Several additional differentially expressed genes were identified in teratomas (EGR1 and MMP7), yolk sac tumors (PTPN13 and FN1), and seminomas (NR6A1, DPPA4, and IRX1). Dynamic computation of interaction networks and mapping to existing pathways knowledge databases revealed a potential role of EGR1 in p21-induced cell cycle arrest and intrinsic chemotherapy resistance of mature teratomas.


Asunto(s)
Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias de Células Germinales y Embrionarias/clasificación , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias/clasificación , Neoplasias/genética , Cromosomas Humanos/genética , Humanos , Masculino , Neoplasias de Células Germinales y Embrionarias/patología
10.
Mol Pharmacol ; 65(4): 906-16, 2004 04.
Artículo en Inglés | MEDLINE | ID: mdl-15044620

RESUMEN

We investigated the mechanisms of MDR1 gene activation by CCAAT/enhancer binding protein beta (C/EBPbeta, or nuclear factor for interleukin 6) in human cancer cells. Transfection of the breast cancer cell line MCF-7 and its doxorubicin-selected variant MCF-7/ADR by either C/EBPbeta or C/EBPbeta-LIP (a dominant-negative form of C/EBPbeta) confirmed their roles in the activation or repression of the endogenous, chromosomally embedded MDR1 gene. Cotransfection experiments with promoter constructs revealed a C/EBPbeta interaction on the MDR1 promoter via the region within -128 to -75. Deletions within the putative AP-1 box (-123 to -111) increased MDR1 promoter activity when stimulated by C/EBPbeta, suggesting that the AP-1 site negatively regulates MDR1 activation by C/EBPbeta. Mutations within the inverted CCAAT box (Y box) (-82 to -73) abolished the C/EBPbeta-stimulated MDR1 promoter activity, indicating that the Y box is required for MDR1 activation by C/EBPbeta. Chromatin immunoprecipitation (ChIP) revealed that C/EBPbeta precipitates a transcription complex containing C/EBPbeta, the MDR1 promoter sequences (-250 to +54), and the hBrm protein. In conclusion, alteration of expression or function of C/EBPbeta plays an important role in MDR1 gene regulation. C/EBPbeta activates the endogenous MDR1 gene of MCF-7 cells, and this activation was associated with a novel C/EBPbeta interaction region within the proximal MDR1 promoter (-128 to -75). The mechanisms of MDR1 activation by C/EBPbeta include C/EBPbeta binding of the chromatin of the MDR1 gene and interactions of C/EBPbeta with the Y box and Y box-associated proteins.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Interleucina-6/metabolismo , Factores de Transcripción , Activación Transcripcional , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/fisiología , Silenciador del Gen , Humanos , Mutación , Factores de Transcripción NFI , Proteínas Nucleares , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-1/fisiología , Células Tumorales Cultivadas , Proteína 1 de Unión a la Caja Y
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