RESUMEN
We have characterized a transgenic mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of multimerized LEF-1 responsive elements. In embryos, EGFP was detected in known sites of Wnt activation, including the primitive streak, mesoderm, neural tube, somites, heart, limb buds, mammary placodes, and whisker follicles. In vitro cultured transgenic embryonic fibroblasts upregulated EGFP expression in response to activation of Wnt signaling by GSK3beta inhibition. Mammary tumor cell lines derived from female LEF-EGFP transgenic mice treated with the carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA) also express EGFP. Thus, this transgenic line is useful for ex vivo and in vitro studies of Wnt signaling in development and cancer.
Asunto(s)
Embrión de Mamíferos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Neoplasias Mamarias Experimentales/metabolismo , Proteínas Wnt/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinógenos , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Proteínas Fluorescentes Verdes/genética , Immunoblotting , Masculino , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Elementos de Respuesta/genética , Células Tumorales Cultivadas , beta Catenina/metabolismoRESUMEN
Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2α and/or CK2α') and two regulatory subunits (CK2ß). It is implicated in every stage of the cell cycle and in the regulation of various intracellular pathways associated with health and disease states. The catalytic subunits have similar biochemical activity, however, their functions may differ significantly in cells and in vivo. In this regard, homozygous deletion of CK2α leads to embryonic lethality in mid-gestation potentially due to severely impaired cell proliferation. To determine the CK2α-dependent molecular mechanisms that control cell proliferation, we established a myoblast-derived cell line with inducible silencing of CK2α and carried out a comprehensive RNA-Seq analysis of gene expression. We report evidence that CK2α depletion causes delayed cell cycle progression through the S-phase and defective response to replication stress. Differential gene expression analysis revealed that the down-regulated genes were enriched in pathways implicated in cell cycle regulation, DNA replication and DNA damage repair. Interestingly, the genes coding for the minichromosome maintenance proteins (MCMs), which constitute the core of the replication origin recognition complex, were among the most significantly down-regulated genes. These findings were validated in cells and whole mouse embryos. Taken together, our study provides new evidence for a critical role of protein kinase CK2 in controlling DNA replication initiation and the expression levels of replicative DNA helicases, which ensure maintenance of proliferative potential and genome integrity in eukaryotic cells.