RESUMO
As part of ongoing studies to obtain a global picture of invasion related events in colorectal liver metastases, here, we report our findings on gene expression of the pro-angiogenic subgroup of chemokines, the CXCL-ELR+ chemokines. Apart from their pro-angiogenic and chemoattractant function, these chemokines appear to also contribute to tumor cell transformation, growth and invasion. In our nude mouse model of colorectal liver metastases, we found CXCL1,2,3,5 and 8 (IL-8) to be up-regulated in the tumor cells of the invasion front as compared to the tumor cells in the inner parts of the tumor. ShRNA mediated down-regulation of the most prominently up-regulated group member, CXCL1/gro-alpha resulted in inhibition of cell viability, invasion and proliferation. In vivo, down-regulation of CXCL1 resulted in a nearly complete prevention of tumor growth in nude mice. Mechanistically, auto-regulatory mechanisms involving NF-kappaB and Akt appear to be involved in pro-tumorigenic functions of CXCL1.
Assuntos
Quimiocina CXCL1/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação para Baixo/genética , Neoplasias Hepáticas/secundário , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Camundongos , Camundongos Nus , NF-kappa B/metabolismo , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para Cima/genéticaRESUMO
Gene trapping is a high-throughput insertional mutagenesis approach that has been primarily used in mouse embryonic stem cells (ESCs). As a high throughput technology, gene trapping helped to generate tenth of thousands of ESC lines harboring mutations in single genes that can be used for making knock-out mice. Ongoing international efforts operating under the umbrella of the International Knockout Mouse Consortium (IKMC; www.knockoutmouse.org) aim to generate conditional alleles for every protein coding gene in the mouse genome by high throughput conditional gene targeting and trapping. Here, we provide protocols for gene trapping in ESCs that can be easily adapted to any other mammalian cell. We further provide protocols for handling and verifying conditional gene trap alleles in ESC lines obtained from the IKMC repositories and describe a highly efficient method for the postinsertional modification of gene trap alleles. More specifically, we describe a protein tagging strategy based on recombinase mediated cassette exchange (RMCE) that enables protein localization and protein-protein interaction studies under physiological conditions.