RESUMO
Cancer is a result of complex changes that occur in normal cells as they transform to become malignant and further when they become metastatic. These changes are not a consequence of a single protein but rather involve multiple proteins that function in pathways and networks. Thus, profiling cancer-associated changes requires simultaneous measurement of many proteins in a single sample. Identifying these changes may lead to the discovery of cancer-associated biomarkers that may assist in diagnosis, prognosis, patient monitoring and possibly for therapeutic purposes. Antibody arrays are a relatively new technology that enables one to perform multiplex high-throughput protein expression profiling. This review describes current technologies in antibody array and assay design, and presents a survey of the current literature on the use of these arrays in cancer research.
Assuntos
Anticorpos/imunologia , Neoplasias/imunologia , Proteômica/métodos , Humanos , Neoplasias/diagnóstico , Análise Serial de Proteínas/métodosRESUMO
Antibody arrays are a promising new tool for mass analysis of protein level changes in cells responding to different stimuli. Here we describe a novel antibody array system called Panorama Ab Microarray Cell Signaling, that contains 224 antibodies spotted on FAST nitrocellulose-coated slides that can detect protein levels as low as a few nanograms per mL. The antibodies spotted are specific for proteins important in various areas of cell signaling such as phosphorylation, cell cycle, apoptosis, nuclear signaling and cytoskeleton proteins. Furthermore, for some of the protein targes, the Panorama Ab Microarray can detect phosphorylated and nonphosphorylated forms of the traget protein. We found that treatment of the slides post-spotting is important for the array performance (ratio of signal to background) and its stability. Panorama Ab Microarray was used to analyze changes in protein expression in F9 embryonic carcinoma cells stimulated to differentiate by all-trans retinoic acid. We found that the level of several proteins, among them cell cycle regulators and kineases, was either up- or down-regulated. For more than ten protein targets, the results obtained by the Panorama Ab Microarray were confirmed by immunoblotting.
Assuntos
Proteoma/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Camundongos , Especificidade de Órgãos , Fosforilação , Análise Serial de Proteínas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Tretinoína/farmacologiaRESUMO
Nuclear retinoic acid (RA) receptors (RARs) are phosphorylated at conserved serine residues located in their N-terminal domain. Phosphorylation of RARgamma2 at these residues is increased in response to RA subsequently to the activation of p38MAPK. We show here that this RA-induced phosphorylation of RARgamma2 resulted from the down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. By overexpressing Akt and by using agents that activated or inhibited the PI3K/Akt pathway, we also demonstrated that the RA-induced down-regulation of the PI3K/Akt pathway targeted not only the phosphorylation of RARgamma2 but also the turnover and transcriptional activity of the receptor. Altogether these data indicate that the PI3K/Akt pathway plays an important role in retinoic acid signaling.