RESUMEN
High content analysis (HCA; also referred to as high content biology) is a quantitative, automated, medium-throughput microscopy approach whereby cell images are segmented into relevant compartments (nuclei, cytoplasm) and the staining in each compartment quantified by computer algorithms. The extraction of quantitative information from the cell image generates a wealth of data which contributes significantly to the acceleration of drug discovery and biological research. Here we have adapted HCA to analyze protein acetylations in the cytoskeleton. This approach yields associative information on the link between acetylation and cytoskeletal organization. The protocol also describes optimization steps for cytoskeletal analysis and its application across different cell types, and HCA platforms. The methods described herein are readily adaptable to non-cytoskeletal acetylations and have been applied to the analysis of transcription factors.
Asunto(s)
Biología Computacional/métodos , Citoesqueleto/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas/análisis , Acetilación , Algoritmos , Células CACO-2 , Línea Celular Tumoral , Citoesqueleto/ultraestructura , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo , Células HCT116 , Células HT29 , Humanos , Células MCF-7 , Microscopía , Proteínas/metabolismoRESUMEN
Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC50 concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors.