9-hydroxystearic acid (9-HSA) is a product of endogenous lipoperoxidation identified in several human cell lines; its administration to HT29, a colon adenocarcinoma cell line, causes growth arrest in G0/G1 and differentiation toward a benign phenotype. The relevant molecular events are the increased transcription of p21WAF1, TGFbeta1, alkaline phosphatase, and subunit alfa5 of integrin. The number of genes whose activity is modified is indeed rather restricted: in particular variations of the activity of genes that regulate the cell cycle, apoptosis and differentiation have been evidenced for several HDAC1 inhibitors, but the mechanisms that induce or repress the selective activation of genes, beginning from the acetylation state of chromatin or of nuclear proteins, are not yet clear. In HT29, both histones H3 and H4 appear to be hyperacetylated as a consequence of 9-HSA administration. The isolation of histones, the identification of their isoforms as well as the determination of their modifications are essential steps of our study. Reverse phase, ion exchange or hydrophylic-interaction liquid chromatography and subsequent identification of each fraction by electrophoresis or mass spectrometry have become urepleaceble approaches for the analysis of protein modification sites. This work proposes an experimental approach for studying the different isoforms through the injection of the mixture of histones, extracted and solubilized in water, in a liquid chromatograph Jasco PU-1585 equipped with a Rheodyne Model 7725i injector, connected to a UV (JascoUV-1575) detector and to a LCQDuo (Thermo Finnigan) mass spectrometer, that is in turn interfaced to an electrospray ionization source (ESI), and equipped with an ion trap analyzer. The analysis of the peaks so obtained has been carried out by employing deconvolution programs that allow the determination of the MW of proteins and characterize and quantify also the acetylated and methylated isoforms.
V. Andrisano, M. Naldi, N. Calonghi, E. Pagnotta, C. Parolin, C. Boga, et al. (2005). IDENTIFICATION OF HISTONE MODIFICATIONS INVOLVED IN HDAC1 INHIBITION BY 9-HSA. SIENA : Tipografia Senese.
IDENTIFICATION OF HISTONE MODIFICATIONS INVOLVED IN HDAC1 INHIBITION BY 9-HSA
ANDRISANO, VINCENZA;NALDI, MARINA;CALONGHI, NATALIA;PAGNOTTA, ELEONORA;PAROLIN, CAROLA ELEONORA;BOGA, CARLA;MASOTTI, LANFRANCO
2005
Abstract
9-hydroxystearic acid (9-HSA) is a product of endogenous lipoperoxidation identified in several human cell lines; its administration to HT29, a colon adenocarcinoma cell line, causes growth arrest in G0/G1 and differentiation toward a benign phenotype. The relevant molecular events are the increased transcription of p21WAF1, TGFbeta1, alkaline phosphatase, and subunit alfa5 of integrin. The number of genes whose activity is modified is indeed rather restricted: in particular variations of the activity of genes that regulate the cell cycle, apoptosis and differentiation have been evidenced for several HDAC1 inhibitors, but the mechanisms that induce or repress the selective activation of genes, beginning from the acetylation state of chromatin or of nuclear proteins, are not yet clear. In HT29, both histones H3 and H4 appear to be hyperacetylated as a consequence of 9-HSA administration. The isolation of histones, the identification of their isoforms as well as the determination of their modifications are essential steps of our study. Reverse phase, ion exchange or hydrophylic-interaction liquid chromatography and subsequent identification of each fraction by electrophoresis or mass spectrometry have become urepleaceble approaches for the analysis of protein modification sites. This work proposes an experimental approach for studying the different isoforms through the injection of the mixture of histones, extracted and solubilized in water, in a liquid chromatograph Jasco PU-1585 equipped with a Rheodyne Model 7725i injector, connected to a UV (JascoUV-1575) detector and to a LCQDuo (Thermo Finnigan) mass spectrometer, that is in turn interfaced to an electrospray ionization source (ESI), and equipped with an ion trap analyzer. The analysis of the peaks so obtained has been carried out by employing deconvolution programs that allow the determination of the MW of proteins and characterize and quantify also the acetylated and methylated isoforms.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
