Histones post-translational modifications in colon cancer cell line determined by HPLC-ESI-MS and Maldi-Tof for the characterization of deacetylase inhibitors

The nucleosome is the basic structural unit of eukariotic chromosomes and consists of a DNA molecule associated with a histone octamer comprised of pairs of the core histones H2A, H2B, H3 and H4. Linker DNA and histone1 to form chromatin join the nucleosomes. Histones play an important role in transcription, DNA replication, DNA repair and recombination. Post-translational modifications of specific residues in the core histones (acetylation, methylation, phosphorylation, etc.) have been demonstrated to be critical to their regulatory function. In particular acetylation is a very specific phenomenon with various isoforms playing distinct roles. Increased acetylation is generally correlated with transcriptionally active or poised genes. Histone deacetylases’ inhibitors (short chain fatty acids, such as sodium butyrate, hydroxamic acids such as thrichostatin A) have been described as potential cancer therapeutics in a variety of preclinical studies1. Inhibitors’ treatment of cells resulted in the increase of highly acetylated isoforms of the histones, which affect gene expression, cell differentiation and apoptosis. It is here described the application of reversed-phase high-pressure liquid chromatography under gradient conditions and mass spectrometry (LC-ESI-MS) to analyse global modification levels of core histones. The LC-MS method was optimised using histones extracted from HT 29 colon cancer cell line2. Histones eluted from the column were detected with Q-Tof and ion trap mass spectrometers with an electrospray source put in parallel and UV detection at 214 nm. These methods were then applied to the characterisation of changes in histone modification in HT29 treated with histone deacetylase inhibitors such as valproic acid, sodium butyrate and 9-hydroxystearic acid. Acetylation were found located in the H4 histone tail by MALDI-Tof analysis of tryptic and arginase peptide digests. 1Rasheed WK, Johnstone RW, Prince H,Histone deacetylase inhibitors in cancer therapy Expert Opin Investig Drugs. 2007 May;16(5):659-78. 2Naldi M, Andrisano V, Fiori J, Calonghi N, Pagnotta E, Parolin C, Pieraccini G,Masotti L. Histone proteins determined in a human colon cancer by high-performance liquid chromatography and mass spectrometry. J Chromatogr A. 2006 Sep 29;1129(1):73-81.