PROTEOMIC ANALYSIS OF POST-TRANSLATIONAL MODIFICATIONS OF HUMAN HISTONES IN CANCER DRUG DISCOVERY

Histone proteins are subject to a range of post-translational modifications in living cells. The combinatorial nature of these modifications reflects in the so called "histone code" that modulates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Histone acetylation and deacetylation are controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs) enzymes. HDAC inhibitors (HDACi) increase acetylation levels, induce open chromatin structure and increased gene transcription, being found to function as potential chemotherapeutic in tumour cell types (1). Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, it is here reported a LC-MS approach for qualitative and quantitative proteomic analysis of histones extracted from human colon cancer cells after treatment with HDACi. LC-MS-based methods were applied in a quantitative proteomic study of the dose and time response effect of HDACi on histone acetylation in HT29 colon cancer cell cultures. A correlation was established between the degree of histone post-translational modifications and the cell cycle effects caused by treatment of HT29 colon cancer cells with selective and non selective HDACi. This correlation affords a mean to better understand the mechanism of action of new, more potent and selective HDACi directly on the cells. On the basis of these studies, LC-MS-based quantitative proteomic analysis of histone post-translational modifications results as a viable approach for functional analysis of colon cancer candidate drugs, such as HDACi.