Histone cell-based assay

The first functional cell-based assay for the determination of pan-histone deacetylase activity was achieved by detecting hyperacetylated histones by Western blotting. The histone acetylation level in cells offers a measure of candidate HDACI activity and has been linked to antiproliferative and cytotoxic effects. Beckers et al. used a cellular histone hyperacetylation assay for quantification of the cellular efficacy of HDACIs using the Cellomics Array Scan II platform in 96 wells [14]. The program... 

It shares homology with the JmjC domains found in the histone lysine demethylases. JMJD6 can demethylate H3 R2 and H4 R3 in biochemical and cell-based assays (42). The recent data that are emerging place histone arginine methylation as a key player of cell growth and cell proliferation processes. 

Fig. 6-20 Forward chemical-genetic screen for inhibitors of protein deacetylation (data from Ref. 80). (a) Overview of cell-based screens of the 1,3-dioxane-based, diversity-oriented synthesis-derived library using antibodies to measure tubulin and histone acetylation, (b) Relative position of selected active compounds in a three-dimensional principal component model computed from five cell-based assay descriptors. AcTubulin-selective (red),...

Commonly, in vitro determination of HDAC activity is a manual assay utilizing a coupled two-step process, including enzymatic deacetylation of a substrate followed by reaction termination and readout [10]. Assays utilize nuclear extracts and substrates containing labeled (radioactive or fluorescent) acetylated histones. For the isotope-based assays, the enzymes are incubated with acetate-radiolabled histones prepared from chicken reticulocytes or chemically [ Hjacetylated peptide substrates, and the enzymatic activity is determined by liquid scintillation counting [11]. Alternatively, histones may be obtained from cells following treatment with [ H]acetyl-CoA [12]. The caveats of these approaches include the variability of prelabeled acetylated core histones within preparations, potential high costs, their labor-intensive nature and the presence of radioactive waste. 

In a cell cycle assay, the percentage of cells in each cell cycle stage is the most crucial measurement. Cells can be classified into Glor G2 phase by their DNA content, the M phase (mitotic or interphase) by the phospho-histone H3 mitotic marker, and the S phase by a pulsed label with dU to detect DNA synthesis. Each cell has four intensity parameters and has to be classified into the Gl, G2, S, or M phase based on these parameters. Because we are potentially dealing with billions of cells, cell cycle classification must be automated. 

Apoptosis and necrosis were detected using the Cell Death Detection EL1SA kit, Roche (Version 11.0), a photometric enzyme-immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone associated DNA fragments after induced cell death. Assay is based on a quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones that allows specific determination of mono- and oligonucleosomes in the cytoplasmatic fraction of the cell. 

In addition to enzyme assays, HDAC inhibitors are usually evaluated in cancer cell lines, where they potently inhibit growth proliferation to provide a cell-based phenotypic readout of activity. The cellular potency of HDAC inhibitors can exceed their activity in enzyme assays, as the precise HDAC isoforms that drive proliferation in a given cell type may be unknown. In any case, growth inhibition data should be supplemented with evidence to confirm that it is due to HDAC inhibition and not off-target effects. Typically, confirmatory evidence involves Western blotting of client proteins such as histones for nuclear HDACs or tubulin for HDAC6 to detect an increase in acetylation levels, or changes in a downstream biomarker such as induction of the cyclin-dependent kinase inhibitor p21. 

Primary screenings of candidate HDACIs are generally performed using in vitro assays consisting of sources of HDACs (i.e. cell extracts or rat liver) and substrates (i.e. radiolabeled acetylated histones or synthetic peptides with a fluorophor). Isoform selective studies are performed using purified recombinant, immunoprecipitated proteins using antibodies against selected HDACs and computer-based virtual prediction assays. 

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