title: Assessment of HDACi-Induced Cytotoxicity.
abstract: The chromatin contains the genetic and the epigenetic information of a eukaryotic organism. Posttranslational modifications of histones, such as acetylation and methylation, regulate their structure and control gene expression. Histone acetyltransferases (HATs) acetylate lysine residues in histones while histone deacetylases (HDACs) remove this modification. HDAC inhibitors (HDACi) can alter gene expression patterns and induce cytotoxicity in cancer cells. Here we provide an overview of methods to determine the cytotoxic effects of HDACi treatment. Our chapter describes colorimetric methods, like trypan blue exclusion test, crystal violet staining, lactate dehydrogenase assay, MTT and Alamar Blue assays, as well as fluorogenic methods like TUNEL staining and the caspase-3/7 activity assay. Moreover, we summarize flow cytometric analysis of propidium iodide uptake, annexin V staining, cell cycle status, ROS levels, and mitochondrial membrane potential as well as detection of apoptosis by Western blot.
authors.affiliation: Department of Pediatric Hematology and Oncology, Children's Clinic, Jena University Hospital, Jena, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Marx-Blümel
authors.affiliation: Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Jena, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Marx
authors.affiliation: Department of Pediatric Hematology and Oncology, Children's Clinic, Jena University Hospital, Jena, Germany.
authors.forename: Marie
authors.initials: M
authors.lastname: Kühne
authors.affiliation: Department of Pediatric Hematology and Oncology, Children's Clinic, Jena University Hospital, Jena, Germany. juergen.sonnemann@med.uni-jena.de.|Klinik für Kinder- und Jugendmedizin, Friedrich-Schiller-Universität Jena, Kochstr. 2, 07745, Jena, Germany. juergen.sonnemann@med.uni-jena.de.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Sonnemann
mesh_terms: D000083:Acetanilides / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3 / Q000235:genetics* / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002843:Chromatin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D003124:Colorimetry / Q000379:methods; D004396:Coloring Agents / Q000737:chemistry; D004789:Enzyme Activation; D005434:Flow Cytometry / Q000379:methods; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015972:Gene Expression Regulation, Neoplastic; D045325:HCT116 Cells; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011499:Protein Processing, Post-Translational; D017382:Reactive Oxygen Species / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D000083:Acetanilides; D000970:Antineoplastic Agents; D002843:Chromatin; D004396:Coloring Agents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D056572:Histone Deacetylase Inhibitors; D017382:Reactive Oxygen Species; C029569:diacetyldichlorofluorescein; C000594735:tenovin-1; D051548:Histone Acetyltransferases; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D006655:Histone Deacetylases; D013890:Thiourea
keywords: Cell death; Colorimetric assay; Flow cytometry; Fluorogenic assay; HDACi; Histone deacetylase inhibitor; Immunoblotting; Toxicity; Viability; Vitality