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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Spiegel S, Milstien S, Grant S (2012) Endogenous modulators and pharmacological inhibitors of histone deacetylases in cancer therapy. Oncogene 31:537–551
Falkenberg KJ, Johnstone RW (2014) Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov 13:673–691
Kwolek-Mirek M, Zadrag-Tecza R (2014) Comparison of methods used for assessing the viability and vitality of yeast cells. FEMS Yeast Res 14:1068–1079
Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G (2011) Cell death assays for drug discovery. Nat Rev Drug Discov 10:221–237
Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol Appendix 3, Appendix 3B
Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89:271–277
Chiba K, Kawakami K, Tohyama K (1998) Simultaneous evaluation of cell viability by neutral red, MTT and crystal violet staining assays of the same cells. Toxicol In Vitro 12:251–258
Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH et al (2009) Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ 16:1093–1107
Collins L, Franzblau SG (1997) Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 41:1004–1009
Gossett DR, Weaver WM, Mach AJ, Hur SC, Tse HT, Lee W et al (2010) Label-free cell separation and sorting in microfluidic systems. Anal Bioanal Chem 397:3249–3267
Jahan-Tigh RR, Ryan C, Obermoser G, Schwarzenberger K (2012) Flow cytometry. J Invest Dermatol 132, e1
Cottet-Rousselle C, Ronot X, Leverve X, Mayol JF (2011) Cytometric assessment of mitochondria using fluorescent probes. Cytometry A 79:405–425
Royall JA, Ischiropoulos H (1993) Evaluation of 2′,7′-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 302:348–355
Vindelov LL, Christensen IJ (1990) A review of techniques and results obtained in one laboratory by an integrated system of methods designed for routine clinical flow cytometric DNA analysis. Cytometry 11:753–770
Ruefli AA, Ausserlechner MJ, Bernhard D, Sutton VR, Tainton KM, Kofler R et al (2001) The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species. Proc Natl Acad Sci U S A 98:10833–10838
Dikalov SI, Harrison DG (2014) Methods for detection of mitochondrial and cellular reactive oxygen species. Antioxid Redox Signal 20:372–382
Balcerczyk A, Soszynski M, Bartosz G (2005) On the specificity of 4-amino-5-methylamino-2′,7′-difluorofluorescein as a probe for nitric oxide. Free Radic Biol Med 39:327–335
Fischer U, Janicke RU, Schulze-Osthoff K (2003) Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ 10:76–100
Kim MY, Zhang T, Kraus WL (2005) Poly(ADP-ribosyl)ation by PARP-1: ‘PAR-laying’ NAD+ into a nuclear signal. Genes Dev 19:1951–1967
McStay GP, Salvesen GS, Green DR (2008) Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ 15:322–331
Pereira NA, Song Z (2008) Some commonly used caspase substrates and inhibitors lack the specificity required to monitor individual caspase activity. Biochem Biophys Res Commun 377:873–877
Sonnemann J, Hartwig M, Plath A, Saravana KK, Müller C, Beck JF (2006) Histone deacetylase inhibitors require caspase activity to induce apoptosis in lung and prostate carcinoma cells. Cancer Lett 232:148–160
Sonnemann J, Marx C, Becker S, Wittig S, Palani CD, Krämer OH, Beck JF (2014) p53-dependent and p53-independent anticancer effects of different histone deacetylase inhibitors. Br J Cancer 110:656–667
Mahmood T, Yang PC (2012) Western blot: technique, theory, and trouble shooting. North Am J Med Sci 4:429–434
Sonnemann J, Trommer N, Becker S, Wittig S, Grauel D, Palani CD, Beck JF (2012) Histone deacetylase inhibitor-mediated sensitization to TRAIL-induced apoptosis in childhood malignancies is not associated with upregulation of TRAIL receptor expression, but with potentiated caspase-8 activation. Cancer Biol Ther 13:417–424
Kroemer G, Galluzzi L, Brenner C (2007) Mitochondrial membrane permeabilization in cell death. Physiol Rev 87:99–163
Luo X, Budihardjo I, Zou H, Slaughter C, Wang X (1998) Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94:481–490
Dzieran J, Beck JF, Sonnemann J (2008) Differential responsiveness of human hepatoma cells versus normal hepatocytes to TRAIL in combination with either histone deacetylase inhibitors or conventional cytostatics. Cancer Sci 99:1685–1692
Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W, Schulte-Hermann R (1995) In situ detection of fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21:1465–1468
Sonnemann J, Gressmann S, Becker S, Wittig S, Schmudde M, Beck JF (2010) The histone deacetylase inhibitor vorinostat induces calreticulin exposure in childhood brain tumour cells in vitro. Cancer Chemother Pharmacol 66:611–616
West AC, Mattarollo SR, Shortt J, Cluse LA, Christiansen AJ, Smyth MJ, Johnstone RW (2013) An intact immune system is required for the anticancer activities of histone deacetylase inhibitors. Cancer Res 73:7265–7276
Kepp O, Senovilla L, Vitale I, Vacchelli E, Adjemian S, Agostinis P et al (2014) Consensus guidelines for the detection of immunogenic cell death. Oncoimmunology 3, e955691
Ginter T, Heinzel T, Krämer OH (2013) Acetylation of endogenous STAT proteins. Methods Mol Biol 967:167–178
Bueno C, Villegas ML, Bertolotti SG, Previtali CM, Neumann MG, Encinas MV (2002) The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions. Photochem Photobiol 76:385–390
Acknowledgement
Christian Marx was funded by RTG 1715 SP13 and Richard-Winter-Stiftung. We would like to thank Harald Schuhwerk for his contribution to the TUNEL protocol.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Marx-Blümel, L., Marx, C., Kühne, M., Sonnemann, J. (2017). Assessment of HDACi-Induced Cytotoxicity. In: Krämer, O. (eds) HDAC/HAT Function Assessment and Inhibitor Development. Methods in Molecular Biology, vol 1510. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6527-4_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6527-4_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6525-0
Online ISBN: 978-1-4939-6527-4
eBook Packages: Springer Protocols