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Assessment of HDACi-Induced Cytotoxicity

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HDAC/HAT Function Assessment and Inhibitor Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1510))

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.

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References

  1. Spiegel S, Milstien S, Grant S (2012) Endogenous modulators and pharmacological inhibitors of histone deacetylases in cancer therapy. Oncogene 31:537–551

    CAS  PubMed  Google Scholar 

  2. Falkenberg KJ, Johnstone RW (2014) Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat Rev Drug Discov 13:673–691

    Article  CAS  PubMed  Google Scholar 

  3. 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

    CAS  PubMed  Google Scholar 

  4. Kepp O, Galluzzi L, Lipinski M, Yuan J, Kroemer G (2011) Cell death assays for drug discovery. Nat Rev Drug Discov 10:221–237

    Article  CAS  PubMed  Google Scholar 

  5. Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol Appendix 3, Appendix 3B

    Google Scholar 

  6. 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

    Article  CAS  PubMed  Google Scholar 

  7. 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

    Article  CAS  PubMed  Google Scholar 

  8. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jahan-Tigh RR, Ryan C, Obermoser G, Schwarzenberger K (2012) Flow cytometry. J Invest Dermatol 132, e1

    Article  CAS  PubMed  Google Scholar 

  12. Cottet-Rousselle C, Ronot X, Leverve X, Mayol JF (2011) Cytometric assessment of mitochondria using fluorescent probes. Cytometry A 79:405–425

    Article  PubMed  Google Scholar 

  13. 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

    Article  CAS  PubMed  Google Scholar 

  14. 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

    Article  CAS  PubMed  Google Scholar 

  15. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dikalov SI, Harrison DG (2014) Methods for detection of mitochondrial and cellular reactive oxygen species. Antioxid Redox Signal 20:372–382

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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

    Article  CAS  PubMed  Google Scholar 

  18. Fischer U, Janicke RU, Schulze-Osthoff K (2003) Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ 10:76–100

    Article  CAS  PubMed  Google Scholar 

  19. 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

    Article  CAS  PubMed  Google Scholar 

  20. 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

    Article  CAS  PubMed  Google Scholar 

  21. 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

    Article  CAS  PubMed  Google Scholar 

  22. 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

    Article  CAS  PubMed  Google Scholar 

  23. 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

    Article  CAS  PubMed  Google Scholar 

  24. Mahmood T, Yang PC (2012) Western blot: technique, theory, and trouble shooting. North Am J Med Sci 4:429–434

    Article  Google Scholar 

  25. 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

    Article  CAS  PubMed  Google Scholar 

  26. Kroemer G, Galluzzi L, Brenner C (2007) Mitochondrial membrane permeabilization in cell death. Physiol Rev 87:99–163

    Article  CAS  PubMed  Google Scholar 

  27. 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

    Article  CAS  PubMed  Google Scholar 

  28. 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

    Article  CAS  PubMed  Google Scholar 

  29. 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

    CAS  PubMed  Google Scholar 

  30. 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

    Article  CAS  PubMed  Google Scholar 

  31. 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

    Article  CAS  PubMed  Google Scholar 

  32. 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

    Article  PubMed  PubMed Central  Google Scholar 

  33. Ginter T, Heinzel T, Krämer OH (2013) Acetylation of endogenous STAT proteins. Methods Mol Biol 967:167–178

    Article  CAS  PubMed  Google Scholar 

  34. 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

    Article  CAS  PubMed  Google Scholar 

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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.

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Authors and Affiliations

  1. Department of Pediatric Hematology and Oncology, Children’s Clinic, Jena University Hospital, Jena, Germany

    Lisa Marx-Blümel, Marie Kühne & Jürgen Sonnemann

  2. Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Jena, Germany

    Christian Marx

  3. Klinik für Kinder- und Jugendmedizin, Friedrich-Schiller-Universität Jena, Kochstr. 2, 07745, Jena, Germany

    Jürgen Sonnemann

Authors
  1. Lisa Marx-Blümel
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  2. Christian Marx
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  3. Marie Kühne
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  4. Jürgen Sonnemann
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Corresponding author

Correspondence to Jürgen Sonnemann .

Editor information

Editors and Affiliations

  1. Institute of Toxicology, University Medical Center Mainz, Mainz, Germany

    Oliver H. Krämer

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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

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  • DOI: https://doi.org/10.1007/978-1-4939-6527-4_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6525-0

  • Online ISBN: 978-1-4939-6527-4

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