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Assessment of Histone Acetylation Levels in Relation to Cell Cycle Phase

Simona Ronzoni1,  Mario Faretta1,  Marco Ballarini1,  PierGiuseppe Pelicci1,  Saverio Minucci2,1

1Department of Experimental Oncology, European Institute of Oncology, Milan, Italy
2Department of Biomolecular Sciences and Biotechnology, University of Milan, Milan, Italy


Publication Name: 
Current Protocols in Cytometry
Unit Number: 
UNIT 7.35
DOI: 
10.1002/0471142956.cy0735s46
Online Posting Date: 
October, 2008
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Abstract

Histone acetylation affects chromatin structural organization, thus regulating gene expression and DNA-related cellular events. Levels of histone acetylation are tightly modulated in normal cells and frequently altered in tumors. Consequently, histone deacetylase inhibitors are currently being tested in clinical trials as anticancer drugs. Presented here is a protocol for measuring the degree of cellular histone tail acetylation, alone or in combination with DNA content to simultaneously evaluate cell ploidy and/or cell cycle progression. The procedure can also be employed to stain peripheral blood samples in order to assess mean histone acetylation levels in patients treated with histone deacetylase inhibitors. Curr. Protocol. Cytom. 46:7.35.1-7.35.8. © 2008 by John Wiley & Sons, Inc.

Keywords: flow cytometry; histone; acetylation; histone deacetylase inhibitors; cell cycle; leukemia; clinical trials

     
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Table of Contents

  • Basic Protocol: Analysis of Histone Acetylation and Cell Cycle
  • Commentary
  • Literature Cited
  • Figures
     
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Materials

Basic Protocol: Analysis of Histone Acetylation and Cell Cycle

 Materials
  • In vitro cultured cells or human peripheral blood cells, untreated or treated with HDAC inhibitors (treatment according to the type of drug employed)
  • Phosphate-buffer saline (PBS; appendix 2A), 4°C
  • 0.1% (w/v) NaCl
  • 1.6% (w/v) NaCl
  • 1% (v/v) formaldehyde/PBS, 4°C
  • 70% (v/v) ethanol, –20°C
  • 1% (w/v) BSA/PBS
  • 0.1% (v/v) Triton X-100/PBS
  • 10% (v/v) normal goat serum (NGS)/PBS
  • Unconjugated primary antibody: 1 mg/ml T52 mouse monoclonal antibody in 1% (w/v) BSA/PBS
  • Secondary antibody: fluorescein isothiocyanate (FITC)-conjugated affinity-purified F(ab¢)2 fragment of goat anti-mouse IgG (Sigma)
  • 2.5 µg/ml propidium iodide (PI) in PBS
  • 1 mg/ml RNase
  • 12 × 75–mm polypropylene tubes
  • Refrigerated centrifuge, 4°C
  • 1.5-ml microcentrifuge tubes
  • Flow cytometer equipped with 488 nm argon laser and a band-pass 530/30-nm optical filter for FITC fluorescence detection (FL1 channel) and a 650-nm long-pass optical filter in front of the PI (FL3 channel) detector.
  • Additional reagents and equipment for counting cells (appendix 3A) and preparing human peripheral blood cells (Strober, 1997)
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Figures

  • Figure 7.35.1
    Correlation of histone acetylation levels to the cell cycle. Biparametric analysis of histone acetylation levels and cell cycle in (A) U937 exponentially growing cells (control) and (B) U937 cells treated with 50 ng/ml of trichostatin A (TSA) for 4 hr. Correlation between histone acetylation mean fluorescence and DNA content in (C) exponentially growing and (D) TSA-treated cells. (E) Ratios of histone acetylation levels in TSA-treated cells and untreated cells were then calculated for each DNA compartment showing increased action of the drug in the early G1-S early and late S-G2M phases. (Figure freely adapted from Ronzoni et al., 2005).

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  • Figure 7.35.2
    Kinetics of histone acetylation and cell cycle progression upon trichostatin A (TSA) treatment. NB4 leukemic cells were treated with the histone deacetylase inhibitor TSA and fixed at different time points to compare histone acetylation and DNA content to untreated cells. (Figure freely adapted from Ronzoni et al., 2005).

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  • Figure 7.35.3
    Peripheral blood samples from patients affected by breast cancer (A,B), colon cancer (C,D), melanomas (E,F), myeloproliferative disease (G), and acute myeloid leukemia (H), treated with the histone deacetylase inhibitor valproic acid. Blood samples were taken before therapy (curve 2) and after 2 weeks of treatment (curve 3; 30 mg/kg/day administered orally). Curve 1 represents the fluorescence level from blank sample. The table reports mean fluorescence values in arbitrary units (AU). (Figure freely adapted from Ronzoni et al., 2005).

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Literature Cited

Literature Cited
    Berger, S.L. 2002. Histone modifications in transcriptional regulation. Curr. Opin. Genet. Dev. 12:142-148.
    Di Croce, L., Buschbeck, M., Gutierrez, A., Joval, I., Morey, L., Villa, R., and Minucci, S. 2004. Altered epigenetic signals in human disease. Cancer Biol. Ther. 3:831-837.
    Grunstein, M. 1997. Histone acetylation in chromatin structure and transcription. Nature 389:349-352.
    Jenuwein, T. and Allis, C.D. 2001. Translating the histone code. Science 293:1074-1080.
    Minucci, S. and Pelicci, P.G. 2006. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. Cancer 6:38-51.
    Minucci, S. and Pelicci, P.G. 2007. Determinants of oncogenic transformation in acute promyelocytic leukemia: The hetero-union makes the force. Cancer Cell 12:1-3.
    Minucci, S., Nervi, C., Lo Coco, F., and Pelicci, P.G. 2001. Histone deacetylases: A common molecular target for differentiation treatment of acute myeloid leukemias Oncogene 20:3110-3115.
    Ronzoni, S., Faretta, M., Ballarini, M., Pelicci, P., and Minucci, S. 2005. New method to detect histone acetylation levels by flow cytometry. Cytometry A 66:52-61.
    Senese, S., Zaragoza, K., Minardi, S., Muradore, I., Ronzoni, S., Passafaro, A., Bernard, L., Draetta, G.F., Alcalay, M., Seiser, C., and Chiocca, S. 2007. Role for histone deacetylase 1 in human tumor cell proliferation. Mol. Cell Biol. 27:4784-4795.
    Strober, W. 1997. Obtaining human peripheral blood cells. Curr. Protoc. Immunol. A.3F.1-A.3F.2.
    Villa, R., De Santis, F., Gutierrez, A., Minucci, S., Pelicci, P.G., and Di Croce, L. 2004. Epigenetic gene silencing in acute promyelocytic leukemia. Biochem. Pharmacol. 68:1247-1254.
     
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