Detection of Histone H2AX Phosphorylation on Ser‐139 as an Indicator of DNA Damage (DNA Double‐Strand Breaks)

Xuan Huang1, H. Dorota Halicka1, Zbigniew Darzynkiewicz1

1 Brander Cancer Research Institute, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.27
DOI:  10.1002/0471142956.cy0727s30
Online Posting Date:  November, 2004
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This unit describes immunocytochemical detection of phosphorylated histone H2AX for revealing the presence of DNA double‐strand breaks. Double‐strand breaks indicate DNA damage induced by ionizing radiation or by treatment with antitumor drugs such as DNA topoisomerase inhibitors. However, double‐strand breaks can also be intrinsic, occurring in healthy, nontreated cells for a variety of reasons, and are generated in the course of DNA fragmentation in apoptotic cells. The unit presents strategies to distinguish radiation‐ or drug‐induced breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes the immunocytochemical detection of histone H2AX phosphorylated on Ser‐139 combined with measurement of DNA content to identify cells that have DNA double‐strand breaks and to concurrently assess their cell cycle phase. The detection is based on indirect immunofluorescence using a FITC‐labeled secondary antibody, and DNA is counterstained with propidium iodide (PI). Cellular RNA, which may be stained by PI, is removed with RNase A.

Keywords: DNA damage; double‐strand breaks; phosphorylated histone; flow cytometry; immunofluorescence

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Table of Contents

  • Basic Protocol 1: Detection of H2AX Phosphorylated on SER‐139 in Relation to the Cell Cycle Phase
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Detection of H2AX Phosphorylated on SER‐139 in Relation to the Cell Cycle Phase

  • Cells
  • Medium
  • Agents expected to induce DSBs
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% methanol‐free formaldehyde (Polysciences) in PBS, 4°C (store ≤2 weeks at 4°C)
  • 70% ethanol, 0° to 4°C
  • 1% (w/v) BSA and 0.2% (v/v) Triton X‐100 in PBS (BSA‐T‐PBS; store ≤2 weeks at 4°C)
  • Unconjugated primary γH2AX antibody: murine monoclonal anti–histone γH2AX antibody (Upstate Biotechnology) or rabbit polyclonal anti–histone γH2AX antibody (Trevigen)
  • FITC‐conjugated secondary antibody, appropriately titered: e.g., polyclonal goat anti‐mouse or anti‐rabbit‐F(ab′)2, depending on the source of the primary antibody
  • Propidium iodide (PI) staining solution: 5 µl/ml PI (Molecular Probes) and 100 µl/ml DNase‐free RNase A (Sigma) in PBS (store ≤2 weeks protected from light at 4°C)
  • 12 × 75–mm polypropylene tubes
  • Flow cytometer with blue‐light (e.g., 488‐nm argon‐ion laser or BG‐12 excitation filter) excitation source and filters for collection of green (530 ± 20 nm) and red (>600 nm) fluorescence
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Literature Cited

Literature Cited
   Anderson, L., Henderson, C., and Adachi, Y. 2001. Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. Mol. Cell Biol. 21:1719‐29.
   Banath, J.P. and Olive, P.L. 2003. Expression of phosphorylated of histone H2AX as a surrogate of cell killing by drugs that create DNA double‐strand breaks. Cancer Res. 63:4347‐50.
   Burma, S., Chen, B.P., Murphy, M., Kurimasa, A., and Chen, D.J. 2001. ATM phosphorylates histone H2AX in response to DNA double‐strand breaks. J. Biol. Chem. 276:42462‐67.
   Celeste, A., Difilippantonio, S., Fernandez‐Capetillo, O., Pilch, D.R., Sedelnikova, O., Eckhaus, M., Ried, T., Bonner, W.M., and Nussenzweig, A. 2003. H2AX haploinsufficiency modifies genomic stabilty and tumor susceptibility. Cell 114:371‐83.
   Crul, M., van Waardenburg, R.C.A.M., Bocxe, S., van Eijndhoven, M.A.J., Pluim, D., Beijnen, J.H., and Schellens, J.H.M. 2003. DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin. Biochem. Pharmacol. 65:275‐282.
   Earnshaw, W.C., Martins, L.M., and Kaufmann, S.H. 1999. Mammalian caspases: Structure, activation, substrates and functions during apoptosis. Annu. Rev. Biochem. 68:383‐424.
   Furuta, T., Takemura, H., Liao, Z.‐Y., Aune, G.J., Redon, C., Sedelnikova, O.A., Pilch, D.R., Rogakou, E.P., Celeste, A., Chen, H.T., Nussenzweig, A., Aladjem, M.I., Bonner, W.M., and Pommier, Y. 2003. Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication‐dependent DNA double‐strand breaks induces by mammalian topoisomerase I cleavage complexes. J. Biol. Chem. 278:20303‐20312.
   Gorczyca, W., Bruno, S., Darzynkiewicz, R.J., Gong, J., and Darzynkiewicz, Z. 1992. DNA strand breaks occurring during apoptosis: Their early in situ detection by the terminal‐deoxynucleotidyl transferase and nick translation and prevention by serine protease inhibitors. Int. J. Oncol. 1:639‐648.
   Huang, X., Okafuji, M., Traganos, F., Luther, E., Holden, E., and Darzynkiewicz, Z. 2004. Assesment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by DNA cross‐linking agent cisplatin. Cytometry 58A:99‐110.
   Huang, X., Traganos, F., and Darzynkiewicz, Z. 2003. DNA damage induced by DNA topoisomerase I‐ or topoisomerase II‐ inhibitors detected by histone H2AX phosphorylation in relation to the cell cycle phase and apoptosis. Cell Cycle 2:614‐619.
   Jackson, S.P. 2001. DNA damage signaling and apoptosis. Biochem. Soc. Transactions 29:655‐61.
   MacPhail, S.H., Banath, J.P., Yu, Y., Chu, E., and Olive, P.L. 2003a. Cell cycle‐dependent expression of phosphorylated histone H2AX: Reduced expression in unirradiated but not X‐irradiated G1‐phase cells. Radiat. Res. 159:759‐67.
   MacPhail, S.H., Banath, J.P., Yu, T.Y., Chu, E.H., Lambur, H., and Olive, P.L. 2003b. Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X‐rays. Int. J. Radiat. Biol. 79:351‐358.
   Marzluff, W.F. and Duronio, R.J. 2002. Histone mRNA expression: Multiple levels of cell cycle regulation and important developmental consequences. Curr. Opin. Cell Biol. 14:692‐699.
   Nazarov, I.B., Smirnova, A.N., Krutilina, R.I., Svetlova, M.P., Solovjeva, L.V., Nikiforov, A.A., Oei, S.‐L., Zalenskaya, I.A., You, P.M., Bradbury, E.M., and Tomilin, N.V. 2003. Dephosphorylation of histone γ‐H2AX during repair of DNA double‐strand breaks in mammalian cells and its inhibition by calyculin A. Radiat. Res. 160:309‐317.
   Park, E.J., Chan, D.W., Park, J.H., Oettinger, M.A., and Kwon, J. 2003. DNA‐PK is activated by nucleosomes and phosphorylated H2AX within the nucleosomes in an acetylation‐dependent manner. Nucleic Acids Res. 31:6819‐6827.
   Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgesser, C.U., Gellert, M., and Bonner, W.M. 2000. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10:886‐985.
   Rogakou, E.P., Boon, C., Redon, C., and Bonner, W.M. 1999. Megabase chromatin domains involved in DNA double‐strand breaks in vivo. J. Cell Biol. 146:905‐916.
   Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., and Bonner, W.M. 1998. DNA double‐stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273:5858‐5868.
   Sedelnikova, O.A., Pilch, D.R., Redon, C., and Bonner, W.M. 2003. Histone H2AX in DNA damage and repair. Canc. Biol. Ther. 2:233‐235.
   Sedelnikova, O.A., Rogakou, E.P., Panuytin, I.G., and Bonner W. 2002. Quantitive detection of 125IUdr‐induced DNA double‐strand breaks with γ‐H2AX antibody. Radiat. Res. 158:486‐492.
   Speit, G. and Hartmann A. 1999. The comet assay (single cell gel test). Meth. Mol. Biol. 113:203‐212.
   Vilenchik, M.M. and Knudson, A.G. 2003. Endogenous DNA double‐strand breaks: Production, fidelity of repair, and induction of cancer. Proc. Natl. Acad. Sci. U.S.A. 100:12871‐12876
   West, M.H. and Bonner, W.M. 1980. Histone 2A, a heteromorphous family of eight protein species. Biochemistry 19:3238‐3245.
   Yoshida, K., Yoshida, S.H., Shimoda, C., and Morita, T. 2003. Expression and radiation‐induced phosphorylation of H2AX in mammalian cells. J. Radiat. Res. (Tokyo), 44:47‐51.
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