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Radiation and Environmental Biophysics

, Volume 54, Issue 4, pp 403–411 | Cite as

Absolute quantification of acetylation and phosphorylation of the histone variant H2AX upon ionizing radiation reveals distinct cellular responses in two cancer cell lines

  • Shun Matsuda
  • Kanji Furuya
  • Masae Ikura
  • Tomonari Matsuda
  • Tsuyoshi IkuraEmail author
Original Paper

Abstract

Histone modifications change upon the cellular response to ionizing radiation, and their cellular amounts could reflect the DNA damage response activity. We previously reported a sensitive and reliable method for the absolute quantification of γH2AX within cells, using liquid chromatography–tandem mass spectrometry (LC/MS/MS). The technique has broad adaptability to a variety of biological systems and can quantitate different modifications of histones. In this study, we applied it to quantitate the levels of γH2AX and K5-acetylated H2AX, and to compare the radiation responses between two cancer cell lines: HeLa and U-2 OS. The two cell lines have distinct properties in terms of their H2AX modifications. HeLa cells have relatively high γH2AX (3.1 %) against the total H2AX even in un-irradiated cells, while U-2 OS cells have an essentially undetectable level (nearly 0 %) of γH2AX. In contrast, the amounts of acetylated histones are lower in HeLa cells (9.3 %) and higher in U-2 OS cells (24.2 %) under un-irradiated conditions. Furthermore, after ionizing radiation exposure, the time-dependent increases and decreases in the amounts of histone modifications differed between the two cell lines, especially at the early time points. These results suggest that each biological system has distinct kinase/phosphatase and/or acetylase/deacetylase activities. In conclusion, for the first time, we have succeeded in simultaneously monitoring the absolute amounts of phosphorylated and acetylated cellular H2AX after ionizing radiation exposure. This multi-criteria assessment enables precise comparisons of the effects of radiation between any biological systems.

Keywords

γH2AX Multiple reaction monitoring/selected reaction monitoring (MRM/SRM) Absolute quantification DNA damage Acetylation Phosphorylation 

Notes

Acknowledgments

This study was supported by KAKENHI (23221006) from the Japan Society for the Promotion of Science (S.M. and T.M.) and Grants-in-Aid for Scientific Research on Innovative Areas (22131001) (K.F. and T.I.).

Supplementary material

411_2015_608_MOESM1_ESM.docx (355 kb)
Supplementary material 1 (DOCX 355 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Shun Matsuda
    • 1
  • Kanji Furuya
    • 2
  • Masae Ikura
    • 3
  • Tomonari Matsuda
    • 1
  • Tsuyoshi Ikura
    • 3
    Email author
  1. 1.Laboratory of Environment Quality Management, Research Center for Environmental Quality ManagementKyoto UniversityOtsuJapan
  2. 2.Laboratory of Cell Cycle Response, Department of Mutagenesis, Radiation Biology CenterKyoto UniversityKyotoJapan
  3. 3.Laboratory of Chromatin Regulatory Network, Department of Mutagenesis, Radiation Biology CenterKyoto UniversityKyotoJapan

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