Skip to main content

Advertisement

Log in

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

  • Original Paper
  • Published:
Radiation and Environmental Biophysics Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Ando M, Yoshikawa K, Iwase Y, Ishiura S (2014) Usefulness of monitoring gamma-H2AX and cell cycle arrest in HepG2 cells for estimating genotoxicity using a high-content analysis system. J Biomol Screen 19:1246–1254. doi:10.1177/1087057114541147

    Article  Google Scholar 

  • Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ (2001) ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 276:42462–42467. doi:10.1074/jbc.C100466200

    Article  Google Scholar 

  • Cha H, Lowe JM, Li H, Lee JS, Belova GI, Bulavin DV, Fornace AJ Jr (2010) Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response. Cancer Res 70:4112–4122. doi:10.1158/0008-5472.CAN-09-4244

    Article  Google Scholar 

  • Chowdhury D, Keogh MC, Ishii H, Peterson CL, Buratowski S, Lieberman J (2005) Gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Mol Cell 20:801–809. doi:10.1016/j.molcel.2005.10.003

    Article  Google Scholar 

  • Douglas P, Zhong J, Ye R, Moorhead GB, Xu X, Lees-Miller SP (2010) Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit and dephosphorylates gamma-H2AX. Mol Cell Biol 30:1368–1381. doi:10.1128/MCB.00741-09

    Article  Google Scholar 

  • Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A, Liloglou T, Venere M, Ditullio RA Jr, Kastrinakis NG, Levy B, Kletsas D, Yoneta A, Herlyn M, Kittas C, Halazonetis TD (2005) Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434:907–913. doi:10.1038/nature03485

    Article  ADS  Google Scholar 

  • Hunt CR, Ramnarain D, Horikoshi N, Iyengar P, Pandita RK, Shay JW, Pandita TK (2013) Histone modifications and DNA double-strand break repair after exposure to ionizing radiations. Radiat Res 179:383–392. doi:10.1667/RR3308.2

    Article  Google Scholar 

  • Ikura T, Tashiro S, Kakino A, Shima H, Jacob N, Amunugama R, Yoder K, Izumi S, Kuraoka I, Tanaka K, Kimura H, Ikura M, Nishikubo S, Ito T, Muto A, Miyagawa K, Takeda S, Fishel R, Igarashi K, Kamiya K (2007) DNA damage-dependent acetylation and ubiquitination of H2AX enhances chromatin dynamics. Mol Cell Biol 27:7028–7040. doi:10.1128/MCB.00579-07

    Article  Google Scholar 

  • Kao J, Milano MT, Javaheri A, Garofalo MC, Chmura SJ, Weichselbaum RR, Kron SJ (2006) Gamma-H2AX as a therapeutic target for improving the efficacy of radiation therapy. Curr Cancer Drug Targets 6:197–205

    Article  Google Scholar 

  • Khoury L, Zalko D, Audebert M (2013) Validation of high-throughput genotoxicity assay screening using gammaH2AX in-cell western assay on HepG2 cells. Environ Mol Mutagen 54:737–746. doi:10.1002/em.21817

    Article  Google Scholar 

  • Macurek L, Lindqvist A, Voets O, Kool J, Vos HR, Medema RH (2010) Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition. Oncogene 29:2281–2291. doi:10.1038/onc.2009.501

    Article  Google Scholar 

  • Matsuda S, Ikura T, Matsuda T (2015) Absolute quantification of gammaH2AX using liquid chromatography-triple quadrupole tandem mass spectrometry. Anal Bioanal Chem. doi:10.1007/s00216-015-8725-z

    MATH  Google Scholar 

  • Moon SH, Nguyen TA, Darlington Y, Lu X, Donehower LA (2010) Dephosphorylation of gamma-H2AX by WIP1: an important homeostatic regulatory event in DNA repair and cell cycle control. Cell Cycle 9:2092–2096

    Article  Google Scholar 

  • Padovani L, Caporossi D, Tedeschi B, Vernole P, Nicoletti B, Mauro F (1993) Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident. Mutat Res 319:55–60

    Article  Google Scholar 

  • Podhorecka M, Skladanowski A, Bozko P (2010) H2AX phosphorylation: its role in DNA damage response and cancer therapy. J Nucleic Acids. doi:10.4061/2010/920161

    Google Scholar 

  • Redon CE, Nakamura AJ, Zhang YW, Ji JJ, Bonner WM, Kinders RJ, Parchment RE, Doroshow JH, Pommier Y (2010) Histone gammaH2AX and poly(ADP-ribose) as clinical pharmacodynamic biomarkers. Clin Cancer Res 16:4532–4542. doi:10.1158/1078-0432.CCR-10-0523

    Article  Google Scholar 

  • Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146:905–916

    Article  Google Scholar 

  • Sanchez-Flores M, Pasaro E, Bonassi S, Laffon B, Valdiglesias V (2015) GammaH2ax assay as DNA damage biomarker for human population studies: defining experimental conditions. Toxicol Sci. doi:10.1093/toxsci/kfv011

    Google Scholar 

  • Sedelnikova OA, Bonner WM (2006) GammaH2AX in cancer cells: a potential biomarker for cancer diagnostics, prediction and recurrence. Cell Cycle 5:2909–2913

    Article  Google Scholar 

  • Smart DJ, Ahmedi KP, Harvey JS, Lynch AM (2011) Genotoxicity screening via the gammaH2AX by flow assay. Mutat Res 715:25–31. doi:10.1016/j.mrfmmm.2011.07.001

    Article  Google Scholar 

  • Stiff T, O’Driscoll M, Rief N, Iwabuchi K, Lobrich M, Jeggo PA (2004) ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res 64:2390–2396

    Article  Google Scholar 

  • Taneja N, Davis M, Choy JS, Beckett MA, Singh R, Kron SJ, Weichselbaum RR (2004) Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy. J Biol Chem 279:2273–2280. doi:10.1074/jbc.M310030200

    Article  Google Scholar 

  • Tsamou M, Jennen DG, Claessen SM, Magkoufopoulou C, Kleinjans JC, van Delft JH (2012) Performance of in vitro gammaH2AX assay in HepG2 cells to predict in vivo genotoxicity. Mutagenesis 27:645–652. doi:10.1093/mutage/ges030

    Article  Google Scholar 

  • Ward IM, Chen J (2001) Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem 276:47759–47762. doi:10.1074/jbc.C100569200

    Article  Google Scholar 

  • Watters GP, Smart DJ, Harvey JS, Austin CA (2009) H2AX phosphorylation as a genotoxicity endpoint. Mutat Res 679:50–58. doi:10.1016/j.mrgentox.2009.07.007

    Article  Google Scholar 

  • Wu J, Clingen PH, Spanswick VJ, Mellinas-Gomez M, Meyer T, Puzanov I, Jodrell D, Hochhauser D, Hartley JA (2013) Gamma-H2AX foci formation as a pharmacodynamic marker of DNA damage produced by DNA cross-linking agents: results from 2 phase I clinical trials of SJG-136 (SG2000). Clin Cancer Res 19:721–730. doi:10.1158/1078-0432.CCR-12-2529

    Article  Google Scholar 

Download references

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.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tsuyoshi Ikura.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 355 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Matsuda, S., Furuya, K., Ikura, M. et al. Absolute quantification of acetylation and phosphorylation of the histone variant H2AX upon ionizing radiation reveals distinct cellular responses in two cancer cell lines. Radiat Environ Biophys 54, 403–411 (2015). https://doi.org/10.1007/s00411-015-0608-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00411-015-0608-3

Keywords

Navigation