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

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• 作者：Shun Matsuda ; Kanji Furuya ; Masae Ikura…
• 关键词：γH2AX ; Multiple reaction monitoring/selected reaction monitoring (MRM/SRM) ; Absolute quantification ; DNA damage ; Acetylation ; Phosphorylation
• 刊名：Radiation and Environmental Biophysics
• 出版年：2015
• 出版时间：November 2015
• 年：2015
• 卷：54
• 期：4
• 页码：403-411
• 全文大小：923 KB
• 参考文献：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 19:1246-254. doi:10.-177/-087057114541147-/span> CrossRef
  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 276:42462-2467. doi:10.-074/?jbc.?C100466200 CrossRef
  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 70:4112-122. doi:10.-158/-008-5472.?CAN-09-4244 CrossRef
  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 20:801-09. doi:10.-016/?j.?molcel.-005.-0.-03 CrossRef
  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 30:1368-381. doi:10.-128/?MCB.-0741-09 CrossRef
  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 434:907-13. doi:10.-038/?nature03485 CrossRef ADS
  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 179:383-92. doi:10.-667/?RR3308.- CrossRef
  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 27:7028-040. doi:10.-128/?MCB.-0579-07 CrossRef
  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 6:197-05CrossRef
  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 54:737-46. doi:10.-002/?em.-1817 CrossRef
  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 29:2281-291. doi:10.-038/?onc.-009.-01 CrossRef
  Matsuda S, Ikura T, Matsuda T (2015) Absolute quantification of gammaH2AX using liquid chromatography-triple quadrupole tandem mass spectrometry. Anal Bioanal Chem. doi:10.-007/?s00216-015-8725-z MATH
  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 9:2092-096CrossRef
  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 319:55-0CrossRef
  Podhorecka M, Skladanowski A, Bozko P (2010) H2AX phosphorylation: its role in DNA damage response and cancer therapy. J Nucleic Acids. doi:10.-061/-010/-20161
  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 16:4532-542. doi:10.-158/-078-0432.?CCR-10-0523 CrossRef
  Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146:905-16CrossRef
  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.-093/?toxsci/?kfv011
  Sedelnikova OA, Bonner WM (2006) GammaH2AX in cancer cells: a potential biomarker for cancer diagnostics, prediction and recurrence. Cell Cycle 5:2909-913CrossRef
  Smart DJ, Ahmedi KP, Harvey JS, Lynch AM (2011) Genotoxicity screening via the gammaH2AX by flow assay. Mutat Res 715:25-1. doi:10.-016/?j.?mrfmmm.-011.-7.-01 CrossRef
  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 64:2390-396CrossRef
  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 radio
• 作者单位：Shun Matsuda (1)
  Kanji Furuya (2)
  Masae Ikura (3)
  Tomonari Matsuda (1)
  Tsuyoshi Ikura (3)
  
  1. Laboratory of Environment Quality Management, Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga, 520-0811, Japan
  2. Laboratory of Cell Cycle Response, Department of Mutagenesis, Radiation Biology Center, Kyoto University, Yoshidakonoecho, Kyoto Sakyo-ku, Kyoto, 606-8501, Japan
  3. Laboratory of Chromatin Regulatory Network, Department of Mutagenesis, Radiation Biology Center, Kyoto University, Yoshidakonoecho, Kyoto Sakyo-ku, Kyoto, 606-8501, Japan
  
• 刊物类别：Physics and Astronomy
• 刊物主题：Physics
  Biophysics and Biomedical Physics
  Effects of Radiation and Radiation Protection
  Environmental Physics
  Environmental Computing and Modeling
  Ecosystems
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-2099

文摘

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