title: Absolute quantification of acetylation and phosphorylation of the histone variant H2AX upon ionizing radiation reveals distinct cellular responses in two cancer cell lines.
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.
authors.affiliation: Laboratory of Environment Quality Management, Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga, 520-0811, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Matsuda
authors.affiliation: Laboratory of Cell Cycle Response, Department of Mutagenesis, Radiation Biology Center, Kyoto University, Yoshidakonoecho, Kyoto Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Kanji
authors.initials: K
authors.lastname: Furuya
authors.affiliation: Laboratory of Chromatin Regulatory Network, Department of Mutagenesis, Radiation Biology Center, Kyoto University, Yoshidakonoecho, Kyoto Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Masae
authors.initials: M
authors.lastname: Ikura
authors.affiliation: Laboratory of Environment Quality Management, Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga, 520-0811, Japan.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: Laboratory of Chromatin Regulatory Network, Department of Mutagenesis, Radiation Biology Center, Kyoto University, Yoshidakonoecho, Kyoto Sakyo-ku, Kyoto, 606-8501, Japan. ikurat@house.rbc.kyoto-u.ac.jp.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ikura
mesh_terms: D000107:Acetylation / Q000528:radiation effects; D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D014644:Genetic Variation / Q000235:genetics / Q000528:radiation effects; D006367:HeLa Cells; D006657:Histones / Q000235:genetics* / Q000528:radiation effects*; D006801:Humans; D009374:Neoplasms, Experimental / Q000473:pathology / Q000503:physiopathology*; D010766:Phosphorylation / Q000528:radiation effects; D011829:Radiation Dosage
chemical_list: C468783:H2AX protein, human; D006657:Histones
keywords: Absolute quantification; Acetylation; DNA damage; Multiple reaction monitoring/selected reaction monitoring (MRM/SRM); Phosphorylation; γH2AX