3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation

Abstract

// Yoshihiko Hagiwara 1, 2, * , Atsuko Niimi 3, * , Mayu Isono 4 , Motohiro Yamauchi 5 , Takaaki Yasuhara 6 , Siripan Limsirichaikul 7 , Takahiro Oike 2 , Hiro Sato 2 , Kathryn D. Held 8, 9 , Takashi Nakano 2, 3 and Atsushi Shibata 1 1 Education and Research Support Center (ERSC), Gunma University, Maebashi 371-8511, Japan 2 Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan 3 Research Program for Heavy Ion Therapy, Division of Integrated Oncology Research, Gunma University Initiative for Advanced Research (GIAR), Maebashi 371-8511, Japan 4 Department of Molecular Metabolic Regulation Research, Sasaki Institute, Tokyo 101-0062, Japan 5 Department of Radiation Biology and Protection, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki 852-8523, Japan 6 Laboratory of Molecular Radiology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan 7 Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand 8 Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114, USA 9 International Open Laboratory, Gunma University Initiative for Advanced Research (GIAR), Gunma 371-8511, Japan * These authors have equally contributed to this work Correspondence to: Atsushi Shibata, email: shibata.at@gunma-u.ac.jp Keywords: clustered DNA double strand break; heavy-ion radiation; carbon-ion therapy; γH2AX foci; RPA foci Received: June 22, 2017      Accepted: November 08, 2017      Published: November 25, 2017 ABSTRACT DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1–2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G 2 -phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm 3 . These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.