https://orcid.org/0000-0001-6625-9785
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Abstract
Purpose
Radiation-induced bystander effect (RIBE) frequently is seen as DNA damage in unirradiated bystander cells, but the repair processes initiated in response to that DNA damage are not well understood. RIBE-mediated formation of micronuclei (MN), a biomarker of persistent DNA damage, was previously observed in bystander normal fibroblast (AG01522) cells, but not in bystander human chondrosarcoma (HTB94) cells. The molecular mechanisms causing this disparity are not clear. Herein, we investigate the role of DNA repair in the bystander responses of the two cell lines.
Methods
Cells were irradiated with X-rays and immediately co-cultured with un-irradiated cells using a trans-well insert system in which they share the same medium. The activation of DNA damage response (DDR) proteins was detected by immunofluorescence staining or Western blotting. MN formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage.
Results
Immunofluorescent foci of γH2AX and 53BP1, biomarkers of DNA damage and repair, revealed a greater capacity for DNA repair in HTB94 cells than in AG01522 cells in both irradiated and bystander populations. Autophosphorylation of ATR at the threonine 1989 site was expressed at a greater level in HTB94 cells compared to AG01522 cells at the baseline and in response to hydroxyurea treatment or exposure to 1 Gy of X-rays. An inhibitor of ATR, but not of ATM, promoted MN formation in bystander HTB94 cells. In contrast, no effect of either inhibitor was observed in bystander AG01522 cells, indicating that ATR signaling might be a pivotal pathway to preventing the MN formation in bystander HTB94 cells. Supporting this idea, we found an ATR-dependent increase in the fractions of bystander HTB94 cells with pRPA2 S33 and RAD51 foci. A blocker of RAD51 facilitated MN formation in bystander HTB94 cells.
Conclusion
Our results indicate that HTB94 cells were likely more efficient in DNA repair than AG01522 cells, specifically via ATR signaling, which inhibited the bystander signal-induced MN formation. This study highlights the significance of DNA repair efficiency in bystander cell responses.
Acknowledgments
We would like to acknowledge Ms. Yoshika Machida (Gunma University) and Mr. Mark E. Essien (Emory University) for laboratory assistance. We thank Dr. Noriyuki Okonogi, Dr. Anggraeini Puspitasari, and Dr. Sandip K. Rath for many helpful discussions throughout the project.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
Notes on contributors
Nho Cong Luong
Nho Cong Luong, PhD, is Postdoctoral Researcher at Department of Radiation Oncology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA. He was a postdoc at Gunma University Initiative for Advanced Research, Gunma University, Gunma, Japan.
Hidemasa Kawamura
Hidemasa Kawamura, MD, PhD, is Professor at Gunma University Heavy Ion Medical Center, Gunma University, Gunma, Japan.
Hiroko Ikeda
Hiroko Ikeda, PhD, is Assistant Professor at Department of Life Science, Faculty of Science and Engineering, Kindai University, Osaka, Japan.
Reiko T. Roppongi
Reiko T. Roppongi, PhD, is Assistant Professor at Gunma University Initiative for Advanced Research, Gunma University, Gunma, Japan.
Atsushi Shibata
Atsushi Shibata, PhD, is Professor at Division of Molecular Oncological Pharmacy, Faculty of Pharmacy, Keio University, Tokyo, Japan.
Jiaxuan Hu
Jiaxuan Hu is a student majoring in Biology at Emory University School of Medicine, Atlanta, GA, USA.
Jinmeng G. Jiang
Jinmeng G. Jiang is a student majoring in Biology at Emory University School of Medicine, Atlanta, GA, USA.
David S. Yu
David S. Yu, MD, PhD, is the Jerome Landry MD Chair in Cancer Research and Professor at Department of Radiation Oncology and Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA, where he also serves as director of the Division of Cancer Biology.
Kathryn D. Held
Kathryn D. Held, PhD, is President of the National Council on Radiation Protection and Measurements (NCRP), Bethesda, MD, USA; Associate Professor and Radiation Biologist at Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA; and Distinguished Visiting Professor at Gunma University Initiative for Advanced Research, Gunma University, Gunma, Japan.