Kobayashi Alisa, Tengku Ahmad Tengku Ahbrizal Farizal, Autsavapromporn Narongchai, Oikawa Masakazu, Homma-Takeda Shino, Furusawa Yoshiya, Wang Jun, Konishi Teruaki
SPICE-BIO Research Core, NIRS-International Open Laboratory, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Anagawa 4-9-1, Inage-ku, Chiba, 263-8555, Japan; Department of Accelerator and Medical Physics, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Anagawa 4-9-1, Inage-ku, Chiba, 263-8555, Japan; Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.
SPICE-BIO Research Core, NIRS-International Open Laboratory, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Anagawa 4-9-1, Inage-ku, Chiba, 263-8555, Japan; Division of Agrotechnology and Biosciences, Malaysian Nuclear Agency, Bangi, 43000, Kajang, Malaysia.
Mutat Res. 2017 Oct;803-805:1-8. doi: 10.1016/j.mrfmmm.2017.06.006. Epub 2017 Jun 23.
Understanding the mechanisms underlying the radiation-induced bystander effect (RIBE) and bi-directional signaling between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to cancer radiotherapy. The present study investigated propagation of RIBE signals between human lung carcinoma A549 cells and normal lung fibroblast WI38 cells in bystander cells, either directly or indirectly contacting irradiated A549 cells. We prepared A549-GFP/WI38 co-cultures and A549-GFP/A549 co-cultures, in which A549-GFP cells stably expressing H2BGFP were co-cultured with either A549 cells or WI38 cells, respectively. Using the SPICE-NIRS microbeam, only the A549-GFP cells were irradiated with 500 protons per cell. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured for up to 24h post-irradiation in three categories of cells: (1) "targeted"/irradiated A549-GFP cells; (2) "neighboring"/non-irradiated cells directly contacting the "targeted" cells; and (3) "distant"/non-irradiated cells, which were not in direct contact with the "targeted" cells. We found that DSB repair in targeted A549-GFP cells was enhanced by co-cultured WI38 cells. The bystander response in A549-GFP/A549 cell co-cultures, as marked by γ-H2AX levels at 8h post-irradiation, showed a decrease to non-irradiated control level when approaching 24h, while the neighboring/distant bystander WI38 cells in A549-GFP/WI38 co-cultures was maintained at a similar level until 24h post-irradiation. Surprisingly, distant A549-GFP cells in A549-GFP/WI38 co-cultures showed time dependency similar to bystander WI38 cells, but not to distant cells in A549-GFP/A549 co-cultures. These observations indicate that γ-H2AX was induced in WI38 cells as a result of RIBE. WI38 cells were not only involved in rescue of targeted A549, but also in the modification of RIBE against distant A549-GFP cells. The present results demonstrate that radiation-induced bi-directional signaling had extended a profound influence on cellular sensitivity to radiation as well as the sensitivity to RIBE.
了解辐射诱导旁观者效应（RIBE）的潜在机制以及受照射癌细胞与其周围未受照射的正常细胞之间的双向信号传导，对于癌症放射治疗具有重要意义。本研究调查了RIBE信号在人肺癌A549细胞与正常肺成纤维细胞WI38细胞之间的传播情况，这些旁观者细胞与受照射的A549细胞直接或间接接触。我们制备了A549-GFP/WI38共培养物和A549-GFP/A549共培养物，其中稳定表达H2BGFP的A549-GFP细胞分别与A549细胞或WI38细胞共培养。使用SPICE-NIRS微束，仅对每个A549-GFP细胞照射500个质子。随后在照射后长达24小时内，对三类细胞中DNA双链断裂（DSB）的标志物γ-H2AX水平进行了测量：（1）“靶向”/受照射的A549-GFP细胞；（2）“相邻”/未受照射且直接接触“靶向”细胞的细胞；（3）“远处”/未受照射且未与“靶向”细胞直接接触的细胞。我们发现，共培养的WI38细胞增强了靶向A549-GFP细胞中的DSB修复。在A549-GFP/A549细胞共培养物中，以照射后8小时的γ-H2AX水平为标志的旁观者反应，在接近24小时时降至未受照射的对照水平，而在A549-GFP/WI38共培养物中，相邻/远处的旁观者WI38细胞在照射后24小时内保持在相似水平。令人惊讶的是，A549-GFP/WI38共培养物中远处的A549-GFP细胞显示出与旁观者WI38细胞相似的时间依赖性，但与A549-GFP/A549共培养物中的远处细胞不同。这些观察结果表明，RIBE导致WI38细胞中诱导产生γ-H2AX。WI38细胞不仅参与了靶向A549细胞的修复，还参与了对远处A549-GFP细胞的RIBE修饰。目前的结果表明，辐射诱导的双向信号传导对细胞对辐射的敏感性以及对RIBE的敏感性产生了深远影响。
