Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Laboratory of Radiation Oncology, Department of Radiation Oncology, Lausanne University Hospital and University of Lausanne, Switzerland; Department of Radiation Oncology, University of California, Irvine, CA 92697-2695, USA.
Radiother Oncol. 2023 Nov;188:109906. doi: 10.1016/j.radonc.2023.109906. Epub 2023 Sep 9.
The impact of radiotherapy (RT) at ultra high vs conventional dose rate (FLASH vs CONV) on the generation and repair of DNA double strand breaks (DSBs) is an important question that remains to be investigated. Here, we tested the hypothesis as to whether FLASH-RT generates decreased chromosomal translocations compared to CONV-RT.
We used two FLASH validated electron beams and high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), employing S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs) in HEK239T cells, to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated after various irradiation doses, dose rates and oxygen tensions (normoxic, 21% O; physiological, 4% O; hypoxic, 2% and 0.5% O). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13 Gy/s) and FLASH (1x10-5x10 Gy/s) dose rates. Related experiments using clonogenic survival and γH2AX foci in the 293T and the U87 glioblastoma lines were also performed to discern FLASH-RT vs CONV-RT DSB effects.
Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Furthermore, RT dose rate modality on U87 cells did not change γH2AX foci numbers at 1- and 24-hours post-irradiation nor did this affect 293T clonogenic survival.
Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.
放射治疗（RT）在超高与常规剂量率（FLASH 与 CONV）下对 DNA 双链断裂（DSBs）的产生和修复的影响是一个尚未解决的重要问题。在此，我们通过测试下述假说，探究 FLASH-RT 是否比 CONV-RT 产生更少的染色体易位：FLASH-RT 生成的染色体易位比 CONV-RT 少。
我们使用两种经过 FLASH 验证的电子束和高通量重连及全基因组易位测序（HTGTS-JoinT-seq），在 HEK239T 细胞中使用金黄色葡萄球菌和化脓性链球菌 Cas9“诱饵”DNA 双链断裂（DSBs），以测量不同照射剂量、剂量率和氧张力（常氧，21%O；生理氧，4%O；低氧，2%和 0.5%O）下，诱饵附近修复和其全基因组易位到“猎物”DSB 的差异。电子照射使用具备 FLASH 能力的瓦里安 Trilogy 和 eRT6/Oriatron 以 CONV（0.08-0.13 Gy/s）和 FLASH（1x10-5x10 Gy/s）剂量率进行。还使用 293T 和 U87 胶质母细胞瘤系中的克隆存活和 γH2AX 焦点相关实验，来辨别 FLASH-RT 与 CONV-RT DSB 效应。
常氧和生理氧照射 HEK293T 细胞增加了易位，但以诱饵附近修复减少为代价，且在 CONV-RT 和 FLASH-RT 之间无法区分。虽然缺氧诱导凋亡时没有观察到染色体易位明显增加，但随着 IR 剂量率调制降低氧张力，并未发现易位水平或其连接结构有显著差异。此外，U87 细胞的 RT 剂量率模式不会改变照射后 1 小时和 24 小时的 γH2AX 焦点数量，也不会影响 293T 细胞的克隆存活。
无论氧张力如何，FLASH-RT 产生的易位和连接结构的水平和比例与 CONV-RT 无法区分。
