PDF(Pubmed)
Abstract：
OBJECTIVE: 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.
METHODS: 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% O2; physiological, 4% O2; hypoxic, 2% and 0.5% O2). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13 Gy/s) and FLASH (1x102-5x106 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.
RESULTS: 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.
CONCLUSIONS: Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.
METHODS: 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% O2; physiological, 4% O2; hypoxic, 2% and 0.5% O2). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13 Gy/s) and FLASH (1x102-5x106 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.
RESULTS: 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.
CONCLUSIONS: Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.
摘要：
目的：超高剂量率（FLASHvsCONV）放疗对DNA双链断裂（DSBs）产生和修复的影响是一个有待研究的重要问题。这里,我们检验了关于FLASH-RT与CONV-RT相比是否产生减少的染色体易位的假设.
方法：我们使用了两个经过FLASH验证的电子束和高通量重组和全基因组易位测序（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（DSB），为了测量诱饵近端修复的差异及其在各种辐照剂量后产生的全基因组易位到“猎物”DSB的差异，剂量率和氧气张力（常氧，21%O2;生理,4%O2;低氧,2%和0.5%O2)。使用具有FLASH功能的VarianTrilogy和eRT6/Oriatron以CONV（0.08-0.13Gy/s）和FLASH（1x102-5x106Gy/s）的剂量率进行电子辐照。还进行了使用293T和U87胶质母细胞瘤细胞系中的克隆形成存活和γH2AX灶的相关实验，以辨别FLASH-RT与CONV-RTDSB的影响。
结果：正常氧和生理氧照射HEK293T细胞以减少诱饵近端修复为代价增加易位，但CONV-RT和FLASH-RT之间没有区别。尽管缺氧诱导的细胞凋亡没有观察到染色体易位的明显增加，氧张力的降低与IR剂量率调节相结合，并未显示易位水平或其连接结构的显着差异。此外，U87细胞上的RT剂量率模式在辐照后1小时和24小时均未改变γH2AX灶的数量，也未影响293T克隆形成的存活。
结论：无论氧张力如何，FLASH-RT以与CONV-RT无法区分的水平和比例产生易位和连接结构。
方法：我们使用了两个经过FLASH验证的电子束和高通量重组和全基因组易位测序（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（DSB），为了测量诱饵近端修复的差异及其在各种辐照剂量后产生的全基因组易位到“猎物”DSB的差异，剂量率和氧气张力（常氧，21%O2;生理,4%O2;低氧,2%和0.5%O2)。使用具有FLASH功能的VarianTrilogy和eRT6/Oriatron以CONV（0.08-0.13Gy/s）和FLASH（1x102-5x106Gy/s）的剂量率进行电子辐照。还进行了使用293T和U87胶质母细胞瘤细胞系中的克隆形成存活和γH2AX灶的相关实验，以辨别FLASH-RT与CONV-RTDSB的影响。
结果：正常氧和生理氧照射HEK293T细胞以减少诱饵近端修复为代价增加易位，但CONV-RT和FLASH-RT之间没有区别。尽管缺氧诱导的细胞凋亡没有观察到染色体易位的明显增加，氧张力的降低与IR剂量率调节相结合，并未显示易位水平或其连接结构的显着差异。此外，U87细胞上的RT剂量率模式在辐照后1小时和24小时均未改变γH2AX灶的数量，也未影响293T克隆形成的存活。
结论：无论氧张力如何，FLASH-RT以与CONV-RT无法区分的水平和比例产生易位和连接结构。
