Kasten-Pisula Ulla, Menegakis Apostolos, Brammer Ingo, Borgmann Kerstin, Mansour Wael Y, Degenhardt Sarah, Krause Mechthild, Schreiber Andreas, Dahm-Daphi Jochen, Petersen Cordula, Dikomey Ekkehard, Baumann Michael
Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg - Eppendorf, Hamburg, Germany.
Radiother Oncol. 2009 Feb;90(2):257-64. doi: 10.1016/j.radonc.2008.10.019. Epub 2008 Nov 27.
Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity. It was the aim of this study to understand the underlying mechanisms.
MATERIALS & METHODS: Experiments were performed with SKX and FaDu, the latter taken as a control of moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts, radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay. For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Double-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and gammaH2AX-foci technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was determined by G01-assay.
Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD(50) for 10 fractions 46.0Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25Gy]) and cell cultures (D(0.01); 7.1 vs. 3.5Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by single-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aberrations when compared to FaDu cells.
The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to preferred usage of other pathways, such as SSA, which prevents efficient endjoining.
鳞状细胞癌（SCC）的特点是具有中等放射敏感性。我们建立了人头颈鳞状细胞癌细胞系SKX，其表现出极高的放射敏感性。本研究的目的是了解其潜在机制。
使用SKX和FaDu进行实验，后者作为中等放射敏感性的对照。细胞系以异种移植物和细胞培养物的形式生长。对于异种移植物，通过局部肿瘤控制试验确定放射敏感性，对于细胞培养物则使用集落形成试验。对于细胞培养物，通过Annexin V染色确定细胞凋亡，通过BrdU标记确定G1期阻滞。通过恒定电场凝胶电泳（CFGE）和γH2AX焦点技术检测双链断裂（DSB）；还通过体外连接试验评估DSB重新连接；通过G01试验确定染色体损伤。
与FaDu相比，SKX细胞在异种移植物（10次分割的TCD(50)为46.0Gy [95%置信区间：39；54 Gy]，而FaDu为18.9 Gy [95%置信区间：13；25Gy]）和细胞培养物（D(0.01)；7.1对3.5Gy）中均表现出极高的放射敏感性。两种细胞系均未显示辐射诱导的细胞凋亡或辐射诱导的永久性G1期阻滞。对于DSB，诱导方面没有差异，但SKX细胞在修复方面表现出更高水平的缓慢修复的DSB和残留DSB。体外DSB修复试验表明，SKX细胞在非同源末端连接（NHEJ）方面存在缺陷，超过40%的DSB通过单链退火（SSA）重新连接。与FaDu细胞相比，SKX细胞的致死性染色体畸变数量也高出两倍。
SCC SKX在体内和体外均表现出的极高放射敏感性可归因于NHEJ缺陷导致的DNA双链断裂修复减少。这种缺陷可能是由于优先使用其他途径，如SSA，从而阻止了有效的末端连接。
