Abstract

Purpose

The classical dogma that restricted the radiation effect to the directly irradiated cells has been challenged by the bystander effect. This off-target phenomenon which was manifested in adjacent cells via signaling of fully exposed cells might be involved in high-dose Grid therapy as well. Here, an in-vitro study was performed to examine the possible extent of carcinoma cells response to the inhomogeneous dose distribution of Grid irradiation in the context of the bystander effect.

Materials and methods

Bystander effect was investigated in human carcinoma cell lines of HeLa and HN5 adjacent to those received high-dose Grid irradiation using ‘medium transfer’ and ‘cell-to-cell contact’ strategies. Based on the Grid peak-to-valley dose profile, medium transfer was exerted from 10 Gy uniformly exposed donors to 1.5 Gy uniformly irradiated recipients. Cell-contact bystander was evaluated after nonuniform dose distribution of 10 Gy Grid irradiation using cloning cylinders. GammaH2AX foci, micronucleus and clonogenic assays besides gene expression analysis were performed.

Results

Various parameters (ɑ/β, D37, D50) extracted from survival curve which fitted to the Linear Quadratic model, verified more radioresistance of HN5. Survival fraction at 2 Gy (SF2) indicated as 0.42 ± 0.06 in HeLa and 0.5 ± 0.03 in HN5. The level of survival decrease, DNA damages and micronucleus of cells located in the Grid shielded areas (1.5 Gy cell-to-cell contact bystander cells) were significantly more than the values obtained from cells which were irradiated by merely uniform dose of 1.5 Gy. The gH2AX foci and micronuclei frequencies were enhanced in cell-contact bystander approximately more than 1.8 times. Relative expression of DNA damage repair pathway genes (Xrcc6 and H2afx) in bystander cells increased significantly. The most cell survival reduction (11.6 times) was revealed in the Grid bystander cells of radioresistant cell line (HN5). No statistically significant difference between 10 Gy uniform beam and Grid non-uniform beam was observed.

Conclusions

Various endpoints confirmed an augmented response of cells in the valley dose region of the Grid block significantly (compared with the cells irradiated by identical dose of uniform beam), suggesting the role of high-dose bystander effect which was more pronounced in resistant carcinoma cell lines. These findings could provide a partial explanation for the Grid beneficial response seen in a number of pre-clinical and clinical studies.

摘要

目的

将辐射效应限制在直接辐射的细胞上的经典教条已经受到旁观者效应的挑战。通过完全暴露的细胞的信号传导在相邻细胞中表现出的脱靶现象也可能与大剂量Grid治疗有关。在这里，进行了一项体外研究，以研究在旁观者效应的背景下癌细胞对Grid辐射不均匀剂量分布的反应的可能程度。

材料与方法：采用“媒介转移”和“细胞间接触”策略，对邻近接受高剂量Grid照射的HeLa和HN5癌细胞系的旁观者效应进行了研究。根据Grid峰谷剂量分布，从10 Gy均匀暴露的供体向1.5 Gy均匀辐照的受体施加了介质转移。在使用克隆圆筒对10 Gy Grid辐射进行不均匀剂量分布之后，评估了细胞接触旁观者。除了基因表达分析外，还进行了GammaH2AX病灶，微核和克隆形成试验。

结果

从生存曲线中提取的各种参数（ɑ/β，D37，D50）与线性二次模型拟合，证明了HN5具有更高的抗辐射性。在2 Gy（SF2）下的存活率在HeLa中为0.42±0.06，在HN5中为0.5±0.03。位于Grid屏蔽区域（1.5 Gy的细胞间接触旁观者细胞）中的细胞的存活率降低，DNA损伤和微核水平明显高于仅以1.5 Gy均匀剂量照射的细胞获得的值。在细胞接触旁观者中，gH2AX焦点和微核频率提高了约1.8倍以上。DNA损伤修复途径基因（Xrcc6和H2afx的相对表达）在旁观者中明显增加。在耐辐射细胞系（HN5）的Grid旁观者细胞中发现最大的细胞存活率降低（11.6倍）。在10 Gy均匀光束和Grid非均匀光束之间没有观察到统计学上的显着差异。

结论

各种终点证实了网格阻滞谷剂量区域中细胞的增强反应显着（与相同剂量的均匀光束照射的细胞相比），表明高剂量旁观者效应的作用在耐药的癌细胞系中更为明显。这些发现可以为在许多临床前和临床研究中看到的Grid有益反应提供部分解释。