Estimation of Biologic and Molecular Aspects of Heavy Charged Particle Therapy for Cancer Cells

碳离子辐射对癌症具有平衡的双重作用：由于剂量的半影和远尾明显而具有有效的剂量定位；由于高相对生物效应（RBE）而具有强大的生物抗癌作用。然而，碳离子辐照的生物学和分子机制尚不清楚。在这项研究中，我们重点研究了碳离子辐照的生物学机制，特别是DNA双链断裂（DSBs）和修复的评估。x射线和碳离子辐照的细胞杀伤效果评估使用集落形成试验。用comat法测定双链断裂量和修复率。在碳离子照射与x射线照射对dsb修复的比较中，碳离子照射细胞中dsb残留程度较重。western blot检测DSB修复相关蛋白的表达水平。应用免疫组织化学方法对DNA修复蛋白Rad51的亚核定位进行检测。x射线照射和碳离子照射后观察到Rad51核病灶形成。碳离子处理细胞的核灶持续到照射后24小时，而x射线照射细胞的核灶在照射后24小时消失一半以上。Rad51反义寡核苷酸被转染后用碳离子照射。转染后的克隆分析显示，在碳离子照射下，Rad51反义寡核苷酸增加了辐射敏感性，而在x射线照射下，它增加了约1.7倍。用彗星法定量分析DSB的诱导和修复。用Rad51反义寡核苷酸处理的细胞与对照寡核苷酸处理的细胞在碳离子辐照后DSB诱导和修复率无差异。这些结果可能表明碳离子诱导的DSBs很难通过同源重组修复。少

Carbon ion irradiation has well-balanced dual actions on cancers : efficient doselocalization due to sharp penumbra and distal taill off in dose, and potent biological anticancer effect due to high relative biological effect (RBE). However, biological and molecular mechanisms of carbon ion irradiation have not been clarified. In this study, we focused on the biologic mechanisms of carbon ion irradiation, especially assessing DNA double strand breaks (DSBs) and repair.Cell-killing effect of X-ray and carbon ion irradiation was assessed using a colony-formation assay. The amount of double strand breaks and repair rate was assessed by comat assay. In the comparison of DSBs repair between carbon ion irradiation and X-ray irradiation, the degree of residual DSBs was severe in carbon ion irradiated cells. The expression level of DSB repair associated protein was assessed by Westem blot analysis. Sub-nuclear localization of DNA repair protein Rad51 were immunohistochemically evaluated via epi … More tluorescence microscopy. Rad51 nuclear foci formation was observed after X-ray irradiation and carbon ion irradiation. These nuclear foci in the cells treated with carbon ion were sustained to 24 hours after irradiation, whereas in the cells treated with X-ray irradiation more than half of nuclear foci were disappeared at 24 hours after irradiation. Rad51 antisense oligonucleotides was transfected and irradiated with carbonions. Clonogenic assay after transfection revealed that Rad51 antisense oligonucleotides increased radiosensitivity very little carbon ion irradiation, whereas ti increased about 1.7 fold in X-irradiation. Comet assay was also performed for quantitative analysis of DSB induction and repair. There was no difference of DSB induction and repair rate after carbon ion irradiation in the cells treated with Rad51 antisense oligonucleotides and with control oligos. These results may suggest that carbon-ion-induced DSBs are difficult to be repaired by homologous recombination. Less