Role of p53 status in combination effect of chemotherapeutic agents and radiation

目的：包括喜树碱在内的拓扑异构酶抑制剂正在被研究为潜在的放射增敏剂。CPT-11是喜树碱的衍生物，可用于临床。本实验研究了CPT-11的活性代谢产物SN-38对4种不同P53状态的未照射和照射小鼠成纤维细胞系的影响，以阐明P53在SN-38辐射增敏作用中的作用。材料和方法：选用遗传背景相同但P53状态不同的4种成纤维细胞系MT158、MT158/neo、MT158/wtP53和MT158/mP53。用SN-38(200 Nm)处理指数生长期细胞，并与药物孵育30min。然后对细胞进行照射(0~12Gy)，并进一步与药物孵育2小时。用常规克隆形成试验测定细胞存活率。用流式细胞仪分析各处理组对细胞周期的影响。用Annexin V法检测细胞的凋亡率。结果：不同细胞系对SN-38的放射敏感性和对SN-38的处理能力无明显差异。照射和SN-38联合作用对所有4种细胞株均表现出超相加作用，与其在G2期的一过性停滞无关，单独、照射或两者合用作用8h后，无论P53状态如何，均可观察到S和G1期细胞百分比的下降。在有无野生型p53的两种细胞系中，细胞凋亡率在处理组和对照组之间均无显著差异。结论：照射与SN-38联合作用对上述4种细胞系均表现出超相加效应，P53的状态与联合作用无关。

Purpose : Topoisomerase inhibitots including camptothecin are being studied as potential radiosensitizers. CPT-11 is a derivative of camptothecin and is clinically available. We investigated here the effects of SN-38 (an active metabolite of CPT-11) on 4 unirradiated and irradiated murine fibroblast cell lines, with different statuses of p53, to clarify the role of p53 in the radiosensitizing activity of SN-38.Materials and Methods : Four fibroblast cell lines, MT158, MT158/neo, MT158/wtp53 and MT158/mp53 with the same genetic background but with different p53 statuses, were used. Exponentially growing cells were treated with SN-38 (200nM) and incubated with the drug for 30 min. Cells were then irradiated (0 to 12 Gy), and further incubated with the drug for 2h. The cell survival rate was determined using a conventional clonogenic assay. The effects of the treatments on the cell cycle were analyzed with flowcytometric assay. Apoptosis after these treatments was also detected by an Annexin V assay.Results : There were no significant differences in sensitivity to radiation or treatment of SN-38 among these cell lines. The combined treatment of irradiation and SN-38 showed supraadditive effects in all 4 cell lines independent of their p Transient arrest in G2 with a decreased percentage of cells in both the S and G1 phases was observed 8 h after treatment with either SN-38 alone, irradiation or their combination, regardless of the p53 status. No significant differences in frequency of apoptosis was observed between treatment and control groups in two cell lines with or without wild type p53.Conclusion : The combination of irradiation and treatment of SN-38 showed supraadditive effects in all 4 cell lines tested here, and the status of p53 did not play a role in the combination effect.