Automatic analysis of chromosomal DNA strand breaks and its application to therapy

染色体DNA链断裂的自动分析及其在治疗中的应用

• 批准号: 10557248
• 负责人: TERAOKA Hirobumi
• 金额: $ 8.32万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10557248/
• 关键词: flow cytometry; DNA strand breaks; SCID; cell cycle; β-ray; DNA double-strand breaks; V(D)J recombination; apoptosis

The purpose of this research is to estimate quantitatively chromosomal DNA strand breaks by flow cytometry. At first, we analyzed relationship between cell cycle progression and DNA strand breaks induced by incorporation of [ィイD13ィエD1H]thymidine into human hemopoietic cell lines, such as HL-60, Molt-4 and Raji. The ends of chromosomal DNA strand breaks in a cell were labeled with the FITC-dUTP/TdT system. In 7.4 kBq [ィイD13ィエD1H]thymidine/ml, cell cycle of HL-60 progressed with a slight increase in the population of S-phase cells, and the relative value of FITC, which reflects the number of DNA strand breaks per cell, were significantly high at 8 and 18 h with decrease to the basal level at 24 h. Analysis of HィイD22ィエD2OィイD22ィエD2-resistant HL-60 variants revealed involvement of reactive oxygen species in the effects of [ィイD13ィエD1H]thymidine incorporation on cell cycle and cell fate. A human glioma cell line, M059J, lacking DNA-PK involved in DNA double-strand break repair showed higher sensitivity to neocarzinostatin (NCS) compared with M059K containing wild-type DNA-PK. Addition of NCS at 50 ng/ml resulted in increase in S-phase cell population at 30 min in M059J with gradual increase in FITC signals. In contrast, there were no significant changes in DNA histogram patterns in M059K in the presence of 50 ng NCS/ml, and transient increase in the relative value of FITC was observed maximally 2 h after the addition. These results demonstrated that the number of chromosomal DNA strand breaks per cell could be estimated semi-quantitatively by flow cytometry. Now it is possible to predict efficacy and side effects of individual therapy with radioactive materials and radiomimetic drugs.

本研究的目的是用流式细胞术定量估计染色体DNA链断裂。首先,我们分析了细胞周期进程和DNA链断裂之间的关系引起的[ィイD13ィエD1H]胸苷对人类造血细胞系,如HL-60 Molt-4 Raji。用FITC-dUTP/TdT系统标记细胞中染色体DNA链断裂的末端。在7.4 kBq[ィイD13ィエD1H]胸苷/毫升,细胞周期的HL-60进展与略有增加的人口s阶段细胞,和FITC的相对价值,它反映了每个细胞的DNA链断裂数,明显高8点和18 h和24小时的基础水平降低。分析hィイD22摊位ィエD2OィイD22摊位ィエD2-resistant HL-60变异显示参与活性氧的影响[ィイD13ィエD1H)胸腺嘧啶核苷掺入对细胞周期和细胞命运。缺乏参与DNA双链断裂修复的DNA- pk的人胶质瘤细胞系M059J与含有野生型DNA- pk的M059K相比，对neocarzinostatatin （NCS）的敏感性更高。添加50 ng/ml的NCS可使M059J在30 min时s期细胞数量增加，FITC信号逐渐增加。相反，在50 ng NCS/ml的作用下，M059K的DNA直方图模式没有明显变化，FITC的相对值在添加后2 h达到最大值。这些结果表明，流式细胞术可以半定量地估计每个细胞染色体DNA链断裂的数量。现在可以预测放射性物质和拟放射性药物单独治疗的疗效和副作用。

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