Induction of Mitochondrial-DNA Mutation by X-ray Irradiation.

之前我们分析了食管肿瘤中线粒体DNA(mtDNA) d环区域的突变，发现了常见的体细胞突变（在34%的肿瘤中）。我们还确定了核基因组的不稳定性，但没有发现体细胞mtDNA突变与核基因组不稳定性之间的任何相关性，这表明食管癌mtDNA的不稳定性可能独立于核基因组的不稳定性。为了了解x射线等诱变剂是否可以诱导mtDNA突变，我们筛选了4种食管癌细胞系KYSE-30、110、150和410以及正常细胞系SuSa/T-n在x射线照射后的mtDNA突变。用x射线照射这些细胞系，每个细胞系的存活率为0.01。采集单细胞菌落后，提取DNA。由于每个细胞中有多达10^3个mtDNA拷贝，并且可能不会在所有mtDNA中发生相同的突变，因此通过评估突变mtDNA与总mtDNA的比例来进行评估。由于mtDNA中具有7个连续C链的D310区域经常出现1个碱基插入或缺失突变，我们使用GeneScan对该区域进行分析，将mtDNA突变比例大于0.3的菌落定义为突变菌落。在SuSa/T-n中，x射线照射后D310长度未见变化，两种食管癌细胞系KYSE-150和410也未见变化改变。另一方面，在食管癌细胞系KYSE-30和110中，11.8%和31.8%的菌落在D310区出现长度变化。由于p53基因在这两种mtDNA突变阳性细胞系中发生突变，而在mtDNA突变阴性细胞系中没有发生突变，p53基因可能在mtDNA稳定性中发挥重要作用。

Previously we analyzed mutations in the D-loop region of mitochondrial DNA(mtDNA) in esophageal tumors and found frequent somatic mutations(in 34 % of tumors) . We also determined nuclear genomic instability, but did not find any correlation between somatic mtDNA mutations and nuclear genomic instability, suggesting that instability of mtDNA in esophageal cancer might be independent of nuclear genomic instability. To know whether mtDNA mutations could be induced by mutageinc agents such as X-ray, we screened for mtDNA mutations in 4 esophageal cancer cell lines, KYSE-30, 110, 150 and 410, and a normal cell line, SuSa/T-n, after X-ray irradiation. These cell lines were irradiated with X-ray at the doses giving survival rate of 0.01 in each cell line. After single-cell colonies were picked up, DNA was extracted. Since there are up to 10^3 copies of mtDNA in each cell and the same mutations may not occur in all mtDNA, evaluation was conducted by assessing the ratio of mutated mtDNA to the total mtDNA. Since the D310 region which has a 7 continuous C stretch in mtDNA frequently showed 1 base insertion or deletion mutations, we analyzed this region by GeneScan, Colonies with more than 0.3 for the proportion of mutated mtDNA were defined as mutant colonies. In SuSa/T-n, no change was observed in D310 length after X-ray irradiation, and two esophageal cancer cell lines, KYSE-150 and 410, also did not exhibit change alteration. On the other hand, 11.8% and 31.8% of colonies from irradiated cells of the esophageal cancer cell lines, KYSE-30 and 110, exhibited length changes in the D310 region. Since the p53 gene was mutated in these two mtDNA-mutation positive cell lines but not in the mtDNA-mutation negative cell lines, the p53 gene might play an important role in mtDNA stability.