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

此前，我们分析了食道肿瘤线粒体DNA(MtDNA)D-loop区域的突变，发现频繁的体细胞突变(在34%的肿瘤中)。我们还检测了核基因组不稳定性，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，提示食道癌mtDNA的不稳定性可能与核基因组不稳定性无关。为了解X射线等诱变剂是否能诱发mtDNA突变，我们对4个食管癌细胞系KYSE-30、110、150、410和正常细胞系Susa/T-n进行了X射线照射后的mtDNA突变筛查。这些细胞系经X射线照射后，每株细胞的存活率为0.01。提取单细胞克隆后，提取DNA。由于每个细胞中有多达10^3个拷贝的mtDNA，并且不是所有的mtDNA都会发生相同的突变，因此通过评估突变的mtDNA占总mtDNA的比例来进行评估。由于线粒体DNA中具有7个连续C延伸的D310区域经常出现1个碱基的插入或缺失突变，我们用GeneScan对该区域进行了分析，突变比例大于0.3的菌落被定义为突变菌落。在Susa/T-n中，X射线照射后D310的长度没有变化，两个食管癌细胞系KYSE-150和410也没有变化。另一方面，食道癌细胞系KYSE-30和110的受照细胞中有11.8%和31.8%的集落在D310区域有长度变化。由于p53基因在这两个mtDNA突变阳性细胞系中发生突变，而在mtDNA突变阴性细胞系中未发生突变，因此p53基因可能在线粒体DNA稳定性中起重要作用。

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.