|Budget Amount *help
¥4,020,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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.