Haploid insufficiency in DNA repair through non-homologous end-joining pathway and its effect on colon carcinogenesis

非同源末端连接途径DNA修复的单倍体不足及其对结肠癌发生的影响

• 批准号: 15590707
• 负责人: OCHIAI Masako
• 金额: $ 2.24万
• 依托单位: National Cancer Center (Research Institute)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590707/
• 关键词: DNA repair through non-homologous end-joining (NHEJ) pathway; haploid insufficiency; colon carcinogenesis; DNA-PKcs mutation; gpt delta mouse; azoxymethane; haploid insufficiency

1. The improvement of detection methods of DNA-PKcs mutationFor typing of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutation to obtain the mice used the following experiments ; the detection methods of DNA-PKcs mutation were improved by newly designing primers, making a fluoresence-labeled oligomer and the application of AFLP analysis using a DNA sequencer.2. The effect of DNA-PKcs deficiency on mutant frequency and mutation spectrum in spontaneous and AOM-induced mutationsThe detection of deletion mutations by Spi^- assay was performed. In spontaneous mutations, no significant difference in mutant frequencies (MF) was detected between homozygotes (scid/scid) and wild type mice (+/+) of DNA-PKcs mutation, but in azoxymethane (AOM)-induced mutation, MF of scid/scid were detected with a higher tendency than those of +/+ (P=0.12). By analysis of mutation spectra, deletion mutations more than 1 kbp (large deletion) in mutants obtained from scid/scid were tended to be more than those from +/+, and in AOM-induced mutation, those from scid/scid were significantly higher than those from +/+.3. The effect of haploid insufficiency of DNA-PKcs in AOM-induced mutation and colon carcinogenesisIn the case of spontaneous mutation, MF obtained from heterozygotes (scid/+) had a higher tendency than those from +/+ in deletion mutations less than 100 bp (small deletion). In the case of AOM-induced mutation, MF from scid/scid, scid/+ and +/+ were 7.3±1.6, 25.9±12.5, 4.7±1.1 per 10^6 plaques. MFs from scid/+ were significantly higher than those from +/+ in small deletions and also large deletions. In DNA repair of small deletions and also large deletions, the possibility was suggested of the presence of haploid insufficiency. This was proceeding histological analysis in colon carcinogenesis.

1. DNA-PKcs突变检测方法的改进为了对获得的小鼠进行DNA依赖性蛋白激酶催化亚基（DNA-PKcs）突变分型，采用以下实验方法：通过设计新的引物，制备荧光标记寡聚体，应用DNA测序仪进行AFLP分析，改进了DNA-PKcs突变检测方法. DNA-PKcs缺陷对自发突变和AOM诱导突变频率和突变谱的影响采用Spi^-法检测缺失突变。在自发突变中，DNA-PKcs突变纯合子（scid/scid）和野生型（+/+）小鼠的突变频率（MF）无显著性差异，但在氧化偶氮甲烷（AOM）诱导突变中，scid/scid小鼠的MF有高于+/+小鼠的趋势（P=0.12）。突变谱分析表明，scid/scid突变体中大于1 kbp的缺失突变（大缺失）多于+/+突变体，AOM诱导突变中scid/scid突变体中大于1 kbp的缺失突变显著多于+/+突变体. DNA-PKcs单倍体不足在AOM诱发突变和结肠癌发生中的作用在自发突变的情况下，在小于100 bp的缺失突变（小缺失）中，杂合子（scid/+）获得的MF比+/+获得的MF有更高的倾向。在AOM诱导突变的情况下，scid/scid、scid/+和+/+的MF分别为7.3±1.6、25.9±12.5和4.7±1.1/10^6噬斑。scid/+基因组的MFs在小缺失和大缺失中均显著高于+/+基因组。在小缺失和大缺失的DNA修复中，可能存在单倍体不足。对结肠癌的发生进行组织学分析。