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

1.DNA-PKcs突变检测方法的改进为获得DNA依赖蛋白激酶催化亚单位(DNA-PKcs)突变的小鼠进行了以下实验：通过设计新的引物、制备荧光标记的寡聚体以及应用DNA测序仪进行AFLP分析，对DNA-PKcs突变的检测方法进行了改进。DNA-PKcs缺陷对自发突变和AOM诱发突变频率和突变谱的影响用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(+/+)的突变频率(Mf)无显著差异，但在偶氮甲烷(AOM)诱导的突变中，SCID/SCID的Mf有高于+/+的趋势(P=0.12)。突变谱分析表明，SCID/SCID突变株的缺失突变大于1kbp(大缺失)的突变数明显多于+/+突变株，而在AOM诱导突变株中，SCID/SCID突变株的缺失突变数显著高于+/+突变株。DNA-PKcs单倍体缺失在AOM诱发突变和结肠癌发生中的作用在自发突变的情况下，缺失突变小于100bp的杂合子(SCID/+)的MF有高于+/+突变的倾向(小缺失)。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、4.7±1.1个/10^6斑块。在小缺失和大缺失中，SCID/+的MF值显著高于+/+。在小缺失和大缺失的DNA修复中，提示存在单倍体不足的可能性。这是在结肠癌发生过程中进行的组织学分析。

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.