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分型dna依赖性蛋白激酶催化亚基（DNA-PKcs）突变获得小鼠使用了以下实验；通过新设计引物、制备荧光标记低聚物以及利用DNA测序仪进行AFLP分析，改进了DNA- pkcs突变的检测方法。DNA-PKcs缺乏对自发性和aom诱导突变的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，纯合子（scid/scid）与野生型小鼠（+/+）DNA-PKcs突变的突变频率（MF）无显著差异，但在偶氮甲烷（AOM）诱导突变中，scid/scid的突变频率（MF）高于+/+ (P=0.12)。通过突变谱分析，scid/scid突变体中大于1 kbp（大缺失）的缺失突变倾向于多于+/+突变体，而在aom诱导突变体中，scid/scid突变体明显高于+/+.3突变体。DNA-PKcs单倍体不足在aom诱导突变和结肠癌发生中的作用在自发突变的情况下，杂合子（scid/+）获得的MF在小于100 bp的缺失突变（小缺失）中比+/+获得的MF有更高的倾向。在aom诱导突变的情况下，来自scid/scid、scid/+和+/+的MF分别为每10^6个斑块7.3±1.6、25.9±12.5和4.7±1.1。在小缺失和大缺失中，scid/+的MFs显著高于+/+。在DNA小缺失和大缺失的修复中，可能存在单倍体缺陷。这是结肠癌变的组织学分析。

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.