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Molecular analysis of the role of p53 in radioadaptive response

p53 在放射适应性反应中作用的分子分析

基本信息

批准号：
15510047
负责人：
TACHIBANA Akira
金额：
$ 2.43万
依托单位：
Kyoto University
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-15510047/
关键词：
radioadaptive response low-dose radiation DNA double-strand break p53 end-joining mutagenesis 突然変異 Hprt遺伝子 multilex PCR

项目摘要

Radioadaptive response is a biological defense mechanism that is induced by low-dose ionizing irradiation for cellular resistance to the genotoxic effects of subsequent irradiation. Nuclear extracts were prepared from unirradiated cells (unirradiated extract), cells exposed to either 2 cGy (2 cGy extract) or 3 Gy (3 Gy extract) of X-rays, and cells pre-exposed with 2 cGy and then challenged with 3 Gy (2 cGy+3 Gy extract). The efficiency of end joining by the 3 Gy extract was somewhat reduced, but the rejoining efficiency of the 2 cGy+3 Gy extract was similar to the efficiency of the unirradiated extract. The fidelity of end-joining was estimated from the frequency of mutant plasmids. The mutant frequency by the 3 Gy extract was higher than the unirradiated level. But, the mutant frequency by the 2 cGy+3 Gy extract was about half of the unirradiated level, indicatinging high fidelity of rejoining. Moreover, we have demonstrated that the radioadaptive response is absent in p53-deficient mouse cells. We examined the end-joining reaction using the extracts isolated from p53-deficient mouse cells irradiated with X-rays with or without pre-irradiation. No change was observed either in the efficiency or in the fidelity of end-joining irrespective of pre-irradiation. Therefore, p53 could be an important regulatory factor of end-joining in radioadaptive response. We further examined mutations induced by ionizing radiation with or without radioadaptive response at the Hprt locus in mouse m5S cells. We analyzed the Hprt gene in 6-thioguanine-resistant mutants by amplification of all the nine exons of the mouse Hprt gene. Our results showed that the proportion of partial deletions was much higher in mutants isolated from the pre-exposed radioadaptive culture than in non-primed irradiated culture.

放射适应性反应是一种生物防御机制，由低剂量电离辐射诱导，用于细胞抵抗后续辐射的基因毒性效应。核提取物由未照射的细胞（未照射提取物）、暴露于 2 cGy（2 cGy 提取物）或 3 Gy（3 Gy 提取物）X 射线的细胞以及预先暴露于 2 cGy 然后用 3 Gy（2 cGy+3 Gy 提取物）激发的细胞制备。 3 Gy 提取物的末端连接效率有所降低，但 2 cGy+3 Gy 提取物的重新连接效率与未辐射提取物的效率相似。根据突变质粒的频率估计末端连接的保真度。 3 Gy 提取物的突变频率高于未照射水平。但是，2 cGy+3 Gy 提取物的突变频率约为未照射水平的一半，表明重新连接的保真度很高。此外，我们已经证明 p53 缺陷的小鼠细胞不存在放射适应性反应。我们使用从经过或不经过预照射的 X 射线照射的 p53 缺陷型小鼠细胞中分离的提取物检查了末端连接反应。无论预照射如何，都没有观察到末端连接的效率或保真度发生变化。因此，p53可能是放射适应性反应中末端连接的重要调节因子。我们进一步检查了小鼠 m5S 细胞中 Hprt 基因座处电离辐射在有或没有放射适应性反应的情况下诱导的突变。我们通过扩增小鼠 Hprt 基因的所有九个外显子来分析 6-硫鸟嘌呤抗性突变体中的 Hprt 基因。我们的结果表明，从预暴露的放射适应性培养物中分离的突变体中部分缺失的比例比未引发的辐射培养物中的要高得多。