The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay
The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay
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DOI：
10.1016/s1383-5718(01)00281-9
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发表时间：
2001-11-15
影响因子：
1.9
通讯作者：
Parry, JM
中科院分区：
文献类型：
--
作者：
Jenkins, GJS;Morgan, C;Parry, JM
我们分析了五个突变热点内的p53基因（密码子175，213，248，249和282）的突变诱导过氧化氢（H2 O2），采用限制性位点突变（RSM）测定。此外，通过RSM测定法分析了覆盖p53基因外显子5-9的非热点密码子（密码子126、153/54、189和外显子9的3 '剪接位点）的其他四个限制性位点的H2 O2诱导的突变。本研究同时分析了两种细胞类型，即原代成纤维细胞和胃癌细胞系。使用RSM分析，H2 O2诱导的突变仅在p53基因的外显子7中检测到。这对两种细胞类型都是如此。这些突变主要在Msp I限制性位点（密码子247/248）中诱导，并且主要是GC到AT的转换（71%）。因此，这些GC到AT的突变可能是由于H2 O2暴露，可能涉及50 HdC加合物，这是已知的诱导C到T的突变时，错误复制。重要的是，这项研究表明，RSM方法能够检测罕见的氧化突变的热点密码子的p53肿瘤抑制基因。因此，这种方法可以允许检测癌前组织中的早期p53突变。(C)2001 Elsevier Science B. V.保留所有权利。
We have analysed five mutation hotspots within the p53 gene (codons 175, 213, 248, 249, and 282) for mutations induced by hydrogen peroxide (H2O2), employing the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5-9 of the p53 gene (codons 126, 153/54, 189 and the 3 ' splice site of exon 9) were analysed by the RSM assay for H2O2-induced mutations. Two cell types were concurrently analysed in this study, i.e. primary fibroblast cells and a gastric cancer cell line. Using the RSM assay, H2O2-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutations were mainly induced in the Msp I restriction site (codon 247/248) and were predominantly GC to AT transitions (71%). Hence these GC to AT mutations were presumably due to H2O2 exposure, possibly implicating the 50HdC adduct, which is known to induce C to T mutations upon misreplication. Importantly, this study demonstrates that the RSM methodology is capable of detecting rare oxidative mutations within the hotspot codons of the p53 tumour suppressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues. (C) 2001 Elsevier Science B.V. All rights reserved.