Department of Radiation Effects Research, Institute for Radiological Science, National Institutes for Quantum Science and Technology, Chiba, Japan.
Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
PLoS One. 2023 Oct 12;18(10):e0292643. doi: 10.1371/journal.pone.0292643. eCollection 2023.
Among the small intestinal tumors that occur in irradiated mice of the established mouse model B6/B6-Chr18MSM-F1 ApcMin/+, loss of heterozygosity analysis can be utilized to estimate whether a deletion in the wild-type allele containing the Adenomatous polyposis coli (Apc) region (hereafter referred to as Deletion), a duplication in the mutant allele with a nonsense mutation at codon 850 of Apc (Duplication), or no aberration (Unidentified) has occurred. Previous research has revealed that the number of Unidentified tumors tends to increase with the radiation dose. In the present study, we investigated the molecular mechanisms underlying the development of an Unidentified tumor type in response to radiation exposure. The mRNA expression levels of Apc were significantly lower in Unidentified tumors than in normal tissues. We focused on epigenetic suppression as the mechanism underlying this decreased expression; however, hypermethylation of the Apc promoter region was not observed. To investigate whether deletions occur that cannot be captured by loss of heterozygosity analysis, we analyzed chromosome 18 using a customized array comparative genomic hybridization approach designed to detect copy-number changes in chromosome 18. However, the copy number of the Apc region was not altered in Unidentified tumors. Finally, gene mutation analysis of the Apc region using next-generation sequencing suggested the existence of a small deletion (approximately 3.5 kbp) in an Unidentified tumor from a mouse in the irradiated group. Furthermore, nonsense and frameshift mutations in Apc were found in approximately 30% of the Unidentified tumors analyzed. These results suggest that radiation-induced Unidentified tumors arise mainly due to decreased Apc expression of an unknown regulatory mechanism that does not depend on promoter hypermethylation, and that some tumors may result from nonsense mutations which are as-yet undefined point mutations.
在已建立的 B6/B6-Chr18MSM-F1 ApcMin/+ 辐射小鼠模型的小肠肿瘤中，可以利用杂合性缺失分析来估计野生型等位基因中是否发生了 Apc 区域的缺失（以下简称缺失），或者是否发生了突变等位基因的重复，该重复在 Apc 的密码子 850 处发生无义突变（重复），或者是否没有发生异常（未识别）。先前的研究表明，未识别肿瘤的数量往往随辐射剂量的增加而增加。在本研究中，我们研究了辐射暴露导致未识别肿瘤类型发展的分子机制。与正常组织相比，未识别肿瘤中的 Apc mRNA 表达水平显著降低。我们专注于表观遗传抑制作为这种表达降低的机制；然而，未观察到 Apc 启动子区域的高甲基化。为了研究是否发生无法通过杂合性缺失分析捕获的缺失，我们使用定制的比较基因组杂交方法分析了染色体 18，该方法旨在检测染色体 18 上的拷贝数变化。然而，未识别肿瘤中 Apc 区域的拷贝数没有改变。最后，使用下一代测序对 Apc 区域进行基因突变分析表明，辐射组小鼠的一个未识别肿瘤中存在大约 3.5 kbp 的小缺失。此外，在大约 30%的分析未识别肿瘤中发现了 Apc 的无义和移码突变。这些结果表明，辐射诱导的未识别肿瘤主要是由于未知调节机制导致的 Apc 表达降低引起的，该机制不依赖于启动子甲基化，并且一些肿瘤可能是由于无义突变引起的，这些无义突变是尚未确定的点突变。
