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Biological role of the regulation of the expressions of transpersonal DNA polymereases by hypoxia

缺氧调节跨个人DNA聚合酶表达的生物学作用

基本信息

批准号：
16590257
负责人：
TAKENAGA Keizo
金额：
$ 1.79万
依托单位：
Chiba Cancer Center Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590257/
关键词：
Hypoxia Mutation HIF-1 Translesional DNA polymerase DNA polymerase ι

项目摘要

AIM : It has been demonstrated that hypoxia/reoxygenation increases mutation frequency in tumor cells, leading to genetic instability. However, its molecular mechanism remains to be obscure. It is known that a large amount of reactive oxygen species is produced in cells after hypoxia/reoxygenation which generate highly mutagenic base 8-oxoG. In this study, we evaluated the possible involvement of error-prone DNA polymerases that can bypass 8-oxoG and proceed translesional DNA replication in hypoxia/reoxygenation-induced mutagenesis. RESULTS : The expressions of error-prone DNA polymerases,η,ι,κ and μ, in HeLa cells were examined by RT-PCR after culturing them under hypoxic conditions (1% O_2) for 6-24 h. The results showed that the expression of DNA polymerase ι (pol ι) gene was significantly up-regulated by hypoxia. Hypoxia mimetic desferrioxamine and CoCl_2 also increased the expression of pol ι gene. Hypoxia up-regulated the pol ι gene in all of 8 human cell lines examined. Luciferase reporter assays revealed that the transcriptional activity of the pol ι gene promoter region (-1345/+419) was increased in response to hypoxia and constitutive active HIF-1 α and that the activity was suppressed by dominant negative HIF-1 α. Furthermore, gel shift and chromatin immunoprecipitation assays demonstrated the importance of the ACGTG sequence in the intron 1 of pol ι gene for HIF-1 responsiveness. On the other hand, it was found that the frequency of mutation was increased in MCF7 cells by hypoxia/reoxygenation, but it was suppressed by treatment of the cells with pol ι -specific small interfering RNA. CONCLUSION : The results suggest that Pol ι is involved in the hypoxia/reoxygenation-induced mutagenesis.

目的：研究表明，缺氧/复氧可增加肿瘤细胞的突变频率，导致遗传不稳定性。然而，其分子机制尚不清楚。众所周知，细胞在缺氧/复氧后会产生大量的活性氧物种，产生高度致突变的碱基8-oxoG。在这项研究中，我们评估了容易出错的DNA聚合酶可能参与了缺氧/复氧诱导的突变中，这些聚合酶可以绕过8-oxoG并进行跨转录DNA复制。结果：在低氧(1%O2)条件下培养6~24 h后，RT-PCR法检测到容易出错的η，ι，κ和μ聚合酶ι和ι的表达，结果表明，缺氧显著上调了DNA聚合酶DNA聚合酶DNA的表达。低氧模拟物去铁胺和CoCl2也能增加POLι基因的表达。低氧可上调所有8种人细胞系中Pollι基因的表达。荧光素酶报告分析显示，POLι基因启动子区域(-1345/+419)的转录活性在低氧和结构性活性HIF-1α的响应下被上调，而该活性被显性阴性的HIF-1α抑制。此外，凝胶漂移和染色质免疫沉淀实验证实了Pollι基因内含子1的ACGTG序列对HIF-1应答的重要性。另一方面，低氧/复氧使MCF7细胞突变频率增加，但用Pollι特异性小干扰RNA处理细胞后，突变频率被抑制。结论：Pol-ι参与了低氧/复氧致突变。