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

目的：研究表明，缺氧/再氧化可增加肿瘤细胞的突变频率，导致遗传不稳定。然而，其分子机制尚不清楚。我们知道，细胞缺氧/再氧化后会产生大量的活性氧，产生高度诱变的碱基8-oxoG。在这项研究中，我们评估了在缺氧/再氧化诱导的诱变过程中，容易出错的DNA聚合酶可能会绕过8-oxoG并进行跨区域DNA复制。结果：用RT-PCR方法检测HeLa细胞在缺氧（1% O_2）条件下培养6 ~ 24 h后易出错DNA聚合酶η、ι、κ和μ的表达，结果表明，缺氧显著上调了DNA聚合酶ι (pol ι)基因的表达。模拟缺氧的去铁胺和CoCl_2也增加了pol ι基因的表达。在所有8个被检测的人类细胞系中，缺氧上调了pol i基因。荧光素酶报告基因分析显示，pol ι基因启动子区域（-1345/+419）的转录活性在缺氧和组成型活性HIF-1 α的作用下增加，而活性被显性阴性HIF-1 α抑制。此外，凝胶转移和染色质免疫沉淀实验表明，pol ι基因内含子1中的ACGTG序列对HIF-1反应性很重要。另一方面，我们发现缺氧/再氧化会增加MCF7细胞的突变频率，但用pol -特异性小干扰RNA处理细胞会抑制突变频率。结论：Pol ι参与了缺氧/再氧化诱导的突变过程。

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.