Kanemaru Yuki, Suzuki Tetsuya, Sassa Akira, Matsumoto Kyomu, Adachi Noritaka, Honma Masamitsu, Numazawa Satoshi, Nohmi Takehiko
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501 Japan ; Division of Toxicology, Department of Pharmacology, Toxicology and Therapeutics, Showa University School of Pharmacy, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-0064 Japan.
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501 Japan ; Present Addresses: Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553 Japan.
Genes Environ. 2017 Jan 7;39:6. doi: 10.1186/s41021-016-0067-3. eCollection 2017.
Interactions between genes and environment are critical factors for causing cancer in humans. The genotoxicity of environmental chemicals can be enhanced via the modulation of susceptible genes in host human cells. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. To better understand the protective roles of Pol κ, we previously engineered two human cell lines either deficient in expression of Pol κ (KO) or expressing catalytically dead Pol κ (CD) in Nalm-6-MSH+ cells and examined cytotoxic sensitivity against various genotoxins. In this study, we set up several genotoxicity assays with cell lines possessing altered Pol κ activities and investigated the protective roles of Pol κ in terms of genotoxicity induced by mitomycin C (MMC), a therapeutic agent that induces bulky DNA adducts and crosslinks in DNA.
We introduced a frameshift mutation in one allele of the thymidine kinase (TK) gene of the KO, CD, and wild-type Pol κ cells (WT), thereby establishing cell lines for the gene mutation assay, namely TK+/- cells. In addition, we formulated experimental conditions to conduct chromosome aberration (CA) and sister chromatid exchange (SCE) assays with cells. By using the WT TK+/- and KO TK+/- cells, we assayed genotoxicity of MMC. In the gene mutation assay, the cytotoxic and mutagenic sensitivities of KO TK+/- cells were higher than those of WT TK+/- cells. MMC induced loss of heterozygosity (LOH), base pair substitutions at CpG sites and tandem mutations at GpG sites in both cell lines. However, the frequencies of LOH and base substitutions at CpG sites were significantly higher in KO TK+/- cells than in WT TK+/- cells. MMC also induced CA and SCE in both cell lines. The KO TK+/- cells displayed higher sensitivity than that displayed by WT TK+/- cells in the SCE assay.
These results suggest that Pol κ is a modulating factor for the genotoxicity of MMC and also that the established cell lines are useful for evaluating the genotoxicity of chemicals from multiple endpoints in different genetic backgrounds of Pol κ.
基因与环境之间的相互作用是人类患癌的关键因素。环境化学物质的遗传毒性可通过宿主人类细胞中易感基因的调节而增强。DNA聚合酶κ（Polκ）是一种特殊的DNA聚合酶，在通过跨损伤DNA合成实现的DNA损伤耐受中起重要作用。为了更好地理解Polκ的保护作用，我们之前构建了两个人类细胞系，在Nalm-6-MSH+细胞中，一个细胞系缺乏Polκ表达（KO），另一个细胞系表达催化失活的Polκ（CD），并检测了它们对各种基因毒素的细胞毒性敏感性。在本研究中，我们利用Polκ活性改变的细胞系建立了几种遗传毒性检测方法，并从丝裂霉素C（MMC）诱导的遗传毒性方面研究了Polκ的保护作用，MMC是一种治疗药物，可在DNA中诱导大的DNA加合物和交联。
我们在KO、CD和野生型Polκ细胞（WT）的胸苷激酶（TK）基因的一个等位基因中引入了移码突变，从而建立了用于基因突变检测的细胞系，即TK+/-细胞。此外，我们制定了实验条件，以便对细胞进行染色体畸变（CA）和姐妹染色单体交换（SCE）检测。通过使用WT TK+/-和KO TK+/-细胞，我们检测了MMC的遗传毒性。在基因突变检测中，KO TK+/-细胞的细胞毒性和诱变敏感性高于WT TK+/-细胞。MMC在两个细胞系中均诱导杂合性缺失（LOH）、CpG位点的碱基对替换以及GpG位点的串联突变。然而，KO TK+/-细胞中LOH和CpG位点碱基替换的频率显著高于WT TK+/-细胞。MMC在两个细胞系中也诱导了CA和SCE。在SCE检测中，KO TK+/-细胞比WT TK+/-细胞表现出更高的敏感性。
这些结果表明，Polκ是MMC遗传毒性的调节因子，并且所建立的细胞系可用于在不同Polκ遗传背景下从多个终点评估化学物质的遗传毒性。
