Hypersensitivity of mouse embryonic fibroblast cells defective for DNA polymerases η, ι and κ to various genotoxic compounds: Its potential for application in chemical genotoxic screening
Hypersensitivity of mouse embryonic fibroblast cells defective for DNA polymerases η, ι and κ to various genotoxic compounds: Its potential for application in chemical genotoxic screening
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DOI：
10.1016/j.dnarep.2017.11.006
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发表时间：
2018-01-01
期刊：
影响因子：
3.8
通讯作者：
Ogawa, Kumiko
中科院分区：
文献类型：
--
作者：
Akagi, Jun-ichi;Yokoi, Masayuki;Ogawa, Kumiko
基因毒性物质引起基因组DNA的修饰，如烷基化、氧化、大体积加合物的形成和链断裂，这可能导致基因结构或数量的突变和改变。大多数点突变是在易出错的修饰核苷酸绕过过程中产生的（翻译DNA合成，TLS）；然而，当TLS失效时，复制分叉在病变处停滞，最终导致更致命的影响，即双链断裂（dsb）的形成。在这里，我们比较了来自野生型和敲除小鼠的胚胎成纤维细胞对各种化合物的敏感性，这些小鼠缺乏三种y家族Us DNA聚合酶（Poll和Pole）中的一种或全部（TKO）。本研究检测的化合物包括基因毒素如甲基甲磺酸盐（MMS）和非基因毒素如氯化铵。我们发现，TKO细胞对大多数测试的基因毒素表现出最高的敏感性，但对非基因毒素不敏感。为了定量评价TKO细胞对不同化学物质的超敏性，我们计算了WT和TKO细胞的一半最大抑制浓度比。10种基因毒素中9种的比值在2.29 ~ 5.73之间，5种非基因毒素的比值在0.81 ~ 1.63之间。此外，MMS处理后，泛素化增殖细胞核抗原和γ - h2ax这两种DNA损伤标志物在TKO细胞中的积累量高于WT细胞。此外，在MMS处理后，与WT细胞相比，TKO细胞的姐妹染色单体交换频率增加。这些结果表明，TKO细胞对基因毒素的超敏反应是由复制叉停滞和随后的DNA双链断裂引起的，因此表明TKO细胞应该有助于评估化学遗传毒性。
Genotoxic agents cause modifications of genomic DNA, such as alkylation, oxidation, bulky adduct formation, and strand breaks, which potentially induce mutations and changes to the structure or number of genes. Majority of point mutations are generated during error-prone bypass of modified nucleotides (translesion DNA synthesis, TLS); however, when TLS fails, replication forks stalled at lesions eventually result in more lethal effects, formation of double-stranded breaks (DSBs). Here we compared sensitivities to various compounds among mouse embryonic fibroblasts derived from wild-type and knock-out mice lacking one of the three Y-family Us DNA polymerases Poll, and Pole) or all of them (TKO). The compounds tested in this study include genotoxins such as methyl methanesulfonate (MMS) and nongenotoxins such as ammonium chloride. We found that TKO cells exhibited the highest sensitivities to most of the tested genotoxins, but not to the non-genotoxins. In order to quantitatively evaluate the hypersensitivity of TKO cells to different chemicals, we calculated ratios of half maximal inhibitory concentration for WT and TKO cells. The ratios for 9 out of 10 genotoxins ranged from 2.29 to 5.73, while those for 5 nongenotoxins ranged from 0.81 to 1.63. Additionally, the two markers for DNA damage, ubiquitylated proliferating cell nuclear antigen and gamma-H2AX after MMS treatment, were accumulated in TKO cells more greatly than in WT cells. Furthermore, following MMS treatment, TKO cells exhibited increased frequency of sister chromatid exchange compared with WT cells. These results indicated that the hypersensitivity of TKO cells to genotoxins resulted from replication fork stalling and subsequent DNA double-strand breaks, thus demonstrating that TKO cells should be useful for evaluating chemical genotoxicity.