Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells.
Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells.
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DOI：
10.18632/oncotarget.10928
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发表时间：
2016-09-13
期刊：
影响因子：
--
通讯作者：
Nakamura J
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文献类型：
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作者：
Hashimoto K;Sharma V;Sasanuma H;Tian X;Takata M;Takeda S;Swenberg JA;Nakamura J
甲磺酸异丙酯 (IPMS) 是甲磺酸酯中最具遗传毒性的化合物。烷基磺酸酯的潜在基因毒性被认为是由于其鸟嘌呤 O6 位的烷基化能力。了解响应 IPMS 诱导的 DNA 损伤而激活的主要修复途径对于分析 IPMS 的潜在遗传毒性非常重要。在本研究中，鸡 DT40 和人 TK6 基于细胞的 DNA 损伤反应 (DDR) 测定表明，与 EMS 或 MMS 相比，FANC 途径功能障碍导致对 IPMS 的敏感性更高。 O6-烷基 dG 主要由甲基鸟嘌呤甲基转移酶 (MGMT) 修复，而异丙基 dG 不太可能是 MGMT 的底物。 IPMS 及其异构体甲磺酸正丙酯 (nPMS) 的细胞毒性潜力的比较表明，异丙基部分避免了 MGMT 的识别，导致更高的细胞毒性。接下来，微核 (MN) 测定表明，FANC 缺陷会增加 DT40 细胞对 IPMS 诱导 MN 的敏感性。用 MGMT 抑制剂 O6-苄基鸟嘌呤 (OBG) 进行预处理，增加了经 nPMS 处理的 DT40 细胞的 MN 频率，但不增加经 IPMS 处理的 DT40 细胞。最后，与野生型细胞相比，IPMS 以时间依赖性方式在 FANC 缺陷细胞中诱导更多双链断裂。总而言之，这些结果表明 IPMS 衍生的 O6-异丙基 dG 逃脱了 MGMT 的识别，并且未修复的 DNA 损伤导致双链断裂，从而导致 MN 诱导。因此，FANC 在防止 IPMS 引起的 MN 诱导和细胞死亡方面发挥着关键作用。
Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS.