Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Sciences and Bioengineering, Beijing University of Technology, Beijing, 100124, P.R., China.
Rapid Commun Mass Spectrom. 2014 Mar 15;28(5):439-47. doi: 10.1002/rcm.6800.
Chloroethylnitrosoureas (CENUs) are important alkylating agents employed for the clinical treatment of cancer. The cellular toxicity of CENUs is primarily due to induction of DNA interstrand crosslinks (ICLs), which has been characterized as l-(3-deoxycytidyl), 2-(l-deoxyguanosinyl)ethane (dG-dC). However, the formation of dG-dC crosslinks can be prevented by O(6) -alkylguanine-DNA alkyltransferase (AGT), which removes the O(6) -chloroethyl group from O(6) -chloroethylguanine (O(6) -ClEt-Gua), and ultimately its increased expression can result in drug resistance. Differing levels of AGT expression can lead to varying amounts of dG-dC crosslinking, which influences the sensitivity of cells to CENUs.
In this work, a sensitive method for the quantitation of dG-dC crosslinks in cellular DNA has been established using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS).
The limit of detection (LOD) and limit of quantitation (LOQ) of the method were determined to be 2 fmol and 8 fmol on-column, respectively, and the recovery ranged from 96% to 105% with the relative standard deviation (RSD) below 5%. Using this method, the levels of dG-dC crosslink induced by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) were determined in NIH/3T3 fibroblasts cells (high level of expression of AGT) and L1210 leukemia cells (low level of expression of AGT). The time-course profile indicated that the levels of dG-dC crosslink uniformly increased in the early incubation period and reached the maximum at 12 h. Subsequently, the amount of dG-dC crosslinking decreased to very low levels presumably owing to the repair of O(6) -ClEt-Gua by AGT. The crosslinking levels in L1210 cells were significantly higher than those in NIH/3T3 cells at each time point. This provides strong evidence that high express of AGT in CENU-resistant cells inhibits the formation of dG-dC crosslinks.
This work will contribute to the further understanding of the drug resistance of CENUs, and will provide a means to evaluate the anticancer activity of new bifunctional anticancer agents.
氯乙基亚硝脲类化合物（CENUs）是一种重要的烷化剂，用于癌症的临床治疗。CENUs 的细胞毒性主要归因于 DNA 链间交联（ICLs）的诱导，其特征为 l-(3-脱氧胞苷基)，2-(l-脱氧鸟苷基)乙烷（dG-dC）。然而，O(6)- 烷基鸟嘌呤-DNA 烷基转移酶（AGT）可以防止 dG-dC 交联的形成，它从 O(6)- 氯乙基鸟嘌呤（O(6)-ClEt-Gua）中去除 O(6)- 氯乙基基团，最终其表达水平的增加会导致药物耐药性。AGT 表达水平的不同会导致不同数量的 dG-dC 交联，从而影响细胞对 CENUs 的敏感性。
本工作采用高效液相色谱/电喷雾串联质谱法（HPLC/ESI-MS/MS）建立了一种定量检测细胞 DNA 中 dG-dC 交联的灵敏方法。
该方法的检测限（LOD）和定量限（LOQ）分别为 2 fmol 和 8 fmol（柱上进样），回收率在 96%至 105%之间，相对标准偏差（RSD）低于 5%。使用该方法，测定了 1-(4-氨基-2-甲基-5-嘧啶基)甲基-3-(2-氯乙基)-3-亚硝脲盐酸盐（ACNU）在 NIH/3T3 成纤维细胞（AGT 高表达）和 L1210 白血病细胞（AGT 低表达）中诱导的 dG-dC 交联水平。时间过程曲线表明，dG-dC 交联水平在早期孵育期均匀增加，并在 12 小时达到最大值。随后，由于 AGT 对 O(6)-ClEt-Gua 的修复，dG-dC 交联的量降低到非常低的水平。L1210 细胞中的交联水平在每个时间点均明显高于 NIH/3T3 细胞。这有力地证明了 CENU 耐药细胞中 AGT 的高表达抑制了 dG-dC 交联的形成。
本工作将有助于进一步了解 CENUs 的耐药性，并为评估新型双功能抗癌药物的抗癌活性提供一种手段。
