Influence of the Expression Level of O6-Alkylguanine-DNA Alkyltransferase on the Formation of DNA Interstrand Crosslinks Induced by Chloroethylnitrosoureas in Cells: A Quantitation Using High-Performance Liquid Chromatography-Mass Spectrometry

Chloroethylnitrosoureas (CENUs), which are bifunctional alkylating agents widely used in the clinical treatment of cancer, exert anticancer activity by inducing crosslink within a guanine-cytosine DNA base pair. However, the formation of dG-dC crosslinks can be prevented by O6-alkylguanine-DNA alkyltransferase (AGT), ultimately leading to drug resistance. Therefore, the level of AGT expression is related to the formation of dG-dC crosslinks and the sensitivity of cells to CENUs. In this work, we determined the CENU-induced dG-dC crosslink in mouse L1210 leukemia cells and in human glioblastoma cells (SF-763, SF-767 and SF-126) containing different levels of AGT using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The results indicate that nimustine (ACNU) induced more dG-dC crosslinks in L1210 leukemia cells than those induced by carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS). This result was consistent with a previously reported cohort study, which demonstrated that ACNU had a better survival gain than BCNU, CCNU and FTMS for patients with high-grade glioma. Moreover, we compared the crosslinking levels and the cytotoxicity in SF-763, SF-767 and SF-126 cells with different AGT expression levels after exposure to ACNU. The levels of dG-dC crosslink in SF-126 cells (low AGT expression) were significantly higher than those in SF-767 (medium AGT expression) and SF-763 (high AGT expression) cells at each time point. Correspondingly, the cytotoxicity of SF-126 was the highest followed by SF-767 and SF-763. The results obtained in this work provided unequivocal evidence for drug resistance to CENUs induced by AGT-mediated repair of DNA ICLs. We postulate that the level of dG-dC crosslink has the potential to be employed as a biomarker for estimating drug resistance and anticancer efficiencies of novel CENU chemotherapies.

氯乙基亚硝脲类（Chloroethylnitrosoureas, CENUs）是一类广泛应用于临床癌症治疗的双功能烷化剂，其通过在鸟嘌呤-胞嘧啶DNA碱基对内诱导交联发挥抗癌活性。然而，O6-烷基鸟嘌呤-DNA烷基转移酶（O6-alkylguanine-DNA alkyltransferase, AGT）可阻断dG-dC交联的形成，最终导致肿瘤细胞产生耐药性。因此，AGT的表达水平与dG-dC交联的形成以及细胞对CENUs的敏感性密切相关。
本研究采用高效液相色谱联用电喷雾电离串联质谱技术，检测了小鼠L1210白血病细胞以及不同AGT表达水平的人胶质母细胞瘤细胞（SF-763、SF-767与SF-126）中CENUs诱导的dG-dC交联水平。结果显示，相较于卡莫司汀（BCNU）、洛莫司汀（CCNU）与福莫司汀（FTMS），尼莫司汀（ACNU）在L1210白血病细胞中诱导产生的dG-dC交联水平更高。这一结果与此前一项队列研究的结论相符，该研究证实对于高级别胶质瘤患者，尼莫司汀的生存获益优于卡莫司汀、洛莫司汀与福莫司汀。
此外，本研究还比较了经尼莫司汀处理后，不同AGT表达水平的SF-763、SF-767与SF-126细胞的交联水平与细胞毒性。在各时间节点下，AGT低表达的SF-126细胞内dG-dC交联水平显著高于AGT中等表达的SF-767细胞与AGT高表达的SF-763细胞。与之对应，SF-126细胞的细胞毒性最高，其次为SF-767与SF-763细胞。
本研究所得结果为AGT介导的DNA链间交联修复所诱导的CENUs耐药性提供了明确的实验证据。本研究推测，dG-dC交联水平有望作为评估新型CENUs化疗方案耐药性与抗癌疗效的生物标志物。