苯丁酸氮芥（CLB）属于氮芥（NMs）类别，它们是高度反应性的双官能烷化剂，是最早开发的化学治疗剂。它们形成DNA链间交联（ICL），导致DNA链分离受阻，抑制DNA代谢的基本过程，如复制和转录。在快速复制的细胞中，例如，肿瘤细胞,这可以诱导细胞死亡。ICL修复的上调被认为是肿瘤细胞对包括NMs在内的ICL诱导细胞抑制剂耐药的关键因素。我们调整了碱性DNA解链测定(rFADU)的自动反向荧光分析,以检测贴壁细胞中的ICL.为了检测单烷基化的DNA碱基，我们建立了LC-MS/MS方法。我们对CLB处理后的五种人类细胞系和外周血单核细胞（PBMC）中的加合物形成和去除进行了比较分析。观察到加合物形成的剂量依赖性增加，并为每个细胞系确定合适的处理浓度，然后用于监测加合物形成的动力学。我们观察到所测试细胞系的修复动力学的显著差异。例如,在A2780细胞中，hTERT永生化VH10细胞，并且在PBMC中，证实了两种主要的单烷基化DNA加合物的时间依赖性修复。关于ICL，在除PBMC外的所有细胞系统中均观察到修复。总之,LC-MS/MS分析结合rFADU技术是研究NM诱导的DNA损伤和修复的分子机制的有力工具。通过将这些方法应用于一系列不同起源和转化状态的人类细胞系统，我们深入了解了不同CLB诱导的DNA损伤的细胞类型特异性修复，这可能有助于确定肿瘤的新耐药机制，并确定治疗干预的分子靶点。
Chlorambucil (CLB) belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents and were the first chemotherapeutic agents developed. They form DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, inhibiting essential processes in DNA metabolism like replication and transcription. In fast replicating cells, e.g., tumor cells, this can induce cell death. The upregulation of ICL repair is thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. To monitor induction and repair of CLB-induced ICLs, we adjusted the automated reversed fluorometric analysis of alkaline DNA unwinding assay (rFADU) for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA bases we established an LC-MS/MS method. We performed a comparative analysis of adduct formation and removal in five human cell lines and in peripheral blood mononuclear cells (PBMCs) after treatment with CLB. Dose-dependent increases in adduct formation were observed, and suitable treatment concentrations were identified for each cell line, which were then used for monitoring the kinetics of adduct formation. We observed significant differences in the repair kinetics of the cell lines tested. For example, in A2780 cells, hTERT immortalized VH10 cells, and in PBMCs a time-dependent repair of the two main monoalkylated DNA-adducts was confirmed. Regarding ICLs, repair was observed in all cell systems except for PBMCs. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of NM-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, we obtained insight into the cell-type specific repair of different CLB-induced DNA lesions, which may help identify novel resistance mechanisms of tumors and define molecular targets for therapeutic interventions.
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