Mechanistic studies on the formation and repair of DNA lesions induced by the clinically-relevant nitrogen mustard chlorambucil

Abstract

Cancer is presently one of the most widespread and at the same time amongst the deadliest diseases known to mankind. The ability of a somatic cell to proliferate indefinitely, which is the case in cancer cells, is due to the accumulation of mutations in specific genes. The mutations necessary for a cell to overcome the inhibition of uncontrolled proliferation mostly arise due to unrepaired DNA damage, which is induced by exogenous as well as endogenous DNA damaging agents. One of the most prominent exogenous DNA damaging agents is chlorambucil (CLB). CLB belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents. In addition, NMs were the first chemotherapeutic agents ever developed. They form DNA monoadducts as well as DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, thus inhibiting essential processes in DNA metabolism like replication and transcription. Especially in fast replicating cells, e.g., tumor cells, the formation of ICLs can induce cell death. The upregulation of ICL repair is also thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. As the DNA adducts induced after the treatment with CLB are thought to be indicative of the treatment outcome of the chemotherapy, sensitive methods for the reliable detection are highly desirable. The central aim of this PhD project was the establishment of a technical platform to detect the main CLB-induced DNA adducts after the treatment of different cell systems of human origin in a highly accurate and quantitative way. In order to monitor the induction and repair of CLB-induced ICLs in cells, the automated reverse fluorometric analysis of alkaline DNA unwinding (rFADU) assay was adjusted for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA nucleobases induced after the treatment with CLB, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established. With the successful establishment of the technical platform, a comparative analysis of adduct formation and removal in five human cell lines (A2780, U2OS, hTERT RPE-1, hTERT VH10, hTERT podocytes) and in human peripheral blood mononuclear cells (PBMCs) was performed after treatment with CLB. Dose-dependent increases in adduct formation were observed in all cell systems, and suitable treatment conditions were identified for each cell line, which were then used for monitoring the kinetics of adduct formation and removal. Time-course experiments revealed significant differences in the repair kinetics of the cell systems 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. In U2OS cells and in hTERT immortalized podocytes, on the other hand, only the repair of N3-CLB-Ade was observed. Regarding ICLs, repair was observed in almost all cell systems, the only exception being PBMCs, as no suitable treatment concentration could be determined for the monitoring of ICLs. Simultaneously performed experiments detecting the proliferation and viability status of the different cell systems during the time-course revealed that the observed removal of the CLB-induced DNA adducts is indeed due to repair of the lesion and not because of selective proliferation or selective cell death. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of CLB-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, intriguing insights into the cell-type specific repair of different CLB-induced DNA lesions was obtained. The methods established and the knowledge gained from the experiments may be used in the future in a clinical setting to make predictions on the efficacy and side effects of CLB treatment of eligible patients. Not only the formation of adducts after treatment with chemotherapeutic agents is important, but also the genetic background of the tumor and normal cells of the patient in question. If, in future studies, specific factors that increase the resistance to CLB can be identified, tumors with defects in these genes can be preferentially treated with CLB and other alkylating crosslinking chemotherapeutic agents. Overall, the long-term perspective of this work is to contribute to the improvement of the efficacy of the chemotherapeutic regimen with CLB and thereby reduce the side effects for the patients.