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Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems
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 proces...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9881385/ https://www.ncbi.nlm.nih.gov/pubmed/36714466 http://dx.doi.org/10.1016/j.toxrep.2023.01.010 |
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author | Krassnig, Sarah Ceylan Mäser, Marina Probst, Nicola Anna Werner, Jens Schlett, Charlotte Schumann, Nina von Scheven, Gudrun Mangerich, Aswin Bürkle, Alexander |
author_facet | Krassnig, Sarah Ceylan Mäser, Marina Probst, Nicola Anna Werner, Jens Schlett, Charlotte Schumann, Nina von Scheven, Gudrun Mangerich, Aswin Bürkle, Alexander |
author_sort | Krassnig, Sarah Ceylan |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9881385 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-98813852023-01-28 Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems Krassnig, Sarah Ceylan Mäser, Marina Probst, Nicola Anna Werner, Jens Schlett, Charlotte Schumann, Nina von Scheven, Gudrun Mangerich, Aswin Bürkle, Alexander Toxicol Rep Article 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. Elsevier 2023-01-20 /pmc/articles/PMC9881385/ /pubmed/36714466 http://dx.doi.org/10.1016/j.toxrep.2023.01.010 Text en © 2023 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Article Krassnig, Sarah Ceylan Mäser, Marina Probst, Nicola Anna Werner, Jens Schlett, Charlotte Schumann, Nina von Scheven, Gudrun Mangerich, Aswin Bürkle, Alexander Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems |
title | Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems |
title_full | Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems |
title_fullStr | Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems |
title_full_unstemmed | Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems |
title_short | Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems |
title_sort | comparative analysis of chlorambucil-induced dna lesion formation and repair in a spectrum of different human cell systems |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9881385/ https://www.ncbi.nlm.nih.gov/pubmed/36714466 http://dx.doi.org/10.1016/j.toxrep.2023.01.010 |
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