title: Measurement of DNA cross-linking in patients on ifosfamide therapy using the single cell gel electrophoresis (comet) assay.
abstract: The single cell gel electrophoresis comet assay has become established as a sensitive technique for measuring DNA strand breaks. The technique has been modified to allow the sensitive detection and quantitation of DNA interstrand cross-linking at the single cell level. Cells are irradiated immediately before analysis to deliver a fixed level of random strand breakage. After embedding of cells in agarose and lysis, the presence of cross-links retards the electrophoretic mobility of the alkaline denatured cellular DNA. Cross-links are, therefore, quantitated as the decrease in the comet tail moment compared with irradiated controls. Using this method, a linear response of cross-linking versus dose of chlorambucil over a wide dose range was demonstrated in human lymphocytes after drug treatment ex vivo. The method was also sensitive enough to determine cross-linking in clinical samples after chemotherapy. For example, crosslinking was observed in the lymphocytes of patients receiving ifosfamide (3 g/m2/day) as a continuous infusion for 3-5 days or as a 3-h infusion daily for 3 days. Cross-links were detected in all patients within 3 h, with no evidence of DNA single strand break formation. In patients receiving continuous infusion, a plateau of cross-linking was reached by 24 h. In the patients receiving ifosfamide over 3 h, a clear decrease in the peak level of cross-linking was observed before subsequent infusions.
authors: Department of Oncology, UCL Medical School, London, United Kingdom.
authors: J M
authors: JM
authors: Hartley
authors: V J
authors: VJ
authors: Spanswick
authors: M
authors: M
authors: Gander
authors: G
authors: G
authors: Giacomini
authors: J
authors: J
authors: Whelan
authors: R L
authors: RL
authors: Souhami
authors: J A
authors: JA
authors: Hartley
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology* / Q000627:therapeutic use; D003432:Cross-Linking Reagents / Q000494:pharmacology* / Q000627:therapeutic use; D004249:DNA Damage / Q000187:drug effects*; D004586:Electrophoresis / Q000379:methods*; D006801:Humans; D007069:Ifosfamide / Q000494:pharmacology* / Q000627:therapeutic use; D008214:Lymphocytes / Q000187:drug effects*; D012512:Sarcoma, Ewing / Q000097:blood / Q000188:drug therapy
chemical_list: D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D007069:Ifosfamide