Mapping DNA Adducts of Mitomycin C and Decarbamoyl Mitomycin C in Cell Lines Using Liquid Chromatography/Electrospray Tandem Mass Spectrometry

Abstract

The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a non-toxic reductive metabolite of MC formed in cells in situ. Several methods have been used for analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex. Development of an assay, based on mass spectrometry is described. DNA from EMT6 mouse mammary tumor cells, Fanconi Anemia -A fibroblasts, normal human fibroblasts, and MCF-7 human breast cancer cells was isolated after MC or DMC treatment of the cells, digested to nucleosides and submitted to liquid chromatography electrospray-tandem mass spectrometry. Two fragments of each parent ion were monitored ("multiple reaction monitoring"; MRM). Identification and quantitative analysis was based on a standard mixture of six adducts, the preparation of which is described here in detail. The lower limit of detection of adducts is estimated as 0.25 picomol. Three initial applications of this method are reported: (i) differential kinetics of adduct repair in EMT6 cells; (ii) analysis of adducts in MC-or DMC-treated Fanconi Anemia cells; and (iii) comparison of the adducts generated by treatment of MCF-7 breast cancer cells with MC and DMC. Notable results are the following: repair removal of the DNA interstrand cross-link and of the two adducts of 2,7-DAM is relatively slow; both MC and DMC generate DNA interstrand cross-links in human fibroblasts, Fanconi Anemia-A fibroblasts and MCF-7 cells as well as EMT6 cells; DMC shows a stereochemical preference of linkage to the guanine-2-amino group opposite from that of MC. This information is available free of charge via the Internet at

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

“…This will enable shuttle vector-based methods, in conjunction with genetic manipulation, for interrogating the roles of BER glycosylases and ALKB family proteins in the removal of interstrand cross-link lesions in cells [164, 175]. Likewise, genetic modulation of these DNA repair enzymes, together with measurements of interstrand cross-links in cellular DNA by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [72, 176–181], also offers the opportunity for exploring the roles in these enzymes in the unhooking and repair of interstrand cross-links. At the same time, it will be important for studies in cellular systems (and cell extracts) to establish how proteins present at a stalled replication fork choreograph the action of NEIL3 and other enzymes involved in cross-link unhooking, and to gather data from living organisms that shed light on the biological roles and potential medicinal relevance of these repair pathways.…”

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

“…The use of a LC-MS/MS-based assay also led to the observation that ICLs induced by mitomycin C could be partially removed in mouse mammary tumor cells. 228 …”

A Quantitative Mass Spectrometry-Based Approach for Assessing the Repair of 8-Methoxypsoralen-Induced DNA Interstrand Cross-Links and Monoadducts in Mammalian Cells

“…Although others and we previously employed LC-MS/MS for detecting DNA ICLs in cells or tissues 24–27 , to our knowledge, this is one of the first few reports about the application of LC-MS/MS for examining the repair of any DNA ICLs. 27 …”