title: Mechlorethamine-induced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells.
abstract: Antitumor nitrogen mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of nitrogen mustards are attributed to their ability to induce DNA-DNA and DNA-protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA-protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC-ESI(+)-MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cysteine thiols within proteins to N-7 positions of guanine in DNA.
authors.affiliation: Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Erin D
authors.initials: ED
authors.lastname: Michaelson-Richie
authors.forename: Xun
authors.initials: X
authors.lastname: Ming
authors.forename: Simona G
authors.initials: SG
authors.lastname: Codreanu
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Loeber
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
authors.forename: Colin
authors.initials: C
authors.lastname: Campbell
authors.forename: Natalia Y
authors.initials: NY
authors.lastname: Tretyakova
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004273:DNA, Neoplasm / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005354:Fibrosarcoma / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008466:Mechlorethamine / Q000494:pharmacology*; D010446:Peptide Fragments / Q000737:chemistry; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D008466:Mechlorethamine