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 which 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.

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

“…The same two peptides were used in our earlier publication investigating replication bypass of DPCs conjugated to the C5 position of thymidine using Cu(I)-catalyzed azide-alkyne cycloaddition (29). In addition, histone H2A (14 kDa), histone H4 (11.5 kDa), and AlkB (22.9 kDa) were chosen for these experiments based on the documented ability of histones and DNA repair proteins to engage in the formation of DNA-protein cross-links (3,4,8,10). We also included enhanced green fluorescent protein (eGFP; 28 kDa), which was used in our earlier study of pyrimidine-conjugated DPCs (29).…”

“…Covalent entrapment of cellular proteins on genomic DNA is a common process that occurs upon exposure to a variety of endogenous and exogenous bis-electrophiles, heavy metals, and free radicals (1)(2)(3)(4)(5)(6)(7). Specifically, common chemotherapeutics such as nitrogen mustards, platinum compounds, and alkylnitrosoureas (3,4,8); environmental carcinogens such as formaldehyde and 1,3-butadiene (5, 9, 10); toxic metals (11)(12)(13); nitric oxide (14); free radicals (15,16); UV light (17); and ionizing radiation (6) mediate the formation of covalent DNAprotein cross-links (DPCs).…”

“…Specifically, common chemotherapeutics such as nitrogen mustards, platinum compounds, and alkylnitrosoureas (3,4,8); environmental carcinogens such as formaldehyde and 1,3-butadiene (5, 9, 10); toxic metals (11)(12)(13); nitric oxide (14); free radicals (15,16); UV light (17); and ionizing radiation (6) mediate the formation of covalent DNAprotein cross-links (DPCs). 3 Mass spectrometry-based proteomic studies have identified a large number of cellular proteins that participate in DPC formation in cells treated with cross-linking agents, including DNA repair proteins, DNA polymerases, transcription factors, and structural proteins such as histones, heat shock proteins, and tubulins (3-6, 8, 10).…”

“…Covalent DPCs have been detected in peripheral blood lymphocytes from breast cancer patients undergoing treatment with cyclophosphamide. 4 Recent studies by Jentsch and co-workers (20,21) and Walter and co-workers (22) have provided evidence that DPCs are subject to proteolytic cleavage to the corresponding DNA-peptide lesions, which are subsequently removed from DNA in a replication-dependent repair process. Thus, it is critically important to investigate the activ-ity and accuracy of DNA polymerases on DNA-protein and DNA-peptide lesions.…”

“…The most common site on DNA that participates in DNAprotein cross-linking mediated by bis-electrophiles is the N7 atom of guanine (3)(4)(5)10). However, to our knowledge, no reports are available in the literature in regard to the replication bypass of N7-guanine adducts, probably because of their labile nature.…”