The antitumor activity of chemotherapeutic nitrogen mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA-DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA-protein cross-linking by nitrogen mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA-protein conjugates are likely to contribute to both target and off-target cytotoxicity of nitrogen mustard drugs. Here, we show that the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of nitrogen mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with the guanine half-mustards of chlorambucil or mechlorethamine revealed the ability of the protein to form either one or two cross-links to guanine. C145A AGT (a variant containing a single point mutation in the protein's active site) was found capable of forming a single guanine conjugate, while cross-linking was virtually abolished upon treatment of the C145A/C150S AGT double mutant with the guanine half-mustards. HPLC-ESI+-MS/MS sequencing of tryptic peptides obtained from the wild-type AGT protein that had been treated with nitrogen mustards in the presence of DNA confirmed that the cross-linking took place between the N7 position of guanine in DNA and two active site residues within the AGT protein (Cys145 and Cys150). The exact chemical structures of AGT-DNA cross-links induced by chlorambucil and mechlorethamine were identified as N-(2-S-cysteinyl]ethyl)-N-(2-[guan-7-yl] ethyl)-p-aminophenylbuyric acid and N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl] ethyl)methylamine, respectively, based upon HPLC-MS/MS analysis of protein hydrolysates in parallel with the corresponding amino acid conjugates prepared synthetically. Mechlorethamine-induced AGT-DNA conjugates were isolated from protein extracts of AGT-expressing CHO cells but not control cells, demonstrating that nitrogen mustards can cross-link the AGT protein to DNA in the presence of other nuclear proteins. Because AGT is overexpressed in many tumor types, further investigations of the potential role of AGT-DNA cross-linking in the antitumor and mutagenic activity of antitumor nitrogen mustards are warranted.
All Science Journal Classification (ASJC) codes