DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells.

DNA-protein cross-links (DPCs) are ubiquitous DNA lesions that are formed when proteins become irreversibly trapped on chromosomal DNA. A variety of physical and chemical agents including formaldehyde,1 ionizing radiation,2 UV light, and common anticancer drugs such as nitrogen mustards and platinum compounds can induce covalent DPCs in cells.3–5 Because of their unusually bulky size and their ability to disrupt dynamic DNA-protein interactions, DPC lesions are thought to interfere with critical cellular processes of DNA replication, transcription, and repair. Although DPCs are hypothesized to contribute to the cytotoxicity and mutagenesis of common cross-linking agents3, their specific contributions to the cellular effects of DNA-alkylating agents are poorly understood because these compounds also induce other types of DNA damage such as interstrand and intrastrand DNA-DNA cross-links and nucleobase monoadducts. For example, 1,2,3,4-diepoxybutane (DEB), the ultimate carcinogenic metabolite of 1,3-butadiene, forms DPCs6–8 but is also capable of generating guanine-guanine and adenine-guanine cross-links,9–11 as well as a number of guanine and adenine monoadducts.12,13 DPCs are estimated to constitute only 1–3% of total DNA damage following exposure to bis-electrophiles and ionizing radiation,2,3,14 making it difficult to evaluate their specific role in the cytotoxic and mutagenic effects of these agents.15,16 Experimental evidence is mounting in support of an important role of DPCs in the biological activity of bis-electrophiles. For example, it has been reported that the cytotoxicity and mutagenicity of several bifunctional alkylation agents including 1,2-dibromoethane, dibromomethane, and DEB, are enhanced in bacteria which over-express human O6-alkylguanine DNA alkyltransferase (AGT) protein due to the formation of toxic AGT-DNA cross-links. 15,16,15,17 AGT is a DNA repair protein that typically protects the human genome from the damaging effects of promutagenic O6-alkylguanine lesions induced by simple alkylating agents.18 During the repair reaction, the O6-alkylguanine nucleotide is flipped out of the base stack into the protein’s active site, where it is subject to nucleophilic attack by the activated side chain thiolate anion of Cys145.19 The irreversible transfer of the O6-alkyl lesion from the alkylated guanine residue to Cys145 of the AGT protein regenerates intact guanine within the DNA duplex. Alkylation at Cys145 destabilizes the protein’s tertiary structure, thereby targeting AGT for ubiquitination and proteosomal degradation.20,21 AGT-mediated enhancement of genotoxicity of bis-electrophiles in bacteria has been attributed to the formation of toxic AGT-DNA cross-links, which have been characterized by gel electrophoresis and mass spectrometry techniques.15,22 Liu and colleagues detected the formation of 1,2-dibromoethane-mediated cross-links between guanine bases in DNA and the catalytic cysteine residue (Cys145) of human AGT.22 Similar results were obtained for 1,2,3,4-diepoxybutane (DEB), which formed butanediol cross-links between the active site cysteine residues of AGT (Cys145 or Cys150) and the N-7 position of guanine in DNA.23 The chemical structure of DEB-induced cross-link has been established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G-BD).23 In theory, AGT-DNA cross-linking can be initiated by DEB reactions with either DNA or the protein to form 2-hydroxy-3,4-epoxybutyl (HEB) intermediates, which can subsequently react with other biomolecules to form covalent DPCs. Kalapila et. al. investigated the sequential order of reactivity of the DEB, protein, and DNA to form DPCs using a gel shift assay.24 Either AGT protein or 35S-labeled DNA duplexes were pre-incubated with DEB for varied lengths of time prior to the addition of the other biomolecule. DPCs were formed regardless of the order of component addition, indicating that cross-linking can originate from DEB reactions at the protein or at the DNA.24 The goal of the present work was to examine the influence of DPC formation on cell survival and mutagenesis by developing a DNA damaging agent that was capable of producing DPCs in chromosomal DNA of intact cells but which, through its design, was unable to produce other types of chromosomal DNA lesions (e.g. monoadducts or DNA-DNA cross-links). Based on the known ability of AGT to become covalently cross-linked to DNA in the presence of bis-electrophiles,15,24 it was selected as the basis for development of a protein reagent capable of selectively inducing DPCs in mammalian cells. Recombinant AGT protein or its variants were treated with DEB to create protein monoepoxides containing DNA-reactive 2-hydroxy-3,4-epoxybutyl (HEB) groups (Scheme 1). AGT treated with 1,2-epoxybut-1-ene (EB) was used as a negative control since the resulting conjugate contains an unreactive double bond instead of the electrophilic epoxide functionality and thus cannot produce DPCs. AGT monoepoxides or the corresponding controls were introduced into mammalian cells via electroporation, and the formation of chromosomal DPCs was demonstrated by mass spectrometry. Furthermore, cell toxicity and mutagenesis were analyzed in cells treated with protein bioconjugates capable of selectively inducing DPCs in the absence of other types of DNA damage. Scheme 1 Experimental strategy for selective introduction of DNA-protein cross-links (DPCs) into mammalian cells.