The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Deri

Article pubs.acs.org/biochemistry A New Cross-Link for an Old Cross-Linking Drug: The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Derived from Abasic Sites in Addition to the Expected Drug-Bridged Cross-Links Maryam Imani Nejad,† Kevin M. Johnson,† Nathan E. Price,† and Kent S. Gates*,†,‡ † Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States ‡ Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States *S Supporting Information ABSTRACT: Nitrogen mustard anticancer drugs generate highly reactive aziridinium ions that alkylate DNA. Mono- adducts arising from reaction with position N7 of guanine residues are the major DNA adducts generated by these agents. Interstrand cross-links in which the drug bridges position N7 of two guanine residues are formed in low yields relative to those of the monoadducts but are generally thought to be central to medicinal activity. The N7-alkylguanine residues generated by nitrogen mustards are depurinated to yield abasic (Ap) sites in duplex DNA. Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine lead to interstrand cross-links of a type not previously associated with this drug. Gel electrophoretic data were consistent with early evolution of the expected drug-bridged cross-links, followed by the appearance of Ap-derived cross-links. The evidence is further consistent with a reaction pathway involving alkylation of a guanine residue in a 5′-GT sequence, followed by depurination to generate the Ap site, and cross-link formation via reaction of the Ap aldehyde residue with the opposing adenine residue at this site [Price, N. E., Johnson, K. M., Wang, J., Fekry, M. I., Wang, Y., and Gates, K. S. (2014) J. Am. Chem. Soc. 136, 3483−3490]. The monofunctional DNA-alkylating agents 2-chloro-N,N-diethylethanamine 5, (2-chloroethyl)ethylsulfide 6, and natural product leinamycin similarly were found to induce the formation of Ap-derived cross-links in duplex DNA. This work provides the first characterization of Ap-derived cross-links at sequences in which a cytosine residue is located directly opposing the Ap site. Cross-linking processes of this type could be relevant in medicine and biology because Ap sites with directly opposing cytosine residues occur frequently in genomic DNA via spontaneous or enzymatic depurination of guanine and N7-alkylguanine residues. itrogen mustards such as mechlorethamine (HN2) were can forge DNA−DNA interstrand cross-links in some the first synthetic anticancer drugs1,2 and remain in sequences via reaction of the Ap aldehyde residue with the N − widespread clinical use.3 6 These bifunctional agents generate exocyclic amino groups of nucleobases such as adenine and aziridinium ions that react with DNA at a variety of locations, guanine on the opposing strand of the DNA duplex (Scheme − including N7-guanine, N3-adenine, N3-cytidine, and the 2).32 40 The cross-linking reactions considered here, involving − phosphodiester linkages of the backbone (Scheme 1).4,7 21 “true” Ap sites, are distinct from those involving oxidized abasic Monoadducts (1 and 2) at guanine residues are the major DNA sites.41,42 Here, we show that Ap sites generated by the nitrogen alkylation products formed by these drugs (Scheme 1).8,10,11 mustard mechlorethamine (HN2) give rise to interstrand cross- Interstrand cross-links (3) are generated in much lower yields links of a type not previously associated with this drug. We (1−10% of total adducts) but are generally believed to be the present gel electrophoretic data that are consistent with an early critical lesions responsible for medicinal activity of the nitrogen evolution of the expected drug-bridged cross-links 3 followed mustards.4,22,23 Cross-link formation by nitrogen mustards can by the appearance of Ap-derived cross-links. The evidence is occur via reactions with two guanine residues in 5′-GNC consistent with a reaction pathway involving alkylation of a − sequences [3 (Scheme 1)];14,24 28 however, there is also guanine residue in a 5′-GT sequence, followed by depurination evidence of G-G cross-link formation at 5′-GC sequences as to generate the Ap site, and cross-link formation via reaction of well as G-A and A-A cross-linking at as-yet-undefined the Ap aldehyde residue with the opposing adenine residue at sequences.12,26,29 The alkylation of guanine and adenine residues by nitrogen Received: October 21, 2016 mustards induces the formation of abasic (Ap) sites in genomic Revised: November 22, 2016 DNA (Scheme 1).9,10,30,31 We recently showed that Ap sites Published: November 29, 2016 © 2016 American Chemical Society 7033 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article Scheme 1 cytosine residues occur readily in genomic DNA via spontaneous depurination of guanine and alkylguanine residues. The processes described here expand the list of mechanisms by which nitrogen mustards and other DNA-alkylating drugs can generate cytotoxic interstrand cross-links. ■ EXPERIMENTAL SECTION Materials and General Procedures. Reagents were purchased from the following suppliers and were of the highest purity available: oligonucleotides from Integrated DNA Technologies (Coralville, IA), uracil DNA glycosylase (UDG) and T4 DNA polynucleotide kinase (T4 PNK) from New England Biolabs (Ipswich, MA), [γ-32P]ATP (6000 Ci/mmol) from PerkinElmer, 19:1 acrylamide/bis-acrylamide (40% solution/electrophoresis) from Fisher Scientific (Waltham, MA), and mechlorethamine hydrochloride and alkylating agents from Sigma-Aldrich (St. Louis, MO). LNM was a gift from Kyowa Hakko Kogyo, Ltd. C-18 Sep-Pak cartridges were purchased from Waters (Milford, MA), and BS Poly Prep columns were obtained from Bio-Rad (Hercules, CA). Quantification of radioactivity in polyacrylamide gels was conducted using a Personal Molecular Imager (Bio-Rad) with Quantity One (version 4.6.5). Representative Procedure for Cross-Link Formation Time Courses by HN2, 5, 6, and LNM. Single-stranded 2′- deoxyoligonucleotides were 5′-labeled using standard procedures.44 Labeled DNA was annealed44 with its complementary strand to give the duplexes shown in Figure 1. In a typical cross- Scheme 2 this site (Scheme 2).35 We further showed that the monofunctional DNA-alkylating agents 2-chloro-N,N-diethylethanamine 5, (2-chloroethyl)ethylsulfide 6, and natural product leinamycin (LNM) similarly induce Ap-derived cross-links in duplex DNA via alkylation and depurination at 5′-GT sequences. Figure 1. DNA sequences used in these studies. Ap-containing duplexes were generated by the action of UDG on the corresponding dU-containing duplex. Cross-link locations are indicated with a red connection. linking reaction, HN2 was introduced into the reaction mixture These results provide the first characterization of Ap-derived as a stock solution in DMF, to give a mixture containing HN2 cross-link formation at sequences in which a cytosine residue (1 mM) and labeled DNA in HEPES buffer (50 mM, pH 7) directly opposes the Ap site. The ability of such sites to containing 100 mM NaCl and 10% (v/v) DMF that was generate interstrand cross-links was by no means certain, incubated at 37 °C for 96 h unless otherwise specified. The because the nature of the directly opposing base can exert DNA was ethanol-precipitated from the reaction mixture,44 significant effects on the structures of Ap-containing duplexes.43 resuspended in formamide loading buffer,44 and loaded onto a Cross-linking reactions of this type could be important in 20% denaturing polyacrylamide gel, and the gel was electro- biology and medicine because Ap sites with directly opposing phoresed for 5 h at 1600 V. The amount of radiolabeled DNA 7034 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article in each band on the gel was measured by phosphorimager containing double-stranded DNA (∼400000 cpm) was analysis. The time course for the formation of the late-forming incubated in HEPES buffer (50 mM, pH 7) containing NaCl cross-link was determined by incubating a solution containing (100 mM) at 37 °C for 120 h. The DNA was ethanol labeled DNA (approximately 100000 cpm) and HEPES buffer precipitated, suspended in formamide loading buffer, and (50 mM, pH 7) containing NaCl (100 mM) and HN2 (1 mM) resolved on a 2 mm thick 20% denaturing polyacrylamide gel. at 37 °C. At specified time points, aliquots (3 μL) were The late-forming cross-linked duplex band was visualized using removed and formamide loading dye was added followed by X-ray film, the band cut out of the gel, and the gel slice crushed, freezing at −20 °C, and gel electrophoretic analysis as described and the gel pieces were vortex-mixed in elution buffer (200 mM above. For cross-link formation by LNM, all conditions were NaCl and 1 mM EDTA) at room temperature for at least 1 h. identical, except LNM was introduced as a stock solution in The mixture was filtered through a Poly-Prep column to acetonitrile, the final concentration of LNM was either 100 or remove gel fragments, and the residue was ethanol precipitated, 500 μM, and β-mercaptoethanol (0.5 or 2.5 mM) was added to redissolved in water, and mixed with 2× oxidation buffer [10 initiate the DNA alkylation reaction.45 μL of a solution composed of 20 mM sodium phosphate (pH Representative Procedure for Preparation of Du- 7.2), 20 mM NaCl, 2 mM sodium ascorbate, and 1 mM H2O2]. plexes Containing Authentic Ap-Derived Cross-Links. A To this mixture was added a solution of iron-EDTA [2 μLof70 · single-stranded, uracil-containing 2′-deoxyoligonucleotide was mM EDTA and 70 mM (NH4)2Fe(SO4)2 6H2O] to start the 5′-labeled using standard procedures,44 annealed with its reaction, and the mixture was vortexed briefly and incubated at complementary strand, and treated with the enzyme UDG room temperature for 5 min before addition of a thiourea stop (50 units/mL, final concentration) to generate the Ap site.

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