1,2,3,4-Diepoxybutane-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

Abstract

1,2,3,4-diepoxybutane (DEB) is the key carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. DEB is a genotoxic bis-electrophile capable of cross-linking cellular biomolecules to form DNA-DNA and DNA-protein cross-links (DPCs). In the present work, mass spectrometry-based proteomics was employed to characterize DEB-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. Over 150 proteins including histones, high mobility group proteins, transcription factors, splicing factors, and tubulins were found among those covalently cross-linked to chromosomal DNA in the presence of DEB. A large portion of the cross-linked proteins are known factors involved in DNA binding, transcriptional regulation, cell signaling, DNA repair, and DNA damage response. HPLC-ESI⁺-MS/MS analysis of total proteolytic digests revealed the presence of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol conjugates, confirming that DEB forms DPCs between cysteine thiols within proteins and the N-7 guanine positions within DNA. However, relatively high concentrations of DEB were required to achieve significant DPC formation, indicating that it is a poor cross-linking agent as compared to antitumor nitrogen mustards and platinum compounds.

Introduction

DNA-Protein cross-links (DPCs) are super-bulky DNA lesions that are formed upon irreversible bonding of proteins to chromosomal DNA in the presence of cross-linking agents. Due to their enormous size, DPCs can distort DNA helix and interfere with normal DNA-protein interactions, blocking DNA replication, transcription, repair, recombination, and chromatin remodeling.¹ DPCs can be induced by various chemical and physical agents including 1,2,3,4-diepoxybutane (DEB),^2,3 endogenous aldehydes,^4,5 ionizing radiation,⁶ UV light,¹ transition metals,⁷ and therapeutic agents including nitrogen mustards,^8-10 platinum drugs,¹¹ and haloethylnitrosoureas.¹² Despite their ubiquitous formation in living cells, structural features and protein composition of DPC lesions are not well established due to their inherent heterogeneity and the difficulty of analyzing macromolecular conjugates containing both proteins and DNA.

1,2,3,4-Diepoxybutane (DEB) is a carcinogenic, clastogenic, and genotoxic diepoxide produced upon metabolic activation of 1,3-butadiene (BD)(Scheme 1). BD is a known animal and human carcinogen present in automobile exhaust, urban air, and cigarette smoke.^13,14 The potent biological activity of DEB is attributed to its ability to cross-link cellular biomolecules. Initial alkylation of adenine and guanine bases in DNA by DEB produces 2-hydroxy-3,4-epoxybut-1-yl (HEB) monoadducts, which can undergo further reactions, such as hydrolysis and alkylation of neighboring nucleobases to form DNA-DNA cross-links.¹⁵ Alternatively, the epoxide groups of 2-hydroxy-3,4-epoxybut-1-yl (HEB) lesions react with nucleophilic amino acid side chains of adjacent proteins, forming bulky DPC lesions (Scheme 1).^3,15

Scheme 1.

Metabolic activation of 1,3-butadiene to 1,2,3,4-diepoxybutane (DEB) and the formation of DNA-protein cross-links by DEB.

DNA-protein cross-linking by DEB was first observed by Jelitto et al.¹⁵ These authors employed alkaline elution methodology to detect DPC formation in liver tissue of BD-exposed B6C3F1 mice.¹⁵ More recently, it has been shown that DEB forms DPC involving specific proteins such as O⁶-alkylguanine DNA-alkyltransferase (AGT), ³ GAPDH,¹⁶ and histones;¹⁷ and that overexpression of these proteins in bacteria can increase the toxicity and mutagenicity of DEB.^16-19 Our laboratory used a mass spectrometry-based approach to demonstrate that in DEB-induced DPC lesions, the N-7 position of guanine in DNA is cross-linked to cysteine side chains within the proteins.³ We further investigated DPC formation by DEB through an affinity capture approach coupled with mass spectrometry.² Biotinylated DNA duplexes were incubated with protein extracts from human cervical carcinoma (HeLa) cells in the presence of DEB, and the resulting DPCs were captured on streptavidine beads and identified by mass spectrometry based proteomics. A total of 39 proteins were identified, including those known to participate in transcriptional regulation (e.g., GAPDH), chromatin remodeling (e.g., actin), and DNA repair (e.g., AGT).² However, these in vitro experiments were limited to synthetic DNA duplexes which cannot model normal DNA-protein interactions observed in cells.

The purpose of the present study was to characterize DNA-protein cross-linking in human fibrosarcoma (HT1080) cells treated with toxic concentrations of DEB. Proteins covalently trapped on DNA were isolated from cells by a modified phenol/chloroform extraction methodology recently developed by our group.⁸ Thermal hydrolysis was used to release DPCs from DNA backbone in the form of protein-guanine conjugates. Proteins participating in cross-linking were separated by SDS-PAGE and identified by mass spectrometry-based proteomics. A total of 152 cross-linked proteins were found, including those known to be involved in transcriptional regulation, apoptosis, DNA repair, DNA damage response, chromatin remodeling, cell motility, and cell signaling. HPLC-ESI⁺-MS/MS analysis of total proteolytic digests revealed that DEB cross-links cysteine thiols within proteins to the N-7 guanine positions within DNA. Comparison of protein lists to those previously generated for mechlorethamine and cisplatin-induced DNA-protein cross-linking in cells⁸ indicates that while some proteins are targeted by all three bis-electrophiles, these cross-linking agents show significant selectivity in regard to the proteins that they cross-link to DNA in human cells.

Experimental Section

Safety statement

DEB is a known carcinogen and must be handled with adequate safety precautions. Phenol and chloroform are toxic chemicals that should be handled in a well-ventilated fume hood with appropriate personal protective equipment.

Chemicals and Reagents

DEB, ammonium bicarbonate, ammonium acetate, phenylmethanesulfonyl fluoride (PMSF), leupeptin, pepstatin, aprotinin, dithiothreitol (DTT), iodoacetamide, chloroform, ribonuclease A, nuclease P1, and alkaline phosphatase were purchased from Sigma (St. Louis, MO). UltraPure™ Buffer-Saturated Phenol was obtained from Invitrogen (Carlsbad, CA). Mass spectrometry-grade trypsin was purchased from Promega (Madison, WI). Proteinase K was obtained from New England Biolabs (Beverly, MA). Primary polyclonal antibodies specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, SC-25778), poly (ADP-ribose) polymerase 1 (PARP, SC-7150), DNA-(apurinic- or apyrimidinic-site) lyase (ref-1, SC-5577), histone-H4 (SC-10810), nucleophosmin (B23, SC-5564), and nucleolin (SC-13057) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cys-N7G-BD and Cys-[¹⁵N]-N7G-BD were prepared as described previously.³

Cell Culture

Human fibrosarcoma (HT1080) cells²⁰ were obtained from the American Type Culture Collection. The cells were maintained as exponentially growing monolayer cultures in Dulbecco’s modified Eagle’s medium supplemented with 9% fetal bovine serum (FBS), in a humidified incubator at 37 °C with 5% CO₂.

Cytotoxicity experiments

Human fibrosarcoma (HT1080) cells were plated in Dulbecco’s modified Eagle’s medium containing 9% FBS at a density of 5 × 10⁵ cells/dish and permitted to adhere overnight. On the following morning, cells (in duplicate) were treated with a range of DEB concentrations for 3 h at 37 °C. Trypan blue exclusion was used to confirm viability. Cells surviving treatment were counted in a hemocytometer, and cytotoxicity was expressed as the number of cells surviving DEB treatment relative to buffer-treated controls.

DPCs isolation from DEB-treated cells

HT1080 cells in culture were treated with DEB (0, 0.05, 0.10, 1.0, or 2.0 mM) for 3 h at 37 °C. Following exposure, the cells were washed with ice cold phosphate-buffered saline (PBS) and re-suspended in PBS to a final density of ~2 × 10⁶ cells/mL. To isolate nuclei, cells were lysed by adding an equal volume of 2× cell lysis buffer (20 mM Tris-HCl/10 mM MgCl₂/2%(v/v) Triton-X100/0.65 M sucrose), incubated on ice for 5 min, and centrifuged at 2000× g for 10 min at 4 °C. The nuclear pellets were re-suspended in a saline-EDTA solution (75 mM NaCl/24 mM EDTA/1% (w/v) SDS, pH 8.0) containing RNase A (10 μg/mL) and a protease inhibitor cocktail (1 mM PMSF; 1 μg/mL pepstatin; 0.5 μg/mL leupeptin; 1.5 μg/mL aprotinin) to a concentration of ~5 × 10⁶ nuclei/mL and incubated for 2 h at 37 °C with gentle shaking. To remove free proteins, nuclear lysates were extracted with Tris-buffer saturated phenol and chloroform, and DPC-containing DNA was precipitated with cold ethanol. DNA amounts and its purity were estimated by UV and subsequently determined by quantitation of dG in enzymatic hydrolysates as described below.

Enzymatic Digestion of DNA and dG Quantitation

To quantify the DNA isolated from HT1080 cells and to detect any RNA contamination, approximately 5 μg aliquot of DNA from each sample was taken and subjected to neutral thermal hydrolysis (1 h at 70 °C) to release protein-guanine conjugates from the DNA backbone. Partially depurinated DNA was digested to 2′-deoxynucleosides in the presence of nuclease P1 (1 U), alkaline phosphatase (10 U), and 45 ng coformycin (to prevent deamination of dA) in 5 mM ZnCl₂/50 mM ammonium acetate (pH 5.3) buffer for 20 h at 37 °C. Enzymatic digests were passed through Amicon Ultra-0.5 mL Centrifugal Filters (10K MWCO, Millipore, Temecula, CA) to remove proteins prior to HPLC-UV analysis.

Quantitative analysis of dG in enzymatic digests was conducted by HPLC-UV on an Agilent Technologies HPLC System (1100 model) equipped with a diode array UV detector and an autosampler. The sample was loaded on a Zorbax SB-C8 column (4.6 × 150 mm, 5 μm, from Agilent Technologies, Palo Alto, CA) was eluted with a gradient of 150 mM ammonium acetate (A) and acetonitrile (B). Solvent composition was held at 0% B for 2 min, followed by a linear increase to 3% B over 13 min, and further to 30% B over 3 min, where it was kept for the final 7 min of HPC run. UV absorbance was monitored at 260 nm. With this method, dG eluted as a sharp peak at ~13.5 min. dG amounts were determined by comparing HPLC peak areas to a calibration curve constructed by injecting known dG amounts.

Mass Spectrometric Identification of Cross-Linked Proteins

To identify cellular proteins that become covalently attached to chromosomal DNA in DEB -treated cells, HT1080 cells (~10⁷ cells, in triplicate) were treated with 2 mM DEB or buffer control for 3 h at 37 °C, and chromosomal DNA containing any covalently cross-linked proteins was isolated by phenol/chloroform extraction and quantified as described above. DNA (26 μg) was subjected to neutral thermal hydrolysis to release protein-guanine conjugates, dried under vacuum, and reconstituted in 1 × NuPAGE Sample Buffer (Invitrogen, Carlsbad, CA). Proteins were separated using NuPAGE® Novex® 12% Bis-Tris Gels (Invitrogen, Carlsbad, CA) and stained with SimplyBlue Safe stain (Invitrogen, Carlsbad, CA). The gel lanes were excised and divided into five sections encompassing the entire molecular weight range, and each section was further diced into ~1 mm pieces. The proteins present within the gel pieces were subjected to in-gel tryptic digestion as described elsewhere.^8,21 In brief, gel pieces were rinsed with 25 mM ammonium bicarbonate, and the protein thiols were subjected to reduction with DTT (300 mM) and alkylation with iodoacetamide. The gel pieces were then dehydrated by incubation with acetonitrile, dried under vacuum, and reconstituted in 25 mM ammonium bicarbonate buffer. Mass spectrometry-grade trypsin (2–3 μg) was added, and the samples were digested overnight at 37 °C. The resulting tryptic peptides were extracted with 60% acetonitrile containing 0.1% aqueous formic acid, evaporated to dryness, and desalted using ZipTip C18 (Millipore, Temecula, CA). Samples were reconstituted in 0.1% formic acid for HPLC–ESI⁺–MS/MS analysis.

HPLC–ESI⁺–MS/MS analyses of tryptic peptides were conducted on a ThermoScientific LTQ Orbitrap Velos mass spectrometer (Thermo Scientific Corp., Waltham, MA) in line with an Eksigent nanoLC 2D HPLC pump, a nanospray source, and an Xcalibur 2.1.0 software for instrument control. Peptide mixtures (8 μL) were loaded onto a Symmetry C18 trapping column (180 μm × 20 mm, Waters, Milford, MA) using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow composition of 95% A and 5% B at 5 μL/min for 3 minutes. Following trapping, the HPLC flow was decreased to 0.3 μL/min and reversed to elute the peptides off the trap column. The mixtures were separated on a capillary column (75 μm ID, 10 cm packed bed, 15 μm orifice) created by hand packing a commercially purchased fused-silica emitter (New Objective, Woburn MA) with Zorbax SB-C18 5 μm separation media (Agilent, Santa Clara, CA). The gradient program started at 5% B, followed by a linear increase to 60% B over 60 min, and further to 95% B in 5 min. Liquid chromatography was carried out at an ambient temperature. Centroid MS-MS scans were acquired using an isolation width of 2.5 m/z, an activation time of 30 ms, an activation Q of 0.25, 35% normalized CID collision energy, and 1 microscan with a max ion time of 100 ms for each MS/MS scan. The mass spectrometer was calibrated prior to each analysis, and the spray voltage was adjusted to assure a stable spray. Typically, the tune parameters were as follows: spray voltage of 1.6 kV, a capillary temperature of 275 °C, and an S-lens RF Level of 50%. Peptide MS/MS spectra were collected using data-dependent scanning, in which one full scan mass spectrum was followed by eight MS/MS spectra. Dynamic exclusion was enabled for 60 s, and singly charged species were excluded from MS/MS.

Spectral data were analyzed using an in-house developed software pipeline “TINT” that linked raw data extraction, database searching, and probability scoring. Raw data were extracted and converted to the mzXML format using ReadW. Spectra that contained fewer than 6 peaks or had less than 20 measured total ion current (TIC) were excluded. Data were processed using the SEQUEST v.27 algorithm²² on a high speed, multiprocessor Linux cluster in the Minnesota Super Computing Institute at the University of Minnesota. Peptide spectra were searched against NCBI derived human protein database v200806 combined with its reversed counterpart, along with common protein contaminants totaling 70,711 entries. Search parameters included trypsin specificity and up to 2 missed cleavage sites. Cysteine carboxamidomethylation (+57.0215 Da) was set as a fixed modification, and methionine oxidation (+15.9949 Da) was set as a variable modification. Precursor mass tolerance was set to 1.25 m/z within the calculated average mass, and fragment ion mass tolerance was set to 0.5 m/z of their monoisotopic mass. The identified peptides were filtered using Scaffold 3 software (Proteome Software, INC., Portland, OR), to a target false discovery rate (FDR) of 5%. The FDR was calculated with the following expression FDR = (2R)/(R+F)*100, where R is the number of passing reversed peptide identifications and F is the number of passing forward (normal orientation) peptide identifications. The second round of filtering removed proteins supported by less than two distinct peptide identifications in the analyses. Indistinguishable proteins were recognized and grouped. Parsimony rules were applied to generate a minimal list of proteins that explained all of the peptides that passed our entry criteria.²³ Any proteins also found in control samples were excluded from the final list.

Western blot analysis of identified proteins

HT1080 cells (~ 10⁷) were treated with DEB (0, 0.05, 0.5, 1.0, or 2.0 mM) for 3 h at 37 °C. Chromosomal DNA, along with any covalently bound proteins, was extracted and quantified as described above. Approximately 100 μg of DNA from each sample was subjected to neutral thermal hydrolysis (1 h at 70 °C) to release protein-guanine conjugates from the DNA backbone. Proteins were separated by NuPAGE® Novex® 12% Bis-Tris Gels (Invitrogen, Carlsbad, CA) and transferred to Trans-blot nitrocellulose membranes (Bio-Rad, Hercules, CA). Following blocking in Tris-buffered saline (TBS) containing 5% (w/v) bovine serum albumin, the membranes were incubated with the primary antibody against the target protein (glyceraldehyde 3-phosphate dehydrogenase (GAPDH), poly(ADP-ribose) polymerase 1 (PARP), DNA-(apurinic- or apyrimidinic-site) lyase (ref-1), histone-H4, nucleophosmin (B23), and nucleolin) for 3 h at room temperature, rinsed with TBS buffer, and incubated overnight at 4 °C with the corresponding alkaline phosphatase-conjugated secondary antibody. The blots were washed and developed with SIGMA Fast BCIP/NBT (Sigma, St. Louis, MO) according to manufacturer’s instructions.

Isotope dilution HPLC–ESI⁺–MS/MS analysis of Cys-N7G-BD in DEB-exposed cells

HT1080 cells (~ 10⁶) were treated with DEB (0, 0.05, 0.1, 1.0, or 2.0 mM for 3 h at 37 °C). Chromosomal DNA containing DPCs was isolated by the phenol/chloroform extraction procedure described above, and DNA samples (50 μg) were subjected to neutral thermal hydrolysis (1 h at 37 °C) to release protein-guanine conjugates from the DNA backbone. Proteins were cleaved with trypsin (10 μg protein in 25 mM ammonium bicarbonate, overnight at 37 °C), and the resulting peptides were further digested to amino acids in the presence of proteinase K (10 μg in 250 μL H₂O, overnight at 37 °C). The digests were spiked with Cys-[¹⁵N]-N7G-BD internal standard (500 fmol), followed by off-line HPLC purification of Cys-N7G-BD. An Agilent Technologies HPLC system (1100 model) incorporating a diode array detector, an autosampler, and a fraction collector was fitted with a Supelcosil LC-18-DB (4.6 × 250 mm, 5 μm) column (Sigma-Aldrich, St. Louis, MO). The column was eluted at a flow rate of 1 mL/min using 15 mM ammonium acetate, pH 4.9 (A) and acetonitrile (B). The solvent composition was changed linearly from 0 to 24% B over 24 min and further to 60% B in 6 min. HPLC fractions containing Cys-N7G-BD (7.5-9 min) were collected, dried under vacuum, and reconstituted in water (25 μL) for HPLC–ESI⁺–MS/MS analysis.

Quantitative analyses of Cys-N7G-BD were conducted with a Thermo Scientific-Dionex-UltiMate® 3000 capillary HPLC system (Thermo Scientific Corp., Waltham, MA) interfaced to a Thermo-Finnigan TSQ Vantage mass spectrometer (Thermo Scientific Corp., Waltham, MA). Chromatographic separation was accomplished with a Phenomonex Synergi Hydro-RP C18 column (250 mm × 0.5 mm, 4 μm) eluted with a gradient of 15 mM ammonium acetate, pH 5.0 (A) and acetonitrile: isopropyl alcohol (1:1) (B) at a flow rate of 10 μL/min. Solvent composition was linearly changed from 4% to 20% in 8 min further and to 30% over 5 min, and brought back to 4% in 3 min. Under these conditions, Cys-N7G-BD and its internal standard (Cys-[¹⁵N]-N7G-BD) eluted at ~ 7 min. Electrospray ionization was achieved at a spray voltage of 3100 V and a capillary temperature of 270 °C. Collision induced dissociation was performed with Ar as a collision gas (1.5 mTorr) at a collision energy of 23 V. Instrument parameters were optimized for maximum response during infusion of a standard solution of Cys-N7G-BD. HPLC-ESI⁺-MS/MS analysis was performed in the selected reaction monitoring mode by following the neutral loss of guanine from protonated molecules of Cys-N7G-BD (m/z 359.0 [M + H]⁺ → 208.2 [M + H – Gua]⁺) and the corresponding mass transition of Cys-[¹⁵N5]-N7G-BD (m/z 364.0 [M + H]⁺ → 208.2 [M + H – ¹⁵N₅ -Gua]⁺). Relative response ratios of the HPLC-ESI⁺-MS/MS peak areas in extracted ion chromatograms corresponding to the analyte and its internal standard were used to obtain concentration dependence curves for Cys-N7G-BD in cells treated with DEB.

Results

Concentration-Dependent Formation of DPCs in Human Cell Cultures Following DEB Treatment

To monitor DPC formation in vivo, human fibrosarcoma (HT1080) cells (~10⁷) were treated with increasing concentrations of DEB (0, 0.05, 0.1, 0.5, 1.0 or 2.0 mM) for 3 h. Chromosomal DNA containing any covalent DPCs was isolated using modified phenol/chloroform extraction as described in our previous publication.⁸ Our earlier studies have established that DEB-mediated DNA-protein cross-linking takes place at the N7 position of guanine to produce hydrolytically labile N7-guanine adducts.³ Therefore, DEB-induced protein-guanine conjugates were released from DNA by thermal hydrolysis and resolved by 12% SDS-PAGE (Scheme 2, Figure 1). The proteins were visualized using SimplyBlue SafeStain (Figure 1A). We found that while some endogenous DPCs were present in untreated cells (lanes 2-3 in Figure 1), a significant increase of protein band intensities was observed in cells treated with 0.1-2 mM DEB (lanes 4–8 in Figure 1A, Figure 1B). In our previous studies, 80- and 20- fold lower concentrations of mechlorethamine and cisplatin, respectively, were required to achieve similar levels of cross-linking (reference⁸ and Ming et al., manuscript in preparation), suggesting that DEB is less efficient at inducing DNA-protein cross-links. Alternatively, DEB-induced DPCs may have shorter half-lives in cells due to spontaneous depurination and/or active repair. On the basis of these results, 2.0 mM DEB was selected for the proteomics experiments.

Scheme 2.

Strategy for the isolation and analysis of DPCs from DEB-treated mammalian cell cultures. Genomic DNA is isolated from control and treated cells using modified phenol-chloroform extraction in the presence of proteasome inhibitors. DPCs are released form the DNA backbone using thermal hydrolysis and are characterized by MS based proteomics, western blotting, and HPLC-ESI-MS/MS of amino acid-nucleobase conjugates in enzymatic digests.

Figure 1.

Concentration-dependent formation of DNA–protein cross-links in human fibrosarcoma (HT1080) cells treated with DEB. Cells were treated with 0–2 mM DEB for 3 h, and chromosomal DNA containing cross-linked proteins was isolated by modified phenol/chloroform extraction in the presence of proteasome inhibitors. Proteins (from 26 μg DNA) were released from DNA by thermal hydrolysisin the form of protein-guanine conjugates, separated by 12% SDS-PAGE, and visualized by staining with SimplyBlue SafeStain (A). Densitometric analysis of protein bands: normalized band intensity values were obtained by subtracting the values observed in controls. Error bars represent the standard error from two independent experiments (B).

Identification of Cross-linked Proteins by Mass Spectrometry-Based Proteomics

To identify the proteins participating in cross-linking, HT1080 cells (~10⁷ cells, in triplicate) were treated with 2.0 mM DEB, while control cells were treated with DEB-free buffer. DPCs were extracted by the modified phenol/chloroform extraction method described above,⁸ and samples corresponding to 26 μg of DNA were subjected to neutral thermal hydrolysis to release protein-guanine conjugates from the DNA backbone (Scheme 2). We have previously shown that this step facilitates the separation and identification of proteins participating in DPC formation by mass spectrometry.²⁴

SDS-PAGE analysis of protein-guanine conjugates in DEB-treated samples (Figure 2) has revealed distinct protein bands (lanes 6-8 in Figure 2), while the untreated samples exhibited only weak protein signals corresponding to endogenous DPCs (lanes 2- 4 in Figure 2). Protein-containing gel lanes were cut into five sections covering the entire molecular weight range 10- 250 kDa (A-E in Figure 2) and excised from the gel. Gel slices were individually subjected to in-gel tryptic digestion, and the resulting peptides were extracted from the gel and subjected to HPLC–ESI⁺–MS/MS analysis for protein identification. MS/MS analysis of tryptic peptides yielded characteristic b- and y-series fragment ions that were used to determine amino acid sequence and to identify the corresponding proteins (see spectra of two representative peptides in Figure 3). Database searching and parsimony analysis of the MS/MS spectral data resulted in identification of 152 proteins participating in cross-linking to the chromosomal DNA from DEB-treated cells (Table 1). Each protein was identified by a minimum of two unique peptides, and any proteins observed in control samples were excluded from the list.

Figure 2.

SDS-PAGE analysis of samples employed in the proteomics studies of DEB-induced DNA-protein cross-linking. HT1080 cells (~10⁷) were treated with 0 (lanes 2, 3, and 4) or 2 mM DEB (lanes 6, 7, and 8) for 3 h. Following modified phenol/chloroform extraction of DNA in the presence of proteasome inhibitors and thermal hydrolysis to release proteins, the cross-linked proteins were separated by 12% SDS-PAGE and visualized by staining with SimplyBlue SafeStain. Proteins present in the 10–250 kDa molecular weight range were excised from the gel, subjected to in-gel tryptic digestion, and analyzed by HPLC–ESI⁺–MS/MS.

Figure 3.

Representative HPLC–ESI⁺–MS/MS spectra of tryptic peptides used in the identification of DPCs involving nucleolin (A) and histone-H4 (B).

Table 1.

Swiss-Prot ID	Identified Protein	% coverage	No. of unique peptides	Total spectra	Primary cellular function	Protein MW (Da)
1 Q13442	28 kDa heat- and acid-stable phosphoprotein	14.9%	2	2	Cell Signalling/Motility/Architecture	20630.8
2 P35613	Basigin	7.0%	2	2		42199.7
3 P25440	Bromodomain-containing protein 2	2.1%	2	2		88063.3
4 P16070	CD44 antigen	6.9%	4	6		81553.4
5 Q07065	Cytoskeleton-associated protein 4	4.7%	2	2		66022.2
6 Q08554	Desmocollin-1	4.5%	4	4		99988.1
7 Q02413	Desmoglein-1	3.1%	2	2		113748.9
8 P06753	Isoform TM30nm of Tropomyosin alpha-3 chain	10.9%	2	2		29033.3
9 Q03252	Lamin-B2	3.5%	2	2		67689.8
10 P55081	Microfibrillar-associated protein 1	6.2%	2	2		51958.7
11 P46821	Microtubule-associated protein 1B	1.2%	2	2		270620.2
12 P19105	Myosin regulatory light chain 12A	18.7%	3	3		19795.3
13 P35579	Myosin-9	1.2%	2	2		226537.5
14 Q09666	Neuroblast differentiation-associated protein AHNAK	0.4%	2	2		629104.4
15 P41219	Peripherin	4.5%	2	3		53652.0
16 Q15149	Plectin-1	0.7%	2	3		531784.5
17 O00592	Podocalyxin	3.9%	2	2		58635.0
18 P12273	Prolactin-inducible protein	13.7%	2	2		16573.1
19 Q13283	Ras GTPase-activating protein-binding protein 1	4.3%	2	2		52162.8
20 P07996	Thrombospondin-1	2.1%	2	2		129381.7
21 Q13428	Treacle protein	1.7%	2	2		152102.5
22 P09493	Tropomyosin alpha-1 chain	6.7%	2	2		32710.0
23 P67936	Tropomyosin alpha-4 chain	9.7%	2	2		28522.4
24 P07951	Tropomyosin beta chain	6.7%	2	2		32851.7
25 Q71U36	Tubulin alpha-1A chain	3.8%	2	2		50135.7
26 Q13885	Tubulin beta-2A chain	5.4%	2	2		49907.1
27 P05556	Integrin beta-1	6.0%	4	4		88415.1
28 Q16643	Drebrin	5.6%	2	2		71428.6
29 Q01469	Fatty acid-binding protein, epidermal	13.3%	2	2		15164.4
30 P14923	Junction plakoglobin	2.8%	2	2		81745.9
31 P62263	40S ribosomal protein S14	15.9%	2	2	Cellular Homeostasis/Cell Cycle	16272.9
32 P60866	40S ribosomal protein S20	19.3%	2	2		13373.0
33 P62851	40S ribosomal protein S25	24.0%	4	5		13743.0
34 P62753	40S ribosomal protein S6	14.1%	3	3		28681.7
35 P08865	40S ribosomal protein SA	13.6%	3	4		32854.1
36 P14625	Endoplasmin	3.7%	3	3		92471.7
37 O60841	Eukaryotic translation initiation factor 5B	6.2%	5	5		138831.5
38 P14314	Glucosidase 2 subunit beta	8.0%	4	4		59425.8
39 P04406	Glyceraldehyde-3-phosphate dehydrogenase	14.9%	3	3		36053.4
40 Q9H1E3	Nuclear ubiquitous casein and cyclin-dependent kinases substrate	22.6%	3	4		27296.7
41 Q14978	Nucleolar and coiled-body phosphoprotein 1	2.7%	2	2		73604.2
42 Q9NR30	Nucleolar RNA helicase 2	3.3%	2	2		87346.0
43 P19338	Nucleolin	18.3%	14	18		76615.9
44 P06748	Nucleophosmin	13.9%	3	5		32575.5
45 P12270	Nucleoprotein TPR	1.4%	2	2		267289.3
46 Q06830	Peroxiredoxin-1	15.1%	3	4		22110.9
47 Q15061	WD repeat-containing protein 43	3.8%	2	3		74890.8
48 Q86VM9	Zinc finger CCCH domain-containing protein 18	6.8%	6	6		106379.8
49 Q01105	Protein SET	7.2%	2	2		33489.4
50 Q8IZQ5	Selenoprotein H	17.2%	2	2		13453.5
51 Q8IYB3	Serine/arginine repetitive matrix protein 1	3.0%	2	2		102337.5
52 Q9UQ35	Serine/arginine repetitive matrix protein 2	6.3%	12	13		299621.6
53 P26583	High mobility group protein B2	11.5%	2	5	DNA Damage Response/DNA Repair	24034.6
54 O15347	High mobility group protein B3	7.0%	2	2		22980.7
55 P07305	Histone H1.0	11.9%	2	2		20864.1
56 Q02539	Histone H1.1	5.6%	2	4		21843.2
57 P16403	Histone H1.2	9.9%	2	3		21365.8
58 Q96QV6	Histone H2A type 1-A	12.2%	2	2		14234.2
59 P62807	Histone H2B type 1-C/E/F/G/I	7.9%	2	3		13906.6
60 Q16695	Histone H3.1t	11.8%	2	2		15508.9
61 P62805	Histone H4	38.8%	4	4		11367.7
62 Q3SYE8	Putative high mobility group protein B3-like-1	20.0%	2	2		14606.9
63 P23246	Splicing factor, proline- and glutamine-rich	4.2%	2	2		76149.5
64 P62988	Ubiquitin	32.9%	2	2		8565.3
65 Q14444	Caprin-1	6.4%	4	4	RNA Processing/mRN A Splicing	78364.4
66 P61978	Heterogeneous nuclear ribonucleoprotein K	5.0%	2	2		50978.5
67 P22626	Heterogeneous nuclear ribonucleoproteins A2/B1	7.1%	2	2		37430.3
68 Q9UKD2	mRNA turnover protein 4 homolog	18.8%	4	4		27561.3
69 P67809	Nuclease-sensitive element-binding protein 1	23.5%	4	7		35923.8
70 O00567	Nucleolar protein 56	5.6%	2	2		66052.0
71 Q9Y2X3	Nucleolar protein 58	4.5%	3	3		59580.2
72 Q9UMY1	Nucleolar protein 7	16.7%	3	3		29426.9
73 Q86U42	Polyadenylate-binding protein 2	9.5%	3	3		32749.5
74 Q7L014	Probable ATP-dependent RNA helicase DDX46	1.0%	2	2		117366.1
75 Q99848	Probable rRNA-processing protein EBP2	15.7%	4	4		34852.9
76 O75526	RNA-binding motif protein, X-linked-like-2	5.6%	2	2		42816.1
77 P49756	RNA-binding protein 25	4.7%	3	3		100189.1
78 Q9Y5S9	RNA-binding protein 8A	21.8%	3	5		19889.4
79 Q9UKM9	RNA-binding protein Raly	5.9%	2	3		32463.9
80 Q9Y3B9	RRP15-like protein	10.3%	3	3		31484.5
81 Q13435	Splicing factor 3B subunit 2	3.0%	2	2		100229.4
82 Q07955	Splicing factor, arginine/serine-rich 1	37.9%	9	20		27745.1
83 O75494	Splicing factor, arginine/serine-rich 13A	8.8%	2	2		31301.7
84 Q8TF01	Splicing factor, arginine/serine-rich 18	3.4%	2	2		92578.3
85 Q01130	Splicing factor, arginine/serine-rich 2	12.7%	2	6		25477.1
86 Q9BRL6	Splicing factor, arginine/serine-rich 2B	7.5%	3	5		32288.5
87 P84103	Splicing factor, arginine/serine-rich 3	26.2%	4	5		19330.0
88 Q13243	Splicing factor, arginine/serine-rich 5	13.6%	3	3		31264.8
89 Q16629	Splicing factor, arginine/serine-rich 7	8.8%	2	2		27367.5
90 Q13242	Splicing factor, arginine/serine-rich 9	17.2%	4	4		25542.9
91 O75683	Surfeit locus protein 6	7.5%	3	3		41451.7
92 P62995	Transformer-2 protein homolog beta	17.0%	5	9		33666.7
93 P08621	U1 small nuclear ribonucleoprotein 70 kDa	12.1%	5	6		51558.4
94 O00566	U3 small nucleolar ribonucleoprotein protein MPP10	7.1%	4	4		78866.8
95 O43290	U4/U6.U5 tri-snRNP-associated protein 1	3.8%	2	2		90257.1
96 O95218	Zinc finger Ran-binding domain-containing protein 2	10.3%	3	3		37405.4
97 P62241	40S ribosomal protein S8	11.5%	2	2	Transcriptional Regulation/Translation	24206.4
98 P46781	40S ribosomal protein S9	9.8%	2	2		22592.5
99 P05387	60S acidic ribosomal protein P2	67.0%	4	5		11665.5
100 Q07020	60S ribosomal protein L18	12.2%	2	2		21635.2
101 P84098	60S ribosomal protein L19	17.9%	3	3		23467.4
102 P35268	60S ribosomal protein L22	18.8%	2	2		14787.3
103 P62829	60S ribosomal protein L23	17.9%	2	2		14865.9
104 P62750	60S ribosomal protein L23a	32.1%	5	9		17696.2
105 P83731	60S ribosomal protein L24	13.4%	2	3		17779.5
106 P47914	60S ribosomal protein L29	14.5%	2	3		17753.0
107 P62899	60S ribosomal protein L31	18.4%	2	2		14463.2
108 P18124	60S ribosomal protein L7	14.1%	4	5		29227.7
109 P39687	Acidic leucine-rich nuclear phosphoprotein 32 family member A	19.7%	6	8		28586.1
110 Q92688	Acidic leucine-rich nuclear phosphoprotein 32 family member B	13.5%	2	5		28788.7
111 P53999	Activated RNA polymerase II transcriptional coactivator p15	29.9%	3	3		14395.9
112 Q9NYF8	Bcl-2-associated transcription factor 1	4.2%	3	3		106125.3
113 P83916	Chromobox protein homolog 1	14.6%	2	2		21418.1
114 Q13185	Chromobox protein homolog 3	13.1%	2	2		20812.0
115 P45973	Chromobox protein homolog 5	10.0%	2	2		22225.6
116 Q5QJE6	Deoxynucleotidyltransferase terminal-interacting protein 2	6.2%	4	5		84471.1
117 P16989	DNA-binding protein A	7.5%	2	2		40089.4
118 Q8WXX5	DnaJ homolog subfamily C member 9	8.9%	2	2		29910.3
119 Q14919	Dr1-associated corepressor	10.2%	2	2		22350.4
120 P29692	Elongation factor 1-delta	8.9%	2	2		31121.9
121 P35269	General transcription factor IIF subunit 1	6.4%	2	2		58241.9
122 Q7Z4V5	Hepatoma-derived growth factor-related protein 2	6.7%	3	3		74318.3
123 Q99729	Heterogeneous nuclear ribonucleoprotein A/B	8.7%	3	3		36225.2
124 Q14103	Heterogeneous nuclear ribonucleoprotein D0	7.3%	2	2		38434.5
125 P17096	High mobility group protein HMG-I/HMG-Y	37.4%	5	14		11676.2
126 P52926	High mobility group protein HMGI-C	56.9%	4	7		11831.9
127 P50502	Hsc70-interacting protein	6.5%	2	2		41332.4
128 P17096-2	Isoform HMGA1b of High mobility group protein HMG-I/HMG-Y	14.6%	2	4		10679.2
129 P43243	Matrin-3	3.8%	2	2		94626.7
130 Q96DR8	Mucin-like protein 1	17.8%	2	2		9039.3
131 Q13765	Nascent polypeptide-associated complex subunit alpha	13.5%	2	3		23383.3
132 P23497	Nuclear autoantigen Sp-100	2.2%		2		100418.8
133 Q9H930	Nuclear body protein SP140-like protein	4.6%	2	2		50075.6
134 P17480	Nucleolar transcription factor 1	3.1%	2	2		89409.6
135 Q99733	Nucleosome assembly protein 1-like 4	9.3%	3	3		42823.9
136 O75475	PC4 and SFRS1-interacting protein	10.0%	4	5		60103.9
137 Q8NC51	Plasminogen activator inhibitor 1 RNA-binding protein	7.6%	3	3		44965.8
138 Q6NZI2	Polymerase I and transcript release factor	15.4%	4	4		43476.5
139 P51531	Probable global transcription activator SNF2L2	1.4%	2	2		181283.0
140 Q8IZL8	Proline-, glutamic acid- and leucine-rich protein 1	4.1%	2	2		119700.6
141 Q9NY61	Protein AATF	6.6%	3	3		63135.0
142 P35659	Protein DEK	6.7%	2	2		42675.9
143 Q96ST2	Protein IWS1 homolog	3.1%	2	2		91956.1
144 P61244	Protein max	14.4%	2	2		18275.3
145 P05109	Protein S100-A8	20.4%	2	2		10835.0
146 P06702	Protein S100-A9	24.6%	2	2		13242.3
147 Q58FF3	Putative endoplasmin-like protein	4.0%	2	2		45859.7
148 Q8WVC0	RNA polymerase-associated protein LEO1	3.0%	2	2		75405.3
149 Q9Y2W1	Thyroid hormone receptor-associated protein 3	5.6%	5	5		108668.9
150 Q14011	Cold-inducible RNA-binding protein	14.5%	2	2		18648.3
151 P35637	RNA-binding protein FUS	7.2%	2	2		53426.0
152 Q15059	Bromodomain-containing protein 3	2.8%	2	2		79542.5

In an effort to better understand the biological significance of DEB-induced DNA-protein cross-linking, the identified proteins were compiled according to their cellular distribution, participation in biological processes, and molecular functions using the GO database available from the European Bioinformatics Institute (http://www.ebi.ac.uk/QuickGO, Figure 4). The majority of the observed proteins (103 total, 67.7%) are classified as known nuclear proteins (Figure 4A), including histones, high mobility group proteins, matrin-3, and tubulin. An additional 28 proteins (18.4%) are classified as cytoplasmic, and 11 (7.2%) are categorized as membrane-bound proteins (Figure 4A). Nuclear proteins are most likely to participate in DPC formation due to their proximity to DNA. However, it is important to note that some of the identified proteins may be present in more than one cellular compartment due to their participation in multiple biological processes (see below).

Figure 4.

GO annotations for proteins involved in DEB-induced DPC formation in human HT1080 cells: (A) cellular distributions, (B) molecular functions, (C) biological processes, and (D) disease associations. The number of proteins falling into each category is labeled on the charts.

In regard to molecular functions, a large portion of the proteins participating in DPC formation by DEB (N = 92, > 60%) are known to participate in binding to DNA and RNA (Figure 4B). This is consistent with many of the identified proteins playing a role in transcriptional regulation (Figure 4C). This group includes high mobility group protein HMG-I (involved in base excision repair),²⁵ Bcl-2 associated transcription factor 1 (plays a role in tumor suppression via the induction of apoptosis),²⁶ and apoptosis-antagonizing transcription factor (functions as a general inhibitor of the histone deacetylase, HDAC1, anti-apoptosis, and response to DNA damage).²⁷ We also observed proteins involved in cell motility/signaling/ architecture (e.g. lamin-B2, tubulin, ras GTPase activating protein, podocalyxin Plectin-1, and tropomyosin) (Table 1 and Figure 4C), and cellular homeostasis/cell cycle (GAPDH, nucleolin, nucleophosmin (B23)) (Table 1 and Figure 4C). An additional 11 proteins (6.4% of total) are involved in RNA processing, including zinc finger Ran-binding domain-containing protein-2, and transformer-2 protein homologue β (Table1 and Figure 4C).

Many of the proteins are counted in multiple GO categories due to their participation in diverse cellular processes. For example, B23 is involved in ribosome biogenesis,²⁸ histone assembly,²⁹ cell proliferation,³⁰ and regulation of p53 tumor suppressor protein (TP53).³¹ As a result, B23 is listed under two GO annotation categories: Cell cycle and apoptosis (Figure 4C). It is also possible that the available GO annotations may not take into account protein’s secondary cellular localizations, biological processes, and molecular functions. However, the prevalence of proteins involved in transcriptional regulation among the cross-linked proteins is consistent with their proximity and direct binding interactions with DNA.

Western Blot Analysis of Cross-Linked Proteins

The identities of a subset of proteins detected by mass spectrometry-based proteomics were further confirmed by western blot analysis using commercial antibodies (Figure 5). HT1080 cells (~10⁷) were treated with increasing concentrations of the DEB (0, 0.05, 0.1, 0.5, 1.0 or 2.0 mM) for 3 h. Chromosomal DNA containing any covalent DPCs was isolated by the modified phenol/chloroform extraction methodology described above. DNA from each sample (100 μg) was subjected to neutral thermal hydrolysis, and the released protein-guanine conjugates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to western blot analysis using commercial antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), poly(ADP-ribose) polymerase 1 (PARP), DNA-(apurinic- or apyrimidinic-site) lyase (ref-1), histone-H4, nucleophosmin (B23), and nucleolin. These proteins were selected based on their detection by mass spectrometry (Table 1) or their previously demonstrated ability to form DPCs in the presence of other bis-electrophiles.^3,8,24 A concentration-dependent formation of DPCs to PARP, nucleolin, and GAPDH was observed (Figure 5). Since these proteins are also targeted by nitrogen mustards and cisplatin, our results suggest that they are particularly susceptible to DPC formation by bis-electrophiles. In contrast, we did not detect Ref1, histone H4, or nucleolin by western blotting (not shown).

Figure 5.

Western blot analysis of DEB-induced DPCs in HT1080 cells. Following treatment with 0, 0.05, 0.5, 1.0, or 2.0 mM DEB, DNA and covalently cross-linked proteins were isolated by phenol/chloroform extraction. Proteins (from 100 μg DNA) were released by thermal hydrolysis, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Western blotting was performed using primary antibodies specific for PARP, nucleolin, and GAPDH. (A) Densitometric analysis of protein bands: normalized band intensity values were obtained by subtracting the values observed in controls. Error bars represent the standard error from two independent experiments (B).

HPLC-ESI⁺-MS/MS Detection of Cys-N7G-BD Conjugates as Evidence for DPC Formation

In order to quantify the formation of covalent DPCs in cells treated with increasing concentrations of DEB, HT1080 cells (~10⁶) were incubated with 0, 0.1, 0.5, 1.0, or 2.0 mM DEB, and the chromosomal DNA was extracted as described above (Scheme 2). Our previous studies conducted with recombinant proteins and cell free extracts^2,3 have revealed that DEB induced cross-linking takes place mainly between the cysteine sulfhydryl side chain within proteins and the N7-position to form 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G- BD) conjugates (Scheme 2), therefore the quantitative HPLC-ESI⁺-MS/MS analyses have focused on Cys-N7G- BD. Equal DNA amounts (50 μg) were taken from each sample and subjected to neutral thermal hydrolysis to release protein-guanine conjugates from the DNA backbone. Proteins were enzymatically digested to amino acids, and the resulting digests were spiked with known amounts of Cys-[¹⁵N]-N7G-BD and subjected to off-line HPLC purification. Conjugate amounts in cells were determined by isotope dilution HPLC-ESI⁺-MS/MS in the selected reaction monitoring mode as described previously.^2,8,24,32 Cys-N7G-BD conjugates were detected in proteolytic hydrolysates of samples from DEB-treated HT1080 cells, but not in untreated cells (Figure 6A). These data confirm that DEB induced DNA-protein cross-linking takes place between the side chain sulfhydryls of cysteine residues in proteins and the N7-position of guanine bases in chromosomal DNA. Furthermore, Cys-N7G-BD amounts in cells were dependent on DEB concentration, reaching the levels of 6 lesions per million normal nucleotides in cells treated with 2 mM DEB (Figure 6B).

Figure 6.

HPLC-ESI⁺-MS/MS analysis of 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G-BD) conjugates in total proteolytic digests. HT1080 cells were exposed to 0, 0.1, 0.5, 1.0, or 2.0 mM DEB for 3 h. Following extraction of DPC-containing chromosomal DNA, equal DNA amounts from each sample were subjected to thermal and enzymatic hydrolysis to release amino acid-nucleobase conjugates. The samples were subjected to offline HPLC to enrich for Cys-N7G-BD prior to HPLC-ESI⁺-MS/MS analysis. Quantification of Cys-N7G-BD was accomplished using isotope dilution with Cys-¹⁵N₅-N7G-BD. Shown are extracted ion chromatograms corresponding to HT1080 cells incubated in the absence of DEB (negative control) and samples treated with 2 mM DEB (A) and concentration dependent formation of Cys-N7G-BD in DEB-treated HT1080 cells (B).

Discussion

Reversible DNA-protein interactions are essential for normal cell function. Chromosomal DNA is packaged in the nucleus by wrapping around histone octamers, and reversible binding of regulatory proteins to their recognition motifs controls DNA replication and transcription. Protein binding also mediates DNA repair and cellular responses to DNA damage. Any interruption of these dynamic interactions is likely to have serious consequences for cell viability and genetic stability.

DNA-protein cross-links are bulky macromolecular conjugates that form when proteins become covalently trapped in the DNA strand. These ubiquitous lesions that can be induced by a variety of chemical and physical drugs, environmental toxins, ionizing radiation, endogenous aldehydes, and free radical generating systems.³³ Covalent DPCs have been shown to accumulate in an age-dependent fashion in brain and heart tissues, probably a result of exposure to endogenous reactive oxygen species, lipid peroxidation products, and transition metals. Due to their considerable size and their pronounced effects on DNA structure and DNA-protein interactions, DPCs are hypothesized to interfere with replication, transcription, and repair, potentially leading to mutations and cell death.¹

Experimental evidence is available in support of a role of DPCs in the biological activity of bis-electrophiles.³³ For example, 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, presumably due to the formation of toxic AGT-DNA cross-links.³⁴ In our recent study, AGT proteins containing DNA-reactive 2-hydroxy-3,4-epoxybutyl groups were introduced into mammalian cell lines, generating covalent AGT-DNA cross-links and inducing statistically significant levels of cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) (Tretyakova et al., submitted for publication). However, the extent of DPC formation in cells and the identities of the participating proteins have not been established, limiting our understanding of the role of DPCs in the biological activity of common antitumor drugs and in the development of cardiovascular disease, cancer, and age-related neurodegeneration.

Recently, our laboratory developed a practical and effective approach for isolating DPC lesions from cultured cells using a modified phenol/chloroform extraction method.⁸ The approach has been successfully used to characterize DPCs induced by antitumor nistrogen mustards and cisplatin in human cells and in blood of cancer patients undergoing chemotherapy (Ming et al., manuscript in preparation and Gherezghiher et al., unpublished observations). In the present work, the same methodology was applied to characterize DNA-protein cross-linking in human HT1080 cells treated with cytotoxic concentrations of DEB, the genotoxic metabolite of 1,3-butadiene (Scheme 1). Following DPC isolation from treated cells, neutral thermal hydrolysis was employed to selectively release DPC from the DNA backbone in the form of protein-guanine conjugates (Scheme 2).⁸ Proteins participating in cross-linking to DNA were identified by mass spectrometry-based proteomics and immunoblotting. Each protein was identified based on sequence of at least two unique peptides, and any proteins present in control samples were excluded.

A total of 152 proteins were found to form cross-links to chromosomal DNA in the presence of DEB (Table 1). These proteins are involved in a variety of cellular functions, including transcriptional regulation (e.g. matrin-3, high mobility group protein B3, and 40S ribosomal protein S20), cell signaling and architecture (e.g. lamin-B2, basigin, and myosin regulatory light chain 12A), regulation of cell cycle (e.g. GAPDH, tubulin beta-2A chain, and nucleolar protein 58), DNA damage response and repair (e.g. histone proteins), and RNA processing (e.g. splicing factor, arginine/serine-rich 3, ATP-dependent RNA helicase, and nucleolar RNA helicase). The identified proteins have been associated with variety of diseases including cancer, renal and urological diseases, cardiovascular diseases, and dermatological disease and conditions as revealed by Ingenuity Pathway Analysis (IPA) (Figure 4D).

Previously, it has been reported that DEB forms DPCs with recombinant AGT,³ GAPDH,¹⁶ and histones. ¹⁷ More recently, our laboratory employed an affinity capture approach to identify 39 proteins which participate in DEB-mediated cross-linking to DNA in cell free protein extracts from HeLa cells.² Only two of the previously identified proteins (GAPDH, and heterogeneous nuclear ribonucleoprotein K)² were also observed in the present study (Table 1), while the remaining proteins were not detected from in vitro cross-linking experiments. This may be partially explained by differences in protein expression profiles between human ovarian carcinoma (HeLa)² and fibrosarcoma (HT1080) cells.³⁵ However, the most dramatic difference between the two studies is the nature of the DNA employed. Our in vitro studies² utilized short synthetic DNA duplexes representing codons 10-15 of K-ras protooncogene (5′-GGA GCT GGT GGC GTAGGC-3′). These duplexes lacked nucleosomal structure and were unlikely to participate in sequence-specific interactions with DNA-binding proteins. In contrast, experiments described in the present report were conducted in intact human cells, where DNA is organized in chromatin and involves specific interactions between DNA and proteins, potentially facilitating DPC formation. Indeed, ~ 10-fold lower concentrations of DEB were required to achieve the same extent of crosslinking in intact cells as compared to in vitro experiments.² Both experiments required millimolar levels of DEB to achieve sufficient numbers of cross-links to enable their MS analysis. Although these concentrations are unlikely to be achieved in vivo, it is expected that the same proteins are targeted following exposure to lower concentratons of DEB.

When the list of proteins identified in the present study of DEB-mediated DNA-protein cross-linking in HT1080 cells (Table 1) is compared to a similar list of mechlorethamine-cross linked proteins,⁸ eighteen proteins are found in common, including, Bcl-2-associated transcription factor 1, B23, matrin-3, high mobility group protein HMG-I/HMG-Y, heterogeneous nuclear ribonucleoprotein A/B, and zinc finger CCCH domain-containing protein 18 (Figure 7A). Similarly, sixty seven proteins identified in the present work, including lamin-B2, nucleolin, myosin-9, matrin-3, histone H4, high mobility group protein HMG-I, AATF, B23, GAPDH, and peroxiredoxin-1 have also been found to form DPCs in HT1080 cells treated with cisplatin (Figure 7B) (Ming et al., manuscript in preparation). The remaining proteins were not common between the three lists, which is not surprising given the distinct mechanisms of cross-link formation by the three bis-electrophiles. In particular, while DEB and nitrogen mustards preferentially target cysteinyl residues within the proteins (Figure 7), cisplatin-mediated DPCs involve lysine and arginine residues (Ming et al., manuscript in preparation).⁸

Figure 7.

Venn diagrams showing the overlaps between proteins that form cross-links to chromosomal DNA in DEB-treated HT1080 cells (2 mM) with proteins that form DPCs in the presence of mechlorethamine (25μM) (A) and proteins that form DPCs in the presence of cisplatin (100 μM) (B).

Due to their unusually bulky nature as compared to the conventional DNA lesions, DPCs are expected to interrupt many critical cellular processes such as DNA replication, DNA repair, recombination, transcription, and chromatin remodeling. Previously, several possible mechanisms for DPCs repair have been proposed including proteolytic degradation, nucleotide excision repair (NER), and homologous recombination (HR). For example, Nakano et al. studied DPC repair in bacteria and mammalian cells and found that NER is involved in the repair of DPCs involving proteins less than12-14 kDa in size in bacteria and less than 8-10 kDa in mammalian cells, whereas HR is responsible for the repair of oversized DPCs.³⁶ This would suggest that the majority of DPCs induced by DEB are repaired by HR, due to their significant size (Table 1).

In conclusion, our study demonstrates that the treatment of human fibrosarcoma cells with DEB induces DNA-protein cross-links to a variety of cellular proteins, including those participating in chromatin remodeling, translation, DNA replication, DNA repair, RNA metabolism, transcriptional regulation, and apoptosis. Although DEB-mediated DNA-protein cross-linking is relatively inefficient as compared to that induced by nitrogen mustards and platinum compounds, the resulting bulky, helix distorting DPC lesions have a considerable potential to interfere with critical cellular processes such as replication and transcription, potentially triggering programmed cell death and/or genotoxic outcomes.

Abbreviations

AGT

O⁶-alkylguanine DNA alkyltansferase

Cys-N7G-BD

1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol

DEB

1,2,3,4-diepoxybutane

DPC

DNA-protein cross-link

DTT

dithiothreitol

FBS

fetal bovine serum

FDR

false discovery rate

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GSH

glutathione

HPLC-ESI⁺-MS/MS

high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

PARP

poly(ADP-ribose) polymerase I

PBS

phosphate-buffered saline

PMSF

phenylmethanesulfonyl fluoride

Ref-1

DNA-(apurinic- or apyrimidinic-site) lyase

TBS

Tris-buffered saline

XRCC-1

x-ray cross-complementing protein I