1,2,3,4-Diepoxybutane-Induced DNAâProtein ... - ACS Publications

Article pubs.acs.org/jpr

1,2,3,4-Diepoxybutane-Induced DNA−Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells Teshome B. Gherezghiher,† Xun Ming,† Peter W. Villalta, Colin Campbell,§ and Natalia Y. Tretyakova*,† †

Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States § Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, United States S Supporting Information *

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 crosslinking 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-7yl)-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. KEYWORDS: DNA−protein cross-links, diepoxybutane, mass spectrometry, proteomics, Western blot

■

INTRODUCTION

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 HEB lesions react with nucleophilic amino acid side chains of adjacent proteins, forming bulky DPC lesions (Scheme 1).3,15 DNA−protein cross-linking by DEB was first observed by Jelitto et al.15 These authors employed alkaline elution

DNA−protein cross-links (DPCs) are superbulky DNA lesions that are formed upon irreversible bonding of proteins to chromosomal DNA in the presence of cross-linking agents. Because of 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. © 2013 American Chemical Society

Received: December 19, 2012 Published: March 18, 2013 2151

dx.doi.org/10.1021/pr3011974 | J. Proteome Res. 2013, 12, 2151−2164

Journal of Proteome Research

Article

Scheme 1. Metabolic Activation of 1,3-Butadiene to 1,2,3,4-Diepoxybutane (DEB) and the Formation of DNA−Protein CrossLinks by DEB

agents show significant selectivity in regard to the proteins that they cross-link to DNA in human cells.

methodology to detect DPC formation in liver tissue of BDexposed B6C3F1 mice.15 More recently, it has been shown that DEB forms DPC involving specific proteins such as O6alkylguanine DNA-alkyltransferase (AGT),3 GAPDH,16 histones,17 and GST;18 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 streptavidin 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 SDSPAGE 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

■

EXPERIMENTAL SECTION

Safety Statement

Caution! 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 f ume 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 BufferSaturated 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-N7GBD 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 2152



Article

CA). Proteins were separated using NuPAGE Novex 12% BisTris 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 LTQ Orbitrap Velos mass spectrometer (Thermo Scientific, Waltham, MA) in line with an Eksigent nanoLC 2D HPLC pump, a nanospray source, and 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 min. 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 i.d., 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 ensure 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

exclusion methodology was used to confirm cell 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 resuspended 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 2000g for 10 min at 4 °C. The nuclear pellets were resuspended 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 (10 K 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 samples were loaded on a Zorbax SBC8 column (4.6 × 150 mm, 5 μm, from Agilent Technologies, Palo Alto, CA) and 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 the HPLC 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, 2153



Article

Scheme 2. Strategy for the Isolation and Analysis of DPCs from DEB-Treated Mammalian Cell Culturesa

a

Genomic DNA is isolated from control and treated cells using modified phenol−chloroform extraction in the presence of proteasome inhibitors. DPCs are released from the DNA backbone using thermal hydrolysis and are characterized by MS based proteomics, Western blotting, and HPLCESI−MS/MS of amino acid−nucleobase conjugates in enzymatic digests.

Isotope Dilution HPLC−ESI+−MS/MS Analysis of Cys-N7G-BD in DEB-Exposed Cells

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.

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. 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 Dionex UltiMate 3000 RSLCnano HPLC system (Thermo Scientific, Waltham, MA) interfaced to a TSQ Vantage mass spectrometer (Thermo Scientific, 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 and further to 30% over 5 min, and brought back to 4% in 3 min. Under these conditions, CysN7G-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

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. 2154



Article

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 crosslinking takes place at the N7 position of guanine to produce hydrolytically labile N7-guanine adducts.3 Therefore, DEBinduced 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 3−4 in Figure 1), a significant increase of protein band intensities was observed in cells treated with 0.1−2 mM DEB (lanes 6−8 in Figure 1A, Figure 1B). In our previous studies, 80- and 20fold lower concentrations of mechlorethamine and cisplatin, respectively, were required to achieve similar levels of crosslinking (ref 8 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. Identification of Cross-Linked Proteins by Mass Spectrometry-Based Proteomics

Figure 1. Concentration-dependent formation of DNA−protein crosslinks 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 hydrolysis in 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).

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 DEBtreated 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 crosslinking 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. 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). A 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. 2155



Article

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 of the DPC fraction (not shown).

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 2−4) or 2 mM DEB (lanes 6−8) for 3 h. Following modified phenol/chloroform extraction of DNA in the presence of proteasome inhibitors and thermal hydrolysis to release the proteins, protein-guanine conjugates were separated by 12% SDSPAGE 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.

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). 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, antiapoptosis, 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 do not take into account protein’s secondary cellular localizations, biological processes, and molecular functions. However, the prevalence of proteins involved in transcriptional regulation

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

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 extracts2,3 have revealed that DEB induced crosslinking takes place mainly between the cysteine sulfhydryl side chain within proteins and the N7-position of guanine in DNA to form 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (CysN7G- BD) conjugates (Scheme 2); therefore, the quantitative HPLC−ESI+−MS/MS analyses have focused on Cys-N7GBD. Equal DNA amounts (50 μg) were taken from each sample and subjected to neutral thermal hydrolysis to release proteinguanine 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. Cys-guanine conjugate amounts in cells were determined by isotope dilution HPLCESI+-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). 2156



Article

Figure 3. Representative HPLC−ESI+−MS/MS spectra of tryptic peptides used in the identification of DPCs involving nucleolin (A) and histoneH4 (B).

■

DISCUSSION

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 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

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 2157



Article

Table 1 Swiss-Prot ID

identified protein

% coverage

no. of unique peptides

total spectra

primary cellular function Cell Signaling/Motility/ Architecture

protein MW (Da)

1

Q13442

28 kDa heat- and acid-stable phosphoprotein

14.9%

2

2

2 3 4 5 6 7 8 9 10 11 12 13 14

P35613 P25440 P16070 Q07065 Q08554 Q02413 P06753 Q03252 P55081 P46821 P19105 P35579 Q09666

7.0% 2.1% 6.9% 4.7% 4.5% 3.1% 10.9% 3.5% 6.2% 1.2% 18.7% 1.2% 0.4%

2 2 4 2 4 2 2 2 2 2 3 2 2

2 2 6 2 4 2 2 2 2 2 3 2 2

42199.7 88063.3 81553.4 66022.2 99988.1 113748.9 29033.3 67689.8 51958.7 270620.2 19795.3 226537.5 629104.4

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

P41219 Q15149 O00592 P12273 Q13283 P07996 Q13428 P09493 P67936 P07951 Q71U36 Q13885 P05556 Q16643 Q01469 P14923 P62263

Basigin Bromodomain-containing protein 2 CD44 antigen Cytoskeleton-associated protein 4 Desmocollin-1 Desmoglein-1 Isoform TM30 nm of Tropomyosin alpha-3 chain Lamin-B2 Microfibrillar-associated protein 1 Microtubule-associated protein 1B Myosin regulatory light chain 12A Myosin-9 Neuroblast differentiation-associated protein AHNAK Peripherin Plectin-1 Podocalyxin Prolactin-inducible protein Ras GTPase-activating protein-binding protein 1 Thrombospondin-1 Treacle protein Tropomyosin alpha-1 chain Tropomyosin alpha-4 chain Tropomyosin beta chain Tubulin alpha-1A chain Tubulin beta-2A chain Integrin beta-1 Drebrin Fatty acid-binding protein, epidermal Junction plakoglobin 40S ribosomal protein S14

4.5% 0.7% 3.9% 13.7% 4.3% 2.1% 1.7% 6.7% 9.7% 6.7% 3.8% 5.4% 6.0% 5.6% 13.3% 2.8% 15.9%

2 2 2 2 2 2 2 2 2 2 2 2 4 2 2 2 2

3 3 2 2 2 2 2 2 2 2 2 2 4 2 2 2 2

53652.0 531784.5 58635.0 16573.1 52162.8 129381.7 152102.5 32710.0 28522.4 32851.7 50135.7 49907.1 88415.1 71428.6 15164.4 81745.9 16272.9

32 33 34 35 36 37 38 39 40

P60866 P62851 P62753 P08865 P14625 O60841 P14314 P04406 Q9H1E3

19.3% 24.0% 14.1% 13.6% 3.7% 6.2% 8.0% 14.9% 22.6%

2 4 3 3 3 5 4 3 3

2 5 3 4 3 5 4 3 4

13373.0 13743.0 28681.7 32854.1 92471.7 138831.5 59425.8 36053.4 27296.7

41 42 43 44 45 46 47 48 49 50 51 52 53

Q14978 Q9NR30 P19338 P06748 P12270 Q06830 Q15061 Q86VM9 Q01105 Q8IZQ5 Q8IYB3 Q9UQ35 P26583

40S ribosomal protein S20 40S ribosomal protein S25 40S ribosomal protein S6 40S ribosomal protein SA Endoplasmin Eukaryotic translation initiation factor 5B Glucosidase 2 subunit beta Glyceraldehyde-3-phosphate dehydrogenase Nuclear ubiquitous casein and cyclin-dependent kinases substrate Nucleolar and coiled-body phosphoprotein 1 Nucleolar RNA helicase 2 Nucleolin Nucleophosmin Nucleoprotein TPR Peroxiredoxin-1 WD repeat-containing protein 43 Zinc finger CCCH domain-containing protein 18 Protein SET Selenoprotein H Serine/arginine repetitive matrix protein 1 Serine/arginine repetitive matrix protein 2 High mobility group protein B2

2.7% 3.3% 18.3% 13.9% 1.4% 15.1% 3.8% 6.8% 7.2% 17.2% 3.0% 6.3% 11.5%

2 2 14 3 2 3 2 6 2 2 2 12 2

2 2 18 5 2 4 3 6 2 2 2 13 5

73604.2 87346.0 76615.9 32575.5 267289.3 22110.9 74890.8 106379.8 33489.4 13453.5 102337.5 299621.6 24034.6

54 55 56

O15347 P07305 Q02539

High mobility group protein B3 Histone H1.0 Histone H1.1

7.0% 11.9% 5.6%

2 2 2

2 2 4

2158

Cellular Homeostasis/Cell Cycle

DNA Damage Response/ DNA Repair

20630.8

22980.7 20864.1 21843.2



Article

Table 1. continued Swiss-Prot ID

identified protein

% coverage


total spectra

2 2 2 2 4

3 2 3 2 4

57 58 59 60 61 62 63 64 65

P16403 Q96QV6 P62807 Q16695 P62805 Q3SYE8 P23246 P62988 Q14444

Histone H1.2 Histone H2A type 1-A Histone H2B type 1-C/E/F/G/I Histone H3.1t Histone H4 Putative high mobility group protein B3-like-1 Splicing factor, proline- and glutamine-rich Ubiquitin Caprin-1

9.9% 12.2% 7.9% 11.8% 38.8% 20.0% 4.2% 32.9% 6.4%

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94

P61978 P22626 Q9UKD2 P67809 O00567 Q9Y2 × 3 Q9UMY1 Q86U42 Q7L014 Q99848 O75526 P49756 Q9Y5S9 Q9UKM9 Q9Y3B9 Q13435 Q07955 O75494 Q8TF01 Q01130 Q9BRL6 P84103 Q13243 Q16629 Q13242 O75683 P62995 P08621 O00566

5.0% 7.1% 18.8% 23.5% 5.6% 4.5% 16.7% 9.5% 1.0% 15.7% 5.6% 4.7% 21.8% 5.9% 10.3% 3.0% 37.9% 8.8% 3.4% 12.7% 7.5% 26.2% 13.6% 8.8% 17.2% 7.5% 17.0% 12.1% 7.1%

95 96

O43290 O95218

97

P62241

Heterogeneous nuclear ribonucleoprotein K Heterogeneous nuclear ribonucleoproteins A2/B1 mRNA turnover protein 4 homologue Nuclease-sensitive element-binding protein 1 Nucleolar protein 56 Nucleolar protein 58 Nucleolar protein 7 Polyadenylate-binding protein 2 Probable ATP-dependent RNA helicase DDX46 Probable rRNA-processing protein EBP2 RNA-binding motif protein, X-linked-like-2 RNA-binding protein 25 RNA-binding protein 8A RNA-binding protein Raly RRP15-like protein Splicing factor 3B subunit 2 Splicing factor, arginine/serine-rich 1 Splicing factor, arginine/serine-rich 13A Splicing factor, arginine/serine-rich 18 Splicing factor, arginine/serine-rich 2 Splicing factor, arginine/serine-rich 2B Splicing factor, arginine/serine-rich 3 Splicing factor, arginine/serine-rich 5 Splicing factor, arginine/serine-rich 7 Splicing factor, arginine/serine-rich 9 Surfeit locus protein 6 Transformer-2 protein homologue beta U1 small nuclear ribonucleoprotein 70 kDa U3 small nucleolar ribonucleoprotein protein MPP10 U4/U6.U5 tri-snRNP-associated protein 1 Zinc finger Ran-binding domain-containing protein 2 40S ribosomal protein S8

98 99 100 101 102 103 104 105 106 107 108 109

P46781 P05387 Q07020 P84098 P35268 P62829 P62750 P83731 P47914 P62899 P18124 P39687

110

Q92688

111

P53999

40S ribosomal protein S9 60S acidic ribosomal protein P2 60S ribosomal protein L18 60S ribosomal protein L19 60S ribosomal protein L22 60S ribosomal protein L23 60S ribosomal protein L23a 60S ribosomal protein L24 60S ribosomal protein L29 60S ribosomal protein L31 60S ribosomal protein L7 Acidic leucine-rich nuclear phosphoprotein 32 family member A Acidic leucine-rich nuclear phosphoprotein 32 family member B Activated RNA polymerase II transcriptional coactivator p15

2

primary cellular function

2 2 2 4

2 2 4

2 2 4

2 2 4

RNA Processing/mRNA Splicing

protein MW (Da) 21365.8 14234.2 13906.6 15508.9 11367.7 14606.9 76149.5 8565.3 78364.4

2 3 3 3 2 4 2 3 3 2 3 2 9 2 2 2 3 4 3 2 4 3 5 5 4

2 3 3 3 2 4 2 3 5 3 3 2 20 2 2 6 5 5 3 2 4 3 9 6 4

50978.5 37430.3 27561.3 35923.8 66052.0 59580.2 29426.9 32749.5 117366.1 34852.9 42816.1 100189.1 19889.4 32463.9 31484.5 100229.4 27745.1 31301.7 92578.3 25477.1 32288.5 19330.0 31264.8 27367.5 25542.9 41451.7 33666.7 51558.4 78866.8

3.8% 10.3%

2 3

2 3

90257.1 37405.4

11.5%

2

2

9.8% 67.0% 12.2% 17.9% 18.8% 17.9% 32.1% 13.4% 14.5% 18.4% 14.1% 19.7%

2 4 2 3 2 2 5 2 2 2 4 6

2 5 2 3 2 2 9 3 3 2 5 8

22592.5 11665.5 21635.2 23467.4 14787.3 14865.9 17696.2 17779.5 17753.0 14463.2 29227.7 28586.1

13.5%

2

5

28788.7

29.9%

3

3

14395.9

2159

4

7

Transcriptional Regulation/ Translation

24206.4



Article

Table 1. continued Swiss-Prot ID 112 113 114 115 116

Q9NYF8 P83916 Q13185 P45973 Q5QJE6

117 118 119 120 121 122 123 124 125 126 127 128

P16989 Q8WXX5 Q14919 P29692 P35269 Q7Z4V5 Q99729 Q14103 P17096 P52926 P50502 P17096−2

129 130 131

P43243 Q96DR8 Q13765

132 133 134 135 136 137

P23497 Q9H930 P17480 Q99733 O75475 Q8NC51

138 139 140 141 142 143 144 145 146 147 148 149 150 151 152

Q6NZI2 P51531 Q8IZL8 Q9NY61 P35659 Q96ST2 P61244 P05109 P06702 Q58FF3 Q8WVC0 Q9Y2W1 Q14011 P35637 Q15059

identified protein

% coverage


total spectra

4.2% 14.6% 13.1% 10.0% 6.2%

3 2 2 2 4

3 2 2 2 5

106125.3 21418.1 20812.0 22225.6 84471.1

7.5% 8.9% 10.2% 8.9% 6.4% 6.7% 8.7% 7.3% 37.4% 56.9% 6.5% 14.6%

2 2 2 2 2 3 3 2 5 4 2 2

2 2 2 2 2 3 3 2 14 7 2 4

40089.4 29910.3 22350.4 31121.9 58241.9 74318.3 36225.2 38434.5 11676.2 11831.9 41332.4 10679.2

3.8% 17.8% 13.5%

2 2 2

2 2 3

94626.7 9039.3 23383.3

2.2% 4.6% 3.1% 9.3% 10.0% 7.6%

2 2 3 4 3

2 2 2 3 5 3

100418.8 50075.6 89409.6 42823.9 60103.9 44965.8

15.4% 1.4% 4.1% 6.6% 6.7% 3.1% 14.4% 20.4% 24.6% 4.0% 3.0% 5.6% 14.5% 7.2% 2.8%

4 2 2 3 2 2 2 2 2 2 2 5 2 2 2

4 2 2 3 2 2 2 2 2 2 2 5 2 2 2

43476.5 181283.0 119700.6 63135.0 42675.9 91956.1 18275.3 10835.0 13242.3 45859.7 75405.3 108668.9 18648.3 53426.0 79542.5

Bcl-2-associated transcription factor 1 Chromobox protein homologue 1 Chromobox protein homologue 3 Chromobox protein homologue 5 Deoxynucleotidyltransferase terminal-interacting protein 2 DNA-binding protein A DnaJ homologue subfamily C member 9 Dr1-associated corepressor Elongation factor 1-delta General transcription factor IIF subunit 1 Hepatoma-derived growth factor-related protein 2 Heterogeneous nuclear ribonucleoprotein A/B Heterogeneous nuclear ribonucleoprotein D0 High mobility group protein HMG-I/HMG-Y High mobility group protein HMGI-C Hsc70-interacting protein Isoform HMGA1b of High mobility group protein HMG-I/HMG-Y Matrin-3 Mucin-like protein 1 Nascent polypeptide-associated complex subunit alpha Nuclear autoantigen Sp-100 Nuclear body protein SP140-like protein Nucleolar transcription factor 1 Nucleosome assembly protein 1-like 4 PC4 and SFRS1-interacting protein Plasminogen activator inhibitor 1 RNA-binding protein Polymerase I and transcript release factor Probable global transcription activator SNF2L2 Proline-, glutamic acid- and leucine-rich protein 1 Protein AATF Protein DEK Protein IWS1 homologue Protein max Protein S100-A8 Protein S100-A9 Putative endoplasmin-like protein RNA polymerase-associated protein LEO1 Thyroid hormone receptor-associated protein 3 Cold-inducible RNA-binding protein RNA-binding protein FUS Bromodomain-containing protein 3

primary cellular function

protein MW (Da)

links.34 In our recent study, AGT proteins containing DNAreactive 2-hydroxy-3,4-epoxybutyl groups were introduced into mammalian cell lines, generating covalent AGT-DNA crosslinks and inducing statistically significant levels of cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) (Tretyakova et al., in press). 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 ap-

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.33,34 For example, the cytotoxicity and mutagenicity of several bifunctional alkylation agents including 1,2-dibromoethane, dibromomethane, and DEB, are enhanced in bacteria which overexpress human O6-alkylguanine DNA alkyltransferase (AGT) protein, presumably due to the formation of toxic AGT-DNA cross2160



Article

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).

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 beta2A 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 are associated with a variety of diseases including cancer, renal, urological, cardiovascular, and dermatological diseases and conditions as revealed by Ingenuity Pathway Analysis (IPA) (Figure 4D). Previously, it has been reported that DEB forms DPCs with human 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) were also observed in the present study (Table 1), while the remaining proteins were not detected from in vitro cross-linking experiments.2 This may be partially explained by differences in protein expression profiles

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.

proach 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 2161



Article

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).

between human ovarian carcinoma (HeLa)20 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, ∼10fold lower concentrations of DEB were required to achieve the same extent of cross-linking 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 mechlorethaminecross-linked proteins,8 18 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 domaincontaining protein 18 (Figure 7A). Similarly, 67 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,8 cisplatin-mediated DPCs involve lysine and arginine residues (Ming et al., manuscript in preparation). Because of 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). 2162



Article

Funding for this research was provided by the National Cancer Institute (CA-100670).

■

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

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 (25 μM) (A) and proteins that form DPCs in the presence of cisplatin (100 μM) (B).

■

(1) Barker, S.; Weinfeld, M.; Murray, D. DNA-protein crosslinks: their induction, repair, and biological consequences. Mutat. Res. 2005, 589, 111−135. (2) Michaelson-Richie, E. D.; Loeber, R. L.; Codreanu, S. G.; Ming, X.; Liebler, D. C.; Campbell, C.; Tretyakova, N. Y. DNA-protein crosslinking by 1,2,3,4-diepoxybutane. J. Proteome Res. 2010, 9, 4356−4367. (3) Loeber, R.; Rajesh, M.; Fang, Q.; Pegg, A. E.; Tretyakova, N. Cross-linking of the human DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of 1,2,3,4-diepoxybutane. Chem. Res. Toxicol. 2006, 19, 645−654. (4) Murata-Kamiya, N.; Kamiya, H. Methylglyoxal, an endogenous aldehyde, crosslinks DNA polymerase and the substrate DNA. Nucleic Acids Res. 2001, 29, 3433−3438. (5) Merk, O.; Speit, G. Significance of formaldehyde-induced DNAprotein crosslinks for mutagenesis. Environ. Mol. Mutagen. 1998, 32, 260−268. (6) Barker, S.; Weinfeld, M.; Zheng, J.; Li, L.; Murray, D. Identification of mammalian proteins cross-linked to DNA by ionizing radiation. J. Biol. Chem. 2005, 280, 33826−33838. (7) Zhitkovich, A.; Voitkun, V.; Kluz, T.; Costa, M. Utilization of DNA-protein cross-links as a biomarker of chromium exposure. Environ. Health Perspect. 1998, 106 (Suppl. 4), 969−974. (8) Michaelson-Richie, E. D.; Ming, X.; Codreanu, S. G.; Loeber, R. L.; Liebler, D. C.; Campbell, C.; Tretyakova, N. Y. Mechlorethamineinduced DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. J. Proteome. Res. 2011, 10, 2785−2796. (9) Baker, J. M.; Parish, J. H.; Curtis, J. P. DNA-DNA and DNAprotein crosslinking and repair in Neurospora crassa following exposure to nitrogen mustard. Mutat. Res. 1984, 132, 171−179. (10) Ewig, R. A.; Kohn, K. W. DNA damage and repair in mouse leukemia L1210 cells treated with nitrogen mustard, 1,3-bis(2chloroethyl)-1-nitrosourea, and other nitrosoureas. Cancer Res. 1977, 37, 2114−2122. (11) Kloster, M.; Kostrhunova, H.; Zaludova, R.; Malina, J.; Kasparkova, J.; Brabec, V.; Farrell, N. Trifunctional dinuclear platinum complexes as DNA-protein cross-linking agent. Biochemistry 2004, 43, 7776−7786. (12) Ewig, R. A.; Kohn, K. W. DNA-protein cross-linking and DNA interstrand cross-linking by haloethylnitrosoureas in L1210 cells. Cancer Res. 1978, 38, 3197−3203. (13) Morrow, N. L. The industrial production and use of 1,3butadiene. Environ. Health Perspect. 1990, 86, 7−8. (14) Hecht, S. S. Tobacco smoke carcinogens and lung cancer. J. Natl. Cancer Inst. 1999, 91, 1194−1210. (15) Jelitto, B.; Vangala, R. R.; Laib, R. J. Species differences in DNA damage by butadiene: role of diepoxybutane. Arch. Toxicol. Suppl. 1989, 13, 246−249. (16) Loecken, E. M.; Guengerich, F. P. Reactions of glyceraldehyde 3-phosphate dehydrogenase sulfhydryl groups with bis-electrophiles

For example, Nakano et al. studied DPC repair in bacteria and in mammalian cells and found that NER is involved in the repair of DPCs involving proteins less than 12−14 kDa in size in bacteria and less than 8−10 kDa in mammalian cells, whereas HR is responsible for the repair of oversized DPCs.33,36 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.

■

ASSOCIATED CONTENT

* Supporting Information S

DEB-induced cell death in HT1080 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Address: University of Minnesota Masonic Cancer Center, Mayo Mail Code 806, 420 Delaware St SE, Minneapolis, MN 55455. Phone: 612-626-3432. Fax: 612-626-5135. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Pratik Jagtap (Minnesota Supercomputing Institute, University of Minnesota) for his help with proteomic data analyses and Bob Carlson (University of Minnesota Masonic Cancer Center) for preparing figures for this manuscript. 2163



Article

conjugates by human O6-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 2004, 17, 972−982. (35) Fountoulakis, M.; Tsangaris, G.; Oh, J.; Maris, A.; Lubec, G. Protein profile of the HeLa cell line. J. Chromatogr.. A 2004, 1038, 247−265. (36) Nakano, T.; Morishita, S.; Katafuchi, A.; Matsubara, M.; Horikawa, Y.; Terato, H.; Salem, A. M.; Izumi, S.; Pack, S. P.; Makino, K.; Ide, H. Nucleotide excision repair and homologous recombination systems commit differentially to the repair of DNA-protein crosslinks. Mol. Cell 2007, 28, 147−158.

produce DNA-protein cross-links but not mutations. Chem. Res. Toxicol. 2008, 21, 453−458. (17) Loecken, E. M.; Dasari, S.; Hill, S.; Tabb, D. L.; Guengerich, F. P. The bis-electrophile diepoxybutane cross-links DNA to human histones but does not result in enhanced mutagenesis in recombinant systems. Chem. Res. Toxicol. 2009, 22, 1069−1076. (18) Thier, R.; Pemble, S. E.; Kramer, H.; Taylor, J. B.; Guengerich, F. P.; Ketterer, B. Human glutathione S-transferase T1−1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4diepoxybutane in Salmonella typhimurium. Carcinogenesis 1996, 17, 163−166. (19) Thier, R.; Muller, M.; Taylor, J. B.; Pemble, S. E.; Ketterer, B.; Guengerich, F. P. Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione S-transferase. Chem. Res. Toxicol. 1995, 8, 465−472. (20) Rasheed, S.; Nelson-Rees, W. A.; Toth, E. M.; Arnstein, P.; Gardner, M. B. Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer 1974, 33, 1027−1033. (21) Shin, N. Y.; Liu, Q.; Stamer, S. L.; Liebler, D. C. Protein targets of reactive electrophiles in human liver microsomes. Chem. Res. Toxicol. 2007, 20, 859−867. (22) Yates, J. R., III; Eng, J. K.; McCormack, A. L. Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases. Anal. Chem. 1995, 67, 3202− 3210. (23) Zhang, B.; Chambers, M. C.; Tabb, D. L. Proteomic parsimony through bipartite graph analysis improves accuracy and transparency. J. Proteome Res. 2007, 6, 3549−3557. (24) Loeber, R. L.; Michaelson-Richie, E. D.; Codreanu, S. G.; Liebler, D. C.; Campbell, C. R.; Tretyakova, N. Y. Proteomic analysis of DNA-protein cross-linking by antitumor nitrogen mustards. Chem. Res. Toxicol. 2009, 22, 1151−1162. (25) Summer, H.; Li, O.; Bao, Q.; Zhan, L.; Peter, S.; Sathiyanathan, P.; Henderson, D.; Klonisch, T.; Goodman, S. D.; Droge, P. HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy. Nucleic Acids Res. 2009, 37, 4371−4384. (26) Liu, H.; Lu, Z. G.; Miki, Y.; Yoshida, K. Protein kinase C delta induces transcription of the TP53 tumor suppressor gene by controlling death-promoting factor Btf in the apoptotic response to DNA damage. Mol. Cell. Biol. 2007, 27, 8480−8491. (27) Xie, J.; Guo, Q. Apoptosis antagonizing transcription factor protects renal tubule cells against oxidative damage and apoptosis induced by ischemia-reperfusion. J. Am. Soc. Nephrol. 2006, 17, 3336− 3346. (28) Maggi, L. B., Jr.; Kuchenruether, M.; Dadey, D. Y.; Schwope, R. M.; Grisendi, S.; Townsend, R. R.; Pandolfi, P. P.; Weber, J. D. Nucleophosmin serves as a rate-limiting nuclear export chaperone for the Mammalian ribosome. Mol. Cell. Biol. 2008, 28, 7050−7065. (29) Okuwaki, M.; Matsumoto, K.; Tsujimoto, M.; Nagata, K. Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone. FEBS Lett. 2001, 506, 272−276. (30) Grisendi, S.; Bernardi, R.; Rossi, M.; Cheng, K.; Khandker, L.; Manova, K.; Pandolfi, P. P. Role of nucleophosmin in embryonic development and tumorigenesis. Nature 2005, 437, 147−153. (31) Bai, R. Y.; Ouyang, T.; Miething, C.; Morris, S. W.; Peschel, C.; Duyster, J. Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway. Blood 2000, 96, 4319− 4327. (32) Loeber, R.; Michaelson, E.; Fang, Q.; Campbell, C.; Pegg, A. E.; Tretyakova, N. Cross-linking of the DNA repair protein O6alkylguanine DNA alkyltransferase to DNA in the presence of antitumor nitrogen mustards. Chem. Res. Toxicol. 2008, 21, 787−795. (33) Ide, H.; Shoulkamy, M. I.; Nakano, T.; Miyamoto-Matsubara, M.; Salem, A. M. Repair and biochemical effects of DNA-protein crosslinks. Mutat. Res. 2011, 711, 113−122. (34) Valadez, J. G.; Liu, L.; Loktionova, N. A.; Pegg, A. E.; Guengerich, F. P. Activation of bis-electrophiles to mutagenic 2164


1,2,3,4-Diepoxybutane-Induced DNAâProtein ... - ACS Publications

Recommend Documents

1,2,3,4-Diepoxybutane-Induced DNAâProtein ... - ACS Publications