Mass Spectrometry Based Proteomics Study of Cisplatin-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells

Abstract

Platinum-based antitumor drugs such as 1,1,2,2-cis-diamminedichloroplatinum(II) (cisplatin), carboplatin, and oxaliplatin are currently used to treat nearly 50% of all cancer cases, and novel platinum based agents are under development. The antitumor effects of cisplatin and other platinum compounds are attributed to their ability to induce interstrand DNA-DNA cross-links, which are thought to inhibit tumor cell growth by blocking DNA replication and/or preventing transcription. However, platinum agents also induce significant numbers of unusually bulky and helix-distorting DNA-protein cross-links (DPCs), which are poorly characterized because of their unusual complexity. We and others have previously shown that model DPCs block DNA replication and transcription and cause toxicity in human cells, potentially contributing to the biological effects of platinum agents. In the present work, we have undertaken a system-wide investigation of cisplatin-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells using mass spectrometry-based proteomics. DPCs were isolated from cisplatin-treated cells using a modified phenol/chloroform DNA extraction in the presence of protease inhibitors. Proteins were released from DNA strands and identified by mass spectrometry-based proteomics and immunological detection. Over 250 nuclear proteins captured on chromosomal DNA following treatment with cisplatin were identified, including high mobility group (HMG) proteins, histone proteins, and elongation factors. To reveal the exact molecular structures of cisplatin-mediated DPCs, isotope dilution HPLC-ESI⁺-MS/MS was employed to detect 1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2′-deoxyguanosine-7-yl)-platinum (II) (dG-Pt-Lys) conjugates between the N7 guanine of DNA and the ε-amino group of lysine. Our results demonstrate that therapeutic levels of cisplatin induce a wide range of DPC lesions, which likely contribute to both target and off target effects of this clinically important drug.

Graphical Abstract

Introduction

DNA-protein cross-links (DPCs) are bulky, helix-distorting lesions that can be induced following exposure to many cytotoxic, mutagenic, and carcinogenic agents including ionizing radiation,¹ transition metals,² and common chemotherapeutic agents such as nitrogen mustards,^3–7 platinum agents,⁸ and alkylnitrosoureas.⁹ These macromolecular lesions block DNA-protein interactions, interfering with basic cellular functions such as DNA replication, transcription, repair, recombination, and chromatin remodeling,^10–13 If left unrepaired, DPCs may result in toxicity and permanent DNA alterations.^10,14

Platinum-based antitumor agents, e.g. 1,1,2,2-cis-diamminedichloroplatinum(II) (cisplatin) and its analog cis-diammine-1,1-cyclobutanedicarboxylate platinum(II) (carboplatin), are highly effective in the treatment of testicular and ovarian malignancies, as well as for chemotherapy of bladder, cervical, head and neck, esophageal, and lung cancer.^15,16 Upon entering cells, cisplatin is spontaneously hydrolyzed,¹⁷ yielding a highly reactive aquated species capable of platinating DNA to form a variety of nucleobase adducts.^18–20 The monofunctional DNA adducts formed initially can further react with neighboring bases to produce intrastrand and interstrand DNA-DNA cross-links.²⁰ Alternatively, the monofunctional adducts can be trapped by nuclear proteins found in a close proximity to chromosomal DNA to form covalent DPCs conjugates (Scheme 1).^20,21 While cisplatin-induced DNA-DNA cross-links, including 1,2-d(GpG) intrastrand cross-links, 1,2-d(ApG) intrastrand cross-links, and 1,3-d(GpNpG) intrastrand cross-links are well characterized and are thought to play a prominent role in their antitumor effects,^20,22–24 relatively little is known about the identities of the corresponding DPC lesions.

Scheme 1.

Formation of DNA-DNA cross-links and DNA-protein cross-links (DPC) by cisplatin.

Several earlier studies have employed biophysical methodologies and western blotting against specific target proteins to show the ability of cisplatin to form DPC lesions.^21,25–29 Because of their inherent limitations, such studies have not been able to reveal the full range of cellular proteins participating in DPC formation by cisplatin or to identify their molecular structures. The main goal of the present work was to conduct a system-wide investigation of cisplatin-mediated DPC formation in cultured human cells. We employed an unbiased mass spectrometry based proteomics approach to identify any human proteins that become trapped on DNA in live cells following treatment with cytotoxic concentrations of cisplatin. In addition, liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI⁺-MS/MS) analysis of total proteolytic digests was employed to determine the chemical structures of the cisplatin-induced conjugates.

Materials and Methods

Safety statement

Phenol and chloroform are toxic chemicals that should be handled with caution in a well-ventilated fume hood with appropriate personal protective equipment. 1,1,2,2-Cis-diamminedichloroplatinum(II) (cisplatin) is toxic and carcinogenic and needs to be treated with extreme caution.

Chemicals and reagents

1,1,2,2-Cis-diamminedichloroplatinum(II) (cisplatin), leupeptin, pepstatin, aprotinin, phenylmethanesulfonyl fluoride (PMSF), dithiothreitol (DTT), iodoacetamide, chloroform, ribonuclease A, nuclease P1, phosphodiesterase I (PDE I), phosphodiesterase II (PDE II), and alkaline phosphatase were purchased from Sigma (St. Louis, MO). Mass spectrometry-grade Trypsin Gold 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), flap endonuclease 1 (Fen-1), nucleoin, actin, poly-(ADP-ribose) polymerase 1 (PARP-1), elongation factor 1α1 (EF-1α1), and DNA-(apurinic- or apyrimidinic-site) lyase (Ref-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies specific for x-ray cross-complementing protein 1 (XRCC-1) and AGT were purchased from Lab Vision/NeoMarkers (Fremont, CA). Alkaline phosphatase-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma (St. Louis, MO). Cis-1,1-diammine-2-(5-amino-5-carboxypentyl)-amino-2-(2′-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) was prepared in our laboratory and purified by HPLC.

Cell culture

Human fibrosarcoma (HT1080) cells³⁰ were obtained from the American Type Cell 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) maintained in a humidified incubator at 37°C with 5% CO₂.

Cisplatin cytotoxicity assay

HT1080 cells were plated in Dulbeccos modified Eagle’s medium containing 9% FBS at a density of 5 × 10⁵ cells/6 cm dish and permitted to adhere overnight. On the following morning, dishes (in triplicate) were treated with cisplatin (0, 5, 10, 50, 100, 250, or 500 μM) for 3 h at 37°C in serum-free growth media. Following treatment, cells were either immediately collected or placed in drug-free serum-containing media and allowed to recover for 18 h. The effect of cisplatin on cell survival was determined by direct cell counting. In brief, after incubation the cells were recovered via treatment with trypsin and resuspended in normal growth media containing trypan blue at a final volume of 1 mL. Cells were counted using a hemocytometer, and cytotoxicity was expressed as the number of cells surviving cisplatin treatment relative to non-drug-treated controls.

Isolation of proteins cross-linked to chromosomal DNA by cisplatin

To analyze DPC formation in mammalian cells exposed to cisplatin, HT1080 cells (10⁷ cells, in triplicate) were treated with increasing concentrations of cisplatin (0, 10, 50, 100, 250, or 500 μM) for 3 h at 37°C. Following exposure, the cells were washed three times with ice cold phosphate-buffered saline (PBS) and DPCs were isolated by a modified phenol/chloroform extraction method as described previously. In brief, cells were recovered from dishes by scraping^6,31,32 and suspended in PBS at a final density of ~2 × 10⁶ cells/mL. To isolate nuclei, cells were lysed by adding an equal volume of 2X 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 2,000 g for 10 min at 4 °C. The nuclear pellet was 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) at a concentration of ~ 5 × 10⁶ nuclei/mL and incubated for 2 h at 37°C with gentle shaking. To isolate chromosomal DNA containing covalent DPCs, nuclear lysates were extracted with two volumes of Tris-buffer saturated phenol, and the resulting white emulsion was centrifuged at 1,000 g for 15 min at room temperature. The aqueous layer and the interface material were subjected to a second extraction with two volumes of Tris buffer saturated phenol:chloroform (1:1). DNA was precipitated from the aqueous/interface layers with cold ethanol. Samples were centrifuged at 4,000 g for 20 min at 4°C, and the resulting DNA pellet was washed with cold 70% ethanol, air dried, and reconstituted in 1 mL Millipore water. DNA concentrations were estimated by UV spectrophotometry. DNA amounts and its purity were determined by quantitation of dG in enzymatic hydrolysates as described previously.⁶ Typical DNA yields were 50 – 75 μg DNA from 10⁷ HT1080 cells.

Mass spectrometric identification of cross-linked proteins

To identify cellular proteins that become covalently attached to chromosomal DNA in cisplatin-treated cells, HT1080 cells (~10⁷ cells, in triplicate) were incubated in serum-free media for 3 h at 37 ºC in the presence or absence of 100 μM cisplatin. Chromosomal DNA containing covalently cross-linked proteins was isolated by a modified phenol/chloroform extraction and quantified as described above. DNA (30 μg) was dissolved in 50 μL of 1X NuPAGE Sample Buffer (Invitrogen, Carlsbad, CA) and heated at 70 °C for 1 h to release the cross-linked proteins. Our earlier studies have revealed that heating releases intact proteins from cisplatin-induced DPCs via platination migration to a neighboring DNA base (Ming and Tretyakova, unpublished observations). The resulting proteins were separated using 12% Tris-HCl Ready Gels (Bio-Rad, Hercules, CA) and stained with SimplyBlue SafeStain (Invitrogen, Carlsbad, CA). Gel regions containing protein bands were divided into five sections encompassing the entire molecular weight range, and the gel sections were further diced into ~1 mm pieces. The proteins present within the gel pieces were subjected to in gel tryptic digestion as described elsewhere.³³ 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) (Promega, Madison, WI) 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, desalted by ZipTip C18 purification (ZipTip C18 Pipette Tips, Millipore, Temecula, CA), and finally reconstituted in 0.1% formic acid (25 μL) prior to mass spectrometry analysis.

Tryptic peptides were analyzed by HPLC-ESI⁺-MS/MS with a Thermo Scientific LTQ Orbitrap Velos mass spectrometer in line with an Eksigent NanoLC-Ultra 2D HPLC system, a nanospray source, and Xcalibur 2.1.0 software for instrument control. Peptide samples (8 μL) were trapped on a 180 μm × 20 mm Symmetry C18 column (Waters, Milford, MA) upon elution with 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 flow was reversed, decreased to 0.3 μL/min, and the peptides were then eluted off the trap column and onto 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 solvent composition was initially set 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 ambient temperature. Centroided 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 mass calibrated prior to each analysis and the spray voltage was adjusted to assure a stable spray. Typical MS 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.

Mass spectral data were analyzed using an in-house developed software pipeline, TINT, which 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 were excluded. Data were searched using the SEQUEST v.27 algorithm^34,35 on a high speed, multiprocessor Linux cluster in the University of Minnesota Super Computing Institute using the human subset consisting of the 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 5 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 10 ppm within the calculated average mass, and fragment ion mass tolerance was set to 10 mmu of their monoisotopic mass. Identified peptides were filtered using Scaffold 3 software (Proteome Software, INC., Portland, OR)³⁶ to a target false discovery rate (FDR) of 5%. The FDR was calculated as follows: 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. A second round of filtering removed proteins supported by less than four distinct peptide identifications in the analyses. Parsimony rules were applied to generate a minimal list of proteins that explained all of the peptides that passed our entry criteria.³⁷ Furthermore, t-test analyses were performed on the total ion counts of the identified proteins to ensure that the levels of proteins captured from treated samples were significantly higher than those in untreated controls. All statistical analyses were conducted in Scaffold version 3.0. The significance level was set at 5% (p < 0.05).

Western blot analysis of identified proteins

HT1080 cells (~10⁷) were treated with cisplatin (0, 10, 50, 100, 250, or 500 μM) for 3 h at 37°C. Chromosomal DNA, along with any covalently bound proteins, was extracted and quantified as described above. Approximately 30 μg of DNA from each sample was subjected to heating (1 h at 70°C) to release the intact proteins via platination transfer. Proteins were separated by 12% SDS-PAGE 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 a target protein 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 the manufacturer’s instructions. The developed blots were scanned as image files. ImageJ software (www.ncbi.nlm.nih.gov) was used to quantify the optical densities of the protein bands. The efficiency of DNA-protein cross-linking was approximated by comparing signal intensities of the protein which was co-purified with chromosomal DNA (corresponding to cross-linked protein) and the intensity of the corresponding protein band present in the whole cell protein lysate (representing total cellular proteins).⁶

HPLC-ESI⁺-MS/MS analysis of dG-Pt-Lys in cells exposed to cisplatin

HT1080 cells (~10⁷) were incubated in serum-free media in the presence or absence of 100 μM cisplatin for 3 h at 37 °C. Chromosomal DNA containing DPCs was isolated using the modified phenol/chloroform extraction procedure described above. DNA (50 μg) was digested with phosphodiesterase I (240 mU), phosphodiesterase II (240 mU), DNase I (120 mU) and alkaline phosphatase (6 U) overnight at 37°C to produce protein-nucleoside conjugates. Samples were dried under vacuum, reconstituted in 25 mM ammonium bicarbonate, and digested to peptides with trypsin (2–3 μg, 37°C overnight). To achieve complete hydrolysis to amino acids, the resulting tryptic peptides were dried under vacuum, reconstituted in water, and digested with proteinase K (20 μg) for 48 h at room temperature. The digest mixtures were subjected to off-line HPLC separation using an Agilent Technologies HPLC system (1100 model) incorporating a diode array detector and 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 dG-Pt-Lys (5–7 min) were collected, dried under vacuum, and reconstituted in 15 mM ammonium acetate (25 μL) for HPLC-ESI⁺-MS/MS analysis (injection volume, 8 μL).

HPLC-ESI⁺-MS/MS analyses of dG-Pt-Lys conjugates were conducted with a Thermo-Finnigan TSQ Vantage mass spectrometer in line with an Eksigent MicroAS autosampler and nanoLC 2D HPLC system, a heated ESI source, and Xcalibur 2.1.0 software for instrument control. Chromatographic separation was accomplished using a Hypercarb HPLC column (100 mm × 0.5 mm, 5 μm, ThermoScientific, Waltharm, MA) eluted with a gradient of 15 mM ammonium acetate (A) and 1:1 acetonitrile:water with 1% formic acid (B) at a flow rate of 13 μL/min. The gradient program began at 2% B, followed by a linear increase to 10% B in 10 min and further to 80% B in 8 min. The column was washed with 80% B for 5 min, and the solvent composition was brought back to 2% B in 6 min. Using this gradient, dG-Pt-Lys eluted at ~17.3 min. ESI was achieved at a spray voltage of 3.2 kV and a capillary temperature of 200°C. CID was performed with Ar as the collision gas (2.0 mTorr) at collision energy of 25 V. MS parameters were optimized for maximum response during infusion of a standard solution of dG-Pt-Lys and may vary slightly between experiments. HPLC-ESI⁺-MS/MS analyses were performed in the selected reaction monitoring (SRM) mode using the mass transitions corresponding to neutral losses of 2′-deoxyribose, ammonia, and dG from protonated molecules of dG-Pt-Lys in a triple quadrupole mass spectrometer (m/z 641.3 [M]⁺ → 508.2 [M–NH₃–deoxyribose+H]⁺, and 340.1 [M–2NH₃–deoxyguanosine]⁺).

Results

Cytotoxicity Experiments

To establish the effects of cisplatin treatment on cellular viability, HT1080 cells were treated with increasing concentrations of the drug (0 – 250 μM) for 3 h. Cell numbers were determined either immediately after treatment (Figure S1) or following overnight incubation in a drug free media (Figure S2). Cytotoxicity was measured as the percentage of cells surviving cisplatin treatment as compared to untreated controls. Treatment with cisplatin for 3 hours had no immediate effect on cell viability (Figure S1), but resulted in a significant decrease in cell numbers if treated cells were left overnight, with approximately 70% cell death observed following treatment with 100 μM cisplatin (Figure S2).

Concentration-dependent DPC formation in cisplatin-treated cells

To investigate DPC formation with increasing concentrations of cisplatin, HT1080 cells were treated with 0, 1, 5, 10, 25, or 50 μM cisplatin for 3 h. DNA was extracted by the modified phenol/chloroform extraction method developed in our laboratory.⁶ Our previously published studies have shown that this methodology removes non-covalently bound proteins, but preserves covalent DNA-protein conjugates.⁶ DNA aliquots from each sample were taken and heated at 70°C to release the cross-linked proteins from DNA via platination migration (Ming and Tretyakova, unpublished observations). SDS-PAGE analysis of the released proteins revealed numerous protein bands present in cisplatin-treated samples (lanes 4–8, Figure 1), although control samples (Lane 3) contained background DPC levels. Significant increase in DPC abundance was observed in cells treated with 50 μM cisplatin, reaching an estimated 8% cross-linking efficiency (Figure 1B).

Figure 1.

Concentration-dependent formation of DPCs in nuclear protein extracts prepared from HeLa human cervical carcinoma cells following exposure to cisplatin. (A) Nuclear protein extracts from HeLa cells (500 μg) and 5′-biotinylated double-stranded oligodeoxynucleotides (3.12 nmol) were incubated in the presence of 0–50 μM Cisplatin. The resulting DPCs were captured on streptavidin beads, and the proteins were resolved on 12% SDS-PAGE. Gels were stained with SilverQuest SilverStain to visualize the cross-linked proteins. (B) Densitometric analysis of protein bands in the 25 – 250 kDa molecular weight range was used to estimate the extent of total protein cross-linking to DNA in the presence of cisplatin. Known amounts of nuclear protein extract were analyzed as a control to estimate the cross-linking efficiency.

Identification of Cross-linked Proteins by Mass Spectrometry-Based Proteomics (Scheme 2)

Scheme 2.

Strategy for the isolation and analysis of DPCs from cisplatin-treated mammalian cell cultures.

To determine the identities of the proteins participating in cisplatin-mediated DPC formation in vivo, HT1080 cells (~ 10⁷ cells, in triplicate) were treated with cisplatin (100 μM), while control cells were incubated in growth media lacking the drug. This concentration was selected based on our preliminary experiments shown in Figure 1 and are approximately 3-fold higher than typical plasma concentrations observed in treated patients. Our cytotoxicity experiments have shown that 3 h treatment with 100 μM cisplatin to did not affect cell viability (Figure S1), although significant cell death was observed if treated cells were left overnight (Figure S2). Following DNA extraction,⁶ the cross-linked proteins were released by heating and resolved by SDS-PAGE, followed by simply blue staining (Figure 2). Numerous protein bands were present in cisplatin-treated samples (lanes 6–8, Figure 2), while the untreated samples exhibited minimal protein bands (lanes 2–4, Figure 2). It should be noted that simply blue staining is significantly less sensitive than silver staining used in experiment shown in Figure 1. Gel regions within the molecular weight range of 10–260 kDa were excised from the gel and subjected to in-gel tryptic digestion, followed by HPLC-ESI⁺-MS/MS analysis of the peptides.³⁸ Protein identification was based on the MS/MS spectra of at least four tryptic peptides, which revealed characteristic b- and y-series fragment ions used to determine their amino acid sequence (see examples in Figure 3).

Figure 2.

SDS-PAGE analysis of samples employed in the proteomics studies of cisplatin-induced DPCs. HT1080 cells (~10⁷ in triplicate) were incubated for 3 h in absence (lanes 2–4) or presence (lanes 6–8) of 100 μM cisplatin, and proteins covalently attached to chromosomal DNA were isolated as described in the Methods section. Proteins were resolved by 12% SDS-PAGE and visualized by staining with SimplyBlue SafeStatin. Molecular weight markers (lanes 1, 5 and 9) were included to permit subsequent recovery of proteins from distinct molecular weight ranges as described in the text.

Figure 3.

Representative HPLC-ESI⁺-MS/MS spectra of tryptic peptides used in the identification of DPCs involving histone H1D (A), HMG B1 (B), and XRCC-6 protein (C).

Database searching and parsimony analysis of the MS/MS spectral data resulted in identification of 256 proteins that co-purified with chromosomal DNA from cisplatin-treated cells (Table 1). All protein identifications were supported by at least four unique peptides. Statistical analyses were conducted to compare proteomics results for treated and untreated samples, and only proteins which exhibited significantly increased total ion counts in treated samples (p < 0.05) were included in the list. The molecular weights of the identified proteins (Table 1) were consistent with their positions on the gel, although some of the proteins were also present in a higher molecular weight fraction, probably due to the accompanied protein-protein cross-linking or proteins with post-translational modifications that affect the charge state of the proteins.

Table 1.

Proteins participating in cross-linking to chromosomal DNA in human fibrosarcoma HT1080 cells treated with cisplatin (100 μM for 3 h).

Swiss-Port ID	Identified Proteins	% Coverage	No. of Unique Peptides	No. of Assigned Spectra	Primary Cellular Function	Protein MW (Da)
P62258	14-3-3 Protein epsilon	24	5	11	Cell Signaling/Motility/Architecture	29175.0
P62736	Actin, aortic smooth muscle	24	8	37		42010.1
P60709	Actin, cytoplasmic 1 (β-actin)	35	7	97		41737.8
Q01518	Adenylyl cyclase-associated protein 1	12	4	9		51855.5
P12814	α-Actinin-1	13	10	19		103061.1
O43707	α-Actinin-4	10	6	11		104857.2
P04083	Annexin A1	37	9	22		38715.9
P07355	Annexin A2	58	15	36		38606.1
P23528	Cofilin-1	45	5	15		18503.2
Q07065	Cytoskeleton-associated protein 4	9	4	7		66022.2
Q16643	Drebrin	14	6	10		71428.6
P14625	Endoplasmin	38	24	80		92471.7
P15311	Ezrin	9	5	9		69414.7
P21333	Filamin-A	3	4	6		280729.4
Q99988	Growth/differentiation factor 15	23	6	12		34154.8
P07900	Heat shock protein HSP 90-α	23	13	45		84663.2
P08238	Heat shock protein HSP 90-β	21	12	75		83267.3
O95373	Importin-7	7	5	15		119519.5
P05787	Keratin, type II cytoskeletal 8	42	18	74		53706.2
P02545	Prelamin-A/C	53	34	93		74140.7
P20700	Lamin-B1	36	16	29		66409.6
Q03252	Lamin-B2	22	13	46		67689.8
Q15185	Prostaglandin E synthase 3	42	6	15		18697.9
P61026	Ras-related protein Rab-10	28	5	9		22542.1
Q13813	Spectrin α chain, brain	37	75	161		284542.7
Q01082	Spectrin β chain, brain 1	27	46	91		274613.4
Q9UH99	SUN domain-containing protein 2	10	4	9		80312.2
P09493	Tropomyosin α-1 chain	14	4	4		32710.0
Q71U36	Tubulin α-1A chain	41	14	53		50135.7
P07437	Tubulin β chain	16	4	20		49670.6
Q13885	Tubulin β-2A	36	11	91		49907.1
P68371	Tubulin β-2C chain	20	6	18		49830.7
Q9BUF5	Tubulin β-6 chain	18	4	6		49857.2
P08670	Vimentin	65	28	990		53652.7
P31946	14-3-3 protein β/α	19	5	10	Cellular Homeostasis/Cell Cycle	28083.1
P62244	40S ribosomal protein S15a	29	4	9		14840.0
P62847	40S ribosomal protein S24	29	4	8		15423.8
P61247	40S ribosomal protein S3a	41	10	23		29945.3
P62753	40S ribosomal protein S6	23	5	11		28681.7
P08865	40S ribosomal protein SA	36	8	15		32854.1
P10809	60 kDa heat shock protein, mitochondrial	23	8	19		61055.7
P11021	78 kDa glucose-regulated protein OS=Homo	23	11	24		72334.7
P84077	ADP-ribosylation factor 1	34	4	8		20697.6
P18085	ADP-ribosylation factor 4	22	4	9		20511.6
P15144	Aminopeptidase N	25	18	40		109542.4
Q9UKV3	Apoptotic chromatin condensation inducer in the nucleus	3	4	7		151887.9
P25705	ATP synthase subunit α, mitochondrial	14	7	10		59752.1
P06576	ATP synthase subunit β, mitochondrial	10	4	7		56560.6
O00571	ATP-dependent RNA helicase DDX3X	9	4	8		73245.8
P27824	Calnexin	23	14	36		67570.2
P27797	Calreticulin	52	18	54		48142.9
Q9UQ88	Cell division protein kinase 11A	14	10	22		90976.0
Q68CQ4	Digestive organ expansion factor homolog	12	7	9		87057.4
P62495	Eukaryotic peptide chain release factor subunit 1	13	4	7		49032.6
Q99613	Eukaryotic translation initiation factor 3 subunit C	6	5	7		105347.2
O60841	Eukaryotic translation initiation factor 5B	12	11	24		138831.5
P02751	Fibronectin	8	10	21		262616.9
P09382	Galectin-1	39	5	11		14715.8
P14314	Glucosidase 2 subunit β	11	5	6		59425.8
P04406	Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)	35	8	18		36053.4
P62826	GTP-binding nuclear protein Ran	28	5	10		24423.1
P11142	Heat shock cognate 71 kDa protein	13	5	16		70899.8
Q1KMD3	Heterogeneous nuclear ribonucleoprotein U-like protein 2	20	13	24		85105.2
Q9Y4L1	Hypoxia up-regulated protein 1	18	12	28		111336.8
P05556	Integrin β-1	16	10	20		88415.1
P05783	Keratin, type I cytoskeletal 18	53	21	61		48059.0
P35580	Myosin-10	8	11	25		229005.3
P35579	Myosin-9	25	38	96		226537.5
P07196	Neurofilament light polypeptide	53	25	69		61517.8
Q14978	Nucleolar and coiled-body phosphoprotein 1	8	5	10		73604.2
Q13823	Nucleolar GTP-binding protein 2	10	5	9		83656.0
Q9Y2X3	Nucleolar protein 58	21	8	17		59580.2
P19338	Nucleolin (C-23)	36	25	305		76615.9
P06748	Nucleophosmin	47	10	52		32575.5
Q99733	Nucleosome assembly protein 1-like 4	21	6	11		42823.9
P62937	Peptidyl-prolyl cis-trans isomerase A	28	5	9		18012.9
Q13427	Peptidyl-prolyl cis-trans isomerase G	5	4	6		88619.0
Q06830	Peroxiredoxin-1	59	9	18		22110.9
P18669	Phosphoglycerate mutase 1	25	4	6		28804.8
P13796	Plastin-2	15	6	10		70292.1
O00622	Protein CYR61	21	8	15		42026.0
P07237	Protein disulfide-isomerase	38	15	38		57118.1
P13667	Protein disulfide-isomerase A4	13	9	17		72934.0
Q58FF3	Putative endoplasmin-like protein	9	4	10		45859.7
Q58FF8	Putative heat shock protein HSP 90-β-2	18	6	11		44350.2
Q58FF7	Putative heat shock protein HSP 90-β-3	20	14	170		68326.5
P14618	Pyruvate kinase isozymes M1/M2	13	5	12		57937.5
P51149	Ras-related protein Rab-7a	33	6	10		23490.0
Q14692	Ribosome biogenesis protein BMS1 homolog	8	6	8		145812.2
Q14137	Ribosome biogenesis protein BOP1	23	12	20		83629.3
Q9Y265	RuvB-like 1	16	6	10		50229.4
P62136	Serine/threonine-protein phosphatase PP1-α catalytic subunit	25	6	9		37513.9
Q9BXP5	Serrate RNA effector molecule homolog	16	11	16		100669.7
Q9NQZ2	Something about silencing protein 10	19	10	35		54559.2
P78371	T-complex protein 1 subunit β	13	4	7		57489.9
P40227	T-complex protein 1 subunit ζ	17	4	7		58025.3
P37802	Transgelin-2	23	4	7		22391.9
P43307	Translocon-associated protein subunit α	20	4	9		32236.0
Q9BV38	WD repeat-containing protein 18	16	4	4		47405.1
Q86VM9	Zinc finger CCCH domain-containing protein 18	11	6	10		106379.8
P23396	40S ribosomal protein S3	35	7	14	DNA Damage Response/DNA Repair	26688.6
Q9Y5B9	FACT complex subunit SPT16	40	41	113		119917.4
Q08945	FACT complex subunit SSRP1	51	29	185		81077.6
P09429	High mobility group protein B1 (HMG B1)	23	5	22		24894.7
P26583	High mobility group protein B2 (HMG B2)	45	11	32		24034.6
P17096	High mobility group protein HMG-I/HMG-Y	45	5	10		11676.2
P16403	Histone H1D	19	5	14		21365.8
Q96QV6	Histone H2A type 1-A	40	5	18		14234.2
Q96A08	Histone H2B type 1-A	28	4	9		14168.0
P33778	Histone H2B type 1-B	36	8	31		13950.8
P62805	Histone H4	54	6	79		11367.7
P09874	Poly [ADP-ribose] polymerase 1 (PARP-1)	4	4	5		113087.8
Q9NY61	Protein AATF	41	17	37		63135.0
B2RPK0	Putative high mobility group protein B1-like 1	32	10	41		24238.8
P23246	Splicing factor, proline- and glutamine-rich	16	7	11		76149.5
Q9UIG0	Tyrosine-protein kinase BAZ1B	5	6	10		170907.0
P13010	X-ray repair cross-complementing protein 5 (XRCC-5 or Ku80/86)	11	4	10		82707.1
P12956	X-ray repair cross-complementing protein 6 (XRCC-6 or Ku70)	27	12	28		69846.4
Q15029	116 kDa U5 small nuclear ribonucleoprotein component	13	9	12	RNA Processing/mRNA Splicing	109438.1
P62081	40S ribosomal protein S7	32	4	8		22127.5
P62913	60S ribosomal protein L11	21	4	8		20253.2
Q08211	ATP-dependent RNA helicase A	8	7	13		140961.5
Q9NVP1	ATP-dependent RNA helicase DDX18	9	5	11		75409.7
O00148	ATP-dependent RNA helicase DDX39	9	4	6		49129.8
Q9H583	HEAT repeat-containing protein 1	3	5	8		242378.8
Q32P51	Heterogeneous nuclear ribonucleoprotein A1-like 2	27	7	17		34225.4
P51991	Heterogeneous nuclear ribonucleoprotein A3	25	8	21		39595.1
O60812	Heterogeneous nuclear ribonucleoprotein C-like 1	29	13	279		32142.7
P38159	Heterogeneous nuclear ribonucleoprotein G	22	9	19		42333.7
P31943	Heterogeneous nuclear ribonucleoprotein H	21	6	15		49229.8
P31942	Heterogeneous nuclear ribonucleoprotein H3	16	4	10		36927.6
P61978	Heterogeneous nuclear ribonucleoprotein K	41	12	24		50978.5
P14866	Heterogeneous nuclear ribonucleoprotein L	16	6	13		64132.8
P52272	Heterogeneous nuclear ribonucleoprotein M	16	9	18		77517.3
O60506	Heterogeneous nuclear ribonucleoprotein Q	15	9	15		69603.5
Q00839	Heterogeneous nuclear ribonucleoprotein U	37	29	78		90585.2
P22626	Heterogeneous nuclear ribonucleoproteins A2/B1	33	10	28		37430.3
P07910	Heterogeneous nuclear ribonucleoproteins C1/C2	48	13	739		33607.5
Q9UKD2	mRNA turnover protein 4 homolog	51	13	37		27561.3
P78316	Nucleolar protein 14	7	5	7		97671.9
O00567	Nucleolar protein 56	24	10	18		66052.0
Q9NR30	Nucleolar RNA helicase 2	14	9	13		87346.0
P12270	Nucleoprotein TPR	16	30	52		267289.3
O00541	Pescadillo homolog	36	19	55		68004.9
Q15365	Poly(rC)-binding protein 1	22	5	8		37498.2
P26599	Polypyrimidine tract-binding protein 1	30	9	20		57222.5
Q9HCG8	Pre-mRNA-splicing factor CWC22 homolog	4	4	11		105470.2
Q92841	Probable ATP-dependent RNA helicase DDX17	9	5	8		72373.0
P17844	Probable ATP-dependent RNA helicase DDX5	13	7	11		69149.7
P46087	Putative ribosomal RNA methyltransferase NOP2	26	18	35		89303.6
Q8IY81	Putative rRNA methyltransferase 3	34	19	40		96560.5
O76021	Ribosomal L1 domain-containing protein 1	18	7	12		54974.7
Q9NW13	RNA-binding protein 28	18	12	26		85739.1
Q9UKM9	RNA-binding protein Raly	37	11	22		32463.9
Q15287	RNA-binding protein with serine-rich domain 1	26	6	30		34209.6
Q9Y3B9	RRP15-like protein	17	5	9		31484.5
Q8IYB3	Serine/arginine repetitive matrix protein 1	8	4	7		102337.5
Q13435	Splicing factor 3B subunit 2	11	9	17		100229.4
Q07955	Splicing factor, arginine/serine-rich 1	39	9	16		27745.1
O75494	Splicing factor, arginine/serine-rich 10	23	5	10		31301.7
P84103	Splicing factor, arginine/serine-rich 3	29	5	11		19330.0
Q08170	Splicing factor, arginine/serine-rich 4	14	7	15		56680.0
Q13243	Splicing factor, arginine/serine-rich 5	19	5	12		31264.8
Q16629	Splicing factor, arginine/serine-rich 7	15	4	6		27367.5
P62995	Transformer-2 protein homolog beta	17	4	10		33666.7
P09661	U2 small nuclear ribonucleoprotein A′	28	5	7		28417.1
O00566	U3 small nucleolar ribonucleoprotein protein MPP10	33	15	38		78866.8
O75643	U5 small nuclear ribonucleoprotein 200 kDa helicase	4	6	11		244513.8
Q96MU7	YTH domain-containing protein 1	9	7	12		84700.6
P62280	40S ribosomal protein S11	42	8	18	Transcriptional Regulation/Translation	18431.3
P62277	40S ribosomal protein S13	30	4	7		17223.3
P62263	40S ribosomal protein S14	36	4	9		16272.9
P62249	40S ribosomal protein S16	45	7	14		16445.9
P08708	40S ribosomal protein S17	55	5	14		15550.5
P62269	40S ribosomal protein S18	36	6	12		17719.3
P15880	40S ribosomal protein S2	23	6	10		31325.2
P62266	40S ribosomal protein S23	34	7	14		15807.7
P62701	40S ribosomal protein S4	21	5	8		29599.3
P46782	40S ribosomal protein S5	35	7	11		22877.0
P62241	40S ribosomal protein S8	42	7	11		24206.4
P46781	40S ribosomal protein S9	53	13	29		22592.5
Q8NHW5	60S acidic ribosomal protein P0-like	18	5	9		34365.1
Q96L21	60S ribosomal protein L10-like	21	5	14		24519.2
P30050	60S ribosomal protein L12	49	6	10		17819.1
P26373	60S ribosomal protein L13	33	7	11		24262.2
P40429	60S ribosomal protein L13a	33	12	26		23577.9
P50914	60S ribosomal protein L14	37	8	22		23432.3
P61313	60S ribosomal protein L15	39	8	18		24146.5
P18621	60S ribosomal protein L17	31	5	11		21397.4
Q07020	60S ribosomal protein L18	35	6	19		21635.2
Q02543	60S ribosomal protein L18a	52	10	23		20762.6
P84098	60S ribosomal protein L19	32	7	24		23467.4
P46778	60S ribosomal protein L21	34	5	12		18565.0
P62829	60S ribosomal protein L23	55	7	21		14865.9
P62750	60S ribosomal protein L23a	33	6	14		17696.2
P83731	60S ribosomal protein L24	41	7	15		17779.5
P61353	60S ribosomal protein L27	36	4	9		15798.4
P46776	60S ribosomal protein L27a	34	5	10		16561.4
P46779	60S ribosomal protein L28	49	8	16		15747.9
P39023	60S ribosomal protein L3	24	9	18		46109.5
P62888	60S ribosomal protein L30	51	4	9		12784.7
P62910	60S ribosomal protein L32	35	4	14		15860.4
P49207	60S ribosomal protein L34	29	5	9		13293.1
Q9Y3U8	60S ribosomal protein L36	35	5	12		12254.2
P61927	60S ribosomal protein L37	20	4	5		11078.2
P61513	60S ribosomal protein L37a	59	5	12		10275.4
P36578	60S ribosomal protein L4	36	13	28		47699.1
P46777	60S ribosomal protein L5	37	9	20		34363.5
Q02878	60S ribosomal protein L6	34	10	26		32729.3
P18124	60S ribosomal protein L7	40	10	22		29227.7
P62424	60S ribosomal protein L7a	40	9	22		29996.3
P62917	60S ribosomal protein L8	22	6	11		28024.8
P32969	60S ribosomal protein L9	55	6	17		21863.7
P11387	DNA topoisomerase 1	6	4	6		90729.7
Q03701	CCAAT/enhancer-binding protein ζ	11	8	14		120992.2
Q13185	Chromobox protein homolog 3	17	4	7		20812.0
O75367	Core histone macro-H2A.1	28	7	14		39618.9
O95602	DNA-directed RNA polymerase I subunit RPA1	4	4	6		194814.5
P68104	Elongation factor 1-α 1 (EF-1α1)	27	9	25		50141.2
P13639	Elongation factor 2 (EF-2)	17	10	24		95340.1
P60842	Eukaryotic initiation factor 4A-I	21	7	12		46155.3
P05198	Eukaryotic translation initiation factor 2 subunit 1	27	8	15		36112.7
P63241	Eukaryotic translation initiation factor 5A-1	39	4	8		16832.7
Q5SSJ5	Heterochromatin protein 1-binding protein 3	16	7	11		61208.7
Q99729	Heterogeneous nuclear ribonucleoprotein A/B	11	4	7		36225.2
O14979	Heterogeneous nuclear ribonucleoprotein D-like	15	4	8		46438.6
Q14103	Heterogeneous nuclear ribonucleoprotein D0	23	8	15		38434.5
P52926	High mobility group protein HMGA2	58	5	17		11831.9
Q12905	Interleukin enhancer-binding factor 2	62	16	80		43062.7
Q12906	Interleukin enhancer-binding factor 3	15	12	18		95338.9
P43243	Matrin-3	50	30	107		94626.7
Q9BQG0	Myb-binding protein 1A	28	31	80		148858.3
Q13765	Nascent polypeptide-associated complex subunit α	26	4	7		23383.3
P17480	Nucleolar transcription factor 1	20	14	33		89409.6
P55209	Nucleosome assembly protein 1-like 1	36	8	24		45375.0
Q9H307	Pinin	37	22	56		81613.2
P51531	Probable global transcription activator SNF2L2	9	10	22		181283.0
Q8IZL8	Proline-, glutamic acid- and leucine-rich protein 1	15	11	22		119700.6
P35659	Protein DEK	22	8	15		42675.9
Q8N7H5	RNA polymerase II-associated factor 1 homolog	15	6	12		59976.0
Q15424	Scaffold attachment factor B1	21	14	30		102642.8
Q92922	SWI/SNF complex subunit SMARCC1	22	18	42		122867.4
Q969G3	SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1	30	11	29		46649.7
Q9Y2W1	Thyroid hormone receptor-associated protein 3	13	12	33		108668.9
P51532	Transcription activator BRG1	11	14	28		184649.6
Q5BKZ1	Zinc finger protein 326	33	14	34		65653.5
Q9NVI7	ATPase family AAA domain-containing protein 3A	7	4	6	unknown	71370.2
P55081	Microfibrillar-associated protein 1	17	5	10		51958.7
Q9Y3T9	Nucleolar complex protein 2 homolog	30	19	70		84907.1
O94880	PHD finger protein 14	6	4	7		100055.1
Q96GQ7	Probable ATP-dependent RNA helicase DDX27	17	12	20		89838.1
Q9H0S4	Probable ATP-dependent RNA helicase DDX47	11	4	9		50648.4
Q9Y4W2	Protein LAS1 homolog	22	13	28		83065.5
Q9BXY0	Protein MAK16 homolog	50	12	30		35370.0
Q5JTH9	RRP12-like protein	5	4	6		143705.1
Q15061	WD repeat-containing protein 43	37	20	106		74890.8

Of the identified proteins listed in Table 1, 126 (49.0%) are classified as nuclear proteins by the GO database available via the European Bioinformatics Institute (http://www.ebi.ac.uk/QuickGO) (Figure 4A). These include high mobility group (HMG) proteins, histone proteins, and elongation factors. This is not unexpected considering that nuclear proteins are either localized in the vicinity of DNA or are directly associated with DNA, increasing their chance to be cross-linked to DNA in the presence of cisplatin. An additional 46 proteins (17.9%) were classified as cytoplasmic, 46 (17.9%) as ribosomal proteins, and 7 (2.7%) as membrane-bound proteins (Figure 3A). It is important to note that many of the identified proteins participate in multiple biological processes, and are subsequently categorized under multiple cellular locations. For example, the 40S ribosomal protein S6,^39–41 40S ribosomal protein S7,³⁹ 40S ribosomal S9,^39,42 60S ribosomal protein L10-like,³⁹ 60S ribosomal protein L13a,^39,43 and 60S ribosomal protein L23a^43,44 have been identified in both the cytoplasm and nucleus of different human cells. DPC-forming proteins were further classified according to their GO annotations relating to their molecular function (Figure 4B) and their participation in biological processes (Figure 4C). We found that the majority of the identified proteins belong to the following three categories: DNA binding (34, or 13.2%), RNA binding (41, or 16.0%), and protein binding (47, or 18.3%) (Table 1 and Figure 4B). Interestingly, 52 of the identified proteins (20.2%) have been reported to play a role in RNA processing or splicing (Figure 4C), including arginine/serine-rich splicing factors, heterogeneous nuclear ribonucleoproteins, and ATP-dependent RNA helicases. An additional 10.5% of proteins (N=27) are involved in transcriptional regulation, including transcription activator BRG 1, matrin-3, and interleukin enhancer-binding factors (Figure 4C and Table 1). This result is not due to RNA contamination as DNA isolated by our phenol/chloroform methodology has minimal RNA contamination as revealed by HPLC-UV analyses of enzymatic digests (see Figure S2).⁶ More likely, in addition to their binding to RNA, these proteins may possess additional DNA-binding capabilities which may be triggered by cisplatin treatment.

Figure 4.

GO annotations for proteins involved in cisplatin-induced DPC formation in human HT1080 cells: cellular distributions (A), molecular functions (B), and biological processes (C). The numbers of proteins in each category is indicated in parentheses.

Western blot analysis of cross-linked proteins

To confirm the results of mass spectrometry analyses and to discover additional proteins participating in DPC formation, proteins co-purified with chromosomal DNA following cisplatin treatment were subjected to western blot analysis using commercial antibodies against EF-1α1, PARP, Ref-1, nucleolin, actin, GAPDH, Fen-1, AGT, and XRCC-1 (Figure 5). These proteins were selected because they were either among the gene products identified by mass spectrometry analyses (EF-1α1, PARP, GAPDH, nucleolin, and actin, Table 1) or have been previously found to form cisplatin-induced DPCs in our earlier studies employing cell free protein extracts (Ref-1, AGT, Fen-1, and XRCC-1).^5,45 Equal amounts (30 μg) of DNA isolated from cisplatin-treated HT1080 cells (10, 50, 100, 250, or 500 μM) were taken and heated with SDS-containing gel loading buffer (1 h at 70°C) to release the proteins (Scheme 2). The resulting proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes for western blot analysis using the specific antibodies mentioned above.

Figure 5.

Western blot analysis of cisplatin-induced DPCs in HT1080 cells. Following treatment with 0 (lane 1), 10 (lane 2), 50 (lane 3), 100 (lane 4), 250 (lane 5), or 500 μM cisplatin (lane 6), DNA and cross-linked proteins were isolated by phenol/chloroform extraction. Samples were normalized for DNA content, proteins from 30 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 EF-1α1, AGT, Fen-1, nucleolin, actin, GAPDH, PARP, Ref-1, andXRCC-1 (A). The efficiency of DPC formation in the presence of cisplatin was estimated by densitometric analysis of protein bands in DPC samples and a whole cell protein lysate control (B).

Western blotting experiments confirmed the identities of five gene products identified from mass spectrometry based proteomics: EF-1α1, PARP, nucleolin, GAPDH, and actin (Figure 5A). In addition, a concentration-dependent DPC formation involving four additional proteins: Ref-1, AGT, Fen-1 and XRCC-1, was observed. Among these, nucleolin displayed the greatest cross-linking efficiency, with approximately 10 % of total protein becoming cross-linked to DNA following treatment with 100 μM cisplatin (Figure 5B).

HPLC-ESI⁺-MS/MS Analysis of dG-Pt-Lys Conjugates as Evidence for DPC Formation

To confirm the formation of covalent DNA-protein conjugates in cisplatin-treated cells, HT1080 cells (~10⁶) were treated with 0 or 100 μM cisplatin, and DPC-containing chromosomal DNA was extracted as described above. Equal DNA amounts were taken from each sample and subjected to enzymatic hydrolysis to yield protein-nucleoside conjugates from the DNA backbone, followed by enzymatic digestion to amino acids in the presence of trypsin and proteinase K. Following offline HPLC enrichment, HPLC-ESI⁺-MS/MS was used to detect covalent cisplatin-induced lysine-guanine conjugates (Figure 6).

Figure 6.

HPLC-ESI⁺-MS/MS analysis of dG-Pt-Lys conjugates in total proteolytic digests of chromosomal DNA recovered from cisplatin-treated cells. HT1080 cells were treated with 100 μM cisplatin for 3 h to induce DNA-protein cross-links. Following extraction of the chromosomal DNA containing covalent DPCs, the cross-linked proteins were subjected to enzymatic hydrolysis to release amino acid-nucleobase conjugates. Synthetic dG-Pt-Lys (A); enzymatic digests of DPC mixtures from HT1080 cells incubated in the absence of cisplatin (B); enzymatic digests of DPC mixtures treated with 100 μM cisplatin (C).

Representative extracted ion chromatograms for HPLC-ESI⁺-MS/MS analysis of dG-Pt-Lys in samples from cisplatin-treated and control HT1080 cells are shown in Figure 5. dG-Pt-Lys was detected in DNA samples from cisplatin-treated cells (Figure 6C), but not in untreated cells (Figure 6B). This conjugate had the same HPLC retention time and the same MS/MS fragmentation pattern as synthetic dG-Pt-Lys standard (Figure 6A). These data indicate that cisplatin-induced DNA-protein cross-linking can take place between the N7 position of guanine in DNA and the side-chain ε-amino group of lysine in proteins, although we cannot exclude the possibility of additional cross-linking via other nucleophilic amino acid side chains such as cysteine, histidine, glutamic acid, and arginine. Our efforts to prepare the corresponding dG-Pt-Cys conjugates were unsuccessful due to their limited stability (results not shown).

Discussion

Previous studies using biophysical methods^25–29 and immunological detection with specific antibodies²⁸ have shown that cisplatin and other platinum compounds are capable of inducing DNA-protein cross-links. These lesions are formed by consecutive platination of proteins and DNA by platinum drugs (Scheme 1). However, to our knowledge, there has been no previous system-wide investigation of the proteins participating in DPC formation in the presence of cisplatin. In the present work, we employed an unbiased mass spectrometry-based approach to identify human proteins that form DPCs in cisplatin treated cells (Scheme 2). We took advantage of a modified phenol/chloroform methodology developed in our laboratory to isolate DPCs from cisplatin-treated cells.⁶ Following extraction, the cross-linked proteins were released from DNA strands by heating. We have previously shown that under these conditions, proteins cross-linked to DNA via platination are displaced by proximal nucleobases on genomic DNA. This forms DNA-DNA cross-links and releases intact proteins, which can be readily identified by mass spectrometry-based proteomics and immunoblotting (Scheme 2).

We found that human fibrosarcoma HT1080 cells treated with pharmacologically relevant concentrations of cisplatin (100 μM) contained DNA-protein cross-links to 256 cellular proteins. Recent studies have measured free cisplatin levels at a concentration of 9.03 μg/mL (~30 μM) after a three hour treatment period of 100 μg/m^2.46 The proteins identified by the mass spectrometry-based proteomics study as being trapped on DNA in the presence of cisplatin (Table 1) participate in a variety of cellular functions including DNA damage response and repair (e.g. HMG proteins, histone proteins, PARP-1, and XRCC proteins), transcriptional regulation (e.g. CCAAT/enhancer-binding protein ζ, Chromobox protein homolog 3, and matrin-3), RNA processing (e.g. ATP-dependent RNA helicase, heterogeneous nuclear ribonucleoproteins, poly(rC)-binding protein 1, and putative rRNA methyltransferase 3), cell signaling and architecture (e.g. actin, keratin, lamin, and vimentin), and regulation of cell cycle (e.g. GAPDH, nucleolin, nucleophosmin, and T-complex proteins). The majority of the identified proteins are known DNA-binding proteins (e.g. HMG proteins, histone proteins, PARP-1, and XRCC), RNA-binding proteins (e.g. heterogeneous nuclear ribonucleoproteins, 40S ribosomal proteins, 60S ribosomal proteins, and arginine/serine-rich splicing factors), and protein-binding proteins (e.g. keratin, lamin, vimentin, and galectin-1) which are present in the nucleus (Figure 4C).

Previous targeted studies employed antibodies against specific proteins have shown that several DNA-binding proteins including HMG 1, 2, and E, cytokeratins, and histones can become cross-linked to DNA in the presence of cisplatin.²⁸ These proteins were also detected in our unbiased proteomics screen of cisplatin-induced DPCs (Table 1). In addition, our system-wide investigation established the identities of many additional nuclear proteins that participate in DNA-protein cross-linking formation in the presence of cisplatin (Table 1) and determined atomic connectivity of the resulting macromolecular conjugates to be between the N7 position of guanine and the ε-amino group of lysine (Figure 6).

Among the proteins identified in the present work (Table 1), 21 proteins (55.2% of all meclorethamine-induced DPCs) were present in both mechlorethamine and cisplatin-treated HT1080 cells.⁶ These proteins included the transcription regulators matrin-3, nucleolar transcription factor 1, and nucleophosmin.⁶ Similarly, 106 proteins (79.1% of all phosphoramide mustard-induced DPCs) were present in both phosphoramide mustard and cisplatin treated HT1080 cells.⁴⁷ The differences in protein targets of cisplatin and nitrogen mustards may result from differences in the respective cross-linking mechanisms associated with DNA/protein platination and alkylation. Furthermore, cisplatin’s ability to react with the lysine, cysteine, and histidine residues of proteins can explain why cisplatin formed DPCs with a greater efficiency than mechlorethamine and phosphoramide mustard and cross-linked a wider range of protein targets.

While the contributions of DNA-protein cross-linking to the biological activity of cisplatin remains to be established, these bulky lesions are expected to block DNA replication, transcription, and repair. We recently reported that proteins conjugated to the N7 position of guanine completely block DNA replication.¹³ The corresponding DNA-peptide conjugates that would form upon proteolytic degradation of DPCs can be bypassed by human translesion synthesis polymerases η and κ with a relatively low efficiency, but with high fidelity.¹³ The yeast metalloprotease Wss1 has been identified as the protease which cleaves the protein constituent of DPC at blocked replisomes.⁴⁸ Recently, the metalloprotease SPRTN has been identified by several laboratories as the mammalian protease responsible for cleaving DPCs at blocked replication forks.^49–51

Cellular repair pathways responsible for the removal of cisplatin-induced DPCs are the subject of intense investigation. It has been proposed that DPCs formed as a consequence of cellular exposure to bifunctional alkylating agents (i.e. formaldehyde) can be repaired by NER,⁵² homologous recombination (HR),⁵³ and proteolytic degradation.⁵⁴ One possible mechanism includes proteolytic degradation of the protein component of DPCs, followed by NER removal of the resulting DNA-peptide lesions.¹⁰ A number of reports are consistent with this hypothesis.^55–57 For example, Reardon and Sancar have shown that 4mer and 12mer peptide-DNA substrates can be excised by nucleotide excision repair in-vitro.⁵⁵ Nakano et al reported that DPCs containing protein constituents smaller than 8 kDa are directly excised by NER in-vitro.⁵³ Similarly, Baker et al⁵⁶ presented evidence that DNA-peptide cross-links were excised by cell free extracts from mammalian cells substantially more efficiently than DNA-protein cross-links.

Conversely, other recent reports suggest that mammalian DPC repair may occur via a pathway(s) distinct from NER. For instance, recent papers provide evidence for a role for homologous recombination in DPC repair.^53,58 Nakano et al failed to detect differences in the kinetics of removal of formaldehyde-induced DPCs when NER-proficient and NER-deficient cells were compared.⁵³ Instead, these authors observed that clones deficient in homologous recombination genes displayed greater hypersensitivity to formaldehyde-induced death than did clones deficient in NER genes.⁵³ Furthermore, a study by Chvalova and colleagues⁵⁹ observed the failure of human NER system to remove proteins cross-linked to DNA by cisplatin, suggesting that another pathway may be important. These discrepancies may indicate that DPC structures and protein identities may affect their repair mechanism, and more than one repair pathway may be required.^10,60 Further studies involving site-specific cisplatin-induced lesions are needed to determine the mechanisms of their repair and their effects on DNA replication.

In conclusion, the present system-wide study demonstrates that DNA-protein cross-links involving a variety of cellular proteins are formed in human fibrosarcoma-derived HT1080 cells following exposure to clinically relevant concentrations of cisplatin.⁶¹ In our experiments, cisplatin was able to cross-link over 250 proteins to chromosomal DNA. Proteins were identified by mass spectrometry-based proteomics, and the identities of several proteins were confirmed by immunological detection. Many of the identified proteins are involved in a variety of cellular processes such as chromatin remodeling, translation, DNA replication, DNA damage response, DNA repair, RNA processing, and transcriptional regulation. If not repaired, these bulky DPC lesions are expected to cause chromosomal double-strand breaks or be misread by DNA polymerases to generate mutations, ultimately triggering programmed cell death or genotoxic outcomes. Ongoing studies with site-specific modified plasmids introduced in mammalian cells with different repair backgrounds are currently underway in our laboratory to obtain additional details on the consequences of DPCs induced by antitumor platinum agents in human cells.

List of Abbreviations

Cisplatin

cis-1,1,2,2-diamminedichloroplatinum (II)

dG-Pt-Cl

cis-1,1-diammine-2-chloro-2-(2′-deoxyguanosine-7-yl)-platinum (II)

dG-Pt-dG

cis-1,1-diammine-2,2-bis-(deoxyguanosine-7-yl)-platinum (II)

dG-Pt-Lys

cis-1,1-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2′-deoxyguanosine-7-yl)-platinum(II)

DEB

1,2,3,4-diepoxybutane

mechlorethamine

bis(2-chloroethyl)methylamine

DPC

DNA-protein cross-link

DTT

dithiothreitol

FBS

fetal bovine serum

FDR

false discovery rate

GSH

glutathione

HPLC-ESI⁺-MS/MS

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

PMSF

phenylmethanesulfonyl fluoride

PARP-1

poly(ADP-ribose) polymerase I

AGT

O⁶-alkylguanine DNA alkyltansferase

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

Ref-1

DNA-(apurinic- or apyrimidinic-site) lyase

XRCC-1

x-ray cross-complementing protein I

Fen-1

flap endonuclease 1

EF 1α1

elongation factor 1α1

HMG

high mobility group protein

DNase I

deoxyribonuclease I

PDE I

phosphodiesterase I

PDE II

phosphodiesterase II

HR

homologous recombination

NER

nucleotide excision repair

SRM

selected reaction monitoring

TIC

total ion current

PBS

phosphate-buffered saline

TBS

tris-buffered saline

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis