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Mutation Research 731 (2012) 75– 84

Contents lists available at SciVerse ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

jo ur n al hom ep a ge: www.elsev ier .com/ locate /molmutC om mun i ty a ddress : www.elsev ier .com/ locate /mutres

Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells

Chunlan Yana,b,1, Zhaojun Chena,b,1, Huanrong Lia,b, Guanglin Zhanga,b, Feng Li c,Penelope J. Duerksen-Hughesd, Xinqiang Zhub,∗, Jun Yanga,e,∗∗

a The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, Chinab Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058, Chinac The First Renmin Hospital, Houma, Shanxi 043000, Chinad Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354, USAe Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310036, China

a r t i c l e i n f o

Article history:Received 12 June 2011Received in revised form 3 November 2011Accepted 16 November 2011Available online 25 November 2011

Keywords:Benzo(a)pyreneProteomicsDNA damage responseAnnexin A1Proteasome

a b s t r a c t

Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to10 �M benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression andalternative splicing. To further expand our knowledge about the DNA damage response (DDR) inducedby BaP, we investigated the nuclear protein expression profiles in HeLa cells treated with different con-centrations of BaP (0.1, 1, and 10 �M) using this proteomics-based 2-D gel electrophoresis assay. Wefound 125 differentially expressed proteins in BaP-treated cells compared to control cells. Among them,79 (63.2%) were down-regulated, 46 (36.8%) were up-regulated; 8 showed changes in the 1 �M and10 �M BaP-treated groups, 2 in the 0.1 �M and 10 �M BaP-treated groups, 4 in the 0.1 �M and 1 �MBaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosenfor liquid chromatography–tandem mass spectrometry (LC–MS/MS) identification, and of these, 39 wereidentified, including subunits of the 26S proteasome and Annexin A1. The functions of some identifiedproteins were further examined and the results showed that they might be involved in BaP-induced DDR.Taken together, these data indicate that proteomics is a valuable approach in the study of environmentalchemical–host interactions, and the identified proteins could provide new leads for better understandingBaP-induced mutagenesis and carcinogenesis.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous envi-ronmental pollutants that can be found in cigarette smoke,cooking and fossil combustion exhaust. Benzo(a)pyrene (BaP) isa model PAH compound, and is classified as a potent carcinogenand/or mutagen, which exhibits strong carcinogenic properties intumor initiation, promotion and progression in humans [1]. Asan indirect-acting genotoxin, BaP has to be metabolically acti-vated by the cytochrome P450 enzymes to form the active formBenzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) [2,3]. TheBaP-induced DNA damage response (DDR) has been extensivelystudied, in an effort to understand the mechanisms of BaP-inducedmutagenesis and carcinogenesis [4,5]. For instance, it is known

∗ Corresponding author. Tel.: +86 571 8820 8146; fax: +86 571 8820 8146.∗∗ Corresponding author at: Department of Toxicology, Hangzhou Normal Univer-

sity School of Public Health, Hangzhou, Zhejiang 310036, China.Tel.: +86 571 8820 8140; fax: +86 571 8820 8140.

E-mail addresses: zhuxq@zju.edu.cn (X. Zhu), gastate@zju.edu.cn (J. Yang).1 These two authors contributed equally to this work.

that BaP exposure can activate the classic p53 pathway, leading toelevated transcription of the p53 gene and subsequent p53 pro-tein accumulation, which in turn up-regulates the cellular p21protein [6]. On the other hand, cadmium, a widespread environ-mental pollutant which is also a cigarette smoke constituent, canenhance the genotoxicity of BaP, by impairing the p53 and p21responses, inhibiting nucleotide excision repair (NER) pathway-dependent DNA repair and overriding G1-S cell cycle arrest inducedby BPDE [7]. In addition, it has been suggested that the excisionrepair cross-complementing 1 (ERCC1) protein could be an impor-tant limiting factor for NER in cells exposed to BaP [8]. Moreover,new molecules that might be involved in DDR have also been iden-tified through the studies of BaP. For example, exposure to UV orBaP induced the up-regulation of three prime exonuclease I (TREX1)and its translocation to the nucleus, while cells deficient in TREX1showed reduced recovery from the UV and BaP-induced replicationinhibition, implicating TREX1 as a novel DNA damage-induciblerepair gene that plays a protective role in the genotoxic stressresponse [9].

In addition to such molecular biological studies, high-throughput technologies have also been applied to examineBaP-induced DDR. Using the RAGE (Rapid Analysis of Gene

0027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2011.11.007

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76 C. Yan et al. / Mutation Research 731 (2012) 75– 84

Expression) technique, Wang et al. analyzed the expression of over1000 genes in BPDE-treated human mammary epithelial HME87cells, and the results showed that many p53-regulated genes aswell as transcription factors ATF3 and E2A were involved in the cel-lular response to DNA damage induced by bulky chemical adducts[10]. Hockley et al. used cDNA microarray to identify those genesthat might participate in cellular responses to BaP or BPDE, andamong those identified, many were predominantly involved in cellcycle regulation, apoptosis, and DNA repair [11]. Many groups havealso used proteomic approaches to investigate BaP- and BPDE-induced DDR. For example, we have shown that BaP exposurecaused expression changes in more than 100 proteins in humanamnion epithelial FL cells, including zinc finger proteins and manyother transcription factors [12]. Using the same cells but exposedto BPDE, Shen et al. found similar changes in proteins involved inthe regulation of transcription, cell cycle, apoptosis, transport, sig-nal transduction, metabolism, etc, as well as eukaryotic translationinitiation factors and components of ubiquitin–proteasome sys-tem [13,14]. Zhao et al. conducted a comparative proteomic studybetween the BPDE-transformed human bronchial epithelial cellline (16HBE-C) and its parental cell line (16HBE) G0/G1 cells. In thisstudy, eukaryotic translation initiation factors as well as ubiquitin-related proteins with changed expression were identified [15]. Inanother proteomic study, Min et al. focused on the oxidative stressinduced by BaP and reported 23 differentially expressed proteinsin A549 cells [16]. These studies have provided useful informationregarding the cellular response to BaP and/or BPDE, thus giving us abetter understanding of the mechanisms underlying the genotoxiceffects of BaP.

However, one disadvantage of these whole-cell proteomicmeasures is their limited ability to detect ‘low-abundance’ pro-teins. Therefore, it is necessary to use subcellular fractionation,or organelle proteomics to identify changes in the expression lev-els of those lower-abundance proteins, such as nuclear proteins,which play pivotal roles in controlling cellular processes, includingmutagenesis and carcinogenesis. Thus, in a previous study, we usedsuch an organelle proteomic method to analyze the nuclear proteinexpression profiles in HeLa cells treated with 10 �M BaP for vari-ous times. We found that the expression levels of many proteinsinvolved in alternative splicing were changed by BaP exposure,and further experiments verified that alternative splicing indeedoccurred in BaP-treated cells for certain genes [17]. A similar phe-nomenon was also observed in cisplatin-treated HeLa cells [18].Together, these data indicated that alternative splicing might bea novel mechanism involved in the DDR, and its function in thisresponse warrants further detailed investigation. In the presentstudy, in order to further expand our knowledge regarding BaP-induced DDR and to identify the underlying novel mechanisms, weexamined the nuclear proteosome response of HeLa cells to dif-ferent concentrations of BaP. As reported here, over 100 proteinsshowed changed expression after BaP exposure, and 39 proteinswere identified by liquid chromatography-tandem mass spectrom-etry (LC–MS/MS). Several of the identified proteins were furtherverified by Western blot analysis, and analyzed for cellular func-tion. Among this group were subunits of the 26S proteasome andAnnexin A1 (ANXA1).

2. Materials and methods

2.1. Cell culture and treatment

Human cervical carcinoma HeLa cells were subcultured in Eagle’s MinimumEssential Medium (MEM, Invitrogen, Carlsbad, CA), containing 10% newborn calfserum, 100 U/ml penicillin, 125 �g/ml streptomycin, 0.03% glutamine, and culti-vated in a 5% CO2 atmosphere at 37 ◦C. BaP (Sigma–Aldrich, Saint Louis, USA) wasdissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution. HeLa cells wereexposed to 0.1, 1, and 10 �M BaP for 6 h, 0.1% DMSO was used as control. After 6 hof incubation, the medium was removed, and cells were harvested.

2.2. Sample preparation

Nuclear proteins were extracted from cells using the nuclear extraction kit(CHEMICON, Cat. No. 2900, USA) following the manufacturer’s instructions. Pro-tein concentrations were determined by the Bradford assay [19]. The samples werethen quick frozen in liquid nitrogen and stored at −80 ◦C for further analysis.

2.3. Two-dimensional gel electrophoresis (2-DE) and image analysis

2-DE was performed using the protocol established in our laboratory [17,20].Briefly, approximately 200 �g of extracted nuclear proteins were applied to lin-ear IPG Readystrips (17 cm; pH 5–8; Bio-Rad, USA) followed by in-gel rehydrationfor 12 h at 20 ◦C. Isoelectric focusing (IEF) was performed using a protein IEF cell(Bio-Rad) under the following conditions: 20 ◦C, 250 V for 30 min; 1000 V for 2 h;10,000 V for 5 h; and 10,000 V until 60,000 Vh was achieved. After the IEF was com-pleted, the individual strips were equilibrated and the proteins were separated in thesecond dimension by vertical 12% SDS-polyacrylamide gel electrophoresis (PAGE).Gels were stained using an improved silver-staining method as described previously[17,20].

Image analysis was conducted following the protocol as described earlier[17,20]. Briefly, the silver-stained 2-DE gels were scanned with a GS-800 calibratedimaging densitometer (Bio-Rad) at a resolution of 300 dots per inch. The imageswere analyzed with PDQuest software 7.4.0 (Bio-Rad). A statistical analysis wasperformed using the Student’s t-test. A p value of <0.05 was considered statisticallysignificant.

2.4. Liquid chromatography and tandem mass spectrometry (LC–MS/MS)

In-gel digestion and LC–MS/MS were performed by Shanghai Applied ProteinTechnology Co. Ltd. (Shanghai, China). Tandem mass spectra were searched usingBioworksBrowser rev. 3.1 software (Thermo Electron, San Jose, CA) against thenonredundant International Protein Index (IPI) human protein database (version3.26, 67,687 entries). The protein identification criteria were based on Delta Cn(≥0.1) and cross-correlation scores (Xcorr, one charge ≥1.9, two charges ≥2.2, threecharges ≥3.75). Only proteins identified by at least two unique peptides or oneunique peptide repeating 4 times were reported.

2.5. Immunoblotting

Immunoblotting analysis was conducted as described before [17,20]. Briefly,30 �g of samples were separated by 12% SDS-PAGE. After electrophoresis, pro-teins were transferred to Immunoblotting PVDF membranes (Bio-Rad) followed byblocking with 5% non-fat skim milk. PVDF membranes were then probed with rab-bit antibodies (anti-ANXA1, anti-NF-�b, anti-�-actin and anti-H3, Bioworld, USA)overnight at 4 ◦C. �-actin was used as a loading control. Finally, the membraneswere incubated by IRDye-conjugated goat anti-rabbit secondary antibody (Cat. No.B81009-02, Li-CoR Biosciences) for 1 h at room temperature. The membrane wasscanned using the Odyssey infrared imaging system (LI-COR Biosciences). Imageswere analyzed by Quantity One 4.6.2 software.

2.6. RNA interference

The siRNAs were purchased from Genepharma Corporation (Shanghai, China).The sense strand of the siRNA was ACUCCAGCGCAAUUUGAUGTT (nucleotides414-432) for human ANXA1. A nonspecific control with nucleotide sequence ofUUCUCCGAACGUGUCACGUTT was used as a negative control (referred to as NS).They were transfected into HeLa cells with the lipofectamine 2000 siRNA mix (Invit-rogen) at a final concentration of 100 nM. After 24 h incubation, gene silencing waschecked with immunoblot and then cells were subjected to BaP treatment.

2.7. Apoptosis assay

An Annexin V-FITC/PI kit (Multiscience, Hangzhou, China) was used to analyzecells for apoptosis. Briefly, cells were washed with phosphate-buffered saline (PBS)and resuspended in 500 �l binding buffer. 5 �l Annexin V-FITC and 10 �l PI wereadded into the buffer and incubated for 5 min in the dark at room temperature. Thecells were then analyzed by flow cytometry (Beackman).

2.8. Immunofluorescence microscopy

The protocol was performed as described previously [20,21]. Briefly, 1 × 105

cells were seeded into a 6-well culture plate containing a glass cover slip in eachwell. After treatment, cells were fixed in 4% paraformaldehyde, permeabilized in0.2% Triton-X 100, and incubated with a mouse monoclonal anti-�H2AX antibody(DAM1782241, Millipore) for 2 h, followed with FITC-conjugated goat-anti-mousesecondary antibody for 1 h. Finally, the coverslips were mounted onto microscopeslides in 90% glycerol and observed with an Olympus AX70 fluorescent microscope(Olympus, Tokyo, Japan). To prevent bias in the selection of cells that display foci,over 800 randomly selected cells were counted. Cells with five or more foci of anysize were classified as positive.

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C. Yan et al. / Mutation Research 731 (2012) 75– 84 77

Fig. 1. Representative 2-DE image of nuclear proteins from HeLa cells. Extracted nuclear proteins were separated on a pH 5–8 IPG linear strip followed by 12% SDS-PAGE,silver staining and analysis using PDQuest software 7.4.0. Arrows indicate the protein spots identified by using LC–MS/MS, and details of the corresponding protein spots arelisted in Table 1.

2.9. Statistical analysis

Statistical analysis was performed using the Student’s t-test. Each experi-ment was conducted at least three times independently. Data are presented asmean ± SEM. A probability level of p < 0.05 was considered significant.

3. Results

3.1. Nuclear protein expression profiles

Nuclear proteins from HeLa cells treated with 0.1, 1 and 10 �MBaP for 6 h were extracted by nuclear extraction kit and separatedby 2-DE. Quantitative spot comparisons were made using the imageanalysis software PDQuest 7.4.0. A representative silver-stainedimage of the nuclear protein expression profile is shown in Fig. 1.Over 700 protein spots per gel in both the BaP-treated and con-trol groups were detected. 125 protein spots showed significantchanges following BaP treatment. Among the 125 protein spots, 79(63.2%) protein spots were down-regulated, and 46 (36.8%) pro-tein spots were up-regulated. 8 protein spots in the 1 �M and10 �M BaP-treated groups, 2 protein spots in the 0.1 �M and 10 �MBaP-treated groups, and 4 protein spots in the 0.1 �M and 1 �MBaP-treated groups showed changes compared to control, whileonly one spot showed changes in all three treatment groups.

3.2. Identification of proteins with altered expression

Among 125 differentially expressed protein spots, 50 proteinspots were selected and excised from the gels, then subjected toLC–MS/MS analysis. A total of 39 proteins were identified, whichwere categorized into different functional classes, such as cellprocesses, DNA-replication, recombination and repair, mRNA tran-scription, pre-mRNA processing, and proteasome, etc. (Table 1). TheMS data for a peptide from the identified ANXA1 protein are shownin Fig. 2.

3.3. Confirmation of altered expression of ANXA1

To verify the proteomic analysis result, the expression levelof ANXA1 was further analyzed in BaP-treated HeLa cells by

immunoblot. As shown in Fig. 3, the expression of ANXA1 was sig-nificantly increased in 1 �M BaP-treated HeLa nuclei as comparedto untreated cells. This result was consistent with the proteomicanalysis. Interestingly, we observed both a significant increase ofANXA1 in nuclei and the simultaneous decrease in cytoplasm afterexposure to BaP (Fig. 3). These data suggest that ANXA1 mighttransfer from cytoplasm into the nuclei in response to BaP, asprevious studies have indicated its involvement in DNA damageresponses [22,23].

3.4. Function of ANXA1 in BaP-induced DDR

In order to investigate the role of ANXA1 in response to BaP inHeLa cells, we inhibited ANXA1 expression with siRNA. HeLa cellswere transfected with ANXA1 siRNA or nonspecific (NS) siRNA, and24 h later the cells were incubated with 1 �M BaP for 6 h. As shownin Fig. 4A, the expression of ANXA1 was almost abolished in ANXA1-siRNA transfected HeLa cells as compared to NS-siRNA transfectedHeLa cells. The effect of ANXA1 on apoptosis was then examined.It was found that ANXA1 siRNA transfection alone caused a slightbut not statistically significant increase in the ratio of apoptoticcells as compared to NS siRNA-transfected cells (Fig. 4B). BaP expo-sure also caused a slight increase in the ratio of apoptotic cellsin both ANXA1 siRNA-transfected and NS siRNA-transfected cells,however, neither was statistically significant (Fig. 4B).

To find out whether ANXA1 is involved in the response to BaP-induced DNA damage, the formation of �H2AX foci, an indicatorof DNA damage, was examined. It was shown that BaP treatmentsignificantly induced �H2AX foci formation in both ANXA1 siRNA-and NS siRNA-transfected HeLa cells (Fig. 4C). Furthermore, theincrease of �H2AX-positive cells in ANXA1 siRNA-transfected cellswas significantly higher compared to NS siRNA-transfected cellsafter BaP exposure (Fig. 4C), indicating a role for ANXA1 in thecellular response to BaP-induced DNA damage in HeLa cells.

3.5. BaP treatment induces changed expression of NF-�B

Notably, many identified proteins were found to be compo-nents of the 26S proteasome (Table 1), which is responsible

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80 C. Yan et al. / Mutation Research 731 (2012) 75– 84

Fig. 2. LC–MS/MS data for the identification of the differentially expressed nuclear protein ANXA1. The reporter ion signal was matched to the sequence of the peptideRKGTDVNVFNTILTTR corresponding to ANXA1 (SWISS-PROT entry: P04083).

Fig. 3. BaP exposure induces changes in ANXA1 expression. (A) Upper panel: enlarged 2-DE images of spot 7204, which was up-regulated in BaP-treated HeLa cell nuclei;middle panel: western blot results showing that the level of ANXA1 was increase in BaP-treated HeLa cell nuclei; lower panel: �-actin was used as loading control; (B) relativeintensity of 2-DE image for spot 7204 and Western blot results for ANXA1; (C) Western blot showed a significant increase of ANXA1 in nuclei and the simultaneous decreasein cytoplasm after exposure to BaP; (D) relative intensity of Western blot results for ANXA1. *p < 0.05.

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C. Yan et al. / Mutation Research 731 (2012) 75– 84 81

Fig. 4. ANXA1 knockdown affects BaP-induced DNA damage. (A) Western blot results showing that ANXA1 expression was inhibited in ANXA1 siRNA-transfected cells after24 h. (B) Apoptosis was assessed by the annexin-V binding assay in ANXA1 siRNA or NS siRNA-transfected HeLa cells treated with BaP (1 �M) for 6 h (n = 3). (C) �H2AX fociformation in ANXA1 siRNA or NS siRNA-transfected HeLa cells treated with BaP (1 �M) for 6 h. Left, representative images of �H2AX foci detected by immunofluorescentmicroscopy; right, �H2AX-positive cells were classified as those with >5 foci/cell, and over 800 cells were counted. *p < 0.05.

for ATP- and ubiquitin-dependent protein degradation in thenucleus and the cytosol [24]. Among them, the 26S protea-some non-ATPase regulatory subunit 10 (p28GANK, also knownas PSMD10, p28 and gankyrin) is involved in the regulationof NF-�B activation, through the retention of it in the cyto-plasm by binding to the NF-�B/RelA and exporting RelA from thenucleus through a chromosomal region maintenance-1 (CRM-1)dependent pathway [25]. Thus, it was of interest to determinewhether the decreased expression of p28GANK due to BaP expo-sure could affect the localization/expression of NF-�B in HeLacells. We found a decrease of NF-�B in the cytoplasm, and aconcomitant increase in the nuclei in BaP-treated cells as com-pared to untreated cells (Fig. 5). This result suggested thatNF-�B was not retained in the cytoplasm due to the reducedexpression of p28GANK, and subsequently, it translocated fromcytoplasm into the nuclei to participate in the cellular responseto BaP.

4. Discussion

BaP, which is widely present in the environment, forms covalentDNA adducts and elicits DNA damage, mutagenesis, and carcino-genesis in mammals [26,27]. However, the underlying mechanismsof BaP-induced mutagenesis and carcinogenesis are not yet fullyunderstood. High-throughput technologies, including proteomics,are powerful methods that can reveal previously unknown or unex-pected associations, and thus are suitable for such mechanisticstudies. In a previous study, using the nuclear proteomic method,we examined the time-response of HeLa cells exposed to BaP,and identified alternative splicing as a novel mechanism for BaP-induced DDR [17]. In the present study, using the same method,we examined the dose-response of HeLa cells to BaP exposure, inan effort to identify new leads for the cellular response to BaP.After exposure to 0.1 �M, 1 �M, and 10 �M of BaP, nuclear pro-tein profiles analysis found 125 proteins with significant changes

Fig. 5. BaP treatment induces a change in NF-�B expression in HeLa cells. (A) Western blot results showed that NF-�B expression was increased in BaP-treated HeLa cellnuclei and decreased in cytoplasm. �-Actin was used as a loading control, and H3 as an indicator for the nuclear proteins. (B) Relative intensity of Western blot results forNF-�B. N, nucleus; C, cytoplasm. *p < 0.05.

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82 C. Yan et al. / Mutation Research 731 (2012) 75– 84

in expression levels, and eventually 39 differentially expressedproteins, which participate in various cellular processes, were suc-cessfully identified (Table 1).

Among the identified proteins, many are also alternative splic-ing proteins, thus confirming the results from our previous study.However, some proteins were not identified in the previous study,including ANXA1. Kassie et al. have shown that ANXA1 was inducedafter feeding a mixture of NNK plus BaP in A/J mice model [28],unfortunately, it is not clear whether ANXA1 is associated withDDR. So in present study, we intend to investigate whether ANXA1is involved in the DDR induced by BaP, and in particular, its function.ANXA1 is the first characterized member of the Annexin superfam-ily, which includes several ubiquitous calcium and phospholipidbinding proteins. ANXA1 is involved in many cellular processesincluding anti-inflammatory mechanisms, signal transduction andapoptosis [29]. We found that the expression of ANXA1 increasedin nuclei after exposure to BaP (Figs. 1 and 3). To investigate thefunctional role of ANXA1 in response to BaP, we inhibited ANXA1expression by siRNA. BaP at concentrations used in this experiment(0.1, 1, and 10 �M) has been shown to induce �H2AX foci, althoughwhether it is physiological relevant is not yet clear [21]. We founda significant increase of �H2AX foci in ANXA1 siRNA-transfectedcells as compared to NS siRNA-transfected cells after BaP exposure(Fig. 4C), which suggests a protective role of ANXA1 against BaP-induced DNA damage. Interestingly, Nair et al. also reported thatover-expression of ANXA1 can protect MCF7 breast cancer cellsagainst heat-induced growth arrest and DNA damage [23], which isconsistent with our results. It has been suggested that BaP-inducedDNA damage could be the result of generated BaP-DNA adductsas well as reactive oxygen species (ROS) [30], thus, it would be ofinterest to know ANXA1 interacts/interferes with which pathway.

Previous studies have also indicated a pro-apoptotic functionfor ANXA1. For instance, the over-expression of ANXA1 promotedapoptosis associated with caspase-3 activation in several types ofcells [31,32]. ANXA1 expression is a contributing factor to its pro-apoptotic effects in prostate cancer [33]. In addition, ANXA1 alsoplays a role in the clearance of apoptotic cells by macrophages[34]. The underlying mechanism for its apoptotic effect probablyis thorough intracellular calcium release and dephosphorylationof BAD, thus enhancing BAD heterodimerization with Bcl-xL andpromoting apoptosis [35,36]. However, there are also reports show-ing an anti-apoptotic effect of ANXA1. For example, Wu et al.demonstrated that ANXA1 rendered monocytic cells resistant toTNF-�-induced apoptosis, and that ANXA1 levels were constitu-tively higher in TNF-� resistant cells than in cells sensitive toTNF-� [37]. This is consistent with the finding that dexamethasonetreatment, through ANXA1-dependent mechanisms, led to resis-tance to doxorubicin and etoposide in prostate cancer cells [38].Thus, it is important to know whether ANXA1 has pro-apoptotic oranti-apoptotic functions during the cellular response to BaP expo-sure. Unfortunately, in our experimental setting, e.g., 1 �M and 6 htreatment, no significant differences in apoptosis were observedbetween ANXA1 siRNA-transfected cells and NS siRNA-transfectedcells (Fig. 4B). The reason for this could be that at this particularconcentration and incubation time, BaP usually does not induce sig-nificant apoptosis [21]. Therefore, further experiments using higherdoses and/or longer incubation times for BaP are required to deter-mine the role of ANXA1 in this type of apoptosis.

Interestingly, we found that the increased nuclear expressionof ANXA1 was associated with decreased cytoplasmic expressionof ANXA1 (Fig. 3), which suggested that ANXA1 might translocatefrom cytoplasm to the nucleus in response to BaP exposure. Thisis also in agreement with many previous reports. For example,treatment with 15 �M MMS or 3 �M As3+ induced the same phe-nomenon [22]. TNF-� treatment also caused ANXA1 to migrate tothe nucleus and/or peri-nucleus region, and this migration can be

inhibited by the over-expression of the anti-apoptotic protein Bcl-2[39]. However, the precise mechanism and function of the nuclearlocalization of ANXA1 requires further examination.

We identified both protein spots 7204 and 8104 as ANXA1(Fig. 1). One possible explanation might be that one of the proteinspots is a cleavage product of ANXA1. Kim et al. reported that phor-bol 12-myristate 13-acetate (PMA) induced the cleavage of ANXA1in HEK293 cells, and the cleaved form of ANX-1 translocated to thenucleus [40]. Sakaguchi et al. also reported that ANXA1 was cleavedsolely at the C-terminal side of Trp12 by cathepsin D in normalhuman keratinocytes (NHK) upon exposure to EGF [41]. Further-more, ubiquitination or SUMOylation of ANXA1 could also causethe appearance of more than one spot for ANXA1 in the 2D gel.

Several of the identified polypeptides are subunits of the 26Sproteasome. The 26S proteasome consists of a 20S proteasome coreand two 19S regulatory subunits, including at least 32 different sub-units [42]. Some subunits are characterized by their ability to cleavepeptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leavinggroup at neutral or slightly basic pH, which have an ATP-dependentproteolytic activity; others are non-ATPase regulatory subunits,which are involved in the ATP-dependent degradation of ubiqui-tinated proteins [42]. One of the identified proteins, p28GANK actsas a chaperone during the assembly of the 26S proteasome, whichis involved in p53-independent apoptosis [43]. It also directly bindsto NF-�B/RelA, resulting in the cytoplasmic retention of NF-�B/RelA[25]. Since BaP caused decreased expression of p28GANK (Fig. 1),we were interested to know how this would affect NF-�B. Westernblot results showed that NF-�B expression increased in the nucleus,along with a concomitant decrease in the cytoplasm after exposureto BaP (Fig. 5). This suggested that due to the decreased expressionof p28GANK, NF-�B may no longer be retained in the cytoplasm,and thus likely translocates from the cytoplasm to the nucleus inorder to function as a regulator of transcription.

Also identified were the 26S proteasome non-ATPase regula-tory subunit 8 and subunit 1, which belong to the 19S regulatorycomponent and are necessary for activation of the CDC28 kinasethat regulates the mitotic cell cycle [44,45]. On the other hand,proteasome subunit beta type 4 and 2 are components of the twoinner rings of the 20S proteasome core, and have proteolytic activity[46,47], while proteasome subunit alpha type 6 and 2 are involvedin directing the assembly of the 20S proteasome core [48]. Theubiquitin–proteasome system is responsible for the eliminationof abnormal proteins and selective destruction of regulatory pro-teins that are involved in many cellular processes, including DDR.For example, Wang et al. reported that oxidative stress inducedby H2O2 resulted in the dissociation of the 20S core particle fromthe 19S regulatory particle, thus leading to the loss of 26S pro-teasome activities and the accumulation of ubiquitinated proteins[49]. The ribonucleotide reductase inhibitor Sml1 was degraded by26S proteasome in response to DNA damage [50]. In addition, theproteasome also targets a double strand break (DSB) repair protein,Mms22, thus linking nuclear proteasomal activity and DSB repair[51].

Other identified proteins may also play important roles in thecellular response to BaP, and further detailed analysis of theseproteins may yield a better understanding of the mechanisms ofBaP-induced carcinogenesis and mutagenesis. For example, pro-teins involved in mRNA processing have also been identified, as inthe previous studies. These identified polypeptides, which functionin various cellular processes including DNA-replication, transcrip-tion, recombination and repair, post-transcriptional modification,etc., are the focus of our future studies.

In summary, our present study employed a nuclear proteomicmethod to analyze the nuclear protein expression profiles inresponse to treatment with various concentrations of BaP. A num-ber of proteins were identified, several of which suggest that

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C. Yan et al. / Mutation Research 731 (2012) 75– 84 83

nuclear proteasome activity might play an important role in theBaP-induced DDR. These data indicate that proteomics-based 2-D gel electrophoresis assay can serve as a valuable technique inthe mechanistic study of the cellular response to environmentalchemicals.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by grants from National Natural Sci-ence Foundation of China (No. 81172692); Zhejiang ProvincialNatural Science Foundation (R2100555); and Ministry of Scienceand Technology, China (2009DFB30390). J. Yang is a recipient ofthe Zhejiang Provincial Program for the Cultivation of High-levelInnovative Health Talents.

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