Mutation Research 731 (2012) 75–84
Contents lists available at SciVerse ScienceDirect
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis journal homepage: www.elsevier.com/locate/molmut Community address: www.elsevier.com/locate/mutres
Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells Chunlan Yan a,b,1 , Zhaojun Chen a,b,1 , Huanrong Li a,b , Guanglin Zhang a,b , Feng Li c , Penelope J. Duerksen-Hughes d , Xinqiang Zhu b,∗ , Jun Yang a,e,∗∗ a
The First Afﬁliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, China Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058, China The First Renmin Hospital, Houma, Shanxi 043000, China d Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354, USA e Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310036, China b c
a r t i c l e
i n f o
Article history: Received 12 June 2011 Received in revised form 3 November 2011 Accepted 16 November 2011 Available online 25 November 2011 Keywords: Benzo(a)pyrene Proteomics DNA damage response Annexin A1 Proteasome
a b s t r a c t Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10 M benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression and alternative splicing. To further expand our knowledge about the DNA damage response (DDR) induced by BaP, we investigated the nuclear protein expression proﬁles in HeLa cells treated with different concentrations of BaP (0.1, 1, and 10 M) using this proteomics-based 2-D gel electrophoresis assay. We found 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 and 10 M BaP-treated groups, 2 in the 0.1 M and 10 M BaP-treated groups, 4 in the 0.1 M and 1 M BaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosen for liquid chromatography–tandem mass spectrometry (LC–MS/MS) identiﬁcation, and of these, 39 were identiﬁed, including subunits of the 26S proteasome and Annexin A1. The functions of some identiﬁed proteins 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 environmental chemical–host interactions, and the identiﬁed proteins could provide new leads for better understanding BaP-induced mutagenesis and carcinogenesis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that can be found in cigarette smoke, cooking and fossil combustion exhaust. Benzo(a)pyrene (BaP) is a model PAH compound, and is classiﬁed as a potent carcinogen and/or mutagen, which exhibits strong carcinogenic properties in tumor initiation, promotion and progression in humans . As an indirect-acting genotoxin, BaP has to be metabolically activated by the cytochrome P450 enzymes to form the active form Benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) [2,3]. The BaP-induced DNA damage response (DDR) has been extensively studied, in an effort to understand the mechanisms of BaP-induced mutagenesis 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 University School of Public Health, Hangzhou, Zhejiang 310036, China. Tel.: +86 571 8820 8140; fax: +86 571 8820 8140. E-mail addresses: [email protected]
(X. Zhu), [email protected]
(J. Yang). 1 These two authors contributed equally to this work. 0027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2011.11.007
that BaP exposure can activate the classic p53 pathway, leading to elevated transcription of the p53 gene and subsequent p53 protein accumulation, which in turn up-regulates the cellular p21 protein . On the other hand, cadmium, a widespread environmental pollutant which is also a cigarette smoke constituent, can enhance the genotoxicity of BaP, by impairing the p53 and p21 responses, inhibiting nucleotide excision repair (NER) pathwaydependent DNA repair and overriding G1-S cell cycle arrest induced by BPDE . In addition, it has been suggested that the excision repair cross-complementing 1 (ERCC1) protein could be an important limiting factor for NER in cells exposed to BaP . Moreover, new molecules that might be involved in DDR have also been identiﬁed through the studies of BaP. For example, exposure to UV or BaP induced the up-regulation of three prime exonuclease I (TREX1) and its translocation to the nucleus, while cells deﬁcient in TREX1 showed reduced recovery from the UV and BaP-induced replication inhibition, implicating TREX1 as a novel DNA damage-inducible repair gene that plays a protective role in the genotoxic stress response . In addition to such molecular biological studies, highthroughput technologies have also been applied to examine BaP-induced DDR. Using the RAGE (Rapid Analysis of Gene
C. Yan et al. / Mutation Research 731 (2012) 75–84
Expression) technique, Wang et al. analyzed the expression of over 1000 genes in BPDE-treated human mammary epithelial HME87 cells, and the results showed that many p53-regulated genes as well as transcription factors ATF3 and E2A were involved in the cellular response to DNA damage induced by bulky chemical adducts . Hockley et al. used cDNA microarray to identify those genes that might participate in cellular responses to BaP or BPDE, and among those identiﬁed, many were predominantly involved in cell cycle regulation, apoptosis, and DNA repair . Many groups have also used proteomic approaches to investigate BaP- and BPDEinduced DDR. For example, we have shown that BaP exposure caused expression changes in more than 100 proteins in human amnion epithelial FL cells, including zinc ﬁnger proteins and many other transcription factors . Using the same cells but exposed to BPDE, Shen et al. found similar changes in proteins involved in the regulation of transcription, cell cycle, apoptosis, transport, signal transduction, metabolism, etc, as well as eukaryotic translation initiation factors and components of ubiquitin–proteasome system [13,14]. Zhao et al. conducted a comparative proteomic study between the BPDE-transformed human bronchial epithelial cell line (16HBE-C) and its parental cell line (16HBE) G0/G1 cells. In this study, eukaryotic translation initiation factors as well as ubiquitinrelated proteins with changed expression were identiﬁed . In another proteomic study, Min et al. focused on the oxidative stress induced by BaP and reported 23 differentially expressed proteins in A549 cells . These studies have provided useful information regarding the cellular response to BaP and/or BPDE, thus giving us a better understanding of the mechanisms underlying the genotoxic effects of BaP. However, one disadvantage of these whole-cell proteomic measures is their limited ability to detect ‘low-abundance’ proteins. Therefore, it is necessary to use subcellular fractionation, or organelle proteomics to identify changes in the expression levels of those lower-abundance proteins, such as nuclear proteins, which play pivotal roles in controlling cellular processes, including mutagenesis and carcinogenesis. Thus, in a previous study, we used such an organelle proteomic method to analyze the nuclear protein expression proﬁles in HeLa cells treated with 10 M BaP for various times. We found that the expression levels of many proteins involved in alternative splicing were changed by BaP exposure, and further experiments veriﬁed that alternative splicing indeed occurred in BaP-treated cells for certain genes . A similar phenomenon was also observed in cisplatin-treated HeLa cells . Together, these data indicated that alternative splicing might be a novel mechanism involved in the DDR, and its function in this response warrants further detailed investigation. In the present study, in order to further expand our knowledge regarding BaPinduced DDR and to identify the underlying novel mechanisms, we examined the nuclear proteosome response of HeLa cells to different concentrations of BaP. As reported here, over 100 proteins showed changed expression after BaP exposure, and 39 proteins were identiﬁed by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Several of the identiﬁed proteins were further veriﬁed by Western blot analysis, and analyzed for cellular function. Among this group were subunits of the 26S proteasome and Annexin A1 (ANXA1).
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. Protein concentrations were determined by the Bradford assay . The samples were then 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 linear IPG Readystrips (17 cm; pH 5–8; Bio-Rad, USA) followed by in-gel rehydration for 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 completed, the individual strips were equilibrated and the proteins were separated in the second 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 calibrated imaging densitometer (Bio-Rad) at a resolution of 300 dots per inch. The images were analyzed with PDQuest software 7.4.0 (Bio-Rad). A statistical analysis was performed using the Student’s t-test. A p value of <0.05 was considered statistically signiﬁcant. 2.4. Liquid chromatography and tandem mass spectrometry (LC–MS/MS) In-gel digestion and LC–MS/MS were performed by Shanghai Applied Protein Technology Co. Ltd. (Shanghai, China). Tandem mass spectra were searched using BioworksBrowser rev. 3.1 software (Thermo Electron, San Jose, CA) against the nonredundant International Protein Index (IPI) human protein database (version 3.26, 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, three charges ≥3.75). Only proteins identiﬁed by at least two unique peptides or one unique 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, proteins were transferred to Immunoblotting PVDF membranes (Bio-Rad) followed by blocking with 5% non-fat skim milk. PVDF membranes were then probed with rabbit 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 membranes were incubated by IRDye-conjugated goat anti-rabbit secondary antibody (Cat. No. B81009-02, Li-CoR Biosciences) for 1 h at room temperature. The membrane was scanned using the Odyssey infrared imaging system (LI-COR Biosciences). Images were 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 (nucleotides 414-432) for human ANXA1. A nonspeciﬁc control with nucleotide sequence of UUCUCCGAACGUGUCACGUTT was used as a negative control (referred to as NS). They were transfected into HeLa cells with the lipofectamine 2000 siRNA mix (Invitrogen) at a ﬁnal concentration of 100 nM. After 24 h incubation, gene silencing was checked 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 analyze cells 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 were added into the buffer and incubated for 5 min in the dark at room temperature. The cells were then analyzed by ﬂow cytometry (Beackman). 2.8. Immunoﬂuorescence microscopy
2. Materials and methods 2.1. Cell culture and treatment Human cervical carcinoma HeLa cells were subcultured in Eagle’s Minimum Essential Medium (MEM, Invitrogen, Carlsbad, CA), containing 10% newborn calf serum, 100 U/ml penicillin, 125 g/ml streptomycin, 0.03% glutamine, and cultivated in a 5% CO2 atmosphere at 37 ◦ C. BaP (Sigma–Aldrich, Saint Louis, USA) was dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution. HeLa cells were exposed to 0.1, 1, and 10 M BaP for 6 h, 0.1% DMSO was used as control. After 6 h of incubation, the medium was removed, and cells were harvested.
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 each well. After treatment, cells were ﬁxed in 4% paraformaldehyde, permeabilized in 0.2% Triton-X 100, and incubated with a mouse monoclonal anti-␥H2AX antibody (DAM1782241, Millipore) for 2 h, followed with FITC-conjugated goat-anti-mouse secondary antibody for 1 h. Finally, the coverslips were mounted onto microscope slides 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 any size were classiﬁed as positive.
C. Yan et al. / Mutation Research 731 (2012) 75–84
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 are listed in Table 1.
2.9. Statistical analysis Statistical analysis was performed using the Student’s t-test. Each experiment was conducted at least three times independently. Data are presented as mean ± 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 M BaP for 6 h were extracted by nuclear extraction kit and separated by 2-DE. Quantitative spot comparisons were made using the image analysis software PDQuest 7.4.0. A representative silver-stained image of the nuclear protein expression proﬁle is shown in Fig. 1. Over 700 protein spots per gel in both the BaP-treated and control groups were detected. 125 protein spots showed signiﬁcant changes following BaP treatment. Among the 125 protein spots, 79 (63.2%) protein spots were down-regulated, and 46 (36.8%) protein spots were up-regulated. 8 protein spots in the 1 M and 10 M BaP-treated groups, 2 protein spots in the 0.1 M and 10 M BaP-treated groups, and 4 protein spots in the 0.1 M and 1 M BaP-treated groups showed changes compared to control, while only one spot showed changes in all three treatment groups.
immunoblot. As shown in Fig. 3, the expression of ANXA1 was signiﬁcantly increased in 1 M BaP-treated HeLa nuclei as compared to untreated cells. This result was consistent with the proteomic analysis. Interestingly, we observed both a signiﬁcant increase of ANXA1 in nuclei and the simultaneous decrease in cytoplasm after exposure to BaP (Fig. 3). These data suggest that ANXA1 might transfer from cytoplasm into the nuclei in response to BaP, as previous studies have indicated its involvement in DNA damage responses [22,23]. 3.4. Function of ANXA1 in BaP-induced DDR
Among 125 differentially expressed protein spots, 50 protein spots were selected and excised from the gels, then subjected to LC–MS/MS analysis. A total of 39 proteins were identiﬁed, which were categorized into different functional classes, such as cell processes, DNA-replication, recombination and repair, mRNA transcription, pre-mRNA processing, and proteasome, etc. (Table 1). The MS data for a peptide from the identiﬁed ANXA1 protein are shown in Fig. 2.
In order to investigate the role of ANXA1 in response to BaP in HeLa cells, we inhibited ANXA1 expression with siRNA. HeLa cells were transfected with ANXA1 siRNA or nonspeciﬁc (NS) siRNA, and 24 h later the cells were incubated with 1 M BaP for 6 h. As shown in Fig. 4A, the expression of ANXA1 was almost abolished in ANXA1siRNA transfected HeLa cells as compared to NS-siRNA transfected HeLa cells. The effect of ANXA1 on apoptosis was then examined. It was found that ANXA1 siRNA transfection alone caused a slight but not statistically signiﬁcant increase in the ratio of apoptotic cells as compared to NS siRNA-transfected cells (Fig. 4B). BaP exposure also caused a slight increase in the ratio of apoptotic cells in 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 BaPinduced DNA damage, the formation of ␥H2AX foci, an indicator of DNA damage, was examined. It was shown that BaP treatment signiﬁcantly induced ␥H2AX foci formation in both ANXA1 siRNAand NS siRNA-transfected HeLa cells (Fig. 4C). Furthermore, the increase of ␥H2AX-positive cells in ANXA1 siRNA-transfected cells was signiﬁcantly higher compared to NS siRNA-transfected cells after BaP exposure (Fig. 4C), indicating a role for ANXA1 in the cellular response to BaP-induced DNA damage in HeLa cells.
3.3. Conﬁrmation of altered expression of ANXA1
3.5. BaP treatment induces changed expression of NF-B
To verify the proteomic analysis result, the expression level of ANXA1 was further analyzed in BaP-treated HeLa cells by
Notably, many identiﬁed proteins were found to be components of the 26S proteasome (Table 1), which is responsible
3.2. Identiﬁcation of proteins with altered expression
Table 1 Proteins with altered expression identiﬁed by LC–MS/MS. Spot no.
Mascot search results Swissprot accession no.
Cell proceedings P51948 3203
CDK-activating kinase assembly factor MAT1 Isoform 1 of Mitotic spindle assembly checkpoint protein MAD1
Involved in cell cycle control and in RNA transcription by RNA polymerase II Component of the spindle-assembly checkpoint that prevents the onset of anaphase until all chromosomes are properly aligned at the metaphase plate Required for the assembly of a functional nuclear pore complex on the surface of chromosomes as nuclei form at the end of mitosis Believed to be a non-apoptotic caspase which is involved in epidermal differentiation Probable protease subunit of the COP9 signalosome complex which involved in various cellular and developmental processes
Isoform 1 of AT-hook-containing transcription factor 1
COP9 signalosome complex subunit 8
COP9 signalosome complex subunit 5 DNA-replication, transcription, recombination and repair 8302 P40937 Replication factor C subunit 5
Pre-mRNA-processing factor 19
DNA-directed RNA polymerase III subunit RPC6
Nuclear protein Hcc-1
RNA binding motif protein 39
TAR DNA-binding protein 43
PR domain zinc ﬁnger protein 8 Isoform alpha-enolase of Alpha-enolase Mothers against decapentaplegic homolog 1
Proliferation-associated protein 2G4
Transcription elongation factor B polypeptide 1
Post-transcriptional modiﬁcation Q5TA45-1 Integrator complex subunit 11 9807
Ratio of spot volume (p-value)
Involved in DNA replication, nucleotide-excision repair, DNA gap ﬁlling Plays a role in DNA double-strand break (DSB) repair and pre-mRNA splicing reaction DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates Binds both single-stranded and double-stranded DNA Involved in mRNA processing DNA and RNA-binding protein which regulates transcription and splicing. May be involved in transcriptional regulation Binds to the myc promoter and acts as a transcriptional repressor Transcriptional modulator activated by BMP (bone morphogenetic proteins) type 1 receptor kinase Inhibits transcription of some E2F1-regulated promoters, probably by recruiting histone acetylase (HAT) activity A general transcription elongation factor that increases the RNA polymerase II transcription elongation past template-encoded arresting sites Involved in the small nuclear RNAs (snRNA) U1 and U2 transcription and in their 3 -box-dependent processing
0.4009(0.0045) 2.8229(0.0321) 0.2735(0.0430)
0.4914(0.0346) 3.1142(0.0180) 2.0399(0.0005) 0.3703(0.0030) 2.3739(0.0132) 0.4501(0.0004)
C. Yan et al. / Mutation Research 731 (2012) 75–84
Necessary for the splicing of pre-mRNA
Binds single-stranded RNA
are components of the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, mediate pre-mRNA alternative splicing regulation
Heterogeneous nuclear ribonucleoprotein H2 Spliceosome RNA helicase DDX39B
Associates with spliced mRNA and not with unspliced pre-mRNA, functions in mRNA export
26S proteasome non-ATPase regulatory subunit 8
Acts as regulatory subunits of the 26 proteasome, involved in the ATP-dependent degradation of ubiquitinated proteins
Isoform 1 of 26S proteasome non-ATPase regulatory subunit 1 26S proteasome non-ATPase regulatory subunit 10 Proteasome subunit beta type-4
Proteasome subunit beta type 2 Proteasome subunit alpha type 6
Proteasome subunit alpha type 2
Isoform 2 of Nucleophosmin
Other protein functions 1003 P06748-2
0.2462(0.0255) Acts as a chaperone during the assembly of the 26S proteasome SMAD1/OAZ1/PSMB4 complex mediates the degradation of the CREBBP/EP300 repressor SNIP1. Has a trypsin-like activity A multicatalytic proteinase complex, has an ATP-dependent proteolytic activity Has a potential regulatory effect on another component(s) of the proteasome complex through tyrosine phosphorylation
0.2783(0.0012) 0.4503(0.00001) 0.4289(0.0022)
Involved in diverse cellular processes such as regulation of tumor suppressors p53/TP53 and ARF Unknown Calcium/phospholipid-binding protein, regulates phospholipase A2 activity
UPF0568 protein C14orf166 Annexin A1
5004 5205 5308
O43402 Q15006 Q8IZQ1-1
G patch domain and KOW motifs-containing protein Ribosomal RNA-processing protein 7 homolog A Neighbor of COX4 Tetratricopeptide repeat protein 35 WD repeat and FYVE domain-containing protein 3
5.92/23.77 6.15/34.83 6.30/39.53
38.57 14.81 0.34
Unknown Unknown Unknown
0.3337(0.0022) 0.4992(0.0107) 0.2636(0.0288)
C. Yan et al. / Mutation Research 731 (2012) 75–84
Proteasome function 1108 P48556
Splicing factor U2AF 65 kDa subunit Isoform 2 of Heterogeneous nuclear ribonucleoprotein A/B Heterogeneous nuclear ribonucleoprotein H
2.2818(0.00006) 3.6577(0.0062) 0.3829(0.0305)
Ratio of spot volume refers to the ratio of relative spot volume of treatment group to control group.
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 peptide RKGTDVNVFNTILTTR 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) relative intensity 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 decrease in cytoplasm after exposure to BaP; (D) relative intensity of Western blot results for ANXA1. *p < 0.05.
C. Yan et al. / Mutation Research 731 (2012) 75–84
Fig. 4. ANXA1 knockdown affects BaP-induced DNA damage. (A) Western blot results showing that ANXA1 expression was inhibited in ANXA1 siRNA-transfected cells after 24 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 foci formation 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 immunoﬂuorescent microscopy; 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 the nucleus and the cytosol . Among them, the 26S proteasome non-ATPase regulatory subunit 10 (p28GANK, also known as PSMD10, p28 and gankyrin) is involved in the regulation of NF-B activation, through the retention of it in the cytoplasm by binding to the NF-B/RelA and exporting RelA from the nucleus through a chromosomal region maintenance-1 (CRM-1) dependent pathway . Thus, it was of interest to determine whether the decreased expression of p28GANK due to BaP exposure could affect the localization/expression of NF-B in HeLa cells. We found a decrease of NF-B in the cytoplasm, and a concomitant increase in the nuclei in BaP-treated cells as compared to untreated cells (Fig. 5). This result suggested that NF-B was not retained in the cytoplasm due to the reduced expression of p28GANK, and subsequently, it translocated from cytoplasm into the nuclei to participate in the cellular response to BaP.
4. Discussion BaP, which is widely present in the environment, forms covalent DNA adducts and elicits DNA damage, mutagenesis, and carcinogenesis in mammals [26,27]. However, the underlying mechanisms of BaP-induced mutagenesis and carcinogenesis are not yet fully understood. High-throughput technologies, including proteomics, are powerful methods that can reveal previously unknown or unexpected associations, and thus are suitable for such mechanistic studies. 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 BaPinduced DDR . In the present study, using the same method, we examined the dose-response of HeLa cells to BaP exposure, in an 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 protein 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 cell nuclei 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 for NF-B. N, nucleus; C, cytoplasm. *p < 0.05.
C. Yan et al. / Mutation Research 731 (2012) 75–84
in expression levels, and eventually 39 differentially expressed proteins, which participate in various cellular processes, were successfully identiﬁed (Table 1). Among the identiﬁed proteins, many are also alternative splicing 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 induced after feeding a mixture of NNK plus BaP in A/J mice model , unfortunately, it is not clear whether ANXA1 is associated with DDR. So in present study, we intend to investigate whether ANXA1 is involved in the DDR induced by BaP, and in particular, its function. ANXA1 is the ﬁrst characterized member of the Annexin superfamily, which includes several ubiquitous calcium and phospholipid binding proteins. ANXA1 is involved in many cellular processes including anti-inﬂammatory mechanisms, signal transduction and apoptosis . We found that the expression of ANXA1 increased in nuclei after exposure to BaP (Figs. 1 and 3). To investigate the functional role of ANXA1 in response to BaP, we inhibited ANXA1 expression by siRNA. BaP at concentrations used in this experiment (0.1, 1, and 10 M) has been shown to induce ␥H2AX foci, although whether it is physiological relevant is not yet clear . We found a signiﬁcant increase of ␥H2AX foci in ANXA1 siRNA-transfected cells as compared to NS siRNA-transfected cells after BaP exposure (Fig. 4C), which suggests a protective role of ANXA1 against BaPinduced DNA damage. Interestingly, Nair et al. also reported that over-expression of ANXA1 can protect MCF7 breast cancer cells against heat-induced growth arrest and DNA damage , which is consistent with our results. It has been suggested that BaP-induced DNA damage could be the result of generated BaP-DNA adducts as well as reactive oxygen species (ROS) , thus, it would be of interest to know ANXA1 interacts/interferes with which pathway. Previous studies have also indicated a pro-apoptotic function for ANXA1. For instance, the over-expression of ANXA1 promoted apoptosis associated with caspase-3 activation in several types of cells [31,32]. ANXA1 expression is a contributing factor to its proapoptotic effects in prostate cancer . In addition, ANXA1 also plays a role in the clearance of apoptotic cells by macrophages . The underlying mechanism for its apoptotic effect probably is thorough intracellular calcium release and dephosphorylation of BAD, thus enhancing BAD heterodimerization with Bcl-xL and promoting apoptosis [35,36]. However, there are also reports showing an anti-apoptotic effect of ANXA1. For example, Wu et al. demonstrated that ANXA1 rendered monocytic cells resistant to TNF-␣-induced apoptosis, and that ANXA1 levels were constitutively higher in TNF-␣ resistant cells than in cells sensitive to TNF-␣ . This is consistent with the ﬁnding that dexamethasone treatment, through ANXA1-dependent mechanisms, led to resistance to doxorubicin and etoposide in prostate cancer cells . Thus, it is important to know whether ANXA1 has pro-apoptotic or anti-apoptotic functions during the cellular response to BaP exposure. Unfortunately, in our experimental setting, e.g., 1 M and 6 h treatment, no signiﬁcant differences in apoptosis were observed between ANXA1 siRNA-transfected cells and NS siRNA-transfected cells (Fig. 4B). The reason for this could be that at this particular concentration and incubation time, BaP usually does not induce signiﬁcant apoptosis . Therefore, further experiments using higher doses and/or longer incubation times for BaP are required to determine the role of ANXA1 in this type of apoptosis. Interestingly, we found that the increased nuclear expression of ANXA1 was associated with decreased cytoplasmic expression of ANXA1 (Fig. 3), which suggested that ANXA1 might translocate from cytoplasm to the nucleus in response to BaP exposure. This is also in agreement with many previous reports. For example, treatment with 15 M MMS or 3 M As3+ induced the same phenomenon . TNF-␣ treatment also caused ANXA1 to migrate to the nucleus and/or peri-nucleus region, and this migration can be
inhibited by the over-expression of the anti-apoptotic protein Bcl-2 . However, the precise mechanism and function of the nuclear localization 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 protein spots is a cleavage product of ANXA1. Kim et al. reported that phorbol 12-myristate 13-acetate (PMA) induced the cleavage of ANXA1 in HEK293 cells, and the cleaved form of ANX-1 translocated to the nucleus . Sakaguchi et al. also reported that ANXA1 was cleaved solely at the C-terminal side of Trp12 by cathepsin D in normal human keratinocytes (NHK) upon exposure to EGF . Furthermore, ubiquitination or SUMOylation of ANXA1 could also cause the appearance of more than one spot for ANXA1 in the 2D gel. Several of the identiﬁed polypeptides are subunits of the 26S proteasome. The 26S proteasome consists of a 20S proteasome core and two 19S regulatory subunits, including at least 32 different subunits . Some subunits are characterized by their ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH, which have an ATP-dependent proteolytic activity; others are non-ATPase regulatory subunits, which are involved in the ATP-dependent degradation of ubiquitinated proteins . One of the identiﬁed proteins, p28GANK acts as a chaperone during the assembly of the 26S proteasome, which is involved in p53-independent apoptosis . It also directly binds to NF-B/RelA, resulting in the cytoplasmic retention of NF-B/RelA . Since BaP caused decreased expression of p28GANK (Fig. 1), we were interested to know how this would affect NF-B. Western blot results showed that NF-B expression increased in the nucleus, along with a concomitant decrease in the cytoplasm after exposure to BaP (Fig. 5). This suggested that due to the decreased expression of p28GANK, NF-B may no longer be retained in the cytoplasm, and thus likely translocates from the cytoplasm to the nucleus in order to function as a regulator of transcription. Also identiﬁed were the 26S proteasome non-ATPase regulatory subunit 8 and subunit 1, which belong to the 19S regulatory component and are necessary for activation of the CDC28 kinase that regulates the mitotic cell cycle [44,45]. On the other hand, proteasome subunit beta type 4 and 2 are components of the two inner rings of the 20S proteasome core, and have proteolytic activity [46,47], while proteasome subunit alpha type 6 and 2 are involved in directing the assembly of the 20S proteasome core . The ubiquitin–proteasome system is responsible for the elimination of abnormal proteins and selective destruction of regulatory proteins that are involved in many cellular processes, including DDR. For example, Wang et al. reported that oxidative stress induced by H2 O2 resulted in the dissociation of the 20S core particle from the 19S regulatory particle, thus leading to the loss of 26S proteasome activities and the accumulation of ubiquitinated proteins . The ribonucleotide reductase inhibitor Sml1 was degraded by 26S proteasome in response to DNA damage . In addition, the proteasome also targets a double strand break (DSB) repair protein, Mms22, thus linking nuclear proteasomal activity and DSB repair . Other identiﬁed proteins may also play important roles in the cellular response to BaP, and further detailed analysis of these proteins may yield a better understanding of the mechanisms of BaP-induced carcinogenesis and mutagenesis. For example, proteins involved in mRNA processing have also been identiﬁed, as in the previous studies. These identiﬁed polypeptides, which function in various cellular processes including DNA-replication, transcription, recombination and repair, post-transcriptional modiﬁcation, etc., are the focus of our future studies. In summary, our present study employed a nuclear proteomic method to analyze the nuclear protein expression proﬁles in response to treatment with various concentrations of BaP. A number of proteins were identiﬁed, several of which suggest that
C. Yan et al. / Mutation Research 731 (2012) 75–84
nuclear proteasome activity might play an important role in the BaP-induced DDR. These data indicate that proteomics-based 2D gel electrophoresis assay can serve as a valuable technique in the mechanistic study of the cellular response to environmental chemicals. Conﬂict of interest The authors declare that there are no conﬂicts of interest. Acknowledgements This work was supported by grants from National Natural Science Foundation of China (No. 81172692); Zhejiang Provincial Natural Science Foundation (R2100555); and Ministry of Science and Technology, China (2009DFB30390). J. Yang is a recipient of the Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents. References  K. Straif, R. Baan, Y. Grosse, B. Secretan, F. El Ghissassi, V. Cogliano, Carcinogenicity of polycyclic aromatic hydrocarbons, Lancet Oncol. 6 (2005) 931–932.  E.G. Hvastkovs, M. So, S. Krishnan, B. Bajrami, M. Tarun, I. Jansson, J.B. Schenkman, J.F. Rusling, Electrochemiluminescent arrays for cytochrome P450-activated genotoxicity screening. DNA damage from benzo[a]pyrene metabolites, Anal. Chem. 79 (2007) 1897–1906.  M. Takaishi, M. Sawada, A. Shimada, J.S. Suzuki, M. Satoh, H. Nagase, Protective role of metallothionein in benzo[a]pyrene-induced DNA damage, J. Toxicol. Sci. 34 (2009) 449–458.  L.J. Lu, R.M. Disher, M.V. Reddy, K. Randerath, 32P-postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4-aminobiphenyl, and benzo(a)pyrene, Cancer Res. 46 (1986) 3046–3054.  A. Tarantini, A. Maitre, E. Lefebvre, M. Marques, C. Marie, J.L. Ravanat, T. Douki, Relative contribution of DNA strand breaks and DNA adducts to the genotoxicity of benzo[a]pyrene as a pure compound and in complex mixtures, Mutat. Res. 671 (2009) 67–75.  S.Y. Park, S.M. Lee, S.K. Ye, S.H. Yoon, M.H. Chung, J. Choi, Benzo[a]pyreneinduced DNA damage and p53 modulation in human hepatoma HepG2 cells for the identiﬁcation of potential biomarkers for PAH monitoring and risk assessment, Toxicol. Lett. 167 (2006) 27–33.  J.J. Mukherjee, S.K. Gupta, S. Kumar, H.C. Sikka, Effects of cadmium(II) on (±)anti-benzo[a]pyrene-7,8-diol-9,10-epoxide-induced DNA damage response in human ﬁbroblasts and DNA repair: a possible mechanism of cadmium’s cogenotoxicity, Chem. Res. Toxicol. 17 (2004) 287–293.  J. Yang, X. Liu, P. Niu, Y. Zou, Z. Gong, J. Yuan, T. Wu, Dynamic changes of XPA, XPC, XPF, XPG and ERCC1 protein expression and their correlations with levels of DNA damage in human bronchial epithelia cells exposed to benzo[a]pyrene, Toxicol. Lett. 174 (2007) 10–17.  M. Christmann, M.T. Tomicic, D. Aasland, N. Berdelle, B. Kaina, Three prime exonuclease I (TREX1) is Fos/AP-1 regulated by genotoxic stress and protects against ultraviolet light and benzo(a)pyrene-induced DNA damage, Nucleic Acids Res. 38 (2010) 6418–6432.  A. Wang, J. Gu, K. Judson-Kremer, K.L. Powell, H. Mistry, P. Simhambhatla, C.M. Aldaz, S. Gaddis, M.C. MacLeod, Response of human mammary epithelial cells to DNA damage induced by BPDE: involvement of novel regulatory pathways, Carcinogenesis 24 (2003) 225–234.  S.L. Hockley, V.M. Arlt, D. Brewer, R. Te Poele, P. Workman, I. Giddings, D.H. Phillips, AHR- and DNA-damage-mediated gene expression responses induced by benzo(a)pyrene in human cell lines, Chem. Res. Toxicol. 20 (2007) 1797–1810.  Z. Gao, J. Jin, J. Yang, Y. Yu, Zinc ﬁnger proteins and other transcription regulators as response proteins in benzo[a]pyrene exposed cells, Mutat. Res. 550 (2004) 11–24.  W. Shen, H. Liu, Y. Yu, Proteomic analysis of cellular responses to different concentrations of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide in human amniotic epithelial FL cells, J. Proteome Res. 6 (2007) 4737–4748.  W. Shen, H. Liu, Y. Yu, Translation initiation proteins, ubiquitin–proteasome system related proteins, and 14-3-3 proteins as response proteins in FL cells exposed to anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, Proteomics 8 (2008) 3450–3468.  P. Zhao, W. Zhong, X. Ying, B. Yao, Z. Yuan, J. Fu, Z. Zhou, Comparative proteomic analysis of anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-transformed and normal human bronchial epithelial G0/G1 cells, Chem. Biol. Interact. 186 (2010) 166–173.  L. Min, S. He, Q. Chen, F. Peng, H. Peng, M. Xie, Comparative proteomic analysis of cellular response of human airway epithelial cells (A549) to benzo(a)pyrene, Toxicol. Mech. Methods 21 (2011) 374–382.
 C. Yan, W. Wu, H. Li, G. Zhang, P.J. Duerksen-Hughes, X. Zhu, J. Yang, Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing, Mutat. Res. 686 (2010) 47–56.  W. Wu, C. Yan, T. Gan, Z. Chen, X. Lu, P.J. Duerksen-Hughes, X. Zhu, J. Yang, Nuclear proteome analysis of cisplatin-treated HeLa cells, Mutat. Res. 691 (2010) 1–8.  M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254.  G. Sun, X. Xu, Y. Wang, X. Shen, Z. Chen, J. Yang, Mycoplasma pneumoniae infection induces reactive oxygen species and DNA damage in A549 human lung carcinoma cells, Infect. Immun. 76 (2008) 4405–4413.  C. Yan, J. Lu, G. Zhang, T. Gan, Q. Zeng, Z. Shao, P.J. Duerksen-Hughes, J. Yang, Benzo[a]pyrene induces complex H2AX phosphorylation patterns by multiple kinases including ATM, ATR, and DNA-PK, Toxicol. In Vitro 25 (2011) 91–99.  F. Hirata, L.M. Thibodeau, A. Hirata, Ubiquitination and SUMOylation of annexin A1 and helicase activity, Biochim. Biophys. Acta 1800 (2010) 899–905.  S. Nair, M.P. Hande, L.H. Lim, Annexin-1 protects MCF7 breast cancer cells against heat-induced growth arrest and DNA damage, Cancer Lett. 294 (2010) 111–117.  C.M. Pickart, R.E. Cohen, Proteasomes and their kin: proteases in the machine age, Nat. Rev. Mol. Cell Biol. 5 (2004) 177–187.  Y. Chen, H.H. Li, J. Fu, X.F. Wang, Y.B. Ren, L.W. Dong, S.H. Tang, S.Q. Liu, M.C. Wu, H.Y. Wang, Oncoprotein p28 GANK binds to RelA and retains NF-kappaB in the cytoplasm through nuclear export, Cell Res. 17 (2007) 1020–1029.  H. Ling, J.M. Sayer, B.S. Plosky, H. Yagi, F. Boudsocq, R. Woodgate, D.M. Jerina, W. Yang, Crystal structure of a benzo[a]pyrene diol epoxide adduct in a ternary complex with a DNA polymerase, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 2265–2269.  F. Perera, D. Tang, R. Whyatt, S.A. Lederman, W. Jedrychowski, DNA damage from polycyclic aromatic hydrocarbons measured by benzo[a]pyrene-DNA adducts in mothers and newborns from Northern Manhattan, the World Trade Center Area, Poland, and China, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 709–714.  F. Kassie, L.B. Anderson, R. Scherber, N. Yu, D. Lahti, P. Upadhyaya, S.S. Hecht, Indole-3-carbinol inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone plus benzo(a)pyrene-induced lung tumorigenesis in A/J mice and modulates carcinogen-induced alterations in protein levels, Cancer Res. 67 (2007) 6502–6511.  L.H. Lim, S. Pervaiz, Annexin 1: the new face of an old molecule, FASEB J. 21 (2007) 968–975.  M.E. Burczynski, T.M. Penning, Genotoxic polycyclic aromatic hydrocarbon ortho-quinones generated by aldo-keto reductases induce CYP1A1 via nuclear translocation of the aryl hydrocarbon receptor, Cancer Res. 60 (2000) 908–915.  R. Debret, H. El Btaouri, L. Duca, I. Rahman, S. Radke, B. Haye, J.M. Sallenave, F. Antonicelli, Annexin A1 processing is associated with caspase-dependent apoptosis in BZR cells, FEBS Lett. 546 (2003) 195–202.  E. Solito, C. de Coupade, S. Canaider, N.J. Goulding, M. Perretti, Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation, Br. J. Pharmacol. 133 (2001) 217–228.  C.W. D’Acunto, B. Fontanella, M. Rodriquez, M. Taddei, L. Parente, A. Petrella, Histone deacetylase inhibitor FR235222 sensitizes human prostate adenocarcinoma cells to apoptosis through up-regulation of Annexin A1, Cancer Lett. 295 (2010) 85–91.  M. Scannell, M.B. Flanagan, A. deStefani, K.J. Wynne, G. Cagney, C. Godson, P. Maderna, Annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages, J. Immunol. 178 (2007) 4595–4605.  E. Solito, A. Kamal, F. Russo-Marie, J.C. Buckingham, S. Marullo, M. Perretti, A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils, FASEB J. 17 (2003) 1544–1546.  H.G. Wang, N. Pathan, I.M. Ethell, S. Krajewski, Y. Yamaguchi, F. Shibasaki, F. McKeon, T. Bobo, T.F. Franke, J.C. Reed, Ca2+ -induced apoptosis through calcineurin dephosphorylation of BAD, Science 284 (1999) 339–343.  Y.L. Wu, X.R. Jiang, D.M. Lillington, A.C. Newland, S.M. Kelsey, Upregulation of lipocortin 1 inhibits tumour necrosis factor-induced apoptosis in human leukaemic cells: a possible mechanism of resistance to immune surveillance, Br. J. Haematol. 111 (2000) 807–816.  M. Carollo, L.N. Parente, D’Alessandro, Dexamethasone-induced cytotoxic activity and drug resistance effects in androgen-independent prostate tumor PC-3 cells are mediated by lipocortin 1, Oncol. Res. 10 (1998) 245–254.  M. Ishido, Overexpression of Bcl-2 inhibits nuclear localization of annexin I during tumor necrosis factor-alpha-mediated apoptosis in porcine renal LLCPK1 cells, Regul. Pept. 124 (2005) 45–51.  Y.S. Kim, J. Ko, I.S. Kim, S.W. Jang, H.J. Sung, H.J. Lee, S.Y. Lee, Y. Kim, D.S. Na, PKCdelta-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate, Eur. J. Biochem. 270 (2003) 4089–4094.  M. Sakaguchi, H. Murata, H. Sonegawa, Y. Sakaguchi, J. Futami, M. Kitazoe, H. Yamada, N.H. Huh, Truncation of annexin A1 is a regulatory lever for linking epidermal growth factor signaling with cytosolic phospholipase A2 in normal and malignant squamous epithelial cells, J. Biol. Chem. 282 (2007) 35679–35686.  D. Ju, L. Wang, X. Mao, Y. Xie, Homeostatic regulation of the proteasome via an Rpn4-dependent feedback circuit, Biochem. Biophys. Res. Commun. 321 (2004) 51–57.
C. Yan et al. / Mutation Research 731 (2012) 75–84
 J. Wang, X.F. Wang, L.G. Zhang, S.Y. Xie, Z.L. Li, Y.J. Li, H.H. Li, F. Jiao, Involvement of the mitochondrial pathway in p53-independent apoptosis induced by p28GANK knockdown in Hep3B cells, Cytogenet. Genome Res. 125 (2009) 87–97.  S. Chaves, C. Baskerville, V. Yu, S.I. Reed, Cks1, Cdk1, and the 19S proteasome collaborate to regulate gene induction-dependent nucleosome eviction in yeast, Mol. Cell. Biol. 30 (2010) 5284–5294.  K. Kominami, G.N. DeMartino, C.R. Moomaw, C.A. Slaughter, N. Shimbara, M. Fujimuro, H. Yokosawa, H. Hisamatsu, N. Tanahashi, Y. Shimizu, et al., Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae, EMBO J. 14 (1995) 3105–3115.  S. Jager, M. Groll, R. Huber, D.H. Wolf, W. Heinemeyer, Proteasome beta-type subunits: unequal roles of propeptides in core particle maturation and a hierarchy of active site function, J. Mol. Biol. 291 (1999) 997–1013.
 W. Heinemeyer, M. Fischer, T. Krimmer, U. Stachon, D.H. Wolf, The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing, J. Biol. Chem. 272 (1997) 25200–25209.  L.A. Elenich, D. Nandi, A.E. Kent, T.S. McCluskey, M. Cruz, M.N. Iyer, E.C. Woodward, C.W. Conn, A.L. Ochoa, D.B. Ginsburg, J.J. Monaco, The complete primary structure of mouse 20S proteasomes, Immunogenetics 49 (1999) 835–842.  X. Wang, J. Yen, P. Kaiser, L. Huang, Regulation of the 26S proteasome complex during oxidative stress, Sci. Signal. 3 (2010) ra88.  B.L. Andreson, A. Gupta, B.P. Georgieva, R. Rothstein, The ribonucleotide reductase inhibitor, Sml1, is sequentially phosphorylated, ubiquitylated and degraded in response to DNA damage, Nucleic Acids Res. 38 (2010) 6490–6501.  S. Ben-Aroya, N. Agmon, K. Yuen, T. Kwok, K. McManus, M. Kupiec, P. Hieter, Proteasome nuclear activity affects chromosome stability by controlling the turnover of Mms22, a protein important for DNA repair, PLoS Genet. 6 (2010) e1000852.