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 Wenyan Shen, Hui Liu, and Yingnian Yu* Department of Pathophysiology, Zhejiang University, School of Medicine, Hangzhou 310058, China Received July 1, 2007
Benzo(a)pyrene is an ubiquitous environmental carcinogen produced during incomplete combustion of organic substances, and anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), is the most carcinogenic form of its ultimate metabolites. The goal of this study was to examine the response of human amniotic epithelial FL cells to increasing concentrations of BPDE and to find potential biomarkers involved in this cellular response. Human amniotic epithelial FL cells were incubated with 0.005, 0.05, and 0.5 µM BPDE to obtain protein extracts which were resolved by two-dimensional electrophoresis (2-DE) and visualized by silver staining. More than 60 protein spots significantly changed after BPDE exposure. Among these, 2 spots were detected only in the exposed group, and 36 spots were upregulated, while 27 spots were down-regulated. These altered spots were excised from the gels and analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS). The analysis led to the identification of 46 proteins affected by BPDE. These proteins were involved in regulation of transcription, cell cycle, cell proliferation, transport, signal transduction, metabolism, and so forth. However, no single protein changed in a dose-dependent manner in all three concentrations. Therefore, the changes of proteomic profiles cannot be considered as only an amplification of low-dose response in the case of high-dose exposure and the cellular responses to different doses of DNA damaging agent may be quite different. These results will aid our understanding of the mechanism of BPDE-induced cell response and provide the possibility of the establishment of potential biomarkers. Keywords: anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide • differentially expressed proteomic profile • two-dimensional electrophoresis • matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry
Introduction Benzo(a)pyrene, an environmental pollutant, is one of the polycyclic aromatic hydrocarbons (PAHs) and is generated by incomplete combustion of organic materials such as gasoline in motor vehicles, coal burning, cooking, and tobacco smoke.1,2 Benzo(a)pyrene is metabolically activated by microsomal enzymes to form the ultimate carcinogen benzo(a)pyrene-7,8dihydrodiol-9,10-epoxide (BPDE).3,4 anti-BPDE is the most carcinogenic form, and its electrophilic species is able to interact with nucleophilic sites on cellular DNA, RNA, and protein resulting in bulky-adduct damage.5 As a result, nucleotide excision repair, translesion DNA synthesis, activation of cell cycle checkpoint pathway, and alterations of gene expression are induced.6–13 Most work has focused on the mutations and carcinogenesis induced by BPDE. For instance, studies have shown that disruption of cellular signaling pathways involved in the regulation of growth and differentiation contributed to the * To whom correspondence should be addressed. Prof. Yingnian Yu, Department of Pathophysiology, Zhejiang University, School of Medicine, Hangzhou 310058, China. Tel/Fax: +86-571-88208209. E-mail: ynyu@ hzcnc.com. 10.1021/pr070406b CCC: $37.00
2007 American Chemical Society
toxicity of BPDE.14,15 Recent reports found that reduced DNA repair of BPDE-induced adducts was associated with an increased risk of breast cancer patients, and the risk may be modulated by XPD polymorphisms.16 “Hot spots” for BPDEinduced mutation exist in the p53 gene due to the preferential binding of BPDE to methylated or hemimethylated, rather than unmethylated CpG sites.17,18 However, diseases are not caused by mutations in a single gene or by exposure to a single environmental agent. Organisms have evolved sophisticated pathways to minimize the biological consequences of environmental insults. These pathways constitute the “environmental response machinery”. As a result, the efficiency of a person’s unique set of environmental response genes is decisive for the risk of developing an illness.19 Therefore, global analysis of cellular response to environmental stressors is strikingly important, and high-throughput technology is needed for such an analysis. Simultaneous measurement of thousands of genes facilitates the uncovering of specific gene expression patterns associated with cellular responses induced by agents such as BPDE. For example, Akerman et al. used an array of 350 human genes and found that those with altered expression after BPDE exposure were involved in cell cycle regulation, glutathione detoxification, apoptosis, and so forth.12 Journal of Proteome Research 2007, 6, 4737–4748 4737 Published on Web 11/01/2007
research articles Luo et al. detected the gene expression pattern using a printed cDNA microarray comprising ∼18 000 human gene/EST sequences and discovered that the altered genes included those involved in cell survival, cell growth, stress response, DNA repair, and so forth.20 However, it is clear that the functional end point of the genetic blueprint lies at the protein level, since cell function is directly regulated through proteins but not through genes or mRNA.21 Furthermore, there is no obvious linear correspondence between mRNA level and protein level.22 As a result, it is of vital importance to identify the changes in protein expression induced by a toxic insult. Proteomic analysis allows simultaneous monitoring of the expression of hundreds and even thousands of proteins in a sample following exposure to a toxicant. Oh et al. investigated the changes in protein expression profiles in Jurkat T-cells following benzo(a)pyrene exposure and found that proteins involved in apoptosis and tumor suppression were upregulated, while proteins involved in energy metabolism, DNA synthesis, and cell structure were down-regulated.23 In addition, we performed proteomic analysis following benzo(a)pyrene exposure in human amniotic epithelial FL cells stably transfected with human CYP1A1 full-length cDNA and identified more than 20 novel proteins that had not been previously connected to a cellular response to DNA-damaging agents involving bulky chemical adduct formation.24 However, there are few reports in proteomics using BPDE as a toxic reagent. In this study, we used the reactive metabolite of benzo(a)pyrene to eliminate the potential confounding effects of differential metabolic activation. The application of different concentrations enables us to further study dose-related effects in environment. Some studies have found that BPDE-induced toxicity and mutagenicity are affected by dose. For example, a dose-related decrease in cell viability was evident at 24 h in TK6 cells exposed to BPDE.12 In addition, exposure of HepG2 cells to BPDE-induced apoptosis was in a dose-dependent fashion, as assessed by DNA fragmentation and cytochrome c release.25 Another study showed that mutant fractions at the thymidine kinase and hypoxanthineguanine phosphoribosyl transferase loci increased with concentrations in BPDE-treated TK6 cells.20 However, responses independent of dose have also been reported. For instance, we found that 0.005 and 0.05 µM BPDE could trigger the endoplasmic reticulum stress in exposed cells, while 0.5 µM BPDE could not induce such a response.26 In these studies mentioned above, the end points used for analysis were all anchored to specific phenotypes; here, we focused on revealing the early cellular protein expression profiles at different concentrations of BPDE. In the present study, we analyzed differential proteomic profiles following BPDE exposure at three concentrations and found more than 60 proteins that showed significant changes. Among them, 46 were successfully identified by mass spectrometry. Further comprehensive characterization of these proteins will lead to a better understanding of the mechanisms of cellular response induced by BPDE, and assist in the development of biomarkers for environment prevention.
Materials and Methods Cell Culture and Treatment. Human amniotic epithelial cell line FL, an epithelial cell line established from human amnion with typical morphology and biomarker expression of epithelial cell was selected as model cell in this study. Serial research 4738
Journal of Proteome Research • Vol. 6, No. 12, 2007
Shen et al. works using this cell line in the in vitro studies of chemical hazardous effects were published. Cells were subcultured in Eagle’s minimum essential medium (MEM) containing 10% newborn bovine serum and supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin. The cultures were maintained in a humidified incubator under 5% CO2 at 37 °C. Before BPDE exposure (NCI Chemical Carcinogen Reference Standard Repository, Kansas City, KS), cells had reached logarithmic growth and all the treatment processes were protected from exposure to light, since BPDE is extremely light sensitive. BPDE was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution and obtained final concentrations of 0.005, 0.05, and 0.5 µM in serum-free MEM at a 1/1000 dilution. The high dose (0.5 µM) used in this study was the lowest BPDE concentration that reduced the cell viability significantly (relative survival of 95.2% ( 2.8% vs control of 100 ( 2.3%, p < 0.05). The medium dose (0.05 µM) was the highest concentration that did not reduce the viability of cells after exposure (relative survival of 99.8% ( 3.0% vs control of 100 ( 2.3%, p > 0.05). The two doses were selected from a cell cytoxicity test by MTT assay, and then another low dose (0.005 µM) was settled. An equal volume of DMSO was added to control cultures. After 2.5 h incubation, the treatment medium was removed, and the cells were rinsed with phosphatebuffered saline (PBS) and then returned to the incubator in fresh whole medium at 37 °C for another 12 h, followed by cell lysis. Sample Preparation. The cells were harvested with 0.02% EDTA and 0.025% trypsin, washed with chilled PBS, and pelleted by centrifugation. The resulting pellets were suspended and lysed in a buffer consisting of 8 M urea, 4% CHAPS, 40 mM Tris base, 2% ampholine, and 65 mM dithiothreitol (DTT), then placed on ice for 1 h. After sonication, the homogenates were centrifuged at 20 000g for 60 min at 4 °C to remove debris. The remaining supernatant was collected, and protein concentration was measured using the Bradford assay.27 Aliquots of protein samples were kept at -70 °C until use. For each group (control or BPDE-treated), three protein samples, obtained from three independent experiments, were prepared and then run independently on analytical 2-DE gels. Two-Dimensional Gel Electrophoresis. 2-DE was generally performed according to the manufacturer’s instructions (Amersham Biosciences, Uppsala, Sweden). In the first dimension, 300 µg of total protein was mixed with a rehydration solution containing 8 M urea, 2% CHAPS, 0.5% immobilized-pHgradient (IPG) buffer (pH 4–7), 18 mM DTT, and a trace of bromophenol blue, to a total volume of 450 µL. Isoelectric focusing (IEF) was carried out using an IPGphor apparatus (Amersham Biosciences) and pH 4–7, 24 cm Immobiline Drystrips (Amersham Biosciences). The focusing protocol was as follows: 50 V active rehydration for 12 h, 1 h at 200 V, 1 h at 500 V, 1 h at 1000 V, 30 min at 1000–8000 V, and 8.5 h at 8000 V, for a total of 70 000–72 000 Vh. Once the IEF was completed, the individual strips were equilibrated for 15 min in an equilibration buffer (50 mM Tris-HCl [pH 8.8], 6 M urea, 30% glycerol, 2% SDS, and a trace of bromophenol blue) containing 1% DTT, with an additional incubation in the same buffer containing 2.5% iodoacetamide for another 15 min. After equilibration, proteins were separated in the second dimension on vertical 12.5% SDS gels using the ETTAN DALT II electrophoresis platform (Amersham Biosciences) with the IPG strips mounted on the top of the gels. SDS-PAGE was run at 2.5 W/gel
5.17
8.16
66198
62255
914
1094
5.14
8.07
8.30
5.64
5.63
4.68
7.76
59492
73028
442
4.66
630
46353
1358
8.04
65678
41413
1294
5.09
336
27248
584
6.41
70248
100418
1206
5.21
1312
169351
1556
9.17
33759
89573
1398
8.65
666
120663
1326
4.29
27871
85708
1324
8.88
36524
94251
698
8.63
492
62290
552
4.74
8.96
9.14
pI
648
65265
31728
234
45388
30
548
MW (Da)
index no.
22/134
8/17
21/76
22/173
21/105
9/34
11/16
13/89
12/47
16/99
10/64
10/25
10/21
23/54
25/80
20/65
25/136
16/52
12/40
10/79
10/27
11/42
peptide matchesa
49
12
30
36
33
30
35
50
29
45
36
49
16
16
40
25
30
26
33
45
20
37
cov (%)b
105
80
113
85
93
80
154
80
69
94
71
135
72
71
86
74
70
70
70
74
66
68
scores
keratin 9
keratin 1
keratin 2 (epidermal ichthyosis bullosa of Siemens) keratin 10
beta-transducin repeat containing protein isoform 1
annexin IV
stratifin (14–3–3 protein sigma) annexin A3
protein kinase C, delta
secernin 1
chloride intracellular channel 1 GDP dissociation inhibitor 2
synapse-associated protein 102 isoform a
microfilament and Actin filament cross-linker protein isoform a
zinc finger protein 268 B
zinc finger protein 658
HIV TAT specific factor 1
zinc finger protein 658B
DNA directed RNA polymerase II polypeptide C zinc finger protein 570
zinc finger protein 658
zinc finger protein 268
description (protein name)
Mascot search results
coding gene symbol
SCRN1
GDI2
NP_006112 NP_000217
KRT9
KRT1
KRT10
KRT2
Development NP_000414 NP_000412
BTRC
ANXA4
ANXA3
NP_378663
NP_001144
NP_005130
Signal transduction NP_997704 PRKCD NP_006245 NP_006133 SFN
NP_055581
NP_001485
0.0180 ( 0.0113
0.0970 ( 0.0135 (0.6245) V 0.0280 ( 0.0090 (induced)
0.1553 ( 0.0268
0.0640 ( 0.0286 (0.6400) V
0.9143 ( 0.2909 (2.6000) v
0.0473 ( 0.0076 (1.9722) v
0.9587 ( 0.0702 (1.7430) v
0.0413 ( 0.0093 (1.6104) v
0.0123 ( 0.0015 (0.5606) V
0.05 µM
0.3310 ( 0.0694 (1.4186) v
0.0663 ( 0.0168 (3.6852) v
0.3553 ( 0.0571 (0.7111) V
0.0497 ( 0.0166 (4.1389) v
0.0207 ( 0.0047 (0.7045) V
0.3083 ( 0.0588 (0.6459)V 0.0857 ( 0.0234 (0.5724) V 0.0590 ( 0.0087 (2.2692) v
0.005 µM
0.2333 ( 0.0542
0.0170 ( 0.0087
0.1000 ( 0.0358
0.4997 ( 0.0872
0.3517 ( 0.0840
0.1977 ( 0.0283
0.0240 ( 0.0010
0.0120 ( 0.0056
0.5500 ( 0.1424
CLIC1
Transport NP_001279
0.0257 ( 0.0055
0.0173 ( 0.0006
MACF1
Cell cycle NP_036222
0.0220 ( 0.0017
0.1363 ( 0.0302
0.0293 ( 0.0049
0.1000 ( 0.0430
0.0430 ( 0.0341
0.0260 ( 0.0026
0.1497 ( 0.0220
0.4773 ( 0.0452
control
Cell proliferation NP_066943 DLG3
ZNF268
ZNF658
HTATSF1
ZNF658B
ZNF570
POLR2C
ZNF658
NP_694422
NP_149350
NP_055315
NP_001027468
NP_653295
NP_116558 NP_002685
NP_149350
Identified Protein Spots Regulation of transcription NP_003406 ZNF268
RefSeq no.
treatedc
Individual spot volume/total volume (%)
0.0867 ( 0.0342 (0.5579) V 0.0943 ( 0.0224 (induced)
0.0237 ( 0.0101 (1.3922) v
0.1047 ( 0.0168 (0.5295) V
0.0280 ( 0.0010 (2.3333) v
0.0340 ( 0.0056 (1.9615) v
0.2717 ( 0.0687 (1.9927) v
0.1140 ( 0.0592 (2.6512) v 0.0757 ( 0.0385 (0.7567) V
0.2403 ( 0.0812 (0.5035) V 0.0850 ( 0.0060 (0.5679) V
0.5 µM
Table 1. Summary of Protein Spots with Expression Changes in FL Cells Exposed to Different Concentrations of BPDE Classified According to Their Roles in Biological Processes
BPDE-Responsive Proteins
research articles
Journal of Proteome Research • Vol. 6, No. 12, 2007 4739
4740
Journal of Proteome Research • Vol. 6, No. 12, 2007
26257
17908
20981
99029
11514
13512
1690
1784
136
1402
1476
1704
36807
770
49894
34451
762
1406
34360
596
41536
16775
474
1308
21365
232
38096
17918
170
1260
31217
808
33746
59859
1720
917
38760
1334
52523
65678
1306
38387
58792
1120
834
66198
1114
840
MW (Da)
index no.
4.80
9.34
5.77
4.81
6.15
5.48
5.89
5.08
6.09
5.05
6.38
5.67
5.38
5.71
5.21
5.00
9.04
4.82
4.90
8.38
5.82
8.07
5.09
8.16
pI
Table 1. Continued
9/59
7/63
14/49
8/40
10/57
10/58
8/15
11/45
10/47
15/93
10/35
23/139
12/53
10/52
7/30
8/40
9/46
10/69
11/53
24/140
10/38
14/69
20/111
16/76
peptide matchesa
46
51
18
51
75
27
28
33
33
48
28
51
57
47
25
47
48
65
42
43
28
38
36
32
cov (%)b
83
67
73
88
105
88
97
96
74
114
97
131
99
89
67
79
83
64
89
90
82
90
108
96
scores
PREDICTED: hypothetical protein XP_379665 huntingtin interacting protein K
NP_001939
NP_005863
NP_005511
NP_003839
NP_003968
NP_006388
NP_002005
NP_000169
NP_000916
NP_000993
NP_003123
NP_005993
NP_057484
XP_379665
NP_003950
Function unknown NP_055070
huntingtin interacting protein-1-related
transmembrane protein 4
dUTP pyrophosphatase (nuclear isoform)
aryl hydrocarbon receptor interacting protein Succinate-CoA ligase, GDP-forming, beta subunit heterogeneous nuclear ribonucleoprotein H1 breast carcinoma amplified sequence 2
ribonuclease HI, large subunit
FK506 binding protein 4
pyruvate dehydrogenase (lipoamide) beta glutathione synthetase
ribosomal protein P0 variant
ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiolesterase) spermidine synthase
HYPK
C9orf109
HIP1R
TMEM4
DUT
BCAS2
HNRPH1
SUCLG2
AIP
RNASEH2A
FKBP4
GSS
PDHB
RPLP0
SRM
UCHL3
MRPL12
NDUFAB1
0.2843 ( 0.0225
0.0903 ( 0.0185
0.2677 ( 0.0481
0.3030 ( 0.0705
0.5247 ( 0.0499
0.2110 ( 0.0183
0.0847 ( 0.0631
0.0280 ( 0.0121
0.0447 ( 0.0225
0.0733 ( 0.0150
0.0163 ( 0.0055
0.1133 ( 0.0284
0.1803 ( 0.0307
0.2420 ( 0.0580
0.1127 ( 0.0329
0.1847 ( 0.0430
1.3763 ( 0.3841
0.9940 ( 0.0661
0.0793 ( 0.0278
NP_775109 Protein biosynthesis NP_001951 EEF1D
0.1780 ( 0.0342
0.0513 ( 0.0047
0.0423 ( 0.0126
0.3180 ( 0.0291
CAPG
Organization NP_001738
control
0.0500 ( 0.0296
KRT6C
KRT2
KRT10
KRT1
coding gene symbol
NP_000414
NP_000412
NP_006112
RefSeq no.
Metabolism NADH dehydrogenase (ubiquinone) 1, NP_004994 alpha/beta subcomplex, 1, 8 kDa mitochondrial ribosomal protein L12 NP_002940
Eukaryotic translation elongation factor 1 delta isoform 2
keratin 6C
gelsolin-like capping protein
keratin 2 (epidermal ichthyosis bullosa of Siemens)
keratin 10
keratin 1
description (protein name)
Mascot search results
0.1390 ( 0.0788 (0.4587) V 0.1537 ( 0.0330 (0.5741) V 0.0393 ( 0.0076 (0.4354) V 0.1907 ( 0.0397 (0.6706) V
0.7990 ( 0.1500 (1.5229)v
0.0733 ( 0.0172 (2.6190) v
0.0597 ( 0.0193 (0.5265) V
0.2050 ( 0.0503 (1.8195) v
0.0603 ( 0.0106 (1.4252) v 0.0757 ( 0.0085 (1.4740) v
0.005 µM
0.0373 ( 0.0199 (0.4133) V
0.1297 ( 0.0595 (1.5315) v
0.0917 ( 0.0168 (1.2500) v 0.0823 ( 0.0169 (1.8432) v
0.3047 ( 0.0535 (1.2590) v
0.8410 ( 0.0430 (0.8461)V
0.3270 ( 0.0656 (1.8370) v 0.1527 ( 0.0264 (0.4801) V
0.05 µM
treatedc
Individual spot volume/total volume (%)
0.5 µM
0.1217 ( 0.0377 (0.5766) V
0.0487 ( 0.0064 (2.9796) v
0.1450 ( 0.0281 (0.8041) V
0.8030 ( 0.3320 (0.5834) V 0.3373 ( 0.0219 (1.8267) v 0.1977 ( 0.0172 (1.7544) v
0.1610 ( 0.0400 (2.0294) v
0.2980 ( 0.0150 (1.6742) v
0.1707 ( 0.0579 (3.4133) v
research articles Shen et al.
29051
31009
34418
1596
1724
1756
4.52
5.25
6.14
4.73
4.87
4.71
5.79
6.23
5.79
5.63
4.52
5.53 5.67
5.54
5.98
6.24
5.31
4.79
5.26
pI
peptide matchesa
cov (%)b scores
RefSeq no.
coding gene symbol
Unidentified Protein Spots
description (protein name)
Mascot search results
0.0583 ( 0.0335
0.0133 ( 0.0084
0.0153 ( 0.0023
0.3680 ( 0.0663
0.3747 ( 0.0738
0.0850 ( 0.0436
0.0120 ( 0.0036
0.0287 ( 0.0087
0.0507 ( 0.0102
0.0223 ( 0.0120
0.0583 ( 0.0475
0.0853 ( 0.0529
0.0473 ( 0.0096
0.1557 ( 0.0032
0.1137 ( 0.0029
0.0367 ( 0.0101
0.1380 ( 0.0200
0.2807 ( 0.0397
control
0.2337 ( 0.1102 (0.6350) V
0.0083 ( 0.0029 (0.6944) V
0.0803 ( 0.0454 (1.3771) v
0.1710 ( 0.0201 (0.6093)V 0.2177 ( 0.0025 (1.5773)v 0.0587 ( 0.0180 (1.6000)v 0.0643 ( 0.0112 (0.5660)V
0.005 µM
0.0767 ( 0.0350 (1.3143)v
0.1597 ( 0.0657 (1.8784) v 0.5097 ( 0.1112 (1.3603) v
0.0710 ( 0.0281 (3.1791) v 0.0593 ( 0.0107 (1.1711)v 0.0143 ( 0.0038 (0.5000) V
0.2987 ( 0.0541 (1.9186) v 0.0357 ( 0.0067 (0.7535) V
0.05 µM
treatedc
Individual spot volume/total volume (%)
a Number of peptides that matched those from the nrNCBI database entry and number of peptides searched. b Percentage of the protein sequence covered by the matched peptides. parentheses denotes the ratio of the relative spot volume of treated and control (treated/control); the upward arrow indicates up-regulation, and the downward arrow indicates down-regulation.
20369
90520
1434
1550
39808
1228
17246
39538
1168
1482
32685
1154
18058
29721
962
1466
34058 21163
915 940
31184
700
44741
35699
592
43870
41367
538
754
18901
122
790
MW (Da)
index no.
Table 1. Continued
c
The number in
0.1897 ( 0.0780 (0.5154) V 0.0407 ( 0.0038 (2.6522) v 0.0490 ( 0.0095 (3.6750)v
0.0180 ( 0.0072 0.0703 ( 0.0513 (0.8242) V
0.0743 ( 0.0140 (0.6540)V 0.2897 ( 0.0126 (1.8608) v
0.5 µM
BPDE-Responsive Proteins
research articles
Journal of Proteome Research • Vol. 6, No. 12, 2007 4741
research articles for 30 min, followed by 15 W/gel until the bromophenol blue front reached the bottom of the gel. Silver Staining and Image Analysis. Silver staining was selected in this study, because it is one of the most sensitive staining methods (200 pg, linear in the range of 0.04–2 ng/mm2) with compatibility to MS and well-developed standardized protocols. It is currently still the most popular and one of the first recommended methods in proteomics research. It was performed following the 2-DE protocol (Amersham Biosciences) with some modifications. Briefly, the gels were fixed for 30 min in 40% (v/v) ethanol and 10% (v/v) acetic acid and sensitized in a solution containing 30% ethanol, 6.8% (w/v) sodium acetate, and 0.2% (w/v) sodium thiosulfate for 30 min. After a distilled water wash, the gels were stained in 0.25% (w/v) silver nitrate solution for 20 min. To remove excess silver nitrate, the gels were then washed twice in water for 1 min each. Development was performed in a solution consisting of 2.5% (w/v) sodium carbonate (anhydrous) and 0.04% (v/v) formaldehyde until the spots achieved the desired intensity. Termination solution containing 1.46% EDTA was used to stop the development, and the stained gels were then washed three times in water for 5 min each. The stained gels were scanned in an UMAX PowerLook1000 Imaging system (Taiwan) and analyzed with Phoretix 2D 6.01 analysis software (Nonlinear Dynamic, Durham, NC). Image analysis included spot detection, spot editing, background subtraction, spot matching, and normalization. The resulting data were exported to Microsoft Excel, and statistical significance was evaluated using Student’s t test. In-Gel Digestion. Spots of interest were manually cut from the 2D gels with a scalpel and transferred to siliconized Eppendorf tubes. The pieces of gel were washed with water and destained in freshly prepared destaining solution (30 mM potassium hexacyanoferrate: 100 mM sodium thiosulfate, 1:1 [v/v]) for 1–2 min until the brown color disappeared. They were rinsed with water and equilibrated in 100 mM ammonium bicarbonate for 5 min. The pieces were then dehydrated with 100% acetonitrile and dried in a vacuum centrifuge (SpeedVac, Thermo Savant, Holbrook, NY) for approximately 20 min. Subsequently, the pieces were covered with a digestion buffer containing 40 mM ammonium bicarbonate, 9% acetonitrile, and 20 µg/mL trypsin (Sigma, proteomics sequencing grade) in an ice-cold bath. After 30 min, the supernatant was removed and replaced with the same buffer but without trypsin and incubated overnight at 37 °C. The liquid was collected, and peptides were extracted twice by adding 5% trifluoroacetic acid (TFA)/50% acetonitrile for 15 min at room temperature, and dried in a vacuum centrifuge. MALDI-TOF Mass Spectrometry Analysis and Database Searching. The peptide extracts were reconstituted in 10 µL of 0.1% TFA. Peptide mixtures (1 µL) were then mixed with an equal volume of 10 mg/mL R-cyano-4-hydroxycinnamic acid (Sigma) saturated with 50% acetonitrile/0.1% TFA and spotted onto a MALDI target plate. The samples were analyzed with a Voyager-DE STR MALDI-TOF mass spectrometer using a delayed ion extraction and ion mirror reflector mass spectrometer (Applied Biosystems, Foster City, CA). The instrument setting was reflector mode with 160 ns delay extraction time, positive polarity, 60–65% grid voltage, and 20 000 V accelerating voltage. Laser shots of 200 per spectrum were used to acquire spectra with a mass range of 1000–4000 Da. The spectra were calibrated externally using P14R and insulin chain B oxidized from bovine pancreas (Sigma). Autolytic peaks of trypsin served as internal standards for mass calibration. For interpretation 4742
Journal of Proteome Research • Vol. 6, No. 12, 2007
Shen et al. of the mass spectra, the monoisotopic peptide mass fingerprinting (PMF) data were used to search for candidate proteins using MASCOT (http://www.matrixscience.com) software. The database (NCBInr) searches were performed using the following parameters: Homo sapiens, trypsin digest (allowed up to 1 missed cleavage), cysteines modified by carbamidomethylation, methionine modified by oxidation, and maximal mass tolerance of 100 ppm. Probability scores greater than 63 were defined as significant (p < 0.05). In cases of low score matching, duplicate or triplicate runs were made to ensure an accurate analysis. Classification of the Identified Proteins. The identified proteins were classified according to their roles in biological process by searching Gene Ontology (http://www.geneontology. org/). In addition, cellular compartments distribution and molecular functions were included. Western Blot Analysis. Aliquots of supernatant containing 50 µg of protein were mixed with equal volumes of 2× sample buffer. The samples were boiled for 5 min and subjected to 12.5% SDS-PAGE. After electrophoresis, the resolved proteins were transferred to nitrocellulose membrane. Membranes were blocked at room temperature for 2 h in TBST (50 mmol/L TrisCl, pH 7.6, 150 mmol/L NaCl, and 0.1% Tween 20) containing 5% nonfat milk to prevent nonspecific binding of reagents, and then incubated with the corresponding antibody (1:1000 dilution) on ice overnight. The membranes were washed three times with TBST for 15 min and incubated with HRPconjugated secondary antibody (1:1500 dilution) for 1 h at room temperature. After that, the membranes were again washed three times in TBST. Then, target protein was detected by ECL. Equal protein loading was confirmed by reprobing the same membranes after stripping with the anti-actin antibody.
Results and Discussion Three pairs of protein samples obtained from each group of BPDE-treated or control cells were subjected to 2-DE, and quantitative spot comparisons were made with the image analysis software. A representative gel image from the control group with silver stain was shown in Figure 1. On the basis of the analysis with Phoretix 2D 6.01 software, 1216 ( 31 protein spots in control, and 1231 ( 58, 1194 ( 33, and 1201 ( 62 spots in cells treated with 0.005, 0.05, and 0.5 µM BPDE, respectively, were detected. Two protein spots were detected only after BPDE exposure (spot 914 in 0.05 and 0.5 µM; spot 915 in 0.5 µM; Figure 2) and no spots were only detected in control group. Moreover, statistical analysis indicated that spot volumes of additional 63 proteins were significantly changed (P < 0.05), among which 37 (11 in 0.005, 16 in 0.05, and 14 in 0.5 µM) were up-regulated and 26 (13 in 0.005, 8 in 0.05, and 11 in 0.5 µM) were down-regulated following BPDE exposure. There was no individual protein spot with altered expression in all three concentrations; however, 10 spots (spots 30, 234, 596, 630, 700, 754, 1294, 1334, 1476, and 1550) showed significant changes in cells exposed to two concentrations of BPDE. Although the proteins that were altered by two treatments all changed in the same direction, no clear dose–response relationship was found. Among the 65 protein spots with expression changes, 46 were successfully identified by MALDI-TOF-based PMF. The remaining 19 protein spots could not be identified, mainly because their abundance was too low to produce a spectrum, or because the database search score was not high enough to yield unambiguous results. The knowledge of relevant genes encoding these proteins was obtained by GeneCards database searching. These identi-
BPDE-Responsive Proteins
Figure 1. 2-DE map indicating protein spots which changed in volume after BPDE exposure in human amniotic epithelial FL cells. Whole cell soluble proteins were separated by 2-DE and visualized by silver staining. Circles indicate proteins only detected in BPDE-treated FL cells; upward- or downward-pointing arrows indicate proteins that were up-regulated or downregulated by BPDE, respectively. The number beside the spot is the index number in the reference gel. The gel is a representation of three independent experiments.
Figure 2. Magnified images of two protein spots detected only in BPDE-treated cells. The arrow indicates the detected protein; “ _” stands for the protein not detected in the control.
fied proteins represented a heterogeneous group and took part in a variety of cellular processes, such as regulation of transcription, cell cycle, cell proliferation, transport, signal transduction, development, organization, metabolism, and unknown functions (Table 1). One induced spot (spot no. 914), which appeared in both 0.05 and 0.5 µM BPDE-exposed cells, had a molecular mass of around 40 kDa on our 2D gels and was identified as a fragment of keratin 1. Among the identified proteins, several categories of proteins seemed of particular interest in the cellular responses to BPDE exposure in human amniotic epithelial cells. Proteins Related to Cytoskeleton. In the current study, the expression levels of keratin 1 (spot no. 1114), keratin 2 (spot nos. 336 and 1306), keratin 6C (spot no. 1720), keratin 9 (spot no.1094), and keratin 10 (spot nos. 630 and 1120) were modulated (Figure 3 and Table 1). Similarly, in Jurkat T-cells treated with benzo(a)pyrene, two cytoskeletal proteins (Actinrelated protein and capping protein [Actin filament]) were down-regulated.23 Keratins make up the largest subgroup of intermediate filament (IF) proteins and are the most abundant proteins in epithelial cells. The major role of keratins is to act as a scaffold that endows epithelial cells with the ability to
research articles
Figure 3. Magnified images of protein spots involved in structural constituents. The oblique arrows indicate the protein spots in control samples and the upward- or downward-pointing arrows indicate proteins that were up-regulated or down-regulated by BPDE, respectively.
sustain mechanical and nonmechanical stresses. In addition to providing structural support, other emerging functions include roles in cell signaling, stress response and apoptosis, regulating the availability of other abundant cellular proteins, and unique roles that are keratin-specific and tissue-specific.28,29 An intact IF network is important in imparting protection to hepatocytes from griseofulvin and microcystin-LR induced stress, as well as Fas and tumor necrosis factor (TNF)-mediated apoptosis.30,31 As for keratin 1, endothelial oxidative stress increased its expression and activated the lectin complement pathway, suggesting a role in the immune response.32 The protein expression level of keratin 9 was elevated in harringtonine-induced K562 apoptosis, which may be related to apoptosis resistance,33 With the use of cultured cells and transgenic mouse models, experiments demonstrated that the expression of keratin 10 inhibited cell cycle progression through its ability to bind and inhibit the activation of Akt and PKC zeta.34–36 Little is known about the specific functions of keratins 2 and 6C except for their structural support role. In addition, a protein (spot no. 914) identified as a fragment of keratin 1 appeared in FL cells treated with 0.05 and 0.5 µM BPDE. It is known that keratins 14, 15, 17–19, and 20 can be degraded by caspase, while keratin 1 is not a good caspase substrate in vitro and in vivo because it lacks the L12 caspase motif.29 Interestingly, a 35 kDa fragment of keratin 1 and its autoantibody were detected in serum and tumor tissue in papillary thyroid carcinoma patients, indicating that collapse of the cytoskeleton may be involved.37 However, the occurrence and significance of the fragment in the present study remains to be determined. Proteins Relevant to Transcription Regulation. Among the differentially expressed proteins, several were relevant to Journal of Proteome Research • Vol. 6, No. 12, 2007 4743
research articles
Figure 4. Magnified images of protein spots involved in regulation of transcription. The oblique arrows indicate the protein spots in control samples, and the upward- or downward-pointing arrows indicate proteins that were up-regulated or downregulated by BPDE, respectively.
transcription regulation, such as zinc finger protein (ZNF) 268 (spot no. 30), ZNF570 (spot no. 552), ZNF658 (spot nos. 234 and 1326), and HIV TAT specific factor 1 (spot no. 1324) (Figure 4 and Table 1). Zinc finger proteins are implicated in both transcriptional regulation and RNA processing.38 They are also involved in developmental and malignant disorders.39 In the current study, ZNF268 was down-regulated after 0.005 and 0.5 µM BPDE exposure, while ZNF268B (spot no.1398) was downregulated after 0.05 µM BPDE; ZNF658B (spot no. 698) was down-regulated after 0.5 µM BPDE treatment, while ZNF658 and ZNF570 were up-regulated after the same exposure. In addition, spot 234, which was down-regulated after 0.005 and 0.5 µM BPDE exposure, was identified as a fragment of ZNF658, suggesting that the up-regulation of ZNF658 may be in part due to the decreased fragmentation of this protein. Consistently, we previously found changes in 8 zinc finger proteins in benzo(a)pyrene-exposed cells.24 Other reports also showed that BPDE can alter the expression level of zinc finger proteins.13,20 Furthermore, alteration of ZNF268 expression was also detected in BPDE-treated TK6 cells using cDNA microarray technology.20 ZNF268 plays a role in early human liver development and the differentiation of blood cells.40,41 It has two splice variants, which have different expression patterns. ZNF268–1 is expressed in normal tissues, whereas ZNF268–2 is expressed in chronic lymphocytic leukemia.41 Little is known about the specific functions of ZNF658, ZNF658B, and ZNF570. Proteins Involved in Protein Metabolism. The expression of several proteins involved in protein metabolism, such as eukaryotic translation elongation factor 1 delta (EEF1D; spot no. 808), ubiquitin carboxyl-terminal esterase L3 (ubiquitin thiolesterase; spot no. 474), mitochondrial ribosomal protein L12 (spot no. 232), and ribosomal protein P0 variant (spot no. 762) was also modulated after BPDE exposure (Figure 5 and Table 1). In this study, EEF1D was up-regulated after exposure to 0.5 µM BPDE. EEF1D is a subunit of eukaryotic translation elongation factor 1, a complex of proteins, which mediate the elongation step of protein synthesis by transferring aminoacyltRNA to 80S ribosomes. EEF1D is a radiation-inducible gene and may participate in G2/M checkpoint.42 It is also a cadmium4744
Journal of Proteome Research • Vol. 6, No. 12, 2007
Shen et al.
Figure 5. Magnified images of protein spots involved in protein metabolism. The oblique arrows indicate the protein spots in control samples, and the upward- or downward-pointing arrows indicate proteins that were up-regulated or down-regulated by BPDE, respectively.
Figure 6. Magnified images of protein spots of chloride intracellular channel 1 (CLIC1), stratifin (SFN) (also called 14-3-3 protein sigma), and aryl hydrocarbon receptor interacting protein (AIP). The oblique arrows indicate the protein spots in control samples, and the upward- or downward-pointing arrows indicate proteins that were up-regulated or down-regulated by BPDE, respectively.
responsive proto-oncogene and causes tumorigenic growth of NIH3T3 cells when overexpressed.43 However, the exact role of EEF1D in BPDE-induced cell response is still unknown, but dysregulated translation and cell cycle may be involved. Other Interesting Proteins. 1. Chloride Intracellular Channel 1 (CLIC1). In the current study, CLIC1 (spot no. 584) was up-regulated with 0.05 µM BPDE exposure (Figure 6 and Table 1). Previously, we have shown, by proteomic analysis, that CLIC1 was enhanced in FL-CYP1A1 cells after exposure to low concentration of benzo(a)pyrene,24 CLIC1 is a member of the chloride ion channel family. The functions of chloride ion channels range from ion homeostasis to cell volume regulation, trans-epithelial transport, and regulation of electrical excitability. They are also involved in modulation of cell cycle, apoptosis, cell adhesion, and cell motility.44 In addition, chloride ions catalyze the formation of cis adducts in the binding of BPDE to nucleic acids, and this may provide a link between CLIC1 and the effect of BPDE.45,46 2. 14-3-3 Protein. A member of 14-3-3 protein family, that is, the expression of stratifin (also called 14-3-3 protein sigma; spot no. 492), was elevated with 0.05 µM BPDE exposure (Figure 6 and Table 1), which was verified by Western blot (Figure 7).
BPDE-Responsive Proteins
research articles
Figure 7. (A) Detection of 14-3-3 protein sigma in FL cell lysates by Western blot. A total of 50 µg of protein from cell lysates was probed with antibody against 14-3-3 protein sigma. The same blot was striped and probed with anti-actin as loading control. (B) Densitometry analysis of 14-3-3 protein sigma expression. Band density was digitized, and mean ( SD of the relative band intensity of 14-3-3 protein sigma (14-3-3 protein sigma/Actin band intensity) from three independent experiments are shown. The point marked (*) indicates P < 0.05 in Student’s t test between 0.05 µM BPDE and DMSO-treated control.
Reports have suggested that 14-3-3 protein sigma may be a tumor suppressor of epithelial cells of multiple origins47,48 and is strongly up-regulated upon exposure to ionizing radiation and DNA-damaging agents in a p53-dependent pathway.49 It is responsible for sequestering the Cdc2–cyclin B1 complex in the cytoplasm and leads to G2 arrest. That is in consistence with the previous report of BPDE-induced G2/M arrest in HME87 cells.50 3. Aryl Hydrocarbon Receptor Interacting Protein (AIP). As a protein for xenobiotic metabolism, AIP (synonym: immunophilin-like X-associated protein 2; spot no. 1260), which was up-regulated following 0.05 µM BPDE exposure, attracted our attention (Figure 6 and Table 1). It is a core component of the aryl hydrocarbon receptor (AhR) complex, which is composed of a 95–105 kDa ligand binding subunit, a dimer of hsp90, and AIP. AIP is able to stabilize the unliganded cytoplasmic AhR and enhance its transcriptional activity.51,52 AhR is a ligandactivated member known to play a major role in toxic effects induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(a)pyrene.53 However, the exact function of AIP in the active metabolite of benzo(a)pyrene–BPDE-induced response remains to be discovered. 4. Glutathione Synthetase (GSS). GSS (spot no. 834) decreases with 0.005 µM BPDE exposure (Table 1); it catalyzes the second and final step in the biosynthesis of glutathione from glutamylcysteine and glycine.54 Intracellular glutathione plays an important role in cellular defenses against oxidative and nitrosative stress as well as against reactive electrophiles,55,56 so its synthesis has received much attention. PAHs decreased intracellular glutathione levels in a time- and dose-dependent manner in the A20.1 cell line, so glutathione depletion may contribute to the overall toxicity of PAHs to lymphocytes.57 Thus, the down-regulation of GSS in amniotic FL cells exposed to BPDE may be a general response to PAH compound. 5. Beta-Transducin Repeat Containing (BTRC). BTRC (spot no. 1312) was elevated by 0.5 µM BPDE exposure (Table 1). BTRC is a component of the SCF (SKP1–CUL1–F-box protein) ubiquitin ligase complex, which mediates the ubiquitination of proteins involved in cell cycle progression, signal transduction, and transcription.58,59 BTRC has a dual role in both Wnt and NF-kappaB signaling pathways, being a negative regulator of Wnt/beta-catenin signaling and a positive regulator of NFkappaB signaling. Interestingly, recent reports showed that BPDE up-regulates NF-kappaB activity and protein level in rat
astrocytes.60 Therefore, the relationship between BTRC and NFkappaB after BPDE exposure in epithelial cells remains to be studied. 6. Annexin III. One of our objects is to find proteins that may serve as new candidate biomarkers for monitoring the exposure of human populations to environmental carcinogens. Interestingly, a protein identified as annexin III (spot 648; Figure 8A and Table 1) exhibited a decreased intracellular content, while it increased in the culture medium after 0.005 µM BPDE exposure in an independent proteomics study (Figure 8B). Its significance and mechanism of action remain to be studied. Limitations and Perspectives. The change of proteome profile described here reflects only a part of the entire change actually happening in cells exposed to BPDE, because many of the protein changes identified by other methods were not found in this study. For example, the well-known increase of p53 protein in BPDE-treated cells was not seen in the present study.50,61 There are several possible reasons for this. First, p53 increases before the time point in our experimental design, and its downstream response occurs just at that time. It is a fact that the protein patterns of cellular response change in waves over time and are not fixed in its developing events, thus, the proteomic observations we found in this study were only one phase of the entire events; a timeresponse relation study is being carried out in this laboratory to validate this suspicion. Second, p53 may has changed at the protein level, but we did not capture it, which may be due to the limitations of 2-DE protocol with silver staining used in this study, that is, its poor dynamic range and bias toward abundant and soluble proteins; therefore, only a fraction of a given proteome can be analyzed. In addition to more efficient staining techniques, representation of lowabundance proteins can be significantly improved if prefractionation of the sample is employed before 2-D separation to reduce the complexity of protein constituents. Recently developed advanced high-resolution 2-DE systems allowing resolution of up to 10 000 protein spots of entire cell lysates in combination with protein identification by new highly sensitive mass spectrometric techniques may be more suitable for a high-throughput screening of different cell situations.62 Third, post-translational modification, especially phosphorylation of p53, is more important than its accumulation in response to DNA damage. However, phosJournal of Proteome Research • Vol. 6, No. 12, 2007 4745
research articles
Shen et al.
Concluding Remarks In conclusion, we have used proteomic technology to analyze global protein expression patterns with low, medium, and high doses of BPDE in human amniotic epithelial FL cells. Although a few proteins were altered in response to two of the concentrations, each dose modified the expression of a unique set of proteins. These results may shed new light on the underlying mechanisms of BPDE-induced cellular responses and also be useful for identification of candidate protein biomarkers of exposure to environmental chemical carcinogens.
Acknowledgment. This work is supported by the National Key Basic Research and Development Program (No. 2002CB512901) of China. We gratefully thank Professor Iain C Bruce (Department of Physiology at Zhejiang University) for his critical review of this manuscript. References Figure 8. Magnified images of intracellular and extracellular annexin III. (A) Down-regulation of annexin III in FL cell lysates after 0.005 µM BPDE exposure. Spot 648, which was downregulated in BPDE-treated group (p < 0.05), was identified as annexin III. (B) Up-regulation of annexin III in secretome of FL cell exposed to 0.005 µM BPDE by an independent proteomics study*. Spot 4304, which was up-regulated in the BPDE-treated group (p < 0.05), was identified as annexin III. *2-D gel electrophoresis and MALDI-TOF mass spectrometry were employed to analyze the proteins secreted/shed into the culture medium of cells exposed to 0.005 µM BPDE. The protein samples were prepared as follows: after 2.5 h exposure, the chemical-containing medium was removed. The cells were recovered in fresh medium containing calf serum at 37 °C for another 12 h. Then, after thorough PBS washing, the cells were further incubated in phenol red-free and serum-free MEM (Gibco) medium for another 6 h. The harvested medium was then filtered through Millipore filters (pore diameter 0.22 µm) and finally concentrated to approximately 0.5 mL for each group with a Centricon Plus-80 (Millipore, Bedford, MA). The proteins in the medium were precipitated with acetone at -20 °C overnight, and the pellets were resuspended and solubilized in 100 µL of lysis buffer. A total of 280 µg of protein samples was then subjected to 2DE-MS-based differential proteomic analysis.
phorylation is generally a substoichiometric reaction indicating that only a small fraction of a given protein is phosphorylated, so special methods need to be used, such as enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, phosphospecific stains, autoradiography, or immunoblotting.63,64 None of the responsive proteins found in this study in response to in vitro exposure to BPDE can be considered or identified as BPDE-specific. We do not believe that there is any BPDE-specific response protein in the cell. The responsive proteins identified in this manuscript after BPDE exposure consisted of unspecific response to cell stress and relative specific response induced by DNA- and protein bulky adduct forming chemicals. Additional parallel works were carried out in this laboratory with additional chemicals such as methylating agent and microcystin that have different properties from that of BPDE. Comprehensive analysis of these results may give valuable information concerning different early response proteins that play special roles against exposure to different types of hazardous chemicals. 4746
Journal of Proteome Research • Vol. 6, No. 12, 2007
(1) Phillips, D. H. Fifty years of benzo(a)pyrene. Nature 1983, 303 (5917), 468–72. (2) Bartsch, H.; Hietanen, E. The role of individual susceptibility in cancer burden related to environmental exposure. Environ. Health Perspect. 1996, 104 (3), 569–77. (3) Conney, A. H.; Chang, R. L.; Jerina, D. M.; Wei, S. J. Studies on the metabolism of benzo[a]pyrene and dose-dependent differences in the mutagenic profile of its ultimate carcinogenic metabolite. Drug Metab. Rev. 1994, 26 (1–2), 125–63. (4) Shimada, T.; Gillam, E. M.; Oda, Y.; Tsumura, F.; Sutter, T. R.; Guengerich, F. P.; Inoue, K. Metabolism of benzo[a]pyrene to trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene by recombinant human cytochrome P450 1B1 and purified liver epoxide hydrolase. Chem. Res. Toxicol. 1999, 12 (7), 623–9. (5) Boysen, G.; Hecht, S. S. Analysis of DNA and protein adducts of benzo[a]pyrene in human tissues using structure-specific methods. Mutat. Res. 2003, 543 (1), 17–30. (6) van Steeg, H. The role of nucleotide excision repair and loss of p53 in mutagenesis and carcinogenesis. Toxicol. Lett. 2001, 120 (1–3), 209–19. (7) Wani, M. A.; El-Mahdy, M. A.; Hamada, F. M.; Wani, G.; Zhu, Q.; Wang, Q. E.; Wani, A. A. Efficient repair of bulky anti-BPDE DNA adducts from non-transcribed DNA strand requires functional p53 but not p21(waf1/cip1) and pRb. Mutat. Res. 2002, 505 (1–2), 13– 25. (8) Zhao, B.; Wang, J.; Geacintov, N. E.; Wang, Z. Poleta, Polzeta and Rev1 together are required for G to T transversion mutations induced by the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts in yeast cells. Nucleic Acids Res. 2006, 34 (2), 417–25. (9) Zhang, Y.; Wu, X.; Guo, D.; Rechkoblit, O.; Geacintov, N. E.; Wang, Z. Two-step error-prone bypass of the (+)- and (-)-trans-antiBPDE-N2-dG adducts by human DNA polymerases eta and kappa. Mutat. Res. 2002, 510 (1–2), 23–35. (10) Shackelford, R. E.; Kaufmann, W. K.; Paules, R. S. Cell cycle control, checkpoint mechanisms, and genotoxic stress. Environ. Health Perspect. 1999, 107 (1), 5–24. (11) Jeffy, B. D.; Chen, E. J.; Gudas, J. M.; Romagnolo, D. F. Disruption of cell cycle kinetics by benzo[a]pyrene: inverse expression patterns of BRCA-1 and p53 in MCF-7 cells arrested in S and G2. Neoplasia 2000, 2 (5), 460–70. (12) Akerman, G. S.; Rosenzweig, B. A.; Domon, O. E.; McGarrity, L. J.; Blankenship, L. R.; Tsai, C. A.; Culp, S. J.; MacGregor, J. T.; Sistare, F. D.; Chen, J. J.; Morris, S. M. Gene expression profiles and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells. Mutat. Res. 2004, 549 (1–2), 43–64. (13) Yu, Z.; Ford, B. N.; Glickman, B. W. Identification of genes responsive to BPDE treatment in HeLa cells using cDNA expression assays. Environ. Mol. Mutagen. 2000, 36 (3), 201–5. (14) Tannheimer, S. L.; Ethier, S. P.; Caldwell, K. K.; Burchiel, S. W. Benzo[a]pyrene- and TCDD-induced alterations in tyrosine phosphorylation and insulin-like growth factor signaling pathways in the MCF-10A human mammary epithelial cell line. Carcinogenesis 1998, 19 (7), 1291–7. (15) Yoshii, S.; Tanaka, M.; Otsuki, Y.; Fujiyama, T.; Kataoka, H.; Arai, H.; Hanai, H.; Sugimura, H. Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene. Mol. Cell. Biol. 2001, 21 (20), 6796–807.
BPDE-Responsive Proteins (16) Shi, Q.; Wang, L. E.; Bondy, M. L.; Brewster, A.; Singletary, S. E.; Wei, Q. Reduced DNA repair of benzo[a]pyrene diol epoxideinduced adducts and common XPD polymorphisms in breast cancer patients. Carcinogenesis 2004, 25 (9), 1695–700. (17) Hussain, S. P.; Amstad, P.; Raja, K.; Sawyer, M.; Hofseth, L.; Shields, P. G.; Hewer, A.; Phillips, D. H.; Ryberg, D.; Haugen, A.; Harris, C. C. Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung. Cancer Res. 2001, 61 (17), 6350–5. (18) Weisenberger, D. J.; Romano, L. J. Cytosine methylation in a CpG sequence leads to enhanced reactivity with Benzo[a]pyrene diol epoxide that correlates with a conformational change. J. Biol. Chem. 1999, 274 (34), 23948–55. (19) Olden, K.; Wilson, S. Environmental health and genomics: visions and implications. Nat. Rev. Genet. 2000, 1 (2), 149–53. (20) Luo, W.; Fan, W.; Xie, H.; Jing, L.; Ricicki, E.; Vouros, P.; Zhao, L. P.; Zarbl, H. Phenotypic anchoring of global gene expression profiles induced by N-hydroxy-4-acetylaminobiphenyl and benzo[a]pyrene diol epoxide reveals correlations between expression profiles and mechanism of toxicity. Chem. Res. Toxicol. 2005, 18 (4), 619–29. (21) Petricoin, E. F.; Zoon, K. C.; Kohn, E. C.; Barrett, J. C.; Liotta, L. A. Clinical proteomics: translating benchside promise into bedside reality. Nat. Rev. Drug Discovery 2002, 1 (9), 683–95. (22) Hegde, P. S.; White, I. R.; Debouck, C. Interplay of transcriptomics and proteomics. Curr. Opin. Biotechnol. 2003, 14 (6), 647–51. (23) Oh, S.; Im, H.; Oh, E.; Lee, J.; Khim, J. Y.; Mun, J.; Kim, Y.; Lee, E.; Kim, J.; Sul, D. Effects of benzo(a)pyrene on protein expression in Jurkat T-cells. Proteomics 2004, 4 (11), 3514–26. (24) Gao, Z.; Jin, J.; Yang, J.; Yu, Y. Zinc finger proteins and other transcription regulators as response proteins in benzo[a]pyrene exposed cells. Mutat. Res. 2004, 550 (1–2), 11–24. (25) Chen, S.; Nguyen, N.; Tamura, K.; Karin, M.; Tukey, R. H. The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis. J. Biol. Chem. 2003, 278 (21), 19526–33. (26) Liu, G.; Yin, Y.; Endoplasmic reticulum stress is involved in N-methyl-N′-nitro-N- nitrosoguanidine, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and mitomycin-induced cellular response in FL cells. Chin. J. Pathophysiol. 2006, 22, 1–6. (27) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–54. (28) Kirfel, J.; Magin, T. M.; Reichelt, J. Keratins: a structural scaffold with emerging functions. Cell. Mol. Life Sci. 2003, 60 (1), 56–71. (29) Coulombe, P. A.; Omary, M. B. ‘Hard’ and ‘soft’ principles defining the structure, function and regulation of keratin intermediate filaments. Curr. Opin. Cell. Biol. 2002, 14 (1), 110–22. (30) Ku, N. O.; Zhou, X.; Toivola, D. M.; Omary, M. B. The cytoskeleton of digestive epithelia in health and disease. Am. J. Physiol. 1999, 277 (6 Pt 1), G1108–37. (31) Zatloukal, K.; Stumptner, C.; Lehner, M.; Denk, H.; Baribault, H.; Eshkind, L. G.; Franke, W. W. Cytokeratin 8 protects from hepatotoxicity, and its ratio to cytokeratin 18 determines the ability of hepatocytes to form Mallory bodies. Am. J. Pathol. 2000, 156 (4), 1263–74. (32) Collard, C. D.; Montalto, M. C.; Reenstra, W. R.; Buras, J. A.; Stahl, G. L. Endothelial oxidative stress activates the lectin complement pathway: role of cytokeratin 1. Am. J. Pathol. 2001, 159 (3), 1045– 54. (33) Li, R.; Liu, X. L.; Du, Q. F.; Zhang, S.; Luo, R. C.; Zhou, S. Y. [Proteome analysis of apoptotic K562 cells induced by harringtonine]. Zhonghua Xueyexue Zazhi 2004, 25 (6), 323–7. (34) Paramio, J. M.; Casanova, M. L.; Segrelles, C.; Mittnacht, S.; Lane, E. B.; Jorcano, J. L. Modulation of cell proliferation by cytokeratins K10 and K16. Mol. Cell. Biol. 1999, 19 (4), 3086–94. (35) Paramio, J. M.; Segrelles, C.; Ruiz, S.; Jorcano, J. L. Inhibition of protein kinase B (PKB) and PKCzeta mediates keratin K10-induced cell cycle arrest. Mol. Cell. Biol. 2001, 21 (21), 7449–59. (36) Santos, M.; Paramio, J. M.; Bravo, A.; Ramirez, A.; Jorcano, J. L. The expression of keratin k10 in the basal layer of the epidermis inhibits cell proliferation and prevents skin tumorigenesis. J. Biol. Chem. 2002, 277 (21), 19122–30. (37) Lucas, S. D.; Ek, B.; Rask, L.; Rastad, J.; Akerstrom, G.; Juhlin, C. Aberrantly expressed cytokeratin 1, a tumor-associated autoantigen in papillary thyroid carcinoma. Int. J. Cancer 1997, 73 (2), 171–7. (38) Urnov, F. D. A feel for the template: zinc finger protein transcription factors and chromatin. Biochem Cell Biol 2002, 80 (3), 321– 33.
research articles (39) Ladomery, M.; Dellaire, G. Multifunctional zinc finger proteins in development and disease. Ann Hum Genet 2002, 66 (Pt 5–6), 331– 42. (40) Sun, Y.; Shao, H.; Li, Z.; Liu, J.; Gao, L.; Peng, X.; Meng, Y.; Li, W. ZNF268, a novel kruppel-like zinc finger protein, is implicated in early human liver development. Int. J. Mol. Med. 2004, 14 (6), 971–5. (41) Krackhardt, A. M.; Witzens, M.; Harig, S.; Hodi, F. S.; Zauls, A. J.; Chessia, M.; Barrett, P.; Gribben, J. G. Identification of tumorassociated antigens in chronic lymphocytic leukemia by SEREX. Blood 2002, 100 (6), 2123–31. (42) Jung, M.; Kondratyev, A. D.; Dritschilo, A. Elongation factor 1 delta is enhanced following exposure to ionizing radiation. Cancer Res. 1994, 54 (10), 2541–3. (43) Joseph, P.; Lei, Y. X.; Whong, W. Z.; Ong, T. M. Oncogenic potential of mouse translation elongation factor-1 delta, a novel cadmiumresponsive proto-oncogene. J. Biol. Chem. 2002, 277 (8), 6131–6. (44) Suh, K. S.; Mutoh, M.; Gerdes, M.; Crutchley, J. M.; Mutoh, T.; Edwards, L. E.; Dumont, R. A.; Sodha, P.; Cheng, C.; Glick, A.; Yuspa, S. H. Antisense suppression of the chloride intracellular channel family induces apoptosis, enhances tumor necrosis factor {alpha}-induced apoptosis, and inhibits tumor growth. Cancer Res. 2005, 65 (2), 562–71. (45) Doan, L.; Yagi, H.; Jerina, D. M.; Whalen, D. L. Chloride ion catalyzed conformational inversion of carbocation intermediates in the hydrolysis of a benzo[a]pyrene 7,8-diol 9,10-epoxide. J. Am. Chem. Soc. 2002, 124 (48), 14382–7. (46) Wolfe, A. R.; Yamamoto, J.; Meehan, T. Chloride ions catalyze the formation of cis adducts in the binding of anti-benzo[a]pyrene diol epoxide to nucleic acids. Proc. Natl. Acad. Sci. U.S.A. 1994, 91 (4), 1371–5. (47) Umbricht, C. B.; Evron, E.; Gabrielson, E.; Ferguson, A.; Marks, J.; Sukumar, S. Hypermethylation of 14–3-3 sigma (stratifin) is an early event in breast cancer. Oncogene 2001, 20 (26), 3348–53. (48) Cheng, L.; Pan, C. X.; Zhang, J. T.; Zhang, S.; Kinch, M. S.; Li, L.; Baldridge, L. A.; Wade, C.; Hu, Z.; Koch, M. O.; Ulbright, T. M.; Eble, J. N. Loss of 14-3-3sigma in prostate cancer and its precursors. Clin. Cancer Res. 2004, 10 (9), 3064–8. (49) Hermeking, H.; Lengauer, C.; Polyak, K.; He, T. C.; Zhang, L.; Thiagalingam, S.; Kinzler, K. W.; Vogelstein, B. 14–3-3 sigma is a p53-regulated inhibitor of G2/M progression. Mol. Cell 1997, 1 (1), 3–11. (50) Wang, A.; Gu, J.; Judson-Kremer, K.; Powell, K. L.; Mistry, H.; Simhambhatla, P.; Aldaz, C. M.; Gaddis, S.; MacLeod, M. C. Response of human mammary epithelial cells to DNA damage induced by BPDE: involvement of novel regulatory pathways. Carcinogenesis 2003, 24 (2), 225–34. (51) Meyer, B. K.; Perdew, G. H. Characterization of the AhR-hsp90XAP2 core complex and the role of the immunophilin-related protein XAP2 in AhR stabilization. Biochemistry 1999, 38 (28), 8907–17. (52) Ma, Q.; Whitlock, J. P. Jr., A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem. 1997, 272 (14), 8878–84. (53) Rowlands, J. C.; Gustafsson, J. A. Aryl hydrocarbon receptormediated signal transduction. Crit Rev Toxicol 1997, 27 (2), 109– 34. (54) Griffith, O. W.; Mulcahy, R. T. The enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase. Adv. Enzymol. Relat. Areas Mol. Biol. 1999, 73, 209–67, xii (55) Meister, A. Glutathione, ascorbate, and cellular protection. Cancer Res. 1994, 54 (7 Suppl.), 1969s–75s. (56) Fahey, R. C.; Sundquist, A. R. Evolution of glutathione metabolism. Adv. Enzymol. Relat. Areas Mol. Biol. 1991, 64, 1–53. (57) Yuan, J. W.; Krieger, J. A.; Maples, K. R.; Born, J. L.; Burchiel, S. W. Polycyclic aromatic hydrocarbons decrease intracellular glutathione levels in the A20.1 murine B cell lymphoma. Fundam. Appl. Toxicol. 1994, 23 (3), 336–41. (58) Maniatis, T. A ubiquitin ligase complex essential for the NFkappaB, Wnt/Wingless, and Hedgehog signaling pathways. Genes Dev. 1999, 13 (5), 505–10. (59) Peters, J. M. Emi1 proteolysis: how SCF(beta-Trcp1) helps to activate the anaphase-promoting complex. Mol. Cell 2003, 11 (6), 1420–1. (60) Weng, M. W.; Hsiao, Y. M.; Chen, C. J.; Wang, J. P.; Chen, W. C.; Ko, J. L. Benzo[a]pyrene diol epoxide up-regulates COX-2 expression through NF-kappaB in rat astrocytes. Toxicol. Lett. 2004, 151 (2), 345–55.
Journal of Proteome Research • Vol. 6, No. 12, 2007 4747
research articles (61) Keshava, C.; Whipkey, D.; Weston, A. Transcriptional signatures of environmentally relevant exposures in normal human mammary epithelial cells: benzo[a]pyrene. Cancer Lett. 2005, 221 (2), 201–11. (62) Wittmann-Liebold, B.; Graack, H. R.; Pohl, T. Two-dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry. Proteomics 2006, 6 (17), 4688–703.
4748
Journal of Proteome Research • Vol. 6, No. 12, 2007
Shen et al. (63) Morandell, S.; Stasyk, T.; Grosstessner-Hain, K.; Roitinger, E.; Mechtler, K.; Bonn, G. K.; Huber, L. A. Phosphoproteomics strategies for the functional analysis of signal transduction. Proteomics 2006, 6 (14), 4047–56. (64) Raggiaschi, R.; Gotta, S.; Terstappen, G. C. Phosphoproteome analysis. Biosci. Rep. 2005, 25 (1–2), 33–44.
PR070406B
