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Diethyl sulfate-induced cell cycle arrest and apoptosis in human bronchial epithelial 16HBE cells
Chemico-Biological Interactions 205 (2013) 81–89
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Diethyl sulfate-induced cell cycle arrest and apoptosis in human bronchial epithelial 16HBE cells Peng Zhao a,b, Juanling Fu a, Biyun Yao a,b, Entan Hu a, Yanchao Song a,b, Lan Mi a,b, Zhenning Li a,b, Hongtao Zhang a,b, Yongrui Jia c, Shiliang Ma c, Wen Chen d, Zongcan Zhou a,⇑ a
Department of Toxicology, Peking University Health Science Center, Beijing 100191, PR China Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Peking University Health Science Center, Beijing 100191, PR China Peking University Medical and Healthy Analytical Center, Beijing 100191, PR China d Department of Toxicology, Faculty of Preventive Medicine, School of Public Health, Sun Yat-sen University, Guangzhou 510080, PR China b c
a r t i c l e
i n f o
Article history: Received 28 April 2013 Received in revised form 18 June 2013 Accepted 24 June 2013 Available online 2 July 2013 Keywords: Diethyl sulfate Apoptosis S arrest G2/M arrest 16HBE cells p53
a b s t r a c t In this study, we investigated the effects of diethyl sulfate (DES) on cell proliferation, cell cycle progression and apoptosis in human bronchial epithelial 16HBE cells. Cells were treated with various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM) for 12, 24 or 36 h. Cell proliferation and apoptosis were determined by MTT assay and flow cytometer, respectively. The results showed that DES inhibited cell proliferation in a dose- and time-dependent manner, and induced significant apoptosis in 16HBE cells. Apoptosis related proteins measurement results revealed that DES-induced apoptosis was concurrent with the increasing of Bax and cleavage fragment caspase-3 and the decreasing of Bcl-2 and full length procaspase-3. When cells were incubated with 2.0 mM of DES for several time intervals, S and G2/M phase accumulation was observed. Further analysis indicated that both DES-induced G1/S transition acceleration and S arrest resulted in S phase accumulation, and that DES-induced G2/M arrest resulted in G2/M phase accumulation. Western blotting results demonstrated that after DES treatment p-chk1 (Ser345) and p-chk2 (Thr68) levels decreased in G1 cells, and increased in S and G2/M cells. In addition, the increasing of chk1 and chk2 were also induced by DES treatment. With the increase in the dose of DES, p53 levels first increased (0.5–4.0 mM) and then decreased (8.0 mM). Down-regulation of p53 by RNA interference increased 4.0 mM of DES-induced apoptosis but did not affect 2.0 mM DES-induced cell cycle arrest. In conclusion, DES inhibits 16HBE cells proliferation in a dose- and time-dependent behavior. Within the sublethal dose, DES induces S and G2/M arrest through activating DNA damage checkpoints. Within the lethal dose, DES induces apoptosis through evoking apoptosis programs. p53 might play an important role in the transition between evoking cell cycle arrest/pro-survival and apoptosis programs upon DES exposure. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diethyl sulfate (DES) is an ethylating agent. Its primary use is as a chemical intermediate in synthesis of ethyl derivatives of phenols, amines, and thiols; as an accelerator in the sulfation of ethylene; and in some sulfonation processes. The routes of potential human exposure to DES are inhalation, ingestion, and dermal contact during its production and use [1]. It has been reported that exposure to DES causes mutations, chromosomal aberrations, and other genetic alterations in various organisms [2]. And moreover, based on sufficient evidence of carcinogenicity from studies in experimental animals, DES is classified as a human probable carcinogen (group 2A) by International Agency for Research on Cancer
⇑ Corresponding author. Tel.: +86 10 82801531; fax: +86 10 62015583. E-mail address: [email protected] (Z. Zhou). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.06.014
[3]. Up to the present, the researches on DES toxicity mechanisms mainly involve DNA and chromosomal changes in mammalian cell lines. Besides DNA and chromosomal damage, other targets and effects might exist [4,5]. One previous study of our laboratory finds that DES can induce cdk2-dependent centrosome abnormal amplification in Chinese hamster lung fibroblasts [6], which indicates that centrosome is one of the subcellular targets of DES toxicity action. Since centrosome amplification is one of the key events of mammalian cell division cycle, the cell cycle progression might be another target. Cell cycle is the basement of growth and development in all living organisms. In eukaryotic cells, DNA damage checkpoint participates in the regulation of cell cycle. It monitors genome integrity, and employs damage sensor proteins, such as ATM and ATR, to detect DNA damage and to initiate signal transduction cascades that employ signal transducers, including chk1 and chk2 Ser/Thr kinases and cdc25 phosphatases. The signal transducers activate
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Fig. 1. Effects of DES on cell proliferation in 16HBE cells. The 16HBE cells were treated with various doses of DES for 0, 12, 24 or 36 h. Cell proliferation was determined by MTT assay. Proliferation rates were normalized with that of DMSO control cells at 0 h. Results are expressed as relative proliferation rates. The value of DMSO control at 0 h was set to 100%; each value represents a mean ± standard error of three experiments. The asterisk indicates a significant difference between DMSO control and DES-treated cells as analyzed by Student’s t-test (⁄P < 0.05, ⁄⁄P < 0.01).
Fig. 2. Effects of DES on apoptosis and necrosis in 16HBE cells. The 16HBE cells were treated with various doses of DES for 36 h. After incubation with DES, the cells were labeled with Annexin V-FITC and PI, and then analyzed by using a flow cytometer. (A) One flow cytometry analysis representative of three individual experiments. Annexin VFITC and PI signals were shown as FL1-H and FL2-H, respectively. The results are presented as the percentages of cells that were early apoptosis (Annexin V+ PI), late apoptosis (Annexin V+ PI+), and necrosis (Annexin V PI+). (B) Results of three individual experiments. Each value represents a mean ± standard deviation of three experiments. The asterisk indicates a significant difference between DMSO control and DES-treated cells as analyzed by Student’s t-test (⁄P < 0.05, ⁄⁄P < 0.01).
p53 and inactivate cdks to inhibit cell cycle progression from G1 to S, DNA replication (S progression), or G2 to mitosis [7–9], so as to allow for repair and prevention of the transmission of damaged or
incompletely replicated chromosomes. If the damage cannot be repaired, the cells will initiate programmed cell death such as apoptosis.
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In this study, we investigated the effects of DES on cell proliferation, cell cycle progression and apoptotic cell death in human bronchial epithelial 16HBE cell line used as a model of DES-induced lung toxicity. Furthermore, in attempt to explore the possible molecular mechanisms of the apoptosis and cell cycle disturbance induced by DES, we determined the cellular levels of some apoptosis related proteins and DNA damage checkpoint proteins after DES treatment.
containing 10% (v/v) FBS. After incubation with DES, the dosing media was removed and the cells were washed twice with prewarmed phosphate buffered saline (PBS). Two hundred microliters of serum-free DMEM medium containing 0.4 mg/ml MTT reagent were then added to each well. Followed incubation with MTT for 3 h, the cells were rinsed twice with PBS, and then exactly 200 ll of DMSO were infused to each well. The plates were read at 570 nm using an ELISA reader (Bio-Rad).
2. Materials and methods
2.3. Measurement of apoptosis by flow cytometry
2.1. Cell culture and reagents
The 16HBE cells were plated in 25 cm2 flasks (5 105 per flask), 36 h later various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM) were applied for 36 h in DMEM medium containing 10% (v/v) FBS. After incubation with DES, the cells were harvested by centrifugation at 800g for 5 min, washed twice with PBS, and labeled with Annexin V-FITC, together with propidium iodide (PI) according to the manufacturer’s instructions (BD Biosciences). Fluorescent signals were measured using a flow cytometer (BD FACSCalibur, Franklin Lakes, NJ, USA). The results are presented as the percentages of cells that were early apoptosis (Annexin V+ PI), late apoptosis (Annexin V+ PI+), or necrosis (Annexin V PI+).
The human bronchial epithelial cell line 16HBE [10,11] was a gift from Dr. D.C. Gruenert (University of California, San Francisco). The 16HBE cells were cultured at 37 °C and 5% CO2 in DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 10 U/ml penicillin and 10 U/ml streptomycin. Reagents used in this study were purchased from Sigma (Sigma–Aldrich, St. Louis, MO, USA) unless specified. 2.2. Cell proliferation assay Inhibition of cell proliferation by DES was measured by MTT assay. Briefly, 16HBE cells were seeded in 96-well culture plates (1 104 per well), 36 h later various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM) were applied for 0, 12, 24 or 36 h in DMEM medium
2.4. Cell cycle analysis The 16HBE cells were plated in 25 cm2 flasks (5 105 per flask), 36 h later treated with DMSO or 2.0 mM (sublethal dose) of DES for
Fig. 3. Effects of DES on the cellular levels of p53, Bax, Bcl-2 and caspase-3 (full length procaspase-3 and cleavage fragment caspase-3 p17/p11) in 16HBE cells. The 16HBE cells were treated with various doses of DES for 36 h. The cellular levels of p53, Bax, Bcl-2 and caspase-3 (full length procaspase-3 and cleavage fragment caspase-3 p17/p11) were determined by Western blotting assay. (A) Representative immunoblot obtained with p53, Bax, Bcl-2 and caspase-3 antibodies. (B) Densitometric analysis of (A). The value of DMSO control was set to 1; each value represents a mean ± standard deviation of three experiments. ⁄P < 0.05, #P < 0.01 compared with DMSO control.
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0, 4, 8, 12, 16, 20, 24, 28 or 32 h. At the end of the treatment, the cells were collected by centrifugation at 800g for 5 min, washed twice with PBS. The cell pellet was resuspended in 70% (v/v) ethanol and stored at 4 °C overnight. The cells were then pelleted and resuspended in 1 ml PBS containing 20 lg/ml RNase A. After incubation with RNase A for 30 min, the cells were stained with PI (20 lg/ml) and analyzed on a flow cytometer (BD FACSCalibur, Franklin Lakes, NJ, USA). The CellQuest software (BD FACSCalibur) was used to evaluate the data.
2.5. Sorting of G1, S and G2/M cells The 16HBE cells were plated in 75 cm2 flasks (1.5 106 per flask), 36 h later treated with DMSO or 2.0 mM of DES for 20 or 28 h. After the treatment, the cells were incubated with Hoechst 33342 (10 lg/ml) for 2 h. After incubation with Hoechst 33342, the cells were collected by centrifugation at 800g for 5 min, and washed twice with PBS, and then resuspended in PBS. Cell density was adjusted to 1 107 per milliliter before cell sorting. FACSVan-
tage DiVa flow cytometer (BD FACSCalibur, Franklin Lakes, NJ, USA) was used to sort G1, S and G2/M cells.
2.6. Western blotting analysis The total cellular proteins were extracted as described previously [12]. Protein concentration was determined using the Bradford protein assay. Cell extracts containing 30 lg of proteins were separated on 12% SDS–PAGE gels and transferred to a nitrocellulose membrane (Pall). After 3 h blocking with 5% (w/v) nonfat milk in TBST (1.5 M NaCl, 20 mM Tris–HCl, 0.05% (v/v) Tween-20, pH 7.4), the nitrocellulose membrane was incubated overnight at 4 °C with p53, Bax, Bcl-2, caspase-3, p-chk1 (Ser345), chk1, pchk2 (Thr68), chk2 and Actin polyclonal antibodies (at 1:500 dilutions) purchased from Cell Signaling Technology. The blots were then washed with TBST prior 2 h incubation at room temperature with horseradish peroxidase conjugated secondary antibody (at 1:10000 dilutions). The protein bands were detected by Western Blotting Luminol Reagent (TIANGEN BIOTECH, Beijing, China) and
Fig. 4. Effects of DES on cell cycle progression in 16HBE cells. The 16HBE cells were treated with 2.0 mM of DES in the presence of 10% (v/v) serum for various time intervals. Cell cycle distribution was determined by flow cytometer. (A–C) Show the G1, S and G2/M fractions, respectively. (D) Cell counting data. (E–G) Calculations of Exit%G1, Exit%S and Exit%G2/M at indicated period in DES-treated and DMSO control cells. Exit% means how many percent of some phase cells enter next phase during a period. Each value represents a mean ± standard deviation of three experiments. The asterisk indicates a significant difference between DMSO control and DES-treated cells as analyzed by Student’s t-test (⁄P < 0.05, ⁄⁄P < 0.01).
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recorded by Kodak films. The images were acquired using an AGFA DuoScan T1200 scanner and analyzed by Quantity One software (Bio-Rad).
2.7. RNA interference (RNAi) experiment The small interfering RNA (siRNA) duplexes were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences of the siRNA oligomer to P53 were: 50 -CUACUUCCUG AAAACAACGdTdT-30 , 50 -CGUUGUUUUCAGGAAGUAGdTdT-30 . The FAM conjugated negative control siRNA oligomer (The sequences were: 50 -UUCUCCGAACGUGUCACGUTT-30 , 50 -ACGUGACACGUUCG GAGAATT-30 ) was used to detect transfection rate. The siRNA transfection was performed using Lipofectamine™ 2000 (Invitrogen) essentially following the manufacturer’s procedure, with 5 ll/well of lipid reagent and 100 pmol/well of siRNA oligomer in a six-well plate. The transfection rate at 24 h after transfection was examined by using fluorescence microscopy (Nikon E400). 2.8. Statistical analysis Data were expressed as the mean ± standard deviation. All statistical analysis was performed by SPSS for windows (SPSS, Inc., Chicago, IL). Student’s t-test was used to compare the statistical significance of the difference in data from the two groups; P < 0.05 was considered statistically significant. All experiments were performed in three times.
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3. Results 3.1. Inhibition of DES on 16HBE cells proliferation The 16HBE cells were treated with various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM) for 0, 12, 24 or 36 h. MTT assay was carried out to determine the effects of DES on 16HBE cells proliferation. As exhibited in Fig. 1, 16HBE cells proliferation was slightly inhibited by 0.5 and 1.0 mM of DES (at 36 h), moderately inhibited by 2.0 mM of DES (at 12, 24 and 36 h), and markedly inhibited by 4.0 and 8.0 mM of DES (at 12, 24 and 36 h). These results indicated that DES inhibited 16HBE cells proliferation in a dose- and timedependent behavior. In addition to a decrease in cell proliferation, the results in Fig. 1 also indicated that cell death was induced by 4.0 (at 24 and 36 h) and 8.0 (at 12, 24 and 36 h) mM of DES. 3.2. DES-induced apoptosis in 16HBE cells The 16HBE cells were treated with various doses of DES for 36 h. DES-induced apoptosis was determined by using a flow cytometer. As shown in Fig. 2, during the observation period, only 4.0 and 8.0 mM of DES induced significant apoptosis. These results indicated that the inhibition effects of 4.0 and 8.0 mM of DES on cell proliferation was partly due to DES-induced apoptosis. To explore the possible molecular mechanisms underlying DESinduced apoptosis, the cellular levels of apoptosis related proteins p53, Bax, Bcl-2 and caspase-3 (including full length procaspase-3 and cleavage fragment caspase-3 p17/p11) were determined by Western blotting assay. As shown in Fig. 3, the cellular levels of
Fig. 5. The cellular levels of p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 in G1, S and G2/M 16HBE cells treated with 2.0 mM of DES at 20 and 28 h. (A) Representative immunoblot obtained with p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 antibodies. (B) Densitometric analysis of (A). The value of DMSO control at 20 h was set to 1; each value represents a mean ± standard deviation of three experiments. ⁄P < 0.05, ⁄⁄P < 0.01 compared with DMSO control at the same time point.
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p53, Bax and cleavage fragment caspase-3 p17/p11 increased significantly when the cells were treated with DES. Compared to the DMSO control cells, the cellular levels of Bcl-2 and full length procaspase-3 decreased in 8.0 mM of DES-treated cells.
3.3. Effects of DES on cell cycle progression in 16HBE cells To observe the effects of DES on cell cycle progression in 16HBE cells, a time-course study was conducted by continuously treating the cells with 2.0 mM of DES for various time intervals and cell cycle distribution profiles were recorded by flow cytometer. As shown in Fig. 4A–C, DES induced S and G2/M phase accumulation in 16HBE cells. S phase accumulation occurred from 12 to 32 h; G2/ M phase accumulation occurred from 24 to 32 h. In order to identify the reasons for the S and G2/M phase accumulation, Exit%, a parameter introduced previously [13], was determined. Exit% may reflect the kinetic profiles of cell cycle progression. The cell counting and Exit% calculating results were shown in Fig. 4D–G. DES inhibited cell proliferation in a time-dependent manner (Fig. 4D) and disrupted cell cycle kinetic characters (Fig. 4E–G). The increas-
ing of Exit%G1 and the decreasing of Exit%S and Exit%G2/M were observed in DES-treated cells, which indicated that DES accelerated the transition of 16HBE cells from G1 to S phase, and arrested S and G2/M progression. All these results demonstrated that both DES-induced G1/S transition acceleration and S arrest resulted in S phase accumulation, and that DES-induced G2/M arrest resulted in G2/M phase accumulation.
3.4. Alterations of DNA damage checkpoint proteins in 16HBE cells after DES treatment To identify whether DNA damage checkpoints are involved in DES-induced cell cycle arrest, the cellular levels of p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 after DES treatment were determined by Western blotting assay when the cells were treated with 2.0 mM of DES at 20 and 28 h. The results were shown in Fig. 5. In DES-treated G1 cells, the levels of p-chk1 (Ser345) and p-chk2 (Thr68) decreased at 20 h, the levels of chk1 increased at 28 h, the levels of chk2 increased significantly at 20 and 28 h. In DES-treated S cells, the levels of p-chk1 (Ser345), chk1 and chk2 in-
Fig. 6. Effects of P53 siRNA transfection on the cellular levels of p53 in 16HBE cells. The 16HBE cells were transfected with P53 siRNA, 24 h later treated with 0, 2.0 or 4.0 mM of DES for 20, 28 or 36 h. After the treatment, the cellular levels of p53 were determined by Western blotting assay. (A) SiRNA transfection rate (above 90%) at 24 h after transfection. (B) and (D) Representative immunoblot obtained with p53 antibody. (C) and (E) Densitometric analysis of (B) and (D), respectively. The value of DMSO-treated 16HBE cells in untransfected (MOCK) group was set to 1; each value represents a mean ± standard deviation of three experiments. ⁄⁄P < 0.01 compared with DMSO. #P < 0.05, ## P < 0.01 compared with MOCK.
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creased significantly at 20 and 28 h, the levels of p-chk2 (Thr68) increased significantly at 20 h. In DES-treated G2/M cells, the cellular levels of p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 increased significantly at 20 and 28 h. These results indicated that DES activated the DNA damage checkpoints in S and G2/M cells. 3.5. Effects of P53 down-regulation by RNAi on DES-induced apoptosis and cell cycle arrest in 16HBE cells To determine the roles of p53 in DES-induced apoptosis and cell cycle arrest, the 16HBE cells were transfected with P53 siRNA, 24 h later treated with 0, 2.0 or 4.0 mM of DES for 20, 28 or 36 h. The apoptosis and cell cycle distribution profiles were determined by flow cytometer. The results were shown in Figs. 6–8. Compared to the untransfected (MOCK) cells, the cellular levels of p53 decreased in both DMSO- and 4.0 mM of DES-treated cells at 36 h (Fig. 6B and C). The down-regulation of p53 by RNAi was concurrent with the significant increasing of apoptosis (both early and late apoptosis) in 4.0 mM of DES-treated cells (Fig. 7). In addition, the expression of p53 was suppressed by RNAi in both DMSO- and 2.0 mM of DES-treated cells at 20 and 28 h (Fig. 6D and E), but the cell cycle distribution was not affected (Fig. 8).
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4. Discussion DES-induced DNA damage and chromosomal aberrations in mammalian cells have been well investigated [2–5]. Besides DNA and chromosome, centrosome was demonstrated to be a subcellular target of DES toxicity action in our previous study [6]. Considering the contribution of centrosome amplification in mammalian cell division cycle, we speculated that cell cycle progression might be another target of DES toxicity action. To verify this hypothesis, we investigated the effects of DES on cell proliferation, cell cycle progression and apoptosis in human bronchial epithelial 16HBE cells. Through continuous observation of the cell cycle distribution, we found that DES induced S and G2/M phase accumulation in 16HBE cells. It is noted that the definitions of cell cycle distribution and cell cycle progression are different. The alteration of cell cycle distribution can only indicate that cell cycle progression might be disrupted. To determine what type of disruption (acceleration or arrest), further analysis is required. As we previously pointed out, the increasing of S phase fraction (S phase accumulation) is maybe due to one of the reasons listed below: (a) S arrest; (b) acceleration of the transition from G1 to S phase; (c) both (a) and (b) [13]. Like-
Fig. 7. Effects of suppression of P53 by RNAi on DES-induced apoptosis in 16HBE cells. The 16HBE cells were transfected with P53 siRNA, 24 h later treated with 0, 2.0 or 4.0 mM DES for 36 h. After the treatment, the cells were labeled with Annexin V-FITC and PI, and then analyzed using a flow cytometer. (A) One flow cytometry analysis representative of three individual experiments. Annexin V-FITC and PI signals were shown as FL1-H and FL2-H, respectively. The results are presented as the percentages of cells that were early apoptosis (Annexin V+ PI), late apoptosis (Annexin V+ PI+), and necrosis (Annexin V PI+). (B) Results of three individual experiments. Each value represents a mean ± standard deviation of three experiments. ⁄P < 0.05, ⁄⁄P < 0.01 compared with DMSO. #P < 0.05, ##P < 0.01 compared with MOCK.
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Fig. 8. Effects of suppression of P53 by RNAi on DES-induced cell cycle arrest in 16HBE cells. The 16HBE cells were transfected with P53 siRNA, 24 h later treated with DMSO or 2.0 mM of DES for 20 or 28 h. Cell cycle distribution was determined by flow cytometer. ⁄⁄P < 0.01 compared with DMSO control at the same time point.
wise, the increasing of G2/M phase fraction (G2/M phase accumulation) is maybe due to G2/M arrest and/or acceleration of the transition from S to G2 phase. Therefore, it is not enough to evaluate the influence of DES on cell cycle kinetics only by using cell cycle distribution results. Exit% is a parameter for evaluating cell cycle kinetics introduced previously, denotes how many percent of some phase cells enter next phase during a period. For example, if Exit%S is 30%, that means 30% of S phase cells entered G2 phase (exited S phase) during a period. Details about Exit% have been well explained in our previous paper [13]. By using parameter Exit%, we identified the reasons for S and G2/M phase accumulation in DES-treated cells. DES accelerated the transition of 16HBE cells from G1 to S phase, and arrested S and G2/M progression. Thus the S phase accumulation was due to DES-induced G1/S transition acceleration and S arrest, the G2/M phase accumulation was due to DES-induced G2/M arrest. The activation of DNA damage checkpoint is one of the mechanisms arresting cell cycle progression. In human cells, chk1 and chk2 are the most important signal transducers with strictly signal transduction function in cell cycle regulation and checkpoint responses. Phosphorylation on Ser345 in chk1 and/or phosphorylation on Thr68 in chk2 are the main characters of DNA damage checkpoint activation [7,9,14]. In the current study, we found that the cellular levels of p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 increased in DES-treated S and G2/M cells, which indicated that the DNA damage checkpoints were activated. The activation of DNA damage checkpoints should be responsible for DES-induced S and G2/M arrest in 16HBE cells. DNA damage checkpoint-mediated cell cycle arrest (S and G2/M arrest) and cdk2-cyclin A-mediated abnormal centrosome amplification [6] might be important mechanisms for DES-induced aneuploidy. In addition, we found that the cellular levels of p-chk1 (Ser345) and p-chk2 (Thr68) decreased in DES-treated G1 cells, which indicated that the DNA damage checkpoint was inactivated. The significance of the inactivation of DNA damage checkpoint in DES-treated G1 cells and the mechanisms of DES-induced G1/S transition acceleration need further investigation.
Besides the activation of DNA damage checkpoint, apoptosis is another DNA damage response reaction. It eliminates heavily damaged or seriously deregulated cells. In this study, only high doses of DES (4.0 and 8.0 mM) induced significant apoptosis in 16HBE cells at 36 h. DES-induced apoptosis was concurrent with the increasing of Bax and the cleavage fragment caspase-3 p17/p11 and the decreasing of Bcl-2 and full length procaspase-3, which indicated that DES induced apoptosis through evoking apoptosis pathway. Based on the detection results of cell cycle and apoptosis, we can conclude that upon DES exposure the activation of DNA damage checkpoint is earlier than the evoking of apoptosis programs. It is well known that p53 is a critical regulator of apoptosis and cell cycle arrest/pro-survival. Upon DNA damage, p53 evokes both cell cycle arrest/pro-survival and apoptosis transcriptional programs. The ultimate cellular outcome depends on the balance of these two programs [15]. With the increase in the dose of DES, the cellular levels of p53 first increased (0.5 to 4.0 mM) and then decreased (8.0 mM). And moreover, down-regulation of p53 by RNAi increased 4.0 mM of DES-induced apoptosis. These results indicated that p53 might act as a protector to promote cell survival when the cells were exposed to DES. It should be noted that this study focuses on exploring the possible mechanisms of the apoptosis and cell cycle disturbance induced by DES in 16HBE cells. The concentrations (2–8 mM) that DES induces those effects are determined through the pre-experiments. They are relatively high. In occupational emergencies and pollution accidents, the primary route of human exposure to DES is inhalation. Because of lacking sufficient relevant toxicokinetics data, it is difficult to achieve the extrapolation between the target concentrations in the bronchial epithelium and the concentrations in the exposure air. And moreover, many factors such as exposure duration, respiratory rate and depth might affect the target concentrations of human bronchial epithelial cells exposure to DES by inhalation. However, it is noteworthy that the workers involved in the production of ethanol by the strong-acid process might be exposed to high concentrations of DES in some cases. Based on the presence of approximately 30% DES in acid extracts, the maxi-
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mum vapor concentration over a spill is calculated as 2000 ppm [1], which is much higher than the lethal concentration (inhalation of 500 ppm of DES for 4 h caused 6/6 deaths in rats) of DES by inhalation [16]. In summary, DES inhibits 16HBE cells proliferation in a doseand time-dependent behavior. Within the sublethal dose, DES induces S and G2/M arrest through activating DNA damage checkpoints. Within the lethal dose, DES induces apoptosis through evoking apoptosis programs. p53 might play an important role in the transition between evoking cell cycle arrest/pro-survival and apoptosis programs upon DES exposure. Conflicts of interest statement The authors declare no conflict of interest. Acknowledgements This work was supported by National Nature Science Foundation of China (Nos. 81001253, 30972502, and 81172693), Beijing Natural Science Foundation (No. 7132122) and Fundamental Research Funds for the Central Universities (No. BMU20090460). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cbi.2013.06.014. References [1] National Toxicology Program, Diethyl sulfate, Rep. Carcinog. 12 (2011) 161– 163. [2] G.R. Hoffmann, Genetic effects of dimethyl sulfate, diethyl sulfate, and related compounds, Mutat. Res. 75 (1980) 63–129.
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[3] International Agency for Research on Cancer, Agents Classified by the IARC Monographs, Volumes 1–100, 2010. Available from: http://monographs.iarc.fr/ ENG/Classification/ClassificationsAlphaOrder.pdf. [4] M. Nüsse, S. Viaggi, S. Bonatti, Induction of kinetochore positive and negative micronuclei in V79 cells by the alkylating agent diethylsulphate, Mutagenesis 4 (1989) 174–178. [5] M. Campagna, P. Beffy, R. Del Carratore, L. Hauri, S. Simi, S. Bonatti, M. Simili, Diethylsulphate and methylnitrosourea affect different targets in Chinese hamster fibroblasts: possible mechanisms of aneuploidy induction by these agents, Mutagenesis 18 (2003) 405–410. [6] E. Hu, J. Fu, P. Zhao, B. Yao, Y. Qi, Z. Yuan, Z. Zhou, Diethyl sulfate induced Cdk2dependent centrosome amplification in CHL cells, Toxicology 275 (2010) 58– 64. [7] A. Sancar, L.A. Lindsey-Boltz, K. Unsal-Kaçmaz, S. Linn, Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints, Annu. Rev. Biochem. 73 (2004) 39–85. [8] A. Poehlmann, A. Roessner, Importance of DNA damage checkpoints in the pathogenesis of human cancers, Pathol. Res. Pract. 206 (2010) 591–601. [9] H.C. Reinhardt, M.B. Yaffe, Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2, Curr. Opin. Cell Biol. 21 (2009) 245–255. [10] A.L. Cozens, M.J. Yezzi, K. Kunzelmann, T. Ohrui, L. Chin, K. Eng, W.E. Finkbeiner, J.H. Widdicombe, D.C. Gruenert, CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells, Am. J. Respir. Cell Mol. Biol. 10 (1994) 38–47. [11] Y. Pang, W. Li, R. Ma, W. Ji, Q. Wang, D. Li, Y. Xiao, Q. Wei, Y. Lai, P. Yang, L. Chen, S. Tang, Y. Lin, Z. Zhuang, Y. Zheng, W. Chen, Development of human cell models for assessing the carcinogenic potential of chemicals, Toxicol. Appl. Pharmacol. 232 (2008) 478–486. [12] 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-epoxidetransformed and normal human bronchial epithelial G0/G1 cells, Chem. Biol. Interact. 186 (2010) 166–173. [13] P. Zhao, W. Zhong, X. Ying, Z. Yuan, J. Fu, Z. Zhou, Manganese chloride-induced G0/G1 and S phase arrest in A549 cells, Toxicology 250 (2008) 39–46. [14] T.H. Stracker, T. Usui, J.H. Petrini, Taking the time to make important decisions: the checkpoint effector kinases Chk1 and Chk2 and the DNA damage response, DNA Repair (Amst) 8 (2009) 1047–1054. [15] X. Zhang, Y. He, K.H. Lee, W. Dubois, Z. Li, X. Wu, A. Kovalchuk, W. Zhang, J. Huang, Rap2b, a novel p53 target, regulates p53-mediated pro-survival function, Cell Cycle 12 (2013) 1279–1291. [16] TOXNET/Hazardous Substances Data Bank (HSDB), DIETHYLSULFATE. Available from: http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB.
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Diethyl sulfate-induced cell cycle arrest and apoptosis in human bronchial epithelial 16HBE cells Peng Zhao a,b, Juanling Fu a, Biyun Yao a,b, Entan Hu a, Yanchao Song a,b, Lan Mi a,b, Zhenning Li a,b, Hongtao Zhang a,b, Yongrui Jia c, Shiliang Ma c, Wen Chen d, Zongcan Zhou a,⇑ a
Department of Toxicology, Peking University Health Science Center, Beijing 100191, PR China Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Peking University Health Science Center, Beijing 100191, PR China Peking University Medical and Healthy Analytical Center, Beijing 100191, PR China d Department of Toxicology, Faculty of Preventive Medicine, School of Public Health, Sun Yat-sen University, Guangzhou 510080, PR China b c
a r t i c l e
i n f o
Article history: Received 28 April 2013 Received in revised form 18 June 2013 Accepted 24 June 2013 Available online 2 July 2013 Keywords: Diethyl sulfate Apoptosis S arrest G2/M arrest 16HBE cells p53
a b s t r a c t In this study, we investigated the effects of diethyl sulfate (DES) on cell proliferation, cell cycle progression and apoptosis in human bronchial epithelial 16HBE cells. Cells were treated with various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM) for 12, 24 or 36 h. Cell proliferation and apoptosis were determined by MTT assay and flow cytometer, respectively. The results showed that DES inhibited cell proliferation in a dose- and time-dependent manner, and induced significant apoptosis in 16HBE cells. Apoptosis related proteins measurement results revealed that DES-induced apoptosis was concurrent with the increasing of Bax and cleavage fragment caspase-3 and the decreasing of Bcl-2 and full length procaspase-3. When cells were incubated with 2.0 mM of DES for several time intervals, S and G2/M phase accumulation was observed. Further analysis indicated that both DES-induced G1/S transition acceleration and S arrest resulted in S phase accumulation, and that DES-induced G2/M arrest resulted in G2/M phase accumulation. Western blotting results demonstrated that after DES treatment p-chk1 (Ser345) and p-chk2 (Thr68) levels decreased in G1 cells, and increased in S and G2/M cells. In addition, the increasing of chk1 and chk2 were also induced by DES treatment. With the increase in the dose of DES, p53 levels first increased (0.5–4.0 mM) and then decreased (8.0 mM). Down-regulation of p53 by RNA interference increased 4.0 mM of DES-induced apoptosis but did not affect 2.0 mM DES-induced cell cycle arrest. In conclusion, DES inhibits 16HBE cells proliferation in a dose- and time-dependent behavior. Within the sublethal dose, DES induces S and G2/M arrest through activating DNA damage checkpoints. Within the lethal dose, DES induces apoptosis through evoking apoptosis programs. p53 might play an important role in the transition between evoking cell cycle arrest/pro-survival and apoptosis programs upon DES exposure. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Diethyl sulfate (DES) is an ethylating agent. Its primary use is as a chemical intermediate in synthesis of ethyl derivatives of phenols, amines, and thiols; as an accelerator in the sulfation of ethylene; and in some sulfonation processes. The routes of potential human exposure to DES are inhalation, ingestion, and dermal contact during its production and use [1]. It has been reported that exposure to DES causes mutations, chromosomal aberrations, and other genetic alterations in various organisms [2]. And moreover, based on sufficient evidence of carcinogenicity from studies in experimental animals, DES is classified as a human probable carcinogen (group 2A) by International Agency for Research on Cancer
⇑ Corresponding author. Tel.: +86 10 82801531; fax: +86 10 62015583. E-mail address: [email protected] (Z. Zhou). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.06.014
[3]. Up to the present, the researches on DES toxicity mechanisms mainly involve DNA and chromosomal changes in mammalian cell lines. Besides DNA and chromosomal damage, other targets and effects might exist [4,5]. One previous study of our laboratory finds that DES can induce cdk2-dependent centrosome abnormal amplification in Chinese hamster lung fibroblasts [6], which indicates that centrosome is one of the subcellular targets of DES toxicity action. Since centrosome amplification is one of the key events of mammalian cell division cycle, the cell cycle progression might be another target. Cell cycle is the basement of growth and development in all living organisms. In eukaryotic cells, DNA damage checkpoint participates in the regulation of cell cycle. It monitors genome integrity, and employs damage sensor proteins, such as ATM and ATR, to detect DNA damage and to initiate signal transduction cascades that employ signal transducers, including chk1 and chk2 Ser/Thr kinases and cdc25 phosphatases. The signal transducers activate
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Fig. 1. Effects of DES on cell proliferation in 16HBE cells. The 16HBE cells were treated with various doses of DES for 0, 12, 24 or 36 h. Cell proliferation was determined by MTT assay. Proliferation rates were normalized with that of DMSO control cells at 0 h. Results are expressed as relative proliferation rates. The value of DMSO control at 0 h was set to 100%; each value represents a mean ± standard error of three experiments. The asterisk indicates a significant difference between DMSO control and DES-treated cells as analyzed by Student’s t-test (⁄P < 0.05, ⁄⁄P < 0.01).
Fig. 2. Effects of DES on apoptosis and necrosis in 16HBE cells. The 16HBE cells were treated with various doses of DES for 36 h. After incubation with DES, the cells were labeled with Annexin V-FITC and PI, and then analyzed by using a flow cytometer. (A) One flow cytometry analysis representative of three individual experiments. Annexin VFITC and PI signals were shown as FL1-H and FL2-H, respectively. The results are presented as the percentages of cells that were early apoptosis (Annexin V+ PI), late apoptosis (Annexin V+ PI+), and necrosis (Annexin V PI+). (B) Results of three individual experiments. Each value represents a mean ± standard deviation of three experiments. The asterisk indicates a significant difference between DMSO control and DES-treated cells as analyzed by Student’s t-test (⁄P < 0.05, ⁄⁄P < 0.01).
p53 and inactivate cdks to inhibit cell cycle progression from G1 to S, DNA replication (S progression), or G2 to mitosis [7–9], so as to allow for repair and prevention of the transmission of damaged or
incompletely replicated chromosomes. If the damage cannot be repaired, the cells will initiate programmed cell death such as apoptosis.
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In this study, we investigated the effects of DES on cell proliferation, cell cycle progression and apoptotic cell death in human bronchial epithelial 16HBE cell line used as a model of DES-induced lung toxicity. Furthermore, in attempt to explore the possible molecular mechanisms of the apoptosis and cell cycle disturbance induced by DES, we determined the cellular levels of some apoptosis related proteins and DNA damage checkpoint proteins after DES treatment.
containing 10% (v/v) FBS. After incubation with DES, the dosing media was removed and the cells were washed twice with prewarmed phosphate buffered saline (PBS). Two hundred microliters of serum-free DMEM medium containing 0.4 mg/ml MTT reagent were then added to each well. Followed incubation with MTT for 3 h, the cells were rinsed twice with PBS, and then exactly 200 ll of DMSO were infused to each well. The plates were read at 570 nm using an ELISA reader (Bio-Rad).
2. Materials and methods
2.3. Measurement of apoptosis by flow cytometry
2.1. Cell culture and reagents
The 16HBE cells were plated in 25 cm2 flasks (5 105 per flask), 36 h later various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM) were applied for 36 h in DMEM medium containing 10% (v/v) FBS. After incubation with DES, the cells were harvested by centrifugation at 800g for 5 min, washed twice with PBS, and labeled with Annexin V-FITC, together with propidium iodide (PI) according to the manufacturer’s instructions (BD Biosciences). Fluorescent signals were measured using a flow cytometer (BD FACSCalibur, Franklin Lakes, NJ, USA). The results are presented as the percentages of cells that were early apoptosis (Annexin V+ PI), late apoptosis (Annexin V+ PI+), or necrosis (Annexin V PI+).
The human bronchial epithelial cell line 16HBE [10,11] was a gift from Dr. D.C. Gruenert (University of California, San Francisco). The 16HBE cells were cultured at 37 °C and 5% CO2 in DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 10 U/ml penicillin and 10 U/ml streptomycin. Reagents used in this study were purchased from Sigma (Sigma–Aldrich, St. Louis, MO, USA) unless specified. 2.2. Cell proliferation assay Inhibition of cell proliferation by DES was measured by MTT assay. Briefly, 16HBE cells were seeded in 96-well culture plates (1 104 per well), 36 h later various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM) were applied for 0, 12, 24 or 36 h in DMEM medium
2.4. Cell cycle analysis The 16HBE cells were plated in 25 cm2 flasks (5 105 per flask), 36 h later treated with DMSO or 2.0 mM (sublethal dose) of DES for
Fig. 3. Effects of DES on the cellular levels of p53, Bax, Bcl-2 and caspase-3 (full length procaspase-3 and cleavage fragment caspase-3 p17/p11) in 16HBE cells. The 16HBE cells were treated with various doses of DES for 36 h. The cellular levels of p53, Bax, Bcl-2 and caspase-3 (full length procaspase-3 and cleavage fragment caspase-3 p17/p11) were determined by Western blotting assay. (A) Representative immunoblot obtained with p53, Bax, Bcl-2 and caspase-3 antibodies. (B) Densitometric analysis of (A). The value of DMSO control was set to 1; each value represents a mean ± standard deviation of three experiments. ⁄P < 0.05, #P < 0.01 compared with DMSO control.
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0, 4, 8, 12, 16, 20, 24, 28 or 32 h. At the end of the treatment, the cells were collected by centrifugation at 800g for 5 min, washed twice with PBS. The cell pellet was resuspended in 70% (v/v) ethanol and stored at 4 °C overnight. The cells were then pelleted and resuspended in 1 ml PBS containing 20 lg/ml RNase A. After incubation with RNase A for 30 min, the cells were stained with PI (20 lg/ml) and analyzed on a flow cytometer (BD FACSCalibur, Franklin Lakes, NJ, USA). The CellQuest software (BD FACSCalibur) was used to evaluate the data.
2.5. Sorting of G1, S and G2/M cells The 16HBE cells were plated in 75 cm2 flasks (1.5 106 per flask), 36 h later treated with DMSO or 2.0 mM of DES for 20 or 28 h. After the treatment, the cells were incubated with Hoechst 33342 (10 lg/ml) for 2 h. After incubation with Hoechst 33342, the cells were collected by centrifugation at 800g for 5 min, and washed twice with PBS, and then resuspended in PBS. Cell density was adjusted to 1 107 per milliliter before cell sorting. FACSVan-
tage DiVa flow cytometer (BD FACSCalibur, Franklin Lakes, NJ, USA) was used to sort G1, S and G2/M cells.
2.6. Western blotting analysis The total cellular proteins were extracted as described previously [12]. Protein concentration was determined using the Bradford protein assay. Cell extracts containing 30 lg of proteins were separated on 12% SDS–PAGE gels and transferred to a nitrocellulose membrane (Pall). After 3 h blocking with 5% (w/v) nonfat milk in TBST (1.5 M NaCl, 20 mM Tris–HCl, 0.05% (v/v) Tween-20, pH 7.4), the nitrocellulose membrane was incubated overnight at 4 °C with p53, Bax, Bcl-2, caspase-3, p-chk1 (Ser345), chk1, pchk2 (Thr68), chk2 and Actin polyclonal antibodies (at 1:500 dilutions) purchased from Cell Signaling Technology. The blots were then washed with TBST prior 2 h incubation at room temperature with horseradish peroxidase conjugated secondary antibody (at 1:10000 dilutions). The protein bands were detected by Western Blotting Luminol Reagent (TIANGEN BIOTECH, Beijing, China) and
Fig. 4. Effects of DES on cell cycle progression in 16HBE cells. The 16HBE cells were treated with 2.0 mM of DES in the presence of 10% (v/v) serum for various time intervals. Cell cycle distribution was determined by flow cytometer. (A–C) Show the G1, S and G2/M fractions, respectively. (D) Cell counting data. (E–G) Calculations of Exit%G1, Exit%S and Exit%G2/M at indicated period in DES-treated and DMSO control cells. Exit% means how many percent of some phase cells enter next phase during a period. Each value represents a mean ± standard deviation of three experiments. The asterisk indicates a significant difference between DMSO control and DES-treated cells as analyzed by Student’s t-test (⁄P < 0.05, ⁄⁄P < 0.01).
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recorded by Kodak films. The images were acquired using an AGFA DuoScan T1200 scanner and analyzed by Quantity One software (Bio-Rad).
2.7. RNA interference (RNAi) experiment The small interfering RNA (siRNA) duplexes were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences of the siRNA oligomer to P53 were: 50 -CUACUUCCUG AAAACAACGdTdT-30 , 50 -CGUUGUUUUCAGGAAGUAGdTdT-30 . The FAM conjugated negative control siRNA oligomer (The sequences were: 50 -UUCUCCGAACGUGUCACGUTT-30 , 50 -ACGUGACACGUUCG GAGAATT-30 ) was used to detect transfection rate. The siRNA transfection was performed using Lipofectamine™ 2000 (Invitrogen) essentially following the manufacturer’s procedure, with 5 ll/well of lipid reagent and 100 pmol/well of siRNA oligomer in a six-well plate. The transfection rate at 24 h after transfection was examined by using fluorescence microscopy (Nikon E400). 2.8. Statistical analysis Data were expressed as the mean ± standard deviation. All statistical analysis was performed by SPSS for windows (SPSS, Inc., Chicago, IL). Student’s t-test was used to compare the statistical significance of the difference in data from the two groups; P < 0.05 was considered statistically significant. All experiments were performed in three times.
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3. Results 3.1. Inhibition of DES on 16HBE cells proliferation The 16HBE cells were treated with various doses of DES (0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM) for 0, 12, 24 or 36 h. MTT assay was carried out to determine the effects of DES on 16HBE cells proliferation. As exhibited in Fig. 1, 16HBE cells proliferation was slightly inhibited by 0.5 and 1.0 mM of DES (at 36 h), moderately inhibited by 2.0 mM of DES (at 12, 24 and 36 h), and markedly inhibited by 4.0 and 8.0 mM of DES (at 12, 24 and 36 h). These results indicated that DES inhibited 16HBE cells proliferation in a dose- and timedependent behavior. In addition to a decrease in cell proliferation, the results in Fig. 1 also indicated that cell death was induced by 4.0 (at 24 and 36 h) and 8.0 (at 12, 24 and 36 h) mM of DES. 3.2. DES-induced apoptosis in 16HBE cells The 16HBE cells were treated with various doses of DES for 36 h. DES-induced apoptosis was determined by using a flow cytometer. As shown in Fig. 2, during the observation period, only 4.0 and 8.0 mM of DES induced significant apoptosis. These results indicated that the inhibition effects of 4.0 and 8.0 mM of DES on cell proliferation was partly due to DES-induced apoptosis. To explore the possible molecular mechanisms underlying DESinduced apoptosis, the cellular levels of apoptosis related proteins p53, Bax, Bcl-2 and caspase-3 (including full length procaspase-3 and cleavage fragment caspase-3 p17/p11) were determined by Western blotting assay. As shown in Fig. 3, the cellular levels of
Fig. 5. The cellular levels of p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 in G1, S and G2/M 16HBE cells treated with 2.0 mM of DES at 20 and 28 h. (A) Representative immunoblot obtained with p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 antibodies. (B) Densitometric analysis of (A). The value of DMSO control at 20 h was set to 1; each value represents a mean ± standard deviation of three experiments. ⁄P < 0.05, ⁄⁄P < 0.01 compared with DMSO control at the same time point.
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p53, Bax and cleavage fragment caspase-3 p17/p11 increased significantly when the cells were treated with DES. Compared to the DMSO control cells, the cellular levels of Bcl-2 and full length procaspase-3 decreased in 8.0 mM of DES-treated cells.
3.3. Effects of DES on cell cycle progression in 16HBE cells To observe the effects of DES on cell cycle progression in 16HBE cells, a time-course study was conducted by continuously treating the cells with 2.0 mM of DES for various time intervals and cell cycle distribution profiles were recorded by flow cytometer. As shown in Fig. 4A–C, DES induced S and G2/M phase accumulation in 16HBE cells. S phase accumulation occurred from 12 to 32 h; G2/ M phase accumulation occurred from 24 to 32 h. In order to identify the reasons for the S and G2/M phase accumulation, Exit%, a parameter introduced previously [13], was determined. Exit% may reflect the kinetic profiles of cell cycle progression. The cell counting and Exit% calculating results were shown in Fig. 4D–G. DES inhibited cell proliferation in a time-dependent manner (Fig. 4D) and disrupted cell cycle kinetic characters (Fig. 4E–G). The increas-
ing of Exit%G1 and the decreasing of Exit%S and Exit%G2/M were observed in DES-treated cells, which indicated that DES accelerated the transition of 16HBE cells from G1 to S phase, and arrested S and G2/M progression. All these results demonstrated that both DES-induced G1/S transition acceleration and S arrest resulted in S phase accumulation, and that DES-induced G2/M arrest resulted in G2/M phase accumulation.
3.4. Alterations of DNA damage checkpoint proteins in 16HBE cells after DES treatment To identify whether DNA damage checkpoints are involved in DES-induced cell cycle arrest, the cellular levels of p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 after DES treatment were determined by Western blotting assay when the cells were treated with 2.0 mM of DES at 20 and 28 h. The results were shown in Fig. 5. In DES-treated G1 cells, the levels of p-chk1 (Ser345) and p-chk2 (Thr68) decreased at 20 h, the levels of chk1 increased at 28 h, the levels of chk2 increased significantly at 20 and 28 h. In DES-treated S cells, the levels of p-chk1 (Ser345), chk1 and chk2 in-
Fig. 6. Effects of P53 siRNA transfection on the cellular levels of p53 in 16HBE cells. The 16HBE cells were transfected with P53 siRNA, 24 h later treated with 0, 2.0 or 4.0 mM of DES for 20, 28 or 36 h. After the treatment, the cellular levels of p53 were determined by Western blotting assay. (A) SiRNA transfection rate (above 90%) at 24 h after transfection. (B) and (D) Representative immunoblot obtained with p53 antibody. (C) and (E) Densitometric analysis of (B) and (D), respectively. The value of DMSO-treated 16HBE cells in untransfected (MOCK) group was set to 1; each value represents a mean ± standard deviation of three experiments. ⁄⁄P < 0.01 compared with DMSO. #P < 0.05, ## P < 0.01 compared with MOCK.
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creased significantly at 20 and 28 h, the levels of p-chk2 (Thr68) increased significantly at 20 h. In DES-treated G2/M cells, the cellular levels of p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 increased significantly at 20 and 28 h. These results indicated that DES activated the DNA damage checkpoints in S and G2/M cells. 3.5. Effects of P53 down-regulation by RNAi on DES-induced apoptosis and cell cycle arrest in 16HBE cells To determine the roles of p53 in DES-induced apoptosis and cell cycle arrest, the 16HBE cells were transfected with P53 siRNA, 24 h later treated with 0, 2.0 or 4.0 mM of DES for 20, 28 or 36 h. The apoptosis and cell cycle distribution profiles were determined by flow cytometer. The results were shown in Figs. 6–8. Compared to the untransfected (MOCK) cells, the cellular levels of p53 decreased in both DMSO- and 4.0 mM of DES-treated cells at 36 h (Fig. 6B and C). The down-regulation of p53 by RNAi was concurrent with the significant increasing of apoptosis (both early and late apoptosis) in 4.0 mM of DES-treated cells (Fig. 7). In addition, the expression of p53 was suppressed by RNAi in both DMSO- and 2.0 mM of DES-treated cells at 20 and 28 h (Fig. 6D and E), but the cell cycle distribution was not affected (Fig. 8).
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4. Discussion DES-induced DNA damage and chromosomal aberrations in mammalian cells have been well investigated [2–5]. Besides DNA and chromosome, centrosome was demonstrated to be a subcellular target of DES toxicity action in our previous study [6]. Considering the contribution of centrosome amplification in mammalian cell division cycle, we speculated that cell cycle progression might be another target of DES toxicity action. To verify this hypothesis, we investigated the effects of DES on cell proliferation, cell cycle progression and apoptosis in human bronchial epithelial 16HBE cells. Through continuous observation of the cell cycle distribution, we found that DES induced S and G2/M phase accumulation in 16HBE cells. It is noted that the definitions of cell cycle distribution and cell cycle progression are different. The alteration of cell cycle distribution can only indicate that cell cycle progression might be disrupted. To determine what type of disruption (acceleration or arrest), further analysis is required. As we previously pointed out, the increasing of S phase fraction (S phase accumulation) is maybe due to one of the reasons listed below: (a) S arrest; (b) acceleration of the transition from G1 to S phase; (c) both (a) and (b) [13]. Like-
Fig. 7. Effects of suppression of P53 by RNAi on DES-induced apoptosis in 16HBE cells. The 16HBE cells were transfected with P53 siRNA, 24 h later treated with 0, 2.0 or 4.0 mM DES for 36 h. After the treatment, the cells were labeled with Annexin V-FITC and PI, and then analyzed using a flow cytometer. (A) One flow cytometry analysis representative of three individual experiments. Annexin V-FITC and PI signals were shown as FL1-H and FL2-H, respectively. The results are presented as the percentages of cells that were early apoptosis (Annexin V+ PI), late apoptosis (Annexin V+ PI+), and necrosis (Annexin V PI+). (B) Results of three individual experiments. Each value represents a mean ± standard deviation of three experiments. ⁄P < 0.05, ⁄⁄P < 0.01 compared with DMSO. #P < 0.05, ##P < 0.01 compared with MOCK.
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Fig. 8. Effects of suppression of P53 by RNAi on DES-induced cell cycle arrest in 16HBE cells. The 16HBE cells were transfected with P53 siRNA, 24 h later treated with DMSO or 2.0 mM of DES for 20 or 28 h. Cell cycle distribution was determined by flow cytometer. ⁄⁄P < 0.01 compared with DMSO control at the same time point.
wise, the increasing of G2/M phase fraction (G2/M phase accumulation) is maybe due to G2/M arrest and/or acceleration of the transition from S to G2 phase. Therefore, it is not enough to evaluate the influence of DES on cell cycle kinetics only by using cell cycle distribution results. Exit% is a parameter for evaluating cell cycle kinetics introduced previously, denotes how many percent of some phase cells enter next phase during a period. For example, if Exit%S is 30%, that means 30% of S phase cells entered G2 phase (exited S phase) during a period. Details about Exit% have been well explained in our previous paper [13]. By using parameter Exit%, we identified the reasons for S and G2/M phase accumulation in DES-treated cells. DES accelerated the transition of 16HBE cells from G1 to S phase, and arrested S and G2/M progression. Thus the S phase accumulation was due to DES-induced G1/S transition acceleration and S arrest, the G2/M phase accumulation was due to DES-induced G2/M arrest. The activation of DNA damage checkpoint is one of the mechanisms arresting cell cycle progression. In human cells, chk1 and chk2 are the most important signal transducers with strictly signal transduction function in cell cycle regulation and checkpoint responses. Phosphorylation on Ser345 in chk1 and/or phosphorylation on Thr68 in chk2 are the main characters of DNA damage checkpoint activation [7,9,14]. In the current study, we found that the cellular levels of p-chk1 (Ser345), chk1, p-chk2 (Thr68) and chk2 increased in DES-treated S and G2/M cells, which indicated that the DNA damage checkpoints were activated. The activation of DNA damage checkpoints should be responsible for DES-induced S and G2/M arrest in 16HBE cells. DNA damage checkpoint-mediated cell cycle arrest (S and G2/M arrest) and cdk2-cyclin A-mediated abnormal centrosome amplification [6] might be important mechanisms for DES-induced aneuploidy. In addition, we found that the cellular levels of p-chk1 (Ser345) and p-chk2 (Thr68) decreased in DES-treated G1 cells, which indicated that the DNA damage checkpoint was inactivated. The significance of the inactivation of DNA damage checkpoint in DES-treated G1 cells and the mechanisms of DES-induced G1/S transition acceleration need further investigation.
Besides the activation of DNA damage checkpoint, apoptosis is another DNA damage response reaction. It eliminates heavily damaged or seriously deregulated cells. In this study, only high doses of DES (4.0 and 8.0 mM) induced significant apoptosis in 16HBE cells at 36 h. DES-induced apoptosis was concurrent with the increasing of Bax and the cleavage fragment caspase-3 p17/p11 and the decreasing of Bcl-2 and full length procaspase-3, which indicated that DES induced apoptosis through evoking apoptosis pathway. Based on the detection results of cell cycle and apoptosis, we can conclude that upon DES exposure the activation of DNA damage checkpoint is earlier than the evoking of apoptosis programs. It is well known that p53 is a critical regulator of apoptosis and cell cycle arrest/pro-survival. Upon DNA damage, p53 evokes both cell cycle arrest/pro-survival and apoptosis transcriptional programs. The ultimate cellular outcome depends on the balance of these two programs [15]. With the increase in the dose of DES, the cellular levels of p53 first increased (0.5 to 4.0 mM) and then decreased (8.0 mM). And moreover, down-regulation of p53 by RNAi increased 4.0 mM of DES-induced apoptosis. These results indicated that p53 might act as a protector to promote cell survival when the cells were exposed to DES. It should be noted that this study focuses on exploring the possible mechanisms of the apoptosis and cell cycle disturbance induced by DES in 16HBE cells. The concentrations (2–8 mM) that DES induces those effects are determined through the pre-experiments. They are relatively high. In occupational emergencies and pollution accidents, the primary route of human exposure to DES is inhalation. Because of lacking sufficient relevant toxicokinetics data, it is difficult to achieve the extrapolation between the target concentrations in the bronchial epithelium and the concentrations in the exposure air. And moreover, many factors such as exposure duration, respiratory rate and depth might affect the target concentrations of human bronchial epithelial cells exposure to DES by inhalation. However, it is noteworthy that the workers involved in the production of ethanol by the strong-acid process might be exposed to high concentrations of DES in some cases. Based on the presence of approximately 30% DES in acid extracts, the maxi-
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mum vapor concentration over a spill is calculated as 2000 ppm [1], which is much higher than the lethal concentration (inhalation of 500 ppm of DES for 4 h caused 6/6 deaths in rats) of DES by inhalation [16]. In summary, DES inhibits 16HBE cells proliferation in a doseand time-dependent behavior. Within the sublethal dose, DES induces S and G2/M arrest through activating DNA damage checkpoints. Within the lethal dose, DES induces apoptosis through evoking apoptosis programs. p53 might play an important role in the transition between evoking cell cycle arrest/pro-survival and apoptosis programs upon DES exposure. Conflicts of interest statement The authors declare no conflict of interest. Acknowledgements This work was supported by National Nature Science Foundation of China (Nos. 81001253, 30972502, and 81172693), Beijing Natural Science Foundation (No. 7132122) and Fundamental Research Funds for the Central Universities (No. BMU20090460). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cbi.2013.06.014. References [1] National Toxicology Program, Diethyl sulfate, Rep. Carcinog. 12 (2011) 161– 163. [2] G.R. Hoffmann, Genetic effects of dimethyl sulfate, diethyl sulfate, and related compounds, Mutat. Res. 75 (1980) 63–129.
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[3] International Agency for Research on Cancer, Agents Classified by the IARC Monographs, Volumes 1–100, 2010. Available from: http://monographs.iarc.fr/ ENG/Classification/ClassificationsAlphaOrder.pdf. [4] M. Nüsse, S. Viaggi, S. Bonatti, Induction of kinetochore positive and negative micronuclei in V79 cells by the alkylating agent diethylsulphate, Mutagenesis 4 (1989) 174–178. [5] M. Campagna, P. Beffy, R. Del Carratore, L. Hauri, S. Simi, S. Bonatti, M. Simili, Diethylsulphate and methylnitrosourea affect different targets in Chinese hamster fibroblasts: possible mechanisms of aneuploidy induction by these agents, Mutagenesis 18 (2003) 405–410. [6] E. Hu, J. Fu, P. Zhao, B. Yao, Y. Qi, Z. Yuan, Z. Zhou, Diethyl sulfate induced Cdk2dependent centrosome amplification in CHL cells, Toxicology 275 (2010) 58– 64. [7] A. Sancar, L.A. Lindsey-Boltz, K. Unsal-Kaçmaz, S. Linn, Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints, Annu. Rev. Biochem. 73 (2004) 39–85. [8] A. Poehlmann, A. Roessner, Importance of DNA damage checkpoints in the pathogenesis of human cancers, Pathol. Res. Pract. 206 (2010) 591–601. [9] H.C. Reinhardt, M.B. Yaffe, Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2, Curr. Opin. Cell Biol. 21 (2009) 245–255. [10] A.L. Cozens, M.J. Yezzi, K. Kunzelmann, T. Ohrui, L. Chin, K. Eng, W.E. Finkbeiner, J.H. Widdicombe, D.C. Gruenert, CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells, Am. J. Respir. Cell Mol. Biol. 10 (1994) 38–47. [11] Y. Pang, W. Li, R. Ma, W. Ji, Q. Wang, D. Li, Y. Xiao, Q. Wei, Y. Lai, P. Yang, L. Chen, S. Tang, Y. Lin, Z. Zhuang, Y. Zheng, W. Chen, Development of human cell models for assessing the carcinogenic potential of chemicals, Toxicol. Appl. Pharmacol. 232 (2008) 478–486. [12] 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-epoxidetransformed and normal human bronchial epithelial G0/G1 cells, Chem. Biol. Interact. 186 (2010) 166–173. [13] P. Zhao, W. Zhong, X. Ying, Z. Yuan, J. Fu, Z. Zhou, Manganese chloride-induced G0/G1 and S phase arrest in A549 cells, Toxicology 250 (2008) 39–46. [14] T.H. Stracker, T. Usui, J.H. Petrini, Taking the time to make important decisions: the checkpoint effector kinases Chk1 and Chk2 and the DNA damage response, DNA Repair (Amst) 8 (2009) 1047–1054. [15] X. Zhang, Y. He, K.H. Lee, W. Dubois, Z. Li, X. Wu, A. Kovalchuk, W. Zhang, J. Huang, Rap2b, a novel p53 target, regulates p53-mediated pro-survival function, Cell Cycle 12 (2013) 1279–1291. [16] TOXNET/Hazardous Substances Data Bank (HSDB), DIETHYLSULFATE. Available from: http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen?HSDB.