Abstract Benzo[a]pyrene (B[a]P) is an environmental carcinogen found in tobacco smoke. It leads to high levels of DNA adducts in the lungs of cigarette smokers contributing to genomic instability. Alterations in the mitotic spindle apparatus play a major role in the generation of genomic instability through promoting chromosome mis-segregation and aneuploidy. To date, the effect of B[a]P exposure on altering the mitotic apparatus in normal lung epithelial cells remains unknown. In our study, BEAS-2B human bronchial epithelial cells were exposed to B[a]P and spindle dynamics were evaluated. Confocal imaging showed that B[a]P exposure significantly alters spindles misorientation, leading to chromosome mis-segregations in the form of chromosome lags and bridges. In addition, centrosome duplication and premature centriole disengagement were induced leading to misaligned and multipolar spindle formation. Comparative genomic analysis of mitotic spindle associated genes, revealed downregulation of AurA-Plk1-AurB signaling cascade by B[a]P. In addition, we analyzed the status of p53 and its downstream p21 in B[a]P-treated cells and showed a suppression of p53-p21 axis. When the extent of DNA damage associated with induced mitotic abnormalities was investigated using γ-H2AX, a significant increase and persistence in DNA damage was observed. Overall, our findings show that B[a]P potently induces mitotic abnormalities, DNA damage, and genetic instability. Aneuploidy, genomic instability, mitotic spindle defects, DNA damage Smoking is the major cause of lung cancer worldwide. Cigarette smoke is a complex chemical mixture containing around 5700 different compounds of which 76 are classified as carcinogenic in laboratory animals or humans (Hecht, 2012). Benzo[a]pyrene (B[a]P) is one of the major carcinogens present in cigarette smoke and, its DNA damaging effect has been shown by the relatively high B[a]P-DNA adduct level in the lungs of cigarette smokers (Izzotti et al., 1991). An early event in carcinogenesis is the induction of the genomic instability phenotype [genomic rearrangements/aneuploidy] which is considered a hallmark of cancer (Negrini et al., 2010). One of the theories proposed explaining the mechanism associated with genomic instability is alterations in mitotic spindle apparatus. Such alterations lead to asymmetric segregation of chromosomes and generation of chromosome instability in preneoplastic and cancer cells. Mitosis is a highly regulated event in which the duplicated genome is compacted into chromatid pairs, captured by kinetochore-microtubules from opposite spindle poles and aligned at the metaphase plate until segregated to form 2 identical daughter cells. Failure of such segregation may result in loss or gain of 1 or more chromosomes leading to aneuploidy—an early event and a hallmark of malignant cells (Durrbaum and Storchova, 2016). Precise control of the cell division plane is achieved through the proper assembly, positioning, and orientation of the centrosomes and the microtubule-based spindle (Bornens, 2012). During the cell cycle, centrosomes duplicate only once during S phase to ensure that at mitotic onset a cell has 2 centrosomes that will form the poles of the mitotic spindle (Nigg, 2007). Centrosome amplification—a frequent event in cancer—is linked to tumorigenesis and aneuploidy (Lingle et al., 1998; Nigg, 2002) and is associated with supernumerary centrosomes and chromosomal instability (Chan, 2011; Lingle et al., 1998; Pihan et al., 1998). These changes may be due to the activation of oncogenic kinases that control centrosome duplication (Lens et al., 2010) and/or the loss of tumor suppressor genes (Fukasawa et al., 1996; Meraldi et al., 2002). Several mechanisms lead to errors in chromosome segregation such as chromosome misalignments (Tame et al., 2016), defective centrosome duplication and overly stable attachments of microtubules to chromosomes (Bakhoum et al., 2009a,b). All these mechanisms result in chromosome lagging (Bakhoum et al., 2009a,b; Ganem et al., 2009; Thompson and Compton, 2008). Previous studies reported that lagging chromosomes induce DNA damage as evidenced by the formation of γ-H2AX foci (Janssen et al., 2011). Mitotic kinases play a major role in orchestrating bipolar spindle establishment, chromosome alignment and segregation. Among them, Aurora kinases play a prominent role as essential regulators of the mitotic spindle (Lens et al., 2010; Vader et al., 2008). Aurora B inhibition allows mitotic progression with mis-segregated chromosomes (Piekorz, 2010), on the other hand, cells lacking Aurora A activity show chromosome misalignment, chromosome segregation and aneuploidy. Recent reports show that abnormal mitosis alone is sufficient to generate DNA damage (Ganem and Pellman, 2012). Whether B[a]P induces mitotic abnormalities that could result in further genomic instability remains unknown. We have previously reported that the cytogenetic genomic instability markers are strong predictors of lung cancer risk with high positive and negative predictive values associated with disease status (El-Zein et al., 2008, 2006, 2014). We showed that the events observed are antecedent to development of lung cancer, not associated with tumor progression and can therefore serve as markers for early detection. We have also reported that genomic instability (in the form of both structural and numerical abnormalities) among lung cancer patients is nonrandom and certain areas in the genome are consistently involved in the tumorigenic process. This in turn may provide a tool for identification of individuals highly susceptible to tobacco carcinogens (Lloyd et al., 2013). Here, we further our understanding of the underlying mechanisms associated with the development of genomic instability in response to exposure to the potent tobacco smoke carcinogen, B[a]P. Our hypothesis is that B(a)P exposure induces genetic instability through alteration in the mitotic spindle apparatus leading to abnormal chromosome segregation and increase in DNA damage. MATERIALS AND METHODS Cells and Cell Culture Human bronchial epithelial cells BEAS-2B were obtained from ATCC, Manassas, VA (Cat# ATCC-CRL-9609). The flasks/dishes/plates were coated with a mixture of 0.01 mg/ml fibronectin (Sigma), 0.03 mg/ml PureCol (Sigma, St. Louis, MO), and 0.01 mg/ml bovine serum albumin (Sigma) dissolved in BEBM at 37 °C for at least 2 h. BEAS-2B cells were cultured in Bronchial Epithelial Growth Medium (BEGM) completed growth medium containing 100 U penicillin/ml and 100 μg/ml streptomycin. BEGM medium is BEBM medium supplement with 0.5 ng/ml human recombinant epidermal growth factor, 50 μg/ml bovine pituitary extract, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10 μg/ml transferrin, 0.5 μg/ml epinephrine, 0.1 ng/ml retinoic % air, and 5% CO2 until reaching 80% confluency. Cells were then harvested and plated for consequent B[a]P exposure. A total of 0.05% trypsin/EDTA solution (HyClone, Pittsburgh, PA) was used to perform cell subculture. Cell Synchronization To synchronize BEAS-2B cells in mitosis, a double thymidine block was used. Cells were incubated in thymidine (2.5 mM) for 24 h at 37 °C and released from thymidine for 14 h, and treated with 2.5 mM thymidine for another 24 h. To obtain cells in early S phase, cells were released in fresh medium for 2 h. Benzo[a]Pyrene Treatment The B[a]P (Sigma cat # B-1760) was solubilized in DMSO and the working solution of 10 µM concentration was prepared in cell culture media. BEAS-2B cells were seeded into 96-well plates at a density of 1 × 104 cells/well and were treated at various time points namely 24, 48, and 72 h. The B[a]P concentration used was selected on the basis of reported literature (Zhu et al., 2014) and our cytotoxicity study using XTT (ATCC). At the 10 µM B[a]P concentration, cytotoxicity analysis using XTT assay for all time points, revealed 85% cell viability (Supplementary Figure 1). The mitotic abnormalities were examined at each time point and the results compared with those of untreated cells (control). The DMSO (0.1% of final volume) was used as a control in all experiments. The treatment was conducted under low light and the experiment was repeated three times. Immunofluorescence and Antibodies for Spindle Apparatus Detection Immunofluorescence was performed following standard protocols (Schafer-Hales et al., 2007; Zhang et al., 2008). Briefly, cells were allowed to adhere overnight on no. 1.5 coverslips placed in tissue culture plates. Cells were fixed in PHEMO buffer, consisting of 3.7% formaldehyde, 0.05% glutaraldehyde, 60 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], 25 mM HEPES, 10 mM EGTA, and 4 mM MgSO4, for 10 min. Cells were then washed in phosphate-buffered saline (PBS) and blocked with 10% bovine serum albumin for 15 min. After being washed with PBS, the coverslips were incubated with the appropriate primary antibody overnight at 4 °C. Cells were washed again with PBS, and the appropriate Alexa Fluor-conjugated secondary antibody was added at 1:500 for 1 h at room temperature. Antibody incubations were then sequentially repeated for each additional primary-secondary-antibody pair. Primary antibodies were used as follows: Monoclonal mouse anti-α-tubulin 1:1000 (Sigma-T-9026) and rabbit polyclonal antipericentrin (Abcam-ab4448, Cambridge, MA). Secondary antibodies were Alexa Fluor 488 or 555 (Invitrogen, Waltham, MA) at a dilution of 1:500 and incubated for 1 h at room temperature. Nuclear staining was performed by incubating cells with DAPI (4′,6-diamidino-2-phenylindole) containing mounting media (Vectashield). Confocal Imaging Image acquisition and analysis For fixed-cell experiments, fluorescence image acquisition was performed using a Nikon A1R confocal imaging system controlled by the Nikon NIS Elements software (Nikon, Melville, NY). The objective lens was an oil immersion Plan-Apo_60 numerical aperture (NA) 1.40 lens (Nikon). Images were acquired as Z-stacks at 0.2-mm intervals and maximum-intensity projections were generated using the NIS Elements software (Nikon). Confocal z stacks were acquired with sections ranging from 0.5 to 1.1 μm. Image acquisition settings were kept constant throughout the experiments. Spindle orientation, centroid measurements, and image analysis Spindle angle measurements were adapted from our previously published method (Thaiparambil et al., 2012). Briefly, the three-dimensional (3D) distance (across the x, y, and z planes) between the 2 spindle poles and the 2D distance (across the x and y planes) of the spindle were measured. The spindle angle was then calculated using the cos−1 (arccosine). Image analysis was done using confocal z stacks with Nikon NIS Elements software (Nikon), using the 3D length tool. Spindle pole centroid measurements were performed using Metamorph software. Images from confocal z stacks of tubulin were first thresholded and then binarized. The binarized image was then outlined by the software, and the centroid was calculated. A line was then drawn between the 2 centroids of the spindle poles (Figure 1B). Figure 1. View largeDownload slide Benzo[a]pyrene exposure results in mis-oriented spindle. A, Immunofluorescence images of BEAS-2B cells with pericentrin (green), tubulin (red) and DNA (DAPI-blue) staining. Confocal z sections of benzo[a]pyrene exposed and unexposed spindles beginning at the top of the spindle and sectioning toward the bottom. Numbers in the top left indicate the z distance from the top. Arrows indicate spindle poles. Scale bar = 5 μm. Control panel shows 2 spindle poles in the same plane at z 5.97, whereas in the benzo[a]pyrene exposed panel, the first pole appears at z 5.37 and the second pole at z 7.76 indicating that the spindle poles are rotated. B, Schematic of how the angle of rotation was determined and calculated. C, Significantly different mean angle of rotation is observed in benzo[a]pyrene-exposed cells when compared with unexposed cells (p < .001). Data are the mean ± SD from 3 independent experiments. Figure 1. View largeDownload slide Benzo[a]pyrene exposure results in mis-oriented spindle. A, Immunofluorescence images of BEAS-2B cells with pericentrin (green), tubulin (red) and DNA (DAPI-blue) staining. Confocal z sections of benzo[a]pyrene exposed and unexposed spindles beginning at the top of the spindle and sectioning toward the bottom. Numbers in the top left indicate the z distance from the top. Arrows indicate spindle poles. Scale bar = 5 μm. Control panel shows 2 spindle poles in the same plane at z 5.97, whereas in the benzo[a]pyrene exposed panel, the first pole appears at z 5.37 and the second pole at z 7.76 indicating that the spindle poles are rotated. B, Schematic of how the angle of rotation was determined and calculated. C, Significantly different mean angle of rotation is observed in benzo[a]pyrene-exposed cells when compared with unexposed cells (p < .001). Data are the mean ± SD from 3 independent experiments. Centrosome disengagement We used pericentrin antibody to stain centrosomes or centrioles (mentioned above) and centriole numbers were analyzed in 100 metaphase cells (mitotic cells) per concentration/time point using confocal microscopy. The number of centrioles was counted to quantify the number of cells with centriole disengagement based on previous reports (Martino et al., 2015). Mitotic cells with engaged centrioles have 2 centrosomes with 2 centrioles each, whereas cells with evidence of disengagement either have 3 centrosomes (1 with 2 centrioles and 2 with single centrioles); or 4 centrosomes each with a single centriole. Multipolar spindles The percentage of multipolar spindles was determined as those that contained more than 2 spindle poles. The additional spindle poles were counted by pericentrin staining in 100 metaphase cells/concentration per time. Real-time PCR In order to identify the genes associated with mitotic spindle defects and chromosome mis-segregation, we designed a custom made 96-well plate (Bio-Rad) on a CFX96 Touch Real-Time (RT) PCR Detection System (Bio-Rad). Genes included on the plate are presented in Supplementary Figure 2. Total RNA was extracted from BEAS-2B cells (QIAGEN Kit# 74104) according to the manufacturer’s instructions. The RT-PCR was performed using the iScript cDNA synthesis kit (Bio-Rad # 1708841) using 500 ng RNA and Sso advanced Universal SYBR Green Supermix (# 172-5271). The following cycle was used: 95 °C for 10 min (1 cycle), 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s for 40 cycles and then, 95 °C for 15 s and 60 °C for 1 min. Specificity of the PCR products was confirmed by melt curve analysis. Data were normalized to Ct values of GAPDH and β-actin from the same sample and the fold-changes in the expression of each gene were calculated using the ΔΔCt method. Analysis of data was performed using CFX Manager Software (Bio-Rad). Protein Extraction and Western Blot Analysis To further confirm the RT-PCR results, we performed Western blot analysis on selected gene products identified from the mitotic spindle plate. Cells were lysed and centrifuged. Samples containing equal amounts of protein (50 μg) were separated by SDS-PAGE and electroblotted onto PVDF membrane (Bio-Rad). Immunoblotting was performed using γ-H2AX, Aurora A, Aurora B, PLK1, p53, p21, cyclin B1, and β-actin (Abcam) as a loading control. After a brief incubation with enhanced chemiluminescence, the blots on the membrane were exposed to X-ray film. Each Western blot analysis was repeated at least 3 times. Assessment of DNA Damage Using γ-H2AX We investigated the extent of DNA damage in B[a]P-treated cells using γ-H2AX (a well-established DNA double strand break marker). γ-H2AX was determined by both Western blot and confocal microscopy. Mouse monoclonal antibody (Mab) to γ-H2AX (3F2) phospho S139+S140d (Abcam-ab22551) was added at 1:500. Nuclear staining was performed using DAPI (4′,6-diamidino-2-phenylindole) containing mounting media (Vectashield). To quantitate γ-H2AX, 100 cells were evaluated and the number of foci/per cell was recorded. In addition the percentage of cells/field containing γ-H2AX foci was recorded. Statistical Analysis Statistical analysis was conducted using Prism 7 (GraphPad Software Inc.). The B[a]P-treated samples were compared with untreated (control) samples by using two-way ANOVA analysis. We used Bonferroni’s multiple comparison test to analyze the significance at each time point after B[a]P exposure. For the spindle angle, the samples were compared by using two-tailed unpaired t test. Differences were considered significant with a p-value <.05. RESULTS Benzo[a]Pyrene Exposure and Spindle Orientation Because spindle orientation plays a major role in chromosome segregation and cytokinesis, we investigated the effect of B[a]P on spindle orientation. Immunofluorescence analysis using confocal planar z sections of tubulin (Figure 1A), shows in control cells a bipolar spindle that was oriented parallel to the substratum, with spindle poles in similar z planes. In contrast, B[a]P-treated mitotic cells had bipolar spindles that appeared to be misoriented relative to the substratum, whereby spindle poles were in drastically different confocal z planes. Quantitation of the angle of spindle rotation shows that in control cells the mean angle of spindle pole orientation was 18°, compared with 38° in benzo[a]pyrene-treated cells (Figs. 1A and 1C) (p < .001). These findings suggest that B[a]P-exposed cells have misoriented spindles relative to the substratum. Benzo[a]Pyrene Induces Centrosome Amplification and Disengagement During mitosis, 2 centrosomes form spindle poles and direct the formation of bipolar mitotic spindles, which is essential for accurate chromosome segregation into daughter cells (Fukasawa et al., 1996). After B[a]P exposure, cells with more than 2 centrosomes were observed (Figure 2A[b]) indicating that B[a]P induces centrosome amplification. Because centrosome amplification can be caused by the splitting of a centriole pair as well as by centrosome re-duplication (Fukasawa, 2007), we evaluated centriole profiles in B[a]P-induced supernumerary centrosomes using pericentrin antibodies. Vehicle-treated mitotic cells contained 2 centrosomes, each with a pair of centrioles. On the other hand, B[a]P-treated cells had undergone a premature centriole disengagement which led to multipolar spindles with single centrioles at individual poles (Figs. 2A[a] and 2A[c]). The majority of (90%) vehicle-treated mitotic cells contained only 2 centrosome spots per cell (Figure 2A[b]—control). However, a significant proportion (20%) of B[a]P-treated mitotic cells had more than 2 spots per cell (Figs. 2A[b] and 2A[c]), strongly suggesting that multipolarity was in fact due to abnormal amplification of centrosomes that clearly contained centrioles. Figure 2. View largeDownload slide Immunofluorescence images of BEAS-2B cells with pericentrin (green), tubulin (red), and DNA (DAPI-blue) staining. Scale bar = 10 μm. A, (a) Unexposed control mitotic cell showing 2 centrosomes located at opposite poles (white arrows, pericentrin column) that produce bipolar spindles (tubulin column) and organize the chromosomes along the metaphase plate (merged column); whereas benzo[a]pyrene exposed mitotic cell shows 1 normal centrosome at 1 pole and disengagement with 2 centrioles (white arrows, pericentrin column) on the opposite pole leading to centrosome disengagement (white arrows, Tubulin column); (b) Centrosome amplification in response to benzo[a]pyrene exposure leading to multipolar spindles; (c) Significantly increased percentage of mitotic abnormalities in the benzo[a]pyrene exposed versus nonexposed cells in the form of centrosome disengagement (p < .001) and multipolar spindle formation (p < .001) at the different time points. Data are the mean ± SE from 3 independent experiments with 100 cells counted per dose. B, (a) Well-aligned metaphase chromosomes in unexposed cells when compared with metaphase chromosome lagging in benzo[a]pyrene exposed cells due to misaligned chromosomes (white arrows); (b) Anaphase chromosome lagging (white arrow) and bridge formation (yellow arrow) in exposed cells; (c) telophase lagging (white arrow) and bridge formation (yellow arrow) in exposed cells; (d) significantly increased percentage of segregation errors in benzo[a]pyrene exposed versus nonexposed cells in the form of metaphase misalignment, anaphase lagging and telophase lagging (p < .001) at the different time points. Data are the mean ± SE from 3 independent experiments with 100 cells counted per dose. Figure 2. View largeDownload slide Immunofluorescence images of BEAS-2B cells with pericentrin (green), tubulin (red), and DNA (DAPI-blue) staining. Scale bar = 10 μm. A, (a) Unexposed control mitotic cell showing 2 centrosomes located at opposite poles (white arrows, pericentrin column) that produce bipolar spindles (tubulin column) and organize the chromosomes along the metaphase plate (merged column); whereas benzo[a]pyrene exposed mitotic cell shows 1 normal centrosome at 1 pole and disengagement with 2 centrioles (white arrows, pericentrin column) on the opposite pole leading to centrosome disengagement (white arrows, Tubulin column); (b) Centrosome amplification in response to benzo[a]pyrene exposure leading to multipolar spindles; (c) Significantly increased percentage of mitotic abnormalities in the benzo[a]pyrene exposed versus nonexposed cells in the form of centrosome disengagement (p < .001) and multipolar spindle formation (p < .001) at the different time points. Data are the mean ± SE from 3 independent experiments with 100 cells counted per dose. B, (a) Well-aligned metaphase chromosomes in unexposed cells when compared with metaphase chromosome lagging in benzo[a]pyrene exposed cells due to misaligned chromosomes (white arrows); (b) Anaphase chromosome lagging (white arrow) and bridge formation (yellow arrow) in exposed cells; (c) telophase lagging (white arrow) and bridge formation (yellow arrow) in exposed cells; (d) significantly increased percentage of segregation errors in benzo[a]pyrene exposed versus nonexposed cells in the form of metaphase misalignment, anaphase lagging and telophase lagging (p < .001) at the different time points. Data are the mean ± SE from 3 independent experiments with 100 cells counted per dose. As tightly regulated centrosome duplication is crucial to proper spindle biogenesis, we examined whether aberrant centrosomes contributed to the formation of multipolar spindles. Normal cells contain 1 (nonduplicated) or 2 (duplicated) centrosomes as shown by pericentrin staining. Multipolar spindles were observed as early as 2 h with a steady increase in number 9%–15% and 17% at 48 and 72 h, respectively, in B[a]P-exposed cells (p < .0001) compared with untreated vehicle control (Figure 2A[c]). Similarly, the majority of vehicle-treated mitotic cells showed only a pair of centrioles at each spindle pole, however, more than a pair of centrioles were present at each spindle pole upon B[a]P treatment. These findings suggest that B[a]P treatment causes centrosome amplification that could in turn nucleate the formation of multipolar spindles. Benzo[a]Pyrene Induces Chromosome Segregation Defects Multiple mitotic defects can increase the frequency of chromosome mis-segregation, resulting in chromosome loss and aneuploidy. To determine the extent of mis-segregation, cells were stained for α-tubulin and DAPI for DNA, and chromosome lags and bridges were recorded. We observed that B[a]P treatment led to a significant increase of misaligned chromosomes at metaphase (Figure 2B[a]) as well as lagging chromosomes and formation of bridges in anaphase and telophase spindle (Figs. 2B[b] and 2B[c]) at each time point compared with untreated vehicle control (p< .0001). Interestingly, our results showed a steady increase in chromosome lagging and bridge formation from 9% at 2 h to > 20% at 48 and 72 h (Figure 2B[d]) suggesting that in the presence of B[a]P, chromosomes were misaligned at metaphase. Benzo[a]Pyrene Downregulates Key Players in Spindle Assembly and Chromosome Separation Pathways Our RT-PCR and Western blot results confirmed a B[a]P induced significant reduction of Aurora A-Aurora B-PLK1 at the 48 and 72 h time points compared with untreated cells suggesting that B[a]P causes chromosome misegregation and cytokinesis failure. In addition, our data showed, a down regulation in the Aurora A-PLK1 cascade, which plays a crucial role in centrosome biogenesis and spindle formation (Joukov et al., 2014) (Figs. 3A and 3B). Figure 3. View largeDownload slide Gene expression profiles of key genes regulating mitotic spindle dynamics using RT2 arrays. A, Benzo[a]pyrene exposure downregulated the expression of Aurora A, Aurora B and PLK1. Aurora A and PLK1 showed close to a 3-fold change in expression; data are the mean ± SE from 3 independent experiments. B, Western blot analysis of Aurora A, Aurora B, and PLK-1 confirmed the RT-PCR data. C, Suppression of p53, p21, and cyclinB1 in exposure to denzo[a]pyrene. D, Western blot analysis of p53, p21, and cyclinB1. Figure 3. View largeDownload slide Gene expression profiles of key genes regulating mitotic spindle dynamics using RT2 arrays. A, Benzo[a]pyrene exposure downregulated the expression of Aurora A, Aurora B and PLK1. Aurora A and PLK1 showed close to a 3-fold change in expression; data are the mean ± SE from 3 independent experiments. B, Western blot analysis of Aurora A, Aurora B, and PLK-1 confirmed the RT-PCR data. C, Suppression of p53, p21, and cyclinB1 in exposure to denzo[a]pyrene. D, Western blot analysis of p53, p21, and cyclinB1. Assessment of DNA Damage and Signaling Pathways Both DNA damage extent (reflected by the number of cells with γ-H2AX foci) and DNA damage intensity (reflected by the number of γ-H2AX foci/cell) were measured in B[a]P-exposed cells. We counted the number of γ-H2AX foci present in each cell nucleus as shown in Figure 4A and quantitated the percentage of cell having γ-H2AX foci or percentage of foci/cell. A significant increase in DNA damage was observed throughout the time points in the B[a]P-treated cells when compared with the untreated controls (p < .0001) (Figs. 4B and 4C). Using Western blot, our data confirmed these findings showing a significant increase of H2AX phosphorylation at 48 and 72 h time points (Figure 3B). Western blot analysis also showed a significant down regulation of p53 and p21 and a progressive degradation of cyclin B1 at 48 and 72 h time points explaining the perpetuation of cells with DNA damage through the cell cycle (Figs. 3C and 3D). The XTT results for cell viability confirmed the survival of cells with DNA damage (Supplementary Figure 1). Figure 4. View largeDownload slide Benzo[a]pyrene-induced DNA damage was measured using γ-H2AX. A, The number of γ-H2AX foci observed in control and exposed cells; scale bar = 10 μm. B, Western blot analysis of γ-H2AX at different time points with a gradual increase in intensity. C, Extent of DNA damage assessed as the percentage of cells with γ-H2AX foci at the different experimental time points; D, intensity of DNA damage assessed as the number of γ-H2AX foci per cells (<10 and 10+) at the different experimental time points. Data are the mean ± SD from 3 independent experiments with 100 cells counted per dose. Figure 4. View largeDownload slide Benzo[a]pyrene-induced DNA damage was measured using γ-H2AX. A, The number of γ-H2AX foci observed in control and exposed cells; scale bar = 10 μm. B, Western blot analysis of γ-H2AX at different time points with a gradual increase in intensity. C, Extent of DNA damage assessed as the percentage of cells with γ-H2AX foci at the different experimental time points; D, intensity of DNA damage assessed as the number of γ-H2AX foci per cells (<10 and 10+) at the different experimental time points. Data are the mean ± SD from 3 independent experiments with 100 cells counted per dose. DISCUSSION Aneuploidy is the most frequently identified genomic abnormality in cancer. Some carcinogens may generate aneuploidy by chemically or physically altering one or more of the chromosomes or the proteins of the spindle apparatus, leading to asymmetric segregation of chromosomes and generation of chromosome instability. In this study, we demonstrated that one of the major tobacco smoke carcinogens, B[a]P induces persistent mitotic abnormalities in human bronchial epithelial cells. Proper spindle orientation is required for development, cell fate, and tissue organization by ensuring an accurate distribution of genetic material and a normal division plane. A recent report by Tame et al. (2016) shows that chromosome misalignment induces spindle-positioning defects that may be due to depletion of kinetochore proteins. Our data demonstrate that B[a]P induces spindle mis-orientation that could interfere with proper alignment of chromosomes and ultimately, leads to chromosome mis-segregation. The link between spindle misorientation and aneuploidy maybe explained by 2 mechanisms: First, spindle orientation and chromosome segregation both require the end-on interactions of a subset of microtubules with other cellular structures (Pease and Tirnauer, 2011). B[a]P induction of misoriented spindle could alter the microtubule plus end regulators and lead to chromosome mis-segregation and aneuploidy. Secondly, spindle misorientation might facilitate the development of aneuploidy through its effects on cytokinesis (Pease and Tirnauer, 2011). Previous studies have demonstrated that spindle misorientation induced by RNAi of APC also causes a failure of cytokinesis, resulting in tetraploidy (Caldwell et al., 2007). This is well correlated with a recent report that spindle misorientation alone is unlikely to be tumorigenic, but it has the potential to synergize with cancer-associated changes to facilitate genomic instability, tissue disorganization, metastasis, and expansion of cancer stem cell compartments (Pease and Tirnauer, 2011). Overall, our result shows that the induction of misoriented spindle might be an early event that leads to B[a]P-induced mitotic abnormalities. We further investigated the effect of B[a]P-induced misoriented spindle on mitosis. Our data show that B[a]P induces aberrant centrosome amplification and multipolar spindles which in turn increases the frequency of aberrant mitoses and chromosome segregation errors in the form of centrosome amplification, disengagement, chromosome lags, and bridges. Such abnormalities correlated well with the observed downregulation of key genes associated with spindle dynamics. An interesting finding was the persistence of the mitotic abnormalities and down regulation of spindle-associated genes, especially Aurora A and B, throughout the experiment suggesting that B[a]P may have a major impact on mitosis. Centrosome abnormalities have been found in various cancers. Our data show that B[a]P induces supernumerary centrosome in BEAS-2B cells that are p53 competent through down regulation which has been previously reported in response to arsenite exposure (Ochi et al., 2003). Previous studies also report that p21 deficiency stimulates centriole overduplication (Duensing et al., 2006) which is supportive of our observations of aberrant centrosome number and suppression of p21 in response to B[a]P exposure. These data suggest that the ability of B[a]P to promote centrosome hyper-amplification and mitotic aberration may contribute to genomic instability and aneuploidy. Polo-like kinase 1 (PLK1) is a key regulator of centrosome maturation (Bettencourt-Dias and Glover, 2007; Gruneberg et al., 2004) and its deregulation is linked to centrosome abnormalities and oncogenesis (Zyss and Gergely, 2009). Our real time and Western blot data suggest that the reduced expression of PLK-1, together with the downregulation of Aurora A and B, result in centrosome disengagement and the formation of aberrant mitotic spindles and chromosome segregation errors. In our study, B[a]P treatment induced lagging chromosome and chromatin bridges in anaphase and telophase. It has been shown that the induction of misoriented spindle could cause merotelic conformation and lead to lagging chromosome in anaphase. Direct analysis of human tumor cell lines with chromosome instability shows that the most common mitotic defect is lagging chromosomes in anaphase (Thompson and Compton, 2008). The mitotic defect in chromosome instability cell lines represents a single chromatid that fail to segregate because the kinetochore is attached to spindle microtubules that are oriented toward opposite spindle poles in a merotelic conformation (Thompson and Compton, 2008). Thus, it is critical that merotelic kinetochores be corrected prior to anaphase onset to ensure error-free chromosome segregation. Merotelic attachments lead to lagging chromosomes at anaphase (Ganem et al., 2009) resulting in anaphase bridges that interfere and delay cytokinesis. If the bridge is resolved and cytokinesis completed, the lagging chromosomes becomes a micronucleus in one of the daughter cells. Alternatively, if the bridge is not resolved, the outcome is cytokinesis failure and polyploidy. In our study, we observed a persistent increase in the number of anaphase and telophase lagging chromosomes and bridges after B[a]P exposure (Figure 2B[b] and 2B[c]) as well as the presence of polyploidy (data have not shown) strongly suggesting the induction of aneuploidy in response to B[a]P. Previous studies showed that prolonged mitotic arrest causes DNA damage, as identified by detection of the phosphorylated histone variant H2AX (γ-H2AX) (Dalton et al., 2007). Our result shows an elevated level of γ-H2AX foci at 48 and 72 h, indicating that the B[a]P-induced DNA damage is unrepaired and could induce genome instability and promote tumorigenesis. The DNA repair is tightly coordinated with cell cycle progression through the activation of orchestrated signaling pathways that are often termed DNA damage checkpoints (Harper and Elledge, 2007; Harrison and Haber, 2006). In response to unrepaired DNA damage, these pathways delay or stop the cell cycle at critical stages before or during DNA replication (G1/S and intra-S checkpoints) and before cell division (G2/M checkpoint), thereby preventing duplication and segregation of damaged DNA. In our study, exposure to B[a]P led to a significant reduction in the level of Aurora A and Aurora B which may have led to mitotic exit delay and mitotic slippage, respectively. Previous studies using Aurora inhibitors showed that inhibition of AURKB alone is sufficient to trigger mitotic slippage (Girdler et al., 2006; Harrington et al., 2004). Another study reports that only when both AURKA and AURKB are deleted, cells exit mitosis without anaphase (Hegarat et al., 2011). Our RT-PCR data show depletion of both AURKA and AURKB and is consistent with the Western blot data revealing downregulation of both Auroras. Therefore, suppressing both AURKA and AURKB could potentially be one of the mechanisms by which BaP-treated cells escape mitosis whereas harboring mitotic abnormalities. Another important observation in our study was that depletion of p53-p21 axis at checkpoints allowing the damaged cells to proceed into the G2 phase. Several studies reported that the loss of p21 generates polyploidy, especially after treatment with DNA damaging agents or irradiation or when the mitotic spindle is disrupted (Bunz et al., 1998; Lanni and Jacks, 1998; Stewart et al., 1999). Loss of p21 prolongs the duration of mitosis and results in severe mitotic defects such as chromosome mis-segregation and cytokinesis failures promoting genomic instability (Kreis et al., 2015; Lo et al., 2012). Because p53-p21 axis acts as a guardian for a faithful G2/M transition through maintaining the G2 checkpoint and centrosome integrity. Interrupting this pathway could induce mitotic slippage and retains mitotic defects leading to additional genomic instability. These reported findings provide a plausible mechanism for the observed genetic instability in our BEAS-2B normal lung epithelial cells in response to B[a]P exposure. In addition, it has been previously reported (Brito and Rieder, 2006) that mitotic slippage occurs via cyclin B destruction at the G2/M checkpoint further allowing damaged cells to proceed into mitosis. This supports our observed reduction in cyclin B1 (by Western blot) and the persistent DNA damage (by γ-H2AX) further contributing to genome instability. The B[a]P is a well-known mutagen (Jeng and Bocca, 2013; Nebert et al., 2013) (Hainaut et al., 2001) that has been implicated in the etiology of lung cancer among cigarette smokers (Hainaut et al., 2001; Petruzzelli et al., 1998; Pfeifer et al., 2002; Smith et al., 2000). Mutations associated with B[a]P exposure have been reported in TP53 in the form of G → T transversions with hotspots in codons 157, 248, and 273 of the gene (Hainaut et al., 2001; Pfeifer et al., 2002). The mutagenic and genotoxic effect of B[a]P are due to the induction of DNA adducts resulting in DNA damage and genetic instability (Moorthy et al., 2015). However, to date, B[a]P-induced genetic instability through alterations in mitotic spindle apparatus has not been reported. Our study shows that this major tobacco carcinogen has the potential to contribute to genomic instability through induction of a cascade of mitotic abnormalities namely: Spindle misorientation, aberrant centrosome amplification, and chromosome mis-segregation. This is further supported by the demonstrated suppression of the Aurora A-Plk1-Aurora B signaling cascade which plays a major role in spindle assembly, centrosome maturation and chromosome segregation. In conclusion, our data provide evidence that exposure of normal human bronchial epithelial cells to B[a]P leads to the induction of alterations in the different components of the mitotic spindle apparatus which in turn generates genomic instability not only through induction of DNA damage but also through induction of chromosome loss and aneuploidy. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING The work was supported by Institutional funds from Houston Methodist Research Institute. REFERENCES Bakhoum S. F., Genovese G., Compton D. A. ( 2009a). Deviant kinetochore microtubule dynamics underlie chromosomal instability. Curr. Biol. 19, 1937– 1942. Google Scholar CrossRef Search ADS Bakhoum S. F., Thompson S. L., Manning A. L., Compton D. A. ( 2009b). Genome stability is ensured by temporal control of kinetochore-microtubule dynamics. Nat. Cell Biol. 11, 27– 35. Google Scholar CrossRef Search ADS Bettencourt-Dias M., Glover D. M. ( 2007). 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Published: Mar 1, 2018
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