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Bisphenol A disrupts mitotic progression via disturbing spindle attachment to kinetochore and centriole duplication in cancer cell lines
Toxicology in Vitro 59 (2019) 115–125
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Bisphenol A disrupts mitotic progression via disturbing spindle attachment to kinetochore and centriole duplication in cancer cell lines
T
Seul Kima,1, Dasom Gwona,1, Jeong Ah Kimb,c, Hanl Choib,d, Chang-Young Janga,
⁎
a
Drug Information Research Institute, College of Pharmacy, Sookmyung Women's University, Seoul 04310, Republic of Korea Hana Academy Seoul, Seoul 03305, Republic of Korea c Department of Chemistry, Sungkyunkwan University, Suwon 16419, Republic of Korea d College of Software, Sungkyungkwan University, Suwon 16419, Republic of Korea b
ARTICLE INFO
ABSTRACT
Keywords: Bisphenol A Mitosis Spindle attachment Multipolar spindle Centriole Chromosome instability
Bisphenol A [BPA, 2,2-bis-(4-hydroxyphenyl)propane] is one of the most prevalent synthetic environmental estrogens; as an endocrine disruptor, it is associated with endocrine-related cancers including breast, ovarian, and prostate. However, the mechanisms by which BPA contributes to carcinogenesis are unclear. This study aims to clarify its toxic effects on mitotic cells and investigate the molecular mechanism. In vitro effects of BPA on mitotic progression were examined by performing experiments on HeLa cells. Proteins involved in mitotic processes were detected by Western blot, live cell imaging, and immunofluorescence staining. The results showed that BPA increased chromosomal instability by perturbing mitotic processes such as bipolar spindle formation and spindle microtubule attachment to the kinetochore. BPA prolonged mitotic progression by disturbing spindle attachment and concomitant activating spindle assembly checkpoint (SAC). Mechanistically, BPA interfered proper localization of HURP to the proximal ends of spindle microtubules, Kif2a to the minus ends of spindle microtubules, and TPX2 on the mitotic spindle. This mislocalization of microtubule associated proteins (MAPs) is postulated to lead to spindle attachment failure. Furthermore, BPA caused multipolar spindle by inducing centriole overduplication and premature disengagement. Although BPA acts as an estrogen receptor (ER) agonist, mitotic defects caused by BPA occurred in an ER-independent manner. Our findings indicate that BPA may stimulate carcinogenesis not only by acting as an endocrine disruptor but also by increasing chromosomal instability during mitosis.
1. Introduction Environmental hormones are deemed endocrine disruptors or hormone mimics, because these chemical substances can enter the body and interfere with the endocrine system in mammals by acting as hormone mimics. As one of prevalent environmental pollutants to exhibit estrogenic activity, bisphenol A [BPA, 2,2-bis-(4-hydroxyphenyl) propane] leaches from coatings on containers into foods or liquids stored therein, as well as from various dental materials (Brotons et al., 1995; Drozdz et al., 2011). It is considered a xenoestrogen, because BPA binds to estrogen receptors (ERs) and acts as an agonist or antagonist in estrogen signaling pathways (Gould et al., 1998). Although BPA activity for ERs is 10,000–100,000-fold weaker than that of estradiol (Welshons
et al., 2006), it can stimulate cellular responses at very low concentrations and concomitantly disrupt the endocrine system (CwiekLudwicka and Ludwicki, 2014). Given that its structure is similar to that of the highly potent estrogen receptor agonist, diethylstilbestrol, BPA is presumed to have effects on hormone sensitive tissues. In this regard, it has been reported that BPA exposure during early development increases the incidence of infertility, genital tract abnormalities, and precocious puberty (LaRocca et al., 2011; Munoz-de-Toro et al., 2005; Wang et al., 2014). Also, BPA has also been shown to play a role in the pathogenesis of endocrine disorders including such metabolic disorders such as polycystic ovary syndrome and hormone-related cancers such as breast and prostate cancer (Diamanti-Kandarakis et al., 2009). Tumorigenesis in humans is a multistep process comprised of steps
Abbreviation: BPA, Bisphenol A [2,2-bis-(4-hydroxyphenyl)propane]; CIN, chromosomal instability; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride; DMSO, dimethyl sulfoxide; ER, estrogen receptor; HRP, horseradish peroxidase; KT, kinetochore; MAP, microtubule associated protein; MT, microtubule; MTOC, microtubule-organizing center; SAC, spindle assembly checkpoint; SDS, sodium dodecyl sulfate ⁎ Corresponding author. E-mail address: [email protected] (C.-Y. Jang). 1 Seul Kim and Dasom Gwon have contributed equally to this study. https://doi.org/10.1016/j.tiv.2019.04.009 Received 30 January 2019; Received in revised form 9 April 2019; Accepted 9 April 2019 Available online 11 April 2019 0887-2333/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. BPA perturbs mitotic progression. (A and B) After treatment with 100 nM BPA for 5 h, HeLa cells were fixed with MeOH and stained with the indicated antibodies. Images are maximum projections from z stacks of representative cells stained for β-tubulin (red) and DNA (blue). Metaphase cells with unaligned chromosomes and mitotic cells with multipolar spindle were quantified and plotted (B). (C and D) HeLa S3 cells were synchronized by double thymidine (C; Thy-Thy) or thymidine-nocodazole (D; Thy-Noc) treatment and then released into fresh media. Cells were harvested at the indicated releasing times. Cells were analyzed by western blotting. Hsp90 served as a loading control. Asy, unsynchronized cells. (E and F) HeLa/GFP-histone H2B cells were transfected with 100 nM BPA for 5 h and time-lapse imaged for GFP-H2B. The duration of the periods from nuclear envelope breakdown (NEB) to the formation of a bipolar spindle/metaphase plate with some unaligned chromosomes (UC) (NEB to UC), from the unaligned state to metaphase (M) (UC to M), and from metaphase to anaphase onset (A) (M to A) were determined for control and ANKRD53-depleted cells (E; n = 50 cells). Images were captured every 3 min to monitor mitotic progression. Still frames were taken from time-lapse movies of representative cells (F). Arrowheads in (F) point to unaligned chromosomes. Error bars, SEM. Scale bars, 5 μm. *, p < 0.01 (two-tailed t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. BAP disrupts spindle attachment to kinetochore. (A and B) The maximum projections from z stacks of representative control or 100 nM BPA-treated cells stained for Mad2 (green), Hec1 (red), and DNA (blue) (A) are shown. Mad2 signals on kinetochores were quantified in five control or BPA-treated cells (A) at prometaphase (PM), metaphase (M), and unaligned chromosomes (UC; n > 50 kinetochores for each quantification). Error bars show SEM. (C and D) The maximum projections from z stacks of representative control or 100 nM BPA-treated cells stained with CREST (green), BubR1 (red), and DNA (blue) are shown. BubR1 signals on kinetochores in (C) were quantified in five control or BPA-treated cells at prometaphase (PM), metaphase (M), and unaligned chromosomes (UC; n > 50 kinetochores for each quantification). AU, arbitrary units. Scale bar, 5 μm. Error bars show SEM. *, p < 0.01 (two-tailed t-test relative to control cells). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 117
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that reflect genetic alterations such as mutations in oncogenes with dominant gain-of-function or tumor suppressor genes with recessive loss-of-function (Sieber et al., 2005). These genetic alterations drive the progressive transformation of normal cells into highly malignant cancer cells by rendering novel capabilities including self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis (Hanahan and Weinberg, 2000). To prevent the accumulation of genetic changes, animal cells have evolved to monitor their genomes before transition to the next cell cycle phase with G1/S and G2/M checkpoints (Sullivan and Morgan, 2007). In addition to DNA damage checkpoints, cells also monitor chromosome segregation during mitosis to ensure the faithful transmission of sister chromatids to two daughter cells with the spindle assembly checkpoint (SAC) (Musacchio and Salmon, 2007). During prometaphase, the centrosome nucleates spindle microtubules (MTs) to search for and capture kinetochores (KTs) on condensed sister chromatids (Pihan, 2013). At the same time, duplicated centrioles separate in opposite directions to form a bipolar spindle which allows for the structural basis of bi-oriented attachments of MTs to KTs (Gadde and Heald, 2004). The major task of SAC is to ensure accurate chromosome segregation by stabilizing proper bi-oriented attachments, in which one sister KT is captured by MTs from one spindle pole and the other sister by those from the opposite pole, and by resolving inappropriate attachments such as syntelic attachments, in which both sister KTs are captured by MTs from the same pole (Kline-Smith and Walczak, 2004; Silva et al., 2011). To this end, Mad2 and BubR1, as SAC components, check spindle MT attachment to KT and tension between sister-KTs, respectively (Skoufias et al., 2001). Consequently, defective mitotic progression increases chromosomal instability (CIN) which is a feature of most human cancers and drives tumor initiation and progression via the acquisition of genomic alterations through a small rate of chromosome gain and loss (Levine and Holland, 2018). In carcinogenesis, a moderate amount of CIN results in aneuploidy and enables a cell to deregulate gene expression, amplify oncogene, delete tumor suppressor gene, and accumulate a number of mutations (Gibbs, 2003). In contrast, large amount of CIN can suppress tumor progression because it is detrimental to the survival pathways and ultimately induces apoptosis (Dabas et al., 2012). In addition, CIN acts as a driving force for metastasis through chronic inflammation caused by micronuclei (Bakhoum et al., 2018). Thus far, both of ERα and ERβ have been found to exhibit mitotic functions distinct from the established role of ERs as transcription factors in that ERβ interacts with the SAC protein, Mad2, and ERα localizes on the spindle and spindle pole to regulate chromosome alignment and spindle dynamics (Poelzl et al., 2000; Zhang et al., 2015). As an ER binding xenoestrogen, BPA also induces cell cycle delay by altering spindle MT organization and chromosome alignment, thereby inducing aneuploidy during meiosis and mitosis (Adamakis et al., 2013; 2016; Can et al., 2005; George et al., 2008; Hunt et al., 2003; Ochi, 1999; Pfeiffer et al., 1997; Santovito et al., 2018). In contrast, exposure of mouse oocytes to BPA in vitro causes meiotic arrest by affecting spindle formation and chromosome alignment, but not aneuploidy (Eichenlaub-Ritter et al., 2008). Furthermore, BPA induces multiple microtubule-organizing centers (MTOCs) and multipolar spindle in mitosis (Adamakis et al., 2016; Ochi, 1999). The purpose of this study was to elucidate the molecular mechanisms underlying the disruption of chromosome alignment and centrosome integrity by BPA. Our data showed that BPA induced multipolar spindles and unaligned chromosomes in mitotic cells by interfering with the attachment of spindle MTs to KTs and the concomitant activation of the SAC. Mechanistically, BPA interferes with the localization of mitotic kinase Plk1 and MAPs, such as HURP, Kif2a, and TPX2. In addition, BPA caused centriole overduplication by increasing the level of SAS-6 protein and premature centriole disengagement. Thus, we conclude that BPA increases CIN by perturbing centrosome integrity and chromosome alignment in both meiosis and mitosis.
2. Materials and methods 2.1. Antibodies and chemicals The anti-HURP sera were raised against full-length recombinant HURP and affinity-purified (Wong and Fang, 2006). Anti-β-tubulin E7 monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank (DSHB, Iowa City, IA, USA). The following antibodies were obtained from commercial sources: anti-Hsp90, anti-CENP-E, antiERα, and anti-Cyclin B1, anti-Cyclin E, anti-Plk1 (Santa Cruz Biotechnology, Dallas, TX, USA); anti-γ-tubulin (Signal-Aldrich, St. Louis, MO, USA); anti-centrin-1, anti-c-NAP1 (Proteintech, Rosemont, IL, USA); anti-PLK4 (Merck Millipore, Darmstadt, Germany); anti-Mad2 (Pierce Antibodies; Thermo Fisher Scientific, Waltham, MA, USA); antiBubR1 (LifeSpan BioSciences, Seattle, WA, USA); anti-CREST (Antibodies Incorporated, Davis, CA, USA); Aurora B (Abcam, Cambridge, UK); anti-phospho-Aurora A (Cell Signaling Technology, Danvers, MA, USA); anti-TPX2 (Thermo Fisher Scientific, Waltham, MA, USA); antiEB1 (LifeSpan BioSciences, Nottingham, UK); anti-Hec1 (GeneTex, Irvine, CA, USA); Alexa Fluor-488 rabbit secondary antibody and Alexa Fluor-594 mouse secondary antibody (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA); and horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA). The following chemicals were obtained from commercial sources: Bisphenol A, dimethyl sulfoxide (DMSO), and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO, USA); Tris base, NaCl, HCl, and sodium dodecyl sulfate (SDS) (Amresco, Solon, OH, USA); paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA); bovine serum albumin (BSA) (BioShop, Burlington, ON, CA); Triton X-100, Mowiol, and N-propyl gallate (Fisher scientific); Bradford protein assay kit (Thermo Fisher Scientific); polyvinylidene difluoride (PVDF) membrane and methanol (MilliporeSigma, Burlington, MA, USA); and protein molecular weight standard for electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA). BPA was dissolved in DMSO at a concentration of 100 μM as a stock solution and diluted with culture medium for use. HeLa, MCF-7, and MDA-MB-231 cells were treated with 100 nM BPA for 5 h. 2.2. Cell culture HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM; WelGENE Inc., Daegu, South Korea) supplemented with 10% fetal bovine serum (FBS; Invitrogen/Thermo Fisher Scientific), penicillin (100 units/mL), and 100 μg/mL streptomycin (Invitrogen/ Thermo Fisher Scientific). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. Immunofluorescence HeLa cells were cultured on cover glasses and fixed with methanol at −20 °C for 30 min. Alternatively, cells were treated with BRB80-T buffer (80 mM PIPES, pH 6.8, 1 mM MgCl2, 5 mM EGTA, and 0.5% Triton X-100) before fixing with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were blocked with PBS-BT (1× PBS, 3% BSA, and 0.1% Triton X-100) for 30 min at room temperature and stained with primary and secondary antibodies diluted in PBS-BT for 30 min at room temperature. After final washes with PBS-BT, cells were mounted with Mowiol mounting solution (20% glycerol, 10% Mowiol, 1.2 M Tris, pH 8.5, 2% N-propyl gallate). Z-image stacks were acquired in 0.2 μm increments with AxioVision 4.8.2 (Carl Zeiss AG, Oberkochen, Germany) under a Zeiss Axiovert 200 M microscope using a 1.4 NA plan-Apo 100× oil immersion lens and an HRm CCD camera. Deconvolved images were obtained using an AutoQuant X3 (AutoQuant Imaging, Inc., Troy, NY, USA). All images are maximum projections from z stacks of representative cells stained for the indicated antigens. 118
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Fig. 3. BPA interferes with localization of microtubule-associated proteins. HeLa cells were fixed at 5 h post-treatment with 100 nM BPA and stained with the indicated antibodies. (A and B) The maximum projections from z stacks of representative control or BPA-treated cells stained for Aurora A, Aurora B, CENP-E or Plk1 (green), βtubulin or Hec1 (red), and DNA (blue) are shown. Plk1 signals on kinetochores were quantified and plotted (B). (C and D) The maximum projections from z stacks of representative control or BPA-treated cells stained for CENP-E, HURP or Kif2a (green), Hec1, EB1, or β-tubulin (red), and DNA (blue) are shown. (E) The maximum projections from z stacks of representative control or BPA-treated cells stained for TPX2 (green), β-tubulin (red), and DNA (blue) are shown. Images were acquired under constant exposure time for the TPX2 channel. Total immunofluorescence intensity for TPX2 on the metaphase spindle (n = 10 cells for each quantification) was quantified and plotted. AU, arbitrary units. Error bars, SEM. Scale bars, 5 μm. *, p < 0.01 (two-tailed t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Images for quantification were acquired under constant exposure in each channel for all of the cells.
aneuploidy (Wheeler et al., 1986). To investigate whether BPA also influences mitosis, we treated HeLa cells with BPA at concentrations ranging from 1 nM to 10 μM for 5 h and found the highest chromosome alignment defects and multipolar spindles in 100 nM BPA-treated cells (Fig. 1A, B). This dosage is less than the reference dose, 0.5 μg/mL, established by United States Environmental Protection Agency and similar to the higher concentration of unconjugated BPA found in human serum, 0.02 μg/mL, established by European Union (Santovito et al., 2018). To further examine the inhibitory effects of BPA on mitotic cell cycle progression, HeLa S3 cells were treated with double thymidine (Thy-Thy) and released into fresh media. Western blot analysis revealed that BPA prolonged the expression of mitotic regulators including cyclin B1, Plk1, and phosphorylated Aurora A after release from double thymidine arrest (Fig. 1C). Consistent with this result, the level of cyclin B1 was also prolonged by BPA after release from TN arrest (Fig. 1D), suggesting that BPA interferes with mitotic progression by perturbing chromosome alignment and bipolar spindle formation. To further investigate the mitotic defects caused by BPA treatment, we observed mitotic cells under a time-lapse experiment after BPA treatment (Fig. 1E, F, Video 1, 2). HeLa cells stably expressing GFP-histone H2B were treated with BPA and imaged every 3 min for 5 h. Consistent with previous data, treatment with BPA prolonged the duration of prometaphase [from nuclear envelope breakdown (NEB) to the formation of a metaphase plate with some unaligned chromosomes (UC)], the unaligned state [from UC to metaphase (M)], and metaphase [from metaphase to anaphase onset (A)]. Thus, we conclude that BPA induces mitotic delay by perturbing chromosome congression and bipolar spindle formation.
2.4. Live cell images For time-lapse microscopy, HeLa cells stably expressing GFP-H2B were plated on a cell imaging dish with a glass bottom and cultured in Leibovitz's L-15 medium (Invitrogen/Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen/Thermo Fisher Scientific) and 2 mM L-glutamine (Invitrogen/Thermo Fisher Scientific). Cells were placed into a growth chamber heated to 37 °C and observed on a Zeiss Axiovert 200 M microscope with a 20× lens. Images were acquired every 3 min for 5 h with AxioVision 4.8.2 (Carl Zeiss AG). 2.5. Western blotting Cells were harvested and lysed with NP-40 lysis buffer (50 mM Hepes, pH 7.4, 200 mM KCl, 0.3% NP-40, 10% glycerol, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, 0.5 μM microcystin, and 10 μg/ml concentrations of leupeptin, pepstatin, and chymostatin) on ice for 10 min and debris was removed by centrifugation. Protein concentrations in total cell lysates were measured using a Bradford protein assay kit before boiling in Laemmli buffer. The lysates were separated by a 12% SDS-PAGE gel and transferred to a PVDF membrane. Protein bands were detected using chemiluminescence blotting substrate (Roche, Basel, Switzerland). HRP-conjugated anti-mouse IgG or anti-rabbit IgG were used as secondary antibodies. 2.6. Colony forming assay
3.2. BPA interferes with spindle MT attachment to KTs and activates the SAC
Cells were treated with 100 nM BPA for five hours, washed with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.2) and trypsinized to produce a single-cell suspension. After counting the number of cells in the cell suspension, 150 cells were plated onto a dish and cultured in fresh media for 14 days to form colonies. The colonies were washed with PBS and stained with glutaraldehyde crystal violet mixture (6.0% glutaraldehyde and 0.5% crystal violet) for 30 min. After removing the glutaraldehyde crystal violet mixture and rinsing with water carefully, the dishes with colonies were dried in normal air at room temperature.
To investigate whether the unaligned chromosomes caused by BPA treatment resulted from a lack of MT attachment to the KT or tension between sister chromatids, we examined the activation of the SAC by analyzing the intensity of Mad2 and BubR1 with immunofluorescence staining. These proteins are KT-associated checkpoint proteins that control the metaphase-to-anaphase transition by inhibiting APC/C E3 ligase and concomitant degradation of securin to ensure chromosome integrity. Whereas Mad2 perceives the attachment between KT and MT, BubR1 acts as a sensor for the tension between sister KTs during mitosis (Musacchio and Salmon, 2007; Skoufias et al., 2001). Therefore, Mad2 and BubR1 are concentrated at KTs in prometaphase and released from KTs in metaphase after the completion of MT attachment to KT and the generation of inter-KT tension by formation of the bi-oriented spindle. Interestingly, Mad2 was absent from the aligned KTs but still resided in unaligned KTs in BPA-treated metaphase cells (Fig. 2A, B), suggesting that BPA interferes with chromosome alignment by interrupting spindle MT attachment to KTs. In contrast, BubR1 was observed in unaligned KTs but not in aligned KTs in BPA-treated metaphase cells (Fig. 2C, D), indicating that the SAC is inactivated by the generation of tension at aligned KTs and that BPA is not associated with spindle dynamics. The accumulation of BubR1 at unaligned KTs resulted from the absence of tension between sister KTs due to attachment failure. Thus, SAC activation delayed mitotic progression in BPA-treated cells.
2.7. Statistical analysis Data were analyzed using Student's t-test. Error bars represent the SE of three independent experiments. A p-value < 0.01 (two-tailed) was considered statistically significant. 3. Results 3.1. BPA causes unaligned chromosomes and multipolar spindles It has been reported that BPA has potential inhibitory effects on meiotic cell cycle progression and disrupts centrosome and spindle integrity (Can et al., 2005; Susiarjo et al., 2007). Furthermore, synthetic estrogens such as diethylstilbestrol are known to be spindle poisons that inhibits mitotic progression and while concurrently inducing 120
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Fig. 4. BPA perturbs centriole duplication cycle. (A and B) HeLa cells were treated with 100 nM BPA for 5 h and stained for centrin-1 (green), γ-tubulin (red), and DNA (blue). Mitotic cells with multipolar spindle or over-duplicated centrioles were quantified and plotted. (C) Five hours after BPA treatment, the cells were harvested and analyzed by western blotting with indicated antibodies. Hsp70 served as a loading control. (D and E) Shown are maximum projections from the deconvolved z stacks of representative HeLa cells stained for Centrin-1 (green), STIL, PLK4, or SAS-6 (red) and DNA (blue). Fluorescence intensity of STIL, PLK4, and SAS-6 was quantified and plotted (E). (F and G) HeLa cells stably expressing GFP-Centrin-1 were treated with 100 nM BPA for 5 h and stained for C-NAP1 (red) and DNA (blue). Cells with premature disengaged centrioles were quantified and plotted (G). Error bars, SEM. Scale bars, 5 μm. *, p < 0.01 (two-tailed t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.3. BPA interrupts proper localization of MT-associated proteins (MAPs) on the mitotic spindle
duplication initiators. We then investigated the effect of BPA treatment on centriole disengagement, as odd number of centrioles were found in overduplicated centrosome (Fig. 4A). As expected, the number of CNAP1 foci increased at centrosome in BPA treated cells (Fig. 4F, G), suggesting that BPA also induces premature centriole disengagement in G2 or early mitosis. Overall, these findings indicate that centriole overduplication and premature disengagement induced by BPA result in multipolar spindle in mitosis.
Given that BPA induces unaligned chromosome (Fig. 1A) and activates the SAC (Fig. 2), we hypothesized that it inhibits regulators of spindle MT attachment to KTs. To test this hypothesis, we investigated the localization of mitotic regulators, which are recruited to mitotic structures such as mitotic spindles, KTs, centromeres, and centrosomes. First, we examined the localization of mitotic kinases because they show distinct localization on mitotic structures. While Aurora A is recruited to centrosomes, Aurora B is concentrated in centromeres during mitosis. Polo-like kinase 1 (Plk1) localizes to centrosomes in early mitosis and transfers to KTs at metaphase and to central spindles at anaphase. Following treatment with BPA, the distinct localization of mitotic kinases was confirmed using immunofluorescence microscopy. Although Aurora A and Aurora B showed normal localization at the spindle pole and centromeres after BPA-treatment, respectively, Plk1 was substantially increased at unaligned KTs (Fig. 3A, B). Next, we examined the localization of plus-end tracking protein (+TIP), EB1, and plus end-directed kinetochore motor, CENP-E, because they are responsible for the congression of polar-localized chromosomes to the metaphase plate (Kapoor et al., 2006). In addition, depletion of CENP-E or EB1 causes a failure of chromosome alignment and generates a few unattached chromosomes around the spindle pole (Putkey et al., 2002). However, BPA did not affect on the localization of CENP-E and EB1 (Fig. 3C), suggesting that BPA does not disrupt the mechanical link between KTs and the plus-end tip of a dynamic MT. third, we confirmed the localization of MAPs on the mitotic spindle that are involved in chromosome congression. HURP (hepatoma upregulated protein)known to localize to the end of KT fiber and regulate chromosome congression (Ye et al., 2011)-was dispersed on the mitotic spindle in BPA-depleted cells (Fig. 3D). Kif2a, which has been identified as a minus-end MT depolymerase and is known to be involved in spindle dynamics and chromosome alignment around the spindle pole (Kwon et al., 2016), was also mislocalized on the mitotic spindle in BPAtreated cells (Fig. 3D). TPX2, a spindle assembly factor with prominent roles in local MT formation around chromatin (Gruss et al., 2002), was decreased on the mitotic spindle in BPA-treated cells (Fig. 3E). Thus, we conclude that BPA perturbs chromosome congression by disrupting the localization of mitotic regulators including Plk1, HUPP, Kif2a, and TPX2.
3.5. BPA stimulates tumorigenesis in both ER-positive and -negative breast cancer cells To investigate whether BPA induces mitotic defects by disrupting the transcriptional activity of ER or non-transcriptional function, we treated BPA to ER-positive or -negative breast cancer cells. As shown in Fig. 5A, BPA induced chromosome congression defects and multipolar spindles in BPA-treated ER-positive MCF-7 cells. Recently, it was reported that ERα localizes to the centrosome in prometaphase and the mitotic spindle in metaphase and regulates chromosome alignment in a transcription-independent manner (Zhang et al., 2015). Therefore, we confirmed the localization of ERα in BPA-treated MCF-7 cells and found that BPA did not affect the recruitment of ERα to the mitotic spindle and the localization of ERα on the spindle (Fig. 5B). Interestingly, BPA also induced chromosome congression defect in ER-negative NDA-MB231 cells (Fig. 5C and D), suggesting that BPA does not act as an endocrine disruptor in mitosis. Consistent with this, BPA increased the number and the size of colonies both in MCF-7 and MDA-MB-231 breast cancer cells (Fig. 5E and F). Taken together, BPA promotes tumor progression by perturbing mitotic progression in an ER-independent manner. 4. Discussion As an endocrine disrupting chemical, BPA is suspected as a potential risk factor for increasing cancer rates involving the human reproductive system, such as breast and prostate cancer (Yang et al., 2006). However, the direct effect of BPA exposure on the stimulation and initiation of cancer and the promotion of cancer aggressiveness has not been fully addressed. Given that ER plays a pivotal role in the growth and maturation of the mammalian oocyte (Susiarjo et al., 2007), brief exposures during fetal development to BPA affect prophase at the onset of oogenesis, chromosome segregation, and the formation of follicles in the female fetus (Hunt et al., 2012). Furthermore, ERα is involved in chromosome alignment and spindle dynamics by stabilizing microtubules during mitosis in an estrogen-independent manner (Zhang et al., 2015). Recently, it has been reported that treatment with lowdose BPA in utero increased the incidence of hepatic tumors or preneoplastic lesions (Weinhouse et al., 2014). Furthermore, delayed chromosome alignment to mitotic equator causes a defective bi-orientation establishment and concomitant chromosome missegregation (Kuniyasu et al., 2018). In this study, we showed that BPA induced chromosome alignment defects by interrupting the proper localization of spindle-associated proteins in an ER-independent manner, suggesting that BPA promotes tumorigenesis both in non-reproductive estrogentarget cells and in non-reproductive estrogen-non-target cells. Although meiotic processes such as bipolar spindle formation, alignment of homologous chromosomes at metaphase I and sister
3.4. BPA perturbs centriole duplication cycle by increasing SAS-6 Because BPA induced multipolar spindle (Figs. 1B, 4B), we sought to examine whether it causes centriole overduplication or pericentriolar material (PCM) fragmentation. We found that ~18% of BPA-treated cells have over 4 centrioles per each cell (Fig. 4A, B), indicating that BPA causes multipolar spindle by inducing centriole overduplication. To determine the mechanism underlying BPA-induced centriole overduplication, we examined the level of three proteins, such as PLK4, STIL, and SAS-6, which are involved in the initiation of centriole duplication (Arquint and Nigg, 2016). Interestingly, the level of PLK4 and STIL slightly increased but SAS-6 substantially increased in BPA-treated cells (Fig. 4C). In accordance with this, the level of SAS-6 markedly increased at centrioles (Fig. 4D, E), suggesting that BPA provokes centriole overduplication by disrupting the level of centriole 122
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Fig. 5. BPA promotes tumorigenesis in an ERα-independent manner. (A) MCF-7 cells were treated with 100 nM BPA and stained for DNA (blue). (B) Shown are maximum projections from the deconvolved z stacks of representative MCF-7 cells stained for ERα (green), β-tubulin (red) and DNA (blue). (C and D) MDA-MB-231 cells were treated with 100 nM BPA and stained for ERα (green), β-tubulin (red) and DNA (blue). Metaphase cells with unaligned chromosome and mitotic cells with multipolar spindle were quantified and plotted (D). (E and F) MCF-7 or MDA-MB-231 cells were treated with 100 nM BPA. 150 cells were incubated for 14 days to form a colony, and were stained with glutaraldehyde crystal violet mixture. The number of colonies in control or BPA-treated cells were counted and plotted (F). Error bars, SEM. *, p < 0.01 (two-tailed t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
chromatids at metaphase II, and chromosome segregation in anaphase I differ from mitotic processes in somatic cell division, the SAC plays as a safe guard mechanism to avoid aneuploidy in meiosis (Vogt et al., 2008). In mitosis, the SAC monitors the attachment of the sister KTs to spindle MTs, that emanated from opposite poles, to establish sister KT
bi-orientation for faithful sister chromatid segregation (Musacchio and Salmon, 2007). However, homologous chromosomes are linked together by synaptonemal complex to form a bivalent which consists of 4 chromatids during meiosis I. In contrast to mitosis and meiosis II, sister KTs attach to MTs from the same pole and establish mono-orientation in 123
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meiosis I (Sun and Kim, 2012; Vogt et al., 2008). Consequently, the SAC discriminates different tension-generating configuration in meiosis I and meiosis II and delays anaphase onset until all chromosomes have achieved the right orientation. In this respect, the canonical chromosome segregation machinery appears to be adapted in meiosis. The molecular mechanism by which BPA induces spindle attachment defect and concomitant SAC activation in meiosis remains to be elucidated. Because of the accurate function in cellular processes, the structure and number of centrosomes are tightly regulated throughout cell cycle (Meraldi and Nigg, 2002). Therefore, the formation of multipolar spindles is involved in causing unequal segregation of the chromosomes because the multipolar division results in aneuploidy. Furthermore, centrosome abnormalities such as centrosome amplification-dependent or –independent multipolarity are frequently observed in carcinoma cells (Lingle et al., 1998; Pihan et al., 1998). In this regard, centrosome hypertrophy may cause mitotic abnormalities and uncontrolled proliferation in carcinogenesis. It has also been reported that BPA induces multipolar spindles in mammalian and plant cells (Adamakis et al., 2016; Ochi, 1999). Recently, it has been established that PLK4, STIL, and SAS-6 play important roles in initiation of centriole duplication (Arquint and Nigg, 2016). Consistent with this, we found that BPA induces centriole overduplication via increasing the level of SAS-6 at the centrosome (Fig. 4C–E). In summary, BPA increases chromosomal instability by disturbing spindle MT attachment to KTs and centriole duplication in an ER-independent manner. Consequently, BPA exposure increases a cancer susceptibility not only in estrogen target cells but also in estrogen nontarget cells. Although we showed the mislocalization of mitotic regulators including Plk1, HURP, Kif2a, and TPX2 caused by BPA-treatment, the direct target proteins of BPA and the detailed mechanisms involved will be determined in further experiments. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.tiv.2019.04.009.
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Author contributions CYJ conceived and supervised the study; SK and DSK designed experiments and performed experiments; JAK and HC conceived the study and analyzed data; CYJ wrote the manuscript. Acknowledgement This study was supported in part by grants from the National Research Foundation of Korea (NRF-2015R1A2A2A01005500 and NRF2018R1D1A1B07050933). References Adamakis, I.D.S., Panteris, E., Cherianidou, A., Eleftheriou, E.P., 2013. Effects of Bisphenol A on the microtubule arrays in root meristematic cells of Pisum sativum L. Mutat. Res. Gen. Tox. En. 750, 111–120. Adamakis, I.D., Panteris, E., Eleftheriou, E.P., 2016. Bisphenol A disrupts microtubules and induces multipolar spindles in dividing root tip cells of the gymnosperm Abies cephalonica. Chemosphere 149, 202–210. Arquint, C., Nigg, E.A., 2016. The PLK4-STIL-SAS-6 module at the core of centriole duplication. Biochem. Soc. T 44, 1253–1263. Bakhoum, S.F., Ngo, B., Laughney, A.M., Cavallo, J.A., Murphy, C.J., Ly, P., Shah, P., Sriram, R.K., Watkins, T.B.K., Taunk, N.K., Duran, M., Pauli, C., Shaw, C., Chadalavada, K., Rajasekhar, V.K., Genovese, G., Venkatesan, S., Birkbak, N.J., McGranahan, N., Lundquist, M., LaPlant, Q., Healey, J.H., Elemento, O., Chung, C.H., Lee, N.Y., Imielenski, M., Nanjangud, G., Pe'er, D., Cleveland, D.W., Powell, S.N., Lammerding, J., Swanton, C., Cantley, L.C., 2018. Chromosomal instability drives metastasis through a cytosolic DNA response. Nature 553, 467–472. Brotons, J.A., Olea-Serrano, M.F., Villalobos, M., Pedraza, V., Olea, N., 1995. Xenoestrogens released from lacquer coatings in food cans. Environ. Health Perspect. 103, 608–612. Can, A., Semiz, O., Cinar, O., 2005. Bisphenol-A induces cell cycle delay and alters centrosome and spindle microtubular organization in oocytes during meiosis. Mol. Hum. Reprod. 11, 389–396. Cwiek-Ludwicka, K., Ludwicki, J.K., 2014. Endocrine disruptors in food contact materials; is there a health threat? Rocz. Panstw. Zakl. Hig. 65, 169–177.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Bisphenol A disrupts mitotic progression via disturbing spindle attachment to kinetochore and centriole duplication in cancer cell lines
T
Seul Kima,1, Dasom Gwona,1, Jeong Ah Kimb,c, Hanl Choib,d, Chang-Young Janga,
⁎
a
Drug Information Research Institute, College of Pharmacy, Sookmyung Women's University, Seoul 04310, Republic of Korea Hana Academy Seoul, Seoul 03305, Republic of Korea c Department of Chemistry, Sungkyunkwan University, Suwon 16419, Republic of Korea d College of Software, Sungkyungkwan University, Suwon 16419, Republic of Korea b
ARTICLE INFO
ABSTRACT
Keywords: Bisphenol A Mitosis Spindle attachment Multipolar spindle Centriole Chromosome instability
Bisphenol A [BPA, 2,2-bis-(4-hydroxyphenyl)propane] is one of the most prevalent synthetic environmental estrogens; as an endocrine disruptor, it is associated with endocrine-related cancers including breast, ovarian, and prostate. However, the mechanisms by which BPA contributes to carcinogenesis are unclear. This study aims to clarify its toxic effects on mitotic cells and investigate the molecular mechanism. In vitro effects of BPA on mitotic progression were examined by performing experiments on HeLa cells. Proteins involved in mitotic processes were detected by Western blot, live cell imaging, and immunofluorescence staining. The results showed that BPA increased chromosomal instability by perturbing mitotic processes such as bipolar spindle formation and spindle microtubule attachment to the kinetochore. BPA prolonged mitotic progression by disturbing spindle attachment and concomitant activating spindle assembly checkpoint (SAC). Mechanistically, BPA interfered proper localization of HURP to the proximal ends of spindle microtubules, Kif2a to the minus ends of spindle microtubules, and TPX2 on the mitotic spindle. This mislocalization of microtubule associated proteins (MAPs) is postulated to lead to spindle attachment failure. Furthermore, BPA caused multipolar spindle by inducing centriole overduplication and premature disengagement. Although BPA acts as an estrogen receptor (ER) agonist, mitotic defects caused by BPA occurred in an ER-independent manner. Our findings indicate that BPA may stimulate carcinogenesis not only by acting as an endocrine disruptor but also by increasing chromosomal instability during mitosis.
1. Introduction Environmental hormones are deemed endocrine disruptors or hormone mimics, because these chemical substances can enter the body and interfere with the endocrine system in mammals by acting as hormone mimics. As one of prevalent environmental pollutants to exhibit estrogenic activity, bisphenol A [BPA, 2,2-bis-(4-hydroxyphenyl) propane] leaches from coatings on containers into foods or liquids stored therein, as well as from various dental materials (Brotons et al., 1995; Drozdz et al., 2011). It is considered a xenoestrogen, because BPA binds to estrogen receptors (ERs) and acts as an agonist or antagonist in estrogen signaling pathways (Gould et al., 1998). Although BPA activity for ERs is 10,000–100,000-fold weaker than that of estradiol (Welshons
et al., 2006), it can stimulate cellular responses at very low concentrations and concomitantly disrupt the endocrine system (CwiekLudwicka and Ludwicki, 2014). Given that its structure is similar to that of the highly potent estrogen receptor agonist, diethylstilbestrol, BPA is presumed to have effects on hormone sensitive tissues. In this regard, it has been reported that BPA exposure during early development increases the incidence of infertility, genital tract abnormalities, and precocious puberty (LaRocca et al., 2011; Munoz-de-Toro et al., 2005; Wang et al., 2014). Also, BPA has also been shown to play a role in the pathogenesis of endocrine disorders including such metabolic disorders such as polycystic ovary syndrome and hormone-related cancers such as breast and prostate cancer (Diamanti-Kandarakis et al., 2009). Tumorigenesis in humans is a multistep process comprised of steps
Abbreviation: BPA, Bisphenol A [2,2-bis-(4-hydroxyphenyl)propane]; CIN, chromosomal instability; DAPI, 4′,6-diamidine-2′-phenylindole dihydrochloride; DMSO, dimethyl sulfoxide; ER, estrogen receptor; HRP, horseradish peroxidase; KT, kinetochore; MAP, microtubule associated protein; MT, microtubule; MTOC, microtubule-organizing center; SAC, spindle assembly checkpoint; SDS, sodium dodecyl sulfate ⁎ Corresponding author. E-mail address: [email protected] (C.-Y. Jang). 1 Seul Kim and Dasom Gwon have contributed equally to this study. https://doi.org/10.1016/j.tiv.2019.04.009 Received 30 January 2019; Received in revised form 9 April 2019; Accepted 9 April 2019 Available online 11 April 2019 0887-2333/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. BPA perturbs mitotic progression. (A and B) After treatment with 100 nM BPA for 5 h, HeLa cells were fixed with MeOH and stained with the indicated antibodies. Images are maximum projections from z stacks of representative cells stained for β-tubulin (red) and DNA (blue). Metaphase cells with unaligned chromosomes and mitotic cells with multipolar spindle were quantified and plotted (B). (C and D) HeLa S3 cells were synchronized by double thymidine (C; Thy-Thy) or thymidine-nocodazole (D; Thy-Noc) treatment and then released into fresh media. Cells were harvested at the indicated releasing times. Cells were analyzed by western blotting. Hsp90 served as a loading control. Asy, unsynchronized cells. (E and F) HeLa/GFP-histone H2B cells were transfected with 100 nM BPA for 5 h and time-lapse imaged for GFP-H2B. The duration of the periods from nuclear envelope breakdown (NEB) to the formation of a bipolar spindle/metaphase plate with some unaligned chromosomes (UC) (NEB to UC), from the unaligned state to metaphase (M) (UC to M), and from metaphase to anaphase onset (A) (M to A) were determined for control and ANKRD53-depleted cells (E; n = 50 cells). Images were captured every 3 min to monitor mitotic progression. Still frames were taken from time-lapse movies of representative cells (F). Arrowheads in (F) point to unaligned chromosomes. Error bars, SEM. Scale bars, 5 μm. *, p < 0.01 (two-tailed t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. BAP disrupts spindle attachment to kinetochore. (A and B) The maximum projections from z stacks of representative control or 100 nM BPA-treated cells stained for Mad2 (green), Hec1 (red), and DNA (blue) (A) are shown. Mad2 signals on kinetochores were quantified in five control or BPA-treated cells (A) at prometaphase (PM), metaphase (M), and unaligned chromosomes (UC; n > 50 kinetochores for each quantification). Error bars show SEM. (C and D) The maximum projections from z stacks of representative control or 100 nM BPA-treated cells stained with CREST (green), BubR1 (red), and DNA (blue) are shown. BubR1 signals on kinetochores in (C) were quantified in five control or BPA-treated cells at prometaphase (PM), metaphase (M), and unaligned chromosomes (UC; n > 50 kinetochores for each quantification). AU, arbitrary units. Scale bar, 5 μm. Error bars show SEM. *, p < 0.01 (two-tailed t-test relative to control cells). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 117
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that reflect genetic alterations such as mutations in oncogenes with dominant gain-of-function or tumor suppressor genes with recessive loss-of-function (Sieber et al., 2005). These genetic alterations drive the progressive transformation of normal cells into highly malignant cancer cells by rendering novel capabilities including self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis (Hanahan and Weinberg, 2000). To prevent the accumulation of genetic changes, animal cells have evolved to monitor their genomes before transition to the next cell cycle phase with G1/S and G2/M checkpoints (Sullivan and Morgan, 2007). In addition to DNA damage checkpoints, cells also monitor chromosome segregation during mitosis to ensure the faithful transmission of sister chromatids to two daughter cells with the spindle assembly checkpoint (SAC) (Musacchio and Salmon, 2007). During prometaphase, the centrosome nucleates spindle microtubules (MTs) to search for and capture kinetochores (KTs) on condensed sister chromatids (Pihan, 2013). At the same time, duplicated centrioles separate in opposite directions to form a bipolar spindle which allows for the structural basis of bi-oriented attachments of MTs to KTs (Gadde and Heald, 2004). The major task of SAC is to ensure accurate chromosome segregation by stabilizing proper bi-oriented attachments, in which one sister KT is captured by MTs from one spindle pole and the other sister by those from the opposite pole, and by resolving inappropriate attachments such as syntelic attachments, in which both sister KTs are captured by MTs from the same pole (Kline-Smith and Walczak, 2004; Silva et al., 2011). To this end, Mad2 and BubR1, as SAC components, check spindle MT attachment to KT and tension between sister-KTs, respectively (Skoufias et al., 2001). Consequently, defective mitotic progression increases chromosomal instability (CIN) which is a feature of most human cancers and drives tumor initiation and progression via the acquisition of genomic alterations through a small rate of chromosome gain and loss (Levine and Holland, 2018). In carcinogenesis, a moderate amount of CIN results in aneuploidy and enables a cell to deregulate gene expression, amplify oncogene, delete tumor suppressor gene, and accumulate a number of mutations (Gibbs, 2003). In contrast, large amount of CIN can suppress tumor progression because it is detrimental to the survival pathways and ultimately induces apoptosis (Dabas et al., 2012). In addition, CIN acts as a driving force for metastasis through chronic inflammation caused by micronuclei (Bakhoum et al., 2018). Thus far, both of ERα and ERβ have been found to exhibit mitotic functions distinct from the established role of ERs as transcription factors in that ERβ interacts with the SAC protein, Mad2, and ERα localizes on the spindle and spindle pole to regulate chromosome alignment and spindle dynamics (Poelzl et al., 2000; Zhang et al., 2015). As an ER binding xenoestrogen, BPA also induces cell cycle delay by altering spindle MT organization and chromosome alignment, thereby inducing aneuploidy during meiosis and mitosis (Adamakis et al., 2013; 2016; Can et al., 2005; George et al., 2008; Hunt et al., 2003; Ochi, 1999; Pfeiffer et al., 1997; Santovito et al., 2018). In contrast, exposure of mouse oocytes to BPA in vitro causes meiotic arrest by affecting spindle formation and chromosome alignment, but not aneuploidy (Eichenlaub-Ritter et al., 2008). Furthermore, BPA induces multiple microtubule-organizing centers (MTOCs) and multipolar spindle in mitosis (Adamakis et al., 2016; Ochi, 1999). The purpose of this study was to elucidate the molecular mechanisms underlying the disruption of chromosome alignment and centrosome integrity by BPA. Our data showed that BPA induced multipolar spindles and unaligned chromosomes in mitotic cells by interfering with the attachment of spindle MTs to KTs and the concomitant activation of the SAC. Mechanistically, BPA interferes with the localization of mitotic kinase Plk1 and MAPs, such as HURP, Kif2a, and TPX2. In addition, BPA caused centriole overduplication by increasing the level of SAS-6 protein and premature centriole disengagement. Thus, we conclude that BPA increases CIN by perturbing centrosome integrity and chromosome alignment in both meiosis and mitosis.
2. Materials and methods 2.1. Antibodies and chemicals The anti-HURP sera were raised against full-length recombinant HURP and affinity-purified (Wong and Fang, 2006). Anti-β-tubulin E7 monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank (DSHB, Iowa City, IA, USA). The following antibodies were obtained from commercial sources: anti-Hsp90, anti-CENP-E, antiERα, and anti-Cyclin B1, anti-Cyclin E, anti-Plk1 (Santa Cruz Biotechnology, Dallas, TX, USA); anti-γ-tubulin (Signal-Aldrich, St. Louis, MO, USA); anti-centrin-1, anti-c-NAP1 (Proteintech, Rosemont, IL, USA); anti-PLK4 (Merck Millipore, Darmstadt, Germany); anti-Mad2 (Pierce Antibodies; Thermo Fisher Scientific, Waltham, MA, USA); antiBubR1 (LifeSpan BioSciences, Seattle, WA, USA); anti-CREST (Antibodies Incorporated, Davis, CA, USA); Aurora B (Abcam, Cambridge, UK); anti-phospho-Aurora A (Cell Signaling Technology, Danvers, MA, USA); anti-TPX2 (Thermo Fisher Scientific, Waltham, MA, USA); antiEB1 (LifeSpan BioSciences, Nottingham, UK); anti-Hec1 (GeneTex, Irvine, CA, USA); Alexa Fluor-488 rabbit secondary antibody and Alexa Fluor-594 mouse secondary antibody (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA, USA); and horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA). The following chemicals were obtained from commercial sources: Bisphenol A, dimethyl sulfoxide (DMSO), and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO, USA); Tris base, NaCl, HCl, and sodium dodecyl sulfate (SDS) (Amresco, Solon, OH, USA); paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA); bovine serum albumin (BSA) (BioShop, Burlington, ON, CA); Triton X-100, Mowiol, and N-propyl gallate (Fisher scientific); Bradford protein assay kit (Thermo Fisher Scientific); polyvinylidene difluoride (PVDF) membrane and methanol (MilliporeSigma, Burlington, MA, USA); and protein molecular weight standard for electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA). BPA was dissolved in DMSO at a concentration of 100 μM as a stock solution and diluted with culture medium for use. HeLa, MCF-7, and MDA-MB-231 cells were treated with 100 nM BPA for 5 h. 2.2. Cell culture HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM; WelGENE Inc., Daegu, South Korea) supplemented with 10% fetal bovine serum (FBS; Invitrogen/Thermo Fisher Scientific), penicillin (100 units/mL), and 100 μg/mL streptomycin (Invitrogen/ Thermo Fisher Scientific). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. Immunofluorescence HeLa cells were cultured on cover glasses and fixed with methanol at −20 °C for 30 min. Alternatively, cells were treated with BRB80-T buffer (80 mM PIPES, pH 6.8, 1 mM MgCl2, 5 mM EGTA, and 0.5% Triton X-100) before fixing with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were blocked with PBS-BT (1× PBS, 3% BSA, and 0.1% Triton X-100) for 30 min at room temperature and stained with primary and secondary antibodies diluted in PBS-BT for 30 min at room temperature. After final washes with PBS-BT, cells were mounted with Mowiol mounting solution (20% glycerol, 10% Mowiol, 1.2 M Tris, pH 8.5, 2% N-propyl gallate). Z-image stacks were acquired in 0.2 μm increments with AxioVision 4.8.2 (Carl Zeiss AG, Oberkochen, Germany) under a Zeiss Axiovert 200 M microscope using a 1.4 NA plan-Apo 100× oil immersion lens and an HRm CCD camera. Deconvolved images were obtained using an AutoQuant X3 (AutoQuant Imaging, Inc., Troy, NY, USA). All images are maximum projections from z stacks of representative cells stained for the indicated antigens. 118
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Fig. 3. BPA interferes with localization of microtubule-associated proteins. HeLa cells were fixed at 5 h post-treatment with 100 nM BPA and stained with the indicated antibodies. (A and B) The maximum projections from z stacks of representative control or BPA-treated cells stained for Aurora A, Aurora B, CENP-E or Plk1 (green), βtubulin or Hec1 (red), and DNA (blue) are shown. Plk1 signals on kinetochores were quantified and plotted (B). (C and D) The maximum projections from z stacks of representative control or BPA-treated cells stained for CENP-E, HURP or Kif2a (green), Hec1, EB1, or β-tubulin (red), and DNA (blue) are shown. (E) The maximum projections from z stacks of representative control or BPA-treated cells stained for TPX2 (green), β-tubulin (red), and DNA (blue) are shown. Images were acquired under constant exposure time for the TPX2 channel. Total immunofluorescence intensity for TPX2 on the metaphase spindle (n = 10 cells for each quantification) was quantified and plotted. AU, arbitrary units. Error bars, SEM. Scale bars, 5 μm. *, p < 0.01 (two-tailed t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Images for quantification were acquired under constant exposure in each channel for all of the cells.
aneuploidy (Wheeler et al., 1986). To investigate whether BPA also influences mitosis, we treated HeLa cells with BPA at concentrations ranging from 1 nM to 10 μM for 5 h and found the highest chromosome alignment defects and multipolar spindles in 100 nM BPA-treated cells (Fig. 1A, B). This dosage is less than the reference dose, 0.5 μg/mL, established by United States Environmental Protection Agency and similar to the higher concentration of unconjugated BPA found in human serum, 0.02 μg/mL, established by European Union (Santovito et al., 2018). To further examine the inhibitory effects of BPA on mitotic cell cycle progression, HeLa S3 cells were treated with double thymidine (Thy-Thy) and released into fresh media. Western blot analysis revealed that BPA prolonged the expression of mitotic regulators including cyclin B1, Plk1, and phosphorylated Aurora A after release from double thymidine arrest (Fig. 1C). Consistent with this result, the level of cyclin B1 was also prolonged by BPA after release from TN arrest (Fig. 1D), suggesting that BPA interferes with mitotic progression by perturbing chromosome alignment and bipolar spindle formation. To further investigate the mitotic defects caused by BPA treatment, we observed mitotic cells under a time-lapse experiment after BPA treatment (Fig. 1E, F, Video 1, 2). HeLa cells stably expressing GFP-histone H2B were treated with BPA and imaged every 3 min for 5 h. Consistent with previous data, treatment with BPA prolonged the duration of prometaphase [from nuclear envelope breakdown (NEB) to the formation of a metaphase plate with some unaligned chromosomes (UC)], the unaligned state [from UC to metaphase (M)], and metaphase [from metaphase to anaphase onset (A)]. Thus, we conclude that BPA induces mitotic delay by perturbing chromosome congression and bipolar spindle formation.
2.4. Live cell images For time-lapse microscopy, HeLa cells stably expressing GFP-H2B were plated on a cell imaging dish with a glass bottom and cultured in Leibovitz's L-15 medium (Invitrogen/Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen/Thermo Fisher Scientific) and 2 mM L-glutamine (Invitrogen/Thermo Fisher Scientific). Cells were placed into a growth chamber heated to 37 °C and observed on a Zeiss Axiovert 200 M microscope with a 20× lens. Images were acquired every 3 min for 5 h with AxioVision 4.8.2 (Carl Zeiss AG). 2.5. Western blotting Cells were harvested and lysed with NP-40 lysis buffer (50 mM Hepes, pH 7.4, 200 mM KCl, 0.3% NP-40, 10% glycerol, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, 0.5 μM microcystin, and 10 μg/ml concentrations of leupeptin, pepstatin, and chymostatin) on ice for 10 min and debris was removed by centrifugation. Protein concentrations in total cell lysates were measured using a Bradford protein assay kit before boiling in Laemmli buffer. The lysates were separated by a 12% SDS-PAGE gel and transferred to a PVDF membrane. Protein bands were detected using chemiluminescence blotting substrate (Roche, Basel, Switzerland). HRP-conjugated anti-mouse IgG or anti-rabbit IgG were used as secondary antibodies. 2.6. Colony forming assay
3.2. BPA interferes with spindle MT attachment to KTs and activates the SAC
Cells were treated with 100 nM BPA for five hours, washed with PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.2) and trypsinized to produce a single-cell suspension. After counting the number of cells in the cell suspension, 150 cells were plated onto a dish and cultured in fresh media for 14 days to form colonies. The colonies were washed with PBS and stained with glutaraldehyde crystal violet mixture (6.0% glutaraldehyde and 0.5% crystal violet) for 30 min. After removing the glutaraldehyde crystal violet mixture and rinsing with water carefully, the dishes with colonies were dried in normal air at room temperature.
To investigate whether the unaligned chromosomes caused by BPA treatment resulted from a lack of MT attachment to the KT or tension between sister chromatids, we examined the activation of the SAC by analyzing the intensity of Mad2 and BubR1 with immunofluorescence staining. These proteins are KT-associated checkpoint proteins that control the metaphase-to-anaphase transition by inhibiting APC/C E3 ligase and concomitant degradation of securin to ensure chromosome integrity. Whereas Mad2 perceives the attachment between KT and MT, BubR1 acts as a sensor for the tension between sister KTs during mitosis (Musacchio and Salmon, 2007; Skoufias et al., 2001). Therefore, Mad2 and BubR1 are concentrated at KTs in prometaphase and released from KTs in metaphase after the completion of MT attachment to KT and the generation of inter-KT tension by formation of the bi-oriented spindle. Interestingly, Mad2 was absent from the aligned KTs but still resided in unaligned KTs in BPA-treated metaphase cells (Fig. 2A, B), suggesting that BPA interferes with chromosome alignment by interrupting spindle MT attachment to KTs. In contrast, BubR1 was observed in unaligned KTs but not in aligned KTs in BPA-treated metaphase cells (Fig. 2C, D), indicating that the SAC is inactivated by the generation of tension at aligned KTs and that BPA is not associated with spindle dynamics. The accumulation of BubR1 at unaligned KTs resulted from the absence of tension between sister KTs due to attachment failure. Thus, SAC activation delayed mitotic progression in BPA-treated cells.
2.7. Statistical analysis Data were analyzed using Student's t-test. Error bars represent the SE of three independent experiments. A p-value < 0.01 (two-tailed) was considered statistically significant. 3. Results 3.1. BPA causes unaligned chromosomes and multipolar spindles It has been reported that BPA has potential inhibitory effects on meiotic cell cycle progression and disrupts centrosome and spindle integrity (Can et al., 2005; Susiarjo et al., 2007). Furthermore, synthetic estrogens such as diethylstilbestrol are known to be spindle poisons that inhibits mitotic progression and while concurrently inducing 120
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Fig. 4. BPA perturbs centriole duplication cycle. (A and B) HeLa cells were treated with 100 nM BPA for 5 h and stained for centrin-1 (green), γ-tubulin (red), and DNA (blue). Mitotic cells with multipolar spindle or over-duplicated centrioles were quantified and plotted. (C) Five hours after BPA treatment, the cells were harvested and analyzed by western blotting with indicated antibodies. Hsp70 served as a loading control. (D and E) Shown are maximum projections from the deconvolved z stacks of representative HeLa cells stained for Centrin-1 (green), STIL, PLK4, or SAS-6 (red) and DNA (blue). Fluorescence intensity of STIL, PLK4, and SAS-6 was quantified and plotted (E). (F and G) HeLa cells stably expressing GFP-Centrin-1 were treated with 100 nM BPA for 5 h and stained for C-NAP1 (red) and DNA (blue). Cells with premature disengaged centrioles were quantified and plotted (G). Error bars, SEM. Scale bars, 5 μm. *, p < 0.01 (two-tailed t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.3. BPA interrupts proper localization of MT-associated proteins (MAPs) on the mitotic spindle
duplication initiators. We then investigated the effect of BPA treatment on centriole disengagement, as odd number of centrioles were found in overduplicated centrosome (Fig. 4A). As expected, the number of CNAP1 foci increased at centrosome in BPA treated cells (Fig. 4F, G), suggesting that BPA also induces premature centriole disengagement in G2 or early mitosis. Overall, these findings indicate that centriole overduplication and premature disengagement induced by BPA result in multipolar spindle in mitosis.
Given that BPA induces unaligned chromosome (Fig. 1A) and activates the SAC (Fig. 2), we hypothesized that it inhibits regulators of spindle MT attachment to KTs. To test this hypothesis, we investigated the localization of mitotic regulators, which are recruited to mitotic structures such as mitotic spindles, KTs, centromeres, and centrosomes. First, we examined the localization of mitotic kinases because they show distinct localization on mitotic structures. While Aurora A is recruited to centrosomes, Aurora B is concentrated in centromeres during mitosis. Polo-like kinase 1 (Plk1) localizes to centrosomes in early mitosis and transfers to KTs at metaphase and to central spindles at anaphase. Following treatment with BPA, the distinct localization of mitotic kinases was confirmed using immunofluorescence microscopy. Although Aurora A and Aurora B showed normal localization at the spindle pole and centromeres after BPA-treatment, respectively, Plk1 was substantially increased at unaligned KTs (Fig. 3A, B). Next, we examined the localization of plus-end tracking protein (+TIP), EB1, and plus end-directed kinetochore motor, CENP-E, because they are responsible for the congression of polar-localized chromosomes to the metaphase plate (Kapoor et al., 2006). In addition, depletion of CENP-E or EB1 causes a failure of chromosome alignment and generates a few unattached chromosomes around the spindle pole (Putkey et al., 2002). However, BPA did not affect on the localization of CENP-E and EB1 (Fig. 3C), suggesting that BPA does not disrupt the mechanical link between KTs and the plus-end tip of a dynamic MT. third, we confirmed the localization of MAPs on the mitotic spindle that are involved in chromosome congression. HURP (hepatoma upregulated protein)known to localize to the end of KT fiber and regulate chromosome congression (Ye et al., 2011)-was dispersed on the mitotic spindle in BPA-depleted cells (Fig. 3D). Kif2a, which has been identified as a minus-end MT depolymerase and is known to be involved in spindle dynamics and chromosome alignment around the spindle pole (Kwon et al., 2016), was also mislocalized on the mitotic spindle in BPAtreated cells (Fig. 3D). TPX2, a spindle assembly factor with prominent roles in local MT formation around chromatin (Gruss et al., 2002), was decreased on the mitotic spindle in BPA-treated cells (Fig. 3E). Thus, we conclude that BPA perturbs chromosome congression by disrupting the localization of mitotic regulators including Plk1, HUPP, Kif2a, and TPX2.
3.5. BPA stimulates tumorigenesis in both ER-positive and -negative breast cancer cells To investigate whether BPA induces mitotic defects by disrupting the transcriptional activity of ER or non-transcriptional function, we treated BPA to ER-positive or -negative breast cancer cells. As shown in Fig. 5A, BPA induced chromosome congression defects and multipolar spindles in BPA-treated ER-positive MCF-7 cells. Recently, it was reported that ERα localizes to the centrosome in prometaphase and the mitotic spindle in metaphase and regulates chromosome alignment in a transcription-independent manner (Zhang et al., 2015). Therefore, we confirmed the localization of ERα in BPA-treated MCF-7 cells and found that BPA did not affect the recruitment of ERα to the mitotic spindle and the localization of ERα on the spindle (Fig. 5B). Interestingly, BPA also induced chromosome congression defect in ER-negative NDA-MB231 cells (Fig. 5C and D), suggesting that BPA does not act as an endocrine disruptor in mitosis. Consistent with this, BPA increased the number and the size of colonies both in MCF-7 and MDA-MB-231 breast cancer cells (Fig. 5E and F). Taken together, BPA promotes tumor progression by perturbing mitotic progression in an ER-independent manner. 4. Discussion As an endocrine disrupting chemical, BPA is suspected as a potential risk factor for increasing cancer rates involving the human reproductive system, such as breast and prostate cancer (Yang et al., 2006). However, the direct effect of BPA exposure on the stimulation and initiation of cancer and the promotion of cancer aggressiveness has not been fully addressed. Given that ER plays a pivotal role in the growth and maturation of the mammalian oocyte (Susiarjo et al., 2007), brief exposures during fetal development to BPA affect prophase at the onset of oogenesis, chromosome segregation, and the formation of follicles in the female fetus (Hunt et al., 2012). Furthermore, ERα is involved in chromosome alignment and spindle dynamics by stabilizing microtubules during mitosis in an estrogen-independent manner (Zhang et al., 2015). Recently, it has been reported that treatment with lowdose BPA in utero increased the incidence of hepatic tumors or preneoplastic lesions (Weinhouse et al., 2014). Furthermore, delayed chromosome alignment to mitotic equator causes a defective bi-orientation establishment and concomitant chromosome missegregation (Kuniyasu et al., 2018). In this study, we showed that BPA induced chromosome alignment defects by interrupting the proper localization of spindle-associated proteins in an ER-independent manner, suggesting that BPA promotes tumorigenesis both in non-reproductive estrogentarget cells and in non-reproductive estrogen-non-target cells. Although meiotic processes such as bipolar spindle formation, alignment of homologous chromosomes at metaphase I and sister
3.4. BPA perturbs centriole duplication cycle by increasing SAS-6 Because BPA induced multipolar spindle (Figs. 1B, 4B), we sought to examine whether it causes centriole overduplication or pericentriolar material (PCM) fragmentation. We found that ~18% of BPA-treated cells have over 4 centrioles per each cell (Fig. 4A, B), indicating that BPA causes multipolar spindle by inducing centriole overduplication. To determine the mechanism underlying BPA-induced centriole overduplication, we examined the level of three proteins, such as PLK4, STIL, and SAS-6, which are involved in the initiation of centriole duplication (Arquint and Nigg, 2016). Interestingly, the level of PLK4 and STIL slightly increased but SAS-6 substantially increased in BPA-treated cells (Fig. 4C). In accordance with this, the level of SAS-6 markedly increased at centrioles (Fig. 4D, E), suggesting that BPA provokes centriole overduplication by disrupting the level of centriole 122
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Fig. 5. BPA promotes tumorigenesis in an ERα-independent manner. (A) MCF-7 cells were treated with 100 nM BPA and stained for DNA (blue). (B) Shown are maximum projections from the deconvolved z stacks of representative MCF-7 cells stained for ERα (green), β-tubulin (red) and DNA (blue). (C and D) MDA-MB-231 cells were treated with 100 nM BPA and stained for ERα (green), β-tubulin (red) and DNA (blue). Metaphase cells with unaligned chromosome and mitotic cells with multipolar spindle were quantified and plotted (D). (E and F) MCF-7 or MDA-MB-231 cells were treated with 100 nM BPA. 150 cells were incubated for 14 days to form a colony, and were stained with glutaraldehyde crystal violet mixture. The number of colonies in control or BPA-treated cells were counted and plotted (F). Error bars, SEM. *, p < 0.01 (two-tailed t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
chromatids at metaphase II, and chromosome segregation in anaphase I differ from mitotic processes in somatic cell division, the SAC plays as a safe guard mechanism to avoid aneuploidy in meiosis (Vogt et al., 2008). In mitosis, the SAC monitors the attachment of the sister KTs to spindle MTs, that emanated from opposite poles, to establish sister KT
bi-orientation for faithful sister chromatid segregation (Musacchio and Salmon, 2007). However, homologous chromosomes are linked together by synaptonemal complex to form a bivalent which consists of 4 chromatids during meiosis I. In contrast to mitosis and meiosis II, sister KTs attach to MTs from the same pole and establish mono-orientation in 123
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meiosis I (Sun and Kim, 2012; Vogt et al., 2008). Consequently, the SAC discriminates different tension-generating configuration in meiosis I and meiosis II and delays anaphase onset until all chromosomes have achieved the right orientation. In this respect, the canonical chromosome segregation machinery appears to be adapted in meiosis. The molecular mechanism by which BPA induces spindle attachment defect and concomitant SAC activation in meiosis remains to be elucidated. Because of the accurate function in cellular processes, the structure and number of centrosomes are tightly regulated throughout cell cycle (Meraldi and Nigg, 2002). Therefore, the formation of multipolar spindles is involved in causing unequal segregation of the chromosomes because the multipolar division results in aneuploidy. Furthermore, centrosome abnormalities such as centrosome amplification-dependent or –independent multipolarity are frequently observed in carcinoma cells (Lingle et al., 1998; Pihan et al., 1998). In this regard, centrosome hypertrophy may cause mitotic abnormalities and uncontrolled proliferation in carcinogenesis. It has also been reported that BPA induces multipolar spindles in mammalian and plant cells (Adamakis et al., 2016; Ochi, 1999). Recently, it has been established that PLK4, STIL, and SAS-6 play important roles in initiation of centriole duplication (Arquint and Nigg, 2016). Consistent with this, we found that BPA induces centriole overduplication via increasing the level of SAS-6 at the centrosome (Fig. 4C–E). In summary, BPA increases chromosomal instability by disturbing spindle MT attachment to KTs and centriole duplication in an ER-independent manner. Consequently, BPA exposure increases a cancer susceptibility not only in estrogen target cells but also in estrogen nontarget cells. Although we showed the mislocalization of mitotic regulators including Plk1, HURP, Kif2a, and TPX2 caused by BPA-treatment, the direct target proteins of BPA and the detailed mechanisms involved will be determined in further experiments. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.tiv.2019.04.009.
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