Chromosomal passenger proteins have emerged as key players in the regulation of mitosis and cytokinesis. Histone deacetylase inhibitors (HDACi) are a class of anticancer drugs that induce aberrant mitosis and can overcome the spindle assembly checkpoint. Here, we investigate the mechanism by which HDACi disrupt normal mitotic progression and checkpoint function. We demonstrate that HDACi treatment temporarily delays mitotic progression through prometaphase due to activation of the spindle assembly checkpoint. Despite failing to congress chromosomes to the metaphase plate, cells aberrantly segregate their chromosomes and exit mitosis to undergo a failed cytokinesis. We show that this premature exit from mitosis is a form of mitotic slippage. Chromosomal passenger proteins fail to accumulate at the centromere following HDACi treatment. This results in inadequate concentrations of chromosomal passenger proteins at the centromere, which are insufficient to regulate the phosphorylation of the kinetochore checkpoint component BubR1, and an inability to maintain the mitotic arrest. Thus, the centromeric accumulation of chromosomal passenger complex components is critical for regulating kinetochore but not centromeric processes, and failure of this accumulation underlies the HDACi-induced mitotic slippage.
Successful cell division requires the accurate duplication and segregation of the genome, mediated by the mitotic spindle. Ensuring the fidelity of mitosis is vital, as defects at any stage can result in aneuploidy that can contribute to cancer development. The spindle assembly checkpoint is the cell cycle control mechanism that acts during mitosis to ensure the fidelity of chromosome segregation by preventing premature entry into anaphase. Unattached kinetochores recruit checkpoint proteins, such as Mad2 and BubR1, which sequester Cdc20 and thereby inhibit APCCdc20-dependent degradation of proteins whose destruction is required for anaphase initiation and the spindle assembly checkpoint is thereby extended (reviewed by Kops et al., 2005).
Chromosomal passenger proteins have emerged as key players in the regulation of mitosis (reviewed by Vagnarelli and Earnshaw, 2004). The chromosomal passenger complex (CPC) includes the protein kinase Aurora B, INCENP, Survivin and Borealin (Adams et al., 2001; Mollinari et al., 2003; Gassmann et al., 2004; Sampath et al., 2004). These proteins associate along the length of the condensing chromosomes in prophase, accumulate at the centromeres in prometaphase and metaphase, and relocate to the central spindle at anaphase onset before concentrating in the midbody during cytokinesis. The complex has a pivotal role in chromosome congression, spindle assembly checkpoint function, chromosome segregation, central spindle formation and cytokinesis (Mackay et al., 1998; Adams et al., 2001; Kaitna et al., 2002; Kallio et al., 2002; Murata-Hori and Wang, 2002; Carvalho et al., 2003; Ditchfield et al., 2003; Hauf et al., 2003; Honda et al., 2003; Gassmann et al., 2004; Lampson et al., 2004). The CPC proteins interact either directly or indirectly with each other, and disrupting the localization of any one protein results in a loss of centromeric and central spindle localization of the other proteins in the complex. Borealin has been shown to bind both Survivin and INCENP, but unlike the other passenger proteins, Borealin appears to have a further function in stabilization of the mitotic spindle (Gassmann et al., 2004).
Histone deacetylase inhibitors (HDACi) have emerged as a class of anticancer drugs that have the desirable characteristics of tumour cell-directed toxicity with little normal cell toxicity (Burgess et al., 2004). The modification of core histones is of fundamental importance to conformational changes of the chromatin, with post-translational dynamic histone acetylation states being regulated by the opposing activities of histone acetyltransferases and histone deacetylases (HDAC). These enzymes play a critical role in a wide variety of biological activities including regulating cell cycle progression and chromatin assembly and architecture. Treatment of human tumour cells with HDACi causes aberrant mitosis with a failure of chromosome congression (Qiu et al., 2000; Taddei et al., 2001; Robbins et al., 2005). In a previous study, we have demonstrated that this phenotype requires the presence of HDACi during S phase, with addition later in the cell cycle failing to generate the aberrant mitosis. S phase HDACi treatment also overcomes the spindle assembly checkpoint, and HDACi-induced mitotic exit is required for the rapid apoptosis observed (Warrener et al., 2003). Investigating the molecular mechanism by which HDACi treatment induces aberrant mitoses; overcomes the spindle assembly checkpoint and causes failure of cytokinesis should define critical components and interactions required for the establishment and/or maintenance of spindle assembly checkpoint arrest.
HDACi treatment delays progression through mitosis
HDACi treatment of synchronized HeLa cells during S phase delayed progression through G2/M. The control population re-entered G1 phase after 10 h, as demonstrated by fluorescence-activated cell sorter and histone H3 Ser10 phosphorylation, a marker of mitosis (Figures 1a and b). HDACi-treated cells remained in the G2/M compartment until at least 15 h. An increase in the subdiploid (<2n) population from 13 h in HDACi-treated cells corresponded to the appearance of the cleaved form of poly(ADP-ribose)polymerase, indicative of apoptosis (Figure 1b). Time-lapse microscopy revealed that HDACi-treated cells remained in mitosis approximately 2.5 times longer than control cells (Figure 1c), consistent with the biochemical analysis.
HDACi treatment disrupts chromosome congression and bipolar spindle formation
The delayed progression through mitosis with HDACi treatment was likely to be a consequence of aberrant mitosis. Immunofluorescence staining of chromosome and spindle structures showed normal chromosome condensation and congression on a bipolar spindle in control cells (Figure 2). HDACi-treated cells, in contrast, displayed a prometaphase configuration with condensed DNA but failure of chromosome alignment on the metaphase plate (Figure 2). This aberrant mitotic phenotype was observed with the HDACi tested. Close examination of the microtubule spindle structure revealed it to be defective in a high proportion of HDACi-treated cells. Spindles rarely displayed the symmetrical fusiform-shaped microtubule arrangement normally seen during mitosis (Figures 2 and 3a). Microtubule bundling at the centrosome appeared less organized and focused (unfocused poles) in over 60% of cells (Figures 2 and 3a, filled arrows). The more aberrant spindles had the least chromosome alignment, suggesting that there were defects in attachment of the spindle microtubules with the chromosomes. Extra γ-tubulin containing microtubule organizing centres (MTOCs) were observed in over 30% of HDACi-treated mitotic cells (Figure 2, open arrows, Figure 3a and Supplementary Figure 1A). However, the majority of cells contained only two pairs of closely spaced centrin foci, and this was unaffected by HDACi treatment (Supplementary Figure 1B). The subsidiary MTOC in the HDACi-treated cells did not contain centrin, and therefore not bona fide centrosomes. The extra MTOCs formed upon HDACi treatment were only detected in mitotic cells, S and G2 phase cells contained only two γ-tubulin foci.
Fluorescence time-lapse microscopy of α-tubulin-cherry fluorescent protein (CFP) HeLa cells showed control cells enter mitosis and nucleate microtubules from two centrosomes, which interdigitated to form the mitotic spindle (Figure 3b and Supplementary Movie 1). In HDACi-treated cells, multiple microtubule foci were observed at the nuclear periphery in the early stages of mitosis. The foci appeared to coalesce to produce two major spindle poles, but failed to establish a symmetrical mitotic spindle (Figure 3b and Supplementary Movie 2).
HDACi treatment overcomes the spindle assembly checkpoint
Quantitative analysis of DNA and spindle morphology of HDACi-treated HeLa cells revealed that they arrested for an extended period in prometaphase (Supplementary Figure 2). Metaphase chromosome congression and normal anaphase with distinct central spindle structures were rarely observed, but readily detected in control cells. The majority of HDACi-treated cells scored for mitotic exit were undergoing cytokinesis with clear evidence of a midbody (Supplementary Figure 3). This aberrant cell division resulted in multinuclear daughter cells, and these aberrant phenotypes were observed with all class of HDACi employed. Time-lapse microscopy of the α-tubulin-CFP HeLa cells in the later stages of mitosis confirmed the aberrant mitotic exit in HDACi-treated cells. Control cells displayed intense central spindle microtubule bundling upon anaphase initiation to form the central spindle, which progressed to form the midbody at telophase (Figure 4 and Supplementary Movie 3). The central spindle bundling of microtubules was rarely observed in HDACi-treated cells, although midbody formation appeared normal (Figure 4 and Supplementary Movie 4).
Delays during mitosis are the result of activation of the spindle assembly checkpoint. To determine whether this checkpoint was responsible for the prometaphase delay with HDACi treatment, Mad2, BubR1 and Bub1 localization was analysed. In prophase and prometaphase cells, Mad2, BubR1 and Bub1 localized as two discrete adjacent foci corresponding to the paired sister chromatid kinetochores in control- and HDACi-treated cells, demonstrating that the spindle assembly checkpoint was activated (Figure 5a). In control cells, these foci were lost and checkpoint inactivation occurred when all chromosomes achieved stable bipolar attachments to the mitotic spindle (Figure 5a, arrow). The Aurora inhibitor ZM447439 has been demonstrated to overcome the spindle assembly checkpoint (Ditchfield et al., 2003). Addition of ZM447439 to HDACi-treated cells entering mitosis resulted in a failure of spindle checkpoint protein BubR1 to localize at the kinetochore, and effectively overcame the HDACi-induced mitotic arrest (Supplementary Figure 4). Thus, HDACi treatment initiates the spindle assembly checkpoint to delay mitotic exit, but the checkpoint is inactivated prematurely.
To determine the extent of premature spindle assembly checkpoint inactivation, synchronized HeLa cells were treated with HDACi and nocodazole to activate the spindle assembly checkpoint, and followed by time-lapse microscopy. Untreated control cells transited mitosis in 1 h, whereas nocodazole-treated cells remained blocked in mitosis for approximately 18 h (Figure 5b). HDACi-treated cells arrested in mitosis for approximately 3 h before exiting, and cells treated with both nocodazole and HDACi exited mitosis with similar kinetics. This clearly demonstrates that HDACi treatment was capable of overriding the spindle assembly checkpoint after an initial delay in mitosis. Premature mitotic exit was visualized using HeLa cells stably expressing H2B-GFP and time-lapse microscopy. Control cells normally condensed their chromosomes, aligned them at metaphase and underwent symmetrical cell division (Figure 5c and Supplementary Movie 5). HDACi-treated cells also condensed their chromosomes but were unable to align these, then eventually exit mitosis and undergo cytokinesis, consequently segregating the chromosomes unequally often into multiple daughter cells, which frequently remerged (Figure 5c and Supplementary Movie 6). This aberrant mitosis was accompanied by eventual cell death in the majority of cells analysed, consistent with previous reports (Warrener et al., 2003).
Chromosomal passenger proteins fail to concentrate at the centromeres in HDACi-treated cells
The mitotic defects observed with HDACi treatment are reminiscent of the phenotypes observed upon depletion of the CPC proteins Aurora B, INCENP, Borealin and Survivin. HDACi treatment, however, did not affect the levels of CPC proteins in mitotic cells (Figure 6a). Similar results were obtained after treatment with different classes of HDACi, and in two other cell lines (HaCaT and A2058) treated with HDACi (data not shown). Mitotic histone H3 Ser10 phosphorylation is catalysed by Aurora B (Crosio et al., 2002), and was equivalent in control- and HDACi-treated cells (Figures 1b and 6a), demonstrating the level of Aurora B kinase activity to be unaffected by HDACi treatment. CENP A phosphorylation was also readily detected in HDACi-treated cells, providing another indicator of normal Aurora B kinase activity at the centromere (Figures 6a and b). The level of CENP A was unaffected by HDACi treatment (data not shown).
Control mitotic and nocodazole arrested cells displayed the characteristic hyperphosphorylated BubR1 band indicative of spindle assembly checkpoint activation, and lost in later mitotic time points corresponding to checkpoint inactivation and mitotic exit (Figures 6a and b; Chan et al., 1999). This corresponded with the loss of CENP A phosphorylation and the degradation of Aurora B. By contrast, BubR1 phosphorylation was undetectable in mitotic HDACi-treated cells despite Aurora B activity at the centromere, marked by phosphorylation of CENP A (Figures 6a and b). HDACi treatment had no effect on INCENP/Aurora B interaction (Figure 6c).
Examination of the localization of Aurora B and the other CPC proteins through mitosis revealed the characteristic strong centromeric accumulation of the CPC proteins in control prometaphase and metaphase cells (Figure 7). In HDACi-treated cells, the CPC failed to concentrate and appeared more diffuse along the chromosome arms (Figures 7 and 8a). The accumulation to the centromeric region was quantified by measuring the ratio of staining intensity of Aurora B, Borealin and Survivin at the centromere and on the chromosome arms (Figure 8b). Control cells displayed a wide range of CPC protein accumulation to the centromeric region, ranging from >3- to 1.25-fold that of the intensity of staining on the chromosome arms. HDACi-treated cells displayed significantly less centromeric staining, with an average 1.2-fold accumulation. Subsequent midbody localization of the CPC proteins was readily detected in control cells and appeared relatively unaffected in HDACi-treated cells, regardless of the HDACi employed (Supplementary Figure 5).
Here we have reported that HDACi treatment during S-phase causes (1) microtubule nucleation at multiple γ-tubulin foci in prophase; (2) aberrant mitotic spindles with ectopic spindle poles, despite no increase in the number of centrosomes; (3) delay in prometaphase; (4) overcoming the spindle assembly checkpoint; (5) exit from mitosis without evidence of metaphase chromosome congression or central spindle formation. These HDACi-induced mitotic defects are also observed with depletion of individual CPC proteins (Wheatley et al., 2001; Carvalho et al., 2003; Ditchfield et al., 2003; Hauf et al., 2003; Lens et al., 2003; Mollinari et al., 2003; Gassmann et al., 2004; Klein et al., 2006). Although HDACi treatment does not affect the levels of the CPC proteins, they fail to accumulate at the centromeres in prometaphase. The failure CPC accumulation at the centromere underlies the mitotic defects following HDACi treatment.
HDACi treatment did not affect the levels of the spindle assembly checkpoint components, Mad2, Bub1 and BubR1, the CPC, or Cdc20 and Cdh1 (unpublished observations), and normal kinetochore localization of Mad2, Bub1 and BubR1 is observed in prometaphase cells following HDACi treatment. However, HDACi-treated cells fail to maintain the arrest and undergo premature mitotic exit, accompanied by the loss of Mad2, Bub1 and BubR1 from the kinetochores. This parallels Survivin depletion, where Mad2 and BubR1 are loaded onto unattached kinetochores, although there is eventual loss of BubR1 kinetochore association and premature mitotic exit (Lens et al., 2003). Kinetochore localization of Mad2, Bub1 and BubR1 is controlled by the CPC (Carvalho et al., 2003; Lens et al., 2003; Morrow et al., 2005). Sufficient CPC must localize to the centromeres in HDACi-treated cells for kinetochore loading of the checkpoint proteins and checkpoint activation. The normal level of CENP A phosphorylation is evidence that sufficient active Aurora B localizes to the centromere in HDACi-treated cells. CENP E association with the kinetochore is also dependent on Aurora B activity (Ditchfield et al., 2003; Mao et al., 2005), and CENP E is lost from the kinetochore after HDACi treatment (Dowling et al., 2005; Robbins et al., 2005). CENP E regulates BubR1 phosphorylation (Lampson et al., 2004; Mao et al., 2005), thus, the lack of phosphorylated BubR1 in HDACi-treated cells is a consequence of reduced kinetochore-associated Aurora B and ensures failure of the spindle assembly checkpoint (Ditchfield et al., 2003; Morrow et al., 2005).
It is possible to reconcile the normal centromeric function of Aurora B, indicated by CENP A phosphorylation, with the apparent failure of Aurora B-dependent kinetochore functions. Some CPCs and kinetochore proteins display highly dynamic associations with the centromeres and kinetochore during mitosis. Centromere-associated Survivin rapidly exchanges with a soluble pool, whereas Aurora B appears to be relatively more stably associated (Murata-Hori and Wang, 2002; Beardmore et al., 2004; Delacour-Larose et al., 2004). In a normal cell, this dynamic binding to the centromere and the relatively higher accumulation of Aurora B would provide a high concentration of the kinase to perform not only its chromatin-associated roles but also its kinetochore-associated functions. In HDACi-treated cells, the reduced accumulation of CPC at the centromere would reduce the amount of Aurora B available to phosphorylate its kinetochore-associated targets, but would have relatively little effect on centromeric targets as we have observed.
The multiple microtubule nucleating γ-tubulin foci and spindle defects may also be a consequence of mislocalized Aurora B-INCENP and the aberrant mitotic spindle structures reiterate depletion of Borealin (Gassmann et al., 2004). HDACi treatment does not affect the assembly of INCENP-Aurora B-containing complexes, and this complex can regulate spindle microtubule assembly through a Ran-independent pathway, and requires only clustering of the complex to produce microtubule asters (Kelly et al., 2007). Thus, the spindle defects may simply reflect a failure to localize the activated INCENP-Aurora B-containing complex to the centromere in mitosis. The lack of central spindle formation in HDACi-treated cells was also reported with depletion of the CPC proteins (reviewed by Vagnarelli and Earnshaw, 2004; Glotzer, 2005).
How does HDACi treatment block CPC accumulation at the centromere in mitosis? Sodium butyrate does not inhibit HDAC6 catalysed α-tubulin deacetylation, and yet ablates centromeric CPC accumulation, demonstrating a lack of involvement of HDAC6 and substrates such as α-tubulin and HSP90 (Hubbert et al., 2002; Verdin et al., 2003; Aoyagi and Archer, 2005). The most likely target is the centromeric chromatin itself. The heterochromatin-binding protein HP1 associates with the centromere and its binding is reduced with chronic HDACi treatment (Taddei et al., 2001). However, its association with the normal mitotic centromere is reduced as a consequence of H3 Ser10 phosphorylation (Fischle et al., 2005; Hirota et al., 2005), making it unlikely to be directly involved in normal CPC accumulation to the centromere during mitosis. HDACi treatment directly modifies the chromatin structure, and this may be sufficient to disrupt the centromeric accumulation of the CPC. The details of the mechanism by which HDACi treatment affects CPC accumulation are as yet unknown.
In conclusion, we have demonstrated that the many mitotic defects observed following HDACi treatment, particularly the spindle assembly checkpoint defect is a direct consequence of the lack of centromeric accumulation of the active CPC in mitosis. The checkpoint arrest cannot be maintained and therefore results in ‘mitotic slippage’ (Weaver and Cleveland, 2005). This describes the failure to maintain the spindle assembly checkpoint resulting in aberrant cell division and describes the premature mitotic exit seen upon HDACi treatment. This study points to the importance of the centromeric accumulation of the active CPC in mitosis, demonstrating that mislocalization of this complex has wide-reaching consequences on mitosis and ultimately viability.
Materials and methods
Cell culture and synchrony
All cells were cultured in a humidified incubator at 37 °C with 5% CO2. HeLa cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% Serum Supreme (Biowhittaker, Walkersville, MD, USA). For time-lapse microscopy, cells were grown in Ham's F-12 medium (Sigma-Aldrich, Castle Hill, NSW, Australia) supplemented as above. Cells were synchronized using 2 mM hydroxyurea as described previously (Qiu et al., 2000). HDACi were added to cells immediately after synchrony release and harvested at the times indicated. HDACi concentrations used were 100 μg ml−1 suberohydroxamic acid (SBHA; Sigma-Aldrich), 10 μM suberoylanilide hydroxamic acid (SAHA; a gift from Victoria Richon, Aton Pharma, Lawrenceville, NJ, USA), 1 μM trichostatin A (Sigma-Aldrich) and 20 mM sodium butyrate (Sigma-Aldrich). ZM447439 was kindly provided by AstraZeneca Pharmaceuticals (Cheshire, UK). Synchrony efficiency was determined by flow cytometric analysis (Warrener et al., 2003).
Indirect immunofluorescence staining was performed as described previously (Warrener et al., 2003) using antibodies against Aurora B (BD Biosciences, North Ryde, NSW, Australia), α-tubulin (Abcam, Redfern, NSW, Australia), γ-tubulin (Sigma-Aldrich), Borealin (a gift from WC Earnshaw, University of Edinburgh, UK), Survivin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), INCENP (Sigma-Aldrich), Mad2 antibody (Covance, Princeton, NJ, USA) and Human autoimmune ACA serum (a gift from R Thomas, University of Queensland, Brisbane, Australia). Antibody binding was detected using appropriate secondary antibodies. Centrin localization was determined using HeLa cells stably expressing centrin tagged with GFP (a gift from M Bornens, Institut Curie, Paris, France). Antibody binding detected using an appropriate secondary antibody and DNA detected with 4′,6′-diamidino-2-phenylindole. Confocal microscopy was carried out on HeLa cells stably expressing histone H2B tagged with GFP (pBOS-H2BGFP, BD Biosciences) using a Zeiss LSM 510 Meta microscope and × 100 objective under the control of AxioVision software.
Chromosome spreads were carried out on mitotic shake off cells. Quantification of CPC staining in regions encompassing the centromere (as determined by ACA staining) and identically sized regions on the chromosome arms were carried out using Image J software. The data are presented as a ratio of centromeric/chromosome arm intensity. For each chromosomal passenger, ten chromosomes in five cells were quantified in three independent experiments.
HeLa cells stably expressing H2B-GFP or CFP-tagged α-tubulin (plasmid gift from R Tsien, University of California, La Jolla, CA, USA) were synchronized and treated with HDACi as described previously. The cells were followed by time-lapse microscopy using a Zeiss Live Cell Observer with a 37 °C incubator and 5% CO2 hood. Images were captured every 6 min.
Cells were lysed in sodium dodecyl sulphate sample buffer and immunoblotting was performed as described previously (Warrener et al., 2003) using the same antibodies described for indirect immunofluorescence and poly(ADP-ribose)polymerase (BD Biosciences), α-tubulin (Sigma), Histone H3, Acetyl-Histone H3 (Lys9) and Phospho Histone H3 (Ser10) (Cell Signaling, Danvers, MA, USA).
chromosomal passenger complex
histone deacetylase inhibitor
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We thank WC Earnshaw, University of Edinburgh, UK, for the Borealin antibody, R Thomas, University of Queensland, for ACA serum and V Richon (Merck) for SAHA. BG is an NHMRC Senior Research Fellow. HB is a Lions Research Fellow. This work was supported by an NHMRC Australia project grant.
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Stevens, F., Beamish, H., Warrener, R. et al. Histone deacetylase inhibitors induce mitotic slippage. Oncogene 27, 1345–1354 (2008). https://doi.org/10.1038/sj.onc.1210779
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