Article |
Address correspondence to Stephen S. Taylor, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK. Tel.: 44-161-275-5100. Fax: 44-161-275-5763. E-mail: stephen.taylor{at}man.ac.uk
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Abstract |
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Key Words: mitosis; spindle checkpoint; chemical biology; aneuploidy; ZM447439
* Abbreviation used in this paper: RNAi, RNA interference.
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Introduction |
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The Ipl1/Aurora family of protein kinases plays multiple roles in mitosis (Bischoff and Plowman, 1999; Giet and Prigent, 1999; Adams et al., 2001a; Nigg, 2001). In budding yeast, Ipl1 ensures accurate chromosome segregation by resolving syntelic orientations, possibly by monitoring tension at centromeres and destabilizing inappropriately bound microtubules (Tanaka et al., 2002). Ipl1 phosphorylates the kinetochore component Ndc10 in vitro, suggesting that it may regulate kinetochoremicrotubule interactions directly (Biggins et al., 1999). However, budding yeast are atypical in that centromeres connect to the unduplicated spindle pole body (SPB) in G1. Because centromeres replicate before SPB duplication, budding yeast cells then enter mitosis with both kinetochores attached to the old SPB (Tanaka et al., 2002). In higher eukaryotes, syntelic orientations are rare during mitosis (Nicklas, 1997), and therefore, it is unclear whether the kinetochoreSPB resolving activity exhibited by Ipl1 is a universal feature of the Aurora kinase family. Further evidence suggesting that Ipl1 monitors tension at centromeres comes from analyzing budding yeast mutants that lack sister chromatid cohesion or enter mitosis without replicating their DNA. In such mutants, anaphase is prevented in an Ipl1-dependent manner (Biggins and Murray, 2001). Because kinetochores in these cells lack sisters, they fail to come under tension despite microtubule attachment, arguing that Ipl1 is required for spindle checkpoint activation in response to loss of tension at centromeres. However, budding yeast kinetochores only attach a single microtubule. Therefore, if the primary role of Ipl1 is to destabilize bound microtubules, the apparent role of Ipl1 in checkpoint activation may be simply a secondary consequence of exposing microtubule binding sites (Tanaka et al., 2002). Furthermore, although the analysis of replication and cohesion mutants suggests that Ipl1 monitors tension at centromeres, these interpretations are complicated by the fact that centromeric localization of the fission yeast Aurora kinase, Ark1, requires sister chromatid cohesion (Morishita et al., 2001). Therefore, there is a clear need to analyze Aurora kinase function under conditions where replication and cohesion are normal.
Higher eukaryotes express two or more Aurora kinases. Aurora A and C localize to spindle poles, and Aurora A is required for bipolar spindle formation in a variety of systems (Bischoff and Plowman, 1999; Giet and Prigent, 1999; Adams et al., 2001a; Nigg, 2001). Inhibition of Aurora B, an inner centromere protein, affects multiple mitotic events including histone H3 phosphorylation, chromosome segregation, and cytokinesis (Bischoff and Plowman, 1999; Giet and Prigent, 1999; Adams et al., 2001a; Nigg, 2001). The role of Aurora B kinase activity has been addressed by ectopically expressing mutants in mammalian cells. However, these studies have yielded conflicting results. In two cases, cells expressing Aurora B K109R completed mitosis, but failed to undergo cytokinesis, suggesting that Aurora B activity is not required for chromosome segregation (Tatsuka et al., 1998; Terada et al., 1998). However, another report indicates that Aurora B K109R prevents chromosome alignment due to the failure of kinetochoremicrotubule interactions (Murata-Hori and Wang, 2002). These experiments are complicated by the fact that expression of wild-type Aurora B can itself affect cell division (Tatsuka et al., 1998), and therefore, it is not clear whether these phenotypes are due to reduced Aurora B kinase activity or the disruption of Aurora B protein complexes. Xenopus cells injected with anti-Aurora B antibodies exit mitosis prematurely, consistent with a role for Aurora B in the spindle checkpoint (Kallio et al., 2002). However, in contrast to Ipl1 deficient strains, mitotic exit also occurred when microtubule polymerization was inhibited, suggesting that Aurora B monitors microtubule attachment, not just tension. Thus, although many roles have been attributed to Aurora B, the emerging picture is confusing, and molecular explanations for these phenotypes are currently lacking.
Aurora A and B are overexpressed in human tumors, and ectopic overexpression in cultured cells leads to transformation, centrosome abnormalities, and aneuploidy (Bischoff et al., 1998; Tatsuka et al., 1998; Zhou et al., 1998; Adams et al., 2001b; Meraldi et al., 2002). In addition, cells overexpressing Aurora A, but not a kinase mutant, readily form tumors in nude mice (Bischoff et al., 1998). Therefore, elevated Aurora kinase activity may promote tumor evolution either by providing a growth advantage or by promoting genetic instability. To develop novel anti-cancer drugs, we have generated small molecule inhibitors of Aurora kinase activity. Here, we describe ZM447439, which selectively inhibits the kinase activity of Aurora A and B. Using ZM447439 as a research tool, we directly address the role of Aurora kinase activity in human cells. We show that inhibition of Aurora kinase activity does not prevent progression through interphase, mitotic entry, bipolar spindle formation, or kinetochoremicrotubule interactions. Rather, Aurora kinase activity is required for correct chromosome alignment and spindle checkpoint function. Using RNA interference (RNAi;* Elbashir et al., 2001), we demonstrate that these phenotypes are due to inhibition of Aurora B, not Aurora A.
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Results |
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Although asynchronous cultures treated with ZM447439 and nocodazole accumulate mitotic cells in a manner similar to cultures treated with nocodazole alone (Fig. 4 C), after a 12-h nocodazole block, mitotic cells released into nocodazole plus ZM447439 exit mitosis faster than cells released into nocodazole alone (Fig. 5 B). This suggests that perhaps after prolonged mitotic arrest, ZM447439 can also compromise the checkpoint induced by microtubule depolymerization. To test this, mitotic HeLa cells were harvested after a 2- or 12-h nocodazole block, then replated for 4 h in either nocodazole alone or nocodazole plus ZM447439. Although 80% of the cells harvested after a 2-h block remained arrested in the presence of ZM447439 and nocodazole, only
20% of the cells isolated after a 12-h block remained arrested. Thus, after prolonged mitotic arrest, ZM447439 can compromise checkpoint arrest induced by microtubule depolymerization. However, what is clearly evident from this analysis is that in the short term, ZM447439-treated cells do undergo mitotic arrest when microtubules are depolymerized, but fail to arrest when microtubules are stabilized (Fig. 5 E).
ZM447439 inhibits kinetochore localization of BubR1, Mad2, and Cenp-E
To gain insight into how ZM447439 compromises chromosome alignment and checkpoint function, we analyzed its effect on the localization of Aurora A, Aurora B, Survivin, the spindle checkpoint components BubR1 and Mad2, and the kinesin-related motor protein Cenp-E. ZM447439 did not prevent localization of Aurora A to spindle poles (unpublished data) or the localization of Aurora B and Survivin to centromeres (Fig. 6 A). However, ZM447439 did reduce kinetochore bound BubR1, Cenp-E, and Mad2 (Fig. 7 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200208091/DC1). Quantitation of pixel intensities shows that ZM447439 reduced kinetochore-associated BubR1 to 10%, both in the presence and absence of microtubule toxins (Fig. 7 B, Fig. S1, and Table SI). ZM447439 reduced kinetochore-bound Mad2 to <10% in prometaphase cells and to 30 and 43% in the presence of nocodazole and paclitaxel, respectively (Fig. S1 C). Kinetochore-bound Cenp-E was reduced to 28 and 23% in cells treated with ZM447439 and ZM447439 plus paclitaxel, respectively. However, in the presence of nocodazole and ZM447439, Cenp-E was only reduced to 59%. Interestingly, inspection of the fluorescence intensity histograms (Fig. S2) shows that in the presence of nocodazole, although ZM447439 reduced kinetochore-bound Cenp-E at the majority of kinetochores, a significant number retained normal Cenp-E levels (30% have a Cenp-E/ACA ratio greater than the mean value for kinetochores in cells treated with nocodazole only). In contrast, no kinetochores had normal BubR1 levels in the presence of ZM447439 and nocodazole (0% have a BubR1/ACA ratio greater than the mean nocodazole only value).
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BubR1 is phosphorylated in response to spindle damage (Chan et al., 1999; Taylor et al., 2001). To determine whether ZM447439 prevents BubR1 phosphorylation, mitotic cells were harvested 9.5 h after release from a G1/S block into various drug combinations. In the presence of ZM447439, the phosphorylated form of BubR1 was not detectable, either in the presence or absence of nocodazole (Fig. 7 D). To rule out the possibility that ZM447439 inhibits BubR1 directly, BubR1 immunoprecipitates were assayed for kinase activity in the presence and absence of ZM447439. Although ZM447439 inhibited Aurora A, it did not inhibit BubR1 (Fig. 7 E).
Repression of Aurora B inhibits kinetochore localization of BubR1, Cenp-E, and Mad2
The ZM447439 data suggest that Aurora kinase activity is required for chromosome alignment and spindle checkpoint function. To determine which Aurora is required for these functions, and to rule out the possibility that the ZM447439 phenotypes might be due to inhibition of another kinase, we repressed Aurora A and B by RNAi (Fig. 8 A). Although control and Aurora A RNAi cultures had robust spindle checkpoints, repression of Aurora B reduced the accumulation of mitotic cells after spindle damage (Fig. 8 B). Repression of Aurora B (but not Aurora A) inhibited kinetochore localization of BubR1, Cenp-E, and Mad2 (Fig. 8 C and Fig. S3). Thus, these observations indicate that the ZM447439-induced phenotypes described above are due to inhibition of Aurora B, not Aurora A or some other kinase.
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BubR1 is required for chromosome alignment
Our observations are consistent with the notion that Aurora B kinase activity regulates the spindle checkpoint, at least in part, by targeting BubR1 to kinetochores. Because BubR1 binds Cenp-E (Chan et al., 1999; Yao et al., 2000), we reasoned that the requirement for Aurora B in promoting correct chromosome alignment might also be mediated, at least in part, via its affect on BubR1. To test this, we used RNAi to determine whether repression of BubR1 inhibited chromosome alignment. Consistent with antibody injection experiments (Chan et al., 1999), repression of BubR1 compromised spindle checkpoint function. In particular, in asynchronous cultures, the number of metaphases was reduced and the anaphases frequently displayed lagging chromosomes (Fig. S4). Furthermore, BubR1 RNAi cultures did not accumulate mitotic cells on exposure to spindle toxins (unpublished data). Strikingly, prometaphase cells in BubR1 RNAi cultures often appeared abnormal with the chromosomes aligned along the length of the spindle rather than at the metaphase plate (Fig. 9 A). Although these chromosomes appear to be attached to the spindle, the mean interkinetochore distance was reduced compared with control cells (Fig. 9 B), consistent with a reduction in pulling forces.
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Discussion |
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Aurora B kinase activity regulates chromosome alignment and the spindle checkpoint
Consistent with previous reports (Adams et al., 2001c; Kallio et al., 2002; Murata-Hori and Wang, 2002), our observations show that Aurora B is required for both spindle checkpoint function and metaphase chromosome alignment in human cells. By using a novel selective protein kinase inhibitor, we have been able to directly address, for the first time, the requirement for Aurora B kinase activity in these processes. Because the phenotypes derived from protein repression and overexpression appear more extensive than those induced by ZM447439, our data demonstrate the usefulness of small molecule inhibitors in dissecting complex cellular processes. Indeed, kinetochore fibers form in the presence of ZM447439, suggesting that Aurora B kinase activity is not required for kinetochoremicrotubule interactions, but rather regulates these interactions to promote correct chromosome alignment. Such a role for Aurora B is entirely consistent with the role Ipl1 plays in budding yeast: Ipl1 is not required for the attachment of chromosomes to the spindle, but rather resolves inappropriate kinetochoremicrotubule interactions to ensure correct bi-orientation (Tanaka et al., 2002).
Ipl1 has also been implicated in spindle checkpoint function (Biggins and Murray, 2001) as well as chromosome alignment. Because Aurora B also promotes chromosome alignment, is it possible that its role in checkpoint activation is a secondary consequence of generating unattached kinetochores, as has been argued for Ipl1 (Tanaka et al., 2002)? If this were the case, we would predict that in the absence of Aurora B kinase activity, BubR1 and Mad2 should localize to kinetochores that lack bound microtubules. However, in the presence of nocodazole and ZM447439, localization of both BubR1 and Mad2 to kinetochores is severely reduced. Thus, in human cells at least, Aurora B kinase activity does appear to be directly required for checkpoint function.
The most striking observation in this work is the differential effect that ZM447439 has on nocodazole- and paclitaxel-induced mitotic arrest. Below, we consider two possible explanations for this observation, one of which is qualitative in nature, the other quantitative. The "qualitative" interpretation is that the checkpoint must be composed of multiple pathways, one of which does not require Aurora B kinase activity. If this is the case, then Aurora B kinase activity is not essential for checkpoint activation in response to loss of kinetochoremicrotubule interactions and/or spindle destruction, but is essential under conditions where kinetochores can capture microtubules, but not correctly align on the spindle. This would indicate that, like Ipl1 in budding yeast (Biggins and Murray, 2001), Aurora B does not directly monitor kinetochoremicrotubule interactions, but rather activates the checkpoint in response to loss of tension at centromeres.
An alternative "quantitative" explanation is that Aurora B simply targets BubR1 and Mad2 to kinetochores irrespective of tension. Although ZM447439 clearly reduces kinetochore-associated BubR1 and Mad2 (by 90 and
70%, respectively), perhaps the residual bound protein is sufficient to sustain mitotic arrest in the absence of kinetochoremicrotubule interactions. If microtubule occupancy is sufficient to then inactivate the remaining bound protein, this may explain why cells cannot arrest in the presence of ZM447439 and paclitaxel. Interestingly, kinetochore-bound Cenp-E is almost twofold higher in cells treated with ZM447439 and nocodazole compared with cells treated with ZM447439 plus paclitaxel. Because Cenp-E is thought to activate the checkpoint via BubR1 (Chan et al., 1999; Yao et al., 2000), perhaps this elevated level of Cenp-E is sufficient to activate the residual BubR1 and thus maintain mitotic arrest in the presence of nocodazole.
At present, it is difficult to imagine how one could distinguish between these two possibilities. Indeed, whether the spindle checkpoint monitors tension, microtubule attachment, or both remains unsolved (Musacchio and Hardwick, 2002). Although evidence from budding yeast suggests that Ipl1 does activate the checkpoint in response to the lack of tension (Biggins and Murray, 2001), there is compelling evidence to argue that the checkpoint in mammalian somatic cells only monitors microtubule attachment (Rieder et al., 1995). Yet mammalian kinetochores are clearly sensitive to changes in tension (Waters et al., 1998), and in particular BubR1, but not Mad2, is recruited to aligned kinetochores after loss of tension (Skoufias et al., 2001; Shannon et al., 2002). However, it is possible that tension and attachment are not separable events in terms of checkpoint function. Indeed, it was shown many years ago that tension stabilizes microtubule attachment (Nicklas and Koch, 1969). Furthermore, our observation showing that BubR1 plays a dual role in congression and checkpoint function indicates that the mechanisms which regulate and monitor chromosome alignment are interweaved at the molecular level.
Aurora inhibitors as potential therapeutic agents
The Auroras represent a new family of protein kinases with oncogenic potential (Bischoff et al., 1998; Tatsuka et al., 1998; Zhou et al., 1998; Adams et al., 2001a). Our observations show that it is possible to selectively inhibit Aurora kinase activity in cells with a small molecule inhibitor. Furthermore, we have shown that relative to cells with a functional p53 response, p53-deficient cells are more likely to continue cell cycle progression in the presence of ZM447439. In addition, cycling cells rapidly lose viability in the presence of ZM447439, whereas nondividing cells retain viability. Together, these observations suggest that Aurora kinase inhibitors may be selectively toxic to proliferating tumor cells, and therefore open up new opportunities to develop novel anti-cancer agents.
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Materials and methods |
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Cell culture
A549, MCF-7, and DLD-1 cells (American Type Culture Collection), TA-HeLa cells (Taylor and McKeon, 1997), U2OS cells stably transfected with pCMVp53 143A, and parental U2OS cells (both from Karen Vousden, Beatson Institute for Cancer Research, Glasgow, UK) were all cultured as described previously (Taylor et al., 2001). Nontransformed human mammary epithelial cells expressing hTERT (CLONTECH Laboratories, Inc.) were cultured in MEGM (Clonetics). Nocodazole, paclitaxel, and thymidine were used as described previously (Taylor et al., 2001). MG132 (Calbiochem) was dissolved in DMSO and used at a final concentration of 20 µM. ZM447439 was dissolved in DMSO at 10 mM and stored at -20°C for up to 9 mo in individual aliquots to avoid freeze-thaw cycles, and was then freshly diluted in media. The IC50 values for Aurora A and B (100 nM) were determined at the Km for ATP (see above). However, because the cellular ATP concentration is
200-fold higher, and because ZM447439 is an ATP competitor, we used ZM447439 at a concentration of 2 µM in all the cell assays unless stated otherwise. DMSO was added to drug-free cultures to account for the solvent.
Cell cycle analysis and cloning assays
DNA content and mitotic index measurements and synchronization of TA-HeLa cells at G1/S using a double thymidine block were done as described previously (Taylor and McKeon, 1997). To determine cloning efficiency, MCF7 cells were plated in phenol red free DME plus 5% stripped serum (HyClone), and were then treated with or without the anti-estrogen ICI 182780 at 1 µM for 48 h. ZM447439 was then added at the indicated concentrations for 72 h. The cells were harvested, washed, and 400 cells plated in each well of a 6-well plate in complete media without ZM447439. After 10 d, the colonies were fixed, stained with crystal violet, and counted. The cloning efficiency represents the number of colonies on ZM447439-treated plates compared with DMSO-treated controls.
Antibody techniques
Immunofluorescence, immunoblotting, and immunoprecipitations were all done as described previously (Taylor et al., 2001) using antibodies against the following: phosphohistone H3 (Upstate Biotechnology); cyclin B1 (Upstate Biotechnology); tubulin (TAT1); centromere/kinetochores (human ACA); BubR1 (SBR1.1); Bub1 (4B12); Aurora A (RAA.1); and Myc-tag (9E10). Aurora B was detected using either the anti-AIM-1 mouse mAb (Transduction Laboratories) or a sheep antihuman Aurora B pAb (unpublished data). For localization of Mad2 and Survivin, we used DLD-1 cell lines stably expressing Myc-tagged hMad2 or hSurvivin ORFs (unpublished data). For IP kinase assays, beads were equilibrated in kinase buffer (10 mM Tris, pH7.5, 5 mM KCl, 1 mM NaF, 0.24 mM DTT, and 2.5 mM MnCl) and were then incubated at RT for 1 h in kinase buffer supplemented with 2.5 µM ATP, 5 µCi [32P]ATP, and 3 µM biotinyl-Ahx-tetra (LRRWSLG) peptide substrate. Reactions were stopped with 20% phosphoric acid, and were then spotted onto P30 filtermat (Whatman). After five washes in 0.5% phosphoric acid, bound radiolabel was quantitated by scintillation counting. Deconvolution microscopy and pixel intensity quantitation were performed as described previously (Taylor et al., 2001). In brief, kinetochore fluorescence values were determined using softWoRx imaging software (Applied Precision). Background readings were subtracted, and the values were then normalized against the ACA signal to account for any variations in staining or image acquisition. SoftWoRx was used to measure interkinetochore distances using either ACA or Bub1 foci as indicated to determine kinetochore position.
Electron microscopy
Cells were fixed with 2.5% glutaraldehyde in phosphate buffer for 2 h at RT, and were then pelleted and post-fixed in 1% osmium tetroxide for 1 h at RT. After washes, samples were stained en bloc in 1% uranyl acetate for 16 h at 4°C, dehydrated with acetone, and then embedded in Spurr's resin. 70-nm sections were cut, stained with uranyl acetate and lead citrate, then examined on an electron microscope (Tecnai 12 BioTWIN; FEI Company).
RNAi
siRNA duplexes (Dharmacon Research) designed to repress Aurora A (5'-AAGCACAAAAGCUUGUCUCCA-3'), Aurora B (5'-AACGCGGCACUUCACAAUUGA-3'), and BubR1 (5'-AACGGGCAUUUGAAUAUGAAA-3') were transfected using OligofectAMINETM (Invitrogen) according to the manufacturer's instructions. In brief, 105 cells were seeded in wells of a 24-well plate 24 h before transfection. siRNA duplexes and OligofectAMINETM were diluted in media, mixed, and incubated for 20 min. siRNA/lipid complexes were then added to cells for 4 h followed by addition of complete media. 24 h later, the cells were replated and then analyzed 4872 h after transfection.
Transient transfections
The human Aurora B ORF (Bischoff et al., 1998) was amplified by PCR, cloned into pcDNA-3 Myc (Taylor and McKeon, 1997), mutated to create K106R, and sequenced, all after standard procedures. Plasmid DNA was then transfected into TA-HeLa cells on coverslips using the ProFection® calcium phosphate kit (Promega). 24 h after transfection, the cells were fixed with 1% formaldehyde in PBS and processed for immunofluorescence as described above.
Online supplemental material
The supplemental figures and tables show (1) the effects of ZM447439 and Aurora B RNAi on Cenp-E and Mad2 localization; (2) the effect of BubR1 RNAi on the spindle checkpoint; and (3) quantitation of the data from the experiment examining the effect of ZM447439 on interkinetochore distance and BubR1 binding. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200208091/DC1
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Acknowledgments |
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C. Ditchfield is funded by AstraZeneca, and V.L. Johnson by Cancer Research UK and The Biotechnology and Biological Sciences Research Council (BBSRC). S.S. Taylor is a BBSRC David Phillips Research Fellow.
Submitted: 15 August 2002
Revised: 24 February 2003
Accepted: 24 February 2003
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