ZM447439, a novel inhibitor of Aurora A and Aurora B
To identify novel Aurora inhibitors, ~250,000 compounds were screened for the ability to inhibit the kinase activity of recombinant human Aurora A against an artificial peptide substrate. One inhibitor identified was further modified to produce ZM447439 (4-(4-(N
A). In in vitro kinase assays using purified recombinant proteins, ZM447439 inhibited Aurora A and B with IC50
values of 110 and 130 nM, respectively ( B). In contrast, the majority of other protein kinases assayed were not inhibited by ZM447439. Based on data from model systems, we predicted that an Aurora inhibitor should prevent cell division and inhibit phosphorylation of histone H3 on serine 10. To determine whether ZM447439 inhibits cell division, a panel of human cell lines was treated with 2 μM ZM447439 for up to 96 h ( C). After 18 h, the vast majority of cells in all the lines had 4N DNA contents. All the lines analyzed then endoreduplicated, accumulating cells with DNA contents greater than 4N, demonstrating that ZM447439 completely inhibits cell division. To determine whether ZM447439 inhibits phosphorylation of histone H3 on serine 10, untreated and ZM447439-treated cells were stained with an anti-phosphohistone H3 antibody (Hsu et al., 2000
). In untreated cells, chromosomes stained positive for phosphohistone H3 ( D). However, after brief exposures to ZM447439, phosphohistone H3 was not detectable, demonstrating that ZM447439 does inhibit mitotic phosphorylation of histone H3. Consistent with previous observations (Adams et al., 2001c
), the lack of histone H3 phosphorylation did not appear to affect chromosome condensation.
Figure 1. ZM447439 inhibits Aurora A and Aurora B. (A) Chemical structure of ZM447439. (B) Table showing the IC50 values (μM) of ZM447439 against a panel of protein kinases. (C) DNA content histograms of HeLa, A549, and HME cells treated with ZM447439 for (more ...)
ZM447439-induced endoreduplication is enhanced in the absence of p53 function
Although a significant fraction of A549 and HME cells endoreduplicated in the presence of ZM447439, after 48 h virtually all the cells arrested with either 4N or 8N DNA contents ( C). In contrast, HeLa cells, which lack a functional p53 response, continued DNA synthesis and rapidly lost viability. This raises the possibility that the 4N/8N arrest exhibited by the A549 and HME cells was not directly due to ZM447439, but was rather due to activation of the p53-dependent post-mitotic checkpoint that occurs after an aberrant mitosis and/or cytokinesis (Andreassen et al., 2001
). Consistently, in the presence of ZM447439, U2OS cells expressing a dominant-negative p53 mutant endoreduplicated efficiently such that by 48 h, 78% had 8N DNA contents (
A). In contrast, only 38% of the p53-proficient parental U2OS cells had 8N DNA contents, demonstrating that p53 does restrain cell cycle progression after ZM447439-induced division failure. To test if cell cycle progression in the presence of ZM447439 may account for why the HeLa cells lost viability, we analyzed the colony-forming potential of cells after a 72-h exposure to ZM447439. MCF7 cells were selected for this experiment, as they can be arrested in G0 by treatment with anti-estrogens. Cells that were growth-arrested during exposure to ZM447439 gave rise to more colonies than those that were proliferating ( B). Consistently, at 1.25 and 2.5 μM ZM447439, the cloning efficiency of the proliferating cells was reduced to below 40%, whereas the cloning efficiency of the growth-arrested cells was largely unaffected ( C). Although continued cell cycle progression in the presence of ZM447439 leads to loss of viability, the p53-deficient U2OS cells did not appear to lose viability as rapidly as the HeLa cells. The reason for this is unclear, but it suggests that p53-independent mechanisms may also affect cell fate after ZM447439-induced tetraploidization.
Figure 2. p53 restrains endoreduplication in the presence of ZM447439. (A) DNA content histograms of U20S cells with (p53+) or without (p53−) a functional p53 response treated with ZM447439 for the times indicated in hours. (B) Growth-arrested or proliferating (more ...)
ZM447439-treated cells enter mitosis, but fail to divide
The observation that ZM447439-treated cells can enter additional S phases without dividing raises two possibilities: either the cells re-replicate their genomes without entering mitosis, or alternatively, the cells enter and exit mitosis without dividing and then enter a second S phase. To distinguish between these two possibilities, we analyzed HeLa cells after release from a G1/S block into fresh medium or media supplemented with either ZM447439 or nocodazole, a spindle toxin that prevents microtubule polymerization ()
. At various times after G1/S, the cells were harvested to determine DNA content, mitotic index, and cyclin B1 levels. DNA content histograms show that the vast majority of control cells divided by 12 h then entered a second S phase such that by 18 h, the majority had DNA contents greater than 2N ( A). Consistently, the mitotic index peaked at 10 h and cyclin B1 levels decreased as the cells completed mitosis (). Nocodazole-treated cells entered mitosis, and then remained arrested with high cyclin B1 levels for the remainder of the experiment. Cells released into ZM447439 progressed through S phase, failed to divide, degraded cyclin B1 normally, and then entered a second S phase (). Significantly, the kinetics with which the mitotic index increased and decreased was very similar to the control culture ( B). Thus, in the presence of ZM447439, HeLa cells enter and exit mitosis normally, but fail to divide. A549 and HME cells exhibited a similar response (unpublished data).
Figure 3. ZM447439-treated cells enter mitosis, but fail to divide. HeLa cells were released from G1/S into various drug combinations, harvested at the times indicated in hours, and then analyzed by flow cytometry and immunoblotting. The data shown are representative (more ...)
ZM447439 inhibits chromosome alignment
The observation that ZM447439-treated cells enter and exit mitosis but fail to divide raises two possibilities: either chromosome segregation takes place normally but cytokinesis fails, or alternatively, chromosome segregation fails, preventing cytokinesis. To determine whether ZM447439 prevents chromosome segregation, we analyzed spindle morphology in ZM447439-treated cultures. In controls, prometaphase rosettes, and metaphase and anaphase spindles were readily apparent ()
. In ZM447439-treated cultures, bipolar spindles were observed and the proportion of cells in prophase appeared normal. However, the proportion of metaphase and anaphase spindles was markedly reduced, indicating that ZM447439 inhibits chromosome segregation ( B). Despite the lack of anaphases, cells treated with ZM447439 alone did not accumulate in mitosis ( C). Cells treated with ZM447439 and nocodazole did however accumulate in mitosis, confirming that ZM447439-treated cells enter and exit mitosis without undergoing chromosome segregation. Chromosome alignment also appeared abnormal in the presence of ZM447439. In particular, the chromosomes either splayed out throughout the cell or, rather than aligning at the spindle equator, lined up along the length of the spindle ( A). In these latter cases, the kinetochores were oriented toward the spindle, suggesting that kinetochore–microtubule interactions were taking place. Indeed, kinetochores and kinetochore fibers were observed in thin sections analyzed by electron microscopy ( D). In 19 control cells, fibers containing multiple microtubules were observed in 12 cases. Eight of these could be traced to kinetochores. In 20 ZM447439-treated cells, fibers containing multiple microtubules were observed in 15 cases, and six were traced to kinetochores. Although we cannot rule out the possibility that ZM447439 effects kinetochore structure and/or microtubule binding capacity, ZM447439 clearly does not prevent kinetochore–microtubule interactions or bipolar spindle formation. However, ZM447439 does inhibit chromosome alignment and segregation. To determine whether ZM447439 prevents sister chromatid separation, we allowed cells to pass through mitosis in the presence of ZM447439, and then analyzed chromosome spreads in the following mitosis. Diplochromosomes were never observed (unpublished data), suggesting that ZM447439 does not prevent loss of sister chromatid cohesion. It is unclear whether ZM447439 directly inhibits cytokinesis or whether cytokinesis is prevented as a secondary consequence of the block to chromosome segregation.
Figure 4. ZM447439 prevents chromosome alignment and segregation. (A) Mitotic DLD-1 cells stained for tubulin, kinetochores/centromeres (ACA, green), and DNA (red). Bottom panels show examples of abnormal prometaphases frequently observed after treatment with ZM447439 (more ...)
ZM447439 compromises spindle checkpoint function
The observations described above present a paradox: chromosome alignment defects should activate the spindle checkpoint, and thus cause mitotic arrest. However, ZM447439-treated cells exit mitosis with normal kinetics ( B), suggesting that ZM447439 may compromise checkpoint function. To test this possibility, we analyzed the effect of ZM447439 on cells after release from a nocodazole block. Nocodazole-arrested mitotic cells were isolated by selective detachment, washed to remove the nocodazole, and were then replated in various drug combinations (
, A–C). At various times the cells were harvested to determine DNA content, mitotic index, and cyclin B1 levels. 2 h after release, the majority of the control cells had completed chromosome segregation and divided ( A). In contrast, ZM447439, nocodazole, and ZM447439 plus nocodazole-treated cells failed to divide (unpublished data). The mitotic index of the control cells was initially high, but then fell to 15% 2 h after release. In contrast, in the continued presence of nocodazole the cells remained arrested in mitosis ( B). Consistently, in control cells, cyclin B1 levels fell after 2 h but remained high in the presence of nocodazole ( C). Strikingly, the mitotic index of ZM447439-treated cells fell extremely rapidly: after 1 h, only 4% of the ZM447439-treated cells were still in mitosis, compared with 67% of the controls ( B). Consistently, cyclin B1 was undetectable after 1 h. Thus, ZM447439-treated cells rapidly exit mitosis, consistent with the notion that ZM447439 overrides the spindle checkpoint.
Figure 5. ZM447439 compromises spindle checkpoint function. (A–C) Mitotic HeLa cells were isolated by selective detachment after a 12-h nocodazole block, replated in various drugs combinations, and then harvested at the times indicated in hours and analyzed. (more ...)
However, asynchronous cultures treated with ZM447439 and nocodazole accumulated cells in mitosis ( C), indicating that ZM447439 does not override the checkpoint under all circumstances. To explain this, we reasoned that perhaps ZM447439 only compromises the checkpoint when microtubules are allowed to polymerize. To test this, we analyzed the effect of ZM447439 on the accumulation of mitotic cells in response to paclitaxel, a spindle toxin that activates the checkpoint by stabilizing microtubules. In the presence of paclitaxel, nocodazole, or nocodazole plus ZM447439, the mitotic index reached ~25% after 6 h ( D). In contrast, the mitotic index of the ZM447439-treated culture remained low at ~7%. Significantly, the mitotic index of the culture treated with paclitaxel plus ZM447439 reached only 12%. To confirm this differential effect, we determined the mitotic index of nocodazole- and paclitaxel-treated cultures over a range of ZM447439 concentrations. E clearly shows that ZM447439 resolves the pharmacological effects nocodazole and paclitaxel have on checkpoint function. Thus, these observations confirm that ZM447439 can efficiently override the spindle checkpoint when microtubules are allowed to polymerize.
Although asynchronous cultures treated with ZM447439 and nocodazole accumulate mitotic cells in a manner similar to cultures treated with nocodazole alone ( C), after a 12-h nocodazole block, mitotic cells released into nocodazole plus ZM447439 exit mitosis faster than cells released into nocodazole alone ( 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 ( 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 (
A). However, ZM447439 did reduce kinetochore bound BubR1, Cenp-E, and Mad2 (
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 ( 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).
Figure 6. ZM447439 does not prevent localization of Aurora B to centromeres. Immunofluorescence images of prometaphase DLD-1 cells stained to detect Aurora B (green), Survivin (red), and DNA (blue) after (A) exposure to ZM447439 for 1 h or (B) transfection of Aurora (more ...)
Figure 7. ZM447439 inhibits kinetochore localization and phosphorylation of BubR1. (A) Projections of deconvolved image stacks showing mitotic DLD-1 cells treated for 1 h with the drugs indicated and then stained to detect kinetochores/centromeres (ACA), BubR1, (more ...)
Although kinetochore-bound BubR1 diminishes after chromosome alignment (Chan et al., 1999
; Taylor et al., 2001
), it is recruited back to metaphase kinetochores after the loss of tension induced by low levels of vinblastine (Skoufias et al., 2001
). To determine whether ZM447439 inhibits the reassociation of BubR1 with aligned kinetochores after loss of tension, we measured BubR1 signal intensity and interkinetochore distance at metaphase kinetochores after a 40-min treatment with either ZM447439 or paclitaxel ( C and Table SII). After paclitaxel treatment, the mean interkinetochore distance was reduced from 0.89 ± 0.20 μm to 0.58 ± 0.15 μm (P < 0.001), and the mean BubR1/ACA fluorescence ratio increased from 2.23 ± 1.21 to 3.86 ± 2.41 (P < 0.001). In contrast, although exposure to ZM447439 reduced the mean interkinetochore distance to 0.55 ± 0.14 μm, the BubR1/ACA fluorescence ratio decreased to 1.11 ± 0 0.88 μm, which is statistically different from the paclitaxel-treated and control cells (P < 0.001).
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 ( 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 ( 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 (
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 ( B). Repression of Aurora B (but not Aurora A) inhibited kinetochore localization of BubR1, Cenp-E, and Mad2 ( 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.
Figure 8. Repression of Aurora B prevents kinetochore localization of BubR1. HeLa and DLD-1 cells were transfected with siRNA duplexes to repress either Aurora A or B. (A) Immunoblots of HeLa cell lysates showing repression of Aurora A and B. (B) Transfected DLD-1 (more ...)
Overexpression of Aurora B K106R mislocalizes the endogenous protein
The Aurora B RNAi phenotypes appear more severe than those induced by ZM447439. In particular, Aurora B RNAi compromised checkpoint arrest in response to nocodazole as well as paclitaxel ( B). Furthermore, the chromosomes were frequently adjacent to the spindle ( D), suggesting the absence of kinetochore–microtubule interactions. Accordingly, the mean interkinetochore distance was markedly reduced ( E). One possible explanation for these differences is that in addition to its catalytic role, Aurora B may also play a structural role at centromeres. Significantly, Aurora B, INCENP, and Survivin form a complex that is required for multiple aspects of mitosis and cytokinesis (Adams et al., 2001a
). Disruption of this complex may have more extensive consequences rather than simply inhibiting Aurora B kinase activity. Consistently, although Survivin localizes to centromeres after ZM447439 treatment ( A), it does not after Aurora B RNAi ( B). If the above argument is valid, ectopic expression of an Aurora B kinase mutant should produce a phenotype similar to ZM447439. However, overexpression of Aurora B K109R abolished kinetochore–microtubule interactions (Murata-Hori and Wang, 2002
), resulting in a phenotype that is more consistent with our RNAi data. Therefore, we tested whether Aurora B localized to centromeres after overexpression of the Aurora B kinase mutant. After transient transfections of HeLa cells, the exogenous wild-type Aurora B localized to centromeres in prometaphase ( C) and midbodies after cell division (unpublished data). In addition, at low levels of expression, the Aurora B K106R mutant also localized to centromeres (note that human Aurora B K106R mutant is equivalent to rat K109R mutant described previously in Murata-Hori and Wang 
). However, in prometaphase cells expressing moderate to high levels of the K106R mutant, Aurora B was not apparent at centromeres ( C). Thus, in contrast to treatment with the Aurora kinase inhibitor, overexpression of an Aurora B kinase mutant prevents localization of the endogenous protein to centromeres.
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 (
A). Although these chromosomes appear to be attached to the spindle, the mean interkinetochore distance was reduced compared with control cells ( B), consistent with a reduction in pulling forces.
Figure 9. Repression of BubR1 prevents chromosome alignment. DLD-1 cells were transfected with siRNA duplexes to repress BubR1 and were then stained to detect Bub1 (green), tubulin (red), and DNA (blue). (A) Examples of abnormal prometaphase cells with chromosomes (more ...)
To rule out the possibility that this alignment defect was simply due to the cells prematurely entering anaphase, we treated control and BubR1 RNAi cultures with the proteosome inhibitor MG132 to prevent anaphase onset. Over a 3-h time course, the number of prophases and anaphases fell to almost zero in both control and BubR1 RNAi cultures (Fig. S4). In control cultures, the proportion of prometaphases remained roughly constant at ~25% and the proportion of metaphases rose to ~66% ( C), consistent with MG132 preventing the metaphase–anaphase transition. However, in BubR1-repressed cultures, the proportion of prometaphases rose from ~32 to ~49%, and the number of metaphases reached only ~44% ( B). Thus, when the metaphase–anaphase transition is blocked downstream of spindle checkpoint activation, repression of BubR1 reduces the accumulation of cells in metaphase, suggesting that BubR1 is indeed required for chromosome alignment.