An indolinone that causes polyploidy
In the course of a synthesis program for novel indolinones, we tested the effect of several compounds on cell proliferation. One of these, Hesperadin (
A; Walter et al., 2002
), had dramatic effects; HeLa cells treated with 50 nM of Hesperadin stopped proliferating but did not stop growing, and over a 6-d period, the cell diameter increased more than sevenfold (from ~20 to >150 μm; B). During this time, the cells acquired enlarged lobed nuclei (see i). FACS®
analysis revealed that the increase in nuclear size correlated with polyploidization, reaching a 32C DNA content on day 3 ( B; unpublished data). At later stages, FACS®
could not be used to analyze these cells, presumably because they had grown too big to enter the measuring capillary.
Figure 1. Hesperadin causes polyploidy in HeLa cells. (A) Chemical structure of Hesperadin. (B) Hesperadin (50 nM) was added to logarithmically growing HeLa cells (day 0). At the indicated time points, the DNA content was determined by flow cytometry, and phase (more ...)
Figure 4. Hesperadin-treated HeLa cells show alignment and segregation defects, but sister chromatid separation is intact. HeLa cells were left untreated or were treated with 50 nM Hesperadin for different periods of time before harvesting by mitotic shake off, (more ...)
Hesperadin causes defects in mitosis and cytokinesis
To analyze how Hesperadin causes polyploidy, we filmed HeLa cells expressing a GFP-tagged version of histone H2B (Kanda et al., 1998
) in the presence of 100 nM Hesperadin (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1
). During mitosis, the cells rounded normally, condensed their chromosomes, and aligned some, but not all, chromosomes on a metaphase-like plate. The cells then exited mitosis in the presence of nonaligned chromosomes. During this time, the chromatin mass appeared to be stretched toward opposite poles, but the chromosomes were not segregated into two distinct masses. The cells attempted cytokinesis, which was accompanied by intense blebbing of the plasma membrane. However, the cleavage furrow ultimately regressed to produce a single cell in which the stretched chromosomal masses decondensed to form an irregularly shaped restitution nucleus.
Hesperadin does not inhibit APC and separase activation
To understand how Hesperadin causes mitotic defects, we arrested HeLa cells by double-thymidine treatment in S phase, released them into 100 nM Hesperadin, and analyzed their progression through mitosis (). Immunoblot analysis revealed that the APC subunit Cdc27 underwent an electrophoretic mobility shift and that the APC substrates cyclin A, cyclin B, securin, and Cdc20 were degraded in mitosis, indicating that APC phosphorylation and activation were not inhibited by Hesperadin ( D; unpublished data). Also, the activation of separase appeared unaffected, as we observed cleavage of separase and the cohesin subunit Scc1 (unpublished data). All of these events were delayed by 60–90 min relative to control cells. Phase contrast microscopy of living cells showed that Hesperadin-treated cells began to round up about 1 h later than control cells (unpublished data), and immunoblotting revealed that mitotic dephosphorylation of Cdk1 also occurred ~60–90 min later in Hesperadin-treated cells (unpublished data), suggesting that the observed delay in APC and separase activation may indirectly be caused by a slight delay in mitotic entry.
Hesperadin inhibits Aurora B function
As a mitotic marker, we also analyzed phosphorylation of serine 10 on histone H3 in the same experiment. We found that immunoblotting yielded a phospho-histone H3 signal in mitotic Hesperadin-treated cells that was greatly reduced relative to controls (
A). Immunofluorescence microscopy confirmed this result ( B). Because mitotic H3-Ser10 phosphorylation depends on Aurora B (Adams et al., 2001a
), Hesperadin appears to inhibit Aurora B function.
Figure 2. Hesperadin inhibits histone H3 phosphorylation and causes midspindle defects. (A) Samples from the same experiment as in (C and D) were analyzed by SDS-PAGE and immunoblotting with anti–phospho-histone H3 antibody. (B) HeLa cells were treated (more ...)
In Caenorhabditis elegans
, Aurora B is required for the formation of a central spindle during anaphase and for stable association of the centralspindlin complex with this structure (for review see Adams et al., 2001a
). Immunofluorescence microscopy revealed that Hesperadin-treated cells in which chromosomes were stretched toward opposite poles, i.e., which had entered anaphase, failed to assemble a central spindle and to properly localize the human centralspindlin subunits CYK-4 and MKLP1 ( C). These findings further indicate that Hesperadin inhibits Aurora B and imply that Aurora B function is also required for central spindle assembly in human cells.
Approximately 250 nM Hesperadin was required to half-maximally inhibit the ability of immunoprecipitated Aurora B to phosphorylate histone H3 ( D). In contrast, dose response experiments on HeLa cells revealed that polyploidization and loss of mitotic histone H3-Ser10 phosphorylation were induced by only 20–100 nM Hesperadin (unpublished data). Currently, we do not know if the different doses that are required to inhibit Aurora B function in cells and in vitro are due to technical differences in the assays used, or due to an intracellular accumulation of Hesperadin. It also remains possible that Aurora B function is inhibited indirectly, e.g., by inhibiting an enzyme required for Aurora B activation. We conclude that Hesperadin inhibits Aurora B function in living cells either directly or indirectly.
Specificity of Hesperadin
When we measured the activity of 25 kinases in the presence of 1 μM Hesperadin, we found that Hesperadin markedly reduced the activity of six kinases (AMPK, Lck, MKK1, MAPKAP-K1, CHK1, and PHK) (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1
). In the same assay, in vitro inhibition of multiple kinases has also been found for other presumably specific inhibitors (Davies et al., 2000
). Inhibition of the MAP kinase kinase (MEK1/MKK1) has been reported to block cells in G2 (Wright et al., 1999
; Hayne et al., 2000
) and could therefore account for the delay in mitotic entry that we observe. However, immunostaining of Hesperadin- and control-treated cells with a phosphospecific antibody for the MKK1 substrates Erk1 and Erk2 did not reveal any difference in staining (unpublished data), suggesting that at the used concentration, Hesperadin does not inhibit MKK1 activity in vivo.
We cannot formally exclude that Hesperadin influences AMPK, CHK1, Lck, MAPKAP-K1/p90Rsk, or PHK activity in vivo. However, based on their known cellular functions, their inhibition is unlikely to cause the phenotype that we describe here. Furthermore, Cdk1/cyclin B was half-maximally inhibited at 2.8 μM, and Cdk2/cyclinE and Cdk4/cyclinD1 at >10 μM ( D; unpublished data), suggesting that Cdks are not inhibited in vivo.
Aurora B RNA interference (RNAi) phenocopies Hesperadin treatment
To further test if the phenotype induced by Hesperadin is due to the inhibition of Aurora B function, we used RNA interference (RNAi). In HeLa cells treated with small inhibitory RNAs (siRNAs) directed against Aurora B, the level of Aurora B protein was substantially reduced, as judged by both immunoblotting (
A) and immunofluorescence ( B), whereas Aurora A levels were affected only to a minor extent ( A). Immunofluorescence microscopy revealed that mitotic cells without detectable Aurora B staining frequently contained chromosomes that were not aligned on the spindle equator ( B) and phospho-histone H3 staining was often reduced or absent ( B). We also observed elongated cells, presumably undergoing anaphase, with some chromosomes that were located at opposite poles but others that were lagging ( C). α-Tubulin staining revealed that these cells did not form a midspindle structure ( C). Instead of being highly localized, as in control anaphase cells, Survivin was diffusely localized in the region between the poles ( C). siRNA cultures also contained enlarged cells with multiple nuclei and micronuclei ( D) that were likely the consequence of cytokinesis defects. Together, these observations reveal that human Aurora B is required for mitotic phosphorylation of histone H3, chromosome alignment, chromosome segregation, formation of a midspindle, and cytokinesis, consistent with earlier observations in C. elegans
embryos and Drosophila melanogaster
(for review see Adams et al., 2001a
). Hesperadin-treated cells exhibit a very similar phenotype ( and ), which is further evidence that Hesperadin inhibits Aurora B function in vivo. We therefore named our inhibitor Hesperadin in reference to the antique goddess of dusk, Hespera, who is the counterpart of Aurora, the goddess of dawn.
Figure 3. Aurora B RNAi in human cells induces a similar phenotype as Hesperadin. (A) HeLa cells were transfected with Aurora B–targeting siRNA duplex (RNAi AurB) or with H2O replacing the siRNA (control). After 50 h, cells were lysed in sample buffer. (more ...)
Aurora B function is not required for chromosome condensation or sister chromatid separation
To investigate the role of Aurora B in chromosome segregation, we performed chromosome spreading of mitotic HeLa cells treated with Hesperadin. Although normal metaphase and anaphase figures (
, a and b) were absent, prometaphase cells showed some degree of chromosome alignment, and chromosomes were frequently bent in the centromeric region ( c, inset, black squares). Such bendings are infrequent in nocodazole-treated cells (compare with A) and therefore are likely to arise from microtubule attachments. This, and other observations (see below), suggests that most chromosomes become attached to the mitotic spindle when Aurora B is inhibited.
Figure 8. Hesperadin quickly overrides the mitotic arrest induced by taxol or monastrol. (A, B, and C) HeLa cells were arrested in nocodazole (330 nM), taxol (10 μM), or monastrol (100 μM). Hesperadin (100 nM) or the solvent DMSO was added, and (more ...)
The overall compaction of chromatin in prometaphase and the association of condensin with chromosomes did not seem to be affected (, c–f; unpublished data), suggesting that Aurora B function is not required for chromosome condensation in human cells. Notably, however, in preanaphase chromosomes, sister chromatids were often less resolved in Hesperadin-treated cells in that the interchromatid distance was smaller than in chromosomes from untreated cells (, compare c with a, insets, arrow). Strikingly, this resolution defect in prometaphase did not preclude the separation of sister chromatids during anaphase (, d–f).
All anaphase figures appeared highly aberrant in chromosome spreads from Hesperadin-treated cells. Although the sister chromatids clearly disjoined, many were found next to each other (, d–f, arrows) and were also in close proximity at the time of chromosome decondensation (). It seems likely that spindle forces were present in Hesperadin-treated cells during anaphase, because some sister chromatids had been pulled to opposite poles ( d, arrowheads). Separated sister chromatids in close proximity, therefore, could have arisen either from unattached chromosomes or from chromosomes whose two sister chromatids were attached to the same spindle pole. Together, these observations suggest that the chromosome segregation defect seen in Hesperadin-treated cells does not result from defects in chromosome condensation or sister chromatid separation.
Aurora B function is required during chromosome attachment to the mitotic spindle
To test if Hesperadin's inhibitory effect on sister chromatid resolution in prometaphase could be responsible for the observed chromosome alignment defect, e.g., by preventing complete resolution of sister kinetochores, we analyzed when in mitosis Hesperadin addition causes chromosome segregation defects. Cells were arrested by nocodazole, and Hesperadin was only added shortly before releasing cells from nocodazole so that cells progressed through prometaphase in the absence of Hesperadin (
A). In this case, the resolution of sister chromatids was not affected, but the cells nevertheless exited mitosis with the same chromosome alignment and segregation defects as described above ( C; see Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1
). In contrast, cells exited mitosis without abnormalities in anaphase or telophase ( D) and with similar kinetics as controls (Fig. S2) when Hesperadin was washed out before the cells were released from nocodazole ( B), i.e., when spindles assembled in the absence of Hesperadin. These observations indicate that the defect in chromosome segregation induced by Hesperadin can be specifically ascribed to the inhibition of Aurora B function during the process of chromosome attachment.
Figure 5. Aurora B function is required at the time of microtubule attachment to kinetochores. (A and C) HeLa cells were arrested in nocodazole. Hesperadin or the solvent DMSO were added shortly before release from nocodazole (outlined in A). Chromosome spreads (more ...)
Aurora B function is required to ensure bipolar attachments before anaphase
To understand the nature of the observed chromosome segregation defect, we filmed Hesperadin-treated living PtK1 cells by differential interference contrast microscopy. At nuclear envelope breakdown (NEB;
, time 0:00), some chromosomes were usually positioned at an equal distance between the two spindle poles and appeared to rapidly acquire a normal bipolar attachment (, time 0:06). However, other chromosomes that were positioned closer to one of the poles at NEB rapidly acquired a monopolar attachment to that pole. Like monooriented chromosomes in untreated cells (Rieder and Salmon, 1998
), these chromosomes exhibited pronounced oscillatory motions toward and away from the pole they were attached to (; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1
The number of bioriented versus monooriented chromosomes varied between different cells (unpublished data), but one striking feature that all cells had in common was that initially monooriented chromosomes did not achieve biorientation until the onset of anaphase. Time-lapse studies revealed that Hesperadin also causes chromosome biorientation defects in human CF-PAC and RPE-1 cells, indicating that this effect is not specific for PtK1 cells (unpublished data). Because cells normally do not enter anaphase in the presence of monooriented chromosomes, Hesperadin could cause a spindle assembly checkpoint defect. The failure to congress all chromosomes might therefore be a secondary consequence of a premature onset of anaphase. To evaluate this possibility, we compared the duration from NEB to the onset of anaphase in Hesperadin-treated or control cells filmed in a 37°C warm room (). Although, on average, Hesperadin-treated cells enter anaphase earlier after NEB than controls (after 16 and 23 min, respectively), most remain in prometaphase longer (16 min on average) than the time it usually takes for chromosomes in untreated cells to become attached in a bipolar fashion (13 min on average). The failure to achieve biorientation therefore cannot be fully attributed to a precocious exit from mitosis.
Time spent in prometaphase by PtK1 cells treated with Hesperadin
Sister chromatids of bioriented chromosomes moved to opposite poles after they disjoined at anaphase onset (, 0:58, arrowheads), demonstrating that they were indeed attached in an amphitelic fashion. At the same time, both sister chromatids of monooriented chromosomes moved to the same pole (, 0:58), suggesting the possibility that they were attached in a syntelic manner. Note that when a monotelic chromosome undergoes anaphase, only the attached chromatid moves poleward whereas the unattached chromatid initially shows no motion (Rieder et al., 1986
; Ault and Rieder, 1992
The chromosome movements observed during prometaphase and anaphase in Hesperadin-treated cells demonstrate that spindle forces are present and acting on the chromosomes. We often found, however, that the sister chromatids of bioriented chromosomes did not move as far apart during anaphase as they do in untreated cells (, 0:58). It is possible that anaphase B spindle elongation does not occur in Hesperadin-treated cells because a spindle midzone is not assembled ( C), or the force-producing mechanism for anaphase A is impaired, or chromosome decondensation occurs prematurely.
Syntelic attachment is more frequent in Hesperadin-treated cells than in control-treated cells
To determine the type of attachment of monooriented chromosomes in Hesperadin-treated cells, we performed deconvolution microscopy on PtK1 or PtK2 cells that were fixed and stained with anti-tubulin antibodies and CREST serum (
; see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1
). We were not able to determine the type of attachment for each chromosome of a prometaphase cell. However, we found, on average, one clearly syntelic chromosome in Hesperadin-treated cells, whereas only one in six control cells contained a clearly syntelic chromosome. Conversely, control cells contained, on average, one chromosome that could clearly be identified as monotelic, whereas this number was reduced sixfold in Hesperadin-treated cells ( C; Fig. S3), indicating that Aurora B function might be required to convert syntelic into monotelic attachment.
Figure 7. Hesperadin-treated PtK cells show a higher frequency of syntelic attachments during prometaphase than control-treated cells. (A and B) PtK1 cell treated with 500 nM Hesperadin for 3 h. Deconvolved images taken at 0.2-μm Z-interval. α-Tubulin (more ...)
Hesperadin treatment rapidly overrides the spindle assembly checkpoint in taxol- and monastrol-treated cells but not in nocodazole-treated cells
To determine if Aurora B function is required for the spindle checkpoint, we treated HeLa cells with nocodazole, which induces a Mad2-dependent mitotic arrest because microtubules are depolymerized and kinetochores are unattached. We then added Hesperadin and followed the cells over time by either chromosome spreading (
A) or time-lapse video light microscopy (unpublished data). We found that nocodazole-treated cells remained arrested in mitosis for at least 3 h (; unpublished data) after addition of Hesperadin.
The spindle checkpoint is also activated by taxol (paclitaxel), which stabilizes microtubules. Surprisingly, when cells arrested with taxol were treated with Hesperadin, they exited mitosis within 1 h (). This observation, and our finding that Aurora B inhibition stabilizes syntelic attachments, raised the possibility that Hesperadin treatment turned off checkpoint signaling in taxol-treated cells because all kinetochores progressively accumulated stably attached microtubules.
To explore this hypothesis further, we created monopolar spindles in cells by treating them with the kinesin Eg5 inhibitor monastrol (Mayer et al., 1999
). Under this condition, cells arrest in mitosis for at least 5 h with ~70% of their chromosomes syntelically monooriented, whereas the remainder are monotelically monooriented (Kapoor et al., 2000
). We found that cells arrested with monastrol exited mitosis within 1 h after addition of Hesperadin (). Moreover, when we added Hesperadin to monastrol-arrested Ptk1 cells, all chromatids moved toward the single pole of the monopolar spindles in anaphase ( E), consistent with the hypothesis that all monotelic chromosomes that are normally found in monastrol-treated cells were converted into syntelic states by Hesperadin treatment. To confirm this notion, we fixed monastrol-treated PtK1 cells 1 h after addition of Hesperadin, chose cells that still exhibited a monoastral spindle, and determined the number of kinetochores that were staining with Mad2 antibodies, e.g., that were presumably unattached. The number of Mad2-positive kinetochores decreased from 6.3 (range 3–9; n
= 12 cells) in monastrol-treated control cells to 1.2 (range 0–5; n
= 25 cells) after 1 h of Hesperadin treatment, indicating that inhibition of Aurora B function might indeed stabilize syntelic attachments.
BubR1 does not localize to kinetochores when cells are treated with Hesperadin and nocodazole
Hesperadin might induce mitotic exit in taxol- or monastrol-treated cells by stabilizing improper microtubule attachments. However, it is possible that Aurora B also has a direct role in the spindle assembly checkpoint, as even under conditions where none of the kinetochores are attached (, nocodazole), cells that are additionally treated with Hesperadin exit mitosis precociously ( D). Recruitment of checkpoint proteins to unattached kinetochores is thought to be necessary to maintain checkpoint signaling (Shah and Cleveland, 2000
). We therefore tested whether inhibition of Aurora B function might impair this recruitment. We found that in the presence of nocodazole and Hesperadin, Mad2 and the motor protein CENP-E were still present at kinetochores. In contrast, kinetochore localization of BubR1 was abolished, and the intensity of Bub1 at kinetochores was diminished (
, A–C). Similar results were obtained in logarithmically growing HeLa cells (see Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200208092/DC1
), where after addition of Hesperadin, Mad2 and CENP-E could be observed at kinetochores in early prometaphase, whereas BubR1 and Bub1 did not localize to kinetochores at any stage of mitosis. Together, these data suggest that Aurora B function is required for efficient kinetochore recruitment of BubR1 and Bub1, which in turn might be necessary for prolonged checkpoint signaling.
Figure 9. BubR1 localization to kinetochores is abolished in HeLa cells treated with nocodazole and Hesperadin. (A) HeLa cells were arrested in mitosis with 10 μM nocodazole and then additionally treated with 100 nM Hesperadin or the solvent DMSO for 2 (more ...)