Current Biology 16, 1194–1200, June 20, 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2006.04.043 Report Mitotic Checkpoint Slippage in Humans Occurs via B Destruction in the Presence of an Active Checkpoint

Daniela A. Brito1 and Conly L. Rieder1,2,* in 17 6 6 min at 37ºC (n = 22), they require 546 6 96 min 1 Department of Biomedical Sciences (n = 18) in the presence of 200 nM nocodazole and 875 6 School of Public Health 130 min (n = 13) in the presence of 3.2 mM nocodazole. State University of New York As with PtK1 cells, RPE1 cells form rudimentary spindles Albany, New York 12222 at the lower (200 nM) but not at the higher (3.2 mM) drug 2 Laboratory of Cell Regulation concentration (Figures 1C and 1D). When the duration of Division of Molecular Medicine in the presence or absence of MTs is compared Wadsworth Center for each cell type, by means of the two-tailed Student’s New York State Department of Health t test, they are found to be statistically different with Albany, New York 12201 p values below the 0.0001 level. As both cell types escape mitosis in the absence of MTs, they begin to violently bleb and thereafter reflatten Summary with multiple micronuclei (e.g., see Figures 2B, 2C, and 2E). These findings are consistent with previous reports In the presence of unattached/weakly attached kineto- that PtK1 and RPE1 cells can exit mitosis without satis- chores, the spindle assembly checkpoint (SAC) delays fying the SAC and also that the duration of the delay is exit from mitosis by preventing the anaphase-promot- related to the drug concentration (reviewed in [6]). ing complex (APC)-mediated proteolysis of cyclin B, a regulatory subunit of cyclin-dependent kinase 1 Bypassing an Unsatisfied SAC Requires (Cdk1). Like all checkpoints, the SAC does not arrest Cyclin B Degradation cells permanently, and escape from mitosis in the If escaping an unsatisfied SAC involves a Cdki or an in- presence of an unsatisfied SAC requires that cyclin hibitory phosphorylation on cyclin B/Cdk1, then early G1 B/Cdk1 activity be inhibited. In yeast [1–5], and likely cells produced by this process should contain high cy- Drosophila [6–8], this occurs through an ‘‘adaptation’’ clin B levels. To test this, we followed RPE1 cells in the process involving an inhibitory phosphorylation on presence of 200 nM nocodazole, fixed them shortly after Cdk1 and/or activation of a cyclin-dependent kinase they had exited mitosis, and stained them for the indi- inhibitor (Cdki). The mechanism that allows vertebrate rect immunofluorescence (IMF) localization of cyclin B. cells to escape mitosis when the SAC cannot be satis- When compared to G2 controls in the same cultures, fied is unknown. To explore this issue, we conducted cells that had escaped mitosis and entered early G1 fluorescence microscopy studies on rat kangaroo without satisfying the SAC appeared depleted of cyclin (PtK) and human (RPE1) cells dividing in the presence B (see Figure S1 in the Supplemental Data available of nocodazole. We find that in the absence of micro- with this article online). To confirm this qualitative find- tubules (MTs), escape from mitosis occurs in the pres- ing, we transfected RPE1 cells with a cyclin B-GFP plas- ence of an active SAC and requires cyclin B destruc- mid and followed them, in the presence of 200 nM (n = 5) tion. We also find that cyclin B is progressively or 3.2 mM (n = 3) nocodazole, as they escaped mitosis. destroyed during the block by a proteasome-depen- Under both conditions, and in all cases, cyclin B fluores- dent mechanism. Thus, vertebrate cells do not adapt cence slowly decayed during the block until it was no to the SAC. Rather, our data suggest that in normal longer detectable by eye, after which the cells exited cells, the SAC cannot prevent a slow but continuous mitosis (Figures 2A–2C). This decay occurred more degradation of cyclin B that ultimately drives the cell rapidly in cells treated with 200 nM nocodazole than out of mitosis. with 3.2 mM nocodazole (Figure 3). Clute and Pines [9] reported a similar progressive decay in GFP-cyclin B Results and Discussion fluorescence intensity when HeLa cells were delayed in mitosis by 10 mM taxol, a drug that stabilizes MTs. In At 37ºC, the duration of mitosis in PtK1 cells (2N = 11), our study, as in theirs, the fluorescence decay was not defined from nuclear envelope breakdown to nuclear due to photobleaching, since the fluorescence intensity envelope reformation, is 33 6 7 min (n = 23). When PtK1 of GFP-cyclin B containing bystander cells in the same cells enter mitosis in the presence of 400 nM nocoda- fields did not change (Figure 3). Thus, in vertebrates, zole, they form a monopolar spindle (Figure 1A) and escape from mitosis when the SAC cannot be satisfied remain in mitosis for 191 6 52 min (n = 22). When PtK1 correlates with the progressive destruction of cyclin B. enter mitosis in 10 mM nocodazole, no spindle MTs are formed (Figure 1B) and they remain in mitosis for 287 6 Bypassing a Functional SAC Does Not Involve 79 min (n = 38), after which they enter the next G1 in Adaptation to the Checkpoint the presence of 22 unattached . Similarly, Our finding that cyclin B is destroyed as vertebrate cells although RPE1 (2N = 46) cells normally complete mitosis bypass a functional SAC implies that escaping mitosis requires cyclin B destruction rather than activation of a Cdki or an inhibitory phosphorylation on cyclin B/Cdk1. *Correspondence: [email protected] However, it is possible that these other escape routes Bypassing the Spindle Assembly Checkpoint 1195

Figure 1. Abnormal Spindles with Mad2- Positive Kinetochores Form in the Presence of Nocodazole PtK1 (A, B) and RPE1 (C, D) cultures were in- cubated with 400 nM (A), 10 mM (B), 200 nM (C), or 3.2 mM nocodazole for 6 hr (A and B), 9 hr (C), or 15 hr (D), respectively, prior to fixation and immunostaining for Mad2 and a-tubulin. First column, DNA; second column, Mad2; third column, a-tubulin; fourth column, merged images. Note that a rudimentary spindle forms in both cell types at the lower but not higher nocodazole concentrations. See text for details. Scale bar equals 5 mm.

are present but masked by cyclin B destruction. To or BubR1), or if one or more of these checkpoint proteins test this possibility, we followed RPE1 and PtK1 cells is degraded during the block. To evaluate this idea, we as they entered mitosis in the presence of nocodazole asked whether escape from mitosis in the presence of and MG132, a potent proteasome inhibitor. Under this nocodazole correlates with the disappearance of one condition, both RPE1 (n = 7) and PtK1 (n = 9) cells failed or more SAC proteins from kinetochores, as occurs to exit mitosis and ultimately died in division after a pro- during a normal mitosis. To this end, we followed noco- longed (>12–15 hr) arrest (Figure S2). To determine dazole-treated YFP-Mad2-expressing PtK2 cells as they whether the failure of cells treated with both nocodazole entered and exited mitosis. As expected, in control and MG132 to escape mitosis is due to toxic effects cells Mad2 fluorescence was not detected on kineto- caused by inhibiting proteolysis, we transfected RPE1 chores just prior to anaphase (n = 14; data not shown). cultures with GFP-cyclin B-D85. This cyclin lacks the In contrast, we always found a variable number of Mad2- N-terminal D-box required for APC-mediated proteoly- positive kinetochores in PtK2 cells (n = 17) as they exited sis [10], yet it binds to Cdk1 and forms an active kinase mitosis in the presence of 10 mM nocodazole (Figure S3). complex [11]. We then followed GFP-cyclin B-D85- To expand this finding, we treated RPE1 cultures with containing cells by phase/fluorescence video LM as 200 nM or 3.2 mM nocodazole for 9 or 15 hr, respectively, they entered mitosis in the presence of 200 nM nocoda- to ensure the presence in each of a cell population that zole. Not surprisingly, we found that cells expressing had overridden the SAC. After fixation, the cultures nondegradable cyclin B invariably remained in mitosis were then stained with antibodies against SAC proteins for >24 hr (Figures 2D and 3), whereas neighboring cells (BubR1, Mad1, and Mad2). Under both conditions, we that were not expressing the GFP construct exited mito- invariably found a dot-like staining pattern associated sis within 8–10 hr (Figure 2E). Thus, in vertebrates, with the chromatin in cells that had escaped mitosis, bypassing an unsatisfied SAC requires cyclin B destruc- as judged by the presence of decondensing chromo- tion, and in these cells, other upstream proteolysis- somes and/or micronuclei (Figures 4A and 4B). When independent ‘‘adaptation’’ pathways are not operative. compared to cells treated with 200 nM nocodazole, the regions of cells that had escaped mito- Mad2 and BubR1 Remain Associated with sis in the presence of 3.2 mM nocodazole stained very Kinetochores as Cells Bypass a Functional SAC intensely for all checkpoint proteins (cf. Figures 4A Cells could escape mitosis without satisfying the SAC if and 4B). This difference can be attributed to the fact kinetochores became progressively modified over time, that the formation of rudimentary spindles and kineto- until they no longer bound one or more kinetochore- chore fibers at the lower drug concentration (Fig- associated checkpoint-signaling proteins (like Mad2 ure 1C) depletes checkpoint proteins from some of the Current Biology 1196

Figure 2. Cyclin B Degradation Is Required for RPE1 Cells to Exit Mitosis in the Presence of an Unsatisfied SAC Selected phase-contrast (top) and fluorescence (bottom) frames from time-lapse video recordings. (A–C) RPE1 cells were transfected with a cyclin B-GFP plasmid and GFP-expressing cells were followed. (A) Non-drug-treated control cell in which cyclin B-GFP accumulates in the nucleus during prophase (25 min) and is then destroyed after the SAC is satisfied and the cell enters anaphase (15–17 min). Insets show centrosomes at a higher magnification. Time, relative to nuclear envelope breakdown, is shown in minutes. Scale bar equals 5 mm. (B and C) GFP-expressing mitotic cells in the presence of 200 nM (B) or 3.2 mM (C) nocodazole. The arrow in each frame marks the cell of interest. Time is shown in minutes. Note that the cyclin B-GFP fluorescence begins to visibly decline throughout the block and is barely detectable near the time the cells finally begin to exit mitosis (360 min in [B] and 860 min in [C]). (D and E) RPE1 cultures were transfected with a GFP-cyclin B-D85 plasmid. 24 hr after transfection, the cultures were incubated with 200 nM nocodazole and areas were subsequently followed by dual-mode time-lapse microscopy. In the same field of view, we could find GFP-express- ing and nonexpressing cells. (D) GFP-expressing cells (e.g., arrow) remained in mitosis until filming was terminated after more than 24 hr, and throughout this time the GFP fluorescence remained relatively level (see Figure 3). (E) In contrast, cells in the same culture that were not transfected with the plasmid (e.g., arrows) escaped mitosis after w8 hr. See also Figure 3. kinetochores. Thus, in vertebrates, exit from mitosis true for PtK1 cells [13], our data imply that it does not oc- when the SAC cannot be satisfied is not due to global cur in RPE1 cells. This is suggested from the fact that changes in kinetochores and/or the destruction of SAC many of the sister kinetochores contained in the restitu- proteins. tion nuclei formed in cells treated with 3.2 mM nocoda- When spindle MT formation is prevented, sister chro- zole appeared closely linked through a single centro- matids in some but not all vertebrate cells disjoin just mere region (Figure 4B). Since chromatid disjunction before the cell escapes mitosis [12]. Although this is normally occurs via the APCCdc20-mediated destruction Bypassing the Spindle Assembly Checkpoint 1197

Figure 3. Cyclin B Proteolysis Occurs throughout a Nocodazole-Induced Block in Mitosis RPE1 cultures were transfected with a plasmid carrying either a degradable or nondegradable (D85) GFP-cyclin B, and progress through mitosis was followed in GFP-expressing cells. Red line, GFP-cyclin B-expressing control cell not treated with nocodazole; orange line, the GFP-cyclin B- expressing cell shown in Figure 2B that was treated with 200 nM nocodazole; green line, the GFP-expressing cell shown in Figure 2C that was treated with 3.2 mM nocodazole. Areas containing mitotic cells were then followed by dual-mode time-lapse microscopy. Fluorescence was mea- sured at each time point in a fixed size area of the cell corresponding to the mitotic spindle region in mitotic cells or the cytoplasm in interphase cells. Fluorescence levels of both mitotic and interphase cells were normalized to the background. Fluorescence of the mitotic cells was further normalized to a transfected interphase bystander cell (black lines) to allow comparison of intensities from independent recordings. The data shown here are representative of 11 control cells, 5 cells expressing GFP-cyclin B and incubated in 200 nM nocodazole, 3 cells expressing GFP-cyclin B and incubated in 3.2 mM nocodazole, and 3 cells expressing nondegradable GFP-cyclin B-D85 and incubated with 200 nM noco- dazole (blue line). Note that the higher the nocodazole concentration, the slower cyclin B is degraded and the longer the cells take to exit mitosis (cf. orange and green lines). Also, degradation of the GFP fluorescence associated with the nondegradable form of cyclin B (blue line) was largely inhibited. Cells transfected with this construct were unable to escape mitosis during the recording interval (up to 1740 min). of securin, this finding suggests that APCCdc20 is inac- Tpx2 peaks during spindle formation and then rapidly tive as RPE1 cells exit mitosis without forming MTs. declines during anaphase/telophase until it is gone in Tpx2, a MT-associated protein required for spindle early G1 [14]. It was recently shown that Tpx2 degrada- formation, is a conspicuous component of S, G2, and tion is controlled by APCCdh1, which, unlike APCCdc20, mitotic, but not G1, cells. Like cyclin B, the level of does not normally become active until anaphase [15].

Figure 4. BubR1, Mad1, and Mad2 Remain Associated with RPE1 Kinetochores as Cells Exit Mitosis in the Presence of an Unsatisfied SAC RPE1 cultures were incubated with 200 nM (A) or 3.2 mM (B) nocodazole for 9 or 15 hr, respectively, before fixation and immunostaining for BubR1 (top), Mad1 (middle), or Mad2 (bottom). Cells that had just exited mitosis (see text for details) were then located and photographed. Left columns in each panel, DNA; middle columns, BubR1, Mad1, or Mad2; right columns, merged images. Scale bar equals 5 mm. Current Biology 1198

Figure 5. Tpx2, an APC Substrate, Is Not Degraded in Cells that Exit Mitosis without Satisfying the SAC (A) In untreated RPE1 cells, Tpx2 (bottom) is found in S/G2 but not G1 nuclei. During mitosis, this MT-associated protein localizes to spindle MTs, while during anaphase it is found on midbody MTs and centrosomes. Tpx2 is largely degraded by the time the nuclear envelope reforms around daughter nuclei during late telophase, although some remains associated with the centrosomes (far right row). (B) After treatment with 3.2 mM nocodazole, Tpx2 (bottom row) is found in the nuclei of G2 and prophase cells. In the absence of spindle MT formation, this protein then remains diffuse in the cytoplasm throughout the prolonged mitosis. However, once the cells exit mitosis and reflatten on the substrate (last three images in each row), it is found concentrated in the G1 micronuclei. Top row, merged images; middle row, DNA; bottom row, Tpx2. Scale bar equals 5 mm.

We therefore asked whether Tpx2 is degraded in cells How Vertebrate Cells Bypass a Functional SAC that have exited mitosis without satisfying the SAC. In the presence of drugs that inhibit MT formation, the For this study, we treated RPE1 cultures with 3.2 mM no- SAC cannot be satisfied, yet many cells ultimately es- codazole for 15 hr and fixed and stained them for Tpx2 cape mitosis and enter the next G1. Here we show that immunofluorescence. As expected, we found intense this escape is not due to adaptation pathways that sup- Tpx2 staining in mitotic cells (Figure 5A). However, un- press cyclin B/Cdk1 activity by, e.g., inhibitory phos- like in early G1 cells in nontreated control cultures that phorylations or activation of a Cdki. Importantly, we are largely depleted of Tpx2 (Figure 5A), high levels of also show that this escape is not due to the depletion this protein were always seen in the micronuclei of cells of Mad2 or BubR1, which remain associated with unat- that had reflattened after bypassing an unsatisfied SAC tached kinetochores as the SAC is bypassed. Instead, (Figure 5B). This result suggests that when the SAC can- we find that in vertebrates, escaping mitosis when in the not be satisfied, APCCdh1 activity remains depressed as presence of a functional SAC requires proteolysis and, cells exit mitosis. more specifically, the destruction of cyclin B. As first Bypassing the Spindle Assembly Checkpoint 1199

reported for treatments that stabilize MTs [9], we also Finally, we found that cells that form rudimentary spin- found that cyclin B levels progressively decline during dles in nocodazole escape mitosis significantly faster a mitotic block induced by drugs that prevent MT than those that cannot generate spindle MTs (Figure 3; assembly. Thus, the key issue for how a vertebrate cell [6, 22]). This positive correlation between the presence escapes mitosis when it cannot satisfy the SAC is how of spindle MTs and an accelerated escape from mitosis it degrades its cyclin B during the block. is difficult to explain by a nonspecific cyclin B destruc- Several recent reports suggest that the checkpoint tion mechanism. proteins Bub1 and BubR1 are degraded in HeLa in a cas- The more attractive mechanism for how cells exit mito- pase-dependent manner during a mitosis prolonged by sis in the presence of a functional SAC is simply that the drugs [16–18]. This, by itself, would lead to checkpoint SAC is not normally 100% effective at blocking all APCs inactivation, APC-mediated cyclin B destruction, and from ubiquinating cyclin B. Under this condition, a con- escape from mitosis. However, these claims are not stant low level of ubiquination and subsequent proteoly- supported by our data that BubR1 remains associated sis would gradually drop the cyclin B level below that with kinetochores even after cells have escaped the needed to maintain the mitotic state. This route provides mitotic block (Figure 4). Moreover, they are based on a simple and elegant mechanism for ultimately overcom- sorting analyses of cell populations, arrested in mitosis ing the mitotic condition before the cell dies, which, at for 24–48 hr, in which the cells are likely dying during least under some conditions, may be important (e.g., mitosis via apoptosis [19, 20]. Instead, our data appear although the value of this feature to multicellular organ- to rule out the possibility that vertebrates escape mito- isms is not readily apparent, in plants it leads to changes sis in the absence of MTs by suddenly inactivating the in ploidy levels and new strains of commercially valuable SAC and thus activating the APCs near the end of crops). This mechanism also provides a straightforward the block. Indeed, when the SAC cannot be satisfied, explanation for why the nocodazole-induced mitotic de- the levels of cyclin B decline gradually and not suddenly. lay seen in normal [23, 24] and some cancer [25–27] cells Furthermore, during and after escaping mitosis in the containing abnormally low levels of Mad2 or BubR1 is absence of MTs, the kinetochores retain high levels of greatly attenuated relative to cells containing normal checkpoint signaling proteins (e.g., Mad2 and BubR1), levels of these checkpoint proteins: since even with nor- the APC substrate Tpx2 is not degraded, and RPE1 cells mal Mad2 or BubR1 concentrations the SAC cannot pro- do not appear to disjoin their chromatids. Concerning hibit a low level ubiquination of cyclin B by the APCCdc20 the last point, it is noteworthy that in vertebrates chro- complex, lowering the concentration further makes the matid disjunction does not occur even in untreated cells SAC even less effective at inhibiting the APCs, and the containing a fully functional spindle until w95% of the more rapidly cyclin B levels would be degraded below cyclin B has been degraded [9, 11]. Moreover, evidence that needed to maintain the mitotic state. is accumulating that this event requires cyclin B destruc- Cells that form rudimentary spindles in response to tion since it does not occur, or is considerably delayed, low nocodazole concentrations are unable to satisfy in non-drug-treated cells expressing a nondegradable the SAC as evidenced by the continuous presence of cyclin B that have presumably satisfied the SAC [11, 21]. Mad2 and BubR1 on some kinetochores after they In lieu of checkpoint inactivation, there are two non- escape mitosis (Figure 4A). However, as noted above, mutually exclusive mechanisms for how cells can es- such cells exit mitosis significantly faster than those cape mitosis in the presence of a functional SAC. The that cannot generate spindle MTs (see also [6, 22, 28]). first is that cyclin B is slowly and constantly degraded Not surprisingly, we find that nocodazole-treated cells in an APC-independent manner until it falls below the containing rudimentary spindles degrade their cyclin B threshold for maintaining the mitotic state. For several much faster than cells lacking MTs (Figure 3). This means reasons this scenario is highly unlikely. First, our data re- that when the SAC cannot be satisfied, the mere pres- veal that the degradation of cyclin B is specific to mito- ence of MTs somehow accelerates the destruction of sis, since it is not seen in G2 GFP-cyclin B-expressing cyclin B. Although the reasons for this are unknown, it bystander cells. Second, the degradation of GFP-cyclin is possible that APCs, which are normally associated B lacking a D-box, and therefore its APC recognition with the spindle [29, 30], work more efficiently when site, occurs extremely slowly even in the presence of attached to MTs. Alternatively, at the lower drug concen- fully functional spindles (Figure 3; [9, 11]). Presumably, trations, many kinetochores become attached to spindle in the absence of MTs, any general cyclin denatur- MTs, which makes them poor checkpoint signalers be- ation/degradation pathway not involving APCs would cause they possess reduced amounts of checkpoint also degrade GFP-cyclin B-D85 at the same rate as de- proteins (e.g., cf. Figures 1A and 1C with 1B and 1D; gradable cyclin. We evaluated this idea by treating RPE1 Figures 4A and 4B; see [31]). Under this condition, the cells expressing GFP-cyclin B with 200 nM nocodazole amount of the APC inhibitor ultimately generated may and 5 mM MG132, and then by asking how quickly be significantly less than that formed in the complete GFP-cyclin B is degraded in the absence of proteasome absence of all MTs, and the higher residual APC activity function. Although this combination of drugs is lethal would produce a more rapid degradation of cyclin B and over prolonged periods (12–15 hr; see Figure S2), a corresponding shorter delay in mitosis. we found that the GFP-cyclin B fluorescence intensity did not decay (and actually slowly increased) over the 5–10 hr we followed the cells (Figure S4). Thus, when Supplemental Data Supplemental Data include four figures and Supplemental Experi- the SAC cannot be satisfied, cyclin B degradation is mental Procedures and can be found with this article online not due to a steady general denaturation of the protein at http://www.current-biology.com/cgi/content/full/16/12/1194/ but instead requires proteasome-mediated proteolysis. DC1/. Current Biology 1200

Acknowledgments BubR1 is a determinant of mitotic progression. Mol. Cell. Biol. 25, 9232–9248. We thank Dr. Jonathon Pines (University of Cambridge, UK) for the 18. Shin, H.J., Baek, K.H., Jeon, A.H., Park, M.T., Lee, S.J., Kang, pCMX cyclin B1-GFP plasmid, Dr. Tim Yen (Fox Chase Cancer Cen- C.M., Lee, H.S., Yoo, S.H., Chung, D.H., Sung, Y.C., et al. ter, Philadelphia, PA) for the anti-BubR1 antibody, Dr. Edward (2003). Dual roles of human BubR1, a mitotic checkpoint kinase, Salmon (University of North Carolina, Chapel Hill, NC) for the in the monitoring of chromosomal instability. Cancer Cell 4, 483– PtK2 YFP-Mad2-expressing cell line and anti-Mad2 antibody, and 497. Dr. Naihan Xu (Hong Kong University, Clear Water Bay, Kowloon) 19. Sherwood, S.W., Sheridan, J.P., and Schimke, R.T. (1994). for the GFP-cyclin B1-D85 construct. We also thank Drs. Helder Induction of apoptosis by the anti-tubulin drug colcemid: re- Maiato and Alexey Khodjakov for help with this project. Supported lationship of mitotic checkpoint control to the induction of by NIH/GMS 40198 to C.L.R. D.A.B. is the recipient of a doctoral apoptosis in HeLa S3 cells. Exp. Cell Res. 215, 373–379. research fellowship SFRH/BD/13663/2003 from Fundac¸a˜ o para a 20. Panvichian, R., Orth, K., Day, M.L., Day, K.C., Pilat, M.J., and Cieˆ ncia e a Tecnologia of Portugal. Pienta, K.J. (1998). Paclitaxel-associated multimininucleation is permitted by the inhibition of caspase activation: a potential Received: January 20, 2006 early step in drug resistance. Cancer Res. 58, 4667–4672. Revised: March 31, 2006 21. Stemmann, O., Zou, H., Gerber, S.A., Gygi, S.P., and Kirschner, Accepted: April 18, 2006 M.W. (2001). Dual inhibition of chromatid separation at meta- Published: June 19, 2006 phase. Cell 107, 715–726. 22. Andreassen, P.R., and Margolis, R.L. (1994). Microtubule depen- dency of p34cdc2 inactivation and mitotic exit in mammalian References cells. J. Cell Biol. 127, 789–802. 23. Michel, L.S., Liberal, V., Chatterjee, A., Kirchwegger, R., Pasche, 1. Rudner, A.D., and Murray, A.W. (1996). The spindle assembly B., Gerald, W., Dobles, M., Sorger, P.K., Murty, V.V., and checkpoint. Curr. Opin. Cell Biol. 8, 773–780. Benezra, R. (2001). MAD2 haplo-insufficiency causes premature 2. Minshull, J., Straight, A., Rudner, A.D., Dernburg, A.F., Belmont, anaphase and chromosome instability in mammalian cells. A., and Murray, A.W. (1996). Protein phosphatase 2A regulates Nature 409, 355–359. MPF activity and sister chromatid cohesion in budding yeast. 24. Dai, W., Wang, Q., Liu, T., Swamy, M., Fang, Y., Xie, S., Mah- Curr. Biol. 6, 1609–1620. mood, R., Yang, Y.M., Xu, M., and Rao, C.V. (2004). Slippage 3. Rudner, A.D., Hardwick, K.G., and Murray, A.W. (2000). Cdc28 of mitotic arrest and enhanced tumor development in mice activates exit from mitosis in budding yeast. J. Cell Biol. 149, with BubR1 haploinsufficiency. Cancer Res. 64, 440–445. 1361–1376. 25. Cahill, D.P., Lengauer, C., Yu, J., Riggins, G.J., Willson, J.K., 4. Novak, B., Toth, A., Csikasz-Nagy, A., Gyorffy, B., Tyson, J.J., Markowitz, S.D., Kinzler, K.W., and Vogelstein, B. (1998). Muta- and Nasmyth, K. (1999). Finishing the cell cycle. J. Theor. Biol. tions of mitotic checkpoint genes in human cancers. Nature 392, 199, 223–233. 300–303. 5. Wolfe, B.A., and Gould, K.L. (2004). Fission yeast Clp1p phos- 26. Kasai, T., Iwanaga, Y., Iha, H., and Jeang, K.T. (2002). Prevalent phatase affects G(2)/M transition and mitotic exit through loss of mitotic spindle checkpoint in adult T-cell leukemia con- Cdc25p inactivation. EMBO J. 23, 919–929. fers resistance to microtubule inhibitors. J. Biol. Chem. 277, 6. Rieder, C.L., and Maiato, H. (2004). Stuck in division or passing 5187–5193. through: what happens when cells cannot satisfy the spindle 27. Takahashi, T., Haruki, N., Nomoto, S., Masuda, A., Saji, S., assembly checkpoint. Dev. Cell 7, 637–651. Osada, H., and Takahashi, T. (1999). Identification of frequent 7. Foley, E., O’Farrell, P.H., and Sprenger, F. (1999). Rux is a cyclin- impairment of the mitotic checkpoint and molecular analysis dependent kinase inhibitor (CKI) specific for mitotic cyclin-Cdk of the mitotic checkpoint genes, hsMAD2 and p55CDC, in complexes. Curr. Biol. 9, 1392–1402. human lung cancers. Oncogene 18, 4295–4300. 8. Foley, E., and Sprenger, F. (2001). The cyclin-dependent kinase 28. Chen, J.G., and Horwitz, S.B. (2002). Differential mitotic re- inhibitor Roughex is involved in mitotic exit in Drosophila. Curr. sponses to microtubule-stabilizing and destabilizing drugs. Biol. 11, 151–160. Cancer Res. 62, 935–938. 9. Clute, P., and Pines, J. (1999). Temporal and spatial control of 29. Tugendreich, S., Tomkiel, J., Earnshaw, W., and Hieter, P. (1995). cyclin B1 destruction in metaphase. Nat. Cell Biol. 1, 82–87. Cdc27Hs colocalizes with Cdc16Hs to the centrosome and 10. Yamano, H., Gannon, J., Mahbubani, H., and Hunt, T. (2004). Cell mitotic spindle and is essential for the metaphase to anaphase cycle-regulated recognition of the destruction box of cyclin B by transition. Cell 81, 261–268. the APC/C in Xenopus egg extracts. Mol. Cell 132, 137–147. 30. Melloy, P.G., and Holloway, S.L. (2004). Changes in the localiza- 11. Chang, D.C., Xu, N., and Luo, K.Q. (2003). Degradation of cyclin tion of the Saccharomyces cerevisiae anaphase-promoting B is required for the onset of anaphase in mammalian cells. complex upon microtubule depolymerizaton and spindle check- J. Biol. Chem. 278, 37865–37873. point activation. Genetics 167, 1079–1094. 12. Rieder, C.L., and Palazzo, R.E. (1992). Colcemid and the mitotic 31. Weaver, B.A.A., and Cleveland, D.W. (2005). Decoding the links cycle. J. Cell Sci. 102, 387–392. between mitosis, cancer, and chemotherapy: the mitotic check- 13. Rieder, C.L., Schultz, A., Cole, R., and Sluder, G. (1994). Ana- point, adaptation, and cell death. Cancer Cell 8, 7–12. phase onset in vertebrate somatic cells is controlled by a check- point that monitors sister kinetochore attachment to the spindle. J. Cell Biol. 127, 301–310. 14. Gruss, O.J., Wittmann, M., Yokoyama, H., Pepperkok, R., Kuffer, T., Sillje, H., Karsenti, E., Mattaj, I.W., and Vernos, I. (2002). Chro- mosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells. Nat. Cell Biol. 4, 871–879. 15. Stewart, S., and Fang, G. (2005). Anaphase-promoting complex/ cyclosome controls the stability of TPX2 during mitotic exit. Mol. Cell. Biol. 25, 10516–10527. 16. Baek, K.H., Shin, H.J., Jeong, S.J., Park, J.W., McKeon, F., Lee, C.W., and Kim, C.M. (2005). Caspases-dependent cleavage of mitotic checkpoint proteins in response to microtubule inhibitor. Oncol. Res. 15, 161–168. 17. Kim, M., Murphy, K., Liu, F., Parker, S.E., Dowling, M.L., Baff, W., and Kao, G.D. (2005). Caspase-mediated specific cleavage of