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• W2076753965 abstract "Current antimitotics work by perturbing spindle assembly, which activates the spindle assembly checkpoint, causes mitotic arrest, and triggers apoptosis. Cancer cells can resist such killing by premature exit, before cells initiate apoptosis, due to a weak checkpoint or rapid slippage. We reasoned blocking mitotic exit downstream of the checkpoint might circumvent this resistance. Using single-cell approaches, we showed that blocking mitotic exit by Cdc20 knockdown slowed cyclin B1 proteolysis, thus allowed more time for death initiation. Killing by Cdc20 knockdown did not require checkpoint activity and can occur by intrinsic apoptosis or an alternative death pathway when Bcl2 was overexpressed. We conclude targeting Cdc20, or otherwise blocking mitotic exit, may be a better cancer therapeutic strategy than perturbing spindle assembly. Current antimitotics work by perturbing spindle assembly, which activates the spindle assembly checkpoint, causes mitotic arrest, and triggers apoptosis. Cancer cells can resist such killing by premature exit, before cells initiate apoptosis, due to a weak checkpoint or rapid slippage. We reasoned blocking mitotic exit downstream of the checkpoint might circumvent this resistance. Using single-cell approaches, we showed that blocking mitotic exit by Cdc20 knockdown slowed cyclin B1 proteolysis, thus allowed more time for death initiation. Killing by Cdc20 knockdown did not require checkpoint activity and can occur by intrinsic apoptosis or an alternative death pathway when Bcl2 was overexpressed. We conclude targeting Cdc20, or otherwise blocking mitotic exit, may be a better cancer therapeutic strategy than perturbing spindle assembly. Drugs targeting spindle proteins, including kinesin-5, Aurora kinases, and Polo-like kinases, were recently developed in the hope of providing less neurotoxic antimitotic drugs. In clinical trials to date, their efficacy seems no better than taxanes and vincas. We provide evidence that an alternative antimitotic strategy, blocking mitotic exit, might be more effective. We found Cdc20 knockdown provides a checkpoint-independent mitotic arrest that kills cancer cells more effectively than spindle-perturbing drugs. We suggest mitotic exit is a promising therapeutic target, and propose a cell-based screening strategy for compounds with this mechanism. These findings are significant from a drug development perspective, and also as a mechanistic step toward understanding how mitotic arrest triggers death, and how some cancer cells resist this trigger. Antimitotic drugs that target microtubule dynamics, including taxanes, vinca alkaloids, and epothilones, are active against a broad range of cancers, but they also cause neurotoxicity, presumably due to perturbation of microtubules in neurons (Jordan and Wilson, 2004Jordan M.A. Wilson L. Microtubules as a target for anticancer drugs.Nat. Rev. Cancer. 2004; 4: 253-265Crossref PubMed Scopus (3235) Google Scholar). In an effort to develop antimitotic drugs lacking this toxicity, small-molecule inhibitors of a number of proteins specific to the mitotic spindle were developed, including the motor protein kinesin-5 (KSP, Eg5, Kif11), Aurora kinases, and Polo-like kinases (Jackson et al., 2007Jackson J.R. Patrick D.R. Dar M.M. Huang P.S. Targeted anti-mitotic therapies: Can we improve on tubulin agents?.Nat. Rev. Cancer. 2007; 7: 107-117Crossref PubMed Scopus (382) Google Scholar). In clinical trials to date, these spindle-specific antimitotic drugs lack neurotoxicity as hoped, but their efficacy against solid tumors seems to be no better than taxanes and vincas, and perhaps not as good. Can we find an antimitotic strategy that not only lacks neurotoxicity, but is also more effective than current strategies at causing regression of solid tumors? We set out to address this question using RNAi knockdown as a surrogate for potential drugs, and comparing efficacy for killing cancer cell lines with representative drugs that interfere with spindle assembly. The net effect of antimitotic drugs is to perturb mitotic spindle assembly, which activates the spindle assembly checkpoint (SAC). After many hours of SAC-induced mitotic arrest, cancer cells either die inside mitosis, or exit mitosis by slippage into a tetraploid G1 state, from which they either die, arrest in G1, or initiate a new round of the cell cycle (Rieder and Maiato, 2004Rieder C.L. Maiato H. Stuck in division or passing through: What happens when cells cannot satisfy the spindle assembly checkpoint.Dev. Cell. 2004; 7: 637-651Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, Gascoigne and Taylor, 2008Gascoigne K.E. Taylor S.S. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs.Cancer Cell. 2008; 14: 111-122Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, Orth et al., 2008Orth J.D. Tang Y. Shi J. Loy C.T. Amendt C. Wilm C. Zenke F.T. Mitchison T.J. Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate.Mol. Cancer Ther. 2008; 7: 3480-3489Crossref PubMed Scopus (88) Google Scholar). Slippage is thought to occur by gradual proteolysis of cyclin B1, which continues slowly even when the SAC is active (Brito and Rieder, 2006Brito D.A. Rieder C.L. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint.Curr. Biol. 2006; 16: 1194-1200Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Cell death occurs mainly via activation of the intrinsic apoptosis (Wang et al., 1999Wang L.G. Liu X.M. Kreis W. Budman D.R. The effect of antimicrotubule agents on signal transduction pathways of apoptosis: A review.Cancer Chemother. Pharmacol. 1999; 44: 355-361Crossref PubMed Scopus (354) Google Scholar, Park et al., 2004Park S.J. Wu C.H. Gordon J.D. Zhong X. Emami A. Safa A.R. Taxol induces caspase-10-dependent apoptosis.J. Biol. Chem. 2004; 279: 51057-51067Crossref PubMed Scopus (180) Google Scholar, Tao et al., 2005Tao W. South V.J. Zhang Y. Davide J.P. Farrell L. Kohl N.E. Sepp-Lorenzino L. Lobell R.B. Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage.Cancer Cell. 2005; 8: 49-59Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, Bergstralh and Ting, 2006Bergstralh D.T. Ting J.P. Microtubule stabilizing agents: Their molecular signaling consequences and the potential for enhancement by drug combination.Cancer Treat. Rev. 2006; 32: 166-179Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), a pathway involving mitochondrial outer membrane permeabilization (MOMP) (Letai, 2008Letai A.G. Diagnosing and exploiting cancer's addiction to blocks in apoptosis.Nat. Rev. Cancer. 2008; 8: 121-132Crossref PubMed Scopus (434) Google Scholar). Failure to initiate apoptosis during or after mitotic arrest appears to be a major factor limiting efficacy of antimitotic drugs because mitotic arrest without subsequent apoptosis is commonly observed following taxane treatment in various cancer cell lines (Shi et al., 2008Shi J. Orth J.D. Mitchison T. Cell type variation in responses to antimitotic drugs that target microtubules and kinesin-5.Cancer Res. 2008; 68: 3269-3276Crossref PubMed Scopus (168) Google Scholar), mouse cancers (Milross et al., 1996Milross C.G. Mason K.A. Hunter N.R. Chung W.K. Peters L.J. Milas L. Relationship of mitotic arrest and apoptosis to antitumor effect of paclitaxel.J. Natl. Cancer Inst. 1996; 88: 1308-1314Crossref PubMed Scopus (265) Google Scholar), and, though data are very limited, human breast cancers, where it correlates with poor tumor responses (Symmans et al., 2000Symmans W.F. Volm M.D. Shapiro R.L. Perkins A.B. Kim A.Y. Demaria S. Yee H.T. McMullen H. Oratz R. Klein P. et al.Paclitaxel-induced apoptosis and mitotic arrest assessed by serial fine-needle aspiration: Implications for early prediction of breast cancer response to neoadjuvant treatment.Clin. Cancer Res. 2000; 6: 4610-4617PubMed Google Scholar). Here, we focus on drug resistance caused by lack of apoptosis downstream of spindle damage; clinical resistance might also arise from mutations that prevent drugs from causing spindle damage, e.g., due to target protein mutations or drug efflux pump expression (Pusztai, 2007Pusztai L. Markers predicting clinical benefit in breast cancer from microtubule-targeting agents.Ann. Oncol. 2007; 18: xii15-xii20PubMed Scopus (69) Google Scholar), from failure of cancer cells to enter mitosis during drug exposure (Baguley et al., 1995Baguley B.C. Marshall E.S. Whittaker J.R. Dotchin M.C. Nixon J. McCrystal M.R. Finlay G.J. Matthews J.H. Holdaway K.M. van Zijl P. Resistance mechanisms determining the in vitro sensitivity to paclitaxel of tumour cells cultured from patients with ovarian cancer.Eur. J. Cancer. 1995; 31A: 230-237Abstract Full Text PDF PubMed Scopus (40) Google Scholar), or other causes. Previous studies provide two mechanistic clues to how cancer cells choose a nonapoptotic outcome following spindle damage and mitotic arrest. First, they may fail to execute apoptosis efficiently due to downregulation of apoptosis pathways. Protection against MOMP at the level of Bcl2 protein family reduces sensitivity to apoptosis promoted by paclitaxel and vinca alkaloids (Tan et al., 2005Tan T.T. Degenhardt K. Nelson D.A. Beaudoin B. Nieves-Neira W. Bouillet P. Villunger A. Adams J.M. White E. Key roles of BIM-driven apoptosis in epithelial tumors and rational chemotherapy.Cancer Cell. 2005; 7: 227-238Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, Deng et al., 2007Deng J. Carlson N. Takeyama K. Dal Cin P. Shipp M. Letai A. BH3 profiling identifies three distinct classes of apoptotic blocks to predict response to ABT-737 and conventional chemotherapeutic agents.Cancer Cell. 2007; 12: 171-185Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, Kutuk and Letai, 2008Kutuk O. Letai A. Alteration of the mitochondrial apoptotic pathway is key to acquired paclitaxel resistance and can be reversed by ABT-737.Cancer Res. 2008; 68: 7985-7994Crossref PubMed Scopus (111) Google Scholar). Second, they may slip out of mitotic arrest before they die; in other words, slippage and apoptosis can be viewed as two competing pathways (Gascoigne and Taylor, 2008Gascoigne K.E. Taylor S.S. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs.Cancer Cell. 2008; 14: 111-122Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). Consistent with slippage protecting cells from death, premature exit from mitotic arrest due to a weakened or ablated SAC is known to decrease sensitivity to spindle-perturbing drugs (Taylor and McKeon, 1997Taylor S.S. McKeon F. Kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage.Cell. 1997; 89: 727-735Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, Shin et al., 2003Shin H.J. Baek K.H. Jeon A.H. Park M.T. Lee S.J. Kang C.M. Lee H.S. Yoo S.H. Chung D.H. Sung Y.C. et al.Dual roles of human BubR1, a mitotic checkpoint kinase, in the monitoring of chromosomal instability.Cancer Cell. 2003; 4: 483-497Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, Tao et al., 2005Tao W. South V.J. Zhang Y. Davide J.P. Farrell L. Kohl N.E. Sepp-Lorenzino L. Lobell R.B. Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage.Cancer Cell. 2005; 8: 49-59Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, Swanton et al., 2007Swanton C. Marani M. Pardo O. Warne P.H. Kelly G. Sahai E. Elustondo F. Chang J. Temple J. Ahmed A.A. et al.Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs.Cancer Cell. 2007; 11: 498-512Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, Gascoigne and Taylor, 2008Gascoigne K.E. Taylor S.S. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs.Cancer Cell. 2008; 14: 111-122Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, Bekier et al., 2009Bekier M.E. Fischbach R. Lee J. Taylor W.R. Length of mitotic arrest induced by microtubule-stabilizing drugs determines cell death after mitotic exit.Mol. Cancer Ther. 2009; 8: 1646-1654Crossref PubMed Scopus (67) Google Scholar). Based on these clues, we reasoned that blocking mitotic exit downstream of the SAC may be a better strategy for killing apoptosis-resistant, slippage-prone, or SAC-defective cancer cells than any of the currently available antimitotic drugs, all of which target spindle assembly. As surrogate for a potential drug that directly blocks mitotic exit, we knocked down Cdc20 using siRNAs. Cdc20 activates the APC/C to trigger cyclin B1 degradation during normal mitosis, and it is sequestered by SAC proteins when the spindle is damaged (Figure 1A) (Musacchio and Hardwick, 2002Musacchio A. Hardwick K.G. The spindle checkpoint: Structural insights into dynamic signalling.Nat. Rev. Mol. Cell Biol. 2002; 3: 731-741Crossref PubMed Scopus (460) Google Scholar, Musacchio and Salmon, 2007Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1617) Google Scholar). Cdc20 must be depleted to less than 5% of its normal levels to arrest cells in mitosis (Wolthuis et al., 2008Wolthuis R. Clay-Farrace L. van Zon W. Yekezare M. Koop L. Ogink J. Medema R. Pines J. Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A.Mol. Cell. 2008; 30: 290-302Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). We tested several siRNA duplexes and hairpin constructs in HeLa cells, and selected two duplexes on the basis of promoting the most robust mitotic arrest, and most efficient knockdown by immunoblotting (Figure 1B). All data shown are for duplex 1, but similar results were obtained using duplex 2. HeLa cells depleted of Cdc20 arrested in mitosis for an average of 18.8 ± 7.3 hr (n = 98) before undergoing death in mitosis (Figure 1D, top panel). Specificity is a major concern for siRNA duplexes; to evaluate this, we performed a RNAi-resistant transgene rescue experiment for duplex 1, using mouse Cdc20 cDNA with two extra silent mutations (mCdc20) as the rescue construct (Figure 1C). In HeLa cells infected with control vector, and transfected with duplex 1, more than 98% underwent prolonged arrest followed by death in mitosis. In cells infected with retrovirus expressing mCdc20, and then transfected with duplex 1, 83% went through mitosis with little or no delay (1.5 ± 0.9 hr, n = 85), divided, did not die, and continued to the next cell cycle (Figure 1D, bottom panel). The remaining 17% that still showed prolonged arrest may not have been infected with the rescue construct. We conclude that the robust arrest and cell death phenotype caused by duplex 1 is specific to knockdown of Cdc20. Duplex 1 also efficiently knocked down Cdc20 in four other cell lines we investigated below (Figure 1E). We next systematically compared the ability to promote death during mitotic arrest between Cdc20 knockdown and treatment with a mitosis-specific kinesin-5 inhibitor, EMD534085 (Orth et al., 2008Orth J.D. Tang Y. Shi J. Loy C.T. Amendt C. Wilm C. Zenke F.T. Mitchison T.J. Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate.Mol. Cancer Ther. 2008; 7: 3480-3489Crossref PubMed Scopus (88) Google Scholar). We made this comparison in five solid-tumor-derived cell lines: four were selected from a larger panel tested previously so as to span the full range of death sensitivity when treated with antimitotic drugs (Shi et al., 2008Shi J. Orth J.D. Mitchison T. Cell type variation in responses to antimitotic drugs that target microtubules and kinesin-5.Cancer Res. 2008; 68: 3269-3276Crossref PubMed Scopus (168) Google Scholar); Bcl2-overexpressing HeLa cells were added as a fifth line with a known mechanism of apoptosis resistance. Because individual cells vary greatly in their kinetics of mitotic arrest and death during mitosis (Gascoigne and Taylor, 2008Gascoigne K.E. Taylor S.S. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs.Cancer Cell. 2008; 14: 111-122Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, Orth et al., 2008Orth J.D. Tang Y. Shi J. Loy C.T. Amendt C. Wilm C. Zenke F.T. Mitchison T.J. Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate.Mol. Cancer Ther. 2008; 7: 3480-3489Crossref PubMed Scopus (88) Google Scholar), we quantified single-cell behavior using time-lapse microscopy. Figures 2A–2E show death kinetics in individual cells by time-lapse phase-contrast imaging, where death was scored by vigorous blebbing followed by cessation of all movement. Time of death was normalized to time of mitotic entry, which was scored by cell rounding. Because both kinesin-5 and Cdc20 are thought to function only in mitosis, and death in both kinesin-5 inhibitor and Cdc20 knockdown only occurred during or after mitotic arrest, normalizing so that T = 0 was the time of mitotic entry conceptually synchronizes all cells at the start of the prodeath stimulus. These data compare four treatments: lamin A/C siRNA alone (to control for the toxicity of siRNA transfection), kinesin-5 inhibitor plus lamin A/C siRNA, Cdc20 siRNA, and kinesin-5 inhibitor plus Cdc20 siRNA. A saturating concentration of kinesin-5 inhibitor was used, so all drug-treated cells that entered mitosis arrested, and none succeeded in executing cytokinesis. For kinesin-5 inhibitor treatment, we observed some death in mitosis, some slippage, and some death after slippage in all lines. These data are reported separately in Table 1. For simplicity, Figures 2A–2E report kinetics of all death, whether it occurred before or after slippage, as cumulative survival curves. For Cdc20 knockdown, we observed no slippage. HeLa was the most death sensitive in our previous profiling experiment (Shi et al., 2008Shi J. Orth J.D. Mitchison T. Cell type variation in responses to antimitotic drugs that target microtubules and kinesin-5.Cancer Res. 2008; 68: 3269-3276Crossref PubMed Scopus (168) Google Scholar). In this line, more than 90% of cells died during mitotic arrest for all treatments except control siRNA alone, and death kinetics were similar in each case (Figure 2A). In moderately resistant MDA-MB-435S, 15% cells slipped out of kinesin-5 inhibitor-induced mitotic arrest and survived, and in highly resistant MCF7 and A549, ∼80% slipped and survived (blue lines in Figures 2B, 2C, and 2D; Table 1). In each of these lines, knockdown of Cdc20 prevented slippage, regardless of whether kinesin-5 inhibitor was present. All Cdc20 knocked-down cells remained arrested in mitosis for the whole time course, and all eventually died (red and green lines in Figures 2B, 2C, and 2D).Table 1Duration of Mitosis and Percentage of Cell Death in Response to Kinesin-5 Inhibitor or Paclitaxel Incubated with Lamin A/C siRNACell LineKinesin-5 InhibitorPaclitaxelDuration of Mitosis ± SD (hr)Death (%)aPercentage of total cell death.D in M (%)bPercentage of death in mitosis.D after E (%)cPercentage of death after mitotic exit.Duration of Mitosis ± SD (hr)Death (%)aPercentage of total cell death.D in M (%)bPercentage of death in mitosis.D after E (%)cPercentage of death after mitotic exit.HeLa19.7 ± 7.7 (n = 62)98.490.38.121.3 ± 6.8 (n = 142)100.0100.00.0MDA-MB-435S23.6 ± 6.3 (n = 66)84.977.37.624.9 ± 7.9 (n = 63)88.971.417.7MCF719.2 ± 6.6 (n = 63)21.08.112.915.7 ± 7.3 (n = 82)13.42.411.0A54917.9 ± 4.0 (n = 76)20.86.514.316.3 ± 4.4 (n = 93)79.65.474.2HeLa Bcl2 OEdHeLa cells overexpressing Bcl2.19.7 ± 8.8 (n = 72)27.815.312.520.1 ± 7.1 (n = 92)52.26.545.7a Percentage of total cell death.b Percentage of death in mitosis.c Percentage of death after mitotic exit.d HeLa cells overexpressing Bcl2. Open table in a new tab The molecular origin of death resistance in MCF7 and A549 is incompletely understood. To compare Cdc20 knockdown with kinesin-5 inhibitor in cells where we know the origin of death resistance, we used a HeLa line that stably overexpresses Bcl2. Bcl2 antagonizes MOMP, and overexpression of Bcl2 and related family members has been widely implicated in apoptosis resistance in cancer (Letai, 2008Letai A.G. Diagnosing and exploiting cancer's addiction to blocks in apoptosis.Nat. Rev. Cancer. 2008; 8: 121-132Crossref PubMed Scopus (434) Google Scholar). More than 70% of HeLa cells overexpressing Bcl2 slipped out of mitotic arrest induced by kinesin-5 inhibitor, and survived (blue line in Figure 2E; Table 1), like the naturally death-resistant cancer lines. Cdc20 knockdown again prevented slippage, and killed all cells that entered mitosis (red and green lines in Figure 2E), though this took ∼2.5-fold longer in time on average than normal HeLa (compare red and green lines in Figures 2A and 2E). These data allow several conclusions. First, Cdc20 knockdown efficiently promotes death during mitotic arrest. In lines that tend to die inside mitosis in kinesin-5 inhibitor, Cdc20 knockdown is equally effective at promoting death, but in lines that tend to slip before they die, it is much more effective. Second, because Cdc20 knockdown blocks slippage, these data allow us to compare the rate of death induction during mitotic arrest among the lines, without the complication of slippage. The median times for induction of death in Cdc20 knockdown were: HeLa 18.0 hr, MDA-MB-435S 24.3 hr, MCF7 39.8 hr, A549 40.0 hr, HeLa overexpressing Bcl2 40.8 hr. Thus, death induction rates during mitotic arrest were ∼2.5-fold faster in the most death-sensitive line compared with the most resistant. This relatively small difference in death induction rate translates into a much larger difference in survival in kinesin-5 inhibitor (∼0% in HeLa compared with >70% in MCF7, A549 and HeLa overexpressing Bcl2) because slippage intervenes to rescue the slower-dying lines, as proposed in the competing pathway model (Gascoigne and Taylor, 2008Gascoigne K.E. Taylor S.S. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs.Cancer Cell. 2008; 14: 111-122Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar). Finally, in HeLa cells Bcl2 overexpression confers strong resistance to kinesin-5 inhibitor, but not to Cdc20 knockdown. We next extended the comparison to paclitaxel, a drug with proven activity in solid tumors (Jordan and Wilson, 2004Jordan M.A. Wilson L. Microtubules as a target for anticancer drugs.Nat. Rev. Cancer. 2004; 4: 253-265Crossref PubMed Scopus (3235) Google Scholar). Again, we used a drug concentration that was saturating for mitotic arrest and failure of cytokinesis in all lines, to avoid complications from drug efflux pump or tubulin isotype differences. Across the panel, addition of Cdc20 knockdown to paclitaxel was always as, or more, efficient than paclitaxel alone at inducing cell death (Figures 2F–2J). In some lines, paclitaxel is more proapoptotic than kinesin-5 inhibitor. The duration of mitotic arrest was essentially the same for both drugs in all lines, and the extra cell death in paclitaxel manifested mostly after slippage (Table 1; Shi et al., 2008Shi J. Orth J.D. Mitchison T. Cell type variation in responses to antimitotic drugs that target microtubules and kinesin-5.Cancer Res. 2008; 68: 3269-3276Crossref PubMed Scopus (168) Google Scholar). In the more death-sensitive lines (HeLa, MDA-MB-435S), paclitaxel and kinesin-5 inhibitor caused death with similar kinetics, and Cdc20 knockdown killed with either the same (HeLa) or somewhat greater (MDA-MB-435S) efficiency. Death-resistant MCF7 cells responded similarly to the two drugs, and in this line Cdc20 knockdown killed with much greater efficiency than either drug. A549 cells were killed more efficiently by paclitaxel than kinesin-5 inhibitor, but Cdc20 knockdown was yet more efficient. HeLa overexpressing Bcl2 was intermediate between MCF7 and A549. Overall, although paclitaxel was somewhat more efficient at promoting killing than kinesin-5 inhibitor in some apoptosis-resistant lines, Cdc20 knockdown was always more efficient than either drug. A priori, we do not expect Cdc20 knockdown to perturb spindle assembly or activate the SAC. To test whether Cdc20 knockdown perturbs spindle assembly, we imaged microtubules live in HeLa stably expressing green fluorescent protein (GFP) β-tubulin (Figure 3). We observed normal bipolar spindles early in the arrest, which gradually became multipolar and abnormal over hours. From these images, it seems likely that the SAC is not activated early in the Cdc20 knockdown arrest, though it may be activated later. Because combining Cdc20 knockdown and kinesin-5 inhibitor showed similar death kinetics to Cdc20 knockdown alone in all lines (red and green lines in Figures 2A–2E), we used this combination in most subsequent experiments. By deliberately activating the SAC, we removed the ambiguity of whether it was activated. Combination with drugs was also more reliable for blocking slippage than Cdc20 knockdown alone in cell lines where transfection efficiency was variable. To determine how Cdc20 knockdown prevents slippage, we imaged cells infected with adenovirus expressing full-length cyclin B1 fused to enhanced green fluorescent protein (EGFP) (Bentley et al., 2007Bentley A.M. Normand G. Hoyt J. King R.W. Distinct sequence elements of cyclin B1 promote localization to chromatin, centrosomes, and kinetochores during mitosis.Mol. Biol. Cell. 2007; 18: 4847-4858Crossref PubMed Scopus (50) Google Scholar). We first confirmed that our cyclin B1-EGFP expression did not affect normal mitosis, duration of drug-induced mitotic arrest, or kinetics of cell death (data not shown). In HeLa, where most cells died in mitosis in kinesin-5 inhibitor, cyclin B1 levels gradually decreased to 30%–60% of the starting value by the time of death (Figure 4A, n > 30). In A549, where most cells slipped out of arrest without dying in kinesin-5 inhibitor, cyclin B1 levels slowly decreased, until they were 0%–10% of the level at the start of mitosis, when the cell slipped by morphological criteria (Figure 4B, n > 40). We observed considerable cell-to-cell variation in the shape and slope of cyclin B1 decrease kinetics, as we might expect because slippage kinetics are highly variable from cell to cell (Gascoigne and Taylor, 2008Gascoigne K.E. Taylor S.S. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs.Cancer Cell. 2008; 14: 111-122Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, Orth et al., 2008Orth J.D. Tang Y. Shi J. Loy C.T. Amendt C. Wilm C. Zenke F.T. Mitchison T.J. Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate.Mol. Cancer Ther. 2008; 7: 3480-3489Crossref PubMed Scopus (88) Google Scholar), but slippage always correlated with the time that cyclin B1 levels were reduced to 0%–10% of their starting value. When Cdc20 was depleted, cyclin B1 levels declined more slowly, especially in A549 (Figures 4B and 4D). In this situation, each time course ended when the cell underwent death in mitosis, which occurred on average 18.8 ± 7.3 hr (n = 98) after mitotic entry in HeLa, and 43.8 ± 16.5 hr (n = 101) in A549. At this time, cyclin B1 levels were 50%–90% of their mitotic entry value in HeLa, and 30%–70% in A549. Similar results were found when we used HeLa and A549 lines stably expressing full-length cyclin-B1-EYFP, suggesting that such degradation kinetics is not specific to adenovirus-mediated expression of cyclin-B1-EGFP (see Figure S1 available online). We conclude that Cdc20 knockdown stabilizes cyclin B1 levels during mitotic arrest more efficiently than SAC activation via kinesin-5 inhibition. This presumably explains why arrest is sustained for longer in Cdc20 knockdown, which gives cells more time to die in mitosis. These data are also consistent with a previous hypothesis that slippage is due to slow proteolysis of cyclin B1 by leaky activity of the APC/CCdc20 - proteasome pathway even when SAC is active (Brito and Rieder, 2006Brito D.A. Rieder C.L. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint.Curr. Biol. 2006; 16: 1194-1200Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), though a potential complication is the recent observation that cyclin B1 turns over with a half life of 1–2 hr, so its gradual loss presumably reflects a balance between synthesis and proteolysis (Nilsson et al., 2008Nilsson J. Yekezare M. Minshull J. Pines J. The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction.Nat. Cell Biol. 2008; 10: 1411-1420Crossref PubMed Scopus (225) Google Scholar). Other mitotic cyclins could potentially contribute to Cdc20 knockdown-mediated mitotic arrest, because depletion of Cdc20 also stabilizes other APC/CCdc20 substrates, for example cyclin A (Sigrist et al., 1995Sigrist S. Jacobs H. Stratmann R. Lehner C.F. Exit from mitosis is regulated by Drosophila fizzy" @default.
• W2076753965 created "2016-06-24" @default.
• W2076753965 creator A5006462973 @default.
• W2076753965 creator A5034492799 @default.
• W2076753965 creator A5059124650 @default.
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• W2076753965 date "2009-10-01" @default.
• W2076753965 modified "2023-10-12" @default.
• W2076753965 title "Evidence that Mitotic Exit Is a Better Cancer Therapeutic Target Than Spindle Assembly" @default.
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