Excess of a Rassf1-targeting microRNA, miR-193a-3p, perturbs cell division fidelity.

BACKGROUND: Several microRNA (miRNA) molecules have emerged as important post-transcriptional regulators of tumour suppressor and oncogene expression. Ras association domain family member 1 (RASSF1) is a critical tumour suppressor that controls multiple aspects of cell proliferation such as cell cycle, cell division and apoptosis. The expression of RASSF1 is lost in a variety of cancers due to the promoter hypermethylation. METHODS: miR-193a-3p was identified as a RASSF1-targeting miRNA by a dual screening approach. In cultured human cancer cells, immunoblotting, qRT-PCR, luciferase reporter assays, time-lapse microscopy and immunofluorescence methods were used to study the effects of excess miR-193a-3p on RASSF1 expression and cell division. RESULTS: Here, we report a new miRNA-mediated mechanism that regulates RASSF1 expression: miR-193a-3p binds directly to RASSF1-3'UTR and represses the mRNA and protein expression. In human cancer cells, excess of miR-193a-3p causes polyploidy through impairment of the Rassf1-Syntaxin 16 signalling pathway that is needed for completion of cytokinesis. In the next cell cycle the miR-193a-3p-overexpressing cells exhibit multipolar mitotic spindles, mitotic delay and elevated frequency of cell death. CONCLUSIONS: Our results suggest that besides epigenetic regulation, altered expression of specific miRNAs may contribute to the loss of Rassf1 in cancer cells and cause cell division errors.

Mitosis as an anti-cancer drug target.

Suppression of cell proliferation by targeting mitosis is one potential cancer intervention. A number of existing chemotherapy drugs disrupt mitosis by targeting microtubule dynamics. While efficacious, these drugs have limitations, i.e. neuropathy, unpredictability and development of resistance. In order to overcome these issues, a great deal of effort has been spent exploring novel mitotic targets including Polo-like kinase 1, Aurora kinases, Mps1, Cenp-E and KSP/Eg5. Here we summarize the latest developments in the discovery and clinical evaluation of new mitotic drug targets.

Loss of p38gamma MAPK induces pleiotropic mitotic defects and massive cell death.

The p38 mitogen-activated protein kinase (p38 MAPK) family, which is comprised of four protein isoforms, p38α, p38ß, p38γ and p38δ, forms one of the key MAPK pathways. The p38 MAPKs are implicated in many cellular processes including inflammation, differentiation, cell growth, cell cycle and cell death. The function of p38 MAPKs in mitotic entry has been well established, but their role in mitotic progression has remained controversial. We identify p38γ MAPK as a modulator of mitotic progression and mitotic cell death. In HeLa cells, loss of p38γ results in multipolar spindle formation and chromosome misalignment, which induce a transient M phase arrest. The majority of p38γ-depleted cells die at mitotic arrest or soon after abnormal exit from M-phase. We show that p38 MAPKs are activated at the kinetochores and spindle poles throughout mitosis by kinase(s) that are stably bound to these structures. Finally, p38γ is required for the normal kinetochore localization of polo-like kinase 1 (Plk1), and this contributes to the activity of the p38 MAPK pathway. Our data suggest a link between mitotic regulation and the p38 MAPK pathway, in which p38γ prevents chromosomal instability and supports mitotic cell viability.

The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis.

The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) as an anti-mitotic flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.

Translating Academic Drug Discovery Into Clinical Development: A Survey of the Awareness of Regulatory Support and Requirements Among Stakeholders in Europe.

Important discoveries by academic drug developers hold the promise of bringing innovative treatments that address unmet medical needs to the market. However, the drug development process has proved to be challenging and demanding for academic researchers, and regulatory challenges are an important barrier to implementing academic findings in clinical practice. European regulators offer varying degrees of support services to help drug developers meet regulatory standards and requirements. "Strengthening Training of Academia in Regulatory Sciences and Supporting Regulatory Scientific Advice" (STARS) is a European Commission-funded consortium aiming to strengthen the training of academics in regulatory science and requirements. Here, we report the results of four surveys that investigated the awareness and utilization of support tools offered by European regulators and identified the regulatory challenges and support needs of researchers. The surveys targeted four main European stakeholders in academic medicines research: academic research groups (706 respondents), academic research centers (99), funding organizations (49), and regulators (22). The results show that while European regulators provide various regulatory support tools, less than half of the responding academic researchers were aware of these tools and many experienced challenges in reaching a sufficient level of regulatory knowledge. There was a general lack of understanding of the regulatory environment that was aggravated by poor communication between stakeholders. The results of this study form a foundation for an improved European medicines regulatory network, in which regulatory challenges faced by academia are tackled.

High frequency of TTK mutations in microsatellite-unstable colorectal cancer and evaluation of their effect on spindle assembly checkpoint.

Frameshift mutations frequently accumulate in microsatellite-unstable colorectal cancers (MSI CRCs) typically leading to downregulation of the target genes due to nonsense-mediated messenger RNA decay. However, frameshift mutations that occur in the 3' end of the coding regions can escape decay, which has largely been ignored in previous works. In this study, we characterized nonsense-mediated decay-escaping frameshift mutations in MSI CRC in an unbiased, genome wide manner. Combining bioinformatic search with expression profiling, we identified genes that were predicted to escape decay after a deletion in a microsatellite repeat. These repeats, located in 258 genes, were initially sequenced in 30 MSI CRC samples. The mitotic checkpoint kinase TTK was found to harbor decay-escaping heterozygous mutations in exon 22 in 59% (105/179) of MSI CRCs, which is notably more than previously reported. Additional novel deletions were found in exon 5, raising the mutation frequency to 66%. The exon 22 of TTK contains an A(9)-G(4)-A(7) locus, in which the most common mutation was a mononucleotide deletion in the A(9) (c.2560delA). When compared with identical non-coding repeats, TTK was found to be mutated significantly more often than expected without selective advantage. Since TTK inhibition is known to induce override of the mitotic spindle assembly checkpoint (SAC), we challenged mutated cancer cells with the microtubule-stabilizing drug paclitaxel. No evidence of checkpoint weakening was observed. As a conclusion, heterozygous TTK mutations occur at a high frequency in MSI CRCs. Unexpectedly, the plausible selective advantage in tumourigenesis does not appear to be related to SAC.

VTT-006, an anti-mitotic compound, binds to the Ndc80 complex and suppresses cancer cell growth in vitro.

Hec1 (Highly expressed in cancer 1) resides in the outer kinetochore where it works to facilitate proper kinetochore-microtubule interactions during mitosis. Hec1 is overexpressed in various cancers and its expression shows correlation with high tumour grade and poor patient prognosis. Chemical perturbation of Hec1 is anticipated to impair kinetochore-microtubule binding, activate the spindle assembly checkpoint (spindle checkpoint) and thereby suppress cell proliferation. In this study, we performed high-throughput screen to identify novel small molecules that target the Hec1 calponin homology domain (CHD), which is needed for normal microtubule attachments. 4 million compounds were first virtually fitted against the CHD, and the best hit molecules were evaluated in vitro. These approaches led to the identification of VTT-006, a 1,2-disubstituted-tetrahydro-beta-carboline derivative, which showed binding to recombinant Ndc80 complex and modulated Hec1 association with microtubules in vitro. VTT-006 treatment resulted in chromosome congression defects, reduced chromosome oscillations and induced loss of inter-kinetochore tension. Cells remained arrested in mitosis with an active spindle checkpoint for several hours before undergoing cell death. VTT-006 suppressed the growth of several cancer cell lines and enhanced the sensitivity of HeLa cells to Taxol. Our findings propose that VTT-006 is a potential anti-mitotic compound that disrupts M phase, impairs kinetochore-microtubule interactions, and activates the spindle checkpoint.

Perturbation of Incenp function impedes anaphase chromatid movements and chromosomal passenger protein flux at centromeres.

Incenp is an essential mitotic protein that, together with Aurora B, Survivin, and Borealin, forms the core of the chromosomal passenger protein complex (CPC). The CPC regulates various mitotic processes and functions to maintain genomic stability. The proper subcellular localization of the CPC and its full catalytic activity require the presence of each core subunit in the complex. We have investigated the mitotic tasks of the CPC using a function blocking antibody against Incenp microinjected into cells at different mitotic phases. This method allowed temporal analysis of CPC functions without perturbation of complex assembly or activity prior to injection. We have also studied the dynamic properties of Incenp and Aurora B using fusion protein photobleaching. We found that in early mitotic cells, Incenp and Aurora B exhibit dynamic turnover at centromeres, which is prevented by the anti-Incenp antibody. In these cells, the loss of centromeric CPC turnover is accompanied by forced mitotic exit without the execution of cytokinesis. Introduction of anti-Incenp antibody into early anaphase cells causes abnormalities in sister chromatid separation through defects in anaphase spindle functions. In summary, our data uncovers new mitotic roles for the CPC in anaphase and proposes that CPC turnover at centromeres modulates spindle assembly checkpoint signaling.

Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint.

Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2-160 microg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound.

Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells.

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20-GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

MYC-Induced miR-203b-3p and miR-203a-3p Control Bcl-xL Expression and Paclitaxel Sensitivity in Tumor Cells.

Taxanes are chemotherapeutic agents used in the treatment of solid tumors, particularly of breast, ovarian, and lung origin. However, patients show divergent therapy responses, and the molecular determinants of taxane sensitivity have remained elusive. Especially the signaling pathways that promote death of the taxane-treated cells are poorly characterized. Here we describe a novel part of a signaling route in which c-Myc enhances paclitaxel sensitivity through upregulation of miR-203b-3p and miR-203a-3p; two clustered antiapoptosis protein Bcl-xL controlling microRNAs. In vitro, the miR-203b-3p decreases the expression of Bcl-xL by direct targeting of the gene's mRNA 3'UTR. Notably, overexpression of the miR-203b-3p changed the fate of paclitaxel-treated breast and ovarian cancer cells from mitotic slippage to cell death. In breast tumors, high expression of the miR-203b-3p and MYC was associated with better therapy response and patient survival. Interestingly, in the breast tumors, MYC expression correlated negatively with BCL2L1 expression but positively with miR-203b-3p and miR-203a-3p. Finally, silencing of MYC suppressed the transcription of both miRNAs in breast tumor cells. Pending further validation, these results may assist in patient stratification for taxane therapy.

Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores.

BACKGROUND: In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the spindle checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression. RESULTS: We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a spindle-checkpoint-mediated mitotic arrest. CONCLUSIONS: Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.

Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis.

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.

The vertebrate Ndc80 complex contains Spc24 and Spc25 homologs, which are required to establish and maintain kinetochore-microtubule attachment.

How kinetochores bind to microtubules and move on the mitotic spindle remain unanswered questions. Multiple systems have implicated the Ndc80/Hec1 (Ndc80) kinetochore complex in kinetochore-microtubule interaction and spindle checkpoint activity. In budding yeast, Ndc80 copurifies with three additional interacting proteins: Nuf2, Spc24, and Spc25. Although functional vertebrate homologs of Ndc80 and Nuf2 exist, extensive sequence similarity searches have not uncovered homologs of Spc24 and Spc25. We have purified the xNdc80 complex to homogeneity from Xenopus egg extracts and identified two novel interacting proteins. Although the sequences have greatly diverged, we have concluded that these are the functional homologs of the yeast Spc24 and Spc25 proteins based on limited sequence similarity, common coiled-coil domains, kinetochore localization, similar phenotypes, and copurification with xNdc80 and xNuf2. Using both RNAi and antibody injection experiments, we have extended previous characterization of the complex and found that Spc24 and Spc25 are required not only to establish, but also to maintain kinetochore-microtubule attachments and metaphase alignment. In addition, we show that Spc24 and Spc25 are required for chromosomal movement to the spindle poles in anaphase.

Evaluation of the antitumor activity of NOV202, a novel microtubule targeting and vascular disrupting agent.

PURPOSE: Overall, ~65% of patients diagnosed with advanced ovarian cancer (OC) will relapse after primary surgery and adjuvant first-line platinum- and taxane-based chemotherapy. Significant improvements in the treatment of OC are expected from the development of novel compounds having combined cytotoxic and antiangiogenic properties that make them effective on refractory tumors. METHODS: Permeability of NOV202 was determined with Caco-2 monolayer assay. The compound's pharmacokinetic profile and plasma:brain distribution were assessed in male C57Bl/6 mice. The compound's impacts on tubulin, microtubules and cell cycle were investigated by using in vitro tubulin polymerization assay, cell-based immunofluorescence and live cell microscopy. The IC50 concentrations of NOV202 were assessed in a panel of eight cancer cell lines. Impact of the compound on vascular tube formation was determined using the StemKit and Chick chorioallantoic membrane assays. The in vivo efficacy of the compound was analyzed with an OC xenograft mouse model. RESULTS: NOV202 was found to suppress cancer cell proliferation at low nanomolar concentrations (IC50 2.3-12.0 nM) and showed equal efficacy between OC cell line A2780 (IC50 2.4 nM) and its multidrug-resistant subline A2780/Adr (IC50 2.3 nM). Mechanistically, NOV202 targeted tubulin polymerization in vitro in a dose-dependent manner and in cells induced an M phase arrest. In vivo, NOV202 caused a dose-dependent reduction of tumor mass in an A2780 xenograft model, which at the highest dose (40 mg/kg) was comparable to the effect of paclitaxel (24 mg/kg). Interestingly, NOV202 exhibited vascular disrupting properties that were similar to the effects of Combretastatin A4. CONCLUSION: NOV202 is a novel tubulin and vascular targeting agent that shows strong anticancer efficacy in cells and OC xenograft models. The finding that the compound induced significantly more cell death in Pgp/MDR1 overexpressing OC cells compared to vincristine and paclitaxel warrants further development of the compound as a new therapy for OC patients with treatment refractory tumors and/or relapsing disease.

Regulation of microtubule stability and mitotic progression by survivin.

Survivin is a member of the inhibitor of apoptosis (IAP) gene family, which has been implicated in both preservation of cell viability and regulation of mitosis in cancer cells. Here, we show that HeLa cells microinjected with a polyclonal antibody to survivin exhibited delayed progression in prometaphase (31.5 +/- 6.9 min) and metaphase (126.8 +/- 73.8 min), as compared with control injected cells (prometaphase, 21.5 +/- 3.3 min; metaphase, 18.9 +/- 4.5 min; P < 0.01). Cells injected with the antibody to survivin displayed short mitotic spindles severely depleted of microtubules and occasionally underwent apoptosis without exiting the mitotic block or thereafter. Forced expression of survivin in HeLa cells profoundly influenced microtubule dynamics with reduction of pole-to-pole distance at metaphase (8.57 +/- 0.21 microm versus 10.58 +/- 0.19 microm; P < 0.0001) and stabilization of microtubules against nocodazole-induced depolymerization in vivo. These data demonstrate that survivin functions at cell division to control microtubule stability and assembly of a normal mitotic spindle. This pathway may facilitate checkpoint evasion and promote resistance to chemotherapy in cancer.

Novel Mad2-targeting miR-493-3p controls mitotic fidelity and cancer cells' sensitivity to paclitaxel.

The molecular pathways that contribute to the proliferation and drug response of cancer cells are highly complex and currently insufficiently characterized. We have identified a previously unknown microRNA-based mechanism that provides cancer cells means to stimulate tumorigenesis via increased genomic instability and, at the same time, evade the action of clinically utilized microtubule drugs. We demonstrate miR-493-3p to be a novel negative regulator of mitotic arrest deficient-2 (MAD2), an essential component of the spindle assembly checkpoint that monitors the fidelity of chromosome segregation. The microRNA targets the 3' UTR of Mad2 mRNA thereby preventing translation of the Mad2 protein. In cancer cells, overexpression of miR-493-3p induced a premature mitotic exit that led to increased frequency of aneuploidy and cellular senescence in the progeny cells. Importantly, excess of the miR-493-3p conferred resistance of cancer cells to microtubule drugs. In human neoplasms, miR-493-3p and Mad2 expression alterations correlated with advanced ovarian cancer forms and high miR-493-3p levels were associated with reduced survival of ovarian and breast cancer patients with aggressive tumors, especially in the paclitaxel therapy arm. Our results suggest that intratumoral profiling of miR-493-3p and Mad2 levels can have diagnostic value in predicting the efficacy of taxane chemotherapy.

Expression of HSF2 decreases in mitosis to enable stress-inducible transcription and cell survival.

Unless mitigated, external and physiological stresses are detrimental for cells, especially in mitosis, resulting in chromosomal missegregation, aneuploidy, or apoptosis. Heat shock proteins (Hsps) maintain protein homeostasis and promote cell survival. Hsps are transcriptionally regulated by heat shock factors (HSFs). Of these, HSF1 is the master regulator and HSF2 modulates Hsp expression by interacting with HSF1. Due to global inhibition of transcription in mitosis, including HSF1-mediated expression of Hsps, mitotic cells are highly vulnerable to stress. Here, we show that cells can counteract transcriptional silencing and protect themselves against proteotoxicity in mitosis. We found that the condensed chromatin of HSF2-deficient cells is accessible for HSF1 and RNA polymerase II, allowing stress-inducible Hsp expression. Consequently, HSF2-deficient cells exposed to acute stress display diminished mitotic errors and have a survival advantage. We also show that HSF2 expression declines during mitosis in several but not all human cell lines, which corresponds to the Hsp70 induction and protection against stress-induced mitotic abnormalities and apoptosis.

Centmitor-1, a novel acridinyl-acetohydrazide, possesses similar molecular interaction field and antimitotic cellular phenotype as rigosertib, on 01910.Na.

Mitosis is an attractive target for the development of new anticancer drugs. In a search for novel mitotic inhibitors, we virtually screened for low molecular weight compounds that would possess similar steric and electrostatic features, but different chemical structure than rigosertib (ON 01910.Na), a putative inhibitor of phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathways. Highest scoring hit compounds were tested in cell-based assays for their ability to induce mitotic arrest. We identified a novel acridinyl-acetohydrazide, here named as Centmitor-1 (Cent-1), that possesses highly similar molecular interaction field as rigosertib. In cells, Cent-1 phenocopied the cellular effects of rigosertib and caused mitotic arrest characterized by chromosome alignment defects, multipolar spindles, centrosome fragmentation, and activated spindle assembly checkpoint. We compared the effects of Cent-1 and rigosertib on microtubules and found that both compounds modulated microtubule plus-ends and reduced microtubule dynamics. Also, mitotic spindle forces were affected by the compounds as tension across sister kinetochores was reduced in mitotic cells. Our results showed that both Cent-1 and rigosertib target processes that occur during mitosis as they had immediate antimitotic effects when added to cells during mitosis. Analysis of Plk1 activity in cells using a Förster resonance energy transfer (FRET)-based assay indicated that neither compound affected the activity of the kinase. Taken together, these findings suggest that Cent-1 and rigosertib elicit their antimitotic effects by targeting mitotic processes without impairment of Plk1 kinase activity.