Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death.     

Suppressors of spindle checkpoint defect (such) mutants identify new mdf-1/MAD1 interactors in Caenorhabditis elegans

The spindle assembly checkpoint (SAC) governs the timing of metaphase-to-anaphase transition and is essential for genome stability. The Caenorhabditis elegans mutant strain gk2 carries a deletion within the mdf-1/MAD1 gene that results in death of the homozygous strain after two or three generations. Here we describe 11 suppressors of the mdf-1(gk2) lethality, 10 identified in an ethyl methanesulfonate (EMS) mutagenesis screen and 1 isolated using the dog-1(gk10) (deletions of guanine-rich DNA) mutator strain. Using time-lapse imaging of early embryonic cells and germline mitotic division, we demonstrate that there are two classes of suppressors. Eight suppressors compensate for the loss of the checkpoint by delaying mitotic progression, which coincides with securin (IFY-1/Pds1) accumulation; three suppressors have normal IFY-1/Pds1 levels and normal anaphase onset. Furthermore, in the class of suppressors with delayed mitotic progression, we have identified four alleles of known suppressors emb-30/APC4 and fzy-1/CDC20, which are components of the anaphase-promoting complex/cyclosome (APC/C). In addition, we have identified another APC/C component capable of bypassing the checkpoint requirement that has not previously been described in C. elegans. The such-1/APC5-like mutation, h1960, significantly delays anaphase onset both in germline and in early embryonic cells.     

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.     

JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules.     

Small interfering RNA screens reveal enhanced cisplatin cytotoxicity in tumor cells having both BRCA network and TP53 disruptions

RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53(+/-) matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity approximately 4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.     

Re-evaluating the role of Tao1 in the spindle checkpoint

The spindle checkpoint restrains anaphase onset and mitotic exit until all chromosomes are stably attached to the mitotic spindle via their kinetochores. The Tao1 protein kinase was recently reported as a novel spindle checkpoint component. When an siRNA was used to repress Tao1, the essential spindle checkpoint component Mad2 failed to localise to kinetochores, and cells rapidly exited mitosis. Tao1 was also shown to interact with BubR1, another essential checkpoint component, and be rapidly degraded after mitosis, a feature typical of many mitotic regulators. Here, we identify four different siRNAs that repress Tao1 protein levels as efficiently as the previously reported siRNA. However, these siRNAs do not override the spindle checkpoint. We also present data indicating that Tao1 does not interact with BubR1 and that it is not rapidly degraded after mitosis. We show that the previously reported siRNA not only represses Tao1 but also dramatically reduces Mad2 protein levels. Crucially, expression of exogenous Mad2, but not Tao1, rescued the spindle checkpoint phenotype induced by this siRNA. Thus, the key functional data implicating Tao1 in the spindle checkpoint can be explained by an off-target siRNA phenomenon that results in Mad2 inhibition. Taken together, our data do not support the notion that Tao1 is a component of the spindle checkpoint.     

Dual inhibition of Kif15 by oxindole and quinazolinedione chemical probes

The mitotic spindle is a microtubule-based machine that segregates a replicated set of chromosomes during cell division. Many cancer drugs alter or disrupt the microtubules that form the mitotic spindle. Microtubule-dependent molecular motors that function during mitosis are logical alternative mitotic targets for drug development. Eg5 (Kinesin-5) and Kif15 (Kinesin-12), in particular, are an attractive pair of motor proteins, as they work in concert to drive centrosome separation and promote spindle bipolarity. Furthermore, we hypothesize that the clinical failure of Eg5 inhibitors may be (in part) due to compensation by Kif15. In order to test this idea, we screened a small library of kinase inhibitors and identified GW108X, an oxindole that inhibits Kif15 in vitro. We show that GW108X has a distinct mechanism of action compared with a commercially available Kif15 inhibitor, Kif15-IN-1 and may serve as a lead with which to further develop Kif15 inhibitors as clinically relevant agents.     

Identification of siRNA delivery enhancers by a chemical library screen

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2–5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.     

Whole genome functional analysis identifies novel components required for mitotic spindle integrity in human cells

Background

The mitotic spindle is a complex mechanical apparatus required for accurate segregation of sister chromosomes during mitosis. We designed a genetic screen using automated microscopy to discover factors essential for mitotic progression. Using a RNA interference library of 49,164 double-stranded RNAs targeting 23,835 human genes, we performed a loss of function screen to look for small interfering RNAs that arrest cells in metaphase.

Results

Here we report the identification of genes that, when suppressed, result in structural defects in the mitotic spindle leading to bent, twisted, monopolar, or multipolar spindles, and cause cell cycle arrest. We further describe a novel analysis methodology for large-scale RNA interference datasets that relies on supervised clustering of these genes based on Gene Ontology, protein families, tissue expression, and protein-protein interactions.

Conclusion

This approach was utilized to classify functionally the identified genes in discrete mitotic processes. We confirmed the identity for a subset of these genes and examined more closely their mechanical role in spindle architecture.     

G1 tetraploidy checkpoint and the suppression of tumorigenesis

Checkpoints suppress improper cell cycle progression to ensure that cells maintain the integrity of their genome. During mitosis, a metaphase checkpoint requires the integration of all chromosomes into a metaphase array in the mitotic spindle prior to mitotic exit. Still, mitotic errors occur in mammalian cells with a relatively high frequency. Metaphase represents the last point of control in mitosis. Once the cell commits to anaphase there are no checkpoints to sense segregation defects. In this context, we will explore our recent finding that non-transformed mammalian cells have a checkpoint that acts subsequent to mitotic errors to block the proliferation of cells that have entered G1 with tetraploid status. This arrest is dependent upon both p53 and pRb, and may represent an important function of both p53 and pRb as tumor suppressors. Further, we discuss the possibility that this mechanism may similarly impose G1 arrest in cells that become aneuploid through errors in mitosis.     

Genome‐wide siRNA screen reveals coupling between mitotic apoptosis and adaptation

The antimitotic anti‐cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint‐dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome‐wide small interfering RNA (siRNA) screen in taxol‐treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31^comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro‐apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.     

Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5

Mitotic kinesins represent potential drug targets for anticancer chemotherapy. Inhibitors of different chemical classes have been identified that target human Eg5, a kinesin responsible for the establishment of the bipolar spindle. One potent Eg5 inhibitor is S-trityl-L-cysteine (STLC), which arrests cells in mitosis and exhibits tumor growth inhibition activity. However, the underlying mechanism of STLC action on the molecular level is unknown. Here, cells treated with STLC were blocked in mitosis through activation of the spindle assembly checkpoint as shown by the phosphorylated state of BubR1 and the accumulation of mitosis specific phosphorylation on histone H3 and aurora A kinase. Using live cell imaging, we observed prolonged mitotic arrest and subsequent cell death after incubation of GFP-alpha-tubulin HeLa cells with STLC. Activated caspase-9 occurred before cleavage of caspase-8 leading to the accumulation of the activated executioner caspase-3 suggesting that STLC induces apoptosis through the intrinsic apoptotic pathway. Proteome analysis following STLC treatment revealed 33 differentially regulated proteins of various cellular processes, 31 of which can be linked to apoptotic cell death. Interestingly, four identified proteins, chromobox protein homolog, RNA-binding Src associated in mitosis 68 kDa protein, stathmin, and translationally controlled tumor protein can be linked to mitotic and apoptotic processes.     

Paclitaxel Induces Primary and Postmitotic G1 Arrest in Human Arterial Smooth Muscle Cells

Paclitaxel (PTX), a microtubule-active drug, causes mitotic arrest leading to apoptosis in certain tumor cell lines. Here we investigated the effects of PTX on human arterial smooth muscle cell (SMC) cells. In SMC, PTX caused both (a) primary arrest in G1 and (b) post-mitotic arrest in G1. Post-mitotic cells were multinucleated (MN) with either 2C (near-diploid) or 4C (tetraploid) DNA content. At PTX concentrations above12 ng/ml, MN cells had 4C DNA content consistent with the lack of cytokinesis during abortive mitosis. Treatment with 6-12 ng/ml PTX yielded MN cells with 2C DNA content. Finally, 1-6 ng/ml of PTX, the lowest concentrations that affected cell proliferation, caused G1 arrest without multinucleation. It is important that PTX did not cause apoptosis in SMC. The absence of apoptosis could be explained by mitotic exit and G1 arrest as well as by low constitutive levels of caspase expression and by p53 and p21 induction. Thus, following transient mitotic arrest, SMC exit mitosis to form MN cells. These post-mitotic cells were subsequently arrested in G1 but maintained normal elongated morphology and were viable for at least 21 days. We conclude that in SMC PTX causes post-mitotic cell cycle arrest rather than cell death.     

Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.     

Rapid functional screening of effective siRNAs against Plk1 and its growth inhibitory effects in laryngeal carcinoma cells

Plk 1 is overexpressed in many human malignancies including laryngeal carcinoma. However, its therapeutic potential has been never examined in laryngeal carcinoma. In the present study, a simple cellular morphology-based strategy was firstly proposed for rapidly screening the effective siRNAs against Plk1. Furthermore, we investigated the effects of Plk1 depletion via a novel identified effective siRNA against Plk1, Plk1 siRNA-607, on human laryngeal carcinoma Hep-2 cells. The results indicated that Plk1 siRNA-607 transfection resulted in a significant inhibition in Plk1 expression in cells, and subsequently caused a dramatic mitotic cell cycle arrest followed by massive apoptotic cell death, and eventually resulted in a significant decrease in growth and viability of the laryngeal carcinoma cells. Taken together, our present study not only suggests a simple strategy for rapidly screening effective siRNAs against Plk1 but also implicates that Plk1 may serve as a potential therapeutic target in human laryngeal carcinoma.     

Targeting mitosis‐regulating genes in cisplatin‐sensitive and ‐resistant melanoma cells: A live‐cell RNAi screen displays differential nucleus‐derived phenotypes

Chemoresistance in malignant melanoma remains an unresolved clinical issue. In the search for novel molecular targets, a live‐cell high‐content RNAi screen based on gene expression data was performed in cisplatin‐sensitive and cisplatin‐resistant MeWo melanoma cells, Mel‐28 cells and a melanocyte cell line. Cells were exposed to 91 siRNAs and distinct nucleus‐derived phenotypes such as cell division, cell death and migration phenotypes were detected by time‐lapse microscopy over 60 h. Using this approach, cisplatin‐sensitive and cisplatin‐resistant melanoma cells were compared by automated image analysis and visual inspection. In cisplatin‐sensitive MeWo melanoma cells, 14 genes were identified that showed distinct phenotype abnormalities after exposure to gene‐specific siRNAs. In cisplatin‐resistant MeWo cells, five genes were detected. Nine genes were detected whose knock‐down led to differential nuclear phenotypes in cisplatin‐sensitive and ‐resistant cells. In Mel‐28 cells, nine genes were identified which induced nuclear phenotypes including all eight genes which were identified in cisplatin‐resistant MeWo cells. An analogous RNAi screen on melanocytes revealed no detectable phenotype abnormalities after RNAi. Pathway analysis showed in cisplatin‐sensitive MeWo cells and Mel‐28 cells an enrichment of at least three genes in major mitotic pathways. We hereby show that siRNA screening may help to identify tumor‐specific genes leading to phenotype abnormalities. These genes may serve as potential therapeutic targets in the treatment of melanoma.     

Incorporation of biaryl units into the 5' and 3' ends of sense and antisense strands of siRNA duplexes improves strand selectivity and nuclease resistance

Small interfering RNA (siRNA) is a noncoding RNA with considerable potential as a new therapeutic drug for intractable diseases. siRNAs can be rationally designed and synthesized if the sequences of the disease-causing genes are known. In this paper, we describe the synthesis and properties of siRNAs modified with biaryl units. We found that incorporation of biaryl units into the 5' and 3' ends of sense and antisense strands of siRNA duplexes improved strand selectivity and nuclease resistance.     

Role of the kinesin-2 family protein, KIF3, during mitosis

During mitosis, kinesin and dynein motor proteins play critical roles in the equal segregation of chromosomes between two daughter cells. Kinesin-2 is composed of two microtubule-based motor subunits, KIF3A/3B, and a kinesin-associated protein known as KAP3, which links KIF3A/3B to cargo that is carried to cellular organelles along microtubules in interphase cells. We have shown here that the kinesin-2 complex is localized with components of the mitotic apparatus such as spindle microtubules and centrosomes. Furthermore, we found that expression of a mutant KIF3B, which is able to associate with KIF3A but not KAP3 in NIH3T3 cells, caused chromosomal aneuploidy and abnormal spindle formation. Our data suggested that the kinesin-2 complex plays an important role not only in interphase but also in mitosis.     

An Extragenic Suppressor of the Mitosis-Defective Bimd6 Mutation of Aspergillus Nidulans Codes for a Chromosome Scaffold Protein

We previously identified a gene, bimD, that functions in chromosome segregation and contains sequences suggesting that it may be a DNA-binding protein. Two conditionally lethal mutations in bimD arrest with aberrant mitotic spindles at restrictive temperature. These spindles have one-third the normal number of microtubules, and the chromosomes never attach to the remaining microtubules. For this reason, we hypothesized that BIMD functioned in chromosome segregation, possibly as a component of the kinetochore. To identify other components that function with bimD, we conducted a screen for extragenic suppressors of the bimD5 and bimD6 mutations. We have isolated seven cold-sensitive extragenic suppressors of bimD6 heat sensitivity that represent three or possibly four separate sud genes. We have cloned one of the suppressor genes by complementation of the cold-sensitive phenotype of the sudA3 mutation. SUDA belongs to the DA-box protein family. DA-box proteins have been shown to function in chromosome structure and segregation. Thus bimD and the sud genes cooperatively function in chromosome segregation in Aspergillus nidulans.     

Cyclin B1 interacts with the BH3-only protein Bim and mediates its phosphorylation by Cdk1 during mitosis

Protracted mitotic arrest leads to cell death; however, the molecular signals that link these distinct processes remain poorly understood. Here we report that the pro-apoptotic BH3-only family member Bim undergoes phosphorylation in K562 cells following treatment with the microtubule targeting agents Taxol and Nocodazole. The phosphorylation of two Bim isoforms, BimEL and BimL, at the mitochondria correlates with mitotic arrest and precedes cell death induced by Taxol. It was also found that Bim undergoes transient phosphorylation during normal mitosis in K562 cells. In addition, siRNA silencing of Bim reduces sensitivity to Taxol-induced cell death. The transition of K562 cells from mitosis to G1 results in the loss of BimEL and BimL phosphorylation and correlates with the degradation of cyclin B1. The Cdk1 inhibitors, RO-3306 and Purvalanol A, block Bim phosphorylation in mitotically arrested cells. Importantly, it was found that cyclin B1 co-immunoprecipitates with endogenous Bim in mitotic extracts. Furthermore, active recombinant Cdk1/cyclin B1 phosphorylates BimEL and BimL in vitro and Serine 44 on BimL has been identified as a Cdk1 phosphorylation site. Collectively, these results suggest that Cdk1/cyclin B1-dependent hyper-phosphorylation of Bim during prolonged mitotic arrest is an important cell death signal.