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In the present study, the development of an efficient and feasible protocol for chromosome preparation from sterlet (A. ruthenus) embryos and larvae was carried out. In the established protocol, the mean efficiency of chromosome extraction ranged from 70 to 100%. The average number of recorded metaphases per slide was between 9 to 15. In general, the most satisfactory results were obtained for embryos at 6 dpf and larvae at the age of up to 7 dph. In the 24 dpf group, chromosome isolation was possible without immersion in spindle poison, however; in successive developmental stages, the minimal immersion time exceeded 1.5 hours, regardless of chorionation. Immersion for 14 hours did not compromise the efficacy of chromosome isolation. In the current study, successful chromosome isolation was determined mainly by hypotonization conditions. Younger developmental stages generally require the shortest hypotonization times, whereas older larvae require longer hypotonization times. The optimal hypotonization period is 5-15 minutes for embryos at 24 dpf, 40 minutes for embryos at 4dpf, and 50-60 minutes for fish at 6 dpf-7 dph. The only exception was the 24 hpf group where only blastula cells were used. An additional overnight fixation step significantly enhanced chromosome quality and supported chromosome counting especially in the 24 dpf group. The quality and quantity of chromosome slides were also significantly determined by tissue type, and the slides prepared from heads (gill cells) produced the best results.

Mesothelioma is a universally lethal cancer lacking effective therapy. The spindle poison vinorelbine exhibits clinical activity in the relapsed setting, and in preclinical models requires BRCA1 to initiate apoptosis. However, the mechanisms underlying this regulation and the clinical implications have not been explored. Here, we show that BRCA1 silencing abrogated vinorelbine-induced cell-cycle arrest, recruitment of BUBR1 to kinetochores, and apoptosis. BRCA1 silencing led to codepletion of MAD2L1 at the mRNA and protein levels consistent with its status as a transcriptional target of BRCA1. Silencing of MAD2L1 phenocopied BRCA1 and was sufficient to confer resistance to vinorelbine. This was recapitulated in cell lines selected for resistance to vinorelbine, which acquired loss of both BRCA1 and MAD2L1 expression. Following ex vivo vinorelbine in 20 primary tumor explants, apoptotic response rate was 59% in BRCA1/MAD2L1-positive explants compared with 0% in BRCA1/MAD2L1-negative explants. In 48 patients, BRCA1 and/or MAD2L1 loss of expression was not prognostic; however, in a subset of patients treated with vinorelbine, survival was shorter for patients lacking BRCA1/MAD2L1 expression compared with double-positive patients (5.9 vs. 36.7 months, P = 0.03). Our data implicate BRCA1/MAD2L1 loss as a putative predictive marker of resistance to vinorelbine in mesothelioma and warrant prospective clinical evaluation.

Exposure to chemical agents is an inevitable consequence of modern society; some of these agents are hazardous to human health. The effects of chemical carcinogens are of great concern in many countries, and international organizations, such as the World Health Organization, have established guidelines for the regulation of these chemicals. Carcinogens are currently categorized into two classes, genotoxic and non-genotoxic carcinogens, which are subject to different regulatory policies. Genotoxic carcinogens are chemicals that exert carcinogenicity via the induction of mutations. Owing to their DNA interaction properties, there is thought to be no safe exposure threshold or dose. Genotoxic carcinogens are regulated under the assumption that they pose a cancer risk for humans, even at very low doses. In contrast, non-genotoxic carcinogens, which induce cancer through mechanisms other than mutations, such as hormonal effects, cytotoxicity, cell proliferation, or epigenetic changes, are thought to have a safe exposure threshold or dose; thus, their use in society is permitted unless the exposure or intake level would exceed the threshold. Genotoxicity assays are an important method to distinguish the two classes of carcinogens. However, some carcinogens have negative results in in vitro bacterial mutation assays, but yield positive results in the in vivo transgenic rodent gene mutation assay. Non-DNA damage, such as spindle poison or topoisomerase inhibition, often leads to positive results in cytogenetic genotoxicity assays such as the chromosome aberration assay or the micronucleus assay. Therefore, mechanistic considerations of tumor induction, based on the results of the genotoxicity assays, are necessary to distinguish genotoxic and non-genotoxic carcinogens. In this review, the concept of threshold of toxicological concern is introduced and the potential risk from multiple exposures to low doses of genotoxic carcinogens is also discussed.

Background Chromosomal instability (CIN) is a common trait of cancer characterised by the continuous gain and loss of chromosomes during mitosis. Excessive levels of CIN can suppress tumour growth, providing a possible therapeutic strategy. The Mps1/TTK kinase has been one of the prime targets to explore this concept, and indeed Mps1 inhibitors synergise with the spindle poison docetaxel in inhibiting the growth of tumours in mice. Methods To investigate how the combination of docetaxel and a Mps1 inhibitor (Cpd-5) promote tumour cell death, we treated mice transplanted with BRCA1−/−;TP53−/− mammary tumours with docetaxel and/or Cpd-5. The tumours were analysed regarding their histopathology, chromosome segregation errors, copy number variations and cell death to understand the mechanism of action of the drug combination. Results The enhanced efficacy of combining an Mps1 inhibitor with clinically relevant doses of docetaxel is associated with an increase in multipolar anaphases, aberrant nuclear morphologies and cell death. Tumours treated with docetaxel and Cpd-5 displayed more genomic deviations, indicating that chromosome stability is affected mostly in the combinatorial treatment. Conclusions Our study shows that the synergy between taxanes and Mps1 inhibitors depends on increased errors in cell division, allowing further optimisation of this treatment regimen for cancer therapy.

The commonly used antimitotic chemotherapeutic agents such as taxol and vinblastine arrest cell cycle progression by disrupting mitotic spindles, and cause cancer cells to undergo apoptosis through 'mitotic catastrophe'. The molecular mechanisms by which these drugs induce apoptosis and their relevance to clinical efficacy are not known. Facilitated by a new spindle poison diazonamide, we found that apoptosis induced by these agents requires death receptor 3 (DR3). Mitotic arrest by these agents induces lysosome-dependent secretion of the DR3 ligand, TL1A. Engagement of TL1A with DR3 stimulates the formation of FADD-containing and caspase-8-containing death-inducing signaling complex (DISC), which subsequently activates apoptosis in cells that express DR3. Expression of DR3 and TL1A correlates with the apoptotic response of human tumor xenograft models and human cancer cell lines to antimitotic drugs, providing further evidence that these drugs kill cancer cells through the DR3/TL1A-mediated pathway. These results suggest that TL1A and DR3 may hold promise to be used as biomarkers for predicting clinical response to antimitotic therapeutics.

// Yu Liu 1, * , Xiao Du 1, * , Shuting Zhang 1 , Yang Liu 1 , Qiaoling Zhang 1 , Qi Yin 1 , Michael A. McNutt 1 and Yuxin Yin 1 1 Institute of Systems Biomedicine, Department of Pathology, School of Basic Medical Sciences, Beijing Key Laboratory of Tumor Systems Biology, Peking-Tsinghua Center for Life Sciences, Peking University Health Science Center, Beijing 100191, China * These authors have contributed equally to this work Correspondence to: Yuxin Yin, email: yinyuxin@hsc.pku.edu.cn Keywords: PTEN; MAD1; spindle assembly checkpoint; mitosis; genome stability Received: November 25, 2016 Accepted: August 04, 2017 Published: August 24, 2017 ABSTRACT The spindle assembly checkpoint (SAC) restrains anaphase progression to ensure all chromosomes attach properly to the spindle. Although SAC timing has been extensively investigated in mitosis, its mechanism of regulation in interphase is unclear. We report that PTEN functions as a crucial activator of SAC timing and protects chromosome segregation under both spindle poison treated and untreated conditions. We show that PTEN physically interacts with MAD1 and promotes its dimerization and localization in the nuclear pore. Consequently, PTEN is important for the formation of the mitotic checkpoint complex (MCC) in interphase. We propose PTEN/MAD1 signaling is essential for maintenance of SAC timing and chromosome integrity.

Spindle poisons are chemotherapeutic drugs used in the treatment of malignant tumors; however, numerous patients develop resistance following chemotherapy. The present study aimed to induce polyploidy in breast cancer cells using the spindle poison nocodazole to investigate the mechanism of polyploid-induced tumor resistance. It was revealed that the spindle poison nocodazole induced apoptosis in HCC1806 cells but also induced polyploidy in MDA-MB-231 cells. The drug sensitivities of the polyploid MDA-MB-231 cells to paclitaxel, docetaxel, epirubicin, 5-fluorouracil and oxaliplatin were lower than those of the original tumor cells; however, the polyploid MDA-MB-231 cells were more sensitive to etoposide than the original tumor cells. The expression of F-box and WD repeat domain containing 7 (FBW7) was decreased, while the expression of MCL1 apoptosis regulator BCL2 family member (MCL-1) and Bcl-2 was increased, and caspase-3/9 and Bax were not expressed in MDA-MB-231 cells. The resistance to docetaxel and etoposide was reversed, but the sensitivity of paclitaxel was not changed following Bcl-2 silencing. The formation of polyploidy in tumors may be one of the molecular mechanisms underlying tumor resistance to spindle poisons. Expression of the Bcl-2 family members, for example FBW7 and MCL-1, plays a key role in apoptosis and the cell escape process that forms polyploid cells. However, Bcl-2 silencing has different reversal effects on different anti-tumor drugs, which requires further investigation.

mTOR is a serine/threonine kinase which acts a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in mitotic arrested cells across a variety of cancer and normal cell lines. Prevention of raptor phosphorylation during mitosis resulted in reactivation of mTORC1 in a rapamycin-sensitive manner. Importantly, expression of a non-phosphorylatable raptor mutant caused a dramatic reduction in cytotoxicity of the spindle poison Taxol. This effect was mediated via degradation of Programmed Cell Death Protein 4 (PDCD4), a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A was able to enhance the effects of taxol and restore sensitivity in Taxol resistant cancer cells. These findings indicate that the mTORC1/S6K/PDCD4/eIF4A axis has a pivotal role in death vs. slippage decision during prolonged mitotic arrest and may be exploited to gain a clinical benefit in treating cancers resistant to anti-mitotic drugs.

Disrupting microtubule dynamics with spindle poisons activates the spindle-assembly checkpoint (SAC) and induces mitotic cell death. However, mitotic exit can occur prematurely without proper chromosomal segregation or cytokinesis by a process termed mitotic slippage. It remains controversial whether mitotic slippage increases the cytotoxicity of spindle poisons or the converse. Altering the SAC induces either mitotic cell death or mitotic slippage. While knockout of MAD2-binding protein p31comet strengthened the SAC and promoted mitotic cell death, knockout of TRIP13 had the opposite effect of triggering mitotic slippage. We demonstrated that mitotic slippage prevented mitotic cell death caused by spindle poisons, but reduced subsequent long-term survival. Weakening of the SAC also reduced cell survival in response to spindle perturbation insufficient for triggering mitotic slippage, of which mitotic exit was characterized by displaced chromosomes during metaphase. In either mitotic slippage or mitotic exit with missegregated chromosomes, cell death occurred only after one cell cycle following mitotic exit and increased progressively during subsequent cell cycles. Consistent with these results, transient inhibition of the SAC using an MPS1 inhibitor acted synergistically with spindle perturbation in inducing chromosome missegregation and cytotoxicity. The specific temporal patterns of cell death after mitotic exit with weakened SAC may reconcile the contradictory results from many previous studies.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that targets substrates for degradation to promote mitotic progression. Here, we show that the DNA damage response protein 53BP1 contains conserved KEN boxes that are required for APC/C-dependent degradation in early mitosis. Mutation of the 53BP1 KEN boxes stabilized the protein and extended mitotic duration, whereas 53BP1 knockdown resulted in a shorter and delayed mitosis. Loss of 53BP1 increased APC/C activity, and we show that 53BP1 is a direct APC/C inhibitor. Although 53BP1 function is not absolutely required for normal cell cycle progression, knockdown was highly toxic in combination with mitotic spindle poisons. Moreover, chemical inhibition of the APC/C was able to rescue the lethality of 53BP1 loss. Our findings reveal a reciprocal regulation between 53BP1 and APC/C that is required for response to mitotic stress and may contribute to the tumor-suppressor functions of 53BP1.

Bystander effect is a known radiobiological effect, widely described using ionizing radiations and which, more recently, has also been related to chemical mutagens. In this study, we aimed to assess whether or not a bystander response can be induced in cultured human peripheral lymphocytes by vincristine, a chemotherapeutic mutagen acting as spindle poison, and by mitomycin-C, an alkylating agent already known to induce this response in human lymphoblastoid cells. Designing a modified ad hoc protocol for the cytokinesis blocked micronucleus (MN) assay, we detected the presence of a dose-dependent bystander response in untreated cultures receiving the conditioned medium (CM) from mitomycin-C (MMC) or vincristine (VCR) treated cultures. In the case of MMC, MN frequencies, expressed as micronucleated binucleates, were: 13.5±1.41 at 6μM, 22±2.12 at 12μM or 28.25±5.13 at 15μM vs. a control value of 4.75±1.59. MN levels for VCR, expressed as micronucleated mononucleates were: 2.75±0.88 at 0.0μM, 27.25±2.30 at 0.4μM, 46.25±1.94 at 0.8μM, 98.25±7.25 at 1.6μM. To verify that no mutagen residual was transferred to recipient cultures together with the CM, we evaluated MN levels in cultures receiving the medium immediately after three washings following the chemical treatment (unconditioned medium). We further confirmed these results using a cell-mixing approach where untreated lymphocytes were co-cultured with donor cells treated with an effect-inducing dose of MMC or VCR. A distinct production pattern of both reactive oxygen species and soluble mediator proteins by treated cells may account for the differences observed in the manifestation of the bystander effect induced by VCR. In fact, we observed an increased level of ROS, IL-32 and TGF-β in the CM from VCR treated cultures, not present in MMC treated cultures.

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches.

Iron oxide nanoparticles are one of the most promising types of nanoparticles for biomedical applications, primarily in the context of nanomedicine-based diagnostics and therapy; hence, great attention should be paid to their bio-safety. Here, we investigate the ability of surface-modified magnetite nanoparticles (MNPs) to produce chromosome damage in human alveolar A549 cells. Compared to control cells, all the applied MNPs increased the level of micronuclei moderately but did not cause structural chromosomal aberrations in exposed cells. A rise in endoreplication, polyploid and multinuclear cells along with disruption of tubulin filaments, downregulation of Aurora protein kinases and p53 protein activation indicated the capacity of these MNPs to impair the chromosomal passenger complex and/or centrosome maturation. We suppose that surface-modified MNPs may act as aneugen-like spindle poisons via interference with tubulin polymerization. Further studies on experimental animals revealing mechanisms of therapeutic-aimed MNPs are required to confirm their suitability as potential anti-cancer drugs.

The BH3-only Bcl-2 subfamily member Bim is a well known apoptosis promoting protein. However, the mechanisms upstream of mitochondrion membrane permeability by which Bim is involved in apoptosis have been poorly investigated, particularly in response to agents capable of interfering with the cytoskeleton architecture and arresting cells in mitosis. Based on the observation that Bim is sequestered on the microtubule-array by interaction with the light chain of dynein, we have investigated upon depolymerisation, whether Bim could be involved in the commitment of apoptosis. With this purpose H460 Non Small Lung Cancer Cells (NSLC) were treated with the microtubule damaging agent combretastatin-A4 (CA-4) (7.5 nM; 8-48 h), and various parameters were investigated. Upon treatment, cells arrested in mitosis and died through a caspase-3-dependent mitotic catastrophe. Transient knock down of Bim drastically reduced apoptosis, indicating that this protein was involved in cell death as induced by microtubules disorganisation. In response to increasing conditions of microtubules depolymerisation, we found that the protein level of Bim was strongly upregulated in a time-dependent manner at transcriptional level. Furthermore, Bim was released from microtubule-associated components. Bim was translocated to mitochondria, even in a condition of protein synthesis inhibition, where it showed a markedly increased interaction with Bcl-2. In turn, the fraction of Bax bound to Bcl-2 decreases in response to treatment, thereby indicating that Bim possibly promotes Bax release from the pro-survival protein Bcl-2. Overall, we demonstrated that Bim is required for the CA-4-induced cell death in the H460 lung cancer cell line via activation of the mitochondrial signalling pathway. Defining the contribution of Bim to the mechanism of apoptosis may offer some different clues in view of developing new strategies for chemotherapy with CA-4, underlining the relevance of the cytoskeleton integrity in the apoptotic response.

The high frequency of false or irrelevant positive results in in vitro mammalian cell genotoxicity tests is a critical concern for regulators. Here, we tested whether such results may be due to the mammalian cells used in the tests being deficient in p53, which is involved in the maintenance of genomic stability. We compared the in vitro responses of two human lymphoblastoid cell lines derived from the same progenitor cell-p53-competent (TK6) and p53-deficient (WTK-1) cells-in a micronucleus (MN) test and a thymidine kinase gene (TK) mutation assay. We tested 14 chemicals including three mutagens and 11 clastogens and spindle poisons. The three mutagens evoked clear positive responses in both assays in both cell lines. The responses to the clastogens and spindle poisons, on the other hand, depended on the assay endpoint and/or the cell line. Most of clastogens and spindle poisons were positive in the MN test in both cell lines. In the TK mutation assay, on the other hand, WTK-1 cells but not TK6 cells detected spindle poisons, which may have been due to the disturbance of the spindle checkpoint and lack of apoptosis in the p53-deficient cells. Some chemicals that induced chromosome aberrations in rodent cells were negative in both TK6 and WTK-1 cells, indicating that a species-specific factor rather than p53 status was associated with the response. In conclusion, the p53 status did not seriously influence the MN test results but it did influence the TK mutation assay results.

The cyclin E–cyclin-dependent kinase 2 (CDK2) complex accelerates entry into the S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. The effect of low molecular weight isoforms of cyclin E (LMW-E) overexpression on mitotic progression and its link to genomic instability were the focus of this study. Here, we show that full-length cyclin E (EL) and LMW-E overexpression impairs the G2-M transition differently by targeting dual-specificity phosphatase Cdc25C activity. We identify Cdc25C as an interaction partner and substrate for cyclin E/CDK2 kinase. Specifically, the cyclin E/CDK2 complex phosphorylates Cdc25C on Ser214, leading to its premature activation, which coincides with higher cyclin B/CDK1 and Polo-like kinase 1 (PLK1) activities in an S-phase–enriched population that result in faster mitotic entry. Whereas EL overexpression leads to hyperactivation of Cdc25C, cyclin B/CDK1, and PLK1 in a G2-M–enriched population, LMW-E overexpression causes premature inactivation of Cdc25C and PLK1, leading to faster mitotic exit. In addition, LMW-E–overexpressing cells showed a reduction in the mitotic index in the presence of a spindle poison and faster degradation of cyclin B, suggesting an increased rate of mitotic slippage and adaptation to the spindle checkpoint. Lastly, downregulation of Cdc25C inhibits LMW-E–mediated chromosome missegregation, anaphase bridges, and centrosome amplification. These results suggest that the high levels of LMW-E isoforms found in breast cancer may contribute to cellular transformation and genomic instability by impairing mitotic progression involving Cdc25C. Cancer Res; 70(12); 5085–95. ©2010 AACR.

When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37°C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.

Spindle poisons elicit various cellular responses following metaphase arrest, but how they relate to long-term clonogenicity has remained unclear. We prepared several HeLa lines in which the canonical apoptosis pathway was attenuated, and compared their acute responses to paclitaxel, as well as long-term fate, with the parental line. Three-nanomolar paclitaxel induced brief metaphase arrest (S-trityl-L-cysteine (STLC), at 1 µM led to much longer arrest (∼20 h) and predominance of subsequent line-specific responses: mitochondrial outer membrane permeabilization (MOMP) in the apoptosis-prone line, or mitotic slippage without obvious MOMP in the apoptosis-reluctant lines. In spite of this, combination with STLC did not lead to a marked difference in clonogenicity between the apoptosis-prone and -reluctant lines, and intriguingly resulted in slightly better clonogenicity than that of cells treated with 3 nM paclitaxel alone. This indicates that changes in the short-term response within 3 possible scenarios — acute MOMP, mitotic slippage or aberrant mitosis ― has only a weak impact on clonogenicity. Our results suggest that once cells have committed to slippage or aberrant mitosis they eventually undergo proliferative death irrespective of canonical apoptosis or p53 function. Consistent with this, cells with irregular DNA contents originating from mitotic slippage or aberrant mitosis were mostly eliminated from the population within several rounds of division after the drug treatment.

The implementation of the REACH regulation has imposed the urgent need of developing alternative testing methods to screen large number of compounds more quickly and at lower costs. In this study, a battery of tests, suitable for reproductive toxicology testing, was developed with the objective of detecting the mechanism of action of estrogenic and xenoestrogenic compounds. With this aim, two compounds known for their estrogenic activity, diethylstilbestrol and 17β-estradiol, were used to set up four different in vitro tests: 1) bovine oocyte in vitro maturation assay, 2) bovine preimplantation embryo in vitro culture assay and 3) MCF-7 and 4) BALB/3T3 cell lines proliferation and cytotoxicity assay, respectively. The results show that this battery of tests allows to identify and to distinguish between two major mechanisms of action of (xeno)estrogenic compounds: the receptor-mediated mechanism and the spindle-poison effect on microtubules polimerization.

The applicability and standard methodology of the rodent bone marrow chromosome aberration test, as used in regulatory toxicology submissions, are described in detail. The purpose of the test is to determine whether the test material causes chromosome breakage in actively dividing cells in the bone marrow. The test is sensitive to DNA-damaging agents (or their active metabolites) that are systemically distributed at adequate levels following exposure. It is used as part of a primary screen when high levels of human exposure are anticipated and as a follow-up test to determine the relevance of in vitro findings, such as in the event of apparent chromosomal effects in an in vitro chromosome aberration or in vitro micronucleus test. The principles of the test and the techniques involved are closely related to those described previously in this volume for the in vitro chromosome aberration and in vivo micronucleus tests. Usually, main group animals are euthanized and samples are taken 12–18 h after treatment; samples from a second group of animals are taken 24 h later in case the test item causes cell-cycle delay or is only slowly absorbed and/or metabolized. A spindle poison is administered to the animals 2–5 h prior to euthanasia to arrest and accumulate cells in metaphase. The bone marrow cells are extracted, swollen by hypotonic treatment, and fixed, and metaphase spreads are made on microscope slides. The spreads are stained and 200 metaphases per animal are analyzed under the code for chromosomal aberrations using high-resolution light microscopy. At the same time, the mitotic index is assessed as a measure of cytotoxic or cytostatic effects.

Regulators of the mitotic spindle apparatus are attractive cellular targets for antitumor therapy. The centrosomal protein transforming acidic coiled coil (TACC) 3 is required for spindle assembly and proper chromosome segregation. In this study, we employed an inducible RNA interference approach to downregulate TACC3 expression. We show that TACC3 knock-down in NIH3T3 fibroblasts caused aneuploidy, but failed to overtly impair mitotic progression. TACC3 depletion rather triggered a postmitotic p53-p21(WAF) pathway and led to a reversible cell cycle arrest. Similar effects were induced by low concentrations of paclitaxel, a spindle poison used in antitumor therapy. Interestingly, however, and unlike in TACC3-proficient cells, paclitaxel was able to induce strong polyploidy and subsequent apoptosis in TACC3-depleted cells. Even though paclitaxel treatment was associated with the activation of the survival kinase Akt and an antiapoptotic expression of cytoplasmic p21(WAF) and cyclin D1, this inhibition of cell death was abrogated by depletion of TACC3. Thus, our data identify TACC3 as a potential target to overcome p21(WAF)-associated protection of transformed cells against paclitaxel-induced cell death.

Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31comet. A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers., Cohesion is associated with many forms of cancer. De Lange et al. show that such cohesion defects can sensitise cells to apoptosis in response to a new APC/C ubiquitin ligase inhibitor, by prolonging mitotic arrest and checkpoint activation due to cohesion fatigue.

Chlorinated organic chemicals are widely used in industry and are present in the environment. Five chlorinated aliphatic hydrocarbons, namely 1-2-dichloroethane, 1,1,2-trichloroethane, trichloroethylene, 2,3-dichlorobutane and 1-chlorohexane were investigated to determine their influence upon the fidelity of cell division in cultured mammalian cells. In order to determine the influence of these chemical compounds upon the fidelity of cell division, a technique known as differential staining of chromosomes and spindle was performed with one genetically engineered cell line and its parental cell line. The genetically engineered cell line used in this study expressed a human P450 enzyme, CYP2E1. Four chemicals, 1-2-dichloroethane, trichloroethylene, 2,3-dichlorobutane and 1-chlorohexane required metabolic bioactivations in order to induce spindle damage in cultured mammalian cells whereas 1,1,2-trichloroethane was a direct-acting spindle poison.

In mammalian cells, DNA replication proceeds according to a precise temporal order during the S phase, but how this program is controlled remains poorly understood. We analyzed the replication-dependent bromodeoxyuridine banding of chromosomes in Chinese hamster cells treated with the spindle poison nocodazole. In these cells, nocodazole induces a transient mitotic arrest, followed by DNA re-replication without intervening cell division. Nuclear fragmentation is often observed in tetraploid derivatives, and previous studies suggest that replication timing of chromosomes could be affected when they are segregated into different micronuclei. Here we show that the onset of replication is frequently asynchronous on individual chromosomes during the re-replication process. Moreover, fluorescence in situ hybridization analysis revealed that replication synchrony is equally altered in fragmented and non-fragmented nuclei, indicating that asynchronous onset of replication is not dependent on physical separation of the chromosomes into isolated compartments. We also show that the ordered program of replication is always preserved along individual chromosomes. Our results demonstrate that the onset of replication of individual chromosomes in the same nuclear compartment can be uncoupled from the time of S-phase entry and from the programmed replication of chromosome sub-domains, revealing that multi-level controls contribute to establish replication timing in mammalian cells.

The fission yeast cps6-153 mutant was originally isolated based on its hypersensitivity to the spindle poison isopropyl N-3-chlorophenyl carbamate (CIPC). The mutant also shows defects in both cell wall integrity and cytokinesis, resulting in the accumulation of unseparated cells with weakened cell walls. The arrested cells display a disoriented alignment of cytoplasmic microtubules. When the mutant cells are cultivated at high temperature (35 degrees C), both cell walls and septa become very thick. Electron microscopy revealed the disorganized structure of the thickened cell walls and septa, in which fibrillar components were not completely masked with an amorphous matrix. rad25+ was cloned from a genomic library by complementation of the mutant phenotypes, suggesting the involvement of Rad25p, one of two 14-3-3 proteins in S. pombe, in the pathway of cell wall integrity and cytokinesis.

Dimethylarsinic acid (DMA) is the most effective inducer of cell-cycle disruption among the arsenic compounds and their metabolites. The present study was conducted to gain further insight into cell-cycle disruption induced by DMA. The inhibition of cell proliferation and the mitotic arrest induced by DMA were significant and dose-dependent in Chinese hamster V79 cells and the two seemed to be closely related. At less than 140 µM the DMA did not inhibit the proliferation of cells, but it significantly induced mitotic arrest. An indirect immunofluorescence assay using anti-α-tubulin antibodies revealed that DMA induced the formation of abnormal spindles in the metaphase cells even at 350 µM with 5 h of treatment. At 1.4 mM DMA no metaphase cells could form a definite spindle structure. The spindle figures were similar to those induced by colchicine (125 nM) or vinblastine (110 nM), major antimitotic agents. In DMA-treated interphase cells, the microtubule networks were indistinguishable from those of normal cells. With the tubulin-assembly assay estimated by turbidity, DMA at less than 200 µM suppressed tubulin assembly in a dose-dependent manner, whereas at more than 700 µM it enhanced tubulin polymerization remarkably with or without addition of excess guanosine-5′-triphosphate. According to the above findings, we discussed the possibility that DMA, a primary metabolite of inorganic arsenic in mammals, is related to arsenic carcinogenicity. Copyright © 2001 John Wiley & Sons, Ltd.

In order to assess the effects of trichlorfon on cell division and on aneuploidy induction, we conducted an in vitro assay for spindle disturbances using V79 cells and an in vivo assay for aneuploidy induction in meiosis of male mice using multicolour fluorescence in situ hybridization (FISH) with epididymal sperm. In the in vitro assay, the chemical caused a concentration-dependent increase in the incidence of initial and full c-mitoses in the dose range 40-120 microg/ml trichlorfon. The mitotic index (MI) was decreased between 40 and 100 microg/ml trichlorfon, whereas at 120 microg/ml the MI was back to the control level, coinciding with the dramatic increase in c-mitoses. The results confirm that trichlorfon is a potent spindle poison in V79 cells. In the in vivo multicolour FISH assay, administration of trichlorfon to male mice at single doses of 200, 300 and 405 mg/kg caused a dose-dependent increase of the frequencies of disomic sperm (0.068, 0.074 and 0.134%, respectively) compared with the corresponding controls (0.046, 0.042 and 0.056%, respectively). The prevalence of X-X-8 and Y-Y-8 sperm suggests that trichlorfon affected chromosome segregation predominantly during the second meiotic division. Diploid sperm were not induced by trichlorfon treatment, indicating that no meiotic block occurred. It is concluded that trichlorfon is a potent spindle poison in V79 cells and induces aneuploidy in mouse spermatocytes during meiosis.

The polychlorinated biphenyls 2,2',5,5'- and 3,3',4, 4'-tetrachlorobiphenyl, 2,3,3',4,4'- and 3,3',4,4', 5-pentachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl were tested for spindle-disturbing activity in V79 Chinese hamster cells. Clones lacking endogenous cytochrome P450 activity or expressing rat CYP1A1 or CYP2B1 were used. Induction of abnormal chromosomal arrangements in mitosis were found to be favoured by o-chlorine substitutions, but not by co-planarity giving affinity, for example, for the Ah receptor and CYP1A isoenzymes. Only 2,2',5, 5'-tetrachloro- and 2,3,3',4,4'-pentachlorobiphenyl gave dose-response curves similar to many other compounds tested in vitro, showing an increase from the background level of 10 to 100% disturbed mitoses with nominal concentrations >10(-6) M, i.e. concentrations far above the total PCB concentrations found in human blood. Cells transfected with rat CYP2B1 were more sensitive to the most active congener, 2,3,3',4,4'-pentachlorobiphenyl, than cells lacking P450 activity or expressing CYP1A1. Induction of abnormal mitosis by PCB metabolites formed by P450 enzymes cannot be excluded, but does not seem likely because of the short treatment time and the reportedly slow metabolism of PCBs. 2,3,3',4, 4'-Pentachlorobiphenyl showed synergistic activity with the potent spindle poison triphenyltin. Inactive concentrations of both agents (10 and 50 nM, respectively) caused abnormal configurations when combined. This is an important finding since exposure to mixtures of compounds is common and it motivates further studies of subthreshold activities of highly lipophilic environmental contaminants.

The proportions of aneuploid/polyploid versus euploid cells formed after treatment with spindle poisons like nocodazole are of course dependent on the relative survival of cells with numerical chromosome aberrations. This work aimed at studying the survival of polyploid cells formed after treatment with a nocodazole concentration sufficient to significantly decrease tubulin polymerization (0.1 microg/ml). First, normal primary lymphocytes were analysed and the following complementary chromosomal parameters were quantified: mitotic index, frequency of abnormal mitoses, polyploid metaphases and apoptotic cells. The results clearly indicate a positive correlation between abnormal mitotic figures, apoptosis and the induction of polyploidy. They therefore led to a single cell approach in which both apoptosis and polyploidy induction could be scored in the same cell. For this purpose, actively proliferating cells are required and two human leukaemic cell lines were used, KS (p53-positive) and K562 (p53-negative), which have a near-triploid karyotype. Cells were separated into an apoptotic and a viable fraction by means of annexin-V staining and flow cytometry. In KS, treatment with nocodazole induced a similar fraction of hexaploid cells in both the viable and apoptotic fraction, but no dodecaploid cells were ever observed. In contrast, a population of dodecaploid cells (essentially viable) was clearly observed in the K562 cell line. The results in KS, as compared with K562, confirm that wild-type p53 can prevent further cycling of polyploid cells by blocking rereplication. The most probable explanation for these data is that not only the mitotic spindle but also interphase microtubules are sensitive to nocodazole treatment. Our data thus strongly suggest that besides the G(1)/S checkpoint under the control of p53, the G(2)/M transition may be sensitive to depolymerization of microtubules, possibly under the control of Cdc2, Bcl-2, Raf-1 and/or Rho.

The marine environment receives a wide variety of chemical inputs, many of which have the potential to damage DNA or interfere with the process of cell division. Here we describe a new assay based on the early embryo and larval stages of a planktonic spawning, tube dwelling marine worm, Pomatoceros lamarckii, which for experimental purposes has the advantage of producing large numbers of ripe gametes throughout the majority of the year. One of the most promising end-points is the use of dividing cells to detect anaphase aberrations such as lagging chromosomes, tripolar anaphases, acentric fragments and chromosome bridges. Apart from the reference mutagens mitomycin C and cyclophosphamide and the well-documented spindle poison colchicine, we tested the fungicide carbendazim, a primary metabolite of the fungicide benomyl, and thiabendazole, a pesticide and antihelminthic drug; both of which are known to act as aneugens in other test systems. In addition we tested sodium hypochlorite, a widely used oxidizing agent and disinfectant, di-butylphthalate, a commercial plasticizer and suspected aneugen, and sodium chloride, a recognized non-genotoxin. Significant increases in the frequency of anaphase abnormalities occurred with most test compounds at relatively low concentrations, confirming the sensitivity of the new assay. Sodium chloride yielded a negative response except at the highest non-relevant concentrations, where some chromatid stickiness was observed. In addition, the developmental consequences of exposure to these compounds were assessed in 4-8 cell embryos and at 48 h once the embryos had metamorphosed into free swimming larvae. Mitotic inhibition and anaphase aberrations were found to be a more sensitive indicator of genotoxic exposure than larval development, although there was a suggestion of a possible mechanistic link between aneugenicity/clastogenicity and larval fitness. The new test assay provides a rapid and inexpensive method for screening chemicals and effluents destined for release into the marine environment for potential gamete effects.

We found that different ploidy effects were induced in four Chinese hamster-derived cell lines (V79-MZ, V79, CHL and CHO-K1) treated through two cell cycles with polycyclic aromatic hydrocarbons in the absence of a metabolic activation system. 5-Bromodeoxyuridine was used to investigate cell cycle delay and sister chromatid exchanges (SCE) induced by the chemicals. Benzo[a]pyrene (BP) induced aneuploidy at 2.5-10 micrograms/ml in V79-MZ cells. 7,12-Dimethylbenz[a]anthracene (DMBA) induced polyploidy at 3.125-6.25 and 6.25-1.25 micrograms/ml in V79-MZ and V79 cells respectively. Higher concentrations caused cell cycle delay and, therefore, did not affect ploidy. BP and DMBA did not induce a significant increase in SCE frequency at the above doses. 3-Methylcholanthrene tested up to its solubility limit (10 micrograms/ml) did not induce numerical aberrations in any cell line. The clastogen mitomycin C, tested up to 0.01 microgram/ml, did not produce numerical aberrations but did significantly increase SCE frequency in all cell lines. The spindle poison colchicine, tested up to 0.1 microgram/ml, induced ploidy changes in the four cell lines that showed different sensitivities. Four cell lines showed no arylhydrocarbon hydroxylase activity, and V79-MZ, but not the other cells lines, showed high glutathione S-transferase activity. Aneuploidy induction by BP and polyploidy induction by DMBA in the absence of S9 mix in vitro have not been described before, and the finding might be due to the effect on tubulin. Due to their specificity and high sensitivity, the V79-MZ and V79 cell lines might be good systems for detecting aneuploidogens.

Spindle poison-based therapy is of only limited benefit in acute myeloid leukemia while lymphoblastic leukemia/lymphoma responds well. In this study, we demonstrated that the spindle assembly checkpoint protein BubR1 was down-regulated in the vast majority of cases of acute myeloid leukemia whereas its expression was high in lymphoblastic cells. Correct function of the spindle assembly checkpoint is pivotal in mediating mitotic delay in response to spindle poisons. Mitotic delay by the spindle assembly checkpoint is achieved by inhibition of anaphase-promoting complex-dependent proteolysis of cyclin B and securin. We demonstrated a link between the repression of the spindle assembly checkpoint protein BubR1 in acute myeloid leukemia and the limited response to spindle poison. In accordance with its established role as an anaphase-promoting complex-inhibitor, we found that repression of BubR1 was associated with enhanced anaphase-promoting complex activity and cyclin B and securin degradation, which leads to premature sister-chromatid separation and failure to sustain a mitotic arrest. This suggests that repression of BubR1 in acute myeloid leukemia renders the spindle assembly checkpoint-mediated inhibition of the anaphase-promoting complex insufficient, which facilitates completion of mitosis in the presence of spindle poison. As both direct and BubR1-mediated restoration of cyclin B expression enhanced response to spindle poison, we propose that the downstream axis of the spindle assembly checkpoint is a promising target for tailored therapies for acute myeloid leukemia.

The application of methods based on in situ hybridization to centromeric regions to cytokinesis-blocked cells provides a convenient way for the analysis of chromosome segregation in interphase cells. In this way, the reciprocal segregation patterns in daughter nuclei can be visualized and most of the problems related to the artefactual loss or gain of chromosomes which flaw other methods are avoided. In this work, the methodology has been applied to human lymphocytes to investigate the influence of donor age on spontaneous malsegregation rates, the occurrence of multiple malsegregation events, and the effect of the cytokinesis-blocking agent cytochalasin B (Cyt B) on spontaneous and induced chromosome malsegregation. The results obtained with 14 male donors, aged 22-57 years, demonstrated a significant (p < 0.001) increase in the frequency of micronuclei and X chromosome missegregation (both non-disjunction and chromosome loss) with the increasing age of the donors. Moreover, a similar association was observed with cultures hybridized with either chromosome 8 or 18 centromere probes, suggesting that the age-related loss of fidelity in chromosome segregation in vitro may be a general trait. The investigation of the distribution of multiple malsegregation events in cultured lymphocytes of eight male and nine female donors, with the simultaneous hybridization with pairs of centromeric probes (for chromosomes X and 8 or X and 18), demonstrated a large excess of multiple events with respect to that expected by random segregation. This fact may highlight the existence of cellular subpopulation(s) prone to malsegregate, or indicate that the malsegregation of one chromosome is able to affect the fidelity of segregation of the other chromosomes. Finally, the possible influence of Cyt B on chemically induced malsegregation has been investigated with the analysis of chromosomes X and 8 signals in nuclei of lymphocyte cultures treated with vinblastine (2.5-20 ng/ml) in the presence and absence of 6 micrograms/ml Cyt B. Vinblastine induced a small increase in hyperploidy of either chromosome X or 8 at 10 ng/ml in cultures treated with Cyt B. Without Cyt B, a significant increase of hyperploidy was only observed at the highest dose assayed (20 ng/ml). This vinblastine dosage had a severe inhibitory effect on cultures treated with Cyt B, where no binucleated cells were detected. At all doses, a relatively greater mitotic index was observed in cultures with Cyt B, suggesting a synergistic effect of this drug with vinblastine. Most notably, at the two highest vinblastine dosages (10 and 20 ng/ml), a large incidence of polyploid nuclei was observed in cytokinesis-blocked cultures, whereas none or far lower increases of polyploidy were found in the absence or Cyt. B. This results provides direct evidence of the potential of Cyt B to indirectly interfere with chromosome misdistribution induced by a spindle poison, to be considered before drawing firm conclusions from kinesis-blocked systems.

Micronuclei (MN) induced by N-methyl-N-nitrosourea (MNU) or vinblastine in cultured mammalian cells were analyzed for the accumulation of p53 by immunocytochemical staining with a p53 monoclonal antibody. Our data showed that MN induced by both agents were p53-negative at early post-treatment times, but became positive at late times. Assuming that most MNU-induced micronuclei reflect DNA damage, and most vinblastine-induced micronuclei reflect damage to the mitotic apparatus, we conclude that p53 accumulation in micronuclei is not triggered by DNA damage per se but instead probably stems from DNA degradation occurring during ageing of micronuclei.

The detection of aneugenic chemicals is important due to the implications of aneuploidy for human health. Aneuploidy can result from chromosome loss or nondisjunction due to chromosome mis-segregation at anaphase. Frequently, aneugens are detected using the in vitro micronucleus assay (IVM), with either centromere or kinetochore labeling. However, this method does not consider nondisjunction, the suggested predominant mechanism of spindle poison induced aneugenicity in primary human lymphocytes. Therefore, the IVM may be relatively insensitive in detecting aneuploidy. To investigate whether chromosome distribution analysis, specifically of nondisjunction, using chromosome-specific centromeric probes provides a more sensitive assay for aneugen detection, six reference aneugens with differing modes of action were tested on human lymphoblastoid TK6 cells. The results show that chromosome loss is a substantial part of the process leading to aneuploidy in TK6 cells. This differs from previous studies on human lymphocytes where nondisjunction has been described as the major mechanism of aneugenicity. However, in the current study more cells and types of aneugenic damage were analyzed. Although compound specific effects on nondisjunction were identified, chromosome distribution analysis did not provide increased sensitivity for the detection of aneugens: For the six reference aneugens examined, chromosome loss was shown at the same concentrations or lower than nondisjunction, even when nondisjunction levels were comparatively high. Therefore, in TK6 cells methods that detect chromosome loss, eg, the IVM, provide a more sensitive technique for the detection of aneugens than the measurement of nondisjunction.

Summary The cell has many mechanisms for protecting the integrity of its genome. These mechanisms are often weakened or absent in many cancers, leading to high rates of chromosomal instability in tumors. Control of the cell cycle is crucial for the function of these checkpoints, and is frequently lost in cancers as well. Overexpression of Cyclin D1 in a large number of breast cancers causes overactivation of the cyclin-dependent kinases, including Cdk2. Constitutive Cdk2 activation through Cyclin D1 generates tumors in mice that are aneuploid and have many characteristics indicative of chromosomal instability. Expression of these complexes in the MCF10A cell line leads to retinoblastoma protein (Rb) hyperphosphorylation, a subsequent increase in proliferation rate, and increased expression of the spindle assembly checkpoint protein Mad2. This results in a strengthening of the spindle assembly checkpoint and renders cells more sensitive to the spindle poison paclitaxel. Constitutive Rb phosphorylation also causes a weakening of the p53-dependent tetraploidy checkpoint. Cells with overactive Cdk2 fail to arrest after mitotic slippage in the presence of paclitaxel or cytokinesis failure during treatment with cytochalasin-B, generating 8N populations. This additional increase in DNA content appears to further intensify the tetraploidy checkpoint in a step-wise manner. These polyploid cells are not viable long-term, either failing to undergo division or creating daughter cells that are unable to undergo subsequent division. This study raises intriguing questions about the treatment of tumors with overactive Cdk2.

Introduction: Cdc20 is a co-activator recruited by the anaphase-promoting complex (APC), a large, multi-subunit complex that functions as an Ubiqutin ligase that targets key substrates for degradation to promote cell cycle progression. During mitosis the APC/Cdc20 complex is inhibited by the spindle assembly checkpoint (SAC) pathway, which ensures the correct chromosome segregation during mitosis. When associated with the mitotic checkpoint complex (MCC), formed by Mad2, BubR1 and Bub3 proteins, Cdc20 cannot activate the APC and cell cycle is arrested. Deregulation of this pathway can result in cells proceeding through mitosis before proper chromosomal attachment to the kinetochore, potentially leading to aneuploidy and cancer. We have found 2 different missense variants in CDC20 (L151R and N331K), in the affected members of two families with malignant ovarian germ cell tumors, a rare non-epithelial neoplastic transformation of ovarian primitive oocyte. Interestingly, several members of L151R family presented with other additional tumors such as colorectal or breast cancers. Purpose: We aim to elucidate the mechanism by which L151R and N331K are able to trigger tumorigenesis, and demonstrate that the disruption of Cdc20 may play an important role in cancer. Methods: The effect of the mutations has been measured through three different cell types: S. cerevisiae carrying the Cdc20N407K (N331K in humans); HeLa cells transfected with Cdc20L151R and Cdc20N331K; and human primary skin fibroblasts from two siblings carrying N331K mutation. Cell cycle progression has been measured in the presence of the spindle poison nocodazole by several methods: checkpoint cell survival assay, western blot of MCC and APC proteins, flow cytometer and time-lapse. The effect of the mutations on the interaction of Cdc20 and its MCC and APC partners has been assessed by immunoprecipitation. Results: S. cerevisiae carrying the Cdc20N407K (N331K in humans) show a clear reduction in viability of the mutant compared to wild-type (wt) after the addition of nocodazole, as a result of mutant cells keep dividing despite microtubule-kinetochore attachment being destabilized, thus causing genomic instability and catastrophe in progeny cells. HeLa cells transfected with Cdc20L151R and Cdc20N331K show a significant increase in the non-phosphorylated forms of Cdc27 and Bubr1 compared to the wt and the empty vector, indicating the abundance of non-mitotic cells increases with the mutated forms of Cdc20. The increased number of polyploidy cells (8n) in the mutants seen by flow cytometry proves that the non-mitotic cells are a result of the mutated Cdc20 overriding mitosis even in the presence of nocodazole. Additionally, time-lapse results fit with a reduced time spent in mitosis by the HeLa cells carrying the mutated forms of Cdc20. Immunoprecipitation of the transfected Cdc20 showed a reduced binding of the mutated proteins to Bubr1, suggesting that the mutated forms of Cdc20 could be over-activating the APC thanks to a reduced affinity with its MCC partner Bubr1. Time-lapse experiments performed in human skin fibroblasts from two patients carrying the N331K mutation in heterozygosity showed similar results in terms of the length of the mitotic arrest in the presence of nocodazole. Last but not least, we are expecting to be able to see the translation of these in vitro functional results at the tumoral level after the WES and cytogenetic analysis of the patient's tumors is finalized. Conclusions: Our preliminary data suggest that L151R and N331K may drive cancer through the insensitivity of Cdc20 to the SAC. To our knowledge this is the first description of CDC20 as a cancer predisposition gene. The rapid division of cancer cells demands that they maintain high APC activity and this may represent an Achilles heel that can be exploited therapeutically. This abstract is also being presented as Poster B25. Citation Format: Ester Castellsague, Jian Carrot-Zhang, Isabelle Gamache, Barbara Rivera, Mohamed Moustafa, David Barford, Jacek Majewski, Jose Teodoro, William David Foulkes. Germ-line mutations in CDC20 result in familial cancers via deregulation of the cell cycle. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR13.

Differential staining of spindle and chromatin was adapted to meiotic cells of male mice. Immunostaining with antitubulin antibodies was developed. Both methods were used to test acrylamide, diazepam and vinblastine for their potential to cause spindle disturbances in male mouse germ cells. The analyses were performed after in vivo treatment, comparing the classical safranin/brilliant blue spindle staining to an immunofluorescent assay. The two staining methods complemented each other. Differential staining seems to be more sensitive to the detection of misplaced chromatin whereas the immunostaining is superior when scoring for spindle structure aberrations. All three chemicals showed spindle activity. Acrylamide mainly caused multipolar spindles which possibly develop from a separation of mother and daughter centrioles during an unspecific meiotic block. Diazepam elevated the level of monopolar spindles and induced the loss of single chromosomes, suggesting an effect on motor proteins. Vinblastine was the strongest spindle poison by far. It produced high numbers of shortened and monopolar spindles as well as multiple chromosome loss.

The search for micronuclei (MN) in binucleated cells is not always the best choice to recognize microtubule-perturbing agents, as they give rise to (micronucleated) mononucleated cells, mainly via mitotic slippage. We therefore treated peripheral lymphocytes with vincristine (VCR), nocodazole (NOC) and colcemid (COL): (i) to quantify the formation of MN in mononucleated cells and the occurrence of abnormal mitoses (c-anaphases, endoreduplicated or tetraploid metaphases); (ii) to investigate the role of cytokinesis inhibition in determining or modulating the cytogenetic effects induced by the spindle poisons (we used either cytochalasin B (cyt B) or latrunculin A, a cytokinesis inhibitor that acts differently as compared with cyt B); (iii) to assess the ploidy of cells bearing MN by fluorescence in situ hybridisation (FISH) analysis; and (iv) to evaluate the levels of the mitotic arrest deficient (MAD2) protein, that blocks the cell at the metaphase-anaphase transition, by immunoblotting. We observed the induction of numerous abnormal mitoses and tetraploid interphase nuclei, as well as of MN in mononucleated cells, a high percentage of which had a diploid complement. We also found that the effects were generally not dose but chemical dependent, where NOC was proven to be more effective than COL and VCR in inducing overall MN formation and, specifically, diploid micronucleated lymphocytes. Aneugens damaged cells to a greater extent in the presence of cytokinesis inhibitors rather than in their absence. MAD2 protein was expressed in controls to an extent reflecting the amount of lymphocytes which were initially in the G2/M transition phase. The same trend was seen in aneugen-treated cells where MAD2 levels decreased with increasing spindle poison concentration. Here, we demonstrate that micronucleated mononucleated cells and aberrant mitoses can be considered useful markers of exposure to aneugens-like spindle poisons causing preferentially, but not exclusively, mitotic slippage. Assessment of MAD2 levels can be used to confirm the cell-damaging activity of the compounds.