Spindle Defects Research Articles

Podophyllotoxin Exposure Causes Spindle Defects and DNA Damage-Induced Apoptosis in Mouse Fertilized Oocytes and Early Embryos.

Podophyllotoxin (PPT) is a kind of lignans extracted from the roots and stems of the genus Podophyllum from the tiller family, and it has been widely used in the treatment of condyloma acuminatum, multiple superficial epithelioma in the clinics. However, PPT has been reported to be toxic and can cause liver defects and other organ poisoning. In addition, emerging evidences also indicate that PPT has reproductive toxicity and causes female reproduction disorders. In this study, we used fertilized oocytes and tried to explore the effects of PPT on the early embryonic development with the mouse model. The results showed that exposure to PPT had negative effects on the cleavage of zygotes. Further analysis indicated that PPT could disrupt the organization of spindle and chromosome arrangement at the metaphase of first cleavage. We also found that PPT exposure to the zygotes induced excessive reactive oxygen species (ROS), suggesting the occurrence of oxidative stress. Moreover, in the PPT-exposed embryos, there was positive γH2A.X and Annexin-V signals, indicating that PPT induced embryonic DNA damage and early apoptosis. In conclusion, our results suggested that PPT could affect spindle formation and chromosome alignment during the first cleavage of mouse embryos, and its exposure induced DNA damage-mediated oxidative stress which eventually led to embryonic apoptosis, indicating the toxic effects of PPT on the early embryo development.

Mitotic Spindle Apparatus Abnormalities in Chronic Obstructive Pulmonary Disease Cells: A Potential Pathway to Lung Cancer.

Chronic obstructive pulmonary disease (COPD) is a long-term lung disease characterized by irreversible lung damage resulting in airflow limitation, abnormal permanent air-space enlargement, and emphysema. Cigarette smoking is the major cause of COPD with 15% to 30% of smokers developing either disease. About 50% to 80% of patients with lung cancer have preexisting COPD and smokers who have COPD are at an increased risk for developing lung cancer. Therefore, COPD is considered an independent risk for lung cancer, even after adjusting for smoking. A crucial early event in carcinogenesis is the induction of the genomic instability through alterations in the mitotic spindle apparatus. To date, the underlying mechanism by which COPD contributes to lung cancer risk is unclear. We hypothesized that tobacco smoke carcinogens induce mitotic spindle apparatus abnormalities and alter expression of crucial genes leading to increased genomic instability and ultimately tumorigenesis. To test our hypothesis, we assessed the genotoxic effects of a potent tobacco-smoke carcinogen [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)] on bronchial epithelial cells from patients with COPD and normal bronchial epithelial cells and identified genes associated with mitotic spindle defects and chromosome missegregation that also overlap with lung cancer. Our results indicate that exposure to NNK leads to a significantly altered spindle orientation, centrosome amplification, and chromosome misalignment in COPD cells as compared with normal epithelial cells. In addition, we identified several genes (such as AURKA, AURKB, and MAD2L2) that were upregulated and overlap with lung cancer suggesting a potential common pathway in the transition from COPD to lung cancer.

Wwc2 Is a Novel Cell Division Regulator During Preimplantation Mouse Embryo Lineage Formation and Oogenesis.

Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.

Orientation of the Mitotic Spindle in Blood Vessel Development.

Angiogenesis requires coordinated endothelial cell specification, proliferation, and collective migration. The orientation of endothelial cell division is tightly regulated during the earliest stages of blood vessel formation in response to morphogenetic cues and the controlled orientation of the mitotic spindle. Consequently, oriented cell division is a vital mechanism in vessel morphogenesis, and defective spindle orientation can perturb the spatial arrangement of daughter cells and consequently contribute to several diseases related to vascular development. Many factors affect endothelial cell proliferation and orientation and therefore blood vessel formation, with the relationship between improper spindle orientation in endothelial cells and various diseases extensively studied. Here we review the molecular mechanisms driving the orientation of endothelial cell division, particularly with respect to the mitotic spindle, and how these processes affect vascular development, disease pathogenesis, and their potential as novel targets.

NUP62 is required for the maintenance of the spindle assembly checkpoint and chromosomal stability.

The nuclear pore protein NUP62 localizes to spindle poles in mitosis and plays a role in maintaining centrosome homeostasis. In this study, we found that NUP62-depleted cells exhibited a defective spindle assembly checkpoint (SAC) and that depletion of NUP62 caused a slight decrease in MAD2 protein levels after nocodazole treatment. However, depletion of NUP62 did not cause a failure in kinetochore localization of the SAC proteins BUBR1, MAD1, and MAD2 in prometaphase. NUP62 depletion slightly prolonged mitotic timing but did not affect cell doubling time. In addition, NUP62 depletion caused a SAC defect and induced aneuploidy in human neural stem cells. Furthermore, overexpression of NUP62Q391P, a mutant protein causing autosomal recessive infantile bilateral striatal necrosis, resulted in a defect in the SAC, indicating that the amino acid residue Q391 in NUP62 is crucial for its effect on the SAC. Overall, we conclude that NUP62 maintains the SAC downstream of kinetochores and thereby ensures maintenance of chromosomal stability.

Abstract 5817: Anti-tropomyosin drugs prevent the rescue of vincristine-induced mitotic spindle defects

Abstract Drugs targeting a major component of the actin filaments of cancer cells, tropomyosin Tpm3.1, synergize with anti-microtubule drugs in neuroblastoma and lung cancer models both in vitro and in vivo and a wide range of other cancer types in vitro. We have determined the mechanism of synergy in HeLa cells to gain insight into the potential interaction of actin filaments and microtubules in the survival and proliferation of cancer cells. HeLa cells exhibit a strong synergistic response to the combined treatment of vincristine (VCR) and anti-Tpm3.1 compounds, marked by an enhanced reduction in cell viability, apoptosis induction and mitotic cell cycle arrest. Tpm3.1 localizes to the cell cortex during mitosis, potentially associating with the microtubule network, particularly the dynein/dynactin complexes responsible for mediating cortical pulling forces during spindle assembly. VCR alone causes supernumerary NuMA organized acentrosomal microtubule organizing centers upon nuclear envelope breakdown, which can be resolved via a clustering mechanism to achieve bipolar cell division. The addition of anti-Tpm3.1 compounds inhibits NuMA-associated clustering in VCR-treated cells, leading to irreparable defects during spindle assembly and thus a largely increased number of cells with multi-polar spindles undergoing mitotic delay and catastrophe. We conclude that actin/Tpm3.1 filaments contribute to the formation of the bipolar spindle and play a critical role in the clustering of acentrosomal microtubule asters. Citation Format: Peter Gunning, Yao Wang, Jeff H. Stear, Ashleigh Swain, Xing Xu, Nicole Bryce, Irina B. Alieva, Vera B. Dugina, Timothy Cripe, Justine Stehn, Edna C. Hardeman. Anti-tropomyosin drugs prevent the rescue of vincristine-induced mitotic spindle defects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5817.

Nonylphenol exposure affects mouse oocyte quality by inducing spindle defects and mitochondria dysfunction

Nonylphenol (NP) is a chemical raw material and intermediate which is mainly used in the production of surfactants, lubricating oil additives and pesticide emulsifiers. NP is reported to be toxic on the immune system, nervous system and reproductive system due to its binding to estrogen receptors. However, the toxicity of NP on mammalian oocyte quality remains unclear. In present study, we explored the effects of NP exposure on mouse oocyte maturation. Our results showed that 4 weeks of NP exposure increased the number of atresia follicles and decreased oocyte developmental competence. Transcriptomic analysis indicated that NP exposure altered the expression of more than 800 genes in oocytes, including multiple biological pathways. Subcellular structure examination indicated that NP exposure disrupted meiotic spindle organization and caused chromosome misalignment. Moreover, aberrant mitochondrial distribution and decreased membrane potential were also observed, indicating that NP exposure caused mitochondria dysfunction. Further analysis showed that NP exposure resulted in the accumulation of reactive oxygen species (ROS), which causes oxidative stress; and the NP-exposed oocytes showed positive Annexin-V signal, indicating the occurrence of early apoptosis. In summary, our results indicated that NP exposure reduced oocyte quality by affecting cytoskeletal dynamics and mitochondrial function, which further induced oxidative stress and apoptosis in mice.

Drug Targeting the Actin Cytoskeleton Potentiates the Cytotoxicity of Low Dose Vincristine by Abrogating Actin-Mediated Repair of Spindle Defects.

Antimicrotubule vinca alkaloids are widely used in the clinic but their toxicity is often dose limiting. Strategies that enhance their effectiveness at lower doses are needed. We show that combining vinca alkaloids with compounds that target a specific population of actin filaments containing the cancer-associated tropomyosin Tpm3.1 result in synergy against a broad range of tumor cell types. We discovered that low concentrations of vincristine alone induce supernumerary microtubule asters that form transient multi-polar spindles in early mitosis. Over time these asters can be reconstructed into functional bipolar spindles resulting in cell division and survival. These microtubule asters are organized by the nuclear mitotic apparatus protein (NuMA)-dynein-dynactin complex without involvement of centrosomes. However, anti-Tpm3.1 compounds at nontoxic concentrations inhibit this rescue mechanism resulting in delayed onset of anaphase, formation of multi-polar spindles, and apoptosis during mitosis. These findings indicate that drug targeting actin filaments containing Tpm3.1 potentiates the anticancer activity of low-dose vincristine treatment. IMPLICATIONS: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.

Oral administration of olaquindox negatively affects oocytes quality and reproductive ability in female mice.

As an effective feed additive in the livestock industry, olaquindox (OLA) has been widely used in domestic animal production. However, it is unclear whether OLA has negative effects on mammalian oocyte quality and fetal development. In this study, toxic effects of OLA were tested by intragastric gavage ICR mice with water, low-dose OLA (5mg/kg/day), or high-dose OLA (60mg/kg/day) for continuous 45 days. Results showed that high-dose OLA gavage severely affected the offspring birth and growth. Significantly, high-dose OLA impaired oocyte maturation and early embryo development, indicated by the decreased percentage of germinal vesicle breakdown, first polar body extrusion and blastocyst formation. Meanwhile, oxidative stress levels were increased in oocytes or ovaries, indexed by the increased levels of ROS, MDA, H2O2, NO, and decreased levels of GSH, SOD, CAT, GSH-Px and GSH-Rd. Furthermore, aberrant mitochondria distribution, defective spindle assembly, abnormal H3K4me2/H3K9me3 levels, increased DNA double-strand breaks and early apoptosis rate, were observed after high-dose OLA gavage. Taken together, our results for the first time illustrated that high-dose OLA gavage led to sub-fertility of females, which means that restricted utilization of OLA as feed additive should be considered.

CENP-W regulates kinetochore-microtubule attachment and meiotic progression of mouse oocytes

Oocyte meiotic maturation failure and unfaithful chromosome segregation are major causes for female infertility. Here, we showed that CENP-W, a relatively novel member of the kinetochore protein family, was expressed in mouse oocytes from the germinal vesicle (GV) to metaphase II (MII) stages. Confocal microscopy revealed that CENP-W was localized in the germinal vesicle in the GV stage, and then became concentrated on kinetochores during oocyte maturation. Knockdown of CENP-W by specific siRNA injection in vitro caused kinetochore-microtubule detachment, resulting in severely defective spindles and misaligned chromosomes, leading to metaphase I arrest and failure of first polar body (PB1) extrusion. Correspondingly, spindle assembly checkpoint (SAC) activation was observed in CENP-W knockdown oocytes even after 10h of culture. Our results suggest that CENP-W acts as a kinetochore protein, which takes part in kinetochore-microtubule attachment, thus mediating the progression of oocyte meiotic maturation.

The FSH-HIF-1α-VEGF Pathway Is Critical for Ovulation and Oocyte Health but Not Necessary for Follicular Growth in Mice.

Follicle-stimulating hormone (FSH)-induced growth of ovarian follicles is independent of follicular vascularization. Recent evidence has indicated that follicular vascularization is critical to ovarian follicle development and survival. FSH, a gonadotropin that induces follicular growth and development, also acts as the major survival factor for antral follicles. FSH has been reported to stimulate angiogenesis in the theca layers mediated in part by the vascular endothelial growth factor A (VEGFA) and the transcription factor hypoxia inducible factor 1α (HIF-1α). However, it remains largely undetermined whether FSH-dependent growth and survival of antral follicles relies on FSH-induced vascularization. Here, we first demonstrated that induction of angiogenesis through the FSH-HIF-1α-VEGFA axis is not required for FSH-stimulated follicular growth in mouse ovary. FSH increased the total number of blood vessels in mouse ovarian follicles, which was correlated with elevated expression of VEGFA and HIF-1α in granulosa cells. In contrast, blocking of follicular angiogenesis using inhibitors against the HIF-1α-VEGFA pathway repressed vasculature formation in follicles despite FSH administration. Interestingly, by measuring follicular size and ovarian weight, we found that the suppression of angiogenesis via HIF-1α-VEGFA pathway did not influence FSH-mediated follicular growth. However, inhibition of FSH-induced follicular vascularization by PX-478, a small-molecule inhibitor that suppresses HIF-1α activity, blocked ovulation and triggered atresia in large follicles. On the other hand, PX-478 injection reduced oocyte quality via impairing the meiotic apparatus, showing a prominently defective spindle assembly and actin dynamics. Collectively, our findings unveiled a vascularization-independent effect of FSH on follicular growth, whereas follicular survival, ovulation, and oocyte development relies on FSH-mediated angiogenesis in the follicles.

Design, synthesis, and evaluation of N-phenyl-4-(2-phenylsulfonamido)-benzamides as microtubule-targeting agents in drug-resistant cancer cells, displaying HDAC inhibitory response

Microtubule-targeting agents (MTA) have enjoyed significant clinical success for decades. However, several mechanisms may cause inactivation of such drugs, leading to acquired resistance in patients treated with them. Therefore, drugs containing a stilbene-like skeleton and possessing dual inhibitory activity may provide a new and differentiated treatment for patients to overcome challenging acquired resistance. A new compound (16c) displays promising anticancer activity with GI50 of 22 ± 2 and 12 ± 0.1 nM in vincristine-resistant nasopharyngeal (KB-Vin) cancer cells and etoposide-resistant nasopharyngeal (KB-7D) cancer cells and is better than vincristine, etoposide, ABT-751, and MS-275. A mechanistic study revealed that 16c interferes with the cell cycle distribution and induces cell cycle arrest at the G2/M phase and severe mitotic spindle defects followed by apoptosis. In addition, it produces much more significant cytotoxicity than vincristine and etoposide in the corresponding resistant cells, indicating that it may be a promising candidate to overcome drug resistance in cancer cells. Compound 16c also displays inhibitory activity against HDAC 1 and HDAC 2 with IC50 values of 1.07 μM, and 1.47 μM, respectively. These findings may lead to a new type of structural motif for future development of drugs that could overcome acquired resistance to MTAs.

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes.

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.

Loss of NKX6.3 During Gastric Carcinogenesis Enhances Genomic Instability and Promote Tumor Development

Background: The maintenance of genomic stability requires correct DNA replication, chromosome segregation, and DNA repair, while defects of these processes result in tumor development or cell death. Here, we investigated whether NKX6.3 plays a role in maintaining the genomic stability of gastric epithelial cells. Methods: qRT-PCR, immunofluorescent, fluorescence in situ hybridization (FISH) and immunoblot were utilized to analyze the copy number alterations (CNAs) and expression levels of CDKN3, DLGAP5, AurkA, TPX2, SPARCL1, and SORBS2 in NKX6.3 depleted cells and gastric cancer tissues. Fluorescence-activated cell sorting was used for cell cycle and DNA repair analysis. DNA fiber assay, BrdU, and EdU assays were performed to analyze DNA replication stress. DNA fragmentation and Comet assays were used for DNA damage analysis. Chromosomal aberrations were analyzed by metaphase spread and mitotic index assays. Xenograft experiments were performed to determine CNAs and expression levels of genes caused by NKX6.3 depletion in vivo. Whole-genome sequencing was performed to analyze CNAs caused by NKX6.3 depletion. Findings: NKX6.3 depleted HFE-145 immortalized gastric epithelial cells induced a rapidly cell cycle with increased S and G2/M phase cell populations, by altering the expression of cell cycle related proteins. During cell cycle, NKX6.3 depletion induced faster fork progression, fork symmetry, longer gap length, mitotic spindle defects, and chromosome mis-segregation. In DNA repair, depletion of NKX6.3 led to an induction of homologous recombination repression. When we investigated whether NKX6.3 was involved in CNAs of gastric epithelial cells, frequently gained or amplified, and lost or deleted broad chromosomal regions were detected in NKX6.3-depleted cells. Hallmark enrichment analyses of NKX6.3-depleted cells revealed gain of CDKN3, DLGAP5, AurkA, and TPX2, and deletion of SORBS2 and SPARCL1. Moreover, NKX6.3-depletion caused mitotic spindle defects and chromosome mis-segregation in vitro and in vivo through the CNA of genes involved in mitotic spindle formation. In 65 human gastric cancers and xenograft mice models for NKX6.3-depleted HFE-145 cells, expression levels of CDKN3, DLGAP5, AurkA, and TPX2 were markedly increased while expression levels of SPARCL1 and SORBS2 were decreased. In NKX6.3-depleted cells, silencing of CDKN3 and DLGAP5 as well as ectopic expression of SPARCL1 and SORBS2 inhibited cell proliferation and colony formation. Interpretation: These results demonstrate that a novel molecular mechanism for gastric carcinogenesis through the induction of genomic instability by abnormal regulation of DNA replication and repair due to NKX6.3-depletion. Thus, we conclude that NKX6.3 may DNA replication and repair regulation for protecting gastric mucosal epithelial cells from genomic instability such as CNAs. Funding Statement: This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017R1D1A1A09000781 to J.H.Y.; 2018R1A2A2A14019713 to W.S.P.). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: Written informed consent was obtained from all subjects. The study was approved by the Institutional Review Board of The Catholic University of Korea, College of Medicine (MC15SISI0015). Animal experiments were performed in mice maintained under pathogen-free conditions based on approval by the Animal Experiment Ethics Committee of the Catholic University of Korea College of Medicine (CUMC-2017-0018-01).

The centriole protein CEP76 negatively regulates PLK1 activity in the cytoplasm for proper mitotic progression.

Polo-like kinase 1 (PLK1) dynamically changes its localization and plays important roles in proper mitotic progression. In particular, strict control of cytoplasmic PLK1 is needed to prevent mitotic defects. However, the regulation of cytoplasmic PLK1 is not fully understood. In this study, we show that CEP76, a centriolar protein, physically interacts with PLK1 and tightly controls the activation of cytoplasmic PLK1 during mitosis in human cells. We found that removal of centrosomes induced ectopic aggregation of PLK1, which is highly phosphorylated, in the cytoplasm during mitosis. Importantly, a targeted RNAi screen revealed that depletion of CEP76 resulted in a similar phenotype. In addition, depletion of CEP76 caused defective spindle orientation and mitotic delay. Moreover, the formation of ectopic PLK1 aggregates and defective spindle orientation were significantly suppressed by the inhibition of PLK1 kinase activity. Overall, these results demonstrate that CEP76 suppresses the aberrant activation of cytoplasmic PLK1 for proper mitotic progression.This article has an associated First Person interview with the first author of the paper.

Subcellular drug targeting illuminates local kinase action.

Deciphering how signaling enzymes operate within discrete microenvironments is fundamental to understanding biological processes. A-kinase anchoring proteins (AKAPs) restrict the range of action of protein kinases within intracellular compartments. We exploited the AKAP targeting concept to create genetically encoded platforms that restrain kinase inhibitor drugs at distinct subcellular locations. Local Kinase Inhibition (LoKI) allows us to ascribe organelle-specific functions to broad specificity kinases. Using chemical genetics, super resolution microscopy, and live-cell imaging we discover that centrosomal delivery of Polo-like kinase 1 (Plk1) and Aurora A (AurA) inhibitors attenuates kinase activity, produces spindle defects, and prolongs mitosis. Targeted inhibition of Plk1 in zebrafish embryos illustrates how centrosomal Plk1 underlies mitotic spindle assembly. Inhibition of kinetochore-associated pools of AurA blocks phosphorylation of microtubule-kinetochore components. This versatile precision pharmacology tool enhances investigation of local kinase biology.

Loss of Ikbkap/Elp1 in mouse oocytes causes spindle disorganization, developmental defects in preimplantation embryos and impaired female fertility

Elongator complexes are well known to be involved in a wide variety of cellular processes; however, their functions in mammalian oocytes have not been characterized. Here, we demonstrated in mice that specific deletion of one of the core subunits, Ikbkap/Elp1, in oocytes resulted in spindle defects and chromosome disorganization without affecting folliculogenesis. In accordance with these findings, we observed that Ikbkap mutant female mice are subfertile. Further analyses uncovered that kinetochore–microtubule attachments are severely compromised in Ikbkap-deficient oocytes. Moreover, we revealed that Ikbkap modulates the acetylation status of α-tubulin in oocytes, which may at least in part mediate the meiotic phenotypes described above by affecting microtubule dynamics and kinetochore function. Finally, we showed that embryos derived from Ikbkap-deficient oocytes exhibit an increased frequency of aneuploidy, digyny, progressive delays in preimplantation development, and severe degeneration before reaching the blastocyst stage. In summary, we identify Ikbkap as an important player in regulating oocyte meiosis by modulating tubulin acetylation for chromosome/spindle organization.

Investigation of mechanisms of toxicity and exclusion by transporters of the preservatives triclosan and propylparaben using batteries of Schizosaccharomyces pombe strains

Triclosan (TCS) and propylparaben (PPB) are antimicrobials widely used. They present many similarities in their applications and also in their human and environmental health risks. In order to investigate the mechanisms of toxic action and the efflux pumps involved in their detoxication, we used a strategy with batteries of Schizosaccharomyces pombe yeast strains, either defective in cell signalling, in detoxification pumps, or in cell surveillance mechanisms. Yeast were exposed up to 20 h in solid medium or in liquid medium in 96-well plates. The mechanisms of action investigated were spindle defects (mph1), stress (pmk1), DNA interference (rad3) or diverse effects (MDR-sup). The efflux pumps investigated were Bfr1, Pmd1, Mfs1 and Caf5 or the Pap1 transcription factor. Here we show that TCS was 75 times more toxic than PPB in the wild type fission yeast. More oxidative stress and less protection by exclusion pumps were observed for TCS than for PPB. The cytotoxicity produced by TCS decreased from bfr1>mfs1>pmd1 > pap1 and caf5A deficient strains. In contrast, cytotoxic concentrations of PPB caused only a mild stress. The protection provided for PPB by the transporters was more marked than for TCS, decreasing from Pmd1, Caf5, Mfs1 and Bfr1. Furthermore, microtubule and DNA interferences were revealed for PPB, according to the cytotoxicity of mph1 and rad3 defective cells, respectively. As both compounds present complex adverse effects at concentrations close to exposure, and their combination clearly causes a strong potentiation, more exhaustive controls and regulations in their use should be considered.

Downregulation of lumican enhanced mitotic defects and aneuploidy in lung cancer cells

ABSTRACT Lumican is overexpressed in lung cancer cells and has been implicated in the pathogenesis of tumorigenesis and regulation of cancer cell invasion. Lumican is robustly associated with the binding of p120-catenin protein to modulate cell metastasis. However, its role in cancer cell proliferation is still unclear. This study investigated the effect of lumican on the cell division including mitosis and cytokinesis in non-small lung cancer cells. We found that the downregulation of lumican prolonged the doubling time of cells and retarded the cell growth in H460 and A549 cells. Along with tubulin, lumican localized to the mitotic spindle and centrosome during the metaphase-anaphase stage. The cell cycle was retained in the G2/M phase after the downregulation of lumican. Interestingly, lumican was found to play important roles in central spindle and midbody formation during cytokinesis. Lumican interacted with the midbody-associated proteins such as MKLP1, Aurora B, and ECT2. Notably, the downregulation of lumican decreased the level of MKLP1 accompanied by the retention of midbody-residual that resulted in multi-nucleated cells. Downregulation of lumican promoted the chromosome missegregation and the increment of the bi-/multinucleated cells. The results of this study indicated that lumican associated with tubulin is crucial for spindle fiber formation and midbody assembly in cell division. Downregulation of lumican displayed the defects in mitotic spindle assembly/dynamics and improper kinetochore-microtubules attachment that led to increase aneuploidy. This emerging property of lumican is suggested to tightly control chromosome segregation during cell division in lung cancer cells. Abbreviations: ESCRT: endosomal sorting complex required for transport; PRC1: protein regulator of cytokinesis 1; Nci: negative control siRNA; Lumi: lumican siRNAs; MKLP1: mitotic kinesin-like protein 1; H460LD and A549LD: H460 and A549 cell lines with less expressed lumican

Prolonged ovarian storage of mature Drosophila oocytes dramatically increases meiotic spindle instability.

Human oocytes frequently generate aneuploid embryos that subsequently miscarry. In contrast, Drosophila oocytes from outbred laboratory stocks develop fully regardless of maternal age. Since mature Drosophila oocytes are not extensively stored in the ovary under laboratory conditions like they are in the wild, we developed a system to investigate how storage affects oocyte quality. The developmental capacity of stored mature Drosophila oocytes decays in a precise manner over 14 days at 25°C. These oocytes are transcriptionally inactive and persist using ongoing translation of stored mRNAs. Ribosome profiling revealed a progressive 2.3-fold decline in average translational efficiency during storage that correlates with oocyte functional decay. Although normal bipolar meiotic spindles predominate during the first week, oocytes stored for longer periods increasingly show tripolar, monopolar and other spindle defects, and give rise to embryos that fail to develop due to aneuploidy. Thus, meiotic chromosome segregation in mature Drosophila oocytes is uniquely sensitive to prolonged storage. Our work suggests the chromosome instability of human embryos could be mitigated by reducing the period of time mature human oocytes are stored in the ovary prior to ovulation.