Multipolar Divisions Research Articles

Dimethyl Sulfoxide Attenuates Ionizing Radiation-induced Centrosome Overduplication and Multipolar Cell Division in Human Induced Pluripotent Stem Cells.

Centrosomes are important organelles for cell division and genome stability. Ionizing radiation exposure efficiently induces centrosome overduplication via the disconnection of the cell and centrosome duplication cycles. Over duplicated centrosomes cause mitotic catastrophe or chromosome aberrations, leading to cell death or tumorigenesis. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into all organs. To maintain pluripotency, PSCs show specific cellular dynamics, such as a short G1 phase and silenced cell-cycle checkpoints for high cellular proliferation. However, how exogenous DNA damage affects cell cycle-dependent centrosome number regulation in PSCs remains unknown. This study used human iPSCs (hiPSCs) derived from primary skin fibroblasts as a PSC model to address this question. hiPSCs derived from somatic cells could be a useful tool for addressing the radiation response in cell lineage differentiation. After radiation exposure, the hiPSCs showed a higher frequency of centrosome overduplication and multipolar cell division than the differentiated cells. To suppress the indirect effect of radiation exposure, we used the radical scavenger dimethyl sulfoxide (DMSO). Combined treatment with radiation and DMSO efficiently suppressed DNA damage and centrosome overduplication in hiPSCs. Our results will contribute to the understanding of the dynamics of stem cells and the assessment of the risk of genome instability for regenerative medicine.

Abstract 3347: KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3347.

The oncogene cyclin D1 promotes bipolar spindle integrity under compressive force.

The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, possibly contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.

Single Cell Whole Genome Measurement of Genomic Heterogeneity and Clonal Evolution in Multiple Myeloma

Background: Sequencing studies have advanced our understanding of the genomic underpinnings of Multiple Myeloma (MM). While whole genome sequencing (WGS) has emerged as a comprehensive approach for profiling the genomic landscape of MM, WGS has limited sensitivity in defining the intrapatient genomic heterogeneity. Single cell RNA approaches can capture phenotypic heterogeneity but fail to recover most of the key genomic drivers. To overcome these historical limitations, we utilized a newer single cell whole genome direct library preparation (scWGS DLP+; Lacks et al. Cell 2019) sequencing technology able to detect early changes and study clonal population dynamics, providing a more precise understanding of the emergence and clinical impact of genomic events during MM evolution. Methods: scWGS DLP+ was applied to CD138 + Plasma Cells (PC) isolated from bone marrow samples of MM patients. Using newly developed computational approaches, we characterized the Single Nucleotide Variants (SNVs), copy number changes and complex Structural Variants (SVs) at a single cell level and use this information to reconstruct the evolutionary histories of each patient's myeloma. Results: We applied DLP to 7 MM patients obtaining 800-1600 single cell whole genomes per patient. All canonical IgH translocations identified by FISH were recovered in the scWGS data. Single cell copy number heterogeneity was observed in all but 1 patient, including heterogeneity affecting known drivers. The number of subclones ranged from 1 to 15, with an average of 6 subclones detected per patient (≥10 cells, ≥5MB distinct segmental difference). This is in striking contrast with bulk WGS where we are usually able to define no more than one or two subclones. Gain of 1q (3 copies) was observed in all patients but was either rare (1-3%, 3 patients) or clonal (>99%, 4 patients), with two clonal 1q patients exhibiting convergent subclonal 1q amplification (>3 copies). Loss of previously gained chromosomes was observed including loss of ancestrally gained chromosome 15 in hypodiploid Patient 2, and sporadic loss of 1q in patients with ubiquitous 1q gain. For 3 of 7 patients, hypodiploidy (HRD) had arisen before the most recent clonal expansion. HRD clones continued to diversify, accruing multiple additional events on HRD chromosomes including large or focal gains, or deletions subsequent to a gain. Interestingly, one case had 1 small hypodiploid clone co-existing with a completely different major clone with 1q and t(14;20) IgH translocation, suggesting the existence of two distinct clonal plasma cell populations (one malignant and one likely bening). Two of the 7 patients harbored both small (<20 cells) whole genome doubled (WGD) subclones, and small numbers of highly aneuploid likely produced by multi-polar cell division post WGD. We explored two patient's evolutionary histories in detail, highlighting the unique resolution of scWGS DLP+ to detect CNV but also SNV, mutational signatures and SVs. Patient 3 exhibited 4 distinct subclones with distant evolutionary origin as evidenced by the relatively small number of shared SNV. APOBEC signature mutations were detected in all 4 subclones suggesting APOBEC mutagenesis was still ongoing across the cell populations in this patient. In Patient 1, a complex SV on chromosome 17 resulted in loss of most of 17q and an amplification of 17q segments. The heterogeneous structure of chromosome 17 across clones suggests a late onset of this complex event and ongoing instability. Chromosome 16 exhibited a pattern of cell specific arm loss likely resulting from telomere loss. Interestingly, a minor clone showed complex amplification of 16p affecting TNFRSF17, suggesting that chromosome 16 is at an earlier stage of a process of instability that starts with cell specific arm loss, and ends with clonal expansion of a complex amplification SV targeting a gene with fitness benefit. Discussion: Applied to Multiple Myeloma, scWGS shows higher clonal heterogeneity than previously described with multiple clones exhibiting diverse genetic alterations. The findings underscore the genomic complexity and dynamic nature of MM, highlighting the need for sensitive techniques like scWGS to capture the full spectrum and chronological order of genomic events.

Diverse microtubule-targeted anticancer agents kill cells by inducing chromosome missegregation on multipolar spindles.

Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration: clinicaltrials.gov, NCT03393741.

Non-canonical functions of a mutant TSC2 protein in mitotic division.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates the mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms of mutations affecting these functions are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with the inhibitor rapamycin. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC.

The fate of extra centrosomes in newly formed tetraploid cells: should I stay, or should I go?

An increase in centrosome number is commonly observed in cancer cells, but the role centrosome amplification plays along with how and when it occurs during cancer development is unclear. One mechanism for generating cancer cells with extra centrosomes is whole genome doubling (WGD), an event that occurs in over 30% of human cancers and is associated with poor survival. Newly formed tetraploid cells can acquire extra centrosomes during WGD, and a generally accepted model proposes that centrosome amplification in tetraploid cells promotes cancer progression by generating aneuploidy and chromosomal instability. Recent findings, however, indicate that newly formed tetraploid cells in vitro lose their extra centrosomes to prevent multipolar cell divisions. Rather than persistent centrosome amplification, this evidence raises the possibility that it may be advantageous for tetraploid cells to initially restore centrosome number homeostasis and for a fraction of the population to reacquire additional centrosomes in the later stages of cancer evolution. In this review, we explore the different evolutionary paths available to newly formed tetraploid cells, their effects on centrosome and chromosome number distribution in daughter cells, and their probabilities of long-term survival. We then discuss the mechanisms that may alter centrosome and chromosome numbers in tetraploid cells and their relevance to cancer progression following WGD.

P-252 A roadmap to normal human embryo development – a timelapse study of embryos resulting in the live birth

Abstract Study question What are the morphokinetic parameters of preimplantation development in normal human embryos? Summary answer We provide a timeline of early developmental events and the incidence of embryo morphological features compatible with childbirth. What is known already Timelapse embryo monitoring provides unique insight into early human development. Implementation of this technique in clinical practice allows for assessing developmental dynamics for improved embryo selection. Several markers and ranking algorithms have been proposed to predict blastulation or implantation outcomes, but only a few studies assessed time-lapse data with live birth as the primary endpoint. Therefore, the comprehensive morphokinetic profile of embryos that gave rise to live birth has not been defined. Study design, size, duration Retrospective observational analysis of 300 time-lapse records of IVF embryos known to develop into healthy newborns. Out of total 73 quantitative/qualitative signs annotated, we focused on 9 developmental events and 14 morphological features and evaluated their timing, duration, and incidence. The influence of potential confounding factors (i.e., egg age, donor egg, donor sperm, sperm quality, and sex of the child) was evaluated by advanced statistical methods. Participants/materials, setting, methods Embryo monitoring was performed by a GERI-timelapse incubator. Image acquisition of individual embryo development lasted 5-6 days, starting 10 minutes after ICSI, which was used as reference time point. Transmitted light images were automatically captured in 5-minute intervals and 7-11 focal planes. The embryo selection for transfer was based solely on its morphology. The annotation data represent the consensus of minimum 3 experienced embryologists. Generalized linear models were used to explore relationships between dataset variables. Main results and the role of chance The average age of female participants was 27.5 years; 71.67% of embryos were derived from young donor eggs without fertility disorders. The severe andrological factor was present in 8% of male patients; donor sperm was used in 23% of cases; 16% of embryos were derived from dysmorphic oocytes. Both sexes were equally represented among analyzed embryos. Our analysis showed that early development was characterized by remarkably short first mitosis (2.68 ± 0.73h) and the interval between the second and third division (t4-t3 = 0.65 ± 0.66h), whereas the third cleavage was less synchronous (t8-t5 = 5.46 ± 4.9h) No association between quantitative signs and confounding factors was found. Interestingly, 2-cell stage embryos often contained multiple nuclei in at least one blastomere (51%), but multinucleation was reduced at the 4-cell stage (8%). Multipolar divisions were absent during zygotic division and rare (2.3%) during the second cleavage. Preterm and partial compaction was observed in 26.6% and 34% of embryos, respectively. Importantly, we noticed that 11.6% of embryos in our dataset deviated from conventional scenario of cleavage dynamics. These “atypical” embryos were characterized by a significantly higher incidence of multipolar divisions during the second cleavage compared to “typical,” regularly dividing embryos. Additionally, the odds of partial compaction increase 10 times in “atypical” embryos. Limitations, reasons for caution The presented analysis only involved embryos confirmed to result in a live birth after transfer. The comparison with data from embryos that did not produce pregnancy was not performed because it would be impossible to discriminate between the role of the embryo and endometrial factors in implantation failure. Wider implications of the findings Human embryos may develop into healthy children despite irregular cell division. A high incidence of partial compaction in “atypical” embryos suggests a self-correction mechanism rescuing their developmental potential. A larger-scale analysis of features compatible with live birth is needed to improve embryo selection criteria for evidence-based decision-making. Trial registration number not applicable

Confinement plus myosin-II suppression maximizes heritable loss of chromosomes, as revealed by live-cell ChReporters.

The mechanical environment of a cell can have many effects, but whether it impacts the DNA sequence of a cell has remained unexamined. To investigate this, we developed a live-cell method to measure changes in chromosome numbers. We edited constitutive genes with GFP or RFP tags on single alleles and discovered that cells that lose Chromosome reporters (ChReporters) become non-fluorescent. We applied our new tools to confined mitosis and to inhibition of the putative tumor suppressor myosin-II. We quantified compression of mitotic chromatin in vivo and demonstrated that similar compression in vitro resulted in cell death, but also rare and heritable ChReptorter loss. Myosin-II suppression rescued lethal multipolar divisions and maximized ChReporter loss during three-dimensional (3D) compression and two-dimensional (2D) lateral confinement, but not in standard 2D culture. ChReporter loss was associated with chromosome mis-segregation, rather than just the number of divisions, and loss in vitro and in mice was selected against in subsequent 2D cultures. Inhibition of the spindle assembly checkpoint (SAC) caused ChReporter loss in 2D culture, as expected, but not during 3D compression, suggesting a SAC perturbation. Thus, ChReporters enable diverse studies of viable genetic changes, and show that confinement and myosin-II affect DNA sequence and mechano-evolution.

Cortical dynein drives centrosome clustering in cells with centrosome amplification.

During cell division, the microtubule nucleating and organizing organelle, known as the centrosome, is a critical component of the mitotic spindle. In cells with two centrosomes, each centrosome functions as an anchor point for microtubules, leading to the formation of a bipolar spindle and progression through a bipolar cell division. When extra centrosomes are present, multipolar spindles form and the parent cell may divide into more than two daughter cells. Cells that are born from multipolar divisions are not viable, and hence clustering of extra centrosomes and progression to a bipolar division are critical determinants of viability in cells with extra centrosomes. We combine experimental approaches with computational modeling to define a role for cortical dynein in centrosome clustering. We show that centrosome clustering fails and multipolar spindles dominate when cortical dynein distribution or activity is experimentally perturbed. Our simulations further reveal that centrosome clustering is sensitive to the distribution of dynein on the cortex. Together, these results indicate that dynein's cortical localization alone is insufficient for effective centrosome clustering and, instead, dynamic relocalization of dynein from one side of the cell to the other throughout mitosis promotes timely clustering and bipolar cell division in cells with extra centrosomes.

Abstract 1556: CDKN3 inhibition enhances the antitumor effect of paclitaxel by promoting abnormal mitosis of cancer cells

Abstract Background: Paclitaxel (PTX) is one of the key drugs in clinical practice for esophageal squamous cell carcinoma (ESCC), however, therapeutic effects of PTX alone are not sufficient. PTX causes activation of spindle assembly checkpoint resulting in mitotic arrest of cancer cells. Recently, the antitumor effect of PTX has been shown to depend on aberrant chromosome segregation and abnormal spindle formation. Whereas, recent reports showed that cyclin dependent kinase inhibitor 3 (CDKN3) was often overexpressed in various human cancer tissues which was attributed to the higher mitotic activity in cancer cells, and loss of CDKN3 induced abnormal mitosis and centrosome overduplication. Therefore, we hypothesized that the combination of PTX and CDKN3 inhibition may promote mitotic errors in cancer cells. The aim of this study is to examine the combined effect of PTX and CDKN3 inhibition on ESCC. Methods: The expression levels of CDKN3 were compared between ESCC cell lines (TE1, TE5, TE6, TE8, TE10, and TE11) and normal esophageal epithelial cell line (EPC2-hTERT). CDKN3 knockdown ESCC cells using shRNA were created. Cell viability assay (WST-1 and clonogenic assay), immunofluorescence microscopy for measuring the number of centrosomes or spindle microtubules, and time-lapse images for assessing mitosis and cell fate were performed. Results: The expression levels of CDKN3 in ESCC cells were high compared to normal esophageal epithelial cells. Here, TE6 and TE10 ESCC cells were selected for further experiments based on higher CDKN3 expression. WST-1 assays showed an increased sensitivity to PTX after CDKN3 knockdown. Clonogenic assay showed reduced clonogenicity with CDKN3 knockdown, which was further reduced when exposed to 1 nM PTX (no interaction by two-way ANOVA analysis). Quantification of centrosomes revealed an increased frequency of centrosome overduplication in cells with CDKN3 knockdown, and it was further increased when treated with 1 nM PTX. Quantification of microtubules revealed CDKN3 knockdown cells with 1nM PTX had irregular multipolar spindle formation in almost half of the cells in mitosis. In time-lapse images, mitotic errors were more frequent ( > 80% in combination vs 54% in PTX vs 32% in CDKN3 knockdown) and most of the daughter cells after abnormal mitoses resulted in cell death in interphase or did not undergo another cell division. The cumulative cell death was significantly increased in combination treatment with PTX and CDKN3 inhibition compared to PTX treatment alone (Hazard ratio 2.22; 95% CI 1.43 - 3.45, p = 0.0004 ) and CDKN3 inhibition alone (Hazard ratio 3.09; 95% CI 1.92 - 4.98, p < 0.0001). Conclusions: CDKN3 knockdown increased the frequencies of centrosome overduplication and multipolar spindle formation when combined with PTX exposure. These mitotic errors caused multipolar divisions and subsequent cell death. Targeting CDKN3 could be a novel anti-cancer strategy. Citation Format: Shigeki Kataoka, Junichi Matsubara, Trang H. Vu, Tomohiro Kondo, Tomoki Saito, Yosuke Mitani, Osamu Kikuchi, Atsushi Yamada, Shinya Ohashi, Manabu Muto. CDKN3 inhibition enhances the antitumor effect of paclitaxel by promoting abnormal mitosis of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1556.

Embryo morphokinetics derived from fresh and vitrified bovine oocytes predict blastocyst development and nuclear abnormalities

Embryo development is a dynamic process and critical stages may go unnoticed with the use of traditional morphologic assessments, especially the timing of embryonic divisions and aberrant zygotic cleavage patterns. Bovine embryo development is impaired after oocyte vitrification, but little is known about the underlying morphokinetic behavior. Here, bovine zygotes from fresh (n = 708) and vitrified oocytes (n = 182) were monitored by time-lapse imaging and the timing and nature of early blastomere divisions were modeled to find associations with blastocyst development at day 8. The predictive potential of morphokinetic parameters was analyzed by logistic regression and receiver operating characteristic curve analysis to determine optimal cut-off values. Lag-phase was highly correlated with embryo development. Remarkably, 100% of zygotes that reached the blastocyst stage showed a lag-phase. Fast first cleavage increased the chance of blastocyst development to 30% with a cut-off of 32 h and 22 min. Aberrant zygotic cleavage events, including multipolar division, unequal blastomere sizes, and membrane ruffling resulted in decreased blastocyst development. Multipolar division leads to uneven blastomeres, which was associated with anuclear and multinuclear blastomeres, indicating genome segregation errors. Moreover, we described for the first time morphokinetics of embryos derived from vitrified bovine oocytes. Vitrification severely affected blastocyst development, although lower cryoprotectant concentration in equilibration solutions seems to be less detrimental for embryo yield. Impaired development was linked to slow cleavages, lower lag-phase incidence, and increased early embryonic arrest. Typically, less than 15% of the embryos produced from vitrified oocytes reached more than eight cells. Interestingly, the rate of abnormal first cleavage events was not affected by oocyte vitrification. In conclusion, time to first cleavage, the presence of a lag-phase, and the absence of aberrant zygotic cleavage were the best predictors of bovine blastocyst development for both fresh and vitrified oocytes.

NuMA deficiency causes micronuclei via checkpoint-insensitive k-fiber minus-end detachment from mitotic spindle poles

Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 Toprevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.

Parental genomes segregate into distinct blastomeres during multipolar zygotic divisions leading to mixoploid and chimeric blastocysts

BackgroundDuring normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres.ResultsContrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest.ConclusionsHeterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF.

Empirical single-cell tracking and cell-fate simulation reveal dual roles of p53 in tumor suppression.

The tumor suppressor p53 regulates various stress responses via increasing its cellular levels. The lowest p53 levels occur in unstressed cells; however, the functions of these low levels remain unclear. To investigate the functions, we used empirical single-cell tracking of p53-expressing (Control) cells and cells in which p53 expression was silenced by RNA interference (p53 RNAi). Here, we show that p53 RNAi cells underwent more frequent cell death and cell fusion, which further induced multipolar cell division to generate aneuploid progeny. Those results suggest that the low levels of p53 in unstressed cells indeed have a role in suppressing the induction of cell death and the formation of aneuploid cells. We further investigated the impact of p53 silencing by developing an algorithm to simulate the fates of individual cells. Simulation of the fate of aneuploid cells revealed that these cells could propagate to create an aneuploid cell population. In addition, the simulation also revealed that more frequent induction of cell death in p53 RNAi cells under unstressed conditions conferred a disadvantage in terms of population expansion compared with Control cells, resulting in faster expansion of Control cells compared with p53 RNAi cells, leading to Control cells predominating in mixed cell populations. In contrast, the expansion of Control cells, but not p53 RNAi cells, was suppressed when the damage response was induced, allowing p53 RNAi cells to expand their population compared with the Control cells. These results suggest that, although p53 could suppress the formation of aneuploid cells, which could have a role in tumorigenesis, it could also allow the expansion of cells lacking p53 expression when the damage response is induced. p53 may thus play a role in both the suppression and the promotion of malignant cell formation during tumorigenesis.

Molecular contribution to embryonic aneuploidy and karyotypic complexity in initial cleavage divisions of mammalian development.

Embryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus or formed a chromatin bridge with neighboring cells. A correlation between a lack of syngamy, multipolar divisions and asymmetric genome partitioning was also revealed, and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors. Depletion of the mitotic checkpoint protein BUB1B (also known as BUBR1) resulted in similarly abnormal nuclear structures and cell divisions, as well as chaotic aneuploidy and dysregulation of the kinase-substrate network that mediates mitotic progression, all before zygotic genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides an explanation for blastomeres with uniparental origins, and substantiates defective checkpoints and likely other maternally derived factors as major contributors to the karyotypic complexity afflicting mammalian preimplantation development.

Midkine inhibitor (iMDK) induces apoptosis of primary effusion lymphoma via G2/M cell cycle arrest

Primary effusion lymphoma (PEL) is an aggressive B-cell non-Hodgkin lymphoma in immunocompromised individuals such as AIDS patients. PEL shows a poor prognosis (median survival time < 6 months) compared with other AIDS-related lymphomas, and is generally resistant to conventional treatments. Novel drugs for PEL treatment are required. Midkine inhibitor (iMDK) was previously found to suppress midkine protein expression. Interestingly, iMDK suppressed cell proliferation in PEL cell lines in a time- and dose-dependent manner, regardless of midkine gene expression. We examined the mechanism of iMDK on PEL. Importantly, iMDK strongly induced cell cycle arrest at the G2/M phase within 12 h of incubation and suppressed the p-CDK1 protein level, which is associated with the cell cycle checkpoint at G2/M, resulting in mitotic catastrophe with observation of multipolar division. After mitotic catastrophe, iMDK-treated PEL showed apoptosis with caspase-3, − 8, and − 9 activation at 24 h incubation. However, iMDK showed no effects on viral protein-activated signaling pathways such as JAK-STAT, PI3K-Akt and NF-κB, and HHV-8/KSHV gene expression in PEL. These results indicate that iMDK is a novel CDK1 inhibitor and a promising lead compound for PEL chemotherapy treatment.

How a cell achieves bipolar spindle assembly with normal or aberrant centrosome numbers

Assembly of a bipolar mitotic spindle is critical for equal partitioning of the genetic material into daughter cells during mitosis. In normal cells, each of the two spindle poles is organized by one centrosome. In many cancer cells, however, there are extra centrosomes, which are often clustered to enable bipolar spindle assembly. This provides an important mechanism for cancer cells to escape fatal multipolar divisions and accumulate genomic heterogeneity through chromosome mis-segregation. To dissect how a cell achieves bipolar spindle assembly with different centrosome numbers, we developed a biophysical model for centrosome movement during spindle assembly.

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis.

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.

Chromosomal instability sensitizes patient breast tumors to multipolar divisions induced by paclitaxel.

Paclitaxel (Taxol) is a cornerstone of cancer treatment. However, its mechanism of cytotoxicity is incompletely understood and not all patients benefit from treatment. We show that patients with breast cancer did not accumulate sufficient intratumoral paclitaxel to induce mitotic arrest in tumor cells. Instead, clinically relevant concentrations induced multipolar mitotic spindle formation. However, the extent of early multipolarity did not predict patient response. Whereas multipolar divisions frequently led to cell death, multipolar spindles focused into bipolar spindles before division at variable frequency, and maintaining multipolarity throughout mitosis was critical to induce the high rates of chromosomal instability necessary for paclitaxel to elicit cell death. Increasing multipolar divisions in paclitaxel resulted in improved cytotoxicity. Conversely, decreasing paclitaxel-induced multipolar divisions reduced paclitaxel efficacy. Moreover, we found that preexisting chromosomal instability sensitized breast cancer cells to paclitaxel. Both genetic and pharmacological methods of inducing chromosomal instability were sufficient to increase paclitaxel efficacy. In patients, the amount of pretreatment chromosomal instability directly correlated with taxane response in metastatic breast cancer such that patients with a higher rate of preexisting chromosomal instability showed improved response to taxanes. Together, these results support the use of baseline rates of chromosomal instability as a predictive biomarker for paclitaxel response. Furthermore, they suggest that agents that increase chromosomal instability or maintain multipolar spindles throughout mitosis will improve the clinical utility of paclitaxel.