Mitotic Events Research Articles

MAD2L2 Dimerization Is Not Essential for Mitotic Regulation.

MAD2L2 is a small HORMA domain protein that plays a crucial role in DNA repair and mitosis. In both TLS and shieldin, the dimerization of MAD2L2 via its HORMA domain is critical for the stability and function of these complexes. However, in mitosis, the dimerization state of MAD2L2 remains unknown. To assess the importance of MAD2L2's dimerization during mitosis, we utilized CRISPR/Cas9 to generate MAD2L2 knockout cells, which were subsequently complemented with MAD2L2 species carrying different dimer-disrupting point mutations. We assessed the ability of these MAD2L2 dimer-disrupting mutants to regulate mitosis by evaluating early mitotic events and mitotic fidelity. Our findings indicate that MAD2L2 can function in its monomeric form during mitosis, suggesting that MAD2L2 homodimerization is dispensable for early mitotic regulation. Furthermore, our results suggest that the binding of CDH1 to MAD2L2 is a key regulating factor in mitosis that may actively prevent the formation of MAD2L2 dimers, thereby shifting the cellular balance toward MAD2L2-CDH1 interaction. Thus, the equilibrium between the monomeric and dimeric forms of MAD2L2 is an important cellular factor regulating the MAD2L2-containing complexes.

PIWIL1 is recruited to centrosomes during mitosis in colorectal cancer cells and is linked to cell cycle progression

PIWI proteins, traditionally associated with germline development, have recently gained attention for their expression in various cancers, including colorectal cancer. However, the molecular mechanisms underlying their reactivation and impact on cancer initiation and progression remain elusive. Here, we found that PIWIL1 is expressed at relatively high levels in CRC-derived samples and cell lines, where it undergoes a dynamic relocalization to the centrosome during mitosis. Knockdown of PIWIL1 induces G2/M arrest associated with disruption of the mitotic spindle and aberrant metaphase events, highlighting its role in cell cycle progression. We also found that the expression of PIWIL1 is lost during the differentiation of Caco-2 cells into enterocytes and that PIWIL1 is expressed in cells at the base of the intestinal crypts in normal human colon tissue, where intestinal stem cells are known to reside. Thus, it is possible that the presence of PIWIL1 in cancer cells reflects a physiological role of this protein in stem cell maintenance, which would argue in favor of the proposed stem cell origin of CRC. Supporting this view, dedifferentiation of human fibroblasts into induced pluripotent stem cells (iPSCs) involves the reactivation of PIWIL2 expression, another member of the PIWI protein family. Overall, our findings suggest a role of PIWIL1 in mediating cell cycle dynamics, both in colorectal cancer cells and possibly also in intestinal stem cells. In a broader aspect, we provide evidence supporting an involvement of PIWI proteins in somatic stem cell maintenance, thus expanding the known non-gonadal functions of this protein family.

Specific Mitotic Events Drive Cytoskeletal Remodeling Required for Left-Right Organizer Development.

Cellular proliferation is vital for tissue development, including the Left-Right Organizer (LRO), a transient organ critical for establishing the vertebrate LR body plan. This study investigates cell redistribution and the role of specific progenitor cells in LRO formation, focusing on cell lineage and behavior. Using zebrafish as a model, we mapped all mitotic events in Kupffer's Vesicle (KV), revealing an FGF-dependent, anteriorly enriched mitotic pattern. With a KV-specific fluorescent microtubule (MT) line, we observed that mitotic spindles align along the KV's longest axis until the rosette stage, spindles that form after spin, and are excluded from KV. Early aligned spindles assemble cytokinetic bridges that point MT bundles toward a tight junction where a rosette will initially form. Post-abscission, repurposed MT bundles remain targeted at the rosette center, facilitating actin recruitment. Additional cells, both cytokinetic and non-cytokinetic, are incorporated into the rosette, repurposing or assembling MT bundles before actin recruitment. These findings show that initial divisions are crucial for rosette assembly, MT patterning, and actin remodeling during KV development.

Micronuclei in 2-cell embryos show higher blastocyst formation rates on human embryonic development

ObjectiveMultinucleated blastomeres at the two-cell stage (2MNB) represent a frequently observed nuclear abnormality in early human embryos. This abnormality has been reported to significantly impact on the embryo’s developmental potential to reach the blastocyst stage. However, our understanding of the embryo’s developmental potential and the morphokinetics of 2MNB remains limited. This study investigates the influence of 2MNB and its subtypes on the blastocyst formation. Study designA non-interventional retrospective study was performed in the Reproductive Medical Center of the First Affiliated Hospital of Hainan Medical University, using a time-lapse incubator. The study involved the evaluation of 4416 embryos, including 628 multinucleated embryos, from 1521 intracytoplasmic sperm injection (ICSI) cycles conducted between October 2019 and October 2021. The morphokinetic characteristics of multinucleated embryos were analyzed. ResultsThe results show multinucleation was the most common abnormal mitotic event during embryo development (14.22 %) in 4416 embryos. A control group of 3210 developmentally normal embryos was used in the study. The multinucleated blastomeres caused a lower blastocyst rate (52.48 % VS 64.02 %) compared to the control group. Whereas, 2MNBcause a higher blastocyst rate thanthemat the 4-cell stage (4MNB) (58.89 % VS 43.64 %). 2MNB can be further be further divided into 2MNB1/2cell and 2MNB2/2cell based on one multinucleated blastomere or two multinucleated blastomere appeared. Time to pronuclei fading (tPNf) is significantly longer in 2MNB2/2cell compared to 2MNB1/2cell. Furthermore, the 2MNB1/2cell embryos were divided into four subgroups (Bi-: two nuclei with almost the same size, Micro-: two nuclei with varying sizes, Poly-: more than two nuclei with almost the same size, and Cluster-: more than two nuclei with varying sizes) based on the number of nuclei and relative size. The results show that the Bi- and Micro- groups had a significantly increased blastocyst rate. The Cluster-, and Poly- groups showed significantly delayed embryonic development compared to normal controls. Bi-group has significant delays at t3, t5, and t8 and the Micro-group had a significant delay only at t8. Conclusion2MNB cause higher blastocyst rate than them at 4MNB. 2MNB1/2cell shows shorter tPNf compared to 2MNB2/2cell. Moreover, the Micro-, Bi- groups had a significantly increased blastocyst rate and different kinetic parameters compared to Cluster-, Poly-groups, suggesting that it is necessary to distinguish the nucleus status within 2MNB to increase the blastocyst rate. When selecting embryos for transformation from the 2MNB1/2cell, Micro- is the best choice.

Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses.

Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase ∼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is ∼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an ∼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.

Mono- and bis(steroids) containing a cyclooctane core: Synthesis, antiproliferative activity, and action on cell cytoskeleton microtubules.

Steroid dimers of natural and synthetic origin possess an unusual and complex molecular architecture that may lead to the realization of peculiar effects in biological systems, in particular in different cancer cell lines. In the present work, diastereoselective ring-opening of mono- and polyoxiranes, containing a cyclooctane core, by azide-anion was performed to yield a series of azidoalcohols with different types of symmetry. The products were involved in copper-catalyzed azyde-alkyne cycloaddition (CuAAC) reaction with ethinylestradiol and ethinyltestosterone, and the resulting steroids and steroid dimers with triazole linkers were screened for their antiproliferative activity via (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay. All the compounds revealed cytotoxicity toward several cancer cell lines. The effect of the most potent compound, containing two estradiol moieties, on the microtubules (MT) dynamics was investigated by immunofluorescent microscopy. The disruption of the majority of interphase cell cytoplasmic MT and mitotic event disturbances in the presence of the studied compound were observed. The latter effect caused the appearance of numerous multinucleated cells.

A Lombard Variety of Sweet Pepper Regulating Senescence and Proliferation: The Voghera Pepper.

Aging and its related disorders are important issues nowadays and the first cause of this physio-pathological condition is the overproduction of ROS. Ascorbic acid is an antioxidant mediator and its anti-aging proprieties are well known. Our previous data demonstrated that Voghera sweet pepper (VP), a distinctive type of pepper cultivated in Italy, is particularly rich in ascorbic acid. Based on these data, the anti-aging effect mediated by extracts of the edible part of VP was evaluated on an in vitro model of both young and old Normal Human Diploid Fibroblasts (NHDF). Using phase contrast microscopy, we observed that VP may help cells in the maintenance of physiological morphology during aging. Cytofluorimetric analyses revealed that VP extracts led to an increase in DNA synthesis and percentage of living cells, linked to a consequent increase in mitotic events. This hypothesis is supported by the enhancement of PCNA expression levels observed in old, treated fibroblasts, corroborating the idea that this extract could recover a young phenotype in adult fibroblasts, confirmed by the study of p16 and p53 expression levels and TEM analyses. Based on these results, we may suppose that VP can lead to the partial recovery of "young-like" phenotypes in old fibroblasts.

The System of Analysis for Mitosis and Meiosis

In cellular biology, mitosis and meiosis are essential processes that control cell division and genetic transmission. Numerous disciplines, including developmental biology, genetics, and cancer research, depend on an understanding of these processes. An innovative method for examining these processes is the Mitosis and Meiosis Analysis System (MMAS), which uses artificial intelligence (AI) methods for automated image analysis. An overview of MMAS, including its history, elements, and uses, is given in this paper. We go over how to accurately detect, classify, and quantify mitotic and meiotic events from microscopy images by integrating machine learning models, deep learning frameworks, and advanced image processing algorithms into MMAS. In addition, we discuss how MMAS is used in developmental biology, genetics, and cancer research, among other fields of cellular biology study. By automating image analysis and offering insightful information about cellular dynamics, MMAS is a potent tool that can speed up cellular biology research and deepen our understanding of basic biological processes.

The System of Analysis for Mitosis and Meiosis

In cellular biology, mitosis and meiosis are essential processes that control cell division and genetic transmission. Numerous disciplines, including developmental biology, genetics, and cancer research, depend on an understanding of these processes. An innovative method for examining these processes is the Mitosis and Meiosis Analysis System (MMAS), which uses artificial intelligence (AI) methods for automated image analysis. An overview of MMAS, including its history, elements, and uses, is given in this paper. We go over how to accurately detect, classify, and quantify mitotic and meiotic events from microscopy images by integrating machine learning models, deep learning frameworks, and advanced image processing algorithms into MMAS. In addition, we discuss how MMAS is used in developmental biology, genetics, and cancer research, among other fields of cellular biology study. By automating image analysis and offering insightful information about cellular dynamics, MMAS is a potent tool that can speed up cellular biology research and deepen our understanding of basic biological processes.

Artificial Intelligence for Meiosis and Mitosis Analysis

In cellular biology, meiosis and mitosis are essential processes that control cell division and replication as well as the transfer of genetic material. A thorough understanding of these intricate processes is essential for many fields, such as cancer research, genetics, and developmental biology. In this study, we suggest building a Mitosis and Meiosis Analysis System (MMAS) that uses artificial intelligence (AI) methods to make automated analysis and meiotic event characterization easier. The MMAS uses machine learning models, deep learning frameworks, and sophisticated image processing algorithms to precisely recognize and categorize various meiotic and mitotic stages from microscopy images. The MMAS seeks to increase the accuracy and efficiency of cellular biology research while streamlining the analysis process and minimizing manual labor by utilizing artificial intelligence. Furthermore, by providing insightful information about the dynamic character of mitotic and meiotic events, the MMAS helps scientists understand the underlying mechanisms and their implications for a range of physiological and pathological conditions. We hope to improve our knowledge of meiosis and mitosis and hasten research findings in cellular biology by putting the MMAS into practice.

Robust spatiotemporal organization of mitotic events in mechanically perturbed C. elegans embryos

Early embryogenesis of the nematode Caenorhabditis elegans progresses in an autonomous fashion within a protective chitin eggshell. Cell-division timing and the subsequent mechanically guided positioning of cells is virtually invariant between individuals, especially before gastrulation. Here, we have challenged this stereotypical developmental program in early stages by mechanically perturbing the embryo without breaking its eggshell. Compressing embryos to about two-thirds of their unperturbed diameter only resulted in markedly slower cell divisions. In contrast, compressing embryos to half of their native diameter frequently resulted in a loss of cytokinesis, yielding a non-natural syncytium that still allowed for multiple divisions of nuclei. Although the orientation of mitotic axes was strongly altered in the syncytium, key features of division timing and spatial arrangement of nuclei remained surprisingly similar to those of unperturbed embryos in the first few division cycles. This suggests that few, very robust mechanisms provide a basic and resilient program for safeguarding the early embryogenesis of C. elegans.

Functions of HP1 in preventing chromosomal instability.

Chromosomal instability (CIN), caused by errors in the segregation of chromosomes during mitosis, is a hallmark of many types of cancer. The fidelity of chromosome segregation is governed by a sophisticated cellular signaling network, one crucial orchestrator of which is Heterochromatin protein 1 (HP1). HP1 dynamically localizes to distinct sites at various stages of mitosis, where it regulates key mitotic events ranging from chromosome-microtubule attachment to sister chromatid cohesion to cytokinesis. Our evolving comprehension of HP1's multifaceted role has positioned it as a central protein in the orchestration of mitotic processes.

Abstract 2117: Elucidation of the fates of CDK2 inhibited aneuploid and residual lung cancers

Abstract Genomic instability is a hallmark of cancer. Unlike normal diploid cells, cancer cells exhibit abnormal centrosome numbers and chromosomal instability. Mitosis with supernumerary centrosomes yields progeny with aberrant chromosome segregation and aneuploidy. While most progeny cells with abnormal chromosomes are eliminated through apoptosis, some have distinct fates with a growth advantage. These cancer cells can acquire resistance to antineoplastic agents. This confers an aggressive tumor biology and unfavorable clinical outcomes. Cyclin-dependent kinase 2 (CDK2) regulates cell cycle progression and the centrosome cycle. We previously reported that CDK2 inhibition prevents supernumerary centrosome clustering, causing multipolar mitosis and anaphase catastrophe in lung and other aneuploid cancers. This was associated with residual transplanted tumors in mice. To elucidate cell fates of progeny with multipolar mitosis after CDK2/9 inhibition with CYC065 (0.2 µM) treatment, a panel of murine and human lung cancer cells underwent time-lapse fluorescent microscopy. This elucidated multipolar mitotic events within the progeny. Intriguingly, distinct outcomes occurred after mitosis including formation of multipolar and multinucleated cells. Cell death of progeny is the predominant fate, but some progeny survive despite continuous CDK2 inhibition. Surprisingly, some of these cells fuse together. Those multinucleated cells can undergo multiple cell cycles without successful cytokinesis. To confirm these effects were through CDK2 inhibition, the selective CDK2 inhibitor Tagtociclib (PF-07104091, at the 2 µM dosage) was used. This treatment statistically-significantly increased multipolar lung cancer cells and increased apoptotic death during time-lapse fluorescent microscopy. Findings were independently validated by CDK2 shRNA knockdown. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent staining revealed the ultrastructure of multinucleated cells. Insights into tumor biology came from intravital imaging of transplanted lung cancer cells in mice. Multipolar and multinuclear cells followed CDK2 antagonism. Incucyte® Live-Cell Analysis System and Artificial Intelligence-based imaging determined distinct growth responses in aneuploid as compared to non-aneuploid lung cancer cells after CDK2 inhibitor treatments. In summary, these findings are translationally relevant. CDK2 inhibition of aneuploid lung cancers yields distinct cell fates. This is linked to resistance to CDK2 antagonism and to formation of residual in vivo tumors. Citation Format: Liliya Tyutyunyk, Zibo Chen, Xiuxia Liu, Yeap Ng, Aayush Bhatawadekar, Kedar Narayan, Roberto Weigert, Xi Liu, Ethan Dmitrovsky. Elucidation of the fates of CDK2 inhibited aneuploid and residual lung cancers [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 2117.

Abstract 847: Association of methylation-based stem cell divisions with lifetime cancer risk and cancer progression

Abstract Cancer initiation arises from the accumulation of somatic mutations acquired during stem cell mitotic events mediated by carcinogenic risk factors. On an ecological level, Tomasetti and Vogelstein (2015) demonstrated that lifetime risk of solid tumor cancer types were strongly correlated with total number of stem cell divisions estimated from tissue-specific cell lines. Recently, several mitotic clocks (MC) have been developed to estimate stem cell division rates (SCDR) based on whole-genome methylation arrays. Here, we compare estimated SCDR for several tissue types with tissue-specific cancer incidence and lifetime risk estimates. Furthermore, we assess the link between SCDR with cancer progression and survival. Array-based methylation data derived from normal adjacent tissue in TCGA was used to estimate SCDR for 15 cancer types. A Pearson correlation test was performed to assess the association between SCDR and cancer-specific incidence and lifetime risk for each cancer type overall and stratified by self-reported race (e.g. White and Black). SCDR was estimated in tumors from TCGA for 4 common cancers and subtypes: breast (BRCA), prostate (PRAD), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC). A Spearman correlation test was used to identify potential trends between SCDR and pathological stage. Cox proportional hazard (PH) models adjusted for age at diagnosis and treatment status (e.g. surgery, radiation, etc.) were used to evaluate the association of time to death with SCDR terciles across different cancer stages. We observed a similar trend between SCDR and overall incidence (cor=0.61, p=0.01), aligning with earlier findings. Stratified-specific trends between cancer incidence and SCDR was consistent for both Black and White populations (cor=0.62 vs 0.66). Tumor analysis revealed a significant correlation between SCDR and pathological stage (e.g. stage 1, 2, and 3/4) for BRCA (p<0.001) and pathological stage (e.g. stage 2, 3 and 4) for PRAD (p<0.001), suggesting its potential as a marker of cancer progression. However, the correlation between LUAD, LUSC and pathological stage was insignificant. Based on Cox PH model, within stage III BRCA, SCDR significantly contributed to explaining variability in survival (p=0.04), emphasizing its potential prognostic value beyond cancer stage. Our study supports earlier findings applying methylation based SCDR estimates and shows that this association is consistent across racial groups. This implies that SCDR may act as a biological marker of a combination of various genetic and environmental risk factors underlying disease etiology. Further analyses will be extended to all cancers in TCGA. Lastly, we demonstrate that MC-based SCDR estimation has the potential to serve as a clinical tool to identify individuals at elevated risk of cancer initiation, track progression, and complement cancer stage for predicting survival. Citation Format: Yanning Wu, Cheryl Thompson, Fredrick Schumacher. Association of methylation-based stem cell divisions with lifetime cancer risk and cancer progression [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 847.

Quantitative Proteomic Analysis Identifying and Evaluating TRAF6 and IL-8 as Potential Diagnostic Biomarkers in Neonatal Patients with Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a common gastrointestinal complication in premature infants, resulting in high morbidity and mortality, and its early detection is crucial for accurate treatment and outcome prediction. Extensive research has demonstrated a clear correlation between NEC and extremely low birth weight, degree of preterm, formula feeding, infection, hypoxic/ischemic damage, and intestinal dysbiosis. The development of noninvasive biomarkers of NEC from stool, urine, and serum has attracted a great deal of interest because to these clinical connections and the quest for a deeper knowledge of disease pathophysiology. Therefore, this study aims to identify protein expression patterns in NEC and discover innovative diagnostic biomarkers. In this study, we recruited five patients diagnosed with NEC and paired necrotic segments of intestinal tissue with adjacent normal segments of intestine to form experimental and control groups. Quantitative proteomics tandem mass tagging (TMT) labeling technique was used to detect and quantify the proteins, and the expression levels of the candidate biomarkers in the intestinal tissues were further determined by quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, Immunofluorescence methods and enzyme-linked immunosorbent assay (ELISA). A total of 6880 proteins were identified and quantified in patients with NEC. A significant disparity in protein expression was observed between necrotic and normal segments of intestinal tissue in NEC patients. A total of 55 proteins were found to be upregulated, and 40 proteins were found to be downregulated in NEC patients when using a p-value of < 0.05, and an absolute fold change of > 1.2 for analysis. GO function enrichment analysis showed the positive regulation of significant biological processes such as mitochondrial organization, vasoconstriction, rRNA catabolism, fluid shear stress response, and glycerol ether biosynthesis processes. Enrichment analysis also revealed essential functions such as ligand-gated ion channel activity, potassium channel activity, ligand-gated cation channel activity, ligand-gated ion channel activity, and ligand-gated channel activity, including molecular functions such as ligand-gated ion channel activity and mitotic events in this comparative group. Significant changes were found in endomembrane protein complex, membrane fraction, mitochondrial membrane fraction, membrane components, membrane intrinsic components, and other localized proteins. Additional validation of intestinal tissue and serum revealed a substantial increase in TRAF6 (tumor necrosis factor receptor-associated factor 6) and IL-8(Interleukin-8, CXCL8). The quantitative proteomic TMT method can effectively detect proteins with differential expression in the intestinal tissues of NEC patients. Proteins TRAF6 and CXCL8/IL-8 are significantly upregulated in the intestinal tissues and serum samples of patients and may serve as valuable predictor factors for NEC's early diagnosis.

Mechanistic insights into DNA damage recognition and checkpoint control in plants.

The plant DNA damage response (DDR) pathway safeguards genomic integrity by rapid recognition and repair of DNA lesions that, if unrepaired, may cause genome instability. Most frequently, DNA repair goes hand in hand with a transient cell cycle arrest, which allows cells to repair the DNA lesions before engaging in a mitotic event, but consequently also affects plant growth and yield. Through the identification of DDR proteins and cell cycle regulators that react to DNA double-strand breaks or replication defects, it has become clear that these proteins and regulators form highly interconnected networks. These networks operate at both the transcriptional and post-transcriptional levels and include liquid-liquid phase separation and epigenetic mechanisms. Strikingly, whereas the upstream DDR sensors and signalling components are well conserved across eukaryotes, some of the more downstream effectors are diverged in plants, probably to suit unique lifestyle features. Additionally, DDR components display functional diversity across ancient plant species, dicots and monocots. The observed resistance of DDR mutants towards aluminium toxicity, phosphate limitation and seed ageing indicates that gaining knowledge about the plant DDR may offer solutions to combat the effects of climate change and the associated risk for food security.

High-volume, label-free imaging for quantifying single-cell dynamics in induced pluripotent stem cell colonies.

To facilitate the characterization of unlabeled induced pluripotent stem cells (iPSCs) during culture and expansion, we developed an AI pipeline for nuclear segmentation and mitosis detection from phase contrast images of individual cells within iPSC colonies. The analysis uses a 2D convolutional neural network (U-Net) plus a 3D U-Net applied on time lapse images to detect and segment nuclei, mitotic events, and daughter nuclei to enable tracking of large numbers of individual cells over long times in culture. The analysis uses fluorescence data to train models for segmenting nuclei in phase contrast images. The use of classical image processing routines to segment fluorescent nuclei precludes the need for manual annotation. We optimize and evaluate the accuracy of automated annotation to assure the reliability of the training. The model is generalizable in that it performs well on different datasets with an average F1 score of 0.94, on cells at different densities, and on cells from different pluripotent cell lines. The method allows us to assess, in a non-invasive manner, rates of mitosis and cell division which serve as indicators of cell state and cell health. We assess these parameters in up to hundreds of thousands of cells in culture for more than 36 hours, at different locations in the colonies, and as a function of excitation light exposure.

Insights into embryonic chromosomal instability: mechanisms of DNA elimination during mammalian preimplantation development.

Mammalian preimplantation embryos often contend with aneuploidy that arose either by the inheritance of meiotic errors from the gametes, or from mitotic mis-segregation events that occurred following fertilization. Regardless of the origin, mis-segregated chromosomes become encapsulated in micronuclei (MN) that are spatially isolated from the main nucleus. Much of our knowledge of MN formation comes from dividing somatic cells during tumorigenesis, but the error-prone cleavage-stage of early embryogenesis is fundamentally different. One unique aspect is that cellular fragmentation (CF), whereby small subcellular bodies pinch off embryonic blastomeres, is frequently observed. CF has been detected in both in vitro and in vivo-derived embryos and likely represents a response to chromosome mis-segregation since it only appears after MN formation. There are multiple fates for MN, including sequestration into CFs, but the molecular mechanism(s) by which this occurs remains unclear. Due to nuclear envelope rupture, the chromosomal material contained within MN and CFs becomes susceptible to double stranded-DNA breaks. Despite this damage, embryos may still progress to the blastocyst stage and exclude chromosome-containing CFs, as well as non-dividing aneuploid blastomeres, from participating in further development. Whether these are attempts to rectify MN formation or eliminate embryos with poor implantation potential is unknown and this review will discuss the potential implications of DNA removal by CF/blastomere exclusion. We will also extrapolate what is known about the intracellular pathways mediating MN formation and rupture in somatic cells to preimplantation embryogenesis and how nuclear budding and DNA release into the cytoplasm may impact overall development.

Abstract PR007: The combination of TRIP13 and Aurora kinase A inhibition caused cell cycle specific DNA damage and death in Rb-deficient cancers

Abstract Many diverse, common, and lethal cancer types have inactivation of the retinoblastoma (RB1, Rb) tumor suppressor pathway including cancers caused by the human papillomavirus (HPV), which causes &amp;gt;5% of all cancers worldwide. Despite therapeutic advances, advanced Rb-deficient cancers are still lethal with therapy-related adverse effects. Moreover, there are no therapies that uniquely target HPV-driven or Rb-deficient cancers. Therefore, biomarker-selected, targeted therapy for Rb-deficient cancers represents both a substantial unmet need and a significant translational knowledge gap. Rb-deficient cancer cells depend on Aurora A kinases to bypass the spindle assembly checkpoint (SAC) and survive due to overexpression of the mitotic checkpoint protein Mad2. Thyroid hormone receptor interactor 13 (TRIP13) catalyzes the ATP-dependent conversion of Mad2 from its active to inactive form, which turns off the SAC. Our lab discovered that the combination of Aurora A inhibition with alisertib plus TRIP13 depletion induced extensive apoptosis selectively in Rb-deficient cancer cells. Moreover, our results demonstrated that the synthetic lethality between Aurora A and TRIP13 reflects mitotic catastrophe. The combination led to highly abnormal mitoses in Rb-deficient cancer cells, evidenced by unaligned, dispersed chromosomes, mitotic spindle defects, and G2/M arrest. Moreover, live cell imaging showed that alisertib treatment increased the duration of first mitoses and induced mitotic cell death, which was even more pronounced in second mitotic events. The combination resulted in both prolonged mitotic arrest and enhanced lethality, indicated by increased mitotic cell death after the first mitoses and interphase cell death following the first mitotic arrest. Furthermore, using degron tagging to rapidly degrade TRIP13, we showed that TRIP13 depletion led to transient mitotic arrest in cells that were in late G2, supporting the role of TRIP13 in regulating the SAC and mitotic progression. Since prolonged mitotic arrest is known to cause DNA damage and both Aurora A and TRIP13 are known to be involved in the DNA damage response, we also tested the effect of the combination treatment on DNA damage. Treating Rb-deficient cancer cells with the combination resulted in robust increases in gamma-H2AX, pTIF1 beta (KAP1, TRIM28), and DNA PKcs. The lethal effects of the combination required mitotic entry as concurrent CDK1 inhibition with RO-3306 prevented apoptosis and DNA damage induced by the combination. The selective effect of the combination in Rb-deficient cancer cells may have a wide therapeutic index resulting in less toxic and more effective therapy. Additionally, targeting mitosis may contribute to a cancer-specific effect due to cancer cells’ high proliferation rate. Our study suggests that targeting TRIP13 and Aurora A led to DNA damage and death specifically during mitosis in Rb-deficient cells. This work has potential to establish a foundation for future clinical trials and may change current clinical practice paradigms. Citation Format: Lacin Yapindi, Soma Ghosh, Tuhina Mazumdar, Faye M. Johnson. The combination of TRIP13 and Aurora kinase A inhibition caused cell cycle specific DNA damage and death in Rb-deficient cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR007.

Cytological and transcriptomic analyses provide insights into the pollen fertility of synthetic allodiploid Brassica juncea hybrids.

Imbalanced chromosomes and cell cycle arrest, along with down-regulated genes in DNA damage repair and sperm cell differentiation, caused pollen abortion in synthetic allodiploid Brassica juncea hybrids. Interspecific hybridization is considered to be a major pathway for species formation and evolution in angiosperms, but the occurrence of pollen abortion in the hybrids is common, prompting us to recheck male gamete development in allodiploid hybrids after the initial combination of different genomes. Here, we investigated the several key meiotic and mitotic events during pollen development using the newly synthesised allodiploid B. juncea hybrids (AB, 2n=2× =18) as a model system. Our results demonstrated the partial synapsis and pairing of non-homologous chromosomes concurrent with chaotic spindle assembly, affected chromosome assortment and distribution during meiosis, which finally caused difference in genetic constitution amongst the final tetrads. The mitotic cell cycle arrest during microspore development resulted in the production of anucleate pollen cells. Transcription analysis showed that sets of key genes regulating cyclin (CYCA1;2 and CYCA2;3), DNA damage repair (DMC1, NBS1 and MMD1), and ubiquitin-proteasome pathway (SINAT4 and UBC) were largely downregulated at the early pollen meiosis stages, and those genes involved in sperm cell differentiation (DUO1, PIRL1, PIRL9 and LBD27) and pollen wall synthesis (PME48, VGDH11 and COBL10) were mostly repressed at the late pollen mitosis stages in the synthetic allodiploid B. juncea hybrids (AB). In conclusion, this study elucidated the related mechanisms affecting pollen fertility during male gametophyte development at the cytological and transcriptomic levels in the synthetic allodiploid B. juncea hybrids.