Chromosome Segregation Errors Research Articles

Statistical image processing quantifies the changes in cytoplasmic texture associated with aging in Caenorhabditis elegans oocytes

BackgroundOocyte quality decreases with aging, thereby increasing errors in fertilization, chromosome segregation, and embryonic cleavage. Oocyte appearance also changes with aging, suggesting a functional relationship between oocyte quality and appearance. However, no methods are available to objectively quantify age-associated changes in oocyte appearance.ResultsWe show that statistical image processing of Nomarski differential interference contrast microscopy images can be used to quantify age-associated changes in oocyte appearance in the nematode Caenorhabditis elegans. Max–min value (mean difference between the maximum and minimum intensities within each moving window) quantitatively characterized the difference in oocyte cytoplasmic texture between 1- and 3-day-old adults (Day 1 and Day 3 oocytes, respectively). With an appropriate parameter set, the gray level co-occurrence matrix (GLCM)-based texture feature Correlation (COR) more sensitively characterized this difference than the Max–min Value. Manipulating the smoothness of and/or adding irregular structures to the cytoplasmic texture of Day 1 oocyte images reproduced the difference in Max–min Value but not in COR between Day 1 and Day 3 oocytes. Increasing the size of granules in synthetic images recapitulated the age-associated changes in COR. Manual measurements validated that the cytoplasmic granules in oocytes become larger with aging.ConclusionsThe Max–min value and COR objectively quantify age-related changes in C. elegans oocyte in Nomarski DIC microscopy images. Our methods provide new opportunities for understanding the mechanism underlying oocyte aging.

Cep57 and Cep57L1 maintain centriole engagement in interphase to ensure centriole duplication cycle.

Centrioles duplicate in interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Codepletion of Cep57 and Cep57L1 induces precocious centriole disengagement in interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation.

Aneuploidy in human eggs: contributions of the meiotic spindle.

Human eggs frequently contain an incorrect number of chromosomes, a condition termed aneuploidy. Aneuploidy affects ∼10–25% of eggs in women in their early 30s, and more than 50% of eggs from women over 40. Most aneuploid eggs cannot develop to term upon fertilization, making aneuploidy in eggs a leading cause of miscarriages and infertility. The cellular origins of aneuploidy in human eggs are incompletely understood. Aneuploidy arises from chromosome segregation errors during the two meiotic divisions of the oocyte, the progenitor cell of the egg. Chromosome segregation is driven by a microtubule spindle, which captures and separates the paired chromosomes during meiosis I, and sister chromatids during meiosis II. Recent studies reveal that defects in the organization of the acentrosomal meiotic spindle contribute to human egg aneuploidy. The microtubules of the human oocyte spindle are very frequently incorrectly attached to meiotic kinetochores, the multi-protein complexes on chromosomes to which microtubules bind. Multiple features of human oocyte spindles favour incorrect attachments. These include spindle instability and many age-related changes in chromosome and kinetochore architecture. Here, we review how the unusual spindle assembly mechanism in human oocytes contributes to the remarkably high levels of aneuploidy in young human eggs, and how age-related changes in chromosome and kinetochore architecture cause aneuploidy levels to rise even higher as women approach their forties.

NCAPH Recruits and Stabilizes GEN1 in Chromatin to Facilitate DNA Interstrand Crosslink Repair

Repairing damaged DNA and removing all physical connections between sister chromosomes is important for proper chromosomal segregation by contributing to chromosomal stability. Here, we show that depletion of NCAPH exacerbates chromosome segregation errors and cytokinesis failure due to sister-chromatid intertwinement, which is distinct form the ultrafine DNA bridges (UFB) induced by DNA inter-strand crosslinks (DNA-ICL). Importantly, we identified an interaction between NCAPH and GEN1 in the chromatin via binding at the N-terminus of NCAPH. DNA-ICL activation, using ICL-inducing agents, increased the expression and interaction between NCAPH and GEN1 in the soluble nuclear and chromatin fractions, indicating that the NCAPH-GEN1 interaction participates in repairing DNA-ICLs. Moreover, NCAPH recruits and stabilizes GEN1 within chromatin at the G2/M phase, and is associated with DNA-ICL induced damage repair. Therefore, NCAPH-GEN1 specifically responds to DNA-ICLs, and NCAPH recruits and stabilizes GEN1 to ensure DNA damage repair and proper chromosome separation.

Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1.

The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology. These defects are dependent on a hitherto unknown chromatin-binding region of LAP1 that we have delineated. LAP1-induced NE abnormalities are efficiently suppressed by expression of wild-type but not ATPase-deficient Torsins. Furthermore, a dominant-negative Torsin induces chromosome segregation defects in a LAP1-dependent manner. These results indicate that association of LAP1 with chromatin in the nucleus can be modulated by Torsins in the perinuclear space, shedding new light on the LAP1-Torsin interplay.

Mechanobiology of the Mitotic Spindle.

The mitotic spindle is a microtubule-based assembly that separates the chromosomes during cell division. As the spindle is basically a mechanical micro machine, the understanding of its functioning is constantly motivating the development of experimental approaches based on mechanical perturbations, which are complementary to and work together with the classical genetics and biochemistry methods. Recent data emerging from these approaches in combination with theoretical modeling led to novel ideas and significant revisions of the basic concepts in the field. In this Perspective, we discuss the advances inthe understanding of spindle mechanics, focusing on microtubule forces that control chromosome movements.

Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

Epithelia are an eminent tissue type and a common driver of tumorigenesis, requiring continual precision in cell division to maintain tissue structure and genome integrity. Mitotic defects often trigger apoptosis, impairing cell viability as a tradeoff for tumor suppression. Identifying conditions that lead to cell death and understanding the mechanisms behind this response are therefore of considerable importance. Here we investigated how epithelia of the Drosophila wing disc respond to loss of Short stop (Shot), a cytoskeletal crosslinking spectraplakin protein that we previously found to control mitotic spindle assembly and chromosome dynamics. In contrast to other known spindle-regulating genes, Shot knockdown induces apoptosis in the absence of Jun kinase (JNK) activation, but instead leads to elevated levels of active p38 kinase. Shot loss leads to double-strand break (DSB) DNA damage, and the apoptotic response is exacerbated by concomitant loss of p53. DSB accumulation is increased by suppression of the spindle assembly checkpoint, suggesting this effect results from chromosome damage during error-prone mitoses. Consistent with DSB induction, we found that the DNA damage and stress response genes, Growth arrest and DNA damage (GADD45) and Apoptosis signal-regulating kinase 1 (Ask1), are transcriptionally upregulated as part of the shot-induced apoptotic response. Finally, co-depletion of Shot and GADD45 induced significantly higher rates of chromosome segregation errors in cultured cells and suppressed shot-induced mitotic arrest. Our results demonstrate that epithelia are capable of mounting molecularly distinct responses to loss of different spindle-associated genes and underscore the importance of proper cytoskeletal organization in tissue homeostasis.

CBIO-24. KINETOCHORE MISREGULATION IN GLIOBLASTOMA AND OTHER CANCERS

Abstract Aneuploidy is a hallmark of most late stage cancers and contributes to tumor cell evolution, invasiveness, therapy resistance, and recurrence. A major cause of aneuploidy is lagging anaphase chromosomes that have merotelic kinetochore (KT) attachments in mitosis. In this scenario, a KT (i.e., the proteinaceous structure assembled onto centromeres in mitosis) is bound to microtubules (MTs) emanating from both spindle poles, causing chromosome mis-segregation. “Background” chromosome missegregation likely contributes to the initiation of multiple cancers, including glioblastoma (GBM), where chr 7 gain and chr 10 loss are early events. However, we have found that in GBM and transformed cells activation of the RAS signaling pathway significantly increases lagging anaphase chromosomes, and, as a result, aneuploidy and tumor heterogeneity. However, the underlying mechanism of RAS-induced chromosome instability has remained elusive. Here we present one possible cause: MAPK-driven KT stress. The hallmarks of KT stress include chronic KT-MT attachment instability in mitosis, dramatic changes in KT morphology, and increases in merotelic KT attachments and chromosome segregation errors. KT stress is lethal to cancer cells. However, stressed cells survive by relying on BUB1B/BubR1 activity to strengthen KT-MT attachments. We first observed KT stress in GBM patient isolates, where ~60–70% display its hallmarks. We subsequently found that RAS or MEK activity is sufficient to induce KT stress in both CNS and non-CNS derived cell types. We propose that KT stress is caused by aberrant mitotic RAS/MAPK activity, which likely targets one or more KT proteins resulting in KT-MT attachment defects and chromosome instability. In addition, Dr. Jun Zhu’s lab has also created a computational classifier that can identify KT stressed cells and tumors based on the expression of 838 genes associated with KT stress, which can be also used to identify orthogonal therapeutic sensitivities. We will present these findings as well at this meeting.

SIRT1 and HSP90alpha Are Functionally Linked and Control Mitotic Chromosome Segregation and Cell Viability in a Subset of Dlbcls

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma in adults, exhibiting highly heterogenous clinical behavior and complex molecular background. In addition to the genetic complexity, different DLBCL subsets are functionally dependent on different survival mechanisms and exhibit distinct metabolic fingerprints. One of DLBCLs subtypes relies on mitochondrial oxidative phosphorylation and nutrient utilization pathways that provide pro-survival advantage independent of B-cell receptor (BCR) signaling. These OxPhos-DLBCLs are resistant to BCR inhibition and remain functionally poorly defined. We and others found that OxPhos-DLBCLs, compared to the BCR-dependent DLBCLs, overexpress heat shock protein HSP90alpha (Br J Haematol 2009; 144:358-66). Expression of multiple heat shock proteins is regulated by the activity of a stress-responsive, acetylation-dependent transcription factor HSF1. Acetylation blocks, whereas deacetylation by sirtuins increases HSF1 activity. We found that HSP90alpha gene/protein expression correlated with SIRT1 protein level in DLBCL cell lines. SIRT1 knockdown or chemical inhibition reduced HSP90alpha expression in a mechanism involving HSF1, whereas HSP90 inhibitor (17AAG) reduced SIRT1 protein stability suggesting a chaperone function of HSP90alpha toward SIRT1 and a functional link between these proteins. We confirmed the SIRT1-HSP90alpha interaction in DLBCL cells using proximity ligation assay (PLA). The number of PLA complexes was significantly higher in OxPhos- (Ly4, K422, Toledo) than BCR- (Ly1, DHL4, Ly7, DHL6) DLBCL cell lines (p<0.001). Importantly, the number of PLA complexes increased markedly in mitotic when compared to interphase cells, indicating that the interaction between these proteins plays a mitosis-specific role. For this reason, we next assessed the chromosome segregation of DLBCL cells in the presence and absence of SIRT1 and/or HSP90alpha activity. Both genetic (siRNA) and chemical (EX-527) inhibition of SIRT1 significantly increased the number of cells with chromosome segregation errors (multipolar spindle formation, anaphase bridges and lagging chromosomes- respectively 50%, 26% and 20% of all observed abnormal mitotic incidents). Similarly, chemical (17AAG) or genetic (siRNA) inhibition of HSP90alpha disturbed chromosome segregation, albeit to a lesser extent than SIRT1 disruption. Concurrent chemical or genetic ablation of SIRT1 and HSP90alpha synergistically increased the number of mitotic cells with chromosome segregation errors in OxPhos-DLBCLs. In the BCR-dependent cell lines, neither of the inhibitors used separately had a significant impact on the number of cells with improper chromosome segregation events, but the simultaneous inhibition of SIRT1 (EX-527) and HSP90alpha (17AAG) significantly increased the number of cells with aberrant mitosis, although to a lesser extent than in OxPhos-DLBCLs (15.4-17.1% for BCR-DLBCL versus 28.37-48% for OxPhos-DLBCL, respectively). Consistent with the postulated role of SIRT1 in chromosome dynamics during mitosis, we found downregulated expression of SIRT1-dependent genes in the recently characterized DLBCL subset characterized by chromosome instability (C2, Chapuy et al., Nat Medicine 2018). While low rates of chromosomal instability induces tumorigenesis, increased chromosomal missegregaton leads to cell death and suppresses cancer development. SIRT1 inhibitors (EX-527, cambinol, tenovin-6) induced dose-dependent cytotoxicity in DLBCL cell lines, while simultaneous addition of HSP90 inhibitor (17AAG) produced synergistic or additive effect in OxPhos-, but not BCR-DLBCLs. Taken together, our findings define a new OxPhos DLBCL-specific pathogenetic mechanism involving SIRT1 and HSP90alpha that regulates chromosome dynamics during mitosis and may be exploited therapeutically. Disclosures Juszczynski: Ryvu Therapeutics: Other: member of advisory board.

Abstract PO-075: Dysfunction of centrosome regulation by BRCA1-containing complex increases tumor heterogeneity

Abstract Centrosomes are the major microtubule-organizing centers in animal cells and are critical for the formation of a bipolar mitotic spindle. Alterations in centrosome number and structure often occur together and are associated with many cancers. Centrosome aberrations results in chromosome segregation errors and abnormal cell division, leading to chromosomal instability (CIN). CIN is a major source of aneuploidy, resulting in tumor heterogeneity. Centrosome aberrations are observed in the early stages of tumorigenesis and are correlated with CIN. Breast cancer gene 1 (BRCA1) is a tumor suppressor that is associated with hereditary breast and ovarian cancer. Functions of BRCA1 in DNA repair, especially homologous recombination, has been focused, whereas BRCA1 regulates centrosome together with BRCA1-associated RING domain protein (BARD1). We identified BRCA1-interacting proteins, Obg-like ATPase 1 (OLA1) and receptor for activated C kinase (RACK1). OLA1 and RACK1 directly bound to BRCA1, BARD1, and γ-tubulin and localized the centrosomes throughout the cell cycle. Overexpression of OLA1 or RACK1 caused centrosome amplification due to centriole overduplication in mammary tissue-derived cells. The number of centrioles in cells was higher in cell lines derived from mammary tissue compared to those derived from other tissues. Knockdown of RACK1 caused abnormal centrosomal localization of BRCA1 and abrogated centriole duplication. Overexpression of RACK1 increased the centrosomal localization of BRCA1. Cancer-derived variants of BRCA1, BARD1, OLA1, and RACK1 abolished or weakened their interaction and function in centrosome duplication. These data suggest that the formation of BRCA1/BARD1/OLA1/RACK1 complex is important for centrosome regulation to maintain genome stability and that dysfunction of this complex will increase tumor heterogeneity that associated with carcinogenesis and cancer progression. Citation Format: Natsuko Chiba. Dysfunction of centrosome regulation by BRCA1-containing complex increases tumor heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-075.

Integrating pathology, chromosomal instability and mutations for risk stratification in early-stage endometrioid endometrial carcinoma

BackgroundRisk stratifications for endometrial carcinoma (EC) depend on histopathology and molecular pathology. Histopathological risk stratification lacks reproducibility, neglects heterogeneity and contributes little to surgical procedures. Existing molecular stratification is useless in patients with specific pathological or molecular characteristics and cannot guide postoperative adjuvant radiotherapies. Chromosomal instability (CIN), the numerical and structural alterations of chromosomes resulting from ongoing errors of chromosome segregation, is an intrinsic biological mechanism for the evolution of different prognostic factors of histopathology and molecular pathology and may be applicable to the risk stratification of EC.ResultsBy analyzing CIN25 and CIN70, two reliable gene expression signatures for CIN, we found that EC with unfavorable prognostic factors of histopathology or molecular pathology had serious CIN. However, the POLE mutant, as a favorable prognostic factor, had elevated CIN signatures, and the CTNNB1 mutant, as an unfavorable prognostic factor, had decreased CIN signatures. Only if these two mutations were excluded were CIN signatures strongly prognostic for outcomes in different adjuvant radiotherapy subgroups. Integrating pathology, CIN signatures and POLE/CTNNB1 mutation stratified stageIendometrioid EC into four groups with improved risk prognostication and treatment recommendations.ConclusionsWe revealed the possibility of integrating histopathology and molecular pathology by CIN for risk stratification in early-stage EC. Our integrated risk model deserves further improvement and validation.

CHK1 monitors spindle assembly checkpoint and DNA damage repair during the first cleavage of mouse early embryos

ObjectivesDNA damage and errors of accurate chromosome segregation lead to aneuploidy and foetal defects. DNA repair and the spindle assembly checkpoint (SAC) are the mechanisms developed to protect from these defects. Checkpoint kinase 1 (CHK1) is reported to be an important DNA damage response protein in multiple models, but its functions remain unclear in early mouse embryos.Materials and MethodsImmunofluorescence staining, immunoblotting and real‐time reverse transcription polymerase chain reaction were used to perform the analyses. Reactive oxygen species levels and Annexin‐V were also detected.ResultsLoss of CHK1 activity accelerated progress of the cell cycle at the first cleavage; however, it disturbed the development of early embryos to the morula/blastocyst stages. Further analysis indicated that CHK1 participated in spindle assembly and chromosome alignment, possibly due to its regulation of kinetochore‐microtubule attachment and recruitment of BubR1 and p‐Aurora B to the kinetochores, indicating its role in SAC activity. Loss of CHK1 activity led to embryonic DNA damage and oxidative stress, which further induced early apoptosis and autophagy, indicating that CHK1 is responsible for interphase DNA damage repair.ConclusionsOur results indicate that CHK1 is a key regulator of the SAC and DNA damage repair during early embryonic development in mice.

Aberrant BUB1 Overexpression Promotes Mitotic Segregation Errors and Chromosomal Instability in Multiple Myeloma.

Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.

Chromosomal instability upregulates interferon in acute myeloid leukemia.

Chromosome instability (CIN) generates genetic and karyotypic diversity that is common in hematological malignancies. Low to moderate levels of CIN are well tolerated and can promote cancer proliferation. However, high levels of CIN are lethal. Thus, CIN may serve both as a prognostic factor to predict clinical outcome and as a predictive biomarker. A retrospective study was performed to evaluate CIN in acute myeloid leukemia (AML). Chromosome mis-segregation frequency was correlated with clinical outcome in bone marrow core biopsy specimens from 17 AML cases. Additionally, we induced chromosome segregation errors in AML cell lines with AZ3146, an inhibitor of the Mps1 mitotic checkpoint kinase, to quantify the phenotypic effects of high CIN. We observed a broad distribution of chromosome mis-segregation frequency in AML bone marrow core specimens. High CIN correlated with complex karyotype in AML, as expected, although there was no clear survival effect. In addition to CIN, experimentally inducing chromosome segregation errors by Mps1 inhibition in AML cell lines causes DNA damage, micronuclei formation, and upregulation of interferon stimulated genes. High levels of CIN appear to be immunostimulatory, suggesting an opportunity to combine mitotic checkpoint inhibitors with immunotherapy in treatment of AML.

Guarding the Genome: CENP-A-Chromatin in Health and Cancer.

Faithful chromosome segregation is essential for the maintenance of genomic integrity and requires functional centromeres. Centromeres are epigenetically defined by the histone H3 variant, centromere protein A (CENP-A). Here we highlight current knowledge regarding CENP-A-containing chromatin structure, specification of centromere identity, regulation of CENP-A deposition and possible contribution to cancer formation and/or progression. CENP-A overexpression is common among many cancers and predicts poor prognosis. Overexpression of CENP-A increases rates of CENP-A deposition ectopically at sites of high histone turnover, occluding CCCTC-binding factor (CTCF) binding. Ectopic CENP-A deposition leads to mitotic defects, centromere dysfunction and chromosomal instability (CIN), a hallmark of cancer. CENP-A overexpression is often accompanied by overexpression of its chaperone Holliday Junction Recognition Protein (HJURP), leading to epigenetic addiction in which increased levels of HJURP and CENP-A become necessary to support rapidly dividing p53 deficient cancer cells. Alterations in CENP-A posttranslational modifications are also linked to chromosome segregation errors and CIN. Collectively, CENP-A is pivotal to genomic stability through centromere maintenance, perturbation of which can lead to tumorigenesis.

Functional genetic variants in centrosome-related genes CEP72 and YWHAG confer susceptibility to gastric cancer.

Structural and numeric centrosome aberrations can induce chromosome segregation errors and promote tumor development and progression. We systematically evaluated associations of 19,603 single nucleotide polymorphisms (SNPs) across 136 centrosome-related genes with gastric cancer (GC) risk using four GWAS datasets with a total of 3771 cases and 5426 controls. We identified two loci at 15p13.3 and 7q11.23 significantly associated with GC risk, whose risk alleles were correlated with increased mRNA expression of CEP72 (P = 7.30 × 10-4) and YWHAG (P = 1.60 × 10-3), respectively. Dual-luciferase reporter assays confirmed that the risk T allele of rs924607 at 15p13.3 significantly increased a promoter activity of the reporter gene, leading to a higher CEP72 expression level. At 7q11.23, the risk haplotype of rs2961037 [G]-rs2961038 [G] significantly elevated an enhancer activity and the expression of YWHAG. Both the mRNA and protein levels of CEP72 and YWHAG were overexpressed in GC tumor tissues compared with peritumor tissues and overexpression of either gene showed an unfavorable prognosis of GC patients. Moreover, knockdown of either CEP72 or YWHAG inhibited GC cell proliferation, migration and invasion and promoted GC cell apoptosis. The genes coexpressed with CEP72 or YWHAG in GC tumor tissues were enriched in the Ras signaling pathway, which was confirmed that knockdown of either one decreased the expression of cyclin D1 but increased the expression of p21 and p27. In conclusion, genetic variants at 15p13.3 and 7q11.23 may confer GC risk via modulating the biological functions of CEP72 and YWHAG, respectively, suggesting the importance of centrosome-regulated genes in GC development.

DNA Replication Stress and Chromosomal Instability: Dangerous Liaisons.

Chromosomal instability (CIN) is associated with many human diseases, including neurodevelopmental or neurodegenerative conditions, age-related disorders and cancer, and is a key driver for disease initiation and progression. A major source of structural chromosome instability (s-CIN) leading to structural chromosome aberrations is “replication stress”, a condition in which stalled or slowly progressing replication forks interfere with timely and error-free completion of the S phase. On the other hand, mitotic errors that result in chromosome mis-segregation are the cause of numerical chromosome instability (n-CIN) and aneuploidy. In this review, we will discuss recent evidence showing that these two forms of chromosomal instability can be mechanistically interlinked. We first summarize how replication stress causes structural and numerical CIN, focusing on mechanisms such as mitotic rescue of replication stress (MRRS) and centriole disengagement, which prevent or contribute to specific types of structural chromosome aberrations and segregation errors. We describe the main outcomes of segregation errors and how micronucleation and aneuploidy can be the key stimuli promoting inflammation, senescence, or chromothripsis. At the end, we discuss how CIN can reduce cellular fitness and may behave as an anticancer barrier in noncancerous cells or precancerous lesions, whereas it fuels genomic instability in the context of cancer, and how our current knowledge may be exploited for developing cancer therapies.

Polo-like kinase 4 and Stromal antigen 3 are not associated with recurrent pregnancy loss caused by embryonic aneuploidy

No genetic association with recurrent pregnancy loss (RPL) caused by embryonic aneuploidy has been found. Recent studies have indicated that the common genetic variant rs2305957, surrounding the PLK4 gene, contributes to mitotic-origin aneuploidy risk during human early embryo development. The decrease in meiosis-specific cohesin causes predivision of sister chromatids in the centromere and chromosome segregation errors. STAG3 is a component of cohesin and is a meiosis-specific gene. Our case-control study included 184 patients with RPL whose previous products of conception (POC) exhibited aneuploidy and 190 fertile control women without a history of miscarriage. We performed a genetic association study to examine the genotype distribution at PLK4 (rs2305957) and STAG3 in patients with RPL caused by aneuploidy compared with controls. Regarding STAG3, SNPs with a minor allele frequency (MAF) threshold > 0.05 that were predicted to be binding sites of transcription factors and that showed significant associations in expression quantitative trait locus (e-QTL) analysis were selected. No significant differences in the MAF or distribution in any model of PLK4 (rs2305957) and 5 selected tag SNPs in STAG3 were found between the patients and controls. A further genome-wide association study is needed since a combination of genetic risk alleles might be useful in predicting future age-dependent RPL caused by aneuploidy.

Prc1-rich kinetochores are required for error-free acentrosomal spindle bipolarization during meiosis I in mouse oocytes

Acentrosomal meiosis in oocytes represents a gametogenic challenge, requiring spindle bipolarization without predefined bipolar cues. While much is known about the structures that promote acentrosomal microtubule nucleation, less is known about the structures that mediate spindle bipolarization in mammalian oocytes. Here, we show that in mouse oocytes, kinetochores are required for spindle bipolarization in meiosis I. This process is promoted by oocyte-specific, microtubule-independent enrichment of the antiparallel microtubule crosslinker Prc1 at kinetochores via the Ndc80 complex. In contrast, in meiosis II, cytoplasm that contains upregulated factors including Prc1 supports kinetochore-independent pathways for spindle bipolarization. The kinetochore-dependent mode of spindle bipolarization is required for meiosis I to prevent chromosome segregation errors. Human oocytes, where spindle bipolarization is reportedly error prone, exhibit no detectable kinetochore enrichment of Prc1. This study reveals an oocyte-specific function of kinetochores in acentrosomal spindle bipolarization in mice, and provides insights into the error-prone nature of human oocytes.

Cell division cycle 23 is required for mouse oocyte meiotic maturation.

Precise regulation of chromosome segregation during oocyte meiosis is of vital importance to mammalian reproduction. Anaphase promoting complex/cyclosome (APC/C) is reported to play an important role in metaphase-to-anaphase transition. Here we report that cell division cycle 23 (Cdc23, also known as APC8) plays a critical role in regulating the oocyte chromosome separation. Cdc23 localized on the meiotic spindle, and microinjection of Cdc23 siRNA caused decreased ratios of metaphase-to-anaphase transition. Loss of Cdc23 resulted in abnormal spindles, misaligned chromosomes, errors of homologous chromosome segregation, and production of aneuploid oocytes. Further study showed that inactivation of spindle assembly checkpoint and degradation of Cyclin B1 and securin were disturbed after Cdc23 knockdown. Furthermore, we found that inhibiting spindle assembly checkpoint protein Msp1 partly rescued the decreased polar body extrusion and reduced the accumulation of securin in Cdc23 knockdown oocytes. Taken together, our data demonstrate that Cdc23 is required for the chromosome segregation through regulating the spindle assembly checkpoint activity, and cyclin B1 and securin degradation in meiotic mouse oocytes.