RESUMEN
Embryonic stem cells (ESCs) can instruct the conversion of differentiated cells toward pluripotency following cell-to-cell fusion by a mechanism that is rapid but poorly understood. Here, we used centrifugal elutriation to enrich for mouse ESCs at sequential stages of the cell cycle and showed that ESCs in S/G2 phases have an enhanced capacity to dominantly reprogram lymphocytes and fibroblasts in heterokaryon and hybrid assays. Reprogramming success was associated with an ability to induce precocious nucleotide incorporation within the somatic partner nuclei in heterokaryons. BrdU pulse-labeling experiments revealed that virtually all successfully reprogrammed somatic nuclei, identified on the basis of Oct4 re-expression, had undergone DNA synthesis within 24 hr of fusion with ESCs. This was essential for successful reprogramming because drugs that inhibited DNA polymerase activity effectively blocked pluripotent conversion. These data indicate that nucleotide incorporation is an early and critical event in the epigenetic reprogramming of somatic cells in experimental ESC-heterokaryons.
Asunto(s)
Replicación del ADN , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Animales , Linfocitos B/citología , Fusión Celular , Núcleo Celular/metabolismo , Reprogramación Celular , Células Madre Embrionarias/citología , Fibroblastos/citología , Humanos , Ratones , Nucleótidos/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.
Asunto(s)
Inestabilidad Cromosómica , Daño del ADN , Duplicación de Gen , Fase S , Tetraploidía , Inestabilidad Cromosómica/genética , Replicación del ADN , Humanos , Cariotipo , Mitosis , Fase S/genéticaRESUMEN
To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation. Using an optimized double-degron system, together with kinase inhibitors to enforce tight inhibition of key proteins, we find that human cells unable to initiate DNA replication prematurely enter mitosis. Preventing DNA replication licensing and/or firing causes prompt activation of CDK1 and PLK1 in S phase. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of mitotic kinases, which induces severe replication stress. Our results demonstrate that, rather than merely a cell cycle output, DNA replication is an integral signaling component that restricts activation of mitotic kinases. DNA replication thus functions as a brake that determines cell cycle duration.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Fase S , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Activación Enzimática , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Quinasa Tipo Polo 1RESUMEN
Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2-phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin-dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phosphorylation.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Mitosis , Proteína Fosfatasa 2/metabolismo , Proteína Quinasa CDC2/genética , Línea Celular , Ciclina A/genética , Ciclina B/genética , Humanos , Proteína Fosfatasa 2/genéticaRESUMEN
Cell-cycle transitions in higher eukaryotes are regulated by different cyclin-dependent kinases (CDKs) and their activating cyclin subunits. Based on pioneering findings that a dominant-negative mutation of CDK1 blocks the cell cycle at G2-M phase, whereas dominant-negative CDK2 inhibits the transition into S phase, a model of cell-cycle control has emerged in which each transition is regulated by a specific subset of CDKs and cyclins. Recent work with gene-targeted mice has led to a revision of this model. We discuss cell-cycle control in light of overlapping and essential functions of the different CDKs and cyclins.
Asunto(s)
Ciclo Celular , Quinasas Ciclina-Dependientes/fisiología , Animales , Ciclinas/fisiología , Marcación de Gen , Humanos , RatonesRESUMEN
Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cromatina/genética , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Interfase/genética , Proteómica/métodos , Inteligencia Artificial , Proteínas de Ciclo Celular/clasificación , Centrifugación , Quinasas Ciclina-Dependientes/genética , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Ontología de Genes , Humanos , Espectrometría de Masas , Modelos Biológicos , Anotación de Secuencia MolecularRESUMEN
Mitotic entry and exit are switch-like transitions that are driven by the activation and inactivation of Cdk1 and mitotic cyclins. This simple on/off reaction turns out to be a complex interplay of various reversible reactions, feedback loops, and thresholds that involve both the direct regulators of Cdk1 and its counteracting phosphatases. In this review, we summarize the interplay of the major components of the system and discuss how they work together to generate robustness, bistability, and irreversibility. We propose that it may be beneficial to regard the entry and exit reactions as two separate reversible switches that are distinguished by differences in the state of phosphatase activity, mitotic proteolysis, and a dramatic rearrangement of cellular components after nuclear envelope breakdown, and discuss how the major Cdk1 activity thresholds could be determined for these transitions.
Asunto(s)
Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/fisiología , Mitosis , Animales , HumanosRESUMEN
Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, inhibition or depletion of neither Fcp1 nor PP2A appears to block dephosphorylation of the bulk of mitotic Cdk1 substrates during mitotic exit. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Mitosis/genética , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas/genética , Proteína Fosfatasa 2/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/genética , Ciclina B/genética , Ciclina B/metabolismo , Redes Reguladoras de Genes/genética , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , División Celular , Centrosoma/metabolismo , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Línea Celular , Humanos , Quinasa Tipo Polo 1RESUMEN
Squamous or epidermoid cancer arises in stratified epithelia but also is frequent in the non-epidermoid epithelium of the lung by unclear mechanisms. A poorly studied mitotic checkpoint drives epithelial cells bearing irreparable genetic damage into epidermoid differentiation. We performed an RNA-sequencing gene search to target unknown regulators of this response and selected the SUMO regulatory protein SENP2. Alterations of SENP2 expression have been associated with some types of cancer. We found the protein to be strongly localised to mitotic spindles of freshly isolated human epidermal cells. Primary cells rapidly differentiated after silencing SENP2 with specific shRNAs. Loss of SENP2 produced in synchronised epithelial cells delays in mitotic entry and exit and defects in chromosomal alignment. The results altogether strongly argue for an essential role of SENP2 in the mitotic spindle and hence in controlling differentiation. In addition, the expression of SENP2 displayed an inverse correlation with the immuno-checkpoint biomarker PD-L1 in a pilot collection of aggressive lung carcinomas. Consistently, metastatic head and neck cancer cells that do not respond to the mitosis-differentiation checkpoint were resistant to depletion of SENP2. Our results identify SENP2 as a novel regulator of the epithelial mitosis-differentiation checkpoint and a potential biomarker in epithelial cancer.
Asunto(s)
Diferenciación Celular , Cisteína Endopeptidasas , Mitosis , Humanos , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Línea Celular Tumoral , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/genética , Homeostasis , Células Epiteliales/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Huso Acromático/metabolismoRESUMEN
In vertebrates Cdk1 is required to initiate mitosis; however, any functionality of this kinase during S phase remains unclear. To investigate this, we generated chicken DT40 mutants, in which an analog-sensitive mutant cdk1 as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. In cells that also lack Cdk2, we find that Cdk1 activity is essential for DNA replication initiation and centrosome duplication. The presence of a single Cdk2 allele renders S phase progression independent of Cdk1, which suggests a complete overlap of these kinases in S phase control. Moreover, we find that Cdk1 inhibition did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition during mitosis of Cdk1 causes rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor Geminin. This study demonstrates essential functions of Cdk1 in the control of S phase, and exemplifies a chemical genetics approach to target cyclin-dependent kinases in vertebrate cells.
Asunto(s)
Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Fase S , Alelos , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Pollos , Inhibidores de Cisteína Proteinasa/farmacología , Geminina , Células HeLa , Humanos , Leupeptinas/farmacología , Modelos Biológicos , Mutación , Purinas/farmacología , RoscovitinaRESUMEN
Degron tags allow the precise and well-controlled analysis of essential genes by rapidly inducing degradation of the protein of interest. This is critical when the consequences of loss of gene function needs to be analyzed in a strictly defined time window such as a specific cell cycle phase. We have recently published the successful application of degron tags to analyze cell cycle genes such as CDC6, CCNA2, and CCNB1. A critical aspect of our approach was to combine two tags to generate a synergy in the degradation dynamics. Here we outline our approach and describe some of the essential steps to generate double-degron-tagged genes in RPE-1 cells. Similar procedures can easily be applied to other cell lines.
Asunto(s)
Ácidos Indolacéticos , Proteínas , Línea Celular , Ácidos Indolacéticos/metabolismo , Proteínas/metabolismo , ProteolisisRESUMEN
Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.
Asunto(s)
Roturas del ADN de Cadena Simple , Reparación del ADN , Glicósido Hidrolasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Línea Celular Tumoral , Pollos , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Glicósido Hidrolasas/deficiencia , Células HeLa , Histonas/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Poli(ADP-Ribosa) Polimerasas/fisiología , Recombinación Genética , Animales , Antígenos Nucleares/fisiología , Camptotecina/farmacología , Línea Celular , Pollos , Proteínas de Unión al ADN/genética , Humanos , Autoantígeno Ku , Poli(ADP-Ribosa) Polimerasas/genética , Inhibidores de Topoisomerasa I , Ubiquitina-Proteína LigasasRESUMEN
Centrosome separation in late G2/ early prophase requires precise spatial coordination that is determined by a balance of forces promoting and antagonizing separation. The major effector of centrosome separation is the kinesin Eg5. However, the identity and regulation of Eg5-antagonizing forces is less well characterized. By manipulating candidate components, we find that centrosome separation is reversible and that separated centrosomes congress toward a central position underneath the flat nucleus. This positioning mechanism requires microtubule polymerization, as well as actin polymerization. We identify perinuclear actin structures that form in late G2/early prophase and interact with microtubules emanating from the centrosomes. Disrupting these structures by breaking the interactions of the linker of nucleoskeleton and cytoskeleton (LINC) complex with perinuclear actin filaments abrogates this centrosome positioning mechanism and causes an increase in subsequent chromosome segregation errors. Our results demonstrate how geometrical cues from the cell nucleus coordinate the orientation of the emanating spindle poles before nuclear envelope breakdown.
Asunto(s)
Actinas/metabolismo , Centrosoma/metabolismo , Segregación Cromosómica/genética , Profase/genética , HumanosRESUMEN
Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Roturas del ADN de Doble Cadena , Reparación del ADN/efectos de los fármacos , Leupeptinas/farmacología , Inhibidores de Proteasoma , Recombinación Genética/efectos de los fármacos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/fisiología , Genes BRCA1 , Células HeLa , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinasa Rad51/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Entry into mitosis is triggered by the activation of cyclin-dependent kinase 1 (Cdk1). This simple reaction rapidly and irreversibly sets the cell up for division. Even though the core step in triggering mitosis is so simple, the regulation of this cellular switch is highly complex, involving a large number of interconnected signalling cascades. We do have a detailed knowledge of most of the components of this network, but only a poor understanding of how they work together to create a precise and robust system that ensures that mitosis is triggered at the right time and in an orderly fashion. In this review, we will give an overview of the literature that describes the Cdk1 activation network and then address questions relating to the systems biology of this switch. How is the timing of the trigger controlled? How is mitosis insulated from interphase? What determines the sequence of events, following the initial trigger of Cdk1 activation? Which elements ensure robustness in the timing and execution of the switch? How has this system been adapted to the high levels of replication stress in cancer cells?
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/genética , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/genética , Replicación del ADN , Mitosis , Proteínas Tirosina Quinasas/genética , Fosfatasas cdc25/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Regulación de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Oocitos/citología , Oocitos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Biología de Sistemas , Factores de Tiempo , Fosfatasas cdc25/metabolismoRESUMEN
Glioma stem cells account for glioblastoma relapse and resistance to conventional therapies, and protein kinases, involved in the regulation of the mitotic machinery (i.e., Aurora kinases), have recently emerged as attractive therapeutic targets. In this study, we investigated the effect of Aurora kinases inhibition in five glioma stem cell lines isolated from glioblastoma patients. As expected, cell lines responded to the loss of Aurora kinases with cytokinesis failure and mitotic exit without cell division. Surprisingly, this resulted in a proliferative arrest in only two of the five cell lines. These sensitive cell lines entered a senescent/autophagic state following aberrant mitotic exit, while the non-sensitive cell lines continued to proliferate. This senescence response did not correlate with TP53 mutation status but only occurred in the cell lines with the highest chromosome content. Repeated rounds of Aurora kinases inhibition caused a gradual increase in chromosome content in the resistant cell lines and eventually caused a similar senescence response and proliferative arrest. Our results suggest that a ploidy threshold is the main determinant of Aurora kinases sensitivity in TP53 mutant glioma stem cells. Thus, ploidy could be used as a biomarker for treating glioma patients with Aurora kinases inhibitors.
RESUMEN
To ensure proper segregation during mitosis, chromosomes must be efficiently captured by spindle microtubules and subsequently aligned on the mitotic spindle. The efficacy of chromosome interaction with the spindle can be influenced by how widely chromosomes are scattered in space. Here, we quantify chromosome-scattering volume (CSV) and find that it is reduced soon after nuclear envelope breakdown (NEBD) in human cells. The CSV reduction occurs primarily independently of microtubules and is therefore not an outcome of interactions between chromosomes and the spindle. We find that, prior to NEBD, an acto-myosin network is assembled in a LINC complex-dependent manner on the cytoplasmic surface of the nuclear envelope. This acto-myosin network remains on nuclear envelope remnants soon after NEBD, and its myosin-II-mediated contraction reduces CSV and facilitates timely chromosome congression and correct segregation. Thus, we find a novel mechanism that positions chromosomes in early mitosis to ensure efficient and correct chromosome-spindle interactions.