ABSTRACT
In prophase of meiosis I, homologous chromosomes pair and become connected by cross-overs. Chiasmata, the connections formed by cross-overs, enable the chromosome pair, called a bivalent, to attach as a single unit to the spindle. When the meiotic spindle forms in prometaphase, most bivalents are associated with one spindle pole and then go through a series of oscillations on the spindle, attaching to and detaching from microtubules until the partners of the bivalent become bioriented-attached to microtubules from opposite sides of the spindle. The conserved kinase, Mps1, is essential for the bivalents to be pulled by microtubules across the spindle in prometaphase. Here we show that MPS1 is needed for efficient triggering of the migration of microtubule-attached kinetochores toward the poles and promotes microtubule depolymerization. Our data support the model Mps1 acts at the kinetochore to coordinate the successful attachment of a microtubule and the triggering of microtubule depolymerization to then move the chromosome.
Subject(s)
Chromosomes/physiology , Prometaphase/physiology , Protein Serine-Threonine Kinases/physiology , Saccharomyces cerevisiae Proteins/physiology , Cell Polarity , Chromosome Pairing , Kinetochores/physiology , Microtubules/physiology , Mutation , Prometaphase/genetics , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , SaccharomycetalesABSTRACT
BACKGROUND/AIM: Accurate regulation of the spindle assembly checkpoint (SAC) and anaphase promoting complex/cyclosome (APC/C) are essential for the correct execution of mitosis. In this work, we focused on MAD2L2 (REV7), a central translesion (TLS) protein, which also functions as a mitotic regulator by inhibiting APC/C in prometaphase. MATERIALS AND METHODS: Using bioinformatics analysis, live cell imaging and APC/C protein binding and degradation assays, we explored the influence of MAD2L2 over-expression in breast cancer. RESULTS: A significant over-expression of MAD2L2 was found in triple negative breast cancers (TNBC), compared to other breast cancers, correlating to poor patient prognosis. We also identified significant over-expression of MAD2L2 in the MDA-MB-157 triple negative (TN) cell line. A high percentage of MDA-MB-157 cells failed to complete mitosis and died during mitosis or shortly after. In addition, these cells completed mitosis at a significantly slower rate than control cells. MDA-MB-157 cells present high levels of mitotic slippage upon nocodazole treatment and acute dysregulation in APC/C function and substrate degradation. Moreover, silencing of MAD2L2 in the MDA-MB-157 cell line improved mitotic phenotypes. CONCLUSION: MAD2L2 over-expression supports the carcinogenic phenotype of MDA-MB-157 cells by promoting uncontrolled mitosis.
Subject(s)
Breast Neoplasms/genetics , Mad2 Proteins/genetics , Mitosis/genetics , Breast Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic/genetics , HeLa Cells , Humans , M Phase Cell Cycle Checkpoints/genetics , Prometaphase/geneticsABSTRACT
The faithful inheritance of chromosomes is essential for the propagation of organisms. In eukaryotes, central to this process is the mitotic spindle. Recently, we have identified TRIM8 as a gene aberrantly expressed in gliomas whose expression reduces the clonogenic potential in the patients' glioma cells. TRIM8 encodes an E3 ubiquitin ligase involved in various pathological processes, including hypertrophy, antiviral defense, encephalopathy, and cancer development. To gain insights into the TRIM8 functions, we characterized the TRIM8 interactome in primary mouse embryonic neural stem cells using proteomics. We found that TRIM8 interacts with KIFC1, and KIF11/Eg5, two master regulators of mitotic spindle assembly and cytoskeleton reorganization. By exploring the TRIM8 role in the mitotic spindle machinery, we showed that TRIM8 localizes at the mitotic spindle during mitosis and plays a role in centrosome separation at the beginning of mitosis with a subsequent delay of the mitotic progression and impact on chromosomal stability.
Subject(s)
Carrier Proteins/metabolism , Chromosomal Instability , Kinesins/metabolism , Nerve Tissue Proteins/metabolism , Spindle Apparatus/metabolism , Ubiquitin-Protein Ligases/metabolism , beta Karyopherins/metabolism , Aneuploidy , Animals , Carrier Proteins/genetics , Cells, Cultured , Embryo, Mammalian , Fibroblasts , HEK293 Cells , Humans , Mice , Micronuclei, Chromosome-Defective , Mitosis , Nerve Tissue Proteins/genetics , Neural Stem Cells , Primary Cell Culture , Prometaphase/genetics , Protein Binding/genetics , ProteomicsABSTRACT
We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI50 compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Curcumin/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Prometaphase/drug effects , Administration, Oral , Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Antineoplastic Agents, Phytogenic/chemistry , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Death/drug effects , Cell Division/drug effects , Cell Division/genetics , Cell Movement/drug effects , Cell Proliferation/drug effects , Cellular Senescence/drug effects , Curcumin/analogs & derivatives , Glutathione S-Transferase pi/antagonists & inhibitors , Glutathione S-Transferase pi/genetics , Glutathione S-Transferase pi/metabolism , Glutathione Transferase/antagonists & inhibitors , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , HEK293 Cells , HeLa Cells , Humans , K562 Cells , Lactoylglutathione Lyase/antagonists & inhibitors , Lactoylglutathione Lyase/genetics , Lactoylglutathione Lyase/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , MCF-7 Cells , Mice, Nude , NAD(P)H Dehydrogenase (Quinone)/antagonists & inhibitors , NAD(P)H Dehydrogenase (Quinone)/genetics , NAD(P)H Dehydrogenase (Quinone)/metabolism , Peroxiredoxins/antagonists & inhibitors , Peroxiredoxins/genetics , Peroxiredoxins/metabolism , Prometaphase/genetics , Reactive Oxygen Species/metabolism , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural cis-elements, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.
Subject(s)
CCCTC-Binding Factor/metabolism , Cell Cycle/genetics , Nucleosomes/physiology , Binding Sites , Cells, Cultured , Chromatin/chemistry , HeLa Cells , Histone Code , Humans , Interphase/genetics , Mitosis/genetics , Nucleotide Motifs , Prometaphase/genetics , Transcription Initiation SiteABSTRACT
During the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in anaphase. The spindle assembly checkpoint (SAC) is a quality control mechanism that monitors proper attachment of microtubules to chromosome kinetochores during prometaphase. Segregation occurs only when each chromosome is bi-oriented with each kinetochore pair attached to microtubules emanating from opposite spindle poles. Overexpression of the protein kinase Aurora A is a feature of various cancers and is thought to enable tumour cells to bypass the SAC, leading to aneuploidy. Here, we took advantage of a chemical and chemical-genetic approach to specifically inhibit Aurora A kinase activity in late prometaphase. We observed that a loss of Aurora A activity directly affects SAC function, that Aurora A is essential for maintaining the checkpoint protein Mad2 on unattached kinetochores and that inhibition of Aurora A leads to loss of the SAC, even in the presence of nocodazole or Taxol. This is a new finding that should affect the way Aurora A inhibitors are used in cancer treatments.This article has an associated First Person interview with the first authors of the paper.
Subject(s)
Aurora Kinase A/genetics , M Phase Cell Cycle Checkpoints/genetics , Mad2 Proteins/genetics , Prometaphase/genetics , Anaphase/genetics , Aurora Kinase A/antagonists & inhibitors , Azepines/pharmacology , Cell Line, Tumor , Chromatids/genetics , Chromosome Segregation/drug effects , Gene Expression Regulation, Enzymologic/genetics , Humans , Kinetochores/drug effects , Microtubules/drug effects , Mitosis/drug effects , Mitosis/genetics , Nocodazole/pharmacology , Paclitaxel/pharmacology , Prometaphase/drug effects , Pyrimidines/pharmacology , Spindle Apparatus/geneticsABSTRACT
ARHGAP19 is a hematopoietic-specific Rho GTPase-activating protein (RhoGAP) that acts through the RhoA/ROCK pathway to critically regulate cell elongation and cytokinesis during lymphocyte mitosis. We report here that, during mitosis progression, ARHGAP19 is sequentially phosphorylated by the RhoA-activated kinases ROCK1 and ROCK2 (hereafter ROCK) on serine residue 422, and by CDK1 on threonine residues 404 and 476. The phosphorylation of ARHGAP19 by ROCK occurs before mitosis onset and generates a binding site for 14-3-3 family proteins. ARHGAP19 is then phosphorylated by CDK1 in prometaphase. The docking of 14-3-3 proteins to phosphorylated S422 protects ARHGAP19 from dephosphorylation of the threonine sites and prevents ARHGAP19 from relocating to the plasma membrane during prophase and metaphase, thus allowing RhoA to become activated. Disruption of these phosphorylation sites results in premature localization of ARHGAP19 at the cell membrane and in its enrichment to the equatorial cortex in anaphase leading to cytokinesis failure and cell multinucleation.
Subject(s)
Cytokinesis/genetics , GTPase-Activating Proteins/genetics , Mitosis/genetics , rhoA GTP-Binding Protein/genetics , 14-3-3 Proteins/genetics , CDC2 Protein Kinase/genetics , Humans , Jurkat Cells , Phosphorylation/genetics , Prometaphase/genetics , Serine/genetics , rho-Associated Kinases/geneticsABSTRACT
Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle regulatory mechanisms and cell cycle progression invariably require synchronization of cell populations at specific cell cycle stages. Several methods have been established to synchronize cells, including serum deprivation, contact inhibition, centrifugal elutriation, and drug-dependent synchronization. Despite potential adverse cellular consequences of synchronizing cells by pharmacological agents, drug-dependent methods can be advantageous when studying later cell cycle events to ensure specific enrichment at selected mitotic stages. This chapter describes protocols used in our laboratory for isolating mitotic mammalian cells in a large-scale manner. In particular, we discuss the technical aspects of adherent or suspension cell isolation, the methods necessary to enrich cells at different mitotic stages and the optimized culture conditions.
Subject(s)
Cell Cycle/physiology , Mitosis/physiology , Anaphase/genetics , Cell Culture Techniques , Cell Cycle/genetics , Fluorescent Antibody Technique , HeLa Cells , Humans , Metaphase/genetics , Mitosis/genetics , Prometaphase/genetics , Telophase/geneticsABSTRACT
TD-60 (also known as RCC2) is a highly conserved protein that structurally resembles the Ran guanine exchange factor (GEF) RCC1, but has not previously been shown to have GEF activity. TD-60 has a typical chromosomal passenger complex (CPC) distribution in mitotic cells, but associates with integrin complexes and is involved in cell motility during interphase. Here we show that TD-60 exhibits GEF activity, in vitro and in cells, for the small GTPase RalA. TD-60 or RalA depletion causes spindle abnormalities in prometaphase associated with abnormal centromeric accumulation of CPC components. TD-60 and RalA apparently work together to contribute to the regulation of kinetochore-microtubule interactions in early mitosis. Importantly, several mitotic phenotypes caused by TD-60 depletion are reverted by the expression of a GTP-locked mutant, RalA (Q72L). The demonstration that a small GTPase participates in the regulation of the CPC reveals a level of mitotic regulation not suspected in previous studies.
Subject(s)
Chromosomal Proteins, Non-Histone/genetics , Guanine Nucleotide Exchange Factors/genetics , Mitosis/genetics , Spindle Apparatus/metabolism , ral GTP-Binding Proteins/genetics , Aurora Kinase B/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Movement , Centromere/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , Guanine Nucleotide Exchange Factors/metabolism , HeLa Cells , Humans , In Vitro Techniques , Inhibitor of Apoptosis Proteins/metabolism , Prometaphase/genetics , Survivin , ral GTP-Binding Proteins/metabolismABSTRACT
Polyoma small T antigen (PyST), an early gene product of the polyoma virus, has been shown to cause cell death in a number of mammalian cells in a protein phosphatase 2A (PP2A)-dependent manner. In the current study, using a cell line featuring regulated expression of PyST, we found that PyST arrests cells in mitosis. Live-cell and immunofluorescence studies showed that the majority of the PyST expressing cells were arrested in prometaphase with almost no cells progressing beyond metaphase. These cells exhibited defects in chromosomal congression, sister chromatid cohesion and spindle positioning, thereby resulting in the activation of the spindle assembly checkpoint. Prolonged mitotic arrest then led to cell death via mitotic catastrophe. Cell cycle inhibitors that block cells in G1/S prevented PyST-induced death. PyST-induced cell death that occurs during M is not dependent on p53 status. These data suggested, and our results confirmed, that PP2A inhibition could be used to preferentially kill cancer cells with p53 mutations that proliferate normally in the presence of cell cycle inhibitors.
Subject(s)
Antigens, Viral, Tumor/metabolism , M Phase Cell Cycle Checkpoints/genetics , Polyomavirus/metabolism , Protein Phosphatase 2/antagonists & inhibitors , Spindle Apparatus/genetics , 3T3 Cells , Animals , Antigens, Viral, Tumor/biosynthesis , Antigens, Viral, Tumor/genetics , Apoptosis/genetics , Cell Cycle/genetics , Cell Line, Tumor , G1 Phase Cell Cycle Checkpoints/genetics , HeLa Cells , Humans , Mice , Mitosis/genetics , Prometaphase/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
An intercentrosomal linker keeps a cell's two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF(ßTrCP) (Skp1-Cul1-F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by ßTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215. We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.
Subject(s)
Antigens/metabolism , Centrioles/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Nerve Tissue Proteins/metabolism , Proteolysis , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , HEK293 Cells , HeLa Cells , Humans , Metaphase/genetics , Phosphorylation , Prometaphase/genetics , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference , RNA, Small Interfering , SKP Cullin F-Box Protein Ligases/genetics , Polo-Like Kinase 1ABSTRACT
In budding yeast, meiotic commitment is the irreversible continuation of the developmental path of meiosis. After reaching meiotic commitment, cells finish meiosis and gametogenesis, even in the absence of the meiosis-inducing signal. In contrast, if the meiosis-inducing signal is removed and the mitosis-inducing signal is provided prior to reaching meiotic commitment, cells exit meiosis and return to mitosis. Previous work has shown that cells commit to meiosis after prophase I but before entering the meiotic divisions. Since the Ndt80 transcription factor induces expression of middle meiosis genes necessary for the meiotic divisions, we examined the role of the NDT80 transcriptional network in meiotic commitment. Using a microfluidic approach to analyze single cells, we found that cells commit to meiosis in prometaphase I, after the induction of the Ndt80-dependent genes. Our results showed that high-level expression of NDT80 is important for the timing and irreversibility of meiotic commitment. A modest reduction in NDT80 levels delayed meiotic commitment based on meiotic stages, although the timing of each meiotic stage was similar to that of wildtype cells. A further reduction of NDT80 resulted in the surprising finding of inappropriately uncommitted cells: withdrawal of the meiosis-inducing signal and addition of the mitosis-inducing signal to cells at stages beyond metaphase I caused return to mitosis, leading to multi-nucleate cells. Since Ndt80 enhances its own transcription through positive feedback, we tested whether positive feedback ensured the irreversibility of meiotic commitment. Ablating positive feedback in NDT80 expression resulted in a complete loss of meiotic commitment. These findings suggest that irreversibility of meiotic commitment is a consequence of the NDT80 transcriptional positive feedback loop, which provides the high-level of Ndt80 required for the developmental switch of meiotic commitment. These results also illustrate the importance of irreversible meiotic commitment for maintaining genome integrity by preventing formation of multi-nucleate cells.
Subject(s)
DNA-Binding Proteins/genetics , Meiosis/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics , Cell Cycle Proteins/biosynthesis , DNA, Fungal/biosynthesis , DNA, Fungal/genetics , DNA-Binding Proteins/biosynthesis , Gametogenesis/genetics , Microfluidics/methods , Prometaphase/genetics , Promoter Regions, Genetic/genetics , Protein Serine-Threonine Kinases/biosynthesis , Saccharomyces cerevisiae Proteins/biosynthesis , Spindle Apparatus/metabolism , Transcription Factors/biosynthesisABSTRACT
Treatment of cancer cells with microtubule inhibitors causes mitotic arrest, which subsequently leads to cell death via activation of the intrinsic apoptotic pathway. Mitotically arrested cells typically display increased phosphorylation (i.e., inactivation) of two key anti-apoptotic proteins, Bcl-2 and Bcl-XL , but the mechanisms that regulate their phosphorylation as well as their role in apoptotic cell death following mitotic arrest are still poorly understood at present, which are the focus of this study. We recently showed that cyclin B1 and cell division cycle 2 (Cdc2) proteins are strongly up-regulated in human breast cancer cells following treatment with nocodazole (a prototypical microtubule inhibitor), and their up-regulation plays a critical role in the development of mitotic prometaphase arrest. In this study, we present evidence showing that the up-regulated cyclin B1/Cdc2 complex in nocodazole-treated human breast cancer cells is also responsible for the increased phosphorylation of Bcl-2 and Bcl-XL . However, only the increased phosphorylation of Bcl-XL , but not the phosphorylation of Bcl-2, contributes to subsequent activation of the intrinsic cell death pathway. In addition, evidence is presented to show that mitotic arrest deficient 2 (MAD2) is a key upstream mediator of the up-regulation of cyclin B1/Cdc2 as well as the subsequent increase in phosphorylationof Bcl-2 and Bcl-XL in nocodazole-treated cancer cells. Together, these results reveal that the up-regulated cyclin B1/Cdc2 complex not only mediates prometaphase arrest in nocodazole-treated cells, but also activates the subsequent intrinsic cell death pathway in these cells via increased phosphorylation of Bcl-XL .
Subject(s)
Apoptosis/genetics , Cell Cycle Checkpoints/genetics , Cyclin B1/genetics , Cyclin B/genetics , Mitosis/genetics , Phosphorylation/genetics , bcl-X Protein/genetics , Apoptosis/drug effects , CDC2 Protein Kinase , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cyclin-Dependent Kinases , Humans , MCF-7 Cells , Mad2 Proteins/genetics , Mitosis/drug effects , Nocodazole/pharmacology , Phosphorylation/drug effects , Prometaphase/drug effects , Prometaphase/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Up-Regulation/drug effectsABSTRACT
The learning of cytyogenetic special of cariotip in children with the bronchial asthma maked by course of the investigation of prometahyases chromosomes of limphocytes of the periferic bloods. The quantity of association of acrocentric chromosome was analised. The 82 children in age 6-18 years old with the bronchial asthma and with the different control were learned by results of asthma--test control. In children with the noncontrol bronchial asthma the big frequency of of association of acrocentric chromosome 13-15 (D-D), 21-22 (G-G) n 13-15--21-22 (D-G) were received. In patients with the bronchial asthma the lover mitotic activity by healthy were marked (P(N) < 0.05). With the degree of activity it was decreased.
Subject(s)
Asthma/genetics , Chromosome Aberrations , Chromosomes, Human, 13-15/genetics , Chromosomes, Human, 21-22 and Y/genetics , Lymphocytes , Prometaphase/genetics , Adolescent , Asthma/pathology , Asthma/prevention & control , Child , Genetic Markers , Humans , Karyotype , Karyotyping , Lymphocytes/pathologyABSTRACT
Cardiac glycosides as inhibitors of the sodium/potassium adenosine triphosphatase (sodium pump) have been reported to block cancer growth by inducing G2/M phase arrest in many cancer cells. However, no detailed studies have been performed to distinguish between these two phases of cardiac glycoside-arrested cells. Furthermore, the underlying mechanisms involved in this cell cycle arrest process are still not known. Here, we report that bufalin and other cardiac glycosides potently induce mitotic arrest by the downregulation of polo-like kinase 1 (Plk1) expression. Live-cell imaging results demonstrate that bufalin-treated cells exhibit a marked delay in entering prophase at an early stage and are then arrested at prometaphase or induced entry into apoptosis. This phenotypic change is attributed to the downregulation of Plk1. We also show that bufalin and the knockdown of sodium pump reduce Plk1, at least in part, through downregulation of the nuclear transcription factors, hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB). These findings suggest that cardiac glycosides induce mitotic arrest and apoptosis through HIF-1α- and NF-κB-mediated downregulation of Plk1 expression, demonstrating that HIF-1α and NF-κB are critical targets of cardiac glycosides in exerting their anticancer action.
Subject(s)
Cardiac Glycosides/pharmacology , Cell Cycle Proteins/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Cycle Proteins/genetics , Cell Division/drug effects , Cell Division/genetics , Cell Line, Tumor , Down-Regulation/drug effects , G2 Phase/drug effects , G2 Phase/genetics , HCT116 Cells , HT29 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Microtubules/genetics , Microtubules/metabolism , NF-kappa B/genetics , Prometaphase/drug effects , Prometaphase/genetics , Prophase/drug effects , Prophase/genetics , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Polo-Like Kinase 1ABSTRACT
One apparent feature of cancerous cells is genomic instability, which may include various types of chromosomal aberrations, such as translocation, aneuploidy, and the presence of micronuclei inside the cells. Mutagenic factors that promote the emergence of genomic instability are recognized as risk factors for the development of human malignancies. In Asia, betel quid (BQ) chewing is one of such risk factors for oral cancer. Areca nut is an essential constitute of BQ and is declared as a group I carcinogen by the International Agency for Research on Cancer. However, the molecular and cellular mechanisms regarding the carcinogenicity of areca nut are not fully explored. Here we reported that arecoline, a major alkaloid of areca nut, could arrest cells at prometaphase with large amounts of misaligned chromosomes. This prometaphase arrest was evidenced by condensed chromosome pattern, increased histone H3 phosphorylation, and accumulation of mitotic proteins, including aurora A and cyclin B(1). To investigate the molecular mechanisms accounting for arecoline-induced prometaphase arrest, we found that arecoline could stabilize mitotic spindle assembly, which led to distorted organization of mitotic spindles, misalignment of chromosomes, and up-regulation of spindle assembly checkpoint (SAC) genes. The SAC proteins BubR1 and Mps1 were differentially modified between the cells treated with arecoline and nocodazole. This together with aurora A overexpression suggested that SAC might be partly suppressed by arecoline. As a result, the arecoline-exposed cells might produce progeny that contained various chromosomal aberrations and exhibited genomic instability.
Subject(s)
Areca/chemistry , Arecoline/pharmacology , Carcinogens/pharmacology , Mouth Neoplasms/chemically induced , Prometaphase/drug effects , Spindle Apparatus/drug effects , Arecoline/adverse effects , Cell Line, Tumor , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cells, Cultured , Genomic Instability/genetics , Histones/metabolism , Humans , Mitosis/drug effects , Mitosis/genetics , Mouth Neoplasms/genetics , Mouth Neoplasms/pathology , Prometaphase/genetics , Spindle Apparatus/genetics , Tumor Suppressor Protein p53/antagonists & inhibitorsABSTRACT
Proper chromosome congression (the process of aligning chromosomes on the spindle) contributes to accurate and faithful chromosome segregation. It is widely accepted that congression requires kinetochore fibres (K-fibres), microtubule bundles that extend from the kinetochores to spindle poles. Here, we demonstrate that chromosomes in human cells co-depleted of HSET (human kinesin-14) and hNuf2 (human Ndc80/Hec1-complex component) can congress to the metaphase plate in the absence of K-fibres. However, the chromosomes are not stably maintained at the metaphase plate under these conditions. Chromosome congression in HSET + hNuf2 co-depleted cells required the plus-end directed motor CENP-E (centromere protein E; kinesin-7 family member), which has been implicated in the gliding of mono-oriented kinetochores alongside adjacent K-fibres. Thus, proper end-on attachment of kinetochores to microtubules is not necessary for chromosome congression. Instead, our data support the idea that congression allows unattached chromosomes to move to the middle of the spindle where they have a higher probability of establishing connections with both spindle poles. These bi-oriented connections are also used to maintain stable chromosome alignment at the spindle equator.
Subject(s)
Chromosomes/metabolism , Kinetochores/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/physiology , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/physiology , Chromosome Segregation/genetics , Chromosome Segregation/physiology , Chromosomes/genetics , Fluorescent Antibody Technique , HeLa Cells , Humans , Kinesins/genetics , Kinesins/metabolism , Kinesins/physiology , Metaphase/genetics , Prometaphase/genetics , Spindle Apparatus/metabolismABSTRACT
The use of prometaphase chromosomes prepared for high-resolution imaging is essential for accurate cytogenetic investigations. The process of Giemsa chromosome banding (G-banding), however, is often time consuming and difficult to standardize owing to differing ambient conditions and inter-operator variability. Consequently, many laboratories currently are introducing automatic metaphase finder and analysis systems to achieve the goals of higher throughput of samples and more consistent chromosome quality. In this context, we investigated the use of automation to improve the G-banding process. We investigated the use of the Shandon Thermo Varistain Gemini automatic stainer to replicate the manual process of G-banding. We compared the current manual method and the automated procedure and found that automation provided equivalent quality, and increased consistency while decreasing the time required and reducing the cost per preparation.
Subject(s)
Automation/methods , Azure Stains/metabolism , Chromosome Banding/methods , Chromosomes, Human/metabolism , Humans , Metaphase/genetics , Prometaphase/geneticsABSTRACT
Karyotype analysis is a widespread procedure in cytogenetics to assess the possible presence of genetics defects. The procedure is lengthy and repetitive, so that an automatic analysis would greatly help the cytogeneticist routine work. Still, automatic segmentation and full disentangling of chromosomes are open issues. We propose an automatic procedure to obtain the separated chromosomes, which are then ready for a subsequent classification step. The segmentation is carried out by means of a space-variant thresholding scheme, which proved to be successful even in presence of hyper- or hypofluorescent regions in the image. Then, the tree of choices to resolve touching and overlapping chromosomes is recursively explored, choosing the best combination of cuts and overlaps based on geometric evidence and image information. We show the effectiveness of the proposed method on routine data acquired with different microscope-camera setup at different laboratories: from 162 images of 117 cells totaling 6683 chromosomes, 94% of the chromosomes were correctly segmented, solving 90% of the overlaps and 90% of the touchings. In order to provide the scientific community with a public dataset, the data used in this paper are available for public download.
Subject(s)
Chromosomes, Human/ultrastructure , Image Processing, Computer-Assisted/methods , Karyotyping/methods , Prometaphase/genetics , Algorithms , Chromosomes, Human/genetics , Cluster Analysis , HumansABSTRACT
The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle-dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction.