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Anticancer Research Oct 2020Accurate regulation of the spindle assembly checkpoint (SAC) and anaphase promoting complex/cyclosome (APC/C) are essential for the correct execution of mitosis. In this...
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.
Topics: Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; M Phase Cell Cycle Checkpoints; Mad2 Proteins; Mitosis; Prometaphase
PubMed: 32988869
DOI: 10.21873/anticanres.14558 -
BMC Cancer Aug 2023HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell...
BACKGROUND
HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model.
METHODS
Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry.
RESULTS
The three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG.
CONCLUSIONS
We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.
Topics: Humans; Glioblastoma; Apoptosis; Cell Line, Tumor; G2 Phase Cell Cycle Checkpoints; Interferon-alpha; Anaphase; Interferon-gamma; Skin Neoplasms
PubMed: 37644431
DOI: 10.1186/s12885-023-11330-2 -
Current Biology : CB Feb 2020Polo-like kinases (PLKs) have numerous roles in both mitosis and meiosis, including functions related to chromosome segregation, cohesin removal, and kinetochore...
Polo-like kinases (PLKs) have numerous roles in both mitosis and meiosis, including functions related to chromosome segregation, cohesin removal, and kinetochore orientation [1-7]. PLKs require specific regulation during meiosis to control those processes. Genetic studies demonstrate that the Drosophila PLK Polo kinase (Polo) is inhibited by the female meiosis-specific protein Matrimony (Mtrm) in a stoichiometric manner [8]. Drosophila Polo localizes strongly to kinetochores and to central spindle microtubules during prometaphase and metaphase I of female meiosis [9, 10]. Mtrm protein levels increase dramatically after nuclear envelope breakdown [11]. We show that Mtrm is enriched along the meiotic spindle and that loss of mtrm results in mislocalization of the catalytically active form of Polo. The mtrm gene is haploinsufficient, and heterozygosity for mtrm (mtrm/) results in high levels of achiasmate chromosome missegregation [8, 12]. In mtrm/ heterozygotes, there is a low level of sister centromere separation, as well as precocious loss of cohesion along the arms of achiasmate chromosomes. However, mtrm-null females are sterile [13], and sister chromatid cohesion is abolished on all chromosomes, leading to a failure to properly congress or orient chromosomes in metaphase I. These data demonstrate a requirement for the inhibition of Polo, perhaps by sequestering Polo to the microtubules during Drosophila melanogaster female meiosis and suggest that catalytically active Polo is a distinct subset of the total Polo population within the oocyte that requires its own regulation.
Topics: Animals; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Drosophila Proteins; Drosophila melanogaster; Female; Meiosis; Protein Serine-Threonine Kinases; Cohesins
PubMed: 32008903
DOI: 10.1016/j.cub.2019.12.027 -
International Journal of Surgical... Apr 2024The identification of mitotic figures is essential for the diagnosis, grading, and classification of various different tumors. Despite its importance, there is a...
The identification of mitotic figures is essential for the diagnosis, grading, and classification of various different tumors. Despite its importance, there is a paucity of literature reporting the consistency in interpreting mitotic figures among pathologists. This study leverages publicly accessible datasets and social media to recruit an international group of pathologists to score an image database of more than 1000 mitotic figures collectively. Pathologists were instructed to randomly select a digital slide from The Cancer Genome Atlas (TCGA) datasets and annotate 10-20 mitotic figures within a 2 mm area. The first 1010 submitted mitotic figures were used to create an image dataset, with each figure transformed into an individual tile at 40x magnification. The dataset was redistributed to all pathologists to review and determine whether each tile constituted a mitotic figure. Overall pathologists had a median agreement rate of 80.2% (range 42.0%-95.7%). Individual mitotic figure tiles had a median agreement rate of 87.1% and a fair inter-rater agreement across all tiles (kappa = 0.284). Mitotic figures in prometaphase had lower percentage agreement rates compared to other phases of mitosis. This dataset stands as the largest international consensus study for mitotic figures to date and can be utilized as a training set for future studies. The agreement range reflects a spectrum of criteria that pathologists use to decide what constitutes a mitotic figure, which may have potential implications in tumor diagnostics and clinical management.
PubMed: 38627896
DOI: 10.1177/10668969241234321 -
Physical Review. E Sep 2023Variation in the chromosome numbers can arise from the erroneous mitosis or fusion and fission of chromosomes. While the mitotic errors lead to an increase or decrease...
Variation in the chromosome numbers can arise from the erroneous mitosis or fusion and fission of chromosomes. While the mitotic errors lead to an increase or decrease in the overall chromosomal substance in the daughter cells, fission and fusion keep this conserved. Variations in chromosome numbers are assumed to be a crucial driver of speciation. For example, the members of the muntjac species are known to have very different karyotypes with the chromosome numbers varying from 2n=70+3B in the brown brocket deer to 2n=46 in the Chinese muntjac and 2n=6/7 in the Indian muntjac. The chromosomal content in the nucleus of these closely related mammals is roughly the same and various chromosome fusion and fission pathways have been suggested as the evolution process of these karyotypes. Similar trends can also be found in lepidoptera and yeast species which show a wide variation of chromosome numbers. The effect of chromosome number variation on the spindle assembly time and accuracy is still not properly addressed. We computationally investigate the effect of conservation of the total chromosomal substance on the spindle assembly during prometaphase. Our results suggest that chromosomal fusion pathways aid the microtubule-driven search and capture of the kinetochore in cells with monocentric chromosomes. We further report a comparative analysis of the site and percentage of amphitelic captures, dependence on cell shape, and position of the kinetochore in respect to chromosomal volume partitioning.
Topics: Animals; Muntjacs; Deer; Mitosis; Microtubules; Kinetochores
PubMed: 37849183
DOI: 10.1103/PhysRevE.108.034401 -
ELife Dec 2020Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by...
Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In , the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Chromosome Segregation; Chromosomes; Meiosis; Oocytes; Protein Phosphatase 2; Protein Serine-Threonine Kinases
PubMed: 33355089
DOI: 10.7554/eLife.65307 -
Cells Jul 2021Tubulin post-translational modifications regulate microtubule properties and functions. Mitotic spindle microtubules are highly modified. While tubulin detyrosination...
Tubulin post-translational modifications regulate microtubule properties and functions. Mitotic spindle microtubules are highly modified. While tubulin detyrosination promotes proper mitotic progression by recruiting specific microtubule-associated proteins motors, tubulin acetylation that occurs on specific microtubule subsets during mitosis is less well understood. Here, we show that siRNA-mediated depletion of the tubulin acetyltransferase in epithelial cells leads to a prolonged prometaphase arrest and the formation of monopolar spindles. This results from collapse of bipolar spindles, as previously described in cells deficient for the mitotic kinase . -depleted mitotic cells have defective recruitment of to centrosomes, defects in centrosome maturation and thus microtubule nucleation, as well as labile microtubule-kinetochore attachments. Spindle bipolarity could be restored, in the absence of , by stabilizing microtubule plus-ends or by increasing activity at centrosomes, demonstrating that the phenotype is not just a consequence of lack of K-fiber stability. We propose that microtubule acetylation of K-fibers is required for a recently evidenced cross talk between centrosomes and kinetochores.
Topics: Acetylation; Acetyltransferases; Animals; Cell Cycle Proteins; Centrosome; Epithelial Cells; LLC-PK1 Cells; Microtubule Proteins; Microtubules; Mitosis; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Signal Transduction; Spindle Apparatus; Swine; Polo-Like Kinase 1
PubMed: 34440628
DOI: 10.3390/cells10081859 -
Nature Communications Jul 2021Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding...
Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes.
Topics: Amino Acid Motifs; Animals; Chromosome Segregation; Meiosis; Mice; Mutation; Oocytes; Phenylalanine; Prometaphase; Protein Binding; Securin; Separase
PubMed: 34262048
DOI: 10.1038/s41467-021-24554-2 -
Genetics Mar 2020Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined...
Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the ribosomal DNA (rDNA). Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Using fluorescence hybridization to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis, while the autosomal-specific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.
Topics: Animals; Centromere; Chromosome Pairing; Chromosome Segregation; DNA, Ribosomal; Drosophila melanogaster; Euchromatin; Heterochromatin; In Situ Hybridization, Fluorescence; Male; Meiosis; Sex Chromosomes; Spermatocytes; X Chromosome; Y Chromosome
PubMed: 31915134
DOI: 10.1534/genetics.119.302936 -
Molecular Oncology Feb 2022The Fanconi anemia (FA) pathway safeguards genomic stability through cell cycle regulation and DNA damage repair. The canonical tumor suppressive role of FA proteins in...
The Fanconi anemia (FA) pathway safeguards genomic stability through cell cycle regulation and DNA damage repair. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established, but their function in mitosis is incompletely understood. Here, we performed a kinome-wide synthetic lethality screen in FANCA fibroblasts, which revealed multiple mitotic kinases as necessary for survival of FANCA-deficient cells. Among these kinases, we identified the depletion of the centrosome kinase SIK2 as synthetic lethal upon loss of FANCA. We found that FANCA colocalizes with SIK2 at multiple mitotic structures and regulates the activity of SIK2 at centrosomes. Furthermore, we found that loss of FANCA exacerbates cell cycle defects induced by pharmacological inhibition of SIK2, including impaired G2-M transition, delayed mitotic progression, and cytokinesis failure. In addition, we showed that inhibition of SIK2 abrogates nocodazole-induced prometaphase arrest, suggesting a novel role for SIK2 in the spindle assembly checkpoint. Together, these findings demonstrate that FANCA-deficient cells are dependent upon SIK2 for survival, supporting a preclinical rationale for targeting of SIK2 in FA-disrupted cancers.
Topics: Cell Cycle; Fanconi Anemia; Fanconi Anemia Complementation Group A Protein; Fanconi Anemia Complementation Group Proteins; Humans; Mitosis; Protein Serine-Threonine Kinases; Synthetic Lethal Mutations
PubMed: 34058059
DOI: 10.1002/1878-0261.13027