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Frontiers in Oncology 2021Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining...
Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis , the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in mice by introducing heterozygosity of the key spindle assembly checkpoint regulator . mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than mice. We utilized hematopoietic stem cell transplantation to propagate AML in irradiated healthy mice to model -deficient AMLs arising in the non-FA population. Compared to cells from mice, those from mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies , show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.
PubMed: 34804941
DOI: 10.3389/fonc.2021.752933 -
Biochemical Society Transactions Dec 2021Ki-67 is highly expressed in proliferating cells, a characteristic that made the protein a very important proliferation marker widely used in the clinic. However, the...
Ki-67 is highly expressed in proliferating cells, a characteristic that made the protein a very important proliferation marker widely used in the clinic. However, the molecular functions and properties of Ki-67 remained quite obscure for a long time. Only recently important discoveries have shed some light on its function and shown that Ki-67 has a major role in the formation of mitotic chromosome periphery compartment, it is associated with protein phosphatase one (PP1) and regulates chromatin function in interphase and mitosis. In this review, we discuss the role of Ki-67 during cell division. Specifically, we focus on the importance of Ki-67 in chromosome individualisation at mitotic entry (prometaphase) and its contribution to chromosome clustering and nuclear remodelling during mitotic exit.
Topics: Chromosomes, Human; Humans; Ki-67 Antigen; Mitosis
PubMed: 34783345
DOI: 10.1042/BST20210717 -
Molecular Cell Dec 2021As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further...
As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.
Topics: Cell Proliferation; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation Sequencing; Colorectal Neoplasms; DNA Topoisomerases, Type I; G1 Phase; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; MTOR Inhibitors; Mitosis; RNA Polymerase II; Transcription, Genetic
PubMed: 34767771
DOI: 10.1016/j.molcel.2021.10.015 -
The Journal of Cell Biology Dec 2021The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the...
The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein's cargo-binding selectivity is through the C-terminal domain (CTD) of its light intermediate chain 1 subunit (LIC1), which binds directly with cargo adaptors. Here we show that mitotic phosphorylation of LIC1-CTD at its three cdk1 sites is required for proper mitotic progression, for dynein loading onto prometaphase kinetochores, and for spindle assembly checkpoint inactivation in human cells. Mitotic LIC1-CTD phosphorylation also engages the prolyl isomerase Pin1 predominantly to Hook2-dynein-Nde1-Lis1 complexes, but not to dynein-spindly-dynactin complexes. LIC1-CTD dephosphorylation abrogates dynein-Pin1 binding, promotes prophase centrosome-nuclear envelope detachment, and impairs metaphase chromosome congression and mitotic Golgi fragmentation, without affecting interphase membrane transport. Phosphomutation of a conserved LIC1-CTD SP site in zebrafish leads to early developmental defects. Our work reveals that LIC1-CTD phosphorylation differentially regulates distinct mitotic dynein pools and suggests the evolutionary conservation of this phosphoregulation.
Topics: Animals; Cell Line, Tumor; Centrosome; Cytoplasmic Dyneins; Dynactin Complex; Evolution, Molecular; Golgi Apparatus; Humans; Interphase; Kinetochores; Metaphase; Microtubule-Associated Proteins; Mitosis; Mutant Proteins; NIMA-Interacting Peptidylprolyl Isomerase; Nuclear Envelope; Phosphorylation; Protein Binding; Protein Subunits; Rats; Zebrafish
PubMed: 34709360
DOI: 10.1083/jcb.202005184 -
Pharmaceutics Sep 2021Ewing's sarcoma, characterized by pathognomonic t (11; 22) (q24; q12) and related chromosomal ETS family translocations, is a rare aggressive cancer of bone and soft...
Ewing's sarcoma, characterized by pathognomonic t (11; 22) (q24; q12) and related chromosomal ETS family translocations, is a rare aggressive cancer of bone and soft tissue. Current protocols that include cytotoxic chemotherapeutic agents effectively treat localized disease; however, these aggressive therapies may result in treatment-related morbidities including second-site cancers in survivors. Moreover, the five-year survival rate in patients with relapsed, recurrent, or metastatic disease is less than 30%, despite intensive therapy with these cytotoxic agents. By using high-throughput phenotypic screening of small molecule libraries, we identified a previously uncharacterized compound (ML111) that inhibited in vitro proliferation of six established Ewing's sarcoma cell lines with nanomolar potency. Proteomic studies show that ML111 treatment induced prometaphase arrest followed by rapid caspase-dependent apoptotic cell death in Ewing's sarcoma cell lines. ML111, delivered via methoxypoly(ethylene glycol)-polycaprolactone copolymer nanoparticles, induced dose-dependent inhibition of Ewing's sarcoma tumor growth in a murine xenograft model and invoked prometaphase arrest in vivo, consistent with in vitro data. These results suggest that ML111 represents a promising new drug lead for further preclinical studies and is a potential clinical development for the treatment of Ewing's sarcoma.
PubMed: 34683845
DOI: 10.3390/pharmaceutics13101553 -
Asian Pacific Journal of Cancer... Sep 2021We aim to enhance the effectiveness of curcumin analog PGV-1 through co-treatment with diosmin, a citrus flavonoid, on 4T1 cells and evaluate the molecular targets...
OBJECTIVE
We aim to enhance the effectiveness of curcumin analog PGV-1 through co-treatment with diosmin, a citrus flavonoid, on 4T1 cells and evaluate the molecular targets underlying its effect on the cell cycle.
METHODS
Cytotoxic effects were performed by MTT assay against 4T1 cells. The May Grünwald-Giemsa staining was used to observe cell cycle arrest. The senescence was assayed with SA-ß-gal staining. Bioinformatic studies were utilized to discover protein targets of PGV-1 and diosmin on triple-negative breast cancer (TNBC) using SwissTargetPrediction, then exploration of protein targets was performed using the TCGA dataset via the UALCAN website. Kaplan-Meier was performed using GraphPad with data from the TCGA dataset via Oncoln. Using MOE 2010, we conducted the binding affinity between PGV-1 and diosmin to protein targets.
RESULTS
PGV-1 and diosmin showed cytotoxic effect with IC50 values of 9 µM and 389 µM, respectively, and the combined cytotoxic assay exhibited a synergistic effect with a combination index (CI) of <1. PGV-arrested 4T1 cells in pro-metaphase and induced mitotic catastrophe, while the combination of diosmin with PGV-1 increased the number of mitotic catastrophes. The SA-ß-gal assay revealed that both compounds were capable of inducing senescence in 4T1 cells. Study bioinformatics and molecular docking showed that PGV-1 and diosmin target cell cycle regulatory proteins in TNBC, namely CDK1, KIF11, and AURKA. Thus, the combination of diosmin and PGV-1 modulating the cell cycle that causes senescence and catastrophic death of 4T1 cancer cells is related to the inhibition of these cell cycle proteins.
CONCLUSION
Diosmin enhances the cytotoxic effect of PGV-1 synergistically on 4T1 cancer cells, which correlates to the increasing senescence and mitotic catastrophe. The synergistic effect of the co-treatment is likely to target AURKA, CDK1, and KIF11. The combination of PGV-1 and diosmin performs a potential as a combinatorial anticancer drug for TNBC.
Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Curcumin; Diosmin; Drug Therapy, Combination; Female; Humans; Mitosis; Triple Negative Breast Neoplasms
PubMed: 34582664
DOI: 10.31557/APJCP.2021.22.9.2929 -
Cell Cycle (Georgetown, Tex.) Oct 2021To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal...
To ensure genome stability, chromosomes need to undergo proper condensation into two linked sister chromatids from prophase to prometaphase, followed by equal segregation at anaphase. Emerging evidence has shown that persistent DNA entanglements connecting the sister chromatids lead to the formation of ultrafine anaphase bridges (UFBs). If UFBs are not resolved soon after anaphase, they can induce chromosome missegregation. PICH (PLK1-interacting checkpoint helicase) is a DNA translocase that localizes on chromosome arms, centromeres and UFBs. It plays multiple essential roles in mitotic chromosome organization and segregation. PICH also recruits other associated proteins to UFBs, and together they mediate UFB resolution. Here, the proposed mechanism behind PICH's functions in chromosome organization and UFB resolution will be discussed. We summarize the regulation of PICH action at chromosome arms and centromeres, how PICH recognizes UFBs and recruits other UFB-associated factors, and finally how PICH promotes UFB resolution together with other DNA processing enzymes.
Topics: Anaphase; Centromere; Chromatids; Chromosome Segregation; Genomic Instability; Humans; Mitosis
PubMed: 34530686
DOI: 10.1080/15384101.2021.1970877 -
Molecular Biology of the Cell Nov 2021The protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important...
The protein Slk19 has been shown to localize to kinetochores throughout mitosis and to the spindle midzone in anaphase. However, Slk19 clearly also has an important role for spindle formation and stabilization in prometaphase and metaphase, albeit this role is unresolved. Here we show that Slk19's localization to metaphase spindles in vivo and to microtubules (MTs) in vitro depends on the MT cross-linking protein Ase1 and the MT cross-linking and stabilizing protein Stu1. By analyzing a mutant that specifically fails to localize to spindles and MTs, we surprisingly found that the presence of Slk19 amplified the amount of Ase1 strongly and that of Stu1 moderately at the metaphase spindle in vivo and at MTs in vitro. Furthermore, Slk19 markedly enhanced the cross-linking of MTs in vitro when added together with Ase1 or Stu1. We therefore suggest that Slk19 recruits additional Ase1 and Stu1 to the interpolar MTs (ipMTs) of metaphase spindles and thus increases their cross-linking and stabilization. This is in agreement with our observation that cells with defective Slk19 localization exhibit shorter metaphase spindles, an increased number of unaligned nuclear MTs, and most likely reduced ipMT overlaps.
Topics: Cell Cycle; Cell Nucleus; Kinetochores; Metaphase; Microtubule-Associated Proteins; Microtubules; Mitosis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus
PubMed: 34495712
DOI: 10.1091/mbc.E21-05-0279 -
Frontiers in Cell and Developmental... 2021The division of one cell into two looks so easy, as if it happens without any control at all. Mitosis, the hallmark of mammalian life is, however, tightly regulated from... (Review)
Review
The division of one cell into two looks so easy, as if it happens without any control at all. Mitosis, the hallmark of mammalian life is, however, tightly regulated from the early onset to the very last phase. Despite the tight control, errors in mitotic division occur frequently and they may result in various chromosomal instabilities and malignancies. The flow of events during mitotic progression where the chromosomes condensate and rearrange with the help of the cytoskeletal network has been described in great detail. Plasma membrane dynamics and endocytic vesicle movement upon deadhesion and reattachment of dividing cells are also demonstrated to be functionally important for the mitotic integrity. Other cytoplasmic organelles, such as autophagosomes and lysosomes, have until recently been considered merely as passive bystanders in this process. Accordingly, at the onset of nuclear envelope breakdown in prometaphase, the number of autophagic structures and lysosomes is reduced and the bulk autophagic machinery is suppressed for the duration of mitosis. This is believed to ensure that the exposed nuclear components are not unintentionally delivered to autophagic degradation. With the evolving technologies that allow the detection of subtle alterations in cytoplasmic organelles, our understanding of the small-scale regulation of intracellular organelles has deepened rapidly and we discuss here recent discoveries revealing unexpected roles for autophagy and lysosomes in the preservation of genomic integrity during mitosis.
PubMed: 34485308
DOI: 10.3389/fcell.2021.727538 -
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