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The Journal of Cell Biology Jan 2020Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect...
Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect from aneuploidy, cells possess mechanisms to delay anaphase onset when Topo II is perturbed, providing additional time for decatenation. Molecular insight into this checkpoint is lacking. Here we present evidence that catalytic inhibition of Topo II, which activates the checkpoint, leads to SUMOylation of the Topo II C-terminal domain (CTD). This modification triggers mobilization of Aurora B kinase from inner centromeres to kinetochore proximal centromeres and the core of chromosome arms. Aurora B recruitment accompanies histone H3 threonine-3 phosphorylation and requires Haspin kinase. Strikingly, activation of the checkpoint depends both on Haspin and Aurora B. Moreover, mutation of the conserved CTD SUMOylation sites perturbs Aurora B recruitment and checkpoint activation. The data indicate that SUMOylated Topo II recruits Aurora B to ectopic sites, constituting the molecular trigger of the metaphase checkpoint when Topo II is catalytically inhibited.
Topics: Aurora Kinase B; Chromosome Segregation; Chromosomes, Human; DNA Topoisomerases, Type II; Diketopiperazines; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Kinetochores; Metaphase; Mitosis; Phosphorylation; Piperazines; Protein Serine-Threonine Kinases; Sumoylation; Topoisomerase II Inhibitors
PubMed: 31712254
DOI: 10.1083/jcb.201807189 -
Frontiers in Bioscience (Landmark... Apr 2023Cholangiocarcinoma (CCA) is a malignant tumor with an increasing incidence worldwide. Although radiation therapy has improved the therapeutic efficiency of CCA...
BACKGROUND
Cholangiocarcinoma (CCA) is a malignant tumor with an increasing incidence worldwide. Although radiation therapy has improved the therapeutic efficiency of CCA treatment, differential expression of genes among cholangiocarcinoma subtypes has been revealed through precise sequencing. However, no specific molecular therapeutic targets or biomarkers have been figured out for use in precision medicine, and the exact mechanism by which antitumorigenic effects occur is still unclear. Therefore, it is necessary to conduct further studies on the development and mechanisms associated with CCA.
METHODS
We examined the clinical data and pathological features of patients with cholangiocarcinomas. We investigated the associations between DNA Topoisomerase II Alpha (TOP2A) expression and patient outcomes, such as metastasis-free survival (MFS) and disease-specific survival (DSS), as well as clinical characteristics and pathological results.
RESULTS
expression was shown to be upregulated in CCA tissue sections by immunohistochemistry staining and data mining. Moreover, we observed that the expression correlated with clinical features, such as the primary tumor stage, histological variants, and patients with hepatitis. Furthermore, high expression of was associated with worse survival outcomes in terms of the overall survival ( < 0.0001), disease-specific survival ( < 0.0001), and metastasis-free survival ( < 0.0001) compared with patients in the low expression group. This indicates that a high level of expression is related to an unfavorable prognosis.
CONCLUSIONS
Our results show that is highly expressed in CCA tissues, and its upregulation is correlated with the primary disease stage and poor prognosis significantly. Consequently, is a prognostic biomarker and a novel therapeutic target for the treatment of CCA.
Topics: Humans; Prognosis; DNA Topoisomerases, Type II; Cholangiocarcinoma; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Biomarkers, Tumor
PubMed: 37114547
DOI: 10.31083/j.fbl2804075 -
Molecules (Basel, Switzerland) Nov 2022Fluoroquinolones (FQs) are synthetic broad-spectrum antimicrobial agents that have been recently repurposed to anticancer candidates. Designing new derivatives of FQs...
Fluoroquinolones (FQs) are synthetic broad-spectrum antimicrobial agents that have been recently repurposed to anticancer candidates. Designing new derivatives of FQs with different moieties to target DNA topoisomerases could improve their anticancer efficacy. The present study aimed to synthesize a novel ciprofloxacin derivative, examine its anticancer activity against HepG2 and A549 cancer cells, and investigate the possible molecular mechanism underlying this activity by examining its ability to inhibit the topo I/II activity and to induce the apoptotic and necro-apoptotic pathways. Molecular docking, cell viability, cell migration, colony formation, cell cycle, Annexin V, lactate dehydrogenase (LDH) release, ELISA, and western blotting assays were utilized. Molecular docking results showed that this novel ciprofloxacin derivative exerted dual topo I and topo II binding and inhibition. It significantly inhibited the proliferation of A549 and HepG2 cancer cells and decreased their cell migration and colony formation abilities. In addition, it significantly increased the % of apoptotic cells, caused cell cycle arrest at G2/M phase, and elevated the LDH release levels in both cancer cells. Furthermore, it increased the expression of cleaved caspase 3, RIPK1, RIPK3, and MLKL proteins. This novel ciprofloxacin derivative exerted substantial dual inhibition of topo I/II enzyme activities, showed antiproliferative activity, suppressed the cell migration and colony formation abilities for A549 and HepG2 cancer cells and activated the apoptotic pathway. In addition, it initiated another backup deadly pathway, necro-apoptosis, through the activation of the RIPK1/RIPK3/MLKL pathway.
Topics: Apoptosis; Ciprofloxacin; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Molecular Docking Simulation; Neoplasms; Protein Kinases
PubMed: 36432094
DOI: 10.3390/molecules27227993 -
Genes Oct 2019Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a... (Review)
Review
Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this 'abortive catalysis' can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones.
Topics: BRCA1 Protein; BRCA2 Protein; Cell Cycle; DNA; DNA Breaks, Double-Stranded; DNA Damage; DNA End-Joining Repair; DNA Repair; DNA Topoisomerases, Type II; DNA-Binding Proteins; Endonucleases; Genome, Human; Humans; MRE11 Homologue Protein; Nuclear Proteins; Phosphoric Diester Hydrolases; Poly-ADP-Ribose Binding Proteins; Transcription Factors
PubMed: 31671674
DOI: 10.3390/genes10110868 -
Cell Cycle (Georgetown, Tex.) Feb 2021DNA Topoisomerase II (TopoII) uses ATP hydrolysis to decatenate chromosomes so that sister chromatids can faithfully segregate in mitosis. When the TopoII enzyme cycle... (Review)
Review
DNA Topoisomerase II (TopoII) uses ATP hydrolysis to decatenate chromosomes so that sister chromatids can faithfully segregate in mitosis. When the TopoII enzyme cycle stalls due to failed ATP hydrolysis, the onset of anaphase is delayed, presumably to allow extra time for decatenation to be completed. Recent evidence revealed that, unlike the spindle assembly checkpoint, this TopoII checkpoint response requires Aurora B and Haspin kinases and is triggered by SUMOylation of the C-terminal domain of TopoII.
Topics: Animals; Aurora Kinase B; Cell Cycle Proteins; DNA Topoisomerases, Type II; Genes, cdc; Humans; Intracellular Signaling Peptides and Proteins; M Phase Cell Cycle Checkpoints; Metaphase; Mitosis; Protein Serine-Threonine Kinases
PubMed: 33459116
DOI: 10.1080/15384101.2021.1875671 -
International Journal of Molecular... Apr 2022It was established that the synthesis of hybrid molecules containing a thiazolidinone and a (2)-2-chloro-3-(4-nitrophenyl)prop-2-ene structural fragments is an effective...
It was established that the synthesis of hybrid molecules containing a thiazolidinone and a (2)-2-chloro-3-(4-nitrophenyl)prop-2-ene structural fragments is an effective approach for the design of potential anticancer agents. Given the results of the previous SAR-analysis, the aim of the study was to synthesize a novel 4-thiazolidinone derivative Les-3331 and investigate its molecular mechanism of action in MCF-7 and MDA-MB-231 breast cancer cells. The cytotoxic properties and antiproliferative potential of Les-3331 were determined. The effect of the tested compound on apoptosis induction and mitochondrial membrane potential was checked by flow cytometry. ELISA was used to determine caspase-8 and caspase-9, LC3A, LC3B, Beclin-1, and topoisomerase II concentration. Additionally, PAMPA, in silico or in vitro prediction of metabolism, CYP3A4/2D6 inhibition, and an Ames test were performed. Les-3331 possesses high cytotoxic and antiproliferative activity in MCF-7 and MDA-MB-231 breast cancer cells. Its molecular mechanism of action is associated with apoptosis induction, decreased mitochondrial membrane potential, and increased caspase-9 and caspase-8 concentrations. Les-3331 decreased LC3A, LC3B, and Beclin-1 concentration in tested cell lines. Topoisomerase II concentration was also lowered. The most probable metabolic pathways and no DDIs risk of Les-3331 were confirmed in in vitro assays. Our studies confirmed that a novel 4-thiazolidinone derivative represents promising anti-breast cancer activity.
Topics: Antineoplastic Agents; Apoptosis; Beclin-1; Breast Neoplasms; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; DNA Topoisomerases, Type II; Female; Humans; Nitrophenols
PubMed: 35456915
DOI: 10.3390/ijms23084091 -
Genes Jan 2020Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos).... (Review)
Review
Next-generation sequencing (NGS) platforms have been adapted to generate genome-wide maps and sequence context of binding and cleavage of DNA topoisomerases (topos). Continuous refinements of these techniques have resulted in the acquisition of data with unprecedented depth and resolution, which has shed new light on in vivo topo behavior. Topos regulate DNA topology through the formation of reversible single- or double-stranded DNA breaks. Topo activity is critical for DNA metabolism in general, and in particular to support transcription and replication. However, the binding and activity of topos over the genome in vivo was difficult to study until the advent of NGS. Over and above traditional chromatin immunoprecipitation (ChIP)-seq approaches that probe protein binding, the unique formation of covalent protein-DNA linkages associated with DNA cleavage by topos affords the ability to probe cleavage and, by extension, activity over the genome. NGS platforms have facilitated genome-wide studies mapping the behavior of topos in vivo, how the behavior varies among species and how inhibitors affect cleavage. Many NGS approaches achieve nucleotide resolution of topo binding and cleavage sites, imparting an extent of information not previously attainable. We review the development of NGS approaches to probe topo interactions over the genome in vivo and highlight general conclusions and quandaries that have arisen from this rapidly advancing field of topoisomerase research.
Topics: DNA Cleavage; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Genome, Human; Genome-Wide Association Study; High-Throughput Nucleotide Sequencing; Humans
PubMed: 31941152
DOI: 10.3390/genes11010092 -
International Journal of Molecular... May 2018Gyrase is a type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme consists of two GyrA and two GyrB subunits. It is believed to introduce... (Review)
Review
Gyrase is a type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme consists of two GyrA and two GyrB subunits. It is believed to introduce negative supercoils into DNA by converting a positive DNA node into a negative node through strand passage: First, it cleaves both DNA strands of a double-stranded DNA, termed the G-segment, and then it passes a second segment of the same DNA molecule, termed the T-segment, through the gap created. As a two-fold symmetric enzyme, gyrase contains two copies of all elements that are key for the supercoiling reaction: The GyrB subunits provide two active sites for ATP binding and hydrolysis. The GyrA subunits contain two C-terminal domains (CTDs) for DNA binding and wrapping to stabilize the positive DNA node, and two catalytic tyrosines for DNA cleavage. While the presence of two catalytic tyrosines has been ascribed to the necessity of cleaving both strands of the G-segment to enable strand passage, the role of the two ATP hydrolysis events and of the two CTDs has been less clear. This review summarizes recent results on the role of these duplicate elements for individual steps of the supercoiling reaction, and discusses the implications for the mechanism of DNA supercoiling.
Topics: Animals; DNA; DNA Gyrase; DNA Topoisomerases, Type II; Humans; Nucleic Acid Conformation; Protein Subunits; Structure-Activity Relationship
PubMed: 29772727
DOI: 10.3390/ijms19051489 -
Frontiers in Immunology 2022Topoisomerase 2β (TOP2B) introduces transient double strand breaks in the DNA helix to remove supercoiling structures and unwind entangled DNA strains. Advances in...
Topoisomerase 2β (TOP2B) introduces transient double strand breaks in the DNA helix to remove supercoiling structures and unwind entangled DNA strains. Advances in genomic technologies have enabled the discovery of novel functions for TOP2B in processes such as releasing of the paused RNA polymerase II and maintaining the genome organization through DNA loop domains. Thus, TOP2B can regulate transcription directly by acting on transcription elongation and indirectly by controlling interactions between enhancer and promoter regions through genome folding. The identification of TOP2B mutations in humans unexpectedly revealed a unique role of TOP2B in B-cell progenitors. Here we discuss the functions of TOP2B and the mechanisms leading to the B-cell development defect in patients with TOP2B deficiency.
Topics: DNA; DNA Topoisomerases, Type II; DNA-Binding Proteins; Humans; Poly-ADP-Ribose Binding Proteins
PubMed: 36045673
DOI: 10.3389/fimmu.2022.982870 -
Science Advances Dec 2023Type II topoisomerases (TOP2) form transient TOP2 cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress. TOP2ccs are covalently linked...
Type II topoisomerases (TOP2) form transient TOP2 cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress. TOP2ccs are covalently linked TOP2-DNA intermediates that are reversible but can be trapped by TOP2 poisons. Trapped TOP2ccs block transactions on DNA and generate genotoxic stress, which are the mechanisms of action of TOP2 poisons. How cells avoid TOP2cc accumulation remains largely unknown. In this study, we uncovered RAD54 like 2 (RAD54L2) as a key factor that mediates a TOP2-specific DNA damage avoidance pathway. RAD54L2 deficiency conferred unique sensitivity to treatment with TOP2 poisons. RAD54L2 interacted with TOP2A/TOP2B and ZATT/ZNF451 and promoted the turnover of TOP2 from DNA with or without TOP2 poisons. Additionally, inhibition of proteasome activity enhanced the chromatin binding of RAD54L2, which in turn led to the removal of TOP2 from chromatin. In conclusion, we propose that RAD54L2-mediated TOP2 turnover is critically important for the avoidance of potential TOP2-linked DNA damage under physiological conditions and in response to TOP2 poisons.
Topics: Poisons; DNA Topoisomerases, Type II; DNA Damage; DNA Repair; DNA; Chromatin
PubMed: 38055811
DOI: 10.1126/sciadv.adi6681