Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.470
Filtrar
1.
Gene ; 754: 144859, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32535049

RESUMO

DNA topoisomerases essentially remove topological strains generated during DNA replication, transcription, DNA repair, and other cytogenetic processes. However, distinct expression level and prognostic significance of individual topoisomerase isoforms in gastric cancer (GC) remain largely unexplored. In this study, we utilized Oncomine and Kaplan-Meier plotter database to detect the mRNA expression level of individual topoisomerase isoforms as well as assess their prognostic significance in GC patients. With the exception of TOP3B and TOP2B, levels of all topoisomerase isoforms were found to be elevated in GC patients when compared to the normal tissues. Elevated expression of TOP1 and TOP1MT was relevant to longer overall survival (OS) in GC and gastric intestinal type adenocarcinoma (GITA) patients, but not in diffuse gastric adenocarcinoma (DFA) patients. Increased expression of TOP2A and TOP2B was related to better OS in GC, as well as in GITA and DFA patients. In contrast, increased expression TOP3A and TOP3B was associated with shorter OS in GC, as well as in GITA and DFA patients. We also applied the Tumor IMmune Estimation Resource (TIMER) tool to assess the correlations between distinct topoisomerase isoforms and the infiltrating immune cell landscape. Furthermore, we found that down-regulating the expression of TOP3A by shRNA significantly inhibited the proliferation and colony formation in GC cells compared to control shRNA treated cells. Thus our study lays the framework for utilizing topoisomerases in better understanding the complexity and heterogeneity of GC and for developing strategies for novel customized therapy in GC patients.


Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias Gástricas/patologia , Adenocarcinoma/enzimologia , Biomarcadores Tumorais/genética , DNA Topoisomerases Tipo II/genética , Precursores Enzimáticos , Perfilação da Expressão Gênica , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Prognóstico , Neoplasias Gástricas/enzimologia , Taxa de Sobrevida
2.
Nat Commun ; 11(1): 2156, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358485

RESUMO

Colorectal cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. and approximately 50% of patients develop metastatic disease (mCRC). Despite our understanding of long non-coding RNAs (lncRNAs) in primary colon cancer, their role in mCRC and treatment resistance remains poorly characterized. Therefore, through transcriptome sequencing of normal, primary, and distant mCRC tissues we find 148 differentially expressed RNAs Associated with Metastasis (RAMS). We prioritize RAMS11 due to its association with poor disease-free survival and promotion of aggressive phenotypes in vitro and in vivo. A FDA-approved drug high-throughput viability assay shows that elevated RAMS11 expression increases resistance to topoisomerase inhibitors. Subsequent experiments demonstrate RAMS11-dependent recruitment of Chromobox protein 4 (CBX4) transcriptionally activates Topoisomerase II alpha (TOP2α). Overall, recent clinical trials using topoisomerase inhibitors coupled with our findings of RAMS11-dependent regulation of TOP2α supports the potential use of RAMS11 as a biomarker and therapeutic target for mCRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Biologia Computacional , DNA Topoisomerases Tipo II/metabolismo , Progressão da Doença , Éxons/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Células HT29 , Humanos , Ligases/metabolismo , Camundongos , Proteínas do Grupo Polycomb/metabolismo , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Inibidores da Topoisomerase/farmacologia
3.
Proc Natl Acad Sci U S A ; 117(8): 4053-4060, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041867

RESUMO

Small molecules can affect many cellular processes. The disambiguation of these effects to identify the causative mechanisms of cell death is extremely challenging. This challenge impacts both clinical development and the interpretation of chemical genetic experiments. CX-5461 was developed as a selective RNA polymerase I inhibitor, but recent evidence suggests that it may cause DNA damage and induce G-quadraplex formation. Here we use three complimentary data mining modalities alongside biochemical and cell biological assays to show that CX-5461 exerts its primary cytotoxic activity through topoisomerase II poisoning. We then show that acquired resistance to CX-5461 in previously sensitive lymphoma cells confers collateral resistance to the topoisomerase II poison doxorubicin. Doxorubicin is already a frontline chemotherapy in a variety of hematopoietic malignancies, and CX-5461 is being tested in relapse/refractory hematopoietic tumors. Our data suggest that the mechanism of cell death induced by CX-5461 is critical for rational clinical development in these patients. Moreover, CX-5461 usage as a specific chemical genetic probe of RNA polymerase I function is challenging to interpret. Our multimodal data-driven approach is a useful way to detangle the intended and unintended mechanisms of drug action across diverse essential cellular processes.


Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Naftiridinas/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Interferência de RNA , Sensibilidade e Especificidade
4.
Nat Commun ; 11(1): 910, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060399

RESUMO

The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm-/- mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm-/- mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/metabolismo , Neoplasias do Timo/epidemiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Camundongos Knockout , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Neoplasias do Timo/genética
5.
Anticancer Res ; 40(2): 807-811, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32014923

RESUMO

BACKGROUND/AIM: Topoisomerase II alpha (TopoIIa) is a critical gene associated with response to chemo-therapeutic agents, such as anthracyclines, especially in breast adenocarcinoma. The aim of this study was to investigate the role of aberrant TopoIIa protein expression in laryngeal squamous cell carcinoma (LSCC). MATERIALS AND METHODS: Fifty (n=50) LSCC cases were enrolled in the study. Immunohistochemistry and a digital image analysis assay were implemented. RESULTS: TopoIIa protein overexpression was observed in 32/50 (64%) cases, whereas low expression rates were detected in 18/50 (36%). TopoIIa overall expression presented strong association with the grade of the examined malignant tissues and borderline association with stage. TopoIIa overexpression correlated also with Human papillomavirus (HPV) positivity. CONCLUSION: TopoIIa overexpression was observed in significant subsets of LSCCs, and correlated predominantly with the grade of differentiation. HPV persistent infection seems to be associated with increased TopoIIa protein expression. TopoIIa expression analysis appears to be critical in identifying sub-groups of patients eligible for specific chemotherapy.


Assuntos
Carcinoma de Células Escamosas/genética , DNA Topoisomerases Tipo II/metabolismo , Neoplasias Laríngeas/genética , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Neoplasias Laríngeas/patologia , Masculino , Pessoa de Meia-Idade
6.
Life Sci ; 248: 117467, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32105706

RESUMO

BACKGROUND: NQO1 protein acts as a cellular protective system, on account of its role as a quinone reductase and redox regulator. Nonetheless, new NQO1 roles are emerging-including its regulation of the cellular proliferation of many tumor cells-and this enzyme has been found to relate to the incidence of various diseases, including chronic myeloid leukemia. However, the mechanisms through which NQO1 influences leukemia progression remain unclear. MARTIAL AND METHODS: The current study looks to name NQO1 as a novel molecular target that modulates DNA synthesis and chronic myeloid leukemia growth. RESULTS AND CONCLUSION: Our results indicate that the frequency of the T allele of NQO1 polymorphism in chronic myeloid leukemia patients is higher than that among healthy East Asian individuals (0.492 vs. 0.419) and much higher than the average level of the general population (0.492 vs. 0.289) (1000 Genomes). Functionally, NQO1 knockdown increases the protein expression of the TOP2A and MCM complex, and consequently promotes DNA synthesis and K562 cell growth. NQO1 knockdown also promotes tumorigenesis in a xenograft model. NQO1 overexpression, on the other hand, was found to have the opposite effects. SIGNIFICANCE: Our results show that NQO1 downregulation promotes K562 cellular proliferation via the elevation of DNA synthesis.


Assuntos
DNA de Neoplasias/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucócitos/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , Adulto , Alelos , Animais , Grupo com Ancestrais do Continente Asiático , Linhagem Celular Tumoral , Proliferação de Células , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA de Neoplasias/biossíntese , Feminino , Xenoenxertos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/etnologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos/patologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Polimorfismo Genético , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
7.
PLoS One ; 15(2): e0228509, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074119

RESUMO

In this study, a drug discovery programme that sought to identify novel dual bacterial topoisomerase II inhibitors (NBTIs) led to the selection of six optimized compounds. In enzymatic assays, the molecules showed equivalent dual-targeting activity against the DNA gyrase and topoisomerase IV enzymes of Staphylococcus aureus and Escherichia coli. Consistently, the compounds demonstrated potent activity in susceptibility tests against various Gram-positive and Gram-negative reference species, including ciprofloxacin-resistant strains. The activity of the compounds against clinical multidrug-resistant isolates of S. aureus, Clostridium difficile, Acinetobacter baumannii, Neisseria gonorrhoeae, E. coli and vancomycin-resistant Enterococcus spp. was also confirmed. Two compounds (1 and 2) were tested in time-kill and post-antibiotic effect (PAE) assays. Compound 1 was bactericidal against all tested reference strains and showed higher activity than ciprofloxacin, and compound 2 showed a prolonged PAE, even against the ciprofloxacin-resistant S. aureus BAA-1720 strain. Spontaneous development of resistance to both compounds was selected for in S. aureus at frequencies comparable to those obtained for quinolones and other NBTIs. S. aureus BAA-1720 mutants resistant to compounds 1 and 2 had single point mutations in gyrA or gyrB outside of the quinolone resistance-determining region (QRDR), confirming the distinct site of action of these NBTIs compared to that of quinolones. Overall, the very good antibacterial activity of the compounds and their optimizable in vitro safety and physicochemical profile may have relevant implications for the development of new broad-spectrum antibiotics.


Assuntos
Antibacterianos/farmacologia , DNA Girase/efeitos dos fármacos , DNA Topoisomerases Tipo II/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologia , Animais , Células CHO , Ciprofloxacino/farmacologia , Cricetulus , DNA Topoisomerases Tipo II/metabolismo , DNA Bacteriano/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Células Hep G2 , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Testes de Toxicidade
8.
Mutat Res ; 849: 503144, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087851

RESUMO

Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.


Assuntos
Reparo do DNA/efeitos dos fármacos , Flavonoides/toxicidade , Genisteína/toxicidade , Quempferóis/toxicidade , Luteolina/toxicidade , Quercetina/toxicidade , Animais , Linhagem Celular , Pontos de Quebra do Cromossomo/efeitos dos fármacos , Cromossomos de Mamíferos/efeitos dos fármacos , DNA/química , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Dexrazoxano/farmacologia , Etoposídeo/toxicidade , Histonas/genética , Histonas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/ultraestrutura , Inibidores da Topoisomerase II/farmacologia , Translocação Genética/efeitos dos fármacos
9.
Nature ; 577(7792): 701-705, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969709

RESUMO

Transcription challenges the integrity of replicating chromosomes by generating topological stress and conflicts with forks1,2. The DNA topoisomerases Top1 and Top2 and the HMGB family protein Hmo1 assist DNA replication and transcription3-6. Here we describe the topological architecture of genes in Saccharomyces cerevisiae during the G1 and S phases of the cell cycle. We found under-wound DNA at gene boundaries and over-wound DNA within coding regions. This arrangement does not depend on Pol II or S phase. Top2 and Hmo1 preserve negative supercoil at gene boundaries, while Top1 acts at coding regions. Transcription generates RNA-DNA hybrids within coding regions, independently of fork orientation. During S phase, Hmo1 protects under-wound DNA from Top2, while Top2 confines Pol II and Top1 at coding units, counteracting transcription leakage and aberrant hybrids at gene boundaries. Negative supercoil at gene boundaries prevents supercoil diffusion and nucleosome repositioning at coding regions. DNA looping occurs at Top2 clusters. We propose that Hmo1 locks gene boundaries in a cruciform conformation and, with Top2, modulates the architecture of genes that retain the memory of the topological arrangements even when transcription is repressed.


Assuntos
DNA Fúngico/química , DNA Super-Helicoidal/química , Genes Fúngicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Cruciforme/química , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Fase G1 , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Mutação , Hibridização de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Fases de Leitura Aberta/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , Fase S , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Genética
10.
BMC Genomics ; 21(1): 25, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31914926

RESUMO

BACKGROUND: DNA double-stranded breaks (DSBs) are potentially deleterious events in a cell. The end structures (blunt, 3'- and 5'-overhangs) at DSB sites contribute to the fate of their repair and provide critical information concerning the consequences of the damage. Therefore, there has been a recent eruption of DNA break mapping and sequencing methods that aim to map at single-nucleotide resolution where breaks are generated genome-wide. These methods provide high resolution data for the location of DSBs, which can encode the type of end-structure present at these breaks. However, genome-wide analysis of the resulting end structures has not been investigated following these sequencing methods. RESULTS: To address this analysis gap, we develop the use of a coverage-normalized cross correlation analysis (CNCC) to process the high-precision genome-wide break mapping data, and determine genome-wide break end structure distributions at single-nucleotide resolution. We take advantage of the single-nucleotide position and the knowledge of strandness from every mapped break to analyze the relative shifts between positive and negative strand encoded break nucleotides. By applying CNCC we can identify the most abundant end structures captured by a break mapping technique, and further can make comparisons between different samples and treatments. We validate our analysis with restriction enzyme digestions of genomic DNA and establish the sensitivity of the analysis using end structures that only exist as a minor fraction of total breaks. Finally, we demonstrate the versatility of our analysis by applying CNCC to the breaks resulting after treatment with etoposide and study the variety of resulting end structures. CONCLUSION: For the first time, on a genome-wide scale, our analysis revealed the increase in the 5' to 3' end resection following etoposide treatment, and the global progression of the resection. Furthermore, our method distinguished the change in the pattern of DSB end structure with increasing doses of the drug. The ability of this method to determine DNA break end structures without a priori knowledge of break sequences or genomic position should have broad applications in understanding genome instability.


Assuntos
Mapeamento Cromossômico/métodos , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Instabilidade Genômica/genética , Humanos
11.
Nat Cell Biol ; 22(2): 175-186, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932739

RESUMO

Mouse embryonic stem cells (ESCs) sporadically transit into an early embryonic-like state characterized by the expression of 2-cell (2C) stage-restricted transcripts. Here, we identify a maternal factor-negative elongation factor A (NELFA)-whose heterogeneous expression in mouse ESCs is coupled to 2C gene upregulation and expanded developmental potential in vivo. We show that NELFA partners with Top2a in an interaction specific to the 2C-like state, and that it drives the expression of Dux-a key 2C regulator. Accordingly, loss of NELFA and/or Top2a suppressed Dux activation. Further characterization of 2C-like cells uncovered reduced glycolytic activity; remarkably, mere chemical suppression of glycolysis was sufficient to promote a 2C-like fate, obviating the need for genetic manipulation. Global chromatin state analysis on NELFA-induced cells revealed decommissioning of ESC-specific enhancers, suggesting ESC-state impediments to 2C reversion. Our study positions NELFA as one of the earliest drivers of the 2C-like state and illuminates factors and processes that govern this transition.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Cromatina/química , Cromatina/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Embrião de Mamíferos , Feminino , Glicólise/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Padrões de Herança , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Família Multigênica , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
12.
Cell Mol Life Sci ; 77(1): 81-91, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31728578

RESUMO

The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATTZnf451-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.


Assuntos
Quebras de DNA , Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Animais , DNA/química , DNA/genética , DNA Topoisomerases Tipo II/química , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/química , Conformação Proteica , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
13.
J Cell Biol ; 219(1)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31712254

RESUMO

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.


Assuntos
Aurora Quinase B/metabolismo , Cromossomos Humanos/genética , DNA Topoisomerases Tipo II/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metáfase , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Sumoilação , Aurora Quinase B/genética , Segregação de Cromossomos , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinetocoros , Fosforilação , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/genética , Inibidores da Topoisomerase II/farmacologia
14.
Eur J Med Chem ; 187: 111960, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31869654

RESUMO

A series of l-lysine-conjugated pyridophenoxazinones 2-5 and 2'-5' were designed and synthesized for developing compounds with multimodal anticancer potentialities. All compounds inhibited the proliferation of a panel of human liquid and solid neoplastic cell lines. 2 and 5 were the most active compounds with IC50 values in the submicromolar range. UV-vis, 1H NMR, unwinding, and docking experiments demonstrated that they intercalate between the middle 5'-GC-3' base pairs with the carboxamide side chain lying into major groove. Charge-transfer contribution to the complex stability, evaluated by ab initio calculations, was found to correlate with cytotoxicity. Relaxation and cleavage assays showed that 2 and 5 selectively target Topo IIα over Topo IIß and stimulate the formation of covalent Topo II-DNA complexes, functioning as poisons. Moreover, compound 5 induced DNA damage and arrested MCF-7 cells at the G2/M phase. Altogether, the work provides interesting structure-activity relationships in the pyridophenoxazinone-l-lysine conjugate series and identifies 5 as a promising candidate for further in vivo evaluation.


Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , DNA de Neoplasias/efeitos dos fármacos , Lisina/farmacologia , Oxazinas/farmacologia , Inibidores da Topoisomerase II/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Modelos Lineares , Lisina/química , Modelos Moleculares , Estrutura Molecular , Oxazinas/química , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química
15.
Int J Mol Sci ; 20(24)2019 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-31817267

RESUMO

Ovarian cancer is the most lethal gynecological malignancy, often because of the frequent insurgence of chemoresistance to the drugs currently used. Thus, new therapeutical agents are needed. We tested the toxicity of 16 new DNA-intercalating agents to cisplatin (cDDP)-sensitive human ovarian carcinoma cell lines and their resistant counterparts. The compounds were the complexes of Pt(II) or Pd(II) with bipyridyl (bipy) and phenanthrolyl (phen) and with four different thiourea ancillary ligands. Within each of the four series of complexes characterized by the same thiourea ligand, the Pd(phen) drugs invariably showed the highest anti-proliferative efficacy. This paralleled both a higher intracellular drug accumulation and a more efficient DNA intercalation than all the other metal-bidentate ligand combinations. The consequent inhibition of topoisomerase II activity led to the greatest inhibition of DNA metabolism, evidenced by the inhibition of the expression of the folate cycle enzymes and a marked perturbation of cell-cycle distribution in both cell lines. These findings indicate that the particular interaction of Pd(II) with phenanthroline confers the best pharmacokinetic and pharmacodynamic properties that make this class of DNA intercalators remarkable inhibitors, even of the resistant cell growth.


Assuntos
Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Substâncias Intercalantes/farmacologia , Fenantrolinas/química , Tioureia/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , DNA/química , DNA/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Feminino , Humanos , Substâncias Intercalantes/química , Ligantes , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Paládio/química , Platina/química
16.
Genes Dev ; 33(23-24): 1751-1774, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31753913

RESUMO

Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.


Assuntos
Instabilidade Genômica , Estruturas R-Loop , Reparo de DNA por Recombinação/genética , Fatores de Transcrição/genética , Acetilação , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Células HEK293 , Células HeLa , Humanos , Transativadores/metabolismo , Fatores de Transcrição/análise , Ubiquitinação , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
17.
Genes (Basel) ; 10(11)2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671531

RESUMO

Type II topoisomerases are ubiquitous enzymes in all branches of life that can alter DNA superhelicity and unlink double-stranded DNA segments during processes such as replication and transcription. In cells, type II topoisomerases are particularly useful for their ability to disentangle newly-replicated sister chromosomes. Growing lines of evidence indicate that eukaryotic topoisomerase II (topo II) activity is monitored and regulated throughout the cell cycle. Here, we discuss the various roles of topo II throughout the cell cycle, as well as mechanisms that have been found to govern and/or respond to topo II function and dysfunction. Knowledge of how topo II activity is controlled during cell cycle progression is important for understanding how its misregulation can contribute to genetic instability and how modulatory pathways may be exploited to advance chemotherapeutic development.


Assuntos
Ciclo Celular/fisiologia , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo II/fisiologia , Animais , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular , Divisão Celular , Cromossomos/metabolismo , DNA/metabolismo , Replicação do DNA/genética , Replicação do DNA/fisiologia , DNA Topoisomerases Tipo II/genética , Células Eucarióticas/metabolismo , Humanos , Mitose/fisiologia , Inibidores da Topoisomerase II
18.
Genes (Basel) ; 10(11)2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671674

RESUMO

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.


Assuntos
Reparo do DNA/genética , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Ciclo Celular/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Genoma Humano/genética , Humanos , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/genética , Diester Fosfórico Hidrolases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fatores de Transcrição/genética
19.
Life Sci ; 239: 117070, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31751580

RESUMO

Doxorubicin (DOX) induced cardiotoxicity is a life-threatening side effect of chemotherapy and decreased cardiac function can present years after treatment. Despite the investigation of a broad range of pharmacologic interventions, to date the only drug shown to reduce DOX-related cardiotoxicity in preclinical studies and limited clinical trials is the iron chelating agent, dexrazoxane (DRZ), although the mechanisms responsible for DRZ mediated protection from DOX related cardiotoxicity remain unclear. Engineered cardiac tissues (ECTs) can be used for tissue repair strategies and as in vitro surrogate models to test cardiac toxicities and preventative countermeasures. Neonatal murine ECTs display cardiotoxicity in response to the environmental toxin, cadmium, and reduced cadmium toxicity with Zinc co-treatment, in part via the induction of the anti-oxidant Metallothionein (MT). We adapted our in vitro ECT model to determine the feasibility of using the ECT approach to investigate DOX-related cardiac injury and DRZ prevention. We found: (1) DOX induced dose and time dependent cell death in ECTs; (2) Zinc did not show protection from DOX cardiotoxicity; (3) MT overexpression induced by Zinc, low dose Cd pretreatment, or MT-overexpression (MT-TG) did not reduce ECT DOX cardiotoxicity; (4) DRZ reduced ECT DOX induced cell death; and (5) The mechanism of DRZ ECT protection from DOX cardiotoxicity was topoisomerase 2B (TOP2B) inhibition rather than reduced reactive oxygen species. Our data support the feasibility of ECTs as an in vitro platform technology for the investigation of drug induced cardiotoxicities including the role of TOP2B in DOX toxicity and DRZ mediated DOX toxicity prevention.


Assuntos
Cardiotoxicidade/metabolismo , Dexrazoxano/farmacologia , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Cardiotoxicidade/prevenção & controle , DNA Topoisomerases Tipo II/metabolismo , Dexrazoxano/metabolismo , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Quelantes de Ferro/farmacologia , Metalotioneína/metabolismo , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Engenharia Tecidual/métodos
20.
Nat Commun ; 10(1): 4846, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649282

RESUMO

DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes-and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage-distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.


Assuntos
Reparo do DNA , DNA Topoisomerases Tipo II/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Antineoplásicos Fitogênicos/farmacologia , Sequência de Bases , Fator de Ligação a CCCTC/genética , DNA/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Etoposídeo/farmacologia , Humanos , Mapeamento de Nucleotídeos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA