RESUMEN
Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system, catalysing the formation of reactive oxygen species that are important in anti-microbial action, but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti-cancer drugs, but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy-related leukaemia by protecting haematopoietic cells from TOP2 poison-mediated genotoxic damage while preserving the anti-cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide-induced TOP2B-DNA covalent complexes and resulting DNA double-strand break formation in primary ex vivo expanded CD34+ progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti-inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons.
Asunto(s)
Daño del ADN , ADN-Topoisomerasas de Tipo II , Etopósido , Peroxidasa , Proteínas de Unión a Poli-ADP-Ribosa , Etopósido/farmacología , Humanos , Peroxidasa/metabolismo , Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Inhibidores de Topoisomerasa II/farmacología , Antineoplásicos/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismoRESUMEN
Anthracyclines, such as doxorubicin (adriamycin), daunorubicin, or epirubicin, rank among the most effective agents in classical anticancer chemotherapy. However, cardiotoxicity remains the main limitation of their clinical use. Topoisomerase IIß has recently been identified as a plausible target of anthracyclines in cardiomyocytes. We examined the putative topoisomerase IIß selective agent XK469 as a potential cardioprotective and designed several new analogs. In our experiments, XK469 inhibited both topoisomerase isoforms (α and ß) and did not induce topoisomerase II covalent complexes in isolated cardiomyocytes and HL-60, but induced proteasomal degradation of topoisomerase II in these cell types. The cardioprotective potential of XK469 was studied on rat neonatal cardiomyocytes, where dexrazoxane (ICRF-187), the only clinically approved cardioprotective, was effective. Initially, XK469 prevented daunorubicin-induced toxicity and p53 phosphorylation in cardiomyocytes. However, it only partially prevented the phosphorylation of H2AX and did not affect DNA damage measured by Comet Assay. It also did not compromise the daunorubicin antiproliferative effect in HL-60 leukemic cells. When administered to rabbits to evaluate its cardioprotective potential in vivo, XK469 failed to prevent the daunorubicin-induced cardiac toxicity in either acute or chronic settings. In the following in vitro analysis, we found that prolonged and continuous exposure of rat neonatal cardiomyocytes to XK469 led to significant toxicity. In conclusion, this study provides important evidence on the effects of XK469 and its combination with daunorubicin in clinically relevant doses in cardiomyocytes. Despite its promising characteristics, long-term treatments and in vivo experiments have not confirmed its cardioprotective potential.
Asunto(s)
Antraciclinas , Quinoxalinas , Inhibidores de Topoisomerasa II , Ratas , Animales , Conejos , Inhibidores de Topoisomerasa II/toxicidad , Inhibidores de Topoisomerasa II/uso terapéutico , Antraciclinas/toxicidad , Antraciclinas/uso terapéutico , Cardiotoxicidad , Daunorrubicina/toxicidad , Daunorrubicina/uso terapéutico , Doxorrubicina/toxicidad , Antibióticos Antineoplásicos/toxicidad , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/uso terapéutico , Daño del ADNRESUMEN
Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA introduces positive helical torsion and supercoiling upstream and negative torsion and supercoiling behind its direction of travel. This can inhibit transcriptional elongation and other processes essential to transcription. In addition, chromatin remodeling associated with gene activation can generate or be hindered by excess DNA torsional stress in gene regulatory regions. These topological challenges are solved by DNA topoisomerases via a strand-passage reaction which involves transiently breaking and re-joining of one (type I topoisomerases) or both (type II topoisomerases) strands of the phosphodiester backbone. This review will focus on one of the two mammalian type II DNA topoisomerase enzymes, DNA topoisomerase II beta (TOP2B), that have been implicated in correct execution of developmental transcriptional programs and in signal-induced transcription, including transcriptional activation by nuclear hormone ligands. Surprisingly, several lines of evidence indicate that TOP2B-mediated protein-free DNA double-strand breaks are involved in signal-induced transcription. We discuss the possible significance and origins of these DSBs along with a network of protein interaction data supporting a variety of roles for TOP2B in transcriptional regulation.
Asunto(s)
ADN-Topoisomerasas de Tipo II , Transcripción Genética , Animales , ADN , Replicación del ADN , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Mamíferos/metabolismo , HumanosRESUMEN
The Mixed-Lineage Leukaemia (MLL/KMT2A) gene is frequently rearranged in childhood and adult acute leukaemia (AL) and in secondary leukaemias occurring after therapy with DNA topoisomerase targeting anti-cancer agents such as etoposide (t-AL). MLL/KMT2A chromosome translocation break sites in AL patients fall within an 8 kb breakpoint cluster region (BCR). Furthermore, MLL/KMT2A break sites in t-AL frequently occur in a much smaller region, or hotspot, towards the 3' end of the BCR, close to the intron 11/exon 12 boundary. These findings have prompted considerable effort to uncover mechanisms behind the apparent fragility of the BCR and particularly the t-AL hotspot. Recent genome-wide analyses have demonstrated etoposide-induced DNA cleavage within the BCR, and it is tempting to conclude that this cleavage explains the distribution of translocation break sites in t-AL. However, the t-AL hotspot and the centre of the observed preferential DNA cleavage are offset by over 250 nucleotides, suggesting additional factors contribute to the distribution of t-AL break sites. We review these recent genomic datasets along with older experimental results, analysis of TOP2 DNA cleavage site preferences and DNA secondary structure features that may lead to break site selection in t-AL MLL/KMT2A translocations.
Asunto(s)
Estudio de Asociación del Genoma Completo , N-Metiltransferasa de Histona-Lisina , Leucemia , Proteína de la Leucemia Mieloide-Linfoide , Humanos , ADN , ADN-Topoisomerasas de Tipo II , Etopósido/farmacología , Leucemia/genética , Translocación Genética , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genéticaRESUMEN
Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.
Asunto(s)
ADN Mitocondrial , Mitocondrias , Humanos , Mitocondrias/metabolismo , ADN Mitocondrial/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Replicación del ADN/genética , ADN-Topoisomerasas/genéticaRESUMEN
The neuroblastoma cell line SH-SY5Y is widely used to study retinoic acid (RA)-induced gene expression and differentiation and as a tool to study neurodegenerative disorders. SH-SY5Y cells predominantly exhibit adrenergic neuronal properties, but they can also exist in an epigenetically interconvertible alternative state with more mesenchymal characteristics; as a result, these cells can be used to study gene regulation circuitry controlling neuroblastoma phenotype. Using a combination of pharmacological inhibition and targeted gene inactivation, we have probed the requirement for DNA topoisomerase IIB (TOP2B) in RA-induced gene expression and differentiation and in the balance between adrenergic neuronal versus mesenchymal transcription programmes. We found that expression of many, but not all genes that are rapidly induced by ATRA in SH-SY5Y cells was significantly reduced in the TOP2B null cells; these genes include BCL2, CYP26A1, CRABP2, and NTRK2. Comparing gene expression profiles in wild-type versus TOP2B null cells, we found that long genes and genes expressed at a high level in WT SH-SY5Y cells were disproportionately dependent on TOP2B. Notably, TOP2B null SH-SY5Y cells upregulated mesenchymal markers vimentin (VIM) and fibronectin (FN1) and components of the NOTCH signalling pathway. Enrichment analysis and comparison with the transcription profiles of other neuroblastoma-derived cell lines supported the conclusion that TOP2B is required to fully maintain the adrenergic neural-like transcriptional signature of SH-SY5Y cells and to suppress the alternative mesenchymal epithelial-like epigenetic state.
Asunto(s)
ADN-Topoisomerasas de Tipo II , Neuroblastoma , Proteínas de Unión a Poli-ADP-Ribosa , Adrenérgicos , Diferenciación Celular , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Humanos , Neuroblastoma/metabolismo , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Human topoisomerase II beta (TOP2B) modulates DNA topology using energy from ATP hydrolysis. To investigate the conformational changes that occur during ATP hydrolysis, we determined the X-ray crystallographic structures of the human TOP2B ATPase domain bound to AMPPNP or ADP at 1.9 Å and 2.6 Å resolution, respectively. The GHKL domains of both structures are similar, whereas the QTK loop within the transducer domain can move for product release. As TOP2B is the clinical target of bisdioxopiperazines, we also determined the structure of a TOP2B:ADP:ICRF193 complex to 2.3 Å resolution and identified key drug-binding residues. Biochemical characterization revealed the N-terminal strap reduces the rate of ATP hydrolysis. Mutagenesis demonstrated residue E103 as essential for ATP hydrolysis in TOP2B. Our data provide fundamental insights into the tertiary structure of the human TOP2B ATPase domain and a potential regulatory mechanism for ATP hydrolysis.
Asunto(s)
Adenosina Trifosfatasas , Adenosina Trifosfato , ADN-Topoisomerasas de Tipo II , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/genética , Humanos , Hidrólisis , Proteínas de Unión a Poli-ADP-RibosaRESUMEN
BACKGROUND: Hoffman syndrome is a syndromic, inborn error of immunity due to autosomal-dominant mutations in TOP2B, an essential gene required to alleviate topological stress during DNA replication and gene transcription. Although mutations identified in patients lead to a block in B-cell development and the absence of circulating B cells, an effect on natural killer (NK) cells was not previously examined. OBJECTIVE: We sought to determine whether disease-associated mutations in TOP2B impact NK-cell development and function. METHODS: Using a knockin murine model and patient-derived induced pluripotent stem cells (iPSCs), we investigated NK-cell development in mouse bone marrow and spleen, and performed immunophenotyping by flow cytometry, gene expression, and functional assessment of cytotoxic activity in murine NK cells, and human IPSC-derived NK cells. RESULTS: Mature NK cells were reduced in the periphery of TOP2B knockin mice consistent with patient reports, with reduced cytotoxicity toward target cell lines. IPSCs were successfully derived from patients with Hoffman syndrome, but under optimal conditions showed reduced cytotoxicity compared with iPSC-derived NK cells from healthy controls. CONCLUSIONS: Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function in murine and human models.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Asesinas Naturales , Animales , Línea Celular , Anomalías Craneofaciales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Deformidades Congénitas de las Extremidades , Ratones , Mutación , Enfermedades de Inmunodeficiencia Primaria , Anomalías UrogenitalesRESUMEN
DNA topoisomerases regulate the topological state of DNA, relaxing DNA supercoils and resolving catenanes and knots that result from biologic processes, such as transcription and replication. DNA topoisomerase II (TOP2) enzymes achieve this by binding DNA and introducing an enzyme-bridged DNA double-strand break (DSB) where each protomer of the dimeric enzyme is covalently attached to the 5' end of the cleaved DNA via an active site tyrosine phosphodiester linkage. The enzyme then passes a second DNA duplex through the DNA break, before religation and release of the enzyme. However, this activity is potentially hazardous to the cell, as failure to complete religation leads to persistent TOP2 protein-DNA covalent complexes, which are cytotoxic. Indeed, this property of topoisomerase has been exploited in cancer therapy in the form of topoisomerase poisons which block the religation stage of the reaction cycle, leading to an accumulation of topoisomerase-DNA adducts. A number of parallel cellular processes have been identified that lead to removal of these covalent TOP2-DNA complexes, facilitating repair of the resulting protein-free DSB by standard DNA repair pathways. These pathways presumably arose to repair spontaneous stalled or poisoned TOP2-DNA complexes, but understanding their mechanisms also has implications for cancer therapy, particularly resistance to anti-cancer TOP2 poisons and the genotoxic side effects of these drugs. Here, we review recent progress in the understanding of the processing of TOP2 DNA covalent complexes, the basic components and mechanisms, as well as the additional layer of complexity posed by the post-translational modifications that modulate these pathways. SIGNIFICANCE STATEMENT: Multiple pathways have been reported for removal and repair of TOP2-DNA covalent complexes to ensure the timely and efficient repair of TOP2-DNA covalent adducts to protect the genome. Post-translational modifications, such as ubiquitination and SUMOylation, are involved in the regulation of TOP2-DNA complex repair. Small molecule inhibitors of these post-translational modifications may help to improve outcomes of TOP2 poison chemotherapy, for example by increasing TOP2 poison cytotoxicity and reducing genotoxicity, but this remains to be determined.
Asunto(s)
Reparación del ADN/fisiología , ADN-Topoisomerasas de Tipo II/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Roturas del ADN/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Reparación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/genética , Humanos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/fisiologíaRESUMEN
Transcription is regulated and mediated by multiprotein complexes in a chromatin context. Transcription causes changes in DNA topology which is modulated by DNA topoisomerases, enzymes that catalyse changes in DNA topology via transient breaking and re-joining of one or both strands of the phosphodiester backbone. Mammals have six DNA topoisomerases, this review focuses on one, DNA topoisomerase II beta (TOP2B). In the absence of TOP2B transcription of many developmentally regulated genes is altered. Long genes seem particularly susceptible to the lack of TOP2B. Biochemical studies of the role of TOP2B in transcription regulated by ligands such as nuclear hormones, growth factors and insulin has revealed PARP1 associated with TOP2B and also PRKDC, XRCC5 and XRCC6. Analysis of publicly available databases of protein interactions confirms these interactions and illustrates interactions with other key transcriptional regulators including TRIM28. TOP2B has been shown to interact with proteins involved in chromosome organisation including CTCF and RAD21. Comparison of publicly available Chip-seq datasets reveals the location at which these proteins interact with genes. The availability of resources such as large datasets of protein-protein interactions, e.g. BioGrid and IntAct and protein-DNA interactions such as Chip-seq in GEO enables scientists to extend models and propose new hypotheses.
Asunto(s)
ADN-Topoisomerasas de Tipo II/fisiología , Proteínas de Unión a Poli-ADP-Ribosa/fisiología , Transcripción Genética/fisiología , Animales , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Unión ProteicaRESUMEN
DNA topoisomerase II (TOP2) poisons induce protein-DNA crosslinks termed TOP2-DNA covalent complexes, in which TOP2 remains covalently bound to each end of an enzyme-induced double-strand DNA break (DSB) via a 5'-phosphotyrosyl bond. Repair of the enzyme-induced DSB first requires the removal of the TOP2 protein adduct, which, among other mechanisms, can be accomplished through the proteasomal degradation of TOP2. VCP/p97 is a AAA ATPase that utilizes energy from ATP hydrolysis to unfold protein substrates, which can facilitate proteasomal degradation by extracting target proteins from certain cellular structures (such as chromatin) and/or by aiding their translocation into the proteolytic core of the proteasome. In this study, we show that inhibition of VCP/p97 leads to the prolonged accumulation of etoposide-induced TOP2A and TOP2B complexes in a manner that is epistatic with the proteasomal pathway. VCP/p97 inhibition also reduces the etoposide-induced phosphorylation of histone H2A.X, indicative of fewer DSBs. This suggests that VCP/p97 is required for the proteasomal degradation of TOP2-DNA covalent complexes and is thus likely to be an important mediator of DSB repair after treatment with a TOP2 poison. SIGNIFICANCE STATEMENT: TOP2 poisons are chemotherapeutic agents used in the treatment of a range of cancers. A better understanding of how TOP2 poison-induced DNA damage is repaired could improve therapy with TOP2 poisons by increasing TOP2 poison cytotoxicity and reducing genotoxicity. The results presented herein suggest that repair of TOP2-DNA covalent complexes involves the protein segregase VCP/p97.
Asunto(s)
Acetanilidas/farmacología , Benzotiazoles/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , ADN/metabolismo , Etopósido/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteína que Contiene Valosina/metabolismo , Adenosina Trifosfato/metabolismo , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Humanos , Hidrólisis , Células K562 , Fosforilación , Pliegue de Proteína , Estabilidad Proteica , Proteína que Contiene Valosina/genéticaRESUMEN
Cardioprotective activity of dexrazoxane (ICRF-187), the only clinically approved drug against anthracycline-induced cardiotoxicity, has traditionally been attributed to its iron-chelating metabolite. However, recent experimental evidence suggested that the inhibition and/or depletion of topoisomerase IIß (TOP2B) by dexrazoxane could be cardioprotective. Hence, we evaluated a series of dexrazoxane analogues and found that their cardioprotective activity strongly correlated with their interaction with TOP2B in cardiomyocytes, but was independent of their iron chelation ability. Very tight structure-activity relationships were demonstrated on stereoisomeric forms of 4,4'-(butane-2,3-diyl)bis(piperazine-2,6-dione). In contrast to its rac-form 12, meso-derivative 11 (ICRF-193) showed a favorable binding mode to topoisomerase II in silico, inhibited and depleted TOP2B in cardiomyocytes more efficiently than dexrazoxane, and showed the highest cardioprotective efficiency. Importantly, the observed ICRF-193 cardioprotection did not interfere with the antiproliferative activity of anthracycline. Hence, this study identifies ICRF-193 as the new lead compound in the development of efficient cardioprotective agents.
Asunto(s)
Cardiotónicos/uso terapéutico , Cardiotoxicidad/tratamiento farmacológico , Piperazinas/uso terapéutico , Inhibidores de Topoisomerasa II/uso terapéutico , Animales , Animales Recién Nacidos , Cardiotónicos/síntesis química , Cardiotónicos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Daunorrubicina/toxicidad , Dicetopiperazinas , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Miocitos Cardíacos/efectos de los fármacos , Piperazinas/síntesis química , Piperazinas/metabolismo , Unión Proteica , Ratas Wistar , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/metabolismoRESUMEN
DNA topoisomerase II beta (TOP2B) has a role in transcriptional regulation. Here, to further investigate transcriptional regulation by TOP2B, we used RNA-sequencing and real-time PCR to analyse the differential gene expression profiles of wild-type and two independent TOP2B-null pre-B Nalm-6 cell lines, one generated by targeted insertion and the other using CRISPR-Cas9 gene editing. We identified carbonyl reductase 1 (CBR1) among the most significantly downregulated genes in these TOP2B-null cells. Reduced CBR1 expression was accompanied by loss of binding of the transcription factors USF2 and MAX to the CBR1 promoter. We describe possible mechanisms by which loss of TOP2B results in CBR1 downregulation. To our knowledge, this is the first report of a link between TOP2B and CBR1.
Asunto(s)
Carbonil Reductasa (NADPH)/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Carbonil Reductasa (NADPH)/metabolismo , Línea Celular , Epigénesis Genética , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Regiones Promotoras GenéticasRESUMEN
DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Nucleósidos/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Sulfonamidas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Línea Celular , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/química , Humanos , Células K562 , Proteínas de Unión a Poli-ADP-Ribosa/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Pirazoles , Pirimidinas , Sulfuros , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
2,6-Diaminopyridine-3,5-bis(thiocyanate) (PR-619) is a broad-spectrum deubiquitinating enzyme (DUB) inhibitor that has been employed in cell-based studies as a tool to investigate the role of ubiquitination in various cellular processes. Here, we demonstrate that in addition to its action as a DUB inhibitor, PR-619 is a potent DNA topoisomerase II (TOP2) poison, inducing both DNA topoisomerase IIα (TOP2A) and DNA topoisomerase IIß (TOP2B) covalent DNA complexes with similar efficiency to the archetypal TOP2 poison etoposide. However, in contrast to etoposide, which induces TOP2-DNA complexes with a pan-nuclear distribution, PR-619 treatment results in nucleolar concentration of TOP2A and TOP2B. Notably, neither the induction of TOP2-DNA covalent complexes nor their nucleolar concentration are due to TOP2 hyperubiquitination since both occur even under conditions of depleted ubiquitin. Like etoposide, since PR-619 affected TOP2 enzyme activity in in vitro enzyme assays as well as in live cells, we conclude that PR-619 interacts directly with TOP2A and TOP2B. The concentration at which PR-619 exhibits robust cellular DUB inhibitor activity (5-20 µM) is similar to the lowest concentration at which TOP2 poison activity was detected (above 20 µM), which suggests that caution should be exercised when employing this DUB inhibitor in cell-based studies.
Asunto(s)
Aminopiridinas/farmacología , Aminopiridinas/envenenamiento , ADN-Topoisomerasas de Tipo II/biosíntesis , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Enzimas Desubicuitinizantes/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Unión a Poli-ADP-Ribosa/biosíntesis , Tiocianatos/farmacología , Tiocianatos/envenenamiento , Células HeLa , Humanos , Células K562 , Proteínas de Unión a Poli-ADP-Ribosa/agonistasRESUMEN
B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization.
Asunto(s)
Linfocitos B/inmunología , ADN-Topoisomerasas de Tipo II/genética , Enfermedades de Inmunodeficiencia Primaria/enzimología , Animales , Diferenciación Celular , ADN-Topoisomerasas de Tipo II/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Mutación , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/inmunología , Enfermedades de Inmunodeficiencia Primaria/fisiopatología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
Topoisomerase II (TOP2) poisons are effective cytotoxic anticancer agents that stabilize the normally transient TOP2-DNA covalent complexes formed during the enzyme reaction cycle. These drugs include etoposide, mitoxantrone, and the anthracyclines doxorubicin and epirubicin. Anthracyclines also exert cell-killing activity via TOP2-independent mechanisms, including DNA adduct formation, redox activity, and lipid peroxidation. Here, we show that anthracyclines and another intercalating TOP2 poison, mitoxantrone, stabilize TOP2-DNA covalent complexes less efficiently than etoposide, and at higher concentrations they suppress the formation of TOP2-DNA covalent complexes, thus behaving as TOP2 poisons at low concentration and inhibitors at high concentration. We used induced pluripotent stem cell (iPSC)-derived human cardiomyocytes as a model to study anthracycline-induced damage in cardiac cells. Using immunofluorescence, our study is the first to demonstrate the presence of topoisomerase IIß (TOP2B) as the only TOP2 isoform in iPSC-derived cardiomyocytes. In these cells, etoposide robustly induced TOP2B covalent complexes, but we could not detect doxorubicin-induced TOP2-DNA complexes, and doxorubicin suppressed etoposide-induced TOP2-DNA complexes. In vitro, etoposide-stabilized DNA cleavage was attenuated by doxorubicin, epirubicin, or mitoxantrone. Clinical use of anthracyclines is associated with cardiotoxicity. The observations in this study have potentially important clinical consequences regarding the effectiveness of anticancer treatment regimens when TOP2-targeting drugs are used in combination. These observations suggest that inhibition of TOP2B activity, rather than DNA damage resulting from TOP2 poisoning, may play a role in doxorubicin cardiotoxicity. SIGNIFICANCE STATEMENT: We show that anthracyclines and mitoxantrone act as topoisomerase II (TOP2) poisons at low concentration but attenuate TOP2 activity at higher concentration, both in cells and in in vitro cleavage experiments. Inhibition of type II topoisomerases suppresses the action of other drugs that poison TOP2. Thus, combinations containing anthracyclines or mitoxantrone and etoposide may reduce the activity of etoposide as a TOP2 poison and thus reduce the efficacy of drug combinations.
Asunto(s)
Antraciclinas/farmacología , Aductos de ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Etopósido/farmacología , Mitoxantrona/farmacología , Cardiotoxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Aductos de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/efectos adversos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células K562 , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Type II topoisomerases catalyze essential DNA transactions and are proven drug targets. Drug discrimination by prokaryotic and eukaryotic topoisomerases is vital to therapeutic utility, but is poorly understood. We developed a next-generation sequencing (NGS) approach to identify drug-resistance mutations in eukaryotic topoisomerases. We show that alterations conferring resistance to poisons of human and yeast topoisomerase II derive from a rich mutational 'landscape' of amino acid substitutions broadly distributed throughout the entire enzyme. Both general and discriminatory drug-resistant behaviors are found to arise from different point mutations found at the same amino acid position and to occur far outside known drug-binding sites. Studies of selected resistant enzymes confirm the NGS data and further show that the anti-cancer quinolone vosaroxin acts solely as an intercalating poison, and that the antibacterial ciprofloxacin can poison yeast topoisomerase II. The innate drug-sensitivity of the DNA binding and cleavage region of human and yeast topoisomerases (particularly hTOP2ß) is additionally revealed to be significantly regulated by the enzymes' adenosine triphosphatase regions. Collectively, these studies highlight the utility of using NGS-based methods to rapidly map drug resistance landscapes and reveal that the nucleotide turnover elements of type II topoisomerases impact drug specificity.
Asunto(s)
Ciprofloxacina/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , Naftiridinas/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Tiazoles/farmacología , Inhibidores de Topoisomerasa II/farmacología , Antibacterianos/farmacología , Antineoplásicos/farmacología , ADN/química , ADN/genética , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/genética , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Type II DNA topoisomerases (EC 5.99.1.3) are enzymes that catalyse topological changes in DNA in an ATP dependent manner. Strand passage reactions involve passing one double stranded DNA duplex (transported helix) through a transient enzyme-bridged break in another (gated helix). This activity is required for a range of cellular processes including transcription. Vertebrates have two isoforms: topoisomerase IIα and ß. Topoisomerase IIß was first reported in 1987. Here we review the research on DNA topoisomerase IIß over the 30 years since its discovery.
Asunto(s)
ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Investigación , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Ciclo Celular/genética , Clonación Molecular , ADN-Topoisomerasas de Tipo II/química , ADN Complementario/química , ADN Complementario/genética , Expresión Génica , Regulación de la Expresión Génica , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Espacio Intracelular/metabolismo , Isoenzimas , Terapia Molecular Dirigida , Unión Proteica , Transporte de Proteínas , Investigación/historia , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/uso terapéutico , Activación TranscripcionalRESUMEN
DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5'-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5'-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5' tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition.