Antifolates can potentiate topoisomerase II inhibitors in vitro and in vivo

AbstractPeposertib (M3814) is a potent and selective DNA-PK inhibitor in early clinical development. It effectively blocks non-homologous end-joining repair of DNA double-strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By suppressing DNA-PK catalytic activity in the presence of DNA DSB, M3814 potentiates ATM/p53 signaling leading to enhanced p53-dependent antitumor activity in tumor cells. Here, we investigated the therapeutic potential of M3814 in combination with DSB-inducing agents in leukemia cells and a patient-derived tumor. We show that in the presence of IR or topoisomerase II inhibitors, M3814 boosts the ATM/p53 response in acute leukemia cells leading to the elevation of p53 protein levels as well as its transcriptional activity. M3814 synergistically sensitized p53 wild-type, but not p53-deficient, AML cells to killing by DSB-inducing agents via p53-dependent apoptosis involving both intrinsic and extrinsic effector pathways. The antileukemic effect was further potentiated by enhancing daunorubicin-induced myeloid cell differentiation. Further, combined with the fixed-ratio liposomal formulation of daunorubicin and cytarabine, CPX-351, M3814 enhanced the efficacy against leukemia cells in vitro and in vivo without increasing hematopoietic toxicity, suggesting that DNA-PK inhibition could offer a novel clinical strategy for harnessing the anticancer potential of p53 in AML therapy.

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Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast

Biochemical Journal ◽

10.1042/bj3240329 ◽

1997 ◽

Vol 324 (1) ◽

pp. 329-339 ◽

Cited By ~ 14

Author(s):

Beatrice A. KELLER ◽

Sandhiya PATEL ◽

L. Mark FISHER

Keyword(s):

Candida Albicans ◽

Topoisomerase Ii ◽

Cloning And Expression ◽

Dna Topoisomerase Ii ◽

Topoisomerase Iiα ◽

Dna Topoisomerase ◽

Topoisomerase Ii Inhibitors ◽

Anti Fungal

Candida albicans topoisomerase II, encoded by the TOP2 gene, mediates chromosome segregation by a double-strand DNA break mechanism and is a potential target for anti-fungal therapy. In this paper, we report the characterization of the C. albicans TOP2 gene and its use to develop a yeast system that allows the identification and study of anti-fungal topoisomerase II inhibitors in vivo. The gene, specifying a 1461-residue polypeptide with only 40% identity with human topoisomerase IIα and β isoforms, was isolated from C. albicans on a 6.3 kb EcoRI fragment that mapped to chromosome 4. It was used to construct a plasmid in which TOP2 expresses a recombinant enzyme (residues 57–1461 of C. albicans topoisomerase II fused to the first five residues of Saccharomyces cerevisiae topoisomerase II) under the control of a galactose-inducible promoter. The plasmid rescued the lethal phenotype of a temperature-sensitive S. cerevisiae DNA topoisomerase II mutant allowing growth at 35 °C. Yeast cells, bearing ISE2 permeability and rad52 double-strand-break-repair mutations the growth of which at 35 °C was dependent on C. albicans topoisomerase II, were killed by the known topoisomerase II inhibitors amsacrine and doxorubicin. Parallel experiments in yeast expressing human topoisomerase IIα allowed the relative sensitivities of the fungal and host topoisomerases to be examined in the same genetic background. To compare the killing in vivo with drug inhibition in vitro, the recombinant C. albicans topoisomerase II protein was expressed and purified to near-homogeneity from S. cerevisiae yielding a 160 kDa polypeptide that displayed the expected ATP-dependent DNA-relaxation and DNA-decatenation activities. The enzyme, whether examined in vitro or complementing in S. cerevisiae, was comparably sensitive to amsacrine and doxorubicin. Our results suggest that potential topoisomerase II-targeting anti-fungal inhibitors can be identified and studied in S. cerevisiae.

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The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146217 ◽

1993 ◽

Vol 51 ◽

pp. 74-75

Author(s):

Jason R. Swedlow ◽

Neil Osheroff ◽

Tim Karr ◽

John W. Sedat ◽

David A. Agard

Keyword(s):

Topoisomerase Ii ◽

Biochemical Characterization ◽

Developmental Cycle ◽

Dna Topoisomerase Ii ◽

Chromosomal Distribution ◽

Dna Topoisomerase ◽

Ideal System ◽

Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

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Limited activity of bacterial DNA topoisomerase II inhibitors against Leishmania donovani and Trypanosoma cruzi amastigotes in vitro

Transactions of the Royal Society of Tropical Medicine and Hygiene ◽

10.1016/0035-9203(88)90017-x ◽

1988 ◽

Vol 82 (6) ◽

pp. 856 ◽

Cited By ~ 15

Author(s):

S.L. Croft ◽

J. Hogg

Keyword(s):

Trypanosoma Cruzi ◽

Topoisomerase Ii ◽

Leishmania Donovani ◽

Dna Topoisomerase Ii ◽

Dna Topoisomerase ◽

Bacterial Dna ◽

Topoisomerase Ii Inhibitors

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The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3197-3207.1994 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3197-3207

Author(s):

P R Caron ◽

P Watt ◽

J C Wang

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Localization ◽

Topoisomerase Ii ◽

Dna Topoisomerase Ii ◽

Dna Topoisomerase ◽

Mutant Proteins ◽

Terminal Domain ◽

Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

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Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy

Journal of Cell Science ◽

10.1242/jcs.104.4.1175 ◽

1993 ◽

Vol 104 (4) ◽

pp. 1175-1185 ◽

Cited By ~ 12

Author(s):

P. Buchenau ◽

H. Saumweber ◽

D.J. Arndt-Jovin

Keyword(s):

Confocal Laser Scanning Microscopy ◽

Laser Scanning ◽

Topoisomerase Ii ◽

Metaphase Plate ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Scanning Microscopy ◽

Confocal Laser

The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. We show that the enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos.

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Effect of etoposide on grass pea DNA topoisomerase II: an in silico, in vivo, and in vitro assessments

Bulletin of the National Research Centre ◽

10.1186/s42269-019-0217-4 ◽

2019 ◽

Vol 43 (1) ◽

Author(s):

Aveek Samanta ◽

Tilak Raj Maity ◽

Sudip Das ◽

Animesh Kumar Datta ◽

Siraj Datta

Keyword(s):

In Silico ◽

Topoisomerase Ii ◽

Cost Effective ◽

Colchicine Treatment ◽

Grass Pea ◽

Dna Topoisomerase ◽

Ammonium Sulphate Precipitation ◽

In Silico Study

Abstract Background Etoposide is one of the most potential anti-cancerous drugs that targets topoisomerase II (topoII) and inhibits its activity by ligation with the DNA molecule. Results In silico study confirmed that the etoposide-binding sites of topoII are conserved among the plants and human. The efficacy of the drug on plant system was initially assessed using germinated grass pea (Lathyrus sativus L.) seedlings (in vivo) in relation to radicle length and mitotic index. The callus system (in vitro) was also used to elucidate the effect of etoposide on callus growth kinetics. Furthermore, it was observed that etoposide able to inhibit the division of polyploid cells induced by colchicine treatment (0.5%, 8 h). To determine the molecular interaction, topoII was isolated from young grass pea leaves using polyethylene glycol fractionation and ammonium sulphate precipitation followed by column chromatography on CM-Sephadex (C-25). The plasmid linearization assays by isolated plant topoII in the presence of etoposide significantly revealed the functional similarity of plants and human topoII. Results indicated that the effect of etoposide on plant topoII is significant. Conclusions This study may pave the way to develop a plant-based assay system for screening the topoisomerase targeted anti-cancerous drugs, as it is convenient and cost-effective.

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Topoisomerase II levels and drug sensitivity in adult acute myelogenous leukemia

Blood ◽

10.1182/blood.v83.2.517.517 ◽

1994 ◽

Vol 83 (2) ◽

pp. 517-530 ◽

Cited By ~ 71

Author(s):

SH Kaufmann ◽

JE Karp ◽

RJ Jones ◽

CB Miller ◽

E Schneider ◽

...

Keyword(s):

Acute Myelogenous Leukemia ◽

Topoisomerase Ii ◽

Drug Sensitivity ◽

Myelogenous Leukemia ◽

Clinical Samples ◽

Immunoperoxidase Staining ◽

Acute Myelogenous ◽

Topo Ii

Abstract The topoisomerase (topo) II-directed agents etoposide, daunorubicin (DNR), and amsacrine (m-AMSA) are widely used in the treatment of acute myelogenous leukemia (AML). In the present study, multiple aspects of topo II-mediated drug action were examined in marrows from adult AML patients. Colony-forming assays revealed that the dose of etoposide, DNR, or m-AMSA required to diminish leukemic colony formation by 90% (LD90) varied over a greater than 20-fold range between different pretreatment marrows. Measurement of nuclear DNR accumulation in the absence and presence of quinidine revealed evidence of P-glycoprotein (Pgp) function in 8 of 82 samples at diagnosis and 5 of 36 samples at first relapse, but the largest quinidine-induced increment in DNR accumulation (< 2-fold) was too small to explain the variations in drug sensitivity. Restriction enzyme-based assays and sequencing of partial topo II alpha and topo II beta cDNAs from the most highly resistant specimens failed to demonstrate topo II gene mutations that could account for resistance. Western blotting of marrow samples containing greater than 80% blasts revealed that the content of the two topo II isoenzymes varied over a greater than 20-fold range, but did not correlate with drug sensitivity in vitro or in vivo. In addition, levels of topo II alpha and topo II beta in 46 of 47 clinical samples were lower than in human AML cell lines. Immunoperoxidase staining showed that these low topo II levels were accompanied by marked cell-to- cell heterogeneity, with topo II alpha being abundant in some blasts and diminished or absent from others. There was a trend toward increasing percentages of topo II alpha-positive cells in pretreatment marrows that contained more S-phase cells. Consistent with this observation, treatment of patients with granulocyte-macrophage colony- stimulating factor for 3 days before chemotherapy resulted in increases in topo II alpha-positive cells concomitant with increases in the number of cells traversing the cell cycle. These observations have implications for the regulation of topo II in AML, for the use of topo II-directed chemotherapy, and for future attempts to relate drug sensitivity to topo II levels in clinical material.

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