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Event: 1252

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Binding to (interferes with) topoisomerase II enzyme

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. More help
Binding, topoisomerase II
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Biological Context

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Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Cell term
eukaryotic cell

Organ term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling).  The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor).  Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description.  To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons.  If a desired term does not exist, a new term request may be made via Term Requests.  Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Process Object Action
DNA topoisomerase II activity abnormal

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
topoisomerase II binding, infant leukaemia MolecularInitiatingEvent Andrea Terron (send email) Open for comment. Do not cite WPHA/WNT Endorsed

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
mammals mammals High NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help

Sex Applicability

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Term Evidence
Mixed High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

Type II topoisomerases are ubiquitous enzymes that are required for proper chromosome structure and segregation and play important roles in DNA replication, transcription, and recombination. Type II topoisomerases change DNA topology by breaking and rejoining double-stranded DNA. These enzymes can introduce or remove supercoils and can separate two DNA duplexes that are intertwined. Type II topoisomerases relax DNA and remove knots and tangles from the genetic material by passing an intact double helix (transport segment) through a transient double-stranded break that they generate in a separate DNA segment (gate segment). Humans encode two closely related isoforms of the type II enzyme. Topoisomerase II Þ is essential for the survival of proliferating cells and topoisomerase II ß plays critical roles during development. However, because these enzymes generate double-stranded DNA breaks during their crucial catalytic functions, the consequences are not only beneficial. Although essential to cell survival, they also pose an intrinsic threat to genomic integrity every time they act. Beyond their critical physiological functions, topoisomerase IIÞ and IIß are the primary targets for some of the most active and widely prescribed drugs currently used for the treatment of human cancers. These agents kill cells by increasing levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, intermediates in the catalytic DNA strand passage reaction. Many chemicals do so by inhibiting the ability of the type II enzymes to ligate cleaved DNAs. When the resulting enzyme-associated DNA breaks are present in sufficient concentrations, they can trigger cell death pathways. Chemicals that target type II enzymes are referred to as topoisomerase II poisons because they convert these indispensable enzymes to potent physiological toxins that generate DNA damage in treated cells. Because the enzyme functions by passing an intact double helix through a transient double-stranded break, any disturbances in its function, e.g. by chemical inhibitors, could have a profound effect on genomic stability, resulting in DNA repair response, gene and chromosomal damage, initiation of apoptosis and ultimate cell death. A double-strand break and error-prone non-homologous end-joining (NHEJ) DNA repair mechanism may lead to gene rearrangements; chromosomal translocations and consequently fusion genes (see Figure 33). A comprehensive description of TopoII enzymes and their functions and derangements could be found in recent review articles (Cowell and Austin 2012; Pendleton et al 2014; Ketron and Osheroff 2014).

Fig.33: TOP2 Poisons, downstream events. TOP2 poisons inhibit the religation step of the TOP2 reaction cycle, leading to accumulation of covalent TOP2-DNA cleavage complexes. These lesions are cytotoxic and lead to activation of the DNA damage response and potentially apoptosis. Alternatively these lesions are repaired, largely through the non-homologous end-joining pathway. Translocations observed in therapy-related leukemia are presumed to occur as a result of mis-repair, joining two heterologous ends. (from Cowell and Austin 2012)

DNA topoisomerase (Top) II enzyme “poisons” disturb the normal TopoII enzyme function and cause a ‘hanging double strand break (DSB)’ at a specified DNA sequence. The above description of the MIE is of significance because there are 3 different kinds of “poisons" of TopoII enzyme, out of which competitive inhibitors prevent the function of the enzyme and cause cell death, whereas other interfacial and covalent inhibitors may cause – depending on the situation – other consequences of DNA damage response including chromosomal rearrangements (Pendleton et L 2014; Lu et al 2015). A further prerequisite for the specific outcome, i.e. creation of chromosomal rearrangement, is that TopoII “poison” has to occur in an especially vulnerable and correct hot spot in the MLL locus in the right target cell vulnerable to transformation.

The MIE, topo II poisons, can occur prenatally i.e. prenatal exposure to topo II poisons. Human embryonic stem cells are more sensitive to topo II inhibition than postnatal CD34+ cells, linking embryonic exposure to topoisomerase II poisons to genomic instability. However, little is know about the nature of the target cell for transformation (Bueno et al. 2011).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

The identification and measurement of the inhibition of TopoII enzymes is made more difficult by the presence of different molecular mechanisms (see above). However, some assays are used in pharmacological research to screen TopoII “poisons”, including cell-free decatenation assay (Schroeter et al., 2015). The most important mode, the cleavage activity of TopoII can be studied in vitro, by using a human recombinant enzyme and an appropriate double-stranded plasmid as a target to quantitate double-strand breaks (Fortune and Osheroff 1998). A cleavage can also be indirectly detected by measuring various indicators of DNA damage response, such as ATM activity, p53 expression, γH2AX or Comet assay (Li et al 2014, Schroeter et al., 2015, Castano et al 2016).

It is useful to note that several chemicals identified as TopoII “poisons”do require metabolic oxidation to become active inhibitors. Etoposide itself is converted via the catechol metabolite to etoposide 3-quinone, which is a covalent TopoII poison (Smith et al 2014), whereas etoposide and its catechol are interfacial inhibitors which bind selectively to interfaces as macromolecular machines assemblewhich bind selectively to interfaces as macromolecular machines assemble. Curcumin is also an active TopoII poison due to its oxidized metabolites (Gordon et al 2015). This fact deserves consideration if a screening for TopoII inhibition is envisaged.

Topoisomerase poisons stabilize the covalent enzyme–DNA complex. There are several key characteristics of this complex: it includes protein covalently bound to DNA as well as a strand break in the DNA substrate, and it is also freely reversible. Accordingly, if the chemical is removed the enzyme rapidly reseals the DNA. Covalent complexes are quantified in two ways: by measuring the levels of protein covalently bound to DNA or by directly assaying for DNA strand breaks in the presence of topoisomerase and test agent or known drug. The assay directly measures DNA strand breaks induced by topoisomerase I in a substrate that carries a strong DNA cleavage site. Similarly, the plasmid linearization assay measures double strand breaks induced in plasmid DNA by topoisomerase II. The Alternate Protocol allows for the visualization of breaks induced on a larger substrate. Different protocols are used to measure the amount of the cleavage complex by determining the levels of topoisomerases that are covalently associated with DNA. Since the covalent complex is a normal step in the topoisomerase reaction, it can be detected (using very sensitive assays) even in the absence of a topoisomerase poison. However, addition of a topoisomerase poison greatly increases the levels of covalent complexes. Protocol and procedure details for mewasuring topoisomerase inhibition are fully reported in Nitiss et al. 2012.

In vivo complex enzyme assay (Rodriguez et al. 2020): hESCs were either immediately lysed in 1 % (w/v) sarkosyl (Sigma L7414). Lysates were processed according to the in vivo complex of enzyme (ICE) assay (Nitiss, Soans, Rogojina, Seth, & Mishina, 2012; Schellenberg et al., 2017). Briefly, sheared samples were centrifuged with a CsCl (Applichem-Panreac, A1098) gradient at 57,000 r.p.m. for 20 h at 25 °C using 3.3 ml 13 x 33 mm polyallomer Optiseal tubes (Beckman Coulter) in a TLN100 rotor (Beckman Coulter). For slot blotting, ICE samples containing 1, 2 or 4 µg of DNA were transferred onto Odyssey Nitrocellulose Membranes (LI-COR Biosciences) using a Bio-Dot SF Microfiltration Apparatus (Biorad). For western blot of ICE, samples were resuspended in 12,000 units of Micrococcal Nuclease (MNase, NEB 0247S), 1 x MNase buffer (NEB, B0247S) and 100 µg / mL BSA (NEB, B9000S), then incubated at 37 °C for 6 h. Samples were run in 10% SDS-PAGE and transferred to Immobilon-FL Transfer Membranes (Millipore). Membranes were blocked for 1 h in Odyssey Blocking Buffer (LI-COR Biosciences), then incubated for 2 h with primary antibodies in the same buffer with additional 0.1% (v/v) TWEEN 20, washed 3x with TBS-0.1%-TWEEN20, incubated with secondary antibodies for 1 h, and finally washed again. Once the membranes were dry, slots were analyzed and quantified in Odyssey CLx using ImageStudio Odyssey CLx Software.

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided).  More help

DNA topoisomerases are ubiquitous enzymes, which control the integrity of double-stranded DNA. They are thus key enzymes at all levels of living organisms. The available evidence suggest that important differences in sensitivity to topoisomerase inhibition  might exist among different cell types, depending on the amount of proliferative burden, of the TopoII enzymes and on physiological repair processes. Mesodermal precursor or hematopoietic stem and progenitor cells (HSPCs) are rapidly dividing cells with a high content of TopoII and for these reasons they can be a sensitive target during a critical developmental window (Hernandez and Menendez 2016).  In addition, evidence from micronuclei assay studies conducted in untreated and chemical-treated foetuses and newborns show that both the baseline and chemically induced micronuclei frequencies are higher in the foetuses and infants than in adults (Udroiu et al 2016). This is possibly indicating a greater sensitivity to genotoxic insult during development which can be due to the higher proliferation rate and lower ability of DNA repair of the hematopoietic stem cells. However, the role that the different microenvironments (foetal liver, infant bone marrow and adult bone marrow) during ontogenesis can exert on cell sensitivity cannot be ruled out (Udroiu et al. 2016). The existence of relevant interspecies differences is unknown, but it cannot be ruled out presently.

References

List of the literature that was cited for this KE description. More help

Alexander FE, Patheal SL, Biondi A, Brandalise S, Cabrera ME, Chan LC, Chen Z, Cimino G, Cordoba JC, Gu LJ, Hussein H, Ishii E, Kamel AM, Labra S, Magalhaes IQ, Mizutani S, Petridou E, de Oliveira MP, Yuen P, Wiemels JL, Greaves MF. Transplacental chemical exposure and risk of infant leukemia with MLL gene fusion. Cancer Res. 2001 Mar 15;61(6):2542-6.

Hande KR. Etoposide: four decades of development of a topoisomerase II inhibitor. Eur. J. Cancer. 1998;34:1514–1521.

Azarova AM, Lin RK, Tsai YC, Liu LF, Lin CP, Lyu YL. Genistein induces topoisomerase IIbeta- and proteasome-mediated DNA sequence rearrangements: Implications in infant leukemia. Biochem Biophys Res Commun. 2010 Aug 13;399(1):66-71. doi: 10.1016/j.bbrc.2010.07.043.

Bandele OJ, Osheroff N. Bioflavonoids as poisons of human topoisomerase II alpha and II beta. Biochemistry. 2007 May 22;46(20):6097-108.

Barjesteh van Waalwijk van Doorn-Khosrovani S, Janssen J, Maas LM, Godschalk RW, Nijhuis JG, van Schooten FJ. Dietary flavonoids induce MLL translocations in primary human CD34+ cells. Carcinogenesis. 2007 Aug;28(8):1703-9.

Castaño J, Herrero AB, Bursen A, González F, Marschalek R, Gutiérrez NC, Menendez P.Expression of MLL.AF4 or 1 AF4.MLL fusions 2 does not impact the efficiency of DNA damage repair. Nucl Acid Res 2016; in press

Cowell IG, Austin CA. Mechanism of generation of therapy related leukemia in response to anti-topoisomerase II agents. Int J Environ Res Public Health. 2012 Jun;9(6):2075-91. doi: 10.3390/ijerph9062075.

Fortune JM, Osheroff  N. Merbarone inhibits the catalytic activity of human topoisomerase IIalpha by blocking DNA cleavage. J Biol Chem. 1998; 273(28): 17643-17650.

Gordon ON, Luis PB, Ashley RE, Osheroff N, Schneider C. Oxidative Transformation of Demethoxy- and Bisdemethoxycurcumin: Products, Mechanism of Formation, and Poisoning of Human Topoisomerase IIβ. Chem Res Toxicol. 2015; 28(5): 989-996. doi: 10.1021/acs.chemrestox.5b00009.

Hernandez Jerez A and Menendez P. Linking pesticide exposure with pediatric leukemia: potential underlying mechanisms. Int J Mol Sci 2016; 17: 461.

Lanoue L, Green KK, Kwik-Uribe C, Keen CL. Dietary factors and the risk for acute infant leukemia: evaluating the effects of cocoa-derived flavanols on DNA topoisomerase activity. Exp Biol Med (Maywood). 2010; 235(1): 77-89. doi: 10.1258/ebm.2009.009184.

Li Z, Sun B, Clewell RA, Adeleye Y, Andersen ME, Zhang Q. Dose-response modeling of etoposide-induced DNA damage response. Toxicol Sci. 2014 Feb;137(2):371-84. doi: 10.1093/toxsci/kft259.

Lopez-Lazaro M, Willmore E, Austin CA. The dietary flavonoids myricetin and fisetin act as dual inhibitors of DNA topoisomerases I and II in cells. Mutat Res. 2010 Feb;696(1):41-7. doi: 10.1016/j.mrgentox.2009.12.010.

Lu C, Liu X, Liu C, Wang J, Li C, Liu Q, Li Y, Li S, Sun S, Yan J, Shao J. Chlorpyrifos Induces MLL Translocations Through Caspase 3-Dependent Genomic Instability and Topoisomerase II Inhibition in Human Fetal Liver Hematopoietic Stem Cells. Toxicol Sci. 2015; 147(2): 588-606. doi: 10.1093/toxsci/kfv153.

Nitiss JLSoans ERogojina ASeth AMishina M. 2012, Topoisomerase assays.Curr Protoc Pharmacol. 2012 Jun;Chapter 3:Unit 3.3.. doi: 10.1002/0471141755.ph0303s57.

Pendleton M, Lindsey RH Jr, Felix CA, Grimwade D, Osheroff N. Topoisomerase II and leukemia. Ann N Y Acad Sci. 2014 Mar;1310:98-110. doi: 10.1111/nyas.12358.

Rodríguez‐Cortez, V C, Menéndez, P, 2020. Genotoxicity of permethrin and clorpyriphos on human stem and progenitor cells at different ontogeny stages: implications in leukaemia development. EFSA supporting publication 2020: 17( 5): EN‐1866. 35 pp. doi: 10.2903/sp.efsa.2020.EN‐1866

Ross JA, Potter JD, Reaman GH, Pendergrass TW, Robison LL. Maternal exposure to potential inhibitors of DNA topoisomerase II and infant leukemia (United States): a report from the Children's Cancer Group. Cancer Causes Control. 1996 Nov;7(6):581-590.

Ross W, Rowe T, Glisson B, Yalowich J, Liu L. Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleavage. Cancer Res. 1984;44:5857–5860.

Sanjuan-Pla A, Bueno C, Prieto C, Acha P, Stam RW, Marschalek R, Menendez P. Revisiting the biology of infant t(4;11)/MLL-AF4+ B-cell acute lymphoblastic leukemia. Blood. 2015; 126(25): 2676-2685 DOI 10.1182/blood-2015-09-667378.

Schellenberg, M. J., Lieberman, J. A., Herrero‐Ruiz, A., Butler, L. R., Williams, J. G. and Muñoz‐Cabello, A. M. Williams, R. S. (2017). ZATT (ZNF451)‐mediated resolution of topoisomerase 2 DNA‐protein cross‐links. Science, 357(6358), 1412–1416. https://doi.org/10.1126/science.aam6468

Schroeter A, Groh IA, Favero GD, Pignitter M, Schueller K, Somoza V, Marko D. Inhibition of topoisomerase II by phase II metabolites of resveratrol in human colon cancer cells. Mol Nutr Food Res. 2015 Oct 12. doi: 10.1002/mnfr.201500352.

Smith NA, Byl JA, Mercer SL, Deweese JE, Osheroff N. Etoposide quinone is a covalent poison of human topoisomerase IIβ. Biochemistry. 2014; 53(19): 3229-3236. doi: 10.1021/bi500421q.

Spector LG, Xie Y, Robison LL, Heerema NA, Hilden JM, Lange B, Felix CA, Davies SM, Slavin J, Potter JD, Blair CK, Reaman GH, Ross JA. Maternal diet and infant leukemia: the DNA topoisomerase II inhibitor hypothesis: a report from the children's oncology group. Cancer Epidemiol Biomarkers Prev. 2005 Mar;14(3):651-655.

Strick R, Strissel PL, Borgers S, Smith SL, Rowley JD. Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia. Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4790-5.

Udroiu I., Sgura A. Genotoxicity sensitivity of the developing hematopoietic system. 2012. Mutation Research 2012; 767: 1-7.

Wilstermann A. M.; Bender R. P.; Godfrey M.; Choi S.; Anklin C.; Berkowitz D. B.; Osheroff N.; Graves D. E. (2007) Topoisomerase II-drug interaction domains: Identification of substituents on etoposide that interact with the enzyme. Biochemistry 46, 8217–8225.