API

Event: 1252

Key Event Title

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Binding to (interferes with) topoisomerase II enzyme

Short name

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Binding, topoisomerase II

Biological Context

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Level of Biological Organization
Molecular

Cell term

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Cell term
eukaryotic cell


Organ term

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Key Event Components

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Process Object Action
DNA topoisomerase II activity abnormal

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
topoisomerase II binding, infant leukaemia MolecularInitiatingEvent

Stressors

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Taxonomic Applicability

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Term Scientific Term Evidence Link
mammals mammals High NCBI

Life Stages

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Life stage Evidence
Embryo High

Sex Applicability

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Term Evidence
Mixed High

Key Event Description

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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

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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. 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 comple. Protocol and procedure details for mewasuring topoisomerase inhibition are fully reported in Nitiss et al. 2012.


Domain of Applicability

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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.


Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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Etoposide is one of the most well studied topoisomerase II targeted agents. It stabilizes covalent topoisomerase II-cleaved DNA complexes (ie cleavage complexes) by interacting at the enzyme-DNA interface in a noncovalent manner (Smith et al. 2014).

Etoposide ( and itsderivatives) stimulate DNA cleavage mediated by yeast topoisomerase II.  As a result of etoposide action, high levels of topoisomerase II-associated DNA breaks accumulate in treated cells (Hande et al. 1998; Ross et al 1984; Wistelrman et al.2007).

Etoposide quinone induces DNA cleavage via an enzyme-mediated mechanism. Control reactions were conducted in the absence of enzyme or drug (DNA Control), in the presence of 30 μM etoposide quinone without enzyme (+EQ −hTIIβ), or in the presence of topoisomerase IIβ without drug (−EQ +hTIIβ). The quinone induced ∼4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was ∼2 times greater against topoisomerase IIβ than it was against topoisomerase IIα, and the drug reacted ∼2–4 times faster with the β isoform. Etoposide quinone induced a higher ratio of double- to single-stranded breaks than etoposide, and its activity was less dependent on ATP (Smith et al. 2014).



Etoposide

Etoposide is one of the most well studied topoisomerase II-targeted agents in clinical use. The drug stabilizes covalent topoisomerase II-cleaved DNA complexes (i.e., cleavage complexes) by interacting at the enzyme–DNA interface in a noncovalent manner. Once the double helix is cut, the drug slips (i.e., intercalates) between the 3′-hydroxyl and the enzyme-linked 5′-phosphate at the cleaved scissile bond and acts as a physical block to topoisomerase II-mediated DNA ligation. Etoposide and other drugs that utilize this mechanism are termed “interfacial topoisomerase II poisons”. The catechol displayed properties that were similar to those of the parent drug and appeared to be an interfacial poison. The properties of the quinone metabolite differed from those of etoposide, and the quinone appeared to function by a different mechanism. Previous studies with quinones and other protein-reactive agents have found that some of these compounds increase levels of topoisomerase II-mediated DNA cleavage by covalently adducting to the enzyme at residues that are distal to the active site.Thus, these agents are termed “covalent topoisomerase II poisons”. It is believed that covalent poisons enhance DNA cleavage, at least in part, by closing the N-terminal gate of the protein. Several lines of evidence suggest that etoposide quinone poisons topoisomerase II by this latter, covalent mechanism (Smith NA, 2014).

 


etoposide quinone

Etoposide metabolites, ie etoposide quinone, is also a potent topoisomerase IIß poisons. The quinone is able to induce about 4 times more enzyme-mediated DNA clevage than does the parent drug. Furthermore, the potency of etoposide quinone was about 2 times greater against topoisomerase IIß than it is agains topoisomerase IIÞ, and it reacts about 2 to 4 time faster with the ß isoform. The quinone metabolite induces a higher ratio of double - to single strand breaks than the parent chemical, and its activity is less dependent on ATP. Whereas etoposide acts as an interfacial topoisomerase II poison, etoposide quinone displayed all of the hallmarks of a covalent poison: the activity of the metabolite was abolished by reducing agents, and the compound inactivated topoisomerase IIβ when it was incubated with the enzyme prior to the addition of DNA​ (Smith et al. 2014)


References

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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.

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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.

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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.

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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.

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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.