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Key Event Title
Binding to (interferes with) topoisomerase II enzyme
|Level of Biological Organization|
Key Event Components
|DNA topoisomerase II activity||abnormal|
Key Event Overview
AOPs Including This Key Event
Key Event Description
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
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
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.
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