Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been proposed that the topoisomerase II (TOP2)beta-DNA covalent complex arrests transcription and triggers 26S proteasome-mediated degradation of TOP2beta. It is unclear whether the initial trigger for proteasomal degradation is due to DNA damage or transcriptional arrest. In the current study we show that the TOP2 catalytic inhibitor 4,4-(2,3-butanediyl)-bis(2,6-piperazinedione) (ICRF-193), which traps TOP2 into a circular clamp rather than the TOP2-DNA covalent complex, can also arrest transcription. Arrest of transcription, which is TOP2beta-dependent, is accompanied by proteasomal degradation of TOP2beta. Different from TOP2 poisons and other DNA-damaging agents, ICRF-193 did not induce proteasomal degradation of the large subunit of RNA polymerase II. These results suggest that proteasomal degradation of TOP2beta induced by the TOP2-DNA covalent complex or the TOP2 circular clamp is due to transcriptional arrest but not DNA damage. By contrast, degradation of the large subunit of RNA polymerase II is due to a DNA-damage signal.
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PMID:The topoisomerase IIbeta circular clamp arrests transcription and signals a 26S proteasome pathway. 1262 7

A Chinese hamster V79 cell-based assay for detection of topoisomerase II (topo II) poisons and catalytic inhibitors has been applied to study two bis(dioxopiperazine)s (ICRF-187 and ICRF-154) and a structurally distinct but related compound, merbarone. All three compounds have been previously characterized as being catalytic inhibitors of DNA topo II based primarily on in vitro studies with purified enzymes. The present studies indicate, to the contrary, that all three compounds are very potent DNA clastogens in V79 cells, by virtue of their ability to produce micronuclei, the formation of which is strongly antagonized under conditions in which DNA topo II is rendered catalytically inactive. None of the compounds could be demonstrated to possess catalytic inhibitory activity in intact V79 cells under the conditions tested. These studies provide biological evidence that bis(dioxopiperazine)s are capable of functional topo II poisoning in intact mammalian cells.
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PMID:Evidence from studies with intact mammalian cells that merbarone and bis(dioxopiperazine)s are topoisomerase II poisons. 1264 37

The bisdioxopiperazines are catalytic inhibitors of eukaryotic type II DNA topoisomerases capable of trapping these enzymes as a salt-stable closed-clamp complex on circular DNA. The various bisdioxopiperazine analogs differ from each other because of structural differences in the linker connecting the two dioxopiperazine rings. Although the composition of this linker region has been found to be important for potency, the structural basis for this is largely unknown. To elucidate the role of the linker region in drug action, we have analyzed the effect of different linker substituents in otherwise identical analogs by studying their interaction with wild-type and mutant human topoisomerase II alpha. Two mutations, L169I and R162Q, displayed differential sensitivity toward closely related analogs, suggesting that the linker region in these compounds plays a highly specific role in protein drug interaction. The finding that the L169I mutation, which probably represents a subtle structural change, was sufficient to confer resistance further emphases the importance of this region of the protein for bisdioxopiperazine inhibition of topoisomerase II. Comparing the sensitivity profiles of different bisdioxopiperazines against wild-type and mutant proteins with that of mitindomide, we observed a spectrum of sensitivity closely resembling that of ICRF-154, a bisdioxopiperazine with no linker substituents. We discuss the implications of these observations for the understanding of the mechanism of bisdioxopiperazine action on topoisomerase II.
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PMID:Probing the role of linker substituents in bisdioxopiperazine analogs for activity against wild-type and mutant human topoisomerase II alpha. 1269 44

DNA topoisomerase I and II have been shown to be modified with a ubiquitin-like protein SUMO in response to their specific inhibitors called 'poisons'. These drugs also damage DNA by stabilizing the enzyme-DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIbeta, but not its isoform IIalpha, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF-193 which does not damage DNA. The beta isoform immunoprecipitated from ICRF-treated cells was modified by multiple modifiers, SUMO-2/3, SUMO-1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF-induced degradation of topoisomerase IIbeta did not occur, suggesting that the SUMO modification pathway is essential for the degradation.
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PMID:The SUMO pathway is required for selective degradation of DNA topoisomerase IIbeta induced by a catalytic inhibitor ICRF-193(1). 1283 72

A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4-/- and KU70-/- cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54-/- cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.
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PMID:Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage. 1284 86

Inactivation of topoisomerase (topo) IIalpha arrests murine embryonic development. In topo IIalpha-depleted embryos, nuclei were partitioned to daughter cells without complete separation and formed an interconnecting droplet-like structure. The present study examined the fates of topo IIalpha-depleted cells with the droplet-like nuclear structure. When the embryos with abnormal nuclei were further incubated, apoptosis was induced along with the formation of fragmented and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive nuclei. ICRF-193 treatment of embryos activated caspases. Apoptosis induced by ICRF-193 was suppressed by z-VAD-fmk, a caspase inhibitor, and pifithrin-alpha, a p53 inhibitor. Moreover, when mitosis was blocked by nocodazole, ICRF-193-induced nuclear abnormalities and apoptosis were abolished. These data suggest that cycling through the M-phase is essential for ICRF-193-induced apoptosis. Nuclear abnormalities similar to those of topo IIalpha-depleted embryos were induced in HeLa cells in which topo IIalpha was knocked down by transfection with short interfering RNA (siRNA) against topo IIalpha, followed by induction of apoptosis. Our results suggest that topo IIalpha-depleted cells with the droplet-like nuclear structure induce apoptosis, which is dependent on caspase and p53 activity during the G1 phase in mammalian cells.
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PMID:Induction of apoptosis by depletion of DNA topoisomerase IIalpha in mammalian cells. 1285 55

The bis-dioxopiperazine ICRF-193 has long time been considered as a pure topoisomerase II catalytic inhibitor able to exert its inhibitory effect on the enzyme without stabilization of the so-called cleavable complex formed by DNA covalently bound to topoisomerase II. In recent years, however, this concept has been challenged, as a number of reports have shown that ICRF-193 really "poisons" the enzyme, most likely through a different mechanism from that shown by the classical topoisomerase II poisons used in cancer chemotherapy. In the present investigation, we have carried out a study of the capacity of ICRF-193 to induce DNA strand breaks, as classical poisons do, in cultured V79 and irs-2 Chinese hamster lung fibroblasts using the comet assay and pulsed-field gel electrophoresis (PFGE). Our results clearly show that ICRF-193 readily induces breakage in DNA through a mechanism as yet poorly understood.
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PMID:DNA strand breaks induced by the anti-topoisomerase II bis-dioxopiperazine ICRF-193. 1456 29

After chromosome replication, the intertwined sister chromatids are disentangled by topoisomerases. The integrity of this process is monitored by the chromatid decatenation checkpoint. Here, we describe small molecule modulators of the human chromatid decatenation checkpoint identified using a cell-based, chemical genetic modifier screen. Similar to 1,2,7-trimethylyxanthine (caffeine), these small molecules suppress the G(2)-phase arrest caused by ICRF-193, a small molecule inhibitor of the enzymatic activity of topoisomerase II. Analysis of specific suppressors, here named suptopins for suppressor of Topoisomerase II inhibition, revealed distinct effects on cell cycle progression, microtubule stability, nucleocytoplasmic transport of cyclin B1, and no effect on the chromatin deacetylation checkpoint induced by trichostatin A. The suptopins provide new molecular tools for dissecting the role of topoisomerases in maintaining genomic stability and determining whether inhibiting the chromatid decatenation checkpoint sensitizes tumor cells to chemotherapeutics.
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PMID:Small molecule modulation of the human chromatid decatenation checkpoint. 1470 Jun 34

Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs. One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis. Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure. Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear. In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA. We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast. Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments. Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation.
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PMID:Dissecting the cell-killing mechanism of the topoisomerase II-targeting drug ICRF-193. 1512 16

In vertebrate cells, DNA double-strand breaks are efficiently repaired by homologous recombination or nonhomologous end-joining (NHEJ). The latter pathway relies on Ku (the Ku70/Ku86 heterodimer), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (Lig4). Here, we show that a human pre-B cell line nullizygous for Lig4 exhibits hypersensitivity to topoisomerase II (Top2) inhibitors, demonstrating a crucial role for the NHEJ pathway in repair of Top2-induced DNA damage in vertebrates. We also show that in the chicken DT40 cell line, all NHEJ mutants (i.e., Ku70-, Lig4-, and DNA-PKcs-null cells) are equally hypersensitive to the Top2 inhibitor ICRF-193, indicating that the drug-induced damage is repaired by NHEJ involving DNA-PKcs. Intriguingly, however, DNA-PKcs-null cells display considerably less severe phenotype than other NHEJ mutants in terms of hypersensitivity to VP-16, a Top2 poison that stabilizes cleavable complexes. The results indicate that two distinct NHEJ pathways, involving or not involving DNA-PKcs, are important for the repair of VP-16-induced DNA damage, providing additional evidence for the biological relevance of DNA-PKcs-independent NHEJ. Our results provide significant insights into the mechanisms of repair of Top2-mediated DNA damage, with implications for chemotherapy involving Top2 inhibitors.
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PMID:Genetic evidence for involvement of two distinct nonhomologous end-joining pathways in repair of topoisomerase II-mediated DNA damage. 1514 50


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