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)

Using semi-quantitative PCR-based approach, we have shown that the breakpoint cluster region of the AML1 gene was associated with the nuclear matrix. We have demonstrated that inhibition of topoisomerase II by etoposide stimulates the appearance of histone gammaH2AX foci, an indicator for the presence of DNA double-strand breaks. Furthermore, the major part of these foci was associated with the nuclear matrix. We also visualized nuclear matrix--associated multiprotein complexes involved in topoisomerase II--induced DNA double-strand break repair. Colocalization studies have demonstrated that these complexes included the principal components of the non-homologous end joining repair system (Ku80, DNA-PKcs and DNA ligase IV). Thus, it is reasonable to suggest that the non-homologous DNA end joining is a possible mechanism of topoisomerase II--induced chromosomal rearrangements.
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PMID:[Illegitimate recombination as a possible mechanism of DNA-topoisomerase II induced chromosomal rearrangements]. 1708 89

Multimodality treatments (i.e. surgery, chemotherapy, and radiotherapy) are recommended for anaplastic thyroid carcinoma (ATC), an extremely lethal human cancer, but to date there is little evidence that such approaches improve survival rates. It is thus necessary to seek new therapeutic tools. Histone deacetylase (HDAC) inhibitors are a promising class of anti-neoplastic agents that induce differentiation and apoptosis. Moreover, they may enhance the cytotoxicity of drugs targeting DNA through acetylation of histones. Using two ATC cell lines (CAL-62 and ARO), we show here that valproic acid (VPA), a clinically available HDAC inhibitor, enhances the activity of doxorubicin, whose anti-tumor properties involve binding to DNA and inhibiting topoisomerase II. A meager 0.7 mM VPA, which corresponds to serum concentrations in patients treated for epilepsy, is able to increase the cytotoxicity of doxorubicin about threefold in CAL-62 cells and twofold in ARO cells. The sensitizing effect, which is through histone acetylation, involves increased apoptosis, which is also shown by the increased caspase 3 activation and the enhancement of doxorubicin-induced G2 cell cycle arrest. These results might offer a rationale for clinical studies of a new combined therapy in an effort to improve the outcome of patients with anaplastic thyroid cancer.
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PMID:Valproic acid, a histone deacetylase inhibitor, enhances sensitivity to doxorubicin in anaplastic thyroid cancer cells. 1708 16

Potentiation is the transition from higher-order, transcriptionally silent chromatin to a less condensed state requisite to accommodating the molecular elements required for transcription. To examine the underlying mechanism of potentiation an approximately 13.7-kb mouse protamine domain of increased nuclease sensitivity flanked by 5' and 3' nuclear matrix attachment regions was defined. The potentiated DNase I-sensitive region is formed at the pachytene spermatocyte stage with the recruitment to the nuclear matrix of a large approximately 9.6-kb region just upstream of the domain. Attachment is then specified in the transcribing round spermatid, recapitulating the organization of the human cluster. In comparison to other modifiers that have no effect, i.e., histone methylation, HP1, and SATB1, topoisomerase engages nuclear matrix binding as minor marks of histone acetylation appear. Reorganization is marked by specific sites of topoisomerase II activity that are initially detected in leptotene-zygotene spermatocytes just preceding the formation of the DNase I-sensitive domain. This has provided a likely model of the events initiating potentiation, i.e., the opening of a chromatin domain.
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PMID:Decondensing the protamine domain for transcription. 1748 71

Chemotherapy has been a major approach to treat cancer. Both constituents of chromatin, chromosomal DNA and the associated chromosomal histone proteins are the molecular targets of the anticancer drugs. Small DNA binding ligands, which inhibit enzymatic processes with DNA substrate, are well known in cancer chemotherapy. These drugs inhibit the polymerase and topoisomerase activity. With the advent in the knowledge of chromatin chemistry and biology, attempts have shifted from studies of the structural basis of the association of these drugs or small ligands (with the potential of drugs) with DNA to their association with chromatin and nucleosome. These drugs often inhibit the expression of specific genes leading to a series of biochemical events. An overview will be given about the latest understanding of the molecular basis of their action. We shall restrict to those drugs, synthetic or natural, whose prime cellular targets are so far known to be chromosomal DNA.
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PMID:Chromatin as a target for the DNA-binding anticancer drugs. 1748 28

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that has recently entered clinical trials as an antitumor compound, based on impressive activities in preclinical models. The present investigations were directed at determining the mechanism of action of this agent. NK314 induced significant G(2) cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G(2) checkpoint pathway was activated in response to 100 nmol/L NK314 in ML-1 human acute myeloid leukemia cells. This was associated with the phosphorylation of the histone variant H2AX, an action that was predominant in the G(2) population, suggesting that double-strand DNA breaks caused cells to activate the checkpoint pathway. Double-strand DNA breaks were visualized as chromosomal aberrations when the G(2) checkpoint was abrogated by 7-hydroxystaurosporine. In vitro assays showed that NK314 inhibited the ability of topoisomerase IIalpha to relax supercoiled DNA and trapped topoisomerase IIalpha in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIalpha, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. These findings support the conclusion that the major mechanism of NK314 is to inhibit topoisomerase IIalpha, an action that leads to the generation of double-strand DNA breaks, which activate the G(2) DNA damage checkpoint pathway.
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PMID:Inhibition of topoisomerase IIalpha and G2 cell cycle arrest by NK314, a novel benzo[c]phenanthridine currently in clinical trials. 1751 99

Drugs developed for the treatment of conditions other than neoplasia can also show promise as potential antitumor agents. The fluoroquinolone antibiotic ciprofloxacin (CPFX) is known to modulate cycle cell progression and apoptosis in cancer cells, and is thought to induce DNA double-strand breaks (DSBs) via topoisomerase II (topo II) inhibition and stabilized cleavage complex (SCC) formation. DSBs trigger Ser-139 phosphorylation of histone H2AX (gammaH2AX) by PI-3-like kinases including ATM; gammaH2AX can serve as a marker of DNA damage when measured in situ using immunocytochemistry and flow cytometry. The aim of the present study was to investigate the relationship between CPFX-mediated DNA damage and induction of apoptosis in human lymphoblastoid cells and phytohaemagglutinin (PHA)-stimulated lymphocytes (Lymphs). Treatment of TK6 cells (wild-type p53) with 100 microg/ml CPFX for 2-10 h produced no increase in gammaH2AX; to the contrary, its level in S phase cells was reduced at 10 h compared to controls. Nevertheless, stabilization of topo IIalpha, ATM Ser-1981 phosphorylation and G(2) arrest was observed in TK6 cells exposed to CPFX for > or = 4 h. However, following 24 h treatment, gammaH2AX was dramatically increased in a sub-population of cells indicating the onset of apoptosis (confirmed by presence of activated caspase 3). CPFX had a similar lack of effect on induction of gammaH2AX at early time points in WTK1 and NH32 cells (devoid of functional p53) and proliferating Lymphs, however, induction of apoptosis was less pronounced than in TK6 cells. Formation of SCC and activation of ATM (but lack of gammaH2AX induction) indicates topo II-mediated chromatin or DNA changes in the absence of DSBs; ATM activation apparently triggers the G(2)M checkpoint leading to G(2) arrest. The subsequent induction of apoptosis appears to be facilitated by functional p53. CPFX may therefore have a potential use as a chemotherapeutic agent in the treatment of lymphoblast-derived cancer.
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PMID:Ciprofloxacin-induced G2 arrest and apoptosis in TK6 lymphoblastoid cells is not dependent on DNA double-strand break formation. 1805 76

The provenance and biochemical roles of eukaryotic MORC proteins have remained poorly understood since the discovery of their prototype MORC1, which is required for meiotic nuclear division in animals. The MORC family contains a combination of a gyrase, histidine kinase, and MutL (GHKL) and S5 domains that together constitute a catalytically active ATPase module. We identify the prokaryotic MORCs and establish that the MORC family belongs to a larger radiation of several families of GHKL proteins (paraMORCs) in prokaryotes. Using contextual information from conserved gene neighborhoods we show that these proteins primarily function in restriction-modification systems, in conjunction with diverse superfamily II DNA helicases and endonucleases. The common ancestor of these GHKL proteins, MutL and topoisomerase ATPase modules appears to have catalyzed structural reorganization of protein complexes and concomitant DNA-superstructure manipulations along with fused or standalone nuclease domains. Furthermore, contextual associations of the prokaryotic MORCs and their relatives suggest that their eukaryotic counterparts are likely to carry out chromatin remodeling by DNA superstructure manipulation in response to epigenetic signals such as histone and DNA methylation.
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PMID:MutL homologs in restriction-modification systems and the origin of eukaryotic MORC ATPases. 1834 80

Double-strand breaks (DSBs) are highly deleterious DNA lesions as they lead to chromosome aberrations and/or apoptosis. The formation of nuclear DSBs triggers phosphorylation of histone H2AX on Ser-139 (defined as gammaH2AX), which participates in the repair of such DNA damage. Our aim was to compare the induction of gammaH2AX in relation to DSBs induced by topoisomerase II (TOPO II) poisons, etoposide (ETOP) and mitoxantrone (MXT), in V79 cells. DSBs were measured by the neutral comet assay, while gammaH2AX was quantified using immunocytochemistry and flow cytometry. Stabilized cleavage complexes (SCCs), lesions thought to be responsible for TOPO II poison-induced genotoxicity, were measured using a complex of enzyme-DNA assay. In the case of ETOP, a no observed adverse effect level (NOAEL) and lowest observed effect level (LOEL) for genotoxicity was determined; gammaH2AX levels paralleled DSBs at all concentrations but significant DNA damage was not detected below 0.5 microg/ml. Furthermore, DNA damage was dependent on the formation of SCCs. In contrast, at low MXT concentrations (0.0001-0.001 microg/ml), induction of gammaH2AX was not accompanied by increases in DSBs. Rather, DSBs were only significantly increased when SCCs were detected. These findings suggest MXT-induced genotoxicity occurred via at least two mechanisms, possibly related to DNA intercalation and/or redox cycling as well as TOPO II inhibition. Our findings also indicate that gammaH2AX can be induced by DNA lesions other than DSBs. In conclusion, gammaH2AX, when measured using immunocytochemical and flow cytometric methods, is a sensitive indicator of DNA damage and may be a useful tool in genetic toxicology screens. ETOP data are consistent with the threshold concept for TOPO II poison-induced genotoxicity and this should be considered in the safety assessment of chemicals displaying an affinity for TOPO II and genotoxic/clastogenic effects.
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PMID:Assessment of DNA double-strand breaks and gammaH2AX induced by the topoisomerase II poisons etoposide and mitoxantrone. 1842 98

This unit describes immunocytochemical detection of phosphorylated histone H2AX for revealing the presence of DNA double-strand breaks. Double-strand breaks indicate DNA damage induced by ionizing radiation or by treatment with antitumor drugs such as DNA topoisomerase inhibitors. However, double-strand breaks can also be intrinsic, occurring in healthy, nontreated cells for a variety of reasons, and are generated in the course of DNA fragmentation in apoptotic cells. The unit presents strategies to distinguish radiation- or drug-induced breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes the immunocytochemical detection of histone H2AX phosphorylated on Ser-139 combined with measurement of DNA content to identify cells that have DNA double-strand breaks and to concurrently assess their cell cycle phase. The detection is based on indirect immunofluorescence using a FITC-labeled secondary antibody, and DNA is counterstained with propidium iodide (PI). Cellular RNA, which may be stained by PI, is removed with RNase A.
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PMID:Detection of histone H2AX phosphorylation on Ser-139 as an indicator of DNA damage (DNA double-strand breaks). 1877 Aug 4

Genistein, a widely consumed bioflavonoid with chemopreventative properties in adults, and etoposide, a commonly prescribed anticancer drug, are well-characterized topoisomerase II poisons. Although both compounds display similar potencies against human topoisomerase IIalpha and IIbeta in vitro and induce comparable levels of DNA cleavage complexes in cultured human cells, their cytotoxic and genotoxic effects differ significantly. As determined by assays that monitored cell viability or the phosphorylation of histone H2AX, etoposide was much more toxic in CEM cells than genistein. Further studies that characterized the simultaneous treatment of cells with genistein and etoposide indicate that the differential actions of the two compounds are not related to the effects of genistein on cellular processes outside of its activity against topoisomerase II. Rather, they appear to result from a longer persistence of cleavage complexes induced by etoposide as compared to genistein. Parallel in vitro studies with purified type II enzymes led to similar conclusions regarding cleavage complex persistence. Isoform-specific differences were observed in vitro and in cells treated with etoposide. To this point, the t 1/2 of etoposide-induced DNA cleavage complexes formed with topoisomerase IIalpha in CEM cells was approximately 5 times longer than those formed with topoisomerase IIbeta. The cytotoxicity of etoposide following four treatment-recovery cycles was similar to that induced by continuous exposure to the drug over an equivalent time period. Taken together, these findings suggest that it may be possible to preferentially target topoisomerase IIalpha with etoposide by employing a schedule that utilizes pulsed drug treatment-recovery cycles.
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PMID:The efficacy of topoisomerase II-targeted anticancer agents reflects the persistence of drug-induced cleavage complexes in cells. 1892 22


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