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)

The Fas/Fas ligand (FasL) pathway is widely involved in apoptotic cell death in lymphoid and nonlymphoid cells. It has recently been postulated that many chemotherapeutic agents also induce cell death by activating the Fas/FasL pathway. In the present study we compared apoptotic pathways induced by anti-Fas or chemotherapeutic agents in the Jurkat human T-cell leukemia line. Immunoblotting showed that treatment of wild-type Jurkat cells with anti-Fas or the topoisomerase II-directed agent etoposide resulted in proteolytic cleavage of precursors for the cysteine-dependent aspartate-directed proteases caspase-3 and caspase-7 and degradation of the caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B1. Likewise, affinity labeling with N-(N(alpha)-benzyloxycarbonylglutamyl-N(epsilon)-biotinyllysyl+ ++)aspartic acid [(2,6-dimethyl-benzoyl)oxy]methyl ketone [Z-EK(bio)D-amok] labeled the same five active caspase species after each treatment, suggesting that the same downstream apoptotic pathways have been activated by anti-Fas and etoposide. Treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to block etoposide-induced apoptosis, raising the possibility that etoposide does not initiate apoptosis through Fas/FasL interactions. To further explore the relationship between Fas- and chemotherapy-induced apoptosis, Fas-resistant Jurkat cells were treated with various chemotherapeutic agents. Multiple independently derived Fas-resistant Jurkat lines underwent apoptosis that was indistinguishable from that of the Fas-sensitive parental cells after treatment with etoposide, doxorubicin, topotecan, cisplatin, methotrexate, staurosporine, or gamma-irradiation. These results indicate that antineoplastic treatments induce apoptosis through a Fas-independent pathway even though Fas- and chemotherapy-induced pathways converge on common downstream apoptotic effector molecules.
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PMID:Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. 924 21

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.
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PMID:Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease. 942 5

Beta-Lapachone a novel topoisomerase inhibitor, has been found to induce apoptosis in various human cancer cells. In this study we report that a dramatic elevation of hydrogen peroxide (H2O2) in human leukemia HL-60 cells following 1 microM beta-lapachone treatment and that this increase was effectively inhibited by treatment with antioxidant N-acetyl-L-cysteine (NAC), ascorbic acid, alpha-tocopherol. NAC strongly prevented beta-lapachone-induced apoptotic characteristics such as DNA fragmentation and apoptotic morphology. However, treatment of HL-60 cells with another topoisomerase inhibitor camptothecin (CPT) did not induce H2O2 production as compared to untreated cells. NAC also failed to block CPT-induced apoptosis. Correlated with these findings, we found that cancer cell lines K562, MCF-7, and SW620, contained high level of intracellular glutathione (GSH), were not elevated in H2O2 and were resistant to apoptosis after treatment with beta-lapachone. In contrast, cancer cell lines such as, HL-60, U937, and Molt-4 which have lower level of GSH, were readily increased of H2O2 and were sensitive to this drug. Furthermore, ectopic overexpression of Bcl-2 in HL-60 cells also attenuated beta-lapachone-induced H2O2 and conferred resistance to beta-lapachone-induced cell death. Beta-Lapachone at the concentration as low as 0.25 microM effectively induced HL-60 cells to undergo monocytic differentiation, as evidenced by CD14 antigenicity and alpha-naphthyl acetate esterase activity. Again, the beta-lapachone-induced monocytic differentiation was suppressed by NAC. These results suggest that intracellular H2O2 generation plays a crucial role in beta-lapachone-induced cell death and differentiation.
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PMID:Involvement of hydrogen peroxide in topoisomerase inhibitor beta-lapachone-induced apoptosis and differentiation in human leukemia cells. 955 79

A type I topoisomerase has been purified to homogeneity from Mycobacterium smegmatis. It is the largest single subunit enzyme of this class having molecular mass of 110 kDa. The enzyme is Mg2+ dependent and can relax negatively supercoiled DNA, catenate, and knot single-stranded DNA, thus having typical properties of type I topoisomerases. Furthermore, the enzyme makes single-stranded nicks and the 5'-phosphoryl end of the nicked DNA gets covalently linked with a tyrosine residue of the enzyme. However, M. smegmatis enzyme shows some distinctive features from the prototype Escherichia coli topoisomerase I. The enzyme is relatively stable at higher temperatures and not inhibited by spermidine. It apparently does not contain any bound Zn2+ and on modification of cysteine residues retains the activity, suggesting the absence of the zinc-finger motif in DNA binding. Partially purified Mycobacterium tuberculosis topoisomerase I exhibits very similar properties with respect to size, stability, and reaction characteristics. Sequence comparison of topoisomerase I from E. coli and M. tuberculosis shows the absence of zinc-finger motifs in mycobacterial enzyme. Using a two-substrate assay system, we demonstrate that the enzyme acts processively at low ionic strength and switches over to distributive mode at high Mg2+ concentration. Significantly, the enzyme activity is stimulated by single strand DNA-binding protein. There is a potential to exploit the characteristics of the enzyme to develop it as a molecular target against mycobacterial infections.
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PMID:DNA topoisomerase I from Mycobacterium smegmatis. An enzyme with distinct features. 959 41

Caspases are aspartate-specific cysteine proteases that play a pivotal role in drug-induced cell death. We designed RT-PCR assays to analyse the expression of CASP-3, CASP-4, CASP-6 and the long and short isoforms of CASP-2 genes in human cells. These genes heterogeneously coexpress in leukemic cell lines and bone marrow samples from patients with de novo acute myelogenous leukemia at diagnosis. Treatment of U937 and HL60 leukemic cells and HT29 colon carcinoma cells with the topoisomerase II inhibitor etoposide upregulates CASP-2 and CASP-3 genes in these cells before inducing their apoptosis. This effect of etoposide is not observed in K562 cells and bcl-2-transfected U937 cells which are less sensitive to drug-induced apoptosis. Nuclear run-on experiments demonstrate that etoposide increases CASP gene transcription in U937 cells, an effect that is prevented by Bcl-2 overexpression. Upregulation of CASP genes is associated with an enhanced synthesis of related procaspases that precedes the appearance of apoptosis markers including caspase-3 activation, poly(ADP-ribose) polymerase cleavage and internucleosomal DNA fragmentation. These results suggest that the ability of tumor cells to upregulate CASP-2 and CASP-3 genes in response to cytotoxic drugs could be predictive of their sensitivity to drug-induced apoptosis.
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PMID:Upregulation of CASP genes in human tumor cells undergoing etoposide-induced apoptosis. 967 9

Chemotherapeutic agents that target topoisomerase I and II set into motion a series of biochemical changes that culminate in cell death, but only under some conditions. The realization that stabilization of covalent topoisomerase-DNA complexes is not sufficient to insure cell death has prompted investigators to examine various aspects of the drug-induced death process itself. Several discrete steps along this pathway have been identified, including (a) the processing of stabilized cleavage complexes into frank DNA strand breaks; (b) sensing of the DNA damage, leading to activation of stress-associated signaling pathways and cell cycle arrest; and (c) activation of a preexisting group of enzymes and enzyme precursors, typified by the cysteine-dependent aspartate-directed proteases (caspases), that catalyze the relatively orderly biochemical cascade of terminal events known as apoptosis. The present review discusses the evidence that these steps occur after treatment with etoposide or camptothecin, the two prototypic topoisomerase poisons that are commonly studied. As in any emerging area, a large number of questions remain to be answered about the process of cell death induced by topoisomerase-directed drugs.
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PMID:Cell death induced by topoisomerase-targeted drugs: more questions than answers. 974 75

Our laboratory has previously reported the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)2tetramer). This transition was induced by DNA positive torsional constraint, generated either by initial supercoiling of the loop or by its thermal fluctuations during topoisomerase relaxation. Taking into account the wrapping of the DNA around the histones into less than a turn, and its negative crossing at the entry-exit, the transition was proposed to involve a 360 degrees rotation of the loop around the particle dyad axis, and the formation of a positive crossing. The tetramer horseshoe-shaped conformation within the octamer further suggested that this process could be mediated by a reorientation of the two sector-like H3-H4 dimers about their H3/H3 interface, which would switch the overall handedness of the proteinaceous superhelix from left to right-handed. We now provide additional evidence for such a contribution of the protein by showing, through gel electrophoresis, topoisomerase relaxation and electron microscopy, that a sterical hindrance at the H3/H3 interface, introduced by covalent linking of bulky adducts through thiol oxidation of H3 cysteine 110, interferes with the transition. Such interference varies, depending on the particular SH-reagent used; but the most remarkable effect was obtained with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB), which displaces the preferred conformation of the tetrasomes from left-handed to semi-right-handed, and at the same time preserves a significant degree of chiral flexibility. DNA contribution was evidenced by a specific fractionation of circular tetrasomes in gel electrophoresis which, together with a different positioning of control and DTNB tetrasomes on linear DNA, pointed to an interdependence between tetrasome conformation and positions. Moreover, linear tetrasomes fluctuate between crossed and uncrossed conformations in a salt-dependent equilibrium which appears to vary with their positions on the DNA. These data suggest a modulatable role of the DNA around the dyad in the transition, depending primarily on its sequence-dependent deformability. This role is played at both levels of H3-H4 dimer reorientation and lateral opening, a mechanism by which the particle may relieve the clash between its entering and exiting DNAs. These properties make the tetrasome an attractive potential intermediate in nucleosome dynamics in vivo, in particular duringX transcriptional activation and elongation.
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PMID:Nucleosome dynamics. Protein and DNA contributions in the chiral transition of the tetrasome, the histone (H3-H4)2 tetramer-DNA particle. 1045 91

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.
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PMID:Anthracyclines trigger apoptosis of both G0-G1 and cycling peripheral blood lymphocytes and induce massive deletion of mature T and B cells. 1076 78

Type IA DNA topoisomerases possess several domains forming a toroidal molecule with a central hole large enough to accommodate single- or double-stranded DNA. The sign inversion model predicts several protein-DNA intermediates, including those in which DNA is trapped within the hole. Opposing cysteine residues were incorporated into two independent domains surrounding the putative DNA binding cavity of E. coli topoisomerase III, creating a molecule that can be covalently closed or opened by oxidizing or reducing the disulfide bond. The formation of the disulfide bond allowed the trapping of single- and double-stranded DNA within the cavity of the enzyme and the identification of other intermediates proposed by the sign inversion model.
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PMID:The mechanism of type IA topoisomerase-mediated DNA topological transformations. 1123 59

1DNA topoisomerase II (topo II) is a nuclear enzyme that modifies DNA topology and also serves as a target to mediate the cytotoxicity of several antineoplastic agents. Several reports have demonstrated that a reduction of topo II is associated with reduced sensitivity to these agents. Topo II exists as two isoforms in mammalian cells: topo IIalpha and topo IIbeta. In MCF-7 cells, the half-life (mean +/- SEM) values of topo IIalpha and topo IIbeta in situ were 6.6 +/- 0.3 and 17.6 +/- 2.3 hr, respectively, as determined by [(35)S]methionine/cysteine pulse-chase analysis. Degradation of topo IIalpha in situ was abrogated by the presence of proteasome inhibitors, and the relative activities were carbobenzoxy-leucyl-leucyl-leucinal (MG132) > carbobenzoxy-leucyl-leucyl-norvalinal (MG115) > ALLN congruent with lactacystin. ATP-dependent degradation of topo IIalpha, but not topo IIbeta, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furthermore, degradation of topo IIalpha was abrogated by the proteasome inhibitors MG132 and MG115, but not by lactacystin, in extracts of asynchronously dividing MCF-7 cells. Finally, degradation of topo IIalpha, but not topo IIbeta, was observed to occur in a cell cycle-dependent fashion, in extracts of synchronized HeLa cells, with maximal loss of the alpha isoform occurring 2 hr after release from mitotic arrest. This degradation of topo IIalpha appeared to be facilitated by an ATP-dependent activity. Furthermore, high molecular weight bands (>200 kDa), which may represent polyubiquitinated-topo IIalpha conjugates, were also detected in extracts of synchronized HeLa cells. This study provides evidence for a role of the ubiquitin-proteasome pathway in the cell cycle-dependent regulation of topo IIalpha expression.
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PMID:Role of proteasomal degradation in the cell cycle-dependent regulation of DNA topoisomerase IIalpha expression. 1127 64


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