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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The breakage/reunion reaction of DNA topoisomerase II (TOP2) can be interrupted by DNA intercalators (e.g., doxorubicin), enzyme binders (e.g., etoposide), or DNA lesions (e.g., abasic sites) to produce TOP2-mediated DNA damage. Here, we demonstrate that thiol alkylation of TOP2 can also produce TOP2-mediated DNA damage. This conclusion is supported by the following observations using purified TOP2: (1) Thiol-reactive quinones were shown to induce TOP2-mediated DNA cleavage. (2) Thiol-reactive compounds such as N-ethylmaleimide (NEM), disulfiram, and organic disulfides [e.g., 2,2'-dithiobis(5-nitropyridine)] were also shown to induce TOP2-mediated DNA cleavage with similar reaction characteristics as thiol-reactive quinones. (3) TOP2-mediated DNA cleavage induced by thiol-reactive quinones was completely abolished using mutant yeast TOP2 with all cysteine residues replaced with alanine (cysteineless TOP2). These results suggest the possibility that cellular DNA damage could occur indirectly through thiolation of a nuclear protein, TOP2. The implications of this reaction in carcinogenesis and apoptotic cell death are discussed.
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PMID:Stimulation of topoisomerase II-mediated DNA damage via a mechanism involving protein thiolation. 1125 51

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

DNA damage in neurons is implicated in the pathogenesis of several neurodegenerative disorders and may also contribute to the often severe neurological complications in cancer patients treated with chemotherapeutic agents. DNA damage can trigger apoptosis, a form of controlled cell death that involves activation of cysteine proteases called caspases. The excitatory neurotransmitter glutamate plays central roles in the activation of neurons and in processes such as learning and memory, but overactivation of ionotropic glutamate receptors can induce either apoptosis or necrosis. Glutamate receptors of the AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) type mediate such physiological and pathological processes in most neurons. We now report that DNA damage can alter glutamate receptor channel activity by a mechanism involving activation of caspases. Whole-cell patch clamp analyses revealed a marked decrease in AMPA-induced currents after exposure of neurons to camptothecin, a topoisomerase inhibitor that induces DNA damage; N-methyl-d-aspartate (NMDA)-induced currents were unaffected by camptothecin. The decrease in AMPA-induced current was accompanied by a decreased calcium response to AMPA. Pharmacological inhibition of caspases abolished the effects of camptothecin on AMPA-induced current and calcium responses, and promoted excitotoxic necrosis. Combined treatment with glutamate receptor antagonists and a caspase inhibitor prevented camptothecin-induced neuronal death. Caspase-mediated suppression of AMPA currents may allow neurons with damaged DNA to withdraw their participation in excitatory circuits and undergo apoptosis, thereby avoiding widespread necrosis. These findings have important implications for treatment of patients with cancer and neurodegenerative disorders.
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PMID:Caspase-mediated suppression of glutamate (AMPA) receptor channel activity in hippocampal neurons in response to DNA damage promotes apoptosis and prevents necrosis: implications for neurological side effects of cancer therapy and neurodegenerative disorders. 1130 Jul 17

Procaspase-2 is one of the cysteine aspartate proteases involved in apoptotic cell death. Alternative splicing of CASP-2 messenger RNA generates a long isoform, procaspase-2L, whose overexpression induces cell death and a truncated isoform, procaspase-2S, whose function remains poorly defined. The present study explored the consequences of procaspase-2S overexpression in U937 human leukemic cells exposed to the topoisomerase II inhibitor etoposide as an apoptotic stimulus. Overexpression of procaspase-2S in U937 cells partially prevented nuclear changes associated with etoposide-induced cell death, as determined by Hoechst 33342 staining of nuclear chromatin and electron microscopy studies. Procaspase-2S also prevented the maturation of apoptotic bodies, delayed phosphatidylserine externalization on the plasma membrane and prevented the cleavage and activation of procaspase-2L. These effects were not observed when the cysteine 289 in the consensus QACRG motif was mutated into a serine. Wild-type procaspase-2S overexpression did not influence the cleavage of procaspase-3, procaspase-7 and poly(ADP-ribose)polymerase nor the fragmentation of nuclear DNA into nucleosome-sized fragments. Altogether, these results indicate that the short isoform of procaspase-2 negatively interferes with selective features of apoptosis, an activity that is suppressed by mutation of the cysteine 289.
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PMID:Modulation of apoptosis by procaspase-2 short isoform: selective inhibition of chromatin condensation, apoptotic body formation and phosphatidylserine externalization. 1131 53

Treatment with the DNA topoisomerase inhibitors etoposide, doxorubicin, and camptothecin, and with the alkylating agents cisplatin and melphalan, caused peroxide accumulation and apoptosis in U-937 human promonocytic cells. Preincubation with the reduced glutathione (GSH) synthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO) always potentiated peroxide accumulation. However, although GSH depletion potentiated the toxicity of cisplatin and melphalan, occasionally switching the mode of death from apoptosis to necrosis, it did not affect the toxicity of the other antitumor drugs. Hypoxia or preincubation with antioxidant agents attenuated death induction, apoptotic and necrotic, by alkylating drugs. The generation of necrosis by cisplatin could not be mimicked by addition of exogenous H(2)O(2) instead of BSO and was not adequately explained by caspase inactivation nor by a selective fall in ATP content. Treatment with cisplatin and melphalan caused a late decrease in mitochondrial transmembrane potential (DeltaPsim), which was much greater during necrosis than during apoptosis. The administration of the antioxidant agents N-acetyl-l-cysteine and butylated hydroxyanisole after pulse treatment with cisplatin or melphalan did not affect apoptosis but attenuated necrosis. Under these conditions, both antioxidants attenuated the necrosis-associated DeltaPsim decrease. These results indicate that oxidation-mediated alterations in mitochondrial function regulate the selection between apoptosis and necrosis in alkylating drug-treated human promonocytic cells.
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PMID:Effect of glutathione depletion on antitumor drug toxicity (apoptosis and necrosis) in U-937 human promonocytic cells. The role of intracellular oxidation. 1160 74

Maleimide, N-ethyl-maleimide (NEM), and N-methyl-maleimide (NMM) were identified as potent catalytic inhibitors of purified human topoisomerase IIalpha, whereas the ring-saturated analog succinimide was completely inactive. Catalytic inhibition was not abrogated by topoisomerase II mutations that totally abolish the effect of bisdioxopiperazine compounds on catalytic inhibition, suggesting a different mode of action by these maleimides. Furthermore, in DNA cleavage assay maleimide and NEM could antagonize etoposide-induced DNA double-strand breaks. Consistently, maleimide could antagonize the effect of topoisomerase II poisons in three different in vivo assays: 1) In an alkaline elution assay maleimide protected against etoposide-induced DNA damage. 2) In a band depletion assay maleimide reduced etoposide-induced trapping of topoisomerase IIalpha and beta on DNA. 3) In a clonogenic assay maleimide antagonized the cytotoxicity of etoposide and daunorubicin on four different cell lines of human and murine origin. at-MDR cell lines with reduced nuclear topoisomerase IIalpha content are fully sensitive to maleimide, indicating that it is not a topoisomerase II poison in vivo. Our finding that topoisomerase II is sensitive to maleimide, NMM, and NEM but insensitive to succinimide demonstrates a strict requirement for the unsaturated ring bond for activity. We suggest that the observed antagonism in vitro and in vivo is caused by covalent modification of topoisomerase II cysteine residues reducing the amount of catalytically active enzyme sensitive to the action of topoisomerase II poisons.
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PMID:Maleimide is a potent inhibitor of topoisomerase II in vitro and in vivo: a new mode of catalytic inhibition. 1196 Nov 42

The electrochemical reduction of beta-lapachone and its 3-sulphonic salt was studied by cyclic, square wave and differential pulse voltammetry in aqueous media using a glassy carbon electrode. These compounds have a wide range of biological activities, including antibacterial, cytotoxic, antifungal, trypanocidal and anticancer action. The reduction of beta-lapachone in the presence of L-cysteine and 2-mercaptoethanol was studied and the results, together with others already published, suggest that the anticancer mechanism of beta-lapachones can be explained via interaction with topoisomerase.
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PMID:Reduction of lapachones and their reaction with L-cysteine and mercaptoethanol on glassy carbon electrodes. 1200 43

In several 'in vitro' models of apoptosis, lysosomal proteolysis has been shown to play an active role in mediating the death signal by cytokines or antiblastic drugs. Depending on the experimental cell model and the cytotoxic stimulus applied, an increased expression and the cytosolic translocation of either cathepsin D or B have been reported in apoptotic cells. We have analysed the involvement of these lysosomal proteases in a canonical apoptotic cell model, namely L929 fibroblasts, in which apoptosis was induced by cytotoxic agents acting through different mechanisms: (i) the cytokine TNFalpha, which triggers the cell suicide via interaction with its membrane receptor, and (ii) the topoisomerase II-inhibitor etoposide (VP16), which directly causes DNA damage. In both cases the activity of cathepsins B and D increased in apoptosing cultures. CA074-Me, a specific inhibitor of cathepsin B, and Leupeptin, a broad inhibitor of serine and cysteine proteases (among which is cathepsin B), did not exert any protection from TNFalpha. In contrast, pre-loading the cells with pepstatin A, a specific inhibitor of cathepsin D, protected L929 cells from TNFalpha cytotoxicity by more than 50%. However, no protection was observed if pepstatin A was added concomitantly with the cytokine. Inhibition of either cathepsin B or D did not impede apoptosis induced by etoposide. Lysosomal integrity was preserved and cathepsin D remained still confined in vesicular structures in apoptotic cells treated with either TNFalpha or etoposide. It follows that proteolysis by cathepsin D is likely to represent an early event in the death pathway triggered by TNFalpha and occurs within the endosomal-lysosomal compartment.
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PMID:Endosomal-lysosomal proteolysis mediates death signalling by TNFalpha, not by etoposide, in L929 fibrosarcoma cells: evidence for an active role of cathepsin D. 1243 11


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