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)

This study shows that not only concanavalin A-stimulated proliferating lymphocytes but also unstimulated mouse splenic lymphocytes are sensitive to the topoisomerase II (topo II) inhibitor teniposide (VM-26). When unstimulated lymphocytes are pretreated with VM-26 for a 2-h period and are then incubated in drug-free medium, cell viability, as determined by trypan blue exclusion, decreases to 40% of the control by 6 h. The drug-treated cultures show two to three times the level of detergent soluble DNA than the control cultures and agarose gel electrophoresis of the soluble DNA shows the presence of oligonucleosomal-sized fragments, a feature considered to be a hallmark of apoptosis. Phase contrast microscopy, Hoechst staining for DNA, and immunofluorescence microscopy of various nuclear and cytoplasmic antigens (nucleolar fibrillarin, snRNP, ubiquitin, vimentin, tubulin) in the VM-26-treated cells characterize the morphological changes during apoptosis of these cells. The role of topo II as the mediator of the VM-26 effects is supported by pulsed field gel electrophoresis, which shows the typical topo II-induced cleavage of supercoiled DNA into loop-sized 300- and 50-kbp fragments. We conclude that the cancer chemotherapeutic agent VM-26 interacts with topo II and induces apoptosis in unstimulated lymphocytes.
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PMID:The topoisomerase II inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes. 131 87

The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A12S in response to dexamethasone. The level of topoisomerase IIalpha begins to decrease steeply within 36 h preceding the onset of DNA fragmentation, whereas its mRNA level is unchanged (Nakajima, T., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., and Oda, K. (1995) Oncogene 10, 651-662). Topoisomerase IIalpha prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dexamethasone for 42 h (the 42-h extract) than in the extract from untreated MA1 cells (the 0-h extract) in an ATP- and ubiquitin-dependent manner. The proteolytic activity for degradation of topoisomerase IIalpha was suppressed specifically by inhibitors for the proteasome and was much reduced in the 42-h extract prepared from MA1-derivative cell lines expressing E1B19k or Bcl-2. The proteolytic activity was lost after fractionation of the 42-h S10 extract into the S70 and P70 fractions by centrifugation at 70,000 x g for 6 h but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase IIalpha could be detected by incubating it in the S70 or S100 extract, which lacks most of the proteasome activity. The ubiquitination activity in S70 prepared from the 42-h extract was 4- to 5-fold higher than that prepared from the 0-h extract. These results suggest that a component(s) in the ubiquitin proteolysis pathway, responsible for ubiquitination and degradation of topoisomerase IIalpha, is activated or induced during the latent phase of E1A-induced apoptosis.
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PMID:Degradation of topoisomerase IIalpha during adenovirus E1A-induced apoptosis is mediated by the activation of the ubiquitin proteolysis system. 879 59

Activation of the transcription factor NF-kappaB by extracellular signals involves its release from the inhibitor protein IkappaBalpha in the cytoplasm and subsequent nuclear translocation. NF-kappaB can also be activated by the anticancer agent camptothecin (CPT), which inhibits DNA topoisomerase (Topo) I activity and causes DNA double-strand breaks during DNA replication to induce S phase-dependent cytotoxicity. Here we show that CPT activates NF-kappaB by a mechanism that is dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. NF-kappaB activation by CPT is dramatically diminished in cytoplasts and in CEM/C2 cells expressing a mutant Topo I protein that fails to bind CPT. This response is intensified in S phase cell populations and is prevented by the DNA polymerase inhibitor aphidicolin. In addition, CPT activation of NF-kappaB involves degradation of cytoplasmic IkappaBalpha by the ubiquitin-proteasome pathway in a manner that depends on the IkappaB kinase complex. Finally, inhibition of NF-kappaB activation augments CPT-induced apoptosis. These findings elucidate the progression of signaling events that initiates in the nucleus with CPT-Topo I interaction and continues in the cytoplasm resulting in degradation of IkappaBalpha and nuclear translocation of NF-kappaB to attenuate the apoptotic response.
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PMID:NF-kappaB activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events. 1073 98

Topoisomerase I-mediated DNA damage induced by camptothecin has been shown to induce rapid small ubiquitin-related modifier (SUMO)-1 conjugation to topoisomerase I. In the current study, we show that topoisomerase II-mediated DNA damage induced by teniposide (VM-26) results in the formation of high molecular weight conjugates of both topoisomerase IIalpha and IIbeta isozymes in HeLa cells. Immunological characterization of these conjugates suggests that both topoisomerase IIalpha and IIbeta isozymes are conjugated to SUMO-1. The involvement of SUMO-1/UBC9 in the modification of topoisomerase II isozymes is also supported by the demonstration of physical interaction between topoisomerase II and SUMO-1/UBC9. Surprisingly, ICRF-193, which does not induce topoisomerase II-mediated DNA damage but traps topoisomerase II into a circular clamp conformation, is also shown to induce similar SUMO-1 conjugation to topoisomerase II isozymes. In addition, we show that both oxidative and heat shock stresses, which can cause protein damage, rapidly increase nuclear SUMO-1 conjugates. These studies raise the question on whether SUMO-1 conjugation to topoisomerases is an indirect result of a DNA damage response or a direct result because of protein conformational changes.
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PMID:SUMO-1 conjugation to human DNA topoisomerase II isozymes. 1086 13

One hundred and fourteen kilobase pairs (kb) of contiguous genomic sequence have been determined immediately distal to the his5 genetic marker located about 0.9 Mb from the centromere on the long arm of Schizosaccharomyces pombe chromosome 2. The sequence is contained in overlapping cosmid clones c16H5, c12D12, c24C6 and c19G7, of which 20 kb are identical to previously reported sequence from clone c21H7. The remaining 93 781 bp of sequence contains 10 known genes (cdc14, cdm1, cps1, gpa1, msh2, pck2, rip1, rps30-2, sad1 and ubl1), 32 open reading frames (ORFs) capable of coding for proteins of at least 100 amino acid residues in length, one 5S rRNA gene, one tRNA(Pro) gene, one lone Tf1-type long terminal repeat (LTR) and one lone Tf2-type LTR. There is a density of one protein-coding gene per 2.2 kb and 22 of the 42 ORFs (52%) incorporate one or more introns. Twenty-one of the novel ORFs show sequence similarities which suggest functions of their products, including a cyclin C, a MADS box transcription factor, mad2-like protein, telomere binding protein, topoisomerase II-associated protein, ATP-dependent DEAH box RNA helicase, G10 protein, ubiquitin-activating e1-like enzyme, nucleoporin, prolyl-tRNA synthetase, peptidylprolyl isomerase, delta-1-pyrroline-5-carboxylate dehydrogenase, protein transport protein, coatomer epsilon, TCP-1 chaperonin, beta-subunit of 6-phosphofructokinase, aminodeoxychorismate lyase, a phosphate transport protein and a thioredoxin.
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PMID:Analysis of 114 kb of DNA sequence from fission yeast chromosome 2 immediately centromere-distal to his5. 1105 21

DNA topoisomerases are double-edged swords. They are essential for many vital functions of DNA during normal cell growth. However, they are also highly vulnerable under various physiological and nonphysiological stresses because of their delicate act on breaking and rejoining DNA. These stresses (e.g. exposure to topoisomerase poisons, acidic pH, and oxidative stresses) can convert DNA topoisomerases into DNA-breaking nucleases, resulting in cell death and/or genomic instability. The importance of topoisomerase-mediated DNA cleavage in tumor cell death and carcinogenesis has been recognized. This review focuses on recent findings concerning the molecular mechanisms of the stress responses to topoisomerase-mediated DNA damage. The involvement of ubiquitin/26S proteasome and SUMO/UBC9 in these processes, as well as the role of topoisomerase cleavable complexes in apoptotic cell death are discussed.
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PMID:Tumor cell death induced by topoisomerase-targeting drugs. 1126 50

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 topoisomerase (topo) I is an essential nuclear protein and a target for anticancer drug camptothecin derivatives. As a nuclear protein, topo I is concentrated in the nucleolus. However, this nucleolar distribution of topo I is dynamic. It has been shown recently that topo I rapidly moves out of the nucleolus (nucleolar delocalization) in response to topo I inhibitors. In the present study, we demonstrated that nucleolar delocalization of topo I is associated with its conjugation by SUMOs (small ubiquitin-like modifiers) in response to the topo I inhibitor topotecan. Time-course experiments revealed that SUMO-topo I conjugation occurred at as early as 5 min after drug treatment, which was earlier than its observed nucleolar delocalization. Furthermore, heat shock blocked sumoylation of topo I; it also blocked the nucleolar delocalization of topo I fusion proteins. UBC9 is an E2 (ubiquitin carrier protein)-conjugating enzyme essential for sumoylation. Although overexpression of wild-type UBC9 enhanced both sumoylation and nuclear delocalization of topo I, overexpression of a UBC9 dominant negative mutant attenuated topo I sumoylation and its nucleolar delocalization. Taken together, our results suggest that sumoylation of topo I might serve as an addressing tag for its nucleolar delocalization in response to topo I inhibitors.
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PMID:Nucleolar delocalization of human topoisomerase I in response to topotecan correlates with sumoylation of the protein. 1170 53

We report a novel nucleolar interaction between the AAA ATPase p97/VCP and the Werner protein (WRNp), a member of the RecQ helicase family. p97/VCP mediates several important cellular functions in eucaryotic cells, including membrane fusion of the endoplasmic reticulum and Golgi and ubiquitin-dependent protein degradation. Mutations in the WRN gene cause Werner syndrome, a genetic disorder characterized by premature onset of aging symptoms, a higher incidence of cancer, and a high susceptibility to DNA damage caused by topoisomerase inhibitors. We observed that both WRNp and valosin-containing protein (VCP) were present in the nucleoplasm and in nucleolar foci in mammalian cells and that WRNp and p97/VCP physically interacted in the nucleoli. Importantly, the nucleolar WRNp/VCP complex was dissociated by treatment with camptothecin, an inhibitor of topoisomerase I, whereas other WRNp-associated protein complexes, such as WRNp/Ku 80, were not dissociated by this drug. Because WRN syndrome cells are sensitive to topoisomerase inhibitors, these observations suggest that the VCP/WRNp interaction plays an important role in WRN biology. We propose a novel role for VCP in the DNA damage response pathway through modulation of WRNp availability.
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PMID:DNA damage modulates nucleolar interaction of the Werner protein with the AAA ATPase p97/VCP. 1293 74

We have previously reported that XK469 inhibited topoisomerase (topo) IIbeta, in Waldenstrom's macroglobulinemia cell line (WSU-WM) however the inhibition alone is not sufficient to induce apoptosis. In this study, the apoptotic potential of XK469 and its mechanism in WSU-WM cell line was investigated. Exposure of WSU-WM cells to XK469 caused a decrease in viable cell number in a dose-dependent manner. In addition, XK469 caused the activation of caspase 3 resulting in subsequent cleavage of PARP. These events were preceded by the release of cytochrome c from the mitochondria to the cytosol. Simultaneous exposure of cells to cyclosporin A prevented the release of cytochrome c to cytosol and reduced the loss of viability. XK469 caused the activation of p53 with up-regulation of p53-dependent proteins such as Bax, p21, Gadd 45 and cyclin B1 in association with G2M arrest. The addition of ubiquitin carboxyl terminal hydrolase (UCH-L1) inhibitor (NaBH4) inhibited up-regulation of p53 and p53 related molecules by XK469 and reduced the loss of viability. Pre-incubation with NOK-1, a monoclonal antibody that prevents Fas-Fas ligand interaction and is inhibitory to Fas signaling interfered with XK469 induced activation of caspase 8 and also reduced the loss of viability. Simultaneous exposure of all three inhibitors (cyclosporin A, NaBH4 and NOK-1) abrogated the toxicity of XK469 by 95%. These data define multiple sequences of biochemical events that mediate cell death induced by XK469. Our study suggests a complex mechanistic cascade of XK469-mediated apoptosis that involves Fas signaling pathway, ubiquitination, p53 activation and cytochrome c release.
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PMID:XK469, a topo IIbeta inhibitor, induces apoptosis in Waldenstrom's macroglobulinemia through multiple pathways. 1461 35


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