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 relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.
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PMID:Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis. 787 55

DNA topoisomerase I isolated from the lower eukaryote Neurospora crassa mitochondria was characterized. Molar mass of the enzyme in the native state is 120 kDa and 60-65 kDa when denatured. The pH optimum of the enzyme is 7.8 and the KCl optimum concentration is 40 mmol/L. This topoisomerase is independent of ATP and Mg2+. N-Ethylmaleimide, 4-chloromercuribenzoate, SDS, guanidinium chloride, polyethylene glycol, heparin and ethidium bromide inhibit its activity, while novobiocin, nalidixic acid, Triton X-100 and chloroquine do not. Polyamines and histone H1 stimulate the topoisomerase activity. We classify this DNA topoisomerase as type I and eukaryotic. Conversion of the topoisomerase to a nonspecific endonuclease at increased temperature is proposed.
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PMID:Characterization of mitochondrial DNA topoisomerase I from Neurospora crassa. 795 26

Topoisomerase II alpha (170 kDa) expressed in human HL-60 cells is heterogeneous in charge. By two-dimensional electrophoresis and chromatofocussing two major subforms with pI of 6.5 and 6.7 can be resolved. By preparative anion-exchange chromatography we separated the known topoisomerase II isoenzymes (170/180 kDa) and in addition a late-eluting 170 kDa form, which has not been described before. The catalytic optimum of this late-eluting form is shifted to pH 9.4. It is more than 100-fold resistant to orthovanadate, amsacrine or etoposide, and has an increased salt stability. SDS-treatment induces covalent attachment of this enzyme fraction to calf thymus DNA in the absence of drug. The latter observations indicate an increase in DNA-binding. In the tightly DNA-bound state the late-eluting enzyme is not targeted by cleavable complex forming drugs. Accordingly, cells may become drug-resistant by expressing this form predominantly.
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PMID:Drug-sensitivity and DNA-binding of a subform of topoisomerase II alpha in resistant human HL-60 cells. 799 49

We have purified the type II DNA topoisomerase from regenerating rat liver. The purified topoisomerase II migrated as two bands with molecular masses of 70 kDa and 55 kDa on SDS-PAGE. Immunoblotting analysis using antiserum against rat topoisomerase II gene product expressed in Escherichia coli suggested that the two bands on SDS-gel are proteolytic products of the intact 173 kDa form. However, these products retained the enzyme activities such as catenation and relaxation of supercoiled circular duplex monomer DNA and unknotting of knotted phage P4 DNA. These results suggest that DNA topoisomerase II consists of different functional domains and that the whole enzyme is not required for its activity. The activities of the purified enzyme were completely inhibited by 1 mM novobiocin, a bacterial gyrase inhibitor. However, no inhibitory effect was observed when another gyrase inhibitor, nalidixic acid was used.
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PMID:Active DNA topoisomerase II with minimum molecular mass from regenerating rat liver. 803 15

The topoisomerase II inhibitor, VP-16 (etoposide), is an important component in many chemotherapeutic regimens. To characterize resistance to this drug, the human melanoma cell line, FEM-X, was selected in multiple steps with VP-16. To prevent the development of typical multidrug resistance, an inhibitor of P-glycoprotein, the tiapamil analog, RO-11-2933, was added to the selections. The resultant clone FVP3 is 56-fold resistant to VP-16 and cross-resistant to doxorubicin (Adriamycin) (9-fold) and VM-26 (27-fold). These cells are also two- to four-fold resistant to m-AMSA, daunorubicin, and mitoxantrone. FVP3 is not resistant to the P-glycoprotein substrates vinblastine, does not express the MDR1 gene at detectable levels, and does not show reduced 3H-VP-16 accumulation. Unlike other cell lines that exhibit resistance to inhibitors of topoisomerase II, FVP3 has the same level of topoisomerase II expression and activity as FEM-X. Using live cells treated with VP-16, band depletion assays and KCI/SDS precipitation assays show that topoisomerase II from FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. This difference in sensitivity to VP-16 is also detected using lysates from disrupted cells, but not with isolated nuclei devoid of cytoplasmic and membrane components. In addition, the topoisomerase II present in nuclear extracts from FVP3 is not resistant to the effects of VP-16 as measured by: (1) inhibition of strand passing activity during decatenation of kinetoplast DNA, (2) drug-induced linearization of plasmid DNA, and (3) immunodepletion by VP-16. These results suggest that some component of the cytoplasm or cellular membranes, or a factor depleted from nuclei during their isolation, is responsible for the resistance to VP-16 in FVP3.
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PMID:Characterization of an unusual mutant of human melanoma cells resistant to anticancer drugs that inhibit topoisomerase II. 809 46

DNA topoisomerase V is a novel prokaryotic enzyme related to eukaryotic topoisomerase I. The enzyme is a type I DNA topoisomerase and is recognized by polyclonal antibody against human topoisomerase I. We describe its purification from the hyperthermophilic methanogen Methanopyrus kandleri. The enzyme has high activity in crude extracts and is present in at least 1,500 copies/cell. Topoisomerase V migrates as a 110-kDa polypeptide in SDS-polyacrylamide gel electrophoresis and as a 142-kDa globular protein in gel filtration. It is active up to at least 100 degrees C on both positively and negatively supercoiled DNA and is not inhibited by single-stranded DNA. The enzyme works from 1 to 650 mM NaCl and up to 3.1 M potassium glutamate. It acts processively at low ionic strength and distributively at high NaCl or KCl concentration. Magnesium is not required and does not stimulate the enzymatic activity. Under DNA denaturing conditions, topoisomerase V catalyzes an unlinking reaction which results in substantial reduction in the linking number of closed circular DNA. The driving force for this process is DNA melting. Camptothecin is not nearly as good an inhibitor for topoisomerase V as it is for eukaryotic topoisomerase I. The unique occurrence of two major type I topoisomerases (reverse gyrase and topoisomerase V) in M. kandleri may shed new light on the evolution of this family of enzymes and supports the concept of a distant but significant relationship between some hyperthermophilic organisms and eukaryotes.
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PMID:Purification and characterization of DNA topoisomerase V. An enzyme from the hyperthermophilic prokaryote Methanopyrus kandleri that resembles eukaryotic topoisomerase I. 810 68

Reverse gyrase, an ATP-dependent topoisomerase that positively supercoils DNA, has been purified to near-homogeneity from the hyperthermophile Methanopyrus kandleri. It migrates on SDS-polyacrylamide gel electrophoresis as two principal bands with apparent molecular masses of 150 and 50 kDa. Both proteins remain associated throughout all chromatographic steps. Transfer of a radioactive phosphate from DNA to the 50-kDa protein and gel retardation experiments indicate that this protein forms the covalent complex with DNA. A blot overlay assay identifies the 150-kDa protein as the potential ATPase. This is the first evidence that a reverse gyrase can be a topoisomerase consisting of two protomers. In analogy with the DNA gyrase A subunit (DNA breakage and reunion activity) and the B subunit (ATPase), the 50- and 150-kDa components of Mka reverse gyrase have been designated the A and B subunits, respectively. Methanopyrus reverse gyrase changes DNA linking number in steps of one and its A subunit covalently binds to the 5'-DNA phosphoryl group. It nicks DNA at sites that predominantly have a cytosine at the -4-position. The same rule was derived previously for monomeric reverse gyrase from sulfur-metabolizing hyperthermophiles and for topoisomerase I from mesophilic bacteria. Based on these results, Mka reverse gyrase is classified as belonging to group A of type I topoisomerases. The structural diversity of type I group A topoisomerases parallels the diversity of type II enzymes and suggests the evolution of an essential function by gene fusion.
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PMID:A reverse gyrase with an unusual structure. A type I DNA topoisomerase from the hyperthermophile Methanopyrus kandleri is a two-subunit protein. 815 33

Pulse treatments of U-937 human promonocytic leukemia cells with the DNA topoisomerase-II inhibitors 4'-(9-acridynilamino)methanesulfon-m-anisidide (amsacrine, mAMSA) or etoposide (VP-16) caused growth inhibition, G2-arrest, increase in cell size and expression of differentiation markers. All these effects were greatly reduced by the presence of 5-10 mM caffeine. In addition, caffeine partially prevented the increase in the number of topoisomerase-DNA cleavable complexes caused by the topoisomerase inhibitors, as determined by SDS/CIK precipitation assays; it caused chromatin condensation, as determined by flow cytometry assays, and interacted with mAMSA in solution, as suggested by spectrophotometric assays. Pulse treatment with caffeine greatly inhibited RNA synthesis but not DNA or protein synthesis, as indicated by labelled precursor incorporation assays. The transcription inhibitor 5,6-dichloro-I-beta-D-ribofuranosylbenzymidazole reduced the mAMSA- and VP-16-produced growth inhibition in a similar manner. It is concluded that RNA synthesis inhibition is one of the possible mechanisms by which caffeine protects cells from the action of topoisomerase-II inhibitors.
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PMID:Caffeine attenuates the action of amsacrine and etoposide in U-937 cells by mechanisms which involve inhibition of RNA synthesis. 820 82

beta-Lapachone is a plant product that has been found to have many pharmacological effects. To date, very little is known about its biochemical target. In this study, we found that beta-lapachone inhibits the catalytic activity of topoisomerase I from calf thymus and human cells. But, unlike camptothecin, beta-lapachone does not stabilize the cleavable complex, indicating a different mechanism of action. beta-Lapachone inhibits topoisomerase I-mediated DNA cleavage induced by camptothecin. Incubation of topoisomerase I with beta-lapachone before adding DNA substrate dramatically increases this inhibition. Incubation of topoisomerase I with DNA prior to beta-lapachone makes the enzyme refractory, and treatment of DNA with beta-lapachone before topoisomerase has no effect. These results suggest a direct interaction of beta-lapachone with topoisomerase I rather than DNA substrate. beta-Lapachone does not inhibit binding of enzyme to DNA substrate. In cells, beta-lapachone itself does not induce a SDS-K(+)-precipitable complex, but it inhibits complex formation with camptothecin. We propose that the direct interaction of beta-lapachone with topoisomerase I does not affect the assembly of the enzyme-DNA complex but does inhibit the formation of cleavable complex.
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PMID:beta-Lapachone, a novel DNA topoisomerase I inhibitor with a mode of action different from camptothecin. 822 54

Anion-exchange chromatography of partially purified human HL-60 topoisomerase II resolves the known alpha (170 kDa) and beta (180 kDa) isoenzymes at 150 mM NaCl and 230 mM NaCl, respectively. An additional topoisomerase II fraction was eluted by > 300 mM NaCl. It could be identified by Western blotting as a late-eluting variant of topoisomerase II alpha, which is functionally altered as compared to the early-eluting form, having the following properties: a shift in the catalytic optimum to pH 9; increased stability in DNA complex formation; approximately 100-fold resistance to orthovanadate; approximately 1000-fold resistance to the cytostatic substances N-[4-(9-acridinylamino)-3-methoxyphenyl]-methanesulphonamide (amsacrine) and the podophyllotoxin etoposide (VP 16). 80% of the late-eluting topoisomerase II alpha could be captured by SDS on calf thymus DNA without further enhancement by drugs. In contrast, the early-eluting topoisomerase II alpha exhibits 10% complex formation with SDS alone, and an increase to 90% complex formation in the presence of drugs. A HL-60 subline (HL-60/R), approximately 1000-fold resistant to etoposide and amsacrine, has equivalent proportions of topoisomerase II alpha and topoisomerase II beta and similar levels of both isoenzymes, as compared to the drug-sensitive HL-60/WT cells. However, determination of the cellular levels of the early-eluting and late-eluting forms of topoisomerase II alpha revealed that the HL-60/R cell line contains approximately 80% of the late-eluting topoisomerase II alpha, whereas the sensitive HL-60/WT cell line contains only 15-20% of this form. The nuclear distribution of the two forms also differs. Sensitive HL-60/WT cells show a diffuse nuclear distribution but in resistant cells the distribution is localized in the nucleoli. Apparently two functionally distinct subforms of topoisomerase II alpha coexist in drug-sensitive and drug-resistant HL-60 cells and changes in their relative levels affect the cellular sensitivity to topoisomerase-II-targeting drugs.
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PMID:A drug-resistant variant of topoisomerase II alpha in human HL-60 cells exhibits alterations in catalytic pH optimum, DNA binding and sub-nuclear distribution. 826 48


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