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)

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.
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PMID:Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. 131 85

The two-year survival rate of patients with small cell lung cancer is less than 10%. The major reason for this poor outcome is the development of drug resistance. Panels of small cell lung cancer cell lines have been established, providing models for the study of drug resistance in this tumour. One such model is the doxorubicin-selected H69AR cell line. H69AR displays the typical multidrug resistance phenotype in that it is cross-resistant to anthracyclines, Vinca alkaloids (e.g., vinblastine) and epipodophyllotoxins (e.g., VP-16). However, H69AR cells do not overexpress P-glycoprotein, the membrane drug efflux pump frequently found on multidrug resistant cells. Some alterations in glutathione levels and associated enzyme activities were found but the data do not support the notion that enhanced drug detoxication is involved in H69AR cell resistance. Fewer drug-induced DNA strand breaks, reduced levels of topoisomerase II, and reduced formation of drug-stabilized DNA/topoisomerase II complexes were observed in H69AR cells. These data implicate topoisomerase II in the resistance phenotype of H69AR cells, but cannot explain H69AR cell resistance to the Vinca alkaloids, which do not have topoisomerase II as a target. Monoclonal antibodies against antigens overexpressed on H69AR cells have been derived and four have been characterized. Immunoscreening of an H69AR cDNA expression library has allowed the identification of one of these antigens as p36 (annexin II), a Ca2+/phospholipid binding protein. Chemosensitizers and novel xenobiotics have been examined for their ability to circumvent the drug resistance of H69AR cells. The limited success of these investigations suggests that innovative approaches may be required. In conclusion, the data obtained with H69AR and other models of small cell lung cancer indicate that multiple mechanisms contribute to drug resistance in this disease.
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PMID:The 1991 Merck Frosst Award. Multidrug resistance in small cell lung cancer. 131 57

Adriamycin, an anticancer agent acting on topoisomerase II, promotes the arrest of cell division and neurite extension in a "neurite-minus" murine neuroblastoma cell line, N1A-103. This morphological differentiation is accompanied by a blockade in the S phase of the cell cycle, modification of the amount of peripherin, and appearance of the beta 7-tubulin isoform. Yet, adriamycin-induced N1A-103 cells fail to express other neuronal markers, such as long-lasting Ca2+ channels, synaptophysin, and the shift in the proportion of the beta'1 tubulin isoform to the beta'2 isoform, whose appearance parallels the terminal differentiation of the wild type neuroblastoma cell line N1E-115. Hence, a comparison of the behavior of these two cell lines leads to the proposal that there are two programs of neuroblastoma differentiation: one where expression is triggered by the arrest of cell division and which is observed in adriamycin-induced N1A-103 variant cells, and the other, presumably occurring further downstream, which would involve further changes in morphogenesis and acquisition of new electrophysiological properties.
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PMID:Adriamycin promotes neurite outgrowth in the "neurite-minus" N1A-103 mouse neuroblastoma cell line. 133 Jun 60

Drugs that interfere with the action of P-glycoprotein (P-gp), the membrane efflux pump responsible for multidrug resistance (MDR), should be valuable in the treatment of patients with drug-resistant cancer. We have used one class of drug, the phenothiazines, to study the structural features required for optimum interference with the function of P-gp. The structure-activity relationships revealed three important components including the hydrophobicity of the tricyclic ring, the length of the alkyl bridge and the charge on the terminal amino group. Trans-flupenthixol is a lead compound that conforms to these structural requirements and demonstrates significant activity as a sensitizer of MDR cell lines to drugs affected by the MDR phenotype. Based on these data, we have proposed a model for the binding of modulators to P-gp and have speculated on the structure of the drug-binding domain. We have developed pre-clinical models of MDR that may help predict clinical activity of chemo-modulators. L1210/VMDRC.06 is a murine lymphocytic leukemia line transformed by a retroviral expression vector containing a full-length cDNA for the human mdr1 gene. K562/VBL1-3 are clones of human myeloid blast cells that were transformed with the same vector. Resistance in these lines is not complicated by changes in the cellular content of glutathione or alterations in topoisomerase II. The transformed L1210 line grows in mice as a slowly proliferating non-metastatic peritoneal implant. Both MDR lines are restored to sensitivity by cyclosporin A or trans-flupenthixol, and the K562 clones are induced to differentiate by hemin. These lines should provide simple, sensitive screens for new drugs for use against cancers expressing P-gp. We have proposed a model to explain how the pumping activity of P-gp is activated in response to toxic drugs. In this schema, basal activity of P-gp is modulated through phosphorylation/dephosphorylation reactions mediated by protein kinase C (PKC) and calcium sensitive phosphatases. In response to the activation of phospholipase C by toxic drugs and the local production of 1,2-diacylglycerol, PKC is translocated to the cell membrane where it phosphorylates P-gp. Following the extrusion of drug from the cell membrane, phospholipase C activity returns to baseline, diacylglycerol is metabolized, PKC returns to the cytosol and serine/threonine phosphatases dephosphorylate P-gp returning it to the basal state.
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PMID:Rational design and pre-clinical pharmacology of drugs for reversing multidrug resistance. 134 93

The effect of the topoisomerase II inhibitor Novobiocin on T-cell mediated cytotoxicity was tested under various assay conditions. When effector cells were class I major histocompatibility complex (MHC)-specific CD8+ cytotoxic T cells (Tc), Novobiocin caused a biphasic pattern of inhibition and the two components of the inhibition could be separated based on Ca2+ requirement. Unseparated populations of class II MHC specific Tc, containing CD4+ and CD8+ effectors gave the same pattern of inhibition. When CD8+ cells were depleted from the latter population of effectors, different patterns of inhibition from those obtained with CD8+ Tc were seen and furthermore the target affected the pattern of inhibition. Overall the results add further support to there being more than one pathway of CD8+ T-cell mediated cytotoxicity and further illustrate differences between CD4+ and CD8+ T-cell mediated cytotoxicity.
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PMID:Inhibition of T-cell mediated cytotoxicity by Novobiocin suggests multiple pathways for both CD4+ and CD8+ cytotoxic T cells. 135 96

The level of expression of cellular proto-oncogens c-myc and c-fos in rat liver has been studied as a function of protein synthesis rate (cycloheximide dose). Activation of proto-oncogens has been established to be initiated by 50% inhibition of nuclear protein synthesis. This promotes a certain level in chromatin structural rearrangements which is manifested, in particular, in decreasing activity of chromatin cleavage by Ca2+, Mg(2+)-DNAase and increasing degree of chromatin condensation. A role of topoisomerase II in chromatin structural rearrangements during proto-oncogen activation is postulated.
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PMID:[Structural changes of chromatin during proto-oncogene activation by cycloheximide: dose-effect]. 145 95

An anucleated cell system has been used for the first time to study mitochondrial topoisomerase activity. Mitochondrial extracts from human blood platelets contained type I topoisomerase. The type I classification was based on ATP-independent activity, inhibition by ATP or camptothecin, and the lack of inhibition by novobiocin. Platelet mitochondrial topoisomerase I relaxation activity was inhibited linearly by increasing concentrations of EGTA. Topoisomerase activity greater than 90% inhibited by 175 microM EGTA was partially restored to 16 and 50% of the initial level of activity by the subsequent addition of 50 and 100 microM Ca2+, respectively. Additionally, results from studies of partially purified platelet mitochondrial topoisomerase I were consistent with the crude extract data. This work supports the hypothesis that platelet mitochondria contain a type I topoisomerase that is biochemically distinct from that previously isolated and characterized from cell nuclei.
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PMID:Mitochondrial DNA topoisomerase I from human platelets. 164 36

Although the stabilization of topoisomerase II cleavable complexes by etoposide (VP-16) has been recognized to be important for cell killing, the lethal events following the formation of cleavable complexes remain to be elucidated. In an attempt to characterize the biochemical requirements for VP-16-induced cytotoxicity, we examined the effects of calcium depletion in Chinese hamster DC3F cells. Four-hour preincubation in calcium-free medium or in complete medium containing 5 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) protected against the cytotoxicity of VP-16. Under these same conditions, the VP-16-induced DNA single-strand break frequency in calcium-depleted cells remained similar to that of control cells. Cell-cycle analysis and thymidine pulse incorporation indicated that calcium depletion did not alter DNA synthesis and cell cycle distribution. Drug-induced cytotoxicity was restored progressively within 4-8 hr after calcium-depleted cells were refed with calcium-containing medium. Calcium depletion also protected against the cytotoxicity of camptothecin, hyperthermia and, to a lesser extent, nitrogen mustard and gamma radiation in DC3F cells. Similar results were obtained in human colon carcinoma HT-29 cells. Our results suggest that topoisomerase II-mediated DNA breaks are only potentially lethal and that calcium-dependent cellular processes are required for the cytotoxicity of topoisomerase inhibitors.
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PMID:Cell death induced by topoisomerase inhibitors. Role of calcium in mammalian cells. 164 24

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.
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PMID:Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity. 215 52


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