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 relationship between DNA topoisomerase II activity and drug resistance was studied in cloned cell lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia; drug resistant P388/ADR/3 (clone 3) and P388/ADR/7 (clone 7) cells are 5- and 10-fold more resistant to ADR than the sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986). Topoisomerase II catalytic activity in crude nuclear extracts was reduced in drug-resistant cells as determined qualitatively by decatenation of kDNA. Using the centrifugal method fo quantitative analysis, topoisomerase II catalytic activity (mean +/- SE) was 81 +/- 10 units/mg total nuclear protein in sensitive cells, 29 +/- 2 units/mg total nuclear protein in resistant clone 3 cells, and 16 +/- 2 units/mg total nuclear protein in resistant clone 7 cells; these differences were highly significant (P less than 0.005). Similarly, quantitative analysis of DNA cleavage activity using 3' 32P-end-labeled pBR322 restriction fragments showed that drug-stimulated topoisomerase II cleavage activity in nuclear extracts from sensitive cells was approximately 1.7- and 2.9-fold greater than that from resistant clone 3 and 7 cells, respectively. Western blot analysis of nuclear extracts from the three cell lines using antibody against the C-terminal half of recombinant-prepared human topoisomerase II polypeptide revealed reduced immunoreactivity of topoisomerase II protein in the drug-resistant cells. These data suggest that reduced topoisomerase II activity in resistant cells, which may represent quantitative reduction of the enzyme, may be another property contributing to multifactorial drug resistance in these cells.
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PMID:Direct correlation between DNA topoisomerase II activity and cytotoxicity in adriamycin-sensitive and -resistant P388 leukemia cell lines. 253 93

A complementary DNA fragment of the human DNA topoisomerase II gene was cloned into a T7 expression vector and overproduced in Escherichia coli. Rabbit polyclonal antibodies were raised against the recombinant topoisomerase II polypeptide which corresponds to the C-terminal one-third of human topoisomerase II polypeptide. Using the antiserum, DNA topoisomerase II levels were measured by immunoblotting human lymphocytes following phytohemagglutinin (PHA) stimulation. Our results showed that the intracellular topoisomerase II but not the topoisomerase I level increased in parallel with the entry of cells into proliferation. At least a 100-fold increase in topoisomerase II was observed at 50 h after PHA stimulation. As topoisomerase II levels increased upon PHA stimulation, DNA damage induced by teniposide (VM26) increased in parallel, as measured by both DNA synthesis inhibition and chromosomal aberrations. However, the damage induced by camptothecin also increased upon PHA stimulation, while the level of topoisomerase I remained relatively constant. Our results suggest that, in addition to cellular contents of topoisomerases, the state of cell proliferation is another important determinant of drug action.
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PMID:Studies of topoisomerase-specific antitumor drugs in human lymphocytes using rabbit antisera against recombinant human topoisomerase II polypeptide. 253 95

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.
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PMID:Human autoantibody to topoisomerase II. 253 10

Among its many properties, amiloride is a DNA intercalator and topoisomerase II inhibitor. Previous work has indicated that the most stable conformation for amiloride is a planar, hydrogen-bonded, tricyclic structure. To determine whether the ability of amiloride to intercalate into DNA and to inhibit DNA topoisomerase II was dependent on the ability to assume a cyclized conformation, we studied the structure-activity relationship for 12 amiloride analogs. These analogs contained structural modifications which could be expected to allow or impede formation of a cyclized conformation. Empirical assays consisting of biophysical, biochemical, and cell biological approaches, as well as computational molecular modeling approaches, were used to determine conformational properties for these molecules, and to determine whether they intercalated into DNA and inhibited topoisomerase II. Specifically, we measured the ability of these compounds to 1) alter the thermal denaturation profile of DNA, 2) modify the hydrodynamic behavior of DNA, 3) inhibit the catalytic activity of purified DNA topoisomerase II in vitro, 4) promote the topoisomerase II-dependent cleavage of DNA, and 5) inhibit functions associated with DNA topoisomerase II in intact cells. Results indicated that only those analogs capable of cyclization could intercalate into DNA and inhibit topoisomerase II. Thus, the ability of amiloride and the 12 analogs studied to intercalate into DNA and to inhibit topoisomerase II appears dependent on the ability to exist in a planar, hydrogen-bonded, tricyclic conformation.
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PMID:DNA intercalation and inhibition of topoisomerase II. Structure-activity relationships for a series of amiloride analogs. 253 4

As a means of gaining additional information on the topoisomerase-mediated cytotoxicity induced by a variety of antibacterial and antitumor compounds we have examined the interaction of the quinolone anti-bacterial agent, norfloxacin, with the bacterial topoisomerase, DNA gyrase. Membrane filtration and spin-column techniques were used to study the binding of [3H]norfloxacin to purified plasmid DNA, DNA gyrase, and complexes formed by adding gyrase to different forms of plasmid DNA. Consistent with previous results (Shen, L. L., and Pernet, A. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 301-311) little [3H]norfloxacin binds to reconstituted gyrase, but significant levels of drug bind nonspecifically to relaxed DNA. However, when DNA and gyrase are incubated together additional norfloxacin binding sites are detectable. These complex-dependent sites are distinguishable from those sites involved in nonspecific DNA binding in that the complex-dependent sites are saturable and they retain bound norfloxacin after centrifuging the complex through a spin column. In addition, extent of binding is influenced by the topological state of DNA used to form the complex. The complex-dependent norfloxacin binding sites are likely involved in the inhibition of the enzyme since saturation of these sites occurs in the same norfloxacin concentration range as the inhibition of DNA supercoiling activity. Moreover, there is a close correlation of norfloxacin-induced DNA breakage with levels of norfloxacin bound to complexes of gyrase and relaxed DNA. These findings provide the first direct correlation of quinolone binding with inhibition of enzyme activity and induction of DNA breakage, and they suggest that the inhibition of DNA gyrase by norfloxacin occurs as a result of binding to a site which appears after the formation of a gyrase-DNA complex.
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PMID:Mechanism of quinolone inhibition of DNA gyrase. Appearance of unique norfloxacin binding sites in enzyme-DNA complexes. 253 29

Abnormal expression of the nuclear-associated enzyme DNA topoisomerase II (topoisomerase II) has been implicated in the in vitro phenotype of radiation hypersensitive ataxia-telangiectasia (A-T) cells and in modifying sensitivity of eukaryotic cells to topoisomerase II-inhibitor drugs [e.g., the DNA intercalator amsacrine (mAMSA)]. To study such relationships, various SV40- and Epstein-Barr Virus-transformed human cell lines derived from normal, A-T, or UV-sensitive xeroderma pigmentosum donors have been assayed for their sensitivity to mAMSA together with direct and indirect measurements of topoisomerase II expression. We report on the identification of an SV40-transformed A-T fibroblast cell line with abnormally high levels of topoisomerase II in nuclear protein extracts as determined by immunoblotting, measurement of kinetoplast DNA decatenation activity, and mAMSA-dependent DNA-protein cross-linking activity in a filter binding assay. Using a flow cytometric assay for the analysis of reactivity of nuclei with a polyclonal antitopoisomerase II antibody, overproduction was found to occur in all phases of the cell cycle. High levels of topoisomerase II were associated with hypersensitivity (5-10-fold) to mAMSA-induced cell cycle delay, cell kill, and DNA strand breakage (assayed under protein-denaturing conditions). Xeroderma pigmentosum (group A) cells demonstrated normal responses to mAMSA. The results provide evidence that cellular potential for the generation of topoisomerase II-dependent DNA damage is a major factor in governing the sensitivity to mAMSA. Furthermore, underexpression of topoisomerase II does not appear to be a primary factor in describing the in vitro A-T phenotype. The findings also relate to how changes in chromatin structure and function may either reflect or dictate the expression of topoisomerase II in human cells.
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PMID:Cellular consequences of overproduction of DNA topoisomerase II in an ataxia-telangiectasia cell line. 253 42

Evidence from several in vitro systems indicates that cellular responses to DNA topoisomerase II-reactive compounds (i.e., the epipodophyllotoxins and intercalating agents) may be affected by the relative rate of proliferation. Using a human head and neck squamous carcinoma cell line 183A, we have investigated the effect of beta-all-trans-retinoic acid (RA), a substance with known antiproliferative effects, on the DNA cleavage and cytotoxic activities of etoposide and 4'-(acridinylamino)methanesulfon-m-anisidide which interact with topoisomerase II. The effect of RA treatment on the activity of X-radiation and bleomycin, both of which produce free radical mediated effects, was also examined. RA treatment (10 to 20 microM for 72 h) does not significantly influence DNA cleavage induced by X-radiation or bleomycin but decreases DNA cleavage and cytotoxicity mediated by etoposide and 4'-(acridinylamino)methanesulfon-m-anisidide. Further, this effect can be demonstrated at a dose of RA that is minimally growth inhibitory. The inhibitory effect of RA appears to be localized to the nucleus given that similar effects on drug-mediated DNA cleavage can be demonstrated in nuclei isolated from RA-treated cells. However, both drug-stimulated DNA cleavage activity and topoisomerase II catalytic activity are approximately equal in crude nuclear extracts of untreated and RA-treated cells. These data suggest that the resistance to topoisomerase II-reactive drugs induced by RA treatment of 183A cells is not mediated through a direct effect on the enzyme, but, instead, is related to other changes in the nuclear milieu occurring in the initial stages of quiescence such as altered chromatin conformation.
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PMID:Effect of retinoic acid on DNA cleavage and cytotoxicity of topoisomerase II-reactive drugs in a human head and neck squamous carcinoma cell line. 253 45

We have determined the nucleotide sequence of the Drosophila DNA topoisomerase II gene. Data from primer extension and S1 nuclease protection experiments were combined with comparisons of genomic and cDNA sequences to determine the structure of the mature messenger RNA. This message has a large open reading frame of 4341 nucleotides. The length of the predicted protein is 1447 amino acids with a molecular weight of 164,424. Topoisomerase II can be divided into three domains: (1) an N-terminal region with homology to the B (ATPase) subunit of the bacterial type II topoisomerase, DNA gyrase; (2) a central region with homology to the A (breaking and rejoining) subunit of DNA gyrase; (3) a C-terminal region characterized by alternating stretches of positively and negatively charged amino acids. DNA topoisomerase II from the fruit fly shares significant sequence homology with those from divergent sources, including bacteria, bacteriophage T4 and yeasts. The location and distribution of homologous stretches in these sequences are analyzed.
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PMID:Structure of the Drosophila DNA topoisomerase II gene. Nucleotide sequence and homology among topoisomerases II. 253 21

The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.
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PMID:DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage. 253 17

In recent years, evidence has accumulated that suggests that mammalian topoisomerase may play a role in the formation of spontaneous or chemically induced sister chromatid exchange (SCE). In microbial systems, nalidixic acid is known to disrupt the function of a topoisomerase-like enzyme, DNA gyrase. To explore the possible relationship to topoisomerase function and SCE formation in mammalian cells, an analog of nalidixic acid with potent topoisomerase II inhibitory activity was selected for examination in a variety of genetic toxicology assays. This analog, CP-67,015, proved to be a positive direct-acting mutagen in the L5178Y/TK+/-, CHO/HGPRT, and V79/HGPRT systems. However, no gene mutational activity was observed using the Ames test in direct plate, mouse and rat metabolic activation, and mouse urine tests. In vitro cytogenetic studies showed strong clastogenic activity in human lymphocytes and in CHO cells. Compound-induced chromosome damage was also observed in vivo in mouse bone marrow cells. Surprisingly, SCE studies in vitro in human lymphocytes or CHO cells showed only slight increases, even at levels producing severe chromosome breakage. Mouse bone marrow showed no significant elevation of SCE following parenteral treatment with CP-67,015. These results, taken together, demonstrate that CP-67,015 is a direct-acting mutagen in mammalian cells with both gene and chromosomal level effects. The relative ineffectiveness in producing SCEs suggests that CP-67,015 may interfere with a DNA replicative/repair process, perhaps by alteration of one or more DNA polymerase activities. This suggestion is based in part on the known effect of the analog nalidixic acid on DNA gyrase in microbial cells and on topoisomerase in mammalian cells. The profile of genetic activity of CP-67,015, coupled with its inhibitory effect on topoisomerase function, gives rise to a model for SCE formation that is based on anomalies of topoisomerase activity during DNA synthesis.
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PMID:Genetic profile of a nalidixic acid analog: a model for the mechanism of sister chromatid exchange induction. 253 98


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