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)

Immature rat thymocytes readily undergo apoptosis following exposure to many different stimuli, including agents which cause DNA damage, such as the topoisomerase II inhibitor etoposide and irradiation. We have shown previously that cells isolated from the immature rat thymus are resistant to the induction of apoptosis by the DNA-damaging agent cis-diamminedichloroplatinum(II) (cisplatin) (D. L. Evans and C. Dive, Cancer Res., 53:2133-2139, 1993). More than 85% of these thymocytes are quiescent. Here, we demonstrate that following purification of the minority subpopulation of thymocytes that are proliferating, a 2-h exposure to 50 microM cisplatin resulted in rapid apoptosis with 66% apoptotic cells by 12 h. In contrast, purified, nonproliferating thymocytes treated with cisplatin exhibited control levels of apoptosis at 12 h. Both proliferating and nonproliferating thymocytes rapidly underwent apoptosis following continuous exposure to methylprednisolone (10 microM) and etoposide (10 microM). The discrepancy in the levels of apoptosis seen in proliferating and quiescent thymocytes in response to cisplatin could not be attributed to changes in total cellular levels of cisplatin or to the number of DNA-platinum adducts which were determined, respectively, by atomic absorption spectrometry and competitive enzyme-linked immunoadsorbent assay. These results imply that in contrast to engagement of thymocyte apoptosis by methylprednisolone and etoposide, where apoptosis was proliferation independent, cisplatin-induced apoptosis depends on the presence of cells in S and G2-M phases of the cell cycle. Moreover, comparison of etoposide and cisplatin responses in thymocytes suggests that DNA damage per se may not be sufficient to induce apoptosis and that the type of DNA damage is important in this regard.
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PMID:Differential sensitivity to the induction of apoptosis by cisplatin in proliferating and quiescent immature rat thymocytes is independent of the levels of drug accumulation and DNA adduct formation. 813 65

Several substituted analogs of 7-(cis-3,5-dimethylpiperazinyl)-6,8-difluoro-5-amino-1-cyclopropyl quinolone were prepared and tested in a DNA cleavage assay with calf thymus topoisomerase II. Positioning of the methyl groups on the C-7 piperazine ring influenced potency against the mammalian enzyme; the cis-3,5-dimethyl configuration did not stimulate cleavage at drug concentrations less than or equal to 2,000 microM, while the trans configuration was active at drug levels as low as 36 microM. Removal of the cis-methyl groups produced a compound that was only sixfold less potent than the antitumor agent etoposide in stimulating enzyme-mediated DNA cleavage. The cis- and trans-methyl substitutions on the piperazine that conferred potency against the mammalian type II enzyme had little effect on bacterial DNA gyrase cleavage activity, suggesting that an asymmetric barrier exists with the mammalian enzyme which influences productive quinolone interaction, favoring the less bulky trans-3,5-dimethylpiperazine substituent at C-7.
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PMID:Placement of alkyl substituents on the C-7 piperazine ring of fluoroquinolones: dramatic differential effects on mammalian topoisomerase II and DNA gyrase. 814 66

Cytotoxic sesterterpenes, manoalide 25-acetals (1a, 1b), seco-manoalide (2), (E)-neomanoalide (3), (Z)-neomanoalide (4), and heteronemin (6), were isolated from the marine sponge Hyrtios erecta (collected at Amami Island, Kagoshima Prefecture, Japan) by bioassay-guided separation and the absolute configurations of these manoalide family members have been determined. Manoalide 25-acetals (1a, 1b) were shown to exhibit in vivo antitumor activity and to inhibit the DNA-relaxing activity of mouse DNA topoisomerase I and the DNA-unknotting activity of calf thymus DNA topoisomerase II.
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PMID:Marine natural products. XXXII. Absolute configurations of C-4 of the manoalide family, biologically active sesterterpenes from the marine sponge Hyrtios erecta. 814 54

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

The antileukemic alkaloid, fagaronine, is a potent differentiation inducer of various hematopoietic cell lines. We show here that fagaronine is a DNA base-pair intercalator with a K(app) of 2.1 x 10(5) M-1 for calf thymus DNA. Fagaronine inhibits the catalytic activity of purified calf thymus topoisomerase I as shown by relaxation of supercoiled plasmid DNA followed by electrophoresis in neutral as well as in chloroquine-containing gels. The catalytic activity of topoisomerase I is inhibited at concentrations above 30 microM. Fagaronine also inhibits the catalytic activity of purified calf thymus topoisomerase II at concentrations above 25 microM as shown by decatenation of kinetoplast DNA. Fagaronine stabilizes the covalent DNA-enzyme reaction intermediate (the cleavable complex) between topoisomerase I and linear pBR322 DNA at concentrations up to 1 microM. Further increase of the fagaronine concentration leads to a progressive decrease in the cleavable complex formation, which is totally inhibited at 100 microM. In contrast, up to 1 microM fagaronine has no effect on cleavable complex formation between purified calf thymus topoisomerase II and linear pBR322 DNA, whereas cleavable complex formation is inhibited at higher concentrations. Exposure to fagaronine results in an increase in DNA-protein complex formation in intact P388 murine leukemia cells. P388CPT5 cells, which have an altered topoisomerase I activity, are 4-fold resistant to the growth inhibitory effects of fagaronine compared to the parental cell line. Similarly, DC-3F/9-OH-E Chinese hamster fibrosarcoma cells, which have an altered topoisomerase II activity, are about 5-fold resistant to the growth inhibitory effects of fagaronine. We conclude that fagaronine is an inhibitor of both DNA topoisomerase I and II and propose that this might play a role in the cytotoxic activity.
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PMID:The antileukemic alkaloid fagaronine is an inhibitor of DNA topoisomerases I and II. 824 Mar 89

We have produced two murine monoclonal antibodies (SWT3D1 and SWR1C2) to a recombinant polypeptide corresponding to the carboxyl-terminal one-third (amino acid 854-amino acid 1447) of human topoisomerase II alpha. Each antibody is able to recognize intact human topoisomerase II using immunoblotting and enzyme-linked immunosorbent assay (ELISA) techniques. Data is presented demonstrating that the antibodies bind specifically to topoisomerase II alpha but do not interact with topoisomerase II beta. The monoclonal antibodies do not recognize murine or calf thymus topoisomerase II indicating that each may bind exclusively to the human enzyme. The topoisomerase II binding sites for each monoclonal antibody have been compared in a competition ELISA. The SWT3D1 antibody had no significant effect on the binding efficiency of biotinylated SWR1C2 antibody. Although SWR1C2 was capable of inhibiting the binding of biotinylated SWT3D1, this only occurred at concentrations approximately 1000-fold higher than those required of SWT3D1 to block binding of itself. These results suggest that SWT3D1 and SWR1C2 do not recognize identical epitopes on topoisomerase II.
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PMID:Isolation and characterization of monoclonal antibodies to a recombinant human topoisomerase II polypeptide. 824 17

CP-115,953 [6,8-difluoro-7-(4'-hydroxyphenyl)-1-cyclopropyl-4- quinolone-3-carboxylic acid] is a novel quinolone that is highly active against topoisomerase II in vitro and in mammalian cells in culture (M. J. Robinson, B. A. Martin, T. D. Gootz, P. R. McGuirk, M. Moynihan, J. A. Sutcliffe, and N. Osheroff, J. Biol. Chem. 266:14585-14592, 1991). However, the features of the drug that contribute to its activity towards mammalian systems have not been characterized. Therefore, CP-115,953 and a series of related quinolones were examined for their activity against calf thymus topoisomerase II and cultured mammalian cells. CP-115,953 stimulated DNA cleavage mediated by the type II enzyme with a potency that was approximately 600-fold greater than that of the antimicrobial quinolone ciprofloxacin and approximately 50-fold greater than that of the antineoplastic drug etoposide. As determined by the ability to enhance enzyme-mediated DNA cleavage, quinolone activity towards calf thymus topoisomerase II was enhanced by the presence of a cyclopropyl group at the N-1 ring position and by the presence of a fluorine at C-8. Furthermore, the 4'-hydroxyphenyl substituent at the C-7 position was critical for the potency of CP-115,953 towards the mammalian type II enzyme. In this regard, the aromatic nature of the C-7 ring as well as the presence and the position of the 4'-hydroxyl group contributed greatly to drug activity. Finally, the cytotoxicity of quinolones in the CP-115,953 series towards mammalian cells paralleled the in vitro stimulation of DNA cleavage by topoisomerase II rather than the inhibition of enzyme-catalyzed DNA relaxation. This correlation strongly suggests that these quinolones promote cell death by converting topoisomerase II to a cellular poison.
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PMID:Drug features that contribute to the activity of quinolones against mammalian topoisomerase II and cultured cells: correlation between enhancement of enzyme-mediated DNA cleavage in vitro and cytotoxic potential. 825 42

Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.
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PMID:Intoplicine (RP 60475) and its derivatives, a new class of antitumor agents inhibiting both topoisomerase I and II activities. 826 12

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

Apoptosis, a major form of cell death in the immune system, is characterized by cell shrinkage, chromatin condensation, and cleavage into nucleosomal fragments. Apoptosis may be the mechanism for the elimination of autoreactive and unselected CD4+ CD8+ thymocytes in the thymus. A large number of diverse agents are capable of inducing apoptosis in immature thymocytes. Rat thymocytes were treated with etoposide, a DNA topoisomerase II reactive agent, or dexamethasone, a glucocorticoid, and separated on discontinuous Percoll gradients. We have identified and isolated a transitional preapoptotic population of thymocytes that exhibited early morphologic and biochemical changes associated with apoptosis. These preapoptotic cells were intermediate in size and density between normal and apoptotic thymocytes and exhibited a decreased surface expression of both CD4 and CD8 molecules compared to control thymocytes. On ultrastructural examination, they were shown to possess sharply defined clumps of condensed chromatin abutting onto the nuclear membrane. These morphologic changes, the first detectable signs of apoptosis, occurred prior to the internucleosomal cleavage of DNA, often regarded as the biochemical hallmark of apoptosis. Nucleosomal fragments of 180 to 200 base pairs or multiples thereof were, however, detected following subsequent dramatic changes in the nuclear structure of these preapoptotic cells that resulted in morphology typical of apoptosis. These results suggest that early critical events in apoptosis precede internucleosomal cleavage of DNA.
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PMID:Identification of a transitional preapoptotic population of thymocytes. 833


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