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)

Oxygen is thought to be involved both directly and indirectly in the mechanisms of action of several anti-cancer agents. We studied the effects of various oxygen concentrations on the cytotoxicities of the following drugs: bleomycin (BLM), etoposide (VP-16), doxorubicin (DOX), and mitomycin C (MMC). Human sarcoma cells, MESSA, were exposed to drug for 1 h at one of several oxygen concentrations: less than 1%, 2.5%, 5%, 21%, and 95%. Cytotoxicity was assessed by cellular incorporation of 3H-thymidine into DNA 5 days after drug exposure. Control experiments varying oxygen concentration without drugs demonstrated toxicity only at the highest concentration (95%). Three different responses of drug sensitivity to varying oxygen tensions were observed. BLM, which has been shown to utilize oxygen as a substrate in generating free radicals and producing DNA scission, demonstrated a progressive increase in cytotoxicity over the entire range of increasing oxygen concentrations. This is consistent with the model of a BLM-cation-oxygen complex and catalytic reduction of oxygen. VP-16, which also produces DNA strand breakage but by interaction with topoisomerase II, exhibited a threshold response. VP-16 toxicity was ameliorated by anoxic conditions (less than 1% O2), but not by oxygen concentrations of 2.5%-95%. The reason for this protective effect of anoxia with VP-16 is not clear. In contrast, acute anoxia had no effect on the cytotoxicities of DOX and MMC. We conclude that acute hypoxia protects cells from both BLM and VP-16 but that the nature of that protection is different. VP-16 toxicity is blunted only by severe anoxia, whereas BLM exhibits a dose response effect over the entire range of oxygen concentrations.
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PMID:Differential protective effects of varying degrees of hypoxia on the cytotoxicities of etoposide and bleomycin. 243 23

CHO-Cdr20 cells are 10-20 times more resistant to killing by cadmium than the parental CHO cells. Resistance has been linked to amplification of the metallothionein genes MT-I and MT-II and their coordinate induction by cadmium and other toxic metals. We studied the roles of the nuclear enzymes topoisomerase I and topoisomerase II in Cd-induced expression of MT-II. Camptothecin-induced DNA strand breakage, mediated by topoisomerase I in cells, increased by approximately 20% when the resistant cells were incubated first with 50 microM Cd and then with camptothecin. Short DNA fragments were enriched in MT-II-hybridizing sequences, indicating that topoisomerase I-associated breakage was directed in part toward the location of induced gene activity. Ten microM camptothecin inhibited Cd-induced accumulation of MT-II mRNA as well as induced and uninduced RNA synthesis in the resistant cells. These data are consistent with the notion that topoisomerase I participates in most or all forms of RNA synthesis. Topoisomerase II inhibitors which trap cleavable complexes (amsacrine, VM-26, VP-16) increased DNA strand breakage at very high concentrations (50-100 microM); the increased breakage appeared to be concentrated near the MT-II gene. This class of inhibitor did not block the accumulation of MT-II message. Novobiocin, a second type of topoisomerase II inhibitor blocked transcription at 300 microM. Merbarone, a novel, third type of topoisomerase II inhibitor, blocked MT-II transcription at 50-100 microM. The latter two inhibited total RNA synthesis in induced, but not uninduced cells. Thus, it is possible that topoisomerase II plays more than one role in transcription and that more than one form of this enzyme is involved.
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PMID:Evidence for the participation of topoisomerases I and II in cadmium-induced metallothionein expression in Chinese hamster ovary cells. 247 39

Resistance to etoposide (VP-16), amsacrine (mAMSA), and doxorubicin (Adriamycin) was studied in two Chinese hamster cell lines primarily selected for resistance to the epipodophyllotoxin. Both lines demonstrated profound resistance to VP-16, and mAMSA stimulated DNA breakage. However, the resistance to mAMSA cytotoxicity in both lines was less than expected from the level of resistance to the effects of topoisomerase II inhibition. Similarly, resistance to the cytotoxicity of high VP-16 concentrations in one of the lines was less than expected from the resistance to inhibition of topoisomerase II. An analysis of the relation of DNA breaks to drug cytotoxicity suggests that cross-resistance to mAMSA was mainly conferred through loss of mAMSA-stimulated, topoisomerase II-mediated DNA breaks. This mechanism also contributed towards reduced VP-16 cytotoxicity. However, our studies suggest that additional mechanisms, independent of resistance to VP-16-mediated topoisomerase II effects, greatly increased the resistance to this agent. Resistance to VP-16 cytotoxicity, not dependent on resistance to drug-mediated DNA cleavage, could be overcome at high drug concentrations in one of the resistant lines and might be responsible for the greater relative resistance to VP-16 than to mAMSA. These findings suggest the presence of two distinct mechanisms of resistance to VP-16 cytotoxicity, one presumably mediated by topoisomerase II and dependent on resistance to drug-mediated DNA scission, and a second mechanism independent of the effects of the drug on topoisomerase II.
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PMID:Topoisomerase II-dependent and -independent mechanisms of etoposide resistance in Chinese hamster cell lines. 253 64

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radiation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.
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PMID:Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains. 253 34

Our human T-cell leukemia line, CEM/VM-1, selected for resistance to VM-26 (teniposide), is cross-resistant to several drugs that interact with topoisomerase II, including VP-16 (etoposide), 4'-(9-acridinylamino)methanesulphon-m-anisidide, daunorubicin, and mitoxantrone. However, in contrast to cell lines exhibiting multidrug resistance (MDR) associated with overexpression of P-glycoprotein, this line is not cross-resistant to the Vinca alkaloids, is not impaired in drug accumulation, and does not overexpress the mdrl gene (Cancer Res., 47: 1297, 5455, 1987). More recently we found that nuclear extracts of these cells exhibit decreased topoisomerase II catalytic and cleavage activity, compared to the drug-sensitive line (Biochemistry, 1988). These results suggest that an alteration in topoisomerase II or a modulator of this enzyme may be responsible for this altered topoisomerase II-form of multidrug resistance (at-MDR). In the present work, we studied the somatic cell genetics of at-MDR. We produced hybrid cell lines by polyethylene glycol-mediated fusion of the CEM/VM-1 line with a hypoxanthine-guanine phosphoribosyl transferase-deficient, ouabain-resistant CEM line (CEM.AG1.OU1.5) that exhibits VM-26 sensitivity. Ten of the hybrid lines that grew in selective medium were randomly chosen for expansion and four were analyzed for both DNA content by flow cytometry and VM-26 sensitivity in a 72-h growth inhibition assay. The hybrid lines all contained approximately 2x DNA compared to unfused controls, indicating that the fusions were successful. The IC50 for VM-26 in 3 of the 4 lines was the same as that of the sensitive controls, ranging from 4.7 to 7.4 x 10(-8) M, and another was 76 x 10(-8) M. These data indicate that drug sensitivity was reconstituted by the hybridization procedure. By comparison, the VM-26 IC50 values in the CEM/VM-1 cells and CEM/VM-1 x CEM/VM-1 control "fusions" were 360 and 750 x 10(-8) M, respectively. To determine whether a topoisomerase II-mediated function was reconstituted in the hybrids, we measured drug-stimulated DNA cleavage ("cleavable complex formation"). Using 32P-labeled pBR322 DNA as substrate with nuclear extracts from drug sensitive cells, 100 microM VM-26 maximally stimulated DNA cleavage by approximately 11-fold compared to no-drug controls.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Genetic characterization of the multidrug-resistant phenotype of VM-26-resistant human leukemic cells. 253 2

Circular plasmid DNA was efficiently converted into huge catenated intranuclear networks by incubation with isolated nuclei in the presence of ATP. The network production is abolished by omission of ATP, strongly inhibited by etoposide (VP-16), but only slightly inhibited by antibody to topoisomerase I, indicating that the major enzyme responsible for catenation is DNA topoisomerase II. Under optimal conditions, a single nucleus incorporates about 4.2 x 10(4) DNA rings into its networks. Under the light microscope, networks retrieved from nuclei appear like spheres of various sizes. Sedimentation analysis showed that most of the networks are composed of thousands of catenated rings, which was confirmed by electron microscopy. Data from experiments that caused partial disruption of the networks were submitted to analysis based on probable models of catenane structure. The results suggest that the predominant pattern is a linear alignment of catenated rings. Similar networks are formed when the nuclear scaffold is incubated with circular DNA in the presence of nuclear extract containing topoisomerase II. Titration experiments showed that the scaffold binds a stoichiometric amount of the substrate and that a critical level of DNA is required for network formation. The results are consistent with the idea that DNA-binding sites are fixed on the scaffold and mediate catenation of bound DNA circles by holding them in close proximity to each other. We propose that catenation by the nuclear scaffold also occurs in intact nuclei, suggesting additional roles for the scaffold in vivo.
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PMID:Incorporation of exogenous circular DNA into large catenated networks in isolated nuclei. Evidence for involvement of the nuclear scaffold. 254 Jan 99

Cells released from quiescence exhibit increased levels of the DNA-modifying enzyme topoisomerase II, a nuclear protein which is also a target for antitumour drugs such as VP-16 (etoposide) and m-AMSA (4',9'-acridinylamino-methanesulfon-m-anisidide). By using Western blotting, DNA-protein crosslinking and drug-induced DNA cleavage to detect topoisomerase II, we show here that oestrogen stimulation of T-47D human breast cancer cells results in increased cellular enzyme content at least 4hr prior to enhancement of DNA synthesis. Taken in conjunction with previous findings, these results suggest that oestrogen enhances topoisomerase II synthesis within a G1-phase cell subset.
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PMID:Mitogen-induced topoisomerase II synthesis precedes DNA synthesis in human breast cancer cells. 254 Jul 40

Several podophyllotoxin-related lignans have been shown to possess significant antifungal activity against a number of filamentous fungi. Initial structure-activity studies indicate this action is sensitive to change at the 4 and 4' positions of the podophyllotoxin skeleton. Good correlation has been observed between antifungal action and the ability to inhibit the relaxation of supercoiled plasmid DNA by a topoisomerase II preparation from Saccharomyces cerevisiae. Etoposide, an inhibitor of mammalian topoisomerase II, is inactive against this yeast enzyme, although good inhibition is shown by amiloride, 4'-(9-acridinylamino)-methanesulphon-m-anisidide (m-AMSA) and novobiocin, known inhibitors of the mammalian enzyme.
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PMID:Topoisomerase II: a potential target for novel antifungal agents. 254 Jul 47

We have found that blockade of the Na+,K+-pump by the cardiac glycoside ouabain protects human A549 and hamster V79 cells from the cytotoxic effects of the topoisomerase II poison etoposide. One thousand-fold higher concentrations of ouabain were required to protect V79 cells compared to A549 cells. Since this difference parallels previously measured differences in pump sensitivity, it suggests that protection is mediated directly through pump blockade. Ouabain affected neither the cellular influx nor efflux of etoposide. However, pump blockade did decrease the formation of etoposide-induced DNA-topoisomerase, II-cleavable complexes, assessed as single and double strand DNA breaks using alkaline and neutral elution. To determine if this decrease were a direct effect of change in ionic environment produced by pump blockade, experiments with isolated nuclei and partially purified topoisomerase II were performed. Etoposide-induced cleavable complex formation and topoisomerase-mediated decatenation were assessed in buffers which mimicked either normal intracellular ionic conditions or those produced by ouabain. Compared to the buffer which resembled the normal intracellular ionic conditions, the buffer that mimicked the conditions produced by pump blockade produced fewer etoposide-mediated cleavable complexes in isolated nuclei and less decatenating activity of partially purified topoisomerase II. These findings demonstrate that inhibition of the Na+,K+-pump causes an alteration in the intracellular ionic environment which decreases the activity of topoisomerase II, thus producing a decrease in etoposide-induced cleavable complex formation and cytotoxicity. Since ionic changes occur inside normal cells during progression through the cell cycle as well as in cells that have undergone transformation, these data suggest that the intracellular ionic environment plays a role in determining the sensitivity of normal and malignant cells to this group of chemotherapeutic agents.
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PMID:Dependence of etoposide-induced cytotoxicity and topoisomerase II-mediated DNA strand breakage on the intracellular ionic environment. 254 16

The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agents appear to be mediated by DNA topoisomerase II. We have purified topoisomerase II to homogeneity both from an epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, and from the wild-type parental cell line. Immunoblots demonstrate similar topoisomerase II content in these two cell lines. The purified enzymes are dissimilar in that DNA cleavage by VpmR-5 topoisomerase II is not stimulated by VP-16 or m-AMSA. Furthermore, the VpmR-5 enzyme is unstable at 37 degrees C. Thus, the drug resistance of VpmR-5 cells appears to result from the presence of an altered topoisomerase II in these cells. Purified topoisomerase II from VPMR-5 and wild-type cells has the same monomeric molecular mass as well as equivalent catalytic activity with respect to decatenation of kinetoplast DNA. Etoposide (VP-16) inhibits the activity of both enzymes. Noncovalent DNA-enzyme complex formation, assayed by nitrocellulose filter binding, is also similar, as is protection from salt dissociation of this complex by ATP and VP-16. The data suggest a model in which the drug-resistant cell line, VpmR-5, has religation activity which is less affected by drug than that of the wild-type cells. Drug effect on DNA religation and catalytic activity are dissociated mechanistically. In addition, under certain circumstances, the "cleavable complex" observed following denaturation of a drug-stabilized DNA-enzyme complex may not adequately reflect the nature of the antecedent lesion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of an altered topoisomerase II from a drug-resistant Chinese hamster ovary cell line. 255 59


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