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 effects of serine phosphorylation on the DNA cleavage/religation equilibrium of topoisomerase II and the sensitivity of the enzyme to antineoplastic drugs were characterized. Both casein kinase II and protein kinase C were used for these studies. Each kinase incorporated a maximum of approximately 1.4 phosphate molecules per homodimer of topoisomerase II. When the enzyme was incubated with both kinases simultaneously, phosphate incorporation increased to approximately 2.6 molecules/homodimer. In the absence of antineoplastic drugs, phosphorylation had only a slight effect on the DNA cleavage/religation equilibrium of topoisomerase II. However, in the presence of etoposide or 4'-(9-acridinylamino)methane-sulfon-m-anisidide, phosphorylation attenuated the ability of drugs to stabilize enzyme-DNA cleavage complexes. Levels of drug-induced DNA cleavage products decreased approximately 33% following phosphorylation of topoisomerase II by casein kinase II, approximately 17% following modification by protein kinase C, and approximately 50% following simultaneous phosphorylation of the enzyme by both kinases. This latter 50% reduction in DNA cleavage products correlated with an approximately 2-fold increase in the apparent first order rate constant for DNA religation mediated by simultaneously modified topoisomerase II. These results strongly suggest that the sensitivity of topoisomerase II toward antineoplastic drugs can be modulated by altering the phosphorylation state of the enzyme.
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PMID:Phosphorylation of topoisomerase II by casein kinase II and protein kinase C: effects on enzyme-mediated DNA cleavage/religation and sensitivity to the antineoplastic drugs etoposide and 4'-(9-acridinylamino)methane-sulfon-m-anisidide. 131 38

Our earlier studies have shown that gossypol [1,1',6,6',7,7'-hexahydroxy-5,5-diisopropyl - 3,3'-dimethyl - (2,2'- binaphthalene)-8,8'-dicarboxyaldehyde], a male contraceptive, inhibits DNA synthesis by decreasing the activities of DNA polymerase alpha and beta, resulting in the arrest of cells in mid-S phase [L.J. Rosenberg, R.C. Adlakha, D.M. Desai, and P.N. Rao, Biochim. Biophys. Acta, 866: 258-267, 1986]. Now we have examined the effects of gossypol on another enzyme of importance to cellular functions, topoisomerase II (topo II). We have determined the consequences of gossypol treatment on 4'-(9-acridinylamino)methane-sulfon-m anisidide (m-AMSA)-induced topoisomerase II-mediated, protein-associated DNA cleavage using the alkaline elution technique. In HeLa cells pretreated with gossypol (3.4-17.5 microM) for 8-16 h we observed a dose- and time-dependent decrease (50-75%) in DNA cleavage compared to that quantified in cells treated with m-AMSA alone. Gossypol by itself did not induce more than 25 rad-equivalents of DNA single-strand breaks even at the highest dose tested (26 microM). [14C]m-AMSA uptake was identical in treated and untreated cells. Pretreatment of cells with another inhibitor of DNA synthesis, thymidine, which blocks cells at G1/S boundary increased the m-AMSA-induced DNA cleavage by 25%, suggesting that the effect of gossypol might be due to the arrest of cells in mid-S phase. In contrast to gossypol's effects on m-AMSA-induced DNA cleavage, m-AMSA-induced cytotoxicity was actually increased in gossypol pretreated cells. Gossypol blocked topo II strand passing activity (decatenation of kinetoplast DNA) of cellular extracts from HeLa cells. The inhibition of this activity by gossypol was synergistic with the inhibition produced by m-AMSA or etoposide. These data suggest that gossypol can both inhibit topo II catalytic activity and interfere with the stabilization of topo II-DNA complex formation by m-AMSA. These data indicate that the magnitude of m-AMSA-induced DNA cleavage may not necessarily parallel the magnitude of m-AMSA-induced cytotoxicity. The cytotoxicity data may rather be explained by an action of gossypol and m-AMSA to block topo II catalytic activity at a point in the enzyme's strand passing cycle prior to cleavage complex formation that might be particularly toxic to cells in S phase. Gossypol should therefore be useful in improving our understanding of the cellular role of topo II and the consequences of interference with topo II activity by active antineoplastic agents.
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PMID:Modulation of 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced, topoisomerase II-mediated DNA cleavage by gossypol. 253 51

Antitumor drugs from many chemical classes have been shown to induce protein-linked DNA breaks in cultured mammalian cells and in vitro in the presence of purified mammalian DNA topoisomerase II. The possibility that mammalian DNA topoisomerase II is an intracellular target which mediates drug-induced DNA breaks is supported by the following studies using 4'-(9-acridinylamino)methane-sulfon-m-anisidide (m-AMSA): (a) a single m-AMSA-dependent DNA cleavage activity copurified with calf thymus DNA topoisomerase II activity at all chromatographic steps of the enzyme purification; (b) m-AMSA-induced DNA cleavage by this purified activity resulted in the covalent attachment of protein to the 5'-ends of the DNA via a tyrosyl phosphate bond. This covalently linked protein has the same reduced molecular weight as purified calf thymus DNA topoisomerase II. The possibility that topoisomerase II-mediated DNA breaks may be responsible for cytotoxicity has also been investigated using a number of m-AMSA-related acridines. The level of topoisomerase II-mediated DNA breaks in vitro strongly correlates with the level of protein-linked DNA breaks in cultured cells and drug-induced cytotoxicity. These results suggest that mammalian DNA topoisomerase II may be a cytotoxic target of antitumor acridines.
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PMID:DNA damage by antitumor acridines mediated by mammalian DNA topoisomerase II. 300 16

Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II. We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573. Selection with doxorubicin frequently resulted in reduced topo II alpha mRNA and protein levels, whereas clones selected with vincristine showed normal levels of topo II alpha. No alterations of topo II beta levels were detected. To determine the contribution of topo II alterations to drug resistance, topo II activity was analysed by the determination of DNA breaks induced by the topo II-inhibiting drug 4'-(9-acridinylamino)methane-sulphon-m-anisidide (m-AMSA) in living cells, as m-AMSA is not affected by the drug efflux mechanism in the SW-1573 cells. The number of m-AMSA-induced DNA breaks correlated well (r = 0.96) with in vitro m-AMSA sensitivity. Drug sensitivity, however, did not always correlate with reduced topo II mRNA or protein levels. In one of the five doxorubicin-selected clones m-AMSA resistance and a reduction in m-AMSA-induced DNA breaks were found in the absence of reduced topo II protein levels. Therefore, we assume that post-translational modifications of topo II also contribute to drug resistance in SW-1573 cells. These results suggest that methods that detect quantitative as well as qualitative alterations of topo II should be used to predict the responsiveness of tumours to cytotoxic agents. The assay we used, which measures DNA breaks as an end point of topo II activity, could be a good candidate.
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PMID:Reduced topoisomerase II activity in multidrug-resistant human non-small cell lung cancer cell lines. 781 46

Aurintricarboxylic acid (ATA) is a polyanionic, polyaromatic compound which has been shown to inhibit apoptotic cell death in various cell types induced by a variety of factors. Since ATA is known to be a general inhibitor of nuclease activities in vitro (ID50S ranging from 2 to 50 microM), the in vivo effects are usually attributed to inhibition of endogenous endonuclease activities. We show herein that ATA is a potent inhibitor of the nuclear enzyme DNA topoisomerase II. ATA inhibits the catalytic activity of purified yeast topoisomerase II with an ID50 of approx. 75nM as measured by relaxation assays. ATA does not stabilize the covalent DNA-topoisomerase II reaction intermediate ("cleavable complex") as do other inhibitors of this enzyme such as 4'-(9-acridinylamino)-methane sulfon-m-anisidide (amsacrime), 4'-demethyl-epipodophyllotoxin-9-(4,6-O-ethylidine-beta-D-gluco pyr anoside) (etoposide) and ellipticines. In contrast, cleavable complex formation induced by amsacrine and etoposide is trongly inhibited in the presence of ATA. ATA also prevents the binding of topoisomerase II to DNA and inhibits topoisomerase II-catalysed ATP hydrolysis. The ability of ATA to interfere with more than one step in t he catalytic cycle of DNA topoisomerase II may explain its unusual potency as an inhibitor of this enzyme. ATA reduces the number of amsacrine-induced DNA-protein complexes in intact DC-3F Chinese hamster fibrosarcoma cells and protects these cells from the cytotoxic action of amsacrine. The effects of ATA on DNA-protein complex formation in living cells appear to be due to the direct interaction of the drug with topoisomerase II, since similar results are found when nuclei from untreated DC-3F cells are exposed to amsacrine after a short preincubation with ATA. Cells resistant to 9-hydroxyellipticine, which have been shown to possess altered topoisomerase II activity, are approx. 5-fold more resistant to ATA than the sensitive parental cells as shown by colony formation essays. We conclude that ATA is a potent inhibitor of topoisomerase II and that the drug interacts with topoisomerase II in living cells. Our findings raise the possibility that the protective effects of ATA towards apoptotic cell death might, at least in part, involve DNA topoisomerase II.
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PMID:Aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells. 785 17

cis-Dichlorodiammineplatinum(II) (CDDP) resistance in L1210/10 murine leukemia cells is multifactorial and involves decreased drug uptake, increased glutathione content, and enhanced DNA repair activity. We show here that 0.35 M NaCl nuclear extracts from L1210/10 cells possess an approximately 3-fold increase in DNA topoisomerase II activity, compared with parental L1210 cells, as measured by decatenation of kinetoplast DNA. No difference in topoisomerase I activity is observed between the two cell lines. Immunoblot analysis of topoisomerase II protein in resistant and sensitive cells suggests that the observed differences in topoisomerase II activity cannot be explained by differences in the level of protein expressed. L1210/10 cells are 2.5-fold more sensitive than L1210 cells to the cytotoxic effects of the topoisomerase II inhibitor 4'-(9-acridylamino)methane-sulfon-m-anisidide. Sequential treatment with 4'-(9-acridyl-amino)methanesulfon-m-anisidide and CDDP leads to an additive cytotoxic effect of the two drugs in sensitive L1210 cells, as determined by colony formation in semi-solid medium. In contrast, the same treatment leads to a supra-additive effect in L1210/10 cells, which strongly suggests a role for topoisomerase II in the CDDP resistance of this cell line.
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PMID:A cisplatin-resistant murine leukemia cell line exhibits increased topoisomerase II activity. 793 22

Pulse field gel electrophoresis using a contour-clamped homogeneous electric field was applied for the analysis of DNA-fragmenting activity of antitumor agents towards human uterine cervix carcinoma HeLa S3 cells. Duocarmycins (DUMs), novel antitumor antibiotics with ultrapotent cell growth-inhibitory activities, caused DNA fragmentation at 10 times their IC50 values at 2 h exposure. At 100 times their IC50 values, the size of the smallest fragments was about 245 kilobase pairs (kbp). DUMA, DUMB1 and DUMB2 exhibited similar DNA fragmentation patterns, suggesting similar action mechanisms. DNA fragmentation was also detected in cells treated with radical producers, intercalators and topoisomerase inhibitors. Two bands of about 1800 and 1500 kbp were commonly detected in the cells treated with DUMs and these agents. In addition, fragments of about 900 kbp were detected in the cells treated with a topoisomerase inhibitor, 4'-(9-acridinylamino)methane-sulfon-m-anisidine, and fragments in the broad size range between 700 and 245 kbp in the cells treated with radical producers, bleomycin and neocarzinostatin. DUMs showed a characteristic DNA fragmentation pattern, since both types of fragments induced by the topoisomerase inhibitor and the radical producers were simultaneously detected, suggesting a novel mode of interaction with DNA. DNA-crosslinking agents and mitotic inhibitors did not induce DNA fragmentation under these conditions. The pulse field gel electrophoresis is potentially useful for characterizing DNA-cleaving activity of various antitumor agents at the cellular level.
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PMID:Analysis of DNA fragmentation in human uterine cervix carcinoma HeLa S3 cells treated with duocarmycins or other antitumor agents by pulse field gel electrophoresis. 844 32

Imidazoacridinones are new antitumor compounds that exhibit strong antitumor effect against solid tumors such as human colon and breast carcinomas. The mechanism of action of imidazoacridinones is unknown, although a similarity in the chemical structure between active imidazoacridinones and mitoxantrone suggests common cellular targets. We show that imidazoacridinones inhibit the catalytic activity of purified topoisomerase II as determined by both relaxation and decatenation assays. All biologically active compounds stimulated the formation of cleavable complexes in vitro, whereas inactive compounds did not. The pattern of DNA cleavage in SV40 DNA was similar to that obtained for 4'-(9-acridinylamino)methane-sulfon-m-aniside, particularly within the matrix-associated region. Significant levels of DNA complexes were observed when DC-3F fibrosarcoma cells were treated with active compounds, whereas negligible amounts of these complexes were induced by inactive analogues. DC-3F/9-OHE cells, which are resistant to other topoisomerase II inhibitors, are 30-125-fold cross-resistant to active imidazoacridinones. The resistance is associated with a reduction in the formation of DNA/protein complexes and is highest for compounds that are potent topoisomerase II inhibitors in vitro. Interestingly, the two most active derivatives, C-1310 and C-1311, were equally cytotoxic toward fast-growing monolayer cultures and cells growing in three dimensions as multicellular spheroids, which have a slower growth fraction. In contrast, 4'-(9-acridinylamino)methanesulfon-m-aniside, mitoxantrone, and doxorubicin were more cytotoxic toward monolayer cultures. Taken together, the results suggest that DNA topoisomerase II is a major cellular target of biologically active imidazoacridinones and that these drugs show both similarities and dissimilarities compared with classic topoisomerase II inhibitors.
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PMID:Inhibition of DNA topoisomerase II by imidazoacridinones, new antineoplastic agents with strong activity against solid tumors. 862 25

To investigate whether mammalian DNA topoisomerase II is directly involved in recombination events, the effects of ICRF-193, a specific catalytic inhibitor on sister chromatid exchange (SCE), were examined in MR-6 cells. ICRF-193 only slightly elevated SCE formation after 3 or 44 h treatments, while VP-16, a cleavable complex forming type of topoisomerase II inhibitor, caused significant enhancement. ICRF-193 had no effect on N-methyl-N'-nitro-N-nitrosoguanidine-induced SCE formation. It would thus appear that the inhibition of topoisomerase II does not affect recombinational repair, and that topoisomerase II inhibitors such as VP-16 and 4'-(9-acridinyl amino) methane sulfon-m-anisidide induce SCE through production of DNA strand breaks, rather than by inhibiting the enzyme activity.
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PMID:Effects of ICRF-193, a catalytic inhibitor of DNA topoisomerase II, on sister chromatid exchange. 930 May 80

Topoisomerase II represents the main target for the antitumour drugs etoposide and amsacrine, which are both used clinically. Previous studies have shown that the glycoside moiety of etoposide is not necessary for cytotoxicity or DNA topoisomerase II inhibition. For this reason, we designed two epipodophyllotoxin derivatives for which the dispensable sugar moiety of etoposide has been replaced by a m-methoxy-methane-sulfonamide-anilino group analogous to the topoisomerase II-targeted domain of amsacrine. We report the synthesis of the hybrid molecules that have the epipodophyllotoxin and anilino groups directly linked (ICP-114) or connected by an ethylene spacer (ICP-147). Plasmid DNA relaxation and kinetoplast DNA decatenation assays were used to evaluate the effects of the drug on the catalytic activity of human topoisomerase II. We found that the hybrid ICP-147 was significantly more potent than both etoposide and amsacrine at stimulating DNA cleavage by the enzyme, whereas the hybrid ICP-114 lacking the linker chain was less potent. ICP-147 produces approximately 3 times more double-stranded breaks than ICP-114, suggesting that an ethylene spacer between the epipodophyllotoxin and amsacrine moieties is highly effective at inhibiting topoisomerase II. Sequencing data also supported the idea that the two moieties of ICP-147 participate to the interaction with topoisomerase II-DNA covalent complexes. Both hybrid compounds are more cytotoxic than etoposide but much less toxic than amsacrine toward L1210 leukemia cells. In addition to its effect on topoisomerase II, ICP-114 can inhibit tubulin polymerization, whereas ICP-147 is almost totally inactive in this assay. The unexpected capacity of ICP-114 to interfere with the polymerization of tubulin suggests that this compound can target tubulin dimers, as it is the case with certain antitumor sulfonamides. The design of etoposide-amsacrine hybrids may thus represent an opportunity for the discovery of dual inhibitors that target both topoisomerase II and tubulin.
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PMID:Design of two etoposide-amsacrine conjugates: topoisomerase II and tubuline polymerization inhibition and relation to cytotoxicity. 1171 34


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