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)

ICRF-187 is a bisdioxopiperazine anticancer drug that inhibits the catalytic activity of DNA topoisomerase (topo) II without stabilizing DNA-topoII cleavable complexes. To better understand the mechanisms of action of and resistance to topoII catalytic inhibitors, human leukemic CEM cells were selected for resistance to ICRF-187. The clones CEM/ICRF-8 and CEM/ICRF-18 are approximately 40- and 69-fold resistant to ICRF-187, and 12- and 67-fold cross-resistant to ICRF-193, respectively, but are sensitive to other topoII catalytic inhibitors (merbarone and aclarubicin), as well as collaterally sensitive to the DNA-topoII complex-stabilizing drug etoposide (VP-16). Both the number of VP-16- induced DNA-topoII complexes formed and the amount of in vitro topoII catalytic activity are enhanced in the drug-resistant cells. The ICRF-187-resistant clones contain approximately 5-fold increase in topoIIalpha protein levels and approximately 2.2-fold increase in topoIIalpha mRNA levels. Furthermore, CEM/ICRF-8 expresses approximately 3.5-fold increase in topoIIalpha promoter activity, suggesting that up-regulation of topoIIalpha in this clone occurs at the transcriptional level. Treatment of the drug-resistant or -sensitive cells with equitoxic doses of merbarone or teniposide results in a G(2)/M arrest. In marked contrast, when treated with equitoxic ICRF-187 doses, the drug-resistant clones exhibit either a transient arrest or completely lack the G(2)/M checkpoint compared with the drug-sensitive cells. This aberrant cell cycle profile is associated with a 48-h delay in drug-induced apoptotic cell death, as revealed by fluorescent-end labeling of DNA and poly (ADP-ribose) polymerase cleavage. In summary, resistance to ICRF-187 in CEM cells is associated with increased levels of catalytically active topoIIalpha and altered G(2)/M checkpoint and apoptotic responses.
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PMID:Selection of human leukemic CEM cells for resistance to the DNA topoisomerase II catalytic inhibitor ICRF-187 results in increased levels of topoisomerase IIalpha and altered G(2)/M checkpoint and apoptotic responses. 1064 39

The topoisomerase II (TOP2)-associated DNA cleavage activity and the DNA sequence preference of 20 antitumor drugs, including 15 TOP2-interacting compounds, have been defined. Four major classes of drugs have been identified: (i) those which enhanced the stabilization of cleavable complexes at a single major site (e.g. amsacrine, doxorubicin), or (ii) at many sites (e.g. etoposide, azatoxin), with chemically related compounds having very similar, although not identical, cleavage patterns (e.g. etoposide, GL331 and Top-53); (iii) those which inhibited DNA breakage (e.g. aclarubicin, actinomycin D); and (iv) those which did not visibly interfere with TOP2-mediated cleavable complexes (e.g. ICRF-187, camptothecin). All drugs tested induced similar overall patterns of sites of preferred DNA cleavage, in the presence either of the two known isoforms, TOP2alpha or TOP2beta, although relative intensities of signals at each position varied. It has been further shown that etoposide and its derivatives blocked the religation step downstream of the DNA cleavage step, whereas amsacrine, ellipticine, azatoxin and genistein acted upstream through enhancement of DNA cleavage. The information provided by this mechanistically based comparison can now be exploited in designing or synthesizing novel TOP2-interacting agents.
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PMID:Differential in vitro interactions of a series of clinically useful topoisomerase-interacting compounds with the cleavage/religation activity of the human topoisomerase IIalpha and IIbeta isoforms. 1088 3

Random mutagenesis of human topoisomerase II alpha cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer > 37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.
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PMID:N-terminal and core-domain random mutations in human topoisomerase II alpha conferring bisdioxopiperazine resistance. 1103 29

The bisdioxopiperazines, including dexrazoxane (ICRF-187), are catalytic or noncleavable complex-forming inhibitors of DNA topoisomerase II that do not produce DNA strand breaks. In this study we show that dexrazoxane inhibits the division of Chinese hamster ovary (CHO) cells resulting in marked increases in cell size (up to 80 microm in diameter), volume (up to 150-fold greater), and ploidy (as high as 32N). This last result indicates that the dexrazoxane-induced DNA reduplication was restricted to once per cell cycle. Kinetic analysis of the flow cytometry data indicated that the conversion between successively higher ploidy levels was progressively slowed at longer times of exposure to dexrazoxane. Both the protein and DNA content of dexrazoxane-treated CHO cells increased linearly over time in the same proportion. Light and electron microscopic studies of dexrazoxane-treated cells showed ring-like multilobulated nuclei. Immunohistochemical staining of dexrazoxane-treated cells showed that F-actin and acetylated alpha-tubulin were present in large, highly organized networks. Immunohistochemical staining of the dexrazoxane-treated CHO cells also showed that the topoisomerase II alpha colocalized with the DNA of the multilobulated nuclei. Staining of gamma-tubulin revealed that the dexrazoxane-treated cells contained multiple centrosomes, indicating that dexrazoxane prevents cytokinesis but not centrosome reduplication. It is concluded that dexrazoxane inhibits CHO cytokinesis in cells by virtue of its ability to inhibit topoisomerase II.
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PMID:The catalytic DNA topoisomerase II inhibitor dexrazoxane (ICRF-187) induces endopolyploidy in Chinese hamster ovary cells. 1104 78

F 11782 is a newly identified catalytic inhibitor of topoisomerases I and II, without any detectable interaction with DNA. This study aimed to establish whether its catalytic inhibition of topoisomerase II was mediated by mechanisms similar to those identified for the bisdioxopiperazines. In vitro combinations of F 11782 with etoposide resulted in greater than additive cytotoxicity in L1210 cells, contrasting with marked antagonism for combinations of etoposide with either ICRF-187 or ICRF-193. All three compounds caused a G2/M blockade of P388 cells after an 18-h incubation, but by 40 h polyploidization was evident only with the bisdioxopiperazines. Gel retardation data revealed that only F 11782, and not the bisdioxopiperazines, was capable of completely inhibiting the DNA-binding activity of topoisomerase II, confirming its novel mechanism of action. Furthermore, unlike ICRF-187 and ICRF-193, the cytotoxicity of F 11782 appeared mediated, at least partially, by DNA damage induction in cultured GCT27 human teratoma cells, as judged by a fluorescence-enhancement assay and monitoring p53 activation. Finally, the major in vivo antitumor activity of F 11782 against the murine P388 leukemia (i.v. implanted) and the B16 melanoma (s.c. grafted) contrasted with the bisdioxopiperazines' general lack of activity. Overall, F 11782 and the bisdioxopiperazines appear to function as quite distinctive catalytic topoisomerase II inhibitors.
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PMID:Characterization of the biological and biochemical activities of F 11782 and the bisdioxopiperazines, ICRF-187 and ICRF-193, two types of topoisomerase II catalytic inhibitors with distinctive mechanisms of action. 1114 91

DNA topoisomerase (topo) IIalpha gene expression or activity is altered in tumor cells selected for resistance to inhibitors of topoII. To better understand the mechanisms by which topoIIalpha expression levels are modulated, we examined topoIIalpha transcriptional regulation in ICRF-187-sensitive and ICRF-187-resistant human leukemic cell lines that express an increased amount of topoIIalpha protein and mRNA. Transient transfections of luciferase reporter plasmids containing either the full-length human topoIIalpha promoter or fragments of it revealed that topoIIalpha transcriptional activity was significantly increased in the drug-resistant CEM/ICRF-8 cells, compared with CEM cells. Specifically, the transcriptional activity of the full-length topoIIalpha promoter (nucleotides -557 to +90) was doubled in CEM/ICRF-8 compared with CEM cells. Serial deletion of the topoIIalpha promoter permitted localization of the region responsible for its up-regulation in the drug-resistant cells between nucleotides -557 and -162, which includes the last three inverted CCAAT elements (ICE) 3 to 5. Note that construction of a point mutation in ICE3 resulted in a significant increase in transcriptional activity of the topoIIalpha promoter in the drug-sensitive CEM cells. In addition, by electrophoretic mobility shift assay, ICE3 was recognized by a protein complex containing NF-YB that was present at reduced levels in the topoIIalpha-overexpressing CEM/ICRF-8 extracts, suggesting that ICE3 plays a negative regulatory role in human topoIIalpha gene expression. This is the first study to show that topoIIalpha transcriptional up-regulation in ICRF-187-resistant cells is mediated in part by altered regulation of the third inverted CCAAT box in the topoIIalpha promoter.
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PMID:Role of an inverted CCAAT element in human topoisomerase IIalpha gene expression in ICRF-187-sensitive and -resistant CEM leukemic cells. 1116 Aug 54

We studied the consequences of interfering with DNA topoisomerase IIalpha (topo IIalpha) activity on melphalan-induced cytotoxicity. In order to accomplish our goal we used three different approaches to interfere with topo IIalpha. These include: i) use of three V79 Chinese hamster lung fibroblast-derived mutant cell lines, V507, V511, and V513 that are dysfunctional in topo IIalpha activity; ii) treatment of cells with etoposide (VP-16) which inhibits topo IIalpha through the formation of DNA-enzyme cleavable complex; and iii) exposure of cells to merbarone or ICRF-187 (Zinecard) that inhibits the activity of topo IIalpha by restricting its access to DNA. Based on clonogenic survival assays, all three approaches resulted in a significant potentiation of cytotoxicity of melphalan suggesting that topo IIalpha plays an important role in processing of DNA damage induced by melphalan. Furthermore, using alkaline elution assay, we show that melphalan-induced DNA cross-link formation and its repair is faster in V511 cells compared to the parental V79 cells. However, melphalan-induced sister chromatid exchanges (SCE) are found to be significantly higher in V511 cells compared to V79 cells. In addition, we find an excellent correlation between melphalan-induced SCE and cytotoxicity. These results could be explained on the assumption that topo IIalpha plays an important role in damage processing through excision repair of melphalan-induced DNA cross-links. However, in the absence of topo IIalpha the damages are primarily processed by recombination repair which may be prone to deleterious genetic alterations resulting in increased lethality as the frequency of recombination increases. In summary, our results demonstrate that: i) topo IIalpha deficiency is associated with increased sensitivity to melphalan; ii) deficiency of topo IIalpha is associated with an increase in melphalan-induced SCE; iii) increase in melphalan-induced SCE is associated with an increase in cytotoxicity; and iv) downregulation of topo IIalpha may be a useful approach to modulate the cytotoxicity of melphalan in combination chemotherapy regimens. These results have several important clinical implications. First, interference with topo IIalpha using agents such as VP-16 or ICRF-187 may provide a useful approach to enhance the efficacy of melphalan in combination chemotherapy regimens. Second, tumors which develop resistance to topo IIalpha-directed drugs due to quantitative or qualitative alterations in topo IIalpha may show increased susceptibility to a chemotherapy regimen containing melphalan.
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PMID:Interference with topoisomerase IIalpha potentiates melphalan cytotoxicity. 1178 94

Dexrazoxane (ICRF-187), which is clinically used to reduce doxorubicin-induced cardiotoxicity, is also a potent catalytic inhibitor of DNA topoisomerase II. In this study we showed that dexrazoxane inhibited the division of neonatal rat ventricular myocytes in culture, and resulted in nuclear multilobulation (demonstrated by three-dimensional reconstruction of confocal images) and marked increases in nuclear size and DNA ploidy levels (as shown by flow cytometry). It was concluded that dexrazoxane interfered with cell division in cardiac myocytes by virtue of its ability to inhibit topoisomerase II.
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PMID:The doxorubicin cardioprotective agent dexrazoxane (ICRF-187) induces endopolyploidy in rat neonatal myocytes through inhibition of DNA topoisomerase II. 1198 69

Merbarone is a catalytic inhibitor of topoisomerase II (topo II) that has been proposed to act primarily by blocking topo II-mediated DNA cleavage without stabilizing DNA-topo II-cleavable complexes. In this study merbarone was used as a model compound to investigate the genotoxic effects of catalytic inhibitors of topo II. The clastogenic properties of merbarone were evaluated using in vitro and in vivo micronucleus (MN) assays combined with CREST staining. For the in vitro MN assay, ICRF-187, a different type of catalytic inhibitor, and etoposide, a topo II poison, were used for comparison. Treatment of TK6 cells with all three of these drugs resulted in highly significant dose-related increases in kinetochore-lacking MN and, to a lesser extent, kinetochore-containing MN. In addition, a good correlation between p53 accumulation and MN formation was seen in the drug-treated cells. A mouse MN assay was performed to confirm that similar DNA-damaging effects would occur in vivo. Bone marrow smears from merbarone-treated B6C3F1 mice showed a dose-related increase in micronucleated polychromatic erythrocytes with a mean of 26 MN per 1000 cells being seen at the 60 mg/kg dose. Almost all MN lacked a kinetochore signal, indicating that merbarone was predominantly clastogenic under these conditions in vivo. The present study clearly shows that merbarone is genotoxic both in vitro and in vivo, and demonstrates the inaccuracy of earlier statements that merbarone and other catalytic inhibitors block the enzymatic activity of topo II without damaging DNA.
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PMID:Catalytic inhibitors of topoisomerase II are DNA-damaging agents: induction of chromosomal damage by merbarone and ICRF-187. 1211 87

We have synthesized two podophyllotoxin-acridine conjugates-pACR6 and pACR8. In these compounds an 9-acridinyl moiety is beta linked to the C4 carbon of the four ring system in 4'-demethylepipodophyllotoxin (epiDPT) via eighter an N-6-aminohexanylamide linker (pACR6) or via an N-8-aminooctanylamide linker containing two more carbon atoms (pACR8). The acridine-linker moiety occupies the position where different glucoside moieties, dispensable for activity, are normally linked to epiDPT in the well known epipodophyllotoxins VP-16 and VM-26. As with VP-16 and VM-26, pACR6 and pACR8 show evidence of being topoisomerase II poisons as they stimulate topoisomerase II mediated DNA cleavage in vitro and induce DNA damage in vivo. This in vivo DNA damage, as well as pACR6/pACR8 mediated cytotoxicity, is antagonized by the catalytic topoisomerase II inhibitors ICRF-187 and aclarubicin, demonstrating that topoisomerase II is a functional biological target for these drugs. Despite their structural similarities, pACR6 was more potent than pACR8 in stimulating topoisomerase II mediated DNA cleavage in vitro as well as DNA damage in vivo and pACR6 was accordingly more cytotoxic towards various human and murine cell lines than pACR8. Further, marked cross-resistance to pACR6 was seen among a panel of multidrug-resistant (MDR) cell lines over-expressing the MDR1 (multidrug resistance protein 1) ABC drug transporter, while these cell lines remained sensitive towards pACR8. pACR8 was also capable of circumventing drug resistance among at-MDR (altered topoisomerase II MDR) cell lines not over-expressing drug transporters, while pACR6 was not. Two resistant cell lines, OC-NYH/pACR6 and OC-NYH/pACR8, were developed by exposure of small cell lung cancer (SCLC) OC-NYH cells to gradually increasing concentrations of pACR6 and pACR8, respectively. Here, OC-NYH/pACR6 cells were found to over-express MDR1 and, accordingly, displayed active transport of 3H-labeled vincristine, while OC-NYH/pACR8 cells did not, further suggesting that pACR6, but not pACR8, is a substrate for MDR1. Our results show that the spatial orientation of podophyllotoxin and acridine moieties in hybrid molecules determine target interaction as well as substrate specificity in active drug transport.
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PMID:Linker length in podophyllotoxin-acridine conjugates determines potency in vivo and in vitro as well as specificity against MDR cell lines. 1237 83


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