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)

Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II alpha and beta isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II alpha are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II beta is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II alpha present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II alpha content is only slightly decreased. On the other hand, the great majority of topoisomerase II beta is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topoisomerase types.
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PMID:Discrete localization of different DNA topoisomerases in HeLa and K562 cell nuclei and subnuclear fractions. 829 28

The topology of DNA is regulated by DNA-topoisomerase enzymes, which induce either transient DNA single-strand breaks (topoisomerase I) or DNA double-strand breaks (topoisomerase II). The action of several anticancer drugs, eg, DNA intercalating agents (ie, anthracyclines, anthracenediones, anthrapyrazoles, amsacrines, and ellipticines) and epipodophyllotoxins, appears to be mediated by the enzyme topoisomerase II alpha. The action of camptothecins is mediated by topoisomerase I. All of these drugs cause the induction of DNA enzyme complexes. Some new topoisomerase inhibitors have instead the ability to inhibit the catalytic activity of the enzyme without inducing the formation of complexes. Inhibition of transcription and DNA replication have been implicated as the most likely mechanisms of cytotoxicity. The drugs stimulate DNA cleavage with a certain degree of sequence specificity, which is typical for each drug or class of drugs. The differences in the location of the cleavage sites may explain the different pharmacologic effects of the various topoisomerase inhibitors. Cellular resistance to topoisomerase inhibitors is due to decreased intracellular drug retention (eg, overexpression of P-170), to a decreased level of topoisomerase enzymes, or to mutations that reduce the drug sensitivity of the enzymes. Synergistic activity of the combination of topoisomerase I and topoisomerase II inhibitors or of topoisomerase inhibitors and radiotherapy has been found to be highly dependent on the sequence used.
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PMID:DNA-topoisomerase inhibitors. 830 35

A drug-resistant human small cell lung cancer cell line, H209/V6, selected in the presence of increasing concentrations of 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16) from parental H209 cells, is 22-, 9-, and 4-fold resistant to VP-16, 4'-(9-acridinyl-amino)methanesulfon-m-anisidide, and doxorubicin, respectively, but not cross-resistant to 1,4-dihydroxy-5,8-bis((2-[(2-hydroxyethyl)amino] ethyl]-amino)-9,10-anthracenedione. These cells do not overexpress P-glycoprotein or the multidrug resistance-associated protein. Immunoblotting demonstrates that H209 cells contain the M(r) 170,000 isoform of topoisomerase II (topo II), while H209/V6 cells have a M(r) 160,000 enzyme but none of the M(r) 170,000 isoform. The cell lines have equal amounts of topo II beta. The H209/V6 cells have a 5-fold decrease in total immunoreactive topo II alpha. The catalytic and VP-16-induced DNA cleavage activities of the topo II present in 0.35 M NaCl nuclear extracts are decreased 2- to 3-fold in the drug-resistant cell line. This decrease in enzymatic activity is not consistent with either the 22-fold VP-16 resistance of the H209/V6 cell line or the approximately 5-fold decrease in immunoreactive topo II alpha in the cells. The M(r) 160,000 isoform from the H209/V6 cell line and the M(r) 170,000 enzyme from the parental cell line were purified so that the enzymatic activity of the 2 isoforms could be evaluated. The catalytic activities of the purified isoforms were found to be very similar. The drug-induced DNA cleavage activity of the M(r) 160,000 enzyme was reduced compared to the M(r) 170,000 enzyme. However, as with the nuclear extracts, the differences in enzymatic activity of the purified enzymes are considerably less than the level of drug resistance. Investigations of the subcellular localization of topo II by immunocytochemical techniques and cytoplasm/nuclear fractionation studies demonstrated that the M(r) 160,000 topo II alpha-related enzyme is primarily localized in the cytoplasm, while the M(r) 170,000 topo II alpha enzyme and topo II beta are located in the nucleus. These data imply that the deleted sequence in the M(r) 160,000 enzyme is not necessary for catalytic activity but is required to facilitate nuclear localization.
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PMID:Altered subcellular distribution of topoisomerase II alpha in a drug-resistant human small cell lung cancer cell line. 830 38

We have studied the genetic alterations acquired during selection of a cloned human leukaemic cell line (CEM/VP-1) that is 15-fold more resistant to the anticancer topoisomerase II-inhibitor etoposide than parental CCRF-CEM cells. CEM/VP-1 cells exhibit an 'atypical MDR' phenotype: cross resistance to other topo II inhibitors (but not Vinca alkaloids) and expression of a drug-resistant topo II activity. Cytogenetic and molecular studies revealed that the cell line carried multiple genetic changes affecting TOP2 genes encoding both topo II alpha and beta isoforms. CEM/VP-1 was diploid, 47,XX,+20, and appears to have been preferentially selected from a 1% diploid subpopulation present in the tetraploid parental cells. The same chromosomal abnormalities were present in resistant and sensitive cells except for an acquired 3p- change most likely deleting one TOP2 beta allele. PCR/DNA sequence analysis and allele-specific hybridisation showed that one of two TOP2 alpha alleles expressed in CEM/VP-1 cells had acquired a Lys-797-->Asn codon change. This mutation lies close to the catalytic Tyr-804 residue of the protein and may interfere with drug-induced trapping of the cleavable complex. Alternatively, it could exert a loss of function phenotype. CEM/VP-1 cells did not exhibit codon 449 or 486 TOP2 alpha mutations in the ATP binding domain reported in two other resistant cell lines. Diploid selection and multiple changes observed in CEM/VP-1 cells appear to be consequences of the recessive phenotype of at-MDR. These results may be useful in approaching the mechanisms of clinical resistance.
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PMID:Novel selection and genetic characterisation of an etoposide-resistant human leukaemic CCRF-CEM cell line. 838 8

DNA topoisomerase (topo) II mediates DNA strand passage in an ATP-dependent reaction. Human cell lines express at least two genetically distinct forms of the enzyme, topo II alpha (p170) and II beta (p180). Previously, we isolated a novel HeLa cDNA clone (CAA5) that partially encodes a protein homologous to topo II alpha (Austin, C.A. and Fisher, L.M. (1990) FEBS Lett. 266, 115-117). In this paper we show that CAA5 encodes a C-terminal segment of human topo II beta. We report here for the first time cDNA clones spanning the entire coding sequence. Overlapping clones specifying the 3' end of the cDNA have been isolated, mapped and sequenced. The missing 5' coding sequence was obtained by an inverse PCR protocol and from a specifically primed cDNA library. Human topo II beta is a 1621 amino acid protein which is closely homologous to topo II alpha in the N-terminal three quarters of its sequence. In contrast, the C-terminal segments of the alpha and beta sequences show considerable divergence suggesting these regions may mediate different cellular functions of the two isoforms. Southern blot analysis of yeast and Drosophila DNA using human alpha and beta specific probes detected a single topo II homologue in these lower eukaryotes. Comparison of the protein sequence for human topo II beta with other type II topoisomerases revealed several conserved motifs and has allowed identification of the likely ATPase- and DNA breakage-reunion domains.
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PMID:Novel HeLa topoisomerase II is the II beta isoform: complete coding sequence and homology with other type II topoisomerases. 838 37

There is accumulating evidence that both type I and type II DNA-topoisomerases play a key role in cellular differentiation. Human HL-60 leukemia cells can be induced to monocytic or granulocytic differentiation with various compounds; amongst them camptothecin, a topoisomerase I inhibitor and VP-16, VM-26 and mitoxantrone, all potent topoisomerase II inhibitors. During HL-60 cell differentiation topoisomerase I activity increases and topoisomerase II activity decreases. The two isoenzymes topoisomerase II alpha and topoisomerase II beta seem to have different physiological functions in highly proliferating cells, G0 cells and differentiated cells as their expression is regulated differently. In concentrations sublethal to undifferentiated cells, m-AMSA, also a potent topoisomerase II inhibitor, is able to prevent DMSO-induced granulocytic HL-60 cell differentiation. In drug-sensitive cells derived from several sources (mouse erythroleukemia, human gastric carcinoma, human leukemia), we found a functional heterogeneity of topoisomerase activity which is altered specifically during cellular differentiation. The isoactivities can be separated by their different pH and salt requirements and they exhibit different sensitivity to topoisomerase II inhibiting drugs. Functional heterogeneity of topoisomerases seems to be a prerequisite to high drug sensitivity of the cells, since drug-resistant sublines in our experiments do not exhibit this heterogeneity. We propose that the topoisomerase II inhibiting drugs which are able to induce differentiation, namely the epipodophyllotoxines VP-16 and VM-26, inhibit subfractions of the topoisomerase II pool which are necessary to maintain the undifferentiated status of the cells. These drugs induce differentiation in concentrations 10-100-fold below the lethal dose, the concentration must be sufficient to inhibit topoisomerase II but well below the concentration to induce the cleavable complex. This might be the reason that anthracyclines with a high DNA binding affinity have low differentiation-inducing capacity.
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PMID:Correlation between the DNA-binding affinity of topoisomerase inhibiting drugs and their capacity to induce hematopoetic cell differentiation. 838 90

Many cytotoxic anticancer drugs act at topoisomerase II (topo II) by stabilizing cleavable complexes with DNA formed by this enzyme. Several cell lines, selected for resistance to topo II-interactive drugs, show decreased expression or activity of topo II, suggesting that such a decrease may be responsible for drug resistance. In the present study, etoposide resistance was used as the selection strategy to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human topo II (alpha form) cDNA. Twelve GSEs were isolated, encoding either peptides corresponding to short segments of the topo II alpha molecule (2.4-6.5% of the protein) or 163- to 220-bp-long antisense RNA sequences. Expression of a GSE encoding antisense RNA led to decreased cellular expression of the topo II alpha protein. Both types of GSE induced resistance to several topo II poisons but not to drugs that do not act at topo II. These results provide direct evidence that inhibition of topo II results in resistance to topo II-interactive drugs, indicate structural domains of topo II capable of independent functional interactions, and demonstrate that expression selection of random fragments constitutes an efficient approach to the generation of GSEs in mammalian cells.
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PMID:Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA. 838 68

Using reverse transcription polymerase chain reaction, we determined mRNA expression of topoisomerase (topo) II alpha and beta in adriamycin- and etoposide-resistant small cell lung cancer sublines, SBC-3/ADM 100 and SBC-3/ETP. The expression of topo II alpha mRNA decreased substantially in SBC-3/ADM 100 and SBC-3/ETP as compared with the parent cell line, SBC-3; 0.71-fold in the former and 0.38-fold in the latter. Similarly, that of topo II beta mRNA decreased to an extent of 0.68-fold in SBC-3/ADM 100 and 0.28-fold in SBC-3/ETP as compared with the parent cell line. SBC-3/ADM 100 and SBC-3/ETP were highly resistant to topo II inhibitors such as daunorubicin, epirubicin, pirarubicin, mitoxantrone, and teniposide. However, SBC-3/ADM 100 showed a less resistance to aclarubicin, and SBC-3/ETP was as sensitive to the drug as was in the parent cell line. The resistance to topo II inhibitors excluding for aclarubicin might be partially explained by the decreased expression of topo II alpha and beta mRNA.
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PMID:[Cytotoxic effect of topoisomerase II inhibitors against adriamycin- and etoposide-resistant small cell lung cancer sublines]. 838 62

cDNA segments for DNA topoisomerase II were amplified from rat brain RNA after reverse transcription by the polymerase chain reaction, using degenerate oligonucleotide primers deduced from the conserved regions of topoisomerase II of higher eukaryotes. The cDNA product from a successful amplification was homogeneous in length but heterogeneous in sequence. Restriction mapping of the cloned cDNA fragments revealed that they consisted of two distinct sequence groups. DNA sequencing of representative clones from each group, designated A and B, showed that they are highly homologous to cDNAs of human topoisomerase II isoforms, alpha and beta, respectively. Northern blot analysis indicated that the transcript level for rat topoisomerase II alpha was high in embryonic brain and in the cerebellum of 2-day newborns, followed by rapid decrease to a undetectable level at 4 weeks after birth. In contrast, rat topoisomerase II beta transcript was present throughout the embryonic and postnatal stages. In the developing cerebellum, cells expressing topoisomerase II alpha were confirmed exclusively to the outer mitotic zone of the external granular layer, whereas the transcript of topoisomerase II beta was detected over the entire cortical region. These results clearly indicate that the isoform alpha is expressed only in proliferating cells. The differential expression of topoisomerase II isozymes was also observed among developed tissues. Therefore, the isozymes are most likely to be involved in the following different physiological processes: topoisomerase II alpha in cell proliferation, and topoisomerase II beta in some processes unrelated to cell proliferation.
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PMID:Molecular cloning of partial cDNAs for rat DNA topoisomerase II isoforms and their differential expression in brain development. 839 28

Human topoisomerase II enzymes are targets for a number of widely used anticancer agents. We have analysed a lung adenocarcinoma cell line CALU3, which has co-amplified topoisomerase II alpha and ERBB2 sequences, for the structure of the amplicon and for expression of both topoisomerase II alpha and beta. The region of chromosome 17q amplified in CALU3 also includes the retinoic acid receptor alpha locus and is therefore similar to the amplicon observed in breast cancers carrying amplified topoisomerase II alpha and retinoic acid receptor sequences. The use of fluorescence in situ hybridisation localises the amplified topoisomerase II alpha sequences to a cluster on one chromosome with single copies localised to others. CALU3 express high levels of topoisomerase II alpha is determined by Western blot, immunofluorescence and enzyme activity. The enzyme activity extracted from CALU3 is sensitive to inhibition by the topoisomerase II poison etoposide. Topoisomerase II beta expression was observed in three lung cancer cell lines including CALU3 and was confined to the nucleoli. Thus, the CALU3 cell line is an ideal model to study the amplification and expression of topoisomerase II alpha in adenocarcinomas.
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PMID:Expression of topoisomerase II alpha and beta in an adenocarcinoma cell line carrying amplified topoisomerase II alpha and retinoic acid receptor alpha genes. 839 10


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