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)

Human cells contain two topoisomerase II isozymes named topo II alpha and topo II beta. The complementary DNAs for both enzymes have been cloned. The topo II alpha and topo II beta complementary DNAs hybridized to unique sequences of human, rodent, and chicken DNAs in Southern blots. The human topo II alpha gene has previously been mapped to chromosome 17. We confirmed the chromosomal location of topo II alpha and mapped the topo II beta gene to chromosome 3. In addition, topo II beta exhibits genetic polymorphism as has been reported for topoisomerases I and II alpha.
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PMID:Topoisomerase II alpha and topoisomerase II beta genes: characterization and mapping to human chromosomes 17 and 3, respectively. 130 26

The cellular content and the cell-cycle distribution of the 170 kD- and 180 kD-isoforms of DNA topoisomerase II were investigated in human tumor cells with specific monoclonal antibodies and by immunofluorescent detection with flow cytometry. Levels of topo II alpha were almost three-fold higher than the beta-isozyme in exponentially growing cells in vitro. In contrast, topo II alpha but not beta, was markedly reduced in plateau-phase cells. Tumor cells from surgical biopsies, mainly in G0/G1 phase, exhibited a 95% beta- versus 5% alpha-isoform expression. These results support the hypothesis that topo II alpha is mainly related to DNA synthesis, and topo II beta to DNA transcription.
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PMID:Topoisomerase II alpha and beta in human tumor cells grown in vitro and in vivo. 133 75

We have examined features of DNA topoisomerase II (topo II) isoforms in human leukemic CCRF-CEM cells and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5. They are about 40- to 50-fold and 150- to > 400-fold resistant to teniposide, respectively, and have increased levels of cross-resistance to other complex-stabilizing topo II inhibitors. Topo II activity in these lines is reduced in proportion to their resistance. However, both sublines carry two identical point mutations in topo II alpha cDNA that have recently been found to confer resistance to etoposide and m-AMSA in transfected yeast cells. Although these data provide a strong molecular basis for this type of multidrug resistance, these findings alone cannot explain the increased level of resistance and cross-resistance, and the further decreased cellular topo II activities in the most resistant CEM/VM-1-5 cells compared to CEM/VM-1 cells. In this study we found that (1) topo II beta is not expressed in CEM/VM-1-5 cells; (2) a 160-kDa protein was consistently detected and coimmunoprecipatated only in nuclear extracts of CEM cells; (3) when nuclear extracts from all three cell lines were incubated at 37 degrees C, an immunoreactive band of 140-kDa appeared by 60-90 min only in samples of CEM cells, not in those of CEM/VM-1 and CEM/VM-1-5 cells; and (4) the in vivo phosphorylation level of topo II alpha protein was decreased > or = 2-fold in both resistant cell lines, compared to that of CEM cells. Thus, cell lines selected for the altered topo II-associated multidrug resistance phenotype may contain multiple alterations in both topo II isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:DNA topoisomerase II expression, stability, and phosphorylation in two VM-26-resistant human leukemic CEM sublines. 757 26

Topoisomerase (topo) inhibitors induce enzyme-linked DNA breaks. Resulting DNA damage can lead to cell cycle arrest and/or cell death by apoptosis. The sensitivity of five human leukemic cell lines to topo I (camptothecin or CPT) and topo II (etoposide or VP-16) inhibitors varied widely (100-fold for CPT and 30-fold for VP-16). Three cell lines were more sensitive (BV173, HL60, U937) and two cell lines were resistant (K562, KCL22) to both drugs. None of these cell lines were selected for drug resistance and overexpressed mdr1 gene. Their sensitivity was not related to their doubling time nor to cell cycle repartition. The initial DNA damage (cleavable complexes) induced by topo I and II inhibitors was measured as DNA-protein crosslinks (DPC) using alkaline elution. Neither DPC level induced by 30-min treatment with CPT or VP-16 nor the levels of topo 1, topo II alpha and topo II beta mRNA were related to sensitivity. Electron microscopy and DNA fragmentation measured by filter elution and agarose gel electrophoresis demonstrated that apoptosis was induced by both drugs in the five cell lines. The kinetics of DNA fragmentation was related to cell sensitivity. At drug concentrations higher than IC50, DNA fragmentation increased very rapidly in the three sensitive, compared with the two resistant, cell lines. Continuous exposure to both drugs induced cell cycle arrest in either G2 or S phase that was related both to cell sensitivity and drug concentration. Comparison between cell lines indicated that the ability of cells to arrest cell cycle in G2 or S phase was related to their drug sensitivity and increased with cell resistance. In a given cell line, cell cycle progression was observed to be progressively inhibited by increasing drug concentrations. Treatment of synchronized cells demonstrated that highly cytotoxic drug concentration induced a complete inhibition of cell cycle progression. Altogether, these data suggest that the ability of leukemic cell lines to regulate cell cycle progression and to trigger apoptosis is more indicative of their sensitivity to topoisomerase poisons than cleavable complexes induced by these drugs.
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PMID:The role of cell cycle regulation and apoptosis triggering in determining the sensitivity of leukemic cells to topoisomerase I and II inhibitors. 759 66

The interaction of topoisomerase II (topo II) with human ribosomal DNA (rDNA) was investigated in vivo using the antitumoral drug VM26, a specific inhibitor of topo II, that stabilizes the transient cleavable complex. rDNA-protein complexes isolated from nucleoli of TG cells were analyzed for double strand breaks with probes that covered almost all intergenic transcribed spacer (IGS) and all transcribed sequences of tandem repeat ribosomal DNA genes. Preferential cleavage sites were present in only a part of nucleolar rDNA, i.e., the transcribed region. Proteins, purified from the same complexes, were analyzed by Western-blot and stained by an antiserum against both topo II forms, showing the presence of topo II beta.
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PMID:Topoisomerase-II-mediated DNA cleavage within the human ribosomal genes. 763 46

We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoisomerase II (topo II), the isoform predominant in proliferating cells. As a prerequisite to making this discovery, it was necessary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isolation procedures. Immunofluorescence analysis of the large chromosomes prepared from Indian Muntjac cells revealed colocalization of MPM-2 and anti-topo II antibodies to the chromosomal centromeres and to the axial regions of the chromosomal arms. For biochemical fractionation studies, large quantities of chromosomes from the P388D1 mouse lymphocyte cell line were isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold fraction identified the major MPM-2-reactive phosphoprotein to be DNA topo II. Using a panel of anti-peptide antibodies specific to the isoforms of topo II, we determined that the major phosphoprotein recognized by MPM-2 is the 170-kDa isoform of topo II, topo II alpha. The 180-kDa isoform, topo II beta, present in the isolated chromosomes in much smaller quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo II proteins may be critical for proper chromosome condensation and segregation.
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PMID:DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2. 769 Sep 61

A human HL-60 leukemia cell line selected for resistance to mitoxantrone, HL-60/MX2, displays cross-resistance only to agents whose cytotoxicities result from interaction with the nuclear enzyme DNA topoisomerase II (topo II). The topo II catalytic activity is reduced 2-fold in the drug-resistant cell line in association with the absence of the M(r) 180,000 isoform of topo II and the finding of novel M(r) 160,000 topo II alpha-related immunoreactive protein in these cells by immunoblot. The topo II alpha (M(r) 170,000) protein levels in nuclear extracts from the HL-60/MX2 cells were noted on average to be approximately 40% lower than in comparable HL-60 nuclei. Studies of the subcellular localization of topo II by immunohistochemical and fractional extraction techniques demonstrated that the M(r) 160,000 topo II alpha-related protein is primarily localized in the cytoplasm. Levels of the 6.3-kilobase topo II alpha mRNA were noted to be reduced 2-fold in the HL-60/MX2 cells in association with the finding of a novel 4.8-kilobase topo II alpha-related mRNA transcript that was present in HL-60/MX2 but not HL-60 cells. The absence of topo II beta protein in nuclear and whole cell extracts from the HL-60/MX2 cells was associated with the virtual absence of detectable topo II beta mRNA in those cells by Northern blot analysis. Using a reverse transcription-PCR assay we were able to demonstrate the presence of very low levels of topo II beta mRNA in HL-60/MX2 cells, representing < 1% of that found in the HL-60 cells. In contrast, the nuclear catalytic activity and cellular mRNA levels of the related nuclear enzyme DNA topoisomerase I were nearly identical in the two cell types. Southern blot analysis of DNA extracted from the drug-sensitive and drug-resistant cells revealed a structural alteration in one topo II alpha allele in the HL-60/MX2 cells, but there was no evidence of rearrangement or hypermethylation of the topo II beta locus. These results indicate that the reduced levels of topo II alpha and beta isoenzymes observed in mitoxantrone-resistant HL-60/MX2 cells are related to changes in the levels of their respective mRNA transcripts. The identification of structural changes in one topo II alpha allele in the HL-60/MX2 cell line suggests that the altered allele may serve as the source of the unique 4.8-kilobase topo II alpha-related mRNA transcript and the M(r) 160,000 protein discovered in those cells.
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PMID:Alterations in the topoisomerase II alpha gene, messenger RNA, and subcellular protein distribution as well as reduced expression of the DNA topoisomerase II beta enzyme in a mitoxantrone-resistant HL-60 human leukemia cell line. 771 79

The binding activities of the 170 kDa and the 180 kDa human topoisomerases II (topo II alpha and topo II beta) to linear DNA fragments with different degrees of curvature were characterized. In gel retardation experiments it was shown that both forms of the enzyme bind preferentially to a curved 287 bp fragment, forming a detectable stable complex. The affinity for straight DNA fragments of similar length is significantly lower. Both a commercially available topo II alpha, isolated from placenta, and topo II alpha and topo II beta purified from nuclear extracts of the Namalwa lymphoma tissue culture line gave similar results. The effects of double-stranded poly[d(A-T)], poly[d(G-C)], supercoiled plasmid DNA and linear Z-DNA on the topo II-complex with curved DNA were analyzed in competition experiments. The hierarchy of affinities of the 180 kDa topo II beta for these DNAs has the order: linear left-handed DNA > supercoiled DNA > or = curved DNA >> poly[d(A-T)] > poly[d(G-C)]. The 170 kDa topo II alpha binds with similar affinity to curved DNA and linear Z-DNA > or = supercoiled DNA >> linear B-DNA. The data imply that human topoisomerase II binding is more sensitive to DNA secondary structure than to DNA sequence per se. The ability of the enzyme to preferentially recognize a wide variety of sequences in unusual secondary structures suggests a mode of targeting the enzyme in vivo to regions of high negative supercoiling.
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PMID:Human 170 kDa and 180 kDa topoisomerases II bind preferentially to curved and left-handed linear DNA. 772 61

Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC4-ADR150 displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation. Not the MDR1 gene, but the MRP gene, was over-expressed in this cell line. The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin. To address this question, changes in expression of topo II alpha/topo II beta, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant). Topo II alpha and II beta mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line. Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance. MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC4-ADR150 cells). Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines. Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines. This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC4 cells upon adriamycin selection.
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PMID:Resistance-associated factors in human small-cell lung-carcinoma GLC4 sub-lines with increasing adriamycin resistance. 772 50

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


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