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)

Western blotting experiments with topoisomerase II antiserum were performed to measure type II topoisomerase content in human tissues. In this study, antiserum produced against the C-terminus of recombinant HeLa topoisomerase II recognized both p170 and p180 forms of the enzyme. Antisera specifically made against p180 selectively recognized that enzyme form. By using these antisera, a remarkable difference in topoisomerase content between the tissues studied was observed. Tissues with a high proportion of cycling cells contained predominantly the p170 form of the enzyme, but also smaller amounts of p180. In terminally differentiated organs or in neoplasms with a low percentage of cycling cells, topoisomerase II immunoprotein was not detected in whole tissue extracts. In term human placenta, this was because this tissue contained predominantly the p180 enzyme form, yet the concentration of this form of the enzyme was apparently in too low a concentration to be detected by the antibody in crude extracts. It was readily observed after concentration by chromatography on hydroxylapatite.
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PMID:The distribution of immunoreactive topoisomerase II Protein in human tissues and neoplasms. 132 31

The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides. Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms. Less homology was seen with the Drosophila, yeast and bacterial type II enzymes. Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme alpha. Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity.
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PMID:Structure and partial amino acid sequence of calf thymus DNA topoisomerase II: comparison with other type II enzymes. 169 76

Murine leukemia L1210 cells selected for progressive resistance to doxorubicin (DOX) display both the multidrug resistant (MDR) phenotype and reductions in drug induced topoisomerase II-mediated DNA cleavage in nuclear extracts (Ganapathi, R.; Grabowski, D.; Ford, J.; Heiss, C.; Kerrigan, D.; Pommier, Y., Cancer Commun. 1:217-224; 1989). The present study was performed to characterize the results of exposure of the sensitive (S) and progressively DOX-resistant (10-fold, R1, and 40-fold, R2) L1210 cells to the topoisomerase II inhibitor, etoposide, and to investigate the modulating effects of the calmodulin inhibitor, trifluoperazine (TFP). Immunoblotting experiments indicated no apparent decrease in the p170 or p180 isoforms of topoisomerase II in the resistant sublines versus parental sensitive cells. Cross-resistance to etoposide (VP-16) was similar to that of DOX (10- and 40-fold). A non-cytotoxic concentration of 5 microM TFP enhanced cell kill 1.5- fold in the sensitive and 3- to 5-fold in the progressively DOX-resistant cells. Accumulation of VP-16 was 30% to 50% lower in the resistant sublines versus similarly treated sensitive cells, and a marked enhancement of drug uptake in the presence of TFP was observed in the sensitive but not in the resistant cells exposed to equivalent extracellular levels of VP-16. Although equimolar concentrations of VP-16 produced fewer DNA single strand breaks (SSB) and DNA protein crosslinks (DPC) in the resistant versus sensitive cells, similar DNA damage was apparent when S and R1, but not R2, cells were treated at VP-16 concentrations that produced equivalent cell death.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Trifluoperazine modulation of resistance to the topoisomerase II inhibitor etoposide in doxorubicin resistant L1210 murine leukemia cells. 199 27

The p170 and p180 forms of topoisomerase II have been compared. The concentration dependence of ATP for catalytic activity of the two forms of the enzyme was identical, and each was equally sensitive to novobiocin. Orthovanadate was found to be a potent inhibitor of catalytic activity of both p170 and p180, with an IC50 value of about 2 microM for each. Under standard reaction conditions, relaxation of supercoiled pBR322 by p180 was highly processive, while p170 performed the same reaction in a distributive manner. The optimal concentration of KCl for catalytic activity of p180 was 20-30 mM higher than that for p170. Comparison of their thermal stability showed that p180 was inactivated at twice the rate of p170. Teniposide and merbarone selectively inhibited catalytic activity of p170, requiring concentrations 3-fold and 8-fold lower, respectively, than those required for equivalent inhibition of p180. Similar selectivity for p170 was seen for teniposide-stimulated DNA cleavage or its inhibition by merbarone. Analysis of sites of DNA cleavage indicated a subset of sites that were either preferred or unique for each of the enzymes. A synthetic oligonucleotide representative of p170 sites selectively inhibited the p170 enzyme. Immunoblotting of p170 and p180 from U937 cells at different stages of proliferation showed that p170 levels declined as the cells reached the plateau phase of growth, while p180 levels were low during rapid proliferation and increased as the growth rate slowed. The data indicate that the p170 and p180 forms of topoisomerase II can be distinguished biochemically, pharmacologically, and by differential cellular regulation.
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PMID:Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II. 255 97

Topoisomerase II was purified from an amsacrine-resistant mutant of P388 leukemia. A procedure has been developed which allows the rapid purification of nearly homogeneous enzyme in quantities sufficient for enzyme studies or production of specific antisera. The purified topoisomerase II migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with apparent molecular masses of 180 (p180) and 170 kDa (p170); both proteins unknotted P4 DNA in an ATP-dependent manner and displayed amsacrine-stimulated covalent attachment to DNA. Staphylococcus V8 protease cleavage patterns of p170 and p180 showed distinct differences. Specific polyclonal antibodies to either p170 or p180 recognized very selectively the form of the enzyme used to generate the antibodies. Immunoblotting with these specific antibodies showed that both p180 and p170 were present in cells lysed immediately in boiling sodium dodecyl sulfate. Comparison of the purified topoisomerase II from amsacrine-resistant P388 with that from amsacrine-sensitive P388 demonstrated that each cell type contained both p180 and p170; however, the relative amounts of the two proteins were consistently different in the two cell types. The data strongly suggest that p170 is not a proteolytic fragment of p180. Thus, P388 cells appear to contain two distinct forms of topoisomerase II.
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PMID:Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme. 282 4

Human cell lines express two genetically distinct isoforms of DNA topoisomerase (topo II) II: topo II alpha (p170) and topo II beta (p180). We detected a higher molecular weight form with an apparent molecular mass of about 190 kDa in M phase-arrested HeLa cells (Kimura, K., Saijo, M., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 1173-1176). In this study we confirmed, using anti-topo II alpha and topo II beta monoclonal antibodies, that this higher molecular weight form is topo II beta and consists of doublet bands around 190 kDa. We confirmed that the doublet bands constituted an M phase-specific phenomenon and were not an artifact of the procedure used to accumulate mitotic cells. Digesting the immunoprecipitated materials from mitotic cell extracts with alkaline phosphatase resulted in the disappearance of the doublet bands and the appearance of the 180-kDa band with the concomitant disappearance of 32P label in the region of the doublet bands. Neither heat-inactivated alkaline phosphatase nor phosphodiesterase affected the doublet bands and the 32P label. Topo II beta in interphase cells was also phosphorylated, but the shift in apparent molecular weight was very slight after alkaline phosphatase digestion. Analysis of the labeled phosphoamino acids present in topo II beta from M phase and logarithmically growing cells indicated that phosphorylation occurred mainly on serine and fairly on threonine residues in both topo II beta isoforms. These results indicated that topo II beta is phosphorylated at specific sites in M phase, resulting in the formation of the doublet bands.
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PMID:Identification of the nature of modification that causes the shift of DNA topoisomerase II beta to apparent higher molecular weight forms in the M phase. 792 18

Endogenous host topoisomerase II acts upon herpes simplex virus type 1 (HSV-1) DNA in infected cells (S.N. Ebert, S.S. Shtrom, and M.T. Muller, J. Virol. 56:4059-4066, 1990), and cleavage is directed exclusively at progeny viral DNA while parental DNA is resistant. To evaluate the possibility that HSV-1 induces topoisomerase II activity which could account for the preferential cleavage of progeny viral DNA, we assessed topoisomerase II cleavage activity on cellular and viral DNA substrates before and after the initiation of viral DNA replication. We show that cleavage of a host gene in mock-infected cells was similar to that observed in HSV-1-infected cells, regardless of whether viral DNA replication had occurred. In addition, quantitative measurements revealed comparable amounts of topoisomerase II activity in infected and mock-infected cells; thus, HSV-1 neither induces nor encodes its own type II topoisomerase and cleavages in vivo are due to a preexisting host topoisomerase. Human cells contain two isozymes of topoisomerase II (p170 and p180), encoded by separate genes. Through the use of isozyme-specific antibodies, we demonstrate that only p170 was found to be cross-linked to HSV-1 DNA even though both forms were present at nearly constant levels in HSV-1-infected cells. Immunofluorescence revealed that by 6 h postinfection, p170 becomes redistributed and localized to sites of active viral DNA synthesis. The data suggest that p170 gains preferential access to replicated viral DNA molecules, which explains why topoisomerase II activity is concentrated on progeny DNA.
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PMID:Association between the p170 form of human topoisomerase II and progeny viral DNA in cells infected with herpes simplex virus type 1. 828 31

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

To further define the nucleic acid determinants of DNA site recognition by mammalian topoisomerase II, base mismatch effects on the enzyme DNA cleavage activity were determined in a 36-bp synthetic oligonucleotide corresponding to SV40 DNA. DNA cleavage sites induced by topoisomerase II without or with the antitumor drugs teniposide, idarubicin, or amsacrine were mapped using sequencing gels. Selected mismatches were studied, and always one of the two strands had the wild-type sequence. The effects of base mismatches were independent from the studied drugs. Mismatches introduced at the -4, -3, -2, or -1 positions, relative to the enzyme cleavage site, often abolished, or much reduced, DNA cleavage, whereas those at +1 and +2 positions often increased DNA breakage or were without influence. Mismatches at more distant positions, e.g., -7, -8, etc., had no effect. Those at positions -5 and -6 sometimes increased cleavage levels. These effects were always observed at sites already cleaved in the wild-type oligomer; new sites of cleavage were not induced by the studied mismatches. These results were obtained both for the native murine topoisomerase II and for the two recombinant human isozymes. No difference between topoisomerases II alpha(p170) and beta(p180) was seen in their response to mismatches. The results demonstrate that topoisomerase II recognition of the DNA site of cleavage requires fully paired nucleotides at the 3' terminus. Nevertheless, similarly to other DNA strand transferase enzymes, both topoisomerase II isoforms may have a sequence-specific nicking activity at the 5' side of unpaired bases.
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PMID:Position-specific effects of base mismatch on mammalian topoisomerase II DNA cleaving activity. 855 69