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)

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

Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.
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PMID:Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. 133 83

DNA was prepared from normal tissue and 19 lung cancer cell lines. Using probes which detect restriction fragment length polymorphisms at both the topoisomerase II alpha and beta loci, heterozygosity was detected at a frequency of 0.17 and 0.37 for the alpha and beta loci, respectively. Southern blot analysis of DNA extracted from lung cancer cell lines detected amplification of both the topoisomerase II alpha and ERBB2 genes in the adenocarcinoma line Calu3. These results indicate that topoisomerase II alpha and ERBB2 may be closely linked on chromosome 17 and coamplified during adenocarcinoma progression. Since topoisomerase II is a target for several anticancer drugs, it will be of interest to study alterations to topoisomerase II genes during tumour development, as these may in part determine the response of the tumour to chemotherapy.
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PMID:Amplification of the topoisomerase II alpha gene in a non-small cell lung cancer cell line and characterisation of polymorphisms at the human topoisomerase II alpha and beta loci in normal tissue. 137 18

Topoisomerases are essential enzymes for DNA metabolism in prokaryotes and eukaryotes. In human cells, DNA topoisomerase II enzyme activity can be modulated by both viral transformation and changes in proliferation status. To identify elements important for regulation of topoisomerase II alpha gene expression, genomic DNA clones covering the 5'-end of the gene were isolated. The intron/exon structure of a 2.5-kilobase region encompassing the translation start site was determined. Transcription was found to initiate at multiple sites clustered around 90 base pairs 5' to the ATG initiation codon. Transient expression of chimeric topoisomerase II-reporter gene constructs in HeLa cells revealed that the 5'-flanking region exhibited promoter activity. The region -90 to -1 upstream of the major transcription start site was shown by deletion analysis to include a promoter. This minimal promoter lacks a TATA box, is moderately GC-rich, and contains a high frequency of CpG dinucleotides; characteristic of a "housekeeping" gene promoter. Maximal promoter activity was observed using a fragment extending to position -562. Putative regulatory elements are contained within and immediately upstream of the minimal promoter region. The regulatory region of the topoisomerase II alpha gene identified here is similar in basic structure to those of the human thymidine kinase and DNA polymerase alpha genes, which are also controlled by proliferation-specific factors.
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PMID:Cloning and characterization of the 5'-flanking region of the human topoisomerase II alpha gene. 138 64

Type II topoisomerases are essential for faithful cell division in all organisms. In human cells, the alpha isozyme of topoisomerase II has been implicated in catalyzing mitotic chromosome segregation via its action as a DNA unlinking enzyme. Here, we have shown that the enzymatic activity of topoisomerase II alpha protein purified from HeLa cell nuclei was strongly enhanced following phosphorylation by protein kinase C. We have investigated the possibility that this kinase is involved in cell cycle phase-specific phosphorylation of topoisomerase II alpha in HeLa cells. Two-dimensional tryptic phosphopeptide mapping revealed that topoisomerase II alpha protein immunoprecipitated from metabolically labeled HeLa cells was differentially phosphorylated during the G2/M phases of the cell cycle. To identify sites of phosphorylation, and the kinase(s) responsible for this modification, oligohistidine-tagged recombinant domains of topoisomerase II alpha protein were overexpressed in Escherichia coli and purified by affinity chromatography. Phosphorylation of a short fragment of the N-terminal ATPase domain of topoisomerase II alpha by protein kinase C in vitro generated two phosphopeptides that co-migrated with prominent G2/M phase-specific phosphopeptides from the HeLa cell-derived topoisomerase II alpha protein. Site-directed mutagenesis studies indicated that phosphorylation of serine 29 generated both of these phosphopeptides. Our results implicate protein kinase C in the cell cycle phase-dependent modulation of topoisomerase II alpha enzymatic activity in human cells.
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PMID:Cell cycle phase-specific phosphorylation of human topoisomerase II alpha. Evidence of a role for protein kinase C. 749 37

Proliferation-linked expression of the nuclear Ki-S1 antigen is a significant prognostic indicator in mammary carcinomas. Here, we show staining of a protein of 170 kd by Ki-S1 antibody in immunoblots of Saccharomyces cerevisiae expressing human topoisomerase II alpha but not in the parental strain. In HL-60 cells containing both isoforms of human topoisomerase II, Ki-S1 antibody binds selectively to the 170-kd isoenzyme in a similar fashion as peptide-antibodies directed against amino acid residues 1 to 15 or 1512 to 1530 of human topoisomerase II alpha. Conversely, antibodies directed against carboxyl-terminal sequences of human topoisomerase II beta selectively stain a 180-kd protein. The immunoreactive pattern of V8 endoproteinase restriction digests of human topoisomerase II alpha was identical for Ki-S1-antibody and peptide-antibodies directed against residues 1512 to 1530 but different for peptide-antibodies directed against residues 1 to 15. The Rf values of the smallest fragment commonly recognized by Ki-S1 antibody and the carboxy terminus-specific peptide-antibody place the Ki-S1 epitope within the last 495 carboxyl-terminal amino acid residues of topoisomerase II alpha.
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PMID:Proliferation-associated nuclear antigen Ki-S1 is identical with topoisomerase II alpha. Delineation of a carboxy-terminal epitope with peptide antibodies. 753 79

V511 and V513 are Chinese hamster cell lines with acquired resistance to topoisomerase II (topo II) directed agents. These cell lines were obtained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently selecting in etoposide (VP-16). We have previously shown that this resistance is not associated with alterations in drug uptake. To elucidate whether any alterations in the functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reaction, single-strand conformational polymorphism analysis, and subsequent sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of the consensus ATP binding sequences (Motif A and Motif B/dinucleotide binding site), the DNA binding domain, and the 5' and 3' flanking regions of the DNA binding position. The V511 cells showed no mutational changes in these regions. However, the topo II alpha gene from V513 showed a point mutation at nucleotide 2552 that resulted in a glycine-to-aspartate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V513 cells is associated with reduced VP-16-induced cleavable complex formation demonstrated by potassium-sodium dodecyl sulfate assay and band-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by interfering with enzyme-DNA binding.
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PMID:A novel point mutation in the 3' flanking region of the DNA-binding domain of topoisomerase II alpha associated with acquired resistance to topoisomerase II active agents. 754 41

Metaphase chromatids are believed to consist of loops of chromatin anchored to a central scaffold, of which a major component is the decatenatory enzyme DNA topoisomerase II. Silver impregnation selectively stains an axial element of metaphase and anaphase chromatids; but we find that in earlier stages of mitosis, silver staining reveals an initially single, folded midline structure, which separates at prometaphase to form two chromatid axes. Inhibition of topoisomerase II prevents this separation, and also prevents the contraction of chromatids that occurs when metaphase is arrested. Immunolocalization of topoisomerase II alpha reveals chromatid cores analogous to those seen with silver staining. We conclude that the chromatid cores in early mitosis form a single structure, constrained by DNA catenations, which must separate before metaphase chromatids can be resolved.
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PMID:A postprophase topoisomerase II-dependent chromatid core separation step in the formation of metaphase chromosomes. 755 88

DNA topoisomerase II is a major protein of the nuclear matrix. The enzyme appears to have a central role in both DNA organization and replication. The importance of nuclear matrix topoisomerase II alpha as a target for certain anticancer agents was evaluated in CEM human leukemia cells. Studies were done to determine the extent to which the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II form covalent enzyme-DNA complexes in whole cells and in the nuclear matrix and nonmatrix fractions of CEM cells that are either sensitive or resistant to topoisomerase II-active anticancer agents. Topoisomerase II alpha was detected in both the high salt-soluble (nonmatrix) and matrix fractions of nuclei from parental CEM cells. Most of the matrix topoisomerase II alpha was tightly bound to DNA in cells incubated with VM-26. In contrast, topoisomerase II beta was detected only in the high salt-soluble (nonmatrix) fraction of the nucleus. The subnuclear distribution of the alpha and beta topoisomerase II isozymes in CEM/VM-1 cells resistant to topoisomerase-active drugs was similar to that in drug-sensitive CEM cells. However, the amount and activity of topoisomerase II alpha in nuclear matrices of CEM/VM-1 cells were decreased 3- to 6-fold relative to that of CEM cells. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function. Furthermore, these results provide evidence that topoisomerase II alpha is the nuclear matrix target for VM-26, and that depletion of the nuclear matrix isozyme contributes to cellular resistance to this anticancer agent.
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PMID:DNA topoisomerase II isozymes involved in anticancer drug action and resistance. 757 48

Topoisomerase II alpha is an essential nuclear enzyme involved in DNA replication and a target for many of the clinically useful antineoplastic agents. In a mitoxantrone-selected human leukemia cell line, HL-60/MX2, cellular topoisomerase II (topo II) catalytic activity is decreased, in association with the finding of reduced nuclear topo II alpha and beta protein levels. In addition, HL-60/MX2 cells contain a novel M(r) 160,000 topo II alpha-related protein that localizes predominantly to the cell cytoplasm (W. G. Harker et al., Biochemistry, 30: 9953-9961, 1991). In these studies, we have investigated the molecular mechanisms underlying the altered expression of the topo II alpha protein(s) in these cells. Three topo II alpha mRNAs, 7.2, 6.3, and 4.8 kb, were identified in the HL-60/MX2 cells, with the 6.3 and 4.8 kb transcripts being present in roughly equivalent amounts, while the 7.2-kb mRNA represents < 7% of the total topo II alpha-specific mRNA. Portions of the 3'-coding and 3'-untranslated regions were found to be missing from the 7.2- and 4.8-kb topo II alpha mRNAs by Northern blot analysis. Sequences encoding the 3' regions of the normal and truncated forms of the topo II alpha enzyme were obtained from the HL-60/MX2 cells through the use of a 3'-rapid amplification of cDNA ends strategy. Approximately 1321 nucleotides are missing from the 3'-coding and 3'-untranslated regions of the 4.8-kb mRNA and are replaced by 122 nucleotides that contain an in-frame stop codon and consensus polyadenylation signal. The translation product of the truncated 4388-bp topo II alpha transcript would have a predicted M(r) of 157,850, with 108 COOH-terminal amino acids being replaced by 13 novel residues. Immunoblot analysis confirmed that amino acids in the COOH-terminal region of topo II alpha were missing from the M(r) 160,000 HL-60/MX2 protein, and antisera generated to a synthetic peptide representing the 13 unique amino acids identified a M(r) 160,000 protein in nuclear extracts from these cells. PCR evaluation of the organization of the 3' region of the topo II alpha gene revealed that the 4.8-kb mRNA found in HL-60/MX2 cells diverges from that of the 6.3-kb mRNA at a consensus exon-intron splice donor site. The 122-bp novel nucleotides identified in the truncated transcript appear to originate from an adjacent intron as a result of altered RNA processing. These studies suggest that as a result of the disruption of the carboxy terminus of the topo II alpha protein and the putative nuclear targeting sequences identified therein, cellular localization of the protein is altered, which may confer a growth advantage for the HL-60/MX2 cells in the presence of mitoxantrone.
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PMID:Selective use of an alternative stop codon and polyadenylation signal within intron sequences leads to a truncated topoisomerase II alpha messenger RNA and protein in human HL-60 leukemia cells selected for resistance to mitoxantrone. 758 37


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