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)

Cytokine stimulation of human umbilical vein endothelial cells (HUVE) induces surface expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin). We previously found that induction of adhesion molecule expression in HUVE is regulated, at least in part, by protein kinase C (PKC) activation, although this is not associated with the expected translocation of PKC from the cytosolic to the particulate fraction. We therefore investigated potential nuclear targets for PKC. Topoisomerase II is localized to the nuclear matrix and has been shown to be phosphorylated, both in vitro and in vivo, by PKC. In HUVE, the topoisomerase II selective inhibitors novobiocin, nalidixic acid, and etoposide prevented cytokine-induced VCAM-1 surface expression, but not E-selectin or ICAM-1 surface expression. Similarly, novobiocin and nalidixic acid reduced the accumulation of VCAM-1 mRNA in response to tumor necrosis factor-alpha treatment of HUVE. The inhibitory effect of the topoisomerase II inhibitors on VCAM-1 expression was not due to non-specific toxicity, as protein synthesis, measured by trichloroacetic acid precipitation of 35S-methionine labeled proteins, and transcription, determined by beta-actin mRNA levels, were not decreased. In contrast to the observed reduction of VCAM-1 mRNA accumulation and surface protein expression, inhibition of topoisomerase II activity enhanced E-selectin mRNA accumulation and surface protein expression in response to tumor necrosis factor-alpha stimulation of HUVE. This work demonstrates that topoisomerase II activity may differentially regulate the expression of adhesion molecules on HUVE.
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PMID:Inhibitors of topoisomerase II prevent cytokine-induced expression of vascular cell adhesion molecule-1, while augmenting the expression of endothelial leukocyte adhesion molecule-1 on human umbilical vein endothelial cells. 752 51

Two kilobase segments of the 5'-untranslated regions of the human and rabbit butyrylcholinesterase (BCHE) genes were characterized. The sequences shared extensive identity except for a 333-base pair (bp) Alu repeat present only in human BCHE. One single transcription start site was found in both genes with the techniques of primer extension, amplification of the 5'-end of mRNA, and RNase protection. Cap sites in human and rabbit BCHE genes were found in strictly homologous positions. In human BCHE, the transcription start site was found 157 bp upstream of Met-28, the translation start site. Potential regulatory elements in both promoters included one AP1 site and multiple sites for topoisomerase, Oct-1 and PEA-3. Transient expression of BCHE-reporter gene constructs showed that a 194-bp fragment of the 5'-flanking region of human BCHE and a 570-bp fragment of rabbit BCHE were sufficient for promoting chloramphenicol acetyltransferase activity in HeLa cells. No consensus TATA and CAAT boxes were found. However, the sequence around the transcription start site exhibited homology with initiator elements found in other TATA-less promoters in developmentally regulated genes.
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PMID:Promoter and transcription start site of human and rabbit butyrylcholinesterase genes. 806 98

Resistance to amsacrine in HL-60/AMSA is 50-100 fold compared to the parental HL-60/S cells. Synthesis and phosphorylation of topoisomerase II (TOPO II) were 2-3 fold lower in HL-60/AMSA compared to HL-60/S cells metabolically labelled with [32P]-orthophosphoric acid or [35S]-L-methionine. Incubating cells in radiolabel-free media following metabolic labelling for 4 hr revealed: (a) dephosphorylation of topoisomerase II at 4 hr was 70% and 20% in HL-60/S and HL-60/AMSA cells, respectively; and (b) degradation of topoisomerase II at 4 hr was 40% and 10% in HL-60/S and HL-60/AMSA cells, respectively, while at 8 hr degradation was 80% and 50% in HL-60/S and HL-60/AMSA cells, respectively. The magnitude of topoisomerase II band depletion in immunoprecipitates of amsacrine-treated cells labelled with [35S]-L-methionine or [32P]-orthophosphoric acid, correlated with the differential amsacrine sensitivity of HL-60/S and HL-60/AMSA cells, suggesting that the amount of newly synthesized and phosphorylated topoisomerase II may be contributing to amsacrine resistance. Thus, the attenuated synthesis and phosphorylation of TOPO II in HL-60/AMSA may contribute to the resistance of these cells to amsacrine.
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PMID:Altered phosphorylation, biosynthesis and degradation of the 170 kDa isoform of topoisomerase II in amsacrine-resistant human leukemia cells. 838 46

Anthracenyl-amino acid and dipeptide conjugates represent new classes of topoisomerase (topo) inhibitors. To investigate the structural basis for their different selectivity against topo I and II and varying potency, the binding of six compounds to d(CGTACG) was studied by molecular modeling. Modeling data were in good agreement with physical data showing that five compounds intercalated DNA with the anthraquinone chromophore orientated in parallel to the long dimension of the d(CpG) base pairs and the amino acid placed in the minor groove. Differences in binding modes emerged which correlated to different biological properties. The amino acid chain of the topo I inhibitor (NU/ICRF 600, gly-phe) extended significantly out from the helical axis horizontal. The amino acid side chains of two topo II inhibitors (NU/ICRF 510, arginine and NU/ ICRF 512, methionine) were inserted into the minor groove, whereas the C-terminal groups (hydrazide) of two potent topo II inhibitors (NU/ICRF 500 and 506, serine) were placed into the minor groove while the amino acid side chains pointed away from the minor groove. These data provide structural information which may prove valuable in rational design of second generation analogs.
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PMID:Molecular modeling of the interaction of anthracenyl-amino acid topoisomerase inhibitors with the DNA sequence d(CGTACG). 891 31

Regulation of topoisomerase II (TOPO II) isozymes alpha and beta is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO IIalpha and TOPO IIbeta proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II (alpha and beta) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIbeta protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIbeta protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 micromol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 micromol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 micromol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIbeta protein levels are posttranscriptionally regulated and that degradation of TOPO IIbeta is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II (alpha + beta) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIbeta may have a specific role in transcription of genes involved in differentiation with RA treatment.
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PMID:Altered expression and activity of topoisomerases during all-trans retinoic acid-induced differentiation of HL-60 cells. 976 71

Msx2 is a homeodomain transcriptional repressor that exerts tissue-specific actions during craniofacial skeletal and neural development. To identify coregulatory molecules that participate in transcriptional repression by Msx2, we applied a Farwestern expression cloning strategy to identify transcripts encoding proteins that bind Msx2. A lambdagt11 expression library from mouse brain was screened with radiolabeled GST-Msx2 fusion protein encompassing the core suppressor domain of Msx2. A cDNA was isolated that encodes a novel protein fragment that binds radiolabeled Msx2. Homeoprotein binding activity was confirmed by Farwestern analysis of the T7-epitope-tagged recombinant protein fragment, and interactions in vitro require Msx2 residues necessary for transcriptional suppression in vivo. On the basis of biochemical analyses, this novel protein was named MINT, an acronym for Msx2-interacting nuclear target protein. The original clone is part of a 12.6 kb transcript expressed at high levels in testis and at lower levels in calvarial osteoblasts and brain. Multiple clones isolated from a mouse testis library were sequenced to construct a MINT cDNA contig of 11 kb. Starting from an initiator Met in good Kozak context, a large nascent polypeptide of 3576 amino acids is predicted, in contiguous open reading frame with the Msx2 interaction domain residues 2070-2394. Protein sequence analysis reveals that MINT has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals. Western blot analysis of fractionated cell extracts reveals that mature approximately 110 kDa (N-terminal) and approximately 250 kDa (C-terminal) MINT protein fragments accumulate in chromatin and nuclear matrix fractions, cosegregating with Msx2 and topoisomerase II. In gel shift assays, the MINT RRM domain selectively binds T- and G-rich DNA sequences; this includes a large G/T-rich inverted repeat element present in the proximal rat osteocalcin (OC) promoter, overlapping three cognates that support OC expression in osteoblasts. MINT and OC mRNAs are reciprocally regulated during differentiation of MC3T3E1 calvarial osteoblasts. Consistent with its proposed role as a nuclear transcriptional factor, transient expression of MINT(1-812) suppresses the FGF/forskolin-activated OC promoter, does not significantly regulate CMV promoter activity, but markedly upregulates the HSV thymidine kinase promoter in MC3T3E1 cells. In toto, these data indicate that the novel nuclear protein MINT binds the homeoprotein Msx2 and coregulates OC during craniofacial development. Msx2 and MINT both target an information-dense, osteoblast-specific regulatory region of the OC proximal promoter, nucleotides -141 to -111. The N-terminal MINT RRM domain represents an authentic dsDNA binding module for this novel vertebrate nuclear matrix protein. Acting as a scaffold protein, MINT potentially exerts both positive and negative regulatory actions by organizing transcriptional complexes in the nuclear matrix.
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PMID:The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates the rat osteocalcin promoter. 1045 62

1DNA topoisomerase II (topo II) is a nuclear enzyme that modifies DNA topology and also serves as a target to mediate the cytotoxicity of several antineoplastic agents. Several reports have demonstrated that a reduction of topo II is associated with reduced sensitivity to these agents. Topo II exists as two isoforms in mammalian cells: topo IIalpha and topo IIbeta. In MCF-7 cells, the half-life (mean +/- SEM) values of topo IIalpha and topo IIbeta in situ were 6.6 +/- 0.3 and 17.6 +/- 2.3 hr, respectively, as determined by [(35)S]methionine/cysteine pulse-chase analysis. Degradation of topo IIalpha in situ was abrogated by the presence of proteasome inhibitors, and the relative activities were carbobenzoxy-leucyl-leucyl-leucinal (MG132) > carbobenzoxy-leucyl-leucyl-norvalinal (MG115) > ALLN congruent with lactacystin. ATP-dependent degradation of topo IIalpha, but not topo IIbeta, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furthermore, degradation of topo IIalpha was abrogated by the proteasome inhibitors MG132 and MG115, but not by lactacystin, in extracts of asynchronously dividing MCF-7 cells. Finally, degradation of topo IIalpha, but not topo IIbeta, was observed to occur in a cell cycle-dependent fashion, in extracts of synchronized HeLa cells, with maximal loss of the alpha isoform occurring 2 hr after release from mitotic arrest. This degradation of topo IIalpha appeared to be facilitated by an ATP-dependent activity. Furthermore, high molecular weight bands (>200 kDa), which may represent polyubiquitinated-topo IIalpha conjugates, were also detected in extracts of synchronized HeLa cells. This study provides evidence for a role of the ubiquitin-proteasome pathway in the cell cycle-dependent regulation of topo IIalpha expression.
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PMID:Role of proteasomal degradation in the cell cycle-dependent regulation of DNA topoisomerase IIalpha expression. 1127 64

The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 microg/ml. GyrA (Leu-55 to Gln-155, Escherichia coli numbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa, with the degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa (70.9 to 88.6%) and E. coli (73.0 to 88.6%) were observed. Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs. The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected. The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 microg/ml) showed a ParC amino acid change (Ser-80-->Arg). In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE. The frequencies of alterations at positions 437, 465, 477, and 485 were higher (P < 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs. An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (0.5 microg/ml) or reserpine (10 microg/ml), was suspected in seven strains. These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S. maltophilia.
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PMID:Topoisomerase II and IV quinolone resistance-determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility. 1185 Feb 46

Hybridization with cDNA arrays was used to obtain expression profiles of 214 protein-tyrosine kinase, protein-tyrosine phosphatase, dual-specific phosphatase, and other genes for kidney carcinomas (KC) and normal kidney tissues of 34 patients and for seven carcinoma cell lines. Computer analysis revealed three clusters of genes coexpressed in KC. A proliferating-cell gene cluster included MET, VIM, MYC, TOP2A, PCNA, etc. A neoangiogenesis and blood-cell gene cluster included LCK, HCK, FGR, MMP9, CSFR1, VEGF, FLT1, and KDR. A cluster corresponding to normal, differentiated kidney cells included ERBB2 (HER2) for receptor protein-tyrosine kinase, several phosphatase genes (PTPRE, PTPRB, DUSP9), and EGF. The results suggested that MET, DUSP9, PCNA, TOP2A, and VIM may serve as diagnostic and prognostic markers in KC. Tubulin and topoisomerase II were assumed to be promising targets for cell proliferation inhibitors in KC.
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PMID:[Molecular portrait of human kidney carcinomas: the gene expression profiling of protein-tyrosine kinases and tyrosine phosphatases which controlled regulatory signals in the cells]. 1206 34

Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits. The CKIIbeta subunit is thought to mediate the tetramer formation and interact with other target proteins. However, its physiological function remains obscure. In this study, point mutants of CKIIbeta that are defective for the L41 binding were isolated by using the reverse two-hybrid system. A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of CKIIbeta are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for CKIIbeta homodimerization. Two point mutants, R75 and R83, of CKIIbeta interacted with L5, topoisomerase IIbeta, and CKBBP1/SAG, but not with the wild-type CKIIbeta. This indicates that CKIIbeta homodimerization is not a prerequisite for its binding to target proteins. These CKIIbeta point mutants may be useful in exploring the biochemical physiological functions of CKIIbeta.
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PMID:Identification of mutations in protein kinase CKIIbeta subunit that affect its binding to ribosomal protein L41 and homodimerization. 1289 90


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