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)

The nuclear enzyme DNA topoisomerase II catalyzes the breakage and resealing of duplex DNA and plays an important role in several genetic processes. It also mediates the DNA cleavage activity and cytotoxicity of clinically important anticancer agents such as etoposide. We have examined the activity of topoisomerase II during the first cell cycle of quiescent BALB/c 3T3 cells following serum stimulation. Etoposide-mediated DNA break frequency in vivo was used as a parameter of topoisomerase II activity, and enzyme content was assayed by immunoblotting. Density-arrested A31 cells exhibited a much lower sensitivity to the effects of etoposide than did actively proliferating cells. Upon serum stimulation of the quiescent cells, however, there was a marked increase in drug sensitivity which began during S phase and reached its peak just before mitosis. Maximal drug sensitivity during this period was 2.5 times greater than that of log-phase cells. This increase in drug sensitivity was associated with an increase in intracellular topoisomerase II content as determined by immunoblotting. The induction of topoisomerase II-mediated drug sensitivity was aborted within 1 h of exposure of cells to the protein synthesis inhibitor cycloheximide, but the DNA synthesis inhibitor aphidicolin had no effect. In contrast to the sensitivity of cells to drug-induced DNA cleavage, maximal cytotoxicity occurred during S phase. A 3-h exposure to cycloheximide before etoposide treatment resulted in nearly complete loss of cytotoxicity. Our findings indicate that topoisomerase II activity fluctuates with cell cycle progression, with peak activity occurring during the G2 phase. This increase in topoisomerase II is protein synthesis dependent and may reflect a high rate of enzyme turnover. The dissociation between maximal drug-induced DNA cleavage and cytotoxicity indicates that the topoisomerase-mediated DNA breaks may be necessary but are not sufficient for cytotoxicity and that the other factors which are particularly expressed during S phase may be important as well.
Mol Cell Biol 1987 Sep
PMID:Topoisomerase-specific drug sensitivity in relation to cell cycle progression. 282 20

A type I topoisomerase has been purified more than 4000-fold from calf thymus mitochondria. The enzyme is membrane associated and is effectively solubilized by 1% Triton X-100 treatment of purified mitochondrial inner membranes. This ATP-independent enzyme relaxes positively and negatively supercoiled DNA with delta LK = 1. At low ionic strength, the native enzyme appears to be a monomer (sedimentation coefficient of 4.3 S and Stokes radius of 34 A), but it can form a weakly associated dimer at higher salt concentrations (sedimentation coefficient of 7.0 S and Stokes radius of 47.5 A). The mitochondrial type I topoisomerase is distinguishable from the nuclear enzyme by its (1) pH profile, (2) thermal stability, (3) response to dimethyl sulfoxide and Berenil, and (4) molecular weight. The mitochondrial enzyme is inhibited by elevated concentrations of the bacterial DNA gyrase inhibitor novobiocin, but not nalidixic or oxolinic acids. Sensitivity to N-ethylmaleimide indicates the importance of cysteine for catalytic activity. It is estimated that there are at least five copies of topoisomerase I per mammalian mitochondrion or a minimum of one to two per mitochondrial genome. In a manner similar to that observed with leukemia (nuclear and mitochondrial), calf thymus (nuclear), and HeLa (nuclear) cell type I topoisomerase, the calf thymus mitochondrial enzyme is inhibited by physiological concentrations of ATP.
Biochemistry 1987 Sep 22
PMID:Purification and characterization of a type I DNA topoisomerase from calf thymus mitochondria. 282 74

We evaluated the ability of the antitumor agent 4-(9-acridinylamino)-methanesulfon-m-anisidide (amsacrine or m-AMSA) and its congener, o-AMSA, to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK+/- -3.7.2C mouse lymphoma cells. These cells permit the recovery of mutants due to single-gene or chromosomal mutation. m-AMSA was highly mutagenic at the tk locus, producing approximately 3000 mutants/10(6) survivors at 10% survival; positive dose range 1-10 ng/ml; o-AMSA produced approximately 1500 mutants/10(6) survivors at 10% survival; positive dose range 0.1-2.5 micrograms/ml. Most of the TK mutants were small colonies, which suggests that m-AMSA and o-AMSA induce primarily chromosomal mutations as opposed to single-gene mutations. The potent clastogenicity of these agents was confirmed by cytogenetic analysis for chromosomal aberrations, which showed that m-AMSA (9 ng/ml, 10% survival) and o-AMSA (1 microgram/ml, 10% survival) produced 383 and 179 aberrations, respectively, per 100 metaphases (background = 3-4/100). The large-colony TK mutant frequencies produced by m-AMSA (67 - 112/10(6) survivors; background = 7/10(6); survival = 63 - 16%) were comparable to the published HPRT mutant frequencies produced by m-AMSA in V79 cells. Novobiocin (50 micrograms/ml), an inhibitor of mammalian DNA topoisomerase II and other enzymes, inhibited the mutagenic effects of m-AMSA, suggesting that DNA topoisomerase II (or another enzyme) may play a role in the mutagenic/clastogenic activity of m-AMSA.
Mutagenesis 1987 Sep
PMID:Mutagenicity of m-AMSA and o-AMSA in mammalian cells due to clastogenic mechanism: possible role of topoisomerase. 283 Apr 52

Recent biochemical and molecular biological data on the composition and structure of the chromosome and the nucleus, combined with observations on the chromosomes of mutant yeast cells and grasshopper neuroblasts, offer new perspectives on mutagen-induced chromosome stickiness and its relation to chromosome breakage. A hypothesis consistent with these data states that chromosome stickiness (i) results from changes in specific non-histone proteins (topoisomerase II and the peripheral proteins) that are integral components of the chromosome and whose function is necessary for separation and segregation of chromatids, the changes being caused either by mutation in structural genes for the proteins (heritable stickiness) or by direct action of mutagens on the proteins (induced stickiness); (ii) occurs in various degrees (slight, moderate, severe, extreme) that are determined by the number of target protein molecules affected, a certain number (threshold) of affected molecules at a given site on a chromosome being required to resist the forces of anaphase movement in order to produce microscopically detectable stickiness; (iii) results from molecular events that can occur at several phases of the cell cycle (including interphase), but can only be recognized at prometaphase, metaphase and anaphase; and (iv) causes chromosome aberrations by the physical stretching and breaking of chromatids at the sticky sites; hence the breakage resulting from stickiness is a secondary effect that requires anaphase movement, in contrast to breakage resulting from direct action of mutagens on DNA.
Mutagenesis 1987 Sep
PMID:Hypothesis: some mutagens directly alter specific chromosomal proteins (DNA topoisomerase II and peripheral proteins) to produce chromosome stickiness, which causes chromosome aberrations. 283 Apr 53

Pleotropic resistant human breast cancer cells (MCF-7), selected for resistance to Adriamycin, were used to study the production of DNA strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity. It was shown that the resistant MCF-7 cell line was cross-resistant to VP-16, and the degree of resistance was found to be 125-200-fold. Alkaline elution studies indicated that the parental cell line was very sensitive to VP-16 which caused extensive DNA strand breakage. In contrast, little DNA strand breakage was detected in the resistant MCF-7 cells, even at very high drug concentrations, indicating a good agreement between strand breaks and cytotoxicity. Further studies indicated that the nuclei isolated from the parental cell line were more resistant to VP-16-induced DNA strand breaks than the intact cells, while the opposite was found in the resistant cell line. In addition, the alkaline elution studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced DNA breaks in nuclei from the resistant cells. In agreement with this result, it was found that nuclear extract from the resistant cells produced 2-3-fold less VP-16-induced DNA breaks than that from the sensitive cells in 32P-end-labeled SV40 DNA. VP-16 uptake and efflux studies indicated that there was a 2-3-fold decrease in net cellular accumulation of VP-16 in the resistant cells. Although the reduced uptake of VP-16 and decreased drug sensitivity of topoisomerase II appear to contribute to the mechanism of action and the development of resistance to VP-16, they do not completely explain the degree of resistance to VP-16 in this multidrug-resistant MCF-7 cell line indicating that other biochemical factors, such as activation of VP-16, are also involved in drug resistance and suggesting that the resistance is multifactorial.
Cancer Res 1988 Sep 15
PMID:DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implications for the mechanism of action. 284 45

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.
J Biol Chem 1988 Sep 05
PMID:Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II. 284 38

Since DNA topoisomerase II (EC 5.99.1.3) is an essential enzyme in yeast, heterologous topoisomerase II gene expression in yeast cells can provide a system for analyzing the structure and function of topoisomerase II genes from other species. A series of yeast expression plasmids was constructed in which segments of the cDNA sequences encoding Drosophila DNA topoisomerase II were inserted under the transcriptional control of yeast GAL1 promoter. Expression of the functional form of Drosophila topoisomerase II cDNA can complement conditionally lethal, temperature-sensitive mutations in the yeast topoisomerase II gene (TOP2), as well as mutations in which the TOP2 locus was disrupted. The survival of these yeast cells depends upon the continuous expression of Drosophila topoisomerase II. Repression of Drosophila gene expression by glucose causes these yeast cells to cease dividing after a few generations. In addition to these genetic complementation data, the expression of the Drosophila topoisomerase II gene in yeast cells with a disruption in TOP2 can also be detected by immunochemical methods with an antibody specific for Drosophila topoisomerase II.
Proc Natl Acad Sci U S A 1988 Sep
PMID:Functional expression of a Drosophila gene in yeast: genetic complementation of DNA topoisomerase II. 284 62

A topoisomerase has been purified from extracts of a topoisomerase I-deficient strain of Escherichia coli based solely on its ability to segregate pBR322 DNA replication intermediates in vitro. This enzyme rapidly decatenated multiply linked form II:form II DNA dimers to form II DNA, provided that the DNA substrate contained single-stranded regions. Efficient relaxation of negatively supercoiled DNA was observed when reaction mixtures were incubated at 52 degrees C, but not at 30 degrees C (the temperature at which decatenation was readily observed). This topoisomerase was insensitive to the DNA gyrase inhibitor norfloxacin and unaffected by antibody directed against topoisomerase I. Relaxation of a unique plasmid topoisomer revealed that this decatenase changed the linking number of the DNA in steps of one and was therefore a type 1 topoisomerase. The cleavage pattern of a fragment of single-stranded phi X174 DNA generated by this decatenase was virtually identical to that reported for topoisomerase III, the least characterized topoisomerase present in E. coli.
J Biol Chem 1988 Sep 15
PMID:Identification of a potent decatenating enzyme from Escherichia coli. 284 17

A novobiocin-resistant BHK cell line, designated as NovrA2, was found to exhibit cross-resistance to other topoisomerase II inhibitors such as 4'-dimethylepipodophyllotoxin-4-(4,6-O-ethylidine-beta-D-glu copyranoside) (VP-16), adriamycin, and 4'-(9-acridinyl-amino)methanesulfon-m-anisidide (m-AMSA), and also to different types of drugs such as vinblastine and arabinocytidine. Nalidixic acid-resistant cells (A2Nalr) of the NovrA2 cell line were phenotypically reverted to novobiocin sensitivity like wild-type cells and were also partially reverted to sensitivity to VP-16 and adriamycin, but not to vinblastine and arabinocytidine. When VP-16 was added to cell culture, the drug-induced DNA strand breaks were much fewer in NovrA2 cells than in BHK cells. This reduced level of strand breaks in NovrA2 cells was not due to reduced drug uptake, because the two cell lines accumulated similar levels of radiolabeled VP-16. VP-16 also induced fewer DNA breaks in isolated nuclei of NovrA2 cells than in those of BHK cells. There was no significant difference in the VP-16-induced DNA cleavage activities of partially purified topoisomerase II from BHK and Novr cells. These results show that the resistance of NovrA2 cells to various drugs is not acquired by a defense mechanism related to membrane permeability and suggest that the resistance of the NovrA2 cells to topoisomerase II inhibitors might be due in part to alteration in a topoisomerase II associated factor(s).
Somat Cell Mol Genet 1988 Sep
PMID:Cross-resistance of novobiocin-resistant BHK cell line to topoisomerase II inhibitors. 284 88

Endogenous topoisomerase II cleavage sites were mapped in the chicken beta A-globin gene of 12- to 14-day embryonic erythrocytes. A major topoisomerase II catalytic site was mapped to the 5' end of the globin gene which contained a nucleosome-free and DNase I-hypersensitive site and additional but minor sites were mapped to the second intron and 3' of the gene to a tissue-specific enhancer. Cleavage sites, mapped in situ by indirect end labeling, were aligned to single-base-pair resolution by comparison to a consensus sequence derived for vertebrate topoisomerase II catalytic sites. In contrast to embryonic erythrocytes, endogenous topoisomerase II cleavages were not detected in erythrocytes from peripheral blood of adult chickens; therefore, as the transcriptional activity of the beta A-globin gene declines during terminal differentiation of erythrocytes, the activity of topoisomerase II in situ declines as well, despite the fact that DNase I hypersensitivity persists. The results showed that DNase I-hypersensitive chromatin can be maintained in the absence of topoisomerase II activity and suggested that topoisomerase II acts at hypersensitive sites because of an inherent attraction to some preexisting combination of DNA sequence or chromatin structure associated with DNase I-hypersensitive regions.
Mol Cell Biol 1988 Sep
PMID:DNase I hypersensitivity is independent of endogenous topoisomerase II activity during chicken erythrocyte differentiation. 285 23


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