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)

Electron microscopy studies demonstrate unequivocally that the observed oligonucleosome-sized secondary DNA fragmentation in human promyelocytic HL-60 cells treated with the topoisomerase inhibitors camptothecin and teniposide is correlated with the morphological changes in cell structure typical of programmed cell death (apoptosis). Since apoptosis has been associated with potential involvement of intracellular signaling linked to the Ca2+/calmodulin and protein kinase C transduction pathways, we also investigated the effects of signaling modulators on camptothecin- and teniposide-induced secondary DNA fragmentation in HL-60 cells. Neither calcium chelators, calcium/calmodulin inhibitors (calmidazolium or cyclosporine A), protein kinase C stimulation by TPA, protein phosphatase inhibition by okadaic acid, protein kinase inhibition by staurosporine, calphostin C, genistein or H7, nor cell cycle alterations by caffeine had any detectable effect. Interestingly, most of these intracellular signaling modulators were able to induce DNA fragmentation in HL-60 cells by themselves. These results may suggest that even though modulation of these signaling pathways was unable to prevent topoisomerase inhibitor-induced apoptosis, their sole deregulations could induce apoptosis in HL-60 cells. In contrast, aphidicolin blocked camptothecin-induced secondary DNA fragmentation, indicating that replication-induced DNA damage is required for camptothecin- but not teniposide-induced secondary DNA fragmentation. Zinc, 3-aminobenzamide, and spermine also modulated both camptothecin- and teniposide-induced secondary DNA fragmentation without significant alteration of topoisomerase-mediated primary DNA strand breaks. Hence, poly(ADP-ribosyl)ation and chromatin structure may be important in modulating oligonucleosome-sized DNA fragmentation associated with apoptosis in HL-60 cells treated with topoisomerase inhibitors.
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PMID:Apoptosis and its modulation in human promyelocytic HL-60 cells treated with DNA topoisomerase I and II inhibitors. 768 16

Yeast mitochondria were found to contain a novel topoisomerase-like activity which required nucleoside di- or tri-phosphates as a cofactor. ADP supported activity as effectively as ATP and the optimal concentration for each was approximately 20 microM. None of the other standard ribo- or deoxyrib-onucleotides could fully substitute for either ADP or ATP. The non-hydrolyzable ATP analogs, adenosine-5'-0-(3-thiotriphosphate) (ATP-gamma-S), adenylyl (beta,gamma-methylene) (AMP-PCP), and andenyl-imidodiphosphate (AMP-PNP) also supported activity suggesting that the nucleotide cofactor regulated topoisomerase activity rather than serving as an energy donor in the reaction. The mitochondrial topoisomerase activity relaxed both positively and negatively supercoiled DNA. It was not inhibited by concentrations of ethidium bromide up to 2 micrograms/ml nor by either nalidixic or oxolinic acids; novobiocin, coumermycin, and berenil inhibited the activity. Genetic and biochemical analysis of the mitochondrial topoisomerase activity indicated that it was not encoded by the nuclear TOP1, TOP2, and TOP3 genes.
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PMID:Evidence for a nucleotide-dependent topoisomerase activity from yeast mitochondria. 775 Jan 44

The rate of relaxation of supercoiled DNA by purified vaccinia topoisomerase I is stimulated 20-fold by 5 mM ATP. A similar effect is elicited by GTP, CTP, UTP, dATP, and adenosine 5'-(beta, gamma-imido)triphosphate. ATP-mediated rate enhancement requires salt as a coactivator. ADP and inorganic pyrophosphate also stimulate relaxation 10-20-fold, whereas AMP and inorganic phosphate have little effect. A model for allosteric activation of topoisomerase by nucleotides is suggested.
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PMID:Stimulation of vaccinia topoisomerase I by nucleoside triphosphates. 796 68

DNA topoisomerases II are enzymes which have been purified from a lot of organisms and have been found to be involved in segregation of chromosomes. The following article reports the analysis of a partially purified DNA topoisomerase II from Sulfolobus (strain B12) a thermophilic archaebacterium which grows at 80 degrees C. The enzyme is composed by two subunits: the A subunit with a molecular mass of 85,000 Da which contains the nicking-closing activity and the B subunit with a molecular mass of 65,000 Da which contains the ATP binding site. The enzyme relaxes negatively as well as positively supercoiled DNA consequently to ATP hydrolysis into ADP (as eukaryotic DNA topoisomerases II and Escherichia coli DNA topoisomerase IV do). DNA relaxation catalyzed by the thermophilic enzyme is inhibited in the presence of both bacterial antibiotics acting at the ATP binding site such as novobiocin and coumermycin A1 at the concentration which was found to inhibit the E. coli type II DNA topoisomerases (DNA gyrase and DNA topoisomerase IV). Based on the relaxation of both negatively and positively supercoiled DNA and the sensitivity to antibiotics such as novobiocin and coumermycin A1, the DNA topoisomerase II isolated from thermophilic archaebacterium shares common characteristics with E. coli DNA topoisomerase II.
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PMID:DNA unknotting activity (DNA topoisomerase II) isolated from a thermophilic archaebacterium Sulfolobus is inhibited by novobiocin. Partial purification, identification of the two subunits and characteristics of the enzyme. 808 47

We previously demonstrated that in murine T cells thermotolerance correlated with heat shock protein 70 (hsp70) synthesis and protection of nuclear type I topoisomerase (topo I). Topo I activity returned to normal levels following heat stress even in cells not rendered thermotolerant by a prior heat shock. Recovery of topo I activity was not dependent on de novo protein synthesis, suggesting that the cell possesses a pathway(s) for refolding this nuclear protein. In this report we demonstrate that topo I and hsc70, the constitutively produced member of the hsp70 family, associated in vivo during heat stress. That this association may play a physiologically important role in protecting topo I activity from heat stress was suggested by the observation that hsc70 protected topo I from heat inactivation in vitro. hsc70 but not actin also reactivated previously heat-denatured topo I in a dose-dependent fashion. However, refolding of heat-denatured topo I by purified hsc70 was inefficient relative to a hsc70-containing cell lysate. Protection from heat inactivation as well as reactivation by hsc70 did not require exogenous ATP. Similarly, reactivation by the cell lysate was not inhibited by ADP or a nonhydrolyzable analogue of ATP. Thus, our studies suggest that nuclear topo I complexes with hsc70 during heat stress, which may explain, at least in part, why hsp70 proteins accumulate in the nucleus, particularly the nucleolus. This interaction may limit heat-induced protein damage and/or accelerate restoration of protein function in an ATP-independent reaction.
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PMID:Heat stress induces hsc70/nuclear topoisomerase I complex formation in vivo: evidence for hsc70-mediated, ATP-independent reactivation in vitro. 812 77

We have studied the nonhistone proteins which are modified by ADP-ribosylation in HeLa cells. When isolated nuclei were incubated with 32P-NAD, the main labeled proteins presented sizes of 170, 116, 70, and 45 kDa. To provide evidence for the identification of the 170-kDa band as DNA topoisomerase II, the enzyme was immunoprecipitated from isolated nuclei incubated with 32P-NAD and a labeled peptide of 170 kDa was observed. The label was sensitive to the action of venom phosphodiesterase which specifically degrades ADP-ribose. ADP-ribosylated proteins were also isolated from HeLa cells by affinity chromatography on boronate-agarose gel. Using a monoclonal antibody against the 170-kDa isoform of topoisomerase II, a single 170-kDa immunoreactive peptide was recognized by Western blot among the retained protein acceptors. When ADP-ribosylation was blocked by treating HeLa cells with 3-aminobenzamide, topoisomerase II was no longer retained on the boronate column. These results provide experimental evidence indicating that DNA topoisomerase II is ADP-ribosylated in HeLa cells. To possibly correlate ADP-ribosylation of nuclear proteins with the extent of DNA damage, permeabilized HeLa cells were incubated with 32P-NAD after treatment with the alkylating agent dimethylsulfate. ADP-ribosylated proteins were isolated by boronate chromatography. A strong increase in the ADP-ribosylation of the poly(ADP-ribose)polymerase was observed, whereas no further modification of topoisomerase II was noted.
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PMID:ADP-ribosylation of nonhistone proteins in HeLa cells: modification of DNA topoisomerase II. 838 22

There is compelling evidence for the central role of oxidative damage in the aging process and for the participation of reactive oxygen species in tumor initiation and promotion. Caloric restriction (CR) or energy restriction retards age-associated increases in mitochondrial free-radical production and reduces the accumulation of oxidatively damaged cell components. CR has also been shown to slow down age-related declines in various repair capabilities, including some types of DNA repair. It is proposed that inhibitors of mitochondrial electron transport and/or uncouplers of oxidative phosphorylation (rotenone, amytal, amiodarone, valinomycin, etc.), when used at extremely low doses, could mimic the effects of CR in model systems. The objective is to lower mitochondrial free-radical production by decreasing the fraction of electron carriers in the reduced state. In addition to a variety of other effects, CR has been shown to increase the rate of apoptosis, particularly in preneoplastic cells, and in general, to promote elevated levels of free glucocorticoids (GCs). GCs are known to induce tissue-specific apoptosis and to upregulate gap-junction-mediated intercellular communication (GJIC). Tumor promoters like phorbol esters have the opposite effect, in that they inhibit both the process of apoptosis and GJIC. The enzyme poly (ADP-ribose) polymerase (PARP) is thought to play a central role in apoptosis, in a manner that has been highly conserved in evolution. There is good evidence that the apoptosis-associated Ca/Mg-dependent DNA endonuclease is maintained in a latent form by being poly (ADP-ribosylated). Apoptosis would require the removal of this polymer from the endonuclease, and, most likely, its removal from topoisomerase II and histone H1 as well. The role of poly (ADP-ribose) in apoptosis, carcinogenesis, and aging could be studied by the use of modulators of PARP activity (3-aminobenzamide, 3-nitrosobenzamide, 1% ethanol, etc.), inhibitors of poly ADP-ribose) glycohydrolase activity (ethacridine, 43 degrees C, etc.), and inhibitors of the PARP-specific protease (interleukin-1 beta converting enzyme (ICE)-like protease). Also, it would be of interest to determine if CR can decrease the half-life of poly (ADP-ribose), upregulate GJIC, and modulate the activities of PARP, the glycohydrolase, and the PARP-specific protease, factors potentially important in these processes.
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PMID:The beneficial effects of dietary restriction: reduced oxidative damage and enhanced apoptosis. 865 88

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.
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PMID:Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases. 897 82

Type II DNA topoisomerases function as homodimeric enzymes in transiently cleaving double-stranded DNA to catalyze unlinking and unknotting reactions. The dimeric enzyme creates a DNA double-strand break by forming a covalent attachment between an active site tyrosine from each monomer and a 5'-phosphate from each strand of DNA. The dimer must be very stable to dissociation or subunit exchange when covalently attached to DNA to prevent directly or indirectly catalyzed rearrangements of the genome. Past studies have indicated conflicting results for the monomer-dimer stability of topoisomerase II in solution. Here, we report results from sedimentation equilibrium studies and two different subunit exchange assays indicating that purified Saccharomyces cerevisiae DNA topoisomerase II exists as a stable dimer in solution, with a Kd estimated to be < or = 10(-11) M. This high dimer stability is not detectably altered by a change of ionic strength or by the presence of ATP, ADP, or DNA.
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PMID:Type II DNA topoisomerase from Saccharomyces cerevisiae is a stable dimer. 916 81

Studies of the biochemical mechanisms evoked by conventional treatments for neoplastic diseases point to apoptosis as a key process for elimination of unwanted cells. Although the pathways through which chemotherapeutics promote cell death remain largely unknown, caspase proteases play a central role in the induction of apoptosis in response to a variety of stimuli including tumor necrosis factor, fas ligand, and growth factor deprivation. In this article, we demonstrate the induction of caspase protease activity in MCF7 human breast carcinoma cells exposed to the topoisomerase inhibitor, etoposide. Caspase protease activity was assessed by incubating cell lysates with the known caspase substrates, acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin or acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin. We observed maximal cleavage of acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin within 6 hr following etoposide addition, a time that precedes cell death. In contrast, acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin was resistant to cleavage activity. This substrate cleavage specificity implies that a caspase-3-like protease is activated in response to DNA damage. Consistent with the lysate protease activity, an intracellular marker of caspase activation, poly-ADP ribose polymerase (PARP), was cleaved in a concentration- and time-dependent manner after etoposide-treatment. PARP cleavage followed caspase activation and reached maximum cleavage between 12 and 16 hr. Incubation of the cells with the peptidic caspase inhibitor z-valine-alanine-asparagine-CH2F prevented caspase activation, inhibited PARP cleavage, and inhibited cell death. Thus, etoposide killing of MCF7 cells requires a caspase-3-like protease.
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PMID:Caspase activation in MCF7 cells responding to etoposide treatment. 949 10


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