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)

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.
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PMID:Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. 284 43

DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 4 degrees C as well as at 30 degrees C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA.
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PMID:Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells. 300 58

A protein kinase activity has been identified that is tightly associated with the purified Drosophila type II DNA topoisomerase. The kinase and topoisomerase activities are not separated when the enzyme is subjected to analytical chromatography (phosphocellulose, single-strand DNA agarose, and Sephacryl S-300) and analytical glycerol gradient sedimentation. These two activities are also inactivated to the same extent by either heat or N-ethylmaleimide treatment. The evidence, however, does not rule out the possibility that the kinase activity resides in a polypeptide other than the topoisomerase polypeptide. The topoisomerase-associated protein kinase activity is not stimulated by Ca2+ or cyclic nucleotides. It shows a broad substrate range, including the DNA topoisomerase itself, casein, phosvitin, and histones. Phosphoamino acid analysis identified phosphoserine and phosphothreonine in polypeptides modified by the topoisomerase-associated protein kinase. No similar activity has been identified previously in Drosophila melanogaster.
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PMID:A protein kinase activity tightly associated with Drosophila type II DNA topoisomerase. 609 62

30 years ago an anthracycline antibiotic was shown to have antineoplastic activity. This led to the development of well over 1000 analogues with a vast spectrum of biochemical characteristics. Many biological actions have been described. The original anthracyclines are active against many types of cancer and are an integral part of several curative combinations. They are ineffective against other tumours. Although some analogues show an altered spectrum of activity or an improved therapeutic index relative to the older agents, it is not clear that cardiotoxicity can be totally avoided with these agents. Primary and secondary resistance to anthracyclines remain major clinical problems. Pharmacokinetic studies have been of limited help in explaining this. Overexpression of a surface-membrane permeability glycoprotein (Pgp) was identified in ovarian cancer of patients who had clinical multidrug resistance in 1985. This led the way for the discovery of a number of resistance mechanisms in vitro. Some of these have been found in more than 1 type of cell line, and more than 1 mechanism may exist in a single cell. Additional resistance proteins have been identified, qualitative and quantitative alterations of topoisomerase II have been described, and some mechanisms in other systems have not yet been identified. Some of these may prove to be important in clinical drug resistance. Drugs such as calcium antagonists and cyclosporin, studied initially for their ability to block the Pgp pump, appear to be heterogeneous in this capacity and may have additional sites of action. It will be critical for clinical studies to define the precise resistance mechanism(s) that must be reversed. To date this has been difficult, even in trials ostensibly dealing with the original Pgp. Liposomes can potentially alter toxicity and target drug delivery to specific sites. In addition, they may permit the use of lipophilic drugs that would otherwise be difficult to administer systemically. Resistant tumours may be sensitive to anthracyclines delivered by liposomes. To reduce cardiac toxicity, administering doxorubicin (adriamycin) by slow infusion through a central-venous line should be considered whenever feasible. Monitoring of cardiac ejection fraction and the use of endomyocardial biopsy will permit patients to be treated safely after they reach the dose threshold at which heart failure begins to be a potential risk. A number of structurally modified anthracyclines with the potential advantages of decreased cardiotoxicity and avoidance of multidrug resistance mechanisms are entering clinical trials. Meanwhile, the vast weight of clinical experience leaves doxorubicin as a well tolerated and effective choice for most potentially anthracycline-sensitive tumours.
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PMID:Anthracycline antibiotics in cancer therapy. Focus on drug resistance. 751 99

Internucleosomal DNA fragmentation and cell death induced by dexamethasone in rat thymocytes were inhibited when cells were cultured in 95% N2/5% CO2 atmosphere, in which oxygen was rapidly reduced to under 0.5%. DNA fragmentation was delayed by a less severe hypoxia in 5% oxygen whilst in cell cultured in high oxygen atmosphere (95% O2) cell death was increased. On the other hand, prolonged oxygen deprivation caused an increase of spontaneous apoptotic cell death. Hypoxia also inhibited DNA fragmentation induced by calcium ionophore A23187, but not by topoisomerase inhibitor camptothecin. These data support the hypothesis of the involvement of oxygen reactive species in calcium-mediated apoptosis and suggest a complex role of oxygen in the modulation of programmed cell death.
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PMID:Oxygen tension influences DNA fragmentation and cell death in glucocorticoid-treated thymocytes. 762 42

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

A common strongly ordered multi-step-pattern of endogenous DNA degradation was induced in rat liver nuclei and intact thymocytes, prepared in the presence of chelating agents and incubated in the presence of CaCl2 and/or MgCl2. It consisted of sequential generation of 0.3 Mbp, then 0.05 Mbp DNA fragments and finally of oligo- and mononucleosomal DNA. Oligonucleosomal DNA was generated when the genome had already been disintegrated into 0.05 Mbp DNA fragments. ZnCl2 completely inhibited advanced genome cleavage to oligo- and mononucleosomal DNA without affecting the initial generation of large DNA fragments. Therefore, the endonucleolytic activity which produce large DNA fragments is different from Ca2+/Mg2+ endonuclease. The similar pattern of DNA degradation was observed in thymocytes treated with dexamethasone and with the topoisomerase II inhibitor VM-26, the agents known to induce apoptosis. The effect of VM-26 strongly suggests the involvement of topoisomerase II in generation of large DNA fragments. Multi-level organization and regulation of the chromatin structure determine the stepwise process of genome degradation. Detachment of chromatin from the nuclear matrix attachment regions may be one of the possible mechanisms of switching off the genome function and triggering the multi-step process of endogenous chromatin degradation thus leading to cell death in terminal differentiation or stress-induced apoptosis.
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PMID:Comparative study of induction of endogenous DNA degradation in rat liver nuclei and intact thymocytes. 774 35

Apoptosis of human B cells and murine T and B cells was analyzed by DNA agarose gel electrophoresis, clamped homogeneous electric field, measurement of cell DNA content by flow cytometry, transmission electron microscopy and by UV microscopy. Apoptosis was induced by etoposide (an inhibitor of topoisomerase II), by the calcium ionophore ionomycin or by cross-linking of membrane immunoglobulins (Ig) with anti-Ig-antibodies. Two types of apoptosis could be defined. Apoptosis resulting in small DNA fragments (180-200 base pairs and multiples thereof) was associated with a typical 'ladder' in agarose gel electrophoresis and a decrease in cell DNA content assessed by flow cytometry. Conversely apoptosis with large DNA fragments (100-150 kilobase pairs) was only demonstrated by clamped homogeneous electric field but was not associated with decreased cell DNA content or the observation of DNA ladders. Nuclear condensation without fragmentation was more frequent when apoptosis generated large DNA fragments. The type of apoptosis appears to be an intrinsic property of each cell type.
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PMID:Apoptosis without decrease of cell DNA content. 779 18

Three photoproducts of merocyanine 540 have been isolated, chemically characterized and synthesized. Two of these photoproducts, merocil and merodantoin, show significant antitumor activity in vitro and in vivo while demonstrating minimal toxicity to normal cells and tissues. Treatment of lymphoma cells with these compounds resulted in a rapid decline in macromolecular synthesis, DNA fragmentation inhibitable by actinomycin D and cycloheximide, and a marked rise in intracellular calcium. In vitro analysis revealed that activity of these compounds is dependent on topoisomerase II. These results are discussed in terms of the novel class of topoisomerase II-dependent compounds for potential use in chemotherapy.
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PMID:Topoisomerase II-dependent novel antitumor compounds merocil and merodantoin induce apoptosis in Daudi cells. 785 88

Little is known about the regulation of apoptosis in fibroblasts although several model systems including serum deprivation and treatment with staurosporine or topoisomerase inhibitors have been used to induce apoptosis in vitro. To validate a reproducible in vitro model for the study of apoptosis in fibroblasts, we cultured density-inhibited monolayer cultures of Balb/c 3T3 fibroblasts in Dulbecco's modified essential medium plus 15% fetal calf serum and then withdrew serum. Time-lapse video microscopy demonstrated that within minutes of serum withdrawal, cells lost substrate attachment and floated to the top of the liquid growth medium. There was a time-dependent increase in the number of non-adherent cells. Some of these cells regained attachment and spread momentarily, but they eventually rounded up and lost attachment permanently. In contrast to serum-containing cultures in which similar morphological changes were followed by mitosis, in serum-free cultures repeated attempts at mitosis were followed by permanent attachment loss and presumably cell death. To assess whether all the non-adherent cells were in fact dead, the percentages of cells that continued to proliferate upon return to serum-supplemented conditions was computed. After various periods of serum starvation a decreasing proportion (approx. 75% at 30 minutes; < 2% at 24 hours) of the non-adherent cells could be rescued by addition of serum. Transmission electron microscopy of cells 3 hours after serum withdrawal showed that the majority (approximately 60%) of non-adherent cells exhibited marked intranuclear chromatin condensation but maintained integrity of cell and nuclear membranes and cell organelles, morphological changes consistent with those of apoptotic cell death. Scanning electron microscopy of cultures 3 hours following serum withdrawal showed rounded cells with marked surface blebbing. Fluorescence and confocal microscopy revealed increased intensity of nuclear staining with DAPI while actin filaments became indistinct or collapsed around the nucleus. After cycloheximide treatment to inhibit protein synthesis, there was no reduction of apoptosis. Gel electrophoresis of DNA from both control and 3 hour-serum-deprived cells showed intact DNA with no oligonucleosomal length fragmentation. After serum withdrawal, intracellular calcium was reduced by about 32% over 5 minutes as measured by fura2 ratio fluorimetry in single cells. Serum-starved cells showed a time-dependent shrinkage in mean cell diameter compared to trypsinized, adherent control cells (at 0 hours, mean diameter = 18.0 microns--viable; at 4 hours, mean diameter = 15.5 microns--apoptotic). Flow cytometric analysis showed increased propidium iodide staining and reduced fluorescein diacetate uptake over 3 hours, changes that were contemporaneous with the reduction of cell diameter.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Serum deprivation induces apoptotic cell death in a subset of Balb/c 3T3 fibroblasts. 792 26


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