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)

Apoptosis is characterized by the nonrandom cleavage of DNA. After continuous treatment of MOLT-4 human T lymphoblastoid cells with the topoisomerase II inhibitor etoposide (50 microM) and the nongenotoxic agent N-methylformamide (300 mM), apoptosis was confirmed by electron microscopy. Analysis of DNA integrity by conventional gel electrophoresis failed to detect internucleosomal DNA cleavage. Resolution of DNA by field inversion gel electrophoresis showed fragments of 50 kilobases (kb). Etoposide induced the transient appearance of an additional DNA band of > 600 kb, which was temporally coincident with DNA-protein complex formation and was rapidly reversible upon drug removal. This DNA band was not observed after N-methylformamide treatment. In situ DNA end-labeling showed the incorporation of biotinylated dUTP into 50-kb DNA fragments but not etoposide-induced DNA fragments of > 600 kb. DNA end-labelling with terminal deoxynucleotidyltransferase was therefore not dependent upon intenucleosomal DNA cleavage, and fragments of approximately 50 kb were characterized by free 3'-OH termini that were not occluded by topoisomerase II protein. Although we considered that topoisomerase II potentially played an active role in the fragmentation of higher order chromatin during apoptosis, the results showed that DNA cleavage by topoisomerase II induced reversible, protein-associated fragments of > 600 kb and not irreversible cleavage to 50-kb fragments. The reversible cleavage of DNA to fragments of > 600 kb appears to be a signal for the engagement of apoptosis and is not an initial step in the sequential unwinding of chromatin.
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PMID:Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis. 774 85

The apoptosis-associated DNA strand breaks were detected in situ, in individual leukemic cells in peripheral blood and bone marrow of over 110 patients with different types of leukemia (ALL, AML, CML in blastic crisis, APL), prior to and during routine chemotherapy. The DNA strand breaks were labeled with digoxigenin- or biotin-conjugated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase, and the cells, counterstained for DNA, were analyzed by bivariate flow cytometry. The proportion of cells with DNA strand breaks prior to therapy, most likely reflecting spontaneous apoptosis, varied from 0.1 to 16%, but in the large majority of cases was below 3%. Administration of drugs of different classes, which included DNA topoisomerase I (Topotecan) and II (mitoxantrone, VP-16) inhibitors, antimetabolite (ara-C) or microtubule poison (Taxol), all triggered the appearance of cells with extensive DNA breakage, typical of apoptosis, to up to 80%. The peak of the response, measured as maximal percent of cells with DNA strand breaks, which varied between individual patients by as much as factor 10, was generally seen between 8 to 24 h after the initial administration of DNA topoisomerase inhibitors, and somewhat later (48-72 h) during the response to Taxol or ara-C. Thus, the data show that the response to treatment with a variety of drugs, in terms of induction of apoptosis, can be conveniently measured by the present method. The prognostic value of the apoptotic index, before, as well as during treatment, is being estimated for each type of leukemia, in the ongoing prospective studies.
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PMID:Apoptotic cell death during treatment of leukemias. 807 83

DNA topoisomerase I inhibitor camptothecin (CAM), topoisomerase II inhibitors teniposide (TN) and amsacrine (m-AMSA) induce apoptosis of HL-60 cells. One of the early events of apoptosis is DNA degradation, which occurs as a result of activation of the specific endonuclease. DNA strand breaks generated during this process were revealed, in the present study, by the in situ nick translation assay which was adapted to flow cytometry. In this assay, the incorporation of biotinylated dUTP by apoptotic cells was detected by the use of fluorescinated avidin, whereas simultaneous staining of DNA with propidium iodide made it possible to correlate the appearance of DNA strand breaks with cell position in the cell cycle. The breaks were detected as early as 90 min after the initial cell contact with CAM, and they were limited to cells in the S phase of the cell cycle. At that early stage of apoptosis DNA was not yet extractable from the cells; the loss of DNA from S-phase cells could not be seen, by flow cytometry, during the initial 2 h of incubation with CAM. DNA strand breaks induced by TN and m-AMSA also occurred preferentially in S-phase cells. The data indicate that DNA strand breaks resulting from activation of endonuclease in HL-60 cells treated with DNA topoisomerase I or II inhibitors can be conveniently measured using the in situ nick translation assay. This assay has certain advantages over other methods of identification of apoptotic cells by flow cytometry, such as providing direct evidence of DNA damage and offering the opportunity to correlate DNA damage with cell position in the cell cycle. The method may be of interest in clinical oncology where testing tumor response (by DNA degradation) to DNA topoisomerase inhibitors or other treatments may be of prognostic value.
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PMID:Apoptosis of S-phase HL-60 cells induced by DNA topoisomerase inhibitors: detection of DNA strand breaks by flow cytometry using the in situ nick translation assay. 838 87

DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays. During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope. In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA. Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle. The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay. Camptothecin, teniposide, and 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle. In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle. The method of detection of DNA strand breaks (3'-hydroxyl termini) in individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker. The protein-associated DNA breaks in the "cleavable" DNA-topoisomerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods.
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PMID:Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. 846 13

A new flow cytometric method is described to detect DNA strand breaks associated with apoptosis, by labeling the 3'-OH termini in the breaks with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase. The method has been applied in studies on leukemic HL-60 and MOLT-4 cell lines to reveal whether it is specific to apoptotic cells, and whether it can be used in the clinic to detect DNA breakage in leukemic cells during chemotherapy. There was labeling of mononuclear cells in peripheral blood of all 11 patients studied during chemotherapy for acute lymphoblastic, acute myelogenous, or chronic myelogenous leukemia (ALL, AML, or CML) in blastic crisis, indicating induced DNA damage; the number of labeled cells increased from 1-8% before treatment up to 80% during the course of treatment. The DNA topoisomerase inhibitors mitoxantrone, VP-16 (etoposide), and m-AMSA (amsacrine) were more effective in inducing DNA breaks than was hydroxyurea or cytosine arabinoside (AraC). Cells with DNA breaks were identified in peripheral blood for up to 5 days following administration of Mitoxantrone and VP-16. In the case of DNA aneuploid leukemias, the DNA breaks were predominant in the aneuploid cell subpopulations, whereas presumably non-neoplastic diploid cells were unlabeled. In one case of ALL there were two distinct subpopulations of aneuploid cells: one responded to the treatment (by DNA breakage) and the other was non-responding. Thus, cells undergoing apoptosis can be detected by this method of labeling DNA strand breaks and the technique is applicable for analysis of response of leukemic cells to chemotherapy. With this method it may be possible to identify tumor cell sensitivity or resistance to particular drugs early in the course of treatment.
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PMID:Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. 848 18

A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.
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PMID:Purification and characterization of human topoisomerase I mutants. 861 7

We related cellular content of DNA topoisomerase (topo) IIalpha and IIbeta with the cell cycle position in proliferating, differentiated, and apoptotic HL-60 cells using two-dimensional flow cytometry. In logarithmically growing HL-60 cells, topo IIalpha increased especially in late S to G2/M phases, although the topo IIbeta level was almost constant throughout the cell cycle. Induction of differentiation by all-trans retinoic acid dramatically reduced the topo IIalpha but not the topo IIbeta level. A new G2/M population containing virtually no topo IIalpha appeared during differentiation and was supposed to be alive and noncycling. Two-dimensional flow cytometry of topo IIalpha or IIbeta staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay showed that one topo IIbeta epitope situated at the C-terminal end decreased specifically in apoptotic HL-60 cells treated with Ara-C, etoposide, and vincristine. The amounts of a topo IIalpha epitope and another topo IIbeta epitope located at a more central portion were almost equal between apoptotic and nonapoptotic cells. Western blot analysis confirmed that topo IIbeta protein was completely degraded into smaller fragments and lost its C-terminal end during apoptosis. On the contrary, a large portion of topo IIalpha remained of its original size, although both topo IIalpha and IIbeta left from the nuclear fraction in apoptotic cells. Confocal laser microscopy showed nuclear localization of topo IIalpha and IIbeta in growing HL-60 cells. Although topo IIalpha and IIbeta were distributed throughout the cell during mitosis, only topo IIalpha was densely concentrated in the mitotic chromosomes. Both enzymes were dissociated from the genomic DNA even at an early phase of apoptosis and completely separated from the propidium iodide signal of DNA in the advanced stage. Chromatin condensation process in apoptosis is therefore completely topo II-independent and obviously differs from the mitotic one.
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PMID:Temporal and spatial distribution of DNA topoisomerase II alters during proliferation, differentiation, and apoptosis in HL-60 cells. 945 72

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle-dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein-dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase beta is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.
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PMID:Changes in organization of Crithidia fasciculata kinetoplast DNA replication proteins during the cell cycle. 981 50

Doxorubicin induces DNA breakage by stabilizing a cleavable topoisomerase II-DNA complex. In contrast, topoisomerase II catalytic inhibitor ICRF-193 and uncoupling inhibitor aclarubicin interfere with the cleavable complex formation. We analysed combination effects of these drugs using two-dimensional flow cytometry of DNA content and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling assay. Both ICRF-193 and aclarubicin attenuated the cytotoxic effect of doxorubicin on HL-60 cells (85% and 46% maximum reduction, respectively), which suggested that doxorubicin exerts its cytotoxic effect at least partially through the topoisomerase II-dependent DNA cleavage. Doxorubicin and ICRF-193 both induced G2 arrest in HL-60 cells, by which they may have reduced the cytotoxic effect of vincristine. Indeed, although ICRF-193 inhibited doxorubicin-induced apoptosis, ICRF-193 and doxorubicin cooperated in arresting HL-60 cells at G2 phase. These results indicated that G2 arrest was caused not only by DNA damage but also through a DNA damage-free, topoisomerase II inactivation-induced pathway. Western blot analysis showed that both types of G2 arrest were mediated by the inhibition of p34cdc2 dephosphorylation.
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PMID:Inhibition of p34cdc2 dephosphorylation in DNA damage- and topoisomerase II inactivation-induced G2 arrests in HL-60 cells. 1035 37

We report studies of schwannomas with a high percentage of MIB-1 positive cells. Thirty-eight specimens from 36 cases of schwannoma in the intracranial and spinal regions comprise the substance of this study. The MIB-1 positive cells were measured using immunohistochemical staining. In nine cases with a positivity index (PI) of 5% or more, immunohistochemical staining using DNA topoisomerase IIalpha (topo-II) and CD68 was performed. In some cases, we also searched for apoptosis with the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method. Three of nine cases with 5% or more positive MIB-1 cells had a very high cellularity with mitotic figures and were considered cellular Schwannomas. Their MIB-1 PI values were 8.21%, 10.00%, and 21.37%. However, the remaining six cases showed little evidence of malignancy. Their PIs were comparatively low, ranging from 5.19% to 8.41%, and the positive findings were localized in many cases. In these cases, we examined the sites where MIB-1 was measured and found that they corresponded to the borderline site between Antoni type A and B patterns and tended to be associated with an infiltration of CD68-positive macrophage. Furthermore, apoptotic cells appeared in the sites. With topo-II staining, the PIs in the same sites of these six cases were low, ranging from 0.78% to 1.93%. This implies that the high MIB-1 PI that was seen in these six cases was caused by reaction of MIB-1 to tumor cells that brought about an abnormality in the cell cycle by degeneration, such as apoptosis. In the site of formation of Antoni type B, MIB-1 may be a false positive in tumors with degenerative findings such as schwannomas. Topo-II was useful in these cases.
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PMID:Analyses of proliferative potential in schwannomas. 1098 8


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