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)

Pretreatment of the human lymphoblastoid cell line CCRF-CEM with 0.02 microM arabinosyl cytosine (ara C) enhances both the cytotoxic and the DNA-damaging effects of etoposide. This concentration of ara C is itself non-cytotoxic and results in no detectable DNA damage as measured by alkaline elution. Ara C pretreatment results in the synchronisation of cells, a 24-h pretreatment resulting in the accumulation of cells in the early S phase. The sensitivity of cells to etoposide-induced cytotoxicity was increased 2.5 times and DNA damage was enhanced 1.66 times by this pretreatment. Maximal potentiation of etoposide-induced DNA damage (2.06-fold increase) was observed after 48 h continuous treatment with ara C, but no further enhancement of cytotoxicity occurred. Cell-cycle analysis demonstrated that 48 h ara C treatment resulted in the accumulation of cells in the late S/G2M phase. Cells returned to a normal cell-cycle distribution within 24 h of the removal of ara C, and the potentiation of etoposide activity was then reduced to a 1.3- to 1.4-fold level. DNA damage induced by etoposide following ara C pretreatment was qualitatively identical to that produced by etoposide alone, suggesting a mechanism involving topoisomerase II. To investigate this possibility, we measured topoisomerase II protein levels by immunoblotting. Measurement of topoisomerase II levels in whole-cell lysates of ara C-pretreated cells showed a 3- to 5-fold increase in topoisomerase levels relative to total protein content. This suggests that elevated enzyme levels may be responsible for the increased sensitivity of ara C-pretreated cells to etoposide.
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PMID:Potentiation of etoposide-induced cytotoxicity and DNA damage in CCRF-CEM cells by pretreatment with non-cytotoxic concentrations of arabinosyl cytosine. 133 70

Hydroxyurea is a potent inhibitor of the enzyme ribonucleotide reductase. Due to its effects on cellular deoxyribonucleotide pools, hydroxyurea can modulate the activity of several pyrimidine and purine antimetabolites. As an inhibitor of DNA repair, it can potentially interact with DNA-damaging agents such as alkylating agents or inhibitors of topoisomerase II. Both cytokinetic and biochemical interactions occur between hydroxyurea and cytarabine (ara-C), which account for their synergistic cytotoxicity. Inhibition of ribonucleotide reductase by hydroxyurea depletes cellular deoxycytidine triphosphate pools, thereby enhancing ara-C uptake and phosphorylation to ara-C triphosphate. In a phase II clinical trial, the combination of hydroxyurea and ara-C produced a 43% response rate in patients with refractory malignant lymphoma. Studies in murine leukemia models have demonstrated therapeutic synergy when hydroxyurea is combined with fluoropyrimidines. High levels of deoxyuridine monophosphate that have been associated with resistance to 5-fluorouracil can be suppressed by hydroxyurea, leading to greater inhibition of thymidylate synthase. Despite the strong biochemical rationale for the use of hydroxyurea and 5-fluorouracil in combination, few clinical trials have been conducted thus far. Antimetabolites and topoisomerase II inhibitors have also been shown to be synergistic in vitro. Hydroxyurea has been shown to enhance the formation of DNA strand breaks produced by amsacrine and to produce synergistic cytotoxicity with etoposide. A phase I clinical trial of these drugs has demonstrated bone marrow suppression to be the major toxicity of the combination. In summary, hydroxyurea has been shown to undergo cytokinetic and biochemical interactions with a number of established antitumor agents. Clinical trials of hydroxyurea in combination with these agents have identified doses and schedules of administration that produce acceptable levels of clinical toxicity and appear feasible for further testing.
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PMID:Laboratory and clinical studies of biochemical modulation by hydroxyurea. 164 59

Defining specific biochemical targets of active antineoplastic agents could aid in discovering better anticancer therapy and more thoroughly understanding the biochemical basis of malignancy. Through a series of cellular and biochemical studies, we and others have identified the nuclear enzyme topoisomerase II as the target of several active agents, including 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA). The interference with topoisomerase II produced by m-AMSA can be quantified in whole cells exposed to m-AMSA by using the alkaline elution technique to measure DNA cleavage. Antimetabolites such as ara-C, hydroxyurea, and 5-azacytidine can augment m-AMSA-induced, topoisomerase II-mediated DNA cleavage and, concurrently, m-AMSA-induced cell killing. Studies in proliferating and quiescent human cells and an m-AMSA-sensitive/resistant human leukemia cell pair further support the hypothesis that a connection exists between topoisomerase II-mediated DNA cleavage and the mechanism by which m-AMSA kills cells. Pharmacologic or hormonal modification of specific biochemical processes critical to drug-induced cytotoxicity may enhance the therapeutic index of clinically useful agents.
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PMID:Intercalator-induced, topoisomerase II-mediated DNA cleavage and its modification by antineoplastic antimetabolites. 242 89

The ability of a noncytotoxic dose of ara-C to modulate the amount of 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA)- or etoposide-induced topoisomerase II-mediated DNA cleavage and cytotoxicity was examined in m-AMSA-sensitive and -resistant HL-60 human leukemia cells. Ara-C pretreatment (0.1 microM x 48 hr) sensitized m-AMSA-sensitive cells to the cytotoxicity and DNA cleavage produced by both m-AMSA and etoposide. The actions of m-AMSA in the m-AMSA-resistant cells were affected minimally by ara-C. By contrast, ara-C enhanced etoposide-induced DNA cleavage and, to an even greater extent, etoposide-induced cytotoxicity in m-AMSA-resistant cells. These cells were only minimally cross-resistant to etoposide. Ara-C did not affect the cellular uptake of m-AMSA or etoposide, the amount of 0.35 M NaCl-extractable nuclear topoisomerase II activity from either cell line, or the ability of this enzyme activity to covalently bind to DNA in the presence of the drugs, m-AMSA- and etoposide-induced DNA cleavage is thought to result from drug-induced stabilization of a topoisomerase II-DNA complex. The ability of ara-C to modulate this effect and associated cytotoxicity appears to be mediated by the effects of ara-C on cellular targets other than topoisomerase II but which are important to topoisomerase II-mediated events, such as protein-associated DNA cleavage. A good candidate for such a target may be cellular chromatin.
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PMID:Effect of 1-beta-D-arabinofuranosylcytosine (ara-C) on nuclear topoisomerase II activity and on the DNA cleavage and cytotoxicity produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide in m-AMSA-sensitive and -resistant human leukemia cells. 282 13

4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) and other DNA intercalating agents produce protein-associated DNA strand breaks, the formation of which are mediated by topoisomerase-like chromosomal proteins. As topoisomerases would be expected to be most active during DNA replication, DNA synthesis inhibitors may alter the sensitivity of cellular DNA to intercalator-induced scission. We report that treatment of L1210 cells with 1-beta-D-arabinofuranosylcytosine (ara-C) (0.1 microM) or hydroxyurea (HU) (0.1 mM) for 18 hr resulted in a 2- to 2.4-fold enhancement of m-AMSA-induced protein-associated DNA single-strand breaks and DNA-protein cross-links as measured by alkaline elution. This enhancement was dependent on the duration of ara-C or HU treatment as well as on the concentration of ara-C or HU. Enhancement did not correlate with any alteration in cellular uptake of intercalator or with ara-C- or HU-induced alterations in the DNA synthetic rate. The DNA within nuclei isolated from ara-C- or HU-treated cells also displayed an enhanced susceptibility to m-AMSA-induced scission. There was a correlation between enhanced single-strand break formation and recruitment of cells into S-phase as well as between single-strand break formation and the production of a hypomethylated state of cellular DNA. Concurrent with the enhancement of m-AMSA-induced cellular DNA effects was a synergistic effect on m-AMSA cytotoxicity by ara-C or HU. This enhancement of intercalator effects was also found for the intercalator Adriamycin. We propose that these sublethal concentrations of ara-C and HU alter chromatin structure possibly via DNA hypomethylation and/or altered DNA-histone interactions so that intercalator-induced DNA effects are enhanced. Alternatively, the topoisomerase-like activity involved in intercalator-induced, protein-associated DNA break production may be increased in the nuclei of ara-C- or HU-treated cells.
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PMID:Enhancement of the DNA breakage and cytotoxic effects of intercalating agents by treatment with sublethal doses of 1-beta-D-arabinofuranosylcytosine or hydroxyurea in L1210 cells. 620 99

We investigated, in a cloned hamster tracheal epithelial cell line HTE-B, the effects of inhibitors of DNA topoisomerase, novobiocin and nalidixic acid; of DNA polymerase, 1-beta-arabinofuranosylcytosine (ara-C) and 2',3'-dideoxythymidine; of ribonucleotide reductase, hydroxyurea; and of poly(ADP-ribose)synthetase, 3-aminobenzamide, upon the removal of benzo[a]pyrene adducted to DNA [B[a]P--DNA]. A substantial reduction in the rate of removal of the polycyclic hydrocarbon-adducts occurred when nalidixic acid was added to the HTE-B cells that had been previously incubated with B[a]P for 8 h. Novobiocin produced a similar, but less marked, effect. The rate of disappearance of the individual B[a]P--DNA adducts was measured by analysis of the h.p.l.c. profiles. Of the 5 major adducts observed under the h.p.l.c. conditions, 4 were reduced in control cells to 30% of the original levels by 24 h after removal of the B[a]P from the medium; adduct 5 was almost completely removed. In the presence of nalidixic acid, during the 24 h repair period, only the removal of adduct 5 was unimpaired; the removal of the other 4 adducts was significantly retarded. On the other hand, 3-aminobenzamide addition did not affect the rate of removal of B[a]P--DNA adducts from the HTE-B cells. We employed the combinations of ara-C and dideoxythymidine or ara-C and hydroxyurea to allow the accumulation of single strand breaks after incubation of the HTE-B cells with B[a]P. These breaks were assayed by alkaline elution analysis. Inclusion of these inhibitors during the 2 h after removal of the B[a]P from the medium resulted in the accumulation of 4-5 single strand breaks/10(10) daltons of HTE-B DNA. This compares with a minimum estimate of the number of adducts removed during this period of 3 adducts/10(7) daltons. This discrepancy may indicate that the majority of lesions are not repaired by a pathway sensitive to polymerase inhibitors. In the presence of 3-aminobenzamide, we routinely observed a 10% increase in the alkaline elution of the DNA obtained from B[a]P-treated cells (1-2 breaks/10(10) daltons). Our results indicate that an excision repair process may be involved in the removal of at least some of the B[a]P-induced damage to DNA. However, the repair of the multiple adducts is complex and may involve pathways other than classical excision repair.
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PMID:The influence of inhibitors on the repair of benzo[a]pyrene-damaged DNA in hamster tracheal epithelial cells. 632 Oct 50

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

A panel of six 'wild type' and three VP-16 resistant small cell lung cancer (SCLC) cell lines is used to evaluate to what extent in vitro sensitivity testing using a clonogenic assay can contribute to combine cytotoxic drugs to regimens with improved efficacy against SCLC. The resistant lines include (a) H69/DAU4, which is classical multidrug resistant (MDR) with a P-glycoprotein efflux pump (b) NYH/VM, which exhibits an altered topoisomerase II (topo II) activity and (c) H69/VP, which is cross-resistant to vincristine, exhibits a reduced drug accumulation as H69/DAU4 but is without P-glycoprotein. 19 anticancer agents were compared in the panel. The MDR lines demonstrated, as expected, cross-resistance to all topo II drugs, but also different patterns of collateral sensitivity to BCNU, cisplatin, ara-C, hydroxyurea, and to the topo I inhibitor camptothecin. The complete panel of nine cell lines clearly demonstrated diverse sensitivity patterns to drugs with different modes of action. Correlation analysis showed high correlation coefficients (CC) among drug analogues (e.g. VP-16/VM-26 0.99, vincristine/vindesine 0.89), and between drugs with similar mechanisms of action (e.g. BCNU/Cisplatin 0.89, VP-16/Doxorubicin 0.92), whereas different drug classes demonstrated low or even negative CC (e.g. BCNU/VP-16 -0.21). When the CC of the 19 drug patterns to VP-16 were plotted against the CC to BCNU, clustering was observed between drugs acting on microtubules, on topo II, alkylating agents, and antimetabolites. In this plot, camptothecin and ara-C patterns were promising by virtue of their lack of cross-resistance to alkylating agents and topo II drugs. Thus, the differential cytotoxicity patterns on this panel of cells can (1) give information about drug mechanism of action, (2) enable the selection and combination of non-cross-resistant drugs, and (3) show where new drugs 'fit in' among established agents.
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PMID:Differential cytotoxicity of 19 anticancer agents in wild type and etoposide resistant small cell lung cancer cell lines. 809 93

Only two classes of chemotherapeutic agents have shown activity in acute myeloid leukemia (AML): ara-C and topoisomerase II reactive agents. Frontline combinations of these agents produce complete response (CR) rates of 70% and long-term event free survival rates of 25%. New agents with different mechanisms of action are being explored. Nucleoside analogs such as chlorodeoxyadenosine (2-CdA) or fludarabine have shown single-agent efficacy and may be synergistic with ara-C. Combination therapy with ara-C and nucleoside analogs have shown promising results both as salvage therapy and in newly diagnosed patients. Combinations of topotecan with ara-C, VP16, and anthracyclines are being pursued, as is testing of other Topo-I inhibitors. Hypomethylating agents (5-azacytidine, decitabine) are showing activity in AML, producing CR rates of 5% to 30% as AML salvage therapy as a single agent, and 40%-60% in combinations. Decitabine may be synergistic with topo I inhibitors, biologic agents, and differentiating agents. Homoharringtonine has modest anti-AML activity, with CR rates of 10% to 30% as salvage therapy. Other classes of agents worthy of continuing investigation are platinum analogs and agents with novel mechanisms of action such as tallimustine.
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PMID:New chemotherapeutic agents in acute myeloid leukemia. 861 70

Antimetabolites and topoisomerase (topo) II-reactive drugs are frequently combined in the therapy of acute leukemia. The two types of agents are thought to be synergistic in their actions against malignant blasts but the mechanism for this synergism is incompletely described. This study sought to determine whether the combination of two rather than one anti-metabolite with the topo II-reactive intercalator mitoxantrone would be greater than the effect of the single antimetabolite ara-C on mitoxantrone's cytotoxic actions. We also aimed to determine a mechanism for synergism should it occur. The model system used was K562 human leukemia cells. The second anti-metabolite selected was F-ara-A, the active form of fludarabine. The resultant combination (F-ara-A, ara-C, and a topo II reactive drug) is one currently being tested against acute myelogenous leukemia in clinical trials. F-ara-A itself had little effect on the cytotoxicity or the topo II-mediated DNA cleaving actions of mitoxantrone, while ara-C potentiated these actions as it does those of other topo II-reactive drugs. Surprisingly F-ara-A enhanced the actions of ara-C on mitoxantrone-associated cytotoxicity by at least an order of magnitude. The effect of the addition of F-ara-A to ara-C on mitoxantrone-induced DNA cleavage was considerably smaller, but present. Antimetabolite treatment did not increase the amount of topo II within cells measured directly by immunoblotting or indirectly by quantifying the maximum number of topo II-DNA complexes stabilized by mitoxantrone. Rather, the anti-metabolites altered the distribution of the cells in the cell cycle. Antimetabolite treatment caused a large increase in S-phase cells, a phase in which cells are more sensitive to topo II-reactive drugs than the associated topo II-mediated DNA cleavage would predict. Therefore, it is likely that this shift in the distribution of the cells within the cell cycle accounts for both the enhanced cytotoxicity of mitoxantrone in antimetabolite pretreated cells and the discrepancy between the magnitude of antimetabolite action on topo II-mediated DNA cleavage.
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PMID:The effect of 9-beta-D-arabinofuranosyl-2-fluoroadenine and 1-beta-D-arabinofuranosylcytosine on the cell cycle phase distribution, topoisomerase II level, mitoxantrone cytotoxicity, and DNA strand break production in K562 human leukemia cells. 864 1


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