Roles of nonhomologous end-joining pathways in surviving topoisomerase II-mediated DNA damage.
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Topoisomerase II is a target for clinically active anticancer drugs. Drugs targeting these enzymes act by preventing the religation of enzyme-DNA covalent complexes leading to protein-DNA adducts that include single- and double-strand breaks. In mammalian cells, nonhomologous repair pathways are critical for repairing topoisomerase II-mediated DNA damage. Because topoisomerase II-targeting agents, such as etoposide, can also induce chromosomal translocations that can lead to secondary malignancies, understanding nonhomologous repair of topoisomerase II-mediated DNA damage may help to define strategies that limit this critical side effect on an important class of anticancer agents. Using Saccharomyces cerevisiae as a model eukaryote, we have determined the contribution of genes required for nonhomologous end-joining (NHEJ) for repairing DNA damage arising from treatment with topoisomerase II poisons, such as etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA). To increase cellular sensitivity to topoisomerase II poisons, we overexpressed either wild-type or drug-hypersensitive alleles of yeast topoisomerase II. Using this approach, we found that yku70 (hdf1), yku80 (hdf2), and other genes required for NHEJ were important for cell survival following exposure to etoposide. The clearest increase in sensitivity was observed with cells overexpressing an etoposide-hypersensitive allele of TOP2 (Ser740Trp). Hypersensitivity was also seen in some end-joining defective mutants exposed to the intercalating agent mAMSA, although the increase in sensitivity was less pronounced. To confirm that the increase in sensitivity was not solely due to the elevated expression of TOP2 or due to specific effects of the drug-hypersensitive TOP2 alleles, we also found that deletion of genes required for NHEJ increased the sensitivity of rad52 deletions to both etoposide and mAMSA. Taken together, these results show a clear role for NHEJ in the repair of DNA damage induced by topoisomerase II-targeting agents and suggest that this pathway may participate in translocations generated by drugs, such as etoposide. (+info)
AClAP, Autonomous hierarchical agglomerative Cluster Analysis based protocol to partition conformational datasets.
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MOTIVATION: Sampling the conformational space is a fundamental step for both ligand- and structure-based drug design. However, the rational organization of different molecular conformations still remains a challenge. In fact, for drug design applications, the sampling process provides a redundant conformation set whose thorough analysis can be intensive, or even prohibitive. We propose a statistical approach based on cluster analysis aimed at rationalizing the output of methods such as Monte Carlo, genetic, and reconstruction algorithms. Although some software already implements clustering procedures, at present, a universally accepted protocol is still missing. RESULTS: We integrated hierarchical agglomerative cluster analysis with a clusterability assessment method and a user independent cutting rule, to form a global protocol that we implemented in a MATLAB metalanguage program (AClAP). We tested it on the conformational space of a quite diverse set of drugs generated via Metropolis Monte Carlo simulation, and on the poses we obtained by reiterated docking runs performed by four widespread programs. In our tests, AClAP proved to remarkably reduce the dimensionality of the original datasets at a negligible computational cost. Moreover, when applied to the outcomes of many docking programs together, it was able to point to the crystallographic pose. AVAILABILITY: AClAP is available at the "AClAP" section of the website http://www.scfarm.unibo.it. (+info)
Zygomycosis in the immunocompromised patient: a case report.
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Zygomycosis is a highly aggressive infection observed in immunocompromised patients, such as those with haematological malignancies. The sites most frequently involved are the sinuses and the lungs. New diagnostic tools and new antifungal treatments are essential in order to diagnose early and treat efficiently infections due to moulds. We report a case of sinusitis due to Absidia corymbifera occurring during chemotherapy-induced bone marrow aplasia in a patient with acute leukaemia. The sinusitis was successfully treated with AmBisome, and surgical debridement. (+info)
Involvement of nucleic acid synthesis in cell killing mechanisms of topoisomerase poisons.
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The primary cytotoxic mechanism of camptothecin has been proposed to involve an interaction between the replication machinery and the camptothecin-mediated topoisomerase I-DNA cleavable complex (Y. H. Hsiang, M.G. Lihou, and L.F. Liu, Cancer Res., 49:5077-5082, 1989). In the present study, we show that killing of V79 cells by the topoisomerase II poisons 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide may involve ongoing RNA synthesis in addition to ongoing DNA synthesis. V79 cells synchronized by mitotic shake-off were treated with topoisomerase poisons in the presence of inhibitors of nucleic acid synthesis. S-Phase V79 cells were more sensitive to the topoisomerase I poison camptothecin and the topoisomerase II poison m-AMSA than G1-phase cells. The greater sensitivity of S-phase cells to killing by m-AMSA and camptothecin was abolished during cotreatment, but not posttreatment, with aphidicolin, suggesting that ongoing DNA synthesis in involved in cell killing by both topoisomerase I and II poisons. Cotreatment with transcription inhibitors, such as 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole or cordycepin, partially protected cells from the cytotoxic effects of m-AMSA but had no effect on camptothecin-mediated cytotoxicity. These results suggest that ongoing RNA transcription may be involved in cell killing by topoisomerase II poisons but not topoisomerase I poisons. Cotreatment with camptothecin reduced m-AMSA-mediated cytotoxicity in G1-phase V79 cells, suggesting a possible antagonism between topoisomerase I and II poisons. This antagonistic effect between topoisomerase I and II poisons could be explained by the strong inhibitory effect of camptothecin on RNA transcription. (+info)
Antagonism between camptothecin and topoisomerase II-directed chemotherapeutic agents in a human leukemia cell line.
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To search for possible synergy between topoisomerase (topo) II-directed chemotherapeutic agents and topo I-directed agents, IL-60 human progranulocytic leukemia cells were incubated with etoposide in the absence or presence of camptothecin (CPT). Treatment of HL-60 cells for 1 h with 15-20 microM etoposide resulted in the death of 99-99.9% of the cells as assessed by colony formation in soft agar. Unexpectedly, simultaneous incubation with 1 microM CPT increased the survival of etoposide-treated cells as much as 30-fold. Inhibition of etoposide cytotoxicity was observed at CPT concentrations as low as 0.01 microM and was one-half maximal at 0.1 microM. CPT also antagonized the cytotoxicity of 4'-(9-acridinylamino)methanesulfon-M-anisidide and daunorubicin, two structurally unrelated topo II-directed agents. Topotecan, a CPT analogue currently undergoing Phase I clinical trials, had a similar effect. Studies using an alkaline unwinding assay (to measure DNA strand breaks) and Western blotting (to assess formation of covalent adducts involving topo II) revealed that CPT did not alter the ability of etoposide to stabilize topo II-DNA adducts. CPT is a potent inhibitor of both DNA and RNA synthesis. To further assess the mechanism by which CPT diminished the cytotoxicity of topo II-directed agents, inhibitors of DNA synthesis or RNA synthesis were substituted for CPT. Aphidicolin, an inhibitor of replicative DNA polymerases, enhanced the survival of etoposide-treated HL-60 cells less than 3-fold. In contrast, inhibitors of RNA synthesis (cordycepin or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) enhanced the survival of etoposide-treated HL-60 cells as much as 20-fold. The potential biological and therapeutic implications of these results are discussed. (+info)
The antileukaemic activity of 5-Aza-2 deoxycytidine (Aza-dC) in patients with relapsed and resistant leukaemia.
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In the present study we demonstrate that Aza-dC in combination with Amsacrine has major antileukaemic properties in patients who have not already received extensive Ara-C therapy. Eight out of 11 patients in their first relapse of acute leukaemia achieved complete remission. Cross resistance between Ara-C and Aza-dC was revealed by the lack of antileukaemic activity in five patients with with Ara-C resistant leukaemia. Combination therapy with Aza-dC/Ams-acrine induced a considerable period of a granulocytopenia (28-35 days), while the toxic effect on erythro- and megakaryopoiesis was comparable to that reported for high dose Ara-C/Amsacrine chemotherapy. Remarkable is the long disappearance time for leukaemic blast cells in bone marrow, i.e. 3-5 weeks in some cases. Analysis of cell membrane markers showed a loss of the early differentiation antigens CD34 and CD33 from leukaemic bone marrow cells after 7 days of Aza-dC treatment, which is suggestive of leukaemic cell differentiation. In the small group of patients tested for DNA hypomethylation no association existed between the degree of hypomethylation and clinical response. Non-haematologic side effects were considerable in patients receiving the highest dosages of Aza-dC and consisted of severe, although usually reversible, gastrointestinal and neurological complications. In comparison with Ara-C, Aza-dC causes less nausea and vomiting and is therefore better tolerated. (+info)
Topoisomerase II and tubulin inhibitors both induce the formation of apoptotic topoisomerase I cleavage complexes.
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Topoisomerase I (Top1) is a ubiquitous enzyme that removes DNA supercoiling generated during transcription and replication. Top1 can be trapped on DNA as cleavage complexes by the anticancer drugs referred to as Top1 inhibitors as well as by alterations of the DNA structure. We reported recently that Top1 cleavage complexes (Top1cc) are trapped during apoptosis induced by arsenic trioxide and staurosporine. In the present study, we generalize the occurrence of apoptotic Top1cc in response to anticancer drugs, which by themselves do not directly interact with Top1: the topoisomerase II inhibitors etoposide, doxorubicin, and amsacrine, and the tubulin inhibitors vinblastine and Taxol. In all cases, the Top1cc form in the early phase of apoptosis and persist throughout the apoptotic process. Their formation is prevented by the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone and the antioxidant N-acetyl-L-cysteine. We propose that the trapping of Top1cc is a general process of programmed cell death, which is caused by alterations of the DNA structure (oxidized bases and strand breaks) induced by caspases and reactive oxygen species. (+info)
Autologous stem cell transplantation after complete remission and first consolidation in acute myeloid leukemia patients aged 61-70 years: results of the prospective EORTC-GIMEMA AML-13 study.
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BACKGROUND AND OBJECTIVES: The optimal post-remission treatment for elderly patients with acute myeloid leukemia (AML) is presently unknown. Recent studies have reported the feasibility of autologous peripheral blood stem cell transplantation (PBSCT) in this population. We evaluate the outcome of this post-remission approach after complete remission (CR) and consolidation in elderly patients included in the EORTC-GIMEMA AML-13 trial. DESIGN AND METHODS: PBSCT after induction and consolidation chemotherapy was evaluated in patients aged 61 to 70 years with a WHO performance status 0-1. The induction therapy was mitoxantrone, etoposide and cytarabine (MICE) with or without granulocyte colony-stimulating factor (G-CSF) during and/or after chemotherapy. The consolidation therapy consisted of non-infusion or infusional idarubicin, etposide and cytarabine (mini-ICE). RESULTS: Sixty-one patients were scheduled for stem cell harvest by leukapheresis after s.c. recombinant human G-CSF administration initiated after hematopoietic recovery from consolidation. Stem cells were effectively harvested from 54 patients. A median of two aphereses (range, 1-5) were performed, resulting in a median collection of 11.7 x 10(8) nucleated cells/kg (range, 2.4-99.8) containing 40.2 x 10(4) CFU-GM/kg (range, 0-786.8), and 5 x 10(6) CD34+ cells/kg (range, 0.1-99.8). For the whole group of 61 patients, the median disease-free survival (DFS) was 1.0 years and the 3-year DFS rate was 21%, while the median overall survival (OS) was 1.4 years and the 3-year OS rate was 32%. A total of 26 patients could not be autografed due to inadequate/no harvest (21 patients), early relapse (3 patients), or treatment refusal (2 patients). Autologous transplantation was performed in 35 patients following conditioning with the BAVC regimen. The median time for granulocyte recovery >0.5 109 yen/L was 24 days and for platelets >20 x10(9)/L was 23 days following transplantation. After a median follow-up of 5.0 years from transplantation, the median DFS and OS were 1.1 and 1.6 years respectively, and the 3-year rates were 28% and 39% respectively. Eight autografted patients were still in continuous complete remission, 22 patients had relapsed and five had died in CR. INTERPRETATION AND CONCLUSIONS: Intensification of remission including autologous PBSCT is feasible in about half of harvested patients aged 61 to 70 years old, and did not improve the general outcome. This shows the limitations of autologous PBSCT and other intensive treatment modalities in elderly AML patients. Key words: acute myeloid leukemia, elderly, autologous stem cell transplantation. (+info)