Novel selective inhibitors for human topoisomerase I, BM2419-1 and -2 derived from saintopin.
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Compounds BM2419-1 and -2 were isolated from a culture broth of a fungus Paecilomyces sp. BM2419. It was shown that these novel compounds were artifacts derived from saintopin, a dual inhibitor of topoisomerase I and II by independent processes. In the human topoisomerase I inhibition assay using the recombinant Saccharomyces cerevisiae, BM2419-1 and -2 inhibited selectively the yeast growth dependent on human topoisomerase I induction with IC50 values of 0.3 ng/ml and 6.0 ng/ml, respectively. (+info)
Detection of poly(ADP-ribose) polymerase cleavage in response to treatment with topoisomerase I inhibitors: a potential surrogate end point to assess treatment effectiveness.
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Cleavage of poly(ADP-ribose) polymerase (PARP) by caspases is a prominent characteristic of apoptosis or programmed cell death shown to be induced by topoisomerase (Topo) inhibitors. Because Topo I inhibitors have been shown to be effective in the treatment of some patients with colon cancer, we considered the possibility of using PARP cleavage as an early predictor of responsiveness to this class of agents. We show cleavage of PARP in response to treatment with Topo I inhibitors in colon cancer both in vitro and in vivo: (a) in vitro in SW480, HCT116, VACO5, VACO6, VACO8, VACO411, VACO425, and VACO451 human colon cancer cell lines treated with topotecan (TPT) or CPT-11; (b) in vivo in SW480, VACO451, and VRC5 colon cancer xenografts grown in athymic mice treated with TPT or CPT-11; and (c) in vivo in colon cancer samples from patients undergoing a Phase II clinical trial with CPT-11. Our results show a strong correlation between percentage of PARP cleavage and percentage of acridine orange-positive cells in colon cancer cell lines treated with 0.1 microM TPT for 24 and 48 h, confirming that PARP cleavage is a useful marker for programmed cell death in colon cancer cell lines. Results from experiments performed on colon cancer xenografts also show an association between PARP cleavage and response to treatment with TPT or CPT-11. The increase of PARP cleavage in xenografts and in clinical samples corresponding to treatment with Topo I inhibitors suggests that this procedure may have early predictive value to assess effectiveness of treatment. These results provide the basis for determining the validity of using PARP cleavage as an early marker of chemotherapeutic effectiveness in human samples. (+info)
Degradation of topoisomerase I induced by topoisomerase I inhibitors is dependent on inhibitor structure but independent of cell death.
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DNA topoisomerase I (top I) is the target of the antitumor drug camptothecin (CPT) and its analogs. CPT induces dose- and time-dependent degradation of top I. Degradation of top I also occurs in a CPT-resistant cell line and, therefore, is not a consequence of cell death. Top I degradation is preceded by the appearance of a high molecular weight ladder of top I immunoreactivity and can be blocked by specific inhibitors of the proteasome. We compared the effects of five top I poisons [CPT, topotecan, 6-N-formylamino-12,13-dihydro-1, 11-dihydroxy-13-(beta-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3, 4-c]carbazole-5,7(6H)-dione (NB506), camptothecin-(para)-4beta-amino-4'-O-demethyl Epipodophyllotoxin (W1), and camptothecin-(ortho)-4beta-amino-4'-O-demethyl Epipodophyllotoxin (W2)] on cleavable complex formation and top I degradation. Although all five drugs induced cleavable complex formation, two of the drugs, NB506 and W1 did not induce top I degradation. (+info)
Inhibition of inducible nitric oxide synthase by beta-lapachone in rat alveolar macrophages and aorta.
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Beta-lapachone, a plant product, has been shown to be a novel inhibitor of DNA topoisomerase. In this study, we performed experiments to examine the effects of beta-lapachone on lipopolysaccharide (LPS)-induced inducible nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages and aortic rings. In alveolar macrophages, incubation with LPS (10 microg ml(-1)) for various time intervals resulted in a significant increase in nitrite production and iNOS protein synthesis, that was inhibited by coincubation with beta-lapachone (1-4.5 microM) without any cytotoxic effects. However, addition of beta-lapachone after induction of NO synthase by LPS failed to affect the nitrite production. Treatment with LPS (10 microg ml(-1)) for 6 h resulted in significant expression of mRNA for iNOS which was significantly inhibited in the presence of beta-lapachone (3 microM) in alveolar macrophages. In endothelium-intact rings of thoracic aorta, beta-lapachone (1 and 3 microM) markedly inhibited the hypocontractility to phenylephrine in aortic rings treated with LPS (10 microg ml(-1)) for 4 h. When beta-lapachone was added 3 h after LPS into the medium, the contractions evoked by phenylephrine were not significantly different in the presence or absence of beta-lapachone. Treatment with LPS (10 microg ml(-1)) for 4 h resulted in a significant increase in iNOS protein synthesis which was inhibited in the presence of beta-lapachone (3 microM), but did not affect the constitutive (endothelial and neuronal) NOS forms in aortic rings. These results indicate that beta-lapachone is capable of inhibiting expression and function of iNOS in rat alveolar macrophages and aortic rings. It is considered that beta-lapachone can be developed as a potential anti-inflammatory agent in the future. (+info)
Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells.
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Stable transfected human p53 (mt/mt) B lymphoma Namalwa variant lines showing differential expression of the Bax-alpha protein were derived under hygromycin selection. Overexpression of Bax-alpha in these variant cells accelerates cell death induced by short or continuous treatments with various concentrations of camptothecin, etoposide, vinblastine and shows no accelerating cell death activity in cis-platinum and paclitaxel-treated cells. Activation of apoptosis and oligonucleosome-sized DNA fragmentation was observed in the variant lines with more pronounced effect in cells containing high level of Bax-alpha protein. These results suggest that increased cell death mediated by anticancer drugs correlates with Bax-alpha level of expression and that Bax-alpha sensitizes Namalwa cells treated at low drug concentrations. The extent of DNA synthesis inhibition following DNA topoisomerase inhibitor treatments was similar in control and all transfected Namalwa cells suggesting that Bax-alpha acts downstream of DNA topoisomerase-mediated DNA strand breaks. To define further the relation between Bax-alpha expression and apoptosis activation, kinetics of caspase activation was measured in drug-treated cells. Caspase activities were measured using specific fluorogenic peptide derivatives DABCYL-YVADAPV-EDANS and Ac-DEVD-AMC, substrates of the caspase 1-like and caspase 3-like families, respectively. In control and Bax-alpha transfected Namalwa cells no increase in caspase 1-like activity was detected following camptothecin and etoposide treatments. In contrast, a significant difference in Ac-DEVD-AMC hydrolysis activity was observed in Bax-alpha transfected Namalwa cells compared to that of control Namalwa cells after camptothecin and etoposide treatment. Increased caspase 3-like activity correlated also with poly(ADPribosyl) polymerase cleavage. Taken together, these results suggest that Bax-alpha sensitize B lymphoma cells to series of anticancer drugs and accelerates the activation of apoptotic protease cascade. (+info)
Escherichia coli DNA topoisomerase I copurifies with Tn5 transposase, and Tn5 transposase inhibits topoisomerase I.
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Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. Genetic evidence suggested that this killing involves titration of E. coli topoisomerase I (Topo I). Here, we present biochemical evidence that supports this model. Tn5 Tnp copurifies with Topo I while nonkilling derivatives of Tnp, Delta37Tnp and Delta55Tnp (Inhibitor [Inh]), show reduced affinity or no affinity, respectively, for Topo I. In agreement with these results, the presence of Tnp, but not Delta37 or Inh derivatives of Tnp, inhibits the DNA relaxation activity of Topo I in vivo as well as in vitro. Other proteins, including RNA polymerase, are also found to copurify with Tnp. For RNA polymerase, reduced copurification with Tnp is observed in extracts from a topA mutant strain, suggesting that RNA polymerase interacts with Topo I and not Tnp. (+info)
Calories from carbohydrates: energetic contribution of the carbohydrate moiety of rebeccamycin to DNA binding and the effect of its orientation on topoisomerase I inhibition.
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BACKGROUND: Only a few antitumor drugs inhibit the DNA breakage-reunion reaction catalyzed by topoisomerase. One is the camptothecin derivative topotecan that has recently been used clinically. Others are the glycosylated antibiotic rebeccamycin and its synthetic analog NB-506, which is presently in phase I of clinical trials. Unlike the camptothecins, rebeccamycin-type compounds bind to DNA. We set out to elucidate the molecular basis of their interaction with duplex DNA, with particular emphasis on the role of the carbohydrate residue. RESULTS: We compared the DNA-binding and topoisomerase-I-inhibition activities of two isomers of rebeccamycin that contain a galactose residue attached to the indolocarbazole chromophore via an alpha (axial) or a beta (equatorial) glycosidic linkage. The modification of the stereochemistry of the chromophore-sugar linkage results in a marked change of the DNA-binding and topoisomerase-I- poisoning activities. The inverted configuration at the C-1' of the carbohydrate residue abolishes intercalative binding of the drug to DNA thereby drastically reducing the binding affinity. Consequently, the alpha isomer has lost the capacity to induce topoisomerase-I-mediated cleavage of DNA. Comparison with the aglycone allowed us to determine the energetic contribution of the sugar residue. CONCLUSIONS: The optimal interaction of rebeccamycin analogs with DNA is controlled to a large extent by the stereochemistry of the sugar residue. The results clarify the role of carbohydrates in stereospecific drug-DNA interactions and provide valuable information for the rational design of new rebeccamycin-type antitumor agents. (+info)
The ATM protein is required for sustained activation of NF-kappaB following DNA damage.
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Cells lacking an intact ATM gene are hypersensitive to ionizing radiation and show multiple defects in the cell cycle-coupled checkpoints. DNA damage usually triggers cell cycle arrest through, among other things, the activation of p53. Another DNA-damage responsive factor is NF-kappaB. It is activated by various stress situations, including oxidative stress, and by DNA-damaging compounds such as topoisomerase poisons. We found that cells from Ataxia Telangiectasia patients exhibit a defect in NF-kappaB activation in response to treatment with camptothecin, a topoisomerase I poison. In AT cells, this activation is shortened or suppressed, compared to that observed in normal cells. Ectopic expression of the ATM protein in AT cells increases the activation of NF-kappaB in response to camptothecin. MO59J glioblastoma cells that do not express the DNA-PK catalytic subunit respond normally to camptothecin. These results support the hypothesis that NF-kappaB is a DNA damage-responsive transcription factor and that its activation pathway by DNA damage shares some components with the one leading to p53 activation. (+info)