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)

DNA damage is believed to be the main cause of the antiproliferative effect of cisplatin, a cornerstone agent in anticancer therapy. However, cisplatin can be expected to react also with nucleophiles other than DNA. Using enucleated cells (cytoplasts) we demonstrate here that cisplatin-induced apoptotic signaling may occur independently of DNA damage. Cisplatin-induced caspase-3 activation in cytoplasts required calcium and the activity of the calcium-dependent protease calpain. It is known that calpain activation may be associated with endoplasmic reticulum (ER) stress, suggesting that the ER is a cytosolic target of cisplatin. Consistent with this hypothesis, cisplatin induced calpain-dependent activation of the ER-specific caspase-12 in cytoplasts as well as in intact cells. Cisplatin also induced increased expression of Grp78/BiP, another marker of ER stress. By contrast, the DNA-damaging topoisomerase II inhibitor etoposide did not induce apoptotic signaling in cytoplasts nor ER stress in intact cells. We have thus identified a novel mechanism of action of cisplatin. The results have implications for the understanding of resistance mechanisms as well as the unique efficiency of this drug.
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PMID:Cisplatin induces endoplasmic reticulum stress and nucleus-independent apoptotic signaling. 1250 15

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.
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PMID:Detailed in vitro pharmacological analysis of FK506-induced neuroprotection. 1287 56

After several weeks of treatment, levels of alanine aminotransferase (ALT) increase in 50% of patients treated with tacrine for Alzheimer's disease. We looked for progressive effects on DNA to explain delayed toxicity. We first studied the in vitro effects of tacrine on DNA replication and topoisomerase-mediated DNA relaxation. We then treated mice with doses of tacrine reproducing the human daily dose on a body area basis and studied the effects of tacrine administration for up to 28 days on hepatic DNA, mitochondrial function, and cell death. In vitro, tacrine impaired DNA polymerase gamma-mediated DNA replication and also poisoned topoisomerases I and II to increase the relaxation of a supercoiled plasmid. In vivo, administration of tacrine markedly decreased incorporation of [(3)H]thymidine into mitochondrial DNA (mtDNA), progressively and severely depleted mtDNA, and partly unwound supercoiled mtDNA into circular mtDNA. Incorporation of [(3)H]thymidine into nuclear DNA (nDNA) was barely decreased, and nDNA levels were unchanged. After 12 to 28 days of treatment, administration of tacrine increased p53, Bax, mitochondrial permeability transition, cytosolic cytochrome c, and caspase-3 activity and triggered hepatocyte apoptosis and/or necrosis. In conclusion, the intercalating drug tacrine poisons topoisomerases and impairs DNA synthesis. Tacrine has been shown to accumulate within mitochondria, and it particularly targets mtDNA. After several weeks of treatment, the combination of severe mtDNA depletion and a genotoxic stress enhancing p53, Bax, and permeability transition trigger hepatocyte necrosis and/or apoptosis.
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PMID:Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver. 1293 98

The correlation between the histological features and clinical outcome remains poor in pediatric intracranial ependymomas. We performed a retrospective study of a group of 31 patients (diagnosed from 1985 to 1995) to assess prognostic implications of the current grading system, of histological and immunohistochemical features, and of ploidy status estimated by flow cytometry. Immunoexpression of a broad spectrum of antigens was evaluated, including MIB-1, topoisomerase-IIalpha, cyclin D1, glial and epithelial proteins (GFAP, EMA, cytokeratins), molecules involved in controlling apoptosis (bcl-2, caspase-3/CPP32), and p53 oncoprotein. Univariate and multivariate statistical analyses were performed to evaluate the influence of each variable on both the progression free survival (PFS) and the overall survival (OS) with at least 7-year follow up. Although we showed a significant correlation between histological grade and prognosis, the current grading system failed in predicting outcome in nearly one third of individual cases. Problems with interpathologist reproducibility were also demonstrated. The extent of surgical resection was the only clinical factor that was associated with survival. Both the PFS and the OS were significantly decreased for the following pathological variables: increased cellularity (>300 nuclei per HPF), mitotic activity of >7 per 10 HPF, increased MIB-1 labeling index (LI), topoisomerase-IIalpha LI, S-phase fraction, and p53 and bcl-2 positivity. Increased cyclin D1 LI was demonstrated to have only a marginally significant impact on PFS. A flow chart modeling was further performed to formulate a scheme for discriminating of prognostic subgroups. Based on that, p53 immunopositivity and/or MIB-1 LI of >5% (after subtotal resection) or MIB-1 LI of >15% (after complete resection) were the strongest indicators of the tumor's aggressive behavior and of a poor prognosis of the disease. Foci of hypercellularity should be specifically looked for in ependymomas for assessing the immunohistochemical studies.
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PMID:Pediatric intracranial ependymomas: prognostic relevance of histological, immunohistochemical, and flow cytometric factors. 1455 80

The main objective of this study was to test the effectiveness of candidate apoptosis inhibitors in limiting chondrocyte apoptosis induced by collagen degradation. Primary human chondrocytes were isolated from normal articular cartilage and grown in monolayer culture. Collagenase was added to the cells in the presence and absence of caspase inhibitors and insulin like growth factor (IGF)-1. The amount of chondrocyte apoptosis was measured using an enzyme linked immunosorbent assay for nucleosomes, a specific and quantitative measure of apoptosis. Chondrocyte apoptosis was induced by collagenase treatment in both a time and dose dependent manner. The non-selective caspase inhibitor Z-VAD, the caspase-3 selective inhibitor Z-DEVD, and the growth factor insulin like growth factor (IGF)-1 inhibited chondrocyte apoptosis induced by collagenase treatment. The caspase-1 selective inhibitor Z-YVAD also blocked chondrocyte apoptosis under these conditions, in contrast to previous studies where caspase-1 inhibition failed to block apoptosis induced by agents such as the topoisomerase inhibitor camptothecin. These data demonstrate that the response of chondrocytes to caspase inhibition may be dependent upon the specific stimulus that initiates apoptosis. Furthermore, these findings suggest that multiple pathways involving both the initiation and execution of programmed cell death are potential targets for chondrocyte apoptosis inhibition therapy.
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PMID:Chondrocyte apoptosis induced by collagen degradation: inhibition by caspase inhibitors and IGF-1. 1465 72

Human cytomegalovirus (HCMV) has many strategies to survive the attack of the host. HCMV infection of host cells induces cellular activation and disturbance of the cell cycle. It is possible that HCMV modulates the behavior of certain cancer cells that are susceptible to HCMV infection. This study was performed to identify the possible mechanism of resistance to apoptotic stimuli in some cancer cell lines by HCMV infection. HCMV-infected cancer cells showed resistance to apoptosis induced by the topoisomerase II inhibitor etoposide. UMG1-2, which constitutively expresses HCMV immediate-early protein-1 (IE1), had resistance to apoptosis induced by etoposide as compared with the parental cell line U373MG. Measurement of caspases activity with fluorogenic substrates in etoposide-treated U373MG and UMG1-2 cells and the direct activation of caspase-3 with peptides containing arginine-glycine-aspartate in U373MG and UMG1-2 cells revealed that the inhibition level of apoptosis by HCMV IE1 would be upstream of caspase-3 in the caspase cascade pathway. Cellular expression of Cdk2 was increased in UMG1- 2 after etoposide treatment while the expression of E2F-1 in UMG1-2 was decreased as compared with that in U373MG. The Cdk2 inhibitor, roscovitine, decreased the resistance to apoptosis on etoposide-treated UMG1-2. These results suggest that aberrant HCMV infection confers resistance to anticancer drugs on some cancer cells and protects cells from apoptosis, possibly due to the deregulation of cyclin-dependent kinase by HCMV immediate-early protein.
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PMID:Human cytomegalovirus (HCMV) IE1 plays role in resistance to apoptosis with etoposide in cancer cell line by Cdk2 accumulation. 1469 46

Poly(ADP-ribose) polymerase-1 is a highly abundant nuclear enzyme implicated in transcription, DNA replication, and DNA repair through binding of nascent RNA and interactions with various factors. We found that purified fractions of recombinant human poly(ADP-ribose) polymerase-1 expressed in Escherichia coli possess yet another activity, a Mg(2+)-dependent DNA supercoil relaxation activity. Cleavage of recombinant poly(ADP-ribose) polymerase-1 by caspase-3, an apoptotic protease, reduced this activity, as did the removal of either of the two zinc finger motifs located in the N-terminal DNA-binding domain of poly(ADP-ribose) polymerase-1. In addition, this activity was separated from E. coli topoisomerase I by gel-filtration column chromatography, suggesting that this activity is specifically associated with poly(ADP-ribose) polymerase-1. Because this relaxation activity did not require ATP and was resistant to VP16, a topoisomerase II inhibitor, this activity is closer to that of topoisomerase I. However, the supercoiled DNA relaxation activity associated with poly(ADP-ribose) polymerase-1 is distinct from that of human or E. coli topoisomerase I, as this activity could not completely remove superhelical tensions from plasmid DNA. Thus, we referred to this activity as topoisomerase I-like activity. This Mg(2+)-dependent DNA supercoil relaxation activity was found to be sensitive to camptothecin, a mammalian topoisomerase I inhibitor.
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PMID:Camptothecin-sensitive relaxation of supercoiled DNA by the topoisomerase I-like activity associated with poly(ADP-ribose) polymerase-1. 1471 57

The ability of melanoma cells to evade engagement of apoptosis plays a significant role in their resistance to chemotherapy. In an attempt to lower the apoptotic threshold of melanoma cells as a possible strategy to increase their drug sensitivity, we generated a hammerhead ribozyme to down-regulate the expression of the anti-apoptotic protein survivin. The JR8 human melanoma cell line was stably transfected with the active ribozyme RZsurv (targeting the 3' end of the GUC294 triplet in the exon 3 of the survivin mRNA) or the catalytically inactive ribozyme mutRZsurv (carrying a mutation in the catalytic core of RZsurv). Two polyclonal cell populations expressing the active (JR8/RZsurv) or the mutant (JR8/mutRZsurv) ribozyme were selected for the study. JR8/RZsurv cells were characterized by a markedly lower survivin protein level than JR8 parental cells, whereas a negligible reduction in survivin expression was observed in JR8/mutRZsurv cells. JR8/RZsurv cells showed a significantly increased sensitivity to the topoisomerase-I inhibitor topotecan (as detected by clonogenic cell survival) compared with JR8/mutRZsurv cells. Moreover, the extent of drug-induced apoptosis (in terms of percentage of apoptotic nuclei and level of caspase-9 and caspase-3 catalytic activity) was significantly greater in JR8/RZsurv than in JR8/mutRZsurv cells. Finally, an increased antitumor activity of oral topotecan was observed in JR8/RZsurv cells grown as xenograft tumors in athymic nude mice compared with JR8/mutRZsurv cells. These results demonstrate that attenuation of survivin expression renders human melanoma cells more susceptible to topotecan-induced apoptosis and more responsive to in vivo treatment, and support the concept that survivin is an attractive target for new therapeutic interventions in melanoma.
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PMID:Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo. 1476 61

Caspases are critical proapoptotic proteases that execute cell death signals by selectively cleaving proteins at Asp residues to alter their function. Caspases trigger apoptotic chromatin degradation by activating caspase-activated DNase and by inactivating a number of enzymes that sense or repair DNA damage. We have identified the mismatch repair protein MLH1 as a novel caspase-3 substrate by screening small pools of a human prostate adenocarcinoma cDNA library for cDNAs encoding caspase substrates. In this report, we demonstrate that human MLH1 is specifically cleaved by caspase-3 at Asp(418) in vitro. Furthermore, MLH1 is rapidly proteolyzed by caspase-3 in cancer cells induced to undergo apoptosis by treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the topoisomerase II inhibitor etoposide, which damages DNA. Importantly, proteolysis of MLH1 by caspase-3 triggers its partial redistribution from the nucleus to the cytoplasm and generates a proapoptotic carboxyl-terminal product. In addition, we demonstrate that a caspase-3 cleavage-resistant D418E MLH1 mutant inhibits etoposide-induced apoptosis but has little effect on TRAIL-induced apoptosis. These results indicate that the proteolysis of MLH1 by caspase-3 plays a functionally important and previously unrecognized role in the execution of DNA damage-induced apoptosis.
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PMID:Proteolysis of the mismatch repair protein MLH1 by caspase-3 promotes DNA damage-induced apoptosis. 1508 50

Topoisomerases are nuclear enzymes that maintain and modulate DNA structure. Inhibitors of topoisomerases like camptothecin (CPT), etoposide, and others are widely used antitumor drugs that interfere with transcription, induce DNA strand breaks, and trigger apoptosis preferentially in dividing cells. Because transcription inhibitors (actinomycin D, galactosamine, alpha-amanitin) sensitize primary hepatocytes to the cytotoxic action of tumor necrosis factor (TNF), we reasoned whether topoisomerase inhibitors would act similarly. CPT alone was not toxic to primary cultured murine hepatocytes. When incubated with CPT, murine hepatocytes displayed an inhibition of protein synthesis and were thereby rendered sensitive to apoptosis induction by TNF. Apoptosis was characterized by morphology (condensed/fragmented nuclei, membrane blebbing), caspase-3-like protease activity, fragmentation of nuclear DNA, and late cytolysis. Hepatocytes derived from TNF receptor-1 knockout mice were resistant to CPT/TNF-induced apoptosis. CPT treatment completely abrogated the TNF-induced NF-kappa B activation, and mRNA expression of the antiapoptotic factors TNF-receptor associated factor 2, FLICE-inhibitory protein, and X-linked inhibitor of apoptosis protein was also inhibited by CPT. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-chloromethylketone (zDEVD-fmk), as well as depletion of intracellular ATP by fructose prevented CPT/TNF-induced apoptosis. In vivo, CPT treatment sensitized mice to TNF-induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies.
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PMID:Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytes in vitro and in vivo to TNF-mediated apoptosis. 1512 60


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