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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several DNA topoisomerase II (Topo II; EC 5.99.1.3) partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes.
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PMID:Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes. 255 12

A human Burkitt's lymphoma cell line (Raji-HN2) made resistant to nitrogen mustard, a bifunctional alkylating agent, was used to study the mechanism of resistance to nitrogen mustard. A comparative study of Raji-HN2 and the parental sensitive Raji cell lines revealed the following: (1) The DNA of Raji-HN2 cells was crosslinked by nitrogen mustard to a lower extent than Raji DNA; (2) once interstrand crosslinks were formed, they were repaired at the same rate in both cell lines; (3) DNA crosslink formation in Raji-HN2, but not in Raji cells, was enhanced by novobiocin, a topoisomerase II inhibitor; (4) Raji-HN2 cells had elevated topoisomerase II activity and were hypersensitive to topoisomerase inhibitors (amsacrine, novobiocin, teniposide); (5) similar amounts of topoisomerase I were found in both cell lines; and (6) the chromatin of Raji-HN2 but not of Raji cells, was hypersensitive to DNase I digestion. The relationship between DNA repair, topoisomerase II activity, chromatin structure and drug resistance is discussed.
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PMID:Elevated topoisomerase II activity and altered chromatin in nitrogen mustard-resistant human cells. 281 39

A human Burkitt lymphoma cell line, Raji-HN2, made 10-fold more resistant to nitrogen mustard (HN2) than the parental Raji cell line, exhibited the following characteristics when compared to the parental Raji cells: (i) decreased HN2-induced DNA interstrand crosslinking; (ii) increased (3-fold) DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] activity; (iii) increased (4- to 11-fold) sensitivity to topoisomerase II inhibitors; (iv) increased (2-fold) glutathione content; and (v) increased (2-fold) cell doubling time. The resistant phenotype was unstable and was maintained by weekly treatment of the cells with HN2. Growing the resistant cells in the absence of HN2 resulted in a time-dependent decrease in both resistance to HN2 and topoisomerase II activity and an increase in DNA interstrand crosslinking induced by HN2. We hypothesize that HN2 resistance is due to enhanced monoadduct repair with resultant decreased DNA crosslinking and that this process is mediated by topoisomerase II.
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PMID:Elevated DNA topoisomerase II activity in nitrogen mustard-resistant human cells. 282 70

A cloned plasmid, pmyc(H-K), containing sequences derived from human c-myc gene replicated in vitro in Raji nuclear extract in a semiconservative manner. Using this system, it was found that phosphatidylinositol and cardiolipin strongly inhibited the replication of pmyc(H-K) in vitro, whereas other phospholipids, i.e., phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and sphingomyelin, had no appreciable effect. The concentrations of phosphatidylinositol and cardiolipin producing 50% inhibition of the replication were 4.6 and 5.4 microM, respectively. Phosphatidylinositol and cardiolipin inhibited the relaxation of pmyc(H-K) supercoiled DNA, but showed little or weaker effects on DNA polymerase alpha and topoisomerase II in Raji nuclear extract. These results suggest that phosphatidylinositol and cardiolipin antagonize the replication of pmyc(H-K) in vitro, through, at least in part, the interaction with topoisomerase I.
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PMID:Phospholipid modulates in vitro replication of autonomous replicating sequence from human cells. 285 2

The studies described below were carried out to analyze the damage induced by DNA active drugs to episomal (Epstein-Barr virus, EBV) DNA in the Raji Burkitt's lymphoma cell line. This work: (i) applies pulsed-field gel electrophoresis (PFGE) techniques to quantify DNA damage on a large (approximately 180 kbp), circular target, (ii) investigates the DNA strand-scission behavior of different classes of drugs on the EBV episome, and (iii) compares EBV episomal damage to that generated in genomic DNA in the Raji cell line. Cells were treated with ionizing radiation to induce random strand scission, and the migration of topological forms of EBV was measured using PFGE. DNA damage induced in the episome by DNA active drugs was then assayed. Three drugs, acting by different types of DNA interactive mechanisms, were used: bleomycin, an intercalative DNA strand-scission agent; and amsacrine (mAMSA) and teniposide (VM26), intercalative and nonintercalative topoisomerase II active drugs, respectively. Rad equivalency of damage was determined by comparing the drug-induced change in percentage of Forms I and III to that generated by ionizing radiation. Additionally, single- and double-strand scission induced in genomic (total cellular) DNA by X-rays, bleomycin, amsacrine, and teniposide were assayed by high-sensitivity alkaline and neutral filter elution techniques. We demonstrate that pulsed-field gel electrophoresis is a useful technique for measuring form conversion in large episomal DNA. While all three drugs effect both episomal and genomic DNA strand scission, bleomycin appears to preferentially damage the EBV episome. The topoisomerase II active drugs mAMSA and VM26 show no evidence of episome-directed damage in this system and, in fact, damage genomic DNA at somewhat higher rates.
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PMID:Damage induced in episomal EBV DNA in Raji cells by antitumor drugs as measured by pulsed field gel electrophoresis. 752 29

The roles of topoisomerases I and II in Epstein-Barr virus (EBV) replication were investigated using Raji cells infected with EBV. The topoisomerase II inhibitor ellipticine inhibited the synthesis of EBV polypeptides at concentrations which did not affect total protein synthesis. Slot blot analysis of total cellular DNA showed that camptothecin and ellipticine inhibited replication of progeny EBV DNA in superinfected Raji cells at concentrations which did not inhibit synthesis of EBV early polypeptides prerequisite for EBV DNA replication. Analysis of the structure of EBV DNA termini demonstrated that both inhibitors affected the replicating EBV DNA. Gardella gel electrophoresis showed that both inhibitors affected the formation of the linear form of EBV DNA. However, restriction analysis of EBV DNA in superinfected Raji cells demonstrated that both inhibitors degraded neither endogenous nor exogenous EBV DNA. Cell viability was not affected by either inhibitor at the concentrations tested. These findings suggest that topoisomerase II is required for expression of the EBV genome and that both topoisomerases I and II are involved in replication of the EBV genome during the lytic phase of the life cycle. The effects of topoisomerase inhibitors on the circular form of EBV DNA during virus replication are discussed.
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PMID:Topoisomerase I and II activities are required for Epstein-Barr virus replication. 840 49

Overexpressed MDM2 inactivates wild-type (wt) p53 in various human tumors. However, whether and how the wild-type p53 can be activated by anticancer drug treatment in the presence of excess MDM2 is still unclear. In the present study, we showed that the topoisomerase II inhibitor of widely used anticancer drugs etoposide and doxorubicin activated wt p53 in BL2, a Burkitt's lymphoma cell line which overexpressed MDM2. Activation of p53 was followed by apoptosis in BL2 cells, while the same drug treatment did not induce apoptosis in Raji cells, another Burkitt's lymphoma cell line which carried mutant p53. Activation of p53 was accompanied by phosphorylation of p53 at Ser-15 and elevated p21 and MDM2, both of which were at least partly blocked by wortmannin, a kinase inhibitor against proteins with a PI3 kinase domain. Although MDM2 protein was rapidly cleaved and degraded after anticancer drug treatment, cotreatment with caspase inhibitor Z-VAD blocked degradation, while wt p53 remained activated, suggesting MDM2 degradation not to be essential for the activation of p53. Treatment with proteasome inhibitor stabilized p53 without being further phosphorylated. This p53 was co-immunoprecipitated with MDM2, but p53 activated by etoposide or doxorubicin barely complexed with MDM2. These results suggest that the wild-type p53 in MDM2-overexpressing cells can be activated by anticancer drugs through phosphorylation of p53, alleviating inhibitory action by MDM2, and activating caspases which in turn downregulates MDM2. The activation of p53 in MDM2-overexpressing tumor cells, which does not require the downregulation of MDM2, may have important implications in cancer therapy.
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PMID:Activation of p53 in MDM2-overexpressing cells through phosphorylation. 1054 21

We have recently identified a novel CCAAT box binding protein (ICBP90) involved in the regulation of topoisomerase IIalpha gene expression. We have observed that it is expressed in non-tumoral proliferating human lung fibroblast cells whereas in HeLa cells, a tumoral cell line, ICBP90 was still present even when cells were at confluence. In the present study, we have determined the ICBP90 gene structure by screening of a human placenta genomic library and PCR analysis. We report that the ICBP90 gene spans about 35.8 kb and contains six coding exons named A to F. In the 5' upstream sequence of the region containing the coding exons, two additional exons (I and II) were found. Additionally, an internal splicing site was found in exon A. A promoter region, including three putative Sp1 binding sites between exons I and A, was identified by transient transfection. Northern blot analysis of several cancer cell lines revealed the existence of two ICBP90 mRNA species of 5.1 and 4.3 kb that are transcribed from the gene. The relative amounts of these mRNAs depended on the cell type. In MOLT-4 cells and Burkitt's lymphoma Raji cells, the 4.3 kb or the 5.1 kb transcripts were mainly observed, respectively. In other cell lines, such as HL-60 cells, chronic myelogenous leukaemia K-562, lung carcinoma A549, HeLa or colorectal SW480, both 4.3 and 5.1 kb forms of ICBP90 mRNA could be detected. Interestingly, western blot analysis showed several ICBP90 protein bands in HeLa but only a single band in MOLT-4 cell extracts. Taken together our results are consistent with the ICBP90 gene exhibiting alternative splicing and promoter usage in a cell-specific manner.
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PMID:Genomic structure and chromosomal mapping of the gene coding for ICBP90, a protein involved in the regulation of the topoisomerase IIalpha gene expression. 1129 Apr 15

The present study assessed the role of adenoviral vector-mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines (n=5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53-transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-beta-Gal-transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols.
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PMID:Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells. 1149 63

Interleukin-24 (IL-24) is a cytokine encoded by a tumor suppressor gene of the IL-10 family, also known as the melanoma differentiation associated gene-7 (Mda-7) and first discovered in human melanoma cells. Mda-7/IL-24 has been shown to inhibit the proliferation of various human tumor cell lines, but its effect on the sensitivity of B cell lymphoma to chemotherapy agents is not yet clear. The present study investigated the effects of Mda-7/IL-24 overexpression on the sensitivity of human B cell lymphoma cells to chemotherapy, as well as its mechanism of action. The sensitivity of stable Mda-7/IL-24 overexpressing Raji and Daudi cells to cis-diamminedichloroplatinum (CDDP), epirubicin and vinblastine (VCR) were assessed by the MTS method, and the IC50 value calculated. Cell apoptosis and the intracellular accumulation of Rhodamine-123 were assayed by flow cytometry. The expression of multidrug resistance gene 1 (MDR1), B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1), topoisomerase II (Topo II) and multidrug resistance-related protein 1 (MRP1) mRNA and protein were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. In addition, western blot analysis was also used to investigate the effect of Mda-7/IL-24 on activity of GTP-RhoA-ERK signaling pathway in Raji and Daudi cells. Growth inhibition and apoptosis rates of Mda-7/IL-24 overexpressing Raji and Daudi cells were higher than those of non-transfected cells and cells transfected with vector alone when treated with CDDP, epirubicin and VCR. The IC50 values of CDDP, epirubicin and VCR were lower for Mda-7/IL-24-overexpressing Raji and Daudi cells than for non-transfected cells and cells transfected with empty vector. Intracellular accumulation of Rhodamine-123 and the expression of Topo II were higher, while the levels of MDR1, BMI and MRP1 mRNA and protein were lower, in Mda-7/IL-24 overexpressing Raji and Daudi cells. Furthermore, the activities of GTP-RhoA-ERK signaling pathway in Raji and Daudi cells were suppressed. These results indicated that Mda-7/IL-24 enhanced the sensitivity of B lymphoma cells to chemotherapy agents by altering the expression of multidrug-resistance genes via downregulating GTP-RhoA-ERK signaling pathway, suggesting that treatment of B cell lymphomas with Mda-7/IL-24 could avoid MDR.
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PMID:Mda-7/IL-24 enhances sensitivity of B cell lymphoma to chemotherapy drugs. 2688 73


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