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)

Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II. We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573. Selection with doxorubicin frequently resulted in reduced topo II alpha mRNA and protein levels, whereas clones selected with vincristine showed normal levels of topo II alpha. No alterations of topo II beta levels were detected. To determine the contribution of topo II alterations to drug resistance, topo II activity was analysed by the determination of DNA breaks induced by the topo II-inhibiting drug 4'-(9-acridinylamino)methane-sulphon-m-anisidide (m-AMSA) in living cells, as m-AMSA is not affected by the drug efflux mechanism in the SW-1573 cells. The number of m-AMSA-induced DNA breaks correlated well (r = 0.96) with in vitro m-AMSA sensitivity. Drug sensitivity, however, did not always correlate with reduced topo II mRNA or protein levels. In one of the five doxorubicin-selected clones m-AMSA resistance and a reduction in m-AMSA-induced DNA breaks were found in the absence of reduced topo II protein levels. Therefore, we assume that post-translational modifications of topo II also contribute to drug resistance in SW-1573 cells. These results suggest that methods that detect quantitative as well as qualitative alterations of topo II should be used to predict the responsiveness of tumours to cytotoxic agents. The assay we used, which measures DNA breaks as an end point of topo II activity, could be a good candidate.
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PMID:Reduced topoisomerase II activity in multidrug-resistant human non-small cell lung cancer cell lines. 781 46

In a series of 60 ALL samples drawn during different stages of the disease we used a cDNA-PCR approach to analyse the relative mRNA levels of the MDR-associated genes encoding mdr1/P-glycoprotein, mrp, and the topoisomerase II isozymes alpha and beta. Expression analysis of the cyclin A gene was included to examine cellular proliferation activity. The expression of gapdh served as an internal standard. Calculating the mean values we found: (i) a distinctly lower mdr1 gene expression in primary ALL and first relapses compared to bone marrow from healthy donors, (ii) no change in mdr1 and mrp, but a decreased topoisomerase II alpha gene expression in first relapses of ALL compared to the primary leukaemia, and (iii) increased mdr1 and mrp levels combined to decreased topoisomerase II alpha levels in recurrent relapses of ALL showing significant correlations (mdr1/mrp: rs = +0.6833, P < 0.05; mdr1/topoII alpha: rs = -0.6727, P < 0.05). The expression of the topoisomerase II alpha gene was correlated to that of cyclin A, indicating a link of its expression to cellular proliferation. Our findings suggest that a multifactorial MDR including mrp appears particularly in recurrent relapses of ALL, which often do not respond to chemotherapy. Nonetheless, some individual samples showed gene expression levels very different from the mean values calculated for a particular state of the leukaemia, indicating the need of an individual expression analysis of MDR-associated genes.
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PMID:Expression of mdr1, mrp, topoisomerase II alpha/beta, and cyclin A in primary or relapsed states of acute lymphoblastic leukaemias. 787 86

The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.
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PMID:Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis. 787 55

The topoisomerase (topo) II-directed agents etoposide, daunorubicin (DNR), and amsacrine (m-AMSA) are widely used in the treatment of acute myelogenous leukemia (AML). In the present study, multiple aspects of topo II-mediated drug action were examined in marrows from adult AML patients. Colony-forming assays revealed that the dose of etoposide, DNR, or m-AMSA required to diminish leukemic colony formation by 90% (LD90) varied over a greater than 20-fold range between different pretreatment marrows. Measurement of nuclear DNR accumulation in the absence and presence of quinidine revealed evidence of P-glycoprotein (Pgp) function in 8 of 82 samples at diagnosis and 5 of 36 samples at first relapse, but the largest quinidine-induced increment in DNR accumulation (< 2-fold) was too small to explain the variations in drug sensitivity. Restriction enzyme-based assays and sequencing of partial topo II alpha and topo II beta cDNAs from the most highly resistant specimens failed to demonstrate topo II gene mutations that could account for resistance. Western blotting of marrow samples containing greater than 80% blasts revealed that the content of the two topo II isoenzymes varied over a greater than 20-fold range, but did not correlate with drug sensitivity in vitro or in vivo. In addition, levels of topo II alpha and topo II beta in 46 of 47 clinical samples were lower than in human AML cell lines. Immunoperoxidase staining showed that these low topo II levels were accompanied by marked cell-to-cell heterogeneity, with topo II alpha being abundant in some blasts and diminished or absent from others. There was a trend toward increasing percentages of topo II alpha-positive cells in pretreatment marrows that contained more S-phase cells. Consistent with this observation, treatment of patients with granulocyte-macrophage colony-stimulating factor for 3 days before chemotherapy resulted in increases in topo II alpha-positive cells concomitant with increases in the number of cells traversing the cell cycle. These observations have implications for the regulation of topo II in AML, for the use of topo II-directed chemotherapy, and for future attempts to relate drug sensitivity to topo II levels in clinical material.
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PMID:Topoisomerase II levels and drug sensitivity in adult acute myelogenous leukemia. 790 87

The induced expression of multiple drug resistance (MDR)-associated genes as a direct response of tumor cells to antineoplastic drugs could be an important factor influencing the success of cancer chemotherapy. We investigated the effects of such compounds on mdr1/P-glycoprotein (P-gp) gene expression and drug sensitivities in the T-lymphoblastoid human cell line CCRF-CEM and MDR sublines. Thereby, we observed that actinomycin D or adriamycin administered at sublethal concentrations induced increases of mdr1 mRNA levels and resistance within 72 h. Furthermore, on leukemia cell samples collected before and after chemotherapy we checked by a complementary DNA polymerase chain reaction (cDNA-PCR) approach for similar alterations in the relative expression levels of the MDR-associated genes (a) mdr1/P-gp (b) mrp (MDR related protein), and (c) the topoisomerase II isoforms alpha and beta. We found a concomitant increase in mdr1 and mrp gene expression combined with a decreased expression of topoisomerase II alpha in the course of the second relapse of an acute lymphoblastic leukemia (ALL). This points to the emergence of at least three different MDR mechanisms in this type of leukemia unresponsive to chemotherapy. A chronic myeloid leukemia (CML) in blast crisis, however, showed combined increases in mdr1 (about 20-fold) and mrp (about four fold) gene expression after intense but unsuccessful chemotherapy over a 6-month period. Our results indicate the occurrence of induced resistance in vitro and in vivo and suggest a contribution of the newly identified ATP-binding cassette (ABC) transporter MRP in MDR.
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PMID:Drug-induced changes in the expression of MDR-associated genes: investigations on cultured cell lines and chemotherapeutically treated leukemias. 791 48

Human cell lines express two genetically distinct isoforms of DNA topoisomerase (topo II) II: topo II alpha (p170) and topo II beta (p180). We detected a higher molecular weight form with an apparent molecular mass of about 190 kDa in M phase-arrested HeLa cells (Kimura, K., Saijo, M., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 1173-1176). In this study we confirmed, using anti-topo II alpha and topo II beta monoclonal antibodies, that this higher molecular weight form is topo II beta and consists of doublet bands around 190 kDa. We confirmed that the doublet bands constituted an M phase-specific phenomenon and were not an artifact of the procedure used to accumulate mitotic cells. Digesting the immunoprecipitated materials from mitotic cell extracts with alkaline phosphatase resulted in the disappearance of the doublet bands and the appearance of the 180-kDa band with the concomitant disappearance of 32P label in the region of the doublet bands. Neither heat-inactivated alkaline phosphatase nor phosphodiesterase affected the doublet bands and the 32P label. Topo II beta in interphase cells was also phosphorylated, but the shift in apparent molecular weight was very slight after alkaline phosphatase digestion. Analysis of the labeled phosphoamino acids present in topo II beta from M phase and logarithmically growing cells indicated that phosphorylation occurred mainly on serine and fairly on threonine residues in both topo II beta isoforms. These results indicated that topo II beta is phosphorylated at specific sites in M phase, resulting in the formation of the doublet bands.
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PMID:Identification of the nature of modification that causes the shift of DNA topoisomerase II beta to apparent higher molecular weight forms in the M phase. 792 18

As an approach to the rational design of combination chemotherapy involving the anti-cancer DNA topoisomerase II poison etoposide (VP-16), we have studied the dynamic changes occurring in small-cell lung cancer (SCLC) cell populations during protracted VP-16 exposure. Cytometric methods were used to analyse changes in target enzyme availability and cell cycle progression in a SCLC cell line, mutant for the tumour-suppressor gene p53 and defective in the ability to arrest at the G1/S phase boundary. At concentrations up to 0.25 microM VP-16, cells became arrested in G2 by 24 h exposure, whereas at concentrations 0.25-2 microM G2 arrest was preceded by a dose-dependent early S-phase delay, confirmed by bromodeoxyuridine incorporation. Recovery potential was determined by stathmokinetic analysis and was studied further in aphidicolin-synchronised cultures released from G1/S and subsequently exposed to VP-16 in early S-phase. Cells not experiencing a VP-16-induced S-phase delay entered G2 delay dependent upon the continued presence of VP-16. These cells could progress to mitosis during a 6-24 h period after drug removal. Cells experiencing an early S-phase delay remained in long-term G2 arrest with greatly reducing ability to enter mitosis up to 24 h after removal of VP-16. Irreversible G2 arrest was delimited by the induction of significant levels of DNA cleavage or fragmentation, not associated with overt apoptosis, in the majority of cells. Western blotting of whole-cell preparations showed increases in topoisomerase II levels (up to 4-fold) attributable to cell cycle redistribution, while nuclei from cells recovering from S-phase delay showed enhanced immunoreactivity with an anti-topoisomerase II alpha antibody. The results imply that traverse of G1/S and early S-phase in the presence of a specific topoisomerase II poison gives rise to progressive low-level trapping of topoisomerase II alpha, enhanced topoisomerase II alpha availability and the subsequent irreversible arrest in G2 of cells showing limited DNA fragmentation. We suggest that protracted, low-dose chemotherapeutic regimens incorporating VP-16 are preferentially active towards cells attempting G1/S transition and have the potential for increasing the subsequent action of other topoisomerase II-targeted agents through target enzyme modulation. Combination modalities which prevent such dynamic changes occurring would act to reduce the effectiveness of the VP-16 component.
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PMID:Etoposide-induced cell cycle delay and arrest-dependent modulation of DNA topoisomerase II in small-cell lung cancer cells. 794 97

Topoisomerase II protein is essential for cell proliferation and is known to exist as a phosphoprotein in cells from both lower and higher eukaryotic species. In this paper, we have investigated the phosphorylation of the alpha isozyme of human topoisomerase II. The topoisomerase II alpha protein was phosphorylated predominantly on serine residues in the human tumor cell lines HeLa and NSCLC-3. Two-dimensional tryptic phosphopeptide mapping studies revealed several sites of phosphorylation in vivo, including a major site that was common to topoisomerase II alpha protein from both HeLa and NSCLC-3 cells. To identify sites of phosphorylation, the regulatory C-terminal domain of human topoisomerase II alpha protein was overexpressed in Escherichia coli as a hexahistidine-tagged fusion protein and purified by nickel chelate chromatography. Tryptic phosphopeptide mapping revealed that casein kinase II phosphorylated the C-terminal domain primarily on 2 serine residues in vitro, which were shown to be sites of modification in vivo. Site-directed mutagenesis studies identified these casein kinase II-specific phosphorylation sites as serine 1524 and serine 1376.
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PMID:Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro. 796 67

Anti-topoisomerase II agents represent a major class of anticancer therapeutic agents. Resistance to this class of agents can be mediated by several possible mechanisms. One mechanism may involve mutations in the structural gene(s) for topoisomerases, altering the drug sensitivity of the enzymes. Several mutations have been described in mammalian cell lines that were selected for resistance to topoisomerase II-targeting drugs such as Adriamycin, etoposide, or amsacrine. The difficulty of performing genetic analysis in mammalian cell lines has complicated the determination of whether the observed mutations are responsible for drug resistance. We have reconstructed, in the yeast topoisomerase II gene, the arginine to glutamine mutation at position 450 of human topoisomerase II alpha that was originally identified by Bugg et al. [Proc. Natl. Acad. Sci. USA 88:7654-7658 (1991)]. Mutation of Lys439, the equivalent amino acid in the yeast protein, to either glutamine or glutamic acid confers resistance to etoposide and amsacrine. Interestingly, in diploid yeast cells the heterozygous mutation can still confer partial drug resistance, compared with a diploid strain that is homozygous for wild-type topoisomerase II. Because mutations in the topoisomerase II gene that can confer dominant resistance to anti-topoisomerase II agents are relatively rare, mutations in the gyrB region may be important in the development of clinical drug resistance.
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PMID:Mutations in the gyrB domain of eukaryotic topoisomerase II can lead to partially dominant resistance to etoposide and amsacrine. 796 59

Studies were done to determine (a) the subcellular distribution of the alpha (170 kDa) and beta (180 kDa) isozymes of topoisomerase II, and (b) the extent to which each isozyme forms complexes with DNA in tumor cells incubated with and without VM-26. Western blotting revealed that topoisomerase II beta was highly unstable during cell fractionation. However, preincubation of human CEM leukemia cells with 5-100 microM VM-26 for 30 min protected the beta isozyme from degradation by progressively increasing the amount of this isoform bound to DNA. The amount of topoisomerase II beta detected in nuclei of CEM cells incubated for 30 min with 25 microM VM-26 was 7-fold greater than in nuclei from untreated control cells. VM-26 also had a protective effect on topoisomerase II beta in HL-60 leukemia and WiDR colon carcinoma cells. In contrast, the intercalating agents mitoxantrone and m-AMSA did not protect topoisomerase II beta from degradation during cell fractionation. The stabilization of topoisomerase II beta by VM-26 allowed subsequent studies of the subcellular distribution of the topoisomerase II isozymes. Both isozymes were detected in the nonmatrix (high salt-soluble) fraction of nuclei from CEM cells, but only topoisomerase II alpha was present in the nuclear matrix. VM-26 stabilized binding of the alpha and beta topoisomerase II isoenzymes to nonmatrix DNA and topoisomerase II alpha to matrix DNA. The differences observed in the subnuclear distribution and DNA binding pattern of the topoisomerase II isozymes support the hypotheses that each isozyme has a distinct cellular function, and that both the alpha and beta isozymes are potential targets for VM-26 in intact cells. In addition, the results demonstrated that pretreatment of various cell lines with VM-26 is a useful way to stabilize topoisomerase II beta during cell fractionation.
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PMID:Subcellular distribution of the alpha and beta topoisomerase II-DNA complexes stabilized by VM-26. 798 Jun 48


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