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)

Chinese hamster lung cells resistant to 9-OH-ellipticine, i.e., DC-3F/9-OH-E cells, are several hundredfold resistant to DNA topoisomerase II inhibitors. According to previous studies, this resistance is associated with reduced topoisomerase II activity (about 4-fold) and decreased capacity of the topoisomerase II inhibitors to induce stabilization of the cleavable complex (about 10-fold). In the present work, an antibody was raised against a fragment of human topoisomerase II alpha. This antibody, which recognizes both isoforms, was used to determine the amounts of topoisomerases II alpha and beta in the sensitive and resistant cells. Northern and immunoblot analyses showed that (i) in the parental DC-3F cells the alpha enzyme is about 20-fold more abundant than the beta enzyme and the enzyme isoforms undergo reciprocal regulation during the cell growth phases, with the expression of the alpha enzyme dropping at the plateau phase while the expression of the beta enzyme increases, and (ii) in the resistant cells the amount of alpha enzyme is about 4-5-fold smaller than that in the sensitive cells, whereas the beta enzyme is almost undetectable. Analysis of DNA restriction sites in several independently selected resistant subclones revealed some rearrangements in the beta gene in two clones. However, these gene alterations did not correlate with changes in the resistance level. The relative contribution of these different changes to the resistance phenotype is discussed.
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PMID:Expression of topoisomerases II alpha and beta in Chinese hamster lung cells resistant to topoisomerase II inhibitors. 807 94

Topoisomerase II alpha (topo II alpha) is a key enzyme in DNA replication and a target for many anti-cancer drugs. High levels are associated with sensitivity to topoisomerase II inhibitors. Because its chromosome location is similar to erbB2 (17q21-22), which is frequently amplified in breast cancer, co-amplification of these genes was assessed. In 117 primary breast cancers, 25 were amplified for erbB2. Three of these cases showed co-amplification of topo II alpha. Topo II beta was not amplified. Four human breast cancer cell lines were assessed for erbB2 and topo II alpha co-amplification. They were also analysed for sensitivity to the topoisomerase inhibitors m-AMSA and mitoxantrone. The most sensitive cell line was SKBr3, which was the only one with erbB2 amplification. Topo II alpha was co-amplified to a similar extent as in tumours. This suggests that patients whose tumours show topo II alpha amplification should be assessed specifically for therapy with topoisomerase inhibitors.
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PMID:Topoisomerase II alpha co-amplification with erbB2 in human primary breast cancer and breast cancer cell lines: relationship to m-AMSA and mitoxantrone sensitivity. 809 76

The DNA topoisomerase enzymes are targets for the cytotoxic effects of a number of anticancer agents termed topoisomerase inhibitors. We have analysed breast cancer biopsy specimens for genetic alterations at and around topoisomerase loci in order to obtain molecular insight into factors which may determine how tumours respond to chemotherapy. We show that of 50 tumours examined, the topoisomerase II alpha locus is co-amplified in 3 cases out of 6 with erbB2 amplification and that amplification can be accompanied by high expression of topoisomerase II alpha. In our attempts to distinguish amplification from aneuploidy and define the limits of amplification, we also observed co-amplification of the retinoic acid-alpha receptor with erbB2 and topoisomerase II alpha in the same three samples. At the topoisomerase I locus on chromosome 20, we observed allelic loss in two out of 17 samples. Genetics abberations at topoisomerase loci, therefore, appear to be relatively common in breast cancer.
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PMID:Co-amplification of erbB2, topoisomerase II alpha and retinoic acid receptor alpha genes in breast cancer and allelic loss at topoisomerase I on chromosome 20. 810 40

The H209/V6 cell line was derived from the H209 small cell lung cancer cell line by selection in etoposide (VP-16). Cytogenetic analysis indicates that the sensitive and resistant cell lines share 20 marker chromosomes and thus are clearly related. However, the H209/V6 cell line has four additional structurally altered chromosomes and a 2 N-modal chromosome number, while the H209 cell line is hypotetraploid (4 N-). H209/V6 cells are cross-resistant to some drugs that interact with topoisomerase II but not mitoxantrone. H209/V6 cells are also not cross-resistant to vincristine, trimetrexate, or cisplatin. The rates of VP-16 efflux are the same in the resistant and sensitive cell lines, which is consistent with the observation that P-glycoprotein mRNA is not detectable in either cell line. Fewer VP-16-induced DNA-protein complexes are observed in H209/V6 cells, and immunoblot analysis shows that levels of topoisomerase II alpha are reduced in H209/V6 cells compared to the sensitive H209 cells. Furthermore, the topoisomerase II alpha-related protein in H209/V6 cells has an increased electrophoretic mobility, with an apparent M(r) of 160,000. The levels of the topoisomerase II alpha 6.1-kilobase mRNA in H209/V6 cells are reduced > 10-fold. In addition, a second topoisomerase II alpha-related mRNA of approximately 4.8 kilobases is observed in H209/V6 cells but not in H209 cells. The quantity and electrophoretic mobility of the M(r) 180,000 topoisomerase II beta protein and its 6.1-kilobase mRNA are the same in the sensitive and resistant cell lines. The topoisomerase II strand-passing activity in H209/V6 nuclear extracts is reduced about 2-fold, but this activity is not more resistant to inhibition by VP-16 than the activity in H209 cells. However, band depletion immunoblot experiments show that the topoisomerase II alpha-related M(r) 160,000 protein in H209/V6 cells is not bound to DNA in the presence of concentrations of VP-16 that deplete the M(r) 170,000 topoisomerase II alpha in H209 cells and the M(r) 180,000 topoisomerase II beta in both the resistant and sensitive cells. We conclude that quantitative and qualitative alterations in topoisomerase II alpha have occurred in H209/V6 cells and are likely to contribute to its resistance phenotype.
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PMID:Altered topoisomerase II alpha in a drug-resistant small cell lung cancer cell line selected in VP-16. 810 87

K562 leukaemia cells were selected for resistance using 0.5 microM etoposide (VP-16). Cloned K/VP.5 cells were 30-fold resistant to growth inhibition by VP-16 and 5- to 13-fold resistant to m-AMSA, adriamycin and mitoxantrone. K/VP.5 cells did not overexpress P-glycoprotein; VP-16 accumulation was similar to that in K562 cells. VP-16-induced DNA damage was reduced in cells and nuclei from K/VP.5 cells compared with K562 cells. Topoisomerase II protein was reduced 3- to 7-fold and topoisomerase II alpha and topoisomerase II beta mRNAs were each reduced 3-fold in resistant cells. After drug removal, VP-16-induced DNA damage disappeared 1.7 times more rapidly and VP-16-induced DNA-topoisomerase II adducts dissociated 1.5 times more rapidly in K/VP.5 cells than in K562 cells. ATP (1 mM) was more effective in enhancing VP-16-induced DNA damage in nuclei isolated from sensitive cells than in nuclei from resistant cells. In addition, ATP (0.3-5 mM) stimulated VP-16-induced DNA-topoisomerase II adducts to a greater extent in K562 nuclei than in K/VP.5 nuclei. Taken together, these results indicate that resistance to VP-16 in a K562 subline is associated with a quantitative reduction in topoisomerase II protein and, in addition, a distinct qualitative alteration in topoisomerase II affecting the stability of drug-induced DNA-topoisomerase II complexes.
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PMID:Altered stability of etoposide-induced topoisomerase II-DNA complexes in resistant human leukaemia K562 cells. 814 56

The cytotoxicity of a class of compounds related to the topoisomerase-II poison amsacrine was investigated against plateau-phase murine Lewis lung carcinoma cells (LLTC), HCT-8 human colon carcinoma cells and other cell lines. Methyl N-[4-(9-acridinylamino)-2-methoxy-phenyl]carbamate hydrochloride and the corresponding demethoxy compound, which contain a methylcarbamate instead of the methylsulphonylamino group, manifested relatively high cytotoxic activity against plateau-phase cells as measured by clonogenic survival. The concentration of drug required for a given cytotoxic effect on plateau-phase cells was about 2 times higher than that required for an equitoxic effect on actively proliferating cells. In contrast, at least 5 times more amsacrine, doxorubicin or etoposide was needed for an equitoxic effect on plateau-phase cells. Cells taken directly from subcutaneous LLTC tumours and exposed to drugs displayed the same differential drug sensitivity to the carbamate compounds, suggesting that the plateau-phase cells provide an appropriate model for cells growing in vivo. The greater cytotoxicity of the carbamate drugs was shown to depend critically on the provision of an energy source such as glucose, suggesting that nutrient starvation both in plateau-phase cells and in tumours induced a glucose-sensitive resistance mechanism. It is suggested that the carbamate analogues of amsacrine recognize a form of topoisomerase II, possibly topoisomerase II beta, the activity of which increases relative to that of topoisomerase II alpha in non-cycling cells, and might be used to devise new strategies for the treatment of solid tumours.
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PMID:Novel carbamate analogues of amsacrine with activity against non-cycling murine and human tumour cells. 819 67

We have produced two murine monoclonal antibodies (SWT3D1 and SWR1C2) to a recombinant polypeptide corresponding to the carboxyl-terminal one-third (amino acid 854-amino acid 1447) of human topoisomerase II alpha. Each antibody is able to recognize intact human topoisomerase II using immunoblotting and enzyme-linked immunosorbent assay (ELISA) techniques. Data is presented demonstrating that the antibodies bind specifically to topoisomerase II alpha but do not interact with topoisomerase II beta. The monoclonal antibodies do not recognize murine or calf thymus topoisomerase II indicating that each may bind exclusively to the human enzyme. The topoisomerase II binding sites for each monoclonal antibody have been compared in a competition ELISA. The SWT3D1 antibody had no significant effect on the binding efficiency of biotinylated SWR1C2 antibody. Although SWR1C2 was capable of inhibiting the binding of biotinylated SWT3D1, this only occurred at concentrations approximately 1000-fold higher than those required of SWT3D1 to block binding of itself. These results suggest that SWT3D1 and SWR1C2 do not recognize identical epitopes on topoisomerase II.
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PMID:Isolation and characterization of monoclonal antibodies to a recombinant human topoisomerase II polypeptide. 824 17

Previous studies using cloned lines of Adriamycin-sensitive and -resistant P388 murine leukemia cells have suggested that a reduction in DNA topoisomerase II alpha (topo II alpha) enzyme activity and protein levels in drug-resistant cell lines (A. M. Deffie, J. K. Batra, and G. J. Goldenberg, Cancer Res., 49: 58-62, 1989) may be due to an allelic mutation in the topo II alpha gene (A. M. Deffie, D. J. Bosman, and G. J. Goldenberg, Cancer Res., 49: 6879-6882, 1989). The drug-resistant cell lines P388/ADR/3 and P388/ADR/7 express a shortened topo II alpha mRNA transcript in addition to the native transcript present in the drug-sensitive P388/4 cell line. Using complementary DNA probes derived from the coding sequence and 3' untranslated region of the native mouse topo II alpha transcript, we have determined that the shorter 4.5-kilobase topo II alpha transcript expressed in the drug-resistant cell lines contains only 3.5-kilobases of topo II sequence from the 5'-terminus onwards. Using a 3'-rapid amplification of cDNA ends strategy, we have cloned cDNAs representing the 3'-termini of both the native and mutant transcripts from both P388/ADR/3 and P388/ADR/7 cells. DNA sequence analysis revealed that the shorter 4.5-kilobase transcript: (a) encodes topoisomerase II alpha until nucleotide position 3494, at which point the sequence diverges for the remaining 956 bases; (b) contains a polyadenylation signal distinct from the native transcript; and (c) contains an open reading frame predicting a truncated topo II alpha fusion protein. Of great interest was the finding that the non-topo II alpha 956-base sequence in the shorter transcript encodes the promoter, exon I, and part of the first intron of the murine retinoic acid receptor alpha gene locus in the antisense orientation, suggesting that a rearrangement on chromosome 11 in the drug-resistant cells led to a gene fusion event between the loci encoding topo II alpha and retinoic acid receptor alpha.
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PMID:Characterization of a DNA topoisomerase IIalpha gene rearrangement in adriamycin-resistant P388 leukemia: expression of a fusion messenger RNA transcript encoding topoisomerase IIalpha and the retinoic acid receptor alpha locus. 826 98

We have examined the effects of a group of DNA topoisomerase II (topo II) inhibitors, merbarone, aclarubicin, SN22995, RP60475F, and fostriecin, in CCRF-CEM cells and two sublines, CEM/VM-1 and CEM/VM-1-5, that were selected for increasing resistance to teniposide (VM-26). The teniposide-resistant sublines have been termed "at-MDR" for altered topo II-associated multidrug resistance. These topo II inhibitors differ from the "classic" inhibitors such as teniposide in that they do not stabilize DNA-topo II complexes. In this study, we found that our at-MDR cell lines express little or no cross-resistance to these "non-classic" topo II inhibitors. Merbarone and SN22995 inhibited VM-26-mediated DNA-topo II complexes in CEM cells only when they were added before VM-26. Since they did not deplete topo II protein, it suggested that these drugs may inhibit topo II activity before the enzyme binds to DNA, thereby preventing stabilization of VM-26-mediated topo II-DNA complexes. Continuous exposure of CEM cells to merbarone, SN22995, or VM-26 caused G2 arrest, as determined by flow cytometry. Likewise, at-MDR cells continuously treated with VM-26 also arrested in G2. By contrast, treatment of at-MDR cells with either merbarone or SN22995 produced a qualitatively different pattern; the at-MDR cells first accumulated in G2 but then escaped the G2 block and proceeded into mitosis with elongated and intertwined chromosomes but failed to divide. Their DNA was re-replicated, however, and the cells eventually accumulated at the 8N DNA stage. Given that both wild-type and mutant topo II alpha alleles are expressed in the at-MDR cells (B. Y. Bugg, M. K. Danks, W. T. Beck, and D. P. Suttle. Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. Proc. Natl. Acad. Sci. USA, 88: 7654-7658, 1991), we hypothesize that drugs such as merbarone may inhibit the activity of wild-type topo II alpha, allowing the aberrant activity of the mutant enzyme to be revealed during chromosome condensation and sister chromatid segregation.
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PMID:Teniposide-resistant CEM cells, which express mutant DNA topoisomerase II alpha, when treated with non-complex-stabilizing inhibitors of the enzyme, display no cross-resistance and reveal aberrant functions of the mutant enzyme. 826 8

Sublines of K562 human leukemia cells were selected for resistance (30- to 80-fold) to etoposide by continuous exposure to 0.5 microM VP-16. Two etoposide-resistant cell lines, K/VP.5 and K/VP.5-1, showed a 5-fold reduction in levels of topoisomerase II alpha protein compared with K562 cells. Northern analysis indicated a 2.5-fold reduction in topoisomerase II alpha mRNA in etoposide-resistant cell lines, due in part to a 1.7-fold decrease in topoisomerase II mRNA stability with no change in transcription rate. Immunoblotting assays of electrophoresed cell lysates from VP-16-treated cells revealed less drug-induced covalent topoisomerase II/DNA adducts in resistant than in sensitive cells, suggesting a functional alteration in resistant cell topoisomerase II. Recent reports of specific topoisomerase II DNA binding sites near the promoter sites of growth response genes and alterations of gene expression in cells treated with topoisomerase II inhibitory drugs led to experiments to determine if the apparent functional alterations of topoisomerase II were accompanied by changes in the regulation of these genes. Therefore, the expression of several growth response genes was compared by northern analysis in parental K562 and both VP-16-resistant cell lines. Basal levels of c-myc were comparable for all three cell lines, but levels of c-jun and c-fos were elevated 2- to 4-fold in VP-16-resistant cell lines. Increased levels of c-fos and c-jun were not a result of altered rates of transcription, as determined by nuclear run-off assays. Exposure of both sensitive and resistant cells to 200 microM VP-16 for 5 hr resulted in no further changes in topoisomerase II mRNA levels but caused an additional 2- to 3-fold elevation in the level of c-jun mRNA, indicating that altered basal levels of this gene were not due to deregulation of this gene. Acquired VP-16 resistance in K/VP.5 and K/VP.5-1 cells was accompanied by reduced levels and altered activities of DNA topoisomerase II as well as changes affecting the expression of genes important for growth and differentiation.
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PMID:Altered gene expression in human leukemia K562 cells selected for resistance to etoposide. 826 50


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