Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P67775 (alpha isoform)
 797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNA topoisomerase I and the isoforms alpha and beta of DNA topoisomerase II were analyzed in different animal cells using a panel of monoclonal antibodies (MoAbs). The beta isoform is a most unstable enzyme. We investigated conditions to stabilize beta isoform because its variability changes according to the derivation of cells. We describe two MoAbs specific to DNA topoisomerase I: the first one recognizes the enzyme in all the species tested including fish; the second one, in contrast, recognizes an epitope present only in mammalian cells. We also found that eight of eight MoAbs against DNA topoisomerase II alpha and five of six against the beta isoform recognize the respective enzymes in all the species tested excluding fish. In addition, MoAbs to the alpha isoform are specific to epitopes not present in the carboxyl third of the enzyme.
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PMID:DNA topoisomerase II beta: stability and distribution in different animal cells in comparison to DNA topoisomerase I and II alpha. 768 76

DNA topoisomerase II is the molecular target of several clinically useful chemotherapeutic drugs. The sensitivity of cells to drugs that target topoisomerase II is dependent on the cellular content of this enzyme. Drug-sensitive cells have elevated amounts of type II topoisomerase. To determine relative amounts of enzyme in malignant neoplasms, we developed an in situ immunohistochemical stain for topoisomerase II. The stain uses either polyclonal or monoclonal antibodies produced against the alpha isoform of the enzyme. Staining can be done on both frozen and formalin-fixed, paraffin-embedded tissues. By using this immunostain, we found marked differences in enzyme content in several human malignancies.
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PMID:Immunohistochemical staining for DNA topoisomerase II in frozen and formalin-fixed paraffin-embedded human tissues. 783 37

To examine the role of DNA topoisomerase II (Topo II) in the mitogenic activation of mouse lymphocytes, we applied the Topo II inhibitor VM26 throughout the stimulation period and monitored morphological and functional parameters of lymphocyte activation. Cell viability and the usual increase in cell size were little affected at doses between 0.05 and 0.5 microM. DNA synthesis, however, was already significantly inhibited at 0.05 microM, with RNA synthesis inhibited to a lesser extent. Light microscope autoradiography showed that a smaller proportion of cells entered S phase, with each S phase cell incorporating less [3H]thymidine. In immunofluorescence studies, the nucleolar antigen fibrillarin was reduced, although only minor effects on the snRNP Sm antigen and the internal component labeled by antibody PI1 were observed. At the electron microscope level, nucleoli were remodeled and chromatin became aggregated. At a high dose of VM26 (5 microM), cells showed the expected high levels of apoptosis and strong inhibition in all activation parameters assayed. The results support the hypothesis that the Topo II beta isoform is involved in the very early phases of lymphocyte activation, with function of the Topo II alpha isoform, which is more sensitive to VM26, being required for progression through S phase.
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PMID:Role of topoisomerase II in the structural and functional evolution of mitogen-stimulated lymphocyte nuclei. 808 36

Antibodies raised against the alpha and beta isoforms of human DNA topoisomerase II were found to react with the corresponding mouse enzymes. By using these antibodies in Western blotting experiments, it was observed that these isoforms are differentially expressed in adult mouse tissues. The alpha isoform is abundant in testis and spleen. The beta isoform is also abundant in spleen and in addition is present in kidney and liver. Type II topoisomerase is low in brain and heart. These results suggest different functions for these two isoforms of DNA topoisomerase II.
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PMID:The distribution of DNA topoisomerase II isoforms in differentiated adult mouse tissues. 824 Dec 59

Site-specific DNA cleavage by topoisomerase II (EC 5.99.1.3) is induced by many antitumour drugs. Although human cells express two genetically distinct topoisomerase II isoforms, thus far the role and determinants of drug-induced DNA cleavage have been examined only for alpha. Here we report the first high-resolution study of amsacrine (mAMSA) induced DNA breakage by human topoisomerase II beta (overexpressed and purified from yeast) and a direct comparison with the recombinant alpha isoform. DNA cleavage in plasmid pBR322 and SV40 DNA was induced by alpha or beta in the absence or presence of the antitumour agent mAMSA, and sites were mapped using sequencing gel methodology. Low-resolution studies indicated that recombinant human alpha promoted DNA breakage at sites akin to those of beta, although some sites were only cleaved by one enzyme and different intensities were observed at some sites. However, statistical analysis of 70 drug-induced sites for beta and 70 sites for alpha revealed that both isoforms share the same base preferences at 13 positions relative to the enzyme cleavage site, including a very strong preference for A at +1. The result for recombinant alpha isoform is in agreement with previous studies using alpha purified from human cell lines. Thus, alpha and beta proteins apparently form similar ternary complexes with mAMSA and DNA. Previous studies have emphasized the importance of DNA topoisomerase II alpha; the results presented here demonstrate that beta is an in vitro target with similar site determinants, strongly suggesting that beta should also be considered a target of mAMSA in vivo.
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PMID:Amsacrine-promoted DNA cleavage site determinants for the two human DNA topoisomerase II isoforms alpha and beta. 898 29

Type II DNA topoisomerase activity is required to change DNA topology. It is important in the relaxation of DNA supercoils generated by cellular processes, such as transcription and replication, and it is essential for the condensation of chromosomes and their segregation during mitosis. In mammals this activity is derived from at least two isoforms, termed DNA topoisomerase II alpha and beta. The alpha isoform is involved in chromosome condensation and segregation, whereas the role of the beta isoform is not yet clear. DNA topoisomerase II beta was first reported in 1987. Here we review the research on DNA topoisomerase II beta over the last 10 years.
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PMID:Eukaryotic DNA topoisomerase II beta. 963 49

Human DNA topoisomerase II is essential for chromosome segregation and is the target for several clinically important anticancer agents. It is expressed as genetically distinct alpha and beta isoforms encoded by the TOP2alpha and TOP2beta genes that map to chromosomes 17q21-22 and 3p24, respectively. The genes display different patterns of cell cycle- and tissue-specific expression, with the alpha isoform markedly upregulated in proliferating cells. In addition to the fundamental role of TOP2alpha and TOP2beta genes in cell growth and development, altered expression and rearrangement of both genes are implicated in anticancer drug resistance. Here, we report the complete structure of the human topoisomerase IIalpha gene, which consists of 35 exons spanning 27.5 kb. Sequence data for the exon-intron boundaries were determined and examined in the context of topoisomerase IIalpha protein structure comprising three functional domains associated with energy transduction, DNA breakage-reunion activity and nuclear localization. The organization of the 3' half of human TOP2beta, including sequence specifying the C-terminal nuclear localization domain, was also elucidated. Of the 15 introns identified in this 20 kb region of TOP2beta, the first nine and the last intron align in identical positions and display the same phases as introns in TOP2alpha. Though their extreme 3' ends differ, the striking conservation suggests the two genes diverged recently in evolutionary terms consistent with a gene duplication event. Access to TOP2alpha and TOP2beta gene structures should aid studies of mutations and gene rearrangements associated with anticancer drug resistance.
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PMID:Molecular cloning and characterization of the human topoisomerase IIalpha and IIbeta genes: evidence for isoform evolution through gene duplication. 1009 62

Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations. This suggests that it is a shift in the equilibrium to an open-clamp state in the enzyme's catalytic cycle caused by a decreased ATP binding by the mutated enzyme that is responsible for bisdioxopiperazine resistance.
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PMID:Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform. 1041 8

The expression of the 170-kDa (alpha) and the 180-kDa (beta) isoforms of DNA topoisomerase II (topo II) was investigated with two specific monoclonal antibodies (MoAbs) in human peripheral blood lymphocytes (PBL), before and after phytohaemoagglutinin (PHA) stimulation. Binding of each MoAb was detected by indirect immunofluorescence labelling and quantified with flow cytometry. In resting PBL, the intensity of immunostaining was very low for both isozymes; however, topo IIbeta-associated immunofluorescence was about 2.5 times significantly higher (P<0.001) than that associated with the alpha isoform. Between 48 and 72 h of PHA stimulation, when the highest percentage of cells in S and G2+M phases was found, the levels of topo IIalpha and beta increased up to about 30 and 10 times the value measured in resting PBL, respectively. Thus, the two isoforms reached comparable immunofluorescence values. At longer stimulation periods (96-120 h), topo IIalpha immunofluorescence was not significantly changed, while that relative to topo IIbeta declined to about 50% of the peak value (P<0.02). At this time however, topo IIalpha-associated immunofluorescence was not significantly different from that related to the beta isozyme. These results suggest that in resting PBL topo IIalpha is required at levels lower than topo IIbeta, while in proliferating lymphocytes both isoforms are expressed to significantly higher levels.
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PMID:Expression of the 170-kDa and 180-kDa isoforms of DNA topoisomerase II in resting and proliferating human lymphocytes. 1046 10

The alpha and beta isoforms of DNA topoisomerase II (topo II) are targets for several widely used chemotherapeutic agents, and resistance to some of these drugs may be associated with reduced nuclear localization of the alpha isoform. Human topo IIalpha contains a strong bipartite nuclear localization signal (NLS) sequence between amino acids 1454 and 1497 (alphaNLS(1454-1497)). In the present study, we show that human topo IIalpha tagged with green fluorescence protein is still detectable in the nucleus when alphaNLS(1454-1497) has been disrupted. Seven additional regions in topo IIalpha containing overlapping potential bipartite NLSs were evaluated for their nuclear targeting abilities using a beta-galactosidase reporter system. A moderately functional NLS was identified between amino acids 1259 and 1296. When human topo IIbeta was examined in a similar fashion, it was found to contain two strongly functional sequences betaNLS(1522-1548) and betaNLS(1538-1573) in the region of topo IIbeta comparable to the region in topo IIalpha that contains the strongly functional alphaNLS(1454-1497). The third, betaNLS(1294-1332), although weaker than the other two beta sequences, is significantly stronger than the analogous alphaNLS(1259-1296). Differences in the NLS sequences of human topo II isoforms may contribute to their differences in subnuclear localization.
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PMID:Sequence determinants of nuclear localization in the alpha and beta isoforms of human topoisomerase II. 1047 18


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