scholarly journals Chromosome damage induced by DNA topoisomerase II inhibitors combined with g-radiation in vitro

1998 ◽  
Vol 21 (3) ◽  
pp. 407-417
Author(s):  
Maria Cristina P. Araújo ◽  
Francisca da Luz Dias ◽  
Andréa O. Cecchi ◽  
Lusânia M.G. Antunes ◽  
Catarina S. Takahashi
Keyword(s):  
Cho Cells ◽  
Topoisomerase Ii ◽  
Chinese Hamster ◽  
The Other ◽  
Additive Effects ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Topoisomerase Ii Inhibitors ◽  
Radiation Induced

Combined radiation and antineoplastic drug treatment have important applications in cancer therapy. In the present work, an evaluation was made of two known topoisomerase II inhibitors, doxorubicin (DXR) and mitoxantrone (MXN), with g-radiation. The effects of DXR or MXN on g-radiation-induced chromosome aberrations in Chinese hamster ovary (CHO) cells were analyzed. Two concentrations of each drug, 0.5 and 1.0 µg/ml DXR, and 0.02 and 0.04 µg/ml MXN, were applied in combination with two doses of g-radiation (20 and 40 cGy). A significant potentiating effect on chromosomal aberrations was observed in CHO cells exposed to 1.0 µg/ml DXR plus 40 cGy. In the other tests, the combination of g-radiation with DXR or MXN gave approximately additive effects. Reduced mitotic indices reflected higher toxicity of the drugs when combined with radiation.

scholarly journals Participation of ATP in the binding of a yeast replicative complex to DNA.

1987 ◽  
Vol 246 (1) ◽  
pp. 213-219 ◽  
Author(s):  
S M Jazwinski
Keyword(s):  
Topoisomerase Ii ◽  
Relative Sensitivity ◽  
The Other ◽  
Functional Assay ◽  
Dna Topoisomerase Ii ◽  
Dna Complexes ◽  
Dna Topoisomerase ◽  
Initial Interaction ◽  
Atp Analogues

The activity that replicates yeast DNA in vitro can be isolated from cells of the budding yeast Saccharomyces in a high-Mr (approximately 2 × 10(6] form. Several lines of evidence indicate that this fraction contains a multiprotein replicative complex. A functional assay has been developed for the analysis of the interaction of the replicating activity with DNA. Binding of the activity required Mg2+, but did not require the addition of ATP or the other ribo- or deoxynucleoside triphosphates. However, the ATP analogues adenosine 5′-[gamma-thio]triphosphate and adenosine 5′-[beta gamma-imido]triphosphate blocked the binding, suggesting that ATP participates in the interaction at some stage. The binding was template (origin)-specific in either the presence or the absence of ATP and the other nucleoside triphosphates; however, ATP stabilized the replicating activity. The preferential inhibition of binding that was observed in the presence of the DNA topoisomerase II inhibitor coumermycin suggests that the requirement for ATP may be at least partially accounted for by the involvement of this enzyme in the initial interaction of the replicating activity with DNA. Finally, the binding was rapid. In contrast, DNA synthesis displayed a lag when assayed directly without first allowing a period for the replicating activity to bind to the DNA. In addition, binding was ‘tight’, as judged by the resistance of the protein–DNA complexes to salt in comparison with the relative sensitivity of binding. The replicating activity was not readily displaced from the complexes by exogenous DNAs, either possessing or lacking yeast origins of replication. The results suggest that the interaction of the replicating activity with the DNA occurs in more than one stage.

Diminished molecular response to doxorubicin and loss of cardioprotective effect of dexrazoxane inEgr-1deficient female mice

2001 ◽  
Vol 79 (6) ◽  
pp. 533-544 ◽  
Author(s):  
Nacéra Saadane ◽  
Ping Yue ◽  
Lesley Alpert ◽  
Benjamin Mitmaker ◽  
Gordon M Kirby ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Topoisomerase Ii ◽  
Molecular Response ◽  
Dna Topoisomerase Ii ◽  
Cardiac Damage ◽  
Dna Topoisomerase ◽  
Female Mice ◽  
Topoisomerase Ii Inhibitors

Doxorubicin (DOX) and VP16 are DNA topoisomerase II inhibitors yet only DOX induces an irreversible cardiotoxicity, likely through DOX-induced oxidative stress. Egr-1 is overexpressed after many stimuli that increase oxidative stress in vitro and after DOX-injection into adult mice in vivo. To investigate Egr-1 function in the heart, we compared the molecular and histological responses of wild type (+/+) and Egr-1 deficient (–/–) female mice to saline, DOX, VP16, the cardioprotectant dexrazoxane (DZR), or DOX+DZR injection. DOX, and to a lesser extent VP16, induced characteristic increases in cardiac muscle and non-muscle genes typical of cardiac damage in +/+ mice, whereas only β-MHC and Sp1 were increased in –/– mice. DZR-alone treated +/+ mice showed increased cardiomyocyte transnuclear width without a change to the heart to body weight (HW/BW) ratio. However, DZR-alone treated –/– mice had an increased HW/BW, increased cardiomyocyte transnuclear width, and gene expression changes similar to DOX-injected +/+ mice. DZR pre-injection alleviated DOX-induced gene changes in +/+ mice; in DZR+DOX injected –/– mice the increases in cardiac and non-muscle gene expression were equal to, or exceeded that, detected after DOX-alone or DZR-alone injections. We conclude that Egr-1 is required for DOX-induced molecular changes and for DZR-mediated cardioprotection.Key words: mice, gene expression, doxorubicin, DNA topoisomerase II inhibitors, cardioprotection.

Multifactorial resistance to antineoplastic agents in drug-resistant P388 murine leukemia, Chinese hamster ovary, and human HeLa cells, with emphasis on the role of DNA topoisomerase II

1992 ◽  
Vol 70 (5) ◽  
pp. 354-364 ◽  
Author(s):  
Abdul M. Deffie ◽  
J. Peter McPherson ◽  
Radhey S. Gupta ◽  
David W. Hedley ◽  
Gerald J. Goldenberg
Keyword(s):  
Hela Cells ◽  
Cho Cells ◽  
Topoisomerase Ii ◽  
Chinese Hamster ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Catalytic Activities ◽  
Topo Ii ◽  
Posttranscriptional Level

The role of DNA topoisomerase II in multifactorial resistance to antineoplastic agents is reviewed. We have previously observed that in Adriamycin (ADR) resistant P388 murine leukemia cells, DNA topoisomerase II enzyme content and cleavage and catalytic activities were all reduced and correlated with drug sensitivity. A subsequent study provided evidence for an allelic mutation of the gene for DNA topoisomerase II as a possible molecular mechanism underlying the enzyme alterations. To ascertain how universal were these observations, a study was undertaken of DNA topoisomerase II (topo II) in other cell lines resistant either to ADR or another topo-II-interactive drug, mitoxantrone. In ADR-resistant Chinese hamster ovary (CHO) cells, topo II cleavage and catalytic activities and the gene product were all reduced; however, only cleavage activity correlated with drug sensitivity. No differences were noted between ADR-sensitive and -resistant CHO cells by Northern or Southern blot analysis, raising the possibility that the enzyme in resistant cells may be regulated at a posttranscriptional level. Findings on a gel retardation or immunoblot band depletion assay showed that the enzyme in CHO/ADR-1 cells failed to bind to the DNA–drug–enzyme complex, suggesting a qualitative as well as quantitative enzyme alteration in those cells. Mitoxantrone-resistant HeLa cells (Mito-1) displayed not only a lower level of cleavage activity but also of enzyme content and catalytic activity, relative to the parental drug-sensitive HeLa cells. As with the CHO cells, no differences were noted between mitoxantrone-sensitive and -resistant HeLa cells on Northern and Southern blot analyses, suggesting that enzyme regulation in these resistant cells may also be at a posttranscriptional level. There was no evidence of enzyme binding to DNA–drug–enzyme complex in resistant HeLa/Mito-1 cells, once again suggesting the presence of a qualitative enzyme alteration. The findings in both ADR-resistant CHO cells and mitoxantrone-resistant HeLa cells do not exclude the possibility that subtle changes in the topoisomerase II gene, such as point mutations, may account for these enzyme changes. The apparent qualitative changes observed in enzyme may result from posttranslational modifications such as phosphorylation.Key words: drug resistance, DNA topoisomerase II, Adriamycin, mitoxantrone.

scholarly journals Suramin is an inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells.

1992 ◽  
Vol 89 (7) ◽  
pp. 3025-3029 ◽  
Author(s):  
K. Bojanowski ◽  
S. Lelievre ◽  
J. Markovits ◽  
J. Couprie ◽  
A. Jacquemin-Sablon ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
Chinese Hamster ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Fibrosarcoma Cells ◽  
Hamster Fibrosarcoma

scholarly journals Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast

1997 ◽  
Vol 324 (1) ◽  
pp. 329-339 ◽  
Author(s):  
Beatrice A. KELLER ◽  
Sandhiya PATEL ◽  
L. Mark FISHER
Keyword(s):  
Candida Albicans ◽  
Topoisomerase Ii ◽  
Cloning And Expression ◽  
Dna Topoisomerase Ii ◽  
Topoisomerase Iiα ◽  
Dna Topoisomerase ◽  
Topoisomerase Ii Inhibitors ◽  
Anti Fungal

Candida albicans topoisomerase II, encoded by the TOP2 gene, mediates chromosome segregation by a double-strand DNA break mechanism and is a potential target for anti-fungal therapy. In this paper, we report the characterization of the C. albicans TOP2 gene and its use to develop a yeast system that allows the identification and study of anti-fungal topoisomerase II inhibitors in vivo. The gene, specifying a 1461-residue polypeptide with only 40% identity with human topoisomerase IIα and β isoforms, was isolated from C. albicans on a 6.3 kb EcoRI fragment that mapped to chromosome 4. It was used to construct a plasmid in which TOP2 expresses a recombinant enzyme (residues 57–1461 of C. albicans topoisomerase II fused to the first five residues of Saccharomyces cerevisiae topoisomerase II) under the control of a galactose-inducible promoter. The plasmid rescued the lethal phenotype of a temperature-sensitive S. cerevisiae DNA topoisomerase II mutant allowing growth at 35 °C. Yeast cells, bearing ISE2 permeability and rad52 double-strand-break-repair mutations the growth of which at 35 °C was dependent on C. albicans topoisomerase II, were killed by the known topoisomerase II inhibitors amsacrine and doxorubicin. Parallel experiments in yeast expressing human topoisomerase IIα allowed the relative sensitivities of the fungal and host topoisomerases to be examined in the same genetic background. To compare the killing in vivo with drug inhibition in vitro, the recombinant C. albicans topoisomerase II protein was expressed and purified to near-homogeneity from S. cerevisiae yielding a 160 kDa polypeptide that displayed the expected ATP-dependent DNA-relaxation and DNA-decatenation activities. The enzyme, whether examined in vitro or complementing in S. cerevisiae, was comparably sensitive to amsacrine and doxorubicin. Our results suggest that potential topoisomerase II-targeting anti-fungal inhibitors can be identified and studied in S. cerevisiae.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

1993 ◽  
Vol 51 ◽  
pp. 74-75
Author(s):  
Jason R. Swedlow ◽  
Neil Osheroff ◽  
Tim Karr ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Topoisomerase Ii ◽  
Biochemical Characterization ◽  
Developmental Cycle ◽  
Dna Topoisomerase Ii ◽  
Chromosomal Distribution ◽  
Dna Topoisomerase ◽  
Ideal System ◽  
Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

Sign in / Sign up

Export Citation Format

Share Document