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)

We show herein that human DNA topoisomerase II beta is functional in yeast. It can complement a yeast temperature-sensitive mutation in topoisomerase II. The effect on human topoisomerase II beta of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase II alpha and wild-type yeast topoisomerase II. A drug permeable yeast strain (JN394 top2-4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase II beta transformants. A parallel analysis on human topoisomerase II alpha transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs. The strain was analysed at 35 degrees C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active. A shuttle vector with either human topoisomerase II beta, human topoisomerase II alpha or yeast topoisomerase II under the control of a GAL1 promoter was used. The key findings were that amsacrine produced comparable levels of cell killing with both alpha and beta, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with alpha than with beta or yeast topoisomerase II. Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II. Suramin, quercetin and genistein showed little cell killing in this system. This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms. This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase II beta or to select for drug-resistance mutations in human topoisomerase II beta.
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PMID:Complementation of temperature-sensitive topoisomerase II mutations in Saccharomyces cerevisiae by a human TOP2 beta construct allows the study of topoisomerase II beta inhibitors in yeast. 902 79

Using confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase II beta (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37 degrees or 42 degrees C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0 degree C in a buffer containing spermine/spermidine/KCl or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0 degree C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37 degrees or 42 degrees C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KCl, the incubation buffer also contained 5 mM Mg++ and the temperature was 37 degrees C. If the stabilization was performed at 42 degrees C, Mg++ failed to maintain the original distribution of DNA topoisomerase II beta, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37 degrees C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37 degrees C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0 degree C incubated nuclei; they showed a certain degree of shrinkage in 0 degree C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37 degrees or 42 degrees C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37 degrees C, but not at 42 degrees C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus.
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PMID:Redistribution of DNA topoisomerase II beta after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy. 908 Apr 8