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Inhibition of topoisomerase II activity and its effect on nucleolar structure and function
M Govoni1, F Farabegoli, A Pession
1Dipartimento di Patologia Sperimentale, Università degli Studi di Bologna, Italy.
Abstract:
The relationship between topoisomerase II activity and ribosomal RNA synthesis was investigated using the antitumoral drug VM26, a specific inhibitor of topoisomerase II. For this purpose TG cells, a human tumor cell line, were cultured in the presence of 2.5 microM VM26 for 1 and 3 h; VM26 reduced the topoisomerase II activity, measured in whole cell extracts. In the presence of VM26 the [3H]uridine incorporation into ribosomal RNA was decreased; electron microscopy investigation of nucleoli showed a segregation of nucleolar components. Because VM26 stabilizes the cleavable complex and inhibits the resealing reaction, thus causing potential cleavage sites, we have analyzed the double-strand breaks caused by the drug treatment in the tandem repeat ribosomal DNA (rDNA) genes, by indirect labeling with two probes recognizing the 5' portion of ETS (BES) and the 3' portion of 28S (LS6BE) transcribed gene. In VM26-treated cells rDNA is fragmented and a topoisomerase II preferential cleavage site is present, localized at 1.85 kb in 28S region from 3' EcoRI site.
Insights
The antitumoral drug VM26 inhibits topoisomerase II activity, decreasing ribosomal RNA synthesis and causing DNA double-strand breaks in ribosomal DNA genes. This reveals a direct link between topoisomerase II function and rRNA production in human tumor cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Topoisomerase II is crucial for DNA topology management.
- Ribosomal RNA (rRNA) synthesis is vital for cell function.
- The impact of topoisomerase II inhibition on rRNA synthesis requires elucidation.
Purpose of the Study:
- To investigate the relationship between topoisomerase II activity and rRNA synthesis.
- To determine the effects of the topoisomerase II inhibitor VM26 on rRNA production and rDNA integrity.
Main Methods:
- Utilized TG cells (human tumor cell line) treated with VM26.
- Measured topoisomerase II activity in cell extracts.
- Assessed [3H]uridine incorporation into rRNA.
- Performed electron microscopy on nucleoli.
- Analyzed double-strand breaks in ribosomal DNA (rDNA) using indirect labeling.
Main Results:
- VM26 significantly reduced topoisomerase II activity.
- VM26 treatment decreased [3H]uridine incorporation into rRNA.
- Nucleoli exhibited segregation of components upon VM26 treatment.
- VM26 induced double-strand breaks in rDNA, with a preferential cleavage site identified.
Conclusions:
- Topoisomerase II activity is directly linked to rRNA synthesis.
- VM26-induced topoisomerase II inhibition disrupts rRNA production and rDNA stability.
- The study identifies a specific topoisomerase II cleavage site within the rDNA locus.