The modulation of DNA topology by topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and has thus become a highly attractive target for chemotherapeutic drugs. However, these drugs are highly toxic, and so new approaches are required. One such strategy is to target topoisomerase II-interacting proteins. Here we report the identification of potential topoisomerase II-associated proteins using immunoprecipitation, followed by 1-D and 2-D gel electrophoresis and MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIa-associated proteins by coimmunoprecipitation and Western blot. Six of the interacting proteins were cellular chaperones, including 3 members of the heat shock protein-90 (Hsp90) family, and so the effect of Hsp90 modulation on the antitumor activity of topoisomerase II drugs was tested using the sulforhodamine B assay, clonogenic assays and a xenograft model. The Hsp90 inhibitors geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and radicicol significantly enhanced the activity of the topoisomerase II poisons etoposide and mitoxantrone in vitro and in vivo. Thus, our method of identifying topoisomerase II-interacting proteins appears to be effective, and at least 1 novel topoisomerase IIa-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy. ' 2005 Wiley-Liss, Inc.Key words: topoisomerase II; Hsp90; protein-protein interactions; chemotherapy Topoisomerase II is an essential nuclear enzyme that modulates DNA topology by generating double stranded breaks, allowing the passage of 1 double strand of DNA through another via an ATPdependent mechanism.1,2 This enzyme is a component of DNA transcription and replication machinery, required for chromosomal segregation and maintenance of the nuclear scaffold.2 Recently, topoisomerase II has also been shown to be required for RNA polymerase II transcription.3 In humans, 2 topoisomerase II isoforms have been identified, topoisomerase IIa 4 and b. 5 The 2 isoforms differ in their cell cycle expression, nuclear localization and tissue specific expression. 6 As type II topoisomerases play such an important role within dividing cells, they are highly attractive targets for chemotherapeutic agents, especially in human cancers.Topoisomerase II can be inhibited via one of two mechanisms: Topoisomerase II poisons act by enhancing DNA cleavage and inhibiting DNA ligation, thus increasing the level of stabilized enzyme-DNA cleavage intermediate complexes. Poisons take advantage of the catalytic mechanism by changing the enzymatic conformation and converting it to a cellular toxin.7 Examples are etoposide (VP16) and mitoxantrone. Catalytic inhibitors, however, target other stages of the topoisomerase II catalytic cycle. The bisdioxopiperazines, e.g. ICRF-159, ''lock'' the ATP-operated clamp of the enzyme, causing death by deprivation of the essential activity of topoisomerase II rath...
Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90–topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.
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