Barker, C.R.; Rackstraw, S.; Hamlett, J.; Pennington, S.R.; Watson, A.J.M.; Jenkins, J.R.
April 2003
Gut;Apr2003 Supplement 1, Vol. 52, pA56
Academic Journal
Background and Aims: Topoisomerase II is an essential enzyme required for the viability of all cells. It plays important roles in DNA replication, recombination, chromosome segregation, mRNA expression, and maintenance of the nuclear scaffold. In humans there are two isoforms of the topoisomerase II enzyme designated α and β. Numerous topoisomerase II inhibitors are used in the treatment of acute cancers for remission induction, as salvage therapy and conditioning therapy for bone marrow transplantation. However, these chemotherapeutic drugs are highly toxic and cells are developing drug resistance. Therefore, it is of great importance to investigate the effect of current topoisomerase II inhibitors upon cells with regard to protein-protein interactions with topoisomerase II. These interacting proteins represent potential chemotherapeutic targets. Methods: Immunoprecipitations using anti-human topoisomerase II rabbit polyclonal antibodies were performed on proteins extracted from a pair of human colon cancer cell lines, HCT116 (WT, p53 KO, isogenic), Following treatment with etoposide (VP16), a topoisomerase II inhibitor. Samples were subjected to 1 and 2 dimensional electrophoresis and associated proteins identified by MALDI-TOF mass spectrometry and confirmed by counter-immunoprecipitations. Newly identified interactions were then disrupted using chemical inhibitors in growth inhibition assays. The effect of the drugs, when used in combination with topoisomerase II inhibitors was also tested. Results: Mass spectrometry and subsequent counterimmunoprecipitations identified heat shock protein 90 (HSP90) to be specifically associated with topoisomerase II. The use of VP16, a topoisomerase II poison, and geldanamycin (GA), an HSP90 inhibitor, when used in combination produced synergistic growth inhibition at concentrations where there is little or no effect when they are used individually. Conclusions: (1) There is a direct physical interaction between...


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