monorden and Carcinoma--Adenosquamous

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 IIalpha-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 IIalpha-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy.

Topics: Adenocarcinoma; Animals; Benzoquinones; Breast Neoplasms; Carcinoma, Adenosquamous; Colonic Neoplasms; DNA Topoisomerases, Type II; Electrophoresis, Gel, Two-Dimensional; Enzyme Inhibitors; Female; HSP90 Heat-Shock Proteins; Humans; Immunoprecipitation; Lactams, Macrocyclic; Lactones; Macrolides; Mice; Mice, Nude; Molecular Chaperones; Neoplasms; Protein Binding; Quinones; Rifabutin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transplantation, Heterologous; Tumor Cells, Cultured