WP1130 activity is not mediated through the loss of Hsp90/Bcr-Abl chaperone association, 20S proteasome inhibition, or increased ROS. (A) K562 cells were incubated with 5μM WP1130 for 30 minutes before detergent-soluble cell extracts were subjected to Hsp90 immunoprecipitation followed by immunoblotting for Bcr-Abl, ubiquitin, or Hsp90. (B) K562 cells were incubated with WP1130 (5μM), H2O2 (500μM), or dithiothreitol (1mM) for 2 hours at 37°C. Cells were then washed and replated in medium containing DCFDA for 20 minutes at 37°C. DCFDA fluorescence was read and used as a measure of ROS production in treated samples. The results represent the average ± SD of triplicate assays. Similar results were obtained in 2 additional CML cell lines, WDT-2 and BV-173. (C left) K562 cells were left untreated or treated with 5μM of the indicated compound for 2 hours at 37°C. Protein lysates were incubated with proteasome substrate and activity was determined by fluorogenic substrate cleavage. (C right) Purified 20S proteasome was incubated with 5μM MG-132, lactacystin, or WP1130 before assaying proteasome activity as described above. The results represent the average ± SD of triplicate assays. Similar results were obtained with 2 additional CML cell lines. (D) K562 cells were treated with 50 nM bortezomib (Bz) or 5μM WP1130 (WP) before equal volumes of lysate from the detergent-soluble, detergent-insoluble, and total cell lysate were immunoblotted for Bcr-Abl (top) or ubiquitin (bottom). Both Bz and WP increased ubiquitin content, but only WP affected Bcr-Abl and detergent-insoluble ubiquitinated protein levels.
