WP1130 inhibits the autoactivation of Bcr/Abl by inducing its rapid down-regulation. (A) Chemical structure of WP1130. The IC50 of Bcr/Abl-expressing cell lines (K562, BV-173) is also noted. (B) The BV173 (wild-type Bcr/Abl) and BV173R (T315I Bcr/Abl mutant) CML cell lines (1 × 106 cells/mL each) were treated with 5 μM imatinib mesylate or WP1130 for 1 hour. Cells were harvested, and the expression of Bcr/Abl, pY-Bcr/Abl, Hck, and pY396-Hck were determined by Western blotting. Actin was blotted as a protein-loading control. Controls were cells exposed to a comparable volume of solvent (DMSO) for 1 hour. (C) Immune-complex kinase assays were performed with Bcr/Abl isolated from K562 cells and incubated with the indicated agent for 30 minutes at 30°C in the presence of unlabeled ATP. Bcr/Abl autophosphorylation was determined by immunoblotting the resolved proteins for phosphotyrosine. (D) K562 CML cells (1 × 106 cells/mL) were treated with 5 μM WP1130 for the indicated times and subjected to Western blotting to assess Bcr/Abl protein expression levels over the 120-minute treatment period (top). Actin was used as a protein-loading control. Real-time PCR analysis of mRNA extracted from the harvested cells (bottom) was carried out to examine the effect of WP1130 on transcription of the bcr/abl gene. Results are expressed as mean ± standard error of 3 independent experiments. (E) K562 CML cells (1 × 106 cells/mL) were treated with 5 mM WP1130 for 1 hour or 2 hours, and the expression of Bcr/Abl, c-Abl, and Bcr proteins in treated and untreated cells was determined by Western blotting. Actin was used as a protein-loading control.
