Figure 5
From: Anomalous constitutive Src kinase activity promotes B lymphoma survival and growth
Dasatinib inhibits constitutive BCR signaling and B lymphoma growth. Bcl-2 and Bcl-x L expression confers resistance to PP2 induced apoptosis in B cell lymphomas. (A) Dasatinib inhibits SFK phosphorylation and phosphorylation of its downstream targets ERK and AKT and reduces the level of Egr-1 in BKS-2 lymphoma cells. BKS-2 cells were treated with or without 100 nM dasatinib for 1 hr. The cell lysates were analyzed by Western blotting for p-SFK, p-ERK and p-AKT. The blots were stripped and re-probed for Egr-1, ERK and β-actin. (B) Dasatinib induced growth inhibition can be partially overcome by PMA or CpG. BKS-2 cells were treated with DMSO, 10 nM dasatinib, 10 nM dasatinib+10 ng/ml PMA or 10 nM dasatinib+1 μg/ml CpG for 48 hrs. The proliferation assay was performed as described in the "Materials and Methods" section. Data points indicate percent control response ± S.E. of triplicate cultures from a representative experiment of three independent experiments. The percent control response is defined as (cpm in dasatinib treated group/cpm in DMSO treated group) × 100 for dasatinib; (cpm in dasatinib+PMA treated group/cpm in the PMA treated group) × 100 for dasatinib+PMA; (cpm in dasatinib+CpG treated group/cpm in CpG treated group) × 100 for dasatinib+CpG. The actual counts for DMSO, PMA and CpG treated groups are 16355 ± 1394, 12939 ± 1345, 89504 ± 9159, respectively. (C) SudHL-4 and CH12.Lx cells were treated with or without 8 μM PP2 for two days. Early apoptotic cells were detected by flow cytometry with AnnexinV-APC and PI staining as described in the "Materials and Methods". (D) Bcl-2 and Bcl-xL protein expression in six B lymphoma cells was determined. Cell lysates were analyzed by Western blotting for Bcl-xL and Bcl-2. The blots were stripped and re-probed for β-actin for loading control. (E) The Bcl-xL expression in WEHI-231 and WEHI-231-Bcl-xL cells (WEHI-231 stably transfected with Bcl-xL). Cell lysates were analyzed by Western blotting for Bcl-xL. Then the blot was stripped and re-probed for β-actin for loading control. (F) Ectopic expression of Bcl-xL makes WEHI-231 cells more resistant to PP2 induced apoptosis. WEHI-231 and WEHI-231-Bcl-xL cells were treated with or without 5 μM PP2 for one day. Early apoptotic cells were analyzed by flow cytometry with Annexin-V-FITC and PI staining as described.