AZD6244 induces cell cycle and growth arrest in DLBCL cell lines, primary cells, and in a SCID xenograft model. (A) All cell lines were treated with the indicated concentrations of AZD6244 for 24 hours. After fixation in 70% ethanol, cells were stained with propidium iodide (final concentration 50 μg/mL) in hypotonic solution containing RNase (180 units/mL) for 30 minutes. Cells were analyzed by flow cytometry. The bar graph shows significant G1 arrest after AZD6244 treatment. (B) AZD6244 induces growth inhibition. All cell lines were treated with variousconcentration (50nM to 400nM) for 24, 48, and 72 hours. Cell growth was measured by MTT assay. P values are inserted for each cell line regarding concentration- and time-dependent comparisons. (C) AZD6244 induces concentration-dependent apoptosis. All cell lines were treated with indicated concentrations of AZD6244 for 48 hours. Apoptosis was measured by annexin V/PI staining by flow cytometry. (D) Apoptotic induction in primary/fresh DLBCL cells afterAZD6244. PBMCs were incubated with the indicated concentrations for 24 (left) or 72 hours (right). Apoptosis was measured by annexin V/PI staining followed by flow cytometric analysis. Data were analyzed by FACS express software. (E) SUDHL-6 cells were subcutaneously injected into left and right dorsal flanks of 7-week-old female SCID mice. When the tumor reached the size of approximately 60-160 mm,³  AZD6244 was administered intraperitoneally every other day at a dose of 10 mg/kg body weight for a total of 3 weeks. Tumors were measured 3 times weekly. Ctrl indicates control; and Hr, hours. *P < .05; **P < .01, and ***P < .001.