LY294002 inhibits survival of AML blasts. (A) AML blasts were thawed and rested for 2 hours in serum-free medium. Viable cells were counted and plated in serum-free medium containing IL-3 (5 ng/mL), SCF (50 ng/mL), and IL-6 (10 ng/mL). Cells were plated in 96-well dishes at 2 million cells per milliliter in medium lacking or containing LY294002 at the indicated concentrations. Cells were incubated for 48 hours and relative cell survival estimated using an XTT assay. For comparison, normal CD34 cells were incubated in similar conditions (▪). (B-C) AML blasts were thawed and rested for 2 hours in serum-free medium. Viable cells were counted and replated in serum-free medium containing IL-3 (5 ng/mL), SCF (50 ng/mL), and IL-6 (10 ng/mL) for 48 hours without or with LY294002 (25 μM) and then stained with propidium iodide and annexin V conjugated to FITC to analyze for apoptosis. Cells were analyzed by 2-color immunofluorescence and data analyzed. (B) A representative analysis of one patient sample. Apoptotic cells are in the bottom right quadrant (PI^-, annexin V⁺). (C) A graphical summary of results for 6 patient samples treated as in panel B. Black speckled bars indicate results for patient samples; these results are compared to MO7e cells (open bars), which do not undergo apoptosis after treatment with LY294002, to show specificity of effect. Error bars for patient samples indicate SDs.