AZD1152-HQPA inhibited the proliferation of leukemia cells. (A) 3H-thymidine uptake study. Cells from various types of human leukemias were cultured in the presence of various concentrations of AZD1152-HQPA (1-100 nM) for 48 hours. Proliferation of leukemia cells was measured by 3H-thymidine uptake (isotope added 6 hours before harvest), and the concentration that induced 50% growth inhibition (IC50) was calculated from dose-response curves. Results represent the mean plus or minus SD of 3 experiments performed in triplicate plate. (B) Clonogenic growth assay. MOLM13 or MV4-11 cells (1 × 105 cells/mL) were added 1:10 to methylcellulose medium H4534, containing 1% methylcellulose, 30% FCS, 1% BSA, 10−4 M mercaptoethanol, 2 mM l-glutamine, 50 ng/mL stem-cell factor, 10 ng/mL GM-CSF, and 10 ng/mL IL-3. Cells were placed in 24-well plates in a volume of 200 μL. Prior to this step, either AZD1152-HQPA (1-10 nM) or control diluent was placed into the wells. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2, and 10 days later colonies were counted. All experiments were done twice using triplicate plates per experimental point. AZD1152-HQPA inhibited phosphorylation of histone H3 (Ser10) in leukemia cells. Error bars represent SD. (C) MOLM13 or MV4-11 cells were exposed to AZD1152-HQPA (10 nM). After 24 hours, p-histone H3-expressing population (percentages are shown) was measured by FACScan (Becton Dickinson, Mountain View, CA). (D) Freshly isolated leukemia cells (case nos. 8-10, Table 1) were exposed to AZD1152-HQPA (3 nM). After 3 hours, p-histone H3–expressing population (percentages are shown) was measured by FACScan. Results represent one of the experiments performed twice in duplicate plate.
