Figure 5

YM155 activates DNA damage response with S phase arrest. (A) Immunoblot verifies the increase in threonine 68 phosphorylation of Chk2. REH, RCH, and SUPB15 cells were treated with increasing concentrations of YM155 (0, 10, 100 nM), doxorubicin (0.1 μg/ml) or dasatinib (100 nM) for 24 h. Whole cell lysates were subjected to immunoblot using antibodies specific for phospho-T68 Chk2, total Chk2, survivin, and Aurora B kinase. (B) Treatment with YM155 causes an increase in γH2AX. REH, RCH, and SUPB15 cells were treated with vehicle or YM155 (10 and 100nM) for 24 h and then stained with γH2AX-FITC and Vibrant DyeCycle Violet Stain. The cells were quantified using FACS/AriaIII for DNA content and FITC. Treatment with YM155 in each cell line showed a dose-dependent increase in γH2AX-FITC, mostly in G1 and S. (C) Comet assays of leukemia cell lines identify DNA damage. REH, RCH, SUPB15, and K562 cells were treated with 100 nM YM155 and 100 nM Dasatinib (excluding REH) for 24 h. Top panel is the box-whisker plot of the percent of DNA in the comet tail after treatment. Bottom panels are photographic representations of the comet assays for each cell line.