Figure 2
Figure 2. Inhibitory effects of KW-2449 on primary AML and colony-forming cells. (A) Ten AML samples consisting of 5 with wild-type FLT3 (Wt-1 to Wt-5), 3 with FLT3/ITD (ITD-1 to ITD-4), and one with both FLT3/ITD and FLT3/KDM (ITD/KDM), were analyzed. Primary AML cells were incubated with or without KW-2449 at 0.1 μM for 6 hours, and then phosphorylation status of FLT3 and STAT5 was analyzed. KW-2449 reduced phosphorylation levels of FLT3 and STAT5 in all AML samples with FLT3 mutations. In the Wt-5 sample, KW-2449 reduced the phosphorylation level of FLT3 but not of STAT5. Vertical lines have been inserted to indicate a repositioned gel lane. (B) AML cells were suspended in methylcellulose semisolid medium containing human stem cell factor, GM-CSF, and interleukin-3 with or without 0.1 μM KW-2449. Colonies were counted after 14 days. Mean treatment/control ratio ± SD from 3 experiments in each sample are shown. In accordance with the down-regulation levels of FLT3 and STAT5 phosphorylations, KW-2449 inhibited the colony formations in all AML samples with FLT3 mutations. In the Wt-5 sample, weak inhibition of the colony formation seems to reflect the sustained STAT5 activation induced by another activation mechanism. (C) Mononuclear cells from human CB were plated in the complete methylcellulose semisolid medium with an increasing concentration of KW-2449. After 14 days of culture, BFU-E, CFU-GM, and CFU-GEMM colonies were counted. Mean total colony numbers ± SD at the indicated concentrations of KW-2449 are shown (n = 5). Although KW-2449 inhibited a total number of colonies in a dose-dependent manner, the Bonferroni test revealed that the statistical significances were found between control and at the 0.5 μM (P < .01), and at the .01 μM and at the 0.5 μM (P < .05). (D) The representative result of the distribution of BFU-E, CFU-GM, and CFU-GEMM colonies from 1 CB sample is shown. Although KW-2449 inhibited the colony formation of CB mononuclear cells in a dose-dependent manner, the distribution of BFU-E, CFU-GM, and CFU-GEMM colonies was not affected by the KW-2449 treatment.

Inhibitory effects of KW-2449 on primary AML and colony-forming cells. (A) Ten AML samples consisting of 5 with wild-type FLT3 (Wt-1 to Wt-5), 3 with FLT3/ITD (ITD-1 to ITD-4), and one with both FLT3/ITD and FLT3/KDM (ITD/KDM), were analyzed. Primary AML cells were incubated with or without KW-2449 at 0.1 μM for 6 hours, and then phosphorylation status of FLT3 and STAT5 was analyzed. KW-2449 reduced phosphorylation levels of FLT3 and STAT5 in all AML samples with FLT3 mutations. In the Wt-5 sample, KW-2449 reduced the phosphorylation level of FLT3 but not of STAT5. Vertical lines have been inserted to indicate a repositioned gel lane. (B) AML cells were suspended in methylcellulose semisolid medium containing human stem cell factor, GM-CSF, and interleukin-3 with or without 0.1 μM KW-2449. Colonies were counted after 14 days. Mean treatment/control ratio ± SD from 3 experiments in each sample are shown. In accordance with the down-regulation levels of FLT3 and STAT5 phosphorylations, KW-2449 inhibited the colony formations in all AML samples with FLT3 mutations. In the Wt-5 sample, weak inhibition of the colony formation seems to reflect the sustained STAT5 activation induced by another activation mechanism. (C) Mononuclear cells from human CB were plated in the complete methylcellulose semisolid medium with an increasing concentration of KW-2449. After 14 days of culture, BFU-E, CFU-GM, and CFU-GEMM colonies were counted. Mean total colony numbers ± SD at the indicated concentrations of KW-2449 are shown (n = 5). Although KW-2449 inhibited a total number of colonies in a dose-dependent manner, the Bonferroni test revealed that the statistical significances were found between control and at the 0.5 μM (P < .01), and at the .01 μM and at the 0.5 μM (P < .05). (D) The representative result of the distribution of BFU-E, CFU-GM, and CFU-GEMM colonies from 1 CB sample is shown. Although KW-2449 inhibited the colony formation of CB mononuclear cells in a dose-dependent manner, the distribution of BFU-E, CFU-GM, and CFU-GEMM colonies was not affected by the KW-2449 treatment.

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