ITD-Flt3 mutations increase Survivin expression in Ba/F3 cells coincident with enhanced cell proliferation and reduction in active caspase-3. (A) Ba/F3 cells transduced with wild-type or ITD-Flt3 were cultured in RPMI-1640 with 1% HI-FBS in the absence of IL-3 for 24 and 48 hours. Survivin protein was determined by Western analysis. Representative data from 1 of 2 experiments are shown. The inset shows the percentage increase in Survivin mRNA expression in Ba/F3 cells ectopically expressing 3 different ITD-Flt3 constructs (N51, N73, and N78) compared with cells expressing wild-type Flt3 as determined by QRT-PCR. QRT-PCR was performed using Platinum SYBR Green qPCR SuperMix UDG. The primers for mouse Survivin were 5′-TGG CAG CTG TAC CTC AAG AA-3′ and 5′-AGC TGC TCA ATT GAC TGA CG-3′. The sequences for the mouse GAPDH primers were 5′-ATG GTG AAG GTC GGT GTG AAC G-3′ and 5′-GTT GTC ATG GAT GAC CTT GGC C-3′. (B) Proliferation of Ba/F3 cells expressing wild-type or ITD-Flt3 after IL-3 withdrawal. One million cells were seeded in RPMI-1640 plus 1% HI-FBS and total cell number was enumerated after 24 and 48 hours using trypan blue. Data shown are mean ± SEM for 1 of 3 experiments with identical results. *P < .05 compared with wild-type Flt3. (C) Percentage of Ba/F3 cells described in panel B in S+G₂/M phase of the cell cycle. Cells were fixed in 1% paraformaldehyde and stained with 1 μg/mL propidium iodide. Cell cycle was analyzed by flow cytometry and ModFit software (Verity Software House, Topsham, ME). Data are mean ± SEM from 3 experiments. *P < .05 compared with wild-type Flt3. (D) Active caspase-3 in Ba/F3 cells expressing wild-type or ITD-Flt3 after IL-3 withdrawal for 48 hours. Cells were fixed in 1% paraformaldehyde and stained in 0.25% Triton X-100/1% BSA/PBS using PE conjugated anti–active caspase-3 antibody (BD Biosciences). Representative histogram for 1 of 2 experiments with identical results is shown. (E) Total cell proliferation and Survivin expression of ITD-Flt3⁺ human acute leukemia MV4-11 cells treated with the ITD-Flt3 inhibitor SU5416 for 72 hours. (F) Survivin expression in human primary ITD-Flt3⁺ or ITD-Flt3⁻ AML cells. Lane 1: ITD-Flt3⁺ AML; normal cytogenetics. Lane 2: ITD-Flt3⁺ AML; normal cytogenetics. Lane 3: ITD-Flt3⁺ AML; normal cytogenetics. Lane 4: ITD-Flt3⁻ AML; normal cytogenetics. Lane 5: ITD-Flt3⁻ MLL (11q23). Lane 6: ITD-Flt3⁻ AML; trisomy 13.