Immature hematopoietic cells expand abnormally in FLT3/ITD mice. (A) Expression of FLT3 in BM from FLT3/ITD mice. (i) Quantitative RT-PCR analysis of FLT3 expression in CD34⁺ MPPs from FLT3^wt/ITD, FLT3^−/ITD, and FLT3^ITD/ITD mice. (ii) Densitometry quantitation of RT-PCR products resulting from amplifying the WT and ITD alleles. Values below the electrophoresis image indicate relative fold changes of mRNA levels normalized to murine S16. Flow cytometry shows that surface FLT3 expression is reduced in FLT3^−/ITD and FLT3^ITD/ITD mice (iii), whereas intracellular expression of FLT3 in FLT3^wt/ITD and FLT3^−/ITD BM is comparable (iv). (Bii) Two- to 3-week-old FLT3^ITD/ITD BM shows the highest level of phospho-STAT5 compared with the age-matched FLT3^−/ITD, FLT3^wt/ITD, and WT control. (ii) Two-month-old FLT3^−/ITD and FLT3^wt/ITD BM cells show increased levels of phospho-STAT5 compared with the WT control. (C-D) Flow cytometric analysis of BM from 2-month-old FLT3^wt/ITD, FLT3^−/ITD mice, and a 3-week-old FLT3^ITD/ITD mouse. Numbers indicate percentages of cells in whole BM. Data were obtained from 5 individual mice in each group. (E) BM from FLT3^−/ITD and FLT3^ITD/ITD mice demonstrates enhanced granulocytic/monocytic colony-forming activity. Methylcellulose-based in vitro colony-forming assay of Lin⁻ BM cells from 2-month-old WT, FLT3^wt/ITD, FLT3^−/ITD mice, and a 3-week-old FLT3^ITD/ITD mouse. A representative of 3 experiments is shown. (F) Hematopoietic progenitors from FLT3/ITD BM have increased PU.1 and decreased GATA-1 expression. Quantitative RT-PCR using CD34⁺ MPP cells from 2- to 3-week-old WT, FLT3^wt/ITD, and FLT3^−/ITD, and FLT3^ITD/ITD mice (i) or 2-month-old WT, FLT3^wt/ITD, and FLT3^−/ITD mice (ii). A representative of 3 experiments is shown. Data are expressed as mean ± SEM (error bars).