Biologic effects of the expression level of the FLT3 transcript. Biologic effects of the FLT3 transcript level were analyzed by using 8 AML samples harboring a normal karyotype. Two—UPNs 2 (FAB, M4) and 119 (M4)—had FLT3/ITD, and 6—UPNs 14 (M2), 78 (M2), 11 (M1), 18 (M5), 23 (M0), and 67 (M1)—had Wt-FLT3. Of these AML samples, UPN-23 had both MLL-TD and N-RAS mutations, but the others did not have FLT3/D835, MLL-TD, p53, or N-RAS mutations. (A) Surface expression level of FLT3 was examined by flow cytometry. There was no marked difference according to the expression level of the FLT3 transcript. Open and filled histograms indicate staining with anti-FLT3 and isotype control, respectively. (B) Immunoblot analysis revealed that the total cellular level of FLT3 protein reflected the expression level of the FLT3 transcript. Each expression level of FLT3 protein was adjusted to that of Actin protein. Ratio indicates the relative expression level of the whole cellular protein (Protein), surface protein (Surface), and the transcript when compared with that of UPN 14. (C) Tyrosine phosphorylation of each FLT3 protein was examined. Overexpressed Wt-FLT3 was phosphorylated as well as FLT3/ITD (upper 2 panels). These phosphorylations were inhibited by treatment with AG1296 for 3 hours (lower 2 panels). (D) Cell viability was measured by using the CellTiter96 Proliferation Assay 72 hours after the addition of AG1296 at the indicated concentration to primary AML cells. The y-axis indicates the ratio of absorbance for AG1296-treated cells to untreated cells. Wt-average indicates the results from UPNs 14 and 78 cases, ITD from UPNs 2 and 119, and Wt-High from UPN-23 and -67.