Factor-independent growth of 32D cells expressing either Flt3-D835Y or Flt3-ITD. (A) Ligand-independent 3H-thymidine incorporation by Flt3-ITD and Flt3-D835Y mutants. 32D cells expressing different Flt3 constructs were starved for 12 hours in medium supplemented with 0.5% serum. Subsequently, cells were exposed to FL or IL-3 or left unstimulated. Data are shown as percentage of thymidine incorporation compared with thymidine incorporation of the respective cell line under IL-3 stimulation. (B) Long-term growth of Flt3-expressing cells. 32D cells containing the indicated Flt3 constructs were seeded at 2 × 105/mL in suspension cultures. Cells were grown in the presence of 10% FCS alone or in combination with FL and were counted until day 6 by trypan blue exclusion method. GF indicates growth factor. (C) Ligand-independent proliferation of cells expressing Flt3 mutants is dependent on Flt3 kinase activity. 3H-thymidine incorporation was analyzed in the presence of the tyrosine kinase inhibitor SU11248 in the presence of FCS only (Flt3-ITD, Flt3-D835Y) or FCS and FL (Flt3-WT). Data represent mean values ± standard deviation (SD) of triplicates.
