CEP-701 inhibits the kinase activity of FLT3/ITD and the proliferation of 32D/FLT3/ITD cells.
(A) 32D/FLT3/ITD cells were treated with CEP-701 at increasing concentrations for 1 hour at 37°C. Cell lysates were immunoprecipitated with anti-FLT3 antibody and immunoblotted with antiphosphotyrosine antibody (top panel). The same membrane was then stripped and probed with anti-FLT3 antibody (bottom panel). (B) Effects of CEP-701 on the growth of 32D/FLT3/ITD cells and 32D/pBabe cells were evaluated by counting the number of viable cells for 11 days. 32D/FLT3/ITD cells were seeded at an initial density of 2 × 105/mL in 10% FCS/RPMI medium supplemented with various concentrations of CEP-701 with and without G-CSF (20 ng/mL). 32D/pBabe cells were seeded at the same density in medium containing IL-3 (1 ng/mL) either with or without CEP-701 (5 nM), or G-CSF. The medium was replenished every other day, and the cell densities were adjusted to 2 × 105/mL. Viable cells were counted on the basis of trypan blue exclusion. Results shown are the means from triplicate assays.
