Assessment of Ras activity and farnesyl protein transferase inhibition in BCR/ABL-BaF3 cells treated with SCH66336.

(A) Levels of activated, GTP-bound Ras protein in BCR/ABL-BaF3 cells was determined by quantifying the association with the effector domain of Raf-1 fused to GST. The high levels of active Ras-GTP protein in BCR/ABL-BaF3 cells were significantly reduced after treatment with 500 nM SCH66336. Expression of a dominant-negative (DN) form of Ras reduced the levels of Ras-GTP to undetectable levels. (B) DNA binding activity of AP-1 transcription factor complex in BaF3 and BCR/ABL-BaF3 cells as determined by electrophoretic mobility shift assay. Lanes 1 and 2: Parental BaF3 cells without IL-3 or treated with 25 ng/mL IL-3 for 30 minutes. Lane 3: BaF3 cells transduced with a retrovirus encoding an activated form of H-Ras (61L). Lane 4: BCR/ABL-BaF3 cells. Lanes 5-7: BCR/ABL-BaF3 cells transduced with a dominant-negative form of H-Ras (lane 5), treated with FTI vehicle DMSO (lane 6), or treated with 1 μM SCH66336 for 48 hours before extract preparation (lane 7). Lanes 8, 9: BaF3 cells transduced with a retrovirus encoding an activated form of H-Ras (61L) and treated with FTI vehicle DMSO (lane 8) or 1 μM SCH66336 (lane 9) for 48 hours before extract preparation. (C) Blot showing cellular inhibition of farnesylation by SCH66336 using a representative protein, DnaJ, which, unlike K-Ras, was not subject to alternative prenylation in the presence of FTI. BaF3 and BCR/ABL-BaF3 cells were incubated in the indicated concentrations of SCH66336, and DnaJ was detected by immunoblotting, as described in “Materials and methods.” Farnesylation of DnaJ resulted in reduced molecular mass due to subsequent processing of the protein. Thus, the “unprocessed” band represents DnaJ that was not farnesylated, whereas the “processed” band represents DnaJ that was farnesylated. Farnesylation was almost completely inhibited in BCR/ABL-BaF3 cells treated with 1 μM SCH66336.