Effects of selective signaling pathway inhibitors or dn- cDNAs on Survivin expression. (A) Ba/F3 cells expressing ITD-Flt3 (N51, N73, and N78) were incubated with 10 and 100 μM PD98059, 10 and 50 μM LY294002, 1 and 10 μM H89, 5 and 50 μM Akt inhibitor, or control DMSO for 24 hours. Ba/F3 cells expressing wild-type Flt3 were included as a control. Survivin expression was determined by Western analysis. Ratio of Survivin/actin relative to DMSO control in ITD-Flt3+ cells determined using densitometer analysis is shown beneath the blot. The selective pathway inhibitors PD-98059 (MAPKp42/44), LY-294002 (PI3-kinase), H89 (PKA), and Akt inhibitor were purchased from Wako Chemicals (Osaka, Japan). (B) dn-Akt or dn-H-Ras cDNAs were transfected into Ba/F3 cells expressing ITD-Flt3 (N51). Ba/F3 cells expressing wild-type Flt3 or ITD-Flt3 were also transfected with vector alone. dn-Akt in pcDNA3 was a kind gift from Drs R. A. Roth and H. Nakshatri (Stanford University School of Medicine, Stanford, CA, and Indiana University School of Medicine, respectively). Transfection of dn-Akt or dn-H-Ras cDNA into Ba/F3 cells expressing Flt3 were performed using a Nucleofector transfection kit (Amaxa, Gaithersburg, MD). Twenty-four hours after transfection, cells were subjected to Western analysis for Survivin. (C) MV4-11 cells were incubated with control DMSO, 50 μM LY290042, or 50 μM Akt inhibitor up to 72 hours in 10% HI-FBS plus RPMI-1640. Cell counts were enumerated by trypan blue exclusion in triplicate. Survivin expression was determined by Western analysis at 24 hours. *P < .05 compared with DMSO control. Representative data for 1 of 2 experiments are shown and are expressed as mean ± SEM.
