Role of pim-1 in the BCR/ABL-mediated leukemogenesis.

(A) FDCP1 cells expressing neomycin-resistance gene (FD/neo) and cells overexpressing mouse pim-1 (FD/mpim44) were infected with retrovirus carrying BCR/ABLWT-IRES-GFP, BCR/ABLΔΔ-IRES-GFP, or IRES-GFP (E). BCR/ABL and pim-1 proteins were detected in growth factor–starved GFP⁺ cells by Western analysis. Actin was detected as control for protein loading. (B) Apoptosis was examined in IL-3–free conditions as described in the legend to Figure 2. (C) Cell proliferation in IL-3–free medium was assessed by trypan blue exclusion. (D) 32Dcl3 cells were infected with retrovirus carrying BCR/ABL or with empty virus (E). After short selection in puromycin freshly established mix populations were infected with retrovirus carrying pim-1(K67M)FLAG-IRES-GFP or IRES-GFP (E). GFP⁺cells were obtained by FACS and BCR/ABL and pim-1(K67M)FLAG were detected in growth factor–starved cells by Western analysis using anti-ABL and anti-FLAG antibodies, respectively. The kinase reactions were performed in anti–pim-1 immunoprecipitates containing [γ-³²P]ATP and histone H1 as a substrate, which were then separated on SDS-PAGE and visualized by autoradiography (bottom box). Apoptosis (E) and proliferation potential (F) in the absence of IL-3 were assessed as described in the legend to Figure 2. Results represent at least 3 experiments.