TEL/AML1 modulates genes with antiapoptotic functions. (A) Gel sections of 2D gels displaying HSP expression and synthesis of siRNA C1–treated (functional) and control REH cells on day 6 of the experiment, the time at which REH cells were depleted of TEL/AML1 (Figure 1B-C). Cells were metabolically labeled with 35S Protein Labeling Mix (MP Biomedicals, Irvine, CA). Cytoplasmic proteins were separated by 2D gel electrophoresis and proteins stained with a 400-nM solution of ruthenium II tris. Fluorography scanning was performed with the FluorImager 595 (GE Healthcare, Fairfield, CT) and autoradiography scanning, with the Phosphorimager SI (GE Healthcare), both at a resolution of 100 μm. Shown is 1 of 2 independent experiments. HSP90 expression was decreased in TEL/AML1-silenced REH cells (top right panel) compared with the control (bottom left panel). Protein synthesis of HSP70 (HSP70 and HSP8A) and HSP90 was decreased in TEL/AML1-silenced REH cells (bottom right panel) compared with control REH cells (bottom left panel), while the expression and synthesis of actin were unchanged. (B) The differences of protein amounts and protein synthesis of HSP90 and HSP70 from the gel presented in panel A were quantified and depicted as arbitrary units (AU). □ represent siRNA C1–treated cells; ▪, the control cells. (C) Immunoblots were performed using cell lysates from siRNA S– or C1–treated REH cells on day 8 of the experiment, and HSP90, HSP70, and survivin expression was detected by anti-HSP90, anti-HSP70 (Santa Cruz Biotechnologies, Santa Cruz, CA), and rabbit antisurvivin (Cell Signaling Technology, Danvers, MA) antibodies, respectively. The same lysate and amount of protein were used for tubulin as loading control. (D) Western blot analysis of cell lysates from TEL/AML1 stably expressing Ba/F3 cells was performed to assess HSP90 and survivin regulation by the chimeric protein. Ba/F3 cells that were transfected with either the empty vector or the GFP vector, as indicated at the bottom of the blot, served as controls. TEL/AML1 was detected by an anti–V5-HRP antibody (Invitrogen). For the detection of HSP90 and survivin, the same antibodies were used as in panel C. Loading control was performed with an anti-p44/p42 MAPK antibody (Cell Signaling Technology). The film was exposed only very shortly in order to visualize the increase in HSP90 and survivin. Upon longer exposure, the difference becomes less distinct due to the large amounts of these highly abundant proteins in all cells (data not shown). (E) TEL/AML1 confers resistance to apoptosis-inducing conditions. Stably TEL/AML1-expressing Ba/F3 cells (a fresh mixture of 3 clones with a fusion gene mRNA expression within the range of primary ALL cells) and controls (vector without insert) were grown at 0.5 × 106/mL in 4 replicates and subjected to either IL3 (WEHI-conditioned medium) or serum plus IL3 starvation (no suppl), or to VP16 (Bristol-Myers Pharmaceuticals, Hounslow, United Kingdom) treatment (1 mg/mL). Apoptosis was assessed by PI staining after 24 and 72 hours. The relative increment of PI-positive cells under apoptosis-inducing conditions was calculated against that of untreated cells. Data in the graph represent mean ± SD of 3 separate experiments. *Statistically significant changes (P < .001).
