Regulation of survivin in CD34⁺ cells by hematopoietic growth factors.

(A) Multivariate flow dot plots for survivin expression (panel 2) versus isotype control (panel 1) in freshly isolated UCB CD34⁺ cells (purity in excess of 95%). Identical results were observed with the use of either an antisurvivin mAb (shown) or a polyclonal antisurvivin antibody. Cell-cycle analysis indicated that 84% of the CD34⁺ cells were in G₀/G₁ and 16% in S+G₂/M. Panels 3 and 5 represent survivin expression in CD34⁺ cells cultured without growth factors for 24 and 48 hours, respectively. Panels 4 and 6 represent survivin expression in CD34⁺ cells cultured with Tpo, SCF, and FL for 24 and 48 hours. Cell-cycle distribution of each cell population is presented below the appropriate panels. All cells were counterstained with anti-CD34 mAb and gated to include only CD34⁺ cells. Cell samples from each population were analyzed for survivin and GAPDH by RT-PCR (35 cycles). Lane 2: freshly harvested UCB CD34⁺ cells. Lane 5: CD34⁺ cells cultured without growth factors for 48 hours. Lane 6: CD34⁺cells cultured in the presence of Tpo, SCF, and FL for 48 hours. Lane C: control sample without RNA template. Data are from 1 UCB sample and representative of 5 experiments. For Western blot analysis, UCB CD34⁺ cells were cultured with or without Tpo, SCF, and FL for 48 hours. Lysates from 1 × 10⁶cells were separated on 10% SDS-PAGE gels and probed with anti–human polyclonal antibody. (B) Multivariate flow dot plots for survivin expression (panel 2) versus isotype control (panel 1) in freshly isolated adult bone marrow CD34⁺ cells (purity in excess of 97%). Identical results were observed with the use of either an antisurvivin mAb (shown) or a polyclonal antisurvivin antibody. Cell-cycle analysis indicated that 87% of the CD34⁺ cells were in G₀/G₁ and 13% in S+G₂/M. Panels 3 and 5 represent survivin expression in CD34⁺ cells cultured in the absence of growth factors for 24 and 48 hours. Panels 4 and 6 represent survivin expression in CD34⁺ cells cultured in the presence of Tpo, SCF, and FL for 24 and 48 hours. Cell-cycle distribution of each cell population is presented below the appropriate panels. All cells were counterstained with anti-CD34 mAb and gated to include only CD34⁺ cells. Cell samples from each population were analyzed for survivin and GAPDH mRNA by RT-PCR (35 cycles). Lane 2: freshly isolated bone marrow CD34⁺ cells. Lanes 3 and 5: CD34⁺ cells cultured without growth factors for 24 and 48 hours, respectively. Lanes 4 and 6: CD34⁺ cells cultured in the presence of Tpo, SCF, and FL for 24 and 48 hours, respectively. Lane C represents control sample without any RNA template. Data are from 1 of 2 identical experiments.