![]() Normal rabbit IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antihuman retinoblastoma protein (Rb) monoclonal antibody (mAb) (G3-245), fluorescein isothiocyanate (FITC)–conjugated anti–underphosphorylated-Rb mAb (clone G99-549), FITC–antihuman cyclin D1/D2/D3 mAb (clone G124-259), FITC–anti-Ki-67 mAb (clone B56), FITC–antimouse Ig, FITC-mouse IgG1 and 7-AAD (Via Probe) were obtained from BD Pharmingen (San Diego, CA). 18 Monoclonal antihuman survivin antibody (6E4) was purchased from Cell Signaling (Beverly, MA). We previously described the specificity of the AF886 survivin antibody for intracellular staining in CD34 + cells. Affinity purified antihuman survivin polyclonal antibody (AF886) and mouse IgG 1 were purchased from R&D Systems (Minneapolis, MN). Recombinant murine GM-CSF was purchased from BioVision (Palo Alto, CA). Recombinant human and mouse stem cell factor (SCF) were a gift from Dr Karl Nocka, UCB Research (Cambridge, MA). ![]() Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (FL), and thrombopoietin (Tpo) were provided by Immunex, Seattle, WA. These studies provide further evidence that survivin up-regulation by growth factors is not a consequence of cell cycle progression and strongly suggest that survivin is an important early event for cell cycle entry by CD34 +cells. An antisense survivin construct decreased total and S-phase CFU-GM. Retrovirus transduction of survivin-internal ribosome entry site–enhanced green fluorescent protein (survivin-IRES-EGFP) in primary mouse marrow cells increased granulocyte macrophage–colony-forming units (CFU-GM) by 1.7- to 6.2-fold and the proportion of CFU-GM in S phase, compared to vector control. Selective inhibition of the PI3-kinase/AKT and mitogen-activated protein kinase (MAPK p42/44) pathways blocked survivin up-regulation by growth factors before arresting cell cycle. Quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) and multivariate flow cytometry demonstrated that Tpo, SCF, and FL increase survivin mRNA and protein in quiescent G 0 CD34 +cells without increasing Ki-67 expression, indicating that cytokine-stimulated up-regulation of survivin in CD34 +cells occurs during G 0, before cells enter G 1. Up-regulation of survivin by thrombopoietin (Tpo), Flt3 ligand (FL), and stem cell factor (SCF) occurred in underphosphorylated-retinoblastoma protein (Rb) positive, Ki-67 negative, and cyclin D negativeCD34 + cells. Survivin expression is coincident with cell cycle progression. Analysis of known human IAPs revealed that only survivin is cytokine regulated in CD34 + cells. Herein, we examined survivin expression in CD34 + cells before and after cell cycle entry and demonstrate a role for survivin in cell cycle regulation and proliferation. We previously reported that survivin is expressed and growth factor regulated in normal adult CD34 + cells. The inhibitor of the apoptosis protein (IAP) survivin is expressed in proliferating cells such as fetal tissues and cancers. ![]()
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