engleski

The role of the nuclear protein kinase B/Akt activation in HL-60 leukemia cells

Introduction: At the cell membrane, phosphoinositide 3-kinase(PI3K)/Akt signaling pathway plays a crucial role in mediating growth factor-induced cell survival and proliferation. However, previous studies have demonstrated a progressive increase in the level and activity of PI3K in nuclei of HL-60 cells differentiated in the presence of all-trans-retinoic acid (ATRA). The aim of the present study is to investigate the changes in subcellular distribution and activities of Akt during agonist-induced differentiation and particular phase of the cell cycle and to determine the role of the nuclear Akt in differentiation and proliferation of HL-60 cells. Materials and methods: HL-60 cells (ECCACC, UK) are maintained in exponential growth and differentiated in the presence of 1 M ATRA, 1, 25% DMSO or 500 nM phorbol miristate acetate (PMA). The expression of CD11b is determined by FACS analysis. Cell lysates and nuclei are analyzed for the presence of phosphorylated and total Akt by Western blot analysis or subjected to Akt kinase assay using Crosstide as the substrate (Matkovic et al, Leukemia, 20:941-951, 2006). Akt protein is down-modulated by commercially available siRNA (Upstate, USA). Cells are synchronized by incubation in the presence of aphidicolin. Results: The Akt-activity is found to be increased in the nuclei and lysates of HL-60 cells incubated in the presence of 1μ M ATRA for 4 days. The kinase assay show no increase in the activity of Akt immunoprecipitated from either postnuclear membranes or cytosol. Time-course study of nuclear Akt-activity in HL-60 cells show progressive increase in the levels of phosphorylated Akt that correlates with an increase in the expression of differentiation marker (CD11b). Down-modulation of Akt protein by siRNA inhibits the expression of Akt protein and decreases the level of CD11b. No increase in the level of the nuclear Akt is observed in HL-60 cells treated with other granulocytic inducers (DMSO) or strong antiproliferative agonists (phorbol miristate acetate, PMA). Preliminary data show a progressive increase in the level of phosphorylated Akt in lysates of aphidicolin-synchronized HL-60 cells. Conclusion: Our results reveal that ATRA increases activity of nuclear Akt in HL-60 cells, and that the activity of Akt is necessary for ATRA-mediated differentiation.

nuclei; protein-kinase B/Akt; HL-60 cells

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