1. by Dana S. Levy, Jason A. Kahana, and Rakesh Kumar
AKT inhibitor, GSK690693, induces growth inhibition and apoptosis in acute lymphoblastic leukemia cell lines
by Dana S. Levy, Jason A. Kahana, and Rakesh Kumar
Blood
Volume 113(8):
February 19, 2009
©2009 by American Society of Hematology

2. Inhibition of ALL cell proliferation by AKT inhibitor.
Inhibition of ALL cell proliferation by AKT inhibitor. ALL cell lines from both T-cell (A) and B-cell (B) origin were treated with varying concentration of GSK for 72 hours. Cell proliferation was measured as described in “Methods.” Graphs represent curve fit analysis of the dose response data for a subset of cell lines analyzed. Please note the relative insensitivity of MN60 and Tanoue cells to GSK (C) Effect of GSK on the proliferation of primary human CD4+ T lymphocytes, mouse thymocytes, and an AKT inhibitor-sensitive T-ALL cell line, A3.
Dana S. Levy et al. Blood 2009;113:
©2009 by American Society of Hematology

3. GSK690693 inhibits AKT signaling in ALL cells.
GSK inhibits AKT signaling in ALL cells. (A) I2.1, I9.2, A3, and Tanoue cells were treated with DMSO or different concentrations of GSK for 6 hours. Cell lysates were analyzed by Western blot analysis for phospho-GSK3β Ser9, phospho-PRAS40 Thr246, phospho-p70S6K Thr421/Ser424, phospho-AKT Ser473, and phospho-AKT Thr308. Total AKT and tubulin are shown as loading controls. (B) GSK inhibits AKT kinase activity in ALL cells. A3 and Tanoue cell lines were treated with DMSO or various concentrations of GSK for 1 hour. AKT was immunoprecipitated from cell lysates with immobilized anti-AKT antibody, and the kinase assay was performed using GSK-3 fusion protein as a substrate. Phosphorylation of GSK3 was measured by Western blot analysis, using phospho-GSK3β antibody. (C) GSK inhibits stimulated AKT kinase activity in ALL cells. Tanoue cells were serum-starved overnight, then treated with DMSO or 1 μM GSK for 1 hour. Cells were treated with 5 ng/mL insulin for various times to stimulate AKT activity. AKT kinase assay was performed.
Dana S. Levy et al. Blood 2009;113:
©2009 by American Society of Hematology

4. GSK690693 induces apoptosis in ALL cells.
GSK induces apoptosis in ALL cells. (A) I2.1, I9.2, A3, and Tanoue cells were treated with DMSO or varying concentrations of GSK for 72 hours, and cell-cycle distribution was determined based on DNA content of propidium iodide-stained populations. (B) Representative histogram of cell-cycle analysis. Histograms illustrate cell cycle profile of DMSO- and GSK treated I2.1 and Tanoue cells. (C) ALL cell lines were treated with DMSO or different concentrations of GSK for 24 hours, and endogenous levels of cleaved caspase-3 were measured. (D) PARP cleavage was determined by Western blot analysis in ALL cell lines treated with DMSO or various concentrations of GSK for 24 hours. (E) ALL cell lines were treated with DMSO or different concentrations of GSK for 24 hours, and the caspase activity was measured using caspase 3/7 assay kit. Data represent relative luminescence signal (mean ± SD) for each cell line. (F) DNA fragmentation was measured in A3, I2.1, and Tanoue cells treated with DMSO or GSK for 72 hours as described in “Methods.” Data represent a comparison between GSK sensitive and insensitive cell lines.
Dana S. Levy et al. Blood 2009;113:
©2009 by American Society of Hematology