GSI and rapamycin treatment in vitro induces massive apoptosis of mouse T-ALL cells and cooperates to suppress mTOR activity. (A) Mouse T-ALL cell lines, 135, 5046, and 5151, were treated with DMSO, 1 μM MRK-003, 10 nM rapamycin, or 1 μM MRK-003 and 10 nM rapamycin for 24 hours. Cells were assayed for DNA content by staining with PI followed by flow cytometry. (B) mTOR activity is ablated when mouse T-ALL lines are treated with GSI and rapamycin. Mouse T-ALL lines, 720 and 5046, were treated with DMSO, 1 μM MRK-003, 10 nM rapamycin, or 1 μM MRK-003 and 10 nM rapamycin, and mTOR kinase activity was assayed by immunoblotting the cell lysates with phospho-p70 S6 kinase antibody (no. 9205; Cell Signaling Technology) after 18 hours. Total p70 S6 kinase was used as a loading control (no. 9202; Cell Signaling Technology). Fold reductions in kinase activity were determined by densitometry and represent ratios (phospho/total) normalized to DMSO-treated samples. (C) At low pharmacologic doses, MRK-003 and rapamycin may have synergistic effects on mouse leukemic growth. Mouse T-ALL lines, 720 and 5046, were treated for 72 hours with 1 nM rapamycin, increasing concentrations of MRK-003 (10⁻⁵ to 10¹ μM), or rapamycin and MRK-003, and growth was assayed by MTT analysis. The figure is a representative of 3 independent experiments using mouse T-ALL cell line 720.