05. But in GC-resistant cell lines, rapamycin augmented the Selleck H 89 effect of G0/G1 arrest significantly, from 45%

to 58% in CEM-C1-15 cells, 50% to 65% in Jurkat cells, and 57% to 75% in Molt-4 cells, p < 0.05 (Figure 3A). Figure 3 The effect of rapamycin and Dex on cell cycles and the cell cycle regulatory proteins. (A) T-ALL cells were incubated for 48 h with rapamycin(10 nM) and/or Dex (1 μM) and the cell cycle phases were analyzed by PI staining. For all experiments, values of triple experiments were shown as mean plus or minus find more SD. * p < 0.05 as compared with control group or Dex group or Rap group except for CEM-C1-15 cells. (B) Cell-cycle proteins were studied. After 48 h exposure to rapamycin and/or Dex, Molt-4 cells were lysed

and extracts were analyzed learn more by Western blotting. R, rapamycin; D, Dex; RD, rapamycin+Dex; and C, control. To evaluate the molecular basis underlying cell cycle arrest, we investigated the expression of cell cycle regulatory proteins. As shown in Figure 3B, both rapamycin and Dex could induce up-regulation of cyclin-dependent kinase (CDK) inhibitors of p21 and p27, and a synergistic effect of induction was detected when using these two drugs together. Rapamycin did not obviously affect the expression of cyclin A, whereas dexamethasone induced cyclin A expession. Rapamycin prevented dexamethasone-induced expression of cyclin A. Cyclin D1 levels were reduced when treated with rapamycin or dexamethasone alone, or in combination. Compared with Dex, rapamycin had a stronger effect on down-regulation of cyclin D1. Rapamycin sensitizes T-ALL cells to Dex-induced apoptosis Cell cycle arrest could not explain the magic effect on growth inhibition of Dex when co-treated with rapamycin. The main mechanism of Dex in the treatment of lymphoid malignancies is to induce apoptotic cell death. We used Annexin V-PI staining to determine the

early stage of apoptosis. Dex, used alone at 1 μM (Dex group), had no apoptotic effect on Jurkat and Molt-4 cells, and there was only a minimal effect on CEM-C1-15 cells at 48 h and a modest effect on CEM-C7-14 cells at 24 h (After 24 h the cells came to the late phase of apoptosis, data not shown.), p > 0.05. Rapamycin, Forskolin in vivo used at 10 nM (Rap group), also had no obvious apoptosis-inducing effect on all 4 cell lines, although at this concentration, significant cell cycle arrest at G1 phase occurred (Figure 3A). However, when combined Dex with rapamycin (Rap+Dex group), a remarkable increase in cell apoptosis was ensued in all 4 cell lines (Figure 4A). Compared with Rap group, the combination treatment group of cells increased the apoptotic rate from 3% to 20% in CEM-C7-14 at 24 h, p < 0.05, from 3% to 16% in CEM-C1-15 cells at 24 h, p < 0.05, from 9% to 18% in Jurkat cells at 72 h, p < 0.05, and from 5% to 14% in Molt-4 cells at 48 h, p < 0.05.