Figure 4. PTEN activity is downregulated by ROS in T-ALL.

(A) Intracellular H₂O₂ levels were assessed by flow cytometry after incubation with the redox-sensitive fluorescent dye DCF-DA. Shown is 1 representative patient sample and 1 representative normal control. (B) Mean intensity of DCF-DA fluorescence was compared between normal thymocytes (n = 7) and T-ALL (n = 5). Data are representative of most T-ALL samples (see Table 1) analyzed in 4 independent experiments. (C) TAIL7 cells previously treated with or without 1 mM H₂O₂ for 30 min were lysed in nonreducing conditions and analyzed for expression of PTEN oxidized and reduced bands. DTT was added where indicated to demonstrate the specificity of the lower, oxidized band. (D and E) PTEN-positive TAIL7, HPB-ALL, or primary T-ALL cells and PTEN-null Jurkat cells were treated for 2 h with 0.5 mM β-ME (D) or for 30 min with 1 mM H₂O₂ (E), and levels of Akt and GSK-3β phosphorylation were determined by immunoblot. (F) TAIL7 and HPB-ALL cells were pretreated for 1 h with 25 μm LY294002 (LY) or with DMSO vehicle control, stimulated with 1 mM H₂O₂ for 30 min, and analyzed for Akt phosphorylation by immunoblot. (G and H) Effects of β-ME (G) or H₂O₂ (H) on PTEN activity were measured by using the indirect PTEN redox assay, as described previously (19). More relative PTEN activity in the assay reflects less PTEN activity in the cell. Data from 2 independent experiments were normalized to the highest value in the control conditions. Values in B, G, and H are mean ± SEM.