Figure 5.

Activation of the ROS-p53 feedback loop by combination treatment of L-asparaginase and chloroquine in ALL cells. (a) Effect of N-acetyl-L-cysteine (NAC) on induction of cell death. REH cells were concurrently treated with 2 mM of NAC with 1 U/ml of L-asp and/or 10 μM of CQ for 48 h. (b) Western blot analysis of L-asp and/or CQ-treated REH cells. (c) Western blot analysis of NAC-treated REH cells. L-asp and/or CQ-treated cells were concurrently treated with 2 mM NAC for 48 h. (d–f) Cellular ROS detection assay (d). Western blot analysis (e) and apoptosis analysis (f) of TP53-knockdown REH cells. (g) Kaplan–Meier overall survival curve in mice. Non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were transplanted with REH-Luc2 cells stably transfected with sh-control or sh-p53 by tail vein injection. These mice were treated as described in Figure 4d (n=5 per group). (f) Cell survival assay in primary ALL samples. ALL cells were purified from the bone marrow of samples from ALL patients shown in Supplementary Table S3. Cells were treated with 1 U/ml of L-asp and/or 10 μM of CQ for 48 h, and viable cells were measured by flow cytometry using Annexin-V staining. (g) Effect of p53 expression on cell survival in ALL cells purified from patient No.7. Cells transduced with Ad-LacZ (as a control) or Ad-p53 were treated with 1 U/ml of L-asp and/or 10 μM of CQ for 48 h. Viable cells were measured as described in h. Data in a, d, f, h, i represent as mean±s.d. (n=3); *P<0.05, ^**P<0.01, ^***P<0.001; NS, not significant. P-values were calculated using two-sided Student's t-test (a, d, f, h, i) and log-rank test (g).