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S. Demir et al.
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Figure 4. APR-246 activity depends on mutant p53. (A-C) Stable lentiviral shRNA-mediated p53 knockdown in TP53mut (RS4;11 and KOPN-8) and TP53wt (NALM- 6) cell lines. Western blot, anti-p53 antibody DO-7, with GAPDH as a loading control, non-transduced cells (ctrl), cells transduced with scrambled control (scr-ctrl) and TP53-specific shRNA (sh-p53). (D-F) Increasing cell death (forward/side scatter criteria, flow cytometry) in control transduced cells and abrogated cell death induction upon p53 knockdown at increasing concentrations of APR-246 (APR, 48 h) in TP53mut but not TP53wt cells. Mean values ± standard deviation (SD) of three independent experiments, each performed in triplicate. Student t-test, *P<0.05; **P<0.01. (G-J) si-RNA-mediated p53 downregulation in TP53mut (KOPN-8), (G) and TP53wt (NALM-6), (I) cells leading to clearly lower cell death induction upon p53 downregulation as compared to higher cell death in control cells (H), while p53 downregulation in TP53wt cells did not affect cell death induction upon APR-246 treatment (J). Mean values ± SD of three independent experiments. Student t-test, *P<0.05; **P<0.01. (K) TP53 mutations identified in primary samples from patients with acute lymphoblastic leukemia (ALL): the mutations were localized in the DNA-binding domain with one splice site mutation (open circle, Patient-1) and three missense mutations (filled circles, Patients -2, -3, -4). (L) No detectable p53 protein in ALL cells from Patient-1 (western blot, anti-p53 antibody DO-7, GAPDH as a loading control), and (M) no APR-246 activity in these cells (Patient-1), in contrast to cell death induction in cases carrying missense hot spot TP53 mutations (N, O, P; Patients-2, -3, -4). Mean values ± SD, measurements performed in triplicate. Student t-test, ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; n.s., not significant.
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