ROS mediate PML NB rearrangements, cell apoptosis, NPM1 mutant oncoprotein degradation, and chemosensitization to DNR upon ATO treatment. (A) Flow cytometric detection of ROS in OCI/AML3 upon ATO treatment (DHE, green histograms, upper; and H2DCFDA, violet histograms, lower) compared with untreated cells (black line). (B) Effect of NAC pretreatment on ATO-induced ROS (as detected by DHE) at 24 hours in OCI/AML3. Histograms show levels of fluorescence of treated cells (red, ATO 3 μM; dashed line, NAC 20 mM; black, NAC + ATO) as compared with untreated cells (blue). (C) PML immunofluorescence staining (PG-M3, secondary goat anti-mouse Alexa-Fluor 568, red) of cytospin preparations of OCI/AML3: untreated cells (NT), 16 hours NAC 20 mM pretreatment (NAC), 8 hours ATO 3 μM treatment (ATO), and NAC pretreatment + ATO treatment (NAC + ATO). NBs/cell count (mean ± SD, on 50 cells) is shown in the inset. (D) Apoptosis assay (Annexin V/7AAD) on OCI/AML3 (left) and primary NPM1-mutated AML cells (n = 3) (right): untreated cells (white), 24-hour ATO 3 μM treatment (black), 16-hour NAC 20 mM pretreatment (checked), and NAC pretreatment + ATO treatment (gray). Means ± SD. Two-tailed paired Student t test P values are indicated. (E) Effect of NAC pretreatment on NPM1-mutant degradation. Western blot analysis of NPMm and NPMwt protein levels in OCI/AML3 (left) and 1 primary NPM1-mutated AML patient sample (pt. 613R, right). Activation of apoptosis (cPARP), p53 upregulation, and DNA damage induction (γH2AX) in OCI/AML3 are also shown (left). Histone H3 or β-actin expression was used, respectively, as loading control. In all cases, membranes probed with the different antibodies were from the same gel. (F) Effect of NAC pretreatment on chemosensitization to DNR by ATO/ATRA. Apoptosis flow cytometric evaluation (7AAD/Annexin V) upon 24-hour DNR treatment (0.05 μM) in OCI/AML3 cell lines pretreated with ATO (3 μM), ATRA (1 μM), and ATO/ATRA for 24 hours, previous incubation with or without NAC 20 mM for 16 hours.
