Figure 4.

Promyelocytic leukemia / retinoic acid receptor α (PML/RARa) expression sensitizes cells to ascorbate treatment. (a) The levels of reactive oxygen species (ROS) content in PR9 and Mock cell lines systems were measured after induction of PML/RARa with ZnSO4. ROS levels were also evaluated after treatment with rotenone/antimycin A, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and ASC as oxidants. In all systems the presence of PML/RARa induced a significantly higher level of ROS production. (b) The level of ROS production was evaluated in NB4, bearing PML/RARa, and U937 cells as control using ZnSO4, Rotenone/Antimicyn A, FCCP and ascorbate (ASC) as oxidant. NB4 cells produced higher level of ROS compared to U937 cells (c) PR9 and Mock cell lines systems were treated with ASC 1 mM for six hours and NRF2 and HO-1 protein expression was evaluated by western blot. In PR9 cells the PML/RARa presence inhibited the expression of NRF2 and its target HO-1 protein. (d) NB4 and U937 cells were treated with 1 mM ASC and evaluated for NRF2 and HO-1 production by western blot. In NB4 cells PML/RARa presence inhibited NRF2 protein and downregulated its target HO-1. (e) NB4 and U937 cells were treated with ASC 1 mM for 24 h and HMOX1 mRNA expression was measured demonstrating NRF2 transcriptional deficiency. (f) Primary blasts from one AML and one APL patient were treated with ASC 3 mM for 24 h: NRF2 was clearly abated in the APL blasts. (g) PR9 and Mock control cells were treated with ZnSO4 100 μM for two hours then with Asc at 1 and 3 mM for 72 h and vitality was assessed by MTS assay. (h) NB4 and U937 cells were treated with increasing concentrations of ASC and viability was assessed using the ATP lite test. (i) NB4 and Oci-AML2 cells were treated with increasing concentrations of ASC and viability was assessed by cytometric analysis using Anexin – PI. *: p < 0.05, **: p < 0.01, ***: p < 0.001 by unpaired t-test. All the experiments were performed in triplicate.