PMC

IRP activity as detected by IRP binding assay with relative densitometric quantification (top) and cell viability as detected by Trypan blue exclusion (bottom) according to iron status. AU: arbitrary units. SV40 hepatocytes were treated for 24 hours with 100 μmol/L Dfo (Dfo), 150 μmol/L FAC (FAC) or vehicle (Control). Cell viability was detected by Trypan blue exclusion and represents the mean ± SD of three independent experiments.

Effect of antioxidants on p53 protein levels in SV40 hepatocytes. After plating, cells were treated for 24 hours with or without NAC and α-Tocopherol, and then 150 μmol/L FAC was added to the culture medium for additional 24 hours. Ct: control, untreated cells, NAC: N-acetyl-cysteine 10 mmol/L, α-Toc: α-Tocopherol 60 μmol/L (+), 120 μmol/L (++). p53 levels AU (arbitrary units): indicates the p53/β-actin ratio as detected by densitometry in two independent experiments. *P < 0.05 vs. untreated cells. **P < 0.05 vs. cells treated with FAC 150 μmol/L alone. The Western blot shown in the lower part of the figure is representative of two independent experiments.

Effect of iron status on the MDM2/p53 pathway in human monocytes. A: Effect of iron status on p53 protein levels. Twenty-four hours after plating, after removal of lymphocytes, cells were treated as indicated for 24 hours. Ct: control, untreated monocytes. H₂O₂ is shown as positive control. β-actin is shown as a loading control. AU (arbitrary units): indicates the p53/β-actin ratio as detected by densitometry. Results are representative of two independent experiments. B: Expression of mRNAs of transferrin receptor-1 (up-regulated by iron depletion), MDM2, p53, and p53 target genes (including Bax, Aldh4a1, Sod2) in monocytes of subjects with C282Y +/+ HH (n = 6, black bars), and subjects with normal iron parameters negative for HFE mutations (n = 6, white bars). *P < 0.05 vs. control subjects. AU: mRNA levels, arbitrary units. C: MDM2 and p53 protein expression, as detected by Western blotting, in subjects with C282Y +/+ HH, and in subjects with normal iron parameters negative for HFE mutations. Three representative subjects are shown for each group. β-actin is shown as a loading control.

Effect of iron status and of modulation of MDM2/p53 on cell proliferation in HepG2 human hepatoblastoma cells. A: Effect of iron status on H³-Thymidine incorporation in HepG2 cells untreated (Control) and treated for 24 hours with 150 μmol/L FAC (FAC) or 100 μmol/L Dfo (Dfo) in the presence or absence of insulin 0.33 μmol/L. Results are mean ± SD of three independent experiments. cpm: counts per minute. *P < 0.05 vs. untreated cells in the absence of insulin. B: Effect of iron status and of MDM2/p53 modulation on cell number in HepG2 cells untreated or treated for 24 hours with 150 μmol/L FAC (FAC) or 80/120 μmol/L Dfo (+/++ Dfo) in the presence or absence of 25 μmol/L nutlin (Nutlin). The horizontal line indicated the number of cells plated at the beginning of the experiment. *P < 0.05 vs. untreated cells in the absence of nutlin, **P < 0.05 vs. untreated cells in the presence of nutlin. C: Effect of iron status and of MDM2/p53 modulation on p53 protein levels in HepG2 cells untreated or treated for 24 hours with 150 μmol/L FAC (FAC) or 120 μmol/L Dfo (Dfo) in the presence or absence of 25 μmol/L nutlin (Nutlin). AU: protein levels normalized for β-actin, arbitrary units. Results are representative of two independent experiments. *P < 0.05 vs. untreated cells in the absence of nutlin, **P < 0.05 vs. untreated cells in the presence of nutlin.

Effect of iron status on the MDM2/p53 pathway in SV40 mouse hepatocytes and in rat liver. A: Effect of Dfo and FAC on MDM2 and p53 protein levels in SV40 hepatocytes. Twenty-four hours after plating, cells were treated with 100 μmol/L Dfo (Dfo) or 150 μmol/L FAC (FAC) or vehicle for 24 hours and then lysed. MDM2, p53, and H-ferritin were detected in cell extracts by immunoblot analysis. The blots were reprobed with the antibody against β-actin as a loading control. B: Dose response relationship between iron availability and p53 protein levels in SV40 hepatocytes. Ct: control, untreated hepatocytes. Cells were treated for 24 hours with the indicated concentrations of FAC and Dfo. The upper part of the figure indicates the p53/β-actin ratio as detected by densitometry. AU: arbitrary units. Results are representative of two independent experiments. In the lower part of the figure the corresponding β-actin normalized MDM2 mRNA levels are shown. Results are representative of two independent experiments. *P < 0.05 vs. untreated cells. C: Effect of iron status on p53 protein and mRNA levels and cell viability in differentiating SV40 hepatocytes, at different days from cell plating. AU: arbitrary units. p53 AU indicates the p53/β-actin ratio as detected by densitometry. p53 mRNA levels were normalized for β-actin. *P < 0.05 vs. untreated cells 24 hours after plating (controls). Results are representative of two independent experiments. D: Effect of iron depletion by Dfo on serum iron, hepatic iron concentration, MDM2 and p53 protein levels in rat liver. White bars: control rats (n = 5), black bars: iron depleted rats (n = 4). Serum iron and hepatic iron concentration were measured by atomic absorption spectrometry. Protein expression of p53 and MDM2, was evaluated by densitometry and normalized for β-actin. AU: arbitrary units. *P ≤ 0.05 vs. control rats.

Effect of iron status on p53 expression is mediated through MDM2. A: Effect of Nutlin-3 on iron mediated regulation of p53 levels. The effect of manipulation of iron status with FAC and Dfo on p53 is compared in cells exposed or not to Nutlin-3. Human monocytes isolated from a healthy subject with normal iron parameters and negative for HFE mutations were cultured for 24 hours after removal of lymphocytes in the presence or in the absence of 25 μmol/L Nutlin-3, 100 μmol/L Dfo, and 150 μmol/L FAC. β-actin is shown as a loading control. p53 levels normalized for β-actin, as detected by densitometry, are shown in the upper part of the figure. Results are representative of two independent experiments. B: Effect of iron status on p53 polyubiquitination during inhibition of proteasomal activity. Human monocytes isolated from a healthy subject with normal iron parameters and negative for HFE mutations were cultured for 24 hours in the presence or not of 25 μmol/L Nutlin-3, 100 μmol/L Dfo, and 150 μmol/L FAC in the presence of the proteasomal inhibitor MG132. Proteins were immunoprecipitated with p53 antibody and pull-downs blotted with antibodies against ubiquitin (upper panel) and p53 (lower panel). The histogram shows the average levels of ubiquitylated/total p53 as determined by densitometric analysis of two independent experiments. *P < 0.05. C: Iron status affects p53 degradation kinetics. Human monocytes isolated from a healthy subject with normal iron parameters and negative for HFE mutations were cultured for 24 hours after removal of lymphocytes in the presence or not of 120 μmol/L Dfo, and 150 μmol/L FAC for 24 hours and then treated with CHX 40 μg/ml for 1 to 8 hours. Western blot analysis and densitometric analysis were performed for p53 levels, which were normalized for β-actin. Thus, relative expression >1 compared with baseline means that protein degradation was slower, whereas relative expression <1 means that protein degradation was faster than that of β-actin. Results represent means and SDs of two independent experiments. *P < 0.05 compared with controls.