FIGURE 2.

Deletion analysis of the GADD45α promoter and assessment of the roles of the transcription factors, p53, Egr1, Oct-1, and Hif-1α, on the up-regulation of GADD45α mRNA after iron depletion. A, following transient transfection of MCF7 cells with promoter deletion constructs, cells were incubated with either control medium or this medium containing 311 (25 μm) for 24 h/37 °C. Luciferase and protein assays were then performed on cell extracts. The activity of the promoter constructs is given as raw light units (RLU)/μg of protein. B, RT-PCR examining the effect of a 24 h/37 °C incubation with control medium or this medium containing 311 (25 μm) on the up-regulation of GADD45α in cells either expressing or not expressing p53, Egr1, Oct-1, or Hif-1α. C, schematic diagram showing the restriction enzyme sites used to prepare GADD45α promoter constructs that were then used to generate the constructs implemented in D. D, luciferase assay was performed using MCF7 cells transiently transfected with promoter deletion constructs to identify the specific consensus sequences responsible for the up-regulation of GADD45α mRNA after iron depletion in the −234 and −81 bp region of the GADD45α promoter. Transfected cells were incubated for 24 h/37 °C with control medium or this medium containing DFO (250 μm) or 311 (25 μm). Results shown are typical gels from three experiments, while the densitometry is mean ± S.D. ***, p < 0.001, relative to the control.