The effect of Fe chelators and DNA-damaging agents on Ndrg1 expression. (A) Densitometric analysis of gene array data demonstrating marked up-regulation of Ndrg1 mRNA expression after incubation of MCF-7 cells with the chelators desferrioxamine (DFO) and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) but not the DNA-damaging agent actinomycin D (Act D). (B) Semi-quantitative RT-PCR demonstrates that Ndrg1 mRNA is markedly up-regulated after incubation of MCF-7 cells with DFO or 311. (C) Chelators up-regulate the expression of Ndrg1 mRNA in multiple cell lines. (D) The p53-independent induction of Ndrg1 mRNA following Fe chelation with DFO or 311 in the H1299 p53 mutant cell line. (E) The chelator DFO, but not Act D, cisplatin (CP), or mitomycin C (MC), increases Ndrg1 mRNA expression. Cells were incubated for 24 hours at 37°C with either 311 (25 μM), DFO (250 μM), Act D (5 nM), CP (20 μM), or MC (30 μM), and the RNA was isolated and then used for (A) gene array analysis using GEArray Q series membranes (Superarray), or (B-E) semiquantitative RT-PCR analysis (see “Materials and methods” for details). (A-D) Densitometry was performed and gene expression then calculated relative to the β-actin control. Results in panel A show a typical experiment from 2 performed, and panels B to E show a typical experiment from 3 performed.