Iron chelators down-regulate cyclin D1 protein but not mRNA expression, an effect that can be reversed by Fe supplementation. Cellular Fe depletion after a 24-hour incubation with DFO (250 μM) or 311 (25 μM) either (A) has no effect on cyclin D1 mRNA; (B) decreases cyclin D1 protein levels; (C) markedly increases both TfR1 mRNA; and (D) markedly increases both TfR1 protein levels. actinomycin D (AD; 9 nM) was used as a DNA-damage control (A-D) and had no effect on cyclin D1 or TfR1 expression. (Ei) Kinetic study over 24 hours with Fe chelators showing that cyclin D1 mRNA levels were not significantly altered compared with cells incubated with control medium, while (Eii) cyclin D1 protein levels are decreased after 14 hours during incubation with chelators. MCF-7 cells were incubated with control medium or medium containing 311 (25 μM) or DFO (250 μM) for up to 24 hours at 37°C. Cells were harvested and RT-PCR and Western analysis performed. (Fi-iv) Reincubation of 311-treated cells with the Fe source FAC restores cyclin D1 protein levels. Cells were preincubated with either medium alone or medium containing 311 (25 μM) for 24 hours at 37°C and then reincubated with FAC (100 μg/mL) in the presence or absence of medium containing 311 (25 μM) for 4 to 24 hours at 37°C. Cells were harvested for Western analysis. Results are a representative experiment from 4 performed.