Cellular Iron Depletion and the Mechanisms Involved in the Iron-dependent Regulation of the Growth Arrest and DNA Damage Family of Genes

Iron plays a crucial part in proliferation while iron deficiency results in G(1)/S arrest, DNA damage, and apoptosis. However, the precise role of iron in cell cycle control remains unclear. We showed that iron depletion using the iron chelators, desferrioxamine (DFO), or 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311), increased the mRNA levels of the growth arrest and DNA damage 45 alpha gene, GADD45 alpha (Darnell, G. and Richardson, D. R. (1999) Blood 94, 781-792). In this study, we examined the effect of iron depletion on up-regulating GADD family members involved in growth control, including cell cycle arrest, apoptosis, and DNA repair, making them therapeutic targets for tumor suppression. We showed the GADD family members were up-regulated by cellular iron depletion. Further, upregulation of GADD45 alpha after iron deprivation was independent of hypoxia-inducible factor-1 alpha (HIF-1 alpha), octamer-1 (Oct-1), p53 and early growth response 1 (Egr1). We then analyzed the regulatory elements responsible for iron depletion-mediated regulation of GADD45 alpha and identified the specific transcription factor/s involved. This region was within -117 bp and -81 bp relative to the start codon where the consensus sequences of three transcription factors are located: the CCAAT-binding factor/nuclear factor-Y (NF-Y), the stabilizing molecule v-MYB and the enhancer, CCAAT enhancer-binding protein (CEBP alpha). Mutation analysis, shRNA studies, Western blotting, and electrophoretic mobility shift assays led to the identification of NF-Y in the transcriptional up-regulation of GADD45 alpha after iron depletion. Furthermore, like GADD45 alpha, NF-YA was upregulated after iron chelation and down-regulated by iron supplementation. These results are important for understanding the mechanisms of iron depletion-mediated cell cycle arrest, DNA damage repair, and apoptosis.