Iron Chelator-Mediated Alterations in Gene Expression: Identification of Novel Iron-Regulated Molecules That Are Molecular Targets of Hypoxia-Inducible Factor-1α and p53

Abstract

Iron deficiency affects 500 million people, yet the molecular role of iron in gene expression remains poorly characterized. In addition, the alterations in global gene expression after iron chelation remain unclear and are important to assess for understanding the molecular pathology of iron deficiency and the biological effects of chelators. Considering this, we assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone) that have markedly different permeability properties. Sixteen genes were significantly regulated by both ligands, whereas a further 50 genes were significantly regulated by either compound. Apart from ironmediated regulation of expression via hypoxia inducible factor-1␣, it was noteworthy that the transcription factor p53 was also involved in iron-regulated gene expression. Examining 16 genes regulated by both chelators in normal and neoplastic cells, five genes (APP, GDF15, CITED2, EGR1, and PNRC1) were significantly differentially expressed between the cell types. In view of their functions in tumor suppression, proliferation, and apoptosis, these findings are important for understanding the selective antiproliferative effects of chelators against neoplastic cells. Most of the genes identified have not been described previously to be iron-regulated and are important for understanding the molecular and cellular effects of iron depletion.Iron deficiency affects approximately 500 million people. However, despite the enormity of this problem, very little is understood concerning the precise molecular roles played by iron in growth, cell-cycle progression, and apoptosis.Iron plays essential roles in cells, including DNA synthesis and cell cycle control (Buss et al., 2003). It is well known that iron deficiency induced by chelators results in a G 1 /S arrest and apoptosis (Buss et al., 2003). The best-characterized role of iron in proliferation involves its function in the rate-limiting step of DNA synthesis catalyzed by ribonucleotide reductase (Buss et al., 2003). Iron has also been shown to regulate the expression of a variety of molecules involved in cell-cycle control (e.g., p21CIP1/WAF1 , GADD45, p53, cyclin D1, etc.) (Gao and Richardson, 2001;Liang and Richardson, 2003) and metastasis suppression (e.g., N-myc downstreamregulated gene-1; NDRG-1) (Le and Richardson, 2004). Because iron is important for mitochondrial heme and iron sulfur cluster synthesis, it is likely that iron chelation will also affect basic mitochondrial metabolism and function.Iron chelators are well known therapeutics for the treatment of iron-overload disease and some of these agents show potential for cancer therapy (Buss et al., 2003;Whitnall et al., 2006 ABBREVIATIONS: 311, 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone; DFO, desferrioxamine; IRP, iron-regulatory protein; IRE, ironresponsive element; UTR, untranslated region; HIF-1, hypoxia-inducible factor-1; HUVEC, human umbilical vein endothelial cell; MEF, murine e...

Cellular Iron Depletion Stimulates the JNK and p38 MAPK Signaling Transduction Pathways, Dissociation of ASK1-Thioredoxin, and Activation of ASK1

“…These chelator concentrations and this incubation period were used because we previously demonstrated that under these conditions the chelators modulate iron-responsive genes (e.g. transferrin receptor 1 (TfR1) (21,30)). The higher concentration of DFO was implemented due to its limited ability to permeate membranes (33,34).…”

“…Microarray Processing and Data Analysis-RNA isolation and microarray processing were performed, as described previously (21,31). The Affymetrix Human Genome U133 Plus 2.0 430 2.0 array (Affymetrix, Santa Clara, CA) was used, which assesses Ͼ47,000 transcripts from over 38,500 genes.…”

“…Protein Extraction and Western Blotting-Protein extraction and Western blotting were performed using well established procedures (21,31). Rabbit anti-human phospho-specific and nonphospho-specific ERK1/2 (catalog no.…”

“…The expression of cyclindependent kinase 2 (cdk2) is also decreased (19). In addition, DFO also affects gene expression mediated by transcription factors, such as hypoxia-inducible factor-1␣ (HIF-1␣) and p53 (21).…”

Nitric Oxide Suppresses Tumor Cell Migration Through N-Myc Downstream Regulated Gene-1 (NDRG1) Expression: the Role of Chelatable Iron

“…An attractive explanation, therefore, for NDRG1 up-regulation by NO would be through IRE-IRP interactions. This was ruled out, however, because the stem-loop structure of the 3Ј-UTR of NDRG1 has been previously examined and determined not to show structural similarities to any other known highly conserved IRE structures (58). It was therefore concluded that it would not be capable of binding IRPs (58,59).…”