Tuning Cell Cycle Regulation with an Iron Key

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1 Cell Cycle ISSN: (Print) (Online) Journal homepage: Tuning Cell Cycle Regulation with an Iron Key Yu Yu, Zaklina Kovacevic & Des R. Richardson To cite this article: Yu Yu, Zaklina Kovacevic & Des R. Richardson (2007) Tuning Cell Cycle Regulation with an Iron Key, Cell Cycle, 6:16, , DOI: /cc To link to this article: Published online: 15 Aug Submit your article to this journal Article views: 610 Citing articles: 126 View citing articles Full Terms & Conditions of access and use can be found at

2 [Cell Cycle 6:16, , 15 August 2007]; 2007 Landes Bioscience Review Tuning Cell Cycle Regulation With an Iron Key Yu Yu Zaklina Kovacevic Des R. Richardson* Iron Metabolism and Chelation Program; Department of Pathology; University of Sydney; Sydney, New South Wales Australia These authors contributed equally to this manuscript. *Correspondence to: Des R. Richardson; Iron Metabolism and Chelation Program; Department of Pathology; University of Sydney; Sydney, New South Wales Australia; Tel.: ; Fax: ; Original manuscript submitted: 06/19/07 Manuscript accepted: 06/19/07 Previously published online as a Cell Cycle E-publication: Key words iron, iron chelators, cell cycle, p53, cyclin D1, p21 CIP1/WAF1 ACKNOWLEDGEMENTS This project was supported by a fellowship and project grants from the National Health and Medical Research Council of Australia, Australian Research Council Discovery Grant, Cancer Institute of New South Wales Foundation grant and grants from Australian Rotary Health Research Fund to D.R.R. Z.K. thanks the Australian Rotary Health Research Fund (Dural Rotary Club) and the Cancer Institute of New South Wales for a Ph.D. Scholarship. Y.Y. appreciates an International Post-Graduate Research Scholarship from the University of Sydney. The authors kindly thank Mr. Yohan Suryo Rahmanto and Miss Danuta Kalinowski for their tremendous assistance with the reference formatting of the article. Abstract Iron (Fe) is essential for cellular metabolism e.g., DNA synthesis and its depletion causes G 1 /S arrest and apoptosis. Considering this, Fe chelators have been shown to be effective anti proliferative agents. In order to understand the anti tumor activity of Fe chelators, the mechanisms responsible for G 1 /S arrest and apoptosis after Fe depletion have been investigated. These studies reveal a multitude of cell cycle control molecules are regulated by Fe. These include p53, p27 Kip1, cyclin D1 and cyclin dependent kinase 2 (cdk2). Additionally, Fe depletion up regulates the mrna levels of the cdk inhibitor, p21 CIP1/WAF1, but paradoxically down regulates its protein expression. This effect could contribute to the apoptosis observed after Fe depletion. Iron depletion also leads to proteasomal degradation of p21 CIP1/WAF1 and cyclin D1 via an ubiquitin independent pathway. This is in contrast to the mechanism in Fe replete cells, where it occurs by ubiquitin dependent proteasomal degradation. Up regulation of p38 mitogen activated protein kinase (MAPK) after Fe depletion suggests another facet of cell cycle regulation responsible for inhibition of proliferation and apoptosis induction. Elucidation of the complex effects of Fe depletion on the expression of cell cycle control molecules remains at its infancy. However, these processes are important to dissect for complete understanding of Fe deficiency and the development of chelators for cancer treatment. Prelude Iron (Fe) is a metal that is vital for the sustenance of life. 1 3 It is an essential component of many proteins and enzymes that are involved in cell growth and replication. 1,3 For instance, Fe plays a crucial role in the conversion of ribonucleotides into deoxyribonucleotides by participating in the rate limiting step of DNA synthesis catalyzed by ribonucleotide reductase (RR). 4 6 Depletion of Fe in cells typically results in a G 1 /S arrest, 7,8 indicating that this metal is essential for cell cycle progression, growth and division. 9,10 Under some conditions of Fe deprivation, a G 2 /M arrest has also been identified. 11 Iron deficiency is a common nutritional problem affecting an estimated 500 million individuals resulting in anemia, lethargy, defective psychomotor development and disturbed cognitive function. 12 Despite this and the fact that Fe depletion induces a G 1 /S arrest and apoptosis, 8,13 it is surprising that little is known concerning the role of Fe in cell cycle regulation. Furthermore, it has become clear that some Fe chelators show promising anti cancer activity by inducing cell cycle arrest and apoptosis. 14,15 However, the mechanisms involved in these effects remain uncertain and important to investigate in terms of understanding chelator structure-activity relationships. In order to understand the role of Fe in cell cycle control, knowledge of its cellular metabolism is essential and this is briefly described below. For more detailed accounts of Fe metabolism and homeostasis, the reader is referred to specialized review articles. 3,16 18 Cellular Iron Metabolism: From Uptake to Storage 2007 LANDES BIOSCIENCE. DO NOT DISTRIBUTE. Inorganic Fe in the diet is largely in its ferric state and is absorbed by enterocytes lining the small intestine. 19 The uptake of Fe is first performed by a reduction step that may be mediated by the enzyme duodenal cytochrome b (Dcytb; Fig. 1A). 19 However, the role of this protein in this process is controversial, as Dcytb knockout mice do not show an Fe deficiency phenotype. 20 Apart from inorganic Fe uptake from the diet, heme may be transported into enterocytes by the recently identified heme carrier protein 1 (HCP1). 21 Again, the function of this molecule in Fe absorption remains unclear, with HCP1 also being suggested to be a folate transporter. 22,23 Irrespective of the transport mechanism, 1982 Cell Cycle 2007; Vol. 6 Issue 16

3 Figure 1. The absorption, transport and metabolism of Fe: (A) Fe is absorbed by enterocytes in the small intestine. In the diet, Fe exists as either inorganic Fe or heme Fe. Inorganic Fe is primarily in the Fe 3+ state which may be converted to Fe 2+ by the postulated ferrireductase, duodenal cytochrome b (Dcytb), although this remains controversial. The Fe 2+ is then transported into enterocytes via divalent metal ion transporter 1 (DMT1). Heme Fe is thought to be transported into the cell by heme carrier protein 1 (HCP1), although again this remains uncertain. Once internalized, heme is metabolized by heme oxygenase to release Fe, carbon monoxide (CO) and bilirubin. Irrespective of whether Fe has been derived from dietary inorganic Fe or heme, once inside the enterocyte, Fe 2+ is either stored in ferritin or transported out of the enterocyte into the blood via the Fe export protein, ferroportin-1. The intracellular ferroxidase, hephaestin, is thought to be involved in this process, although its exact contribution remains unclear. Once at the surface of the enterocyte, Fe 2+ is converted back to Fe 3+ by the serum ferroxidase, ceruloplasmin. The Fe 3+ then binds to the serum Fe transport molecule, transferrin (Tf). (B) Cells which require Fe express the transferrin receptor 1 (TfR1) on their surface, which binds two molecules of Tf. The Tf TfR1 complex is then internalized by receptor mediated endocytosis. Once in the endosome, the ph decreases, allowing the Fe 3+ to dissociate from the Tf-TfR1 complex. The endosomal ferrireductase, six transmembrane epithelial antigen of the prostate 3 (Steap3), is thought to convert Fe 3+ to Fe 2+ in the endosome, allowing Fe 2+ to be transported out of the endosome by DMT1. Once in the cell, Fe 2+ can be stored in ferritin or it can enter the poorly characterized intracellular labile Fe pool where it is used in the synthesis of various proteins and enzymes such as ribonucleotide reductase (RR) etc. heme enters the cell and is then metabolized by heme oxygenase, leading to the liberation of Fe, bilirubin and carbon monoxide (Fig. 1A). 24 After transport through the apical membrane of the enterocyte and depending upon the body s requirement, some Fe is stored in the protein, ferritin. 25,26 Iron that is not stored within ferritin is released by enterocytes into the bloodstream via a poorly understood mechanism (Fig. 1A). The intracellular ferroxidase, hephaestin, plays some role in this process, 27 while the transporter ferroportin 1, is known to export cellular Fe into the circulation through the basolateral membrane. 28 Hepcidin is a peptide hormone secreted by the liver and is involved in maintaining Fe homeostasis. 29 It has been shown to downregulate ferroportin 1 by causing its internalization and degradation. 29 Indeed, this is one step of a homeostatic loop of Fe regulation where an excess of Fe in the diet stimulates hepcidin secretion. Hepcidin then reduces Fe uptake into the bloodstream and consequently promotes Fe storage in ferritin. 26 Another recently identified protein involved in Fe homeostasis is hemojuvelin (HJV), that is highly expressed in skeletal muscle and liver. 30,31 When it is mutated, HJV has been identified as the gene responsible for juvenile hemochromatosis. 30 Mice with mutated HJV fail to express hepcidin, leading to severe Fe overload. 31 These observations suggest HJV plays an essential role in regulating hepcidin expression in the Fe sensing pathway. 31 Once the Fe in its ferrous state (Fe +2 ) has been transported by ferroportin 1 to the surface of the enterocyte, it is thought to be converted to the ferric form (Fe +3 ) by the copper containing serum ferroxidase, ceruloplasmin (Fig. 1A). 26,27 Ceruloplasmin may directly interact with ferroportin 1 to achieve this reaction, although the mechanism involved remains unclear. Serum transferrin (Tf) then binds the Fe +3 with high affinity. 32 Two Fe +3 atoms become bound to Tf, which then binds to the transferrin receptor 1 (TfR1) that is found on the surface of most cells and is involved in internalized Fe uptake. 33 The uptake of Fe occurs following the binding of two Tf molecules to the TfR1. 32 The entire complex is then internalized via receptor mediated endocytosis (Fig. 1B). 9,26,32,34 Following internalization of the Tf TfR1 complex, the ph within the endosome declines due to the presence of a proton pump in the endosomal membrane. 35 The so formed acidic environment (ph 5.5) then allows the Fe +3 atoms to dissociate from the complex. 35 The Fe +3 is subsequently reduced to the ferrous state (Fe +2 ) possibly by a ferrireductase known as the six transmembrane epithelial antigen of the prostate 3 (Steap3) 36 and is then transported out of the endosome into the cytoplasm via the divalent metal Cell Cycle 1983

4 transporter 1 (DMTI; Fig. 1B). 18,35 37 Apo Tf bound to the TfR1 is then recycled back to the cell surface rather than being degraded (Fig. 1B). 26 A second Tf receptor (TfR2) gene has been cloned and results in the generation of two transcripts, a and b. 38 The TfR2a leads to a membrane bound protein that binds Tf, but at a lower affinity than TfR1. 38,39 The TfR2a appears to play a role in sensing Fe levels rather than being involved in quantitative Fe uptake. 16,40 In fact, mutation of this protein leads to Fe loading and hemochromatosis. 41 Consistent with the role of TfR1 expression in quantitative Fe uptake, TfR1 is well known to be regulated by intracellular Fe concentration, 18,42 while TfR2 is not. 39 TfR2 appears to be regulated as a function of the cell cycle, with the highest expression in late G 1 phase and no expression in G 0 /G Interestingly, CHO cells expressing TfR2a developed into tumors in nude mice, whereas CHO control cells did not. 39 While TfR2 is involved in Tf binding and Fe uptake, 38 it cannot compensate for TfR1 mediated Fe uptake, since TfR1 knockout mice are not viable. 43,44 Once in the cytoplasm, Fe is thought to enter the poorly characterized intracellular labile pool and can either be used in the production of new Fe containing proteins and enzymes such as RR and hemoglobin etc, or it can be stored in ferritin. 17,26 The labile Fe pool was once thought to be composed of low M r complexes, 45 but more recent studies suggest that active protein protein and/or organelle interactions such as contact of the Tf containing endosome with the mitochondrion may be involved A significant amount of metabolically active Fe is found in the mitochondrion where it participates in the synthesis of Fe S clusters or heme. 49,50 In mammalian cells, Fe is thought to be transported into the mitochondrion by the membrane bound Fe transporter, mitoferrin. 51 Disruption of yeast mitoferrin homologs, MRS3 and MRS4, was found to cause defective mitochondrial Fe uptake, leading to defects in mitochondrial Fe metabolism and Fe S cluster formation. 51 Hence, mitoferrin appears to play an essential role in mitochondrial Fe uptake. 51 The storage of some Fe in the mitochondrion is mediated by mitochondrial ferritin. 52 Recently, it was found that overexpression of mitochondrial ferritin alters cellular Fe homeostasis causing a cytosolic Fe deficiency, increasing TfR1 expression and leading to enhanced Fe uptake from Tf. 53 The role of Fe in a variety of metabolic processes necessitates that its cellular levels are tightly controlled. Two iron regulatory proteins (IRP1 and IRP2) have been identified that play important roles in regulating Fe homeostasis. 17,54 Both are mrna binding proteins which play an important role in the post transcriptional control of a variety of molecules involved in Fe homeostasis 18,34,42 and potentially cell cycle regulation. 55 The IRPs bind to stem loop structures known as Fe responsive elements (IRE) found in the untranslated regions (UTRs) of the mrna of molecules involved in Fe metabolism e.g., ferritin, TfR1, ferroportin 1, DMT1 etc. 34,42 The RNA binding activity of the two IRPs are regulated by Fe via different mechanisms. In the case of IRP1, when cells are Fe replete, an Fe S cluster forms within the protein that inhibits RNA binding activity. 17 In contrast, in the case of IRP2, high Fe levels result in its proteasomal degradation. 56 During Fe deprivation, IRP RNA binding activity is high, leading to the binding of IRPs to the IRE on the 5' UTR of ferritin mrna resulting in inhibition of its translation. 55 This leads to less Fe storage and the use of Fe for metabolic requirements. On the other hand, the binding of IRPs to IREs in the 3' UTR of TfR1 mrna makes it more stable and less susceptible to degradation leading to greater TfR1 expression. 55 Subsequently, this promotes the uptake of more Fe via the binding of Tf to the TfR1. It follows that removal of Fe from the hypothetical intracellular labile Fe pool will result in increased activity of the IRPs, leading to the altered expression of a range of molecules possessing IREs. In contrast, when cells are Fe replete, this leads to decreased IRP RNA binding activity and an opposite scenario develops, leading to decreased TfR1 expression and increased Fe storage in ferritin. 17 The recent identification of an IRE within the 3' UTR of cell division cycle 14A (CDC14A) mrna implies a potential role for this molecule in the cell cycle arrest observed after Fe depletion and this is discussed later in the section on CDC14A. Considering the importance of Fe in vital cellular processes such as DNA synthesis, it is not surprising that the cell cycle is tuned to Fe availability. For instance, it is well known that TfR1 expression is increased during the S phase of the cell cycle. 57 This is presumably due to the Fe requirement of RR that is critical for DNA synthesis. 4,6 In agreement with this, it has been reported that depression of TfR1 expression leads to G 1 arrest and an alteration in the expression of genes that regulate the cell cycle. 58 The essential requirement of Fe for cellular metabolism has led to investigations on the use of Fe chelators as anti cancer agents. 2,59 By understanding the effects of these agents on cell cycle control, the rationale design of more active compounds can be envisaged. Iron as Possible Therapeutic Strategy for Cancer Treatment Compared to normal cells, neoplastic cells require greater amount of Fe because generally they proliferate at a greater rate than their normal counterparts. 10,15 This is reflected by the higher expression of TfR1 32,60 and the higher rate of Fe uptake from Tf in cancer cells. 61,62 Furthermore, neoplastic cells express high levels of RR 63,64 making them more susceptible to the action of Fe chelators than normal cells. 10,65 Early studies showed that the clinically used Fe chelator, desferrioxamine (DFO), had some activity at inhibiting the growth of neuroblastoma and leukemia in cell culture and clinical trials This was despite the fact that this chelator suffers from limited membrane permeability. 71,72 Considering that DFO was developed specifically for the treatment of Fe loading diseases such as b thalassemia major and not cancer treatment, its activity in these later trials was encouraging. 15 Indeed, there has been continuing efforts to improve the potency and selectivity of Fe chelators against cancer cells (reviewed in refs. 2, 15, 59 and 73). Among these ligands are 3 aminopyridine 2 carboxaldehyde thiosemicarbazone (Triapine ), 74 Tachpyridine, 75 O Trensox 76 and hybrid chelators derived from pyridoxal isonicotinoyl hydrazone (PIH) 77,78 and thiosemicarbazones The success of Fe chelators as potential anti tumor agents is marked by the entry of Triapine into phase I and II clinical trials alone or in combination with a range of chemotherapeutics. 85,86 In fact, recent results with this agent have shown that it can successfully reduce white blood cell counts by 50% in leukemia patients. 87 A structurally different ligand known as Tachpyridine is also currently in pre-clinical development with the National Cancer Institute. 88 Another series of chelators are those of the PIH class that led to the development of the highly permeable ligand, 2 hydroxy 1 napthylaldehyde isonicotinoyl hydrazone (311). 79,89 This compound shows much greater Fe chelation efficacy and anti tumor activity than DFO. 72,90,91 Studies with 311 and its derivatives 83,92 led to the 1984 Cell Cycle 2007; Vol. 6 Issue 16

5 Figure 2. Summary of the cell cycle in normal Fe replete cells. The cell cycle consists of four main phases: G 1, S, G 2 and M phase. Under normal conditions in Fe replete cells, the progression of the cell cycle is controlled by a number of molecules including the cyclins A, B, D and E, as well as the cyclin dependent kinases (cdks). Cyclin D1 forms a complex with cdk4, while cyclin E binds with cdk2, allowing them to become active enzymes. These complexes are then involved in hyper-phosphorylation of the retinoblastoma susceptibility gene product (prb), which allows it to release the transcription factor, E2F1. Once free, E2F1 is able to translocate to the nucleus where it mediates the transcription of a range of genes vital for S phase progression. One of the most important mediators of this G 1 /S checkpoint is p53, which is able to cause G 1 /S arrest under conditions of cell stress or DNA damage. One function of p53 is to transactivate the expression of the cdk inhibitor, p21 CIP1/WAF1, which then inhibits the activity of cyclin D1/cdk4 and cyclin E/cdk2 complexes, thereby preventing entry into S phase. However, the activity of p21 CIP1/WAF1 can be paradoxical and under some conditions can aid in cell cycle progression (see section on p21 CIP1/WAF1 ). In addition, p53 is also able to inhibit cyclins A and B leading to G 2 /M arrest. retinoblastoma susceptibility gene product (prb) The cdks are dependent on cyclins to modulate their phosphorylation activity. 97,98 For example, cyclin D1 binds to cdk4/6, while cyclin E binds to cdk2 leading to an activation of kinase activity (Fig. 2). 97,98,100 Together, these molecules allow the progression of the cell cycle from G 1 to S phase (Fig. 2). 97,98,100 The S phase is regulated by cyclin A, while the M phase is controlled in part by cyclin B (Fig. 2). 97,98 It is the up regulation and degradation of the cyclins and their subsequent interaction with cdks that mediate progression through the cell cycle. 97,100 There are a number of major checkpoints in the cell cycle that are present at G 1 /S, S, G 2 /M and M phase (Fig. 2). 97,100 These checkpoints are important to determine whether cells will proceed to the next phase of the cycle. 97,98,100 One important regulator of the cell cycle is p53, which is involved in both the G 1 /S and the G 2 /M checkpoints (Fig. 2). 98 In fact, p53 functions to arrest cells following DNA damage and initiate a repair mechanism, or if the DNA is beyond repair, it will activate an apoptotic pathway. 97,100 In response to DNA damage, p53 will transactivate one of its downstream targets, p21 CIP1/WAF1, which subsequently inhibits cyclin D/cdk4/6 and the cyclin E/cdk2 complexes resulting in G 1 /S arrest (Fig. 2). 97,100 Other targets of p53 include cell cycle regulatory molecules such as growth arrest and DNA damage inducible 45 alpha (GADD45a), which is induced upon DNA damage and can arrest the cell cycle (see section on the GADD family). 97,100 Another key regulator of the cell cycle is prb, which controls entry of cells into S phase. 97,100 When hypo phosphorylated, prb binds to and represses the transcription factor E2F1 (Fig. 2). 98 Under normal conditions, accumulating cdk cyclin D and E complexes function to hyper phosphorylate prb, resulting in the release of E2F1, which leads to the expression of S phase genes and subsequent entry into S phase. 34 The role of Fe in regulating some of these effector molecules that play roles in cell cycle control are discussed below. development of a highly potent series of Fe chelators derived from di 2 pyridylketone thiosemicarbazone (DpT). 59,84,93,94 A number of these ligands have also demonstrated marked in vitro and in vivo anti tumor activity 82,84,93,94 and are currently being developed as novel anti cancer agents. In an attempt to understand the effects of Fe chelators on cellular proliferation and in order to develop more effective ligands, a number of studies have examined the roles of Fe in regulating the cell cycle. 95 The Cell Cycle: A Brief Overview As normal cells grow and divide, they progress through the cell cycle in a regulated manner. 96 The main groups of molecules involved in the regulation of the cell cycle include the cyclins, cyclin dependent kinases (cdks), cyclin dependent kinase inhibitors (e.g., p21 CIP1/WAF1 ) and tumor suppressor genes such as p53 and the The Molecular Targets of Iron Chelators and their Effects on the Cell Cycle A mechanism by which Fe chelators exert their anti proliferative effects on tumors is by targeting molecules that are critical in regulating progression of the cell cycle. 101,102 Some of these include: RR, 5,91 cyclins, 101,103 cdks, 101,103 p53, 101, p21 CIP1/WAF1, 101,107 p27 Kip1, GADD45a, 72 hypoxia inducible factor 1a (HIF 1a), N myc downstream regulatory gene 1 (Ndrg 1) 102 and prb. 98,111 By altering the expression and/or function of the above molecules, Fe depletion is able to effectively inhibit the growth of tumor cells. Ribonucleotide reductase. Ribonucleotide reductase is an enzyme that catalyses the de novo biosynthesis of deoxyribonucleotides essential for DNA replication, cell cycle progression and cellular repair. 6 This process requires a tyrosyl free radical which acts to reduce the corresponding ribonucleotides to deoxyribonucleotides. 112 Hence, structurally, RR consists of a radical generator and a reductase Cell Cycle 1985

6 The enzyme is classified into three classes based on different cofactors, such as Fe or cobalamin for their catalytic activity. 6 Class I enzymes are found practically in all eukaryotic organisms and in some prokaryotes and viruses. This class is further divided into three subclasses (Ia, Ib and Ic) based on polypeptide sequence homology and allosteric behaviour. 114 Human RR is a tetramer that belongs to class Ia and consists of two non-identical homodimers, R1 and either R2 or p53r The R1 protein contains the active sites and binding sites for allosteric effectors, while the R2 subunit contains one di nuclear Fe centre and one stable tyrosyl radical per monomer that is vital for enzymatic activity. 112 The R2 subunit is necessary for housekeeping DNA synthesis that is essential for DNA replication during the S/G 2 phases. In contrast, the p53r2 subunit supplies dntps for DNA repair after DNA damage in G 0 /G 1 phase cells in a p53 dependent manner. 115 It has been reported that there is also an additional p53 independent induction of p53r2, because cells with mutated p53 still express this molecule in response to DNA damaging agents. 116 In fact, p53r2 can be a transcriptional target of the p53 family member, p Both the R2 and p53r2 subunits possess an Fe binding site that is important for their enzymatic function. 118 Since the reduction of ribonucleotides is the rate limiting step of DNA synthesis, inactivation of RR has a number of consequences, such as inhibition of DNA synthesis, cell proliferation and DNA repair, leading to cell cycle arrest and apoptosis. 119 On the other hand, increased RR activity has been associated with malignant transformation and tumor cell growth, 119 making RR an important but largely ignored target for anti cancer agents. As Fe is required for the enzymatic activity of RR, Fe chelation is well known to effectively inhibit the activity of this enzyme. 5,91,120 Triapine is one of the most potent inhibitors of RR that targets the R2 subunit of the enzyme. 5,121 However, more recent data also found that Triapine indiscriminately inhibits the p53r2 subunit as well. 118 Iron-depletion Iron depletion affects multiple molecular targets involved in cell cycle control besides RR and these are described below. Cyclins and cdks. As described above, cyclins and cdks are critical for the normal progression through the cell cycle (Fig. 2). 96,99 It is the regulated alterations in the availability and activity of these molecules that governs the transition between the different phases of the cycle. 96 Iron depletion mediated by chelators was found to affect the expression of several cyclins and cdks. 101,103,122 Specifically, in SK N MC neuroepithelioma cells, Fe depletion markedly reduced the expression of cyclins D1, D2 and D3, while having a lesser effect on decreasing cyclin A and B levels. 101 This latter reduction in cyclin A protein in tumor cells is in good agreement with the results from normal T lymphocytes, where there was a decrease in cyclin A protein and its kinase activity after incubation with DFO. 7 Iron depletion has also been shown to decrease the expression of cdk2 101,120 or cdk4 103 protein depending on the cell type and experimental conditions. Studies examining the effect of DFO in neuroblastoma cells have found that protein levels and kinase activity of p34 cdc2 is decreased. 8 This is of significance, as p34 cdc2 functions in the G 2 /M and potentially G 1 /S phase transitions, being the catalytic subunit that complexes with cyclin A, B and E. 123,124 The effect of Fe depletion on p34 cdc2 may explain, at least in part, the G 1 /S and G 2 /M arrest observed after Fe chelation under some experimental conditions. A recent study has identified that the mechanism of the Fe depletion mediated reduction in cyclin D1 protein expression is a result of proteasomal degradation, there being no decrease in cyclin D1 mrna levels (Fig. 3). 125 However, in contrast to the ubiquitin dependent pathway of cyclin D1 degradation that regulates the expression of this molecule under Fe replete conditions, Fe depletion induced an ubiquitin independent pathway of proteasomal degradation (Fig. 3). 125 Importantly, the effects of Fe chelators on cyclin D1 expression were found to be due to Fe depletion, as the supplementation of Fe was able to reverse these effects. 125 Considering the rate limiting role that cyclin D1 plays in G 1 /S progression, 96 its regulation by Fe appears to be important for preventing entrance into the S phase, where Fe is essential for RR activity 4 and thus, DNA synthesis. It has been suggested that the Fe depletion mediated down regulation of cyclin D1 leads to decreased phosphorylation of prb that may be, in part, responsible for the G 1 /S arrest observed. The importance of regulating cyclin D1 in terms of cell cycle control is obvious from studies demonstrating that over expression of cyclin D1 releases cells from their normal controls and acts as an oncogene. 126,127 In fact, pharmacological targeting of cyclin D1 may lead to novel anti tumor agents 128 and the fact that Fe chelators markedly decrease the expression of this molecule may be important in their anti tumor activity. Interestingly, in contrast to other cyclins, cyclin E protein expression was found to be elevated in response to Fe depletion in neuroepithelioma cells. 101,120 This paradoxical response may reflect an attempt by the cell to maintain cell cycle progression after Fe chelation. However, because cyclin E requires cdk2 for its activity and since the expression of the latter is reduced by Fe depletion, 101 the increase in cyclin E does not overcome the G 1 /S arrest after Fe chelation. 101,103,111 In fact, several studies have shown that following Fe depletion, prb becomes hypo phosphorylated leading to G 1 /S arrest. 111,129 These studies demonstrate that expression of critical effectors of the cell cycle such as some cyclins and cdks are affected by intracellular Fe levels, providing another level of control on cell cycle progression. prb. prb is an important molecule that mediates progression of the cells from G 1 to the S phase of the cell cycle. As already discussed, the regulation of prb during the cell cycle is through phosphorylation of the protein by cyclin dependent kinases (cdks) (Fig. 2). 130 Since the expression of cyclin D1 and cdk2 are reduced during Fe depletion, 101,120,125 this will prevent the formation of cyclin cdk complexes leading to hypo phosphorylation of prb that will contribute to G 1 /S arrest (Fig. 4). 101 Indeed, a number of investigators have reported that Fe chelation resulted in the decrease of hyper phosphorylated prb 101,111 in neuroepithelioma cells, 101 human breast cancer cells 103 and T lymphocytes. 111 Hypo phosphorylation of prb during mid to late G 1 phase by cdk4 or cdk6 cyclin D complexes prevents the release of transcription factor E2F1 from prb. 130,131 The release of E2F1 is necessary to transcribe genes critical for cell cycle progression, such as cyclin A and cyclin E. 131 Hence, Fe availability results in alterations in prb phosphorylation that may play a role in the G 1 /S arrest observed after Fe depletion. p53. One of the most well known and important regulators of the cell cycle is p53. 10,129 This transcription factor has a multitude of molecular targets and plays a critical role in the G 1 /S checkpoint (Fig. 2). 129 p53 is activated in response to cellular stress or DNA damage and functions to initiate repair mechanisms or, when the damage is irreparable, apoptotic pathways. 101,104,105,132 A number of studies have reported elevated levels of p53 protein expression following Fe depletion. 105,106,133 This increase appears to be at the post transcriptional level as there is no change in p53 mrna after Fe depletion. 101,105 In some studies, Fe depletion 1986 Cell Cycle 2007; Vol. 6 Issue 16

7 Figure 3. Iron depletion induces Ub independent degradation of cyclin D1 and p21 CIP1/WAF1. Iron depletion results in decreased expression of the key cell cycle regulators, cyclin D1 and p21 CIP1/WAF1. When cells are Fe replete, the traditional pathway of cyclin D1 and p21 CIP1/ WAF1 degradation involves ubiqutination of these proteins, allowing them to be transported to the proteasome for degradation. Upon Fe depletion, an Ub independent pathway leads to the degradation of both cyclin D1 and p21 CIP1/WAF1 by the proteasome. Iron depletion also reduces the transport of p21 CIP1/WAF1 from the nucleus to the cytoplasm, further reducing the translation of this protein. Furthermore, cyclin D1 and p21 CIP1/ WAF1 compete for the proteasomal binding site, suggesting that decreased cyclin D1 expression may lead to increased p21 CIP1/WAF1 degradation and vice versa. resulted in the upregulation of p53 expression only in cells expressing the wild type molecule, 106 while in other investigations, p53 protein levels increased after Fe chelation irrespective of whether it was mutated or not. 105 In whole cell systems, Fe depletion was able to induce p53 transactivational activity and sequence specific DNA binding in a dose and time dependent manner. 106,133 Further studies have revealed that several mechanisms may be involved in the activation of p53 by Fe chelation. These include: (1) an increase in p53 protein expression; 106 (2) increased conversion of latent p53 to its active DNA binding form; 104 (3) phosphorylation of p53 at serine 15 which increases its stability and prevents proteasomal degradation by mdm The increased p53 phosphorylation at this site may indicate up regulation of ataxia telangiectasia mutated (ATM) and/or ATM Rad3 related (ATR) after Fe depletion; 104 and (4) other molecules that are also targets of Fe depletion, such as the transcription factor, hypoxia inducible factor 1a (HIF 1a), can also increase p53 expression. 98 While Fe depletion results in an increase in p53 protein expression and transcriptional activity, it is unclear which of its molecular targets are affected. It is known that the expression of both p21 CIP1/WAF1 and GADD45 mrna are increased after Fe chelation, but this occurs not only in cells with native p53, but also in those with mutant p This indicates that p53 independent up regulation of these genes can occur after Fe depletion. p21 CIP1/WAF1. The progression of the cell cycle is a strictly regulated process in which a number of crucial molecules are involved. 99 One such protein is p21 CIP1/WAF1, a cdk inhibitor that is involved in regulating the cell cycle Specifically, when over expressed, p21 CIP1/WAF1 binds to the cyclin E/cdk2 complex, preventing prb phosphorylation (Fig. 2) 137,138 and preventing the G 1 /S phase transition leading to cell cycle arrest. 139 In addition, p21 CIP1/WAF1 can also prevent DNA replication and affect other transcription factors involved in cell cycle progression, such as E2F1 and c myc Interestingly, and paradoxically, when expressed at very low levels, p21 CIP1/WAF1 is required for the assembly of cyclin D/cdk complexes and is therefore an important component of cell cycle progression. 143,144 Furthermore, p21 CIP1/WAF1 has been shown to directly inhibit caspase 3 activation, thereby preventing apoptosis. 143,144 Indeed, down regulation of p21 CIP1/WAF1 in tumor cells was found to lead to increased apoptosis. 145 Although often activated by p53 in response to cellular stress and/ or DNA damage, p21 CIP1/WAF1 can also be induced by p53 independent pathways involving other transcription factors such as AP2, Sp1 or Sp3. 146,147 More recently, it was discovered that p21 CIP1/WAF1 mrna can be markedly up regulated by Fe depletion using chelators such as 311 and DFO by a p53 independent pathway. 72,148 At the same time, it was found that Fe depletion actually decreased the expression of p21 CIP1/WAF1 protein. 107 Subsequent supplementation of these cells with Fe restored p21 CIP1/WAF1 protein levels, demonstrating that the effect observed was due to Fe depletion. 107,148 It has also been shown that the Fe depletion mediated decrease in p21 CIP1/WAF1 protein was due to two mechanisms: (1) a decrease in nuclear translocation of p21 CIP1/WAF1 mrna to the cytosol and (2) ubiquitin independent proteasomal degradation leading to reduced p21 CIP1/WAF1 protein levels (Fig. 3). 107,148 Interestingly, Fe depletion appears to have similar effects on both cyclin D1 and p21 CIP1/WAF1 protein expression, where it induces ubiquitin independent degradation (Fig. 3). 107,125 The precise ubiquitin independent pathway of p21 CIP1/WAF1 degradation has not been identified. However, it may be mediated by NAD(P)H:quinone oxidoreductase or antizyme 150 that are responsible for degradation of other proteins by this process. Of interest, it has been previously demonstrated that cyclin D1 and p21 CIP1/WAF1 compete for the C8a subunit binding site of the proteasome (Fig. 3). 151 Therefore, the down regulation of cyclin D1 and p21 CIP1/WAF1 following Fe depletion may facilitate degradation of each of these molecules via this pathway. 151 Under very different experimental conditions, Gazitt and colleagues 152 identified that HL60 leukemia cells need to be Fe replete to transcribe p21 CIP1/WAF1 when induced by the phorbol ester, phorbol myristate acetate (PMA). In these studies, Fe deprivation induced by DFO blocked PMA induced differentiation and induced S phase arrest and apoptosis. 152 Clearly, the difference on whether there is up or down regulation of p21 CIP1/WAF1 mrna after Fe depletion can be ascribed to the experimental conditions used Cell Cycle 1987

8 in each case. The biological effects of PMA are numerous and do not relate to a physiological or pharmacologically induced state in humans. Furthermore, the effect of PMA on p21 CIP1/WAF1 has only been found in HL60 cells. 152 In contrast, the effects of Fe depletion on up regulating p21 CIP1/WAF1 mrna have been observed in a variety of cell types 72,92,101,107,148,153 and are relevant to the pharmacological effects of chelators as anti tumor agents. 75,84,93 Examination of the Fe chelator mediated downregulation of p21 CIP1/WAF1 is important, as apart from being a cdk inhibitor and positive regulator of the cell cycle, this protein also has anti apoptotic activity. It is known that high p21 CIP1/WAF1 expression in some cancers may provide a growth advantage capable of subverting apoptosis induced by DNA damaging chemotherapeutics. 143 In fact, by decreasing p21 CIP1/WAF1 expression using anti sense oligonucleotides, cancer cell apoptosis can be induced. 143,154 Hence, p21 CIP1/WAF1 has been proposed as a target for developing novel anti cancer agents. 143 Since Fe chelators effectively inhibit p21 CIP1/WAF1 expression, this is important for understanding, at least in part, their marked anti tumor activity and ability to induce apoptosis. However, the effect of chelators at inhibiting cancer proliferation and inducing apoptosis is probably due to their influence on multiple molecular targets. This may explain the high anti tumor activity of some chelators and their ability to overcome resistance to standard chemotherapeutic agents. 84,91 p27 Kip1. Another cdk inhibitor that is regulated by Fe depletion is p27 Kip1. This was first shown by Wang and colleagues 108,109 using the chelator mimosine and then confirmed by others. 155,156 The upregulation of p27 Kip1 by Fe deprivation occurred at both the mrna and protein levels. 108 It was suggested that Fe depletion also increased the expression of transforming growth factor b1 (TGF b1). 156 Interestingly, when this factor was neutralized using an TGF b1 antibody, it prevented the upregulation of p27 Kip1 (Fig. 4). 156 In a later more comprehensive study, it was shown that the p170 subunit of the eukaryotic initiation factor 3 (eif3) was down regulated by Fe depletion and that this allowed increased p27 Kip1 expression (Fig. 4). 155 The significance of this observation is that the regulation of translation generally occurs at the initiation step which requires multiple eif s and the ribosome. This is of interest, as it suggests that p170 is an early response gene to Fe deprivation that regulates the translation of a subset of mrnas. 155 This later finding may have wider implications for understanding the changes in the expression of proteins after Fe depletion. The GADD family. The GADD group of genes constitutes a small family of stress response molecules comprised of GADD34, GADD45 and GADD153. The expression of these genes is often increased when cells are subjected to a stress such as nutrient deprivation (e.g., glucose, glutamine, zinc) or exposed to DNA damaging agents (e.g., peroxynitrite) 160 which may cause cell cycle arrest and/or apoptosis. The GADD45 group of genes plays an important role in the G 2 /M checkpoint and apoptosis. 161 This family of genes encodes three structurally related proteins, GADD45a, GADD45b and GADD45g. 161 However, only GADD45a has been shown to activate p53 dependent G 2 /M arrest and inhibit cdc2 kinase. 161 Neither GADD45b nor GADD45g have been shown to be downstream targets of p Interestingly, GADD45a is also known to interact with key cell cycle regulatory molecules, such as p21 CIP1/WAF1, 162 cdc2/cyclin B1 163 and p38 mitogen activated protein kinase (MAPK; see section below on p38 MAPK). 164 In fact, the cellular function of GADD45a is dependent on its interacting partner. 164 For example, interaction between GADD45a and p38 MAPK has been shown to play a pivotal role in preventing oncogene induced growth in part by regulating p Studies using transfected cells suggest that GADD34 and GADD153 appear to have a direct role in initiating apoptosis rather than inducing cell cycle arrest. 165,166 Overexpression of each GADD gene causes growth inhibition and/or apoptosis, while combined overexpression of the three GADD genes leads to synergistic or cooperative effects on anti proliferative activity. 167 Cellular Fe depletion mediated by DFO or 311 has been shown to cause a pronounced concentration and time dependent increase in the expression of GADD45 mrna after 20 h of incubation in three different cell lines, BE 2 neuroblastoma, SK N MC neuroepithelioma and K562 erythroleukemia. 72 This effect was reversible after the removal of the ligands and also the Fe(III) complexes of DFO and 311 had no effect on GADD45 mrna levels, suggesting that Fe depletion was necessary to increase GADD45 mrna. 72 Recent studies from our laboratory also indicate that GADD153 mrna is also increased upon Fe depletion (Siafakas R, Fu D, Richardson DR, unpublished observations). However, interestingly, there was no appreciable increase in the GADD45 protein level in cells after Fe depletion, although only a single incubation time (30 h) was assessed 101 and further studies are required. Similar to the effect observed after Fe depletion, both GADD45 and GADD153 mrnas have been shown to be up regulated during hypoxia. 168 This suggests a potential role for the transcription factor HIF 1a in the up regulation of these genes that may be activated by both hypoxia and Fe depletion via prolyl hydroxylases (see section on HIF-1a). Several studies have reported that after Fe depletion there are alterations to the level of GADD45 interacting partners, such as p and cdk2/cyclin B. 101 It can be hypothesized that GADD45 may mediate growth arrest through inhibiting the activity of cyclin B and cdk2 163 and studies have shown that Fe depletion decreases the expression of these regulatory molecules. 101 p38 MAPK. The signaling molecule p38 MAPK is one of the three members of the MAPK family which also includes extracellular signal regulated kinase (ERK) and c Jun N terminal protein kinase/ stress activated protein kinase (JNK/SAPK). 170 These proteins are activated by a variety of environmental stresses and inflammatory cytokines and affect processes such as cell differentiation and apoptosis. 164 Studying the effect of DFO, Lee et al. showed that Fe depletion strongly activated p38 MAPK and ERK, but did not activate JNK. 169 In addition, the selective p38 MAPK inhibitor, SB203580, and ERK inhibitor, PD98059, protected cells against Fe chelator induced death in oral keratinocytes and cancer cells. 169 This suggests that p38 and ERK MAPKs are potential mediators of cell death induced by Fe deprivation. It is of interest to note that p38 mediated growth inhibition has been suggested to involve activation of p and decrease cyclin D1 expression, 172 both of which also occur upon Fe depleti on. 101,103,105,125 However, the effect of p38 MAPK on decreasing cyclin D1 expression was thought to be via decreased cyclin D1 transcription, although a post transcriptional mechanism that led to decreased protein levels was not excluded. 172 Clearly, as noted previously, Fe depletion leads to decreased cyclin D1 protein levels due to proteasomal degradation. 125 Later studies by Moon and colleagues 173 using vascular smooth muscle cells demonstrated that the p38 MAPK pathway participated in p21 CIP1/WAF1 induction after metal chelation, which consequently leads to a decrease of cyclin 1988 Cell Cycle 2007; Vol. 6 Issue 16

9 Figure 4. The demonstrated and potential effects of iron (Fe) deprivation on cell cycle arrest, apoptosis, metastasis and growth suppression. Iron regulatory protein (IRP) RNA binding activity and the expression of transforming growth factor b (TGF b1), p53, p38 mitogen activated protein kinase (MAPK) and hypoxia inducible factor 1a (HIF 1a) are increased upon Fe depletion. Increased RNA binding activity of the IRPs acts to stabilize CDC14A mrna, which may promote its translation, although an elevation in CDC14 protein after Fe depletion has not been shown. CDC14A can de phosphorylate cdk substrates and may act as another link to mediate the effect of Fe depletion on cell cycle progression. Iron depletion also leads to increased expression of TGF b1, which may up regulate the cdk inhibitor, p27 Kip1, under some conditions. Additionally, reduced expression of the p170 subunit of the eukaryotic initiation factor 3 (eif3) upon Fe depletion also increases p27 Kip1 expression. The reduction in expression of some cyclins such as cyclin D1 and cyclin dependent kinase (cdk) 2 and/or 4 results in hypo phosphorylation of the retinoblastoma susceptibility product (prb). This leads to reduced release of the E2F1 transcription factor from prb, preventing transactivation of genes essential for cell cycle progression. Upregulation of p53 may lead to the increased expression of its downstream targets such as growth arrest and DNA damage inducible gene 45a (GADD45a) or cause apoptosis, although these effects are yet to be established. The depletion of Fe also mimics the hypoxic state, leading to increased expression of HIF 1a, which acts on its downstream targets, p53, BNIP3 and N myc downstream regulated gene 1 (Ndrg 1) etc. The increased expression of BNIP3 can lead to apoptosis through the mitochondrial pathway by increasing the expression of pro apoptotic molecules. Increased Ndrg 1 expression is known to result in the inhibition of tumor growth and metastasis. Reduction of p21 CIP1/WAF1 protein expression may also be potentially involved in mediating apoptosis observed upon Fe depletion. Iron depletion also up regulates p38 mitogen activated protein kinase (MAPK) which could be involved in cell cycle regulation and/or apoptosis. D1/cdk4 and cyclin E/cdk2 complexes. Further studies are obviously essential to determine the role of p38 MAPK in the Fe depletion mediated decrease in cyclin D1 125 expression that could be important in mediating the cell cycle arrest observed. HIF 1a. Hypoxia inducible factor 1 (HIF 1) is a transcription factor that is activated under hypoxic conditions and acts to initiate a signaling pathway leading to cell survival. 174,175 This protein is composed of two subunits, a constitutively expressed b subunit and an a subunit which is regulated by the hypoxic state. 176 Under conditions of normal oxygen tension and Fe levels, HIF 1a is regulated by prolyl hydroxylase enzymes 177,178 which allow its binding to the von Hippel Lindau (VHL) protein. This protein activates a ubiquitin E3 ligase resulting in the subsequent degradation of HIF 1a. 174,175,178 However, under conditions of oxygen deprivation and/or Fe depletion, prolyl hydroxylases fail to function, leading to the accumulation of HIF 1a in the cell. 177,178 HIF 1a is then able to translocate to the nucleus where it binds to HIF 1b to form the HIF 1 complex. 176,179 Once assembled, HIF 1 can regulate a number of genes by binding to their hypoxia responsive element (HRE) located in the promoter or enhancer. 180 For example, TfR1 is transcriptionally upregulated by HIF 1 180,181 which then functions to increase intracellular Fe levels. Also targeted by this transcription factor is Ndrg 1 (see section on Ndrg-1), 182 which has a role in differentiation 183 and may be involved in regulating the cell cycle. 184 Under conditions of severe hypoxia, HIF 1 can also induce apoptotic pathways by stabilizing p53 expression 185 and up regulating pro apoptotic factors such as BNIP3 (Fig. 4). 186,187 Interestingly, BNIP3 can also be induced in a HIF 1 independent manner in response to Fe depletion. 188,189 This is thought to be mediated by the transcription factor, pleomorphic adenoma gene like 2 (PLAGL2). 188,189 Cells treated with DFO respond by increasing the nuclear expression of PLAGL2 188 that is then able to induce BNIP3 expression in a HIF 1 independent manner, by increasing BNIP3 promoter activity. 189 Indeed, transfection of mouse Balb/c3T3 fibroblasts with PLAGL2 inhibited proliferation and led to the induction of apoptosis, perhaps as a result of BNIP3 activation. 189 It is important to note that Fe depletion does not cause apoptosis via BNIP3 alone, as BNIP3 sirna transfected cells incubated with Fe chelators also undergo apoptosis. 189 This indicates that Fe depletion activates a number of different apoptotic pathways and these are further discussed in the section on apoptosis. The depletion of intracellular Fe has crucial repercussions, resulting in the activation of HIF 1a and its numerous down stream targets, ultimately leading to cell cycle arrest, apoptosis, metastasis suppression and inhibition of growth (Fig. 4). 190 Although some of the effects of HIF 1a up regulation are growth and angiogenesis (e.g., through vascular endothelial factor 1), potent Fe chelators have been shown to override this and activate apoptotic pathways that are induced in part by HIF 1a. 101,190 Ndrg 1. N myc downstream regulated gene 1 (Ndrg 1) is also known as differentiation related gene 1 (Drg 1) and Cap 43 and is a recently identified metastasis suppressor gene that plays roles in cell differentiation and proliferation , Iron depletion markedly up regulates Ndrg 1 mrna and protein expression in a number of neoplastic cell types, and hence, may be significant for understanding the mechanisms involved in the inhibition of proliferation by Fe chelators. 102,110 High expression of Ndrg 1 is associated with greater survival and less aggressive tumors in prostate cancer patients. 192 There is also a significant inverse correlation of Ndrg 1 expression with depth of invasion in pancreatic adenocarcinoma patients. 194 Moreover, Cell Cycle 1989