Biochimica et Biophysica Acta 1603 (2002) 31 – 46

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Review
The role of iron in cell cycle progression and the proliferation
of neoplastic cells
Nghia T.V. Le, Des R. Richardson *
The Iron Metabolism and Chelation Group, The Heart Research Institute, 145 Missenden Rd, Camperdown, Sydney, New South Wales, 2050 Australia
Received 3 May 2002; accepted 6 June 2002

Abstract

Iron (Fe) is an obligate requirement for life and it is well known that Fe depletion leads to G1/S arrest and apoptosis. These facts, together
with studies showing that Fe chelators can inhibit the growth of aggressive tumours such as neuroblastoma, suggest that Fe-deprivation may be
an important therapeutic strategy. To optimise the anti-proliferative effects of Fe chelators, the role of Fe in cell cycle control requires intense
investigation. For many years, Fe chelators were known to prevent the activity of the R2 subunit of ribonucleotide reductase (RR) that catalyzes
the conversion of ribonucleotides into deoxyribonucleotides (dNTPs) for DNA synthesis. In addition, Fe depletion may also inhibit the newly
identified p53-inducible form of this molecule called p53R2. This protein has the same Fe-binding sites as found in R2, and its activity is
thought to supply dNTPs for the critical process of DNA repair. Iron chelation also causes hypophosphorylation of the retinoblastoma protein
(pRb) and decreases the expression of cyclins A, B and D, which are vital for cell cycle progression. Other regulatory molecules whose
expression is affected by Fe depletion include p53 and hypoxia inducible factor-1a (HIF-1a). The levels of p53 increase following Fe chelation
via the ability of HIF-1a to bind and stabilize p53. The activity of HIF-1a is controlled by an Fe-dependent enzyme known as HIF-a prolyl
hydroxylase (PH). Chelation of Fe from this enzyme inhibits its activity, leading to stabilization of HIF-1a and the subsequent effects on
downstream targets critical for angiogenesis and tumour growth. The levels of p53 may also increase after Fe chelation by phosphorylation of
this protein at serine-15 and -37. This prevents the interaction of p53 with murine double minute-2 (mdm-2) and its degradation. Iron chelation
also markedly increases the mRNA levels of the p53-inducible cyclin-dependent kinase (cdk) inhibitor, p21WAF1/CIP1. Surprisingly, the increase
in p21WAF1/CIP1 mRNA was not reciprocated at the protein level, and this may result in cell cycle dysregulation. This review will focus on the
molecular mechanisms induced following Fe chelation and the role of Fe in cell cycle progression.
D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Iron; Iron chelation; Iron chelator; Iron metabolism; Cancer; p53; p21; Ribonucleotide reductase

1. Introduction: iron as a molecular target for anti-
cancer agents
A major challenge facing researchers is the development
of effective anti-cancer drugs that show high selectivity
against tumour cells compared to normal cells. Compound-
ing this problem is the emergence of tumours that are
unresponsive to radiation and chemotherapeutic treatments.
As a consequence, novel strategies for cancer therapy must
be sought.
One such approach involves the targeting of intracellular
iron (Fe) to induce cell cycle arrest and apoptosis (for
reviews see Refs. [1 –3]). Indeed, Fe is an absolute require-
ment for proliferation, as Fe-containing proteins catalyze
*
Corresponding author. Tel.: +61-2-9550-3560; fax: +61-2-9550-3302.
E-mail address: d.richardson@hri.org.au (D.R. Richardson).
key reactions involved in oxygen sensing [4,5], energy
metabolism, respiration, folate metabolism (e.g. serine
hydroxymethyltransferase expression; [6]) and DNA syn-
thesis (e.g. ribonucleotide reductase (RR) [7]). In fact,
without Fe, cells are unable to proceed from the G1 to the
S phase of the cell cycle [7].
Before describing the effects of Fe depletion on the
molecular control of the cell cycle, we will discuss the
mechanisms involved in the uptake and intracellular metab-
olism of Fe.
1.1. Iron transport, uptake and metabolism by cells
Iron is transported in the serum bound to the protein
transferrin (Tf) [8,9]. The uptake of Fe into cells involves
binding of Tf to the transferrin receptor 1 (TfR1). This
complex is then internalised by receptor-mediated endocy-
tosis and the Fe released from Tf by endosomal acidification

0304-419X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 1 9 X ( 0 2 ) 0 0 0 6 8 - 9
32 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46
(for reviews see Refs. [8– 11]). Recently, another TfR has
been identified and is known as TfR2 [12,13]. However, Tf
has a lower affinity for TfR2 than TfR1, and the exact
function of TfR2 remains unclear [12,13].
Iron released from Tf in the endosome is transported
through the endosomal membrane by Nramp2 (also known
as the divalent metal ion transporter 1; DMT1; [14,15]; for
review see Ref. [9]). Once Fe has entered the cell, it is
incorporated into vital Fe-containing molecules (e.g. haem
and non-haem Fe-containing proteins) and/or stored in the
Fe-storage protein, ferritin (for review see Ref. [16]).
Ferritin is a polymeric protein consisting of 24 subunits of
two types, a heavy (H) subunit of 21 kDa and a light (L)
subunit of 19 kDa [10,16]. At present, the intermediate(s)
involved in intracellular Fe transport from Nramp2 to
ferritin or to the haem synthesis pathway [17] remains
unknown. Likely molecular candidates for intracellular Fe
transport include low-Mr Fe complexes [18] or specific Fe-
binding chaperones [19].
The regulation of intracellular Fe homeostasis is largely
controlled by two mRNA-binding molecules known as iron-
regulatory protein-1 and -2 (IRP1 and IRP2; [10,20]). The
mRNAs of certain molecules involved in Fe metabolism
including ferritin, TfR1 and Nramp2 [9,15] contain hairpin-
loop structures in their 5V- or 3V-untranslated regions
(UTRs), called iron-responsive elements (IREs). The IRPs
can bind to IREs and either stabilize the mRNA against
degradation or inhibit translation [10,20]. For instance, in
ferritin mRNA, the IRE is located in the 5V UTR, and IRP –
RNA-binding inhibits translation, thereby decreasing Fe
storage. However, in TfR1 mRNA the IREs (five hairpin
loops) are located in the 3V UTR, and IRP – RNA-binding
confers stability to the mRNA, enhancing translation and Fe
uptake via the TfR1 [10,20]. Therefore, the IRP –RNA-
binding mechanism is crucial in regulating cellular Fe
homeostasis.
Neoplastic cells, as compared to normal cells, have
significantly higher levels of TfR1 [21] and take up Fe
rapidly from Tf [7,22 –24]. This is exploited clinically by
67
Ga radioimaging, where 67Ga binds to Tf and is taken up
by tumour cells that express much higher TfR1 levels than
normal cells [21,25,26].
The marked Fe requirement of tumour cells results in
their greater sensitivity to Fe chelators relative to normal
cells [7,27,28]. In addition, antibodies that inhibit Fe uptake
by preventing the binding of Tf to the TfR1 can halt tumour
cell growth in vitro [29] and in vivo [30]. Significantly,
these antibodies can stop cellular proliferation in the
absence [29] and presence of Fe chelators [31,32].
The importance of Fe as a likely molecular target for
anti-cancer agents is exemplified by the fact that nitric oxide
(NO) produced by activated macrophages acts as a cytotoxic
effector molecule via its ability to bind Fe [33,34]. More-
over, NO acts similarly to chelators [35] to induce Fe release
from cells [36 –38] and to inhibit Fe uptake from Tf [39,40].
These effects of NO on Fe metabolism may contribute to the
subsequent inhibition of energy metabolism and DNA syn-
thesis [33,34].
1.2. The iron-containing enzyme, RR, is an important
molecular target for iron chelators
The fact that Fe is critical for both cellular growth and the
activity of the Fe-containing enzyme RR means that it is an
important target for anti-tumour drugs including Fe chelators
[1]. RR is the rate-limiting enzyme involved in the conver-
sion of ribonucleotides into deoxyribonucleotides (dNTPs)
for DNA synthesis. This enzyme consists of two protein
subunits, designated as R1 and R2 [41,42]. The R1 subunit
contains a catalytic site that is involved in the binding of
ribonucleotides for reduction to dNTPs [41,42]. In the
absence of a constant supply of Fe to R2, the R1 subunit is
inactive and thus, RR cannot function [41,42]. The activity
of RR is dependent on Fe since the R2 subunit contains a
tyrosyl radical that requires Fe for stabilization [42].
The potential of RR as a therapeutic target for the
treatment of cancer is illustrated by the cytotoxic drug
hydroxyurea (HU) that acts to scavenge the tyrosyl radical
of this enzyme [43,44]. However, HU has limited potency
due to its short half-life, low affinity for RR, and the fact
that resistance develops against this drug [45 – 47]. In
comparison to several key enzymes, RR shows the greatest
increase in activity in tumour compared to normal cells
[48,49]. This may partly explain the greater sensitivity of
neoplastic cells to Fe chelators and RR inhibitors, e.g. HU
and TriapineR [50 –52]. Therefore, Fe chelation may pro-
vide an alternative mechanism to inhibit RR activity in HU-
resistant tumours. In fact, studies in vitro have shown that
some chelators that are RR inhibitors can overcome HU-
resistance via their ability to bind Fe [53].
In contrast to the R2 subunit, R1 is constantly expressed
throughout the cell cycle [54]. Synthesis of R2 is initiated
during S-phase where the protein is rapidly produced for
DNA synthesis [54]. Following S-phase, R2 is then quickly
degraded to barely detectable levels following M-phase
[41]. Thus, the regulation of RR involves the control of
R2 expression and degradation. The effect of Fe chelators
on R2 expression has not been studied in detail, particularly
in terms of the differences in expression between normal
and neoplastic cells.
1.3. A novel p53-inducible form of RR (p53R2): another
molecular target for iron chelators?
While the function of RR is to ensure that the cellular
levels of dNTPs are adequate for DNA synthesis during S-
phase [41,42], little is known about the supply of dNTPs to
cells that are undergoing DNA repair. The recent discovery
of a stress-inducible RR subunit called p53-inducible R2
(p53R2) has provided insight into this process [55,56]. In
contrast to R2, expression of p53R2 can be induced via p53
following DNA damage by UV-light, gamma-irradiation and
 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46 33

adriamycin treatment [55,56]. The RR activity of p53R2 may
not be important for the maintenance of a dNTP pool, but for
the urgent supply of dNTPs for DNA repair [56,57]. It
remains unknown whether Fe chelation can induce p53R2
to compensate for the decreased R2 activity after Fe deple-
tion. However, intriguingly, it is known that p53R2 can
substitute for R2 to form an active complex with R1 [57].
The mouse R2 subunit has been shown to bind Fe(III)
through Glu-170, Glu-233, Glu-267 (bidentate) and His-270
[58]. These residues are highly conserved in a wide variety
of organisms, indicating their key roles in RR activity [58].
By sequence comparison, we identified that the same Fe-
coordination sites in human R2 are also found in human
p53R2 (Fig. 1). This is an interesting observation, as it may
suggest that the enzymatic activity of this molecule will be
affected by Fe chelators. Hence, because p53R2 activity
could be important for the production of dNTPs for DNA
repair [56,57], Fe chelators may effectively inhibit this
process. Therefore, this could partly explain why DNA-
damaging agents such as doxorubicin are more effective at
inhibiting cancer cell proliferation in the presence of Fe
chelators [59].
2. Effect of iron chelators on tumour growth
Many studies have shown that the anti-proliferative
effects of Fe deprivation on cancer cells in vitro [60,61],
in vivo [60 – 63] and in a number of clinical trials [64 – 69].
Indeed, the aggressive RR inhibitor, Triapine (3-aminopyr-
idine-carboxaldehyde thiosemicarbazone) [51,52], is a tri-
dentate Fe chelator that is currently in Phase I combination
studies and Phase II single agent trials (Vion Pharmaceut-
icals Ltd—www.vionpharm.com/). The role of Fe chelators
as anti-proliferative agents has previously been reviewed
[1 –3,70] and the reader is referred to these publications for
a more detailed analysis.
Currently, Fe chelators that show much greater anti-
proliferative activity than DFO in vitro are in development.
Some of these include aroylhydrazones such as 2-hydroxy-
1-naphthylaldehyde isonicotinoyl hydrazone and its ana-
logues [28,71 – 74], Triapine [51,52], and tachpyridine
(N,NV,NW-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclo-
hexane) [75,76]. Comprehensive studies of these chelators
in vivo are underway, and are essential to judge the efficacy
and toxicity of these drugs.

Fig. 1. Sequence alignment of the human ribonucleotide reductase subunit, R2, and the p53-inducible R2 subunit. Both human R2 and p53R2 contain the amino
acid residues (denoted by *) critical for binding Fe(III), namely Glu-169, Glu-232, Glu-266 and His-269 [58]. Therefore, p53R2 activity may be susceptible to
Fe chelators and could inhibit the production of deoxyribonucleotides that are essential for DNA repair (see text for details). Note that mouse R2 has one more
amino acid in its sequence compared to human R2. Hence, in the mouse, the R2 Fe-binding sites are at Glu-170, Glu-233, Glu-267 and His-270 [58].
34 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46
Considering the great potential of Fe chelators at inhibit-
ing the proliferation of tumour cells, further studies are
required to understand the molecular basis of their effects on
proliferation. In the present review, we summarize the
molecular mechanisms of regulating cell cycle progression
and then discuss the effects of Fe chelators on these
processes.
3. The cell cycle
While traditionally it was thought that the anti-prolifer-
ative effect of Fe chelation was solely related to the
inhibition of RR, there is growing evidence that this may
not be the only molecular target. Several studies have shown
that Fe chelation affects the expression of proteins critical
for cell cycle progression [73,74,77,78]. In addition, Fe
chelation can also induce the tumour suppressor protein p53
[79 – 82] that transactivates genes involved in cell cycle
arrest and apoptosis. Before describing the effects of chela-
tors on the cell cycle, we will briefly describe the major
molecules involved in regulating this critical process.
3.1. Cyclins and cyclin-dependent kinases (cdks)
The cell cycle consists of five distinct phases, G0, G1, S,
G2 and M (Fig. 2A). In the last 15 years, significant advances
have been made in understanding the molecular mechanisms
involved in cell cycle control (for a detailed review see Ref.
[83]). Cell cycle progression depends on the activity of the
cdks. The active forms of these kinases occur as hetero-
dimers that are composed of a regulatory subunit called a
cyclin, and its catalytic counterpart, the cdk (Fig. 2A) [83].
The cyclin – cdk complexes are activated by phosphor-
ylation via cyclin-activating kinases (CAKs) [84]. Cyclins
facilitate cell cycle progression and are regarded as positive
regulators of the cycle [84]. Indeed, progression through G1
is mediated by the expression of cyclin D, E and A [83],
while S and M phase progression are characterised by the
appearance of cyclin A and B, respectively [83] (Fig. 2A).
An important regulatory mechanism performed by cdk
molecules involves the phosphorylation of the retinoblas-
toma protein (pRb) [83]. This molecule mediates progres-
sion of cells from G1 to the S phase of the cell cycle. The
hyperphosphorylation of pRb occurs following the forma-
tion of cdk4 – or cdk6 –cyclin D complexes during mid to
late G1 phase (Fig. 2A) [83]. This phosphorylation event
results in the release of transcription factor, E2F1, from pRb,
which then transcribes genes critical for cell cycle progres-
sion (e.g. dihydrofolate reductase, cyclin A and cyclin E)
(Fig. 2B) [83].
3.2. cdk inhibitors
The activity of cyclin– cdk complexes are affected by
cdk inhibitors (CKI) that are classed into two families.
These are the inhibitors of cdk4 (INK4) or the kinase
inhibitor proteins (CIP/KIP) (for a detailed review see
Ref. [84]). The CIP/KIP family of inhibitors consists of
three proteins: namely p21WAF1/CIP1, p27KIP1 and p57KIP2.
These molecules prevent cell cycle progression and are
commonly regarded as negative regulators of the cycle.
The CIP/KIP inhibitors exert their influence during most
periods of the cell cycle by binding directly to the cdk/cyclin
complex to inhibit their activity [84].
There are numerous mechanisms involved in the regu-
lation of CIP/KIP inhibitors. For instance, p21WAF1/CIP1 may
be maintained at low levels by proteasomal protein degra-
dation [85]. However, during periods of stress, the expres-
sion of p21WAF1/CIP1 can be induced by p53 to cause G1/S
arrest [86]. In contrast to p21WAF1/CIP1, the inhibitory effects
of the KIP proteins involve the modulation of their cdk/
cyclin-binding affinities via phosphorylation [87,88]. Phos-
phorylated KIP inhibitors have lower affinities for their
respective cdks and readily dissociate for degradation
[87,88].
The notion that KIP/CIP inhibitors are solely negative
regulators of the cell cycle has recently been challenged.
Several studies have shown that the activity of cdk4 or -6
during mid-G1 phase not only requires cyclin D, but also
p21WAF1/CIP1 or p27KIP1 [89 – 92]. These studies demon-
strate that KIP/CIP inhibitors are required for the assembly,
nuclear accumulation and stabilization of the cdk/cyclin
complex. In addition, the sequestration of p21WAF1/CIP1 or
p27KIP1 during mid-G1 may allow or enhance the hyper-
phosphorylation of pRb by cdk2– cyclin E [89– 91]. At
present, the mechanisms that govern whether a KIP/CIP
CKI becomes a positive or negative cell cycle regulator are
unclear.
In contrast to the KIP/CIP family of CKIs, the inhibitory
activities of INK4 are restricted to cdk4 and cdk6 [93]. As a
consequence, the INK4 family is thought to play a major
role in G1/S arrest. This family of CKI includes p15INK4B,
p16INK4A, p18INK4C and p19INK4D [93].
3.3. The p53 tumour suppressor protein
The p53 tumour suppressor protein plays a pivotal role in
preventing cancer development by acting as a critical tran-
scription factor to induce cellular arrest and/or apoptosis
[94 – 98]. There are many stress factors that can initiate the
stabilization, accumulation and activation of p53. These
include DNA damage, decreased dNTP levels, hypoxia,
loss of a cell survival signal, oncogene activation, telomere
attrition, abnormal cell growth and, more recently, Fe
chelation [79,82,99,100]. Once activated, p53 can initiate
the transcription and subsequent expression of various
downstream genes that commit the cell to differentiation,
senescence, DNA repair, cellular arrest and/or apoptosis
(Table 1) (for review see Ref. [100]).
As a consequence of its function, p53 transcription,
translation, protein stabilization, subcellular localization
 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46 35

Fig. 2. (A) The cell cycle. (B) Some of the critical molecular events involved in G1/S progression. Cells enter the cycle by expressing cyclin D at G1 which
binds cdk4 or -6 to form a catalytic complex involved in the phosphorylation of the retinoblastoma protein (pRb). This latter molecule acts as a suppressor of
the E2F1 family of transcription factors. Phosphorylation of pRb leads to the dissociation of E2F that promotes the expression of a variety of genes important
for S-phase entry. One of the genes upregulated by E2F1 is cyclin E. This protein when associated with cdk2 further hyperphosphorylates pRb to ‘‘drive’’ cells
into S-phase.

and activation are tightly regulated. A molecule that regu-
lates the level of p53 is the murine double minute-2 (mdm-
2) protein that acts as a ubiquitin ligase to mediate p53
degradation [101]. The expression of this protein is con-
trolled by p53 to form an auto-regulatory feedback loop
(Fig. 3). Due to this loop, any increase in p53 results in
increased mdm-2 expression [101]. This control of p53
activity ensures that correct activation and stabilization of
this protein only occurs as a result of cellular stress. In
addition, mdm-2 not only inhibits p53 transcriptional activ-
ity, but also targets the protein [102] and itself for cytoplas-
mic export, followed by degradation via the ubiquitin
pathway [101,103].
There are several pathways that can activate and stabilize
p53 (Fig. 3). A p53 response can be initiated after DNA
damage, for instance when cells are exposed to ionising
radiation. As a consequence of DNA damage, the ataxia
telangiectasia mutated (ATM) protein and check-point kin-
ase 2 (CHK2) phosphokinase are expressed to stabilize p53
by phosphorylation [104]. In a similar manner to the ATM
36 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46

Table 1
p53-inducible proteins involved in apoptosis, cell arrest and DNA repair
p53-inducible molecules Comments References
BAX Well-characterised [160]
pro-apoptotic protein.

BAX is inactivated by bcl-2.

Overexpression of BAX
induces mitochondrial
apoptosis.

NOXA Member of the bcl [161]

(for damage) family of pro- and
anti-apoptotic proteins.

Cells exposed to X-ray

irradiation express
NOXA to induce
mitochondrial apoptosis.

PUMA Consists of an alpha [162,163]

(p53 up-regulated and beta form with
modulator of similar apoptotic functions.
apoptosis)
Localizes to mitochondria
to induce apoptosis.

p53AIP1 Pro-apoptotic protein. [164]

(p53 mediated
apoptosis inducing Localizes to mitochondria
protein 1) to induce apoptosis.

Requires p53 to be
phosphorylated at serine 46.

p53 DINP1 Apoptosis induced after [165]

(p53-dependent double-stranded DNA
damage-inducible breaks via p53AIP1
nuclear protein 1) expression.

This protein is associated

with the phosphorylation
of p53 at serine 46 to
initiate apoptosis.

p21WAF1/CIP1 Cell cycle inhibitor of [86]

the CIP/KIP family.

Can arrest cell during all

stages of the cell cycle.

GADD45 Protein causes cellular arrest [166]

and DNA excision repair.

p53R2 Shares 80% homology to R2. [56,57]

(p53-inducible R2)
This protein is probably
required for ribonucleotide
reductase activity during
DNA repair.

MDM-2 Involved in the targeting [101,167]

of p53 via ubiquitination for
proteasomal degradation.
 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46
pathway, other forms of DNA damage (e.g. chemotherapeu-
tic drugs, ultraviolet light or protein kinase inhibitors) can
stabilize p53 by phosphorylation via the ataxia telangiecta-
sia-related (ATR) phosphokinase (Fig. 3) [105]. Alterna-
tively, several oncogenes (e.g. c-myc and ras) can increase
the levels of p53 via the expression of the alternative
reading frame of the INK4A locus (ARF) protein
([106,107]; for detailed review see Ref. [108]).
As a consequence of cellular damage, either p53 or
mdm-2 can be post-translationally modified to stabilize the
former for nuclear accumulation. For example, p53 is often
phosphorylated at serine-15 and/or -37 to prevent its
association with mdm-2, thus inhibiting its degradation in
cells [105]. Another reported mechanism that inhibits
mdm-2 activity involves its rapid phosphorylation by
ATM following exposure to radiation [109]. The phosphor-
ylation of mdm-2 may prevent its association with p53,
thus allowing nuclear accumulation of this latter protein
[109]. In contrast, the ARF protein (p14ARF) binds to
mdm-2, resulting in nucleolar sequestration that inhibits
p53 degradation [110,111] (Fig. 3). Moreover, the ARF
protein in conjunction with mdm-2 can bind to E2F1 to
inhibit its transactivation role and results in cellular arrest
[112].
4. Effect of iron chelation on the expression of cell cycle
control molecules
4.1. Effect of iron deprivation on cyclins and cdks
Several investigators examining a variety of tumours,
including neuroepithelioma cells, breast cancer cells, leu-
kemia cells and Kaposi’s sarcoma cells, have examined
the effects of Fe chelation on the expression of cyclin A,
B, E, and D, cdk2 and cdk4 [74,77,78,113]. Iron chela-
tion caused a marked decrease in the levels of cyclin D1,
D2 and D3, while the expression of cyclin A and B were
also reduced but to a lesser extent [74,77,78]. Importantly,
in contrast to the chelators, their Fe complexes had no
effect on the expression of these molecules, suggesting
that Fe depletion was critical [74]. Using gene array
technology, Alcantara et al. [113] showed that incubation
of HL-60 leukaemia cells with DFO resulted in a marked
decrease in the mRNA levels of cyclin A, while the
expression of other cyclins such as cyclin D3 and E1
were only slightly decreased. Hence, there appears to be
some differences in cyclin expression in response to Fe
chelation, which may reflect variations in cell cycle
control between cell types.
The expressions of cdk2 mRNA [113] and protein [74]
are also decreased after Fe chelation in leukemia and
neuroepithelioma cells, respectively, while cdk4 protein
levels were not affected in this latter cell type [74]. In
addition to this, there was a marked increase in cyclin E
expression in neuroepithelioma cells after Fe depletion [74].
37
Normally, during the G1-S phases, cyclin D and cyclin E are
bound to cdk4 and cdk2, respectively (Fig. 2A), and act to
phosphorylate pRb (for review see Ref. [83]). Reduction in
the levels of these cyclins would result in G1/S arrest and
hypophosphorylation of pRb. Indeed, several investigators
were able to demonstrate hypophosphorylation of pRb after
Fe chelation [74,77,114].
Apart from the effects on pRb, the marked decrease in
cdk2 levels after incubation with Fe chelators [74] would
prevent the formation of the cyclin A – cdk2 complex that is
essential for G1/S progression (Fig. 2A) [115]. It has been
shown that DFO reduces cdk1 expression that is vital for the
G2/M transition [116]. Hence, the decrease in the levels of
cdk1 and cdk2 may explain why a G1/S arrest, and more
rarely, a G2/M arrest can be observed after exposure to
chelators [72,117]. Other cell cycle molecules that showed
decreased expression after exposure to Fe chelators include
cyclin A and B [74], which are required for cdk1 and cdk2
activity during S, G2 and M progression (Fig. 2A).
The observed decrease in the D-type cyclin family and
increase in cyclin E after Fe chelation [74] deserve
discussion. The difference in the response of these latter
cyclins to Fe chelators was not unexpected, as both
regulate different aspects of G1 progression (Fig. 2A)
[118]. After combination with the relevant cdks, cyclin E
is thought to act on pRb, but only after the D-type cyclins
[115,119,120]. In fact, cdk4– cyclin D complexes phos-
phorylate pRb, releasing E2F transcription factors that act
to increase cyclin E transcription (Fig. 2B) [115,119,120].
Kung et al. [122] have shown that inhibition of DNA
synthesis using the S phase inhibitor aphidicolin [121]
results in cyclin B accumulation. These authors propose
that inhibition of cell cycle progression can dissociate
normally coupled cell cycle events, which has implications
for cytotoxicity [121,122]. Similarly, the increase in cyclin
E protein levels may possibly reflect dysregulation of the
cell cycle [74]. Alternatively, and more likely, the Fe
chelators may inhibit progression through G1 at approx-
imately the G1/S transition [74], when cyclin E protein
levels are at their maximum and cyclin D levels have fallen
[119].
4.2. Effect of iron deprivation on cdk inhibitors
The effect of Fe chelators on cell cycle inhibitory
molecules has only been superficially studied. Previous
investigations in this field have focussed on the effects of
Fe chelators on the more well-characterised cdk inhibitors
(e.g. p16INK4A, p21WAF1/CIP1, p27KIP1) [73,74,80,81,123].
Investigations using neuroepithelioma, neuroblastoma, leu-
kemia and breast cancer cells demonstrated a large increase
in p21WAF1/CIP1 mRNA after Fe chelation, which was not
reciprocated at the protein level [73,74,80,81,123].
Studies with the proteasomal inhibitor, lactacystin, dem-
onstrated restoration of p21WAF1/CIP1 protein expression
after chelation using DFO in human myelocytic leukaemia
38 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46

Fig. 3. Schematic illustration of the stabilization of p53 after DNA damage or oncogene expression. DNA damage stabilizes p53 by the expression of ATM or
ATR phosphokinases. These enzymes phosphorylate p53 at serine-15 and -37 to prevent mdm-2 from sequestering p53 for exportation and degradation.
Alternatively, the detection of abnormal proliferation or expression of various oncogenes leads to expression of the alternative reading frame of the INK4A
locus (ARF) protein. This molecule directly binds to mdm-2 to inhibit the targeted degradation of p53. Once stabilized, p53 is subjected to phosphorylation,
acetylation and sumolyation to enhance or regulate its transactivational abilities. p53 can transactivate a variety of molecules involved in cellular differentiation,
apoptosis, DNA repair and cell cycle arrest (see Table 1).

(ML-1) cells [80]. In addition to this, following Fe chelation
and lactacystin treatment, p21WAF1/CIP1 was ubiquitinated
[81]. This suggested that after Fe chelation the levels of
p21WAF1/CIP1 could be controlled via ubiquitination for
proteasomal degradation [80,81]. However, the fact that
the p21WAF1/CIP1 mRNA level was markedly increased
while its protein level was reduced, is totally unexpected.
This observation was paradoxical, as it could be predicted
that an increase in p21WAF1/CIP1 protein levels may play an
important role in the G1/S arrest observed after exposure to
chelators.
In contrast to the studies described above [73,74,
80,81,123], incubation of HL-60 leukemia cells with DFO
and the differentiation agent, phorbol myristate acetate
(PMA), results in decreased p21WAF1/CIP1 mRNA levels
relative to PMA alone [124]. The different effect of DFO
on p21WAF1/CIP1 expression in this cell line may relate to the
use of PMA that acts to induce protein kinase C-h activity
[125]. In fact, it has been shown that the transcription of
protein kinase C-h is inhibited by Fe chelators in HL-60
cells and can be restored using an Fe source [126]. Hence,
the results in PMA-treated cells could represent a unique
effect of Fe-deprivation on p21WAF1/CIP1 expression that
cannot be extrapolated to other cell types not treated with
this agent.
4.3. Effect of iron deprivation on p53 and its downstream
targets
Several studies have examined the effects of Fe chelation
on p53 and its downstream targets p21CIP1/WAF1 and
GADD45 [73,74,76,79,82,123]. Data from these studies
showed increased p53 protein expression after chelation
[74,76,79,123], although p53-DNA-binding activity was
not directly assessed.
In a cell-free system, p53 appears to be subject to redox
regulation [127]. Oxidants or metal chelators disrupt wild-
type p53 conformation and decrease its DNA-binding
activity in lysates [128 –131]. In marked contrast, using
whole cell systems, it has been shown that the Fe chelator,
1,10-phenanthroline, can induce p53 transactivational activ-
ity as well as sequence-specific DNA-binding in a dose-
dependent manner [131]. In addition, this chelator induced
the expression of several known p53-target genes including
p21WAF1/CIP1 and mdm-2, but not Bax, GADD45 or the
proliferating cell nuclear antigen (PCNA) [131]. Surpris-
ingly, the increase in p53 activity induced by 1,10-phenan-
throline was not due to elevated p53 mRNA or protein
levels [131]. Presumably, the increase in p53 activity in
these latter experiments may be due to an increase in the
conversion of latent p53 to its active DNA-binding form.
 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46 39

Recent studies have suggested that several mechanisms may
be involved in increasing p53-transcriptional activity after
Fe chelation (see Sections 4.3.1 –4.3.3 below).
4.3.1. Phosphorylation of p53 at serine-15 may prevent
mdm-2-mediated degradation after iron chelation: the
effects of chelators at increasing p53 levels is different
from that found after DNA damage
Ashcroft et al. [123] have demonstrated that p53 was
phosphorylated at serine-15 but not serine-20 after incuba-
tion of DFO with normal MRC-5 fibroblasts and neoplastic
cells (RKO colon cancer cells, MCF7 breast cancer cells and
U2OS osteosarcoma cells). These results suggested that Fe-
chelator-induced stabilization of p53 may be related to the
up-regulation of the ATM and/or ATR activity, both of
which phosphorylate p53 at serine-15 (Fig. 4) [104]. Con-
sidering this, later studies showed that DFO activated the
ATR-signalling pathway that phosphorylates p53 at serine-
15 and -37 [132].
Interestingly, it has been shown that the DNA-damaging
agents, actinomycin D (ActD) and camptothecin (Campt),
have different effects to DFO on p53 phosphorylation [123].
The Campt treatment induced phosphorylation of p53 at
serine-15 and -20, while no phosphorylation was detected
with ActD [123]. Since ActD, Campt and DFO all increased
p53 protein levels, these studies suggested the agents act via
different mechanisms to stabilize the protein. Indeed, there
are at least two mechanisms for disrupting mdm-2 function,
namely phosphorylation of p53 to prevent mdm-2 binding
[133,134] and increased ARF expression [108]. The results
of Ashcroft et al. [123] suggest that DFO may stabilize p53
by this former mechanism.
Although treatment of normal and neoplastic cells with
DFO, ActD and Campt caused a robust increase in p53
protein levels, only ActD and Campt resulted in an increase
in the mRNA and protein levels of p21WAF1/CIP1 [123].
Further studies examining other molecules transactivated by
p53 showed that DFO only induced mdm-2 in fibroblasts.
This result was in contrast to ActD, which markedly
induced mdm-2 protein levels in normal and tumour cells
[123]. Since p53 transcriptional activity depends on both
elevation of protein levels and a shift from latent to the
DNA-binding form of p53 [135], these data suggest that
DFO increased the expression of latent p53 [100].
4.3.2. Iron chelation increases the levels of hypoxia-
inducible factor-a (HIF-1a) that stabilizes p53
It has been shown that hypoxia, cobalt chloride or
chelation of Fe from cells by DFO resulted in the rapid
accumulation of HIF-1a that then stabilized p53 [82] (Figs.
4 and 5). The mechanism of stabilization of p53 by HIF-1a
involves the direct interaction of these two molecules [82].
Increased p53 stability may then initiate G1/S arrest and
apoptosis via downstream effectors [136].
HIF is a heterodimer composed of a and h subunits that
plays a central role in oxygen homeostasis (for review see
Ref. [137]). Its transcriptional targets include genes with
critical roles in erythropoiesis, energy metabolism, vaso-

Fig. 4. Schematic illustration demonstrating the effect of Fe chelation on the induction of p53-dependent and -independent pathways and their failure to result
in an increase in p21WAF1/CIP1 protein levels. Iron chelation can result in elevated p53 protein levels via the hypoxia inducible factor-1a (HIF-1a) that binds to
p53 to stabilize it. In addition to this mechanism, Fe chelation may induce the phosphorylation of p53 at serine-15 and -37. This prevents association with
mdm-2 that sequesters p53 for proteasomal degradation. Furthermore, Fe depletion can inhibit ribonucleotide reductase (RR) activity that results in reduced
deoxyribonucleotide (dNTP) levels that may act to increase p53 transcriptional activity. The increase in p53 results in transactivation of its downstream target,
p21WAF1/CIP1. The increase in p21WAF1/CIP1 mRNA does not result in an increase in its protein levels, perhaps due to increased proteasomal degradation. The
nature of the p53-independent pathway involved in increasing p21WAF1/CIP1 expression remains unknown. Possible transactivating candidate molecules that can
bind to the p21WAF1/CIP1 promoter include AP-2, Sp1, Sp3, BRACA1 and E2F1 (see text for details).
40 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46

motor function and angiogenesis [137]. Under conditions of
hypoxia, HIF-1a binds to the aryl hydrocarbon receptor
nuclear translocator (ARNT; also known as HIF-1h) to
activate expression of genes important in cell survival.
Alternatively, HIF-1a can bind to p53 and promote p53-
dependent activities, e.g. apoptosis [138]. Whether HIF-1a
could be involved in either function may be controlled by its
phosphorylation state [138]. Phosphorylation of HIF-1a
results in the binding to ARNT, while the dephosphorylated
form of HIF-1a becomes bound to p53 and mediates
apoptosis [138].
Recently, the mechanism of how HIF-1a becomes
stabilized under hypoxia or Fe-depletion has become clear.
Both Ivan et al. [4] and Jaakkola et al. [5] have charac-
terized the activity of an Fe-dependent HIF-a prolyl
hydroxylase (PH) that hydroxylates HIF-1a at proline-
564 (Fig. 5). This then mediates the ubiquitination of
HIF-1a for proteasomal degradation. When cells are Fe-
replete and under normal oxygen tension, the hydroxyla-
tion of proline-564 of HIF-1a alters its conformation to
allow binding to the von Hippel –Lindau protein (pVHL)
[4,5]. Subsequently, the pVHL molecule organizes the
assembly of a complex that activates the ubiquitin E3
ligase. This enzyme then ubiquitinates the bound HIF-1a
to target it for proteasomal degradation [4,5]. Thus, Fe
depletion or hypoxia prevents hydroxylation of proline-564
to stabilize the HIF-1a protein that subsequently leads to
increased p53 levels (Fig. 5). This important discovery
indicates that Fe is critical for both the hypoxic response
[4,5] and cell cycle control.
The stabilization of p53 following HIF-1a accumulation
may also be a response to limit the transactivational activity
of HIF-1a [139] (Fig. 5). However, it is unclear whether this
repression of HIF-1a transactivational activity is relevant
after Fe deprivation. This is because the downstream targets
of HIF-1a such as vascular endothelial growth factor-1
(VEGF1) and erythropoietin (EPO) increase following Fe
chelation [140,141] (Fig. 5).
4.3.3. Can iron chelation increase p53 transcriptional
activity by depletion of deoxyribonucleotide pools?
Apart from the mechanisms described above, decreased
dNTP levels are known to increase the transcriptional
activity of p53 (Fig. 4). In fact, previous studies have
suggested that p53 can act as a metabolic sensor [99]. When
nucleotide pools are disturbed by a variety of inhibitors, this
results in a reversible Go/G1 cell cycle arrest associated with
induction of p53 and p21WAF1/CIP1 [99]. Since Fe chelators
decrease RR activity and subsequently dNTP concentrations
[53,142,143], this may be important in terms of the signal-
ling involved in increasing p53 levels (Fig. 4). It is interest-
ing to note that the phosphorylation state of p53 is the same
after Fe chelation as found in cells treated with the potent
RR inhibitor, HU, and hypoxia [132]. This could indicate
that the p53-signalling pathways induced due dNTP deple-
tion, hypoxia and Fe deprivation may be similar.

Fig. 5. Schematic illustration of the downstream effects of the transcription factors, HIF-1a and PLAGL2, following Fe chelation. Cellular Fe depletion
prevents hydroxylation of HIF-1a by an Fe-dependent enzyme called HIF-a prolyl-hydroxylase. Hydroxylation of HIF-1a by this protein occurs in Fe-replete
and oxygenated cells. This results in binding to the pVHL for ubiquitination and proteasomal degradation. The inhibition of HIF-a prolyl hydroxylase by Fe-
chelation allows the stabilization and accumulation of HIF-1a. Once expressed, HIF-1a can initiate the transcription of genes involved in neovascularization,
oxygen transport and Fe uptake, namely, VEGF1, EPO and TfR1, respectively. Iron depletion can also increase TfR1 expression via the IRP – IRE mechanism.
Up-regulation of HIF-1a expression following Fe chelation stabilizes p53 (see Section 1.1). The expression of p53 may then allow transactivation of various
genes involved in cellular arrest, DNA repair or apoptosis (see Table 1). The p53 protein may inhibit the transcriptional role of HIF-1a. Iron chelation can also
induce the expression of the transcription factor, PLAGL2, which then transactivates the pro-apoptotic protein Nip3 (see text for details).
 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46
4.4. Iron deprivation induces a p53-independent increase in
p21WAF1/CIP1 and GADD45 mRNA levels
Recently, growing evidence has indicated that Fe chela-
tion can mediate a p53-independent response to cause cell
cycle arrest and apoptosis in cells lacking functional p53
[53,73,74,76]. The p53-independent responses induced by
Fe-depletion resulted in the up-regulation of GADD45 and
p21WAF1/CIP1 mRNA levels in SK-N-MC neuroepithelioma
and K562 erythroleukemia cells that have mutant p53
[73,74] (Fig. 4). Surprisingly, despite a relatively large
increase in mRNA levels there was no change in both
GADD45 or p21WAF1/CIP1 protein expression [74] (Fig. 4).
The transcription factors responsible for activating the
p53-independent increase in p21WAF1/CIP1 expression fol-
lowing Fe chelation [73,74] are unknown at present. How-
ever, numerous transcription factors such as specificity
protein (Sp) 1 and 3, activating protein 2 (AP2), breast
and ovarian cancer susceptibility gene product (BRACA1)
and E2F1, have DNA-binding sites within the p21WAF1/CIP1
promoter (for a detailed review, see Ref. [144]). It is already
known that the Sp1 transcription factor can induce p21WAF1/
CIP1
mRNA expression following exposure to okadaic acid
and PMA [145]. In contrast to Sp1 that has its own trans-
activation sequence within the p21WAF1/CIP1 promoter, p53
homologues like p73 can transactivate the p21WAF1/CIP1
gene using the p53 protein consensus sequence [146,147].
Contrary to the p21WAF1/CIP1 gene, GADD45 has an AP1
site, and AP1 is the only transcription factor-binding site
other than that of p53 which initiates its transcription [148].
At present, the mechanism(s) by which Fe chelation induces
cell cycle arrest in cells without wild-type p53 remains
unclear and warrants further investigation.
5. Iron-regulated expression of N-myc and c-myc proto-
oncogenes
5.1. N-myc
Apart from the effect of Fe on p53, the influence of
intracellular Fe levels on N-myc and c-myc expression have
also been investigated. The N-myc gene has some sequence
homology to c-myc, and is known to play an important role
in the pathogenesis of the aggressive childhood cancer,
neuroblastoma [149 – 151]. The amplification of N-myc
correlates with rapid NB progression [149 – 151]. Further-
more, it is worthy to note that secretion of high levels of
serum ferritin exist in NB patients with advanced disease,
and this is actually used as a prognostic indicator [151,152].
These facts are of interest, since transfection of cells with
copies of the related c-myc gene resulted in overexpression
of ferritin-H chain in the SW-613-S human carcinoma cell
type [153]. Hence, these observations could indicate a
possible role of N-myc in neuroblastoma cell Fe metabo-
lism.
41
Recent studies by Fan et al. [154] have added further
evidence to support a link between N-myc and Fe metab-
olism. These authors showed that Fe chelation using DFO
resulted in down-regulation of N-myc protein levels [154].
In contrast, the expression of other oncogenes such as c-fos
was increased, while housekeeping genes remained
unchanged [154]. Nuclear run-on experiments and transient
reporter gene expression studies suggested that the decrease
in N-myc expression occurred at the level of transcription
initiation and by inhibiting N-myc promoter activity [154].
5.2. c-myc
Studies using peripheral blood mononuclear cells have
shown that chelators decrease c-myc expression and this can
be prevented by the addition of Fe [155]. At the mRNA
level, c-myc expression in HL-60 cells has been shown to
increase and then subsequently decrease after Fe chelation
using DFO [156]. More recently, studies by Wu et al. [157]
have demonstrated that c-myc coordinately regulates genes
controlling intracellular Fe levels, namely the ferritin H-
subunit and IRP2. In fact, c-myc was shown to repress
ferritin-H chain expression and stimulate the transcription of
IRP2. Interestingly, the down-regulation of ferritin-H chain
expression appeared to be required for cell transformation
[157]. These changes in ferritin-H and IRP2 expression may
be necessary for maintaining intracellular Fe levels that are
required for critical molecules such as RR. However, it is of
interest that transfection of c-myc into other cell types
resulted in increased ferritin-H chain expression [153].
Collectively, these results suggest that c-myc plays a mod-
ulatory role in controlling the expression of ferritin-H.
In summary, these studies examining the roles of N-myc
and c-myc suggest some linkage between the metabolism of
Fe and the role of proto-oncogenes in controlling cellular
proliferation. However, these results are preliminary, and
require further investigation.
6. PLAGL2: an iron-regulated zinc finger protein is
involved in apoptosis
Furukawa et al. [158] have recently identified the pleo-
morphic adenoma gene like 2 (PLAGL2) after screening Fe-
deficient inducible cDNA from the mouse macrophage cell
line RAW264.7. This molecule contained seven C2H2 zinc
finger motifs and was induced when cells were incubated
under hypoxic conditions or with DFO [158]. Moreover,
PLAGL2 appeared to act as a transcription factor through
the HIF-1 responsive element [158,159]. Treatment of cells
with DFO induced apoptosis, nuclear accumulation of
PLAGL2 and an increase in the expression of the pro-
apoptotic factor Nip3 [159] (Fig. 5). While the Nip3
promoter contained an hypoxia-responsive element, the
transcription of this gene was activated independent of
HIF-1a. In addition, it was suggested that PLAGL2 acted
42 N.T.V. Le, D.R. Richardson / Biochimica et Biophysica Acta 1603 (2002) 31–46
as a tumour suppressor in association with HIF-1a [159].
Hence, in combination with the induction of p53 and its
possible role in cell cycle arrest and apoptosis, PLAGL2
represents a novel apoptotic pathway that is activated after
Fe chelation (Fig. 5).
7. Summary
The emergence of tumours that are resistant to conven-
tional therapies is of great concern. Since cancer cells have a
higher Fe requirement than their normal counterparts, they
are susceptible to the effects of Fe chelation. At present, the
exact molecular mechanisms that result in cell cycle arrest
after Fe chelation have only been superficially assessed.
However, the studies performed have revealed a glimpse of
the role of Fe in complex biological processes such as
oxygen sensing and DNA synthesis. Indeed, we now know
that the expression of critical molecules involved in cell
cycle progression and proliferation such as p53, HIF-1a,
p21WAF1/CIP1, N-myc and c-myc are regulated by intra-
cellular Fe levels. Further examination of the role that Fe
plays in cell cycle progression and proliferation may be vital
in terms of designing and developing Fe chelators as
effective anti-tumour agents.
Acknowledgements
The authors are grateful to Mr. David Lovejoy and Ms.
Juliana Kwok for helpful comments on the manuscript prior
to submission. D.R.R. thanks the National Health and
Medical Research Council and Australian Research Council
for grant and fellowship funding. The Heart Research
Institute is also thanked for financial support.
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