|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 781-792
The Potential of Iron Chelators of the Pyridoxal Isonicotinoyl
Hydrazone Class as Effective Antiproliferative Agents III: The Effect
of the Ligands on Molecular Targets Involved in Proliferation
By
G. Darnell and
D.R. Richardson
From the Department of Medicine, University of Queensland, Royal
Brisbane Hospital, Brisbane, Queensland, Australia.
 |
ABSTRACT |
We have identified specific iron (Fe) chelators of the pyridoxal
isonicotinoyl hydrazone (PIH) class that are far more effective ligands
than desferrioxamine (DFO; Richardson et al, Blood 86:4295, 1995; Richardson and Milnes, Blood 89:3025, 1997). In the
present study, we have compared the effect of DFO and one of the most active chelators (2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; 311) on molecular targets involved in proliferation. This was performed
to further understand the mechanisms involved in the antitumor activity
of Fe chelators. Ligand 311 was far more active than DFO at increasing
Fe release from SK-N-MC neuroepithelioma and BE-2 neuroblastoma cells
and preventing Fe uptake from transferrin. Like DFO, 311 increased the
RNA-binding activity of the iron-regulatory proteins (IRPs). However,
despite the far greater Fe chelation efficacy of 311 compared with DFO,
a similar increase in IRP-RNA binding activity occurred after 2 to 4 hours of incubation with either chelator, and the binding activity was
not inhibited by cycloheximide. These results suggest that,
irrespective of the Fe chelation efficacy of a ligand, an increase
IRP-RNA binding activity occurred via a time-dependent step that did
not require protein synthesis. Further studies examined the effect of
311 and DFO on the expression of p53-transactivated genes that are crucial for cell cycle control and DNA repair, namely WAF1,
GADD45, and mdm-2. Incubation of 3 different cell lines
with DFO or 311 caused a pronounced concentration- and time-dependent
increase in the expression of WAF1 and GADD45 mRNA, but not mdm-2 mRNA. In accordance with the distinct differences in Fe chelation efficacy and antiproliferative activity of DFO and 311, much higher
concentrations of DFO (150 µmol/L) than 311 (2.5 to 5 µmol/L) were
required to markedly increase GADD45 and WAF1 mRNA levels. The increase
in GADD45 and WAF1 mRNA expression was seen only after 20 hours of incubation with the chelators and was reversible after removal of the
ligands. In contrast to the chelators, the Fe(III) complexes of DFO and
311 had no effect on increasing GADD45 and WAF1 mRNA levels, suggesting
that Fe chelation was required. Finally, the increase in GADD45 and
WAF1 mRNAs appeared to occur by a p53-independent pathway in SK-N-MC
and K562 cells, because these cell lines lack functional p53. Our
results suggest that GADD45 and WAF1 may play important roles in the
cell cycle arrest observed after exposure to these chelators.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
A WIDE VARIETY OF studies have suggested
that cancer cell proliferation can be inhibited using a range of
specific iron (Fe) chelating agents.1-6 The ability of
these ligands to inhibit growth reflects the well-known importance of
Fe in a variety of crucial metabolic pathways, including DNA synthesis
and ATP production.6 The most well-characterized Fe
chelator in terms of its antiproliferative activity is desferrioxamine
(DFO; Fig 1), the drug used to treat Fe overload
disease. DFO has been shown to be effective in vitro at inhibiting the
growth of a number of neoplastic cell types, with the most well-studied
tumors being neuroblastoma7-10 and leukemia.11
These in vitro studies have encouraged limited clinical trials in which
DFO has shown promise both as a single agent12,13 or in
combination with other cytotoxic drugs.14
The antiproliferative effect of DFO prompted us to investigate the
antineoplastic activity of Fe chelators of the pyridoxal isonicotinoyl
hydrazone (PIH) class. These compounds have a high affinity and
specificity for Fe that is similar to that found for DFO and much
greater than that of EDTA.15,16 Moreover, PIH and some of
its analogues have been shown to possess marked Fe chelation efficacy
in a wide variety of biological systems both in vitro and in
vivo.17-20 Considering their high Fe chelation efficacy, in
recent studies we have screened the antineoplastic activity of 3 groups
of ligands based on PIH.21,22 From these investigations,
one of the most active Fe chelators identified was
2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311; Fig
1).21,22
At present, very little is known concerning the molecular mechanisms by
which Fe chelators inhibit cellular proliferation and the cell cycle.
It has been well documented that some Fe chelators can inhibit the
activity of ribonucleotide reductase, the critical enzyme involved in
the conversion of ribonucleotides to
deoxyribonucleotides.23,24 The reduction in
deoxyribonucleotide concentrations is thought to prevent DNA synthesis,
which then results in G1/S arrest.3,10,22,25,26 Apart from this, nothing is known concerning why some Fe chelators are
more effective antiproliferative agents than others.
In an initial attempt to further elucidate the molecular targets of Fe
chelators, we have compared the effect of DFO, which shows relatively
low antiproliferative efficacy, with that of 311.21,22 In
these studies, we have compared the Fe chelation efficacy of these
ligands and their effect on the RNA-binding activity of the
iron-regulatory proteins (IRPs). The IRPs are key regulators of
intracellular Fe metabolism and control the expression of a number of
proteins, including those that are involved in Fe uptake (the
transferrin receptor [TfR]) and Fe storage (ferritin-H and -L
subunits).27-29 We have also examined the effect of DFO and
311 on the expression of a number of genes that are thought to play key
roles in cellular proliferation and the regulation of the cell cycle.
These include the p53-responsive genes WAF1 (wild-type p53
activating fragment 1 gene), GADD-45 (growth arrest and DNA
damage gene), and human mdm-2 (murine double minute gene). WAF-1 is a potent universal inhibitor of cyclin-dependent
kinases30 and can induce a G1/S
arrest31-33 and possibly a G2/M
arrest.34 GADD-45 is induced upon DNA damage, can arrest
the cell cycle, and is also involved in DNA nucleotide excision
repair.33,35 On the other hand, p53-mediated
transactivation of mdm-2 results in a feedback control
mechanism of p53 activity.33,36,37 Interestingly, both the
expression of WAF1 and GADD45 can also be controlled by
p53-independent pathways.38,39
Our present studies clearly demonstrate that 311 rapidly chelates
intracellular Fe and prevents Fe uptake from transferrin (Tf) at very
low concentrations, being far more effective than DFO. Despite the far
greater Fe chelation efficacy of 311 compared with DFO, the increase in
IRP-RNA binding activity of the IRPs occurred by similar kinetics and
was not inhibited by cycloheximide. These latter data suggest that,
irrespective of Fe chelation efficacy, the increase in IRP-RNA binding
activity was a time-dependent event that did not require protein
synthesis. In agreement with the marked differences in Fe chelation
efficacy and antiproliferative activity of 311 compared with DFO, a
pronounced increase in WAF1 and GADD45 mRNA levels was observed at a
DFO concentration of 150 µmol/L, whereas 311 was effective at
concentrations as low as 2.5 to 5 µmol/L. The results suggest that
the increase in GADD45 and WAF1 mRNA expression may be important in the
cell cycle arrest observed after exposure to potent Fe chelators.
| |
MATERIALS AND METHODS |
Preparation of 311 and its iron(III) complex.
Chelator 311 was synthesized by Schiff base condensation between
2-hydroxy-1-naphthylaldehyde and isonicotinic acid hydrazide using
standard procedures.40 DFO was purchased from Ciba-Geigy Pharmaceutical Co (Summit, NJ). Chelator 311 and its Fe(III) complex ([Fe(311)2] (NO3)·5H2O) were
characterized by a combination of elemental analysis, infrared
spectroscopy, NMR spectroscopy, and x-ray
crystallography.41 The Fe complex of DFO (ferrioxamine) was
prepared as reported in previous studies.42 Chelator 311 was dissolved in dimethyl sulphoxide (DMSO) as a 10 mmol/L stock solution immediately before an experiment and then diluted in 10%
fetal calf serum (FCS) so that the final concentration of DMSO was
equal to or less than 0.5% (vol/vol).21 All incubations of
cells with the chelators were performed in 10% FCS.
Cell culture.
The human BE-2 neuroblastoma cell line was kindly provided by Dr Greg
Anderson (Queensland Institute for Medical Research, Herston, Brisbane,
Queensland). The SK-N-MC cell line (American Type Culture Collection
[ATCC], Rockville, MD) was originally classified as a neuroblastoma,
but it has been recently reclassified as a neuroepithelioma, a closely
related, primitive neuroectodermal malignancy. The human K562
erythroleukemia cell line was also from the ATCC. The SK-N-MC cell line
was grown in Eagle's modified minimum essential medium (MEM; GIBCO
BRL, Sydney, Australia) containing 10% FCS (Commonwealth Serum
Laboratories, Melbourne, Australia), 1% (vol/vol) nonessential amino
acids (GIBCO), 2 mmol/L L-glutamine (Sigma Chemical Co, St Louis, MO),
100 µg/mL of streptomycin (GIBCO), 100 U/mL penicillin (GIBCO), and
0.28 µg/mL of fungizone (Squibb Pharmaceuticals, Montréal,
Quebec, Canada). The BE-2 and K562 cell lines were grown in RPMI
(GIBCO) containing 10% FCS and the supplements described above for
MEM. Cells were grown in an incubator (Forma Scientific, Marietta,
OH) at 37°C in an humidified atmosphere of 5%
CO2/95% air and subcultured as described
previously.43 Cellular growth and viability were monitored
using phase-contrast microscopy and trypan blue staining.
Protein labeling.
Apotransferrin (Sigma Chemical Co) was prepared and labeled with
59Fe (as ferric chloride in 0.1 mol/L HCl; Dupont NEN,
Boston, MA) to produce
59Fe2-transferrin (59Fe-Tf) using
standard procedures.43
Metabolic labeling.
Labeling of cellular proteins with 3H-leucine (52 Ci/mmol;
Dupont NEN) was estimated after precipitation with trichloroacetic acid
(TCA). Briefly, after the required exposure period to cycloheximide, 3H-leucine (1 µCi/mL) was added and the cells were
incubated for 1 hour at 37°C. The petri dishes were then placed on
ice and washed four times with ice-cold Hanks' balanced salt solution
(BSS; GIBCO BRL), and the cells were detached from the plate using 1 mmol/L EDTA in Ca/Mg-free saline. The cells were then pelleted by
centrifugation, the supernatant was removed, and the pellet was frozen
at  70°C. After thawing the cells on ice, 1 mL of ice-cold
20% TCA was added and the solution was then vortexed and kept on ice
for 1 hour with periodic vortex mixing. This solution was then
centrifuged at 15,000 rpm for 15 minutes at 4°C and the supernatant
was removed. The pellet was then washed twice with 1 mL of ice-cold
10% TCA and dissolved in 0.5 mL of 1 mol/L NaOH. After transfering
this solution to scintillation tubes, 3 mL of scintillant was added and
the radioactivity was measured on a  -scintillation counter (LKB
Wallace, Turku, Finland).
Iron uptake and iron efflux experiments.
The effect of chelators on 59Fe uptake from
59Fe-Tf and 59Fe release from prelabeled cells
was studied using standard methods reported previously.21
IRP gel-retardation assay.
The gel-retardation assay was used to measure the interaction between
the IRPs and iron-responsive element (IRE) using
established techniques.44,45 Briefly, after incubation with
medium alone (control) or medium containing ferric ammonium citrate
(100 µg/mL; Aldrich Ltd., Sydney, Australia) or the chelators, 2 to 5 × 106 cells were washed with ice-cold
phosphate-buffered saline (PBS) and lysed at 4°C in ice-cold Munro
extraction buffer (10 mmol/L HEPES, pH 7.6, 3 mmol/L MgCl2,
40 mmol/L KCl, 5% glycerol, 1 mmol/L dithiothreitol, and 0.5% Nonidet
P-40). After lysis, the samples were then centrifuged at
10,000g for 3 minutes at 4°C to remove nuclei and the
supernatant was stored at 70°C. Frozen cytoplasmic extracts
were thawed on ice and then centrifuged at 15,000 rpm for 10 minutes at
4°C. The protein concentration of the supernatant was determined
using the Bio-Rad protein assay (Bio-Rad Ltd, Hercules, CA). Samples of cytoplasmic extracts were diluted to a
protein concentration of 100 µg/mL in Munro buffer without Nonidet
P-40, and 1-µg aliquots were analyzed for IRP by incubation with 0.1 ng (~1 × 105 cpm) of 32P-labeled pGL66
RNA transcript (pGL66 was kindly provided by Dr Elizabeth Leibold,
University of Utah, Salt Lake City, UT). The riboprobe was transcribed
in vitro from linearized plasmid templates with SP6 RNA polymerase in
the presence of  -32P UTP (Dupont, NEN) using the Promega
Riboprobe In Vitro Transcription Kit (Promega, Madison, WI). The probe
was subsequently purified on a 6% urea/polyacrylamide gel
electrophoresis (PAGE) gel. To form RNA-protein complexes,
cytoplasmic extracts containing 1 µg of protein were incubated for 10 minutes at room temperature with the 32P-labeled riboprobe.
Unprotected probe was degraded by incubation with 1 U of RNAse T1 for
10 minutes at room temperature. Heparin (Sigma) at a final
concentration of 5 mg/mL was then added and incubated with the extract
for another 10 minutes at room temperature to exclude nonspecific
binding. RNA-protein complexes were analyzed in 6% nondenaturing
polyacrylamide gels at 4°C. Gels were dried, covered in plastic
film, and exposed to Kodak XAR films (Eastman Kodak, Rochester,
NY) at 70°C with an intensifying screen.
Northern blot analysis.
Northern blot analysis was performed by isolating total RNA using the
Total RNA Isolation Reagent from Advanced Biotechnologies Ltd (Surrey,
UK). The RNA (15 µg) was heat-denatured at 90°C for 2 minutes in
RNA-loading buffer and then loaded onto a 1.2% agarose-formaldehyde gel. After electrophoresis, RNA was transferred to a nylon membrane (GeneScreen; New England Nuclear, Boston, MA) in 10× SSC using the capillary blotting method. The RNA was then cross-linked to the
membrane using a UV-crosslinker (UV Stratalinker 1800; Stratagene Ltd,
La Jolla, CA).
The membranes were hybridized with probes specific for the human TfR,
-actin, N-myc, WAF1, GADD-45, and mdm-2. The TfR probe consisted of
a 2.8-kb coding region from the human TfR cDNA cloned into pCD-TR1
(kindly supplied by Dr L.C. Kühn, Swiss Institute for
Experimental Cancer Research, Epalinges, Switzerland). The -actin
probe consisted of a 1.4-kb fragment from human -actin cDNA cloned
into pBluescript SK- (ATCC; catalogue no. 37997). The N-myc probe was a
1-kb fragment from human N-myc cDNA cloned into pBR322 (ATCC; catalogue
no. 41011). The WAF1 probe consisted of a 1-kb fragment from pSXV
(ATCC; catalogue no. 79928). The GADD45 probe consisted of a 760-bp
fragment from human GADD45 cDNA cloned into pHu145B2 (kindly supplied
by Dr Albert Fornace, National Cancer Institute, National Institutes of
Health, Bethesda, MD). The human mdm-2 probe was a 1.5-kb
fragment derived from the full-length human mdm-2 cDNA cloned into the
pGEM vector (kindly supplied by Dr Andreas Evdokiou, Royal Adelaide
Hospital, Adelaide, South Australia).
Hybridization of probes to the membranes and their subsequent washing
were performed as described by Mahmoudi and Lin46 using a
Hybaid Shake and Stack Hybridization oven (Hybaid Ltd, Middlesex, UK).
Membranes were then wrapped in plastic film and exposed to Kodak XAR
films at 70°C with an intensifying screen. Probes were
stripped from the nylon membrane by boiling in a solution containing 10 mmol/L Tris-HCl (pH 7.0), 1 mmol/L EDTA (pH 8.0), and 1% sodium
dodecyl sulfate (SDS) for 15 to 30 minutes, as described by the
membrane manufacturer. Densitometric data were collected with a Laser
Densitometer and analyzed by Kodak Biomax I Software (Kodak
Ltd, Rochester, NY).
| |
RESULTS |
The effect of DFO and 311 on iron release from the SK-N-MC and BE-2
cell lines.
The effect of DFO and 311 concentration on Fe mobilization from the
SK-N-MC and BE-2 cell lines were examined after a 3-hour (Fig 2A) and 24-hour (Fig 2B) labeling
period with 59Fe-Tf (0.75 µmol/L), followed by 3 hours of
reincubation in the presence and absence of the chelators (0.2 to 50 µmol/L). Chelator 311 was far more effective than DFO and showed
similar activity in both cell types markedly increasing
59Fe release (Fig 2A). Increasing the concentration of 311 from 2.5 µmol/L up to 50 µmol/L did not increase 59Fe
release (Fig 2A), suggesting that the chelator depleted the 59Fe pool that it had targeted. However, increasing the
concentration of DFO from 2.5 µmol/L up to 50 µmol/L resulted in
additional 59Fe release, but even at a DFO concentration of
50 µmol/L, the percentage of 59Fe released from SK-N-MC
and BE-2 cells (12% and 29%, respectively) was less than that
observed at a 311 concentration of 2.5 µmol/L (Fig 2A).
View larger version (20K):
[in this window]
[in a new window]
| Fig 2.
The effect of the concentration of DFO or 311 on
59Fe efflux from the SK-N-MC and BE-2 cell lines that have
been labeled with 59Fe-transferrin (0.75 µmol/L) for (A)
3 hours or (B) 24 hours and then reincubated in the presence of the
chelators (0.2 to 50 µmol/L) for 3 hours at 37°C. Results are
means of duplicate determinations in a typical experiment of two
experiments performed.
|
|
Comparing DFO and 311-mediated 59Fe release after a 3-hour
(Fig 2A) and 24-hour (Fig 2B) labeling time, it is obvious that, after
a 24-hour label, the percentage of cellular 59Fe released
was far less (Fig 2B). For example, even at a 311 concentration of 50 µmol/L, 59Fe efflux from SK-N-MC and BE-2 cells was equal
to 11% and 14%, respectively (Fig 2B). In contrast to the effect of
311, which showed similar activity in each cell type, DFO was far more
effective at increasing 59Fe release from BE-2 cells
compared with the SK-N-MC cell line after both a 3-hour (Fig 2A) and
24-hour (Fig 2B) labeling period.
The effect of DFO and 311 on iron uptake from Tf by the SK-N-MC and
BE-2 cell lines.
To determine the ability of DFO and 311 to inhibit 59Fe
uptake from 59Fe-Tf (0.75 µmol/L), cells were incubated
with the chelators (0.2 to 50 µmol/L) and 59Fe-Tf (0.75 µmol/L) for 3 hours (Fig 3A) or 24 hours
at 37°C (Fig 3B). Chelator 311 markedly decreased 59Fe
uptake from 59Fe-Tf in a similar way for both the SK-N-MC
and BE-2 cell lines after the 3-hour and 24-hour labeling times (Fig 3A
and B). In contrast, DFO was far less effective than 311 at preventing
59Fe uptake from 59Fe-Tf, especially in SK-N-MC
cells after both 3 and 24 hours of incubation (Fig 3A and B).
View larger version (22K):
[in this window]
[in a new window]
| Fig 3.
The effect of the concentration of DFO or 311 on iron
uptake from 59Fe-transferrin (0.75 µmol/L) by BE-2 and
SK-N-MC cells over (A) 3 hours and (B) 24 hours of incubation at
37°C. After this incubation, the cells were washed four times with
ice-cold BSS and incubated with pronase (1 mg/mL) at 30 minutes for
4°C.21,43 Results are means of duplicate determinations
in a typical experiment of two experiments performed.
|
|
The effect of DFO and 311 on the RNA-binding activity of the IRPs.
An important effect of intracellular Fe depletion using DFO is the
activation of RNA-binding activity of the IRPs. Although the effect of
DFO on the RNA-binding activity of this protein has been well
characterized,28 the effect of other Fe chelators such as
311 have not been examined. In initial experiments, we examined the
effect of range concentrations of DFO (0.5 to 150 µmol/L) and 311 (0.5 to 25 µmol/L) on IRP activation over 6 and 20 hours of
incubation using the SK-N-MC and BE-2 cell lines
(Fig 4). From Fig 4 it is obvious that one
major IRP-IRE band is present, because human IRP1-IRE and IRP2-IRE
complexes comigrate in nondenaturing polyacrylamide gels.47
The IRP-RNA binding activity after exposure to DFO or 311 was far less
pronounced after 6 hours compared with 20 hours of incubation in
SK-N-MC cells, whereas in the BE-2 cell line it was similar (Fig 4). In
the SK-N-MC cell line after 6 and 20 hours of incubation, and in the
BE-2 cell line after 20 hours of incubation, 311 was more effective on
a molar basis than DFO at increasing RNA-binding activity of the IRPs.
For example, after 20 hours of incubation of SK-N-MC cells with 1 µmol/L 311, IRP-RNA binding activity was very similar to that found
after incubation with 50 µmol/L DFO (Fig 4). These results agree with our studies showing marked differences in Fe chelation efficacy between
DFO and 311 (Figs 2 and 3). However, after 6 hours of incubation of
BE-2 cells with DFO or 311, there was no difference in their ability to
increase IRP-RNA binding activity, which was only slightly increased
over the control at all chelator concentrations (Fig 4).
View larger version (53K):
[in this window]
[in a new window]
| Fig 4.
The effect of the concentration of DFO (0.5 to 150 µmol/L) or 311 (0.5 to 25 µmol/L) on the RNA-binding activity of
the IRPs from the SK-N-MC and BE-2 cell lines after 6 and 20 hours of
incubation at 37°C. This result is a typical experiment from three
performed for each time point.
|
|
As expected, after 6 hours of incubation with the Fe donor ferric
ammonium citrate (FAC), there was a significant decrease in IRP-RNA
binding activity in both SK-N-MC and BE-2 cells compared with the
controls (Fig 4). In contrast, after 20 hours of incubation with FAC,
the decrease in IRP-RNA binding activity was not so obvious when
compared with the relevant control (Fig 4). This may be because the
IRP-RNA binding activity of the controls is far less after 20 hours
compared with 6 hours of incubation, suggesting that the control cells
are Fe-replete after the longer incubation.
Further studies examined the dependence of incubation time with 311 and
DFO on the RNA-binding activity of the IRPs (data not shown). Despite
marked differences in the Fe chelation efficacy between 311 and DFO
(Figs 2 and 3), activation of the IRPs was observed after 2 to 4 hours
of incubation with both these chelators in BE-2 and SK-N-MC cells (for
example, see Fig 6).
To determine if 311 (10 or 25 µmol/L) or DFO (50 or 150 µmol/L)
could remove Fe from the FeS cluster of IRP1, we examined the ability
of the chelators to increase IRP-RNA binding activity by incubating DFO
or 311 with lysates prepared from cells pretreated with FAC (100 µg/mL) or 311 (25 µmol/L) for 20 hours
(Fig 5). Using lysates prepared from cells
preincubated with FAC for 20 hours, the IRPs only weakly bind the
32P-labeled IRE compared with lysates derived from cells
that had been preincubated with 311 (25 µmol/L; Fig 5). These data
suggest that, as observed previously,28,29 after
preincubation of cells with FAC, much of the IRP1 present has an FeS
cluster. Incubation of these lysates for 30 minutes at 4°C with 311 or DFO had no effect at increasing the RNA-binding activity of the IRPs
(Fig 5), suggesting that neither chelator could directly remove Fe from
the Fe-S cluster of IRP1. Longer incubation periods with the chelators
(up to 20 hours) also did not increase IRP-RNA binding activity (data
not shown).
View larger version (30K):
[in this window]
[in a new window]
| Fig 5.
The effect on IRP-RNA-binding activity of incubating cell
lysates with either Munro buffer (control) or Munro buffer containing
either 311 (10 or 25 µmol/L) or DFO (50 or 150 µmol/L) for 30 minutes at 4°C. The lysates were derived from cells pretreated for
20 hours at 37°C with either FAC (100 µg/mL) or 311 (25 µmol/L). The result is a typical experiment from two experiments
performed.
|
|
To determine if protein synthesis was necessary to increase IRP-RNA
binding activity, we examined the ability of cycloheximide to inhibit
3H-leucine incorporation into protein and prevent the
increase in the RNA-binding activity of the IRPs during incubation with 311 in human SK-N-MC cells (Fig 6A and B).
Cycloheximide (40 µg/mL) markedly reduced the incorporation of
3H-leucine into protein after all incubation periods from 1 to 20 hours (Fig 6A) without having any effect on cellular viability. However, this agent had little to no appreciable effect on the increase
in RNA-binding activity of the IRPs in the presence of 311 in this cell
line (Fig 6B). These results suggest that protein synthesis was not
responsible for the increase in RNA-binding activity of the IRPs after
exposure to 311. Very similar results were also seen for the BE-2 cell
line, and cycloheximide also had little effect on IRP-RNA binding
activity during incubation of cells with DFO (data not shown).
View larger version (26K):
[in this window]
[in a new window]
| Fig 6.
The effect of the protein synthesis inhibitor,
cycloheximide (40 µg/mL), on (A) 3H-leucine incorporation
into protein over 1 to 20 hours and (B) on the RNA-binding activity of
the IRPs during 1 to 20 hours of incubation with control medium
(control) or 311 (25 µmol/L). The result in (A) is the mean ± SD of
5 replicates in a typical experiment from two experiments performed.
The result in (B) is a typical experiment from four experiments
performed.
|
|
Effect of DFO and 311 on the expression of genes involved in iron
metabolism, cell proliferation, and the cell cycle.
The TfR is necessary for the proliferation of most cells, because this
molecule is involved in the uptake of Fe from Tf.29 Although it is well known that TfR mRNA expression can be markedly upregulated by DFO, very little is understood concerning the effect of
311, which has antiproliferative activity. Hence, it was important to
understand the effect of 311 on TfR mRNA levels. After 20 hours of
incubation with increasing concentrations of DFO (0.5 to 150 µmol/L)
or 311 (0.5 to 25 µmol/L), TfR mRNA levels increased in a
concentration-dependent manner, with 311 being more effective than DFO
on a molar basis (Fig 7 I-B). In contrast,
incubation of cells with FAC (100 µg/mL) decreased TfR expression
compared with the control. The increase in TfR mRNA levels after
exposure to DFO and 311 was also time-dependent, with increased
expression being observed only after 2 hours of incubation with the
chelators (Fig 8B).
View larger version (28K):
[in this window]
[in a new window]
| Fig 7.
The effect of the concentration of DFO or 311 on mRNA
levels of the TfR, N-myc, WAF1, -actin, mdm-2, and GADD-45 in BE-2
neuroblastoma cells. (I-A) Ethidium bromide staining of the agarose
gel; (I-B) TfR; (I-C) N-myc; (I-D) WAF1; (I-E) -actin. (II-A)
Ethidium bromide staining of the agarose gel; (II-B) mdm-2; (II-C)
GADD45; (II-D) -actin. The result shown is a typical experiment from
three experiments performed.
|
|
View larger version (60K):
[in this window]
[in a new window]
| Fig 8.
The effect of incubation time (1 to 20 hours) with DFO
(150 µmol/L) or 311 (25 µmol/L) on mRNA levels of the TfR, N-myc,
WAF1, GADD45, mdm-2, and -actin in BE-2 neuroblastoma cells. (I-A)
Ethidium bromide staining of the agarose gel; (I-B) TfR; (I-C) N-myc;
(I-D) WAF1; (I-E) GADD45; (I-F) -actin. The result illustrated is a
typical experiment from three experiments performed.
|
|
Increased expression of the proto-oncogene N-myc has been
suggested to play a role in the malignant behavior of
neuroblastoma.48 In several reports using neuroblastoma
cells, treatment of a number of cell lines with DFO resulted in a
decrease in the expression of N-myc mRNA, and this was thought
to be due to a decrease in N-myc
transcription.49,50 However, in our experiments using the
BE-2 neuroblastoma cell line, neither DFO nor 311 had any effect on
N-myc mRNA levels (Figs 7 I-C and 8C).
Although it is well known that DFO, 311, and other Fe chelators can
cause cell cycle arrest,3,10,22,25 very little is understood concerning the mechanisms responsible for this activity. As
part of our initial investigation into the molecular events involved in
the antiproliferative effects of Fe chelators, we have examined the
effects of 311 and DFO concentration on WAF1, GADD45, and human mdm-2
mRNA levels. These latter genes play crucial roles in cell cycle
control and the repair of DNA damage.33 Interestingly, both
Fe chelators caused an obvious concentration-dependent increase in the
levels of WAF1 and GADD45 mRNA in BE-2 cells (Fig 7 I-D and II-C).
Examining WAF1 and GADD45 mRNA levels, it is important to note that DFO
only caused a marked increase in expression at a concentration of 150 µmol/L, whereas 311 increased the levels of these transcripts at
concentrations as low as 2.5 to 5 µmol/L (Fig 7 I-D and II-C).
The increase in the levels of both the WAF1 and GADD45 transcripts in
the presence of the chelators was time-dependent and only became
appreciable after 20 hours of incubation of BE-2 cells with DFO (150 µmol/L) or 311 (25 µmol/L; Fig 8D and E). In contrast to the clear
concentration- and time-dependent increase in WAF1 and GADD45
transcripts after exposure to DFO and 311, no such relationships were
obvious for mdm-2 mRNA levels (Fig 7 II-B and data not shown). The
elevation in the levels of WAF1 and GADD45 transcripts may be very
important in terms of explaining the arrest in the cell cycle, which is
known to occur after exposure to both DFO and 311.9,10,22
Exactly comparable results to those observed for the BE-2 cell line in
Figs 7 and 8 were also observed for the SK-N-MC and K562 cell lines
(data not shown).
Further studies were designed to determine if the increase in the
levels of WAF1 and GADD45 transcripts observed after 20 hours of
exposure to chelators could be reversed by removing DFO (150 µmol/L)
and 311 (25 µmol/L), extensively washing the cells (3 washes of the
monolayer followed by two 30-minute incubations in MEM), and then
reincubating them for 20 hours in the presence of medium containing 100 µg/mL of FAC as an Fe source (Fig 9). As
shown above, 20 hours of incubation with DFO or 311 caused a marked
increase in the levels of TfR, WAF1, and GADD45 mRNAs when compared
with the relevant controls (Fig 9). After removal of the chelators and
reincubation in the presence of FAC (100 µg/mL), there was a decrease
in the levels of WAF1, GADD45, and TfR mRNA transcripts in both the
BE-2 and SK-N-MC cell lines, whereas no such change was observed in the
level of mdm-2 mRNA (Fig 9). It is of interest to note that
reincubation of SK-N-MC cells in the presence of FAC caused a much
larger decrease in WAF1 and GADD45 mRNA levels than that found for the
BE-2 cell line (Fig 9).
View larger version (41K):
[in this window]
[in a new window]
| Fig 9.
The effect on mRNA levels of the TfR, mdm-2, WAF1,
GADD45, and -actin after exposure of SK-N-MC and BE-2 cells to DFO
(150 µmol/L) or 311 (25 µmol/L) for 20 hours, and also cells
exposed to these agents for 20 hours followed by reincubation for 20 hours in medium containing FAC (100 µg/mL). (A) Ethidium bromide
staining of the agarose gel; (B) TfR; (C) mdm-2; (D) WAF1; (E) GADD45;
(F) -actin. The result shown is a typical experiment from two
experiments performed.
|
|
To determine that the increase in WAF1 and GADD45 mRNA levels were due
to the ability of DFO and 311 to bind Fe, the Fe(III) complexes of
these ligands (DFO-Fe, 311-Fe) were prepared and then incubated with
SK-N-MC cells for 20 hours (Fig 10). In
contrast to DFO (150 µmol/L) and 311 (25 µmol/L) that increased the
levels of the TfR, GADD45, and WAF1 mRNAs when compared with the
controls, the Fe complexes of these ligands at the same concentration
had no appreciable effect (Fig 10). Very similar results were also seen
for the BE-2 cell line (data not shown).
View larger version (41K):
[in this window]
[in a new window]
| Fig 10.
The effect of DFO, 311, and their iron(III) complexes
(DFO-Fe or 311-Fe) on the levels of TfR, WAF1, GADD45, and -actin
mRNAs in SK-N-MC cells. (A) Ethidium bromide staining of the agarose
gel; (B) TfR; (C) WAF1; (D) GADD45; (E) -actin. The result shown is
a typical experiment from two experiments performed.
|
|
It is well known that DFO is an inhibitor of ribonucleotide reductase
activity, and our previous studies have demonstrated that 311 can
markedly inhibit 3H-thymidine incorporation, being far more
effective than DFO.22 Considering that both GADD45 and WAF1
can be transactivated by p53 and that the levels of p53 can be
increased by a decline in deoxyribonucleotide levels,51 we
examined the effect of the potent ribonucleotide reductase inhibitor,
hydroxyurea (HU), on TfR, mdm-2, WAF1, GADD45, and -actin mRNA
levels in BE-2 cells (Fig 11). As found
for 311 (25 µmol/L) and DFO (150 µmol/L), both WAF1 and GADD45 mRNA
levels were increased after exposure to HU, whereas little effect was
observed on the level of mdm-2 mRNA (after normalization to -actin;
Fig 11C). Interestingly, as found in a lymphoblastic leukemic cell
line,47 high concentrations of HU (50 and 150 µmol/L)
were found to increase TfR mRNA levels (Fig 11B).
View larger version (44K):
[in this window]
[in a new window]
| Fig 11.
The effect of the concentration of HU on mRNA levels of
the TfR, mdm-2, WAF1, GADD45, and -actin in BE-2 neuroblastoma
cells. (A) Ethidium bromide staining of the agarose gel; (B) TfR; (C)
mdm-2; (D) WAF1; (E) GADD45; (F) -actin. The result shown is a
typical experiment from two experiments performed.
|
|
| |
DISCUSSION |
Although treatment of cells with Fe chelators is known to inhibit
ribonucleotide reductase activity26 and result in a
G1/S block,3,10,22 very little is known about
the changes in gene expression that may play an important role in cell
cycle arrest. WAF1 is a universal inhibitor of cyclin-dependent kinases
and has been shown to be crucial in terms of mediating cell cycle arrest at G1/S.30-32 In addition, GADD45 is
induced upon DNA damage and can also arrest the cell
cycle.33,35 In the present study, we clearly demonstrate a
concentration-dependent increase in WAF1 and GADD45 mRNA expression
after exposure to both DFO and 311 (Figs 7 I-D and II-C and 9D and E),
which was reversible after these chelators were removed (Fig 9).
Importantly, there was a marked difference in the concentration of DFO
and 311 that were required to increase WAF1 and GADD45 mRNA levels (Fig
7 I-D and II-C). For example, the concentrations of DFO and 311 required to obtain a marked increase in WAF1 and GADD45 transcripts
were 150 µmol/L and 2.5 to 5 µmol/L, respectively. This variation
in activity corresponds to the differences in Fe chelation efficacy (Figs 2 and 3) and antiproliferative effects of these
chelators.21,22
The effect of the ligands at increasing GADD45 and WAF1 mRNA levels
were very similar in 3 different human cell lines, namely SK-N-MC
neuroepithelioma cells, BE-2 neuroblastoma cells, and K562
erythroleukemia cells. Previous studies have demonstrated that SK-N-MC
cells do not have functional p53, probably due to a deletion in the
gene,52,53 and it is unknown whether the BE-2 cell line has
wild-type p53. In addition, it has been demonstrated that the K562 cell
line does not express wild-type p53 at the mRNA or protein
level,54,55 whereas we have observed a marked increase in
GADD45 and WAF1 mRNA levels in K562 cells after exposure to 311 and DFO
(data not shown). Hence, at least for the SK-N-MC and K562 cell lines,
the increase in GADD45 and WAF1 mRNA levels after exposure to chelators
may be via a p53-independent pathway. Indeed, whereas it is well known
that p53 activates transcription of WAF1 and GADD45 by its binding to
the p53-binding site in their respective promoters, transcriptional
activation of these genes can also occur by a p53-independent
process.38,39,56,57 However, the mechanisms
involved in p53-independent regulation of WAF1 and GADD45 expression
are not well understood and may occur by multiple pathways, depending
on the stimulus.39
Complexation of DFO or 311 with Fe prevented the effect of these
chelators at increasing the expression of GADD45 and WAF1 mRNAs (Fig
10). These results suggest that the effect of 311 and DFO at increasing
the levels of these latter transcripts were due to their ability to
chelate Fe from cells. In addition, the Fe complexes of both 311 and
DFO did not inhibit cellular proliferation, in marked contrast to the
ligands.41 It can be speculated that the effect of these
chelators at increasing the levels of GADD45 and WAF1 mRNAs may be due
to intracellular Fe chelation, which inhibits ribonucleotide reductase
activity and decreases deoxyribonucleotide levels. Our previous studies
with 311 and DFO have shown that both chelators inhibited the
incorporation of 3H-thymidine into DNA, but 311 was far
more effective.22 It has been demonstrated that a decline
in the intracellular concentration of deoxyribonucleotides increases
p53 levels that can transactivate WAF1 and GADD45.51
However, whether a similar stimulus can increase WAF1 and GADD45 mRNA
levels by p53-independent pathways remains unclear. The fact that
treatment of cells with the potent ribonucleotide reductase inhibitor
HU also increased WAF1 and GADD45 mRNA levels (Fig 11) lends support to
our suggestion that inhibition of ribonucleotide reductase activity may
be a regulatory step. As a working hypothesis that will be tested in
future studies, we suggest that the ligands examined in the present
investigation may act by chelating intracellular Fe, which then results
in a decrease in ribonucleotide reductase activity. This decrease in
enzyme activity causes a decline in deoxyribonucleotide levels that
leads to an increase in the expression of WAF1 and GADD45 that act to
inhibit the cell cycle. Obviously, other mechanisms of regulation are
also possible, and further studies are essential to establish the
precise molecular processes involved.
The Fe chelation experiments performed in the present study directly
demonstrate the much greater efficacy of 311 compared with DFO in terms
of its ability to increase Fe release and inhibit Fe uptake from Tf
(Figs 2 and 3). Our studies have shown that both 311 and DFO are far
less effective at mobilizing 59Fe from cells that have been
labeled with 59Fe-Tf for 24 hours compared with 3 hours
(Fig 2A and B). Because a greater proportion of Fe is found in ferritin
after long labeling periods with Tf than short incubation
times,22 these results suggest that both DFO and 311 chelate a labile form of 59Fe. We have also found that
there was variation in the ability of DFO to chelate Fe from the
SK-N-MC and BE-2 cell lines, in contrast to 311, which was equally
efficient in both (Figs 2 and 3). This may be due to differences in the
ability of DFO or its Fe complex to permeate the cell membrane or may
suggest a difference in the Fe metabolism between these cell types.
Because chelator 311 was more effective than DFO in terms of its
ability to chelate Fe from BE-2 and especially SK-N-MC cells (see Figs
2 and 3), we compared the efficacy of each chelator at activating the
RNA-binding activity of the IRPs. Both DFO and 311 took 2 to 4 hours to
increase the RNA-binding activity of the IRPs (Fig 6 and data not
shown). These results suggest that, irrespective of Fe chelation
efficacy of the ligands, there was a time-dependent step that was
required before IRP-RNA binding activity can increase. The protein
synthesis inhibitor cycloheximide had little to no effect on the
increase in the IRP-RNA binding activity in the presence of 311, suggesting that de novo protein synthesis was not required to increase
RNA-binding activity. At present, the evidence regarding how an
increase in IRP-RNA binding activity occurs in the presence of DFO is
controversial, with several studies being performed in mouse
Ltk cells.45,58 Using cycloheximide as the protein synthesis inhibitor, one group of investigators suggested that protein synthesis was necessary to increase IRP-RNA binding activity in the presence of DFO.45 In contrast, Pantopolous et al,58 using the same cell line, have shown that, in the presence of DFO, cycloheximide did not prevent
activation of IRP-1 but completely blocked the appearance of IRP2-RNA
binding activity. These latter results have led to the suggestion that
the Fe-S cluster in IRP1 can be removed by some uncharacterized
mechanism to generate an active RNA-binding protein.28
Further studies are obviously essential to determine the process
responsible for the increase in IRP-RNA binding activity in the
presence of Fe chelators.
Neither DFO nor 311 when added to lysates from Fe-replete cells
increased the RNA-binding activity of the IRPs (Fig 5). This result
indicated that neither chelator acted directly on the cluster to remove
Fe and increase RNA-binding activity. Therefore, the activation of
RNA-binding activity of the IRPs by these chelators may be due to an
indirect effect caused by the depletion of Fe in a labile intracellular
pool. Indeed, we have shown that both 311 and DFO act on the same or a
similar labile pool of Fe that markedly decreases as the incubation
time with Tf increased (Fig 2A and B). Our present results using 311 and DFO are in agreement with previous investigations in which very
high concentrations of DFO (10 mmol/L) added to purified IRP1 only
resulted in a slight increase in RNA-binding activity.59
Moreover, the results concur with studies showing that the Fe-S cluster
of IRP1 is highly stable, requiring high concentrations of ferricyanide
and EDTA to remove it.60
In summary, we have demonstrated that 311 is a highly effective Fe
chelator compared with DFO. Despite the far greater Fe chelation
efficacy of 311 compared with DFO, the increase in IRP-RNA binding
activity in the presence of these chelators occurred by similar
kinetics and was not inhibited by cycloheximide. Our studies demonstrate that DFO and 311 markedly increase WAF1 and GADD45 mRNA
levels in a concentration- and time-dependent manner. However, only
very low concentrations of 311 (2.5 to 5 µmol/L) compared with DFO
(150 µmol/L) were required to induce this effect, in direct
accordance with the difference in the Fe chelation efficacy and
antiproliferative activity of these ligands. We suggest that, because
both WAF1 and GADD45 are critical in cell cycle arrest, the increased
expression of these molecules may play an important role in the
antiproliferative activity of these chelators.21,22
| |
ACKNOWLEDGMENT |
Dr Greg Anderson and Lex Cowley are thanked for their generous
assistance in setting up the IRP gel retardation assay.
| |
FOOTNOTES |
Submitted November 30, 1998; accepted March 16, 1999.
Supported by a project grant from the National Health and Medical
Research Council of Australia (Grant No. 970360), the Kathleen Cuningham Breast Cancer Research Foundation, and a Research
Fellowship/Senior Research Fellowship from the Department of Medicine,
University of Queensland (to D.R.R.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to D.R. Richardson, PhD, Department of
Medicine, Clinical Sciences Building Floor C, Royal Brisbane Hospital,
Brisbane, Australia 4029; e-mail:
D.Richardson{at}medicine.herston.uq.edu.au.
| |
REFERENCES |
1.
Hoffbrand AV, Ganeshaguru K, Hooton JWL, Tatersall MHN:
Effect of iron deficiency and desferrioxamine on DNA synthesis in human cells.
Br J Haematol
33:517, 1976[Medline]
[Order article via Infotrieve]
2.
Bergeron RJ, Cavanaugh PF Jr, Kline SJ, Hughes RG Jr, Elliot GT, Porter CW:
Antineoplastic and antiherpetic activity of spermidine catecholamide iron chelators.
Biochem Biophys Res Commun
121:848, 1984[Medline]
[Order article via Infotrieve]
3.
Hoyes KP, Hider RC, Porter JB:
Cell cycle synchronization and growth inhibition by 3-hydroxypyridin-4-one iron chelators in leukemic cell lines.
Cancer Res
52:4591, 1992[Abstract/Free Full Text]
4.
Seligman PA, Schleicher RB, Siriwardana G, Domenico J, Gelfand EW:
Effects of agents that inhibit cellular iron incorporation on bladder cell proliferation.
Blood
82:1608, 1993[Abstract/Free Full Text]
5.
Torti SV, Torti FM, Whitman SP, Brechbiel MW, Park G, Planap RP:
Tumor cell cytotoxicity of a novel metal chelator.
Blood
92:1384, 1998[Abstract/Free Full Text]
6.
Richardson DR:
Potential of iron chelators as effective anti-proliferative agents.
Can J Physiol Pharmacol
75:1164, 1997[Medline]
[Order article via Infotrieve]
7.
Becton DL, Bryles P:
Deferoxamine inhibition of human neuroblastoma viability and proliferation.
Cancer Res
48:7189, 1988
8.
Blatt J, Taylor SR, Stitely S:
Mechanism of antineuroblastoma activity of deferoxamine in vitro.
J Lab Clin Med
112:433, 1988 |
TOP
ABSTRACT
INTRODUCTION