[CANCER RESEARCH 52, 4591-4599. September I, 1992]
Cell Cycle Synchronization and Growth Inhibition by 3-Hydroxypyridin-4-one Iron
Chelators in Leukemia Cell Lines *
Katharine P. Hoyes,2 Robert C. Hider, and John B. Porter3
Department of Haematology, University College and Middlesex School of Medicine ¡K.P. H., J. B. P.], and the Department of Pharmaceutical Chemistry, Kings
College [R. C. H.], London, United Kingdom
ABSTRACT
The effect of bidentate 3-hydroxypyridin-4-one
(HPO) iron chelators
on cell cycle arrest with subsequent cycle synchronization
has been
compared with that of the hexadentate desferrioxamine (DFO) in K562
and Daudi cells. The relationships between chelator concentration and
inhibition of growth, DNA synthesis and ribonucleotide reductase, and
phase of cell cycle arrest have also been explored. HPOs and DFO
arrest the cell cycle in a dose-dependent manner causing a blockade at
the Gi-S border after 24 h at concentrations above 30 MMiron-binding
equivalents. This is associated with reduced ribonucleotide reductase
activity and concomitant cessation of DNA synthesis and growth. When
the chelator is subsequently removed, HPO-treated cells synchronously
cascade into S phase, unlike DFO-treated cells which resume cycling in
a nonsynchronous manner. Chelator concentrations of approximately 25
«
M and 3 MMiron-binding equivalents inhibited growth, DNA synthesis,
and ribonucleotide reductase activity by 50% in K562 and Daudi cells,
respectively. Concentrations less than 10 MM iron-binding equivalents
inhibited K562 cell growth without an effect on DNA synthesis but with
accumulation of cells in <•_>
and M phases. These results suggest that
HPOs have advantages over DFO as cell cycle synchronization agents
and may be useful adjuncts in cell cycle-specific treatment regimens.
INTRODUCTION
Proliferating cells have an essential requirement for iron, and
deprivation of this element has profound effects on cell growth
and division (1-3). The withholding of iron from cells has been
proposed as a strategy for the treatment of neoplastic disease
(4). In particular, iron-chelating agents, such as DFO,4 have
been demonstrated to inhibit the proliferation of normal human
hemopoietic cells (5), a variety of malignant cell lines (6-8), and
human bone marrow neuroblastoma cells (9). Additionally
DFO has been shown to have antitumour activity in acute neo
natal leukemia (10). Furthermore, the microbial siderophore
parabactin has been shown to be a potent cell cycle synchroni
zation agent, and this has led to the suggestion that iron che
lators may be used to obtain a population of synchronized pro
liferating cells with enhanced sensitivity to cell cycle-specific
antitumour agents (11).
Received 5/17/91; accepted 6/18/92.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
'The work is supported by the National Institutes of Health Grant RO1-HL42800-01.
2 Present address: Department of Experimental Radiation Oncology, Paterson
Institute for Cancer Research, Wilmslow Road, Manchester M20 9BX, United
Kingdom.
3 To whom requests for reprints should be addressed, at Department of Hae
matology, University College and Middlesex School of Medicine, 98 Chenies
Mews. London WC1E 6HX, United Kingdom.
4 The abbreviations used are: DFO, desferrioxamine; HPO. 3-hydroxypyridin4-one; FO, ferrioxamine; CP94, l,2-diethyl-3-hydroxypyridin-4-one; UK, United
Kingdom; PBS, phosphate-buffered saline; IBE, iron-binding equivalents: ICso,
50% inhibitory concentration; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; DTT. dithiothreitol; CP20.L 1,1,2-dimethyl-3-hydroxypyridin-4-one;
CP24, l-methyl-2-buty!-3-hydroxypyridin-4-one; CP40, l-methyl-2-hydroxyethyl3-hydroxypyridin-4-one: CP93, l-ethyl-2-methyl-3-hydroxypyridin-4-one;
CP96,
l-ethyl-2-methoxyethyl-3-hydroxypyridin-4-one.
The HPOs are a series of iron chelators which exhibit high
specificity and selectivity for iron(III). These compounds have
rapid access across cell membranes due to their low molecular
weight and neutral charge in both the iron-free and iron-complexed forms (12). By contrast, DFO has a high molecular
weight, is relatively hydrophilic, and may consequently enter
cells more slowly. In addition, DFO and its iron-complexed
form FO bear a net positive charge which favors the accumu
lation of FO within the cell cytosol (13). As a result the HPOs
may distribute more freely within intracellular compartments,
conferring a greater potential to act as cell cycle synchroniza
tion agents than DFO.
There remains some debate over the exact mechanisms by
which iron chelators exert their antiproliferative effects. Incu
bation of cells with micromolar concentrations of DFO leads to
cessation of DNA synthesis (14) and a blockade in cell cycle at
the Gj-S border (5). Evidence suggests that competition with
ribonucleotide reductase for iron is the mechanism of action
(15). This enzyme is responsible for the free radical-mediated
reduction of ribonucleotides to deoxyribonucleotides, the ratelimiting step in DNA synthesis, having a constant requirement
for iron and oxygen for regeneration of the tyrosine free radical
essential for its activity (16). Although there has been convinc
ing demonstration of the effects of DFO on both ribonucleotide
reductase activity in vitro (14) and intracellular levels of deoxyribonucleoside triphosphates (15,17), direct in vivo effects on
ribonucleotide reductase have not been demonstrated with
DFO. Furthermore, DFO has also been demonstrated to inhibit
cellular proliferation independently of DNA synthesis in some
cellular models (18, 19), suggesting that other iron-dependent
cellular mechanisms may be involved (20).
If chelators are to be used as modulators of tumor-growth or
as adjunctive treatment with cell cycle-specific chemotherapeutic agents, then an understanding of their actions on cell pro
liferation and the mechanisms involved is necessary to maxi
mize such effects. In this paper we have therefore compared the
effects of one of the most active HPOs, CP94 (21, 22), with
DFO on reversible cell cycle arrest and subsequent cycle syn
chronization. We have further examined how the concentration
of the chelators determines their relative effects on the cell
cycle, cell proliferation, and ribonucleotide reductase activity.
MATERIALS
AND METHODS
Chemicals. The JV-alkyl-3-hydroxypyridin-4-one chelators were syn
thesized as previously described (23). Purity was confirmed by 'H
NMR, elemental analysis, and reverse-phase high-pressure liquid chromatography. Desferrioxamine was purchased from Ciba-Geigy (Basel,
Switzerland). The structure of CP94 and DFO is shown in Fig. 1.
RPMI 1640 medium and fetal calf serum were from Gibco (Paisley,
UK). All radiochemicals were from Amersham (Amersham, UK).
Aquasol was from New England Nuclear (Stevenage, UK). Affigel 601
was purchased from Bio-Rad (Hemel Hempstead, UK), and all other
chemicals were of analytical grade.
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CELL CYCLE SYNCHRONIZATION
BY HYDROXYPYRIDINONES
washed again, and RNA was digested with 0.5 mg/ml of RNase by
incubation at 37°Cfor 30 min. After further washing, the cellular DNA
content was quantified by staining with 50 /¿g/mlof propidium iodide.
Analysis was carried out using an EPICS CS flow cytometer (Coulter).
Control cells were used as a diploid standard to establish a consistent
location (channel number) of the G0-G] peak with the G2-M peak being
twice this number. The proportion of cells in each cell cycle phase
was obtained from planimatric analysis of the DNA histograms, assum
ing a Gaussian distribution of the GI and G2 + M peaks and attributing
the remaining area to cells in S phase as described by Barlogie et al.
(24).
The proportion of mitotic and polyploid forms was determined by
cytological analysis of cytospin cell preparations stained with hematoxylin.
Ribonucleotide ReducÃ-aseAssay. Ribonucleotide reducÃ-aseactivity
was assessed in intact cells by the method of Wright et al. (25). In brief,
'CH3CH,
O
(CH2)2
+NH/
NH
X(CH2),
N
HO
(CH2)2
NH
X(CH2)5
O
HO
CH,
N
N
D
HO
300 -
DFO
Fig. 1. The structure of CP94 and DFO.
CONTROL
200 -
In some experiments the iron chelators were saturated to 100% with
ferrous ammonium sulfate to produce FO and CP94-Fe, the iron-complexed forms of DFO and CP94, respectively.
Cell Culture. The human K562 erythroleukemia and Daudi Burkitt's lymphoma cell lines were cultured at 37°Cin RPMI 1640 medium
with 5% (v/v) heat-inactivated fetal calf serum under CO2:air (1:19).
Cells were kept at densities between 0.3 x lO-Vml and 0.6 x lOVml to
ensure that exponential growth was maintained.
Effect of Iron Chelators on Cell Proliferation. Desferrioxamine and
3-hydroxypyridin-4-one iron chelators were added to cells in exponen
tial growth from a 1Ox-concentrated stock solution in PBS. The same
volume of PBS was added to control cultures. The concentrations of
chelators added to the culture medium are expressed as IBE for each
chelator. The hexadentate chelator, DFO. coordinates iron in a 1:1
ratio, whereas the bidentate HPOs coordinate in a 3:1 ratio. Hence, 3
mol of hydroxypyridin-4-one bind the equivalent iron to 1 mol of DFO.
Growth curves were constructed by incubating cells with the chelators for up to 96 h and sampling at 24-h intervals to measure cell
number and viability. Cell number was determined using an electronic
particle counter (Coulter STK-R), and viability was assessed by fluo
rescence microscopy using ethidium bromide and acridine orange via
bility stain. Ethidium bromide selectively enters dead cells which then
fluoresce an orange red color, while acridine orange is taken up by
viable cells which appear a light green color under UV light.
The IC50 was defined as the concentration of chelator necessary' to
inhibit cell growth by 50% at 72 h when compared with control cells.
Clonogenic Assay. K562 or Daudi cells were incubated in the pres
ence or absence of 100 MMIBE CP94 or DFO for 24 h. The cells were
then washed twice with fresh chelator-free medium and resuspended to
4 cells/ml. Aliquots (100 M!)were plated in triplicate 96-well microtiter
plates to give a final concentration of 0.4 cells/well, as described by
Bergeron and Ingeno (11). The plates were incubated at 37°Cunder
CU2:air (1:19). Aft .- 7 days of incubation the plates were examined
with a stereo microscope. Colonies of more than 50 cells/well were
scored as having been cloned from a single cell.
Incorporation of Radioisotopes. Following treatment with the che
lators, 200 M!of cell suspensions were plated in a 96-well microtiter
plate and pulsed with 1 nC\ of [3H]thymidine (23 Ci/mmol), [3H]uridine
(17.8 Ci/mmol), or [3H]leucine (142 Ci/mmol) for l h to assess cellular
DNA, RNA, and protein synthesis, respectively. Cells were harvested
onto glass fiber filters with an Automash 2000 (Dynatech). Incorpora
tion of -'H label was measured by liquid scintillation counting after
addition of Aquasol scintillation fluid.
Flow Cytometry. Cells were stained for DNA cell cycle analysis by
flow cytometry as described previously (6). Briefly, cells were washed
with PBS and fixed in ice-cold 70% ethanol for 1 h. Then cells were
100 -
20
40
60
80
100
300 -
O
120
CP94
200 -
er
LU
O 100 -
20
300 -
40
60
80
100
120
DFO
A
200 -
100 -
20
40
DNA
60
80
100
120
CONTENT
Fig. 2. Flow cytometric profiles of K562 cells stained with propidium iodide
after 24-h exposure to 100 MMIBE CP94 or DFO.
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CELL CYCLE SYNCHRONIZATION BY HYDROXYPYR1DINONES
Table 1 Effects ofCP94 and DFO on K562 cell cycle kinetics
K562 cells were incubated with DFO, CP94, or their preformed iron-saturated
complexes, ferrioxamine (FO) and CP94-Fe, respectively, in the concentrations
shown for 24 h. Cell cycle status was assessed by flow cytometry after staining
with propidium iodide. Results are expressed as the percentage of cells in each
phase of the cell cycle.
phaseGO-GI47of cells in
Chelator concentration
IBE)ControlDFO31033100CP9431033100FO,
(MM
1°46
±
320±
±349
455±
±2*61
2C45
±
323±
120±
±218
±221
146±
±223
554±
±322
±2"66±3C48 ±316+
1*20
±234
I"29±
124±
±316
±2*34
1*31
±
±320
±415
±2*31
330+
149±
±221
100CP94-Fe,
100%
±3S24
±1G2-M26
±3
" Mean ±SEM of five independent experiments in triplicate.
"P< 0.05 by Student's / test.
CP< 0.001 by Student's t test.
prior to addition to the cell cultures, totally abrogated the in
hibitory effects of the chelators on cell cycle.
In K562 cells treated for 24 h with chelator concentrations
below 10 MMIBE, there was a significant accumulation of cells
in the G2-M phase. In separate experiments, cytospins were
prepared from cells which had been exposed to low concentra
tions of DFO or CP94 for 24 h. Cytological examination after
staining with hematoxylin revealed a higher proportion of both
polynuclear and mitotic cells in the chelator-treated popula
tions than in the control population (Table 3).
Cell Cycle Synchronization. K562 and Daudi cells in expo
nential growth were cultured with 100 MMor 33 MMIBE chelator, respectively. After 24 h the cells were washed and recultured in chelator-free medium. At 4-h intervals cell aliquots
were taken and fixed in preparation for cell cycle analysis. Fig.
3 shows the percentage of K562 and Daudi cells in each phase
of the cell cycle for the 24 h following reculture as compared to
controls. The blockade of cell cycle by both compounds was
reversible and, on reculture, cells recommenced cycling after a
lag phase of approximately 4 to 8 h relative to controls. CP94treated cells cascaded back into cycle in a synchronized manner
and remained synchronized throughout the remainder of the
cells were washed and resuspended at 107/ml in permeabilization buffer
Table 2 Effects of DFO and CP94 on Daudi cell cycle kinetics
containing 1% Tween 80, 0.25 M sucrose, 50 min HEPES, and 2 ITIM
Daudi cells were incubated with CP94, DFO, or their performed ion complexes
DTT. Cells were incubated at 22°Cfor 30 min, centrifuged, and resus
for 24 h. Cell cycle status was assessed by flow cytometry after staining with
pended in fresh permeabilization buffer at 2.5 x 107/ml. Aliquots cor
propidium iodide. The results are expressed as for Table 5.
responding to 5 x IO6 cells were added to assay tubes containing reac
phaseGO-GI48of cells in
Chelator concentration
tion buffer to give a final volume of 300 n\ containing 0.167 Msucrose,
IBE)ControlDFO31033100CP9431033100FO,
(MM
50 mM HEPES (pH 7.2), 2 ITIMATP, 8 HIMMgCl2, 6 mvi DTT, 0.67%
±3"57
Tween 80, and 0.05 ¿¡Ci
of [14C]CDP (545 mCi/mmol). The assays were
±222
222±
incubated at 37°Cin a shaking waterbath for 25 min, and the reaction
was terminated by boiling. Nucleotides were converted to nucleosides
by treatment with 2 mg/tube of Crotalus atrox venom, dissolved in 0.1
M HEPES (pH 8.0) and 10 HIMMgCl2, for 2 h at 37°C.Again this
reaction was terminated by boiling. Following the addition of 0.5 ml of
distilled water to each tube, heat-precipitated material was removed by
centrifugation, and the supernatant was passed down an 8- x 150-mm
Affigel 601 column to separate deoxycytidine from cytidine. The deoxycytidine was eluted from the column with 0.1 MHEPES (pH 8.0), and
4 ml of Aquasol liquid scintillation fluid were added. Radioactivity was
determined with an LKB liquid scintillation spectrophotometer.
Statistical Analysis. The Student / test (nonpaired) was used to iden
tify significant differences between sample means. The IC50 concentra
tions were calculated by extrapolating from a least-squares linear re
gression line relating chelator concentration to the percentage of
growth inhibition for each experiment.
RESULTS
Effect of Chelators on Cell Cycle Kinetics. Fig. 2 shows typ
ical DNA histograms obtained by flow cytometry of K562 cells
cultured for 24 h in the presence or absence of 100 MMIBE
CP94 or DFO. In control cultures approximately 50% of cells
were in the G, phase, with the remainder equally distributed
through S and G2 + M phases; however, the chelator-treated
cell populations exhibited a block in late-G! phase with a con
comitant decrease in the proportion of cells in the S and G2 +
M phases. The blockade of cell cycle was dose dependent and
plateaued at concentrations equivalent to 100 MM(Table 1).
Daudi cells were markedly more sensitive to chelator-induced
cell cycle arrest than were K562 cells, with accumulation of cells
in the G, phase at concentrations as low as 3 MMIBE and
maximal arrest at 33 MMIBE (Table 2). Saturation of the che
lators with equimolar amounts of ferrous ammonium sulfate,
±457
±264
1*65±2C54
±
±222
122±
±222
±221
±355
126±
±366±2C65±2C48
±223
123±
±225
±222
113±
2*12±2C24
±
120±
±212±
1"13±
Ie23
±249
100CP94-Fe,
±223
±322
100%
±3S25
±2G2-M22 ±3
" Mean ±SEM of 3 separate experiments in triplicate.
* P < 0.05 by Student's t test.
c P < 0.001.
Table 3 Effect of chelators at low concentrations on the percentage of mitotic
and polynuclear forms
K562 cells (3 x 105/ml) were incubated for 24 h with CP94 or DFO. The
proportion of mitotic and polynuclear cells was assessed by Cytological examina
tion after staining with hematoxylin.
Chelator
concentration
IBE)ControlDFO
(MM
3
10CP94
mitotic
cells3.92
0.3"5.79
±
±0.2*
0.4C8.07
7.58
±
polynuclear
cells0.25
of cells in
phase26.10
G2-M
±0.11.73
±2.129.04
±0.1'1.28
±0.7
30.07
1.2C30.13
±
±0.1*1.57
±0.3*
+ 0.1'
3
1.12 + 0.2''%
8.83 ±0.7r%of
10%of
" Mean ±SEM of 2 independent experiments in duplicate.
* P < 0.001 by Student's t test.
'•
P 0.05 by Student's ( test.
±0.1C
27.64 ±0.6
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CELL CYCLE SYNCHRONIZATION
a)
BY HYDROXYPYRIDINONES
b)
70
60 -
50
40-
20
16
20
24
12
16
20
24
70
16
O
4
8
12
16
TIME AFTER RECULTURE
20
24
20
24
(hours)
4
8
TIME AFTER
12
16
RECULTURE
20
24
(hours)
Fig. 3. The recovery of K562 (a) and Daudi (b) cells from cell cycle arrest by CP94 or DFO. K562 cells (a) or Daudi cells (b) (3 x 105/ml) were incubated with 100
MMIBE (K562) or 33 MMIBE (Daudi) CP94 (•),DFO (D), or PBS for controls (x) for 24 h, washed, and recultured in chelator-free medium. Aliquots were taken at
4-h intervals for cell cycle analysis. The values shown are the means of duplicate observations from a representative experiment. Three additional experiments gave
similar results.
cycle. In contrast cells which had been incubated with DFO
cell cultures which had been preincubated for 24 h with 100 /UM
reentered the cycle in a nonsynchronized manner, and cell cycle IBE DFO or CP94. No washing step was included. While the
kinetics was the same as control cells by 12 h.
addition of iron had no effects on the cycling of control cells,
chelator-treated cells recommenced cycling after a lag period of
A less disruptive method of achieving cell cycle synchroniza
tion may be to add iron directly to chelator-treated cultures to 4 to 6 h. As observed previously, CP94-treated cells recom
overcome the chelator-induced cellular iron deprivation. This
menced cycling in a synchronous manner; however, no cycle
synchronization was observed in DFO-treated cells (data not
hypothesis was tested by adding iron, in the form of ferric
ammonium citrate, to a final concentration of 110 MMto K562
shown).
4594
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CELL CYCLE SYNCHRONIZATION
In order to assess whether synchronization is a unique prop
erty of CP94 or common to all the HPO series, we screened five
other HPO chelators of varying lipid solubility for their ability
to synchronize the cell cycle. The compounds tested were
CP20,L1, CP24, CP40, CP93, and CP96. We found all of these
compounds to be potent cell cycle synchronization agents. Fur
thermore, there was no correlation between lipophilicity and
the ability to arrest or synchronize the cell cycle (Fig. 4).
Effect of Chelators on Cell Viability. When K562 and Daudi
cells were treated for 24 h with CP94 or DFO , there was no
significant loss of cell viability at any chelator concentration
(Table 4). These data were confirmed by cloning assays in which
the number of colonies cloned from a single K.562 cell after
24-h treatment with 100 MMIBE CP94 and DFO was found to
be 93 ±3% and 91 ±1% of control values, respectively. Sim
ilarly, the number of colonies formed in Daudi cell clonogenic
assays after 24-h treatment with CP94 or DFO, respectively,
was 92 ±3% and 89 ±2% of controls. However, unlike K562
BY HYDROXYPYR1DINONES
Table 4 Effect of DFO and CP94 on the viability of K562 and Daudi cells
Cells were cultured in the presence of increasing concentrations of DFO and
CP94. At the times indicated, viability was assessed by fluorescence microscopy
with ethidium bromide and acridine orange.
% of viable cells
Chelator concentration
IBE)ControlDFO3103350CP943103350Daudi24
(UM
h97
h96
±4"95
±294
±291
±290
±392
194±
188±
±483
±2"68
Ie94±
h95
h96
194±
193±
±293
195±
±294
±395
±292
±290
1*79
±
Ie93±
192±
185
±
±494
3<'80±4C67
±
±291
192±
±291
194±
±IeK56224 ±272
±372
" Mean ±SEM for three independent experiments.
* P < 0.001 by Student's / test.
c P < 0.05 by Student's t test.
±389
384±
Ie81±
±4C
a)DFO
0.0
24
48
TIME
72
(hours)
1.4 -i
9G
TIME
(HOURS)
Fig. 5. Growth curves for K562 cells. K562 cells (1 x lOVml) were cultured
with increasing concentrations of DFO (a) or CP94 (A) for up to 96 h. At 24-h
intervals, cells were counted with an electronic particle counter. The results are
the mean of 2 separate experiments in triplicate.
O
4
8
12
16
TIME AFTER RECULTURE (hours)
Fig. 4. K562 cell cycle synchronization by 3-hydroxypyridin-4-ones. K562 cells
(3 X 105/ml) were incubated for 24 h with 100 UMIBE CP20 (•),CP24 (*), CP40
(O), CP93 (C) or CP96 (+). The cells were then washed in chelator-free medium,
and aliquots were taken at 4-h intervals. The values shown are the means of
duplicate observations from a representative experiment. Two other experiments
gave similar results.
colonies, the number of cells in the chelator-treated Daudi col
onies was consistently less than controls.
After 96-h incubation with the chelators, K562 cell viability
was maintained at above 90% at concentrations up to 10 MM
IBE (Table 4). Above this concentration cultures contained 15
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CELL CYCLE SYNCHRONIZATION
1.6-
a) DFO
those shown in Fig. la, both CP94 and DFO caused a dosedependent reduction in pHjthymidine uptake by K562 and
Daudi cells (Fig. Ib). While incorporation of [3H]thymidine by
control K562 cultures was 6462 cpm/105 cells, concentrations
of 33 MMIBE CP94 or DFO caused a reduction in [3H]thymidine uptake of more than 90%. Lower doses caused more mod
erate decreases in uptake and, at concentrations of 10 MMor
less, there was no apparent reduction in cellular pHjthymidine
incorporation despite the inhibition of cell growth observed at
these concentrations.
Incorporation of pHJthymidine by Daudi cells was reduced
by more than 90% from 5442 cpm/105 cells in control popula
tions to less than 500 cpm/105 cells in cells treated with CP94
CONTROL
14 1.2ce
m
CD
1.0080.61 OjiM
LU
u
0.4 •¿o
30nM
0.200
24
48
TIME
72
or DFO at concentrations above 6 MMIBE; at lower doses
pHjthymidine incorporation was reduced to a lesser extent.
In parallel experiments to those in Fig. 7, treatment of K562
and Daudi cells with concentrations of CP94 or DFO sufficient
to inhibit [3H]thymidine incorporation by more than 90% had
no significant effect on RNA or protein synthesis as measured
by uptake of [-'HJuridine and pHJIeucine, respectively (Table 5).
Ribonucleotide ReducÃ-aseActivity. The activity of ribonucleotide reducÃ-ase was determined in K562 cells after 24-h
treatment with a range of concentrations of CP94 or DFO. As
shown in Fig. 8, there was a dose-related reduction in enzyme
activity with both chelators. Enzyme activity was decreased by
20% from control values of 222,982 ±14,254 cpm/106 cells at
concentrations of 10 MMIBE with both CP94 and DFO and by
more than 90% to 19,874 ±3,567 cpm/106 cells at concentra
tions of 100 MMIBE. Quantification of DNA synthesis, as mea
sured by cellular incorporation of [3H]thymidine, in the same
experiments revealed no reduction in thymidine uptake at 10
MMIBE, but uptake was reduced by more than 90% at 33 MM
IBE (data not shown).
9G
(hours)
b)CP94
CONTROL
ce
LU
en
O
24
48
TIME
BY HYDROXYPYRIDINONES
72
(hours)
Fig. 6. Growth cunes for Daudi cells. Daudi cells (3 x lOVml) were cultured
with increasing concentrations of DFO (a) or CP94 (ft) for up to 96 h. The data
represent the mean of 2 separate experiments in triplicate.
DISCUSSION
Iron is required for a wide variety of cellular functions. It is
conceivable that individual cellular pathways may have varying
sensitivity to the actions of iron chelators, depending on the
availability of iron to chelation and the rate of turnover of the
iron-dependent enzymes involved. There has been interest in
the use of iron chelators as antineoplastic agents (8, 10) because
of their well-documented effects on cell growth (26) and DNA
synthesis (14). However, the effects on reversal of cell cycle
arrest and the subsequent potential for cell cycle synchroniza
tion have not been studied extensively. Furthermore, the effect
K562 and Daudi cells, respectively, the iron chelators inhibited
proliferation in a dose-dependent manner. At concentrations of of HPOs on the latter has not been described. In order to
10 MMIBE CP94 or DFO, the number of K562 cells at 96 h was optimize the use of iron chelators as putative anticancer agents,
reduced to 75 to 80% of control and, at 50 MMIBE, there were it is important to understand the mechanisms by which they
only 5 to 15% ofthat in control cultures (Fig. 5). Daudi cells affect various aspects of cell function and how different cell
were markedly more sensitive to growth inhibition by the che
types may vary in their sensitivity to such agents.
lators, and maximal growth inhibition was observed at 30 MM
The results of these experiments show that reversible cell
cycle arrest followed by cell cycle synchronization occurs with
(Fig. 6).
The percentage of inhibition of cell growth at each chelator
the bidentate HPO iron chelators at concentrations at which
concentration is summarized in Fig. la. From these data the cell viability is preserved and are achievable clinically. The
mean concentrations required to inhibit proliferation by 50% length of preincubation with iron chelators is critical. In the
(IC50) were determined to be 21.2 ±2.7 MMand 24.9 ±4.3 MM majority of experiments shown, cells were incubated with che
IBE for CP94 and DFO, respectively, in K562 cells and 3.1 ± lators for 24 h, roughly equivalent to one cell cycle in the cells
0.2 MMand 3.06 ±0.3 MMIBE, respectively, in Daudi cells.
studied, in order to produce cell cycle arrest in G, prior to
Effect of Chelators on DNA, RNA, and Protein Synthesis.
synchronous return into S phase following the removal of
DNA, RNA, and protein synthesis were indirectly quantified by HPOs (Fig. 3). When cells were incubated for longer periods,
measuring cellular incorporation of ['Hjthymidine, [3H]uri- cell death ensued with both HPOs and DFO (Table 4). Incuba
dine, and ['Hjleucine, respectively. In the same experiments as tion with chelators for periods significantly shorter than one
to 20% nonviable cells at 96 h. The Daudi cells remained more
than 90% viable at concentrations of 3 MMIBE; at higher con
centrations, viability was significantly reduced (Table 4).
Effect of Chelator Concentration on Cell Growth. K562 and
Daudi cells were cultured at starting concentrations of 0.1 to
0.3 x IO6 cells/ml for 96 h, and the effect of CP94 and DFO on
cell growth and viability was examined. Whereas cell number in
control cultures increased to 1.4 x 106/ml and 1.6 x 106/ml for
4596
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CELL CYCLE SYNCHRONIZATION
BY HYDROXYPYR1D1NONES
100 -
Fig. 7. Effect of CP94 and DFO on growth
(a) and [3H]thymidine (¿>)
incorporation in
K562 and Daudi cells. K562 (
) and
Daudi (
) cells were cultured in the
presence of increasing concentrations of CP94
(•)or DFO (D) where the concentration is in
iron-binding equivalents (¡BE). After 96 h,
cells
weremeasured
counted after
and athymidine
incorpora
tion was
1-h pulse with
1 /<''»
of pH]thymidine. Points, mean of three in
dependent experiments in triplicate and ex
pressed as a percentage of control values; bars,
SEM.
30
40
50
0
10
CHELATOR
CHELATOR
CONCENTRATION
20
30
CONCENTRATION
40
50
((iM IBE)
((,M IBE)
Table 5 Cellular incorporation of[3H]thymidine, [3H]uridine, and (3HJIeucine
by KS62 and Daudi cells after 72-h exposure to DFO or CP94
Cells were cultured for 72 h with 50 UMIBE CP94 or DFO and pulsed for the
final hour with 1 MCi of ¡3H]thymidine,['Hjuridine, or [3H]leucine per ID5 cells.
Furthermore, any iron free HPOs will diffuse from the cell
rapidly due to their neutral charge and low molecular weight,
unlike the positively charged DFO where egress across cell
membranes is predicted to be slower (13).
of|3H]Thymidine5853
(cpm)
Chelator concentration
The above hypothesis assumes that the observed cell cycle
IBE)K-562
(MM
arrest was due to inhibition of ribonucleotide reducÃ-ase.It has
been suggested that other mechanisms may be responsible for
±860"
±5
±225
Control
665 ±50*
the antiproliferative effects of iron chelators (20, 28). In this
3340 ±892
1228 ±197
50 MMDFO
28*4417
578 ±
3928
200393
±
1123
±273290
CP94Daudi
50 MM
paper we have shown that ribonucleotide reducÃ-aseis inhibited
in intact cells by 50% at 33 MMby both DFO and HPOs (Fig. 8).
±467
±18
±52
Control
As ihe aclivily of ribonucleolide reducÃ-aseis known lo vary with
455 ±91*
184647
603 ±
30 MMDFO
552 ±176
the cell cycle and in particular to be so low as to be virtually
13
±66*|3H]Uridine4788
±237|'H|Leucine935
377 ±120
30 MMCP94Uptake
undelectable in the G, phase (29), it is not possible lo say wilh
" Mean ±SEM of cpm from 3 independent experiments in triplicate.
* P < 0.001 by Student's t test.
certainty that the low ribonucleotide reducÃ-aseaclivily observed
after 24-h incubalion with chelators is directly due to inhibition
cell cycle resulted in lower proportions of cells arrested in GÌ by iron chelation or secondary lo cell cycle arresi by anolher
mechanism. However, we have shown lhal inhibilion of ribo
(data not shown).
nucleolide reducÃ-aseactivity occurs at a chelalor concenlralion
The finding that DFO does not synchronize cells so effec
(33 MMIBE in K562 cells) al which cell cycle kinetics (Table 1)
tively as the HPOs, even though cell cycle arrest is achieved
and viability (Table 4) are similar to those of conlrol cells, and
with equal effect, requires explanation. DFO is a hydrophilic
compound (Apart = 0.001) with a hexadentate structure and a il is probable lhal ribonucleolide reducÃ-aseinhibilion precedes
molecular weight of 657. By contrast, the bidentate HPO che- cell cycle arresi in the G! phase.
The actions of iron chelalors on ihe cell cycle also need lo be
lators are more lipophilic [the Kpart of CP94 = 0.84 (21)] and of
lower molecular weight (MT CP94 = 241). Consequently it is considered in ihe conlexl of Iheir effecl on cell growih and
viabilily, as well as on DNA, RNA, and prolein synlhesis. Il has
predicted that this group of compounds would have greater
access to intracellular iron pools than DFO. Furthermore a been observed previously lhat the concenlralion of DFO re
significant correlation between antiproliferative activity and li- quired to arrest the cell cycle is above thai required to slow cell
pophilicity has been demonstrated for a range of bacterial sid- growih significanlly (18). Our resulls confirm ihese observalions for DFO (Fig. 7a; Table 1). The HPOs are similar in Ihis
erophores (27). However, under the conditions of the synchro
nization experiment where initial incubation with chelators is respecl wilh significanl inhibilion of K562 cell growth occur
over a 24-h period, DFO is as equally capable of arresting the ring at 3 to 10 MMbul significanl cell cycle arresi in G! noi being
cell cycle as the HPOs. Presumably therefore, the 24-h period
observed below 33 MMin K562 cells.
Al ihe low concentrations of chelators (3 lo 10 MMIBE of
of incubation is sufficient for DFO to permeate the cell to cause
cell cycle arrest. However, because of their bidentate nature, the CP94 or DFO) where K562 cell growih is partially inhibiled
HPOs will tend to dissociate into 2:1, 1:1 complexes and free (Fig. la), a proportion of cells was noi arresled in Gt, as seen al
chelate at low concentrations more readily than DFO (12). higher chelalor concenlralions (Table 1), but in G2 + M phases
Following the removal of chelator, HPO iron complexes may (Table 3). This was associated with an increase in mitotic
dissociate, unlike DFO complexes which are relatively stable.
andpolynuclear forms (Table 3) without a fall in cell viability.
4597
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CELL CYCLE SYNCHRONIZATION
100 -
O
<
N
Z
LU
Z
g
40 -
H
CD
X
z
20 -
20
CHELATOR
40
60
CONCENTRATION
1 00
80
(|iM
IBE)
Fig. 8. Reduction of ribonucleotide reducÃ-aseactivity in K562 cells after 24-h
incubation with DFO or CP94. K562 cells (3 x lOVml) were incubated for 24 h
with increasing concentrations of CP94 (•)or DFO (D), where the concentration
is expressed in IBE. Ribonucleotide reducÃ-aseactivity was assessed in cell pellets
as described in the text. Results represent data from two independent experi
ments.
Additionally there was no significant inhibition of ribonucle
otide reducÃ-ase(Fig. 8) or [sH]thymidine (Fig. Ib) incorpora
tion into K562 cells at these low concentrations. A similar
observation has been made by Bomford et al. (18) who reported
that the concentration of DFO required to inhibit the K562 cell
cycle and DNA synthesis was significantly above that required
to slow cell growth. These results suggest that the effects on cell
polyploidy and arrest in G2 + M seen at low concentrations of
chelator are through a different mechanism to that of ribonu
cleotide reducÃ-aseinhibition. This could occur by iron depriva
tion from a variety of enzyme systems necessary for cell
function and growth. Clearly, however, protein synthesis is not
affected globally at these low concentrations (Table 5), indicat
ing that such inhibition would have to be specifically on irondependent molecules.
One such system may be the mitochondrial respiratory en
zymes (30). Included in this group are several enzymes contain
ing non-heme-bound iron in the form of iron-sulfur clusters. In
particular, the citric acid cycle enzyme aconitase has been im
plicated as a target enzyme in the inhibition of tumorcell pro
liferation by cytotoxic activated macrophages by selective iron
depletion (31) and may be a target for chelator action. This may
lead to functional incompetence of the citric acid cycle enzyme
system which could result in inhibition of cytokinesis causing
the observed arrest of the cell cycle in the G2 + M phase.
These observations have implications for the use of ironchelating agents as cell cycle synchronization agents prior to
BY HYDROXYPYRIDINONES
chemotherapy of neoplasia (32, 33). Bergeron and Ingeno (11)
have demonstrated that preincubation with the catecholate
chelator parabactin prior to addition of 1-^-o-arabinofuranosylcytosine or Adriamycin potentiated the cidal effects of these
compounds on LI210 cells. Furthermore, Estrov et al. (IO)
have demonstrated an apparent synergism between DFO and
l-/3-D-arabinofuranosylcytosine when used in the treatment of
neonatal acute leukemia. The finding that, unlike DFO, bidentate HPOs can synchronize the cell cycle after removal of the
chelator suggests that these compounds have a potential clinical
role as adjuncts to cell cycle-specific chemotherapy that is not
available to DFO. In addition, short-term exposure of cells (up
to 24 h) to the HPOs does not lead to a loss of viability (Table
4), suggesting that use of these compounds will not lead to any
significant damage to quiescent cells, while the selectivity of
phase-specific agents for cycling cells may be enhanced. For this
to be useful therapeutically, an effect should be demonstrable at
concentrations that are achievable in vivo without undue toxicity. The concentration necessary to achieve cell cycle synchro
nization may vary for different cell types. The Daudi cell line is
considerably more sensitive to the effects of iron deprivation
than the K562 cell line, as seen with the inhibition of the cell
cycle (Table 2), cell proliferation and thymidine incorporation
(Fig. 7), and cell cycle synchronization (Fig. 3b). This could be
due to differences in iron requirement, iron utilization, or transferrin receptor expression on different cell types. Such differ
ences could be exploited to enhance the treatment of particular
tumorcell types. The concentration at which cell cycle synchro
nization occurred in the Daudi cell line (33 MMIBE; equivalent
to a plasma concentration of 99 MM)was well within the plasma
levels of 132 MMthat have been achieved in clinical studies with
one of the HPOs following p.o. administration (34).
Further work is required to investigate how cells differ in
their relative sensitivities to cell cycle synchronization with bidentate HPOs. Additional studies on the use of these com
pounds with cell cycle-specific chemotherapeutic agents would
elucidate whether the hydroxypyridin-4-ones were significantly
more effective in this respect than other iron-chelating agents
such as DFO or parabactin.
ACKNOWLEDGMENTS
The authors would like to thank A. Pizzey and N. S. B. Thomas for
their help and advice with cell cycle analysis.
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4599
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Cell Cycle Synchronization and Growth Inhibition by
3-Hydroxypyridin-4-one Iron Chelators in Leukemia Cell Lines
Katharine P. Hoyes, Robert C. Hider and John B. Porter
Cancer Res 1992;52:4591-4599.
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