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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3593-3603
H-Ferritin Subunit Overexpression in Erythroid Cells Reduces the
Oxidative Stress Response and Induces Multidrug Resistance Properties
By
Silvina Epsztejn,
Hava Glickstein,
Virginie Picard,
Itzchak N. Slotki,
William Breuer,
Carole Beaumont, and
Z. Ioav Cabantchik
From the Department of Biological Chemistry, Institute of Life
Sciences, Hebrew University of Jerusalem, Jerusalem, Isreal; Nephrology
Unit, Shaare Zedek Medical Center, Jerusalem, Israel;
Génétique et Pathologie Moleculaire de
l'Hématopoiese, Faculté Xavier Bichat, Paris, France.
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ABSTRACT |
The labile iron pool (LIP) of animal cells has been implicated in
cell iron regulation and as a key component of the oxidative-stress response. A major mechanism commonly implied in the downregulation of
LIP has been the induced expression of ferritin (FT), particularly the
heavy subunits (H-FT) that display ferroxidase activity. The effects of
H-FT on LIP and other physiological parameters were studied in murine
erythroleukemia (MEL) cells stably transfected with H-FT subunits.
Clones expressing different levels of H-FT displayed similar
concentrations of total cell iron (0.3 ± 0.1 mmol/L) and of
reduced/total glutathione. However, with increasing H-FT levels the
cells expressed lower levels of LIP and reactive oxygen species (ROS)
and ensuing cell death after iron loads and oxidative challenges. These
results provide direct experimental support for the alleged roles of
H-FT as a regulator of labile cell iron and as a possible attenuator of
the oxidative cell response. H-FT overexpression was of no apparent
consequence to the cellular proliferative capacity. However,
concomitant with the acquisition of iron and redox regulatory
capacities, the H-FT-transfectant cells commensurately acquired
multidrug resistance (MDR) properties. These properties were identified
as increased expression of MDR1 mRNA (by reverse transcription
polymerase chain reaction [RT-PCR]), P-glycoprotein (Western
immunoblotting), drug transport activity (verapamil-sensitive drug
efflux), and drug cytotoxicity associated with increased MDR1 or PgP.
Although enhanced MDR expression per se evoked no significant changes
in either LIP levels or ROS production, it might be essential for the
survival of H-FT transfectants, possibly by expediting the export of
cell-generated metabolites.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
A VARIETY OF PHYSIOLOGICAL and pathological conditions
are attributed to metabolically active forms of cell iron associated with the labile iron pool (LIP).1 Elevated levels of LIP
are commonly assumed to compromise cell integrity by metal-catalyzed formation of reactive oxygen species (ROS).1-5 Regulatory
mechanisms have therefore been invoked in maintaining LIP at relatively
low levels.6,7 The major one has been attributed to
iron-responsive proteins (IRP) that purportedly sense cytosolic LIP
levels and translationally control the expression of the major cell
iron supplier transferrin receptor (TfR) and the iron withdrawing
protein ferritin (FT).6,7 This apparently passive mode of
LIP controlling its own levels is allegedly complemented by an
"active" mode in which LIP is transcriptionally modulated,
primarily via expression of FT.8 Of the two FT subunits,
expression of the ferroxidase-carrying heavy subunits (H-FT) has been
shown to be the most responsive to various chemical challenges and
inducers.9,14 However, because only recently
has a method for assessing LIP become available,15-17 it
has not been possible to ascertain whether induced H-FT expression reflected rather than affected the cell LIP levels. That pertains equally to experimental and pathological conditions,18,19
including the proposed H-FT modulation of LIP induced by myc
oncogene.12,13
In this work we attempted to assess whether induced H-FT expression, by
downregulating LIP levels, can in fact confer upon cells a first line
of defense against chemically induced oxidative stress. Stable
transfectants expressing H-FT in a manner independent of labile cell
iron or IRP and at levels higher than untransfected cells were
difficult to obtain with a variety of cell lines. This was overcome
with murine erythroleukemia (MEL),20 which provided various
clones with demonstrably higher H-FT levels and a commensurate capacity
for withdrawing iron from the LIP, both under steady-state conditions
and after acute iron loads.20,21 Whereas the proliferative capacity of the clones remained unchanged, the capacity to withstand oxidative damage when challenged with pro-oxidants increased with H-FT
expression. However, with H-FT overexpression the cells concomitantly acquired multidrug resistance (MDR) properties. The latter were discovered in the clones during the assessment of LIP levels using the
hydrophobic precursor of the fluorescent metalosensor calcein, namely
the nonfluorescent acetomethoxy derivative calcein-AM. That
probe,22,23 as well as many other hydrophobic probes, has
been identified previously as a possible substrate of ATP-dependent MDR
pumps.24-26 The MDR character associated with H-FT
overexpression was reflected both functionally, as resistance to drugs
and verapamil-sensitive drug pumping activity, and structurally, as
increased MDR1a mRNA levels and membrane P-glycoprotein (PgP). Possible
linkages between H-FT, LIP, and MDR functions are explored and discussed.
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MATERIALS AND METHODS |
Materials.
Calcein, calcein-AM, and Rhodamine 123 were procured from Molecular
Probes (Eugene, OR). [3H]-thymidine was from the
Radiochemical Centre (Amersham Life Science Ltd, Little Chalfont,
UK). Iron nitrate (Spectrosol grade) standard was from BDH
Chemicals Ltd (Pode, Dorset, UK). Unless specified
otherwise, all other chemicals were from Sigma Chemical Co (St Louis,
MO) or best available grade. SIH (salicylaldehyde hydrazone) was a kind
gift of Prof Prem Ponka (McGill University, Montreal, Canada).
Cells.
The cell lines used in this study were of murine erythroid leukemia
(MEL) origin stably transfected with the mouse H-FT gene, as described
earlier.20,21 The properties of additional H-FT transfectants are described. All the MEL cells were grown in minimum essential medium, Earle's salts base, (MEM-Eagle)
supplemented with 10% fetal calf serum (FCS) (both from Biological
Industries, Beth Haemek, Israel). They were periodically reselected for
neomycin-resistance in the presence of G418 (800 µg/mL) and assayed
for mycoplasma by a kit procured from Biological Industries. The number
of cells were estimated in a Coulter Cell Counter (Coulter Electronics Ltd, Harpenden, Herts, UK). Cell volume was determined as described elsewhere.15 The human erythroleukemia K562 cells used were wild type and stable transfected with a retroviral construct pLMDR1L6 carrying the human MDR gene (kindly provided by Dr Igor Roninson, Chicago, IL).27 The cells were grown as described for MEL
cells except in Dulbecco-MEM medium (DMEM).
Cell proliferation.
Cells were plated in octaplicate in 96-well culture plates (Nunc,
Roskilde, Denmark) at a density of 25,000 cells/mL growth medium. The
plates were analyzed daily for cell count as described above and for
metabolic activity in the presence of Alamar Blue (5% final) (Almog
Diagnostic, Rishon Letzion, Israel)28 and reading the
fluorescence (after 4-hour incubation in culture conditions) in a BMG
Fluorostar plate reader (BMG, Offenburg, Germany) at 540 nm exc and 590 nm emi.
Immunodetection of proteins.
Aliquots of 1 to 2 × 106 cells washed with isotonic
buffer were extracted with 150 µL of Tris-HCl 10 mmol/L, NaCl 150 mmol/L, Triton X-100 0.5% (Boehringer Mannheim, Mannheim,
Germany), NaN3 0.25 pH 7.4, and a cocktail of
antiproteases (P-8340 Sigma, St Louis, MO) and phenylmethyl sulfonyl
fluoride (PMSF) 250 µmol/L. The extract was centrifuged at
8,500g for 5 minutes at 5°C and the supernate frozen at
 20°C after sampling for protein determination (BCA method;
Sigma). Samples of 30 to 40 µg protein were loaded per lane.
Ferritin (H and L), P-glycoprotein, and actin were detected on
immunoblots of samples run on Laemmli sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE: 6% for PgP and
12% for H and L ferritin and actin). The primary antibodies used were
monoclonal mouse antihuman PgP (C-219; Signet Laboratories Inc, Dedham,
MA), monoclonal antiactin (IgM) (Amersham), and rabbit antimouse H and
L ferritin (kindly provided by Dr Paolo Santambrogio, DIBIT, Milano,
Italy). The secondary antibodies were goat antimouse or goat antirabbit
gamma globulin conjugated to horseradish peroxidase (Jackson
Immunoresearch, West Grove, PA) and they were used in conjunction with
enhanced chemiluminescence (ECL) detection (Radiochemical Centre,
Amersham) and densitometry tracing.
Reverse transcription polymerase chain reaction (RT-PCR) of H-FT and
MDR1a (modified from Orly et al29).
RNA was isolated from exponentially growing MEL cells using Ultraspec
RNA isolation reagent (Biotec Laboratories, Houston, TX). Total RNA
(0.4 µg) was reverse transcribed (75 minutes, 42°C) using 500 ng
polydeoxy thymidine [(pd(T)]12-18 primers (Pharmacia, Uppsala, Sweden) and 0.25 U avian myeloblast virus (AMV)
reverse transcriptase (RT) (Promega) in a 0.02 mL reaction containing 1 × PCR buffer and 20 U RNAsin ribonuclease inhibitor (both from Promega). The RT reaction was terminated by heating for 5 minutes at
95°C and 0.08 mL of a mixture containing 1 × PCR buffer, 2.5 mmol/L MgCl2, and 1.5 U Taq DNA polymerase (TaqZol; Tal-Ron
Ltd, Rehovot, Israel) was added to the RT RNA. The appropriate
oligonucleotide primers were added last, and PCR was performed for up
to 30 cycles (Mastercycler; Eppendorf, Hamburg, Germany) using the
following temperatures: denaturing, 94°C (1 minute), annealing
59°C (2 minutes), extension, 72°C (3 minutes). Samples (0.01 mL) were removed from the PCR reactions after cycles 15 to 20 for H-FT,
and after cycles 25 to 30 for MDR and L7. The primers used were: Mouse
H-FT1 (access. # X12812): sense, 5'-GGAGTTGTATGCCTCCTAC-3';
antisense, 5'-GAGATATTCTGCCATGCC-3'); expected product 430 bp, spanning sequence 147-576. Mouse MDR1A (access. # M33581): sense,
5'-GGATCAGTGTTTCTAGAT-3'; antisense, 5'-CTGTATCCAGAGCTGATG-3'): expected product 352 bp,
spanning sequence 3398-3749. Mouse ribosomal L7 protein, which served
as the reference control (access. # M29016): sense,
5'-CAGATCTTCAATGGCACC-3'; antisense,
5'-GCAGATGATGCCAAACTTAC-3'); expected product 213 bp, spanning sequence 436-648. Tracking buffer containing Ficoll 15%, 100 mmol/L EDTA, 5 × TAE buffer, and Xylene Cyanol Blue dye, was added to the PCR products (1/5 volume), and the products were separated
on a 1.6% agarose gel containing 1 × TAE buffer. The gel was
stained for 30 minutes in SYBR-gold nucleic acid stain as per
manufacturer's instructions (Molecular Probes) and photographed under
ultraviolet (UV) illumination. The relative band intensities were
quantified by scanning-densitometry. The PCR cycle number that
generated product quantities representing the up-slope of the curve was
used for comparisons.
Cell iron.
Total cell iron was measured in triplicate samples of 5 × 106 cells suspended in 1 mL HBS buffer (150 mmol/L NaCl, 20 mmol/L HEPES). In method 1,30 the cells
were mixed with an equal volume of an acid mixture (3 N HCl, 10%
trichloroacetic acid, and 3% thioglycolic acid) and incubated for 2 hours at 37°C, cooled, centrifuged at 3,000 rpm for 30 minutes and
mixed with 0.5 mL batophenan troline sulconate (BPS)
(0.045% in 4.5 N Na-acetate, 0.2% thioglycolic acid), and the light
absorption of the pink solution was read at 535 nm in a Spectronic 3000 UV-VIS Photodiode Spectrophotometer (Milton Roy, Oostende, Belgium). In
method 2, 1 mL of the cell suspension was supplemented with 2 mL
concentrated nitric acid, incubated for 30 minutes at 37°C, and the
supernates used for assessing iron in a Zeeman Atomic Absorption
Spectrometer (Spectra AA-300; Varian Instruments, Victoria, Australia).
Calibrations in the range 5 to 20 ng Fe/mL were performed automatically
by the instrument.
Cell LIP.15
A suspension of 1 × 106 cells/mL in HBS was incubated
alone (control) or with ferrous ammonium sulfate (FAS) 20 µmol/L for 10 minutes at 37°C, washed, resuspended again in HBS, and
reincubated alone or with H2O2 (5 µmol/L for
20 minutes at 37°C). After washing and resuspension in DMEM medium
supplemented with 20 mmol/L HEPES and 1 mg/mL bovine serum albumin
(BSA), the cells were loaded with CA-AM (0.125 µmol/L) for 5 minutes
at 37°C, washed twice with medium, and processed for LIP
measurements as described in detail previously.15
Cell glutathione (GSH). Total levels.31
Samples of 3 × 106 cells were pelleted by
centrifugation and resuspended in 0.5 mL cold 0.6% sulfosalicylic acid
made up in distilled water containing 0.5 mmol/L Na-EDTA. After 1 hour
in ice, the cell extract was spun down at 14,000 rpm for 15 minutes at
4°C, the supernate transferred to an Eppendorf tube and a 50 µL
aliquot added to a cuvette containing 60 µg DTNB, 200 µg NADPH, and
1 unit of GSH reductase in 1 mL phosphate-buffered saline (PBS) at room
temperature. The reaction rate that is proportional to the
concentration of total cell GSH was monitored by absorption at 412 nm
in a Spectronic 3000 UV-VIS spectrophotometer.
GSH.32,33
Cell GSH was monitored with monochlorobimane (MCB). The reaction of MCB
with cell GSH is catalyzed by GSH-S-transferase leading to the in situ
quantitative formation of a fluorescence adduct that is retained within
the cells.33,34 Aliquots of the various cell clones (3 × 105 cells into 200 µL of HBS) were supplemented
with 20 µmol/L MCB (final concentration) and fluorescence was
assessed for approximately 1.5 hours at 37°C using a Fluorostar
Fluorescence plate reader (BMG, Offenburg, Germany) (Ex395 and Em 470 nm). The maximum fluorescence represented the amount of GSH per sample.
Cell ROS production.35
Method A. Samples of cells (1 × 106/mL) suspended in
DMEM (no phenol red) and supplemented with 20 mmol/L Na-HEPES and 1%
BSA were exposed to 10 µmol/L CDCF-DA
(2'-7'-carboxy-dichlorofluorescin diacetate) for 20 minutes
at 37°C. The reaction was stopped by rapid centrifugation and
resuspension in HBS with or without FAS 20 µmol/L for 10 minutes, at
37°C. The basal- and H2O2- (0 to 20 µmol/L) induced conversion of the nonfluorescent
2'-7'-carboxy-dichlorofluorescein to
2'-7'-carboxy-dichlorofluorescein was followed with time by analyzing the fluorescence of 10,000 living cells by
fluorescence-activated cell sorting (FACS) (Becton Dickinson FACS
caliber, Immunocytometry systems [BDIS]; Becton Dickinson, Mountain
View, CA). Values of median fluorescence intensity were
used. Experiments were run on triplicate samples.
Method B. Cells, as in A, were preincubated with or without FAS (20 µmol/L) for 10 minutes at 37°C, washed and subsequently exposed
to CDCF-DA in the FACS tubes submerged in a 37°C water bath. The
fluorescence intensity profiles were followed by FACS at 10 minute
intervals (while keeping the cells at 37°C). At 30 minutes, the
cells were supplemented with H2O2 (5 to 250 µmol/L).
ROS and cell death.
Samples of different clones (1 × 106 cells/mL)
suspended in HBS were incubated in the presence of different
concentrations of H2O2 (0 to 1 mmol/L) for 30 minutes at 37°C. The reaction was stopped by rapid centrifugation
and resuspension of the cells in growth medium. After 24 to 48 hours in
full culture medium conditions, the cells were centrifuged and
resuspended in PBS containing 2% FCS and propidium iodide (10 µg/mL
per 1 × 106 cells). Samples of cells were analyzed by
flow cytometry ( FACS) (Becton Dickinson) and the values of the median
fluorescence intensity were taken for assessing the percent of dead
cells in a given cell population (10,000 cells).36,37
MDR properties drug cytotoxicity.
Drug susceptibility to colchicine and vinblastine was
followed by incubating cells (50 to 100,000 cells/mL) in culture medium with the indicated concentrations of drug for 48 to 72 hours (all assays done in triplicate in either 24- or 96-well culture plates; Nunc). Cell viability was assessed by incubating the cells for an
additional 4- to 5-hour period in growth medium with the respective additive. For [3H]-thymidine (1 µCi/mL) incorporation
into nucleic acids,38 cell samples were harvested and
processed with a Matrix Cell Harvester (Packard Instruments, Meriden,
CT). For metabolic activity, cells exposed to 5% Alamar Blue were
analyzed as described above.
Drug transport (CA-AM and Rhodamine 123).
CA-AM (100 nmol/L) uptake and conversion into fluorescent CA were
followed on line and in parallel in four stirred, thermostated (37°C) cuvettes placed in a four-cell turret of a PTI fluorescence station (PTI, Offenburg, Germany). Fluorescence
measurements (485 nm excitation and 517 nm emission) were obtained
sequentially for each cuvette at 2-second intervals and
computer-recorded. Aliquots of 1 × 106 cells/mL were
suspended in DMEM medium (no phenol red) or PBS supplemented with 5 mmol/L glucose at 37°C and, at the indicated time (approximately 10 minutes), either verapamil (5 to 10 µmol/L) or cyclosporin (8 µmol/L) was added. Cell-associated calcein fluorescence was also
followed by FACS (fluorescein settings) by reading 10,000 cells at
different time intervals of exposure of cells to CA-AM (± verapamil
or cyclosporin). Rhodamine 123 uptake from a 5 µmol/L solution was
followed in a similar manner except that aliquots of cells were washed
with ice-cold PBS medium and the fluorescence of 10,000 cells estimated
by FACS at room temperature.
Transport of probes was also assessed by FACS. For that, 1 mL aliquots
containing 106 cells were rapidly centrifuged and
resuspended in the same volume of HBS containing 5 mmol/L glucose
either alone or with cyclosporin 10 µmol/L at 37°C. Aliquots of
100 µL were transferred into 900 µL of ice-cold HBS for zero
fluorescence readings. To the remaining suspension R123 or CA-AM was
added to final concentrations of 5 µmol/L or 125 nmol/L respectively.
At the indicated times, further 100 µL samples were transferred
rapidly into 900 µL of ice-cold HBS to stop probe uptake. For each
timed sample 10,000 cells were analyzed.
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RESULTS |
Basic properties of the H-FT-expressing clones.
In an earlier characterization of MEL cells transfected with the mouse
H-FT gene mutated in the iron-responsive element (IRE) we determined
that the steady-state LIP levels were reduced in a manner commensurate
with the levels of expressed H-FT protein.21 Concomitant
with the downmodulation of LIP there was an activation of
IRP20 and an increase in transferrin-receptor-mediated
iron uptake (Glickstein and Cabantchik, unpublished observations). In
this work we performed a more complete characterization of the clones
with the aim of assessing the functional consequences of increased H-FT
expression and reduction in LIP. These properties were initially
studied in four clones expressing various levels of H-FT and LIP and
cultured in equivalent conditions. We assessed the short-term and
long-term consequences of H-FT overexpression, with particular emphasis
on the capacity of the cells to respond to oxidative stress. The
various parameters associated with labile and total cell iron and the
reductive capacity of the cells in resting conditions are depicted in
Table 1. The total cell iron concentration
was determined on the basis of amount of acid-extracted cell iron
measured either by atomic absorption or colorimetrically in conjunction
with volume measurements of the cells. The latter was taken as the
small solute accessible space of the cell.15 Although the
LIP levels of the various clones grown in equivalent conditions were
significantly different, their total iron content was apparently
similar. Importantly, the steady-state LIP levels in all the clones
represented only a minor fraction (less than 1%) of the total cell
iron.
Under resting conditions, namely when unchallenged with pro-oxidants or
exogenously added inducers of ROS formation, the various clones
displayed similar levels of reductive power, as reflected in their GSH
and GSH/GSSG contents (Table 1). The total GSH (GSH + GSSG) levels
could be reproducibly determined in cell extracts by the DTNB-GSSG
Reductase Recycling Assay.31,32 However, determination of
either GSH or GSSG in cell extracts might give inconsistent results,
apparently because of partial oxidation of GSH during or after cell
disruption. Therefore, we determined the in situ GSH cell levels with
the fluorogenic reagent monochlorobimane (MCB), that reacts with cell
SH-containing substances in a manner that is specific and quantitative
for GSH, as it is catalyzed by GSH-S-transferase.33,34
To assess whether the forms of iron found in the LIP were in fact redox
active, we imposed on the cells a minor oxidative stress in the form of
H2O2 (0 to 20 µmol/L for 20 minutes) and followed several properties in control cells and cells acutely loaded
with Fe(II). The acute iron load was imposed to assess the alleged H-FT
capacity to prevent iron in the LIP from engaging in ROS formation. The
latter, as well as LIP, was measured during or immediately after the
chemical treatments. The long-term effects of the acute chemical
treatments were assessed in terms of cell death 24 to 48 hours later,
because the acute treatments did not lead to immediate cell death. ROS
levels were followed by FACS analysis of CDCF generated in cells via
oxidation of a permeant nonfluorescent precursor.35 We
ascertained that the loading of the precursor was not rate limiting for
the assay. Under the experimental conditions used, less than 1% of the
precursor was oxidized by the cells. The results depicted in Fig
1A show that ROS production was
significantly lower in H-FT-overexpressing clones (6 and 12) at all
concentrations of H2O2 used. This was reflected
in both the rates of CDCF oxidation and the levels of product attained.
However, the differences among the clones were more accentuated after
preloading the cells with Fe(II), as manifested primarily in the low
H-FT-expressing clone 16 and nontransfected wild type. This is
particularly evident in the contribution of the preloaded Fe(II) to the
ROS forming capacity of the various clones, as given in the legend to
Fig 1. This is also evident in the levels of basal ROS formation (no
H2O2 added), which was relatively lower in the
high H-FT-expressing clones. As a major fraction of the ROS formation
could be inhibited by pretreating cells with iron
chelators,1,3 it is implied that LIP plays a major role in
catalyzing ROS formation.
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| Fig 1.
In situ tracing of ROS formation in MEL cells. The
various MEL clones were treated with the indicated concentration of
H2O2 after loading with the nonfluorescent
carboxy-2',7'-di-Cl-fluorescein (CDCF) permeant analog
(CDCFDA). The latter is converted intracellularly into the fluorescent
analog by reacting with ROS in a metal-dependent fashion. Cells were
preincubated for 10 minutes with FAS (20 µmol/L) (A, bottom) or
buffered saline (B, top) and washed. Cell fluorescence was analyzed by
FACS at different times after addition of H2O2.
Data (n = 4 experiments run in triplicate samples) are given in terms
of mean rates of fluorescence change with time (AFU/min = arbitrary
fluorescence units/min) with SEM of less than 8% the indicated points
in the graph. The increment in the fluorescence intensity  attributable to FAS ( = A to B) (in AFU/min) was at 0, 10, and 20 µmol/L H2O2 respectively for wt: 0.31, 0.85, and 1.1; for cl-16: 0.45, 0.78, and 0.83; for cl-12: 0.18, 0.12, and
0.32; and for cl-6: 0.11, 0.10, and 0.10. ANOVA paired analysis of the
n = 4 experiments showed statistically significant differences
(P < .05) between data points at given
H2O2 concentrations when cl-6 or cl-12 were
compared with either cl-16 or wt but not when they were compared with
each other.
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The increased ROS production in various clones was also assessed in
terms of breakdown of the cell permeability barrier to the fluorogenic
propidium iodide, after cell exposure to a wide range of
H2O2 concentrations (Fig
2). When fluorescence is monitored by
flow-cell cytometry, it provides a measure for the number of damaged or
dead cells in a given cell population.36,37 No attempts were made in this study to distinguish between necrosis and apoptosis. In general, no apparent cell damage was detected immediately after the
chemical treatments were applied to cells. Cell damage or death was
detectable only 24 hours later and it was highly dependent on the
degree of the imposed oxidative stress, as given by the concentration
of H2O2. The technique was not sufficiently
sensitive for detecting significant changes after short cell treatments with  20 µmol/L H2O2. For stronger
treatments, cell damage was consistently lower the higher the
H-FT-expression capacity of the clone. Even with the highest
concentration of H2O2 used the apparent plateau
level attained was significantly lower in the H-FT-overexpressing
clones.
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| Fig 2.
ROS-induced cell death. The various MEL clones were
treated with H2O2 for 20 minutes at room
temperature and analyzed by FACS for viability (by propidium iodide) 48 hours later. Data are given in terms of units of fluorescence (= % dead cells) per total number of cells (live + dead) for one out of
four independent experiments. ANOVA paired analysis for n = 4 set of
data points at each H2O2 concentration were
significantly different at P < .01 for all data analyzed.
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Because changes in LIP were implicated in myc-induced H-FT expression
and cell growth,12,13 we assessed the possibility that H-FT
overexpressers might display restrictive growth capacity. As observed
in Fig 3, the growth rates of the various
clones displaying the largest differences in H-FT levels were not
significantly different than the untransfected (wild type) parent line.
The same result was obtained whether proliferation was measured in terms of cell number or cell metabolic activity.
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| Fig 3.
Growth rates of H-FT-overexpressing clones. The various
MEL clones (wt, cl-6 and cl-16) were seeded at the same density (25,000 cells/mL) and their growth rate followed daily for 4 days either by
cell count (top) or with Alamar Blue (4-hour development). Data are
given as mean of octaplicates ± SE.
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LIP, ROS, and MDR.
In the course of assessing LIP with calcein we consistently encountered
relatively lower fluorescence levels in H-FT-expressing clones, as
observed in Fig 4 (left). The fluorescent
calcein is intracellularly generated from its nonfluorescent
acetomethoxy precursor calcein-AM. The latter has been used previously
as a hydrophobic substrate of various drug pumps that are known to confer MDR properties upon cells.22,23 This fortuitous
finding led us to explore the possibility that H-FT-transfected cells might have also acquired an MDR phenotype, independent of the neo
resistance gene acquired, which differs from MDR. As depicted in Fig 4
(left), the time-dependent rise in cell fluorescence reflected both
CA-AM net uptake into cells (ingress and egress) and intracellular
de-esterification into the carboxylated (impermeant) fluorescent
calcein. Addition of either cyclosporin or verapamil, two well-known
blockers of the PgP pump and reversers of MDR,39,40 fully
normalized the uptake to levels found in the wild type or the low
H-FT-expressing clone 16 (Fig 4, left). These results led us to imply
the involvement of an MDR pump. The ratios between the rates of
fluorescence changes before and after addition of the MDR reversers
were in the range of 3 to 10 for high H-FT expressers as compared with
1 to 2 for low expressers. The wide range of changes in ratio values
between experiments was apparently associated with the age or metabolic
state of the culture. However, the reversal was consistently three- to
fourfold higher in the high as compared with low H-FT expressers.
Because probenecid,23 a known blocker of the MRP-type of
drug pump26 hardly affected the uptake profiles of
calcein-AM (not shown) when used up to 10 mmol/L, we deduced that the
MDR character displayed by the clones was likely to be associated with
a PgP-type pump. As drug pumps have been implicated in some modes of
multidrug resistance,25,26 we compared the clones in terms
of susceptibility to two classes of hydrophobic cytotoxic drugs, the
neutral colchicine, and the cationic vinblastine (Fig 4, right). Using
Alamar Blue (AB) fluorescence for assessing cell metabolic activity and
viability,38 we found the H-FT-overexpressing clones to be
three- to sevenfold more resistant to the drugs in a 48-hour
cytotoxicity test. Similar results were obtained using (3H)thymidine incorporation into nucleic acids (Glickstein
and Cabantchik, unpublished observations).
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| Fig 4.
MDR properties of H-FT-transfected MEL. Left: CAAM
uptake into MEL cells and its reversal by verapamil (VP) and
cyclosporin (CS). Cells (1 × 106/mL) were suspended in
MEM medium at 37°C, supplemented with CA-AM (125 nmol/L) and
fluorescence recorded in parallel with time. Verapamil 15 µmol/L
(ver) or cyclosporin 6 µmol/L addition is indicated by the arrow. The
relative rates of CA-AM entry into cells after and before addition of
verapamil were: wt: 1.2; cl-6: 2.8; cl-12: 1.8; cl-16: 1.6. Cl-6
displayed ×3 and ×5 higher IC50 towards vinblastine and
colchicine, respectively. Right: viability profiles. The plots depict
the fluorescence intensity (540 to greater than 590 nm) of Alamar Blue
(AB = 5%) for each cell system at the indicated concentration of the
drug (total exposure time to drug: 48 hours and to Alamar Blue: 4 hours; 7 × 103 cells/well; n = 3). The average of n = 3 ± SE was plotted against the respective inhibitor concentration and
analyzed by NLSQ (best fits depicted over the experimental points)
yielding the following IC50 (µmol/L) for colchicine and
vinblastine, respectively: wt (150 ± 47 and 8 ± 1.5); cl-6 (700 ± 23 and 23 ± 1); cl-12 (230 ± 14 and 12 ± 1); and cl-16 (100 ± 23 and 8 ± 1).
|
|
The characterization of the MDR phenotype of the two transfectants
carrying extreme levels of H-FT expression, cl-6 and cl-16, were also
expanded to other potential substrates and to temperatures in which
ATP-driven pumps are inoperative (Fig 5).
Using the cationic-hydrophobic substrate Rhodamine 123,41
we found that the uptake of the dye at 25°C was in fact similar and
virtually unaffected by cyclosporin in either clone. However, at
37°C, the uptake was apparently lower in both clones than at the
lower temperature, due to the operation of a highly
temperature-dependent MDR efflux pump. The effect of the putative pump
on drug uptake and the reversal by cyclosporin were more pronounced in
the high H-FT expresser cl-6 than in the cl-16 low H-FT expresser.
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| Fig 5.
Uptake of Rhodamine 123 in H-FT-transfected clones. Role
of temperature. Uptake of Rhodamine 123 (5 µmol/L in a buffered salt
solution) into 106/mL cells was performed at the two
indicated temperatures. At different times aliquots were transferred to
an ice-cold buffered salt solution, and samples were aliquoted for FACS
analysis (104 cells per sample). The median fluorescence
values (given as arbitrary units, au) are plotted as a function of time
(of n = 3 representative experiments).
|
|
Because MDR pumping activity is highly dependent on factors affecting
the metabolic status of cells24 and those might differ in
the various transgene H-FT clones, we assessed directly the level of
implicated PgP pump in the nuclei-free fraction of the cells. We
included in this study additional H-FT transfectants, so as to
establish possible correlations between H-FT and PgP levels. The H-FT
and PgP levels were compared with those of actin, which was taken as a
constitutive and, presumably, housekeeping marker of cell constituents.
As observed in Fig 6, PgP was definitely expressed in all the H-FT transfectants. The three lowest
H-FT-expressing clones cl-16, cl-11, and cl-2, were in fact also the
lowest expressers of PgP. Analysis of the correspondence between the
relative levels of PgP and H-FT in all the transfectants and the
wild-type clones showed no linear relationship, but an apparent
hyperbolic, possibly saturative trend (Fig 6, center). To ascertain
that the higher levels of PgP protein resulted from increased
expression of the MDR1a gene, we assessed the mRNA levels of H-FT,
MDR1a, and L7 ribosomal protein (as a putative housekeeping marker) by
RT-PCR, using specific primers to the respective mRNA sequences. As
depicted in Fig 6 (bottom) for cl-6 and wild-type MEL cells, the mRNA
levels of H-FT, and in particular MDR1a, were clearly higher in cl-6.
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| Fig 6.
(top) Western immunoblots of cell lysates isolated from
H-FT-transfected clones. Depicted are the ECL exposures of the
trans-blots of SDS-PAGE of the respective samples (same protein load on
gels) derived from the different clones using the following antibodies:
anti-PgP mouse monoclonal, rabbit antimouse H-FT and anti-L-FT
antibodies, rabbit antiactin and the respective goat antimouse or
antirabbit IgG conjugated to HRP. The samples were run in parallel on
separate gels, but the exposure times differed for the different
antibodies used. The numbers above the bands represent the values of
the densitometry tracings using the intensity of cl-16 for
normalization (center). Correlation between H-FT and PgP levels of
expression in MEL clones. The densitometry tracings of the immunoblots
shown on the top and others (not shown) were normalized to the values
obtained in cl-16 for both H-FT and MDR. The denstity values of each
pair (n = 3) were from parallel SDS-PAGE runs originating from the
same cell samples normalized to that of actin. The mean OD values are
given as symbols and the ± SE as bars both PgP and H-FT, respectively
(bottom). RT-PCR of H-FT, MDR1, and L7 ribosomal mRNAs of a high H-FT
expresser and the wild-type untransfected clone. The propidium iodide
stains are of the various mRNA samples analyzed by RT-PCR as described
in Methods. For the relatively abundant H-FT message the system
saturated at cycle 18, whereas for MDR1a and L7 it had to be run at
higher cycle number. MDR1a was essentially undetected in the wild type
(wt).
|
|
The possibility that PgP activity might be essential or favorable for
the survival of H-FT overexpressers was given preliminary consideration. As an initial attempt we opted for inhibiting the PgP
pump activity with agents that compromise cell viability only in a
limited fashion. Cell viability was assessed after 24-hour exposure of
cells to either 20 µmol/L verapamil or cyclosporin. The results shown
in Fig 7 indicate that cell growth was
significantly, but not substantially, inhibited by verapamil in all
clones except wild type. However, more significant and substantial
differences were obtained with cyclosporin, which differentially
reduced cell growth in the H-FT transfectants in the rank order of H-FT
expression.
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| Fig 7.
Differential susceptibility of H-FT-transfected MEL
clones to blockers of the PgP (MDR1) transporter. The various MEL
clones were exposed to 20 µmol/L of verapamil or 5 µmol/L
cyclosporin and assayed for metabolic activity 24 hours later by the
Alamar Blue method, as described in Fig 3. The fluorescence intensity
relative to the untreated control of each clone is depicted as a
function of the treatment (± SE, n = 3), with * denoting
statistically significant differences from control.
|
|
The reciprocal phenomenon, namely a modification of LIP or ROS
production by MDR itself, was explored in clones of K562 transfected with the human MDR1 gene. When compared with wild-type K562 cells by
the verapamil reversal test (as shown in Fig 4 for MEL
cells), functional PgP was 4- to 5-fold higher in the MDR-expressing
clone. The steady-state LIP levels were not significantly altered by the transfected MDR1, as the analyzed free Fe values in the respective clones were 80 ± 9 nmol/L (n = 6) and 84 ± 8 (n = 4). Moreover, ROS production assessed by CDCF after hydroperoxide was not modified by
MDR presence (not shown).
| |
DISCUSSION |
The present study addressed the possible roles of FT as a modulator of
the cell LIP and the cell response to oxidative stress. We based the
study on MEL clones that expressed a relatively wide range of H-FT
levels, up to fivefold higher than the wild-type control. The fact that
H-FT overexpression led to a demonstrably lower cell LIP (Table 1), to
higher IRP activation,20 and to an increased
Tf-TfR-mediated iron uptake (Glickstein and Cabantchik, unpublished
information), indicated that at least some of the expressed H-FT was
functional. This is also supported by measurements of iron levels found
associated with H-FT in the various clones acutely exposed to permeant
iron salts.20 However, the fact that the clones showed no
differences in total cell iron accumulation might indicate that factors
other than H-FT per se contributed to the cell iron maintenance. One
such factor might be L-FT, whose reduced expression in
H-FT-overexpressing clones,20 could slow down stable iron
core formation in FT polymers.13,14 That is in line with
our previous finding that some of the labeled iron taken up by H-FT in
the transfected cells was chelatable, implying that a fraction of the
H-FT-associated iron might have a labile character.21
However, an additional and equally plausible factor might be associated
with an increased H-FT turnover in overexpressing clones, a phenomenon
we observed in cells with reduced or depleted LIP levels (Glickstein
and Cabantchik, unpublished information).
The downmodulation of LIP by H-FT overexpression also had no apparent
impact on the cell-reductive capacity, as measured by the GSH/GSSG
levels (Table 1). However, the steady-state LIP levels largely dictated
the response of the cells to H2O2 challenges, as shown by various parameters. First, clones with higher H-FT and
lower LIP levels showed proportionally lower ROS production in the face
of the pro-oxidant challenges (Fig 1 and Fig 3). Second, although the
oxidative cell response was augmented by an acute rise in LIP (Fig 1),
the response was more attenuated in the H-FT-expressing clones. Third,
ROS formation could be blocked by membrane-permeating iron chelators,
as shown previously for other cells1,3 and also with MEL
cells (Epsztejn and Cabantchik, unpublished observations). Finally, the
long-term cell damage or death induced by H2O2
was significantly lower in the H-FT-overexpressing clones (Fig 3). Taken together, the studies indicate that the levels of ROS production and cell damage caused by H2O2 were correlated
with the LIP levels in the H-FT-overexpressing clones. The possibility
that factors other than LIP might have also compensated for the changes
in ROS production, such as protective antioxidant enzymes, cannot be
dismissed. However, the similar levels of GSH/GSSG among the clones
would indicate otherwise. Moreover, because the proliferative capacity
of the various clones was essentially similar (Fig 2), it appears that
twofold differences in LIP levels might not be sufficient for limiting
the rate of growth of cells. Thus, the results of this study might be
relevant for understanding the implied protective role of H-FT in
various cell stress-adaptive responses42-44 and
cytokine14 and oncogene action.12-14 However, the role of H-FT in those phenomena awaits direct assessment of the LIP
and its correlation to H-FT and ROS production.
An unexpected feature detected in the H-FT-overexpressing clones was
an increase in MDR properties. Those were reflected both functionally
and structurally as associated with increased PgP or MDR expression in
all the H-FT-transfected clones (Fig 4 to 7). Although the
quantitative correlation between the levels of expression of the H-FT
and MDR genes was hyperbolic, possibly saturative (Fig 6, center), it
was striking when compared on the basis of the highest and lowest H-FT
expressers, cl-6 and cl-16 (Figs 5 and 6). It can therefore be surmised
that the acquisition of the MDR character was not associated with the
transfection per se but with the transfected gene. This raises the
possibility that PgP might confer upon the H-FT overexpressers some
essential cell property that H-FT per se might compromise. In support
of this hypothesis, we found that classical blockers of PgP such as
verapamil or cyclosporin differentially affected the survival of the
H-FT/MDR overexpressers (Fig 7). Particularly interesting was the
correlation found between levels of H-FT overexpressed in the clones
and their susceptibility to cyclosporin. Clearly, that phenomenon will
have to be assessed with more specific reverses of the MDR pump.
Although various mechanisms of induced expression of MDR genes have
been described,25,26,45-51 including those invoking stress and metals,48,52-54 it is still unclear if and how H-FT per
se might lead to induction of MDR. A pertinent property linking MDR and
FT might be associated with a factor causing similar lens defects
leading to cataract formation in both transgenic mice expressing
MDR155 and humans displaying increased L-FT
expression as a result of a mutation in IRP.56 PgP and
possibly other MDR pumps have been regarded as physiological membrane
cleansing mechanisms,25,26 although the natural substrates
have not been identified. Previous studies have also suggested putative
associations of FT with membranes. Recent work has indicated that PgP
localizes in unique lipidic patches in the membrane,57,58
possibly associated with cholesterol export from
cells.59-62 In this context, it is conceivable that, in
some overexpressing clones, H-FT-associated ferroxidase activity may
cause direct or indirect chemical changes to the membrane. MDR could
then be involved in the removal of the products of these changes.
However, all these facts provide only circumstantial evidence for the
alleged association between two distinct functions whose individual,
let alone combined, physiological roles have yet to be determined.
| |
ACKNOWLEDGMENT |
We thank Drs Paolo Arosio and Paolo Santambrogio from DIBIT, San
Raffaele Scientific Institute, Milano, Italy, for their assistance with
the FT measurements and for provisions of invaluable antibodies and Dr
Igor Roninson for kindly supplying the K562 MDR cell line.
| |
FOOTNOTES |
Submitted February 16, 1999; accepted July 13, 1999.
Supported in part by the Israel Research Fund (ZIC), an EMBO Short term
fellowship (VP), the Biomed II Program (ZIC and CB), a collaborative
INSERM/CNR agreement (CB), and the Mirsky Fund for Cancer Research (INS).
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 correspondence to Z. Ioav Cabantchik, Department of Biological
Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem,
Jerusalem 91904, Israel.
| |
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K. Sakamoto, K. Iwasaki, H. Sugiyama, and Y. Tsuji
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Mol. Biol. Cell,
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[Abstract]
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F. Zhang, W. Wang, Y. Tsuji, S. V. Torti, and F. M. Torti
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W. Aung, S. Hasegawa, T. Furukawa, and T. Saga
Potential role of ferritin heavy chain in oxidative stress and apoptosis in human mesothelial and mesothelioma cells: implications for asbestos-induced oncogenesis
Carcinogenesis,
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[Abstract]
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K. Iwasaki, K. Hailemariam, and Y. Tsuji
PIAS3 Interacts with ATF1 and Regulates the Human Ferritin H Gene through an Antioxidant-responsive Element
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T. Prasad, A. Chandra, C. K. Mukhopadhyay, and R. Prasad
Unexpected Link between Iron and Drug Resistance of Candida spp.: Iron Depletion Enhances Membrane Fluidity and Drug Diffusion, Leading to Drug-Susceptible Cells
Antimicrob. Agents Chemother.,
November 1, 2006;
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[Abstract]
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G. Nie, G. Chen, A. D. Sheftel, K. Pantopoulos, and P. Ponka
In vivo tumor growth is inhibited by cytosolic iron deprivation caused by the expression of mitochondrial ferritin
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K. Iwasaki, E. L. MacKenzie, K. Hailemariam, K. Sakamoto, and Y. Tsuji
Hemin-Mediated Regulation of an Antioxidant-Responsive Element of the Human Ferritin H Gene and Role of Ref-1 during Erythroid Differentiation of K562 Cells.
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J. A. Carlyon, D. Ryan, K. Archer, and E. Fikrig
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Infect. Immun.,
November 1, 2005;
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[Abstract]
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C. Xie, N. Zhang, H. Zhou, J. Li, Q. Li, T. Zarubin, S.-C. Lin, and J. Han
Distinct Roles of Basal Steady-State and Induced H-Ferritin in Tumor Necrosis Factor-Induced Death in L929 Cells
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B. D. Schneider and E. A. Leibold
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M. Goralska, B. L. Holley, and M. C. McGahan
Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain
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H. Bowen, T. E. Biggs, E. Phillips, S. T. Baker, V. H. Perry, D. A. Mann, and C. H. Barton
c-Myc Represses and Miz-1 Activates the Murine Natural Resistance-associated Protein 1 Promoter
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September 13, 2002;
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B. Corsi, A. Cozzi, P. Arosio, J. Drysdale, P. Santambrogio, A. Campanella, G. Biasiotto, A. Albertini, and S. Levi
Human Mitochondrial Ferritin Expressed in HeLa Cells Incorporates Iron and Affects Cellular Iron Metabolism
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F. M. Torti and S. V. Torti
Regulation of ferritin genes and protein
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C. Ferreira, P. Santambrogio, M.-E. Martin, V. Andrieu, G. Feldmann, D. Henin, and C. Beaumont
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M. Goralska, B. L. Holley, and M. C. McGahan
Overexpression of H- and L-Ferritin Subunits in Lens Epithelial Cells: Fe Metabolism and Cellular Response to UVB Irradiation
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July 1, 2001;
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A. Cozzi, B. Corsi, S. Levi, P. Santambrogio, A. Albertini, and P. Arosio
Overexpression of Wild Type and Mutated Human Ferritin H-chain in HeLa Cells. IN VIVO ROLE OF FERRITIN FERROXIDASE ACTIVITY
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TOP
ABSTRACT
INTRODUCTION