1 Department of Neuroscience and Anatomy, Pennsylvania State University College
of Medicine, Milton S. Hershey Medical Center, Hershey, PA 17033, USA
2 Department of Biochemistry and Molecular Biology, Pennsylvania State
University College of Medicine, Milton S. Hershey Medical Center, Hershey, PA
17033, USA
3 Department of Pathology, Pennsylvania State University College of Medicine,
Milton S. Hershey Medical Center, Hershey, PA 17033, USA
* Authors for correspondence (e-mail: jrc3{at}psu.edu , mfried{at}psu.edu and kjm13{at}psu.edu )
Accepted 26 February 2002
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Summary |
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Key words: Iron, Oxidative damage, DNA protection, Nuclear translocation
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Introduction |
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Most vertebrate ferritins occur as hollow, spherical assemblies of 24
protein subunits (aggregate Mr 450,000). As many as
4500 iron atoms can be accommodated within a ferritin assembly
(Aisen and Listowsky, 1980
).
Ferritin assemblies comprise two functionally and genetically distinct subunit
types: H (heavy) and L (light), which are present in varying ratios in
different tissues. Subunits of type L contribute to the nucleation of the iron
core, but lack the ferroxidase activity necessary for uptake of ferrous
(Fe2+) iron. Subunits of type H possess ferroxidase activity and
promote rapid uptake and oxidation of ferrous iron
(Lawson et al., 1989
).
Ferritin has been observed in the nucleus of rat hepatocytes
(Smith et al., 1990), chicken
corneal epithelial cells (Cai et al.,
1997
; Cai et al.,
1998
), the human K562 cell line
(Pountney et al., 1999
) and
rodent neurons during development and after hypoxic ischemic insult
(Cheepsunthorn et al., 1998
;
Cheepsunthorn et al., 2001
).
There is little agreement between these studies either about the mechanism of
action of ferritin in the nucleus or the mechanism by which it enters the
nucleus. In corneal epithelial cells, the nuclear expression pattern of
ferritin is developmentally regulated (Cai
et al., 1997
), whereas in hepatocytes the ferritin in the nucleus
is thought to follow iron passively across a concentration gradient after iron
overload (Smith et al., 1990
).
Recently, Cai and Linsenmayer (Cai and
Linsenmayer, 2001
) suggested that nuclear localization of ferritin
involved tissue-specific mechanisms. Functionally, the presence of ferritin in
the nucleus in corneal epithelial cells protects DNA from UV damage
(Cai et al., 1998
), but
ferritin in the nuclei of hepatocytes is hypothesized to be a catalyst for
hydroxyl radical formation during toxic, carcinogenic and aging processes
(Smith et al., 1990
).
In this study, we demonstrate that ferritin is present in the nucleus of
human astrocytoma cells in vivo and a human SW1088 astrocytoma cell line.
Normally astrocytes do not contain ferritin in either their nuclei or
cytoplasm. Some cytoplasmic staining for L-ferritin has been observed in
diseased states (Connor, 1994;
Connor and Menzies, 1995
). We
established a cell culture model using the SW1088 cells to elucidate the
mechanism for ferritin uptake into the nucleus, in order to begin to identify
factors that regulate the concentration of ferritin in the nucleus.
Subsequently, we used a supercoil strand break assay and the cell culture
model to test directly the hypothesis that ferritin protects DNA from
iron-induced oxidative damage.
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Materials and Methods |
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SW1088 cell culture
Human astrocytoma SW1088 cells (HTB-12; ATCC) were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum (BioCell), 4 mM
L-glutamine (Sigma) and antibiotics [100 U/ml penicillin and 1 ng/ml
streptomycin (Gibco)]. Cultures were maintained in 75 mm and 150 mm culture
flasks and passaged every 5 days. For immunohistochemical analyses,
astrocytoma cells were plated on 12 mm poly-L-lysine-coated glass coverslips
at a density of 1.5x104 cells. Cells were fixed with 4%
paraformaldehyde and nonspecific antibody reactions were blocked by incubation
for 30 minutes in 5% non-fat dry milk. The cells were incubated with the
monoclonal anti-human rH-ferritin antibody (HO2; 1:250) followed by a Texas
Red-conjugated IgG secondary (Sigma) at a dilution 1:100. Control
immunoreactions were performed without the primary antibody. Nuclei were
visualized using DAPI (100 ng/ml; Molecular Probes). DNA synthesis was
detected using the 5-bromo-2'-deoxyuridine (BrdU) Labeling and Detection
Kit (Boehringer Mannheim). Results were visualized by fluorescence
microscopy.
Myc-ferritin constructs and cellular localization of Myc tagged
H-ferritin
The human H-ferritin cDNA was inserted 5' of the Myc segment of the
Myc containing vector pcDNA3 (Invitrogen). The H-ferritin Myc construct was a
generous gift from Dwight Stambolian (University of Pennsylvania). SW1088
cells at 50-60% confluence were transfected with the construct using the
Lipofectamine transfection reagent (Boehringer Mannheim). Cells were grown in
standard culture conditions for 12 hours, fixed with 4% paraformaldehyde and
the Myc epitope visualized with the Ab-1 anti-Myc antibody (Calbiochem; 1:250)
and FITC-conjugated IgG secondary (Sigma; 1:100). Nuclear localization of
staining was verified using DAPI.
Iron chelation in SW1088 cells
The iron chelator deferoxamine (DFO; Sigma) was used to examine the effect
of iron chelation on the presence of ferritin in nuclei of astrocytoma cells.
Cells were plated in the presence of 100 µM DFO for 6, 12, 24, 48 and 72
hours. Immunohistochemical and uptake studies were routinely performed on
cells treated with DFO for 72 hours. The Live/Dead Stain (Molecular Probes)
was used to demonstrate that the DFO-treated cells were still viable.
Cell stressors
Astrocytoma cells were treated with 100 µM DFO for 72 hours to minimize
ferritin expression in the cells. After this treatment, the cultures were
rinsed with Hanks balanced salts solution and incubated for 12 hours in
standard medium alone (control) or medium containing ferric ammonium citrate
(FAC; 0, 50, 100, 200 µM), hydrogen peroxide (H2O2;
0, 50, 100, 300 µM), tumor necrosis factor (TNF
; 1, 10, 50
ng/ml) or interleukin 1ß (IL-1ß; 50, 100, 200 U/ml). Treatments were
performed in triplicate and nuclear and cytosolic fractions were obtained
separately for each trial.
Isolation of nuclear and cytosolic fractions
Nuclear and cytosolic fractions of harvested astrocytoma cells were
isolated according to standard methods
(Abmayr and Workman, 1997). The
cultures were rinsed with Hanks Balanced Salts Solution, trypsinized and the
cells collected by centrifugation. The pelleted cells were rinsed with 5 ml
0.1 M phosphate-buffered saline (PBS) and collected by centrifugation at 1850
g for 5 minutes. The cell pellets were resuspended in five
pelleted cell volumes (p.c.v.) of hypotonic buffer [10 mM Hepes (pH 7.9), 1.5
mM MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT]. The resuspended
cells were collected by centrifugation for 5 minutes at 1850
g. These cells were lysed by resuspension in five p.c.v. of
hypotonic solution for 10 minutes on ice and homogenized using a Dounce
homogenizer. The cells were lysed with 20 up-and-down pestle strokes of a type
B pestle and cell lysis was verified by light microscopy. The nuclei were
collected by centrifugation for 15 minutes at 3300 g. The
supernatant was collected for cytosolic analysis.
Relative amounts of ferritin in nuclear and cytosolic extracts
The relative amounts of H-ferritin in the cytosolic and nuclear extracts
were determined by western immunoblot analysis. Equal amounts of nuclear (5
µg/50 µl) or cytosolic (10 µg/50 µl) proteins from control and
experimental groups were examined by 15% SDS-PAGE. Proteins were transferred
to a nitrocellulose membrane. H-and L-rich ferritins (0.5 ng/50 µl) were
added as standards and also served as positive and negative controls for the
antibodies. Membranes were blocked with 5% non-fat dry milk, washed in
TBS-Tween (Tris-buffered saline/0.2% Tween-20), and probed with mouse
anti-human rH-ferritin (HS-59) at a dilution of 1:1500, overnight at 4°C.
The blots were washed and incubated with goat anti-mouse IgG conjugated with
peroxidase for the SW1088 cell extracts, at a dilution of 1:5000 for 60
minutes at room temperature. The immunoreaction was visualized using
chemiluminescent detection (ECL+Plus, Amersham). Autoradiography films were
scanned and the band intensities for each experiment were assessed using the
image analysis software program Collage (Fotodyne). The threshold value for
each analysis was set to zero. S/N values [area intensity(pixel
intensityxbackground)/background pixel intensity] for each band were
obtained. The S/N values were converted to percent of control (12 hour media
replete samples) to compare experiments between the three trials for each
experimental manipulation. Each experiment was repeated three times and a
separate immunoblot analysis performed each time.
Proteins and antibodies
The human recombinant ferritins used in this study (rH, rL and 222) and
ferritin subunit-specific monoclonal antibodies were generously supplied by
Paolo Arosio (Milan, Italy). The specificity of the HS-59 and HO2 monoclonal
antibodies to the H-ferritin subunit has been well characterized
(Cavanna et al., 1983;
Luzzago et al., 1986
;
Ruggeri et al., 1992
). The
HS-59 antibody was used to detect the denatured form of H-ferritin present in
the western blot and paraffin wax-embedded tumor sections as a result of
sample processing. The 222 ferritin is an H-ferritin with amino acid
substitutions E62K and H65G that diminish the ferroxidase activity
(Levi et al., 1988
). The
recombinant proteins have proper assembly, folding and functional properties
(Lawson et al., 1989
;
Levi et al., 1987
;
Levi et al., 1989
). The
monoclonal GFAP antibody was obtained from Boehringer Mannheim, the Mouse IgG
from Santa Cruz Biotechnologies, (500 µg/ml) and the monoclonal
neurofilament antibody from AMAC (#0168). Crystalline bovine serum albumin,
chymotrypsin, horse spleen apoferritin, transferrin and ovalbumin were
obtained from Sigma. CAP protein was purified from E. coli strain
pp47 containing the plasmid pHA5 according to the method of
(Fried and Crothers, 1983
).
The ferritins (5 mg/ml) and BSA (10 mg/ml) were labeled with
fluorescein-5-EX-succinimidyl ester (Molecular Probes) according to the
manufacturer's protocol.
Characterization of ferritin nuclear import
Nuclear import of ferritin was measured using digitonin
(Aldrich)-permeabilized cells (Adam et al.,
1990; Cserpan and Udvardy,
1995
). To demonstrate ferritin uptake, cells were permeabilized
with digitonin (40 µg/ml) for 5 minutes in transport buffer (20 mM Hepes,
pH 7.3, 100 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate,
1 mM EGTA, 2 mM DTT and 1 µg/ml each aprotinin, leupeptin and pepstatin)
while on ice. Excess digitonin was removed with three rinses in transport
buffer. Fluorescein (FITC)-labeled recombinant H-ferritin was added to control
and DFO-treated astrocytoma cells for 30 or 60 minutes in standard media.
Cells were exposed to a range of concentrations of FITC-ferritin (10 nM, 100
nM, 1 µM, 5 µM, 10 µM) and 5 µM was selected as the optimal
concentration, based on detectability and cell viability. To demonstrate that
the permeabilization of the cells with digitonin did not affect nuclear
membrane integrity, we performed the following control experiments in parallel
cultures: cultures were treated with TRITC-dextran (70 kDa; Molecular Probes 5
µM final concentration), 222 mutant ferritin, L-rich ferritin, BSA (Sigma)
or transferrin (Sigma). The latter were added at the same concentration as
that used for ferritin.
Once conditions in which a fluorescently tagged ferritin could enter the
nucleus were established, the effect of cellular iron status on ferritin
nuclear transport was investigated. Cells were exposed for 72 hours to either
standard media or media containing the iron chelator deferoxamine (100 µM).
To control for a nonspecific deferoxamine effect, DFO and FAC were combined in
a 1:1 molar ratio to form ferroxiamine B, which blocks the iron chelating
effect of deferoxamine (Bergamini et al.,
1999; Gutteridge et al.,
1994
). After 72 hours of treatment, FITC-rH ferritin (5 µM) was
added to the media for 1 hour. The cells were then rinsed briefly in PBS, and
viewed with a confocal microscope. These experiments were performed three
times each.
To identify the mechanism for rH-ferritin nuclear translocation ferritin
nuclear uptake was monitored under the following conditions: temperature
variation (4°C and 37°C), nuclear pore receptor inhibition [wheat germ
agglutinin (WGA); 200 µg/ml], ATP depletion (25 U/ml apyrase; Sigma), or
ATP repletion (apyrase followed by addition of ATP)
(Adam et al., 1990;
Adam and Adam, 1994
;
Duverger et al., 1995
). For
each of these conditions, cells were first depleted of iron by exposure to DFO
(100 µM) for 72 hours and then permeabilized with digitonin (40 µg/ml).
Fluorescein-labeled rH-ferritin (5 µM in transport buffer) was added to the
cells after each treatment for 60 minutes at 37°C. To study the effect of
nuclear pore inhibition on ferritin translocation to the nucleus, WGA was
added to the culture medium (200 µg/ml) for 10 minutes on ice in transport
buffer. Excess WGA was removed by three washes in transport buffer. To
determine if ATP is required for ferritin nuclear translocation, ATP was
depleted by apyrase treatment (25 U/ml) in transport buffer for 15 minutes at
37°C. As a control for the ATP depletion experiments, after apyrase
treatment, ATP was reintroduced in a parallel set of cells by incubating them
in an ATP regeneration system (9 mM ATP, 20 mM phosphocreatine and 20-100 U/ml
creatine kinase) in the presence of FITC-rH ferritin
(Newmeyer et al., 1986
). In
addition, because nuclear transport is temperature dependent, an additional
set of uptake studies was performed at 4°C. To determine if cytosolic
factors were required for translocation of ferritin to the nucleus, cells were
exposed to N-ethylamleimide (NEM) (Adam et
al., 1990
). Permeabilized cells were treated with 2 mM NEM
(Aldrich) for 10 minutes at 4°C in transport buffer lacking DTT
(Adam et al., 1990
;
Duverger et al., 1995
). At the
end of the incubation in FITC-rH-ferritin, cells were rinsed three times in
transport buffer containing DTT, fixed in 4% paraformaldehyde and examined by
fluorescence and confocal microscopy. Each experiment was repeated three
times.
Ferritin/DNA crosslinking studies
Human astrocytoma cells (SW1088) were grown to 80% confluence in 75
cm2 flasks in the presence or absence of 100 µM deferoxamine for
72 hours. The cells were rinsed and permeabilized with 40 µg/ml digitonin
for 5 minutes on ice. The permeabilization step is necessary for exogenous
ferritin to enter the cell. 125I-rH-ferritin was added to the
medium for 30 minutes at 30°C. The media was replaced with media
containing 1% formaldehyde or standard media alone and the flasks placed at
4°C for 4 days (Solomon et al.,
1988). After incubation at 4°C the cells were harvested
mechanically and DNA-protein complexes isolated. Samples (50 µl) were
analyzed by slot blot analysis and autoradiography. Intensity of each band was
measured using the image analysis program Collage and normalized for DNA
concentrations.
Supercoil relaxation assay
The DNA used for the supercoil assays was the plasmid pUC19
(Yanisch-Perron et al., 1985).
Plasmid pUC19 was propagated in DH5a cells, and the covalently closed circular
fraction purified by two cycles of centrifugation in CsCl density gradients.
Reaction mixtures (50 µl) contained supercoiled pUC19 DNA (10 nM) dissolved
in 10 mM Tris, 100 mM KCl, (pH 7.4), 50 µM FeCl3, and 10 mM
H2O2, plus varying amounts of proteins (both ferritins
and non-ferritins were tested). Reaction mixtures were assembled as follows.
The DNA was dissolved in 10 mM Tris, 100 mM KCl, (pH 7.4). The protein of
interest was added, in amounts sufficient to give the desired final
concentration, and the sample was incubated at room temperature for 15
minutes. FeCl3 was added to the sample and, following a 15-minute
incubation, H2O2 were added to the mixture. The
assembled samples were incubated for 1 hour at 37°C. Reactions were
terminated by the addition of 25 µl of 4 M urea, 50% sucrose, 50 mM EDTA
and 0.1% Bromophenol Blue. Aliquots were subjected to electrophoresis on 1.5%
agarose gels, and the mole fractions of superhelical and relaxed forms
measured by densitometry of photographic negatives of the gels after staining
with Ethidium Bromide (0.5 µg/ml).
Whole cell damage assay
To examine the potential DNA protective effects of ferritin in live cells,
comparisons were made between DFO-treated cells (control) and DFO-treated
cells incubated with WGA. DFO treatment for 72 hours resulted in a loss of
detectable ferritin in the nucleus. After DFO treatment, WGA was added to the
cells at 200 µg/ml for 10 minutes on ice in transport buffer. WGA blocks
the relocalization of actively transported proteins to the nucleus. Cells were
incubated for 30 or 60 minutes in 100 µM hydrogen peroxide or ferric
ammonium citrate in standard culture conditions (standard conditions contain 5
µM iron). Cells were then fixed with 4% paraformaldehyde and ssDNA
strand-breaks were detected using the TUNEL assay (Cell Death Kit, Boehringer
Mannheim). The TUNEL assay uses terminal deoxynucleotidyl transferase to
introduce fluorescein-dUTP into partially degraded DNA that can result from
oxidative damaging reagents. Nuclei were counterstained with DAPI (100 ng/ml).
Results were ranked according to labeling intensity and scored by double-blind
analysis.
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Results |
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Immunohistochemical analysis of ferritin nuclear localization in cell
culture
A cell culture model using the human astrocytoma SW1088 cell line was
established to manipulate ferritin experimentally in the nucleus. To establish
the fundamental conditions under which ferritin is present in the nucleus, the
cells were first examined immunohistochemically. Human astrocytoma cells
(SW1088) stained with anti-human rH-ferritin monoclonal antibody display
intense immunohistochemical nuclear staining and light cytoplasmic staining
(Fig. 2A). Staining was not
detected in control sections (data not shown). The cultures were triple
stained with rH-ferritin, DAPI and BrdU. The triple stain confirmed the
presence of ferritin in the cell nuclei
(Fig. 2A,B) and also indicated
that ferritin was present in the nucleus of both BrdU-positive and
BrdU-negative cells (compare Fig.
2C,D), but is not associated with condensed chromatin of actively
mitotic cells (Fig. 2E,F).
Almost all of the cells observed in these cultures were H-ferritin nuclear
positive and 50% of these cells are BrdU positive. Treatment with 100 µM
DFO routinely results in loss of detectable nuclear staining of ferritin in at
least 80% of the cells (Fig.
2G,H). The ferritin nuclear immunostaining reappears when normal
medium is reapplied (Fig.
2I,J). The Live/Dead cell death assay (Molecular Probes)
demonstrated that 95% of the cells were viable after 72 hours of DFO treatment
(data not shown).
|
Fluctuations in nuclear ferritin concentrations in response to iron,
H2O2 and cytokines
The results reported in Fig.
2 identified a treatment paradigm wherein ferritin levels in cell
nuclei appear to increase or decrease based on a non-quantitative
immunohistochemical detection method. With this basic model established, the
next set of experiments were designed to test the hypothesis that the amount
of ferritin found in the nucleus could be altered by changing culture
conditions. To test this hypothesis, astrocytoma cells were plated in DFO to
remove ferritin from the nucleus. The cells were then exposed to standard
medium (control) or media supplemented with FAC, TNF, IL-1ß or
H2O2. Addition of FAC in the media resulted in a
significant increase in ferritin protein levels over control in both the
cytosolic and nuclear extracts at each FAC concentration
(Fig. 3A). The cytokines
TNF
and IL-1ß are known to increase ferritin levels in cells
(Fahmy and Young, 1993
;
Kwak et al., 1995
;
Miller et al., 1991
;
Torti and Torti, 1994
) and
were used to determine how increases in ferritin not directly related to iron
would alter nuclear ferritin levels. Treatment with either 10 or 50 ng/ml
TNF
resulted in a significant increase in nuclear but a decrease in
cytosolic ferritin levels (Fig.
3B). IL-1ß treatment, in general had less of an effect than
treatment with TNF
, but did result in a slight (but statistically
significant) increase in nuclear ferritin at 50 U/ml
(Fig. 3C). However, IL-1ß
treatment at greater concentrations resulted in slightly but significantly
decreased nuclear ferritin levels compared with control, without changing the
cytosolic ferritin levels (Fig.
3C). Astrocytoma cells exposed to hydrogen peroxide
(Fig. 3D) had significantly
higher ferritin protein levels in the nucleus at 100 µM
H2O2 when compared with control nuclear levels.
Cytosolic levels did not vary from control levels at any
H2O2 concentration.
|
Demonstration of ferritin nuclear translocation
To demonstrate directly that ferritin enters the nucleus, FITC-conjugated
ferritin was added to digitonin permeabilized cells. A TRITC-dextran complex
(70 kDa) was one approach used to demonstrate that digitonin permeabilization
did not affect the integrity of the nuclear membrane
(Fig. 4A). FITC-rH ferritin did
not enter the nuclei of cells in standard culture conditions but, rather,
remained in the cytoplasm even after 60 minutes of exposure
(Fig. 4B). If cells were first
treated with deferoxamine, FITC-rH-ferritin was found in their nuclei within
60 minutes of exposure (Fig.
4C). Translocation of ferritin to the nucleus did not occur in the
presence of iron saturated DFO (Fig.
4D). BSA-FITC (Fig.
4E) and Tf-FITC (data not shown) conjugates were also used as
controls in this study. Both of these proteins enter the digitonin
permeabilized cells but both remained within the cytoplasm. These proteins
provide additional evidence that the digitonin treatment did not affect the
integrity of the nuclear membrane and further suggest specificity of ferritin
translocation. The specificity of ferritin uptake was further examined using
rL-ferritin and the ferroxidase mutant rH-ferritin 222, respectively, under
the same conditions as in Fig.
4C. The FITC-222 ferroxidase mutant entered cell nuclei
(Fig. 4F), suggesting the
iron-binding status of ferritin may not be a factor in ferritin uptake because
the ability of the 222 mutant to store iron is compromised
(Levi et al., 1988). FITC-rL
ferritin entered the cell but not the nucleus
(Fig. 4G). The failure of the
rL-ferritin conjugate to enter the nucleus indicates subunit preference for
uptake and is a further control that demonstrates the integrity of the nuclear
membrane. As an additional control for the uptake studies that have to this
point used permeabilized cells, we transfected astrocytoma cells with Myc
epitope-tagged rH-ferritin and detected Myc-tagged H-ferritin in the cell
nuclei and cytoplasm (Fig.
4H).
|
Mechanisms controlling ferritin nuclear import
Having demonstrated ferritin translocation to the nucleus, the next set of
experiments were designed to test the hypothesis that ferritin uptake into the
nucleus is active rather than passive. The combination of DFO pretreatment and
digitonin was used in these experiments. FITC-rH ferritin was imported to the
nucleus in DFO-treated permeabilized cells
(Fig. 4C,
Fig. 5A). In the presence of
WGA, which blocks import through nuclear pores, FITC-rH ferritin accumulates
in the cytoplasm, near the nuclear envelope, but does not translocate to the
nucleus (Fig. 5B). To determine
if nuclear import of FITC-ferritin is energy dependent, uptake studies were
performed at 4°C on DFO-treated, digitonin permeabilized cells (already
described). At this temperature, nuclear transport of ferritin was markedly
reduced compared with controls (37°C)
(Fig. 5C). Nuclear uptake of
ferritin was also decreased in the presence of apyrase, an ATP-hydrolyzing
enzyme (Fig. 5D). In the
presence of an ATP regeneration system, nuclear uptake of ferritin was
re-established (Fig. 5E).
|
These experiments indicated that the uptake of ferritin was active and
occurred via the nuclear pore. However, those experiments did not address
whether cytosolic factors were required for ferritin nuclear translocation.
Consequently, cell cultures were exposed to NEM which inactivates nuclear
import of NLS-bearing proteins (Adam et
al., 1990; Duverger et al.,
1995
). NEM did not inhibit translocation of ferritin into the
nucleus (Fig. 5F).
Ferritin/DNA interactions in cultured SW108 cells
Having established ferritin translocation to the nucleus is regulated and
characterized the import mechanism, we designed experiments to test the
hypothesis that ferritin in the nucleus interacts with DNA. Formaldehyde
crosslinking techniques were used to test the hypothesis that ferritin binds
DNA in cell culture (Solomon et al.,
1988). Astrocytoma cell cultures were DFO treated and the cells
were permeabilized with digitonin before adding 125I-rH ferritin to
the culture medium. Control cultures were not treated with DFO. Protein-DNA
complexes were isolated by precipitation of DNA. 125I-labeled
ferritin crosslinked to DNA. DFO pretreatment increased the amount of ferritin
that was crosslinked (Fig.
6A,B). A group of control and DFO-treated cells subjected to
125I-rH-ferritin but not exposed to formaldehyde showed very low to
no detectable ferritin associated with the DNA
(Fig. 6C).
|
Ferritin protects DNA from iron-induced oxidative damage
The close proximity of nuclear H-ferritin to DNA
(Fig. 6), suggests that one
possible function of this protein may be a DNA protectant. Many soluble iron
species catalyze the formation of hydroxyl radicals, which react readily with
nucleic acids, giving a spectrum of products
(Henle et al., 1996;
Imlay and Linn, 1988
).
Breakage of the DNA backbone as a consequence of hydroxyl radical attack on
deoxyribose moieties (Floyd,
1990
; Tachon,
1989
) is particularly easy to detect using a superhelical DNA
relaxation assay (Tachon,
1989
). This assay was used to determine whether ferritin is
capable of protecting DNA from iron-catalyzed damage
(Fig. 7A). Exposure of a sample
of DNA to 10 mM H2O2 and 50 µM FeCl3 for
60 minutes at 37°C resulted in the conversion of the form I monomer to
form II (relaxed circular DNA); in some cases a small amount of linear (form
III) DNA was also produced. DNA damage was only observed when both ferric
chloride and hydrogen peroxide were present. Neither reagent alone damaged the
DNA to a detectable extent (data not shown). Addition of increasing amounts of
recombinant H-ferritin to the reaction mixture, prior to the sequential
addition of FeCl3 and H2O2, resulted in the
preservation of an increasing fraction of supercoiled DNA. The incubation of
ferritin and hydrogen peroxide with DNA (in the absence of added
FeCl3) resulted in no detectable damage to the DNA (data not
shown). A qualitatively similar DNA relaxation was obtained when 200 µM
FeSO4 was substituted for the
FeCl3/H2O2 solution and this relaxation was
also prevented by the inclusion of recombinant H-ferritin in the reaction
mixture (data not shown). An additional observation in these studies is that
the electrophoretic mobilities of the protected DNAs were reduced in a
ferritin concentration-dependent manner
(Fig. 7A, lane 7). Such
mobility shifts are consistent with the formation of protein-DNA complexes
(Fried and Crothers, 1981
;
Fried and Garner, 1997
).
|
To determine whether the ability of ferritin to protect DNA from
iron-induced oxidative damage is related to its ability to bind iron,
supercoil relaxation experiments were carried out with recombinant H-ferritin,
recombinant 222 ferritin, horse spleen apoferritin (Sigma; 84% L-subunit,
iron poor) and several non-ferritin proteins
(Fig. 7B). All ferritins
protected DNA to some degree, although ferritins with reduced ferroxidase
activity (the L-subunit-rich spleen apoferritin and the mutant 222 ferritin)
were significantly less effective. Transferrin was also tested in this assay
at concentrations (10-90 µM) that were more than adequate to bind the 50
µM iron present in the reaction (Fig.
7B). Transferrin did not offer DNA protection at any concentration
tested. In addition, bovine serum albumin, chymotrypsin and ovalbumin did not
significantly inhibit iron/H2O2-induced DNA damage. The
addition of non-ferritin proteins in this assay demonstrated that not only was
the iron-binding capacity of ferritin required for DNA protection, but protein
mass was not responsible for the DNA protection that was observed. Because the
non-ferritin proteins used as controls do not bind DNA, we also used a
DNA-binding protein, E. coli cAMP receptor protein (CAP), as a
control. No more than 30% of the DNA was protected by CAP at concentrations
sufficient to coat the DNA (>2 µM), whereas a similar concentration of
ferritin afforded nearly 100% protection
(Fig. 7B). Finally, DNA
protection requires incubation of ferritin with FeCl3 for at least
15 minutes, prior to addition of H2O2. If the two
reagents are added simultaneously, no reduction in DNA damage is observed
(data not shown). Taken together, these results support the notion that
ferritin protects DNA and that the degree of protection is at least
qualitatively related to the iron-binding activity of the ferritin.
Ferritin protects DNA in live SW1088 cells
We attempted to develop a cell culture model to test the ability of
ferritin to protect DNA by taking advantage of our observation that ferritin
can be removed from the cell nucleus with deferoxamine treatment and that
translocation of ferritin into the nucleus can be blocked by treatment with
wheat germ agglutinin (WGA). In this model, astrocytoma cells were treated
with DFO followed by digitonin treatment to decrease ferritin expression in
the cells, as described earlier. The DFO-containing medium was then replaced
with media containing WGA, followed by either 100 µM
H2O2 or 100 µM FAC. FAC and
H2O2 were used as cell stressors and these
concentrations were chosen because they resulted in increased nuclear ferritin
concentrations (Fig. 3A,D). The
control for this experiment was media that did not contain WGA. The results
indicate that when WGA is present in the media (blocking the translocation of
nuclear ferritin), there is an increase in the number of TUNEL-positive cells
compared with the number found in the non-WGA-treated cultures when the cells
are exposed to H2O2
(Fig. 8A). The addition of iron
(FAC) to the media did not affect the number of TUNEL-positive cells
regardless of the presence or absence of WGA
(Fig. 8B). Although WGA will
block the movement of all actively transported proteins into the nucleus, the
data still indicate that those cells that do not have nuclear ferritin are
susceptible to DNA damage after hydrogen peroxide treatment.
|
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Discussion |
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Because there are specific iron-binding sites on DNA
(Henle et al., 1999), a
mechanism for iron transport into the nucleus, presumably involving a protein
(because `free iron' is not thought to be present in biological systems), must
exist. A mechanism for iron transport into rat liver nuclei has been
identified (Gurgueira and Meneghini,
1996
) and recently the divalent metal transport protein (DMT1) has
been demonstrated in the nucleus of some neural cells
(Roth et al., 2000
).
Lactoferrin in the only other iron-binding protein besides ferritin that has
been demonstrated in cell nuclei
(Garré et al., 1992
;
He and Furmanski, 1995
).
Ferritin is unique because it can play a role in both iron delivery and iron
sequestration. In this study we demonstrate that ferritin is present in nuclei
of human astrocytoma cells both in tissue and in cell culture. These
observations extend the original observations of cells that contain ferritin
in the nucleus and expand the notion that ferritin is present in the nucleus
to protect cells from u.v. damage (Cai et
al., 1998
) to the more general concept of protection from
iron-induced oxidative damage. Our DNA protection assays are the first to test
directly the hypothesis that DNA is protected by ferritin and our studies may
suggest a potential role for nuclear ferritin in the growth of tumor
cells.
Modulation of relative amounts of ferritin in the nucleus and
cytoplasm
The cell culture system was developed to characterize the conditions under
which ferritin was present in the nucleus. Ferritin was present in the
astrocytoma cells from the time that they were initially plated. The
appearance of ferritin in the nucleus could be demonstrated
immunohistochemically, with uptake of exogenously applied fluorescently tagged
ferritin and epitope-tagged endogenously synthesized ferritin. The presence of
ferritin in the nucleus was not dependent on whether the cells were
synthesizing DNA because it is found in the nucleus of both BrdU-positive and
-negative cells. However, ferritin is not present in cells that were actively
dividing. Iron chelation with DFO resulted in loss of ferritin from the
nucleus to below immunodetectable levels. The crosslinking studies indicate
that ferritin is associated with DNA. The amount of ferritin associated with
DNA can be increased if the cells are first treated with DFO and then exposed
to control media, which is consistent with the quantitative and fluorescently
labeled ferritin uptake studies. These studies show that the presence of
ferritin in the nucleus is not dependent on the state of the cell's growth
with respect to cell division and proliferation, but is dependent upon iron
availability
To determine if iron is the sole modulator of the amount of ferritin in the
nucleus, we exposed cells to peroxide and cytokines as forms of biological
stress. Our results show that stress-induced changes in the relative
concentrations of ferritin are most evident in nuclei, and less evident in
cytoplasm. The increase in nuclear ferritin with 100 µM
H2O2 correlates well with the finding of maximal DNA
nicking at this concentration of peroxide
(Luo et al., 1994). These
results support our hypothesis that the increase of ferritin in the nucleus is
an attempt to sequester iron and prevent DNA-Fe2+ interactions with
H2O2 that can result in double strand breaks and cell
death (Stevens and Kalkwarf,
1990
). At concentrations of H2O2 higher than
100 µM (Luo et al., 1994
),
there was decreased DNA damage and we found no significant increase in
ferritin (nuclear or cytoplasmic) over control levels. These latter results
suggest that cell damage at this concentration of peroxide is not occurring
within the nucleus and thus the lack of DNA damage and lack of nuclear
ferritin response are consistent.
Cytokine exposure can also be a form of stress to a cell and TNF and
IL-1ß have been shown to increase H-ferritin biosynthesis selectively
relative to L-ferritin (Fahmy and Young,
1993
; Kwak et al.,
1995
; Miller et al.,
1991
; Torti and Torti,
1994
). These cytokines induced significant changes in nuclear
levels of ferritin. The lowest concentration of IL-1ß resulted in a
small, but significant increase in nuclear ferritin, whereas the two higher
concentrations decreased nuclear ferritin levels. The two higher
concentrations of TNF
resulted in a significant increase in nuclear
ferritin and a decrease in cytosolic ferritin. These data indicate that the
amount of ferritin translocated to the nucleus depends on the nature of the
stressor. Even small alterations in ferritin levels may have a large
physiological impact, as one ferritin molecule has the capacity to take up
4500 atoms of iron.
Ferritin nuclear translocation
Having established that the concentration of ferritin in the nucleus could
be manipulated, studies were undertaken to examine the mechanism by which
ferritin entered the nucleus. Permeabilization of the cells with digitonin was
necessary for exogenous ferritin to enter the cell. The permeabilization step
was also necessary for WGA, the nuclear pore inhibitor, to enter the cell.
Several controls, such as dextran, transferrin, BSA and even the L-rich
subunit of ferritin were included to demonstrate that permeabilization with
digitonin did not alter the integrity of the nuclear membrane. The inclusion
of L-rich ferritin not only supported the data that the nuclear membrane was
still intact in our studies, but also revealed that there is selective
ferritin subunit transport into the nucleus. The H-ferritin mutant 222, which
is structurally similar to H-chain ferritin but lacks a ferroxidase center
(Levi et al., 1988), is also
translocated. This latter result suggests that selected ferritin uptake may be
due to protein sequence or structure and not iron uptake efficiency of the
ferritin. In addition to the exogenously applied labeled proteins, a Myc
epitope-tagged ferritin was also translocated to the cell nucleus, indicating
that translocation of ferritin to the nucleus is a normal process of viable
cells.
The nuclear pore complex (NPC) provides the sole avenue for macromolecular
transport between the nucleus and cytoplasm
(Davis, 1995). The movement of
ferritin from the cytoplasm to the nucleus could occur either by passive
diffusion or active transport through the NPC. The preference for H-ferritin
subunit for transport suggests that ferritin transport into the nucleus is
selective. Such selectivity is a hallmark of a facilitated process, as opposed
to passive diffusion. The mechanism of pore-mediated protein import requires
two steps: docking and translocation. The docking of the import complex
[nuclear-targeted proteins and NPC receptors (importins)] can be blocked by
the addition of wheat germ agglutinin (WGA), while the translocation process
can be inhibited by ATP depletion and temperature changes
(Adam and Adam, 1994
;
Newmeyer et al., 1986
;
Richardson et al., 1988
).
H-ferritin nuclear entry was blocked by WGA and translocation of H-ferritin
into the nucleus was blocked at 4°C and by depletion of ATP. Combined,
these results are consistent with models in which H-ferritin is actively
transported into the nucleus through the NPC. The inability of NEM to inhibit
ferritin translocation to the nucleus indicates that a NLS bearing cytosolic
factor is not involved in translocating ferritin into the nucleus
(Duverger et al., 1995
).
DNA protection by ferritin
The ability of ferritin to sequester iron may allow it to protect DNA from
iron-induced oxidative damage. The supercoil assay results show that ferritin
can act on iron to reduce its ability to catalyze oxidative damage, even when
DNA is present as a competing ligand for iron. Addition of other proteins,
including CAP (at concentrations that coat DNA) and transferrin (at
concentrations to bind the iron in the system), did not protect DNA to the
same extent as ferritin. Thus, coating the DNA, as a large protein like
ferritin might do to some extent, is not an effective protection mechanism.
The conclusions from the transferrin results suggest that the iron bound to
transferrin may still be available to peroxide for redox reactions and/or that
some affinity of the protein for DNA is necessary for protection. Our
crosslinking studies show that ferritin interacts with DNA. A report exists
that nuclear ferritin binds a region of the ß-globin promoter
(Broyles et al., 2001;
Pountney et al., 1999
). The
order of addition experiments indicate that the ferritin-iron interactions
require time to complete, as DNA protection requires pre-incubation of
ferritin with FeCl3 prior to the addition of
H2O2. Several previous studies have implicated ferritin
as a source of hydroxyl radical-production, either through the oxidation of
iron after sequestration, or after iron release from the molecule
(Reif, 1992
;
Reif et al., 1988
;
Samokyszyn et al., 1988
).
However, the absence of DNA cleavage in reaction mixtures containing ferritin,
DNA and H2O2, but not FeCl3, described above,
is most consistent with the notion that on interaction with ferritin, the iron
becomes unavailable to participate in H2O2-dependent DNA
cleavage. In addition, the absence of DNA cleavage in reaction mixtures
containing ferritin, DNA, FeCl3 but not H2O2,
suggest that the reactions associated with iron uptake by ferritin do not
damage the DNA. The increased DNA damage seen in the WGA-treated cells in
response to hydrogen peroxide (Fig.
8A) further supports the concept that ferritin protects DNA, and
extends our in vitro findings to a cell culture system. That induction of DNA
damage requires peroxide is to be expected, as significant damage to the cell
would not occur solely in the presence of an iron source without an oxidative
stressor. In a previous report, we have shown that iron loading of astrocytes
in culture does not induce damage in the absence of peroxide
(Robb and Connor, 1998
). These
results are consistent with the idea that ferritin in the nucleus is
associated with protection of DNA. A caveat in the cell culture model is that
translocation of all nuclear-targeted factors that require active transport
are inhibited by WGA. However, the deferoxamine pre-treatment that was
required to decrease nuclear ferritin to below detectable levels so that the
protection studies could be initiated should affect primarily only proteins
transcriptionally and translationally regulated by iron. Thus, while the cell
culture experiments do not provide direct evidence of DNA protection by
ferritin, the experiment was designed so that ferritin is absent at times when
DNA damage is induced. Taken together, these data strongly suggest that
nuclear ferritin acts as a DNA protectant.
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Conclusions |
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Acknowledgments |
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