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Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85721
3To whom correspondence and reprint requests should be addressed at Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • p53 • zinc deficiency • HepG2 cells • tumor suppressor gene.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Hollstein et al. 1991
). It has been called the "guardian of the genome"
(Lane 1992
) because of its ability to cause cell cycle
arrest in response to certain types of DNA damage, thereby allowing DNA
repair to occur before cell cycle progression (Kastan et al. 1991
). Furthermore, after DNA damage in some cell types, p53
can trigger the genetically altered cells to be eliminated by inducing
apoptosis (Lowe et al. 1993
). In addition to its
DNA-damage response, p53 is also involved in the response by
abnormal or stress conditions such as hypoxia (Graeber et al. 1994
), oxidative stress (Yin et al. 1998
), the
presence of genotoxic chemicals (Sun et al. 1995
) and
depletion of ribonucleotides (Linke at al. 1996
).
Therefore, normal p53 expression and function are crucial to prevent
the propagation of genetically damaged cells and to prevent
proliferation of cells under stress conditions.
p53 is a 393-amino acid transcription factor that binds its consensus
DNA sequence through high affinity interactions to control the
transcription of several sets of genes. Some of the genes regulated by
p53 include regulators of apoptosis such as Bax-1 and also inhibitors
of cell cycle progression such as p21waf-1 and
GADD45. p53 can also transactivate genes involved in the metabolism of
reactive oxygen species (Yin et al. 1998
). In addition
to its transactivation properties, p53 can repress a number of positive
regulators of cell growth or survival, such as c-fos, c-jun and
bcl-2 [reviewed in Donehower and Bradley (1993)
].
The earliest report to show that suboptimal zinc and apoptosis may be
linked was published in 1977 by Elmes who found significantly increased
numbers of apoptotic bodies in the small intestine of
zinc-deficient rats (Elmes 1977
). Subsequently,
several investigators have used zinc-depleted culture medium or
zinc chelators to show that low cellular zinc induced apoptosis in
several different cell lines [reviewed in Fraker and Telford (1996)
]. These data led to speculation that a chelatable or
depletable pool of intracellular zinc may influence the propensity of
cells to undergo apoptosis; however, the mechanism(s) responsible for
the induction of apoptosis remains unclear.
Because of the prevalence of marginal zinc deficiency in certain
subpopulations of the United States (Sandstead 1995
),
these studies were designed to investigate how compromised cellular
zinc status affects the expression of the p53 tumor suppressor gene. We
utilized two different methods to deplete cellular zinc in HepG2 cells.
Compared with the alternative of exposing cells directly to a chelator,
we believe that culturing cells in a low zinc medium more closely
resembles physiologic conditions. We considered the findings of
numerous other investigators as well as extensive efforts in our own
laboratory (Wu et al. 1998
) in establishing these
models. An essential criterion for a successful model is that
zinc-dependent processes such as growth must not be impaired. The
systems that we chose for these studies depleted cellular zinc levels
without affecting cell morphology or growth. Therefore, our system may
reflect a state of marginal zinc deficiency.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human hepatoblastoma cell line, HepG2, was purchased from the American Type Culture Collection (Rockville, MD). Cell culture reagents were purchased from Life Technologies (Grand Island, NY). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM)4 containing 10% fetal bovine serum (FBS) and antibiotics (100,000 U/L penicillin and 100 mg/L streptomycin). Medium was replaced every 2 d; ~6.5 d of culture constituted one passage. At the end of passage 80, nearly confluent cells were subcultured at a ratio of 1:8 for the initiation of experimental treatments.
Two different methods were utilized to deplete cellular zinc levels for
these studies. For the first method, cells were cultured in the
specially formulated medium Opti-MEM (Life Technologies). This
medium contains supplemented growth factors and was designed for
experiments in which serum-free or serum-reduced conditions may
be desirable such as DNA transfection experiments. We obtained a
customized Zn-free Opti-MEM formulation from Life Technologies and
have used this method to deplete HepG2 cellular zinc in these as well
as previous studies. Appropriate growth conditions for this medium have
been determined and described previously (Wu et al. 1999
). Briefly, HepG2 cells were cultured in Opti-MEM with
1% FBS, and various levels of zinc (0–16 µmol/L)
were added. Cellular zinc responded in a dose-dependent manner and
no morphological differences were apparent among the cells cultured in
different levels of zinc. Therefore, our zinc-depleted (ZD) cells
were cultured in Opti-MEM plus 1% FBS. This medium contained 0.4
µmol/L zinc. For the zinc-adequate group (ZA),
ZnSO4 was added to the ZD medium to 4.0
µmol/L, the level in normal HepG2 medium with 10%
FBS. In the third group (ZDA), ZD cells were replenished for the last
24 h with the ZA medium to examine the specificity of the zinc
effects.
The second strategy for zinc depletion utilized a divalent
ion–chelating resin with a high affinity for zinc. Chelex 100 resin
(BioRad, Richmond, CA) was used to deplete zinc from FBS before the FBS
was combined with DMEM. The resin was mixed with FBS at a ratio of 1:4
and shaken for 2 h at 4°C as described previously (Flynn and Yen 1981
, Messer et al. 1982
). Chelex resin
was separated from FBS by centrifugation followed by filtration through
a 0.4-µm filter for sterilization and removal of
residual Chelex resin. The amount of zinc in chelexed sera was not
above the background level (1.0 µmol/L) of detection
by flame atomic absorption spectrophotometry. Therefore, DMEM with 10%
chelexed FBS was termed the zinc-free basal treatment medium.
However, for cells cultured in the zinc-free basal medium, growth
was slowed slightly by the low level of zinc compared with cells grown
in regular medium. Studies in our laboratory were performed with
increasing amounts of media zinc to establish the optimal growth
conditions for HepG2 cells (Wu et al. 1999
). From these
studies, it was determined that the zinc-free basal medium
supplemented with 0.4 µmol/L ZnSO4 was
suitable to deplete cellular zinc without affecting overall growth;
thus it was used as the Zn-depleted medium (ZD). The
Zn-adequate (ZA4) medium contained 4.0 µmol/L
ZnSO4 added to the zinc-free basal medium, and the
medium of the Zn-supplemented group (ZA16) contained 16
µmol/L ZnSO4 added to the zinc-free
basal medium. The ZA medium was used as a comparison to standard
culture medium, and ZA16 was used as a representative of human plasma
Zn levels. Cells were grown for 6.5 d (one passage) in their
respective treatment media and then harvested.
Determination of cellular DNA and zinc levels.
Cells and media were collected from each tissue culture plate and
centrifuged at 500 x g for 5 min at 4°C. Cell
pellets were then washed two times with PBS, resuspended in 1.5 mL PBS
and sonicated. An aliquot of the sonicant was used to measure cellular
zinc by flame atomic absorption spectrophotometry (Hitachi, San Jose,
CA) as previously described (Wu et al. 1999
).
The zinc concentration of the cell samples was determined on the basis
of standard curves generated with certified zinc reference solutions
(Fisher Scientific, Fair Lawn, NJ). In addition, the certified zinc
solutions were compared with bovine Liver Standard Reference (U.S.
Department of Commerce, National Institute of Standards, Gaithersburg,
MD). Appropriate blanks were employed for all measurements. An aliquot
of the sonicant was also used to measure cellular DNA content by the
method of Williams et al. (1986)
. Cellular zinc was
expressed per DNA because a linear relationship between cellular DNA
and cell numbers was previously established.
HepG2 nuclear extracts.
Nuclear extracts were prepared as previously described by
Schreiber et al. (1989)
with slight modifications. Cells
were harvested with trypsin-EDTA, and 1 mL ice-cold
Tris-buffered saline (TBS) was added to each flask to collect the
cells. Cells were centrifuged at 1500 x g for 5
min at 4°C. Pelleted cells were then washed with 10 mL ice-cold
TBS, shaken vigorously for 30 s and spun as described above
followed by an additional wash. TBS was removed and the cell pellet was
resuspended in 5 mL ice-cold buffer A [10 mmol/L HEPES, pH 7.9, 10
mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol
(DTT)] with freshly added DTT and protease inhibitors [0.5 mmol/L
phenylmethylsulfonylfluoride (PMSF), 0.5 mg/L leupeptin, 1 mg/L
pepstatin A, 1 mmol/L benzamidine-HCl] and mixed gently by pipetting.
Cells were allowed to swell by incubating on ice for 15 min; then 312
µL of 10% NP-40 solution was added for each 5 mL of
buffer A, and tubes were vortexed vigorously for 20 s. Samples
were transferred to 1.5-mL microfuge tubes and spun at 16,000 x g for 50 s at 4°C. The supernatant fraction
composed of cytoplasm and RNA was discarded, and the nuclear pellet was
resuspended in ice-cold buffer C (20 mmol/L HEPES pH 7.9, 0.4 mol/L
NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, 0.5 mg/L leupeptin, 1
mg/L pepstatin A, 1 mmol/L benzamidine-HCl) by vigorously shaking at
4°C for 15 min on a shaking platform. Nuclear extract was centrifuged
at 16,000 x g for 15 min at 4°C and the
supernatant fraction was frozen in aliquots at -80°C. Protein
concentrations were determined by the method of Lowry (1951)
.
Western blot analysis.
Nuclear extract (20 µg) was combined with an equal volume of sample loading buffer (20% glycerol, 10% 2-mercaptoethanol, 5% SDS, 200 mmol/L Tris-HCl, pH 6.7, 0.01% bromophenol blue), boiled for 3 min and then subjected to 7.5% SDS-polyacrylamide electrophoresis. After electrophoresis, gels were equilibrated briefly in transfer buffer (20% methanol, 192 mmol/L glycine, 25 mmol/L Tris-aminomethane, 0.05% SDS) before transfer onto nitrocellullose membranes. Transfer was performed at 30 V overnight at 4°C. Equal loading of samples was verified by staining a duplicate gel with Coomassie Brilliant Blue R-250 and scanning with a laser densitometer to compare optical density units between lanes. After blocking [5% (wt/v) nonfat dried milk, 10 mmol/L Tris-HCl pH 8.0, 150 mmol/L NaCl, 0.05% Tween 20] for at least 1 h, blots were washed twice in TBST (10 mmol/L Tris-HCl pH 8.0, 150 mmol/L NaCl, 0.05% Tween 20) for 10 min each. Blots were then incubated with mouse anti-p53 antibody (Clone PAb421; Oncogene Research Products, Cambridge, MA) diluted to 1 mg/L in TBST 0.5% milk at room temperature for 1 h, followed by two 10 min washes in TBST. Incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Santa Cruz Biotechnology, Santa Cruz, CA), diluted to 0.1 mg/L in TBST, was for 30 min followed by a 10 min wash in TBST and two 10 min washes in TBS (no Tween 20). Autoradiography was performed utilizing enhanced chemiluminescence according to the manufacturer’s instructions (Amersham, Arlington Heights, IL). p53 bands were verified by running p53-GST fusion protein (Oncogene Research Products) or A431 cell nuclear extract (Santa Cruz Biotechnology) on a lane in each gel. Blots were also stained with Amido black and photographed to document equivalent protein loading. Laser densitometry (Molecular Dynamics, Sunnyvale, CA) was used to quantify p53 bands after establishing linearity curves.
RNase protection assays.
Total cellular RNA was isolated from cells using a RNaqueous kit
(Ambion, Austin, TX) according to the manufacturer’s instructions, and
the integrity of each sample was checked by electrophoresis. The
abundance of p53, metallothionein (MT)-II, ß-actin and cyclophilin
mRNA was measured by RNase Protection Assays (RPA). The p53 human probe
antisense template was purchased from Ambion. The template consisted of
a 300-bp cDNA fragment of the human p53 tumor suppressor gene, spanning
exons 2, 3 and 4. The human cyclophilin probe (Ambion), which protects
a fragment 103 nt in length, was used as an internal reference for
normalization in the Chelex experiments. For the Chelex study, we also
measured mRNA abundance of the zinc-responsive MT-II gene as an
assessment of cellular zinc status. The human MT-II antisense
template was prepared by reverse transcriptase-polymerase chain
reaction (RT-PCR), as previously described (Sullivan and Cousins 1997
, Wu et al. 1998
). A pair of human MT
primers, MT5 and MT3, corresponding to the 5' and 3' regions of the
human MT-II cDNA (Sullivan and Cousins 1997
) were
synthesized. RT-PCR products were cloned into pGEM-T PCR
cloning vector (Promega, Madison, WI). Plasmid DNA was isolated
from a correct clone, which contained a MT-II cDNA fragment in the
antisense orientation with respect to the T7 promoter. A pair of
primers (puc/M13F and Rev-T), which correspond to upstream/downstream
vector sequences, were used to prepare the final cDNA template for
MT-II RPA probe synthesis in a PCR reaction. The resulting fragment
was 354 nt in length and the PCR product was used to synthesize labeled
probe. The RNA probe transcribed from the T7 promoter was 288 nt in
length, which contained 201 nt of human MT-II antisense sequence.
ß-Actin riboprobe was synthesized from pTRI-ß-actin template
(Ambion) and was used as the internal control for the Opti-MEM
experiments. All RNA probes for p53, MT-II, ß-actin, cyclophilin
and RNA century size markers (Ambion) were synthesized using the
MAXI-script in vitro transcription system kit (Ambion) with T7 RNA
polymerase. These probes were labeled at predetermined specific
activities by changing the ratio of 
-P32-UTP to cold UTP
in the labeling reaction to provide roughly comparable band intensities
in the final RPA gel.
RPA were done using the RPA-II Kit (Ambion). HepG2 RNA (40 µg) was combined with a molar excess of labeled RNA probe. The RNA and labeled antisense probes were coprecipitated with ammonium acetate and ethanol and then resuspended in hybridization buffer at 43.5°C for 10 h. RNase cocktail was then added and samples were incubated at 37°C for 30 min. The RNase digestion was then stopped by adding inactivation/precipitation buffer and samples were precipitated at -80°C. Protected fragments were separated by PAGE on 6% acrylamide, 8 mol/L urea gels. Controls for the digestion step were included in which yeast RNA replaced HepG2 RNA. No protected bands appeared in these lanes, indicating that the digestions were complete. Conversely, controls were also included in which reactions were prepared as the other samples but were not digested with RNase. Without digestion, only full-length probes were observed. RPA gels were dried and exposed to film. Band intensities of protected signals were quantified by a laser densitometer (Molecular Dynamics). The relative mRNA abundance in each sample was expressed as the arbitrary units of the p53 or MT-II band per arbitrary unit of cyclophilin or ß-actin in the same RPA reaction.
Statistics.
Values are means ± SEM. Differences were considered
significant at P < 0.05. The data were analyzed
using one-way ANOVA and Duncan’s new multiple range test
(Jaccard and Becker 1990
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HepG2 cells were cultured for nearly one passage in two types of
zinc-reduced media. Culture in both types of zinc-deficient
media resulted in significant reductions in cellular zinc. Cellular
zinc levels were expressed per cellular DNA to correct for any
differences in cell numbers between plates. Growth was not affected by
the medium zinc concentration because no significant differences in DNA
were observed among treatment groups (data not shown). Cellular zinc
was reduced to 27 or 64% in the Opti-MEM and Chelex studies,
respectively, compared with their zinc-adequate controls
(Figs. 1
,
2
). In the Opti-MEM experiments, culturing ZD cells for the last
24 h in ZA medium (ZDA cells) resulted in a significant increase
in cellular zinc to a level ~80% of the ZA cells (Fig. 1)
. For the
Chelex study, cells cultured in medium containing 16
µmol/L Zn (ZA16) had significantly higher cellular zinc
than both ZA4 and ZD cells (Fig. 2)
. Because Chelex can reportedly bind
copper under certain conditions, we also analyzed cellular copper level
but found no differences among groups (data not shown).
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p53 mRNA abundance was higher in the ZD cells in both studies. The p53
mRNA abundance in the ZD cells of the Opti-MEM study was almost
twofold higher than the level found in ZA cells (Fig. 3
). Similarly, ZD cells in the Chelex study exhibited >100% higher
levels of p53 mRNA compared with their ZA4 controls (Fig. 4
). Zinc replenishment of ZD cells (ZDA cells) in the Opti-MEM study
resulted in p53 mRNA levels not different from those of ZA controls
(Fig. 3)
. Nevertheless, the p53 mRNA abundance was significantly
different between the ZA16 and ZD cells (Fig. 4)
. The mRNA abundance of
the zinc-responsive MT-II is also shown for the Chelex study.
MT-II mRNA levels were drastically lower in the ZD cells than those
found in ZA4 cells (Fig. 5
). In addition, MT-II was also sensitive to increasing levels of
cellular zinc because MT-II mRNA was >100% higher in ZA16 cells
than in ZA4 cells (Fig. 5)
.
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Western blot analysis of nuclear extracts was used to quantitate
nuclear p53 protein levels in each of the studies. In the Opti-MEM
study, nuclear p53 protein in ZD cells was significantly higher than in
both ZA and ZDA cells (Fig. 6
). One-day zinc replenishment resulted in reducing p53 to levels
lower than both ZA and ZD cells (Fig. 6)
. However, no significant
differences were detected in nuclear p53 protein among the treatment
groups in the Chelex study (ZA, 100 ± 16%; ZD, 95 ± 10%;
ZA16, 107 ± 19%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and HepG2 cells
(Wu et al. 1999
). Previous work in our laboratory
(Wu et al. 1999
) demonstrated that HepG2 cells could be
cultured in Zn-free Opti-MEM with 1% FBS for one passage without
altering growth or morphology. Therefore, the combination of
Zn-free Opti-MEM and 1% FBS constituted our ZD medium. For the ZA
treatment group, zinc in the form of ZnSO4 was
added so that the level of zinc in ZA medium was equal to that found in
Opti-MEM with 10% FBS. Therefore, the only difference between ZA
and ZD media was zinc concentration. Because Opti-MEM had been used
successfully to examine zinc-regulated expression of
metallothionein (Palmiter 1994
) and apolipoprotein
A-I (Wu et al. 1999
), we chose to use Opti-MEM
to examine the response of p53 to alterations in cellular zinc.
For the second study, we utilized Chelex 100, a divalent ion–chelating
resin that has been used extensively in biological research for
numerous applications (Flynn and Yen 1981
, Messer et al. 1982
, Prasad et al. 1996
). To avoid
exposing cells directly to the chelating resin, FBS was incubated with
Chelex and then removed before FBS was used for the cell culture
medium. This provided an effective yet noninvasive technique to control
zinc concentrations in medium because serum is the major source of zinc
in most culture media. A separate experiment demonstrated that HepG2
cells cultured in ZA4 treatment medium (medium containing Chelexed FBS
and ZnSO4 added as a supplement) for three
consecutive passages did not appear to be morphologically distinct from
control cells cultured in normal medium (data not shown). Even though
Chelex has a high affinity for zinc, it has also been reported to
sequester other divalent metals under certain conditions (Prasad et al. 1996
). Because of the divalent properties of copper and
the interrelationships of copper and zinc, we also measured
intracellular copper levels in each group. No significant differences
in cellular copper existed (data not shown). Although we acknowledge
that other divalent ions besides zinc may be affected by Chelex, levels
of the ions would be the same for all groups. Therefore, we believe our
findings are due to differences in cellular zinc status. As an
additional marker of cellular zinc status, we also measured MT-II
mRNA abundance in the Chelex study. The regulation of the MT genes by
zinc has been well documented and is mediated by metal responsive
elements located within their promoters (Hamer 1986
).
Indeed, we found that MT-II mRNA abundance was related to cellular
zinc levels because ZA4 MT-II mRNA was dramatically higher than ZD
cells and ZA16 MT-II mRNA abundance was significantly higher than
ZA4.
HepG2 cells have been used extensively as a model with which to study
p53 because of several key attributes that make them distinct from many
other hepatic cell lines. Of seven hepatic cell lines recently
examined, the hepatoblastoma-derived HepG2 cells were the only cell
line in which the p53 gene and its expression appeared to be unaltered
(Bressac et al. 1990
). Other reports have indeed
confirmed that HepG2 cells express a wild-type p53 protein
(Hsu et al. 1993
) that can be activated to elicit normal
p53 function (Muller et al. l977). Because of these characteristics and
our ability to deplete cellular zinc from HepG2 cells, we believe this
model could provide important information about how a nutrient such as
zinc may affect the expression of p53.
In the current studies, reductions in cellular zinc were associated
with an increase in cellular p53 mRNA abundance regardless of the
method used to deplete cellular zinc. Zinc depletion in the
Opti-MEM study (reduced to 27% of controls) was slightly more
drastic than in the Chelex study (64% of controls). It is possible
that the efficiency of zinc depletion between the two studies is
different because of differences in the serum concentrations used;
however, we have not determined the mechanism. Of interest was that the
p53 mRNA was higher relative to controls (nearly 200%) in the
Opti-MEM study compared with the Chelex study (>100%).
Replenishing zinc levels in ZD cells in the Opti-MEM study reduced
p53 mRNA to control levels even though zinc was replenished to only
80% of controls. For the Chelex study, culturing cells in ZA16 media
did not significantly affect p53 mRNA abundance even though MT-II
mRNA was elevated by the increase in zinc. Relative differences in
cellular zinc from controls were comparable for the ZA16 and ZD cells
(137 and 64%, respectively) in the Chelex study; however, the
regulation of p53 mRNA may be more sensitive to decreases in cellular
zinc than to increases in cellular zinc. Because zinc is relatively
nontoxic, it would seem unlikely that p53 expression would be affected
by moderate increases in cellular zinc. This is supported by the
findings that moderately high and very high levels of zinc are required
to modulate apoptosis (Fraker and Telford 1996
).
However, because p53 is a stress-response factor, one or more types
of cellular stress (e.g., oxidative stress, DNA damage) may have been
imposed by the level of depletion in our studies and up-regulated
p53 expression.
Although data from both of our studies showed that p53 mRNA was higher
in zinc-depleted cells, the mechanism responsible for these changes
remains to be determined. Several investigators have reported that zinc
deficiency is capable of inducing cellular oxidative stress, depending
on the severity of the deficiency and the model or cell line used
[reviewed by Bray and Bettger (1990)
]. One factor that
we suspect may be involved in the observed changes for p53 expression
is the well-described nuclear factor (NF)-
B. Because NF-B is
sensitive to cellular reactive oxygen species [reviewed by
Schreck et al. (1992)
], and has been shown to bind to
and regulate p53 promoter activity (Wu and Lozano 1994
),
it would seem to be a likely candidate as a regulatory factor in these
conditions. Electrophoretic mobility shift assays and transfection
experiments using p53 promoter sequences must be performed under these
zinc-deficient conditions to identify regulatory factors and
elements affected by zinc status.
Nuclear p53 protein levels were also higher in the ZD cells of the
Opti-MEM study. It is interesting that the increase in nuclear p53
protein did not affect cell growth. As postulated in a recent review by
Meplan et al. (1999)
, several aspects of p53 may be
affected by a deficiency in cellular zinc. The authors point out that
p53 is sensitive to oxidative stress because of key cysteine residues
present in both the zinc coordination motif as well as the
DNA-binding domain. Studies using metal chelators as well as
oxidizing agents have shown that zinc chelation (Verhaegh et al. 1998
) and oxidation (Hainaut and Milner 1993
)
cause p53 to adopt a "mutant-like" form with decreased
DNA-binding activity. It should also be noted that p53 also
exhibits functions other than suppressing growth, including the
regulation of genes involved in the oxidative stress response as well
as other cytotoxic stresses. The finding that there was no significant
difference in p53 protein between ZA and ZD cells of the Chelex
experiments was interesting and somewhat surprising in light of the
Opti-MEM data. Perhaps these findings were related to a less
drastic zinc depletion in the Chelex ZD cells (64% of controls)
compared with the ZD cells in the Opti-MEM study (27% of
controls). Indeed, the increase in p53 mRNA was less dramatic in Chelex
ZD cells compared with Opti-MEM ZD cells, and this smaller change
in mRNA may be responsible at least in part for the lack of change in
the p53 protein level. A distinct post-transcriptional regulatory
mechanism, one that is less sensitive to zinc deprivation, may also be
involved in controlling nuclear p53 protein levels.
Cellular zinc is thought to exist either in stable, fixed pools or in
more dynamic, labile pools. Many researchers believe that these labile
pools regulate important cellular processes, including gene expression,
and are influenced by zinc supplementation or deprivation. In a recent
study, cultured cells were exposed to a membrane-permeable chelator
with a high affinity for zinc; p53 conformation was altered and
DNA-binding activity was decreased (Verhaegh et al. 1998
). At this point, however, it is difficult to determine
whether these labile pools of zinc may be affected differently
depending on whether a cell-permeable chelator or a strategy such
as the one utilized for these studies is used to deplete cellular zinc.
Obviously, integrating findings from studies using
membrane-permeable chelators with our present findings is
difficult, and each strategy has distinct advantages. However, we
believe our strategy of depleting zinc in the media or sera, the
extracellular environment of these cells, better reflects suboptimal
plasma zinc levels, a common characteristic of dietary zinc deficiency.
To the best of our knowledge, this is the first reported use of a
nonchelator strategy (which did not involve the culture of cells with
chelators) to examine how depletion of cellular zinc affects p53 mRNA
and protein levels.
In these studies, we examined the effects of zinc depletion on p53 mRNA abundance and nuclear p53 protein levels. Using the human hepatoblastoma HepG2 cell line, which expresses moderate levels of wild-type p53, we showed that zinc depletion increases p53 mRNA abundance. Zinc depletion in the Opti-MEM study showed a concomitant increase in nuclear p53 protein levels. Furthermore, zinc repletion of zinc-deficient cells resulted in a return of p53 mRNA abundance to control levels and lower levels of p53 protein than that of controls in the Opti-MEM study. On the basis of these results, we propose that the expression of p53 may be responsive to cellular zinc status under certain conditions. One potential implication for this study is that zinc deficiency may reduce the ability of p53 to protect cells from carcinogenic compounds or conditions such as radiation. On the basis of these studies, it appears that zinc depletion alters normal p53 expression; work from other laboratories indicates that zinc is crucial to maintain wild-type p53 conformation and DNA-binding activity. Therefore, compromised cellular zinc status may possibly enhance the susceptibility of an organism to cancer by attenuating the tumor-suppressive activity of p53.
|
FOOTNOTES |
|---|
2 Supported by the Arizona Disease Control Research Commission (S.K.R.) and funds from the University of Arizona Agricultural Experiment Station (K.Y.L.).
4 Abbreviations used: DMEM, Dulbecco’s modified Eagle medium; DTT, dithiothreitol; FBS, fetal bovine serum; MT, metallothionein; NF, nuclear factor; PMSF, phenylmethylsulfonylflouride; RPA, ribonuclease protection assays;
RT-PCR, reverse transcriptase-polymerase chain reaction; TBS,Tris-buffered saline; ZA, zinc-adequate; ZD, zinc-deficient; ZA4, zinc-adequate media containing 4.0 µmol/L Zn;
ZA16, zinc-adequate media containing 16 µmol/L Zn; ZDA, zinc-replenished.
Manuscript received December 16, 1999. Initial review completed February 4, 2000. Revision accepted March 23, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. Bray T. M., Bettger W. J. The physiological role of zinc as an antioxidant. Free Radic. Biol. Med. 1990;8:281-291[Medline]
2.
Bressac B., Galvin K. M., Liang T. J., Isselbacher K. J., Wands J. R., Ozturk M. Abnormal structure and expression of p53 in human hepatocellular carcinoma. Proc. Natl. Acad. Sci. U.S.A. 1990;87:1973-1977
3. Donehower L. A., Bradley A. The tumor suppressor p53. Biochim. Biophys. Acta 1993;1155:181-205[Medline]
4. Elmes M. Apoptosis in the small intestine of zinc deficient and fasted rats. J. Pathol. 1977;123:219-224[Medline]
5. Flynn A., Yen B. R. Mineral deficiency effects on the generation of cytotoxic T-cells and T-helper cell factors in vitro. J. Nutr. 1981;111:907-913
6. Fraker P. J., Telford W. Regulation of apototic events by zinc. Berdanier C. D. eds. Nutrients and Gene Expression, Clinical Aspects 1996:189-208 CRC Press Boca Raton, FL.
7.
Graeber T. G., Peterson J. F., Tsai M., Monica K., Fornace A .J., Jr, Giaccia A. J. Hypoxia induces accumulation of p53 protein, but activation of a G-1 phase checkpoint by low oxygen conditions is independent of p53 status. Mol. Cell. Biol. 1994;14:6264-6277
8.
Greenblatt M. S., Bennett W. P., Hollstein M., Harris C. C. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54:4855-4878
9.
Hainaut P., Milner J. Redox modulation of p53 conformation and sequence-specific DNA binding in vitro. Cancer Res 1993;53:4469-4473
10. Hamer D. H. Metallothionein. Annu. Rev. Biochem. 1986;55:913-951[Medline]
11.
Hollstein M., Sidransky D., Vogelstein B., Harris C. C. p53 mutations in human cancers. Science (Washington, DC) 1991;253:49-53
12.
Hsu I. C., Tokiwa T., Bennett W., Metcalf R. A., Welsh J. A., Sun T., Harris C. C. p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines. Carcinogenesis 1993;14:987-992
13. Jaccard J., Becker M. A. Statistics for the Behavioral Sciences 2nd ed. 1990:304-329 Wadsworth Publishing Belmont, CA.
14. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 in the cellular reponse to DNA damage. Cancer Res 1991;51:6304-6311[Medline]
15. Lane D. P. Cancer. p53, guardian of the genome. Nature (Lond.) 1992;358:15-16[Medline]
16.
Linke S. P., Clarkin K. C., DiLeonardo A., Tsou A., Wahl G. M. A reversible, p53-dependent GO/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage. Genes Dev 1996;10:934-947
17. Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A., Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature (Lond.) 1993;362:847-849[Medline]
18.
Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265-275
19. Meplan C., Richard M.-J., Hainaut P. Redox signalling and transition metals in the control of the p53 pathway. Biochem. Pharmacol. 1999;59:25-33
20. Messer H. H., Murray E. J., Goebel N. K. Removal of trace metals from culture media and sera for in vitro deficiency studies. J. Nutr. 1982;112:652-657
21. Muller M., Strand S., Hug H., Heinemann E. M., Walczak H., Hofmann W. J., Stremmel W., Krammer P. H., Galle P. R. Drug-induced apoptosis in hepatoma cells is mediated by the the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53. J. Clin. Investig. 1997;99:403-413[Medline]
22.
Palmiter R. D. Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc. Natl. Acad. Sci. U.S.A. 1994;91:1219-1223
23. Prasad A. S., Beck F.W.J., Endre L., Handschu W., Kukuruga M., Kumar G. Zinc deficiency affects cell cycle and deoxythymidine kinase gene expression in HUT-78 cells. J. Lab. Clin. Med. 1996;128:51-60[Medline]
24. Sandstead H. H. Is zinc deficiency a public health problem?. Nutrition 1995;11:87-92[Medline]
25. Schreck R., Albermann K., Baeuerle P. A. Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free Radic. Res. Commun. 1992;17:221-237[Medline]
26.
Schreiber E., Matthias P., Muller M. M., Schaffner W. Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res 1989;17:6419-6421
27.
Sullivan V. K., Cousins R. J. Competitive reverse transcriptase-polymerase chain reaction shows that dietary zinc supplementation in humans increases monocyte metallothionein mRNA levels. J. Nutr. 1997;127:694-698
28. Sun X., Shimizu H., Yamamoto K.-I. Identification of a novel p53 promoter element involved in genotonic stress-inducible p53 gene expression. Mol. Cell. Biol. 1995;15:4489-4496[Abstract]
29. Verhaegh G. W., Parat M.-O., Richard M.-J., Hainaut P. Modulation of p53 protein conformation and DNA-binding activity by intracellular chelation of zinc. Mol. Carcinog. 1998;21:205-214[Medline]
30. Williams D. L., Newman T. C., Shelness G. S., Gordon D. A. Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes. Methods Enzymol 1986;128:671-689[Medline]
31.
Wu H., Lozano G. NF-B activation of p53. A potential mechanism for suppressing cell growth in response to stress. J. Biol. Chem. 1994;269:20067-20074
32.
Wu J.Y.J., Reaves S. K., Wang Y. R., Wu Y., Lei P. P., Lei K. Y. Zinc deficiency decreases plasma level and hepatic mRNA abundance of apolipoprotein A-I in rats and hamsters. Am. J. Physiol. 1998;275:C1516-C1525
33.
Wu J.Y.J., Wu Y., Reaves S. K., Wang Y. R., Lei P. P., Lei K. Y. Apolipoprotein A-I gene expression is regulated by cellular zinc status in HepG2 cells. Am J. Physiol. 1999;277:C537-C544
34. Yin Y., Terauchi Y., Soloman G. G., Aizawa S., Rangarajan P. N., Yazaki Y., Kadowaki T., Barrett J. C. Involvement of p85 in p53-dependent apoptotic response to oxidative stress. Nature (Lond.) 1998;391:707-710[Medline]
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