Metallothionein Is Part of a Zinc-scavenging Mechanism for Cell
Survival under Conditions of Extreme Zinc Deprivation*
David A.
Suhy
§,
Kathryn D.
Simon¶,
Daniel I. H.
Linzer
, and
Thomas V.
O'Halloran
¶
From the
Department of Biochemistry, Molecular
Biology, and Cell Biology and the ¶ Department of Chemistry,
Northwestern University, Evanston, Illinois 60208
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ABSTRACT |
Metallothionein (MT) is a small cysteine-rich
protein thought to play a critical role in cellular detoxification of
inorganic species by sequestering metal ions that are present in
elevated concentrations. We demonstrate here that metallothionein can
play an important role at the other end of the homeostatic spectrum by
scavenging an essential metal in a mouse fibroblast cell line that has
been cultured under conditions of extreme zinc deprivation (LZA-LTK
). These cells unexpectedly produce
constitutively high levels of metallothionein mRNA; however, the MT
protein accumulates only when high concentrations of zinc are provided
in the media. Until this MT pool is saturated, no measurable zinc
remains in the external media. In this case, zinc deprivation leads to
amplification of the MT gene locus in the LZA-LTK
cell
line. Furthermore, the intracellular zinc levels in the fully adapted
cells remain at the normal level of 0.4 fmol zinc/cell, even when
extracellular zinc concentration is decreased by 2 orders of magnitude
relative to normal media.
 |
INTRODUCTION |
Zinc is an ubiquitous and essential component in biological
systems. Iron is the only other transition metal that is more abundant
in humans; however, if one subtracts the amount of iron in hemoglobin,
zinc becomes the most abundant transition metal (1). Zinc has been
identified as a central component of over 300 enzymes and plays an
essential structural function in an entire class of transcription
factors. The biological essentiality of zinc implies the existence of
homeostatic mechanisms that regulate its absorption, distribution,
cellular uptake, and excretion (2). However, until recently little was
known about how these processes occur within the cell or about the
molecules that mediate their action (3).
Metallothionein (MT)1 is an
abundant zinc-binding protein and one of the few eukaryotic proteins
identified as having an essential role in heavy metal detoxification.
Copious levels of MT protein and mRNA are found in organisms and
tissues exposed to high levels of zinc or cadmium (4). Transcriptional
activation of the MT genes in response to high concentrations of these
potentially toxic metals is mediated through trans-acting proteins that
bind to DNA regulatory elements located upstream of the MT gene coding sequences (5-8). These metal-responsive elements (MREs) are necessary and sufficient to confer zinc- and cadmium-responsive activation of MT
genes (9). Each MT protein molecule may bind up to seven atoms of
either zinc or cadmium, where each metal is tetrahedrally coordinated
to cysteine residues. Because newly synthesized MT proteins sequester
the inducing metals, elevated tissue concentrations of the ions are
often detected upon metal exposure (10-12). Conversely, MT protein and
mRNA levels generally decrease in a dose-dependent manner in tissues of animals fed zinc-restrictive diets (13-15). Additional studies showed that moderate maternal zinc deficiency in
rats during pregnancy and lactation results in the reduced expression
of MT in the livers of their pups (12, 16-18). Such results have led
to suggestions that the MT protein may serve to buffer intracellular
zinc levels (12).
We have described the methods used to generate a cell line derived from
mouse fibroblasts (L-M(TK
)), herein designated as
LTK
, which survives conditions of extreme zinc
deprivation (19). Previous attempts at establishing similar
zinc-deficient cell lines by other laboratories were unsuccessful in
producing a system that would survive beyond a few passages of growth
in medium containing less than 0.1 µM zinc (20-24). This
cell line, designated as low zinc-adapted (LZA-LTK
)
survives continuous passage in media that contain less than 60 nM zinc and thus provides a useful system to study
fundamental components of the cellular machinery responsible for zinc
homeostasis. Furthermore, despite continuous culture in these
conditions of extreme zinc deprivation, these cells remarkably
maintained intracellular zinc at levels comparable with the parental
LTK
cell line grown in normal media containing 1-5
µM zinc (19).
In this paper, we present evidence that MT plays a role in the
maintenance of intracellular zinc at the standard 0.4 fmol zinc/cell
level in the LZA-LTK
cells, thus identifying a
zinc-scavenging role for the protein. Unlike the studies in which
animals were fed zinc-restrictive diets (12, 16-18), MT mRNA is
not degraded in the LZA-LTK
cell line; instead, it is
constitutively present at high levels. Our results suggest that the
overexpression of MT is one step that enhances the survival of the
LZA-LTK
cells in conditions of extreme zinc deprivation.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
BALB/c 3T3 and LTK
cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
calf serum, 5 mM glutamine, and 30,000 units/liter of
penicillin/streptomycin (all manufactured by Life Technologies, Inc.).
Batch treatments of Chelex (Bio-Rad) were used to strip the media and
other solutions of divalent cations. The Chelex resin was prepared
according to the manufacturer's instructions with neutralization
of the resin to physiological pH and multiple washings with 0.25 M HEPES, pH 7.4, before its addition to media (at 40 g/liter) and other solutions (at 20 g/liter) with constant stirring at
4 °C for 20-24 h. Aliquots of the solutions were removed prior to
and after Chelex treatment, and the concentrations of divalent cations
in the samples were determined using an inductively coupled
plasma-atomic emission spectrophotometer (ICP-AES) on an Atomscan 25 (Thermo Jarrell Ash) with Thermospec software (version 5.02). Divalent
cations other than zinc were restored to their normal concentrations by
the addition of appropriate metal chloride salts. Sterilization of the
media following Chelex treatment was accomplished by filtering through
a 0.2 µM Nylon Media Plus filter unit (Nalgene).
Measurement of Intracellular Zinc--
LZA-LTK
cells were passaged into zinc-deficient medium supplemented with
various concentrations of zinc chloride from a 5 mM stock
solution. Following growth, the media were decanted and saved for zinc
analysis by ICP-AES. The cells were washed three times with
metal-stripped 1× phosphate-buffered saline (PBS) (136 mM
NaCl, 2.6 mM KCl, 10.14 mM
Na2HPO4, 1.76 mM
KH2PO4, pH 7.4) to remove the contaminating
traces of zinc. The cells were scraped into metal-stripped 1× PBS and
were pelleted at 14,000 × g for 1 min. The cell pellet
was dried overnight in a 65 °C oven, digested with 150 µl of
concentrated nitric acid at 80 °C for 15 min, and diluted to 1.5 ml
with distilled H2O prior to analysis on the ICP-AES.
RNA and DNA Filter Hybridization--
RNA was isolated from cell
cultures lysed with guanidinium thiocyanate by sedimentation through a
cesium chloride cushion (25). Equivalent amounts of RNA were
fractionated on formaldehyde-agarose gels (26), transferred to
nitrocellulose, and hybridized with radiolabeled cDNA probes for
mouse MT-I (a gift from Dr. Richard Palmiter) or GAPDH (a gift from Dr.
Richard Morimoto). Genomic DNA was purified from cells lysed with
SDS/proteinase K solution by phenol/chloroform extraction (27). DNA
digested with EcoRI was fractionated by agarose gel
electrophoresis, transferred to nitrocellulose, and probed with
radiolabeled mouse MT-I or GAPDH cDNA. The nitrocellulose filters
were washed and exposed to x-ray film at
70 °C with an
intensifying screen.
[35S]Cysteine Labeling of MT
Proteins--
LZA-LTK
cells were grown in various
concentrations of zinc ([Zn]media) and labeled with 10 µCi of [35S]cysteine (Amersham Pharmacia Biotech) for
24 h. The radioactive media were decanted, and the cells were
washed several times with metal-stripped 1× PBS. Cells were scraped
into a lysis solution and were vigorously vortexed (27). After brief
centrifugation to rid the extract of membranes, the soluble extract was
carboxymethylated with 12.5 mM iodoacetate, 50 mM Tris, pH 8.0, at 50 °C for 15 min (28). Equivalent
amounts of protein per lane, as determined by the Bradford method, were
electrophoresed utilizing Tricine-based PAGE (29). Following
electrophoresis, the gels were fixed, treated with En3hance
(DuPont), and dried prior to autoradiography.
Subcellular Fractionation--
LZA-LTK
cells that
had been grown in various [Zn]media were washed with
metal-stripped 1× PBS. Cells were scraped into metal-stripped 1× PBS,
pelleted by centrifugation, and resuspended in 0.25 M sucrose buffered with 5 mM MOPS, pH 7.4. Whole cell zinc
levels were measured by ICP-AES using an aliquot of the resuspended
pellet that had been digested with nitric acid. Other cell aliquots
were passed three times through a stainless steel syringe filter holder that was lined with two polycarbonate 16.0-µm filters (Osmonics).
-Hexosaminidase activity assays, which test for the integrity or
latency of the organelles, were conducted on aliquots of each homogenate (30). Differential velocity sedimentation resulted in four
fractions: 1) P1 (pellet from homogenate spun at 1,000 × g, 10 min), which typically contains nuclei and unbroken
cells; 2) P2 (pellet from the P1 supernatant spun at 10,000 × g, 10 min) containing large intracellular membranes; 3) P3
(pellet from the P2 supernatant spun at 100,000 × g,
60 min), which typically contains smaller intracellular membranes and
vesicles; and 4) the soluble fraction or cytosol (the supernatant from
the 100,000 × g, 60-min spin) (31). As with the total
intracellular zinc measurements, the pellets from each of the fractions
were dried overnight, dissolved in concentrated nitric acid, diluted in
distilled H2O, and analyzed for zinc content by
ICP-AES.
Gel Filtration Column Chromatography--
Cytosols from
subcellular fractionation experiments were chromatographed on a G-75
Sephadex superfine size exclusion column (Amersham Pharmacia Biotech)
utilizing 20 mM Tris, pH 7.8, as the running buffer. In the
cadmium saturation analyses, a portion of the cytosol or purified horse
MT (Sigma) was incubated with a 5-fold molar excess of cadmium
chloride, relative to zinc concentration of the sample, in the presence
of 5 mM
-mercaptoethanol at 37 °C for 1 h
immediately prior to sample application to the column (32, 33). Zinc
and/or cadmium content of the resultant fractions was measured via
ICP-AES.
DNA Transfection and Reporter Construct Assays--
RSV-CAT and
MRE-
Geo (a gift of Richard Palmiter) plasmids were transiently
transfected into cells via the DEAE-dextran method (34). After 3 days
of growth, cells were harvested and split into samples for the reporter
construct assays. The Hoechst 33258 staining assay was used to measure
the amounts of DNA per plate of cells (7).
-Galactosidase activity
from the MRE-
Geo was measured as cleavage of an
O-nitrophenyl
-galactopyranoside substrate and expressed
as A405 per µg of DNA from the harvested
cells. CAT activity from the RSV-CAT construct was measured as the
percentage of acetylation of a radiolabeled chloramphenicol substrate
(35).
 |
RESULTS |
LZA-LTK
Cells Maintain Basal Zinc Levels When Grown
in Zinc-deficient Conditions and Are Efficient Scavengers of the
Metal--
The ability of the LTK
and
LZA-LTK
cell lines to accumulate Zn(II) was
examined in response to the addition of a wide range of zinc
concentrations in external media ([Zn]media).
LTK
cells and LZA-LTK
cells were seeded
into unmanipulated media and zinc-stripped media (19), respectively,
supplemented with various concentrations of ZnCl2.
Following growth to near confluency, samples of the media were
collected, and the cells were washed several times with PBS, which had
been treated with Chelex to remove residual traces of zinc. Cells from
each plate were harvested, counted, and then lysed in concentrated
nitric acid. The amount of zinc per cell was determined at each
[Zn]media by ICP-AES. Basal levels of intracellular zinc
in the parental LTK
cells were measured to be 0.44 ± 0.09 fmol zinc/cell in n = 4 experiments (Fig.
1A). There was little increase
in intracellular zinc levels in LTK
cells until
[Zn]media exceeded 20 µM. A 3.5-fold
increase of intracellular zinc concentration was observed in the
presence of the maximum amount of zinc added into the medium (50 µM [Zn]media). Consistent with
previous observations (19), LZA-LTK
cells grown in
unsupplemented zinc-deficient medium maintained basal intracellular
zinc levels (0.54 ± 0.13 fmol zinc/cell n = 4 experiments) similar to those measured in the LTK
cells
(Fig. 1A). As [Zn]media was increased,
however, the intracellular zinc levels within the LZA-LTK
cells increased substantially, reaching a maximal level of 8.68 ± 0.86 fmol zinc/cell when [Zn]media was 3.5 µM (a 16-fold increase). Additional zinc supplemented
into the media, up to 50 µM, resulted in no further
increase in intracellular zinc levels.

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Fig. 1.
Measurements of intracellular zinc from
LZA-LTK and LTK cells grown in various
[Zn]media. A, after 3 days of growth,
LZA-LTK (open circles) and
LTK cells (filled circles) cells
were harvested, counted, and measured for intracellular metal content
by ICP-AES. S.D. was calculated from n = 3 plates
at each metal concentration. B, the zinc remaining in the
spent media from the LZA-LTK (open
circles) and LTK cells (filled
circles) was determined by ICP-AES. The straight
dashed line, with a slope of 1, represents the
amount of zinc that would be present in the media if growth of the
cells did not perturb the zinc concentrations.
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The concentration of zinc remaining in the spent medium following
growth was also measured by ICP-AES (Fig. 1B). The
dashed line represents the concentration of zinc expected in
spent media if the growth of the cells did not significantly perturb
[Zn]media; this was the case with the LTK
cell line. Intriguingly, no measurable zinc was observed in the spent
media from LZA-LTK
cells grown with
[Zn]media < 3.5 µM. Furthermore, there was
a striking relationship between the amount of intracellular zinc in
LZA-LTK
cells and the concentration of extracellular zinc
remaining in spent media (Fig. 1, A and B).
Although measurable amounts of metal were added, zinc was undetectable
in spent media until the intracellular zinc levels in the
LZA-LTK
cells reached the "plateau" level of
approximately 8.6 fmol zinc/cell. One interpretation of these data is
that zinc-scavenging mechanisms are present in the
LZA-LTK
cells that enhance uptake and intracellular
sequestration of zinc from the extracellular medium and that this
mechanism fills the zinc storage molecules with all available
environmental zinc until these sites are saturated.
High Constitutive Levels of MT RNA Are Present in
LZA-LTK
Cells at All
[Zn]media--
Intracellular metal concentrations of
zinc or cadmium are often directly proportional to the amount of MT
protein present within cells (36-38). In the LTK
cells,
the rate of increase in intracellular zinc levels as a function of
[Zn]media is similar to previously published patterns for
metal-induced MT gene transcription (39). However, the
LZA-LTK
cells exhibit a strikingly different
intracellular zinc profile in response to increased extracellular zinc.
MT mRNA levels were therefore examined in both cell lines to
determine what role, if any, MT plays in assisting the cell to scavenge
zinc from the media. As expected from previous studies, MT mRNA
levels rise in LTK
cells in response to elevated
[Zn]media (>20 µM) in a
dose-dependent manner that is presumably mediated through
transcriptional activation of the MREs (Fig.
2A). A 5-fold induction of MT
mRNA was detected between LTK
cells grown in the
lowest and highest [Zn]media (Fig. 2B). In contrast, high, constitutive levels of MT mRNA were present in LZA-LTK
cells regardless of the external
[Zn]media (Fig. 2A). There were no significant
[Zn]media-dependent changes in the high
levels of MT mRNA in LZA-LTK
cells (Fig.
2B).

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Fig. 2.
MT mRNA levels in LTK or
LZA-LTK cells that were grown in various
[Zn]media. A, each lane
contains 10 µg of total RNA, which was separated on
formaldehyde-agarose gels. Following transfer to nitrocellulose, the
blot was hybridized with 32P-radiolabeled MT-I or GAPDH
cDNA. B, quantitative PhosphorImager analyses of MT
mRNA levels from the LTK (filled
circles) and LZA-LTK (open
circles) cell lines. The hybridization intensity of the
radiolabeled GAPDH provided the basis of normalization between lanes
within each set.
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MT Protein Levels Do Not Correspond to MT mRNA Levels in
LZA-LTK
Cells--
To determine if the constitutively
elevated MT mRNA levels in the LZA-LTK
cells result
in correspondingly high steady-state MT protein levels, metabolic
labeling studies were conducted. The high cysteine content of MT and
relatively low molecular weight (~10 kDa) make the protein readily
detectable in [35S]cysteine labeling experiments.
LTK
and LZA-LTK
cells were grown in
[Zn]media similar to those used in the RNA and
intracellular zinc measurements, and [35S]cysteine was
added 24 h prior to harvesting to achieve steady-state labeling.
Extracts were carboxymethylated to prevent oxidation of MT sulfhydryl
groups (28), and equivalent amounts of protein were loaded and resolved
on Tricine-based SDS-PAGE gels (29). In the parental LTK
cell line (Fig. 3A), increases
in [Zn]media lead to increases in MT as well as increases
in intracellular zinc and MT RNA levels (Figs. 1A and
2B, respectively). Each of these parameters remained low
until [Zn]media was 20 µM or greater. The
increase in MT protein levels occured at lower [Zn]media
in the LZA-LTK
cell line and paralleled the rise in
intracellular zinc in LZA-LTK
cells at each
[Zn]media (Fig. 1B). Grown at a wide range of
[Zn]media, the amount of MT protein in cells grown in the
lower zinc concentrations was relatively low. It is possible that
differential translation rates of the MT mRNA might contribute to
the differences in MT protein abundance in the LZA-LTK
cell line at the different [Zn]media conditions. However,
it is more likely that differences in the rates of degradation of the
apoprotein and the metal-bound protein account for the observed steady-state protein levels. Specifically, Klaassen et al.
(40) have shown in proteolysis experiments performed in lysosomal
extracts that apo-MT, MT complexed with Cd(II), or MT complexed with
Zn(II) had rates of degradation of 50, 200, 35, and 20 pmol/mg of
lysosomal protein/min, respectively. Thus, in the absence of enough
saturating metal to stabilize the protein, it is expected that apo-MT
will be rapidly proteolyzed in the LZA-LTK
cells.

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Fig. 3.
MT protein levels from LTK and
LZA-LTK cells grown at various
[Zn]media. Following steady-state labeling with 10 µCi of [35S]cysteine for 24 h, LTK
(A, filled circles) and
LZA-LTK cells (B, open
circles) were harvested, and extracts were prepared.
Following carboxymethylation, equivalent amounts of protein per lane
were separated on Tricine-based polyacrylamide gels. The gels were
treated with En3hance prior to autoradiography. The data
from quantification of the gels by scanning densitometry are shown
below each autoradiogram.
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To test whether restriction of zinc availability leads to rapid
proteolytic turnover of MT protein, LZA-LTK
cells grown
in 20 µM [Zn]media and labeled for 24 h with [35S]cysteine were exposed to the intracellular
metal-ion chelator TPEN or the extracellular agents EDTA and EGTA for
various lengths of times. Dose-dependent and
time-dependent degradation of MT protein occurred in cells
treated with the intracellular chelator TPEN (Fig.
4). The t1/2 of MT
in cells treated with TPEN was approximately 90 min, while the
t1/2 for MT in the untreated cells was greater than the 4-h experiment. Other labeled proteins were not affected by
TPEN, suggesting that MT degradation is due to metal chelation and not
general degradation of all proteins. EDTA and EGTA, unable to traverse
the plasma membrane because of negatively charged moieties, had no
effect on the labeled MT although both have high affinities for
zinc.

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Fig. 4.
Effect of chelators of extracellular and
intracellular metals on MT levels in LZA-LTK cells grown
in 20 µM
[Zn]media. Following steady-state labeling with 10 µCi of [35S]cysteine, cells were treated with chelators
for the indicated times, the cells were harvested, and extracts were
prepared. Following carboxymethylation, equivalent amounts of protein
per lane were separated on a Tricine-based polyacrylamide gel. The gel
was treated with En3hance prior to autoradiography.
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The Majority of Zinc Accumulated by LZA-LTK
Cells Is Associated with MT--
To determine if MT protein was
directly involved in the increased zinc, the distribution of
intracellular zinc accumulation in the LZA-LTK
cell line
at various [Zn]media concentrations was examined.
Cells grown in various [Zn]media were subjected to
subcellular fractionation following disruption of the plasma membrane
by extrusion through polycarbonate membranes with pore sizes slightly
smaller than the diameters of the cells. Latency, a measurement of the
structural integrity of cellular organelles, was examined by
-hexosaminidase enzyme activity measurements on aliquots from the
postnuclear supernatants (30). Only samples with greater than 90%
latency were used in subsequent procedures to ensure that the
distribution of zinc among the fractions was not altered by
contamination from disrupted organelles. Additionally, a wide variety
of buffers were utilized to ensure that zinc was not being chelated
from the organelle fractions (data not shown). Subcellular
fractionation by differential velocity centrifugation was used to
partition the homogenate into four fractions (31). ICP-AES analyses
(Fig. 5A and Table
I) demonstrated that the majority of the
zinc taken up by the cells at increasing [Zn]media is
found in the cytosol. Additionally, there were small, but measurable,
increases in the other membranous compartments (Table I).

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Fig. 5.
Subcellular fractionation of
LZA-LTK cells grown up in various
[Zn]media, homogenized by pressure filtration, and then
separated into rough subcellular compartments by differential velocity
centrifugation. A, the amount of zinc within each
fraction was determined by ICP-AES and normalized to a per cell basis.
P1 is pellet from a low speed spin, typically containing nuclei, large
membranes, and unbroken cells; P2 and P3 are pellets from supernatants
obtained at increased spinning speeds, containing organelles,
intracellular membranes, and vesicles. The supernatant obtained from
the P3 spin is labeled cytosol. Cytosol fractions were split
into two halves and were further separated by gel filtration.
B, chromatographic profiles (Sephadex G-75) of the zinc
content of one-half of the cytosolic fraction measured by ICP-AES.
C, the other half of the cytosol was incubated with a 5-fold
molar excess of cadmium (as compared with zinc) in the presence of
thiols for 1 h and then separated on the G-75 Sephadex column. The
resultant fractions were measured for cadmium content by ICP-AES.
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The cytosolic fraction was further resolved by gel filtration
chromatography. The majority of zinc in the cytosolic fraction was
associated with a single peak that increased in parallel with [Zn]media (Fig. 5B) and eluted with an apparent molecular
mass of 10,000 daltons. This peak coincides with the elution profile of
purified cadmium-saturated MT (data not shown). The problems associated
with the detection of MT by Western blot analysis have been well
documented, and specific modifications have been suggested to improve
the performance of these assays (41). Even employing these changes, we
were still unable to detect MT by Western analysis in fractions
obtained by gel filtration or from commercially available purified MT
(data not shown). However, competitive displacement of MT-bound zinc by
Cd(II), which relies upon a rapid exchange of Cd(II) into the protein,
has frequently been used to quantify MT (42). Aliquots of the cytosols
were incubated with excess cadmium in the presence of thiols followed
by gel filtration chromatography. Zinc was completely removed from each
of the 10,000 molecular weight fractions and replaced with cadmium
(Fig. 5C). The zinc displaced from MT (data not shown),
along with excess cadmium, could be detected in the flow-through
fractions (peaks 28-33). These data indicate that the principle
zinc-binding species in the 10,000 fraction is metallothionein.
MT mRNA Levels Do Not Rapidly Increase in LTK
Cells Transferred to Zinc-deficient Medium--
It is possible that
the LZA-LTK
cell line results from the selection of a
subset of cells that grow in conditions of low zinc by increasing MT
expression. Alternatively, higher MT mRNA and protein levels may
result in most or all LTK
cells as a rapid response to
conditions of zinc deprivation. A rapid induction of MT in response to
zinc deficiency would suggest that overexpression of MT is a primary
response to deal with the starvation conditions. If the levels of MT
mRNA remain relatively constant in the initial hours of exposure to
zinc deficiency, then the high constitutive levels of MT mRNA
probably represent a long term adaptation of the LZA-LTK
cell line and suggest that other components of zinc homeostasis machinery are altered to respond to the zinc-deficient conditions. Northern blot analysis was performed on mRNA from LTK
cells isolated after transfer of the cells into zinc-deficient medium
for several days. Individual plates were harvested on each of the first
3 days. On the third day, an additional plate of the LTK
cells was passaged into several more plates containing zinc-deficient medium for time points extending to 124 h. No significant increase in MT mRNA occurred (Fig. 6),
indicating that increased MT expression is not a simple adaptive
response of LTK
cells to low zinc conditions and is
likely to have arisen from a genetic event.

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Fig. 6.
MT mRNA levels of LTK cells
after their transfer into zinc-deficient media. Each lane contains
10 µg of total RNA harvested at the indicated times, which was
separated on formaldehyde-agarose gels and then transferred to
nitrocellulose. The blots were probed with radiolabeled cDNA of
mouse MT-I or GAPDH, the latter used as a control for the amount of
total RNA loaded per lane.
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MT MRE Elements Are Not Constitutively Activated in
LZA-LTK
Cells--
Increased metallothionein gene
expression in cell lines adapted to growth in media containing high
concentrations of zinc and cadmium has been shown to result, in part,
from increased binding of factors to the metal-responsive elements
(43). Thus, we tested the possibility that the long term adaptation
leading to metallothionein message elevation in LZA-LTK
cells may be the result of strong constitutive activation of the MT
promoter through the metal-responsive elements. An MRE-driven
-galactosidase reporter construct (MRE-
Geo) (7) was transiently transfected into the LTK
and LZA-LTK
cell
lines, and
-galactosidase activity was examined as a function of
increasing [Zn]media. Differences in transfection
efficiencies between the two cell lines were controlled by
cotransfection with a RSV-CAT construct, which is under the regulation
of the strong, constitutively active Rous sarcoma virus long terminal
repeat (44). In the parental LTK
cell line,
zinc-dependent activation of the MRE-
Geo construct (Fig.
7A) paralleled the Northern
blot results in Fig. 2, indicating that the increase of metallothionein
message in this case is probably mediated through the metal-responsive
elements. In the LZA-LTK
cell line, however, there was no
indication of
-galactosidase activity for any of the
[Zn]media conditions (Fig. 7A). While CAT
activity assays demonstrated that the LZA-LTK
cells had
lowered transfection efficiencies relative to the LTK
cells (data not shown), it was not sufficiently decreased to account for the lack of
-galactosidase activity. At 50 µM [Zn]media, LTK
cells
possessed 100-fold more
-galactosidase activity than the LZA-LTK
cells grown at similar [Zn]media.
The lack of responsive transfected MRE elements led us to examine
other mechanisms for elevation of MT mRNA.

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Fig. 7.
Induction of the
MRE- Geo reporter construct in
LZA-LTK and LTK cells in response to
elevated [Zn]media. MRE- Geo and RSV-CAT plasmids
were transiently co-transfected into cells by the DEAE-dextran method.
After 3 days of growth, the cells were harvested and split into three
samples for the measurement of the DNA content, -galactosidase
activity, and CAT activity. A, -galactosidase activity
(normalized per µg of DNA from harvested cells) from
LTK (filled circles) and
LZA-LTK (open circles) cells, which
were grown for 24 h in increasing [Zn]media and then
co-transfected with 10 µg each of MRE- Geo and RSV-CAT plasmids.
B, -galactosidase activity of LTK
(filled circles) and LZA-LTK
(open circles) cells grown for 24 h at 50 µM [Zn]media and then co-transfected with
10 µg of RSV-CAT and increasing amounts of MRE- Geo.
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MT Gene Copy Number Is Amplified in LZA-LTK
Cells--
Another mechanism that can lead to increased levels of
specific mRNAs, selective gene amplification, was also examined.
Cell lines, including LTK
cells, have been shown to
amplify the copy number of the MT locus in response to continuous
exposure to high concentrations of cadmium or copper (27, 45-48).
Increased amounts of MT protein produced from the amplified genes
provide a selective advantage to the cell by binding and sequestering
toxic metals. To determine if the copy number of the MT-I gene had been
increased in the LZA-LTK
cell line relative to the
parental LTK
cells, Southern blot analysis was performed.
Fig. 8 shows an autoradiograph of
EcoRI-digested total DNA isolated from LZA-LTK
and LTK
cell lines and probed with radiolabeled MT-I
cDNA. The radiolabeled MT-I cDNA hybridized to a band of
approximately 4 kilobase pairs, a size consistent with other studies of
the murine MT-I gene locus isolated by EcoRI digestion (27).
Quantitative PhosphorImager (Molecular Dynamics, Inc.) analysis
showed that the LZA-LTK
cell line had 7-10 times the
number of the MT-I gene copies relative to the parental
LTK
counterpart, clearly demonstrating MT-I gene
amplification in the former. Analysis of the copy number of the GAPDH
gene indicated an equivalent number of that gene in both cell lines
(data not shown).

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Fig. 8.
Copy number of the MT-I gene in genomic DNA
isolated from LZA-LTK and LTK cells.
The DNA was digested with EcoRI and separated on a 0.75%
agarose gel in the quantities listed. Following transfer to
nitrocellulose, the blot was hybridized with
32P-radiolabeled MT-I or GAPDH cDNA. Quantitative
analysis of the blot was conducted on a PhosphorImager. Kb,
kilobase pairs.
|
|
The clear cut amplification of the MT locus has important implications
for metal-responsive regulation. If genomic amplification of the MT
locus in the LZA-LTK
cell line leads to multiple endogenous
MREs, these could sequester a limiting MRE-binding factor and thus
prevent zinc-responsive expression from the transfected reporter
construct (Fig. 7A). Alternatively, the overexpression of
the metallothionein protein in the LZA-LTK
cell line may
abrogate activation of the metal-responsive element regulatory system.
Since the high constitutive levels of MT would immediately bind labile
metal as it entered the cell, the availability of the cellular Zn(II)
would be limited and perhaps prevented from reaching levels necessary
to activate MRE-binding factors such as MTF-1. Experiments were
conducted in which increasing amounts of the MRE-
Geo plasmid were
transfected into the parental LTK
and LZA-LTK
cell lines grown in 50 µM [Zn]media.
Increased
-galactosidase activity was observed as the amount of
plasmid transfected into the LTK
cell line was increased
(Fig. 7B), indicating that increased levels of MRE from the
transfected constructs can recruit the requisite zinc-responsive
activating factors. Conversely, there was a lack of increased
-galactosidase activity in LZA-LTK
cells grown in 50 µM [Zn]media as the amount of transfected
construct was increased. Although it is possible that the endogenous,
amplified MREs within the LZA-LTK
cell line monopolize
MRE-binding activation factors such as MTF-1 and thus deprive the
transfected constructs, we consider it more likely that the
overexpressed MT protein suppresses labile intracellular zinc to levels
below the threshold needed to activate MTF-1. Supporting evidence for
this conclusion comes from previous studies, which demonstrated that
cell lines in which the MT locus had been amplified 10-12-fold still
retain the ability to induce metallothionein by heavy metals at the
transcriptional level (49, 50). Furthermore, transgenic mice that carry
56 additional copies of the mouse MT-I locus also retain the ability to
induce MT protein levels in response to high levels of zinc and cadmium
(51, 52). The simplest interpretation is that the high constitutive
levels of MT mRNA in the LZA-LTK
cells are not
regulated at the level of MRE/MTF-1-dependent transcription.
LTK
Status of the LZA-LTK
Cells Does
Not Contribute to Their Survivability in Zinc-deficient
Conditions--
Experiments were conducted to determine if the
LTK
cells have a proliferative advantage in
zinc-deficient media because of their independence from thymidine
kinase, an enzyme that has been reported to be extremely sensitive to
zinc-deficient conditions (53, 54). The parental L cell line is
dependent on this enzyme for efficient growth. Fresh aliquots of both
the L-M(TK
) (ATCC CCL 1.3) and L (ATCC CCL 1) cell lines
were obtained through the American Type Culture Collection (Rockville,
MD) and established as continuous cultures in unmanipulated media. Each
cell line was then transferred into the zinc-stripped media (<60
nM zinc) and was passaged every fourth day in the media.
Periodically, extracts generated from the cells were measured for
intracellular zinc content. The levels of the metal immediately dropped
to 0.1 fmol zinc/cell in each cell line within 24 h of being
placed into the zinc-deficient media (Fig.
9). Unlike the previously cited attempts
at establishing a cell line in zinc-deficient conditions, neither
culture stopped proliferating, although the morphology of both cell
lines did rapidly change to an elongated, spindly appearance as
reported for the LTK
cells (19). Furthermore, both cell
lines were able to regain the original level of 0.4 fmol zinc/cell
after 36 days of continuous culture. These results indicate the
thymidine kinase status of the LTK
cells does not affect
their ability to proliferate in zinc-deficient media. These findings
also demonstrate the reproducibility of the selection process for cells
that grow in a low zinc medium.

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Fig. 9.
Analysis of intracellular zinc levels of LTK-
and L cells after their transfer into zinc-deficient media.
LTK (filled circles) and L
(open triangles) cell cultures were established
from frozen ATCC stocks in unmanipulated 10% calf serum/Dulbecco's
modified Eagle's medium prior to exposure to the zinc-deficient media.
During the course of the experiment, the cultures were passaged every
fourth day. Periodically, the cells were harvested, counted, and
measured for intracellular metal content by ICP-AES. S.D. was
calculated from n = 3 plates at each time point.
|
|
 |
DISCUSSION |
Upon encountering conditions of extreme zinc deficiency, mouse
fibroblasts undergo a series of changes that ultimately allow them to
survive deprivation of this essential nutrient. One dramatic difference
observed between the cells that have been subjected to prolonged zinc
deficiency (LZA-LTK
) and the parental LTK
cells is a large increase in basal MT mRNA levels. The
opposite was anticipated, because MT gene transcription is normally
induced by high, not low, concentrations of heavy metals and because
the only known role attributed to MT is in metal ion detoxification. Furthermore, high levels of the cysteine-rich MT protein would be
expected to compete with cellular zinc-binding molecules for the
limited available supply of the metal. Clearly, MT is not simply
sequestering or irreversibly tying up zinc.
Three key biochemical observations establish that MT protein
participates in a zinc-scavenging system in the LZA-LTK
cell line. First, LZA-LTK
cells grown at the highest
[Zn]media tested accumulate more than 6 times the amount
of intracellular zinc than their LTK
counterparts (8.6 fmol of zinc/cell versus 1.4 fmol of zinc/cell). Fractionation experiments led us to conclude that >90% of the zinc is
associated with MT protein. Second, intracellular zinc levels increase
in the LZA-LTK
cells in response to the addition of very
small amounts of zinc into the zinc-deficient medium. For example, a
2.5-fold increase in intracellular zinc was measured in the
LZA-LTK
cell line upon the addition of 400 nM
zinc into the growth medium. Conversely, intracellular zinc does not
change in the parental LTK
cell line until
[Zn]media exceeds 20 µM. This threshold
value of 20 µM [Zn]media corresponds to the
concentration of zinc at which increases in MT mRNA and protein
levels are first detected in the parental LTK
cells. A
2.5-fold increase in intracellular zinc was not observed in the
parental LTK
cell line until [Zn]media
reached 40 µM, or 100 times the level of
[Zn]media required for a similar increase of
intracellular zinc in LZA-LTK
cells. Third, the amounts
of MT protein and intracellular zinc correlate well with the observed
depletion of extracellular zinc in the spent growth medium in the
LZA-LTK
cell line. MT protein levels and intracellular
zinc levels rose in parallel with increasing [Zn]media.
No measurable zinc remained in the spent growth medium until
intracellular MT levels reached a plateau at 3.5 µM
[Zn]media. Thus, in these zinc-limiting conditions, MT
appears to act as a labile sponge or a buffering component that
facilitates cellular retention of the available portion of zinc that
exceeds the basal cellular requirements of ~0.4 fmol zinc/cell. Once
this pool of MT protein is saturated with the metal, then external zinc
is readily measured in the spent growth medium.
Recent genetic studies have provided complementary evidence that MT is
able to exert a cytoprotective role in organisms exposed to zinc
deficiency. Transgenic mouse embryos that express multiple copies of
the MT gene demonstrated a greater resistance to developmental defects
caused by zinc deficiency than did wild-type embryos (55). Furthermore,
mouse strains in which the MT-I and MT-II alleles were disrupted
demonstrated a much greater sensitivity to the phenotypic effects of
zinc deficiency, such as malformed kidney structures, than their
wild-type counterparts (56). These authors suggested that MT-bound zinc
may serve as a reservoir that can be accessed and utilized at times of
zinc deficiency. However, we propose that MT also plays a role in
scavenging zinc from extracellular sources when intracellular supplies
of this metal are low. Indeed, MT may bind zinc entering the cell and
subsequently chaperone the metal to specific storage sites or proteins
in a manner that is analogous to the protein metallochaperone, Atx1,
which delivers copper to specific intracellular vesicle proteins (57).
It is clear that zinc-laden MT can readily donate its metal cargo to restore the function of inactive apoenzymes in vitro,
including alcohol dehydrogenase, aldolase, thermolysin, alkaline
phosphatase, carbonic anhydrase, TFIIIA, and Sp1 (22, 58-60). To date,
however, there is no evidence for direct MT interaction with specific
partners. This leaves open the question of a metallochaperone function
for MT.
Amplification of the MT gene locus in the LZA-LTK
cell
line explains, at least in part, the high levels of MT mRNA. Cell
lines or organisms that have increased copy number of specific genes can be selected by environmental stress (61). Numerous studies have
demonstrated that amplification of the MT gene locus in mammalian and
yeast cells can be selected by continuous exposure to high concentrations of cadmium, copper, or metal-based drugs such as cisplatin (27, 45, 47, 62). In these cases, cells containing the
amplified genes produce elevated levels of MT protein compared with
those that contain only a single copy of the gene. Under conditions of
exposure to high levels of heavy metals, abundant MT provides a
cytoprotective role by sequestering the potentially harmful ions from
the rest of the cell. We propose that elevated levels of MT can also
provide a zinc-scavenging mechanism and thus a selection for
amplification of the MT gene in cells exposed to zinc-deficient
conditions. Yet, mechanisms in addition to gene amplification cannot be
ruled out to explain the high levels of MT mRNA in response to the
zinc-deficient conditions. For instance, it has been recently
demonstrated that interleukin-1
-induced MT-I gene expression is
markedly enhanced in rats fed zinc-restrictive diets (15). Additional
processes may also participate in elevation of MT mRNA levels in
the zinc-deprived cell lines.
A Model for the Zinc-scavenging Role of MT in Conditions of Extreme
Zinc Deprivation--
Regardless of the mechanism by which the
LZA-LTK
cells accumulate high levels of MT mRNA, an
increase in the steady state concentration of apo-MT is expected. The
apoprotein is predicted to be rapidly proteolyzed in vivo
when [Zn]media levels are insufficient to saturate the MT
protein pool. Yet even at low [Zn]media, small "steady-state" levels of apo-MT may persist and bind zinc as it is
transported in from the environment, trapping the ion within the cell.
Since MT is being continuously produced from the large, constitutive MT
mRNA pool in the LZA-LTK
cells, enough molecules of
apo-MT are present at any given time in the cell to retain additional
zinc as it becomes available (Fig. 10).
Although the assiduous production and degradation of MT may seem like a
futile and energetically unfavorable cycle, it apparently provides a
mechanism by which the LZA-LTK
cells can efficiently
scavenge zinc from the environment, thus permitting survival under
conditions in which the availability of the ion is severely
restricted.

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Fig. 10.
Model of zinc ion homeostasis in the
LZA-LTK cell line. Zinc ions are represented by the
shaded circles, and apo-MT was drawn to resemble
a cylinder on a revolver. The packed
bundles represent transporter proteins that have been
identified in mammalian or yeast cells (24, 64, 67, 68).
Black boxes represent putative interactions of
zinc-loaded MT that may donate its cargo to nascent, zinc-requiring
polypeptides or to cellular compartments responsible for the storage of
loosely complexed zinc. See text for the discussion of the interactions
between the components.
|
|
This amplification of the MT gene locus is the result of a long term
process that requires many cell doublings. Increased MT levels may not
be utilized by a cell to deal with short term metal ion deficiencies.
Indeed, no appreciable increase in MT mRNA levels was noted in the
parental LTK
cells after transfer into zinc-deficient
media, even over a period of several days. Since BALB/c 3T3 cells were
unable to tolerate even one passage in the zinc-deficient medium
without entering quiescence (19), the LTK
cell line may
have characteristics in addition to the ability to amplify the MT gene
locus that allow survival and that are not shared by all immortalized
mouse cell lines. One difference between the BALB/c 3T3 and
LTK
cell lines is that the latter lacks functional
thymidine kinase, an enzyme that has previously been reported to be
extremely sensitive to zinc-deficient conditions (53, 54). However,
both L and L-M(TK
) cell lines had similar intracellular
zinc profiles when exposed to zinc-deficient conditions, suggesting
that the ability to utilize functional thymidine kinase does not affect
the ability of these two cell lines to proliferate in zinc-deficient
media. Consistent with the requirement of events in addition to MT
amplification, the constitutive overexpression of either MT-I or MT-III
was insufficient for long term survival of baby hamster kidney cells, a
line that also lacks thymidine kinase, under zinc-restrictive
conditions (63). These results lead us to conclude that high levels of MT expression alone are not responsible for the robustness of the
adapted L cells in response to zinc limitation.
Thus, in addition to increasing the steady-state concentration of
apo-MT, it is likely that the LZA-LTK
cell line
up-regulates other components of the zinc homeostasis machinery (Fig.
10) in response to the zinc-deficient conditions. For example, a family
of homologous mammalian proteins, designated as ZnT-1, ZnT-2, ZnT-3,
and ZnT-4, thought to encode zinc transport proteins, has recently been
identified (24, 64-66). ZnT-1, the first member isolated, appears to
be responsible for pumping excess zinc out (efflux) of the cell. ZnT-2
encodes a protein that sequesters zinc into intracellular storage
compartments. However, none of these proteins appears to play an
important role in shuttling zinc into the cell. Eide and co-workers
(67, 68) have recently identified two genes in Saccharomyces
cerevisiae, designated as ZRT1 and ZRT2
(zinc-regulated transporters),
which appear to encode zinc transporters of a high affinity and low
affinity uptake system, respectively, the former of which is
up-regulated under conditions of zinc deficiency.
Zinc is an essential structural component in a wide variety of
proteins, and therefore the maintenance of appropriate intracellular zinc levels is critical for cell survival. Although the exact function
of the metallothionein proteins remains elusive (69), one proposed role
for the protein is to maintain cellular homeostasis by metal ion
detoxification, because transcription of MT can be induced by high
levels of heavy metals. We have shown that the amplification of the MT
gene locus can occur in response to conditions of extreme zinc
deprivation. This finding represents the first example of increased MT
expression in response to low concentrations of metals and implies that
MT can act as a survival factor by assisting cells in sequestering
limited supplies of available zinc from the environment. The derivation
and characterization of this cell line should also be useful in
identifying other components of the zinc homeostasis system.
 |
ACKNOWLEDGEMENTS |
We thank Richard Palmiter for the generous
gift of the mouse MT-I cDNA and the MRE-
Geo plasmid;
Richard Morimoto for the GAPDH cDNA; and members of the
O'Halloran group, in particular Rama Dwevedi, Sarwar Nasir, and
Christoph Farhni for helpful discussions. Additionally, we thank Kelly
Mayo, Richard Gaber, Val Culotta, and Karla Kirkegaard for constructive
discussions and critical reading of this manuscript.
 |
FOOTNOTES |
*
This work was supported by the Robert H. Lurie Cancer Center
Northwestern University Grant P30 CA60553 and National Institute of
Health Grants R01 DK52627 and R01 GM38784 (to T. V. O.) and R01
HD29962 (to D. I. H. L.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Microbiology and Immunology, Stanford
University, Palo Alto, CA 94305-5124.
To whom correspondence should be addressed. Tel.:
847-491-5060; Fax: 847-491-7713; E-mail: t-ohalloran{at}nwu.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
MT, metallothionein;
ICP-AES, inductively coupled plasma-atomic emission
spectroscopy;
GAPDH, glyceraldehyde-2-phosphate dehydrogenase;
MOPS, 3-(N-morpholino)propanesulfonic acid;
TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine;
MRE, metal-responsive element;
PBS, phosphate-buffered saline;
RSV, Rous sarcoma virus;
CAT, chloramphenicol acetyltransferase;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
 |
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