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The Influence of Zinc on Caspase-3 and DNA Breakdown in Cultured Human Retinal Pigment Epithelial Cells
John P. M. Wood, DPhil;
Neville N. Osborne, PhD, DSc
Arch Ophthalmol. 2001;119:81-88.
ABSTRACT
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Objectives To investigate the role of extracellular zinc on the death process of
cultured human retinal pigment epithelial (RPE) cells.
Methods Confluent cells on borosilicate glass coverslips were treated with substances
in serum-free growth medium for various times and were analyzed for death
by means of changes in morphologic features, numbers of attached cells, and
terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate
nick-end labeling (TUNEL) procedure. Some cultures were also exposed to experimental
ischemia (defined as a lack of oxygen, glucose, and serum). Electrophoresis
and Western blotting and enzyme assays were used to investigate changes in
expression of the protease enzyme, caspase-3.
Results Experimental ischemia caused death of RPE cells. Zinc sulfate had no
effect on these cells at low concentrations (100 pmol/L to 10 nmol/L), but
protected them at higher concentrations ( 10 µmol/L) and appeared
to exacerbate cell death at still greater concentrations. Moreover, zinc compounds
(>10 µmol/L) also induced death of cells in control cultures that could
be blocked by zinc chelators and partially by the caspase-3 inhibitor, DEVD-FMK.
Zinc also increased the amount of the active form of caspase-3 in RPE cells.
Conclusions Zinc salts protect RPE cells from experimental ischemia–induced
death at low concentrations (100 pmol/L-10 nmol/L). However, at higher concentrations,
zinc causes cell death and alters the cellular level of caspase-3. These observations
are consistent with the death process being apoptosis.
Clinical Relevance Zinc supplements are taken by many individuals. Low doses of zinc can
protect RPE cells against ischemic-type insults as may occur in certain ocular
complaints. Furthermore, high concentrations of zinc can damage RPE cells.
Because zinc ions are known to be taken up by RPE cells from the choroidal
circulation, the actual therapeutic dose taken by patients is critical.
INTRODUCTION
ZINC IS one of the most abundant trace elements in nervous tissues,
including the neural retina and the associated retinal pigment epithelium
(RPE).1-2 Zinc ions bind to and
contribute to the actions of more than 400 metalloproteins that play a part
in nucleic acid and protein synthesis, energy metabolism, and intracellular
signaling.3 By far the most studied roles for
zinc in mammalian cells, however, are those involving maintenance of cellular
antioxidation states, by acting on catalase, copper-zinc superoxide dismutase,
metallothionein, and some nicotinamide adenine dinucleotide phosphate oxidases.3 Deficiency of zinc ions causes night blindness, impaired
dark adaptation, and reduced visual acuity that can be reversed by dietary
supplementation of this ion.4
Contradictory roles for zinc have been described in the induction, maintenance,
and inhibition of apoptotic cell death.1, 5-6
Apoptosis is a form of programmed death that is under tight genetic control
and that involves removal of damaged or superfluous cells without inflammation,
eg, during aging and development.7-9
Apoptosis is characterized by cytoplasmic and nuclear shrinkage and precise
internucleosomal chromatin cleavage.10-11
It has been shown in different cells that zinc supplementation prevents apoptosis
induced by a wide variety of agents, and that cells grown under conditions
of zinc deficiency or in the presence of zinc chelators can undergo this death
process spontaneously.1, 5 It is
thought that zinc acts as an inhibitor of the endonuclease that is responsible
for apoptotic DNA degradation.12 Furthermore,
it has been reported recently that zinc acts as a potent inhibitor of the
protease caspase-3 (CPP32/yama/apopain).13
This protease is frequently activated in mammalian cell apoptosis to cleave
key cellular proteins, leading to completion of the apoptotic process once
the cell has been committed to die.14-16
Evidence exists, however, that zinc may actually induce apoptosis at physiologic
concentrations, and this has been demonstrated in pancreatic acinar cells17 and mouse thymocytes.18-20
It is now believed, therefore, that the concentration of this ion is important
in determining whether it acts as an activator or inhibitor of the death process.
The RPE forms a cellular monolayer that lies immediately posterior and
in juxtaposition to the retina, playing a vital role in the maintenance of
the normal functioning of this tissue. Damage to the RPE will necessarily
affect the retina and may lead to its degeneration secondary to an initial
insult. It has been suggested that RPE cells die by apoptosis in conditions
such as age-related macular degeneration, retinal-choroidal ischemia, or proliferative
vitreoretinopathy and as a result of intense light damage.21-24
Moreover, in vitro, RPE cell apoptosis can be induced by nutrient deprivation
(experimental ischemia),25-26
inhibition of glutathione biosynthesis,27 or
hypoxia.28 Therefore, any compounds that prevent
or reduce incidences of RPE apoptosis, or indeed any other modes of RPE cell
death, should be identified to preserve retinal function in relevant ocular
disease states.
Zinc is known to be involved in the maintenance of RPE cell antioxidant
levels,29-30 and it is also suspected
that oxidative insults may contribute to the development of diseases such
as age-related macular degeneration.31-33
Dietary zinc supplementation has therefore been suggested as a measure to
counteract ocular damage resulting from this and other similar conditions.33-35 Because raised intracellular
levels of oxidative agents (reactive oxygen species) are also involved in
death processes,36 zinc may have a protective
effect on death of RPE cells. Our aims, therefore, were to investigate the
effects of zinc on experimental ischemia–induced death of cultured human
RPE cells. Furthermore, a range of zinc concentrations were investigated to
see whether zinc had any detrimental effects on RPE cells.
MATERIALS AND METHODS
MATERIALS
Deoxyribonuclease 1, proteinase K, and biotin-16-2'-deoxyuridine-5'-triphosphate
(biotin-16-dUTP) were obtained from Boehringer Mannheim (Lewes, England).
Terminal deoxynucleotidyl transferase was obtained from Promega (Southampton,
England) and avidin-biotin complex kits from Vector Laboratories (Peterborough,
England). Fetal bovine serum (approved by the European Community), Hams F-10,
amphotericin B (Fungizone), glutamine, 0.25% (weight to volume) trypsin solution,
and 24-multiwell plates (NUNC) were from GIBCO (Paisley, Scotland); 25- and
75-cm2 tissue culture flasks were obtained from Falcon (Oxford,
England). Monoclonal anti–caspase-3 antibody was obtained from Signal
Transduction Labs (distributed by Affinity, Exeter, England). The caspase-3
activity assay kit was obtained from Clontech Labs Inc (Palo Alto, Calif),
including the caspase-3 inhibitor DEVD-FMK. All other chemicals were obtained
from Sigma-Aldrich (St Louis, Mo).
HUMAN RPE CULTURE
Postmortem donor human eyes (donors aged 7, 26, 48, 54, and 58 years)
were obtained without their cornea (for transplantation purposes) from Bristol
Eye Bank, Bristol, England, up to 48 hours after enucleation. These were used
immediately or stored for up to 24 hours at 4°C. All culture work was
undertaken in a sterile laminar flow hood (ICN Flow, Thame, England). Cultures
of RPE cells were prepared and characterized by labeling for cytokeratin (KG 8.13) as described previously.37 Culture
medium consisted of Hams-F10 supplemented with 10% (volume-volume) fetal bovine
serum, 0.4% glucose, 2-mmol/L glutamine, amphotericin B (25 µg/mL),
and gentamicin (100 µg/mL). Primary cultures were grown in 25-cm2 culture flasks and passaged in a ratio of 1:3, and thereafter, in
75-cm2 flasks. While growing, cultures were kept in an incubator
at 35.5°C, with saturating humidity and an atmosphere of 5% carbon dioxide
to 95% air. After reaching the third passage, some cells were transferred
to 13-mm glass coverslips in 24-well plates at an approximate seeding density
of 2.0 x 104 cells per well.
EXPERIMENTAL ISCHEMIA
Confluent RPE cells on coverslips were transferred to serum-free culture
medium for 4 hours before the experiments. Cultures were then transferred
in fresh serum-free medium to a sealed glass chamber with saturating humidity,
within the normal incubator, which was linked to a reservoir of oxygen-free
gas mixture (95% nitrogen to 5% carbon dioxide). This delivered anoxic conditions
to the cells. Once the cultures were inside, the chamber was completely sealed,
and the anoxic gas mixture passed through for 3 hours to ensure that all oxygen
was removed. Hypoglycemia was achieved by incubating the cells with serum-free
medium lacking additional glucose. The combination of anoxic and hypoglycemic
conditions was defined as experimental ischemia.
EXPERIMENTAL DESIGN
To assess whether zinc could protect against apoptotic death that was
induced by experimental ischemia, increasing concentrations of zinc sulfate
were added to cells that were to be deprived of nutrients for 48 or 72 hours.
To see the effect of zinc on untreated cultures, zinc sulfate or other zinc
salts also were added at different concentrations to control cells for varying
periods. In other instances, different compounds were coincubated with zinc
(fetal calf serum, melatonin, flupirtine gluconate, EDTA, diethyldithiocarbamate
[DDCA], and cycloheximide) in serum-free medium at the concentrations described.
To determine the effectiveness of caspase-3 inhibition in the prevention of
cell death, cultures were preincubated with DEVD-FMK for 24 hours before insult
(10 or 100 µmol/L).
At the appropriate times, coverslips were removed and cells were fixed
with 4% paraformaldehyde in phosphate-buffered saline solution (137-mmol/L
sodium chloride, 5.4-mmol/L potassium chloride, 1.28-mmol/L sodium dihydrogen
phosphate, and 7-mmol/L disodium hydrogen phosphate [pH, 7.4]) for 30 minutes.
Some cells were visualized with a solution of toluidine blue (0.5% toluidine
blue, 0.5% thionine, and 1% sodium tetraborate) to enable culture density
(total number of cells per square millimeter) to be determined. Other cells
were analyzed for DNA degradation by staining with the terminal deoxynucleotidyl
transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)
method. A single culture density determination was obtained by using a standard
hemocytometer to count and then average the amount of cells from 4 different
fields. Statistical analyses were performed by using the paired t test to compare experimental counts with those obtained from parallel
control cultures. A value of P<.05 was considered
significant.
Other cultures were assessed for activity and expression of the enzyme
caspase-3 using a spectrophotometric assay and using electrophoresis and Western
blotting analysis, respectively. The cells that were analyzed for caspase-3
expression and activity were grown to confluence in 75-cm2 culture
flasks for use.
For each set of analyses, cultures derived from different donors were
used in a randomized way, ensuring that at least 3 different cell lines were
represented in each case. Values presented for n
in each case refer to replicates from different experiments.
ASSESSMENT OF DNA BREAKDOWN USING THE TUNEL METHOD
This method was performed as described previously.37
Briefly, cells were treated with 1% hydrogen peroxide, and then the free DNA
ends were labeled by incubating in buffer (30-mmol/L Tris hydrochloride [pH,
7.2] containing 140-mmol/L sodium cacodylate and 1-mmol/L cobalt chloride)
along with 0.25 U/µL of terminal deoxynucleotidyl transferase and 10-µmol/L
biotin-16-dUTP. The reaction was stopped by washing (2 x 15 minutes)
in saline sodium citrate buffer (30-mmol/L sodium citrate with 300-mmol/L
sodium chloride). After an additional wash in 2% bovine serum albumin (in
phosphate-buffered saline solution) for 15 minutes, stained nuclei were visualized
using an avidin-biotin-peroxidase complex kit with 3-3'-diaminobenzidine
and 0.1% hydrogen peroxide as substrates. The number of cells with positive
TUNEL findings was determined as for the total counts with a hemocytometer.
CASPASE-3 ACTIVITY ASSAY
After the appropriate drug incubation times, adherent cells were washed
and harvested in phosphate-buffered saline solution (35.5°C) using a cell
scraper. Cell counting was used to determine that aliquots of 2 x 106 cells were assayed in each case. These were collected by centrifugation
(80g for 8 minutes at 4°C). Cells were then lysed
and incubated with the reaction substrate (peptide with the sequence DEVD,
conjugated to p-nitroanilide) for 1 hour at 37°C
in a reaction buffer as described in the manufacturer's instructions and by
Gurtu et al.38 Samples were read on a spectrophotometer
at a wavelength of 405 nm. The change in optical density values in the apoptotic
samples relative to control samples indicates the increase in DEVD-dependent
caspase-3 activity in these samples.
ELECTROPHORESIS AND WESTERN BLOTTING
Confluent RPE cells grown in 75-cm2 flasks were harvested
as per the cells assayed for caspase-3 activity. Cells were sonicated in buffer
(20-mmol/L Tris hydrochloride [pH, 7.4] containing 2-mmol/L EDTA, 0.5-mmol/L
ethyleneglycoltetracetic acid, and 0.1-mmol/L phenylmethylsulfonyl fluoride).
An equal volume of sample buffer 62.5-mmol/L Tris hydrochloride [pH, 7.4]
containing 4% sodium dodecyl sulfate, 10% glycerol, 10%  -mercaptoethanol,
and 0.002% bromophenol blue) was added, and samples were boiled for approximately
3 minutes. An aliquot was taken for the determination of protein concentration
using the method of Bradford.39 Electrophoresis
of samples was performed using the method of Laemmli40
with 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate. Proteins
were transferred to nitrocellulose41 and blots
were stained as previously described37 for
the presence of caspase-3.
RESULTS
The RPE cells died when subjected to a deprivation of oxygen, glucose,
and serum from their growth medium (experimental ischemia) (Table 1). As reported previously,26
after 72 hours of experimental ischemia, culture density was reduced and nuclear
labeling by means of the TUNEL procedure increased when compared with untreated
cells.
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Table 1. Effects of Experimental Ischemia and Substances on Induction
of RPE Cell Apoptosis After 72 Hours*
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Zinc had no obvious effect on cultures at low concentrations (100 pmol/L
to 10 nmol/L), but protected cells from experimental ischemia (48 or 72 hours)
at higher concentrations (10 µmol/L) as indicated by a greater density
of cells associated with the coverslips (Figure 1A) and a reduced amount of TUNEL-positive nuclei (Figure 1B). At concentrations higher than
10 µmol/L, zinc exacerbated cell death caused by experimental ischemia.
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Figure 1. Graphs showing the effect of zinc
sulfate on cells undergoing death caused by experimental ischemia for 48 or
72 hours. A, Change in cell culture density as the concentration of zinc sulfate
is increased. B, Change in the percentage of nuclei with positive results
of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate
nick-end labeling (TUNEL) as the zinc salt is increased. Data are expressed
as mean ± SEM, with 8 determinations for each data point.
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When control cultures were treated with concentrations of zinc sulfate
higher than 10 µmol/L, large numbers of cells died (Figure 2 and Figure 3).
Cell shrinkage and TUNEL assessment suggested this death process to be apoptosis
(Figure 2). As can be seen in Table 1, the effects of other zinc salts
(zinc acetate and zinc chloride) did not vary significantly from those of
zinc sulfate in this regard.
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Figure 2. Application of morphologic analysis
(A and B) and results of terminal deoxynucleotidyl transferase–mediated
deoxyuridine triphosphate nick-end labeling (TUNEL) assay (C and D) to human
retinal pigment epithelial (RPE) cells (donor aged 54 years; third passage)
after treatment for 72 hours with zinc sulfate, 100 µmol/L, to indicate
the presence of dying cells. Control cells show no death (A and C). Shrunken
cells (B) and cells with TUNEL-positive nuclei (D; arrows) are seen after
treatment with the zinc salt. E, Positive control for the detection of chromatin
degradation by means of the TUNEL reaction with cells treated for 15 minutes
at 37°C with deoxyribonuclease 1. F, Third-passage cells stained for the
uniform localization of cytokeratin (KG 8.13). Scale bar indicates
10 µm.
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Figure 3. Graphs showing the effect of increasing
concentrations of zinc sulfate on density of cells in culture (A) and percentage
of cells exhibiting nuclei with positive results of terminal deoxynucleotidyl
transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL)
(B) during a time-course of 72 hours. As the concentration of the zinc salt
is increased, cells are lost (A), and there is an increase in the TUNEL labeling
of the ones that remain (B). Controls indicate untreated cells that remain
unchanged in each case. Data are expressed as mean ± SEM, with 8 determinations
for each data point.
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The mechanism of cell death induced by zinc sulfate appeared to differ
from that of experimental ischemia, as substances that have been shown previously
to attenuate the latter process had no effect on zinc-induced cell death (Table 1) (100-µmol/L flupirtine,
100-µmol/L melatonin, and 10% [volume-volume] fetal calf serum). The
cation chelator, EDTA, and to a lesser extent, the zinc chelator, DDCA, significantly
attenuated RPE cell death induced by zinc, however (Table 1).
Caspase-3 activity (Figure 4)
was detectable in control cells that had been incubated in serum-free medium
for 24 hours. The amount of activity increased optimally by approximately
10-fold in RPE cells treated with 10-µmol/L zinc sulfate for 24 hours
(Table 2). Greater concentrations
of zinc sulfate (10 mmol/L) produced less of a measurable amount of caspase-3.
Significantly, experimental ischemia had no effect on caspase-3 (Table 2). Western blot analysis revealed
an increased presence of the 17-kd active protein for caspase-3 after incubation
of cultures with 100-µmmol/L zinc sulfate for 6 and then 24 hours, compared
with cells incubated in just serum-free medium or medium containing 10% (volume-volume)
fetal bovine serum (Figure 5). Furthermore,
there was no appearance of the 17-kd immunoreactive protein for caspase-3
in cells subjected to experimental ischemia for 24 hours (Figure 5). Coincubation of 100-µmol/L cycloheximide also had
no effect on the death of RPE cells induced by 100-µmol/L zinc sulfate
(Table 1). Zinc-induced death
but not experimental ischemia–induced cell death could be partially
ameliorated by preincubation of the cells with DEVD-FMK, a specific and irreversible
caspase-3 inhibitor, at concentrations of 100 µmol/L and, to a lesser
extent, 10 µmol/L (Table 1).
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Figure 4. Graph showing activity assay measurements
for the change in amount of caspase-3 after treatment of cultured human retinal
pigment epithelial cells with increasing concentrations of zinc sulfate. The
assay detected caspase-3 activity in 2 x 106 adherent cells
after treatment as outlined in the Materials and Methods section. Since there
is a linear relationship between the amount of caspase-3 present and the absorbance
at 405 nm, it is evident that there is a 10-fold increase over control (untreated)
levels (shaded bar) in activity of the enzyme at zinc sulfate concentrations
of up to 10 µmol/L. Results are expressed as mean ± SEM, with
5 determinations for each data point.
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Table 2. Amount of Caspase-3 Present in RPE Cell Extracts After Various
Treatments*
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Figure 5. Western blots showing the increased
presence of caspase-3 in samples derived from cultured human retinal pigment
epithelial cells that have been treated with zinc sulfate, 100 µmol/L.
Lane 1, control cells; lane 2, cells plus zinc sulfate at a concentration
of 100 µmol/L for 6 hours; lane 3, cells plus zinc sulfate at a concentration
of 100 µmol/L for 24 hours; lane 4, cells plus 10% (volume to volume)
fetal bovine serum for 24 hours; and lane 5, cells subjected to oxygen, glucose,
and serum deprivation for 24 hours. Molecular weight of detected caspase-3,
17 kd.
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COMMENT
Previous studies have shown that human RPE cells subjected to experimental
ischemia die by means of a process that is characteristic of apoptosis.25-26,37 This is because cells
treated in this way exhibit a shrunken appearance and have nuclei that stain
positively for the TUNEL reaction. Furthermore, apoptosis-related genes have
also been reported to be affected, consistent with this idea.25
It is believed, however, that the differences between distinct types of cell
death are not so clear.42 Indeed, many of the
events that have been ascribed previously to a particular mode of death have
now been shown to be less specific. Thus, although the data described herein
suggest that the death process involved is apoptosis, this cannot be stated
definitively.
The present data show that zinc sulfate has contrasting effects on cultured
human RPE cells, depending on the concentrations used. Previous reports have
demonstrated that exogenously applied zinc can have biphasic effects on nonneuronal
cells by either initiating or counteracting apoptosis.5-6,18, 43
The concentrations of zinc required to cause or prevent apoptosis, however,
seem to be variable and may depend on the cell type under investigation. In
mouse thymocytes, for example, high zinc concentrations (10 µmol/L to
1 mmol/L) inhibit serum-free medium- and dexamethasone-induced apoptosis,
whereas zinc at concentrations of less than 10 µmol/L induces apoptosis.18, 20, 43
The mechanism by which zinc induces death of RPE cells cannot be inferred
from the present data. Each of the zinc salts tested had the same ability
to initiate death of RPE cells in culture, however, confirming that it was
zinc and not the respective anion in each case that was responsible. It is
also likely that zinc is equally effective at entering RPE cells in each case.
This has been described in intact erythrocytes, which accumulated zinc sulfate,
zinc acetate, or zinc chloride from an incubation medium at the same rate.44 It is known that intracellular zinc can modulate
expression and activity of immediate early gene products1
such as c-fos or c-jun and
that these have been correlated with apoptosis, particularly in neurons.45 Furthermore, protein kinases such as protein kinase
C have high-affinity binding sites for zinc.46
This cation has a biphasic effect on protein kinase C, acting as a stimulator
at concentrations lower than 10 µmol/L and an inhibitor at higher amounts.1 The latter effect may well explain the toxic effects
of zinc that are reported in the present study, as it has already been shown
that inhibition of protein kinase C with staurosporine or polymyxin B sulfate
can cause apoptosis of human RPE cells,37 and
with hypericin can cause apoptosis of bovine RPE cells.47
It has been thought that the primary mode of action for zinc as an attenuator
of apoptosis is to inhibit the endonuclease that is responsible for internucleosomal
DNA degradation5-6,12
usually but not always accompanying this death process.48
A number of recent observations have suggested, however, that zinc may have
other cellular targets during apoptosis.49-50
One such enzyme that is activated by zinc at submicromolar concentrations
is the DNA repair enzyme poly-(adenosine diphosphate)-ribose polymerase (PARP).51 When PARP is degraded during the early stages of
apoptosis, the cell loses its capacity to repair DNA strand damage. Increases
in intracellular zinc concentrations, such as in the present study, will therefore
increase the antiapoptotic activity of PARP. Zinc is also responsible for
the activity of many antioxidant or metabolic enzymes in the RPE such as catalase,
alkaline phosphatase,  -mannosidase, and metallothionein.30
Because increases in intracellular levels of pro-oxidants are known to be
associated with apoptosis, raising the cellular supply of zinc to the level
where its action on the antioxidant status becomes optimal could counteract
cell-death processes. Furthermore, zinc has also been reported to potently
inhibit the protease enzyme, caspase-3,13 which
is involved in processing of the apoptotic signals in the regulation stages
of this type of cell death.15 It is unlikely
that this action accounts for the counteraction of experimental ischemia–induced
apoptosis in the present study, however, as there is no increase in the active
form of caspase-3 during this process (Table 2), and this cell death cannot be blocked by the caspase-3
inhibitor (Table 1).
Increases in the amount of caspase-3 present in cultures after zinc
treatment (Figure 4 and Table 2) confirm that this enzyme can be
induced in an active form during RPE cell death. A small quantity of caspase-3
is also present in control cultures incubated in serum-free medium for 24
hours, and this may reflect the low incidence of death of these cells when
incubated without serum, as shown previously.26
Because experimental ischemia did not give rise to any changes in caspase-3,
and because the irreversible caspase-3 inhibitor, DEVD-FMK, partially attenuated
zinc- but not ischemia-induced death, then it is clear that both death processes
involve distinct pathways. It is obvious, therefore, that distinct pathways
are involved in apoptotic death of certain cells and that caspase-3 does not
participate in all of these. This has been suggested previously.52
The data shown in Figure 5
indicate that detection of the active 17-kd caspase-3 enzyme is possible after
6 and 24 hours of treatment of the cultures with zinc sulfate at concentrations
of 100 µmol/L. However, the toxic effects of zinc sulfate are not blocked
in the presence of the protein synthesis inhibitor cycloheximide. Caspase-3
is therefore not produced, de novo, during this mode of death. These results
are explained by the cellular presence of procaspase-3, an inactive precursor
enzyme, which is cleaved in the early stages of an apoptotic insult by other
proteases (eg, caspase-9) to release an active 17-kd form.15
It is suspected that without a signal telling the cells to enter a death pathway,
procaspase-3 is present but inactive. In agreement with the assay data for
caspase-3, the active form of this enzyme could not be detected after 24 hours
of experimental ischemia. These data also confirm that nutrient deprivation–induced
death of RPE cells does not involve this protease. The fact that substances
that have been shown previously to block experimental ischemia–induced
death in human RPE cultures (eg, serum, melatonin, and flupirtine)25-26 have no effect on death induced by
zinc also confirms that both insults must involve distinct mechanisms.
In the present investigation, coincubation of cultures with zinc sulfate
and the metal-chelating agent EDTA led to a complete counteraction of toxic
effects. The marked decrease in RPE cell death in such instances indicates
that zinc uptake is critical for toxic events to occur, as EDTA will not pass
the plasma membrane and so must have exerted its effects extracellularly.
The more readily membrane-permeable chelator, DDCA, was not quite so effective
in counteracting cell death as EDTA. In this instance it is possible that
the chelator exerted its protective effect on RPE cells by binding extracellular
zinc, as did EDTA. Moreover, because it is known that intracellular entry
of zinc chelators will promote cell death,6, 20, 53
then it is also possible that in the present study the effect of DDCA was
manifested as an intracellular toxic effect. However, previous data (J.W.,
unpublished data, January 1998) have indicated that DDCA is not toxic to RPE
cells alone, and so the difference in counteracting zinc-induced RPE cell
death in the present study was probably caused by a greater zinc-chelating
property for EDTA.
It is obvious that the cellular effects of zinc, nutrient deprivation,
and pathways leading to death are extremely complex events that are likely
specific for cell type and paradigm. It must be borne in mind, therefore,
that the effects of zinc reported herein are relevant only for RPE cells that
have been cultured and treated under the conditions described. This means
that the effects may differ in different strains of RPE cells, in cells cultured
in different ways, under different conditions of assay, or for RPE cells in
situ. Generally, RPE cells in culture have been shown to perform a number
of in vivo functions readily, such as ingestion and degradation of photoreceptor
outer segments54-55 and uptake
of important ions such as zinc,56 and so studies
on these cells provide useful data for the understanding of their in situ
functioning. Furthermore, the use of donors of different ages for establishment
of the RPE cells used herein reveals consistency in the data, suggesting further
relevance.
With these points in mind, then, it can be stated that the present studies
show zinc to protect against or induce RPE cell death that is characteristic
of apoptosis, according to its concentration. Further studies will determine
the effects of zinc on RPE cells in vivo. It has also been reported previously
that zinc has a role in ischemic, necrotic-type cell death in the brain57-58 and the retina.59
All of these results implicate zinc involvement in cellular death processes.
Further experiments will have to be undertaken to describe the mechanism of
damage and the role of caspase-3. Indeed, a more detailed analysis of the
interplay between cultured RPE cells and zinc will need to be performed, and
this will have to be correlated with in situ data. The present data should,
however, be taken into account by individuals taking high-dose dietary supplements
of zinc, because this ion obviously can be specifically harmful to certain
cells, under certain conditions, at elevated concentrations.
AUTHOR INFORMATION
Accepted for publication June 22, 2000.
The financial assistance of The National Eye Research Centre, Bristol,
England, is gratefully acknowledged.
Reprints: Neville N. Osborne, PhD, DSc, The Nuffield Laboratory of
Ophthalmology, University of Oxford, Walton Street Oxford, OX2 6AW, England
(e-mail: neville.osborne{at}eye.ox.ac.uk or john.wood{at}eye.ox.ac.uk).
From the Nuffield Laboratory of Ophthalmology, University of Oxford,
Oxford, England.
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